Electrodes Modified with the Phase Transition Polymer and Heme

Nov 22, 2005 - An electrode was modified with a phase transition polymer, poly(N-isopropylacrylamide), and the polymer was further modified with a ...
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Langmuir 2006, 22, 478-483

Electrodes Modified with the Phase Transition Polymer and Heme Peptide: Biocatalysis and Biosensing with Tunable Activity and Dynamic Range Kikuo Komori, Kazutake Takada, and Tetsu Tatsuma* Institute of Industrial Science, UniVersity of Tokyo, Komaba, Meguro-ku, Tokyo 153-8505, Japan ReceiVed September 6, 2005. In Final Form: October 19, 2005 An electrode was modified with a phase transition polymer, poly(N-isopropylacrylamide), and the polymer was further modified with a peroxidase model compound, heme peptide (HP). As the polymer layer shrank at temperatures above 30-40 °C, the catalytic activity of the HP molecules for H2O2 reduction improved, and simultaneously, the number of HP molecules that can communicate electrochemically with the electrode increased. As a result, the catalytic current for H2O2 reduction in the shrunken state was 4 times larger than that in the swollen state. This reversible change was exploited for tuning the sensitivity and dynamic range of the HP electrode in H2O2 biosensing. The dynamic range in inhibition-based biosensing of imidazole derivatives was also tunable.

Introduction Activity control of biocatalysts, such as an enzyme, by using intelligent materials which respond to external signals and stimuli, including changes in temperature,1-6 light,7-13 pH,14-16 and electric field,17 has been under intense study. By combining the activity control system with electrochemistry, one could expect development of a bio-electrocatalytic device of which activity can be regulated by an external signal or stimulus. A biosensor with tunable sensitivity and/or dynamic range could also be developed. Nevertheless, only a few such devices have been developed so far. Most of the devices have been based on controlled diffusion of the substrate to a biocatalyst, rather than regulation of its intrinsic activity. Kubo et al.17 have electrochemically controlled diffusion of the substrate to an enzymemodified electrode. Willner et al.11 have reported on control of substrate diffusion to an enzyme-modified electrode by incident light. These diffusion-based controls are, however, expected to perturb mass transfer of other molecules like cofactors and * To whom correspondence should be addressed. E-mail: tatsuma@ iis.u-tokyo.ac.jp. (1) Dong, L. C.; Hoffman, A. S. J. Controlled Release 1986, 4, 223-227. (2) Shiroya, T.; Yasui, M.; Fujimoto, K.; Kawaguchi, H. Colloids Surf., B 1995, 4, 275-285. (3) Shiroya, T.; Yasui, M.; Fujimoto, K.; Kawaguchi, H. Colloids Surf., B 1997, 8, 311-319. (4) Stayton, P. S.; Shimoboji, T.; Long, C.; Chilkoti, A.; Harris, J. M.; Hoffman, A. S. Nature 1995, 378, 472-474. (5) Ding, Z.; Long, C. J.; Hayashi, Y.; Bulmus, E. V.; Hoffman, A. S.; Stayton, P. S. Bioconjugate Chem. 1999, 10, 395-400. (6) Shimoboji, T.; Larenas, E.; Fowler, T.; Hoffman, A. S.; Stayton, P. S. Bioconjugate Chem. 2003, 14, 517-525. (7) Bieth, J.; Vratsanos, S. M.; Wassermann, N.; Erlanger, B. F. Proc. Natl. Acad. Sci. U.S.A. 1969, 64, 1103-1106. (8) Karube, I.; Nakamoto, Y.; Namba, K.; Suzuki, S. Biochim. Biophys. Acta 1976, 429, 975-981. (9) Westmark, P. R.; Kelly, J. P.; Smith, B. D. J. Am. Chem. Soc. 1993, 115, 3416-3419. (10) Willner, I.; Rubin, S. Angew. Chem., Int. Ed. 1996, 35, 367-385. (11) Willner, I.; Willner, B. Bioelectrochem. Bioenerg. 1997, 42, 43-57. (12) Shimoboji, T.; Larenas, E.; Fowler, T.; Kulkarni, S.; Hoffman, A. S.; Stayton, P. S. Proc. Natl. Acad. Sci. U.S.A. 2002, 99, 16592-16596. (13) Komori, K.; Yatagai, K.; Tatsuma, T. J. Biotechnol. 2004, 108, 11-16. (14) Ishihara, K.; Kobayashi, M.; Ishimaru, N.; Shinohara, I. Polym. J. 1984, 16, 625-631. (15) Kokufuta, E.; Sodeyama, T.; Katano, T. J. Chem. Soc., Chem. Commun. 1986, 9, 641-642. (16) Kokufuta, E.; Zhang, Y.-Q.; Tanaka, T. J. Biomater. Sci., Polym. Ed. 1994, 6, 35-40. (17) Kubo, M.; Karube, I.; Suzuki, S. Biochem. Biophys. Res. Commun. 1976, 69, 731-736.

