Hydrogen Peroxide Sensor Based on Coimmobilized Methylene Green and Horseradish Peroxidase in the Same Montmorillonite-Modified Bovine Serum Albumin−Glutaraldehyde Matrix on a Glassy Carbon Electrode Surface

Abstract

A new approach to construct a second-generation amperometric biosensor is described. The classical dye methylene green as a probing-needle mediator and horseradish peroxidase as a base enzyme were coimmobilized in the same montmorillonite-modified bovine serum albumin (BSA)−glutaraldehyde matrix to construct a H₂O₂ sensor. The immobilization matrix was formed from the pretreated sodium montmorillonite colloid in which the enzyme and the cross-linker were dissolved. Immobilization of methylene green from the dye mother solution was attributed to the adsorption function of the montmorillonite, whereas immobilization of horseradish peroxidase was attributed to the cross-linking function of the BSA−glutaraldehyde as usual. Cyclic voltammetry and potentiostatic measurements indicated that methylene green efficiently mediated electrons from the base electrode to the enzyme in the matrix. The sensor responded rapidly to low H₂O₂ concentration and achieved 95% of the steady-state current in less than 20 s, with a detection limit of 4.0 × 10^-7 M H₂O₂.