Monitoring and modelling the glutamine metabolic pathway: a review and future perspectives.

BACKGROUND: Analysis of the glutamine metabolic pathway has taken a special place in metabolomics research in recent years, given its important role in cell biosynthesis and bioenergetics across several disorders, especially in cancer cell survival. The science of metabolomics addresses the intricate intracellular metabolic network by exploring and understanding how cells function and respond to external or internal perturbations to identify potential therapeutic targets. However, despite recent advances in metabolomics, monitoring the kinetics of a metabolic pathway in a living cell in situ, real-time and holistically remains a significant challenge. AIM: This review paper explores the range of analytical approaches for monitoring metabolic pathways, as well as physicochemical modeling techniques, with a focus on glutamine metabolism. We discuss the advantages and disadvantages of each method and explore the potential of label-free Raman microspectroscopy, in conjunction with kinetic modeling, to enable real-time and in situ monitoring of the cellular kinetics of the glutamine metabolic pathway. KEY SCIENTIFIC CONCEPTS: Given its important role in cell metabolism, the ability to monitor and model the glutamine metabolic pathways are highlighted. Novel, label free approaches have the potential to revolutionise metabolic biosensing, laying the foundation for a new paradigm in metabolomics research and addressing the challenges in monitoring metabolic pathways in living cells.

Multivariate curve Resolution-Alternating least squares coupled with Raman microspectroscopy: new insights into the kinetic response of primary oral squamous carcinoma cells to cisplatin.

Raman MicroSpectroscopy (RMS) is a powerful label-free tool to probe the effects of drugs at a cellular/subcellular level. It is important, however, to be able to extract relevant biochemical and kinetic spectroscopic signatures of the specific cellular responses. In the present study, a combination of Multivariate Curve Resolution-Alternating Least Squares (MCR-ALS) and Principal Component Analysis (PCA) is used to analyse the RMS data for the example of exposure of primary Oral Squamous Carcinoma Cells (OSCC) to the chemotherapeutic agent cisplatin. Dosing regimens were established by cytotoxicity assays, and the effects of the drug on cellular spectral profiles were monitored from 16 to 72 hours post-exposure using an apoptosis assay, to establish the relative populations of viable (V), early (EA) and late apoptotic/dead (LA/D) cells after the drug treatment. Based on a kinetic model of the progression from V > EA > D, MCR-ALS regression analysis of the RMS responses was able to extract spectral profiles associated with each stage of the cellular responses, enabling a quantitative comparison of the response rates for the respective drug treatments. Moreover, PCA was used to compare the spectral profiles of the viable cells exposed to the drug. Spectral differences were highlighted in the early stages (16 hours exposure), indicative of the initial cellular response to the drug treatment, and also in the late stages (48-72 hours exposure), representing the cell death pathway. The study demonstrates that RMS coupled with multivariate analysis can be used to quantitatively monitor the progression of cellular responses to different drugs, towards future applications for label-free, in vitro, pre-clinical screening.

A simple polystyrene microfluidic device for sensitive and accurate SERS-based detection of infection by malaria parasites.

Early and accurate detection of infection by pathogenic microorganisms, such as Plasmodium, the causative agent of malaria, is critical for clinical diagnosis and ultimately determines the patient's outcome. We have combined a polystyrene-based microfluidic device with an immunoassay which utilises Surface-Enhanced Raman Spectroscopy (SERS) to detect malaria. The method can be easily translated to a point-of-care testing format and shows excellent sensitivity and specificity, when compared to the gold standard for laboratorial detection of Plasmodium infections. The device can be fabricated in less than 30 min by direct patterning on shrinkable polystyrene sheets of adaptable three-dimensional microfluidic chips. To validate the microfluidic system, samples of P. falciparum-infected red blood cell cultures were used. The SERS-based immunoassay enabled the detection of 0.0012 ± 0.0001% parasitaemia in a P. falciparum-infected red blood cell culture supernatant, an ∼7-fold higher sensitivity than that attained by most rapid diagnostic tests. Our approach successfully overcomes the main challenges of the current Plasmodium detection methods, including increased reproducibility, sensitivity, and specificity. Furthermore, our system can be easily adapted for detection of other pathogens and has excellent properties for early diagnosis of infectious diseases, a decisive step towards lowering their high burden on healthcare systems worldwide.

