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Surface Modification of a Self-Assembled Ferredoxin Monolayer on a Gold Substrate by CHAPS Jong Bum Lee, Soong Ho Um, Jeong-Woo Choi, and Kee-Kahb Koo* Department of Chemical and Biomolecular Engineering, Sogang University, Seoul 121-742, Korea Received May 29, 2003. In Final Form: July 18, 2003 A self-assembled ferredoxin monolayer on a gold substrate was prepared, and surface modification to remove the protein aggregates was performed with a zwitterionic surfactant, (3-[(3-cholamidopropyl) dimethylammonio]-1-propanesulfonate) (CHAPS). It was found that CHAPS segregates most of the ferredoxin aggregates nonspecifically adsorbed on the substrate. The atomic force microscopy image of the self-assembled ferredoxin monolayer taken from the substrate with CHAPS treatment shows that the size of the ferredoxin clusters is about 10-20 nm, which is on the order of clusters of 2-3 ferredoxin molecules. On the other hand, the size of ferredoxin aggregates without CHAPS treatment was measured to be about 100 nm. The current-voltage relationship of the self-assembled ferredoxin monolayer with CHAPS treatment was confirmed to remain intact by means of cyclic voltammetry measurements. Those results demonstrate that the elimination of the protein aggregates nonspecifically adsorbed on an inorganic surface is possible without losing the electrochemical property of protein molecules and thus this technique would be useful to improve the long-term stability of biomolecular electronic devices.
Introduction Current study on the fabrication of biomolecular electronic devices as an alternative to silicon technology facing the process limit in miniaturization of integrated circuits shows the possibility for the realization of molecular-sized circuitry soon.1-10 For instance, various artificial biomolecular photodiodes or electronic diodes have been proposed by a biomimetic approach using metalloproteins and their current-voltage characteristics and photoswitching effect have been successfully demonstrated.10-13 In the fabrication of artificial bioelectronic devices, the self-assembly technique for the molecular patterning of metalloproteins is commonly adopted due to the stable and uniform surface structure and relatively easy modification of functionalities. Preparation of thin films by the self-assembly technique is very important because the performance of the device is dependent on the characteristics of the molecular pattern formed on the substrate. In particular, aggregation of proteins on the substrate is an unavoidable problem because protein aggregates usually hinder the orientation of proteins which is important in a biomolecular device. Therefore, this problem should be eliminated for the long(1) Weetall, H. Biotechnol. Prog. 1999, 15, 963-963. (2) Krysinki, P.; Tien, H.; Ottova, A. Biotechnol. Prog. 1999, 15, 974990. (3) Bae, Y.-S.; Yang, J.; Jin, S.-I.; Lee, S.-Y.; Park, C.-H. Biotechnol. Prog. 1999, 15, 971-973. (4) Willner, I.; Willner, B. Biotechnol. Prog. 1999, 15, 991-1002. (5) Roberto, C.; Ross, R.; Giuseppe, M.; Adriana, B. Physica E 2002, 13, 1229-1235. (6) Ben-Jacob, E.; Hermon, Z.; Caspi, S. Phys. Lett. A 1999, 263, 199-202. (7) Chaki, N. K.; Vijayamohanan, K. Biosens. Bioelectron. 2002, 17, 1-12. (8) Willner, I. Nat. Biotechnol. 2001, 19, 1023-1024. (9) Willner, I.; Willner, B. Trends Biotechnol. 2001, 19, 222-230. (10) Choi, J.-W.; Park, S.-J.; Nam, Y.-S.; Lee, W.-H.; Fujihira, M. Colloids Surf., B 2002, 23, 295-303. (11) Choi, J.-W.; Nam, Y.-S.; Kong, B.-S.; Choi, H.-G.; Lee, W.-H.; Fujihira, M. Colloids Surf., B 2002, 23, 263-271. (12) Koyama, K.; Yamaguchi, N.; Miyasaka, T. Science 1994, 265, 762-765. (13) Choi, J.-W.; Park, S.-J.; Yoo, C.-J.; Oh, S.-Y.; Lee, W.-H. Mol. Cryst. Liq. Cryst. 2002, 377, 253-256.
