Raman Characterization of Monolayers Formed from Mixtures of

Mar 31, 2010 - ... and APY monolayers is deprotonated. The structure of the MTR monolayer practically does not depend on the pH of the surrounding sol...
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Raman Characterization of Monolayers Formed from Mixtures of Sodium 2-Mercaptoethanesulfonate and Various Aromatic Mercapto-Derivative Bases Andrzej Kudelski* Faculty of Chemistry, UniVersity of Warsaw, Pasteur 1, PL-02-093 Warsaw, Poland ReceiVed: January 8, 2010; ReVised Manuscript ReceiVed: March 15, 2010

Metal electrodes covered with organic (mono)layers containing π-delocalized structures have many potential applications, for example, in construction of bioelectronic elements with high efficiency of electron transfer. In this contribution, a silver surface was modified with mixed monolayers formed from sodium 2-mercaptoethanesulfonate (MES) and four model (stable and easily available) aromatic thiols with strong basic properties: 4,6-diamino-2-mercaptopyrimidine (APY), 1H-1,2,4-triazole-3-thiol (HTR), 4-methyl-1,2,4-triazole3-thiol (MTR), and 3-amino-1,2,4-triazole-5-thiol (ATR). The structure of the formed monolayers was determined from surface-enhanced Raman scattering (SERS) measurements. These studies showed that, despite significant differences in the molecular structures, MES is a very promising candidate for making mixed monolayers with mercapto-derivative aromatic bases that are relatively homogeneous (without large onecomponent domains) in broad pH range. At high pH, in a one-component ATR monolayer, a significant amount of molecules are in the anionic form and adopt a flat orientation versus the metal surface. At the same pH, in mixed ATR+MES monolayers, a significantly larger part of ATR molecules than for the respective one-component monolayer is adsorbed in the neutral form with the aromatic ring(s) oriented perpendicularly to the metal surface. Also in the mixed APY+MES and HTR+MES monolayers, a significantly smaller part of HTR or APY molecules than for the respective one-component HTR and APY monolayers is deprotonated. The structure of the MTR monolayer practically does not depend on the pH of the surrounding solution. Increase of the ratio of acidic dissociation and reorientation of ATR molecules from the perpendicular to the parallel orientation is also observed during storage of the respective one-component and mixed monolayers in water, phosphorus buffers, and in the solutions of model peptides (bovine serum albumin or laccase). In some cases, the reorientation of ATR molecules forming the linkage monolayer when immersed in the peptide solution is very large. Significant spectral changes during soaking in water and solutions of model peptides has also been observed for APY monolayers. All studied mixed monolayers practically prevent the direct adsorption of peptides on the metal surface for at least 30 min. 1. Introduction The controlled modification of metal surfaces with monolayers formed from various organic compounds is the field of intensive investigations because of many potential applications of such systems. For example, modification of metal surfaces with organic monolayers may provide a microenvironment on metal surfaces similar to biological membranes, which allows adsorption of active (not denatured) enzymes on metal electrodes.1-6 The immobilization of active enzymes onto metal surfaces modified with organic so-called linkage layers forms the basis for a range of biosensors, biofuel cell elements, and other bioelectronics devices.4-6 Mercapto-derivative compounds are among the most successful chemicals employed for formation of such linkage layers6-10 because they react chemically with many metals (e.g., Ag, Au, Pt, Cu), forming very stable metal-sulfur bonds.11-13 Linkage layers are usually formed from alkanethiols, ω-substituted alkanethiols, and their mixtures (for some applications, mixed monolayers are better than those formed from only one compound14,15). In many practical applications, when high efficiency of an electron transfer between an enzyme molecule and the metal substrate must be obtained (e.g., in biofuel cell elements), the linkage layers * E-mail: [email protected]. Phone: +48-228220211, x278. Fax: +48-228225996.

containing aromatic compounds seem to be significantly better than those formed only from aliphatic species.16-19 Previous study of mixed monolayers formed from 4-mercaptobenzoic acid and model mercapto-derivative aromatic bases shows that it is often difficult to form a mixed monolayer (contains both acidic and basic compounds in a comparable amount) of which composition does not change significantly during partial desorption of molecules forming the monolayer (e.g., during soaking of such systems in water) or when the pH of the ambient solution is changed.20 In this contribution, the composition and the structure of various mixed monolayers are analyzed. The studied monolayers were formed from sodium 2-mercaptoethanesulfonate (MES), which is a very stable and commercially available sodium salt of the sulfonate-derivative thiol, and four commonly used (due to their stability and commercial availability) mercapto-derivative aromatic bases: 4,6-diamino-2mercaptopyrimidine (APY), 1H-1,2,4-triazole-3-thiol (HTR), 4-methyl-1,2,4-triazole-3-thiol (MTR), and 3-amino-1,2,4-triazole-5-thiol (ATR). Scheme 1 presents molecular structures of all of these compounds. Although mixed monolayers studied in this contribution have not yet been used practically, they may be potentially useful in many practical applications, since they contain aromatic moieties, which significantly facilitate an electron transfer via the monolayer, and charged sulfonate groups, which allow for strong electrostatic interactions with

10.1021/jp100196x  2010 American Chemical Society Published on Web 03/31/2010

Monolayers from Aromatic Mercapto-Derivative Bases

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SCHEME 1: Molecular Structures of MES, APY, HTR, MTR, and ATR

