A Pyruvate Oxidase Electrode Based on an Electrochemically Deposited Redox Polymer

A redox polymer-modified, multilayer biosensor for the determination of pyruvate and phosphate in oxygen-free samples has been developed. The new, highly conductive redox polymer was produced by potentiostatic copolymerization (+1.4 V vs. Ag/AgCl) of Os(bipy)2pyCl-modified pyrrole monomer (6×10–3 mol L–1) and thiophene (1×10–3 mol L–1) on top of a platinized glassy-carbon electrode. The redox polymer-coated platinum black layer with increased active surface area permitted the adsorption of pyruvate oxidase as the biological recognition element and efficient electron transfer from enzyme-bound FAD-groups to the electrode. Pyruvate was detected at anodic potentials (350–500 mV) in oxygen-free solution in the presence of phosphate as the cosubstrate with a linear range from 0.02×10–3 to 0.3×10–3 mol L–1. A sensitivity as high as 0.2 A cm–2 mol–1 L was obtained. Phosphate was measured similarly between 0.02×10–3 and 0.5×10–3 mol L–1 in the presence of pyruvate as co-substrate. The sensitivity of the sensor dropped to about 12 % after 10 days. Since interference by ascorbate, due to the high formal potential of the used Os(bipy)2pyCl-group, could be a problem in real samples, coverage of the adsorbed enzyme by a polycationic size exclusion layer of polypyrrole was investigated. Compared to former enzyme electrodes utilizing pyruvate oxidase, the new approach offered an unprecedentedly high sensitivity, O2- and thiamindiphosphate-independent operation and presented a large step towards electrochemical pyruvate determination in vivo.