(164e) 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.