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

The electrochemical detection for horseradish peroxidase–cosubstrate–H2O2 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 H2O2 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).