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Recoverable hybrid enzymatic biofuel cell with molecular oxygen-independence
Author’s Accepted Manuscript Recoverable Hybrid enzymatic biofuel cell with molecular oxygen-independence You Yu, Miao Xu, Lu Bai, Lei Han, Shaojun Dong
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S0956-5663(15)30318-3 http://dx.doi.org/10.1016/j.bios.2015.07.070 BIOS7888
To appear in: Biosensors and Bioelectronic Received date: 10 February 2015 Revised date: 11 July 2015 Accepted date: 30 July 2015 Cite this article as: You Yu, Miao Xu, Lu Bai, Lei Han and Shaojun Dong, Recoverable Hybrid enzymatic biofuel cell with molecular oxygen-independence, Biosensors and Bioelectronic, http://dx.doi.org/10.1016/j.bios.2015.07.070 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Recoverable Hybrid Enzymatic Biofuel Cell with Molecular Oxygen-independence You Yu a, b†, Miao Xu a, b†, Lu Bai a, Lei Han a, b, Shaojun Dong a, b,* a
State Key Laboratory of Electroanalytical Chemistry, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun, Jilin, 130022; b
University of Chinese Academy of Sciences, Beijing, 100049 Corresponding author email: [email protected] †These authors contributed equally to this work.
Abstract Enzymatic biofuel cells (EBFCs) have drawn great attentions because of its potential in energy conversion. However, designing of highly efficient EBFCs which can adapt to the anaerobic system is still a great challenge. In this study, we propose a novel hybrid enzymatic biofuel cell (HEBFC) which was fabricated by a glucose dehydrogenase modified bioanode and a solid-state silver oxide/silver (Ag2O/Ag) cathode. The as-assembled HEBFC exhibited an open circuit potential of 0.59 V and a maximum power output of 0.281 mW cm-2 at 0.34 V in air saturated buffer. Especially, due to the introduction of Ag2O/Ag, our HEBFC could also operate under anaerobic condition, while the maximum power output would reach to 0.275 mW cm-2 at 0.34 V. Furthermore, our HEBFC had stable cycle operation and could keep high power output for a certain time as the result of the regeneration of Ag2O. Our work provides a new concept to develop EBFCs for efficient energy conversion in the future. 1
Keywords: biofuel cell; molecular oxygen- independence; recoverable; Ag2O/Ag
1. Introduction Enzymatic biofuel cells (EBFCs), which employ enzymes as the biocatalyst to harvest energy from numerous kinds of fuels, have attracted substantial research efforts in recent years (Heller 2004). As one important type of the green energy devices, EBFCs usually show high efficiency and specificity compared with the noble metal catalysts-based fuel cells as the result of using the enzymes as bioelectrocatalysts (Cracknell et al. 2008). As more and more significant improvements concerned on the EBFCs are achieved, EBFCs have already been successfully applied in several ways like healthcare devices (Diamond et al. 2008), self-power sensors for poisonous ions (Wen et al. 2011), implanted devices in living creatures (Katz and MacVittie 2013) and several photoelectrochemical devices (Zhang et al. 2014). For examples, Wang and his co-workers had used epidermal EBFCs to harvest energy from human perspiration and detect the healthy conditions by the power output at the same time (Jia et al. 2013). Moreover, Katz et al. had used EBFCs to assemble electrochemical memristor devices for the biocomputing systems (MacVittie and Katz 2014). Up to now, except biocatalysts, which include bilirubin oxidase (Mano and Edembe 2013), laccase (Agnès et al. 2014) and horseradish peroxidase (Agnès et al. 2013), EBFC devices have also utilized various types of materials as cathodic 2
catalysts, for instance, noble metal like Pt black (Jia et al. 2013) or some hybrid materials such as functional TiO2 supported Pt nanoparticles (Wen et al. 2010), cobalt oxide nanoparticles on graphene nanosheets (Co3O4/GN) (Hsieh et al. 2012) and graphene assembled by Fe/Pt nanoparticles (GN/FePt) (Guo and Sun 2012). Most of the cathodic catalysts above are based on the oxygen reduction. The performances of these molecular oxygen-dependent EBFCs rely on the content of oxygen in the solution tremendously; even worse, they cannot adapt to the anaerobic systems such as Saccharomyces cerevisiae. In Saccharomyces cerevisiae, glucose would change into ethanol by the saccharomyces sereviciae (Aiba et al. 1968). In order to monitor this process, the EBFCs as self-powered sensors should work well in this anaerobic system, but most conventional EBFCs cannot work even for regular operating. Further for the implantable applications, EBFCs have already been implanted in vivo or fruits such as grape (Mano et al. 2003), rat (Castorena-Gonzalez et al. 2013; Cheng et al. 2013), insect (Rasmussen et al. 2012) and snail (Halamkova et al. 2012) successfully. However, the concentration of oxygen in the blood or juice always limits the performances of the implanted EBFCs, these devices even need some oxygen from outside. Furthermore, healthcare devices, which are important EBFCs` applications, would be used to monitor the lactate after anaerobic exercises to analyze the health of human. Therefore, molecular oxygen-independent EBFCs, which could operate for long under less dissolved oxygen content, are urgent for the implantable devices. To assemble a molecular oxygen-independent EBFC, a novel strategy is 3
proposed in this study. As known, metal oxide can accept electrons then transform to metal subsequently (Desilvestro 1990). Hence the metal oxide has potential to take the place of the oxygen as an electron acceptor at the cathode (Jiang et al. 2012). So we may combine a bioanode modified by enzymes with the metal oxide cathode to fabricate
a
hybrid
enzymatic
biofuel
cell
(HEBFC)
with
molecular
oxygen-independence. Moreover metal oxide/metal as a redox couple can be converted to each other through electrochemical processes (Therese and Kamath 2000), it means that the HEBFCs assembled with metal oxide cathode will be recoverable by the interconversion of the metal oxide/metal. Among the various kinds of metal oxide/metal couples, the silver oxide/silver (Ag2O/Ag) redox couple was chosen as the cathode for the HEBFC, because this redox couple possessed several advantages (Xie et al. 2013). Firstly, Ag2O and Ag could remain stable under neutral pH and ambient temperature for a long time (Priya et al. 2009), which was significant for EBFCs. Secondly, the Ag2O/Ag is favorable to gain electrons because of its higher reduction potential than that of other electron acceptors in anaerobic system (such as SO42- and CO2) (Han et al. 2014). Thirdly, the reduction potential of Ag2O/Ag (0.342 V vs SHE) was close to that of O2/OH- (0.401 V vs SHE) at pH = 7. Besides, Ag is cheaper than other noble metals like gold or platinum, it was important for further applications with broader prospect. Herein, a silver foil was directly electrochemically oxidized under a potentiostatical process to prepare Ag2O/Ag cathode. This Ag2O/Ag cathode has a high reduction potential, a short generating time and shows high cycle stability after 4
regeneration. In this context, we combine the Ag2O/Ag cathode with a bioanode modified by glucose dehydrogenase to fabricate a molecular oxygen-independent recoverable HBFC, which can operate well no matter oxygen existed or not. This HEBFC showed an excellent power output, contributed by the improved electronic transfer rate at the Ag2O/Ag cathode. Overall, this HEBFC may introduce a new type of EBFCs for energy recovery and further applications in the anaerobic system.
