Shedding enhanced light on biological and biomedical systems: the application of surface enhanced Raman spectroscopy
I.T. Shadi Cardiac Electrophysiology, National Heart and Lung Institute, Faculty of Medicine, Imperial College London, St Marys Campus, London, W2 1NY Email:
[email protected]
R. Goodacre Manchester interdisciplinary Biocentre, School of Chemistry, the University of Manchester, 131 Princess Road, Manchester, M1 7DN Email:
[email protected]
Freeman Dyson once said that “Surprises in science often arise from new tools rather than from new concepts” (1) and this is very pertinent to the (bio)analytical science which goes through very exciting developmental stages from inception to real world applications. Raman spectroscopy, since its discovery by C.V Raman in 1928 (2), who received the Nobel Prize in Physics for this discovery, has proven to be a formidable and potent analytical tool with an extraordinary number of applications in multiple disciplines. In the last 35 years the pace of change for this technique has been rapid where it has endured a remarkable journey. Raman spectroscopy is now well established as a contemporary and as a complementary technique to much of the analytical instrumentation currently available with applications in, for example, forensic science, archaeology, environmental monitoring, pharmaceutical analysis, clinical medicine, biomedical analysis and space exploration. Raman spectroscopy is becoming an analytical tool employed in all areas of science as shown by applications of both normal Raman and SERS (Surface Enhanced Raman Spectroscopy) reported in the literature, and some of these will be highlighted below. Every compound has its own unique Raman spectrum, which can be used for sample identification, detection and quantification. The differences in energy between the incident photons (usually provided by a laser) and the scattered photons correspond to vibrations in the molecule or crystal, and provide a “fingerprint” of the sample’s composition and molecular structure. There are several advantages of Raman spectroscopy many of which are significant in the life sciences. These often include minimal sample preparation, the ability to observe the Raman spectra of analytes in aqueous
solutions, as water is a poor Raman scatterer, in situ analysis is generally straightforward; Raman spectroscopy can also be used to investigate analytes through e.g. glass and fibre optics which can be employed for remote sampling. Raman spectroscopy can be used for the analysis of organic and inorganic materials giving vibrational bands belonging to symmetrical vibrations (generally very weak in infrared spectroscopy). In addition, Raman bands of solids are usually sharper than infrared bands making the spectra more information rich and easier to interpret. Most of the disadvantages of Raman spectroscopic methods arise directly from the fact that Raman scattering is a rather weak effect, which requires intense laser excitation sources and sensitive (CCD) detectors which in turn has led to the, relative (and arguably) high costs of Raman instrumentation. The later is one of the main obstacles to the widespread application of Raman spectroscopy for routine biological analysis. This situation is now changing as lasers and detector costs have fallen and instrument performance has steadily improved, with concomitant improvements in spectral resolution and several vendors now offering portable easy to deploy systems. When light is scattered from a molecule the vast majority of photons are elastically scattered; this is termed “Rayleigh scattered light”. The scattered photons have the same energy (frequency) and, therefore, wavelength, as the incident photons. A small fraction of light (approximately 1 in 107 photons) is scattered at optical frequencies different from, and usually lower than, the frequency of the incident photons. The process leading to this inelastic scatter is termed the Raman effect (Fig.1).
Fig.1.
A cartoon showing the Raman effect. Incident light is shown in green (hv0) and Raman scattering in red (h(v0±v))
The advantages of Raman spectroscopy include the following:
Visible light is often used to excite samples. This facilitates the use of microscopes and fibre optic cables in Raman experiments. Can be applied to many chemical problems (see Table 1). No (or very little) sample preparation is required. In situ real time measurements can be undertaken. Non-destructive and non-intrusive sampling.
