Mediated reagentless enzyme inhibition electrodes

We have investigated the use of the copper-containing oxygenase enzymes, laccase, tyrosinase and ceruloplasmin as reagentless enzyme activity sensors. The system is based on the mediated reduction of oxygen by the enzymes co- immobilized in an osmium redox polymer hydrogel on glassy carbon electrode surfaces. Both laccase and tyrosinase present rapid homogeneous second-order rate constants for the interaction with a model monomer, [Os(2,2'- bipyridine)₂(N-methylimidazole)Cl]⁺ (OsMelm). Ceruloplasmin rates are several orders of magnitude slower and no catalytic currents are observed upon co-immobilization of this enzyme in the redox hydrogel. The activity of the immobilized laccase and tyrosinase sensors is shown to be influenced by the enzyme loading in the deposition solution, the electrolyte pH and ionic strength. The immobilized sensors can be utilized for the detection of modulators of enzyme activity, such as the respiratory poison azide. Reproducible inhibition curves can be obtained by normalization of the sensor response. The resulting enzyme inhibition biosensors can detect levels of azide as low as 1 μM in solution and may be useful as an early warning sensor fro the presence of such respiratory toxins.