Surface-Enhanced Raman Spectroscopy and Density Functional

Article pubs.acs.org/JPCC

Surface-Enhanced Raman Spectroscopy and Density Functional Theory Calculations of a Rationally Designed Rhodamine with Thiol Groups at the Xanthene Ring Svetlana Brem and Sebastian Schlücker* Department of Chemistry and Center for Nanointegration Duisburg-Essen (CENIDE), University of Duisburg-Essen, Universitätsstrasse 5, 45141 Essen, Germany S Supporting Information *

ABSTRACT: Rhodamines are widely used dyes in fluorescence and surface-enhanced Raman spectroscopy (SERS). The latter requires adsorption of the dye onto the surface of plasmonic nanostructures, a process which requires attractive molecule− surface interactions. Here, we report an experimental SERS and computational density functional theory (DFT) study investigating the role of thiol functionalization at the xanthene ring of the rhodamine in the adsorption onto gold nanoparticles. For this purpose, a new bisthiolated rhodamine derivative was rationally designed and synthesized via a PPh3/I2 reduction route. The introduction of two thiol moieties directly at the xanthene ring provides the shortest possible distance between the molecular π-system and the metal surface for maximum SERS enhancement combined with the strong Au−S interaction for chemisorption. The comparison of experimental SERS spectra obtained from gold nanostars and a film of gold nanoparticles with results from DFT calculations (molecular electrostatic potential, normal modes) suggests adsorption via the thiol groups at the xanthene moiety.

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INTRODUCTION Rhodamines (Figure 1) are xanthene-type dyes that are widely used in fluorescence spectroscopy and microscopy.1 A prominent representative is Alexa 488 (compound 4 in Figure 1), a bis-sulfonated and therefore water-soluble derivative. In comparison with fluorescein isothiocyanate (FITC), Alexa 488 is more photostable and less sensitive to pH and yields a stronger fluorescence signal.2 Rhodamine 6G (3 in Figure 1) is a commonly used dye in surface-enhanced Raman scattering (SERS), surface-enhanced resonance Raman scattering (SERRS), and tip-enhanced Raman scattering (TERS).3−6 High photostability in combination with large Raman scattering cross sections due to the conjugated π-system of the chromophore and additional signal increases due to molecular electronic resonances make rhodamine 6G an ideal candidate for single-molecule SERS.7 In addition to label-free SERS/ SERRS and TERS, dyes are also used as Raman reporter molecules in targeted SERS-based approaches using molecularly functionalized plasmonically active colloids as labels. An SERS label or nanotag8,9 consists of a noble-metal nanoparticle and Raman reporters adsorbed onto its surface. Raman reporters/dyes should have the following properties: a large Raman scattering cross section for achieving high SERS signal contributions, a functional group such as a thiol or amino moiety for strong adsorption (chemisorption) to the metal surface, and a dense surface packing for maximum surface coverage to increase the overall SERS signal intensity. In the © XXXX American Chemical Society

case of direct covalent bioconjugation of the target-specific ligands, they must contain a second functional group such as a carboxylic acid. Alternatively, a protective shell such as a silica coating around the SERS nanotag can be used for stabilization and subsequent bioconjugation. Although the Raman reporter is a major factor that influences the properties of SERS tags, its role has surprisingly not been studied as intensively as the influence of the metal substrates. Rational design and systematic characteristic investigations on new Raman reporters are rarely reported. For example, Graham and co-workers introduced benzotriazole derivatives for use as dyes in SERRS on silver colloids.10 Chang and Olivo along with their coworkers presented cyanine and triphenylmethine reporters comprising fluorescent cyanine and triphenylmethine covalently conjugated to a lipoic acid spacer (cyclic disulfide) for strong binding to gold surfaces.11,12 Lambert and Schlücker along with their co-workers presented the design and synthesis of Raman reporter molecules comprising olefin or alkyne moieties with strong and characteristic vibrational Raman bands for use in tissue imaging.13 Detty and Kircher along with their co-workers designed and synthesized chalcogenopyryliumbased dyes for highly sensitive SERRS.14 These studies show Received: February 15, 2017 Revised: May 15, 2017 Published: June 19, 2017 A

