Lactate‐dehydrogenase‐based biosensors for glyoxylate and NADH determination: A novel principle of analyte recyling

Two types of bienzyme electrodes based on the oxidation of glyoxylate to oxalate by NAD+ in the presence of lactate dehydrogenase (LDH) are described. The enzymes were entrapped in gelatin membranes and combined with an oxygen probe. A sequence sensor using LDH coupled with oxalate oxidase is useful for the determination of glyoxylate between 1 × 10−5 and 2.5 × 10−3 M. The sensor performance was optimized with respect to pH and cofactor concentration. As a novel approach to internal analyte recycling in biosensors, the LDH‐catalyzed recycling of NADH/NAD+ during the parallel oxidation of glyoxylate and reduction of pyruvate is utilized in a sensor for highly sensitive NADH measurement. Lactate monooxygenase was coupled as an auxiliary enzyme to indicate the lactate formed from pyruvate. The calibration curve of the proposed sensor is linear between 7 × 10−6 and 1.8 × 10−3 M NADH in the absence of glyoxylate (i.e., without analyte recycling) and between 3 × 10−8 and 2 × 10−6 M in the presence of glyoxylate (i.e., with analyte recycling).