Unraveling the toxicity of tire wear contamination in three freshwater species: From chemical mixture to nanoparticles.

Tire wear particle (TWP) contamination is of growing concern as recent studies show the ubiquity and toxicity of this contaminant in various environmental compartments. The multidimensional aspect of TWPs makes it difficult to assess toxicity and predict impacts on ecosystems, as it combines a complex mixture of chemicals and can release micro- and nanoparticles when suspended in water. Our work aimed to shed light on the toxicity of the different components of TWP leachate, namely, the dissolved chemicals and the nanoparticle fractions, on three freshwater model species of different trophic levels: Chlorella vulgaris, Lemna minor, and Daphnia magna. Acute toxicity was observed for all three fractions in D. magna, and an additive effect was observed between the nanoparticles and dissolved chemicals. L. minor experienced phytotoxicity from the dissolved chemicals only with a decrease up to 50% in photosynthesis efficiency parameters. C. vulgaris showed minor signs of toxicity on apical endpoints in response to each of the fractions. Our study highlights that nanoparticles from TWP leachate that were mostly overlooked in several previous studies are as toxic as dissolved chemicals for the filter-feeder species D. magna, and we also show the toxicity to photosynthesis in aquatic plants.

Design, Development and Validation of a Knee Brace to Standardize the US Imaging Evaluation of Knee Osteoarthritis.

OBJECTIVE: A repeatable and reliable follow-up of knee injuries would be desirable to prevent delayed diagnosis and to monitor the efficacy of the applied treatment over time. Ultrasound (US) techniques are an attractive option to this purpose, since they are safe, low-cost and non-invasive. However, its use in the clinical practice is limited by the high dependency on the operator's experience. Hence, the objective of this study is to provide a standardization of the US image acquisition process for knee osteoarthritis (OA) allowing an extended clinical use of US technologies in this domain. METHODS: Clinical specifications were provided by expert musculoskeletal radiologists thus identifying the subject poses and the US probe positions needed to evaluate the cartilage structure, signs of synovitis and joint effusion. Such considerations were used to derive the technical requirements needed for the development of a wearable brace equipped with specific openings to guide the correct placement of the probe. The feasibility of the developed wearable brace was tested on three healthy volunteers, which were asked to acquire informative US images, similar to the reference images performed by the musculoskeletal radiologist. RESULTS: Thanks to the knee brace, the untrained subjects were able to self-acquire informative B-mode images comparable to the corresponding images acquired by an expert clinician. DISCUSSION/CONCLUSION: The use of a knee brace intended for knee OA US diagnosis demonstrated the possibility to standardize the acquisition protocol and make its application achievable also for untrained subjects, representing a key step toward tele-ultrasonography.

Development and validation of low-intensity pulsed ultrasound systems for highly controlled in vitro cell stimulation.

This work aims to describe the development and validation of two low-intensity pulsed ultrasound stimulation systems able to control the dose delivered to the biological target. Transducer characterization was performed in terms of pressure field shape and intensity, for a high-frequency range (500 kHz to 5 MHz) and for a low-frequency value (38 kHz). This allowed defining the distance, on the beam axis, at which biological samples should be placed during stimulation and to exactly know the intensity at the target. Carefully designed retaining systems were developed, for hosting biological samples. Sealing tests proved their impermeability to external contaminants. The assembly/de-assembly time of the systems resulted ~3 min. Time-domain acoustic simulations allowed to precisely estimate the ultrasound beam within the biological sample chamber, thus enabling the possibility to precisely control the pressure to be transmitted to the biological target, by modulating the transducer's input voltage. Biological in vitro tests were also carried out, demonstrating the sterility of the system and the absence of toxic and inflammatory effects on growing cells after multiple immersions in water, over seven days.

Raman imaging as a new analytical tool for the quality control of the monitoring of osteogenic differentiation in forming 3D bone tissue.

