Role of probe design and bioassay configuration in surface enhanced Raman scattering based biosensors for miRNA detection.

The accurate design of labelled oligo probes for the detection of miRNA biomarkers by Surface Enhanced Raman Scattering (SERS) may improve the exploitation of the plasmonic enhancement. This work, thus, critically investigates the role of probe labelling configuration on the performance of SERS-based bioassays for miRNA quantitation. To this aim, highly efficient SERS substrates based on Ag-decorated porous silicon/PDMS membranes are functionalized according to bioassays relying on a one-step or two-step hybridization of the target miRNA with DNA probes. Then, the detection configuration is varied to evaluate the impact of different Raman reporters and their labelling position along the oligo sequence on bioassay sensitivity. At high miRNA concentration (100-10 nM), a significantly increased SERS intensity is detected when the reporters are located closer to the plasmonic surface compared to farther probe labelling positions. Counterintuitively, a levelling-off of the SERS intensity from the different configurations is recorded at low miRNA concentration. Such effect is attributed to the increased relative contribution of Raman hot-spots to the whole SERS signal, in line with the electric near field distribution simulated for a simplified model of the Ag nanostructures. However, the beneficial effect of reducing the reporter-to-surface distance is partially retained for a two-step hybridization assay thanks to the less sterically hindered environment in which the second hybridization occurs. The study thus demonstrates an improvement of the detection limit of the two-step assay by tuning the probe labelling position, but sheds at the same time light on the multiple factors affecting the sensitivity of SERS-based bioassays.

Chemometric Differentiation of Sole and Plaice Fish Fillets Using Three Near-Infrared Instruments.

Fish species substitution is one of the most common forms of fraud all over the world, as fish identification can be very challenging for both consumers and experienced inspectors in the case of fish sold as fillets. The difficulties in distinguishing among different species may generate a "grey area" in which mislabelling can occur. Thus, the development of fast and reliable tools able to detect such frauds in the field is of crucial importance. In this study, we focused on the distinction between two flatfish species largely available on the market, namely the Guinean sole (Synaptura cadenati) and European plaice (Pleuronectes platessa), which are very similar looking. Fifty fillets of each species were analysed using three near-infrared (NIR) instruments: the handheld SCiO (Consumer Physics), the portable MicroNIR (VIAVI), and the benchtop MPA (Bruker). PLS-DA classification models were built using the spectral datasets, and all three instruments provided very good results, showing high accuracy: 94.1% for the SCiO and MicroNIR portable instruments, and 90.1% for the MPA benchtop spectrometer. The good classification results of the approach combining NIR spectroscopy, and simple chemometric classification methods suggest great applicability directly in the context of real-world marketplaces, as well as in official control plans.

Catalytic wet air oxidation of d-glucose by perovskite type oxides (Fe, Co, Mn) for the synthesis of value-added chemicals.

The conversion of common biomasses derived, as d-glucose, into value-added chemicals has received highest attention in the last few years. Among all processes, the catalytic wet air oxidation (CWAO) of derived biomasses using noble metal-based heterogeneous catalytic systems has been investigated. Nevertheless, the redox effect of such catalysts on such bio-compounds has still to be investigated in detail. In the present work, the activity for the conversion of d-glucose into C6 aldaric acid, lactic acid and levulinic acid of some perovskite type oxides (LaBO3; B: Fe, Co, Mn) characterized by noticeable catalytic properties and stability under hydrothermal conditions, have been investigated. The influence of the reaction temperature and the effect of the catalytic properties on the distribution of the liquid products have been studied. In the best conditions, 50.3 mol.% and 69.5 mol.% of lactic and levulinic acid have been obtained by using LaCoO3 and LaMnO3, respectively. Apart from the oxidative effect, the affinity hydrogen allowed the catalytic conversion of some key intermediates, such as pyruvic aldehyde and hydroxymethylfurfural, into the desired products. LaMnO3, which has resulted to be the most oxidizable/reducible catalyst at low temperatures, has shown the best catalytic activity among the studied catalysts, promoting the conversion of hydroxymethylfurfural to levulinic acid and giving overall the highest yield.

