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Label-free surface-enhanced Raman scattering (SERS) has recently gained attention in the field of liquid biopsy as a rapid and relatively inexpensive technique that could significantly ease clinical diagnosis and prognosis by investigating a biofluid sample with a laser. Indeed, SERS spectra provide information about a set of metabolites present in the analysed biofluid, thereby offering biochemical insight into specific health conditions. Ergothioneine plays a key role since it is one of the few metabolites in biofluids that are detectable by label-free SERS. In the past decade, many studies characterizing biofluids or other biological samples have unknowingly linked this amino acid with crucial metabolic processes, including inflammation, in a plethora of diseases. However, since the SERS spectrum of ergothioneine has been reported only recently, most past studies inadvertently assigned what are now recognized as the spectral features of this compound to other molecules. The purpose of the present review is to summarize and re-evaluate these studies in the light of the recent SERS characterization of ergothioneine so as to better recognize the role of ergothioneine in many clinical conditions.

Intense label-free surface-enhanced Raman scattering (SERS) spectra of serum samples were rapidly obtained on Ag plasmonic paper substrates upon 785 nm excitation. Spectra from the hepatocellular carcinoma (HCC) patients showed consistent differences with respect to those of the control group. In particular, uric acid was found to be relatively more abundant in patients, while hypoxanthine, ergothioneine, and glutathione were found as relatively more abundant in the control group. A repeated double cross-validation (RDCV) strategy was applied to optimize and validate principal component analysis-linear discriminant analysis (PCA-LDA) models. An analysis of the RDCV results indicated that a PCA-LDA model using up to the first four principal components has a good classification performance (average accuracy was 81%). The analysis also allowed confidence intervals to be calculated for the figures of merit, and the principal components used by the LDA to be interpreted in terms of metabolites, confirming that bands of uric acid, hypoxanthine, ergothioneine, and glutathione were indeed used by the PCA-LDA algorithm to classify the spectra. Supplementary Information The online version contains supplementary material available at 10.1007/s00216-020-03093-7.

Surface-enhanced Raman scattering (SERS) spectra of faecal samples can be obtained by adding AuNP to their methanol extracts according to the reported protocol, and display bands that are due to bilirubin-like species but also to xanthine and hypoxanthine, two metabolic products secreted by gut bacteria. A total of 27 faecal samples from three different groups, i.e. coeliac patients (n = 9), coeliac patients on gluten-free diet (n = 10) and a control group (n = 8), were characterized with both SERS spectroscopy and 16S rRNA sequencing analysis. Significant differences are present between SERS spectra of coeliac patients and those on gluten-free diet, with a marked increase in the relative intensity of both xanthine and hypoxanthine for the latter. Interestingly, these differences do not correlate with bacterial composition as derived from 16S rRNA sequencing.

Label-free SERS is a powerful bio-analytical technique in which molecular fingerprinting is combined with localized surface plasmons (LSPs) on metal surfaces to achieve high sensitivity. Silver and gold colloids are among the most common nanostructured substrates used in SERS, but since protein-rich samples such as serum or plasma can hinder the SERS effect due to protein–substrate interactions, they often require a deproteinization step. Moreover, SERS methods based on metal colloids often suffer from a poor reproducibility. Here, we propose a paper-based SERS sampling method in which unprocessed human serum samples are first soaked on paper strips (0.4 × 2 cm2), and then mixed with colloidal silver nanoparticles by centrifugation to obtain a Centrifugal Silver Plasmonic Paper (CSPP). The CSPP methodology has the potential to become a promising tool in bioanalytical SERS applications: it uses common colloidal substrates but without the need for sample deproteinization, while having a good reproducibility both in terms of overall spectral shape (r > 0.96) and absolute intensity (RSD < 10%). Moreover, this methodology allows SERS analysis more than one month after serum collection on the paper strip, facilitating storage and handling of clinical samples (including shipping from clinical sites to labs).

Surface-enhanced Raman scattering (SERS) is a powerful and sensitive technique for the detection of fingerprint sig-nals of molecules and for the investigation of a series of surface chemical reactions. Many studies introduced quantita-tive applications of SERS in various fields and several SERS methods have been implemented for each specific applica- tion, 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 high-ly- 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 interlabora-tory study (ILS) frequently performed to ascertain the quality of a single analytical method. The idea of an ILS of quan- tification with SERS arose within the framework of Working Group 1 (WG1) of the COST Action BM1401 Ra-man4Clinics32 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 41 researchers. In this study we tried to define a methodology to assess the reproducibility and trueness of a quantitative SERS method, and to compare differ- ent 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.

Gingival crevicular fluid (GCF) is an interesting biofluid reflecting the physiological and pathological states of a single dental element. Due to this unique feature, in recent years, metabolomic analysis of GCF has gained attention as a biometric tool for the diagnosis and therapy of periodontal disease. Traditional methods are, however, too slow, cumbersome and expensive for a health-care routine. Surface enhanced Raman scattering (SERS) can offer rapid and label-free detailed molecular fingerprints that can be used for biofluid analysis. Here we report the first SERS characterization of GCF using an easy and quick sample preparation. The dominant features in the SERS spectrum of GCF are ascribed to very few metabolites, in particular to uric acid, hypoxanthine, glutathione and ergothioneine. Additionally, we succeeded in differentiating between the SERS signal of GCF collected from healthy volunteers and the one collected from patients with periodontal disease.