inhibitors. Therefore, those systems have aimed solely at activitycontrolled catalysis, but not at sensors with tunable dynamic range. In the meantime, most of other activity control systems, which are not associated with electrochemistry, are also based on the control of substrate diffusion. Previously, we have controlled the activity of an enzyme and enzyme model immobilized on electrodes by photoisomerizable compounds13 and thermoresponsive polymers such as poly(Nisopropylacrylamide) [poly(NIPA)]18-20 and poly(NIPA) gel.21,22 Heme peptide (HP; Figure 1a), which is a peroxidase model compound, has a structure and a catalytic activity (i.e., H2O2 reduction) similar to those of the active site of enzyme peroxidase. The active site of HP is, however, not fully covered with an insulating polypeptide, so the activity of HP can easily be controlled by changing the microenvironment of the active site with the phase transition materials.20,22 For the same reason, electron exchange between HP molecules and an electrode is possible, either directly23-27 or via self-mediation.27 HP is therefore compatible with electrochemical systems, and has been applied to electrochemical biosensors for H2O225,28-30 and inhibitors such as cyanide,27 imidazole,27,31-33 histamine,27,31 histidine,31-33 and urocanic acid.34 (18) Tatsuma, T.; Mori, H.; Takahashi, H.; Fujishima, A. Electrochem. SolidState Lett. 2001, 4, E5-E7. (19) Komori, K.; Matsui, H.; Tatsuma, T. Bioelectrochemistry 2005, 65, 129134. (20) Komori, K.; Takada, K.; Tatsuma, T. Anal. Sci. 2005, 21, 351-353. (21) Tatsuma, T.; Watanabe, Y.; Oyama, N. Electrochem. Solid-State Lett. 1998, 1, 136-138. (22) Tatsuma, T.; Fujimoto, Y.; Oyama, N. Electrochem. Solid-State Lett. 2000, 3, 283-285. (23) Santucci, R.; Reinhard, H.; Brunori, M. J. Am. Chem. Soc. 1988, 110, 8536-8537. (24) Razumas, V. J.; Gudavicius, A. V.; Kazlauskaite, J. D.; Kulys, J. J. J. Electroanal. Chem. 1989, 271, 155-160. (25) Tatsuma, T.; Watanabe, T. Anal. Chem. 1991, 63, 1580-1585. (26) Ruzgas, T.; Gaigalas, A.; Gorton, L. J. Electroanal. Chem. 1999, 469, 123-131. (27) Komori, K.; Takada, K.; Tatsuma, T. J. Electroanal. Chem. 2005, 585, 89-96. (28) Gorton, L.; Bremle, G.; Cso¨regi, E.; Jo¨nsson-Pettersson, G.; Persson, B. Anal. Chim. Acta 1991, 249, 43-54. (29) Razumas, V.; Kazlauskaite, J.; Ruzgas, T.; Kulys, J. Bioelectrochem. Bioenerg. 1992, 28, 159-176. (30) Razumas, V.; Kazlauskaite, J.; Vidziunaite, R. Bioelectrochem. Bioenerg. 1996, 39, 139-143. (31) Tatsuma, T.; Watanabe, T. Anal. Chem. 1992, 64, 143-147. (32) Tatsuma, T.; Buttry, D. A. Anal. Chem. 1997, 69, 887-893. (33) Tatsuma, T.; Mori, H.; Fujishima, A. Chem. Commun. 1999, 2395-2396.

10.1021/la052425a CCC: $33.50 © 2006 American Chemical Society Published on Web 11/22/2005

Electrodes Modified with Polymer and Heme Peptide

Figure 1. (a) Structure of the heme undecapeptide and (b) illustration of the poly(N-isopropylacrylamide-co-heme peptide)-modified electrode.

