Electrochemical Monitoring of DNA-Protein Interactions At Modified Electrodes

April 29, 2011

Keeshan Williams (presenting author)

Department of Chemical and Biological Engineering

6 Metrotech Center

Brooklyn, NY 11201

Kwilli03@students.poly.edu

The Polytechnic Institute of NYU

Department of Chemical and Biological Engineering

6 Metrotech Center

Brooklyn, NY 11201

Additional author: Dr. Rastislav Levicky

Re: AIChE Annual Conference – Section: Interfacial Aspects of Nanosensors

Electrochemical Monitoring of DNA-Protein Interactions

Electrochemical interrogation of electro-active, self assembled DNA monolayers can provide precise information on the total number of molecules on the electrode surface. Moreover, electrochemical methods are useful for capturing the motions of biomacromolecular species at interfaces, including double-and single-stranded DNA. As one example, alternating current voltammetry (ACV) can capture the thermal motions of electroactively-labeled DNA chains tethered to gold surfaces, before and after association with a transcription factor protein. As shown previously by others, the faradaic current resulting from collisions of the DNA labels with the electrode surface tapers off at higher frequencies, resulting in a frequency-dependent tracking of chain dynamics [figure1] and enabling detection of protein binding. Upon binding, the shape of the protein-DNA complex is significantly altered from that of the original DNA layer (rigid rod) and, as such, affects the ability of the DNA-protein complex to undergo electron transfer interactions with the surface. Thus, by examining ACV traces at a judiciously selected frequency information can be obtained on protein-DNA interactions from changes in the faradaic current. In addition, protein association also impacts the redox potential of the electrochemical label, providing independent confirmation of the interaction. In this work, we consider how these different electrochemical signatures complement each other to provide a detailed description of the interaction between a bacteriophage transcription factor and its cognate DNA site. In addition, in situations when continuous monitoring of the biomolecular layer is required, practical limitations such as susceptibility of redox labels towards degradation must be recognized and calibrated for if necessary. In the case of ferrocene-based labels, for example, oxidation causes the ferrocene moiety to switch to the ferrocenium cation which is vulnerable to attack by nucleophilic species. This irreversible degradation leads to gradual loss of signal with the number of redox cycles performed. The loss of signal can be partly compensated for through changes in experimental conditions and/or signal processing methods. This presentation will discuss the fabrication of DNA-based sensors, electrochemical signatures of protein-DNA interactions under varying experimental conditions, as well as the practical considerations involved with signal maintenance.

Figure 1. The thermal motions of surface bound dsDNA can be discerned by recording ACV scans at several frequencies.