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H2O2 biofuel cell operating under physiological conditions
Electrochemistry Communications 34 (2013) 105–108
Contents lists available at ScienceDirect
Electrochemistry Communications journal homepage: www.elsevier.com/locate/elecom
Short communication
A double-walled carbon nanotube-based glucose/H2O2 biofuel cell operating under physiological conditions Charles Agnès, Bertrand Reuillard, Alan Le Goff, Michael Holzinger, Serge Cosnier ⁎ Département de Chimie Moléculaire UMR-5250, ICMG FR-2607, CNRS Université Joseph Fourier, BP-53, 38041 Grenoble, France
a r t i c l e
i n f o
Article history: Received 18 April 2013 Received in revised form 15 May 2013 Accepted 15 May 2013 Available online 30 May 2013 Keywords: Carbon nanotubes Peroxidase Glucose oxidase Biofuel cells Hydrogen peroxide
a b s t r a c t We report the first example of a glucose/H2O2 biofuel cell operating at 5 mM glucose under air at 37 °C in 0.14 M NaCl. At a bienzymatic cathode, the direct wiring of horseradish peroxidase (HRP) achieves the electrocatalytic reduction of H2O2 concomitantly produced during the oxidation of glucose by glucose oxidase (GOx) in the presence of O2. At the bienzymatic anode, the indirect wiring of glucose oxidase achieves electrocatalytic glucose oxidation while catalase removes trace of H2O2 and, in combination with GOx, traces of O2. The bienzymatic catalysis involved GOx at both electrodes. The biofuel cell exhibits OCV of 450 mV and maximum power output of 30 μW cm−2 at 0.3 V in 5 mM glucose and 0.14 M NaCl at 37 °C. This represents a novel alternative to the use of copper oxidases in conventional glucose/O2 biofuel cells for implantable applications. © 2013 Elsevier B.V. All rights reserved.
1. Introduction Enzymatic biofuel cells rely on the oxidation of substrate such as hydrogen or glucose and reduction of oxygen to harvest energy from complex media [1–3]. In particular, glucose biofuel cells (BFCs) represent a promising alternative to supply energy from living organisms to implanted electronic devices [4–8]. Owing to their ability to achieve electrocatalytic reduction of oxygen at low overpotentials, laccases and bilirubin oxidases are multicopper oxidases that have been mostly employed for BFCs cathodes. Nevertheless, the resulting biocathodes suffer from instability over time due to the short life times of these “blue” copper oxidases or their deactivation by inhibitors like chloride ions. Investigations are therefore focused on new sources of copper enzymes with enhanced stability [9,10]. A more promising alternative, recently initiated by W. Schuhmann et al., consists to replace the conventional use of laccase or bilirubine oxidase by an elegant bienzymatic system based on the combination of glucose oxidase (GOx) and horseradish peroxidase (HRP) [11]. GOx produces H2O2 from the oxidation of glucose, and HRP achieves the electrocatalytic reduction of H2O2 into water at a low overpotential. High-performance GBFCs are mostly based on carbon nanotubes as electrode materials due to their high specific surface and their excellent electrochemical properties for the transfer of electrons with enzymes [12–15]. In this context, we investigated the co-adsorption of GOx and HRP on double-walled carbon nanotube (DWCNT) cathodes. By wiring GOx at the bioanode using mediated electron transfer via naphthoquinone [15], we have designed a novel type of glucose/ ⁎ Corresponding author. Fax: +33 4 7651 4267. E-mail address: [email protected] (S. Cosnier). 1388-2481/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.elecom.2013.05.018
H2O2 biofuel cell that is able to harvest energy in physiological conditions. 2. Experimental 2.1. Methods and instrumentation All reagents, HRP (193 U mg−1), GOx (139 U mg−1), catalase (2000 U mg−1) and 1,4-naphthoquinone were purchased from Aldrich. DWCNTs (>90% purity) were purchased from Nanocyl and used as received. The electrochemical experiments were carried out in a threeelectrode electrochemical cell using a Biologic VMP3 Multi Potentiostat. A platinum grid was used as the counter electrode and a saturated calomel electrode (SCE) served as reference electrode. All potentials given in this work are referred to the Saturated Calomel Electrode (SCE). All experiments were conducted in phosphate-buffered saline solutions at pH 7.4 (PBS: 0.01 M phosphate buffer, 0.0027 M potassium chloride and 0.137 M sodium chloride). The morphology of the DWCNT electrodes was investigated by SEM using an ULTRA 55 FESEM based on the GEMENI FESEM column with beam booster (Nanotechnology Systems Division, Carl Zeiss NTS GmbH, Germany) and tungsten gun. UV-visible spectra were recorded with a CARRY 1 spectrophotometer with a quartz cuvette (1 cm depth). Ten milligrams of DWCNT were sonicated 10 min in 500 ml of ultra-pure water, then different load of enzymes (GOx or HRP) were added to this solution, at different enzyme/DWCNT ratios. After 1 h of stirring, solutions were centrifuged at 10,000 rpm during 10 min. Then the supernatant was analyzed to estimate the enzyme adsorption.