Figure 1. In situ SERS spectra of MES adsorbed on a roughened silver substrate from a 1 mM MES aqueous solution buffered with phosphorus buffers to (a) pH 2.0, (b) pH 3.4, (c) pH 5.3, (d) pH 6.7, (e) pH 8.1, (f) pH 10.4, (g) pH 11.5, and (h) pH 12.5. The total concentration of phosphate species in the solution was 0.1 M. Spectra were scaled and vertically shifted for clarity.

the charged groups of coadsorbed peptides. This contribution shows that in the wide pH range studied (2-12.5) MES and all studied aromatic bases formed mixed monolayers having a similar surface concentration for both compounds. The temporal evolutions of the structure of formed monolayers during soaking in water and in solutions of model peptides (bovine serum albumin and laccase) have also been investigated. The stability of studied mixed monolayers in the peptide solutions was not worse than the stability of the one-component monolayers formed from respective aromatic bases. 2. Experimental Section Thiols (APY, MTR, and ATR from Aldrich and HTR and MES from Fluka), peptides (laccase from the fungus Trametes Versicolor, lyophilized powder, 23.1 U/mg, from Fluka; and bovine serum albumin >99% from Sigma), silver electrodes (>99.99% from Mennica Panstwowa), and all inorganic compounds (analytical reagent grade, POCH) were used as received from commercial suppliers. Solutions were prepared with water with a resistivity of 18.0 MΩ cm (purification was carried out with a Millipore ultrapure water system). The pH was controlled with phosphorus buffers prepared from H3PO4, NaH2PO4, Na2HPO4, and Na3PO4. To obtain sufficiently enhanced intensity of the SERS bands, silver substrates were electrochemically roughened by three successive oxidation-reduction cycles in a 0.1 M KCl aqueous solution from -0.3 to 0.3 to -0.3 V at a sweep rate of 5 mV s-1. The cycling was finished at -0.3 V, and after that, the Ag electrode was kept for 30 s at this potential. The roughening procedure was carried out in a conventional three-electrode cell with a large platinum sheet as the counter-electrode and a 0.1 M KCl AgCl/Ag electrode as the reference (all potentials are

referred to the potential of this electrode). Then, the working electrode was removed at an open circuit potential and very carefully rinsed with water. On such silver substrates, organic species were adsorbed. All SERS measurements except desorption experiments and experiments in the peptide solutions were carried out at the silver substrates immersed in the solution of the respective thiol (or mixture of thiols). Raman spectra have been recorded with an ISA T64000 (Jobin Yvon) Raman spectrometer equipped with a thermoelectrically cooled CCD detector, 600 grooves/mm holographic grating, a Kaiser SuperNotch-Plus holographic filter, and an Olympus BX40 microscope with a 50× long distance objective. A Spectra-Physics model 2018-RM mixed argon/krypton ion laser provided excitation radiation of 647.1 nm. Scanning electron microscopic (SEM) examinations of roughened silver substrates were performed utilizing the SEM functions of a Microlab 350 (Thermo VG Scientific) equipped with a Duo-STEM detector (STEM, Hitachi S-5500, 15 kV). 3. Results 3.1. One-Component Monolayers. Figure 1 shows SERS spectra measured from a silver substrate immersed in a 1 mM MES aqueous solution buffered at various pH with phosphorus buffers (the total concentration of phosphate species in the solution was 0.1 M). From the analysis of the blank spectra measured at a roughened silver substrate immersed in pure (without MES) phosphorus buffers, one can conclude that the contribution of phosphate ions to the spectra presented in Figure 1 can be neglected (for more details, see the Supporting Information). The SERS spectrum of MES monolayer is dominated by the band at 792 cm-1, attributed to the C-S stretching vibration (where sulfur is from the sulfone group).21,22

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The other well visible bands are at 704, 1040, 1065, and 1292 cm-1. The band at 704 cm-1 is also due to the C-S stretching vibration; however, in this case, sulfur is from the thiol group. The 704 cm-1 band is characteristic for chemisorbed MES molecules having a trans conformation of the Ag-S-C-C chain (the C-S stretching vibration characteristic for the gauche conformer of the chain gives the band at 638 cm-1).21,22 The doublet at 1040/1065 cm-1 is attributed to the symmetric νs(SO3-) stretchingsthe component at higher wavenumber (also observed for the polycrystalline salt) is due to -SO3- groups strongly interacting with metal cations that have lost some hydration water molecules.21,22 The band at 1292 cm-1 is due to the antisymmetric νas(SO3-) stretching.21,22 As can be seen from Figure 1, except for the doublet at 1040/1065 cm-1, the

Kudelski measured spectrum practically does not depend on the pH of the surrounding solution. The largest change in the ratio of relative intensities of the components at 1040/1065 cm-1 is observed at pH higher than 3.4 (see Figure 1), when the protonation of the sulfonic group is impossible. On the other hand, increase of the relative intensity of the 1065 cm-1 component and decrease of the relative intensity of the 1040 cm-1 component, analogous to that presented in Figure 1, is observed when the concentration of Na+ cations in the surrounding solution increases.21,22 Therefore, the increase of the concentration of Na+ cations in the solution having higher pH is probably the main reason for the observed differences between spectra measured at various pH’s (the concentrations of Na+ ions in the ambient solution changes from ca. 0.05 M for

Figure 2. In situ SERS spectra of HTR, ATR, MTR, and APY adsorbed on roughened silver substrates from respective 1 mM aqueous solution buffered with phosphorus buffers to (a) pH 2.0, (b) pH 3.4, (c) pH 5.3, (d) pH 6.7, (e) pH 8.1, (f) pH 10.4, (g) pH 11.5, and (h) pH 12.5. The total concentration of phosphate species in the solution was 0.1 M. Spectra were scaled and vertically shifted for clarity.