Intense SERS spectra of the natural amino acid ergothioneine (ERG) are obtained on different substrates upon 785 nm excitation. A characteristic spectral pattern with a distinctive intense band at 480–486 cm−1 is conserved when substrates of different type and characteristics are used. On the basis of available literature, we propose ERG is adsorbed on the metal surface in its thiolate form via the sulphur and heterocyclic nitrogen. The same spectral pattern is obtained in SERS spectra of filtered erythrocytes lysates, confirming the presence of ERG in those cells. The occurrence of ERG bands in label-free SERS spectra of serum and plasma reported in literature by different authors is discussed, highlighting the importance of this amino acid for the interpretation of SERS spectra of these biofluids.

Raman hyperspectral imaging is an emerging practice in biological and biomedical research for label free analysis of tissues and cells. Using this method, both spatial distribution and spectral information of analyzed samples can be obtained. The current study reports the first Raman microspectroscopic characterisation of colon tissues from patients with Coeliac Disease (CD). The aim was to assess if Raman imaging coupled with hyperspectral multivariate image analysis is capable of detecting the alterations in the biochemical composition of intestinal tissues associated with CD. The analytical approach was based on a multi-step methodology: duodenal biopsies from healthy and coeliac patients were measured and processed with Multivariate Curve Resolution Alternating Least Squares (MCR-ALS). Based on the distribution maps and the pure spectra of the image constituents obtained from MCR-ALS, interesting biochemical differences between healthy and coeliac patients has been derived. Noticeably, a reduced distribution of complex lipids in the pericryptic space, and a different distribution and abundance of proteins rich in beta-sheet structures was found in CD patients. The output of the MCR-ALS analysis was then used as a starting point for two clustering algorithms (k-means clustering and hierarchical clustering methods). Both methods converged with similar results providing precise segmentation over multiple Raman images of studied tissues.

Objective Separately addressing the fatigue resistance (ISO 14801, evaluation of final product) and aging behavior (ISO 13356, standardized sample) of oral implants made from yttria-stabilized zirconia proved to be insufficient in verifying their long-term stability, since (1) implant processing is known to significantly influence transformation kinetics and (2) aging, up from a certain level, is liable to decrease fatigue resistance. Therefore, the aim of this investigation was to apply a new testing protocol considering environmental conditions adequately inducing aging during dynamic fatigue. Methods Zirconia implants were dynamically loaded (107 cycles), hydrothermally aged (85°, 60 days) or subjected to both treatments simultaneously. Subsequent, monoclinic intensity ratios (Xm) were obtained by locally resolved X-ray microdiffraction (μ-XRD2). Transformation propagation was monitored at cross-sections by μ-Raman spectroscopy and scanning electron microscopy (SEM). Finally, implants were statically loaded to fracture. Linear regression models (fracture load) and mixed models (Xm) were used for statistical analyses. Results All treatments resulted in increased fracture load (p ≤ 0.005), indicating the formation of transformation induced compressive stresses around surface defects during all treatment modalities. However, only hydrothermal and combinational treatment were found to increase Xm (p < 0.001). No change in Xm was observed for solely dynamically loaded samples (p ≥ 0.524). Depending on the variable observed, a monoclinic layer thickness of 1–2 μm (SEM) or 6–8 μm (Raman spectroscopy) was measured at surfaces exposed to water during treatments. Significance Hydrothermal aging was successfully induced during dynamic fatigue. Therefore, the presented setup might serve as reference protocol for ensuring pre-clinically long-term reliability of zirconia oral implants.

Non-alcoholic fatty liver disease (NAFLD) is a chronic disorder progressing from an initial benign accumulation of fat (NAFL) towards steatohepatitis (NASH), a degenerative form that can lead to liver cirrhosis and cancer. The development of non-invasive, rapid and accurate method to diagnose NASH is of high clinical relevance. Surface-enhanced Raman spectroscopy (SERS) of plasma was tested as a method to distinguish NAFL from NASH. SERS spectra from plasma of female patients diagnosed with NAFL (n = 32) and NASH (n = 35) were obtained in few seconds, using a portable Raman spectrometer. The sample consisted of 5 μL of biofluid deposited on paper coated with Ag nanoparticles. The spectra show consistent differences between the NAFL and NASH patients, with the uric acid/hypoxanthine band area ratio statistically different (p-value

A crystal of synthetic chromite FeCr2O4 has been annealed in air at 700 °C for 50 days at room pressure in order to study physical-chemical changes. After the annealing treatment, Scanning Electron Microscopy (SEM) images of the polished surface of the sample showed areas of different composition. Detailed Raman mapping revealed that the annealed chromite undergoes an oxidation process, proceeding inwards from the outer surface and leading to the transformation of chromite to magnetite, and ultimately to hematite. The oxidation process also leads to the formation of trellis-like lines, arguably via stress-related mechanisms associated with the phase transformation and consequent volume changes. The present study confirms the possibility of non-stoichiometry or structural defects causing slight structural asymmetries and the formation of new modes, resulting in a valuable support in characterizing both natural and artificial material. The potential of Raman spectroscopy in the identification and spatial resolution of mineralogical phases, is also highlighted.

