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Direct Observation of Self-Assembled Monolayers, Ion Complexation, and Protein Conformation at the Gold/ Water Interface: An FTIR Spectroscopic Approach Martha Liley, Thomas A. Keller, Claus Duschl, and Horst Vogel* Laboratoire de Chimie Physique des Polyme` res et Membranes, Swiss Federal Institute of Technology Lausanne, CH-1015 Ecublens, Switzerland Received March 10, 1997. In Final Form: May 29, 1997X A simple approach to the in situ measurement of infrared spectra of self-assembled monolayers (SAMs) on gold in an aqueous environment is described. A thin gold film (3-5 nm) on the surface of a germanium internal reflection element is used as a substrate for the self-assembly of a monolayer of a chelating thioalkane. High signal-to-noise spectra are obtained, allowing identification of the chemical groups present in the SAM. Complexation of ions by the SAM can be monitored, and the secondary conformation of his-tagged proteins bound to the SAM can be observed.
Introduction The use of self-assembled monolayers (SAMs) of alkanethiols on gold for the production of well-defined organic surfaces has been remarkably successful. Of particular interest is their application to biological systems. Numerous studies using gold surfaces modified with thiols or disulfides have investigated: the adsorption of proteins at surfaces (biocompatibility);1 the formation of supported lipid layers and the incorporation of biological molecules in these layers;2,3 and the immobilization of proteins via molecular recognition.4 However, few techniques are available for the study of these processes in situ on gold surfaces in aqueous media. In particular, structural information about the chemical and biological species at the surface is missing. We describe in this article a new approach to the study of SAMs on gold surfaces in aqueous environments: the use of attenuated total internal reflection (ATR) Fourier transform infrared (FTIR) spectroscopy. This technique is based on the use of extremely thin films (3-5 nm) of gold deposited on germanium ATR elements, as shown in Figure 1. The use of thin gold films (5-10 nm thick) as substrates for self-assembled monolayers (SAMs) has been described by DiMilla et al.,5 who demonstrated that the SAMs formed on these substrates are comparable if not superior to those formed on bulk gold substrates and can be investigated using UV/vis spectroscopy. In the application described here, the relative transparency of these gold films in the infrared is exploited: internal reflection of the infrared beam at the germanium/gold interface produces an evanescent field which penetrates through the gold film and into the aqueous phase on the other side.6 This allows sampling of SAMs at the gold/water interface. Multiple reflections increase the intensity of the spectrum of the SAM.7,8 The ATR configuration was used to investigate SAMs of chelator thioalkane (CTA) analogous to the surfaces * To whom correspondence should be addressed. E-mail:
[email protected]. X Abstract published in Advance ACS Abstracts, August 15, 1997. (1) Prime, K. L.; Whitesides, G. M. Science 1991, 252, 1164. (2) Terrettaz, S.; Stora, T.; Duschl, C.; Vogel, H. Langmuir 1993, 9, 1361. (3) Plant, A. L.; Gueguetchgeri, M.; Yap, W. Biophys. J. 1994, 67, 1126. (4) Spinke, J.; Liley, M.; Guder, H.-J.; Angermaier, L.; Knoll, W. Langmuir 1993, 9, 1821. (5) DiMilla, P. A.; Folkers, J. P.; Biebuyck, H. A.; Ha¨rter, R.; Lopez, G. P.; Whitesides, G. M. J. Am. Chem. Soc. 1994, 116, 2225. (6) From the optical constants of bulk gold in the IR, we calculate the exponential decay length of an IR wave in gold at around 1700 cm-1 (6 µm) to be approximately 28 nm.
S0743-7463(97)00258-8 CCC: $14.00
Figure 1. (Top) Experimental configuration used for this study. A germanium ATR element is coated with a thin layer (approximately 3 nm) of gold on one face. This face is pressed against a Teflon cell to form a watertight seal. The gold film is used as a substrate for self-assembly of thiol monolayers. The cell is filled with aqueous solution: in this case, since the spectral region of interest was 1800-1400 cm-1, D2O was used. (Bottom) The structures of the chemical compounds used. Compound 1, (NR,NR-bis(carboxymethyl)-Nω-[[[(11-mercaptoundecanoyl)glycyl]glycyl]glycyl]-L-lysine (“chelator thioalkane” or CTA)) formed SAMs at the gold surface by adsorption from D2O. Compound 2, (NR-NR-bis(carboxymethyl)-L-lysine (lysineNTA)) was used for comparison purposes. At the pD used, both compounds were zwitterionic, with the carboxyl groups deprotonated and the tertiary amine protonated.