Lipids in a Nutshell: Quick Determination of Lipid Content in Hazelnuts with NIR Spectroscopy.

Hazelnuts (Corylus avellana L.) are among the most consumed dry fruits all over the world. Their commercial quality is defined, above all, by origin and dimension, as well as by lipid content. Evaluation of this parameter is currently performed with chemical methods, which are expensive, time consuming, and complex. In the present work, the near-infrared (NIR) spectroscopy, using both a benchtop research spectrometer and a retail handheld instrument, was evaluated in comparison with the traditional chemical approach. The lipid content of hazelnuts from different growing regions of origin (Italy, Chile, Turkey, Georgia, and Azerbaijan) was determined with two NIR instruments: a benchtop FT-NIR spectrometer (Multi Purpose Analyser-MPA, by Bruker), equipped with an integrating sphere and an optic fibre probe, and the pocket-sized, battery-powered SCiO molecular sensor (by Consumer Physics). The Randall/Soxtec method was used as the reference measurement of total lipid content. The collected NIR spectra were inspected through multivariate data analysis. First, a Principal Component Analysis (PCA) model was built to explore the information contained in the spectral datasets. Then, a Partial Least Square (PLS) regression model was developed to predict the percentage of lipid content. PCA showed samples distributions that could be linked to their total crude fat content determined with the Randall/Soxtec method, confirming that a trend related to the lipid content could be detected in the spectral data, based on their chemical profiles. PLS models performed better with the MPA instrument than SCiO, with the highest R2 of prediction (R2PRED = 0.897) achieved by MPA probe, while this parameter for SCiO was much lower (R2PRED = 0.550). Further analyses are necessary to evaluate if more acquisitions may lead to better performances when using the SCiO portable spectrometer.

Real-Time Monitoring of the In Situ Microfluidic Synthesis of Ag Nanoparticles on Solid Substrate for Reliable SERS Detection.

A sharpened control over the parameters affecting the synthesis of plasmonic nanostructures is often crucial for their application in biosensing, which, if based on surface-enhanced Raman spectroscopy (SERS), requires well-defined optical properties of the substrate. In this work, a method for the microfluidic synthesis of Ag nanoparticles (NPs) on porous silicon (pSi) was developed, focusing on achieving a fine control over the morphological characteristics and spatial distribution of the produced nanostructures to be used as SERS substrates. To this end, a pSi membrane was integrated in a microfluidic chamber in which the silver precursor solution was injected, allowing for the real-time monitoring of the reaction by UV-Vis spectroscopy. The synthesis parameters, such as the concentration of the silver precursor, the temperature, and the flow rate, were varied in order to study their effects on the final silver NPs' morphology. Variations in the flow rate affected the size distribution of the NPs, whereas both the temperature and the concentration of the silver precursor strongly influenced the rate of the reaction and the particle size. Consistently with the described trends, SERS tests using 4-MBA as a probe showed how the flow rate variation affected the SERS enhancement uniformity, and how the production of larger NPs, as a result of an increase in temperature or of the concentration of the Ag precursor, led to an increased SERS efficiency.

Differentiation between Fresh and Thawed Cephalopods Using NIR Spectroscopy and Multivariate Data Analysis.

The sale of frozen-thawed fish and fish products, labeled as fresh, is currently one of the most common and insidious commercial food frauds. For this reason, the demand of reliable tools to identify the storage conditions is increasing. The present study was performed on two species, commonly sold in large-scale distribution: Cuttlefish (Sepia officinalis) and musky octopus (Eledone spp.). Fifty fresh cephalopod specimens were analyzed at refrigeration temperature (2 ± 2 °C), then frozen at -20 °C for 10 days and finally thawed and analyzed again. The performance of three near-infrared (NIR) instruments in identifying storage conditions were compared: The benchtop NIR Multi Purpose Analyzer (MPA) by Bruker, the portable MicroNIR by VIAVI and the handheld NIR SCiO by Consumer Physics. All collected spectra were processed and analyzed with chemometric methods. The SCiO data were also analyzed using the analytical tools available in the online application provided by the manufacturer to evaluate its performance. NIR spectroscopy, coupled with chemometrics, allowed discriminating between fresh and thawed samples with high accuracy: Cuttlefish between 82.3-94.1%, musky octopus between 91.2-97.1%, global model between 86.8-95.6%. Results show how food frauds could be detected directly in the marketplace, through small, ultra-fast and simplified handheld devices, whereas official control laboratories could use benchtop analytical instruments, coupled with chemometric approaches, to develop accurate and validated methods, suitable for regulatory purposes.