term stability of a proposed device. Protein aggregates are commonly observed at the isoelectric point of a protein sample, in which the solubility of the protein drops down rapidly. Accordingly, the change of pH could lead to the dissolution of the protein in solution.14 However, the pH alteration often transforms the three-dimensional folding structure into a linear amino acid sequence and subsequently the protein is deactivated. As an effort for the control of the size of aggregates during the pattern formation of protein onto a metal substrate, Choi et al.10,13 have used a porous cellulose membrane with a pore size of 200 nm as a pattern mask. They have shown that the size of the cytochrome c aggregates, which is about 300 nm without a mask, is controlled to be about the pore size of the membrane. However, the size of the cytochrome c molecule is generally on the order of 5 nm and thus their attempt to eliminate the protein aggregates is not successful. The segregation problem of proteins may be resolved by a surfactant.15-18 Harder et al.17 and Sigal et al.18 have reported the interaction between proteins and a solid surface with self-assembled specific functional groups. They found that a number of functional groups have an ability to prevent nonspecific adsorption of sample proteins on the solid support. However, the deformation of the three-dimensional structure of the protein and the loss of biological activity are often observed due to the strong affinity between protein molecules and the surfactant.19-21 (14) Scopes, R. K. Protein Purification: Principle and Practice; Springer-Verlag: New York, 1994. (15) Kimitto, A.; Holzenburg, A.; Ford, R. C. J. Biol. Chem. 1997, 272, 19497-19501. (16) Crispin, D. J.; Street, G.; Valey, J. E. Food Chem. 2001, 72, 355-362. (17) Harder, P.; Grunze, M.; Dahint, R.; Whitesides, G. M.; Laibinis, P. E. J. Phys. Chem. B 1998, 102, 426-436. (18) Sigal, G. B.; Mrksich, M.; Whitesides, G. M. J. Am. Chem. Soc. 1998, 120, 3464-3473. (19) Green, R. J.; Su, T. J.; Lu, J. R.; Webster, J. R. P. J. Phys. Chem. B 2001, 105, 9331-9338. (20) Poumier, F.; Schaaf, P.; Voegel, J. C. Langmuir 1999, 15, 62996303. (21) Wahlgren, M.; Arnebrant, T. Colloids Surf., B 1996, 6, 63-69.
10.1021/la034933v CCC: $25.00 © 2003 American Chemical Society Published on Web 09/18/2003
Surface Modification of Self-Assembled Monolayer
When a surfactant is applied in the fabrication of biomolecular electronic devices, protein aggregates should be removed without pH alteration in order to keep optimal activity of the protein. In this point of view, study on the interaction of a surfactant with self-assembled protein aggregates has not appeared to date. Ferredoxin, an Fe-S redox metalloprotein, is composed of 97 amino acids, its molecular weight is 11 000 Da, and it has optimal activity at pH ) 7.0. The ferredoxin molecule has an ellipsoidal shape with a size of 5 nm. Recently, we have investigated the interaction between ferredoxin and a surfactant in Trizmer buffer solution by dynamic light scattering analysis.22 Two sizes of ferredoxin aggregates were found to exist mainly in the solution without addition of any surfactant: 10 nm sized clusters, which seem to be on the order of a few ferredoxin molecules, and 120 nm sized aggregates. However, it was found that the ferredoxin aggregates are gradually segregated and ferredoxin can exist as a monomer in the solution when the ferredoxin aggregates are treated with a zwitterionic surfactant, CHAPS (3-[(3-cholamidopropyl) dimethylammonio]-1propanesulfonate).23-26 As the molar ratio of CHAPS to ferredoxin was increased up to 200, only 10 nm sized ferredoxin clusters were found to exist in the solution.22 In this paper, we report the fabrication method of the self-assembled ferredoxin monolayer onto a gold substrate by considering the molecular structure of ferredoxin. Successful elimination of the ferredoxin aggregates from the substrate by the CHAPS treatment is demonstrated by providing high-resolution atomic force microscopy (AFM) images of nanosized ferredoxin clusters on the (111) surface of the gold substrate. Finally, we show that the current-voltage relationship of the ferredoxin monolayer, which is the essential property as a molecular electronic diode, is not degraded by CHAPS treatment. Experimental Section Materials. All chemicals were used as received unless stated otherwise. Ferredoxin from Spinacia oleracea (lot number 71K7008), 11-mercaptoundecanoic acid (MUA), and 