Monolayers from Aromatic Mercapto-Derivative Bases experiments carried out at pH 2 to ca. 0.3 M for experiments carried out at pH 12.5). The temporal evolution of a SERS spectrum during storage of an MES monolayer on silver in water was studied in detail in our previous work.21 These studies showed that the MES monolayer immersed in water undergoes a very slow rearrangement process leading to an increase in the ratio of relative intensities of the ν(C-S) gauche band vs the ν(C-S) trans one (for these bands, sulfur is from the thiol group) and increase in the relative intensity of the ν(C-H) band at 2933 cm-1, which also suggests increasing of the molar ratio of MES molecules in the gauche conformation. Probably, the relative surface concentration of the gauche conformer increases when the surface concentration of MES molecules decreases because the gauche conformer needs more space on the surface than the trans one. The rearrangement is very slow, and during the desorption times analyzed in this contribution (up to 30 min), only a small percentage of MES molecules changes its conformation.21 Figure 2 shows SERS spectra of monolayers formed on silver from four model mercapto-derivative aromatic bases (HTR, MTR, ATR, and APY). Analogously to MES, the monolayers were formed from 1 mM aqueous solutions of respective thiols buffered at various pH’s with phosphorus buffers (the total concentration of phosphate species in the solution was 0.1 M). As can be concluded from the comparison of blank SERS spectra measured at a roughened silver substrate immersed in pure (without an aromatic compound) phosphorus buffers (for more details, see the Supporting Information) and the SERS spectra presented in Figure 2, no significant contribution of phosphate ions to the spectra presented in Figure 2 is observed. The aromatic compounds used in this contribution have been studied by many groups, and therefore, their vibrational spectra have already been carefully analyzed. The detailed vibrational assignments based on the density functional theory (DFT) calculations are available in the literature for HTR23 and MTR.24,25 For APY, the results of DFT calculations are available for similar compounds: 2-mercaptopyrimidine26,27 and 4,6dimethyl-2-mercaptopyrimidine.28 The assignments of SERS bands for ATR have been made by Wrzosek and Bukowska29 (this group has also carried out detailed DFT calculations for ATR and complexes of ATR with silver cations30). The vibrations of studied compounds are usually very delocalized, which means there are many “significant” (larger that 5%) PED contributions. For example, there are five “significant” contributions to the vibration of HTR giving the band at 1312 cm-1: three different C-N stretching vibrations (with PED values of 33, 13, and 9%) and two in-plane N-C-H bending vibrations (with PED values of 18 and 7%).23 To the vibration responsible for the HTR band at 1458 cm-1, there are even six “significant” contributions with PED values of 18, 17, 16, 8, 8, and 7%.23 Since knowledge of the detailed assignment of all bands visible in the spectra presented in Figure 2 is not required for the further discussion, and since these vibrations are usually very delocalized, the PED values for the vibrations of studied compounds are not reproduced in this contribution. Only the assignments for bands revealing very large changes during carried out experiments is given, and for other details, the reader is referred to the original contributions.23-29 As can be seen in Figure 2, for monolayers formed from ATR, one can observe a significant dependence of the recorded spectra on the pH of the ambient solution. The spectrum measured at silver substrate immersed in alkali ATR solution is dominated by the band at 1355 cm-1, whereas in measurements carried

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Figure 3. In situ SERS spectra measured at the silver surface immersed in (a) 10-6 M and (b) 10-3 M ATR aqueous solution. Spectra were scaled and vertically shifted for clarity.

out in acidic or neutral ATR solutions this spectral region is dominated by the band at 1324 cm-1. The SERS spectrum dominated by the band at 1355/1360 cm-1 can also be recorded in experiments carried out in neutral solution, provided the ATR surface coverage is low. Such a situation occurs, for example, when the concentration of ATR in the solution is very low and its adsorption leads to the submonolayer coverage (for an example, see Figure 3) or after partial desorption of ATR in water (for an example, see Figure 4) or a respective buffer solution. ATR molecules having the orientation of the triazole ring closer to the perpendicular may be undoubtedly more densely packed on the metal surface than ATR molecules which are (practically) laying flat on the metal surface. Therefore, the only explanation for the results of the above described experiments is that, when the surface concentration of ATR decreases, the triazole ring of ATR adopts a more parallel orientation versus the metal surface. Suggested reorientation of ATR molecules is also supported by more detailed analysis, in which the character of strongly enhanced SERS bands is taken into account. Recently, Wrzosek et al. carried out DFT calculations for ATR anion interacting with the silver cations.30 They found that the vibration responsible for the 1355/1360 cm-1 band (very strong in experiments with submonolayer coverage and/or carried out in alkali solutions) is mainly (PET ) 71%) due to the in-plane bending involving the Ag-N bond and neighboring N-C and N-H bonds (nitrogen is from the deprotonated amino group which directly interacts with the silver surface).30 Such bidentate bonding must force the ATR molecule to adopt a flat orientation versus the metal surface. According to SERS surface selection rules, the normal modes with polarizability derivative components perpendicular to the metal surface should dominate the SERS spectra.31,32 This means that for triazole ring oriented perpendicular to the metal surface more effectively enhanced should be bands due to the stretching and in-plane bending

