Raman Spectroscopy Applications in Grapevine: Metabolic Analysis of Plants Infected by Two Different Viruses.

Grapevine is one of the most cultivated fruit plant among economically relevant species in the world. It is vegetatively propagated and can be attacked by more than 80 viruses with possible detrimental effects on crop yield and wine quality. Preventive measures relying on extensive and robust diagnosis are fundamental to guarantee the use of virus-free grapevine plants and to manage its diseases. New phenotyping techniques for non-invasive identification of biochemical changes occurring during virus infection can be used for rapid diagnostic purposes. Here, we have investigated the potential of Raman spectroscopy (RS) to identify the presence of two different viruses, grapevine fan leaf virus (GFLV) and grapevine rupestris stem pitting-associated virus (GRSPaV) in Vitis vinifera cv. Chardonnay. We showed that RS can discriminate healthy plants from those infected by each of the two viruses, even in the absence of visible symptoms, with accuracy up to 100% and 80% for GFLV and GRSPaV, respectively. Chemometric analyses of the Raman spectra followed by chemical measurements showed that RS could probe a decrease in the carotenoid content in infected leaves, more profoundly altered by GFLV infection. Transcriptional analysis of genes involved in the carotenoid pathway confirmed that this biosynthetic process is altered during infection. These results indicate that RS is a cutting-edge alternative for a real-time dynamic monitoring of pathogens in grapevine plants and can be useful for studying the metabolic changes ensuing from plant stresses.

Efficacy, biocompatibility and degradability of carbon nanoparticles for photothermal therapy of lung cancer.

Aim: To investigate near infrared-induced phototoxicity toward lung cancer cells, and the biodegradability and effect on immune cells of glucose-derived carbon nanoparticles (CNPs). Methods: The human A549 lung adenocarcinoma cell line was used as a model to study the phototoxicity of CNPs. The biodegradability and the effect on immune cells was demonstrated in primary human neutrophils and macrophages. Results: Near infrared-activated CNPs elicited rapid cell death, characterized by the elevation of heat shock proteins and the induction of DNA damage. CNPs were found to be noncytotoxic toward primary human macrophages and were susceptible to biodegradation when cocultured with human neutrophils. Conclusions: Our results identify CNPs as promising platforms for photothermal therapy of lung cancer.

Migration study of organotin compounds from food packaging by surface-enhanced Raman scattering.

The potential of surface-enhanced Raman scattering (SERS) has been investigated for the rapid analysis of two representative organotin compounds (OTCs): dibutyltin maleate (DTM) and tributyltin chloride (TBT), after migration tests from polyvinyl chloride (PVC), as a model food packaging material in aqueous food simulant (acetic acid 3% w/v). OTCs, often used as heat stabilizers additives for PVC, are classified as endocrine disrupting chemicals (EDCs) and their migration potential has to be controlled in compliance with the normative prescriptions for food contact materials. In this study, colloidal silver nanoparticles (AgNPs) were applied as liquid SERS substrate for direct-in-liquid analysis of food simulant after standardized migration tests of PVC samples spiked with OTCs. Promising results were obtained, reaching detection limits below the permitted limits for the considered OTCs (i.e. 0.15 mg/l): DTM and TBT were detected down to 0.01 mg/l and 0.08 mg/l, respectively. Calibration curves were calculated for standard solutions of DTM and TBT in the dynamic range between 0 and 1 mg/l (reduced χ2 = 0.8), and 0.5-5 mg/l (reduced χ2 = 0.2), respectively. Migrated TBT and DTM were detected in the food simulant, specifically identified and quantified by SERS, with a measurement uncertainty around 10% in all cases. In particular, it was found that TBT can migrate in higher amount compared to DTM when the PVC film is in contact with a slightly acidic matrix. These results were further confirmed by inductively coupled plasma-mass spectrometry and UV-Vis spectroscopy. In the present study, direct-in-liquid SERS approach showed to be very promising because it provides a fast response and it allows to overcome most of the common drawbacks of solid SERS substrates due to inhomogeneity problems and low repeatability.

In situ seed-growth synthesis of silver nanoplates on glass for the detection of food contaminants by surface enhanced Raman scattering.

Seed-growth synthesis is a common strategy to prepare silver nanoplates, whose peculiar plasmonic features can be exploited for surface enhanced Raman scattering (SERS) applications. Here we describe the fabrication and characterization of SERS chips using a peculiar in situ seed growth method, yielding a dense layer of nano-objects directly on a glass slide. In this way, geometric features (i.e. shape and dimensions) of the nano-objects can be tuned by controlling the growth time, obtaining a high concentration of hot spots on the surface. In particular, the SERS response of four kinds of chips were investigated to define the best SERS configuration in terms of size of the silver nano-objects, excitation wavelength and homogeneity of the SERS response. Silver nano-plates with a seeded growth time of 60 min demonstrated remarkable results both in terms of plasmonic enhancement, with an enhancement factor (EF) of 2 × 105 using a 532 nm laser excitation, and good homogeneity of the SERS response with intra- and inter-maps RSD of 10% and 5%, respectively. In order to demonstrate application of these chips for real sample analysis, an analytical procedure for the detection of a model pesticide, i.e. thiram fungicide, was developed and applied to its detection on green apples peels. SERS measurements on 60 min seeded growth silver nano-plates chip coupled with a multivariate PLS approach demonstrated high accuracy and repeatability for thiram detection in food matrix within the European law limits.

