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Direct electron transfer catalyzed by bilirubin oxidase for air breathing gas-diffusion electrodes
Electrochemistry Communications 13 (2011) 247–249
Contents lists available at ScienceDirect
Electrochemistry Communications j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / e l e c o m
Direct electron transfer catalyzed by bilirubin oxidase for air breathing gas-diffusion electrodes Gautam Gupta, Carolin Lau, Vijaykumar Rajendran, Frisia Colon, Brittany Branch, Dmitri Ivnitski, Plamen Atanassov ⁎ Center for Emerging Energy Technologies, Department of Chemical and Nuclear Engineering, University of New Mexico, Albuquerque, NM 87131, United States
a r t i c l e
i n f o
Article history: Received 18 November 2010 Received in revised form 22 December 2010 Accepted 29 December 2010 Available online 4 January 2011 Keywords: Bilirubin oxidase Direct electron transfer Oxygen reduction Biofuel cell Gas-diffusion electrode
a b s t r a c t A gas-diffusion electrode based on hydrophobized carbon black composite for enhanced enzymatic oxygen reduction is presented for the possible application as a biocathode in enzymatic fuel cells. Immobilized bilirubin oxidase (BOD) from Myrothecium verrucaria reduces oxygen in a 4-electron mechanism, as shown by Tafel kinetics and shows evidence of direct electron transfer. Under air-breathing conditions the galvanostatic polarization results in an open circuit potential of + 0.65 V (vs. Ag/AgCl, pH 7) and in + 0.5 V at an applied current density of 0.35 mA cm−2 which is 3 times higher compared to aqueous oxygen supply. © 2011 Elsevier B.V. All rights reserved.
1. Introduction Introducing nano-structured materials has benefited biofuel cells by allowing biocatalytic operations to take place closer to the redox potential of the enzymes [1–3]. The most attractive enzymes for oxygen bio-cathode development are laccase and bilirubin oxidase (BOD). Although laccase has been found to be a very efficient biocatalyst, the inhibitory effect of chloride ions in physiological solutions has prompted the use of BOD as an alternative. This is of particular importance for implantable biofuel cells, or cells utilizing physiological fluids, that contain chloride ions [1, 2, 4–11]. BOD belongs to the group of multicopper oxidases and catalyzes the oxidation of substances, such as bilirubin to biliverdin with the concurrent reduction of oxygen to water [2, 9, 12–19]. Most of the reports on bio-cathodes for enzymatic reduction of oxygen are based on mediated mechanisms using ferrocene [20], metallocyanides [21], and osmium redox polymer [22, 23]. Achieving direct electron transfer (DET) between redox-enzymes and electrode is attractive due to simplicity, easiness of miniaturization, higher redox potential, and high power-output. In DET the electrons are transferred directly from the electrode to the substrate molecule (or vise versa) via the active site of the enzyme, thus avoiding the use of mediator molecules that normally impose stringent conditions on the resulting electrode potential and the electrode design itself, especially when used in
⁎ Corresponding author. Tel.: +1 505 277 2640; fax: +1 505 277 5433. E-mail address: [email protected] (P. Atanassov). 1388-2481/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.elecom.2010.12.024
biofuel cells. Direct electrochemistry of multicopper oxidases, e.g. laccase, has been known for over 30 years [24, 25]. DET of BOD on different carbon electrodes has been demonstrated in several publications [16, 26–28] and has been previously reviewed [29]. The very low solubility and small diffusion coefficients of dissolved oxygen in aqueous solutions limit the biocatalytic ORR. Engineering a gas-diffusion electrode exposes the enzyme to the three phase interphase, namely, the current collecting solid phase for DET, the liquid phase for efficient proton transfer, and the gas phase for effective oxygen transport. The gas-diffusion layer consists of hydrophobic gas channels for accelerated gas (e.g. oxygen) transport, therefore the rate of enzymatic reaction becomes diffusion-controlled [30–33]. In this study, we report a gas-diffusion BOD catalyzed cathode for the bioelectroreduction of oxygen at neutral pH based on DET. The gas-diffusion cathode consists of two layers; (i) a gas diffusion layer made of hydrophobized carbon black and (ii) a catalytic layer made of untreated hydrophilic carbon black. The second layer contains the physically adsorbed enzymes and provides a porous, three-dimensional, conductive matrix which is at the same time electrochemically accessible for the electrolyte. 2. Experimental BOD (Myrothecium verrucaria) was purchased from Sigma (St. Louis, MO, USA). Hydrophobized carbon black was prepared following a previously reported procedure [30], and contained 35% by weight of Teflon. The gas-diffusion electrode (GDE), as shown in Fig. 1 (insert) was prepared by pressing 10 mg hydrophobized carbon black (Vulcan
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G. Gupta et al. / Electrochemistry Communications 13 (2011) 247–249
Fig. 1. Cyclic voltammogram of carbon composite electrode (––) with and (∙∙∙) without BOD, 0.1 M phosphate buffer oxygen saturated, 20 mV s−1. The inserted image shows a scheme of the gas-diffusion electrode with (1) agarose gelled electrolyte, (2) Nylon membrane, (3) catalytic layer and (4) gas-diffusion layer.
