A.Yu. Rubina

Development of hydrogel biochip for in vitro allergy diagnostics

A hydrogel biochip was developed for the simultaneous quantitative determination of sIgE for 21 allergens and total IgE in human serum. The biochips are manufactured by photoinduced copolymerization of different molecules (allergens and antibodies) with gel-forming monomers resulting in the formation of three-dimensional hydrogel elements (1nl gel drops). After incubation of the biochip with the serum, the results are visualized using fluorescently labeled anti-IgE antibodies. Using biochips, serum samples from allergic patients and healthy donors were analyzed and good correlation with the results obtained using commercial EIA test systems of generally recognized quality (Dr. Fooke Laboratorien GmbH, Germany) was observed.

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.

Development of a biochip with an internal calibration curve for quantitating two forms of the prostate-specific antigen

Three-dimensional gel-based biological microchips were developed for simultaneous quantitation of total (PSAtot) and free (PSAfree) forms of the prostate-specific antigen in human serum in the “one patient, one biochip” format. A method not demanding construction of calibration curves prior to the assay was applied to quantitation of PSAtot and PSAfree. In addition to gel elements with immobilized antibodies against PSAtot and PSAfree, the biochip contains elements with immobilized PSA at different concentrations, forming an internal calibration curve. Data are processed and interpreted with the special-purpose ImaGelAssay program. The sensitivity of the assay is 0.3 ng/ml for PSAtot and 0.2 ng/ml for PSAfree. The variation coefficient for measurements with one biochip series does not exceed 10%. The correlation coefficients between the estimates obtained for human sera by the biochip assay and by conventional ELISA were 0.988 for PSAtot and 0.987 for PSAfree.

Gel-Based Microchips: History and Prospects

The review describes the history of formation and development of the microchip technology and its role in the human genome project in Russia. The main accent was done on the three-dimensional gel-based microchips developed at the Center of Biological Microchips headed by A.D. Mirzabekov since 1988. The gel-based chips of the last generation, IMAGE chips (Immobilized Micro Array of Gel Elements), have a number of advantages over the previous models. The microchips are manufactured by photoinitiated copolymerization of gel components and immobilized molecules (DNA, proteins, and ligands). This ensures an even distribution of the immobilized probe throughout the microchip gel element with a high yield (about 50% for oligonucleotides). The use of methacrylamide as a main component of the polymerization mixture resulted in a substantial increase of gel porosity without affecting its mechanical properties and stability; this allowed one to work with the DNA fragments of up to 500 nt in length, as well as with quite large protein molecules. At present, the gel-based microchips are widely applied to solve different problems. The generic microchips containing a complete set of possible hexanucleotides are used to reveal the DNA motifs binding with different proteins and to study the DNA–protein interactions. The oligonucleotide microchips are a cheap and reliable diagnostic tool designed for mass application. Biochips have been developed for identification of the tuberculosis pathogen and its antibiotic-resistant forms; of orthopoxviruses, including the smallpox virus; of the anthrax pathogen; and chromosomal rearrangements in leukemia patients. The protein microchips can be adapted for further use in proteo-mics. Bacterial and yeast cells were also immobilized in the gel, maintaining their viability, which opens a wide potential for creating biosensors on the basis of microchips.

Protein Microchips in Quantitative Assays for Tumor Markers

Diagnosing malignant tumors is a major problem in oncology. The use of microchips in clinical testing makes it possible to detect several tumor markers in parallel without consuming large volumes of samples and expensive reagents. The goal of this study was to develop a quantitative immunoassay for some markers of commonly occurring tumors using three-dimensional hydrogel-based protein microchips designed at the Engelhardt Institute of Molecular Biology (EIMB), Russian Academy of Sciences. Microchips with immobilized antibodies were effective in detecting a number of tumor markers at clinically relevant concentrations in patient and donor sera.

A Fluorescent Dye with Low Specificity to DNA Nucleotide Sequences: Quantitative Assessment of Oligonucleotides Immobilized in Microchip Gel Pads

To assess the DNA amount in samples (e.g., in biological microchip gel pads) by means of fluorescent dyes, one should use the dyes whose fluorescence weakly depends on DNA composition and structure. With the ImD-310 dye created for this purpose, we have analyzed the staining of single- and double-stranded oligo- and polynucleotides of different nucleotide composition, length, and concentration both in solution and being immobilized in biological microchip gel pads. It turned out that ImD-310 has no pronounced specificity to the single- and double-stranded nucleotide sequences, while the intensity of fluorescence for the dye complexes with d(A)8, d(T)8, d(C)8, and d(G)8 at high temperatures (50°C) differs by less than 25%. A linear correlation has been established between the intensity of fluorescence and the amount of oligonucleotides immobilized on a biological microchip. The plots of the intensity of fluorescence against the concentration of NaCl and the temperature were obtained. By using a generic microchip containing all 4096 hexamer oligonucleotides, it has been determined that the dye has no distinct specificity to any certain motifs of the nucleotide sequence. Thus, ImD-310 may serve as an efficient fluorescent probe to quickly estimate the amount of oligonucleotides immobilized in a microchip, in an electrophoretic gel, etc.

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.