An amperometric sensor for the detection of pyruvate in biological fluids was formed by modifying the tip of a 0.25 mm gold wire with a layer of electrically "wired" recombinant pyruvate oxidase (POP). The sensor did not require O2 for its operation. The electroactive area of the tip of the microwire was increased by electrodeposition of platinum black. The POP was adsorbed on the platinum black and then "wired" with the cross-linked, subsequently deposited poly(4-vinylpyridine), part of the pyridine functions of which were complexed with [Os(bpy)2Cl](+/2+) and part quaternized with 2-bromoethylamine. In the resulting thin layer the POP was well "wired". When the electrode was poised at 0.4 V vs Ag/ AgCl, the sensitivity at pH 6 was 0.26 A cm(-2) M(-1) and the current increased linearly with the pyruvate concentration through the 2 x 10(-6) - 6 x 10(-4) M range. Thiamine diphosphate, flavin adenine dinucleotide, and MgCl2 were not required for the assay, but stabilized the stored enzyme electrode. Placement of a dialysis membrane (MWCO 3500) on the electrode alleviated the severe interference of ascorbate. In calf serum, the detection limit was 30 microM, suggesting that the electrode might be used in the continuous monitoring of pyruvate in hypoxic organs.
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