In this study, adipose-derived stem cells (ASCs) are used to produce 3D bone grafts. The safety and the feasibility of using these bone grafts have been already showed and quality controls are already implemented. However, a cheaper, fast and non-destructive technique is required to monitor the osteogenic differentiation process. Here, the use of Raman imaging to monitor the synthesis of the extracellular matrix and its progressive mineralization occurring during the osteogenic differentiation process is investigated for the first time on a 3D in forming bone tissue. The attention was focused on Raman bands related to this matrix belonging to phosphate, phenylalanine and hydroxyproline, which are very distinctive and intense. The kinetic of the osteogenic differentiation process was first compared between a 2D and a 3D forming bone tissue. It was observed that the kinetics of the osteogenic differentiation process is slower in 3D in forming bone tissue. In a second step, an evaluation of the reliability of the Raman imaging method was performed including a study of the influence of the harvest biopsies position on the forming 3D bone tissue. The repeatability and the specificity of this method were also demonstrated. In a last step, several batches of ASCs were cultured and analyzed in 3D at different time points using Raman imaging. From the mean Raman spectra, mineral to matrix ratios (MTMR) were determined and used to evaluate the formation of mineral deposits accompanying the extracellular matrix synthesis which is indicative of an ongoing osteogenic differentiation process. These ratios peaked between the day 35 and 49. This observation was very interesting since it corresponds to the time at which the 3D bone grafts are used for the patient surgery. To conclude, Raman imaging allowed fast acquisition and time-resolved monitoring in vitro of the mineralization of extracellular matrix during osteogenic differentiation.

Quantitation of active pharmaceutical ingredient through the packaging using Raman handheld spectrophotometers: A comparison study.

Handheld Raman spectroscopy is actually booming. Recent devices improvements aim at addressing the usual Raman spectroscopy issues: fluorescence with shifted-excitation Raman difference spectroscopy (SERDS), poor sensitivity with surface enhanced Raman scattering (SERS) and information only about the sample surface with spatially offset Raman spectroscopy (SORS). While qualitative performances of handheld devices are generally well established, the quantitative analysis of pharmaceutical samples remains challenging. The aim of this study was to compare the quantitative performances of three commercially available handheld Raman spectroscopy devices. Two of them (TruScan and IDRaman mini) are equipped with a 785 nm laser wavelength and operate in a conventional backscattering mode. The IDRaman has the Orbital Raster Scanning (ORS) option to increase the analyzed surface. The third device (Resolve) operates with an 830 nm laser wavelength both in backscattering and in SORS modes. The comparative study was carried out on ibuprofen-mannitol-microcrystalline cellulose ternary mixtures. The concentration of ibuprofen ranged from 24 to 52% (w/w) while the proportions of the two excipients were varied to avoid cross-correlation as much as possible. Analyses were performed either directly through a glass vial or with the glass vial in an opaque polypropylene flask, using a validated FT-NIR spectroscopy method as a reference method. Chemometric analyses were carried out with the Partial Least Squares Regression (PLS-R) algorithm. The quantitative models were validated using the total error approach and the ICH Q2 (R1) guidelines with ±â€¯15% as acceptance limits.

A novel GPR55-mediated satiety signal in the oval Bed Nucleus of the Stria Terminalis.

Nestled within feeding circuits, the oval (ov) region of the Bed Nucleus of the Stria Terminalis (BNST) may be critical for monitoring energy balance through changes in synaptic strength. Here we report that bidirectional plasticity at ovBNST GABA synapses was tightly linked to the caloric state of male rats, seesawing between long-term potentiation (iLTP, fed) and depression (iLTD, food restricted). L-α-lysophosphatidylinositol (LPI) acting on GPR55 receptors and 2-arachidonoylglycerol (2-AG) through CB1R were respectively responsible for fed (iLTP) and food restricted (iLTD) states. Thus, we have characterized a potential gating mechanism within the ovBNST that may signal metabolic state within the rat brain feeding circuitry.

Ultrasound Acoustic Radiation Force Impulse imaging for High Intensity Focused Ultrasound focal spot localization.

Focal spot precise localization highly contributes to the accuracy and safety of High Intensity Focused Ultrasound (HIFU) therapies, and it is usually performed by means of Magnetic Resonance-Acoustic Radiation Force Impulse imaging (MR-ARFI). Acoustic Radiation Force Impulse imaging using ultrasound (US-ARFI) is herein proposed as a valid alternative to MR-ARFI for an accurate and non-destructive detection of the focal spot position during the pre-treatment phase. To this aim, a system composed of a HIFU transducer for generating the acoustic radiation force and a 2D confocal ultrasound probe for measuring the induced micro-displacement have been used. Then, an algorithm based on the Normalized Cross Correlation was implemented for the creation of a displacement map in which the highest displacement area, corresponding to the focal spot region, is unequivocally visualized. The feasibility of the proposed USARFI method for HIFU focal spot localization was successfully demonstrated in a tissue mimicking phantom model.

