NANO LETTERS
Preferential Adsorption of Horse Heart Cytochrome c on Nanometer-Scale Domains of a Phase-Separated Binary Self-Assembled Monolayer of 3-Mercaptopropionic Acid and 1-Hexadecanethiol on Au(111)
2002 Vol. 2, No. 9 1021-1025
Daisuke Hobara,† Shin-ichiro Imabayashi,‡ and Takashi Kakiuchi*,† Department of Energy and Hydrocarbon Chemistry, Graduate School of Engineering, Kyoto UniVersity, Kyoto 606-8501, Japan, and Department of Chemistry and Biotechnology, Faculty of Engineering, Yokohama National UniVersity, Yokohama 240-8501, Japan Received July 8, 2002; Revised Manuscript Received July 26, 2002
ABSTRACT Cytochrome c preferentially adsorbs on the nanometer-scale 3-mercaptopropionic acid (MPA) domains of the phase-separated binary selfassembled monolayers (SAMs) of MPA and 1-hexadecanethiol on Au(111), which has been imaged by scanning tunneling microscopy (STM). The amount of the adsorbed cytochrome c estimated by STM increases with increasing the ratio of MPA of the binary SAMs and agrees well with that estimated by cyclic voltammetry of the cytochrome c-adsorbed Au(111).
The present paper aims to show the two-dimensional distribution of cytochrome c adsorbed on phase-separated binary self-assembled monolayers (SAMs) of thiol derivatives1-10 on the nanometer scale. Adsorption and covalent immobilization of protein molecules on the binary SAMs have been studied to control the coverage,11-15 orientation,16-18 activity,19 molecular recognition,20,21 lateral steric interaction,22 and electron-transfer properties23-25 of the proteins. The elucidation of the location and the density of the protein molecules at the surfaces is important to understanding the microenvironment of the proteins, which affects their conformation, enzymatic activity,19 and other properties.23 Few studies, however, have been reported to show the twodimensional distribution of protein molecules at the binary SAMs on the nanometer scale,13,20,26 whereas the micrometerscale two-dimensional distribution of patterned protein monolayers has been observed using scanning electron microscopy.27-32 The binary SAMs formed by the coadsorption of 3-mercaptopropionic acid (MPA) and 1-hexadecanethiol (HDT) on Au(111) is a typical system that exhibits phase separation * Corresponding author. Telephone: +81-75-753-5528, Fax: +8175-753-3360; E-mail:
[email protected]. † Kyoto University. ‡ Yokohama National University. 10.1021/nl0256863 CCC: $22.00 Published on Web 08/14/2002
© 2002 American Chemical Society
on the nanometer scale.4,8,33-35 Scanning tunneling microscopy (STM) measurements of the binary SAMs revealed that two different types of domains mainly consist of either MPA or HDT.4 Because cytochrome c strongly adsorbs on the surfaces of the single component SAMs of the COOHterminated alkanethiol,24,36-45 the binary SAMs are expected to provide the surfaces where cytochrome c preferentially adsorbs on the nanometer-scale domains of MPA. In the present paper, the binary SAMs of MPA and HDT have been used to control the two-dimensional distribution of the adsorbed cytochrome c on the nanometer scale. The preferential adsorption of cytochrome c was confirmed by STM, X-ray photoelectron spectroscopy (XPS), and cyclic voltammetry of the cytochrome c-adsorbed Au(111). Horse heart cytochrome c (Sigma, type VI) was purified chromatographically as described elsewhere.46 Au(111) films were vapor deposited on freshly cleaved mica sheets (Nilaco Co.) at less than 3 × 10-6 Torr.47,48 A binary SAM of MPA (Dojin Chemical Laboratory) and HDT (Aldrich Chemical Co.) was formed on Au(111) by immersing a Au substrate in the mixed thiol solution for more than 24 h, followed by rinsing with ethanol and drying in air.4 The total concentration of thiols was 1.0 × 10-3 mol dm-3. The composition of the binary monolayers was changed by varying the ratio,
