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Surface Reaction of Sulfur-Containing Amino Acids on Cu(110) Jeong Won Kim,*,† Young Mi Lee,†,‡ Seung Mi Lee,† Min Jung Son,† Hyeseung Kang,‡ and Yongsup Park‡ †
Korea Research Institute of Standards and Science, Daejeon 305-340, Korea, and ‡Department of Physics and Research Institute for Basic Sciences, Kyung Hee University, 1 Hoegi-dong, Dongdaemun, Seoul 130-701, Korea Received November 29, 2009. Revised Manuscript Received February 12, 2010
Adsorption behaviors of sulfur-containing amino acids, cysteine, methionine, and cystine molecules on Cu(110) surface were studied by core level photoelectron spectroscopy using synchrotron radiation. We found the following through the systematic comparisons of core level peaks such as S 2p, N 1s, and O 1s from different amino acids. At low coverage regimes, all the molecules form two distinct thiolate species, and their S 2p binding energy difference was about 0.9 eV. The relative populations of the two thiolates were different for different molecules and their coverage, which is due to the different bond strength of the sulfur-containing functional groups. At high coverage regimes, only cysteine molecules form zwitterionic state, which is related to the molecular ordering on Cu(110) surface.
Introduction Manipulation of functional molecules on a solid surface is one of the most important goals for nanoscale surface science and technology. The adsorption and possible self-assembly of biomolecules on a well-defined crystalline surface is the first step toward this direction, leading to the recent focus on structural and functional characterization of biomaterials on various surfaces.1,2 Detailed study of chemical reactions at the interface between biomolecules and inorganic substrate would eventually open a new scheme for building biochips and chemical sensors. For instance, amino acids containing various functional groups are able to play an important role in modification of inorganic templates and stepwise chemical reactions at the surface. Figure 1 shows the molecular structures of cysteine, methionine, and cystine which contain sulfur atoms in common. Cysteine [HOOC-CH(NH2)CH2-SH] contains a sulfhydryl group (-SH). A pair of sulfhydryl group forms a disulfide bond that provides a binding force for folding between different cysteine sections of a protein chain. Methionine has a methyl sulfide group (-SCH3). Cystine is composed of a dimer form of two cysteine molecules that are joined by the disulfide bond (-S-S-) through oxidation. Thus, it is interesting to compare the chemical reaction of sulfur atoms with a metal surface that are contained in different functional groups. The three S-containing molecules considered here have exhibited a various attractive features on clean metal surfaces. For example, the cysteine molecules forms a short-range order on Au(110) surface, which is believed to be a consequence of homochiral dimer interaction3 and unidirectional molecular growth.4,5 On Ag(111) surface, the methionine molecules form *To whom correspondence should be addressed. E-mail: jeongwonk@ kriss.re.kr. (1) Barlow, S. M.; Raval, R. Surf. Sci. Rep. 2003, 50, 201. (2) Smith, R. K.; Lewis, P. A.; Weiss, P. S. Prog. Surf. Sci. 2004, 75, 1. (3) K€uhnle, A.; Linderoth, T. R.; Besenbacher, F. J. Am. Chem. Soc. 2003, 125, 14680. (4) K€uhnle, A.; Molina, L. M.; Linderoth, T. R.; Hammer, B.; Besenbacher, F. Phys. Rev. Lett. 2004, 93, 086101. (5) Cossaro, A.; Terreni, S.; Cavalleri, O.; Prato, M.; Cvetko, D.; Morgante, A.; Floreano, L.; Canepa, M. Langmuir 2006, 22, 11193. (6) Schiffrin, A.; Riemann, A.; Auw€arter, W.; Pennec, Y.; Weber-Bargioni, A.; Cvetko, D.; Cossaro, A.; Morgante, A.; Barth, J. V. Proc. Natl. Acad. Sci. U.S.A. 2007, 104, 5279.
