Development of a third generation biosensor to determine hydrogen peroxide based on a composite of soybean peroxidase/chemically reduced graphene oxide deposited on glassy carbon electrodes

Abstract

A third generation enzymatic biosensor is developed to determine H2O2. The biosensor is based on the use of a composite obtained from soybean peroxidase enzyme and chemically reduced graphene oxide deposited at glassy carbon electrodes. Experiments were carried out in 0.1 M phosphate buffer solutions, pH 7.0.Cyclic voltammograms of the biosensor show two reduction peaks centered at about 0.15 and −0.4 V, respectively. A quasi-reversible redox couple is defined in the region of potentials of the first peak, which is assigned to the reduction/oxidation of compound II involved in the catalytic cycle of peroxidases. The surface concentration of the electroactive enzyme was (1.1 ± 0.2) × 10−10 mol cm−2. Values of 0.33 and 0.04 s−1 were determined for the cathodic charge transfer coefficient and the heterogeneous electron transfer rate constant, respectively. UV–vis spectroscopy and scanning electron microscopy (SEM) were used to demonstrate the chemical reduction of graphene oxide. Electrochemical impedance spectroscopy was used to characterize the different stages in the process of the electrode surface modification. Amperometric measurements were performed at a potential of −0.090 V vs. Ag/AgCl. Current responses were linear in the concentration range from 1.5 × 10−7 to 3.0 × 10−6 M. The limit of detection, limit of quantification, reproducibility, and repeatability were 5 × 10−8 M, 1.5 × 10−7 M, 9%, and 4%, respectively. The biosensor was stable during five days. The Michaelis Menten apparent constant was 1.6 × 10−6 M. The presence of uric acid, glucose, dopamine and ascorbic acid do not interfere in the determination of H2O2. The biosensor was applied to the determination of H2O2 in commercial contact lens care solutions. Good accuracy and recovery parameters were obtained.