1-ethyl-3(3-dimethylaminopropyl)carbodiimide (EDC) were purchased from Sigma. CHAPS was purchased from Fluka, and ethanol was obtained from Hayman. Trizma buffer (pH 7.5) and phosphate buffer (pH 7.4) from Sigma were used. Gold substrates, prepared by e-beam evaporation of 1000 Å of gold onto the clean and polished (100) plane of a silicon singlecrystal wafer primed with an adhesion layer of about 100 Å of titanium, were purchased from Inostek (Korea). They were cleaned by dipping into piranha solution with a 3:1 (v/v) mixture of sulfuric acid and hydrogen peroxide for 10 min to remove organic residues on the surface and finally were rinsed with deionized water (DI water). The surface morphology and X-ray diffraction pattern of the gold film were investigated by atomic force microscopy (Park Scientific Instruments, Autotube CP) and an X-ray diffractometer (Rigaku, D/MAX-1C) with a monochromated beam of Cu KR, respectively. Figure 1 shows the polycrystalline gold film with grain sizes of about 200-300 nm and the X-ray diffraction pattern exhibiting a strong (111) texture. Methods. In the present experiments, self-assembled ferredoxin monolayers on the gold substrates were prepared by four steps as shown in Figure 2. (22) Lee, J. B.; Um, S. H.; Choi, J.-W.; Koo, K.-K. Colloids Surf., B, in press. (23) Kimitto, A.; Holzenburg, A.; Ford, R. C. J. Biol. Chem. 1997, 272, 19497-19501. (24) Carla, E. G.; Arnouldus, W. P. V.; Willem, N. Langmuir 2000, 16, 4853-4858. (25) Amitabha, C.; Harikumar, K. G. FEBS Lett. 1996, 391, 199202. (26) Buckley, J. J.; Wellaufer, D. B. J. Chromatogr., A 1991, 464, 61-71.
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Figure 1. AFM image and XRD pattern of the bare gold substrate used. Immobilization of MUA onto the Au Substrate. The gold plates were placed in a flat-bottomed cylindrical reactor with a Teflon slab with four supporting legs. MUA (5 mM) in ethanol was charged and reacted for 2 h at room temperature. The gold substrates modified with carboxyl groups were taken from the reactor and were washed with ethanol followed by DI water. The MUA/Au substrates were then dried with a gentle stream of high-purity N2. Linking of EDC to the MUA/Au Substrate. In order to activate the reaction of MUA with ferredoxin, EDC was taken as a coupling agent. The MUA-modified Au substrates and 10% (w/v) EDC were introduced into a reaction vessel containing 100 mM phosphate buffer solution (pH 7.4), and the reaction mixture was kept for 2 h at room temperature. The EDC-linked MUA/Au substrates were removed from the vessel and washed with phosphate buffer solution. Finally the substrates were washed by DI water and then dried with a gentle stream of high-purity N2. Water contact angle measurements were performed with a contact angle goniometer (Rame-Hart Inc., model 100) to confirm the attachment of MUA and EDC onto the gold surface. The attachment of the MUA and EDC moieties on the gold surface was further examined by X-ray photoelectron spectroscopy (XPS). X-ray photoelectron spectra of the samples were recorded on a ESCALAB 250 XPS spectrometer (VG Scientifics) with Al KR excitation (1486.6 eV). Formation of the Self-Assembled Ferredoxin Monolayer on the EDC/MUA/Au Substrate. Ferredoxin solution (9.8 µM) was prepared using 10 mM phosphate buffer for 10 h at room temperature, and the EDC/MUA/Au substrates were inserted into the solution for the preparation of the self-assembled ferredoxin monolayer. The MUA/Au substrates with the selfassembled ferredoxin monolayer were washed with DI water and dried with a gentle stream of high-purity N2. AFM images of the ferredoxin monolayer with its aggregates on the gold substrate were obtained for the comparison with those of the samples treated with CHAPS. Segregation of Ferredoxin Aggregates from the Surface of the Ferredoxin/MUA/Au Substrate. To remove ferredoxin aggregates from the surface of the ferredoxin-adsorbed MUA/Au substrates, the substrates were introduced into the Trizma buffer solution with 19.6 mM CHAPS. The molar ratio of CHAPS to
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Lee et al. Table 1. Average Values of the Water Contact Angles Measured for Surfaces of Bare and Chemically Treated Gold Substrates surfaces
θ/deg
bare gold substrate Au/MUA Au/MUA/EDC Au/MUA/ferredoxin (without CHAPS treatment) Au/MUA/ferredoxin (with CHAPS treatment)
84 14 56 64 62
Figure 3. Measured values of the water contact angles for the gold substrates modified by MUA, EDC, and ferredoxin.
