Discrimination of bacteria and bacteriophages by Raman spectroscopy and surface-enhanced Raman spectroscopy

Detection of pathogenic organisms in the environment presents several challenges due to the high cost and long times typically required for identification and quantification. Polymerase chain reaction (PCR) based methods are often hindered by the presence of polymerase inhibiting compounds and so direct methods of quantification that do not require enrichment or amplification are being sought. This work presents an analysis of pathogen detection using Raman spectroscopy to identify and quantify microorganisms without drying. Confocal Raman measurements of the bacterium Escherichia coli and of two bacteriophages, MS2 and PRD1, were analyzed for characteristic peaks and to estimate detection limits using traditional Raman and surface-enhanced Raman spectroscopy (SERS). MS2, PRD1, and E. coli produced differentiable Raman spectra with approximate detection limits for PRD1 and E. coli of 10(9) pfu/mL and 10(6) cells/mL, respectively. These high detection concentration limits are partly due to the small sampling volume of the confocal system but translate to quantification of as little as 100 bacteriophages to generate a reliable spectral signal. SERS increased signal intensity 10(3) fold and presented peaks that were visible using 2-second acquisitions; however, peak locations and intensities were variable, as typical with SERS. These results demonstrate that Raman spectroscopy and SERS have potential as a pathogen monitoring platform.     

Discrimination of bacteria using surface-enhanced Raman spectroscopy

Raman spectroscopy has recently been shown to be a potentially powerful whole-organism fingerprinting technique and is attracting interest within microbial systematics for the rapid identification of bacteria and fungi. However, while the Raman effect is so weak that only approximately 1 in 10(8) incident photons are Raman scattered (so that collection times are in the order of minutes), it can be greatly enhanced (by some 10(3)-10(6)-fold) if the molecules are attached to, or microscopically close to, a suitably roughened surface, a technique known as surface-enhanced Raman scattering (SERS). In this study, SERS, employing an aggregated silver colloid substrate, was used to analyze a collection of clinical bacterial isolates associated with urinary tract infections. While each spectrum took 10 s to collect, to acquire reproducible data, 50 spectra were collected making the spectral acquisition times per bacterium approximately 8 min. The multivariate statistical techniques of discriminant function analysis (DFA) and hierarchical cluster analysis (HCA) were applied in order to group these organisms based on their spectral fingerprints. The resultant ordination plots and dendrograms showed correct groupings for these organisms, including discrimination to strain level for a sample group of Escherichia coli, which was validated by projection of test spectra into DFA and HCA space. We believe this to be the first report showing bacterial discrimination using SERS.     

Discrimination between ricin and sulphur mustard toxicity in vitro using Raman spectroscopy.

A Raman spectroscopy cell-based biosensor has been proposed for rapid detection of toxic agents, identification of the type of toxin and prediction of the concentration used. This technology allows the monitoring of the biochemical properties of living cells over long periods of time by measuring the Raman spectra of the cells non-invasively, rapidly and without use of labels (Notingher et al. 2004 doi:10.1016/j.bios.2004.04.008). Here we show that this technology can be used to distinguish between changes induced in A549 lung cells by the toxin ricin and the chemical warfare agent sulphur mustard. A multivariate model based on principal component analysis (PCA) and linear discriminant analysis (LDA) was used for the analysis of the Raman spectra of the cells. The leave-one-out cross-validation of the PCA-LDA model showed that the damaged cells can be detected with high sensitivity (98.9%) and high specificity (87.7%). High accuracy in identifying the toxic agent was also found: 88.6% for sulphur mustard and 71.4% for ricin. The prediction errors were observed mostly for the ricin treated cells and the cells exposed to the lower concentration of sulphur mustard, as they induced similar biochemical changes, as indicated by cytotoxicity assays. The concentrations of sulphur mustard used were also identified with high accuracy: 93% for 200 microM and 500 microM, and 100% for 1,000 microM. Thus, biological Raman microspectroscopy and PCA-LDA analysis not only distinguishes between viable and damaged cells, but can also discriminate between toxic challenges based on the cellular biochemical and structural changes induced by these agents and the eventual mode of cell death.     