In the case of the poly(NIPA) gel-modified electrode incorporating HP,22 the catalytic current of H2O2 reduction could be reversibly controlled by the volume phase transition of the gel (i.e., swelling and/or shrinking) induced by temperature changes. The current changes have been ascribed to association and dissociation of the HP molecules or changes in their microenvironment, both caused by the phase transition of the gel, but not to changes in the substrate diffusion. However, the current changes were small, by a factor of ca. 1.5, so the dynamic range in the biosensing was not tunable. In addition, the gel sometimes peeled off from the electrode surface with repeated swellingshrinking cycles. These problems have been believed to arise from local restriction of swelling and shrinking because of the cross-link of polymer chains, which gives rise to distortion in the gel. In this study, to address these problems, we employed a polymer without a cross-link (Figure 1b) instead of the crosslinked gels. The HP activity was reversibly controlled by a factor of 4, and the rate-determining step for the H2O2 reduction process was switched between the catalytic reaction and the diffusion of H2O2. On the basis of this effect, a reversible control of dynamic ranges for biosensing of inhibitors as well as H2O2 is now possible.

Langmuir, Vol. 22, No. 1, 2006 479 no)propyl]carbodiimide (EDC) was added to the cast solution [poly(NIPA)-HP-modified electrodes 2 and 3, respectively]. To remove unreacted HP and EDC from the polymer membrane, the modified electrodes were immersed in the phosphate buffer at 25 and 45 °C for 2 h each. HP should have been covalently linked to the polymer via an amide bond between NAS and amino groups of the terminal valine and lysine residues. When EDC was used, the HP linked to the polymer should have further bound to other HP molecules via amide bonds between the amino groups and carboxyl groups of the glutamate residue and the heme moiety. A HP monolayer-modified electrode for comparative experiments was prepared as follows. An ITO-coated glass plate (area of ∼0.50 cm2) treated with a 1.0 M sodium hydroxide aqueous solution was immersed in a 2.5% acetic acid aqueous solution containing 2.5% 3-(aminopropyl)triethoxysilane (APTES) and then treated with a 2.5% glutaraldehyde (GA) aqueous solution for 12 h. After each treatment, the electrode was thoroughly rinsed with distilled water. A 30 µL aliquot of a 1.0 mM HP aqueous solution was cast onto the electrode surface and stored at 4 °C for 12 h. The solution was removed, and the electrode was thoroughly rinsed with water. HP was immobilized through the GA and APTES onto the ITO surface. Surface coverages of HP on the electrodes were determined by spectrophotometric measurements (MCPD-3000 and MC2530, Otsuka Electronics Co. Ltd.) at 25 °C with a molar extinction coefficient  of 176 mM-1 cm-1 for the Soret band.35,36 Electrochemical Measurements. Electrochemical measurements were performed in a 0.067 M phosphate buffer solution (pH 7.4) with a batch system. A Ag|AgCl|KCl sat. and a coiled platinum wire were used as reference and counter electrodes, respectively. The catalytic activity of the poly(NIPA)-HP-modified electrode toward H2O2 reduction was evaluated by amperometry with an LC-4C potentiostat (BAS). After the working electrode was polarized at 150 mV and a steady current was obtained, the H2O2 solution was added into the electrolyte solution followed by stirring for 30 s, and the steady-state reduction current was recorded. An inhibition effect on the catalytic reaction was measured as follows. After the working electrode was polarized at 150 mV, H2O2 (final concentration of 10 µM) and an inhibitor (imidazole or histamine) were successively added to the electrolyte solution. Each addition was followed by stirring for 30 s. The inhibition ratio was determined from decreases in the H2O2 reduction current. For cyclic voltammetry measurements, an HSV-100 potentiostat (Hokuto Denko) was employed.

