Cysteine Chemisorption on Cu(110) - American Chemical Society

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(S)-Cysteine Chemisorption on Cu(110), from the Gas or Liquid Phase: An FT-RAIRS and XPS Study E. Mateo Marti,† Ch. Methivier, and C. M. Pradier* Laboratoire de Physico-Chimie des Surfaces, CNRS (UMR 7045), Ecole Nationale Supe´ rieure de Chimie de Paris, 11 rue Pierre et Marie Curie, 75005 Paris, France Received April 27, 2004. In Final Form: August 26, 2004 (S)-Cysteine has been deposited on a Cu(110) surface from sublimation of a crystalline phase. The surface was characterized by Fourier transform reflection absorption infrared spectroscopy (FT-RAIRS) during exposure and compared to the same copper surface after immersion into cysteine solutions at various pH values. X-ray photoelectron spectroscopy (XPS) measurements provided a chemical characterization of the surface at certain stages. The combination of these two techniques highlighted the importance of the cysteine “source” for the adsorbed form of the molecules and the mode of interaction. The zwitterionic amino acid was found to be predominant after adsorption at pH values close to the isoelectrice point (IEP) of the molecule but also when the layer was formed in the vapor phase. This state was very sensitive to the atmosphere, contained an excess of hydroxyls, and/or underwent reduction into the anionic form when in contact with water or air. Weakly bound cysteine or cystine molecules, formed in the adsorbed phase, were considered to explain the average thickness of the adsorbed layer that was close to 20 Å. As expected, immersion in very acidic or very basic solutions led to cationic and anionic forms, respectively.

1. Introduction The adsorption of biomolecules on metal surfaces is a relevant topic nowadays, leading to important applications in biomaterials, biocorrosion, biosensors, and biocompatibility; when having an asymmetric carbon atom, they may be used to make chiral surfaces of fundamental interest in chemical biology and pharmacology1-3 and induce specific chemical or biological responses. Studying biomolecule-surface bonding will improve knowledge in the surface modification field and surface design with specific adsorption properties. Proteins themselves are known to play a major role in fouling deposits such as those formed in food industry and, more generally, in any biological medium. The adsorption of proteins at solid-liquid interfaces has been well described in several recent papers;4-7 the role of electrostatic or hydrophobic/hydrophilic interactions was exemplified by data at various pH values. However, little was said about the nature of the peptide or amino acid that was really involved in the binding with the solid surface. Using amino acids or peptides to gain information on the mode of protein adsorption is a common strategy. Lieberg et al. in the 1980s characterized the adsorption of glycine, histidine, and other related complex molecules on evaporated gold and copper films.8-11 Since that time, adsorption experiments, often coupled with theoretical investigations, provided better insight into the adsorption * Corresponding author. E-mail: claire-marie-pradier@ enscp.jussieu.fr. † Present address: Centro de Astrobiologia (CSIC-INTA). Carretera de Ajalvir, Km. 4, 28850 Torrejon de Ardoz, Madrid, Spain. (1) Ratner, B. D.; Hornell, T. A.; Shuttleworth, D.; Thomas, H. R. J. Colloid Interface Sci. 1981, 83, 630. (2) Sundgren, J. E.; Bodo, P.; Lundstro¨m, I. J. Colloid Interface Sci. 1986, 110, 9. (3) Barlow, S. M.; Raval, R. Surf. Sci. Rep. 2003, 50, 201-341. (4) Omanovic, S.; Roscoe, S. G. J. Colloid Interface Sci. 2000, 227, 452-460. (5) Murray, B. S.; Cros, L. Colloids Surf., B 1998, 10, 227-241. (6) Fukuzaki, S.; Urano, H.; Nagata, K. J. Ferment. Bioeng. 1995, 80, 6-11. (7) Moulton, S. E.; Barisci, J. N.; Bath, A.; Stella, R.; Wallace, G. G. J Colloid Interface Sci. 2003, 261, 312-319.

forms of amino acids on various metallic surfaces. Imamura et al. made clear the role of acidic residues in the protein adsorption on stainless steel surfaces.12,13 Jurkiewicz et al. established a sequence of adsorption energy for tyrosine, histidine, and serine on gold.14 The adsorption of (S)-histidine on Cu(110) was shown to adopt a configuration allowing interaction of both the carboxylate and the dehydrogenated N of the cycle on the side chain in interaction with the surface.15 It has been reported that the interaction of several amino acids (alanine, glycine, and proline) with metals may occur via either the NHx (amino) or COO- (carboxylate) end groups, depending on the conditions of interaction and on the substrate. The side chain of the amino acid can also play an important role; as an example, for a sulfurcontaining amino acid like cysteine, the strong affinity of sulfur to different metals is a key factor.11 Cysteine bears three main functional groups, which are the potential binding sites, the amino and carboxylate functionalities, and the terminal thiol group; among the 20 amino acids, cysteine is the only one having a thiol side group, and beside the well-known affinity of thiol groups for some metals, those may undergo dimerization via the oxidation of two thiol groups. The SH group of cysteine is also capable of donating and accepting intramolecular hydrogen bonds, hence playing a role in the stabilization of the secondary structure of the proteins.16 Cysteine is often on the outer side of proteins, being a potential link to anchor these proteins to inorganic or organic supports. (8) Lieberg, B.; Lundstro¨m, I.; Wu, C. R.; Salaneck, W. R. J. Colloid Interface Sci. 1985, 108, 123-132. (9) Lieberg, B.; Carlsson, C.; Lundstro¨m, I. J. Colloid Interface Sci. 1987, 120, 64-75. (10) Ihs, A.; Liedberg, B.; Uvdal, K.; To¨rnkvist, C.; Bodo¨, P.; Lundstro¨m, I. J. Colloid Interface Sci. 1990, 140, 192-206. (11) Ihs, A.; Lieberg, B. J. Colloid Interface Sci. 1991, 144, 282-292. (12) Imamura, K.; Mimura, T.; Okamoto, M.; Sakiyama, T.; Nakanishi, K. J. Colloid Interface Sci. 2000, 229, 237-246. (13) Imamura, K.; Kawasaki, Y.; Awadzu, T.; Sakiyama, T.; Nakanishi, K. J. Colloid Interface Sci. 2003, 267, 294-301. (14) Slojkowska, R.; Jurkiewicz-Herbich, M. Colloids Surf., A 2001, 178, 325-336. (15) Marti, E. M.; Me´thivier, C.; Dubot, P.; Pradier, C. M. J. Phys. Chem. 2003, 107, 10785-10792. (16) Quiam, W.; Krimm, S. Biopolymers 1992, 37, 1503.

