Author’s Accepted Manuscript Enzyme orientation for direct electron transfer in an enzymatic fuel cell with alcohol oxidase and laccase electrodes. Andrés A. Arrocha, Ulises Cano-Castillo, Sergio A. Aguila, Rafael Vazquez-Duhalt www.elsevier.com/locate/bios
PII: DOI: Reference:
S0956-5663(14)00418-7 http://dx.doi.org/10.1016/j.bios.2014.06.009 BIOS6839
To appear in: Biosensors and Bioelectronic Received date: 24 March 2014 Revised date: 24 May 2014 Accepted date: 3 June 2014 Cite this article as: Andrés A. Arrocha, Ulises Cano-Castillo, Sergio A. Aguila and Rafael Vazquez-Duhalt, Enzyme orientation for direct electron transfer in an enzymatic fuel cell with alcohol oxidase and laccase electrodes., Biosensors and Bioelectronic, http://dx.doi.org/10.1016/j.bios.2014.06.009 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.
Enzyme orientation for direct electron transfer in an enzymatic fuel cell with alcohol oxidase and laccase electrodes. Andrés A. Arrocha1, Ulises Cano-Castillo3, Sergio A. Aguila2 and Rafael Vazquez-Duhalt1,2* 1 2
Instituto de Biotecnologia UNAM. Cuernavaca, Mor. Mexico
Centro de Nanociencias y Nanotecnología UNAM. Ensenada, Baja California. 3
Instituto de Investigaciones Eléctricas, Cuernavaca, Mor. Mexico.
*Corresponding author:
Prof. Rafael Vazquez-Duhalt Department of Bionanotechnology Center for Nanosciences and Nanotechnology Km 107 carretera Tijuana-Ensenada Ensenada, Baja California, 22860 Mexico Email: [email protected]
1
Abstract A new full enzymatic fuel cell was built and characterized. Both enzymatic electrodes were molecularly oriented to enhance the direct electron transfer between the enzyme active site and the electrode surface. The anode consisted in immobilized alcohol oxidase on functionalized carbon nanotubes with 4-azidoaniline, which acts as active-site ligand to orientate the enzyme molecule. The cathode consisted of immobilized laccase on functionalized graphite electrode with 4-(2-aminoethyl) benzoic acid. The enzymatic fuel cell reaches 0.5 V at open circuit voltage with both, ethanol and methanol, while in short circuit the highest current intensity of 250 A cm-2 was obtained with methanol. Concerning the power density, the methanol was the best substrate reaching 60 W cm-2, while with ethanol 40 W cm-2 were obtained.
Highlights
A new full enzymatic fuel cell was built and characterized. Alcohol oxidase served as anodic biocatalyst, while laccase as cathodic ones. Enzymes were molecularly oriented on the electrode to enhance electron transfer. The molecules were oriented by substrate-like electrode functionalization.
Keywords: Alcohol oxidase, Enzymatic electrodes, Fuel cell, Laccase, Molecular orientation.
2
1. Introduction The generation of electricity from renewable and environmentally friendly techniques, and the development of devices capable of meeting the needs of wireless sensors and remote monitoring, have motivated the development of different technologies to use the energy stored in organic compounds such as glucose, fructose, ethanol, methanol and glycerol (Sundmacher, 2010). In recent decades, enzymatic fuel cells have generated increasing interest because they are devices that have different potential applications such as communications, bioelectronics and medical purposes (Willner et al., 2007; Ivanov et al., 2010; Osman et al., 2011; Xia et al., 2013; Lang et al., 2014). Instead of metallic catalysts, enzymatic fuel cells use enzymes as biocatalysts to carry out the oxidation of the substrate at the anode, releasing electrons that travel through an electrical circuit to reach the cathode. The protons produced by the oxidation of the substrates are combined with an oxidant (commonly oxygen) to form a product (commonly water)(Meredith and Minteer, 2012). The ultimate goal of the design of enzymatic fuel cells is to reach efficiencies that are only limited by thermodynamics and not by engineering issues. The electron transfer between the redox active site of the enzyme and the electrode surface is a key issue that should be considered in the design of enzymatic fuel cells. Enzyme orientation and electron connectivity are of immense importance in direct electron transfer. The enzyme molecular orientation on the electrode surface, and the use of computational techniques to carry out this orientation, have recently been reviewed (Vazquez-Duhalt et al., 2014). Several reports on different enzyme orientation techniques, allowing a better electron transfer reflected in higher current and power densities, are available in the literature (Martinez-Ortiz et al., 2011, Demin and Hall, 2009).
3
However, it is well know that the vast majority of the oxido-reductase enzymes cannot achieve a direct electron transfer when they are randomly immobilized on the electrode surface (Ramanavicius and Ramanaviciene, 2009). Several strategies have been proposed to induce the electron transfer between enzyme molecules and electrodes: (i) The enzyme entrapment in a polymeric conductor matrix, (ii) contacting the enzyme redox center through nanostructures such as carbon nanotubes and metal nanoparticles, and (iii) the enzyme reconstitution on the redox cofactor, which is previously covalently immobilized on the electrode surface (Willner et al., 2009). The first two of these strategies are the most widely used but they have the disadvantage of a low enzyme orientation. Nevertheless, the low direct electron transfer of the randomly orientated enzyme molecules could be compensated by a high enzyme loading on the electrode surface (Deng et al., 2010; Reuillard et al., 2013). On the other hand, the electron transfer between the redox site of the enzyme and the electrode surface can be enhanced by the adequate orientation of the enzyme molecule. This orientation could be obtained by functionalizing the electrode surface with a substrate-like compound that interacts with the redox site of the enzyme as ligand (Blanford et al., 2007; VazDominguez et al., 2008; Martinez-Ortiz et al., 2011). This approach could be combined with the use of nanotubes, such as MWCNT, to improve the ligand-enzyme interaction (Meredith et al., 2011) . For alcohol bio-batteries (with ethanol or methanol as fuel), alcohol dehydrogenase (ADH) is the most recurrent enzyme in the literature. In most of cases, the NADH-dependent ADH is used, and there are few reports available in the literature using an ADH containing PQQ and heme prosthetic groups (Yakushi and Matsushita, 2010). The advantage of the ADH-PQQ is the presence of an exposed heme that allows direct electron transfer, as demonstrated in an
4
hybrid fuel cell with a platinum cathode (Aquino Neto et al., 2013). Enzymatic biofuel cell in which both, anode and cathode, electrodes were powered by ethanol has been described (Ramanavicius et al. 2008). The anode of this enzymatic fuel cell was based on immobilized quino-hemoprotein-alcohol dehydrogenase, while the cathode on co-immobilized alcohol oxidase (AOx) and microperoxidase. More recently, an alcohol oxidase (AOx) based biosensor, in which the enzyme was immobilized on a gold-MWCNT-Nafion-polientilenimine electrode, showed a direct electron transfer from the FAD to the matrix of the electrode (Das and Goswami, 2013). In the present study we report the design of an enzymatic fuel cell with molecularly orientated alcohol oxidase and laccase. The electrodes were prepared by the “substrate-like linkers” approach using 4-azidoaniline for the alcohol oxidase anode and benzoic acid for the laccase cathode, which emulates the natural ligands of both enzymes. This is, in our knowledge, the first enzymatic fuel cell built with an alcohol oxidase anode and a laccase cathode.
2. Materials and methods 2.1 Chemicals and reagents 4-Azidoaniline hydrochloride, N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDC), 2-(N-morpholine) ethanosulfonic acid (MES), 4-[2-aminoethyl] benzoic acid hydrochloride (AEBA), and the 2,2-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid) (ABTS) were purchased from Sigma-Aldrich Chemical Corporation (St. Louis, MI, USA). Sodium hydroxide, potassium phosphate monobasic, sodium phosphate dibasic, nitric acid, isopropanol, methanol, and acetone were obtained from J.T. Baker (Phillipsburg, NJ, USA), 5
Toluene was purchased from Fisher Scientific (Fair Lawn, NJ, USA) and tetrahydrofuran from Burdick & Jackson (Muskegon, MI, USA).
