Single-Molecule SERS Spectroscopy (original) (raw)

Single-Molecule Raman Spectroscopy – Fact or Fiction?

CHIMIA

Single-molecule detection represents the ultimate sensitivity limit in chemical analysis. Spectroscopic studies may even allow identifying the chemical structure of a single molecule, offering far-reaching opportunities in basic and applied research. Recent advances have allowed detection and dynamic studies of single molecules under both cryogenic and ambient conditions [l]. Most of these studies are based on laser-induced fluorescence, a method that provides ultra-high sensitivity but is limited in the amount of molecular information. Vibrational spectroscopy, for example Raman spectroscopy, would be a preferred method for single-molecule studies because of the very high chemical information content. Raman scattering, however, is a very weak effect, with cross sections between 10-30 cm 2 and 10-25 cm 2 per molecule, the larger values occuring only under favorable resonance Raman conditions. Such small Raman cross sections require a large number of molecules to achieve adequate conversion rates from excitation laser photons to Raman photons, thereby making single-molecule Raman spectroscopy 'science fiction'. This situation is dramatically improved if surface-enhanced Raman scattering (SERS) is used. The exciting phenomenon of a strongly increased Raman signal from molecules attached to metallic nanostructures was discovered in 1977 by Van Duyne, Jeanmaire, Albrecht and Creighton [2]. Very recently, and almost simultaneously, two groups, the one of Kathrin Kneipp and the other of Shuming Nie, unexpectedly observed enhancement factors much larger than the ensemble-aver

Single Molecule Detection Using Surface-Enhanced Raman Scattering (SERS)

Physical Review Letters, 1997

By exploiting the extremely large effective cross sections ( 10 -17 –10 -16 cm 2 /molecule) available from surface-enhanced Raman scattering (SERS), we achieved the first observation of single molecule Raman scattering. Measured spectra of a single crystal violet molecule in ...

Single molecule surface-enhanced Raman spectroscopy in nanogap structures

2009

A general overview of the field of single-molecule (SM) surface-enhanced Raman spectroscopy (SERS) as it stands today is provided. After years of debates on the basic aspects of SM-SERS, the technique is emerging as a well-established subfield of spectroscopy and SERS. SM-SERS is allowing the observation of subtle spectroscopic phenomena that were not hitherto accessible. Examples of the latter are natural isotopic substitutions in single molecules, observation of the true homogeneous broadening of Raman peaks, Raman excitation profiles of individual molecules, and SM electrochemistry. With background examples of the contributions produced by our group, properly interleaved with results by other practitioners in the field, we present some of the latest developments and promising new leads in this new field of spectroscopy. 65 Annu. Rev. Phys. Chem. 2012.63:65-87. Downloaded from www.annualreviews.org by Victoria University of Wellington on 04/10/12. For personal use only. Click here for quick links to Annual Reviews content online, including: • Other articles in this volume • Top cited articles • Top downloaded articles • Our comprehensive search Further ANNUAL REVIEWS

Online Fluorescence Suppression in Modulated Raman Spectroscopy

Analytical Chemistry, 2010

Label-free chemical characterization of single cells is an important aim for biomedical research. Standard Raman spectroscopy provides intrinsic biochemical markers for noninvasive analysis of biological samples but is often hindered by the presence of fluorescence background. In this paper, we present an innovative modulated Raman spectroscopy technique to filter out the Raman spectra from the fluorescence background. The method is based on the principle that the fluorescence background does not change whereas the Raman scattering is shifted by the periodical modulation of the laser wavelength. Exploiting this physical property and importantly the multichannel lock-in detection of the Raman signal, the modulation technique fulfills the requirements of an effective fluorescence subtraction method. Indeed, once the synchronization and calibration procedure is performed, minimal user intervention is required, making the method online and less time-consuming than the other fluorescent suppression methods. We analyze the modulated Raman signal and shifted excitation Raman difference spectroscopy (SERDS) signal of 2 µm-sized polystyrene beads suspended in a solution of fluorescent dye as a function of modulation rate. We show that the signal-to-noise ratio of the modulated Raman spectra at the highest modulation rate is 3 times higher than the SERDS one. To finally evaluate the real benefits of the modulated Raman spectroscopy, we apply our technique to Chinese hamster ovary cells (CHO). Specifically, by analyzing separate spectra from the membrane, cytoplasm, and nucleus of CHO cells, we demonstrate the ability of this method to obtain localized sensitive chemical information from cells, away from the interfering fluorescence background. In particular, statistical analysis of the Raman data and classification using PCA (principal component analysis) indicate that our method allows us to distinguish between different cell locations with higher sensitivity and specificity, avoiding potential misinterpretation of the data obtained using standard background procedures.

