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A novel electrochemical biosensing platform for nicotinamide adenine dinucleotide (NAD+)-dependent dehydrogenase catalysis was designed using the nitrogen-doped graphene (NG), which had properties similar to NADH dehydrogenase (CoI). NG mimicked flavin mononucleotide (FMN) in CoI and efficiently catalyzed NADH oxidation. NG also acted as an electron transport "bridge" from NADH to the electrode due to its excellent conductivity. In comparison with a bare gold electrode, an 800mV decrease in the overpotential for NADH oxidation and CoI-like behavior were observed at NG-modified electrode, which is the largest decrease in overpotential for NADH oxidation reported to date. The catalytic rate constant (k) for the CoI-like behavior of NG was estimated to be 2.3×105M−1s−1, which is much higher than that of other previously reported FMN analogs. The Michaelis-Menten constant (Km) of NG was 26μM, which is comparable to the Km of CoI (10μM). Electrodes modified with NG and NG/gold nanoparticals/formate dehydrogenase (NG/AuNPs/FDH) showed excellent analytical performance for the detection of NADH and formate. This electrode fabrication strategy could be used to create a universal biosensing platform for developing NAD+-dependent dehydrogenase biosensors and biofuel cells. [ABSTRACT FROM AUTHOR]