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one-dimensional self-assembled structure through dimeric interaction.6 On Cu(110), the cysteine molecules also showed several kinds of long-range orders.7 All these molecules have zwitterionic configuration on the surface that facilitates intermolecular hydrogen bond formation. However, detailed chemical reactions of amino acids on a transition metal surface as a function of coverage have scarcely been reported. In this work, we report a core level photoelectron spectroscopy study of the initial stages of adsorption of three different S-containing amino acid molecules on a Cu(110) using synchrotron radiation. The characteristic chemical shifts and relative intensity changes of S 2p, N 1s, and O 1s core level peaks were monitored upon adsorption on the Cu(110) to find chemical changes of functional groups in each amino acid. At low coverage, all the molecules showed the formation of two different types of thiolates and carboxylate by deprotonation. However, the evolution of detailed molecular species is different for different molecules. As the coverage increases, only the cysteine molecules formed zwitterions. Evidences for second layer formation above the first layer became apparent at high coverage.
Experimental Section A Cu(110) crystal surface was cleaned by repeated cycles of sputtering by 1.5 keV Arþ ion and annealing at 450 °C by current through Ta wires wrapped around the sample. The cleanliness of the surface was checked by the absence of impurities such as S and C in survey or narrow-scan photoelectron spectra. The amino acids were commercially purchased (Sigma-Aldrich, >98% purity) and evaporated from glass cells heated to 80-90 °C and differentially pumped. The depositions were made while the clean Cu(110) surface was kept at room temperature under the pressure of 10-8 Torr. All measurements were performed in an analysis chamber with the base pressure of 2 10-10 Torr at 7B1 beamline in Pohang Acceleration Laboratory (PAL). The photoelectron intensity at surface normal direction was recorded using an electron energy analyzer equipped with a 2D CCD detector (PHOIBOS 150, SPECS GmbH). The photoelectron peaks for S 2p, N 1s, O 1s, and C 1s core levels from the adsorbed molecules were recorded by using photon energies selected for best surface sensitivity. The photon energy used was between 250 and 620 eV (7) Kim, J. W.; Hwang, H.-N.; Hwang, C.-C. J. Phys. Chem. C 2007, 111, 13192.
Published on Web 03/19/2010
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Figure 1. Molecular structures of S-containing amino acids used in the experiment.
Figure 2. S 2p core level spectra and their peak analysis of cysteine, methionine, and cystine molecules adsorbed on Cu(110) surface as a function of relative molecular coverage. The S1 and S2 denote two different thiolate species formed by the reaction with substrate, while S3 denotes the sulfur-containing group without interaction to the substrate. and was calibrated in situ using Au 4f7/2 peak from a clean Au film. The C 1s peaks are not shown because they did not exhibit any significant chemical changes upon adsorption. The total energy resolution of the spectra ranged from 0.4 to 0.7 eV depending on the photon energy. No spectral change was observed during the data acquisition, which might be caused by X-ray beam irradiation as reported elsewhere.8-10
Results and Discussion S 2p Core Level Spectra. Figure 2 shows a series of S 2p core level spectra of (a) cysteine, (b) methionine, and (c) cystine on a Cu(110) surface as a function of molecular coverage. The molecular coverage here is a value relative to the reference saturation coverage of chemisorbed layer, which was formed by annealing thick layer of adsorbed molecules at around 100 °C for a short period of time. We assumed that this layer corresponds to one monolayer of molecules on the Cu(110) surface. The relative coverage values were then calculated by comparing the S 2p peak intensities of adsorbed molecules with respect to the Cu 3p peak intensities of substrate. Selected core level peaks of each molecule were fitted with Voigt functions after Shirley-type background subtraction. The S 2p doublet peaks exhibit an equivalent spin-orbit splitting of 1.18 eV with a branching ratio of 2:1. However, the full widths at half-maximum (fwhm) of the S 2p doublets varied within the range of 0.6-1.0 depending on the type of chemical species on the surface. Especially, the existence of broadened S3 component suggests that many different inter(8) Cavalleri, O.; Gonella, G.; Terreni, S.; Vignolo, M.; Pelori, P.; Floreano, L.; Morgante, A.; Canepa, M.; Rolandi, R. J. Phys.: Condens. Matter 2004, 16, S2477. (9) Gonella, G.; Terreni, S.; Cvetko, D.; Cossaro, A.; Mattera, L.; Cavalleri, O.; Rolandi, R.; Morgante, A.; Floreano, L.; Canepa, M. J. Phys. Chem. B 2005, 109, 18003. (10) Stampfl, A. P. J.; Chen, C. H.; Wang, S. C.; Huang, M. L.; Klauser, R. J. Electron Spectrosc. Relat. Phenom. 2005, 144, 417.
