2.3. Enzyme Immobilization
The procedure used is the same in all cases. The PANI film was conditioned at pH 7.0 by immersion in pH 7.0 0.1 M phosphate buffer, then afterwards, an AOX solution of appropriate concentration (10 μL) was deposited on the PANI film, and left to dry 30 min. The PANI film with immobilised AOX was then stored at 4 °C for further use.
2.4. Biosensor Construction
The PANI film with immobilized AOX was constructed as a visual biosensor in the form of a dip stick test as shown in Figure 1, where the AOX/PANI film was connected to a cellulose paper as a handle for easy use using transparent plastic tape. In this dip-stick format, the biosensor could be easily used, just by dipping the biosensor into a sample solution for a few seconds (±5 s), then the color change of the biosensor could be detected visually using the naked eye for ethanol concentrations >1% (as this level is the allowable ethanol content in fermented beverages), and for quantitative measurement the color change could be further determined using color image analysis.