Evaluation of Selenomethionine Entrapped in Nanoparticles for Oral Supplementation Using In Vitro, Ex Vivo and In Vivo Models.

Selenium methionine (SeMet) is an essential micronutrient required for normal body function and is associated with additional health benefits. However, oral administration of SeMet can be challenging due to its purported narrow therapeutic index, low oral bioavailability, and high susceptibility to oxidation. To address these issues, SeMet was entrapped in zein-coated nanoparticles made from chitosan using an ionic gelation formulation. The high stability of both the SeMet and selenomethionine nanoparticles (SeMet-NPs) was established using cultured human intestinal and liver epithelial cells, rat liver homogenates, and rat intestinal homogenates and lumen washes. Minimal cytotoxicity to Caco-2 and HepG2 cells was observed for SeMet and SeMet-NPs. Antioxidant properties of SeMet were revealed using a Reactive Oxygen Species (ROS) assay, based on the observation of a concentration-dependent reduction in the build-up of peroxides, hydroxides and hydroxyl radicals in Caco-2 cells exposed to SeMet (6.25-100 µM). The basal apparent permeability coefficient (Papp) of SeMet across isolated rat jejunal mucosae mounted in Ussing chambers was low, but the Papp was increased when presented in NP. SeMet had minimal effects on the electrogenic ion secretion of rat jejunal and colonic mucosae in Ussing chambers. Intra-jejunal injections of SeMet-NPs to rats yielded increased plasma levels of SeMet after 3 h for the SeMet-NPs compared to free SeMet. Overall, there is potential to further develop SeMet-NPs for oral supplementation due to the increased intestinal permeability, versus free SeMet, and the low potential for toxicity.

Formulation, Characterisation and Evaluation of the Antihypertensive Peptides, Isoleucine-Proline-Proline and Leucine-Lysine-Proline in Chitosan Nanoparticles Coated with Zein for Oral Drug Delivery.

Isoleucine-Proline-Proline (IPP) and Leucine-Lysine-Proline (LKP) are food-derived tripeptides whose antihypertensive functions have been demonstrated in hypertensive rat models. However, peptides display low oral bioavailability due to poor intestinal epithelial permeability and instability. IPP and LKP were formulated into nanoparticles (NP) using chitosan (CL113) via ionotropic gelation and then coated with zein. Following addition of zein, a high encapsulation efficiency (EE) (>80%) was obtained for the NP. In simulated gastric fluid (SGF), 20% cumulative release of the peptides was achieved after 2 h, whereas in simulated intestinal fluid (SIF), ~90% cumulative release was observed after 6 h. Higher colloidal stability (39−41 mV) was observed for the coated NP compared to uncoated ones (30−35 mV). In vitro cytotoxicity studies showed no reduction in cellular viability of human intestinal epithelial Caco-2 and HepG2 liver cells upon exposure to NP and NP components. Administration of NP encapsulating IPP and LKP by oral gavage to spontaneously hypertensive rats (SHR) attenuated systolic blood pressure (SBP) for 8 h. This suggests that the NP provide appropriate release to achieve prolonged hypotensive effects in vivo. In conclusion, chitosan-zein nanoparticles (CZ NP) have potential as oral delivery system for the encapsulation of IPP and LKP.

Quantitative Raman Analysis of Carotenoid Protein Complexes in Aqueous Solution.

