Aromatic Amino Acid Monolayers Sandwiched between Gold and Silver

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Aromatic Amino Acid Monolayers Sandwiched between Gold and Silver: A Combined Tip-Enhanced Raman and Theoretical Approach† Tanja Deckert-Gaudig,‡ Eva Rauls,§ and Volker Deckert*,‡,⊥ Institute for Photonic Technology (IPHT), Albert-Einstein-Strasse 9, D-07745 Jena, Germany, Institut fu¨r Theoretische Physik, UniVersita¨t Paderborn, Pohlweg 55, D-33098 Paderborn, Germany, and Institut fu¨r Physikalische Chemie, UniVersita¨t Jena, Lessingstrasse 10, D-07743 Jena, Germany ReceiVed: October 13, 2009; ReVised Manuscript ReceiVed: NoVember 25, 2009

Tip-enhanced Raman scattering (TERS) measurements and theoretical calculations of the aromatic amino acids phenylalanine, tyrosine, and tryptophan sandwiched between gold and silver layers are presented and discussed. Atomically flat micrometer-sized gold nanoplates were chosen as substrates and covered with a monolayer of the respective amino acid. On the basis of spectral data, the carboxylate and amino groups were determined to be the preferential moieties attached to the metals in the cavity. The experimental observation of benzene rings oriented parallel to the gold surface and the influence of the silver tip are discussed and compared with theoretical calculations. Finally, it is shown that TERS allows a clear distinction between phenylalanine and tryptophan moieties from a mixture on the substrates; hence, demonstrating the selectivity of the method on the nanometer scale without further need of any labeling techniques. 1. Introduction When a molecule is adsorbed onto a rough metal surface such as gold or silver, the Raman signal intensity can be enhanced up to several orders of magnitude. This phenomenon of surfaceenhanced Raman scattering (SERS) can be utilized to characterize a variety of inorganic, organic, and biological samples with limits of detection even down to single molecules.1-6 Disadvantages of this technique are the lack of high lateral resolution and the varying enhancement across the metal surface. Due to the inhomogeneous metal surface (each silver nanoparticle contains numerous nonequivalent chemisorption sites), differing results on measurements on, for instance, the very same protein are reported.7 Those restricting attributes can be overcome by turning the metal-adsorbate upside down, as realized in tip-enhanced Raman scattering (TERS).8-14 In TERS, the SERS-active particle is attached to the tip of an atomic force microscope (AFM) that is coupled to a Raman spectrometer. The reduction to only one enhancing unit guarantees a constant signal enhancement for the whole experiment. While scanning the tip across the sample surface, information on the topography as well as Raman spectra can be recorded on every distinct point on the sample, simultaneously. Here, a lateral resolution down to 20 nm and better, depending only on the grain size of the deposited particle on the tip, can be reached. Such a high lateral resolution is required if compartments of biological samples such as cells, viruses, proteins, and DNA/RNA strands are the target of research.15-19 For sequencing macromolecular chains such as DNA or peptide strands, it is crucial that the strands are oriented homogenously on the substrate to make sure that equal units deliver (nearly) equal TERS spectra. Recently, it was shown †

Part of the “Martin Moskovits Festschrift”. * Corresponding author. Phone: +49-3641-206 113. Fax: +49-3641206 139. E-mail: [email protected]. ‡ IPHT. § Universita¨t Paderborn. ⊥ Universita¨t Jena.

