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High performance bioanode based on direct electron transfer of fructose dehydrogenase at gold nanoparticle-modified electrodes
Electrochemistry Communications 11 (2009) 668–671
Contents lists available at ScienceDirect
Electrochemistry Communications journal homepage: www.elsevier.com/locate/elecom
High performance bioanode based on direct electron transfer of fructose dehydrogenase at gold nanoparticle-modified electrodes Kenichi Murata, Masato Suzuki, Kazuki Kajiya, Nobuhumi Nakamura *, Hiroyuki Ohno Department of Biotechnology and Life Science, Tokyo University of Agriculture and Technology, 2-24-16 Nakacho, Koganei, Tokyo 184-8588, Japan
a r t i c l e
i n f o
Article history: Received 1 December 2008 Received in revised form 22 December 2008 Accepted 9 January 2009 Available online 14 January 2009 Keywords: Fructose dehydrogenase Gold nanoparticles Nanostructured electrode Direct electron transfer reaction Bioelectrocatalysis Biofuel cells
a b s t r a c t The direct electron transfer reaction of fructose dehydrogenase (FDH) from Gluconobacter sp. on alkanethiol-modified gold nanoparticles (AuNPs) was examined. AuNP-modified electrodes were simply fabricated by depositing citrate-reduced gold nanoparticles onto a gold electrode and carbon fiber paper and then covering the surface with a self-assembled monolayer of alkanethiols. The immobilization of AuNPs provided a large effective surface area for the adsorption of FDH. Catalytic oxidation currents based on the direct electron transfer reaction of FDH were observed from a potential about 100 mV (vs. Ag/AgCl, 3 M NaCl) in the presence of D-fructose without a mediator. The current density reached as high as 14.3 ± 0.93 mA/cm2 (at +500 mV), which was achieved in the presence of 200 mM D-fructose by immobilization of FDH on 2-mercaptoethanol-modified AuNP/carbon fiber paper electrodes. Ó 2009 Elsevier B.V. All rights reserved.
1. Introduction The direct electron transfer (DET) reaction of redox enzymes and electrodes has gained considerable attention in recent years for applications in biosensors and biofuel cells [1–4]. To obtain a high current density electrode the following requirements must be satisfied: (i) a high density of enzymes at electrodes, (ii) a proper orientation of enzymes at the electrode surface, and (iii) a sufficient mass transfer of substrates to enzymes at electrodes. Therefore, suitable electrodes and immobilizations for individual redox enzymes are required. Fructose dehydrogenase (FDH; EC 1.1.99.11) from Gluconobacter sp. is a membrane-bound flavohemo-protein [5–7], which consists of three tightly bound units of 67.0 kDa (flavin-domain), 50.8 kDa (heme c-domain) and 19.7 kDa (peptide of unknown function) [5]. The DET reaction of FDH has been examined at various electrodes including nano-carbon materials [7–15]. Carbon and metal nanoparticles are desirable materials for the immobilization of redox enzymes due to their large surface area and high conductivity [1,13,16]. Recently, Kamitaka et al. reported a high current density of 8 mA cm 2 by using Ketjen black-modified glassy carbon electrodes [13]. In this study, we investigated the DET reaction of FDH at a gold nanoparticle (AuNP)-modified electrode with a large effective surface area to obtain a high density of FDH. We fabricated an AuNP-
* Corresponding author. Tel./fax: +81 42 388 7482. E-mail address: [email protected] (N. Nakamura). 1388-2481/$ - see front matter Ó 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.elecom.2009.01.011
modified electrode in a simple manner by casting only citrate-reduced AuNPs onto supporting electrodes of gold electrode or carbon fiber paper. The use of gold nanoparticles is also advantageous because the surface of gold can be easily modified with the direct linkage of a thiol functional group [17]. To achieve a favorable orientation of FDH at AuNPs, we used a self-assembled monolayer (SAM) of x-functionalized alkylthiols on the AuNPmodified electrodes. 2. Materials and methods 2.1. Materials FDH from Gluconobacter sp. (Toyobo Co., Japan) was used as received. D-Fructose (Wako, Japan) was used as the substrate for FDH. 3-Mercaptopropionic acid (MPA, from Dojindo, Japan), 2mercaptoethanol (MET, from Kanto Chemical Co., Japan), and 2aminoethanthiol (AET, from Wako) were purchased and used without further purification. Carbon fiber paper (TGP-H-090) was obtained from Toray Industries, Inc. (Japan). 2.2. Preparation of AuNPs AuNPs were prepared following a procedure described by Frens [18]. Briefly, 12.5 mL of 38.8 mM sodium citrate solution (Wako) was added to 125 mL of boiling 1.0 mM HAuCl4 (Wako) with vigorous stirring. After the appearance of a deep red color, boiling and stirring were continued for 15 min. The solution was then allowed