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molecular interactions could exist in molecular multilayer. At the early stage of cysteine adsorption, a single peak is observed at the S 2p3/2 binding energy of 161.5 ( 0.1 eV (S1) as shown in the bottom spectrum of Figure 2a. As the cysteine coverage increases, a new peak appears at 162.3 ( 0.1 eV (S2), which later dominates the S 2p3/2 spectrum. Above one monolayer, a peak at 164.2 ( 0.2 eV (S3) appears as seen in the top spectrum of Figure 2a. In the case of methionine adsorption shown in Figure 2b, the binding energies of all three components (S1-S3) are almost the same as in cysteine case, but their relative intensities are different at different coverage. Similar to the cysteine case, the binding energies of S1 and S2 components indicate that they are originated from strong chemisorption. It is believed that the cleavage of S-CH3 bond and rebonding to Cu substrate atoms are what actually happens for methionine at low coverages. However, the S2 intensity remains very low throughout the coverage window, and the high-binding energy peak S3 appears even in low-coverage regime, which becomes predominant at high coverages.1 Figure 2c shows the situation for the cystine adsorption. Core level peaks for chemical species similar to the previous two cases were clearly observed but with different relative intensities. The S1 component is obviously dominant from the low coverage and remains so even at high coverage, while the S3 component remains weak throughout. The S1 and S2 components at low binding energies are attributed to two different chemisorbed species originated from the dissociation of disulfide bonds and rebonding to Cu. It is clear from Figure 2a-c that the coexistence of the three different peaks at high coverage is common for all three S-containing amino acids considered here. Similar behaviors were reported for dimethyl disulfide adsorption on a Au surface,11 (11) Noh, J.; Jang, S.; Lee, D.; Shin, S.; Ko, Y. J.; Ito, E.; Joo, S.-W. Curr. Appl. Phys. 2007, 7, 605.
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Figure 3. Relative intensity changes of S 2p components for the three molecules in Figure 1 as a function of relative molecular coverage. The spans, at a given coverage, of the differently shaded areas, are proportional to the relative amounts of the S1, S2, and S3 species at that coverage.
Figure 4. N 1s core level spectra of cysteine, methionine, and cystine molecules adsorbed on Cu(110) surface. The low-binding energy peak at 399.6 eV corresponds to -NH2 and the high-binding energy one at 401.7 eV to -NH3þ configuration.
