Amphiphilic Polymer Mediators Promoting Electron Transfer on

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Amphiphilic Polymer Mediators Promoting Electron Transfer on Bioanodes with PQQ-Dependent Glucose Dehydrogenase Yasuo Nakashima, Norihiro Mizoshita, Hiromitsu Tanaka, and Yuichiro Nakaoki Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.6b03145 • Publication Date (Web): 13 Nov 2016 Downloaded from http://pubs.acs.org on November 19, 2016

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Amphiphilic Polymer Mediators Promoting Electron Transfer on Bioanodes with PQQ-Dependent Glucose Dehydrogenase Yasuo Nakashima,†,§ Norihiro Mizoshita,*,‡,§ Hiromitsu Tanaka,‡ and Yuichiro Nakaoki*,† †Aisin Cosmos R&D Co., Ltd., Kisarazu, Chiba 292-0818, Japan. ‡Toyota Central R&D Labs., Inc., Nagakute, Aichi 480-1192, Japan.

ABSTRACT: Redox-active phenazinium salts bonded to amphiphilic polymer backbones are demonstrated to function as high-performance electron-transfer mediators in enzymatic bioanodes applicable to biofuel cells. The redox-active moieties could be easily tethered to the electrodes by physical adsorption of the hydrophobic regions of the polymer backbones onto the electrode surface. On the other hand, long hydrophilic chains were essential to ensure high mobility of the redox-active moieties in aqueous solutions and to enhance their electron-transfer properties. We found that an amphiphilic mediator with a linear polymer backbone exhibited stable adsorption behavior onto the electrode surface and generated high bioelectrocatalytic current (>1.8 ± 0.32 mA/cm2) in the presence of PQQ-dependent glucose dehydrogenase and an

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aqueous solution of glucose fuel. This current was more than two times higher than that of an electrode treated with a low molecular weight phenazinium salt. Moreover, the bioelectrode modified with the polymer mediator retained the high electrocatalytic current after 10 exchanges of the glucose fuel. The mediator-modified bioelectrodes are expected to be useful for various bio-related energy and electronic devices.


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INTRODUCTION Recently, a variety of electronic devices based on organic electroactive materials have been developed utilizing merits of organic materials such as diversity and tunability of molecular structures, flexibility, and high processibility. In these devices, enhancement of charge transfer and injection at electrode surfaces is the key to exhibit significant performances.1 Electronic devices using biomolecules such as enzymes and DNA have also been fabricated for biochemical sensing and energy production.2 In this case, formation of efficient charge transfer paths around the electrode surfaces is needed because large amounts of insulating organic frameworks are necessarily involved in the bio-related materials. Biofuel cells that generate electricity through biological reactions have great potential as energy devices due to the high abundance of biomass fuels as well as their numerous advantages. In particular, biofuel cells are very safe and environmentally benign, can be operated under moderate conditions, are applicable to implantable medical devices, and can be down-scaled easily.3–6 In general, the oxidation of renewable energy sources (sugars, alcohols, etc.) at an anode is coupled with the reduction of oxygen at a cathode by using enzymes or microorganisms as electrode catalysts. The performance of bioelectrodes used in biofuel cells has steadily improved with the development and application of new materials including porous carbon electrodes,7–11 conductive auxiliaries,12–18 functionalized mediators,19–23 and immobilized enzymes.24–27 In enzymatic biofuel cells, the current density depends strongly on electrontransfer processes between enzymatic reaction sites and electrode surfaces via redox-active mediators. Redox-active low molecular weight compounds, such as NADH, ferrocenes, and naphthoquinone derivatives, are incorporated into electrode as a highly diffusive mediator,