K. Murata et al. / Electrochemistry Communications 11 (2009) 668–671
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to cool to room temperature. The particle diameter of the AuNPs was estimated to be about 15 nm from the UV-vis spectrum of the solution [19]. 2.3. Preparation of AuNP-modified electrodes To increase the number of particles per volume, the AuNP solution was centrifuged (10,000g, 30 min) in 1.5 ml Eppendorf tubes; then, 98% of the remaining supernatant volume was thrown away. A polycrystalline gold electrode (u = 1.6 mm) was polished with water on aluminum oxide lapping film sheets, and then 1 ll of the concentrated AuNPs was dropped onto the surface of the electrode, followed by being dried in air. To increase the number of particles at the electrode, this procedure was repeated three times. In experiments using a carbon fiber paper electrode, the electrodes with a surface area of 0.25 cm2 were first anodized at +2.0 V for 15 s followed by cathodization at 1.1 V for 15 s in 100 mM phosphate buffer (pH 7.0) to obtain a hydrophilic surface. Twenty microliters of the concentrated AuNPs were dropped onto the hydrophilic surface of the carbon fiber paper, and then the electrode was dried in air. This procedure was repeated four times. 2.4. Preparation of FDH-immobilized electrodes The AuNP-modified and unmodified gold electrodes were immersed in a 20 mM aqueous solution of thiols, MET, MPA, and AET, for 1 h at room temperature. The resulting electrodes were thoroughly rinsed with water to remove physically adsorbed thiol molecules. Then they were immersed in a 1.7 mg/ml FDH solution of 100 mM acetate buffer solution (pH 5.0) at room temperature. As mentioned below, the adsorption of FDH was completed within 20 min and 2 h for AuNP-unmodified and -modified electrodes, respectively. 2.5. Electrochemical measurement Electrochemical experiments were carried out with an ALS Electrochemical Analyzer (Model 702B). All experiments were performed using a three-electrode cell with Ag/AgCl (3 M NaCl) as a reference electrode and a Pt mesh electrode as a counter electrode. All measurements were carried out at room temperature.
3. Results and discussion 3.1. DET reaction at polycrystalline gold electrode Cyclic voltammetry measurements of FDH at SAM-modified polycrystalline gold electrodes (AuE) were performed. The type of thiols to build SAMs influence the electronic coupling between FDH and an electrode: the distance between the redox active site of FDH and the electrode can be controlled by using different electrode modifiers. In this context, we chose the short chain thiols with various termination groups, such as MPA, AET, and MET. Fig. 1A shows cyclic voltammograms of FDH at SAM-modified and -unmodified AuE. In the absence of D-fructose, no redox response of FDH itself was observed at any electrode. When D-fructose was added into the buffer solution, well-defined catalytic currents were observed. No catalytic current was observed at the FDH-unmodified electrodes (data not shown). These results indicate that FDH electrochemically catalyzed the oxidation of D-fructose through direct electron transfer between the active center of FDH and the electrodes. As shown in Fig. 1A, the catalytic oxidation current began to increase at a potential around 100 mV (vs. Ag/ AgCl). This result agrees with the previous reports on the DET reaction of FDH at SAM-modified gold electrodes [12] and carbon-
Fig. 1. (A) Cyclic voltammograms of a FDH-modified bare and thiol-modified AuE at pH 5 in the presence of 200 mM D-fructose, at a scan rate of 10 mV s 1. (B) pH dependence of catalytic current at +500 mV obtained from cyclic voltammetry for FDH-modified MET-AuNP/AuE in the presence of 200 mM D-fructose.
