Review

pubs.acs.org/CR

Single-Molecule Chemistry with Surface- and Tip-Enhanced Raman Spectroscopy † ‡ † † † Alyssa B. Zrimsek, Naihao Chiang, Michael Mattei, Stephanie Zaleski, Michael O. McAnally, † † † † ‡ § Craig T. Chapman, Anne-Isabelle Henry, George C. Schatz, and Richard P. Van Duyne*, , , † ‡ § Department of Chemistry, Applied Physics Program, and Biomedical Engineering, Northwestern University, Evanston, Illinois 60208, United States

ABSTRACT: Single-molecule (SM) surface-enhanced Raman spectroscopy (SERS) and tip-enhanced Raman spectroscopy (TERS) have emerged as analytical techniques for characterizing molecular systems in nanoscale environments. SERS and TERS use plasmonically enhanced Raman scattering to characterize the chemical information on single molecules. Additionally, TERS can image single molecules with subnanometer spatial resolution. In this review, we cover the development and history of SERS and TERS, including the concept of SERS hot spots and the plasmonic nanostructures necessary for SM detection, the past and current methodologies for verifying SMSERS, and investigations into understanding the signal heterogeneities observed with SMSERS. Moving on to TERS, we cover tip fabrication and the physical origins of the subnanometer spatial resolution. Then, we highlight recent advances of SMSERS and TERS in fields such as electrochemistry, catalysis, and SM electronics, which all benefit from the vibrational characterization of single molecules. SMSERS and TERS provide new insights on molecular behavior that would otherwise be obscured in an ensemble- averaged measurement.

CONTENTS 3. Single-Molecule and High-Resolution TERS P 3.1. Background and Principles P 1. Introduction B 3.2. AFM and STM Tip Fabrication R 1.1. Background B 3.3. Spatial Resolution S 1.2. Scope of Review C 3.3.1. Early Development of Imaging/Environ- 2. Single-Molecule SERS C mental Control for Single-Molecule TERS S 2.1. Hot Spots and Plasmonic Nanostructures C 3.3.2. Single-Molecule TER Imaging with Sub-5 2.1.1. Enhancement Factor Distributions D nm Resolution S 2.1.2. Super-Localization of SMSERS Hot Spots E 3.3.3. Theoretical Explanations for High Spatial 2.1.3. Rational Design of SMSERS Substrates F fi Resolution T 2.2. Veri cation of SMSERS G 3.4. Single-Molecule TERS Studies U 2.2.1. Early Strategies G 3.5. Applications of Single-Molecule TERS and 2.2.2. Current Methodologies H TERS Nanoscopy V 2.2.3. Reliable Sample Preparation I 3.5.1. Electrochemical TERS V 2.3. Nonresonant SMSERS J 3.5.2. Biological Samples: Amyloid Fibrils and 2.4. Signal Fluctuations and Heterogeneity in DNA Sequencing W SMSERS J 3.5.3. Low-Dimensional Materials W 2.4.1. Blinking J 3.5.4. TERS Investigations of Single-Molecule 2.4.2. Spectral Wandering K Electronics X 2.4.3. Distribution in the Anti-Stokes-to-Stokes 4. Conclusion and Outlook X Scattering Ratio L Author Information Y 2.4.4. Pressure-Induced Blue Shift of Vibra- Corresponding Author Y tional Modes M ORCID Y 2.4.5. Excitation-Wavelength Dependence of Notes Y SMSERS M Biographies Y 2.4.6. Summary N 2.5. Chemistry at the Single-Molecule Limit N 2.5.1. Electrochemical SMSERS N Special Issue: Super-Resolution and Single-Molecule Imaging 2.5.2. Observing Catalytic Reactions O Received: August 15, 2016 2.5.3. Additional Applications of SMSERS O

© XXXX American Chemical Society A DOI: 10.1021/acs.chemrev.6b00552 Chem. Rev. XXXX, XXX, XXX−XXX Chemical Reviews Review

Acknowledgments Z visualized in Figure 1A. The Stokes and anti-Stokes scattering are References Z symmetric about the Rayleigh (i.e., elastic) scattering (v =0tov =

1. INTRODUCTION Molecular systems and nanoscale environments are neither static nor simple. Ensemble averaging obscures the vast complexity and heterogeneity of all accessible molecular microstates. Important information with regard to site-specific behavior, distributions in molecular dynamics and interactions, and chemistry cannot be obtained on the ensemble level. Over the past several decades, several analytical techniques have been used to overcome the effects of ensemble averaging, including single-molecule − fluorescence spectroscopies,1 5 scanning probe microscopies − (SPM),6 8 and force spectroscopies, such as magnetic and − optical tweezers.9 11 While many of these single-molecule (SM) techniques can provide information about the molecular electronic states, surface topography, or even the behavior of a single molecule under stretching or torsional mechanical force, most are limited by their lack of chemical specificity. Unlike the aforementioned techniques, Raman spectroscopy can access the chemical content of a molecular system through the observation of molecular vibrations. As such, Raman scattering provides great chemical specificity and yields what is often referred to as a chemical or vibrational “fingerprint”. In 1997, Nie and Emory12 and Kneipp et al.13 both claimed SM detection of resonant dyes by surface-enhanced Raman spectroscopy (SERS). These first observations of single-molecule SERS (SMSERS) opened new possibilities toward obtaining the vibrational spectrum of a single molecule. SMSERS is ideal for improving the fundamental understanding of SERS mechanisms and probing single- Figure 1. (A) Energy diagram showing the spectroscopic transitions molecule chemistries. involved in Rayleigh, Raman, and resonance Raman scattering. Two Despite the chemical sensitivity of SERS, it cannot, by itself, pathways can be found in Raman: either the scattered photon has a provide subnanometer spatial resolution. Overcoming the lower energy than the adsorbed photon (Stokes scattering) or, inversely, diffraction limit of light and achieving simultaneous subnan- the scattered photon has a higher energy than the scattered photon ometer spatial resolution with SERS is a major challenge for (anti-Stokes scattering). (B) Schematic drawing (not to scale) of a investigating complex molecular systems and their environments plasmonic nanosphere when the electric field component of incident at the SM level. SPM is commonly used to study surface-bound light induces an oscillation of the electron cloud at a frequency defined single molecules with atomic resolution,14 but SPM alone cannot by the nanosphere size and shape, known as the localized surface capture detailed chemical information such as adsorbate− plasmon resonance. adsorbate interactions and adsorbate−surface interactions except for few-atom molecules using inelastic electron tunneling spectroscopy at few to milli-kelvin temperatures.15 In contrast, 0). Under equilibrium conditions, the intensity of the anti-Stokes tip-enhanced Raman spectroscopy (TERS) combines the normal Raman scattering is related to the populations of excited imaging ability of SPM, achieving subnanometer spatial vibrational states, which follow a Boltzmann distribution. resolution, along with the single-molecule chemical sensitivity Raman spectroscopy provides detailed chemical and structural − ffi provided by SERS.16 21 Both SMSERS and TERS are ideal information on molecular systems. The e ciency of the inelastic approaches for obtaining deeper insight into the mechanisms of scattering process, however, is very low. To enhance the inelastic SERS, the behaviors of single molecules on surfaces and in scattering probability, resonance Raman (RR) scattering occurs plasmonic cavities, and chemistries obscured by ensemble when the incident laser is near an electronic transition of the molecule of interest, increasing the signal by an additional factor averaging. − of 102−106.22 25 1.1. Background To achieve single-molecule detection with Raman spectros- Raman spectroscopy is a method for studying the inelastic copy requires the large signal enhancements of SERS. The two scattering of light. An excitation photon induces a dipole in the main mechanisms of signal enhancement are the electromagnetic molecule, yielding inelastic light scattering. Stokes scattering (EM) mechanism and the chemical enhancement (CE) occurs when the scattered photon is lower in energy than the mechanism. Enhancement factors (EF) are used to quantify incident photon by a quantum (or more) of vibrational energy the signal enhancement and are generally defined as the ratio of (positive Raman shift relative to excitation), leaving the molecule the normalized SERS signal over the normal Raman signal of the in the first vibrational state (v =0tov = 1). Anti-Stokes scattering same molecule, as follows occurs when the scattered photon is higher in energy than the INSERS/ SERS incident photon (negative Raman shift relative to excitation), EFSERS = leaving the molecule in the ground state (v =1tov = 0), as INNRS/ NRS (1)

B DOI: 10.1021/acs.chemrev.6b00552 Chem. Rev. XXXX, XXX, XXX−XXX Chemical Reviews Review

Figure 2. (A) High-resolution TEM image of a SMSERS-active nanoparticle dimer with correlated Raman scattering in panel B. The optical properties of the nanoparticle aggregated in panel A obtained from 3D calculations using DDA are provided in panels C and D. (C) Extinction efficiency (blue | |4 | |4 trace) and maximum SERS enhancement (red trace) for each wavelength calculated as Eloc / E0 . (D) Contour plot of the enhancement. The maximum enhancement value is 3.9 × 108 at 532 nm in the crevice sites of the nanoparticle dimer junction. Adapted with permission from ref 26. Copyright 2008 American Chemical Society. where ISERS and INRS are the intensities of the SERS and normal 1.2. Scope of Review Raman spectroscopy (NRS) signals, respectively, and NSERS and In this review, we describe progress in SMSERS and TERS NNRS are the number of molecules contributing to the SERS and toward the ultimate goal of understanding the fundamental NRS signals, respectively. behavior of single molecules on surfaces with high chemical The EM mechanism of SERS relies on a phenomenon known sensitivity and spatial resolution. The first half of this review will as the localized surface plasmon resonance (LSPR). The LSPR is focus on SMSERS, with an emphasis on the development and the collective oscillation of surface conduction electrons of understanding of the phenomena underlying SMSERS, which includes hot spots and plasmonic substrate design, signal noble-metal plasmonic nanoparticles that are smaller in size fl compared to the wavelength of incoming light. A physically uctuations and heterogeneity, and verifying SM detection. Then, we will highlight the application of SMSERS toward intuitive diagram of the LSPR is shown in Figure 1B. The LSPR fi studying chemistry at the single-molecule limit. The second half creates regions of intense EM eld about a nanoparticle surface of this review will focus on the current imaging capability of − or tip sample junction, known as hot spots, which can produce TERS and its applications to various SM studies, which include 26−31 EFs ranging from 105 to 1010. The EM mechanism SM electronics and isomerization processes. To orient the reader combined with RR is the most common strategy used to achieve and provide an accurate account of the field, selected TERS single-molecule sensitivity with SERS/TERS or, more accurately, experiments that were performed on low-dimensional materials surface/tip-enhanced resonance Raman spectroscopy (SERRS/ and biological samples are described, as they show comparable TERRS). For simplicity, however, we will use the terms SERS/ lateral resolution to SM systems. For a thorough coverage of the fi TERS throughout the remainder of this review. eld of SMSERS and TERS, we direct the reader to other relevant reviews on SERS and TERS EFs,38,39 characterizing hot The CE mechanism is typically the smallest contributor to the − spots,27 and TERS setups and applications,40 42 including signal enhancement, with EFs on the order of 10−100. The CE is 43 − catalysis. postulated to arise from two main processes:32 37 (1) enhance- ments from charge transfer resonances between the molecule 2. SINGLE-MOLECULE SERS and the SERS substrate33 and (2) nonresonant changes in the molecular polarizability35,36 upon surface binding. Combining 2.1. Hot Spots and Plasmonic Nanostructures the chemical contribution with EM enhancement is a strategy for After the first demonstrations of SMSERS, early studies set out to boosting the signal of nonresonant molecules for SMSERS. understand the specific nanoparticle structures giving rise to SM Additional background relevant to SMSERS and TERS will be detection, including the connection with SERS hot spots. These provided as necessary throughout this review. investigations focused on the most commonly used SMSERS

C DOI: 10.1021/acs.chemrev.6b00552 Chem. Rev. XXXX, XXX, XXX−XXX Chemical Reviews Review substrates: salt-aggregated Ag nanoparticles (e.g., Lee and Meisel important consideration for fundamental and analytical SERS Ag colloids).12,23,26,44 Nie and Emory collected atomic force studies and quantifying EFs. To investigate this further, Fang and microscopy (AFM) images of particles resulting in SMSERS, but co-workers54 used photochemical hole burning on a Ag film over it was unclear whether aggregation of the nanoparticles was nanosphere (AgFON) SERS substrate to compare the necessary or if a single “hot” particle was trapped within an percentage of molecules contributing to the SERS signal with aggregate.12 A subsequent study correlated SMSERS-active respect to the contributions from the EM enhancement. nanoparticles with AFM and found that all of the SERS-active Essentially, laser pulses were used to photochemically damage nanoparticles were aggregates,44 supporting the importance of molecules adsorbed to the AgFON. Molecules in the highest aggregation to obtain SMSERS detection. AFM correlation, enhancing regions (i.e., experiencing the highest EFs) are however, provided only limited structural information for photodamaged first, resulting in a loss of signal. As the electric elucidating the nature of the SMSERS hot spots. fields created by the laser pulses are increased, molecules in Camden et al. completed the first comprehensive character- increasingly lower enhancing regions will be subsequently ization of hot spots using a correlated experimental and photodamaged, leading to further decreases in the SERS signal. theoretical approach. After locating SMSERS-active nanoparticle By monitoring the signal loss with respect to the fields created by aggregates, they conducted high-resolution transmission elec- the laser pulses, they found that ∼25% of the SERS signal is tron microscopy (HRTEM) to image the aggregate structures, generated by less than 0.01% of the molecules on the substrate followed by theoretical calculations to model the EM field surface, which were located in the highest enhancing regions of enhancements of the aforementioned structures.26 Figure 2 up to 109. This study highlights the importance of molecular shows correlated (A) HRTEM images, (B) SMSERS spectrum, location in SMSERS experiments, but it was focused on EF and (C and D) discrete dipole approximation (DDA) distributions across the entire SERS substrate with ensemble fi | |4 | calculations of the EM eld enhancement calculated as Eloc / coverage. For SMSERS, a molecule is only detectable when |4 E0 . The DDA calculations indicate that the dimer structure in located in a hot spot; thus, it is important to consider the EF panel A can provide enhancements of 108 at 532 nm excitation in distribution within an individual hot spot. the crevice sites of the nanoparticle dimer junction (i.e., hot Interrogation of individual hot spots was performed by Le Ru spots). These results have been corroborated in multiple and co-workers for the hot spot created by a nanoparticle gap, 26,27,31 studies. theoretically55 and experimentally.38 In both studies, they found It is important to remember that all of these studies were the EF probability distribution of the nanoparticle gaps followed performed on electronic resonance with the molecule of interest. a long-tail distribution with an average EM enhancement of ∼107 25 Rhodamine 6G (R6G), for example, has a RR cross section and a maximum as high as ∼1010−1011.56,57 EFs as low as ∼107− integrated over all observable modes (514 nm excitation) of 2.3 108 were found to be sufficient to observe the SER scattering of a −22 2 −1 × 10 cm ·molecule compared with a nonresonant molecule single resonant molecule, but nonresonant molecules require −28 −30 2 −1 at ∼10 −10 cm ·molecule . Assuming the total enhance- higher EFs (∼109−1011), as visualized in Figure 3. These EF 6 ment is a multiplication of the RR contributions (up to 10 for values are assuming typical experimental conditions with no 8 R6G) and the EM enhancement (10 ), these values are in good additional contributions from chemical enhancements. 14 agreement with the total EFs of 10 , claimed in earlier An important take-away of these studies is that the calculated 12,13 studies. EF is critically affected by molecular location with respect to the Hot spots in plasmonic structures are not exclusive to hot spots of a SERS-active substrate. A difference of only 2 nm in nanoparticle junctions. The EM enhancement of individual the location of the molecule can result in an order of magnitude nanoparticles can be increased with the introduction of sharp features. For example, EFs at the corners of nanobars31 have been calculated to reach as high as 105, the tips of nanopyramids45 as high as 108, and the sharp tips of Au nanostars46 as high as ∼109− 1010. We can therefore define hot spots as local areas of intense (105−1010)EMfield occurring at sharp edges, nanoparticle gaps and crevices, or other geometries with a sharp nanoroughness (typically <10 nm). A demonstration of SM sensitivity from individual nano- particles was performed with SERS substrates fabricated by − nanosphere lithography (NSL).46 50 The NSL substrates consisted of an array of nanopyramids with an average nanoparticle distance of 83 ± 20 nm and sharp tips (Figure 6B).51 The large distances observed between the nanoparticles indicate that the SM sensitivity is the result of individual nanopyramids and not nanoparticle junctions or nanogaps (i.e., gaps <10 nm). The highest enhancements of the nanopyramids have been shown with DDA calculations to occur at the tip and Figure 3. A typical long-tail probability distribution for the SERS − can be as high as 108.47,51 53 While the understanding of the enhancement factor [p(F)] for a SMSERS-active substrate. Molecules with differential Raman scattering cross sections of ∼10−27 cm2·sr−1· plasmonic structures that are SMSERS-active has improved, − molecule 1 are observable as single molecules with EFs ∼ 108 under challenges in the rational design and reproducibility of SERS typical experimental conditions, while nonresonant molecules with substrates still remain and will be addressed in section 2.1.3. differential Raman scattering cross sections ≈10−30 cm2·sr−1·molecule−1 2.1.1. Enhancement Factor Distributions. Enhancement require typical enhancements of ∼1011, at the high end of the factors are not uniform across a SERS substrate or even within an distribution. Adapted with permission from ref 56. Copyright 2009 individual hot spot. This implies that molecular positioning is an American Chemical Society.

