Quantitative label-free RNA detection using surface-enhanced Raman spectroscopy

Surface-Enhanced Raman Spectroscopy (SERS) was performed to detect label-free RNA. We defined conditions which make it possible to probe the four bases of RNA, in single strands of polyadenosine (pA), polyuridine (pU), polycytosine (pC) and polyguanosine (pG). We therefore present below a quantitative analysis of mixtures of non-hybridized single strands, based on the deconvolution of the SERS mixture spectrum into the relative contributions of the SERS spectra of each constituent.     

Quantitative histochemical analysis of human artery using Raman spectroscopy.

We have developed a method for using near infrared Raman spectroscopy to quantitatively analyze the histochemical composition of human artery. The main contributors to bands observed in the Raman spectra of normal and atherosclerotic aorta are the proteins collagen and elastin, cholesterol lipids, and calcium hydroxyapatite. The Raman scattering cross-sections of different bands for these components have been determined in order to understand their relative contributions to the Raman spectra of biological tissue. The Raman signal is observed to behave linearly with the concentration of the components, even in a highly scattering medium such as a powder. Using these data, we have developed a linear model that can be used to extract the quantitative contribution of an individual component to the spectrum of a mixture. The model has been applied to several mixtures of known composition of tissue constituents in order to evaluate its precision and accuracy. The calculated fit coefficients from the spectra are in agreement with the measured values within experimental uncertainties. The spectra of different types of atherosclerotic aorta have also been modeled, and we have extracted quantitative information regarding the relative concentration of biological constituents in atherosclerotic aorta.     

Quantitative monitoring of yeast fermentation using Raman spectroscopy

Compared to traditional IR methods, Raman spectroscopy has the advantage of only minimal interference from water when measuring aqueous samples, which makes this method potentially useful for in situ monitoring of important industrial bioprocesses. This study demonstrates real-time monitoring of a Saccharomyces cerevisiae fermentation process using a Raman spectroscopy instrument equipped with a robust sapphire ball probe. A method was developed to correct the Raman signal for the attenuation caused by light scattering cell particulate, hence enabling quantification of reaction components and possibly measurement of yeast cell concentrations. Extinction of Raman intensities to more than 50 % during fermentation was normalized with approximated extinction expressions using Raman signal of water around 1,627 cm^?1 as internal standard to correct for the effect of scattering. Complicated standard multi-variant chemometric techniques, such as PLS, were avoided in the quantification model, as an attempt to keep the monitoring method as simple as possible and still get satisfactory estimations. Instead, estimations were made with a two-step approach, where initial scattering correction of attenuated signals was followed by linear regression. In situ quantification measurements of the fermentation resulted in root mean square errors of prediction (RMSEP) of 2.357, 1.611, and 0.633 g/L for glucose, ethanol, and yeast concentrations, respectively.     

Towards single-microorganism detection using surface-enhanced Raman spectroscopy

The identification and discrimination of microorganisms is important not only for clinical reasons but also for pharmaceutical clean room production and food-processing technology. Vibrational spectroscopy such as IR, Raman, and surface-enhanced Raman scattering (SERS) can provide a rapid ‘fingerprint’ on the chemical structure of molecules and is used to obtain a ‘fingerprint’ from microorganisms as well. Because of the requirement that a single bacterium cell and noble metal nanoparticles must be in close contact and the lack of a significant physical support to hold nanoparticles around the single bacterium cell, the acquisition of SERS spectra for a single bacterium using colloidal nanoparticles could be a challenging task. The feasibility of SERS for identification down to a single bacterium is investigated. A Gram-negative bacterium, Escherichia coli, is chosen as a model for the investigation. Because the adsorption of silver nanoparticles onto the bacterial cell is an exclusive way for locating nanoparticles close to the bacterium cell, the absorption characteristics of silver nanoparticles with different surface charges are investigated. It is demonstrated that the citrate-reduced colloidal silver solution generates more reproducible SERS spectra. It is found that E. coli cells aggregate upon mixing with silver colloidal solution, and this may provide an additional benefit in locating the bacterial cell under a light microscope. It is also found that a laser wavelength in the UV region could be a better choice for the study due to the shallow penetration depth. It is finally shown that it is possible to obtain SERS spectra from a single cell down to a few bacterial cells, depending on the aggregation properties of bacterial cells for identification and discrimination.     

Quantitative surface-enhanced Raman spectroscopy

The purpose of this tutorial review is to show how surface-enhanced Raman (SERS) and resonance Raman (SERRS) spectroscopy have evolved to the stage where they can be used as a quantitative analytical technique. SER(R)S has enormous potential for a range of applications where high sensitivity needs to be combined with good discrimination between molecular targets, particularly since low cost, compact spectrometers can read the high signal levels that SER(R)S typically provides. These advantages over conventional Raman measurements come at the cost of increased complexity and this review discusses the factors that need to be controlled to generate stable and reproducible SER(R)S calibrations.     