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2.2. Preparation of DWCNT electrodes For bioanode electrodes, after DWCNT sonication, 2 mg GOx, 1 mg catalase and 1 mg 1,4-naphthoquinone (NQ) were added and stirred for 1 h. Two hundred microliters were drop casted on a carbon cloth and dried at 40 °C. Then, 20 μl of a 0.2% solution of Nafion® (diluted 10 times from a Nafion® 117 solution, from Sigma-Aldrich, ~ 5% in a mixture of lower aliphatic alcohols and water) was drop-casted on the electrode surface and dried. For biocathode electrodes, the same procedure was used. Instead of GOx, NQ and catalase, we used 2 mg of HRP and 0.3 mg of GOx.
3. Results and discussion 3.1. Fabrication and characterization of DWCNT bioelectrodes The DWCNT-based bioelectrodes were prepared in several steps. First, the adsorption of the different enzymes was monitored and optimized using UV-visible spectroscopy. Monitoring the activity in solution of the different enzymes allows the estimation of the enzyme/DWCNT ratio necessary to maximize the amount of adsorbed enzymes on DWCNTs. Fig. S1 shows the percentage of adsorbed enzymes towards the enzyme/DWCNT ratio for HRP and GOx. We estimated the optimized ratio to be 0.2 (w/w) for HRP and for GOX. Dispersions of bio-functionalized DWCNTs were then carefully deposited on carbon cloth by controlled drop casting. A solution of Nafion is finally casted on the DWCNT film to improve the mechanical stability of the film and preventing any leakage of enzymes and mediators. A schematic representation of the glucose/H2O2 BFC and a photograph of the flexible bioelectrode are displayed in Fig. 1A and B. SEM image of the DWCNT surface from Fig. 1C shows the highly porous structure of the DWCNT film containing thin bundles with diameters between 3 and 5 nm. The biocathode is based on the production of H2O2 via O2 reduction by GOx during glucose oxidation. The direct wiring of HRP on DWCNTs triggers the reduction of H2O2 at low overpotentials. We first investigated the direct wiring of HRP with DWCNTs individually. Although HRP can directly be wired to carbon nanotubes for the bioelectrocatalytic reduction of H2O2, this reduction process is faced to the electrocatalytic oxidation of H2O2 on the electrode at high potential [11]. Upon addition of increasing amounts of H2O2, the catalytic cathodic current, measured by potentiostating the electrode at 0.2 V for 5 min, reflects the reduction of H2O2 by wired HRP. Fig. 2A shows the current response of the DWCNT/HRP cathode as a function of H2O2 concentration. The error bars correspond to the values recorded for each concentration by three different bioelectrodes
illustrating the reproducibility of the measurements and of the bioelectrode fabrication. This evolution indicates a typical enzymatic dependence for the directly wired HRP [16]. The direct electron transfer of the FeIII/FeIV center of the HRP heme cofactor is responsible for the H2O2 reduction according to the Eq. (1) −
þ
H2 O2 þ 2e þ 2H →2H2 OðelectroenzymaticÞ
ð1Þ
It is noteworthy that no reduction currents were observed on non-functionalized DWCNTs at this potential. On the contrary, as depicted in curve (a) of Fig. 2B, DWCNTs shows catalytic properties towards H2O2 oxidation according to Eq. (2) −
þ
H2 O2 →O2 þ 2e þ 2H
ð2Þ
