Food Quality and Safety
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HIGHLIGHTS
Paper published in Food Microbiology
Rapid and sensitive detection of viable Listeria monocytogenes in food products by a filtration-based protocol and qPCR. Alejandro Garrido-Maestu,Sarah Azinheiro, Joana Carvalho, Marta Prado. Volume 73, August 2018, Pages 254–263.
Paper published in Microchemical Journal
Novel approach for accurate minute DNA quantification on microvolumetric solutions
JoanaCarvalho,RenatoNegrinho,SarahAzinheiro, AlejandroGarrido-Maestu,JorgeBarros-Velázquez.MartaPrado.Volume 138,May 2018,Pages 540-549.
Published a Research Article in Front. Sustain. Food Syst.
SarahAzinheiro,JoanaCarvalho,MartaPrado and AlejandroGarrido-Maestu. 21 February 2018.
Paper published in Analytica chimica Acta.
JoanaCarvalho,GemaPuertas,JoãoGaspar,
SarahAzinheiro,LorenaDiéguez,
AlejandroGarrido Maestu, ManuelVázquez,
JorgeBarros-Velázquez,SusanaCardoso,MartaPrado.
Volume 1020, 22 August 2018, Pages 30-40
Scientific Opinion !
The EFSA (European Food Safety Authority) has recently published a scientific opinion on an “Updated quantitative risk assessment (QRA) of the BSE risk posed by processed animal protein (PAP)” & We are happy to mention that Our Food Quality & Safety research Group leader Dr.Marta Prado has participated in this activity Find the links at The Wiley Online Library (WOL) & The EFSA website
Published a Research Article in Food Control
Author: Alejandro Garrido-Maestu, PabloFuciños, SarahAzinheiro, CarlaCarvalho, JoanaCarvalho, MartaPrado
Food Control 99 , pp. 79-83, 2019.
Book Chapter in collaboration with David Tomás Fornés, Lead Scientist in the Microbial and Molecular Analytics group at Nestlé Research Center (Lausanne, Switzerland). !!!!!! Foodborne Bacterial Pathogens
The Use of Multiplex Real-Time PCR for the Simultaneous Detection of Foodborne Bacterial Pathogens In: Bridier A. (eds) Foodborne Bacterial Pathogens. Methods in Molecular Biology, vol 1918. Humana Press, New York, NY
Reliable Nano & Micro-solutions for Food Safety & Quality Analysis
The main objective of the Food & Quality Research Group is the development of analytical approaches based on the combination of molecular biology (mainly DNA based methodology) and nano and microfabrication technology in order to provide the food industry and control laboratories with reliable analytical tools.
Following this objective, the methodology is based on working on very specific analytical needs and on using a modular approach for each of the steps of the analytical process. This approach, help us to evaluate and to choose the best method in each case, to have a sound integrated final product, and at the same time a wide-range of intermediate products that can be used by themselves to solve specific analytical challenges.
Figure 1 summarizes the overall approach and research lines. Our topics of interest involve the detection of foodborne pathogens, the detection of allergenic ingredients in food products and food authenticity.
RESEARCH LINES
- Sample preparation:
Sample preparation is the series of steps required to transform a sample to a form suitable for analysis, the reliability of the conclusions drawn from food analysis greatly depends upon on this step. We work on: (i) the development of pre-treatment steps in order to overcome some of the limitations associated with food analysis and (ii) on the development of tailored, miniaturized, automatized and faster sample preparation techniques. Microscale solid phase extraction (µSPE) is used for on-chip DNA extraction and purification, being possible to put in contact a higher volume of initial binding material with the solid phase and recover the DNA in a lower volume during the elution phase. This feature allows to concentrate the DNA when minute amounts are present in the sample (e.g. olive oil, wine), for complex matrixes such as processed foodstuff and for environmental samples (e.g. water samples).
- Alternative DNA amplification methods:
Food & Quality Safety Research Group is working on new amplification techniques, in their combination with NPs and on the evaluation of DNA based analytical methods for food analysis. We work on isothermal amplification techniques, such as Loop-Mediated Isothermal Amplification (LAMP), and Recombinase Polymerase Amplification (RPA), specially interesting for miniaturization purposes. Other alternative techniques currently being used include Ligation Chain Reaction (LCR) which allows to distinguish very closely related organisms and high similar DNA sequences.
- Nanoparticle-assisted DNA analysis:
The use of nanomaterials for DNA analysis has the potential of providing increased sensitivity, multiplexing capabilities, and reduced costs. Exploiting the features of nanoparticles (NPs) is considered to be a good alternative to foster the potential of diagnostics and analytical method development. NPs, such as gold NPs (AuNPs) and gold nanorods (AuNRs) are being used for DNA detection taking advantage of their optical properties.
Food Quality and Safety research team (picture from April 2019)
FUNDED PROJECTS
DEVELOPMENT OF A SYSTEM OF EARLY DETECTION OF THE ZEBRA MUSSEL THROUGH ANALYSIS OF ENVIRONMENTAL DNA
Cooperation Agreement with Confederación hidrográfica del Guadalquivir (CHG) Spain.
The objective of this cooperation agreement is the development of a micro Total Analysis Systems (µTAS) and optimized protocol for the detection of zebra mussel, an invasive species affecting numerous river basins worldwide, through environmental DNA (eDNA).
NANOEATERS: Valorization and transfer of NANOtechnologies to EArly adopTERS of the Euroregion Galicia-Norte Portugal-Use Case 2: Olive Oil Characterization
The FQ&S research group participates on the NANOEATERS project, on the Use Case 2: Olive Oil characterization on collaboration with the University of Vigo (Spain).
The main objective of use case 2 is the characterization of Extra virgin olive oil produced in Galicia, and the development of analytical approaches that would allow the differentiation of such olive oil from the one produced elsewhere.
N2020- NBFS-FOODSAFE
Innovative sample preparation and detection methods for foodborne pathogens, allergenic ingredients and food authenticity assessment are being developed. In parallel, new smart packaging and delivery systems are being designed and validated.
PORTGRAPHE
Control of Port and Douro Wines authenticity using graphene DNA sensors project
Time Frame: June 2018 to June 2021
The main objective of the project is the development, test and in-house validation of a miniaturized DNA sensing device for varietal discrimination of grapes, wines, musts, and grape juice in order to ensure the authenticity of wine from Port and Douro DOP. With this objective the participating teams will combine their expertise for the development of a miniaturized analytical device composed of 3 modules namely: a DNA extraction and purification module, an isothermal DNA amplification module, and a DNA Biosensors based on field-effect transistors (FETs) made using single layer graphene (SLG) for varietal discrimination.
NanoBioSensor
Development of nanosensors to evaluate the microbiological quality of fruit-based products
Time Frame: July 2018 to June 2021
A micro-total analysis system will be developed by the participating team to enable faster and better control of selected fruit-based products, reducing the analysis time from 7 days to few hours with greater sensitivity than conventional methods. This device will be designed and manufactured combining the latest developments in molecular biology, microfluidics and electronics.
SF4SF Smart Factoy for Safe Foods
Project funded by CDTI, Ministry of Industry of Spain.
The project aims the integration of emerging technologies for the detection, removal and food hazards management in food processing plants. The role of the group is the development of analytical methods based on DNA detection from gluten producing cereals and Listeria monocytogenes in fish products.
Innovative Consumer OrieNted Safe Solutions (ICONSS. Project_ 20431) Funding from EITFood BP2020
The ICONSS project objective is the development of an integrated solution targeting consumers and food operators. This integrated solution is made up of two modules: the first one, an app that will look at nutritional aspects for vulnerable population groups and create a food box from retailers ready to pick up or for home delivery, to minimize exposure in indoor environments particularly for vulnerable people. The second module, also connected with an app, will be a miniaturized device for fast, reliable and easy to use testing of workers and surfaces to ensure the safety in food-related environments, with special emphasis on retailers. This miniaturized kit and device will include sampling, a simplified RNA extraction and isothermal amplification and detection, ready to be used on decentralized settings with minimum interaction from users, and giving a simple and easy to read answer.
Webpage https://iconss.eu/
CRISPR technology against the COVID-19 pandemic. The DETECTR of SARS-CoV-2 (project n. 480) funding from FCT (Portugal)
COVID-19 pandemic is still on-going, affecting 4 million people. The viral detection relies on RT-qPCR that is sensitive and specific, but takes long time to results, needs special equipment and trained personnel. Thus, there is a need for methods equally sensitive and specific. DETECTR technology, combining RPA and CRISPR-Cas12a can fulfill these requirements. We will use it to develop a multiplex lateral flow method for naked-eye detection of the virus in only 30 min.
LAMP‐Light in the diagnosis of COVID‐19 (project n. 165) funding from FCT (Portugal)
SARS‐CoV‐2 virus is rapidly spreading. The standard to detect it is RT‐qPCR, but the method is lengthy and needs expensive equipment. In this project we will develop a SARS‐CoV‐2 detection methodology based on RT‐LAMP. The technique has a fast turnaround time, does not need specialized equipment, and detection can be performed by naked‐eye. Thus the proposed method has great potential to enhance diagnostics throughput, reduce time of analysis, and improve implementation of appropriate measures.
PUBLICATIONS
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2022
Pastrana C, Guerreiro JRL, Elumalai M, Carpena-Torres C, Crooke A, Carracedo G, Prado M, Huete-Toral F.
Dual-Mode Gold Nanoparticle-Based Method for Early Detection of Acanthamoeba Journal Article
International Journal of Molecular Sciences, 23 , pp. 14877, 2022.
@article{C2022,
title = {Dual-Mode Gold Nanoparticle-Based Method for Early Detection of Acanthamoeba},
author = {Pastrana C, Guerreiro JRL, Elumalai M, Carpena-Torres C, Crooke A, Carracedo G, Prado M, Huete-Toral F.},
url = {https://doi.org/10.3390/ijms232314877},
doi = {10.3390/ijms232314877},
year = {2022},
date = {2022-11-28},
journal = {International Journal of Molecular Sciences},
volume = {23},
pages = {14877},
abstract = {Acanthamoeba keratitis is an aggressive and rapidly progressing ocular pathology whose main risk factor is the use of contact lenses. An early and differential diagnosis is considered the main factor to prevent the progression and improve the prognosis of the pathology. However, current diagnosis techniques require time, complex and costly materials making an early diagnosis challenging. Thus, there is a need for fast, accessible, and accurate methods for Acanthamoeba detection by practitioners for timely and suitable treatment and even for contact lens user as preventive diagnosis. Here, we developed a dual-mode colorimetric-based method for fast, visual, and accurate detection of Acanthamoeba using gold nanoparticles (AuNPs). For this strategy, AuNPs were functionalized with thiolated probes and the presence of target Acanthamoeba genomic sequences, produce a colorimetric change from red to purple. This approach allows the detection of 0.02 and 0.009 μM of the unamplified Acanthamoeba genome by the naked eye in less than 20 min and by color analysis using a smartphone. Additionally, real samples were successfully analyzed showing the potential of the technology considering the lack of point-of-care tools that are mostly needed.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}Acanthamoeba keratitis is an aggressive and rapidly progressing ocular pathology whose main risk factor is the use of contact lenses. An early and differential diagnosis is considered the main factor to prevent the progression and improve the prognosis of the pathology. However, current diagnosis techniques require time, complex and costly materials making an early diagnosis challenging. Thus, there is a need for fast, accessible, and accurate methods for Acanthamoeba detection by practitioners for timely and suitable treatment and even for contact lens user as preventive diagnosis. Here, we developed a dual-mode colorimetric-based method for fast, visual, and accurate detection of Acanthamoeba using gold nanoparticles (AuNPs). For this strategy, AuNPs were functionalized with thiolated probes and the presence of target Acanthamoeba genomic sequences, produce a colorimetric change from red to purple. This approach allows the detection of 0.02 and 0.009 μM of the unamplified Acanthamoeba genome by the naked eye in less than 20 min and by color analysis using a smartphone. Additionally, real samples were successfully analyzed showing the potential of the technology considering the lack of point-of-care tools that are mostly needed.Joana Carvalho, Andrey Ipatov, Laura Rodriguez-Lorenzo, Alejandro Garrido-Maestu, Sarah Azinheiro, Begoña Espiña, Jorge Barros-Velázquez, Marta Prado
Microchemical Journal, 183 , pp. 108115, 2022.
@article{Carvalho2022b,
title = {Towards on-site detection of gluten-containing cereals with a portable and miniaturized prototype combining isothermal DNA amplification and naked eye detection},
author = {Joana Carvalho, Andrey Ipatov, Laura Rodriguez-Lorenzo, Alejandro Garrido-Maestu, Sarah Azinheiro, Begoña Espiña, Jorge Barros-Velázquez, Marta Prado},
url = {https://doi.org/10.1016/j.microc.2022.108115.},
doi = {10.1016/j.microc.2022.108115.},
year = {2022},
date = {2022-11-09},
journal = {Microchemical Journal},
volume = {183},
pages = {108115},
abstract = {Miniaturization enables the development of portable devices for rapid on-site DNA analysis, providing cheaper and more convenient systems for target DNA detection in resource-limited settings. Miniaturized devices for DNA-based analysis are highly promising analytical tools for different aims, in particular for food quality and safety analysis in the different points of the food value chain DNA amplification is a key step for the development of highly specific and sensitive detection platforms. Although PCR remains the most widely adopted DNA amplification technique, interest for isothermal DNA amplification approaches, such as loop-mediated isothermal amplification (LAMP) and recombinase polymerase amplification (RPA), has grown exponentially when considering miniaturization. In this work, a portable and miniaturized prototype has been developed to perform isothermal DNA amplification by LAMP and RPA, containing an integrated heating system, and including naked-eye detection, therefore not requiring any additional equipment for the amplification reactions. Prototype performance was evaluated and successfully demonstrated for these amplification techniques, with different detection chemistries being explored. As a proof-of-concept, performance of the integrated heating system was compared to conventional equipment for the detection of gluten-containing cereals in food products, showing similar results in terms of amplification time, specificity, and sensitivity. Amplification by RPA achieved the lowest detectable concentration, presenting a limit of detection of 0.0180 ng µL-1, being more sensitive than LAMP by one order of magnitude, and enabling amplification in less than 20 min.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}Miniaturization enables the development of portable devices for rapid on-site DNA analysis, providing cheaper and more convenient systems for target DNA detection in resource-limited settings. Miniaturized devices for DNA-based analysis are highly promising analytical tools for different aims, in particular for food quality and safety analysis in the different points of the food value chain DNA amplification is a key step for the development of highly specific and sensitive detection platforms. Although PCR remains the most widely adopted DNA amplification technique, interest for isothermal DNA amplification approaches, such as loop-mediated isothermal amplification (LAMP) and recombinase polymerase amplification (RPA), has grown exponentially when considering miniaturization. In this work, a portable and miniaturized prototype has been developed to perform isothermal DNA amplification by LAMP and RPA, containing an integrated heating system, and including naked-eye detection, therefore not requiring any additional equipment for the amplification reactions. Prototype performance was evaluated and successfully demonstrated for these amplification techniques, with different detection chemistries being explored. As a proof-of-concept, performance of the integrated heating system was compared to conventional equipment for the detection of gluten-containing cereals in food products, showing similar results in terms of amplification time, specificity, and sensitivity. Amplification by RPA achieved the lowest detectable concentration, presenting a limit of detection of 0.0180 ng µL-1, being more sensitive than LAMP by one order of magnitude, and enabling amplification in less than 20 min.Foteini Roumani, Jorge Barros-Velázquez, Alejandro Garrido-Maestu, Marta Prado
Food Control, 144 , 2022.
@article{Roumani2022b,
title = {Real-time PCR, and Recombinase Polymerase Amplification combined with SYBR Green I for naked-eye detection, along with Propidium Monoazide (PMA) for the detection of viable patulin-producing fungi in apples and by-products},
author = {Foteini Roumani, Jorge Barros-Velázquez, Alejandro Garrido-Maestu, Marta Prado},
url = {https://doi.org/10.1016/j.foodcont.2022.109347},
doi = {10.1016/j.foodcont.2022.109347},
year = {2022},
date = {2022-09-02},
journal = {Food Control},
volume = {144},
abstract = {Nowadays, the application of rapid molecular-based methods such as PCR/qPCR and isothermal techniques like Recombinase Polymerase Amplification (RPA), has increased in order to overcome some of the drawbacks of traditional culture-based methods due to their high sensitivity and specificity. One of the main limitations of these techniques is their inability to differentiate between live and dead microorganisms. Patulin is a mycotoxin typically produced by fungi belonging to Penicillium spp. that exerts acute and chronic toxic effects to humans and animals. In the present study, one real-time PCR assay, and a RPA coupled with SYBR Green I for naked-eye detection, were developed for the detection of patulin-producing fungi. Primers and a probe were designed based on the idh gene of the patulin metabolic pathway. Furthermore, propidium monoazide was implemented in the DNA extraction protocol for the specific detection of viable fungi. The developed assays were able to detect down to 1.25 pg/μL (qPCR) and 23.8 pg/μL (RPA-SG) of pure P. expansum DNA. Finally, when artificially inoculated apples, and by-products, were analysed the LOD50 of the qPCR was found to be 8.1 × 103 spores/5 g of food sample. In the case of the RPA-SG, the determined LOD50 was 5.8 × 104 spores/5 g.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}Nowadays, the application of rapid molecular-based methods such as PCR/qPCR and isothermal techniques like Recombinase Polymerase Amplification (RPA), has increased in order to overcome some of the drawbacks of traditional culture-based methods due to their high sensitivity and specificity. One of the main limitations of these techniques is their inability to differentiate between live and dead microorganisms. Patulin is a mycotoxin typically produced by fungi belonging to Penicillium spp. that exerts acute and chronic toxic effects to humans and animals. In the present study, one real-time PCR assay, and a RPA coupled with SYBR Green I for naked-eye detection, were developed for the detection of patulin-producing fungi. Primers and a probe were designed based on the idh gene of the patulin metabolic pathway. Furthermore, propidium monoazide was implemented in the DNA extraction protocol for the specific detection of viable fungi. The developed assays were able to detect down to 1.25 pg/μL (qPCR) and 23.8 pg/μL (RPA-SG) of pure P. expansum DNA. Finally, when artificially inoculated apples, and by-products, were analysed the LOD50 of the qPCR was found to be 8.1 × 103 spores/5 g of food sample. In the case of the RPA-SG, the determined LOD50 was 5.8 × 104 spores/5 g.Azinheiro, S., Roumani, F., Prado, M. , Garrido-Maetsu, A.
Rapid Same-Day Detection of Listeria monocytogenes, Salmonella spp., and Escherichia coli O157 by Colorimetric LAMP in Dairy Products. Journal Article
Food Anal. Methods (2022), 2022.
@article{Azinheiro2022b,
title = {Rapid Same-Day Detection of Listeria monocytogenes, Salmonella spp., and Escherichia coli O157 by Colorimetric LAMP in Dairy Products.},
author = {Azinheiro, S., Roumani, F., Prado, M. , Garrido-Maetsu, A.},
url = {https://doi.org/10.1007/s12161-022-02345-9},
doi = {10.1007/s12161-022-02345-9},
year = {2022},
date = {2022-06-18},
journal = {Food Anal. Methods (2022)},
abstract = {Foodborne illnesses are being reported everyday; thus, there is an obvious need for faster and sensitive methodologies to detect foodborne pathogens in order to assure the safeness of foods. In the present study, the detection of L. monocytogenes, Salmonella spp., and E. coli O157 was performed combining a multiplex short enrichment of 7 h in Tryptic Soy Broth, with a colorimetric LAMP-based naked-eye detection. The LAMP reaction obtained a similar sensitivity to that of real-time PCR. The methodology was evaluated in UHT, fresh and raw milk were tested, achieving a LoD95 of 1.6 CFU/25 mL for Salmonella spp. and E. coli O157 respectively without matrix interference, and for L. monocytogenes, the LoD95 was calculated to be 79.0 CFU/25 mL, showing some interference when a higher natural microflora was present in the sample. The methodology can be applied in the food industry with reliability, as the evaluation obtained a k index of 1.0 for L. monocytogenes and Salmonella spp. and 0.94 for E. coli O157.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}Foodborne illnesses are being reported everyday; thus, there is an obvious need for faster and sensitive methodologies to detect foodborne pathogens in order to assure the safeness of foods. In the present study, the detection of L. monocytogenes, Salmonella spp., and E. coli O157 was performed combining a multiplex short enrichment of 7 h in Tryptic Soy Broth, with a colorimetric LAMP-based naked-eye detection. The LAMP reaction obtained a similar sensitivity to that of real-time PCR. The methodology was evaluated in UHT, fresh and raw milk were tested, achieving a LoD95 of 1.6 CFU/25 mL for Salmonella spp. and E. coli O157 respectively without matrix interference, and for L. monocytogenes, the LoD95 was calculated to be 79.0 CFU/25 mL, showing some interference when a higher natural microflora was present in the sample. The methodology can be applied in the food industry with reliability, as the evaluation obtained a k index of 1.0 for L. monocytogenes and Salmonella spp. and 0.94 for E. coli O157.Agnes Purwidyantri, Andrey Ipatov, Telma Domingues, Jérôme Borme, Marco Martins, Pedro Alpuim, Marta Prado
Programmable graphene-based microfluidic sensor for DNA detection Journal Article
Sensors and Actuators B: Chemical, 367 , pp. 132044, 2022.
@article{Purwidyantri2022,
title = {Programmable graphene-based microfluidic sensor for DNA detection},
author = {Agnes Purwidyantri, Andrey Ipatov, Telma Domingues, Jérôme Borme, Marco Martins, Pedro Alpuim, Marta Prado},
url = {https://doi.org/10.1016/j.snb.2022.132044},
doi = {10.1016/j.snb.2022.132044},
year = {2022},
date = {2022-05-26},
journal = {Sensors and Actuators B: Chemical},
volume = {367},
pages = {132044},
abstract = {This study presents the development of a lab-on-a-chip (LoC) by integrating a graphene field-effect transistor (FET) chip with a programmable microfluidic device for DNA detection. The real-time biochemical events on the graphene FET chip were monitored through Dirac voltage shift data from the portable graphene curve reader with changes dependent on the fluidic flow into the sensing interface by a fully automated programmable microfluidic system. High sensitivity with high reliability can be obtained with a nine-graphene sensor layout on a single chip. The portable graphene curve reader also provides a tunable electrical parameter setup and straightforward data acquisition. Fluidic control was performed through a multi-position valve, allowing sequential commands for liquid injection into the polydimethylsiloxane (PDMS) flow cell mounted on the sensing chip. The flow cell design with impinging jet geometry and the microfluidic system packaging offer high precision and portability as a less laborious and low-cost sensing setup. The merged system allows for various functionalities, including probe DNA (pDNA) immobilization, a blocking step, and DNA hybridization with stable signal output autonomously, even in a long-run experimental setup. As a DNA sensor, the proposed prototype has demonstrated a high sensitivity of ~44 mV/decade of target DNA concentration, with an outstanding limit of detection (LoD) of ~0.642 aM, making it one of the most sensitive sensors reported up to date. The programmable device has demonstrated essential versatilities for biomolecular detection in a fully portable and automated platform.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}This study presents the development of a lab-on-a-chip (LoC) by integrating a graphene field-effect transistor (FET) chip with a programmable microfluidic device for DNA detection. The real-time biochemical events on the graphene FET chip were monitored through Dirac voltage shift data from the portable graphene curve reader with changes dependent on the fluidic flow into the sensing interface by a fully automated programmable microfluidic system. High sensitivity with high reliability can be obtained with a nine-graphene sensor layout on a single chip. The portable graphene curve reader also provides a tunable electrical parameter setup and straightforward data acquisition. Fluidic control was performed through a multi-position valve, allowing sequential commands for liquid injection into the polydimethylsiloxane (PDMS) flow cell mounted on the sensing chip. The flow cell design with impinging jet geometry and the microfluidic system packaging offer high precision and portability as a less laborious and low-cost sensing setup. The merged system allows for various functionalities, including probe DNA (pDNA) immobilization, a blocking step, and DNA hybridization with stable signal output autonomously, even in a long-run experimental setup. As a DNA sensor, the proposed prototype has demonstrated a high sensitivity of ~44 mV/decade of target DNA concentration, with an outstanding limit of detection (LoD) of ~0.642 aM, making it one of the most sensitive sensors reported up to date. The programmable device has demonstrated essential versatilities for biomolecular detection in a fully portable and automated platform.Chih-Hsien Hsu, Akhilesh Kumar Gupta, Agnes Purwidyantri, Briliant Adhi Prabowo, Ching-Hsiang Chen, Chi-Cheng Chuang, Ya-Chung Tian, Yu-Jen Lu and Chao-Sung Lai
Sensing Alzheimer's Disease Utilizing Au Electrode by Controlling Nanorestructuring Journal Article
CHEMOSENSORS, 10 (3) (94), 2022.
@article{Hsu2022,
title = {Sensing Alzheimer's Disease Utilizing Au Electrode by Controlling Nanorestructuring},
author = {Chih-Hsien Hsu, Akhilesh Kumar Gupta, Agnes Purwidyantri, Briliant Adhi Prabowo, Ching-Hsiang Chen, Chi-Cheng Chuang, Ya-Chung Tian, Yu-Jen Lu and Chao-Sung Lai},
doi = {10.3390/chemosensors10030094},
year = {2022},
date = {2022-04-12},
journal = {CHEMOSENSORS},
volume = {10 (3)},
number = {94},
abstract = {This paper reports the development of Alzheimer's disease (AD) sensor through early detection of amyloid-beta (A beta) (1-42) using simple nanorestructuring of Au sheet plate by oxidation-reduction cycle (ORC) via the electrochemical system. The topology of Au substrates was enhanced through the roughening and Au grains grown by a simple ORC technique in aqueous solutions containing 0.1 mol/L KCl electrolytes. The roughened substrate was then functionalized with the highly specific antibody beta-amyloid A beta (1-28) through HS-PEG-NHS modification, which enabled effective and direct detection of A beta (1-42) peptide. The efficacy of the ORC method had been exhibited in the polished Au surface by approximately 15% larger electro-active sites compared to the polished Au without ORC. The ORC polished structure demonstrated a rapid, accurate, precise, reproducible, and highly sensitive detection of A beta (1-42) peptide with a low detection limit of 10.4 fg/mL and a wide linear range of 10(-2) to 10(6 )pg/mL. The proposed structure had been proven to have potential as an early-stage Alzheimer's disease (AD) detection platform with low-cost fabrication and ease of operation.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}This paper reports the development of Alzheimer's disease (AD) sensor through early detection of amyloid-beta (A beta) (1-42) using simple nanorestructuring of Au sheet plate by oxidation-reduction cycle (ORC) via the electrochemical system. The topology of Au substrates was enhanced through the roughening and Au grains grown by a simple ORC technique in aqueous solutions containing 0.1 mol/L KCl electrolytes. The roughened substrate was then functionalized with the highly specific antibody beta-amyloid A beta (1-28) through HS-PEG-NHS modification, which enabled effective and direct detection of A beta (1-42) peptide. The efficacy of the ORC method had been exhibited in the polished Au surface by approximately 15% larger electro-active sites compared to the polished Au without ORC. The ORC polished structure demonstrated a rapid, accurate, precise, reproducible, and highly sensitive detection of A beta (1-42) peptide with a low detection limit of 10.4 fg/mL and a wide linear range of 10(-2) to 10(6 )pg/mL. The proposed structure had been proven to have potential as an early-stage Alzheimer's disease (AD) detection platform with low-cost fabrication and ease of operation.Foteini Roumani, Cristina Rodrigues, Jorge Barros-Velázquez, Alejandro Garrido-Maestu & Marta Prado
FOOD ANALYTICAL METHODS, 2022.
@article{Roumani2022,
title = {Development of a Panfungal Recombinase Polymerase Amplification (RPA) Method Coupled with Lateral Flow Strips for the Detection of Spoilage Fungi},
author = {Foteini Roumani, Cristina Rodrigues, Jorge Barros-Velázquez, Alejandro Garrido-Maestu & Marta Prado },
doi = {10.1007/s12161-022-02242-1},
year = {2022},
date = {2022-03-21},
journal = {FOOD ANALYTICAL METHODS},
abstract = {Recombinase Polymerase Amplification combined with Lateral Flow (RPA-LF) detection was reported to be a good alternative to traditional culture-based methods, as well as other molecular techniques such as qPCR, PCR and Loop-Mediated Isothermal Amplification. The reasons for this are its simplicity, high sensitivity/specificity and ability for point of care (POC) applications. In this study, an RPA-LF assay was developed and evaluated for the detection of spoilage fungi in fruit-based products. In this sense, the universal primers ITS3 and ITS4 were labelled with digoxigenin and biotin, respectively. The method proved to be very sensitive with the ability to detect down to 1.2 pg/µL of pure fungal DNA. Furthermore, when the assay was applied in artificially contaminated samples, low detection limits were determined, in particular 1.0 CFU/50 g and 45.7 spores/50 g for yeasts and moulds, respectively. Overall, a rapid (24–48 h) and reliable assay was developed for the detection of fungi that could be applied for POC applications.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}Recombinase Polymerase Amplification combined with Lateral Flow (RPA-LF) detection was reported to be a good alternative to traditional culture-based methods, as well as other molecular techniques such as qPCR, PCR and Loop-Mediated Isothermal Amplification. The reasons for this are its simplicity, high sensitivity/specificity and ability for point of care (POC) applications. In this study, an RPA-LF assay was developed and evaluated for the detection of spoilage fungi in fruit-based products. In this sense, the universal primers ITS3 and ITS4 were labelled with digoxigenin and biotin, respectively. The method proved to be very sensitive with the ability to detect down to 1.2 pg/µL of pure fungal DNA. Furthermore, when the assay was applied in artificially contaminated samples, low detection limits were determined, in particular 1.0 CFU/50 g and 45.7 spores/50 g for yeasts and moulds, respectively. Overall, a rapid (24–48 h) and reliable assay was developed for the detection of fungi that could be applied for POC applications.Julio Vacacela, Anna-Lisa Schaap-Johansen, Patricia Manikova, Paolo Marcatili, Marta Prado, Yi Sun, Jon Ashley
The Protein-Templated Synthesis of Enzyme-Generated Aptamers Journal Article
ANGEWANDTE CHEMIE-INTERNATIONAL EDITION, (e202201061), 2022.
@article{Vacacela2022,
title = {The Protein-Templated Synthesis of Enzyme-Generated Aptamers},
author = {Julio Vacacela, Anna-Lisa Schaap-Johansen, Patricia Manikova, Paolo Marcatili, Marta Prado, Yi Sun, Jon Ashley},
url = {10.1002/anie.202201061},
year = {2022},
date = {2022-03-13},
journal = {ANGEWANDTE CHEMIE-INTERNATIONAL EDITION},
number = {e202201061},
abstract = {Inspired by the chemical synthesis of molecularly imprinted polymers, we demonstrated for the first time, the protein-target mediated synthesis of enzyme-generated aptamers (EGAs). We prepared pre-polymerisation mixtures containing different ratios of nucleotides, an initiator sequence and protein template and incubated each mixture with terminal deoxynucleotidyl transferase (TdT). Upon purification and rebinding of the EGAs against the target, we observed an enhancement in binding of templated-EGAs towards the target compared to a non-templated control. These results demonstrate the presence of two primary mechanisms for the formation of EGAs, namely, the binding of random sequences to the target as observed in systematic evolution of ligands by exponential enrichment (SELEX) and the dynamic competition between TdT enzyme and the target protein for binding of EGAs during synthesis. The latter mechanism serves to increase the stringency of EGA-based screening and represents a new way to develop aptamers that relies on rational design.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}Inspired by the chemical synthesis of molecularly imprinted polymers, we demonstrated for the first time, the protein-target mediated synthesis of enzyme-generated aptamers (EGAs). We prepared pre-polymerisation mixtures containing different ratios of nucleotides, an initiator sequence and protein template and incubated each mixture with terminal deoxynucleotidyl transferase (TdT). Upon purification and rebinding of the EGAs against the target, we observed an enhancement in binding of templated-EGAs towards the target compared to a non-templated control. These results demonstrate the presence of two primary mechanisms for the formation of EGAs, namely, the binding of random sequences to the target as observed in systematic evolution of ligands by exponential enrichment (SELEX) and the dynamic competition between TdT enzyme and the target protein for binding of EGAs during synthesis. The latter mechanism serves to increase the stringency of EGA-based screening and represents a new way to develop aptamers that relies on rational design.Alejandro Garrido-Maestu, Marta Prado
Naked-eye detection strategies coupled with isothermal nucleic acid amplification techniques for the detection of human pathogens Journal Article
COMPREHENSIVE REVIEWS IN FOOD SCIENCE AND FOOD SAFETY, 2022.
@article{Garrido-Maestu2022,
title = {Naked-eye detection strategies coupled with isothermal nucleic acid amplification techniques for the detection of human pathogens},
author = {Alejandro Garrido-Maestu, Marta Prado},
url = {https://doi.org/10.1111/1541-4337.12902},
doi = {10.1111/1541-4337.12902},
year = {2022},
date = {2022-02-04},
journal = {COMPREHENSIVE REVIEWS IN FOOD SCIENCE AND FOOD SAFETY},
abstract = {Nucleic acid amplification-based techniques have gained acceptance by the scientific, and general, community as reference methodologies for many different applications. Since the development of the gold standard of these techniques, polymerase chain reaction (PCR), back in the 1980s many improvements have been made, and alternative techniques emerged reporting improvements over PCR. Among these, isothermal amplification approaches resulted of particular interest as could overcome the need of specialized equipment to accurately control temperature changes, but it was after year 2000 that these techniques have flourished in a huge number of novel alternatives with many different degrees of complexities and requirements. An added value is their possibility to be combined with many different naked-eye detection strategies, simplifying the resources needed, allowing to reduce cost, and serving as the basis for novel developments of lab-on-chip systems, and miniaturized devices, for point-of-care testing. In this review, we will go over different types of naked-eye detection strategies, combined with isothermal amplification. This will provide the readers up-to-date information for them to select the most appropriate strategies depending on the particular needs and resources for their experimental setup.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}Nucleic acid amplification-based techniques have gained acceptance by the scientific, and general, community as reference methodologies for many different applications. Since the development of the gold standard of these techniques, polymerase chain reaction (PCR), back in the 1980s many improvements have been made, and alternative techniques emerged reporting improvements over PCR. Among these, isothermal amplification approaches resulted of particular interest as could overcome the need of specialized equipment to accurately control temperature changes, but it was after year 2000 that these techniques have flourished in a huge number of novel alternatives with many different degrees of complexities and requirements. An added value is their possibility to be combined with many different naked-eye detection strategies, simplifying the resources needed, allowing to reduce cost, and serving as the basis for novel developments of lab-on-chip systems, and miniaturized devices, for point-of-care testing. In this review, we will go over different types of naked-eye detection strategies, combined with isothermal amplification. This will provide the readers up-to-date information for them to select the most appropriate strategies depending on the particular needs and resources for their experimental setup.Sarah Azinheiro, Joana Carvalho, Pablo Fuciños, Lorenzo Pastrana, Marta Prado, Alejandro Garrido-Maestu
Short pre-enrichment and modified matrix lysis. A comparative study towards same-day detection of Listeria monocytogenes Journal Article
LWT, 154 , 2022.
@article{Azinheiro2022,
title = {Short pre-enrichment and modified matrix lysis. A comparative study towards same-day detection of Listeria monocytogenes},
author = {Sarah Azinheiro, Joana Carvalho, Pablo Fuciños, Lorenzo Pastrana, Marta Prado, Alejandro Garrido-Maestu},
url = {https://doi.org/10.1016/j.lwt.2021.112900},
doi = {10.1016/j.lwt.2021.112900},
year = {2022},
date = {2022-01-15},
journal = {LWT},
volume = {154},
abstract = {Listeria monocytogenes is a foodborne pathogen of particular concern in ready-to-eat foods. Different methodologies have been published in recent years, in order to reduce the time of analysis of this pathogen but, best case scenario, all these methods allow for “next-day detection”. There is permanent need in the food industry for faster methods capable of providing accurate results, without compromising the safety of the consumers, in order to cope with nowadays' intensive production system. In the present study, two sample treatment protocols namely, short pre-enrichment and matrix lysis, were compared to determine their suitability for “same-day detection” of L. monocytogenes. Both methodologies were sensitive, specific and accurate (100%), and allowed to obtain results in one working day. In addition to this, the results obtained matched those expected as observed by the Cohen's k value obtained (1.00). However, a the major difference was observed in their limit of detection (LOD95), as the short pre-enrichment allowed to detect L. monocytogenes in samples inoculated below 10 CFU/25 g, while the matrix lysis remained in the range of 105 CFU/25 g. These results indicate that whenever a “zero tolerance” is required, the short pre-enrichment protocol must be selected.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}Listeria monocytogenes is a foodborne pathogen of particular concern in ready-to-eat foods. Different methodologies have been published in recent years, in order to reduce the time of analysis of this pathogen but, best case scenario, all these methods allow for “next-day detection”. There is permanent need in the food industry for faster methods capable of providing accurate results, without compromising the safety of the consumers, in order to cope with nowadays' intensive production system. In the present study, two sample treatment protocols namely, short pre-enrichment and matrix lysis, were compared to determine their suitability for “same-day detection” of L. monocytogenes. Both methodologies were sensitive, specific and accurate (100%), and allowed to obtain results in one working day. In addition to this, the results obtained matched those expected as observed by the Cohen's k value obtained (1.00). However, a the major difference was observed in their limit of detection (LOD95), as the short pre-enrichment allowed to detect L. monocytogenes in samples inoculated below 10 CFU/25 g, while the matrix lysis remained in the range of 105 CFU/25 g. These results indicate that whenever a “zero tolerance” is required, the short pre-enrichment protocol must be selected. -
2021
Foteini Roumani, Sarah Azinheiro, Cristina Rodrigues, Jorge Barros-Velázquez, Alejandro Garrido-Maestu, Marta Prado
Development of a real-time PCR assay with an internal amplification control for the detection of spoilage fungi in fruit preparations Journal Article
Food Control, 135 (108783), 2021.
@article{Roumani2021b,
title = {Development of a real-time PCR assay with an internal amplification control for the detection of spoilage fungi in fruit preparations},
author = {Foteini Roumani, Sarah Azinheiro, Cristina Rodrigues, Jorge Barros-Velázquez, Alejandro Garrido-Maestu, Marta Prado},
url = {https://doi.org/10.1016/j.foodcont.2021.108783},
doi = {10.1016/j.foodcont.2021.108783},
year = {2021},
date = {2021-12-24},
journal = {Food Control},
volume = {135},
number = {108783},
abstract = {Fungal spoilage of food commodities is still of great concern for food industry due to increased costs regarding food waste and product recalls. Traditional culture-based methods are laborious and require huge amounts of reagents and media. In addition, results are available after seven days rendering these techniques unsuitable for the intense production that exists nowadays. As a result, there is a need for faster and more sensitive methods for fungal detection. The aim of this study was the development and evaluation of a method (enrichment, sample treatment, DNA extraction, and qPCR) for the fast detection of spoilage fungi in fruit preparations. In this sense, a set of universal primers was selected and a hydrolysis probe was designed in-house. In addition, a non-competitive internal amplification control was included in the assay to eliminate false negative results due to reaction inhibition. It was demonstrated that the method could reliably detect yeasts with a LOD95 of 1.0 CFU/50 g. Regarding moulds detection, two different enrichment times were examined, namely 24 h and 48 h in order to increase the sensitivity. The obtained LOD95 values were 123.5 spores/50 g and 37.1 spores/50 g for 24 h and 48 h respectively. During the method evaluation all the performance parameters resulted in values higher than 85.0% and the Cohen's k was determined to be above 0.86 for yeasts and moulds. Overall, a reliable and sensitive next-day detection method for fungi was achieved.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}Fungal spoilage of food commodities is still of great concern for food industry due to increased costs regarding food waste and product recalls. Traditional culture-based methods are laborious and require huge amounts of reagents and media. In addition, results are available after seven days rendering these techniques unsuitable for the intense production that exists nowadays. As a result, there is a need for faster and more sensitive methods for fungal detection. The aim of this study was the development and evaluation of a method (enrichment, sample treatment, DNA extraction, and qPCR) for the fast detection of spoilage fungi in fruit preparations. In this sense, a set of universal primers was selected and a hydrolysis probe was designed in-house. In addition, a non-competitive internal amplification control was included in the assay to eliminate false negative results due to reaction inhibition. It was demonstrated that the method could reliably detect yeasts with a LOD95 of 1.0 CFU/50 g. Regarding moulds detection, two different enrichment times were examined, namely 24 h and 48 h in order to increase the sensitivity. The obtained LOD95 values were 123.5 spores/50 g and 37.1 spores/50 g for 24 h and 48 h respectively. During the method evaluation all the performance parameters resulted in values higher than 85.0% and the Cohen's k was determined to be above 0.86 for yeasts and moulds. Overall, a reliable and sensitive next-day detection method for fungi was achieved.Foteini Roumani, Saioa Gómez, Cristina Rodrigues, Jorge Barros-Velázquez, Alejandro Garrido-Maestu, Marta Prado
Food Control, 135 (108784), 2021.
@article{Roumani2021c,
title = {Development and evaluation of a real-time fluorescence, and naked-eye colorimetric, loop-mediated isothermal amplification-based method for the rapid detection of spoilage fungi in fruit preparations},
author = {Foteini Roumani, Saioa Gómez, Cristina Rodrigues, Jorge Barros-Velázquez, Alejandro Garrido-Maestu, Marta Prado},
url = {https://doi.org/10.1016/j.foodcont.2021.108784},
doi = {10.1016/j.foodcont.2021.108784},
year = {2021},
date = {2021-12-24},
journal = {Food Control},
volume = {135},
number = {108784},
abstract = {DNA-based techniques like PCR/qPCR have been applied for the detection of microorganisms in order to provide faster results, due to their high sensitivity and specificity, when compared to culture-based techniques. However, isothermal amplification techniques like Loop-mediated isothermal amplification (LAMP) have emerged lately allowing the simplification of the assays, and reduction of costs. The aim of this study was the development, and evaluation, of a panfungal LAMP assay (LAMP- 18S) for the fast detection of spoilage fungi in fruit preparations. In this sense, a set of primers was newly designed. Two different detection strategies were examined, namely real-time fluorescence and naked-eye colour detection. In addition, the results were compared against another published panfungal LAMP (LAMP-POW) and a qPCR assay, which served as the reference method. The developed method showed high sensitivity being able to detect down to 1.4 pg/reaction for yeasts and 170 pg/reaction for moulds. The addition of an enrichment step significantly reduced the LOD as the method could reliably detect yeasts with a LOD95 of 3.1 and 3.0 CFU/50 g for the fluorescent and colorimetric assay, respectively. The fluorescent assay showed “substantial agreement” with the reference method, while the colorimetric assay was “almost in complete concordance” with the reference method. Overall, a reliable and sensitive next-day detection method for fungi was achieved.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}DNA-based techniques like PCR/qPCR have been applied for the detection of microorganisms in order to provide faster results, due to their high sensitivity and specificity, when compared to culture-based techniques. However, isothermal amplification techniques like Loop-mediated isothermal amplification (LAMP) have emerged lately allowing the simplification of the assays, and reduction of costs. The aim of this study was the development, and evaluation, of a panfungal LAMP assay (LAMP- 18S) for the fast detection of spoilage fungi in fruit preparations. In this sense, a set of primers was newly designed. Two different detection strategies were examined, namely real-time fluorescence and naked-eye colour detection. In addition, the results were compared against another published panfungal LAMP (LAMP-POW) and a qPCR assay, which served as the reference method. The developed method showed high sensitivity being able to detect down to 1.4 pg/reaction for yeasts and 170 pg/reaction for moulds. The addition of an enrichment step significantly reduced the LOD as the method could reliably detect yeasts with a LOD95 of 3.1 and 3.0 CFU/50 g for the fluorescent and colorimetric assay, respectively. The fluorescent assay showed “substantial agreement” with the reference method, while the colorimetric assay was “almost in complete concordance” with the reference method. Overall, a reliable and sensitive next-day detection method for fungi was achieved.Azinheiro, S., Ghimire, D., Carvalho, J., Prado, M., & Garrido-Maestu, A. (2022)
Next-day detection of viable Listeria monocytogenes by multiplex reverse transcriptase real-time PCR Journal Article
Food Control, 133 (PA) , 2021.
@article{Azinheiro2021c,
title = {Next-day detection of viable Listeria monocytogenes by multiplex reverse transcriptase real-time PCR},
author = {Azinheiro, S., Ghimire, D., Carvalho, J., Prado, M., & Garrido-Maestu, A. (2022)},
url = {https://doi.org/10.1016/j.foodcont.2021.108593},
doi = {10.1016/j.foodcont.2021.108593},
year = {2021},
date = {2021-10-01},
journal = {Food Control},
volume = {133 (PA)},
abstract = {Listeria monocytogenes continues to be a major challenge for the food industry due to its ubiquity and difficulty to be eliminated from processing facilities. The DNA amplification-based methods can overcome limitations of culture-based methods; however, due to the stability of the DNA, false positive results may occur, associated to the presence of harmless, dead microorganisms. The incorrect assessment of the results will have a huge impact on the producers in terms of stopping the production/distribution, leading to economic losses. In the current study, a multiplex RT-qPCR method was developed. The detection of mRNA allows for the specific detection of live bacteria. Additionally, two genetic targets (hly and actA) were co-amplified for improved specificity, along with an Internal Amplification Control to rule out false negative results due to reaction inhibition. The optimized methodology was evaluated in smoked salmon samples inoculated with different combinations and concentrations of live and dead bacteria. The method demonstrated high sensitivity (LOD50/LOD95 of 1.2/5.1 cfu/25 g). The performance of the method was compared against the reference standard ISO 11290, for which a Cohen's k of 0.94 was obtained, being interpreted as “almost complete concordance” among both methods. In addition, other parameters evaluated included the relative sensitivity, specificity, accuracy, as well as the positive and negative predictive values, all being above 90%. This next-day methodology can significantly reduce the time of analysis of culture-based methods and can specifically detect live L. monocytogenes.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}Listeria monocytogenes continues to be a major challenge for the food industry due to its ubiquity and difficulty to be eliminated from processing facilities. The DNA amplification-based methods can overcome limitations of culture-based methods; however, due to the stability of the DNA, false positive results may occur, associated to the presence of harmless, dead microorganisms. The incorrect assessment of the results will have a huge impact on the producers in terms of stopping the production/distribution, leading to economic losses. In the current study, a multiplex RT-qPCR method was developed. The detection of mRNA allows for the specific detection of live bacteria. Additionally, two genetic targets (hly and actA) were co-amplified for improved specificity, along with an Internal Amplification Control to rule out false negative results due to reaction inhibition. The optimized methodology was evaluated in smoked salmon samples inoculated with different combinations and concentrations of live and dead bacteria. The method demonstrated high sensitivity (LOD50/LOD95 of 1.2/5.1 cfu/25 g). The performance of the method was compared against the reference standard ISO 11290, for which a Cohen's k of 0.94 was obtained, being interpreted as “almost complete concordance” among both methods. In addition, other parameters evaluated included the relative sensitivity, specificity, accuracy, as well as the positive and negative predictive values, all being above 90%. This next-day methodology can significantly reduce the time of analysis of culture-based methods and can specifically detect live L. monocytogenes.Azinheiro, S., Roumani, F., Carvalho, J., Prado, M., & Garrido-Maestu, A.
Suitability of the MinION long read sequencer for semi-targeted detection of foodborne pathogens Journal Article
Analytica Chimica Acta, 1184 , 2021.
@article{Azinheiro2021b,
title = { Suitability of the MinION long read sequencer for semi-targeted detection of foodborne pathogens},
author = {Azinheiro, S., Roumani, F., Carvalho, J., Prado, M., & Garrido-Maestu, A. },
url = {https://doi.org/10.1016/j.aca.2021.339051},
doi = {10.1016/j.aca.2021.339051},
year = {2021},
date = {2021-09-10},
journal = {Analytica Chimica Acta},
volume = {1184},
abstract = {Foodborne pathogens are still a significant source of morbidity and mortality worldwide. In addition to this the current methodologies to track these microorganisms cannot cope with the current intensive production systems, thus novel methods are of outmost importance. DNA-based methods have already demonstrated suitable to address this issue, but most of them are targeted methods such as real-time PCR (qPCR), meaning that one will only find what is looking for, thus taking the risk of missing relevant pathogens in a given sample. To overcome this limitation we have developed an easy-to-implement methodology which enables the detection of several pathogens simultaneously by using long-read Next Generation Sequencing (NGS) with MinION. The method was named “semi-targeted” due to the combination of a non-targeted detection method, NGS, with the usage of selective media in order to partially eliminate non-pathogenic interfering bacteria. To this end, we included an enrichment step for the recovery of different pathogens, namely Salmonella Enteritidis and Typhimurium, Listeria monocytogenes and Escherichia coli O157:H7, after DNA extraction and library preparation, the samples were analyzed with MinION implementing the low-cost Flongle Flow Cells. The methodology was successfully evaluated in spiked milk samples with an excellent agreement with the results obtained by qPCR and culture-based methods. The method can provide accurate results after only 2 h of sequencing. Sample multiplexing, along with the lower cost of the Flongle Flow Cells and the reduced price of the MinION platform, make the assay cost-effective that is of importance for the food industry. Starting the method with a classical microbiological approach, the enrichment, the method is easy to implement in testing laboratories, it provides flexibility in terms of potential pathogens to be detected, and the positive results can be easily confirmed following culture-based, or other type, of confirmation procedures.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}Foodborne pathogens are still a significant source of morbidity and mortality worldwide. In addition to this the current methodologies to track these microorganisms cannot cope with the current intensive production systems, thus novel methods are of outmost importance. DNA-based methods have already demonstrated suitable to address this issue, but most of them are targeted methods such as real-time PCR (qPCR), meaning that one will only find what is looking for, thus taking the risk of missing relevant pathogens in a given sample. To overcome this limitation we have developed an easy-to-implement methodology which enables the detection of several pathogens simultaneously by using long-read Next Generation Sequencing (NGS) with MinION. The method was named “semi-targeted” due to the combination of a non-targeted detection method, NGS, with the usage of selective media in order to partially eliminate non-pathogenic interfering bacteria. To this end, we included an enrichment step for the recovery of different pathogens, namely Salmonella Enteritidis and Typhimurium, Listeria monocytogenes and Escherichia coli O157:H7, after DNA extraction and library preparation, the samples were analyzed with MinION implementing the low-cost Flongle Flow Cells. The methodology was successfully evaluated in spiked milk samples with an excellent agreement with the results obtained by qPCR and culture-based methods. The method can provide accurate results after only 2 h of sequencing. Sample multiplexing, along with the lower cost of the Flongle Flow Cells and the reduced price of the MinION platform, make the assay cost-effective that is of importance for the food industry. Starting the method with a classical microbiological approach, the enrichment, the method is easy to implement in testing laboratories, it provides flexibility in terms of potential pathogens to be detected, and the positive results can be easily confirmed following culture-based, or other type, of confirmation procedures.Guerreiro, J. R. L., Ipatov, A., Carvalho, J., Toldrà, A., & Prado, M.
Amplified plasmonic and microfluidic setup for DNA monitoring Journal Article
Microchimica Acta, (2021), (326), 2021.
@article{Guerreiro2021,
title = {Amplified plasmonic and microfluidic setup for DNA monitoring},
author = {Guerreiro, J. R. L., Ipatov, A., Carvalho, J., Toldrà, A., & Prado, M.},
url = {https://doi.org/10.1007/s00604-021-04983-y},
doi = {10.1007/s00604-021-04983-y},
year = {2021},
date = {2021-09-07},
journal = {Microchimica Acta, (2021)},
number = {326},
abstract = {Plasmonic nanosensors for label-free detection of DNA require excellent sensing resolution, which is crucial when monitoring short DNA sequences, as these induce tiny peak shifts, compared to large biomolecules. We report a versatile and simple strategy for plasmonic sensor signal enhancement by assembling multiple (four) plasmonic sensors in series. This approach provided a fourfold signal enhancement, increased signal-to-noise ratio, and improved sensitivity for DNA detection. The response of multiple sensors based on AuNSpheres was also compared with AuNRods, the latter showing better sensing resolution. The amplification system based on AuNR was integrated into a microfluidic sequential injection platform and applied to the monitoring of DNA, specifically from environmental invasive species—zebra mussels. DNA from zebra mussels was log concentration-dependent from 1 to 1 × 106 pM, reaching a detection limit of 2.0 pM. In situ tests were also successfully applied to real samples, within less than 45 min, using DNA extracted from zebra mussel meat. The plasmonic nanosensors’ signal can be used as a binary output (yes/no) to assess the presence of those invasive species. Even though these genosensors were applied to the monitoring of DNA in environmental samples, they potentially offer advantage in a wide range of fields, such as disease diagnostics.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}Plasmonic nanosensors for label-free detection of DNA require excellent sensing resolution, which is crucial when monitoring short DNA sequences, as these induce tiny peak shifts, compared to large biomolecules. We report a versatile and simple strategy for plasmonic sensor signal enhancement by assembling multiple (four) plasmonic sensors in series. This approach provided a fourfold signal enhancement, increased signal-to-noise ratio, and improved sensitivity for DNA detection. The response of multiple sensors based on AuNSpheres was also compared with AuNRods, the latter showing better sensing resolution. The amplification system based on AuNR was integrated into a microfluidic sequential injection platform and applied to the monitoring of DNA, specifically from environmental invasive species—zebra mussels. DNA from zebra mussels was log concentration-dependent from 1 to 1 × 106 pM, reaching a detection limit of 2.0 pM. In situ tests were also successfully applied to real samples, within less than 45 min, using DNA extracted from zebra mussel meat. The plasmonic nanosensors’ signal can be used as a binary output (yes/no) to assess the presence of those invasive species. Even though these genosensors were applied to the monitoring of DNA in environmental samples, they potentially offer advantage in a wide range of fields, such as disease diagnostics.Azinheiro, S., Roumani, F., Rodríguez-Lorenzo, L., Carvalho, J., Prado, M., & Garrido-Maestu, A.
Food Control (2022), 132 , 2021.
@article{Azinheiro2021,
title = {Combination of Recombinase Polymerase Amplification with SYBR Green I for naked-eye, same-day detection of Escherichia coli O157:H7 in ground meat},
author = {Azinheiro, S., Roumani, F., Rodríguez-Lorenzo, L., Carvalho, J., Prado, M., & Garrido-Maestu, A.},
url = {https://doi.org/10.1016/j.foodcont.2021.108494},
doi = {10.1016/j.foodcont.2021.108494},
year = {2021},
date = {2021-08-21},
journal = {Food Control (2022)},
volume = {132},
abstract = {Escherichia coli O157 continues to be the most prevalent serotype among the Shiga toxin-producing E. coli infection cases confirmed in Europe. The reference methodology to detect this pathogen is lengthy and time consuming, thus we sought to develop a novel method that has low instrumentation requirement, and allowed naked-eye detection. Isothermal amplification of bacterial DNA was performed by Recombinase Polymerase Amplification, and the addition of SYBR Green I (RPA-SG), which allowed the visualization of results with naked-eye under a UV lamp. The results obtained in spiked ground meat samples by RPA-SG compared favorably to qPCR (relative sensitivity, specificity and accuracy higher than 90%, and Cohen's k of 0.81), with a limit of detection of 19 cfu/25 g. The novel methodology outperformed a culture-based approach, where none of the typical colonies were confirmed as O157 due to high concentration of interfering microorganisms. These results were obtained in one working day (same-day detection), having an average time to completion of about 5 h, including enrichment, DNA extraction, amplification and detection.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}Escherichia coli O157 continues to be the most prevalent serotype among the Shiga toxin-producing E. coli infection cases confirmed in Europe. The reference methodology to detect this pathogen is lengthy and time consuming, thus we sought to develop a novel method that has low instrumentation requirement, and allowed naked-eye detection. Isothermal amplification of bacterial DNA was performed by Recombinase Polymerase Amplification, and the addition of SYBR Green I (RPA-SG), which allowed the visualization of results with naked-eye under a UV lamp. The results obtained in spiked ground meat samples by RPA-SG compared favorably to qPCR (relative sensitivity, specificity and accuracy higher than 90%, and Cohen's k of 0.81), with a limit of detection of 19 cfu/25 g. The novel methodology outperformed a culture-based approach, where none of the typical colonies were confirmed as O157 due to high concentration of interfering microorganisms. These results were obtained in one working day (same-day detection), having an average time to completion of about 5 h, including enrichment, DNA extraction, amplification and detection.Shambhavi Yadav, Joana Carvalho, Isabel Trujillo and Marta Prado
Microsatellite Markers in Olives (Olea europaea L.): Utility in the Cataloging of Germplasm, Food Authenticity and Traceability Studies Journal Article
Foods 2021, 10 (8), pp. 1907, 2021.