XC72R, Cabot, MA, USA, with 35% wt. Teflon®) to form a hydrophobic layer of 1 cm2 on one side of a nickel mesh (Alfa Aesar, MA, USA). 20 mg of a 1:1 mixture of carbon black and hydrophobized carbon black was placed on top of the hydrophobic layer and pressed at 15 Kpsi for 5 min to form the hydrophilic (catalytic) layer. The BOD enzyme solution (0.6 mg in 60 μl buffer) was spread evenly on the hydrophilic layer and allowed to dry for 30 min at room temperature. The enzyme electrode with the catalytic layer facing a protective nylon membrane (Pall, MI, USA) was placed over the pre-formed agar gel electrolyte presoaked in 0.1 M phosphate buffer solution at pH 7. All electrochemical measurements were performed in 0.1 M phosphate buffer at pH 7 in a three electrode configuration with Ag/AgCl reference and nickel mesh counter electrodes using a EG&G potentiostat (Model 263A, Princeton, NJ). 3. Results and discussion Cyclic voltammetric experiments were carried out under N2 and O2 saturated conditions, to determine the on-set potential for oxygen electroreduction by scanning the potential between −0.8 V and +0.8 V at a rate of 5 mV s−1. Fig. 1 shows the cyclic voltammogram of the GDE electrode with and without enzymes. The ORR on-set potential was around + 0.55 V (vs. Ag/AgCl) which agrees with literature data of approximately + 0.5 V (vs. Ag/AgCl, +0.7 V vs. NHE at pH 7.4) [26] whereas a half wave potential of + 0.31 V (vs. Ag/AgCl) is slightly lower compared to a previously reported value of +0.4 V [27] and + 0.35 V [34] (vs. Ag/AgCl). These results together with an obtained OCP of +0.65 V (vs. Ag/AgCl), which is close to the redox potential of the enzyme, clearly indicate the DET phenomenon of the BOD enzyme with the porous carbon electrode. The factors controlling the kinetics of electron transfer are determined by the difference in reduction potentials between the donor and acceptor redox centers and the distance between the centers [35]. Both, the reduction potential and distance, are modulated by pH and the surrounding protein environment [12, 36]. This hypothesis is tested in the gas-diffusion electrodes we have fabricated, and Fig. 3 summarizes the pH dependence of OCP and current density. Experiments were done both under galvanostatic mode with the OCPs measured at zero current density, and the current densities measured at +0.5 V applied potential to study the influence of pH on the BODcatalyzed oxygen reduction. Fig. 2 (left axis) depicts a linear pH influence on the OCPs with a slope of 43 mV/pH. In the literature, comparable data show similar behavior of the half-wave potential in the pH range from 3 to 7 with about 30 mV/pH and 21 mV/pH for free and immobilized BOD, respectively [34]. This implies a more complex
Fig. 2. Influence of pH on the (left) open circuit potential and the (right) current density at an applied potential of + 0.5 V; 0.1 M phosphate buffer pH 7 oxygen saturated.
