Team:Mingdao/Demonstrate
Glucose oxidase (GOX) and alcohol oxidase (AOX) are oxidoreductase enzymes which catalyze the oxidation of glucose and alcohol, respectively. When applied to an electrochemical analyzer, the electrode covered with a given enzyme will generate current signal, which intensity is corresponding to the concentration of substrates. Before this application, the enzyme’s reduction potential need to be determined and set up a program to convert the current intensity to a digital number revealing the real concentration of a sample measured. For example, in the blood glucose meter, the reduction potential of GOX is -97 ± 3 mV (Anal Chem. 2014) and set on the analyzer to facilitate the redox reaction between GOX enzyme and the substrate, glucose. Unfortunately, the reduction potential of AOX is not well studied. Therefore, we have to determine the reduction potential of AOX prior to applying on the blood alcohol meter we want to create.
Cyclic voltammetry is a potentiodynamic electrochemical measurement, in which the working electrode potential is ramped linearly versus time. The ramping potential move forward to the maximum and backward to the minimum, the cycle continues until the end of the experiment. In a CV experiment, electrochemical properties of a chemical molecule in the solution can be studied.
To analyze the electrochemical properties of AOX, a cyclic voltammetry experiment was performed to determine the reduction potential of AOX. 50µl of the K2HPO4 solution containing the AOX enzyme displayed on the surface of E. coli was put onto the test strip. Then, 20µl of 10% alcohol was added and mixed in the solution. As shown in Figure 2, the result indicated that a current peak (the current values at 2.0E-05~2.2E-05) was induced around the voltage of -1200 mV, implying that the AOX enzyme did undergo redox reaction. But the current was not much significantly changed when responding to the different concentrations of alcohol.
To improve the redox reaction, we added 200 µl of potassium ferricyanide (K3[Fe(CN)6]) as electron mediators to facilitate the transfer of electrons to electrodes. When mixed with various concentration of ethanol, the electric currents rose significantly (i.e., the higher current intensity between 2.6E-05~2.9E-05) and dose-dependently upon the increasing concentrations of alcohol around the voltage of -1200 mV (Figure 2).
Figure 3: The current strength of AOX enzyme with different concentration of ethanol in an amperometry experiment.
Amperometry is a way to detect the change of electric current along with the time (IT) in a given voltage. Followed the CV experiment, we’d like to know the current variation dependent on the different concentration of ethanol with the electric potential set at -1200 mV. As Figure 3 showed, the increased currents were measured along with the three different concentrations of ethanol (i.e., 0.385%, 0.741% and 1.071%), indicating the enhanced AOX enzyme activity was responding to the increasing concentration of ethanol.
Figure 2: The electrochemical properties of AOX enzyme with different concentration of ethanol in a cyclic voltammetry experiment.
Based on the data above, we plotted a concentration calibration curve (Figure 4) with a mathematical equation, which could be applied to estimate unknown concentration of alcohol samples using current strength.
Figure 4: The concentration calibration curve based on the current strength reaction with ethanol.
Figure 5: The prototype of our new product – IGEM BLOOD ALCOHOL METER (iMeter)
Based on the mathematical formula obtained from the concentration calibration curve, we are ready to create a test strip with AOX to measure alcohol concentration and apply it to be a blood alcohol meter. We called it IGEM BLOOD ALCOHOL METER (iMeter).
This work was done by the collaboration with BIONIME Corp. who is an innovator working on Blood Glucose Meter design and develop. We thank the executive vice president, Dr. Wiley Chung (Shie-Shiun Jung), went to Mingdao High School to help us develop the blood alcohol meter. From him, we not only learned the electrochemical chemistry, but also completed the amazing experiments in our lab.