@article{Yadav2021,
title = {Microsatellite Markers in Olives (Olea europaea L.): Utility in the Cataloging of Germplasm, Food Authenticity and Traceability Studies},
author = {Shambhavi Yadav, Joana Carvalho, Isabel Trujillo and Marta Prado},
url = {https://doi.org/10.3390/foods10081907},
doi = {10.3390/foods10081907},
year = {2021},
date = {2021-08-17},
journal = {Foods 2021},
volume = {10},
number = {8},
pages = {1907},
abstract = {The olive fruit, a symbol of Mediterranean diets, is a rich source of antioxidants and oleic acid (55–83%). Olive genetic resources, including cultivated olives (cultivars), wild olives as well as related subspecies, are distributed widely across the Mediterranean region and other countries. Certain cultivars have a high commercial demand and economical value due to the differentiating organoleptic characteristics. This might result in economically motivated fraudulent practices and adulteration. Hence, tools to ensure the authenticity of constituent olive cultivars are crucial, and this can be achieved accurately through DNA-based methods. The present review outlines the applications of microsatellite markers, one of the most extensively used types of molecular markers in olive species, particularly referring to the use of these DNA-based markers in cataloging the vast olive germplasm, leading to identification and authentication of the cultivars. Emphasis has been given on the need to adopt a uniform platform where global molecular information pertaining to the details of available markers, cultivar-specific genotyping profiles (their synonyms or homonyms) and the comparative profiles of oil and reference leaf samples is accessible to researchers. The challenges of working with microsatellite markers and efforts underway, mainly advancements in genotyping methods which can be effectively incorporated in olive oil varietal testing, are also provided. Such efforts will pave the way for the development of more robust microsatellite marker-based olive agri-food authentication platforms.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}The olive fruit, a symbol of Mediterranean diets, is a rich source of antioxidants and oleic acid (55–83%). Olive genetic resources, including cultivated olives (cultivars), wild olives as well as related subspecies, are distributed widely across the Mediterranean region and other countries. Certain cultivars have a high commercial demand and economical value due to the differentiating organoleptic characteristics. This might result in economically motivated fraudulent practices and adulteration. Hence, tools to ensure the authenticity of constituent olive cultivars are crucial, and this can be achieved accurately through DNA-based methods. The present review outlines the applications of microsatellite markers, one of the most extensively used types of molecular markers in olive species, particularly referring to the use of these DNA-based markers in cataloging the vast olive germplasm, leading to identification and authentication of the cultivars. Emphasis has been given on the need to adopt a uniform platform where global molecular information pertaining to the details of available markers, cultivar-specific genotyping profiles (their synonyms or homonyms) and the comparative profiles of oil and reference leaf samples is accessible to researchers. The challenges of working with microsatellite markers and efforts underway, mainly advancements in genotyping methods which can be effectively incorporated in olive oil varietal testing, are also provided. Such efforts will pave the way for the development of more robust microsatellite marker-based olive agri-food authentication platforms.Yadav, S.; Carvalho, J.; Trujillo, I.; Prado, M.
Microsatellite Markers in Olives (Olea europaea L.): Utility in the Cataloging of Germplasm, Food Authenticity and Traceability Studies Journal Article
Foods, 10 (1907), 2021.
@article{Yadav2021b,
title = {Microsatellite Markers in Olives (Olea europaea L.): Utility in the Cataloging of Germplasm, Food Authenticity and Traceability Studies},
author = {Yadav, S.; Carvalho, J.; Trujillo, I.; Prado, M.},
url = {https://doi.org/10.3390/foods10081907},
doi = {10.3390/foods10081907},
year = {2021},
date = {2021-08-17},
journal = {Foods},
volume = {10},
number = {1907},
abstract = {The olive fruit, a symbol of Mediterranean diets, is a rich source of antioxidants and oleic acid (55–83%). Olive genetic resources, including cultivated olives (cultivars), wild olives as well as related subspecies, are distributed widely across the Mediterranean region and other countries. Certain cultivars have a high commercial demand and economical value due to the differentiating organoleptic characteristics. This might result in economically motivated fraudulent practices and adulteration. Hence, tools to ensure the authenticity of constituent olive cultivars are crucial, and this can be achieved accurately through DNA-based methods. The present review outlines the applications of microsatellite markers, one of the most extensively used types of molecular markers in olive species, particularly referring to the use of these DNA-based markers in cataloging the vast olive germplasm, leading to identification and authentication of the cultivars. Emphasis has been given on the need to adopt a uniform platform where global molecular information pertaining to the details of available markers, cultivar-specific genotyping profiles (their synonyms or homonyms) and the comparative profiles of oil and reference leaf samples is accessible to researchers. The challenges of working with microsatellite markers and efforts underway, mainly advancements in genotyping methods which can be effectively incorporated in olive oil varietal testing, are also provided. Such efforts will pave the way for the development of more robust microsatellite marker-based olive agri-food authentication platforms.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}The olive fruit, a symbol of Mediterranean diets, is a rich source of antioxidants and oleic acid (55–83%). Olive genetic resources, including cultivated olives (cultivars), wild olives as well as related subspecies, are distributed widely across the Mediterranean region and other countries. Certain cultivars have a high commercial demand and economical value due to the differentiating organoleptic characteristics. This might result in economically motivated fraudulent practices and adulteration. Hence, tools to ensure the authenticity of constituent olive cultivars are crucial, and this can be achieved accurately through DNA-based methods. The present review outlines the applications of microsatellite markers, one of the most extensively used types of molecular markers in olive species, particularly referring to the use of these DNA-based markers in cataloging the vast olive germplasm, leading to identification and authentication of the cultivars. Emphasis has been given on the need to adopt a uniform platform where global molecular information pertaining to the details of available markers, cultivar-specific genotyping profiles (their synonyms or homonyms) and the comparative profiles of oil and reference leaf samples is accessible to researchers. The challenges of working with microsatellite markers and efforts underway, mainly advancements in genotyping methods which can be effectively incorporated in olive oil varietal testing, are also provided. Such efforts will pave the way for the development of more robust microsatellite marker-based olive agri-food authentication platforms.Elumalai, M., Ipatov, A., Carvalho, J., Guerreiro, J., Prado, M.
Dual colorimetric strategy for specific DNA detection by nicking endonuclease-assisted gold nanoparticle signal amplification Journal Article
Anal Bioanal Chem, 2021.
@article{Elumalai2021,
title = {Dual colorimetric strategy for specific DNA detection by nicking endonuclease-assisted gold nanoparticle signal amplification},
author = {Elumalai, M., Ipatov, A., Carvalho, J., Guerreiro, J., Prado, M.},
url = {https://doi.org/10.1007/s00216-021-03564-5},
doi = {10.1007/s00216-021-03564-5},
year = {2021},
date = {2021-08-10},
journal = {Anal Bioanal Chem},
abstract = {The continuous spread of invasive alien species, as zebra mussel (Dreissena polymorpha), is a major global concern and it is urgent to stop it. Early stages of an invasion are crucial and challenging; however, detection tools based on environmental DNA analysis are promising alternatives. We present an alternative DNA target amplification strategy for signal enhancement followed by dual-mode colorimetric naked eye and optical smartphone analysis for the early detection of zebra mussel environmental DNA. Target amplification was designed based on the nicking endonuclease probe cleavage upon probe and complementary target hybridization. The cleaved/intact probe interacts with DNA-modified nanoparticles for colorimetric detection. We have demonstrated that enzyme amplification strategy enhanced 12-fold the sensitivity by naked eye detection, achieving a detection limit of ~8 nM (4.48×1010 copies) in controlled conditions, whereas target in complex environmental samples allowed the detection of 22.5 nM (1.26×1011 copies). Competitive assays also showed that the system can discriminate specific zebra mussel DNA sequences from other DNA sequences. Additionally, smartphone analysis for DNA quantification further improved the sensitivity of its detection by 130-fold, more than 2 orders of magnitude, when applied to environmental samples. The limit of detection to 0.17 nM (9.52×108 copies) is based on RGB coordinates, which is especially relevant to monitor early aggregation stages, being more accurate and reducing naked eye detection subjectivity. DNA extracted from zebra mussel meat, zebra mussel contaminated river water, and non-contaminated river water samples were successfully tested. Dual-mode colorimetric detection is useful in field analysis without the need for expensive laboratory equipment.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}The continuous spread of invasive alien species, as zebra mussel (Dreissena polymorpha), is a major global concern and it is urgent to stop it. Early stages of an invasion are crucial and challenging; however, detection tools based on environmental DNA analysis are promising alternatives. We present an alternative DNA target amplification strategy for signal enhancement followed by dual-mode colorimetric naked eye and optical smartphone analysis for the early detection of zebra mussel environmental DNA. Target amplification was designed based on the nicking endonuclease probe cleavage upon probe and complementary target hybridization. The cleaved/intact probe interacts with DNA-modified nanoparticles for colorimetric detection. We have demonstrated that enzyme amplification strategy enhanced 12-fold the sensitivity by naked eye detection, achieving a detection limit of ~8 nM (4.48×1010 copies) in controlled conditions, whereas target in complex environmental samples allowed the detection of 22.5 nM (1.26×1011 copies). Competitive assays also showed that the system can discriminate specific zebra mussel DNA sequences from other DNA sequences. Additionally, smartphone analysis for DNA quantification further improved the sensitivity of its detection by 130-fold, more than 2 orders of magnitude, when applied to environmental samples. The limit of detection to 0.17 nM (9.52×108 copies) is based on RGB coordinates, which is especially relevant to monitor early aggregation stages, being more accurate and reducing naked eye detection subjectivity. DNA extracted from zebra mussel meat, zebra mussel contaminated river water, and non-contaminated river water samples were successfully tested. Dual-mode colorimetric detection is useful in field analysis without the need for expensive laboratory equipment.Joana Carvalho, Shambhavi Yadav, Alejandro Garrido-Maestu, Sarah Azinheiro, Isabel Trujillo, Jorge Barros-Velázquez, Marta Prado
FOOD CHEMISTRY: MOLECULAR SCIENCES, 3 (100038), 2021.
@article{Carvalho2021b,
title = {Evaluation of simple sequence repeats (SSR) and single nucleotide polymorphism (SNP)-based methods in olive varieties from the Northwest of Spain and potential for miniaturization},
author = {Joana Carvalho, Shambhavi Yadav, Alejandro Garrido-Maestu, Sarah Azinheiro, Isabel Trujillo, Jorge Barros-Velázquez, Marta Prado},
url = {https://doi.org/10.1016/j.fochms.2021.100038},
doi = {10.1016/j.fochms.2021.100038},
year = {2021},
date = {2021-08-07},
journal = {FOOD CHEMISTRY: MOLECULAR SCIENCES},
volume = {3},
number = {100038},
abstract = {Miniaturization of DNA-based techniques can bring interesting advantages for food analysis, such as portability of complex analytical procedures. In the olive oil industry, miniaturization can be particularly interesting for authenticity and traceability applications, through in situ control of raw materials before production and/or the final products. However, variety identification is challenging, and implementation on miniaturized settings must be carefully evaluated, starting from the selected analytical approach. In this work, SSR- and SNP-based genotyping strategies were investigated for the identification and differentiation of two olive varieties from the Northwest of Spain. For the selected SNPs two genotyping methods were tested: real-time allele-specific PCR and high resolution melting analysis. These methods were compared and evaluated regarding their potential for integration in a microfluidic device. Both SNP-based methods proved to be successful for identification of the selected varieties, however real-time allele-specific PCR was the one that achieved the best results when analyzing mixtures, allowing the identification of both monovarietal samples and mixtures of the varieties tested with up to 25%.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}Miniaturization of DNA-based techniques can bring interesting advantages for food analysis, such as portability of complex analytical procedures. In the olive oil industry, miniaturization can be particularly interesting for authenticity and traceability applications, through in situ control of raw materials before production and/or the final products. However, variety identification is challenging, and implementation on miniaturized settings must be carefully evaluated, starting from the selected analytical approach. In this work, SSR- and SNP-based genotyping strategies were investigated for the identification and differentiation of two olive varieties from the Northwest of Spain. For the selected SNPs two genotyping methods were tested: real-time allele-specific PCR and high resolution melting analysis. These methods were compared and evaluated regarding their potential for integration in a microfluidic device. Both SNP-based methods proved to be successful for identification of the selected varieties, however real-time allele-specific PCR was the one that achieved the best results when analyzing mixtures, allowing the identification of both monovarietal samples and mixtures of the varieties tested with up to 25%.Carvalho, J.; Yadav, S.; Garrido-Maestu, A.G.; Azinheiro, S.; Trujillo, I.; Barros-Velázquez, J.; Prado, M.
Food Chemistry: Molecular Sciences, 3 , 2021.
@article{Carvalho2021,
title = {Evaluation of simple sequence repeats (SSR) and single nucleotide polymorphism (SNP)-based methods in olive varieties from the Northwest of Spain and potential for miniaturization},
author = {Carvalho, J.; Yadav, S.; Garrido-Maestu, A.G.; Azinheiro, S.; Trujillo, I.; Barros-Velázquez, J.; Prado, M. },
url = {https://doi.org/10.1016/j.fochms.2021.100038},
doi = {10.1016/j.fochms.2021.100038},
year = {2021},
date = {2021-07-31},
journal = { Food Chemistry: Molecular Sciences},
volume = {3},
abstract = {Miniaturization of DNA-based techniques can bring interesting advantages for food analysis, such as portability of complex analytical procedures. In the olive oil industry, miniaturization can be particularly interesting for authenticity and traceability applications, through in situ control of raw materials before production and/or the final products. However, variety identification is challenging, and implementation on miniaturized settings must be carefully evaluated, starting from the selected analytical approach. In this work, SSR- and SNP-based genotyping strategies were investigated for the identification and differentiation of two olive varieties from the Northwest of Spain. For the selected SNPs two genotyping methods were tested: real-time allele-specific PCR and high resolution melting analysis. These methods were compared and evaluated regarding their potential for integration in a microfluidic device. Both SNP-based methods proved to be successful for identification of the selected varieties, however real-time allele-specific PCR was the one that achieved the best results when analyzing mixtures, allowing the identification of both monovarietal samples and mixtures of the varieties tested with up to 25%.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}Miniaturization of DNA-based techniques can bring interesting advantages for food analysis, such as portability of complex analytical procedures. In the olive oil industry, miniaturization can be particularly interesting for authenticity and traceability applications, through in situ control of raw materials before production and/or the final products. However, variety identification is challenging, and implementation on miniaturized settings must be carefully evaluated, starting from the selected analytical approach. In this work, SSR- and SNP-based genotyping strategies were investigated for the identification and differentiation of two olive varieties from the Northwest of Spain. For the selected SNPs two genotyping methods were tested: real-time allele-specific PCR and high resolution melting analysis. These methods were compared and evaluated regarding their potential for integration in a microfluidic device. Both SNP-based methods proved to be successful for identification of the selected varieties, however real-time allele-specific PCR was the one that achieved the best results when analyzing mixtures, allowing the identification of both monovarietal samples and mixtures of the varieties tested with up to 25%.Maryam Naseri ,Arnab Halder ,Mohsen Mohammadniaei ,Marta Prado ,Jon Ashley ,Yi Sun
A multivalent aptamer-based electrochemical biosensor for biomarker detection in urinary tract infection Journal Article
Electrochimica Acta (2021), 389 , 2021.
@article{Naseri2021b,
title = {A multivalent aptamer-based electrochemical biosensor for biomarker detection in urinary tract infection},
author = {Maryam Naseri ,Arnab Halder ,Mohsen Mohammadniaei ,Marta Prado ,Jon Ashley ,Yi Sun},
url = {https://doi.org/10.1016/j.electacta.2021.138644},
doi = {10.1016/j.electacta.2021.138644},
year = {2021},
date = {2021-05-23},
journal = { Electrochimica Acta (2021)},
volume = {389},
abstract = {Lactoferrin is a multifunctional protein of the transferrin family and is known as a biomarker for various clinical diseases including urinary tract infection (UTI). However, wide concentration range of lactoferrin in urine samples due to the high interpatient variations, requires a more practical biosensor. In this article, we used a novel multivalent aptamer immobilized on the surface of screen-printed gold electrode (aptamer/SPGE) to develop the first electrochemical aptasensor for label-free detection of lactoferrin with wide dynamic detection range and high sensitivity. The performance of the fabricated biosensor was tested using electrochemical impedance spectroscopy and differential pulse voltammetry. The multivalent aptamer as the bioreceptor with high affinity and good specificity against human lactoferrin, acts to enhance the electrochemical signals and widen the working window. The aptamer/SPGE demonstrates superior sensing performances for lactoferrin in buffer solution, with a wide linear range of 10 to 1300 ng/mL with LOD of 0.9 ng/mL, as well as high selectivity, and excellent reproducibility. Besides, the constructed aptasensor was successfully applied to quantify lactoferrin concentrations in spiked urine solutions. Owing to excellent sensitivity, ease of miniaturization, simple sensing procedure, low-cost, and fast response, the proposed electrochemical aptasensor indicates a great potential towards the development of lactoferrin analysis systems, which would be helpful in the early diagnosis of UTI.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}Lactoferrin is a multifunctional protein of the transferrin family and is known as a biomarker for various clinical diseases including urinary tract infection (UTI). However, wide concentration range of lactoferrin in urine samples due to the high interpatient variations, requires a more practical biosensor. In this article, we used a novel multivalent aptamer immobilized on the surface of screen-printed gold electrode (aptamer/SPGE) to develop the first electrochemical aptasensor for label-free detection of lactoferrin with wide dynamic detection range and high sensitivity. The performance of the fabricated biosensor was tested using electrochemical impedance spectroscopy and differential pulse voltammetry. The multivalent aptamer as the bioreceptor with high affinity and good specificity against human lactoferrin, acts to enhance the electrochemical signals and widen the working window. The aptamer/SPGE demonstrates superior sensing performances for lactoferrin in buffer solution, with a wide linear range of 10 to 1300 ng/mL with LOD of 0.9 ng/mL, as well as high selectivity, and excellent reproducibility. Besides, the constructed aptasensor was successfully applied to quantify lactoferrin concentrations in spiked urine solutions. Owing to excellent sensitivity, ease of miniaturization, simple sensing procedure, low-cost, and fast response, the proposed electrochemical aptasensor indicates a great potential towards the development of lactoferrin analysis systems, which would be helpful in the early diagnosis of UTI.Roumani, F.; Azinheiro, S.; Sousa, H.; Sousa, A.; Timóteo, M.; Varandas, T.; Fonseca-silva, D.; Baldaque, I.; Carvalho, J.; Prado, M.; Garrido-Maestu
Viruses 2021, 2021.
@article{Roumani2021,
title = {Optimization and Clinical Evaluation of a Multi-Target Loop-Mediated Isothermal Amplification Assay for the Detection of SARS-CoV-2 in Nasopharyngeal Samples},
author = {Roumani, F.; Azinheiro, S.; Sousa, H.; Sousa, A.; Timóteo, M.; Varandas, T.; Fonseca-silva, D.; Baldaque, I.; Carvalho, J.; Prado, M.; Garrido-Maestu},
url = {https://doi.org/10.3390/v13050940},
doi = {10.3390/v13050940},
year = {2021},
date = {2021-05-16},
journal = {Viruses 2021},
abstract = {SARS-CoV-2 is the coronavirus responsible for COVID-19, which has spread worldwide, affecting more than 200 countries, infecting over 140 million people in one year. The gold standard to identify infected people is RT-qPCR, which is highly sensitive, but needs specialized equipment and trained personnel. The demand for these reagents has caused shortages in certain countries. Isothermal nucleic acid techniques, such as loop-mediated isothermal amplification (LAMP) have emerged as an alternative or as a complement to RT-qPCR. In this study, we developed and evaluated a multi-target RT-LAMP for the detection of SARS-CoV-2. The method was evaluated against an RT-qPCR in 152 clinical nasopharyngeal swab samples. The results obtained indicated that both assays presented a “good concordance” (Cohen’s k of 0.69), the RT-LAMP was highly specific (99%) but had lower sensitivity compared to the gold standard (63.3%). The calculated low sensitivity was associated with samples with very low viral load (RT-qPCR Cq values higher than 35) which may be associated with non-infectious individuals. If an internal Cq threshold below 35 was set, the sensitivity and Cohen’s k increased to 90.9% and 0.92, respectively. The interpretation of the Cohen’s k for this was “very good concordance”. The RT-LAMP is an attractive approach for frequent individual testing in decentralized setups.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}SARS-CoV-2 is the coronavirus responsible for COVID-19, which has spread worldwide, affecting more than 200 countries, infecting over 140 million people in one year. The gold standard to identify infected people is RT-qPCR, which is highly sensitive, but needs specialized equipment and trained personnel. The demand for these reagents has caused shortages in certain countries. Isothermal nucleic acid techniques, such as loop-mediated isothermal amplification (LAMP) have emerged as an alternative or as a complement to RT-qPCR. In this study, we developed and evaluated a multi-target RT-LAMP for the detection of SARS-CoV-2. The method was evaluated against an RT-qPCR in 152 clinical nasopharyngeal swab samples. The results obtained indicated that both assays presented a “good concordance” (Cohen’s k of 0.69), the RT-LAMP was highly specific (99%) but had lower sensitivity compared to the gold standard (63.3%). The calculated low sensitivity was associated with samples with very low viral load (RT-qPCR Cq values higher than 35) which may be associated with non-infectious individuals. If an internal Cq threshold below 35 was set, the sensitivity and Cohen’s k increased to 90.9% and 0.92, respectively. The interpretation of the Cohen’s k for this was “very good concordance”. The RT-LAMP is an attractive approach for frequent individual testing in decentralized setups.Carvalho J and Maestu AG and Azinheiro S and Fuciños P and Velázquez J B and Miguel RJ De and Gros V and Prado M
Faster monitoring of the invasive alien species ( IAS ) Dreissena polymorpha in river basins through isothermal amplification Journal Article
Scientific Reports, 11 (10175), 2021.
@article{J2021b,
title = {Faster monitoring of the invasive alien species ( IAS ) Dreissena polymorpha in river basins through isothermal amplification},
author = {Carvalho J and Maestu AG and Azinheiro S and Fuciños P and Velázquez J B and Miguel RJ De and Gros V and Prado M},
url = {https://doi.org/10.1038/s41598-021-89574-w},
doi = {10.1038/s41598-021-89574-w},
year = {2021},
date = {2021-05-13},
journal = {Scientific Reports},
volume = {11},
number = {10175},
abstract = {Zebra mussel (Dreissena polymorpha) is considered as one of the 100 most harmful IAS in the world. Traditional detection methods have limitations, and PCR based environmental DNA detection has provided interesting results for early warning. However, in the last years, the development of isothermal amplification methods has received increasing attention. Among them, loop-mediated isothermal amplification (LAMP) has several advantages, including its higher tolerance to the presence of inhibitors and the possibility of naked-eye detection, which enables and simplifies its potential use in decentralized settings. In the current study, a real-time LAMP (qLAMP) method for the detection of Dreissena polymorpha was developed and tested with samples from the Guadalquivir River basin, together with two real-time PCR (qPCR) methods using different detection chemistries, targeting a specific region of the mitochondrial gene cytochrome C oxidase subunit I. All three developed approaches were evaluated regarding specificity, sensitivity and time required for detection. Regarding sensitivity, both qPCR approaches were more sensitive than qLAMP by one order of magnitude, however the qLAMP method proved to be as specific and much faster being performed in just 9 min versus 23 and 29 min for the qPCR methods based on hydrolysis probe and intercalating dye respectively.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}Zebra mussel (Dreissena polymorpha) is considered as one of the 100 most harmful IAS in the world. Traditional detection methods have limitations, and PCR based environmental DNA detection has provided interesting results for early warning. However, in the last years, the development of isothermal amplification methods has received increasing attention. Among them, loop-mediated isothermal amplification (LAMP) has several advantages, including its higher tolerance to the presence of inhibitors and the possibility of naked-eye detection, which enables and simplifies its potential use in decentralized settings. In the current study, a real-time LAMP (qLAMP) method for the detection of Dreissena polymorpha was developed and tested with samples from the Guadalquivir River basin, together with two real-time PCR (qPCR) methods using different detection chemistries, targeting a specific region of the mitochondrial gene cytochrome C oxidase subunit I. All three developed approaches were evaluated regarding specificity, sensitivity and time required for detection. Regarding sensitivity, both qPCR approaches were more sensitive than qLAMP by one order of magnitude, however the qLAMP method proved to be as specific and much faster being performed in just 9 min versus 23 and 29 min for the qPCR methods based on hydrolysis probe and intercalating dye respectively.Ipatov, A and Garrido- Maestu, A and Guerreiro, J R and Purwidyantri, A and Azinheiro, S and Carvalho, J and Romani, F and Elumalai, M and Prado, M
Application of Omics-based Miniaturized Systems in Food Quality and Safety Book Chapter
2021, ISBN: 978-1-78801-884-5.
@inbook{Ipatov2021,
title = {Application of Omics-based Miniaturized Systems in Food Quality and Safety},
author = {Ipatov, A and Garrido- Maestu, A and Guerreiro, J R and Purwidyantri, A and Azinheiro, S and Carvalho, J and Romani, F and Elumalai, M and Prado, M},
url = {https://doi.org/10.1039/9781839163005},
doi = {10.1039/9781839163005-00222},
isbn = {978-1-78801-884-5},
year = {2021},
date = {2021-03-26},
journal = {Foodomics: Omic Strategies and Applications in Food Science},
abstract = {Miniaturized devices are revolutionizing food analysis. Such devices have a number of advantages, namely low consumption of sample and reagents, portability, low-energy consumption, rapid response and multiplexing capabilities, that have fostered their development towards marketable products. An increasing number of microfluidic-based devices are being released to the market, providing enormous possibilities for application in the omics sciences. One of the most interesting, and yet most challenging applications of miniaturized devices in food monitoring, is the development of hand-held devices that can enable in situ analysis at different points of the food value chain. The main objectives are to ensure the health of consumers, to easily determine whether a food product has been subjected to cross-contamination and, simultaneously, to identify how and when this cross-contamination occurred in order to establish the proper corrective measures. Herein, we focus mainly on the development of tools for food monitoring, particularly for protein- and DNA-based analysis, including the main fabrication processes and materials. Sample preparation and pre-treatment is highlighted as a challenging and fundamental step of the analytical procedure. Particularly in the case of foodborne pathogens, in which sample treatment is considered the main bottleneck for fast and in situ analysis. Nano-labelled and non-labelled detection strategies are discussed together with the main advantages and disadvantages of both for targeted analysis. Although there are several interesting developments of miniaturized devices that can contribute to omics analysis via non-targeted approaches, this chapter focuses mainly on the latest developments towards targeted analysis.},
keywords = {},
pubstate = {published},
tppubtype = {inbook}
}Miniaturized devices are revolutionizing food analysis. Such devices have a number of advantages, namely low consumption of sample and reagents, portability, low-energy consumption, rapid response and multiplexing capabilities, that have fostered their development towards marketable products. An increasing number of microfluidic-based devices are being released to the market, providing enormous possibilities for application in the omics sciences. One of the most interesting, and yet most challenging applications of miniaturized devices in food monitoring, is the development of hand-held devices that can enable in situ analysis at different points of the food value chain. The main objectives are to ensure the health of consumers, to easily determine whether a food product has been subjected to cross-contamination and, simultaneously, to identify how and when this cross-contamination occurred in order to establish the proper corrective measures. Herein, we focus mainly on the development of tools for food monitoring, particularly for protein- and DNA-based analysis, including the main fabrication processes and materials. Sample preparation and pre-treatment is highlighted as a challenging and fundamental step of the analytical procedure. Particularly in the case of foodborne pathogens, in which sample treatment is considered the main bottleneck for fast and in situ analysis. Nano-labelled and non-labelled detection strategies are discussed together with the main advantages and disadvantages of both for targeted analysis. Although there are several interesting developments of miniaturized devices that can contribute to omics analysis via non-targeted approaches, this chapter focuses mainly on the latest developments towards targeted analysis.Prabowo BA, Purwidyantri A, Liu B, Lai HC, Liu KC
Gold nanoparticle-assisted plasmonic enhancement for DNA detection on a graphene-based portable surface plasmon resonance sensor Journal Article
Nanotechnology, 32 (9), 2021.
@article{BA2021,
title = {Gold nanoparticle-assisted plasmonic enhancement for DNA detection on a graphene-based portable surface plasmon resonance sensor},
author = {Prabowo BA, Purwidyantri A, Liu B, Lai HC, Liu KC},
url = {https://doi.org/10.1088/1361-6528/abcd62},
doi = {10.1088/1361-6528/abcd62},
year = {2021},
date = {2021-02-26},
journal = {Nanotechnology},
volume = {32},
number = {9},
abstract = {The impact of different gold nanoparticle (GNP) structures on plasmonic enhancement for DNA detection is investigated on a few-layer graphene (FLG) surface plasmon resonance (SPR) sensor. Two distinct structures of gold nano-urchins (GNu) and gold nanorods (GNr) were used to bind the uniquely designed single-stranded probe DNA (ssDNA) of Mycobacterium tuberculosis complex DNA. The two types of GNP-ssDNA mixture were adsorbed onto the FLG-coated SPR sensor through the π-π stacking force between the ssDNA and the graphene layer. In the presence of complementary single-stranded DNA, the hybridization process took place and gradually removed the probes from the graphene surface. From SPR sensor preparation, the annealing process of the Au layer of the SPR sensor effectively enhanced the FLG coverage leading to a higher load of the probe DNA onto the sensing interface. The FLG was shown to be effective in providing a larger surface area for biomolecular capture due to its roughness. Carried out in the DNA hybridization study with the SPR sensor, GNu, with its rough and spiky structures, significantly reinforced the overall DNA hybridization signal compared with GNr with smooth superficies, especially in capturing the probe DNA. The DNA hybridization detection assisted by GNu reached the femtomolar range limit of detection. An optical simulation validated the extreme plasmonic field enhancement at the tip of the GNu spicules. The overall integrated approach of the graphene-based SPR sensor and GNu-assisted DNA detection provided the proof-of-concept for the possibility of tuberculosis disease screening using a low-cost and portable system to be potentially applied in remote or third-world countries.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}The impact of different gold nanoparticle (GNP) structures on plasmonic enhancement for DNA detection is investigated on a few-layer graphene (FLG) surface plasmon resonance (SPR) sensor. Two distinct structures of gold nano-urchins (GNu) and gold nanorods (GNr) were used to bind the uniquely designed single-stranded probe DNA (ssDNA) of Mycobacterium tuberculosis complex DNA. The two types of GNP-ssDNA mixture were adsorbed onto the FLG-coated SPR sensor through the π-π stacking force between the ssDNA and the graphene layer. In the presence of complementary single-stranded DNA, the hybridization process took place and gradually removed the probes from the graphene surface. From SPR sensor preparation, the annealing process of the Au layer of the SPR sensor effectively enhanced the FLG coverage leading to a higher load of the probe DNA onto the sensing interface. The FLG was shown to be effective in providing a larger surface area for biomolecular capture due to its roughness. Carried out in the DNA hybridization study with the SPR sensor, GNu, with its rough and spiky structures, significantly reinforced the overall DNA hybridization signal compared with GNr with smooth superficies, especially in capturing the probe DNA. The DNA hybridization detection assisted by GNu reached the femtomolar range limit of detection. An optical simulation validated the extreme plasmonic field enhancement at the tip of the GNu spicules. The overall integrated approach of the graphene-based SPR sensor and GNu-assisted DNA detection provided the proof-of-concept for the possibility of tuberculosis disease screening using a low-cost and portable system to be potentially applied in remote or third-world countries.Purwidyantri, A.; Tian, Y.-C.; Saputra, G.M.A.; Prabowo, B.A.; Liu, H.-L.; Yang, C.-M.; Lai, C.-S.
Gold Nanoframe Array Electrode for Straightforward Detection of Hydrogen Peroxide Journal Article
Chemosensors, 9 (37), 2021.
@article{Purwidyantri2021b,
title = {Gold Nanoframe Array Electrode for Straightforward Detection of Hydrogen Peroxide},
author = {Purwidyantri, A.; Tian, Y.-C.; Saputra, G.M.A.; Prabowo, B.A.; Liu, H.-L.; Yang, C.-M.; Lai, C.-S.},
url = {https://doi.org/10.3390/chemosensors9020037},
doi = {10.3390/chemosensors9020037},
year = {2021},
date = {2021-02-16},
journal = {Chemosensors},
volume = {9},
number = {37},
abstract = {The nanostructuring of a sensing membrane is performed through colloidal nanosphere lithography (NSL) techniques with a tiny polystyrene nanobead template 100 nm in size. The solvent ratio adjustment has been proven to be effective in assisting the monolayer deposition of small templating particles with minimal defects. Two distinct structures, namely, a billowy gold nanostructure (BGN) where the nanobead template is left unetched and a gold nanoframe array (GNA) with a regular ring-like structure after template removal, are used for the extended-gate field-effect transistor (EGFET) electrodes. The GNA structure generates an electroactive surface area significantly (~20%) larger than its geometrical area as well as a greater surface roughness than the BGN. When integrated with the portable constant voltage–constant current (CVCC) FET circuitry for pH screening to determine the optimized measurement conditions for H2O2 sensing, the GNA sensing membrane also shows more improved Nernstian sensitivity at ~50 mV/pH than the BGN electrode. The more optimized sensitivity is then proven using the GNA in the detection of H2O2, the most common representative reactive oxygen species (ROS) involved in the environment, food, and neurodegenerative diseases, such as Parkinson´s and Alzheimer´s diseases. The GNA electrode has a sensitivity of 70.42 mV/log µM [H2O2] and a limit of detection (LoD) of 1.183 µM H2O2. The integrated ion sensing system employing unique, highly ordered gold array gate electrodes and a portable CVCC circuit system has shown a stable real-time output voltage signal, representing an alternative to bulky conventional FET devices for potential on-site H2O2 detection.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}The nanostructuring of a sensing membrane is performed through colloidal nanosphere lithography (NSL) techniques with a tiny polystyrene nanobead template 100 nm in size. The solvent ratio adjustment has been proven to be effective in assisting the monolayer deposition of small templating particles with minimal defects. Two distinct structures, namely, a billowy gold nanostructure (BGN) where the nanobead template is left unetched and a gold nanoframe array (GNA) with a regular ring-like structure after template removal, are used for the extended-gate field-effect transistor (EGFET) electrodes. The GNA structure generates an electroactive surface area significantly (~20%) larger than its geometrical area as well as a greater surface roughness than the BGN. When integrated with the portable constant voltage–constant current (CVCC) FET circuitry for pH screening to determine the optimized measurement conditions for H2O2 sensing, the GNA sensing membrane also shows more improved Nernstian sensitivity at ~50 mV/pH than the BGN electrode. The more optimized sensitivity is then proven using the GNA in the detection of H2O2, the most common representative reactive oxygen species (ROS) involved in the environment, food, and neurodegenerative diseases, such as Parkinson´s and Alzheimer´s diseases. The GNA electrode has a sensitivity of 70.42 mV/log µM [H2O2] and a limit of detection (LoD) of 1.183 µM H2O2. The integrated ion sensing system employing unique, highly ordered gold array gate electrodes and a portable CVCC circuit system has shown a stable real-time output voltage signal, representing an alternative to bulky conventional FET devices for potential on-site H2O2 detection.Carvalho J and Diéguez L and Ipatov A and Guerreiro J R and Garrido-Maestu A and Azinheiro S and Prado M
Single-use microfluidic device for purification and concentration of environmental DNA from river water Journal Article
Talanta, 226 , 2021.
@article{J2021,
title = {Single-use microfluidic device for purification and concentration of environmental DNA from river water},
author = {Carvalho J and Diéguez L and Ipatov A and Guerreiro J R and Garrido-Maestu A and Azinheiro S and Prado M},
url = {https://doi.org/10.1016/j.talanta.2021.122109},
doi = {10.1016/j.talanta.2021.122109},
year = {2021},
date = {2021-02-04},
journal = {Talanta},
volume = {226},
abstract = {Purification and concentration of DNA is a critical step on DNA-based analysis, which should ensure efficient DNA isolation and effective removal of contaminants that may interfere with downstream DNA amplification. Complexity of samples, minute content of target analyte, or high DNA fragmentation greatly entangles the success of this step. To overcome this issue, we designed and fabricated a novel miniaturized disposable device for a highly efficient DNA purification. The microfluidic device showed binding efficiency and elution yield of 90.1% and 86.7%, respectively. Moreover, the effect of DNA fragmentation, a parameter that has not been previously addressed, showed a great impact in the recovery step. The microfluidic system integrated micropillars with chitosan being used as the solid-phase for a pH-dependent DNA capture and release. We have showed the potential of the device in the successful purification of environmental DNA (eDNA) from river water samples contaminated with Dreissena polymorpha, an invasive alien species responsible for unquestionable economic and environmental consequences in river water basins. Additionally, the device was also able to concentrate the DNA extract from highly diluted samples, showing promising results for the early detection of such invasive species, which may allow prompt measures for a more efficient control in affected areas. Suitability for integration with downstream DNA analysis was also demonstrated through qPCR analysis of the samples purified with the microfluidic device, allowing detection of the target species even if highly diluted.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}Purification and concentration of DNA is a critical step on DNA-based analysis, which should ensure efficient DNA isolation and effective removal of contaminants that may interfere with downstream DNA amplification. Complexity of samples, minute content of target analyte, or high DNA fragmentation greatly entangles the success of this step. To overcome this issue, we designed and fabricated a novel miniaturized disposable device for a highly efficient DNA purification. The microfluidic device showed binding efficiency and elution yield of 90.1% and 86.7%, respectively. Moreover, the effect of DNA fragmentation, a parameter that has not been previously addressed, showed a great impact in the recovery step. The microfluidic system integrated micropillars with chitosan being used as the solid-phase for a pH-dependent DNA capture and release. We have showed the potential of the device in the successful purification of environmental DNA (eDNA) from river water samples contaminated with Dreissena polymorpha, an invasive alien species responsible for unquestionable economic and environmental consequences in river water basins. Additionally, the device was also able to concentrate the DNA extract from highly diluted samples, showing promising results for the early detection of such invasive species, which may allow prompt measures for a more efficient control in affected areas. Suitability for integration with downstream DNA analysis was also demonstrated through qPCR analysis of the samples purified with the microfluidic device, allowing detection of the target species even if highly diluted.Purwidyantri A and Domingues T and Borme J and Guerreiro J R and Ipatov A and Abreu C M and Martins M and Alpuim P and Prado M
Influence of the Electrolyte Salt Concentration on DNA Detection with Graphene Transistors Journal Article
Biosensors, 11 , pp. 24, 2021.
@article{A2021,
title = {Influence of the Electrolyte Salt Concentration on DNA Detection with Graphene Transistors},
author = {Purwidyantri A and Domingues T and Borme J and Guerreiro J R and Ipatov A and Abreu C M and Martins M and Alpuim P and Prado M},
url = {https://doi.org/10.3390/bios11010024},
doi = {10.3390/bios11010024},
year = {2021},
date = {2021-01-17},
journal = {Biosensors},
volume = {11},
pages = {24},
abstract = {Liquid-gated Graphene Field-Effect Transistors (GFET) are ultrasensitive bio-detection platforms carrying out the graphene’s exceptional intrinsic functionalities. Buffer and dilution factor are prevalent strategies towards the optimum performance of the GFETs. However, beyond the Debye length (λD), the role of the graphene-electrolytes’ ionic species interactions on the DNA behavior at the nanoscale interface is complicated. We studied the characteristics of the GFETs under different ionic strength, pH, and electrolyte type, e.g., phosphate buffer (PB), and phosphate buffer saline (PBS), in an automatic portable built-in system. The electrostatic gating and charge transfer phenomena were inferred from the field-effect measurements of the Dirac point position in single-layer graphene (SLG) transistors transfer curves. Results denote that λD is not the main factor governing the effective nanoscale screening environment. We observed that the longer λD was not the determining characteristic for sensitivity increment and limit of detection (LoD) as demonstrated by different types and ionic strengths of measuring buffers. In the DNA hybridization study, our findings show the role of the additional salts present in PBS, as compared to PB, in increasing graphene electron mobility, electrostatic shielding, intermolecular forces and DNA adsorption kinetics leading to an improved sensitivity.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}Liquid-gated Graphene Field-Effect Transistors (GFET) are ultrasensitive bio-detection platforms carrying out the graphene’s exceptional intrinsic functionalities. Buffer and dilution factor are prevalent strategies towards the optimum performance of the GFETs. However, beyond the Debye length (λD), the role of the graphene-electrolytes’ ionic species interactions on the DNA behavior at the nanoscale interface is complicated. We studied the characteristics of the GFETs under different ionic strength, pH, and electrolyte type, e.g., phosphate buffer (PB), and phosphate buffer saline (PBS), in an automatic portable built-in system. The electrostatic gating and charge transfer phenomena were inferred from the field-effect measurements of the Dirac point position in single-layer graphene (SLG) transistors transfer curves. Results denote that λD is not the main factor governing the effective nanoscale screening environment. We observed that the longer λD was not the determining characteristic for sensitivity increment and limit of detection (LoD) as demonstrated by different types and ionic strengths of measuring buffers. In the DNA hybridization study, our findings show the role of the additional salts present in PBS, as compared to PB, in increasing graphene electron mobility, electrostatic shielding, intermolecular forces and DNA adsorption kinetics leading to an improved sensitivity.Roumani F and Azinheiro S and Carvalho J and Prado M and Garrido-maestu A
Food Control, 125 , 2021.
@article{F2021,
title = {Loop-mediated isothermal amplification combined with immunomagnetic separation and propidium monoazide for the specific detection of viable Listeria monocytogenes in milk products , with an internal amplification control},
author = {Roumani F and Azinheiro S and Carvalho J and Prado M and Garrido-maestu A},
url = {https://doi.org/10.1016/j.foodcont.2021.107975},
doi = {doi.org/10.1016/j.foodcont.2021.107975},
year = {2021},
date = {2021-01-15},
journal = {Food Control},
volume = {125},
abstract = {Nowadays, the most widely accepted rapid methods in food microbiology rely on nucleic acid amplification such as PCR/real-time PCR. A major claimed limitation of these methods is their incapacity to differentiate among viable and non-viable microorganisms. In the present study we report the development of a novel multiplex loop-mediated isothermal amplification method which, by combining immunomagnetic separation, to concentrate and purify the bacteria, along with propidium monoazide to block the amplification of DNA from non-viable microorganisms. The method allowed to specifically detect viable Listeria monocytogenes present in milk products. We designed an internal amplification control to rule out false negative results due to reaction inhibition, which is differentiated from L. monocytogenes by a simple melt curve analysis. Overall, the methodology provided results higher than 95% in terms of sensitivity, specificity and accuracy, as well as a Cohen's k of 0.97, reaching a limit of detection of 2.7 cfu/25 g. In samples inoculated with up to 106-107 cfu of dead microorganisms, the method demonstrated capable of effectively eliminating undesired amplification.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}Nowadays, the most widely accepted rapid methods in food microbiology rely on nucleic acid amplification such as PCR/real-time PCR. A major claimed limitation of these methods is their incapacity to differentiate among viable and non-viable microorganisms. In the present study we report the development of a novel multiplex loop-mediated isothermal amplification method which, by combining immunomagnetic separation, to concentrate and purify the bacteria, along with propidium monoazide to block the amplification of DNA from non-viable microorganisms. The method allowed to specifically detect viable Listeria monocytogenes present in milk products. We designed an internal amplification control to rule out false negative results due to reaction inhibition, which is differentiated from L. monocytogenes by a simple melt curve analysis. Overall, the methodology provided results higher than 95% in terms of sensitivity, specificity and accuracy, as well as a Cohen's k of 0.97, reaching a limit of detection of 2.7 cfu/25 g. In samples inoculated with up to 106-107 cfu of dead microorganisms, the method demonstrated capable of effectively eliminating undesired amplification.Jon Ashley, Anna-Lisa Schaap-Johansen, Mohsen Mohammadniaei, Maryam Naseri, Paolo Marcatili, Marta Prado, Yi Sun
Terminal deoxynucleotidyl transferase-mediated formation of protein binding polynucleotides Journal Article
Nucleic Acids Research, 2021.
@article{and Anna-Lisaand Mohsenand MaryamNaseri2021,
title = {Terminal deoxynucleotidyl transferase-mediated formation of protein binding polynucleotides},
author = {Jon Ashley, Anna-Lisa Schaap-Johansen, Mohsen Mohammadniaei, Maryam Naseri, Paolo Marcatili, Marta Prado, Yi Sun},
url = { https://doi.org/10.1093/nar/gkaa1263},
doi = {10.1093/nar/gkaa1263},
year = {2021},
date = {2021-01-04},
journal = {Nucleic Acids Research},
abstract = {Terminal deoxynucleotidyl transferase (TdT) enzyme plays an integral part in the V(D)J recombination, allowing for the huge diversity in expression of immunoglobulins and T-cell receptors within lymphocytes, through their unique ability to incorporate single nucleotides into oligonucleotides without the need of a template. The role played by TdT in lymphocytes precursors found in early vertebrates is not known. In this paper, we demonstrated a new screening method that utilises TdT to form libraries of variable sized (vsDNA) libraries of polynucleotides that displayed binding towards protein targets. The extent of binding and size distribution of each vsDNA library towards their respective protein target can be controlled through the alteration of different reaction conditions such as time of reaction, nucleotide ratio and initiator concentration raising the possibility for the rational design of aptamers prior to screening. The new approach, allows for the screening of aptamers based on size as well as sequence in a single round, which minimises PCR bias. We converted the protein bound sequences to dsDNA using rapid amplification of variable ends assays (RAVE) and sequenced them using next generation sequencing. The resultant aptamers demonstrated low nanomolar binding and high selectivity towards their respective targets.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}Terminal deoxynucleotidyl transferase (TdT) enzyme plays an integral part in the V(D)J recombination, allowing for the huge diversity in expression of immunoglobulins and T-cell receptors within lymphocytes, through their unique ability to incorporate single nucleotides into oligonucleotides without the need of a template. The role played by TdT in lymphocytes precursors found in early vertebrates is not known. In this paper, we demonstrated a new screening method that utilises TdT to form libraries of variable sized (vsDNA) libraries of polynucleotides that displayed binding towards protein targets. The extent of binding and size distribution of each vsDNA library towards their respective protein target can be controlled through the alteration of different reaction conditions such as time of reaction, nucleotide ratio and initiator concentration raising the possibility for the rational design of aptamers prior to screening. The new approach, allows for the screening of aptamers based on size as well as sequence in a single round, which minimises PCR bias. We converted the protein bound sequences to dsDNA using rapid amplification of variable ends assays (RAVE) and sequenced them using next generation sequencing. The resultant aptamers demonstrated low nanomolar binding and high selectivity towards their respective targets. -
2020
Akhilesh Kumar Gupta, Chih-Hsien Hsu, Agnes Purwidyantri, Briliant Adhi Prabowo, Kuo-Ping Chiu, Ching-Hsiang Chen, Ya-Chung Tian, Chao-SungLai
ZnO-Nanorod processed PC-SET as the light-harvesting model for plasmontronic fluorescence Sensor Journal Article
Sensors and Actuators B: Chemical, 307 , pp. 127597, 2020.
@article{Gupta2020,
title = {ZnO-Nanorod processed PC-SET as the light-harvesting model for plasmontronic fluorescence Sensor},
author = {Akhilesh Kumar Gupta, Chih-Hsien Hsu, Agnes Purwidyantri, Briliant Adhi Prabowo, Kuo-Ping Chiu, Ching-Hsiang Chen, Ya-Chung Tian, Chao-SungLai},
url = {https://doi.org/10.1016/j.snb.2019.127597},
doi = {10.1016/j.snb.2019.127597},
year = {2020},
date = {2020-12-19},
journal = {Sensors and Actuators B: Chemical},
volume = {307},
pages = {127597},
abstract = {This paper reports the combined plasmon coupled - surface energy transfer (PC-SET) and a distance-dependent model constructed by gold nanoparticles (GNPs) over zinc oxide nanorod (ZnO-NR) as a robust and tunable plasmontronic fluorescence regime for the detection of rhodamine 6G (R6G). Further, the deposition of metal created extraordinary contact through ZnO-NR utilizing a rapid thermal process (RTP) allowing the interaction of plasmon-coupled nature and surface energy transfer from the donor (R6G) to the acceptor (ZnO). The percentage of energy transfer efficiency continuously decreased with the increment of GNPs size, shown by 72.93, 67.52 and 47.86%, corresponding to the increase of the distance between the donor and acceptor of 63.03, 67.25, and 82.49 Å, respectively. In other words, the efficiency of PC-SET complied the 1/d4 distance dependence model between donor and acceptor molecules with the detection of long-distance ranges from 46.95 to 120 Å. These findings suggest that PC-SET process has a more realistic agreement with experimental outcomes and highly supports quenching efficiency impacts related to the size of GNPs, in which the smaller size of NPs causes’ greater effectiveness towards challenges in light harvest enhanced sensing system.
Keywords: ZnO-nanorods; PC-SET model; Light-harvesting; GNPs size effect; Schottky barrier height (SBH); Plasmontronic-fluorescence sensor},
keywords = {},
pubstate = {published},
tppubtype = {article}
}This paper reports the combined plasmon coupled - surface energy transfer (PC-SET) and a distance-dependent model constructed by gold nanoparticles (GNPs) over zinc oxide nanorod (ZnO-NR) as a robust and tunable plasmontronic fluorescence regime for the detection of rhodamine 6G (R6G). Further, the deposition of metal created extraordinary contact through ZnO-NR utilizing a rapid thermal process (RTP) allowing the interaction of plasmon-coupled nature and surface energy transfer from the donor (R6G) to the acceptor (ZnO). The percentage of energy transfer efficiency continuously decreased with the increment of GNPs size, shown by 72.93, 67.52 and 47.86%, corresponding to the increase of the distance between the donor and acceptor of 63.03, 67.25, and 82.49 Å, respectively. In other words, the efficiency of PC-SET complied the 1/d4 distance dependence model between donor and acceptor molecules with the detection of long-distance ranges from 46.95 to 120 Å. These findings suggest that PC-SET process has a more realistic agreement with experimental outcomes and highly supports quenching efficiency impacts related to the size of GNPs, in which the smaller size of NPs causes’ greater effectiveness towards challenges in light harvest enhanced sensing system.
Keywords: ZnO-nanorods; PC-SET model; Light-harvesting; GNPs size effect; Schottky barrier height (SBH); Plasmontronic-fluorescence sensorEspiña, Begoña, Prado, Marta, dos Santos, Marilia B., Salonen, Laura M., Queirós, Raquel B., Fernandes, Soraia P.S., Vial, Stephanie and Martins, Verónica C.
2 New techniques in environment monitoring". Climate Change and Marine and Freshwater Toxins Book Chapter
pp. 35-106, Berlin, Boston: De Gruyter, 2020.
@inbook{Espiña2020b,
title = {2 New techniques in environment monitoring". Climate Change and Marine and Freshwater Toxins},
author = {Espiña, Begoña, Prado, Marta, dos Santos, Marilia B., Salonen, Laura M., Queirós, Raquel B., Fernandes, Soraia P.S., Vial, Stephanie and Martins, Verónica C.},
url = {https://doi.org/10.1515/9783110625738-002},
doi = {10.1515/9783110625738-002},
year = {2020},
date = {2020-12-16},
pages = {35-106},
publisher = {Berlin, Boston: De Gruyter},
keywords = {},
pubstate = {published},
tppubtype = {inbook}
}A Garrido-Maestu and S Azinheiro and F Roumani and J Carvalho and M Prado
Front. Microbiol, 11 , pp. 1-8, 2020.
@article{Prado2020b,
title = {Application of Short Pre-enrichment, and Double Chemistry Real-Time PCR, Combining Fluorescent Probes and an Intercalating Dye, for Same-Day Detection and Confirmation of Salmonella spp. and Escherichia coli O157 in Ground Beef and Chicken Samples},
author = {A Garrido-Maestu and S Azinheiro and F Roumani and J Carvalho and M Prado},
url = {https://doi.org/10.3389/fmicb.2020.591041},
doi = {10.3389/fmicb.2020.591041},
year = {2020},
date = {2020-10-09},
journal = {Front. Microbiol},
volume = {11},
pages = {1-8},
abstract = {Molecular methods, particularly those based on real-time PCR (qPCR), have become a popular approach to detect pathogens in food samples. This technique may take advantage of hydrolysis fluorescent probes for increased specificity. Even though suitable, this approach loses the capacity of performing result confirmation by melt curve analysis. In the current study, we developed an alternative approach, combining fluorescent probes along with an intercalating dye (SYBR Green) in order to simultaneously detect, and confirm the result, of two foodborne pathogens (Salmonella spp. and Escherichia coli O157). This new approach named double chemistry qPCR was combined with a short pre-enrichment in order to obtain a multiplex “same-day” detection method for the selected pathogens. The evaluation of the novel method in spiked food samples (ground beef and chicken breast) obtained values of relative sensitivity, specificity, and accuracy higher than 95%, and Cohen’s kappa of 0.92, with a Limit of Detection95 below 5 cfu/25 g, demonstrating its reliability. In addition to this, the method was challenged by inoculating heat-stressed bacteria as well as dead ones. It was observed that it was also possible to detect stressed bacteria with an initial inoculation level below 10 cfu/25 g. Also, it was noticed that high initial concentration of either pathogen (higher than 104 cfu/25 g) was needed in order to generate false positive results due to the presence of dead bacteria, thus the method presents potential for its application in the specific detection of live microorganisms.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}Molecular methods, particularly those based on real-time PCR (qPCR), have become a popular approach to detect pathogens in food samples. This technique may take advantage of hydrolysis fluorescent probes for increased specificity. Even though suitable, this approach loses the capacity of performing result confirmation by melt curve analysis. In the current study, we developed an alternative approach, combining fluorescent probes along with an intercalating dye (SYBR Green) in order to simultaneously detect, and confirm the result, of two foodborne pathogens (Salmonella spp. and Escherichia coli O157). This new approach named double chemistry qPCR was combined with a short pre-enrichment in order to obtain a multiplex “same-day” detection method for the selected pathogens. The evaluation of the novel method in spiked food samples (ground beef and chicken breast) obtained values of relative sensitivity, specificity, and accuracy higher than 95%, and Cohen’s kappa of 0.92, with a Limit of Detection95 below 5 cfu/25 g, demonstrating its reliability. In addition to this, the method was challenged by inoculating heat-stressed bacteria as well as dead ones. It was observed that it was also possible to detect stressed bacteria with an initial inoculation level below 10 cfu/25 g. Also, it was noticed that high initial concentration of either pathogen (higher than 104 cfu/25 g) was needed in order to generate false positive results due to the presence of dead bacteria, thus the method presents potential for its application in the specific detection of live microorganisms.L Wu and A Teixeira and A Garrido-Maestu and L Muinelo-Romay and L Lima and LL Santos and M Prado and L Diéguez
Profiling DNA mutation patterns by SERS fingerprinting for supervised cancer classification Journal Article
Biosens. Bioelectron, 165 , 2020.
@article{Wu2020,
title = {Profiling DNA mutation patterns by SERS fingerprinting for supervised cancer classification},
author = {L Wu and A Teixeira and A Garrido-Maestu and L Muinelo-Romay and L Lima and LL Santos and M Prado and L Diéguez},
url = {https://doi.org/10.1016/j.bios.2020.112392},
doi = {10.1016/j.bios.2020.112392},
year = {2020},
date = {2020-10-01},
journal = {Biosens. Bioelectron},
volume = {165},
abstract = {Profiling DNA mutation patterns for cancer classification plays an essential role in precision and personalized medicine. Conventional PCR-based mutation assay is limited by the extensive labour on target amplification. We herein create an amplification-free surface enhanced Raman spectroscopy (SERS) biochip which enables direct and simultaneous identification of multiple point mutations in tumor cells. Without pre-amplifying the target sequences, the SERS assay reads out the presence of cellular mutations through the interpretation of Raman fingerprints. The SERS sensor is integrated into a microfluidic chip, achieving one-step multiplex analysis within 40 min. Importantly, by combining SERS spectra encoding technique with supervised learning algorithm, a panel of nucleotide mixtures can be well distinguished according to their mutation profiles. We initially demonstrate an excellent levels of classification in samples from colorectal cancer and melanoma cell lines. For final clinical validation, the system performance is verified by classifying cancer patient samples, which shows an accuracy above 90%. Due to the simplicity and rapidness, the SERS biosensor is expected to become a promising tool for clinical point-of-care diagnosis towards precision medicine.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}Profiling DNA mutation patterns for cancer classification plays an essential role in precision and personalized medicine. Conventional PCR-based mutation assay is limited by the extensive labour on target amplification. We herein create an amplification-free surface enhanced Raman spectroscopy (SERS) biochip which enables direct and simultaneous identification of multiple point mutations in tumor cells. Without pre-amplifying the target sequences, the SERS assay reads out the presence of cellular mutations through the interpretation of Raman fingerprints. The SERS sensor is integrated into a microfluidic chip, achieving one-step multiplex analysis within 40 min. Importantly, by combining SERS spectra encoding technique with supervised learning algorithm, a panel of nucleotide mixtures can be well distinguished according to their mutation profiles. We initially demonstrate an excellent levels of classification in samples from colorectal cancer and melanoma cell lines. For final clinical validation, the system performance is verified by classifying cancer patient samples, which shows an accuracy above 90%. Due to the simplicity and rapidness, the SERS biosensor is expected to become a promising tool for clinical point-of-care diagnosis towards precision medicine.Filipa M.C. Freitas, Miguel A. Cerqueira, Catarina Gonçalves, Sarah Azinheiro, Alejandro Garrido-Maestu, António A. Vicente, Lorenzo M. Pastrana, José A. Teixeira, Michele Michelin
Green synthesis of lignin nano- and micro-particles: Physicochemical characterization, bioactive properties and cytotoxicity assessment Journal Article
International Journal of Biological Macromolecules, 163 , pp. 1798-1809, 2020.
