Immobilisation of protein macromolecules in biochip cells made of different polymers

Microarrays with immobilised protein probes are used in the analysis of protein samples. Selection of materials for biochip fabrication, functionalisation of the carrier surface, obtaining ordered cell matrices, immobilisation of protein molecular probes in cells, achieving high sensitivity of protein sample analysis are the key tasks of biochip technology. The following methodological approaches were used in this work. To preserve affinity properties, protein probes were immobilised in biochip cells under ‘soft’ conditions, after cell preparation. In order to achieve high concentration and prostanse accessibility, probes were immobilised in three-dimensional cells obtained from dynamically mobile brush polymers fixed on the substrate only at one end. The cell matrix was obtained on the substrate surface by photoinduced radical polymerisation of monomers with reactive chemical groups, photolithographically, according to the photomask template. We carried out a comparative analysis of polymer brush structures prepared on a polybutylene terephthalate substrate by photoinduced radical polymerisation. These structures consisted of links of one or more monomers. The influence of the method of activation of reactive groups on the polymer chains on the efficiency of immobilisation of molecular protein probes in the cells was investigated. The influence of the composition of acrylate monomers, from which the cells were obtained, on the specific binding of response proteins to protein probes immobilised in biochip cells was studied. A new method of biochip fabrication was developed. Substrates made of photoactive polybutylene terephthalate were coated with a thin layer of photoactive polymer polyvinyl acetate. The cells, which were obtained by photopolymerisation of monomers on the modified substrate, did not degrade or peel from the surface in aqueous solutions. The substrates coated with polyvinyl acetate do not adsorb proteins. Streptavidin and human immunoglobulins were used as models of protein probes, and biotinylated goat immunoglobulins and goat antibodies against human immunoglobulins were used as response proteins. The study found that polymers with irregular structure promoted higher concentration of protein probes and their uniform distribution within the cells, which positively influenced the efficiency of specific binding to response proteins. Biochips with cells of their brush polymers on black polybutylene terephthalate substrate appear promising for further improvement for use in immunofluorescence analysis of protein targets for the development of ‘lab-on-a-chip’ microanalysis technologies.