used in immobilized metal ion affinity chromatography (IMAC).9,10 In IMAC, chelating groups at a surface are used to complex metal ions. Free coordination sites remain in the complex formed and are available for complexation (7) Harrick, N. J. Internal reflection spectroscopy; Interscience: New York, 1967. (8) Similar approaches using metal films on ATR elements have been described for electrochemical measurements. See for example: Hatta, A.; Chiba, Y.; Suetaka, W. Appl. Surf. Sci. 1986, 25, 327 and references therein. (9) Porath, J.; Carlsson, J.; Olsson, I.; Belfrage, G. Nature 1975, 258, 598. (10) Hochuli, E. In Genetic Engineering; Setlow, J. K., Ed.; Plenum Press: New York, 1990; Vol. 12, p 87.
© 1997 American Chemical Society
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with electron donors, such as histidine groups at protein surfaces. The addition of a sequence of histidines (a ‘histidine tag’ or ‘his-tag’) to a given protein by genetic engineering allows immobilization and thus purification of the protein in a one-step process. The use of an IMACbased approach for the immobilization of proteins at gold surfaces and at the air/water interface was recently described by several authors.11-13 This approach has advantages over other immobilization methods used: firstly, the histidine tag can be inserted at a defined site on the protein, allowing its well-defined orientation at the surface. In addition, the immobilization is completely reversible on addition of a competitive ligand such as imidazole or on removal of the metal ion by EDTA complexation. Subsequent incubation of the CTA SAM with histidinetagged protein (a his-tagged Fab fragment) led to its reversible immobilization at the gold surface. The selfassembled CTA monolayers and the binding of histidinetagged protein to them have been described in more detail in a previous publication.11 Experimental Section Gold Films. Germanium ATR elements (60 × 10 × 4 mm3 with an internal angle of incidence of 45° and 11 internal reflections) were coated with a thin film of gold (approximately 3 nm) on one face by thermal evaporation at a pressure below 5 × 10-6 mbar. Before coating, they were first modified either by silanization with 3-(mercaptopropyl)trimethoxysilane or by evaporation of 0.5-1 nm of chromium onto one face of the element. This modification improves the wetting of the surface by the gold film, thus improving the adherence of the film and producing continuous thin gold films rather than isolated gold droplets. After cooling under vacuum for about 15 min, the ATR elements were removed from the vacuum chamber and immediately pressed against a Teflon cell, to produce a watertight compartment which was filled with aqueous (D2O) solution. Spectra of the bare gold surface in D2O were collected at once and used as references for subsequent CTA spectra. CTA Monolayers. The synthesis of CTA has been described in a previous paper.11 CTA monolayers were formed on gold surfaces by self-assembly from a 0.16 mM solution of CTA in D2O. After 2 h the gold surface was washed extensively with D2O. For the complexation of Ni2+, CTA layers were incubated with a solution of NiSO4 (50 mM) for 1 min and then washed with D2O. Spectra of CTA monolayers were recorded in deuterated buffer (20 mM sodium phosphate, 250 mM NaCl, pD 7.5). His-Tagged Fab Fragment. The anti-lysozyme Fab fragment D1.3 bearing a hexahistidine extension at the C-terminus of the heavy chain was a gift from W. Schiweck and Prof. A. Skerra. The Fab fragment was equilibrated in deuterated buffer for a period of several days. The absence of an amide II peak in the spectral region 1510-1580 cm-1 was indicative of quasicomplete exchange of amide protons for deuterons (data not shown). Buffer solutions were 20 mM sodium phosphate, 250 mM NaCl, pD 7.5 in D2O unless otherwise stated. Infrared spectra were recorded using a Bruker IFS 28 spectrometer. One thousand scans were recorded at 4 cm-1 resolution with triangular apodization and two levels of zero filling. Water vapor bands were subtracted from the spectra, which were also baseline corrected. None of the spectra were smoothed. Transmission spectra in D2O solution were collected using CaF2 windows and a 50 µm lead spacer.
