Alcohol dehydrogenase amperometric biosensor based on a colloidal gold–carbon nanotubes composite electrode

A novel ethanol biosensor based on the bulk incorporation of alcohol dehydrogenase (ADH) into a colloidal gold (Aucoll)–multiwalled carbon nanotubes (MWCNTs) composite electrode using Teflon as binding material is reported. The composite Aucoll–MWCNTs–Teflon electrode exhibited significantly improved electrooxidation of NADH when compared with other carbon composite electrodes, including those based on carbon nanotubes. Amperometric measurements for NADH at +0.3 V showed significant differences in sensitivity between Aucoll–MWCNTs–Teflon and MWCNTs–Teflon composite electrodes. Incorporation of ADH into the bulk electrode material allowed the construction of a mediatorless ethanol biosensor. Both the enzyme loading and the NAD+ concentration in solution were optimized. The ADH–Aucoll–MWCNTs–Teflon biosensor allowed a limit of detection for ethanol of 4.7 μmol l−1, which is remarkably better than those reported for other CNTs-based ADH biosensors. The apparent Michaelis–Menten constant was 4.95 mmol l−1, which is much lower than that reported by immobilization of ADH onto a gold electrode. Both repeatability of the ethanol amperometric measurements, reproducibility with different biosensors, lifetime and storage ability can be, in general, advantageously compared with other ADH–CNTs biosensors. The biosensor was applied for the rapid determination of ethanol in commercial and certified beer samples.