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.

FTIR Spectroscopy to Reveal Lipid and Protein Changes Induced on Sperm by Capacitation: Bases for an Improvement of Sample Selection in ART.

Although being a crucial step for Assisted Reproduction Technologies (ART) success, to date sperm selection is based only on morphology, motility and concentration characteristics. Considering the many possible alterations, there is a great need for analytical approaches allowing more effective sperm selections. The use of Fourier Transform Infrared (FTIR) may represent an interesting possibility, being able to reveal many macromolecular changes in a single measurement in a nondestructive way. As a proof of concept, in this observational study, we used a FTIR approach to reveal features related to sperm quality and chemical changes promoted by in vitro capacitation. We found indication that α-helix content is increased in capacitated sperm, while high percentages of the ß-structures seem to correlate to poor-quality spermatozoa. The most interesting observation was related to the lipid composition, when measured as CH2/CH3 vibrations (ratio 2853/2870), which resulted in being strongly influenced by capacitation and well correlated with sperm motility. Interestingly, this ratio is higher than 1 in infertile samples, suggesting that motility is related to sperm membranes stiffness and lipid composition. Although further analyses are requested, our results support the concept that FTIR can be proposed as a new smart diagnostic tool for semen quality assessment in ART.

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.

FTIR investigation of the secondary structure of type I collagen: New insight into the amide III band.

This work presents a thorough study on the Amide III band in fibrous proteins using Fourier Transformed Infrared Spectroscopy (FTIR). Type I collagen was chosen as a model for this family of proteins, not only because of its important role in mammalian tissues, but also for its involvement in several pathologies. In order to disclose the conformational information contained in the collagen bands, the spectral characteristics of Amide III of type I collagen were related to the ones of Amide I band, performing experiments of thermal denaturation of the protein in acidic solution. Data acquired allowed to observe the protein unfolding and retrieve information about its structural arrangements during the thermal cycle. Taking as guideline the well-known behaviour of the Amide I band, we correlated the structural changes deducible from Amide I analysis with the ones detectable for Amide III band, by exploiting three spectral analysis techniques, namely 2D-correlation analysis, second derivative analysis, and peak-fitting. This approach enabled us to jointly support the obtained results and finally to assign the components of the Amide III of a typical fibrous protein, such as type I collagen, to its characteristic secondary structure.

In vitro FTIR microspectroscopy analysis of primary oral squamous carcinoma cells treated with cisplatin and 5-fluorouracil: a new spectroscopic approach for studying the drug-cell interaction.

In the present study, human primary oral squamous carcinoma cells treated with cisplatin and 5-fluorouracil were analyzed, for the first time, by in vitro FTIR Microspectroscopy (FTIRM), to improve the knowledge on the biochemical pathways activated by these two chemotherapy drugs. To date, most of the studies regarding FTIRM cellular analysis have been executed on fixed cells from immortalized cell lines. FTIRM analysis performed on primary tumor cells under controlled hydrated conditions provides more reliable information on the biochemical processes occurring in in vivo tumor cells. This spectroscopic analysis allows to get on the same sample and at the same time an overview of the composition and structure of the most remarkable cellular components. In vitro FTIRM analysis of primary oral squamous carcinoma cells evidenced a time-dependent drug-specific cellular response, also including apoptosis triggering. Furthermore, the univariate and multivariate analyses of IR data evidenced meaningful spectroscopic differences ascribable to alterations affecting cellular proteins, lipids and nucleic acids. These findings suggest for the two drugs different pathways and extents of cellular damage, not provided by conventional cell-based assays (MTT assay and image-based cytometry).

A new light on Alkaptonuria: A Fourier-transform infrared microscopy (FTIRM) and low energy X-ray fluorescence (LEXRF) microscopy correlative study on a rare disease.