@article{Freitas2020,
title = {Green synthesis of lignin nano- and micro-particles: Physicochemical characterization, bioactive properties and cytotoxicity assessment},
author = {Filipa M.C. Freitas, Miguel A. Cerqueira, Catarina Gonçalves, Sarah Azinheiro, Alejandro Garrido-Maestu, António A. Vicente, Lorenzo M. Pastrana, José A. Teixeira, Michele Michelin},
url = {https://doi.org/10.1016/j.ijbiomac.2020.09.110.},
doi = {10.1016/j.ijbiomac.2020.09.110.},
year = {2020},
date = {2020-09-29},
journal = {International Journal of Biological Macromolecules},
volume = {163},
pages = {1798-1809},
abstract = {Lignin particles (LPs) have gained prominence due to their biodegradability and bioactive properties. LP production at nano and micro scale produced from organosolv lignin and the understanding of size's effect on their properties is unexplored. This work aimed to produce and characterize lignin nanoparticles and microparticles using a green synthesis process, based on ethanol-solubilized lignin and water. Spherical shape LPs, with a mean size of 75 nm and 215 nm and with a low polydispersity were produced, as confirmed by transmission electron microscopy and dynamic light scattering. LPs thermal stability improved over raw lignin, and the chemical structure of lignin was not affected by the production method. The antimicrobial tests proved that LPs presented a bacteriostatic effect on Escherichiacoli and Salmonella enterica. Regarding the antioxidant potential, LPs had a good antioxidant activity that increased with the reaction time and LPs concentration. LPs also presented an antioxidant effect against intracellular ROS, reducing the intracellular ROS levels significantly. Furthermore, the LPs showed a low cytotoxic effect in Caco-2 cell line. These results showed that LPs at different scales (nano and micro) present biological properties and are safe to be used in different high value industrial sectors, such as biomedical, pharmaceutical and food.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}Lignin particles (LPs) have gained prominence due to their biodegradability and bioactive properties. LP production at nano and micro scale produced from organosolv lignin and the understanding of size's effect on their properties is unexplored. This work aimed to produce and characterize lignin nanoparticles and microparticles using a green synthesis process, based on ethanol-solubilized lignin and water. Spherical shape LPs, with a mean size of 75 nm and 215 nm and with a low polydispersity were produced, as confirmed by transmission electron microscopy and dynamic light scattering. LPs thermal stability improved over raw lignin, and the chemical structure of lignin was not affected by the production method. The antimicrobial tests proved that LPs presented a bacteriostatic effect on Escherichiacoli and Salmonella enterica. Regarding the antioxidant potential, LPs had a good antioxidant activity that increased with the reaction time and LPs concentration. LPs also presented an antioxidant effect against intracellular ROS, reducing the intracellular ROS levels significantly. Furthermore, the LPs showed a low cytotoxic effect in Caco-2 cell line. These results showed that LPs at different scales (nano and micro) present biological properties and are safe to be used in different high value industrial sectors, such as biomedical, pharmaceutical and food.S Azinheiro and J Carvalho and M Prado and A Garrido-Maestu
Foods, 9 , 2020.
@article{Azinheiro2020,
title = {Application of Recombinase Polymerase Amplification with Lateral Flow for a Naked-Eye Detection of Listeria monocytogenes on Food Processing Surfaces},
author = {S Azinheiro and J Carvalho and M Prado and A Garrido-Maestu},
url = {https://doi.org/10.3390/foods9091249},
doi = {10.3390/foods9091249},
year = {2020},
date = {2020-09-07},
journal = {Foods},
volume = {9},
abstract = {The continuous contamination of foods with L. monocytogenes, highlights the need for additional controls in the food industry. The verification of food processing plants is key to avoid cross-contaminations, and to assure the safety of the food products. In this study, a new methodology for the detection of L. monocytogenes on food contact surfaces was developed and evaluated. It combines Recombinase Polymerase Amplification (RPA) with the lateral flow (LF) naked-eye detection. Different approaches for the recovery of the bacteria from the surface, the enrichment step and downstream analysis by RPA-LF were tested and optimized. The results were compared with a standard culture-based technique and qPCR analysis. Sampling procedure with sponges was more efficient for the recovery of the bacteria than a regular swab. A 24 h enrichment in ONE broth was needed for the most sensitive detection of the pathogen. By RPA-LF, it was possible to detect 1.1 pg/µL of pure L. monocytogenes DNA, and the complete methodology reached a LoD50 of 4.2 CFU/cm2 and LoD95 of 18.2 CFU/cm2. These results are comparable with the culture-based methodology and qPCR. The developed approach allows for a next-day detection without complex equipment and a naked-eye visualization of the results.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}The continuous contamination of foods with L. monocytogenes, highlights the need for additional controls in the food industry. The verification of food processing plants is key to avoid cross-contaminations, and to assure the safety of the food products. In this study, a new methodology for the detection of L. monocytogenes on food contact surfaces was developed and evaluated. It combines Recombinase Polymerase Amplification (RPA) with the lateral flow (LF) naked-eye detection. Different approaches for the recovery of the bacteria from the surface, the enrichment step and downstream analysis by RPA-LF were tested and optimized. The results were compared with a standard culture-based technique and qPCR analysis. Sampling procedure with sponges was more efficient for the recovery of the bacteria than a regular swab. A 24 h enrichment in ONE broth was needed for the most sensitive detection of the pathogen. By RPA-LF, it was possible to detect 1.1 pg/µL of pure L. monocytogenes DNA, and the complete methodology reached a LoD50 of 4.2 CFU/cm2 and LoD95 of 18.2 CFU/cm2. These results are comparable with the culture-based methodology and qPCR. The developed approach allows for a next-day detection without complex equipment and a naked-eye visualization of the results.S Azinheiro and J Carvalho and M Prado
Multiplex Detection of Salmonella spp., E. coli O157 and L. monocytogenes by qPCR Melt Curve Analysis in Spiked Infant Formula Journal Article
Microorganisms, 8 , 2020.
@article{Azinheiro2020c,
title = {Multiplex Detection of Salmonella spp., E. coli O157 and L. monocytogenes by qPCR Melt Curve Analysis in Spiked Infant Formula},
author = {S Azinheiro and J Carvalho and M Prado},
url = {https://doi.org/10.3390/microorganisms8091359},
doi = {10.3390/microorganisms8091359},
year = {2020},
date = {2020-09-04},
journal = {Microorganisms},
volume = {8},
abstract = {Food poisoning continue to be a threat in the food industry showing a need to improve the detection of the pathogen responsible for the hospitalization cases and death. DNA-based techniques represent a real advantage and allow the detection of several targets at the same time, reducing cost and time of analysis. The development of new methodology using SYBR Green qPCR for the detection of L. monocytogenes, Salmonella spp. and E. coli O157 simultaneously was developed and a non-competitive internal amplification control (NC-IAC) was implemented to detect reaction inhibition. The formulation and supplementation of the enrichment medium was also optimized to allow the growth of all pathogens. The limit of detection (LoD) 95% obtained was <1 CFU/25 g for E. coli O157, and 2 CFU/25 g for Salmonella spp. and L. monocytogenes and regarding the multiplex detection a LoD 95% of 1.7 CFU/25 g was observed. The specificity, relative sensitivity and accuracy of full methodology were 100% and the use of the NC-IAC allowed the reliability of the results without interfering with the sensitivity of the methodology. The described study proved to obtain results comparable to those of probe-based qPCR, and more economically than classical high resolution melting qPCR, being both important aspects for its implementation in the food industry.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}Food poisoning continue to be a threat in the food industry showing a need to improve the detection of the pathogen responsible for the hospitalization cases and death. DNA-based techniques represent a real advantage and allow the detection of several targets at the same time, reducing cost and time of analysis. The development of new methodology using SYBR Green qPCR for the detection of L. monocytogenes, Salmonella spp. and E. coli O157 simultaneously was developed and a non-competitive internal amplification control (NC-IAC) was implemented to detect reaction inhibition. The formulation and supplementation of the enrichment medium was also optimized to allow the growth of all pathogens. The limit of detection (LoD) 95% obtained was <1 CFU/25 g for E. coli O157, and 2 CFU/25 g for Salmonella spp. and L. monocytogenes and regarding the multiplex detection a LoD 95% of 1.7 CFU/25 g was observed. The specificity, relative sensitivity and accuracy of full methodology were 100% and the use of the NC-IAC allowed the reliability of the results without interfering with the sensitivity of the methodology. The described study proved to obtain results comparable to those of probe-based qPCR, and more economically than classical high resolution melting qPCR, being both important aspects for its implementation in the food industry.AJN Oliveira and J de Wild and K Oliveira and BA Valença and JP Teixeira and JRL Guerreiro and S Abalde-Cela and TS Lopes and RM Ribeiro and JM V Cunha and MA Curado and M Monteiro and A Violas and AG Silva and M Prado and PA Fernandes and B Vermang and PMP Salomé
Encapsulation of Nanostructures in a Dielectric Matrix Providing Optical Enhancement in Ultrathin Solar Cells Journal Article
Sol. RRL., 4 , 2020.
@article{Oliveira2020,
title = {Encapsulation of Nanostructures in a Dielectric Matrix Providing Optical Enhancement in Ultrathin Solar Cells},
author = {AJN Oliveira and J de Wild and K Oliveira and BA Valença and JP Teixeira and JRL Guerreiro and S Abalde-Cela and TS Lopes and RM Ribeiro and JM V Cunha and MA Curado and M Monteiro and A Violas and AG Silva and M Prado and PA Fernandes and B Vermang and PMP Salomé},
url = {https://doi.org/10.1002/solr.202000310},
doi = {10.1002/solr.202000310},
year = {2020},
date = {2020-08-29},
journal = {Sol. RRL.},
volume = {4},
abstract = {The incorporation of nanostructures in optoelectronic devices for enhancing their optical performance is widely studied. However, several problems related to the processing complexity and the low performance of the nanostructures have hindered such actions in real‐life devices. Herein, a novel way of introducing gold nanoparticles in a solar cell structure is proposed in which the nanostructures are encapsulated with a dielectric layer, shielding them from high temperatures and harsh growth processing conditions of the remaining device. Through optical simulations, an enhancement of the effective optical path length of approximately four times the nominal thickness of the absorber layer is verified with the new architecture. Furthermore, the proposed concept in a Cu(In,Ga)Se2 solar cell device is demonstrated, where the short‐circuit current density is increased by 17.4%. The novel structure presented in this work is achieved by combining a bottom‐up chemical approach of depositing the nanostructures with a top‐down photolithographic process, which allows for an electrical contact.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}The incorporation of nanostructures in optoelectronic devices for enhancing their optical performance is widely studied. However, several problems related to the processing complexity and the low performance of the nanostructures have hindered such actions in real‐life devices. Herein, a novel way of introducing gold nanoparticles in a solar cell structure is proposed in which the nanostructures are encapsulated with a dielectric layer, shielding them from high temperatures and harsh growth processing conditions of the remaining device. Through optical simulations, an enhancement of the effective optical path length of approximately four times the nominal thickness of the absorber layer is verified with the new architecture. Furthermore, the proposed concept in a Cu(In,Ga)Se2 solar cell device is demonstrated, where the short‐circuit current density is increased by 17.4%. The novel structure presented in this work is achieved by combining a bottom‐up chemical approach of depositing the nanostructures with a top‐down photolithographic process, which allows for an electrical contact.António J. N. Oliveira, Jessica de Wild, Kevin Oliveira, Beatriz A. Valença, Jennifer P. Teixeira, Joana R. L. Guerreiro, Sara Abalde-Cela, Tomás S. Lopes, Rodrigo M. Ribeiro, José M. V. Cunha, Marco A. Curado, Margarida Monteiro, André Violas, Ana Gomes Silva, Marta Prado, Paulo A. Fernandes, Bart Vermang, Pedro M. P. Salomé
Encapsulation of Nanostructures in a Dielectric Matrix Providing Optical Enhancement in Ultrathin Solar Cells Journal Article
RRL Solar, 4 (11), 2020.
@article{Oliveira2020b,
title = {Encapsulation of Nanostructures in a Dielectric Matrix Providing Optical Enhancement in Ultrathin Solar Cells},
author = {António J. N. Oliveira, Jessica de Wild, Kevin Oliveira, Beatriz A. Valença, Jennifer P. Teixeira, Joana R. L. Guerreiro, Sara Abalde-Cela, Tomás S. Lopes, Rodrigo M. Ribeiro, José M. V. Cunha, Marco A. Curado, Margarida Monteiro, André Violas, Ana Gomes Silva, Marta Prado, Paulo A. Fernandes, Bart Vermang, Pedro M. P. Salomé},
url = {https://doi.org/10.1002/solr.202000310},
doi = {10.1002/solr.202000310},
year = {2020},
date = {2020-08-29},
journal = {RRL Solar},
volume = {4},
number = {11},
abstract = {The incorporation of nanostructures in optoelectronic devices for enhancing their optical performance is widely studied. However, several problems related to the processing complexity and the low performance of the nanostructures have hindered such actions in real-life devices. Herein, a novel way of introducing gold nanoparticles in a solar cell structure is proposed in which the nanostructures are encapsulated with a dielectric layer, shielding them from high temperatures and harsh growth processing conditions of the remaining device. Through optical simulations, an enhancement of the effective optical path length of approximately four times the nominal thickness of the absorber layer is verified with the new architecture. Furthermore, the proposed concept in a Cu(In,Ga)Se2 solar cell device is demonstrated, where the short-circuit current density is increased by 17.4%. The novel structure presented in this work is achieved by combining a bottom-up chemical approach of depositing the nanostructures with a top-down photolithographic process, which allows for an electrical contact.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}The incorporation of nanostructures in optoelectronic devices for enhancing their optical performance is widely studied. However, several problems related to the processing complexity and the low performance of the nanostructures have hindered such actions in real-life devices. Herein, a novel way of introducing gold nanoparticles in a solar cell structure is proposed in which the nanostructures are encapsulated with a dielectric layer, shielding them from high temperatures and harsh growth processing conditions of the remaining device. Through optical simulations, an enhancement of the effective optical path length of approximately four times the nominal thickness of the absorber layer is verified with the new architecture. Furthermore, the proposed concept in a Cu(In,Ga)Se2 solar cell device is demonstrated, where the short-circuit current density is increased by 17.4%. The novel structure presented in this work is achieved by combining a bottom-up chemical approach of depositing the nanostructures with a top-down photolithographic process, which allows for an electrical contact.S Azinheiro and K Kant and MA Shahbazi and A Garrido-Maestu and M Prado and L Diéguez
A smart microfluidic platform for rapid multiplexed detection of foodborne pathogens Journal Article
Food Control, 114 , 2020.
@article{Azinheiro2020b,
title = {A smart microfluidic platform for rapid multiplexed detection of foodborne pathogens},
author = {S Azinheiro and K Kant and MA Shahbazi and A Garrido-Maestu and M Prado and L Diéguez},
url = {https://doi.org/10.1016/j.foodcont.2020.107242},
doi = {10.1016/j.foodcont.2020.107242},
year = {2020},
date = {2020-08-01},
journal = {Food Control},
volume = {114},
abstract = {Rapid and sensitive detection of foodborne pathogens in food industry is of high importance in day-to-day practice to ensure safe food. To address this issue, multiple foodborne pathogens are targeted for rapid identification based in DNA amplification. A 3D PDMS sponge was fabricated using salt crystals as scarifying mold and functionalized with a ligand, apolipoprotein-H (ApoH), to test bacterial capturing for both Gram positive (L. monocytogenes) and negative bacteria (Salmonella spp.), in a microfluidic device. Pure culture of both pathogens in a range of ~10–105 CFU/mL were tested and the application of the developed automated pre-concentration protocol in real samples was verified using spiked surface samples after swab sampling. Bacterial DNA was extracted directly from the sponge and used for Real Time quantitative Polymerase Chain Reaction (qPCR) detection. The sponges did not show any significant resistance to sample flow and could easily be incorporated in a microfluidic device. A capture efficiency above 70% was observed for both targeted (Gram positive and Gram negative) pathogens and a Limit of Detection (LoD) in the range of 103 and 104 CFU/mL was obtained for Salmonella spp. and L. monocytogenes, respectively. Using this approached, we are able to perform multiplexed (Gram positive and Gram negative) capturing and reduce the enrichment time compared to the gold standard plate culture (over 1-day) method. The use of a 3D sponge for direct capturing of multiplexed pathogen on microfluidic device, followed by qPCR detection is an efficient and versatile method to stratify the presence of bacteria. This approach and methodology has potential to be integrated in full automatized device and used as point of need (PoN) system for foodborne pathogen stratification in food packaging/production industries.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}Rapid and sensitive detection of foodborne pathogens in food industry is of high importance in day-to-day practice to ensure safe food. To address this issue, multiple foodborne pathogens are targeted for rapid identification based in DNA amplification. A 3D PDMS sponge was fabricated using salt crystals as scarifying mold and functionalized with a ligand, apolipoprotein-H (ApoH), to test bacterial capturing for both Gram positive (L. monocytogenes) and negative bacteria (Salmonella spp.), in a microfluidic device. Pure culture of both pathogens in a range of ~10–105 CFU/mL were tested and the application of the developed automated pre-concentration protocol in real samples was verified using spiked surface samples after swab sampling. Bacterial DNA was extracted directly from the sponge and used for Real Time quantitative Polymerase Chain Reaction (qPCR) detection. The sponges did not show any significant resistance to sample flow and could easily be incorporated in a microfluidic device. A capture efficiency above 70% was observed for both targeted (Gram positive and Gram negative) pathogens and a Limit of Detection (LoD) in the range of 103 and 104 CFU/mL was obtained for Salmonella spp. and L. monocytogenes, respectively. Using this approached, we are able to perform multiplexed (Gram positive and Gram negative) capturing and reduce the enrichment time compared to the gold standard plate culture (over 1-day) method. The use of a 3D sponge for direct capturing of multiplexed pathogen on microfluidic device, followed by qPCR detection is an efficient and versatile method to stratify the presence of bacteria. This approach and methodology has potential to be integrated in full automatized device and used as point of need (PoN) system for foodborne pathogen stratification in food packaging/production industries.Alejandro Garrido-Maestu, Sarah Azinheiro, Pablo Fuciños, Joana Carvalho, Marta Prado
Food Microbiology, 92 , pp. 103570, 2020.
@article{Garrido-Maestu2020c,
title = {Comparative study of multiplex real-time recombinase polymerase amplification and ISO 11290-1 methods for the detection of Listeria monocytogenes in dairy products},
author = {Alejandro Garrido-Maestu, Sarah Azinheiro, Pablo Fuciños, Joana Carvalho, Marta Prado},
url = {https://doi.org/10.1016/j.fm.2020.103570},
doi = {10.1016/j.fm.2020.103570},
year = {2020},
date = {2020-06-20},
journal = {Food Microbiology},
volume = {92},
pages = {103570},
abstract = {Dairy products have been implicated in foodborne infections caused by different bacterial pathogens. Among them, Listeria monocytogenes is of particular concern due to its ubiquity, resistance to sanitation processes and high mortality rates resulting from infection. These issues make the development of novel methods for the rapid detection of this bacterium of high interest. The evaluation of a novel multiplex real-time Recombinase Polymerase Amplification method including an internal amplification control is reported in the present work. The method performance was compared to that of the European reference method (ISO 11290-1) for the detection of the species in samples from 40 commercial products, including 14 UHT milk samples, 16 hard cheese samples, 6 infant dairy preparation samples and 4 fresh cheese samples. A limit of detection below 10 cfu/25 g or mL sample was achieved, and values higher than 90% were obtained for relative sensitivity, specificity, accuracy, positive and negative predictive values and the index (kappa) of concordance. Analysis was achieved within one working day, compared to the six days required using the ISO method. Moreover, slight modification of the ISO 11290-1 method to include secondary enrichment in half Fraser broth resulted in the confirmation of all positive samples.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}Dairy products have been implicated in foodborne infections caused by different bacterial pathogens. Among them, Listeria monocytogenes is of particular concern due to its ubiquity, resistance to sanitation processes and high mortality rates resulting from infection. These issues make the development of novel methods for the rapid detection of this bacterium of high interest. The evaluation of a novel multiplex real-time Recombinase Polymerase Amplification method including an internal amplification control is reported in the present work. The method performance was compared to that of the European reference method (ISO 11290-1) for the detection of the species in samples from 40 commercial products, including 14 UHT milk samples, 16 hard cheese samples, 6 infant dairy preparation samples and 4 fresh cheese samples. A limit of detection below 10 cfu/25 g or mL sample was achieved, and values higher than 90% were obtained for relative sensitivity, specificity, accuracy, positive and negative predictive values and the index (kappa) of concordance. Analysis was achieved within one working day, compared to the six days required using the ISO method. Moreover, slight modification of the ISO 11290-1 method to include secondary enrichment in half Fraser broth resulted in the confirmation of all positive samples.Lu, Yu-Jen and Purwidyantri, Agnes and Liu, Hui-Ling and Wang, Le-Wen and Shih, Cheng-Ye and Pijanowska, Dorota G. and Yang, Chia-Ming
Photoelectrochemical Detection of β-amyloid Peptides by a TiO2 Nanobrush Biosensor Journal Article
IEEE Sensors Journal, 20 (12), pp. 6248-6255, 2020.
@article{Lu2020,
title = {Photoelectrochemical Detection of β-amyloid Peptides by a TiO2 Nanobrush Biosensor},
author = {Lu, Yu-Jen and Purwidyantri, Agnes and Liu, Hui-Ling and Wang, Le-Wen and Shih, Cheng-Ye and Pijanowska, Dorota G. and Yang, Chia-Ming},
doi = {10.1109/JSEN.2020.2976561},
year = {2020},
date = {2020-06-15},
journal = {IEEE Sensors Journal},
volume = {20},
number = {12},
pages = {6248-6255},
abstract = {A simple, facile and cost-effective nanostructuring technique is proposed to construct a photoelectrochemical (PEC) sensor via the solution-based hydrothermal growth of a TiO 2 nanobrush (TiO 2 NB). It is demonstrated that the control of the TiO 2 seed solution ratio, process temperature and duration significantly contributed to the final morphological characteristics of the rutile TiO 2 NB, as confirmed by the X-ray diffraction (XRD) analysis. By carrying out a simple and inexpensive fabrication involving no additional surface modification materials, the proposed TiO 2 NB enabled the enhancement of the surface area by up to 162% in comparison with its actual geometric area. The photoactivity achieved by applying UV-range light was drastically improved, and photocurrents could potentially be utilized to enhance the redox activity on the interface. The system was used to detect β-amyloid (Aβ) 1-28 peptides, one of the most crucial biomarkers of patients with Alzheimer's disease (AD), and demonstrated an excellent biocompatibility caused by the straightforward self-assembled monolayers (SAMs) modified on its surface. For the detection of a wide range of Aβ 1-28 peptides, the constructed TiO 2 NB photoelectrochemical (PEC) sensor exhibited a great sensitivity of 114.8 μ A/(ng . mL -1 ) and limit of detection (LoD) of 26.3 ng.mL -1 , facilitating a simple, label-free, rapid, sensitive and noninvasive method to overcome the limitations of conventional techniques used for AD diagnosis.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}A simple, facile and cost-effective nanostructuring technique is proposed to construct a photoelectrochemical (PEC) sensor via the solution-based hydrothermal growth of a TiO 2 nanobrush (TiO 2 NB). It is demonstrated that the control of the TiO 2 seed solution ratio, process temperature and duration significantly contributed to the final morphological characteristics of the rutile TiO 2 NB, as confirmed by the X-ray diffraction (XRD) analysis. By carrying out a simple and inexpensive fabrication involving no additional surface modification materials, the proposed TiO 2 NB enabled the enhancement of the surface area by up to 162% in comparison with its actual geometric area. The photoactivity achieved by applying UV-range light was drastically improved, and photocurrents could potentially be utilized to enhance the redox activity on the interface. The system was used to detect β-amyloid (Aβ) 1-28 peptides, one of the most crucial biomarkers of patients with Alzheimer's disease (AD), and demonstrated an excellent biocompatibility caused by the straightforward self-assembled monolayers (SAMs) modified on its surface. For the detection of a wide range of Aβ 1-28 peptides, the constructed TiO 2 NB photoelectrochemical (PEC) sensor exhibited a great sensitivity of 114.8 μ A/(ng . mL -1 ) and limit of detection (LoD) of 26.3 ng.mL -1 , facilitating a simple, label-free, rapid, sensitive and noninvasive method to overcome the limitations of conventional techniques used for AD diagnosis.Garrido-Maestu A and Azinheiro S and Carvalho J and Espiña B and Prado M
Food Sci. Technol., (57), pp. 4143–4151, 2020.
@article{Prado2020b,
title = {Evaluation and implementation of commercial antibodies for improved nanoparticle-based immunomagnetic separation and real-time PCR for faster detection of Listeria monocytogenes},
author = {Garrido-Maestu A and Azinheiro S and Carvalho J and Espiña B and Prado M},
url = {https://doi.org/10.1007/s13197-020-04450-1},
doi = {10.1007/s13197-020-04450-1},
year = {2020},
date = {2020-04-24},
journal = {Food Sci. Technol.},
number = {57},
pages = {4143–4151},
abstract = {L. monocytogenes continues to be a major health issue in Europe, as well as worldwide. Faster methods, not only for detection, but also for sample preparation are of great interest particularly for this slow-growing pathogen. Immunomagnetic separation has been previously reported to be an effective way to concentrate bacteria, and remove inhibitors. In the present study, different commercial antibodies were evaluated to select the most appropriate one, in order to develop a highly specific method. Additionally, magnetic nanoparticles, instead of microparticles, were selected due to their reported advantages (higher surface-volume ration and faster kinetics). Finally, the separation protocol, with a calculated capture efficiency of 95%, was combined with real-time PCR for highly sensitive detection of the concentrated bacteria. The optimized IMS-qPCR allowed to reduce hands-on time in the sample treatment, without affecting the overall performance of the method as a very low limit of detection was still obtained (9.7 CFU/ 25 g) with values for sensitivity, specificity, accuracy, positive and negative predictive values of 100%, resulting in a kappa index of concordance of 1.00. These results were obtained in spiked food samples of different types (chicken, fish, milk, hard and fresh cheese), further demonstrating the applicability of the optimized methodology presented.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}L. monocytogenes continues to be a major health issue in Europe, as well as worldwide. Faster methods, not only for detection, but also for sample preparation are of great interest particularly for this slow-growing pathogen. Immunomagnetic separation has been previously reported to be an effective way to concentrate bacteria, and remove inhibitors. In the present study, different commercial antibodies were evaluated to select the most appropriate one, in order to develop a highly specific method. Additionally, magnetic nanoparticles, instead of microparticles, were selected due to their reported advantages (higher surface-volume ration and faster kinetics). Finally, the separation protocol, with a calculated capture efficiency of 95%, was combined with real-time PCR for highly sensitive detection of the concentrated bacteria. The optimized IMS-qPCR allowed to reduce hands-on time in the sample treatment, without affecting the overall performance of the method as a very low limit of detection was still obtained (9.7 CFU/ 25 g) with values for sensitivity, specificity, accuracy, positive and negative predictive values of 100%, resulting in a kappa index of concordance of 1.00. These results were obtained in spiked food samples of different types (chicken, fish, milk, hard and fresh cheese), further demonstrating the applicability of the optimized methodology presented.A Teixeira and JL Paris and F Roumani and L Diéguez and M Prado and B Espiña and S Abalde-Cela and A Garrido-Maestu and L Rodriguez-Lorenzo
Multifuntional gold nanoparticles for the SERS detection of pathogens combined with a LAMP-in-microdroplets approach Journal Article
Materials (Basel), 13 , 2020.
@article{Teixeira2020,
title = {Multifuntional gold nanoparticles for the SERS detection of pathogens combined with a LAMP-in-microdroplets approach},
author = {A Teixeira and JL Paris and F Roumani and L Diéguez and M Prado and B Espiña and S Abalde-Cela and A Garrido-Maestu and L Rodriguez-Lorenzo},
url = {https://doi.org/10.3390/ma13081934},
doi = {10.3390/ma13081934},
year = {2020},
date = {2020-04-20},
journal = {Materials (Basel)},
volume = {13},
abstract = {We developed a droplet-based optofluidic system for the detection of foodborne pathogens. Specifically, the loop-mediated isothermal amplification (LAMP) technique was combined with surface-enhanced Raman scattering (SERS), which offers an excellent method for DNA ultradetection. However, the direct SERS detection of DNA compromises the simplicity of data interpretation due to the variability of its SERS fingerprints. Therefore, we designed an indirect SERS detection method using multifunctional gold nanoparticles (AuNPs) based on the formation of pyrophosphate generated during the DNA amplification by LAMP. Towards this goal, we prepared multifunctional AuNPs involving three components with key roles: (1) thiolated poly(ethylene glycol) as stabilizing agent, (2) 1-naphthalenethiol as Raman reporter, and (3) glutathione as a bioinspired chelating agent of magnesium (II) ions. Thus, the variation in the SERS signal of 1-naphthalenethiol was controlled by the aggregation of AuNPs triggered by the complexation of pyrophosphate and glutathione with free magnesium ions. Using this strategy, we detected Listeria monocytogenes, not only in buffer, but also in a food matrix (i.e., ultra-high temperaturemilk) enabled by the massive production of hotspots as a result of the self-assemblies that enhanced the SERS signal. This allowed the development of a microdroplet-LAMP-SERS platform with isothermal amplification and real-time identification capabilities.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}We developed a droplet-based optofluidic system for the detection of foodborne pathogens. Specifically, the loop-mediated isothermal amplification (LAMP) technique was combined with surface-enhanced Raman scattering (SERS), which offers an excellent method for DNA ultradetection. However, the direct SERS detection of DNA compromises the simplicity of data interpretation due to the variability of its SERS fingerprints. Therefore, we designed an indirect SERS detection method using multifunctional gold nanoparticles (AuNPs) based on the formation of pyrophosphate generated during the DNA amplification by LAMP. Towards this goal, we prepared multifunctional AuNPs involving three components with key roles: (1) thiolated poly(ethylene glycol) as stabilizing agent, (2) 1-naphthalenethiol as Raman reporter, and (3) glutathione as a bioinspired chelating agent of magnesium (II) ions. Thus, the variation in the SERS signal of 1-naphthalenethiol was controlled by the aggregation of AuNPs triggered by the complexation of pyrophosphate and glutathione with free magnesium ions. Using this strategy, we detected Listeria monocytogenes, not only in buffer, but also in a food matrix (i.e., ultra-high temperaturemilk) enabled by the massive production of hotspots as a result of the self-assemblies that enhanced the SERS signal. This allowed the development of a microdroplet-LAMP-SERS platform with isothermal amplification and real-time identification capabilities.Agnes Purwidyantri, Myrtha Karina, Chih-Hsien Hsu, Yoice Srikandace, Briliant Adhi Prabowo, and Chao-Sung Lai
Facile Bacterial Cellulose Nanofibrillation for the Development of a Plasmonic Paper Sensor Journal Article
ACS Biomaterials Science & Engineering , 6 (5), pp. 3122-3131, 2020.
@article{Purwidyantri2020,
title = {Facile Bacterial Cellulose Nanofibrillation for the Development of a Plasmonic Paper Sensor},
author = {Agnes Purwidyantri, Myrtha Karina, Chih-Hsien Hsu, Yoice Srikandace, Briliant Adhi Prabowo, and Chao-Sung Lai},
doi = {10.1021/acsbiomaterials.9b01890},
year = {2020},
date = {2020-04-16},
journal = {ACS Biomaterials Science & Engineering },
volume = {6},
number = {5},
pages = {3122-3131},
abstract = {In this present work, a plasmonic sensor is developed through an extremely cheap cellulose-based source, widely known as a food product, nata de coco (NDC). Capturing its interesting features, such as innate surface roughness from naturally grown cellulose during its fermentation period, the engineering and modulation of NDC fibril size and properties were attempted through a high-pressure homogenization (HPH) treatment to obtain highly dense nanofibrils. After the transformation into a thin, paper-sheet form through a casting process, the homogenized bacterial cellulose (HBC) resulting from HPH was compared with the normally agitated bacterial cellulose (BC) pulp and decorated with silver nanoparticles (AgNPs) to produce plasmonic papers, for further application as surface-enhanced Raman scattering (SERS) substrate. As demonstrated in the measurement of Rhodamine 6G (R6G) molecule, the plasmonic HBC paper sheet provided more prominent SERS signals than the plasmonic BC due to its high surface roughness and improved textural properties from the nanofibrillation process favoring better adsorption of AgNPs and effective SERS hotspots generation. The plasmonic HBC obtained a 2 order higher estimated SERS enhancement factor over the plasmonic BC with a limit of detection of approximately 92 fM. Results denote that the proposed approach provides a new, green-synthesis route toward the exploration of biodegradable sources integrated into an inexpensive and simple nanostructuring process for the production of flexible, paper-based, plasmonic sensors.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}In this present work, a plasmonic sensor is developed through an extremely cheap cellulose-based source, widely known as a food product, nata de coco (NDC). Capturing its interesting features, such as innate surface roughness from naturally grown cellulose during its fermentation period, the engineering and modulation of NDC fibril size and properties were attempted through a high-pressure homogenization (HPH) treatment to obtain highly dense nanofibrils. After the transformation into a thin, paper-sheet form through a casting process, the homogenized bacterial cellulose (HBC) resulting from HPH was compared with the normally agitated bacterial cellulose (BC) pulp and decorated with silver nanoparticles (AgNPs) to produce plasmonic papers, for further application as surface-enhanced Raman scattering (SERS) substrate. As demonstrated in the measurement of Rhodamine 6G (R6G) molecule, the plasmonic HBC paper sheet provided more prominent SERS signals than the plasmonic BC due to its high surface roughness and improved textural properties from the nanofibrillation process favoring better adsorption of AgNPs and effective SERS hotspots generation. The plasmonic HBC obtained a 2 order higher estimated SERS enhancement factor over the plasmonic BC with a limit of detection of approximately 92 fM. Results denote that the proposed approach provides a new, green-synthesis route toward the exploration of biodegradable sources integrated into an inexpensive and simple nanostructuring process for the production of flexible, paper-based, plasmonic sensors.A Garrido-Maestu and S Azinheiro and J Carvalho and P Fuciños and M Prado
Optimized sample treatment, combined with real-time PCR, for same-day detection of E. coli O157 in ground beef and leafy greens Journal Article
Food Control, 108 , 2020.
@article{Garrido-Maestu2020,
title = {Optimized sample treatment, combined with real-time PCR, for same-day detection of E. coli O157 in ground beef and leafy greens},
author = {A Garrido-Maestu and S Azinheiro and J Carvalho and P Fuciños and M Prado},
url = {https://doi.org/10.1016/j.foodcont.2019.106790},
doi = {10.1016/j.foodcont.2019.106790},
year = {2020},
date = {2020-02-01},
journal = {Food Control},
volume = {108},
abstract = {E. coli O157 is the most commonly identified serotype among Shiga like-toxin E. coli infections. Current reference methodologies to detect these bacteria are reliable but take several days to provide a definitive result, as they require selective enrichment, molecular detection of the bacteria, followed by plate confirmation. Even alternative methods need extensive sample enrichment in order to increase the amount of bacteria to detectable numbers, resulting in “next-day detection” methodologies, as it is not possible to reach the final result within the same day of the start of the analysis. In the current study, the sample treatment was optimized in such a way that after a short enrichment (3 h) followed by an enzymatic treatment, DNA extraction and amplification by real-time PCR, it was possible to detect down to 3.9 and 3.3 CFU/25 g of ground beef and leafy greens respectively, achieving a combined LOD95 of 3.6 CFU/25 g. This sample treatment optimization allowed us to develop a real “same-day detection” method, being this a major advantage over other previously published methodologies. Additionally, the analysis of spiked samples provided excellent results (90%) for the relative sensitivity, specificity and accuracy; along with the positive and negative predictive values. Finally, the Cohen's k also obtained a value of 1, indicating that the results obtained with the novel method were in “almost complete concordance” with the expected values. It is worth to highlight that the performance parameters fulfill the requirements of the NordVal regulation for the validation of alternative methods.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}E. coli O157 is the most commonly identified serotype among Shiga like-toxin E. coli infections. Current reference methodologies to detect these bacteria are reliable but take several days to provide a definitive result, as they require selective enrichment, molecular detection of the bacteria, followed by plate confirmation. Even alternative methods need extensive sample enrichment in order to increase the amount of bacteria to detectable numbers, resulting in “next-day detection” methodologies, as it is not possible to reach the final result within the same day of the start of the analysis. In the current study, the sample treatment was optimized in such a way that after a short enrichment (3 h) followed by an enzymatic treatment, DNA extraction and amplification by real-time PCR, it was possible to detect down to 3.9 and 3.3 CFU/25 g of ground beef and leafy greens respectively, achieving a combined LOD95 of 3.6 CFU/25 g. This sample treatment optimization allowed us to develop a real “same-day detection” method, being this a major advantage over other previously published methodologies. Additionally, the analysis of spiked samples provided excellent results (90%) for the relative sensitivity, specificity and accuracy; along with the positive and negative predictive values. Finally, the Cohen's k also obtained a value of 1, indicating that the results obtained with the novel method were in “almost complete concordance” with the expected values. It is worth to highlight that the performance parameters fulfill the requirements of the NordVal regulation for the validation of alternative methods. -
2019
L Rodriguez-Lorenzo and A Garrido-Maestu and AK Bhunia and B Espiña and M Prado and L Diéguez and S Abalde-Cela
Gold Nanostars for the Detection of Foodborne Pathogens via Surface-Enhanced Raman Scattering Combined with Microfluidics Journal Article
ACS Appl. Nano Mater., 2 (10), pp. 6081–6086, 2019.
@article{Abalde-Cela2019,
title = {Gold Nanostars for the Detection of Foodborne Pathogens via Surface-Enhanced Raman Scattering Combined with Microfluidics},
author = {L Rodriguez-Lorenzo and A Garrido-Maestu and AK Bhunia and B Espiña and M Prado and L Diéguez and S Abalde-Cela},
url = {https://doi.org/10.1021/acsanm.9b01223},
doi = {10.1021/acsanm.9b01223},
year = {2019},
date = {2019-09-20},
journal = {ACS Appl. Nano Mater.},
volume = {2},
number = {10},
pages = {6081–6086},
abstract = {Herein, we demonstrated the potential of surface-enhanced Raman scattering (SERS) spectroscopy combined with microfluidics for the detection and discrimination of foodborne pathogens. SERS-tagged gold nanostars were functionalized with a monoclonal antibody specific for Listeria monocytogenes. In the presence of L. monocytogenes, a SERS signal corresponding to the SERS tag paired to the antibody was detected in real time and in continuous flow, enabling the discrimination of L. monocytogenes and Listeria innocua in just 100 s. To the best of our knowledge, this is the first time that SERS tags have been used for the in-flow detection of living organisms.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}Herein, we demonstrated the potential of surface-enhanced Raman scattering (SERS) spectroscopy combined with microfluidics for the detection and discrimination of foodborne pathogens. SERS-tagged gold nanostars were functionalized with a monoclonal antibody specific for Listeria monocytogenes. In the presence of L. monocytogenes, a SERS signal corresponding to the SERS tag paired to the antibody was detected in real time and in continuous flow, enabling the discrimination of L. monocytogenes and Listeria innocua in just 100 s. To the best of our knowledge, this is the first time that SERS tags have been used for the in-flow detection of living organisms.Carla Carvalho Joana Carvalho Marta Prado Alejandro Garrido-Maestu Sarah Azinheiro
Food Science, 84 (7), pp. 1881-1887, 2019.
@article{Garrido-Maestu2019b,
title = {Combination of Immunomagnetic separation and real-time Recombinase Polymerase Amplification (IMS-qRPA) for specific detection of Listeria monocytogenes in smoked salmon samples},
author = {Carla Carvalho Joana Carvalho Marta Prado Alejandro Garrido-Maestu Sarah Azinheiro},
url = {https://doi.org/10.1111/1750-3841.14662},
doi = {10.1111/1750-3841.14662},
year = {2019},
date = {2019-07-02},
journal = {Food Science},
volume = {84},
number = {7},
pages = {1881-1887},
abstract = {Nowadays, Listeria monocytogenes continues to be a major health issue. Therefore, improvements in the speed and reliability of its detection are still needed. In the present study, the combination of real‐time Recombinase Polymerase Amplification (qRPA) with immunomagnetic separation (IMS) is described. The proposed methodology was tested against a real‐time PCR method, and was successfully applied to 50 smoked salmon samples spiked at levels ranging from 2 to 9.3 × 102 cfu/25 g. L. monocytogenes was detected after a 24 hr pre‐enrichment, which represents a great improvement over other previously published RPA methods. Additionally, the evaluation of the method reported a Limit of dDetection 50 (LoD50) of 6.3 cfu/25 g, along with relative sensitivity, specificity and accuracy values higher than 90%. Finally, the index of kappa concordance was calculated to be 0.93 which is interpreted as “almost complete concordance” between the reference and alternative method. Overall, the described methodology proved to be faster, specific, and as sensitive as other methods based on RPA or real‐time PCR.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}Nowadays, Listeria monocytogenes continues to be a major health issue. Therefore, improvements in the speed and reliability of its detection are still needed. In the present study, the combination of real‐time Recombinase Polymerase Amplification (qRPA) with immunomagnetic separation (IMS) is described. The proposed methodology was tested against a real‐time PCR method, and was successfully applied to 50 smoked salmon samples spiked at levels ranging from 2 to 9.3 × 102 cfu/25 g. L. monocytogenes was detected after a 24 hr pre‐enrichment, which represents a great improvement over other previously published RPA methods. Additionally, the evaluation of the method reported a Limit of dDetection 50 (LoD50) of 6.3 cfu/25 g, along with relative sensitivity, specificity and accuracy values higher than 90%. Finally, the index of kappa concordance was calculated to be 0.93 which is interpreted as “almost complete concordance” between the reference and alternative method. Overall, the described methodology proved to be faster, specific, and as sensitive as other methods based on RPA or real‐time PCR.Carla Carvalho Joana Carvalho Marta Prado Alejandro Garrido-Maestu Sarah Azinheiro
Food Science, 84 (7), pp. 1881-1887, 2019.
@article{Garrido-Maestu2019bc,
title = {Combination of Immunomagnetic separation and real-time Recombinase Polymerase Amplification (IMS-qRPA) for specific detection of Listeria monocytogenes in smoked salmon samples},
author = {Carla Carvalho Joana Carvalho Marta Prado Alejandro Garrido-Maestu Sarah Azinheiro},
url = {https://doi.org/10.1111/1750-3841.14662},
doi = {10.1111/1750-3841.14662},
year = {2019},
date = {2019-07-02},
journal = {Food Science},
volume = {84},
number = {7},
pages = {1881-1887},
abstract = {Nowadays, Listeria monocytogenes continues to be a major health issue. Therefore, improvements in the speed and reliability of its detection are still needed. In the present study, the combination of real‐time Recombinase Polymerase Amplification (qRPA) with immunomagnetic separation (IMS) is described. The proposed methodology was tested against a real‐time PCR method, and was successfully applied to 50 smoked salmon samples spiked at levels ranging from 2 to 9.3 × 102 cfu/25 g. L. monocytogenes was detected after a 24 hr pre‐enrichment, which represents a great improvement over other previously published RPA methods. Additionally, the evaluation of the method reported a Limit of dDetection 50 (LoD50) of 6.3 cfu/25 g, along with relative sensitivity, specificity and accuracy values higher than 90%. Finally, the index of kappa concordance was calculated to be 0.93 which is interpreted as “almost complete concordance” between the reference and alternative method. Overall, the described methodology proved to be faster, specific, and as sensitive as other methods based on RPA or real‐time PCR.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}Nowadays, Listeria monocytogenes continues to be a major health issue. Therefore, improvements in the speed and reliability of its detection are still needed. In the present study, the combination of real‐time Recombinase Polymerase Amplification (qRPA) with immunomagnetic separation (IMS) is described. The proposed methodology was tested against a real‐time PCR method, and was successfully applied to 50 smoked salmon samples spiked at levels ranging from 2 to 9.3 × 102 cfu/25 g. L. monocytogenes was detected after a 24 hr pre‐enrichment, which represents a great improvement over other previously published RPA methods. Additionally, the evaluation of the method reported a Limit of dDetection 50 (LoD50) of 6.3 cfu/25 g, along with relative sensitivity, specificity and accuracy values higher than 90%. Finally, the index of kappa concordance was calculated to be 0.93 which is interpreted as “almost complete concordance” between the reference and alternative method. Overall, the described methodology proved to be faster, specific, and as sensitive as other methods based on RPA or real‐time PCR.SarahAzinheiro CarlaCarvalho JoanaCarvalho MartaPrado Alejandro Garrido-Maestu PabloFuciños
Specific detection of viable Salmonella Enteritidis by phage amplification combined with qPCR (PAA-qPCR) in spiked chicken meat samples Journal Article
Food Control, 99 , pp. 79-83, 2019.
@article{Garrido-Maestu2019b,
title = {Specific detection of viable Salmonella Enteritidis by phage amplification combined with qPCR (PAA-qPCR) in spiked chicken meat samples},
author = {SarahAzinheiro CarlaCarvalho JoanaCarvalho MartaPrado Alejandro Garrido-Maestu PabloFuciños},
url = {https://doi.org/10.1016/j.foodcont.2018.12.038},
doi = {10.1016/j.foodcont.2018.12.038},
year = {2019},
date = {2019-05-01},
journal = {Food Control},
volume = {99},
pages = {79-83},
abstract = {Serovar Enteritidis represents 45.7% of all Salmonella reported human cases identified in Europe. Additionally, “minced meat and meat preparations from poultry” have a high level of non-compliance, regarding Salmonella regulation.In the current study, a novel method based on the amplification of the Salmonella bacteriophage vB_SenS_PVP-SE2, coupled with real-time PCR (qPCR), was developed and evaluated, for the rapid detection of viable Salmonella Enteritidis in chicken samples. The results obtained indicated that the qPCR method could detect down to 0.22 fg/μL of pure virus DNA and a concentration of viral particles of 103 pfu/mL. After a short bacterial recovery step, the addition of bacteriophages to spiked chicken samples indicated that 8 cfu/25 g could be detected within 10 h, including the time for DNA extraction and qPCR analysis. Additionally, the evaluation of the performance parameters: relative sensitivity, specificity, accuracy, positive and negative predictive values, and index kappa of concordance, obtained values higher than 92%, and the acceptability limit values were within the limits. All these results demonstrate that the proposed methodology is a powerful tool for the rapid detection of viable Salmonella Enteritidis.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}Serovar Enteritidis represents 45.7% of all Salmonella reported human cases identified in Europe. Additionally, “minced meat and meat preparations from poultry” have a high level of non-compliance, regarding Salmonella regulation.In the current study, a novel method based on the amplification of the Salmonella bacteriophage vB_SenS_PVP-SE2, coupled with real-time PCR (qPCR), was developed and evaluated, for the rapid detection of viable Salmonella Enteritidis in chicken samples. The results obtained indicated that the qPCR method could detect down to 0.22 fg/μL of pure virus DNA and a concentration of viral particles of 103 pfu/mL. After a short bacterial recovery step, the addition of bacteriophages to spiked chicken samples indicated that 8 cfu/25 g could be detected within 10 h, including the time for DNA extraction and qPCR analysis. Additionally, the evaluation of the performance parameters: relative sensitivity, specificity, accuracy, positive and negative predictive values, and index kappa of concordance, obtained values higher than 92%, and the acceptability limit values were within the limits. All these results demonstrate that the proposed methodology is a powerful tool for the rapid detection of viable Salmonella Enteritidis.
SarahAzinheiro CarlaCarvalho JoanaCarvalho MartaPrado Alejandro Garrido-Maestu PabloFuciños
Specific detection of viable Salmonella Enteritidis by phage amplification combined with qPCR (PAA-qPCR) in spiked chicken meat samples Journal Article
Food Control, 99 , pp. 79-83, 2019.
@article{Garrido-Maestu2019d,
title = {Specific detection of viable Salmonella Enteritidis by phage amplification combined with qPCR (PAA-qPCR) in spiked chicken meat samples},
author = {SarahAzinheiro CarlaCarvalho JoanaCarvalho MartaPrado Alejandro Garrido-Maestu PabloFuciños},
url = {https://doi.org/10.1016/j.foodcont.2018.12.038},
doi = {10.1016/j.foodcont.2018.12.038},
year = {2019},
date = {2019-05-01},
journal = {Food Control},
volume = {99},
pages = {79-83},
abstract = {Serovar Enteritidis represents 45.7% of all Salmonella reported human cases identified in Europe. Additionally, “minced meat and meat preparations from poultry” have a high level of non-compliance, regarding Salmonella regulation.In the current study, a novel method based on the amplification of the Salmonella bacteriophage vB_SenS_PVP-SE2, coupled with real-time PCR (qPCR), was developed and evaluated, for the rapid detection of viable Salmonella Enteritidis in chicken samples. The results obtained indicated that the qPCR method could detect down to 0.22 fg/μL of pure virus DNA and a concentration of viral particles of 103 pfu/mL. After a short bacterial recovery step, the addition of bacteriophages to spiked chicken samples indicated that 8 cfu/25 g could be detected within 10 h, including the time for DNA extraction and qPCR analysis. Additionally, the evaluation of the performance parameters: relative sensitivity, specificity, accuracy, positive and negative predictive values, and index kappa of concordance, obtained values higher than 92%, and the acceptability limit values were within the limits. All these results demonstrate that the proposed methodology is a powerful tool for the rapid detection of viable Salmonella Enteritidis.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}Serovar Enteritidis represents 45.7% of all Salmonella reported human cases identified in Europe. Additionally, “minced meat and meat preparations from poultry” have a high level of non-compliance, regarding Salmonella regulation.In the current study, a novel method based on the amplification of the Salmonella bacteriophage vB_SenS_PVP-SE2, coupled with real-time PCR (qPCR), was developed and evaluated, for the rapid detection of viable Salmonella Enteritidis in chicken samples. The results obtained indicated that the qPCR method could detect down to 0.22 fg/μL of pure virus DNA and a concentration of viral particles of 103 pfu/mL. After a short bacterial recovery step, the addition of bacteriophages to spiked chicken samples indicated that 8 cfu/25 g could be detected within 10 h, including the time for DNA extraction and qPCR analysis. Additionally, the evaluation of the performance parameters: relative sensitivity, specificity, accuracy, positive and negative predictive values, and index kappa of concordance, obtained values higher than 92%, and the acceptability limit values were within the limits. All these results demonstrate that the proposed methodology is a powerful tool for the rapid detection of viable Salmonella Enteritidis.
Joana Guerreiro Sandra Carvalho Sara Abalde-Cela Marta Prado Lorena Diéguez R L Lei Wu Alejandro Garrido-Maestu
Amplification-free SERS analysis of DNA mutation in cancer cells with single-base sensitivity Journal Article
Nanoscale, 11 (16), pp. 7781-7789, 2019.
@article{Wu2019,
title = {Amplification-free SERS analysis of DNA mutation in cancer cells with single-base sensitivity},
author = {Joana Guerreiro Sandra Carvalho Sara Abalde-Cela Marta Prado Lorena Diéguez R L Lei Wu Alejandro Garrido-Maestu},
url = {https://doi.org/10.1039/C9NR00501C},
doi = {10.1039/C9NR00501C},
year = {2019},
date = {2019-04-01},
journal = {Nanoscale},
volume = {11},
number = {16},
pages = {7781-7789},
abstract = {Accurate and sensitive identification of DNA mutations in tumor cells is critical to the diagnosis, prognosis and personalized therapy of cancer. Conventional polymerase chain reaction (PCR)-based methods are limited by the complicated amplification process. Herein, an amplification-free surface enhanced Raman spectroscopy (SERS) approach which directly detects point mutations in cancer cells has been proposed. A highly sensitive and uniform SERS substrate was fabricated using gold@silver core–shell nanorods, achieving an enhancement factor of 1.85 × 106. By combining the SERS-active nanosubstrate with molecular beacon probes, the limit of detection reached as low as 50 fM. To enable parallel analysis and automated operation, the SERS sensor was integrated into a microfluidic chip. This novel chip-based assay was able to differentiate between mutated and wild-type KRAS genes among a variety of other nucleic acids from cancer cells in 40 min. Owing to the simple operation and fast analysis, the SERS-based DNA assay chip could potentially provide insights into clinical cancer theranostics in an easy and inexpensive manner at the point of care.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}Accurate and sensitive identification of DNA mutations in tumor cells is critical to the diagnosis, prognosis and personalized therapy of cancer. Conventional polymerase chain reaction (PCR)-based methods are limited by the complicated amplification process. Herein, an amplification-free surface enhanced Raman spectroscopy (SERS) approach which directly detects point mutations in cancer cells has been proposed. A highly sensitive and uniform SERS substrate was fabricated using gold@silver core–shell nanorods, achieving an enhancement factor of 1.85 × 106. By combining the SERS-active nanosubstrate with molecular beacon probes, the limit of detection reached as low as 50 fM. To enable parallel analysis and automated operation, the SERS sensor was integrated into a microfluidic chip. This novel chip-based assay was able to differentiate between mutated and wild-type KRAS genes among a variety of other nucleic acids from cancer cells in 40 min. Owing to the simple operation and fast analysis, the SERS-based DNA assay chip could potentially provide insights into clinical cancer theranostics in an easy and inexpensive manner at the point of care.Joana Guerreiro Sandra Carvalho Sara Abalde-Cela Marta Prado Lorena Diéguez R L Lei Wu Alejandro Garrido-Maestu
Amplification-free SERS analysis of DNA mutation in cancer cells with single-base sensitivity Journal Article
Nanoscale, 11 (16), pp. 7781-7789, 2019.
@article{Wu2019b,
title = {Amplification-free SERS analysis of DNA mutation in cancer cells with single-base sensitivity},
author = {Joana Guerreiro Sandra Carvalho Sara Abalde-Cela Marta Prado Lorena Diéguez R L Lei Wu Alejandro Garrido-Maestu},
url = {https://doi.org/10.1039/C9NR00501C},
doi = {10.1039/C9NR00501C},
year = {2019},
date = {2019-04-01},
journal = {Nanoscale},
volume = {11},
number = {16},
pages = {7781-7789},
abstract = {Accurate and sensitive identification of DNA mutations in tumor cells is critical to the diagnosis, prognosis and personalized therapy of cancer. Conventional polymerase chain reaction (PCR)-based methods are limited by the complicated amplification process. Herein, an amplification-free surface enhanced Raman spectroscopy (SERS) approach which directly detects point mutations in cancer cells has been proposed. A highly sensitive and uniform SERS substrate was fabricated using gold@silver core–shell nanorods, achieving an enhancement factor of 1.85 × 106. By combining the SERS-active nanosubstrate with molecular beacon probes, the limit of detection reached as low as 50 fM. To enable parallel analysis and automated operation, the SERS sensor was integrated into a microfluidic chip. This novel chip-based assay was able to differentiate between mutated and wild-type KRAS genes among a variety of other nucleic acids from cancer cells in 40 min. Owing to the simple operation and fast analysis, the SERS-based DNA assay chip could potentially provide insights into clinical cancer theranostics in an easy and inexpensive manner at the point of care.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}Accurate and sensitive identification of DNA mutations in tumor cells is critical to the diagnosis, prognosis and personalized therapy of cancer. Conventional polymerase chain reaction (PCR)-based methods are limited by the complicated amplification process. Herein, an amplification-free surface enhanced Raman spectroscopy (SERS) approach which directly detects point mutations in cancer cells has been proposed. A highly sensitive and uniform SERS substrate was fabricated using gold@silver core–shell nanorods, achieving an enhancement factor of 1.85 × 106. By combining the SERS-active nanosubstrate with molecular beacon probes, the limit of detection reached as low as 50 fM. To enable parallel analysis and automated operation, the SERS sensor was integrated into a microfluidic chip. This novel chip-based assay was able to differentiate between mutated and wild-type KRAS genes among a variety of other nucleic acids from cancer cells in 40 min. Owing to the simple operation and fast analysis, the SERS-based DNA assay chip could potentially provide insights into clinical cancer theranostics in an easy and inexpensive manner at the point of care.Rui Campos, Jérôme Borme, Joana Rafaela Guerreiro, George Machado Jr., Maria Fátima Cerqueira, Dmitri Y. Petrovykh, and Pedro Alpuim
Attomolar Label-Free Detection of DNA Hybridization with Electrolyte-Gated Graphene Field-Effect Transistors Journal Article
ACS Sensors, 4 (2), pp. 286-293, 2019.
@article{Campos2019bb,
title = {Attomolar Label-Free Detection of DNA Hybridization with Electrolyte-Gated Graphene Field-Effect Transistors},
author = {Rui Campos, Jérôme Borme, Joana Rafaela Guerreiro, George Machado Jr., Maria Fátima Cerqueira, Dmitri Y. Petrovykh, and Pedro Alpuim},
url = {https://doi.org/10.1021/acssensors.8b00344},
doi = {10.1021/acssensors.8b00344},
year = {2019},
date = {2019-01-23},
journal = {ACS Sensors},
volume = {4},
number = {2},
pages = {286-293},
abstract = {In this work, we develop a field-effect transistor with a two-dimensional channel made of a single graphene layer to achieve label-free detection of DNA hybridization down to attomolar concentration, while being able to discriminate a single nucleotide polymorphism (SNP). The SNP-level target specificity is achieved by immobilization of probe DNA on the graphene surface through a pyrene-derivative heterobifunctional linker. Biorecognition events result in a positive gate voltage shift of the graphene charge neutrality point. The graphene transistor biosensor displays a sensitivity of 24 mV/dec with a detection limit of 25 aM: the lowest target DNA concentration for which the sensor can discriminate between a perfect-match target sequence and SNP-containing one.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}In this work, we develop a field-effect transistor with a two-dimensional channel made of a single graphene layer to achieve label-free detection of DNA hybridization down to attomolar concentration, while being able to discriminate a single nucleotide polymorphism (SNP). The SNP-level target specificity is achieved by immobilization of probe DNA on the graphene surface through a pyrene-derivative heterobifunctional linker. Biorecognition events result in a positive gate voltage shift of the graphene charge neutrality point. The graphene transistor biosensor displays a sensitivity of 24 mV/dec with a detection limit of 25 aM: the lowest target DNA concentration for which the sensor can discriminate between a perfect-match target sequence and SNP-containing one. -
2018
Marta Prado Rodríguez Alejandro Garrido-Maestu David Tomás Fornés
The Use of Multiplex Real-Time PCR for the Simultaneous Detection of Foodborne Bacterial Pathogens Book Chapter
Methods in Molecular Biology, 1918 , pp. 35-45, Humana Press, New York, NY, 2018.