DOI: 10.1021/acs.jpcc.7b01504 J. Phys. Chem. C XXXX, XXX, XXX−XXX

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The Journal of Physical Chemistry C

in an oven at 35 °C. The obtained film was incubated with 1 mM dye solution for 24 h, rinsed with water and ethanol, and air-dried before the SERS experiments. UV/Vis Absorption and Fluorescence Spectroscopy. Absorption spectra were recorded with a JASCO V-630 spectrometer and the fluorescence spectra with a SpectraMax M5e instrument. For each sample, the absorption measurements were performed with a cuvette and a sample concentration of 1 × 10−5 M in methanol. First, a baseline was determined by running a scan without a sample. Scans were performed in the range of 300−700 nm. Fluorescence spectra were recorded with the excitation wavelengths obtained from the corresponding absorption spectra. For all measurements, the scan was performed in 10 steps. The fluorescence lifetime imaging microscopy (FLIM) experiments were performed with a fluorescence microscope (model Leica TCS SP8 SMD) on aqueous solutions of the dyes in the concentration range from 10−6 to 10−4 M. The excitation wavelengths were obtained from the corresponding absorption spectra. The scan resolution was set to 154 × 154 pixels, and each sample was scanned three times for 60 s at different positions in the probe. Overall, the concentrations were chosen such that they were high enough for obtaining maximum fluorescence intensities but low enough to avoid possible aggregation of the molecules or other molecular effects that might affect the optical characteristics. Raman Instrumentation. Raman experiments were carried out with a confocal Raman microscope (WITec, alpha 300 R, 30 cm focal length, 600 grooves/mm grating spectrometer) equipped with an electron-multiplying charge-coupled device (EM-CCD; Andor, Newton DU970N-BV-353). Radiation from the 632.8 nm line from a He/Ne laser was focused on the sample with a 10× microscope objective (Olympus, numerical aperture (NA) 0.25). SERS experiments on Au nanostars in suspension were performed with the Raman microscope and a laser power of 12 mW at the sample (quartz cuvette). The spectra were recorded for 30 s. Confocal Raman mapping experiments were performed with a 50× objective (Olympus, NA 0.7) and a He/Ne laser (λex = 632.8 nm) with a laser power of 49 μW at the sample. The integration time per pixel was 1 s (30 × 22 pixels). Data Processing of SERS Spectra. Original spectra (raw data, unprocessed) are shown in Figures 5 and 6. For the generation of the SERS false-color maps in Figure 8, the corresponding SERS spectra were baseline corrected using Whittaker−Henderson smoothing (Eilers, P. H. C.; Boelens, H. F. M. Baseline correction with asymmetric least-squares smoothing; Leiden University Medical Centre Report 1; Leiden, The Netherlands, 2005; p 1). Organic Syntheses. Organic solvents were dried and distilled under an argon atmosphere prior to use. Solvent mixtures are reported as volume/volume ratios. The standard Schlenk line technique was used where required. 1H and 13C NMR spectra were recorded on a Bruker DMX300 spectrometer. ESI-mass spectra were obtained by an amaZon SL instrument from Bruker. The samples were injected (flow injection method) at a concentration of 1 μM. The details of the organic syntheses are summarized in the Supporting Information. DFT Calculations. DFT calculations of the molecular geometry, vibrational spectra (including IR and Raman

the importance of the design and synthesis of bright Raman reporters with unique vibrational fingerprints. Herein we report the rational design and synthesis of a new thiolated rhodamine derivative (compound 1 in Figure 1) as a

Figure 1. Molecular structures of the rationally designed thiolated rhodamine (1), 5,6-carboxyrhodamine (2), rhodamine 6G (3), and Alexa 488 (4).

Raman dye for chemisorption to gold surfaces. Raman dye 1 can adsorb via the thiol moieties at the xanthene ring or via the carboxylic group at the phenyl ring. We employed SERS and density functional theory (DFT) calculations for investigating the binding of the thiolated rhodamine (1) to the surface of gold nanoparticles.