conjugated electron/proton transfer mechanism at the T1 copper center, which should be close to a Nernst-factor of 60 mV/pH for a one-proton-one-electron exchange. Speculations on enzyme purity, different enzyme isoforms, changing substrate pH optima [26] and influences of immobilization procedures can be made. Fig. 2 (right axis) illustrates the optimum neutral pH for BOD catalysis, measured as current density at an applied potential of 0.5 V vs. Ag/AgCl. This agrees with findings by Shleev et al. who determined two pH maxima at pH 4.5 and pH 7 and current densities at zero potential of 20 μA cm−2 and 15 μA cm−2, respectively [26]. In contrast our gas-diffusion electrode resulted in much higher current densities of 0.3 mA cm−2 (at 0.5 V applied potential, pH 7). To study the Faradaic oxygen reduction current of BOD modified cathodes galvanostatic polarization curves were obtained (Fig. 3) with open (air breathing condition) and blocked air supply. At a potential of + 0.5 V a current density of 350 μA cm−2 is about 3 times higher in air breathing mode compared to a purely aqueous oxygen supply. Furthermore the addition of physiologically relevant concentration of chloride ions (150 mM KCl) on BOD catalysis shows almost no inhibition on the polarization behavior. The results were found in concurrence with earlier reports on the non-influence of chloride ions on BOD-catalyzed ORR at cathodes for biofuel cells operated under physiological conditions. Also chloride ions do not inhibit the DET characteristics of the BOD enzyme [22, 29] and the wide operational pH range (5–8) makes the BOD enzyme an excellent contender
Fig. 3. Polarization curves obtained for BOD on the gas-diffusion electrode in (▲▼) air breathing mode in the (▼) absence and (▲) presence of 150 mM KCl and with a blocked gas-diffusion layer in (●) oxygen and (⋄) nitrogen saturated buffer (0.1 M PB, pH 7). (Insert) Tafel plot.
G. Gupta et al. / Electrochemistry Communications 13 (2011) 247–249
for implantable devices [37]. For high overpotentials η, the Tafel behavior was analyzed according to η = a+ b log(i) by plotting the overpotential as a function of the current density i, with the slope b = −(2.3RT)/(Fαn) and the intercept a = (2.3RT) log (i0)/(Fα) [38]; α is the charge transfer coefficient, i0 is the exchange current density, n is the numbers of electrons per molecule and R, F, and T are the gas constant, Faraday constant and temperature, respectively. In bioelectrocatalysis similar Tafel slopes for adsorbed laccase on carbon of 15, 30 and 60 mV/ dec (corresponding to four-, two-, and one-electron mechanisms) have been reported [39] and a more complex value of 67 mV/dec has been observed for CueO [40]. Steady-state polarization dependencies obtained with BOD-catalyzed ORR in gas-diffusion “air-breathing” mode (Fig. 3) are characterized by a Tafel slope of 30 mV/dec (Fig. 3 insert). Whether this can be interpreted as a “2-electron-transfer” while assuming α=0.5 or a “4-electron transfer” while assuming α being far from 0.5 remains outside of the goals of this communication. Tafel slopes are difficult to interpret in conjunction with the reaction mechanism for ORR catalysts as studied as Pt. Even for such well established 4-electron ORR catalysts both theoretical and experimental papers suggest 60 and 120 mV/dec depending on conditions and current density [41–43]. Direct relationship of the ORR mechanism on BOD and its Tafel behavior remains a task for an upcoming study.
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4. Conclusions This paper demonstrates a biofuel cell cathode design based on direct (mediator-less) oxygen reduction by BOD incorporated into the catalytic layer of a gas-diffusion electrode of hydrophobic type operating in a passive, “air-breathing” mode. This presents a successful application of a BOD electrode as a cathode for the biofuel cell based on DET between BOD and a composite surface architecture of the gasdiffusion carbon matrix. The open circuit potential of the BOD cathode was +0.65 V vs. Ag/AgCl, which is close to the redox potential of the enzyme itself. The BOD-catalyzed gas-diffusion electrode did not show any significant influence on the polarization curve in the presence of chloride ions in the supporting electrolyte, thus allowing the use of such cathodes under physiological conditions. Higher current densities with a minimal overpotential are achieved using the gas-diffusion electrode design of this new BOD-catalyzed gas-diffusion cathode based on the DET approach that offers promising solutions for generations of new classes of membrane-less biofuel cells. Acknowledgements Support from the ONR (N00140210169) and AFOSR (FA9550-061-0264) is gratefully acknowledged.
