Effect of the Electrostatic Interaction on the Redox Reaction of

In contrast, the value of ket of cyt c on the SAM of MES is pH-independent at 100 ... Bo Jin , Gui-Xia Wang , Diego Millo , Peter Hildebrandt , and Xi...
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Effect of the Electrostatic Interaction on the Redox Reaction of Positively Charged Cytochrome c Adsorbed on the Negatively Charged Surfaces of Acid-Terminated Alkanethiol Monolayers on a Au(111) Electrode Shin-ichiro Imabayashi,*,† Takahiro Mita,† and Takashi Kakiuchi*,‡ Department of Chemistry and Biotechnology, Faculty of Engineering, Yokohama National University, Yokohama 240-8501, Japan, and Department of Energy and Hydrocarbon Chemistry, Graduate School of Engineering, Kyoto University, Kyoto 615-8510, Japan Received August 10, 2004. In Final Form: November 18, 2004 The electrochemical properties of cytochrome c (cyt c) adsorbed on mixed self-assembled monolayers (SAMs) of 2-mercaptoethanesulfonate (MES)/2-mercaptoethanol (MEL) are compared with those on singlecomponent SAMs of MES, MEL, and mercaptopropionic acid (MPA), using cyclic voltammetry and potentialmodulated UV-vis reflectance spectroscopy. The rate constant of electron transfer (ET), ket, of cyt c adsorbed on the SAM of MPA decreases from 1450 ( 210 s-1 at pH 7 to 890 ( 100 s-1 at pH 9. In contrast, the value of ket of cyt c on the SAM of MES is pH-independent at 100 ( 15 s-1. Those facts suggest that a large negative charge density on the SAM surface slows down the ET between cyt c and the electrode. The surface charge density of the SAM affects also the amount of electroactive cyt c, Γe, which decreases from 10.0 ( 1.0 to 5.3 ( 1.1 pmol cm-2 with increasing pH from 7 to 9 on the SAM of MPA. Similarly, the ket of cyt c adsorbed on the mixed SAMs of MES/MEL sharply decreases from 900 ( 300 s-1 to 110 s-1 as the surface mole fraction of MES increases beyond 0.5, suggesting the presence of a negative surface charge threshold beyond which the rate of ET of cyt c is dramatically lowered. The decrease in the ket on the SAMs at high negative charge densities probably results from the confinement of adsorbed cyt c by the strong electrostatic force to an orientation that is not optimal for the ET reaction.

Introduction Self-assembled monolayers (SAMs) of ω-substituted alkanethiols have been widely used for studying the electron transfer (ET) reactions of redox proteins and enzymes.1-39 Among those SAMs, carboxyl-terminated SAMs on gold electrodes have been most commonly used * To whom correspondence should be addressed: S. Imabayashi ([email protected]) and T. Kakiuchi (kakiuchi@ sun.scl.kyoto-u.ac.jp). † Yokohama National University. ‡ Kyoto University. (1) Kinnear, K. T.; Monbouquette, H. G. Langmuir 1993, 9, 22552257. (2) Willner, I.; Lapidot, N.; Riklin, A.; Kasher, R.; Zahavy, E.; Katz, E. J. Am. Chem. Soc. 1994, 116, 1428-1441. (3) Jiang, L.; McNeil, C. J.; Cooper, J. M. Chem. Commun. 1995, 1293-1294. (4) Creager, S.; Olsen, K. G. Anal. Chim. Acta 1995, 307, 277-298. (5) Rubin, S.; Chow, J. T.; Ferraris, J. P.; Zawodzinski, T. A., Jr. Langmuir 1996, 12, 363-370. (6) Purves, J. T.; Vogt, T.; Sligar, S. G.; Knoll, W. Book of Abstracts, 231th ACS National Meeting, San Francisco, 1997; American Chemical Society: Washington, DC, 1997. (7) Madoz, J.; Kuznetzov, B. A.; Medrano, F. J.; Garcia, J. L.; Fernandez, V. M. J. Am. Chem. Soc. 1997, 119, 1043-1051. (8) Katz, E.; Riklin, A.; Shabtai, V. H.; Willner, I.; Bu¨ckmann, A. F. Anal. Chim. Acta 1999, 385, 45-58. (9) Guiomar, A. J.; Guthrie, J. T.; Evans, S. D. Langmuir 1999, 15, 1198-1207. (10) Zimmermann, H.; Lindgren, A.; Schuhmann, W.; Gorton, L. Chem.sEur. J. 2000, 6, 592-599. (11) Schuhmann, W.; Zimmermann, H.; Habermuller, K.; Laurinavicius, V. Faraday Discuss. 2000, 116, 245-255. (12) Willner, I.; Katz, E. Angew. Chem., Int. Ed. 2000, 39, 11811218. (13) Madoz-Gu´rpide, J.; Abad, J. M.; Ferna´ndez-Recio, J.; Ve´lez, M.; Va´zques, L.; Go´mez-Moreno, C.; Ferna´ndez, V. M. J. Am. Chem. Soc. 2000, 122, 9808-9817. (14) Kang, C.; Kim, S.; Shin, H. Anal. Sci. 2001, 17, a13-a16. (15) Glenn, J. D. H.; Bowden, E. F. Chem. Lett. 1996, 399-400. (16) Gaspar, S.; Zimmermann, H.; Gazaryan, I.; Cso¨regi, E.; Schuhmann, W. Electroanalysis 2001, 13, 284-288. (17) Tominaga, M.; Taniguchi, I. Chem. Lett. 2001, 704-705.