In vivo detection of dysplastic tissue by Raman spectroscopy

The detection of dysplasia and early cancer is important because of the improved survival rates associated with early treatment of cancer. Raman spectroscopy is sensitive to the changes in molecular composition and molecular conformation that occur in tissue during carcinogenesis, and recent developments in fiber-optic probe technology enable its application as an in vivo technique. In this study, the potential of Raman spectroscopy for in vivo classification of normal and dysplastic tissue was investigated. A rat model was used for this purpose, in which dysplasia in the epithelium of the palate was induced by topical application of the carcinogen 4-nitroquinoline 1-oxide. High quality in vivo spectra of normal and dysplastic rat palate tissue, obtained using signal integration times of 100 s were used to create tissue classification models based on multivariate statistical analysis methods. These were tested with an independent set of in vivo spectra, obtained using signal collection times of 10 s. The best performing model, in which signal variance due to signal contributions of the palatal bone was eliminated, was able to distinguish between normal tissue, low-grade dysplasia, and high-grade dysplasia/carcinoma in situ with a selectivity of 0.93 and a sensitivity of 0.78 for detecting low-grade dysplasia and a specificity of 1 and a sensitivity of 1 for detecting high-grade dysplasia/ carcinoma in situ.     

Oxygenation monitoring of tissue vasculature by resonance Raman spectroscopy

Resonance Raman spectroscopy offers a mechanism for the noninvasive measurement of in vivo and in situ hemoglobin oxygen saturation (HbO(2)Sat) in living tissue. Clinically informative signals can be provided by resonance enhancement with deep violet excitation. It is notable that fluorescence does not significantly degrade the quality of the signals. During the controlled hemorrhage and resuscitation of rats, signal intensity ratios of oxy- vs. deoxyhemoglobin from sublingual mucosa correlated with co-oximetry values of blood withdrawn from a central venous catheter. The spectroscopic application described here has potential as a noninvasive method for the diagnosis of clinical shock and guidance of its therapy.     

Discrimination of serum Raman spectroscopy between normal and colorectal cancer using selected parameters and regression-discriminant analysis

Raman spectroscopy of tissues has been widely studied for the diagnosis of various cancers, but biofluids were seldom chosen as the analyte because of the low concentration. Herein, serum of 30 normal people, 46 colon cancer, and 44 rectum cancer patients were measured using Raman spectra and analyzed. The information of Raman peaks (intensity and width) and that of the fluorescence background (baseline function coefficients) were selected as parameters for statistical analysis. Principal component regression (PCR) and partial least square regression (PLSR) were used on the selected parameters separately to see the diagnosing performance of the parameters. PCR performed better than PLSR in our spectral data. Then linear discriminant analysis (LDA) was used on the principal components (PCs) of the two regression methods on the selected parameters, and the diagnostic accuracy were 88% and 83%. The conclusion is that the selected parameters can maintain the information of the original spectra well and Raman spectroscopy of serum has the potential for the diagnosis of colorectal cancer.     

Determination of water concentration in brain tissue by Raman spectroscopy

Brain edema is one of the most common morbidity factors in patients with intracranial neoplasms and cerebrovascular pathology. Monitoring of intracranial pressure gives only an indirect and global measure of brain swelling. We have made an assessment of the applicability of Raman spectroscopy as an alternative method for assessing brain edema, which measures the water concentration in the tissue directly. Partial least-squares models were developed on the basis of Raman spectra measured in the 2600-3800-cm(-1) region, which predict the water fraction of brain tissue in the 0.75-0.95 range, with an accuracy better than 0.01.     

Noninvasive Raman spectroscopy of human tissue in vivo

We report the first transcutaneous Raman spectrum of human bone in vivo obtained at skin-safe laser illumination levels. The spectrum of thumb distal phalanx was obtained using spatially offset Raman spectroscopy (SORS), which provides chemically specific information on deep layers of human tissue, well beyond the reach of existing comparative approaches. The spectroscopy is based on collecting Raman spectra away from the point of laser illumination using concentric rings of optical fibers. As a generic analytical tool this approach paves the way for a range of uses including disease diagnosis, noninvasive probing of pharmaceutical products, biofilms, catalysts, paints, and in dermatological applications.     

Detection of anions by normal Raman spectroscopy and surface-enhanced Raman spectroscopy of cationic-coated substrates

The use of normal Raman spectroscopy and surface-enhanced Raman spectroscopy (SERS) of cationic-coated silver and gold substrates to detect polyatomic anions in aqueous environments is examined. For normal Raman spectroscopy, using near-infrared excitation, linear concentration responses were observed. Detection limits varied from 84 ppm for perchlorate to 2600 ppm for phosphate. In general, detection limits in the ppb to ppm concentration range for the polyatomic anions were achieved using cationic-coated SERS substrates. Adsorption of the polyatomic anions on the cationic-coated SERS substrates was described by a Frumkin isotherm. The SERS technique could not be used to detect dichromate, as this anion reacted with the coatings to form thiol esters. A competitive complexation method was used to evaluate the interaction of chloride ion with the cationic coatings. Hydrogen bonding and pi-pi interactions play significant roles in the selectivity of the cationic coatings.     

