Modification of an enzyme electrode by electrodeposition of hydroquinone for use as the anode of a glucose fuel cell

Applied Surface Science 258 (2012) 6321–6325

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Modification of an enzyme electrode by electrodeposition of hydroquinone for use as the anode of a glucose fuel cell Takashi Kuwahara, Hiraku Yamazaki, Mizuki Kondo, Masato Shimomura ∗ Department of Bioengineering, Faculty of Engineering, Nagaoka University of Technology, 1603-1, Kamitomioka-machi, Nagaoka 940-2188, Japan

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Article history: Received 23 January 2012 Received in revised form 1 March 2012 Accepted 7 March 2012 Available online 15 March 2012 Keywords: Biofuel cell Electrodeposition Electron transfer Glucose oxidase Hydroquinone

a b s t r a c t An electrode having immobilized glucose oxidase (GOx) was modified with polyhydroquinone (PHQ), which was employed as an electron-transferring mediator, by a simple electrochemical method and used as the anode of a glucose fuel cell. The GOx-immobilized electrode was fabricated by attaching polyallylamine (PAAm) and then GOx covalently onto a gold electrode covered with a monolayer formed with 3-mercaptopropionic acid. Subsequently, the GOx-immobilized electrode (GOx/PAAm electrode) was modified with PHQ by electrodeposition of hydroquinone. The cyclic voltammogram of the modified electrode (PHQ/GOx/PAAm electrode) in a phosphate buffer solution (0.10 M, pH 7.0) showed redox peaks due to the electrodeposited PHQ, whereas no redox peaks were found for the GOx/PAAm electrode in the buffer solution containing p-benzoquinone (BQ). The onset potential of glucose oxidation with the PHQ/GOx/PAAm electrode became ca. 0.2 V more negative than that observed with the GOx/PAAm electrode in the presence of BQ. The glucose fuel cell equipped with the PHQ/GOx/PAAm electrode as an anode gave a 3 times larger power output than the cell with the GOx/PAAm electrode using dissolved quinone as the mediator. © 2012 Elsevier B.V. All rights reserved.

1. Introduction Enzyme electrodes, which combine biocatalytic and electrochemical reactions, have been investigated extensively for the purpose of using them for biosensors and biofuel cells [1–5]. Especially, redox enzymes, such as glucose oxidase (GOx) [6–9] and alcohol dehydrogenase [10–12], have been employed frequently as components of the enzyme electrodes. Since enzymes have no electron-conducting property, it is difficult to transfer electrons directly between the enzymes and electrodes. From the viewpoint of overcoming this difficulty, an effort has been made to fabricate the enzyme electrodes with some additive compounds having a redox property to mediate the electron transfer [13–16]. It is well known that quinones [17–19] and metal complexes [3,20–22] effectively act as an electron-transferring mediator. In addition, various approaches have been attempted to immobilize these compounds on the enzyme electrode. As for the immobilizing technique, as well as for enzyme immobilization, adsorption [23,24], enclosing [25–27] and covalent binding [28–32] methods have been investigated. Of these methods, however, an optimum procedure has not yet been established and, therefore, it is still

∗ Corresponding author. Tel.: +81 258 47 9404; fax: +81 258 47 9404. E-mail address: [email protected] (M. Shimomura). 0169-4332/$ – see front matter © 2012 Elsevier B.V. All rights reserved. doi:10.1016/j.apsusc.2012.03.030

under investigation how to design the enzyme electrode incorporating the electron-transferring mediator. The electrodeposition technique has been adopted to polymerize thiophenes [33,34], pyrroles [35–37] and anilines [38,39] because of such advantages as simplicity and cost-effectiveness. The polymerization can be readily performed by applying an appropriate voltage on an electrode in a monomer solution, where the resulting polymer, in general, is deposited onto the electrode within a short time. Thus, the electrodeposition is a useful method of modifying electrodes. The electrodeposition of quinones is noteworthy because they are expected to function as the electron-transferring mediator mentioned above. For example, hydroquinone (HQ) which is of a reduced form of the simplest quinone has been electrodeposited, and the electrode modified with polyhydroquinone (PHQ) has been obtained [40]. In addition, the anthraquinone including an aniline structure [41,42] and the benzoquinone having specific functional groups [43,44] have been electrodeposited to give polymeric forms. The authors have been interested in the electrodeposition of quinones as the method of immobilizing the electron-transferring mediator on the enzyme electrodes. In the present study, a GOx-immobilized electrode was modified with PHQ by the electrodeposition of HQ. To begin with, the redox behavior of the PHQ on the electrode was investigated electrochemically. Then, glucose oxidation was examined with the modified electrode to clarify the role of PHQ in electron transfer between GOx and the electrode