for the study of the ET reactions of cytochrome c (cyt c)20-39 with the expectation that lysine-rich positively charged cyt c molecules are electrostatically stabilized on those (18) Chen, X.; Discher, B. M.; Pilloud, D. L.; Gibney, B. R.; Moser, C. C.; Dutton, P. L. J. Phys. Chem. B 2002, 106, 617-624. (19) Scheller, F. W.; Wollenberger, U.; Lei, C.; Jin, W.; Ge, B.; Lehmenn, C.; Lisdat, F.; Fridman, V. Rev. Mol. Biotech. 2002, 82, 411424. (20) Kuznetsov, B. A.; Byzova, N. A.; Shumakovich, G. P. J. Electroanal. Chem. 1994, 371, 85-92. (21) Knoll, W.; Pirwitz, G.; Tamada, K.; Offenha¨usser, A.; Hara, M. J. Electronal. Chem. 1997, 438, 199-205. (22) Fedurco, M. Coord. Chem. Rev. 2000, 209, 263-331. (23) Avila, A.; Gregory, B. W.; Niki, K.; Cotton, T. M. J. Phys. Chem. B 2000, 104, 2759-2766. (24) Dick, L. A.; Haes, A. J.; Van Duyne, R. P. J. Phys. Chem. B 2000, 104, 11752-11762. (25) Wackerbarth, H.; Murgida, D. H.; Oellerich, S.; Do¨pner, S.; Rivas, L.; Hildebrandt, P. J. Mol. Struct. 2001, 563, 51-59. (26) Murgida, D. H.; Hildebrandt, P. J. Phys. Chem. B 2002, 106, 12814-12819. (27) Khoshtariya, D. E.; Wei, J.; Liu, H.; Yue, H.; Waldeck, D. H. J. Am. Chem. Soc. 2003, 125, 7704-7714. (28) Tarlov, M. J.; Bowden, E. F. J. Am. Chem. Soc. 1991, 113, 18471849. (29) Collison, M.; Bowden, E. F.; Tarlov, M. J. Langmuir 1992, 8, 1247-1250. (30) Clark, R. A.; Bowden, E. F. Langmuir 1993, 13, 559-565. (31) Song, S.; Clark, R. A.; Bowden, E. F.; Tarlov, M. J. J. Phys. Chem. 1993, 97, 6564-6572. (32) Nahir, T. M.; Bowden, E. F. J. Electronal. Chem. 1996, 410, 9-13. (33) Kasmi, A. E.; Wallace, J. M.; Bowden, E. F.; Binet, S. M.; Linderman, R. J. J. Am. Chem. Soc. 1998, 120, 225-226. (34) Leopold, M. C.; Bowden, E. F. Langmuir 2002, 18, 2239-2245. (35) Feng, Z. Q.; Imabayashi, S.; Kakiuchi, T.; Niki, K. J. Electronal. Chem. 1995, 394, 149-154. (36) Imabayashi, S.; Mita, T.; Iida, M.; Feng, Z. Q.; Niki, K.; Kakiuchi, T. Denki Kagaku 1997, 65, 467-470. (37) Arnold, S.; Feng, Z. Q.; Kakiuchi, T.; Knoll, W.; Niki, K. J. Electroanal. Chem. 1997, 438, 91-97. (38) Feng, Z. Q.; Imabayashi, S.; Kakiuchi, T.; Niki, K. J. Chem. Soc., Faraday Trans. 1997, 93, 1367-1370. (39) Hobara, D.; Imabayashi, S.; Kakiuchi, T. Nano Lett. 2002, 2, 1021-1025.