D DOI: 10.1021/acs.chemrev.6b00552 Chem. Rev. XXXX, XXX, XXX−XXX Chemical Reviews Review change in the EF, which can ultimately dictate whether a aggregates, later work by Willets et al. combined super- molecule is detected or not. It is also difficult to distinguish localization SERS with correlated scanning electron microscopy between multiple molecules experiencing a weaker EF and a (SEM) nanoparticle structure measurements.66 The authors single molecule experiencing a higher EF. The large intensity reported an excellent agreement between hot spot junctions of distributions observed for SM events spanning as high as 3 orders Ag nanoparticle aggregates and the SERS centroid positions of magnitude for R6G and just over 2 orders of magnitude for when the centroid positions of the R6G SERS signal were crystal violet (CV) at 532 nm excitation58 are a consequence of overlaid on SEM images (Figure 4).68 Interestingly, the spatial the EF distribution across a SERS substrate. Thus, molecular binding affinity is an important consideration when using two analytes to verify SM detection, as we will discuss in section 2.2. All of these findings imply that molecular binding affinity and positioning of molecules within a hot spot are important considerations for fundamental and analytical SERS studies and quantifying EFs. A second consideration is the influence of enhancement gradients in the hot spot on the observed molecular behaviors. For example, molecular diffusion across EM fields on the nanoparticle surface has been proposed as a mechanism for the origin of blinking (i.e., the on−off cycling of SMSERS − signal).59 61 A wavelength-dependent near-field enhancement has also been demonstrated to strongly contribute to the anti- Stokes-to-Stokes intensity ratios of SMs.62 Accordingly, under- standing the role of molecular coupling with variable optical near fields is essential for accurately describing molecular behaviors and should be pursued in future theoretical models. 2.1.2. Super-Localization of SMSERS Hot Spots. Spatially resolving the location of a single molecule on a nanoparticle would aid in understanding the coupling between a single molecule and the optical fields of a nanoparticle and how the Figure 4. (A) Rhodamine 6G SERS signal intensity spatial map and (B) location and density of nanoparticle hot spots affect the overall SEM image of the Ag colloidal trimer data from which panel A was optical response of the molecule. Obtaining nanometer scale collected. (C) Overlay of panels A and B. Adapted with permission from spatial resolution, however, from optical spectroscopies like ref 68. Copyright 2011 American Chemical Society. SERS is restricted by the diffraction limit, or approximately half of the emission wavelength (∼250 nm with 532 nm excitation). origin of the SERS centroids suggests that the molecule is One means of achieving subdiffraction-limited resolution is to fit confined to the surface of a single nanoparticle within an the diffraction-limited emission from a single emitter, such as a aggregate system. Using the isotopologue approach for SMSERS noble-metal nanoparticle aggregate labeled with a SERS-active can further illuminate the behavior of distinct molecules on a probe, using super-resolution imaging techniques. Super- nanoparticle surface: Weber, Willets, and their co-workers used resolution fluorescence techniques, including photoactivated this approach to prove that observed shifts in the centroid localization microscopy (PALM), stochastic optical reconstruc- position from previous work were due to movement of R6G tion microscopy (STORM), and stimulated emission depletion molecules on the surface and not molecular reorientation or (STED) microscopy, have demonstrated sub-20-nm resolution random nanoparticle emission.69 Using super-localization SERS of fluorescent molecules in biologically relevant systems.63,64 to watch the centroid positions of distinct molecules on a single Willets et al. first combined principles of super-resolution nanoparticle aggregate with sub-5-nm spatial precision lends fluorescence microscopy with SERS, coining the term super- itself to exploring the nanoscale reactivity of nanoparticles, which localization SERS,65,66 where they imaged the spatial profile of has been applied to studying the site specificity of electro- R6G SMSERS signal on single Ag colloidal nanoparticle chemical reactions70,71 and will be discussed in section 2.5.1. aggregates with sub-15-nm spatial precision.67 The position of Super-localization SERS is an extremely promising means of the R6G SERS emission was determined by fitting the signal to a imaging single molecules on nanoparticle surfaces with sub-5-nm two-dimensional Gaussian point spread function (PSF), with the spatial precision. However, there are challenges with regard to peak of the fitdefined as the centroid position. The emission of accurately modeling the position of a SERS-active emitter that the R6G molecule can be readily separated from any background must be addressed in order to interpret the results.72 As emission, potentially originating from the citrate molecules described above, the position of an emitter is most commonly capping the Ag colloidal nanoparticles. The authors found that modeled with a two-dimensional (2D) Gaussian PSF, where the the spread of centroid positions of the SMSERS-active regions on emitter position is approximated to be the peak position of the fit. a single nanoparticle aggregate (20−100 nm) was much greater Despite its utility in determining centroid positions, the 2D than that of the expected nanoparticle aggregate hot spot Gaussian PSF simply models the symmetric shape of a junction size (1−10 nm). They concluded that the larger diffraction-limited spot and is not an accurate physical SMSERS active area was caused by the R6G molecule being representation of the emitter, especially in the case of a SERS- located between multiple hot spots, greatly extending the active molecule adsorbed on a nanoparticle surface. Additionally, SMSERS-active area on the nanoparticle aggregate. experimental conditions such as the microscope objective used In order to precisely elucidate and understand the spatial can induce aberrations in the emission and therefore skew the origin of the SERS centroid positions on single Ag nanoparticle calculated centroid positions.73 Accurately modeling molecule−

E DOI: 10.1021/acs.chemrev.6b00552 Chem. Rev. XXXX, XXX, XXX−XXX Chemical Reviews Review plasmon coupling is not trivial; one must take into account that and costly. The multistep, top-down fabrication requires electron higher-order aggregates (e.g., dimers, trimers, tetramers) or beam lithography and alignment with nanoscale precision. On elongated nanoparticles such as nanorods have multiple plasmon the other hand, NSL used to fabricate arrays of nanopyramids is a modes. Alternative PSFs have been explored and compared to facile, cost-effective, and easily tunable approach to prepare the standard 2D Gaussian PSF in an attempt to model the nanoparticles. However, due to the simplicity of NSL, nano- complexity of plasmon−molecule coupling and the resulting particles are subject to packing defects due to small variations in − SERS emission spatial profile. For example, PSFs derived from a nanosphere size.47 49,52,53 In NSL, nanospheres are dried into a 2D centroid/single dipole and an intensity-weighted sum of close-packed monolayer on glass coverslips, onto which a layer of three orthogonal dipoles were both used to model the centroid metal is deposited. Removal of the nanospheres leaves behind an positions of SERS emission on Ag colloidal nanoparticles in the array of nanopyramids created in the interstitial sites of the close- 74 many- and single-molecule regimes. As shown in Figure 5, the packed monolayer (Figure 6B). Outside of NSL and nano- antenna fabrication, other efforts have focused on improving the reproducibility of chemically synthesized Ag nanoparticles. One common strategy is to use DNA self-assembly and other linking molecules77 to form controlled nanoparticle dimers or larger aggregates.78,79 Another strategy to improve reproducibility is gap-mode SERS (GM-SERS), a simple nanoparticle-based analog to TERS (section 3). In GM-SERS, the analyte of interest is adsorbed onto a smooth metallic thin film, upon which nanoparticles are deposited. The molecules of interest are consequently located in the film−nanoparticle gap, as shown in Figure 6D.81 The benefit of GM-SERS is that it does not require the random aggregation of nanoparticles, which improves substrate reproducibility, assuming the nanoparticles are monodisperse in size. An additional advantage of GM-SERS is the possibility to observe Figure 5. AFM images of Ag colloidal nanoparticle aggregates and SERS fi fi spatial intensity maps that indicate the average intensity of all fitted site-speci cchemistryonthin lm surfaces that are not 84−86 centroids. (A and D) AFM images with the three-dipole φ-fit estimate inherently plasmonic. GM-SERS has recently been used (green dashed line) with the same scale bar. (B and E) SERS spatial to study single-molecule host−guest interactions, which we intensity maps using the 2D Gaussian PSF model. (C and F) Spatial discuss in detail in section 2.5.3. intensity maps using the three-dipole model. A white x on panels C and Increasing the hot spot density of SERS substrates is also an F indicates the average position of the 2D Gaussian PSF. Panels A−C − important parameter in substrate design. For aggregated Ag shows an example of single-molecule coverage, and panels D F shows colloids, typically only 1% of the nanoparticles are SMSERS- many-molecule coverage. The scale bars in panels B, C, E, and F 26 represent 10 nm. Adapted with permission from ref 74. Copyright 2013 active, making the collection of statistics in SMSERS studies American Chemical Society. challenging. Attempts to improve hot spot density include fabricating close-packed monolayers of Ag83 (Figure 6F) and Au87 nanoparticles, templated Ag nanocube arrays,88 and fits of the 2D Gaussian and three-dipole-derived PSF have stark synthesizing porous Au−Ag nanospheres80 (Figure 6C). Porous differences in the single-molecule regime, whereas the high Au−Ag nanoparticles, for example, create a high density of hot molecule coverage is very similar. Overall, the three-dipole PSF spots across the nanoparticle surface through the introduction of was the most successful in providing a precise fit, albeit more computationally demanding than the other fits. This study nanoscale features. The single particles were reported to have EFs on the order of ∼107 compared with “smooth” single, highlights that the choice of PSF model will alter the spatial ∼ 4 28,80,89−91 profile of the emission centroid and that performing correlated spherical nanoparticles at 10 . structural measurements is crucial to gain a complete under- Another major challenge is the controllable positioning of standing of how molecule−plasmon interactions effect the molecules within the plasmonic hot spot. One strategy fi addressing this challenge was the fabrication of cethylrimethy- observed emission centroid pro le. Improving the ability to 82 accurately model SERS-active molecule interactions with lamonium bromide (CTAB)-coated bipyramids (Figure 6E). plasmonic nanoparticle systems could help extend super- The CTAB bilayer prevents the adsorption of positively charged localization SERS to studying chemical reactions at the few- to dyes (crystal violet in this study) to the nanoparticle surface single-molecule level with sub-5-nm spatial precision. except on the tip, where CTAB was not present. In principle, this 2.1.3. Rational Design of SMSERS Substrates. design would only allow the binding of molecules to the regions The design 82 of new substrates for SMSERS has focused primarily on of highest enhancement. They observed that every crystal improving the reproducibility of nanoparticle size, shape, and violet molecule experienced an EF close to the maximum value degree of aggregation, increasing hot spot density, and expected for the bipyramid tips, suggesting the success of this controlling the positioning of molecules into a hot spot. strategy. This bilayer approach, however, is not necessarily Lithographically prepared substrates such as nanoantennas75,76 adaptable to gap-containing nanoparticles and is highly depend- (Figure 6A) and nanopyramid arrays (Figure 6B)51 are one ent on the analyte’s adsorption affinity. More recently, a avenue to improve substrate reproducibility. Nanoantennas have plasmonic substrate was designed to reversibly trap single been reported to have EFs of up to ∼1013 due to the combination molecules in hot spots.92 The substrate was fabricated through of an optimized local EM field enhancement (1011) and antenna the electrostatic self-assembly of Au nanoparticles onto a Au- and directionality (102),75,76 with excellent control over hot spot silica-coated silicon platform. The hot spots were isolated by positioning. However, nanoantenna fabrication is complicated using a thermoresponsive polymer, which acts as a gate for

F DOI: 10.1021/acs.chemrev.6b00552 Chem. Rev. XXXX, XXX, XXX−XXX Chemical Reviews Review

Figure 6. (A) Lithographically prepared nanoantennas with directional Raman scattering,76 (B) a tunable nanopyramid array fabricated by nanosphere lithography,51 (C) porous Au−Ag nanospheres with increased hot spot density due to the introduction of nanoroughness,80 (D) schematic of a GM- SERS experiment with Au nanoparticles located over a Au film,81 (E) TEM image of CTAB-stabilized bipyramids,82 and (F) a close-packed monolayer of Ag nanospheres on a quartz substrate with an image area of 1 × 0.7 μm2.83 The inset shows that the interparticle gap is regulated by the thiolate chain length. Adapted with permission from refs 76, 51, 80, 81, 82, and 83, respectively. Copyright 2013, 2013, 2016, 2016, 2011, and 2015, respectively, American Chemical Society. trapping the molecules through the heating and cooling of the consequence, ultralow concentrations as a justification for substrate. SMSERS are inadequate. This is further highlighted by Darby Over the past several years, great strides have been made and Le Ru, who found that the dilution procedure used to toward improving SERS substrates. The majority of SMSERS prepare samples could artificially create the appearance of experiments, however, still rely on the random aggregation of Ag SMSERS due to the competition between molecule diffusion and nanoparticles. As the field moves forward, utilizing more adsorption to the nanoparticles.95 Instead, most strategies have reproducible nanoparticle substrates will allow for the systematic employed ultralow concentrations with additional protocols to improvement of our fundamental understanding of SERS prove SMSERS ranging from the observation of fluctuations to a mechanisms and provide more reliable means of interpreting statistical analysis. chemistry at the single-molecule level. In addition, the ability to As an alternative to using ultralow concentrations to prove increase hot spot density while simultaneously controlling the SMSERS, dramatic fluctuations in the SERS signal were viewed positioning of molecules within those hot spots will greatly as validation of SM detection in many early studies. Fluctuations improve the collection of statistics for SMSERS. include spectral wandering,58,96 large relative intensity fluctua- 97 12,59,98−102 2.2. Verification of SMSERS tions of particular vibration modes, and blinking. Nie and Emory, for example, noted that the Raman signals would 2.2.1. Early Strategies. The first SMSERS studies adapted suddenly disappear or change during a period of continuous proof methodologies from SM fluorescence. SM detection by illumination.12 This blinking behavior, or the on−offfluctuation fluorescence spectroscopy is unequivocally proven through the of the SERS signal, was initially used as a verification of SM observation of photon antibunching, which is based on the detection.59,103 Andersen et al., however, demonstrated that Ag phenomenon that single molecules cannot emit two fluorescence colloids exhibit blinking that is independent of the molecule photons simultaneously.93 However, this strategy is not possible being probed and that this phenomenon was observed in both for SMSERS because the lifetime of Raman scattering is short the presence and absence of a probe molecule.104 A second study − − (<10 14 s) relative to that of fluorescence (10 9 s).94 Another observed large spectral fluctuations for an amorphous carbon strategy to confirm SM detection, also common in SM layer deposited on SERS substrates.105 Furthermore, blinking has fluorescence, is to sufficiently dilute the target molecule been observed at surface coverage above the SM level.60,96 While concentration to achieve, on average, one or fewer molecules fluctuations are a characteristic behavior of single molecules, they within the probe volume. While this is an effective strategy for do not exclude the possibility of multiple molecules or other fluorescence spectroscopy, the addition of metallic nanoparticles phenomena104,105 causing the fluctuations. Therefore, the use of complicates the process. Unequivocally proving SMSERS a fluctuation argument is inadequate evidence for SM detection. detection using only ultralow concentrations would require Another indirect strategy to verify SM detection is the knowledge of the contributions from molecular binding affinity observation of a polarization-dependent SER signal.12 This, and diffusion, molecule and nanoparticle concentrations, the however, requires the ability to deconvolute the polarization position of the molecule relative to plasmonic hot spots, and the dependence of the plasmonic structures from that of the SER number of nanoparticle hot spots within the probe volume. As a scattering of the analyte.

