Immobilized enzyme electrode for the determination of l-lactate in food samples

A sensitive immobilized enzyme electrode for l-lactate is described. Toluidine blue O (TBO) and lactate dehydrogenase (LDH) were co-immobilized onto the surface of a graphite electrode by means of a dialysis membrane. l-lactate is oxidized to pyruvate by LDH in the presence of nicotinamide adenine dinucleotide NAD+ which is then reduced to NADH. The NADH formed is electrocatalytically oxidized by TBO and the reduced mediator is detected at 0 V vs. a AgAgCl reference electrode. Different dialysis membranes were tested in order to improve stability. The most stable enzyme electrode was fabricated with a benzoylated membrane, showing a half-life of eight days. This amperometric electrode responds fast and linearly to l-lactate in a concentration range 10−6 − 6 × 10−5 M. The limit of detection is 4 × 10−7M. The enzyme electrode was applied to the determination of l-lactate in food samples. Results were compared to those obtained using a spectrophotometric method, showing a good agreement.