[Biophysical methods for biochip analysis. Use of wide field digital fluorescent microscopy].

This paper discusses an issue on the development of biophysical methods for biochip analysis. A scheme and construction of a biochip analyzer based on wide-field digital fluorescence microscopy are described. The analyzer is designed to register images of biological microchips labeled with fluorescent dyes. The device developed is useful for high-sensitive throughput recording analyses by biochips after interaction of immobilized probes with fluorescently labeled sample molecules as well as it provides the higher rate of the analysis compared to laser scanning devices. With this analyzer a scope where biological microchips can be applied becomes wider, the development of new protocols of the analyses is possible and standard analyses run faster with the use of biochips, the expenses for the analysis performance can be reduced.

Kinetic effects on signal normalization in oligonucleotide microchips with labeled immobilized probes.

Among various factors affecting operation of oligonucleotide microchips, the variations in concentration and in homogeneous distribution of immobilized probes over the cells are one of the most important. The labeling of immobilized probes ensures the complete current monitoring on the probe distribution and is reliable and convenient. Using hydrogel-based oligonucleotide microchips, the applicability of Cy3-labeled immobilized probes for quality control and signal normalization after hybridization with Cy5-labeled target DNA was investigated. This study showed that proper signal normalization should be different in thermodynamic conditions and in transient regime with hybridization far from saturation. This kinetic effect holds for both hydrogel-based and surface oligonucleotide microchips. Besides proving basic features, the technique was assessed on a sampling batch of 50 microchips developed for identifying mutations responsible for rifampicin and isoniazid resistance of Mycobacterium tuberculosis.

Comparison of surface and hydrogel-based protein microchips.

Protein microchips are designed for high-throughput evaluation of the concentrations and activities of various proteins. The rapid advance in microchip technology and a wide variety of existing techniques pose the problem of unified approach to the assessment and comparison of different platforms. Here we compare the characteristics of protein microchips developed for quantitative immunoassay with those of antibodies immobilized on glass surfaces and in hemispherical gel pads. Spotting concentrations of antibodies used for manufacturing of microchips of both types and concentrations of antigen in analyte solution were identical. We compared the efficiency of antibody immobilization, the intensity of fluorescence signals for both direct and sandwich-type immunoassays, and the reaction-diffusion kinetics of the formation of antibody-antigen complexes for surface and gel-based microchips. Our results demonstrate higher capacity and sensitivity for the hydrogel-based protein microchips, while fluorescence saturation kinetics for the two types of microarrays was comparable.

Kinetics of hybridization on surface oligonucleotide microchips: theory, experiment, and comparison with hybridization on gel-based microchips.

The optimal design of oligonucleotide microchips and efficient discrimination between perfect and mismatch duplexes strongly depend on the external transport of target DNA to the cells with immobilized probes as well as on respective association and dissociation rates at the duplex formation. In this paper we present the relevant theory for hybridization of DNA fragments with oligonucleotide probes immobilized in the cells on flat substrate. With minor modifications, our theory also is applicable to reaction-diffusion hybridization kinetics for the probes immobilized on the surface of microbeads immersed in hybridization solution. The main theoretical predictions are verified with control experiments. Besides that, we compared the characteristics of the surface and gel-based oligonucleotide microchips. The comparison was performed for the chips printed with the same pin robot, for the signals measured with the same devices and processed by the same technique, and for the same hybridization conditions. The sets of probe oligonucleotides and the concentrations of probes in respective solutions used for immobilization on each platform were identical as well. We found that, despite the slower hybridization kinetics, the fluorescence signals and mutation discrimination efficiency appeared to be higher for the gel-based microchips with respect to their surface counterparts even for the relatively short hybridization time about 0.5-1 hour. Both the divergence between signals for perfects and the difference in mutation discrimination efficiency for the counterpart platforms rapidly grow with incubation time. In particular, for hybridization during 3 h the signals for gel-based microchips surpassed their surface counterparts in 5-20 times, while the ratios of signals for perfect-mismatch pairs for gel microchips exceeded the corresponding ratios for surface microchips in 2-4 times. These effects may be attributed to the better immobilization efficiency and to the higher thermodynamic association constants for duplex formation within gel pads.

[Biochip development for polymorphism analysis in biotransformation system genes].

Large-scale population researches, diagnostics of genetic predisposition to multifactorial diseases, screening of the polymorphic loci associated with individual sensitivity to pharmaceutical preparations, require the development of effective, exact and rapid methods of analysis for detection of many mutations simultaneously. One of the most perspective methods to solve these problems is a method of allele-specific hybridization with biochips. Taking the analysis of mutations in genes CYP1A1, CYP2D6, GSTM1, GSTT1, NAT2, CYP2C9, CYP2C19 and MTHFR as an example we showed the efficiency of using the approach for identification of individual genetic polymorphism. We believe that the biochips can be also a convenient tool in pharmacogenetics researches.