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The adsorption of cysteine on different metals, mainly on gold, and also on evaporated copper surfaces, has been reported in the literature using several surface science techniques; it has also been studied in solution using electrochemical techniques, sometimes associated with microscopy, thus bringing additional information about the influence of pH and/or concentration on the binding process and structure layer. Liedberg and Ihs found that (S)-cysteine binds to a gold surface via the SH group, whereas a more complex adsorption process seems to occur on copper.11 Recent scanning tunneling microscopy (STM) studies on Au(110) suggest that the molecular binding to the gold surface is associated with local surface restructuring;17 the structure of a self-assembled monolayer (SAM) of (S)-cysteine on a Au(111) surface was investigated in solution at pH 1 by electrochemical STM: the adlayer structure of (S)-cysteine molecules on Au(111) originates from strong Au-S binding plus intermolecular interactions;18 highly ordered and stable monolayers giving the same two-dimensional periodicity and structure have been obtained on a Au(111) electrode in aqueous solution.19 The strong chemisorption of cysteine via the S atom on Au(111) was confirmed by a density functional theory (DFT) study that made clear a hybridization between the S 2p orbitals and the Au d bands.20 Most of these studies were focused on the characterization of (S)-cysteine adlayers on gold, and there are still questions about the adsorption of cysteine on copper, for instance, its chemical form, the nature of the chemical groups involved in the adsorption, and the orientation of the molecule. This prompted us to investigate the interaction of (S)-cysteine with a well defined copper surface both from solutions at various pH values and under vacuum conditions. In this paper, we report an original combination of in situ and ex situ data obtained upon the adsorption of the amino acid (S)-cysteine on a Cu(110) surface. Before characterizing the copper surfaces modified by (S)-cysteine under various conditions, we prepared solutions of cysteine, adjusted them at three pH values, and characterized them by IR in the attenuated total reflection (ATR) mode. These preliminary analyses provided us with reference spectra for the molecule at pH values acidic, basic, or close to its isoelectric point. Moreover, in this mode of analysis, the band intensities were not dependent on the orientation of the molecule in contrast to the reflection absorption infrared spectroscopy (RAIRS) mode which implies surface selection rules. A second series of data were obtained by polarization modulation reflection absorption infrared spectroscopy (PM-RAIRS) measurements on the Cu(110) surface after immersion of the copper sample into solutions at pH values equal to the ones of the solutions analyzed before. A punctual X-ray photoelectron spectroscopy (XPS) analysis of a sample, after drying, was also performed. Eventually, the Cu(110) surface was characterized by reflection absorption infrared spectroscopy (RAIRS) and X-ray photoelectron spectroscopy (XPS) during exposure to (S)-cysteine and under ultrahigh vacuum (UHV) conditions, respectively. RAIRS, either with polarization modulation or not, is a particularly appropriate technique for detecting changes (17) Ku¨hnle, A.; Linderoth, T. R.; Hammer, B.; Besenbacher, F. Nature 2002, 415, 891-893. (18) Xu, Q.-M.; Wan, L.-J.; Wang, C.; Bai, C.-L.; Wang, Z.-Y.; Nozawa, T. Langmuir 2001, 17, 6203-6206. (19) Zhang, J.; Chi, Q.; Nielsen, J. U.; Friis, E. P.; Andersen, J. E. T.; Ulstrup, J. Langmuir 2000, 16, 7229-7237. (20) Felice, R. D.; Selloni, A.; Molinari, E. J. Phys. Chem. B 2003, 107, 1151-1156.