Results and Discussion The compounds used in this study are shown in Figure 1. SAMs were formed by adsorption of compound 1, (NR,NR-bis(carboxymethyl)-Nω-[[[(11-mercaptoundecanoyl)(11) Keller, T. A.; Duschl, C.; Kro¨ger, D.; Se´vin-Landais, A.-F.; Vogel, H.; Cervigni, S. E.; Dumy, P. Supramol. Sci. 1995, 2, 155. (12) Sigal, G. B.; Bamdad, C.; Barberis, A.; Strominger, J.; Whitesides, G. M. Anal. Chem. 1996, 68, 490. (13) Schmitt, L.; Bohanon, T. M.; Denzinger, S.; Ringsdorf, H.; Tampe´, R. Angew. Chem., Int. Ed. Engl. 1996, 35, 317.
Figure 2. Spectra of CTA SAMs and lysine-NTA in solution. No attempt was made to correct for solution concentration. No comparison should be made between the intensities of different spectra. (A) Spectra of a CTA SAM on a gold surface and of lysine-NTA in solution. Transmission spectra were collected of lysine-NTA in deuterated buffer solution. The CTA monolayer spectra were collected in buffer, and spectra of the original bare gold surface in buffer were used as a reference. The scale bar refers to the absorbance units of the CTA SAM. (B) Same as in part A, but after complexation of Ni2+. Transmission spectra of lysine-NTA in deuterated buffer containing NiSO4 (20 mM sodium phosphate, 250 mM NaCl, 20 mM NiSO4, pD 7.5). CTA SAMs after incubation with NiSO4.
glycyl]glycyl]glycyl]-L-lysine (“chelator thioalkane” or CTA)) onto the gold surface from D2O. For comparison purposes, solution measurements were also performed with the CTA headgroup, compound 2 (NR,NR-bis(carboxymethyl)-L-lysine (lysine-NTA)). The spectrum obtained on formation of a SAM of CTA on a gold surface, and the transmission spectrum of lysine-NTA in D2O are shown in Figure 2A. Peaks were assigned by reference to literature spectra of NTA.14 At the pH used, both CTA and lysine-NTA were zwitterionic, with the carboxylic groups deprotonated and the tertiary amine protonated.15 The lysine-NTA spectrum in the absence of nickel ions has two major peaks at 1625 and at 1402 cm-1. These are assigned to the carboxylate asymmetric (νasym) and symmetric stretches (νsym), respectively. (The peak at 1730 cm-1 is assigned to a minor component of protonated carboxylic acid groups.) The spectrum of the CTA SAM at the gold surface retains the two major peaks: the peak at 1624 cm-1 is attributed to the carboxylate asymmetric stretch and the amide I′ band of the four peptide bonds in the CTA spacer; the peak at 1403 cm-1 is attributed to the carboxylate symmetric stretch, while the new peak at around 1467 cm-1 is attributed to the amide II′ bands of the (deuterated) peptide bonds with a contribution from the δ(CH2) modes. Incubation of the SAM with a solution of NiCl2 or NiSO4 led to complexation of the nickel by the CTA. The spectrum of lysine-NTA in D2O in the presence of Ni2+ ions and the spectrum of the CTA SAM after incubation with NiSO4 (14) Tomita, Y.; Ueno, K. Bull. Chem. Soc. Jpn. 1963, 36, 1069. (15) Nakamoto, K.; Morimoto, Y.; Martell, A. J. Am. Chem. Soc. 1962, 84, 2081.
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Figure 3. Spectra of the Fab fragment in solution and adsorbed to the CTA monolayer in the amide I spectral region. Transmission spectra of the Fab were collected using a 6 µM solution in deuterated buffer. ATR spectra were collected using the nickel-charged CTA SAMs, which were incubated with a 1 µM solution of Fab in deuterated buffer for 30 min. The spectrum shown is the difference spectrum between a nickel-charged CTA SAM with Fab and the same SAM without Fab. Absorbance units are given for the ATR spectrum of Fab on the CTA surface. The transmission spectrum of Fab in deuterated buffer was normalized to the same peak intensity.