Experimental Section

Results and Discussion

Preparation of Poly(NIPA)-HP-Modified Electrodes. An indium-tin oxide (ITO)-coated glass plate treated with a 1.0 M sodium hydroxide aqueous solution was immersed in toluene containing 2.5% triethoxyvinylsilane for 12 h. After the electrode was thoroughly rinsed with acetone, an insulating adhesive gasket was placed on the surface to define the area where the polymer is to be prepared (area of ∼0.50 cm2). A 30 µL aliquot of aqueous solution containing 950 mM NIPA, 50 mM N-acryloxysuccinimide (NAS, Acros), 17.5 mM ammonium persulfate as an initiator, and 33.1 mM N,N,N′,N′-tetramethylethylenediamine as an accelerator was applied onto the electrode surface and stored under N2 for 2 h to obtain the poly(NIPA-co-NAS)-modified electrode. The electrode was immersed in an aqueous solution at 4 °C for 12 h to swell the polymer membrane. Then it was transferred to a 0.067 M phosphate buffer solution (pH 7.4) at 45 °C and left for 2 h to shrink so that species that were not bound to the electrode surface were removed. The thickness of the polymer membrane was determined to be 0.5-1.0 mm in the swollen state on the basis of its photograph. The polymer bound to the electrode was modified with HP by casting 30 µL of a 1.0 mM heme undecapeptide (Sigma) aqueous solution to the poly(NIPA-co-NAS)-modified electrode in the shrunken state and storing it at 4 °C for 2 days [poly(NIPA)-HPmodified electrode 1]. In the case of preparation of electrodes with more concentrated HP, 10 and 100 mM 1-ethyl-3-[3-(dimethylami-

Spectroscopic Characterization of the Poly(NIPA)-HP Films. Typical temperature dependences of the film thickness and absorbance (500 nm) of the poly(NIPA)-HP membrane of electrode 1 were examined (Figure 2). The temperature was increased from 20 to 50 °C. Each datum was collected ∼30 min after the temperature was increased by 5 °C. The film thickness was measured on the basis of the corresponding photograph. As Figure 2 shows, the film shrank gradually from 30 to 40 °C. Simultaneously, the absorbance increased and the film became opaque. In general, poly(NIPA) dissolves in water at temperatures below the lower critical solution temperature (LCST) due to hydrophobic hydration and precipitates at temperatures above the LCST due to hydrophobic interaction between isopropyl moieties of poly(NIPA).37-40 The shrinking of the film should also be due to the hydrophobic interaction. Formation of aggregated microdomains due to the interaction, which scatter

(34) Tatsuma, T.; Okamura, K.; Komori, K.; Fujishima, A. Anal. Chem. 2002, 74, 5154-5156.

(35) Wilson, M. T.; Ranson, R. J.; Masiakowski, P.; Czarnecka, E.; Brunori, M. Eur. J. Biochem. 1977, 77, 193-199. (36) Razumas, V.; Kazlauskaite, J.; Ruzgas, T.; Kulys, J. Bioelectrochem. Bioenerg. 1992, 28, 159-176. (37) Heskins, H.; Gullet, J. E. J. Micromol. Sci. Chem. 1968, A2, 1441-1455. (38) Taylor, L. D.; Cerankowski, L. D. J. Polym. Sci. Polym. Chem. 1975, 13, 2551-2570. (39) Schild, H. G. Prog. Polym. Sci. 1992, 17, 163-249. (40) Feil, H.; Bae, Y. H.; Feijen, J.; Kim, S. W. Macromolecules 1993, 26, 2496-2500.

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Komori et al.

Figure 2. (a) Temperature-dependent polymer layer thickness normalized at 20 °C and (b) absorbance changes (500 nm) of poly(NIPA)-HP-modified electrode 1 in a 0.067 M phosphate buffer solution (pH 7.4).

light, should be responsible for the decrease in the film transparency. The gradual decrease in the absorbance above 40 °C may be explained in terms of gradual growth and fusion of the domains. Although domain formation may be accompanied by physical cross-linking of polymers, it is reversible. The fact that the phase transition temperature appeared to be broad (30-40 °C) could be ascribed to the variety of the polymer lengths. It should be emphasized that repeated swelling and shrinking of the polymer membrane (at least seven times) did not give rise to peeling of the film from the electrode surface, in contrast to the previous gel-modified electrode.22 Mechanical stress generated in our film by the volume changes should easily be relaxed since there are no chemical cross-linking points. Absorption spectra of poly(NIPA)-HP-modified electrodes 1-3 were examined at 25 °C where the poly(NIPA)-HP films were in the swollen state. Assuming that the molar extinction coefficient, , of the immobilized HP at the peak wavelength is equal to that of dissolved HP ( ) 176 mM-1),35,36 values for surface coverage of HP, Γsp, for poly(NIPA)-HP-modified electrodes 1-3 were determined to be (1.8 ( 0.5) × 10-11 (mean ( standard error, n ) 6), (1.4 ( 0.1) × 10-10 (n ) 10), and (1.9 ( 0.7) × 10-9 mol cm-2 (n ) 10), respectively. In addition, the Γsp of the HP monolayer-modified electrode was (2.5 ( 0.4) × 10-11 mol cm-2 (n ) 7). Peak wavelengths of the Soret band were also compared. Monomeric HP at 1 µM in a 0.067 M phosphate buffer solution (pH 7.4) gave the absorption peak corresponding to the Soret band at ca. 398 nm. HP has been reported to form dimers and oligomers through coordination of the amino group of the lysine and the terminal valine residues to the heme of another HP molecule in higher-pH solutions.35 It also forms a µ-oxo dimer through coordination of the sixth coordination sites of the hemes via oxygen at higher HP concentrations41 [>3 µM in a 0.067 M phosphate buffer solution (pH 7.4)]. As a consequence, the Soret band is red-shifted by, for instance, ca. 7 nm at 40 µM (pH 7.4). The absorbance peak of electrode 1 was observed at ca. 400 nm in both swollen and shrunken states, indicating that HP molecules in the polymer film mainly exist in the monomeric form. In the case of electrodes 2 and 3, the peaks were found at 408.5 and 410.8 nm, respectively, at 25 °C. These results suggest that HP molecules aggregated at least partially, likely because HP molecules were concentrated in those films. Since the molar extinction coefficient of HP lowers as it aggregates, Γsp values for electrodes 2 and 3 might be underestimated. Temperature Dependences of H2O2 Reduction at Poly(NIPA)-HP-Modified Electrodes. Amperometric measure(41) Urry, D. W. J. Am. Chem. Soc. 1967, 89, 4190-4196.