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in the adsorbed chemical groups and providing information about the geometrical orientation of the adsorbed molecules relative to the surface. Auger electron spectroscopy (AES) was systematically performed before and after dosing the (S)-cysteine, to check the copper surface cleanliness and chemical composition, respectively. 2. Experimental Section IR-ATR. Solutions (10 mM) of (S)-cysteine were prepared from Milli-Q water, leading to pH 5.4. The solution pH was adjusted by the addition of HCl (37% from Prolabo, France) or KOH (37% from Prolabo, France) to reach values of 1.0 or 10.5, respectively. These three solutions were successively deposited on an ATR ZnSe crystal; spectra were recorded by adding 32 scans, at an 8 cm-1 resolution, and using a deuterated triglycine sulfate (DTGS) detector. PM-RAIRS. The copper sample was placed in the external beam of a Fourier transform infrared (FTIR) instrument, Nicolet Nexus spectrometer, and the reflected light was focused on a nitrogen-cooled mercury cadmium telluride (MCT) detector. A ZnSe grid polarizer and a ZnSe photoelastic modulator to modulate the incident beam between p and s polarizations (HINDS Instruments, PEM 90, modulation frequency ) 37 kHz) were placed prior to the sample. The detector output was sent to a two-channel electronic device that generates the sum and difference interferograms. Those were processed and Fouriertransformed to lead to the PM-RAIRS signal ∆R/R ) (Rp - Rs)/ (Rp + Rs). All reported spectra were recorded at an 8 cm-1 resolution by the coaddition of 64 scans, using modulation of polarization enabled to perform rapid analyses of the samples after immersion without purging the atmosphere and without requiring a reference spectrum. Spectrum Processing. In PM-IRAS experiments, the spectrum of the interface is superimposed to the second order Bessel function.21 The baseline was hence systematically corrected by subtracting a spline line, a polynomial fit recommended for curved baselines. Adsorption of (S)-Cysteine from Aqueous Solutions. Solutions (2 mM) of (S)-cysteine were prepared similarly the the above method. Before immersion into the (S)-cysteine solutions, the Cu(110) crystal was polished on diamond paste down to 0.5 µm grade and cleaned under a flow of pure H2 at 873 K for 90 min. The copper sample was immersed in the fresh (S)-cysteine solutions for 10 min, copiously rinsed in solution at a pH equal to the immersion one, dried by blowing clean air, and analyzed in the air by PM-RAIRS. Adsorption of (S)-Cysteine under Low Pressure. RAIRS spectra were recorded using a Nicolet magna 550 FTIR spectrometer equipped with a liquid-nitrogen-cooled MCT wide-band detector with a spectral range of 650-4000 cm-1. The spectrometer was interfaced to the ultrahigh vacuum (UHV) chamber with ZnSe windows. The Cu(110) single crystal was mounted in a multitechnique (UHV) chamber, with RAIRS, low-energy electron diffraction (LEED), and AES facilities and a base pressure of 1 × 10-9 mbar. The Cu(110) crystal was cleaned by cycles of Ar+ ion sputtering, flashing, and annealing to ∼900 K. The surface structure and cleanliness were monitored by LEED and AES before and after the adsorption experiments. (S)-cysteine (>99%) was obtained from the Fluka Chemical Company and used without further purification. It was contained in a small electrically heated glass tube, separated from the main vacuum chamber by a gate valve, and differentially pumped by a turbomolecular pump. Before sublimation, the (S)-cysteine powder was outgassed at 360 K; it was then heated to 375 K and dosed into the chamber. The dosing pressure was maintained at around 2 × 10-8 mbar during the RAIRS measurements. The RAIRS spectra were recorded in situ, throughout a continuous dosing regime, and ratioed against a reference background single beam spectrum recorded on the clean Cu(110) crystal. All spectra were obtained at an 8 cm-1 resolution, the by coaddition of 256 scans. (21) Buffeteau, T.; Desbat, B.; Turlet, J. M. Appl. Spectrosc. 1991, 45, 380.

(S)-Cysteine Chemisorption on Cu(110)

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Figure 1. IR-ATR spectra of (S)-cysteine solutions at pH 1.0, 5.4, and 10.4.

Figure 2. RAIRS spectra of the Cu(110) surface after the adsorption of (S)-cysteine in solutions at pH 1.0, 5.4, and 10.5.

XPS Analyses. XPS analyses of the Cu(110) surface, after immersion, rinsing and, drying or after adsorption at low pressure and transfer in the air, were carried out on a VG ESCALAB Mk II spectrometer, using the Al KR X-ray source (1486.6 eV). A 20 eV pass energy was applied for analyzing the following core level spectra: Cu 2p, CuLVV, O 1s, C 1s, S 2p, and N 1s. Analyses were performed at two takeoff angles, 90 and 45°. The binding energies were calibrated against the binding energy of Au 4f7/2 and Cu 2p3/2; with this calibration, the low-energy carbon peak, attributed to hydrocarbon contamination, was measured at 285.0 ( 0.1 eV. The sensitivity factors of the elements were taken in ref 22.

3.1. Different Forms of (S)-Cysteine. (S)-Cysteine has three ionization constants, the corresponding pK values being 1.71, 8.33, and 10.28; the first one is related to the carboxyl group, the second one, to the SH group, and the third one, to the NH3+ group. The possible ionic forms of the cysteine are thus, when increasing the pH, NH3+/SH/COOH, NH3+/SH/COO-, NH3+/S-/COO-, and NH2/SH/COO-. Each of the ionic forms of cysteine can be distinguished by its characteristic infrared frequencies. The IEP of cysteine is equal to 5.02.23,24 For a better identification of the vibrational groups and further discussion of the orientation of the molecule, the IR-ATR spectra of cysteine solutions at three pH values, 1.0, 5.4, and 10.5, were recorded. A careful analysis of the IR spectra was done in the 1200-1800 cm-1 region, where significant changes were observed (see Figure 1); absorption bands are thus appearing, with no restriction due to surface selection rules or shift due to interaction with the metal. The spectrum of the (S)-cysteine in an acidic solution (lower line) shows several intense absorption bands in the 1200-1800 cm-1 region. Two bands, at 1520 and 1640 cm-1, can be ascribed to the symmetric and asymmetric deformation modes of NH3+. Noticeable is the absorption band at 1735 cm-1 due to CdO stretch. However, a weak absorption at 1400 cm-1, the wavenumber of the COOsymmetric stretch, indicates that a fraction of the carboxylic groups remain deprotonated. The very weak signal at 2550 cm-1, attributed to the SH stretch, shows that this vibration has a very low extinction coefficient. At that very low pH, all sulfur atoms are indeed expected to