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resulting in the current densities of 0.01 to 1 mA/cm2 order. However, it is difficult to apply them to long-term uses because of the elimination from electrode surfaces. Therefore, rational design of electron-transfer mediators is of great importance for enhancing and stabilizing the bioelectrocatalytic current.3,19 Various approaches have been used to facilitate mediated electron-transfer processes in bioelectrodes.28–32 Willner et al. reported electrical wiring in enzyme electrodes by reconstitution of glucose oxidase onto an Au electrode modified with a monolayer of cofactors, which resulted in efficient electrical contact with the electrode and effective stimulation of the bioelectrocatalyzed oxidation of glucose.28,29 Hatazawa and Kano et al. immobilized enzymes and mediators onto carbon electrodes with a polyion complex in order to retain their biological and electrochemical activities.30 Yamaguchi et al. reported immobilization of hydroquinone on carbon black via a polymer grafting method for application in a high-surface-area biofuel cell electrode.31 Heller et al. enhanced electron transport by using a redox hydrogel having long tethers between the redox centers and polymer backbones.32 To achieve efficient electron transfer in bioelectrodes, immobilization of mediators onto electrode surfaces should be made compatible with high accessibility of the mediators to the active sites of the enzymes. Immobilization of catalytic systems onto electrodes is also essential for ensuring reusability of the electrodes in practical applications. Here, we report enhancement of the electron-transfer efficiency in bioanodes by introducing amphiphilic polymer backbones to a redox-active mediator compound. A schematic diagram of our proposed bioanode is presented in Figure 1. The molecular-scale thin coating with the mediators is designed to facilitate electron transfer from the substrate/enzyme complex to the electrode at the proximity of the electrode surface. The electrode is equipped with a


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bioelectrocatalyst based on a PQQ-dependent glucose dehydrogenase (PQQ-GDH), which is often used in biosensors and biofuel cells.24,27,33,34 Our strategy is to impart the amphiphilic mediators tethered to the electrodes with high electron-transfer activities by giving different roles to the hydrophobic and hydrophilic chains of the backbones. The hydrophobic chain is expected to stably adsorb onto the hydrophobic surfaces of the carbon-based electrodes, which tether redox-active mediator moieties in the proximity of the electrode surface. The immobilization of mediators by physical adsorption also allows for simple preparation of the bioelectrodes and easy control over the mediator density. In contrast, the hydrophilic flexible chain is mobile in aqueous solutions; therefore, the mediator moieties can exhibit high mobility, including shuttling motion between the electrode surfaces and the active sites of the enzymes, despite being tethered to the electrode surfaces. In this work, we demonstrate that properly designed amphiphilic polymer mediators containing redox-active phenazinium salt moieties (Figure 2) can be stably fixed onto electrode surfaces by physical adsorption, and that they exhibit high electron-transfer activities. A low molecular weight phenazinium salt, 1-methoxy-5-methylphenazinium methyl sulfate (mPMS), is known as a redox-active compound applicable to various electron-transfer systems.35–37 In the present GDH-based electrodes, mPMS is preliminarily confirmed to exhibit higher electron-transfer activity than conventional low molecular weight mediators such as 1,4-naphthoquinone and vitamin K. In order to examine the roles of the polymer backbones, we prepared phenazinium salt-containing polymers with linear (LP-1 and LP-2) and branched (BP-1) backbones having polyethylene glycol (PEG) chains as hydrophilic and flexible linkers. The amphiphilic polymer mediators are shown to work efficiently at a low concentration, and the bioelectrode modified with LP-1 retains high electrocatalytic currents even after ten fuel exchanges.


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EXPERIMENTAL SECTION Materials and methods. All reagents and solvents for synthesis of polymer mediators were purchased from Aldrich, Tokyo Chemical Industry, and Gelest, and used without further purification. Low molecular weight phenazinium salt, mPMS, was purchased from Dojindo Laboratories. The redox potential of mPMS was –0.11 V (vs. Ag|AgCl). Coenzyme pyrroloquinoline quinone, PQQ, was purchased from Sigma-Aldrich. The redox potential of PQQ was –0.21 V (vs. Ag|AgCl). The enzyme was prepared from recombinant E.coli, for comparing with mutants in further study. The 20 µM holo-PQQ-GDH was reconstituted in a reconstitution solution containing 1 mM CaCl2 and 55 µM PQQ for 30 min on an ice bath before measuring the catalytic current. The carbon cloth electrode was purchased from Tsukuba Materials Information Laboratory, Ltd. The surface area of the carbon cloth was estimated to be 0.1–0.2 m2/g. 1H NMR spectra were measured using a Jeol JNM-ECX400P spectrometer, UVvis absorption spectra were recorded using a Jasco V-670 spectrometer or a Beckman Coulter DU800 spectrometer, and cyclic voltammograms were measured using a BAS ALS/H CH Instruments Model 600C electrochemical analyzer. Construction of the expression plasmid for the PQQ-GDH gene. The PQQ-GDH expression vector was constructed by amplifying the PQQ-GDH structural gene and inserting it into pET22b (Novagen). Genomic DNA from A. calcoaceticus (NBRC; 12552) was prepared using genomic DNA purification kits (Promega) according to the manufacturer’s instructions. Recombinant DNA techniques and expression of the recombinant gene were performed in E. coli DH5α and BL21(DE3), respectively. The complete A. calcoaceticus PQQ-GDH gene, which is


