Name: manufacturing of a nafion-coated, reduced graphene oxide/polyaniline chemiresistive sensor to monitor ph in real-time during microbial fermentation
Uploaded: 2019-01-07T14:00:00
Description: 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

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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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). ...

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