10.1021/la047992x CCC: $30.25 © 2005 American Chemical Society Published on Web 01/20/2005

Redox Reaction of Adsorbed Cytochrome c

SAMs as in the case of the protein-protein association between cyt c and its protein reaction partners, cyt c oxidase, cyt c reductase, or cyt c peroxidase.40-42 However, the foregoing studies suggest that the strong electrostatic immobilization of cyt c on a negatively charged electrode surface does not necessarily lead to efficient ET between cyt c and the electrode.23,33,37 The standard rate constant of the heterogeneous ET, ket, of cyt c adsorbed on the COOH-terminated SAMs decreases as the pH of the solution increases from 6 to 9,23,37 giving rise to the deprotonation of the carboxyl groups.43,44 The ket on the SAM prepared from a mixed thiol solution of mercaptoundecanoic acid (MUA) and decanethiol at a mole fraction of MUA of 0.8 is about 5 times greater than that on a single-component SAM of MUA at the same pH.37 Bowden et al. reported a 5-fold increase of the ket of horse heart cyt c adsorbed on the mixed SAMs of MUA and mercaptooctanol in comparison with the ket on the single-component SAM of MUA.33 The high negative charge density on carboxyl-terminated SAMs thus appears to deteriorate the ET rate between cyt c and electrodes. The aim of the present work is to quantitatively evaluate the effect of the negative surface charge on the ET of cyt c adsorbed on acid-terminated SAM surfaces. For this purpose, we used mixed SAMs composed of 2-mercaptoethanesulfonate (MES) and 2-mercaptoethanol (MEL), where the sulfonic acid groups on the MES SAM are fully dissociated in the wide pH range.37 Compared with the carboxyl-terminated SAM, little is known on the redox reactions of cyt c adsorbed on sulfonate-terminated SAMs. While cyt c is known to adsorb on the sulfonate-terminated SAMs,45,46 no attempt has been made to measure ket values of cyt c on this SAM. In the present study, we investigated the ET reactions of cyt c adsorbed on the mixed SAMs in comparison with those on single-component SAMs of MES, MEL, and 3-mercaptopropionic acid (MPA) using cyclic voltammetry and potential-modulated UV-vis reflectance spectroscopy (electroreflectance spectroscopy, ER). We will show the presence of a threshold value of the surface negative charge density above which the rate of the ET of cyt c is significantly reduced. Experimental Section Reagents. MPA, MES, and MEL were purchased from Dojindo Laboratory Co., Aldrich, and Tokyo Kasei, respectively, and were used as received. Cytochrome c (Sigma type VI, horse heart) was purified with a cation exchange column (Whatman CM-52) and stored at 4 °C. Water was distilled and purified with a Milli-Q system (Millipore Co.). All other chemicals were of reagent grade and used without further purification. Gold substrates were prepared by vapor deposition of gold (99.99% purity) onto freshly cleaved mica.47 Sample Preparation. A single-component SAM of MPA, MES, or MEL was formed by placing a gold substrate in a 1 mmol dm-3 ethanolic solution of the thiol for 20 h. Mixed SAMs of MES and MEL were prepared by the immersion of gold substrates in a mixed ethanolic solution of the corresponding alkanethiols at a total thiol concentration of 1 mmol dm-3 for 25 (40) Waldmeyer, B.; Bechtold, R.; Bosshard, H. R.; Poulos, T. L. J. Biol. Chem. 1982, 257, 6073-6076. (41) Bond, A. M. Inorg. Chim. Acta 1994, 226, 293-340. (42) Rieder, R.; Bosshard, H. R. J. Biol. Chem. 1980, 255, 47324739. (43) Creager, S. E.; Clarke, J. Langmuir 1994, 10, 3675-3683. (44) Kakiuchi, T.; Iida, M.; Imabayashi, S.; Niki, K. Langmuir 2000, 16, 5397-5401. (45) Chen, X.; Ferrigno, R.; Young, J.; Whitesides, G. M. Langmuir 2002, 18, 7009-7015. (46) Du, Y.-Z.; Saavedra, S. S. Langmuir 2003, 19, 6443-6448. (47) Imabayashi, S.; Iida, M.; Hobara, D.; Feng, Z. Q.; Niki, K.; Kakiuchi, T. J. Electroanal. Chem. 1997, 428, 33-38.