2.2 Enzymes Alcohol oxidase from Pichia pastoris was purchased from Sigma-Aldrich (21 U/mg and 38 µg/µl). Laccase from Coriolopsis gallica UAMH 8260 was obtained and purified as previously described (Tinoco et al., 2001). The purified preparation showed a specific activity of 313 U/mg and a purity of 81% estimated by electrophoresis. 2.3 Electrode and fuel cell materials Nafion solution (5%) was purchased from Fuel Cell Scientific, LLC (Stoneham, MA, USA). SIGRACET graphite laminated sheets (GDL 35 BC), used as electrode, were obtained from SGL Carbon Group (Wiesbaden, Germany). Carboxylated Multi-Walled Carbon Nanotubes (C-MWCNT) from US Research Nanomaterials Inc. (Outside diameter of 5-15 nm, inside diameter 3-5 nm, length 50 µm) (Houston, TX, USA), Nafion 232 membranes (50µm) were purchased from DuPont. 2.4 Determination of enzymatic activity and linker affinity Alcohol oxidase (AOx) activity was measured spectrophotometrically in a 1 ml reaction mixture containing 100 mM potassium phosphate buffer (pH 7.5), 0.1% (v/v) methanol, 2 mM ABTS, and 2.5 units of horseradish peroxidase. The amount of H2O2 produced is estimated monitoring the ABTS-diradical production at 405 nm (
405
= 36,800 M-1 cm-1). The protein
6
content was determined by the Bradford method with the BioRad protein reagent and measured at 595 nm. In order to prove that the linker (4-azidoaniline) interacts as ligand in the AOx active site, the enzyme (5 µM in monomer basis) was incubated for 12 h with different concentration of 4azidoanile (from 0 to 47 mM), and the changes in the absorbance spectrum were recorded. The spectra were obtained by scanning the absorbance from 300 to 600 nm in a Perkin Elmer Lambda 25 UV/VIS spectrophotometer. 2.5 Electrochemical experiments Cyclic Voltammetry experiments were performed with a Solartron-1287 potensiostat using a three electrodes configuration: a saturated calomel electrode (SCE) as a reference electrode in close proximity to the working electrode volume using a Luggin capillary; a platinum mesh as a counter electrode; and the different preparations of AOx electrodes as working electrodes, as well as the respective controls. All voltage potentials from the cyclic voltammetry experiments are referred to the SCE (KCl sat.) electrode. All the literature reported data was converted to the SCE electrode scale. All the anode cyclic voltammetry experiments where done under nitrogen purge. 2.6 Preparation of oriented enzymatic electrodes The enzymes were immobilized in an oriented fashion on graphite sheets. Before AOx immobilization, the graphite sheets electrodes (1 cm2) were successively cleaned with dichloromethane, tetrahydrofuran and water, and dried. Then, the clean graphite electrodes were carboxylated under nitric acid reflux for 2 h at 80°C.
7
On the other hand, C-MWCNT were functionalized with 4-azidoaniline creating an amide bond between the amino group of the 4-azidoaniline and the carboxylic group of CMWCNT. The functionalization was performed dissolving 5 mg of C-MWCNT, 5 mg of 4azidoaniline and 5 mg of EDC in dimethylsulfoxide, and the reaction was kept during 4 h. The functionalized nanotubes (ANA-MWCNT) were washed three times with 13 ml of phosphate buffer and centrifuged at 9000 rpm during 10 min. Then, the enzyme was immobilized on the ANA-MWCNT as follows: An aliquot of 15 µl of AOx was homogenized with 60 µl of ANAMWCNT, previously sonicated, and incubated during 12 h to ensure the formation of the AOxANA complex. Once the incubation was completed, 15 µl of Nafion (5% w/w) were added and homogenized. Twenty-two µl of the suspension were applied on the graphite electrode and dried under darkness. The oriented enzymatic electrodes (AOx-ANA-MWCNT) were then washed and stored in phosphate buffer 100 mM at pH 7.5. The same procedure was performed for control electrodes without the addition of 4-azidoanile to the C-MWCNT. The oriented laccase electrodes were fabricated also by the “substrate-like linker” approach with 4-(2-aminoethyl) benzoic acid (AEBA) and according to the protocol previously published (Martinez-Ortiz et al., 2011). 2.7 Fuel cell experiments Semi-enzymatic fuel cells were assayed in order to evaluate the performance of AOx anode without the limitations of laccase cathode. An electrochemical cell was built in acrylic hardware with a graphite sheet (3 cm x 2 cm) containing 20% platinum as cathode, and anodes with different preparations of immobilized AOx (1 cm2).
8
The full enzymatic fuel cell included the two oriented enzymatic electrodes, AOx-ANAMWCNT anode and laccase cathode. The anodic chamber contained 100 mM phosphate buffer (pH 7.5) and the cathodic chamber was filled with 50 mM succinate buffer at pH 4.5. The chambers were separated by a 50 µm Nafion 232 membrane. Anodic chamber was aerated with nitrogen and the cathodic chamber with oxygen. The cell performance was measured using a potensiostat (Solartron-1287) and the polarimetric curves at 10 mVs-1 where obtained. The cell was stabilized before measures at open circuit potential (OCP) during 2 h before measurements.
3. Results and discussions.
3.1 Linker for enzyme orientation The “substrate-like linker” approach was selected for the molecular orientation of the alcohol oxidase (AOx) on the electrode surface. Sodium azide has been reported as able to bind the FAD cofactor pocket of AOx (Bringer et al., 1979). Thus we have selected an aromatic azide derivative as linker in order to orientate the enzyme molecule and to assure the electron transfer. The 4-azidoaniline was selected because it contains an azide group that can interact with the enzyme and an amino group in para position to be covalently attached to the carboxylated surface of graphite electrode. The interaction of 4-azidoaniline as an active-site ligand with the AOx molecule was spectrophotometrically analyzed. The AOx absorbance spectra with different amounts of ligand revealed the formation of a complex that modifies the absorbance of the FAD binding pocket of AOx (Fig. 1A). A pronounced increase in the AOx absorbance between 340 and 600 nm, with a maximal increase at 450 nm, was observed after subtraction of background 9
spectrum of 4-azidoaniline alone. This absorbance increase at 450 nm is proportional to the amount of 4-azidoaniline, fitting a hyperbolic curve (Fig. 1B). This change of absorbance spectrum in the presence of 4-azidoaniline strongly suggests the compound interaction as ligand in the FAD protein pocket. The incubation for 12 h was enough to reach the complex equilibrium. Using a concentration range from 0 to 47 mM of 4-azidoaniline, the calculated dissociation constant (Kd) was 17.3 (±4.8) mM, which implies affinity for this ligand. Thus, AOx-azidoaniline complex could be used for the orientation of AOx molecules on the graphite electrode. The azide molecule binds strongly to the isoalloxazine moiety of the flavin as demonstrated by crystallography in the case of NADH oxidase from Streptococcus pyogenes (PDB 2BCP). The isoalloxazine ring of the flavin, which is involved in catalysis, offers several possibilities for interaction with various compounds. Its amphipathic property due to the hydrophobicity of the xylene moiety and the hydrophilic pyrimidine ring that is relatively electron-deficient and able to form hydrogen bridges (Ghisla and Massey, 1986). Most of the flavoproteins so far studied show a channel lying over the face of the flavin, which permits access of the substrate. In addition, crystallographic structures show that the flavin 8-position are exposed to solvent. On the other hand, 8-azido-FAD, has been used for reconstituting both apo proteins; amino acid oxidase and glucose oxidase (Fitzpatrick et al., 1985). The binding is specific and very tight, so that only a stoichiometric amount of reagent is necessary. Nevertheless, still studies should be conducted to elucidate the interaction between FAD and 4azidoaniline.