xsol MPA () cMPA/(cMPA + cHDT)), of HDT and MPA in the solution, where ci (i ) MPA or HDT) denotes the molar concentration of i. The ratio of MPA adsorbed on the surface, xsurf MPA () QMPA/(QMPA + QHDT)), was estimated from the charge under the voltammetric peaks of the reductive desorption49,50 of MPA (QMPA) and HDT (QHDT).4 Cytochrome c was adsorbed on the SAMs by immersing the SAM-formed substrates in a 3.0 × 10-2 mol dm-3 phosphate buffer solution (pH 7.0) containing 1.0 mol dm-4 cytochrome c for 10 min, followed by thorough rinsing with the buffer solution and purified water. Cyclic voltammograms were recorded in the buffer solution, which contains no cytochrome c. STM measurements were made under ambient conditions using NanoScope E (Digital Instruments) equipped with a low current converter (model: CSTMLC, Digital Instruments). Mechanically cut Pt/Ir tips were used for STM imaging. XPS measurements were made using model 5500 MT (ULVAC-PHI Co., Ltd.) with Mg KR radiation (15 kV, 400 W). The residual gas pressure in the chamber during the data acquisition was 5 × 10-9 Torr. Figure 1a shows an STM image of the phase-separated binary SAM of MPA and HDT formed from the mixed surf solution of xsol MPA ) 0.94 (corresponding to xMPA ) ∼0.5) on Au(111). The bright and the dark regions in Figure 1a correspond to the domains that are mainly composed of HDT and MPA, respectively. 4 The height difference between the two types of the domains is ∼0.5 nm.4 Figure 1b shows an STM image taken after adsorption of cytochrome c on the binary SAM. Cytochrome c molecules appeared as bright spots that were absent before the adsorption. The height of the spots was 1.6 ( 0.3 nm, which is much larger than that of the HDT domains observed in the image of the binary SAM (Figure 1a). The average size of a spot was 5.3 ( 1.1 nm in diameter. Both size and height of the spots are comparable to those of a cytochrome c molecule51,52 and consistent with the previous results of STM images of cytochrome c on electrode surfaces.53-57 The distribution of cytochrome c on the binary SAM was not uniform (Figure 1b). The cytochrome c molecules were found to form domains having a size of several tens of nanometers, which is comparable to the MPA domains of the binary SAM (Figure 1a). Figure 1 suggests that the cytochrome c molecules are located predominantly on the domains composed mainly of MPA and are not present on the HDT domains. Figure 2 shows STM images of cytochrome c adsorbed on the binary SAMs having different xsurf MPA. The bright spots similar to those observed in Figure 1b are clearly seen in Figure 2. The total area of the bright spots increased with increasing xsurf MPA (see Figure 4), indicating a larger total amount of adsorbed cytochrome c on the surface at larger xsurf MPA. This is consistent with the increase of the XPS signals in the N 1s region of the cytochrome c-adsorbed binary SAMs shown in Figure 3; the XPS signal from N 1s is attributed to that from the peptide chain of adsorbed proteins and is proportional to the average amount of adsorbed proteins.27,38,58,59 Figures 2 and 3 support that cytochrome c molecules are located only on the MPA 1022
Figure 1. STM images of (a) the binary SAM of MPA and HDT on Au(111) and (b) cytochrome c adsorbed on the binary SAM of MPA and HDT. The binary SAMs were formed from a solution of sol surf xMPA ) 0.94 (corresponding to xMPA ) 0.54). Bias voltage: 1.5 V; setpoint: (a) 10 pA, (b) 80 pA.
