Missing Amide I Mode in Gap-Mode Tip-Enhanced Raman Spectra of

Oct 1, 2012 - amide I mode, which is widely used to identify secondary structure motifs of proteins, is not visible in gap-mode TERS. Aromatic modes a...
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Missing Amide I Mode in Gap-Mode Tip-Enhanced Raman Spectra of Proteins Carolin Blum, Thomas Schmid,† Lothar Opilik,† Norman Metanis, Simon Weidmann, and Renato Zenobi* Department of Chemistry and Applied Biosciences, Laboratory of Organic Chemistry, ETH Zurich, 8093 Zurich, Switzerland S Supporting Information *

ABSTRACT: Tip-enhanced Raman spectroscopy is a surface sensitive analytical method that combines the advantages of scanning probe microscopy and Raman spectroscopy. It holds great promises for imaging of biological samples with high spatial resolution (10−50 nm), well below the optical diffraction limit. It offers the opportunity to directly localize and identify proteins and their conformation in a complex (e.g., native) environment. Tip-enhanced Raman (TER) spectra in the socalled “gap-mode” configuration with a metal tip in scanning tunnelling feedback with a metal substrate coated with different proteins (bovine serum albumin, immunoglobulin G, trypsin, and β-lactoglobulin) as well as of model octapeptides (with and without an aromatic amino acid residue) are presented. The goal was to determine if it is possible to reliably assign marker bands for proteins and if different secondary structures of proteins can be distinguished in their gap-mode TER spectra as reliably as by IR and conventional Raman spectroscopy. It is shown that contrary to the presented conventional Raman spectra of proteins the amide I mode, which is widely used to identify secondary structure motifs of proteins, is not visible in gap-mode TERS. Aromatic modes are prominent and can be used as reliable marker bands for imaging of proteins in a complex environment.

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each were shown by Opilik et al.5 IR spectroscopy and conventional Raman spectroscopy studies have shown that a correlation exists between the dominant secondary structure motif found in the protein crystal structure and the spectral positions of the amide modes.1,3,6 The amide I mode is most often used for determining the dominant secondary structure motif of the protein. It mainly consists of the CO stretching vibration (80%) with contributions from the C−N stretching and C−C−N deformation modes.7 α-Helical secondary structures cause an amide I mode in the range of 1650−1655 cm−1, β-sheets in the range of 1665−1670 cm−1, and random coils >1680 cm−1 in both Raman and IR spectra.7,8 The amide II mode appears at ∼1550 cm−1 and is an out-of-phase combination of NH in-plane bending modes (60%) and C−N stretching modes (40%). However, it is not as widely used for determining the secondary structure, probably because it is caused by more than one functional group.6 The amide III mode is also sensitive to the secondary structure of the protein and is mainly caused by the in-phase combination of the NH in-plane bending vibrations with the C−N stretching vibrations. Because it is found in the same spectral region as the CH2 wagging modes (∼1280 cm−1), determining the secondary structure relying solely on this mode is problematic.6 By analyzing proteins (bovine serum albumin (BSA), immunoglobulin G (IgG), trypsin, and β-lactoglobulin) with

INTRODUCTION Proteins are essential macromolecules found in all organisms that participate in nearly every cellular process. For example, they help building up the cell structure, pump ions through the cell membrane, transport metabolites into and out of the cell, and catalyze different chemical reactions.1 For understanding their biological role, it would be highly beneficial to not only locate them in their native environment but to simultaneously access chemical information and thereby information about their unique structure. Tip-enhanced Raman spectroscopy (TERS) is a surface-sensitive analytical method that combines the advantages of two techniques: the high lateral resolution of scanning probe microscopy, such as scanning tunnelling microscopy (STM), and the chemical information provided by Raman spectroscopy.2 A very sharp metal or metal-coated tip is brought into feedback with the sample under investigation. By illuminating this tip with a focused laser beam, a combination of the so-called lightning rod effect (the crowding of electromagnetic field lines at a sharp end), the optical antenna effect, and the excitation of surface plasmons on the metal surface cause a strongly confined and enhanced field at the sharp tip end.3 This causes a strongly enhanced Raman signal from the molecules in the vicinity of the tip. Thus TERS has the potential to probe the surface of many biological samples and to directly image the distribution of, for example, proteins in a biological membrane, with a typical resolution of 10−50 nm, well below the optical diffraction limit.4 First, highresolution images of biological samples − in this case model lipid domains − with 128 × 128 pixels with 47 nm side length © XXXX American Chemical Society