Direct quantification of sulfur dioxide in wine by Surface Enhanced Raman Spectroscopy.

A rapid Surface Enhanced Raman Spectroscopy (SERS) method to detect SO2 in wine is presented, exploiting the preferential binding of silver nanoparticles (AgNPs) with sulfur-containing species. This interaction promotes the agglomeration of the AgNPs and inducing the formation of SERS "hot spots" responsible for SO2 signals enhancement. For increasing SO2 concentrations from 0 to100 mg/l in wine simulant, SERS intensity showed an increasing trend, following a Langmuir absorption function (R2 = 0.94). Due to the wine matrix variability, a standard additions method was then employed for quantitative analysis in red and white wines. This method does not require the SO2 separation but only a matrix pre-cleaning by solid phase extraction. The limit of detection (LOD) was defined for each wine tested, ranging from 0.6 mg/l to 9.6 mg/l. The results obtained were validated by comparison with the International Organization of Vine and Wine method (OIV-MA-AS323-04A).

Flexible and Transparent Substrates Based on Gold Nanoparticles and TiO2 for in Situ Bioanalysis by Surface-Enhanced Raman Spectroscopy.

Flexible and transparent substrates are emerging as low cost and easy-to-operate support for surface-enhanced Raman spectroscopy (SERS). In particular, in situ SERS detection approach for surface characterization in transmission modality can be efficiently employed for non-invasive analysis of non-planar surfaces. Here we propose a new methodology to fabricate a homogenous, transparent, and flexible SERS membrane by the assistance of a thin TiO2 porous layer deposited on the PDMS surface, which supports the uniform loading of gold nanoparticles over large area. The substrate was first characterized for homogeneity, sensitivity and repeatability using a model molecule for SERS, i.e., 7-mercapto-4-methylcoumarin. Satisfactory intra-substrate uniformity and inter-substrates repeatability was achieved, showing an RSD of 10%, and an analytical sensitivity down to 10 nM was determined with an EF of 3.4 × 105 ± 0.4 × 105. Furthermore, SERS detection of pyrimethanil (PMT), a commonly employed pesticide in crops for human consumption, was performed in situ, exploiting the optical transparency of the device, using both model surfaces and non-flat bio-samples. PMT contamination at the phytochemical concentration levels corresponding to commonly used infield doses was successfully detected on the surface of the yellow Ficus benjiamina leaves, supporting the use of this substrate for food safety in-field application.

Nondestructive Raman Spectroscopy as a Tool for Early Detection and Discrimination of the Infection of Tomato Plants by Two Economically Important Viruses.

Global population forecasts dictate a rapid adoption of multifaceted approaches to fulfill increasing food requirements, ameliorate food dietary value and security using sustainable and economically feasible agricultural processes. Plant pathogens induce up to 25% losses in vegetable crops and their early detection would contribute to limit their spread and economic impact. As an alternative to time-consuming, destructive, and expensive diagnostic procedures, such as immunological assays and nucleic acid-based techniques, Raman spectroscopy (RS) is a nondestructive rapid technique that generates a chemical fingerprinting of a sample, at low operating costs. Here, we assessed the suitability of RS combined to chemometric analysis to monitor the infection of an important vegetable crop plant, tomato, by two dangerous and peculiarly different viral pathogens, Tomato yellow leaf curl Sardinia virus (TYLCSV) and Tomato spotted wilt virus (TSWV). Experimentally inoculated plants were monitored over 28 days for symptom occurrence and subjected to RS analysis, alongside with measuring the virus amount by quantitative real-time PCR. RS allowed to discriminate mock inoculated (healthy) from virus-infected specimens, reaching an accuracy of >70% after only 14 days after inoculation for TYLCSV and >85% only after 8 days for TSWV, demonstrating its suitability for early detection of virus infection. Importantly, RS also highlighted spectral differences induced by the two viruses, providing specific information on the infecting agent.

Authentication of cocoa bean shells by near- and mid-infrared spectroscopy and inductively coupled plasma-optical emission spectroscopy.

The aim of this study was to evaluate the efficacy of a multi-analytical approach for origin authentication of cocoa bean shells (CBS). The overall chemical profiles of CBS from different origins were characterized using diffuse reflectance near-infrared spectroscopy (NIRS) and attenuated total reflectance mid-infrared spectroscopy (ATR-FT-IR) for molecular composition identification, as well as inductively coupled plasma-optical emission spectroscopy (ICP-OES) for elemental composition identification. Exploratory chemometric techniques based on Principal Component Analysis (PCA) were applied to each single technique for the identification of systematic patterns related to the geographical origin of samples. A combination of the three techniques proved to be the most promising approach to establish classification models. Partial Least Squares-Discriminant Analysis modelling of fused PCA scores of three independent models was used and compared with single technique models. Improved classification of CBS samples was obtained using the fused model. Satisfactory classification rates were obtained for Central African samples with an accuracy of 0.84.