Figure 3. Temperature dependences of the steady-state cathodic current densities for 10 µM H2O2 reduction at (A) the poly(NIPAco-NAS)-modified (a), the HP monolayer-modified (b), and the poly(NIPA)-HP-modified (electrode 1, c) electrodes and those at (B) electrodes 2 (d) and 3 (e) at 150 mV vs Ag|AgCl in an air-saturated 0.067 M phosphate buffer solution (pH 7.4).

ments were performed for the HP-modified electrodes at 150 mV in an air-saturated 0.067 M phosphate buffer solution (pH 7.4). Upon an injection of H2O2 to the solution after the current reached a steady state, a cathodic current was observed. This current was likely due to electron transfer from the electrode to HP.25 Possible reaction mechanisms were proposed as follows.25

ferric HP + H2O2 f compound I + H2O

(1)

compound I + H+ + e- f compound II

(2)

compound II + H+ + e- f ferric HP + H2O

(3)

where compounds I and II are oxidized complexes of HP, in which FeIV of heme is coordinated with oxygen. The steadystate currents of poly(NIPA)-HP electrode 1 and the HP monolayer- and the poly(NIPA-co-NAS)-modified electrodes for the H2O2 reduction (final concentration of 10 µM) are plotted against temperature in Figure 3A. The catalytic current was observed at both HP-modified electrodes, but it was not the case for the poly(NIPA-co-NAS)-modified electrode (plot a). These results indicate that the HP molecules bound to the polymer catalyzes H2O2 reduction as do those bound directly to the electrode surface. In the case of the directly modified HP electrode (plot b), the catalytic current gradually increased as the temperature increased. This result suggests that the catalytic activity of HP (i.e., rate of reaction 1) and/or the diffusion rate of H2O2 increases with temperature. On the other hand, in the case of electrode 1 (plot c), the cathodic current response increased far more sharply in the temperature range from 30 to 40 °C. This change is likely due to the phase transition of the polymer, since it took place

Electrodes Modified with Polymer and Heme Peptide

Figure 4. Dependences of the steady-state cathodic current densities on H2O2 concentration for poly(NIPA)-HP-modified electrode 1 in an air-saturated 0.067 M phosphate buffer solution (pH 7.4) at (a) 25 and (b) 45 °C. The electrode potential was 150 mV vs Ag|AgCl.

