Direct comparison of different protocols to obtain surface enhanced Raman spectra of human serum.

Label-free Surface Enhanced Raman Spectroscopy (SERS) is a rapid technique that has been extensively applied in clinical diagnosis and biomedicine for the analysis of biofluids. The purpose of this approach relies on the ability to detect specific "metabolic fingerprints" of complex biological samples, but the full potential of this technique in diagnostics is yet to be exploited, mainly because of the lack of common analytical protocols for sample preparation and analysis. Variation of experimental parameters, such as substrate type, laser wavelength and sample processing can greatly influence spectral patterns, making results from different research groups difficult to compare. This study aims at making a step toward a standardization of the protocols in the analysis of human serum samples with Ag nanoparticles, by directly comparing the SERS spectra obtained from five different methods in which parameters like laser power, nanoparticle concentration, incubation/deproteinization steps and type of substrate used vary. Two protocols are the most used in the literature, and the other three are "in-house" protocols proposed by our group; all of them are employed to analyze the same human serum sample. The experimental results show that all protocols yield spectra that share the same overall spectral pattern, conveying the same biochemical information, but they significantly differ in terms of overall spectral intensity, repeatability, and preparation steps of the sample. A Principal Component Analysis (PCA) was performed revealing that protocol 3 and protocol 1 have the least variability in the dataset, while protocol 2 and 4 are the least repeatable.

Spectroscopic insights into peri-implant mucositis and peri-implantitis: unveiling peri-implant crevicular fluid profiles using surface enhanced Raman scattering.

The precise identification and differentiation of peri-implant diseases, without the need for intrusive procedures, is crucial for the successful clinical treatment and overall durability of dental implants. This work introduces a novel approach that combines surface-enhanced Raman scattering (SERS) spectroscopy with advanced chemometrics to analyse peri-implant crevicular fluid (PICF) samples. The primary purpose is to offer an unbiased evaluation of implant health. A detailed investigation was performed on PICF samples obtained from a cohort of patients exhibiting different levels of peri-implant health, including those with healthy implants, implants impacted by peri-implantitis, and implants with peri-implant mucositis. The obtained SERS spectra were analysed using canonical-powered partial least squares (CPPLS) to identify unique chemical characteristics associated with each inflammatory state. Significantly, our research findings unveil the presence of a common inflammatory SERS spectral pattern in cases of peri-implantitis and peri-implant mucositis. Furthermore, the SERS-based scores obtained from CPPLS were combined with established clinical scores and subjected to a linear discriminant analysis (LDA) classifier. Repeated double cross-validation was used to validate the method's capacity to discriminate different implant conditions. The integrated approach showcased high sensitivity and specificity and an overall balanced accuracy of 92%, demonstrating its potential to serve as a non-invasive diagnostic tool for real-time implant monitoring and early detection of inflammatory conditions.

Spectroscopic investigation of faeces with surface-enhanced Raman scattering: a case study with coeliac patients on gluten-free diet.

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.

The key role of ergothioneine in label-free surface-enhanced Raman scattering spectra of biofluids: a retrospective re-assessment of the literature.

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.

Label-free Surface Enhanced Raman Scattering (SERS) on Centrifugal Silver Plasmonic Paper (CSPP): A Novel Methodology for Unprocessed Biofluids Sampling and Analysis.

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).

Label-free analysis of gingival crevicular fluid (GCF) by surface enhanced Raman scattering (SERS).

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.

Repeated double cross-validation applied to the PCA-LDA classification of SERS spectra: a case study with serum samples from hepatocellular carcinoma patients.

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.

Ergothioneine, a dietary amino acid with a high relevance for the interpretation of label-free surface enhanced Raman scattering (SERS) spectra of many biological samples.

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.

Danon Disease-Associated LAMP-2 Deficiency Drives Metabolic Signature Indicative of Mitochondrial Aging and Fibrosis in Cardiac Tissue and hiPSC-Derived Cardiomyocytes.

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' 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.

Reliability of an injection-moulded two-piece zirconia implant with PEKK abutment after long-term thermo-mechanical loading.