Stabilized zirconia exhibits unsurpassed mechanical properties and biocompatibility, making it an indispensable ceramic material for biomedical implants. One of the most problematic features of stabilized zirconia has been its low-temperature degradation, which is attributed to the observed transformation of its crystalline structure from tetragonal to monoclinic phase. The presence of monoclinic phases, therefore, is a red-flag for the impending catastrophic breakdown of its mechanical properties. In this work, we utilize surface-enhanced Raman spectroscopy (SERS) with colloidal gold nanostars with mean diameter of 78 +/- 13 nm (measured from tip to tip across the nanostar) as substrate. The nanostars have localized surface plasmon resonance at 690 nm. Spectral maps on clean and nanostar-covered surfaces were obtained exactly at the same position using con focal Raman spectroscopy. Comparison of the two maps shows that there are more monoclinic phases detected in the nanostar-covered surface possibly due to the "lightning rod" effect in the nanostar tips. SERS of solid zirconia has not been demonstrated elsewhere and our results could provide early evidence of the effectivity of the technique even on non-porous materials. With further improvement in sensitivity, SERS can be a promising technique for the early detection of monoclinic phase in zirconia-based implants.

International audience; 40 years ago, Garvie and his Australian co-workers reported that the stress induced transformation of metastable tetragonal zirconia grains to the monoclinic symmetry could give rise to a powerful toughening mechanism. Their results even led them to consider zirconia systems as analogues of certain steels. This seminal paper generated extraordinary excitement in the ceramic community and it is still the subject of extensive research. Transformation toughening is widely used in zirconia materials and results in an increase in strength and toughness when compared to non-transformable ceramics, but the implementation into strong, tough, and sufficiently stable materials has not been fully reached. Zirconia ceramics generally fail at low strains with a much larger scatter in the strength values than metals and require statistical approaches to failure. Here we describe in details the mechanical behavior laws of ceria-doped zirconia composites exhibiting a high degree of stress-induced transformation. They present, to some extent, mechanical behavior analogous to a metal, displaying, i) significant amount of transformation-induced plasticity without damage, ii) very high flaw

Danon disease is a severe X-linked disorder caused by deficiency of the lysosome-associated membrane protein-2 (LAMP-2). Clinical manifestations are phenotypically diverse and consist of hypertrophic and dilated cardiomyopathies, skeletal myopathy, retinopathy, and intellectual dysfunction. Here, we investigated the metabolic landscape of Danon disease by applying a multi-omics approach and combined structural and functional readouts provided by Raman and atomic force microscopy. Using these tools, Danon patient-derived cardiac tissue, primary fibroblasts, and human induced pluripotent stem cells differentiated into cardiomyocytes (hiPSC-CMs) were analyzed. Metabolic profiling indicated LAMP-2 deficiency promoted a switch toward glycolysis accompanied by rerouting of tryptophan metabolism. Cardiomyocytes&rsquo, energetic balance and NAD+/NADH ratio appeared to be maintained despite mitochondrial aging. In turn, metabolic adaption was accompanied by a senescence-associated signature. Similarly, Danon fibroblasts appeared more stress prone and less biomechanically compliant. Overall, shaping of both morphology and metabolism contributed to the loss of cardiac biomechanical competence that characterizes the clinical progression of Danon disease.

Modern diagnostic tools ever aim to reduce the amount of analyte and the time needed for obtaining the result. Surface-enhanced Raman spectroscopy is a method that could satisfy both of these requirements, provided that for each analyte an adequate substrate is found. Here we demonstrate the ability of gold-sputtered silicon nanowires (SiNW) to bind p-mercaptobenzoic acid in 10−3, 10−4 and 10−5 M and adenine in 30 and 100 μM concentrations. Based on the normal mode analysis, presented here for the first time, the binding of p-mercaptobenzoic acid is deduced. The intensity enhancement of the 1106 cm−1 band is explained by involvement of the C S stretching deformation, and the appearance of the broad 300 cm−1 band attributed to S Au stretching mode. Adenine SERS spectra demonstrate the existence of the 7H tautomer since the strongest band observed is at 736 cm−1. The adenine binding is likely to occur in several ways, because the number of observed bands in the 1200–1600 cm−1 interval exceeds the number of observed bands in the normal Raman spectrum of the free molecule.

Duncan Graham opened a general discussion of the paper by Pavel Matousek: Regarding the temperature measurements, how did you do them? Stokes/anti-Stokes measurement? The values are not what I'd expect and seem lower. What was the wavelength and power of the laser and did you calculate what you thought the temperature measurement should be based on that? How far does the temperature gradient reach? The scattering absorption ration changes with nanoparticle size and composition. Have you thought about using more highly absorbing nanoparticles such as hollow gold nanoshells?