based electrodes [7,8,13–15]. When the MET-modified electrode was used, a current density was the highest among several SAMmodified electrodes (Fig. 1A). The difference in the obtained current densities for several thiols should originate from the orientation and/or stability of FDH at the SAM surfaces. To examine the useable pH range of the electrode, an analysis of the pH dependence of catalytic currents for the FDH-immobilized MET-AuE, which was prepared in 100 mM acetate buffer solution at pH 5.0, was performed (Fig. 1B). The catalytic current was measured at +500 mV. The optimum pH was about 6. It was higher (about 1– 2 pH units) than those at edge-plane [14] and basal-plane [7] highly oriented pyrolytic graphite electrodes and that for enzymatic reaction in solution. It was reported that the FDH complex decomposes and its activity losses in neutral and alkaline solutions [7]. It is suggested that the adsorption of FDH to an MET-modified electrode might prevent the decomposition. 3.2. DET reaction at AuNP-modified electrodes An effective surface area of an AuNP/AuE was investigated by cyclic voltammetry measurement in 0.5 M H2SO4 [20]. The real surface area of the AuNP/AuE was determined to be 2.32 cm2 and about 65 times as large as that of the bare AuE (0.0356 cm2). Fig. 2A shows typical voltammetric curves obtained at the FDH adsorbed MET-AuNP/AuE in 100 mM acetate buffer solution (pH 5.0). The catalytic current was observed only when fructose dehydrogenase was adsorbed at the electrode. These results indicate
670
K. Murata et al. / Electrochemistry Communications 11 (2009) 668–671
Fig. 3. Cyclic voltammograms of a FDH-modified MET-AuNP/carbon fiber paper electrodes at pH 5 in the presence of 200 mM D-fructose in quiet solution (1) and with stirring at 800 rpm (2), and absence of D-fructose in quiet solution (3), at a scan rate of 10 mV s 1.
electrolysis of D-fructose. Fig. 3 shows the cyclic voltammogram of FDH at an MET-AuNP/CFP. The current density reached a value as high as about 14.3 ± 0.93 mA cm 2 (at +500 mV) in the presence of 200 mM D-fructose at room temperature. This is the highest value ever reported for a bioanode based on both DET-type [13] and mediated electron transfer-type [21] bioelectrocatalysis as far as we know. The catalytic current obtained was not influenced even when the solution was stirred by a magnetic stirring bar. This indicates that the mass transfer of D-fructose to the nanostructured electrode surface is sufficient, and that the total process for the bioelectrolysis of D-fructose was controlled by enzyme kinetics. Fig. 2. (A) Cyclic voltammograms of a FDH-modified (solid line) and -unmodified (dotted line) MET-AuNP/AuE at pH 5 in the presence of 200 mM D-fructose, at a scan rate of 10 mV s 1. The electrodes were immersed in a FDH solution for (a) 8, (b) 18, (c) 36, (d) 64, (e) 102, (f) 120 min. (B) pH dependence of catalytic current at +500 mV obtained from cyclic voltammetry for FDH-modified MET-AuNP/AuE in the presence of 200 mM D-fructose, at the first cycle (circle) and fifth cycle (triangle).
