Direct electrocatalytic determination of dissolved peroxidases

A novel voltammetric approach is described for the quantification of dissolved redox enzymes. The measurement of horseradish peroxidase and microperoxidase is based on the electrocatalytic current generated when the enzyme reduces hydrogen peroxide to water and oxidises at low potential a mediator which is regenerated in a cathodic electrode reaction. Since the mediator regeneration is sufficiently fast, the overall reaction rate is determined by the enzyme activity. As a consequence stationary current-time curves are obtained in dependence on the activity of peroxidase in solution. For the chemical modification the electrode was modified with 1-(N,N-dimethylamine)-4-(4-morpholine)be a mediator of low solubility in reduced form and with favourably low formal potential. At a working potential of -50 mV vs, 0.1 M Ag/AgCl cathodic steady-state currents were obtained after injection of peroxidase in the presence of 1 mmol/l hydrogen peroxide. The detection limit for horseradish peroxidase and microperoxidase-11 were 10 ng/ml (250 pmol/l) and 20 ng/ml (10 nmol/l), respectively.