BACKGROUND: Alkaptonuria (AKU) is an ultra-rare disease associated to the lack of an enzyme involved in tyrosine catabolism. This deficiency results in the accumulation of homogentisic acid (HGA) in the form of ochronotic pigment in joint cartilage, leading to a severe arthropathy. Secondary amyloidosis has been also unequivocally assessed as a comorbidity of AKU arthropathy. Composition of ochronotic pigment and how it is structurally related to amyloid is still unknown. METHODS: We exploited Synchrotron Radiation Infrared and X-Ray Fluorescence microscopies in combination with conventional bio-assays and analytical tools to characterize chemical composition and morphology of AKU cartilage. RESULTS: We evinced that AKU cartilage is characterized by proteoglycans depletion, increased Sodium levels, accumulation of lipids in the peri-lacunar regions and amyloid formation. We also highlighted an increase of aromatic compounds and oxygen-containing species, depletion in overall Magnesium content (although localized in the peri-lacunar region) and the presence of calcium carbonate fragments in proximity of cartilage lacunae. CONCLUSIONS: We highlighted common features between AKU and arthropathy, but also specific signatures of the disease, like presence of amyloids and peculiar calcifications. Our analyses provide a unified picture of AKU cartilage, shedding a new light on the disease and opening new perspectives. GENERAL SIGNIFICANCE: Ochronotic pigment is a hallmark of AKU and responsible of tissue degeneration. Conventional bio-assays have not yet clarified its composition and its structural relationship with amyloids. The present work proposes new strategies for filling the aforementioned gap that encompass the integration of new analytical approaches with standardized analyses.

Pitfalls and promises in FTIR spectromicroscopy analyses to monitor iron-mediated DNA damage in sperm.

Many drugs, chemicals, and environmental factors can impair sperm functionality by inducing DNA damage, one of the important causes of reduced fertility potential. The use of vibrational spectromicroscopy represents a promising approach for monitoring DNA integrity in sperm, although some limitations exist, depending from the experimental conditions. Here, we report that when using FTIR spectromicroscopy to reveal oxidative stress mediated by Fenton's reaction on hydrated sperm samples, DNA damage interpretation is partially compromised by unexpected cell surface precipitates. The precipitates give a broad band in the 1150-1000cm(-1) infrared region, which partially covers one of the signatures of DNA (phosphate stretching bands), and are detected as iron and oxygen containing material when using XRF spectroscopy. On the other hand, the analyses further support the potential of FTIR spectromicroscopy to reveal cellular oxidative damage events such as lipid peroxidation, protein misfolding and aggregations, as well as DNA strain breaks.

Contribution of Ribonucleic Acid (RNA) to the Fourier Transform Infrared (FTIR) Spectrum of Eukaryotic Cells.

We report on an optimized protocol for the digestion of cellular RNA, which minimally affects the cell membrane integrity, maintaining substantially unaltered the vibrational contributions of the other cellular macromolecules. The design of this protocol allowed us to collect the first Fourier transform infrared (FTIR) spectra of intact hydrated B16 mouse melanoma cells deprived of RNA and to highlight the in-cell diagnostic spectral features of it. Complementing the cellular results with the FTIR analysis of extracted RNA, ds-DNA, ss-cDNA and isolated nuclei, we verified that the spectral component centered at ∼1220 cm-1 is a good qualitative and semiquantitative marker of cellular DNA, since it is minimally affected by cellular RNA removal. Conversely, the band centered at ∼1240 cm-1, conventionally attributed to RNA, is only a qualitative marker of it, since its intensity is majorly influenced by other macromolecules containing diverse phosphate groups, such as phospholipids and phosphorylated proteins. On the other hand, we proved that the spectral contribution centered at ∼1120 cm-1 is the most reliable indicator of variations in cellular RNA levels, that better correlates with cellular metabolic activity. The achievement of these results have been made possible also by the implementation of new methods for baseline correction and automated peak fitting, presented in this paper.

Vibrational mapping of sinonasal lesions by Fourier transform infrared imaging spectroscopy.

Fourier transform infrared imaging (FTIRI) is a powerful tool for analyzing biochemical changes in tumoral tissues. The head and neck region is characterized by a great variety of lesions, with different degrees of malignancy, which are often difficult to diagnose. Schneiderian papillomas are sinonasal benign neoplasms arising from the Schneiderian mucosa; they can evolve into malignant tumoral lesions (squamous cell carcinoma). In addition, they can sometimes be confused with the more common inflammatory polyps. Therefore, an early and definitive diagnosis of this pathology is mandatory. Progressing in our research on the study of oral cavity lesions, 15 sections consisting of inflammatory sinonasal polyps, benign Schneiderian papillomas, and sinonasal undifferentiated carcinomas were analyzed using FTIRI. To allow a rigorous description of these pathologies and to gain objective diagnosis, the epithelial layer and the adjacent connective tissue of each section were separately investigated by following a multivariate analysis approach. According to the nature of the lesion, interesting modifications were detected in the average spectra of the different tissue components, above all in the lipid and protein patterns. Specific band-area ratios acting as spectral markers of the different pathologies were also highlighted.