Minute samples of micrometer dimensions can be examined. Spectra can be collected through glass windows and via fibre optics. Aqueous and non-aqueous solutions can be examined. Samples can be solids, liquids, gases; transparent or opaque. Spectra are, usually, well resolved with high information content. Chemometrics can assist in complex analysis. Compact portable Raman systems are now available (example e.g. from Ahura, Delta Nu and Ocean Optics). Variants of conventional Raman spectroscopy have evolved and in the last few years novel emerging techniques have been reported for, example, deep non invasive Raman scattering, SERS (surface enhanced Raman scattering), TERS-AFM (tip enhanced Raman scattering – atomic force microscopy) and SESORS (Surface enhanced spatially offset Raman spectroscopy).
Applications include:
Quality control of hard disk manufacturers; for example, in analysis of diamond films. Pharmaceutical - quality control, polymorphism, mapping formulations, reaction monitoring, combinational chemistry, fermentation broths. Environmental monitoring. Forensic analysis - drug abuse, explosives. Minerals, pigments and dyestuffs – ancient artefact authentication. Reaction monitoring - polymerisation processes in situ - polymer composition and structure. Semiconductors. Single molecule detection. Biochemistry – e.g., SERS in single living cells. Disease diagnosis – e.g., HIV, cancer.
Raman spectroscopy has evolved to include several variants of the normal dispersive technique and most notably two of these are SERS and the emerging technique TERS-AFM. From the perspective of pharmaceutical analysis Raman scattering has enabled the rapid noninvasive volumetric analysis of pharmaceutical formulations which could lead to many important applications in pharmaceutical settings, including imaging and the quantitative analysis of pharmaceutical tablets and capsules in process and quality control (3, 4). Recently there have been major advances in the area of deep non-invasive characterisation of diffusely scattering samples at depths not accessible by conventional Raman spectroscopy (>200 µm). As a consequence examples of emerging research activities include non-invasive diagnosis of bone disease and cancer, rapid quality control of pharmaceutical formulations and security screening of explosives and counterfeit drugs through unopened translucent bottles (5).
Table 1. Application of vibrational spectroscopy to chemical problems. Gas
Liquid
Solid
Finger Print
IR, Raman Microwave
IR, Raman MS NMR UV-Vis
Functional Groups
IR, Raman MS NMR
IR, Raman MS NMR
IR, Raman Microwaves Electron diffraction
IR, Raman MS NMR
IR, Raman MS X-Ray diff UV-Vis IR, Raman MS NMR Mössbauer IR, Raman MS NMR Mössbauer
t
Molecular Symmetry
Bond Distances Bond Angles
Electronic Structure
IR, Raman Microwaves Electron diffraction
EXAFS LCNMR
UV-Vis UPS ESR Resonance Raman
UV-Vis UPS ESR
X-Ray diff Neutron diffraction
UV-Vis UPS ESR Mössbauer NQR X-Ray diffraction Neutron diffraction
There are many variations of conventional Raman spectroscopy and attention is drawn to two variants, resonance Raman (RS) and SERS. SERS which was first reported by Fleishman et al. in 1974 (6) overcomes the relative low sensitivity of Raman scattering with the added advantage of fluorescence quenching. SERS occurs for a very large number of molecules adsorbed on or near to the surface of metals in a variety of morphologies. The largest enhancements (with surface enhancement factors (SEF) of 107– 1014 approaching single molecule detection are often claimed in the literature (7)) are observed when using visible light for the coinage metals silver, gold and copper roughened with features of submicrometre dimensions (between 20 – 300 nm). Detailed and some relatively conclusive descriptions of SERS models and mechanisms have been given. In these SERS models, two types of mechanisms are widely discussed: the electromagnetic enhancement effect (EM), which considers the change of the local electric field around the adsorbed molecule; the second phenomenon, the chemical enhancement (CE) mechanism, primarily involves a charge transfer (CT) state between the surface complex of the adsorbed molecule and a metal surface. There is still disagreement over the relative and absolute enhancement factors for each