be under the SH form. The less intense signal, at 1340 cm-1, is attributed to the CH2 wagging mode. At very low pH, the predominating form is likely to be NH3+/SH/COOH with some NH3+/SH/COO- also being present. At pH 5.4 (intermediate spectrum of Figure 1), the main change is the disappearance of the band at 1740, showing that all acidic groups are now deprotonated. The net increase of the band at ∼1600 confirms the increase of the fraction of carboxylates. The decrease of the absorption band at 1520 cm-1 shows the deprotonation of a fraction of the NH3+ groups; a weak signal at 2550 cm-1 still appears on the spectrum, indicating the presence of SH groups. The dominating form is now NH3+/SH/COO- with some NH2/SH/COO-. In a basic solution, pH 10.5 (upper spectrum), the spectrum is dominated by intense bands at 1407 and 1560-1600 cm-1; the former can be easily ascribed to the COO- symmetric stretch, while the latter, shifted to a lower frequency compared to the previous spectrum, is a combination of the NH2 deformation, at ∼1580 cm-1, and the COO- asymmetric stretch, at ∼1600 cm-1. The NH3+ symmetric stretch vibration, at 1525 cm-1, has now disappeared. As expected at this elevated pH, no absorption could be detected at 2550 cm-1, the S-H stretching frequency. The dominating form, identified by its IR spectrum, is thus anionic, NH2/S-/COO-. These assignments have been done following vibrational data in the literature.11,25-27. 3.2. Adsorption of (S)-Cysteine from Solutions at Various pH Values. PM-RAIRS Analyses. RAIRS spectra of the Cu(110) surface after 10 min of immersion in (S)-cysteine solutions at pH 1.0, 5.4, and 10.5, rinsing, and drying are shown in Figure 2; immersions were stopped after 10 min, having checked that longer times did not lead to more intense IR absorption signals, but to a risk of surface corrosion. After immersion in an acidic cysteine solution, pH 1.0 (lower spectrum), the predominant feature is the strong ν(CdO) stretching vibration at 1735 cm-1; another band related to the carboxylic functional group is the ν(CsO) one at 1260 cm-1. Two less intense signals, at 1625 and 1495 cm-1, respectively, can be assigned to the deformation modes δas(NH3+) and δs(NH3+) of an ammonium functional group. A weak, but

(22) Scofield, J. H. J. Electron Spectrosc. Relat. Phenom. 1976, 8, 129. (23) Garfinkel, D.; Edsall, J. T. 1958, 80, 3823-3826. (24) Edsall, J. T.; Wyman, J. Biophysical Chemistry; Academic Press: New York, 1958; Vol. 1.

(25) Pearson, J. F.; Slifkin, M. A. Spectrochim. Acta 1972, 28A, 24032417. (26) Stewart, S.; Fredericks, P. M. Spectrochim. Acta 1999, 55A, 1641. (27) Cooper, E.; Krebs, F.; Smith, M. D.; Raval, R. J. Electron Spectrosc. Relat. Phenom. 1993, 64/65, 469-475.

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still detectable, absorption band appears on the spectrum at 1404 cm-1, proving that some cysteine molecules bear a carboxylate group. Finally, the rather broad signal, centered at 1625 cm-1, may include a contribution from δas(COO-) but also from the scissor deformation of NH2 groups. This seems to be corroborated by some absorption at 1080 cm-1, an NH2 deformation frequency. The weak band, at 1050 cm-1, is attributed to the CsN stretch. Observing all these bands proves that, even at pH 1.0, (S)-cysteine is adsorbed on copper under three possible forms, the cationic NH3+/S-/COOH, likely to be the most abundant, but also the zwitterionic one, NH3+/S-/COO-, and probably the neutral one, NH2/S-/COOH, with Sstanding for S protonated or S bound to copper. However, the SsH stretching mode at ∼2550 cm-1, expected at this low pH value (see the spectrum of Figure 1), is not visible, suggesting that the molecule is bound to the copper surface via the deprotonated sulfur atom or that there is some dimerization of the cysteine molecules into cystine; the formation of disulfide by adsorption of thiols on metal surfaces was demonstrated by several authors;28,29 the formation of cystine from cysteine is also known to be rapid in the presence of copper.30,31 The COOH group, not involved in the binding of the molecule, is likely to be placed in such a way that the CdO is close to the normal to the surface, as deduced from the intense band at 1735 cm-1. In a solution at pH 5.4, a value close to the isoelectric point (IEP) of cysteine, the concentration of zwitterionic cysteine molecules is known to be at a maximum and the concentration of cationic cysteine molecules, if there are any, should be equal to the concentration of anionic ones. The Cu(110) RAIRS spectrum following (S)-cysteine adsorption at pH 5.4 shows, as expected, the almost disappearance of the CdO and CsO stretching modes (intermediate spectrum). The carboxylate functionality is now responsible for the main features in the spectra, the νas(COO-) and νs(COO-) modes appearing at 1610 and 1395 cm-1, respectively. The very high intensity of the former band is likely to be due to a contribution of the NH3+ groups’ asymmetric deformation mode at ∼1630 cm-1; the symmetric deformation mode, expected at ∼1500 cm-1, is hardly observed, which suggests that the NH3+ does not adopt a position normal to the surface; this fact could suggest a reorientation of the molecule on the surface. Noticeable is the increase in the absorption at 1080 cm-1 that reflects a higher fraction of NH2 groups. Here again, the absence of any absorption signal at 2550 cm-1 probes the absence, or very low amount, of SsH groups. Finally, the RAIRS spectrum of (S)-cysteine adsorbed on a Cu(110) surface from a solution at pH 10.5 is slightly different (upper spectrum). There are three intense bands; the first one at 1407 cm-1 is attributed to the νs(COO-) mode, while the corresponding asymmetric mode is seen at ∼1606 cm-1; observing both the symmetric and asymmetric COO- stretches suggests that the carboxylate group is slightly tilted with the two oxygen atoms not equidistant to the surface. Moreover, the strong decrease of the absorption at ∼1600 cm-1 can be interpreted as proof of the disappearance of NH3+ groups to the benefit of NH2. Finally, a new signal has appeared at 1465 cm-1 that may

