Non-invasive analysis of turbid samples using deep Raman spectroscopy

Raman spectroscopy has recently seen major advances in the area of deep non-invasive characterisation of diffusely scattering samples; this progress is underpinned by the emergence of spatially offset Raman spectroscopy and associated renaissance of transmission Raman spectroscopy permitting the characterisation of diffusely scattering samples at depths not accessible by conventional Raman spectroscopy. 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. This article reviews this field focusing on recent developments with high societal relevance.     

Non-invasive detection of cocaine dissolved in beverages using displaced Raman spectroscopy

We demonstrate the potential of Raman spectroscopy to detect cocaine concealed inside transparent glass bottles containing alcoholic beverages. A clear Raman signature of cocaine with good signal-to-noise was obtained from a ∼300 g solution of adulterated cocaine (purity 75%) in a 0.7 L authentic brown bottle of rum with 1 s acquisition time. The detection limit was estimated to be of the order of 9 g of pure cocaine per 0.7 L (∼0.04 moles L⁻¹) with 1 s acquisition time. The technique holds great promise for the fast, non-invasive, detection of concealed illicit compounds inside beverages using portable Raman instruments, thus permitting drug trafficking to be combated more effectively.     

Remote Raman and fluorescence studies of mineral samples

In the present study, we investigated remote laser-induced fluorescence (LIF), at a distance of 4.8 m, of a variety of natural minerals and rocks, and Hawaiian Ti (Cordyline terminalis) plant leaves. These minerals included calcite cleavage, calcite onex and calcite travertine, gypsum, fluorapatite, Dover flint and chalk, chalcedony and nephelene syenite, and rubies containing rock. Pulsed laser excitation of the samples at 355 and 266 nm often resulted in strong fluorescence. The LIF bands in the violet-blue region at approximately 413 and approximately 437 nm were observed only in the spectrum of calcite cleavage. The green LIF bands with band maxima in the narrow range of approximately 501-504 nm were observed in the spectra of all the minerals with the exception of the nephelene syenite and ruby rocks. The LIF red bands were observed in the range approximately 685-711 nm in all samples. Excitation with 532 nm wavelength laser gave broad but relatively low fluorescence background in the low-frequency region of the Raman spectra of these minerals. One microsecond signal gating was effective in removing nearly all background fluorescence (with peak at approximately 610 nm) from calcite cleavage Raman spectra, indicating that the fluorescence was probably from long-lifetime inorganic phosphorescence.     

Rapid in situ detection of street samples of drugs of abuse on textile substrates using microRaman spectroscopy

Trace amounts of street samples of cocaine hydrochloride and N-methyl-3,4-methylenedioxy-amphetamine (MDMA) on natural and synthetic textiles were successfully detected in situ using confocal Raman microscopy. The presence of some excipient bands in the spectra of the drugs did not prevent the unambiguous identification of the drugs. Raman spectra of the drugs were readily obtained without significant interference from the fibre substrates. Interfering bands arising from the fibre natural or synthetic polymer structure and/or dye molecules did not overlap with the characteristic Raman bands of the drugs. If needed, interfering bands could be successfully removed by spectral subtraction. Also, Raman spectra could be acquired from drug particles trapped between the fibres of highly fluorescent textile specimens. The total acquisition time of the spectra of the drug particles was 90 s accomplished non-destructively and without detachment from their substrates. Sample preparation was not required and spectra of the drugs could be obtained non-invasively preserving the integrity of the evidential material for further analysis.     

The use of fluorescence quenchers in resonance Raman spectroscopy

We discuss methods of increasing the resonance Raman signal to noise in the presence of a strong fluorescence background. By considering the time dependence of the competing processes, we conclude that for many commonly encountered systems the Raman signal can be enhanced over the fluorescence by introducing a quencher that decreases the lifetime of the radiative electronic state. The efficacy of this approach is demonstrated by the appearance of resonance Raman from fluorescein in water with the progressive addition of KI.     

Analysis of high-explosive samples by Fourier transform Raman spectroscopy

The application of spectroscopic techniques to the detection and identification of explosive materials is of considerable importance in forensic investigations. However, forensic scientists do not routinely use conventional Raman spectroscopy in their analysis. This is due to the problems of high background scatter and time-consuming sample alignment. The development of Fourier transform Raman spectroscopy has overcome these problems. Using this technique three samples of different batches of Semtex labelled Semtex A, Semtex B and Semtex C were analysed. Semtex A was found to contain the explosive cyclotrimethylenetrinitramine (RDX), Semtex B contained pentaerythritol tetranitrate (PETN) and Semtex C contained a mixture of RDX and PETN.     