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Contents lists available at ScienceDirect
Electrochemistry Communications j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / e l e c o m
Direct electron transfer catalyzed by bilirubin oxidase for air breathing gas-diffusion electrodes Gautam Gupta, Carolin Lau, Vijaykumar Rajendran, Frisia Colon, Brittany Branch, Dmitri Ivnitski, Plamen Atanassov ⁎ Center for Emerging Energy Technologies, Department of Chemical and Nuclear Engineering, University of New Mexico, Albuquerque, NM 87131, United States
a r t i c l e
i n f o
Article history: Received 18 November 2010 Received in revised form 22 December 2010 Accepted 29 December 2010 Available online 4 January 2011 Keywords: Bilirubin oxidase Direct electron transfer Oxygen reduction Biofuel cell Gas-diffusion electrode
a b s t r a c t A gas-diffusion electrode based on hydrophobized carbon black composite for enhanced enzymatic oxygen reduction is presented for the possible application as a biocathode in enzymatic fuel cells. Immobilized bilirubin oxidase (BOD) from Myrothecium verrucaria reduces oxygen in a 4-electron mechanism, as shown by Tafel kinetics and shows evidence of direct electron transfer. Under air-breathing conditions the galvanostatic polarization results in an open circuit potential of + 0.65 V (vs. Ag/AgCl, pH 7) and in + 0.5 V at an applied current density of 0.35 mA cm−2 which is 3 times higher compared to aqueous oxygen supply. © 2011 Elsevier B.V. All rights reserved.
1. Introduction Introducing nano-structured materials has benefited biofuel cells by allowing biocatalytic operations to take place closer to the redox potential of the enzymes [1–3]. The most attractive enzymes for oxygen bio-cathode development are laccase and bilirubin oxidase (BOD). Although laccase has been found to be a very efficient biocatalyst, the inhibitory effect of chloride ions in physiological solutions has prompted the use of BOD as an alternative. This is of particular importance for implantable biofuel cells, or cells utilizing physiological fluids, that contain chloride ions [1, 2, 4–11]. BOD belongs to the group of multicopper oxidases and catalyzes the oxidation of substances, such as bilirubin to biliverdin with the concurrent reduction of oxygen to water [2, 9, 12–19]. Most of the reports on bio-cathodes for enzymatic reduction of oxygen are based on mediated mechanisms using ferrocene [20], metallocyanides [21], and osmium redox polymer [22, 23]. Achieving direct electron transfer (DET) between redox-enzymes and electrode is attractive due to simplicity, easiness of miniaturization, higher redox potential, and high power-output. In DET the electrons are transferred directly from the electrode to the substrate molecule (or vise versa) via the active site of the enzyme, thus avoiding the use of mediator molecules that normally impose stringent conditions on the resulting electrode potential and the electrode design itself, especially when used in
⁎ Corresponding author. Tel.: +1 505 277 2640; fax: +1 505 277 5433. E-mail address: [email protected] (P. Atanassov). 1388-2481/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.elecom.2010.12.024
biofuel cells. Direct electrochemistry of multicopper oxidases, e.g. laccase, has been known for over 30 years [24, 25]. DET of BOD on different carbon electrodes has been demonstrated in several publications [16, 26–28] and has been previously reviewed [29]. The very low solubility and small diffusion coefficients of dissolved oxygen in aqueous solutions limit the biocatalytic ORR. Engineering a gas-diffusion electrode exposes the enzyme to the three phase interphase, namely, the current collecting solid phase for DET, the liquid phase for efficient proton transfer, and the gas phase for effective oxygen transport. The gas-diffusion layer consists of hydrophobic gas channels for accelerated gas (e.g. oxygen) transport, therefore the rate of enzymatic reaction becomes diffusion-controlled [30–33]. In this study, we report a gas-diffusion BOD catalyzed cathode for the bioelectroreduction of oxygen at neutral pH based on DET. The gas-diffusion cathode consists of two layers; (i) a gas diffusion layer made of hydrophobized carbon black and (ii) a catalytic layer made of untreated hydrophilic carbon black. The second layer contains the physically adsorbed enzymes and provides a porous, three-dimensional, conductive matrix which is at the same time electrochemically accessible for the electrolyte. 2. Experimental BOD (Myrothecium verrucaria) was purchased from Sigma (St. Louis, MO, USA). Hydrophobized carbon black was prepared following a previously reported procedure [30], and contained 35% by weight of Teflon. The gas-diffusion electrode (GDE), as shown in Fig. 1 (insert) was prepared by pressing 10 mg hydrophobized carbon black (Vulcan
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G. Gupta et al. / Electrochemistry Communications 13 (2011) 247–249
Fig. 1. Cyclic voltammogram of carbon composite electrode (––) with and (∙∙∙) without BOD, 0.1 M phosphate buffer oxygen saturated, 20 mV s−1. The inserted image shows a scheme of the gas-diffusion electrode with (1) agarose gelled electrolyte, (2) Nylon membrane, (3) catalytic layer and (4) gas-diffusion layer.