Indeed, when no HRP is present, H2O2 is oxidized at DWCNTs with current densities reaching 78 μA cm−2 at 0.45 V in 1 mM H2O2 solution. When HRP is adsorbed on DWCNTs (curve (b) from Fig. 2B), H2O2 reduction occurs with a cathodic current density of 120 mA cm−2 at 0.2 V in 1 mM H2O2. The open-circuit potential (OCP) of 380 mV for these electrodes thus results from both processes, the bioelectrocatalytic reduction of H2O2 and the electrocatalytic oxidation. By subtracting the H2O2 oxidation currents, we estimated the onset potential for the bioelectrocatalytic reduction of H2O2 by HRP at 440 mV vs SCE (dashed curve, Fig. 2B). These experiments underline the fact that the H2O2 production has to be limited to avoid as possible its direct oxidation by DWCNTs. Therefore, we carefully adjusted the amount of GOx on DWCNT biocathodes to restrict the production of H2O2. Taking into account the respective GOx and HRP activities, an HRP/GOx ratio (w/w) less than 7 led to a H2O2 overproduction and a loss of HRP activity. Fig. 3A shows the polarization curves, performed for the different types of bioelectrodes. Each current value reported on the polarization curve corresponds to the catalytic current obtained using chronoamperometry at different potentials. In presence of glucose (5 mM) and oxygen, the polarization curve for the biocathode with the co-immobilized HRP and GOx exhibits a maximum current density reaching 115 μA cm−2 (Fig. 3A, curve a). This corresponds to the production of H2O2 by GOx, which is then reduced by HRP. The complete 4H+/4e− reduction of O2 into water is described in Eqs. (1) and (3): −
þ
O2 þ 2e þ 2H →H2 O2 ðenzymaticÞ
ð3Þ
Curve (b) shows the residual anodic current in 5 mM glucose and oxygen-saturated PBS solution when GOx is immobilized without HRP. Owing to the absence of HRP, H2O2, produced by GOx, is directly oxidized by DWCNTs as previously mentioned. It is noteworthy that
Fig. 1. (A) Schematic representation of the GBFC. (B) Image of the bioelectrode: a 1-cm2 DWCNT film deposited on carbon cloth. (C) SEM micrograph of the DWCNT film deposited on carbon cloth.
C. Agnès et al. / Electrochemistry Communications 34 (2013) 105–108
107
no anodic current was observed at lower potential corroborating the absence of electrocatalytic oxidation of glucose through the direct wiring of GOx on DWCNTs. Curve (c) shows the polarization curve for the bioanode based on the simultaneous confinement of GOx and naphthoquinone (NQ) in the CNT matrix. While NQ allows the electrical connection of GOx [15], catalase is also adsorbed at these electrodes to remove excess of H2O2. In addition, the combined action of catalase and GOx for the reduction of O2 traces can create localized anaerobic conditions for efficient electrocatalytic oxidation of glucose [15–17] according to Eq. (4): −
þ
glucose→gluconolactone þ 2e þ 2H
ð4Þ
These bioanodes exhibit an OCP of −0.05 V at 5 mM glucose, which is closed to the redox potential of NQ, and a maximum current density of 106 μA cm−2 at 5 mM glucose and 140 mM Cl−. The polarization curves indicate that DWCNT bioanodes are the limiting bioelectrode in this system. It should be noted that percentages of error are higher in the case of the bienzymatic DWCNT/HRP/GOx biocathodes. This may ascribed to the complexity of the mechanism of oxygen reduction performed by two different enzymes immobilized in the DWCNT film. These electrodes were further studied in a GBFC configuration. 3.2. The glucose/H2O2 biofuel cell
Fig. 2. (A) Calibration curve for a DWCNT/HRP cathode at increasing H2O2 concentrations. (B) Electrocatalytic properties towards H2O2 oxidation and reduction at (♦) DWCNT electrodes and (■)DWCNT/HRP electrodes in 1 mM H2O2. Dashed line: mathematical subtraction of H2O2 oxidation currents from bioelectrocatalytic H2O2 reduction currents. All values were measured after a 5-min discharge under stirring at constant potentials in PBS at 37 °C. The error bars correspond to the values recorded for three bioelectrodes.
