Co-immobilized L-lactate oxidase and L-lactate dehydrogenase on a film mounted on oxygen electrode for highly sensitive L-lactate determination

Covalent immobilization of L-lactate oxidase (LOD) with L-lactate dehydrogenase (LDH) on a film tightly bound to an oxygen electrode, for rapid and sensitive L-lactate measurements, is described. Regeneration of L-lactate by substrate recycling provided an amplification of the sensor response, making it possible to decrease the detection limit of L-lactate from 10 μM to 20 nM. The apparent K m for L-lactate with the LOD-LDH coupled reaction was 1 μM, compared with 3 mM when utilizing only LOD. Linearity was obtained from 20 to 300 nM with both enzymes, whereas with LOD alone it was from 10 μM to 1 mM. Optimization of the biosensor was obtained with an increase in LOD and LDH film loading and low L-lactate concentration. The enzymes covalently bound to the film stabilized the biosensor (half life 8 weeks) for over 400 measurements. Low L-lactate excreted by E. coli bacteria metabolism can be assayed in turbid culture medium without pretreatment by the amplified L-lactate detection.