Sequence and temperature influence on kinetics of DNA strand displacement at gold electrode surfaces

Understanding complex contributions of surface environment to tethered nucleic acid sensing experiments has proven challenging, yet it is important because it is essential for interpretation and calibration of indispensable methods, such as microarrays. We investigate the effects of DNA sequence and solution temperature gradients on the kinetics of strand displacement at heated gold wire electrodes, and at gold disc electrodes in a heated solution. Addition of a terminal double mismatch (toehold) provides a reduction in strand displacement energy barriers sufficient to probe the secondary mechanisms involved in the hybridization process. In four different DNA capture probe sequences (relevant for the identification of genetically modified maize MON810), all but one revealed a high activation energy up to 200 kJ/mol during hybridization, that we attribute to displacement of protective strands by capture probes. Protective strands contain 4 to 5 mismatches to ease their displacement by the surface-confined probes at the gold electrodes. A low activation energy (30 kJ/mol) was observed for the sequence whose protective strand contained a toehold and one central mismatch, its kinetic curves displayed significantly different shapes, and we observed a reduced maximum signal intensity as compared to other sequences. These findings point to potential sequence-related contributions to oligonucleotide diffusion influencing kinetics. Additionally, for all sequences studied with heated wire electrodes, we observed a 23 K lower optimal hybridization temperature in comparison with disc electrodes in heated solution, and greatly reduced voltammetric signals after taking into account electrode surface area. We propose that thermodiffusion due to temperature gradients may influence both hybridization and strand displacement kinetics at heated microelectrodes, an explanation supported by computational fluid dynamics. DNA assays with surface-confined capture probes and temperature gradients should not neglect potential influences of thermodiffusion as well as sequence-related effects. Furthermore, studies attempting to characterize surface-tethered environments should consider thermodiffusion if temperature gradients are involved.