that similar alignment of various single amino acids could be achieved if atomically flat gold or silver nanoplates were the substrate of choice.20,21 As a result, TERS spectra of the respective amino acid were lacking the spectral variations typical for small numbers of molecules (e.g., see ref 19) and clearly revealed the interactions of the carboxyl and amino moieties with the metal surfaces. Even a small peptide has been immobilized in such a way, and the structural composition could be determined without decomposition.22 Until now, much research has been done on SERS on aromatic amino acids; see, for example, refs 23-29 for the adsorbate-surface interactions. Until now, however, no information has been available on the behavior of phenylalanine (Phe), tyrosine (Tyr) and tryptophan (Trp) monolayers sandwiched in the gap of gold and silver layers. Such information, however, would be crucial for a discussion of TER spectra of single-peptide or protein strands. In this paper, we present the results of TERS measurements on such systems and correlate them with theoretical calculations delivering insight into the orientation of those aromatic molecules within such a gold-silver gap. 2. Experimental Methods The gold nanoplates were synthesized and deposited on glass slides as previously described.20 Monolayers of the amino acids were immobilized by immersing the substrates in a 4 × 10-4 molar solution (water/ethanol 1:1 for Phe and Trp; water/HCl for Tyr) for several hours. After washing and drying, the samples were used for the specific experiments without further treatment. TERS measurements were performed as previously described and can be reviewed together with the setup in the literature.20,30 In short, a 530 nm laser light (laser power after the last lens: Phe, P ) 630 µW; Trp, P ) 950 µW; Tyr, P ) 820 µW; Phe + Trp, P ) 630 µW) was focused through a 60× oil immersion objective (N.A. ) 1.45), and the sample, on the silver-covered (20 nm) AFM silicon tip. To rule out tip contamination, reference measurements next to the gold nanoplates were carried out. Reference spectra are shown for the experiments of Tyr

10.1021/jp9098045  2010 American Chemical Society Published on Web 12/22/2009

TERS of Sandwiched Amino Acid Monolayers

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Figure 2. TERS spectra of Phe on a smooth, gold nanoplate, λ ) 530 nm, P ) 630 µW, tacq ) 10 s. Orange highlighted bands are discussed in the text; for the entire assignment, see Table 1.

Figure 1. (a) SEM image of a typical distribution of gold nanoplates on a glass slide and (b) AFM topography of gold nanoparticles used in the experiments.

and the Phe + Trp mixture. For neat Phe and Trp, the TERS spectra showed a somewhat higher background, and baseline correction using the reference spectrum was used. In the case of Tyr and the Phe/Trp mixture, no further data treatment (smoothing, etc.) was applied. The enhancement factors were estimated by means of the signal-to-noise ratio assuming a tip diameter of 20 nm and laser diameter of 1 µm. The duty cycle of the oscillating tip was disregarded. Since the parameters are very conservative, the obtained enhancement factors can be considered as values that are achieved at least. 3. Theoretical Modeling To understand the adsorption mechanism of the molecules on the gold surface in detail and comprehend the influence of the tip on an atomic scale, we performed density functional theory (DFT) calculations using the Vienna Ab Initio Simulation Package31 of DFT and the PW91 functional32 to model the electron exchange and correlation within the generalized gradient approximation. The electron-ion interaction is described by the projector-augmented wave method.33 The Au(111) surface was modeled with four atomic layers in a slab model with (5 × 6) surface unit cells, ensuring the molecule-molecule interactions due to periodic images to be negligible. In the case of molecules weakly bonded to each other or to the surface, dispersion interaction (not accounted for in DFT) may contribute a sizable

percentage of the total interaction energy.34 To assess the influence of these van der Waals interactions on the adsorption energetics, we have extended our description by a semiempirical approach based on the London dispersion formula to include the dispersion interaction.35 This approach has proven to be successful in the description of similar systems.36-39 Surface and molecules were completely relaxed, in addition to the lowest Au layer. For the calculation of the sandwiched molecular structures, the silver tip has been added to these relaxed structures. Due to its large size compared to the molecules, the silver tip has been modeled as a flat slab, that is, two layers of Ag at a defined distance from the substrate. Since there is only a negligible mismatch in the lattice constants of Au and Ag, modeling the two metals with the same lattice constant (i.e. that of Au) did not induce any noticeable stress in the Ag layers. Upon further relaxation of the structures, the silver atoms were kept fixed, and full relaxation was allowed for the molecule and the topmost three gold layers. 4. Results and Discussion In Figure 1a, a scanning electron microscopy (SEM) image shows a typical spreading of gold nanoplates on a glass cover slide. The triangular and hexagonal nanoparticles have a size distribution around 4 µm and serve as atomically flat substrates for the presented TERS experiments. The thickness of these nanoparticles was determined by AFM to be around 20-35 nm. An AFM image of such nanoplates (obtained during the phenylalanine experiment) is given in Figure 1b as representative for all gold nanoparticles employed in this work. Phenylalanine. Figure 2 provides background-corrected TERS spectra of phenylalanine adsorbed on a gold nanoplate.