Received: July 10, 2012 Revised: September 27, 2012

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different secondary structures by TERS, we systematically investigated whether it is possible to assign typical marker bands of proteins in their TER spectra that would enable labelfree imaging experiments of proteins in a complex environment with a spatial resolution well below the optical diffraction limit. Additionally, we investigated if and how TER spectra of proteins provide the information necessary to investigate their secondary structure. This would in the future offer the opportunity to directly survey proteins and their conformation, for example, in a cell membrane, and learn about the relation between their conformation, localization, and biological function.

(HOBt) were obtained from Peptides International. The solvents N-methyl-2-pyrrolidone (NMP), N,N-dimethylformamide (DMF), dichloromethane (DCM), acetonitrile (ACN), and N,N-diisopropylethyl amine (DIEA) were purchased with HPLC-grade quality from Sigma Aldrich or Applied Biosystems Europe. Trifluoroacetic acid (TFA) was obtained from Halocarbon Products. Octapeptide Synthesis. Octapeptides (1 abd 2) differ at only one position in the middle of their sequences Glu-Lys-SerXaa-Leu-Arg-Val-Gly; where Xaa = Ala (1) or Phe (2) (Scheme 1). The exact procedure of their synthesis is described in the Supporting Information.

EXPERIMENTAL SECTION Experimental Setup. Confocal Raman and TERS measurements were performed on a commercial combined atomic force (AFM)/STM/Raman microscope (NTEGRA Spectra Upright, NT-MDT) that has been previously described by Stadler et al.9 We utilized a HeNe laser with an excitation wavelength of 632.8 nm. A maximum laser power of ∼2 mW was used for the bulk confocal Raman measurements. For the TERS measurements, the laser power was adjusted by means of neutral density filters to either 47 or 230 μW. (See Table S1 in the Supporting Information.) Acquisition times for the confocal Raman measurements were between 8 and 20 min (except for the very weak Raman scatterer BSA, there it was 1 h) and between 2 and 10 min for the TERS measurements. (See Table S1 in the Supporting Information.) Each measurement was performed several times to ensure reproducibility. This includes the use of different tips, measurements on different sample positions, and control experiments with the used tip on an empty substrate (to check for tip cleanliness) as well as measurements with the laser focused on the sample while the tip was retracted (to check for possible far-field signal contributions). (See Figure S1 in the Supporting Information.) TERS Tips. Silver wires (diameter 0.25 mm, 99.99% purity, Sigma Aldrich) were electrochemically etched at a voltage of 8 V in a 1:1 (v/v) mixture of perchloric acid (70%, VWR) and methanol (p.a., Sigma Aldrich), as previously described by Stadler et al.5,9 The resulting silver tips were rinsed with methanol and checked for their sharpness with a Nikon 360x stereo microscope. Afterward the tip was mounted in a customized NT-MDT tip holder and brought into STM feedback with the sample surface. The focused laser beam was scanned over the tip while spectra were collected at every pixel. In this way, a spectral intensity map was obtained that allowed aligning the laser to the most strongly enhancing spot of the tip. In a next step, the focused laser was fixed to this position and only the sample was moved by means of a piezoelectric scanner.9 Samples. Chemicals. Trypsin, β-lactoglobulin, IgG, phenylalanine (Phe), and Phe-Phe were purchased from Sigma Aldrich. BSA was purchased from Fluka Fine Chemicals. A NANOpure Diamond (18.2 MΩ·cm) water purification system was used to obtain ultrapure water. All Fmoc-amino acids were obtained from Novabiochem (Merck) or Bachem with the following side-chain protecting groups: Glu(OtBu), Lys(Boc), Ser(tBu), and Arg(Pbf) (Boc = tert-butyloxycarbonyl; tBu = tert-butyl; Pbf = 2,2,4,6,7-pentamethyl-dihydrobenzofuran-5sulfonyl). Fmoc-Gly-Wang resin was obtained from Novabiochem. 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU) and hydroxybenzotriazole