Neurotensin and dynorphin Bi-Directionally modulate CeA inhibition of oval BNST neurons in male mice.

Neuropeptides are often co-expressed in neurons, and may therefore be working together to coordinate proper neural circuit function. However, neurophysiological effects of neuropeptides are commonly studied individually possibly underestimating their modulatory roles. Here, we triggered the release of endogenous neuropeptides in brain slices from male mice to better understand their modulation of central amygdala (CeA) inhibitory inputs onto oval (ov) BNST neurons. We found that locally-released neurotensin (NT) and dynorphin (Dyn) antagonistically regulated CeA inhibitory inputs onto ovBNST neurons. NT and Dyn respectively increased and decreased CeA-toovBNST inhibitory inputs through NT receptor 1 (NTR1) and kappa opioid receptor (KOR). Additionally, NT and Dyn mRNAs were highly co-localized in ovBNST neurons suggesting that they may be released from the same cells. Together, we showed that NT and Dyn are key modulators of CeA inputs to ovBNST, paving the way to determine whether different conditions or states can alter the neuropeptidergic regulation of this particular brain circuit.

Development of a SERS strategy to overcome the nanoparticle stabilisation effect in serum-containing samples: Application to the quantification of dopamine in the culture medium of PC-12 cells.

The analysis of serum samples by surface-enhanced Raman spectroscopy (SERS) has gained ground over the last few years. However, the stabilisation of colloids by the proteins contained in these samples has restricted their use in common practice, unless antibodies or aptamers are used. Therefore, this work was dedicated to the development of a SERS methodology allowing the analysis of serum samples in a simple and easy-to-implement way. This approach was based on the pre-aggregation of the colloid with a salt solution. Gold nanoparticles (AuNPs) were used as the SERS substrate and, owing to its physiopathological importance, dopamine was chosen as a model to implement the SERS approach. The presence of this neurotransmitter could be determined in the concentration range 0.5-50 ppm (2.64-264 µM) in the culture medium of PC-12 cells, with a R2 of 0.9874, and at even lower concentrations (0.25 ppm, 1.32 µM) in another matrix containing fewer proteins. Moreover, the effect of calcium and potassium on the dopamine exocytosis from PC-12 cells was studied. Calcium was shown to have a predominant and dose-dependant effect. Finally, PC-12 cells were exposed to dexamethasone in order to increase their biosynthesis and release of dopamine. This increase was monitored with the developed SERS approach.

Critical review of surface-enhanced Raman spectroscopy applications in the pharmaceutical field.

Surface-enhanced Raman spectroscopy (SERS) is a sensitive analytical tool used in the pharmaceutical field in recent years. SERS keeps all the advantages of classical Raman spectroscopy while being is more sensitive allowing its use for the detection and the quantification of low-dose substances contained in pharmaceutical samples. However, the analytical performance of SERS is limited due to the difficulty to implement a quantitative methodology correctly validated. Nevertheless, some studies reported the development of SERS quantitative methods especially in pharmaceutical approaches. In this context, this review presents the main concepts of the SERS technique. The different steps that need to be applied to develop a SERS quantitative method are also deeply described. The last part of the present manuscript gives a critical overview of the different SERS pharmaceutical applications that were developed for a non-exhaustive list of pharmaceutical compounds with the aim to highlights the validation criteria for each application.

GSK2878175, a pan-genotypic non-nucleoside NS5B polymerase inhibitor, in healthy and treatment-naïve chronic hepatitis C subjects.