Quantitative surface-enhanced Raman spectroscopy of dipicolinic acid--towards rapid anthrax endospore detection

Dipicolinic acid (DPA) is an excellent marker compound for bacterial spores, including those of Bacillus anthracis (anthrax). Surface-enhanced Raman spectroscopy (SERS) potentially has the sensitivity and discrimination needed for trace DPA analysis, but mixing DPA solutions with citrate-reduced silver colloid only yielded measurable SERS spectra at much higher (>80 ppm) concentrations than would be desirable for anthrax detection. Aggregation of the colloid with halide salts eliminated even these small DPA bands but aggregation with Na2SO4(aq) resulted in a remarkable increase in the DPA signals. With sulfate aggregation even 1 ppm solutions gave detectable signals with 10 s accumulation times, which is in the sensitivity range required. Addition of CNS- as an internal standard allowed quantitative DPA analysis, plotting the intensity of the strong DPA 1010 cm(-1) band (normalised to the ca. 2120 cm(-1) CNS- band) against DPA concentration gave a linear calibration (R2 = 0.986) over the range 0-50 ppm DPA. The inclusion of thiocyanate also allows false negatives due to accidental deactivation of the enhancing medium to be detected.     

Quantitative determination of naltrexone and beta-naltrexol in human plasma using electron detection.

A gas-liquid chromatographic method is described for the determination of naltrexone and beta-naltrexol in human plasma following derivatization with pentafluoropropionic anhydride using electron capture detection. The lower sensitivity of the method for absolute standards is 5-10 pg. Following an acute 100-mg dose to a subject, peak levels of naltrexone of 15 ng/ml at 2 h and of beta-naltrexol 84 ng/ml at 4 h were observed. The levels of both compounds decreased by 24 h after the dose: naltrexone to 2.9 ng/ml and beta-naltrexol to 25 ng/ml. Following chronic administration for two weeks of 100 mg per day the peak levels of naltrexone and betanaltrexol increased to 26.9 and 131 ng/ml at 2 h, respectively, but by 24 h both compounds were at levels similar to those following a single dose. Thus no accumulation of either drug ro metabolite in the plasma was seen following chronic naltrexone administration.     

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.     

Direct detection of 8-oxo-deoxyguanosine using UV resonance Raman spectroscopy

8-oxo-deoxyguanosine (8-oxo-dG) is a major oxidative lesion in DNA and is responsible for mutation and cancer. Current techniques for detecting 8-oxo-dG are indirect methods. Thus, development of new methodologies is needed to directly detect such oxidative lesions. In this article, we have used ultraviolet resonance Raman (UVRR) spectroscopy as a novel analytical technique for the detection of 8-oxo-dG. Here, the UVRR spectrum of 8-oxo-dG was acquired and compared to that of deoxyguanosine (dG) and deoxyadenosine (dA). Data analysis shows a distinct UVRR spectrum of 8-oxo-dG with characteristic peaks. Detection of 8-oxo-dG was easily achieved from a mixture with dG. These results reveal that UVRR spectroscopy shows promise as a direct method for detecting 8-oxo-dG.     

Hemoglobin conformation detection by Raman spectroscopy on single human red blood cells captured in a microfluidic chip

The Raman spectrum of a single erythrocyte captured by a microfluidic chip was recorded to determine the conformation of hemoglobin under conditions similar to the hemodynamics of a blood vessel. Amplitude changes in the Raman spectrum at 1355, 1375, 1552, 1620, 1585 and 1637 cm?1 reflect changes in pO₂ due to O₂ binding to hemoglobin heme.     

Surface-enhanced Raman spectroscopy of blood plasma and serum using Ag and Au nanoparticles: a systematic study

Surface-enhanced Raman spectroscopy (SERS) is a good candidate for the development of fast and easy-to-use diagnostic tools, possibly used on biofluids in point-of-care or screening tests. In particular, label-free SERS spectra of blood serum and plasma, two biofluids widely used in diagnostics, could be used as a metabolic fingerprinting approach for biomarker discovery. This study aims at a systematic evaluation of SERS spectra of blood serum and plasma, using various Ag and Au aqueous colloids, as SERS substrates, in combination with three excitation lasers of different wavelengths, ranging from the visible to the near-infrared. The analysis of the SERS spectra collected from 20 healthy subjects under a variety of experimental conditions revealed that intense and repeatable spectra are quickly obtained only if proteins are filtered out from samples, and an excitation in the near-infrared is used in combination with Ag colloids. Moreover, common plasma anticoagulants such as EDTA and citrate are found to interfere with SERS spectra; accordingly, filtered serum or heparin plasma are the samples of choice, having identical SERS spectra. Most bands observed in SERS spectra of these biofluids are assigned to uric acid, a metabolite whose blood concentration depends on factors such as sex, age, therapeutic treatments, and various pathological conditions, suggesting that, even when the right experimental conditions are chosen, great care must be taken in designing studies with the purpose of developing diagnostic tests.     

