Temperature-Induced Reversible Change in the Redox Response in

Polymers in Ionic Liquids: Dawn of Neoteric Solvents and Innovative Materials. Takeshi Ueki , Masayoshi Watanabe. Bulletin of the Chemical Society of ...
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Langmuir 2004, 20, 8786-8791

Temperature-Induced Reversible Change in the Redox Response in Phenothiazine-Labeled Poly(ethoxyethyl glycidyl ether) and Its Application to the Thermal Control of the Catalytic Reaction of Glucose Oxidase Naotaka Nakadan, Shin-ichiro Imabayashi,* and Masayoshi Watanabe* Department of Chemistry and Biotechnology, Yokohama National University, 79-5 Tokiwadai, Hodogaya-ku, Yokohama 240-8501, Japan Received May 7, 2004. In Final Form: July 22, 2004 Novel redox-active thermosensitive polymers (phenothiazine-labeled poly(ethoxyethyl glycidyl ether), PT-PEEGE), composed of a polyoxyalkylene backbone, ethoxyethoxymethyl side chains, and an electroactive phenothiazine end group, were prepared by base-catalyzed anionic ring-opening polymerization of ethoxyethyl glycidyl ether monomer in the presence of 10-(2-hydroxyethyl)phenothiazine. Phase separation of a 1.0 mmol dm-3 (0.33 wt %) PT-PEEGE aqueous solution occurs at 28 °C. While the phase separation temperature (Tc) is almost constant in the concentration range above 1.0 mmol dm-3, it increases at below 1.0 mmol dm-3. A 10-fold decrease in the oxidation current of PT-PEEGE is observed above Tc and reflects the decrease in the apparent concentration of electroactive PT-PEEGE due to the phase separation. The redox response mainly comes from PT-PEEGE molecules in the dilute phase, resulting from the phase separation, and the half-wave potential and peak separation are independent of the phase separation. This thermally induced change in the redox response is reversible and is applied for the thermal control of the electrocatalytic reaction of glucose oxidase (GOx). The catalytic current in the presence of PT-PEEGE as an electron mediator decreases at temperatures higher than Tc. This originates from the phase separation of PT-PEEGE, and PT-PEEGE molecules which remained to be soluble participate in the electrocatalytic reactions of GOx as mediators.

1. Introduction Electrical communication between enzymes and electrodes makes it possible to transform the enzymatic reaction into electrochemical responses and supports research efforts toward developing enzyme-based applications such as biosensors1-4 and biofuel cells.5-10 Glucose oxidase (GOx) (E.C. 1.1.3.4), which catalyzes the electron transfer (ET) from glucose to oxygen accompanying the production of gluconic acid and hydrogen peroxide, has been widely used for sensing the blood sugar level,11 and its enzymatic reaction is considered to be a candidate for the anode reaction of biofuel cells.5,9,10 Shuttling electrons between the FAD groups and the electrode, freely diffusing redox mediators with more positive redox potential than that of FAD, has been frequently used.12-16 * To whom correspondence may be addressed: S. Imabayashi (e-mail, [email protected]; fax, +81-45-339-3942) and M. Watanabe (e-mail, [email protected]; fax, +81-45-339-3955). (1) Frew, J. E.; Hill, H. A. O. Anal. Chem., 1987, 59, 933A. (2) Schuhmann, W. Biosens. Bioelectron. 1995, 10, 181-193. (3) Ohara T. J.; Rajagopalan, R.; Heller A. Anal. Chem., 1994, 66, 2451-2457. (4) Willner, I.; Katz, E. Angew. Chem., Int. Ed. 2000, 39, 1180-1218. (5) Delaney, G. M.; Benetto, H. P.; Mason, J. R.; Roller, S. D.; Stirling, J. L.; Thurston, C. F. J. Chem. Technol. Biotechnol. 1983, 34B, 13-27. (6) Tayhas, G.; Palmore, R.; Bertschy, H.; Bergens, S. H.; Whitesides, G. M. J. Electroanal. Chem. 1998, 443, 155-161. (7) Chen, T.; Barton, S. C.; Binyamin, G.; Gao, Z.; Zhang, Y.; Kim, H.-H.; Heller, A. J. Am. Chem. Soc. 2001, 123, 8630-8631. (8) Ikeda, T.; Kano, K. J. Biosci. Bioeng. 2001, 92, 9-18. (9) Mano, N.; Mao, F.; Heller, A. J. Am. Chem. Soc. 2003, 125, 65886594. (10) Katz, E.; Willner, I. J. Am. Chem. Soc. 2003, 125, 6803-6813. (11) Milardovic´, S.; Kruhak, I.; Ivekovic´, D.; Rumenjak, V.; Tkalcˇec, M.; Grabaric´, B. S. Anal. Chim. Acta 1997, 350, 91-96. (12) Cass, A. E. G.; Davis, G.; Francis, G. D.; Hill, H. A. O.; Aston, W. J.; Higgins, I. J.; Plotkin, E. V.; Scott, L. D. L.; Turner, A. P. F. Anal. Chem. 1984, 56, 667-671. (13) Fultz, M. L.; Durst R. A. Anal. Chim. Acta 1982, 130, 1-18.