XC72R, Cabot, MA, USA, with 35% wt. Teflon®) to form a hydrophobic layer of 1 cm2 on one side of a nickel mesh (Alfa Aesar, MA, USA). 20 mg of a 1:1 mixture of carbon black and hydrophobized carbon black was placed on top of the hydrophobic layer and pressed at 15 Kpsi for 5 min to form the hydrophilic (catalytic) layer. The BOD enzyme solution (0.6 mg in 60 μl buffer) was spread evenly on the hydrophilic layer and allowed to dry for 30 min at room temperature. The enzyme electrode with the catalytic layer facing a protective nylon membrane (Pall, MI, USA) was placed over the pre-formed agar gel electrolyte presoaked in 0.1 M phosphate buffer solution at pH 7. All electrochemical measurements were performed in 0.1 M phosphate buffer at pH 7 in a three electrode configuration with Ag/AgCl reference and nickel mesh counter electrodes using a EG&G potentiostat (Model 263A, Princeton, NJ). 3. Results and discussion Cyclic voltammetric experiments were carried out under N2 and O2 saturated conditions, to determine the on-set potential for oxygen electroreduction by scanning the potential between −0.8 V and +0.8 V at a rate of 5 mV s−1. Fig. 1 shows the cyclic voltammogram of the GDE electrode with and without enzymes. The ORR on-set potential was around + 0.55 V (vs. Ag/AgCl) which agrees with literature data of approximately + 0.5 V (vs. Ag/AgCl, +0.7 V vs. NHE at pH 7.4) [26] whereas a half wave potential of + 0.31 V (vs. Ag/AgCl) is slightly lower compared to a previously reported value of +0.4 V [27] and + 0.35 V [34] (vs. Ag/AgCl). These results together with an obtained OCP of +0.65 V (vs. Ag/AgCl), which is close to the redox potential of the enzyme, clearly indicate the DET phenomenon of the BOD enzyme with the porous carbon electrode. The factors controlling the kinetics of electron transfer are determined by the difference in reduction potentials between the donor and acceptor redox centers and the distance between the centers [35]. Both, the reduction potential and distance, are modulated by pH and the surrounding protein environment [12, 36]. This hypothesis is tested in the gas-diffusion electrodes we have fabricated, and Fig. 3 summarizes the pH dependence of OCP and current density. Experiments were done both under galvanostatic mode with the OCPs measured at zero current density, and the current densities measured at +0.5 V applied potential to study the influence of pH on the BODcatalyzed oxygen reduction. Fig. 2 (left axis) depicts a linear pH influence on the OCPs with a slope of 43 mV/pH. In the literature, comparable data show similar behavior of the half-wave potential in the pH range from 3 to 7 with about 30 mV/pH and 21 mV/pH for free and immobilized BOD, respectively [34]. This implies a more complex
Fig. 2. Influence of pH on the (left) open circuit potential and the (right) current density at an applied potential of + 0.5 V; 0.1 M phosphate buffer pH 7 oxygen saturated.