Rationale for 101410^{14}1014 enhancement factor in single molecule Raman spectroscopy

We extend the Purcell's original idea [Phys. Rev. 69, 682 (1946)] on modification of photon spontaneous emission rate to modification of photon spontaneous scattering rate. We find the interplay of local incident field enhancement and local density of photon states enhancement in close proximity to a silver nanoparticle may result in up to 10 14 -fold rise of Raman scattering crosssection. Thus single molecule Raman detection is found to be explained by consistent quantum electrodynamic description without any chemical mechanism involved. A model of the so-called "hot points" in surface enhanced spectroscopy has been elaborated as local areas with high Q-factor at incident and scattered (emitted) light frequencies. For verification of the model we consider further experiments including transient Raman experiments to clarify incident field enhancement and scanning near-field optical mapping of local density of photon states. 33.20.Fb Since the discovery of molecular scattering of light with individual signatures of specific bondings in 1928 [1], vibrational spectroscopy has become the routine analytical tool in molecular physics and chemistry. Discovery of the giant enhanced Raman signals promoted by nanotextured metal surfaces and metal nanoparticles [2] stimulated search for extreme Raman spectroscopy sensitivity and has resulted in pioneering works reported on single molecule Raman signatures. In spite of challenging experimental records, a consistent theory explaining up to 10 14 enhancement factors documented has not been developed to date and the observation of single molecule Raman signals remains unexplained. Typically, local incident field enhancement factor [5] is considered as the major contribution to SERS signals giving factors up to 10 6 [5] for most favorable combination of a metal nanobody shape, a molecule location and incident light frequency. Further enhancement factors are searched for among chemical mechanisms [5]. Notably, the theory is essentially reduced to classical electromagnetism with no quantum electrodynamics (QED) involved.

Bi-analyte single molecule SERS technique with simultaneous spatial resolution

Phys. Chem. Chem. Phys., 2011

The simultaneous combination on CCD detectors of both spectral and spatial information is used in the framework of the single molecule (SM) bi-analyte Surface-Enhanced Raman Scattering (SERS) technique, to provide a new level of understanding on the origins of SM-spectra, as well as reveal the advantages and limitations of the statistical identification of SM-events. A new and deeper interpretation of the roots of the inhomogeneous broadening of single molecule Raman peaks can be uncovered, as well as the origin of Surface-Enhanced Fluorescence (SEF) emission by single molecules. In this manner, subtler aspects of SM-SERS spectroscopy can be revealed by the additional presence of spatial information on the localization of single molecules producing the signal. The spatial information is normally lost through the standard binning of CCD cameras for spectroscopy, which only emphasizes the spectral dimension of the problem. This novel extension of the bi-analyte SM-SERS method should contribute to the furtherance of the technique, and several of its fundamental aspects are discussed in detail.

