Andrei Alexandrovich Stomakhin

Hydrogel-based protein and oligonucleotide microchips on metal-coated surfaces: enhancement of fluorescence and optimization of immunoassay.

Manufacturing of hydrogel-based microchips on metal-coated substrates significantly enhances fluorescent signals upon binding of labeled target molecules. This observation holds true for both oligonucleotide and protein microchips. When Cy5 is used as fluorophore, this enhancement is 8-10-fold in hemispherical gel elements and 4-5-fold in flattened gel pads, as compared with similar microchips manufactured on uncoated glass slides. The effect also depends on the hydrophobicity of metal-coated substrate and on the presence of a layer of liquid over the gel pads. The extent of enhancement is insensitive to the nature of formed complexes and immobilized probes and remains linear within a wide range of fluorescence intensities. Manufacturing of gel-based protein microarrays on metal-coated substrates improves their sensitivity using the same incubation time for immunoassay. Sandwich immunoassay using these microchips allows shortening the incubation time without loss of sensitivity. Unlike microchips with probes immobilized directly on a surface, for which the plasmon mechanism is considered responsible for metal-enhanced fluorescence, the enhancement effect observed using hydrogel-based microchips on metal-coated substrates might be explained within the framework of geometric optics.

Quantification of target proteins using hydrogel antibody arrays and MALDI time-of-flight mass spectrometry (A2M2S)

Mass spectrometry-based analysis techniques are widely applied in proteomics. This study presents a novel method for quantitative multiplex candidate protein profiling. It applies immunocapture of differentially labeled protein complements on hydrogel antibody arrays and subsequent quantification by MS. To make this approach quantitative a labeling approach was devised. The impact of labeling on the antibody/antigen interaction was assessed in detail by surface plasmon resonance. Owing to there solution by mass more than two protein samples can be compared simultaneously. Direct labeling of crude samples such as sera was developed and so enables the absolute quantification of target proteins straight from crude samples without a protein purification step. It was used to measure the concentration of apolipoprotein A-1 in serum. This method has been termed A2M2S for Affinity Array sand MALDI Mass Spectrometry.

Protein microchips: analysis of the expression of the recombinant barstar.

Protein microchips are becoming an effective tool for basic research and practical applications. However, widespread use of protein microchips is limited by sophisticated technology of their production, which is due to the necessity of maintaining the biological function of proteins during microchip manufacturing. The manufacturing of three-dimensional hydrogel protein microchips on the basis of polymerization immobilization technology developed in the Institute of Molecular Biology, Russian Academy of Sciences, is described and the proteins are shown to maintain their biochemical properties. The hydrogel protein microchips were used for the first time for quantitative assay of the expression of recombinant proteins in cells that opens wide perspectives for the application of protein chips in proteomics.