Carbon cloth bioelectrodes were placed in front of each other in a solution of 5 mM glucose under air in 140 mM NaCl. Fig. 3B shows the 5-min discharge curves performed under stirring at constant potentials. Both polarization and power curves are depicted in Fig. 3C. The GBFC exhibits an excellent OCV of 0.45 V, which approximately corresponds to the sum of the OCP values for each bioelectrode. In addition, a maximum current density of 127 μA cm−2 and a maximum
Fig. 3. (A) Polarization curves for (a) DWCNT/GOx/HRP biocathodes in air-saturated 5 mM glucose solution, (b) a DWCNT/GOx electrode in oxygen-saturated 5 mM glucose solution and (c) a DWCNT/NQ/GOx/Catalase bioanode in air-saturated 5 mM glucose solution. (B) Five-minute discharges for GBFC at constant potentials under stirring in an air-saturated 5 mM glucose solution. (C) Polarization and power curves for the GBFC. All values were measured after a 5-min discharge under stirring at constant potentials in 5 mM glucose PBS solutions at 37 °C. The error bars correspond to the values recorded for three bioelectrodes.
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power output of 30 μW cm−2 at 0.3 V were recorded. It is noteworthy that the NaCl concentrations have negligible influence on GBFC performances. In particular, this membraneless GBFC shows no power decrease in the absence or in the presence of 0.14 M NaCl. 4. Conclusion This novel type of GBFC is the first example of a biofuel cell taking advantage of the enzymatic activity of GOx for both oxidation of glucose and reduction of O2 into H2O2. In combination with the efficient wiring of HRP on DWCNT films deposited on flexible carbon cloth, these devices achieve excellent OCV values of 0.45 V and maximum power output of 30 μW cm−2 in 5 mM glucose solutions. Taking into account the stability and availability of these enzymes and the flexibility of the carbon-based electrodes, these GBFCs represent a promising alternative for in vivo applications of GBFCs. Supplementary data to this article can be found online at http:// dx.doi.org/10.1016/j.elecom.2013.05.018. Acknowledgments The authors thank the ANR Emergence-2010 EMMA-043-02 and the ANR Investissements d'avenir-Nanobiotechnologies-10-IANN-0-02 programs for partial funding. The “Région Rhones-Alpes” is acknowledged for the PhD funding of B. Reuillard.
References [1] D. Leech, P. Kavanagh, W. Schuhmann, Electrochimica Acta 84 (2012) 223. [2] J.A. Cracknell, K.A. Vincent, F.A. Armstrong, Chemical Reviews 108 (2008) 2439. [3] M. Cooney, V. Svoboda, C. Lau, G. Martin, S. Minteer, Energy & Environmental Science 1 (2008) 320. [4] L. Halámková, J. Halámek, V. Bocharova, A. Szczupak, L. Alfonta, E. Katz, Journal of the American Chemical Society 134 (2012) 5040. [5] M. Rasmussen, R.E. Ritzmann, I. Lee, A.J. Pollack, D. Scherson, Journal of the American Chemical Society 134 (2012) 1458. [6] K. MacVittie, J. Halamek, L. Halámková, M. Southcott, W.D. Jemison, R. Lobel, E. Katz, Energy & Environmental Science 6 (2013) 81. [7] A. Zebda, S. Cosnier, J.