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Deckert-Gaudig et al.

TABLE 1: Band Assignment for TERS, SERS and Raman Spectra of Phenylalanine TERS (Au plate, this work) 1625 1613, 1610 1593, 1564 1516 1498, 1480-1467 1444-1424 1391 1344 1327, 1318, 1260, 1250 1214, 1197 1165 1006, 1009 976 774 655 595

SERS (Ag colloid)25,28,57

Raman (solid)55,56

assignment -

1602 1586

1602 1586

1437 1411

1447 1353 1336 1308 1216, 1183 1157 1037 1004 951 852 832 747 622 605 527

1309, 1251 1212 1036 1001 930 830 722 620 545

The spectra were recorded on six adjacent positions with a distance of 60 nm and reveal the uniform immobilization of the amino acid on the gold surface. With the parameters given in the Experimental section, the enhancement factor was estimated to be at least 5 × 104. Surprisingly, the characteristic ring breathing mode at 1006 cm-1 is hardly visible. With a digression to the physical background of surface-enhanced Raman spectroscopy, this phenomenon should be elucidated. From SERS experiments, it is known that two mechanisms contribute to the signal enhancement:40 the electromagnetic part arises from surface plasmon resonances (excited by the incident laser light) on the metal surfaces, which is a very strong component of the electric field normal to the metal surface. For flat surfaces, these surface plasmons can usually not be excited. Very large enhancements can also be achieved in the gap between metal nanoparticles, as realized in single-molecule detection.41-43 The second (smaller) contributions are chargetransfer interactions between the metal and adsorbate. According to the SERS selection rules, in such a case, molecules possessing polarizability tensors in the direction of the surface normal should experience the highest change in electron distribution. Such an alteration may cause different enhancements for different vibrational modes, and one often observes intense usually Raman forbidden modes. If strong metal-adsorbate bonds are formed, this chemisorption can even provide new electronic transitions for electrons from metal to adsorbate and the reverse.44-46 The charge-transfer mechanism can be regarded as a resonance Raman effect in which the incoming photon produces the resonant transfer of an electron from the metal to the adsorbed molecule. Under these circumstances, two groups postulated the generation of radicals that are found to be a good explanation for spectral features in SERS spectra of aromatic molecules. See, for example, refs 27 and 47-49. The effect of both mechanisms on the vibrations of amino acids have been studied in detail for R-phenylglycine on Ag colloids, revealing that the charge-transfer mechanism plays an important role.50,51 The spectroscopic results led to the conclusion that the phenyl ring was oriented perpendicular to the metal surface, which is consistent with the SERS selection rules. Elongating this molecule with a CH2 group leads to phenylalanine, which lies nearly flat on the Ag surface, as can be concluded from its SERS spectra.25,26 For an understanding of

CdC str, COO str asym CdC str, NH3+ def asym CdC str, COO- str asym, NH3+ def sym NH3+ def sym CH2 sciss CH2 sciss, NH3+ str COO- str sym CN str, CH bend CH2 wagg CC (ring) breath, NH3+ rock CCN str asym CH (ring) def i p CC (ring) breath str sym C-COO- str CCN str, CC str CH2 rock COO- sciss ring def, COO-, wagg ring twist COO- wagg

these spectra, the authors draw comparisons with benzene, in which the most intense ring vibration (around 992 cm-1) was observed for flat benzene rings on the silver surface, too.45 Addition to density functional theory calculations of minimized energy structures confirmed the experimental observations. Similar results were also reported from benzoic acid derivatives earlier.52 To understand the present tip-enhanced Raman spectra, one must consider the direction of the electric fields (E-field) at the tip. In a back-reflection TERS setup, an electric field is generated on illumination of the tip in the laser focus. The important E-vector components are almost parallel to the main propagation of the incident beam and the z-axis of the tip. Molecules in the close proximity of the tip experience a constant electric field, resulting in a signal intensity increase of modes parallel to the tip. Modes that lie in the plane of the substrate can be enhanced only weakly, since the tangential E-field is comparatively small.9,53 With these considerations, the low intensity of the ring band in the TERS spectra of phenylalanine can be clarified assuming a parallel orientation of the molecules on the smooth gold surface. Here, the interactions of the ring happen via the π-orbitals of the ring, as established for several benzene derivatives.45,54 The signals for the carboxyl (1593, 774 cm-1) as well as the amino group (1564, 1516 cm-1) are enhanced and point toward a direct contact of these moieties with the metal in the cavity. Signals at 1613, 1593, and 1214 cm-1 can be assigned to CdC double bonds (partial overlap with ν(NH3+)) and document a coordination of the phenyl ring with the metals.28 In Table 1, a band assignment in comparison with common Raman55,56 and SERS25,28,57 measurements are given. For the assignment, data of infrared (IR) measurements58 were retrieved, too, because Raman forbidden bands can become active in the vicinity of a rough metallic surface because of a lowering of the symmetry.40 The remaining variation of signal intensities, shifts, and shapes in the TERS experiments can be explained with changes in the actual number of molecules underneath the tip and a slight change in their spatial arrangement, depending on the tip’s distance. Theoretical calculations with four layers of gold match very well with these experimental results and are depicted in Figure 3. The modeling revealed that the additional silver tip only