Discrimination between virgin and recycled polystyrene containers by Fourier transform infrared spectroscopy and principal component analysis

A rapid and reliable method for discriminating virgin and recycled expanded polystyrene (EPS) containers was developed using Fourier transform infrared spectroscopy combined with principal component analysis. Standard normal variate, first and second‐order derivative spectra were compared for the discrimination results. The results show that carbonyl region (1780‐1620 cm^?1) spectra using first derivative transformation give the optimum classification results. In addition, the carbonyl compounds in EPS containers were detected to clarify the chemical difference between virgin and recycled containers, with a higher concentration of carbonyl compounds observed in recycled EPS containers. The combination of carbonyl region of Fourier transform infrared spectroscopy with chemometrics proved to be a promising method to discriminate virgin and recycled EPS containers, which could function as an additional tool for quality control of plastics.     

Multicomponent blood analysis by near-infrared Raman spectroscopy

We demonstrate the use of Raman spectroscopy to measure the concentration of many important constituents (analytes) in serum and whole blood samples at physiological concentration in vitro across a multipatient data set. A near-infrared (830-nm) diode laser generates Raman spectra that contain superpositions of Raman signals from different analytes. Calibrations for glucose, cholesterol, urea, and other analytes are developed by use of partial least-squares cross validation. We predict six analytes in serum with significant accuracy in a 66-patient data set, using 60-s spectra. The calibrations are shown to be fairly robust against system drift over the span of seven weeks. In whole blood, a preliminary analysis yields accurate predictions of some of the same analytes and also hematocrit. The results hold promise for potential medical applications.     

Monitoring cellular behaviour using Raman spectroscopy for tissue engineering and regenerative medicine applications

Raman spectroscopy has been used to determine the chemical composition of materials for over 70 years. Recent spectacular advances in laser and CCD camera technology creating instruments with higher sensitivity and lower cost have initiated a strong resurgence in the technique, ranging from fundamental research to process control methodology. One such area of increased potential is in tissue engineering and regenerative medicine (TERM), where autologous cell culture, stem cell biology and growth of human cells on biomaterial scaffolds are of high importance. Traditional techniques for the in vitro analysis of biochemical cell processes involves cell techniques such as fixation, lysis or the use of radioactive or chemical labels which are time consuming and can involve the perpetuation of artefacts. Several studies have already shown the potential of Raman spectroscopy to provide useful information on key biochemical markers within cells, however, many of these studies have utilised micro- or confocal Raman to do this, which are not suited to the rapid and non-invasive monitoring of cells. For this study a versatile fit-for-purpose Raman spectrometer was used, employing a macro-sampling optical platform (laser spot size 100 μm at focus on the sample) to discriminate between different TERM relevant cell types and viable and non-viable cells. The results clearly show that the technique is capable of obtaining Raman spectra from live cells in a non-destructive, rapid and non-invasive manner, however, in these experiments it was not possible to discriminate between different cell lines. Despite this, notable differences were observed in the spectra obtained from viable and non-viable cells, showing significant changes in the spectral profiles of protein, DNA/RNA and lipid cell constituents after cell death. It is evident that the method employed here shows significant potential for further utilisation in TERM, providing data directly from live cells that fits within a quality assurance framework and provides the opportunity to analyse cells in a non-destructive manner.     

Determination of nanotubes properties by Raman spectroscopy

The basic concepts and characteristics of Raman spectra from single-wall carbon nanotubes (SWNTs, both isolated and bundled) are presented. The physical properties of the SWNTs are introduced, followed by the conceptual framework and characteristics of their Raman spectra. Each Raman feature, namely the radial breathing mode, the tangential G band, combination modes and disorder-induced bands are discussed, addressing their physical origin, as well as their capability for characterizing SWNT properties.     

Single-pollen analysis by laser-induced breakdown spectroscopy and Raman microscopy

The application of laser-induced breakdown spectroscopy to the analysis of single biological microparticles (bioaerosols) is described, exemplified here for a range of pollens. Spectra were recorded by exposure of the pollen to a single laser pulse from a Nd:YAG laser (lambda = 1064 nm, Ep approximately 30 mJ). The intensities of the single-pulse laser-induced breakdown spectra fluctuated dramatically, but an internal signal calibration procedure was applied that referenced elemental line intensities to the carbon matrix of the sample (represented by molecular bands of CN and C2). This procedure allowed us to determine relative element concentration distributions for the different types of pollen. These pollens exhibited some distinct concentration variations, for both major and minor (trace) elements in the biomatrix, through which ultimately individual pollens might be identified and classified. The same pollen samples were also analyzed by Raman microscopy, which provided molecular compositional data (even with spatial resolution). These data allowed us to distinguish between biological and nonbiological specimens and to obtain additional classification information for the various pollen families, complementing the laser-induced breakdown spectroscopy measurement data.     

