Reagentless amperometric biosensors highly sensitive to hydrogen peroxide, glucose and lactose based on N-methyl phenazine methosulfate incorporated in a Nafion film as an electron transfer mediator between horseradish peroxidase and an electrode

Reagentless biosensors highly sensitive to hydrogen peroxide, glucose and lactose have been developed by immobilizing horseradish peroxidase (HRP), glucose oxidase (GOD) and β-galactosidase (GAL) on Nafion-N-methyl phenazine methosulfate modified electrode. The cationic exchange of a perfluorosulfonic acid cation-exchange polymer (Nafion) film coated on a glassy carbon electrode was used to provide high local concentrations of the N-methyl phenazine ion in the Nafion film via a process of ion exchange from the solution. The incorporated N-methyl phenazine ions displayed an electrochemical behavior different from that in aqueous solution. Cyclic voltammetry and chronamperometry were employed to demonstrate the suitability of electron transfer between immobilized HRP and a glassy carbon electrode via N-methyl phenazine methosulfate (NMP) in a Nafion film. A hydrogen peroxide biosensor, prepared by immobilization of HRP alone, provided a detection limit of 75 nM. Comparison of the NMP-mediated biosensor to the mediatorless biosensor indicated that the high sensitivity of the biosensor to hydrogen peroxide arose from the high efficiency of bioelectrocatalytic reduction of hydrogen peroxide via NMP incorporated in the Nafion film. Coimmobilization of HRP and GOD was employed to establish the feasibility of highly effective bienzyme-based biosensors for low glucose concentrations. Addition of GAL to the glucose biosensor provided a sensitive response to lactose, which illustrated the suitability of trienzyme-based biosensors. Performance and characteristics of the biosensors were evaluated with respect to response time, detection limit, selectivity, and dependence on applied potential, thickness of the Nafion film, temperature and pH as well as operating and storage stability.