ARTICLE pubs.acs.org/JPCC
Single-Molecule Tip-Enhanced Raman Spectroscopy Matthew D. Sonntag, Jordan M. Klingsporn, Luis K. Garibay,† John M. Roberts, Jon A. Dieringer, Tamar Seideman, Karl A. Scheidt, Lasse Jensen,‡ George C. Schatz, and Richard P. Van Duyne* Northwestern University, Department of Chemistry, 2145 Sheridan Road, Evanston, Illinois 60208, United States
bS Supporting Information ABSTRACT: An existence proof for single-molecule tipenhanced Raman spectroscopy (SMTERS) is given using the frequency domain approach involving the two isotopologues of Rhodamine 6G (R6G) that were previously employed for singlemolecule surface-enhanced Raman spectroscopy (SMSERS). A combination of experimental and theoretical studies provides a detailed view of the isotopic response of R6Gd0 and R6Gd4 in the 600 800 cm1 region. The single-molecule nature of the TERS experiment is confirmed through two lines of evidence. First, the vibrational signature of only one isotopologue at a time was observed from multiple TER spectra. Second, the spectral wandering of the 610 cm1 mode of R6Gd0 was less than (4 cm1, which in turn is less than the 10 cm1 isotopic shift so that no confusion in assignment resulted. As a consequence, the total TERS enhancement factor can now be accurately established as EFTERS = 1.0 1013 because only one molecule at a time is measured. Furthermore, EFTERS can be partitioned into an electromagnetic contribution of 106 and a molecule-localized resonance Raman contribution of 107.
and new modes were observed whose origin was not identified.18 Additionally, it has been pointed out that small sample sizes (∼100 events) are not sufficient to define a Poisson distribution.19 Other reports pointing toward SMTERS have presented analyses of intensity fluctuations without involving Poissonian statistics but cite discrete signal loss events, a broad intensity distribution, and fluctuations in the peak location.6,20,21 A slightly different method, fishing mode TERS (FM-TERS), has recently claimed single-molecule observation based on changes in line shape and peak location that were correlated with jumps in conductance through the tipsample junction.21 However, these fluctuations of intensity and peak location, by themselves, do not rigorously demonstrate single-molecule behavior.22 With respect to TERS, the enhanced field in the tipsample junction depends sensitively on the position of the molecule relative to the tip apex, and such variations would make quantization of intensities impossible and fluctuations in intensity common.23 The most convincing demonstration of SMTERS was reported in ultrahigh vacuum (UHV) by imaging single molecules with a STM before collecting their Raman spectra.24 Under ambient conditions, the water meniscus formed between the tip and sample makes molecular resolution impossible due to surface diffusion. In our judgment, rigorous validation of SMTERS in ambient requires an alternative approach. The approach we are examining here to establish SMTERS unambiguously is an adaption of the bianalyte or isotopologue
1. INTRODUCTION Since its development just over 10 years ago, tip-enhanced Raman spectroscopy (TERS) has emerged as a promising approach to obtain chemical information on the nanometer length scale.13 Excitation of the localized surface plasmon resonance (LSPR) of a Ag or Au tip under side illumination leads to both field localization on the order of tens of nanometers and Raman enhancement in the 106 to 109 range.46 These attributes allow TERS to overcome the diffraction limited spatial resolution and low sensitivity of normal Raman spectroscopy. The promise of TERS lies in its ability to provide a combination of spectroscopic and spatially resolved chemical information from molecules on a surface. Recently, TERS has been successfully demonstrated in a variety of areas of nanoscale analysis including quantum dots,7 carbon nanotubes,810 DNA bases,1113 viruses,14 and so on. TERS not only provides an excellent tool for nanoscale vibrational spectroscopy but also allows for more fundamental research into increasing its sensitivity to the ultimate level single-molecule detection. Here we demonstrate single-molecule TERS (SMTERS) by using the isotopologue approach to verify unambiguously single-molecule detection via frequency rather than intensity fluctuations.1517 To date, several papers have suggested that SMTERS is plausible based on a statistical analysis of the fluctuations in both peak intensity and spectral position. One interpretation of the fluctuations in peak intensity suggested that the signal was indicative of a Poisson distribution of intensities, with the isolated peaks corresponding to the number of molecules probed.5 However, the Raman spectra presented differ from previous literature reports, r 2011 American Chemical Society