Towards detection and identification of circulating tumour cells using Raman spectroscopy

Body fluids are easily accessible and contain valuable indices for medical diagnosis. Fascinating tools are tumour cells circulating in the peripheral blood of cancer patients. As these cells are extremely rare, they constitute a challenge for clinical diagnostics. In this contribution we present the Raman spectroscopic-based identification of different single cells in suspension that are found in peripheral blood of cancer patients including healthy cells like leukocytes and erythrocytes, and tumour cells like leukaemic cells and cells originating from solid tumours. Leukocytes and erythrocytes were isolated from the peripheral blood of healthy donors while myeloid leukaemia cells (OCI-AML3) and breast carcinoma derived cells (MCF-7, BT-20) were obtained from cell cultures. A laser emitting 785 nm light was used for optical trapping the single cells in the laser focus and to excite the Raman spectrum. Support vector machines were applied to develop a supervised classification model with spectra of 1210 cells originating from three different donors and three independent cultivation batches. Distinguishing tumour cells from healthy cells was achieved with a sensitivity of >99.7% and a specificity of >99.5%. In addition, the correct cell types were predicted with an accuracy of approximately 92%.     

Resonance Raman spectroscopy of red blood cells using near-infrared laser excitation

Resonance Raman spectra of oxygenated and deoxygenated functional erythrocytes recorded using 785 nm laser excitation are presented. The high-quality spectra show a mixture of enhanced A_1g, A_2g, B_1g, B_2g, E_u and vinyl modes. The high sensitivity of the Raman system enabled spectra from four oxygenation and deoxygenation cycles to be recorded with only 18 mW of power at the sample over a 60-minute period. This low power prevented photo-/thermal degradation and negated protein denaturation leading to heme aggregation. The large database consisting of 210 spectra from the four cycles was analyzed with principal components analysis (PCA). The PC1 loadings plot provided exquisite detail on bands associated with the oxygenated and deoxygenated states. The enhancement of a band at 567 cm⁻¹, observed in the spectra of oxygenated cells and the corresponding PC1 loadings plot, was assigned to the Fe–O₂ stretching mode, while a band appearing at 419 cm⁻¹ was assigned to the Fe–O–O bending mode based on previous studies. For deoxygenated cells, the enhancement of B_1g modes at 785 nm excitation is consistent with vibronic coupling between band III and the Soret transition. In the case of oxygenated cells, the enhancement of iron-axial out-of-plane modes and non-totally symmetric modes is consistent with enhancement into the y,z-polarized transition centered at 785 nm. The enhancement of non-totally symmetric B_1g modes in oxygenated cells suggests vibronic coupling between band IV and the Soret band. This study provides new insights into the vibrational dynamics, electronic structure and resonant enhancement of heme moieties within functional erythrocytes at near-IR excitation wavelengths. Part of this work was first presented at SPEC 2006, Shedding New Light on Disease: Optical Diagnosis for the New Millennium, held at Heidelberg, Germany, on 20–24 May 2006.     

Quantitative analysis of diphenhydramine hydrochloride in pharmaceutical wafers using near infrared and Raman spectroscopy

Wafers with varying concentrations of diphenhydramine hydrochloride (DPH-HCl) as active pharmaceutical ingredient (API) were prepared and their near infrared (NIR) and Raman spectra recorded. The purpose of this study was to compare the suitability of these two vibrational spectroscopic techniques for the quantification of DPH-HCl in pharmaceutical wafers. Partial least squares (PLS1) calibration models with different data pretreatments were tested. Both NIR and Raman spectroscopy proved to be suitable to predict DPH-HCl contents at lower concentration ranges. At higher concentrations, interference by crystallization processes was observed. For investigating the general applicability of the quantification methods, two commercially available products were examined.     

Picomolar detection of carcinoembryonic antigen in whole blood using microfluidics and surface‐enhanced Raman spectroscopy

Carcinoembryonic antigen (CEA) is a wide‐spectrum biomarker. Clinically, we generally use serum sample to detect CEA, which needs to be centrifuged to pretreat the raw blood sample. In this study, we realized direct CEA detection in raw blood samples exploiting microfluidics. The LOD was as low as 10^?12 M.     

