By modifying the gold microelectrode surface with a thin layer of PEDOT made in an organic solvent, we can get a higher surface area and boost the sensor sensitivity. The microelectrode procedure is a rapid analysis of antioxidants in various media. This can be applied to different contexts, ranging from monitoring beverages through to an immediate assessment of the status of hospitalized patients.
Use a suitable potentiostat to run cyclic voltammetry as the electrochemical technique of interest. Turn on the potentiostat and the computer attached to it. To test the communication between the computer and the instrument, start the software, then under the setup menu, select the hardware test command.
After hearing some sounds from the potentiostat, the hardware test results are shown in a separate window. Click OK and continue running the experiment. Sometimes by clicking the hardware test command, a link failed error appears.
Check the connection and port settings. After testing the connections, click on the setup menu, choose technique, and from the opening window, choose the cyclic voltammetry method. Then go back to the setup menu and click on parameters to enter the appropriate experimental parameters.
For example, to run 0.1 molar EDOT electropolymerization in an organic electrolyte on the bare gold microelectrode, set the initial potential to negative 0.3 volts, final potential to negative 0.3 volts, high potential to 1.2 volts, number of segments to eight, scan rates to 100 millivolts per second, and direction to positive. After entering the appropriate cyclic voltammetry parameters, prepare three electrode setups in a glass electrochemical cell, including a working electrode, a reference electrode, and a platinum wire counter electrode. Pass these clean and dried electrodes through the holes of an electrode holder attached to a stand, then place the holder above the electrochemical cell to insert the electrodes in the target solution or sample.
Ensure that there are no bubbles on the electrode surfaces. If there are bubbles, remove the electrodes, rinse them with deionized water again, and pat dry with a tissue. Then place the electrodes back into the stand holder and in the solution.
If there are bubbles around the reference electrode, tap the tip gently. If there are bubbles around the counter electrode after starting the scan, clean the counter electrode. If the cyclic voltammetry scan becomes noisy, clean the electrode surface and check the system connections, wires, and clips.
After ensuring that all the three wire connections for reference, working, and counter electrodes are correctly connected, start the experiment by clicking on run. To pretreat the gold microelectrode, polish it for 30 seconds with Alumina Slurry on an Alumina polishing pad placed on a glass polishing plate applying circular and eight-shaped hand motions, then rinse the microelectrode with deionized water. Insert it in a glass vile containing 15 milliliters of absolute ethanol and ultra sonicate for two minutes.
Next, rinse the microelectrode with ethanol and water and ultra sonicate it again for four minutes in deionized water to remove excess Alumina from the electrode surface. Finally, remove additional impurities by cycling in 0.5 molar sulfuric acid for 20 segments between negative 0.4 and positive 1.6 volt potentials at a scan rate of 50 millivolts per second. Ensure there are two clear peaks due to the formation and reduction of gold oxide at consistent anodic and cathodic potentials each time the electrode is cleaned in sulfuric acid.
To prepare 0.1 molar EDOT in an organic solution, first transfer one milliliter of the prepared 0.1 molar lithium perchlorate solution into an electrochemical cell. Then using a micropipette, add 10.68 microliters of the EDOT monomer to the electrochemical cell. To electropolymerize EDOT on the bare gold microelectrode surface, insert all electrode setups in the solution and run cyclic voltammetry.
Then using scanning electron microscopy, characterize the surface of this modified electrode. Rinse the electrodes with deionized water and dry them with a tissue. Then to use this PEDOT modified gold microelectrode for sensing purposes, acclimatize its surface to an aqueous solution by immersing the electrode in a 0.1 molar sodium perchlorate solution and running cyclic voltammetry scans.
Then after rinsing with deionized water, immerse this organically PEDOT-modified and acclimatized microelectrode in a phosphate buffer solution and run cyclic voltammetry to generate a background scan. Finally, take the electrode out of the buffer solution and without rinsing, immediately insert it into uric acid or milk solutions for running cyclic voltammetry scans. Next, to prepare 0.01 molar EDOT in an aqueous acetonitrile solution, use a micropipette and add 10.68 microliters of EDOT to one milliliter of acetonitrile in a glass vial, then add nine milliliters of deionized water to the vial to prepare 10 milliliters of 0.01 molar EDOT solution.
Add 110 milligrams of lithium perchlorate powder to the prepared EDOT solution to obtain a 0.1 molar lithium perchlorate solution and mix gently. Transfer the prepared solution to the electrochemical cell. Insert the electrodes in the aqueous acetonitrile solution and electropolymerize 0.01 molar EDOT on the electrode surface by running cyclic voltammetry, then characterize the surface of this modified electrode by scanning electron microscopy.
Analysis of uric acid content in regular fresh milk using the PEDOT sensor synthesized in the organic solution resulted in a 28.4 nano ampere anodic peak current at 0.35 volts, which is equivalent to a concentration of 82.7 micromoles. The second large oxidation peak in the cyclic voltammetry scan of regular milk at 0.65 volts is related to oxidizable compounds including electroactive amino acids such as cysteine, tryptophan, and tyrosine. The cyclic voltammetry scans obtained for caramel and white chocolate milk samples show a clear peak at 0.36 volts for uric acid, along with an additional peak at 0.5 volts that can be related to the presence of vanillic acid, one of the ingredients of flavored milk.
Cyclic voltammetry of the Belgian chocolate milk sample demonstrates a catechin oxidation peak at 0.26 volts and a catechin reduction peak at 0.22 volts. The 0.3 volts peak current appearing as a sharp peak at the tail of the catechin peak is due to uric acid oxidation. Cyclic voltammetry of Colombian espresso milk sample exhibits broad anodic and cathodic peak currents at 0.35 volts and 0.23 volts respectively due to the major phenolic antioxidants in coffee, namely chlorogenic and caffeic acids.
Cleaning and perturating the electrode is the most important part of this experiment that can affect the obtained current signals. The more detailed analysis of antioxidant compounds is needed, then we can turn to chromatographic methods such as LCMS. However, this is time consuming and is not needed for every sample.