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EXPERIMENTAL DETAILS All starting materials were obtained from commercial sources and used without further purification, unless otherwise stated: gold(III) chloride trihydrate (HAuCl4; ≥99.9%, SigmaAldrich), hexadecyltrimethylammonium bromide (CTAB; ≥96%, Fluka), sodium borohydride (NaBH4; 96%, SigmaAldrich), ascorbic acid (AA; ≥99.0%, AppliChem), trisodium citrate dihydrate (AppliChem), hexadecyltrimethylammonium chloride (CTAC; >95.0%, TCI), 3-aminophenol (VWR Chemicals, 98%), ammonia (Alfa Aesar), benzene-1,2,4tricarboxylic anhydride (VWR Chemicals, 98%), chlorosulfonic acid (Fluka, 98%), dichloromethane (VWR Chemicals, p.a.), ethyl acetate (Bernd Kraft), dimethylformamide (VWR Chemicals), dimethyl sulfoxide (Sigma-Aldrich), sulfuric acid (Merck, 95%), fuming sulfuric acid (Merck, 65%), ethanol (Fisher Chemicals, p.a.), acetonitrile (Fisher Chemicals, p.a.), methanol (Fisher Chemicals, p.a.), RBS 35 concentrate (SigmaAldrich). Sample Preparation for SERS. For colloidal SERS experiments, 50 μL of 1 mM EtOH/H2O rhodamine solution was mixed with Au nanostars (AuNSs; λmax = 645 nm) in 1 mL of HCl (pH 0) or NaOH (pH 14) or 1 mL of water (pH 6−7), and the mixture was incubated for 20 h at 4 °C without shaking. The molecularly functionalized metal colloids were centrifuged (380 g, 40 min) and redispersed in 500 μL of H2O. For SERS experiments, CTAB-stabilized AuNPs were dropped on a silicon wafer cleaned by RBS, followed by drying B


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Figure 2. Reaction scheme for the synthesis of the thiolated rhodamine (1).

intensities), and molecular electrostatic potential of the cationic rhodamines 1 and 2 were performed with Gaussian 03 at the B3LYP/6-31G(d,p) level of theory (Frisch, M.; et.al. Gaussian 03, revision C.02) We obtained equilibrium structures by ensuring that no negative/imaginary frequencies were observed.

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RESULTS AND DISCUSSION Synthesis of Xanthene-Based Raman Dyes. The thiolated rhodamine (1) (Figure 2, right) was designed for Table 1. Optical Parameters of Thiolated Rhodamine (1), 5,6-Carboxyrhodamine (2), and Rhodamine 6G (3) dye CRh-SH (1) CRh (2) R 6G (3)

τ (ns) ε (L·mol−1·cm−1)

rel intens (RFU)

λabs (nm)

λem (nm)

3.81

5000

857

498

525

3.81 4.08

16000 10900

3730 2300

508 528

539 565

Figure 5. Calculated Raman spectra of rhodamines 1 and 2.

functionalization, in particular bioconjugation of the entire SERS nanotag. Figure 2 summarizes the synthesis of the thiolated rhodamine (1). The first step is the synthesis of 5,6carboxyrhodamine (2) via the reaction between 2 equiv of 3aminophenol with 1 equiv of benzene-1,2,4-tricarboxylic anhydride in the presence of a catalytic amount of concentrated sulfuric acid15 involving two electrophilic aromatic substitutions and two condensation steps. An isomeric mixture was obtained with 66% yield after purification. In the second step, 5,6carboxyrhodamine (2) was exposed to chlorosulfonic acid at 80 °C before fuming sulfuric acid was added. The resulting sulfonated rhodamine (5) was not isolated since it is not stable at ambient conditions.16 Finally, the thiolated rhodamine (1) was obtained in 38% yield by reduction of 5 with triphenylphosphine and iodine in DMF at 80 °C. Overall, the development of the synthesis of the thiolated rhodamine (1) was accompanied by several failures, in particular with respect to finding an efficient way for introducing thiol moieties via the reduction of sulfonyl groups (−SO3X; X = H or Cl). For example, the combined use of PPh3 and I2 in the last step of the synthesis is important because the strong reducing agent PPh3 alone does not react directly with sulfonic acids. In contrast, combination of PPh3 and I2 reduces both sulfonic acids (−SO3H) and sulfonyl chlorides (−SO2Cl).17,18 The proposed reaction mechanism of the reduction by PPh3/I2 starts with formation of iodotriphenylphosphonium iodide, which then deprotonates the sulfonic acid.17 The sulfonic acid of the rhodamine derivative 5 is presumably deprotonated. The second sulfonyl group was activated by SO2Cl, which could also be reduced under the same conditions.18 In addition to the mixed sulfonic acid/sulfonyl chloride rhodamine derivative 5, we also tried to obtain the intermediate disulfonyl chloride rhodamine; 16 however, in contrast to 5, isolation by

Figure 3. UV/vis absorption spectrum (black) and emission spectrum (red) of the thiolated rhodamine (1).