are shown in Figure 2B. Large changes in the spectra may be observed: in the lysine-NTA spectrum a new peak appears at 1592 cm-1 and is attributed to the asymmetric stretch of the nickel-complexed carboxylate group.14 The uncomplexed asymmetric and symmetric carboxylate peaks are shifted slightly to 1628 and 1410 cm-1, respectively. Similarly, for the CTA SAM, a new peak appears in the spectrum at around 1591 cm-1. The three peaks present in the previous CTA SAM spectrum (without Ni2+) are now found at 1625, 1472, and 1406 cm-1. Thus we are able to observe the characteristic carboxylate and peptide peaks of CTA in the SAM. The spectra observed correspond very well with the transmission spectra of the headgroup and with the literature spectra of NTA in solution.14 Complexation of Ni2+ by the SAM can clearly be observed. Subsequent adsorption of the histidine-tagged Fab fragment to the gold surface was also investigated. In this case, the spectral region 1700-1600 cm-1 is of special interest, as it corresponds to the amide I peak of the protein. The form and position of the amide I peak are very sensitive to the secondary structure of the protein in solution and can be used to detect changes in this conformation.16,17 The spectrum obtained for the Fab fragment at the CTA SAM was compared with the transmission spectrum of the same Fab fragment in D2O buffer (Figure 3). Both spectra have a broad triangular peak with a maximum at 1636 cm-1 characteristic of the β-sheet conformation of Fab fragments.18 A small dif(16) Goormaghtigh, E.; Cabiaux, V.; Ruysschaert, J.-M. In Subcellular Biochemistry; Hilderson, H. J., Ralston, G. B., Eds.; Plenum Press: New York, 1994; Vol. 23, p 329. (17) Byler, D. M.; Susi, H. Biopolymers 1986, 25, 469. (18) Buijs, J.; Norde, W.; Lichtenbelt, J. W. T. Langmuir 1996, 12, 1605.
Letters
ference in the intensities centred around 1660 cm-1 can be observed, but in general the two spectra are almost identical. Thus we conclude that this approach allows us to obtain good protein IR spectra with protein solutions of relatively low concentration. (The solution used for the ATR spectrum contained 0.05 mg/mL protein, compared to the 10-30 mg/mL normally used for transmission IR spectra of proteins.) In addition, the secondary structure of the Fab fragment undergoes no significant changes on binding to the CTA SAM. Previous surface plasmon resonance studies of protein binding to CTA SAMs on gold showed the binding to be completely reversible on incubation of the surface with 500 mM imidazole.11 Our infrared measurements on the gold-coated ATR element demonstrated that a small fraction of the protein (not more than 15%) was irreversibly bound to the surface (data not shown). This we attribute to defects in the gold film. For IR measurements on gold in the ATR configuration, the gold film must be thin enough that the evanescent field at the gold/water interface has a high intensity, while the reflectivity of the gold/germanium interface must also be high enough that a significant IR intensity arrives at the detector. The optimal film thickness depends on experimental parameters, e.g. detector sensitivity and number of reflections at the gold/germanium interface. In addition, for optimal SAMs, the film must adhere well to the germanium substrate and form a continuous smooth gold surface. Scanning electron microscopy and conductivity measurements of the layer indicate that the layers used in this study probably contain a significant number of holes comprising perhaps as much as 15-20% of the surface area. Thus, we attribute the nonspecific binding of the his-tagged Fab fragment to the surface to interactions between the protein and the area of the germanium substrate that is not covered with gold. Optimization of the experimental parameters and the gold layer thickness to produce gold layers with a minimum number of holes is in progress. Conclusions In conclusion, we have shown that ATR elements, when coated with a gold film a few nanometers thick, can be used for infrared studies of self-assembled monolayers on gold in aqueous solution. This approach compares favorably with the external IR reflection-absorption spectroscopy used for studies of metal electrode surfaces.19 Experimentally, our approach facilitates adsorption/ desorption studies. More importantly it has a high sensitivity, and thus does not require the use of voltage or polarization modulation techniques. Acknowledgment. This work was supported by the Swiss National Science Foundation (SPP Biotechnology, 5002-235180). The authors thank W. Schiweck and Prof. A. Skerra, TH Darmstadt, Germany, for the generous gift of the histidine-tagged Fab fragment. T.A.K. thanks the SPP for a postdoctoral fellowship (5002-38275). LA970258B (19) Seki, H. IBM J. Res. Dev. 1993, 37, 227.