domains. Further evidence for the preferential adsorption of cytochrome c comes from the comparison of the STM data with those from cyclic voltammetry of the cytochrome c-adsorbed Au(111) electrodes. The electrode reaction of cytochrome c at gold surfaces modified with two different thiol derivatives is sensitive to the composition24,25,60-62 and the mixing states of the two thiol derivatives.63 Cyclic voltammograms for the cytochrome c-adsorbed Au(111) modified with the binary SAMs of MPA and HDT showed anodic and cathodic peaks at 50 mV, which are originated from the redox reaction of Nano Lett., Vol. 2, No. 9, 2002
Figure 2. STM images of cytochrome c adsorbed on the binary SAMs of MPA and HDT. The binary SAMs were formed from solutions sol surf surf sol sol ) 0.90 (corresponding to xMPA ) 0.20), (b) xMPA ) 0.94 (corresponding to xMPA ) 0.54), and (c) xMPA ) 0.95 (corresponding of (a) xMPA surf ) 0.77). Bias voltage: (a) 1.7 V, (b) 1.5 V, (c) 1.5 V; setpoint: (a) 80 pA, (b) 80 pA, (c) 50 pA. to xMPA
Figure 3. XPS signal intensity from N 1s region of the cytochrome c-adsorbed Au(111) modified with the binary SAMs of MPA and surf HDT as a function of xMPA .
cytochrome c adsorbed on COOH-terminated alkanethiol SAMs.24,36-39,41,43-45,64 The amount of the adsorbed cytochrome c was estimated from the peak area of the voltammograms by assuming a one-electron reaction and plotted against xsurf MPA in Figure 4. The amount of the adsorbed cytochrome c was also estimated from the STM images and plotted in Figure 4. Total area of the bright spots was obtained by the binarization of the image, assuming 10 nm2 molecules-1 of the occupied area for an adsorbed cytochrome c.38 The estimation at xsol MPA > 0.95 was not possible due to the low contrast of the image, which is probably caused by covering almost all the imaged area with cytochrome c. The amount of the adsorbed cytochrome c estimated from the STM images well agrees with that from cyclic voltammetry (Figure 4); the bright spots in the STM images are ascribed to electrochemically active cytochrome c molecules preferentially adsorbed on the MPA domains of the binary SAMs. The amount of the adsorbed cytochrome c estimated by cyclic voltammetry levels off at xsurf MPA > 0.77 (Figure 4), while the XPS intensity of the adsorbed cytochrome c increases beyond xsurf MPA > 0.77. The difference between the voltammetry and XPS results indicates that the amount of the electrochemically active cytochrome c does not increase Nano Lett., Vol. 2, No. 9, 2002
Figure 4. Amount of the adsorbed cytochrome c on the binary SAMs of MPA and HDT estimated from the STM images (b) and surf the cyclic voltammograms (O) as a function of xMPA . The adsorbed amount was estimated from the STM images by assuming that one cytochrome c molecule occupies 10 nm2. The inset shows typical voltammograms of the adsorbed cytochrome c on the binary surf SAMs (xMPA ) 0.11, 0.54, 0.77). The voltammograms were recorded at a scan rate of 50 mV s-1.
in proportion to the total amount of the adsorbed cytochrome c when xsurf MPA > 0.77. The adsorbed amount estimated by cyclic voltammetry (Figure 4) indicates that only a certain fraction of adsorbed cytochrome c molecules are in the orientation suitable for the electron-transfer reaction44,65,66 24 even at xsol MPA ) 1.00. The adsorbed amount estimated by voltammetry showed a large scattering at xsurf MPA ) 0.92, as shown in Figure 4. The SAMs contain only ∼8% of HDT at xsurf MPA ) 0.92. Such a small amount of HDT might play an important role in the cytochrome c binding. Recent studies have shown that the electron-transfer rate of cytochrome c adsorbed on binary SAMs composed of HS(CH2)10COOH and HS(CH2)9CH324 or HS(CH2)7OH and HS(CH2)10COOH25 becomes larger than that observed for cytochrome c adsorbed on the singlecomponent HS(CH2)10COOH SAM. The amount of cytochrome c adsorbed on the binary SAM of HS(CH2)10OH and HS(CH2)13COOH also increased compared to that obtained 1023