Figure 4. DFT-optimized geometries and molecular electrostatic potentials (MEPs) of rhodamines 1 and 2.

use as a trifunctional Raman reporter in SERS labels: (1) The chromophore comprises an extended conjugated π-system with a high Raman scattering cross section, while (2) the thiol moieties are the surface-seeking groups for chemisorption onto the surface of noble-metal nanoparticles, in particular gold colloids. (3) The carboxylic acids can be used for further C


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The Journal of Physical Chemistry C Table 2. Assignment for Normal Modes of Thiolated Rhodamine (CRh-SH, 1) and 5,6-Carboxyrhodamine (CRh, 2) CRh (2) relative wavenumber (cm−1) (DFT)

tentative band assignment

CRh-SH (1) relative wavenumber (cm−1) (DFT)

tentative band assignment

730

δ (CCC) phenyl

769 936

δ (CCC) phenyl δ (COC) Xa δ (CCC) Xa

768 918, 934

964

δ (CCC) Xa

1022

νs (CCO) phenyl ring breathing phenyl, νa (COC) Xa νa (CC) phenyl νs (CC) Xa νa (CC) Xa + δ (CCC) Xa νs (CC) Xa νs (CC) Xa νa (CC) Xa νs (CC) phenyl δ (NH) νs (CC) Xa νs (CO) carboxyl

1097

δ (CCC) Xa δ (CSH) + νs (CC) Xa + δ (CCC) Ph νa (COC) Xa + νa (CC) Xa + δ (CSH) + (NH) rock νs (CC) phenyl + δ (CCC) Xa νs (CC) Xa + (NH) rock + νs (CC)

732

1199 1232 1340 1395 1456 1550 1569 1581 1659 1697 1712 1841

1107 1226 1343 1369 1392 1446 1447 1508 1535 1576 1636

ring breathing phenyl νa (CC) phenyl νa (CC) Xa + δ (CCC) Xa νs (CC) Xa νs (CC) phenyl νa (CC) Xa δ (CCC) Xa νa (CC) phenyl δ (CCC) Xa δ (NH) + νa (CC) Xa

1657 1690 1835

νs (CC) Xa νs (CC) Xa νs (CO) carboxyl

Figure 7. SEM image of a gold nanoparticle film on the silicon substrate.

precipitation failed in this case. Additionally, also the reduction of the corresponding disulfonic acid (Alexa 488 (4)) to the dithiol was tested. The dye Alexa 488 was successfully synthesized2 and used for the reduction reaction by sodium borohydride. However, this approach yielded a complex product mixture, and the final product could not be isolated with the desired purity. Next we tried the reduction of sulfonic acids to thiols by lithium aluminum hydride in ether.19,20 Unfortunately, this approach is not useful for rhodamine dyes due to their low solubility in nonpolar solvents. For polar solvents, such as ethanol, we demonstrate that the reduction of sulfonic acid groups in Alexa 488 by lithium aluminum hydride is not efficient. This may have several reasons. One reason for the low reactivity of the sulfonic acid may be the poor ability of the hydroxy moiety to act as a leaving group. Moreover, the sulfonic acid is such a strong acid that it protonates the nucleophilic reducing agent. The resulting sulfonate anion (−SO3−) may prevent the nucleophilic attack due to electro-

Figure 6. SERS spectra of CRh-SH (1) and CRh (2) adsorbed on gold nanostars (λmax = 645 nm), 633 nm excitation wavelength in water (left) at pH 1, 6, and 14. D


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Figure 8. (A) SERS false-color image for CRh-SH (1) at 1200 cm−1. (B) Average SERS spectrum of CRh-SH (1) on the AuNP@Si substrate with the standard deviation (gray). (C) SERS false-color image for rhodamine 6G (3) at 1200 cm−1. (D) Average SERS spectrum of rhodamine 6G (3) on the AuNP@Si substrate with the standard deviation (gray).

