Microfluidics for the deposition of density gradients of immobilized oligonucleotide probes; developing surfaces that offer spatial control of the stringency of DNA hybridization

A method for the development of continuous density gradients of immobilized oligonucleotide probes (20mer) along the length of microfluidic channels is demonstrated. The development of continuous density gradients was achieved using variable electrokinetic transport of probes in hybrid glass–polydimethylsiloxane microfluidic chips. The probes were terminated with an amine functional group, and were delivered by electrokinetic pumping to the flat glass channel wall after it had been densely coated with covalently immobilized aldehyde groups. This method provided probe immobilization densities ranging from 4.5(±0.8) × 1013 to 2.5(±0.8) × 1011 molecules cm−2, with longitudinal dilution and differential mass transport of the injected plug of probes being the primary factors responsible for the gradient of density. The utility of the resulting density gradient of immobilized probes to control the selectivity of hybridization was demonstrated at room temperature by discrimination between a fully complementary oligonucleotide target, and a target strand containing 3 base pair mismatches (3 BPM) based on the spatial pattern of hybridization for sub-picomole quantities of targets. Single nucleotide polymorphism (SNP) discrimination was possible when temperature control was implemented to improve resolution of the mismatch discrimination, allowing SNP discrimination at 35 °C with a contrast ratio of almost 5 to 1.

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► Mass transport reaction kinetics generate a density gradient of immobilized probes.
► Targets with similar sequence identified by spatial pattern of hybridization.
► Interactions of probes with surface ameliorated by temperature or use of formamide.