but there are differences even for adsorption on Au(110),9 where only two peaks at 161.95 and 164.3 eV are observed. On Cu surfaces, on the other hand, three different S 2p peaks were clearly observed. Methyl sulfide derivatives are the only case where various thiolate species appear with substrate temperature variation.12,13 The relative intensity variations of S1-S3 components for the molecules as a function of coverage are displayed in Figure 3. Since the binding energies of the three components are almost identical irrespective of the adsorbed molecules, one can assume that each three components for different molecules have the same origins. As previously mentioned, the S1 and S2 components can be attributed to the thiolate formation through the interaction of the S atoms of the molecules with the Cu atoms of the substrate. The S3 component at the higher binding energy is attributed to the second layer molecules that are basically intact.7 Because postannealing at 100 °C eliminates other peaks but S1,7 it is clear that the most stable species on the Cu(110) surface is S1. The relative intensities of S2 and S3 peaks showed a strong dependence on the type of adsorbed molecules. For cysteine, a stepwise change in relative intensities of S1-S3 components appeared sequentially with increasing coverage. In other molecules, however, the relatively intensity of S2 component remained low throughout the coverage window. In addition, the S3 peak appeared from the early stage, indicating that the second layer was formed even before the first layer was completed. We believe this is due to the difference in relative reactivity of S-containing groups in different molecules, which we discuss shortly. (12) Kariapper, M. S.; Grom, G. F.; Jackson, G. J.; McConville, C. F.; Woodruff, D. P. J. Phys.: Condens. Matter 1998, 10, 8661. (13) Lai, Y. H.; Yeh, C. T.; Cheng, S. H.; Liao, P.; Hung, W. H. J. Phys. Chem. B 2002, 106, 5438.
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N 1s and O 1s Core Level Spectra. Figure 4 shows a series of N 1s core level spectra of the molecules on Cu(110). As seen in Figure 4a, the N 1s peak shows only a single component at 399.6 ( 0.2 at low cysteine coverage, while the second component at 401.7 ( 0.2 eV appears at more than a half monolayer. Eventually the high-binding energy component becomes dominant at high coverages. In contrast, other two molecules in Figure 4b,c showed N 1s peaks with a single component at 399.7 ( 0.3 eV throughout the coverage window. The position and width of these peaks are similar to those of low-binding energy component of cysteine in Figure 4a, which led us to believe that the chemical environment of N atoms for the component at 399.7 eV is nearly identical. Previous results suggest this peak is due to NH2 configuration, while the peak at 401.7 eV in Figure 4a is due to NH3þ configuration.8,9,14 Therefore, we conclude that the NH2 group in cysteine molecules transform to NH3þ configuration via protonation on the Cu(110) surface. The NH2 groups in methionine and cystine molecules remain unchanged. The O 1s core level peaks for the three molecules are shown in Figure 5 along with the fitting results at the highest coverage. Cysteine and methionine in parts a and b of Figure 5, respectively, show a single peak at 531.5 ( 0.2 eV at low coverages. Then, the spectra become broader with increasing coverage, which indicates that a new component appears at the highest coverage from the fitting results. On the other hand, Figure 5c shows that the cystine O 1s peak is already quite broad at low coverage. However, the fitting results at high coverages are almost identical for all three molecules as seen in the top spectra in Figure 5. The main component of the peak corresponds to the formation of COO(14) Marti, E. M.; Methivier, C.; Pradier, C. M. Langmuir 2004, 20, 10223.
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Figure 5. O 1s core level spectra of cysteine, methionine, and cystine molecules adsorbed on Cu(110) surface. From the peak fitting, the high binding energy component at high coverages indicates the evolution of COOH, whereas the major low binding one still remains in the form of hydroxylate (COO-).