Received: October 17, 2011 Revised: November 18, 2011 Published: December 09, 2011 478
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technique introduced to prove the existence of single-molecule surface-enhanced Raman spectroscopy (SMSERS).19,25,26 The bianalyte method involves the competition of two different analyte molecules for the adsorption site (viz. hot spot) that generates SMSERS. As a consequence, the interpretation of bianalyte data must contend with the different Raman cross sections, electronic absorption spectra, and surface binding affinities of the analytes. Such complications are eliminated by using two isotopologues as the analytes. Application of the isotopologue approach to SMTERS relies on the ability to identify uniquely each isotopologue by distinct vibrational bands. To accomplish this, the isotope shift in the selected vibrational band must be larger than the frequency excursions caused by spectral wandering. Under ambient conditions, a meniscus of water forms between the tip and underlying surface, allowing for transport of molecules into and out of the enhancing region of the tip. With sufficiently low surface concentrations of both molecules, it is possible to observe only one molecule at a time moving through the electromagnetic hot spot defined by the tipsample junction. As the surface concentration of molecules decreases, the number of molecules moving through the hot spot will decrease and observation of the distinct vibrational features of each isotopologue will be possible. We characterize the vibrations of both R6Gd0 and R6Gd4 with ensemble-averaged TERS in combination with TDDFT calculations. Using this analysis, we determine which vibrations are affected by isotopic substitution and therefore allow for unambiguous identification of each isotopologue.
Figure 1. Schematic of the experimental setup consisting of a homebuilt optical microscope and a commercial STM.
Instrumentation. TER spectra were collected on a homebuilt microscope. A schematic of the optical microscope is shown in Figure 1. In brief, a 532 nm laser (Spectra Physics Excelsior, 100 mW) was fiber-coupled to the optical microscope via a single-mode optical fiber. The laser light was passed through a filter to remove Raman light generated by the fiber (MaxLine laser line 532, Semrock) and polarizer to achieve the desired p polarization. The incident light was focused onto the tip sample junction through an aspheric lens (f = 13.86 mm, NA = 0.18, Geltech Aspheric Lens) at an angle of 55 relative to the tip axis. Inelastic scattered light was collected through the same lens, filtered to remove residual laser light (RazorEdge long-pass 532, Semrock), and fiber-coupled to a 1/3-m spectrometer (SP2300, Princeton Instruments). The Raman light was dispersed by a 1200 groove/mm grating and collected on a thermoelectrically cooled CCD (PIXIS 400, Princeton Instruments). The tip approach and tunneling parameters were operated by a commercial STM system (Molecular Imaging) controlled by RHK electronics. TERS characterization of individual isotopologues was conducted on Ag films. The experimental conditions are: λex = 532 nm, incident power (Iex) = 0.50.7 mW, taq = 3 10 s, bias (V) = 50500 mV, and tunneling current (Ic) = 35 nA. Computational Modeling. The electronic structure calculations presented in this work have been performed using the NWChem program package.28 The ground-state equilibrium geometries and normal modes of R6G-d0 and R6G-d4 were determined using the B3LYP functional and a 6-311G* basis set. A scaling of the frequencies by 0.98 was introduced to account for missing anharmonicity in the simulations. To simulate the resonance Raman scattering (RRS) spectra, we used Heller’s time-dependent theory wherein29,30
