Reflection Anisotropy Spectroscopy Study of the Adsorption of Sulfur

Department of Physics, OliVer Lodge Laboratory, UniVersity of LiVerpool, U.K., ... School of Clinical Sciences, UniVersity of LiVerpool, U.K. and Scho...
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Langmuir 2006, 22, 3413-3420

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Reflection Anisotropy Spectroscopy Study of the Adsorption of Sulfur-Containing Amino Acids at the Au(110)/Electrolyte Interface Rozenn LeParc,† Caroline I. Smith,† M. Consuelo Cuquerella,† Rachel L. Williams,‡ David G. Fernig,§ Clive Edwards,§ David S. Martin,† and Peter Weightman*,† Department of Physics, OliVer Lodge Laboratory, UniVersity of LiVerpool, U.K., Clinical Engineering, School of Clinical Sciences, UniVersity of LiVerpool, U.K. and School of Biological Sciences, UniVersity of LiVerpool, U.K. ReceiVed September 22, 2005. In Final Form: January 12, 2006 Protein interactions with surfaces are key to understanding the behavior of implantable medical devices. The optical technique of reflection anisotropy spectroscopy (RAS) has considerable potential for the study of interactions between important biological molecules and surfaces. This study used RAS to investigate the adsorption of S amino acids onto Au(110) in a liquid environment under different conditions of potential and pH. Certain spectral features can be associated with the Au(110), as reported previously, while other features are assigned to bonds between the amino acids and the Au surface. The RA spectra are shown to be influenced by the structure of the amino acid, the solution pH, and the applied electrode potential. This work has assigned the negative feature at 2.5 eV to the Au-thiolate, bond while the positive feature at 2.5 eV is assigned to the disulfide bond. The broad spectral feature at 3.5 eV is attributed to the Au-amino interaction, while the sharper feature at slightly higher energy is associated with the Au-carboxylate interaction. Sulfur-containing amino acids are frequently found on the outside of protein molecules and could be used to anchor the protein to the surface.

Introduction It is well established that the interface between implanted biomaterials and the biological environment is critically important in determining the success or failure of a medical devices.1 The key to controlling these interfacial reactions is through a greater understanding of the interactions of proteins with biomaterial surfaces that subsequently influence the cellular response to implanted devices. Proteins are complex macromolecules that have hydrophobic and hydrophilic regions of positive or negative charge; their interactions with surfaces will depend on these characteristics, as well as the properties of the surface. Protein adsorption to a surface is a dynamic process, and the characteristics of the protein layer that is important for subsequent cellular interactions with the material include the type of protein adsorbed, the amount adsorbed, the reversibility, and the structure and orientation of the adsorbed macromolecules. All amino acids in the protein structure have unique functional groups that determine the protein structure and their behavior at the solid/ aqueous interface. Cysteine has a highly reactive sulfur-containing functional group, with the thiol of cysteine often found as a disulfide bond to another cysteine to form cystine (Figure 1). The thiol of cysteine, disulfide of cystine, and the thioether of methionine are often found on the surface of native proteins and provide a means to anchor proteins to surfaces. Sulfur-containing groups are known to interact strongly with gold surfaces,2 and it is expected that the primary adsorption of these amino acids onto the Au(110) surface will be via the S groups. In this study, reflection anisotropy spectroscopy (RAS) was used to determine the adsorption of the S-containing amino acids cysteine, cystine, and methionine onto the Au(110) surface in * To whom correspondence should be addressed. [email protected]. Fax: +44-151-794-3441. † Department of Physics. ‡ Clinical Engineering. § School of Biological Sciences.

E-mail:

(1) Castner, D. G.; Ratner, B. D. Surf. Sci. 2002, 500, 28. (2) Smith, R. K.; Lewis, P. A.; Weiss, P. S. Prog. Surf. Sci. 2004, 75, 1.

