Manufacturing of a Nafion-coated, Reduced Graphene Oxide/Polyaniline Chemiresistive Sensor to Monitor pH in Real-time During Microbial Fermentation

This technology can help us to answer key questions in a field of bioprocess technology, such as biomask conversion, or the production of biogas from organic waste. The main advantage of this chemiresistive pH sensing is that the electrodes are small, is the tube replicate in large number, and work without reference electrode. The implications of this technique extends to multiple microreactors, because pH is an important parameter.

Conventional pH electrodes are too expensive or too large to be used in a microreactor screening platform. This method can provide insight into multiple permutation passes on a small scale. It can also be applied in other chemical or biological passes where pH measurements are important.

First, add three grams of graphite to 69 milliliters of concentrated sulfuric acid, and stir the solution until the graphite has completely dispersed. Add 1.5 grams of sodium nitrite. After one hour of stirring, place the container in an ice bath.

Add nine grams of potassium permanganate to the dispersion, and remove the container from the ice bath. After allowing the solution to warm to room temperature, add 138 milliliters of Milli-Q water drop-wise. Then add 420 milliliters of Milli-Q water and maintain the temperature at 90 degrees Celsius for 15 minutes using a hot plate.

Add 7.5 milliliters of 30%hydrogen peroxide to the hot dispersion. After transferring the dispersion to a centrifuge tube, collect the product by centrifugation at 10, 000 times G for 20 minutes. After discarding the supernatant, wash the pellet four times with warm double-distilled water, and two times with 10%hydrochloric acid.

Finally, wash the pellet two times with ethanol and dry it at 50 degrees Celsius in the oven. Disperse 10 milligrams of graphite oxide in 10 milliliters of Milli-Q water, and then sonicate the dispersion in an ultrasonic bath for six hours. Following ultrasonication, remove the unexfoliated graphite oxide flakes by centrifugation for 30 minutes at 2700 times G.After centrifugation, discard the solid particles, and use the supernatant for further experiments.

To prepare the working solution, dilute the graphene oxide stock solution two-fold with Milli-Q water. Now add two microliters of the graphene oxide working solution onto top of an exposed interdigitated gold electrode. After drop casting, dry the graphene oxide electrode at room temperature for 12 hours.

Insert the electrode in a PDMS-electrode holder. Place the other part of the electrode holder, which serves as a solution reservoir, on top of the electrode. Assemble the holders by clipping the two parts together using two paper clips.

Next, pipette 300 microliters of 2 molar phosphate buffer in the reservoir. Then place the reference and counter electrodes in the solution in such a way that they are placed close to the surface of the graphene oxide film. Connect the electrodes with a potentiostat, connected to a computer for data acquisition.

Use cyclic voltammetry for the electrochemical reduction, and select the appropriate potential range and scan rate. Cycle the voltage over the electrode 10 times between zero to minus 1.2 volts. After the experiment, remove the electrode from the holder, and repeatedly wash it with Milli-Q water.

Then dry the electrode in an oven at 101 degrees Celsius for 12 hours. When the electrode is dry, remove it from the oven and allow it to cool down to room temperature. Then measure the conductivity of the ErGO electrode with a multimeter.

Prepare a 10 millimolar solution of aniline monomer for the polyaniline functionalization, by dissolving five microliters of 10 millimolar aniline in five milliliters of one molar sulfuric acid. Add 300 microliters of aniline monomer to the solution reservoir. Then place the ErGO deposited electrode into the electrode holder as previously described.

Use cyclic voltammetry for the electropolymerization of aniline to functionalize the ErGO into ErGO-PA, and select the appropriate potential range and scan rate. Cycle the voltage over the electrode 50 times, between zero to 9 volts. After the polyaniline deposition, remove the electrode and repeatedly wash it with Milli-Q water.

Then dry the electrode at 80 degrees Celsius in the oven for 12 hours. When the electrode is dry, remove it from the oven and allow it to cool down to room temperature before measuring the conductivity of the electrode with a multimeter. Next, add 2 molar sodium hydroxide to Britton-Robinson buffer solution, until the pH is five.

To prepare a Britton-Robinson universal buffer solution, mix 04 moles of phosphoric acid, 04 moles of acetic acid, and 04 moles of boric acid, in 8 liters of Milli-Q water. Then, add Milli-Q water until the final volume is one liter. After conditioning the electrode in the pH five buffer solution, measure its resistance in solutions of different pH, by first dipping it directly in the buffer solution.

Then connect the other part of the electrode to a computer-controlled potentiostat for data acquisition. Choose amperometry current versus time curve from the list of techniques, and apply a 100 millivolt potential difference to the electrode. After the measurements, dry the electrode at room temperature for 12 hours.

Add five microliters of five weight-percent nafion on top of the ErGO-PA electrode, and dry the electrode at room temperature for 12 hours. After the nafion coating, heat the electrode in the buffer solution at pH five for 24 hours before pH measurements. After conditioning in the pH five buffer solution, remove the nafion-coated ErGO-PA electrode and measure the resistance of the electrode from pH four to nine, as previously described.

Add 9.3 grams of M17 powder to 250 milliliters of demineralized water. Slowly agitate the solution until the powder dissolves completely. Then autoclave the solution at 121 degrees Celsius for 15 minutes.

Next, add 50 milliliters of the sterilized M17 medium in a 250 milliliter sterilized flask with a magnetic stir bar. Add 8 milliliters of autoclaved 1 molar glucose solution to the medium. Then, inoculate the solution with 10 microliters of L.lactis culture, previously grown in the same culture medium.

Place the flask with the inoculated culture medium on a magnetic stir plate in an incubation oven at 30 degrees Celsius for 18 hours while stirring. Monitor the pH during incubation. Place the ErGO-PA-NA electrode into the L.lactis culture and close it with a cotton plug.

Then place the set up into a thermostat at 30 degrees Celsius to grow L.lactis. Following this, apply 100 millivolts to the electrode and measure the current against time. Take 5 milliliter samples at different time points to measure offline the optical density at 600 nanometers, and the pH with a conventional glass electrode.

Continue the measurements until the optical density of the culture becomes constant, indicating that the bacteria are not growing anymore. The appearance of a strong reduction peak around minus 1.0 volts illustrated the reduction of graphene oxide to ErGO. The intensity of the peak depends on the number of graphene oxide layers on the electrode.

When the ErGO-PA electrode was placed in pH four to nine buffer solutions, the current increased with increasing pH due to the doping and de-doping of holes during the protonation deprotonation process. The response of the electrode was immediately stable when the addition of sodium hydroxide stopped at a particular pH. The conductivity of the electrode was not affected much by the nafion coating.

But a few ohms of difference in the resistance value occurred, and changed the base current value of the ErGO-PA electrode. Similar to the ErGO-PA electrode, the resistance of the ErGO-PA-NA electrode changed when the pH of the buffer solution changed from four to nine. Once the growth of L.lactis started, the current of the ErGO-PA-NA electrode decreased gradually, and then accelerated during the exponential growth phase, reaching a stable value at the end of the growth.

The final value of the current is comparable to the current value of the ErGO-PA-NA electrode tested in buffer solution. While attempting this procedure, it is important to remember to completely cover the gold electrodes with the graphene oxide. This technology paved the way for researchers in the field of process control, to manufacture and use small pH electrodes for biological and chemical systems.

Don't forget that working with concentrated sulfuric acid, potassium permanganate, and hydrogen peroxide can be extremely hazardous. This procedure must be performed in a fume hood.