Polymer redox mediators, instead of low-molecularweight mediators, were employed to avoid the leakage of mediators from sensor systems.17 But the use of polymer mediators resulted in the reduction of catalytic current due to their slow diffusion or loss of the electrochemical activity induced by conformational changes. We have investigated the mediator properties of phenothiazinelabeled poly(ethylene oxide) (PT-PEO) for the electrocatalytic reaction of GOx.18 The catalytic current of the GOx/ PT-PEO mixed systems decreased with increasing the molecular weight of PT-PEO, which reflects the reductions in the diffusion coefficient of PT-PEO and the intermolecular ET rate from FADH2/ FADH to PT+-PEO. One advantage of the polymer mediator systems, however, is that it affords polymer-based functions to mediators. We tried to elaborate thermally switchable mediators by replacing the PEO part of PT-PEO with a thermosensitive polymer. The aqueous solutions of thermosensitive polymers exhibit thermally induced phase separation into dilute and concentrated phases. As the polymers are phase separated out in the concentrated phase, the aqueous polymer solution turns from transparent into turbid above the cloud point (Tc).19,20 (14) Roller, S. D.; Bennetto, H. P.; Delaney, G. M.; Mason, J. R.; Stirling, J. L.; Thurston, C. F. J. Chem. Technol. Biotechnol., 1984, 34B, 3-12. (15) Williams, D. L.; Doig, A. R., Jr.; Korosi, A. Anal. Chem. 1970, 42, 118-121. (16) Nakabayashi, Y.; Omayu, A.; Yagi, S.; Nakamura, K.; Motonaka, J. Anal. Sci. 2001, 17, 945. (17) Schuhmann, W.; Wohlschla¨ger, H.; Lammert, R.; Schmidt, H.L.; Lo¨ffler, V.; Wiemho¨fer, H.-D.; Go¨pel, W. Sens. Actuators, B 1990, 1, 571-574. (18) Ban, K.; Ueki, T.; Tamada, Y.; Saito, T.; Imabayashi, S.; Watanabe, M.; Electrochem. Commun. 2001, 3, 649-653. (b) Imabayashi, S.; Ban, K.; Ueki, T.; Watanabe, M. J. Phys. Chem. B 2003, 107, 88348839. (c) Ban, K.; Ueki, T.; Tamada, Y.; Saito, T.; Imabayashi, S.; Watanabe, M. Anal. Chem. 2003, 75, 910-917.