Figure 2. Scheme for the preparation of the self-assembled ferredoxin monolayer onto the gold substrate by the selfassembly technique. ferredoxin was taken to be 200. Detailed experiments on the interaction between ferredoxin and CHAPS in the buffer solution are given elsewhere.22 The buffer solution containing CHAPS and the ferredoxin aggregates/MUA/Au substrates was stirred for 48 h with the aid of a small magnetic stirrer at room temperature. Finally, the ferredoxin/MUA/Au substrates were taken from the vessel and washed with copious amounts of Trizma buffer followed by DI water. The water-washed ferredoxin/MUA/ Au substrates were then dried with a gentle stream of highpurity N2. AFM images of the ferredoxin monolayer with CHAPS treatment were obtained for comparison with those of samples without CHAPS treatment. Redox Potential Measurements of Ferredoxin by Cyclic Voltammetry. For the measurements of the redox potential of ferredoxin, we prepared the electrode system which was composed of the ferredoxin-modified MUA/Au substrate as the working electrode, a Ag/AgCl electrode as the reference, and a platinum electrode as the counter. The reduction-oxidation spectra of ferredoxin were obtained using a cyclic voltammetry measurement system (Zahner, model IM6) in Trizma buffer solution.
Results and Discussion Confirmation of Immobilization of MUA and EDC onto the Gold Surface. To confirm the attachment of MUA and EDC onto the gold surface, water contact angles were measured for the surfaces of bare and chemically treated gold substrates as given in Table 1. In this table, values of the water contact angle represent the average values taken from 10 different regions on each sample at
10 s after water-dropping (see Figure 3). Those data show that the contact angle for the bare gold surface is very high as expected and that for the substrate with the carboxyl groups of MUA is the lowest. The contact angle measured on the EDC-modified MUA/Au substrate is about 56° due to hydrophilic nitro groups and hydrophobic methyl groups. The values measured on the surface of ferredoxin aggregates without CHAPS treatment and those measured on the ferredoxin monolayer treated with CHAPS are about 64° and 62°, respectively. Those results indicate that the surface of the ferredoxin-adsorbed MUA/ Au substrate might be a little hydrophobic compared with that of the EDC/MUA/Au substrate because the upper part of the ferredoxin surface is composed of hydrophobic amino acid groups (Ala 41, Arg 40, Cys 39, Cys 44, Val 22) and hydrophilic amino acid groups (Ser 43, Asp 21, Asp 60, Glu 30, Asp 26). X-ray photoelectron spectra also provide clear evidence for the surface modification reaction of the gold substrates. Figure 4 shows the high-resolution core level spectra in the N 1s and S 2p regions for various surfaces studied. The signals of S 2p photoelectrons on the surfaces of the substrates indicate that Au substrates are modified efficiently with MUA. The signal of the N 1s photoelectrons at 398 eV (binding energy) is shown to appear on the surface of the EDC/MUA/Au substrate, whereas any peaks for the N 1s photoelectrons are not observed from the MUA-coated Au surface. The binding energies of the collected spectra were calibrated against the Au 4f photoelectron signal as a reference (87.7 eV), and they are summarized in Table 2. Those data show that the surface is exactly treated with EDC. Confirmation of Self-Assembly of Ferredoxin Molecules onto the Surface of the EDC/MUA/Au Substrate. Ferredoxin has four lysine groups: Lys 91 and Lys 92 are located inside the ferredoxin molecule, and Lys 50 and Lys 52 are on the surface of the bottom side of the molecule. Therefore, Lys 50 and Lys 52 may be suitable to couple with the EDC-modified MUA/Au substrate through the reaction between amide groups of
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Figure 4. N 1s (top) and S 2p (bottom) maximum of the target compounds studied by XPS. The x-axis gives the binding energy in eV. The spectral intensities along the y-axis are in arbitrary units. Table 2. XPS Results of Au Substrates Modified with MUA and EDC S 2p surfaces
Au 4f 5/2
∆I(ref-Au 4f)
Au/MUA Au/MUA/EDC
84.60 85.70
-3.1 -2.0
N 1s
2p1/2
2p3/2
398.73
160.34 161.18
158.95 160.09
ferredoxin and carboxylic acid groups of MUA. This suggests that ferredoxin molecules can be arranged with a configuration in which the active sites are closely perpendicular to the planar surface of the substrate. Furthermore, two-dimensionally oriented alignment of ferredoxin molecules may be possible on the inorganic substrate, if their aggregates are removed efficiently. Here we demonstrate the self-assembled ferredoxin monolayer on the gold substrate prepared by the procedure given in Figure 2. Figure 5A shows the AFM image of a bare gold substrate with the grain size of 200 nm. Figure 5B is the image of the ferredoxin aggregates on the substrate (1 µm × 1 µm pixels) with no CHAPS treatment. This figure shows that the size of the ferredoxin aggregates is on the order of 100 nm. Figure 5C shows the AFM image of the ferredoxin monolayer self-assembled on the gold substrate with CHAPS treatment. The uniform and dense
Figure 5. AFM images of the bare gold substrate (A) and the ferredoxin monolayer on the gold surface without CHAPS treatment (B) and with CHAPS treatment (C).