To date, in spite of their toxicity, the plasmatic concentration of most chemotherapeutic drugs is difficult to monitor in oncological patients, because their quantitative determination is expensive and time consuming. This contribution reports a first attempt for the direct quantitative determination of a chemotherapeutic drug in human serum samples by means of Surface Enhanced Raman Spectroscopy (SERS). In this study, SERS substrates constituted by Au nanoparticles deposited on paper by a simple dipping method have been used for rapid (few minutes) analysis of diluted human serum spiked with different concentrations of methotrexate, MTX. The drug concentrations were chosen in a range designed to cover typical therapeutic plasmatic values (from nanomolar to millimolar) in oncological patients, and the pertinent calibration was obtained by Partial Least-Squares Regression (PLSR). Stability selection was employed to evaluate the capability of the PLSR model to accurately predict and extract spectral variations correlated to MTX concentration. Such a quantitative determination is crucial for frequent, and hence adherent, therapeutic drug monitoring, TDM, of chemiotherapic drugs, given their heavy side effects. Its low cost, rapid response and the possibility of obtaining spectra with simple and compact instruments, make SERS particularly apt for implementing effective TDM. The promising results obtained in the analytical validation indicate which steps are to be taken on the way toward a clinical validation with real samples from oncological patients, for MTX as well as for other chemotherapeutic drugs. © 2016 The Royal Society of Chemistry.

In clinical practice, one objective is to obtain diagnostic information while minimizing the invasiveness of the tests and the pain for the patients. To this end, tests based on the interaction of light with readily available biofluids including blood, urine, or saliva are highly desirable. In this review we examine the state of the art regarding the use of surface-enhanced Raman spectroscopy (SERS) to investigate biofluids, focusing on diagnostic applications. First, a critical evaluation of the experimental aspects involved in the collection of SERS spectra is presented; different substrate types are introduced, with a clear distinction between colloidal and non-colloidal metal nanostructures. Then the effect of the excitation wavelength is discussed, along with anomalous bands and artifacts which might affect SERS spectra of biofluids. The central part of the review examines the literature available on the SERS spectra of blood, plasma, serum, urine, saliva, tears, and semen. Finally, diagnostic applications are critically discussed in the context of the published evidence; this section clearly reveals that SERS of biofluids is most promising as a rapid, cheap, and non-invasive tool for mass screening for cancer.

Surface-enhanced Raman scattering (SERS) spectra were obtained from urine samples from subjects diagnosed with prostate cancer as well as from healthy controls, using Au nanoparticles as substrates. Principal component analysis (PCA) of the spectral data, followed by linear discriminant analysis (LDA), leads to a classification model with a sensitivity of 100 %, a specificity of 89 %, and an overall diagnostic accuracy of 95 %. Even considering the very limited number of samples involved in this report, preliminary results from this approach are extremely promising, encouraging further investigation.

Therapeutic drug monitoring (TDM) for anticancer drug imatinib has been suggested as the best way to improve the treatment response and minimize the risk of adverse reactions in chronic myelogenous leukemia (CML) and gastrointestinal stromal tumor (GIST) patients. TDM of oncology treatments with standard analytical methods, such as liquid chromatography-tandem mass spectrometry (LC-MS/ MS) is, however, complex and demanding. This paper proposes a new method for quantitation of imatinib in human plasma, based on surface enhanced raman spectroscopy (SERS) and multivariate calibration using partial least-squares regression (PLSR). The best PLSR model was obtained with three latent variables in the range from 123 to 5000 ng/mL of imatinib, providing a standard error of prediction (SEP) of 510 ng/mL. The method was validated in accordance with international guidelines, through the estimate of figures of merit, such as precision, accuracy, systematic error, analytical sensitivity, limits of detection, and quantitation. Moreover, the feasibility and clinical utility of this approach have also been verified using real plasma samples taken from deidentified patients. The results were in good agreement with a clinically validated LC-MS/MS method. The new SERS method presented in this preliminary work showed simplicity, short analysis time, good sensitivity, and could be considered a promising platform for TDM of imatinib treatment in a point-of-care setting.

Surface-enhanced Raman spectroscopy (SERS) is a promising and emerging technique to analyze the cellular environment. We developed an alternative, rapid and label-free SERS-based method to get information about the cellular environment by analyzing cells lysates, thus avoiding the need to incorporate nanoparticles into cells. Upon sonicating and filtrating cells, we obtained lysates which, mixed with Au or Ag nanoparticles, yield stable and repeatable SERS spectra, whose overall profile depends on the metal used as substrate, but not on the buffer used for the lysis process. Bands appearing in these spectra were shown to arise mostly from the cytosol and were assigned to adenine, guanine, adenosine and reduced glutathione (GSH). Spectral differences among various cell types also demonstrated that this approach is suitable for cell type identification.

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.

