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.

Increase in newly diagnosed type 1 diabetes and serological evidence of recent SARS-CoV-2 infection: Is there a connection?

Several studies have investigated the correlation between the COVID-19 pandemic and the onset of type 1 diabetes (T1D) in children, reporting an increased incidence of T1D and severe diabetic ketoacidosis (DKA). This study aimed to investigate the infection by SARS-CoV-2 in children with newly-diagnosed T1D to explore a possible link between SARS-CoV-2 infection, T1D and DKA. Thirty-nine children with a T1D new onset between October 15, 2020, and April 15, 2021, were enrolled. SARS-CoV-2 infection was investigated through a polymerase chain reaction on the nasal swab, dosage of specific antibodies, and an anamnestic question form. Nine (23%) of them had antibodies directed toward SARS-CoV-2, and five (12%) had a history of recent SARS-CoV-2 infection in themselves or in their family. No molecular swabs were positive. Compared to the general pediatric population, the overall incidence of COVID-19 was 5.6 times higher in the T1D patients' group (p < 0.00001). Referring only to the cases in the metropolitan area, we find a net increase in the incidence of T1D compared to the 5 years preceding our study, by 50% compared to the same months in 2016/2017 and 2017/2018, by 69% compared to 2018/2019 and by 77% compared to 2019/2020. The same trend was observed regarding DKA cases. The attributable risk of the pandemic cohort compared to the previous year is 44%. The abnormal disproportion of SARS-CoV-2 infection between children with T1D and the pediatric reference population, with a ratio of 5.6, appears to support the causative role of SARS-CoV-2 in triggering the immune response underlying diabetes, as often described for other viral infections. The difficulty accessing care services during the pandemic, with a consequent diagnosis delay, does not justify the increase in observed T1D cases, which could to be directly linked to the pandemic. The acceleration of the immune process provoked by SARS-CoV-2 may play a suggestive role in the development of T1D with DKA. Multicenter studies are needed to deepen and fully understand the pathophysiological link between SARS-CoV-2 and the onset of T1D in children.

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.

Bispyrene Functionalization Drives Self-Assembly of Graphite Nanoplates into Highly Efficient Heat Spreader Foils.

Thermally conductive nanopapers fabricated from graphene and related materials are currently showing great potential in thermal management applications. However, thermal contacts between conductive plates represent the bottleneck for thermal conductivity of nanopapers prepared in the absence of a high temperature step for graphitization. In this work, the problem of ineffective thermal contacts is addressed by the use of bifunctional polyaromatic molecules designed to drive self-assembly of graphite nanoplates (GnP) and establish thermal bridges between them. To preserve the high conductivity associated to a defect-free sp2 structure, non-covalent functionalization with bispyrene compounds, synthesized on purpose with variable tethering chain length, was exploited. Pyrene terminal groups granted for a strong π-π interaction with graphene surface, as demonstrated by UV-Vis, fluorescence, and Raman spectroscopies. Bispyrene molecular junctions between GnP were found to control GnP organization and orientation within the nanopaper, delivering significant enhancement in both in-plane and cross-plane thermal diffusivities. Finally, nanopapers were validated as heat spreader devices for electronic components, evidencing comparable or better thermal dissipation performance than conventional Cu foil, while delivering over 90% weight reduction.

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.

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.

Osteoporosis-related variations of trabecular bone properties of proximal human humeral heads at different scale lengths.

Osteoporosis (OP) is a skeletal disorder responsible for the weakening of the bone structure and, consequently, for an increased fracture risk in the elderly population. In the past, bone mineral density (BMD) variation was considered the best OP indicator, but recently the focus has shifted toward the variation of microstructural bone parameters. This work is based on the characterisation of 8-mm cylindrical biopsies harvested from proximal humeral heads belonging to healthy and osteoporotic patients, in order to assess the OP-related variations of bone properties at different scale lengths. In particular, bone biopsies underwent micro-computed tomography analysis to study the most relevant features of bone architecture and extrapolate the tissue mineral density (TMD) value of bone trabeculae. Compression tests and nanoindentations were performed to investigate the macro- and micromechanical properties of bone biopsies, respectively. In addition, XRD analyses were performed to obtain the mean hydroxyapatite (HA) crystallite size, while Raman spectroscopy investigated the collagen secondary structure. Thermogravimetric analysis was performed to evaluate the ratio between organic and inorganic phases. From the obtained results, OP samples showed a more anisotropic and less interconnected structure responsible for reduced compression strength. From this, it can be supposed that OP caused an alteration of bone structure that led to inferior macroscopic mechanical properties. Furthermore, OP samples possessed higher TMD and bigger HA crystals that are correlated to an increase of the hardness value obtained by means of nanoindentation. This less controlled HA crystal growth is probably due to an alteration of the organic matrix structure, as revealed by the increase of the random coil contribution in the Raman spectra of the OP bone. This higher crystal content led to an increase in trabecular density and hardness. In conclusion, the obtained data showed that OP affects bone properties at different scale lengths causing an alteration of its morphological, structural and mechanical features.