formed by the loss of an acidic hydrogen in the carboxylic groups. The higher binding energy component at high coverage indicates a formation of different configuration for O atom. This usually happens by the protonation of the carboxylate and formation of COOH in which two different kinds of O atoms coexist on the Cu(110) surface. The hydroxyl group gives the high binding energy oxygen peak, and the carbonyl oxygen in COOH gives low binding energy peak at the same position as the carboxylate oxygen.7,9,15 Reactivity and Molecular Ordering on Cu(110). It is clear from Figures 2 and 3 that the S1 component appears first as the molecules begin to be adsorbed on the Cu(110) surface. It comes from a type of thiolate that is the most stable and would occupy a kinetically favorable site. The S2 component is another type of thiolate which is usually formed at a later stage. The relative intensity of S2 from adsorbed methionine and cystine is always lower than that of S1. The cysteine molecule has exposed -SH terminal group. The methionine and cystine, on the other hand, have -SCH3 and -S-S- groups, respectively, surrounded by neighboring alkyl groups. Thus, one can imagine that the S2 species would be formed at a relatively hindered site on Cu(110). At the early stage of molecular adsorption at room temperature, the impinging molecules can easily diffuse on the surface to find the most stable adsorption site so that they form a S1 thiolate. As the coverage increases, the second type of thiolate S2 begins to form, which is not easily accessible for relatively large functional groups of methionine and cystine. This is why the relative intensity of S2 species on methionine and cystine is always low. In order to compare the energetics of thiolate formation from the three different amino acids, theoretical calculations were performed within the framework of density functional theory.16 (15) Huang, J. Y.; Huang, H. G.; Lin, K. Y.; Liu, Q. P.; Sun, Y. M.; Xu, G. Q. Surf. Sci. 2004, 549, 255. (16) Our quantum mechanical calculations used generalized gradient approximations and the revised PBE exchange-correlation functional [Hammer, B. H.; Hansen, L. B.; Norskov, J. K. Phys. Rev. B 1999, 59, 7413]. All electron KohnSham wave functions were expanded in a local atomic basis set with each basis function defined numerically on an atomic-centered spherical-polar mesh with a cutoff of 4.4 Å. The double numeric polarized basis set was used for all elemental atoms. Geometries were optimized until the maximum force on each atom was less than 0.002 hartree/Å, the maximum displacement was less than 0.005 Å, and the total energy change was less than 10-5 hartrees.
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Table 1. Calculated Bond Strengths of Free-Standing and CuAdsorbed Molecules bond strength (eV) free molecule in presence of Cu adatom difference
cysteine (S-H)
methionine (S-CH3)
cystine (S-S)
4.77 3.39
3.98 1.87
2.88 0.58
1.38
2.11
2.30
Free-standing molecules and Cu-adsorbed structures near the S-containing functional group have been optimized in order to get the reasonable geometry and total energies. Then, the bond strengths were calculated as the total energy difference between the equilibrium structure and the structure with corresponding interatomic distances of 8 A˚, as listed in Table 1.17 The bond strength of each group is largely reduced by the interaction with Cu atom. However, the difference in S-H bond strength between free cysteine molecule and in presence of Cu is relatively small compared to other molecules. This means that the methionine and cystine can easily form a completely dissociated thiolate (S1-like species) compared to cysteine as long as it is kinetically accessible. This result is consistent with Figures 2 and 3, where the S1 component is predominant throughout the coverage range for methionine and cystine, whereas the S2 component in cysteine becomes largest at high coverage regimes. The binding energy of S2 at 162.3 eV implies the bonding is chemisorptions,12,18 which seems to be kinetically favorable configuration for cysteine when neighboring adsorption sites are moderately populated. In fact, the kinetic term was not taken into account here as we used a free Cu atom for coordination. For instance, more crowded geometry around the S-containing groups of methionine and cystine molecules (-SCH3 and -S-S-) compared to cysteine (-SH) may hamper easy pathway to the stable configuration. This is one of the reasons why the S2 and S3 components appear even at low coverages, but their intensities remain low. At low coverages, (17) Note that here our calculations provide only a guideline in order to estimate the relative chemical reactivity among three different molecules. More sophisticated calculations about many different cases of surface geometries with consideration of kinetic effects would give more realistic pictures of experiments, which are beyond the scope of this article. (18) Uvdal, K.; Bod, P.; Liedberg, B. J. Colloid Interface Sci. 1992, 149, 162.