Zirconia-based composites were developed through an innovative processing route able to tune compositional and microstructural features very precisely. Fully-dense ceria-stabilized zirconia ceramics (84 vol% Ce-TZP) containing equiaxed alumina (8 vol%Al 2 O 3 ) and elongated strontium hexa-aluminate (8 vol% SrAl 12 O 19 ) second phases were obtained by conventional sintering. This work deals with the effect of the zirconia stabilization degree (CeO 2 in the range 10.0–11.5 mol%) on the transformability and mechanical properties of Ce-TZP-Al 2 O 3 -SrAl 12 O 19 materials. Vickers hardness, biaxial flexural strength and Single-edge V-notched beam tests revealed a strong influence of ceria content on the mechanical properties. Composites with 11.0 mol% CeO 2 or above exhibited the classical behaviour of brittle ceramics, with no apparent plasticity and very low strain to failure. On the contrary, composites with 10.5 mol% CeO 2 or less showed large transformation-induced plasticity and almost no dispersion in strength data. Materials with 10.5 mol% of ceria showed the highest values in terms of biaxial bending strength (up to 1.1 GPa) and fracture toughness (>10 MPa√m). In these ceramics, as zirconia transformation precedes failure, the Weibull modulus was exceptionally high and reached a value of 60, which is in the range typically reported for metals. The results achieved demonstrate the high potential of using these new strong, tough and stable zirconia-based composites in structural biomedical applications. Statement of Significance Yttria-stabilized (Y-TZP) zirconia ceramics are increasingly used for developing metal-free restorations and dental implants. Despite their success related to their excellent mechanical resistance, Y-TZP can undergo Low Temperature Degradation which could be responsible for restoration damage or even worst the failure of the implant. Current research is focusing on strategies to improve the LTD resistance of Y-TZP or to develop alternative composites with better stability in vivo . In this work the mechanical characterization of a new type of very-stable zirconia-based composites is presented. These materials are composed of ceria-stabilized zirconia (84 vol%Ce-TZP) containing two second phases (α-alumina and strontium hexa-aluminate) and exhibit exceptional strength, toughness and ductility, which may allow the processing of dental implants with a perfect reliability and longer lifetime.

In this work, we present a systematic study on solid Surface Enhanced Raman Scattering (SERS) substrates consisting of Au and Ag nanoparticles (NPs) loaded on filter paper with the dip-coating method. The aim of this work is to explore how a series of parameters (e.g., concentration of colloidal solution, different porosity of filter paper, and the presence of an aggregating agent) affects the analytical performance of paper-based SERS substrates. All the substrates developed in this study have been analyzed with two non-resonant probe molecules, 4-mercaptobenzoic acid (4-MBA) and adenine, in terms of (i) inter-sample repeatability, (ii) intra-sample repeatability, (iii) sensitivity, and (iv) overall SERS performance in terms of analyte quantification. Moreover, the issue of how to evaluate the repeatability for a solid SERS substrate is carefully discussed.

An interesting nanodrug delivery system is polyelectrolyte multilayer-coated nanogold. For better understanding of the binding of polycations or the counter-indicative deposition of polyanions on the citrate-stabilized gold nanoparticles, we used a surface-enhanced Raman spectroscopy to characterize the orientation of the polyions towards the gold surface. It was found that poly-allylamine replaces citrate molecules while the polyanion, poly-styrene sulfonate, intercalates in the citrate shell. One of the major obstacles for polyelectrolyte-coated nanogold is its tendency to agglomerate in the presence of high ion concentration as present, e.g., in blood. A novel encapsulation protocol for polyelectrolyte multilayer coating of gold nanoparticles was developed to successfully overcome this drawback. Moreover, electrostatic functionalization of the polyelectrolyte shell with a model target molecule for cancer, folic acid, induced a significant increase in the particle uptake in folate-receptor over-expressing breast cancer cell lines, VP 229 and MDA MB 231, compared to non-targeted particles or cells (non-activated macrophages) not expressing the folate receptor.

Bronchopulmonary sequestration (BPS) involves abnormal pulmonary tissue lacking trancheobronchial connection and having a separate vascular supply that arises from the thoracic or abdominal aorta. For the first time, BPS is studied by Raman and Fourier-transform infrared (FTIR) imaging. Cryosections were mounted on calcium fluoride substrates from tissue samples of two BPS patients. Raman images were collected at a step size of 66–100 µm to assess the whole tissue section, and at a lower step size of 10 µm to resolve details in selected areas. FTIR images were collected at resolutions of 63 and 4 µm per pixel. Data sets were segmented by cluster analyses, and the spectra of each cluster were compared. The spectra revealed higher red blood cell content and lower collagen and lipid content in BPS than in an additional tissue section from the BPS margin. Comparison with previous Raman and FTIR images of congenital cystic adenomatoid malformations (CCAM) and normal lung tissue suggested that the marginal tissue sample of the BPS patient contains CCAM. We conclude that CCAM and BPS are biochemically distinct, which enable the differential diagnosis of lung malformations by specific vibrational spectroscopic fingerprints. Copyright © 2008 John Wiley & Sons, Ltd.