G DOI: 10.1021/acs.chemrev.6b00552 Chem. Rev. XXXX, XXX, XXX−XXX Chemical Reviews Review

× −7 Figure 7. Histograms of spectral counts for bianalyte SERS experiments conducted with CV-d0 and R6G-d0 in data set A at 1 10 M and data set B at 1 × 10−8 M overall concentration. Data sets C and D are analogous multianalyte experiments to data sets A and B, respectively, but with all four analytes fi (CV-d0, CV-d12, R6G-d0, and R6G-d4) summing to the same overall concentration. The columns lled with slashed lines in the multianalyte data sets show the sum of the isotopologue events within the column, displaying them as though they were a bianalyte experiment. It was determined using the isotopologue internal standards that data sets A and C are at the few-molecule level, while data sets B and D are at the SM level. Adapted with permission from ref 58. Copyright 2016 American Chemical Society.

Others have provided evidence for SM detection using a electronic resonances, and Raman scattering cross sections, while combination of ultralow analyte concentration and a statistical bianalyte partners will not have identical values of these analysis of the SERS intensities.13 At ultralow concentrations the properties. number of molecules that bind to the nanoparticle surface should The ratio of counts of individual analyte events to events with follow a Poisson distribution. The Poisson distribution assumed both analytes is used to verify SM detection. This ratio can be that the difference in the SER intensity between events could be described using a Poisson binomial distribution.58,96 At attributed to zero, one, two, or three molecules being probed. sufficiently low coverage, the probability that a molecule is However, it was demonstrated that a valid SMSERS Poisson bound to the nanoparticle aggregate follows a Poisson distribution requires the variation in intensity between SERS distribution. Once bound, the probability that the molecule events to be less than a factor of 2, and a minimum of 10 000 detected is either analyte 1 or analyte 2 follows a binomial sample events needs to be recorded,55,106 which is a highly distribution. Therefore, the overall probability that analyte 1 impractical feat for SMSERS. Furthermore, the variation in and/or analyte 2 will be detected in a given spectrum is the intensity between SM events has been shown to span as many as product of both distributions 3 orders of magnitude, considerably greater than a factor of e−α 2.55,58,96 As a consequence, the existence a Poisson distribution P(,αβnn , ,)= [][(1)] αβαβnn12− 12 !! of SERS intensities alone is not a valid proof of SM detection. nn12 (2) 2.2.2. Current Methodologies. In 2006, Le Ru et al. α where is the average number of molecules per spectrum, and n1 introduced a bianalyte approach for proving SM detection. This and n2 are the number of analyte 1 and analyte 2 molecules approach involves dosing the SERS-active substrate with two detected in the SER spectrum, respectively. P is the probability analytes at equimolar concentrations and determining whether α that n1 analyte 1 and n2 analyte 2 molecules at -coverage will be the SERS events contain a single analyte or a combination of both detected for a given spectrum. The probability that a given 107 analytes. While the bianalyte approach is a useful means of molecule detected in the spectrum corresponds to analyte 1 or proving SMSERS detection, there can be experimental problems, analyte 2 is designated with an empirical variable β. The value of such as different Raman scattering cross sections or molecular β is a function of the binding affinities, concentrations, and affinity for the SERS-active substrate.58 An alternative of the Raman cross sections of analytes 1 and 2. bianalyte approach is the isotopologue approach. The isotopo- In order to test the influence of β on the overall proof of SM logue approach uses a molecule and its isotopically edited analog detection with bianalyte pairs, a multianalyte experiment was 23,96 as a bianalyte pair (i.e., isotopologues). This approach is conducted using R6G and CV isotopologues (R6G-d0/R6G-d4 58 advantageous because isotopologues inherently have identical and CV-d0/CV-d12). This allows for the direct comparison of surface binding affinities, extinction coefficients for molecular statistics for the bianalyte pair with the corresponding

H DOI: 10.1021/acs.chemrev.6b00552 Chem. Rev. XXXX, XXX, XXX−XXX Chemical Reviews Review

Figure 8. (Top) Visual representation of two dilution methods for preparing SMSERS samples that result in drastically different molecular coverage. The large dilution factors (left) are a common approach reported for preparing SMSERS samples but result in unreliable molecular coverage. Half−half dilution (right) is the method proposed by Darby et al. to achieve uniform molecular coverage. (Bottom) The effect of different sample dilution procedures on SMSERS experiments using R6G isotopologues at 10 nM. Scatter plots of the intensity for the isotopic-sensitive R6G mode (∼600 and 610 cm−1) are shown. The samples were prepared in panel A by premixing the two dyes and then diluting them using HHD, in panel B by premixing the dyes and then diluting them using a LDF of 100×, and in panel C by adding the dyes individually with a LDF of 100×. The inset shows a schematic of the expected molecular coverage based on the dilution procedure used. Below the scatter plots are representative SERS spectra indicated by the numbers 1− 6 circled on plots A−C. Adapted with permission from ref 95. Copyright 2014 American Chemical Society. isotopologue statistics as an internal standard. Figure 7 shows thresholds for verifying SM detection.58 Appropriate implemen- two representative bianalyte data sets A and B and their tation of SM proofs will greatly advance the field of SMSERS. analogous multianalyte data sets C and D, respectively. All of the 2.2.3. Reliable Sample Preparation. Along with a rigorous data sets were collected with equimolar mixtures of R6G and CV, statistical analysis, proper sample preparation is essential to where data sets B and D were at 1 × 10−8 M and data sets A and C ensure consistent coverage of molecules on the SERS substrates. were at 1 × 10−7 M overall concentration. In both A and B, the A typical SMSERS experiment involves incubating resonant dye majority of spectra showed individual analyte character, but R6G molecules at ultralow concentrations (typically nanomolar or was observed an order of magnitude more frequently than CV, picomolar) with Ag colloidal nanoparticles. Salt solutions such as demonstrating the different detection probabilities of the NaCl or KCl are used to aggregate the colloids, creating hot spots analytes. As shown in analogous multianalyte data sets C and for EFs on the order of 107−108.12,13,23,58,95,96 Darby and Le Ru D, despite both A and B having primarily individual analyte investigated the effect of sample preparation on SMSERS character, only data set B was at the SM level. Spectra with both experiments for dye/colloidal mixtures.95 Figure 8 demonstrates isotopologues were observed more frequently than those of the two tested dilution procedures: (1) large dilution factors (LDF) individual isotopologues in data set C (analogous to data set A), and (2) half−half dilution (HHD). Due to the competition but not in data set D (analogous to data set B). These results between molecular diffusion and adsorption, LDFs lead to demonstrate that bianalyte partners require careful statistical uneven nanoparticle coverage and the appearance of SM analysis in order to rigorously prove SM detection. detection when it is not actually achieved. Premixing the dyes Recently, a SMSERS study using porphycene isotopologues and using HHDs is recommended to obtain uniform coverage. (Pc-d0 and Pc-d12) to verify SM detection observed the Figure 8A shows experimental results from samples prepared 108 preferential observation of Pc-d0 over Pc-d12. They observed using HHD. Figure 8B shows samples prepared with premixing a similar discrepancy in counts between the isotopologues as of the dyes and a 100× dilution factor. Figure 8C shows samples discussed above for bianalyte partners, showing the importance in which the dyes were added to the colloid mixture sequentially of careful analysis for every SM proof. Zrimsek et al. further with a 100× dilution factor. The overall SERS signal intensities in discussed experimental considerations for SM proofs, including A are weaker than in B and C, suggesting more uniform coverage, correcting for differences in analyte behavior through altering the as indicated in the schematic representations. Most strikingly, the analyte concentration ratios and defining more rigorous majority of events in Figure 8C show SERS spectra with strong

I DOI: 10.1021/acs.chemrev.6b00552 Chem. Rev. XXXX, XXX, XXX−XXX Chemical Reviews Review signals of only one isotopologue, when the majority of spectra fluctuations, such as blinking and spectral wandering and the should have had both analytes. This work illustrates that the heterogeneity of SMSERS events. proper dilution procedure is crucial to obtain the appropriate 2.4.1. Blinking. SMSERS signal fluctuations were originally molecular coverage and accurate verification of SM detection. used to validate SM detection. Despite the fact that this idea was Since a simple dilution procedure can significantly influence later disproven,60,96,104,105 the origin of signal fluctuations in molecular coverage and EF distributions exist across a SERS SMSERS spectra is still a highly debated topic in the SERS substrate or hot spot (discussed in section 2.1.1), all studies community. Blinking is described as the on−off cycling of the should clearly define their experimental procedures to ensure SMSERS signal and can last from milliseconds to mi- − accurate interpretation of results and reproducibility. Other nutes.59,98 102 The roles of illumination power,102 electrolyte practical considerations when preparing SMSERS samples are concentration,102 temperature,61,113 EM mechanism,114,115 and the choice of bianalyte pair for proving SM detection,58 analyte excitation wavelength60,99 have all been explored in relation to concentration, type of SERS substrate, the method of applying blinking behavior, some of which we will discuss further in this the analytes to the SERS substrate (e.g., solution-phase section. incubation,12,13,95,96 spin-coating,51 or Langmuir−Blodgett film Itoh and co-workers conducted a quantitative analysis of deposition109,110), and laser power to limit photodegradation.57 blinking in SERS and surface-enhanced fluorescence (SEF) using For example, SMSERS spectra of R6G have been observed down Ag nanoparticle dimers functionalized with R6G.114 They to 5 nW excitation.111 observed that the SERS and SEF signals varied from dimer to 2.3. Nonresonant SMSERS dimer but could be divided into three main categories: (1) stable, (2) fluctuating, and (3) intermittent. Stable refers to a SERS Achieving SMSERS of nonresonant molecules with Raman cross − − − − signal with minimal changes in intensity throughout the entire sections around 10 29−10 30 cm2·sr 1·molecule 1 requires SERS 9− 11 temporal trajectory. Fluctuating refers to a SERS signal that substrates to support EFs on the order of 10 10 . Nonresonant blinks continuously, and intermittent refers to a SERS signal that SMSERS detection has been observed for 1,2-bis(4-pyridyl)- switches between periods of being stable and blinking. ethylene (BPE) in combination with benzotriazole (BZT) dye as Correlated SER, SEF, plasmon resonance spectra, and temporal a bianalyte partner to verify SM detection.56 For BPE, EFs of trajectories of the SERS and SEF were collected. The authors ∼109 were necessary to achieve SM detection. In this study, the used intense NIR laser pulses to induce fluctuations in an authors also attempted SM detection of adenine, but it was only otherwise stable signal. These laser pulses induced a 50 nm blue possible in the rare cases when EFs reached as high as 1011. This shift in the LSPR, which coincided with the onset of the unstable study demonstrates that SMSERS detection of nonresonant SERS signal. On the basis of these results and their reproduction molecules is possible; however, the rarity of observing a SM event of the SERS and SEF signals using the EM mechanism (see ref due to the high EF requirements makes these experiments very 114 for more details on their quantitative analysis), they challenging. Therefore, in order to expand the scope of molecules proposed two insights into blinking behavior. First, an unstable that can be studied with SMSERS, it is necessary to gain adsorption of R6G to the Ag nanoparticles causes instability in additional enhancement from avenues besides molecular the SERS and SEF signals. Unstable adsorption can alter the resonance. distance between the molecule and the nanoparticle surface One strategy is to use highly enhancing SERS substrates with overtime, leading to changes in the EF that the molecule EFs of 109 or higher, such as the nanoantennas discussed in experiences and, therefore, the signal. Second, changes to the section 2.1.3. Wang and co-workers claimed that the nano- 75,76 plasmon resonance of the Ag nanoparticles can induce blinking antennas had EFs as high as ∼1013, combining an optimized because it also changes the EF. Due to these insights, they suggest local EM field enhancement (1011) and antenna directionality that the underlying cause of blinking can be attributed to (102), which would allow the detection of nonresonant single photoinduced effects, such as thermal heating of nanoparticle hot molecules. Another strategy is to take advantage of the chemical spots leading to plasmon resonance shifts, diffusion of the enhancement (CE) mechanism in addition to the EM mechanism. The CE mechanism in SERS is generally accepted molecule around the hot spot, and/or photobleaching. to contribute enhancements of 10−100 and is based on the Kitahama and co-workers analyzed the bright (on) and dark (off) states of SERS blinking for thiacyanine using a power law electronic interactions between a molecule and a plasmonic 98−100 surface. These chemical effectsincludechargetransfer analysis. Power law statistics have been used to analyze − processes32 34,112 or changes to the polarizability of a molecule long-range ordered nonexponential behavior in quantum dots − fl when it binds to a substrate surface.35 37 Theoretical calculations (QD). The power law for blinking of a single QD uorescence have also found that by tailoring the molecule with different can be explained by a distribution in the passage time required for functional groups, thereby altering the energy difference between a random walker to return to its starting point, which is the highest occupied molecular orbital (HOMO) of the metal approximated using a one-dimensional random walk model. and the lowest unoccupied molecular orbital (LUMO) of the Kitahama et al. postulate that blinking statistics in SERS will be analyte, the chemical enhancement can surpass 103.36 Utilization similar due to the random molecular walk of an adsorbed of CE in combination with EM enhancement, may help probe a molecule on the nanoparticle surface. Indeed, they observed that wider spectrum of molecular systems with SMSERS. the power law reproduced the probability distribution of the occurrence of the bright and dark states over time. Next, the 2.4. Signal Fluctuations and Heterogeneity in SMSERS authors monitored the power law exponent as they swept the One advantage of studies at the SM limit is that they provide a excitation wavelength across the nanoparticle aggregate LSPR. viable route for investigating principle mechanisms of the SERS When excited at the LSPR wavelength of the nanoparticle enhancement, the fundamental properties of spectroscopic aggregate or at high laser power, the power law exponent of the phenomena, and single molecular properties otherwise obscured bright and dark blinking events approach −1.5, similar to what is by ensemble averaging. We will discuss several recent studies expected for QD blinking. When excited off the LSPR focused on improving our fundamental understanding of signal wavelength and at lower excitation laser powers, the power law