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approximately 1.4-kb in length (Gene ID; X15871), was cloned via PCR-amplification using the following primers (restriction sites used for cloning are underlined and the restriction enzymes are indicated in parentheses): sense, 5′-GAGCATATGAATAAACATTTATTGGC-3′ (NdeI) and antisense, 5′-CTAGGATCCTTAGTGGTGGTGGTGGTGGTGCGC-3′(BamH I). Overexpression of recombinant enzyme. Culture of E. coli BL21(DE3) cells harboring pET22b(+)-GDH (40 mL) was grown in LB medium containing ampicillin with shaking at 37 °C until the optical density at 600 nm reached 0.1. Expression of the PQQ-GDH was then induced by adding 0.01 mM isopropyl-β-D-thiogalactopyranoside into the medium and further incubating at 28 °C overnight. The cells were collected by centrifugation and stored at –20 °C for subsequent purification. Purification of recombinant enzyme. The frozen cells (5 g) were resuspended in 15 mL of phosphate buffered saline (PBS) and disrupted by sonication on ice. Cell debris was removed by centrifugation at 7855 × g, 4 °C, for 10 min. The supernatant, which contains recombinant PQQGDH, was filtered using a cellulose acetate 0.45 µm filter (ADVANTEC) and loaded onto a TALON (Clontech) column equilibrated with PBS (Bio-Rad) containing 50 mM NaCl. The column was washed with washing buffer (PBS (Bio-Rad) containing 50 mM NaCl, 10 mM imidazole). Proteins were eluted with elution buffer (PBS (Bio-Rad) containing 50 mM NaCl, 150 mM imidazole). After purification, the buffer in the eluted sample was replaced by 10 mM Tris HCl pH 7.5 and 0.1 mM CaCl2 using PD-10 (GE Healthcare). Synthesis of polymer mediators. Polymer mediators with amphiphilic polymer backbones were synthesized by modifying hydroxyl terminal groups of nonionic polymer surfactants. The synthesis of amphiphilic polymer mediators is outlined in Scheme 1. Commercially available


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amphiphilic polymers BrijS10 (Aldrich; Mn ~711), BrijS2 (Aldrich; main component: diethylene glycol octadecyl ether), and CMS-221 (Gelest; molecular weight ~4000, non-siloxane: 20–25 wt%) were used for the synthesis of LP-1, LP-2, and BP-1, respectively. The terminal hydroxyl groups of the hydrophilic moieties were transformed into 1-phenazinyl ether via tosylated compounds. Then, the 5-position of the phenazine ring was methylated using methyl trifluoromethanesulfonate. Reference polymer SCP-1 was synthesized by conventional radical polymerization of a corresponding methacrylate of 1-alkoxyphenazine, followed by the methylation of the phenazine moiety. Experimental details are provided in the Supporting Information. Evaluation of adsorption behavior of mediators onto electrodes. The mediator compounds (1.0 mg) diluted in acetonitrile (10 mg/mL) were deposited onto the carbon cloth electrode (3 cm2, ~ 87 mg). The solvent was removed by drying in vacuum for more than 20 min. The carbon electrodes incorporating the mediators were repeatedly washed with a 1 M phosphate buffer solution by vortexing for 1 min. The UV-vis absorbance of the washing solutions was measured to determine the amounts of the desorbed mediators, and the remaining rates of the mediators were estimated from the desorption rates. Electrochemical measurements. The carbon cloth electrode (electrode size: 0.5 cm2, ~ 14.5 mg) incorporating 26 nmol mediators were electrochemically characterized using cyclic voltammetry (CV). Ag|AgCl (saturated NaCl) and platinum were used as the reference electrode and the counter electrode, respectively. The CV measurements of the mediator-modified electrodes were performed in 1 M phosphate buffer solution at pH 7.0. For the measurements of catalytic currents, the mediator-modified electrodes were treated with 0.52 nmol holo-PQQ-GDH and the CV measurements were performed in 1 M phosphate buffer solution at pH 7.0 in the