2. EXPERIMENTAL SECTION Syntheses. The syntheses of both R6Gd0 and R6Gd4 are based on conditions given by Zhang27 and have been reported elsewhere.15 Standard solutions (104 to 107 M in EtOH) of R6Gd0 and R6Gd4 were created and analyzed by UVvis absorbance spectroscopy to quantify concentration. The spectrophotometer consisted of a white light source (FO Lite, World Precision Industries) fiber-coupled to a cuvette holder (CUV, Ocean Optics) with the output fiber-coupled to a visible light spectrometer (SD2000, Ocean Optics). Sample Preparation I (TERS Characterization of Isotopologues). Smooth silver films were prepared by electron beam deposition (AXXIS, Kurt J. Lesker) of 200 nm of silver at a rate of 2 Å/s onto a glass slide. These Ag films were incubated in 3 104 M ethanolic solutions of either R6Gd0 or R6Gd4 for at least 4 h and rinsed with ethanol prior to use to achieve monolayer coverage. Sample Preparation II (Low Adsorbate Coverage). Ag films prepared as described above were used in SMTERS experiments. The Ag films were incubated in an equimolar ethanolic R6Gd0 and R6Gd4 (5 107 M each, total 1 106 dye) and rinsed thoroughly with ethanol prior to use to achieve submonolayer coverage. Tip Preparation. The Ag tips used in this experiment were prepared through electrochemical etching similar to the method described by Zhang et al.6 In brief, a mixture of perchloric acid (70%, Aldrich) and ethanol in a volume ratio of 1:4 was used as the etching solution. A platinum ring with diameter 20 mm was used as the negative electrode and positioned at the surface of the etching solution. The silver wire (99.99%, Aldrich) diameter 0.25 mm was employed as the positive electrode, and a constant voltage of 1.6 V was applied. The circuit was manually disconnected after drop off of the lower part of the wire. The tips were rinsed with Milli-Q water, followed by ethanol after etching.
ααβ ¼ 479
∑n μ0nα μ0nβ i
Z ∞ 0
Æf jin ðtÞæeiðEL þ νi0 Þt Γn t dt
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Figure 2. Visible absorption spectra and structure of (A) R6G-d4 and (B) R6G-d0. No perturbation of the electronic structure is observed upon deuteration of the molecule.
where EL is the energy of the incident light, n is the electronic state, μ0n is the electronic transition dipole moment, νi0 is the vibrational energy of state |f>, and |in(t)> is the wavepacket corresponding to the time-dependent nuclear wave function of electronic state n. A homogeneous broadening, Γn, of 500 cm1, was used in all simulations. The overlap between the initial and final wavepacket was obtained analytically using the independent mode displaced harmonic oscillator (IMDHO) method. This model accounts for vibronic coupling effects, but solvent effects in the calculations were not included. The dimensionless displacements were obtained by finite differentiation of the excitedstate energy in the FranckCondon region. A solvent shift was applied to the excitation energy so as to match the experimental result.
Figure 3. Experimental ensemble TER and simulated normal Raman spectra of R6Gd4 on (A) silver film engaged and retracted (B) gasphase TDDFT analysis; R6Gd0 on (C) silver film engaged and retracted (D) gas-phase TDDFT analysis. TER spectra acquired with λex = 532 nm, taq ≈ 3 s, and Iex ≈ 330 μW. Note that the ensemble TER spectra of each individual isotopologue were taken with different tips.
discussed in what follows. For example, an additional peak appears in the R6Gd4 spectrum at ∼1350 cm1, which is not present in R6Gd0 spectrum. However, this peak is often obscured due to the presence of two other peaks in the region of 13301360 cm1. A more convenient point of contrast is the 610 cm1 mode in R6Gd0, which shifts to ∼600 cm1 in the R6Gd4 spectrum. When the tip is retracted several micrometers from the surface, no far field spectrum is observed despite longer acquisition times (1 min). An estimate of the relative enhancement factor can be achieved by assuming that the far field signal is below the noise. The laser spot size is ∼12 μm2 and (assuming monolayer coverage) the packing density is 5 1013 molecules/cm2. The localization length associated with the tip-enhanced field can be estimated by L = (2Rd)1/2 where R is the radius of curvature of the tip and d is the tipsample separation.32 Here R is ∼160 nm as estimated from SEM and d is 1 nm, leading to a field localization length of ∼18 nm. The peak intensity for the 600 cm1 mode in the near field is 80 ADU mW1 s1, whereas the peak intensity in the far field with the tip retracted is below the noise level (∼1 ADU mW1 s1), leading to a relative enhancement factor of 1.0 106. In the original SMSERS reports, the enhancement factor necessary to reach single-molecule detection was ∼1014.33,34 For Rhodamine 6G, the use of excitation at the molecular resonance (λex = 532 nm) provides a resonance Raman contribution leading to an enhancement on the order of 107.35 To reach the appropriate level of enhancement, an electromagnetic enhancement