10
3.2 Alcohol oxidase anode The FAD deep localization inside the protein moiety could difficult the ligand interaction. In order to improve the ligand interaction with the FAD pocket, carboxylated multiwalled carbon nanotubes (C-MWCNT) were first functionalized with 4-azidoaniline (ANA) and then immobilized on the graphite electrode. These electrodes containing functionalized nanotubes were then treated with AOx to form the enzymatic anode (AOx-ANA-MWCNT). As controls, two other electrodes were prepared: One with the AOx directly and randomly immobilized on the graphite surface by enzyme adsorption on the Nafion, and another with AOx immobilized on the electrode containing MWCNT but without ANA. These two latter electrodes could be considered as having the protein randomly oriented. The AOx-ANA-MWCNT electrode preserves the more positive peak but translocated to 150 mV, which is 350 mV (Fig. 2), more negative that the 175 mV reported by Das and Goswami (2013). For fuel cell applications, a more negative anode potential favors a larger potential difference. In addition, the functionalized nanotubes increase the current generation probably due to a better molecular orientation of the enzyme. On the other hand, the electrode prepared with AOx directly adsorbed on Nafion (AOx(a) and AOx(b) in Fig. 2) showed a low increase of the voltammetric response at -500 mV in the presence of methanol, while the other randomly oriented AOx electrode containing carbon nanotubes (AOx-MWCNT) showed the poorest response to the methanol. Thus, the fact that the AOx-ANA-MWCNT electrode generated the most important response to the presence of methanol in the medium with a 3 mA increase with respect to the buffer (Fig 2), evidences the importance of the orientation of enzyme molecule for the electronic transfer between the enzyme active site and the electrode surface.
11
The AOx-ANA-MWCNT heterogeneous electron transfer rate constant was calculated, from triplicate electrodes, employing the equation; Ks=Amax/Q. The obtained Ks value was 0.98 (±0.15), which implies a good contact between the 4-azidoaniline functionalized MWCNT and the AOx molecule. On the other hand, the concentration of electroactive species on the electrode was estimated using the equation Г = Q/nFA, where Q is the electrode charge in coulombs obtained from the integrated area of the cyclic voltammetry experiments, n the number of electrons (n = 1), F is the Faraday constant, and 0.368 cm2 the exposed area of the electrode. The obtained value of Г = 1.5 nM cm-2 is consistent with the 1.9 nM deposited onto the electrode. The response to increasing concentrations of methanol and ethanol of the oriented enzymatic electrode AOx-ANA-MWCNT was determined (Fig. 3). The AOX-ANA-MWCNT electrode showed high current densities and a well-defined peak at -63 mV which shifted 90 mV from the peak registered without substrate (Fig. 2). The AOx oxidation of methanol could be thermodynamically favored at this higher potential. The potential increase for alcohol oxidation in the presence of MWCNT has been reported for other oxidases such as aryl alcohol oxidase on reticulated vitreous carbon electrodes with poly-neutral red as mediator (Barsan and Brett, 2008; Munteanu et al., 2008). From the saturation curves of AOx-ANA-MWCNT (Fig. 3), the methanol plot showed an estimated Kd of 4.9 (±1.4) mM and a maximal current density (Amax) of 667 (±32) µA cm-2. As expected, the estimated Kd for ethanol is 6.6 (±1.8) mM (Fig 3 A) that is consistent with the substrate affinity of AOx for ethanol, which is reported to be 20% lower when compared to those for methanol (Barsan and Brett, 2008) . Nevertheless, the current generated by ethanol of 897 (±60.9) µA cm-2 is higher than those obtained using methanol. This could be originated by the methanol transformation to form formaldehyde, which beside to be AOx substrate it is a strong AOx inactivator (Hopkins and Muller, 1987). However, the
12
sensitivity of both electrodes is the same, 136 µA cm-2 mM-1, indicating no differences between ethanol and methanol in terms of recognition and response. In order to examine the performance of the different AOx electrode preparations, experiments were performed with a hybrid fuel cell with a platinum cathode (Fig 4). The cell was a low potential one with 0.2 Volts, but notably the AOx-ANA-MWCNT overcome the randomly orientated preparations with 60 W cm-2 and 700 A cm-2 at short-circuit. It is important to point out that the AOx electrode could accomplished oxidation of methanol on the cell 35 W cm-2 and 500 A cm-2 at short-circuit. These experiments clearly show that the electrode with orientated enzyme molecules is better for the current and power generation. 3.3 Enzymatic fuel cell alcohol oxidase - laccase Finally, a full enzymatic fuel cell was constructed and tested (Fig. 5). Both, enzymatic electrodes, cathode an anode, were molecularly oriented to enhance the direct electron transfer between the active-site of the enzyme and the electrode surface. The enzymatic fuel cell consisted in an AOx-ANA-MWCNT anode and an oriented laccase-AEBA cathode, previously characterized (Martinez-Ortiz et al., 2011). The enzymatic fuel cell reaches 0.5 V in open circuit with both, ethanol and methanol. However, in short circuit a difference of 50 A cm-2 is found, with a highest current intensity of 250 A cm-2 obtained with methanol (Fig. 6). The values obtained in this work are in agreement with those obtained from enzymes with an embedded cofactor such as alcohol dehydrogenase and aldehyde dehydrogenase dependent of PQQ with and without MWCNT (Aquino et al., 2013). Concerning the power density, the methanol was the best substrate reaching 60 W cm-2, while with ethanol only 40 W cm-2were obtained (Fig. 6). These values are similar to those 13
obtained from a hybrid fuel cell with platinum cathode (Fig. 4). This power density is significantly higher than these obtained from a non-oriented enzymatic fuel cell with alcohol dehydrogenase - alcohol oxidase:microperoxidase (1.5 W cm-1) (Ramanavicius et al. 2008). Maximal current densities of 250 µA cm-2 for methanol and 180 µA cm-2 ethanol were obtained. Higher power densities have been also obtained with methanol, when compared with ethanol, in fuel cells with alcohol dehydrogenase electrodes (Akers et al 2005). For both substrates, a cell efficiency of 40% was estimated using the equation
cell =
[Pmax/(OCV SCC)], where OCV is the
open circuit voltage, SC is the short-circuit current, and Pmax is the maximal power. This efficiency value is superior than reported by (Katz et al., 1999). Using alcohols as fuels, the enzymatic fuel cells with alcohol dehydrogenase (ADH) reached power densities between 32-1000 W cm-2 (Aquino Neto et al., 2013; Kim et al., 2013). The performance of our oriented enzymatic fuel cell (AOx-ANA-MWCNT and laccase-AEBA) is still far from those obtained with alcohol dehydrogenase dependent of NAD+ , in which a power density of around 1000 W cm-2 has been obtained (Akers et al., 2005). However, these results should be taken cautiously because the constant addition of the cofactor NAD+ to the anodic chamber could keep high current generation. It is important to point out that the constant use of cofactor on a bio-battery is an operational disadvantage. In our enzymatic fuel cell, the AOx posses a prosthetic group (FAD) deeply embedded on the protein and there is no need for cofactor addition.
14
4. Conclusions Our results show that the enzyme molecular orientation on the electrode surface improves the direct electron transfer. Alcohol oxidase and laccase can be immobilized in an oriented fashion on graphite electrodes by means of a substrate-like linker, showing better performance than the randomly immobilized preparations. The full enzymatic fuel cell, built with an AOxANA-MWCNT anode and laccase-AEBA cathode, reaches 0.5 V in open circuit with both, ethanol and methanol, while in short circuit the highest current intensity of 250 A cm-2 was obtained with methanol. Concerning the power density, the methanol was the best substrate reaching 60 W cm-2, while with ethanol 40 W cm-2 were obtained. The results clearly show that the electrodes with orientated enzyme molecules are better for the current and power generation when compared a non-oriented systems. Finally, in our knowledge, this is the first enzymatic fuel cell built with an alcohol oxidase anode and a laccase cathode.