be ascribed to a combination of NH deformation and CH2 scissoring. One can conclude that (S)-cysteine mostly exists in its anionic form, NH2/SH/COO-. The absence of any SH signal does not tell anything about the binding site, since SH is not expected to exist after immersion at that pH. The continuous shift to higher wavenumbers and the intensity decrease of the CH symmetric and asymmetric stretching vibrations, when the pH decreases, reflect a loss in the ordering of the adsorbed layer.32 XPS Analysis after Immersion in an Acidic Solution. Figure 3 shows the C 1s, O 1s, N 1s, and S 2p XPS spectra of the copper sample after immersion in a (S)cysteine solution at pH 1.0, rinsing, drying, and transfer in the air. The Cu 2p3/2 peaks and CuLVV signals (spectra not shown) were observed at EB ) 932.8 eV and KE ) 918.4 eV, respectively, values showing that the copper surface is still in its metallic state. The C 1s peak has been curvefitted using the VG Eclipse software. It was best-fitted with three contributions: the first peak, at the lowest binding energy 284.9 ( 0.2 eV, is assigned to the carbon bound only to C or H; the second peak, at 286.4 ( 0.2 eV, is attributed to the carbon in the C-N bond; and the third peak, at the highest binding energy 288.4 ( 0.2 eV, is attributed to the carbon in COOH or COO-.33 The O 1s peak was broad, fitted with two contributions at 531.6 ( 0.2 and 533.1 ( 0.2 eV, corresponding respectively to the OH and to the double-bonded oxygen of the acidic group or to the oxygen in hydroxyls and in water molecules.34 Note that the O/S intensity ratio, corrected by the Scofield factors, IO/IS ) 4, shows an excess of oxygen (the expected value for the cysteine molecule is 2), which indicates the presence of adsorbed oxygen, OH groups, or water on the surface. We rather exclude the first hypothesis, since the O 1s peak could not be fitted with a contribution at ∼530530.5 eV. The N 1s peak was asymmetric and broad, fitted with a main peak centered at 399.9 ( 0.2 eV, as expected for nitrogen in NH2, and a shoulder at 401.1 ( 0.2 eV that we attribute to nitrogen in NH3+; hence, though immersion was performed in a very acidic medium, in contrast to the PM-RAIRS data, the XPS analysis tends to show that the major fraction of the adsorbed molecules are in a COOH/ NH2 form. The sulfur peak was best-fitted with two peaks, of similar intensities, centered at 162.4 and 163.5 eV, values characteristic of sulfur bound to a metal and sulfur in physisorbed thiol or disulfides, respectively.35 This suggests that part of the cysteine molecules may not be bound via the sulfur atom to the metal. Another possibility would be the formation of disulfide under X-ray during spectra acquisition.36 A peak was detected at 202 eV that shows the presence of Cl on the surface; its intensity, corrected by the Scofield factor, was half that of nitrogen. This is obviously resulting from the surface treatment in acidic solution. The thickness of the organic layer could be calculated from the Cu 2p3/2 peak intensities on this sample and on a clean copper one and assuming that the attenuation length obeys the equation given by Whitesides et al. for alkanethiolates37

(28) Fenter, P.; Eberhardt, A.; Eisenberger, P. Science 1994, 266, 1216. (29) Wan, L.-J.; Hara, Y.; Noda, H.; Osawa, M. J. Phys. Chem. B 1998, 102, 5943-5946. (30) Cavallini, D.; Marco, C. D.; Dupre, S.; Rotilio, G. Arch. Biochem. Biophys. 1969, 130, 354. (31) Gurd, F. R. N.; Wilcox, P. E. Adv. Protein Chem. 1956, 11, 335.

The thickness was calculated to be equal to 22 Å.

λ (Å) ) 9.0 + 0.022KE (eV) ) 21 Å for Cu 2p3/2

(32) Nuzzo, R. G.; Dubois, L. H.; Allara, D. L. J. Am. Chem. Soc. 1990, 112, 558-569. (33) Boonaert, C. J. P.; Dufreˆne, Y. F.; Derclaye, S. R.; Rouxhet, P. G. Colloids Surf., B 2001, 22, 171-182.


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Figure 3. XPS spectra after the adsorption of (S)-cysteine in solution at pH 1.0.

Figure 4. RAIRS spectra of the Cu(110) surface during (S)cysteine exposure, P ) 10-8 mbar.

