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 beta-galactosidase (GAL) on Nafion-N -methyl phenazine methosulfate modified electrode. The cationic exchan ge of a perfluorosulfonic acid cation-exchange polymer (Nafion) film c oated on a glassy carbon electrode was used to provide high local conc entrations of the N-methyl phenazine ion in the Nafion film via a proc ess of ion exchange from the solution. The incorporated N-methyl phena zine ions displayed an electrochemical behavior different from that in aqueous solution. Cyclic voltammetry and chronamperometry were employ ed to demonstrate the suitability of electron transfer between immobil ized HRP and a glassy carbon electrode via N-methyl phenazine methosul fate (NMP) in a Nafion film. A hydrogen peroxide biosensor, prepared b y immobilization of HRP alone, provided a detection limit of 75 nM. Co mparison of the NMP-mediated biosensor to the mediatorless biosensor i ndicated that the high sensitivity of the biosensor to hydrogen peroxi de arose from the high efficiency of bioelectrocatalytic reduction of hydrogen peroxide via NMP incorporated in the Nafion film. Coimmobiliz ation of HRP and GOD was employed to establish the feasibility of high ly effective bienzyme-based biosensors for low glucose concentrations. Addition of GAL to the glucose biosensor provided a sensitive respons e to lactose, which illustrated the suitability of trienzyme-based bio sensors. Performance and characteristics of the biosensors were evalua ted with respect to response time, detection limit, selectivity, and d ependence on applied potential, thickness of the Nafion film, temperat ure and pH as well as operating and storage stability.