Carbon-felt-based bioelectrocatalytic flow-detectors: optimization of the adsorption conditions of horseradish peroxidase and thionine onto carbon-felt for highly sensitive amperometric determination of H2O2.

Horseradish peroxidase (HRP) and thionine (Th) were co-adsorbed onto a porous carbon-felt (CF) from a mixed solution of HRP and Th to fabricate a CF-based bioelectrocatalytic flow-detector for H(2)O(2). The resulting HRP and Th co-adsorbed CF (HRP/Th-CF) was successfully used as a working electrode unit of a highly sensitive amperometric flow-H(2)O(2)-biosensor. Differing from the ordinary mediator-based HRP biosensors, which detect mediated currents (i.e., the reduction current of the oxidized form of the mediators), the present HRP/Th-CF-based H(2)O(2)-biosensor directly detects the reduction current of the oxidative HRP-intermediates (i.e., compound I and II) based on a direct electron transfer (DET) between a HRP-redox active heme center and the CF electrode, because co-adsorbed Th together with HRP facilitate the DET. Various adsorption conditions (i.e., HRP concentration, Th concentration, ionic strength, pH, adsorption time) were optimized to obtain excellent sensor performances. When an air-saturated 0.1 M phosphate buffer (pH 7.0) was used as a carrier at 3.25 ml/min and the applied potential of - 0.05 V vs. Ag/AgCl, the HRP/Th-CF-based flow-H(2)O(2)-biosensor exhibited excellent analytical performances (i.e., sensitivity, 3.55 µA/µM; linear range, 0.1 to 10 µM; lower detection limit, 0.03 µM; K(m)(app), 36.2 µM; I(max), 158 µM; sample throughput, ca. 60 samples/h).