2451
Electric Field Induced Melting: Effect of Non-Specifically Absorbed DNA

Wednesday, 16 May 2018
Ballroom 6ABC (Washington State Convention Center)
R. M. West and W. Hetrick (University of San Francisco)
Electric-field induced melting of surface-bound DNA can be carried out using electrochemical detection without parallel spectroscopic monitoring. With proper selection of applied potentials, ionic strength, and DNA coverage, discrimination of single mismatches based on the kinetics of DNA melting can be carried out quickly and reproducibly. Electrodes are prepared by the sequential exposure to thiol-modified oligonucleotides, mercaptohexanol, and finally hybridization with complimentary target oligonucleotides. The target is subsequently melted off the electrode by applying a sufficiently negative potential. The mechanism of e-melting is up for debate but likely involves the electrostatic repulsion between DNA’s phosphate backbone and the electrode surface, destabilization of the base pair hydrogen bonding or pi stacking, or some combination of these effects and others.

The mercaptohexanol passivation layer plays an important role for electrochemical DNA sensing, particularly when the effects of electric field are crucial to the detection scheme as is the case in electrochemical melting. The mercaptohexanol layer, or some other alkyl thiol layer is almost universally used to remove non-specifically absorbed DNA from the surface and prevent further non-specific absorption. Physisorbed oligonucleotides that are not removed by the passivation layer are expected to have ill-defined interaction energies with the surface and will be expelled from the surface at potentials and rates different than the hybridized target oligonucleotides. Additionally, a uniform mercaptohexanol layer is necessary for a uniform electric field. In this work, we examine the extent to which non-specific absorption occurs even when the mercaptohexanol layer is present. We examine how non-specific absorption effects the melting behavior, including the melting potential, melting rate, and the reproducibility. Finally, we compare various methods for mitigating non-specific absorption. These methods include the use of surfactants during electrode modification, applications of potential pulse sequences to destabilize and remove physisorbed oligomers, and the use of elevated temperatures.