Microfluidic Device for the Discrimination of Single-Nucleotide Polymorphisms in DNA Oligomers Using Electrochemically Actuated Alkaline Dehybridization

Abstract

This work describes an integrated microfluidic (mu-fl) device that can be used to effect separations that discriminate single-nucleotide polymorphisms (SNP) based on kinetic differences in the lability of perfectly matched (PM) and mismatched (MM) DNA duplexes during alkaline dehybridization. For this purpose a 21-base single-stranded DNA (ssDNA) probe sequence was immobilized on agarose-coated magnetic beads, that in turn can be localized within the channels of a poly(dimethylsiloxane) microfluidic device using an embedded magnetic separator. The PM and MM ssDNA targets were hybridized with the probe to form a mixture of PM and MM DNA duplexes using standard protocols, and the hydroxide ions necessary for mediating the dehybridization were generated electrochemically in situ by performing the oxygen reduction reaction (ORR) using O2 that passively permeates the device at a Pt working electrode (Pt-WE) embedded within the microfluidic channel system. The alkaline DNA dehybridization process was followed using fluorescence microscopy. The results of this study show that the two duplexes exhibit different kinetics of dehybridization, rate profiles that can be manipulated as a function of both the amount of the hydroxide ions generated and the mass-transfer characteristics of their transport within the device. This system is shown to function as a durable platform for effecting hybridization/dehybridization cycles using a nonthermal, electrochemical actuation mechanism, one that may enable new designs for lab-on-a-chip devices used in DNA analysis.

Genotyping by Alkaline Dehybridization Using Graphically Encoded Particles

“…Increasing the pH of the surrounding medium results in dehybridization of the target T1 from both sides, but the rate at which T1 dehybridizes is expected to be faster for the MM case. [17] Within an appropriate time scale and pH range, the difference in dehybridization kinetics provides an easily measured temporal difference in the fluorescence intensity between the two sides (Figure 1 g step (ii)) which evolves until both sides dehybridize completely (Figure 1 g step (iii)). Dehybridization of the other homozygote (T3) provides a fluorescence difference with opposite sign, while a heterozygous sample (i.e., a mixture of hybridized T1 and T3) is expected to produce little or no difference between the two sides.…”

“…We have previously shown, however, that SNPs can be discriminated by the difference in kinetics exhibited in the dehybridization of PM and MM DNA duplexes in an alkaline solution. [17] In the current work we show a significant improvement in that alkaline dehybridization protocol, one that may be combined with the use of an encoded hydrogel particle array to perform SNP discrimination in a versatile, nonenzymatic and isothermal technique. Encoded hydrogel particles were fabricated in a microfluidic system using stop-flow lithography (SFL) such that each particle included two spatially separated ASO probe regions.…”

“…We have previously shown, however, that SNPs can be discriminated by the difference in kinetics exhibited in the dehybridization of PM and MM DNA duplexes in an alkaline solution. [17] …”

“…They found that electric field (300 Vm −1 ) and temperature (<37 °C) generated in this particular experiment were insufficient for driving DNA dehybridization, and disregarded the possibility of alkaline discrimination because the pH range over which thermodynamic discrimination may be performed is quite narrow (ΔpH <0.3). We have previously shown, however, that SNPs can be discriminated by the difference in kinetics exhibited in the dehybridization of PM and MM DNA duplexes in an alkaline solution 17…”

Peroxidase Activity of DNA Aptamer–Pt Complexes Prepared with Cisplatin

“…DNA can recognize not only a complementing sequence of DNA [8,9], but also low-molecular-weight molecules [10,11] and proteins [12,13]. Such a DNA, DNA aptamer, can bind a variety of target molecules [10][11][12][13] and animal cells [14,15] with high affinity and specificity.…”