Nitrogen-doped graphene approach to enhance the performance of a membraneless enzymatic biofuel cell

Abstract

Heteroatom-doping of pristine graphene is an effective route for tailoring new characteristics in terms of catalytic performance which opens up potentials for new applications in energy conversion and storage devices. Nitrogen-doped graphene (N-graphene), for instance, has shown excellent performance in many electrochemical systems involving oxygen reduction reaction (ORR), and more recently glucose oxidation. Owing to the excellent {\mathrm{H}}_2{\mathrm{O}}_2 sensitivity of N-graphene, the development of highly sensitive and fast-response enzymatic biosensors is made possible. However, a question that needs to be addressed is whether or not improving the anodic response to glucose detection leads to a higher overall performance of enzymatic biofuel cell (eBFC). Thus, here we first synthesized N-graphene via a catalyst-free single-step thermal process, and made use of it as the biocatalyst support in a membraneless eBFC to identify its role in altering the performance characteristics. Our findings demonstrate that the electron accepting nitrogen sites in the graphene structure enhances the electron transfer efficiency between the mediator (redox polymer), redox active site of the enzymes, and electrode surface. Moreover, the best performance in terms of power output and current density of eBFCs was observed when the bioanode was modified with highly doped N-graphene.

Keywords

enzymatic fuel cell / nitrogen-doped graphene / reduced graphene oxide / catalyst-free synthesis

Introduction

Enzymatic biofuel cells (eBFCs) offer unique features in comparison to chemical fuel cells such as operation at room temperature and near neutral pH, ease of miniaturization, cheap organic fuels, small environmental footprint, etc [¹–³]. An eBFC operation is dependent upon enzymatic biocatalysts that are often immobilized on the bioanode and biocathode. On the bioanode side enzymes such as glucose oxidase (GO_X) catalyze the oxidation of biofuels such as glucose [⁴,⁵], fructose [⁶,⁷], or alcohols [⁸], while an oxidant, e.g. oxygen (O₂), is reduced enzymatically at the biocathode. In glucose eBFCs the overall cell reaction is the consumption of glucose and oxygen and the production of gluconolactone as well as hydrogen peroxide and water. Depending on the application, many configurations of eBFCs have been proposed over the years, which can be categorized mainly as either with or without membrane. In general, in membrane-containing cells the bioanode and biocathode are separated by a polymeric layer preferably closer to the cathodic side. The role of membrane is often attributed to the regulation of ionic transfer between the bioanodes and biocathodes, and preventing the diffusion of reaction byproducts from reaching the opposite side of the cell. However, this is often accompanied with higher internal ohmic resistance and power loss, and many attempts have been made to safely remove the membrane while maintaining cell stability and enzymatic activity for longer operating cycles.

Graphene and its derivatives possess unique features such as high surface area, metallic electrical conductivity, ease of synthesis and functionalization, which enable their use for biosensing as well as energy-related applications. Over the years, researchers have recognized chemical functionalization of graphene as an effective tool to tailor new features, especially catalytic performance toward oxygen reduction reaction (ORR) [⁹], hydrogen evolution reaction (HER) [¹⁰], and oxygen evolution reaction (OER) [¹¹]. Engineering these reactions is of huge importance for the state-of-the-art battery and fuel cell design, as well as electrosynthesis of biofuels. Generally, heteroatom-doped graphene such as boron and nitrogen-doped graphene (N-graphene), exhibits different properties than pristine graphene in terms of charge distribution of carbon atoms, leading to energetically activated regions with catalytic performance on graphene structure [¹²]. Wang et al. [¹³], were among the first group to investigate the role of N-graphene on glucose sensitivity in a typical GO_X-based biosensor. Their study has shown that nitrogen sites on graphene structure drastically enhance amperometric response in glucose monitoring. These results open up great potentials for developing next generation amperometric glucose biosensors with high sensitivity and fast response.

In an eBFC, often the cathodic reaction is the rate limiting step, meaning that enhancing the overall cell performance by improving anodic reaction or glucose sensitivity is only a possibility. Thus, in this study, we turned our attention to the impact of N-graphene as the support material for bioanode on the overall eBFC performance. First, N-graphene was synthesized via thermal reduction of graphite oxide (GO) in the presence of melamine as the nitrogen precursor. Next, carbon paper substrates were modified with N-graphene and reduced graphene oxide (rGO), and their electrochemical communication with GO_X/redox polymer hydrogel as the biocatalyst was studied. Finally, in conjunction with a bilirubin oxidase (BO_X) biocathode, we fabricated a membraneless eBFC to reveal the impact of N-graphene on the cell overall performance.