@inbook{Garrido-Maestu2018b,
title = {The Use of Multiplex Real-Time PCR for the Simultaneous Detection of Foodborne Bacterial Pathogens},
author = {Marta Prado Rodríguez Alejandro Garrido-Maestu David Tomás Fornés},
url = {https://doi.org/10.1007/978-1-4939-9000-9_3},
doi = {10.1007/978-1-4939-9000-9_3},
year = {2018},
date = {2018-12-23},
booktitle = {Methods in Molecular Biology},
volume = {1918},
pages = {35-45},
publisher = {Humana Press, New York, NY},
abstract = {Foodborne pathogens continue to be a major health issue worldwide. Culture-dependent methodologies are still considered the gold-standard to perform pathogen detection and quantification. These methods present several drawbacks, such as being time-consuming and labor-intensive. The implementation of real-time PCR has allowed to overcome these limitations and even reduce costs associated with the analyses, due to the possibility of simultaneously and accurately detecting several pathogens in one single assay, with results comparable to those obtained by classical approaches. In this chapter a protocol for the simultaneous detection of two of the most important foodborne pathogens, Salmonella spp. and Listeria monocytogenes, is described.},
keywords = {},
pubstate = {published},
tppubtype = {inbook}
}Foodborne pathogens continue to be a major health issue worldwide. Culture-dependent methodologies are still considered the gold-standard to perform pathogen detection and quantification. These methods present several drawbacks, such as being time-consuming and labor-intensive. The implementation of real-time PCR has allowed to overcome these limitations and even reduce costs associated with the analyses, due to the possibility of simultaneously and accurately detecting several pathogens in one single assay, with results comparable to those obtained by classical approaches. In this chapter a protocol for the simultaneous detection of two of the most important foodborne pathogens, Salmonella spp. and Listeria monocytogenes, is described.Marta Prado Rodríguez Alejandro Garrido-Maestu David Tomás Fornés
The Use of Multiplex Real-Time PCR for the Simultaneous Detection of Foodborne Bacterial Pathogens Book Chapter
Methods in Molecular Biology, 1918 , pp. 35-45, Humana Press, New York, NY, 2018.
@inbook{Garrido-Maestu2018bc,
title = {The Use of Multiplex Real-Time PCR for the Simultaneous Detection of Foodborne Bacterial Pathogens},
author = {Marta Prado Rodríguez Alejandro Garrido-Maestu David Tomás Fornés},
url = {https://doi.org/10.1007/978-1-4939-9000-9_3},
doi = {10.1007/978-1-4939-9000-9_3},
year = {2018},
date = {2018-12-23},
booktitle = {Methods in Molecular Biology},
volume = {1918},
pages = {35-45},
publisher = {Humana Press, New York, NY},
abstract = {Foodborne pathogens continue to be a major health issue worldwide. Culture-dependent methodologies are still considered the gold-standard to perform pathogen detection and quantification. These methods present several drawbacks, such as being time-consuming and labor-intensive. The implementation of real-time PCR has allowed to overcome these limitations and even reduce costs associated with the analyses, due to the possibility of simultaneously and accurately detecting several pathogens in one single assay, with results comparable to those obtained by classical approaches. In this chapter a protocol for the simultaneous detection of two of the most important foodborne pathogens, Salmonella spp. and Listeria monocytogenes, is described.},
keywords = {},
pubstate = {published},
tppubtype = {inbook}
}Foodborne pathogens continue to be a major health issue worldwide. Culture-dependent methodologies are still considered the gold-standard to perform pathogen detection and quantification. These methods present several drawbacks, such as being time-consuming and labor-intensive. The implementation of real-time PCR has allowed to overcome these limitations and even reduce costs associated with the analyses, due to the possibility of simultaneously and accurately detecting several pathogens in one single assay, with results comparable to those obtained by classical approaches. In this chapter a protocol for the simultaneous detection of two of the most important foodborne pathogens, Salmonella spp. and Listeria monocytogenes, is described.SarahAzinheiro AlejandroGarrido-Maestu JorgeBarros-Velázquez MartaPrado JoanaCarvalho RenatoNegrinho
Elsevier, 21 (21), pp. 424-431, 2018.
@article{JoanaCarvalho2018b,
title = {Data on minute DNA quantification on microvolumetric solutions: comparison of mathematical models and effect of some compounds on the DNA quantification accuracy},
author = {SarahAzinheiro AlejandroGarrido-Maestu JorgeBarros-Velázquez MartaPrado JoanaCarvalho RenatoNegrinho},
url = {https://www.sciencedirect.com/science/article/pii/S2352340918311764},
doi = {https://doi.org/10.1016/j.dib.2018.09.098},
year = {2018},
date = {2018-10-02},
journal = {Elsevier},
volume = {21},
number = {21},
pages = {424-431},
abstract = {This article contains data related to the research article entitled “Novel approach for accurate minute DNA quantification on microvolumetric solutions” (Carvalho et al., 2018). The combination of PicoGreen® with a microvolume fluorospectrometer is a popular DNA quantification method due to its high sensitivity and minimal consumption of sample, being commonly used to evaluate the performance of microfluidic devices designed for DNA purification. In this study, the authors present data related with the effect of DNA fragmentation level. The present data article includes the data used on the precision evaluation, in terms of repeatability, of the mathematical models developed to obtain the standards curve for salmon sperm DNA (low molecular weight). In addition, results related with the effect of some compounds on the DNA quantification accuracy using λDNA are presented.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}This article contains data related to the research article entitled “Novel approach for accurate minute DNA quantification on microvolumetric solutions” (Carvalho et al., 2018). The combination of PicoGreen® with a microvolume fluorospectrometer is a popular DNA quantification method due to its high sensitivity and minimal consumption of sample, being commonly used to evaluate the performance of microfluidic devices designed for DNA purification. In this study, the authors present data related with the effect of DNA fragmentation level. The present data article includes the data used on the precision evaluation, in terms of repeatability, of the mathematical models developed to obtain the standards curve for salmon sperm DNA (low molecular weight). In addition, results related with the effect of some compounds on the DNA quantification accuracy using λDNA are presented.SarahAzinheiro AlejandroGarrido-Maestu JorgeBarros-Velázquez MartaPrado JoanaCarvalho RenatoNegrinho
Elsevier, 21 (21), pp. 424-431, 2018.
@article{JoanaCarvalho2018bc,
title = {Data on minute DNA quantification on microvolumetric solutions: comparison of mathematical models and effect of some compounds on the DNA quantification accuracy},
author = {SarahAzinheiro AlejandroGarrido-Maestu JorgeBarros-Velázquez MartaPrado JoanaCarvalho RenatoNegrinho},
url = {https://www.sciencedirect.com/science/article/pii/S2352340918311764},
doi = {https://doi.org/10.1016/j.dib.2018.09.098},
year = {2018},
date = {2018-10-02},
journal = {Elsevier},
volume = {21},
number = {21},
pages = {424-431},
abstract = {This article contains data related to the research article entitled “Novel approach for accurate minute DNA quantification on microvolumetric solutions” (Carvalho et al., 2018). The combination of PicoGreen® with a microvolume fluorospectrometer is a popular DNA quantification method due to its high sensitivity and minimal consumption of sample, being commonly used to evaluate the performance of microfluidic devices designed for DNA purification. In this study, the authors present data related with the effect of DNA fragmentation level. The present data article includes the data used on the precision evaluation, in terms of repeatability, of the mathematical models developed to obtain the standards curve for salmon sperm DNA (low molecular weight). In addition, results related with the effect of some compounds on the DNA quantification accuracy using λDNA are presented.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}This article contains data related to the research article entitled “Novel approach for accurate minute DNA quantification on microvolumetric solutions” (Carvalho et al., 2018). The combination of PicoGreen® with a microvolume fluorospectrometer is a popular DNA quantification method due to its high sensitivity and minimal consumption of sample, being commonly used to evaluate the performance of microfluidic devices designed for DNA purification. In this study, the authors present data related with the effect of DNA fragmentation level. The present data article includes the data used on the precision evaluation, in terms of repeatability, of the mathematical models developed to obtain the standards curve for salmon sperm DNA (low molecular weight). In addition, results related with the effect of some compounds on the DNA quantification accuracy using λDNA are presented.JoãoGaspar SarahAzinheiro LorenaDiéguez AlejandroGarrido-Maestu ManuelVázquez JorgeBarros-Velázquez SusanaCardoso MartaPradoa JoanaCarvalho GemaPuertas
Highly efficient DNA extraction and purification from olive oil on a washable and reusable miniaturized device Journal Article
Analytica Chimica Acta, 1020 , pp. 30-40, 2018.
@article{JoanaCarvalho2018b,
title = {Highly efficient DNA extraction and purification from olive oil on a washable and reusable miniaturized device},
author = {JoãoGaspar SarahAzinheiro LorenaDiéguez AlejandroGarrido-Maestu ManuelVázquez JorgeBarros-Velázquez SusanaCardoso MartaPradoa JoanaCarvalho GemaPuertas},
url = {https://doi.org/10.1016/j.aca.2018.02.079},
doi = {https://doi.org/10.1016/j.aca.2018.02.079},
year = {2018},
date = {2018-08-22},
journal = {Analytica Chimica Acta},
volume = {1020},
pages = {30-40},
abstract = {Sample preparation from complex matrixes with minute DNA content could highly benefit from the miniaturization of solid phase extraction (SPE) based devices due to an increased surface area-to-volume ratio. However, the adaptation of “bench-top” based protocols for DNA purification to miniaturized devices is not as straightforward as it might seem, and several issues need to be considered. A careful evaluation of DNA extraction and purification protocols needs to be performed, taking into account the complexity of such samples, and in order to facilitate the integration with the subsequent step, normally DNA amplification. In this work a washable and reusable miniaturized device for DNA purification based on microscale solid phase extraction (μSPE), containing a commercial disposable silica membrane as the solid phase for DNA capture, was developed. The DNA purification protocol was firstly optimized by testing a set of different conditions, including buffer composition in all three steps of analysis and incubation during the elution step, with the objective of increasing the DNA yield and facilitating the integration in a miniaturized setting. This protocol was then tested with olive oil samples, including a pretreatment step also developed and optimized in this work. DNA analysis of olive oil samples is of high interest for the detection of fraudulent adulteration with oil from other seeds and for cultivar identification. The results were compared with the commercial NucleoSpin® Food kit regarding efficiency and purity of the DNA extract, by estimating the DNA yield and evaluating the absorbance ratios A260/280 and A260/230. The miniaturized DNA purification device showed better performance than the commercial kit tested, making this method a very promising sample preparation approach for olive oil and other samples with minute DNA content.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}Sample preparation from complex matrixes with minute DNA content could highly benefit from the miniaturization of solid phase extraction (SPE) based devices due to an increased surface area-to-volume ratio. However, the adaptation of “bench-top” based protocols for DNA purification to miniaturized devices is not as straightforward as it might seem, and several issues need to be considered. A careful evaluation of DNA extraction and purification protocols needs to be performed, taking into account the complexity of such samples, and in order to facilitate the integration with the subsequent step, normally DNA amplification. In this work a washable and reusable miniaturized device for DNA purification based on microscale solid phase extraction (μSPE), containing a commercial disposable silica membrane as the solid phase for DNA capture, was developed. The DNA purification protocol was firstly optimized by testing a set of different conditions, including buffer composition in all three steps of analysis and incubation during the elution step, with the objective of increasing the DNA yield and facilitating the integration in a miniaturized setting. This protocol was then tested with olive oil samples, including a pretreatment step also developed and optimized in this work. DNA analysis of olive oil samples is of high interest for the detection of fraudulent adulteration with oil from other seeds and for cultivar identification. The results were compared with the commercial NucleoSpin® Food kit regarding efficiency and purity of the DNA extract, by estimating the DNA yield and evaluating the absorbance ratios A260/280 and A260/230. The miniaturized DNA purification device showed better performance than the commercial kit tested, making this method a very promising sample preparation approach for olive oil and other samples with minute DNA content.JoãoGaspar SarahAzinheiro LorenaDiéguez AlejandroGarrido-Maestu ManuelVázquez JorgeBarros-Velázquez SusanaCardoso MartaPradoa JoanaCarvalho GemaPuertas
Highly efficient DNA extraction and purification from olive oil on a washable and reusable miniaturized device Journal Article
Analytica Chimica Acta, 1020 , pp. 30-40, 2018.
@article{JoanaCarvalho2018d,
title = {Highly efficient DNA extraction and purification from olive oil on a washable and reusable miniaturized device},
author = {JoãoGaspar SarahAzinheiro LorenaDiéguez AlejandroGarrido-Maestu ManuelVázquez JorgeBarros-Velázquez SusanaCardoso MartaPradoa JoanaCarvalho GemaPuertas},
url = {https://doi.org/10.1016/j.aca.2018.02.079},
doi = {https://doi.org/10.1016/j.aca.2018.02.079},
year = {2018},
date = {2018-08-22},
journal = {Analytica Chimica Acta},
volume = {1020},
pages = {30-40},
abstract = {Sample preparation from complex matrixes with minute DNA content could highly benefit from the miniaturization of solid phase extraction (SPE) based devices due to an increased surface area-to-volume ratio. However, the adaptation of “bench-top” based protocols for DNA purification to miniaturized devices is not as straightforward as it might seem, and several issues need to be considered. A careful evaluation of DNA extraction and purification protocols needs to be performed, taking into account the complexity of such samples, and in order to facilitate the integration with the subsequent step, normally DNA amplification. In this work a washable and reusable miniaturized device for DNA purification based on microscale solid phase extraction (μSPE), containing a commercial disposable silica membrane as the solid phase for DNA capture, was developed. The DNA purification protocol was firstly optimized by testing a set of different conditions, including buffer composition in all three steps of analysis and incubation during the elution step, with the objective of increasing the DNA yield and facilitating the integration in a miniaturized setting. This protocol was then tested with olive oil samples, including a pretreatment step also developed and optimized in this work. DNA analysis of olive oil samples is of high interest for the detection of fraudulent adulteration with oil from other seeds and for cultivar identification. The results were compared with the commercial NucleoSpin® Food kit regarding efficiency and purity of the DNA extract, by estimating the DNA yield and evaluating the absorbance ratios A260/280 and A260/230. The miniaturized DNA purification device showed better performance than the commercial kit tested, making this method a very promising sample preparation approach for olive oil and other samples with minute DNA content.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}Sample preparation from complex matrixes with minute DNA content could highly benefit from the miniaturization of solid phase extraction (SPE) based devices due to an increased surface area-to-volume ratio. However, the adaptation of “bench-top” based protocols for DNA purification to miniaturized devices is not as straightforward as it might seem, and several issues need to be considered. A careful evaluation of DNA extraction and purification protocols needs to be performed, taking into account the complexity of such samples, and in order to facilitate the integration with the subsequent step, normally DNA amplification. In this work a washable and reusable miniaturized device for DNA purification based on microscale solid phase extraction (μSPE), containing a commercial disposable silica membrane as the solid phase for DNA capture, was developed. The DNA purification protocol was firstly optimized by testing a set of different conditions, including buffer composition in all three steps of analysis and incubation during the elution step, with the objective of increasing the DNA yield and facilitating the integration in a miniaturized setting. This protocol was then tested with olive oil samples, including a pretreatment step also developed and optimized in this work. DNA analysis of olive oil samples is of high interest for the detection of fraudulent adulteration with oil from other seeds and for cultivar identification. The results were compared with the commercial NucleoSpin® Food kit regarding efficiency and purity of the DNA extract, by estimating the DNA yield and evaluating the absorbance ratios A260/280 and A260/230. The miniaturized DNA purification device showed better performance than the commercial kit tested, making this method a very promising sample preparation approach for olive oil and other samples with minute DNA content.Sarah Azinheiro Pablo Fuciños Joana Carvalho Marta Prado Alejandro Garrido-Maestu
Food Chemistry, Volume 246 , pp. Pages 156–163, 2018.
@article{Garrido-Maestu2017b,
title = {Highly sensitive detection of gluten-containing cereals in food samples by real-time Loop-mediated isothermal AMPlification (qLAMP) and real-time polymerase chain reaction (qPCR)},
author = {Sarah Azinheiro Pablo Fuciños Joana Carvalho Marta Prado Alejandro Garrido-Maestu},
url = {https://doi.org/10.1016/j.foodchem.2017.11.005},
year = {2018},
date = {2018-04-25},
journal = {Food Chemistry},
volume = {Volume 246},
pages = {Pages 156–163},
abstract = {The treatment of gluten-related disorders is based on a lifelong, and strict, gluten-free diet. Thus, reliable and sensitive methods are required to detect the presence of gluten contamination. Traditional techniques rely on the detection of these proteins based on specific antibodies, but recent approaches go for an indirect route detecting the DNA that indicates the presence of cereals with gluten content. In the current study two different DNA amplification techniques, real-time PCR (qPCR) and real-time Loop-mediated isothermal AMPlification (qLAMP), were evaluated for their capability to detect and quantify gluten. Different detection strategies, based on these DNA amplification techniques, were tested. Even though good specificity results were obtained with the different approaches, overall qPCR proved more sensitive than qLAMP. This is the first study reporting a qLAMP based-method for the detection of gluten-containing cereals, along with its evaluation in comparison with qPCR.Keywords: Real-time Loop-mediated isothermal AMPlification (qLAMP); Real-time PCR (qPCR); α2-gliadin; Gluten; Gluten-free food},
keywords = {},
pubstate = {published},
tppubtype = {article}
}The treatment of gluten-related disorders is based on a lifelong, and strict, gluten-free diet. Thus, reliable and sensitive methods are required to detect the presence of gluten contamination. Traditional techniques rely on the detection of these proteins based on specific antibodies, but recent approaches go for an indirect route detecting the DNA that indicates the presence of cereals with gluten content. In the current study two different DNA amplification techniques, real-time PCR (qPCR) and real-time Loop-mediated isothermal AMPlification (qLAMP), were evaluated for their capability to detect and quantify gluten. Different detection strategies, based on these DNA amplification techniques, were tested. Even though good specificity results were obtained with the different approaches, overall qPCR proved more sensitive than qLAMP. This is the first study reporting a qLAMP based-method for the detection of gluten-containing cereals, along with its evaluation in comparison with qPCR.Keywords: Real-time Loop-mediated isothermal AMPlification (qLAMP); Real-time PCR (qPCR); α2-gliadin; Gluten; Gluten-free food
Sarah Azinheiro Pablo Fuciños Joana Carvalho Marta Prado Alejandro Garrido-Maestu
Food Chemistry, Volume 246 , pp. Pages 156–163, 2018.
@article{Garrido-Maestu2017bd,
title = {Highly sensitive detection of gluten-containing cereals in food samples by real-time Loop-mediated isothermal AMPlification (qLAMP) and real-time polymerase chain reaction (qPCR)},
author = {Sarah Azinheiro Pablo Fuciños Joana Carvalho Marta Prado Alejandro Garrido-Maestu},
url = {https://doi.org/10.1016/j.foodchem.2017.11.005},
year = {2018},
date = {2018-04-25},
journal = {Food Chemistry},
volume = {Volume 246},
pages = {Pages 156–163},
abstract = {The treatment of gluten-related disorders is based on a lifelong, and strict, gluten-free diet. Thus, reliable and sensitive methods are required to detect the presence of gluten contamination. Traditional techniques rely on the detection of these proteins based on specific antibodies, but recent approaches go for an indirect route detecting the DNA that indicates the presence of cereals with gluten content. In the current study two different DNA amplification techniques, real-time PCR (qPCR) and real-time Loop-mediated isothermal AMPlification (qLAMP), were evaluated for their capability to detect and quantify gluten. Different detection strategies, based on these DNA amplification techniques, were tested. Even though good specificity results were obtained with the different approaches, overall qPCR proved more sensitive than qLAMP. This is the first study reporting a qLAMP based-method for the detection of gluten-containing cereals, along with its evaluation in comparison with qPCR.Keywords: Real-time Loop-mediated isothermal AMPlification (qLAMP); Real-time PCR (qPCR); α2-gliadin; Gluten; Gluten-free food},
keywords = {},
pubstate = {published},
tppubtype = {article}
}The treatment of gluten-related disorders is based on a lifelong, and strict, gluten-free diet. Thus, reliable and sensitive methods are required to detect the presence of gluten contamination. Traditional techniques rely on the detection of these proteins based on specific antibodies, but recent approaches go for an indirect route detecting the DNA that indicates the presence of cereals with gluten content. In the current study two different DNA amplification techniques, real-time PCR (qPCR) and real-time Loop-mediated isothermal AMPlification (qLAMP), were evaluated for their capability to detect and quantify gluten. Different detection strategies, based on these DNA amplification techniques, were tested. Even though good specificity results were obtained with the different approaches, overall qPCR proved more sensitive than qLAMP. This is the first study reporting a qLAMP based-method for the detection of gluten-containing cereals, along with its evaluation in comparison with qPCR.Keywords: Real-time Loop-mediated isothermal AMPlification (qLAMP); Real-time PCR (qPCR); α2-gliadin; Gluten; Gluten-free food
Joana Carvalho Pablo Fuciños Marta Prado Alejandro Garrido-Maestu Sarah Azinheiro
Food Control, Volume 86 , pp. Pages 27–34, 2018.
@article{Garrido-Maestu2017bb,
title = {Development and evaluation of loop-mediated isothermal amplification, and Recombinase Polymerase Amplification methodologies, for the detection of Listeria monocytogenes in ready-to-eat food samples},
author = {Joana Carvalho Pablo Fuciños Marta Prado Alejandro Garrido-Maestu Sarah Azinheiro},
url = {https://doi.org/10.1016/j.foodcont.2017.11.006},
year = {2018},
date = {2018-04-01},
journal = {Food Control},
volume = {Volume 86},
pages = {Pages 27–34},
abstract = {Listeriosis continues to be a major health issue. This is demonstrated by the fact that, even though efforts have been made, its incidence does not decrease. Furthermore, in Europe, over 2014 a 30% increase was reported respect to 2013. In the present study two isothermal DNA amplification methods, one based on Loop-mediated isothermal AMPlification (qLAMP), and the other on Recombinase Polymerase Amplification (RPA), were developed and extensively evaluated. Both techniques demonstrated their reliability to detect Listeria monocytogenes in different types of foods. The method included a two-step enrichment, which additionally reduces the chances of detecting dead bacteria. Over the evaluation with pure bacterial DNA, the qLAMP and RPA methods resulted 10 to 100 times less sensitive than qPCR (with two different detection chemistries), but when tested in real food samples the results showed very good concordance with those obtained by qPCR and by selective agar plating (index kappa of concordance between 0.90 and 0.95). Additionally, a very low limit of detection (below 10 CFU/25 g) was obtained. Thus the optimal performance of both isothermal techniques, and their adequacy for their implementation in the food industry, was demonstrated.Keywords: qLAMP; RPA; qPCR; hly; Listeria monocytogenes},
keywords = {},
pubstate = {published},
tppubtype = {article}
}Listeriosis continues to be a major health issue. This is demonstrated by the fact that, even though efforts have been made, its incidence does not decrease. Furthermore, in Europe, over 2014 a 30% increase was reported respect to 2013. In the present study two isothermal DNA amplification methods, one based on Loop-mediated isothermal AMPlification (qLAMP), and the other on Recombinase Polymerase Amplification (RPA), were developed and extensively evaluated. Both techniques demonstrated their reliability to detect Listeria monocytogenes in different types of foods. The method included a two-step enrichment, which additionally reduces the chances of detecting dead bacteria. Over the evaluation with pure bacterial DNA, the qLAMP and RPA methods resulted 10 to 100 times less sensitive than qPCR (with two different detection chemistries), but when tested in real food samples the results showed very good concordance with those obtained by qPCR and by selective agar plating (index kappa of concordance between 0.90 and 0.95). Additionally, a very low limit of detection (below 10 CFU/25 g) was obtained. Thus the optimal performance of both isothermal techniques, and their adequacy for their implementation in the food industry, was demonstrated.Keywords: qLAMP; RPA; qPCR; hly; Listeria monocytogenes
Joana Carvalho Pablo Fuciños Marta Prado Alejandro Garrido-Maestu Sarah Azinheiro
Food Control, Volume 86 , pp. Pages 27–34, 2018.
@article{Garrido-Maestu2017be,
title = {Development and evaluation of loop-mediated isothermal amplification, and Recombinase Polymerase Amplification methodologies, for the detection of Listeria monocytogenes in ready-to-eat food samples},
author = {Joana Carvalho Pablo Fuciños Marta Prado Alejandro Garrido-Maestu Sarah Azinheiro},
url = {https://doi.org/10.1016/j.foodcont.2017.11.006},
year = {2018},
date = {2018-04-01},
journal = {Food Control},
volume = {Volume 86},
pages = {Pages 27–34},
abstract = {Listeriosis continues to be a major health issue. This is demonstrated by the fact that, even though efforts have been made, its incidence does not decrease. Furthermore, in Europe, over 2014 a 30% increase was reported respect to 2013. In the present study two isothermal DNA amplification methods, one based on Loop-mediated isothermal AMPlification (qLAMP), and the other on Recombinase Polymerase Amplification (RPA), were developed and extensively evaluated. Both techniques demonstrated their reliability to detect Listeria monocytogenes in different types of foods. The method included a two-step enrichment, which additionally reduces the chances of detecting dead bacteria. Over the evaluation with pure bacterial DNA, the qLAMP and RPA methods resulted 10 to 100 times less sensitive than qPCR (with two different detection chemistries), but when tested in real food samples the results showed very good concordance with those obtained by qPCR and by selective agar plating (index kappa of concordance between 0.90 and 0.95). Additionally, a very low limit of detection (below 10 CFU/25 g) was obtained. Thus the optimal performance of both isothermal techniques, and their adequacy for their implementation in the food industry, was demonstrated.Keywords: qLAMP; RPA; qPCR; hly; Listeria monocytogenes},
keywords = {},
pubstate = {published},
tppubtype = {article}
}Listeriosis continues to be a major health issue. This is demonstrated by the fact that, even though efforts have been made, its incidence does not decrease. Furthermore, in Europe, over 2014 a 30% increase was reported respect to 2013. In the present study two isothermal DNA amplification methods, one based on Loop-mediated isothermal AMPlification (qLAMP), and the other on Recombinase Polymerase Amplification (RPA), were developed and extensively evaluated. Both techniques demonstrated their reliability to detect Listeria monocytogenes in different types of foods. The method included a two-step enrichment, which additionally reduces the chances of detecting dead bacteria. Over the evaluation with pure bacterial DNA, the qLAMP and RPA methods resulted 10 to 100 times less sensitive than qPCR (with two different detection chemistries), but when tested in real food samples the results showed very good concordance with those obtained by qPCR and by selective agar plating (index kappa of concordance between 0.90 and 0.95). Additionally, a very low limit of detection (below 10 CFU/25 g) was obtained. Thus the optimal performance of both isothermal techniques, and their adequacy for their implementation in the food industry, was demonstrated.Keywords: qLAMP; RPA; qPCR; hly; Listeria monocytogenes
Joana Carvalho Marta Prado Alejandro Garrido-Maestu Sarah Azinheiro
Rapid and sensitive detection of viable Listeria monocytogenes in food products by a filtration-based protocol and qPCR Journal Article
Food Microbiology, 73 , pp. 254-263, 2018.
@article{Garrido-Maestu2018b,
title = {Rapid and sensitive detection of viable Listeria monocytogenes in food products by a filtration-based protocol and qPCR},
author = {Joana Carvalho Marta Prado Alejandro Garrido-Maestu Sarah Azinheiro},
url = {https://doi.org/10.1016/j.fm.2018.02.004},
year = {2018},
date = {2018-02-07},
journal = {Food Microbiology},
volume = {73},
pages = {254-263},
abstract = {Listeria monocytogenes continues to be one of the most important foodborne pathogens worldwide, either due to its incidence and/or to its high mortality rate. In the present study, a filtration-based protocol was applied for the screening of viable bacteria. Additionally, a complete method (enrichment, filtration, DNA extraction and real-time PCR detection) was evaluated in order to determine the capacity of this protocol to detect viable L. monocytogenes in food samples. A new multiplex qPCR detection system was designed, including an internal amplification control, both targets were detected with hydrolysis probes. It was demonstrated that the method could reliably detect this pathogen, reaching a limit of detection of 9.5 cfu/25 g. The evaluation of the relative sensitivity, specificity, accuracy, positive and negative predictive values, as well as the index kappa of concordance obtained values higher than 90.0% after 24 h sample enrichment. Furthermore, it was demonstrated that with a secondary enrichment step, the limit of the detection could be further decreased to 4.6 cfu/25 g without significantly affecting the performance parameters. The present study demonstrates the reliability of the proposed methodology for the detection of viable L. monocytogenes, and the possibility of its direct implementation for routine analyses in the food industry.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}Listeria monocytogenes continues to be one of the most important foodborne pathogens worldwide, either due to its incidence and/or to its high mortality rate. In the present study, a filtration-based protocol was applied for the screening of viable bacteria. Additionally, a complete method (enrichment, filtration, DNA extraction and real-time PCR detection) was evaluated in order to determine the capacity of this protocol to detect viable L. monocytogenes in food samples. A new multiplex qPCR detection system was designed, including an internal amplification control, both targets were detected with hydrolysis probes. It was demonstrated that the method could reliably detect this pathogen, reaching a limit of detection of 9.5 cfu/25 g. The evaluation of the relative sensitivity, specificity, accuracy, positive and negative predictive values, as well as the index kappa of concordance obtained values higher than 90.0% after 24 h sample enrichment. Furthermore, it was demonstrated that with a secondary enrichment step, the limit of the detection could be further decreased to 4.6 cfu/25 g without significantly affecting the performance parameters. The present study demonstrates the reliability of the proposed methodology for the detection of viable L. monocytogenes, and the possibility of its direct implementation for routine analyses in the food industry.Joana Carvalho Marta Prado Alejandro Garrido-Maestu Sarah Azinheiro
Rapid and sensitive detection of viable Listeria monocytogenes in food products by a filtration-based protocol and qPCR Journal Article
Food Microbiology, 73 , pp. 254-263, 2018.
@article{Garrido-Maestu2018d,
title = {Rapid and sensitive detection of viable Listeria monocytogenes in food products by a filtration-based protocol and qPCR},
author = {Joana Carvalho Marta Prado Alejandro Garrido-Maestu Sarah Azinheiro},
url = {https://doi.org/10.1016/j.fm.2018.02.004},
year = {2018},
date = {2018-02-07},
journal = {Food Microbiology},
volume = {73},
pages = {254-263},
abstract = {Listeria monocytogenes continues to be one of the most important foodborne pathogens worldwide, either due to its incidence and/or to its high mortality rate. In the present study, a filtration-based protocol was applied for the screening of viable bacteria. Additionally, a complete method (enrichment, filtration, DNA extraction and real-time PCR detection) was evaluated in order to determine the capacity of this protocol to detect viable L. monocytogenes in food samples. A new multiplex qPCR detection system was designed, including an internal amplification control, both targets were detected with hydrolysis probes. It was demonstrated that the method could reliably detect this pathogen, reaching a limit of detection of 9.5 cfu/25 g. The evaluation of the relative sensitivity, specificity, accuracy, positive and negative predictive values, as well as the index kappa of concordance obtained values higher than 90.0% after 24 h sample enrichment. Furthermore, it was demonstrated that with a secondary enrichment step, the limit of the detection could be further decreased to 4.6 cfu/25 g without significantly affecting the performance parameters. The present study demonstrates the reliability of the proposed methodology for the detection of viable L. monocytogenes, and the possibility of its direct implementation for routine analyses in the food industry.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}Listeria monocytogenes continues to be one of the most important foodborne pathogens worldwide, either due to its incidence and/or to its high mortality rate. In the present study, a filtration-based protocol was applied for the screening of viable bacteria. Additionally, a complete method (enrichment, filtration, DNA extraction and real-time PCR detection) was evaluated in order to determine the capacity of this protocol to detect viable L. monocytogenes in food samples. A new multiplex qPCR detection system was designed, including an internal amplification control, both targets were detected with hydrolysis probes. It was demonstrated that the method could reliably detect this pathogen, reaching a limit of detection of 9.5 cfu/25 g. The evaluation of the relative sensitivity, specificity, accuracy, positive and negative predictive values, as well as the index kappa of concordance obtained values higher than 90.0% after 24 h sample enrichment. Furthermore, it was demonstrated that with a secondary enrichment step, the limit of the detection could be further decreased to 4.6 cfu/25 g without significantly affecting the performance parameters. The present study demonstrates the reliability of the proposed methodology for the detection of viable L. monocytogenes, and the possibility of its direct implementation for routine analyses in the food industry.Marta Prado Sarah Azinheiro Joana Carvalho and Alejandro Garrido-Maestu.
Frontiers in sustainable food systems, 2018.
@article{Azinheiro2018,
title = {Evaluation of different genetic targets for Salmonella enterica serovar Enteriditis and Typhimurium, using Loop-mediated isothermal AMPlification for detection in food samples},
author = {Marta Prado Sarah Azinheiro Joana Carvalho and Alejandro Garrido-Maestu.},
editor = {United States Joshua B. Gurtler Agricultural Research Service (USDA)},
url = {https://www.frontiersin.org/articles/10.3389/fsufs.2018.00005/abstract},
doi = {doi: 10.3389/fsufs.2018.00005},
year = {2018},
date = {2018-02-05},
journal = {Frontiers in sustainable food systems},
abstract = {Salmonella Enteritidis and Salmonella Typhimurium continue to be the most frequently identified serovars among confirmed cases of salmonellosis. In the current study different genetic targets (safA, sdf I, STM4497 and typh) were compared, attending to their specificity and sensitivity in pure cultures and in spiked samples, in order to determine their capacity to accurately identify them by loop-mediated isothermal amplification (LAMP). For the genes selected to detect Enteritidis, both performed equally well regarding their specificity, but safA proved more sensitive than Sdf I; minor differences were observed among these genes when analyzing spiked food samples. Regarding the targets for Typhimurium, STM4497 and typh, the former demonstrated to be more specific and sensitive, both when analyzing pure cultures as well as spiked samples. These results highlight the importance of an adequate evaluation of the genetic targets selected, before their implementation for routine analyses.
Keywords: Salmonella enteritidis, SAFA, Sdf I, Salmonella typhimurium, STM4497, typh, LAMP, Characterization methods},
keywords = {},
pubstate = {published},
tppubtype = {article}
}Salmonella Enteritidis and Salmonella Typhimurium continue to be the most frequently identified serovars among confirmed cases of salmonellosis. In the current study different genetic targets (safA, sdf I, STM4497 and typh) were compared, attending to their specificity and sensitivity in pure cultures and in spiked samples, in order to determine their capacity to accurately identify them by loop-mediated isothermal amplification (LAMP). For the genes selected to detect Enteritidis, both performed equally well regarding their specificity, but safA proved more sensitive than Sdf I; minor differences were observed among these genes when analyzing spiked food samples. Regarding the targets for Typhimurium, STM4497 and typh, the former demonstrated to be more specific and sensitive, both when analyzing pure cultures as well as spiked samples. These results highlight the importance of an adequate evaluation of the genetic targets selected, before their implementation for routine analyses.
Keywords: Salmonella enteritidis, SAFA, Sdf I, Salmonella typhimurium, STM4497, typh, LAMP, Characterization methodsMarta Prado Sarah Azinheiro Joana Carvalho and Alejandro Garrido-Maestu.
Frontiers in sustainable food systems, 2018.
@article{Azinheiro2018b,
title = {Evaluation of different genetic targets for Salmonella enterica serovar Enteriditis and Typhimurium, using Loop-mediated isothermal AMPlification for detection in food samples},
author = {Marta Prado Sarah Azinheiro Joana Carvalho and Alejandro Garrido-Maestu.},
editor = {United States Joshua B. Gurtler Agricultural Research Service (USDA)},
url = {https://www.frontiersin.org/articles/10.3389/fsufs.2018.00005/abstract},
doi = {doi: 10.3389/fsufs.2018.00005},
year = {2018},
date = {2018-02-05},
journal = {Frontiers in sustainable food systems},
abstract = {Salmonella Enteritidis and Salmonella Typhimurium continue to be the most frequently identified serovars among confirmed cases of salmonellosis. In the current study different genetic targets (safA, sdf I, STM4497 and typh) were compared, attending to their specificity and sensitivity in pure cultures and in spiked samples, in order to determine their capacity to accurately identify them by loop-mediated isothermal amplification (LAMP). For the genes selected to detect Enteritidis, both performed equally well regarding their specificity, but safA proved more sensitive than Sdf I; minor differences were observed among these genes when analyzing spiked food samples. Regarding the targets for Typhimurium, STM4497 and typh, the former demonstrated to be more specific and sensitive, both when analyzing pure cultures as well as spiked samples. These results highlight the importance of an adequate evaluation of the genetic targets selected, before their implementation for routine analyses.
Keywords: Salmonella enteritidis, SAFA, Sdf I, Salmonella typhimurium, STM4497, typh, LAMP, Characterization methods},
keywords = {},
pubstate = {published},
tppubtype = {article}
}Salmonella Enteritidis and Salmonella Typhimurium continue to be the most frequently identified serovars among confirmed cases of salmonellosis. In the current study different genetic targets (safA, sdf I, STM4497 and typh) were compared, attending to their specificity and sensitivity in pure cultures and in spiked samples, in order to determine their capacity to accurately identify them by loop-mediated isothermal amplification (LAMP). For the genes selected to detect Enteritidis, both performed equally well regarding their specificity, but safA proved more sensitive than Sdf I; minor differences were observed among these genes when analyzing spiked food samples. Regarding the targets for Typhimurium, STM4497 and typh, the former demonstrated to be more specific and sensitive, both when analyzing pure cultures as well as spiked samples. These results highlight the importance of an adequate evaluation of the genetic targets selected, before their implementation for routine analyses.
Keywords: Salmonella enteritidis, SAFA, Sdf I, Salmonella typhimurium, STM4497, typh, LAMP, Characterization methodsSarah Azinheiro Alejandro Garrido-Maestu Jorge Barros-Velázquez Marta Prado Joana Carvalho Renato Negrinho
Novel approach for accurate minute DNA quantification on microvolumetric solutions Journal Article
Microchemical Journal, 2018.
@article{Carvalho2018c,
title = {Novel approach for accurate minute DNA quantification on microvolumetric solutions},
author = {Sarah Azinheiro Alejandro Garrido-Maestu Jorge Barros-Velázquez Marta Prado Joana Carvalho Renato Negrinho},
url = {https://doi.org/10.1016/j.microc.2018.02.001},
year = {2018},
date = {2018-02-03},
journal = {Microchemical Journal},
abstract = {The optimization and evaluation of the performance of microfluidic devices for DNA purification requires the use of a reliable DNA quantification method. The samples collected from these devices usually have small volumes and, in the case of food, forensic and environmental applications, these samples are also complex, frequently containing highly fragmented DNA and minute concentrations. Therefore, combining a PicoGreen® assay with a microvolume fluorospectrometer is a popular technique for DNA quantification, providing highly sensitive quantification with minimal consumption of sample. However, this method has limitations, such as being affected by the degree of fragmentation of DNA and by the presence of some compounds commonly used in DNA extraction and purification protocols. In this work, these limitations and their influence on the accuracy of the quantification method were evaluated. Low molecular weight salmon sperm DNA was selected, being less purified and more fragmented than the λDNA standard most frequently used. It was shown that the standard curves generated with λDNA were not suitable for the quantification of fragmented DNA, such as DNA from highly processed samples and/or samples that have been exposed to harsh environments. In addition, a mathematical model was developed to find a better adjustment for the standard curve, for the salmon sperm DNA samples. This approach can be used as a tool to overcome important limitations found in this quantification method, allowing to include more data in the standard curve or test different mathematical models to better fit the standards data.Keywords
DNA quantification; PicoGreen; μTAS; DNA analysis; DNA purification; μSPE},
keywords = {},
pubstate = {published},
tppubtype = {article}
}The optimization and evaluation of the performance of microfluidic devices for DNA purification requires the use of a reliable DNA quantification method. The samples collected from these devices usually have small volumes and, in the case of food, forensic and environmental applications, these samples are also complex, frequently containing highly fragmented DNA and minute concentrations. Therefore, combining a PicoGreen® assay with a microvolume fluorospectrometer is a popular technique for DNA quantification, providing highly sensitive quantification with minimal consumption of sample. However, this method has limitations, such as being affected by the degree of fragmentation of DNA and by the presence of some compounds commonly used in DNA extraction and purification protocols. In this work, these limitations and their influence on the accuracy of the quantification method were evaluated. Low molecular weight salmon sperm DNA was selected, being less purified and more fragmented than the λDNA standard most frequently used. It was shown that the standard curves generated with λDNA were not suitable for the quantification of fragmented DNA, such as DNA from highly processed samples and/or samples that have been exposed to harsh environments. In addition, a mathematical model was developed to find a better adjustment for the standard curve, for the salmon sperm DNA samples. This approach can be used as a tool to overcome important limitations found in this quantification method, allowing to include more data in the standard curve or test different mathematical models to better fit the standards data.Keywords
DNA quantification; PicoGreen; μTAS; DNA analysis; DNA purification; μSPE
Sarah Azinheiro Alejandro Garrido-Maestu Jorge Barros-Velázquez Marta Prado Joana Carvalho Renato Negrinho
Novel approach for accurate minute DNA quantification on microvolumetric solutions Journal Article
Microchemical Journal, 2018.
@article{Carvalho2018f,
title = {Novel approach for accurate minute DNA quantification on microvolumetric solutions},
author = {Sarah Azinheiro Alejandro Garrido-Maestu Jorge Barros-Velázquez Marta Prado Joana Carvalho Renato Negrinho},
url = {https://doi.org/10.1016/j.microc.2018.02.001},
year = {2018},
date = {2018-02-03},
journal = {Microchemical Journal},
abstract = {The optimization and evaluation of the performance of microfluidic devices for DNA purification requires the use of a reliable DNA quantification method. The samples collected from these devices usually have small volumes and, in the case of food, forensic and environmental applications, these samples are also complex, frequently containing highly fragmented DNA and minute concentrations. Therefore, combining a PicoGreen® assay with a microvolume fluorospectrometer is a popular technique for DNA quantification, providing highly sensitive quantification with minimal consumption of sample. However, this method has limitations, such as being affected by the degree of fragmentation of DNA and by the presence of some compounds commonly used in DNA extraction and purification protocols. In this work, these limitations and their influence on the accuracy of the quantification method were evaluated. Low molecular weight salmon sperm DNA was selected, being less purified and more fragmented than the λDNA standard most frequently used. It was shown that the standard curves generated with λDNA were not suitable for the quantification of fragmented DNA, such as DNA from highly processed samples and/or samples that have been exposed to harsh environments. In addition, a mathematical model was developed to find a better adjustment for the standard curve, for the salmon sperm DNA samples. This approach can be used as a tool to overcome important limitations found in this quantification method, allowing to include more data in the standard curve or test different mathematical models to better fit the standards data.Keywords
DNA quantification; PicoGreen; μTAS; DNA analysis; DNA purification; μSPE},
keywords = {},
pubstate = {published},
tppubtype = {article}
}The optimization and evaluation of the performance of microfluidic devices for DNA purification requires the use of a reliable DNA quantification method. The samples collected from these devices usually have small volumes and, in the case of food, forensic and environmental applications, these samples are also complex, frequently containing highly fragmented DNA and minute concentrations. Therefore, combining a PicoGreen® assay with a microvolume fluorospectrometer is a popular technique for DNA quantification, providing highly sensitive quantification with minimal consumption of sample. However, this method has limitations, such as being affected by the degree of fragmentation of DNA and by the presence of some compounds commonly used in DNA extraction and purification protocols. In this work, these limitations and their influence on the accuracy of the quantification method were evaluated. Low molecular weight salmon sperm DNA was selected, being less purified and more fragmented than the λDNA standard most frequently used. It was shown that the standard curves generated with λDNA were not suitable for the quantification of fragmented DNA, such as DNA from highly processed samples and/or samples that have been exposed to harsh environments. In addition, a mathematical model was developed to find a better adjustment for the standard curve, for the salmon sperm DNA samples. This approach can be used as a tool to overcome important limitations found in this quantification method, allowing to include more data in the standard curve or test different mathematical models to better fit the standards data.Keywords
DNA quantification; PicoGreen; μTAS; DNA analysis; DNA purification; μSPE
-
2017
Joana Carvalho Sara Abalde-Cela Enrique Carbó-Argibay Lorena Dieguez Marek Piotrowski Yury Kolen'ko Marta Prado V Alejandro Garrido-Maestu Sarah Azinheiro
Frontiers in Microbiology In Press, 2017.
@article{Garrido-Maestu2017bc,
title = {Combination of microfluidic loop-mediated isothermal amplification with gold nanoparticles for rapid detection of Salmonella spp. in food samples},
author = {Joana Carvalho Sara Abalde-Cela Enrique Carbó-Argibay Lorena Dieguez Marek Piotrowski Yury Kolen'ko Marta Prado V Alejandro Garrido-Maestu Sarah Azinheiro},
url = {https://www.frontiersin.org/articles/10.3389/fmicb.2017.02159/abstract},
doi = {doi: 10.3389/fmicb.2017.02159},
year = {2017},
date = {2017-10-20},
journal = {Frontiers in Microbiology In Press},
abstract = {Foodborne diseases are an important cause of morbidity and mortality. According to the World Health Organization, there are 31 main global hazards, which caused in 2010 600 million foodborne illnesses and 420000 deaths. Among them, Salmonella spp. is one of the most important human pathogens, accounting for more than 90000 cases in Europe and even more in the United States per year. In the current study we report the development, and thorough evaluation in food samples, of a microfluidic system combining loop-mediated isothermal amplification with gold nanoparticles (AuNPs). This system is intended for low-cost, in-situ, detection of different pathogens, as the proposed methodology can be extrapolated to different microorganisms. A very low limit of detection (10 cfu/ 25 g) was obtained. Furthermore, the evaluation of spiked food samples (chicken, turkey, egg products), completely matched the expected results, as denoted by the index kappa of concordance (value of 1.00). The results obtained for the relative sensitivity, specificity and accuracy were of 100 % as well as the positive and negative predictive values.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}Foodborne diseases are an important cause of morbidity and mortality. According to the World Health Organization, there are 31 main global hazards, which caused in 2010 600 million foodborne illnesses and 420000 deaths. Among them, Salmonella spp. is one of the most important human pathogens, accounting for more than 90000 cases in Europe and even more in the United States per year. In the current study we report the development, and thorough evaluation in food samples, of a microfluidic system combining loop-mediated isothermal amplification with gold nanoparticles (AuNPs). This system is intended for low-cost, in-situ, detection of different pathogens, as the proposed methodology can be extrapolated to different microorganisms. A very low limit of detection (10 cfu/ 25 g) was obtained. Furthermore, the evaluation of spiked food samples (chicken, turkey, egg products), completely matched the expected results, as denoted by the index kappa of concordance (value of 1.00). The results obtained for the relative sensitivity, specificity and accuracy were of 100 % as well as the positive and negative predictive values.Joana Carvalho Sara Abalde-Cela Enrique Carbó-Argibay Lorena Dieguez Marek Piotrowski Yury Kolen'ko Marta Prado V Alejandro Garrido-Maestu Sarah Azinheiro
Frontiers in Microbiology In Press, 2017.
@article{Garrido-Maestu2017bf,
title = {Combination of microfluidic loop-mediated isothermal amplification with gold nanoparticles for rapid detection of Salmonella spp. in food samples},
author = {Joana Carvalho Sara Abalde-Cela Enrique Carbó-Argibay Lorena Dieguez Marek Piotrowski Yury Kolen'ko Marta Prado V Alejandro Garrido-Maestu Sarah Azinheiro},
url = {https://www.frontiersin.org/articles/10.3389/fmicb.2017.02159/abstract},
doi = {doi: 10.3389/fmicb.2017.02159},
year = {2017},
date = {2017-10-20},
journal = {Frontiers in Microbiology In Press},
abstract = {Foodborne diseases are an important cause of morbidity and mortality. According to the World Health Organization, there are 31 main global hazards, which caused in 2010 600 million foodborne illnesses and 420000 deaths. Among them, Salmonella spp. is one of the most important human pathogens, accounting for more than 90000 cases in Europe and even more in the United States per year. In the current study we report the development, and thorough evaluation in food samples, of a microfluidic system combining loop-mediated isothermal amplification with gold nanoparticles (AuNPs). This system is intended for low-cost, in-situ, detection of different pathogens, as the proposed methodology can be extrapolated to different microorganisms. A very low limit of detection (10 cfu/ 25 g) was obtained. Furthermore, the evaluation of spiked food samples (chicken, turkey, egg products), completely matched the expected results, as denoted by the index kappa of concordance (value of 1.00). The results obtained for the relative sensitivity, specificity and accuracy were of 100 % as well as the positive and negative predictive values.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}Foodborne diseases are an important cause of morbidity and mortality. According to the World Health Organization, there are 31 main global hazards, which caused in 2010 600 million foodborne illnesses and 420000 deaths. Among them, Salmonella spp. is one of the most important human pathogens, accounting for more than 90000 cases in Europe and even more in the United States per year. In the current study we report the development, and thorough evaluation in food samples, of a microfluidic system combining loop-mediated isothermal amplification with gold nanoparticles (AuNPs). This system is intended for low-cost, in-situ, detection of different pathogens, as the proposed methodology can be extrapolated to different microorganisms. A very low limit of detection (10 cfu/ 25 g) was obtained. Furthermore, the evaluation of spiked food samples (chicken, turkey, egg products), completely matched the expected results, as denoted by the index kappa of concordance (value of 1.00). The results obtained for the relative sensitivity, specificity and accuracy were of 100 % as well as the positive and negative predictive values.Azinheiro Carvalho Prado P ; S ; J ; M Garrido-Maestu A.; Fuciños
Food Control, 80 , pp. Pages 297-306, 2017.
@article{Garrido-Maestu2017d,
title = {Systematic loop-mediated isothermal amplification assays for rapid detection and characterization of Salmonella spp., Enteritidis and Typhimurium in food samples},
author = {Azinheiro Carvalho Prado P ; S ; J ; M Garrido-Maestu A.; Fuciños},
url = {http://www.sciencedirect.com/science/article/pii/S0956713517302542?via%3Dihub},
doi = {https://doi.org/10.1016/j.foodcont.2017.05.011},
year = {2017},
date = {2017-10-01},
journal = {Food Control},
volume = {80},
pages = {Pages 297-306},
abstract = {AbstractEuropean Authorities have made a great effort to decrease the incidence of salmonellosis, but yearly thousands of cases are still reported, being most of them associated with serovars Enteritidis and Typhimurium. In the current study a set of methods for fast detection of these pathogens was developed and evaluated. The methods were based on loop-mediated isothermal amplification due to its advantages. The methods targeted three genes, invA, safA and STM4497, and each one of them was evaluated independently so that they can be targeted directly or in a sequential mode: first screening for the genus Salmonella and secondly on typing those positive samples. In this process, the results were compared against qPCR. The methods were able to detect <10 cfu/25 g, making them suitable for official analyses, and food industry self-monitoring. Of most importance, the limit of detection, relative sensitivity, specificity and accuracy, positive and negative predictive values and the index kappa of concordance, were determined, being all higher than 97%. This demonstrates the reliability of the methods described in this study, which may be comparable with classical culture/serotyping of Salmonella but allowing a much faster response in case of positive results. Finally, a mathematical model was implemented to fit the data recorded by the qPCR thermocycler, allowing a more consistent determination of the parameters describing the qLAMP process, which may be easily implemented in other assays where accurate determination of Tt is needed for quantification purposes. Keywords: qLAMP; qPCR; Salmonella spp.; S. Enteritidis; S. Typhimurium},
keywords = {},
pubstate = {published},
tppubtype = {article}
}AbstractEuropean Authorities have made a great effort to decrease the incidence of salmonellosis, but yearly thousands of cases are still reported, being most of them associated with serovars Enteritidis and Typhimurium. In the current study a set of methods for fast detection of these pathogens was developed and evaluated. The methods were based on loop-mediated isothermal amplification due to its advantages. The methods targeted three genes, invA, safA and STM4497, and each one of them was evaluated independently so that they can be targeted directly or in a sequential mode: first screening for the genus Salmonella and secondly on typing those positive samples. In this process, the results were compared against qPCR. The methods were able to detect <10 cfu/25 g, making them suitable for official analyses, and food industry self-monitoring. Of most importance, the limit of detection, relative sensitivity, specificity and accuracy, positive and negative predictive values and the index kappa of concordance, were determined, being all higher than 97%. This demonstrates the reliability of the methods described in this study, which may be comparable with classical culture/serotyping of Salmonella but allowing a much faster response in case of positive results. Finally, a mathematical model was implemented to fit the data recorded by the qPCR thermocycler, allowing a more consistent determination of the parameters describing the qLAMP process, which may be easily implemented in other assays where accurate determination of Tt is needed for quantification purposes.
Keywords: qLAMP; qPCR; Salmonella spp.; S. Enteritidis; S. Typhimurium
Azinheiro Carvalho Prado P ; S ; J ; M Garrido-Maestu A.; Fuciños
Food Control, 80 , pp. Pages 297-306, 2017.
@article{Garrido-Maestu2017h,
title = {Systematic loop-mediated isothermal amplification assays for rapid detection and characterization of Salmonella spp., Enteritidis and Typhimurium in food samples},
author = {Azinheiro Carvalho Prado P ; S ; J ; M Garrido-Maestu A.; Fuciños},
url = {http://www.sciencedirect.com/science/article/pii/S0956713517302542?via%3Dihub},
doi = {https://doi.org/10.1016/j.foodcont.2017.05.011},
year = {2017},
date = {2017-10-01},
journal = {Food Control},
volume = {80},
pages = {Pages 297-306},
abstract = {AbstractEuropean Authorities have made a great effort to decrease the incidence of salmonellosis, but yearly thousands of cases are still reported, being most of them associated with serovars Enteritidis and Typhimurium. In the current study a set of methods for fast detection of these pathogens was developed and evaluated. The methods were based on loop-mediated isothermal amplification due to its advantages. The methods targeted three genes, invA, safA and STM4497, and each one of them was evaluated independently so that they can be targeted directly or in a sequential mode: first screening for the genus Salmonella and secondly on typing those positive samples. In this process, the results were compared against qPCR. The methods were able to detect <10 cfu/25 g, making them suitable for official analyses, and food industry self-monitoring. Of most importance, the limit of detection, relative sensitivity, specificity and accuracy, positive and negative predictive values and the index kappa of concordance, were determined, being all higher than 97%. This demonstrates the reliability of the methods described in this study, which may be comparable with classical culture/serotyping of Salmonella but allowing a much faster response in case of positive results. Finally, a mathematical model was implemented to fit the data recorded by the qPCR thermocycler, allowing a more consistent determination of the parameters describing the qLAMP process, which may be easily implemented in other assays where accurate determination of Tt is needed for quantification purposes. Keywords: qLAMP; qPCR; Salmonella spp.; S. Enteritidis; S. Typhimurium},
keywords = {},
pubstate = {published},
tppubtype = {article}
}AbstractEuropean Authorities have made a great effort to decrease the incidence of salmonellosis, but yearly thousands of cases are still reported, being most of them associated with serovars Enteritidis and Typhimurium. In the current study a set of methods for fast detection of these pathogens was developed and evaluated. The methods were based on loop-mediated isothermal amplification due to its advantages. The methods targeted three genes, invA, safA and STM4497, and each one of them was evaluated independently so that they can be targeted directly or in a sequential mode: first screening for the genus Salmonella and secondly on typing those positive samples. In this process, the results were compared against qPCR. The methods were able to detect <10 cfu/25 g, making them suitable for official analyses, and food industry self-monitoring. Of most importance, the limit of detection, relative sensitivity, specificity and accuracy, positive and negative predictive values and the index kappa of concordance, were determined, being all higher than 97%. This demonstrates the reliability of the methods described in this study, which may be comparable with classical culture/serotyping of Salmonella but allowing a much faster response in case of positive results. Finally, a mathematical model was implemented to fit the data recorded by the qPCR thermocycler, allowing a more consistent determination of the parameters describing the qLAMP process, which may be easily implemented in other assays where accurate determination of Tt is needed for quantification purposes.
Keywords: qLAMP; qPCR; Salmonella spp.; S. Enteritidis; S. Typhimurium
Prado Wenger Y ; M ; J Vial S.; Berrahal
Single-Step DNA Detection Assay Monitoring Dual-Color Light Scattering from Individual Metal Nanoparticle Aggregates Journal Article
ACS Sensors, 2 (2) , pp. pp 251–256, 2017.
