The sensitivity continuously increased with the increase in enzyme concentration when the enzyme concentration is increased from 0.01% to 0.1%. However, at the same time, the linear range becomes smaller with an increase in enzyme concentration and in particular the colour change at 1.0% of ethanol was low, so that is not clearly visible to the naked eye for detection. Since the biosensor constructed using 0.1% enzyme solution showed a much higher sensitivity, in particular intense colour changes at 1% of ethanol for easy naked eye inspection, this level it was chosen for performing further experiments.
A study of the influence of the pH of the 0.1 M phosphate buffer on biosensor response was performed with the optimised AOX/PANI film as biosensor. The pH values of buffer solution were chosen in the range of 6.0–8.0, since it is known that the pH of AOX extracted from Pichia pastoris is 5.5–8.5 [39]. The results show an increase in the ∆ mean of RGB values up to pH 7.0, above which the response decreases slightly (Figure 4). This also similar with previous studies that have shown that the highest enzyme activities and stabilities are achieved at physiological pH values [14,38].