Carotenoids are naturally abundant, fat-soluble pigmented compounds with dietary, antioxidant and vision protection advantages. The dietary carotenoids, Beta Carotene, Lutein, and Zeaxanthin, complexed with in bovine serum albumin (BSA) in aqueous solution, were explored using Raman spectroscopy to differentiate and quantify their spectral signatures. UV visible absorption spectroscopy was employed to confirm the linearity of responses over the concentration range employed (0.05-1 mg/mL) and, of the 4 Raman source wavelengths (785 nm, 660 nm, 532 nm, 473 nm), 532 nm was chosen to provide the optimal response. After preprocessing to remove water and BSA contributions, and correct for self-absorption, a partial least squares model with R2 of 0.9995, resulted in an accuracy of the Root Mean Squared Error of Prediction for Beta Carotene of 0.0032 mg/mL and Limit of Detection 0.0106 mg/mL. Principal Components Analysis clearly differentiated solutions of the three carotenoids, based primarily on small shifts of the main peak at ~1520 cm-1. Least squares fitting analysis of the spectra of admixtures of the carotenoid:protein complexes showed reasonable correlation between norminal% and fitted%, yielding 100% contribution when fitted with individual carotenoid complexes and variable contributions with multiple ratios of admixtures. The results indicate the technique can potentially be used to quantify the carotenoid content of human serum and to identify their differential contributions for application in clinical analysis.

Raman Spectroscopy of Carotenoid Compounds for Clinical Applications-A Review.

Carotenoid compounds are ubiquitous in nature, providing the characteristic colouring of many algae, bacteria, fruits and vegetables. They are a critical component of the human diet and play a key role in human nutrition, health and disease. Therefore, the clinical importance of qualitative and quantitative carotene content analysis is increasingly recognised. In this review, the structural and optical properties of carotenoid compounds are reviewed, differentiating between those of carotenes and xanthophylls. The strong non-resonant and resonant Raman spectroscopic signatures of carotenoids are described, and advances in the use of Raman spectroscopy to identify carotenoids in biological environments are reviewed. Focus is drawn to applications in nutritional analysis, optometry and serology, based on in vitro and ex vivo measurements in skin, retina and blood, and progress towards establishing the technique in a clinical environment, as well as challenges and future perspectives, are explored.

Estimating the Analytical Performance of Raman Spectroscopy for Quantification of Active Ingredients in Human Stratum Corneum.

Confocal Raman microscopy (CRM) has become a versatile technique that can be applied routinely to monitor skin penetration of active molecules. In the present study, CRM coupled to multivariate analysis (namely PLSR-partial least squares regression) is used for the quantitative measurement of an active ingredient (AI) applied to isolated (ex vivo) human stratum corneum (SC), using systematically varied doses of resorcinol, as model compound, and the performance is quantified according to key figures of merit defined by regulatory bodies (ICH, FDA, and EMA). A methodology is thus demonstrated to establish the limit of detection (LOD), precision, accuracy, sensitivity (SEN), and selectivity (SEL) of the technique, and the performance according to these key figures of merit is compared to that of similar established methodologies, based on studies available in literature. First, principal components analysis (PCA) was used to examine the variability within the spectral data set collected. Second, ratios calculated from the area under the curve (AUC) of characteristic resorcinol and proteins/lipids bands (1400-1500 cm-1) were used to perform linear regression analysis of the Raman spectra. Third, cross-validated PLSR analysis was applied to perform quantitative analysis in the fingerprint region. The AUC results show clearly that the intensities of Raman features in the spectra collected are linearly correlated to resorcinol concentrations in the SC (R2 = 0.999) despite a heterogeneity in the distribution of the active molecule in the samples. The Root Mean Square Error of Cross-Validation (RMSECV) (0.017 mg resorcinol/mg SC), The Root Mean Square of Prediction (RMSEP) (0.015 mg resorcinol/mg SC), and R2 (0.971) demonstrate the reliability of the linear regression constructed, enabling accurate quantification of resorcinol. Furthermore, the results have enabled the determination, for the first time, of numerical criteria to estimate analytical performances of CRM, including LOD, precision using bias corrected mean square error prediction (BCMSEP), sensitivity, and selectivity, for quantification of the performance of the analytical technique. This is one step further towards demonstrating that Raman spectroscopy complies with international guidelines and to establishing the technique as a reference and approved tool for permeation studies.