XPS analysis at 45° was performed, and the intensity ratios were also calculated. The N/S value was close to unity whatever the takeoff angles, whereas the O/S ratio passed from 4.5 to 3 when the angle changed from 90 to 45°; this simply shows that there is, on average, no stratification between the N and S atoms but that oxygen is closer to the copper surface than sulfur. 3.3. Adsorption of (S)-Cysteine from the Gas Phase. In Situ RAIRS Analysis. Figure 4 shows the development of the RAIRS spectra with increasing ex(34) Slaughter, A. R.; Banna, M. S. J. Phys. Chem. 1988, 92, 2165. (35) Nuzzo, R. G.; Zegarski, B. R.; Dubois, L. H. J. Am. Chem. Soc. 1987, 109, 733-740. (36) Zharnikov, M.; Frey, S.; Heister, K.; Grunze, M. Langmuir 2000, 16, 2697-2705. (37) Laibinis, P. E.; Bain, C. B.; Whitesides, G. M. J. Phys. Chem. 1991, 95, 7017-7021.

posure of (S)-cysteine on Cu(110) at room temperature. No evolution of the spectra was observed after 30 min. Assignment of the IR bands from the spectra has been made following data from the literature and from our adsorption experiments in solutions (see Table 1). All these bands can be correlated to the functionalities of the molecule, suggesting that the latter is adsorbed intact on the copper surface. Moreover, no changes in the nature of the molecular forms or in their orientation seem to occur when passing from a low to a high surface coverage. The RAIR spectra clearly show the absence of any ν(CdO) stretching vibration at ∼1740 cm-1 and the appearance of a vibration at 1396 cm-1 that can be attributed to the symmetric stretching mode of COO-; the most intense band, at 1652 cm-1, is very likely due to the asymmetric deformation mode of NH3+ groups. Even though the exact form of the amino group is not straightforward on the basis of the RAIRS spectra, NH2 and NH3+ having very close deformation vibration frequencies, the absence of the strong NH2 scissoring feature, due to be at ∼1580 cm-1, and the presence of the δs(NH3+) at 1510 cm-1 support our previous assignment. We thus conclude that the (S)-cysteine is mainly adsorbed on copper in the zwitterionic form. Once the chemical form of (S)-cysteine has been identified, the adsorption geometry can be discussed from a detailed analysis of the COO-, NH3+, CH, CH2, and SH RAIRS signals. The metal-surface selection rule implies that only vibrational modes corresponding to a transition dipole moment having a component perpendicular to the metal surface are infrared active and therefore appear in the RAIRS spectrum. Let’s consider first the NH3+ group: The spectrum of the Cu(110) surface, after (S)-cysteine adsorption, is dominated by a strong band at 1652 cm-1 assigned to its asymmetric deformation mode. The NH3+ asymmetric and symmetric stretching bands are weak but still observed in the RAIRS spectra at ∼3144 and

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Table 1. Main Infrared Peaks and Their Assignments for (S)-Cysteine on Cu(110) at 300 K (S)-cysteine/Cu(110) (low pressure)

assignment

(S)-cysteine on Cu(110), from solution pH 1.0 pH 5.4 pH 10.5

3144 2992 2957 2908 2870

νas(NH3 νs(NH3+) νas(CH2) ν(CH) νs(CH2) ν(SH)

1652

δas(NH3+) νas(COO-) +)

1735 1625 1590

1630 1606 1508

1510

δs(NH3

1495

1423 1396 1340

sciss(CH2) νs(COO-) wag(CH2)

1216 1130

rock(NH3+)

1426 1404 1339 1315 1260 1211 1125 1080 1050 882

1061 900

assignment

+)

ν(CN)

∼2992 cm-1, respectively. Also remarkable is the presence of a strong-medium intensity band at 1061 cm-1, which could be attributed to the C-N stretch. This analysis suggests that the ammonium group will be oriented slightly tilted from the normal to the surface, allowing the δ(NH3+) symmetric and asymmetric deformation modes to be active (1510 and 1652 cm-1, respectively), and in agreement with a strong-medium intensity band for the C-N stretching. Regarding the carboxylate group, the two expected vibrational modes are the asymmetric νas(COO-) stretch and the symmetric νs(COO-) stretch, whose intensities are strong on the infrared spectra of the (S)-cysteine solutions. On the RAIRS spectra, only the νs(COO) stretch at ∼1396 cm-1 is observed, suggesting that the two oxygen atoms are equidistant to the metal surface, probably involved in the binding process, and making the νas(COO-) stretch dipole inactive. The plane of the carboxylate group must be slightly tilted from the normal to the surface to explain the medium intensity of the νs(COO-) band. The absence of the S-H mode stretching at 2550 cm-1 in the spectra of Figure 4 is a strong indication of the binding of (S)-cysteine to the Cu(110) surface via the sulfur atom, as has already been observed for cysteine adsorbed on gold or on copper11,38,39 and confirming that the sulfur atom is one of the coordination sites of (S)-cysteine. Strong interactions between two cysteine molecules18,19 and the formation of a disulfide on the surface may also occur.29 The C-H region stretching region, 2800-3000 cm-1, exhibits several weak-medium bands in the 2870-2957 cm-1 region that can include the stretching modes of the CH and CH2 groups. The presence of these stretching vibrations is in agreement with the CH and CH2 groups being slightly tilted from the normal to the surface; other vibrational modes, such as the CH2 scissors at 1423 cm-1 and the CH2 wagging at 1340 cm-1, can also be observed, which corroborates our proposed geometry. Auger Analysis. After interaction of (S)-cysteine for 2, 5, 7, 10, and 14 min and gas evacuation, the copper surface was analyzed by Auger spectroscopy. The copper peaks are progressively attenuated, while the signals at (38) Uvdal, K.; Bodo¨, P.; Lieberg, B. J. Colloid Interface Sci. 1992, 149, 162. (39) Dodero, G.; Michieli, L. D.; Cavalleri, O.; Rolandi, R.; Oliveri, L.; Dacca`, A.; Parodi, R. Colloids Surf., A 2000, 175, 121-128.

