Evaluation of seven cosubstrates in the quantification of horseradish peroxidase enzyme by square wave voltammetry

The electrochemical detection for horseradish peroxidase-cosubstrate-H 2O2 systems was optimized. o-Phenilendiamine, phenol, hydroquinone, pyrocatechol, p-chlorophenol, p-aminophenol and 3,3′-5,5′-tetramethylbenzidine were evaluated as cosubstrates of horseradish peroxidase (HRP) enzyme. Therefore, the reaction time, the addition sequence of the substrates, the cosubstrate:H2O2 ratio and the electrochemical techniques were elected by one-factor optimization assays while the buffer pH, the enzymatic activity and cosubstrate and H 2O2 concentrations for each system were selected simultaneously by response surface methodology. Then, the calibration curves for seven horseradish peroxidase-cosubstrate-H2O2 systems were built and the analytic parameters were analyzed. o-Phenilendiamine was selected as the best cosubstrate for the HRP enzyme. For this system the reaction time of 60 s, the phosphate buffer pH 6.0, and the concentrations of 2.5 × 10-4 mol L-1 o-phenilendiamine and of 1.25 × 10-4 mol L-1 H2O2 were chosen as the optimal conditions. In these conditions, the calibration curve of horseradish peroxidase by square wave voltammetry showed a linearity range from 9.5 × 10-11 to 1.9 × 10-8 mol L-1 and the limit of detection of 3.8 × 10-11 mol L-1 with RSD% of 0.03% (n = 3). © 2011 Elsevier B.V. All rights reserved.