GSK2878175 is a potent, pan-genotypic, non-nucleoside, nonstructural protein 5B palm polymerase inhibitor being developed for the treatment of chronic hepatitis C (CHC). A first-in-human, randomized, placebo-controlled, dose escalation study, evaluated the safety and pharmacokinetics of GSK2878175 administered as single and repeat oral doses (once daily for 14 days) to healthy volunteers. A separate proof-of-concept, placebo-controlled, repeat dose (once daily for 2 days) study evaluated the safety, pharmacokinetics and antiviral activity of GSK2878175 monotherapy in treatment-naïve, noncirrhotic, subjects with hepatitis C virus (HCV) genotype 1 [1a and 1b], 2, or 3. No deaths or SAEs were reported in either study, and treatment was well-tolerated. Across all the HCV genotypes, GSK2878175 monotherapy at doses of 10, 30 or 60 mg once daily for 2 days produced a statistically significant multilog reduction (P<.001) in plasma HCV RNA log10 IU/mL from Baseline to 24, 48 and 72 hours after the first dose of GSK2878175 compared to placebo. The reduction in HCV RNA was sustained for a prolonged period across all of the active treatment groups, consistent with the long apparent half-life of GSK2878175 that was observed (mean t1/2 range: 60-63 hours in the CHC subjects). In summary, GSK2878175, when administered to healthy subjects and subjects with CHC, did not reveal any safety concerns that would limit or preclude further clinical development. GSK2878175 monotherapy across a wide dose range produced substantial reduction in HCV RNA, irrespective of HCV genotype. The results from these studies support further evaluation of GSK2878175-based regimens.

A Key Role for Neurotensin in Chronic-Stress-Induced Anxiety-Like Behavior in Rats.

Chronic stress is a major cause of anxiety disorders that can be reliably modeled preclinically, providing insight into alternative therapeutic targets for this mental health illness. Neuropeptides have been targeted in the past to no avail possibly due to our lack of understanding of their role in pathological models. In this study we use a rat model of chronic stress-induced anxiety-like behaviors and hypothesized that neuropeptidergic modulation of synaptic transmission would be altered in the bed nucleus of the stria terminalis (BNST), a brain region suspected to contribute to anxiety disorders. We use brain slice neurophysiology and behavioral pharmacology to compare the role of locally released endogenous neuropeptides on synaptic transmission in the oval (ov) BNST of non-stressed (NS) or chronic unpredictably stressed (CUS) rats. We found that in NS rats, post-synaptic depolarization induced the release of vesicular neurotensin (NT) and corticotropin-releasing factor (CRF) that co-acted to increase ovBNST inhibitory synaptic transmission in 59% of recorded neurons. CUS bolstered this potentiation (100% of recorded neurons) through an enhanced contribution of NT over CRF. In contrast, locally released opioid neuropeptides decreased ovBNST excitatory synaptic transmission in all recorded neurons, regardless of stress. Consistent with CUS-induced enhanced modulatory effects of NT, blockade of ovBNST NT receptors completely abolished stress-induced anxiety-like behaviors in the elevated plus maze paradigm. The role of NT has been largely unexplored in stress and our findings highlight its potential contribution to an important behavioral consequence of chronic stress, that is, exaggerated avoidance of open space in rats.

Development of an analytical method for crystalline content determination in amorphous solid dispersions produced by hot-melt extrusion using transmission Raman spectroscopy: A feasibility study.

The development of a quantitative method determining the crystalline percentage in an amorphous solid dispersion is of great interest in the pharmaceutical field. Indeed, the crystalline Active Pharmaceutical Ingredient transformation into its amorphous state is increasingly used as it enhances the solubility and bioavailability of Biopharmaceutical Classification System class II drugs. One way to produce amorphous solid dispersions is the Hot-Melt Extrusion (HME) process. This study reported the development and the comparison of the analytical performances of two techniques, based on backscattering and transmission Raman spectroscopy, determining the crystalline remaining content in amorphous solid dispersions produced by HME. Principal Component Analysis (PCA) and Partial Least Squares (PLS) regression were performed on preprocessed data and tended towards the same conclusions: for the backscattering Raman results, the use of the DuoScan™ mode improved the PCA and PLS results, due to a larger analyzed sampling volume. For the transmission Raman results, the determination of low crystalline percentages was possible and the best regression model was obtained using this technique. Indeed, the latter acquired spectra through the whole sample volume, in contrast with the previous surface analyses performed using the backscattering mode. This study consequently highlighted the importance of the analyzed sampling volume.

Global approach for the validation of an in-line Raman spectroscopic method to determine the API content in real-time during a hot-melt extrusion process.