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Figure 4. Temporal evolution of SERS spectra during desorption of (a1, a2) ATR and (b1, b2) APY molecules. (a1, b1) SERS spectra of ATR and APY monolayers in contact with a 1 mM solution of the respective thiol, respectively. (a2, b2) SERS spectra of ATR and APY monolayers after 10 min of soaking in pure water, respectively. Spectra were shifted and normalized according to the intensity of the stronger band.

vibrations, whereas for triazole ring oriented parallel to the metal surface out-of-plane (and also vibrations involving just formed bonds with the metal surface) should be more effectively enhanced. As can be seen in Figure 2, the SERS spectra of ATR adsorbed from acidic solutions are dominated by the bands at 480, 1324, 1436, and 1486 cm-1. All of these bands are due to the stretching and in-plane bending vibrations,30 and their high relative intensity supports the hypothesis about the perpendicular (or close to perpendicular) orientation of ATR triazole ring versus the metal surface in such experimental conditions. Since SERS spectra obtained in very diluted neutral ATR solutions are very similar to spectra measured in more concentrated alkali ATR solutions, one can also conclude that ATR molecules laying parallel on the metal surface are probably deprotonated even in neutral solution. Similar results and conclusions have been recently published by Wrzosek and Bukowska.29 For APY adsorbed on the silver surface, the most pronounced pH-induced change of the SERS spectrum is a very strong band at 1372 cm-1 emerging at high pH (see Figure 2). Analogously to ATR, the APY SERS spectrum dominated by the band at 1372 cm-1 can also be recorded in experiments carried out in neutral solution, provided the APY surface coverage is low. Figure 4 shows an example of how the relative intensity of the 1372 cm-1 band significantly increases due to APY partial desorption (similar changes were observed in experiments carried out in a buffer solution). As for ATR, APY molecules having the orientation of the triazole ring closer to the perpendicular may be more densely packed on the metal surface than APY molecules in the more flat orientation versus the metal

Kudelski surface. Therefore, observed spectral changes induced by the decrease of the APY surface concentration suggest that the APY molecules adopt a more flat orientation versus the metal surface. However, because for APY the spectral change is significantly smaller than for ATR (see Figures 2, 3, and 4), one may assume that the change of the orientation of the triazole ring of APY is significantly smaller than that for ATR. Since the spectral variations induced by the changes of pH and the thiol concentration in the ambient solution are analogous for APY and ATR, one may assume that both changes have a similar mechanism: acidic dissociation of the amino group at high pH and bidentate bonding of ATR or APY molecules to the metal surface, which force these molecules to adopt a flat orientation versus the metal surface. Probably very strong bands at ca. 1355-1372 cm-1, emerging in ATR and APY SERS spectra at high pH in both cases, are due to very similar vibration (in-plane bending involving the Ag-N bond and neighboring N-C and N-H bonds). As can be seen in Figure 2, there are also some differences between APY spectra measured at pH 2.0 and 3.4; for example, in the spectrum measured at pH 2, one can notice a well visible band at 691 cm-1, which is hardly seen in the spectrum measured at pH 3.4. One can suppose that these spectral variations are probably induced by the protonation of the amino groups of APY. However, since the changes observed at low pH are not so well reproducible as those observed at high pH, and this kind of change is not pertinent to the discussion on rearrangements of mixed monolayers, this problem is not discussed in detail here in this contribution. For HTR adsorbed on the silver surface, the most pronounced pH-induced change of the SERS spectrum is observed in the doublet 1286/1312 cm-1 (see Figure 2). Chemisorbed HTR can act as a proton donor (an acid) or as a proton acceptor (a base). For HTR in the aqueous solution, the reaction constants of acid and base dissociation have not been found. However, for unsubstituted 1,2,4-triazole (AH), the pKa of the reaction AH / A- + H+ (characterizing acidity) is 10.04, whereas the pKa of the reaction AH2+ / AH + H+ (characterizing basicity) is 2.45.33 It means that, for 1,2,4-triazole in the aqueous solution, the concentration of the neutral form is equal to the concentration of the anion at pH 10.04, whereas the concentration of the neutral form is equal to the concentration of the cation at pH 2.45. HTR contains also the SH group, which however is deprotonated when forming S-Ag binding, so the possible dissociations of chemisorbed HTR should be analogous to those of AH. As can be seen in Figure 2, in experiments carried out in alkali solution, the relative intensity of the band at 1286 cm-1 is roughly equal to the relative intensity of the band at 1312 cm-1 obtained in experiments carried out in acidic solution. Therefore, one can assume that the SERS cross sections of the 1286 and 1312 cm-1 bands are roughly the same and when both bands have the same intensity, the surface concentration of dissociated and nondissociated HTR molecules should be the same. Hence, the value of pH at which this happens may be interpreted as the pKa value of adsorbed HTR molecules. As can be seen from Figure 2, for HTR, the relative intensities of both bands are equal in experiments carried out for pH’s between 7 and 8. This is close to the pH value at which the ratio of the acidic dissociation of the triazole moiety in the aqueous solution should change the most (pH ≈ 10); hence, one can suppose that the acidic dissociation of HTR is responsible for the spectral change presented in Figure 2, and that the HTR spectrum dominated by the band at 1286 cm-1 is characteristic for HTR chemisorbed in the anionic form, whereas the spectrum dominated by the band at 1312 cm-1 (containing, however, also the