10.1021/la0488654 CCC: $27.50 © 2004 American Chemical Society Published on Web 09/03/2004

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Scheme 1. Procedure for Preparation of PT-PEEGE

We report here the synthesis and basic characterization of the thermosensitive redox mediator, which is a polyoxyalkylene-based thermosensitive polymer with a phenothiazine end group (phenothiazine-labeled poly(ehtoxyethyl glycidyl ether), PT-PEEGE). PT-PEEGE can work as a mediator for GOx at below Tc. The redox activity of PT-PEEGE drastically decreased at above Tc, leading to the thermal switching of the electrocatalytic reaction of GOx with PT-PEEGE mediators. 2. Experimental Section 2.1. Preparation of Thermosensitive Polymer. Scheme 1 shows the synthetic procedure of PT-PEEGE. The polymer was synthesized by base-catalyzed anionic ring-opening polymerization of ethoxyethyl glycidyl ether in the presence of 10-(2hydroxyethyl)phenothiazine as a starting substance. The Tc of a series of thermosensitive polymers found in our laboratory depends on the hydrophobicity of the side-chain groups.21 An ethoxyethyl group was selected as the side-chain group so as to adjust the Tc of polymer to the optimum temperature range of GOx.22 Phenothiazine introduced at one end of the polymer was proved to work as a redox mediator for GOx.18 We used 10-(2hydroxyethyl)phenothiazine as a starting substance in this study, instead of phenothiazine used in the previous studies,18 to make the initiation reaction sufficiently faster than the chainpropagation reaction, which is the necessary condition for living polymerization. 10-(2-Hydroxyethyl)phenothiazine.23 Phenothiazine (0.125 mol) dissolved in 100 mL of THF was added to 400 mL of THF solution containing NaH suspension (0.125 mol), and the mixture was refluxed for 2 h. The reaction mixture was cooled to 0 °C and transferred into an autoclave. After the addition of ethylene oxide (0.25 mol), the mixture was stirred at 0 °C for 3 h. Finally, the reaction was quenched by adding saturated aqueous NH4Cl, followed by extraction of the product with dichloromethane. The extract was washed twice with water and then dried over MgSO4. After the solvent evaporation, the obtained purple oil was purified by column chromatography, yielding 10-(2-hydroxyethyl)phenothiazine as a purple solid, yield 20%. 1H NMR (δ from TMS in CDCl3): 2.1 (1H, t, OH), 3.9 (2H, t, NCH2CO), 4.1 (2H, t, NCCH2O), 6.9 (4H, m, Harom), 7.2 (4H, m, Harom). Polymerization of Ethoxyethyl Glycidyl Ether.22 10-(2Hydroxyethyl)phenothiazine (3.4 mmol) and potassium hydroxide (1.7 mmol) were dissolved in 80 mL of diethylene glycol dimethyl ether in an autoclave. To remove water that is a byproduct of the alcoxylation reaction, the solution was stirred for 3 h at 50 °C under reduced pressure. After ethoxyethyl glycidyl ether was added, synthesized from epichlorohydrin by the Williamson condensation reaction,24 the mixture was further stirred for 24 h at 110 °C under anhydrous nitrogen atmosphere. The reaction mixture was neutralized with 1 wt % sulfuric acid until its pH changed from 5 to 6. To remove ionic impurities, the mixture was stirred with an acid adsorbent for 30 min and with a base (19) Schild, H. G. Prog. Polym. Sci. 1992, 17, 163-249. (20) Aoshima, S.; Oda, H.; Kobayashi, E. J. Polym. Sci., Part A: Polym. Chem. 1992, 30, 2407-2413. (21) Aoki, S.; Koide, A.; Imabayashi, S.; Watanabe, M. Chem. Lett. 2002, 11, 1128-1129. (22) Enzyme Catalog: TOYOBO Enzymes; Toyobo Co., Ltd.: Osaka, Japan, 1998; p 112 (23) Albagli, D.; Bazan, G.; Wrighton, M. S.; Schrock, R. R. J. Am. Chem. Soc. 1992, 114, 4156-4158. (24) Motogami, K.; Kono, M.; Mori, S.; Watanabe, M. Electrochim. Acta 1992, 37, 1725-1727.