Figure 1. Structure of cysteine, cystine, and methionine.

a liquid environment. The optical technique of RAS has considerable potential for the study of interactions between important biological molecules and surfaces.3 It is one of the few surface science techniques able to bridge the gap between the UHV and ambient environments, and the technique has been shown to provide information on the orientation of adsorbed molecules.4-7 Recent studies have shown that RAS can yield insight into the metal/liquid interface8-11 and to the interactions between molecules in solution and metal surfaces.12,13 The strategy is to determine the differences between the RAS of the adsorbed molecules and the clean Au(110) surface as a function of the applied voltage and to compare these differences for the three amino acids, cysteine, cystine, and methionine, in (3) Weightman, P.; Martin, D. S. In Surfaces and Interfaces for Biomaterials; Vadgama, P., Ed.; Woodhead Publishing Ltd: Cambridge, England, 2005. (4) Frederick, B. G.; Power, J. R.; Cole, R. J.; Perry, C. C.; Chen, Q.; Haq, S.; Bertrams, Th.; Richardson, N. V.; Weightman, P. Phys. ReV. Lett. 1998, 80, 4490. (5) Frederick, B. G.; Cole, R. J.; Power, J. R.; Perry, C. C.; Chen, Q.; Richardson, N. V.; Weightman, P.; Verdozzi, C.; Jennison, D. R.; Schultz, P. A.; Sears, M. P. Phys. ReV. B 1998, 58, 10883. (6) Power, J. R.; Weightman, P.; Bose, S.; Shkrebtii, A. I.; Del Sole, R. Phys. ReV. Lett. 1998, 80, 3133. (7) P. Weightman Phys. Status Solidi A 2001, 188, 1443. (8) Sheriden, B.; Martin, D. S.; Power, J. R.; Barrett, S. D.; Smith, C. I.; Lucas, C. A.; Nichols, R. J.; Weightman, P. Phys. ReV. Lett. 2000, 85, 4618. (9) Mazine, V.; Borensztein, Y.; Cagnon, L.; Allongue, P. Phys. Status Solidi A 1999, 175, 311. (10) Mazine, V.; Borensztein, Y. Phys. ReV. Lett. 2002, 88, 147403. (11) Weightman, P.; Smith, C. I.; Martin, D. S.; Lucas, C. A.; Nichols, R. J.; Barrett, S. D. Phys. ReV. Lett. 2004, 92, 199707. (12) Smith, C. I.; Maunder, A. J.; Lucas, C. A.; Nichols, R. J.; Weightman, P. J. Electrochem. Soc. 2003, 150, E233. (13) Smith, C. I.; Dolan, G. J.; Farrell, T.; Maunder, A. J.; Fernig, D. G.; Edwards, C.; Weightman, P. J. Phys.: Condens. Mater. 2004, 16, S4385.

10.1021/la052584u CCC: $33.50 © 2006 American Chemical Society Published on Web 02/22/2006

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order to gain insight into their binding modes by exploiting the different coordination of the S groups in the three molecules. There are other possible binding mechanisms involving the amine and carboxyl groups, and the sensitivity of RA spectra to the molecular adsorption geometry combined with differences in the molecular structure of the amino acids makes it possible to obtain insight into how the adsorption of these molecules varies with the potential of the gold substrate and the pH of the aqueous buffer. Experimental Section A Au(110) single crystal (purity 99.999%) of 10 mm diameter and 2 mm thickness with an exposed area of 0.5 cm2 was used in all experiments. The Au(110) was prepared by polishing with successively smaller diamond paste down to 0.25 µm, being cleaned in an ultrasonic bath, followed by flame annealing to preserve the (1 × 2) reconstruction,14 and then being covered with ultrapure water (Millipore Q system 18 MΩ‚cm) before being transfered into the electrochemical cell. All electrolyte solutions were prepared from 0.1 M H2SO4 (BDH, Aristar), NaH2PO4, and K2HPO4 (BDH, Analar) dissolved in ultrapure water. The 1 mM amino acid solutions were prepared from L-cystine, L-cysteine, and L-methionine (Flucka) in ultrapure water without further purification. The electrochemical cell used was a typical homemade threeelectrode cell with a platinum counter electrode and a saturated calomel electrode (SCE) as the reference. A silica strain free disk was used as the window. The potentiostat used was an Autolab PGSTAT 30 with General Purpose Electrochemical Software (GPES) from Eco Chemie. The solutions were purged with argon prior to use. The RAS technique has recently been reviewed.15 The instrument used in this work follows Aspnes design16 and operated in the range 1.5-5.5 eV, whereby the measured RAS signal is given by {Re}