that FDH electrochemically catalyzed the oxidation of D-fructose through the direct electron transfer with AuNPs. The current density at +500 mV for an MPA-AuE and an MPA-AuNP/AuE were 0.17 ± 0.01 mA and 3.25 ± 0.18 mA, respectively. Therefore the current density for an MPA-AuNP/AuE was about 19 times higher than that for a polycrystalline gold electrode. The current density depended on the time for the immobilization of FDH. It reached a maximum value after about 2 h, although it takes only 20 min when using MET-AuE. The slow adsorption behavior for METAuNP/AuE could be explained due to the nanostructure of the AuNPs, as reported at Ketjen black [13]. Fig. 2B shows the pH dependence of catalytic currents of the FDH adsorbed at an MET-AuNP/AuE, which was prepared in a 100 mM acetate buffer solution at pH 5.0. At the first cycle, a high catalytic current density was observed over a broad pH range, although it gradually decreased with successive scans on neutral or slightly basic solutions, pH 7–8 (Fig. 2B). The oxidative activity of FDH buried into the nanostructure of AuNPs would be insusceptible to pH change. The voltammetric response at pH 5.0 remained at 92% of the original signal after the electrode was continuously potential scanned for 30 cycles (70 min). This indicated that the AuNP electrode had a good stability and reproducibility. To further increase effective surface area, AuNPs were immobilized into a carbon fiber paper (CFP). The CFP has a large surface area and thus could improve the current density based on a bio-
4. Conclusion We fabricated AuNP-mobilized electrodes by a simple method in order to investigate the DET reaction of FDH. A current density as high as 14.3 ± 0.93 mA cm 2 was achieved in the presence of 200 mM D-fructose by immobilization of FDH on MET-modified AuNP/carbon fiber paper electrodes. For the DET reaction of FDH, the AuNP-modified electrodes satisfy the following requirements: (i) a high density of FDH at the electrodes, (ii) a proper orientation of FDH at the electrode surface, and (iii) a sufficient mass transfer of D-fructose. Therefore, we can conclude that the AuNP-immobilized electrodes are promising for high performance biofuel cells, third generation biosensors, and bioreactors. It is useful to deposit AuNPs onto mesoporous carbon electrodes for increasing the effective surface area of electrodes. This methodology could be applied to other mesoporous electrodes due to the simplicity of the deposition. Acknowledgment K.M. acknowledges the financial support of the Japan Society for the Promotion of Science (Research Fellowship for Young Scientists). References [1] J.A. Cracknell, K.A. Vincent, F.A. Armstrong, Chem. Rev. 108 (2008) 2439. [2] L. Gorton, A. Lindgren, T. Larsson, F.D. Munteanu, T. Ruzgas, I. Gazaryan, Anal. Chim. Acta 400 (1999) 91. [3] S. Shleev, J. Tkac, A. Christenson, T. Ruzgas, A.I. Yaropolov, J.W. Whittaker, L. Gorton, Biosens. Bioelectron. 20 (2005) 2517. [4] J. Hirst, Biochim. Biophys. Acta 1757 (2006) 225. [5] M. Ameyama, E. Shinagawa, K. Matsushita, O. Adachi, J. Bacteriol. 145 (1981) 814.
K. Murata et al. / Electrochemistry Communications 11 (2009) 668–671 [6] O. Adachi, Y. Ano, H. Toyama, K. Matsushita, Biooxidation with PQQ- and FADdependent dehydrogenase, in: R.D. Schmid, V.B. Urlacher (Eds.), Modern Biooxidation: Enzymes, Reactions and Applications, Wiley-VCH, Weinheim, 2007, pp. 1–41. [7] M. Tominaga, C. Shirakihara, I. Taniguchi, J. Electroanal. Chem. 610 (2007) 1. [8] T. Ikeda, F. Matsushita, M. Senda, Biosens. Bioelectron. 6 (1991) 299. [9] G.F. Khan, H. Shinohara, Y. Ikariyama, M. Aizawa, J. Electroanal. Chem. 315 (1991) 263. [10] J. Parellada, E. Domínguez, V.M. Fernández, Anal. Chim. Acta 330 (1996) 71. [11] S. Yabuki, F. Mizutani, Electroanalysis 9 (1997) 23. [12] E.E. Ferapontova, L. Gorton, Bioelectrochemistry 66 (2005) 55. [13] Y. Kamitaka, S. Tsujimura, K. Kano, Chem. Lett. 36 (2007) 218.
