Ebook: Applications of Raman Spectroscopy to Biology
Raman spectroscopy has been known and used as a technique for 80 years, originally for the study of inorganic substances. Recent advances in underlying technology, such as lasers, detectors, filters and components, have transformed the technique into a very effective modern tool for studying complex biological problems. Professor Mahmoud Ghomi (of the University of Paris XIII) has edited this book on the applications of Raman spectroscopy to biology, covering in a readily accessible way the area from basic studies to the diagnosis of disease. The early chapters provide background information on basic principles underlying the main Raman methods covered in the book, with information on Surface-Enhanced Raman Scattering (SERS) and Surface-Enhanced Fluorescence (SEF), as well as giving accounts of applications to biomolecular and cellular investigations. Among the topics covered are studies of drugs and their complexes with biomolecules on nanoparticles, application of SERS to blood analysis, studies of single cells and of applications to human cancer diagnostics.This will be a useful book for experimental scientists in academic, governmental, industrial and clinical environments and for those entering the field of biomolecular spectroscopy.
From a fundamental point of view, Raman spectroscopy is based on the inelastic scattering of light by matter in one of its phases: gas, liquid and solid. The scattered light can have an energy (or a frequency) either higher, or lower than that of the incident light, giving rise to anti-Stokes or Stokes Raman effects, respectively. In the framework of quantum physics, the interaction of the light (photons) with matter, induces transitions between its vibrational states. Consequently, the bands observed on Raman spectra (representing the scattering intensity versus wavenumber, i.e. inverse of wavelength, routinely expressed in cm-1 units) reflect in fact these vibrational transitions.
An important fact in the field of molecular Raman spectroscopy is that the Raman spectrum of a molecule and its conformational properties are closely related. This is of a fundamental interest in the case of organic molecules, in which rotations around the chemical bonds (torsions), generally lead to the generation of a variety of conformers. Many of these conformers may coexist at normal conditions (temperature and pressure), because of the small amount of energy separating them in the molecular energy landscapes. Conformational transitions may be induced by varying temperature, pressure, or other physicochemical parameters, and can be detected by Raman spectroscopy through the variation of Raman intensity and/or vibrational wavenumbers. This is the reason why we generally speak of Raman conformational markers, which recall the Raman bands considered as the fingerprints of different conformations. This spectroscopic tool can also be used to recognize the interaction sites of two molecular partners, through the changes observed in the intensity or wavenumbers of the molecular groups involved in the interactions. Simultaneously, one can also follow the conformational changes of interacting molecular partners by means of their Raman conformational markers.
The application of Raman spectroscopy to biological molecules has rapidly grown up since the early 1970s, presumably because of the arrival of lasers (monochromatic and coherent sources of light) and modern dispersive spectrometers. The first period of this application was completely devoted to the constitution of a spectral database recorded from different important biological molecules (proteins, nucleic acids, phospholipids, etc) in aqueous solutions, i.e. natural medium of biological molecules and systems. Then the time arrived for studying more complex systems such as cells and tissues. This was especially made possible by the setup of microRaman spectrometers. Furthermore, the progress in microcomputer manufacturing together with the elaboration of rapid mathematical and statistical algorithms, could offer to Raman spectroscopy the possibility of acting as a powerful imaging tool. A Raman image, collected from a complex biological system, i.e. cell or tissue, can provide useful information on the localization or distribution of different constituting molecules. This is the point where global information (image) can also carry local (molecular) information. It was also natural to use Raman spectroscopy as a tool for diagnosis, for instance in analyzing metabolic effects in biological fluids, or alterations induced in tissues upon tumor formation, such as changes in DNA, protein and lipid conformations, or molecular degradation. Thanks to the continuing technical progress, portable miniature Raman spectrometers can be used in the future as a non-invasive tool for medical diagnosis.
Raman spectroscopy is widely recognized as a label-free technique; the collected signal in a spectrometer directly arises from the vibrational motions of different chemical groups of the analyzed molecule. Moreover, the contribution of water to Raman signal is rather low compared to infrared absorption. The weakness of this technique arises from its low intensity: only one scattered photon out of one million incident ones, contributes to the Raman scattering process. As a consequence, the samples used for recording Raman spectra are generally much more concentrated than those needed for other optical spectroscopic methods such as fluorescence and circular dichroism. To overcome this difficulty, two different approaches have been developed during the last years. The first one is the resonance Raman spectroscopy (RRS), which is based on the excitation of a sample by means of a monochromatic light, of which the wavelength falls within its molecular absorption spectrum. This phenomenon leads to a considerable enhancement of Raman intensity, and allows the sample concentration to be decreased. However, the absorption of light by biological molecules corresponding to their absorption spectra, generally located in the ultra-violet range of electromagnetic radiation, can cause irreversible damages due to their degradation. The second possibility is to use the Raman signal enhancement effect by the so-called plasmon resonance, when the analyzed molecule is adsorbed on the surface of a noble metal, i.e. gold or silver, colloid or nanoparticle. This method, known as surface enhanced Raman spectroscopy (SERS) has found a large audience for analyzing the biological molecules. SERS signal was shown to be enhanced up to several orders of magnitude, depending on the molecular affinity for binding to the metal surface, as well as on the molecular group involved in the adsorption. This technique is gradually becoming a routine tool in pharmacology and molecular biology for analyzing molecular samples at very low concentrations.