mechanism. This is mainly due to the fact that EM and CT models are both critical roughness-based models (8). The discovery of SERS impacted on surface science and vibrational spectroscopy because of its extremely high and unique surface sensitivity. It is well known that the most important component of any reliable and general surface analytical method is its sensitivity. Raman scattering is a second order process, with very low cross sections. Hence the corresponding surface Raman intensities expected for a monolayer of adsorbates are typically less than 1 count per second (cps) when using standard Raman spectrometer systems. This low detection sensitivity is no longer a disadvantage; SERS can be employed since the signal from adsorbates on a SERS active surface is sufficiently strong. This phenomenon makes possible the development of in situ diagnostic probes for detailed molecular structure and orientation of surface species, and this is widely applicable to electrochemical, biological and other ambient interfaces. Interest in SERS has grown considerably as reflected in the thousands of papers published in the literature since its discovery. A recent book entitled “SERS and Pharmaceuticals” by Schlücker et al. presents SERS data on several classes of drugs including antipyretics, analgesics, antimalarials, anticarcinogenics and antimutagenics (9). Recently a novel variant of the SERS technique named SESORS (Surface enhanced spatially offset Raman spectroscopy) has been reported in the literature (10). In 2011 Stone and colleagues demonstrated for the first time that multiplexed surface enhanced Raman scattering (SERS) signals could be recovered, non-invasively, from a depth of 20 mm in tissues and reconstructed to produce a false colour image. Four unique ‘flavours’ of SERS nanoparticles (NPs) were injected into a porcine tissue block at the corners of a 10 mm square. A transmission Raman data cube was acquired and the signals were reconstructed using the unique peak intensities of each of the nanoparticles. A false colour image of the relative signal levels was produced, demonstrating the capability of multiplexed imaging of SERS nanoparticles using deep Raman spectroscopy. It was also demonstrated that Raman signals from SERS nanoparticles could be recovered non-invasively from samples 45–50 mm thick. This is a significant achievement in the ability to detect and identify vibrational fingerprints within tissue. This provides an opportunity to adapt these particles and technique for potential clinical applications for disease diagnosis where a tumour may not be readily accessible or surgery is too invasive. Another exciting emerging technique is the combined TERS-AFM which has recently shown great promise (11, 12). This has the potential to transform spectroscopic research and sample characterisation in many varied fields, including biology, semiconductors, and nanomaterials. Tipenhanced Raman mapping is a powerful technique that offers rich chemical information and high spatial resolution. The coupled TERS–AFM approach is now proving itself as a unique mapping tool with the capability of yielding a wealth of chemical and surface topological information simultaneously as demonstrated in the characterisation of membrane bound proteins and carbon nanotubes with very high spacial resolutions of between ∼ 20-50 nm. In recent years multivariate chemometrics coupled with SERS analysis have been shown to provide additional information by employing mathematically-driven data extraction methods (13). These approaches are valuable as they move the Raman analyst from a ‘stare and compare’ mode to a more objective conclusion. For example in a clinical study (reported in 2009) Principal Component