Surface-Enhanced Raman Spectroscopy (SERS) is a label-free technique that enables quick monitoring of substances at low concentrations in biological matrices. These advantages make it an attractive tool for the development of point-of-care tests suitable for Therapeutic Drug Monitoring (TDM) of drugs with a narrow therapeutic window, such as chemotherapeutic drugs, immunosuppressants, and various anticonvulsants. In this article, the current applications of SERS in the field of TDM for cancer therapy are discussed in detail and illustrated according to the different strategies and substrates. In particular, future perspectives are provided and special concerns regarding the standardization of self-assembly methods and nanofabrication procedures, quality assurance, and technology readiness are critically evaluated.

Richard Dluhy opened a general discussion of the paper by Duncan Graham: In your example of a heterogeneous solution-based assay for multicomponent analysis, what is the concentration of the target fungal ssPCR DNA that is used, and how do you manage the kinetics of the reaction such that the target reaches the probe in a time frame appropriate for a clinical assay?

A frequent quantification of drugs concentrations in plasma of patients subject to chemotherapy is seldom performed, mostly because the standard methods (Gas or Liquid Chromatography coupled with Mass Spectroscopy) are expensive and time consuming. In this paper we report the approach pursued in one of the research units of the EU project RAMAN4CLINICS to tackle the problem of a low cost, time adherent quantification of drugs used for oncological patients using a Surface Enhanced Raman Scattering (SERS) spectroscopy. More specifically, the issues concerning the repeatability of the nanostructured substrates will be presented and some promising results to increase the selectivity of the measures toward specific drugs will be discussed, with examples concerning one cytotoxic agent, Irinotecan and one kinase inhibitor, Sunitinib.

Silver-coated porous silicon and polydimethylsiloxane (PDMS) are systematically analyzed as substrates for surface-enhanced Raman scattering (SERS). They were selected as representative metal-dielectric nanostructures characterized by different morphology and substrate dielectric constant which is reflected in the electromagnetic near-field intensity spectra. The study is conducted using 4-mercaptobenzoic acid as probe molecule with the aim to compare the scattering efficiency and the homogeneity of the Raman signal on the selected substrates. A larger SERS enhancement is evidenced for the silicon-based plasmonic nanostructures (>106, despite the electronic off-resonant excitation of the analyte). On the other hand, the silvered elastomeric substrates, characterized by a good Raman efficiency, show good repeatability featured by a low inter- and intrasubstrate standard deviation of the SERS signal intensity, which make them suitable for quantitative analysis. The influence of the excitation wavelength on such properties is deeply discussed taking into account experimental results and 3D modeling.

The emission spectrum of α-alumina in the spectral range between 13900 and 14250 cm−1 is characterized by several bands which are much weaker than the two intense and sharp R1 and R2 bands appearing at 14400 and 14430 cm−1, respectively; these latter are known as Ruby lines and they are the emission bands used in the Ruby laser. Furthermore these bands shift in frequency with stress (Piezo-Spectroscopic effect). In this paper, for the first time, the stress-dependent peak frequency of the weaker bands in the 13900–14250 cm−1 range is calibrated, and the results are presented as Piezo-Spectroscopic coefficients. The calibration is performed by reporting the frequency shift of each investigated band as function of varying stresses. The stresses, residual in nature, are obtained by fabricating composite materials where α-alumina is mixed in various amounts with (i) Ceria-Stabilized Tetragonal Zirconia Polycrystals (Ce-TZP) and (ii) silicon carbide, SiC. The composite materials are prepared at high temperature (1500°–1800°C); due to the difference in thermal expansion, upon cooling to room temperature α-alumina develops compressive and tensile stresses, when mixed with Ce-TZP and SiC, respectively. The stress values necessary for the calibration are obtained from the frequency shift of the R2 band, using its well-established Piezo-Spectroscopic coefficient (7.6 cm−1/GPa). Then the newly obtained Piezo-Spectroscopic coefficients of the bands in the 13900–14250 cm−1 range are tested to retrieve the stresses in two sets of composites; finally the stress values are compared with those obtained in the same samples from the frequency shift of the R2 band. The comparison shows a very good agreement, thus providing evidence that the bands in the 13900–14250 cm−1 range can be used to monitor stresses in α-alumina-based materials.

Using the technique of fluorescence piezospectroscopy, we determine the distribution of thermal residual stresses across the edges of three laminated alumina/zirconia composites. We develop a methodology for separating the measured stress state into microstresses that result from grain-to-grain thermal mismatch and macrostresses that result from lamination-induced thermal mismatch between individual plies. Comparison between the measured edge-stress distributions and those calculated based on a simple force-superposition model shows good agreement, indicating that the laminate system is well approximated as linear elastic. Given the experimental confirmation of significant edge stresses in multi-ply laminates, the possibility of failure initiating at composite edges must be considered in the design of surface-compressed laminate structures with the aim of mediating the detrimental effect of surface flaws.