Comprehensive study on the degradation of ochratoxin A in water by spectroscopic techniques and DFT calculations.

Ochratoxin A (OTA) is one of the most important dietary risk factors and is classified as a possible carcinogen to humans. Assessing the conditions to remove it from foodstuffs in a simple and effective way is of the utmost importance. OTA behaviour in water in the pH range 1.0-12.5 was elucidated to investigate the conditions for irreversible toxicity inactivation of OTA. The results indicate that four forms, from neutral to trianionic, intervene depending on the pH. pK a1,2 were rigorously established by independent spectroscopic techniques to overcome the scarcity of literature. Then, Density Functional Theory (DFT) calculations were used to determine the most probable degradation mechanism and this was confirmed by fluorescence spectroscopy. At pH 12.5, hydrolyzation of the lactone ring starts in less than one hour, but only after two hours does the degradation process lead to fragmentation. After one week this process is not yet completed. The reaction products occurring upon re-acidification were also investigated. OTA degradation is still reversible if acidic conditions are promptly restored, yielding again a hazardous molecule. However, degradation becomes irreversible after fragmentation. This finding suggests proceeding with due caution if a base is exploited to remove the toxin.

Influence of the long-range ordering of gold-coated Si nanowires on SERS.

Controlling the location and the distribution of hot spots is a crucial aspect in the fabrication of surface-enhanced Raman spectroscopy (SERS) substrates for bio-analytical applications. The choice of a suitable method to tailor the dimensions and the position of plasmonic nanostructures becomes fundamental to provide SERS substrates with significant signal enhancement, homogeneity and reproducibility. In the present work, we studied the influence of the long-range ordering of different flexible gold-coated Si nanowires arrays on the SERS activity. The substrates are made by nanosphere lithography and metal-assisted chemical etching. The degree of order is quantitatively evaluated through the correlation length (ξ) as a function of the nanosphere spin-coating speed. Our findings showed a linear increase of the SERS signal for increasing values of ξ, coherently with a more ordered and dense distribution of hot spots on the surface. The substrate with the largest ξ of 1100 nm showed an enhancement factor of 2.6 · 103 and remarkable homogeneity over square-millimetres area. The variability of the signal across the substrate was also investigated by means of a 2D chemical imaging approach and a standard methodology for its practical calculation is proposed for a coherent comparison among the data reported in literature.

Detection of insect's meal in compound feed by Near Infrared spectral imaging.

Insects have recently emerged as a new protein source for both food and feed. Some studies have already demonstrated that insects' meal can be successfully added to animal feed without threaten animals' growth indices. However, effective and validated tests to individuate insects' meal in feed are strongly needed to meet traceability and safety concerns and to support the European legislation under development. Spectroscopic techniques represent valuable rapid and non-destructive methods that can be applied for in-situ analysis in feed production plants or in farms. In this work a Fourier Transform Near Infrared spectroscopy imaging (FT NIR) as a potential screening method for the detection and quantification of insects' meal in feed is presented. Discriminant analysis was used for the automatic recognition of insects' meal fragments into the feed matrix. Moreover, the possibility to quantify insect's meal in feed sample was successfully tested. The proposed method is a rapid and green strategy for feed contamination screening analysis.

Species-specific detection of processed animal proteins in feed by Raman spectroscopy.

The existing European Regulation (EC n° 51/2013) prohibits the use of animals meals in feedstuffs in order to prevent Bovine Spongiform Encephalopathy infection and diffusion, however the legislation is rapidly moving towards a partial lifting of the "feed ban" and the competent control organisms are urged to develop suitable analytical methods able to avoid food safety incidents related to animal origin products. The limitations of the official methods (i.e. light microscopy and Polymerase Chain Reaction) suggest exploring new analytic ways to get reliable results in a short time. The combination of spectroscopic techniques with optical microscopy allows the development of an individual particle method able to meet both selectivity and sensitivity requirements (0.1%w/w). A spectroscopic method based on Fourier Transform micro-Raman spectroscopy coupled with Discriminant Analysis is here presented. This approach could be very useful for in-situ applications, such as customs inspections, since it drastically reduces time and costs of analysis.

Controlling protected designation of origin of wine by Raman spectroscopy.

In this paper, a Fourier Transform Raman spectroscopy method, to authenticate the provenience of wine, for food traceability applications was developed. In particular, due to the specific chemical fingerprint of the Raman spectrum, it was possible to discriminate different wines produced in the Piedmont area (North West Italy) in accordance with i) grape varieties, ii) production area and iii) ageing time. In order to create a consistent training set, more than 300 samples from tens of different producers were analyzed, and a chemometric treatment of raw spectra was applied. A discriminant analysis method was employed in the classification procedures, providing a classification capability (percentage of correct answers) of 90% for validation of grape analysis and geographical area provenance, and a classification capability of 84% for ageing time classification. The present methodology was applied successfully to raw materials without any preliminary treatment of the sample, providing a response in a very short time.