Supplementary Materials: Automated Bioanalyzer Based on Amperometric Enzymatic Biosensors for the Determination of Ethanol in Low ‐ Alcohol Beers

In this work, a new automated bioanalyzer based on the use of enzymatic biosensors as amperometric detectors is reported. This automatic bioanalyzer is configurable both as continuous flow and flow injection analysis systems and enables both on-line and off-line monitoring of ethanol in low-alcohol beer to be performed. The attractive analytical and operational characteristics demonstrated by the automated bioanalyzer make it a promising, simple, rapid, and reliable tool for quality control of this beverage in the beer industry, either during the manufacturing process or in the final product. Moreover its applicability to the analysis of the ethanol content in different non-alcoholic beers working at different modes was successfully demonstrated.


Sampling Unit Design
The optimized variables were membrane pore diameter, number of membranes equipped in the device, and contact surface between acceptor and donor solutions. As expected, the larger number of membranes, the smaller pore size, and the smaller contact surface between donor and acceptor solutions meant that a lower amount of ethanol reached the detector. Accordingly, a lower sensitivity and a wider linear range were obtained (Table S1). Thus, a contact surface diameter of 10 mm and one PTFE membrane of 0.45 μm pore diameter were chosen for further work since they provided an adequate sensitivity for the analysis of non-alcoholic beers and beers with an ethanol concentration below 1.0% (v/v).

Electrical Design
In the bioanalyzer, two parts should be controlled automatically: the amperometric detector, which applies a constant potential during the measurement and acquires the intensity values of current generated in the biosensor response, and an active interface or manager of the instrument, which allows the control of electronically active components of the flow system of the bioanalyzer (valves and pumps) to be carried out. There is also a computer-amperometric detector connection that enables the different experimental variables applied by the amperometric detector to be controlled and the intensity current data generated to be recorded; one active computer-interface connection, which enables the manager to act on the various electronic components of the bioanalyzer; and a computer for acquiring and processing the data transmitted by the bioanalyzer and for controlling the amperometric detector and the active interface. Moreover, as well as being configured as a feedback system controller, needed in an automated instrument for decision making depending on the amperometric signals obtained, the computer is the part of the instrument that allows operator interaction with the bioanalyzer and the real-time display of the information collected during the monitoring process of the parameter under study.
Therefore, considering the elements that should constitute the control system of the bioanalyzer, the installation of the equipment components required an automatic operation of the prototype, which was carried out. As in Figure S4a, the amperometric detector and the printed circuit manager plate that controls the solenoid valves and peristaltic pumps were placed inside the instrument. The connections and the switch to turn on the instrument are placed outside the device ( Figure S4b). Connection with the computer is made with a USB connector type. Finally, the equipment is powered with a power supply of 24 V.

Software Applications
For the operation of the automated bioanalyzer, software applications were developed, which allowed the instrument to be controlled, the analytical information to be visualized, and a real feedback system to be obtained with respect to the registered information. With the objective of using the same prototype in both on-line and off-line analysis, the development of two softwares was realized.
Considering firstly the control software for on-line analysis, the user interface will allow the operator to display the recording parameters such as the working potential, the time interval for recording data of amperometric current, and the time for the analysis. A process indicator, which comprises a simplified diagram of the flow system and allows the analysis to be tracked; the faceplate, in which the current-time record is displayed in real time; the calculated ethanol concentration in the beer sample; and the concentration of the standard solution used as a reference are shown ( Figure S5). To control the developed application, a set of configuration parameters, from which it is possible to program a method of analysis and operation for the bioanalyzer, was established. Thus, the analysis and decision making by the system are performed in predefined conditions. Depending on the recorded results and considering the parameters established from the configuration file of the application, the computer will send specific orders to the active interface so that the required operations at any time were performed.
The configuration parameters depended in a first stage on the working mode (CFA or FIA) and, thereafter, on the analysis phase at which the action is being performed (baseline stabilization or measurement). Thus, the values that have been introduced in the file of configuration parameters, characteristic of this bioanalyzer, consider the dimensions of the flow system, the working flow rate, and the behavior of biosensors, so that the current stabilization and separation of the signals are output in an efficient manner to obtain analytical measurements with adequate accuracy and reproducibility.
Once the measurement stage is finished, appropriate changes in the valve module are performed by the system, so that the stabilization stage restarts to carry out the next measurement once the baseline is reached again. Depending on the analysis method and configuration parameters established, it will correspond to the measurement of the standard solution or the sample.
With respect to the acquisition and data processing for the on-line application, such information is stored in measurement files ( Figure S6). These files provide different information depending on the monitoring mode of the analytical signals. An example of a measurement file of the CFA mode, in which sample measurements have been recorded every second, is shown in Figure S6a. In Figure  S6b a measurement file of the FIA mode is displayed, showing, for each measurement, date, time, ethanol concentration, amperometric peak current, and type of measurement (standard solution or sample and, in the second case, which replica it is). The control software for the off-line application has a user interface, which will allow the operator to display, in addition the information commented above for the on-line application, a control panel from which it is possible to configure with which solution (standard or beer) each entry of the valve module in the sample selector is occupied, the number of replicas to be measured of each sample, and the number of sample measurements to be carried out before the system orders the bioanalyzer to be recalibrated ( Figure S7). As shown in the figure, the application was developed for an eight-entries sample selector.  To control this application, a specific configuration file for this type of analysis was included, from which the analysis method to be performed is established so that the data introduced in such a file will appear in the control panel of entries of the user interface. Regarding the acquisition and data processing obtained by the system for the off-line application, in addition to the analytical information indicated above for the FIA mode of the on-line application, statistical analysis of each sample was included in the measurement file. Thus, the mean value, standard deviation, and relative standard deviation of the results were included ( Figure S8).