Finite element simulations of tip-enhanced Raman and fluorescence spectroscopy

Finite element electromagnetic simulations of scanning probe microscopy tips and substrates are presented. The enhancement of the scattered light intensity is found to be as high as 10(12) for a 20 nm radius gold tip, and tip-substrate separation of 1 nm. Molecular resolution imaging (< 1 nm) is achievable, even with a relatively large radius tip (20 nm). We also make predictions for imaging in aqueous environments, noting a sizable red shift of the spectral peaks. Finally, we discuss signal levels, and predict that high-speed Raman mapping should be possible with gold substrates and a small tip-substrate separation (< 4 nm).     

Assessment of the ageing of triterpenoid paint varnishes using fluorescence,Raman and FTIR spectroscopy

The assessment of the influence of natural and artificial ageing on the spectrofluorescence of triterpenoid varnishes dammar and mastic is the focus of this work. Both Fourier transform infrared (FTIR) microscopy using attenuated total reflectance and Raman spectroscopy have been employed for complementary molecular analysis of samples. Synchronous fluorescence spectroscopy, excitation emission spectroscopy, and statistical analysis of data have been used to monitor changes in the optical properties of varnish samples. Assessment of naturally and artificially aged samples using excitation emission spectroscopy suggests that extensive exposure to visible light does not lead to easily appreciable differences in the fluorescence of mastic and dammar; cluster analysis has been used to assess changes, which occur with artificial ageing under visible light, indicating that differences in the fluorescence spectra of aged triterpenoids may be insufficient for their discrimination. The results highlight significant differences between the initial fluorescence of films of dammar and mastic and the fluorescence, which develops with ageing and oxidation, and specific markers, which change with ageing in FTIR and Raman spectra, have been identified.     

Characterization of thalidomide using Raman spectroscopy

Thalidomide is a potent anticancer therapeutic drug whose mechanism of action has not yet been elucidated. In this report, experimental Raman spectroscopy is used to determine and characterize the vibrational frequencies of the drug. These normal modes are then compared to their quantum mechanical counterparts, which have been computed using density functional theory. Upon analysis of the spectra, we found that there was a high level of agreement between the wavenumbers. As such, this spectroscopic technique may be a viable tool for examining the way in which this drug interacts with its target molecules.     

Picosecond time-resolved multiplex CARS spectroscopy using optical Kerr gating

We have developed a new spectroscopic system for picosecond time-resolved coherent anti-Stokes Raman scattering (CARS) measurements. Using the optical Kerr gating method in conjunction with a nanosecond laser-based CARS system, a time resolution of 1 ps has been achieved. All-trans retinal in 1-butanol has been measured. The observed time-resolved CARS spectra show changes in the 0–10 ps time range, which are ascribed to the photoisomerization dynamics of all-trans retinal in solution.     

Polymer crystallinity studied using Raman spectroscopy

The application of Raman spectroscopy to the study of crystallinity in polymers has been examined. In particular, Fourier transform (FT)-Raman spectroscopy has been applied in a number of studies in recent years to investigate crystallinity in a variety of polymers. The polymers discussed in this review are polyethylene, polystyrene, poly(ether ether ketone), polyamides, poly(ethylene terephthalate), elastomers, liquid crystalline polymers, inorganic polymers and certain polymer blends.     

A new aptameric biosensor for cocaine based on surface-enhanced Raman scattering spectroscopy

The present study reports the proof of principle of a reagentless aptameric sensor based on surface-enhanced Raman scattering (SERS) spectroscopy with "signal-on" architecture using a model target of cocaine. This new aptameric sensor is based on the conformational change of the surface-tethered aptamer on a binding target that draws a certain Raman reporter in close proximity to the SERS substrate, thereby increasing the Raman scattering signal due to the local enhancement effect of SERS. To improve the response performance, the sensor is fabricated from a cocaine-templated mixed self-assembly of a 3'-terminal tetramethylrhodamine (TMR)-labeled DNA aptamer on a silver colloid film by means of an alkanethiol moiety at the 5' end. This immobilization strategy optimizes the orientation of the aptamer on the surface and facilitates the folding on the binding target. Under optimized assay conditions, one can determine cocaine at a concentration of 1 muM, which compares favorably with analogous aptameric sensors based on electrochemical and fluorescence techniques. The sensor can be readily regenerated by being washed with a buffer. These results suggest that the SERS-based transducer might create a new dimension for future development of aptameric sensors for sensitive determination in biochemical and biomedical studies.     

