We have developed a novel mediated biochemical oxygen demand (BOD) biosensor based on immobilized Bacillus subtilis (B. subtilis) on three-dimensional (3D) porous graphene-polypyrrole (rGO-PPy) composite. The 3D porous rGO-PPy composite was prepared using hydrothermal method following with electropolymerization. Then the 3D porous rGO-PPy composite was used as a support for immobilizing negatively charged B. subtilis denoted as rGO-PPy-B through coordination and electrostatic interaction. Further, the prepared rGO-PPy-B was used as a microbial biofilm for establishing a mediated BOD biosensor with ferricyanide as an electronic acceptor. The indirect determination of BOD was performed by electrochemical measuring ferrocyanide generated from a reduced ferricyanide mediator using interdigited ultramicroelectrode array (IUDA) as the working electrode. The experimental results suggested a good linear relationship between the amperometric responses and BOD standard concentrations from 4 to 60 mg/L, with a limit detection of 1.8 mg/L (S/N ≥ 3). The electrochemical measurement of real water samples showed a good agreement with the conventional BOD₅ method, and the good anti-interference as well as the long-term stability were well demonstrated, indicating that the proposed mediated BOD biosensor in this study holds a potential practical application of real water monitoring.
Keywords: 3D porous graphene-polypyrrole composite; electrochemical measurement; immobilized B. subtilis; mediated BOD biosensor.