@article{Vial2017,
title = {Single-Step DNA Detection Assay Monitoring Dual-Color Light Scattering from Individual Metal Nanoparticle Aggregates},
author = {Prado Wenger Y ; M ; J Vial S.; Berrahal},
url = {http://pubs.acs.org/doi/abs/10.1021/acssensors.6b00737},
doi = {10.1021/acssensors.6b00737},
year = {2017},
date = {2017-01-23},
journal = {ACS Sensors},
volume = {2 (2)},
pages = {pp 251–256},
abstract = {Efficiently detecting DNA sequences within a limited time is vital for disease screening and public health monitoring. This calls for a new method that combines high sensitivity, fast read-out time, and easy manipulation of the sample, avoiding the extensive steps of DNA amplification, purification, or grafting to a surface. Here, we introduce photon cross-correlation spectroscopy as a new method for specific DNA sensing with high sensitivity in a single-step homogeneous solution phase. Our approach is based on confocal dual-color illumination and detection of the scattering intensities from individual silver nanoparticles and gold nanorods. In the absence of the target DNA, the nanoparticles move independently and their respective scattering signals are uncorrelated. In the presence of the target DNA, the probe-functionalized gold and silver nanoparticles assemble via DNA hybridization with the target, giving rise to temporal coincidence between the signals scattered by each nanoparticle. The degree of coincidence accurately quantifies the amount of target DNA. To demonstrate the efficiency of our technique, we detect a specific DNA sequence of sesame, an allergenic food ingredient, for a range of concentration from 5 pM to 1.5 nM with a limit of detection of 1 pM. Our method is sensitive and specific enough to detect single nucleotide deletion and mismatch. With the dual-color scattering signals being much brighter than fluorescence-based analogs, the analysis is fast, quantitative, and simple to operate, making it valuable for biosensing applications.KEYWORDS: DNA detection; biosensing; dynamic light scattering; metal nanoparticles; optical scattering; plasmonics},
keywords = {},
pubstate = {published},
tppubtype = {article}
}Efficiently detecting DNA sequences within a limited time is vital for disease screening and public health monitoring. This calls for a new method that combines high sensitivity, fast read-out time, and easy manipulation of the sample, avoiding the extensive steps of DNA amplification, purification, or grafting to a surface. Here, we introduce photon cross-correlation spectroscopy as a new method for specific DNA sensing with high sensitivity in a single-step homogeneous solution phase. Our approach is based on confocal dual-color illumination and detection of the scattering intensities from individual silver nanoparticles and gold nanorods. In the absence of the target DNA, the nanoparticles move independently and their respective scattering signals are uncorrelated. In the presence of the target DNA, the probe-functionalized gold and silver nanoparticles assemble via DNA hybridization with the target, giving rise to temporal coincidence between the signals scattered by each nanoparticle. The degree of coincidence accurately quantifies the amount of target DNA. To demonstrate the efficiency of our technique, we detect a specific DNA sequence of sesame, an allergenic food ingredient, for a range of concentration from 5 pM to 1.5 nM with a limit of detection of 1 pM. Our method is sensitive and specific enough to detect single nucleotide deletion and mismatch. With the dual-color scattering signals being much brighter than fluorescence-based analogs, the analysis is fast, quantitative, and simple to operate, making it valuable for biosensing applications.KEYWORDS: DNA detection; biosensing; dynamic light scattering; metal nanoparticles; optical scattering; plasmonics
Prado Wenger Y ; M ; J Vial S.; Berrahal
Single-Step DNA Detection Assay Monitoring Dual-Color Light Scattering from Individual Metal Nanoparticle Aggregates Journal Article
ACS Sensors, 2 (2) , pp. pp 251–256, 2017.
@article{Vial2017b,
title = {Single-Step DNA Detection Assay Monitoring Dual-Color Light Scattering from Individual Metal Nanoparticle Aggregates},
author = {Prado Wenger Y ; M ; J Vial S.; Berrahal},
url = {http://pubs.acs.org/doi/abs/10.1021/acssensors.6b00737},
doi = {10.1021/acssensors.6b00737},
year = {2017},
date = {2017-01-23},
journal = {ACS Sensors},
volume = {2 (2)},
pages = {pp 251–256},
abstract = {Efficiently detecting DNA sequences within a limited time is vital for disease screening and public health monitoring. This calls for a new method that combines high sensitivity, fast read-out time, and easy manipulation of the sample, avoiding the extensive steps of DNA amplification, purification, or grafting to a surface. Here, we introduce photon cross-correlation spectroscopy as a new method for specific DNA sensing with high sensitivity in a single-step homogeneous solution phase. Our approach is based on confocal dual-color illumination and detection of the scattering intensities from individual silver nanoparticles and gold nanorods. In the absence of the target DNA, the nanoparticles move independently and their respective scattering signals are uncorrelated. In the presence of the target DNA, the probe-functionalized gold and silver nanoparticles assemble via DNA hybridization with the target, giving rise to temporal coincidence between the signals scattered by each nanoparticle. The degree of coincidence accurately quantifies the amount of target DNA. To demonstrate the efficiency of our technique, we detect a specific DNA sequence of sesame, an allergenic food ingredient, for a range of concentration from 5 pM to 1.5 nM with a limit of detection of 1 pM. Our method is sensitive and specific enough to detect single nucleotide deletion and mismatch. With the dual-color scattering signals being much brighter than fluorescence-based analogs, the analysis is fast, quantitative, and simple to operate, making it valuable for biosensing applications.KEYWORDS: DNA detection; biosensing; dynamic light scattering; metal nanoparticles; optical scattering; plasmonics},
keywords = {},
pubstate = {published},
tppubtype = {article}
}Efficiently detecting DNA sequences within a limited time is vital for disease screening and public health monitoring. This calls for a new method that combines high sensitivity, fast read-out time, and easy manipulation of the sample, avoiding the extensive steps of DNA amplification, purification, or grafting to a surface. Here, we introduce photon cross-correlation spectroscopy as a new method for specific DNA sensing with high sensitivity in a single-step homogeneous solution phase. Our approach is based on confocal dual-color illumination and detection of the scattering intensities from individual silver nanoparticles and gold nanorods. In the absence of the target DNA, the nanoparticles move independently and their respective scattering signals are uncorrelated. In the presence of the target DNA, the probe-functionalized gold and silver nanoparticles assemble via DNA hybridization with the target, giving rise to temporal coincidence between the signals scattered by each nanoparticle. The degree of coincidence accurately quantifies the amount of target DNA. To demonstrate the efficiency of our technique, we detect a specific DNA sequence of sesame, an allergenic food ingredient, for a range of concentration from 5 pM to 1.5 nM with a limit of detection of 1 pM. Our method is sensitive and specific enough to detect single nucleotide deletion and mismatch. With the dual-color scattering signals being much brighter than fluorescence-based analogs, the analysis is fast, quantitative, and simple to operate, making it valuable for biosensing applications.KEYWORDS: DNA detection; biosensing; dynamic light scattering; metal nanoparticles; optical scattering; plasmonics
-
2016
Fernandez-Arguelles Diéguez Fuciños Vial Oliveira Reis Boehme B ; M T ; L ; P ; S ; J M ; R L ; K Prado M.; Espiña
Detection of Foodborne Pathogens Using Nanoparticles. Advantages and Trends Book Chapter
Antimicrobial Food Packaging, pp. pp.183-202, Jorge Barros-Velazquez, 1st Edition, 2016, ISBN: 978-0-12-800723-5.
@inbook{Prado2016bc,
title = {Detection of Foodborne Pathogens Using Nanoparticles. Advantages and Trends},
author = {Fernandez-Arguelles Diéguez Fuciños Vial Oliveira Reis Boehme B ; M T ; L ; P ; S ; J M ; R L ; K Prado M.; Espiña},
url = {https://www.sciencedirect.com/science/book/9780128007235},
isbn = {978-0-12-800723-5},
year = {2016},
date = {2016-12-01},
booktitle = {Antimicrobial Food Packaging},
journal = {Academic Press, London},
pages = {pp.183-202},
publisher = {Jorge Barros-Velazquez},
edition = {1st Edition},
abstract = {Due to their critical impact on public health, the detection of foodborne pathogens in food and water is an important issue for both, the food industry and control authorities. Fast and reliable analytical methods are needed in order to ensure the health of consumers, to easily determine whether a food product has been subjected to cross-contamination, and, simultaneously, to identify how and when this cross-contamination occurred in order to establish the proper corrective actions.Recent developments in nanotechnology are greatly impacting the advancement of some analytical techniques, including foodborne pathogen detection. Herein, the advantages and the recent applications of nanoparticles (NPs) in foodborne pathogen detection are reviewed. The most frequently used NPs in this context (gold nanoparticles, quantum dots, and magnetic nanoparticles) are described, and their applicability for food analysis is emphasized. Finally, this chapter provides an overview of the use of micro and nanofluidics, combined with nanoparticles, for food pathogen analysis, and the integration of nanomaterial-based sensors for pathogen detection in food packaging systems.},
keywords = {},
pubstate = {published},
tppubtype = {inbook}
}Due to their critical impact on public health, the detection of foodborne pathogens in food and water is an important issue for both, the food industry and control authorities. Fast and reliable analytical methods are needed in order to ensure the health of consumers, to easily determine whether a food product has been subjected to cross-contamination, and, simultaneously, to identify how and when this cross-contamination occurred in order to establish the proper corrective actions.Recent developments in nanotechnology are greatly impacting the advancement of some analytical techniques, including foodborne pathogen detection. Herein, the advantages and the recent applications of nanoparticles (NPs) in foodborne pathogen detection are reviewed. The most frequently used NPs in this context (gold nanoparticles, quantum dots, and magnetic nanoparticles) are described, and their applicability for food analysis is emphasized. Finally, this chapter provides an overview of the use of micro and nanofluidics, combined with nanoparticles, for food pathogen analysis, and the integration of nanomaterial-based sensors for pathogen detection in food packaging systems.Vial Rivas Calo-Mata Barros-Velázquez I ; S ; J ; P ; J Prado M.; Ortea
Advanced DNA- and Protein-based Methods for the Detection and Investigation of Food Allergens Journal Article
Critical Reviews in Food Science and Nutrition, 56 (Issue 15), pp. 2511-2542, 2016.
@article{Prado2016b,
title = {Advanced DNA- and Protein-based Methods for the Detection and Investigation of Food Allergens},
author = {Vial Rivas Calo-Mata Barros-Velázquez I ; S ; J ; P ; J Prado M.; Ortea},
url = {http://www.tandfonline.com/doi/full/10.1080/10408398.2013.873767},
doi = {10.1080/10408398.2013.873767},
year = {2016},
date = {2016-11-17},
journal = {Critical Reviews in Food Science and Nutrition},
volume = {56},
number = {Issue 15},
pages = {2511-2542},
abstract = {Currently, food allergies are an important health concern worldwide. The presence of undeclared allergenic ingredients or the presence of traces of allergens due to contamination during food processing poses a great health risk to sensitized individuals. Therefore, reliable analytical methods are required to detect and identify allergenic ingredients in food products. The present review addresses the recent developments regarding the application of DNA- and protein-based methods for the detection of allergenic ingredients in foods. The fitness-for-purpose of reviewed methodology will be discussed, and future trends will be highlighted. Special attention will be given to the evaluation of the potential of newly developed and promising technologies that can improve the detection and identification of allergenic ingredients in foods, such as the use of biosensors and/or nanomaterials to improve detection limits, specificity, ease of use, or to reduce the time of analysis. Such rapid food allergen test methods are required to facilitate the reliable detection of allergenic ingredients by control laboratories, to give the food industry the means to easily determine whether its product has been subjected to cross-contamination and, simultaneously, to identify how and when this cross-contamination occurred.Keywords: Allergens, DNA based methods, proteomics, allergen detection, qPCR, biosensors, nanoparticles},
keywords = {},
pubstate = {published},
tppubtype = {article}
}Currently, food allergies are an important health concern worldwide. The presence of undeclared allergenic ingredients or the presence of traces of allergens due to contamination during food processing poses a great health risk to sensitized individuals. Therefore, reliable analytical methods are required to detect and identify allergenic ingredients in food products. The present review addresses the recent developments regarding the application of DNA- and protein-based methods for the detection of allergenic ingredients in foods. The fitness-for-purpose of reviewed methodology will be discussed, and future trends will be highlighted. Special attention will be given to the evaluation of the potential of newly developed and promising technologies that can improve the detection and identification of allergenic ingredients in foods, such as the use of biosensors and/or nanomaterials to improve detection limits, specificity, ease of use, or to reduce the time of analysis. Such rapid food allergen test methods are required to facilitate the reliable detection of allergenic ingredients by control laboratories, to give the food industry the means to easily determine whether its product has been subjected to cross-contamination and, simultaneously, to identify how and when this cross-contamination occurred.Keywords: Allergens, DNA based methods, proteomics, allergen detection, qPCR, biosensors, nanoparticles
Vial Rivas Calo-Mata Barros-Velázquez I ; S ; J ; P ; J Prado M.; Ortea
Advanced DNA- and Protein-based Methods for the Detection and Investigation of Food Allergens Journal Article
Critical Reviews in Food Science and Nutrition, 56 (Issue 15), pp. 2511-2542, 2016.
@article{Prado2016d,
title = {Advanced DNA- and Protein-based Methods for the Detection and Investigation of Food Allergens},
author = {Vial Rivas Calo-Mata Barros-Velázquez I ; S ; J ; P ; J Prado M.; Ortea},
url = {http://www.tandfonline.com/doi/full/10.1080/10408398.2013.873767},
doi = {10.1080/10408398.2013.873767},
year = {2016},
date = {2016-11-17},
journal = {Critical Reviews in Food Science and Nutrition},
volume = {56},
number = {Issue 15},
pages = {2511-2542},
abstract = {Currently, food allergies are an important health concern worldwide. The presence of undeclared allergenic ingredients or the presence of traces of allergens due to contamination during food processing poses a great health risk to sensitized individuals. Therefore, reliable analytical methods are required to detect and identify allergenic ingredients in food products. The present review addresses the recent developments regarding the application of DNA- and protein-based methods for the detection of allergenic ingredients in foods. The fitness-for-purpose of reviewed methodology will be discussed, and future trends will be highlighted. Special attention will be given to the evaluation of the potential of newly developed and promising technologies that can improve the detection and identification of allergenic ingredients in foods, such as the use of biosensors and/or nanomaterials to improve detection limits, specificity, ease of use, or to reduce the time of analysis. Such rapid food allergen test methods are required to facilitate the reliable detection of allergenic ingredients by control laboratories, to give the food industry the means to easily determine whether its product has been subjected to cross-contamination and, simultaneously, to identify how and when this cross-contamination occurred.Keywords: Allergens, DNA based methods, proteomics, allergen detection, qPCR, biosensors, nanoparticles},
keywords = {},
pubstate = {published},
tppubtype = {article}
}Currently, food allergies are an important health concern worldwide. The presence of undeclared allergenic ingredients or the presence of traces of allergens due to contamination during food processing poses a great health risk to sensitized individuals. Therefore, reliable analytical methods are required to detect and identify allergenic ingredients in food products. The present review addresses the recent developments regarding the application of DNA- and protein-based methods for the detection of allergenic ingredients in foods. The fitness-for-purpose of reviewed methodology will be discussed, and future trends will be highlighted. Special attention will be given to the evaluation of the potential of newly developed and promising technologies that can improve the detection and identification of allergenic ingredients in foods, such as the use of biosensors and/or nanomaterials to improve detection limits, specificity, ease of use, or to reduce the time of analysis. Such rapid food allergen test methods are required to facilitate the reliable detection of allergenic ingredients by control laboratories, to give the food industry the means to easily determine whether its product has been subjected to cross-contamination and, simultaneously, to identify how and when this cross-contamination occurred.Keywords: Allergens, DNA based methods, proteomics, allergen detection, qPCR, biosensors, nanoparticles
GROUP LEADER
THE TEAM
Alejandro Garrido
Staff Researcher
Ana Ribeiro
Research Fellow
Andrey Ipatov
Research Fellow
Prabir Kumar Kulabhusan
Research Fellow
Sarah Azinheiro
PhD Student
FORMER GROUP MEMBERS
Agnes Purwidyantri
Research Fellow
Foteini Roumani
Research Fellow
Carlos Carpena
visitor from Ocupharm diagnostics S.L.
Joana Carvalho
Laboratory Assistant
Joana Guerreiro
Research Fellow
Jon Ashley
Research Fellow
Karola Böhme (Research Fellow)
Master student ( February – September 2017)
University de Santiago de Compostela
Visitor (June 2017)
Sofia Granja Martins
Summer Student (July – September 2017)
María Leyva (Scientific Visitor)
Anna Toldrà Filella
Visitor from IRTA
Cristina Pastrana
visitor from Ocupharm diagnostics S.L.
Shambhavi Yadav
Scientific Associate
Monisha Elumalai
Research Fellow
Andrey Ipatov
Research Fellow
NEWS & EVENTS
New funded projects!
-
PORT GRAPHE – Control of Port and Douro Wines authenticity using graphene DNA sensors project(Project Time Frame: June 2018 to June 2021)
-
Nano BioSensor – Development of nanosensors to evaluate the microbiological quality of fruit-based products(Project Time Frame: July 2018 to June 2021)
Reliable Nano & Micro-solutions for Food Safety & Quality Analysis
The main objective of the Food & Quality Research Group is the development of analytical approaches based on the combination of molecular biology (mainly DNA based methodology) and nano and microfabrication technology in order to provide the food industry and control laboratories with reliable analytical tools.
Following this objective, the methodology is based on working on very specific analytical needs and on using a modular approach for each of the steps of the analytical process. This approach, help us to evaluate and to choose the best method in each case, to have a sound integrated final product, and at the same time a wide-range of intermediate products that can be used by themselves to solve specific analytical challenges.
Figure 1 summarizes the overall approach and research lines. Our topics of interest involve the detection of foodborne pathogens, the detection of allergenic ingredients in food products and food authenticity.
RESEARCH LINES
- Sample preparation:
Sample preparation is the series of steps required to transform a sample to a form suitable for analysis, the reliability of the conclusions drawn from food analysis greatly depends upon on this step. We work on: (i) the development of pre-treatment steps in order to overcome some of the limitations associated with food analysis and (ii) on the development of tailored, miniaturized, automatized and faster sample preparation techniques. Microscale solid phase extraction (µSPE) is used for on-chip DNA extraction and purification, being possible to put in contact a higher volume of initial binding material with the solid phase and recover the DNA in a lower volume during the elution phase. This feature allows to concentrate the DNA when minute amounts are present in the sample (e.g. olive oil, wine), for complex matrixes such as processed foodstuff and for environmental samples (e.g. water samples).
- Alternative DNA amplification methods:
Food & Quality Safety Research Group is working on new amplification techniques, in their combination with NPs and on the evaluation of DNA based analytical methods for food analysis. We work on isothermal amplification techniques, such as Loop-Mediated Isothermal Amplification (LAMP), and Recombinase Polymerase Amplification (RPA), specially interesting for miniaturization purposes. Other alternative techniques currently being used include Ligation Chain Reaction (LCR) which allows to distinguish very closely related organisms and high similar DNA sequences.
- Nanoparticle-assisted DNA analysis:
The use of nanomaterials for DNA analysis has the potential of providing increased sensitivity, multiplexing capabilities, and reduced costs. Exploiting the features of nanoparticles (NPs) is considered to be a good alternative to foster the potential of diagnostics and analytical method development. NPs, such as gold NPs (AuNPs) and gold nanorods (AuNRs) are being used for DNA detection taking advantage of their optical properties.
DEVELOPMENT OF A SYSTEM OF EARLY DETECTION OF THE ZEBRA MUSSEL THROUGH ANALYSIS OF ENVIRONMENTAL DNA
Cooperation Agreement with Confederación hidrográfica del Guadalquivir (CHG) Spain.
The objective of this cooperation agreement is the development of a micro Total Analysis Systems (µTAS) and optimized protocol for the detection of zebra mussel, an invasive species affecting numerous river basins worldwide, through environmental DNA (eDNA).
NANOEATERS: Valorization and transfer of NANOtechnologies to EArly adopTERS of the Euroregion Galicia-Norte Portugal-Use Case 2: Olive Oil Characterization
The FQ&S research group participates on the NANOEATERS project, on the Use Case 2: Olive Oil characterization on collaboration with the University of Vigo (Spain).
The main objective of use case 2 is the characterization of Extra virgin olive oil produced in Galicia, and the development of analytical approaches that would allow the differentiation of such olive oil from the one produced elsewhere.
N2020- NBFS-FOODSAFE
Innovative sample preparation and detection methods for foodborne pathogens, allergenic ingredients and food authenticity assessment are being developed. In parallel, new smart packaging and delivery systems are being designed and validated.
PORTGRAPHE
Control of Port and Douro Wines authenticity using graphene DNA sensors project
Time Frame: June 2018 to June 2021
The main objective of the project is the development, test and in-house validation of a miniaturized DNA sensing device for varietal discrimination of grapes, wines, musts, and grape juice in order to ensure the authenticity of wine from Port and Douro DOP. With this objective the participating teams will combine their expertise for the development of a miniaturized analytical device composed of 3 modules namely: a DNA extraction and purification module, an isothermal DNA amplification module, and a DNA Biosensors based on field-effect transistors (FETs) made using single layer graphene (SLG) for varietal discrimination.
NanoBioSensor
Development of nanosensors to evaluate the microbiological quality of fruit-based products
Time Frame: July 2018 to June 2021
A micro-total analysis system will be developed by the participating team to enable faster and better control of selected fruit-based products, reducing the analysis time from 7 days to few hours with greater sensitivity than conventional methods. This device will be designed and manufactured combining the latest developments in molecular biology, microfluidics and electronics.
SF4SF Smart Factoy for Safe Foods
Project funded by CDTI, Ministry of Industry of Spain.
The project aims the integration of emerging technologies for the detection, removal and food hazards management in food processing plants. The role of the group is the development of analytical methods based on DNA detection from gluten producing cereals and Listeria monocytogenes in fish products.
PUBLICATIONS
-
2021
Foteini Roumani, Sarah Azinheiro, Cristina Rodrigues, Jorge Barros-Velázquez, Alejandro Garrido-Maestu, Marta Prado
Development of a real-time PCR assay with an internal amplification control for the detection of spoilage fungi in fruit preparations Journal Article
Food Control, 135 (108783), 2021.
@article{Roumani2021b,
title = {Development of a real-time PCR assay with an internal amplification control for the detection of spoilage fungi in fruit preparations},
author = {Foteini Roumani, Sarah Azinheiro, Cristina Rodrigues, Jorge Barros-Velázquez, Alejandro Garrido-Maestu, Marta Prado},
url = {https://doi.org/10.1016/j.foodcont.2021.108783},
doi = {10.1016/j.foodcont.2021.108783},
year = {2021},
date = {2021-12-24},
journal = {Food Control},
volume = {135},
number = {108783},
abstract = {Fungal spoilage of food commodities is still of great concern for food industry due to increased costs regarding food waste and product recalls. Traditional culture-based methods are laborious and require huge amounts of reagents and media. In addition, results are available after seven days rendering these techniques unsuitable for the intense production that exists nowadays. As a result, there is a need for faster and more sensitive methods for fungal detection. The aim of this study was the development and evaluation of a method (enrichment, sample treatment, DNA extraction, and qPCR) for the fast detection of spoilage fungi in fruit preparations. In this sense, a set of universal primers was selected and a hydrolysis probe was designed in-house. In addition, a non-competitive internal amplification control was included in the assay to eliminate false negative results due to reaction inhibition. It was demonstrated that the method could reliably detect yeasts with a LOD95 of 1.0 CFU/50 g. Regarding moulds detection, two different enrichment times were examined, namely 24 h and 48 h in order to increase the sensitivity. The obtained LOD95 values were 123.5 spores/50 g and 37.1 spores/50 g for 24 h and 48 h respectively. During the method evaluation all the performance parameters resulted in values higher than 85.0% and the Cohen's k was determined to be above 0.86 for yeasts and moulds. Overall, a reliable and sensitive next-day detection method for fungi was achieved.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}Fungal spoilage of food commodities is still of great concern for food industry due to increased costs regarding food waste and product recalls. Traditional culture-based methods are laborious and require huge amounts of reagents and media. In addition, results are available after seven days rendering these techniques unsuitable for the intense production that exists nowadays. As a result, there is a need for faster and more sensitive methods for fungal detection. The aim of this study was the development and evaluation of a method (enrichment, sample treatment, DNA extraction, and qPCR) for the fast detection of spoilage fungi in fruit preparations. In this sense, a set of universal primers was selected and a hydrolysis probe was designed in-house. In addition, a non-competitive internal amplification control was included in the assay to eliminate false negative results due to reaction inhibition. It was demonstrated that the method could reliably detect yeasts with a LOD95 of 1.0 CFU/50 g. Regarding moulds detection, two different enrichment times were examined, namely 24 h and 48 h in order to increase the sensitivity. The obtained LOD95 values were 123.5 spores/50 g and 37.1 spores/50 g for 24 h and 48 h respectively. During the method evaluation all the performance parameters resulted in values higher than 85.0% and the Cohen's k was determined to be above 0.86 for yeasts and moulds. Overall, a reliable and sensitive next-day detection method for fungi was achieved.Foteini Roumani, Saioa Gómez, Cristina Rodrigues, Jorge Barros-Velázquez, Alejandro Garrido-Maestu, Marta Prado
Food Control, 135 (108784), 2021.
@article{Roumani2021c,
title = {Development and evaluation of a real-time fluorescence, and naked-eye colorimetric, loop-mediated isothermal amplification-based method for the rapid detection of spoilage fungi in fruit preparations},
author = {Foteini Roumani, Saioa Gómez, Cristina Rodrigues, Jorge Barros-Velázquez, Alejandro Garrido-Maestu, Marta Prado},
url = {https://doi.org/10.1016/j.foodcont.2021.108784},
doi = {10.1016/j.foodcont.2021.108784},
year = {2021},
date = {2021-12-24},
journal = {Food Control},
volume = {135},
number = {108784},
abstract = {DNA-based techniques like PCR/qPCR have been applied for the detection of microorganisms in order to provide faster results, due to their high sensitivity and specificity, when compared to culture-based techniques. However, isothermal amplification techniques like Loop-mediated isothermal amplification (LAMP) have emerged lately allowing the simplification of the assays, and reduction of costs. The aim of this study was the development, and evaluation, of a panfungal LAMP assay (LAMP- 18S) for the fast detection of spoilage fungi in fruit preparations. In this sense, a set of primers was newly designed. Two different detection strategies were examined, namely real-time fluorescence and naked-eye colour detection. In addition, the results were compared against another published panfungal LAMP (LAMP-POW) and a qPCR assay, which served as the reference method. The developed method showed high sensitivity being able to detect down to 1.4 pg/reaction for yeasts and 170 pg/reaction for moulds. The addition of an enrichment step significantly reduced the LOD as the method could reliably detect yeasts with a LOD95 of 3.1 and 3.0 CFU/50 g for the fluorescent and colorimetric assay, respectively. The fluorescent assay showed “substantial agreement” with the reference method, while the colorimetric assay was “almost in complete concordance” with the reference method. Overall, a reliable and sensitive next-day detection method for fungi was achieved.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}DNA-based techniques like PCR/qPCR have been applied for the detection of microorganisms in order to provide faster results, due to their high sensitivity and specificity, when compared to culture-based techniques. However, isothermal amplification techniques like Loop-mediated isothermal amplification (LAMP) have emerged lately allowing the simplification of the assays, and reduction of costs. The aim of this study was the development, and evaluation, of a panfungal LAMP assay (LAMP- 18S) for the fast detection of spoilage fungi in fruit preparations. In this sense, a set of primers was newly designed. Two different detection strategies were examined, namely real-time fluorescence and naked-eye colour detection. In addition, the results were compared against another published panfungal LAMP (LAMP-POW) and a qPCR assay, which served as the reference method. The developed method showed high sensitivity being able to detect down to 1.4 pg/reaction for yeasts and 170 pg/reaction for moulds. The addition of an enrichment step significantly reduced the LOD as the method could reliably detect yeasts with a LOD95 of 3.1 and 3.0 CFU/50 g for the fluorescent and colorimetric assay, respectively. The fluorescent assay showed “substantial agreement” with the reference method, while the colorimetric assay was “almost in complete concordance” with the reference method. Overall, a reliable and sensitive next-day detection method for fungi was achieved.Azinheiro, S., Ghimire, D., Carvalho, J., Prado, M., & Garrido-Maestu, A. (2022)
Next-day detection of viable Listeria monocytogenes by multiplex reverse transcriptase real-time PCR Journal Article
Food Control, 133 (PA) , 2021.
@article{Azinheiro2021c,
title = {Next-day detection of viable Listeria monocytogenes by multiplex reverse transcriptase real-time PCR},
author = {Azinheiro, S., Ghimire, D., Carvalho, J., Prado, M., & Garrido-Maestu, A. (2022)},
url = {https://doi.org/10.1016/j.foodcont.2021.108593},
doi = {10.1016/j.foodcont.2021.108593},
year = {2021},
date = {2021-10-01},
journal = {Food Control},
volume = {133 (PA)},
abstract = {Listeria monocytogenes continues to be a major challenge for the food industry due to its ubiquity and difficulty to be eliminated from processing facilities. The DNA amplification-based methods can overcome limitations of culture-based methods; however, due to the stability of the DNA, false positive results may occur, associated to the presence of harmless, dead microorganisms. The incorrect assessment of the results will have a huge impact on the producers in terms of stopping the production/distribution, leading to economic losses. In the current study, a multiplex RT-qPCR method was developed. The detection of mRNA allows for the specific detection of live bacteria. Additionally, two genetic targets (hly and actA) were co-amplified for improved specificity, along with an Internal Amplification Control to rule out false negative results due to reaction inhibition. The optimized methodology was evaluated in smoked salmon samples inoculated with different combinations and concentrations of live and dead bacteria. The method demonstrated high sensitivity (LOD50/LOD95 of 1.2/5.1 cfu/25 g). The performance of the method was compared against the reference standard ISO 11290, for which a Cohen's k of 0.94 was obtained, being interpreted as “almost complete concordance” among both methods. In addition, other parameters evaluated included the relative sensitivity, specificity, accuracy, as well as the positive and negative predictive values, all being above 90%. This next-day methodology can significantly reduce the time of analysis of culture-based methods and can specifically detect live L. monocytogenes.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}Listeria monocytogenes continues to be a major challenge for the food industry due to its ubiquity and difficulty to be eliminated from processing facilities. The DNA amplification-based methods can overcome limitations of culture-based methods; however, due to the stability of the DNA, false positive results may occur, associated to the presence of harmless, dead microorganisms. The incorrect assessment of the results will have a huge impact on the producers in terms of stopping the production/distribution, leading to economic losses. In the current study, a multiplex RT-qPCR method was developed. The detection of mRNA allows for the specific detection of live bacteria. Additionally, two genetic targets (hly and actA) were co-amplified for improved specificity, along with an Internal Amplification Control to rule out false negative results due to reaction inhibition. The optimized methodology was evaluated in smoked salmon samples inoculated with different combinations and concentrations of live and dead bacteria. The method demonstrated high sensitivity (LOD50/LOD95 of 1.2/5.1 cfu/25 g). The performance of the method was compared against the reference standard ISO 11290, for which a Cohen's k of 0.94 was obtained, being interpreted as “almost complete concordance” among both methods. In addition, other parameters evaluated included the relative sensitivity, specificity, accuracy, as well as the positive and negative predictive values, all being above 90%. This next-day methodology can significantly reduce the time of analysis of culture-based methods and can specifically detect live L. monocytogenes.Azinheiro, S., Roumani, F., Carvalho, J., Prado, M., & Garrido-Maestu, A.
Suitability of the MinION long read sequencer for semi-targeted detection of foodborne pathogens Journal Article
Analytica Chimica Acta, 1184 , 2021.
@article{Azinheiro2021b,
title = { Suitability of the MinION long read sequencer for semi-targeted detection of foodborne pathogens},
author = {Azinheiro, S., Roumani, F., Carvalho, J., Prado, M., & Garrido-Maestu, A. },
url = {https://doi.org/10.1016/j.aca.2021.339051},
doi = {10.1016/j.aca.2021.339051},
year = {2021},
date = {2021-09-10},
journal = {Analytica Chimica Acta},
volume = {1184},
abstract = {Foodborne pathogens are still a significant source of morbidity and mortality worldwide. In addition to this the current methodologies to track these microorganisms cannot cope with the current intensive production systems, thus novel methods are of outmost importance. DNA-based methods have already demonstrated suitable to address this issue, but most of them are targeted methods such as real-time PCR (qPCR), meaning that one will only find what is looking for, thus taking the risk of missing relevant pathogens in a given sample. To overcome this limitation we have developed an easy-to-implement methodology which enables the detection of several pathogens simultaneously by using long-read Next Generation Sequencing (NGS) with MinION. The method was named “semi-targeted” due to the combination of a non-targeted detection method, NGS, with the usage of selective media in order to partially eliminate non-pathogenic interfering bacteria. To this end, we included an enrichment step for the recovery of different pathogens, namely Salmonella Enteritidis and Typhimurium, Listeria monocytogenes and Escherichia coli O157:H7, after DNA extraction and library preparation, the samples were analyzed with MinION implementing the low-cost Flongle Flow Cells. The methodology was successfully evaluated in spiked milk samples with an excellent agreement with the results obtained by qPCR and culture-based methods. The method can provide accurate results after only 2 h of sequencing. Sample multiplexing, along with the lower cost of the Flongle Flow Cells and the reduced price of the MinION platform, make the assay cost-effective that is of importance for the food industry. Starting the method with a classical microbiological approach, the enrichment, the method is easy to implement in testing laboratories, it provides flexibility in terms of potential pathogens to be detected, and the positive results can be easily confirmed following culture-based, or other type, of confirmation procedures.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}Foodborne pathogens are still a significant source of morbidity and mortality worldwide. In addition to this the current methodologies to track these microorganisms cannot cope with the current intensive production systems, thus novel methods are of outmost importance. DNA-based methods have already demonstrated suitable to address this issue, but most of them are targeted methods such as real-time PCR (qPCR), meaning that one will only find what is looking for, thus taking the risk of missing relevant pathogens in a given sample. To overcome this limitation we have developed an easy-to-implement methodology which enables the detection of several pathogens simultaneously by using long-read Next Generation Sequencing (NGS) with MinION. The method was named “semi-targeted” due to the combination of a non-targeted detection method, NGS, with the usage of selective media in order to partially eliminate non-pathogenic interfering bacteria. To this end, we included an enrichment step for the recovery of different pathogens, namely Salmonella Enteritidis and Typhimurium, Listeria monocytogenes and Escherichia coli O157:H7, after DNA extraction and library preparation, the samples were analyzed with MinION implementing the low-cost Flongle Flow Cells. The methodology was successfully evaluated in spiked milk samples with an excellent agreement with the results obtained by qPCR and culture-based methods. The method can provide accurate results after only 2 h of sequencing. Sample multiplexing, along with the lower cost of the Flongle Flow Cells and the reduced price of the MinION platform, make the assay cost-effective that is of importance for the food industry. Starting the method with a classical microbiological approach, the enrichment, the method is easy to implement in testing laboratories, it provides flexibility in terms of potential pathogens to be detected, and the positive results can be easily confirmed following culture-based, or other type, of confirmation procedures.Guerreiro, J. R. L., Ipatov, A., Carvalho, J., Toldrà, A., & Prado, M.
Amplified plasmonic and microfluidic setup for DNA monitoring Journal Article
Microchimica Acta, (2021), (326), 2021.
@article{Guerreiro2021,
title = {Amplified plasmonic and microfluidic setup for DNA monitoring},
author = {Guerreiro, J. R. L., Ipatov, A., Carvalho, J., Toldrà, A., & Prado, M.},
url = {https://doi.org/10.1007/s00604-021-04983-y},
doi = {10.1007/s00604-021-04983-y},
year = {2021},
date = {2021-09-07},
journal = {Microchimica Acta, (2021)},
number = {326},
abstract = {Plasmonic nanosensors for label-free detection of DNA require excellent sensing resolution, which is crucial when monitoring short DNA sequences, as these induce tiny peak shifts, compared to large biomolecules. We report a versatile and simple strategy for plasmonic sensor signal enhancement by assembling multiple (four) plasmonic sensors in series. This approach provided a fourfold signal enhancement, increased signal-to-noise ratio, and improved sensitivity for DNA detection. The response of multiple sensors based on AuNSpheres was also compared with AuNRods, the latter showing better sensing resolution. The amplification system based on AuNR was integrated into a microfluidic sequential injection platform and applied to the monitoring of DNA, specifically from environmental invasive species—zebra mussels. DNA from zebra mussels was log concentration-dependent from 1 to 1 × 106 pM, reaching a detection limit of 2.0 pM. In situ tests were also successfully applied to real samples, within less than 45 min, using DNA extracted from zebra mussel meat. The plasmonic nanosensors’ signal can be used as a binary output (yes/no) to assess the presence of those invasive species. Even though these genosensors were applied to the monitoring of DNA in environmental samples, they potentially offer advantage in a wide range of fields, such as disease diagnostics.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}Plasmonic nanosensors for label-free detection of DNA require excellent sensing resolution, which is crucial when monitoring short DNA sequences, as these induce tiny peak shifts, compared to large biomolecules. We report a versatile and simple strategy for plasmonic sensor signal enhancement by assembling multiple (four) plasmonic sensors in series. This approach provided a fourfold signal enhancement, increased signal-to-noise ratio, and improved sensitivity for DNA detection. The response of multiple sensors based on AuNSpheres was also compared with AuNRods, the latter showing better sensing resolution. The amplification system based on AuNR was integrated into a microfluidic sequential injection platform and applied to the monitoring of DNA, specifically from environmental invasive species—zebra mussels. DNA from zebra mussels was log concentration-dependent from 1 to 1 × 106 pM, reaching a detection limit of 2.0 pM. In situ tests were also successfully applied to real samples, within less than 45 min, using DNA extracted from zebra mussel meat. The plasmonic nanosensors’ signal can be used as a binary output (yes/no) to assess the presence of those invasive species. Even though these genosensors were applied to the monitoring of DNA in environmental samples, they potentially offer advantage in a wide range of fields, such as disease diagnostics.Azinheiro, S., Roumani, F., Rodríguez-Lorenzo, L., Carvalho, J., Prado, M., & Garrido-Maestu, A.
Food Control (2022), 132 , 2021.
@article{Azinheiro2021,
title = {Combination of Recombinase Polymerase Amplification with SYBR Green I for naked-eye, same-day detection of Escherichia coli O157:H7 in ground meat},
author = {Azinheiro, S., Roumani, F., Rodríguez-Lorenzo, L., Carvalho, J., Prado, M., & Garrido-Maestu, A.},
url = {https://doi.org/10.1016/j.foodcont.2021.108494},
doi = {10.1016/j.foodcont.2021.108494},
year = {2021},
date = {2021-08-21},
journal = {Food Control (2022)},
volume = {132},
abstract = {Escherichia coli O157 continues to be the most prevalent serotype among the Shiga toxin-producing E. coli infection cases confirmed in Europe. The reference methodology to detect this pathogen is lengthy and time consuming, thus we sought to develop a novel method that has low instrumentation requirement, and allowed naked-eye detection. Isothermal amplification of bacterial DNA was performed by Recombinase Polymerase Amplification, and the addition of SYBR Green I (RPA-SG), which allowed the visualization of results with naked-eye under a UV lamp. The results obtained in spiked ground meat samples by RPA-SG compared favorably to qPCR (relative sensitivity, specificity and accuracy higher than 90%, and Cohen's k of 0.81), with a limit of detection of 19 cfu/25 g. The novel methodology outperformed a culture-based approach, where none of the typical colonies were confirmed as O157 due to high concentration of interfering microorganisms. These results were obtained in one working day (same-day detection), having an average time to completion of about 5 h, including enrichment, DNA extraction, amplification and detection.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}Escherichia coli O157 continues to be the most prevalent serotype among the Shiga toxin-producing E. coli infection cases confirmed in Europe. The reference methodology to detect this pathogen is lengthy and time consuming, thus we sought to develop a novel method that has low instrumentation requirement, and allowed naked-eye detection. Isothermal amplification of bacterial DNA was performed by Recombinase Polymerase Amplification, and the addition of SYBR Green I (RPA-SG), which allowed the visualization of results with naked-eye under a UV lamp. The results obtained in spiked ground meat samples by RPA-SG compared favorably to qPCR (relative sensitivity, specificity and accuracy higher than 90%, and Cohen's k of 0.81), with a limit of detection of 19 cfu/25 g. The novel methodology outperformed a culture-based approach, where none of the typical colonies were confirmed as O157 due to high concentration of interfering microorganisms. These results were obtained in one working day (same-day detection), having an average time to completion of about 5 h, including enrichment, DNA extraction, amplification and detection.Shambhavi Yadav, Joana Carvalho, Isabel Trujillo and Marta Prado
Microsatellite Markers in Olives (Olea europaea L.): Utility in the Cataloging of Germplasm, Food Authenticity and Traceability Studies Journal Article
Foods 2021, 10 (8), pp. 1907, 2021.
@article{Yadav2021,
title = {Microsatellite Markers in Olives (Olea europaea L.): Utility in the Cataloging of Germplasm, Food Authenticity and Traceability Studies},
author = {Shambhavi Yadav, Joana Carvalho, Isabel Trujillo and Marta Prado},
url = {https://doi.org/10.3390/foods10081907},
doi = {10.3390/foods10081907},
year = {2021},
date = {2021-08-17},
journal = {Foods 2021},
volume = {10},
number = {8},
pages = {1907},
abstract = {The olive fruit, a symbol of Mediterranean diets, is a rich source of antioxidants and oleic acid (55–83%). Olive genetic resources, including cultivated olives (cultivars), wild olives as well as related subspecies, are distributed widely across the Mediterranean region and other countries. Certain cultivars have a high commercial demand and economical value due to the differentiating organoleptic characteristics. This might result in economically motivated fraudulent practices and adulteration. Hence, tools to ensure the authenticity of constituent olive cultivars are crucial, and this can be achieved accurately through DNA-based methods. The present review outlines the applications of microsatellite markers, one of the most extensively used types of molecular markers in olive species, particularly referring to the use of these DNA-based markers in cataloging the vast olive germplasm, leading to identification and authentication of the cultivars. Emphasis has been given on the need to adopt a uniform platform where global molecular information pertaining to the details of available markers, cultivar-specific genotyping profiles (their synonyms or homonyms) and the comparative profiles of oil and reference leaf samples is accessible to researchers. The challenges of working with microsatellite markers and efforts underway, mainly advancements in genotyping methods which can be effectively incorporated in olive oil varietal testing, are also provided. Such efforts will pave the way for the development of more robust microsatellite marker-based olive agri-food authentication platforms.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}The olive fruit, a symbol of Mediterranean diets, is a rich source of antioxidants and oleic acid (55–83%). Olive genetic resources, including cultivated olives (cultivars), wild olives as well as related subspecies, are distributed widely across the Mediterranean region and other countries. Certain cultivars have a high commercial demand and economical value due to the differentiating organoleptic characteristics. This might result in economically motivated fraudulent practices and adulteration. Hence, tools to ensure the authenticity of constituent olive cultivars are crucial, and this can be achieved accurately through DNA-based methods. The present review outlines the applications of microsatellite markers, one of the most extensively used types of molecular markers in olive species, particularly referring to the use of these DNA-based markers in cataloging the vast olive germplasm, leading to identification and authentication of the cultivars. Emphasis has been given on the need to adopt a uniform platform where global molecular information pertaining to the details of available markers, cultivar-specific genotyping profiles (their synonyms or homonyms) and the comparative profiles of oil and reference leaf samples is accessible to researchers. The challenges of working with microsatellite markers and efforts underway, mainly advancements in genotyping methods which can be effectively incorporated in olive oil varietal testing, are also provided. Such efforts will pave the way for the development of more robust microsatellite marker-based olive agri-food authentication platforms.Yadav, S.; Carvalho, J.; Trujillo, I.; Prado, M.
Microsatellite Markers in Olives (Olea europaea L.): Utility in the Cataloging of Germplasm, Food Authenticity and Traceability Studies Journal Article
Foods, 10 (1907), 2021.
@article{Yadav2021b,
title = {Microsatellite Markers in Olives (Olea europaea L.): Utility in the Cataloging of Germplasm, Food Authenticity and Traceability Studies},
author = {Yadav, S.; Carvalho, J.; Trujillo, I.; Prado, M.},
url = {https://doi.org/10.3390/foods10081907},
doi = {10.3390/foods10081907},
year = {2021},
date = {2021-08-17},
journal = {Foods},
volume = {10},
number = {1907},
abstract = {The olive fruit, a symbol of Mediterranean diets, is a rich source of antioxidants and oleic acid (55–83%). Olive genetic resources, including cultivated olives (cultivars), wild olives as well as related subspecies, are distributed widely across the Mediterranean region and other countries. Certain cultivars have a high commercial demand and economical value due to the differentiating organoleptic characteristics. This might result in economically motivated fraudulent practices and adulteration. Hence, tools to ensure the authenticity of constituent olive cultivars are crucial, and this can be achieved accurately through DNA-based methods. The present review outlines the applications of microsatellite markers, one of the most extensively used types of molecular markers in olive species, particularly referring to the use of these DNA-based markers in cataloging the vast olive germplasm, leading to identification and authentication of the cultivars. Emphasis has been given on the need to adopt a uniform platform where global molecular information pertaining to the details of available markers, cultivar-specific genotyping profiles (their synonyms or homonyms) and the comparative profiles of oil and reference leaf samples is accessible to researchers. The challenges of working with microsatellite markers and efforts underway, mainly advancements in genotyping methods which can be effectively incorporated in olive oil varietal testing, are also provided. Such efforts will pave the way for the development of more robust microsatellite marker-based olive agri-food authentication platforms.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}The olive fruit, a symbol of Mediterranean diets, is a rich source of antioxidants and oleic acid (55–83%). Olive genetic resources, including cultivated olives (cultivars), wild olives as well as related subspecies, are distributed widely across the Mediterranean region and other countries. Certain cultivars have a high commercial demand and economical value due to the differentiating organoleptic characteristics. This might result in economically motivated fraudulent practices and adulteration. Hence, tools to ensure the authenticity of constituent olive cultivars are crucial, and this can be achieved accurately through DNA-based methods. The present review outlines the applications of microsatellite markers, one of the most extensively used types of molecular markers in olive species, particularly referring to the use of these DNA-based markers in cataloging the vast olive germplasm, leading to identification and authentication of the cultivars. Emphasis has been given on the need to adopt a uniform platform where global molecular information pertaining to the details of available markers, cultivar-specific genotyping profiles (their synonyms or homonyms) and the comparative profiles of oil and reference leaf samples is accessible to researchers. The challenges of working with microsatellite markers and efforts underway, mainly advancements in genotyping methods which can be effectively incorporated in olive oil varietal testing, are also provided. Such efforts will pave the way for the development of more robust microsatellite marker-based olive agri-food authentication platforms.Elumalai, M., Ipatov, A., Carvalho, J., Guerreiro, J., Prado, M.
Dual colorimetric strategy for specific DNA detection by nicking endonuclease-assisted gold nanoparticle signal amplification Journal Article
Anal Bioanal Chem, 2021.
@article{Elumalai2021,
title = {Dual colorimetric strategy for specific DNA detection by nicking endonuclease-assisted gold nanoparticle signal amplification},
author = {Elumalai, M., Ipatov, A., Carvalho, J., Guerreiro, J., Prado, M.},
url = {https://doi.org/10.1007/s00216-021-03564-5},
doi = {10.1007/s00216-021-03564-5},
year = {2021},
date = {2021-08-10},
journal = {Anal Bioanal Chem},
abstract = {The continuous spread of invasive alien species, as zebra mussel (Dreissena polymorpha), is a major global concern and it is urgent to stop it. Early stages of an invasion are crucial and challenging; however, detection tools based on environmental DNA analysis are promising alternatives. We present an alternative DNA target amplification strategy for signal enhancement followed by dual-mode colorimetric naked eye and optical smartphone analysis for the early detection of zebra mussel environmental DNA. Target amplification was designed based on the nicking endonuclease probe cleavage upon probe and complementary target hybridization. The cleaved/intact probe interacts with DNA-modified nanoparticles for colorimetric detection. We have demonstrated that enzyme amplification strategy enhanced 12-fold the sensitivity by naked eye detection, achieving a detection limit of ~8 nM (4.48×1010 copies) in controlled conditions, whereas target in complex environmental samples allowed the detection of 22.5 nM (1.26×1011 copies). Competitive assays also showed that the system can discriminate specific zebra mussel DNA sequences from other DNA sequences. Additionally, smartphone analysis for DNA quantification further improved the sensitivity of its detection by 130-fold, more than 2 orders of magnitude, when applied to environmental samples. The limit of detection to 0.17 nM (9.52×108 copies) is based on RGB coordinates, which is especially relevant to monitor early aggregation stages, being more accurate and reducing naked eye detection subjectivity. DNA extracted from zebra mussel meat, zebra mussel contaminated river water, and non-contaminated river water samples were successfully tested. Dual-mode colorimetric detection is useful in field analysis without the need for expensive laboratory equipment.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}The continuous spread of invasive alien species, as zebra mussel (Dreissena polymorpha), is a major global concern and it is urgent to stop it. Early stages of an invasion are crucial and challenging; however, detection tools based on environmental DNA analysis are promising alternatives. We present an alternative DNA target amplification strategy for signal enhancement followed by dual-mode colorimetric naked eye and optical smartphone analysis for the early detection of zebra mussel environmental DNA. Target amplification was designed based on the nicking endonuclease probe cleavage upon probe and complementary target hybridization. The cleaved/intact probe interacts with DNA-modified nanoparticles for colorimetric detection. We have demonstrated that enzyme amplification strategy enhanced 12-fold the sensitivity by naked eye detection, achieving a detection limit of ~8 nM (4.48×1010 copies) in controlled conditions, whereas target in complex environmental samples allowed the detection of 22.5 nM (1.26×1011 copies). Competitive assays also showed that the system can discriminate specific zebra mussel DNA sequences from other DNA sequences. Additionally, smartphone analysis for DNA quantification further improved the sensitivity of its detection by 130-fold, more than 2 orders of magnitude, when applied to environmental samples. The limit of detection to 0.17 nM (9.52×108 copies) is based on RGB coordinates, which is especially relevant to monitor early aggregation stages, being more accurate and reducing naked eye detection subjectivity. DNA extracted from zebra mussel meat, zebra mussel contaminated river water, and non-contaminated river water samples were successfully tested. Dual-mode colorimetric detection is useful in field analysis without the need for expensive laboratory equipment.Joana Carvalho, Shambhavi Yadav, Alejandro Garrido-Maestu, Sarah Azinheiro, Isabel Trujillo, Jorge Barros-Velázquez, Marta Prado
FOOD CHEMISTRY: MOLECULAR SCIENCES, 3 (100038), 2021.
@article{Carvalho2021b,
title = {Evaluation of simple sequence repeats (SSR) and single nucleotide polymorphism (SNP)-based methods in olive varieties from the Northwest of Spain and potential for miniaturization},
author = {Joana Carvalho, Shambhavi Yadav, Alejandro Garrido-Maestu, Sarah Azinheiro, Isabel Trujillo, Jorge Barros-Velázquez, Marta Prado},
url = {https://doi.org/10.1016/j.fochms.2021.100038},
doi = {10.1016/j.fochms.2021.100038},
year = {2021},
date = {2021-08-07},
journal = {FOOD CHEMISTRY: MOLECULAR SCIENCES},
volume = {3},
number = {100038},
abstract = {Miniaturization of DNA-based techniques can bring interesting advantages for food analysis, such as portability of complex analytical procedures. In the olive oil industry, miniaturization can be particularly interesting for authenticity and traceability applications, through in situ control of raw materials before production and/or the final products. However, variety identification is challenging, and implementation on miniaturized settings must be carefully evaluated, starting from the selected analytical approach. In this work, SSR- and SNP-based genotyping strategies were investigated for the identification and differentiation of two olive varieties from the Northwest of Spain. For the selected SNPs two genotyping methods were tested: real-time allele-specific PCR and high resolution melting analysis. These methods were compared and evaluated regarding their potential for integration in a microfluidic device. Both SNP-based methods proved to be successful for identification of the selected varieties, however real-time allele-specific PCR was the one that achieved the best results when analyzing mixtures, allowing the identification of both monovarietal samples and mixtures of the varieties tested with up to 25%.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}Miniaturization of DNA-based techniques can bring interesting advantages for food analysis, such as portability of complex analytical procedures. In the olive oil industry, miniaturization can be particularly interesting for authenticity and traceability applications, through in situ control of raw materials before production and/or the final products. However, variety identification is challenging, and implementation on miniaturized settings must be carefully evaluated, starting from the selected analytical approach. In this work, SSR- and SNP-based genotyping strategies were investigated for the identification and differentiation of two olive varieties from the Northwest of Spain. For the selected SNPs two genotyping methods were tested: real-time allele-specific PCR and high resolution melting analysis. These methods were compared and evaluated regarding their potential for integration in a microfluidic device. Both SNP-based methods proved to be successful for identification of the selected varieties, however real-time allele-specific PCR was the one that achieved the best results when analyzing mixtures, allowing the identification of both monovarietal samples and mixtures of the varieties tested with up to 25%.Carvalho, J.; Yadav, S.; Garrido-Maestu, A.G.; Azinheiro, S.; Trujillo, I.; Barros-Velázquez, J.; Prado, M.
Food Chemistry: Molecular Sciences, 3 , 2021.
@article{Carvalho2021,
title = {Evaluation of simple sequence repeats (SSR) and single nucleotide polymorphism (SNP)-based methods in olive varieties from the Northwest of Spain and potential for miniaturization},
author = {Carvalho, J.; Yadav, S.; Garrido-Maestu, A.G.; Azinheiro, S.; Trujillo, I.; Barros-Velázquez, J.; Prado, M. },
url = {https://doi.org/10.1016/j.fochms.2021.100038},
doi = {10.1016/j.fochms.2021.100038},
year = {2021},
date = {2021-07-31},
journal = { Food Chemistry: Molecular Sciences},
volume = {3},
abstract = {Miniaturization of DNA-based techniques can bring interesting advantages for food analysis, such as portability of complex analytical procedures. In the olive oil industry, miniaturization can be particularly interesting for authenticity and traceability applications, through in situ control of raw materials before production and/or the final products. However, variety identification is challenging, and implementation on miniaturized settings must be carefully evaluated, starting from the selected analytical approach. In this work, SSR- and SNP-based genotyping strategies were investigated for the identification and differentiation of two olive varieties from the Northwest of Spain. For the selected SNPs two genotyping methods were tested: real-time allele-specific PCR and high resolution melting analysis. These methods were compared and evaluated regarding their potential for integration in a microfluidic device. Both SNP-based methods proved to be successful for identification of the selected varieties, however real-time allele-specific PCR was the one that achieved the best results when analyzing mixtures, allowing the identification of both monovarietal samples and mixtures of the varieties tested with up to 25%.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}Miniaturization of DNA-based techniques can bring interesting advantages for food analysis, such as portability of complex analytical procedures. In the olive oil industry, miniaturization can be particularly interesting for authenticity and traceability applications, through in situ control of raw materials before production and/or the final products. However, variety identification is challenging, and implementation on miniaturized settings must be carefully evaluated, starting from the selected analytical approach. In this work, SSR- and SNP-based genotyping strategies were investigated for the identification and differentiation of two olive varieties from the Northwest of Spain. For the selected SNPs two genotyping methods were tested: real-time allele-specific PCR and high resolution melting analysis. These methods were compared and evaluated regarding their potential for integration in a microfluidic device. Both SNP-based methods proved to be successful for identification of the selected varieties, however real-time allele-specific PCR was the one that achieved the best results when analyzing mixtures, allowing the identification of both monovarietal samples and mixtures of the varieties tested with up to 25%.Maryam Naseri ,Arnab Halder ,Mohsen Mohammadniaei ,Marta Prado ,Jon Ashley ,Yi Sun
A multivalent aptamer-based electrochemical biosensor for biomarker detection in urinary tract infection Journal Article
Electrochimica Acta (2021), 389 , 2021.
@article{Naseri2021b,
title = {A multivalent aptamer-based electrochemical biosensor for biomarker detection in urinary tract infection},
author = {Maryam Naseri ,Arnab Halder ,Mohsen Mohammadniaei ,Marta Prado ,Jon Ashley ,Yi Sun},
url = {https://doi.org/10.1016/j.electacta.2021.138644},
doi = {10.1016/j.electacta.2021.138644},
year = {2021},
date = {2021-05-23},
journal = { Electrochimica Acta (2021)},
volume = {389},
abstract = {Lactoferrin is a multifunctional protein of the transferrin family and is known as a biomarker for various clinical diseases including urinary tract infection (UTI). However, wide concentration range of lactoferrin in urine samples due to the high interpatient variations, requires a more practical biosensor. In this article, we used a novel multivalent aptamer immobilized on the surface of screen-printed gold electrode (aptamer/SPGE) to develop the first electrochemical aptasensor for label-free detection of lactoferrin with wide dynamic detection range and high sensitivity. The performance of the fabricated biosensor was tested using electrochemical impedance spectroscopy and differential pulse voltammetry. The multivalent aptamer as the bioreceptor with high affinity and good specificity against human lactoferrin, acts to enhance the electrochemical signals and widen the working window. The aptamer/SPGE demonstrates superior sensing performances for lactoferrin in buffer solution, with a wide linear range of 10 to 1300 ng/mL with LOD of 0.9 ng/mL, as well as high selectivity, and excellent reproducibility. Besides, the constructed aptasensor was successfully applied to quantify lactoferrin concentrations in spiked urine solutions. Owing to excellent sensitivity, ease of miniaturization, simple sensing procedure, low-cost, and fast response, the proposed electrochemical aptasensor indicates a great potential towards the development of lactoferrin analysis systems, which would be helpful in the early diagnosis of UTI.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}Lactoferrin is a multifunctional protein of the transferrin family and is known as a biomarker for various clinical diseases including urinary tract infection (UTI). However, wide concentration range of lactoferrin in urine samples due to the high interpatient variations, requires a more practical biosensor. In this article, we used a novel multivalent aptamer immobilized on the surface of screen-printed gold electrode (aptamer/SPGE) to develop the first electrochemical aptasensor for label-free detection of lactoferrin with wide dynamic detection range and high sensitivity. The performance of the fabricated biosensor was tested using electrochemical impedance spectroscopy and differential pulse voltammetry. The multivalent aptamer as the bioreceptor with high affinity and good specificity against human lactoferrin, acts to enhance the electrochemical signals and widen the working window. The aptamer/SPGE demonstrates superior sensing performances for lactoferrin in buffer solution, with a wide linear range of 10 to 1300 ng/mL with LOD of 0.9 ng/mL, as well as high selectivity, and excellent reproducibility. Besides, the constructed aptasensor was successfully applied to quantify lactoferrin concentrations in spiked urine solutions. Owing to excellent sensitivity, ease of miniaturization, simple sensing procedure, low-cost, and fast response, the proposed electrochemical aptasensor indicates a great potential towards the development of lactoferrin analysis systems, which would be helpful in the early diagnosis of UTI.Roumani, F.; Azinheiro, S.; Sousa, H.; Sousa, A.; Timóteo, M.; Varandas, T.; Fonseca-silva, D.; Baldaque, I.; Carvalho, J.; Prado, M.; Garrido-Maestu
Viruses 2021, 2021.
