[Immobilization of Protein Macromolecules in Biochip Cells Made of Various Polymers].

Abstract

Microarrays with immobilized protein probes are used to analyze protein samples. Selection of materials for biochip fabrication, functionalization of the carrier surface, construction of ordered cell matrices, immobilization of protein molecular probes in cells, and improvement of the sensitivity of protein sample analysis are key tasks of biochip technology. The following methodological approaches were used in this work. To preserve affinity of protein probes, mild conditions were used to immobilize the probes in biochip cells. To achieve a high concentration and spatial accessibility of the probes, three-dimensional cells to immobilize the probes were made of dynamically mobile polymer brushes, in which only one end of a polymer molecule is attached to the substrate. A cell array was formed on a substrate surface by photoinduced radical polymerization of monomers with reactive chemical groups (photolithography), according to a photomask template. A comparative analysis was performed for polymer brush structures prepared on a polybutylene terephthalate substrate by photoinduced radical polymerization. The structures consisted of units formed by one or more monomers. The efficiency of protein probe immobilization in cells was studied as dependent on the method used to activate the reactive groups in polymer chains. The composition of acrylate monomers used to form biochip cells was tested for the effect on the specific binding of response proteins to protein probes immobilized in the cells. A new method of biochip fabrication was developed. Substrates made of nonphotoactive polybutylene terephthalate were coated with a thin layer of the photoactive polymer polyvinyl acetate. Cells obtained by monomer photopolymerization on the modified substrate showed no degradation or detachment from the surface in aqueous solutions. Substrates coated with polyvinyl acetate did not adsorb proteins. Streptavidin and human immunoglobulins were used as model protein probes, and biotinylated goat immunoglobulins and goat antibodies against human immunoglobulins served as response proteins. Polymers with irregular structures were found to promote a higher concentration of protein probes and their uniform distribution within the cells, thus ensuring a higher efficiency of specific binding with response proteins. Biochips with cells made of brush polymers on black polybutylene terephthalate substrates were found to be promising for further improvement, aiming to employ them in immunofluorescence analyses of target proteins and to develop the lab-on-a-chip microanalysis technologies.