Samples of strontium hexaluminate (SrAl12O19), both pure and mixed with different amounts of ceria-stabilized zirconia (CE-TZP), have been prepared via a chemical route. The fluorescence spectrum of SrAl12O19 is similar to that of ruby and yttrium aluminium garnet (YAG); consequently, this similarity has been attributed to the presence of a trace impurity of Cr3+ ions. The most-intense fluorescence peak of SrAl12O19 exhibits a dependence on stress that is given by the average uniaxial piezospectroscopic coefficient ( = 1.5 cm-1·GPa-1). The piezospectroscopic coefficient has been used to convert to stress the fluorescence frequency shift of SrAl12O19 mixed with zirconia (ZrO2). The stress, as a function of the volume fraction of ZrO2, resides between the Hashin-Shtrikman bounds but is best reproduced by an effective medium approximation.

The stresses of laminate structures obtained by joining single layers of pure alumina (A), pure yttria-stabilized tetragonal zirconia, 3Y-TZP (Z), and an intimate mixture of alumina and zirconia (AZ) have been determined by fluorescence (in alumina) and Raman (in zirconia) piezospectroscopy. Three symmetrical stacking sequences were examined, namely, A/Z/A, A/AZ/A, and AZ/Z/AZ, with the aim of designing structures where the higher coefficient of thermal expansion (CTE) of zirconia could be used to induce compressive stress in the external layers (and ensuing tensile stress in the central layer). Two experimental sessions, 6 years apart, were conducted on the same samples, also taking care to record the spectra from the same locations; during the time elapsed between the two sessions, the samples were kept at room temperature and humidity. The stress values in alumina obtained during the more recent session were markedly different from those observed in the first session. Monoclinic zirconia (m-zirconia) was absent in all samples in the first session, whereas up to 25 vol% zirconia could be observed during the second session. m-Zirconia could only be observed in AZ layers and not in Z layers, irrespective of the position in the stacking sequence. It was concluded that 3Y-TZP underwent spontaneous tetragonal-to-monoclinic (t–m) transformation, that is, “aging,” when mixed with alumina at the grain-size level. Aging occurred only where pristine t-zirconia was subject to tensile stresses larger than ∼400 MPa.

Single phase homogeneous CeO 2 –ZrO 2 solid solutions with various compositions were synthesized using a citrate complexation route. Investigation of the sintering behaviour disclosed important modifications of the textural and, in particular, structural properties, which apparently create a strongly defective structure that could explain the unusual redox properties of these catalytic systems.

Photo-stimulated luminescence spectroscopy (PSLS) can be used as a non-destructive tool to gain quantitative information about the stress and strain in the thermally grown oxide (TGO) formed beneath a thermal barrier coating (TBC) and possibly assess damage. In response to the increasing popularity of this technique, a ‘round-robin’ test has been initiated to compare methods of luminescence collection using different instruments as well as various spectral analysis methods. The interim results of this ‘round-robin’ are reported here with a series of PSLS measurements and analyses performed at four different laboratories on the same set of electron beam physical vapor deposited (EB-PVD) TBC samples, having undergone various oxidation treatments. In addition, a set of standard electronic spectra (collected from one group) were circulated for analysis in order to compare the curve-fitting methods used by each group. Apart from sample variations, there was very good agreement between the results obtained in the different laboratories, and there were no systematic differences in the data, indicating that present collection and analysis methods are comparable.

Raman spectroscopy has been used to obtain Raman spectra of yttria-stabilized tetragonal zirconia subject to surface nitridation induced by contact with zirconium nitride. Raman spectra recorded from regions at increasing distance from the source of nitridation have been used to obtain diffusion profiles from samples treated at different times and temperatures. The coupling of X-ray diffraction data previously taken and of the Raman spectra shows that in the samples there is a two-phase region (tetragonal + cubic) near the nitrided surface and that, at larger distance inside the samples, there is only one phase (tetragonal). Fitting of the diffusive profiles in the single-phase tetragonal region with an appropriate diffusion function allows the determination of the diffusion coefficient of nitrogen in tetragonal zirconia which is expressed in terms of the preexponential factor, D0= (3.98 ± 0.5) × 10−3 cm2/s, and the activation energy, Q= 170 ± 10 kJ/mol.

Hierarchical cluster analysis (HCA) is extensively used for the analysis of hyperspectral data. In this work, hyperspectral data sets obtained from Raman maps were analyzed using an alternative mode of cluster analysis, clustering “images” instead of spectra, under the assumption that images showing similar spatial distributions are related to the same chemical species. Such an approach was tested with two Raman maps: one simple “test map” of micro-crystals of four different compounds for a proof of principle and a map of a biological tissue (i.e., cartilage) as an example of chemically complex sample. In both cases, the “image-clustering” approach gave similar results as the traditional HCA, but at lower computational effort. The alternative approach proved to be particularly helpful in cases, as for the cartilage tissue, where concentration gradients of chemical composition are present. Moreover, with this approach, yielded information about correlation between bands in the average spectrum makes band assignment and spectral interpretation easier.