{ }

{

}

r[11h0] - r[001] ∆r ) {Re}2 r r

Figure 2. RA spectra from the Au(110) in 0.1 M NaH2PO4/ K2HPO4 at -0.6 (- - -), -0.4 (×), -0.2 (4), 0 (s), 0.2 ([), 0.4 (O), and 0.6 V (- ‚).

Figure 3. RA Spectra of Au(110) (thin solid line), Au(110) + L-cysteine at -0.6 (- - -), -0.4 (×), -0.2 (4), 0 (solid line), 0.2 ([), 0.4 (O), and 0.6 V (- ‚) in 0.1 M NaH2PO4/K2HPO4.

(1)

RA spectra were collected for the bare gold surface when it was held at potentials of 0, 0.2, 0.4, 0.6, 0, -0.2, -0.4, and -0.6 V vs SCE. To collect the RA spectra of the amino acids, a solution of each amino acid was added to the electrolyte at 0 V vs SCE in the electrochemical cell to give a final concentration of 0.1 mM. Spectra were collected for the same range of potentials. Individual experiments were performed with the solution pH at 1.2, 7.1, or 8.4. The spectrum of Au(110) at each potential was subtracted from the spectrum of the amino acid adsorbed on Au(110) in order to obtain the spectrum for each amino acid at each potential and pH.

Results The RA spectra of the Au(110) surface as a function of the applied voltage in the range -0.6 to 0.6 V is shown in Figure 2. It is well known17 that in an electrochemical environment the Au(110) adopts a (1 × 2) reconstruction at 0 V, and the spectrum corresponding to 0 V in Figure 2 is very similar to that obtained in UHV from the Au(110) (1 × 2) surface.15,18 As the potential is made more positive, the surface undergoes a phase transition to the (1 × 1) structure, and as it is made more negative, there is a phase transition to the (1 × 3) structure.17 There are accompanying strong changes in the RA spectra as a function of voltage (Figure 2). RAS is extremely sensitive to the (14) Kolb, D. M. In Structure of Electrified Interfaces; Lipkowski, J., Ross, P. N., Eds.; VCH Publishers: New York, 1993. (15) Weightman, P.; Martin, D. S.; Cole, R. J.; Farrell, T. Rep. Prog. Phys. 2005, 68, 1251. (16) Aspnes, D. E.; Harrison, J. P.; Studna, A. A.; Florez, L. T. J. Vac. Sci. Technol. A 1988, 6, 132. (17) Magnussen, O. M.; Wiechers, J.; Behm, R. J. Surf. Sci. 1993, 289, 139. (18) Stahrenberg, K.; Herrmann, Th.; Esser, N.; Richter, W. Phys. ReV. B 2001, 65, 35407.