static repulsion. These unsuccessful approaches finally lead us to exchange of the sulfonic acid group by a more reactive group. The replacement of hydrogen to form RSO2−OY should lead to the activation of the OH group, where Y is an electronwithdrawing group, and hence, OY− will be a good leaving group in the nucleophilic attack. The synthesis of aromatic sulfonyl chlorides21 is well established, but there are only a few reports about the sulfonyl chloride rhodamine derivative.16 Absorption and Fluorescence Spectroscopy. Table 1 summarizes the characteristic optical parameters of the new thiolated rhodamine (CRh-SH, 1) compared to 5,6-carboxyrhodamine (CRh, 2) and rhodamine 6G (R 6G, 3). The molar extinction coefficients as well as the relative fluorescence intensities differ by about a factor of 3, with 5,6-carboxyrhodamine (2) being the brightest dye. A blue shift in the electronic absorption (λabs) and fluorescence emission (λem) spectra in the series R 6G (3) to CRh (2) to CRh-SH (1) is observed due to the different substitution patterns. Figure 3 shows the UV/vis absorption and fluorescence spectra of the thiolated rhodamine (1). They exhibit the expected mirror symmetry for absorption and emission profiles. The fluorescence lifetimes in Table 1, determined from a monoexponential fit to the corresponding FLIM transients, are in the range of about 4 ns for all three dyes. We performed a concentration-dependent series of FLIM experiments (dilution from 1 mM to 1 μM) to examine a possible relationship between changes in the fluorescence lifetime depending on the concentration. For both 5,6-carboxyrhodamine (2) and rhodamine 6G (3), fluorescence quenching was detected for concentrations in the range from 10−3 to 10−4 M. Interestingly, no quenching was detectable at all for the thiolated rhodamine (1). The strong fluorescence signal of the rhodamines in solution can be employed for monitoring the adsorption kinetics on the

metal surfaces and thereby quantifying the binding affinity in terms of the rate constant for adsorption (kads). We assume that the fluorescence of the adsorbed dye molecules is quenched22 and that only nonadsorbed dye molecules are detectable. A kinetic analysis indicates that the thiolated rhodamine (1) has a kads comparable to those of 5,6-carboxyrhodamine and rhodamine 6G (Supporting Information, Table S1). However, these kinetic experiments do not provide details on the functional groups involved in the adsorption process. We therefore performed DFT calculation in combination with SERS experiments. DFT Calculations. DFT calculations on 5,6-carboxyrhodamine (2) and the thiolated rhodamine (1) were performed to investigate potential sites of adsorption to metal surfaces. Figure 4 shows their calculated structures together with the molecular electrostatic potentials. The main plane of both molecules is the xanthene moiety, while the phenyl ring is perpendicular to it. The false-color map of the electrostatic potential in Figure 4 highlights regions within the molecules with positive (blue) and negative (red) potentials. The light-blue regions in the false-color map of the molecular electrostatic potential of 5,6-carboxyrhodamine (2) (Figure 4, left) comprise the two amino groups and oxygen at the xanthene ring. Binding of rhodamine to gold nanoparticles therefore typically occurs via the lone pair electrons of nitrogen (amino groups) or oxygen (xanthene ring moiety).23 Upon introduction of the two thiol groups at the xanthene ring, the sign and the spatial distribution of the molecular electrostatic potential change significantly (Figure 4, right) due to the lone pairs and the high polarizability of the sulfur atoms. Specifically, the negative molecular electrostatic potential suggests binding of the thiolated rhodamine (1) to bare gold surfaces via the thiol moieties and/or via the carboxylate at the lower phenyl ring. Bare gold and silver surfaces are present in many E