J DOI: 10.1021/acs.chemrev.6b00552 Chem. Rev. XXXX, XXX, XXX−XXX Chemical Reviews Review deviated from −1.5. They propose that the high EM fields Additional proposed mechanisms for blinking include (1) − created when excited at the LSPR wavelength restrain the thermal diffusion in-and-out of the hot spot,59 61 (2) thermally molecule by a surface-plasmon-enhanced optical trapping stimulated molecular reorientation and chemical processes,83 (3) potential well around the nanoparticle junction. Subsequent photoionization due to charge transfer states,102 and (4) studies by Kitahama and co-workers observed that a truncated metastable and nonemissive states of the molecule.117 On the power law reproduces the probability distribution of dark SERS basis of the current studies, the overall consensus for the origin of events versus their duration time.98,99 They proposed that the blinking is thermally stimulated movement of the molecules on truncation of the power law indicates that a high-energy barrier the nanoparticle surface, likely accompanied by photoinduced must be overcome for the molecule to undergo a transition from effects such as electron transfer or other chemical processes. the nonemissive state to the emissive state and that fast random 2.4.2. Spectral Wandering. Spectral wandering is the shift molecular walking (i.e., surface diffusion into the nanoparticle in frequency or spectral position for a particular vibrational junction) helps to overcome this energy barrier. The energy mode. The isotopically sensitive phenyl band at 600 and 610 −1 barrier is suggested to originate from the coupling of multipolar cm for SM events of undeuterated R6G (R6G-d0) and its deuterated isotopologue (R6G-d4), respectively, have been surface plasmon resonances around the nanoparticle junction. 51,58,96 This research highlights the importance of considering both the observed in multiple studies, showing that the peak center ± −1 bright and dark SERS states to fully explain SERS blinking can shift within a range of 5cm from event to event. Similarly, spectral wandering was explored by Etchegoin and Le Ru for Nile behavior. 118 In addition to on−off cycling, blinking behavior includes Blue (NB) and Rhodamine 800 at 77 K. Using a high- resolution grating, the authors were able to observe inhomoge- sudden, sharp changes in the Raman peak intensities and their −1 ratios.116 For example, in CV the low-frequency region of the neous broadening of the 590 and 2226 cm modes of NB and spectra (<1100 cm−1) was shown to fluctuate more dramatically Rhodamine 800, respectively. Figure 10 shows the individual SM than the high-frequency region of the spectra.101 In R6G, it was observed that the on−off cycling of the 615 and 774 cm−1 modes would blink in tandem, while the intensity of the remaining modes was maintained.102 These fluctuations can be visualized in Figure 9, which shows the Raman intensity observed over time for R6G. Lombardi et al. proposed that this rapid blinking behavior for only select modes in SMSERS is strongly correlated to vibronic coupling and that the steadier signals, which fade in and out gradually, are governed by Franck−Condon factors.116

Figure 10. Inhomogeneous broadening observed for (A) the ∼590 cm−1 peak of NB and (B) the ∼2226 cm−1 mode of Rhodamine 800. The spectra were collected at 77 K with 633 nm excitation and a high- resolution 2400 lines/mm grating. Individual single-molecule events are shown to fall within the average signal of over 7500 spectra (black trace) for both NB and Rhodamine 800. Adapted with permission from ref 118. Copyright 2010 American Chemical Society.

events and average spectra for (A) NB and (B) Rhodamine 800. Multiple SM spectra of the ∼590 cm−1 mode for NB are shown to fit within the average of 7500 spectra collected, signifying that the average SERS signal is the average of a population of many fl molecules. As expected, the SM peaks appear within a range of Figure 9. Representative time-dependent SERS intensity uctuations of ∼ − −1 a single R6G molecule illuminated with a power of 10 W/cm2. Each row 584 596 cm with the majority of events centered at 590 −1 ∼ −1 is a color-coded spectrum based on the intensity. The on−off cycling of cm . The same trend is shown for the 2226 cm cyano bond −1 (CN) mode of Rhodamine 800. In a subsequent study, it was the 615 and 774 cm modes can be seen to blink in tandem. Adapted − with permission from ref 116. Copyright 2001 American Chemical shown that the peak shifts for the ∼2226 cm 1 mode can also be Society. attributed to natural isotopic substitutions from 12Cto13C and

K DOI: 10.1021/acs.chemrev.6b00552 Chem. Rev. XXXX, XXX, XXX−XXX Chemical Reviews Review

14Nto15N.119 These studies show that small frequency shifts observed in SMSERS spectra are generally attributed to different local environments and isotopic effects.119 Additional studies have observed homogeneous broadening120 and overtone and combination bands121 with SMSERS. 2.4.3. Distribution in the Anti-Stokes-to-Stokes Scat- tering Ratio. Broad distributions in the anti-Stokes-to-Stokes scattering ratio (aS/S) have been observed in SMSERS − studies.62,122 124 Anti-Stokes scattering depends on the population of the excited vibrational states. Increases in the anti-Stokes scattering intensity (i.e., increases in aS/S) can be attributed to a few factors, including vibrational pumping, heating, and resonance effects, that variably enhance the Stokes and anti-Stokes scattering. Several studies have focused on understanding how these effects can lead to fluctuations in the anti-Stokes scattering intensity that cannot be accounted for by thermal population of the excited vibrational states − alone.62,122 125 Vibrational pumping is the increase of the population of excited states through Stokes scattering.125 The first conclusive demonstration of vibrational pumping in SERS was done by Maher et al., who used temperature scans to separate out the contributions of resonance effects and heating from those of vibrational pumping.125 Building off this study, Galloway and co- workers, used low temperatures (77 K) and high power densities (4.48 × 108 W·m−2) to verify the effect of vibrational pumping on the aS/S ratio.124 They showed that even though both NB and CV Stokes signals were present for many events, the anti-Stokes scattering could often be observed for only NB or only CV in the Figure 11. Histogram of counts for the aS/S ratio for the 198 and 219 same, simultaneously collected spectrum. This result suggests −1 ff cm modes of (a) brilliant green and (b) crystal violet, respectively, that molecules can experience di erent contributions from plotted as a normalized κ value. The κ value is the aS/S ratio of a SM vibrational pumping. While vibrational pumping is an important divided by the average aS/S ratio of the ensemble. Adapted with consideration at cryogenic temperatures, it is not a main permission from ref 122. Copyright 2012 American Chemical Society. contributor to the aS/S ratio at room temperature, which is dominated by thermal population or resonance affects. Two subsequent studies investigated the role of local EM fields and co-workers also investigated the possibility that local heating and plasmonic heating on the aS/S ratio of SMs. dos Santos and contributed to aS/S ratio based on two assumptions:62 (1) co-workers studied the aS/S ratio for brilliant green (BG) and molecules residing in higher EM fields will give higher SERS CV shown in Figure 11.122 They used a normalized κ value, signals and (2) molecules in more intense local EM fields defined as the aS/S ratio of a SM divided by the ensemble- experience a greater extent of heating. Essentially, this means that averaged aS/S ratio. The mean κ values were 1.00 and 1.04 for higher SERS intensity would be correlated with greater heating BG and CV, respectively. The equal aS/S ratios for the SM and and a higher aS/S ratio. A positive correlation was found, ensemble regime show that the average SERS signal is the suggesting that heating is observed, but since it was only partially combination of contributions from all the individual hot spots correlated, the heterogeneous enhancement of the anti-Stokes illuminated during the SERS experiment. They also found that and Stokes scattering is believed to be the main contributor. the distribution in κ values was the same for both analytes. As a Another recent study by dos Santos et al. approaches this consequence, the variability of the aS/S ratio for different SM phenomenon from a different perspective and uses the aS/S ratio events is expected to result from nonequivalent enhancement of of single CV molecules to probe the aggregation state of the Ag the Stokes or anti-Stokes scattering. colloids.123 The experimental results were interpreted within the This conclusion was later supported by an investigation by framework of generalized Mie theory (GMT) simulations. Figure fi fi Pozzi et al., where the distribution of aS/S ratios for R6G was 12 shows the electric eld enhancement pro les (E/E0 as a found to span more than 1.5 orders of magnitude.62 Using finite- function of incident wavelength) for the hot spots of Ag difference time-domain (FDTD) simulations of a nanoparticle nanospheres 25 nm in diameter. With increasing aggregation aggregate, the theoretical EF as a function of the scattered state, the resonances are red-shifted and the maximum E/E0 photon wavelength was calculated for both crevice sites of a decreases. From these simulations, it is expected that for a Ag nanoparticle junction. In each crevice, two locations at a distance dimer at 633 nm excitation the anti-Stokes scattering would be of 1 nm were calculated (i.e., four locations total). A molecule enhanced relative to the Stokes scattering. For larger aggregates, diffusing 1 nm in the first crevice site would see a decrease in the the Stokes scattering would be preferentially enhanced. They aS/S ratio from 1.9 to 1.6 and in the second crevice site a decrease tested these predictions by using different KBr concentrations to from 0.17 to 0.09. Considering all four locations in the single influence the aggregation state of the Ag nanoparticles. Small nanoparticle junction, the ratio of the anti-Stokes-to-Stokes aggregates (i.e., dimers and trimers) dominated with [KBr] ≤ 7.5 scattering could vary by a factor as high as 21, providing further mM, and larger aggregates dominated at [KBr] > 7.5 mM. At low evidence for the role of local EM fields on the aS/S ratio. Pozzi KBr concentrations (2.5 mM), the anti-Stokes SERS signal was

L DOI: 10.1021/acs.chemrev.6b00552 Chem. Rev. XXXX, XXX, XXX−XXX Chemical Reviews Review

fi Figure 12. (A) Generalized Mie theory simulations for the local eld enhancement (E/E0) at the junctions between the nanoparticles. The colors in the plots represent different aggregation states of 25 nm radius Ag nanospheres as indicated in the schematic. (B) Stimulated SERS enhancement factor profiles as a function of the Raman shift for molecules located in the nanoparticle junctions. Solid lines indicate excitation at 633 nm and dashed lines indicate 785 nm. (C) SERS fluctuation of crystal violet aggregated with 2.5 mM KBr at 633 nm excitation (left) and 785 nm excitation (right). Strong fluctuations are observed at 633 nm. SERS signal is not observed at 785 nm excitation. Adapted with permission from ref 123. Copyright 2016 American Chemical Society. observed at 633 nm excitation but not at 785 nm, as seen in diamond-anvil cell (DAC) to accommodate the high pressures, Figure 12C. As the KBr concentration was increased, the which resulted in ∼25% compression of the sample at the highest nanoparticle resonances red-shifted due to the formation of pressure of 4 GPa. SM detection was verified using the R6G larger nanoparticle aggregates. As a result, the SERS intensities isotopologues. As shown in Figure 13A, the brightness of each with 785 nm excitation increased, while the SERS intensities with pixel in the scan correlates to the integrated intensity of the 633 nm excitation decreased. Furthermore, the anti-Stokes signal ∼1650 cm−1 R6G mode (in-plane stretching of the xanthene at 633 nm excitation only extended out to −600 cm−1, while at moiety), which shifts ∼5cm−1·GPa−1. An initial, rapid drop in 785 nm the Raman shifts were visible out to −800 cm−1. Their SERS intensity at 1.5 GPa is believed to result from a pressure- experimental results are in good agreement with the model induced destruction of numerous hot spots by compression of presented in Figure 12, where, for larger aggregates, the plasmon the sample. With the surviving hot spots, the authors were able to resonance broadens. These works suggest that the local structure observe the effect of pressure on the SERS spectra of R6G. They of nanoparticle aggregates can be probed with SMSERS because, observed a broadening in line width and a blue shift in peak as previously discussed, the SMSERS signal is strongly frequency for the ensemble-averaged 1650 cm−1 mode shown in dependent on the local field properties of the nanostructure. Figure 13B. Figure 13C shows the histogram of the ∼1650 cm−1 2.4.4. Pressure-Induced Blue Shift of Vibrational peak location for the SMSERS spectra at different pressures. The Modes. The ability to study single molecules under high magnitude of the blue shift varied between SM events, as pressure would deepen our understanding of the broadening and expected, and became more pronounced as the environment was blue shifting of vibrational modes in Raman spectroscopy. further compressed under pressure. The line width increase in Pressure-induced blue shifts of molecular vibrations can arise the ensemble spectra of R6G results from the variability in blue from anharmonic coupling between intramolecular vibrations shift from molecule to molecule. Each molecule is located in a and the environment. When molecules are present in disordered different hot spot structure (i.e., environment), leading to media, such as Ag colloids under pressure, individual molecules differences in anharmonic coupling at each molecular site and, will experience different anharmonic coupling to the environ- therefore, different blue shifts. Using insights from SMSERS, the ment, resulting in varied pressure-induced blue shifts for the pressure-dependent ensemble SERS measurements were ex- vibrational modes. Fu and Dlott investigated the effect of plained by the anharmonic shifts of various molecule−environ- pressure on vibrational modes in SERS and SMSERS from 1 to 4 ment interactions. GPa.126 For these studies, the SERS substrate consisted of 2.4.5. Excitation-Wavelength Dependence of SMSERS. aggregated Ag nanoparticles embedded in a poly(vinyl alcohol) The excitation-wavelength dependence of SMSERS was matrix, shown in Figure 13A. The substrate was then placed in a characterized using a tunable optical parametric oscillator to

M DOI: 10.1021/acs.chemrev.6b00552 Chem. Rev. XXXX, XXX, XXX−XXX Chemical Reviews Review

Figure 14. Overlay of the ensemble-averaged surface absorbance spectrum of R6G on Ag with the SMSERS REP. The single-molecule data points were obtained from the sum of all the observed Raman modes and were fit to a Lorentzian function (red). The error bars represent one standard deviation from the mean. The molecular absorption spectrum of R6G on Ag (black) was fit by the sum of two Gaussian distributions. The SM REP is narrower than the ensemble- average analogue, as expected. Adapted with permission from ref 127. Copyright 2009 American Chemical Society.

of photochemical, plasmonic near field, and chemical (e.g., charge transfer) effects and is highly molecule/system depend- ent, complicating our fundamental understanding. For example, there are several proposed mechanisms for the origin of blinking, ranging from thermally stimulated molecular diffusion to the formation of charge-transfer states, but robust theoretical models describing these mechanisms are still insufficient. Determining the role of each of these factors in signal fluctuations requires isolating the individual contributions. This may include tailoring molecular systems to isolate molecular diffusion, charger transfer effects, etc. In addition, correlated nanoparticle structure, theory, and SMSERS signal can help elucidate the various contributions from plasmonic near fields to SMSERS signal fluctuations. 2.5. Chemistry at the Single-Molecule Limit Now that SMSERS has become a relatively well-established Figure 13. (A) Photograph (top left) and Raman microscope images of the SERS substrate placed in a diamond-anvil cell. The pressures are technique, researchers have extended the technique and have indicated in each scan beginning and ending with atmospheric pressure. begun answering questions about molecular reactivity at its most The brightness at each pixel corresponds to the integrated intensity of fundamental limit. To this end, SMSERS has been applied to the ∼1650 cm−1 mode of R6G. (B) Ensemble average of many SMSERS monitoring catalytic and electrochemical reactions, observing spectra of the R6G isotopologues, indicating the blue shift and peak single-molecule tautomerization, and more, as we will discuss in broadening at higher pressures. (C) Histograms of the peak location for this section. the SMSERS events at different pressures. Adapted with permission 2.5.1. Electrochemical SMSERS. As the study of electro- from ref 126. Copyright 2015 American Chemical Society. chemical reactions shrinks to the nanoscale regime,128,129 there has been a recent spike of interest in monitoring the finely tune the excitation wavelength across the molecular electrochemistry of single molecules or single nanoparticles resonance of a single R6G molecule (i.e., ∼500−575 nm).127 The with optical spectroscopy.130 This is achieved by monitoring the Raman excitation profiles (REPs) for ensemble R6G (black line) signal of an optically active redox probe as a function of applied and a SM of R6G (black circles) are shown in Figure 14. The SM potential. In particular, the advantages of utilizing SMSERS to REP was obtained from the sum of all the observed Raman study nanoscale electrochemistry are that single-molecule modes and was fit to a Lorentzian function (red), producing a detection is readily attainable compared to traditional current fwhm of ∼400 cm−1. As expected, the SM REP is narrower than detection techniques, the SMSERS signal does not require the ensemble and was dominated by homogeneous broadening. additional amplification, SMSERS provides rich chemical This study was the first example of a high-density REP (i.e., finely information, and, during the electrochemical reaction, it allows tuned wavelength scan) across the molecular resonance of a SM. one to monitor structural changes of a molecule in close Collecting a distribution of REPs with correlated nanoparticle proximity to a nanostructured metallic surface. SMSERS was first structures could help improve our understanding of how local combined with electrochemistry in 2010 (EC-SMSERS), where a environments influence the energy, line width, and population bianalyte SMSERS proof was done by adsorbing R6G and Nile distribution of SMs that cannot be observed with ensemble Blue (NB) on a Ag mirror−Ag nanoparticle substrate working averaging. electrode.131 As the potential was swept positive from −0.5 to 2.4.6. Summary. While numerous studies have focused on −0.1 V, the 2H+,2e− oxidation of NB was indicated by the explaining the fluctuations observed in SMSERS spectra, there is appearance of the NB signal, and upon the negative sweep, the still much to be learned. Their origin appears to be a convolution reduction of NB was indicated by the loss of the NB signal. The