Scheme 1. Octapeptides (1 and 2) Used in This Studya

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a

Peptides differ at only one position in their sequences Glu-Lys-SerXaa-Leu-Arg-Val-Gly, where Xaa = Ala (1), Phe (2).

Sample Substrates. For the STM-TERS measurements, template-stripped Au substrates were produced as previously described.10 For the confocal bulk Raman measurements, glass coverslips were cleaned in a piranha solution (H2SO4/H2O2, 3:1 v/v), rinsed with ethanol (Scharlau S. L.) and water, and dried in a stream of nitrogen. TERS Sample Preparation. For preparing the trypsin (7 μM), β-lactoglobulin (7 μM), IgG (0.7 μM), Ala-octapeptide (1 mM), Phe-octapeptide (1 mM), phenylalanine (1 mM), and Phe-Phe (1 mM) samples, we deposited 40 μL of aqueous solution (concentration as indicated) on a freshly prepared template stripped Au substrate and dried in a desiccator. For preparing the BSA sample, a 0.5 wt % aqueous solution of BSA was deposited on a uniform polydimethylsiloxane stamp. After an incubation time of 30 min, the stamp was rigorously rinsed with water and dried in a stream of nitrogen. Then, the stamp was gently placed on a freshly cleaved template stripped Au substrate and carefully removed 1 min later. Sample Preparation for Conventional Raman Spectroscopy. Some crystals of the bulk material were deposited on a clean glass coverslip and measured directly by focusing the laser beam onto the crystal. Data Processing. Spikes due to cosmic rays at long measurement times were removed manually from the spectra. Otherwise, we show untreated raw data. No background subtraction or smoothing was performed. This ensures that no conclusions are drawn that might be caused by artifacts B

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1656−1669 cm−1 are prominent modes in all confocal bulk Raman spectra shown. As expected, the spectral position of the amide I mode varies according to the secondary structure that is dominant in the corresponding protein. For BSA, whose secondary structure is dominated by α-helical structures, the amide I mode is found at 1656 cm−1, whereas for the β-sheetdominated β-lactoglobulin, IgG, and trypsin, it is found at 1665 and 1669 cm−1, respectively. All values are in very good agreement with band positions found in the literature for these proteins. (See Table S2 in the Supporting Information.)15 For two reasons, Phe-Phe was measured for comparison: first, a dipeptide is the simplest possible model system for a protein, and second, it does not possess any kind of secondary structure because it is too short to form intramolecular hydrogen bonds. Phe contains no amide group and is shown as a “negative control”. Their confocal bulk Raman spectra, both dominated by the aromatic ring breathing mode at 1004 cm−1, are shown in Figure 1a (red and dark red). Phe does not show any prominent band >1630 cm−1, whereas the amide I mode for Phe-Phe is clearly visible at 1688 cm−1. The TER spectra of the proteins, Phe-Phe, and Phe are shown in Figure 1b. They are, in general, less rich in spectral bands than the confocal bulk Raman spectra. The prominent modes for all TER spectra are the aromatic ring breathing mode of the benzene ring at 1004 cm−1, the CH deformation modes at 1450 cm−1, and another benzene ring vibration at 1600 cm−1. The most remarkable observation in the TER spectra is that the amide I mode is not observable! No mode is visible above 1630 cm−1. Also, the TER spectrum of the dipeptide Phe-Phe that was measured for comparison does not show any significant mode in the spectral range in which the amide I mode is expected. This implies that although it is possible to assign protein marker bands for TERS (e.g., the prominent aromatic modes) it does not seem to be possible with gapmode TERS to distinguish any kind of secondary structure of proteins. Note that the control experiments clearly show that the observed signals are not due to a far-field contribution but are caused by the tip enhancement. (See Figure S1 in the Supporting Information.) In the following, the dominant aromatic modes and possible reasons for the missing amide I mode in the TER spectra are discussed in detail. Note that in all TER spectra a broad background is present (that is often subtracted in other studies in the literature16). It is mainly caused by two factors: the first factor is not fully understood, but the observed unspecific broad background is often explained by a plasmon-dependent photoluminesence of the rough metal surface (or the tip−sample gap).16,17 Second, the enhanced and confined electromagnetic field in direct proximity to the tip can cause sample degradation.3 For long measurement times (in the range of minutes) as used in this study, the Raman signals of the resulting carbonaceous species average to two broad bands centered around 1350 and 1580 cm−1.18 These two main sources of background sum up to the observed background but do not obscure the sharp TER signals of the intact material.10 Model Octapeptides. Because the aromatic modes seem to dominate the spectrum, two model octapeptides that only differ at one position in the middle of their sequence were synthesized to investigate the influence of the aromatic amino acids on the TER spectra: the “Ala-octapeptide” Glu-Lys-SerAla-Leu-Arg-Val-Gly (1) and the “Phe-octapeptide” Glu-LysSer-Phe-Leu-Arg-Val-Gly (2). (See Scheme 1.) This sequence

