Technology focus UNIVERSITY OF SALFORD INDUSTRIAL CENTRE/ A. STERNBERG / SCIENCE PHOTO LIBRARY PHOTO SCIENCE / STERNBERG A. CENTRE/ INDUSTRIAL SALFORD OF UNIVERSITY

60 | Chemistry World | April 2008 www.chemistryworld.org Raman reinvented Raman spectroscopy is no longer an insensitive technique. Tom Westgate finds out how this advancing technology offers new possibilities in biology and security

‘You need an information- hospital, and in airport security. The fortunate discoverer of so- rich technology to explore the In short Drug companies have taken an called surface-enhanced Raman nanoworld,’ says Renato Zenobi of  The sensitivity of active interest in developing Raman spectroscopy (SERS) was Martin the ETH Zurich, the Swiss Federal Raman spectroscopy as an anti-counterfeiting measure. Fleischmann, who would later Institute of Technology. You might has been multiplied by Scientists from Pfizer and the become embroiled in the cold be forgiven for not immediately the development and use Science and Technology Facilities fusion controversy. Fleischmann thinking of Raman spectroscopy as of substrates and tips Council (STFC) Rutherford and his collaborators confirmed the an example of such a technology, but that enhance the optical Appleton Laboratory in Oxfordshire, signals were enhanced by a factor Zenobi and many other researchers signal UK, have collaborated to develop of a million. Raman had, until then, around the world would disagree.  Raman offers the a Raman-based method that can been burdened by its insensitivity ‘There are existing tools available, advantage of identifying quantify the active pharmaceutical to very low levels of a sample. Three such as electron microscopy, atomic as well as detecting the ingredient within intact drug decades later enhancements as high force microscopy and scanning presence of a molecule capsules. as 14 orders of magnitude have been tunnelling microscopy, but they  The technique is The technique is based on reported, allowing the spectrum of a give you no chemical information already being used to spatially offset Raman spectroscopy single molecule to be collected. – a microscopy image can show you develop security and (SORS). An offset detector picks up For the SERS effect to be beautiful structures, but you don’t anti-counterfeiting photons that have travelled through observed, the molecule must be know what they are,’ he says. devices. the drug. Last year scientists adsorbed to the surface of a colloidal Zenobi’s team is developing new  Researchers are reported that the technique could be nanostructure, generally of gold techniques to increase the sensitivity now able to use it used to analyse drugs within their or silver atoms. When the laser of Raman. He suggests that of all the to investigate large packaging (see Chemistry World, hits the metal substrate it creates techniques that provide chemical biomolecules and probe March 2007, p9) and STFC scientists an enhanced local optical field. information, Raman is the one that living cells are investigating its use in a wider The molecule therefore feels an also offers high spatial resolution. range of applications, including the enhanced excitation which boosts And in recent decades, the detection of explosives in containers the intensity of the Raman signal. development of lasers and CCD and the non-invasive diagnosis of Katrin Kneipp of Harvard (charge coupled device) detectors bone disease and cancer. University and Massachusetts has produced step changes and Institute of Technology, in the US, brought smaller, faster instruments. Coming to the surface has been investigating SERS for Modified Raman techniques have For Raman spectroscopy to reach over 20 years. She believes SERS has already reached the fundamental into the realm of single molecule changed the perception of Raman. limit of chemical detection – single detection required the accidental ‘It was never used as a tool for trace molecules. Now, its ability to identify discovery, in 1974, that molecules of detection because the Raman effect single biomolecules and cells could pyridine on a rough silver surface is too weak’ says Kneipp. ‘Now, since see Raman taking a frontline role gave an unexpectedly strong Raman SERS, people look at Raman in a new in the molecular biology lab or scattering signal. way – as a sensitive tool.’ www.chemistryworld.org Chemistry World | April 2008 | 61 Technology focus

