The multiwalled carbon nanotubes/N-butyl-N-methyl-pyrolydinium-bis(trifluoromethylsulfonyl)imide [C4mpyr][NTf2] ionic liquid (MWCNTs/IL) modified glassy carbon (GC) electrode has been utilized as a platform to immobilize electrogenerated NAD+ oxidation products (Ox-P(NAD+)). During potential cycling, the adenine moiety of NAD+ molecule is oxidized and gives rise to generation of a redox active system that shows great electrocatalytic activity toward NADH oxidation. The cyclic voltammetric results indicated the ability of MWCNTs/IL/Ox-P(NAD+) modified GC electrode to catalyze the oxidation of NADH at a very low potential (0.05 V vs. Ag/AgCl) and subsequently, a substantial decrease in the overpotential by about 600 mV compared with the bare GC electrode. This modified electrode thus allowed highly sensitive amperometric detection of NADH with a very low limit of detection (2 × 10−8 mol L−1), low applied potential (+0.05 V) at concentration range up to 4.2 × 10−5 mol L−1 and minimum of surface fouling. High ability of MWCNTs/IL/Ox-P(NAD+) to promote electron transfer between NADH and the electrode suggested a new promising biocompatible platform for development of dehydrogenase-based amperometric biosensors. With alcohol dehydrogenase (ADH) as a model enzyme, ethanol sensing ability of the proposed system was examined. The amperometric response of the biosensor increased linearly with increasing ethanol concentration in two concentration ranges, 5 × 10−6–6 × 10−5 and 6 × 10−5–9 × 10−4 mol L−1 with detection limit of 5 × 10−7 mol L−1 and rapid response of 10 s. Furthermore, the interference effects of redox active species, such as ascorbic acid, uric acid, glucose and acetaminophen for the proposed biosensor are negligible. Finally, the ability of the proposed biosensor for detection of ethanol in real complex samples was successfully demonstrated.
