Elsevier

Biosensors and Bioelectronics

Volume 47, 15 September 2013, Pages 184-189
Biosensors and Bioelectronics

Spontaneous modification of carbon surface with neutral red from its diazonium salts for bioelectrochemical systems

https://doi.org/10.1016/j.bios.2013.02.051Get rights and content

Highlights

  • A new and simple method for neutral red (NR) surface modification.

  • Covalently graft NR onto carbon surfaces was achieved by spontaneous reduction of in situ generated NR diazonium salts.

  • The NR-modified electrodes showed a good stability when stored in PBS solution in the dark.

  • The NR-modified electrodes produced 3.63±0.36 times higher current than the unmodified electrodes.

Abstract

This study introduces a novel and simple method to covalently graft neutral red (NR) onto carbon surfaces based on spontaneous reduction of in situ generated NR diazonium salts. Immobilization of neutral red on carbon surface was achieved by immersing carbon electrodes in NR–NaNO2–HCl solution. The functionalized electrodes were characterized by cyclic voltammetry (CV), atomic force microscope (AFM), and X-ray photoelectron spectroscopy (XPS). Results demonstrated that NR attached in this way retains high electrochemical activity and proved that NR was covalently bound to the carbon surface via the pathway of reduction of aryl diazonium salts. The NR-modified electrodes showed a good stability when stored in PBS solution in the dark. The current output of an acetate-oxidising microbial bioanode made of NR-modified graphite felts were 3.63±0.36 times higher than the unmodified electrodes, which indicates that covalently bound NR can act as electron transfer mediator to facilitate electron transfer from bacteria to electrodes.

Introduction

Bioelectrochemical systems (BESs) which use bacteria as catalysts to drive oxidation and/or reduction reactions at solid-state electrodes are considered as a novel, promising technology for wastewater treatment, power production and valuable chemicals production (Rabaey, 2010). However, at present, the technology appears to be constrained by low current density mainly resulting from the low rate of extracellular electron transfer between bacteria and electrode. It has been previously shown that immobilization of electron transfer mediators, such as neutral red (NR) (Park and Zeikus, 1999, Park et al., 2000, Wang et al., 2011), methylene blue (MB) (Popov et al., 2012), anthraquinone-1,6-disulfonic acid (AQDS) (Lowy and Tender, 2008, Lowy et al., 2006), and 1,4-naphthoquinone (NQ) (Lowy et al., 2006), onto electrode surface is an efficient way to facilitate electron transfer from bacteria to electrode. Among those mediators, NR was considered as one of the ideal mediators for BES anodes as it possesses a redox potential of −0.325 V vs. normal hydrogen electrode (NHE) which is rather close to the −0.32 V (vs. NHE) of NAD+/NADH couple (Park and Zeikus, 1999). Since the latter locates at the low-potential end of the electron transfer chain in microbial respiration, a NR-immobilized electrode has the potential to harvest most of the energy released from the microbial oxidation of organic substrates.

So far, two methods have been employed to immobilize NR to carbon electrodes. The first is a chemical method which grafts NR to carbon surface through the amidation reaction between carboxylated carbon and the primary amine of NR (Jeykumari and Narayanan, 2007, Wang et al., 2011). NR immobilized in this way is tightly anchored to the carbon surface as the linkage is an amido bond. However, the disadvantage of this method is that large amount of nitric/sulfuric acids and organic solvents are required to carboxylate the carbon surface and set off the amidation reaction, respectively. Electropolymerization of NR is the other method to immobilize NR to carbon surface, which is generally carried out by means of cyclic voltammetry (CV) between −0.2 V and 1.2 V vs. SCE in a NR solution with sodium nitrate (Chen and Gao, 2007b). Polymerized NR (PNR) retains the electrochemical properties of the NR monomer as well as enhances the conductivity of the electrode. Hence, it has been used as an insoluble redox mediator for sensors and biosensors (Pauliukaite and Brett, 2008). Comparing to the amidation reaction method, this procedure is much simpler and more environmentally friendly. However, the modification system is complex as a potentiostat, 3-electrode system and an external power source are required. Moreover, since the PNR is just deposited and not chemically bound to the electrode surface, the electrical connection is not as strong as for covalent bonding. Therefore, new, simple and environmentally friendly methods need to be developed.

Electrochemical reduction of in situ generated aryl diazonium salts has been widely used to covalently modify carbon surfaces (Chehimi, 2012, Gooding, 2008). Aryl diazonium salts (N≡N+3Ar3R) can be easily and rapidly prepared in one step from a wide range of aromatic amines. Electrochemical reduction of diazonium salts can be carried out either by CV or poising the electrode potential. However, it has been recently found that some diazonium salts could be reduced spontaneously by the electrode substrates (Mahouche-Chergui et al., 2011). Among them, one of the electron transfer mediators, anthraquinone (AQ), has been covalently bound to the carbon surface using this method (Aulenta et al., 2011, Seinberg et al., 2008).

Since there is a primary aromatic amine group in the NR molecule and the structure of NR is similar to AQ, we hypothesized that NR could be covalently bound to carbon surfaces via spontaneous reduction of its diazonium salts. This work shows experimental evidence that NR can be covalently grafted to glassy carbon surfaces via spontaneous reduction as well as electrochemical reduction. Our results also revealed that significantly enhanced current densities were obtained for acetate-oxidising microbial bioanodes made of graphite felt modified with NR via spontaneous reduction of its in situ generated diazonium salts. Compared with the two previously mentioned methods, spontaneous modification provides a simpler and more environmentally friendly route to covalently graft neutral red to carbon surface.

Section snippets

Electrodes and electrode pre-treatment

Two types of carbon electrodes were used, glassy carbon (GC) plates (HTW, Germany, 20 mm×10 mm×2 mm with an effective surface area of 20 mm×10 mm) and graphite felts (Morgan, Australia, 40 mm×30 mm×2 mm). GC electrodes were used to study the mechanism of the modification procedure, and graphite felts were utilized as a BES anode for current generation test. Before modification, GC plates were polished successively in 1.0, 0.3, and 0.05 µm alumina slurries for 3 minutes at each grade. The electrodes were

Surface modification of GC with neutral red

To check whether NR was covalently bound to the GC surface, GC plates with different modification methods were tested by CV in oxygen free PBS solution. Fig. 2 shows the CV curves obtained. As can be seen from the figure, bare electrodes and electrodes immersed in NR–HCl solutions did not show any obvious reversible redox peaks, while electrodes immersed in NR–NaNO2 and NR–NaNO2–HCl solutions all showed an obvious pair of symmetric peaks with a midpoint redox potential around −0.55 V (vs

Conclusion

Immobilization of neutral red onto carbon electrodes was achieved via spontaneous reduction of in situ generated NR diazonium salts. The immobilized NR could withstand sonication in water, ethanol and acetonitrile rinses, which indicates a covalent bonding between NR moieties and the electrode surface. The formation of NR diazonium salts was essential for the NR immobilization, indicating that that NR was covalently grafted to the electrode surface via the pathway of reduction of aryl diazonium

Acknowledgments

The authors would like to acknowledge Prof. Korneel Rabaey and Dr. Jens Kroemer at CEMES for being the driver of this research area, Prof. Jurg Keller at AWMC for his continuous mentorship and Prof. Justin Gooding at UNSW for providing facilities and support towards electrode modifications and XPS analysis. This work was performed in part at the Queensland node of the Australian National Fabrication Facility, a company established under the National Collaborative Research Infrastructure

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