Reagentless enzyme electrode based on phenothiazine mediation of horseradish peroxidase for subnanomolar hydrogen peroxide determination

The development and characterization of a highly sensitive enzyme immobiliz ed carbon based electrode for the determination of subnanomolar concentrati ons of hydrogen peroxide in aqueous samples is described. The biosensor con sists of horseradish peroxidase (HRP) immobilized in solid carbon paste alo ng with a suitable redox mediator. The latter allows the acceleration of th e electroreduction of HRP in the presence of hydrogen peroxide. Several phe nothiazines as mediators are investigated in a comparative manner and with respect to dimethylferrocene using cyclic voltammetry and amperometry. Inso lubilization of the HRP in the solid carbon paste is achieved by cross-link ing the enzyme with glutaraldehyde and bovine serum albumin. Several experi mental parameters such as pH, mediator and enzyme content are considered. T he hydrogen peroxide determination is better carried out in 0.1 M acetate b uffer, pH 4.5, by amperometry at an applied potential of 0.0 V versus Ag/Ag Cl, 3 M NaCl concentration and by using the phenothiazine base as redox med iator. The biosensor response is linear over the concentration range 2 nM-1 0 mu M with a detection limit of 1 nM. The linear range of the hydrogen per oxide response without a mediator in the biosensor is found between 2 and 4 0 mu M. The biosensor can be used for more than 180 measurements. Additiona l modification of the electrode by incorporation of Nafion SAC-13 micropart icles in the solid carbon paste allows detection of concentrations of hydro gen peroxide as low as 0.1 nM.