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Figure 1. Cyclic voltammograms of cyt c adsorbed on MES/Au mica (a,b) and MPA/Au mica (c,d) measured at the sweep rate of 0.2 V s-1 in 0.01 mol dm-3 sodium phosphate buffer at pH 7 (a,c) and pH 9 (b,d). The ionic strengths of the buffer were 0.03 (solid curves) and 0.13 (dashed curves) mol dm-3. Electrode area, 0.5 cm2. ( 5 h. The substrate was taken out from the thiol solution, rinsed with ethanol, and then dried in air. The composition of the mixed SAM was controlled by altering the mixing ratio of the mixed soln thiol solution, χMES . A small amount of 10 mmol dm-3 sodium phosphate buffer (pH ) 7) containing 200 µmol dm-3 cyt c was put on the top of a thiol-modified gold substrate and left for 1 h. The substrate was then rinsed with the buffer to remove loosely adsorbed cyt c. Electrochemical Measurements. The cell configurations for evaporated gold substrates used in the electrochemical measurements and the procedure for evaluating the ket of cyt c adsorbed on the SAMs by ER spectroscopy have been described previously.38,47,48 All electrochemical measurements of cyt c were performed at 25 ( 2 °C in a deaerated 10 mmol dm-3 sodium phosphate buffer. The ionic strength was adjusted by adding an appropriate amount of NaCl. All potential values were referred to a Ag|AgCl|saturated KCl reference electrode.

Results and Discussion Redox Properties of Cyt c Adsorbed on SingleComponent SAMs of MES and MPA. Figure 1 shows cyclic voltammograms (CVs) of cyt c adsorbed on MES/Au mica (a,b) and MPA/Au mica (c,d) measured at the sweep rate of 0.2 V s-1 in 0.01 mol dm-3 sodium phosphate buffer at pH 7 (a,c) and pH 9 (b,d) whose ionic strength is 0.03 mol dm-3 (solid curves) or 0.13 mol dm-3 (dashed curves). The voltammetric responses were stable for repeated voltammetric measurements in the buffer for at least 3 h. The peak currents were proportional to the sweep rate in both SAM systems, as expected for the redox response of adsorbed redox species.49 The midpoint potential, E1/2, was 55 ( 10 mV for cyt c/MPA/Au and was 58 ( 3 mV for cyt c/MES/Au, values that are close to the E1/2 value of cyt c in an aqueous solution, 65 mV.50,51 The amount of (48) Feng, Z. Q.; Sagara, T.; Niki, K. Anal. Chem. 1995, 67, 35643570. (49) Bard, A. J.; Faulkner, L. R. Electrochemical Methods, 2nd. ed.; John Wiley & Sons: New York, 2001; Chapter 14. (50) Heineman, W. R.; Norris, B. J.; Goelz, J. E. Anal. Chem. 1975, 47, 79-84. (51) Dolla, A.; Blanchard, L.; Guerlesquin, F.; Bruschi, M. Biochimie 1994, 76, 471-479.

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Imabayashi et al. Table 1. Midpoint Potential (E1/2), Surface Coverage (Γe), and Rate Constant of Electron Transfer from the Electrode (ket) of Cyt c Adsorbed on Gold Electrodes in 0.01 mol dm-3 Sodium Phosphate Buffer SAM MPA MES MEL soln c MES/MEL (χMES ) (0.50) (0.75) (0.88) (0.92)

Figure 2. ER spectra of cyt c adsorbed on MES/Au mica (solid line) and MPA/Au mica (dashed line) measured in 0.01 mol dm-3 sodium phosphate buffer. Modulation amplitude, 50 mV; frequency, 14 Hz; monitored potential, -0.05 V; electrode area, 3.0 cm2.