N DOI: 10.1021/acs.chemrev.6b00552 Chem. Rev. XXXX, XXX, XXX−XXX Chemical Reviews Review authors observed slight differences in the bulk surface cyclic weighted SMSERS emission position shifts as a function of voltammogram versus the single-molecule histogram, which potential. This finding suggests that single NB molecules are demonstrates the power of using SMSERS to study single- being oxidized or reduced at unique potentials based on their molecule electrochemistry. In later work, the authors extended location on the electrode. Later work from Weber et al. further the study to high-resolution SMSERS spectra of NB by observing confirmed the site-specific redox activity of NB on Ag changes in the NB vibrational frequencies as a function of applied nanoparticles when performing super-localization EC-SERS potential.132 Observed NB vibrational mode shifts versus along with spatially correlated SEM.70 At highly reducing reduction potential were attributed to reorientation of the NB potentials, the SERS emission is centered at junctions between molecule in the electrochemical double layer and the orientation nanoparticles and localized close to the center of the nanoparticle of NB on the Ag nanoparticle surface. EC-SMSERS was also aggregates at more oxidizing potentials. The change in centroid implementedinastudybyWangetal.tounderstand position as a function of applied potential implies that higher photoelectrochemical charge transfer dynamics in hemin, an energies are required to reduce NB molecules in junctions iron protoporphyrin, where it was found that local thermal between Ag nanoparticles, corroborating the concept of site- fluctuations govern electron transfer dynamics in the proto- specific redox potentials on nanoparticles first postulated by porphyrin system.133 Wilson and Willets. Overall, the use of EC-SMSERS and super- More recently, EC-SMSERS was implemented to study single, localization EC-SERS demonstrates the power of SERS to study heterogeneous electron transfer events of R6G in nonaqueous site-specific electrochemical reactions at the few- to single- conditions.134 Using a sample consisting of R6G-physisorbed Ag molecule level. nanoparticle aggregates covalently tethered to an ITO coverslip, 2.5.2. Observing Catalytic Reactions. As demonstrated by SMSERS detection was first validated by the isotopologue EC-SMSERS studies, SMSERS is a powerful tool for monitoring proof.96 Then, the electrochemical conversion of a single R6G chemical reactions. In particular, SERS has been recently applied molecule to its neutral radical form was observed by stepping the to study heterogeneous catalytic reactions because SERS-active potential of the working electrode from 0 to −1.2 V in −0.1 V nanoparticles can serve both as the catalyst material and Raman- steps and monitoring the SMSERS spectra as a function of enhancing substrate. Many recent studies have focused on applied potential. The potential at which the R6G SMSERS examining the model dimerization reaction of p-nitrothiophenol signal was no longer observed was counted as a SMSERS loss (p-NTP) or p-aminothiophenol (p-ATP) to 4,4-dimercaptoazo- − event and indicated the reduction of R6G to its neutral radical benzene (DMAB).43,136 147 Recently, efforts have pushed form. However, the majority of SMSERS signal loss events did toward studying the DMAB formation reaction at the single- not have a subsequent oxidative signal return, indicative of molecule limit. The Deckert group explored the effect of p-NTP electrochemically induced desorptive losses. This issue can be coverage on Au nanoparticles and how this affects the DMAB addressed using a molecule covalently tethered to the Ag formation yield. Further, they explore the fate of a single p-NTP nanoparticle surface, as demonstrated in recent work from the molecule if it cannot react with another molecule and dimerize to Willets group.71 Despite the occurrence of electrochemical form DMAB.147 At incubation concentrations greater than 5 × desorptive losses, the total SMSERS loss events followed the 10−8 Mofp-NTP, the coverage of p-NTP on the Au nanoparticle profile of a surface voltammogram of R6G on Ag. Interestingly, surface is sufficiently dense such that dimerization to DMAB still there were a small number of SMSERS signal loss events that occurs upon illumination. Below 5 × 10−8 M, the coverage of p- occurred in the underpotential region of the surface voltammo- NTP is too sparse and there is no apparent DMAB formation on gram, which are attributed to site-specific reduction potentials on the Au nanoparticle surface; the authors therefore assume that the Ag nanoparticle surface. Observing single-electron transfer there are one or two molecules in the Au nanoparticle hot spot with EC-SMSERS strongly demonstrates the electrochemical junction. Upon sample illumination, there is the appearance of heterogeneity of nanoparticles and elucidates the importance of new vibrational modes, indicating the cleavage of the nitro group site-specific electrochemical activity on surfaces. of p-NTP to form thiophenol (TP) (Figure 15). They rationalize The Willets group has exploited the 5−10 nm spatial precision the formation of TP via plasmon-induced hot-electron of super-localization SERS, as discussed in section 2.1.2,to generation, providing sufficient energy to cleave the nitro understand the spatial dependence of electrochemical events on group from the p-NTP molecule. This work demonstrates the Ag colloidal nanoparticles.70,135 They implemented NB power of monitoring single-molecule reactions on a plasmonic physisorbed onto Ag colloidal nanoparticle aggregates to observe nanoparticle surface using SERS. In a separate study, Choi et al. the 2H+,2e− reduction reaction of NB, where the SERS intensity demonstrated the heterogeneity of reactivity of single pairs of p- is lost and returns with NB reduction and oxidation, respectively. NTP molecules dimerizing to DMAB, which they attributed to Three different concentration regimes were studied: single- the relative location of the p-NTP molecules within the hot spot molecule coverage, few-molecule coverage, and high molecule junction.137 They demonstrated “hot” and “mild” photo- coverage. At single-molecule coverage, the fitted SMSERS switching regimes, as defined by two unique types of temporal emission centroid displays two unique reduction potentials fluctuations in the DMAB signal. In future work, SMSERS that are observed upon subsequent potential sweeps, which studies of catalytic reactions could be more rigorously performed correspond to two distinct positions of the molecule on the Ag using isotopically substituted molecules like p-ATP or p-NTP,136 nanoparticle aggregate. This result suggests that the reduction where the dimerization of an isotope pair occurs to form a single potential of NB is dependent on its location on the Ag molecule. nanoparticle surface. However, performing multiple potential 2.5.3. Additional Applications of SMSERS. In addition to sweep cycles at single-molecule coverage was challenging, due to potentially monitoring local temperature and observing single factors such as the small number of potential sweep cycles where catalytic reactions, SMSERS can also be used to observe transient the molecule is emissive. The authors then confirmed the site- intermediates, such as the rare cis tautomeric form of specific behavior of NB electrochemistry by performing the same porphycene, as studied by Gawinkowski and co-workers.108 experiment at few-molecule coverage, where the intensity- SMSERS detection of porphycene was proven using the

O DOI: 10.1021/acs.chemrev.6b00552 Chem. Rev. XXXX, XXX, XXX−XXX Chemical Reviews Review

temperature. Instead, they attribute the tautomerization to a hot- spot-dominated effect. This work demonstrates the potential for studying similar molecules such as substituted porphycenes and porphyrins with SMSERS and how nanoparticle hot spot structure can affect the chemical behavior of molecules. SMSERS can also monitor host−guest interactions, as demonstrated by Sigle and co-workers.81 They implement the host molecule cucurbit[7]uril (CB[7]) as a means of detecting single guest molecules within a Au mirror−Au nanoparticle junction. Cucurbit[n]urils (CB[n]s) are macrocyclic molecules with a barrel-like structure consisting of a glycouril monomer unit where [n] is the total number of monomers in the CB[n] molecule.148 CB[n]s are ideal for sensing applications because a single molecule binds in the CB[n] cavity volume and the CB[n] subsequently undergoes structural changes when a guest molecule enters the CB[n] cavity, which is readily detectable using Raman spectroscopy.149 In the CB[7] SMSERS experi- ment, a Au mirror is incubated with CB[7] at submonolayer coverage and Au nanoparticles are then drop-cast onto the sample in a gap-mode SERS configuration (Figure 6D). The authors claim bianalyte SMSERS spectra of either the empty CB[7] or filled CB[7] with eight different molecules [methyl viologen (MV), adamantane, or ferrocene derivatives] were acquired. Binding events were indicated by the appearance of the − −1 Figure 15. (a) Time-dependent SERS spectra of reacting 10 9 M p- guest molecule SERS spectra, as well as a −5 and 10 cm shift in −1 nitrothiophenol (p-NTP) on Au nanoparticles with Pex = 3 mW after 2 the 440 cm mode of CB[7] (Figure 16). Despite the clear min (black trace), 6 min (pink trace), and 30 min (red trace). Over time, distinction between empty and filled CB[7] used to claim two peaks associated with thiophenol (TP) appear and the major peak bianalyte SMSERS detection, nearly half of complex binding −1 (∼1340 cm )ofp-NTP disappears. (b and c) High coverage SERS events detected were both filled and unfilled CB[7] for all guest spectra of TP and p-NTP, respectively. Reproduced from ref 147 with molecules studied, indicating the presence of at least one CB[7] permission. Copyright 2015 the Royal Society of Chemistry. molecule in the Au mirror−nanoparticle junction. While the molecular coverage of the SERS-active substrate needs to be isotopologue method. Statistically valid SMSERS detection optimized in order to rigorously demonstrate SMSERS, this work is a promising step forward toward using SMSERS to probe occurred when the total isotopologue concentration of 1:1 150 −9 −10 biologically relevant molecules including cellular metabolites porphycene-d0:porphycene-d12 was 10 or 10 M. It is interesting to note that in each SMSERS histogram collected, (e.g., pyruvate and lactate), proteins, and DNA. more porphycene-d0 counts were measured than porphycene- ff ffi d12, indicating that the d0 had a higher surface di usion coe cient 3. SINGLE-MOLECULE AND HIGH-RESOLUTION TERS on the nanoparticle surface. This result highlights the importance SMSERS has been routinely achieved under favorable con- of taking into account the subtle differences in molecular ff ditions, allowing the exploration of the fundamental mechanisms properties (e.g., Raman scattering cross section, di usion of SERS and spectroscopic phenomena and the investigation of coefficient) when conducting a statistically valid SMSERS 58 molecular reactivity. Combining SMSERS with super-resolution proof. The authors also found that the porphycene molecules microscopy has successfully overcome the optical diffraction exhibit 100 times higher photostability when adsorbed onto limit; however, the super-resolution signals originate from the silver and gold nanoparticles as compared to solution-phase fi fl heterogeneity of the near eld rather than from the physical uorescence measurements. They attribute the higher photo- environment in which the molecules are located. The direct stability of the adsorbed porphycene, as compared to the free imaging of surface-bound molecules with the capability of molecule, to the decreased lifetime of the lowest excited singlet controlling hot spot location can be achieved using the plasmonic state, which decreases the probability of intersystem crossing to ’ properties of a SPM tip made or coated with a plasmonic metal, the molecule s triplet states. known as TERS. After proving SM detection, the authors sought to observe the cis tautomeric form of porphycene, which had previously only 3.1. Background and Principles been observed on Cu(110) single crystals using STM. They rely In 1985, Wessel first proposed the utilization of a plasmonic on DFT calculations to predict that the major changes between nanoparticle as an STM probe.151 The nanoparticle amplifies and −1 fi fi the d0 and d12 spectra are a shift in the Raman band at 180 cm con nes the electromagnetic eld of incoming photon excitation (trans) to the blue by 20 cm−1 (cis) and the appearance of a to a small volume, defined by the tip−sample junction of the Raman peak at 1420 cm−1 upon formation of the cis form of the STM. This idea, now commonly known as TERS, integrated the d12 isotopologue. Time trajectories of a single nanoparticle high chemical sensitivity of SERS with the high precision spatial aggregate were taken of both isotopologues, and instances of control of STM. However, the experimental realization of TERS trans−cis−trans switching were observed, where the theoretically was not demonstrated until decades later when several groups predicted peak shifts and new modes were observed. They find independently reported Raman spectra of surface-bound that the tautomerization is independent of temperature, as they molecules with plasmonically enhanced fields from metal or observe the cis form at both low temperature and at room metal-coated tips (Figure 17).16,17,19,20,152 Early TERS experi-

P DOI: 10.1021/acs.chemrev.6b00552 Chem. Rev. XXXX, XXX, XXX−XXX Chemical Reviews Review

Figure 16. (a) (Top) Schematic of the distribution of the underlying composition of the CB[7] and MV2+·CB[7] mixture in the nanoparticle on mirror (NPoM) probes showing three observable events. (Bottom) Representative SERS spectra of the characteristic CB[7] Raman modes at 440 and 830 cm−1 (CB[7] modes are gray dashed lines and MV2+ modes are red dotted lines). Each spectrum corresponds to (i) NPoM with purely unfilled CB[7] (green), (ii) a mix of filled and unfilled CB[7] (orange), and (iii) only filled CB[7] (blue). (b) Statistical representation of the number of events observed. Total representation of events observed, where 300−400 NPoMs with sufficiently strong SERS signals enabled identification of the relevant modes. (c) Comparison of the number of times the signal from filled CB[7] is observed for single-molecule events only and mixed events as a function of decreasing order of binding affinities of the guests for CB[7]. The overlaid black trace is the log of binding strengths of the guest molecules. Adapted with permission from ref 81. Copyright 2016 American Chemical Society.