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surface. The modified electrode was used as the anode of a glucose fuel cell, and the performance of the fuel cell was compared with that of the cell using dissolved p-benzoquinone (BQ) as the mediator. 2. Experimental 2.1. Reagents and materials GOx (EC 1.1.3.4, from Aspergillus species) was supplied by Toyobo Co., which had an activity of 162 U/mg. Peroxidase (POx, EC 1.11.1.7, from horseradish) was supplied by Sigma Chemical Co., which had an activity of 52 U/mg. 3-Mercaptopropionic acid (MPA), 1-cyclohexyl-3-(2-morpholinoethyl) carbodiimide methop-toluenesulfonate (CMC, used as a condensing reagent) and Nafion 112 (0.002 in. thick) were purchased from Aldrich Chemical Co. HQ and d-glucose were obtained from Wako Pure Chemical Ind. Polyallylamine (PAAm, molecular weight 60,000) and BQ were from Nitto Boseki Co. and Nacalai Tesque, Inc., respectively. All other chemicals were of analytical grade, which were used without further purification. 2.2. Electrode fabrication The GOx-immobilized electrode bearing PHQ was prepared through the following three steps: (1) Modification of a gold electrode with PAAm. A gold electrode deposited on an alumina plate (1.0 cm × 2.5 cm) was cleaned by immersion in a piranha solution (H2 SO4 /30% H2 O2 = 3/1) for 15 min at room temperature and rinsed with distilled water. Then the working area of the electrode was adjusted to 0.50 cm × 0.50 cm by masking with a Kapton film. The masked electrode was immersed in an ethanol solution of MPA (10 mM) for 30 min at room temperature, washed with distilled water to remove unbound MPA, and air-dried. Subsequently, the electrode (MPA electrode) was immersed in an aqueous solution containing PAAm (10 mg/mL) and CMC (10 mM) for 2 h, and then washed with distilled water. The electrode treated thus (PAAm electrode) was stored for the next step in a phosphate buffer solution (0.10 M, pH 7.0) at room temperature. (2) Immobilization of GOx on the PAAm electrode. The PAAm electrode was immersed in an aqueous solution containing GOx (5 mg/mL) and CMC (50 mM) for 24 h at 4 ◦ C. The electrode treated thus (GOx/PAAm electrode) was washed with distilled water and then stored for the next step in a phosphate buffer solution (0.10 M, pH 7.0) at 4 ◦ C. (3) Electrodeposition of HQ on the GOx/PAAm electrode. A thin layer of PHQ was formed on the GOx/PAAm electrode by electrodeposition of HQ by use of a potentiostat/galvanostat (Hokuto Denko Corp. HA-150G). The electrodeposition was continued by chronoamperometry at 2.0 V vs. a saturated calomel electrode (SCE) in a phosphate buffer solution (0.10 M, pH 7.0) containing HQ (10 mM) until the amount of passed charge reached to 1.0 C. The electrode modified thus with PHQ (PHQ/GOx/PAAm electrode) was washed with distilled water and stored in a phosphate buffer solution (0.10 M, pH 7.0). 2.3. Electrode characterization The quantities of the MPA, PAAm, GOx and PHQ on the PHQ/GOx/PAAm electrode were determined with a quartz crystal microbalance (QCM). AT-cut quartz crystal units (fundamental frequency 6 MHz) obtained from Hokuto Denko Corp. were used for the QCM measurement.