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presence of 0–200 mM glucose. All of the CV measurements were carried out at room temperature. Cyclic voltammograms at the second cycle were used for evaluations unless otherwise stated.

RESULTS AND DISCUSSION Synthesis of polymer mediators. Electron-transfer mediators with amphiphilic polymer backbones LP-1, LP-2, and BP-1 were synthesized by modifying terminal hydroxyl groups of commercially available amphiphilic polymers. Scheme 1 shows the outline of the synthesis of the amphiphilic polymer mediators with a hydrophobic R group. The terminal hydroxyl groups were transformed into p-toluenesulfonate with tosyl chloride (TsCl). The tosylated compounds were then reacted with 1-hydroxyphenazine. The 5-position of the phenazine ring was methylated with methyl trifluoromethanesulfonate (TfOMe) to afford the products. LP-1 and BP1 with PEG linkers were obtained as a paste-like dark red solid, while LP-2 with a short oxyethylene chain was obtained as a dark red powder. Side chain polymer SCP-1 with numerous phenazinium salt moieties was also prepared as a reference polymer having no hydrophilic chains. SCP-1 was synthesized by conventional radical polymerization of a corresponding methacrylate of 1-alkoxyphenazine, followed by the methylation of the phenazine moiety. The polymer mediators exhibited two UV-vis absorption bands at 512 and 384 nm in acetonitrile solutions, which is similar to those exhibited in a solution of mPMS in acetonitrile. Immobilization of mediators. Immobilization of the mediators onto the carbon electrodes was performed by physical adsorption through hydrophobic interactions. The mediators were deposited onto the electrodes as solutions in acetonitrile, and the solvent was moderately


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removed. Considering the effective surface area of the carbon cloth, the deposited mediators (1.0 mg mediators for an 87 mg electrode) can fully cover the hydrophobic carbon surface (Supporting Information, Figure S1). The carbon electrodes incorporating the mediators were repeatedly washed with a 1 M phosphate buffer solution to examine the stability of the physical adsorption of the mediators. Figure 3a shows the remaining rates of mPMS and LP-1 after washing treatments and photographs of the washing solutions. The low molecular weight mediator mPMS was easily eluted into the buffer solution by repeated washing, and the remaining rate was less than 40% after washing five times. In contrast, only slight coloration of the washing solutions was observed for LP-1, and the remaining rate was more than 90% after 10 washes. These results indicate that the hydrophobic chain of the polymer backbone of LP-1 promotes the stable physical adsorption of LP-1 onto the carbon electrode surface. The remaining rates of the mediators after five washes are compared in Figure 3b. The polymer mediators exhibited higher remaining rates than mPMS. In particular, linear polymer mediators LP-1 and LP-2, whose hydrophobic region consists of a long alkyl chain, showed stable adsorption behavior, and their remaining rates were more than 90%. LP-2, which has a short oxyethylene chain, showed the highest remaining rate probably due to its high hydrophobicity. It should be noted that LP-1 can be stably fixed onto the electrode despite being soluble in water due to its long PEG linker. Once the hydrophobic interaction between the carbon electrode surface and the long alkyl chain became valid, the physically adsorbed state was shown to be stably retained in aqueous systems. Branched polymer mediator BP-1 also has a large hydrophobic region consisting of polydimethylsiloxane; however, the remaining rate after the washing was lower than those of the linear mediators. This is probably because the hydrophilic and hydrophobic moieties of BP-1 are not separated spatially into two blocks. Stable