Analysis (PCA) was shown to be invaluable in the determination of post-transplantation rejection. Heart transplantation is the last resort therapy for patients with end-stage heart failure and its efficacy relies on the successful management of recipient immune responses (14). Endomyocardial biopsy is the current standard of care for post-transplantation rejection surveillance of cardiac allografts. Like other biopsy procedures, endomyocardial biopsy is invasive and subject to sampling errors and a less invasive and real-time in vivo diagnostic method is highly desirable. Optical techniques capable of in vivo and molecular-specific detection of disease markers are a continual necessity with the goal of replacing (highly) invasive biopsy procedures. Raman spectroscopy, with its chemical and molecular specificity, as well as its intrinsic detection capability, without the need for exogenous labelling, presents an attractive opportunity for minimally invasive in vivo diagnostics. It has been demonstrated that employing PCA of direct optical detection of specific biomolecules that distinguish a diseased state using Raman spectroscopy, demonstrates its potential for in vivo molecular diagnostics without the need for exogenous labelling. Raman spectroscopy can potentially become a clinically viable tool for minimally invasive, molecularspecific, in vivo diagnostics. The above are only two examples from the many being reported in the literature that demonstrate the potential of Raman spectroscopy as a molecular-specific optical biopsy tool for biomedicine. The direct optical detection of specific biomolecules that distinguish a diseased state using Raman spectroscopy demonstrates its potential for in vivo molecular diagnostics (15). Kinetic investigations employing SERS have also been reported (16, 17) along with acquisition of SERS spectra of intracellular and extracellular bacterial locations (18). SERS has seen much significant advancement in recent years and interest in this field, and the related TERS, is growing rapidly. The application of this technique is extremely diverse and is now being developed for applications in a host of multi-scientific disciplines. Detection and identification of single molecules represents the final goal of trace analysis and is of great scientific and practical interest in many fields, such as physics, chemistry, biology, medicine, pharmacology, materials, and environmental science. Fluorescence and SERS are the spectroscopic techniques which have achieved single molecule sensitivity. In a number of studies it has been demonstrated that SERS has enabled acquisition of spectra at concentrations of analyte as low as femto and atto molar (19-21). SERS has in recent years become one of the most popular research areas of investigation in Raman spectroscopy from both a theoretical and practical laboratory investigative point of view. One indicator of the foregoing statement is the publication of a special issue of the Journal of Raman Spectroscopy (8): The discovery of SERS has had far-reaching consequences in both fundamental and applied research. It not only provides a stimulus for the study of enhanced optical scattering from interfaces, but also opened up a new field of surface-enhanced spectroscopy that includes surface enhanced second-harmonic generation (SE-SHG), surface enhanced infrared spectroscopy (SEIRS), surface-enhanced fluorescence (SEF) and surface-enhanced sum frequency generation (SE-SFG). It offers an ultrasensitive and in situ diagnostic probe for the determination of the detailed structure and orientation of molecules on the surface that is widely applicable to biological, electrochemical, catalytic and other ambient interfaces. In summary, it has been widely demonstrated that SERS has greatly advanced and gained wider application over the last few decades. The renewal of interest has been promoted significantly by the development of Raman instrumentation, laser technology, usability of instrumentation, and by the development of nanoscience. These have all contributed to the explosion of SERS papers that have been published since the mid 1990s. SERS is among the most interesting and complicated