Comparison of Vibrational Spectroscopic Techniques for Quantification of Water in Natural Deep Eutectic Solvents.

Vibrational spectroscopic techniques, i.e., attenuated total reflectance infrared (ATR-IR), near infrared spectroscopy (NIRS) and Raman spectroscopy (RS), coupled with Partial Least Squares Regression (PLSR), were evaluated as cost-effective label-free and reagent-free tools to monitor water content in Levulinic Acid/L-Proline (LALP) (2:1, mol/mol) Natural Deep Eutectic Solvent (NADES). ATR-IR delivered the best outcome of Root Mean Squared Error (RMSE) of Cross-Validation (CV) = 0.27% added water concentration, RMSE of Prediction (P) = 0.27% added water concentration and mean % relative error = 2.59%. Two NIRS instruments (benchtop and handheld) were also compared during the study, respectively yielding RMSECV = 0.35% added water concentration, RMSEP = 0.56% added water concentration and mean % relative error = 5.13% added water concentration, and RMECV = 0.36% added water concentration, RMSEP = 0.68% added water concentration and mean % relative error = 6.23%. RS analysis performed in quartz cuvettes enabled accurate water quantification with RMECV = 0.43% added water concentration, RMSEP = 0.67% added water concentration and mean % relative error = 6.75%. While the vibrational spectroscopic techniques studied have shown high performance in relation to reliable determination of water concentration, their accuracy is most likely related to their sensitivity to detect the LALP compounds in the NADES. For instance, whereas ATR-IR spectra display strong features from water, Levulinic Acid and L-Proline that contribute to the PLSR predictive models constructed, NIRS and RS spectra are respectively dominated by either water or LALP compounds, representing partial molecular information and moderate accuracy compared to ATR-IR. However, while ATR-IR instruments are common in chemistry and physics laboratories, making the technique readily transferable to water quantification in NADES, Raman spectroscopy offers promising potential for future development for in situ, sample withdrawal-free analysis for high throughput and online monitoring.

Monitoring stem cell differentiation using Raman microspectroscopy: chondrogenic differentiation, towards cartilage formation.

Mesenchymal Stem Cells (MSCs) have the ability to differentiate into chondrocytes, the only cellular components of cartilage and are therefore ideal candidates for cartilage and tissue repair technologies. Chondrocytes are surrounded by cartilage-like extracellular matrix (ECM), a complex network rich in glycosaminoglycans, proteoglycans, and collagen, which, together with a multitude of intracellular signalling molecules, trigger the chondrogenesis and allow the chondroprogenitor to acquire the spherical morphology of the chondrocytes. However, although the mechanisms of the differentiation of MSCs have been extensively explored, it has been difficult to provide a holistic picture of the process, in situ. Raman Micro Spectroscopy (RMS) has been demonstrated to be a powerful analytical tool, which provides detailed label free biochemical fingerprint information in a non-invasive way, for analysis of cells, tissues and body fluids. In this work, RMS is explored to monitor the process of Mesenchymal Stem Cell (MSC) differentiation into chondrocytes in vitro, providing a holistic molecular picture of cellular events governing the differentiation. Spectral signatures of the subcellular compartments, nucleolus, nucleus and cytoplasm were initially probed and characteristic molecular changes between differentiated and undifferentiated were identified. Moreover, high density cell micromasses were cultured over a period of three weeks, and a systematic monitoring of cellular molecular components and the progress of the ECM formation, associated with the chondrogenic differentiation, was performed. This study shows the potential applicability of RMS as a powerful tool to monitor and better understand the differentiation pathways and process.

Exploiting fourier transform infrared and Raman microspectroscopies on cancer stem cells from oral squamous cells carcinoma: new evidence of acquired cisplatin chemoresistance.