2. Material and methods 2.1 Chemicals Sodium hydroxide (NaOH) and nitric acid (HNO3) were purchased from Beijing Chemical Works, P. R. China. Multi-walled carbon nanotubes (MWNTs) (80% purity, diameter 20-50 nm) were purchased from Shenzhen Nanotech. Port. Co. Ltd. (Shenzhen, China). Silver foil (99.99%), β-D-(+)-glucose, chitosan (CS), Meldola`s blue (MDB), glucose dehydrogenase (GDH) (E.C. 1.1.1.47, initial activity of 235.3 U mg-1 from Pseudomonas sp.) and Nafion membrane were obtained from Sigma and used as received. NADH and NAD+ were purchased from the Gen-view Scientific Inc. A 0.10 M HAc-NaAc buffer (pH = 7.0) was employed as the supporting electrolyte. All other chemicals were of analytical grade and all aqueous solutions were prepared with ultrapure water (>18.25 MΩ cm) obtained from Millipore system. 2.2 Modification of bioanode Glassy carbon electrode (GCE, 2.6 mm in diameter, Tokai Carbon Co., Japan) was polished sequentially with 1.0 and 0.3 µm alumina slurry and washed 5
ultrasonically in water and ethanol for several minutes, respectively. MWNTs was treated by well-established methods with slight modification, MWNTs was dispersed in 30% HNO3 and then refluxed for 24 h at 140 °C to acquire carboxylic group functionalized MWNTs. 10 µL of MWNTs suspension (2 mg mL−1) was spread onto a GCE to dry, then the electrode was immersed into 0.5 mM MDB for 30 min to achieved MDB-MWNTs/GCE. 5 µL GDH (1 mg mL−1) was coated on the MDB-MWNTs/GCE, which was dried at 4 °C overnight and achieved GDH/MDB-MWNTs/GCE. After that, 10 μL of 1% CS solution was spread onto the electrode surface to form a film (noted as CS/GDH/MDB-MWNTs/GCE). 2.3 Electrochemical synthesis of silver oxide/silver (Ag2O/Ag) cathode The Ag2O/Ag cathode was prepared by electrochemical method according to our previous work (Han et al. 2014). Prior to electrochemical oxidation, the silver foil was polished and sonicated with acetone, ethanol, and distilled water sequentially. Then it was immersed into the aqueous solution containing 1 M NaOH. The depositions were performed potentiostatically at 0.2 V for 1 h at room temperature. After that, it was rinsed with distilled water and dried in the air. When the Ag2O/Ag cathode was discharged for a certain degree, the silver foil would be re-oxidized into the aqueous solution containing 1 M NaOH after getting out from the compartment of the hybrid biofuel cell. Then the regenerated Ag2O/Ag cathode was combined with the bioanode to fabricate a recoverable cell. 2.4 Apparatus The X-ray diffraction (XRD) measurements were performed on a D8 Focus 6
diffractometer (Bruker) with Cu Kα radiation (λ = 0.15405 nm) in the range of 10-80º (2θ). Scanning electron microscopy (SEM) measurements were made on a XL30 ESEM with an accelerating voltage of 10 kV to determine the morphology of products. Cyclic voltammetry (CV) and linear sweep voltammetry (LSV) were performed with an electrochemical analyzer (CHI 832, Shanghai, China). The polarization curves (U-I) were achieved by the LSV measurement at scan rate 1 mv s-1. A three-electrode system was used including a platinum flat as the counter electrode and the Ag/AgCl (saturated KCl) as reference electrode, respectively. The operation of the biofuel cell was performed at 37 °C, other experiments were carried out at room temperature (22 °C).