Figure 4. RA Spectra of L-cysteine after subtraction of the Au(110) spectra at the corresponding potential at -0.6 (- - -), -0.4 (×), -0.2 (4), 0 (s), 0.2 ([), 0.4 (O), and 0.6 V (- ‚).

morphology of the Au(110) surface8-11,15 and in particular the presence of monatomic steps,11,15 factors which are difficult to control in the preparation of specimens. This work focuses on the interpretation of the RAS of the adsorbed amino acids in order to gain insight into the mechanism of molecular adsorption from their different molecular structures and variations in the spectra with voltage and pH. Figure 3 shows that there are strong changes in the evolution of the RAS of L-cysteine on Au(110) as a function of applied potential. The magnitude of these changes is enhanced when the spectra are corrected for the voltage dependence of the RAS of the clean Au surface by subtracting the RAS of the Au(110) at a particular potential from that of the L-cysteine/Au(110) obtained at the same potential (Figure 4). The spectra shown in Figures 5-7 have been derived using the same subtraction procedure described for Figure 4, and the analysis that follows concentrates on these difference spectra obtained for each of the three molecules as a

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Figure 5. Comparison of the L-cysteine (s), L-cystine ([), and L-methionine (O) RA spectra after subtraction of the Au(110) spectra at the corresponding potential as a function of potential at pH 1.2.

function of applied potential and pH. It is implicit in this approach that any changes induced in the underlying RAS of Au by the adsorption of the molecules are small compared to the dependence of the molecular contribution to the RAS on voltage and pH. Figure 5 shows that at pH 1.2 the RA spectra of cysteine and cystine have the same profile for all applied voltages with the spectra of cystine being consistently shifted to more negative values. At -0.6 V, the spectrum falls to a pronounced negative peak at 2.5 eV and then rises smoothly to a broad positive feature at ∼3.7 eV. As the potential is increased to more positive values, the negative peak reduces in intensity, a positive peak develops at ∼2.7 eV, and the positive feature at 3.7 eV sharpens into a

peak. By +0.6 V, the two positive peaks at 2.5 and 3.5 eV dominate the spectrum. Apart from the absence of the strong negative feature at 2.5 eV, the RAS of methionine resembles that of the other two molecules at -0.6 V. As the voltage is made more positive, the broad feature at ∼3.5 eV in the methionine spectrum sharpens and a positive peak develops at 2.7 eV, as found for the other molecules but not as pronounced. At +0.2 and +0.4 V, the spectra of all three molecules have identical shapes, though they differ in intensities. As the potential is increased from 0 V, the RAS of methionine develops a weak negative feature at 2.5 eV, which by +0.6 V resembles that observed in the RAS of cysteine and cystine at -0.6 V.

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Figure 6. Comparison of the RA spectra after subtraction of the Au(110) spectra at the corresponding potential of L-cysteine (s), L-cystine ([), and L-methionine (O) as a function of potential at pH 7.1.

At pH 7.1 (Figure 6), the RAS of cysteine and cystine are virtually identical for all voltages. At -0.6 V, the RAS of methionine is very similar to that of the other two molecules, and this similarity increases as the voltage is made more positive until the spectra of all three molecules are essentially identical at -0.2 V. As the voltage increases further to +0.4 V, the RAS of methionine diverges from that of the other two molecules with a strengthening of the strong negative peak at ∼2.5 eV that is characteristic of the spectra of the other two molecules at the more negative voltages at both pH 7.1 and 1.2. As the voltage is increased from -0.6 to +0.2 V, the RAS of all three molecules show the same trends as observed at pH 1.2, namely the growth

of a positive peak at ∼2.7 eV and a sharpening of the broad positive feature at ∼3.7 eV. At more positive voltages, the RAS of methionine shows a progressive weakening of both the negative feature at 2.5 eV and the positive peaks at 2.7 and 3.7 eV, and the spectrum becomes dominated by the 2.7 eV peak and a slightly stronger peak that grows at ∼3.2 eV as the voltage increases from 0 to +0.6 V. From 0 to +0.6 V, the RAS of all three molecules show a reduction in the intensity of the strong peak at 3.7 eV with the development of a pronounced dip immediately preceding this feature. A comparison of the spectral changes that occur in the RAS of the three molecules as a function of the applied voltage at pH 8.4 is shown in Figure 7. At -0.6 and -0.4

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Figure 7. Comparison of the RA spectra after subtraction of the Au(110) spectra at the corresponding potential of L-cysteine (s), L-cystine ([), and L-methionine (O) as a function of potential at pH 8.4.

