3.3. Analytical Properties of the AOX/PANI Biosensor
3.3.1. Response Time
The response time of the biosensor was counted (using a digital stopwatch with naked eye inspection), as a time needed to change the colour after the biosensor reacted with ethanol solution, just by dipping it into sample solution. The response time was found to be 5 s, which is a quite rapid response for this type of the biosensor. This might be due to the thin film (0.7 μm) used in this biosensor as well as the nanofibres of the PANI film, which make the color change really fast compared to other AOX based biosensors [20–24]. In addition, the response time of the biosensor was also recorded using a digital video camera (Canon PowerShot A4000) for quantifying the colour changes over time. It also found, in good agreement with the above procedure, that 5 s was 90% of the response time. Thus, for the response time, 5 s was used for further experiments.
3.3.2. Reproducibility
In order to examine the reproducibility of the biosensor, the biosensor response to ethanol was recorded under the same experimental conditions. The biosensors’ response toward 1% of ethanol was tested as given in Figure 5, which shows good reproducibility (R.S.D. = 1.6%). This reproducibility is higher when compared with other reported AOX-based biosensors [20–24]. Since, the biosensor is intended for use as a one shot or disposable test, this study demonstrated a very small variation between produced biosensors in terms of response toward ethanol.
Most biosensor assemblies based on AOX consist of bi-enzyme designs where, together with the AOX, a peroxidase is used for hydrogen peroxide detection. Redox hydrogels are commonly used for this type of biosensor construction, but since this procedure implies manual mixing of both enzymes together with the cross-linker, problems may appear in the optimization procedure and reproducibility of these one-layer bi-enzyme biosensors [40,41]. The advantages of this newly developed biosensor assembly consist of its simple construction procedure and good reproducibility (0.6%). Another advantage of these new AOX/PANI biosensors is that they can be used for visual detection. Thus, the biosensor showed a sufficiently good reproducible behaviour and can be used for practical measurements of ethanol.