Characterization of amino acids using Raman spectroscopy

A key process in the development of new drugs is elucidation of the interaction between the drug molecule and the target protein. Such knowledge then makes it possible to make systematic structural modifications of the drug molecule to optimize the interaction. Many analytical techniques can be applied to proteins in solution such as circular dichroism, ultraviolet, and fluorescence spectroscopy but these all have limitations. In this paper, we investigate the feasibility of using relatively simple, visible light Raman spectroscopic methods to investigate amino acids and related biopolymers.     

Coherent amplification in fluorescent dye solutions. III. On the merits of inverse Raman spectroscopy for the rejection of sample fluorescence

The value of the method of inverse Raman spectroscopy for the rejection of sample fluorescence has been investigated using synchronously-pumped mode-locked lasers. It is found that a rejection in excess of 10³ is obtained for a CS₂: ethanol solvent mixture (1:9) containing known amounts of fluorescent impurities. This marked fluorescence rejection arises from the inverse Raman process when the lasers lie to long wavelength of the electronic band origin and from the use of picosecond lasers whose pulse durations are markedly less than the lifetime of the fluorescing state.     

Rapid monitoring of antibiotics using Raman and surface enhanced Raman spectroscopy

Comparatively few studies have explored the ability of Raman spectroscopy for the quantitative analysis of microbial secondary metabolites in fermentation broths. In this study we investigated the ability of Raman spectroscopy to differentiate between different penicillins and to quantify the level of penicillin in fermentation broths. However, the Raman signal is rather weak, therefore the Raman signal was enhanced using surface enhanced Raman spectroscopy (SERS) employing silver colloids. It was difficult by eye to differentiate between the five different penicillin molecules studied using Raman and SERS spectra, therefore the spectra were analysed by multivariate cluster analysis. Principal components analysis (PCA) clearly showed that SERS rather than the Raman spectra produced reproducible enough spectra to allow for the recovery of each of the different penicillins into their respective five groups. To highlight this further the first five principal components were used to construct a dendrogram using agglomerative clustering, and this again clearly showed that SERS can be used to identify which penicillin molecule was being analysed, despite their molecular similarities. With respect to the quantification of penicillin G it was shown that Raman spectroscopy could be used to quantify the amount of penicillin present in solution when relatively high levels of penicillin were analysed (>50 mM). By contrast, the SERS spectra showed reduced fluorescence, and improved signal to noise ratios from considerably lower concentrations of the antibiotic. This could prove to be advantageous in industry for monitoring low levels of penicillin in the early stages of antibiotic production. In addition, SERS may have advantages for quantifying low levels of high value, low yield, secondary metabolites in microbial processes.     

Fast Raman spectroscopy of cytochrome c using intracavity resonance Raman amplification

The resonance Raman spectrum of cytochrome c (5 × 10^?4 M) was obtained using the method of resonance Raman amplification (RRA) in a dye laser resonator in 30 ns. The second harmonic of a Q-switched Nd glass laser was used as an excitation source and the spectra were recorded using a photographic plate.     

Raman amplification spectroscopy using pulsed dye lasers

A picosecond laser system consisting of a mode-locked argon-ion laser synchronously pumping two dye lasers is used for studies of Raman amplification spectra. The two dye laser beams, one kept constant in frequency while the other is tunable, coincide in the Raman sample. Recording the gain or the loss in intensity of one of the lasers as a function of frequency difference produces the Raman spectrum. Good signal to noise ratios have been obtained for a variety of liquids and solids. Fluorescing samples can be studied in the Inverse Raman method where the loss on the higher frequency laser is monitored.     

A Rheumatoid arthritis study using Raman spectroscopy

Rheumatoid arthritis (RA) is characterized by chronic inflammation of the joints and can lead to a progressive destruction of articular cartilage and bone. In this study, the specificity and sensitivity of the RA diagnostic methods based on the receiver-operating characteristic curves for monitoring C-reactive protein (CRP) and rheumatoid factor (RF) were compared with the Raman spectroscopic diagnostic method developed in this work. Sera from 24 patients with rheumatoid arthritis and from 16 healthy individuals were analyzed to assess the biochemical composition and presence of inflammatory activity by the aforementioned methods. By comparing with the clinical results for specificity and sensitivity from the RF and CRP tests, we show that the overall results from the newly developed Raman method were significantly better, with a specificity of 96%, a sensitivity of 88%, and correctly identifying 92% of the RA and healthy individuals, while the RF test gave a specificity of 100% and a sensitivity of 54%, and the CRP test gave a specificity of 87% and a sensitivity of 58%, respectively.