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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.

The kinetics of hybridization on the oligonucleotide microchip with gel pads is studied both theoretically and experimentally. The monitoring of kinetics was performed with the measurements of fluorescence intensity produced by the labeled target oligonucleotides. As is shown, the hybridization time depends on the stability of the formed duplexes, the concentrations of target and probe oligonucleotides, and the diffusion of target oligonucleotides in solution and gel pad. The initial stage of hybridization is determined by the flow of target oligonucleotides from solution, then, followed by the diffusive propagation with approximately constant concentration of oligonucleotides at the boundary of gel pad and, finally, by the exponential saturation. The theoretical predictions of hybridization kinetics reveal a good correspondence with the experimental results and may be used for the choice of the optimal hybridization conditions. The possible applications of kinetic hybridization curves to the discrimination problems and assessment of diffusion coefficients in gel pads are briefly discussed. Finally, we discuss the relationships between the binding kinetics and the general functioning of biomolecular microchips.

Parallel thermodynamic analysis of the coaxial stacking effect of two bases localized in one strand of DNA duplexes has been performed. Oligonucleotides were immobilized in an array of three-dimensional polyacrylamide gel pads of microchips (MAGIChips'). The stacking effect was studied for all combinations of two bases and assessed by measuring the increase in melting temperature and in the free energy of duplexes formed by 5mers stacked to microchip-immobilized 10mers. For any given interface, the effect was studied for perfectly paired bases, as well as terminal mismatches, single base overlaps, single and double gaps, and modified terminal bases. Thermodynamic parameters of contiguous stacking determined by using microchips closely correlated with data obtained in solution. The extension of immobilized oligonucleotides with 5,6-dihydroxyuridine, a urea derivative of deoxyribose, or by phosphate, decreased the stacking effect moderately, while extension with FITC or Texas Red virtually eliminated stacking. The extension of the immobilized oligonucleotides with either acridine or 5-nitroindole increased stacking to mispaired bases and in some GC-rich interfaces. The measurements of stacking parameters were performed in different melting buffers. Although melting temperatures of AT- and GC-rich oligonucleotides in 5 M tetramethylammonium chloride were equalized, the energy of stacking interaction was significantly diminished.

A method of immobilizing oligonucleotides on the surface of a polyethylene terephthalate (PET) substrate has been developed. The effectiveness of the complementary binding of target oligonucleotide to oligonucleotide-probes depending on the long spacers of oligonucleotide-probes that bind to the substrate was investigated. It was shown that an increase in the spacer length leads to an increase in the hybridization signal. [ABSTRACT FROM AUTHOR]