1606 1570

1403

1465 1420 1407

1104 1080 1050 882

1080 1050 877

ν(CdO) δas(NH3+) νas(COO-) sciss(NH2) δs(NH3+) NH2 bend sciss(CH2) νs(COO-) wag(CH2) bend(CH) ν(CsO) ν(CsO) (acid dimers?) rock(NH3+) δ(NH2) ν(CsN) ν(CsC)

Figure 5. Auger peak-to-peak intensity ratios, N380eV/Cu920eV, C270eV/Cu920eV, and S162eV/Cu920eV, after (S)-cysteine exposure.

274, 400, and 150 eV increase, showing the uptake of carbon, nitrogen, and sulfur on the surface. The evolution of the Auger peak-to-peak intensity ratios, N380eV/Cu920eV, C270eV/Cu920eV, and S162eV/Cu920eV, is shown in Figure 5. They did not significantly vary after 10-15 min of exposure, whereas in situ Fourier transform reflection absorption infrared spectroscopy (FT-RAIRS) showed a net increase of the bands up to 30 min. This is likely to be due to a partial desorption of cysteine upon gas evacuation or, more likely, to a position of the sample, closer to the molecular beam that may explain a quicker saturation of the surface. XPS Analysis after Adsorption from the Gas Phase. An XPS analysis of the copper sample was performed after the adsorption of (S)-cysteine in the gas phase (P ) 5 × 10-8 mbar, 25 min) and transfer of the sample in the air. The C 1s, O 1s, N 1s, and S 2p XPS spectra of the copper sample are shown in Figure 6. The Cu 2p3/2 peaks and CuLVV signals were again observed binding energies characteristic of the copper metallic state. The C 1s peak was best-fitted with three contributions, at 285.1, 286.1, and 288.2 eV, that can be assigned to carbon in CH, in C-N, and in carboxylic or carboxylate groups, respectively. The N 1s peak was slightly asymmetric, with a main peak centered at 399.9 eV, a value


Langmuir, Vol. 20, No. 23, 2004 10229

Figure 6. XPS spectra after the adsorption of (S)-cysteine in the gas phase.

4. Discussion and Conclusions

Table 2. XPS (θ) Normalized Intensity Ratios in (S)-Cysteine/Cu(110) 45° 90°

O/S

N-CH/S

N/S

1.96 1.96

0.93 1.2

0.76 0.78

close to the one reported for N 1s in ammonia adsorbed on copper,40 and a shoulder at 401.0 eV that can be attributed to N in NH3+.41 The oxygen peak also shows a unique contribution at 531.60 eV, which is comparable to that recorded for the carboxylate group in glycine42 or (S)-cysteine.38 XPS tends to show that, after adsorption in the gas phase and transfer in the air, (S)-cysteine on Cu(110) is mainly in a COO-/ NH2 form. The sulfur peak could be, here again, decomposed into two peaks, one, the most intense, at 162.0 eV, a value close to that of sulfur 2p in organosulfurs and alkylthiols adsorbed on gold,43 and this time, a smaller one at 163.9 eV, suggesting that some molecules are weakly bound.39 The thickness of the molecular layer, calculated from the attenuation of the copper signal, was found to be equal to 15 Å. Table 2 shows the ratios of the normalized XPS intensities, O/S ∼ 2, N/S ∼ 0.8, N-CH/S ∼ 1. These are in good agreement with the composition of the cysteine molecule. XPS analysis at 45° was performed, and the intensity ratios were also calculated. Noticeable is the very close values of the O/S and N/S ratios whatever the takeoff angles; this simply shows that, on average, there is no stratification between the O and S atoms.

The adsorption of (S)-cysteine from aqueous solutions at various pH values, or from low pressure (vacuum conditions), has been characterized by FT-RAIRS and XPS spectroscopy. The type of (S)-cysteine “source”, aqueous solutions at various pH values or sublimation of a crystalline powder, seems to influence the form of the adsorbed amino acid as well as its binding mode and geometry. To clarify the discussion, Table 3 summarizes the main RAIRS and XPS data. After immersion in aqueous solutions, the adsorbed form of (S)-cysteine very much depends on the pH value. We have clearly identified four different chemical forms of (S)-cysteine, zwitterionic, predominant from a solution at a pH close to the IEP, as expected, cationic at low pH, neutral at low pH, and anionic at high pH, besides the possible deprotonation of the SH group. As a matter of fact, RAIRS spectra never show any SH mode; this is a strong indication of the involvement of sulfur either in a bonding with the metallic surface or in the dimerization of the cysteine molecules. Further XPS analysis of the surface after immersion in an acid solution confirms the presence of dimerized or weakly bound cysteine molecules as well as of extra OH groups in the adsorbed layer. This was suggested both by the intense contribution at 531.6 eV and by the O/S ratio equal to 4. The absence of any stratification between O and S atoms on the surface can be explained by the presence of adsorbed OH and of cysteine or cystine molecules not bound to the surface via sulfur atoms. The layer thickness, around 20 Å, is another