3. Discussion and results In this work, a novel HEBFC consisting of an enzymatic electrode and a Ag2O/Ag electrode was fabricated as shown in Scheme 1. When the HEBFC was discharged, GDH catalyzed the oxidation of glucose to D-glucono-δ-lactone, which generating electrons and transferring to the bioanode. Then, the electrons flowed through an external circuit to the cathode. Even under anaerobic condition, the solid state Ag2O/Ag electrode could efficiently accept the electrons by Ag2O reduced to Ag, which was confirmed by the obvious color change from black to gray white (Figure S1). The phenomenon indicated that the HEBFC was molecular oxygen-independent. The Ag2O/Ag cathode was prepared by potentiostatically electrochemical oxidation method (Figure S2). After HEBFC was discharged completely, the obtained Ag 7
electrode could be easily recovered to its original form by another electrochemical oxidation. After 1 hour re-oxidized process, the regenerated Ag2O/Ag electrode could be used in the HEBFC once again. Therefore, the fabricated HEBFC not only could work under anaerobic condition, but also had the property of regeneration just to show the recoverability. To acquire the molecular oxygen-independent recoverable HEBFC, a Ag2O/Ag electrode was used as the cathode for the HEBFC. Recently, Criddle`s group and Cui`s group prepared a Ag2O/Ag cathode for a microbial battery (Xie et al. 2013), in that experiment, silver nanoparticles were mixed with conductive carbon black then coated onto a carbon cloth, and finally oxidized at 1 mA cm-2. Despite the assembled battery exhibited excellent performance, the complex method to manufacture the cathode still need be improved. Herein, we fabricated the cathode by a more simple way. A silver foil was directly electrochemically oxidized under a potentiostatical process at 0.2 V for 1 h to get the Ag2O/Ag cathode (Figure S2). This Ag2O/Ag cathode has a higher reduction potential and a shorter generating time, while the pretreatment of the Ag foil was simple and time-saving. Moreover, because we used the Ag foil as the electrode substrate and synthetize Ag2O directly, the electroconductivity of the electrode should be better. Furthermore, the Ag2O/Ag electrode can easily regenerated by fast electrochemical oxidation after the assembled HEBFC has been totally discharged. The structure and morphology of the Ag2O/Ag cathode were examined by X-ray diffraction (XRD), scanning electron microscopy (SEM) and Energy Dispersive X-ray 8
Spectrometry (EDX), respectively. Figure 1A showed the XRD pattern of the as-prepared Ag2O/Ag electrode. Compared with Figure S3, we could observe that after electrochemical oxidation two typical diffraction peaks located at 32.9° and 55.0° appeared, which corresponded to the (111) plane and (220) of cubic phase Ag2O (JCPDS 41-1104), respectively. While other diffraction peaks could be indexed to the (111), (200), (220) and (311) planes of the Ag substrate (JCPDS 65-2871). No other diffraction peaks were detected in the as-prepared electrode except the peaks of Ag2O and Ag. In other words, the Ag electrode (Figure S3a) converted to Ag2O/Ag electrode (Figure 1A and Figure S3b) successfully by electrochemical oxididation. And after running in HEBFC, Ag2O was reduced to Ag (Figure S3c). Meanwhile, when the Ag2O on the electrode was consumed completely, the color changed from black to gray white (Figure S1). Figure 1B and Figure S4 exhibited the surface morphology of the electrode in different states characterized by SEM. Compared with the fresh Ag foil (Figure S4A), the polished one (Figure S4B) was much smoother. By electrochemical oxidization, the obtained Ag2O/Ag electrode (Figure 1B and Figure S4C) showed irregular particles with size of 500 nm. However, once Ag2O was completely reduced after running in HEBFC, the large particles disappeared and the surface became much rougher (Figure S4D). Moreover, once the reduced Ag2O/Ag electrode was regenerated by second electrochemical oxidation (Figure S4E), and then reduced completely again (Figure S4F) after running in HEBFC, its surface would show the same morphology with the first time. In addition, the corresponding EDX spectra displayed that the Ag2O/Ag electrode could reduce to Ag electrode when 9
it worked as cathode (Figure S5). On the basis of the results above, it could confirm that the Ag2O/Ag electrode was successfully prepared by the anodization of Ag foil. Moreover, Ag2O acted as an electron acceptor and reduced to Ag when the HEBFC was discharged. Then the following potentiostatical electrochemical oxidation process could get another fresh Ag2O/Ag electrode. So the Ag2O/Ag electrode could be applied under anaerobic condition and has regeneration ability in the meantime. The electrochemical activity of the as-prepared Ag2O/Ag cathode was evaluated by linear sweep voltammetry (LSV) in a three-electrode system (Figure S6A). We found that the reduction potential of Ag2O/Ag electrode was initiated at 0.39 V under ambient air atmosphere and its reduction current was increased along with potential decreasing approached to 4.8 mA cm-2 at 0.1 V. In contrast, the oxygen reduction current at Ag electrode and Pt electrode might be ignored in the same potential range, which obviously indicated the superior electrochemical activity of Ag2O/Ag electrode. Furthermore, in the case of a N2-saturated solution, the reduction current of Ag2O/Ag electrode only slightly decreased. These results suggested that Ag2O/Ag electrode could be used in the absence and presence of oxygen. By all accounts, the Ag2O/Ag electrode was a propitious candidate to be the cathode in the HEBFC system owning to its simple preparation method, regeneration ability and better electrochemical activity under both ambient air atmosphere and anaerobic condition. In addition, we investigated the electrochemical performance of the bioanode. GDH, which was capable of catalyzing glucose to D-glucono-δ-lactone selectively, was fabricated as the biocatalytic anode. To acquire the lower catalytic potential, a 10