Monolayers from Aromatic Mercapto-Derivative Bases 1286 cm-1 component) is characteristic for not dissociated chemisorbed HTR. This assumption is strongly supported by the measurements of normal Raman spectrum of HTR dissolved in pure water and in a NaOH aqueous solution.23 Pergolese and Bigotto found that, like in the above described SERS measurements, the Raman spectrum of the HTR solution in pure water is dominated by the band at 1304 cm-1 and the band at 1274 cm-1 is weak, whereas in the spectrum measured for HTR dissolved in the NaOH solution the band at 1278 cm-1 is significantly more intensive than the band at 1336 cm-1.23 For dense monolayers, the negative charge created during dissociation of a part of HTR makes its further dissociation (creation of further anions) more difficult, and hence, the ratio of dissociation changes between two given values (e.g., from 0.1 to 0.9) in the wider pH range for adsorbed molecules than for molecules in the solution (therefore, significant SERS spectral changes are observed in a wider pH range than for Raman bulk experiments in the solution). As can be seen in Figure 2, except for the bands at 1286 and 1312 cm-1, the other pH-induced spectral changes for HTR are significantly smaller than in the case of ATR and APY monolayers. It supports the hypothesis that for ATR and APY the acidic dissociation of the amino group may be a key factor causing adoption by these molecules of a flat orientation versus the metal surface at high pH. Moreover, it also suggests that the possible dissociation-induced changes of the HTR orientation versus the metal surface are relatively small. For MTR monolayers, the measured SERS spectra practically do not depend on the pH of the ambient solution (see Figure 2). The lack of the pH-induced reorientation of the MTR monolayer is probably caused by the lack of the dissociation of adsorbed MTR molecules, for which the acidic dissociation even of the triazole ring is impossible due to the substitution of the acidic proton from the triazole ring by the methyl group (see Scheme 1). Practical independence of the SERS spectra of adsorbed MTR on the pH of the surrounding solution also supports the hypothesis that the pH-induced spectral changes observed for the ATR, APY, and HTR monolayers are caused by the pH-induced dissociation of these molecules. In the SERS technique, the Raman scattering cross section of some adsorbed molecules is significantly (even more than 7 orders of magnitude) larger than the Raman scattering cross section of the same molecules in the solution.34-37 This effect is mainly a result of the enhancement of the electric field at the metal surface.34-37 Therefore, to obtain high efficiency of Raman scattering for adsorbed molecules, the electromagnetic nanoresonators must be formed on the metal surface (in other words, the metal substrate must be nanostructured). Figure 5 shows the scanning electron microscope picture of the silver surface after electrochemical roughening used in this study. As has been shown in many previous contributions, the SERS enhancement factors at various places of nanostructured silver surfaces may differ by many orders of magnitude (enhancement factors are especially large for molecules located in the slits between silver nanoclusters).34,38-40 However, since in standard SERS experiments the Raman signal is collected from largesrelative to the typical sizes of metal nanoclusterssareas, relatively good reproducibility of intensities of measured SERS spectra can be obtained when the spectra are measured on silver substrates electrochemically nanostructured using the same roughening procedure, in the same electrochemical cell, and immersed in relatively concentrated (to obtain a surface covering factor close to 1, at which all adsorption sites on the metal surface are occupied) adsorbate solution.20

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Figure 5. Scanning electron microscopic (SEM) images of roughened silver substrates.

For a given experimental condition (laser power, focus area, the spherical angle from which the signal is collected, etc.), the intensity of the measured SERS band for densely packed homogeneous monolayers is proportional to the product of a number of scatterers and SERS cross section at the wavenumber of a given strong characteristic Raman band, σ. Since some molecules adsorb preferentially on highly SERS active areas,41 this statement often is not pertinent for submonolayers and for inhomogeneous monolayers. In a previous contribution, we showed that, during adsorption of APY, HTR, MTR, and ATR on silver surfaces from their 1 mM aqueous solutions, dense monolayers are formed.20 Also, MES forms dense monolayers when adsorbed on silver from 1 mM aqueous solution.42 For each one-component monolayer, a series (at least 30) of SERS spectra were measured. In more than 80% of experiments, the measured intensities of the strongest SERS bands were in the range of (30% from the average value. The ratio of relative intensities (taken as a median value in the series) of the characteristic SERS band for MES at 792 cm-1 and characteristic SERS bands for MTR (at 1351 cm-1), APY (at 918 cm-1), HTR (at 1312 cm-1), or ATR (at 1486 cm-1) was determined as equal to 1:1.12, 1:0.94, 1:0.7, and 1:0.56, respectively. Gui et al. showed that in dense monolayers surface concentrations of MES and simple model aromatic thiols (thiophenol and benzyl mercaptan) do not differ by more than a factor of 2.42 It means that the average SERS cross sections at the wavenumbers of the above mentioned strong characteristic Raman band should not differ significantly from the determined ratios of SERS intensities. Since the bonding of all compounds under study is realized in a similar way, probably neither of the compounds

