Enzyme Electrode for Amplification of NAD+/NADH Using Glycerol Dehydrogenase and Diaphorase with Amperometric Detection

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Abstract
An enzyme electrode is made from a glassy carbon electrode covered with a gelatin membrane containing entrapped glycerol dehydrogenase (GDH) and diaphorase, and protected with a dialysis membrane. Based on amplification by the recycling reaction catalyzed by the two-enzyme systems, NAD+ and NADH can be determined with 800–1200 times higher sensitivity than for the same electrode in a substrate sensing mode when the flow rate was 0.08 ml/min. The detection limit was about 0.03 μM for NADH. The amplification factors were around 1000 for 0.08 ml/min, with quite large variations between electrodes. They had decreased to about 70% of the original value after 7 days. The biosensor is intended for detection in immunoassays with alkaline phosphatase as a marker.