redox mediator MDB was adsorbed on the MWNTs via π–π interaction on the bioanode. As shown in Figure S7, cyclic voltammetry (CV) showed the MDB– MWNTs exhibited two pairs of redox waves, which were ascribed to the redox processes of MDB. And the oxidation of NADH was obvious by potential lower than -0.2V. When the GDH was immobilized on the MDB-MWNTs modified GCE, named GDH/MDB-MWNTs/GCE. The bioanode exhibited electrocatalytic activity for the oxidation of glucose. A catalytic wave at the GDH/MDB-MWNTs/GCE was obvious in the presence of 30 mM glucose, and the current density reached 4.1 mA cm-2 at -0.01 V (Figure S6B). The electrochemical catalytic activity toward glucose oxidation suggested that this composite electrode could be used as bioanode for EBFCs. According to the above results, we could evidently find the onset reduction potential of Ag2O/Ag electrode (0.39 V) was much higher than that of the bioanode. Thus, combining Ag2O/Ag cathode and bioanode to assemble a HEBFC and produce electric power was feasible. In this HEBFC, a nafion membrane was needed to avoid the interaction between the cathode and NAD+. As depicted in Figure 1C, the open circuit potential (OCP) of the HEBFC was 0.59 V, and the maximum power output approached to 0.281 mW cm-2 at 0.34 V in the presence of 30 mM glucose and 10 mM NAD+ under ambient air. On the contrary to the conventional EBFCs, the maximum power output of this HEBFC reached 0.275 mW cm-2 at 0.34 V in a N2-saturated atmosphere, which was similar to that under ambient air. The two OCP values were equal no matter the oxygen existed or not. For comparison, the HEBFCs were assembled by the same bioanode and different cathodes, which were pure Ag 11
electrode and Pt electrode. And their maximum power output values were only 0.018 mW cm-2 and 0.041 mW cm-2, respectively. The performances of these two HEBFCs were both significantly lower than the HEBFC with Ag2O/Ag cathode. So the Ag foil, which was treated as the electrode, had little contributions to the power output of the HEBFC, especially in a N2-saturated atmosphere. It was further confirmed that the Ag2O/Ag cathode did improve the performance of the HEBFC. To test its recoverable character, the HEBFC was discharged continuously in stirring 0.10 M HAc-NaAc buffer containing 10 mM NAD+ and 30 mM glucose at a maximum power output under ambient air. As expected, the HEBFC was discharged stably with slight decline of the power output. However the power output of the HEBFC was sharply decreased to zero suddenly, where the Ag2O converted to Ag totally (Figure S8). In the experiment of cycle stability, when the power output fallen to near 65% of maximum power output, the Ag2O/Ag cathode was regenerated to recover the HEBFC. Although the HAc-NaAc buffer had the low buffering capacity, the operation of HEBFC proceeded smoothly for long. As shown in Figure 2, we could obviously observe that the power output might still return to 99% of maximum power output and operated for another ~6 hours with high power output after regeneration. The possible reasons for it might be the slight disturbance of the electrode interface caused by low current and less amount of generated gluconic acid in this HEBFC system. This recoverable property proved that this HEBFC had the possibility to be used in further applications. Additionally, considering the possible interaction between the HEBFC and some ions (e.g. phosphate or halide ions) in some 12
other systems, the investigations aiming at improving the stability of Ag2O and increasing the operated time are still underway.
4. Conclusion In summary, we have successfully fabricated a novel recoverable and molecular oxygen-independent HEBFC composed of the GDH immobilized on the MDB-MWNTs modified bioanode and solid state Ag2O/Ag cathode. Because of the outstanding electrochemical properties of the introduced Ag2O/Ag, which was prepared by simple electrochemical oxidation method, the constructed HEBFC could possess a high OCP of 0.59 V and maximum power output of 0.281 mW cm -2 at 0.34 V in air saturated buffer. The results were significantly superior to the other HEBFC, by using Ag or Pt as cathode. Meanwhile, under anaerobic condition the maximum power output of the HEBFC could still reach 0.275 mW cm-2 at 0.34 V on account of Ag2O acting as a good electrons acceptor. Furthermore, our HEBFC had recoverable cycle stability and might operate with high power output for a certain time. Undoubtedly, our work provided a new strategy for developing EBFC, which can operate under anaerobic environment in the future.
Acknowledgments This work was supported by the National Natural Science Foundation of China (No. 21375123), the 973 Project (No. 2011CB911002) and the Ministry of Science and Technology of China (No. 2013YQ170585).
References Agnès, C., Cosnier, S., 2014. Energ. Environ. Sci. 7, 1884-1888. 13
Agnès, C., Cosnier, S., 2013. Electrochem. Commun. 34, 105-108. Aiba, S., Shoda, M., Nagatani, M., 1968. Biotechnol. Bioeng. 10, 845-864. Castorena-Gonzalez, J.A., Foote, C., MacVittie, K., Halámek, J., Halámková, L., Martinez-Lemus, L.A., Katz, E., 2013. Electroanalysis 25, 1579-1584. Cheng, H., Yu, P., Lu, X., Lin, Y., Ohsaka, T., Mao, L., 2013. The Analyst 138, 179-185. Cracknell, J.A., Vincent, K.A., Armstrong, F.A., 2008. Chem. Rev. 108, 2439-2461. Desilvestro, J., 1990. J. Electrochem. Soc. 137, 5C-22C. Diamond, D., Coyle, S., Scarmagnani, S., Hayes, J., 2008. Chem. Rev. 108, 652-679. Guo, S., Sun, S., 2012. J. Am. Chem. Soc. 134, 2492-2495. Halamkova, L., Katz, E., 2012. J. Am. Chem. Soc. 134, 5040-5043. Han, L., Guo, S., Xu, M., Dong, S., 2014. Chem. Commun. 50, 13331-13333. Heller, A., 2004. Phys. Chem. Chem. Phys. 6, 209-216. Hsieh, C.-T., Lin, J.-S., Chen, Y.-F., Teng, H., 2012. J. Phys. Chem. C 116, 15251-15258. Jia, W., Wang, J., 2013. Angew. Chem. Int. Ed. 52, 7233-7236. Jiang, J., Li, Y., Liu, J., Huang, X., Yuan, C., Lou, X.W., 2012. Adv. Mater. 24, 5166-5180. Katz, E., MacVittie, K., 2013. Energ. Environ. Sci. 6, 2791-2803. MacVittie, K., Katz, E., 2014. Chem. Commun. 50, 4816-4819. Mano, N., Edembe, L., 2013. Biosens. Bioelectron. 50, 478-485. Mano, N., Mao, F., Heller, A., 2003. J. Am. Chem. Soc. 125, 6588-6594. Priya, R., Baiju, K.V., Shukla, S., Biju, S., Reddy, M.L.P., Patil, K., Warrier, K.G.K., 2009. J. Phys. Chem. C 113, 6243-6255. Rasmussen, M., Ritzmann, R.E., Lee, I., Pollack, A.J., Scherson, D., 2012. J. Am. Chem. Soc. 134, 1458-1460. Therese, G.H.A., Kamath, P.V., 2000. Chem. Mater. 12, 1195-1204. Wen, D., Deng, L., Guo, S., Dong, S., 2011. Anal. Chem. 83, 3968-3972. Wen, D., Deng, L., Zhou, M., Guo, S., Shang, L., Xu, G., Dong, S., 2010. Biosens. Bioelectron. 25, 1544-1547. Xie, X., Ye, M., Hsu, P.C., Liu, N., Criddle, C.S., Cui, Y., 2013. Proc. Natl. Acad. Sci. U. S. A. 110, 15925-15930. Zhang, J., Su, Y., Zhu, Y., Yun, J., Yang, X., 2014. New J. Chem. 38(6), 2300-2304.