In this contribution, we investigated whether surface-enhanced Raman scattering (SERS) of serum can be a candidate method for detecting "luminal A" breast cancer (BC) at different stages. We selected three groups of participants aged over 50 years: 20 healthy women, 20 women with early localized small BC, and 20 women affected by BC with lymph node involvement. SERS revealed clear spectral differences between these three groups. A predictive model using principal component analysis (PCA) and linear discriminant analysis (LDA) was developed based on spectral data, and its performance was estimated with cross-validation. PCA-LDA of SERS spectra could distinguish healthy from BC subjects (sensitivity, 92 %; specificity, 85 %), as well as subjects with BC at different stages, with a promising diagnostic performance (sensitivity and specificity, ≥80 %; overall accuracy, 84 %). Our data suggest that SERS spectroscopy of serum, combined with multivariate data analysis, represents a minimally invasive, easy to use, and fast approach to discriminate healthy from BC subjects and even to distinguish BC at different clinical stages.

Le spectre Raman de nanofibres de fullerene a ete acquis avec differentes densites de puissance laser incidentes afin d'evaluer son influence sur la position du pic du mode Ag(2) de la molecule de fullerene. Une procedure pour mesurer le diametre du faisceau laser et la densite de puissance sur l'echantillon dans un microscope Raman est propose. Les incertitudes du processus de mesure et d'etalonnage du spectrometre sont egalement presentes.

The development of nanotechnological devices and their clinical application in medicine has become increasingly important, especially in the context of targeted and personalized therapy. This is particularly important in cancer therapy, where antitumor drugs are highly cytotoxic and often exert their therapeutic effect at concentrations close to systemic toxicity. In the last years a growing number of studies has started to report the use of plasmonic nanoprobes in the field of theranostics, broadening the application of vibrational spectroscopies like Raman scattering and surface enhanced Raman scattering (SERS) in biomedicine. The present work aims to identify and characterize the vibrational profiles of a widely used anticancer drug, irinotecan (CPT-11). With a rational approach, SERS experiments have been performed on this analyte employing both Au and Ag colloids, starting from simple aqueous solutions up to albumin mixtures. A major step forward for drug detection in albumin solutions has been taken with the adoption of a simple deproteinization strategy, and a two-in-one-step separation and identification by coupling thin layer chromatography, TLC, with SERS (TLC-SERS). The latter has revealed to be a valid system for protein separation and simultaneous analyte detection, showing a potential to become an innovative, sensitive and low cost method for antineoplastic drug profiling in patients' body fluids.

Stabilized zirconia is a ceramic material known for its superior mechanical properties and biocompatibility, and has been widely employed as a biomaterial (Chevalier, Biomaterials 27(4):535–543, 2006) – most recently including dental and spinal implants (Lughi and Sergo, Dent Mater Off Publ Acad Dent Mater 26(8):807–820, 2010). However, the properties of zirconia ceramic may be reduced when it contacts humid environments. Low-Temperature Degradation (LTD) – associated to a phase transformation from tetragonal to monoclinic, and thus to a loss of mechanical properties (Lughi and Sergo, Dent Mater Off Publ Acad Dent Mater 26(8):807–820, 2010; Chevalier et al. Annu Rev Mater Res 37(1):1–32, 2007) – makes detection of low levels of monoclinic a critical aspect in developing and engineering zirconia-based biomaterials. In this work, detection of extremely little amounts of monoclinic phase is demonstrated on implant prototypes by combining Raman spectroscopy and multivariate analysis. Implant prototypes made of Yttria-Stabilized Tetragonal Zirconia were subjected to pin-on-disk tests, leaving distinct wear marks on the samples surface. Arrays of Raman spectra were collected in areas across the pristine and the worn surface, using a 785 nm laser with a 50× magnification objective, 0.75 NA. The spectral array was treated by means of standard univariate analysis and by multivariate analysis based on Principal Component Analysis (PCA). The results of the multivariate analysis are represented as maps. Figure 59.1 shows the scores corresponding to the loadings of the relevant component, i.e. the one that most emphasize the presence of monoclinic phase. Note that individual Raman spectrum would suggest that the sample is purely tetragonal. However, PCA results will reveal presence of traces of monoclinic phase of less than 1 vol% (Fig. 59.2). Detection of very low amounts of monoclinic phase, indicating incipient t-m transformation, was made possible by using PCA on Raman spectral arrays. This is a promising approach for early detection of LTD, a key aspect in the development of zirconia-based biomaterials.

The effect of systematic modifications in the chemistry of the phase-boundary film on the macroscopic mechanical properties of Si3N4-matrix composites was investigated. Model composite materials, containing SiC or WC platelets, were prepared and only the bulk anion composition of the glassy-SiO2 intergranular phase was varied by adding increasing amounts of fluorine to the material. Detailed material characterizations by high-resolution electron microscopy (HREM) and Raman spectroscopy on both undoped and F-doped composites allowed to derive a structural model of the phase-boundary film as well as to evaluate the average microscopic stresses acting on it. In addition, high-temperature internal friction measurements provided an estimate of the grain-boundary relaxation temperature as a function of the F content. Noticeable variations of both elastic modulus and fracture energy of the composite were detected upon F addition, which were related to a spontaneous process of phase-boundary microcracking upon cooling. A threshold of the F-content was found for microcrack formation and its existence is theoretically explained according to a percolation process of non-bridged SiO4-tetrahedra, which arises from the incorporation of F into the intergranular film network.