introduced by data processing. Igor Pro (Version 6.22 A, Wavemetrics) was used for visualizing the data. Spectra are offset and/or multiplied along the y axis for better visibility.

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RESULTS AND DISCUSSION Confocal and Tip-Enhanced Raman Spectra of Proteins. The main goals of this study were to investigate whether and how it is possible to assign marker bands for proteins with TERS and if it is possible to distinguish their secondary structure due to the position of the amide I mode in the spectra. We therefore chose proteins of different secondary structure for this investigation: BSA as a representative for a protein mainly composed of α-helical structures11 and IgG, βlactoglobulin, and trypsin as representatives for proteins mainly composed of β-sheets.6,7,12−14 To have reference Raman spectra measured on the same instrument under the same conditions to compare the obtained TER spectra with, bulk confocal Raman spectra of all proteins (and for comparison, also of Phe and Phe-Phe) were measured first. The resulting spectra are shown in Figure 1a. The observed spectral band

Figure 1. (a) Bulk confocal Raman and (b) tip-enhanced Raman spectra of BSA (yellow), β-lactoglobulin (blue), IgG (gray), trypsin (green), Phe (red), and Phe-Phe (dark red). Modes that become relevant during the discussion in the main text are highlighted in gray and red.

pattern of the proteins is in very good agreement (deviation less than 3 cm−1 in most cases) with that of reference Raman spectra from the literature. (See Table S2 in the Supporting Information.)11−15 Bands caused by the aromatic amino acids (the tyrosine doublet at 830 and 850 cm−1, the aromatic ring breathing mode of Phe at 1004 cm−1, etc.), the amide III mode at ∼1242 cm−1 (together with CH2 wagging modes), the CH2 deformation mode at 1450 cm−1, and the amide I mode at C