Detecting single molecules ever higher SERS enhancements PNAS holds fundamental interest for and single molecule detection is chemists and physicists, and to be ‘rather a distraction.’ Goodacre able to identify them from a Raman says he doesn’t care how strong spectrum makes SERS a promising the enhancement is, as long as the technology for security applications, analysis time is brought down to a or, as Kneipp puts it: ‘whenever few seconds, and the results can be you want not only to detect a single reproduced. molecule, but also find out: what Goodacre uses SERS to collect molecule?’ ‘fingerprint’ Raman spectra of As well as picking up spectra bacteria, which he uses to identify from single molecules, Kneipp unknown organisms. He thinks this is applying SERS to biochemical technology could be used to detect analysis. Her team has introduced contaminants in manufacturing or gold nanoclusters, measuring healthcare settings. But interpreting about 50 nm, into live cells. They the spectra is ‘incredibly difficult’, have been able to record the SERS says Goodacre. So reproducibility signatures arising from molecules is vital if the fingerprints are to be on the surfaces of the particles in checked against those from known different compartments of the cell. bacteria. ‘The particles can be considered as ‘The laser spot is about the same tiny nanosensors’ said Kneipp. ‘We width as a bacterium, which means can use them to probe the chemistry we can get a single cell spectrum,’ in the live cell.’ he says. ‘That’s very exciting, but the The rich variety of chemistry in bacteria themselves look different different cellular compartments Before this promise can Intensity: the electrical at the single cell level. So what we’d produces spectra that are difficult become reality, however, SERS field is enhanced up to 50 like to do is to illuminate a wider to interpret. To make sense of researchers must address the times (shown in red) by area, and collect a kind of average the complex data, Kneipp’s question of reproducibility in their gold disks spectrum. In doing that, you actually group compares the differences measurements. The enhancement reduce the reproducibility problem.’ between spectra obtained as effect is strongest at a few ‘hot Kneipp is hopeful that the the nanoparticles move around spots’ on the surface, which have ever-increasing understanding the cell. As well as leading to an particular nanoscale features. But of the nanoworld will bring the understanding of the cellular the number and location of hot spots necessary improvements to SERS chemistry, Kneipp and others is difficult to predict when making a substrate reliability, but Goodacre believe that the technique has new substrate. is tackling the problem through a potential for use in nanoscale ‘machine learning’ design process. biosensors that could be implanted Speeding up In collaboration with Ewan Blanch, and used to measure, for example, For Roy Goodacre of the University also at Manchester, Goodacre diabetes patients’ glucose levels. of Manchester, UK, the focus on applies mathematical algorithms to the many variables in the synthesis The Raman Effect of colloidal SERS substrates. Goodacre and Blanch hope to be The Raman effect was first vibrational state in two ways able to create a design template for