Figure 4. Influence of the Ag tip on the total energy of the three molecular adsorbed amino acids.

Figure 3. Model of the theoretical calculated orientation of Phe on gold (Au111) with a Au-tip distance of (a) 6.5 and (b) 11.5 Å.

slightly influences the orientation of the immobilized Phe on the gold surface. As can be seen in Figure 3, in the most favorable adsorption geometry of Phe, the phenyl ring is flat; that is, almost parallel to the Au surface in one direction and 18° tilted in the direction of the attached CH2 group. The adsorption energy amounts to 1.65 eV in this position (calculated without Ag tip). An adsorption geometry with the phenyl ring rotated by about 90° is also found to be stable, but has an adsorption energy of only 1.52 eV. Reorientation can, thus, be assumed to be a process that can be activated at low cost. Including the tip into the calculations reveals that there is nearly no influence up to a tip-surface distance of about 9 Å. Approaching the Ag surface, however, to as close as 6.5 Å (Figure 3a), the molecule is pressed even flatter

Figure 5. TERS spectra of Tyr on a smooth, gold nanoplate, λ ) 530 nm, P ) 820 µW, tacq ) 10 s. Lowermost spectrum shows the reference spectrum on glass. The green highlighted lines are discussed in the text; for the entire assignment, see Table 2.

to the substrate. The largest tilt angle between the phenyl ring and the surface is reduced to only 6°. The total energy of the system is reduced further, since especially a dispersive interaction between tip and molecule and for small distances also between tip and surface contributes nonnegligibly to it.

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Figure 6. Model of the theoretical calculated orientation of Tyr on gold (Au111) with a Au-tip distance of (a) 6.5 and (b) 11.5 Å.

Deckert-Gaudig et al. For different tip-surface distances, the change in energy induced by the silver tip is illustrated in Figure 4. For the smallest distance of 6.5 Å, the energy gain amounts to 2.15 eV. Lines corresponding to tyrosine and tryptophan are displayed, too, and are discussed in the appropriate chapters. Tyrosine. The studies were extended to tyrosine, which has an additional hydroxyl group (-OH) in the para position of the phenyl ring. In Figure 5, six TERS spectra of Tyr on adjacent positions, each with a distance of 100 nm on a single gold nanoplate, are presented. Because of the low solubility of Tyr in water, the solution was acidified with HCl; thus, the protonated species was investigated. The bottom spectrum refers to the reference spectrum next to the gold plate, proving the purity of the tip. The enhancement factor was estimated to be at least 3 × 105. The Tyr spectra look more homogeneous as compared to the previously discussed Phe spectra. This observation points toward a more stable coordination of the molecules on the gold surface and are left unperturbed by the approaching tip. This observation can be explained by considering the additional coordination center in terms of the hydroxyl group. In both Raman and SERS spectra, Tyr shows a Fermi resonance at 845 and 828 cm-1 instead of a single ring breathing mode, such as benzene or monosubstituted derivatives.55 In the present TERS spectra, this doublet signal can hardly be detected and is once more a strong indication for a horizontal coordination of the species on the flat, gold surface. The enhanced functional groups are, as expected, those of the carboxyl (around 1700 cm-1) and the amino group (1628, 1519 cm-1). Those observations are in good agreement with the recent literature.25,26,28 In Table 2, the experimental data of the TERS measurements are compared with Raman55,59 and SERS25,26,28 data. In single-molecule SERS experiments of Tyr on immobilized Ag particles, spectral fluctuations were assigned to a change of different metastable chemisorbed states with different spectral characteristics.27,60 On the basis of ab initio calculations and their SERS spectroscopical results, the authors conclude that not only tyrosine anions but also phenoxyl radicals are present. As outlined above, those radicals can only be generated if a charge-transfer process between sample and metal is involved. For the current TERS experiments, the presence of such tyrosyl radicals (νCdC (1577, 1535), νC-O (1513 cm-1))61 could be neither