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octapeptide, the aromatic ring breathing mode at 1003 cm−1 and the in-plane C−H deformation mode of a monosubstituted benzene ring at 1031 cm−1 are clearly visible, whereas both are not observable for the Ala-octapeptide, which does not contain an aromatic amino acid residue. The CH2 deformation modes at ∼1450 cm−1 were the only visible modes in the Alaoctapeptide spectra in several measurements. Their intensity was always comparable to the intensity of this mode in the Pheoctapeptide spectra. Equal aquisition time and laser power were used for both measurements shown; see Table S1 in the Supporting Information. Overall, these measurements confirm: (a) Bands assignable to aromatic amino acids (here Phe) dominate both the confocal Raman and the TER spectrum. (b) Aromatic modes are selectively enhanced in TERS. (c) Nonaromatic peptides can also be detected with TERS and mainly show the CH2 deformation modes. (d) Most importantly: In both the TER spectrum of the aromatic peptide as well as in the TER spectrum of the nonaromatic peptide the amide I mode cannot be observed. Why Is the Amide I Mode Missing? Often, a change in the secondary structure of the corresponding protein (e.g., denaturation to a random coil) is used as an explanation for an amide I mode that exhibits less intensity or is missing.20,21 However, the amide I mode itself is mainly caused by the carbonyl stretching vibration (with contributions from C−N stretching and C−C−N deformation modes) and should be visible even in the absence of any secondary structure (see PhePhe).7 Additionally, Sane et al. showed that the Raman scattering efficiency of different secondary structures (including unordered structure) varies by a factor of less than two.22 Therefore, a possible change in the secondary structure that might result in a different Raman scattering efficiency cannot be used as an argument for the absence of the amide I mode. For the following two reasons, we are convinced that our proteins remained in the native state without degradation/ denaturation: First, matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS) experiments were performed (data not shown) to check for such a possible degradation of the proteins during sample preparation. The proteins were clearly intact after sample deposition and did not decompose. Second, it has been shown in a thorough study by Zhang et al.23 that a drop-drying deposition of proteins onto Au substrates (the exact same method we use in this study) with a subsequent Raman measurement does not cause denaturing. The protein spectra they measured are the same as the ones attributed to the protein in its native state and clearly differ from the denatured spectrum. Zhang et al. also performed MALDI-MS measurements after the Raman measurements that show that the protein did not decompose.23 In this context, a pH dependence as observed by Iosin et al.21 can also be excluded because we are working in the dry state (although the proteins are probably still well hydrated23). A strong direct interaction of the protein backbone with the Au substrate could cause a change in the amide I mode. However, we are not working at monolayer coverage, and proteins are far too bulky such that all amide groups could simultaneously interact with the surface in such a way that this mode is invisible. With multilayers present, orientation effects can also be excluded as a possible reason for the absence of the amide I band: the molecules should be randomly oriented with respect to the surface and the tip.

of eight amino acids was chosen to ensure water solubility of the octapeptides and that they are long enough to contain a sufficient number of amide groups (compared with Phe-Phe that only contains one). Their bulk confocal Raman spectra are shown in Figure 2a. The modes resulting from the aromatic

Figure 2. (a) Bulk confocal Raman and (b) tip-enhanced Raman spectra of the Phe-octapeptide (purple) and the Ala-octapeptide (blue). In panel a, modes are highlighted that are only visible in the Phe-octapeptide. The amide I mode is highlighted in red. In panel b, dominant modes and the spectral range in which the amide I mode is expected are highlighted.

amino acid residue can easily be identified: they are only visible in the spectrum of the Phe-octapeptide. A comparison with the set of spectral bands observed by Guicheteau et al.19 for the confocal Raman spectrum of Phe enables an assignment of all modes that are only present in the Phe-octapeptide and thus characteristic for the Phe residue: 622 (ring deformation mode of a monosubstituted benzene ring), 1003 (symmetric ring breathing mode), 1031 (in-plane C−H deformation modes of monosubstituted benzene), 1206 (C6H5−C vibration of the ring, phenyl-C stretching), 1586, and 1604 cm−1 (ring stretching, doublet for benzene derivatives, three or more coupled CC stretching modes). The aromatic ring breathing mode at 1003 cm−1 is the most prominent mode of the confocal Phe-octapeptide spectrum. However, other modes, like the CH2 deformation modes at 1450 cm−1, are nearly as intense. For both octapeptides, the amide I mode is prominent at 1668 cm−1. For the Ala-octapeptide, it is one of the most intense modes in the spectrum, along with the CH 2 deformation modes at 1450 cm−1. The corresponding TER spectra of the two octapeptides are shown in Figure 2b. In both spectra, the CH2 deformations at ∼1450 cm−1 are clearly visible. As expected for the PheD

