Observation of electron transfer between bacteria and high conductivity graphene–PEDOT composites

Abstract

The direct observation of electron transfer in a system comprising bacteria and a conductive substrate is reported. P. aeruginosa is shown to reduce high conductivity, electroactive and biocompatible graphene–PEDOT composites.

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Understanding and controlling the interaction of biological matter with synthetic materials is increasing in importance, driven by society's progression towards improved healthcare, a cleaner environment, and sustainable energy production. Of popular interest is the study of biological matter with surfaces and/or coatings, where the central theme of the studies to date has been how (human or animal) cells interact with engineered materials; from implants¹ to wound dressings² to bio-sensors.³ In parallel with this is the research field of anti-bacterial and anti-biofouling surfaces; surfaces which are designed to resist or inhibit attachment of cells or bacteria (in some cases deliberately killing the bacteria).^4,5 A renewed area of research is related to surfaces engineered to enhance bacterial attachment,⁶ where the creation of a viable bacterial biofilm on the surface is promoted. When the bacteria are electroactive, their attachment can lead to the exchange of electrical charge from the bacteria to the surface. It is this mechanism that forms the fundamental basis for the concept of Microbial Fuel Cells (MFCs), a bio-electrochemical system.⁷ In a similar manner, sensing devices have been envisaged that make use of the electron transfer to detect the presence of bacteria at a surface.⁸

Conducting polymers, and composites thereof,^9,10 are a class of materials that possess desirable electrical properties; being highly conductive,¹¹ electroactive,³ semi-metallic,¹² and electrocatalytic,¹³ to name but a few. Properties such as these allow conducting polymers to be employed in a range of sensor applications (such as electrochemical transistors^14,15). In the scientific literature, a relatively small number of studies exist regarding enhanced bacterial interaction with conducting polymers,^16–20 in contrast to the numerous studies on enhanced interactions between conducting polymers and cells (see pioneering work by Wallace and co-workers for more details^21,22). Thus far, the attachment of bacteria to a conducting polymer substrate is limited to observation by fluorescence or electron microscopy.^16–20 The electrical interaction (electron transfer) is later inferred from the fact that an electrical current can be generated when complete MFCs are fabricated. In this study we directly observe the electron transfer from bacteria to the substrate through the changing optical and electrical properties of high conductivity graphene-conducting polymer composites. The transfer of electrons electrochemically reduces the conducting polymer, leading to the modified properties that allow for direct and simple macroscopic observation.

In the study of Pernites et al.,¹⁹ bacterial attachment (E. coli) was observed on doped electropolymerised polythiophene, without reference to any polymer electrical properties. Wang et al.¹⁸ reported the concept of 2D materials (graphene) and conducting polymers (poly(3,4-ethylenedioxythiophene), PEDOT) as a multilayer thin film for attachment of bacteria for MFCs. In their study the electrode consisted of a carbon paper substrate, coated with a layer of reduced graphene oxide, and over-coated with electropolymerised PEDOT. Electrical interaction and viability of the bacteria with/on the conductive substrate was inferred from the generation of an electrical current when a complete MFC was constructed. Despite recent studies such as these (and others), direct evidence of electron transfer has yet to be demonstrated.

Recently, Vucaj et al.²³ fabricated nanocomposite thin films combining surfactant-exfoliated graphene²⁴ and high conductivity PEDOT synthesised via vapour phase polymerisation (VPP).¹¹ Such high conductivity VPP-PEDOT is biocompatible.²⁵ Furthermore, PEDOT is electroactive, undergoing changes in its optical and electrical properties upon direct interaction (chemically or electrochemically) with reducing or oxidising species.^26,27 Incorporating graphene into the PEDOT matrix prepared through vapour phase polymerization has two important effects. Firstly, it increases the mechanical properties of the films making the graphene–PEDOT composite more robust. Secondly, the performance of the PEDOT film with graphene in electrocatalytic studies is significantly enhanced.

Traditional electrode materials such as metals and carbon paper do not exhibit such behaviour. Herein, it is envisaged that the high electrical conductivity (σ ∼ 2000 S cm⁻¹) of this electroactive and biocompatible material will allow for direct observation of electron transfer between a bacterial system and the substrate. The use of the term ‘bacterial system’ implies that the mechanism of electron transfer may either be directly between bacteria and substrate, or mediated indirectly by biological processes.

To demonstrate the bacterial biocompatibility of these graphene–PEDOT composite substrates, thin films supported on non-conducting glass were prepared which were subsequently exposed to cultures of Pseudomonas aeruginosa. Using fluorescent staining, (Fig. 1), the viability of the P. aeruginosa cells was demonstrated by the large number of green-stained bacteria. This implies that the viability of P. aeruginosa on the high conductivity, electroactive material is good. Approximately 30% of the bacterial cells appeared to be non-viable (Fig. S1†). This was more than compensated for by the high attachment density relative to glass controls. Herein is the first demonstration of bacterial viability on a conducting polymer-based material suitable for use as a complete electrode (previous reports, such as Wang et al., report attachment only without viability information, while Pernites et al. use low conductivity conducting polymers).