subjects in nanoscience. Scientific advances are ongoing in almost all areas of SERS including the development of the latest SERS techniques examples of which are given in this article. References: 1. Madou, M.J. Fundamentals of Microfabrication: The Science of Minaturisation. CRC Press, 2nd edition. 752 (2002). 2. Raman, C.V. & Krishnan, K.S. The Raman Effect. Nature., 122, 12 (1928). 3. Buckley, K. & Matousek, P. Recent Advances in the Application of Transmission Raman Spectroscopy to Pharmaceutical Analysis. J. Biomed. Anal., 25; 55 (4):645-652 (2011). 4. Hargreaves, M.D., Macleod, N.A, Smith, M.R., Andrews, D., Hammond.S.V, & Matousek, P. Characterisation of Transmission Raman Spectroscopy for Rapid Quantitative Analysis of Intact Multi-Component Pharmaceutical Capsules. J. Pharm. and Biomed. Anal., 54, 3, 463-468 (2011). 5. Buckley, K. & Matousek, P. Non Invasive Analysis of Turbid Samples using Deep Raman Spectroscopy. Analyst., 7; 136 (15): 3039-3050 (2011). 6. Fleischmann, M., Hendra. P.J. & McQuillan. A.J. Raman Spectra of Pyridine Adsorbed at Silver Electrode. Chem. Phys. Lett., 26 (2): 163–166 (1974). 7. Le Ru, E.C., Blackie, E., Meyer, M. & Etchegoin, P.G. Surface Enhanced Raman Scattering Enhancement factor: A Comprehensive Study. J. Phys. Chem. C., 111 (37), 13794-13803 (2007). 8. Tian, Z. Q. (Guest Editor). Special Issue: Surface enhanced Raman spectroscopy. J. Raman Spectrosc., 36, 465-748 (2005). 9. Pînzaru, S. C. & Pavel, I. E. SERS and Pharmaceuticals, in Surface Enhanced Raman Spectroscopy: Analytical, Biophysical and Life Science Applications (ed S. Schlücker), WileyVCH Verlag GmbH & Co. KGaA, Weinheim, Germany. ch6. (2010). 10. Stone, N., Kerssens, M., Lloyd, G.R., Faulds. F., Graham. D. & Matousek, P. Surface Enhanced Spatially Offset Raman Spectroscopic (SESORS) Imaging – The Next Dimension, Chem. Sci., 2, 776-780 (2011). 11. Böhme, R., Cialla, D., Richter, M., Rösch, P., Popp, J. & Deckert, V. Biochemical Imaging Below the Diffraction Limit – Probing Cellular Membrane Related Structures by TipEnhanced Raman Spectroscopy (TERS). J. Biophotonics; SPECIAL ISSUE: Advanced Micro and Nanoscopy for Biomedicine., 3(7): 455–461 (2010). 12. Chan, A.K.L. & Kazarian, S.G. Tip-Enhanced Raman mapping with Top-Illumination AFM. Nanotechnology., 22, 175701 (2011). 13. Cheung, W., Shadi, I.T. ., Xu, Y. & Goodacre, R. Quantitative Analysis of the Banned Food Dye Sudan-1 Using Surface Enhanced Raman Scattering with Multivariate Chemometrics. J. Phys. Chem. C., 114(16): 7285-7290 (2010). 14. Chung, Y.G., Tu, Q., Cao, D., Harada, S., Eisen, H.J., Chang, C. Raman Spectroscopy Detects Cardiac Allograft Rejection with Molecular Specificity. Clinical and Translational Science., 2 (3), 206–210 (2009). 15. Ellis, D.I. & Goodacre, R. Metabolic fingerprinting in disease diagnosis: biomedical applications of infrared and Raman spectroscopy. Analyst., 131, 875-885 (2006). 16. Hollywood, K., Shadi, I.T. & Goodacre, R. Monitoring the Succinate Dehydrogenase Activity Isolated from Mitochondria by Surface Enhanced Raman Scattering. J. Phys. Chem. C., 114(16): 7308-7313 (2010). 17. Shadi, I.T., Chowdhry, B.Z., Snowden, M.J. &Withnall, R. Analysis of the Conversion of Indigo into Indigo Carmine Dye using SERRS. Chem. Comm., 1436-1437 (2004).
18. Jarvis, R., Law, N., Shadi, I.T., O'Brien, P., Lloyd, J. & Goodacre, R. Surface-Enhanced Raman Scattering from Intracellular and Extracellular Bacterial Locations. Anal. Chem., 80 (17): 6741-6746 (2008). 19. Kneipp, K., Kneipp, H., Itzkan, I., Dasari, R. R. & Feld, M.S. Ultrasensitive Chemical Analysis by Raman spectroscopy, Chem. Rev., 99, 2957-2975 (1999). 20. Garrell, R.L. Anal. Chem., Surface-enhanced Raman spectroscopy, 61, 401A - 411A (1989). 21. Shadi, I.T., Chowdhry, B.Z., Snowden, M.J. & Withnall, R. Quantitative Detection of Alcian Blue 8GX in the Low Concentration Range Using Surface-Enhanced Resonance Raman Spectroscopy. J. Applied. Spec., 54, 384 - 389 (2000).
50 word biography for each author Iqbal T Shadi holds a PhD in Biological Chemistry and has developed analytical and imaging techniques, using laser Raman spectroscopy, applying SERS to solve chemical and biological problems. As a consequence he has reported novel tangible working models in the literature for potential widespread multi- disciplinary scientific applications. His most recent positions have been at the Manchester Interdisciplinary Biocentre (The University of Manchester) and Imperial College London (NHLI, Faculty of Medicine).
Roy Goodacre is Professor of Biological Chemistry at The University of Manchester. The research group’s (http://www.biospec.net/) interests are broadly within bioanalytical chemistry, and in the application of a combination of a variety of modern analytical techniques (including Raman, IR and MS) and advanced chemometrics and machine learning to the explanatory analysis of complex biological systems within a metabolomics context.