References Akers N.L., Moore C.M., Minteer S.D., 2005. Electrochim. Acta 50, 2521-2525. Aquino Neto S., Suda E.L., Xu S., Meredith M.T., De Andrade A.R., Minteer S.D., 2013, Electrochim. Acta 87, 323-329. Barsan M.M., Brett C.M.A., 2008. Talanta 74, 1505-1510. Blanford C.F., Heath R.S., Armstrong F.A., 2007. Chem. Commun. 1710-1712. Bringer S., Sprey B., Sahm H., 1979. Eur. J. Biochem. 101, 563-570. Das M., Goswami P., 2013. Bioelectrochemistry 89, 19-25.
15
Demin S., and Hall E.A.H., 2009. Bioelectrochemistry 76, 19-27. Deng L., Shang L., Wen D., Zhai J., Dong S., 2010. Biosens. Bioelectron. 26, 70-73. Fitzpatrick P.F., Ghisla S., Massey V., 1985. J. Biol. Chem. 260, 8483-8491. Ghisla S., Massey V., 1986. Biochem. J. 239,1-12. Hopkins T.R., Muller F., 1987. Biochemistry of Alcohol Oxidase, in: H. Verseveld and J. A. Duine (Eds.), Microbial Growth on C1 Compounds, Springer Netherlands. pp. 150-157. Ivanov I., Vidaković-Koch T., Sundmacher K., 2010. Energies 3, 803-846. Katz E., Filanovsky B., Willner I., 1999. New J. Chem. 23, 481-487. Kim Y.H., Campbell E., Yu J., Minteer S.D., Banta S., 2013. Angew. Chem. Int. Ed. 52, 14371440. Lang Q.,Yin L., Shi J., Li L., Xia L., Liu A., 2014. Biosens. Bioelectron. 51, 158-163. Martinez-Ortiz J., Flores R., Vazquez-Duhalt R., 2011. Biosens. Bioelectron. 26, 2626-2631. Meredith M.T., Minteer S.D., 2012. Ann. Rev. Anal. Chem. 5, 157-179. Meredith M.T., Minson M., Hickey D., Artyushkova K., Glatzhofer D.T., Minteer S.D., 2011. ACS Catalysis 1, 1683-1690. Munteanu F.-D., Ferreira P., Ruiz-Dueñas F.J., Martínez A.T., Cavaco-Paulo A., 2008. J. Electroanal. Chem. 618, 83-86. Osman M.H., Shah A.A., Walsh F.C., 2011. Biosens. Bioelectron. 26, 3087-3102. Ramanavicius A., Ramanaviciene A., 2009. Fuel Cells 9, 25-36. Ramanavicius A., Kausaite A., Ramanaviciene A., 2008. Biosens. Bioelectron. 24, 761-766. Reuillard B., Le Goff A., Agnes C., Holzinger M., Zebda A., Gondran C., Elouarzaki K., Cosnier S., 2013. Phys. Chem. Chem. Phys. 15, 4892-4896.
16
Sundmacher K., 2010. Ind. Eng. Chem. Res. 49, 10159-10182. Tinoco R., Pickard M.A., Vazquez-Duhalt R., 2001. Lett. Appl. Microbiol. 32, 331-335. Vaz-Dominguez C., Campuzano S., Rüdiger O., Pita M., Gorbacheva M., Shleev S., Fernandez V.M., De Lacey A.L., 2008. Biosens. Bioelectron. 24, 531-537. Vazquez-Duhalt R., Aguila S.A., Arrocha A.A., Ayala M., 2013. ChemElectroChem, 1, 496-513 Willner I., Willner B., Katz E., 2007. Bioelectrochemistry 70, 2-11. Willner I., Yan Y.M., Willner B., Tel-Vered R., 2009. Fuel Cells 9, 7-24. Xia, L. Liang B., Liang Li L., Tang X., Palchetti I., Mascini M., Liua A., 2013. Biosens. Bioelectron. 44, 160-163. Yakushi T., Matsushita K., 2010. Appl. Microbiol. Biotechnol. 86, 1257-1265.
17
Figure legends
Figure 1. Change of the alcohol oxidase spectrum in the presence of 4-azidoaniline. (A) AOx incubated with different concentrations of 4-azidoaniline. (B) Absorbance at 450 nm vs 4azidoaniline concentration plot. The determined value of Kd is 17.3 (±4.8) mM. Figure 2. Cyclic voltagramms at 50 mV s-1 of different preparations of alcohol oxidase immobilized on graphite electrodes. (a) In the absence of methanol and (b) in the presence of 212 mM methanol. AOx, electrodes prepared by alcohol oxidase absorption on Nafion. AOxMWCNT, electrodes prepared by carbon nanotubes immobilized on the graphite surface and then alcohol oxidase randomly immobilized. AOx-ANA-MWCNT, molecularly oriented electrodes prepared with functionalized carbon nanotubes with 4-azidoaniline covalently immobilized on the graphite surface and alcohol oxidase immobilized by azo affinity. Figure 3. Representative saturation plots using AOx-ANA-MWCNT electrode obtained from cyclic voltammetry experiments at 50 mV s-1 with successive additions of alcohol. (A) Ethanol response at - 93 mV, and (B) methanol response at -63 mV. Figure 4. Potentiodynamic (A) and power curves (B) of three different electrode preparations in a hybrid fuel cell. The anodic chamber (AOx) was fueled with 200 mM methanol on nitrogen purged 100 mM phosphates buffer (pH 7.5). The cathodic chamber (Pt) was bubbled with oxygen and contained 100 mM phosphate buffer (pH 7.5). Figure 5. Scheme of the enzymatic fuel cell with molecularly oriented alcohol oxidase and laccase electrodes. Figure 6. Potentiodynamic and power curves for AOx-ANA-MWCNT vs lacasse-AEBA. The speed scan was performed at 10 mV s-1. The anodic chamber (AOx) was fueled with 200 mM ethanol (solid lines) or methanol (dotted lines) on nitrogen purged 100 mM phosphates buffer (pH 7.5). The cathodic chamber (laccase) was bubbled with oxygen and contained 50 mM succinate buffer (pH 4.5) was used for lacasse cathode.
18
0.6
A
0.2
0.1
0.0 340
B Absorbance at 450 nm
Absorbance units
0.3
0.5
0.4
0.3
0.2 0.0
400
460
520
Wavelength (nm)
580
0
10
20
30
40
50
4-Azidoaniline (mM)
Figure 1.
19
30
mA cm-2
20 10 0 -10
AOx-ANA-MWCNT(a) AOx-ANA-MWCNT(b) AOx-MWCNT(a) AOx-MWCNT(b) AOx(a) AOx(b)
-20 -30 -0.8
-0.6
-0.4
-0.2
0
0.2
0.4
0.6
0.8
Volts
Figure 2.
20
Current density (μAmpers/cm2)
1000
A
800 600 400 200 0
Current density (μAmpers/cm2)
0 800
10
20
30
40
Ethanol (mM)
50
60
70
B
600
400
200
0 0
10
20
30
40
50
60
Methanol (mM)
Figure 3.
21
0.25
70
AOx-ANA-MWCNT AOx-Nf AOx-MWCNT
A 0.2
AOx-ANA-MWCNT AOx-Nf AOx-MWCNT
60
B
0.15
µWatts
Volts
50
0.1
40 30 20
0.05
10 0
0 0
200
400
µAmpers
600
800
0
200
400
600
800
µAmpers
Figure 4.
22
eanode
cathode
H+
O2
Alcohol H+ Aldehyde
H2O
H+
Laccase Alcohol oxidase
Figure 5.