Since the Food and Drug Administration (FDA) published a guidance based on the Process Analytical Technology (PAT) approach, real-time analyses during manufacturing processes are in real expansion. In this study, in-line Raman spectroscopic analyses were performed during a Hot-Melt Extrusion (HME) process to determine the Active Pharmaceutical Ingredient (API) content in real-time. The method was validated based on a univariate and a multivariate approach and the analytical performances of the obtained models were compared. Moreover, on one hand, in-line data were correlated with the real API concentration present in the sample quantified by a previously validated off-line confocal Raman microspectroscopic method. On the other hand, in-line data were also treated in function of the concentration based on the weighing of the components in the prepared mixture. The importance of developing quantitative methods based on the use of a reference method was thus highlighted. The method was validated according to the total error approach fixing the acceptance limits at ±15% and the α risk at ±5%. This method reaches the requirements of the European Pharmacopeia norms for the uniformity of content of single-dose preparations. The validation proves that future results will be in the acceptance limits with a previously defined probability. Finally, the in-line validated method was compared with the off-line one to demonstrate its ability to be used in routine analyses.

Free energy profiles for two ubiquitous damaging agents: methylation and hydroxylation of guanine in B-DNA.

DNA methylation and hydroxylation are two ubiquitous reactions in DNA damage induction, yet insights are scarce concerning the free energy of activation within B-DNA. We resort to multiscale simulations to investigate the attack of a hydroxyl radical and of the primary diazonium onto a guanine embedded in a solvated dodecamer. Reaction free energy profiles characterize two strongly exergonic processes, yet allow unprecedented quantification of the barrier towards this damage reaction, not higher than 6 kcal mol-1 and sometimes inexistent, and of the exergonicities. In the case of the [G(C8)-OH]˙ intermediate, we challenge the functional dependence of such simulations: recently-proposed functionals, such as M06-2X and LC-BLYP, agree on a ∼4 kcal mol-1 barrier, whereas the hybrid GGA B3LYP functional predicts a barrier-less pathway. In the long term, multiscale approaches can help build up a unified panorama of DNA lesion induction. These results stress the importance of DFT/MM-MD simulations involving new functionals towards the sound modelling of biomolecule damage even in the ground state.

Fixed 50:50 mixture of nitrous oxide and oxygen to reduce lumbar-puncture-induced pain: a randomized controlled trial.

BACKGROUND AND PURPOSE: Lumbar puncture (LP) has been frequently performed for more than a century. This procedure is still stressful and often painful. The aim of the study was to evaluate the efficacy of a fixed 50% nitrous oxide-oxygen mixture compared to placebo to reduce immediate procedural pain and anxiety during LP. METHODS: A randomized controlled trial was conducted involving adults who needed a cerebrospinal fluid analysis. Patients were randomly assigned to inhale either a fixed 50% nitrous oxide-oxygen mixture (50% N2 O-O2 ) or medical air (22% O2 -78% N2 ). Cutaneous application of a eutectic mixture of local anaesthetics was systematically done and all LPs were performed with pencil point 25G needles (20G introducer needle). The primary end-point was the maximal pain level felt by the patient during the procedure, the maximal anxiety level being a secondary outcome, both measured using a numerical rating scale (0-10). RESULTS: A total of 66 consecutive patients were randomized. The analysis was intention to treat. The maximal pain was 4.9 ± 2.7 for the 33 patients receiving air and 2.7 ± 2.7 for the 33 receiving 50% N2 O-O2 (P = 0.002). Similarly, the maximal LP-induced anxiety was 4.5 ± 3.1 vs. 2.6 ± 2.6 (P = 0.009), respectively. The number needed to treat to avoid one patient undergoing significant pain (pain score ≥ 4/10) was 2.75. Body mass index >25 kg/m2 was significantly associated with higher pain intensity (P = 0.03). No serious adverse events were attributable to 50% N2 O-O2 inhalation. CONCLUSIONS: Inhalation of a fixed 50% N2 O-O2 mixture is efficient to reduce LP-induced pain and anxiety.

Urbanization impact on sulfur content of groundwater revealed by the study of urban speleothem-like deposits: Case study in Paris, France.