Figure 17. (A) Schematic view of an early scanning probe Raman microscope used for AFM−TERS from the Van Duyne group. (B) AFM−TERS of − 2+ ′ trans-1,2-bis(4-pyridyl)ethylene (BPE) adsorbed to a Ag NSL-substrate with 632.8 nm laser excitation. (C) AFM TERS of Fe(bpy)3 (bpy = 2,2 - bipyridine) adsorbed to a Ag NSL-substrate with 514.5 nm laser excitation. Figure adapted from ref 152. ments were performed on scanning probe microscopes (SPMs) is limited to transparent samples. Later on, refractive side- mounted on inverted optical microscopes in which the laser was illumination optics10,153,154 and parabolic mirrors155,156 were focused through the microscope objective below the sample and incorporated, enabling TERS on opaque samples. collected through the same optical path. While this approach Of the independently published original demonstrations of utilizes the high numerical aperture of the optical microscope, it TERS at the turn of the century, several were performed on an

Q DOI: 10.1021/acs.chemrev.6b00552 Chem. Rev. XXXX, XXX, XXX−XXX Chemical Reviews Review

AFM platform.16,17,20,152 Since its inception, AFM−TERS has rently, the most accurate method of determining the near-field enjoyed widespread use in spectroscopic characterization of large area is through TER imaging, which will be discussed in section molecules, especially biomolecules40,157 and low-dimensional 3.3. 158−160 materials with nanoscale resolution. Due to the popularity 3.2. AFM and STM Tip Fabrication for studying such systems, commercial AFM−TERS systems Arguably the most challenging experimental aspect of TERS is (e.g., the Nano-Raman system from Horiba) have recently been the reliable fabrication of highly enhancing tips. Particularly in developed. While TER imaging of small molecules is generally STM, the fabrication of a tip that yields high TERS enhancement performed with STM systems, AFM−TERS has been applied to and high-resolution STM imaging can be difficult and some small molecular systems, including those relevant to irreproducible. A comprehensive survey of all methods for tip molecular electronics,161 art conservation,162 and electro- 163 fabrication, conditioning, and preservation is beyond the scope of chemistry. this review. Instead, we will briefly survey the most common Similar to SERS, the physics responsible for the enhancements methods and those with which the highest TERS spatial of the Raman scattering in TERS is mainly the EM and CE resolution was obtained. We refer the interested reader to a mechanisms. The EM mechanism derived from classical recent review by Huang and co-workers exclusively devoted to electrodynamics is the dominant factor and thus will be discussed the subject of TERS tips.170 in this section; however, it does not adequately explain the In STM-TERS, the most frequently used method for tip recently reported subnanometer resolution of TERS. The ff fabrication is electrochemical etching of a Ag or Au wire. For their ongoing e orts on integrating quantum mechanics into TERS submolecular resolution UHV-TERS work, the Dong group theories will be discussed in section 3.3.3. etched a 0.3 mm diameter Ag wire centered inside a Pt ring In TERS, a noble-metal tip mostly made of, or coated with, Ag counter electrode immersed in 1:4 perchloric acid:methanol. A 1 or Au serves as an analog to the enhancing nanostructures in V potential was applied to the Ag wire, which was etched for ∼8 SERS. The sharp geometry of the tip apex acts as the source of a min.18,21,171 The Van Duyne group used a similar etching LSPR, which generates an intense and highly localized fi method for their 4 nm resolution TERS imaging of a dynamic electromagnetic eld. As the plasmonic tip approaches the phase boundary,41 etching a 0.25 mm Ag wire in 1:4 perchloric noble-metal surface and is brought within close proximity (∼1−2 ff acid:ethanol with a 1.6 V applied potential versus an ethanolic nm), the gap-mode e ect between the tip and surface further Ag/AgCl reference, as shown in Figure 18. A comparable increases the electromagnetic field intensity and confinement. Roughly speaking, the field enhancement in this narrow active region of the coupled tip−substrate follows a d−10 dependence, where d is the distance between tip and substrate.164,165 EFs of resonant dye molecules exceeding 107 have been − demonstrated in TERS,166 168 which provides sufficient sensitivity for SM detection if the molecule of interest has a strong electronic transition aligning with the incoming photon excitation. Quantification of EFs in TERS is important in order to understand the fundamental effects of the local electromagnetic field on the system. This is typically done with the same equation as used in SERS (eq 1) with slight modification Figure 18. (A) Schematic of a standard etching procedure for Ag TERS IN/ EF = nf nf tips. A Ag wire is centered in a Pt ring counter electrode at the surface of IN/ (3) a 4:1 ethanol:HClO4 solution, and 1.6 V is applied to the Ag wire until ff ff the bottom of the wire drops off. (B) SEM image of a typical Ag TERS fi fi tip. where Inf and Iff are the intensities of the near- eld and far- eld Raman signals, respectively, and Nnf and Nff the number of molecules contributing to the near-field and far-field signals, procedure is generally used for etching Au. For example, the respectively. Since the far-field Raman signal originating from all Pettinger group etches 0.25 mm diameter Au wire in 1:1 molecules within the laser spot always contributes to the total HCl:ethanol at a potential of 2.4 V versus SCE.172 There are TERS signal, Inf cannot be directly measured. However, Inf can be countless variations of the electrochemical etching fabrication calculated by the difference of the signal from when the tip is strategy for TERS tips and all have been used with varying engaged to when the tip is retracted from the substrate, as Inf = degrees of success. Implementation of potentiostatic control with I − I . In the cases of submonolayer to single-molecule current cutoffs and optical feedback for monitoring the etching TERS ff − coverage, the far-field signal is not strong enough to be detected. process can help prevent overetching.173 175 Additionally, In this respect, the noise level can be utilized as an upper bound pulsed dc etching, ac etching, and etching within a thin film of for the far-field intensity, and consequently, the EF will be solution are options for controlling tip shape and morphol- 174,176,177 underestimated. Nff can be obtained by using the SPM image to ogy. Automation techniques have also been employed estimate the surface coverage of the analyte while factoring in the in an attempt to increase etching throughput and reproduci- area of the laser spot. Due to the fact that there is no identical bility.174 SPM probe, the enhancing region of the tip−sample junction is In AFM−TERS, the most common fabrication method for fi not well-de ned. Nnf can only be estimated, except in the case of a producing tips is thermal evaporation of Ag or Au onto a 40,163,178,179 SM, in which Nnf = 1. Pettinger et al. proposed to approximate commercial Si or SiN AFM probe. The deposition 169 Nnf on the basis of half the radius of the curvature of the tip; thickness for Ag varies widely in the literature, generally in the however, this is not accurate because recent experimental results range of 20−70 nm.40,179 However, Au AFM tips are used far less have demonstrated subnanometer lateral resolution.18,21 Cur- frequently in the AFM−TERS literature, with the optimal

R DOI: 10.1021/acs.chemrev.6b00552 Chem. Rev. XXXX, XXX, XXX−XXX Chemical Reviews Review thickness reportedly in the range of 50−80 nm.163,178 Aluminum Additionally, a several nanometer thick water meniscus on tips have also been fabricated using thermal evaporation for surfaces has been measured by AFM at moderate humidity.191 deep-ultraviolet (UV) TERS studies.180 Some common Other than introducing contaminants through diffusion, the alternative methods for AFM−TERS tip fabrication include presence of unwanted water may alter the molecule−surface − functionalizing AFM tips with single Au nanoparticles180 183 and interactions that otherwise would not be present under a electrodeposition of Ag and Au.178,184,185 The use of controlled environment. commercially available Au-coated AFM tips for TERS has also In 2007, the Pettinger group reported the design and been reported.161,186 Tuning-fork-based AFM (shear force operation of the first home-built TERS instrument under microscopy, SFM) has also been extensively used as a TERS ultrahigh vacuum (UHV) conditions.156 All necessary optical platform. In general, tip fabrication for SFM experiments is elements were mounted on a rigid frame containing the STM identical to those discussed above for STM experiments, with the inside the UHV chamber. In 2008, the same group demonstrated resulting tip then attached to a tuning fork.155,158,187 Batch the first correlated UHV-TERS and STM mapping of a single fabrication methods have also been developed to increase tip Brilliant Cresyl Blue (BCB) molecule, reporting a lateral reproducibility. A collaborative effort between the Oh group and resolution of 15 nm (Figure 20A).192 When sample preparations the Novotny group has led to a promising template-stripping are performed under UHV, atomically pristine surfaces are method for SFM probes.188,189 Large-scale fabrication methods achieved with only the molecule(s) of interest present. Utilizing such as this are promising techniques for expanding the utility of the benefits of working in a UHV environment, the Van Duyne TERS toward routine and commercial use (Figure 19). group demonstrated detection of multiple vibrational modes in UHV-TERS of copper phthalocyanine (CuPc) on Ag(111) concurrent with molecular-resolution STM imaging (Figure 20B,C).173 Furthermore, Klingsporn et al. demonstrated UHV- TERS of immobilized R6G molecules on Ag(111) with liquid He cooling. The cryogenic temperature not only minimized the surface diffusion of the adsorbed R6G molecules, but it also increased the vibrational dephasing time, which resulted in narrower line widths of the observed Raman modes. Addition- ally, the spectral shifts observed in the low-temperature UHV- TERS were further analyzed by potential energy distributions of the corresponding vibrational modes, which allowed accurate determinations of the absorption geometry of R6G on Ag(111) at 19 K.192 Since then, UHV has been the desired environment for high-resolution TERS studies on molecular sys- tems.18,21,193,194 3.3.2. Single-Molecule TER Imaging with Sub-5 nm Resolution. After the first demonstration of single-molecule TERS imaging in 2008,192 the lateral resolution of TERS on molecular systems has been pushed to below 5 nm under UHV Figure 19. (A) SEM image showing massively parallel Au tip formation conditions. In UHV, correlated STM and TERS imaging have on a silicon wafer, where ∼1.5 million nominally identical tips can be been applied to three distinct surface-bound molecular systems: fabricated over a single wafer. (B) Close-up image of a single 200 nm (1) an isolated molecule,21 (2) two distinct, adjacent analytes,18 thick gold tip resting in the inverted silicon mold after lift-off. The tips and (3) a dynamic molecular phase boundary.195 Dong and co- can be stored over an extended period of time without degradation. (C) workers first demonstrated subnanometer lateral resolution on SEM image after template stripping with epoxy and a thin tungsten wire. 21 fi an isolated porphyrin molecule at 80 K. The obtained TERS (D) The radius of the tip is 10 nm, suitable for high-resolution near- eld images allow for direct visualization of the inner structure of a imaging. Adapted with permission from ref 188. Copyright 2012 American Chemical Society. single molecule through optical spectroscopy (Figure 21A). In 2015, the same group, with the same instrument, unambiguously distinguished two distinct adjacent porphyrin molecules on a 3.3. Spatial Resolution Ag(111) substrate by the difference in vibrational fingerprints in 3.3.1. Early Development of Imaging/Environmental the TERS line scan (Figure 21B).18 Control for Single-Molecule TERS. An early example of Also performed under UHV conditions, a dynamic molecular TERS imaging on single-walled carbon nanotubes (SWNTs) was phase boundary has been imaged by TERS at room temper- reported by the Novotny group, wherein the authors ature.195 In this study, the condensed phase and the 2D gas phase demonstrated simultaneous AFM topographic and TER imaging of a perylene diimide derivative coexist on a Ag(100) substrate. constructed by integrating the 1596 cm−1 G′ band of SWNT at Collected TERS images exhibit ∼4 nm lateral resolution over the each pixel.190 They reported ∼25 nm lateral resolution in the dynamic phase boundary (Figure 21C). Here, the measured TER image. Since then, significant improvements on the lateral resolution is a convolution of the intrinsic TERS resolution and resolution of TERS have been demonstrated, making TERS an molecular diffusion on the phase boundary. ideal tool for single-molecule chemical imaging. TERS has set the stage for future molecular-resolution For molecular systems, however, TERS under ambient chemical imaging studies. Clearly, the original description of conditions often results in spurious signals in Raman spectra or lateral resolution derived purely from classical electrodynamics, rough features in SPM topographic images. SPM samples in where Pettinger and co-workers estimated that the lateral ambient environments are easily exposed to airborne contam- resolution is about half of the radius of the curvature of the tip,169 inants, which may interfere with the interpretation of data. is insufficient to explain sub-5-nm resolution. A detailed

S DOI: 10.1021/acs.chemrev.6b00552 Chem. Rev. XXXX, XXX, XXX−XXX Chemical Reviews Review

Figure 20. (A) (top) TERS image of 568−572 cm−1 Raman mode of a single BCB molecule on Au(111) and (bottom) x, y cross sections of the TERS intensity. Adapted with permission from ref 192. Copyright 2008 American Physical Society. (B) Molecular-resolution topographic STM images of a copper phthalocyanine (CuPc) adlayer on Ag(111) with the tip illuminated by a 633 nm continuous-wave laser and (C) corresponding UHV-TERS (green) plotted with a TD-DFT-simulated spectrum (black). Adapted by permission from ref 173. Copyright 2012 American Chemical Society.

Figure 21. (A) Subnanometer resolution TERS image of an isolated porphyrin molecule at 80 K. Adapted with permission from ref 21. Copyright 2013 Nature Publishing Group. (B) ∼0.5 nm resolution TERS mapping of adjacent porphyrin molecules at 80 K. Adapted with permission from ref 18. Copyright 2015 Nature Publishing Group. (C) TER imaging of a dynamic 2D phase boundary at room temperature. Adapted with permission from ref 195. Copyright 2016 American Chemical Society. discussion of the recent attempts to explain this high-resolution of plasmonic enhancement beyond the well-established EM with an advanced theoretical approach is presented in the mechanism.28 In this section, we discuss the ongoing theoretical following section. investigations into the high-lateral resolution of TERS. To do 3.3.3. Theoretical Explanations for High Spatial this, we will divide the redevelopment of plasmonic enhance- Resolution. As discussed in the previous section, Zhang et ment in UHV-TERS as either invoking optomechanical cavities al.21 and Jiang et al.18 have demonstrated subnanometer lateral or more deeply exploring plasmonic enhancement effects, such resolutioninUHV-TERS.Inaneffort to explain this as the image field effect,196 electric field gradients,197 and extraordinary lateral resolution, the theoretical community has nonresonant chemical enhancements.198 vigorously pursued several concepts. As the papers discussed in Theoretical approaches previously applied to SERS have been the previous section have shown,18,21,195 extracting the most implemented to gain an understanding about TERS resolution, molecular information requires a combined theoretical and including the image field effect,196 electric field gradient experimental approach. An interesting idea behind high- effects,197 and nonresonant chemical effects.198 The image field resolution TERS experiments originates from a reconsideration effect of SERS199 was applied as a self-interaction of the