adsorbent for 1 h, respectively. The filtrate was evaporated under reduced pressure, yielding PT-PEEGE as a light-yellow viscous liquid. 1H NMR (δ from TMS in CDCl3): 1.2 (xH, t, OCH3), 3.63.8 (yH, m, OCH2CHO, OCH2CH2OCH2C), 3.9 (2H, t, NCH2CO), 4.1 (2H, t, NCCH2O), 6.9 (4H, m, Harom), 7.1 (4H, m, Harom). The number of protons at δ ) 1.2 and 3.6, x and y, are 3(n - 1) and 9(n - 1), respectively, where n is the degree of polymerization of PEEGE. The number average molecular weight (Mn) of PT-PEEGE was calculated from the ratio of integrated signal intensities between PT protons (δ ) 6.9 and 7.1 ppm) and side chain end methyl protons (δ ) 1.2 ppm). The molecular weight distribution (Mw/ Mn) of PT-PEEGE was determined by gel permeation chromatography (GPC). The Mn and Mw/Mn values were 3150 and 1.52 for PT-PEEGE3000, 5990 and 1.28 for PT-PEEGE6000, and 9360 and 1.33 for PT-PEEGE9000, respectively. 2.2. Electrochemical Measurements. We used a conventional three-electrode cell equipped with a glassy carbon working electrode (geometrical area 0.071 cm2), a Ag|AgCl|saturated NaCl reference electrode (0.197 V vs NHE), and a Pt wire auxiliary electrode. A glassy carbon electrode was polished with alumina powder (0.05 µm diameter) and sonicated in pure water prior to use. Sodium acetate buffer (0.05 mol dm-3, pH 5.1) deaerated by N2 purge for 20 min was used for all electrochemical measurements. Cyclic voltammograms (CVs) were recorded at the scan rate of 10 mV s-1 using a BAS-CV-50W electrochemical analyzer, and the diffusion coefficient (D) of PT-PEEGE was determined from the scan rate dependence of the anodic peak current.25 All measured potentials were converted to the values versus Ag|AgCl|saturated KCl (0.199 V vs NHE) reference electrode. 2.3. Determination of Cloud Point, Tc. An optical transmittance curve of 0.05 mol dm-3 sodium acetate buffer (pH 5.1) containing PT-PEEGE was monitored at 500 nm with a heating rate of 1 °C min-1 using a Shimazu UV-2400PC spectrophotometer. The Tc was determined as the temperature where the optical transmittance was 50%.

3. Results and Discussion 3.1. Phase Separation Temperature of PT-PEEGE. The relationship between Tc and the concentration of PTPEEGE solution is shown in Figure 1. The Tc values of 1 mmol dm-3 PT-PEEGE solution are 27.9 °C for PTPEEGE3000, 31.5 °C for PT-PEEGE6000, and 33.4 °C for PT-PEEGE9000. The Tc of 1 mmol dm-3 PT-PEEGE3000 is lower by 12 °C than that of 1 mmol dm-3 aqueous solution of PEEGE3000, which was prepared by anionic ringopening polymerization of ethoxyethyl glycidyl ether using phenol as a starting substance.22 The low-temperature shift of Tc is attributable to the greater hydrophobicity of the phenothiazine group than the phenyl group. The reduction in the hydrophobic effect of the PT group is responsible for the high-temperature shift of Tc with increasing the Mn of PT-PEEGE. While the Tc is constant at concentrations higher than 1.0 mmol dm-3 for all PT-PEEGE, the Tc increases in the concentration range below 1.0 mmol dm-3. The magnitude of the Tc increase is greater for PT-PEEGE with lower Mn and the Tc of PT-PEEGE 3000 reaches up to 45 °C at 50 µmol dm-3, suggesting that the intermolecular aggregation (25) Bard, A. J.; Faulkner, L. R. Electrochemical Methods, 2nd ed.; John Wiley & Sons: New York, 2001; Chapter 6.

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Figure 1. Dependence of Tc on the polymer concentration for PT-PEEGE3000 (9), PT-PEEGE6000 (b), and PT-PEEGE9000 (2) in 0.05 mol dm-3 sodium acetate buffer (pH 5.1).