-P. Alcaraz, M. Holzinger, A. Le Goff, C. Gondran, F. Boucher, F. Giroud, K. Gorgy, H. Lamraoui, P. Cinquin, Scientific Reports 3 (2013) 1516. [8] P. Cinquin, C. Gondran, F. Giroud, S. Mazabrard, A. Pellissier, F. Boucher, J.-P. Alcaraz, K. Gorgy, F. Lenouvel, S. Mathé, P. Porcu, S. Cosnier, PLoS One 5 (2010) e10476. [9] B. Reuillard, A. Le Goff, C. Agnès, A. Zebda, M. Holzinger, S. Cosnier, Electrochemistry Communications 20 (2012) 19. [10] L. Edembe, S. Gounel, M. Cadet, F. Durand, N. Mano, Electrochemistry Communications 23 (2012) 80. [11] W. Jia, C. Jin, W. Xia, M. Muhler, W. Schuhmann, L. Stoica, Chemistry A European Journal 18 (2012) 2783. [12] M. Holzinger, A. Le Goff, S. Cosnier, Electrochimica Acta 82 (2012) 179. [13] F. Gao, L. Viry, M. Maugey, P. Poulin, N. Mano, Nature Communications 1 (2010) 2. [14] A. Zebda, C. Gondran, A. Le Goff, M. Holzinger, P. Cinquin, S. Cosnier, Nature Communications 2 (2011) 370. [15] B. Reuillard, A. Le Goff, C. Agnès, M. Holzinger, A. Zebda, C. Gondran, K. Elouarzaki, S. Cosnier, Physical Chemistry Chemical Physics 15 (2013) 4892. [16] W. Jia, S. Schwamborn, C. Jin, W. Xia, M. Muhler, W. Schuhmann, L. Stoica, Physical Chemistry Chemical Physics 12 (2010) 10088. [17] N. Plumeré, J. Henig, W.H. Campbell, Analytical Chemistry 84 (2012) 2141.
Contents lists available at ScienceDirect
Electrochemistry Communications journal homepage: www.elsevier.com/locate/elecom
Short communication
A double-walled carbon nanotube-based glucose/H2O2 biofuel cell operating under physiological conditions Charles Agnès, Bertrand Reuillard, Alan Le Goff, Michael Holzinger, Serge Cosnier ⁎ Département de Chimie Moléculaire UMR-5250, ICMG FR-2607, CNRS Université Joseph Fourier, BP-53, 38041 Grenoble, France
a r t i c l e
i n f o
Article history: Received 18 April 2013 Received in revised form 15 May 2013 Accepted 15 May 2013 Available online 30 May 2013 Keywords: Carbon nanotubes Peroxidase Glucose oxidase Biofuel cells Hydrogen peroxide
a b s t r a c t We report the first example of a glucose/H2O2 biofuel cell operating at 5 mM glucose under air at 37 °C in 0.14 M NaCl. At a bienzymatic cathode, the direct wiring of horseradish peroxidase (HRP) achieves the electrocatalytic reduction of H2O2 concomitantly produced during the oxidation of glucose by glucose oxidase (GOx) in the presence of O2. At the bienzymatic anode, the indirect wiring of glucose oxidase achieves electrocatalytic glucose oxidation while catalase removes trace of H2O2 and, in combination with GOx, traces of O2. The bienzymatic catalysis involved GOx at both electrodes. The biofuel cell exhibits OCV of 450 mV and maximum power output of 30 μW cm−2 at 0.3 V in 5 mM glucose and 0.14 M NaCl at 37 °C. This represents a novel alternative to the use of copper oxidases in conventional glucose/O2 biofuel cells for implantable applications. © 2013 Elsevier B.V. All rights reserved.