Zirconia implants are appreciated in some clinical indications in light of their aesthetic appearance and good biocompatibility. The aim of this work was to evaluate the performance of a newly developed two-piece zirconia/polyether ketone ketone (PEKK) implant-abutment combination after long-term cyclic loading in a hydrothermal environment, using a new protocol adapted from two available ISO standards. Sixteen implants (n = 8/group) were embedded according to ISO 14801 and divided into two groups: implants in the Observational Group (OG) were cyclically loaded for 60 days (98 N, 10 million loading cycles, 2 Hz) in 85 °C water in a chewing simulator, while non-loaded/non-aged implants (as-received) constituted the Control Group (CG). After 4.7 million loading cycles, one OG implant fractured in the chewing simulator. The surviving implants were compared to CG implants by X-ray diffraction (XRD) to investigate potential ageing as suggested by ISO 13356, but also µ-Raman spectroscopy, Focused-Ion-Beam - Scanning-Electron-Microscopy (FIB-SEM), and load-to-fracture. Ageing was shown to have limited influence on the evaluated zirconia implant, with increased monoclinic content after loading/ageing being to a shallow transformed zone of ~2 µm at the implant surface. However, OG implants showed a significantly decreased fracture load of 751 ± 231 N (CG: 995 ± 161 N; p = .046). These values enable clinical application, but the fact that one failure was recorded during cyclic fatigue along with the significant decrease in strength after cyclic loading/ageing suggest that there may be room for further optimization of especially the PEKK abutment. Furthermore, good agreement was observed between the fracture modes of the implant that failed during the cyclic fatigue experiment and the in vivo failure of one implant during pre-clinical trials, validating the interest of the in vitro protocol used in this work to check the reliability of zirconia implant.

Differentiation between stages of non-alcoholic fatty liver diseases using surface-enhanced Raman spectroscopy.

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 <0.001) between the two groups. The average figures of merit for a diagnostic test based on these ratios, as derived from a repeated 4-fold cross-validation of a logistic regression model, are all between 0.73 and 0.79, with an average area under the curve of 0.81. We conclude that SERS may be a reliable and rapid method to discriminate NAFLD from NASH.

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.

Fine Determination of Monoclinic Phase in Zirconia-Based Implants: A Surface-Enhanced Raman Spectroscopy (SERS) Study.

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 confocal 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.

Label-Free Quantification of Anticancer Drug Imatinib in Human Plasma with Surface Enhanced Raman Spectroscopy.

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.

Potential use of MCR-ALS for the identification of coeliac-related biochemical changes in hyperspectral Raman maps from pediatric intestinal biopsies.

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.

Binding of p-mercaptobenzoic acid and adenine to gold-coated electroless etched silicon nanowires studied by surface-enhanced Raman scattering.

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 10-5M and adenine in 30 and 100µM 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 1106cm-1 band is explained by involvement of the CS stretching deformation, and the appearance of the broad 300cm-1 band attributed to SAu stretching mode. Adenine SERS spectra demonstrate the existence of the 7H tautomer since the strongest band observed is at 736cm-1. The adenine binding is likely to occur in several ways, because the number of observed bands in the 1200-1600cm-1 interval exceeds the number of observed bands in the normal Raman spectrum of the free molecule.

Optimization and Characterization of Paper-Made Surface Enhanced Raman Scattering (SERS) Substrates with Au and Ag NPs for Quantitative Analysis.

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.

Long-term stability of an injection-molded zirconia bone-level implant: A testing protocol considering aging kinetics and dynamic fatigue.

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 1-2µm (SEM) or 6-8µm (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.

Towards long lasting zirconia-based composites for dental implants: Transformation induced plasticity and its consequence on ceramic reliability.

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 (84vol% Ce-TZP) containing equiaxed alumina (8vol%Al2O3) and elongated strontium hexa-aluminate (8vol% SrAl12O19) second phases were obtained by conventional sintering. This work deals with the effect of the zirconia stabilization degree (CeO2 in the range 10.0-11.5mol%) on the transformability and mechanical properties of Ce-TZP-Al2O3-SrAl12O19 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 11.0mol% CeO2 or above exhibited the classical behaviour of brittle ceramics, with no apparent plasticity and very low strain to failure. On the contrary, composites with 10.5mol% CeO2 or less showed large transformation-induced plasticity and almost no dispersion in strength data. Materials with 10.5mol% of ceria showed the highest values in terms of biaxial bending strength (up to 1.1GPa) and fracture toughness (>10MPa√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 (84vol%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.

Potential of Surface Enhanced Raman Spectroscopy (SERS) in Therapeutic Drug Monitoring (TDM). A Critical Review.

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.