Stimulated Raman excited fluorescence spectroscopy and imaging

Nature Photonics, 2019

Powerful optical tools have revolutionized science and technology. The prevalent fluorescence detection offers superb sensitivity down to single molecules but lacks sufficient chemical information 1-3. In contrast, Raman-based vibrational spectroscopy provides exquisite chemical specificity about molecular structure, dynamics and coupling, but is notoriously insensitive 3-5. Here, we report a hybrid technique of stimulated Raman excited fluorescence (SREF) that integrates superb detection sensitivity and fine chemical specificity. Through stimulated Raman pumping to an intermediate vibrational eigenstate, followed by an upconversion to an electronic fluorescent state, SREF encodes vibrational resonance into the excitation spectrum of fluorescence emission. By harnessing the narrow vibrational linewidth, we demonstrated multiplexed SREF imaging in cells, breaking the 'colour barrier' of fluorescence. By leveraging the superb sensitivity of SREF, we achieved all-far-field single-molecule Raman spectroscopy and imaging without plasmonic enhancement, a long-soughtafter goal in photonics. Thus, through merging Raman and fluorescence spectroscopy, SREF would be a valuable tool for chemistry and biology. To merge the advantages from both the worlds of Raman and fluorescence, our idea is to develop a new hybrid spectroscopy by encoding vibrational features onto the fluorescence spectrum. Before we reach the optimized design, we have gone through a series of theoretical considerations and experimental refinement (Fig. 1). Whereas linear fluorescence spectroscopy excites the electronic transition directly (Fig. 1a), nonlinear fluorescence excitation can employ one or more virtual states as intermediates, thus potentially probing more states (Fig. 1b). However, conventional nonlinear fluorescence still lacks chemical specificity, owing to the extremely short-lived virtual states (that is, large energy uncertainty). We reason that, if a long-lived vibrational eigenstate with a well-defined energy level can mediate a multiphoton fluorescence excitation process, the intermediate vibrational information can then be encoded into the fluorescence excitation spectrum. Indeed, such doubleresonance spectroscopy was explored decades ago in which an infrared (IR) pulse excites an intermediate vibrational transition, followed by a visible pulse to excite the fluorescence (Fig. 1c) 6. Despite being a powerful approach to investigate the vibrational dynamics of chromophores 7,8 , the strong IR absorption in water and poor spatial resolution are intrinsically unfavourable for applications in biological systems. Moreover, the reported sensitivity is still several orders away from single molecules. Considering stimulated Raman scattering (SRS) is complementary to IR excitation by offering much higher spatial resolution and avoiding water absorption, we take a different approach, stimulated Raman-excited fluorescence (SREF), by harnessing two

Single-Molecule Tip-Enhanced Raman Spectroscopy

The Journal of Physical Chemistry C, 2012

An existence proof for single molecule tip-enhanced Raman spectroscopy (SMTERS) is given using the frequency domain approach involving the two isotopologues of Rhodamine 6G (R6G) that were previously employed for single molecule surface-enhanced Raman spectroscopy (SMSERS).

Surface Enhanced Raman Spectroscopy for Single Molecule Protein Detection

Scientific Reports, 2019

A two-step process of protein detection at a single molecule level using SeRS was developed as a proofof-concept platform for medical diagnostics. First, a protein molecule was bound to a linker in the bulk solution and then this adduct was chemically reacted with the SeRS substrate. traut's Reagent (tR) was used to thiolate Bovine serum albumin (BSA) in solution followed by chemical cross linking to a gold surface through a sulfhydryl group. A Glycine-TR adduct was used as a control sample to identify the protein contribution to the SER spectra. Gold SERS substrates were manufactured by electrochemical deposition. Solutions at an ultralow concentration were used for attaching the tR adducts to the SeRS substrate. Samples showed the typical behavior of a single molecule SeRS including spectral fluctuations, blinking and Raman signal being generated from only selected points on the substrate. The fluctuating SER spectra were examined using Principle Component Analysis. This unsupervised statistics allowed for the selecting of spectral contribution from protein moiety indicating that the method was capable of detecting a single protein molecule. Thus we have demonstrated, that the developed two-step methodology has the potential as a new platform for medical diagnostics.