23
0.6
70
0.5
60
Volts
40 0.3 30 0.2
µWatts
50
0.4
20
0.1
10
0.0
0 0
50
100
150
200
250
µAmpers
Figure 6.
24
PII: DOI: Reference:
S0956-5663(14)00418-7 http://dx.doi.org/10.1016/j.bios.2014.06.009 BIOS6839
To appear in: Biosensors and Bioelectronic Received date: 24 March 2014 Revised date: 24 May 2014 Accepted date: 3 June 2014 Cite this article as: Andrés A. Arrocha, Ulises Cano-Castillo, Sergio A. Aguila and Rafael Vazquez-Duhalt, Enzyme orientation for direct electron transfer in an enzymatic fuel cell with alcohol oxidase and laccase electrodes., Biosensors and Bioelectronic, http://dx.doi.org/10.1016/j.bios.2014.06.009 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.
Enzyme orientation for direct electron transfer in an enzymatic fuel cell with alcohol oxidase and laccase electrodes. Andrés A. Arrocha1, Ulises Cano-Castillo3, Sergio A. Aguila2 and Rafael Vazquez-Duhalt1,2* 1 2
Instituto de Biotecnologia UNAM. Cuernavaca, Mor. Mexico
Centro de Nanociencias y Nanotecnología UNAM. Ensenada, Baja California. 3
Instituto de Investigaciones Eléctricas, Cuernavaca, Mor. Mexico.
*Corresponding author:
Prof. Rafael Vazquez-Duhalt Department of Bionanotechnology Center for Nanosciences and Nanotechnology Km 107 carretera Tijuana-Ensenada Ensenada, Baja California, 22860 Mexico Email: [email protected]
1
Abstract A new full enzymatic fuel cell was built and characterized. Both enzymatic electrodes were molecularly oriented to enhance the direct electron transfer between the enzyme active site and the electrode surface. The anode consisted in immobilized alcohol oxidase on functionalized carbon nanotubes with 4-azidoaniline, which acts as active-site ligand to orientate the enzyme molecule. The cathode consisted of immobilized laccase on functionalized graphite electrode with 4-(2-aminoethyl) benzoic acid. The enzymatic fuel cell reaches 0.5 V at open circuit voltage with both, ethanol and methanol, while in short circuit the highest current intensity of 250 A cm-2 was obtained with methanol. Concerning the power density, the methanol was the best substrate reaching 60 W cm-2, while with ethanol 40 W cm-2 were obtained.
Highlights
A new full enzymatic fuel cell was built and characterized. Alcohol oxidase served as anodic biocatalyst, while laccase as cathodic ones. Enzymes were molecularly oriented on the electrode to enhance electron transfer. The molecules were oriented by substrate-like electrode functionalization.
Keywords: Alcohol oxidase, Enzymatic electrodes, Fuel cell, Laccase, Molecular orientation.
2
1. Introduction The generation of electricity from renewable and environmentally friendly techniques, and the development of devices capable of meeting the needs of wireless sensors and remote monitoring, have motivated the development of different technologies to use the energy stored in organic compounds such as glucose, fructose, ethanol, methanol and glycerol (Sundmacher, 2010). In recent decades, enzymatic fuel cells have generated increasing interest because they are devices that have different potential applications such as communications, bioelectronics and medical purposes (Willner et al., 2007; Ivanov et al., 2010; Osman et al., 2011; Xia et al., 2013; Lang et al., 2014). Instead of metallic catalysts, enzymatic fuel cells use enzymes as biocatalysts to carry out the oxidation of the substrate at the anode, releasing electrons that travel through an electrical circuit to reach the cathode. The protons produced by the oxidation of the substrates are combined with an oxidant (commonly oxygen) to form a product (commonly water)(Meredith and Minteer, 2012). The ultimate goal of the design of enzymatic fuel cells is to reach efficiencies that are only limited by thermodynamics and not by engineering issues. The electron transfer between the redox active site of the enzyme and the electrode surface is a key issue that should be considered in the design of enzymatic fuel cells. Enzyme orientation and electron connectivity are of immense importance in direct electron transfer. The enzyme molecular orientation on the electrode surface, and the use of computational techniques to carry out this orientation, have recently been reviewed (Vazquez-Duhalt et al., 2014). Several reports on different enzyme orientation techniques, allowing a better electron transfer reflected in higher current and power densities, are available in the literature (Martinez-Ortiz et al., 2011, Demin and Hall, 2009).
3
However, it is well know that the vast majority of the oxido-reductase enzymes cannot achieve a direct electron transfer when they are randomly immobilized on the electrode surface (Ramanavicius and Ramanaviciene, 2009). Several strategies have been proposed to induce the electron transfer between enzyme molecules and electrodes: (i) The enzyme entrapment in a polymeric conductor matrix, (ii) contacting the enzyme redox center through nanostructures such as carbon nanotubes and metal nanoparticles, and (iii) the enzyme reconstitution on the redox cofactor, which is previously covalently immobilized on the electrode surface (Willner et al., 2009). The first two of these strategies are the most widely used but they have the disadvantage of a low enzyme orientation. Nevertheless, the low direct electron transfer of the randomly orientated enzyme molecules could be compensated by a high enzyme loading on the electrode surface (Deng et al., 2010; Reuillard et al., 2013). On the other hand, the electron transfer between the redox site of the enzyme and the electrode surface can be enhanced by the adequate orientation of the enzyme molecule. This orientation could be obtained by functionalizing the electrode surface with a substrate-like compound that interacts with the redox site of the enzyme as ligand (Blanford et al., 2007; VazDominguez et al., 2008; Martinez-Ortiz et al., 2011). This approach could be combined with the use of nanotubes, such as MWCNT, to improve the ligand-enzyme interaction (Meredith et al., 2011) . For alcohol bio-batteries (with ethanol or methanol as fuel), alcohol dehydrogenase (ADH) is the most recurrent enzyme in the literature. In most of cases, the NADH-dependent ADH is used, and there are few reports available in the literature using an ADH containing PQQ and heme prosthetic groups (Yakushi and Matsushita, 2010). The advantage of the ADH-PQQ is the presence of an exposed heme that allows direct electron transfer, as demonstrated in an
4
hybrid fuel cell with a platinum cathode (Aquino Neto et al., 2013). Enzymatic biofuel cell in which both, anode and cathode, electrodes were powered by ethanol has been described (Ramanavicius et al. 2008). The anode of this enzymatic fuel cell was based on immobilized quino-hemoprotein-alcohol dehydrogenase, while the cathode on co-immobilized alcohol oxidase (AOx) and microperoxidase. More recently, an alcohol oxidase (AOx) based biosensor, in which the enzyme was immobilized on a gold-MWCNT-Nafion-polientilenimine electrode, showed a direct electron transfer from the FAD to the matrix of the electrode (Das and Goswami, 2013). In the present study we report the design of an enzymatic fuel cell with molecularly orientated alcohol oxidase and laccase. The electrodes were prepared by the “substrate-like linkers” approach using 4-azidoaniline for the alcohol oxidase anode and benzoic acid for the laccase cathode, which emulates the natural ligands of both enzymes. This is, in our knowledge, the first enzymatic fuel cell built with an alcohol oxidase anode and a laccase cathode.
2. Materials and methods 2.1 Chemicals and reagents 4-Azidoaniline hydrochloride, N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDC), 2-(N-morpholine) ethanosulfonic acid (MES), 4-[2-aminoethyl] benzoic acid hydrochloride (AEBA), and the 2,2-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid) (ABTS) were purchased from Sigma-Aldrich Chemical Corporation (St. Louis, MI, USA). Sodium hydroxide, potassium phosphate monobasic, sodium phosphate dibasic, nitric acid, isopropanol, methanol, and acetone were obtained from J.T. Baker (Phillipsburg, NJ, USA), 5
Toluene was purchased from Fisher Scientific (Fair Lawn, NJ, USA) and tetrahydrofuran from Burdick & Jackson (Muskegon, MI, USA).
