Bi-enzyme amperometric d-lactate sensor using macromolecular NAD+

A bi-enzyme sensor for the determination of d-lactate was developed. The d-lactate dehydrogenase, NADH oxidase and NAD+ linked to dextran were confined in the reaction chamber defined by the surface of a flat platinum electrode and a dialysis membrane or entrapped in a membrane made of photocrosslinkable poly(vinyl alcohol) bearing styrylpyridinium groups in the betaine form. The measurement was conducted using hexacyanoferrate(III) as an electron relay. Using free enzymes the sensor gave a linear response on the range 10 /gmM-1 mM and can be reused 30 times. Using entrapped enzymes the relationship between steady state current and the concentration of d-lactate was found to be linear between 10 μM to 1 mM with a response time of 3 min. The half life time is respectively 1 month and 4 months when the biosensor is stored at 25 °C and 4 °C. The biosensor can be reused more than 1000 times without addition of NAD+. Validation of the assay was made on yoghurt, milk and cheese by comparison with a standard spectrophotometric method.