Highly stable amperometric biosensor for ethanol based on Meldolaʹs blue adsorbed on silica gel modified with niobium oxide

A reagentless amperometric biosensor sensitive to ethanol was developed. This sensor comprises a carbon paste electrode modified with alcohol dehydrogenase (ADH), nicotinamide adenine dinucleotide (NAD+) cofactor and Meldolaʹs blue (MB) adsorbed on silica gel coated with niobium oxide. The amperometric response was based on the electrocatalytic properties of MB to oxidize NADH, which was generated in the enzymatic reaction of ethanol with NAD+ under catalysis of ADH. The dependence on the biosensor response was investigated in terms of pH, supporting electrolyte, ionic strength, ADH and NAD+ amounts and working potential. The biosensor showed an excellent operational stability (95% of the activity was maintained after 300 determinations) and storage stability (allowing measurements for over than 3 months, when stored in a refrigerator). The proposed biosensor also presented good sensitivity allowing ethanol quantification at levels down to 8.0×10−6 mol l−1. Moreover, the biosensor showed a wide linear response range (from 0.1 to 10 mmol l−1 for ethanol). These favorable characteristics allowed its application for direct measurements of ethanol in a great variety of alcoholic beverages, including beer, wine and spirits. The precision and recovery data shown by the proposed biosensor may give reliable results for real complex matrices.