Fourier transform infrared microspectroscopy reveals biochemical changes associated with glioma stem cell differentiation.

According to the cancer stem cell theory malignant glioma is incurable because of the presence of the cancer stem cells - a subpopulation of cells that are resistant to therapy and cause the recurrence of a tumor after surgical resection. Several protein markers of cancer stem cell were reported but none of those is fully reliable to grade the content of stem cells in a tumor. Hereby we propose Fourier transform infrared (FTIR) microspectroscopy as an alternative, labelfree, non-damaging and fast method to identify glioma stem cells based on their own spectral characteristics. The analysis of FTIR data revealed that in NCH421k cells, a model of glioma stem cells, the relative content of lipids is higher than in their all-trans retinoic acid-differentiated counterparts. Moreover, it has been assessed that stem cells have more rigid cellular membranes and more phosphorylated proteins, whereas after differentiation glycogen level increases. The ability of FTIR to estimate the content of stem cells in a heterogeneous sample, on the base of the identified spectral markers, and to classify stem and non-stem cells into two separate populations was probed. Although it was not possible to calculate the exact percentage of each subpopulation, we could clearly see that with the increasing amount of differentiated cells in a sample, more hits occupy the PC space previously identified as a space of differentiated cells. The present study is therefore an initial step towards the development of a FTIR based protocol in clinical practice to estimate the content of stem cells in a tumor sample.

Time-resolved FT-IR microspectroscopy of protein aggregation induced by heat-shock in live cells.

Maintaining the correct folding of cellular proteins is essential for preserving cellular homeostasis. Protein dishomeostasis, aberrant protein folding, and protein aggregation are indeed involved in several diseases including cancer, aging-associated, and neurodegenerative disorders. Accumulation of protein aggregates can also be induced from a variety of stressful conditions, such as temperature increase or oxidative stress. In this work, we monitored by Fourier transform-infrared (FT-IR) microspectroscopy the response of live breast cancer MCF-7 and mammary breast adenocarcinoma MDA-MB 231 cell lines to severe heat-shock (HS), caused by the rise of the cellular medium temperature from 37 ± 0.5 °C to 42 ± 0.5 °C. Through the study of the time-evolution of the second derivatives of the spectra and by the 2D correlation analysis of FT-IR absorbance data, we were able to identify a common sudden heat-shock response (HSR) among the two cell lines. The hyperfluidization of mammalian cell membranes, the transient increment of the signal lipids, as well as the alteration of proteome profile were all monitored within the first 40 min of stress application, while the persistent intracellular accumulation of extended ß-folded protein aggregates was detected after 40 min up to 2 h. As a whole, this paper offers a further prove of the diagnostic capabilities of FT-IR microspectroscopy for monitoring in real-time the biochemical rearrangements undergone by live cells upon external stimulation.

Apoptotic pathways of U937 leukemic monocytes investigated by infrared microspectroscopy and flow cytometry.

Apoptosis is a strictly regulated cell death mechanism that plays a pivotal role in the normal evolution of multicellular organisms. Its misregulation has been associated with many diseases, making its early and reliable detection a key point for modern cellular biology. In this paper, we propose the use of infrared microspectroscopy (IRMS) as a label-free methodology for the detection of apoptotic-related biochemical processes induced on U937 leukemic monocytes by serum starvation and CCCP-exposure. The spectroscopic results are in agreement with parallel Flow Cytometry (FC) experiments, where plasma membrane integrity and mitochondrial activity were assessed. Spectroscopic outcomes complement FC data and allow drawing a more complete picture of the apoptotic pathways. In particular, we established that the two apoptosis-inducing treatments, cell starvation and CCCP exposure, affect the cell cycle in a different way. With the former, cell death is preceded by a cell cycle arrest, whereas the latter causes an increased cell cycle progression. Spectral data demonstrate that for both conditions apoptosis proceeds through the accumulation of lipid droplets within cells. Moreover, we were able to establish a spectral marker for DNA condensation/fragmentation: the enhancement of the PhI band component centred at ~1206 cm(-1), which is more sensitive than the relative intensity of the PhII band to which phospholipids and carbohydrates also contribute significantly. In conclusion, we demonstrate that the intrinsic multi-parametric nature of IRMS and its application on cells under physiological conditions can be well exploited for the investigation of apoptotic pathways.