@article{Roumani2021,
title = {Optimization and Clinical Evaluation of a Multi-Target Loop-Mediated Isothermal Amplification Assay for the Detection of SARS-CoV-2 in Nasopharyngeal Samples},
author = {Roumani, F.; Azinheiro, S.; Sousa, H.; Sousa, A.; Timóteo, M.; Varandas, T.; Fonseca-silva, D.; Baldaque, I.; Carvalho, J.; Prado, M.; Garrido-Maestu},
url = {https://doi.org/10.3390/v13050940},
doi = {10.3390/v13050940},
year = {2021},
date = {2021-05-16},
journal = {Viruses 2021},
abstract = {SARS-CoV-2 is the coronavirus responsible for COVID-19, which has spread worldwide, affecting more than 200 countries, infecting over 140 million people in one year. The gold standard to identify infected people is RT-qPCR, which is highly sensitive, but needs specialized equipment and trained personnel. The demand for these reagents has caused shortages in certain countries. Isothermal nucleic acid techniques, such as loop-mediated isothermal amplification (LAMP) have emerged as an alternative or as a complement to RT-qPCR. In this study, we developed and evaluated a multi-target RT-LAMP for the detection of SARS-CoV-2. The method was evaluated against an RT-qPCR in 152 clinical nasopharyngeal swab samples. The results obtained indicated that both assays presented a “good concordance” (Cohen’s k of 0.69), the RT-LAMP was highly specific (99%) but had lower sensitivity compared to the gold standard (63.3%). The calculated low sensitivity was associated with samples with very low viral load (RT-qPCR Cq values higher than 35) which may be associated with non-infectious individuals. If an internal Cq threshold below 35 was set, the sensitivity and Cohen’s k increased to 90.9% and 0.92, respectively. The interpretation of the Cohen’s k for this was “very good concordance”. The RT-LAMP is an attractive approach for frequent individual testing in decentralized setups.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}SARS-CoV-2 is the coronavirus responsible for COVID-19, which has spread worldwide, affecting more than 200 countries, infecting over 140 million people in one year. The gold standard to identify infected people is RT-qPCR, which is highly sensitive, but needs specialized equipment and trained personnel. The demand for these reagents has caused shortages in certain countries. Isothermal nucleic acid techniques, such as loop-mediated isothermal amplification (LAMP) have emerged as an alternative or as a complement to RT-qPCR. In this study, we developed and evaluated a multi-target RT-LAMP for the detection of SARS-CoV-2. The method was evaluated against an RT-qPCR in 152 clinical nasopharyngeal swab samples. The results obtained indicated that both assays presented a “good concordance” (Cohen’s k of 0.69), the RT-LAMP was highly specific (99%) but had lower sensitivity compared to the gold standard (63.3%). The calculated low sensitivity was associated with samples with very low viral load (RT-qPCR Cq values higher than 35) which may be associated with non-infectious individuals. If an internal Cq threshold below 35 was set, the sensitivity and Cohen’s k increased to 90.9% and 0.92, respectively. The interpretation of the Cohen’s k for this was “very good concordance”. The RT-LAMP is an attractive approach for frequent individual testing in decentralized setups.Carvalho J and Maestu AG and Azinheiro S and Fuciños P and Velázquez J B and Miguel RJ De and Gros V and Prado M
Faster monitoring of the invasive alien species ( IAS ) Dreissena polymorpha in river basins through isothermal amplification Journal Article
Scientific Reports, 11 (10175), 2021.
@article{J2021b,
title = {Faster monitoring of the invasive alien species ( IAS ) Dreissena polymorpha in river basins through isothermal amplification},
author = {Carvalho J and Maestu AG and Azinheiro S and Fuciños P and Velázquez J B and Miguel RJ De and Gros V and Prado M},
url = {https://doi.org/10.1038/s41598-021-89574-w},
doi = {10.1038/s41598-021-89574-w},
year = {2021},
date = {2021-05-13},
journal = {Scientific Reports},
volume = {11},
number = {10175},
abstract = {Zebra mussel (Dreissena polymorpha) is considered as one of the 100 most harmful IAS in the world. Traditional detection methods have limitations, and PCR based environmental DNA detection has provided interesting results for early warning. However, in the last years, the development of isothermal amplification methods has received increasing attention. Among them, loop-mediated isothermal amplification (LAMP) has several advantages, including its higher tolerance to the presence of inhibitors and the possibility of naked-eye detection, which enables and simplifies its potential use in decentralized settings. In the current study, a real-time LAMP (qLAMP) method for the detection of Dreissena polymorpha was developed and tested with samples from the Guadalquivir River basin, together with two real-time PCR (qPCR) methods using different detection chemistries, targeting a specific region of the mitochondrial gene cytochrome C oxidase subunit I. All three developed approaches were evaluated regarding specificity, sensitivity and time required for detection. Regarding sensitivity, both qPCR approaches were more sensitive than qLAMP by one order of magnitude, however the qLAMP method proved to be as specific and much faster being performed in just 9 min versus 23 and 29 min for the qPCR methods based on hydrolysis probe and intercalating dye respectively.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}Zebra mussel (Dreissena polymorpha) is considered as one of the 100 most harmful IAS in the world. Traditional detection methods have limitations, and PCR based environmental DNA detection has provided interesting results for early warning. However, in the last years, the development of isothermal amplification methods has received increasing attention. Among them, loop-mediated isothermal amplification (LAMP) has several advantages, including its higher tolerance to the presence of inhibitors and the possibility of naked-eye detection, which enables and simplifies its potential use in decentralized settings. In the current study, a real-time LAMP (qLAMP) method for the detection of Dreissena polymorpha was developed and tested with samples from the Guadalquivir River basin, together with two real-time PCR (qPCR) methods using different detection chemistries, targeting a specific region of the mitochondrial gene cytochrome C oxidase subunit I. All three developed approaches were evaluated regarding specificity, sensitivity and time required for detection. Regarding sensitivity, both qPCR approaches were more sensitive than qLAMP by one order of magnitude, however the qLAMP method proved to be as specific and much faster being performed in just 9 min versus 23 and 29 min for the qPCR methods based on hydrolysis probe and intercalating dye respectively.Ipatov, A and Garrido- Maestu, A and Guerreiro, J R and Purwidyantri, A and Azinheiro, S and Carvalho, J and Romani, F and Elumalai, M and Prado, M
Application of Omics-based Miniaturized Systems in Food Quality and Safety Book Chapter
2021, ISBN: 978-1-78801-884-5.
@inbook{Ipatov2021,
title = {Application of Omics-based Miniaturized Systems in Food Quality and Safety},
author = {Ipatov, A and Garrido- Maestu, A and Guerreiro, J R and Purwidyantri, A and Azinheiro, S and Carvalho, J and Romani, F and Elumalai, M and Prado, M},
url = {https://doi.org/10.1039/9781839163005},
doi = {10.1039/9781839163005-00222},
isbn = {978-1-78801-884-5},
year = {2021},
date = {2021-03-26},
journal = {Foodomics: Omic Strategies and Applications in Food Science},
abstract = {Miniaturized devices are revolutionizing food analysis. Such devices have a number of advantages, namely low consumption of sample and reagents, portability, low-energy consumption, rapid response and multiplexing capabilities, that have fostered their development towards marketable products. An increasing number of microfluidic-based devices are being released to the market, providing enormous possibilities for application in the omics sciences. One of the most interesting, and yet most challenging applications of miniaturized devices in food monitoring, is the development of hand-held devices that can enable in situ analysis at different points of the food value chain. The main objectives are to ensure the health of consumers, to easily determine whether a food product has been subjected to cross-contamination and, simultaneously, to identify how and when this cross-contamination occurred in order to establish the proper corrective measures. Herein, we focus mainly on the development of tools for food monitoring, particularly for protein- and DNA-based analysis, including the main fabrication processes and materials. Sample preparation and pre-treatment is highlighted as a challenging and fundamental step of the analytical procedure. Particularly in the case of foodborne pathogens, in which sample treatment is considered the main bottleneck for fast and in situ analysis. Nano-labelled and non-labelled detection strategies are discussed together with the main advantages and disadvantages of both for targeted analysis. Although there are several interesting developments of miniaturized devices that can contribute to omics analysis via non-targeted approaches, this chapter focuses mainly on the latest developments towards targeted analysis.},
keywords = {},
pubstate = {published},
tppubtype = {inbook}
}Miniaturized devices are revolutionizing food analysis. Such devices have a number of advantages, namely low consumption of sample and reagents, portability, low-energy consumption, rapid response and multiplexing capabilities, that have fostered their development towards marketable products. An increasing number of microfluidic-based devices are being released to the market, providing enormous possibilities for application in the omics sciences. One of the most interesting, and yet most challenging applications of miniaturized devices in food monitoring, is the development of hand-held devices that can enable in situ analysis at different points of the food value chain. The main objectives are to ensure the health of consumers, to easily determine whether a food product has been subjected to cross-contamination and, simultaneously, to identify how and when this cross-contamination occurred in order to establish the proper corrective measures. Herein, we focus mainly on the development of tools for food monitoring, particularly for protein- and DNA-based analysis, including the main fabrication processes and materials. Sample preparation and pre-treatment is highlighted as a challenging and fundamental step of the analytical procedure. Particularly in the case of foodborne pathogens, in which sample treatment is considered the main bottleneck for fast and in situ analysis. Nano-labelled and non-labelled detection strategies are discussed together with the main advantages and disadvantages of both for targeted analysis. Although there are several interesting developments of miniaturized devices that can contribute to omics analysis via non-targeted approaches, this chapter focuses mainly on the latest developments towards targeted analysis.Prabowo BA, Purwidyantri A, Liu B, Lai HC, Liu KC
Gold nanoparticle-assisted plasmonic enhancement for DNA detection on a graphene-based portable surface plasmon resonance sensor Journal Article
Nanotechnology, 32 (9), 2021.
@article{BA2021,
title = {Gold nanoparticle-assisted plasmonic enhancement for DNA detection on a graphene-based portable surface plasmon resonance sensor},
author = {Prabowo BA, Purwidyantri A, Liu B, Lai HC, Liu KC},
url = {https://doi.org/10.1088/1361-6528/abcd62},
doi = {10.1088/1361-6528/abcd62},
year = {2021},
date = {2021-02-26},
journal = {Nanotechnology},
volume = {32},
number = {9},
abstract = {The impact of different gold nanoparticle (GNP) structures on plasmonic enhancement for DNA detection is investigated on a few-layer graphene (FLG) surface plasmon resonance (SPR) sensor. Two distinct structures of gold nano-urchins (GNu) and gold nanorods (GNr) were used to bind the uniquely designed single-stranded probe DNA (ssDNA) of Mycobacterium tuberculosis complex DNA. The two types of GNP-ssDNA mixture were adsorbed onto the FLG-coated SPR sensor through the π-π stacking force between the ssDNA and the graphene layer. In the presence of complementary single-stranded DNA, the hybridization process took place and gradually removed the probes from the graphene surface. From SPR sensor preparation, the annealing process of the Au layer of the SPR sensor effectively enhanced the FLG coverage leading to a higher load of the probe DNA onto the sensing interface. The FLG was shown to be effective in providing a larger surface area for biomolecular capture due to its roughness. Carried out in the DNA hybridization study with the SPR sensor, GNu, with its rough and spiky structures, significantly reinforced the overall DNA hybridization signal compared with GNr with smooth superficies, especially in capturing the probe DNA. The DNA hybridization detection assisted by GNu reached the femtomolar range limit of detection. An optical simulation validated the extreme plasmonic field enhancement at the tip of the GNu spicules. The overall integrated approach of the graphene-based SPR sensor and GNu-assisted DNA detection provided the proof-of-concept for the possibility of tuberculosis disease screening using a low-cost and portable system to be potentially applied in remote or third-world countries.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}The impact of different gold nanoparticle (GNP) structures on plasmonic enhancement for DNA detection is investigated on a few-layer graphene (FLG) surface plasmon resonance (SPR) sensor. Two distinct structures of gold nano-urchins (GNu) and gold nanorods (GNr) were used to bind the uniquely designed single-stranded probe DNA (ssDNA) of Mycobacterium tuberculosis complex DNA. The two types of GNP-ssDNA mixture were adsorbed onto the FLG-coated SPR sensor through the π-π stacking force between the ssDNA and the graphene layer. In the presence of complementary single-stranded DNA, the hybridization process took place and gradually removed the probes from the graphene surface. From SPR sensor preparation, the annealing process of the Au layer of the SPR sensor effectively enhanced the FLG coverage leading to a higher load of the probe DNA onto the sensing interface. The FLG was shown to be effective in providing a larger surface area for biomolecular capture due to its roughness. Carried out in the DNA hybridization study with the SPR sensor, GNu, with its rough and spiky structures, significantly reinforced the overall DNA hybridization signal compared with GNr with smooth superficies, especially in capturing the probe DNA. The DNA hybridization detection assisted by GNu reached the femtomolar range limit of detection. An optical simulation validated the extreme plasmonic field enhancement at the tip of the GNu spicules. The overall integrated approach of the graphene-based SPR sensor and GNu-assisted DNA detection provided the proof-of-concept for the possibility of tuberculosis disease screening using a low-cost and portable system to be potentially applied in remote or third-world countries.Purwidyantri, A.; Tian, Y.-C.; Saputra, G.M.A.; Prabowo, B.A.; Liu, H.-L.; Yang, C.-M.; Lai, C.-S.
Gold Nanoframe Array Electrode for Straightforward Detection of Hydrogen Peroxide Journal Article
Chemosensors, 9 (37), 2021.
@article{Purwidyantri2021b,
title = {Gold Nanoframe Array Electrode for Straightforward Detection of Hydrogen Peroxide},
author = {Purwidyantri, A.; Tian, Y.-C.; Saputra, G.M.A.; Prabowo, B.A.; Liu, H.-L.; Yang, C.-M.; Lai, C.-S.},
url = {https://doi.org/10.3390/chemosensors9020037},
doi = {10.3390/chemosensors9020037},
year = {2021},
date = {2021-02-16},
journal = {Chemosensors},
volume = {9},
number = {37},
abstract = {The nanostructuring of a sensing membrane is performed through colloidal nanosphere lithography (NSL) techniques with a tiny polystyrene nanobead template 100 nm in size. The solvent ratio adjustment has been proven to be effective in assisting the monolayer deposition of small templating particles with minimal defects. Two distinct structures, namely, a billowy gold nanostructure (BGN) where the nanobead template is left unetched and a gold nanoframe array (GNA) with a regular ring-like structure after template removal, are used for the extended-gate field-effect transistor (EGFET) electrodes. The GNA structure generates an electroactive surface area significantly (~20%) larger than its geometrical area as well as a greater surface roughness than the BGN. When integrated with the portable constant voltage–constant current (CVCC) FET circuitry for pH screening to determine the optimized measurement conditions for H2O2 sensing, the GNA sensing membrane also shows more improved Nernstian sensitivity at ~50 mV/pH than the BGN electrode. The more optimized sensitivity is then proven using the GNA in the detection of H2O2, the most common representative reactive oxygen species (ROS) involved in the environment, food, and neurodegenerative diseases, such as Parkinson´s and Alzheimer´s diseases. The GNA electrode has a sensitivity of 70.42 mV/log µM [H2O2] and a limit of detection (LoD) of 1.183 µM H2O2. The integrated ion sensing system employing unique, highly ordered gold array gate electrodes and a portable CVCC circuit system has shown a stable real-time output voltage signal, representing an alternative to bulky conventional FET devices for potential on-site H2O2 detection.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}The nanostructuring of a sensing membrane is performed through colloidal nanosphere lithography (NSL) techniques with a tiny polystyrene nanobead template 100 nm in size. The solvent ratio adjustment has been proven to be effective in assisting the monolayer deposition of small templating particles with minimal defects. Two distinct structures, namely, a billowy gold nanostructure (BGN) where the nanobead template is left unetched and a gold nanoframe array (GNA) with a regular ring-like structure after template removal, are used for the extended-gate field-effect transistor (EGFET) electrodes. The GNA structure generates an electroactive surface area significantly (~20%) larger than its geometrical area as well as a greater surface roughness than the BGN. When integrated with the portable constant voltage–constant current (CVCC) FET circuitry for pH screening to determine the optimized measurement conditions for H2O2 sensing, the GNA sensing membrane also shows more improved Nernstian sensitivity at ~50 mV/pH than the BGN electrode. The more optimized sensitivity is then proven using the GNA in the detection of H2O2, the most common representative reactive oxygen species (ROS) involved in the environment, food, and neurodegenerative diseases, such as Parkinson´s and Alzheimer´s diseases. The GNA electrode has a sensitivity of 70.42 mV/log µM [H2O2] and a limit of detection (LoD) of 1.183 µM H2O2. The integrated ion sensing system employing unique, highly ordered gold array gate electrodes and a portable CVCC circuit system has shown a stable real-time output voltage signal, representing an alternative to bulky conventional FET devices for potential on-site H2O2 detection.Carvalho J and Diéguez L and Ipatov A and Guerreiro J R and Garrido-Maestu A and Azinheiro S and Prado M
Single-use microfluidic device for purification and concentration of environmental DNA from river water Journal Article
Talanta, 226 , 2021.
@article{J2021,
title = {Single-use microfluidic device for purification and concentration of environmental DNA from river water},
author = {Carvalho J and Diéguez L and Ipatov A and Guerreiro J R and Garrido-Maestu A and Azinheiro S and Prado M},
url = {https://doi.org/10.1016/j.talanta.2021.122109},
doi = {10.1016/j.talanta.2021.122109},
year = {2021},
date = {2021-02-04},
journal = {Talanta},
volume = {226},
abstract = {Purification and concentration of DNA is a critical step on DNA-based analysis, which should ensure efficient DNA isolation and effective removal of contaminants that may interfere with downstream DNA amplification. Complexity of samples, minute content of target analyte, or high DNA fragmentation greatly entangles the success of this step. To overcome this issue, we designed and fabricated a novel miniaturized disposable device for a highly efficient DNA purification. The microfluidic device showed binding efficiency and elution yield of 90.1% and 86.7%, respectively. Moreover, the effect of DNA fragmentation, a parameter that has not been previously addressed, showed a great impact in the recovery step. The microfluidic system integrated micropillars with chitosan being used as the solid-phase for a pH-dependent DNA capture and release. We have showed the potential of the device in the successful purification of environmental DNA (eDNA) from river water samples contaminated with Dreissena polymorpha, an invasive alien species responsible for unquestionable economic and environmental consequences in river water basins. Additionally, the device was also able to concentrate the DNA extract from highly diluted samples, showing promising results for the early detection of such invasive species, which may allow prompt measures for a more efficient control in affected areas. Suitability for integration with downstream DNA analysis was also demonstrated through qPCR analysis of the samples purified with the microfluidic device, allowing detection of the target species even if highly diluted.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}Purification and concentration of DNA is a critical step on DNA-based analysis, which should ensure efficient DNA isolation and effective removal of contaminants that may interfere with downstream DNA amplification. Complexity of samples, minute content of target analyte, or high DNA fragmentation greatly entangles the success of this step. To overcome this issue, we designed and fabricated a novel miniaturized disposable device for a highly efficient DNA purification. The microfluidic device showed binding efficiency and elution yield of 90.1% and 86.7%, respectively. Moreover, the effect of DNA fragmentation, a parameter that has not been previously addressed, showed a great impact in the recovery step. The microfluidic system integrated micropillars with chitosan being used as the solid-phase for a pH-dependent DNA capture and release. We have showed the potential of the device in the successful purification of environmental DNA (eDNA) from river water samples contaminated with Dreissena polymorpha, an invasive alien species responsible for unquestionable economic and environmental consequences in river water basins. Additionally, the device was also able to concentrate the DNA extract from highly diluted samples, showing promising results for the early detection of such invasive species, which may allow prompt measures for a more efficient control in affected areas. Suitability for integration with downstream DNA analysis was also demonstrated through qPCR analysis of the samples purified with the microfluidic device, allowing detection of the target species even if highly diluted.Purwidyantri A and Domingues T and Borme J and Guerreiro J R and Ipatov A and Abreu C M and Martins M and Alpuim P and Prado M
Influence of the Electrolyte Salt Concentration on DNA Detection with Graphene Transistors Journal Article
Biosensors, 11 , pp. 24, 2021.
@article{A2021,
title = {Influence of the Electrolyte Salt Concentration on DNA Detection with Graphene Transistors},
author = {Purwidyantri A and Domingues T and Borme J and Guerreiro J R and Ipatov A and Abreu C M and Martins M and Alpuim P and Prado M},
url = {https://doi.org/10.3390/bios11010024},
doi = {10.3390/bios11010024},
year = {2021},
date = {2021-01-17},
journal = {Biosensors},
volume = {11},
pages = {24},
abstract = {Liquid-gated Graphene Field-Effect Transistors (GFET) are ultrasensitive bio-detection platforms carrying out the graphene’s exceptional intrinsic functionalities. Buffer and dilution factor are prevalent strategies towards the optimum performance of the GFETs. However, beyond the Debye length (λD), the role of the graphene-electrolytes’ ionic species interactions on the DNA behavior at the nanoscale interface is complicated. We studied the characteristics of the GFETs under different ionic strength, pH, and electrolyte type, e.g., phosphate buffer (PB), and phosphate buffer saline (PBS), in an automatic portable built-in system. The electrostatic gating and charge transfer phenomena were inferred from the field-effect measurements of the Dirac point position in single-layer graphene (SLG) transistors transfer curves. Results denote that λD is not the main factor governing the effective nanoscale screening environment. We observed that the longer λD was not the determining characteristic for sensitivity increment and limit of detection (LoD) as demonstrated by different types and ionic strengths of measuring buffers. In the DNA hybridization study, our findings show the role of the additional salts present in PBS, as compared to PB, in increasing graphene electron mobility, electrostatic shielding, intermolecular forces and DNA adsorption kinetics leading to an improved sensitivity.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}Liquid-gated Graphene Field-Effect Transistors (GFET) are ultrasensitive bio-detection platforms carrying out the graphene’s exceptional intrinsic functionalities. Buffer and dilution factor are prevalent strategies towards the optimum performance of the GFETs. However, beyond the Debye length (λD), the role of the graphene-electrolytes’ ionic species interactions on the DNA behavior at the nanoscale interface is complicated. We studied the characteristics of the GFETs under different ionic strength, pH, and electrolyte type, e.g., phosphate buffer (PB), and phosphate buffer saline (PBS), in an automatic portable built-in system. The electrostatic gating and charge transfer phenomena were inferred from the field-effect measurements of the Dirac point position in single-layer graphene (SLG) transistors transfer curves. Results denote that λD is not the main factor governing the effective nanoscale screening environment. We observed that the longer λD was not the determining characteristic for sensitivity increment and limit of detection (LoD) as demonstrated by different types and ionic strengths of measuring buffers. In the DNA hybridization study, our findings show the role of the additional salts present in PBS, as compared to PB, in increasing graphene electron mobility, electrostatic shielding, intermolecular forces and DNA adsorption kinetics leading to an improved sensitivity.Roumani F and Azinheiro S and Carvalho J and Prado M and Garrido-maestu A
Food Control, 125 , 2021.
@article{F2021,
title = {Loop-mediated isothermal amplification combined with immunomagnetic separation and propidium monoazide for the specific detection of viable Listeria monocytogenes in milk products , with an internal amplification control},
author = {Roumani F and Azinheiro S and Carvalho J and Prado M and Garrido-maestu A},
url = {https://doi.org/10.1016/j.foodcont.2021.107975},
doi = {doi.org/10.1016/j.foodcont.2021.107975},
year = {2021},
date = {2021-01-15},
journal = {Food Control},
volume = {125},
abstract = {Nowadays, the most widely accepted rapid methods in food microbiology rely on nucleic acid amplification such as PCR/real-time PCR. A major claimed limitation of these methods is their incapacity to differentiate among viable and non-viable microorganisms. In the present study we report the development of a novel multiplex loop-mediated isothermal amplification method which, by combining immunomagnetic separation, to concentrate and purify the bacteria, along with propidium monoazide to block the amplification of DNA from non-viable microorganisms. The method allowed to specifically detect viable Listeria monocytogenes present in milk products. We designed an internal amplification control to rule out false negative results due to reaction inhibition, which is differentiated from L. monocytogenes by a simple melt curve analysis. Overall, the methodology provided results higher than 95% in terms of sensitivity, specificity and accuracy, as well as a Cohen's k of 0.97, reaching a limit of detection of 2.7 cfu/25 g. In samples inoculated with up to 106-107 cfu of dead microorganisms, the method demonstrated capable of effectively eliminating undesired amplification.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}Nowadays, the most widely accepted rapid methods in food microbiology rely on nucleic acid amplification such as PCR/real-time PCR. A major claimed limitation of these methods is their incapacity to differentiate among viable and non-viable microorganisms. In the present study we report the development of a novel multiplex loop-mediated isothermal amplification method which, by combining immunomagnetic separation, to concentrate and purify the bacteria, along with propidium monoazide to block the amplification of DNA from non-viable microorganisms. The method allowed to specifically detect viable Listeria monocytogenes present in milk products. We designed an internal amplification control to rule out false negative results due to reaction inhibition, which is differentiated from L. monocytogenes by a simple melt curve analysis. Overall, the methodology provided results higher than 95% in terms of sensitivity, specificity and accuracy, as well as a Cohen's k of 0.97, reaching a limit of detection of 2.7 cfu/25 g. In samples inoculated with up to 106-107 cfu of dead microorganisms, the method demonstrated capable of effectively eliminating undesired amplification.Jon Ashley, Anna-Lisa Schaap-Johansen, Mohsen Mohammadniaei, Maryam Naseri, Paolo Marcatili, Marta Prado, Yi Sun
Terminal deoxynucleotidyl transferase-mediated formation of protein binding polynucleotides Journal Article
Nucleic Acids Research, 2021.
@article{and Anna-Lisaand Mohsenand MaryamNaseri2021,
title = {Terminal deoxynucleotidyl transferase-mediated formation of protein binding polynucleotides},
author = {Jon Ashley, Anna-Lisa Schaap-Johansen, Mohsen Mohammadniaei, Maryam Naseri, Paolo Marcatili, Marta Prado, Yi Sun},
url = { https://doi.org/10.1093/nar/gkaa1263},
doi = {10.1093/nar/gkaa1263},
year = {2021},
date = {2021-01-04},
journal = {Nucleic Acids Research},
abstract = {Terminal deoxynucleotidyl transferase (TdT) enzyme plays an integral part in the V(D)J recombination, allowing for the huge diversity in expression of immunoglobulins and T-cell receptors within lymphocytes, through their unique ability to incorporate single nucleotides into oligonucleotides without the need of a template. The role played by TdT in lymphocytes precursors found in early vertebrates is not known. In this paper, we demonstrated a new screening method that utilises TdT to form libraries of variable sized (vsDNA) libraries of polynucleotides that displayed binding towards protein targets. The extent of binding and size distribution of each vsDNA library towards their respective protein target can be controlled through the alteration of different reaction conditions such as time of reaction, nucleotide ratio and initiator concentration raising the possibility for the rational design of aptamers prior to screening. The new approach, allows for the screening of aptamers based on size as well as sequence in a single round, which minimises PCR bias. We converted the protein bound sequences to dsDNA using rapid amplification of variable ends assays (RAVE) and sequenced them using next generation sequencing. The resultant aptamers demonstrated low nanomolar binding and high selectivity towards their respective targets.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}Terminal deoxynucleotidyl transferase (TdT) enzyme plays an integral part in the V(D)J recombination, allowing for the huge diversity in expression of immunoglobulins and T-cell receptors within lymphocytes, through their unique ability to incorporate single nucleotides into oligonucleotides without the need of a template. The role played by TdT in lymphocytes precursors found in early vertebrates is not known. In this paper, we demonstrated a new screening method that utilises TdT to form libraries of variable sized (vsDNA) libraries of polynucleotides that displayed binding towards protein targets. The extent of binding and size distribution of each vsDNA library towards their respective protein target can be controlled through the alteration of different reaction conditions such as time of reaction, nucleotide ratio and initiator concentration raising the possibility for the rational design of aptamers prior to screening. The new approach, allows for the screening of aptamers based on size as well as sequence in a single round, which minimises PCR bias. We converted the protein bound sequences to dsDNA using rapid amplification of variable ends assays (RAVE) and sequenced them using next generation sequencing. The resultant aptamers demonstrated low nanomolar binding and high selectivity towards their respective targets. -
2020
Akhilesh Kumar Gupta, Chih-Hsien Hsu, Agnes Purwidyantri, Briliant Adhi Prabowo, Kuo-Ping Chiu, Ching-Hsiang Chen, Ya-Chung Tian, Chao-SungLai
ZnO-Nanorod processed PC-SET as the light-harvesting model for plasmontronic fluorescence Sensor Journal Article
Sensors and Actuators B: Chemical, 307 , pp. 127597, 2020.
@article{Gupta2020,
title = {ZnO-Nanorod processed PC-SET as the light-harvesting model for plasmontronic fluorescence Sensor},
author = {Akhilesh Kumar Gupta, Chih-Hsien Hsu, Agnes Purwidyantri, Briliant Adhi Prabowo, Kuo-Ping Chiu, Ching-Hsiang Chen, Ya-Chung Tian, Chao-SungLai},
url = {https://doi.org/10.1016/j.snb.2019.127597},
doi = {10.1016/j.snb.2019.127597},
year = {2020},
date = {2020-12-19},
journal = {Sensors and Actuators B: Chemical},
volume = {307},
pages = {127597},
abstract = {This paper reports the combined plasmon coupled - surface energy transfer (PC-SET) and a distance-dependent model constructed by gold nanoparticles (GNPs) over zinc oxide nanorod (ZnO-NR) as a robust and tunable plasmontronic fluorescence regime for the detection of rhodamine 6G (R6G). Further, the deposition of metal created extraordinary contact through ZnO-NR utilizing a rapid thermal process (RTP) allowing the interaction of plasmon-coupled nature and surface energy transfer from the donor (R6G) to the acceptor (ZnO). The percentage of energy transfer efficiency continuously decreased with the increment of GNPs size, shown by 72.93, 67.52 and 47.86%, corresponding to the increase of the distance between the donor and acceptor of 63.03, 67.25, and 82.49 Å, respectively. In other words, the efficiency of PC-SET complied the 1/d4 distance dependence model between donor and acceptor molecules with the detection of long-distance ranges from 46.95 to 120 Å. These findings suggest that PC-SET process has a more realistic agreement with experimental outcomes and highly supports quenching efficiency impacts related to the size of GNPs, in which the smaller size of NPs causes’ greater effectiveness towards challenges in light harvest enhanced sensing system.
Keywords: ZnO-nanorods; PC-SET model; Light-harvesting; GNPs size effect; Schottky barrier height (SBH); Plasmontronic-fluorescence sensor},
keywords = {},
pubstate = {published},
tppubtype = {article}
}This paper reports the combined plasmon coupled - surface energy transfer (PC-SET) and a distance-dependent model constructed by gold nanoparticles (GNPs) over zinc oxide nanorod (ZnO-NR) as a robust and tunable plasmontronic fluorescence regime for the detection of rhodamine 6G (R6G). Further, the deposition of metal created extraordinary contact through ZnO-NR utilizing a rapid thermal process (RTP) allowing the interaction of plasmon-coupled nature and surface energy transfer from the donor (R6G) to the acceptor (ZnO). The percentage of energy transfer efficiency continuously decreased with the increment of GNPs size, shown by 72.93, 67.52 and 47.86%, corresponding to the increase of the distance between the donor and acceptor of 63.03, 67.25, and 82.49 Å, respectively. In other words, the efficiency of PC-SET complied the 1/d4 distance dependence model between donor and acceptor molecules with the detection of long-distance ranges from 46.95 to 120 Å. These findings suggest that PC-SET process has a more realistic agreement with experimental outcomes and highly supports quenching efficiency impacts related to the size of GNPs, in which the smaller size of NPs causes’ greater effectiveness towards challenges in light harvest enhanced sensing system.
Keywords: ZnO-nanorods; PC-SET model; Light-harvesting; GNPs size effect; Schottky barrier height (SBH); Plasmontronic-fluorescence sensorEspiña, Begoña, Prado, Marta, dos Santos, Marilia B., Salonen, Laura M., Queirós, Raquel B., Fernandes, Soraia P.S., Vial, Stephanie and Martins, Verónica C.
2 New techniques in environment monitoring". Climate Change and Marine and Freshwater Toxins Book Chapter
pp. 35-106, Berlin, Boston: De Gruyter, 2020.
@inbook{Espiña2020b,
title = {2 New techniques in environment monitoring". Climate Change and Marine and Freshwater Toxins},
author = {Espiña, Begoña, Prado, Marta, dos Santos, Marilia B., Salonen, Laura M., Queirós, Raquel B., Fernandes, Soraia P.S., Vial, Stephanie and Martins, Verónica C.},
url = {https://doi.org/10.1515/9783110625738-002},
doi = {10.1515/9783110625738-002},
year = {2020},
date = {2020-12-16},
pages = {35-106},
publisher = {Berlin, Boston: De Gruyter},
keywords = {},
pubstate = {published},
tppubtype = {inbook}
}A Garrido-Maestu and S Azinheiro and F Roumani and J Carvalho and M Prado
Front. Microbiol, 11 , pp. 1-8, 2020.
@article{Prado2020b,
title = {Application of Short Pre-enrichment, and Double Chemistry Real-Time PCR, Combining Fluorescent Probes and an Intercalating Dye, for Same-Day Detection and Confirmation of Salmonella spp. and Escherichia coli O157 in Ground Beef and Chicken Samples},
author = {A Garrido-Maestu and S Azinheiro and F Roumani and J Carvalho and M Prado},
url = {https://doi.org/10.3389/fmicb.2020.591041},
doi = {10.3389/fmicb.2020.591041},
year = {2020},
date = {2020-10-09},
journal = {Front. Microbiol},
volume = {11},
pages = {1-8},
abstract = {Molecular methods, particularly those based on real-time PCR (qPCR), have become a popular approach to detect pathogens in food samples. This technique may take advantage of hydrolysis fluorescent probes for increased specificity. Even though suitable, this approach loses the capacity of performing result confirmation by melt curve analysis. In the current study, we developed an alternative approach, combining fluorescent probes along with an intercalating dye (SYBR Green) in order to simultaneously detect, and confirm the result, of two foodborne pathogens (Salmonella spp. and Escherichia coli O157). This new approach named double chemistry qPCR was combined with a short pre-enrichment in order to obtain a multiplex “same-day” detection method for the selected pathogens. The evaluation of the novel method in spiked food samples (ground beef and chicken breast) obtained values of relative sensitivity, specificity, and accuracy higher than 95%, and Cohen’s kappa of 0.92, with a Limit of Detection95 below 5 cfu/25 g, demonstrating its reliability. In addition to this, the method was challenged by inoculating heat-stressed bacteria as well as dead ones. It was observed that it was also possible to detect stressed bacteria with an initial inoculation level below 10 cfu/25 g. Also, it was noticed that high initial concentration of either pathogen (higher than 104 cfu/25 g) was needed in order to generate false positive results due to the presence of dead bacteria, thus the method presents potential for its application in the specific detection of live microorganisms.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}Molecular methods, particularly those based on real-time PCR (qPCR), have become a popular approach to detect pathogens in food samples. This technique may take advantage of hydrolysis fluorescent probes for increased specificity. Even though suitable, this approach loses the capacity of performing result confirmation by melt curve analysis. In the current study, we developed an alternative approach, combining fluorescent probes along with an intercalating dye (SYBR Green) in order to simultaneously detect, and confirm the result, of two foodborne pathogens (Salmonella spp. and Escherichia coli O157). This new approach named double chemistry qPCR was combined with a short pre-enrichment in order to obtain a multiplex “same-day” detection method for the selected pathogens. The evaluation of the novel method in spiked food samples (ground beef and chicken breast) obtained values of relative sensitivity, specificity, and accuracy higher than 95%, and Cohen’s kappa of 0.92, with a Limit of Detection95 below 5 cfu/25 g, demonstrating its reliability. In addition to this, the method was challenged by inoculating heat-stressed bacteria as well as dead ones. It was observed that it was also possible to detect stressed bacteria with an initial inoculation level below 10 cfu/25 g. Also, it was noticed that high initial concentration of either pathogen (higher than 104 cfu/25 g) was needed in order to generate false positive results due to the presence of dead bacteria, thus the method presents potential for its application in the specific detection of live microorganisms.L Wu and A Teixeira and A Garrido-Maestu and L Muinelo-Romay and L Lima and LL Santos and M Prado and L Diéguez
Profiling DNA mutation patterns by SERS fingerprinting for supervised cancer classification Journal Article
Biosens. Bioelectron, 165 , 2020.
@article{Wu2020,
title = {Profiling DNA mutation patterns by SERS fingerprinting for supervised cancer classification},
author = {L Wu and A Teixeira and A Garrido-Maestu and L Muinelo-Romay and L Lima and LL Santos and M Prado and L Diéguez},
url = {https://doi.org/10.1016/j.bios.2020.112392},
doi = {10.1016/j.bios.2020.112392},
year = {2020},
date = {2020-10-01},
journal = {Biosens. Bioelectron},
volume = {165},
abstract = {Profiling DNA mutation patterns for cancer classification plays an essential role in precision and personalized medicine. Conventional PCR-based mutation assay is limited by the extensive labour on target amplification. We herein create an amplification-free surface enhanced Raman spectroscopy (SERS) biochip which enables direct and simultaneous identification of multiple point mutations in tumor cells. Without pre-amplifying the target sequences, the SERS assay reads out the presence of cellular mutations through the interpretation of Raman fingerprints. The SERS sensor is integrated into a microfluidic chip, achieving one-step multiplex analysis within 40 min. Importantly, by combining SERS spectra encoding technique with supervised learning algorithm, a panel of nucleotide mixtures can be well distinguished according to their mutation profiles. We initially demonstrate an excellent levels of classification in samples from colorectal cancer and melanoma cell lines. For final clinical validation, the system performance is verified by classifying cancer patient samples, which shows an accuracy above 90%. Due to the simplicity and rapidness, the SERS biosensor is expected to become a promising tool for clinical point-of-care diagnosis towards precision medicine.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}Profiling DNA mutation patterns for cancer classification plays an essential role in precision and personalized medicine. Conventional PCR-based mutation assay is limited by the extensive labour on target amplification. We herein create an amplification-free surface enhanced Raman spectroscopy (SERS) biochip which enables direct and simultaneous identification of multiple point mutations in tumor cells. Without pre-amplifying the target sequences, the SERS assay reads out the presence of cellular mutations through the interpretation of Raman fingerprints. The SERS sensor is integrated into a microfluidic chip, achieving one-step multiplex analysis within 40 min. Importantly, by combining SERS spectra encoding technique with supervised learning algorithm, a panel of nucleotide mixtures can be well distinguished according to their mutation profiles. We initially demonstrate an excellent levels of classification in samples from colorectal cancer and melanoma cell lines. For final clinical validation, the system performance is verified by classifying cancer patient samples, which shows an accuracy above 90%. Due to the simplicity and rapidness, the SERS biosensor is expected to become a promising tool for clinical point-of-care diagnosis towards precision medicine.Filipa M.C. Freitas, Miguel A. Cerqueira, Catarina Gonçalves, Sarah Azinheiro, Alejandro Garrido-Maestu, António A. Vicente, Lorenzo M. Pastrana, José A. Teixeira, Michele Michelin
Green synthesis of lignin nano- and micro-particles: Physicochemical characterization, bioactive properties and cytotoxicity assessment Journal Article
International Journal of Biological Macromolecules, 163 , pp. 1798-1809, 2020.
@article{Freitas2020,
title = {Green synthesis of lignin nano- and micro-particles: Physicochemical characterization, bioactive properties and cytotoxicity assessment},
author = {Filipa M.C. Freitas, Miguel A. Cerqueira, Catarina Gonçalves, Sarah Azinheiro, Alejandro Garrido-Maestu, António A. Vicente, Lorenzo M. Pastrana, José A. Teixeira, Michele Michelin},
url = {https://doi.org/10.1016/j.ijbiomac.2020.09.110.},
doi = {10.1016/j.ijbiomac.2020.09.110.},
year = {2020},
date = {2020-09-29},
journal = {International Journal of Biological Macromolecules},
volume = {163},
pages = {1798-1809},
abstract = {Lignin particles (LPs) have gained prominence due to their biodegradability and bioactive properties. LP production at nano and micro scale produced from organosolv lignin and the understanding of size's effect on their properties is unexplored. This work aimed to produce and characterize lignin nanoparticles and microparticles using a green synthesis process, based on ethanol-solubilized lignin and water. Spherical shape LPs, with a mean size of 75 nm and 215 nm and with a low polydispersity were produced, as confirmed by transmission electron microscopy and dynamic light scattering. LPs thermal stability improved over raw lignin, and the chemical structure of lignin was not affected by the production method. The antimicrobial tests proved that LPs presented a bacteriostatic effect on Escherichiacoli and Salmonella enterica. Regarding the antioxidant potential, LPs had a good antioxidant activity that increased with the reaction time and LPs concentration. LPs also presented an antioxidant effect against intracellular ROS, reducing the intracellular ROS levels significantly. Furthermore, the LPs showed a low cytotoxic effect in Caco-2 cell line. These results showed that LPs at different scales (nano and micro) present biological properties and are safe to be used in different high value industrial sectors, such as biomedical, pharmaceutical and food.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}Lignin particles (LPs) have gained prominence due to their biodegradability and bioactive properties. LP production at nano and micro scale produced from organosolv lignin and the understanding of size's effect on their properties is unexplored. This work aimed to produce and characterize lignin nanoparticles and microparticles using a green synthesis process, based on ethanol-solubilized lignin and water. Spherical shape LPs, with a mean size of 75 nm and 215 nm and with a low polydispersity were produced, as confirmed by transmission electron microscopy and dynamic light scattering. LPs thermal stability improved over raw lignin, and the chemical structure of lignin was not affected by the production method. The antimicrobial tests proved that LPs presented a bacteriostatic effect on Escherichiacoli and Salmonella enterica. Regarding the antioxidant potential, LPs had a good antioxidant activity that increased with the reaction time and LPs concentration. LPs also presented an antioxidant effect against intracellular ROS, reducing the intracellular ROS levels significantly. Furthermore, the LPs showed a low cytotoxic effect in Caco-2 cell line. These results showed that LPs at different scales (nano and micro) present biological properties and are safe to be used in different high value industrial sectors, such as biomedical, pharmaceutical and food.S Azinheiro and J Carvalho and M Prado and A Garrido-Maestu
Foods, 9 , 2020.
@article{Azinheiro2020,
title = {Application of Recombinase Polymerase Amplification with Lateral Flow for a Naked-Eye Detection of Listeria monocytogenes on Food Processing Surfaces},
author = {S Azinheiro and J Carvalho and M Prado and A Garrido-Maestu},
url = {https://doi.org/10.3390/foods9091249},
doi = {10.3390/foods9091249},
year = {2020},
date = {2020-09-07},
journal = {Foods},
volume = {9},
abstract = {The continuous contamination of foods with L. monocytogenes, highlights the need for additional controls in the food industry. The verification of food processing plants is key to avoid cross-contaminations, and to assure the safety of the food products. In this study, a new methodology for the detection of L. monocytogenes on food contact surfaces was developed and evaluated. It combines Recombinase Polymerase Amplification (RPA) with the lateral flow (LF) naked-eye detection. Different approaches for the recovery of the bacteria from the surface, the enrichment step and downstream analysis by RPA-LF were tested and optimized. The results were compared with a standard culture-based technique and qPCR analysis. Sampling procedure with sponges was more efficient for the recovery of the bacteria than a regular swab. A 24 h enrichment in ONE broth was needed for the most sensitive detection of the pathogen. By RPA-LF, it was possible to detect 1.1 pg/µL of pure L. monocytogenes DNA, and the complete methodology reached a LoD50 of 4.2 CFU/cm2 and LoD95 of 18.2 CFU/cm2. These results are comparable with the culture-based methodology and qPCR. The developed approach allows for a next-day detection without complex equipment and a naked-eye visualization of the results.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}The continuous contamination of foods with L. monocytogenes, highlights the need for additional controls in the food industry. The verification of food processing plants is key to avoid cross-contaminations, and to assure the safety of the food products. In this study, a new methodology for the detection of L. monocytogenes on food contact surfaces was developed and evaluated. It combines Recombinase Polymerase Amplification (RPA) with the lateral flow (LF) naked-eye detection. Different approaches for the recovery of the bacteria from the surface, the enrichment step and downstream analysis by RPA-LF were tested and optimized. The results were compared with a standard culture-based technique and qPCR analysis. Sampling procedure with sponges was more efficient for the recovery of the bacteria than a regular swab. A 24 h enrichment in ONE broth was needed for the most sensitive detection of the pathogen. By RPA-LF, it was possible to detect 1.1 pg/µL of pure L. monocytogenes DNA, and the complete methodology reached a LoD50 of 4.2 CFU/cm2 and LoD95 of 18.2 CFU/cm2. These results are comparable with the culture-based methodology and qPCR. The developed approach allows for a next-day detection without complex equipment and a naked-eye visualization of the results.S Azinheiro and J Carvalho and M Prado
Multiplex Detection of Salmonella spp., E. coli O157 and L. monocytogenes by qPCR Melt Curve Analysis in Spiked Infant Formula Journal Article
Microorganisms, 8 , 2020.
@article{Azinheiro2020c,
title = {Multiplex Detection of Salmonella spp., E. coli O157 and L. monocytogenes by qPCR Melt Curve Analysis in Spiked Infant Formula},
author = {S Azinheiro and J Carvalho and M Prado},
url = {https://doi.org/10.3390/microorganisms8091359},
doi = {10.3390/microorganisms8091359},
year = {2020},
date = {2020-09-04},
journal = {Microorganisms},
volume = {8},
abstract = {Food poisoning continue to be a threat in the food industry showing a need to improve the detection of the pathogen responsible for the hospitalization cases and death. DNA-based techniques represent a real advantage and allow the detection of several targets at the same time, reducing cost and time of analysis. The development of new methodology using SYBR Green qPCR for the detection of L. monocytogenes, Salmonella spp. and E. coli O157 simultaneously was developed and a non-competitive internal amplification control (NC-IAC) was implemented to detect reaction inhibition. The formulation and supplementation of the enrichment medium was also optimized to allow the growth of all pathogens. The limit of detection (LoD) 95% obtained was <1 CFU/25 g for E. coli O157, and 2 CFU/25 g for Salmonella spp. and L. monocytogenes and regarding the multiplex detection a LoD 95% of 1.7 CFU/25 g was observed. The specificity, relative sensitivity and accuracy of full methodology were 100% and the use of the NC-IAC allowed the reliability of the results without interfering with the sensitivity of the methodology. The described study proved to obtain results comparable to those of probe-based qPCR, and more economically than classical high resolution melting qPCR, being both important aspects for its implementation in the food industry.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}Food poisoning continue to be a threat in the food industry showing a need to improve the detection of the pathogen responsible for the hospitalization cases and death. DNA-based techniques represent a real advantage and allow the detection of several targets at the same time, reducing cost and time of analysis. The development of new methodology using SYBR Green qPCR for the detection of L. monocytogenes, Salmonella spp. and E. coli O157 simultaneously was developed and a non-competitive internal amplification control (NC-IAC) was implemented to detect reaction inhibition. The formulation and supplementation of the enrichment medium was also optimized to allow the growth of all pathogens. The limit of detection (LoD) 95% obtained was <1 CFU/25 g for E. coli O157, and 2 CFU/25 g for Salmonella spp. and L. monocytogenes and regarding the multiplex detection a LoD 95% of 1.7 CFU/25 g was observed. The specificity, relative sensitivity and accuracy of full methodology were 100% and the use of the NC-IAC allowed the reliability of the results without interfering with the sensitivity of the methodology. The described study proved to obtain results comparable to those of probe-based qPCR, and more economically than classical high resolution melting qPCR, being both important aspects for its implementation in the food industry.AJN Oliveira and J de Wild and K Oliveira and BA Valença and JP Teixeira and JRL Guerreiro and S Abalde-Cela and TS Lopes and RM Ribeiro and JM V Cunha and MA Curado and M Monteiro and A Violas and AG Silva and M Prado and PA Fernandes and B Vermang and PMP Salomé
Encapsulation of Nanostructures in a Dielectric Matrix Providing Optical Enhancement in Ultrathin Solar Cells Journal Article
Sol. RRL., 4 , 2020.
@article{Oliveira2020,
title = {Encapsulation of Nanostructures in a Dielectric Matrix Providing Optical Enhancement in Ultrathin Solar Cells},
author = {AJN Oliveira and J de Wild and K Oliveira and BA Valença and JP Teixeira and JRL Guerreiro and S Abalde-Cela and TS Lopes and RM Ribeiro and JM V Cunha and MA Curado and M Monteiro and A Violas and AG Silva and M Prado and PA Fernandes and B Vermang and PMP Salomé},
url = {https://doi.org/10.1002/solr.202000310},
doi = {10.1002/solr.202000310},
year = {2020},
date = {2020-08-29},
journal = {Sol. RRL.},
volume = {4},
abstract = {The incorporation of nanostructures in optoelectronic devices for enhancing their optical performance is widely studied. However, several problems related to the processing complexity and the low performance of the nanostructures have hindered such actions in real‐life devices. Herein, a novel way of introducing gold nanoparticles in a solar cell structure is proposed in which the nanostructures are encapsulated with a dielectric layer, shielding them from high temperatures and harsh growth processing conditions of the remaining device. Through optical simulations, an enhancement of the effective optical path length of approximately four times the nominal thickness of the absorber layer is verified with the new architecture. Furthermore, the proposed concept in a Cu(In,Ga)Se2 solar cell device is demonstrated, where the short‐circuit current density is increased by 17.4%. The novel structure presented in this work is achieved by combining a bottom‐up chemical approach of depositing the nanostructures with a top‐down photolithographic process, which allows for an electrical contact.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}The incorporation of nanostructures in optoelectronic devices for enhancing their optical performance is widely studied. However, several problems related to the processing complexity and the low performance of the nanostructures have hindered such actions in real‐life devices. Herein, a novel way of introducing gold nanoparticles in a solar cell structure is proposed in which the nanostructures are encapsulated with a dielectric layer, shielding them from high temperatures and harsh growth processing conditions of the remaining device. Through optical simulations, an enhancement of the effective optical path length of approximately four times the nominal thickness of the absorber layer is verified with the new architecture. Furthermore, the proposed concept in a Cu(In,Ga)Se2 solar cell device is demonstrated, where the short‐circuit current density is increased by 17.4%. The novel structure presented in this work is achieved by combining a bottom‐up chemical approach of depositing the nanostructures with a top‐down photolithographic process, which allows for an electrical contact.António J. N. Oliveira, Jessica de Wild, Kevin Oliveira, Beatriz A. Valença, Jennifer P. Teixeira, Joana R. L. Guerreiro, Sara Abalde-Cela, Tomás S. Lopes, Rodrigo M. Ribeiro, José M. V. Cunha, Marco A. Curado, Margarida Monteiro, André Violas, Ana Gomes Silva, Marta Prado, Paulo A. Fernandes, Bart Vermang, Pedro M. P. Salomé
Encapsulation of Nanostructures in a Dielectric Matrix Providing Optical Enhancement in Ultrathin Solar Cells Journal Article
RRL Solar, 4 (11), 2020.
@article{Oliveira2020b,
title = {Encapsulation of Nanostructures in a Dielectric Matrix Providing Optical Enhancement in Ultrathin Solar Cells},
author = {António J. N. Oliveira, Jessica de Wild, Kevin Oliveira, Beatriz A. Valença, Jennifer P. Teixeira, Joana R. L. Guerreiro, Sara Abalde-Cela, Tomás S. Lopes, Rodrigo M. Ribeiro, José M. V. Cunha, Marco A. Curado, Margarida Monteiro, André Violas, Ana Gomes Silva, Marta Prado, Paulo A. Fernandes, Bart Vermang, Pedro M. P. Salomé},
url = {https://doi.org/10.1002/solr.202000310},
doi = {10.1002/solr.202000310},
year = {2020},
date = {2020-08-29},
journal = {RRL Solar},
volume = {4},
number = {11},
abstract = {The incorporation of nanostructures in optoelectronic devices for enhancing their optical performance is widely studied. However, several problems related to the processing complexity and the low performance of the nanostructures have hindered such actions in real-life devices. Herein, a novel way of introducing gold nanoparticles in a solar cell structure is proposed in which the nanostructures are encapsulated with a dielectric layer, shielding them from high temperatures and harsh growth processing conditions of the remaining device. Through optical simulations, an enhancement of the effective optical path length of approximately four times the nominal thickness of the absorber layer is verified with the new architecture. Furthermore, the proposed concept in a Cu(In,Ga)Se2 solar cell device is demonstrated, where the short-circuit current density is increased by 17.4%. The novel structure presented in this work is achieved by combining a bottom-up chemical approach of depositing the nanostructures with a top-down photolithographic process, which allows for an electrical contact.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}The incorporation of nanostructures in optoelectronic devices for enhancing their optical performance is widely studied. However, several problems related to the processing complexity and the low performance of the nanostructures have hindered such actions in real-life devices. Herein, a novel way of introducing gold nanoparticles in a solar cell structure is proposed in which the nanostructures are encapsulated with a dielectric layer, shielding them from high temperatures and harsh growth processing conditions of the remaining device. Through optical simulations, an enhancement of the effective optical path length of approximately four times the nominal thickness of the absorber layer is verified with the new architecture. Furthermore, the proposed concept in a Cu(In,Ga)Se2 solar cell device is demonstrated, where the short-circuit current density is increased by 17.4%. The novel structure presented in this work is achieved by combining a bottom-up chemical approach of depositing the nanostructures with a top-down photolithographic process, which allows for an electrical contact.S Azinheiro and K Kant and MA Shahbazi and A Garrido-Maestu and M Prado and L Diéguez
A smart microfluidic platform for rapid multiplexed detection of foodborne pathogens Journal Article
Food Control, 114 , 2020.
@article{Azinheiro2020b,
title = {A smart microfluidic platform for rapid multiplexed detection of foodborne pathogens},
author = {S Azinheiro and K Kant and MA Shahbazi and A Garrido-Maestu and M Prado and L Diéguez},
url = {https://doi.org/10.1016/j.foodcont.2020.107242},
doi = {10.1016/j.foodcont.2020.107242},
year = {2020},
date = {2020-08-01},
journal = {Food Control},
volume = {114},
abstract = {Rapid and sensitive detection of foodborne pathogens in food industry is of high importance in day-to-day practice to ensure safe food. To address this issue, multiple foodborne pathogens are targeted for rapid identification based in DNA amplification. A 3D PDMS sponge was fabricated using salt crystals as scarifying mold and functionalized with a ligand, apolipoprotein-H (ApoH), to test bacterial capturing for both Gram positive (L. monocytogenes) and negative bacteria (Salmonella spp.), in a microfluidic device. Pure culture of both pathogens in a range of ~10–105 CFU/mL were tested and the application of the developed automated pre-concentration protocol in real samples was verified using spiked surface samples after swab sampling. Bacterial DNA was extracted directly from the sponge and used for Real Time quantitative Polymerase Chain Reaction (qPCR) detection. The sponges did not show any significant resistance to sample flow and could easily be incorporated in a microfluidic device. A capture efficiency above 70% was observed for both targeted (Gram positive and Gram negative) pathogens and a Limit of Detection (LoD) in the range of 103 and 104 CFU/mL was obtained for Salmonella spp. and L. monocytogenes, respectively. Using this approached, we are able to perform multiplexed (Gram positive and Gram negative) capturing and reduce the enrichment time compared to the gold standard plate culture (over 1-day) method. The use of a 3D sponge for direct capturing of multiplexed pathogen on microfluidic device, followed by qPCR detection is an efficient and versatile method to stratify the presence of bacteria. This approach and methodology has potential to be integrated in full automatized device and used as point of need (PoN) system for foodborne pathogen stratification in food packaging/production industries.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}Rapid and sensitive detection of foodborne pathogens in food industry is of high importance in day-to-day practice to ensure safe food. To address this issue, multiple foodborne pathogens are targeted for rapid identification based in DNA amplification. A 3D PDMS sponge was fabricated using salt crystals as scarifying mold and functionalized with a ligand, apolipoprotein-H (ApoH), to test bacterial capturing for both Gram positive (L. monocytogenes) and negative bacteria (Salmonella spp.), in a microfluidic device. Pure culture of both pathogens in a range of ~10–105 CFU/mL were tested and the application of the developed automated pre-concentration protocol in real samples was verified using spiked surface samples after swab sampling. Bacterial DNA was extracted directly from the sponge and used for Real Time quantitative Polymerase Chain Reaction (qPCR) detection. The sponges did not show any significant resistance to sample flow and could easily be incorporated in a microfluidic device. A capture efficiency above 70% was observed for both targeted (Gram positive and Gram negative) pathogens and a Limit of Detection (LoD) in the range of 103 and 104 CFU/mL was obtained for Salmonella spp. and L. monocytogenes, respectively. Using this approached, we are able to perform multiplexed (Gram positive and Gram negative) capturing and reduce the enrichment time compared to the gold standard plate culture (over 1-day) method. The use of a 3D sponge for direct capturing of multiplexed pathogen on microfluidic device, followed by qPCR detection is an efficient and versatile method to stratify the presence of bacteria. This approach and methodology has potential to be integrated in full automatized device and used as point of need (PoN) system for foodborne pathogen stratification in food packaging/production industries.Alejandro Garrido-Maestu, Sarah Azinheiro, Pablo Fuciños, Joana Carvalho, Marta Prado
Food Microbiology, 92 , pp. 103570, 2020.