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Figure Captions
Scheme 1. Schematic illustration for the operating principle of the hybrid enzymatic biofuel cell (HEBFC) based on the bioanode and Ag2O/Ag cathode.
Figure 1. (A) XRD pattern and (B) SEM image of the as-prepared Ag2O/Ag cathode. (C) The power output of the HEBFC with Ag2O/Ag cathode in the N2-saturated atmosphere (a) and Ag2O/Ag cathode (b), Ag cathode (c) Pt cathode (d) in quiescent air saturated acetate buffer containing 10 mM NAD+, 30 mM glucose at 37 °C.
15
Figure 2. The ratio of the power output (P/P0) of the HEBFC changed with operation time. The HEBFC was discharged at +0.35 V with the regeneration process in 0.1 M buffer containing 10 mM NAD+ and 30 mM glucose under ambient air.
16
www.elsevier.com/locate/bios
PII: DOI: Reference:
S0956-5663(15)30318-3 http://dx.doi.org/10.1016/j.bios.2015.07.070 BIOS7888
To appear in: Biosensors and Bioelectronic Received date: 10 February 2015 Revised date: 11 July 2015 Accepted date: 30 July 2015 Cite this article as: You Yu, Miao Xu, Lu Bai, Lei Han and Shaojun Dong, Recoverable Hybrid enzymatic biofuel cell with molecular oxygen-independence, Biosensors and Bioelectronic, http://dx.doi.org/10.1016/j.bios.2015.07.070 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Recoverable Hybrid Enzymatic Biofuel Cell with Molecular Oxygen-independence You Yu a, b†, Miao Xu a, b†, Lu Bai a, Lei Han a, b, Shaojun Dong a, b,* a
State Key Laboratory of Electroanalytical Chemistry, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun, Jilin, 130022; b
University of Chinese Academy of Sciences, Beijing, 100049 Corresponding author email: [email protected] †These authors contributed equally to this work.
Abstract Enzymatic biofuel cells (EBFCs) have drawn great attentions because of its potential in energy conversion. However, designing of highly efficient EBFCs which can adapt to the anaerobic system is still a great challenge. In this study, we propose a novel hybrid enzymatic biofuel cell (HEBFC) which was fabricated by a glucose dehydrogenase modified bioanode and a solid-state silver oxide/silver (Ag2O/Ag) cathode. The as-assembled HEBFC exhibited an open circuit potential of 0.59 V and a maximum power output of 0.281 mW cm-2 at 0.34 V in air saturated buffer. Especially, due to the introduction of Ag2O/Ag, our HEBFC could also operate under anaerobic condition, while the maximum power output would reach to 0.275 mW cm-2 at 0.34 V. Furthermore, our HEBFC had stable cycle operation and could keep high power output for a certain time as the result of the regeneration of Ag2O. Our work provides a new concept to develop EBFCs for efficient energy conversion in the future. 1
Keywords: biofuel cell; molecular oxygen- independence; recoverable; Ag2O/Ag
1. Introduction Enzymatic biofuel cells (EBFCs), which employ enzymes as the biocatalyst to harvest energy from numerous kinds of fuels, have attracted substantial research efforts in recent years (Heller 2004). As one important type of the green energy devices, EBFCs usually show high efficiency and specificity compared with the noble metal catalysts-based fuel cells as the result of using the enzymes as bioelectrocatalysts (Cracknell et al. 2008). As more and more significant improvements concerned on the EBFCs are achieved, EBFCs have already been successfully applied in several ways like healthcare devices (Diamond et al. 2008), self-power sensors for poisonous ions (Wen et al. 2011), implanted devices in living creatures (Katz and MacVittie 2013) and several photoelectrochemical devices (Zhang et al. 2014). For examples, Wang and his co-workers had used epidermal EBFCs to harvest energy from human perspiration and detect the healthy conditions by the power output at the same time (Jia et al. 2013). Moreover, Katz et al. had used EBFCs to assemble electrochemical memristor devices for the biocomputing systems (MacVittie and Katz 2014). Up to now, except biocatalysts, which include bilirubin oxidase (Mano and Edembe 2013), laccase (Agnès et al. 2014) and horseradish peroxidase (Agnès et al. 2013), EBFC devices have also utilized various types of materials as cathodic 2
catalysts, for instance, noble metal like Pt black (Jia et al. 2013) or some hybrid materials such as functional TiO2 supported Pt nanoparticles (Wen et al. 2010), cobalt oxide nanoparticles on graphene nanosheets (Co3O4/GN) (Hsieh et al. 2012) and graphene assembled by Fe/Pt nanoparticles (GN/FePt) (Guo and Sun 2012). Most of the cathodic catalysts above are based on the oxygen reduction. The performances of these molecular oxygen-dependent EBFCs rely on the content of oxygen in the solution tremendously; even worse, they cannot adapt to the anaerobic systems such as Saccharomyces cerevisiae. In Saccharomyces cerevisiae, glucose would change into ethanol by the saccharomyces sereviciae (Aiba et al. 1968). In order to monitor this process, the EBFCs as self-powered sensors should work well in this anaerobic system, but most conventional EBFCs cannot work even for regular operating. Further for the implantable applications, EBFCs have already been implanted in vivo or fruits such as grape (Mano et al. 2003), rat (Castorena-Gonzalez et al. 2013; Cheng et al. 2013), insect (Rasmussen et al. 2012) and snail (Halamkova et al. 2012) successfully. However, the concentration of oxygen in the blood or juice always limits the performances of the implanted EBFCs, these devices even need some oxygen from outside. Furthermore, healthcare devices, which are important EBFCs` applications, would be used to monitor the lactate after anaerobic exercises to analyze the health of human. Therefore, molecular oxygen-independent EBFCs, which could operate for long under less dissolved oxygen content, are urgent for the implantable devices. To assemble a molecular oxygen-independent EBFC, a novel strategy is 3