Oral Squamous Cells Carcinoma (OSCC) is characterised by the risk of recurrence and the onset of a refractoriness response to chemotherapy drugs. These phenomena have been recently related to a subpopulation of Cancer Stem Cells (CSCs), which have either an innate or acquired drug resistance, triggered by chemotherapy treatments. In this light, to precisely target chemotherapy regimens, it is essential to improve knowledge on CSCs, with a particular focus on their molecular features. In this work, a subpopulation of CSCs, isolated by tumour sphere formation from primary OSCC cells, were treated with cisplatin for 16, 24 and 48 hours and analysed by infrared absorption and Raman microspectroscopies. CSC spectral data were compared with those obtained in previous work, for primary OSCC cells treated under the same conditions. Routine viability/apoptosis cell-based assays evidenced in CSCs and primary OSCCs, a similar degree of sensitivity to the drug at 24 hours, while a reversion of the conventional monotonic time response exhibited by OSCCs was shown by CSCs at 48 hours. This peculiar time response was also supported by the analysis of IR and Raman data, which pinpointed alterations in the lipid composition and DNA conformation in CSCs. The results obtained suggest that CSCs, although sharing with OSCC cells a similar sensitivity to cisplatin, display the onset of a mechanism of chemoresistance and enrichment of resistant CSCs as a result of drug treatment, shedding new light on the severe issue of refractoriness of some patients to chemotherapy conventionally used for OSCC.

Comparison of Raman and attenuated total reflectance (ATR) infrared spectroscopy for water quantification in natural deep eutectic solvent.

Natural deep eutectic solvents (NADES) are ionic solutions, of great interest for extraction from biomass, biocatalysis, and nanoparticle synthesis. They are easily synthesised and eco-friendly, have low volatility and high dissolution power, and are biodegradable. However, water content in NADES is a critical parameter, affecting their optimal use and extraction efficiency. Vibrational spectroscopic techniques are rapid, label-free, non-destructive, non-invasive, and cost-effective analytical tools that can probe the molecular composition of samples. A direct comparison between a previous study using attenuated total reflectance infrared (ATR-IR) spectroscopy for water quantification in NADES and the same investigation performed with Raman spectroscopy is presently reported. Three NADES systems, namely betaine-glycerol (BG), choline chloride-glycerol (CCG), and glucose-glycerol (GG), containing a range of water concentrations between 0% (w/w) and 40% (w/w), have been analysed with Raman spectroscopy coupled to partial least squares regression multivariate analysis. The values of root mean square error of cross-validation (RMSECV) obtained from analysis performed on the pre-processed spectra over the full spectral range (150-3750 cm-1) are respectively 0.2966% (w/w), 0.4703% (w/w), and 0.2351% (w/w) for BG, GG, and CCG. While the direct comparison to previous ATR-IR results shows essentially similar outcomes for BG, the RMSECV is 33.14% lower and 65.84% lower for CG and CCG. Furthermore, mean relative errors obtained with Raman spectroscopy, and calculated from a set of samples used as independent samples, were 1.452% (w/w), 1.175% (w/w), and 1.188% (w/w). Ultimately, Raman spectroscopy delivered performances for quantification of water in NADES with similar accuracy to ATR-IR. The present demonstration clearly highlights the potential of Raman spectroscopy to support the development of new analytical protocols in the field of green chemistry.

Confocal Raman Spectroscopic Imaging for Evaluation of Distribution of Nano-Formulated Hydrophobic Active Cosmetic Ingredients in Hydrophilic Films.