1. Introduction Enzymatic biofuel cells rely on the oxidation of substrate such as hydrogen or glucose and reduction of oxygen to harvest energy from complex media [1–3]. In particular, glucose biofuel cells (BFCs) represent a promising alternative to supply energy from living organisms to implanted electronic devices [4–8]. Owing to their ability to achieve electrocatalytic reduction of oxygen at low overpotentials, laccases and bilirubin oxidases are multicopper oxidases that have been mostly employed for BFCs cathodes. Nevertheless, the resulting biocathodes suffer from instability over time due to the short life times of these “blue” copper oxidases or their deactivation by inhibitors like chloride ions. Investigations are therefore focused on new sources of copper enzymes with enhanced stability [9,10]. A more promising alternative, recently initiated by W. Schuhmann et al., consists to replace the conventional use of laccase or bilirubine oxidase by an elegant bienzymatic system based on the combination of glucose oxidase (GOx) and horseradish peroxidase (HRP) [11]. GOx produces H2O2 from the oxidation of glucose, and HRP achieves the electrocatalytic reduction of H2O2 into water at a low overpotential. High-performance GBFCs are mostly based on carbon nanotubes as electrode materials due to their high specific surface and their excellent electrochemical properties for the transfer of electrons with enzymes [12–15]. In this context, we investigated the co-adsorption of GOx and HRP on double-walled carbon nanotube (DWCNT) cathodes. By wiring GOx at the bioanode using mediated electron transfer via naphthoquinone [15], we have designed a novel type of glucose/ ⁎ Corresponding author. Fax: +33 4 7651 4267. E-mail address: [email protected] (S. Cosnier). 1388-2481/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.elecom.2013.05.018
H2O2 biofuel cell that is able to harvest energy in physiological conditions. 2. Experimental 2.1. Methods and instrumentation All reagents, HRP (193 U mg−1), GOx (139 U mg−1), catalase (2000 U mg−1) and 1,4-naphthoquinone were purchased from Aldrich. DWCNTs (>90% purity) were purchased from Nanocyl and used as received. The electrochemical experiments were carried out in a threeelectrode electrochemical cell using a Biologic VMP3 Multi Potentiostat. A platinum grid was used as the counter electrode and a saturated calomel electrode (SCE) served as reference electrode. All potentials given in this work are referred to the Saturated Calomel Electrode (SCE). All experiments were conducted in phosphate-buffered saline solutions at pH 7.4 (PBS: 0.01 M phosphate buffer, 0.0027 M potassium chloride and 0.137 M sodium chloride). The morphology of the DWCNT electrodes was investigated by SEM using an ULTRA 55 FESEM based on the GEMENI FESEM column with beam booster (Nanotechnology Systems Division, Carl Zeiss NTS GmbH, Germany) and tungsten gun. UV-visible spectra were recorded with a CARRY 1 spectrophotometer with a quartz cuvette (1 cm depth). Ten milligrams of DWCNT were sonicated 10 min in 500 ml of ultra-pure water, then different load of enzymes (GOx or HRP) were added to this solution, at different enzyme/DWCNT ratios. After 1 h of stirring, solutions were centrifuged at 10,000 rpm during 10 min. Then the supernatant was analyzed to estimate the enzyme adsorption.
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2.2. Preparation of DWCNT electrodes For bioanode electrodes, after DWCNT sonication, 2 mg GOx, 1 mg catalase and 1 mg 1,4-naphthoquinone (NQ) were added and stirred for 1 h. Two hundred microliters were drop casted on a carbon cloth and dried at 40 °C. Then, 20 μl of a 0.2% solution of Nafion® (diluted 10 times from a Nafion® 117 solution, from Sigma-Aldrich, ~ 5% in a mixture of lower aliphatic alcohols and water) was drop-casted on the electrode surface and dried. For biocathode electrodes, the same procedure was used. Instead of GOx, NQ and catalase, we used 2 mg of HRP and 0.3 mg of GOx.