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several nanometers,27−29 the enhancement should also reach the peptide backbone. Second, even for the investigated Alaoctapeptide that does not contain any bulky aromatic side chain, the amide I mode is not visible. In conclusion, steric shielding effects cannot be used as an argument for the absence of the amide I mode in TERS. Third, an electromagnetic shielding effect of the side-chains could play a role because the aromatic rings or the heteroatoms in the side-chain might have a higher Raman cross-section and preferably interact with the incoming photons. Once again, the Ala-peptide can be used as a negative indicator for this argument because no aromatic ring is present and nearly no heteroatoms in the side chains that could justify the absence of the amide I mode. Overall, the side chains might influence the resulting spectra but cannot be the only reason for the absence of the amide I mode. Iosin et al. used Au nanoparticles of different shape for SERS but were also unable to observe the amide I mode of BSA (however, they could clearly observe it in the confocal bulk Raman spectrum).30 They observed a dominance of the aromatic modes. Contrary to these two studies, the amide I mode was clearly visible in other studies, for example, in the SERS experiments performed by David et al.20 In general, it remains unclear why some groups observe the amide I mode in SERS and others do not. Overall, it is possible to assign marker bands for proteins (e.g., the aromatic modes) that should enable imaging experiments. However, although the assignment of marker bands for proteins is possible, the results presented here indicate that it will be difficult or impossible to derive their secondary structure from TERS. The long measurement times used in this study (in the range of minutes per pixel) might give the impression that imaging is out of reach. However, the focus of an imaging experiment is not on achieving the best spectrum possible − as it was the case in this study − but on obtaining a sufficient signal-to-noise ratio for the marker band. This is possible with much shorter measurement times (in the range of several seconds); therefore, imaging is definitely within reach.

Because gap-mode STM-TERS measurements were performed, the tunnel current flowing between the tip and the Au surface could also influence the corresponding TER spectrum, as observed for different bias voltages in fishing-mode TERS by Liu et al.24 However, contrary to the study by Liu et al., the feedback settings were not changed between the different TERS measurements presented in this work. Additionally, changes in the feedback to increase/decrease the tunnel current did not have any influence on the resulting spectra. (See Figure S2 in the Supporting Information.) Another reason for the absence of the amide I mode could be a too small signal-to-noise ratio, causing a disappearance of the amide I mode in the noise and a dominance of the aromatic modes. A recent study by our group has shown that the band intensity ratios can change dramatically when comparing confocal Raman and TERS measurements. The enhancement of a mode is influenced by the polarizability of the molecule, how good the incident laser wavelength and the scattered Raman signal overlap with the surface plasmon resonance of the tip, the orientation of the molecule relative to the polarization of the electromagnetic field, and its symmetry.10 The special geometry of an enhancing metal tip (here Ag) in feedback with a conducting metal surface (here Au) that is used in this study (so-called gap-mode configuration) that leads to a strongly confined and enhanced field underneath the tip whose field lines are oriented perpendicular to the sample surface could play a significant role and lead to special selection rules for the observed modes of the molecules underneath the tip. The influence of this gap-mode geometry should therefore be compared against TERS measurements on nonmetal surfaces; this is part of developments in our group. Comparison with SERS Data. TERS relies on similar enhancement effects as surface-enhanced Raman spectroscopy (SERS). For comparison, SERS experiments on nanorough silver surfaces with the same proteins were therefore additionally performed. The exact procedure was previously described.10 However, it was rather difficult to probe proteins because they appeared to be very sensitive to decomposition. For the cases where the protein survived, the resulting spectra are shown in Figure S3 in the Supporting Information. As in the TER spectra, the prominent modes result from aromatic vibrations. The amide I mode is missing in the shown SER spectra, as in the TER spectra (see above). Overall, the SER spectra are very similar to the obtained TER spectra. This finding corroborates the notion that the electromagnetic enhancement in TERS and SERS (as opposed to confocal Raman spectroscopy) seems to play a significant role for the disappearance of the amide I mode, as it influences the band ratios that are observed in the spectra.25 Missing amide I modes have sometimes also been discussed in the SERS literature. Contradictory information is found in the SERS literature on the absence/presence of the amide I mode, which will be illustrated with the example of BSA: Chumanov et al. used silver hydrosols to study the SER spectrum of BSA and were not able to observe an amide I mode.26 They explain its absence by a possible shielding of the peptide bands by the side chains of amino acid residues, which increase the distance from the metal surface and therefore hinder the enhancement of the amide I vibration. However, there are several indicators that this steric argument cannot be the only reason for the absence of the amide I mode: First, the size of the side chains is on the order of a few angstroms. Because the enhancement is typically assumed to decay after