Experimental

Materials and chemicals

Glucose oxidase (GO_X, from ‘Aspergillusniger’, EC 1.1.3.4, Type X-S), tetrabutylammonium bromide (TBAB), (6-bromohexyl)ferrocene, and melamine were purchased from Sigma-Aldrich. Citric acid, sodium phosphate dibasic anhydrous, and dextrose (D-glucose) anhydrous were purchased from Fisher Scientific. LPEI (MW 100,000) and ethylene glycol diglycidyl ether (EGDGE) were ordered from Polyscience Inc., Warrington, PA. Bilirubin oxidase (BO_X from ‘Myrothecium sp.’, EC 1.3.3.5) was purchased from Amano Enzyme Inc. Carbon paper MGL280 (non-wet proofed) was purchased from Fuel Cell Earth. Graphite oxide, produced by modified Hummer’s method, was obtained from ACS Material. All chemicals were used as received without further purification.

TBAB-modified Nafion was synthesized following the reported procedure in Refs. [¹⁴,¹⁵]. Anthracene-modified multi-walled carbon nanotubes (An-MWCNTs) were modified as described in Refs. [¹⁶–¹⁸]. The electron transfer in the bioanode is mediated by hexylferrocenyl-LPEI (Fc-C₆-LPEI) redox polymer. The synthesis of Fc-C₆-LPEI described in Refs. [¹⁹,²⁰]. In short, 300 \mathrm{mg} of linear polyethylenimine were added to10 \mathrm{mg} of acetonitrile. The solution was then heated to reflux for approximately 10 min. Next, 380 \mathrm{mg} of (6-bromohexyl)ferrocene in 2 \mathrm{mL} of ethanol were added to the mixture and heated to reflux solvent for about 12 h. Afterwards, the polymer was extracted under reduced pressure, and the remaining residues were rinsed by diethyl ether which helps to remove the ferrocenyl impurities.

Instrumentation

CHI 660E potentiostat (CH Instrument, USA) was utilized for electrochemical analysis. Electrocatalysis investigation of bioelectrodes was conducted in a standard three-electrode setup where a saturated calomel electrode (SCE) and a platinum mesh (1 cm² projected surface area) were used as reference and counter electrodes, respectively. All the electrochemical tests were performed in a 0.2 M citrate/phosphate buffer solution (pH 7.0) as the supporting electrolyte, and at room temperature (21°C). Current and power densities were normalized with respect to the projected surface area of the anode. Raman spectroscopy was carried out using RenishawinVia Reflex Raman, with a 532 nm wavelength laser.

Bioelectrode fabrication

The bioelectrodes were fabricated following the procedures reported in Refs. [²¹,²²]. To prepare 3 cm² of BO_X/An-MWCNT/TBAB-modified Nafion cathodes, 1.5 \mathrm{mg} of BO_X was dissolved in 75 \mathrm{\mu L} of 0.2 M citrate/phosphate buffer (pH 7.0). Next, 7.5 \mathrm{mg} of An-MWCNTs were added to the solution and vortex mixed for 1 min, followed by sonication for 15 s. This step was repeated three times to obtain a homogenized enzymatic paste. Then, 25 \mathrm{\mu L} of TBAB-modified Nafion was added to the mixture, followed by three steps of vortex mixing and sonication. The resulting paste was then coated on the carbon paper electrodes using a brush, and dried at room temperature under positive air flow for about 4 h. Fabrication of 6 cm² of GO_X/Fc-C₆-LPEI bioanode starts with preparing an enzymatic redox hydrogel. Briefly, 210 \mathrm{\mu L} of 10 mg/mL hexylferrocenyl-LPEI polymer in DI water was mixed with 90 \mathrm{\mu L} of 10 mg/mL GO_X solution in DI water. Then, 11.25 \mathrm{\mu L} of EGDGE solution (10% v/v in DI water) was added to the mixture, and vortex mixed for about 30 s. The hydrogel was then pipetted on carbon paper electrodes, and left at room temperature for 24 h to dry. Before coating with hydrogel, the carbon paper substrates were modified with 100 \mathrm{\mu L} of 10 mg/mL (in ethanol) rGO or functionalized graphene solution. This procedure involves drop casting the solution on to the substrate, and drying it at room temperature for 12 h.

