Kinetics of Hybridization on Surface Oligonucleotide Microchips: Theory, Experiment, and Comparison with Hybridization on Gel-Based Microchips

Abstract

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.

Sensitive and Multiplexed On‐chip microRNA Profiling in Oil‐Isolated Hydrogel Chambers

“…[5a,15] Meanwhile for some research platforms, attaining specific miRNA detection (<15% cross reactivity) has required complicated and customized probe designs for each miRNA target. [8d,16] Here, it was postulated that higher association constants for nucleic acid binding in the gel environment relative to a surface [17] would enable imposition of stringent incubation conditions for specificity without precluding high sensitive detection. Indeed, high specificities were achieved (maximum cross reactivity of 11.3%) by simply tuning salt concentration of the hybridization buffer (250 mM NaCl), probe concentration (5 µM inside gel) and by using a high (55°C) assay temperature, which was chosen based on sequence T m (Figure 2, see supporting information).…”

On-Chip Hybridization Kinetics for Optimization of Gene Expression Experiments

“…Equation 2 is true also in case of diffusion-limited kinetics in spite of a different interpretation of effective rate constants (14). …”

DNA microarrays in the clinic: infectious diseases

“…(20) Direct comparison of hybridization on flat microarrays and three-dimensional microchips shows that, although hybridization on flat microarrays is slightly faster, gel-based microchips discriminate between perfect and imperfect duplexes more efficiently and generate 5-to 20-fold stronger signals. (21) Because of the high water content, high porosity and complete isolation of gel pads from each other, they can function as reaction tubes of nanoliter volume. In particular, this concept has been used in the development of the so-called ''on-chip PCR'' procedure, which combines PCR amplification both inside the pads and in overlay solution with hybridization with immobilized probes (Fig.…”