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[14] Y. Kamitaka, S. Tsujimura, N. Setoyama, T. Kajino, K. Kano, Phys. Chem. Chem. Phys. 9 (2007) 1793. [15] M. Tominaga, S. Nomura, I. Taniguchi, Biosens. Bioelectron. 24 (2009) 1184. [16] E. Katz, L. Sheeney-Haj-Ichia, I. Willner, Angew. Chem. Int. Ed. 43 (2004) 3292. [17] J.C. Love, L.A. Estroff, J.K. Kriebel, R.G. Nuzzo, G.M. Whitesides, Chem. Rev. 105 (2005) 1103. [18] G. Frens, Nat. Phys. Sci. 241 (1973) 20. [19] W. Haiss, N.T.K. Thanh, J. Aveyard, D.G. Fernig, Anal. Chem. 79 (2007) 4215. [20] S. Trasatti, O.A. Petrii, Pure Appl. Chem. 63 (1991) 711. [21] H. Sakai, T. Nakagawa, Y. Tokita, T. Hatazawa, T. Ikeda, S. Tsujimura, K. Kano, Energy Environ. Sci. 2 (2009) 133.
Contents lists available at ScienceDirect
Electrochemistry Communications journal homepage: www.elsevier.com/locate/elecom
High performance bioanode based on direct electron transfer of fructose dehydrogenase at gold nanoparticle-modified electrodes Kenichi Murata, Masato Suzuki, Kazuki Kajiya, Nobuhumi Nakamura *, Hiroyuki Ohno Department of Biotechnology and Life Science, Tokyo University of Agriculture and Technology, 2-24-16 Nakacho, Koganei, Tokyo 184-8588, Japan
a r t i c l e
i n f o
Article history: Received 1 December 2008 Received in revised form 22 December 2008 Accepted 9 January 2009 Available online 14 January 2009 Keywords: Fructose dehydrogenase Gold nanoparticles Nanostructured electrode Direct electron transfer reaction Bioelectrocatalysis Biofuel cells
a b s t r a c t The direct electron transfer reaction of fructose dehydrogenase (FDH) from Gluconobacter sp. on alkanethiol-modified gold nanoparticles (AuNPs) was examined. AuNP-modified electrodes were simply fabricated by depositing citrate-reduced gold nanoparticles onto a gold electrode and carbon fiber paper and then covering the surface with a self-assembled monolayer of alkanethiols. The immobilization of AuNPs provided a large effective surface area for the adsorption of FDH. Catalytic oxidation currents based on the direct electron transfer reaction of FDH were observed from a potential about 100 mV (vs. Ag/AgCl, 3 M NaCl) in the presence of D-fructose without a mediator. The current density reached as high as 14.3 ± 0.93 mA/cm2 (at +500 mV), which was achieved in the presence of 200 mM D-fructose by immobilization of FDH on 2-mercaptoethanol-modified AuNP/carbon fiber paper electrodes. Ó 2009 Elsevier B.V. All rights reserved.