CORBIS reported 80 years ago in a when they are hit by a photon optimal substrates for any given paper in Nature on 31 March – by either increasing or sample. 1928. Chandrasekhara Vankata decreasing in energy. At room Raman went on to win the temperature, most molecules Hot tip 1930 Nobel Prize in physics for are in a low energy, or ground There may be only a few enhancing his discovery. The technique state, so a photon excites the sites on a typical SERS substrate, identifies a molecule’s structure molecule and is scattered at so ‘why not bring the hot site to the by probing its vibrational lower energy than the incident sample?’ asks Zenobi. This is the energies. Each molecule has its photon. This reduction in energy principle of tip enhanced Raman own fingerprint of vibrational is known as the Stokes shift. spectroscopy (TERS) which uses energy states – depending on its C V Raman If a molecule is in a a modified AFM (atomic force atomic make-up, and how the vibrationally excited state, the microscopy) tip to enhance the atoms bond to each other. The change in wavelength of the scattered photon will be at Raman effect. When light from a laser photons derives from a change higher energy than the incident The tip is made ‘hot’ by source is shone onto a sample to in the vibrational energy of the photon – a change known as the evaporating a thin silver layer on be identified, the vast majority molecule when the photon hits anti-Stokes shift. Both Stokes to it, which forms colloidal islands. of photons are scattered by the it. This energy exchange can be and anti-Stokes spectra contain ‘Every time you touch the sample sample at the same wavelength used to determine the molecular inverted versions of the same with the tip the signal can be several as the incident light. But a tiny structure. frequency information. And the orders of magnitude stronger than proportion of photons (around Chemical bonds have specific ratio of anti-Stokes to Stokes normal Raman,’ Zenobi says. He uses one in every 100 million) stretching frequencies that can intensity at any vibrational TERS to examine single molecules are scattered at a different be quantified via this photon- frequency can be used as a and also bacteria. wavelength – a phenomenon molecule energy exchange. highly accurate and non-contact It has another advantage over known as Raman scattering. Molecules can change their measure of temperature. SERS because the scanning tip can provide spatial resolution down to 62 | Chemistry World | April 2008 www.chemistryworld.org Technology focus Smaller, faster, simpler There is an increasing UK-based Horiba Jobin Yvon commercial drive to make Raman has focused on speed, and accessible to users with little introduced two new fast Raman experience and expertise. scanning technologies. SWIFTM AHURA SCIENTIFIC AHURA Ocean Optics’ new Raman and DuoScanTM. SWIFT spectra kit, for example, has (scanning with incredibly fast been designed to include all the times) can obtain an image with necessary components in one 50 000 spectra within case. The ‘plug-and-play’ kit 6 minutes, while DuoScanTM uses includes a sensitive spectrometer, specialised scanning hardware to laser and fibre optic probe and enable the image pixel size to be features. chosen. The company says that a Perkin Elmer, meanwhile, has combination of both technologies recently launched a bench-top Ahura Scientific’s handheld Raman system, TruScan could, for example, measure whole RamanStation 400, which is pharmaceutical tablets within ten intended to provide powerful Its DXR SmartRaman measurements are not necessary. minutes. spectroscopy at the push of a spectrometer has been developed The microscope is designed for ChemImage, in the US, button. It has no moving parts in specifically for quality control routine and rapid analysis to has developed a wide field the optical pathway, which makes laboratories. It is designed to identify particles with micrometre hyperspeed imaging platform acquiring the spectrum much take measurements on samples resolution. (Whip) technology, which can faster and simplifies the optical directly through glass or plastic Bruker Optics recently produce the first real-time Raman set-up. packaging, to save time and introduced a fully automated movies. And out of the lab, Ahura Thermo Fisher Scientific avoid contamination. And the Fourier transform Raman Scientific, also in the US, has has just launched two new DXR Raman microscope is fully spectrometer – to determine the introduced hand-held Raman technologies that also bring automated, so calibration and isomer ratios of the chemical spectrometers for security, routine Raman analysis into new set-up steps that would otherwise content of a sample – for routine, military and forensic settings laboratory settings. require an expert to optimise the non-destructive analysis. (see page 8).

10 or 20nm. The AFM is also used you can never know if attaching a the standard toolkit of techniques. to locate a point of interest. ‘Say large fluorophore alters the cellular But Blanch thinks differently. ‘We are you have a bacterium, you can park function in some way,’ he says. In not able to get a pretty PDB [protein the AFM tip on its tail,’ he says. An comparison, TERS allows the study data bank] structure yet,’ he admits. optical microscope is used to line up of real systems, and although the ‘But we can still get useful information the sample, tip and laser, then the tip itself can adversely affect the from Raman that biologists and Raman data can be collected. sample, it avoids complicated or biochemists actually find important.’ Zenobi’s team has reported TERS invasive sample preparation. Blanch’s team uses a technique signals in very dilute samples of TERS does not completely known as Raman optical activity dye. The signals were observed escape the reproducibility problem, (ROA) to gain insights into chiral in spots with relatively large gaps however. The tips are unstable, and structural features of proteins and between them, suggesting that single they are difficult to manufacture other biomolecules. ‘Basically, ROA molecules were responsible. And, so they are equally ‘hot’ every time. is the chiral form of Raman,’ says thanks to TERS, his group is at the ‘We have tiny But Zenobi hopes the engineering Blanch. ‘In normal Raman you don’t forefront of studying the chemical challenges that prevent them being really care what the polarisation state composition of sub-cellular parts. nanosensors mass-produced can be overcome. of the incident or scattered light is. But ‘There are very clever techniques to that can probe the polarisation state also contains a observe nanoscale components, such Chiral clarity lot of information.’ as fluorescent tagging, but you can the chemistry For most structural biologists, x-ray Different chiral forms of a only look at what you tagged. Also, in a live cell’ crystallography and NMR make up molecule scatter light with different