2.2 Enzymes Alcohol oxidase from Pichia pastoris was purchased from Sigma-Aldrich (21 U/mg and 38 µg/µl). Laccase from Coriolopsis gallica UAMH 8260 was obtained and purified as previously described (Tinoco et al., 2001). The purified preparation showed a specific activity of 313 U/mg and a purity of 81% estimated by electrophoresis. 2.3 Electrode and fuel cell materials Nafion solution (5%) was purchased from Fuel Cell Scientific, LLC (Stoneham, MA, USA). SIGRACET graphite laminated sheets (GDL 35 BC), used as electrode, were obtained from SGL Carbon Group (Wiesbaden, Germany). Carboxylated Multi-Walled Carbon Nanotubes (C-MWCNT) from US Research Nanomaterials Inc. (Outside diameter of 5-15 nm, inside diameter 3-5 nm, length 50 µm) (Houston, TX, USA), Nafion 232 membranes (50µm) were purchased from DuPont. 2.4 Determination of enzymatic activity and linker affinity Alcohol oxidase (AOx) activity was measured spectrophotometrically in a 1 ml reaction mixture containing 100 mM potassium phosphate buffer (pH 7.5), 0.1% (v/v) methanol, 2 mM ABTS, and 2.5 units of horseradish peroxidase. The amount of H2O2 produced is estimated monitoring the ABTS-diradical production at 405 nm (
405
= 36,800 M-1 cm-1). The protein
6
content was determined by the Bradford method with the BioRad protein reagent and measured at 595 nm. In order to prove that the linker (4-azidoaniline) interacts as ligand in the AOx active site, the enzyme (5 µM in monomer basis) was incubated for 12 h with different concentration of 4azidoanile (from 0 to 47 mM), and the changes in the absorbance spectrum were recorded. The spectra were obtained by scanning the absorbance from 300 to 600 nm in a Perkin Elmer Lambda 25 UV/VIS spectrophotometer. 2.5 Electrochemical experiments Cyclic Voltammetry experiments were performed with a Solartron-1287 potensiostat using a three electrodes configuration: a saturated calomel electrode (SCE) as a reference electrode in close proximity to the working electrode volume using a Luggin capillary; a platinum mesh as a counter electrode; and the different preparations of AOx electrodes as working electrodes, as well as the respective controls. All voltage potentials from the cyclic voltammetry experiments are referred to the SCE (KCl sat.) electrode. All the literature reported data was converted to the SCE electrode scale. All the anode cyclic voltammetry experiments where done under nitrogen purge. 2.6 Preparation of oriented enzymatic electrodes The enzymes were immobilized in an oriented fashion on graphite sheets. Before AOx immobilization, the graphite sheets electrodes (1 cm2) were successively cleaned with dichloromethane, tetrahydrofuran and water, and dried. Then, the clean graphite electrodes were carboxylated under nitric acid reflux for 2 h at 80°C.
7
On the other hand, C-MWCNT were functionalized with 4-azidoaniline creating an amide bond between the amino group of the 4-azidoaniline and the carboxylic group of CMWCNT. The functionalization was performed dissolving 5 mg of C-MWCNT, 5 mg of 4azidoaniline and 5 mg of EDC in dimethylsulfoxide, and the reaction was kept during 4 h. The functionalized nanotubes (ANA-MWCNT) were washed three times with 13 ml of phosphate buffer and centrifuged at 9000 rpm during 10 min. Then, the enzyme was immobilized on the ANA-MWCNT as follows: An aliquot of 15 µl of AOx was homogenized with 60 µl of ANAMWCNT, previously sonicated, and incubated during 12 h to ensure the formation of the AOxANA complex. Once the incubation was completed, 15 µl of Nafion (5% w/w) were added and homogenized. Twenty-two µl of the suspension were applied on the graphite electrode and dried under darkness. The oriented enzymatic electrodes (AOx-ANA-MWCNT) were then washed and stored in phosphate buffer 100 mM at pH 7.5. The same procedure was performed for control electrodes without the addition of 4-azidoanile to the C-MWCNT. The oriented laccase electrodes were fabricated also by the “substrate-like linker” approach with 4-(2-aminoethyl) benzoic acid (AEBA) and according to the protocol previously published (Martinez-Ortiz et al., 2011). 2.7 Fuel cell experiments Semi-enzymatic fuel cells were assayed in order to evaluate the performance of AOx anode without the limitations of laccase cathode. An electrochemical cell was built in acrylic hardware with a graphite sheet (3 cm x 2 cm) containing 20% platinum as cathode, and anodes with different preparations of immobilized AOx (1 cm2).
8
The full enzymatic fuel cell included the two oriented enzymatic electrodes, AOx-ANAMWCNT anode and laccase cathode. The anodic chamber contained 100 mM phosphate buffer (pH 7.5) and the cathodic chamber was filled with 50 mM succinate buffer at pH 4.5. The chambers were separated by a 50 µm Nafion 232 membrane. Anodic chamber was aerated with nitrogen and the cathodic chamber with oxygen. The cell performance was measured using a potensiostat (Solartron-1287) and the polarimetric curves at 10 mVs-1 where obtained. The cell was stabilized before measures at open circuit potential (OCP) during 2 h before measurements.
3. Results and discussions.
3.1 Linker for enzyme orientation The “substrate-like linker” approach was selected for the molecular orientation of the alcohol oxidase (AOx) on the electrode surface. Sodium azide has been reported as able to bind the FAD cofactor pocket of AOx (Bringer et al., 1979). Thus we have selected an aromatic azide derivative as linker in order to orientate the enzyme molecule and to assure the electron transfer. The 4-azidoaniline was selected because it contains an azide group that can interact with the enzyme and an amino group in para position to be covalently attached to the carboxylated surface of graphite electrode. The interaction of 4-azidoaniline as an active-site ligand with the AOx molecule was spectrophotometrically analyzed. The AOx absorbance spectra with different amounts of ligand revealed the formation of a complex that modifies the absorbance of the FAD binding pocket of AOx (Fig. 1A). A pronounced increase in the AOx absorbance between 340 and 600 nm, with a maximal increase at 450 nm, was observed after subtraction of background 9
spectrum of 4-azidoaniline alone. This absorbance increase at 450 nm is proportional to the amount of 4-azidoaniline, fitting a hyperbolic curve (Fig. 1B). This change of absorbance spectrum in the presence of 4-azidoaniline strongly suggests the compound interaction as ligand in the FAD protein pocket. The incubation for 12 h was enough to reach the complex equilibrium. Using a concentration range from 0 to 47 mM of 4-azidoaniline, the calculated dissociation constant (Kd) was 17.3 (±4.8) mM, which implies affinity for this ligand. Thus, AOx-azidoaniline complex could be used for the orientation of AOx molecules on the graphite electrode. The azide molecule binds strongly to the isoalloxazine moiety of the flavin as demonstrated by crystallography in the case of NADH oxidase from Streptococcus pyogenes (PDB 2BCP). The isoalloxazine ring of the flavin, which is involved in catalysis, offers several possibilities for interaction with various compounds. Its amphipathic property due to the hydrophobicity of the xylene moiety and the hydrophilic pyrimidine ring that is relatively electron-deficient and able to form hydrogen bridges (Ghisla and Massey, 1986). Most of the flavoproteins so far studied show a channel lying over the face of the flavin, which permits access of the substrate. In addition, crystallographic structures show that the flavin 8-position are exposed to solvent. On the other hand, 8-azido-FAD, has been used for reconstituting both apo proteins; amino acid oxidase and glucose oxidase (Fitzpatrick et al., 1985). The binding is specific and very tight, so that only a stoichiometric amount of reagent is necessary. Nevertheless, still studies should be conducted to elucidate the interaction between FAD and 4azidoaniline.
