The authors acknowledge the University of Groningen for financial support.

1. Preparation of Graphite Oxide
NOTE: Graphite oxide is prepared according to Hummers&#39; method10,11.
<ol>
	<li>Add 3 g of graphite into 69 mL of concentrated H2SO4 and stir the solution until the graphite has completely dispersed. Add 1.5 g of sodium nitrite and leave it for 1 h while stirring. Then, place the container in an ice bath.</li>
	<li>Add 9 g of potassium permanganate into the dispersion and remov...

<ol>
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W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polyaniline+functionalized+electrochemically+reduced+graphene+oxide+chemiresistive+sensor+to+monitor+the+pH+in+real+time+during+microbial+fermentations.">Polyaniline functionalized electrochemically reduced graphene oxide chemiresistive sensor to monitor the pH in real time during microbial fermentations.</a> Sensors and Actuators B Chemical. 264, 38-44 (2018).</li><li>Sha, R., Komori, K., Badhulika, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Amperometric+pH+Sensor+Based+on+Graphene-Polyaniline+Composite.">Amperometric pH Sensor Based on Graphene-Polyaniline Composite.</a> IEEE Sensors Journal. 17 (16), 5038-5043 (2017).</li><li>Huai-Ping, C., Xiao-Chen, R., Ping, W., Shu-Hong, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flexible+graphene-polyaniline+composite+paper+for+high-performance+supercapacitor.">Flexible graphene-polyaniline composite paper for high-performance supercapacitor.</a> Energy &amp; Environmental Science. 6, 1185-1191 (2013).</li><li>Xiang, J., Drzal, L. 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The authors have nothing to disclose.

It is essential that the GO layers completely cover the gold electrode wires after the deposition of GO. If the gold electrodes are not covered with GO, polyaniline will not only deposit on ERGO but also on the visible gold electrode wires directly. Deposition of polyaniline on the gold electrode wires may have implications on the performance of the electrode. After the reduction of GO to ERGO, the electrode is dried at 100 &#176;C to strengthen the bonding between the ERGO layer and the gold electrode wires. The resista...

pH plays a vital role in many chemical and biological processes. Even small changes in the pH value alter the process and adversely affect the outcome of the process. Hence, it is necessary to monitor and control the pH value during every stage of experiments. The glass-based pH electrode has been successfully used to monitor pH in many chemical and biological processes, although the use of a glass electrode poses several limitations to measuring pH. The glass-based pH electrode is relatively large, fragile, and small leakages of the electrolyte into the sample are possible. Furthermore, the electrode and electronics are relatively expensive for applications in 96-well screening fermentation systems. Moreover, the electrochemical sensors are invasive and consume the sample. Hence, it is more advantageous to use non-invasive, reference-less sensors.
Nowadays, miniaturized reaction systems are favored in many chemical engineering and biotechnology applications as these microsystems provide enhanced process control, along with many other advantages over their macro system analogs. To monitor and control the parameters in a miniaturized system is a challenging task as the sizes of the sensor to measure, for instance, pH and O2, need to be minimized as well. The successful production of microreactors for biological systems require different kinds of analytical tools for process monitoring. Hence, the development of smart microsensors plays a significant role in carrying out biological processes in microreactors.
Recently, there have been several attempts to develop smart pH sensors using chemiresistive sensing materials like carbon nanotubes and conducting polymers1. These chemiresistive sensors require no reference electrode and are easy to integrate with electronic circuits. Successful chemiresistive sensors make it possible to produce smart sensors that are cost-effective and easy to manufacture, require a small volume for testing, and are non-invasive.
Here, we report a method to develop an electrode with polyaniline-functionalized, electrochemically reduced graphene oxide. The chemiresistive electrode operates as a pH sensor during an L. lactis fermentation. L. lactis is a lactic-acid-producing bacterium used in food fermentation and food preservative processes. During fermentation, the production of lactic acid lowers the pH, and the bacterium stops growing at a low pH2,3,4.
A fermentation medium is a complex chemical environment that contains peptides, salts, and redox molecules which tend to interfere with the sensor surface5,6,7,8,9. This study shows that a pH sensor based on chemiresistive material with a proper surface protection layer could be used to measure pH in this kind of complex fermentation media. In this study, we successfully use Nafion as the protection layer for polyaniline-coated, electrochemically reduced graphene oxide to measure the pH in real-time during an L. lactis fermentation.