3. Results and discussion 3.1. Fabrication and characterization of DWCNT bioelectrodes The DWCNT-based bioelectrodes were prepared in several steps. First, the adsorption of the different enzymes was monitored and optimized using UV-visible spectroscopy. Monitoring the activity in solution of the different enzymes allows the estimation of the enzyme/DWCNT ratio necessary to maximize the amount of adsorbed enzymes on DWCNTs. Fig. S1 shows the percentage of adsorbed enzymes towards the enzyme/DWCNT ratio for HRP and GOx. We estimated the optimized ratio to be 0.2 (w/w) for HRP and for GOX. Dispersions of bio-functionalized DWCNTs were then carefully deposited on carbon cloth by controlled drop casting. A solution of Nafion is finally casted on the DWCNT film to improve the mechanical stability of the film and preventing any leakage of enzymes and mediators. A schematic representation of the glucose/H2O2 BFC and a photograph of the flexible bioelectrode are displayed in Fig. 1A and B. SEM image of the DWCNT surface from Fig. 1C shows the highly porous structure of the DWCNT film containing thin bundles with diameters between 3 and 5 nm. The biocathode is based on the production of H2O2 via O2 reduction by GOx during glucose oxidation. The direct wiring of HRP on DWCNTs triggers the reduction of H2O2 at low overpotentials. We first investigated the direct wiring of HRP with DWCNTs individually. Although HRP can directly be wired to carbon nanotubes for the bioelectrocatalytic reduction of H2O2, this reduction process is faced to the electrocatalytic oxidation of H2O2 on the electrode at high potential [11]. Upon addition of increasing amounts of H2O2, the catalytic cathodic current, measured by potentiostating the electrode at 0.2 V for 5 min, reflects the reduction of H2O2 by wired HRP. Fig. 2A shows the current response of the DWCNT/HRP cathode as a function of H2O2 concentration. The error bars correspond to the values recorded for each concentration by three different bioelectrodes
illustrating the reproducibility of the measurements and of the bioelectrode fabrication. This evolution indicates a typical enzymatic dependence for the directly wired HRP [16]. The direct electron transfer of the FeIII/FeIV center of the HRP heme cofactor is responsible for the H2O2 reduction according to the Eq. (1) −
þ
H2 O2 þ 2e þ 2H →2H2 OðelectroenzymaticÞ
ð1Þ
It is noteworthy that no reduction currents were observed on non-functionalized DWCNTs at this potential. On the contrary, as depicted in curve (a) of Fig. 2B, DWCNTs shows catalytic properties towards H2O2 oxidation according to Eq. (2) −
þ
H2 O2 →O2 þ 2e þ 2H
ð2Þ
Indeed, when no HRP is present, H2O2 is oxidized at DWCNTs with current densities reaching 78 μA cm−2 at 0.45 V in 1 mM H2O2 solution. When HRP is adsorbed on DWCNTs (curve (b) from Fig. 2B), H2O2 reduction occurs with a cathodic current density of 120 mA cm−2 at 0.2 V in 1 mM H2O2. The open-circuit potential (OCP) of 380 mV for these electrodes thus results from both processes, the bioelectrocatalytic reduction of H2O2 and the electrocatalytic oxidation. By subtracting the H2O2 oxidation currents, we estimated the onset potential for the bioelectrocatalytic reduction of H2O2 by HRP at 440 mV vs SCE (dashed curve, Fig. 2B). These experiments underline the fact that the H2O2 production has to be limited to avoid as possible its direct oxidation by DWCNTs. Therefore, we carefully adjusted the amount of GOx on DWCNT biocathodes to restrict the production of H2O2. Taking into account the respective GOx and HRP activities, an HRP/GOx ratio (w/w) less than 7 led to a H2O2 overproduction and a loss of HRP activity. Fig. 3A shows the polarization curves, performed for the different types of bioelectrodes. Each current value reported on the polarization curve corresponds to the catalytic current obtained using chronoamperometry at different potentials. In presence of glucose (5 mM) and oxygen, the polarization curve for the biocathode with the co-immobilized HRP and GOx exhibits a maximum current density reaching 115 μA cm−2 (Fig. 3A, curve a). This corresponds to the production of H2O2 by GOx, which is then reduced by HRP. The complete 4H+/4e− reduction of O2 into water is described in Eqs. (1) and (3): −
þ
O2 þ 2e þ 2H →H2 O2 ðenzymaticÞ
ð3Þ
Curve (b) shows the residual anodic current in 5 mM glucose and oxygen-saturated PBS solution when GOx is immobilized without HRP. Owing to the absence of HRP, H2O2, produced by GOx, is directly oxidized by DWCNTs as previously mentioned. It is noteworthy that
Fig. 1. (A) Schematic representation of the GBFC. (B) Image of the bioelectrode: a 1-cm2 DWCNT film deposited on carbon cloth. (C) SEM micrograph of the DWCNT film deposited on carbon cloth.