Innovative Coatings Based On Peppermint Essential Oil on Titanium and Steel Substrates: Chemical and Mechanical Protection Ability.

A coating that was made of peppermint essential oil was obtained on different metal substrates: Ti6Al4V alloy (mechanically polished and chemically etched) and 316L stainless steel (mechanically polished and mechanically ground). The final aim is to get a multifunctional (chemical and mechanical) protection of metal surfaces in contact with water media. The coatings were characterized by means of fluorescence microscopy, contact angle measurements, and Fourier Transformed Infrared spectroscopy (FTIR) spectroscopy. The chemical stability of the coatings was tested by means of soaking in water for different times (up to seven days) and washing with different alkaline or acidic solutions. The mechanical adhesion of the coating was tested by tape adhesion test (before and after soaking) and scratch tests to verify whether it has protection ability with respect to the metal substrate. All of the performed characterizations show that the coatings are chemically stable on all of the substrates and are nor dissolved or removed by water during soaking or by alkaline solutions during washing. The adhesion is high and classified as 4B or 5B (on the chemically etched or mechanically ground substrates) according to ASTM D3359-97, depending on the substrate roughness, both before and after soaking. In the case of scratch test (up to 10 N), the coating is not removed and it has a protection action that is able to avoid the surface damage, even if the substrate has a plastic deformation.

Label-Free SERS Discrimination and In Situ Analysis of Life Cycle in Escherichia coli and Staphylococcus epidermidis.

Surface enhanced Raman spectroscopy (SERS) has been proven suitable for identifying and characterizing different bacterial species, and to fully understand the chemically driven metabolic variations that occur during their evolution. In this study, SERS was exploited to identify the cellular composition of Gram-positive and Gram-negative bacteria by using mesoporous silicon-based substrates decorated with silver nanoparticles. The main differences between the investigated bacterial strains reside in the structure of the cell walls and plasmatic membranes, as well as their biofilm matrix, as clearly noticed in the corresponding SERS spectrum. A complete characterization of the spectra was provided in order to understand the contribution of each vibrational signal collected from the bacterial culture at different times, allowing the analysis of the bacterial populations after 12, 24, and 48 h. The results show clear features in terms of vibrational bands in line with the bacterial growth curve, including an increasing intensity of the signals during the first 24 h and their subsequent decrease in the late stationary phase after 48 h of culture. The evolution of the bacterial culture was also confirmed by fluorescence microscope images.

SERS-active metal-dielectric nanostructures integrated in microfluidic devices for label-free quantitative detection of miRNA.

In this work, SERS-based microfluidic PDMS chips integrating silver-coated porous silicon membranes were used for the detection and quantitation of microRNAs (miRNAs), which consist of short regulatory non-coding RNA sequences typically over- or under-expressed in connection with several diseases such as oncogenesis. In detail, metal-dielectric nanostructures which provide noticeable Raman enhancements were functionalized according to a biological protocol, adapted and optimized from an enzyme-linked immunosorbent assay (ELISA), for the detection of miR-222. Two sets of experiments based on different approaches were designed and performed, yielding a critical comparison. In the first one, the labelled target miRNA is revealed through hybridization to a complementary thiolated DNA probe, immobilized on the silver nanoparticles. In the second one, the probe is halved into shorter strands (half1 and half2) that interact with the complementary miRNA in two steps of hybridization. Such an approach, taking advantage of the Raman labelling of half2, provides a label-free analysis of the target. After suitable optimisation of the procedures, two calibration curves allowing quantitative measurements were obtained and compared on the basis of the SERS maps acquired on the samples loaded with several miRNA concentrations. The selectivity of the two-step assay was confirmed by the detection of target miR-222 mixed with different synthetic oligos, simulating the hybridization interference coming from similar sequences in real biological samples. Finally, that protocol was applied to the analysis of miR-222 in cellular extracts using an optofluidic multichamber biosensor, confirming the potentialities of SERS-based microfluidics for early-cancer diagnosis.