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immobilization of mediators through non-covalent interactions is advantageous for simple preparation of modified electrodes and easy control over the mediator density on the electrode surfaces. Electrochemical properties and electron-transfer activities. The polymer mediators immobilized onto the electrode exhibited reversible electrochemical responses for the voltage scans in a 1 M phosphate buffer solution. Figure 4 shows the cyclic voltammograms of mPMS and the polymer mediators incorporated onto the carbon electrode. The cyclic voltammogram of the low molecular weight mediator mPMS was sharp and reversible with a small potential difference (0.03 V) between the oxidation and reduction processes at around –0.1 V (vs. Ag|AgCl). The polymer mediators also exhibited reversible oxidation and reduction currents for the voltage scans. These results indicate that electron transfer between the mediator moieties and the carbon electrode takes place sufficiently even when the hydrophobic regions of the polymer backbones are adsorbed onto the electrode surface. However, the cyclic voltammograms of the polymer mediators were broader than that of mPMS. For LP-1 having a long oxyethylene chain, the potential difference between the oxidation and reduction processes was maintained to be 0.03 V although the peak broadening occurred. LP-2, which has a short oxyethylene moiety and a long alkyl chain, exhibited broad and slightly weak electrical responses. A large difference between the anodic and cathodic peak potentials was observed for BP-1 (0.15 V) and SCP-1 (0.14 V). Such behavior is probably due to the kinetic restriction of the electron transfer by the large hydrophobic backbones. The mediator compounds including mPMS also exhibited small peaks at around –0.3 V. These peaks are attributable to a small amount of phenazines formed by demethylation of the 5-methylphenazinium salts.


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The redox potentials of the polymer mediators were positively shifted relative to that of mPMS. The redox potentials estimated from the midpoints of the anodic and cathodic peak potentials are listed in Table 1. The redox potential of mPMS was –0.11 V, and those of the amphiphilic polymer mediators were higher by ~ 0.1 V. The shift of the potential is probably due to the aggregation of the phenazinium salt moieties concentrated on the electrode surface, resulting in the lowering of the LUMO level. This was supported by a slight red shift of the UV-vis absorption bands due to the formation of thin films of the polymer mediators (Supporting Information, Figure S2). We also found that the positive potential shift was reduced by the dilution of the polymer mediators with anionic surfactants (Supporting Information, Table S1), which suggests that the aggregation of the mediator moieties caused the shifts in their redox potentials. In future work, the positive potential shift should be suppressed because it can lead to a decrease in the operational voltage of a biofuel cell. The bioanodes modified with the amphiphilic polymer mediators and PQQ-GDH exhibited high bioelectrocatalytic currents due to the oxidation of glucose. Figure 5a–e shows the cyclic voltammograms of the carbon electrodes incorporating PQQ-GDH and the mediators in the presence of glucose (50 mM). The catalytic currents of the electrodes modified with LP-1 and BP-1 having long PEG chains were much higher than that of mPMS. On the other hand, incorporation of LP-2 and SCP-1 in the electrode led to the decrease in the catalytic currents. Table 1 gives the catalytic currents obtained at a potential of 0.4 V (vs. Ag|AgCl). The catalytic current of LP-1 was 0.90 ± 0.16 mA (apparent current density: 1.8 ± 0.32 mA/cm2), which is more than two times higher than that of mPMS (0.38 ± 0.07 mA). The use of BP-1 was also effective for enhancing the catalytic currents (0.50 ± 0.12 mA). In contrast, the catalytic current for LP-2 with a short oxyethylene chain was 0.11 ± 0.02 mA. These results indicate that