Film-forming systems are highly relevant to the topical administration of active ingredients (AI) to the body. Enhanced contact with the skin can increase the efficacy of delivery and penetration during prolonged exposure. However, after the evaporation of volatile solvents to form a thin film, the distribution of the ingredient should remain homogenous in order to ensure the effectiveness of the formula. This is especially critical for the use of hydrophobic molecules that have poor solubility in hydrophilic films. In order to address this concern, hydroxyphenethyl esters (PHE) of Punica granatum seed oil were prepared as a nanosuspension stabilised by poloxamers (NanoPHE). NanoPHE was then added to a formulation containing polyvinyl alcohol (PVA) as a film forming agent, Glycerol as a plasticiser and an antimicrobial agent, SepicideTM HB. Despite their reliability, reference methods such as high-performance liquid chromatography are increasingly challenged due to the need for consumables and solvents, which is contrary to current concerns about green industry in the cosmetics field. Moreover, such methods fail to provide spatially resolved chemical information. In order to investigate the distribution of ingredients in the dried film, Confocal Raman imaging (CRI) coupled to Non-negatively Constrained Least Squares (NCLS) analysis was used. The reconstructed heat maps from a range of films containing systematically varying PHE concentrations highlighted the changes in spectral contribution from each of the ingredients. First, using NCLS scores it was demonstrated that the distributions of PVA, Glycerol, SepicideTM HB and PHE were homogenous, with respective relative standard deviations (RSD) of 3.33%, 2.48%, 2.72% and 6.27%. Second, the respective relationships between ingredient concentrations in the films and their Raman responses, and the spectral abundance were established. Finally, a model for absolute quantification for PHE was be constructed using the percentage of spectral abundance. The prepared %w/w concentrations regressed against predicted %w/w concentrations, displaying high correlation (R2 = 0.995), while the Root Mean Squared Error (0.0869% w/w PHE) confirmed the precision of the analysis. The mean percent relative error of 3.75% indicates the accuracy to which the concentration in dried films could be determined, further supporting the suitability of CRI for analysis of composite solid film matrix. Ultimately, it was demonstrated that nanoformulation of hydrophobic PHE provides homogenous distribution in PVA based film-forming systems independent of the concentration of NanoPHE used in the formula.

In Situ Water Quantification in Natural Deep Eutectic Solvents Using Portable Raman Spectroscopy.

Raman spectroscopy is a label-free, non-destructive, non-invasive analytical tool that provides insight into the molecular composition of samples with minimum or no sample preparation. The increased availability of commercial portable Raman devices presents a potentially easy and convenient analytical solution for day-to-day analysis in laboratories and production lines. However, their performance for highly specific and sensitive analysis applications has not been extensively evaluated. This study performs a direct comparison of such a commercially available, portable Raman system, with a research grade Raman microscope system for the analysis of water content of Natural Deep Eutectic Solvents (NADES). NADES are renewable, biodegradable and easily tunable "green" solvents, outcompeting existing organic solvents for applications in extraction from biomass, biocatalysis, and nanoparticle synthesis. Water content in NADES is, however, a critical parameter, affecting their properties, optimal use and extraction efficiency. In the present study, portable Raman spectroscopy coupled with Partial Least Squares Regression (PLSR) is investigated for rapid determination of water content in NADES samples in situ, i.e., directly in glassware. Three NADES systems, namely Betaine Glycerol (BG), Choline Chloride Glycerol (CCG) and Glucose Glycerol (GG), containing a range of water concentrations between 0% (w/w) and 28.5% (w/w), were studied. The results are directly compared with previously published studies of the same systems, using a research grade Raman microscope. PLSR results demonstrate the reliability of the analysis, surrendering R2 values above 0.99. Root Mean Square Errors Prediction (RMSEP) of 0.6805%, 0.9859% and 1.2907% w/w were found for respectively unknown CCG, BG and GG samples using the portable device compared to 0.4715%, 0.3437% and 0.7409% w/w previously obtained by analysis in quartz cuvettes with a Raman confocal microscope. Despite the relatively higher values of RMSEP observed, the comparison of the percentage of relative errors in the predicted concentration highlights that, overall, the portable device delivers accuracy below 5%. Ultimately, it has been demonstrated that portable Raman spectroscopy enables accurate quantification of water in NADES directly through glass vials without the requirement for sample withdrawal. Such compact instruments provide solvent and consumable free analysis for rapid analysis directly in laboratories and for non-expert users. Portable Raman is a promising approach for high throughput monitoring of water content in NADES that can support the development of new analytical protocols in the field of green chemistry in research and development laboratories but also in the industry as a routine quality control tool.

Comparability of Raman Spectroscopic Configurations: A Large Scale Cross-Laboratory Study.