in the same temperature range. The typical current ratio, Imax/Imin, of electrode 1 was found to be ca. 4, which is much larger than that of the reported value for the cross-linked poly(NIPA)-HP gel-modified electrode (ca. 1.5).22 This difference could be ascribed to the flexibility of the polymer used in this study, which is not chemically cross-linked. As Figure 3A shows, the cathodic current response of electrode 1 was twice as large as that of the HP monolayer-modified electrode above the phase transition temperature. However, the Γsp value of the former is slightly smaller than that of the latter as mentioned above. In addition, considering the thickness of the HP-modified poly(NIPA) film (Figure 2), the HP monolayermodified electrode is also advantageous in electron transfer from the electrode to HP molecules. Those contradictory results suggest that H2O2 is concentrated or the activity of HP (i.e., rate of reaction 1) is enhanced in the shrunken polymer film. Another possibility is that direct immobilization of HP onto the electrode surface decreases its activity due to steric hindrance. When the cathodic current responses to H2O2 of the poly(NIPA-co-NAS)modified electrode and a bare electrode without HP were measured at -100 mV at various temperatures (25-45 °C), these electrodes exhibited almost the same values (several tens of nanoamperes per square centimeter). These results suggest that the polymer film does not concentrate H2O2 in itself. Control of the Sensitivity and Dynamic Range for H2O2 Reduction. The cathodic current of electrode 1 for H2O2 reduction increased in proportion to the H2O2 concentration up to 30 µM below the phase transition temperature (25 °C) (Figure 4, curve a). This indicates that the reaction of HP with H2O2 (reaction 1) is the rate-determining step. In contrast, at higher H2O2 concentrations, the cathodic current was almost independent of the H2O2 concentrations. This suggests that reactions 2 and 3, in which H2O2 does not participate, are the rate-determining steps. In addition, there might be an effect of decomposition of HP and the polymer. On the other hand, above the phase transition temperature (45 °C), the reduction current was saturated at a lower concentration, ca. 10 µM (curve b). This was caused because the current in the proportional region improved by the phase transition, while that in the saturated region remained constant. Thus, we conclude that the sensitivity and the dynamic range of electrode 1 for H2O2 reduction can be controlled by a change in temperature. To determine lower concentrations of H2O2 with higher sensitivity, the temperature should be increased across the phase transition temperature, whereas for higher concentrations, the temperature should be lowered. Effect of HP Concentration. Properties of poly(NIPA)HP-modified electrodes 2 and 3 were also examined at different

Langmuir, Vol. 22, No. 1, 2006 481

Figure 5. Changes in current densities for H2O2 (1 µM) reduction at (a) the HP-monolayer-modified electrode and (b) poly(NIPA)HP-modified electrode 1 in a 0.067 phosphate buffer solution (pH 7.4) at 25 and 45 °C. The electrode potential was 150 mV vs Ag|AgCl.

temperatures. The cathodic currents for H2O2 (final concentration of 10 µM) clearly changed upon the phase transition of the polymer at 30-40 °C as observed for electrode 1 (Figure 3B). As the HP concentrations increased in the polymer film (electrode 1 < electrode 2 < electrode 3), the cathodic currents were found to be increased and then decreased. This result could be rationalized by taking into account aggregation of HP molecules at higher concentrations as described above, which hinders H2O2 from approaching the active site of HP. In addition, if the polymer enhances the activity of HP, the effect might be suppressed by the aggregation. Cyclability. The cathodic current responses of electrode 1 and the HP monolayer-modified electrode to 1 µM H2O2 were repeatedly measured at 25 and 45 °C (Figure 5). In the case of the HP monolayer-modified electrode, the cathodic currents gradually decreased, likely due to inactivation of the HP by H2O2 and/or detachment of the HP molecules from the electrode. In addition, as mentioned above, reversible changes in the catalytic current upon temperature switching were not evident. In the case of electrode 1, the catalytic currents reversibly changed at least four cycles upon successive temperature switching without an apparent decrease in the catalytic current. As for inactivation of HP, it has been pointed out that attack of compound I, which is generated by reaction 1, against another HP molecule plays a certain role.42 The polymer could interfere with the attack process. Moreover, the activity of an enzyme has been reported to be preserved by a certain type of polymer.43 Mechanisms of the Response Change. The current changes observed at H2O2 concentrations lower than 10 µM accompanying the phase transition of the polymer are supposed to be induced by changes in the following factors: (1) the partition coefficient and/or the diffusion rate of H2O2 in the polymer layer, (2) the catalytic activity of HP (i.e., rate constant of eq 1), and (3) the number of HP molecules which can receive electrons from the electrode (eqs 2 and 3). As mentioned above, since the H2O2 reduction current of the poly(NIPA-co-NAS)-modified electrode was found to be virtually the same as that of a bare electrode, the partition coefficient and the diffusion rate of H2O2, described in issue 1, are believed to have little influence on the current changes. Issue 2 is associated with changes in the intrinsic catalytic activity of HP arising from microscopic changes in the structure and environment in the vicinity of the HP active site. A concentration increase in, for instance, amide and hydrophobic (42) Spector, A.; Zhou, W.; Ma, W. C.; Chignell, C. F.; Reszka, K. J. Exp. Eye Res. 2000, 71, 183-194. (43) Calvo, E. J.; Danilowicz, C.; Diaz, L. J. Chem. Soc., Faraday Trans. 1993, 89, 377-384.