Figure 2. Cyclic voltammograms of 5 mmol dm-3 PTPEEGE3000 measured at a glassy carbon electrode in 0.05 mol dm-3 sodium acetate buffer (pH 5.1) at a scan rate of 10 mV s-1: temperature, 25 (solid line) and 40 °C (dashed line).

of polymer molecules through hydrophobic interaction tends to be less significant at a lower concentration and a higher Mn of PT-PEEGE. 3.2. Temperature and Concentration Dependence of the Redox Response of PT-PEEGE. Figure 2 shows CVs of 5.0 mmol dm-3 PT-PEEGE3000 measured at 25 (solid line) and 40 °C (dashed line) that are below and above Tc (28 °C), respectively. A pair of CV peaks with the peak separation of 68 mV appeared at 600 mV for both temperatures, corresponding to a quasi-reversible redox response of the phenothiazine groups. The peak current, however, decreased by 10-fold from 1.1 × 10-5 to 1.2 × 10-6 A, while temperature increased from 25 to 40 °C. The D of PT-PEEGE3000 was determined to be 2.6 × 10-6 cm2 s-1 at 25 °C from the sweep rate dependence of the anodic peak current and is comparable to that of PT-PEO with the Mn of 1000 (2.4 × 10-6 cm2 s-1).18 Considering that the molecular weight of the monomer unit for PEEGE is three times larger than that for PEO, the D value seems to be governed by the length of the polymer main chain, not by the Mn value. The hydrodynamic radius of the PTPEEGE3000 molecule is calculated to be 0.84 nm from the D value using the Stokes-Einstein equation. At temperatures higher than Tc, the polymer solution is phase separated into the dilute and concentrated phases. In the concentrated phase, PT-PEEGE chains transformed from a coil to a globule state and simultaneously aggregated through hydrophobic interaction. The average D and diameter of PT-PEEGE3000 aggregates at 40 °C

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Figure 3. Oxidation peak currents in CV for 0.1 (9), 0.5 (b), and 1.0 (2) mmol dm-3 PT-PEEGE3000 as a function of temperature. CVs were measured at a glassy carbon electrode in 0.05 mol dm-3 sodium acetate buffer (pH 5.1) at a scan rate of 10 mV s-1. Arrows indicate Tc values of PT-PEEGE.

were estimated by dynamic light scattering to be ca. 1.0 × 10-7 cm2 s-1 and 60 nm, respectively. As the diameter of the aggregates is ca. 70 times greater than the hydrodynamic radius of the PT-PEEGE molecule, it seems that most of the hydrophobic PT groups are incorporated into the aggregates and are difficult to electrically communicate with the electrode. Figure 3 shows the oxidation peak current for the PT end group as a function of temperature, measured at three different PT-PEEGE3000 concentrations. With increasing temperature, the peak current started to decrease around the Tc and converged to a similar value above 40 °C for 0.5 and 1.0 mmol dm-3 of PT-PEEGE3000. The peak current for 0.1 mmol dm-3 PT-PEEGE3000 is almost constant in the whole temperature range examined. It is interesting to note that the peak currents for all three concentrations were approximately equal above 45 °C. The plots in Figure 1 correspond to the low concentration edge of the phase diagram of aqueous PT-PEEGE solutions and suggest that a part of the PT-PEEGE molecules are not aggregated and remain in the dilute phase even at temperatures above Tc. Assuming that the PT-PEEGE molecules in the dilute phase mainly participate in the redox response, the characteristics of Figure 3 that are the gradual decrease in the peak current at temperatures above Tc, the same peak current for all three concentrations above 45 °C, and the almost constant current for 0.1 mmol dm-3 PT-PEEGE3000 is qualitatively understood. However, a possibility remains that a part of the PT groups in the aggregates retain the electrochemical activity, because the current calculated by using the concentration of PT-PEEGE3000 in the dilute phase, which is obtained from Figure 1, is lower than the measured value in the temperature range between 30 and 40 °C. In contrast to the gradual change in the current, the optical transmittance sharply changed from 100 to 0% in a narrow temperature region (2-3 °C) around Tc, which reflects the formation of PT-PEEGE aggregates.22 To clarify the effect of the phase separation on the redox response of PT-PEEGE, similar measurements to those in Figure 3 were carried out for PT-PEO, poly(ethylene oxide) having a phenothiazine group at one end. The Mn and Mw/ Mn values of PT-PEO were 2530 and 1.19, respectively. The Tc of PEO is reported to be higher than 100 °C,26 and thus, its phase separation does not occur in (26) Bailey, F. E.; Callard, R. W. J. Appl. Polym. Sci. 1959, 1, 56-62.

