Immobilization of methylene blue using alpha-zirconium phosphate and its application within a reagentless amperometric hydrogen peroxide biosensor

A novel H2O2 sensor was constructed by employing methylene blue (MB) incorporated in alpha-zirconium phosphate as an electron transfer mediator. Methylene blue was intercalated into a alpha-zirconium phosphate modified horseradish peroxidase (HRP) immobilization matrix cross-linked on a glassy carbon electrode surface via bovine serum albumin (BSA)-glutaraldehyde. Such a coimmobilization matrix of the mediator and enzyme was formed from alpha-zirconium phosphate-MB inclusion colloid in which horseradish peroxidase was dissolved. Intercalation of methylene blue in layered alpha-zirconium phosphate was investigated by X-ray power diffraction (XRD), IR spectra and electrochemical measurements. Methylene blue immobilized in this way underwent a quasi-reversible electrochemical redox reaction. Cyclic voltammetry and amperometric measurements demonstrated that methylene blue coimmobilized with HRP displayed good stability and efficiently shuttled electrons between HRP and the electrode. The sensor responded rapidly and linearly to H2O2 over the range from 2.0 x 10(-6) to 5.0 x 10(-3) mol/L, with a detection limit of 3.0 x 10(-7) mol/L.