A bienzyme modified carbon paste electrode for amperometric detection of pyruvate

Abstract

Recombinant pyruvate oxidase from Lactobacillus plantarum (PyrOD LP) and pyruvate oxidase from Pediococcus sp. (PyrOD PS) were co-immobilized with horseradish peroxidase (HRP) in an organic carbon paste modified with trehalose 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(II), however, with a much lower sensitivity in comparison to the PyrOD LP modified CPEs. Pyruvate could be detected under Flow Injection Analysis (FIA) conditions in the range between 5 μM and 5 mM on PyrOD LP modified CPEs containing selected additives, at potentials around 0 mV versus the Ag/AgCl/0.1 M KCl reference electrode. Pyruvate can be selectively determined in the presence of α-ketoglutarate, α-ketocaproate, l-lactate, uric acid and reduced glutathione. Phosphoenolpyruvate, α-ketobutyrate, cysteine and NADH cause a small, significant bias. The operational stability of the PyrOD LP/HRP modified CPEs can be considerably improved by co-immobilization of poly- l-arginine and further by covering the electrode surface by a dialysis membrane. The PyrOD LP/HRP modified CPEs can be used to determine pyruvate selectively in mammalian cell cultivation media.