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Figure 6. Cyclic voltammograms of poly(NIPA)-HP-modified electrode 3 in a 0.067 phosphate buffer solution (pH 7.4) at (a) 25 and (b) 45 °C. The scan rate is 5 mV/s.

isopropyl moieties by shrinking of the polymer (Figure 1b) could be responsible for an increase in the activity. In fact, the catalytic activity for H2O2 reduction of peroxidase model compounds has been known to be enhanced by formation of a hydrogen bond between the NH site of the proximal imidazole, which coordinates to the heme (Figure 1a), and an electrophilic site of another molecule [e.g., carbonyl site in the case of poly(NIPA)].44 Although the association of HP molecules has also been reported to change the catalytic activity,22 it might scarcely contribute to the system presented here because the Soret absorption peak for HP was not shifted by the phase transition, as discussed above. The description of issue 3 suggests the possibility that electron transfer from the electrode to HP, which is either direct or indirect through electron self-exchange, is suppressed in the swollen state since the distance from the electrode to HP molecules and that between HP molecules increase. If this holds in the system presented here, it should be reflected by weakened redox responses of HP (FeII/III couple)23,24,26,27 in the cyclic voltammetry (CV) of the swollen film. However, both electrodes 1 and 2 exhibited no sufficiently clear responses most probably due to low surface coverage. In the case of electrode 3, the peak currents at 45 °C were ca. 1.5 times higher than those at 25 °C at a scan rate of 5 mV/s (Figure 6). As the scan rate increased, so did the peak currents at 25 and 45 °C. Finally, at a scan rate higher than 300 mV/s, almost the same currents were observed, suggesting that the rates of electron transfer via the electron self-exchange were also similar. Since the difference in the current observed in CV (at 5 mV/s) was lower than that determined by amperometry (i.e., factor of 3; Figure 3B, plot e), effect 2 likely contributes to the current changes in addition to effect 3. Thus, the changes in the current responses at H2O2 concentrations lower than 10 µM are likely due to the changes in both the catalytic activity (reaction 1) and the number of electrochemically accessible HP molecules. Although the latter should also be reflected in the currents at H2O2 concentrations higher than 100 µM, the currents were almost independent of the temperature (Figure 4). This might suggest that the catalytic activity of HP (reactions 2 and 3) at 25 °C might be somewhat larger than that at 45 °C so that the difference in the number of electrochemically accessible HP molecules is offset. Responses to Inhibitors for HP. The catalytic activity of HP is inhibited by a ligand such as imidazole, because it coordinates to the sixth coordination site of ferric HP, where H2O2 reduction is catalyzed. By measuring the inhibition effect on a HP-modified electrode, one can determine a concentration of the inhibitor.25,31 In this case, the dynamic range of the inhibitor sensing depends on the total activity of HP molecules immobilized on the electrode. (44) Traylor, T. G.; Popovitz-Biro, R. J. Am. Chem. Soc. 1988, 110, 239-243.

Komori et al.

Figure 7. Dependences of the inhibition ratio of electrode 1 (a and b) and electrode 2 (c and d) at 150 mV vs Ag|AgCl on the imidazole concentration in an air-saturated 0.067 M phosphate buffer solution (pH 7.4) containing 10 µM H2O2 at (a and c) 25 and (b and d) 45 °C.

From the current responses to H2O2 before and after addition of an inhibitor (i0 and i, respectively), the % inhibition value R is determined from the following equation.

R ) 100(i0 - i)/i0

(4)

When reaction 1 determines the overall rate of H2O2 reduction (reactions 1-3), eq 4 is rewritten in the following form27,31,45

R ) 100/(1/KappCi + 1)

(5)

Kapp ) K/(k1Γd/D + 1)