V, the RAS of cysteine and cystine are virtually identical, as found for these voltages at the other two pH’s. As the voltage is increased, the same changes occur in the RAS of these molecules as are observed at pH 1.2 and 7.1, namely a reduction in the strength of the negative peak at 2.5 eV, the growth of the positive peak at 2.5 eV, and the sharpening of the positive feature at 3.7 eV. The dip that occurs in the spectra of cysteine and cystine immediately preceding the positive 3.7 eV peak and which is first observed at +0.6 V at pH 1.2 and at +0.2 V at pH 7.1 now appears at 0 V at pH 8.4. The RAS of methionine at -0.6 V shows the negative peak at 2.5 eV that is characteristic of the spectra of the other two molecules at negative voltages, but at

this pH, it differs from the other spectra in that the positive peaks at 2.5 and 3.7 eV do not develop as the voltage increases. However, there is a sudden change in the spectral shape of the RAS of this molecule when the voltage is increased to +0.6 V when the spectrum becomes dominated by a strong broad feature between 2.5 and 4.5 eV. Finally, Figure 8 shows the dependence of the RAS intensity and the reflectivity, r, of Au(110) + L-cysteine at 2.5 eV as the potential is cycled between -0.6 and 0.6 V. The changes in the reflectivity are small, as expected, since this quantity is dominated by the bulk optical response. The RAS, however, is a sensitive monitor of the optical response of the surface, and the large

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Figure 8. The intensity of the RAS signal at 2.5 eV Re(∆r/r) (]) and the reflectivity r (solid line) measured as a function of applied electrode potential for Au(110) + L-cysteine in 0.1 M NaH2HPO4/ K2HPO4.

changes observed in the RAS as the voltage is varied will be discussed in what follows.

Discussion The calculation of the RA spectra of adsorbed molecules on surfaces is not only very difficult but also requires a good knowledge of the geometry of the adsorption site and the nature of the local electronic structure.15 Under these circumstances, it is not possible to interpret the spectral features in the RAS of these amino acids from first principles, and therefore, it is better to concentrate on associating changes in the RAS induced by variations in pH and voltage with likely changes in molecular orientation and binding, bearing in mind the different geometries of the three molecules. The charge on the functional groups will depend on the pH of the solution, and at different applied potentials, the driving force for adsorption will vary. Analysis of the spectra clearly demonstrates that these conditions influence the shape of the RA spectra for each amino acid. Noting that the spectra of cysteine and cystine are almost identical for all voltages at neutral pH (Figure 6) and for negative potentials at pH’s 1.2 and 8.4 (Figures 5 and 7) suggests that these results arise from the formation of thiolate (Au-S) bonds between these amino acids and the Au surface.19-21 The thiol of cysteine readily dissociates at the Au surface to produce a Au-S bond.19,22-24 The disulfide of cystine similarly readily dissociates to produce a thiolate bond between the Au and S atoms25-27 so the spectra of the two molecules adsorbed on the Au surface are identical and have the same shape as the spectrum previously reported for cysteine adsorbed on the Au(110) (1 × 2) surface in UHV.28 Furthermore, with the sulfur preferring to bind to the low-coordinated Au sites, clusters formed between cysteine molecules stabilized by intermolecular hydrogen bonding between carboxylate and amino groups rotate off the [11h0] direction to (19) Fawcett, W. R.; Fedurco, M.; Zova´cˇova´, K.; Borkowska, Z. J. Electroanal. Chem. 1994, 368, 265. (20) Dodero, G.; De Michieli, L.; Cavalleri, O.; Rolandi, R.; Oliveri, L.; Dacca`, A.; Parodi, R. Colloids Surf., A 2000, 175, 121. (21) Cohen-Atiya, M.; Mandler, D. J. Electroanal. Chem. 2003, 500-551, 267. (22) Ku¨hnle, A.; Linderoth, T. R.; Hammer, B.; Besenbacher, F. Nature 2002, 415, 891. (23) Shin, T.; Kim, K. N.; Lee, C. W.; Shin, S. K.; Kang, H. J. Phys. Chem. B 2003, 107, 11674. (24) Cavalleri, O.; Gonella, G.; Terreni, S.; Vignolo, M.; Floreano, L.; Morgante, A.; Canepa, M.; Rolandi, R. Phys. Chem. Chem. Phys. 2004, 6, 4042. (25) Gro¨nbeck, H.; Curioni, A.; Andreoni, W.J. Am. Chem. Soc. 2000, 122, 3839. (26) Di Felice, R.; Selloni, A.; Molinari, E. J. Phys. Chem. B 2003, 107, 1151. (27) Hager, G.; Brolo, A. G. J. Electroanal. Chem. 2003, 550-551, 291. (28) Isted, G. E.; Martin, D. S.; Smith, C. I.; LeParc, R.; Cole, R. J.; Weightman, P. Phys. Status Solidi C 2005, 12, 4012.