surface morphology of the self-assembled ferredoxin monolayer clearly shows that the size of the cluster is about 10-20 nm, indicating that the CHAPS removed most of the nonspecifically adsorbed ferredoxin molecules from the surface of the inorganic substrate. Speculation on the Redox Property of the SelfAssembled Ferredoxin Monolayer with CHAPS Treatment. The cyclic voltammogram measured for the ferredoxin-coated gold surface with CHAPS treatment in Trizma buffer solution (pH 7.5) is shown in Figure 6. The region of applied voltage in this experiment was between -0.8 and -0.3 V with a rate of 20 mV/s. The reduction potential at -0.64 V was generated between the redox molecule and the gold electrode, and when the voltage was reversed, the asymmetric oxidation potential at -0.47 V was obtained. This result is close to the standard redox potential of native ferredoxin (-0.6 V vs Ag|AgCl|KCl(sat)27-29). In a control experiment, we obtained no characteristic peak of the bare gold electrode or free ferredoxin in the buffer solution. Also, there was no (27) Christopher, M. A. B.; Maria, O. B. Electrochemistry: Principle, Methods, and Applications; Oxford University Press: New York, 1994. (28) Shabtai, V. H.; Katz, E.; Willner, I. J. Am. Chem. Soc. 1997, 119, 8121-8122. (29) Nassar, A. F.; Rusling, J. F.; Tominaga, M.; Yanagimoto, J.; Nakashima, N. J. Electroanal. Chem. 1996, 416, 183-185.
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Figure 6. Cyclic voltammogram measured for the ferredoxinadsorbed gold surface with CHAPS treatment in Trizma buffer solution (pH 7.5).
difference in the cyclic voltammograms for the ferredoxin monolayer with and without CHAPS treatments in Trizma buffer solution. This result indicates that CHAPS does not deteriorate the redox property of ferredoxin. Conclusions The self-assembled ferredoxin monolayer on the gold substrate was successfully prepared with the aid of a zwitterionic surfactant, CHAPS. It was found that CHAPS readily segregates the ferredoxin aggregates which are nonspecifically adsorbed on the gold substrate and elimi-
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nates most of them when the molar ratio of CHAPS to ferredoxin is 200. The AFM image of the ferredoxin monolayer on the gold substrate with CHAPS treatment clearly shows the size of the ferredoxin clusters is about 10-20 nm. This value indicates that the ferredoxin monolayer consists of clusters composed of a few ferredoxin molecules. In contrast, the size of ferredoxin aggregates with no CHAPS treatment was about 100 nm. The current-voltage relationship obtained with the cyclic voltammetry measurements for the ferredoxin monolayer on the gold surface with CHAPS treatment demonstrates that the protein molecules on the surface remain electrically intact. Therefore, it is concluded that CHAPS does not affect inherent properties of the ferredoxin molecules and the oriented arrangement of the ferredoxin monomers on the MUA-modified gold substrate seems to be achieved as designed to couple the amide groups in ferredoxin molecules to the carboxylic acid groups from MUA. In summary, we demonstrate a new method to remove protein aggregates from a self-assembled protein monolayer on a metal substrate without damage to the currentvoltage property of the metalloprotein required in the application of biomolecular electronic devices. Similar experiments on the interaction of some redox proteins with CHAPS are underway for the fabrication of biomolecular devices with diverse proteins by the self-assembly technique. Acknowledgment. This work was supported by Korea Research Foundation Grant (KRF-2002-005-D00003). LA034933V