TABLE 2: Band Assignment for TERS, SERS, and Raman Spectra of Tyrosine TERS (Au plate, this work) 1727, 1714, 1691 1628, 1612 1589, 1563 1535, 1519 1491, 1476 1458, 1441 1412 1379 1345 1315, 1299 1264 1231 1210, 1198 1159 1097 1058, 1043 856, 828 688

SERS (Ag colloid)25,26,28 1613 1577 1505

Raman (solid)55,59 1616 1604, 1598 1437

1410 1326 1304

1370 1329

1199 1176

1263 1251 1213, 1200 1179, 1153

931 847, 829 721 620

1046 989, 982, 971 845, 828 797, 714 641

assignment COOH str asym CdC str, NH3+ def asym CdC str, NH3+ sciss CO str asym, CdC str sym, NH3+ def sym CH2 sciss CC (ring) str, NH3+ str COOH/COO- str sym CH (chain) bend CN str, CH bend COH str, CH2 wagg COH str, CH2 twist OH (ring) def CH (ring) str sym, NH3+ rock CH (ring) bend, CCN str asym NH3+ rock CN str C-COO- str ring breath, Fermi doublet CdC twist, COOH/COO- def COO- wagg, ring def


Figure 7. TERS spectra (background-corrected) of Trp on a smooth gold nanoplate; λ ) 530 nm, P ) 950 µW, tacq ) 10 s. Blue highlighted bands are discussed in the text; for the entire assignment, see Table 3.

definitely proven nor excluded. The experimental conditions were too different from the SERS studies in ref 61 to enable a direct comparison of the values.

J. Phys. Chem. C, Vol. 114, No. 16, 2010 7417 From SERS studies of Tyr on Ag colloids, it is known that the theoretically calculated wavenumbers of three modes at 1378, 1505, and 1607 cm-1 are pH-dependent and blueshifted with increasing pH value.26 The authors assign that to chargetransfer interactions of different ionic states of Tyr on the Ag+covered silver surface. They state that coordination via nitrogen (NH3+), oxygen (from COO-), and π-electrons (from the phenyl ring) is most reasonable according to different quantum chemical models. On the other hand, the authors did not decide whether the ring was parallel or tilted to the Ag surface. As previously mentioned, we assume a flat coordinated tyrosine from our TERS spectra. To further strengthen this assumption, theoretical calculations were elaborated. In Figure 6, the calculated orientation of tyrosine in the gold-silver sandwich is depicted. One can see clearly that the results match nicely with the experiment. As revealed by the calculations, the carboxyl group reacts sensitively toward a vertical tip movement. The largest variations in shape and intensity in the spectra were those of the carboxyl modes around 1714 cm-1. Since the signal intensity was low, it cannot be decided whether those variations arise from the common tip-enhanced Raman spectroscopic fluctuations or from the theoretically predicted effect. Finally, the hydroxyl groups should be addressed, too. In contrast to common Raman spectroscopy, νCO (1535, 1519 cm-1) and νCOH (around 1300, 1231 cm-1) modes are active and enhanced, leading to the conclusion that a direct contact with the metal layers is present, stabilizing the system in the gap with respect to perturbations, in contrast to phenylalanine, which is immobilized only from one side. Similarly to Phe, the Tyr molecule prefers a flat adsorption geometry; compare Figure 6. The adsorption energy is 1.63 eV in this position. The similarity of the Tyr energy to the one of Phe can be understood, since the adsorption geometry consists of two parts: first, the covalent bond formed via the amine group to the Au atom, which is the same for both molecules; and second, the dispersive interaction between the phenyl ring and