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Figure 6. In situ SERS spectra measured from roughened silver substrate immersed in MES+HTR, MES+ATR, MES+MTR, or MES+APY solution. The concentrations of both MES and the aromatic compound were 1 mM. The solution was buffered with phosphorus buffers to (a) pH 2.0, (b) pH 3.4, (c) pH 5.3, (d) pH 6.7, (e) pH 8.1, (f) pH 10.4, (g) pH 11.5, and (h) pH 12.5. The total concentration of phosphate species in the solution was 0.1 M. Spectra were scaled and vertically shifted for clarity.

in the studied mixed monolayers is adsorbed preferentially at especially SERS-active sites (“hot spots”), and hence, estimated ratios of SERS cross sections may be used for rough estimation of the composition of studied mixed monolayers. 3.2. Two-Component Monolayers. Figure 6 shows SERS spectra measured on the silver substrate immersed in an aqueous solution containing both MES and the aromatic mercaptoderived base. The concentrations of both organic compounds in the ambient solution were 1 mM. The solution was buffered at various pH’s with phosphorus buffers. The total concentration of phosphate species in the solution was 0.1 M. As can be seen in Figure 6, in all measured spectra, the relative intensities of

the strongest SERS band characteristic for MES (at 792 cm-1) and the strong SERS band characteristic for the aromatic base (1351, 918, 1286/1312, and 1486 cm-1 for MTR, APY, HTR, and ATR, respectively) do not usually differ by more than a factor of 3. Taking into account that for these compounds SERS cross sections at the above mentioned characteristic wavenumbers are not significantly different (see the previous chapter), one may deduce that for all studied mixed monolayers both MES and the aromatic base are present on the silver surface in a significant amount (>10%). The spectra presented in Figure 6 illustrate both irreproducibility of a single experiment and the (possible) dependence of measured spectra on the pH of the

Monolayers from Aromatic Mercapto-Derivative Bases ambient solution. Only in the case of about 80% measurements, the measured ratio of the relative intensities of SERS bands due to MES and HTR/ATR/MTR/APY does not change by more than a factor of 1.6 from the average value. Taking this into account, one may conclude that the composition of the mixed monolayers formed from a 1:1 mixture of MES and HTR/ATR/ MTR/APY does not depend significantly on the pH of the surrounding solution. Many mixed monolayers are inhomogeneous; in other words, they contain one-compound domains. When the mixed monolayer is composed of large one-compound domains, its Raman spectrum must be practically a superposition of the spectra of the monolayers formed from the individual components. As can be seen from the comparison of spectra presented in Figures 1, 2, and 6, for MES+HTR, MES+ATR, and MES+APY monolayers, the spectra measured at high pH are significantly different than the superposition of the spectra of the respective onecomponent monolayers measured at the same pH. For example, in the series of SERS spectra of the HTR monolayer, the component at 1286 cm-1, which is characteristic for HTR chemisorbed in the anionic form, begins to dominate the 1286/ 1312 cm-1 doublet at significantly lower pH than in the series of spectra measured for the mixed MES+HTR monolayer (compare Figures 2 and 6). The sulfonic group in the MES molecule is undoubtedly dissociatedsnegatively chargedsin the alkali solution (since sulfonic acids are very strong, one may suppose that the sulfonic group is dissociatedsnegatively chargedsin the whole pH range studied). The negative charge of dissociated MES should hinder creation of the negative charge in the coadsorbed neighboring molecules, which explains hindering of the acidic dissociation of HTR in the mixed monolayer. Analogous hindering of the acidic dissociation of aromatic bases is also observed in the mixed monolayers containing ATR and APY (compare the respective spectra measured at pH 11.5 or at pH 12.5 in Figures 2 and 6). A significant influence of MES on the dissociation ratio of the coadsorbed HTR, ATR, or APY suggests that in these mixed monolayers the nearest surrounding of the molecules of the aromatic base is significantly different than that in the onecomponent monolayers, which means that molecules of HTR, ATR, or APY do not form large one-component islands in the mixed monolayers with MES. For the MES+MTR monolayers (similarly to the MTR monolayersssee the previous paragraph), no significant pHinduced change of the SERS spectra is observed (compare the spectra presented in Figures 1, 2, and 6) and at every pH the spectrum of the MES+MTR monolayer is practically a superposition of the spectra of the one-component MES and MTR monolayers. Such a result does not give us any suggestion about the structure of the formed monolayer, since structural information may only be deduced when the spectrum of the mixed monolayer is not a superposition of the spectra of the monolayers formed from the individual components. There are many parameters which are important in practical applications of linkage monolayers. The most important ones are the stability of the monolayer structure during soaking in water and solutions containing various physiological salts and biomolecules, the monolayer integrity, and the actual structure of the monolayer in the experimental conditions. It is also important to determine how the structure of a monolayer (and for mixed monolayers its composition) changes during immersion in various solutions. From measurements carried out for the mixed monolayers studied in this contribution soaked in pure water and in neutral (pH ≈ 7) phosphorus buffer, one may