Synthesis of N-graphene

N-graphene was synthesized via a simple one-step thermal annealing of GO in the presence of various amounts of melamine acting as the nitrogen doping precursor [²³,²⁴]. Briefly, GO and melamine powder (1:5 and 1:50 mass ratios) were ground together with a pestle in a mortar for about 5 min. Afterwards, the mixture was loaded in to a crucible with lid and placed in the center of a chemical vapor deposition (CVD) furnace. Initially, the furnace was purged with Ar (argon) for 1 h to ensure the removal of O₂ in the reaction tube. Then, the temperature was raised at a rate of 5°C/min, and maintained at 900°C for 30 min. Finally the furnace was slowly cooled down to the room temperature under Ar atmosphere. To synthesize rGO, the above steps were repeated without the addition of melamine.

Results and discussion

Graphene functionalization

Raman spectroscopy is recognized as a non-destructive technique for characterizing carbon materials [²⁵]. The Raman spectra of GO, as-synthesized rGO and N-graphene were obtained, and the peak position and intensities were analyzed. As shown in Fig. 1, the Raman spectra demonstrate noticeable peaks at ca. 1580 and 1350 {\mathrm{cm}}^{\mathrm{-1}} corresponding to G and D band, respectively. Sheng et al. [²⁴] have shown that increasing the melamine content ratio of the precursor from 1:5 to 1:50 increases the N-doping level from ca. 6.6% to 8.4% at the same synthesis temperature of 800°C. Higher doping level is often accompanied by more defects such as holes and generation of edges in the graphene structure, leading to an enhanced relative intensity of the D band [²³,²⁶]. This is consistent with the results observed in Fig. 1, where the I_{\mathrm{D}}/I_{\mathrm{G}} is increasing as the doping level increases. Moreover, analyzing the peak position of G band demonstrates a downshift from ca. 1586 {\mathrm{cm}}^{\mathrm{-1}} for GO to 1584 and 1583 {\mathrm{cm}}^{\mathrm{-1}} for N-graphene (1:5) and (1:50), respectively. This downshift of G position has been recognized as a sign for successful N-doping of graphene structure [²³,²⁷].

The role of N-graphene on eBFC performance

The typical mechanism of glucose oxidation by GO_X enzymes is as follows [²¹]:

(1){\mathrm{GO}}_{\mathrm{X}}\mathrm{(FAD)+Glucose}\to {\mathrm{GO}}_{\mathrm{X}}\left({\mathrm{FADH}}_{\mathrm{2}}\right)+\mathrm{Gluconolactone},

(2){\mathrm{GO}}_{\mathrm{X}}\left({\mathrm{FADH}}_{\mathrm{2}}\right)+{\mathrm{O}}_{\mathrm{2}}\to {\mathrm{GO}}_{\mathrm{X}}{\mathrm{(FAD)+H}}_{\mathrm{2}}{\mathrm{O}}_{\mathrm{2}},

where flavin adenine dinucleotide (FAD) acts as the redox co-factor of GO_X. In this reaction scheme, parasitic {\mathrm{H}}_2{\mathrm{O}}_2 results from an incomplete oxidation of glucose, and is a common byproduct of catalysis by oxidase enzymes. The accumulation of {\mathrm{H}}_2{\mathrm{O}}_2 can negatively affect the enzymatic activity and stability of the electrode especially in long operating cycles. One approach to alleviate this problem is the introduction of {\mathrm{H}}_2{\mathrm{O}}_2 reducing agents to the bioelectrodes. Prussian blue, for instance, can catalyze the reduction of {\mathrm{H}}_2{\mathrm{O}}_2, and has been incorporated into enzymatic bioanodes for enhancing glucose sensitivity in biosensing applications [²⁸–³⁰]. Regarding eBFCs, the {\mathrm{H}}_2{\mathrm{O}}_2 reducing agents may be combined with cathodic catalyst to protect the cathodic environment from peroxide contamination or deactivation of the cathodic enzymes, especially in a membraneless configuration. For instance, our group recently introduced a novel membraneless biobattery by using a Prussian blue modified cathode that can act both as a rechargeable material and reductant of the {\mathrm{H}}_2{\mathrm{O}}_2 diffused from the bioanode [³¹]. N-graphene can also dramatically increase the {\mathrm{H}}_2{\mathrm{O}}_2 electrocatalyis, and thus enhance the biosensing ability of GO_X-based electrodes in terms of glucose detection [¹³]. However, as mentioned earlier, the rate limiting step in the overall eBFC reaction is the cathodic reaction, meaning that the bioanode improvement may have less impact on the overall performance which is addressed in the remaining sections.