3. RESULTS AND DISCUSSION The visible absorption spectrum as well as the structure of the isotopologues are given in Figure 2. Both the line shape and the absorbance maxima are identical, indicating that isotopic substitution does not perturb the electronic structure of the molecule. The absorbance spectra exhibit a major peak at 527 nm. Laser excitation at 532 nm provides an additional resonant enhancement. Therefore, the measurements are more rigorously termed tip-enhanced resonance Raman spectroscopy (TERRS); however, in agreement with previous literature, we will refer to this study as TERS. The ensemble-averaged TER spectra of a monolayer of R6Gd4 and R6Gd0 with both the tip engaged and retracted on a Ag film are shown in Figure 3A,C, respectively. Each isotopologue spectrum was taken on different days and thus utilized different tips, accounting for the differences in the intensity of the TER signal. The experimental spectrum of the isotopologues matches well with previous literature reports.31 Several subtle differences in both peak intensity and frequency are observed upon isotopic substitution, the most prominent of which are 480
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Figure 5. (A) Zoom of the low wavenumber region of the experimental spectra presented in Figure 4 for (a) R6Gd4, (b) both isotopologues, (c) R6Gd0, and (d) tip retracted. Spectra acquired with λex = 532 nm, taq = 10 s, and Iex ≈ 520 μW and tunneling conditions of 3 nA and 500 mV. (B) Fits of the individual data points to Lorentzian line shapes, further illustrating the frequency shift between the isotopologues. (C) Zoom of the low-wavenumber region of the simulated spectra shown in Figure 3 for (a) R6Gd4 and (b) R6Gd0.
Figure 4. (A) Time series waterfall plot of spectra taken continuously with a single tip. The false color represents signal intensity where red is highest and blue is lowest. (B) Three time slices extracted from time series waterfall plot shown in panel A, where (a) is R6Gd4, (b) is both isotopologues, (c) is R6Gd0, and (d) corresponds to retracted tip. Spectra acquired with λex = 532 nm, taq = 10 s, and Iex ≈ 520 μW and tunneling conditions of 3 nA and 500 mV.
interactions. However, the roughly 10 cm1 shift of the 610 cm1 peak upon deuteration is seen in both experiment and simulation and allows for unequivocal identification of the isotopically labeled probe molecules. Because of its proximity to other peaks, the presence or absence of the 1350 cm1 resonance is not always evident. Therefore, the vibrational resonance at 600 cm1 is used for differentiating spectra in the subsequent SMTERS measurements. Prominent changes in the vibrational modes upon isotopic substitution were analyzed by comparing experimental data to vibrational mode simulations. Analysis of the simulation results shows that vibrational resonances with energies that do not shift significantly upon isotopic substitution do not involve the motion of protons on the isolated ring of the molecule. Those resonances in the spectrum for which a frequency shift is observed do involve motion of the protons on the pendent benzene ring, as expected. Results of the ensemble TERS and simulated Raman measurements were used in the analysis of SMTERS events. Figure 4A shows a waterfall plot of the Raman scattering as a function of time. Large intensity fluctuations of the signal are evident throughout the spectrum. Fluctuations in the frequency of
of roughly 107 is necessary for single-molecule detection; however, it has been reported that single-molecule detection for resonant dye molecules can be achieved with an electromagnetic enhancement of ∼106.24 Our calculated relative enhancement factor reflects the fact that we are within the necessary total enhancement necessary for single-molecule detection. A similar calculation of the enhancement factor using the integrated peak intensities would yield a more meaningful answer. However, the lack of signal in the far field spectrum makes this difficult to accomplish and in any case would yield a higher enhancement factor. The simulated Raman spectrum of both isotopologues is presented in Figure 3B,D, where we use time-dependent density functional theory (TDDFT). Small shifts are observed between the experimental and theoretical vibrational frequencies (
