Biosensoring of hydrogen peroxide using new methylene blue N incorporated in a montmorillonite-modified horseradish peroxidase immobilization matrix as an electron shuttle

A novel H2O2 sensor is described. New methylene blue N, a cation dye mediator, was incorporated in the montmorillonite-horseradish peroxidase (HRP) hydrogel cross-linked on a glassy carbon electrode surface via bovine serum albumin (BSA)-glutaraldehyde to construct the sensor. The interaction of the dye with sodium montmorillonite colloidal particles was investigated by employing visible spectrometry, X-ray photoelectron spectroscopy (XPS), and electrochemical measurements. The coimmobilization matrix of the mediator and the enzyme was formed from the pretreated sodium montmorillonite colloid in which horseradish peroxidase was dissolved. Immobilization of new methylene blue N involved its interaction with the montmorillonite colloidal particles while that of horseradish peroxidase involved the cross-linking via BSA-glutaraldehyde as usual. Cyclic voltammetry and amperometric measurements demonstrated that new methylene blue N coimmobilized with HRP in this way displayed good stability and efficiently shuttled electrons between immobilized HRP and the electrode. The sensor responded rapidly to H2O2 in the linear range from 5.0 μM to 2.2 mM with detection limit of 6.0 × 10−7 M.