T DOI: 10.1021/acs.chemrev.6b00552 Chem. Rev. XXXX, XXX, XXX−XXX Chemical Reviews Review molecular dipole with an image dipole in the substrate.196 The future theoretical work that must overcome the nonphysical higher lateral resolution coming from self-interaction relies on consequences discussed above. multiple elastic scattering events within the Ag tip−substrate gap 3.4. Single-Molecule TERS Studies and thus has a higher spatial dependence. While this theory does Aside from TERS mapping of individual molecules, several other provide a higher lateral resolution than that predicted by methods have been used to obtain TERS spectra of single conventional EM mechanisms, it does not account for the molecules and study molecular properties without the effects of subnanometer resolution observed.196 Meanwhile, the effect of fi ensemble averaging. Using an isotopologue method analogous to electric eld gradients on lateral resolution in TERS was explored SMSERS studies,96 Sonntag et al. presented a frequency-domain by Meng et al., who considered a system of meso-tetra(3,5-di-tert- proof of single-molecule TERS (SMTERS).207 TERS spectra of butylphenyl)porphyrin (H2TBPP) molecules interacting with a protonated R6G (R6G-d ) and deuterated R6G (R6G-d ) 178 fi 0 4 plasmonic tip. The authors show that electric eld gradients adsorbed on a polycrystalline silver film were acquired using a can lead to higher TERS lateral resolution than predicted by side illumination ambient STM TERS system with an electro- traditional EM mechanisms. However, both the image dipole and chemically etched silver tip. In the majority of spectra, one of the the electric field gradient effects rely on effects in a spatial regime two isotopologues was observed. Few of the acquired spectra 200,201 where quantum plasmonic effects dominate. exhibited contributions from both molecules, verifying the SM The optomechanical cavity mechanism for UHV-TERS was nature of the measurement. Furthermore, the spectral wandering −1 explored by Roelli et al., and they observed nonlinear plasmonic of the 612 cm phenyl ring breathing mode of R6G-d0 (602 −1 ± −1 enhancement within certain physical limits, such as high power cm for R6G-d4) was found to be 4cm , indicating that there densities.202 Applying the theory of optomechanical cavities to is no spectral overlap between the two isotopologues in the SERS and TERS relies on a coherent coupling between the distinguishing region. In the follow-up work, Sonntag et al. molecular vibrations (mechanical oscillator) and the localized investigated the nature of relative intensity fluctuations in surface plasmon (electromagnetic cavity). The molecular SMTERS spectra.97 In some spectra, the low wavenumber vibration alters the plasmon such that the instantaneous plasmon modes (e.g., 608 and 771 cm−1) are the most intense, while in occupancy changes and then acts back on the molecular others, the higher wavenumber modes (e.g., 1362 and 1651 − vibration. If the incident laser intensity approaches a parametric cm 1) are strongest (Figure 22). Molecular orientation effects on instability threshold, at which the rate of backaction amplification exceeds that of intrinsic damping, a strong nonlinear Raman response is observed. This nonlinear Raman response is a possible explanation of the high lateral resolution demonstrated in TERS. However, the ∼2 × 106 W/cm2 power density giving rise to this effect exceeds power densities used in <5 nm TERS reports, which are in the range 102−105 W/cm2.18,21,157,195,203 Stimulated Raman scattering (SRS) has also been suggested as a concept to account for the exceptional TERS lateral resolution.197,204,205 These reports develop a theory of SRS where a Raman pump field induces an optical plasmonic response and the electric field of this spatially confined plasmon is then taken as the Raman probe field. Using the higher-order confinement from SRS, Duan et al. calculated higher lateral resolution in TERS by the nonlinear Raman effects compared to normal SERS effects.204 However, UHV-TERS experiments were done using laser excitation on resonance with the molecule being studied, which leads to dispersive line shapes in resonant femtosecond stimulated Raman scattering (FSRS) experiments due to the interference of multiple Feynman pathways for the − 206 light matter interaction. Thus, the explanation of high- Figure 22. Selected ambient SMTERS spectra of R6G highlighting resolution TERS by SRS suggests discrepancies with the relative intensity fluctuations observed in the SM regime. In most experiments performed. spectra in the time series, the low wavenumber modes (e.g., 608 cm−1) It is clear that explaining the physical origins of the high lateral have the highest intensity (blue spectrum). However, in some cases (red resolution of TERS imaging in the subnanometer limit is not yet spectrum), the high wavenumber modes are strongest. Adapted with complete, and there exists ample opportunity for new develop- permission from ref 97. Copyright 2013 American Chemical Society. ments. Existing theoretical studies, while convincing on some levels, all achieve their high lateral resolution at the expense of the relative intensity were neglected due to the rotational physicality. Specifically, optomechanical cavities suggest exper- invariance of the Raman tensor for resonant excitation. imental power regimes that are experimentally inaccessible, SRS Additionally, covariance and cross-correlation analysis revealed mechanisms lead to unobserved experimental signatures, and fl − that all modes uctuated completely independently, further previous plasmonic enhancement mechanisms196 198 rely on ruling out orientation or adsorption geometry as the cause of spatial regimes that have stronger quantum plasmonic relative intensity fluctuation. Time-dependent density functional effects.200,201 Thus, further work on high-resolution TERS theory (TDDFT) calculations of the resonance Raman spectra of theories are necessary to fully understand the mechanism(s) R6G revealed that fluctuations in the excited state geometry, responsible for the experimentally observed resolution. The energy, and lifetime could reproduce the experimental results studies analyzed in this section provide a solid foundation for remarkably well.97 Thus, the combination of SMTERS measure-

U DOI: 10.1021/acs.chemrev.6b00552 Chem. Rev. XXXX, XXX, XXX−XXX Chemical Reviews Review ments and TDDFT is a powerful tool that can reveal electrochemical systems) and dynamics at ultrafast time scales fundamental properties of single molecules that are obscured will offer an unprecedented view of fundamental heterogeneities by averaging in ensemble measurements. at the single-molecule level. However, the methods commonly The fundamental interactions of single molecules with used for extracting information about single-molecule structure, plasmons in molecular junctions can also be interrogated using environment, and dynamics from SMTERS results are still TERS. Using cross-correlational analysis, El-Khoury et al. incomplete. For example, a recent work by Jiang and co-workers examined the relationship between the intensities of the revealed through TDDFT that the orientation of the electronic vibrational bands of 4,4′-dimercaptostilbene (DMS) and the transition dipole of a molecule in the tip−sample can significantly intensity of scattering from the charge transfer (CT) plasmon of impact the appearance of the Raman spectrum.195 Thus, cross- the molecular junction in the conducting limit.208 Similar to what correlation analysis and current TDDFT methods may not 97 was observed by Sonntag et al., each of the totally symmetric ag provide a complete picture of the nanoscale processes that modes was found to fluctuate completely independent of all contribute to SMTERS spectra. Further development of other modes and of the scattering from the CT plasmon. In theoretical methods is needed to improve our understanding contrast, the nontotally symmetric bu modes strongly correlate of the origins of characteristic SMTERS behaviors. 208 with each other and with the CT plasmon. The correlation of 3.5. Applications of Single-Molecule TERS and TERS the nontotally symmetric modes with the CT plasmon was Nanoscopy attributed to perturbation of the vibronic coupling terms of the Raman tensor by the tunneling plasmon. Since the intensities of The single-molecule sensitivity and nanoscale resolution of the totally symmetric mode can be described solely by the TERS discussed in the previous sections have been exploited in a Franck−Condon term of the induced dipole, modulation of the variety of practical systems. In this section, we will discuss recent (higher order) Herzberg−Teller term by the CT plasmon affects applications of TERS for studying electrochemical systems, 208 biomolecules, low-dimensional materials, heterogeneous catal- only the nontotally symmetric bu modes. Therefore, SMTERS studies of molecules in plasmonic junctions can reveal detailed ysis, and single-molecule electronics. Since TERS is a relatively fundamental information about the interaction between the CT new research field, several of the experiments discussed in this plasmon and the vibrational modes of the molecule bridging the section are still proof-of-concept. Nevertheless, these results junction. serve as cornerstones and open up new frontiers of SM research In order to extract additional fundamental information about with the ability to interrogate physiochemical phenomena with the structure, environment, and properties of single molecules, unprecedented spatial resolution. TERS at cryogenic temperatures can be employed to slow down 3.5.1. Electrochemical TERS. A nanoscale understanding of molecular motion to measurable time scales. Park et al. recently electrochemical processes, including heterogeneities in electro- used variable-temperature TERS and micro-Raman to observe chemical behavior across an electrode surface, is critical to rapid changes in molecular orientation and elucidate detailed understanding electrocatalysis, biological electron transfer, and information about intramolecular vibrational coupling in energy production and storage. TERS is an ideal tool for malachite green (MG).209 TERS line widths were found to be elucidating structure−activity relationships in electrochemical significantly narrower than those measured with micro-Raman at systems at the nanoscale. Electrochemical TERS (EC-TERS) all temperatures (e.g., 3.5 cm−1 for TERS versus 12 cm−1 for was first demonstrated on an AFM platform by Kurouski et al. 163,210 micro-Raman at 90 K) due to the absence of inhomogeneous and on an STM platform by Zeng et al. Kurouski et al. 163 broadening in SM and small-ensemble measurements. The published the first TERS study of a redox reaction. Using an temperature dependence of the TERS and micro-Raman line electrochemical AFM (EC-AFM) platform, the authors inves- widths and frequencies were fit to Arrhenius functions, and tigated the redox behavior of Nile Blue (NB) spontaneously information about intramolecular coupling was extracted. The adsorbed onto an indium−tin oxide (ITO) film. TERS spectra authors determined that the Arrhenius behavior of the line were acquired with a Au tip (70 nm Au on Si) using 632.8 nm widths emerges from vibrational dephasing resulting from excitation. The spectrum of NB was monitored during cyclic coupling of the observed Raman modes with low-frequency voltammetry in Tris buffer (50 mM Tris +50 mM NaCl at pH 7), torsional modes of MG. Further, fundamental parameters scanning from 0 to −0.6 V versus Ag/AgCl and back with a scan governing this intramolecular coupling were extracted from the rate of 10 mV/s. As the potential was swept from 0 to −0.6 V, the Arrhenius model, including activation energy, lifetime of the resonant oxidized form (NBOX) was converted to the non- exchange mode, and exchange coupling strength. The activation resonant reduced form (NBRED) and the intensity of the 591 − barrier was indeed found to match the frequencies of the cm 1 mode decreased. Upon scanning the potential back to 0 V 209 torsional modes of MG, rather than substrate phonons. versus Ag/AgCl, NBRED was oxidized back to NBOX and the Additionally, using symmetry arguments similar to those above, signal increased back to its original intensity.163 In Figure 23, Park et al. and co-workers used cross-correlation analysis of TERS spectra acquired at various potentials during the TERS modes at low temperature to observe pinwheel-like voltammogram are shown. By integrating the intensity of the − rotational motion of MG on the surface that is only observable 591 cm 1 mode, TERS voltammograms were constructed. when the molecular motions are slowed down to a measurable Remarkably, some TERS voltammograms, such as the one time scale.97,208,209 Therefore, variable-temperature TERS is a shown in Figure 23c, exhibit steplike features indicative of few- or powerful tool for obtaining molecular information on time scales single-molecule behavior. However, in other locations on the inaccessible by room-temperature continuous wave measure- sample, the TERS voltammogram did not contain steps, ments. demonstrating a nonuniform surface coverage of NB across the SMTERS therefore has the flexibility to study single-molecule ITO surface.163 This work demonstrated the potential of TERS processes in a range of environments (e.g., ambient, UHV) and for studying redox reactions at the nanoscale, probing few- or temperatures (e.g., room temperature, cryogenic temperatures). single-molecule behavior across a nonuniform surface coverage Further extension of TERS to study chemical reactions (e.g., in inaccessible by SERS.

V DOI: 10.1021/acs.chemrev.6b00552 Chem. Rev. XXXX, XXX, XXX−XXX Chemical Reviews Review

characterized redox molecule (e.g., NB) are needed to fully integrate nanoscale TERS mapping into electrochemical measurements. Further, a microscopic model must be developed in order to explain the steplike behavior in TERS voltammo- grams. 3.5.2. Biological Samples: Amyloid Fibrils and DNA Sequencing. TERS is an inherently label-free, nondestructive chemical examination tool for complex biological systems with SM resolution. Specifically, TERS has been applied to various biological samples, such as viruses, cells, DNA, RNA, and − amyloid fibrils.211 214 Identification of individual nucleobases on RNA and DNA strands has been demonstrated with exceptional spatial resolution, providing new insights into biological processes.215,216 For example, the termination group of DNA fragments has recently been interrogated by Lipiec et al. with TERS.217 Here, the authors irradiated a thin film of DNA in aqueous solution with a UV source before AFM and TERS measurement. They were able to unambiguously identify double- strand breaks of DNA from the observed TERS spectrum and hence proposed that the C−O double band cleavage was caused by the UV radiations. Moreover, TERS has unraveled the surface structure and composition of insulin fibrils. Instead of the expected dominance of β-sheets formation, only about one-third of the insulin fibrils’ surfaces was composed by β-sheets. The remainder was identified as a mixture of disordered proteins and α-helix.212 Thanks to its high spatial resolution, TERS can ff provide new insights into biological processes. Figure 23. (a) Selected TERS spectra of NB at di erent potentials along 3.5.3. Low-Dimensional Materials. The robustness and the voltammogram in panel b. (b) Voltammogram of NB spontaneously adsorbed onto an ITO working electrode at ∼0.02 monolayer coverage. high Raman cross section of low-dimensional materials, such as (c) TERS voltammogram produced by plotting the integrated intensity graphene, MoS2, and carbon nanotubes, makes them ideal targets of the 591 cm−1 mode as a function of potential. Steplike functions are for imaging by TERS. After the first TERS imaging on SWNTs observed in the TERS voltammogram, indicative of few-molecule with ∼25 nm resolution reported by the Novotny group,159 behavior. The cathodic trace (blue dots) and anodic trace (red dots) are carbon nanotubes remain one of the most common samples ff o set for clarity. Adapted with permission from ref 163. Copyright 2015 studied due to its high Raman cross section and robustness under American Chemical Society. high power laser illumination. Kawata and co-workers have demonstrated sub-2-nm resolution TERS images of SWNT Simultaneously, Zeng et al. demonstrated TERS of (4′- under ambient conditions (Figure 24A).203 Peica et al. have (pyridine-4-yl)biphenyl-4-yl)methanethiol (4-PBT) self-as- identified SWNT within a carbon nanotube bundle containing 218 sembled onto a Au(111) electrode in 0.1 M NaClO4 (pH 3 seven nanotubes. Furthermore, Yano et al. utilized TERS to 210 and 10). The electrochemically etched TERS tip was insulated monitor the mechanical properties of carbon nanotubes by using with polyethylene glue to prevent Faradaic current at the tip from the AFM tip to increase the pressure applied to the nanotube, interfering with the tunneling current. Interestingly, the authors which caused a ∼ 10 cm−1 shift in the G band.219 In the most observed no Faradaic processes attributed to 4-PBT, but they did observe changes in the TERS spectrum of 4-PBT as a function of potential at pH 10. With the sample held at −700 mV versus Pt, the 1592 cm−1 mode is slightly visible as a shoulder of the 1601 cm−1 mode. In contrast, the 1592 cm−1 mode shifts to slightly lower frequency and is clearly resolved at 300 mV versus Pt. No changes in the 4-PBT spectra were observed using TERS at pH 3 or SERS on Au nanoparticles at pH 10.210 The authors proposed that the shift in frequency observed for the 1592 cm−1 mode was due to protonation of the pyridine moiety terminating the 4-PBT SAM, demonstrating the advantage of TERS over SERS as a site- specific, sensitive probe of local surface chemistry. While this work was the pioneering effort to integrate TERS into an EC- STM environment to interrogate a well-defined electrode surface, no electron transfer processes were probed spectroscopi- Figure 24. (A) The 1.7 nm spatial resolution TERS of single carbon nanotubes. Adapted with permission from ref 203. Copyright 2014 cally. Overall, more work is necessary to realize the full potential Nature Publishing Group. (B) Molecular-resolution topographic STM of TERS for studying nanoscale electrochemical processes. In imaging and UHV-TERS of graphene nanoribbons (GNRs) fabricated order to fully exploit the nanoscale capabilities of TERS, aspects on Au(111) by the on-surface polymerization technique under UHV of both pioneering EC-TERS studies must be combined. conditions. Adapted with permission from ref 222. Copyrights 2014 Ultimately, a well-defined electrode [e.g., Au(111)] and well- American Chemical Society.