@article{Garrido-Maestu2020c,
title = {Comparative study of multiplex real-time recombinase polymerase amplification and ISO 11290-1 methods for the detection of Listeria monocytogenes in dairy products},
author = {Alejandro Garrido-Maestu, Sarah Azinheiro, Pablo Fuciños, Joana Carvalho, Marta Prado},
url = {https://doi.org/10.1016/j.fm.2020.103570},
doi = {10.1016/j.fm.2020.103570},
year = {2020},
date = {2020-06-20},
journal = {Food Microbiology},
volume = {92},
pages = {103570},
abstract = {Dairy products have been implicated in foodborne infections caused by different bacterial pathogens. Among them, Listeria monocytogenes is of particular concern due to its ubiquity, resistance to sanitation processes and high mortality rates resulting from infection. These issues make the development of novel methods for the rapid detection of this bacterium of high interest. The evaluation of a novel multiplex real-time Recombinase Polymerase Amplification method including an internal amplification control is reported in the present work. The method performance was compared to that of the European reference method (ISO 11290-1) for the detection of the species in samples from 40 commercial products, including 14 UHT milk samples, 16 hard cheese samples, 6 infant dairy preparation samples and 4 fresh cheese samples. A limit of detection below 10 cfu/25 g or mL sample was achieved, and values higher than 90% were obtained for relative sensitivity, specificity, accuracy, positive and negative predictive values and the index (kappa) of concordance. Analysis was achieved within one working day, compared to the six days required using the ISO method. Moreover, slight modification of the ISO 11290-1 method to include secondary enrichment in half Fraser broth resulted in the confirmation of all positive samples.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}Dairy products have been implicated in foodborne infections caused by different bacterial pathogens. Among them, Listeria monocytogenes is of particular concern due to its ubiquity, resistance to sanitation processes and high mortality rates resulting from infection. These issues make the development of novel methods for the rapid detection of this bacterium of high interest. The evaluation of a novel multiplex real-time Recombinase Polymerase Amplification method including an internal amplification control is reported in the present work. The method performance was compared to that of the European reference method (ISO 11290-1) for the detection of the species in samples from 40 commercial products, including 14 UHT milk samples, 16 hard cheese samples, 6 infant dairy preparation samples and 4 fresh cheese samples. A limit of detection below 10 cfu/25 g or mL sample was achieved, and values higher than 90% were obtained for relative sensitivity, specificity, accuracy, positive and negative predictive values and the index (kappa) of concordance. Analysis was achieved within one working day, compared to the six days required using the ISO method. Moreover, slight modification of the ISO 11290-1 method to include secondary enrichment in half Fraser broth resulted in the confirmation of all positive samples.Lu, Yu-Jen and Purwidyantri, Agnes and Liu, Hui-Ling and Wang, Le-Wen and Shih, Cheng-Ye and Pijanowska, Dorota G. and Yang, Chia-Ming
Photoelectrochemical Detection of β-amyloid Peptides by a TiO2 Nanobrush Biosensor Journal Article
IEEE Sensors Journal, 20 (12), pp. 6248-6255, 2020.
@article{Lu2020,
title = {Photoelectrochemical Detection of β-amyloid Peptides by a TiO2 Nanobrush Biosensor},
author = {Lu, Yu-Jen and Purwidyantri, Agnes and Liu, Hui-Ling and Wang, Le-Wen and Shih, Cheng-Ye and Pijanowska, Dorota G. and Yang, Chia-Ming},
doi = {10.1109/JSEN.2020.2976561},
year = {2020},
date = {2020-06-15},
journal = {IEEE Sensors Journal},
volume = {20},
number = {12},
pages = {6248-6255},
abstract = {A simple, facile and cost-effective nanostructuring technique is proposed to construct a photoelectrochemical (PEC) sensor via the solution-based hydrothermal growth of a TiO 2 nanobrush (TiO 2 NB). It is demonstrated that the control of the TiO 2 seed solution ratio, process temperature and duration significantly contributed to the final morphological characteristics of the rutile TiO 2 NB, as confirmed by the X-ray diffraction (XRD) analysis. By carrying out a simple and inexpensive fabrication involving no additional surface modification materials, the proposed TiO 2 NB enabled the enhancement of the surface area by up to 162% in comparison with its actual geometric area. The photoactivity achieved by applying UV-range light was drastically improved, and photocurrents could potentially be utilized to enhance the redox activity on the interface. The system was used to detect β-amyloid (Aβ) 1-28 peptides, one of the most crucial biomarkers of patients with Alzheimer's disease (AD), and demonstrated an excellent biocompatibility caused by the straightforward self-assembled monolayers (SAMs) modified on its surface. For the detection of a wide range of Aβ 1-28 peptides, the constructed TiO 2 NB photoelectrochemical (PEC) sensor exhibited a great sensitivity of 114.8 μ A/(ng . mL -1 ) and limit of detection (LoD) of 26.3 ng.mL -1 , facilitating a simple, label-free, rapid, sensitive and noninvasive method to overcome the limitations of conventional techniques used for AD diagnosis.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}A simple, facile and cost-effective nanostructuring technique is proposed to construct a photoelectrochemical (PEC) sensor via the solution-based hydrothermal growth of a TiO 2 nanobrush (TiO 2 NB). It is demonstrated that the control of the TiO 2 seed solution ratio, process temperature and duration significantly contributed to the final morphological characteristics of the rutile TiO 2 NB, as confirmed by the X-ray diffraction (XRD) analysis. By carrying out a simple and inexpensive fabrication involving no additional surface modification materials, the proposed TiO 2 NB enabled the enhancement of the surface area by up to 162% in comparison with its actual geometric area. The photoactivity achieved by applying UV-range light was drastically improved, and photocurrents could potentially be utilized to enhance the redox activity on the interface. The system was used to detect β-amyloid (Aβ) 1-28 peptides, one of the most crucial biomarkers of patients with Alzheimer's disease (AD), and demonstrated an excellent biocompatibility caused by the straightforward self-assembled monolayers (SAMs) modified on its surface. For the detection of a wide range of Aβ 1-28 peptides, the constructed TiO 2 NB photoelectrochemical (PEC) sensor exhibited a great sensitivity of 114.8 μ A/(ng . mL -1 ) and limit of detection (LoD) of 26.3 ng.mL -1 , facilitating a simple, label-free, rapid, sensitive and noninvasive method to overcome the limitations of conventional techniques used for AD diagnosis.Garrido-Maestu A and Azinheiro S and Carvalho J and Espiña B and Prado M
Food Sci. Technol., (57), pp. 4143–4151, 2020.
@article{Prado2020b,
title = {Evaluation and implementation of commercial antibodies for improved nanoparticle-based immunomagnetic separation and real-time PCR for faster detection of Listeria monocytogenes},
author = {Garrido-Maestu A and Azinheiro S and Carvalho J and Espiña B and Prado M},
url = {https://doi.org/10.1007/s13197-020-04450-1},
doi = {10.1007/s13197-020-04450-1},
year = {2020},
date = {2020-04-24},
journal = {Food Sci. Technol.},
number = {57},
pages = {4143–4151},
abstract = {L. monocytogenes continues to be a major health issue in Europe, as well as worldwide. Faster methods, not only for detection, but also for sample preparation are of great interest particularly for this slow-growing pathogen. Immunomagnetic separation has been previously reported to be an effective way to concentrate bacteria, and remove inhibitors. In the present study, different commercial antibodies were evaluated to select the most appropriate one, in order to develop a highly specific method. Additionally, magnetic nanoparticles, instead of microparticles, were selected due to their reported advantages (higher surface-volume ration and faster kinetics). Finally, the separation protocol, with a calculated capture efficiency of 95%, was combined with real-time PCR for highly sensitive detection of the concentrated bacteria. The optimized IMS-qPCR allowed to reduce hands-on time in the sample treatment, without affecting the overall performance of the method as a very low limit of detection was still obtained (9.7 CFU/ 25 g) with values for sensitivity, specificity, accuracy, positive and negative predictive values of 100%, resulting in a kappa index of concordance of 1.00. These results were obtained in spiked food samples of different types (chicken, fish, milk, hard and fresh cheese), further demonstrating the applicability of the optimized methodology presented.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}L. monocytogenes continues to be a major health issue in Europe, as well as worldwide. Faster methods, not only for detection, but also for sample preparation are of great interest particularly for this slow-growing pathogen. Immunomagnetic separation has been previously reported to be an effective way to concentrate bacteria, and remove inhibitors. In the present study, different commercial antibodies were evaluated to select the most appropriate one, in order to develop a highly specific method. Additionally, magnetic nanoparticles, instead of microparticles, were selected due to their reported advantages (higher surface-volume ration and faster kinetics). Finally, the separation protocol, with a calculated capture efficiency of 95%, was combined with real-time PCR for highly sensitive detection of the concentrated bacteria. The optimized IMS-qPCR allowed to reduce hands-on time in the sample treatment, without affecting the overall performance of the method as a very low limit of detection was still obtained (9.7 CFU/ 25 g) with values for sensitivity, specificity, accuracy, positive and negative predictive values of 100%, resulting in a kappa index of concordance of 1.00. These results were obtained in spiked food samples of different types (chicken, fish, milk, hard and fresh cheese), further demonstrating the applicability of the optimized methodology presented.A Teixeira and JL Paris and F Roumani and L Diéguez and M Prado and B Espiña and S Abalde-Cela and A Garrido-Maestu and L Rodriguez-Lorenzo
Multifuntional gold nanoparticles for the SERS detection of pathogens combined with a LAMP-in-microdroplets approach Journal Article
Materials (Basel), 13 , 2020.
@article{Teixeira2020,
title = {Multifuntional gold nanoparticles for the SERS detection of pathogens combined with a LAMP-in-microdroplets approach},
author = {A Teixeira and JL Paris and F Roumani and L Diéguez and M Prado and B Espiña and S Abalde-Cela and A Garrido-Maestu and L Rodriguez-Lorenzo},
url = {https://doi.org/10.3390/ma13081934},
doi = {10.3390/ma13081934},
year = {2020},
date = {2020-04-20},
journal = {Materials (Basel)},
volume = {13},
abstract = {We developed a droplet-based optofluidic system for the detection of foodborne pathogens. Specifically, the loop-mediated isothermal amplification (LAMP) technique was combined with surface-enhanced Raman scattering (SERS), which offers an excellent method for DNA ultradetection. However, the direct SERS detection of DNA compromises the simplicity of data interpretation due to the variability of its SERS fingerprints. Therefore, we designed an indirect SERS detection method using multifunctional gold nanoparticles (AuNPs) based on the formation of pyrophosphate generated during the DNA amplification by LAMP. Towards this goal, we prepared multifunctional AuNPs involving three components with key roles: (1) thiolated poly(ethylene glycol) as stabilizing agent, (2) 1-naphthalenethiol as Raman reporter, and (3) glutathione as a bioinspired chelating agent of magnesium (II) ions. Thus, the variation in the SERS signal of 1-naphthalenethiol was controlled by the aggregation of AuNPs triggered by the complexation of pyrophosphate and glutathione with free magnesium ions. Using this strategy, we detected Listeria monocytogenes, not only in buffer, but also in a food matrix (i.e., ultra-high temperaturemilk) enabled by the massive production of hotspots as a result of the self-assemblies that enhanced the SERS signal. This allowed the development of a microdroplet-LAMP-SERS platform with isothermal amplification and real-time identification capabilities.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}We developed a droplet-based optofluidic system for the detection of foodborne pathogens. Specifically, the loop-mediated isothermal amplification (LAMP) technique was combined with surface-enhanced Raman scattering (SERS), which offers an excellent method for DNA ultradetection. However, the direct SERS detection of DNA compromises the simplicity of data interpretation due to the variability of its SERS fingerprints. Therefore, we designed an indirect SERS detection method using multifunctional gold nanoparticles (AuNPs) based on the formation of pyrophosphate generated during the DNA amplification by LAMP. Towards this goal, we prepared multifunctional AuNPs involving three components with key roles: (1) thiolated poly(ethylene glycol) as stabilizing agent, (2) 1-naphthalenethiol as Raman reporter, and (3) glutathione as a bioinspired chelating agent of magnesium (II) ions. Thus, the variation in the SERS signal of 1-naphthalenethiol was controlled by the aggregation of AuNPs triggered by the complexation of pyrophosphate and glutathione with free magnesium ions. Using this strategy, we detected Listeria monocytogenes, not only in buffer, but also in a food matrix (i.e., ultra-high temperaturemilk) enabled by the massive production of hotspots as a result of the self-assemblies that enhanced the SERS signal. This allowed the development of a microdroplet-LAMP-SERS platform with isothermal amplification and real-time identification capabilities.Agnes Purwidyantri, Myrtha Karina, Chih-Hsien Hsu, Yoice Srikandace, Briliant Adhi Prabowo, and Chao-Sung Lai
Facile Bacterial Cellulose Nanofibrillation for the Development of a Plasmonic Paper Sensor Journal Article
ACS Biomaterials Science & Engineering , 6 (5), pp. 3122-3131, 2020.
@article{Purwidyantri2020,
title = {Facile Bacterial Cellulose Nanofibrillation for the Development of a Plasmonic Paper Sensor},
author = {Agnes Purwidyantri, Myrtha Karina, Chih-Hsien Hsu, Yoice Srikandace, Briliant Adhi Prabowo, and Chao-Sung Lai},
doi = {10.1021/acsbiomaterials.9b01890},
year = {2020},
date = {2020-04-16},
journal = {ACS Biomaterials Science & Engineering },
volume = {6},
number = {5},
pages = {3122-3131},
abstract = {In this present work, a plasmonic sensor is developed through an extremely cheap cellulose-based source, widely known as a food product, nata de coco (NDC). Capturing its interesting features, such as innate surface roughness from naturally grown cellulose during its fermentation period, the engineering and modulation of NDC fibril size and properties were attempted through a high-pressure homogenization (HPH) treatment to obtain highly dense nanofibrils. After the transformation into a thin, paper-sheet form through a casting process, the homogenized bacterial cellulose (HBC) resulting from HPH was compared with the normally agitated bacterial cellulose (BC) pulp and decorated with silver nanoparticles (AgNPs) to produce plasmonic papers, for further application as surface-enhanced Raman scattering (SERS) substrate. As demonstrated in the measurement of Rhodamine 6G (R6G) molecule, the plasmonic HBC paper sheet provided more prominent SERS signals than the plasmonic BC due to its high surface roughness and improved textural properties from the nanofibrillation process favoring better adsorption of AgNPs and effective SERS hotspots generation. The plasmonic HBC obtained a 2 order higher estimated SERS enhancement factor over the plasmonic BC with a limit of detection of approximately 92 fM. Results denote that the proposed approach provides a new, green-synthesis route toward the exploration of biodegradable sources integrated into an inexpensive and simple nanostructuring process for the production of flexible, paper-based, plasmonic sensors.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}In this present work, a plasmonic sensor is developed through an extremely cheap cellulose-based source, widely known as a food product, nata de coco (NDC). Capturing its interesting features, such as innate surface roughness from naturally grown cellulose during its fermentation period, the engineering and modulation of NDC fibril size and properties were attempted through a high-pressure homogenization (HPH) treatment to obtain highly dense nanofibrils. After the transformation into a thin, paper-sheet form through a casting process, the homogenized bacterial cellulose (HBC) resulting from HPH was compared with the normally agitated bacterial cellulose (BC) pulp and decorated with silver nanoparticles (AgNPs) to produce plasmonic papers, for further application as surface-enhanced Raman scattering (SERS) substrate. As demonstrated in the measurement of Rhodamine 6G (R6G) molecule, the plasmonic HBC paper sheet provided more prominent SERS signals than the plasmonic BC due to its high surface roughness and improved textural properties from the nanofibrillation process favoring better adsorption of AgNPs and effective SERS hotspots generation. The plasmonic HBC obtained a 2 order higher estimated SERS enhancement factor over the plasmonic BC with a limit of detection of approximately 92 fM. Results denote that the proposed approach provides a new, green-synthesis route toward the exploration of biodegradable sources integrated into an inexpensive and simple nanostructuring process for the production of flexible, paper-based, plasmonic sensors.A Garrido-Maestu and S Azinheiro and J Carvalho and P Fuciños and M Prado
Optimized sample treatment, combined with real-time PCR, for same-day detection of E. coli O157 in ground beef and leafy greens Journal Article
Food Control, 108 , 2020.
@article{Garrido-Maestu2020,
title = {Optimized sample treatment, combined with real-time PCR, for same-day detection of E. coli O157 in ground beef and leafy greens},
author = {A Garrido-Maestu and S Azinheiro and J Carvalho and P Fuciños and M Prado},
url = {https://doi.org/10.1016/j.foodcont.2019.106790},
doi = {10.1016/j.foodcont.2019.106790},
year = {2020},
date = {2020-02-01},
journal = {Food Control},
volume = {108},
abstract = {E. coli O157 is the most commonly identified serotype among Shiga like-toxin E. coli infections. Current reference methodologies to detect these bacteria are reliable but take several days to provide a definitive result, as they require selective enrichment, molecular detection of the bacteria, followed by plate confirmation. Even alternative methods need extensive sample enrichment in order to increase the amount of bacteria to detectable numbers, resulting in “next-day detection” methodologies, as it is not possible to reach the final result within the same day of the start of the analysis. In the current study, the sample treatment was optimized in such a way that after a short enrichment (3 h) followed by an enzymatic treatment, DNA extraction and amplification by real-time PCR, it was possible to detect down to 3.9 and 3.3 CFU/25 g of ground beef and leafy greens respectively, achieving a combined LOD95 of 3.6 CFU/25 g. This sample treatment optimization allowed us to develop a real “same-day detection” method, being this a major advantage over other previously published methodologies. Additionally, the analysis of spiked samples provided excellent results (90%) for the relative sensitivity, specificity and accuracy; along with the positive and negative predictive values. Finally, the Cohen's k also obtained a value of 1, indicating that the results obtained with the novel method were in “almost complete concordance” with the expected values. It is worth to highlight that the performance parameters fulfill the requirements of the NordVal regulation for the validation of alternative methods.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}E. coli O157 is the most commonly identified serotype among Shiga like-toxin E. coli infections. Current reference methodologies to detect these bacteria are reliable but take several days to provide a definitive result, as they require selective enrichment, molecular detection of the bacteria, followed by plate confirmation. Even alternative methods need extensive sample enrichment in order to increase the amount of bacteria to detectable numbers, resulting in “next-day detection” methodologies, as it is not possible to reach the final result within the same day of the start of the analysis. In the current study, the sample treatment was optimized in such a way that after a short enrichment (3 h) followed by an enzymatic treatment, DNA extraction and amplification by real-time PCR, it was possible to detect down to 3.9 and 3.3 CFU/25 g of ground beef and leafy greens respectively, achieving a combined LOD95 of 3.6 CFU/25 g. This sample treatment optimization allowed us to develop a real “same-day detection” method, being this a major advantage over other previously published methodologies. Additionally, the analysis of spiked samples provided excellent results (90%) for the relative sensitivity, specificity and accuracy; along with the positive and negative predictive values. Finally, the Cohen's k also obtained a value of 1, indicating that the results obtained with the novel method were in “almost complete concordance” with the expected values. It is worth to highlight that the performance parameters fulfill the requirements of the NordVal regulation for the validation of alternative methods. -
2019
L Rodriguez-Lorenzo and A Garrido-Maestu and AK Bhunia and B Espiña and M Prado and L Diéguez and S Abalde-Cela
Gold Nanostars for the Detection of Foodborne Pathogens via Surface-Enhanced Raman Scattering Combined with Microfluidics Journal Article
ACS Appl. Nano Mater., 2 (10), pp. 6081–6086, 2019.
@article{Abalde-Cela2019,
title = {Gold Nanostars for the Detection of Foodborne Pathogens via Surface-Enhanced Raman Scattering Combined with Microfluidics},
author = {L Rodriguez-Lorenzo and A Garrido-Maestu and AK Bhunia and B Espiña and M Prado and L Diéguez and S Abalde-Cela},
url = {https://doi.org/10.1021/acsanm.9b01223},
doi = {10.1021/acsanm.9b01223},
year = {2019},
date = {2019-09-20},
journal = {ACS Appl. Nano Mater.},
volume = {2},
number = {10},
pages = {6081–6086},
abstract = {Herein, we demonstrated the potential of surface-enhanced Raman scattering (SERS) spectroscopy combined with microfluidics for the detection and discrimination of foodborne pathogens. SERS-tagged gold nanostars were functionalized with a monoclonal antibody specific for Listeria monocytogenes. In the presence of L. monocytogenes, a SERS signal corresponding to the SERS tag paired to the antibody was detected in real time and in continuous flow, enabling the discrimination of L. monocytogenes and Listeria innocua in just 100 s. To the best of our knowledge, this is the first time that SERS tags have been used for the in-flow detection of living organisms.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}Herein, we demonstrated the potential of surface-enhanced Raman scattering (SERS) spectroscopy combined with microfluidics for the detection and discrimination of foodborne pathogens. SERS-tagged gold nanostars were functionalized with a monoclonal antibody specific for Listeria monocytogenes. In the presence of L. monocytogenes, a SERS signal corresponding to the SERS tag paired to the antibody was detected in real time and in continuous flow, enabling the discrimination of L. monocytogenes and Listeria innocua in just 100 s. To the best of our knowledge, this is the first time that SERS tags have been used for the in-flow detection of living organisms.Carla Carvalho Joana Carvalho Marta Prado Alejandro Garrido-Maestu Sarah Azinheiro
Food Science, 84 (7), pp. 1881-1887, 2019.
@article{Garrido-Maestu2019b,
title = {Combination of Immunomagnetic separation and real-time Recombinase Polymerase Amplification (IMS-qRPA) for specific detection of Listeria monocytogenes in smoked salmon samples},
author = {Carla Carvalho Joana Carvalho Marta Prado Alejandro Garrido-Maestu Sarah Azinheiro},
url = {https://doi.org/10.1111/1750-3841.14662},
doi = {10.1111/1750-3841.14662},
year = {2019},
date = {2019-07-02},
journal = {Food Science},
volume = {84},
number = {7},
pages = {1881-1887},
abstract = {Nowadays, Listeria monocytogenes continues to be a major health issue. Therefore, improvements in the speed and reliability of its detection are still needed. In the present study, the combination of real‐time Recombinase Polymerase Amplification (qRPA) with immunomagnetic separation (IMS) is described. The proposed methodology was tested against a real‐time PCR method, and was successfully applied to 50 smoked salmon samples spiked at levels ranging from 2 to 9.3 × 102 cfu/25 g. L. monocytogenes was detected after a 24 hr pre‐enrichment, which represents a great improvement over other previously published RPA methods. Additionally, the evaluation of the method reported a Limit of dDetection 50 (LoD50) of 6.3 cfu/25 g, along with relative sensitivity, specificity and accuracy values higher than 90%. Finally, the index of kappa concordance was calculated to be 0.93 which is interpreted as “almost complete concordance” between the reference and alternative method. Overall, the described methodology proved to be faster, specific, and as sensitive as other methods based on RPA or real‐time PCR.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}Nowadays, Listeria monocytogenes continues to be a major health issue. Therefore, improvements in the speed and reliability of its detection are still needed. In the present study, the combination of real‐time Recombinase Polymerase Amplification (qRPA) with immunomagnetic separation (IMS) is described. The proposed methodology was tested against a real‐time PCR method, and was successfully applied to 50 smoked salmon samples spiked at levels ranging from 2 to 9.3 × 102 cfu/25 g. L. monocytogenes was detected after a 24 hr pre‐enrichment, which represents a great improvement over other previously published RPA methods. Additionally, the evaluation of the method reported a Limit of dDetection 50 (LoD50) of 6.3 cfu/25 g, along with relative sensitivity, specificity and accuracy values higher than 90%. Finally, the index of kappa concordance was calculated to be 0.93 which is interpreted as “almost complete concordance” between the reference and alternative method. Overall, the described methodology proved to be faster, specific, and as sensitive as other methods based on RPA or real‐time PCR.Carla Carvalho Joana Carvalho Marta Prado Alejandro Garrido-Maestu Sarah Azinheiro
Food Science, 84 (7), pp. 1881-1887, 2019.
@article{Garrido-Maestu2019bc,
title = {Combination of Immunomagnetic separation and real-time Recombinase Polymerase Amplification (IMS-qRPA) for specific detection of Listeria monocytogenes in smoked salmon samples},
author = {Carla Carvalho Joana Carvalho Marta Prado Alejandro Garrido-Maestu Sarah Azinheiro},
url = {https://doi.org/10.1111/1750-3841.14662},
doi = {10.1111/1750-3841.14662},
year = {2019},
date = {2019-07-02},
journal = {Food Science},
volume = {84},
number = {7},
pages = {1881-1887},
abstract = {Nowadays, Listeria monocytogenes continues to be a major health issue. Therefore, improvements in the speed and reliability of its detection are still needed. In the present study, the combination of real‐time Recombinase Polymerase Amplification (qRPA) with immunomagnetic separation (IMS) is described. The proposed methodology was tested against a real‐time PCR method, and was successfully applied to 50 smoked salmon samples spiked at levels ranging from 2 to 9.3 × 102 cfu/25 g. L. monocytogenes was detected after a 24 hr pre‐enrichment, which represents a great improvement over other previously published RPA methods. Additionally, the evaluation of the method reported a Limit of dDetection 50 (LoD50) of 6.3 cfu/25 g, along with relative sensitivity, specificity and accuracy values higher than 90%. Finally, the index of kappa concordance was calculated to be 0.93 which is interpreted as “almost complete concordance” between the reference and alternative method. Overall, the described methodology proved to be faster, specific, and as sensitive as other methods based on RPA or real‐time PCR.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}Nowadays, Listeria monocytogenes continues to be a major health issue. Therefore, improvements in the speed and reliability of its detection are still needed. In the present study, the combination of real‐time Recombinase Polymerase Amplification (qRPA) with immunomagnetic separation (IMS) is described. The proposed methodology was tested against a real‐time PCR method, and was successfully applied to 50 smoked salmon samples spiked at levels ranging from 2 to 9.3 × 102 cfu/25 g. L. monocytogenes was detected after a 24 hr pre‐enrichment, which represents a great improvement over other previously published RPA methods. Additionally, the evaluation of the method reported a Limit of dDetection 50 (LoD50) of 6.3 cfu/25 g, along with relative sensitivity, specificity and accuracy values higher than 90%. Finally, the index of kappa concordance was calculated to be 0.93 which is interpreted as “almost complete concordance” between the reference and alternative method. Overall, the described methodology proved to be faster, specific, and as sensitive as other methods based on RPA or real‐time PCR.SarahAzinheiro CarlaCarvalho JoanaCarvalho MartaPrado Alejandro Garrido-Maestu PabloFuciños
Specific detection of viable Salmonella Enteritidis by phage amplification combined with qPCR (PAA-qPCR) in spiked chicken meat samples Journal Article
Food Control, 99 , pp. 79-83, 2019.
@article{Garrido-Maestu2019b,
title = {Specific detection of viable Salmonella Enteritidis by phage amplification combined with qPCR (PAA-qPCR) in spiked chicken meat samples},
author = {SarahAzinheiro CarlaCarvalho JoanaCarvalho MartaPrado Alejandro Garrido-Maestu PabloFuciños},
url = {https://doi.org/10.1016/j.foodcont.2018.12.038},
doi = {10.1016/j.foodcont.2018.12.038},
year = {2019},
date = {2019-05-01},
journal = {Food Control},
volume = {99},
pages = {79-83},
abstract = {Serovar Enteritidis represents 45.7% of all Salmonella reported human cases identified in Europe. Additionally, “minced meat and meat preparations from poultry” have a high level of non-compliance, regarding Salmonella regulation.In the current study, a novel method based on the amplification of the Salmonella bacteriophage vB_SenS_PVP-SE2, coupled with real-time PCR (qPCR), was developed and evaluated, for the rapid detection of viable Salmonella Enteritidis in chicken samples. The results obtained indicated that the qPCR method could detect down to 0.22 fg/μL of pure virus DNA and a concentration of viral particles of 103 pfu/mL. After a short bacterial recovery step, the addition of bacteriophages to spiked chicken samples indicated that 8 cfu/25 g could be detected within 10 h, including the time for DNA extraction and qPCR analysis. Additionally, the evaluation of the performance parameters: relative sensitivity, specificity, accuracy, positive and negative predictive values, and index kappa of concordance, obtained values higher than 92%, and the acceptability limit values were within the limits. All these results demonstrate that the proposed methodology is a powerful tool for the rapid detection of viable Salmonella Enteritidis.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}Serovar Enteritidis represents 45.7% of all Salmonella reported human cases identified in Europe. Additionally, “minced meat and meat preparations from poultry” have a high level of non-compliance, regarding Salmonella regulation.In the current study, a novel method based on the amplification of the Salmonella bacteriophage vB_SenS_PVP-SE2, coupled with real-time PCR (qPCR), was developed and evaluated, for the rapid detection of viable Salmonella Enteritidis in chicken samples. The results obtained indicated that the qPCR method could detect down to 0.22 fg/μL of pure virus DNA and a concentration of viral particles of 103 pfu/mL. After a short bacterial recovery step, the addition of bacteriophages to spiked chicken samples indicated that 8 cfu/25 g could be detected within 10 h, including the time for DNA extraction and qPCR analysis. Additionally, the evaluation of the performance parameters: relative sensitivity, specificity, accuracy, positive and negative predictive values, and index kappa of concordance, obtained values higher than 92%, and the acceptability limit values were within the limits. All these results demonstrate that the proposed methodology is a powerful tool for the rapid detection of viable Salmonella Enteritidis.
SarahAzinheiro CarlaCarvalho JoanaCarvalho MartaPrado Alejandro Garrido-Maestu PabloFuciños
Specific detection of viable Salmonella Enteritidis by phage amplification combined with qPCR (PAA-qPCR) in spiked chicken meat samples Journal Article
Food Control, 99 , pp. 79-83, 2019.
@article{Garrido-Maestu2019d,
title = {Specific detection of viable Salmonella Enteritidis by phage amplification combined with qPCR (PAA-qPCR) in spiked chicken meat samples},
author = {SarahAzinheiro CarlaCarvalho JoanaCarvalho MartaPrado Alejandro Garrido-Maestu PabloFuciños},
url = {https://doi.org/10.1016/j.foodcont.2018.12.038},
doi = {10.1016/j.foodcont.2018.12.038},
year = {2019},
date = {2019-05-01},
journal = {Food Control},
volume = {99},
pages = {79-83},
abstract = {Serovar Enteritidis represents 45.7% of all Salmonella reported human cases identified in Europe. Additionally, “minced meat and meat preparations from poultry” have a high level of non-compliance, regarding Salmonella regulation.In the current study, a novel method based on the amplification of the Salmonella bacteriophage vB_SenS_PVP-SE2, coupled with real-time PCR (qPCR), was developed and evaluated, for the rapid detection of viable Salmonella Enteritidis in chicken samples. The results obtained indicated that the qPCR method could detect down to 0.22 fg/μL of pure virus DNA and a concentration of viral particles of 103 pfu/mL. After a short bacterial recovery step, the addition of bacteriophages to spiked chicken samples indicated that 8 cfu/25 g could be detected within 10 h, including the time for DNA extraction and qPCR analysis. Additionally, the evaluation of the performance parameters: relative sensitivity, specificity, accuracy, positive and negative predictive values, and index kappa of concordance, obtained values higher than 92%, and the acceptability limit values were within the limits. All these results demonstrate that the proposed methodology is a powerful tool for the rapid detection of viable Salmonella Enteritidis.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}Serovar Enteritidis represents 45.7% of all Salmonella reported human cases identified in Europe. Additionally, “minced meat and meat preparations from poultry” have a high level of non-compliance, regarding Salmonella regulation.In the current study, a novel method based on the amplification of the Salmonella bacteriophage vB_SenS_PVP-SE2, coupled with real-time PCR (qPCR), was developed and evaluated, for the rapid detection of viable Salmonella Enteritidis in chicken samples. The results obtained indicated that the qPCR method could detect down to 0.22 fg/μL of pure virus DNA and a concentration of viral particles of 103 pfu/mL. After a short bacterial recovery step, the addition of bacteriophages to spiked chicken samples indicated that 8 cfu/25 g could be detected within 10 h, including the time for DNA extraction and qPCR analysis. Additionally, the evaluation of the performance parameters: relative sensitivity, specificity, accuracy, positive and negative predictive values, and index kappa of concordance, obtained values higher than 92%, and the acceptability limit values were within the limits. All these results demonstrate that the proposed methodology is a powerful tool for the rapid detection of viable Salmonella Enteritidis.
Joana Guerreiro Sandra Carvalho Sara Abalde-Cela Marta Prado Lorena Diéguez R L Lei Wu Alejandro Garrido-Maestu
Amplification-free SERS analysis of DNA mutation in cancer cells with single-base sensitivity Journal Article
Nanoscale, 11 (16), pp. 7781-7789, 2019.
@article{Wu2019,
title = {Amplification-free SERS analysis of DNA mutation in cancer cells with single-base sensitivity},
author = {Joana Guerreiro Sandra Carvalho Sara Abalde-Cela Marta Prado Lorena Diéguez R L Lei Wu Alejandro Garrido-Maestu},
url = {https://doi.org/10.1039/C9NR00501C},
doi = {10.1039/C9NR00501C},
year = {2019},
date = {2019-04-01},
journal = {Nanoscale},
volume = {11},
number = {16},
pages = {7781-7789},
abstract = {Accurate and sensitive identification of DNA mutations in tumor cells is critical to the diagnosis, prognosis and personalized therapy of cancer. Conventional polymerase chain reaction (PCR)-based methods are limited by the complicated amplification process. Herein, an amplification-free surface enhanced Raman spectroscopy (SERS) approach which directly detects point mutations in cancer cells has been proposed. A highly sensitive and uniform SERS substrate was fabricated using gold@silver core–shell nanorods, achieving an enhancement factor of 1.85 × 106. By combining the SERS-active nanosubstrate with molecular beacon probes, the limit of detection reached as low as 50 fM. To enable parallel analysis and automated operation, the SERS sensor was integrated into a microfluidic chip. This novel chip-based assay was able to differentiate between mutated and wild-type KRAS genes among a variety of other nucleic acids from cancer cells in 40 min. Owing to the simple operation and fast analysis, the SERS-based DNA assay chip could potentially provide insights into clinical cancer theranostics in an easy and inexpensive manner at the point of care.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}Accurate and sensitive identification of DNA mutations in tumor cells is critical to the diagnosis, prognosis and personalized therapy of cancer. Conventional polymerase chain reaction (PCR)-based methods are limited by the complicated amplification process. Herein, an amplification-free surface enhanced Raman spectroscopy (SERS) approach which directly detects point mutations in cancer cells has been proposed. A highly sensitive and uniform SERS substrate was fabricated using gold@silver core–shell nanorods, achieving an enhancement factor of 1.85 × 106. By combining the SERS-active nanosubstrate with molecular beacon probes, the limit of detection reached as low as 50 fM. To enable parallel analysis and automated operation, the SERS sensor was integrated into a microfluidic chip. This novel chip-based assay was able to differentiate between mutated and wild-type KRAS genes among a variety of other nucleic acids from cancer cells in 40 min. Owing to the simple operation and fast analysis, the SERS-based DNA assay chip could potentially provide insights into clinical cancer theranostics in an easy and inexpensive manner at the point of care.Joana Guerreiro Sandra Carvalho Sara Abalde-Cela Marta Prado Lorena Diéguez R L Lei Wu Alejandro Garrido-Maestu
Amplification-free SERS analysis of DNA mutation in cancer cells with single-base sensitivity Journal Article
Nanoscale, 11 (16), pp. 7781-7789, 2019.
@article{Wu2019b,
title = {Amplification-free SERS analysis of DNA mutation in cancer cells with single-base sensitivity},
author = {Joana Guerreiro Sandra Carvalho Sara Abalde-Cela Marta Prado Lorena Diéguez R L Lei Wu Alejandro Garrido-Maestu},
url = {https://doi.org/10.1039/C9NR00501C},
doi = {10.1039/C9NR00501C},
year = {2019},
date = {2019-04-01},
journal = {Nanoscale},
volume = {11},
number = {16},
pages = {7781-7789},
abstract = {Accurate and sensitive identification of DNA mutations in tumor cells is critical to the diagnosis, prognosis and personalized therapy of cancer. Conventional polymerase chain reaction (PCR)-based methods are limited by the complicated amplification process. Herein, an amplification-free surface enhanced Raman spectroscopy (SERS) approach which directly detects point mutations in cancer cells has been proposed. A highly sensitive and uniform SERS substrate was fabricated using gold@silver core–shell nanorods, achieving an enhancement factor of 1.85 × 106. By combining the SERS-active nanosubstrate with molecular beacon probes, the limit of detection reached as low as 50 fM. To enable parallel analysis and automated operation, the SERS sensor was integrated into a microfluidic chip. This novel chip-based assay was able to differentiate between mutated and wild-type KRAS genes among a variety of other nucleic acids from cancer cells in 40 min. Owing to the simple operation and fast analysis, the SERS-based DNA assay chip could potentially provide insights into clinical cancer theranostics in an easy and inexpensive manner at the point of care.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}Accurate and sensitive identification of DNA mutations in tumor cells is critical to the diagnosis, prognosis and personalized therapy of cancer. Conventional polymerase chain reaction (PCR)-based methods are limited by the complicated amplification process. Herein, an amplification-free surface enhanced Raman spectroscopy (SERS) approach which directly detects point mutations in cancer cells has been proposed. A highly sensitive and uniform SERS substrate was fabricated using gold@silver core–shell nanorods, achieving an enhancement factor of 1.85 × 106. By combining the SERS-active nanosubstrate with molecular beacon probes, the limit of detection reached as low as 50 fM. To enable parallel analysis and automated operation, the SERS sensor was integrated into a microfluidic chip. This novel chip-based assay was able to differentiate between mutated and wild-type KRAS genes among a variety of other nucleic acids from cancer cells in 40 min. Owing to the simple operation and fast analysis, the SERS-based DNA assay chip could potentially provide insights into clinical cancer theranostics in an easy and inexpensive manner at the point of care.Rui Campos, Jérôme Borme, Joana Rafaela Guerreiro, George Machado Jr., Maria Fátima Cerqueira, Dmitri Y. Petrovykh, and Pedro Alpuim
Attomolar Label-Free Detection of DNA Hybridization with Electrolyte-Gated Graphene Field-Effect Transistors Journal Article
ACS Sensors, 4 (2), pp. 286-293, 2019.
@article{Campos2019bb,
title = {Attomolar Label-Free Detection of DNA Hybridization with Electrolyte-Gated Graphene Field-Effect Transistors},
author = {Rui Campos, Jérôme Borme, Joana Rafaela Guerreiro, George Machado Jr., Maria Fátima Cerqueira, Dmitri Y. Petrovykh, and Pedro Alpuim},
url = {https://doi.org/10.1021/acssensors.8b00344},
doi = {10.1021/acssensors.8b00344},
year = {2019},
date = {2019-01-23},
journal = {ACS Sensors},
volume = {4},
number = {2},
pages = {286-293},
abstract = {In this work, we develop a field-effect transistor with a two-dimensional channel made of a single graphene layer to achieve label-free detection of DNA hybridization down to attomolar concentration, while being able to discriminate a single nucleotide polymorphism (SNP). The SNP-level target specificity is achieved by immobilization of probe DNA on the graphene surface through a pyrene-derivative heterobifunctional linker. Biorecognition events result in a positive gate voltage shift of the graphene charge neutrality point. The graphene transistor biosensor displays a sensitivity of 24 mV/dec with a detection limit of 25 aM: the lowest target DNA concentration for which the sensor can discriminate between a perfect-match target sequence and SNP-containing one.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}In this work, we develop a field-effect transistor with a two-dimensional channel made of a single graphene layer to achieve label-free detection of DNA hybridization down to attomolar concentration, while being able to discriminate a single nucleotide polymorphism (SNP). The SNP-level target specificity is achieved by immobilization of probe DNA on the graphene surface through a pyrene-derivative heterobifunctional linker. Biorecognition events result in a positive gate voltage shift of the graphene charge neutrality point. The graphene transistor biosensor displays a sensitivity of 24 mV/dec with a detection limit of 25 aM: the lowest target DNA concentration for which the sensor can discriminate between a perfect-match target sequence and SNP-containing one. -
2018
Marta Prado Rodríguez Alejandro Garrido-Maestu David Tomás Fornés
The Use of Multiplex Real-Time PCR for the Simultaneous Detection of Foodborne Bacterial Pathogens Book Chapter
Methods in Molecular Biology, 1918 , pp. 35-45, Humana Press, New York, NY, 2018.
@inbook{Garrido-Maestu2018b,
title = {The Use of Multiplex Real-Time PCR for the Simultaneous Detection of Foodborne Bacterial Pathogens},
author = {Marta Prado Rodríguez Alejandro Garrido-Maestu David Tomás Fornés},
url = {https://doi.org/10.1007/978-1-4939-9000-9_3},
doi = {10.1007/978-1-4939-9000-9_3},
year = {2018},
date = {2018-12-23},
booktitle = {Methods in Molecular Biology},
volume = {1918},
pages = {35-45},
publisher = {Humana Press, New York, NY},
abstract = {Foodborne pathogens continue to be a major health issue worldwide. Culture-dependent methodologies are still considered the gold-standard to perform pathogen detection and quantification. These methods present several drawbacks, such as being time-consuming and labor-intensive. The implementation of real-time PCR has allowed to overcome these limitations and even reduce costs associated with the analyses, due to the possibility of simultaneously and accurately detecting several pathogens in one single assay, with results comparable to those obtained by classical approaches. In this chapter a protocol for the simultaneous detection of two of the most important foodborne pathogens, Salmonella spp. and Listeria monocytogenes, is described.},
keywords = {},
pubstate = {published},
tppubtype = {inbook}
}Foodborne pathogens continue to be a major health issue worldwide. Culture-dependent methodologies are still considered the gold-standard to perform pathogen detection and quantification. These methods present several drawbacks, such as being time-consuming and labor-intensive. The implementation of real-time PCR has allowed to overcome these limitations and even reduce costs associated with the analyses, due to the possibility of simultaneously and accurately detecting several pathogens in one single assay, with results comparable to those obtained by classical approaches. In this chapter a protocol for the simultaneous detection of two of the most important foodborne pathogens, Salmonella spp. and Listeria monocytogenes, is described.Marta Prado Rodríguez Alejandro Garrido-Maestu David Tomás Fornés
The Use of Multiplex Real-Time PCR for the Simultaneous Detection of Foodborne Bacterial Pathogens Book Chapter
Methods in Molecular Biology, 1918 , pp. 35-45, Humana Press, New York, NY, 2018.
@inbook{Garrido-Maestu2018bc,
title = {The Use of Multiplex Real-Time PCR for the Simultaneous Detection of Foodborne Bacterial Pathogens},
author = {Marta Prado Rodríguez Alejandro Garrido-Maestu David Tomás Fornés},
url = {https://doi.org/10.1007/978-1-4939-9000-9_3},
doi = {10.1007/978-1-4939-9000-9_3},
year = {2018},
date = {2018-12-23},
booktitle = {Methods in Molecular Biology},
volume = {1918},
pages = {35-45},
publisher = {Humana Press, New York, NY},
abstract = {Foodborne pathogens continue to be a major health issue worldwide. Culture-dependent methodologies are still considered the gold-standard to perform pathogen detection and quantification. These methods present several drawbacks, such as being time-consuming and labor-intensive. The implementation of real-time PCR has allowed to overcome these limitations and even reduce costs associated with the analyses, due to the possibility of simultaneously and accurately detecting several pathogens in one single assay, with results comparable to those obtained by classical approaches. In this chapter a protocol for the simultaneous detection of two of the most important foodborne pathogens, Salmonella spp. and Listeria monocytogenes, is described.},
keywords = {},
pubstate = {published},
tppubtype = {inbook}
}Foodborne pathogens continue to be a major health issue worldwide. Culture-dependent methodologies are still considered the gold-standard to perform pathogen detection and quantification. These methods present several drawbacks, such as being time-consuming and labor-intensive. The implementation of real-time PCR has allowed to overcome these limitations and even reduce costs associated with the analyses, due to the possibility of simultaneously and accurately detecting several pathogens in one single assay, with results comparable to those obtained by classical approaches. In this chapter a protocol for the simultaneous detection of two of the most important foodborne pathogens, Salmonella spp. and Listeria monocytogenes, is described.SarahAzinheiro AlejandroGarrido-Maestu JorgeBarros-Velázquez MartaPrado JoanaCarvalho RenatoNegrinho
Elsevier, 21 (21), pp. 424-431, 2018.
@article{JoanaCarvalho2018b,
title = {Data on minute DNA quantification on microvolumetric solutions: comparison of mathematical models and effect of some compounds on the DNA quantification accuracy},
author = {SarahAzinheiro AlejandroGarrido-Maestu JorgeBarros-Velázquez MartaPrado JoanaCarvalho RenatoNegrinho},
url = {https://www.sciencedirect.com/science/article/pii/S2352340918311764},
doi = {https://doi.org/10.1016/j.dib.2018.09.098},
year = {2018},
date = {2018-10-02},
journal = {Elsevier},
volume = {21},
number = {21},
pages = {424-431},
abstract = {This article contains data related to the research article entitled “Novel approach for accurate minute DNA quantification on microvolumetric solutions” (Carvalho et al., 2018). The combination of PicoGreen® with a microvolume fluorospectrometer is a popular DNA quantification method due to its high sensitivity and minimal consumption of sample, being commonly used to evaluate the performance of microfluidic devices designed for DNA purification. In this study, the authors present data related with the effect of DNA fragmentation level. The present data article includes the data used on the precision evaluation, in terms of repeatability, of the mathematical models developed to obtain the standards curve for salmon sperm DNA (low molecular weight). In addition, results related with the effect of some compounds on the DNA quantification accuracy using λDNA are presented.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}This article contains data related to the research article entitled “Novel approach for accurate minute DNA quantification on microvolumetric solutions” (Carvalho et al., 2018). The combination of PicoGreen® with a microvolume fluorospectrometer is a popular DNA quantification method due to its high sensitivity and minimal consumption of sample, being commonly used to evaluate the performance of microfluidic devices designed for DNA purification. In this study, the authors present data related with the effect of DNA fragmentation level. The present data article includes the data used on the precision evaluation, in terms of repeatability, of the mathematical models developed to obtain the standards curve for salmon sperm DNA (low molecular weight). In addition, results related with the effect of some compounds on the DNA quantification accuracy using λDNA are presented.SarahAzinheiro AlejandroGarrido-Maestu JorgeBarros-Velázquez MartaPrado JoanaCarvalho RenatoNegrinho
Elsevier, 21 (21), pp. 424-431, 2018.
@article{JoanaCarvalho2018bc,
title = {Data on minute DNA quantification on microvolumetric solutions: comparison of mathematical models and effect of some compounds on the DNA quantification accuracy},
author = {SarahAzinheiro AlejandroGarrido-Maestu JorgeBarros-Velázquez MartaPrado JoanaCarvalho RenatoNegrinho},
url = {https://www.sciencedirect.com/science/article/pii/S2352340918311764},
doi = {https://doi.org/10.1016/j.dib.2018.09.098},
year = {2018},
date = {2018-10-02},
journal = {Elsevier},
volume = {21},
number = {21},
pages = {424-431},
abstract = {This article contains data related to the research article entitled “Novel approach for accurate minute DNA quantification on microvolumetric solutions” (Carvalho et al., 2018). The combination of PicoGreen® with a microvolume fluorospectrometer is a popular DNA quantification method due to its high sensitivity and minimal consumption of sample, being commonly used to evaluate the performance of microfluidic devices designed for DNA purification. In this study, the authors present data related with the effect of DNA fragmentation level. The present data article includes the data used on the precision evaluation, in terms of repeatability, of the mathematical models developed to obtain the standards curve for salmon sperm DNA (low molecular weight). In addition, results related with the effect of some compounds on the DNA quantification accuracy using λDNA are presented.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}This article contains data related to the research article entitled “Novel approach for accurate minute DNA quantification on microvolumetric solutions” (Carvalho et al., 2018). The combination of PicoGreen® with a microvolume fluorospectrometer is a popular DNA quantification method due to its high sensitivity and minimal consumption of sample, being commonly used to evaluate the performance of microfluidic devices designed for DNA purification. In this study, the authors present data related with the effect of DNA fragmentation level. The present data article includes the data used on the precision evaluation, in terms of repeatability, of the mathematical models developed to obtain the standards curve for salmon sperm DNA (low molecular weight). In addition, results related with the effect of some compounds on the DNA quantification accuracy using λDNA are presented.JoãoGaspar SarahAzinheiro LorenaDiéguez AlejandroGarrido-Maestu ManuelVázquez JorgeBarros-Velázquez SusanaCardoso MartaPradoa JoanaCarvalho GemaPuertas
Highly efficient DNA extraction and purification from olive oil on a washable and reusable miniaturized device Journal Article
Analytica Chimica Acta, 1020 , pp. 30-40, 2018.
@article{JoanaCarvalho2018b,
title = {Highly efficient DNA extraction and purification from olive oil on a washable and reusable miniaturized device},
author = {JoãoGaspar SarahAzinheiro LorenaDiéguez AlejandroGarrido-Maestu ManuelVázquez JorgeBarros-Velázquez SusanaCardoso MartaPradoa JoanaCarvalho GemaPuertas},
url = {https://doi.org/10.1016/j.aca.2018.02.079},
doi = {https://doi.org/10.1016/j.aca.2018.02.079},
year = {2018},
date = {2018-08-22},
journal = {Analytica Chimica Acta},
volume = {1020},
pages = {30-40},
abstract = {Sample preparation from complex matrixes with minute DNA content could highly benefit from the miniaturization of solid phase extraction (SPE) based devices due to an increased surface area-to-volume ratio. However, the adaptation of “bench-top” based protocols for DNA purification to miniaturized devices is not as straightforward as it might seem, and several issues need to be considered. A careful evaluation of DNA extraction and purification protocols needs to be performed, taking into account the complexity of such samples, and in order to facilitate the integration with the subsequent step, normally DNA amplification. In this work a washable and reusable miniaturized device for DNA purification based on microscale solid phase extraction (μSPE), containing a commercial disposable silica membrane as the solid phase for DNA capture, was developed. The DNA purification protocol was firstly optimized by testing a set of different conditions, including buffer composition in all three steps of analysis and incubation during the elution step, with the objective of increasing the DNA yield and facilitating the integration in a miniaturized setting. This protocol was then tested with olive oil samples, including a pretreatment step also developed and optimized in this work. DNA analysis of olive oil samples is of high interest for the detection of fraudulent adulteration with oil from other seeds and for cultivar identification. The results were compared with the commercial NucleoSpin® Food kit regarding efficiency and purity of the DNA extract, by estimating the DNA yield and evaluating the absorbance ratios A260/280 and A260/230. The miniaturized DNA purification device showed better performance than the commercial kit tested, making this method a very promising sample preparation approach for olive oil and other samples with minute DNA content.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}Sample preparation from complex matrixes with minute DNA content could highly benefit from the miniaturization of solid phase extraction (SPE) based devices due to an increased surface area-to-volume ratio. However, the adaptation of “bench-top” based protocols for DNA purification to miniaturized devices is not as straightforward as it might seem, and several issues need to be considered. A careful evaluation of DNA extraction and purification protocols needs to be performed, taking into account the complexity of such samples, and in order to facilitate the integration with the subsequent step, normally DNA amplification. In this work a washable and reusable miniaturized device for DNA purification based on microscale solid phase extraction (μSPE), containing a commercial disposable silica membrane as the solid phase for DNA capture, was developed. The DNA purification protocol was firstly optimized by testing a set of different conditions, including buffer composition in all three steps of analysis and incubation during the elution step, with the objective of increasing the DNA yield and facilitating the integration in a miniaturized setting. This protocol was then tested with olive oil samples, including a pretreatment step also developed and optimized in this work. DNA analysis of olive oil samples is of high interest for the detection of fraudulent adulteration with oil from other seeds and for cultivar identification. The results were compared with the commercial NucleoSpin® Food kit regarding efficiency and purity of the DNA extract, by estimating the DNA yield and evaluating the absorbance ratios A260/280 and A260/230. The miniaturized DNA purification device showed better performance than the commercial kit tested, making this method a very promising sample preparation approach for olive oil and other samples with minute DNA content.JoãoGaspar SarahAzinheiro LorenaDiéguez AlejandroGarrido-Maestu ManuelVázquez JorgeBarros-Velázquez SusanaCardoso MartaPradoa JoanaCarvalho GemaPuertas
Highly efficient DNA extraction and purification from olive oil on a washable and reusable miniaturized device Journal Article
Analytica Chimica Acta, 1020 , pp. 30-40, 2018.
@article{JoanaCarvalho2018d,
title = {Highly efficient DNA extraction and purification from olive oil on a washable and reusable miniaturized device},
author = {JoãoGaspar SarahAzinheiro LorenaDiéguez AlejandroGarrido-Maestu ManuelVázquez JorgeBarros-Velázquez SusanaCardoso MartaPradoa JoanaCarvalho GemaPuertas},
url = {https://doi.org/10.1016/j.aca.2018.02.079},
doi = {https://doi.org/10.1016/j.aca.2018.02.079},
year = {2018},
date = {2018-08-22},
journal = {Analytica Chimica Acta},
volume = {1020},
pages = {30-40},
abstract = {Sample preparation from complex matrixes with minute DNA content could highly benefit from the miniaturization of solid phase extraction (SPE) based devices due to an increased surface area-to-volume ratio. However, the adaptation of “bench-top” based protocols for DNA purification to miniaturized devices is not as straightforward as it might seem, and several issues need to be considered. A careful evaluation of DNA extraction and purification protocols needs to be performed, taking into account the complexity of such samples, and in order to facilitate the integration with the subsequent step, normally DNA amplification. In this work a washable and reusable miniaturized device for DNA purification based on microscale solid phase extraction (μSPE), containing a commercial disposable silica membrane as the solid phase for DNA capture, was developed. The DNA purification protocol was firstly optimized by testing a set of different conditions, including buffer composition in all three steps of analysis and incubation during the elution step, with the objective of increasing the DNA yield and facilitating the integration in a miniaturized setting. This protocol was then tested with olive oil samples, including a pretreatment step also developed and optimized in this work. DNA analysis of olive oil samples is of high interest for the detection of fraudulent adulteration with oil from other seeds and for cultivar identification. The results were compared with the commercial NucleoSpin® Food kit regarding efficiency and purity of the DNA extract, by estimating the DNA yield and evaluating the absorbance ratios A260/280 and A260/230. The miniaturized DNA purification device showed better performance than the commercial kit tested, making this method a very promising sample preparation approach for olive oil and other samples with minute DNA content.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}Sample preparation from complex matrixes with minute DNA content could highly benefit from the miniaturization of solid phase extraction (SPE) based devices due to an increased surface area-to-volume ratio. However, the adaptation of “bench-top” based protocols for DNA purification to miniaturized devices is not as straightforward as it might seem, and several issues need to be considered. A careful evaluation of DNA extraction and purification protocols needs to be performed, taking into account the complexity of such samples, and in order to facilitate the integration with the subsequent step, normally DNA amplification. In this work a washable and reusable miniaturized device for DNA purification based on microscale solid phase extraction (μSPE), containing a commercial disposable silica membrane as the solid phase for DNA capture, was developed. The DNA purification protocol was firstly optimized by testing a set of different conditions, including buffer composition in all three steps of analysis and incubation during the elution step, with the objective of increasing the DNA yield and facilitating the integration in a miniaturized setting. This protocol was then tested with olive oil samples, including a pretreatment step also developed and optimized in this work. DNA analysis of olive oil samples is of high interest for the detection of fraudulent adulteration with oil from other seeds and for cultivar identification. The results were compared with the commercial NucleoSpin® Food kit regarding efficiency and purity of the DNA extract, by estimating the DNA yield and evaluating the absorbance ratios A260/280 and A260/230. The miniaturized DNA purification device showed better performance than the commercial kit tested, making this method a very promising sample preparation approach for olive oil and other samples with minute DNA content.Sarah Azinheiro Pablo Fuciños Joana Carvalho Marta Prado Alejandro Garrido-Maestu
Food Chemistry, Volume 246 , pp. Pages 156–163, 2018.
@article{Garrido-Maestu2017b,
title = {Highly sensitive detection of gluten-containing cereals in food samples by real-time Loop-mediated isothermal AMPlification (qLAMP) and real-time polymerase chain reaction (qPCR)},
author = {Sarah Azinheiro Pablo Fuciños Joana Carvalho Marta Prado Alejandro Garrido-Maestu},
url = {https://doi.org/10.1016/j.foodchem.2017.11.005},
year = {2018},
date = {2018-04-25},
journal = {Food Chemistry},
volume = {Volume 246},
pages = {Pages 156–163},
abstract = {The treatment of gluten-related disorders is based on a lifelong, and strict, gluten-free diet. Thus, reliable and sensitive methods are required to detect the presence of gluten contamination. Traditional techniques rely on the detection of these proteins based on specific antibodies, but recent approaches go for an indirect route detecting the DNA that indicates the presence of cereals with gluten content. In the current study two different DNA amplification techniques, real-time PCR (qPCR) and real-time Loop-mediated isothermal AMPlification (qLAMP), were evaluated for their capability to detect and quantify gluten. Different detection strategies, based on these DNA amplification techniques, were tested. Even though good specificity results were obtained with the different approaches, overall qPCR proved more sensitive than qLAMP. This is the first study reporting a qLAMP based-method for the detection of gluten-containing cereals, along with its evaluation in comparison with qPCR.Keywords: Real-time Loop-mediated isothermal AMPlification (qLAMP); Real-time PCR (qPCR); α2-gliadin; Gluten; Gluten-free food},
keywords = {},
pubstate = {published},
tppubtype = {article}
}The treatment of gluten-related disorders is based on a lifelong, and strict, gluten-free diet. Thus, reliable and sensitive methods are required to detect the presence of gluten contamination. Traditional techniques rely on the detection of these proteins based on specific antibodies, but recent approaches go for an indirect route detecting the DNA that indicates the presence of cereals with gluten content. In the current study two different DNA amplification techniques, real-time PCR (qPCR) and real-time Loop-mediated isothermal AMPlification (qLAMP), were evaluated for their capability to detect and quantify gluten. Different detection strategies, based on these DNA amplification techniques, were tested. Even though good specificity results were obtained with the different approaches, overall qPCR proved more sensitive than qLAMP. This is the first study reporting a qLAMP based-method for the detection of gluten-containing cereals, along with its evaluation in comparison with qPCR.Keywords: Real-time Loop-mediated isothermal AMPlification (qLAMP); Real-time PCR (qPCR); α2-gliadin; Gluten; Gluten-free food
Sarah Azinheiro Pablo Fuciños Joana Carvalho Marta Prado Alejandro Garrido-Maestu
Food Chemistry, Volume 246 , pp. Pages 156–163, 2018.