electroactive cyt c, Γe, estimated from the peak area was 5.9 ( 1.2 pmol cm-2 for cyt c/MES/Au and was 10.0 ( 1.0 pmol cm-2 for cyt c/MPA/Au. The latter value is slightly smaller than the surface coverage of electrostatically attached cyt c on the SAMs of COOH-terminated alkanethiols with less than 11 methylene units, 13.5 pmol cm-2.22 An increase in the ionic strength of the phosphate buffer from 0.03 to 0.13 mol dm-3 at pH 7 resulted in a 5-fold decrease in the peak area of cyt c for both SAMs as shown by the dashed curves in Figure 1a,c. The redox response did not recover by the subsequent replacement of the solution with the buffer, indicating that cyt c molecules desorbed from the acid-terminated SAM surfaces presumably because of the decrease in the electrostatic interaction. Figure 2 shows the real part of ER spectra for cyt c adsorbed on the two SAMs of MES and MPA at pH 7. The R, β, and Soret bands appeared at 540, 520, and 420 nm in the ER spectra, respectively, which agree well with the difference absorption spectrum between the reduced and oxidized states of cyt c.52 This agreement suggests that the structure surrounding the heme in the electroactive cyt c was not significantly altered upon the adsorption. The values of ket evaluated from the frequency dependence of the ER response at pH 7 are summarized in Table 1. There exists a large difference in the ket values between cyt c/MES/Au (100 ( 15 s-1) and cyt c/MPA/Au (1450 ( 210 s-1). The latter value is comparable to the values reported for cyt c adsorbed on COOH-terminated SAMs with a similar chain length.38,53 In contrast with the present result, no CV peak was observed for cyt c adsorbed on the SAM of mercaptoundecane sulfonate, which was ascribed to an orientation of adsorbed cyt c that is inappropriate for the ET between the heme and the electrode.45 pH Dependence of the Redox Properties of Cyt c Adsorbed on Single-Component SAMs of MES and MPA. We studied the pH dependence of the redox reactions of cyt c adsorbed on the SAMs in the pH range between 6 and 9 where there is no appreciable change in the conformation of cyt c.54 With the increase in pH from 7 to 9, the ket for cyt c/MPA/Au decreased approximately (52) Sagara, T.; Murakami, H.; Igarashi, S.; Sato, H.; Niki, K. Langmuir 1991, 7, 3190-3196. (53) Ruzgas, T.; Wong, L.; Gaigalas, A. K.; Vilker, V. L. Langmuir 1998, 14, 7298-7305. (54) Scott, R. A.; Mauk, A. G. In Cytochrome c: A Multidisiplinary Approach; University Science Books: Sausalito, CA, 1996; Chapter 19.

E1/2 (mV)a

Γe (×10-12 mol cm-2)a

ket (s-1)b

55 ( 10 10.0 ( 1.0 (pH 7) 1450 ( 210 (pH 7) 53 ( 11 5.3 ( 1.1 (pH 9) 890 ( 100 (pH 9) 58 ( 3 5.9 ( 1.2 (pH 7,9) 100 ( 15 (pH 7, 9) 56 ( 10 3.5 ( 0.4 (pH 7) 800 (pH 7) 58 ( 15 58 60 60

5.1 ( 0.5 (pH 7) 8.6 (pH 7) 6.9 (pH 7) 7.7 (pH 7)

1050 (pH 7) 550 (pH 7) 1100 (pH 7) 110 (pH 7)

a Determined from CV measurements. b Determined from the dependence of the ER response on the frequency of potential modulation. c Mole fraction of MES in the mixed thiol solution for preparing SAMs in which the total thiol concentration is 1 mmol dm-3.