1. Introduction The direct electron transfer (DET) reaction of redox enzymes and electrodes has gained considerable attention in recent years for applications in biosensors and biofuel cells [1–4]. To obtain a high current density electrode the following requirements must be satisfied: (i) a high density of enzymes at electrodes, (ii) a proper orientation of enzymes at the electrode surface, and (iii) a sufficient mass transfer of substrates to enzymes at electrodes. Therefore, suitable electrodes and immobilizations for individual redox enzymes are required. Fructose dehydrogenase (FDH; EC 1.1.99.11) from Gluconobacter sp. is a membrane-bound flavohemo-protein [5–7], which consists of three tightly bound units of 67.0 kDa (flavin-domain), 50.8 kDa (heme c-domain) and 19.7 kDa (peptide of unknown function) [5]. The DET reaction of FDH has been examined at various electrodes including nano-carbon materials [7–15]. Carbon and metal nanoparticles are desirable materials for the immobilization of redox enzymes due to their large surface area and high conductivity [1,13,16]. Recently, Kamitaka et al. reported a high current density of 8 mA cm 2 by using Ketjen black-modified glassy carbon electrodes [13]. In this study, we investigated the DET reaction of FDH at a gold nanoparticle (AuNP)-modified electrode with a large effective surface area to obtain a high density of FDH. We fabricated an AuNP-
* Corresponding author. Tel./fax: +81 42 388 7482. E-mail address: [email protected] (N. Nakamura). 1388-2481/$ - see front matter Ó 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.elecom.2009.01.011
modified electrode in a simple manner by casting only citrate-reduced AuNPs onto supporting electrodes of gold electrode or carbon fiber paper. The use of gold nanoparticles is also advantageous because the surface of gold can be easily modified with the direct linkage of a thiol functional group [17]. To achieve a favorable orientation of FDH at AuNPs, we used a self-assembled monolayer (SAM) of x-functionalized alkylthiols on the AuNPmodified electrodes. 2. Materials and methods 2.1. Materials FDH from Gluconobacter sp. (Toyobo Co., Japan) was used as received. D-Fructose (Wako, Japan) was used as the substrate for FDH. 3-Mercaptopropionic acid (MPA, from Dojindo, Japan), 2mercaptoethanol (MET, from Kanto Chemical Co., Japan), and 2aminoethanthiol (AET, from Wako) were purchased and used without further purification. Carbon fiber paper (TGP-H-090) was obtained from Toray Industries, Inc. (Japan). 2.2. Preparation of AuNPs AuNPs were prepared following a procedure described by Frens [18]. Briefly, 12.5 mL of 38.8 mM sodium citrate solution (Wako) was added to 125 mL of boiling 1.0 mM HAuCl4 (Wako) with vigorous stirring. After the appearance of a deep red color, boiling and stirring were continued for 15 min. The solution was then allowed
K. Murata et al. / Electrochemistry Communications 11 (2009) 668–671
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to cool to room temperature. The particle diameter of the AuNPs was estimated to be about 15 nm from the UV-vis spectrum of the solution [19]. 2.3. Preparation of AuNP-modified electrodes To increase the number of particles per volume, the AuNP solution was centrifuged (10,000g, 30 min) in 1.5 ml Eppendorf tubes; then, 98% of the remaining supernatant volume was thrown away. A polycrystalline gold electrode (u = 1.6 mm) was polished with water on aluminum oxide lapping film sheets, and then 1 ll of the concentrated AuNPs was dropped onto the surface of the electrode, followed by being dried in air. To increase the number of particles at the electrode, this procedure was repeated three times. In experiments using a carbon fiber paper electrode, the electrodes with a surface area of 0.25 cm2 were first anodized at +2.0 V for 15 s followed by cathodization at 1.1 V for 15 s in 100 mM phosphate buffer (pH 7.0) to obtain a hydrophilic surface. Twenty microliters of the concentrated AuNPs were dropped onto the hydrophilic surface of the carbon fiber paper, and then the electrode was dried in air. This procedure was repeated four times. 2.4. Preparation of FDH-immobilized electrodes The AuNP-modified and unmodified gold electrodes were immersed in a 20 mM aqueous solution of thiols, MET, MPA, and AET, for 1 h at room temperature. The resulting electrodes were thoroughly rinsed with water to remove physically adsorbed thiol molecules. Then they were immersed in a 1.7 mg/ml FDH solution of 100 mM acetate buffer solution (pH 5.0) at room temperature. As mentioned below, the adsorption of FDH was completed within 20 min and 2 h for AuNP-unmodified and -modified electrodes, respectively. 2.5. Electrochemical measurement Electrochemical experiments were carried out with an ALS Electrochemical Analyzer (Model 702B). All experiments were performed using a three-electrode cell with Ag/AgCl (3 M NaCl) as a reference electrode and a Pt mesh electrode as a counter electrode. All measurements were carried out at room temperature.