10
3.2 Alcohol oxidase anode The FAD deep localization inside the protein moiety could difficult the ligand interaction. In order to improve the ligand interaction with the FAD pocket, carboxylated multiwalled carbon nanotubes (C-MWCNT) were first functionalized with 4-azidoaniline (ANA) and then immobilized on the graphite electrode. These electrodes containing functionalized nanotubes were then treated with AOx to form the enzymatic anode (AOx-ANA-MWCNT). As controls, two other electrodes were prepared: One with the AOx directly and randomly immobilized on the graphite surface by enzyme adsorption on the Nafion, and another with AOx immobilized on the electrode containing MWCNT but without ANA. These two latter electrodes could be considered as having the protein randomly oriented. The AOx-ANA-MWCNT electrode preserves the more positive peak but translocated to 150 mV, which is 350 mV (Fig. 2), more negative that the 175 mV reported by Das and Goswami (2013). For fuel cell applications, a more negative anode potential favors a larger potential difference. In addition, the functionalized nanotubes increase the current generation probably due to a better molecular orientation of the enzyme. On the other hand, the electrode prepared with AOx directly adsorbed on Nafion (AOx(a) and AOx(b) in Fig. 2) showed a low increase of the voltammetric response at -500 mV in the presence of methanol, while the other randomly oriented AOx electrode containing carbon nanotubes (AOx-MWCNT) showed the poorest response to the methanol. Thus, the fact that the AOx-ANA-MWCNT electrode generated the most important response to the presence of methanol in the medium with a 3 mA increase with respect to the buffer (Fig 2), evidences the importance of the orientation of enzyme molecule for the electronic transfer between the enzyme active site and the electrode surface.
11
The AOx-ANA-MWCNT heterogeneous electron transfer rate constant was calculated, from triplicate electrodes, employing the equation; Ks=Amax/Q. The obtained Ks value was 0.98 (±0.15), which implies a good contact between the 4-azidoaniline functionalized MWCNT and the AOx molecule. On the other hand, the concentration of electroactive species on the electrode was estimated using the equation Г = Q/nFA, where Q is the electrode charge in coulombs obtained from the integrated area of the cyclic voltammetry experiments, n the number of electrons (n = 1), F is the Faraday constant, and 0.368 cm2 the exposed area of the electrode. The obtained value of Г = 1.5 nM cm-2 is consistent with the 1.9 nM deposited onto the electrode. The response to increasing concentrations of methanol and ethanol of the oriented enzymatic electrode AOx-ANA-MWCNT was determined (Fig. 3). The AOX-ANA-MWCNT electrode showed high current densities and a well-defined peak at -63 mV which shifted 90 mV from the peak registered without substrate (Fig. 2). The AOx oxidation of methanol could be thermodynamically favored at this higher potential. The potential increase for alcohol oxidation in the presence of MWCNT has been reported for other oxidases such as aryl alcohol oxidase on reticulated vitreous carbon electrodes with poly-neutral red as mediator (Barsan and Brett, 2008; Munteanu et al., 2008). From the saturation curves of AOx-ANA-MWCNT (Fig. 3), the methanol plot showed an estimated Kd of 4.9 (±1.4) mM and a maximal current density (Amax) of 667 (±32) µA cm-2. As expected, the estimated Kd for ethanol is 6.6 (±1.8) mM (Fig 3 A) that is consistent with the substrate affinity of AOx for ethanol, which is reported to be 20% lower when compared to those for methanol (Barsan and Brett, 2008) . Nevertheless, the current generated by ethanol of 897 (±60.9) µA cm-2 is higher than those obtained using methanol. This could be originated by the methanol transformation to form formaldehyde, which beside to be AOx substrate it is a strong AOx inactivator (Hopkins and Muller, 1987). However, the
12
sensitivity of both electrodes is the same, 136 µA cm-2 mM-1, indicating no differences between ethanol and methanol in terms of recognition and response. In order to examine the performance of the different AOx electrode preparations, experiments were performed with a hybrid fuel cell with a platinum cathode (Fig 4). The cell was a low potential one with 0.2 Volts, but notably the AOx-ANA-MWCNT overcome the randomly orientated preparations with 60 W cm-2 and 700 A cm-2 at short-circuit. It is important to point out that the AOx electrode could accomplished oxidation of methanol on the cell 35 W cm-2 and 500 A cm-2 at short-circuit. These experiments clearly show that the electrode with orientated enzyme molecules is better for the current and power generation. 3.3 Enzymatic fuel cell alcohol oxidase - laccase Finally, a full enzymatic fuel cell was constructed and tested (Fig. 5). Both, enzymatic electrodes, cathode an anode, were molecularly oriented to enhance the direct electron transfer between the active-site of the enzyme and the electrode surface. The enzymatic fuel cell consisted in an AOx-ANA-MWCNT anode and an oriented laccase-AEBA cathode, previously characterized (Martinez-Ortiz et al., 2011). The enzymatic fuel cell reaches 0.5 V in open circuit with both, ethanol and methanol. However, in short circuit a difference of 50 A cm-2 is found, with a highest current intensity of 250 A cm-2 obtained with methanol (Fig. 6). The values obtained in this work are in agreement with those obtained from enzymes with an embedded cofactor such as alcohol dehydrogenase and aldehyde dehydrogenase dependent of PQQ with and without MWCNT (Aquino et al., 2013). Concerning the power density, the methanol was the best substrate reaching 60 W cm-2, while with ethanol only 40 W cm-2were obtained (Fig. 6). These values are similar to those 13
obtained from a hybrid fuel cell with platinum cathode (Fig. 4). This power density is significantly higher than these obtained from a non-oriented enzymatic fuel cell with alcohol dehydrogenase - alcohol oxidase:microperoxidase (1.5 W cm-1) (Ramanavicius et al. 2008). Maximal current densities of 250 µA cm-2 for methanol and 180 µA cm-2 ethanol were obtained. Higher power densities have been also obtained with methanol, when compared with ethanol, in fuel cells with alcohol dehydrogenase electrodes (Akers et al 2005). For both substrates, a cell efficiency of 40% was estimated using the equation
cell =
[Pmax/(OCV SCC)], where OCV is the
open circuit voltage, SC is the short-circuit current, and Pmax is the maximal power. This efficiency value is superior than reported by (Katz et al., 1999). Using alcohols as fuels, the enzymatic fuel cells with alcohol dehydrogenase (ADH) reached power densities between 32-1000 W cm-2 (Aquino Neto et al., 2013; Kim et al., 2013). The performance of our oriented enzymatic fuel cell (AOx-ANA-MWCNT and laccase-AEBA) is still far from those obtained with alcohol dehydrogenase dependent of NAD+ , in which a power density of around 1000 W cm-2 has been obtained (Akers et al., 2005). However, these results should be taken cautiously because the constant addition of the cofactor NAD+ to the anodic chamber could keep high current generation. It is important to point out that the constant use of cofactor on a bio-battery is an operational disadvantage. In our enzymatic fuel cell, the AOx posses a prosthetic group (FAD) deeply embedded on the protein and there is no need for cofactor addition.
14
4. Conclusions Our results show that the enzyme molecular orientation on the electrode surface improves the direct electron transfer. Alcohol oxidase and laccase can be immobilized in an oriented fashion on graphite electrodes by means of a substrate-like linker, showing better performance than the randomly immobilized preparations. The full enzymatic fuel cell, built with an AOxANA-MWCNT anode and laccase-AEBA cathode, reaches 0.5 V in open circuit with both, ethanol and methanol, while in short circuit the highest current intensity of 250 A cm-2 was obtained with methanol. Concerning the power density, the methanol was the best substrate reaching 60 W cm-2, while with ethanol 40 W cm-2 were obtained. The results clearly show that the electrodes with orientated enzyme molecules are better for the current and power generation when compared a non-oriented systems. Finally, in our knowledge, this is the first enzymatic fuel cell built with an alcohol oxidase anode and a laccase cathode.