with cytochrome c adsorbed on the single-component HS(CH2)13COOH SAM.59 These results suggest that the subtle difference in the orientation of the adsorbed cytochrome c influences the electron-transfer reaction. This may also be related to a recent suggestion that the defects in the SAM can affect the electron transfer of the adsorbed cytochrome c.59 In conclusion, the STM imaging of cytochrome c adsorbed on phase-separated binary SAMs of MPA and HDT elucidated the preferential adsorption of adsorbed cytochrome c molecules. The selective adsorption of protein molecules on binary SAMs such as those reported using micrometer-scale patterning of SAMs27-30 can be achieved using phaseseparated binary SAMs on the nanometer scale. Because the mixing state and the nanometer structure of the binary SAMs can be controlled by changing the combination of the thiol derivatives and the preparation of the SAMs,3,5,9,67 the selective adsorption of proteins on binary SAMs can be applied to the nanometer-scale design of biosensing systems.68-71 Acknowledgment. This work was partially supported by a Grant-in-Aid for Encouragement of Young Scientists No. 12750731 from the Ministry of Education, Culture, Sports, Science and Technology of Japan (D.H.) and a grant from CREST of JST (Japan Science and Technology) (D.H. and T.K.). References (1) Stranick, S. J.; Parikh, A. N.; Tao, Y.-T.; Allara, D. L.; Weiss, P. S. J. Phys. Chem. 1994, 98, 7636. (2) Tamada, K.; Hara, M.; Sasabe, H.; Knoll, W. Langmuir 1997, 13, 1558. (3) Ishida, T.; Mizutani, W.; Tokumoto, H.; Hokari, H.; Azehara, H.; Fujihira, M. Appl. Surf. Sci. 1998, 130-132, 786. (4) Hobara, D.; Ota, M.; Imabayashi, S.; Niki, K.; Kakiuchi, T. J. Electroanal. Chem. 1998, 444, 113. (5) Hobara, D.; Ueda, K.; Imabayashi, S.; Yamamoto, M.; Kakiuchi, T. Electrochemistry 1999, 67, 1218. (6) Chen, S.; Li, L.; Boozer, C. L.; Jiang, S. Langmuir 2000, 16, 9287. (7) Sawaguchi, T.; Sato, Y.; Mizutani, F. J. Electroanal. Chem. 2001, 496, 50. (8) Munakata, H.; Kuwabata, S.; Ohko, Y.; Yoneyama, H. J. Electroanal. Chem. 2001, 496, 29. (9) Hobara, D.; Kakiuchi, T. Electrochem. Commun. 2001, 3, 154. (10) Shimazu, K.; Kawaguchi, T.; Isomura, T. J. Am. Chem. Soc. 2002, 124, 652. (11) Prime, K. L.; Whitesides, G. M. J. Am. Chem. Soc. 1993, 115, 10714. (12) Mrksich, M.; Sigal, G. B.; Whitesides, G. M. Langmuir 1995, 11, 4383. (13) Patel, N.; Davies, M. C.; Hartshorne, M.; Heaton, R. J.; Roberts, C. J.; Tendler, S. J. B.; Williams, P. M. Langmuir 1997, 13, 6485. (14) Sigal, G. B.; Mrksich, M.; Whitesides, G. M. J. Am. Chem. Soc. 1998, 120, 3464. (15) Kro¨ger, D.; Liley, M.; Schiweck, W.; Skerra, A.; Vogel, H. Biosens. Bioelectron. 1999, 14, 155. (16) Ta, T. C.; McDermott, M. T. Anal. Chem. 2000, 72, 2627. (17) Madoz-Gu´rpide, J.; Abad, J. M.; Ferna´ndez-Recio, J.; Ve´lez, M.; Va´zquez, L.; Go´mez-Moreno, C.; Ferna´ndez, V. M. J. Am. Chem. Soc. 2000, 122, 9808. (18) Tronin, A.; Edwards, A. M.; Wright, W. W.; Vanderkooi, J. M.; Blasie, J. K. Biophys. J. 2002, 82, 996. (19) Guiomar, A. J.; Guthrie, J. T.; Evans, S. D. Langmuir 1999, 15, 1198. (20) Haussling, L.; Michel, B.; Ringsdorf, H.; Rohrer, H. Angew. Chem., Int. Ed. Engl. 1991, 30, 569. (21) Nelson, K. E.; Gamble, L.; Jung, L. S.; Boeckl, M. S.; Naeemi, E.; Golledge, S. L.; Sasaki, T.; Castner, D. G.; Campbell, C. T.; Stayton, P. S. Langmuir 2001, 17, 2807. 1024
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