Enzymatic biocatalytic performance

At first, we investigated the electrocatalysis of enzymatic bioanodes toward oxidizing glucose by modifying them with rGO and N-graphene with two different doping levels of nitrogen atoms. As shown in Fig. 2(a), the catalytic current dramatically increases, and the onset potential slightly decreases by incorporating rGO and N-graphene as the support material for GO_X immobilization. This is mainly attributed to a higher peroxide sensitivity of graphene doped bioanodes [¹³]. Moreover, heteroatom-doped graphene offers superior electronic conductivity which may enhance the communication between the active sites of the enzyme, mediator, and electrode surface. To investigate this phenomenon, cyclic voltammetry in the absence of glucose was applied to the N-graphene modified electrode with high nitrogen content and the results were compared to bare carbon paper substrate (blank). As illustrated in Fig. 2(b), the redox pair in both cases may correspond to the redox reaction of Fc-C₆-LPEI between the active site of GO_X and the electrode surface. Clearly, applying N-graphene increases the rate of this reaction, signifying a superior electron transfer efficiency.

In the next section we investigated the overall performance of eBFC by combining the modified bioanodes with a BO_X/An-MWCNT/TBAB-modified Nafion biocathode. However, before proceeding, the fabricated biocathodes were studied using cyclic voltammetry to ensure the immobilization of the BO_X enzymes and their proper biocatalytic performance. As shown in Fig. 3, the addition of {\mathrm{O}}_2 to the electrolyte significantly enhances the cathodic current which is a signal for ORR being catalyzed by BO_X enzymes.

eBFC performance

The eBFC studied here had a membraneless configuration, since we anticipated the majority of {\mathrm{H}}_2{\mathrm{O}}_2 forming at the bioanode would be reduced by N-graphene, enabling less peroxide crossover to the biocathode. Moreover, removing the membrane lowers the internal ohmic resistance, leading to a more efficient performance. Figure 4 demonstrates the power output of various eBFCs corresponding to different bioanode modifications. These power curves were obtained by sweeping voltage between the open circuit voltage (OCV) of the cell and 0 at a scan rate of 1 mV/s. Before the linear polarization tests, the OCV of the eBFC setup was measured by maintaining the cell at zero current until a stable voltage over time are observed. Prior to the performance tests, the electrolyte was injected with {\mathrm{O}}_2 for half an hour to ensure the presence of enough oxidant for cathodic reaction ({\mathrm{O}}_2-reducing BO_X).

As evident by the results in Fig. 4, the eBFCs modified with N-graphene demonstrate higher maximum power output and current density compared to rGO and plain carbon paper (blank). Moreover, increasing the N-content of graphene structure further enhances the power output, illustrating the role of electron accepting nitrogen atoms in improving the performance characteristics. The reaction scheme (1) and (2), suggests that as we increase the {\mathrm{O}}_2 content of electrolyte comparably more {\mathrm{H}}_2{\mathrm{O}}_2 is produced by GO_X enzymes. Our experimental setup mimics this condition where bioelectrodes were placed in the {\mathrm{H}}_2{\mathrm{O}}_2 saturated environment. On the other hand, as mentioned earlier, N-graphene exhibits superior {\mathrm{H}}_2{\mathrm{O}}_2 sensitivity over graphene [¹³]. Thus, we may conclude that the majority of the power increase by N-doping of graphene stems from its electron accepting properties and catalytic performance toward reducing {\mathrm{H}}_2{\mathrm{O}}_2.

Table 1 summarizes the results by comparing the performance of N-graphene modified eBFCs versus rGO and unmodified case (blank). As seen, N-graphene is a superior choice over rGO and bare carbon paper for biocatalyst support, mainly due to higher {\mathrm{H}}_2{\mathrm{O}}_2 sensitivity and better electron transfer efficiency between enzymes, redox mediator, and electrode surface. Moreover, N-graphene (1:50) corresponding to a higher level of nitrogen doping, exhibits the maximum increase in power (by ca. 41%) and current density, illustrating the role of electron accepting nitrogen sites in enhancing eBFC’s power output.

Conclusions

In this work, our aim was to identify the role of heteroatom-doped graphene and the mechanisms by which it may impact the eBFC overall performance. As a proof-of-concept, N-graphene widely used as ORR catalyst in batteries and fuel cells was chosen, and its impact was investigated on eBFC’s operation with a membraneless design. The results of the present work are of high practical importance for the design and optimization of self-powered biosensors, eBFCs, and implantable enzymatic devices. For instance, the peroxide formation in implantable devices may hinder the application of conventional eBFCs, as the peroxide might be toxic both toward the enzymatic bioelectrodes and living tissue cells. Additionally, peroxide accumulation diminishes the life time of both cathodic and anodic enzymes, leading to destabilized performance. By incorporating the {\mathrm{H}}_2{\mathrm{O}}_2 reducing agent in the eBFC design we may alleviate some of these issues, and ensure a safer operation of membraneless configurations.

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