Development of a Reagentless Amperometric Ethanol Biosensor Based on Yeast Alcohol Dehydrogenase and its Coenzyme, NAD+, Coimmobilized on a Carbon Nanotubes- modified Electrode

A reagentless amperometric ethanol biosensor was fabricated by modifying a glassy carbon (GC) electrode with a thin film of multi-walled carbon nanotubes (MWNTs) and depositing yeast alcohol dehydrogenase (YADH) and its coenzyme, nicotinamide adenine dinucleotide (NAD⁺), on the surface of the modified electrode. The enzyme was immobilized on the modified electrode using two techniques: adsorption and covalent attachment. Biosensors based on graphite and carbon nanofibers (CNFs) were also fabricated in a similar manner except that the enzyme was only adsorbed to the electrode surface.

The performance of the biosensors was assessed using a number of analytical techniques. Cyclic voltammetry was employed to determine the peak potential of NADH oxidation for each biosensor. Amperometric measurements were then conducted at or near the peak potential and the current response of each biosensor to successive ethanol additions was evaluated. The two MWNT-based biosensors to successive ethanol additions was evaluated. The two MWNT-based biosensors with adsorbed and covalently attached YADH were subjected to more detailed analysis including evaluation of stability, reusability and linear concentration range.

The MWNT-based biosensor was found to exhibit a much higher current response to ethanol than the graphite- and CNF-based biosensors at a working potential of +0.3 V (vs. Ag/AgCl). In addition, it displayed a relatively quick and stable response to individual ethanol additions. Both the adsorbed and covalently attached MWNT-biosensors had large linear concentration ranges, excellent stability and similar reusabilities.