The variable configuration of Raman spectroscopic platforms is one of the major obstacles in establishing Raman spectroscopy as a valuable physicochemical method within real-world scenarios such as clinical diagnostics. For such real world applications like diagnostic classification, the models should ideally be usable to predict data from different setups. Whether it is done by training a rugged model with data from many setups or by a primary-replica strategy where models are developed on a 'primary' setup and the test data are generated on 'replicate' setups, this is only possible if the Raman spectra from different setups are consistent, reproducible, and comparable. However, Raman spectra can be highly sensitive to the measurement conditions, and they change from setup to setup even if the same samples are measured. Although increasingly recognized as an issue, the dependence of the Raman spectra on the instrumental configuration is far from being fully understood and great effort is needed to address the resulting spectral variations and to correct for them. To make the severity of the situation clear, we present a round robin experiment investigating the comparability of 35 Raman spectroscopic devices with different configurations in 15 institutes within seven European countries from the COST (European Cooperation in Science and Technology) action Raman4clinics. The experiment was developed in a fashion that allows various instrumental configurations ranging from highly confocal setups to fibre-optic based systems with different excitation wavelengths. We illustrate the spectral variations caused by the instrumental configurations from the perspectives of peak shifts, intensity variations, peak widths, and noise levels. We conclude this contribution with recommendations that may help to improve the inter-laboratory studies.

Surface Enhanced Raman Spectroscopy for Quantitative Analysis: Results of a Large-Scale European Multi-Instrument Interlaboratory Study.

Surface-enhanced Raman scattering (SERS) is a powerful and sensitive technique for the detection of fingerprint signals of molecules and for the investigation of a series of surface chemical reactions. Many studies introduced quantitative applications of SERS in various fields, and several SERS methods have been implemented for each specific application, ranging in performance characteristics, analytes used, instruments, and analytical matrices. In general, very few methods have been validated according to international guidelines. As a consequence, the application of SERS in highly regulated environments is still considered risky, and the perception of a poorly reproducible and insufficiently robust analytical technique has persistently retarded its routine implementation. Collaborative trials are a type of interlaboratory study (ILS) frequently performed to ascertain the quality of a single analytical method. The idea of an ILS of quantification with SERS arose within the framework of Working Group 1 (WG1) of the EU COST Action BM1401 Raman4Clinics in an effort to overcome the problematic perception of quantitative SERS methods. Here, we report the first interlaboratory SERS study ever conducted, involving 15 laboratories and 44 researchers. In this study, we tried to define a methodology to assess the reproducibility and trueness of a quantitative SERS method and to compare different methods. In our opinion, this is a first important step toward a "standardization" process of SERS protocols, not proposed by a single laboratory but by a larger community.

Potential of Raman spectroscopy for the analysis of plasma/serum in the liquid state: recent advances.

There is compelling evidence in the literature to support the application of Raman spectroscopy for analysis of bodily fluids in their native liquid state. Naturally, the strategies described in the literature for Raman spectroscopic analysis of liquid samples have advantages and disadvantages. Herein, recent advances in the analysis of plasma/serum in the liquid state are reviewed. The potential advantages of Raman analysis in the liquid form over the commonly employed infrared absorption analysis in the dried droplet form are initially highlighted. Improvements in measurement protocols based on inverted microscopic geometries, clinically adaptable substrates, data preprocessing and analysis and applications for routine monitoring of patient health as well as therapeutic administration are reviewed. These advances suggest that clinical translation of Raman spectroscopy for rapid biochemical analysis can be a reality. In the future, this method will prove to be highly beneficial to clinicians for rapid screening and monitoring of analytes and drugs in the biological fluids, and to the patients themselves, enabling early treatment, before the disease becomes symptomatic, allowing early recovery.

Quantification of low-content encapsulated active cosmetic ingredients in complex semi-solid formulations by means of attenuated total reflectance-infrared spectroscopy.