LeParc et al.

allow the S atoms to bind at bridge sites between the first and second layer of gold atoms.22 Thus, at negative voltages at pH 7.1, it is anticipated that cysteine and the disassociated cystine bind to the Au surface with identical geometries involving a Au-S bond (sharp, negative feature at 2.5 eV) and electrostatic interaction with the Au and amino group (broad, positive feature at 3.5 eV). As the applied potential is taken positive, it is expected that the positively charged protonated amino group is repelled from the surface and the negatively charged carboxylate group is attracted to the surface.29 At pH 7.1, there is a significant change in the spectral shape of the RAS of these two molecules on increasing the potential from -0.2 to 0 V. The negative peak at 2.5 eV decreases in intensity, a positive peak arises at ∼2.7 eV, and the broad feature at 3.5 eV increases in intensity, sharpens up, and moves to higher energy. This characteristic change in spectral profile is attributed to a transition from a geometry in which the Au-S bond dominates, with a secondary contribution from the Au-NH3+ group to one in which the Au-S bond changes and the Au-OCO takes over from the Au-NH3+. In particular, the change in the spectral region between 1.5 and 3.0 eV of the adsorbed cysteine and cystine at pH 7.1, as the voltage is varied from -0.2 to +0.2 V, is associated with the oxidation of the surface thiolate species to form a disulfide bond. It is suggested that the sharp positive peak at ∼2.7 eV is a characteristic feature of the presence of the disulfide bond and it is reasonable to attribute the change in sign of the RAS as the thiolate bond is replaced by the disulfide band to the difference in site preference on the Au surface of these two species.27 Furthermore, it is possible that the intense peak at 3.6 eV at 0 V at pH 7.1 arises from the interaction of the carboxylate group with the Au(110) surface. This association is consistent with the observation that this characteristic peak appears at the same potential at pH 8.4 (Figure 7) where the charge on the carboxylate group will also be negative as at pH 7.1 but that it does not appear until higher applied potentials, +0.2 and +0.4 V, at pH 1.2 where the carboxylate group is expected to be neutral (Figure 5). Figures 5-7 show that for the spectral regions 1.5-3.0 eV the RAS of adsorbed cysteine and cystine are similar for all three pH’s at -0.6 V and are also similar, though with a very different line shape, at +0.6 V. This difference in line shape is associated with the change from bonding by the thiolate species to bonding involving the disulfide bond. However, the potential at which the characteristic change in spectral profile associated with this change in bonding occurs is dependent on pH, i.e., +0.6 V at pH 1.2, +0.2 V at pH 7.1, and 0 V at pH 8.4. This variation is consistent with the expectation that the redox potential for cysteine oxidation to cystine will be an important factor in the adsorption of these two amino acids. In a KClO4 solution (pH 7), the oxidation occurs at +0.25 V vs SCE, while in NaOH solution (pH 12), the oxidation occurs at a much lower potential -0.2 V vs SCE.27,30-32 It has been suggested21,27 that the reaction mechanism for the adsorption of alkanethiols on Au is a two-step process where the first step is the oxidative adsorption of the thiol onto Au followed by the discharging process as in