TABLE 3: Band Assignment for TERS, SERS and Raman Spectra of Tryptophan TERS (Au plate, this work)

SERS (Ag colloid)24,28

Raman (solid)24

assignment

1637-1622 1596, 1581, 1575 1558, 1533, 1522, 1509, 1484

1621, 1616 1605, 1583 1556, 1549 1477, 1460 1442, 1423 1410, 1405 1362, 1348 1339 1306 1278 1253, 1232 1213

1622 1581 1561, 1492 1463 1455, 1429

COO- str asym CdC (indole ring) str, NH3+ sciss CdC (indole ring) str, NH3+ def CH2 sciss NH (indole ring) str, CH2 sciss COO- str sym CH bend, CH2 wagg CH (indole ring) str, CN str CH bend, CH2 wagg CH2 bend CH (indole ring) rock CH str (pyrrole ring), C-COO- str CH2 def, CH (benzene ring) sciss CH bend, NH wagg CH (pyrrole ring) sciss CH (chain) bend, NH3+ rock NH3+ def, CH (indole ring) str indole ring breath sym CH2 def, CN str CH (benzene ring) def C-COO- str., NH3+ rock, CH2 rock CH (indole ring) bend CH (benzene ring) bend NH3+ def, benzene ring bend CH2 rock, C-COO- str CdC (indole) breath, COO- def CH (indole ring) breath sym benzene ring wagg indole ring def, CN str indole ring str

1455, 1443, 1430, 1423 1404, 1392 1369, 1363, 1348 1332 1322, 1305 1275, 1269 1246, 1238, 1227 1217 1194, 1183, 1166, 1153 1123, 1117, 1104 1092 1068, 1061, 1051 1041 1007 971 936 876 863 839, 830

1143 1094 1054 1013, 1009 974 927 876 866 806

764 758 710 685

1364, 1344 1333 1320 1283 1254, 1238 1213 1164, 1154 1121 1078 1069 1010 990 965 930 875 866, 848 840 803 766 755 744 706

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the Au surface, which is only slightly varied by the OH group in the Tyr molecule. A rotated form has been found to be stable, too, but with an adsorption energy of 1.47 eV, which is slightly less favorable. Including the tip leads to a rotation of the carboxyl group to a more horizontal alignment, whereas that of the phenyl ring is negligible. When compared to Phe, a slightly larger energy gain due to the additional tip interaction is found for the smallest distances; see Figure 4. Tryptophan. To complete the series of aromatic amino acids, the orientation of tryptophan (Trp) in the gold-silver gap was investigated. TERS spectra of a monoalayer immobilized on a single gold nanoplate were recorded on points with a distance of 380 nm and are given in Figure 7 (background-corrected). The enhancement factor was estimated to be at least 7 × 104. The signal of the ring breathing mode for the indole ring was expected around 1013 cm-1.24 Again, the band could not be detected with the expected intensity in our experiment, most likely for the same reasons as discussed for Phe and Tyr previously: a parallel orientation of the indole ring on the atomically flat gold surface, orthogonal to the E-vector in the TERS experiments. In SERS experiments with silver sol aggregates of different sizes, Trp can attach on the silver surface in different orientations. For small, silver nanoparticles, the authors found that Trp seemed to lie flatly on the silver surface, visible in indole signals of only weak intensity or even absent ones.23 In the present TERS experiments, the enhanced indole signals (1509, 1455, 1246, 685 cm-1) reveal interactions of the pyrrole as well as the benzene unit with the metals. From TERS experiments of histidine, which possesses two nitrogens in the aromatic ring, it is known that the ring attaches preferentially with the nitrogen atom, whose lone electron pair is not donated to the delocalized π-system. In this case, the nitrogen acts as a ligand.21 In the indole ring of tryptophan, the free electron pair of the nitrogen does not participate directly in the aromatic system. Therefore, a coordination of this nitrogen to the gold surface is possible and completes the interactions of the entire ring system. In SERS experiments of Trp- and Trp-containing peptides, the authors found that Trp binds to the metal surface via the NH of the indole ring under certain circumstances.62 These results confirm the assumption given above. In Table 3, the TERS values together with those from SERS24,28 and from Raman data24 and their assignment are listed. The enhancement of νCOO- around 1637 and 1403 cm-1 explains the interactions of this terminal group with the silver in the cavity. This was to be expected regarding the so-far carried out TERS measurements on histidine and cystine, in which both amino acids showed enhanced signals of the carboxyl group.20,21 The same is valid for the protonated amino group (NH3+), whose signals were recorded around 1575 and 1104 cm-1. To analyze the orientation of Trp in the gold-silver sandwich, again, theoretical calculations were performed and are shown in Figure 8. It is obvious that a flat coordination of the molecule is energetically favored and is hardly perturbed by the tip. In addition, the Trp molecule adsorbs parallel to the Au surface, as shown in Figure 8. Its adsorption energy was calculated as 2.02 eV and, thus, is slightly larger than those of Phe and Tyr. This difference can be explained by the larger ring structure, which can introduce a larger dispersive interaction on attaching horizontal to the substrate. Consequently, for this molecule, no rotated geometry is found to be stable. All other orientations of the ring structure relax to the flat orientation.

