Amperometric Biosensor for Hydrogen Peroxide Based on Immobilization of Horseradish Peroxidase on Methylene Blue Modified Graphite Electrode

A methylene blue modified graphite electrode was first prepared by adsorbing the molecules on a spectrographic graphite disk electrode saturated with wax. Then, a novel hydrogen peroxide biosensor was fabricated based on immobilization of horseradish peroxidase on the methylene blue modified graphite electrode by cross-linking with glutaraldehyde. This electrode exhibited excellent electrochemical activity in aqueous solutions, and the electron transfer rate constant for the adsorbed methylene blue was determined as 0.12 s–1. It was demonstrated that the modified methylene blue molecules could shuttle electrons between immobilized HRP and graphite substrate very well for H2O2 sensing. The characteristics of this sensor were evaluated with respect to applied potential, pH and temperature. A detection limit of 3.0×10–6 mol/L hydrogen peroxide was examined at simple experimental conditions. Dopamine and ascorbic acid did not interfere with the determination. This sensor was also stable, reproducible and outstanding for long-term use.