SU-8 bonding protocol for the fabrication of microfluidic devices dedicated to FTIR microspectroscopy of live cells.

Here we present a new bonding protocol for SU-8 negative tone photoresist that exploits the chemical modifications induced in the resin by exposure to 254 nm (UVC) light. Fourier Transform Infrared microspectroscopy (µ-FTIR) was used to carry out a thorough study on the chemical processes and modifications occurring within the epoxy resin by exposure to 365 nm and 254 nm light. In particular, we established that UVC light promotes the opening of the epoxy rings bypassing the post-exposure bake. The possibility to promote a further activation of the resin, already patterned with standard UV lithography, was exploited to produce closed microfluidic devices. Specifically, we were able to fabricate fluidic chips, characterized by broadband transparency from mid-IR to UV and long term stability in continuous flow conditions. CaF2 was used as substrate, coated by sputtering with a nanometric silicon film, in order to make surface properties of this material more suitable for standard fabrication processes with respect to the original substrate. The fabricated microfluidic chips were used to study by µ-FTIR the biochemical response of live breast cancer MCF-7 cells to osmotic stress and their subsequent lysis induced by the injection of deionized water in the device. µ-FTIR analyses detected fast changes in protein, lipid and nucleic acid content as well as cytosol acidification.

Determination of cell cycle phases in live B16 melanoma cells using IRMS.

The knowledge of cell cycle phase distribution is of paramount importance for understanding cellular behaviour under normal and stressed growth conditions. This task is usually assessed using Flow Cytometry (FC) or immunohistochemistry. Here we report on the use of FTIR microspectroscopy in Microfluidic Devices (MD-IRMS) as an alternative technique for studying cell cycle distribution in live cells. Asynchronous, S- and G0-synchronized B16 mouse melanoma cells were studied by running parallel experiments based on MD-IRMS and FC using Propidium Iodide (PI) staining. MD-IRMS experiments have been done using silicon-modified BaF2 devices, where the thin silicon layer prevents BaF2 dissolution without affecting the transparency of the material and therefore enabling a better assessment of the Phosphate I (PhI) and II (PhII) bands. Hierarchical Cluster Analysis (HCA) of cellular microspectra in the 1300-1000 cm(-1) region pointed out a distribution of cells among clusters, which is in good agreement with FC results among G0/G1, S and G2/M phases. The differentiation is mostly driven by the intensity of PhI and PhII bands. In particular, PhI almost doubles from the G0/G1 to G2/M phase, in agreement with the trend followed by nucleic acids during cellular progression. MD-IRMS is then proposed as a powerful method for the in situ determination of the cell cycle stage of an individual cell, without any labelling or staining, which gives the advantage of possibly monitoring specific cellular responses to several types of stimuli by clearly separating the spectral signatures related to the cellular response from those of cells that are normally progressing.

New half sandwich Ru(II) coordination compounds for anticancer activity.

With the aim of expanding the structure-activity relationship investigation, the series of Ru(II) half sandwich coordination compounds of the type [Ru([9]aneS3)(chel)(L)](n+) previously described by us (where [9]aneS3 is the neutral face-capping ligand 1,4,7-trithiacyclononane, chel is a neutral or anonic chelating ligand, L = Cl(-) or dmso-S, n = 0-2) was extended to 1,4,7-triazacyclononane ([9]aneN3). In addition, new neutral N-N, and anionic N-O and O-O chelating ligands, i.e. dach (trans-1,2-diaminocyclohexane), pic(-) (picolinate), and acac(-) (acetylacetonate), were investigated in combination with both [9]aneS3 and [9]aneN3. Overall, ten new half-sandwich complexes were prepared and fully characterized and their chemical behaviour in aqueous solution was established. The single-crystal X-ray structures of eight of them, including the versatile precursor [Ru([9]aneN3)(dmso-S)(2)Cl]Cl (9), were also determined. The results of in vitro antiproliferative tests performed on selected compounds against MDA-MB-231 human mammary carcinoma cells confirmed that, in this series, only compounds that hydrolyse the monodentate ligand at a reasonable rate show moderate activity, provided that the chelate ligand is a hydrogen bond donor.