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 dehy drogenase (LDH) on a film tightly bound to an oxygen electrode, for ra pid and sensitive L-lactate measurements, is described. Regeneration o f L-lactate by substrate recycling provided an amplification of the se nsor response, making it possible to decrease the detection limit of L -lactate from 10 mu M to 20 nM. The apparent K-m for L-lactate with th e LOD-LDH coupled reaction was 1 mu M, compared with 3 mM when utilizi ng only LOD. Linearity was obtained from 20 to 300 nM with both enzyme s, whereas with LOD alone it was from 10 mu M to 1 mM. Optimization of the biosensor was obtained with an increase in LOD and LDH film loadi ng and low L-lactate concentration. The enzymes covalently bound to th e him stabilized the biosensor (half life 8 weeks) for over 400 measur ements. Low L-lactate excreted by E. coli bacteria metabolism can be a ssayed in turbid culture medium without pretreatment by the amplified L-lactate detection. (C) 1996 Elsevier Science Limited