C. Agnès et al. / Electrochemistry Communications 34 (2013) 105–108
107
no anodic current was observed at lower potential corroborating the absence of electrocatalytic oxidation of glucose through the direct wiring of GOx on DWCNTs. Curve (c) shows the polarization curve for the bioanode based on the simultaneous confinement of GOx and naphthoquinone (NQ) in the CNT matrix. While NQ allows the electrical connection of GOx [15], catalase is also adsorbed at these electrodes to remove excess of H2O2. In addition, the combined action of catalase and GOx for the reduction of O2 traces can create localized anaerobic conditions for efficient electrocatalytic oxidation of glucose [15–17] according to Eq. (4): −
þ
glucose→gluconolactone þ 2e þ 2H
ð4Þ
These bioanodes exhibit an OCP of −0.05 V at 5 mM glucose, which is closed to the redox potential of NQ, and a maximum current density of 106 μA cm−2 at 5 mM glucose and 140 mM Cl−. The polarization curves indicate that DWCNT bioanodes are the limiting bioelectrode in this system. It should be noted that percentages of error are higher in the case of the bienzymatic DWCNT/HRP/GOx biocathodes. This may ascribed to the complexity of the mechanism of oxygen reduction performed by two different enzymes immobilized in the DWCNT film. These electrodes were further studied in a GBFC configuration. 3.2. The glucose/H2O2 biofuel cell
Fig. 2. (A) Calibration curve for a DWCNT/HRP cathode at increasing H2O2 concentrations. (B) Electrocatalytic properties towards H2O2 oxidation and reduction at (♦) DWCNT electrodes and (■)DWCNT/HRP electrodes in 1 mM H2O2. Dashed line: mathematical subtraction of H2O2 oxidation currents from bioelectrocatalytic H2O2 reduction currents. All values were measured after a 5-min discharge under stirring at constant potentials in PBS at 37 °C. The error bars correspond to the values recorded for three bioelectrodes.
Carbon cloth bioelectrodes were placed in front of each other in a solution of 5 mM glucose under air in 140 mM NaCl. Fig. 3B shows the 5-min discharge curves performed under stirring at constant potentials. Both polarization and power curves are depicted in Fig. 3C. The GBFC exhibits an excellent OCV of 0.45 V, which approximately corresponds to the sum of the OCP values for each bioelectrode. In addition, a maximum current density of 127 μA cm−2 and a maximum
Fig. 3. (A) Polarization curves for (a) DWCNT/GOx/HRP biocathodes in air-saturated 5 mM glucose solution, (b) a DWCNT/GOx electrode in oxygen-saturated 5 mM glucose solution and (c) a DWCNT/NQ/GOx/Catalase bioanode in air-saturated 5 mM glucose solution. (B) Five-minute discharges for GBFC at constant potentials under stirring in an air-saturated 5 mM glucose solution. (C) Polarization and power curves for the GBFC. All values were measured after a 5-min discharge under stirring at constant potentials in 5 mM glucose PBS solutions at 37 °C. The error bars correspond to the values recorded for three bioelectrodes.
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power output of 30 μW cm−2 at 0.3 V were recorded. It is noteworthy that the NaCl concentrations have negligible influence on GBFC performances. In particular, this membraneless GBFC shows no power decrease in the absence or in the presence of 0.14 M NaCl. 4. Conclusion This novel type of GBFC is the first example of a biofuel cell taking advantage of the enzymatic activity of GOx for both oxidation of glucose and reduction of O2 into H2O2. In combination with the efficient wiring of HRP on DWCNT films deposited on flexible carbon cloth, these devices achieve excellent OCV values of 0.45 V and maximum power output of 30 μW cm−2 in 5 mM glucose solutions. Taking into account the stability and availability of these enzymes and the flexibility of the carbon-based electrodes, these GBFCs represent a promising alternative for in vivo applications of GBFCs. Supplementary data to this article can be found online at http:// dx.doi.org/10.1016/j.elecom.2013.05.018. Acknowledgments The authors thank the ANR Emergence-2010 EMMA-043-02 and the ANR Investissements d'avenir-Nanobiotechnologies-10-IANN-0-02 programs for partial funding. The “Région Rhones-Alpes” is acknowledged for the PhD funding of B. Reuillard.
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