The activity of immobilized GOx was measured at 30 ◦ C by a colorimetric method according to the procedure of Trinder [45]. This method included the reaction of hydrogen peroxide, produced in oxidation of glucose by GOx, with phenol and 4-aminoantipyrine in the presence of POx to yield a colored product. The activity was determined from a standard curve based on the absorbance at 505 nm due to the colored product, which was measured on a Shimadzu UV-3100 PC spectrometer. The electrode surface was observed by scanning electron microscopy (SEM) on a JEOL JSM-6301F scanning microscope. The sample for SEM was coated with gold (10 nm thick) by ion sputtering. The infrared (IR) spectrum of the electrode surface was measured on a JEOL JIR-7000 FT-IR spectrometer. 2.4. Electrochemical measurements Electrochemical properties of the electrodes were investigated with a conventional three-electrode cell connected to the potentiostat/galvanostat equipped with an arbitrary function generator (Hokuto Denko Corp. HB-105A). A platinum plate and SCE were used as a counter electrode and reference electrode, respectively. In advance of the measurement, the electrolyte solution in the cell was purged thoroughly with N2 gas. All the measurements were conducted under N2 atmosphere at 25 ◦ C. A fundamental glucose fuel cell was constructed by use of the PHQ/GOx/PAAm electrode as an anode. The performance of the fuel cell was examined in a two-compartment cell, in which the compartments were separated by Nafion 112. Platinum black was used as a cathode, which had been activated by 10 cycles of potential sweep from −0.5 V to 0.5 V vs. SCE at a scan rate of 50 mV/s in a H2 SO4 solution (0.10 M) and then rinsed thoroughly with distilled water [46,47]. A phosphate buffer solution (0.10 M, pH 7.0) containing d-glucose (1.0 M) was placed in the anodic compartment and kept under N2 atmosphere during the examination. The cathodic compartment was filled with a phosphate buffer solution (0.10 M, pH 7.0) and kept under O2 atmosphere. The power output of the fuel cell was evaluated by measuring the currents at arbitrary potentials. 3. Results and discussion 3.1. Characteristics of the PHQ/GOx/PAAm electrode Fig. 1 shows the SEM image of the surface of the PHQ/GOx/PAAm electrode, together with that of the GOx/PAAm electrode. It can be seen in Fig. 1(a) that the GOx/PAAm electrode had a smooth surface similar to that of a bare gold electrode. In contrast, as seen in Fig. 1(b), the PHQ/GOx/PAAm electrode had a considerably porous surface due to the morphology of electrodeposited PHQ. The quantities of MPA, PAAm, GOx and PHQ on the PHQ/GOx/PAAm electrode were determined by the QCM method as shown in Table 1. The quantity of GOx was 1.75 ␮g (7.0 ␮g/cm2 ), which was 15 times as large as that estimated on the assumption that a monolayer of close-packed GOx molecules occupied the electrode area (0.25 cm2 ). Thus, GOx was immobilized on the electrode in considerably large quantity, and this result suggested the significant role of PAAm in GOx immobilization. In comparison, GOx Table 1 Quantities of MPA, PAAm, GOx and PHQ on the PHQ/GOx/PAAm electrode. Component

[Function]

Quantity (␮g)

MPA PAAm GOx PHQ

[Linker between Au and PAAm] [Linker to immobilize GOx] [Biocatalyst for glucose oxidation] [Electron-transferring mediator]

0.02 0.29 1.75 0.85

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As shown in Fig. 2(b), little decrease in GOx activity was observed by applying 2.0 V vs. SCE until the passed charge reached to 1.0 C, which was the same condition as for the electrodeposition of HQ. This result suggests that MPA, PAAm and GOx on the electrode were neither eliminated nor denatured in the course of the electrodeposition of HQ. Therefore, the decreased activity of GOx on the PHQ/GOx/PAAm electrode can be attributed to a hindrance of the GOx reaction by the deposited PHQ in spite of its porous structure.