In vivo glucose measurement by surface-enhanced Raman spectroscopy

This paper presents the first in vivo application of surface-enhanced Raman scattering (SERS). SERS was used to obtain quantitative in vivo glucose measurements from an animal model. Silver film over nanosphere surfaces were functionalized with a two-component self-assembled monolayer, and subcutaneously implanted in a Sprague-Dawley rat such that the glucose concentration of the interstitial fluid could be measured by spectroscopically addressing the sensor through an optical window. The sensor had relatively low error (RMSEC = 7.46 mg/dL (0.41 mM) and RMSEP = 53.42 mg/dL (2.97 mM).     

Coded aperture Raman spectroscopy for quantitative measurements of ethanol in a tissue phantom

Coded aperture spectroscopy allows for sources of large étendue to be efficiently coupled into dispersive spectrometers by replacing the traditional input slit with a patterned mask. We describe a coded aperture spectrometer optimized for Raman spectroscopy of diffuse sources, (e.g., tissue). We provide design details of the Raman system, along with quantitative estimation results for ethanol at non-toxic levels in a lipid tissue phantom. With 60 mW of excitation power at 808 nm, leave-one-out and blind cross-validation of partial least squares (PLS) regression models achieve r(2) > 0.98. Leave-one-out cross-validation demonstrates prediction errors of <15% at the common legal limit for intoxication (17.4 mmol/L = 0.08% by vol) and the best blind cross-validation achieves <12% error at this concentration.     

Determination of technetium and its speciation by surface-enhanced Raman spectroscopy

Technetium-99 (Tc) is an important radionuclide of concern, and there is a great need for its detection and speciation analysis in the environment. For the first time, we report that surface-enhanced Raman spectroscopy (SERS) is capable of detecting an inorganic radioactive anion, pertechnetate (TcO4-), at approximately 10(-7) M concentration levels. The technique also allows the detection of various species of Tc such as oxidized Tc(VII) and reduced and possibly complexed Tc(IV) species by use of gold nanoparticles as a SERS substrate. The primary Raman scattering band of Tc(VII) occurs at about 904 cm-1, whereas reduced Tc(IV) and its humic and ethylenediaminetetraacetic acid (EDTA) complexes show scattering bands at about 866 and 870 cm-1, respectively. Results also indicate that Tc(IV)-humic complexes are unstable and reoxidize to TcO4- upon exposure to oxygen. This study demonstrates that SERS could potentially offer a new tool and opportunity in studying Tc and its speciation and interactions in the environment at low concentrations.     

Progress in Raman spectroscopy in the fields of tissue engineering, diagnostics and toxicological testing

This review summarises progress in Raman spectroscopy and its application in diagnostics, toxicological testing and tissue engineering. Applications of Raman spectroscopy in cell biology are in the early stages of development, however, recent publications have demonstrated its utilisation as a diagnostic and development tool with the key advantage that investigations of living cells can be performed non-invasively. Some of the research highlighted here demonstrates the ability of Raman spectroscopy to accurately characterise cancer cells and distinguish between similar cell types. Many groups have used Raman spectroscopy to study tissues, but recently increased effort has gone into single cell analysis of cell lines; the advantages being that cell lines offer ease of handling and increased reproducibility over tissue studies and primary cells. The main goals of bio-Raman spectroscopy at this stage are twofold. Firstly, the aim is to further develop the diagnostic ability of Raman spectroscopy so it can be implemented in a clinical environment, producing accurate and rapid diagnoses. Secondly, the aim is to optimise the technique as a research tool for the non-invasive real time investigation of cell/material interactions in the fields of tissue engineering and toxicology testing.     

Raman spectroscopy and imaging of graphene

Graphene has many unique properties that make it an ideal material for fundamental studies as well as for potential applications. Here we review recent results on the Raman spectroscopy and imaging of graphene. We show that Raman spectroscopy and imaging can be used as a quick and unambiguous method to determine the number of graphene layers. The strong Raman signal of single layer graphene compared to graphite is explained by an interference enhancement model. We have also studied the effect of substrates, the top layer deposition, the annealing process, as well as folding (stacking order) on the physical and electronic properties of graphene. Finally, Raman spectroscopy of epitaxial graphene grown on a SiC substrate is presented and strong compressive strain on epitaxial graphene is observed. The results presented here are highly relevant to the application of graphene in nano-electronic devices and help in developing a better understanding of the physical and electronic properties of graphene. This article is published with open access at Springerlink.com