Discrimination between perfect and mismatched duplexes with oligonucleotide gel microchips: role of thermodynamic and kinetic effects during hybridization.

The efficiency of discrimination between perfect and mismatched duplexes during hybridization on microchips depends on the concentrations of target DNA in solution and immobilized probes, buffer composition, and temperature of hybridization and is determined by both thermodynamic relationships and hybridization kinetics. In this work, optimal conditions of discrimination were studied using hybridization of fluorescently labeled target DNA with custom-made gel-based oligonucleotide microchips. The higher the concentration of immobilized probes and the higher the association constant, the higher the concentration of the formed duplexes and the stronger the corresponding fluorescence signal, but, simultaneously, the longer the time needed to reach equilibrium. Since mismatched duplexes hybridize faster than their perfect counterparts, perfect-to-mismatch signal ratio is lower in transient regime, and short hybridization times may hamper the detection of mutations. The saturation time can be shortened by decreasing the probe concentration or augmenting the gel porosity. This improves the detection of mutations in transient regime. It is shown that the decrease in the initial concentration of oligonucleotide probes by an order of magnitude causes only 1.5-2.5-fold decrease of fluorescence signals after hybridization of perfect duplexes for 3-12 h. At the same time, these conditions improve the discrimination between perfect and mismatched duplexes more than two-fold. A similar improvement may be obtained using an optimized dissociation procedure.

Hydrogel drop microchips with immobilized DNA: properties and methods for large-scale production.

Although gel-based microchips offer significant advantages over two-dimensional arrays, their use has been impeded by the lack of an efficient manufacturing procedure. Here we describe two simple, fast, and reproducible methods of fabrication of DNA gel drop microchips. In the first, copolymerization method, unsaturated groups are chemically attached to immobilized molecules, which are then mixed with gel-forming monomers. In the second, simpler polymerization-mediated immobilization method, aminated DNA without prior modification is added to a polymerization mixture. Droplets of polymerization mixtures are spotted by a robot onto glass slides and the slides are illuminated with UV light to induce copolymerization of DNA with gel-forming monomers. This results in immobilization of DNA within the whole volume of semispherical gel drops. The first method can be better controlled while the second one is less expensive, faster, and better suited to large-scale production. The microchips manufactured by both methods are similar in properties. Gel elements of the chip are porous enough to allow penetration of DNA up to 500 nucleotides long and its hybridization with immobilized oligonucleotides. As shown with confocal microscope studies, DNA is hybridized uniformly in the whole volume of gel drops. The gels are mechanically and thermally stable and withstand 20 subsequent hybridizations or 30-40 PCR cycles without decrease in hybridization signal. A method for quality control of the chips by staining with fluorescence dye is proposed. Applications of hydrogel microchips in research and clinical diagnostics are summarized.

Hydrogel-based protein microchips: manufacturing, properties, and applications.

Here a simple, reproducible, and versatile method is described for manufacturing protein and ligand chips. The photo-induced copolymerization of acrylamide-based gel monomers with different probes (oligonucleotides, DNA, proteins, and low-molecular ligands) modified by the introduction of methacrylic groups takes place in drops on a glass or silicone surface. All probes are uniformly and chemically fixed with a high yield within the whole volume of hydrogel semispherical chip elements that are chemically attached to the surface. Purified enzymes, antibodies, antigens, and other proteins, as well as complex protein mixtures such as cell lysates, were immobilized on a chip. Avidin- and oligohistidine-tagged proteins can be immobilized within biotin- and Ni-nitrilotriacetic acid-modified gel elements. Most gel-immobilized proteins maintain their biological properties for at least six months. Fluorescence and chemiluminescence microscopy were used as efficient methods for the quantitative analysis of the microchips. Direct on-chip matrix-assisted laser desorption ionization-time of flight mass spectrometry was used for the qualitative identification of interacting molecules and to analyze tryptic peptides after the digestion of proteins in individual gel elements. We also demonstrate other useful properties of protein microchips and their application to proteomics and diagnostics.

[Dye with low specificity to nucleotide sequences of DNA: use for assessing the quantity of oligonucleotides, immobilized in cells of biological microchips]. / Kasitel' s nizkoi spetsifichnost'iu k nukleotidnoi posledovatel'nosti DNK: primenenie dlia ostenki kolichestva oligonukleotidov, immobilizovannykh v iacheikakh biologicheskikh mikrochipov.

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 degrees 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.