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.

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.

Characterization of white wines from north-eastern Italy with surface-enhanced Raman spectroscopy.

In the present study, label-free SERS spectroscopy is applied as a useful analytical technique for white wine characterization. 180 samples of three white wines varieties from northeastern Italy, Sauvignon Blanc, Ribolla Gialla and Friulano, collected from three different Italian producers from 2016 vintage, have been analyzed using Ag citrate-reduced colloids and a portable Raman instrument with a 785 nm laser. A PCA of SERS spectra showed that discrimination between wines and wineries is possible. Main spectral differences are due to adenine, carboxylic acids and glutathione, with their ratio changing among different wine types and producers. A robust version of the Soft Independent Modelling of Class Analogy (SIMCA) method was used to model the class space of each wine and to perform the classification among the different categories, yielding overall efficiencies between 87 and 93%. These results are extremely encouraging and open the way to the application of this SERS protocol as a wine identification assay.

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.