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amphiphilic polymer mediators LP-1 and BP-1 with long PEG chains can significantly facilitate electron-transfer processes from the active site of the enzyme to the electrode surface. The current density for LP-1 was comparable to the highest current density of recent studies based on PQQ-GDH27,38 although higher current densities have been achieved by using other enzymatic reaction systems such as FAD-dependent glucose dehydrogenase.39 Figure 5f shows the relationships between glucose concentration and catalytic currents for the bioanode incorporating mPMS or LP-1. The catalytic currents are saturated at glucose concentrations of 50–100 mM. The catalytic current for LP-1 is higher than that for mPMS over a wide range of glucose concentrations. In order to examine the enzyme activity and the working of the mediators, the apparent Michaelis–Menten constant (Km,app) was estimated from the catalytic currents at various glucose concentrations using the Hanes–Woolf plots (Supporting Information, Figure S3). The Km,app values for mPMS and LP-1 were calculated to be 15.6 and 16.7 mM, respectively, indicating that the nature of the PQQ-GDH was almost maintained in the presence of LP-1. On the other hand, the maximum catalytic current calculated for LP-1 (1.76 mA) was 3.6 times higher than that for mPMS (0.49 mA). These results verify that the enhancement of catalytic currents by the application of LP-1 is due to not the difference in the diffusion behavior of glucose but the high electron-transfer activity of the amphiphilic mediator at the electrode surface. Although the low molecular weight mediator mPMS is highly diffusive, the localization of the redox-active moieties of LP-1 and BP-1 near the electrode surface due to being tethered with the polymer backbones is thought to be more effective for enhancing the catalytic currents. We also confirmed that the bioelectrocatalytic current for an electrode treated with a 30-fold higher amount of mPMS, most of which dissolved into the aqueous phase, was ~ 0.7 mA. This value was still lower than that of the electrode incorporating a small amount of LP-1. The


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bioelectrocatalytic currents induced by the incorporation of LP-1 (~50 µg/cm2) are comparable to those reported for bioanodes immobilizing organic and organometallic mediators (100–130 µg/cm2).31,32 Figure 6 schematically illustrates plausible electron-transfer behavior in anodes modified with the polymer mediators. Amphiphilic mediators LP-1 and BP-1, which have long PEG chains, exhibited efficient electron transfer from the enzyme to the electrode surface. The redox-active phenazinium salt moieties are highly mobile in the aqueous solutions; therefore, the mediators can transfer electrons via a shuttling motion, as illustrated in Figure 1. The length of the PEG chain consisting of ~ 10 repeating units is ~ 3 nm, whereas the diameter of the PQQ-GDH is estimated to be ~ 5 nm.40–42 Considering the size of the polymer mediators including hydrophobic regions, the size matching of the mediators and the enzyme contributes to the induction of the efficient electron transfer (Figure 6a). The hydrophobic regions anchor the mediators onto the electrode surface. LP-1 has a hydrophobic–hydrophilic diblock structure, whereas BP-1 is branched with two or three hydrophilic PEG chains on the hydrophobic main chain, which may slightly weaken the physical adsorption onto the electrode, as mentioned above. In contrast, LP-2, which has a short oxyethylene chain (~ 0.6 nm) and SCP-1, which has no long hydrophilic chains, exhibited low bioelectrocatalytic currents. Electron transfer between the reaction site of the enzyme and the mediators is thought to be inhibited due to the shortage of the linker length, although electron transfer between the mediators and the electrode surface occurs as was confirmed by the measured cyclic voltammograms (Figure 6b). The phenazinium moieties of LP-2 and SCP-1 are probably immobile after adsorption onto the electrode and not accessible to the reaction center of the enzyme. For the previously reported redox-active polymers, charge hopping and/or collisional electron transfer between redox centers have been