In situ Raman analyses of the soot oxidation reaction over nanostructured ceria-based catalysts.

To reduce the emissions of internal combustion engines, ceria-based catalysts have been widely investigated as possible alternatives to the more expensive noble metals. In the present work, a set of four different ceria-based materials was prepared via hydrothermal synthesis, studying the effect of Cu and Mn as dopants both in binary and ternary oxides. In situ Raman analyses were carried out to monitor the behaviour of defect sites throughout thermal cycles and during the soot oxidation reaction. Despite ceria doped with 5% of Cu featured the highest specific surface area, reducibility and amount of intrinsic and extrinsic defects, a poor soot oxidation activity was observed through the standard activity tests. This result was confirmed by the calculation of soot conversion curves obtained through a newly proposed procedure, starting from the Raman spectra collected during the in situ tests. Moreover, Raman analyses highlighted that new defectiveness was produced on the Cu-doped catalyst at high temperature, especially after soot conversion, while a slight increase of the defect band and a total reversibility were observed in case of the ternary oxide and pure/Mn-doped ceria, respectively. The major increment was related to the extrinsic defects component; tests carried out in different atmospheres suggested the assignment of this feature to vacancy-free sites containing oxidized doping cations. Its increase at the end of the tests can be an evidence of peroxides and superoxides deactivation on catalysts presenting excessive oxygen vacancy concentrations. Instead, ceria doped with 5% of Mn exhibited the best soot oxidation activity, thanks to an intermediate density of oxygen vacancies and to its well-defined morphology.

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.

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.

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.

Catalytic Oxidation of CO and Soot over Ce-Zr-Pr Mixed Oxides Synthesized in a Multi-Inlet Vortex Reactor: Effect of Structural Defects on the Catalytic Activity.

In the present work, ceria, ceria-zirconia (Ce = 80 at.%, Zr = 20 at.%), ceria praseodymia (Ce = 80 at.%, Pr = 20 at.%) and ceria-zirconia-praseodymia catalysts (Ce = 80 at.%, Zr = 10 at.% and Pr = 10 at.%) have been prepared by the multi-inlet vortex reactor (MIVR). For each set of samples, two inlet flow rates have been used during the synthesis (namely, 2 ml min-1, and 20 ml min-1) in order to obtain different particle sizes. Catalytic activity of the prepared materials has been investigated for CO and soot oxidation reactions. As a result, when the catalysts exhibit similar crystallite sizes (in the 7.7-8.8 nm range), it is possible to observe a direct correlation between the Ov/F2g vibrational band intensity ratios and the catalytic performance for the CO oxidation. This means that structural (superficial) defects play a key role for this process. The incorporation of Zr and Pr species into the ceria lattice increases the population of structural defects, as measured by Raman spectroscopy, according to the order: CeO2 < Ce80Zr20 < Ce80Zr10Pr10 < Ce80Pr20. On the other hand, the presence of zirconium and praseodymium into the ceria lattice does not have a direct beneficial effect on the soot oxidation activity for these catalysts, in contrast with nanostructured ones (e.g., Ce-Zr-O nanopolyhedra, Ce-Pr-O nanocubes) described elsewhere (Andana et al. Appl. Catal. B 197: 125-137, 2016; Piumetti et al., Appl Catal B 180: 271-282, 2016).

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.

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.

The interaction of H2O2 with TiAlPO-5 molecular sieves: probing the catalytic potential of framework substituted Ti ions.

A combination of EPR, ENDOR and HYSCORE experiments has been used to investigate the reactivity of hydrogen peroxide with TiAlPO-5 materials under both hydrated and anhydrous conditions. Superoxide radical anions, generated upon reaction, are used as paramagnetic probes to investigate the nature and reactivity of framework incorporated Ti ions. Super hyperfine interactions with (27)Al, (31)P and (1)H are resolved, which allow a detailed mapping of the local environment of the adsorbed O2(-) ions. Evidence is provided for the first time of a specific redox activity associated with Ti ions incorporated at framework Al sites of TiAlPO materials.