References Akers N.L., Moore C.M., Minteer S.D., 2005. Electrochim. Acta 50, 2521-2525. Aquino Neto S., Suda E.L., Xu S., Meredith M.T., De Andrade A.R., Minteer S.D., 2013, Electrochim. Acta 87, 323-329. Barsan M.M., Brett C.M.A., 2008. Talanta 74, 1505-1510. Blanford C.F., Heath R.S., Armstrong F.A., 2007. Chem. Commun. 1710-1712. Bringer S., Sprey B., Sahm H., 1979. Eur. J. Biochem. 101, 563-570. Das M., Goswami P., 2013. Bioelectrochemistry 89, 19-25.
15
Demin S., and Hall E.A.H., 2009. Bioelectrochemistry 76, 19-27. Deng L., Shang L., Wen D., Zhai J., Dong S., 2010. Biosens. Bioelectron. 26, 70-73. Fitzpatrick P.F., Ghisla S., Massey V., 1985. J. Biol. Chem. 260, 8483-8491. Ghisla S., Massey V., 1986. Biochem. J. 239,1-12. Hopkins T.R., Muller F., 1987. Biochemistry of Alcohol Oxidase, in: H. Verseveld and J. A. Duine (Eds.), Microbial Growth on C1 Compounds, Springer Netherlands. pp. 150-157. Ivanov I., Vidaković-Koch T., Sundmacher K., 2010. Energies 3, 803-846. Katz E., Filanovsky B., Willner I., 1999. New J. Chem. 23, 481-487. Kim Y.H., Campbell E., Yu J., Minteer S.D., Banta S., 2013. Angew. Chem. Int. Ed. 52, 14371440. Lang Q.,Yin L., Shi J., Li L., Xia L., Liu A., 2014. Biosens. Bioelectron. 51, 158-163. Martinez-Ortiz J., Flores R., Vazquez-Duhalt R., 2011. Biosens. Bioelectron. 26, 2626-2631. Meredith M.T., Minteer S.D., 2012. Ann. Rev. Anal. Chem. 5, 157-179. Meredith M.T., Minson M., Hickey D., Artyushkova K., Glatzhofer D.T., Minteer S.D., 2011. ACS Catalysis 1, 1683-1690. Munteanu F.-D., Ferreira P., Ruiz-Dueñas F.J., Martínez A.T., Cavaco-Paulo A., 2008. J. Electroanal. Chem. 618, 83-86. Osman M.H., Shah A.A., Walsh F.C., 2011. Biosens. Bioelectron. 26, 3087-3102. Ramanavicius A., Ramanaviciene A., 2009. Fuel Cells 9, 25-36. Ramanavicius A., Kausaite A., Ramanaviciene A., 2008. Biosens. Bioelectron. 24, 761-766. Reuillard B., Le Goff A., Agnes C., Holzinger M., Zebda A., Gondran C., Elouarzaki K., Cosnier S., 2013. Phys. Chem. Chem. Phys. 15, 4892-4896.
16
Sundmacher K., 2010. Ind. Eng. Chem. Res. 49, 10159-10182. Tinoco R., Pickard M.A., Vazquez-Duhalt R., 2001. Lett. Appl. Microbiol. 32, 331-335. Vaz-Dominguez C., Campuzano S., Rüdiger O., Pita M., Gorbacheva M., Shleev S., Fernandez V.M., De Lacey A.L., 2008. Biosens. Bioelectron. 24, 531-537. Vazquez-Duhalt R., Aguila S.A., Arrocha A.A., Ayala M., 2013. ChemElectroChem, 1, 496-513 Willner I., Willner B., Katz E., 2007. Bioelectrochemistry 70, 2-11. Willner I., Yan Y.M., Willner B., Tel-Vered R., 2009. Fuel Cells 9, 7-24. Xia, L. Liang B., Liang Li L., Tang X., Palchetti I., Mascini M., Liua A., 2013. Biosens. Bioelectron. 44, 160-163. Yakushi T., Matsushita K., 2010. Appl. Microbiol. Biotechnol. 86, 1257-1265.
17
Figure legends
Figure 1. Change of the alcohol oxidase spectrum in the presence of 4-azidoaniline. (A) AOx incubated with different concentrations of 4-azidoaniline. (B) Absorbance at 450 nm vs 4azidoaniline concentration plot. The determined value of Kd is 17.3 (±4.8) mM. Figure 2. Cyclic voltagramms at 50 mV s-1 of different preparations of alcohol oxidase immobilized on graphite electrodes. (a) In the absence of methanol and (b) in the presence of 212 mM methanol. AOx, electrodes prepared by alcohol oxidase absorption on Nafion. AOxMWCNT, electrodes prepared by carbon nanotubes immobilized on the graphite surface and then alcohol oxidase randomly immobilized. AOx-ANA-MWCNT, molecularly oriented electrodes prepared with functionalized carbon nanotubes with 4-azidoaniline covalently immobilized on the graphite surface and alcohol oxidase immobilized by azo affinity. Figure 3. Representative saturation plots using AOx-ANA-MWCNT electrode obtained from cyclic voltammetry experiments at 50 mV s-1 with successive additions of alcohol. (A) Ethanol response at - 93 mV, and (B) methanol response at -63 mV. Figure 4. Potentiodynamic (A) and power curves (B) of three different electrode preparations in a hybrid fuel cell. The anodic chamber (AOx) was fueled with 200 mM methanol on nitrogen purged 100 mM phosphates buffer (pH 7.5). The cathodic chamber (Pt) was bubbled with oxygen and contained 100 mM phosphate buffer (pH 7.5). Figure 5. Scheme of the enzymatic fuel cell with molecularly oriented alcohol oxidase and laccase electrodes. Figure 6. Potentiodynamic and power curves for AOx-ANA-MWCNT vs lacasse-AEBA. The speed scan was performed at 10 mV s-1. The anodic chamber (AOx) was fueled with 200 mM ethanol (solid lines) or methanol (dotted lines) on nitrogen purged 100 mM phosphates buffer (pH 7.5). The cathodic chamber (laccase) was bubbled with oxygen and contained 50 mM succinate buffer (pH 4.5) was used for lacasse cathode.
18
0.6
A
0.2
0.1
0.0 340
B Absorbance at 450 nm
Absorbance units
0.3
0.5
0.4
0.3
0.2 0.0
400
460
520
Wavelength (nm)
580
0
10
20
30
40
50
4-Azidoaniline (mM)
Figure 1.
19
30
mA cm-2
20 10 0 -10
AOx-ANA-MWCNT(a) AOx-ANA-MWCNT(b) AOx-MWCNT(a) AOx-MWCNT(b) AOx(a) AOx(b)
-20 -30 -0.8
-0.6
-0.4
-0.2
0
0.2
0.4
0.6
0.8
Volts
Figure 2.
20
Current density (μAmpers/cm2)
1000
A
800 600 400 200 0
Current density (μAmpers/cm2)
0 800
10
20
30
40
Ethanol (mM)
50
60
70
B
600
400
200
0 0
10
20
30
40
50
60
Methanol (mM)
Figure 3.
21
0.25
70
AOx-ANA-MWCNT AOx-Nf AOx-MWCNT
A 0.2
AOx-ANA-MWCNT AOx-Nf AOx-MWCNT
60
B
0.15
µWatts
Volts
50
0.1
40 30 20
0.05
10 0
0 0
200
400
µAmpers
600
800
0
200
400
600
800
µAmpers
Figure 4.
22
eanode
cathode
H+
O2
Alcohol H+ Aldehyde
H2O
H+
Laccase Alcohol oxidase
Figure 5.
23
0.6
70
0.5
60
Volts
40 0.3 30 0.2
µWatts
50
0.4
20
0.1
10
0.0
0 0
50
100
150
200
250
µAmpers
Figure 6.
24
