BIENZYME AMPEROMETRIC D-LACTATE SENSOR USING MACROMOLECULAR NAD(+)

A bi-enzyme sensor for the determination of D-lactate was developed. T he D-lactate dehydrogenase, NADH oxidase and NAD(+) linked to dextran were confined in the reaction chamber defined by the surface of a nat platinum electrode and a dialysis membrane or entrapped in a membrane made of photocrosslinkable poly(vinyl alcohol) bearing styrylpyridiniu m groups in the betaine form. The measurement was conducted using hexa cyanoferrate(III) as an electron relay. Using free enzymes the sensor gave a linear response on the range 10 mu M(-1) mM and can be reused 3 0 times. Using entrapped enzymes the relationship between steady state current and the concentration of D-lactate was found to be linear bet ween 10 mu M 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 degrees C and 4 degrees C. The biosensor can be reused more than 10 00 times without addition of NAD(+). Validation of the assay was made on yoghurt, milk and cheese by comparison with a standard spectrophoto metric method.