Temperature-Induced Change in Redox Response

Figure 4. Oxidation peak currents in CV for 0.1 (9), 0.5 (b), and 1.0 (2) mmol dm-3 PT-PEO2500 as a function of temperature. CVs were measured at a glassy carbon electrode in 0.05 mol dm-3 sodium acetate buffer (pH 5.1) at a scan rate of 10 mV s-1.

Figure 5. Oxidation peak currents in CV for 0.5 mmol dm-3 PT-PEEGE3000 (9), PT-PEEGE6000 (b), and PT-PEEGE9000 (2) as a function of temperature. CVs were measured at a glassy carbon electrode in 0.05 mol dm-3 sodium acetate buffer (pH 5.1) at a scan rate of 10 mV s-1. The inset shows the plots for the current normalized by the maximum value in the measured temperature region. Arrows indicate Tc values of PT-PEEGE.

the measured temperature range. In contrast to PTPEEGE, only a slight increase in the peak current was observed for PT-PEO with increasing temperature, as shown in Figure 4. The fact that no current decrease was observed for PT-PEO indicates that the current decrease in Figure 3 reflects the phase separation of PT-PEEGE chains. The product of the viscosity of water and the D value of PT-PEO calculated from the peak current is constant in the studied temperature range, revealing the hydrodynamic radius of PT-PEO chain is almost constant. The slight increase in the peak current, therefore, originates from the increase in the D value of PT-PEO accompanying the reduction in the viscosity of water with the temperature increase. Figure 5 represents the CV peak current vs temperature plots as a function of the Mn of PT-PEEGE. The change in the peak current accompanying the phase separation was sharper for PT-PEEGE with higher Mn as shown in the inset where the peak current is normalized by its maximum value in the measured temperature region. The concentration of PT-PEEGE in the dilute phase, which is mainly responsible for the CV response above Tc, is lower for PT-PEEGE with higher Mn as expected from the less

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Figure 6. Cyclic voltammograms of GOx (9 µmol dm-3)/PTPEEGE3000 (0.5 mmol dm-3) mixed system in the presence (solid line, 50 mmol dm-3) and absence (dotted line) of glucose measured at a glassy carbon electrode in 0.05 mol dm-3 sodium acetate buffer (pH 5.1) at a scan rate of 10 mV s-1.

Figure 7. Catalytic currents of GOx (9 µmol dm-3)/glucose (50 mmol dm-3)/PT-PEEGE3000 mixed system measured at 25 (b), 40 (1), and 50 °C (×) as a function of the PT-PEEGE concentration. The catalytic current was measured at a scan rate of 10 mV s-1 in 0.05 mol dm-3 sodium acetate buffer (pH 5.1).