64 | Chemistry World | April 2008 www.chemistryworld.org Technology focus

as in situ diagnostic probes,’ says Blanch. Meanwhile, advances like these in the understanding of the mechanisms behind Alzheimer’s onset could help with development of inhibitors. Looking further ahead, Goodacre predicts Raman use will grow in the pharmaceutical sector as

COLIN CUTHBERT / SCIENCE PHOTO LIBRARY PHOTO SCIENCE / CUTHBERT COLIN tighter regulations over metabolic manufacturing of natural products come into force. Raman fits the bill for monitoring these fermentation processes as it is a non-invasive technique which doesn’t suffer from water interference, unlike its rival near-IR. Zenobi, meanwhile, is confident that single molecule TERS will be ‘hugely successful’ in nanoscience, particularly since nanostructures such as carbon nanotubes have strong Raman signals. ‘People are making nanowires out of carbon nanotubes and complicated systems from single molecules, but what you really have on the surface is polarisation, and also respond A three dimensional in protein folding. When this not well known,’ he says. Zenobi in different ways to circularly Raman map of a process goes wrong it can result also believes it will be possible to polarised light. So ROA can give rich nanoindentation, or in structures like amyloid plaques observe reactions on catalytic metal insights into the structural folds hardness, test on silicon – the insoluble protein aggregates surfaces, using TERS to identify and loops that define the function implicated in the development of reacting chemical species. ‘We have of biomolecules. Chiral features diseases such as Alzheimer’s. ROA started to study this. If we could do such as an alpha helix, a beta sheet, it, I’d be getting a call from or a certain RNA fold can be easily Sweden.’ recognised in the fingerprint. According to Blanch has shown that ROA Blanch, the basic is a valuable tool for analysing tools needed to unfolded proteins. He describes realise Raman’s these macromolecules as ‘nebulous, potential are spaghetti type structures’ that are already in very difficult to crystallise so were place. Some often ignored by biologists. ‘But they technological make up 20 to 40 per cent of our development protein, so they’re doing something.’ is needed Their unfolded nature makes before Raman these proteins flexible, and less scanners specialised. They are thought to are seen at be employed by some enzymes to airports and in make them adaptable to a variety of Electron micrograph hospitals, but substrates. ‘ROA has proven useful of a gold colloid, used more important because we can say more than just: in surface enhanced is improving that’s an unfolded structure. You Raman spectroscopy our understanding can get different types of unfolded (SERS) of what it takes to structure and we can look at the get a reliable result and differences,’ says Blanch. understand the data. ‘That Another advantage of Raman should make all these methods more techniques, according to Blanch, useful to biologists and chemists, is that these studies can be government regulators and the rest performed in conditions that are of us,’ he says. close to ‘real life’. Raman and ROA investigations can be performed on proteins in have led Blanch and Tom Westgate is a science writer solution, and can be used to monitor his colleagues to suggest based in Manchester, UK structural changes brought on by that PPII has a critical, changes in pH or temperature. intermediate role in these One of the disordered structures formation processes. Further reading Faraday Discuss., 2006, 132, 3 (DOI: 10.1039/ Blanch has studied – polyproline II ‘Other people are looking b600675m) helix (PPII) – has been recognised at the next step, which is to W H Zhang et al, J. Phys. Chem. C, 2007, 111,

KATRIN KNEIPP KATRIN as having an important role use Raman and IR [infrared] 1733 (DOI: 10.1021/jp064740r) 66 | Chemistry World | April 2008 www.chemistryworld.org