Au + RSH f Au-SR- + H+

(2)

(29) Di Felice, R.; Selloni, A. J. Chem. Phys. 2004, 120, 4906. (30) Fawcett, W. R.; Fedurco, M.; Zova´cˇova´, K.; Borkowska, Z. Langmuir 1994, 10, 912. (31) Fawcett, W. R.; Fedurco, M.; Zova´cˇova´, K.; Borkowska, Z. J. Electroanal. Chem. 1994, 368, 275. (32) Xie, Q.; Zhang, Y.; Yuan, Y.; Guo, Y.; Wang, X.; Yaao, S. J. Electroanal. Chem. 2000, 484, 41.

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Au-SR- + H+ f Au-SR + 1/2H2

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(3)

This explains why at pH 1.2 the bulk concentration of the acidic solution gives sufficient protons to favor reaction 3 forming the thiolate bonds necessary to produce the characteristic RA spectra of the thiolate. For cysteine, the Au-thiolate was observed to form much easier at pH 1.2 and at a lower potential than at higher pH values. For example, in the pH 7.1 and 8.4 solutions, the addition of cysteine at 0 V did not show signs of the Authiolate until taken to -0.6 V; however, it formed immediately at 0 V at pH 1.2. To determine how the changes in cysteine and cystine spectra are related, the addition of the molecules was carried out at the extreme voltages of -0.6 and +0.6 V in a pH 7.1 solution. The RAS recorded at these two voltages had the same spectral profile as when the molecules were added at 0 V and the potential taken to (0.6 V. It is interesting to observe how the spectral features change at intermediate potentials dependent on the potential at which the molecule was added. When the molecules were added at -0.6 V and the potential cycled to 0.6 V, the Au-thiolate persisted for longer before oxidation to a disulfide, and when the disulfide had formed, it persisted to more negative potentials before the thiolate reformed in comparison with the addition of the molecules at +0.6 V. This is demonstrated in Figure 8 where the peak at 2.5 eV is monitored with respect to the voltage applied to the L-cysteine/Au(110) as a function of time. Cycling between voltages produces a reversible cycle with a hystersis between the positive-to-negative and the negative-to-positive parts of the cycle. Thus, there is a smoother transition between the thiolate structure and the disulfide as the potential goes from negative to positive than for the reverse reaction. It is suggested that the shoulder on the reverse reaction is associated with the breaking of the disulfide bond. The S-C bond in methionine will not dissociate and form S-Au bonds in the same way as the S-H and S-S bonds of the other two molecules. At pH 1.2 and -0.6 V (no negative feature at 2.5 eV), it is reasonable to suppose that the molecules adsorb via the positively charged and protonated NH3+ group, as demonstrated by the broad feature at 3.5 eV. This view is consistent with the RAS of the three molecules under these conditions (Figure 5) since the difference between the RAS of methionine and the other two molecules between 1.5 and 3.0 eV is associated with the absence of Au-S bonds in the former. The identical form of the RAS of all three molecules from 3.0 eV onward is consistent with view that the spectral shape in this region arises from bonds between the amino groups and the Au surface. As the potential is made more positive at pH 1.2, the broad feature in the RAS of methionine associated with bonding to the amino group is transformed into a sharp peak at 3.8 eV which has been earlier associated with bonding to the carboxylate group. The changes in spectral shape of the RAS above 3 eV and in particular the strength and shape of the 3.8 eV peak have the same dependence on applied voltage for all three molecules at pH 1.2 and 7.1. It is noted that the RAS of all three molecules have identical shapes for energies above 3 eV at +0.4 V at pH 1.2 and 0 V at pH 7.1. The difference in applied voltage at which these similarities occur is consistent with the expected changes in the charges on the amino and carboxylate groups at the two pH’s. This provides a good understanding of the spectral changes that occur in the RAS of these molecules above 3 eV as the bonding changes from the amino to the carboxylate groups at pH 1.2 and 7.1.