to two-thirds, whereas the ket for cyt c/MES/Au is almost constant in the same pH range (Table 1). The surface pK value of MPA SAM is 8.0.44 This result suggests that the negative charges on the SAM surface are responsible for the decrease in ket. The negative charges at SAM surfaces affect not only the ket but also the Γe value as shown in the voltammograms of Figure 1b,d. The Γe value for cyt c/MPA/Au decreased from 10.0 ( 1.0 pmol cm-2 at pH 7 (c) to 5.3 ( 1.1 at pH 9 (d), and the latter value is close to the Γe value for cyt c/MES/Au. Interestingly, the Γe value was restored to the initial value when the pH was brought back to 7 by replacing the solution with one at pH 7. This means that the decrease in the Γe with the solution pH is not caused by desorption of cyt c from the SAM surface. Three possible reasons for the pH dependence of Γe in the cyt c/MPA/Au system are the change in the cyt c conformation, the change in the orientation of adsorbed cyt c, and the alteration of the ET pathway between the heme and the electrode surface. The “gating mechanism”55 was proposed for the ET reaction of cyt c adsorbed on COOH-terminated SAMs, in which the rearrangement of cyt c from the stable binding orientation to the orientation optimal for efficient ET is required prior to the ET event.23 In the case of the ET through the SAMs of COOH-terminated alkanethiols with fewer than 6 methylene units, the apparent ket value is independent of the chain length38 and the rearrangement of the cyt c orientation is considered to be the rate-limiting step. For the MPA SAM at higher pH values and for the MES SAM, it is highly probable that the rearrangement of cyt c, whose large dipole moment is estimated to be 325 D,56 in an electric field generated by the surface charge of the SAM becomes more difficult and the fraction of electroactive cyt c decreases, resulting in the decrease in ket at higher pH values. On the other hand, the net positive charge on the cyt c surface decreases from +9 to +7 with increasing the solution pH from 7 to 9.57 This can lead to the decrease in electrostatic interaction between cyt c and the electrode. In the present results shown in Table 1, with raising the pH from 7 to 9, the ket for cyt c/MES/Au remained constant and the ket for cyt c/MPA/Au decreased. The ET rate does (55) Hoffman, B. M.; Ratner, M. A. J. Am. Chem. Soc. 1987, 109, 6237-6243. (56) Koppenol, W. H.; Margoliash, E. J. Biol. Chem. 1982, 257, 44264437. (57) Mattew, J. B.; Friend, S. B.; Boteiho, L. H.; Lehman, L. D.; Hanania, G. I. H.; Gurd, F. R. N. Biochem. Biophys. Res. Commun. 1978, 81, 416-421.

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Figure 4. Dependence of the midpoint potential (a), surface coverage (a), and ET rate (b) on χsoln MES for cyt c adsorbed on the mixed SAMs of MEL and MES/Au mica measured in 0.01 mol dm-3 sodium phosphate buffer.

Figure 3. Cyclic voltammograms of cyt c adsorbed on MEL/ Au mica (a) and a mixed SAM of MEL and MES (χsoln MES ) 0.75)/ Au mica (b) measured at the sweep rate of 0.2 V s-1 in 0.01 mol -3 dm sodium phosphate buffer. The ionic strengths of the buffer were 0.03 (solid curves) and 0.13 (dashed curves) mol dm-3. Electrode area, 0.5 cm2.

not, therefore, seem to reflect the change in the positive net charge of cyt c from +9 to +7 in the pH range studied. Redox Properties of Cyt c Adsorbed on Mixed SAMs of MES and MEL at Different Negative Surface Charge Densities. To further elucidate the effect of the charge density at the SAM surface on the ET rate of cyt c, we examined the redox response of cyt c on mixed SAMs composed of a thiol with an ionized tail group, MES, and a diluent thiol without charge, MEL, at varied mixing ratios. When a methyl-terminated alkanethiol was used as the diluent thiol, no redox response of cyt c was obtained at a mole fraction of the diluent thiol in solution of greater than 0.5, probably due to the denaturation of adsorbed cyt c on the hydrophobic surfaces.37 On the other hand, it was reported that short-chain OH-terminated thiolmodified electrodes gave a quasi-reversible CV peak for cyt c in the aqueous solution.58 In fact, Figure 3a shows that cyt c adsorbed on the SAM of MEL gives a redox peak at 56 ( 10 mV. The Γe value on the MEL SAM was 3.5 ( 0.4 pmol cm-2, which is smaller than those on the singlecomponent SAMs of MES and MPA, though 90% of the response was retained after the ionic strength was raised to 0.13 mol dm-3. The mechanism of the adsorption of cyt c on the MEL SAM is thus likely to be different from that on the negatively charged SAMs. The ket for the cyt c/MEL/ Au was 800 s-1 at pH 7. Figure 3b displays CVs of cyt c immobilized on a mixed SAM of MES and MEL prepared from the mixed thiol solution in 0.01 mol dm-3 phosphate buffer at pH 7 at the mole fraction of MES in the mixed thiol solution, χsoln MES, of 0.75. The ionic strength dependence of the redox response (solid CV vs dashed CV) indicates the electrostatic nature of the cyt c adsorption. The formal potential was 58 mV (58) Terrttaz, S.; Cheng, J.; Miller, C. J.; Guiles, R. D. J. Am. Chem. Soc. 1996, 118, 7857-7858.