Table 3. Species Identified on the Surface from FT-RAIRS and XPS Data from aqueous solution surface species identified from FT-RAIRS data from XPS

pH 1.0

pH 5.4

pH 10.5

COOH/SH/NH3+ + COO-/SH/NH3+ + COOH/SH/NH2 NH3+ ≈ NH2 excess of OH ∃ weakly bond S SWB ≈ SS-Au

COO-/SH/NH3+ + COO-/SH/NH2

COO-/S-/NH2

from gas phase

NH2 + NH3+ no excess of O ∃ weakly bond S SWB < SS-Au

10230


Figure 7. Probable orientation of (S)-cysteine on Cu(110) after adsorption in the gas phase.

proof of the presence of more than one monolayer of (S)cysteine on the surface. (S)-cysteine has been successfully deposited on Cu(110) under UHV conditions; the in situ RAIRS analysis reveals that the molecule is adsorbed intact, mostly in the zwitterionic form. Conversely, the XPS spectrum shows that a fraction of zwitterionic molecules were reduced into anionic ones. We believe this is related to the X-ray exposure or to the transfer in the air, containing water molecules, causing a reduction of NH3+ into NH2. Let’s add that observing such an effect of water is in agreement with the fact that, even after adsorption in solution at very low pH, a significant fraction of the molecules were not positively charged but kept an amine group. The XPS analysis also shows that the COO- group and S atoms are, on average, at the same distance from the surface. The combination of FT-RAIRS data and XPS analysis at 90 and 45° takeoff angles allows us to propose a configuration of the molecule with two binding sites, the COOgroup and the S atom as represented in Figure 7. The carboxylate group interacts with the two oxygen atoms equidistant to the surface. Note however that the calculation of the thickness leads to a value again higher than that expected for a cysteine monolayer; rather tightly bound (resisting rinsing or UHV conditions) cysteine molecules are likely to form multilayers. The thickness is lower than that after adsorption in solution, in agreement with the smaller contribution of the XPS S 2p peak at high binding energy. The formation of multilayers of another amino acid, glycine, was evidenced on Cu(110)44 and Pt(111),45 when substrates were held below room temperature. Multilayers of cysteine were also observed (40) Hussla, I.; Seki, H.; Chuang, T. J.; Gortel, Z. W.; Kreuzer, H. J.; Piercy, P. Phys. Rev. B: Condens. Matter 1985, 32, 3498. (41) Clark, D. T.; Peeling, J.; Colling, L. Biochim. Biophys. Acta 1976, 453, 533. (42) Uvdal, K.; Bodo, P.; His, A.; Lieberg, B.; Salaneck, W. R. J. Colloid Interface Sci. 1990, 140, 207. (43) Bain, C. D.; Biebuyck, H. A.; Whitesides, G. M. Langmuir 1989, 5, 723.

Mateo Marti et al.

on Cu(110), upon immersion at room temperature, and explained by the formation of a complex ionic structure involving copper surface atoms or dissolved copper ions.10 The building of a thick molecular layer may also be explained by the easy formation of cystine molecules. Note that this is not contradictory to the geometry deduced from the RAIRS data (Figure 7), since the major fraction of the signal originates from the layer directly linked to the metal surface.46 Finally, it is interesting to make a comparison between the analysis for the adsorption of (S)-cysteine on copper under vacuum conditions and in solution at pH 5.4, as the cysteine mainly exists in the zwitterionic form in both cases. Under vacuum conditions, the molecule interacts with the copper surface through the sulfur atom and the two oxygens of the carboxylate group placed equidistant to the surface and the ammonium group adopts a normal position to the surface; conversely, after adsorption in solution, the interaction likely also proceeds via the sulfur atom, but the two oxygens of the carboxylate group are not equidistant to the surface any longer and the orientation of the NH3+ group is not normal to the surface, as indicated by the weak symmetric deformation; in this case, we rather suggest that the molecule is attached to the surface through the sulfur atoms and the rest of the molecule is like a chain orientated far away from the surface. This could be related to strong interactions between the cysteine chains and water molecules in solution or to the presence of Cl-, detected by XPS, on the surface that decreases the number of sites available. Hence, we could conclude that different conditions of adsorption influence the orientation of the molecule on the surface and the chemical groups involved in the bonding. RAIRS data for cysteine on copper, adsorbed either from the liquid or gas phase, enable us to make clear the influence of the conditions of (S)-cysteine “admission” onto the form and geometry of the molecules. The combination of RAIRS and XPS data was necessary to clearly identify the nature of the adsorbed species. We would like to emphasize that this comparison of data after different experimental procedures of adsorption puts an accent on the importance of the latter and also shows the easy change of phase for such a nonrigid molecule like cysteine. It also suggests the possible multiple biological mechanisms involving that molecule like intra- and intermolecular bonds within proteins. Acknowledgment. The CNRS is gratefully acknowledged for the research grant of E.M.M. LA048952W (44) Barlow, S. M.; Kitching, K. J.; Haq, S.; Richardson, N. V. Surf. Sci. 1998, 401, 322-335. (45) Lo¨fgren, P.; Krozer, A.; Lausmaa, J.; Kasemo, B. Surf. Sci. 1997, 370, 277-292. (46) Greenler, G. J. Chem. Phys. 1966, 44, 310.

Cysteine Chemisorption on Cu(110) - American Chemical Society

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