W DOI: 10.1021/acs.chemrev.6b00552 Chem. Rev. XXXX, XXX, XXX−XXX Chemical Reviews Review

Figure 25. (a) I is an average of all 190 on-state TERS spectra and II is a typical off-state TERS spectrum. Each of III, IV, and V correspond to an average ≥ ff ν −1 of 20 on-state spectra. VI and VII are calculated on- and o -state spectra, respectively. (b) The 8a band of 4bipy (1609 cm ) is seen to change with the bias voltage (current set-point = 5 nA). Inset: current versus bias curve for 4bipy obtained with a mechanical break-junction setup. (c) This diagram illustrates why 1609 cm−1 band splitting is proportional to the applied bias voltage. Adapted with permission from ref 223. Copyright 2011 Nature Publishing Group. recent TERS study of SWNTs, Dong and co-workers reported and shifted due to loss of symmetry. Further structural changes spectral changes due to strain.220 were observed for the on state as a function of bias voltage. − Two-dimensional materials are another popular system of Notably, 1609 cm 1 ring breathing mode reversibly splits into a study for TERS. It has been shown that TERS is capable of doublet as the bias is changed from 0.01 to 0.5 V. However, in its detecting small defects and contaminations in graphene sheets, current implementation, fishing-mode TERS (FM-TERS) which may alter the electronic properties of the sheets. The cannot be used to directly correlate conductance measurements intensity of the graphene D band at 1350 cm−1 is significantly with molecular structure for a discrete SM junction. Instead, the 1 increased in the presence of defects and at the edges of graphene s TERS acquisition time averages over several hundred ON− sheets.221 At the same time, in-plane vibrations (G band) were OFF events, providing the average structure of a SM junction. found to be weakly enhanced because their orientations are not The discrepancy between typical TERS acquisition times and the aligned with the enhanced plasmonic field of the tip−sample frequency of junction formation/breaking is a significant junction. In 2014, the Wolf group reported a TERS study of challenge that must be met before FM-TERS can be used to graphene nanoribbons prepared on a Au(111) surface under understand the relationship between structure and conductance UHV conditions (Figure 24B).222 Molecular-resolution imaging in molecular junctions. provided topographical characterization during the polymer- Recent work by El-Khoury et al. has taken steps toward ization process and corroborates the signal detected in UHV- overcoming this limitation at the expense of structural 161 − TERS. The submolecular resolution of TERS provides new information. Using an AFM TERS platform, with 0.1 s capabilities in studying the relationship between the mechanical acquisition times, the authors observed a broad background peak and electronic properties of carbon-based low-dimensional in spectra, corresponding to the on state of the junction materials. comprised of a gold tip and silver sample bridged by a 3.5.4. TERS Investigations of Single-Molecule Elec- biphenyldithiol (BPDT) molecule. The background peak was tronics. The possibility of combining TERS with STM break attributed to scattering from conducting plasmons across the junction measurements makes TERS a natural tool for studying molecular junction. However, the molecular structure in the ON SM electronics. In recent years, two groups have demonstrated state was not easily distinguishable above the broadened the potential of TERS for studying the structure of molecular background when adding many spectra together. TERS − trajectories (intensity−time transients) collected for BPDT break junctions, in which the tip sample junction is bridged by a − conducting molecule.161,223 Liu et al. acquired TERS spectra resemble current time transients typically collected in STM concurrently with conductance measurements in the “fishing- break junction measurements. However, even with a 0.1 s 223 acquisition time, several tens of formation/breaking events are mode” STM configuration. Fishing-mode STM monitors averaged over in each TERS spectrum. Nonetheless, the current jumps associated with the formation of SM break observation of direct scattering from the junction plasmon junctions as in conventional STM break junction techniques but upon shorting through single BPDT molecules reveals the uses extremely slow feedback to maintain a stable tip−sample potential of TERS in probing the structural and electronic distance within a few angstroms before breaking the junction. properties of molecular break junctions. Further improving This allows conductance measurements to be made for long acquisition time and collection efficiency will allow for the direct periods of time while a tip−sample distance suitable for TERS is ′ correlation of the structure and conductance of a single maintained. By acquiring TERS spectra of 4,4 -bipyridine molecular junction. (4bipy) simultaneously with conductance measurements, Liu et al. could elucidate differences in the structure of 4bipy when the junction was formed (ON state) versus broken (OFF state), 4. CONCLUSION AND OUTLOOK as shown in Figure 25.223 In the ON state, several of the Raman Over the past 2 decades, SMSERS and TERS have become modes of 4bipy, particularly the 1227 cm−1 mode, are broadened established techniques for characterizing molecular systems in

X DOI: 10.1021/acs.chemrev.6b00552 Chem. Rev. XXXX, XXX, XXX−XXX Chemical Reviews Review nanoscale environments. They combine the inherent chemical AUTHOR INFORMATION selectivity of Raman spectroscopy with plasmonically enhanced Corresponding Author signals to achieve the ultimate limit of detectiona single *E-mail: [email protected]. molecule. Moreover, TERS is capable of both chemical specificity and imaging of single molecules with high lateral resolution. ORCID Throughout this review, we have discussed the development and Michael O. McAnally: 0000-0002-8681-2952 understanding of the phenomena underlying SMSERS and high- Craig T. Chapman: 0000-0001-5349-5359 resolution TERS. For SMSERS, we covered hot spots and EF Richard P. Van Duyne: 0000-0001-8861-2228 distributions, the rational design of plasmonic substrates, the Notes rigorous verification of SM detection, and our current under- The authors declare no competing financial interest. standing of signal fluctuations and heterogeneity. For TERS, we covered AFM and STM tip fabrication, the physical origins of the Biographies high spatial resolution, including recent theoretical explanations, Alyssa B. Zrimsek received her B.A. degree in chemistry and art from and fundamental SM studies with TERS. Washington & Jefferson College in 2011. As part of her undergraduate To continue advancing our fundamental understanding of the work, she interned in the Analytical Lab for Art Conservation at the SERS and TERS phenomena, future SM studies should improve Philadelphia Museum of Art and conducted summer research under the the reproducibility of nanoparticle and tip fabrication for direction of Prof. Richard P. Van Duyne at Northwestern University. enhancing the Raman signal, develop methods for controlling She returned to Northwestern to pursue her graduate work in the Van molecular location, and increase hot spot density. Using more Duyne group, where she recently received her Ph.D. from Northwestern reproducible plasmonic structures would allow us to systemati- University working on single-molecule surface-enhanced Raman cally investigate the SERS mechanisms and adsorbate−surface spectroscopy. interactions. Moreover, coupling controlled molecular location Naihao Chiang received his B.S. degree in chemistry, physics, and with higher hot spot densities would increase the statistical mathematics/economics from the University of Southern California in power of SM studies, thereby strengthening experimental 2010. He is currently pursuing his Ph.D. in applied physics, at reproducibility and the interpretation of single-molecule Northwestern University, jointly advised by Richard P. Van Duyne and chemistry. Similarly, applying rigorous SM proofs in future Tamar Seideman. His research interest is focused on the development of investigations of single-molecule chemistry will increase the ultrahigh-vacuum tip-enhanced Raman spectroscopy and its applica- robustness of SMSERS and TERS as analytical techniques. tions. Furthermore, future studies should develop and improve the Michael Mattei received his B.S. degree in chemistry from Bucknell theoretical models used to describe observed SM behaviors. University in 2013. He is currently pursuing his Ph.D. in the Van Duyne Another challenge for SMSERS and TERS is broadening the group at Northwestern University. His research interests include tip- scope of studiable molecules. Expanding the molecular “toolbox” enhanced Raman spectroscopy and single-molecule electrochemistry. will generalize these techniques for new and exciting applications. ff Stephanie Zaleski received her B.A. in biochemistry from Barnard For instance, we highlight in this review recent e orts toward College in 2011. She recently received her Ph.D. in chemistry from studying biologically relevant systems and nonresonant mole- Northwestern University under the supervision of Richard P. Van cules with SMSERS and TERS. It also appears natural to use the Duyne, where her graduate work focused on using surface-enhanced added high resolution of TERS to follow reactions at the Raman spectroscopy to investigate single-electron transfer reactions and 219 nanoscale. For example, dimerization reactions have been to develop novel noninvasive molecular sensing methodologies. She studied by TERS. While several steps have now been taken recently began her postdoctoral fellowship with the Department of toward the monitoring of chemical reactions with TERS, the full Scientific Research at the Metropolitan Museum of Art. potential of TERS to monitor single-molecule chemical reactions Michael O. McAnally received his B.S. degree in chemistry and has yet to be realized. Recent developments in high-resolution mathematics from the University of WisconsinEau Claire in 2011. He 21,195 UHV-TERS also suggest that it should be possible to is currently pursuing his Ph.D. at Northwestern University, jointly achieve <1 nm resolution in monitoring chemical events for advised by Richard P. Van Duyne and George C. Schatz. His research individual molecules in future studies. interests include investigating the dynamics of coupled molecule− Due to great strides in the fundamentals of SERS and TERS, plasmon systems with plasmonically enhanced coherent Raman SM detection is now widely accepted and is being used to study scattering, probing the mechanism of atomic layer deposition in early relevant chemistries at the SM level. Electrochemistry, site- growth cycles by surface-enhanced Raman scattering, and using specific catalysis, and SM electronics are a few fields covered in coherent Raman scattering to understand fundamental Raman this review where the vibrational characterization of single scattering properties. molecules has provided new insight that cannot be gained from Craig T. Chapman received his Ph.D. in chemistry from the University an ensemble-averaged experiment. In addition, single to few of Oregon in 2010 under the supervision of Jeffrey A. Cina, studying molecule level studies have been used to observe rare transient quantum dynamics and the theory of ultrafast nonlinear spectroscopy. intermediates and monitor host−guest interactions. Future He then performed postdoctoral research on the electronic structure endeavors with SMSERS and TERS have the potential to answer and charge transfer dynamics of fullerene derivatives with Xiaosong Li at questions in photochemistry, plasmon-driven chemistry, site- the University of Washington. He is currently a postdoc with George C. specific heterogeneous catalysis, and more. These are only a few Schatz at Northwestern University. His research is concerned with the of the exciting new prospects for application of SM detection that photoinduced dynamical properties of excitonic and plasmonic systems. can benefit from the combination of site-specific chemical and Anne-Isabelle Henry is a research assistant professor at Northwestern structural information. University. She joined the Department of Chemistry after completing

Y DOI: 10.1021/acs.chemrev.6b00552 Chem. Rev. XXXX, XXX, XXX−XXX Chemical Reviews Review her doctoral thesis on the rational design and intrinsic properties of (3) Kinkhabwala, A.; Yu, Z.; Fan, S.; Avlasevich, Y.; Mullen, K.; nanocrystal superlattices. She has extensive experience working with Moerner, W. E. Large single-molecule fluorescence enhancements − plasmonic nanomaterials, from their synthesis, assembly, and structure− produced by a bowtie nanoantenna. Nat. Photonics 2009, 3, 654 657. ̈ ̊ function relationships to implementing strategies for harvesting their (4) Kuhn, S.; Hakanson, U.; Rogobete, L.; Sandoghdar, V. Enhance- ment of Single-Molecule Fluorescence Using a Gold Nanoparticle as an potential as optical sensors for defense and healthcare applications. Optical Nanoantenna. Phys. Rev. Lett. 2006, 97, 017402. Lately, her research has focused on the development of nanoplasmonic (5) Moerner, W. E.; Fromm, D. P. Methods of single-molecule sensors for bioanalyte detection using surface-enhanced Raman fluorescence spectroscopy and microscopy. Rev. Sci. Instrum. 2003, 74, spectroscopy (SERS). She is also actively involved in managing research 3597−3619. programs involving SERS biosensing and tip-enhanced Raman- (6) Betzig, E.; Chichester, R. J. Single molecules observed by near-field spectroscopy-enabled nanoscale imaging. She holds a B.S. (2002) and scanning optical microscopy. Science 1993, 262, 1422−1422. a M.S. (2004) from University of Paris-Sud (Orsay, France) and a Ph.D. (7) Gimzewski, J. K.; Joachim, C. Nanoscale Science of Single (2008) in physical chemistry (summa cum laude) from Pierre and Marie Molecules Using Local Probes. Science 1999, 283, 1683−1688. Curie University (Paris, France). (8) Xie, X. S.; Dunn, R. C. Probing single molecule dynamics. Science 1994, 265, 361−361. George C. Schatz is the Morrison Professor of Chemistry at (9) Celedon, A.; Nodelman, I. M.; Wildt, B.; Dewan, R.; Searson, P.; Northwestern University. He received his undergraduate degree at Wirtz, D.; Bowman, G. D.; Sun, S. X. Magnetic Tweezers Measurement Clarkson University and Ph.D. at Caltech. He was a postdoc at MIT, and of Single Molecule Torque. Nano Lett. 2009, 9, 1720−1725. has been at Northwestern since 1976. Schatz is a member of the (10) Hayazawa, N.; Tarun, A.; Inouye, Y.; Kawata, S. Near-field National Academy of Sciences and has been Editor-in-Chief of the enhanced Raman spectroscopy using side illumination optics. J. Appl. Journal of Physical Chemistry since 2005. He is a theoretician who studies Phys. 2002, 92, 6983−6986. the optical, structural, and thermal properties of nanomaterials, (11) Neuman, K. C.; Nagy, A. Single-molecule force spectroscopy: including plasmonic nanoparticles, DNA and peptide nanostructures, optical tweezers, magnetic tweezers and atomic force microscopy. Nat. − and carbon-based materials, with applications in chemical and biological Methods 2008, 5, 491 505. (12) Nie, S.; Emory, S. R. Probing Single Molecules and Single sensing, electronic and biological materials, and solar energy. Nanoparticles by Surface-Enhanced Raman Scattering. Science 1997, Richard P. Van Duyne is the Charles E. and Emma H. Morrison 275, 1102−1106. Professor of Chemistry and Professor of Biomedical Engineering and (13) Kneipp, K.; Wang, Y.; Kneipp, H.; Perelman, L. T.; Itzkan, I.; Applied Physics at Northwestern University. He received his B.S. (1967) Dasari, R. R.; Feld, M. S. Single Molecule Detection Using Surface- − at Rensselaer Polytechnic Institute and Ph.D. (1971) at the University of Enhanced Raman Scattering (SERS). Phys. Rev. Lett. 1997, 78, 1667 North Carolina, Chapel Hill, both in chemistry. He is a member of the 1670. National Academy of Sciences, the American Academy of Arts and (14) Gross, L.; Mohn, F.; Moll, N.; Liljeroth, P.; Meyer, G. The Chemical Structure of a Molecule Resolved by Atomic Force Sciences, and the American Institute of Medical and Biological Microscopy. Science 2009, 325, 1110−1114. Engineers. He is known for the discovery of surface-enhanced Raman (15) Ho, W. Single-molecule chemistry. J. Chem. Phys. 2002, 117, spectroscopy (SERS), the invention of nanosphere lithography (NSL), 11033−11061. and development of ultrasensitive nanosensors based on localized (16) Anderson, M. S. Locally enhanced Raman spectroscopy with an surface plasmon resonance (LSPR) spectroscopy. atomic force microscope. Appl. Phys. Lett. 2000, 76, 3130−3132. (17) Hayazawa, N.; Inouye, Y.; Sekkat, Z.; Kawata, S. Metallized tip ACKNOWLEDGMENTS amplification of near-field Raman scattering. Opt. Commun. 2000, 183, 333−336. M.O.M., G.C.S. and R.P.V.D. acknowledge support from the (18) Jiang, S.; Zhang, Y.; Zhang, R.; Hu, C.; Liao, M.; Luo, Y.; Yang, J.; National Science Foundation Center for Chemical Innovation Dong, Z.; Hou, J. Distinguishing adjacent molecules on a surface using dedicated to Chemistry at the Space−Time Limit (CaSTL) plasmon-enhanced Raman scattering. Nat. Nanotechnol. 2015, 10, 865− (Grant CHE-1414466). A.B.Z. and R.P.V.D acknowledge 869. support from the National Science Foundation (CHE- (19) Pettinger, B.; Picardi, G.; Schuster, R.; Ertl, G. Surface enhanced 1506683). M.M., C.T.C., A.-I.H., G.C.S., and R.P.V.D. acknowl- Raman spectroscopy: Towards single molecular spectroscopy. Electro- ffi fi chemistry (Tokyo, Jpn.) 2000, 68, 942−949. edge support from the Air Force O ce of Scienti c Research (20) Stöckle, R. M.; Suh, Y. D.; Deckert, V.; Zenobi, R. Nanoscale MURI (FA9550-14-1-0003). N.C., G. C. S., and R.P.V.D. ffi chemical analysis by tip-enhanced Raman spectroscopy. Chem. Phys. acknowledge support from the Department of Energy O ce of Lett. 2000, 318, 131−136. Basic Energy Sciences (SISGR Grant DE-FG02-09ER16109). In (21) Zhang, R.; Zhang, Y.; Dong, Z. 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AE DOI: 10.1021/acs.chemrev.6b00552 Chem. Rev. XXXX, XXX, XXX−XXX