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

Summary

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.

Abstract

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 Ω/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 Ω to 288.6 Ω (5.9 Ω 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.

Introduction

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-wel....

Protocol

1. Preparation of Graphite Oxide

NOTE: Graphite oxide is prepared according to Hummers' method^10,11.

1. 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.
2. Add 9 g of potassium permanganate into the dispersion and remove the container from the ice bath. Allow the solution to warm up to room temperature.
3. First, add 138 mL of distilled water dropwise. Then, c....

Results

The appearance of a strong reduction peak around -1.0 V (Figure 3) illustrated the reduction of GO to ERGO^12,13,14,22. 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 .......

Discussion

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 °C to strengthen the bonding between the ERGO layer and the gold electrode wires. The resista.......

Materials

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