Table 1. Summary of the Assignment of Peaks in the RA Spectrum energy (eV)

associated bond/interaction

2.5 (negative feature) 2.7 (positive feature) 3.5 3.6

-S-Au(110) surface -S-Au(110) surface H3N+‚‚‚Au(110) surface COO-‚‚‚Au(110) surface

Considering the RAS of methionine in the spectral range from 1.5 to 3.0 eV, it is noted that the RAS of all three molecules are identical at -0.2 V at pH 7.1 (Figure 6), and from the deductions made earlier, this implies that in these conditions the S group of methionine is bound to the Au surface. It is suggested that this occurs via a change in the shape of the C-S-C group (thioether) such that the lone pair of electrons on the S bonds to the Au. The systematics of the changes in spectral shape in this region indicate that the S does not bond to the Au at pH 1.2 until a positive potential of +0.6 V is reached. The spectral changes that occur between -0.2 and +0.2 V at pH 7.1 for the other two molecules, most notably the transformation of the negative feature at 2.5 eV into a strong positive peak at ∼2.7 eV were interpreted as arising from the formation of disulfide bonds, presumably mediated by the close proximity of adsorbed cysteine clusters. Consequently, it is not surprising that similar changes, and in particular the formation of the positive peak at ∼2.7 eV, do not occur in the RAS of methionine as the C-S-C bond does not dissociate. Furthermore, the fact that the spectral features at positive potentials for the methionine at pH 7.1, and even more obviously at pH 8.4, are relatively weak could be due to the adsorption of the methionine in a less-ordered manner. The origin of many of the features of the RAS of the three molecules can be explained by considering the changes that occur in the spectral shapes as a function of pH and applied voltage, and this has provided some insight into the nature of the bonding to the Au surface. However, it has not been possible to account for the differences between the RAS of methionine and the other two molecules at pH 8.4 or for the differences between the RAS of cysteine and cystine at positive voltages at pH 8.4 (Figure 7). It may be that at high pH there is a difference in the orientation of the surface clusters. Such a difference in orientation could give rise to different molecular geometries when the disulfide bonds are formed at positive voltages. The data clearly show that the properties of the amino acids, the surface, and the surrounding aqueous environment all influence the orientation of the molecules as they bind to the surface. These S-containing groups may be available on the surface of proteins that interact with biomaterial surfaces when implanted and thus lead to changes in the properties of the adsorbed protein layer. More work is required to develop this technique to study larger proteins, but this work demonstrates the importance of RAS to study the interaction of biological molecules with surfaces under aqueous conditions.

Conclusions This study shows that sulfur-containing amino acids adsorb through an anisotropic organization on Au(110) resulting in a strong change in the Au(110) RA spectrum. Furthermore, the orientation of the adsorbed amino acids is influenced by the structure of the amino acids, the properties of the surface, and the pH of the environment.

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This work has assigned the negative feature at 2.5 eV to the Au-thiolate bond, while the positive feature at 2.5 eV is assigned to the disulfide bond. The broad spectral feature at 3.5 eV is attributed to the Au-amino interaction, while the sharper feature at slightly higher energy is associated with the Au-carboxylate interaction (Table 1).

LeParc et al.

Acknowledgment. R.L. and M.C.C. acknowledge the support of the EU through a Marie Curie Fellowship. The work was also supported by the UK EPSRC.

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