3. Results and discussion 3.1. DET reaction at polycrystalline gold electrode Cyclic voltammetry measurements of FDH at SAM-modified polycrystalline gold electrodes (AuE) were performed. The type of thiols to build SAMs influence the electronic coupling between FDH and an electrode: the distance between the redox active site of FDH and the electrode can be controlled by using different electrode modifiers. In this context, we chose the short chain thiols with various termination groups, such as MPA, AET, and MET. Fig. 1A shows cyclic voltammograms of FDH at SAM-modified and -unmodified AuE. In the absence of D-fructose, no redox response of FDH itself was observed at any electrode. When D-fructose was added into the buffer solution, well-defined catalytic currents were observed. No catalytic current was observed at the FDH-unmodified electrodes (data not shown). These results indicate that FDH electrochemically catalyzed the oxidation of D-fructose through direct electron transfer between the active center of FDH and the electrodes. As shown in Fig. 1A, the catalytic oxidation current began to increase at a potential around 100 mV (vs. Ag/ AgCl). This result agrees with the previous reports on the DET reaction of FDH at SAM-modified gold electrodes [12] and carbon-
Fig. 1. (A) Cyclic voltammograms of a FDH-modified bare and thiol-modified AuE at pH 5 in the presence of 200 mM D-fructose, at a scan rate of 10 mV s 1. (B) pH dependence of catalytic current at +500 mV obtained from cyclic voltammetry for FDH-modified MET-AuNP/AuE in the presence of 200 mM D-fructose.
based electrodes [7,8,13–15]. When the MET-modified electrode was used, a current density was the highest among several SAMmodified electrodes (Fig. 1A). The difference in the obtained current densities for several thiols should originate from the orientation and/or stability of FDH at the SAM surfaces. To examine the useable pH range of the electrode, an analysis of the pH dependence of catalytic currents for the FDH-immobilized MET-AuE, which was prepared in 100 mM acetate buffer solution at pH 5.0, was performed (Fig. 1B). The catalytic current was measured at +500 mV. The optimum pH was about 6. It was higher (about 1– 2 pH units) than those at edge-plane [14] and basal-plane [7] highly oriented pyrolytic graphite electrodes and that for enzymatic reaction in solution. It was reported that the FDH complex decomposes and its activity losses in neutral and alkaline solutions [7]. It is suggested that the adsorption of FDH to an MET-modified electrode might prevent the decomposition. 3.2. DET reaction at AuNP-modified electrodes An effective surface area of an AuNP/AuE was investigated by cyclic voltammetry measurement in 0.5 M H2SO4 [20]. The real surface area of the AuNP/AuE was determined to be 2.32 cm2 and about 65 times as large as that of the bare AuE (0.0356 cm2). Fig. 2A shows typical voltammetric curves obtained at the FDH adsorbed MET-AuNP/AuE in 100 mM acetate buffer solution (pH 5.0). The catalytic current was observed only when fructose dehydrogenase was adsorbed at the electrode. These results indicate
670
K. Murata et al. / Electrochemistry Communications 11 (2009) 668–671
Fig. 3. Cyclic voltammograms of a FDH-modified MET-AuNP/carbon fiber paper electrodes at pH 5 in the presence of 200 mM D-fructose in quiet solution (1) and with stirring at 800 rpm (2), and absence of D-fructose in quiet solution (3), at a scan rate of 10 mV s 1.