<table>
	<tbody>
		<tr>
			<td>Graphite flakes</td>
			<td>Sigma Aldrich</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Sulfuric acid (H2SO4)</td>
			<td>Merck</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium nitrite (NaNO2)</td>
			<td>Sigma Aldrich</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium permanganate (KMnO4)</td>
			<td>Sigma Aldrich</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>30 % H2O2</td>
			<td>Sigma Aldrich</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>HCL</td>
			<td>Merck</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Aniline</td>
			<td>Sigma Aldrich</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>5wt % Nafion</td>
			<td>Sigma Aldrich</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>M17 powder</td>
			<td>BD Difco</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphoric acid (H3PO4)</td>
			<td>Sigma Aldrich</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Boric acid (HBO3)</td>
			<td>Merck</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Acetic acid</td>
			<td>Merck</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Hydroxide</td>
			<td>Sigma Aldrich</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium dihydrogen phosphate</td>
			<td>Sigma Aldrich</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Dipostassium hydrogen phosphate</td>
			<td>Sigma Aldrich</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Au Interdigitated electrodes</td>
			<td>BVT technology - CC1 W1</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Potentiostat</td>
			<td>CH Instruments Inc (CH-600, CH-700)</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

manufacturing of a nafion-coated, reduced graphene oxide/polyaniline chemiresistive sensor to monitor ph in real-time during microbial fermentation

Here, we report the engineering of a solid-state micro pH sensor based on polyaniline-functionalized, electrochemically reduced graphene oxide (ERGO-PA). Electrochemically reduced graphene oxide acts as the conducting layer and polyaniline acts as a pH-sensitive layer. The pH-dependent conductivity of polyaniline occurs by doping of holes during protonation and by the dedoping of holes during deprotonation. We found that an ERGO-PA solid-state electrode was not functional as such in fermentation processes. The electrochemically active species that the bacteria produce during the fermentation process interfere with the electrode response. We successfully applied Nafion as a proton-conducting layer over ERGO-PA. The Nafion-coated electrodes (ERGO-PA-NA) show a good sensitivity of 1.71 &#937;/pH (pH 4 - 9) for chemiresistive sensor measurements. We tested the ERGO-PA-NA electrode in real-time in the fermentation of Lactococcus lactis. During the growth of L. lactis, the pH of the medium changed from pH 7.2 to pH 4.8 and the resistance of the ERGO-PA-NA solid-state electrode changed from 294.5 &#937; to 288.6 &#937; (5.9 &#937; per 2.4 pH unit). The pH response of the ERGO-PA-NA electrode compared with the response of a conventional glass-based pH electrode shows that reference-less solid-state microsensor arrays operate successfully in a microbiological fermentation.

The appearance of a strong reduction peak around -1.0 V (Figure 3) illustrated the reduction of GO to ERGO12,13,14,22. The intensity of the peak depends on the number of GO layers on the electrode. A thick black film completely covered the gold wires on the electrode. At that point, the two insulated gold electrodes were conductive ...

Watch this Scientific Journal Video about Manufacturing of a Nafion-coated, Reduced Graphene Oxide/Polyaniline Chemiresistive Sensor to Monitor pH in Real-time During Microbial Fermentation at JoVE.co

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

Here, we report the protocol for the fabrication of a Nafion-coated, polyaniline-functionalized, electrochemically reduced graphene oxide chemiresistive micro pH sensor. This chemiresistor-based, solid-state micro pH sensor can detect pH changes in real-time during a Lactococcus lactis fermentation process.

Here, we report the engineering of a solid-state micro pH sensor based on polyaniline-functionalized, electrochemically reduced graphene oxide (ERGO-PA). ...

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.

manufacturing-nafion-coated-reduced-graphene-oxidepolyaniline

Research

JoVE Journal

Bioengineering

8.3K 조회수.  University of Groningen. 이 기술은 바이오 마스크 변환이나 유기 폐기물의 바이오 가스 생산과 같은 바이오 공정 기술 분야의 주요 질문에 답하는 데 도움이 될 수 있습니다. 이 화학 성 pH 센싱의 주요 장점은 전극이 작고 튜브가 많은 수의 복제되어 참조 전극없이 작동한다는 것입니다. pH가 중요한 매개 변수이기 때문에 이 기술의 의미는 여러 미세 반응기로 확장됩니다.기존의 pH 전극은 마이?...

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