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The Journal of Physical Chemistry C experimental configurations such as metal films,24 metal tips, including those for tip-enhanced Raman spectroscopy,25 and metal colloids generated by laser ablation.26 Adsorption sites of the thiolated rhodamine (1) onto metal surfaces can be probed by vibrational spectroscopy, in particular SERS. We therefore calculated the vibrational Raman spectrum of 1 and that of 5,6carboxyrhodamine (2) as a reference without thiol groups. Figure 5 shows the characteristic fingerprint regions for both molecules. The most important bands in the Raman spectra are located in two regions: 900−1100 and 1200−1700 cm−1. The latter contains dominant, intense Raman bands due to the large electronic polarizability of the xanthene moiety. Specifically, these bands can be assigned to xanthene ring stretching, in-plane C−H stretching, xanthene ring in-plane deformation, and C−N stretching motions (Table 2). In this region, the wavenumber values for normal modes of the thiolated rhodamine (1) are significantly lower compared to the corresponding ones for 5,6-carboxyrhodamine (2) due to the relatively large mass of the sulfur atoms. In contrast, this effect is less pronounced in the region 900−1100 cm−1, which comprises less intense Raman bands (Figure 5) assigned to C− C−C bending motions of the xanthene moiety and C−O−C stretchings of the thiolated rhodamine (Table 2). The normal modes of the thiolated rhodamine (1) at 918 and 1020 cm−1 involve sulfur atoms (Table 2) and are therefore particularly promising candidates for examining the adsorption of 1 onto metal surfaces by SERS. Although the Raman intensities of these bands in the conventional Raman spectra are very small (Figure 5), their intensity can be significantly enhanced by performing SERS. SERS Spectroscopy Using Au Nanostars and AuNP Films. Figure 6 shows pH-dependent SERS spectra recorded for the rhodamine derivatives 1 and 2 using Au nanostars27 as the colloidal SERS substrate. Most notably, the relatively strong Raman band at ca. 1200 cm−1 is present in the SERS spectra of both molecules at all pH values. A direct comparison of the DFT-calculated Raman spectra (gas phase, harmonic approximation, no metal) in Figure 5 with experimental SERS spectra (condensed phase, molecule−metal interaction, anharmonicity) in Figure 6 should be performed with caution. Weak bands in the conventional Raman spectrum can be significantly enhanced in the SERS spectrum due to the SERS selection rules and charge transfer processes.28 The following assignment of the experimental SERS spectra is based on our DFT calculations (Figure 5 and Table 2) and assignments made in previous work.29 In the wavenumber region around the pHindependent Raman band at ca. 1200 cm−1 in the experimental SERS spectra (Figure 6), several vibrational modes occur. The most dominant Raman band in the DFT-calculated normal Raman spectra of both molecules (Figure 5) is a combination of phenyl ring breathing and xanthene stretching (Table 2, 1232 cm−1 for 2 and 1226 cm−1 for 1). We therefore conclude that this Raman peak is pH-independent because it involves both the phenyl and xanthene rings. In the following, we focus on the identification of enhanced Raman marker bands in the experimental SERS spectra which are indicative of the corresponding adsorption sites. The SERS spectrum of 5,6-carboxyrhodamine (2) recorded under acidic conditions (pH 1, Figure 6, top right) is dominated by intense Raman bands in the region of ca. 1200−1650 cm−1. On the basis of our DFT calculation, we assign these bands to vibrational modes from the xanthene ring and NH groups (see Table 2). Therefore, we conclude that the xanthene ring moiety

is the dominant adsorption site at acidic pH. Upon switching from acidic to basic conditions, significant spectral changes are observed: the SERS spectrum of 5,6-carboxyrhodamine (2) recorded at pH 14 (Figure 6, bottom right) exhibits several additional Raman bands at ca. 1335, 1415, 1473, and 1593 cm−1. On the basis of our DFT calculation, we assign these bands to the phenyl ring (Table 2, 1340 cm−1) and the xanthene ring (Table 2, 1581 cm−1). We cannot conclusively assign the two Raman bands at 1415 and 1473 cm−1 since the calculated normal Raman spectrum of 2 exhibits only one vibrational mode in the wavenumber region of 1400−1480 cm−1 (Table 2, 1456 cm−1, xanthene ring) and other candidates, in particular COO− groups, are not included the DFT calculation (protonated form, COOH). The appearance of a strong Raman band from the phenyl ring at 1335 cm−1 at basic pH suggests that in this case adsorption via the COO− group of the phenyl ring occurs. The SERS spectrum of 5,6carboxyrhodamine (2) recorded at pH 6 (Figure 6, middle right) is dominated by the pH-independent Raman band at ca. 1196 cm−1 and the CC stretching band of the xanthene ring at ca. 1368 cm−1 (see Table 2). The Raman bands in the region of 1400−1650 cm−1 are not well resolved. This may be indicative of multiple molecular orientations on the metal surface. The SERS spectrum of the thiolated rhodamine (1) recorded under acidic conditions (pH 1, Figure 6, top left) exhibits the pH-independent Raman band of the xanthene ring at ca. 1203 cm−1 and several peaks of the xanthene moiety in the region of ca. 1300−1650 cm−1. At pH 6 (Figure 6, middle left), two new strong Raman bands at 938 and 1019 cm−1 together with the pH-independent Raman band at 1192 cm−1 and several bands in the region of 1300−1650 cm−1 are observed. On the basis of the DFT calculations (Table 2), the two new strong Raman bands at 938 and 1020 cm−1, which only show up in the experimental SERS spectrum at pH 6, involve the two thiols of 1: C−S−H bending of the two thiol groups coupled with xanthene C−C stretching and phenyl deformation motions (918 and 934 cm−1) as well as C−S−H bending coupled with NH rocking and xanthene C−C and C−O stretching (1022 cm−1). The Raman band at 1338 cm−1, which is also observed for 5,6-carboxyrhodamine (2) at basic pH (Figure 6, bottom right), is assigned to a phenyl mode. Therefore, we conclude that at pH 6 the thiolated rhodamine (1) adsorbs dominantly via the thiol groups, with some contributions via the COO− groups at the phenyl ring. At pH 14 (Figure 6, bottom left), the thiol-related Raman bands at 938 and 1020 cm−1 vanish, while the phenyl peak (1330 cm−1) is still detectable. The peaks in the wavenumber region of 1400−1600 cm−1 are not resolved. This may be indicative of multiple molecular orientations on the metal surface. This pH-dependent SERS experiment clearly demonstrates that the newly synthesized thiolated rhodamine (1) dominantly binds via the thiol groups to the gold surface. An accurate comparison of SERS intensities for two different dyes using metal colloids is typically problematic since both molecules may induce different extents of aggregation. As long as the exact composition of the colloid in terms of monomer and cluster percentages is not known from experiment, one should be careful with quantitative statements. For comparing the SERS signal brightness of the newly synthesized thiolated rhodamine (1) with the popular Raman dye rhodamine 6G (3), we employed a particle-on-a-film substrate for which aggregation should not be critical. Specifically, we tested silicon wafers covered with highly spherical Au nanoparticles30,31 as F