proposed in this study. As known, metal oxide can accept electrons then transform to metal subsequently (Desilvestro 1990). Hence the metal oxide has potential to take the place of the oxygen as an electron acceptor at the cathode (Jiang et al. 2012). So we may combine a bioanode modified by enzymes with the metal oxide cathode to fabricate
a
hybrid
enzymatic
biofuel
cell
(HEBFC)
with
molecular
oxygen-independence. Moreover metal oxide/metal as a redox couple can be converted to each other through electrochemical processes (Therese and Kamath 2000), it means that the HEBFCs assembled with metal oxide cathode will be recoverable by the interconversion of the metal oxide/metal. Among the various kinds of metal oxide/metal couples, the silver oxide/silver (Ag2O/Ag) redox couple was chosen as the cathode for the HEBFC, because this redox couple possessed several advantages (Xie et al. 2013). Firstly, Ag2O and Ag could remain stable under neutral pH and ambient temperature for a long time (Priya et al. 2009), which was significant for EBFCs. Secondly, the Ag2O/Ag is favorable to gain electrons because of its higher reduction potential than that of other electron acceptors in anaerobic system (such as SO42- and CO2) (Han et al. 2014). Thirdly, the reduction potential of Ag2O/Ag (0.342 V vs SHE) was close to that of O2/OH- (0.401 V vs SHE) at pH = 7. Besides, Ag is cheaper than other noble metals like gold or platinum, it was important for further applications with broader prospect. Herein, a silver foil was directly electrochemically oxidized under a potentiostatical process to prepare Ag2O/Ag cathode. This Ag2O/Ag cathode has a high reduction potential, a short generating time and shows high cycle stability after 4
regeneration. In this context, we combine the Ag2O/Ag cathode with a bioanode modified by glucose dehydrogenase to fabricate a molecular oxygen-independent recoverable HBFC, which can operate well no matter oxygen existed or not. This HEBFC showed an excellent power output, contributed by the improved electronic transfer rate at the Ag2O/Ag cathode. Overall, this HEBFC may introduce a new type of EBFCs for energy recovery and further applications in the anaerobic system.
2. Material and methods 2.1 Chemicals Sodium hydroxide (NaOH) and nitric acid (HNO3) were purchased from Beijing Chemical Works, P. R. China. Multi-walled carbon nanotubes (MWNTs) (80% purity, diameter 20-50 nm) were purchased from Shenzhen Nanotech. Port. Co. Ltd. (Shenzhen, China). Silver foil (99.99%), β-D-(+)-glucose, chitosan (CS), Meldola`s blue (MDB), glucose dehydrogenase (GDH) (E.C. 1.1.1.47, initial activity of 235.3 U mg-1 from Pseudomonas sp.) and Nafion membrane were obtained from Sigma and used as received. NADH and NAD+ were purchased from the Gen-view Scientific Inc. A 0.10 M HAc-NaAc buffer (pH = 7.0) was employed as the supporting electrolyte. All other chemicals were of analytical grade and all aqueous solutions were prepared with ultrapure water (>18.25 MΩ cm) obtained from Millipore system. 2.2 Modification of bioanode Glassy carbon electrode (GCE, 2.6 mm in diameter, Tokai Carbon Co., Japan) was polished sequentially with 1.0 and 0.3 µm alumina slurry and washed 5
ultrasonically in water and ethanol for several minutes, respectively. MWNTs was treated by well-established methods with slight modification, MWNTs was dispersed in 30% HNO3 and then refluxed for 24 h at 140 °C to acquire carboxylic group functionalized MWNTs. 10 µL of MWNTs suspension (2 mg mL−1) was spread onto a GCE to dry, then the electrode was immersed into 0.5 mM MDB for 30 min to achieved MDB-MWNTs/GCE. 5 µL GDH (1 mg mL−1) was coated on the MDB-MWNTs/GCE, which was dried at 4 °C overnight and achieved GDH/MDB-MWNTs/GCE. After that, 10 μL of 1% CS solution was spread onto the electrode surface to form a film (noted as CS/GDH/MDB-MWNTs/GCE). 2.3 Electrochemical synthesis of silver oxide/silver (Ag2O/Ag) cathode The Ag2O/Ag cathode was prepared by electrochemical method according to our previous work (Han et al. 2014). Prior to electrochemical oxidation, the silver foil was polished and sonicated with acetone, ethanol, and distilled water sequentially. Then it was immersed into the aqueous solution containing 1 M NaOH. The depositions were performed potentiostatically at 0.2 V for 1 h at room temperature. After that, it was rinsed with distilled water and dried in the air. When the Ag2O/Ag cathode was discharged for a certain degree, the silver foil would be re-oxidized into the aqueous solution containing 1 M NaOH after getting out from the compartment of the hybrid biofuel cell. Then the regenerated Ag2O/Ag cathode was combined with the bioanode to fabricate a recoverable cell. 2.4 Apparatus The X-ray diffraction (XRD) measurements were performed on a D8 Focus 6
diffractometer (Bruker) with Cu Kα radiation (λ = 0.15405 nm) in the range of 10-80º (2θ). Scanning electron microscopy (SEM) measurements were made on a XL30 ESEM with an accelerating voltage of 10 kV to determine the morphology of products. Cyclic voltammetry (CV) and linear sweep voltammetry (LSV) were performed with an electrochemical analyzer (CHI 832, Shanghai, China). The polarization curves (U-I) were achieved by the LSV measurement at scan rate 1 mv s-1. A three-electrode system was used including a platinum flat as the counter electrode and the Ag/AgCl (saturated KCl) as reference electrode, respectively. The operation of the biofuel cell was performed at 37 °C, other experiments were carried out at room temperature (22 °C).