dependence of Tc on the PT-PEEGE concentration in Figure 1. This fact would be reflected in the sharp current change. 3.3. Electrocatalytic Reaction of GOx Mixed with PT-PEEGE as a Redox Mediator. Figure 6 shows CVs of GOx (9 µmol dm-3) mixed with PT-PEEGE3000 (0.5 mmol dm-3) in the presence of 0.05 mol dm-3 glucose at 25 and 50 °C. A typical catalytic wave with a sigmoidal shape27,28 appeared at each temperature, suggesting that PT-PEEGE3000 can work as a mediator for GOx. The catalytic current, icat, increased with the glucose concentration and saturated at glucose concentrations higher than 0.02 mol dm-3 at 25 °C (data not shown), revealing 0.05 mol dm-3 glucose corresponds to the substratesaturated condition. The catalytic current at 50 °C is significantly smaller than that at 25 °C. Figure 7 represents the icat at 0.65 V, which is 50 mV more positive than the redox potential of PT-PEEGE and corresponds to the diffusion-limited condition, as a function of the concentration of PT-PEEGE3000. The linear relationship between the icat and the PT-PEEGE concen(27) Bard, A. J.; Faulkner, L. R. Electrochemical Methods, 2nd ed.; John Wiley & Sons: New York, 2001; Chapter 12. (28) Liaudet, E.; Battaglini, F.; Calvo, E. J. J. Electroanal. Chem. 1990, 293, 55-68.

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Figure 8. Catalytic currents of GOx (9 µmol dm-3)/glucose (50 mmol dm-3)/PT-PEO2500 mixed system measured at 25 (b), 40 (1), and 50 °C (×) as a function of the PT-PEO concentration. The catalytic current was measured at a scan rate of 10 mV s-1 in 0.05 mol dm-3 sodium acetate buffer (pH 5.1).

tration at 25 °C suggests that the electrocatalytic reaction of GOx is not saturated against the mediator concentration. Under the present condition, the icat is described by eq 1.28 1/2

icat ) FA(2DPT-PEEGEket[GOx]) [PT-PEEGE] (1) where F is Faraday’s constant, A is the electrode surface area, DPT-PEEGE is the diffusion coefficient of PT-PEEGE, and ket is the ET rate constant between PT+-PEEGE and FADH2/FADH. At 25 °C that is below Tc, the ket is estimated to be 2.6 × 105 s-1 mol-1 dm3, independent of PT-PEEGE3000 concentrations, from the slope of the linear icat vs [PTPEEGE] plot using eq 1 and the DPT-PEEGE value obtained above. The obtained ket value is half of that for PT-PEO with a similar chain length, revealing that the ET between PT-PEEGE and GOx occurs effectively in the coil state and is not hindered by the bulky side chains. At 40 or 50 °C, the plot is linear only in the extremely dilute concentration and deviated downward from the linearity in the concentration range where the majority of PTPEEGE molecules are phase separated to form the aggregates, judging from Figure 2. The higher the concentration of PT-PEEGE, the larger deviation from the linearity was observed. The icat was constant at PTPEEGE concentrations higher than 0.3 mmol dm-3 at 40 °C and 0.15 mmol dm-3 at 50 °C. The ratio of the icat values at 25, 40, and 50 °C for 0.5 mmol dm-3 of PT-PEEGE3000 in Figure 7 (1.0:0.58:0.22) agrees well with the ratio of the CV peak currents at the three temperatures for 0.5 mmol dm-3 of PT-PEEGE3000 in Figure 3 (1.0:0.55:0.22). This supports that the decrease in the icat originates from the phase separation of PTPEEGE and that only PT-PEEGE molecules remained to be electroactive participated in the electrocatalytic reactions of GOx as mediators. Figure 8 shows the results of similar measurements to those in Figure 7 done for the GOx/PT-PEO mixed system. The linear icat vs [PT-PEO] plots were obtained for all three temperatures, indicating that the deviation from the linearity observed at 40 and 50 °C in Figure 7 does not come from the denaturation of GOx and is correlated with the phase separation of PT-PEEGE. The larger icat was obtained at the higher temperature for all PT-PEO concentrations. Equation 1 reveals that the slope of the icat vs [PT-PEO] plot is proportional to the value of (DPT-PEO ket)1/2 and its ratio at 25, 40, and 50 °C is 1:1.5:1.7.

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Figure 9. Temperature dependences of the catalytic current for GOx/glucose/PT-PEEGE3000 (9), PT-PEEGE6000 (b), and PT-PEEGE9000 (2) mixed systems measured in 0.05 mol dm-3 sodium acetate buffer (pH 5.1) at a scan rate of 10 mV s-1. The inset shows the plots for the catalytic current normalized by the maximum value in the measured temperature region. The concentrations of GOx, glucose, and PT-PEEGE were 9 µmol dm-3, 50 mmol dm-3, and 0.5 mmol dm-3, respectively. Arrows indicate Tc values of PT-PEEGE.

