(503d) DNA-DNA Competitive Surface-Based Hybridization Through Target Displacement Under Constant Surface Potential Bias

DNA-DNA biosensor devices use single-stranded DNA as surface-tethered "probes" that interact, through base-sequence specific recognition, with DNA or RNA "targets" from solution. These types of DNA layers can also be used to study DNA-drug or DNA-protein interactions. A central limiting factor in DNA-DNA hybridization is the poor fundamental understanding of how target sequences compete for probes and of how the complex interfacial environment biases the hybridization kinetics and thermodynamics. In this work, a mixed SAM of a 20 bp probe sequence (RP1) and Mercaptohexanol passivator is end tethered to a polycrystalline gold electrode, at known probe coverages. Four 10 bp sequences are used (PM, MM1, MM2, MM3) one complementary and the remaining three with geometrically varied SNPs. Kinetics are tracked using electrochemically labeled DNA, at known hydrodynamic conditions. Different RP1-PM layers at the same coverage are allowed to reach equilibrium, at which time: (1) dehybridization is monitored in a target-free buffer (2) equal concentrations of the perfect match and mismatch sequences are introduced in clean buffer. The resulting dehybridization and displacement kinetics when compared show the effect of SNP position on hybridization. A new kinetic model was developed to include the rate of displacement, for all sequences at fixed counterion concentrations C_K+ (0.1 and 1.0 mol L^-1) and fixed moderate probe coverage S₀ (5.0 × 10¹² sites cm^-2). These conditions where targets compete for probe binding sites are representative of microarray platforms, and could account for long equilibrium times or kinetically frustrated states. It is hoped that an improved understanding of how such different DNA targets compete for a specific surface-bound probe is expected to lead to a better diagnostic tool, with more reliable data interpretation algorithms that will improve accuracy of genotyping, functional genomics, DNA based forensics, pathogen detection, drug development, and personalized medicine applications.