INL, Creating value at nanoscale

Nanomedicine Biofunctionalization of surfaces with nm/submicrometer-resolution using one-photon and multi-photon excitation of proteins

With a beam of UV laser light we are able to open disulphide bonds in most SS-containing proteins. If this happens at or close to a thiol reactive surface, such as thiol derivatized glass, quartz or a gold surface, the protein immobilizes onto the surface. Since this happens where the UV photons are present, the size of the focal spot determines where immobilization takes place. We are able to control this process such that spot size is ~3-5 micron. The process is relatively fast, being determined by physical chemical parameters as well as the light fluency. Currently we are operating with 100 ms illumination per spot. With a pitch of 10 microns and spot size of 5 micron, this allows for about 40.000 spots per mm². We have verified that Fab anti prostate specific antigen can be immobilized with our technology and still remains biologically active.

Over the last year we have studied a highly interesting extension to this approach – we are inserting spatial masks in the UV beam prior to focusing on the image plane. Theory predicts that we should obtain an intensity pattern corresponding to the Fourier transform of the spatial mask. We do see patterns similar to what theory predicts – but not identical. Using a simple mask with 8x 1mm sized holes, in a single shot we produce multiple spots (25-36) with immobilized protein with a spot size of ~700 nm.  With this spot density, we can populate 1 mm² with more than 1 million sensor spots, which represents an improvement of 10 fold over existing commercially available high density protein arraying methods. Our approach bypasses the use of micro dispenser techniques – and the technical difficulties associated with the use of such. It is simple, and fast.

Together with ICFO, The Institute of Photonic Sciences (Spain), we are now attempting to immobilize proteins in spatial regions no larger than 20nm using multi-photon excitation