Electrochemical Melting of DNA: Optimization and Application

Abstract

A biosensor is an analytical device that can detect, quantitate, and/or characterize biologically-relevant molecules, often utilizing biologically-derived recognition elements, such as DNA oligomers or enzymes. Applications in point-of-care diagnostics, drug development, and forensic toxicology require portable, rapid, reliable, and easy-to-use devices. Electrochemistry provides a promising platform for such devices due to its potential for parallel measurements, its sensitivity, and its ease-of-miniaturization. In this work, gold electrodes modified with self-assembled monolayers of DNA are utilized to study the electric field assisted melting of double-stranded DNA (i.e. “e-melting”). We have previously demonstrated the ability to detect single mismatches in short DNA oligomers using this approach (J. Electrochem. Soc. 2019 volume 166, issue 4, B236-B242). Broadly, this project aims to (1) develop a better understanding of the mechanism of e-melting and (2) correlate e-melting behavior (kinetics, dependence on applied potential, ionic strength, etc.) to DNA structure and stability. Such analysis has the potential to provide fast and portable screening of biological samples for specific sequences, presence of mismatches, and other mutations. In addition, this method could provide a new platform for fundamental biophysical studies aimed at understanding the behavior of DNA in high electric fields. We will present several key improvements to the procedure for formation of the DNA monolayers using a potential pulse routine during electrode modification. This procedure has now replaced the “passive” method used in previous work and has decreased the amounts of reagents, decreased the reaction time, and increased reproducibility of the DNA surface coverage. This has made possible a correlation between the electrochemical signal (peak height obtained from voltammetric measurements) and the DNA surface coverage (DNA molecules per cm2). Additionally, improvements to the electrochemical melting routine itself will also be presented.These improvements in the methodology allows more controlled and reproducible studies, decreasing the signal-to-noise, and increasing sensitivity to small changes in DNA stability or structure. Results from the application of this method towards the study of interactions between DNA and small molecules, specifically cisplatin, will also be presented. Cisplatin has a strong affinity for DNA: it binds to the N7 of two neighboring purines with preference to neighboring guanines. This binding causes a kink in the DNA which destabilizes it. This research will contribute to a better understanding of the way in which small molecules, like cisplatin, affect the stability of DNA and how such interactions are affected by high electric fields. Ultimately, these studies may provide the foundation upon which future electrochemical biosensors can be developed.