conjugated electron/proton transfer mechanism at the T1 copper center, which should be close to a Nernst-factor of 60 mV/pH for a one-proton-one-electron exchange. Speculations on enzyme purity, different enzyme isoforms, changing substrate pH optima [26] and influences of immobilization procedures can be made. Fig. 2 (right axis) illustrates the optimum neutral pH for BOD catalysis, measured as current density at an applied potential of 0.5 V vs. Ag/AgCl. This agrees with findings by Shleev et al. who determined two pH maxima at pH 4.5 and pH 7 and current densities at zero potential of 20 μA cm−2 and 15 μA cm−2, respectively [26]. In contrast our gas-diffusion electrode resulted in much higher current densities of 0.3 mA cm−2 (at 0.5 V applied potential, pH 7). To study the Faradaic oxygen reduction current of BOD modified cathodes galvanostatic polarization curves were obtained (Fig. 3) with open (air breathing condition) and blocked air supply. At a potential of + 0.5 V a current density of 350 μA cm−2 is about 3 times higher in air breathing mode compared to a purely aqueous oxygen supply. Furthermore the addition of physiologically relevant concentration of chloride ions (150 mM KCl) on BOD catalysis shows almost no inhibition on the polarization behavior. The results were found in concurrence with earlier reports on the non-influence of chloride ions on BOD-catalyzed ORR at cathodes for biofuel cells operated under physiological conditions. Also chloride ions do not inhibit the DET characteristics of the BOD enzyme [22, 29] and the wide operational pH range (5–8) makes the BOD enzyme an excellent contender
Fig. 3. Polarization curves obtained for BOD on the gas-diffusion electrode in (▲▼) air breathing mode in the (▼) absence and (▲) presence of 150 mM KCl and with a blocked gas-diffusion layer in (●) oxygen and (⋄) nitrogen saturated buffer (0.1 M PB, pH 7). (Insert) Tafel plot.
G. Gupta et al. / Electrochemistry Communications 13 (2011) 247–249
for implantable devices [37]. For high overpotentials η, the Tafel behavior was analyzed according to η = a+ b log(i) by plotting the overpotential as a function of the current density i, with the slope b = −(2.3RT)/(Fαn) and the intercept a = (2.3RT) log (i0)/(Fα) [38]; α is the charge transfer coefficient, i0 is the exchange current density, n is the numbers of electrons per molecule and R, F, and T are the gas constant, Faraday constant and temperature, respectively. In bioelectrocatalysis similar Tafel slopes for adsorbed laccase on carbon of 15, 30 and 60 mV/ dec (corresponding to four-, two-, and one-electron mechanisms) have been reported [39] and a more complex value of 67 mV/dec has been observed for CueO [40]. Steady-state polarization dependencies obtained with BOD-catalyzed ORR in gas-diffusion “air-breathing” mode (Fig. 3) are characterized by a Tafel slope of 30 mV/dec (Fig. 3 insert). Whether this can be interpreted as a “2-electron-transfer” while assuming α=0.5 or a “4-electron transfer” while assuming α being far from 0.5 remains outside of the goals of this communication. Tafel slopes are difficult to interpret in conjunction with the reaction mechanism for ORR catalysts as studied as Pt. Even for such well established 4-electron ORR catalysts both theoretical and experimental papers suggest 60 and 120 mV/dec depending on conditions and current density [41–43]. Direct relationship of the ORR mechanism on BOD and its Tafel behavior remains a task for an upcoming study.
References [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] [16] [17] [18] [19] [20] [21] [22]
4. Conclusions This paper demonstrates a biofuel cell cathode design based on direct (mediator-less) oxygen reduction by BOD incorporated into the catalytic layer of a gas-diffusion electrode of hydrophobic type operating in a passive, “air-breathing” mode. This presents a successful application of a BOD electrode as a cathode for the biofuel cell based on DET between BOD and a composite surface architecture of the gasdiffusion carbon matrix. The open circuit potential of the BOD cathode was +0.65 V vs. Ag/AgCl, which is close to the redox potential of the enzyme itself. The BOD-catalyzed gas-diffusion electrode did not show any significant influence on the polarization curve in the presence of chloride ions in the supporting electrolyte, thus allowing the use of such cathodes under physiological conditions. Higher current densities with a minimal overpotential are achieved using the gas-diffusion electrode design of this new BOD-catalyzed gas-diffusion cathode based on the DET approach that offers promising solutions for generations of new classes of membrane-less biofuel cells. Acknowledgements Support from the ONR (N00140210169) and AFOSR (FA9550-061-0264) is gratefully acknowledged.
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