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Immunochemistry & Immunodiagnostics: Research Projects PDF Print E-mail

Microarrays: Tiny and shining

P. Petrou, C. Mastichiadis, I. Christofidis, S. Kakabakos, M. Chatzichristidi, A. Douvas, P. Argitis and K. Misiakos
The steadily growing demand for methods and materials appropriate to create patterns of biomolecules for bioanalytical applications was the driving force for the development of a new photolithographic method for patterning biomolecules onto a silicon surface coated with a polymeric layer of high protein binding capacity. The method was developed in collaboration with the Microelectronics Institute (M. Chatzichristidi, A. Douvas, P. Argitis, K. Misiakos). The patterning process does not affect the polymeric film and the activity of the immobilized onto the surface biomolecules. Therefore, it permits sequential immobilization of different biomolecules on spatially distinct areas on the same solid support. The polymeric layer is based on a commercially available photoresist that is cured at high temperature in order to provide a stable substrate for creation of protein microarrays by the developed photolithographic process. Following this photolithographic procedure onto the polymeric layer coated silicon surface, protein spots with diameters ranging from 2 (see photo) to 50 microns were created with excellent intra-and inter-spot homogeneity. This patterning methodology is expected to considerably facilitate the fabrication of dense protein microarrays for bioanalytical applications. Apart from the development of patterning procedure, effort was also devoted to the improvement of the detection sensitivity of fluorescent labels. A factor limiting the detection sensitivity when fluorescent labels are used is the phenomenon of fluorescence self-quenching observed when a relatively high number of fluorescent compounds is introduced into recognition molecule. By applying a simple treatment after the completion of the immunoreactions it was possible to lift off the self-quenching effect observed when highly labeled antibodies are used as labels in immunoassays. The treatment consisted in incubation of the solid-phase immobilized labeled molecules with a glycerin solution followed by 15-min incubation at 37 °C. The glycerin/thermal treatment resulted in significant increase of the fluorescence signal measured directly onto the solid surface. In addition, the fluorescence self-quenching was completely removed for labeling ratios up to 14.0. As a result, the assay sensitivity was improved 2-4 times when antibodies with molar ratio higher than 5.6 were used. The treatment worked equally well in flat surfaces (e.g. slides, silicon wafers) and in microtitration wells and can been applied to numerous other labels. The picture shows microscope fluorescence images of spots of rabbit gamma globulins on a silicon wafer after reaction with fluorescein labeled sheep anti-rabbit IgG antibodies with molar ratios 5.6 (lower part) and 14.0 (upper part) prior to (left part) and after treatment (right part). (Results published in Anal. Chem. 2007 and Biosens. Bioelectron. 2007)

Freedom to move
P.Petrou, S. Kakabakos, P. Bayiati, A. Tserepi, K. Misiakos and E. Gogolides
Droplet-based microfluidics is a fascinating emerging field with an expanding spectrum of potential applications in drug discovery, diagnostics, and health care. Electrowetting on dielectric is one of the most promising actuation approaches and has been used for droplet creation, cutting, and merging, for droplet transport, dispensing, microinjecting, and deposition, for the realization of variable focal length lenses, for optical actuation, for flow control valves, and even for the fabrication of video speed electronic displays. Electrowetting is based on the combination of good insulating properties of dielectric materials with the hydrophobicity of a top layer, usually a fluoropolymer. Fluorocarbon films can be easily deposited in high-density plasmas using fluorocarbon gases such us C4F8, CHF3, C2F6, and CF4. The use of such films for electrowetting applications has been demonstrated in collaboration with the Microelectronics Institute. The surface physical and chemical properties were studied and correlated with the electrowetting behavior of the samples. The protein adsorption capacity of the films was also determined. It was found that plasma-deposited fluorocarbon films of appropriate chemical composition (high F/C ratio) in combination with silicon nitride, a material of high dielectric constant, can be used for successful transport of biological samples based on electrowetting since they present negligible protein adsorption capacity. (Results published in Microelectron. Eng.2007 & J. Appl. Phys. 2007)