A bienzyme-poly-(o-phenylenediamine)-modified carbon paste electrode for the amperometric detection of L-lactate

A reagentless lactate amperometric biosensor, constructed by immobilising the enzymes glutamic pyruvic transaminase (GPT) and L-lactate dehydrogenase (LDH) together with its cofactor nicotinamide adenine dinucleotide (NAD(+)) in carbon paste using an electropolymerised poly(o-phenylenediamine) (PPD) film, is described. The response of the electrode is based on the electrocatalytic oxidation, at low applied potentials, of the enzymatically produced NADH by the PPD conducting film. This sensor construction was optimised, the final biosensor contained 80 units of LDH, 5 units of GPT and 0.1 mg of NAD(+) mg(-1) of carbon paste. Interference by direct electrochemical oxidation of easily oxidizable substances such as uric acid, L-ascorbic acid, L-cysteine, glutathione and paracetamol, are drastically reduced by covering the PPD-modified electrode with a second electrochemically synthesized, non-conducting poly(o-aminophenol) film. The bienzyme-double polymeric layer-modified electrode at an applied potential of 0 V (vs. Ag/AgCl), gives a linear response for lactate in the range 6x10(-7) M8.5x10-5 M and a response time of 80 s. This sensor was used for the quantification of lactic acid in cider. Results compared favourably with a standard spectrophotometric method.