Surface-enhanced Raman scattering (SERS) is a specific technique of Raman spectroscopy when signal enhancement of several orders is achieved for numerous molecules placed in the closest vicinity of certain rough metal surfaces. Despite of some experimental difficulties and undesirable concomitant effects, this technique provides numerous promises for both highly specific and sensitive measurements. This chapter reviews the state-of-the-art in the field of SERS applications in biomolecular, cellular and biomedical studies. Prior to this, the reader is familiarized with the basics of SERS phenomenon and its experimental aspects including the most common or promising types of SERS-active substrates.
Localized surface plasmon resonances supported on metal nanoparticles cause an enhancement of the Raman and fluorescence spectra. While SERS is principally due to the first layer of molecules deposited on the metal surface, SEF experiments need to be successful to establish a distance between fluorophore and nanoparticles. This distance can be fixed by placement of a new molecule named spacer or by aggregation of the fluorescent molecule. The anti tumoral drugs emodin and hypericin belong to the group of natural anthraquinones dyes and both present and acid-base equilibrium. At several pHs molecules aggregate making possible some molecules to be situated at a certain distance of the nanoparticles surface and so to obtain fluorescence enhancement. SEF and SERS experiments recorded simultaneously permit to obtain vibrational and electronic information at the same time. Thus the aggregation, ionization and molecular structure of emodin and hypericin can be investigated. The binding of the dyes to biomolecules can also be studied through these spectroscopic techniques enhanced by metal nanoparticles.
Raman scattering was shown to be an efficient tool for analyzing the interactions of natural (phosphate) and chemically modified phosphorothioate oligodeoxynucleotides with an amphipathic cationic peptide adopting an α-helix structure in aqueous environment. Examination of the Raman spectra recorded from different partners and their complexes, leads us to conclude that these interactions cannot be interpreted within a restricted electrostatic framework. The cationic peptide can undergo an order-to-disorder transition upon interaction with oligodeoxynucleotides. Furthermore, it can interact with different structural fragments of their partners (nucleoside, backbone). However, these interactions appear to be sequence-dependent and stronger with natural oligodeoxynucleotides compared to their chemically modified analogues.
Over the last decade, there is a surge on researchers utilizing Surface Enhanced Raman Scattering (SERS) spectroscopy for blood analysis for various biomedical applications. This chapter starts with a brief review of SERS substrates and Raman instrumentation for SERS detection. Then four types of SERS based blood analysis methodologies are discussed with application examples including (1) label-free SERS methods for detection of intrinsic ingredients and exogenous substances in blood, sera, and plasma; (2) detection of circulating tumor cells in blood by SERS tags for cancer diagnosis; (3) labeling-free SERS analyses of human serum and plasma for disease diagnoses; and (4) combining membrane electrophoresis for serum protein separation with SERS analysis for cancer detection. At the end, we present a new technology based on electroporation for nanoparticle delivery into blood cells for intracellular SERS analysis.
Single cell studies for biomedical applications gain increased importance. Raman spectroscopy, especially when combined with optical tweezers, has proven to be a valuable tool for cancer cell screening, malaria detection, stem cell research as well as bacterial identification only to name a few applications. Here, basic principles behind methods, experimental design and parameters as well as expected results will be discussed. The goal is to give insight into experimental possibilities but also shortcomings that have to be dealt with regarding single cell Raman spectroscopy.
An overview will be presented of the development and application of Raman microspectroscopy to the study of living cells. A brief history of key events will be succeeded by a description of state-of-the-art instrumentation. An example of a protocol for the conversion of measured data to real data will be discussed. Data-analysis techniques, which are of profound importance to analyse large hyperspectral datasets, will be presented and compared. Data-analysis approaches highlight the potential for data-mining in hyperspectral Raman microscopy and spectral imaging in general. The development of micro-bioreactors, which are efficiently coupled to Raman microspectrometers enable monitoring of the cell- and tissue development under close to in vivo conditions.
Cancer poses a massive global health burden with rates set to double over the next decade. In the UK, it is estimated that 42% of the population will experience a cancer diagnosis in their lifetime and for 64% of these, cancer will cause their death. Surgical excision holds the greatest chance of cure for solid tumours yet once spread beyond the primary site has occurred survival rates decrease rapidly. Early detection of cancer allows early intervention with, potentially, the greatest chance of cure. Raman spectroscopy has the ability to detect biochemical alterations in cells prior to any morphological changes a pathologist would identify. Cancer is believed to have genetic and environmental components to its aetiology. These factors depend on site and cancer biology. Many authors have explored the potential of Raman spectroscopy to discriminate between cancerous, non-cancerous and dysplastic (precancerous) cells. Such work has been undertaken using live cells, formalin-fixed cells, pellets and scaffolds as well as whole tissues and body fluids. Raman based technologies can be used as a screening tool for at risk populations. Another avenue for Raman would be in surgical theatre assessing margins at the time of an operation thus optimsing conservation of normal tissue whilst removing all the cancer. A further breakthrough would be the ability of Raman spectroscopy to diagnose cancer through a blood sample. The advantage this would confer would be greatly enhanced if the biological properties of the cancer could be recognized, it may be then possible to offer bespoke treatment, thus keeping some patients under surveillance whilst others have graded treatment ranging from surgery, radiotherapy to chemotherapy and combination therapies.