0.15

2

Current (mA/cm )

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35

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Fig. 1. SEM images of the surfaces of GOx/PAAm (a) and PHQ/GOx/PAAm (b) electrodes.

immobilization was attempted with the MPA electrode without PAAm, giving the result that the quantity of the immobilized GOx was only 0.13 ␮g (0.5 ␮g/cm2 ). The activities of GOx on the GOx/PAAm and PHQ/GOx/PAAm electrodes are compared in Fig. 2. It can be seen that the latter was 20 mU (11 U/mg), which corresponded to 57% of the former.

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20 15 10

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5

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0 a

b

c

Fig. 2. Activity of GOx on GOx/PAAm (a and b) and PHQ/GOx/PAAm (c) electrodes. The activity (b) was measured after applying 2.0 V vs. SCE to the GOx/PAAm electrode until the passed charge reached to 1.0 C.

-0.4

-0.2 0 0.2 Applied potential (V vs. SCE)

0.4

Fig. 3. Cyclic voltammograms measured with gold (a), PAAm (b), GOx/PAAm (c) and PHQ/GOx/PAAm (d) electrodes at a scan rate of 20 mV/s in a phosphate buffer solution (0.10 M, pH 7.0). The data (a), (b) and (c) were obtained by adding BQ (1.0 mM) to the buffer solution.

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3

b

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Transmittance

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3.2. Electrochemical properties of the PHQ/GOx/PAAm electrode Fig. 3 shows the cyclic voltammograms measured with the gold, PAAm, GOx/PAAm and PHQ/GOx/PAAm electrodes in a phosphate buffer solution of pH 7.0. In the case of the measurements with the gold, PAAm or GOx/PAAm electrodes, 1.0 mM of BQ was added into the buffer solution. Since the PHQ/GOx/PAAm electrode gave the peaks of redox current at around 0 V vs. SCE as shown in Fig. 3(d), the electrochemical redox activity of quinone/hydroquinone was kept even in the PHQ electrodeposited on the GOx/PAAm electrode. Because of the broad redox peaks, the PHQ seems to have a complicated chemical structure. As suggested from the IR spectrum in Fig. 4, it is likely that the oxygen atoms in the PHQ is present in the form of ether bonds, as well as hydroxyl and carbonyl groups, though it has been considered that ether bonds is not formed by electrodeposition of HQ [44]. Thus, the PHQ on the PHQ/GOx/PAAm electrode is not of a simple structure like polyphenylene but contains ether linkages between phenyl rings. On the other hand, as shown in Fig. 3(b), the PAAm electrode gave an oxidation current at a higher potential and a reduction current at a lower 0.3

potential than the case of the PHQ/GOx/PAAm electrode. In addition, no redox peaks due to the dissolved BQ were observed with the GOx/PAAm electrode whereas the redox peaks were observed clearly with the MPA electrode, as well as a bare gold electrode. These results suggest that PAAm and GOx on the GOx/PAAm electrode prevent BQ from diffusing to the gold layer of the electrode. In contrast, the electrodeposited PHQ may have located close to the gold layer to be involved in the electrochemical redox reaction with ease. Fig. 5 shows the relation between glucose oxidation current and applied potential for the GOx/PAAm and PHQ/GOx/PAAm electrodes in a phosphate buffer solution of pH 7.0. In the case of the measurement with the GOx/PAAm electrode, 1.0 mM of BQ was added into the buffer solution. It was clearly found that the onset potential of glucose oxidation with the PHQ/GOx/PAAm electrode was ca. 0.2 V more negative than that observed with the GOx/PAAm electrode. The difference in the onset potential can be related to the redox behavior of the electrodeposited PHQ and dissolved BQ, which will affect the open circuit voltage (Voc ) of the glucose fuel cells using the enzyme electrodes as anodes. Therefore, the PHQ/GOx/PAAm electrode was expected as a promising candidate for the anode of the glucose fuel cell.

b

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3.3. Performance of the glucose fuel cell equipped with the PHQ/GOx/PAAm electrode as an anode

2

Current (mA/cm )

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Fig. 6. Relation between power output and cell voltage for glucose fuel cells using GOx/PAAm (a) and PHQ/GOx/PAAm (b) electrodes as anodes in a phosphate buffer solution (0.10 M, pH 7.0) containing d-glucose (1.0 M). The data for the cell using GOx/PAAm electrode (b) was obtained by adding BQ (1.0 mM) to the buffer solution.