Attenuated total reflectance-infrared (ATR-IR) spectroscopy is a robust tool for molecular characterisation of matter. Applied to semi-solid formulations, it enables rapid and reliable data collection without pre-analytical requirements. Based on nano-encapsulated Omegalight®, a skin-lightening active cosmetic ingredient (ACI), incorporated in a hydrogel, it is first demonstrated that, despite the high water content and the chemical complexity of the samples (i.e. number of ingredients), the spectral features of the ACI can be detected and monitored. Secondly, with a total of 105 samples divided into a training set (n = 60) and an unknown set (n = 45) covering a 0.5% w/w-5% w/w concentration range, the study further investigates the quantitative performance of ATR-IR coupled with partial least squares regression (PLSR). Through a step-by-step approach in testing different cross-validation protocols, accuracy (root mean square error of cross-validation (RMSECV)) and linearity between the experimental and predicted concentrations (R2) of ATR-IR are consistently evaluated to be respectively 0.097% (w/w) and 0.995 with a lower LOD = 0.067% (w/w). Subsequently, further evaluation of the accuracy (relative error of the predicted concentration compared with the true value, expressed as %) of the analysis was undertaken with the 45 unknown samples that were defined as unknown and analysed by PLSR. The outcome of the analysis demonstrates the ruggedness and the consistency of the determination performed using the ATR-IR data. With an average relative error of 2.5% w/w and only 5 samples out of 45 blind samples exhibiting a relative error above the 5% threshold, high accuracy quantification of the nano-encapsulated ACI can be unambiguously achieved by means of the label-free and non-destructive technique of ATR-IR spectroscopy. Ultimately, the study demonstrates that the analytical capabilities of ATR-IR hold significant potential for applications in the cosmetics industry, and although the path remains long, the present study is one step further to support validation of the technique, albeit for the specific case of Omegalight®.

Graphene Nanoflake Uptake Mediated by Scavenger Receptors.

The biological interactions of graphene have been extensively investigated over the last 10 years. However, very little is known about graphene interactions with the cell surface and how the graphene internalization process is driven and mediated by specific recognition sites at the interface with the cell. In this work, we propose a methodology to investigate direct molecular correlations between the biomolecular corona of graphene and specific cell receptors, showing that key protein recognition motifs, presented on the nanomaterial surface, can engage selectively with specific cell receptors. We consider the case of apolipoprotein A-I, found to be very abundant in the graphene protein corona, and observe that the uptake of graphene nanoflakes is somewhat increased in cells with greatly elevated expression of scavenger receptors B1, suggesting a possible mechanism of endogenous interaction. The uptake results, obtained by flow cytometry, have been confirmed using Raman microspectroscopic mapping, exploiting the strong Raman signature of graphene.

Vibrational Spectroscopy for In Vitro Monitoring Stem Cell Differentiation.

Stem cell technology has attracted considerable attention over recent decades due to its enormous potential in regenerative medicine and disease therapeutics. Studying the underlying mechanisms of stem cell differentiation and tissue generation is critical, and robust methodologies and different technologies are required. Towards establishing improved understanding and optimised triggering and control of differentiation processes, analytical techniques such as flow cytometry, immunohistochemistry, reverse transcription polymerase chain reaction, RNA in situ hybridisation analysis, and fluorescence-activated cell sorting have contributed much. However, progress in the field remains limited because such techniques provide only limited information, as they are only able to address specific, selected aspects of the process, and/or cannot visualise the process at the subcellular level. Additionally, many current analytical techniques involve the disruption of the investigation process (tissue sectioning, immunostaining) and cannot monitor the cellular differentiation process in situ, in real-time. Vibrational spectroscopy, as a label-free, non-invasive and non-destructive analytical technique, appears to be a promising candidate to potentially overcome many of these limitations as it can provide detailed biochemical fingerprint information for analysis of cells, tissues, and body fluids. The technique has been widely used in disease diagnosis and increasingly in stem cell technology. In this work, the efforts regarding the use of vibrational spectroscopy to identify mechanisms of stem cell differentiation at a single cell and tissue level are summarised. Both infrared absorption and Raman spectroscopic investigations are explored, and the relative merits, and future perspectives of the techniques are discussed.