Silver-doped keratin nanofibers preserve a titanium surface from biofilm contamination and favor soft-tissue healing.

Peri-implantitis is a severe condition affecting the success of transmucosal dental implants: tissue healing is severely limited by the inflammatory processes that come about to control homeostasis in the surrounding tissues. The main cause of peri-implantitis is bacterial biofilm infection; gingival fibroblasts play a pivotal role in regulating the inflammatory cascades. A new technology aimed at preventing bacterial colonization of titanium (Ti) implants, and enhancing the spread of gingival fibroblasts, is presented. Using electro-spinning, mirror-polished Ti disks were uniformly coated with keratin fibers obtained from discarded wool via sulfitolysis. The keratin-coated surfaces were then doped with silver (Ag) to introduce antibacterial properties, using different concentrations of silver nitrate as a precursor (0.01, 0.05 and 0.1 M). The resulting specimens were characterized in terms of morphology and chemical composition by FESEM, FTIR and XPS, revealing silver concentrations between 1.7 and 1.9%. Silver release into the medium was evaluated in the presence of cells (α-MEM) or bacteria (LB) by ICP; release was 0.2-1.4 mg l-1 for α-MEM, and 10-40 mg l-1 for LB. The antibacterial properties of the Ag-doped specimens were tested against a multidrug-resistant Staphylococcus aureus biofilm through morphology (FESEM) and metabolic assay (XTT); reduction in viability was significant (p < 0.05; >80% reduction within 72 h). Lastly, the cytocompatibility of the specimens was confirmed using human primary gingival fibroblasts, whose viability, spread and matrix deposition were found to be comparable to those of untreated Ti polished controls (p > 0.05). Thus, Ag surface enrichment was effective in reducing viability and maturation of S. aureus biofilm, without compromising human cell viability. Moreover, cell spread was found to be very sensitive to keratin fiber stimulation. The strategy thus appears to be very promising to introduce surface features in line with the main requirements for transmucosal dental implants.

Immobilization of Oligonucleotides on Metal-Dielectric Nanostructures for miRNA Detection.

The development of nanostructured metal-dielectric materials, suitable for biodetection based on surface plasmon resonance and surface enhanced Raman scattering (SERS), requires the refinement of proper biological protocols for their effective exploitation. In this work, the immobilization of DNA probes on nanostructured metal-dielectric/semiconductor substrates has been optimized, to develop a bioassay for the detection of miRNA. To ensure a broad relevance, the proposed biological protocol was applied to different silver-decorated functional supports: porous silicon (pSi), TiO2 nanotube arrays, and polydimethylsiloxane (PDMS). The efficiency and the stability of the substrates were carefully analyzed by Raman spectroscopy and electron microscopy after the incubation in buffers with the appropriate combination of pH, ionic strength, and surfactant content. The customized protocol, initially developed on multiwell plates, was transferred and refined on the nanostructured substrates. The nonspecific interaction of the biological species with the surface was evaluated and reduced thanks to a tailored surface pretreatment. SERS analysis was applied to check the immobilization of DNA probes on pretreated samples. Silvered PDMS-supported pSi membranes, the most promising substrates in terms of stability, were subjected to further optimizations. Concentrations, volume, and duration of incubations were finely adapted with respect to the surface probe density and to the corresponding hybridization of the complementary miRNA. The optimized ELISA-like assay shows sensitivities comparable to those of commercial plates for the detection of miRNA222 (LOD: 485 pM), paving the way for the application of the developed protocol on metal-dielectric/semiconductor nanostructures for ultrasensitive SERS biosensing applications.

Role of ice structuring proteins on freezing-thawing cycles of pasta sauces.