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Figure 6. Schematic diagram for possible cysteine adsorption configurations on a Cu(110) surface with coverage. At initial stage of adsorption, all the functional groups interact directly with the substrate. As the coverage increases, the protonation of amino group takes place, getting up from surface. Finally multilayer species begin to be generated.
more than one Cu atom per molecule might be involved in the initial coordination, which was not considered in the calculations. Indeed, such a situation is more probable in cysteine than in other molecules as the S-containing groups are surrounded by other bulky groups in methionine and cystine. This is the reason for the exclusively high population of S1 for cysteine adsorption at low coverages. Among the S-containing amino acids considered here, only cysteine forms ordered rearrangement on the Cu(110) surface.7 The unique behavior of the cysteine comes from a sequential reaction process outlined above while other molecules show multilayer formation from the beginning. The concerted reaction behavior of methionine and cystine is somewhat similar to the case of cysteine on Au(110)9,17 where mixed types of S species are formed from the very early stage. One of the sequential reaction schemes with increasing coverage is displayed in Figure 6 for cysteine on Cu(110) surface, where we only consider the chemical changes of cysteine molecules on a Cu substrate ignoring a detailed atomic geometry. At low coverages (far left side of Figure 6), the thiol and carboxylic groups undergo deprotonation generating thiolate and carboxylate that are observed by S 2p in Figure 2 and O 1s in Figure 5, respectively. These two ionic forms of functional groups interact more or less directly with the substrate. The amino group also takes part in the interaction in the form of NH2 as observed by N 1s in Figure 4. Thus, all the functional groups of the cysteine molecule are involved in the coordination to Cu substrate atoms at the beginning. As the molecular population on the surface increases (middle of Figure 6), the direct bonds of carboxylic and amino groups to the Cu become weak, and some of the carboxylic and/or amino groups gain H atoms back as evidenced by the appearance of the high binding energy peak in the N 1s core level (Figure 4a) and the asymmetric tail in
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the O 1s core level (Figure 5a). An ordered structure of cysteine was formed on Cu(110) at this stage as it is the only molecule that forms a zwitterionic species. It is similar to the methionine/ Ag(111) case6 where the surface consists of complete combination of carboxylate and ammonium groups that form one-dimensional self-assembled structures via chemical transformation of neutral molecules to a zwitterionic state such as R-(NH3)þ(COO)-. The zwitterionic formation is a strong driving force for intermolecular interactions through something like hydrogen bonds between molecules. This extra interaction of surface molecules may be related with the long-range ordering of any amino acid molecules. It could be argued that the cysteine molecules on Au(110)9 are adsorbed in a concerted manner, and a proper interaction between molecules for ordering could be hindered. This is why reported molecular orderings on a well-known system like cysteine/Au(110) are very few. Above one monolayer of cysteine adsorption (right side of Figure 6), a second layer is formed as evidenced by the S3 component in Figure 2a. The observation that the highest binding energy components of S 2p, N 1s, and O 1s peaks are quite broad suggests that various additional intermolecular complexes and substrate interaction might exist. Some of these may consist of crossed bonds between NH3þ and COOgroups of first and second layer molecules.
Conclusions We have measured the core level photoelectron spectra of three different S-containing amino acids molecules including cysteine, methionine, and cystine adsorbed on Cu(110) surface. All the molecules showed three distinct S 2p peaks, and their O 1s peak analysis revealed formation of carboxylate. The detailed chemical configuration and relative population of S-containing functional groups depend on the relative reactivity of -SH, -SCH3, and -S-S- in good agreement with the calculation results for bond strengths. The sulfhydryl group in cysteine molecule has the lowest energy gain by the bonding to a Cu atom, which results in a relatively high population of the second thiolate species (S2) with increasing coverage. On the other hand, the population of S1 is relatively large throughout the coverage range in methionine and cystine adsorption cases due to the relatively high bond strengths between S-containing groups and Cu. The analysis of N 1s and O 1s core level peaks suggested that the zwitterionic state such as (NH3)þ(COO)- formation is possible only in adsorbed cysteine molecules, which is closely related to the molecular ordering at the surface. Acknowledgment. This work is supported by National Research Foundation through the Nano R&D Program (Grant 2008-04406) and grant (KRF-2008-314-C00169). Y.P. and H.K. acknowledge support by the KRISS Yeon-Hak Research Program and by the NRF (Grant 2009-067309). Experiments at PAL were supported in part by MEST and POSTECH.
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