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proposed, which has been confirmed by evaluating their apparent electron diffusion coefficients.32,43,44 In contrast, such charge exchanges between redox sites would be inactive for the polymers with phenazinium salt moieties. For example, when a 30-fold amount of LP-1 was loaded onto the carbon electrode, the bioelectrocatalytic current was less than half of that shown in Table 1. We also preliminarily synthesized several polymethacrylates and polysiloxanes having phenazinium salts on all of the repeating units in order to increase the density of redoxactive sites. However, they showed weak electrochemical responses in cyclic voltammetry. Therefore, the amphiphilic polymer mediators with phenazinium moieties should be used as molecular-scale thin layer coating of the electrode. The proper design of the mediators with amphiphilic polymer backbones led to the induction of both high electron-transfer activities and stable adsorption onto the hydrophobic surfaces of the electrodes. Durability for fuel exchange. With applications of the polymer mediators to an anode of biofuel cells in mind, we examined the durability of the carbon electrode modified with LP-1 for the exchange of the buffer solutions containing glucose fuel. In practical applications of biofuel cells, it is desirable to reuse the cells by refilling the fuel; therefore, a mediated electron-transfer system localized onto the electrode surface is required for operation after the fuel exchange. Figure 7 plots the bioelectrocatalytic currents of the carbon electrode modified with LP-1 or mPMS for the number of fuel exchanges. The glucose solution was replaced with a fresh one every 10 min. For the electrode modified with mPMS, the catalytic current gradually decreased with subsequent fuel exchanges. After 10 exchanges, the current decreased to less than a tenth of its initial value. This is probably due to the gradual elution of the mediators into the buffer solution and deactivation of the mediator incorporated in the carbon material. In contrast, the electrode modified with LP-1 maintained high catalytic currents of ~ 1 mA after the ten fuel


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exchanges, although the current fluctuated somewhat from wash to wash. This result verifies the reusability of the mediator moieties tethered to the electrode surface via the amphiphilic polymer backbone. Such bioelectrodes are promising for applications in practical biofuel cells. On the other hand, the chemical stability of the present phenazinium salt-based mediators is insufficient for long-term operation tests, because demethylation of the 5-methyl group in the phenazinium moiety gradually takes place under neutral and higher pH conditions45 or under continuous electrical stimulation over several hours. We confirmed by NMR measurements that the 5methyl group of LP-1 adsorbed onto a carbon electrode was eliminated after continuous voltage scans for 4 hours. Enhancement of the chemical stability of the redox active moiety is underway.

CONCLUSIONS In this study, we presented a successfully designed surface-modified bioanodes using electrontransfer mediators with amphiphilic polymer backbones. The hydrophobic and hydrophilic regions of the backbones played important roles in the stable adsorption onto the carbon electrode and the maintenance of the high mobility of the redox-active moiety, respectively. Immobilization of the amphiphilic mediators onto carbon electrodes led to the induction of high bioelectrocatalytic currents of over 1.8 ± 0.32 mA/cm2. The bioelectrodes were also shown to be reusable for at least ten cycles. These bioelectrocatalytic performances are comparable to those for previously reported bioelectrodes with immobilized mediators; therefore, further enhancement of bioelectrocatalytic currents with long-term stability can be expected by applying the present surface design using amphiphilic mediators to high surface area electrodes such as mesoporous carbons and carbon nanotube assemblies. The present molecular design of the


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electron-transfer mediators may be applicable to other redox-active compounds. The design of the electrodes using amphiphilic mediators should also be useful for other types of bio-related devices such as biosensors. The construction of high-power biofuel cells through application of amphiphilic polymer mediators is our next challenge.

ASSOCIATED CONTENT Supporting Information. Supporting Information available: detailed syntheses of polymer mediators, supplementary UVvis spectra, and electrochemical data. This material is available free of charge via the Internet at http://pubs.acs.org.

AUTHOR INFORMATION Corresponding Authors *Norihiro Mizoshita. E-mail: [email protected] *Yuichiro Nakaoki. E-mail: [email protected] Author Contributions §These authors contributed equally.

ACKNOWLEDGMENT


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The PQQ-GDH expression plasmid was kindly provided by Dr. Yasushi Shigemori of Aisin Seiki Co., Ltd. We thank Ms. Yuka Isezaki of Aisin Cosmos R&D Co., Ltd. for appropriate advice and technical assistance.