On the other hand, the ratio of the (DPT-PEO)1/2 values at 25, 40, and 50 °C is estimated to be 1:1.1:1.3 from Figure 4, suggesting that the ET rate from FAD to PT+-PEO also slightly alters with temperature. Figure 9 compares the temperature dependence of the icat value for the three PT-PEEGE molecules with different molecular weights. The icat value at 25 °C is larger for the PT-PEEGE with lower Mn. The decrease in the icat value with increasing Mn is significantly larger than that in the current at 25 °C with Mn in Figure 5, suggesting that not only DPT-PEEGE but also ket values decrease with increasing the Mn of PT-PEEGE. The normalized icat vs temperature plots in the inset of Figure 9 clearly indicate that the sharper icat transition was observed for the PT-PEEGE with higher Mn, which agrees well with the manner of the change in the peak current for PT-PEEGE oxidation in Figure 5. The reversible change in the redox response of PT-PEEGE due to the phase separation realizes the thermoswitching of the electrocatalytic reaction of GOx. 4. Conclusion We have found that the redox properties of PT-PEEGE can be reversibly altered by the change in temperature. This is caused by conversion of the polymer in an aqueous solution from a hydrated coil to a collapsed, hydrophobic globule, resulting in the phase separation of the polymer solution into the dilute and concentrated phases. The PTPEEGE molecules in the dilute phase mainly contribute to the redox response and thus the apparent concentration of electroactive PT-PEEGE decreases above Tc. PT-PEEGE can work as a mediator for the ET between GOx and the electrode and the thermally induced reversible change in the redox response of PT-PEEGE realizes the thermoswitching of the electrocatalytic reaction of GOx. Hoffman et al. have reported that conjugation of the stimuli-sensitive polymers including thermosensitive polymers near or within the active recognition sites of an antibody, enzymes, and affinity proteins can lead to stimuli control of the protein’s recognition process.29-31 We previously found that PT groups bonded to lysine residues on (29) Stayton, P. S.; Shimoboji, T.; Long, C.; Chilkoti, A.; Chen, G.; Harris, J. M.; Hoffman, A. S. Nature 1995, 378, 472-474. (30) Ding, Z.; Fong, R. B.; Long, C. J.; Stayton, P. S.; Hoffman, A. S. Nature 2001, 411, 59-62.

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the GOx surface via the PEO chain effectively mediate the fast ET between the electrode and the FAD center.18 From the above reports, we expect to fabricate GOx hybrids with thermally controllable self-electrocatalytic activity by replacing PT-PEO with PT-PEEGE. The electrocatalytic reaction of GOx-(PT-PEEGE) hybrids, in which PT(31) Hoffman, A. S.; Stayton, P. S.; Bulmus, V.; Chen, G.; Chen, J.; Cheung, C.; Chilkoti, A., Ding, Z.; Dong, L.; Fong, R.; Lackey, C. A.; Long, C. J.; Miura, M.; Morris, J. E.; Murthy, N.; Nabeshima, Y.; Park, T. G.; Press: O. W.; Shimoboji, T.; Shoemaker, S.; Yang, H. J.; Monji, N.; Nowinski, R. C.; Cole, C. A.; Priest, J. H.; Harris, J. M.; Nakamae, K.; Nishino, T.; Miyata, T. J. Biomed. Mater. Res. 2000, 52, 577-586.

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PEEGE molecules are covalently bonded to lysine residues on GOx, is now under investigation. Acknowledgment. This research was supported in part by Grant-in-Aid for Scientific Research on Priority Areas (A) “Molecular Synchronization for Design of New Materials System” (No. 404/1167234) from the Ministry of Education, Culture, Sports, Science, and Technology, Japan. LA0488654