and does not significantly depend on the SAM composition as shown in Figure 4a (O). The convex-shaped relationship of Γe vs χsoln MES having the maximum of the Γe value 8.6 pmol cm-2 at χsoln MES ) 0.75 in Figure 4a (9) suggests the existence of the surface negative charge density that is optimal for the adsorption of cyt c in an electroactive state. Figure 4b shows the ket for cyt c adsorbed on the mixed SAMs of MES and MEL as a function of χsoln MES. The ER levels agreed with the difference spectra at all χsoln MES absorption spectrum between the reduced and oxidized states of cyt c (data not shown). The ket values determined from the frequency dependence of the ER response at χsoln MES < 0.9 were comparable to that for cyt c on the singlecomponent SAM of MEL. However, at χsoln MES > 0.9, the ket dramatically decreased to 110 s-1, which is comparable to the value obtained above for the single-component SAM of MES. This sharp decrease in ket indicates the presence of the threshold level of the negative surface charge density above which the ET rate of cyt c dramatically decreased. We estimated the surface mole fraction of MES, χsurf MES, from the double layer capacitance (Cdl) of mixed SAMs. As evidenced from the CVs in Figures 1 and 3, the Cdl for cyt c/MES/Au (17.6 ( 0.3 µF cm-2) is greater than that for cyt c/MEL/Au (12.1 ( 0.4 µF cm-2). Assuming that the Cdl of the mixed SAMs is described by Frumkin’s two-parallelplate model,59 which has been successfully used to describe the adsorption of organic molecules on electrodes,60 surf MES surf Ctotal ) CMEL dl dl (1 - χMES) + Cdl χMES

where CMEL and CMES are the Cdl values of the singledl dl component SAMs of MEL and MES, respectively. The ket vs χsurf MES plot (Figure 5b) was derived from the relationship and χsoln between Ctotal dl MES (Figure 5a). The sharp decrease in ket occurs around χsurf MES ) 0.5. Assuming that the mixed SAM forms a (x3 × x3)R30° layer with the coverage of 7.7 × 10-10 mol cm-2,61 the threshold negative surface (59) Frumkin, A. N. Z. Phys. 1926, 35, 792-802. (60) Damaskin, B. B.; Petrii, O. A.; Batrakov, V. V. In Adsorption of Organic Compounds on Electrodes; Plenum Press: New York, 1971. (61) Finklea, H. O. In Electroanalytical Chemistry, Vol. 19; Bard, A. J., Rubinstein, I., Eds.; Marcel Dekker: New York, 1996; p 161.

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Conclusion Understanding the adsorbed state and the redox properties of proteins on SAM surfaces is important in the application of SAMs for biomaterials, biosensors, and bioanalytical systems. SAMs having ionizable surface groups have been widely used for the electrostatic immobilization of proteins on the SAM.15-19,22,62-65 This work points out that although the negative charge on the SAM surface is necessary for the electrostatic adsorption of cyt c, excess surface negative charge can be detrimental to the ET between cyt c and the electrode. This reduction of the ET rate probably results from the confinement by the strong electrostatic force of adsorbed cyt c to an orientation that is different from the optimal electronic coupling geometry. Taking into account the inhomogeneous and sparse distribution of charged amino acid residues on the interaction domain of the protein reaction partners for cyt c, SAM systems having an optimal surface charge distribution are decisive to model the biological ET reactions of cyt c on the SAMs.

surf Figure 5. Plots of Cdl vs χsoln MES (a) and ket vs χMES (b) for cyt c adsorbed on mixed SAMs of MEL and MES/Au mica measured in 0.01 mol dm-3 sodium phosphate buffer.

χsurf MES

charge density to retard the ET of cyt c at ) 0.5 is estimated to be 3.8 × 10-10 mol cm-2, which corresponds to the negative charge density on the MPA surface at the surface pK value, pH 8.

Acknowledgment. This work was supported by a Grant-in-Aid for Scientific Research (No. 14205120) and a Grant-in-Aid for Exploratory Research (No. 15655008) (T.K.). This work was partly supported by the Casio Science Promotion Foundation (S.I.). LA047992X (62) Wadu-Mesthrige, K.; Amro, N. A.; Liu, G.-Y. Scanning 2000, 22, 380-388. (63) Narvaez, A.; Suarez, G.; Popescu, I. C.; Katakis, I.; Dominguez, E. Biosens. Bioelectron. 2000, 15, 43-52. (64) Du, Y.-Z.; Saavedra, S. S. Langmuir 2003, 19, 6443-6448. (65) Zhou, D.; Wang, X.; Birch, L.; Rayment, T.; Abell, C. Langmuir 2003, 19, 10557-10562.