DNA detection by dye labeled oligonucleotides using surface enhanced Raman spectroscopy

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Infrared spectroscopy in hemodialysis: reagent-free monitoring of patient detoxification by infrared spectroscopy

A method for monitoring hemodialysis based on quantitative infrared spectroscopic determination of the molecules dialyzed from patient blood is reported. The measurements are reagent-free and aim at real-time and in-line monitoring of the hemodialysis patient. A flow cell using attenuated total reflection infrared spectroscopy is coupled downstream of the dialysis filter unit. A calibration model has been developed from real hemodialysis samples analyzed by chemical reference analysis and from artificially mixed dialysis samples. The infrared monitoring of hemodialysis includes quantitative determination of urea as the lead substance, as well as glucose, lactate, and creatinine, all at a precision only limited by the chemical reference analysis. The flow cell can be fitted to all standard hemodialysis systems. Preliminary tests with hemodialysis patients have demonstrated that detoxification can be clearly monitored. Furthermore, these experiments demonstrate that a wide, real-time control of the patient’s physiological parameters is possible with this method, which could lead to increased patient safety.     

Discrimination of zone-specific spectral signatures in normal human prostate using Raman spectroscopy

The prostate gland is the most common site of pathology in human males. Using the urethra as an anatomical reference point, it can be divided into three distinct zones known as the transition zone (TZ), peripheral zone (PZ) and central zone (CZ). The pathological conditions of benign prostatic hypertrophy and/or prostate adenocarcinoma are highly prevalent in this gland. This preliminary study set out to determine whether biochemical intra-individual differences between normal prostate zones could be identified using Raman spectroscopy with subsequent exploratory analyses. A normal (benign) prostate transverse tissue section perpendicular to the rectal surface and above the verumontanum was obtained in a paraffin-embedded block. A 10-μm-thick slice was floated onto a gold substrate, de-waxed and analysed using Raman spectroscopy (200 epithelial-cell and 140 stromal spectra/zone). Raman spectra were subsequently processed in the 1800-367 cm(-1) spectral region employing principal component analysis (PCA) to determine whether wavenumber-intensity relationships expressed as single points in hyperspace might reveal biochemical differences associated with inter-zone pathological susceptibility. Visualisation of PCA scores plots and their corresponding loadings plots highlighted 781 cm(-1) (cytosine/uracil) and 787 cm(-1) (DNA) as the key discriminating factors segregating PZ from less susceptible TZ and CZ epithelia (P < 0.001). Conversely, 1459 cm(-1) (lipids and proteins) and 1003 cm(-1) (phenylalanine) were identified as the key biochemical factor distinguishing TZ from CZ epithelia (P < 0.05). All stromal zones were discriminated by the protein/lipid region (1459 cm(-1) and 1100 cm(-1)) with DNA/RNA region (781 cm(-1) and 787 cm(-1)) only highlighted between PZ and CZ (P < 0.05). This novel approach identifies biochemical markers that may have aetiological functional roles towards susceptibility of human prostate zones to specific pathological conditions.     

Quantitative aspects of near-infrared Fourier transform Raman spectroscopy

Three fundamental behaviors of vibrational spectroscopy data manipulation routinely associated with Fourier transform infrared (FTIR) spectroscopy are evaluated for near-infrared (NIR) Fourier transform Raman spectroscopy. Spectral reproducibility, spectral subtraction and sensitivity are examined relative to the NIR FT-Raman experiment. Quantitative predictive ability is compared for identical sets of samples containing mixtures of the three xylene isomers. Partial least-squares analysis is used to compare predictive ability. IR performance is found to be better than Raman, though the potential for method development using NIR FT-Raman is shown to be quite promising.     

Quantitative determination of terbutaline in human plasma after administration of bambuterol using coupled columns and electrochemical detection

Summary A method is presented for quantitative determination of terbutaline after administration of its prodrug Bambuterol. Terbutaline is extracted from plasma by liquid-solid extraction on small C₁₈-cartridges. The extract is then analysed by coupled column liquid chromatography with amperometric detection. To inhibit the esterase catalyzed hydrolysis of bambuterol to terbutaline an analogue of bambuterol is added to the plasma sampling tubes. The within-day variation on spiked samples were 2.1% and 2.4% at 8.0 and 40.0 nmol/L respectively. The between-day variation on spiked samples (8.0 nmol/L) was 3.6% and on authentic samples 5.9% at the 11 nmol/L level. The absolute recovery was in the range 83–94% for terbutaline and the internal standard. The limit of quantitation was set at 4.0 nmol/L (C_v=3.4%, n=46). Subsidiary of AB Astra.