Fig. 4. IR spectrum of the PHQ/GOx/PAAm electrode surface.

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Vcell ( V )

500

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Applied potential (V vs. SCE) Fig. 5. Relation between glucose oxidation current and applied potential at a scan rate of 10 mV/s for GOx/PAAm (a) and PHQ/GOx/PAAm (b) electrodes in a phosphate buffer solution (0.10 M, pH 7.0) containing d-glucose (1.0 M). The data for the GOx/PAAm electrode (a) was obtained by adding BQ (1.0 mM) to the buffer solution.

A glucose fuel cell was constructed by use of the PHQ/GOx/PAAm electrode as an anode, and the performance of the fuel cell was compared with that of the cell using the GOx/PAAm electrode alternatively in the presence of 1.0 mM of BQ. To our knowledge, no investigation had ever been conducted into the performance of the glucose fuel cell equipped with the GOx-immobilized anode bearing PHQ as an electron-transferring mediator. Fig. 6 shows the relation between power output (Pcell ) and cell voltage (Vcell ) of the Table 2 Performance of glucose fuel cells using the GOx/PAAm and PHQ/GOx/PAAm electrodes as anodes. Anode used

Voc (V)

Isc (␮A/cm2 )

Pmax (␮W/cm2 )

GOx/PAAm electrode PHQ/GOx/PAAm electrode

0.22 0.37

58 76

1.0 2.7

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fuel cells, in which it was seen that each fuel cell gave a power output due to glucose in the anodic compartment. As expected, the glucose fuel cell using the PHQ/GOx/PAAm electrode generated a higher power than that using the GOx/PAAm electrode in the presence of dissolved BQ. The maximum power output (Pmax ) by the fuel cell using the former electrode was ca. 3 times as large as that by the cell using the latter. It can be seen from Table 2 that both the short circuit current (Isc ) and the open circuit voltage (Voc ) of the fuel cell using the PHQ/GOx/PAAm electrode exceeded those of the cell using the GOx/PAAm electrode. Thus, the electrodeposition of HQ brought about a marked performance of the GOX/PAAm electrode as the anode of the fuel cell. 4. Conclusions A novel GOx-immobilized electrode bearing PHQ was fabricated with the aid of electrodeposition. The cyclic voltammogram of the GOx-immobilized electrode showed redox peaks due to the electrodeposited PHQ. It was confirmed through the glucose oxidation with the GOx-immobilized electrode that the PHQ well functioned as an electron-transferring mediator which combined the biocatalytic reaction of immobilized GOx and the electrochemical reaction on the electrode surface. A glucose fuel cell was constructed by use of the GOx-immobilized electrode as an anode to give the Voc of 0.37 V and the Pmax of 2.7 ␮W/cm2 -anode. The present study demonstrates that the electrodeposition is a simple and effective method of modifying enzyme electrodes with an electron-transferring mediator. A number of quinone derivatives are known to participate in redox reactions in vivo and available for use as the mediator. Therefore, the performance of enzyme electrodes would be achieved by use of the quinone having an appropriate redox potential and by increasing the rate of the reaction between the quinone and the enzyme on the electrodes. Acknowledgment This work was partly supported by a Grant-in-Aid for Scientific Research (B) (No. 23360302) from the Ministry of Education, Culture, Sport, Science and Technology of Japan, which is gratefully acknowledged. References [1] M. Zayats, B. Willner, I. Willner, Electroanalysis 20 (2008) 583–601. [2] S. Tsujimura, M. Fujita, H. Tatsumi, K. Kano, T. Ikeda, Phys. Chem. Chem. Phys. 3 (2001) 1331–1335. [3] N. Mano, F. Mao, A. Heller, J. Am. Chem. Soc. 125 (2003) 6588–6594. [4] S. Topcagic, S.D. Minteer, Electrochim. Acta 51 (2006) 2168–2172. [5] T. Kuwahara, K. Oshima, M. Shimomura, S. Miyauchi, Synth. Met. 152 (2005) 29–32.

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