3. Discussion and results In this work, a novel HEBFC consisting of an enzymatic electrode and a Ag2O/Ag electrode was fabricated as shown in Scheme 1. When the HEBFC was discharged, GDH catalyzed the oxidation of glucose to D-glucono-δ-lactone, which generating electrons and transferring to the bioanode. Then, the electrons flowed through an external circuit to the cathode. Even under anaerobic condition, the solid state Ag2O/Ag electrode could efficiently accept the electrons by Ag2O reduced to Ag, which was confirmed by the obvious color change from black to gray white (Figure S1). The phenomenon indicated that the HEBFC was molecular oxygen-independent. The Ag2O/Ag cathode was prepared by potentiostatically electrochemical oxidation method (Figure S2). After HEBFC was discharged completely, the obtained Ag 7
electrode could be easily recovered to its original form by another electrochemical oxidation. After 1 hour re-oxidized process, the regenerated Ag2O/Ag electrode could be used in the HEBFC once again. Therefore, the fabricated HEBFC not only could work under anaerobic condition, but also had the property of regeneration just to show the recoverability. To acquire the molecular oxygen-independent recoverable HEBFC, a Ag2O/Ag electrode was used as the cathode for the HEBFC. Recently, Criddle`s group and Cui`s group prepared a Ag2O/Ag cathode for a microbial battery (Xie et al. 2013), in that experiment, silver nanoparticles were mixed with conductive carbon black then coated onto a carbon cloth, and finally oxidized at 1 mA cm-2. Despite the assembled battery exhibited excellent performance, the complex method to manufacture the cathode still need be improved. Herein, we fabricated the cathode by a more simple way. A silver foil was directly electrochemically oxidized under a potentiostatical process at 0.2 V for 1 h to get the Ag2O/Ag cathode (Figure S2). This Ag2O/Ag cathode has a higher reduction potential and a shorter generating time, while the pretreatment of the Ag foil was simple and time-saving. Moreover, because we used the Ag foil as the electrode substrate and synthetize Ag2O directly, the electroconductivity of the electrode should be better. Furthermore, the Ag2O/Ag electrode can easily regenerated by fast electrochemical oxidation after the assembled HEBFC has been totally discharged. The structure and morphology of the Ag2O/Ag cathode were examined by X-ray diffraction (XRD), scanning electron microscopy (SEM) and Energy Dispersive X-ray 8
Spectrometry (EDX), respectively. Figure 1A showed the XRD pattern of the as-prepared Ag2O/Ag electrode. Compared with Figure S3, we could observe that after electrochemical oxidation two typical diffraction peaks located at 32.9° and 55.0° appeared, which corresponded to the (111) plane and (220) of cubic phase Ag2O (JCPDS 41-1104), respectively. While other diffraction peaks could be indexed to the (111), (200), (220) and (311) planes of the Ag substrate (JCPDS 65-2871). No other diffraction peaks were detected in the as-prepared electrode except the peaks of Ag2O and Ag. In other words, the Ag electrode (Figure S3a) converted to Ag2O/Ag electrode (Figure 1A and Figure S3b) successfully by electrochemical oxididation. And after running in HEBFC, Ag2O was reduced to Ag (Figure S3c). Meanwhile, when the Ag2O on the electrode was consumed completely, the color changed from black to gray white (Figure S1). Figure 1B and Figure S4 exhibited the surface morphology of the electrode in different states characterized by SEM. Compared with the fresh Ag foil (Figure S4A), the polished one (Figure S4B) was much smoother. By electrochemical oxidization, the obtained Ag2O/Ag electrode (Figure 1B and Figure S4C) showed irregular particles with size of 500 nm. However, once Ag2O was completely reduced after running in HEBFC, the large particles disappeared and the surface became much rougher (Figure S4D). Moreover, once the reduced Ag2O/Ag electrode was regenerated by second electrochemical oxidation (Figure S4E), and then reduced completely again (Figure S4F) after running in HEBFC, its surface would show the same morphology with the first time. In addition, the corresponding EDX spectra displayed that the Ag2O/Ag electrode could reduce to Ag electrode when 9
it worked as cathode (Figure S5). On the basis of the results above, it could confirm that the Ag2O/Ag electrode was successfully prepared by the anodization of Ag foil. Moreover, Ag2O acted as an electron acceptor and reduced to Ag when the HEBFC was discharged. Then the following potentiostatical electrochemical oxidation process could get another fresh Ag2O/Ag electrode. So the Ag2O/Ag electrode could be applied under anaerobic condition and has regeneration ability in the meantime. The electrochemical activity of the as-prepared Ag2O/Ag cathode was evaluated by linear sweep voltammetry (LSV) in a three-electrode system (Figure S6A). We found that the reduction potential of Ag2O/Ag electrode was initiated at 0.39 V under ambient air atmosphere and its reduction current was increased along with potential decreasing approached to 4.8 mA cm-2 at 0.1 V. In contrast, the oxygen reduction current at Ag electrode and Pt electrode might be ignored in the same potential range, which obviously indicated the superior electrochemical activity of Ag2O/Ag electrode. Furthermore, in the case of a N2-saturated solution, the reduction current of Ag2O/Ag electrode only slightly decreased. These results suggested that Ag2O/Ag electrode could be used in the absence and presence of oxygen. By all accounts, the Ag2O/Ag electrode was a propitious candidate to be the cathode in the HEBFC system owning to its simple preparation method, regeneration ability and better electrochemical activity under both ambient air atmosphere and anaerobic condition. In addition, we investigated the electrochemical performance of the bioanode. GDH, which was capable of catalyzing glucose to D-glucono-δ-lactone selectively, was fabricated as the biocatalytic anode. To acquire the lower catalytic potential, a 10
