Operation of a miniature redox hydrogel-based pyruvate sensor in undiluteddeoxygenated calf serum

An amperometric sensor for the detection of pyruvate in biological fluids w as formed by modifying the tip of a 0.25-mm gold wire with a layer of elect rically "wired" recombinant pyruvate oxidase (POP). The sensor did not requ ire O-2 for its operation. The electroactive area of the tip of the microwi re was increased by electrodeposition of platinum black. The POP was adsorb ed on the platinum black and then "wired" with the cross-linked, subsequent ly deposited poly(4-vinylpyridine), part of the pyridine functions of which were complexed with [Os(bpy)(2)Cl](+/2+) and part quaternized with 8-bromo ethyl-amine, 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 concentr ation through the 2 x 10(-6)-6 x 10(-4) M range. Thiamine diphosphate, flav in adenine dinucleotide, and MgCl2 were not required for the assay, but sta bilized 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 mu M, suggesting that the electrode might be used in the continuous monitoring of pyruvate in hypoxic organs.