electrolysis of D-fructose. Fig. 3 shows the cyclic voltammogram of FDH at an MET-AuNP/CFP. The current density reached a value as high as about 14.3 ± 0.93 mA cm 2 (at +500 mV) in the presence of 200 mM D-fructose at room temperature. This is the highest value ever reported for a bioanode based on both DET-type [13] and mediated electron transfer-type [21] bioelectrocatalysis as far as we know. The catalytic current obtained was not influenced even when the solution was stirred by a magnetic stirring bar. This indicates that the mass transfer of D-fructose to the nanostructured electrode surface is sufficient, and that the total process for the bioelectrolysis of D-fructose was controlled by enzyme kinetics. Fig. 2. (A) Cyclic voltammograms of a FDH-modified (solid line) and -unmodified (dotted line) MET-AuNP/AuE at pH 5 in the presence of 200 mM D-fructose, at a scan rate of 10 mV s 1. The electrodes were immersed in a FDH solution for (a) 8, (b) 18, (c) 36, (d) 64, (e) 102, (f) 120 min. (B) pH dependence of catalytic current at +500 mV obtained from cyclic voltammetry for FDH-modified MET-AuNP/AuE in the presence of 200 mM D-fructose, at the first cycle (circle) and fifth cycle (triangle).
that FDH electrochemically catalyzed the oxidation of D-fructose through the direct electron transfer with AuNPs. The current density at +500 mV for an MPA-AuE and an MPA-AuNP/AuE were 0.17 ± 0.01 mA and 3.25 ± 0.18 mA, respectively. Therefore the current density for an MPA-AuNP/AuE was about 19 times higher than that for a polycrystalline gold electrode. The current density depended on the time for the immobilization of FDH. It reached a maximum value after about 2 h, although it takes only 20 min when using MET-AuE. The slow adsorption behavior for METAuNP/AuE could be explained due to the nanostructure of the AuNPs, as reported at Ketjen black [13]. Fig. 2B shows the pH dependence of catalytic currents of the FDH adsorbed at an MET-AuNP/AuE, which was prepared in a 100 mM acetate buffer solution at pH 5.0. At the first cycle, a high catalytic current density was observed over a broad pH range, although it gradually decreased with successive scans on neutral or slightly basic solutions, pH 7–8 (Fig. 2B). The oxidative activity of FDH buried into the nanostructure of AuNPs would be insusceptible to pH change. The voltammetric response at pH 5.0 remained at 92% of the original signal after the electrode was continuously potential scanned for 30 cycles (70 min). This indicated that the AuNP electrode had a good stability and reproducibility. To further increase effective surface area, AuNPs were immobilized into a carbon fiber paper (CFP). The CFP has a large surface area and thus could improve the current density based on a bio-
4. Conclusion We fabricated AuNP-mobilized electrodes by a simple method in order to investigate the DET reaction of FDH. A current density as high as 14.3 ± 0.93 mA cm 2 was achieved in the presence of 200 mM D-fructose by immobilization of FDH on MET-modified AuNP/carbon fiber paper electrodes. For the DET reaction of FDH, the AuNP-modified electrodes satisfy the following requirements: (i) a high density of FDH at the electrodes, (ii) a proper orientation of FDH at the electrode surface, and (iii) a sufficient mass transfer of D-fructose. Therefore, we can conclude that the AuNP-immobilized electrodes are promising for high performance biofuel cells, third generation biosensors, and bioreactors. It is useful to deposit AuNPs onto mesoporous carbon electrodes for increasing the effective surface area of electrodes. This methodology could be applied to other mesoporous electrodes due to the simplicity of the deposition. Acknowledgment K.M. acknowledges the financial support of the Japan Society for the Promotion of Science (Research Fellowship for Young Scientists). References [1] J.A. Cracknell, K.A. Vincent, F.A. Armstrong, Chem. Rev. 108 (2008) 2439. [2] L. Gorton, A. Lindgren, T. Larsson, F.D. Munteanu, T. Ruzgas, I. Gazaryan, Anal. Chim. Acta 400 (1999) 91. [3] S. Shleev, J. Tkac, A. Christenson, T. Ruzgas, A.I. Yaropolov, J.W. Whittaker, L. Gorton, Biosens. Bioelectron. 20 (2005) 2517. [4] J. Hirst, Biochim. Biophys. Acta 1757 (2006) 225. [5] M. Ameyama, E. Shinagawa, K. Matsushita, O. Adachi, J. Bacteriol. 145 (1981) 814.
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