Raman Spectroscopy on Biological samples

Raman spectroscopy is a well established technique for studying a wide range of materials at a molecular level. It relies on the light scattering exerted by molecular vibrations when the light from a source, typically a laser source, impinges on the investigated sample. More in details, the molecular vibrations cause the laser light to be shifted in frequency of an amount equal to the frequency of the vibrations. As a result, a typical Raman spectrum is composed by several (and sometimes, by a large number of) peaks corresponding to the Raman shifts characteristic of the molecular vibrations. In this way Raman spectroscopy, like other spectroscopic techniques (e.g. Infra-Red absorption spectroscopy), is capable to provide a "fingerprint" of the investigated sample, as each molecule and/or macro-molecule holds its own vibrations and frequencies. In our laboratory Raman spectroscopy is largely used for the analysis of biological materials [1-3], ranging from more simple molecules such as amino acids, to larger proteins investigation, and arriving to entire cells analysis. The study of cells is a very challenging task, since one cell include a large variety of biomolecules, from proteins to lipids, from DNA/RNA bases to enzymes, primary metabolites and saccharides. Consequently the Raman spectrum of cells is a very rich one, with many peaks and bands that can easily overlap each other and that require refined mathematical tools for their deconvolution and separation [4]. In the past we have combined microRaman spectroscopy and cells micromanipulation (more in details cells microinjections) to study the nanotoxic effects of metal nanoparticles (NPs) [5]. Very small amounts of silver and iron-oxide NPs have been injected into HeLa cells; NPs solutions and microinjections have been adjusted so that to deliver 10-50NPs to each single cell. After a short incubation time, the injected cells have been studied at the single cell level by means of Raman micro-spectroscopy, thus highlighting the short-time cellular response to NPs inclusion. We have found that, despite the small amount of NPs, cells lower their glycogen levels to provide energy to the Glutathione pathway for controlling the reactive oxygen species (ROS) effects. The increase of ROS levels is a well known effect of NPs, due to their high surface reactivity. In this study principal component analysis (PCA) has been employed to discriminate between injected and non-injected cells and to highlight the differences due to the presence of nanoparticles.
 


Also more fundamental studies are carried out for testing multivariate mathematical procedures proposed as tools for retrieving information on the amount of chemical species. One challenge in Raman spectroscopy applied to biological and medical problems is not only the identification of chemical species but above all to find out how much these species are present. That is, from a spectroscopic point of view, to relate the peak intensities to chemical concentrations. We are using a simple biological model for validating these mathematical procedures. We have collected a Raman database of the 20 standard amino acids; then we are mixing them up in well defined concentrations and analyzing the resulting mixtures. In this way we can go from easy-to-test mixtures, such as binary mixtures, to more complicated ones, in which at least 6 constituents (amino acids) are mixed together. And further we can consider the natural occurring "mixtures", i.e. peptides, polypeptides and proteins, which are larger and larger assemblies of the same 20 constituting unit-blocks, i.e. amino acids. As an example we have shown that extended multiplicative signal correction (EMSC), earlier proposed also for retrieving quantitative biochemical information of the samples, show strong limitations in real complex biological systems, due to macromolecular vibrations occurring in complex large molecules (such as peptides and proteins) but not present in simple mixtures of amino acids [6]. In this context we have also developed a freeware package for Raman spectra processing and analysis, called RamanToolSet [7]. The other aspect under investigation is the use of metal nanostructures as plasmonic resonators capable to enhance the Raman signal, thus giving rise to SERS, surface enhanced Raman scattering. More details concerning this research line can be found in the "Plasmonic Nanodevices" section of our website.  



REFERENCES
[1] G. Das, F. Mecarini, F. Gentile, F. De Angelis, H.G. Mohan Kumar, P. Candeloro, C. Liberale, G. Cuda, E. Di Fabrizio, "Nano-patterned SERS substrate: Application for protein analysis vs. temperature", Biosensors and Bioelectronics 24, 1693-1699 (2009)
[2] G. Das, R. La Rocca, T. Lakshmikanth, F. Gentile, R. Tallerico, L.P. Zambetti, J. Devitt, P. Candeloro, F. De Angelis, E. Carbone, E. Di Fabrizio, "Monitoring human leukocyte antigen class I molecules by micro-Raman spectroscopy at single cell level", Journal of Biomedical Optics 15(2), 27007 (2010)
[3] G. Das, A. Nicastri, M.L. Coluccio, F. Gentile, P. Candeloro, G. Cojoc, C. Liberale, F. De Angelis, E. Di Fabrizio, "FT-IR, Raman, RRS Measurements and DFT Calculation for Doxorubicin", Microscopy Research and Technique 73, 991 (2010)
[4] G. Das, F. Gentile, M.L. Coluccio, A.M. Perri, A. Nicastri, F. Mecarini, G. Cojoc, P. Candeloro, C. Liberale, F. De Angelis, E. Di Fabrizio, "Principal component analysis based methodology to distinguish protein SERS spectra", Journal of Molecular Structure 993, 500-505 (2011)
[5] P. Candeloro, L. Tirinato, N. Malara, A. Fregola, E. Casals, V. Puntes, G. Perozziello, F. Gentile, M. L. Coluccio, G. Das, C. Liberale, F. De Angelis, E. Di Fabrizio, "Nanoparticle microinjection and Raman spectroscopy as tools for nanotoxicology studies", Analyst 136, 4402 (2011)
[6] P. Candeloro, E. Grande, R. Raimondo, D. Di Mascolo, F. Gentile, M.L. Coluccio, G. Perozziello, N. Malara, M. Francardi, E. Di Fabrizio, "Raman database of Amino Acids solutions: a critical study of Extended Multiplicative Signal Correction", Analyst DOI:10.1039/C3AN01665J
[7] RamanToolSet is a free software for Raman spectra processing and analysis available at: https://my.cloudme.com/patrizio.candeloro/RamanToolSet