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.

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.

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.

Leakage radiation interference microscopy.

We present a proof of principle for a new imaging technique combining leakage radiation microscopy with high-resolution interference microscopy. By using oil immersion optics it is demonstrated that amplitude and phase can be retrieved from optical fields, which are evanescent in air. This technique is illustratively applied for mapping a surface mode propagating onto a planar dielectric multilayer on a thin glass substrate. The surface mode propagation constant estimated after Fourier transformation of the measured complex field is well matched with an independent measurement based on back focal plane imaging.

A fluorescent one-dimensional photonic crystal for label-free biosensing based on BLOCH surface waves.

A one-dimensional photonic crystal (1DPC) based on a planar stack of dielectric layers is used as an optical transducer for biosensing, upon the coupling of TE-polarized Bloch Surface Waves (BSW). The structure is tailored with a polymeric layer providing a chemical functionality facilitating the covalent binding of orienting proteins needed for a subsequent grafting of antibodies in an immunoassay detection scheme. The polymeric layer is impregnated with Cy3 dye, in such a way that the photonic structure can exhibit an emissive behavior. The BSW-coupled fluorescence shift is used as a means for detecting refractive index variations occurring at the 1DPC surface, according to a label-free concept. The proposed working principle is successfully demonstrated in real-time tracking of protein G covalent binding on the 1DPC surface within a fluidic cell.

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.

A polymer-based functional pattern on one-dimensional photonic crystals for photon sorting of fluorescence radiation.

In this work we introduce the use of a patterned polymer-based surface functionalization of a one-dimensional photonic crystal (1DPC) for controlling the emission direction of fluorescent proteins (ptA) via coupling to a set of two Bloch Surface Waves (BSW). Each BSW dispersion branch relates to a micrometric region on the patterned 1DPC, characterized by a well defined chemical characteristic. We report on the enhanced and spatially selective excitation of fluorescent ptA, and on the spatially-resolved detection of polarized emitted radiation coupled to specific BSW modes. As a result, we provide an optical multiplexing technique for the angular separation of fluorescence radiated from micrometric regions having different surface properties, even in the case the emitting labels are spectrally identical. This working principle can be advantageously extended to a multi-step nanometric relief structure for self-referencing biosensing or frequency-multiplexed fluorescence detection.

Guided Bloch surface waves on ultrathin polymeric ridges.

We present a direct evidence of Bloch surface waves (BSWs) waveguiding on ultrathin polymeric ridges, supported by near-field measurements. It is demonstrated that near-infrared BSWs sustained by a silicon-based multilayer can be locally coupled and guided through dielectric ridges of nanometric thickness with low propagation losses. Using a conventional prism-based configuration, we demonstrate a wavelength-selective BSW coupling inside and outside the ridge. Such a result can open interesting opportunities in surface wave-mediated sensing applications, where light could be selectively coupled in specific regions defined by nanometric reliefs.

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.

Reverse Micelle Strategy for the Synthesis of MnO x -TiO2 Active Catalysts for NH3-Selective Catalytic Reduction of NO x at Both Low Temperature and Low Mn Content.

MnO x -TiO2 catalysts (0, 1, 5, and 10 wt % Mn nominal content) for NH3-SCR (selective catalytic reduction) of NO x have been synthesized by the reverse micelle-assisted sol-gel procedure, with the aim of improving the dispersion of the active phase, usually poor when obtained by other synthesis methods (e.g., impregnation) and thereby lowering its amount. For comparison, a sample at nominal 10 wt % Mn was obtained by impregnation of the (undoped) TiO2 sample. The catalysts were characterized by using an integrated multitechnique approach, encompassing X-ray diffraction followed by Rietveld refinement, micro-Raman spectroscopy, N2 isotherm measurement at -196 °C, energy-dispersive X-ray analysis, diffuse reflectance UV-vis spectroscopy, temperature-programmed reduction technique, and X-ray photoelectron spectroscopy. The obtained results prove that the reverse micelle sol-gel approach allowed for enhancing the catalytic activity, in that the catalysts were active in a broad temperature range at a substantially low Mn loading, as compared to the impregnated catalyst. Particularly, the 5 wt % Mn catalyst showed the best NH3-SCR activity in terms of both NO x conversion (ca. 90%) and the amount of produced N2O (ca. 50 ppm) in the 200-250 °C temperature range.

Experimental determination of the sensitivity of Bloch surface waves based sensors.