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The Journal of Physical Chemistry C Notes

SERS substrate (Figure 7). A second advantage of this approach is that the gap distances between the particles and the film are relatively homogeneous. This should yield a relatively uniform plasmonic enhancement for molecules located in the gap. Figure 8 depicts false-color maps based on the integrated Raman intensities of the marker band of the thiolated rhodamine (1) (Figure 8A) and rhodamine 6G (3) (Figure 8C) at ca. 1200 cm−1. All spectra were baseline corrected as described in the Experimental Details. The average SERS spectrum of the thiolated rhodamine (1) (Figure 8B) exhibits the same Raman bands at ca. 1020, 1200, and 1360 cm−1 as in the case of the Au nanostars (Figure 6). Again, the presence of the Raman band at 1020 cm−1 indicates the binding via the thiol groups (Table 2 and Figure 6, middle left). The false-color images in Figure 8A,C show the high spectral uniformity of the SERS signal across the films. The relatively low signal intensities in Figures 8B,D are due to the combination of a low laser power at the sample (70 μW) and a 1 s integration time per pixel. On average, rhodamine 6G (3) is about 30% brighter compared to the newly synthesized thiolated rhodamine (1).

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank the Werdelmann Foundation for financial support (Ph.D. fellowship for S.B.). We also thank F. Selbach for providing the superspherical AuNPs, M. König for help with the data processing and providing the Au nanostars, and B. Walkenfort for SEM imaging.

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CONCLUSIONS A rationally designed rhodamine dye with two thiol moieties at the xanthene ring is presented. The synthesis of 1 is based on the reduction of a bis-sulfonyl-substituted 5,6-carboxyrhodamine by PPh3/I2. The introduction of the two thiol groups at the xanthene ring does not alter the fluorescence lifetime, but leads to a reduction in the fluorescence brightness and a blue shift in the absorption and emission spectra compared with those of rhodamine 6G (3) and 5,6-carboxyrhodamine (2). DFT calculations of the molecular electrostatic potential theoretically predict the adsorption of the bisthiolated rhodamine dyes to metals via the thiol groups and/or the carboxylate of the phenyl ring. The calculated normal modes predict the presence of Raman marker bands which are indicative of surface adsorption via the thiol groups. The enhancement of these Raman bands in the experimental SERS spectrum was confirmed by two different plasmonic nanostructures: gold nanostars and a film of spherical gold nanoparticles on a silicon wafer. We expect that the thiolated rhodamine (1) should be particularly useful for (single-molecule) SERS and TERS experiments which employ bare gold surfaces, i.e., in situations where the thiol binding dominates over the electrostatic molecule−metal interactions.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jpcc.7b01504.

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REFERENCES

Synthesis of Au nanostars, superspherical Au nanoparticles, and the rhodamine dyes and NMR data (PDF)

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Corresponding Author

*E-mail: [email protected]. ORCID

Sebastian Schlücker: 0000-0003-4790-4616 G


Article

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Surface-Enhanced Raman Spectroscopy and Density Functional

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