redox mediator MDB was adsorbed on the MWNTs via π–π interaction on the bioanode. As shown in Figure S7, cyclic voltammetry (CV) showed the MDB– MWNTs exhibited two pairs of redox waves, which were ascribed to the redox processes of MDB. And the oxidation of NADH was obvious by potential lower than -0.2V. When the GDH was immobilized on the MDB-MWNTs modified GCE, named GDH/MDB-MWNTs/GCE. The bioanode exhibited electrocatalytic activity for the oxidation of glucose. A catalytic wave at the GDH/MDB-MWNTs/GCE was obvious in the presence of 30 mM glucose, and the current density reached 4.1 mA cm-2 at -0.01 V (Figure S6B). The electrochemical catalytic activity toward glucose oxidation suggested that this composite electrode could be used as bioanode for EBFCs. According to the above results, we could evidently find the onset reduction potential of Ag2O/Ag electrode (0.39 V) was much higher than that of the bioanode. Thus, combining Ag2O/Ag cathode and bioanode to assemble a HEBFC and produce electric power was feasible. In this HEBFC, a nafion membrane was needed to avoid the interaction between the cathode and NAD+. As depicted in Figure 1C, the open circuit potential (OCP) of the HEBFC was 0.59 V, and the maximum power output approached to 0.281 mW cm-2 at 0.34 V in the presence of 30 mM glucose and 10 mM NAD+ under ambient air. On the contrary to the conventional EBFCs, the maximum power output of this HEBFC reached 0.275 mW cm-2 at 0.34 V in a N2-saturated atmosphere, which was similar to that under ambient air. The two OCP values were equal no matter the oxygen existed or not. For comparison, the HEBFCs were assembled by the same bioanode and different cathodes, which were pure Ag 11
electrode and Pt electrode. And their maximum power output values were only 0.018 mW cm-2 and 0.041 mW cm-2, respectively. The performances of these two HEBFCs were both significantly lower than the HEBFC with Ag2O/Ag cathode. So the Ag foil, which was treated as the electrode, had little contributions to the power output of the HEBFC, especially in a N2-saturated atmosphere. It was further confirmed that the Ag2O/Ag cathode did improve the performance of the HEBFC. To test its recoverable character, the HEBFC was discharged continuously in stirring 0.10 M HAc-NaAc buffer containing 10 mM NAD+ and 30 mM glucose at a maximum power output under ambient air. As expected, the HEBFC was discharged stably with slight decline of the power output. However the power output of the HEBFC was sharply decreased to zero suddenly, where the Ag2O converted to Ag totally (Figure S8). In the experiment of cycle stability, when the power output fallen to near 65% of maximum power output, the Ag2O/Ag cathode was regenerated to recover the HEBFC. Although the HAc-NaAc buffer had the low buffering capacity, the operation of HEBFC proceeded smoothly for long. As shown in Figure 2, we could obviously observe that the power output might still return to 99% of maximum power output and operated for another ~6 hours with high power output after regeneration. The possible reasons for it might be the slight disturbance of the electrode interface caused by low current and less amount of generated gluconic acid in this HEBFC system. This recoverable property proved that this HEBFC had the possibility to be used in further applications. Additionally, considering the possible interaction between the HEBFC and some ions (e.g. phosphate or halide ions) in some 12
other systems, the investigations aiming at improving the stability of Ag2O and increasing the operated time are still underway.
4. Conclusion In summary, we have successfully fabricated a novel recoverable and molecular oxygen-independent HEBFC composed of the GDH immobilized on the MDB-MWNTs modified bioanode and solid state Ag2O/Ag cathode. Because of the outstanding electrochemical properties of the introduced Ag2O/Ag, which was prepared by simple electrochemical oxidation method, the constructed HEBFC could possess a high OCP of 0.59 V and maximum power output of 0.281 mW cm -2 at 0.34 V in air saturated buffer. The results were significantly superior to the other HEBFC, by using Ag or Pt as cathode. Meanwhile, under anaerobic condition the maximum power output of the HEBFC could still reach 0.275 mW cm-2 at 0.34 V on account of Ag2O acting as a good electrons acceptor. Furthermore, our HEBFC had recoverable cycle stability and might operate with high power output for a certain time. Undoubtedly, our work provided a new strategy for developing EBFC, which can operate under anaerobic environment in the future.
Acknowledgments This work was supported by the National Natural Science Foundation of China (No. 21375123), the 973 Project (No. 2011CB911002) and the Ministry of Science and Technology of China (No. 2013YQ170585).
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Figure Captions
Scheme 1. Schematic illustration for the operating principle of the hybrid enzymatic biofuel cell (HEBFC) based on the bioanode and Ag2O/Ag cathode.
Figure 1. (A) XRD pattern and (B) SEM image of the as-prepared Ag2O/Ag cathode. (C) The power output of the HEBFC with Ag2O/Ag cathode in the N2-saturated atmosphere (a) and Ag2O/Ag cathode (b), Ag cathode (c) Pt cathode (d) in quiescent air saturated acetate buffer containing 10 mM NAD+, 30 mM glucose at 37 °C.
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Figure 2. The ratio of the power output (P/P0) of the HEBFC changed with operation time. The HEBFC was discharged at +0.35 V with the regeneration process in 0.1 M buffer containing 10 mM NAD+ and 30 mM glucose under ambient air.
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