The freezing of the food is one of the most important technological developments for the storage of food in terms of quality and safety. The aim of this work was to study the role of an ice structuring protein (ISP) on freezing-thawing cycles of different solutions and commercial Italian pasta sauces. Ice structuring proteins were related to the modification of the structure of ice. The results showed that the freezing time of an aqueous solution containing the protein was reduced to about 20% with respect to a pure water solution. The same effect was demonstrated in sugar-containing solutions and in lipid-containing sauces. The study proved a specific role of ISP during thawing, inducing a time decrease similar to that of freezing and even more important in the case of tomato-based sauces. This work demonstrated the role of ISP in the freezing-thawing process, showing a significant reduction of processing in the freezing and thawing phase by adding the protein to pure water and different sugar-, salt- and lipid-containing solutions and commercial sauces, with considerable benefits for the food industry in terms of costs and food quality.

Discrimination between washed Arabica, natural Arabica and Robusta coffees by using near infrared spectroscopy, electronic nose and electronic tongue analysis.

BACKGROUND: The aim of this study is to investigate the feasibility of a 'holistic' approach, using near infrared (NIR) spectroscopy and electronic devices (electronic nose and electronic tongue), as instrumental tools for the classification of different coffee varieties. Analyses were performed on green coffee, on ground roasted coffee and on coffee beverage. Principal component analysis was applied on spectral and sensory data to uncover correlations between samples and variables. After variable selection, linear discriminant analysis was used to classify the samples on the basis of the three coffee classes: Robusta, natural Arabica and washed Arabica. RESULTS: Linear discriminant analysis demonstrates the practicability of this approach: the external test set validation performed with NIR data showed 100% of correctly classified samples. Moreover, a satisfying percentage of correct classification in cross-validation was obtained for the electronic devices: the average values of correctly classified samples were 81.83% and 78.76% for electronic nose and electronic tongue, respectively. CONCLUSION: NIR spectroscopy was shown to be a very reliable and useful tool to classify coffee samples in a fast, clean and inexpensive way compared to classical analysis, while the electronic devices could assume the role of investigating techniques to depict the aroma and taste of coffee samples.

SERS active silver nanoparticles synthesized by inkjet printing on mesoporous silicon.

Inkjet printing technique is exploited for the synthesis of Ag nanoparticles (NPs) patterned on electrochemically etched silicon-based substrates. The nanostructure morphology, here analyzed by scanning electron microscopy, is dictated by the ink composition and the printing parameters. Under suitable excitation conditions, resonant surface-enhanced Raman scattering (SERS) performed on such metal-dielectric nanostructures can approach single-molecule detection as recently demonstrated on silvered porous silicon synthesized by immersion plating. PACS: 78.67.Bf; 78.30.-j.

Protein immobilization on nanoporous silicon functionalized by RF activated plasma polymerization of Acrylic Acid.

Plasma Enhanced Chemical Vapor Deposition (PECVD) technique is used to polymerize Acrylic Acid for the surface functionalization of porous silicon samples with different pore dimensions. The polymer shows free COOH groups also at the pores inner surface, suitable for the immobilization of fluorescent labeled Protein A. The stability of the polymer, its role in the protection from aging of the porous matrix and the efficiency of the functionalization for the binding of protein A have been characterized by ATR-FTIR, SEM, Optical Contact Angle and Fluorescence Microscopy. The polymerization process is well controllable and suitable for the functionalization of porous silicon leaving free carboxylic groups at the surface ready for the immobilization of biochemical species for sensing applications.

Post-curing conversion kinetics as functions of the irradiation time and increment thickness.

OBJECTIVE: This study evaluated the variation of conversion degree (DC) in the 12 hours following initial photoactivation of a low-shrinkage composite resin (Venus Diamond). MATERIAL AND METHODS: The conversion degree was monitored for 12 hours using Attenuated Total Reflection (ATR) F-TIR Spectroscopy. The composite was placed in 1 or 2 mm rings and cured for 10 or 20 seconds with a LED lamp. ATR spectra were acquired from the bottom surface of each sample immediately after the initial photoactivation (P=0), 30 minutes (P=0.5) and 12 hours after photoactivation (P=12) in order to obtain the DC progression during the post-curing period. Interactions between thickness (T), irradiation time (I) and post-curing (P) on the DC were calculated through ANOVA testing. RESULTS: All the first order interactions were statistically significant, with the exception of the T-P interaction. Furthermore, the shift from P=0 to P=0.5 had a statistically higher influence than the shift from P=0.5 to P=12. The post-curing period played a fundamental role in reaching higher DC values with the low-shrinkage composite resin tested in this study. Moreover, both the irradiation time and the composite thickness strongly influenced the DC. CONCLUSIONS: Increased irradiation time may be useful in obtaining a high conversion degree (DC) with a low-shrinkage nano-hybrid composite resin, particularly with 2 mm composite layers.