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CONCLUSIONS The TER spectra of several proteins (BSA, IgG, β-lactogloublin, and trypsin) were studied to answer the question if marker bands can be assigned that could be possibly used for biological TERS imaging experiments. Additionally, it was investigated if the secondary structure of different proteins can be distinguished based on their TER spectra. It was found that the TER spectra of proteins were less rich compared with their confocal Raman spectra and that they were dominated by aromatic modes if aromatic amino acid residues were present. The amide I mode, however, could not be observed. The dominance of aromatic modes in the TER spectra was confirmed by studying two model octapeptides. Possible reasons for the absence of the amide I mode were discussed, for example, a too small signal-to-noise ratio or a possible shielding of the protein/peptide backbone by the amino acid side chains. The amide I mode was also not observed in our own SERS measurements on nanorough silver surfaces.

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ASSOCIATED CONTENT

S Supporting Information *

Measurement parameters of the bulk confocal Raman and the TERS measurements; TER spectrum of trypsin, control experiment, and Raman spectrum spectrum with the laser focused on the sample and the tip retracted; Band assignment E

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(24) Liu, Z.; Ding, S.-Y.; Chen, Z.-B.; Wang, X.; Tian, J.-H.; Anema, J. R.; Zhou, X.-S.; Wu, D.-Y.; Mao, B.-W.; Xu, X.; Bin Ren; Tian, Z.-Q. Nat. Commun. 2011, 2, 305−311. (25) Yeo, B.-S.; Stadler, J.; Schmid, T.; Zenobi, R.; Zhang, W. Chem. Phys. Lett. 2009, 472, 1−13. (26) Chumanov, G.; Efremov, R.; Nabiev, I. R. J. Raman Spectrosc. 1990, 21, 43−48. (27) Yano, T.-A.; Ichimura, T.; Taguchi, A.; Hayazawa, N.; Verma, P.; Inouye, Y.; Kawata, S. Appl. Phys. Lett. 2007, 91, 121101. (28) Pettinger, B.; Domke, K. F.; Zhang, D.; Picardi, G.; Schuster, R. Surf. Sci. 2009, 603, 1335−1341. (29) Cançado, L. G.; Jorio, A.; Ismach, A.; Joselevich, E.; Hartschuh, A.; Novotny, L. Phys. Rev. Lett. 2009, 103, 186101. (30) Iosin, M.; Toderas, F.; Baldeck, P. L.; Astilean, S. J. Mol. Struct. 2009, 924−926, 196−200.

of the bulk confocal Raman bands for the proteins trypsin, IgG, BSA and β-lactoglobulin; investigation of the influence of the tunnel current between tip and sample for Phe-Phe; SER spectra of IgG, trypsin, Phe-octapeptide, Phe-Phe, and Phe; and octapeptide synthesis. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Author Contributions †

These authors contributed equally to this work.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS C.B. thanks the Stiftung Stipendien-Fonds des Verbandes der chemischen Industrie e.V. (funding) and the German National Academic Foundation (ideational promotion) for scholarships. Johannes Stadler is thanked for his advice.

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