Detection of glucose in water solution for several different concentrations has been performed with the purpose to determine the sensitivity of Near Infrared Bloch Surface Waves (lambda = 1.55microm) upon refractive index variations of the outer medium. TE-polarized electromagnetic surface waves are excited by a prism on a silicon nitride multilayer, according to the Kretschmann configuration. The real-time reflectance changes induced by discrete variations in glucose concentration has been revealed and analyzed. Without using any particular averaging strategy during the measurements, we pushed the device detection limit down to a glucose concentration of 2.5mg/dL, corresponding to a minimum detectable refractive index variation of the water solution as low as 3.8.10(-6).

Fast optical vapour sensing by Bloch surface waves on porous silicon membranes.

The coupling of optical Bloch surface waves at the truncated end of one dimensional porous silicon photonic crystals is exploited for fast vapour sensing. Self-standing multilayered membranes bound to transparent substrates were fabricated by electrochemical etching and used in an attenuated total reflection configuration to resonantly excite the surface waves and perform real-time sensing.

High resolution capabilities of all-silica cantilevered probes for near-field optical microscopy.

We report on the possibility of performing Near-field Scanning Optical Microscopy in illumination mode by means of microfabricated, metal-coated silica probes based on transparent cantilevers. A low spring constant silica cantilever hosts a silica tip at its end showing an hyperbolic profile and a circular symmetry. After evaporation of 100 nm of aluminium on the tip and the cantilever we processed the tip apex by means of a FIB, thus obtaining either a probe apex with an optical aperture or an apertureless probe having a thin metal layer on the top. An excellent quality of near-field images of samples showing sub-wavelength features is obtained in both case. In particular, the apertureless probe allows highly resolved topographical and optical images to be collected at the same time. This work further demonstrates that the use of completely transparent, metal-coated cantilevers greatly simplify the light injection into the probe and the fabrication process consequently.

Tips and Tricks for the Surface Engineering of Well-Ordered Morphologically Driven Silver-Based Nanomaterials.

Particularly-shaped silver nanostructures are successfully applied in many scientific fields, such as nanotechnology, catalysis, (nano)engineering, optoelectronics, and sensing. In recent years, the production of shape-controlled silver-based nanostructures and the knowledge around this topic has grown significantly. Hence, on the basis of the most recent results reported in the literature, a critical analysis around the driving forces behind the synthesis of such nanostructures are proposed herein, pointing out the important role of surface-regulating agents in driving crystalline growth by favoring (or opposing) development along specific directions. Additionally, growth mechanisms of the different morphologies considered here are discussed in depth, and critical points highlighted.

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.

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.

Synthesis of thermoelectric magnesium-silicide pastes for 3D printing, electrospinning and low-pressure spray.

In this work, eco-friendly magnesium-silicide (Mg2Si) semiconducting (n-type) thermoelectric pastes for building components concerning energy-harvesting devices through 3D printing, spray and electrospinning were synthetized and tested for the first time. The Mg2Si fine powders were obtained through the combination of ball milling and thermal annealing under Ar atmosphere. While the latter process was crucial for obtaining the desired Mg2Si phase, the ball milling was indispensable for homogenizing and reducing the grain size of the powders. The synthetized Mg2Si powders exhibited a large Seebeck coefficient of ~ 487 µV/K and were blended with a polymeric solution in different mass ratios to adjust the paste viscosity to the different requirements of 3D printing, electrospinning and low-pressure spray. The materials produced in every single stage of the paste synthesis were characterized by a variety of techniques that unequivocally prove their viability for producing thermoelectric parts and components. These can certainly trigger further research and development in green thermoelectric generators (TEGs) capable of adopting any form or shape with enhanced thermoelectric properties. These green TEGs are meant to compete with common toxic materials such as Bi2Te3, PbTe and CoSb that have Seebeck coefficients in the range of ~ 290-700 µV/K, similar to that of the produced Mg2Si powders and lower than that of 3D printed bulk Mg2Si pieces, measured to be ~ 4866 µV/K. Also, their measured thermal conductivities proved to be significantly lower (~ 0.2 W/mK) than that reported for Mg2Si (≥ 4 W/mK). However, it is herein demonstrated that such thermoelectric properties are not stable over time. Pressureless sintering proved to be indispensable, but difficultly achievable by long thermal annealing (even above 32 h) in inert atmosphere at 400 °C, at least for bulk Mg2Si pieces constituted by a mean grain size of 2-3 µm. Hence, for overcoming this sintering challenge and become the silicide's extrusion viable in the production of bulk thermoelectric parts, alternative pressureless sintering methods will have to be further explored.