@article{Garrido-Maestu2017bd,
title = {Highly sensitive detection of gluten-containing cereals in food samples by real-time Loop-mediated isothermal AMPlification (qLAMP) and real-time polymerase chain reaction (qPCR)},
author = {Sarah Azinheiro Pablo Fuciños Joana Carvalho Marta Prado Alejandro Garrido-Maestu},
url = {https://doi.org/10.1016/j.foodchem.2017.11.005},
year = {2018},
date = {2018-04-25},
journal = {Food Chemistry},
volume = {Volume 246},
pages = {Pages 156–163},
abstract = {The treatment of gluten-related disorders is based on a lifelong, and strict, gluten-free diet. Thus, reliable and sensitive methods are required to detect the presence of gluten contamination. Traditional techniques rely on the detection of these proteins based on specific antibodies, but recent approaches go for an indirect route detecting the DNA that indicates the presence of cereals with gluten content. In the current study two different DNA amplification techniques, real-time PCR (qPCR) and real-time Loop-mediated isothermal AMPlification (qLAMP), were evaluated for their capability to detect and quantify gluten. Different detection strategies, based on these DNA amplification techniques, were tested. Even though good specificity results were obtained with the different approaches, overall qPCR proved more sensitive than qLAMP. This is the first study reporting a qLAMP based-method for the detection of gluten-containing cereals, along with its evaluation in comparison with qPCR.Keywords: Real-time Loop-mediated isothermal AMPlification (qLAMP); Real-time PCR (qPCR); α2-gliadin; Gluten; Gluten-free food},
keywords = {},
pubstate = {published},
tppubtype = {article}
}The treatment of gluten-related disorders is based on a lifelong, and strict, gluten-free diet. Thus, reliable and sensitive methods are required to detect the presence of gluten contamination. Traditional techniques rely on the detection of these proteins based on specific antibodies, but recent approaches go for an indirect route detecting the DNA that indicates the presence of cereals with gluten content. In the current study two different DNA amplification techniques, real-time PCR (qPCR) and real-time Loop-mediated isothermal AMPlification (qLAMP), were evaluated for their capability to detect and quantify gluten. Different detection strategies, based on these DNA amplification techniques, were tested. Even though good specificity results were obtained with the different approaches, overall qPCR proved more sensitive than qLAMP. This is the first study reporting a qLAMP based-method for the detection of gluten-containing cereals, along with its evaluation in comparison with qPCR.Keywords: Real-time Loop-mediated isothermal AMPlification (qLAMP); Real-time PCR (qPCR); α2-gliadin; Gluten; Gluten-free food
Joana Carvalho Pablo Fuciños Marta Prado Alejandro Garrido-Maestu Sarah Azinheiro
Food Control, Volume 86 , pp. Pages 27–34, 2018.
@article{Garrido-Maestu2017bb,
title = {Development and evaluation of loop-mediated isothermal amplification, and Recombinase Polymerase Amplification methodologies, for the detection of Listeria monocytogenes in ready-to-eat food samples},
author = {Joana Carvalho Pablo Fuciños Marta Prado Alejandro Garrido-Maestu Sarah Azinheiro},
url = {https://doi.org/10.1016/j.foodcont.2017.11.006},
year = {2018},
date = {2018-04-01},
journal = {Food Control},
volume = {Volume 86},
pages = {Pages 27–34},
abstract = {Listeriosis continues to be a major health issue. This is demonstrated by the fact that, even though efforts have been made, its incidence does not decrease. Furthermore, in Europe, over 2014 a 30% increase was reported respect to 2013. In the present study two isothermal DNA amplification methods, one based on Loop-mediated isothermal AMPlification (qLAMP), and the other on Recombinase Polymerase Amplification (RPA), were developed and extensively evaluated. Both techniques demonstrated their reliability to detect Listeria monocytogenes in different types of foods. The method included a two-step enrichment, which additionally reduces the chances of detecting dead bacteria. Over the evaluation with pure bacterial DNA, the qLAMP and RPA methods resulted 10 to 100 times less sensitive than qPCR (with two different detection chemistries), but when tested in real food samples the results showed very good concordance with those obtained by qPCR and by selective agar plating (index kappa of concordance between 0.90 and 0.95). Additionally, a very low limit of detection (below 10 CFU/25 g) was obtained. Thus the optimal performance of both isothermal techniques, and their adequacy for their implementation in the food industry, was demonstrated.Keywords: qLAMP; RPA; qPCR; hly; Listeria monocytogenes},
keywords = {},
pubstate = {published},
tppubtype = {article}
}Listeriosis continues to be a major health issue. This is demonstrated by the fact that, even though efforts have been made, its incidence does not decrease. Furthermore, in Europe, over 2014 a 30% increase was reported respect to 2013. In the present study two isothermal DNA amplification methods, one based on Loop-mediated isothermal AMPlification (qLAMP), and the other on Recombinase Polymerase Amplification (RPA), were developed and extensively evaluated. Both techniques demonstrated their reliability to detect Listeria monocytogenes in different types of foods. The method included a two-step enrichment, which additionally reduces the chances of detecting dead bacteria. Over the evaluation with pure bacterial DNA, the qLAMP and RPA methods resulted 10 to 100 times less sensitive than qPCR (with two different detection chemistries), but when tested in real food samples the results showed very good concordance with those obtained by qPCR and by selective agar plating (index kappa of concordance between 0.90 and 0.95). Additionally, a very low limit of detection (below 10 CFU/25 g) was obtained. Thus the optimal performance of both isothermal techniques, and their adequacy for their implementation in the food industry, was demonstrated.Keywords: qLAMP; RPA; qPCR; hly; Listeria monocytogenes
Joana Carvalho Pablo Fuciños Marta Prado Alejandro Garrido-Maestu Sarah Azinheiro
Food Control, Volume 86 , pp. Pages 27–34, 2018.
@article{Garrido-Maestu2017be,
title = {Development and evaluation of loop-mediated isothermal amplification, and Recombinase Polymerase Amplification methodologies, for the detection of Listeria monocytogenes in ready-to-eat food samples},
author = {Joana Carvalho Pablo Fuciños Marta Prado Alejandro Garrido-Maestu Sarah Azinheiro},
url = {https://doi.org/10.1016/j.foodcont.2017.11.006},
year = {2018},
date = {2018-04-01},
journal = {Food Control},
volume = {Volume 86},
pages = {Pages 27–34},
abstract = {Listeriosis continues to be a major health issue. This is demonstrated by the fact that, even though efforts have been made, its incidence does not decrease. Furthermore, in Europe, over 2014 a 30% increase was reported respect to 2013. In the present study two isothermal DNA amplification methods, one based on Loop-mediated isothermal AMPlification (qLAMP), and the other on Recombinase Polymerase Amplification (RPA), were developed and extensively evaluated. Both techniques demonstrated their reliability to detect Listeria monocytogenes in different types of foods. The method included a two-step enrichment, which additionally reduces the chances of detecting dead bacteria. Over the evaluation with pure bacterial DNA, the qLAMP and RPA methods resulted 10 to 100 times less sensitive than qPCR (with two different detection chemistries), but when tested in real food samples the results showed very good concordance with those obtained by qPCR and by selective agar plating (index kappa of concordance between 0.90 and 0.95). Additionally, a very low limit of detection (below 10 CFU/25 g) was obtained. Thus the optimal performance of both isothermal techniques, and their adequacy for their implementation in the food industry, was demonstrated.Keywords: qLAMP; RPA; qPCR; hly; Listeria monocytogenes},
keywords = {},
pubstate = {published},
tppubtype = {article}
}Listeriosis continues to be a major health issue. This is demonstrated by the fact that, even though efforts have been made, its incidence does not decrease. Furthermore, in Europe, over 2014 a 30% increase was reported respect to 2013. In the present study two isothermal DNA amplification methods, one based on Loop-mediated isothermal AMPlification (qLAMP), and the other on Recombinase Polymerase Amplification (RPA), were developed and extensively evaluated. Both techniques demonstrated their reliability to detect Listeria monocytogenes in different types of foods. The method included a two-step enrichment, which additionally reduces the chances of detecting dead bacteria. Over the evaluation with pure bacterial DNA, the qLAMP and RPA methods resulted 10 to 100 times less sensitive than qPCR (with two different detection chemistries), but when tested in real food samples the results showed very good concordance with those obtained by qPCR and by selective agar plating (index kappa of concordance between 0.90 and 0.95). Additionally, a very low limit of detection (below 10 CFU/25 g) was obtained. Thus the optimal performance of both isothermal techniques, and their adequacy for their implementation in the food industry, was demonstrated.Keywords: qLAMP; RPA; qPCR; hly; Listeria monocytogenes
Joana Carvalho Marta Prado Alejandro Garrido-Maestu Sarah Azinheiro
Rapid and sensitive detection of viable Listeria monocytogenes in food products by a filtration-based protocol and qPCR Journal Article
Food Microbiology, 73 , pp. 254-263, 2018.
@article{Garrido-Maestu2018b,
title = {Rapid and sensitive detection of viable Listeria monocytogenes in food products by a filtration-based protocol and qPCR},
author = {Joana Carvalho Marta Prado Alejandro Garrido-Maestu Sarah Azinheiro},
url = {https://doi.org/10.1016/j.fm.2018.02.004},
year = {2018},
date = {2018-02-07},
journal = {Food Microbiology},
volume = {73},
pages = {254-263},
abstract = {Listeria monocytogenes continues to be one of the most important foodborne pathogens worldwide, either due to its incidence and/or to its high mortality rate. In the present study, a filtration-based protocol was applied for the screening of viable bacteria. Additionally, a complete method (enrichment, filtration, DNA extraction and real-time PCR detection) was evaluated in order to determine the capacity of this protocol to detect viable L. monocytogenes in food samples. A new multiplex qPCR detection system was designed, including an internal amplification control, both targets were detected with hydrolysis probes. It was demonstrated that the method could reliably detect this pathogen, reaching a limit of detection of 9.5 cfu/25 g. The evaluation of the relative sensitivity, specificity, accuracy, positive and negative predictive values, as well as the index kappa of concordance obtained values higher than 90.0% after 24 h sample enrichment. Furthermore, it was demonstrated that with a secondary enrichment step, the limit of the detection could be further decreased to 4.6 cfu/25 g without significantly affecting the performance parameters. The present study demonstrates the reliability of the proposed methodology for the detection of viable L. monocytogenes, and the possibility of its direct implementation for routine analyses in the food industry.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}Listeria monocytogenes continues to be one of the most important foodborne pathogens worldwide, either due to its incidence and/or to its high mortality rate. In the present study, a filtration-based protocol was applied for the screening of viable bacteria. Additionally, a complete method (enrichment, filtration, DNA extraction and real-time PCR detection) was evaluated in order to determine the capacity of this protocol to detect viable L. monocytogenes in food samples. A new multiplex qPCR detection system was designed, including an internal amplification control, both targets were detected with hydrolysis probes. It was demonstrated that the method could reliably detect this pathogen, reaching a limit of detection of 9.5 cfu/25 g. The evaluation of the relative sensitivity, specificity, accuracy, positive and negative predictive values, as well as the index kappa of concordance obtained values higher than 90.0% after 24 h sample enrichment. Furthermore, it was demonstrated that with a secondary enrichment step, the limit of the detection could be further decreased to 4.6 cfu/25 g without significantly affecting the performance parameters. The present study demonstrates the reliability of the proposed methodology for the detection of viable L. monocytogenes, and the possibility of its direct implementation for routine analyses in the food industry.Joana Carvalho Marta Prado Alejandro Garrido-Maestu Sarah Azinheiro
Rapid and sensitive detection of viable Listeria monocytogenes in food products by a filtration-based protocol and qPCR Journal Article
Food Microbiology, 73 , pp. 254-263, 2018.
@article{Garrido-Maestu2018d,
title = {Rapid and sensitive detection of viable Listeria monocytogenes in food products by a filtration-based protocol and qPCR},
author = {Joana Carvalho Marta Prado Alejandro Garrido-Maestu Sarah Azinheiro},
url = {https://doi.org/10.1016/j.fm.2018.02.004},
year = {2018},
date = {2018-02-07},
journal = {Food Microbiology},
volume = {73},
pages = {254-263},
abstract = {Listeria monocytogenes continues to be one of the most important foodborne pathogens worldwide, either due to its incidence and/or to its high mortality rate. In the present study, a filtration-based protocol was applied for the screening of viable bacteria. Additionally, a complete method (enrichment, filtration, DNA extraction and real-time PCR detection) was evaluated in order to determine the capacity of this protocol to detect viable L. monocytogenes in food samples. A new multiplex qPCR detection system was designed, including an internal amplification control, both targets were detected with hydrolysis probes. It was demonstrated that the method could reliably detect this pathogen, reaching a limit of detection of 9.5 cfu/25 g. The evaluation of the relative sensitivity, specificity, accuracy, positive and negative predictive values, as well as the index kappa of concordance obtained values higher than 90.0% after 24 h sample enrichment. Furthermore, it was demonstrated that with a secondary enrichment step, the limit of the detection could be further decreased to 4.6 cfu/25 g without significantly affecting the performance parameters. The present study demonstrates the reliability of the proposed methodology for the detection of viable L. monocytogenes, and the possibility of its direct implementation for routine analyses in the food industry.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}Listeria monocytogenes continues to be one of the most important foodborne pathogens worldwide, either due to its incidence and/or to its high mortality rate. In the present study, a filtration-based protocol was applied for the screening of viable bacteria. Additionally, a complete method (enrichment, filtration, DNA extraction and real-time PCR detection) was evaluated in order to determine the capacity of this protocol to detect viable L. monocytogenes in food samples. A new multiplex qPCR detection system was designed, including an internal amplification control, both targets were detected with hydrolysis probes. It was demonstrated that the method could reliably detect this pathogen, reaching a limit of detection of 9.5 cfu/25 g. The evaluation of the relative sensitivity, specificity, accuracy, positive and negative predictive values, as well as the index kappa of concordance obtained values higher than 90.0% after 24 h sample enrichment. Furthermore, it was demonstrated that with a secondary enrichment step, the limit of the detection could be further decreased to 4.6 cfu/25 g without significantly affecting the performance parameters. The present study demonstrates the reliability of the proposed methodology for the detection of viable L. monocytogenes, and the possibility of its direct implementation for routine analyses in the food industry.Marta Prado Sarah Azinheiro Joana Carvalho and Alejandro Garrido-Maestu.
Frontiers in sustainable food systems, 2018.
@article{Azinheiro2018,
title = {Evaluation of different genetic targets for Salmonella enterica serovar Enteriditis and Typhimurium, using Loop-mediated isothermal AMPlification for detection in food samples},
author = {Marta Prado Sarah Azinheiro Joana Carvalho and Alejandro Garrido-Maestu.},
editor = {United States Joshua B. Gurtler Agricultural Research Service (USDA)},
url = {https://www.frontiersin.org/articles/10.3389/fsufs.2018.00005/abstract},
doi = {doi: 10.3389/fsufs.2018.00005},
year = {2018},
date = {2018-02-05},
journal = {Frontiers in sustainable food systems},
abstract = {Salmonella Enteritidis and Salmonella Typhimurium continue to be the most frequently identified serovars among confirmed cases of salmonellosis. In the current study different genetic targets (safA, sdf I, STM4497 and typh) were compared, attending to their specificity and sensitivity in pure cultures and in spiked samples, in order to determine their capacity to accurately identify them by loop-mediated isothermal amplification (LAMP). For the genes selected to detect Enteritidis, both performed equally well regarding their specificity, but safA proved more sensitive than Sdf I; minor differences were observed among these genes when analyzing spiked food samples. Regarding the targets for Typhimurium, STM4497 and typh, the former demonstrated to be more specific and sensitive, both when analyzing pure cultures as well as spiked samples. These results highlight the importance of an adequate evaluation of the genetic targets selected, before their implementation for routine analyses.
Keywords: Salmonella enteritidis, SAFA, Sdf I, Salmonella typhimurium, STM4497, typh, LAMP, Characterization methods},
keywords = {},
pubstate = {published},
tppubtype = {article}
}Salmonella Enteritidis and Salmonella Typhimurium continue to be the most frequently identified serovars among confirmed cases of salmonellosis. In the current study different genetic targets (safA, sdf I, STM4497 and typh) were compared, attending to their specificity and sensitivity in pure cultures and in spiked samples, in order to determine their capacity to accurately identify them by loop-mediated isothermal amplification (LAMP). For the genes selected to detect Enteritidis, both performed equally well regarding their specificity, but safA proved more sensitive than Sdf I; minor differences were observed among these genes when analyzing spiked food samples. Regarding the targets for Typhimurium, STM4497 and typh, the former demonstrated to be more specific and sensitive, both when analyzing pure cultures as well as spiked samples. These results highlight the importance of an adequate evaluation of the genetic targets selected, before their implementation for routine analyses.
Keywords: Salmonella enteritidis, SAFA, Sdf I, Salmonella typhimurium, STM4497, typh, LAMP, Characterization methodsMarta Prado Sarah Azinheiro Joana Carvalho and Alejandro Garrido-Maestu.
Frontiers in sustainable food systems, 2018.
@article{Azinheiro2018b,
title = {Evaluation of different genetic targets for Salmonella enterica serovar Enteriditis and Typhimurium, using Loop-mediated isothermal AMPlification for detection in food samples},
author = {Marta Prado Sarah Azinheiro Joana Carvalho and Alejandro Garrido-Maestu.},
editor = {United States Joshua B. Gurtler Agricultural Research Service (USDA)},
url = {https://www.frontiersin.org/articles/10.3389/fsufs.2018.00005/abstract},
doi = {doi: 10.3389/fsufs.2018.00005},
year = {2018},
date = {2018-02-05},
journal = {Frontiers in sustainable food systems},
abstract = {Salmonella Enteritidis and Salmonella Typhimurium continue to be the most frequently identified serovars among confirmed cases of salmonellosis. In the current study different genetic targets (safA, sdf I, STM4497 and typh) were compared, attending to their specificity and sensitivity in pure cultures and in spiked samples, in order to determine their capacity to accurately identify them by loop-mediated isothermal amplification (LAMP). For the genes selected to detect Enteritidis, both performed equally well regarding their specificity, but safA proved more sensitive than Sdf I; minor differences were observed among these genes when analyzing spiked food samples. Regarding the targets for Typhimurium, STM4497 and typh, the former demonstrated to be more specific and sensitive, both when analyzing pure cultures as well as spiked samples. These results highlight the importance of an adequate evaluation of the genetic targets selected, before their implementation for routine analyses.
Keywords: Salmonella enteritidis, SAFA, Sdf I, Salmonella typhimurium, STM4497, typh, LAMP, Characterization methods},
keywords = {},
pubstate = {published},
tppubtype = {article}
}Salmonella Enteritidis and Salmonella Typhimurium continue to be the most frequently identified serovars among confirmed cases of salmonellosis. In the current study different genetic targets (safA, sdf I, STM4497 and typh) were compared, attending to their specificity and sensitivity in pure cultures and in spiked samples, in order to determine their capacity to accurately identify them by loop-mediated isothermal amplification (LAMP). For the genes selected to detect Enteritidis, both performed equally well regarding their specificity, but safA proved more sensitive than Sdf I; minor differences were observed among these genes when analyzing spiked food samples. Regarding the targets for Typhimurium, STM4497 and typh, the former demonstrated to be more specific and sensitive, both when analyzing pure cultures as well as spiked samples. These results highlight the importance of an adequate evaluation of the genetic targets selected, before their implementation for routine analyses.
Keywords: Salmonella enteritidis, SAFA, Sdf I, Salmonella typhimurium, STM4497, typh, LAMP, Characterization methodsSarah Azinheiro Alejandro Garrido-Maestu Jorge Barros-Velázquez Marta Prado Joana Carvalho Renato Negrinho
Novel approach for accurate minute DNA quantification on microvolumetric solutions Journal Article
Microchemical Journal, 2018.
@article{Carvalho2018c,
title = {Novel approach for accurate minute DNA quantification on microvolumetric solutions},
author = {Sarah Azinheiro Alejandro Garrido-Maestu Jorge Barros-Velázquez Marta Prado Joana Carvalho Renato Negrinho},
url = {https://doi.org/10.1016/j.microc.2018.02.001},
year = {2018},
date = {2018-02-03},
journal = {Microchemical Journal},
abstract = {The optimization and evaluation of the performance of microfluidic devices for DNA purification requires the use of a reliable DNA quantification method. The samples collected from these devices usually have small volumes and, in the case of food, forensic and environmental applications, these samples are also complex, frequently containing highly fragmented DNA and minute concentrations. Therefore, combining a PicoGreen® assay with a microvolume fluorospectrometer is a popular technique for DNA quantification, providing highly sensitive quantification with minimal consumption of sample. However, this method has limitations, such as being affected by the degree of fragmentation of DNA and by the presence of some compounds commonly used in DNA extraction and purification protocols. In this work, these limitations and their influence on the accuracy of the quantification method were evaluated. Low molecular weight salmon sperm DNA was selected, being less purified and more fragmented than the λDNA standard most frequently used. It was shown that the standard curves generated with λDNA were not suitable for the quantification of fragmented DNA, such as DNA from highly processed samples and/or samples that have been exposed to harsh environments. In addition, a mathematical model was developed to find a better adjustment for the standard curve, for the salmon sperm DNA samples. This approach can be used as a tool to overcome important limitations found in this quantification method, allowing to include more data in the standard curve or test different mathematical models to better fit the standards data.Keywords
DNA quantification; PicoGreen; μTAS; DNA analysis; DNA purification; μSPE},
keywords = {},
pubstate = {published},
tppubtype = {article}
}The optimization and evaluation of the performance of microfluidic devices for DNA purification requires the use of a reliable DNA quantification method. The samples collected from these devices usually have small volumes and, in the case of food, forensic and environmental applications, these samples are also complex, frequently containing highly fragmented DNA and minute concentrations. Therefore, combining a PicoGreen® assay with a microvolume fluorospectrometer is a popular technique for DNA quantification, providing highly sensitive quantification with minimal consumption of sample. However, this method has limitations, such as being affected by the degree of fragmentation of DNA and by the presence of some compounds commonly used in DNA extraction and purification protocols. In this work, these limitations and their influence on the accuracy of the quantification method were evaluated. Low molecular weight salmon sperm DNA was selected, being less purified and more fragmented than the λDNA standard most frequently used. It was shown that the standard curves generated with λDNA were not suitable for the quantification of fragmented DNA, such as DNA from highly processed samples and/or samples that have been exposed to harsh environments. In addition, a mathematical model was developed to find a better adjustment for the standard curve, for the salmon sperm DNA samples. This approach can be used as a tool to overcome important limitations found in this quantification method, allowing to include more data in the standard curve or test different mathematical models to better fit the standards data.Keywords
DNA quantification; PicoGreen; μTAS; DNA analysis; DNA purification; μSPE
Sarah Azinheiro Alejandro Garrido-Maestu Jorge Barros-Velázquez Marta Prado Joana Carvalho Renato Negrinho
Novel approach for accurate minute DNA quantification on microvolumetric solutions Journal Article
Microchemical Journal, 2018.
@article{Carvalho2018f,
title = {Novel approach for accurate minute DNA quantification on microvolumetric solutions},
author = {Sarah Azinheiro Alejandro Garrido-Maestu Jorge Barros-Velázquez Marta Prado Joana Carvalho Renato Negrinho},
url = {https://doi.org/10.1016/j.microc.2018.02.001},
year = {2018},
date = {2018-02-03},
journal = {Microchemical Journal},
abstract = {The optimization and evaluation of the performance of microfluidic devices for DNA purification requires the use of a reliable DNA quantification method. The samples collected from these devices usually have small volumes and, in the case of food, forensic and environmental applications, these samples are also complex, frequently containing highly fragmented DNA and minute concentrations. Therefore, combining a PicoGreen® assay with a microvolume fluorospectrometer is a popular technique for DNA quantification, providing highly sensitive quantification with minimal consumption of sample. However, this method has limitations, such as being affected by the degree of fragmentation of DNA and by the presence of some compounds commonly used in DNA extraction and purification protocols. In this work, these limitations and their influence on the accuracy of the quantification method were evaluated. Low molecular weight salmon sperm DNA was selected, being less purified and more fragmented than the λDNA standard most frequently used. It was shown that the standard curves generated with λDNA were not suitable for the quantification of fragmented DNA, such as DNA from highly processed samples and/or samples that have been exposed to harsh environments. In addition, a mathematical model was developed to find a better adjustment for the standard curve, for the salmon sperm DNA samples. This approach can be used as a tool to overcome important limitations found in this quantification method, allowing to include more data in the standard curve or test different mathematical models to better fit the standards data.Keywords
DNA quantification; PicoGreen; μTAS; DNA analysis; DNA purification; μSPE},
keywords = {},
pubstate = {published},
tppubtype = {article}
}The optimization and evaluation of the performance of microfluidic devices for DNA purification requires the use of a reliable DNA quantification method. The samples collected from these devices usually have small volumes and, in the case of food, forensic and environmental applications, these samples are also complex, frequently containing highly fragmented DNA and minute concentrations. Therefore, combining a PicoGreen® assay with a microvolume fluorospectrometer is a popular technique for DNA quantification, providing highly sensitive quantification with minimal consumption of sample. However, this method has limitations, such as being affected by the degree of fragmentation of DNA and by the presence of some compounds commonly used in DNA extraction and purification protocols. In this work, these limitations and their influence on the accuracy of the quantification method were evaluated. Low molecular weight salmon sperm DNA was selected, being less purified and more fragmented than the λDNA standard most frequently used. It was shown that the standard curves generated with λDNA were not suitable for the quantification of fragmented DNA, such as DNA from highly processed samples and/or samples that have been exposed to harsh environments. In addition, a mathematical model was developed to find a better adjustment for the standard curve, for the salmon sperm DNA samples. This approach can be used as a tool to overcome important limitations found in this quantification method, allowing to include more data in the standard curve or test different mathematical models to better fit the standards data.Keywords
DNA quantification; PicoGreen; μTAS; DNA analysis; DNA purification; μSPE
-
2017
Joana Carvalho Sara Abalde-Cela Enrique Carbó-Argibay Lorena Dieguez Marek Piotrowski Yury Kolen'ko Marta Prado V Alejandro Garrido-Maestu Sarah Azinheiro
Frontiers in Microbiology In Press, 2017.
@article{Garrido-Maestu2017bc,
title = {Combination of microfluidic loop-mediated isothermal amplification with gold nanoparticles for rapid detection of Salmonella spp. in food samples},
author = {Joana Carvalho Sara Abalde-Cela Enrique Carbó-Argibay Lorena Dieguez Marek Piotrowski Yury Kolen'ko Marta Prado V Alejandro Garrido-Maestu Sarah Azinheiro},
url = {https://www.frontiersin.org/articles/10.3389/fmicb.2017.02159/abstract},
doi = {doi: 10.3389/fmicb.2017.02159},
year = {2017},
date = {2017-10-20},
journal = {Frontiers in Microbiology In Press},
abstract = {Foodborne diseases are an important cause of morbidity and mortality. According to the World Health Organization, there are 31 main global hazards, which caused in 2010 600 million foodborne illnesses and 420000 deaths. Among them, Salmonella spp. is one of the most important human pathogens, accounting for more than 90000 cases in Europe and even more in the United States per year. In the current study we report the development, and thorough evaluation in food samples, of a microfluidic system combining loop-mediated isothermal amplification with gold nanoparticles (AuNPs). This system is intended for low-cost, in-situ, detection of different pathogens, as the proposed methodology can be extrapolated to different microorganisms. A very low limit of detection (10 cfu/ 25 g) was obtained. Furthermore, the evaluation of spiked food samples (chicken, turkey, egg products), completely matched the expected results, as denoted by the index kappa of concordance (value of 1.00). The results obtained for the relative sensitivity, specificity and accuracy were of 100 % as well as the positive and negative predictive values.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}Foodborne diseases are an important cause of morbidity and mortality. According to the World Health Organization, there are 31 main global hazards, which caused in 2010 600 million foodborne illnesses and 420000 deaths. Among them, Salmonella spp. is one of the most important human pathogens, accounting for more than 90000 cases in Europe and even more in the United States per year. In the current study we report the development, and thorough evaluation in food samples, of a microfluidic system combining loop-mediated isothermal amplification with gold nanoparticles (AuNPs). This system is intended for low-cost, in-situ, detection of different pathogens, as the proposed methodology can be extrapolated to different microorganisms. A very low limit of detection (10 cfu/ 25 g) was obtained. Furthermore, the evaluation of spiked food samples (chicken, turkey, egg products), completely matched the expected results, as denoted by the index kappa of concordance (value of 1.00). The results obtained for the relative sensitivity, specificity and accuracy were of 100 % as well as the positive and negative predictive values.Joana Carvalho Sara Abalde-Cela Enrique Carbó-Argibay Lorena Dieguez Marek Piotrowski Yury Kolen'ko Marta Prado V Alejandro Garrido-Maestu Sarah Azinheiro
Frontiers in Microbiology In Press, 2017.
@article{Garrido-Maestu2017bf,
title = {Combination of microfluidic loop-mediated isothermal amplification with gold nanoparticles for rapid detection of Salmonella spp. in food samples},
author = {Joana Carvalho Sara Abalde-Cela Enrique Carbó-Argibay Lorena Dieguez Marek Piotrowski Yury Kolen'ko Marta Prado V Alejandro Garrido-Maestu Sarah Azinheiro},
url = {https://www.frontiersin.org/articles/10.3389/fmicb.2017.02159/abstract},
doi = {doi: 10.3389/fmicb.2017.02159},
year = {2017},
date = {2017-10-20},
journal = {Frontiers in Microbiology In Press},
abstract = {Foodborne diseases are an important cause of morbidity and mortality. According to the World Health Organization, there are 31 main global hazards, which caused in 2010 600 million foodborne illnesses and 420000 deaths. Among them, Salmonella spp. is one of the most important human pathogens, accounting for more than 90000 cases in Europe and even more in the United States per year. In the current study we report the development, and thorough evaluation in food samples, of a microfluidic system combining loop-mediated isothermal amplification with gold nanoparticles (AuNPs). This system is intended for low-cost, in-situ, detection of different pathogens, as the proposed methodology can be extrapolated to different microorganisms. A very low limit of detection (10 cfu/ 25 g) was obtained. Furthermore, the evaluation of spiked food samples (chicken, turkey, egg products), completely matched the expected results, as denoted by the index kappa of concordance (value of 1.00). The results obtained for the relative sensitivity, specificity and accuracy were of 100 % as well as the positive and negative predictive values.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}Foodborne diseases are an important cause of morbidity and mortality. According to the World Health Organization, there are 31 main global hazards, which caused in 2010 600 million foodborne illnesses and 420000 deaths. Among them, Salmonella spp. is one of the most important human pathogens, accounting for more than 90000 cases in Europe and even more in the United States per year. In the current study we report the development, and thorough evaluation in food samples, of a microfluidic system combining loop-mediated isothermal amplification with gold nanoparticles (AuNPs). This system is intended for low-cost, in-situ, detection of different pathogens, as the proposed methodology can be extrapolated to different microorganisms. A very low limit of detection (10 cfu/ 25 g) was obtained. Furthermore, the evaluation of spiked food samples (chicken, turkey, egg products), completely matched the expected results, as denoted by the index kappa of concordance (value of 1.00). The results obtained for the relative sensitivity, specificity and accuracy were of 100 % as well as the positive and negative predictive values.Azinheiro Carvalho Prado P ; S ; J ; M Garrido-Maestu A.; Fuciños
Food Control, 80 , pp. Pages 297-306, 2017.
@article{Garrido-Maestu2017d,
title = {Systematic loop-mediated isothermal amplification assays for rapid detection and characterization of Salmonella spp., Enteritidis and Typhimurium in food samples},
author = {Azinheiro Carvalho Prado P ; S ; J ; M Garrido-Maestu A.; Fuciños},
url = {http://www.sciencedirect.com/science/article/pii/S0956713517302542?via%3Dihub},
doi = {https://doi.org/10.1016/j.foodcont.2017.05.011},
year = {2017},
date = {2017-10-01},
journal = {Food Control},
volume = {80},
pages = {Pages 297-306},
abstract = {AbstractEuropean Authorities have made a great effort to decrease the incidence of salmonellosis, but yearly thousands of cases are still reported, being most of them associated with serovars Enteritidis and Typhimurium. In the current study a set of methods for fast detection of these pathogens was developed and evaluated. The methods were based on loop-mediated isothermal amplification due to its advantages. The methods targeted three genes, invA, safA and STM4497, and each one of them was evaluated independently so that they can be targeted directly or in a sequential mode: first screening for the genus Salmonella and secondly on typing those positive samples. In this process, the results were compared against qPCR. The methods were able to detect <10 cfu/25 g, making them suitable for official analyses, and food industry self-monitoring. Of most importance, the limit of detection, relative sensitivity, specificity and accuracy, positive and negative predictive values and the index kappa of concordance, were determined, being all higher than 97%. This demonstrates the reliability of the methods described in this study, which may be comparable with classical culture/serotyping of Salmonella but allowing a much faster response in case of positive results. Finally, a mathematical model was implemented to fit the data recorded by the qPCR thermocycler, allowing a more consistent determination of the parameters describing the qLAMP process, which may be easily implemented in other assays where accurate determination of Tt is needed for quantification purposes. Keywords: qLAMP; qPCR; Salmonella spp.; S. Enteritidis; S. Typhimurium},
keywords = {},
pubstate = {published},
tppubtype = {article}
}AbstractEuropean Authorities have made a great effort to decrease the incidence of salmonellosis, but yearly thousands of cases are still reported, being most of them associated with serovars Enteritidis and Typhimurium. In the current study a set of methods for fast detection of these pathogens was developed and evaluated. The methods were based on loop-mediated isothermal amplification due to its advantages. The methods targeted three genes, invA, safA and STM4497, and each one of them was evaluated independently so that they can be targeted directly or in a sequential mode: first screening for the genus Salmonella and secondly on typing those positive samples. In this process, the results were compared against qPCR. The methods were able to detect <10 cfu/25 g, making them suitable for official analyses, and food industry self-monitoring. Of most importance, the limit of detection, relative sensitivity, specificity and accuracy, positive and negative predictive values and the index kappa of concordance, were determined, being all higher than 97%. This demonstrates the reliability of the methods described in this study, which may be comparable with classical culture/serotyping of Salmonella but allowing a much faster response in case of positive results. Finally, a mathematical model was implemented to fit the data recorded by the qPCR thermocycler, allowing a more consistent determination of the parameters describing the qLAMP process, which may be easily implemented in other assays where accurate determination of Tt is needed for quantification purposes.
Keywords: qLAMP; qPCR; Salmonella spp.; S. Enteritidis; S. Typhimurium
Azinheiro Carvalho Prado P ; S ; J ; M Garrido-Maestu A.; Fuciños
Food Control, 80 , pp. Pages 297-306, 2017.
@article{Garrido-Maestu2017h,
title = {Systematic loop-mediated isothermal amplification assays for rapid detection and characterization of Salmonella spp., Enteritidis and Typhimurium in food samples},
author = {Azinheiro Carvalho Prado P ; S ; J ; M Garrido-Maestu A.; Fuciños},
url = {http://www.sciencedirect.com/science/article/pii/S0956713517302542?via%3Dihub},
doi = {https://doi.org/10.1016/j.foodcont.2017.05.011},
year = {2017},
date = {2017-10-01},
journal = {Food Control},
volume = {80},
pages = {Pages 297-306},
abstract = {AbstractEuropean Authorities have made a great effort to decrease the incidence of salmonellosis, but yearly thousands of cases are still reported, being most of them associated with serovars Enteritidis and Typhimurium. In the current study a set of methods for fast detection of these pathogens was developed and evaluated. The methods were based on loop-mediated isothermal amplification due to its advantages. The methods targeted three genes, invA, safA and STM4497, and each one of them was evaluated independently so that they can be targeted directly or in a sequential mode: first screening for the genus Salmonella and secondly on typing those positive samples. In this process, the results were compared against qPCR. The methods were able to detect <10 cfu/25 g, making them suitable for official analyses, and food industry self-monitoring. Of most importance, the limit of detection, relative sensitivity, specificity and accuracy, positive and negative predictive values and the index kappa of concordance, were determined, being all higher than 97%. This demonstrates the reliability of the methods described in this study, which may be comparable with classical culture/serotyping of Salmonella but allowing a much faster response in case of positive results. Finally, a mathematical model was implemented to fit the data recorded by the qPCR thermocycler, allowing a more consistent determination of the parameters describing the qLAMP process, which may be easily implemented in other assays where accurate determination of Tt is needed for quantification purposes. Keywords: qLAMP; qPCR; Salmonella spp.; S. Enteritidis; S. Typhimurium},
keywords = {},
pubstate = {published},
tppubtype = {article}
}AbstractEuropean Authorities have made a great effort to decrease the incidence of salmonellosis, but yearly thousands of cases are still reported, being most of them associated with serovars Enteritidis and Typhimurium. In the current study a set of methods for fast detection of these pathogens was developed and evaluated. The methods were based on loop-mediated isothermal amplification due to its advantages. The methods targeted three genes, invA, safA and STM4497, and each one of them was evaluated independently so that they can be targeted directly or in a sequential mode: first screening for the genus Salmonella and secondly on typing those positive samples. In this process, the results were compared against qPCR. The methods were able to detect <10 cfu/25 g, making them suitable for official analyses, and food industry self-monitoring. Of most importance, the limit of detection, relative sensitivity, specificity and accuracy, positive and negative predictive values and the index kappa of concordance, were determined, being all higher than 97%. This demonstrates the reliability of the methods described in this study, which may be comparable with classical culture/serotyping of Salmonella but allowing a much faster response in case of positive results. Finally, a mathematical model was implemented to fit the data recorded by the qPCR thermocycler, allowing a more consistent determination of the parameters describing the qLAMP process, which may be easily implemented in other assays where accurate determination of Tt is needed for quantification purposes.
Keywords: qLAMP; qPCR; Salmonella spp.; S. Enteritidis; S. Typhimurium
Prado Wenger Y ; M ; J Vial S.; Berrahal
Single-Step DNA Detection Assay Monitoring Dual-Color Light Scattering from Individual Metal Nanoparticle Aggregates Journal Article
ACS Sensors, 2 (2) , pp. pp 251–256, 2017.
@article{Vial2017,
title = {Single-Step DNA Detection Assay Monitoring Dual-Color Light Scattering from Individual Metal Nanoparticle Aggregates},
author = {Prado Wenger Y ; M ; J Vial S.; Berrahal},
url = {http://pubs.acs.org/doi/abs/10.1021/acssensors.6b00737},
doi = {10.1021/acssensors.6b00737},
year = {2017},
date = {2017-01-23},
journal = {ACS Sensors},
volume = {2 (2)},
pages = {pp 251–256},
abstract = {Efficiently detecting DNA sequences within a limited time is vital for disease screening and public health monitoring. This calls for a new method that combines high sensitivity, fast read-out time, and easy manipulation of the sample, avoiding the extensive steps of DNA amplification, purification, or grafting to a surface. Here, we introduce photon cross-correlation spectroscopy as a new method for specific DNA sensing with high sensitivity in a single-step homogeneous solution phase. Our approach is based on confocal dual-color illumination and detection of the scattering intensities from individual silver nanoparticles and gold nanorods. In the absence of the target DNA, the nanoparticles move independently and their respective scattering signals are uncorrelated. In the presence of the target DNA, the probe-functionalized gold and silver nanoparticles assemble via DNA hybridization with the target, giving rise to temporal coincidence between the signals scattered by each nanoparticle. The degree of coincidence accurately quantifies the amount of target DNA. To demonstrate the efficiency of our technique, we detect a specific DNA sequence of sesame, an allergenic food ingredient, for a range of concentration from 5 pM to 1.5 nM with a limit of detection of 1 pM. Our method is sensitive and specific enough to detect single nucleotide deletion and mismatch. With the dual-color scattering signals being much brighter than fluorescence-based analogs, the analysis is fast, quantitative, and simple to operate, making it valuable for biosensing applications.KEYWORDS: DNA detection; biosensing; dynamic light scattering; metal nanoparticles; optical scattering; plasmonics},
keywords = {},
pubstate = {published},
tppubtype = {article}
}Efficiently detecting DNA sequences within a limited time is vital for disease screening and public health monitoring. This calls for a new method that combines high sensitivity, fast read-out time, and easy manipulation of the sample, avoiding the extensive steps of DNA amplification, purification, or grafting to a surface. Here, we introduce photon cross-correlation spectroscopy as a new method for specific DNA sensing with high sensitivity in a single-step homogeneous solution phase. Our approach is based on confocal dual-color illumination and detection of the scattering intensities from individual silver nanoparticles and gold nanorods. In the absence of the target DNA, the nanoparticles move independently and their respective scattering signals are uncorrelated. In the presence of the target DNA, the probe-functionalized gold and silver nanoparticles assemble via DNA hybridization with the target, giving rise to temporal coincidence between the signals scattered by each nanoparticle. The degree of coincidence accurately quantifies the amount of target DNA. To demonstrate the efficiency of our technique, we detect a specific DNA sequence of sesame, an allergenic food ingredient, for a range of concentration from 5 pM to 1.5 nM with a limit of detection of 1 pM. Our method is sensitive and specific enough to detect single nucleotide deletion and mismatch. With the dual-color scattering signals being much brighter than fluorescence-based analogs, the analysis is fast, quantitative, and simple to operate, making it valuable for biosensing applications.KEYWORDS: DNA detection; biosensing; dynamic light scattering; metal nanoparticles; optical scattering; plasmonics
Prado Wenger Y ; M ; J Vial S.; Berrahal
Single-Step DNA Detection Assay Monitoring Dual-Color Light Scattering from Individual Metal Nanoparticle Aggregates Journal Article
ACS Sensors, 2 (2) , pp. pp 251–256, 2017.
@article{Vial2017b,
title = {Single-Step DNA Detection Assay Monitoring Dual-Color Light Scattering from Individual Metal Nanoparticle Aggregates},
author = {Prado Wenger Y ; M ; J Vial S.; Berrahal},
url = {http://pubs.acs.org/doi/abs/10.1021/acssensors.6b00737},
doi = {10.1021/acssensors.6b00737},
year = {2017},
date = {2017-01-23},
journal = {ACS Sensors},
volume = {2 (2)},
pages = {pp 251–256},
abstract = {Efficiently detecting DNA sequences within a limited time is vital for disease screening and public health monitoring. This calls for a new method that combines high sensitivity, fast read-out time, and easy manipulation of the sample, avoiding the extensive steps of DNA amplification, purification, or grafting to a surface. Here, we introduce photon cross-correlation spectroscopy as a new method for specific DNA sensing with high sensitivity in a single-step homogeneous solution phase. Our approach is based on confocal dual-color illumination and detection of the scattering intensities from individual silver nanoparticles and gold nanorods. In the absence of the target DNA, the nanoparticles move independently and their respective scattering signals are uncorrelated. In the presence of the target DNA, the probe-functionalized gold and silver nanoparticles assemble via DNA hybridization with the target, giving rise to temporal coincidence between the signals scattered by each nanoparticle. The degree of coincidence accurately quantifies the amount of target DNA. To demonstrate the efficiency of our technique, we detect a specific DNA sequence of sesame, an allergenic food ingredient, for a range of concentration from 5 pM to 1.5 nM with a limit of detection of 1 pM. Our method is sensitive and specific enough to detect single nucleotide deletion and mismatch. With the dual-color scattering signals being much brighter than fluorescence-based analogs, the analysis is fast, quantitative, and simple to operate, making it valuable for biosensing applications.KEYWORDS: DNA detection; biosensing; dynamic light scattering; metal nanoparticles; optical scattering; plasmonics},
keywords = {},
pubstate = {published},
tppubtype = {article}
}Efficiently detecting DNA sequences within a limited time is vital for disease screening and public health monitoring. This calls for a new method that combines high sensitivity, fast read-out time, and easy manipulation of the sample, avoiding the extensive steps of DNA amplification, purification, or grafting to a surface. Here, we introduce photon cross-correlation spectroscopy as a new method for specific DNA sensing with high sensitivity in a single-step homogeneous solution phase. Our approach is based on confocal dual-color illumination and detection of the scattering intensities from individual silver nanoparticles and gold nanorods. In the absence of the target DNA, the nanoparticles move independently and their respective scattering signals are uncorrelated. In the presence of the target DNA, the probe-functionalized gold and silver nanoparticles assemble via DNA hybridization with the target, giving rise to temporal coincidence between the signals scattered by each nanoparticle. The degree of coincidence accurately quantifies the amount of target DNA. To demonstrate the efficiency of our technique, we detect a specific DNA sequence of sesame, an allergenic food ingredient, for a range of concentration from 5 pM to 1.5 nM with a limit of detection of 1 pM. Our method is sensitive and specific enough to detect single nucleotide deletion and mismatch. With the dual-color scattering signals being much brighter than fluorescence-based analogs, the analysis is fast, quantitative, and simple to operate, making it valuable for biosensing applications.KEYWORDS: DNA detection; biosensing; dynamic light scattering; metal nanoparticles; optical scattering; plasmonics
-
2016
Fernandez-Arguelles Diéguez Fuciños Vial Oliveira Reis Boehme B ; M T ; L ; P ; S ; J M ; R L ; K Prado M.; Espiña
Detection of Foodborne Pathogens Using Nanoparticles. Advantages and Trends Book Chapter
Antimicrobial Food Packaging, pp. pp.183-202, Jorge Barros-Velazquez, 1st Edition, 2016, ISBN: 978-0-12-800723-5.
@inbook{Prado2016bc,
title = {Detection of Foodborne Pathogens Using Nanoparticles. Advantages and Trends},
author = {Fernandez-Arguelles Diéguez Fuciños Vial Oliveira Reis Boehme B ; M T ; L ; P ; S ; J M ; R L ; K Prado M.; Espiña},
url = {https://www.sciencedirect.com/science/book/9780128007235},
isbn = {978-0-12-800723-5},
year = {2016},
date = {2016-12-01},
booktitle = {Antimicrobial Food Packaging},
journal = {Academic Press, London},
pages = {pp.183-202},
publisher = {Jorge Barros-Velazquez},
edition = {1st Edition},
abstract = {Due to their critical impact on public health, the detection of foodborne pathogens in food and water is an important issue for both, the food industry and control authorities. Fast and reliable analytical methods are needed in order to ensure the health of consumers, to easily determine whether a food product has been subjected to cross-contamination, and, simultaneously, to identify how and when this cross-contamination occurred in order to establish the proper corrective actions.Recent developments in nanotechnology are greatly impacting the advancement of some analytical techniques, including foodborne pathogen detection. Herein, the advantages and the recent applications of nanoparticles (NPs) in foodborne pathogen detection are reviewed. The most frequently used NPs in this context (gold nanoparticles, quantum dots, and magnetic nanoparticles) are described, and their applicability for food analysis is emphasized. Finally, this chapter provides an overview of the use of micro and nanofluidics, combined with nanoparticles, for food pathogen analysis, and the integration of nanomaterial-based sensors for pathogen detection in food packaging systems.},
keywords = {},
pubstate = {published},
tppubtype = {inbook}
}Due to their critical impact on public health, the detection of foodborne pathogens in food and water is an important issue for both, the food industry and control authorities. Fast and reliable analytical methods are needed in order to ensure the health of consumers, to easily determine whether a food product has been subjected to cross-contamination, and, simultaneously, to identify how and when this cross-contamination occurred in order to establish the proper corrective actions.Recent developments in nanotechnology are greatly impacting the advancement of some analytical techniques, including foodborne pathogen detection. Herein, the advantages and the recent applications of nanoparticles (NPs) in foodborne pathogen detection are reviewed. The most frequently used NPs in this context (gold nanoparticles, quantum dots, and magnetic nanoparticles) are described, and their applicability for food analysis is emphasized. Finally, this chapter provides an overview of the use of micro and nanofluidics, combined with nanoparticles, for food pathogen analysis, and the integration of nanomaterial-based sensors for pathogen detection in food packaging systems.Vial Rivas Calo-Mata Barros-Velázquez I ; S ; J ; P ; J Prado M.; Ortea
Advanced DNA- and Protein-based Methods for the Detection and Investigation of Food Allergens Journal Article
Critical Reviews in Food Science and Nutrition, 56 (Issue 15), pp. 2511-2542, 2016.
@article{Prado2016b,
title = {Advanced DNA- and Protein-based Methods for the Detection and Investigation of Food Allergens},
author = {Vial Rivas Calo-Mata Barros-Velázquez I ; S ; J ; P ; J Prado M.; Ortea},
url = {http://www.tandfonline.com/doi/full/10.1080/10408398.2013.873767},
doi = {10.1080/10408398.2013.873767},
year = {2016},
date = {2016-11-17},
journal = {Critical Reviews in Food Science and Nutrition},
volume = {56},
number = {Issue 15},
pages = {2511-2542},
abstract = {Currently, food allergies are an important health concern worldwide. The presence of undeclared allergenic ingredients or the presence of traces of allergens due to contamination during food processing poses a great health risk to sensitized individuals. Therefore, reliable analytical methods are required to detect and identify allergenic ingredients in food products. The present review addresses the recent developments regarding the application of DNA- and protein-based methods for the detection of allergenic ingredients in foods. The fitness-for-purpose of reviewed methodology will be discussed, and future trends will be highlighted. Special attention will be given to the evaluation of the potential of newly developed and promising technologies that can improve the detection and identification of allergenic ingredients in foods, such as the use of biosensors and/or nanomaterials to improve detection limits, specificity, ease of use, or to reduce the time of analysis. Such rapid food allergen test methods are required to facilitate the reliable detection of allergenic ingredients by control laboratories, to give the food industry the means to easily determine whether its product has been subjected to cross-contamination and, simultaneously, to identify how and when this cross-contamination occurred.Keywords: Allergens, DNA based methods, proteomics, allergen detection, qPCR, biosensors, nanoparticles},
keywords = {},
pubstate = {published},
tppubtype = {article}
}Currently, food allergies are an important health concern worldwide. The presence of undeclared allergenic ingredients or the presence of traces of allergens due to contamination during food processing poses a great health risk to sensitized individuals. Therefore, reliable analytical methods are required to detect and identify allergenic ingredients in food products. The present review addresses the recent developments regarding the application of DNA- and protein-based methods for the detection of allergenic ingredients in foods. The fitness-for-purpose of reviewed methodology will be discussed, and future trends will be highlighted. Special attention will be given to the evaluation of the potential of newly developed and promising technologies that can improve the detection and identification of allergenic ingredients in foods, such as the use of biosensors and/or nanomaterials to improve detection limits, specificity, ease of use, or to reduce the time of analysis. Such rapid food allergen test methods are required to facilitate the reliable detection of allergenic ingredients by control laboratories, to give the food industry the means to easily determine whether its product has been subjected to cross-contamination and, simultaneously, to identify how and when this cross-contamination occurred.Keywords: Allergens, DNA based methods, proteomics, allergen detection, qPCR, biosensors, nanoparticles
Vial Rivas Calo-Mata Barros-Velázquez I ; S ; J ; P ; J Prado M.; Ortea
Advanced DNA- and Protein-based Methods for the Detection and Investigation of Food Allergens Journal Article
Critical Reviews in Food Science and Nutrition, 56 (Issue 15), pp. 2511-2542, 2016.
@article{Prado2016d,
title = {Advanced DNA- and Protein-based Methods for the Detection and Investigation of Food Allergens},
author = {Vial Rivas Calo-Mata Barros-Velázquez I ; S ; J ; P ; J Prado M.; Ortea},
url = {http://www.tandfonline.com/doi/full/10.1080/10408398.2013.873767},
doi = {10.1080/10408398.2013.873767},
year = {2016},
date = {2016-11-17},
journal = {Critical Reviews in Food Science and Nutrition},
volume = {56},
number = {Issue 15},
pages = {2511-2542},
abstract = {Currently, food allergies are an important health concern worldwide. The presence of undeclared allergenic ingredients or the presence of traces of allergens due to contamination during food processing poses a great health risk to sensitized individuals. Therefore, reliable analytical methods are required to detect and identify allergenic ingredients in food products. The present review addresses the recent developments regarding the application of DNA- and protein-based methods for the detection of allergenic ingredients in foods. The fitness-for-purpose of reviewed methodology will be discussed, and future trends will be highlighted. Special attention will be given to the evaluation of the potential of newly developed and promising technologies that can improve the detection and identification of allergenic ingredients in foods, such as the use of biosensors and/or nanomaterials to improve detection limits, specificity, ease of use, or to reduce the time of analysis. Such rapid food allergen test methods are required to facilitate the reliable detection of allergenic ingredients by control laboratories, to give the food industry the means to easily determine whether its product has been subjected to cross-contamination and, simultaneously, to identify how and when this cross-contamination occurred.Keywords: Allergens, DNA based methods, proteomics, allergen detection, qPCR, biosensors, nanoparticles},
keywords = {},
pubstate = {published},
tppubtype = {article}
}Currently, food allergies are an important health concern worldwide. The presence of undeclared allergenic ingredients or the presence of traces of allergens due to contamination during food processing poses a great health risk to sensitized individuals. Therefore, reliable analytical methods are required to detect and identify allergenic ingredients in food products. The present review addresses the recent developments regarding the application of DNA- and protein-based methods for the detection of allergenic ingredients in foods. The fitness-for-purpose of reviewed methodology will be discussed, and future trends will be highlighted. Special attention will be given to the evaluation of the potential of newly developed and promising technologies that can improve the detection and identification of allergenic ingredients in foods, such as the use of biosensors and/or nanomaterials to improve detection limits, specificity, ease of use, or to reduce the time of analysis. Such rapid food allergen test methods are required to facilitate the reliable detection of allergenic ingredients by control laboratories, to give the food industry the means to easily determine whether its product has been subjected to cross-contamination and, simultaneously, to identify how and when this cross-contamination occurred.Keywords: Allergens, DNA based methods, proteomics, allergen detection, qPCR, biosensors, nanoparticles
GROUP LEADER
THE TEAM
Alejandro Garrido
Staff Researcher
Joana Guerreiro
Research Fellow
Monisha Elumalai
Research Fellow
Andrey Ipatov
Research Fellow
Jon Ashley
Research Fellow
Foteini Roumani
Research Fellow
Sarah Azinheiro
Research Laboratory Assistant
Joana Carvalho
Research Laboratory Assistant
Carlos Carpena
visitor from Ocupharm diagnostics S.L.
Shambhavi Yadav
Scientific Associate
FORMER GROUP MEMBERS
Karola Böhme (Research Fellow)
Master student ( February – September 2017)
University de Santiago de Compostela
Visitor (June 2017)
Sofia Granja Martins
Summer Student (July – September 2017)
María Leyva (Scientific Visitor)
Anna Toldrà Filella
Visitor from IRTA
Cristina Pastrana
visitor from Ocupharm diagnostics S.L.
RESEARCH
HIGHLIGHTS
Paper published in Food Microbiology
Rapid and sensitive detection of viable Listeria monocytogenes in food products by a filtration-based protocol and qPCR. Alejandro Garrido-Maestu, , Sarah Azinheiro, Joana Carvalho, Marta Prado. Volume 73, August 2018, Pages 254–263.
Published a Research Article in Front. Sustain. Food Syst.
SarahAzinheiro,JoanaCarvalho,MartaPrado and AlejandroGarrido-Maestu. 21 February 2018.
Paper published in Microchemical Journal
Novel approach for accurate minute DNA quantification on microvolumetric solutions JoanaCarvalho,RenatoNegrinho,SarahAzinheiro,AlejandroGarrido-Maestu,JorgeBarros-Velázquez.MartaPrado.Volume 138, May 2018, Pages 540-549.
Paper published in Analytica chimica Acta.
JoanaCarvalho, GemaPuertas, JoãoGaspar, SarahAzinheiro, LorenaDiéguez, AlejandroGarrido-Maestu, ManuelVázquez,JorgeBarros-Velázquez,SusanaCardoso,MartaPrado.Volume 1020, 22 August 2018, Pages 30-40
NEWS & EVENTS
Author: Alejandro Garrido-Maestu, PabloFuciños, SarahAzinheiro, CarlaCarvalho, JoanaCarvalho, MartaPrado
Food Control 99 , pp. 79-83, 2019.
Book Chapter in collaboration with David Tomás Fornés, Lead Scientist in the Microbial and Molecular Analytics group at Nestlé Research Center (Lausanne, Switzerland). !!!!!! Foodborne Bacterial Pathogens
The Use of Multiplex Real-Time PCR for the Simultaneous Detection of Foodborne Bacterial Pathogens In: Bridier A. (eds) Foodborne Bacterial Pathogens. Methods in Molecular Biology, vol 1918. Humana Press, New York, NY
New funded projects!
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PORT GRAPHE – Control of Port and Douro Wines authenticity using graphene DNA sensors project(Project Time Frame: June 2018 to June 2021)
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Nano BioSensor – Development of nanosensors to evaluate the microbiological quality of fruit-based products(Project Time Frame: July 2018 to June 2021)