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Figure 7. Temporal evolution of SERS spectra of MES+ATR monolayer. (a) Spectrum measured for the monolayer immersed in a 1 mM MES + 1 mM ATR solution. (b) SERS spectrum of the MES+ATR monolayer after 30 min of soaking in pure water. (c) SERS spectrum of the MES+ATR monolayer after 30 min of soaking in a 0.8 mg cm-3 laccase solution. (d) SERS spectrum of the MES+ATR monolayer after 5 min of soaking in a 0.8 mg cm-3 BSA solutionsan example of the exceptionally large spectral change.

conclude that, after the studied soaking times (up to 30 min), the ratios of the relative intensities of the strongest band characteristic for MES (at 792 cm-1) and the strongest band characteristic for the aromatic bases change in the SERS spectra of mixed monolayers less than during changing of the pH of the surrounding solution (see, for example, spectral changes for the MES+ATR system presented in Figure 7). It means that neither MES nor the aromatic base is desorbed preferentially and that the chemical composition of studied monolayers does not change significantly during soaking in water. However, when the surface concentration of molecules forming the monolayer decreases, the relative intensities and positions of some SERS bands due to the adsorbed aromatic bases undoubtedly change (see Figure 7, spectra a and b). The rearrangement rate of the mixed monolayer during soaking in water seems to be smaller than that of the monolayer formed from only aromatic bases; however, due to some irreproducibility of such kinetic experiments (local irreproducibility of the substrate surface is illustrated in Figure 5), this observation should be treated only as a suggestion (the possible kinetic differences are not significantly larger than the irreproducibility of experiments). The integrity and the structure of linkage monolayers undoubtedly influence the structure and the catalytic properties of layers of attached biomolecules.43,44 Therefore, investigation of the degree to which the linkage monolayer prevents direct adsorption of various biomolecules from the ambient solution on the metal surface and the determination of the actual changes in the structure of linkage monolayers caused by the interaction with various biomolecules are often of great interest for practical reasons. In this contribution, the changes in the structure of the

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SCHEME 2: Schematic Illustration of the Squeezing Effect

studied mixed monolayers induced by model peptides (BSA and laccase) present in the ambient solution were analyzed. The major bands in the SERS spectrum of BSA directly adsorbed on the silver surface are at 1149, 1273, 1350, 1385, 1433, 1454, and 1505 cm-1.45,46 Laccase is photocatalytically decomposed on the SERS-active silver surface to the hydrogenated amorphous carbon, and the SERS spectrum recorded from the silver substrate immersed in the laccase solution is dominated by two strong broad bands characteristic for the carbon clusters at ca. 1355-1380 and 1580-1585 cm-1.45,47 The present studies show that, in the SERS spectra measured after 30 min of soaking of all studied mixed monolayers in the 0.8 mg cm-3 BSA or laccase solutions (the experiments were carried out for peptide solutions in water and in the buffer with pH 6.7), there is no significant contribution from peptide molecules. Spectrum c in Figure 7 shows an example result for the MES+ATR monolayer immersed for 30 min in a laccase solution. It means that during this time period BSA and laccase do not replace thiol molecules on a significant part of the silver surface. In some cases (the discussion of observed irreproducibility is given below), immersion in the peptide solution causes very large spectral changes; however, even then bands from coadsorbed peptides are not observed. An example of such large peptide-induced spectral changes for the ATR+MES monolayer immersed in the BSA solution is shown in Figure 7 (see spectrum d). Observed peptide-induced spectral changes prove that peptides are either linked to the modified metal substrate via the thiol linkage monolayer or can directly interact with the metal surface only at some small parts of the silver surface (small enough to not produce a noticeable contribution to the measured SERS spectra). Despite the presence of peptide molecules in the vicinity of the silver surface, they do not observably contribute to the measured SERS spectrum. It is due to the sensitivity of SERS enhancement to the surface proximity: in the SERS effect, the efficiency of Raman scattering from molecules directly interacting with the metal surface is increased much more significantly (in typical SERS experiments, usually more than 2-3 orders of magnitude) than for molecules in the next layer. Therefore, the measured SERS spectrum is dominated by the contribution from molecules directly interacting with the metal surface.3,34-37 As mentioned above, in some cases, immersion of the ATR+MES mixed monolayers in the peptide solution induces large spectral changes (see Figure 7). Observed spectral changes suggest reorientation of a significant part of ATR molecules (which significantly contribute to the measured SERS spectrum) from the standing up to the flat orientation versus the metal surface, which is connected with the acidic dissociation of ATR molecules. The observed reorientation may be explained by the “squeezing” (see Scheme 2) of the thiol monolayer by the coadsorbed (directly interacting with the metal surface or linked via the linkage monolayer) peptide molecules or by the change in the local dissociation ratio of adsorbed ATR molecules (and