Temporal trends of elements in Turin (Italy) atmospheric particulate matter from 1976 to 2001.

The temporal trends of major, minor and trace elements in the total atmospheric particulate sampled in the urban area of Turin (Italy) were determined for the following years: 1976, 1986, 1996 and 2001. The wavelength dispersive X-ray fluorescence (WD-XRF) technique was adopted to determine the concentrations of Ba, Br, Ca, Cl, Cr, Cu, Fe, K, Mg, Mn, Ni, Pb, S, Ti and Zn. A smaller number of samples was also analysed by ICP atomic emission spectroscopy (ICP-AES) and the results were compared with those obtained by WD-XRF to confirm their validity. A clear seasonal pattern with higher concentrations of the aforementioned elements in the cold periods was observed. Moreover, a change in the chemical composition of atmospheric particulate matter was evidenced, particularly between the first (1976 and 1986) and the last (1996 and 2001) years. This change can be attributed both to the greater contribution of Pb and Br to atmospheric pollution in the past and, in recent years, to the higher level of pollutants associated with increased vehicular traffic and industrial activities. The application of chemometric techniques (Principal Component Analysis and Cluster Analysis) allowed us to speculate about the main emitting sources influencing the total atmospheric particulate in these years.

Direct patterning of silver particles on porous silicon by inkjet printing of a silver salt via in-situ reduction.

We have developed a method for obtaining a direct pattern of silver nanoparticles (NPs) on porous silicon (p-Si) by means of inkjet printing (IjP) of a silver salt. Silver NPs were obtained by p-Si mediated in-situ reduction of Ag+ cations using solutions based on AgNO3 which were directly printed on p-Si according to specific geometries and process parameters. The main difference with respect to existing literature is that normally, inkjet printing is applied to silver (metal) NP suspensions, while in our experiment the NPs are formed after jetting the solution on the reactive substrate. We performed both optical and scanning electron microscopes on the NPs traces, correlating the morphology features with the IjP parameters, giving an insight on the synthesis kinetics. The patterned NPs show good performances as SERS substrates.

Switching of fluorescence mediated by a peroxynitrite-glutathione redox reaction in a porous silicon nanoreactor.

A nanostructured porous silicon chip functionalized with dichlorofluorescein is employed as a nanoreactor to respond to Reactive Oxygen Species (ROS) and to real-time studying redox reactions.

New-generation curing units and short irradiation time: the degree of conversion of microhybrid composite resin.

This in vitro study investigated the depth of cure of a microhybrid composite resin when cured with reduced times of exposure to three commercially available curing lights. Different sample thicknesses (1, 2, and 3 mm) were light cured in high intensity polymerization mode (2,400 mW/cm² for 5, 10, 15, and 20 seconds; 1,100 mW/cm² for 10, 20, 30, and 40 seconds; and 1,100 mW/cm² for 10, 20, 30, and 40 seconds, respectively). The degree of conversion (%) at the bottom of each sample was measured by Attenuated Total Reflection Fourier Transform Infrared (ATR F-TIR) analysis after each polymerization step. Data were analyzed by ANOVA for repeated measures, showing the degree of conversion was not influenced by the curing light employed (P = .622) but was significantly influenced by the thickness of composite resin (P < .05). Variations in the degree of conversion vs the shorter irradiation time permitted (T1) were not significant among different lamps but were significant among different thicknesses. The depth of cure of microhybrid composite resin appears not to be influenced by the curing light employed. Increased irradiation time significantly increases the degree of conversion. Thickness strongly influences depth of cure.