Fig. 1 P. aeruginosa viability after adhesion to (A) a glass reference, and (B) a graphene–PEDOT substrate. The bacteria show high attachment density and viability on the biocompatible graphene–PEDOT when compared to the glass reference. This graphene–PEDOT thin film has an as-prepared conductivity of ca. 2000 S cm⁻¹.

The electroactive behaviour of conducting polymers is commonly used in electrochromic devices where the optical properties of the polymer can be electrochemically modified.^28,29 This modification arises from the addition or removal of electrons from the polymer's conjugated chain, via reduction or oxidation respectively. In the case of PEDOT, it can be switched between a dark blue state (relatively lower conductivity) and a transparent state (relatively higher conductivity) when transitioning from the reduced to oxidised state.¹¹ This behaviour is used to directly observe the interaction between the attached bacteria and the graphene–PEDOT. Electron transfer could be directly between the bacteria and substrate, or indirect via chemical mediators produced by the bacteria.

As shown in Fig. 2, the composite thin film exposed to P. aeruginosa for two weeks appears darker in colour (Fig. 2B) with a significant increase in the sheet resistance (Fig. 2D). For a constant film thickness, this equates to a change in electrical conductivity from 2000 S cm⁻¹ to 730 S cm⁻¹ after exposure to the bacteria. Importantly, the culture media was buffered at neutral pH, which avoids any chemical oxidation or reduction³⁰ of the graphene–PEDOT that would influence the measured properties.

Fig. 2 (A–C) Optical and (D) electrical properties of graphene–PEDOT thin films before and after P. aeruginosa attachment, and after subsequent redox reaction with AgNO₃. The (A to B) darkening of the conductive film, with (D) a concomitant increase (×2.7) of the measured sheet resistance, indicates the substrate has been reduced by the bacteria. The bacteria-reduced graphene–PEDOT can subsequently facilitate further redox reactions, where the graphene–PEDOT oxidises to become (C) more transparent and (D) lower in sheet resistance. In (C) the top portion of the graphene–PEDOT was exposed to AgNO₃.

The change in sheet resistance and colour of the graphene–PEDOT are direct observations of the reduction of the graphene–PEDOT being due to the presence of bacteria. This reduction mechanism has implications for a range of applications, including but not limited to (i) sensors, (ii) MFCs and (iii) controlled chemical release. In the simplest form, monitoring of the electrical resistance and/or optical absorbance of the graphene–PEDOT allows for detection of the bacteria present on or near the substrate. Extending this, the electrons which have been transferred to (and hence stored within) the graphene–PEDOT can then be used to generate an electrical current (thus creating an MFC). To demonstrate the presence of stored electrons, the bacteria-reduced graphene–PEDOT in Fig. 2B was subsequently exposed to a solution of AgNO₃. Similar to that shown by Lee et al. for Au on reduced PEDOT,³¹ a redox reaction occurs where the Ag⁺ reduces to Ag⁰ while the graphene–PEDOT oxidises (optical changes shown in Fig. 2C, with a sheet resistance in agreement with that before bacterial attachment).

Studies where the P. aeruginosa were prevented from attaching to the electrode were also undertaken in order to determine the mechanism of electron transfer. Similar changes to the measured sheet resistance and colour of the film were observed as for the direct attachment. This suggests that the mechanism of electron transfer is predominately by chemical mediators rather than directly between the bacteria and substrate. Proximity to the electrode interface should enhance diffusion.

Finally, electron transfer from the bacteria to the graphene–PEDOT leads to its reduction through neutralisation of the positive charge carrier along the conjugated chain. In doing so, the electrostatic force holding the counter anion (in this case tosylate) within the graphene–PEDOT is lost, allowing the anion to diffuse into solution. Through judicious choice of the doping anion molecule in the composite, its selective released into solution can be used to inhibit or enhance bacterial processes. For example, anions could be chosen to directly interact with the specific acyl-homoserine lactone (AHL) autoinducers that facilitate communication among bacteria in a biofilm.³²

Conclusions

The transfer of electrons from a bacterial system to a conductive substrate by utilising the electroactive properties of high conductivity graphene–PEDOT composite thin films was demonstrated. Graphene–PEDOT with an electrical conductivity of ca. 2000 S cm⁻¹ was shown to promote attachment of viable bacteria (P. aeruginosa). The bacterial system transferred electrons to the graphene–PEDOT, ultimately reducing this substrate, as observed by a darkening in colour and a more than doubling of the measured sheet resistance. Such high conductivity, electroactive and biocompatible materials open the pathway to a range of bacteria-sensing devices, MFCs, and controlled release coatings.

Acknowledgements

S.M.N. acknowledges the support from the Australian Research Council through grant FT100100177.

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Footnote

† Electronic supplementary information (ESI) available: (1) Experimental methods, including synthesis of the surfactant stabilised graphene dispersions, composite substrate preparation using vapour phase polymerisation, bacterial attachment, cell viability, AgNO₃ redox reaction, and electrical measurements. See DOI: 10.1039/c5ra08720a

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