Kudelski hence their reorientation) induced by the charged parts of the peptide located very close to the metal surface. The “squeezing” effect appears, since, when the peptide molecules are bonded to the metal surface, their shape does not always fit to the local topology of the metal substrate and the most “jutting” parts of peptide molecules locally “squeeze” the thiol monolayer. It is very interesting why very large spectral changes are observed only in some cases. Probably such irreproducibility has the same origin as strong spectral fluctuations reported during measurements of SERS spectra of carbon clusters deposited on the electrochemically roughened silver substrates: domination of the measured spectrum by the contribution from species adsorbed on so-called SERS “hot spots”, which are only a very small part of the illuminated surface.37,47,48 As reported previously, this effect allows for measurements of SERS spectra from only a few clusters, despite large surface densities of analyzed species and in spite of using a standard optical Raman microscope, which collects a Raman signal from about 1 µm2 of the surface.37,47,48 Very large SERS enhancement factors are observed, especially for molecules located in the slits between silver clusters. Probably, accessibility of such slits to the large peptide molecules strongly depends on their actual morphology and hence is noticeably different at various places of the surface and strongly depends even on small variations in the roughing procedure (for typical local morphology of the used silver substrates, see Figure 5). 4. Conclusion In this contribution, the composition of monolayers formed from MES and some aromatic mercapto-derived basis is analyzed. The presented results show that MES is a very promising candidate for making mixed monolayers containing mercapto-derivative aromatic compounds, since such structures are formed in the broad pH range from 1 mM/1 mM mixtures of MES and all studied aromatic mercapto-derivative bases. At some conditions, the SERS spectra of MES+ATR, APY, or HTR mixed monolayers are clearly different than the superposition of the spectra of the individual compounds, which suggests that a large percentage of aromatic base molecules interact specifically with MES. Moreover, this also suggests that the structures of formed mixed monolayers are relatively homogeneous. For example, in one-component ATR, APY, or HTR monolayers immersed in a buffer with high pH, a significant amount of molecules is in the anionic form. For mixed (with MES) monolayers immersed in the same buffer solution, a larger part of ATR, APY, or HTR molecules than for the respective one-component monolayer is adsorbed in the neutral form. Measurements of the temporal evolution of the SERS spectra during soaking of mixed MES+ATR and MES+APY monolayers in water revealed that during partial desorption of molecules forming the monolayer the average orientation of the aromatic mercapto-derivative base molecules becomes more parallel to the metal surface, and the ratio of the acidic dissociation of the aromatic amino compound increases. Reorientation of the ATR molecules from the vertical to the horizontal orientation is sometimes very fast when the peptide molecules are coadsorbed on the studied mixed MES+ATR monolayers. This effect may be caused by the “squeezing” of the monolayer by the peptide fragments close to the bonding places or by the change in the local dissociation ratio of adsorbed aromatic thiol molecules (and hence their reorientation) induced by the charged parts of the peptide located very close to the metal surface. Studied mixed monolayers practically prevent direct adsorption of peptides on the significant part of the metal

Monolayers from Aromatic Mercapto-Derivative Bases surface for at least 30 min. The ratio of the surface concentration of MES and the aromatic base component does not change significantly even after 30 min of soaking, which suggests that none of the compounds of the mixed monolayer are preferentially desorbed. Acknowledgment. The author is grateful to Prof. Maria Janik-Czachor and Dr. Marcin Pisarek from the Institute of Physical Chemistry, PAS, for the SEM images of roughened silver surfaces. This work was financed by the Ministry of Science and Higher Education (Poland) from funds for scientific research in years 2008-2010 as Project No. N N204 010435. Supporting Information Available: SERS blank spectra measured at a roughened silver substrate immersed in various phosphorus buffers. This material is available free of charge via the Internet at http://pubs.acs.org. References and Notes (1) Bernad, S.; Leygue, N.; Korri-Youssoufi, H.; Lecomte, S. Eur. Biophys. J. 2007, 36, 1039. (2) Grochol, J.; Dronov, R.; Lisdat, F.; Hildebrandt, P.; Murgida, D. H. Langmuir 2007, 23, 11289. (3) Kudelski, A. Vib. Spectrosc. 2005, 39, 200. (4) Gooding, J. J.; Mearns, F.; Yang, W.; Liu, J. Electroanalysis 2003, 15, 81. (5) Gooding, J. J.; Pugliano, L.; Hibbert, D. B.; Erokhin, P. Electrochem. Commun. 2000, 2, 217. (6) Ostuni, E.; Yan, L.; Whitesides, G. M. Colloids Surf., B 1999, 15, 3. (7) Boozer, C.; Yu, Q. M.; Chen, S. F.; Lee, C. Y.; Homola, J.; Yee, S. S.; Jiang, S. Y. Sens. Actuators, B 2003, 90, 22. (8) Li, L. Y.; Chen, S. F.; Jiang, S. Y. Langmuir 2003, 19, 2974. (9) Patel, N.; Davies, M. C.; Harshorne, M.; Heaton, R. J.; Roberts, C. J.; Tendler, S. J. B.; Williams, P. M. Langmuir 1997, 13, 6485. (10) Ostuni, E.; Grzybowski, B. A.; Mrksich, M.; Roberts, C. S.; Whitesides, G. M. Langmuir 2003, 19, 1861. (11) Whitesides, G. M.; Laibinis, P. E. Langmuir 1990, 6, 87. (12) Evans, S. D.; Ulman, A. Chem. Phys. Lett. 1990, 170, 462. (13) Nuzzo, R. G.; Dubois, L. H.; Allara, D. L. J. Am. Chem. Soc. 1990, 112, 558. (14) Ulman, A. Chem. ReV. 1996, 96, 1533.

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