Amperometric DNA quantification based on the use of peroxidase-mercaptopropionic acid-modified gold electrodes

Abstract

An electrochemical biosensor, constructed by immobilization of the enzyme horseradish peroxidase (HRP) by cross-linking atop a 3- mercaptopropionic acid (MPA) self-assembled monolayer modified gold electrode, was employed for the amperometric detection of ss- and ds-calf thymus DNA. The catalytic reduction of methylene blue (MB) at the biosensor in the presence of H2O2 was used as the analytical signal, and both batch and flow injection modes were evaluated. Experimental variables concerning both, the biosensor design and the detection process were investigated for an optimum analytical performance in both cases. The biosensor response to calf-thymus ds-DNA was approximately 3-fold lower than that to ss-DNA, indicating a much more favorable binding event of MB with free guanine bases in the ss-DNA. A linear calibration graph for calf-thymus ss-DNA over the 0.8-7.2 mg L −1 concentration range was obtained. Fifty successive injections of 6 mg L −1 ss-DNA yielded a RSD value for ip of 5.5% under flow injection conditions. No significant worsening in the analytical characteristics towards ss-DNA determination was observed under the hydrodynamic conditions. As a potential application of the FI methodology, the possibility of sulfonamides detection through their competition with MB for the DNA binding sites was demonstrated. © 2008 Elsevier B.V. All rights reserved.