Speleothem-like deposits that develop underground in urban areas are an archive of the environmental impact of anthropic activities that has been little studied so far. In this paper, the sulfate content in shallow groundwater from northern Paris (France) is compared with the sulfur content in two 300-year-old urban carbonate deposits that grew in a historical underground aqueduct. The present-day waters of the aqueduct have very high sulfur and calcium contents, suggesting pollution from gypsum dissolution. However, geological gypsum levels are located below the water table. Sulfur content was measured by micro-X-ray fluorescence in these very S-rich carbonate deposits (0.5 to 1% of S). A twofold S increase during the second half of the 1800s was found in both samples. These dates correspond to two major periods of urbanization above the site. We discus three possible S sources: anthropic sources (industries, fertilizers…), volcanic eruptions and input within the water through gypsum brought for urbanization above the studied site (backfill with quarry waste) since the middle of the 19th century. For the younger second half of the studied section, S input from gypsum brought during urbanization was confirmed by the study of isotopic sulfur composition (δ34S=+15.2‰ at the top). For the oldest part, several sulfur peaks could be related to early industrial activity in Paris, that caused high local air pollution, as reported in historical archives but also to historical gypsum extraction. This study provides information on the origin and timing of the very high SO42- levels measured nowadays within the shallow groundwater, thus demonstrating the interest in using carbonate deposits in urban areas as a proxy for the history of urbanization or human activities and their impact on water bodies.

Monitoring of anatabine release by methyl jasmonate elicited BY-2 cells using surface-enhanced Raman scattering.

A new application of surface-enhanced Raman scattering (SERS) in the field of plant material analysis is proposed in this study. The aim was to monitor the release of anatabine by methyl jasmonate (MeJa) elicited Bright Yellow-2 (BY-2) cells. Gold nanoparticles (AuNps) were used as SERS substrate. The first step was to study the SERS activity of anatabine in a complex matrix comprising the culture medium and BY-2 cells. The second step was the calibration. This one was successfully performed directly in the culture medium in order to take into account the matrix effect, by spiking the medium with different concentrations of anatabine, leading to solutions ranging from 250 to 5000µgL(-1). A univariate analysis was performed, the intensity of a band situated at 1028cm(-1), related to anatabine, was plotted against the anatabine concentration. A linear relationship was observed with a R(2) of 0.9951. During the monitoring study, after the MeJa elicitation, samples were collected from the culture medium containing BY-2 cells at 0, 24h, 48h, 72h and 96h and were analysed using SERS. Finally, the amount of anatabine released in the culture medium was determined using the response function, reaching a plateau after 72h of 82µg of anatabine released/g of fresh weight (FW) MeJa elicited BY-2 cells.

A simple calibration approach based on film-casting for confocal Raman microscopy to support the development of a hot-melt extrusion process.

When developing a new formulation, the development, calibration and validation steps of analytical methods based on vibrational spectroscopy are time-consuming. For each new formulation, real samples must be produced and a "reference method" must be used in order to determine the Active Pharmaceutical Ingredient (API) content of each sample. To circumvent this issue, the paper presents a simple approach based on the film-casting technique used as a calibration tool in the framework of hot-melt extrusion process. Confocal Raman microscopic method was successfully validated for the determination of itraconazole content in film-casting samples. Then, hot-melt extrusion was carried out to produce real samples in order to confront the results obtained with confocal Raman microscopy and Ultra High Performance Liquid Chromatography (UHPLC). The agreement between both methods was demonstrated using a comparison study based on the Bland and Altman's plot.

A simple approach for ultrasensitive detection of bisphenols by multiplexed surface-enhanced Raman scattering.

Bisphenol A (BPA) is well known for its use in plastic manufacture and thermal paper production despite its risk of health toxicity as an endocrine disruptor in humans. Since the publication of new legislation regarding the use of BPA, manufacturers have begun to replace BPA with other phenolic molecules such as bisphenol F (BPF) and bisphenol B (BPB), but there are no guarantees regarding the health safety of these compounds at this time. In this context, a very simple, cheap and fast surface-enhanced Raman scattering (SERS) method was developed for the sensitive detection of these molecules in spiked tap water solutions. Silver nanoparticles were used as SERS substrates. An original strategy was employed to circumvent the issue of the affinity of bisphenols for metallic surfaces and the silver nanoparticles surface was functionalized using pyridine in order to improve again the sensitivity of the detection. Semi-quantitative detections were performed in tap water solutions at a concentrations range from 0.25 to 20 µg L(-1) for BPA and BPB and from 5 to 100 µg L(-1) for BPF. Moreover, a feasibility study for performing a multiplex-SERS detection of these molecules was also performed before successfully implementing the developed SERS method on real samples.