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.
Rubina
A Yu
AY
Center for Biological Microchips, Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, 32 ul. Vavilova, 119991 Moscow, Russia. arubina@eimb.ru
Pan'kov
S V
SV
Dementieva
E I
EI
Pen'kov
D N
DN
Butygin
A V
AV
Vasiliskov
V A
VA
Chudinov
A V
AV
Mikheikin
A L
AL
Mikhailovich
V M
VM
Mirzabekov
A D
AD
D000178:Acrylamides / Q000737:chemistry; D004247:DNA / Q000737:chemistry*; D006824:Hybridization, Genetic; D020136:Hydrogel, Polyethylene Glycol Dimethacrylate / Q000138:chemical synthesis*; D020411:Oligonucleotide Array Sequence Analysis / Q000295:instrumentation* / Q000379:methods*; D009841:Oligonucleotides / Q000138:chemical synthesis* / Q000737:chemistry*; D010777:Photochemistry; D011108:Polymers / Q000737:chemistry; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013050:Spectrometry, Fluorescence
D000178:Acrylamides; D009841:Oligonucleotides; D011108:Polymers; D020136:Hydrogel, Polyethylene Glycol Dimethacrylate; D004247:DNA