Deckert-Gaudig et al.

Figure 8. Model of the theoretical calculated orientation of Trp on gold (Au111) with a Au-tip distance of (a) 6.5 and (b) 11.5 Å.

Including the Ag tip, the dispersive interaction, again, but also the interaction with the oxygen atom seem to lower the energy. In the closest distance of tip and substrate, the energy is lowered by 2.7 eV (compare to Figure 4). An important aspect of the TERS technique, in addition to the previously discussed orientation of neat molecular layers, is the distinction of mixed components. Therefore, finally, an experiment to distinguish Trp and Phe with TERS spectroscopy from a mixture adsorbed onto a gold nanoplate was performed. In Figure 9, the TERS spectra of this experiment are displayed. The points where the measurements were taken on the gold



Figure 9. TERS spectra of a mixture of Phe and Trp adsorbed onto a gold nanoplate, λ ) 530 nm, P ) 630 µW, tacq ) 20 s. Lowermost spectrum shows reference spectrum on glass. Blue highlighted bands correspond to Trp (W); orange, to Phe (F).

surface had a distance of 145 nm. The enhancement factor was estimated to at least 3 × 105. Once more, one can see the uniform spectra along a line on the gold surface. Signals highlighted in blue can be assigned to Trp (W), and orange highlighted, to Phe (F), respectively. Bands in the region around 1600 cm-1 (ν(CdO, CdC)) and 1430 cm-1 (ν(CH2, NH3+)) overlap and cannot be distinguished due to identical functional groups. The reference spectrum (bottom) was, as usual, taken close to the gold nanoparticle to exclude tip contamination. Although a qualitative distinction of the two amino acids is straightforward, a quantitative assumption is more difficult. The vibrational modes of Trp appear to be more enhanced as compared to those of Phe. This fact could be a result of either stronger interaction of Trp with the metal or simply by the fact that more Trp than Phe molecules were attached to the gold surface. At present, this cannot be solved in one experiment because many parameters have to be taken into account to compare signal intensities (e.g., substrate thickness, local distribution, adhesion, etc.). 5. Conclusion In the presented work, TERS spectra of the aromatic amino acids phenylalanine, tyrosine, and tryptophan immobilized on single gold nanoplates have been presented. In contrast to observations in Raman or SERS, in all TERS spectra, the characteristic ring breathing modes of the aromatic unit of the molecules were hardly detected. This fact can be readily explained by the unique geometric rigidity of the TERS setup that is comparable to single-crystal Raman experiments and is consistent with aromatic rings lying flat on the gold surface. The main interaction of the molecules within the gold-silver sandwich occurs via the carboxyl and amino moieties and can be perceived in the enhanced signal intensities in the TERS spectra. The experimental results were supported by theoretical calculations of the molecules within the metal gap. To the best of our knowledge, such a comparison has not been done before and provides insight into the behavior of biomolecules between two metal layers. The immobilization of a combination of phenylalanine and tryptophan on ultraflat gold nanoplates enables TERS to distinguish characteristic moieties of both amino acids on the surface and proves the capability of this technique to analyze complex molecules and even mixtures.

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JP9098045

Aromatic Amino Acid Monolayers Sandwiched between Gold and Silver

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