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Table 1. Adsorption behavior of the modified mediators onto the carbon electrode and bioelectrocatalytic currents generated by the addition of glucose −−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−− Remaining ratea Redox potentialb Catalytic currentc (%) / V vs. Ag|AgCl / mA −−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−− mPMS LP-1

37 ± 15 94 ± 5

–0.11 –0.01

0.38 ± 0.07 0.90 ± 0.16

LP-2 99 ± 2 0.01 0.11 ± 0.02 BP-1 74 ± 7 –0.01 0.50 ± 0.12 SCP-1 85 ± 6 –0.04 0.14 ± 0.05 −−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−− a After 5 washes with a 1 M sodium phosphate buffer solution. b The redox potentials were estimated from the midpoints of the anodic and cathodic peak potentials. c At 0.4 V vs. Ag|AgCl after the addition of glucose (50 mM) in a 1 M sodium phosphate buffer solution.


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R

TsCl

OH

O

R

OTs

O

NaOH aq., THF

n

Ts =

n

O S O

CH3

OH N

K2CO3

N

R

O

O

DMF

n

TfOMe R

Tf =

N

N

N

N+ Me

O

O

CH2Cl2

n

O S CF3 O

TfO

Scheme 1. Chemical modification of terminal hydroxyl groups of hydrophilic chains.

e－

glucose PQQ -GDH

gluconolactone PQQ

electron transfer

anode

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

M

shuttling mediator moiety

hydrophilic flexible chain hydrophobic chain

Figure 1. Schematic illustration of desirable electron-transfer behavior via amphiphilic polymer mediators on a bioanode.


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mPMS

LP-1: n ~ 10

M M

LP-2: n ~ 2

Me Me Si O Me

Me Si O m Me

Me Si O

Me Si Me n Me O

m/n ~ 15

O

M N N+ CH3

M

BP-1

M M M M M M M M M M

~9

TfO

SCP-1

Figure 2. Chemical structures of electron-transfer mediators. The “M”s in the schematics indicate mediator moieties and the red and blue lines represent hydrophobic and hydrophilic chains, respectively.


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Langmuir

Figure 3. (a) Remaining rates of LP-1 and mPMS as a function of the number of washes with a phosphate buffer solution. (b) Remaining rates of mediators after 5 washes.


27

Langmuir

8 .0 0 E -00.8 4 6 .0 0 E -00.6 4

LP-1 mPMS

4 .0 0 E -00.4 4

BP-1 SCP-1 LP-2

Current / mA

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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2 .0 0 E -00.2 4 0 .0 0 E +00.0 0 -2 .0 0 E -0-0.2 4 -4 .0 0 E -0-0.4 4 -6 .0 0 E -0-0.6 4 -8 .0 0 E -0-0.8 4 -0.5 -0.4 -0.3 -0.2 -0.1 0

0.1 0.2 0.3 0.4 0.5

Potential / V vs. Ag|AgCl

Figure 4. Cyclic voltammograms of carbon electrodes incorporating mediators in a 1 M sodium phosphate buffer solution (pH 7.0), obtained at a scan rate of 20 mV/s.


28

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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

Figure 5. Cyclic voltammograms of carbon electrodes incorporating mediators and PQQ-GDH in a 1 M sodium phosphate buffer solution (pH 7.0), obtained at a scan rate of 20 mV/s in the presence of 50 mM glucose (solid line) or absence of glucose (dashed line). (a) mPMS, (b) LP1, (c) LP-2, (d) BP-1, (e) SCP-1. (f) Catalytic currents (at 0.4 V vs. Ag|AgCl) as a function of the glucose concentration for the carbon electrodes incorporating mPMS or LP-1.


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Langmuir

(a)

(b) e－

e－ PQQ -GDH

PQQ

PQQ -GDH

e－

M

e－

LP-2

anode

anode

PQQ -GDH

PQQ

M

LP-1

PQQ

PQQ -GDH

e－

PQQ

e－

M

M

M M M M M M M M M M

BP-1

SCP-1

Figure 6. Schematic illustration of electron-transfer behavior of polymer mediators (a) with and (b) without long hydrophilic chains.

1.5

Catalytic current / mA

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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1.0

LP-1

0.5

mPMS

0.0

0

1

2

3

4

5

6

7

8

9 10

Number of times

Figure 7. Catalytic currents for carbon electrodes incorporating LP-1 and mPMS as a function of the number of fuel exchanges.


30

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Langmuir

Table of Contents Graphic


31

Amphiphilic Polymer Mediators Promoting Electron Transfer on

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