A bienzyme modified carbon paste electrode for amperometric detection of pyruvate

Recombinant pyruvate oxidase from Lactobacillus plantarum (PyrOD(LP)) and p yruvate oxidase from Pediococcus sp. (PyrOD(PS)) were co-immobilized with h orseradish peroxidase (HRP) in an organic carbon paste modified with trehal ose or lactitol alone or in combination with cationic poly-L-amino acids to prepare pyruvate sensitive electrodes. The PyrOD(LP) modified carbon paste electrodes (CPEs) detect pyruvate in the absence of the cofactors thiamine pyrophosphate and Mg(II), which have no significant effect on the pyruvate response. PyrOD(PS) modified carbon pastes respond to pyruvate only after the coimmobilization of thiamine pyrophosphate and Mg(LT), however, with a much lower sensitivity in comparison to the PyrOD(LP) modified CPEs. Pyruva te could be detected under Flow Injection Analysis (FIA) conditions in the range between 5 mu M and 5 mM on PyrOD(LP) modified CPEs containing selecte d additives, at potentials around 0 mV versus the Ag/AgCl/0.1 M KCl referen ce electrode. Pyruvate can be selectively determined in the presence of alp ha-ketoglutarate, alpha-ketocaproate, L-lactate, uric acid and reduced glut athione. Phosphoenolpyruvate, alpha-ketobutyrate, cysteine and NADH cause a small, significant bias. The operational stability of the PyrOD(LP)/HRP mo dified CPEs can be considerably improved by coimmobilization of poly-L-argi nine and further by covering the electrode surface by a dialysis membrane. The PyrOD(LP)/HRP modified CPEs can be used to determine pyruvate selective ly in mammalian cell cultivation media. (C) 1999 Elsevier Science B.V. All rights reserved.