(6)

where Ci is the inhibitor concentration, K is the constant for formation of the species of HP and the inhibitor ()[HP-inhibitor]/ [HP][inhibitor]), Kapp is the apparent formation constant, k1 is the rate constant of reaction 1, Γ is the intrinsic surface coverage of HP, d is the diffusion layer thickness, and D is the diffusion coefficient of H2O2. From eq 5, it is obvious that R ∼ 0% when KappCi , 1 and that R ∼ 100% when KappCi . 1. The midpoint of the dynamic range, where R ) 50%, is given when Ci ) 1/Kapp. Thus, 1/Kapp defines whether the dynamic range of the response to the inhibitor is in a higher- or lower-concentration region. The 1/Kapp value is, in turn, dependent on k1Γd/D, as eq 6 indicates. The 1/Kapp value is minimized to be 1/K when k1Γd/D , 1 (i.e., when the H2O2 reduction is kinetically controlled). On the other hand, as the value of k1Γd/D increases and the system becomes a diffusion-controlled one, the 1/Kapp value becomes larger than 1/K. Thus, the 1/Kapp value, and hence the dynamic range, can be controlled by varying the k1Γ value. Actually, we have shown that the control is possible by changing the intrinsic Γ value for a peroxidase electrode45 and a HP electrode.27 Although reversible control has not yet been achieved, it would be possible with the system presented here. Incidentally, the inhibition ratio of the HP-modified electrode without the polymer for 1 mM imidazole was independent of the temperature over the range from 25 to 45 °C.20 In the presence of 10 µM H2O2, the % inhibition values of poly(NIPA)-HP-modified electrodes 1-3 for imidazole were examined at 25 and 45 °C. The H2O2 reduction currents were found to be inhibited by imidazole in all cases. It should be emphasized that the dynamic range shift accompanied the phase transition only at electrode 2 (Figure 7). This is explained in terms of the switching between kinetic control and diffusion control of the system, as described below. (45) Tatsuma, T.; Oyama, N. Anal. Chem. 1996, 68, 1612-1615.

Electrodes Modified with Polymer and Heme Peptide

The Kapp value of electrode 1 was evaluated to be ca. 6700 M-1 at both 25 and 45 °C, and was in agreement with that of the HP monolayer-modified electrode (7700 M-1),27 at which H2O2 reduction was kinetically controlled. These results indicate that, at both temperatures, the total catalytic activity (i.e., k1Γ) of electrode 1 is so low that the dynamic range cannot be controlled by changing the activity through phase transition. In contrast, the Kapp value of electrode 2 was 3700 M-1 at 25 °C and 970 M-1 at 45 °C; it was controlled by the phase transition. The Kapp value at 45 °C was approximately equal to the value reported for a HP multilayer-modified electrode without the polymer (1000 M-1),27 where the rate-determining step was diffusion of H2O2. Thus, it can be concluded that, in the case of electrode 2, the H2O2 reduction can be switched between kinetically controlled and diffusion-controlled processes by changing the activity through phase transition. As a result, the dynamic range for inhibition-based imidazole sensing can also be controlled. We have also examined histamine (MW ) 111), which is larger than imidazole (MW ) 68), as an inhibitor for electrode 2. As in the case of imidazole, the dynamic range at 45 °C shifted to the higher-concentration region relative to that at 25 °C. The Kapp values were found to be 1700 and 500 M-1 at 25 and 45 °C, respectively. On the other hand, the Kapp values of the HP monolayer- and multilayer-modified electrodes were 1100 and 90 M-1, respectively.27 The differences in the Kapp values

Langmuir, Vol. 22, No. 1, 2006 483

might be indicative of concentration of histamine in the poly(NIPA) layer, possibly due to interaction between the amino group of histamine and the amide bond of poly(NIPA). In any event, the polymer does not interfere with mass transfer of imidazole and histamine. Thus, the dynamic range of the poly(NIPA)-HP-modified electrode for the inhibitor sensing was shown to be controlled by a change in temperature. For the inhibitor at lower concentrations, measurements should be carried out below the phase transition temperature, whereas that at higher concentrations should be measured above the transition temperature. It has been reported that poly(NIPA) incorporated with acrylic acid or azobenzene derivatives shows a phase transition effect in response to pH changes15,39,46 or light,47,48 respectively. By using these polymers, the activity of the biocatalyst is expected to be controlled. Acknowledgment. This work was supported in part by a Grant-in-Aid for Scientific Research on Priority Areas (Area 417, Grant 14050028) from the Ministry of Education, Culture, Sports, Science and Technology of Japan. LA052425A (46) Chen, G.; Hoffman, A. S. Nature 1995, 373, 49-52. (47) Suzuki, A.; Tanaka, T. Nature 1990, 346, 345-347. (48) Irie, M. AdV. Polym. Sci. 1993, 110, 49-65.