Molecular beacon decorated silver nanowires for quantitative miRNA detection by a SERS approach.

Advantages of biosensors based on surface enhanced Raman scattering (SERS) rely on improved sensitivity and specificity, and suited reproducibility in detecting a target molecule that is localized in close proximity to a SERS-active surface. Herein, a comprehensive study on the realization of a SERS biosensor designed for detecting miRNA-183, a miRNA biomarker that is specific for chronic obstructive pulmonary disease (COPD), is presented. The used strategy exploits a signal-off mechanism by means of a labelled molecular beacon (MB) as the oligonucleotide biorecognition element immobilized on a 2D SERS substrate, based on spot-on silver nanowires (AgNWs) and a multi-well low volume cell. The MB was properly designed by following a dedicated protocol to recognize the chosen miRNA. A limit of detection down to femtomolar concentration (3 × 10-16 M) was achieved and the specificity of the biosensor was proved. Furthermore, the possibility to regenerate the sensing system through a simple procedure is shown: with regeneration by using HCl 1 mM, two detection cycles were performed with a good recovery of the initial MB signal (83%) and a reproducible signal after hybridization.

Impact of seed amplification assay and surface-enhanced Raman spectroscopy combined approach on the clinical diagnosis of Alzheimer's disease.

BACKGROUND: The current diagnosis of Alzheimer's disease (AD) is based on a series of analyses which involve clinical, instrumental and laboratory findings. However, signs, symptoms and biomarker alterations observed in AD might overlap with other dementias, resulting in misdiagnosis. METHODS: Here we describe a new diagnostic approach for AD which takes advantage of the boosted sensitivity in biomolecular detection, as allowed by seed amplification assay (SAA), combined with the unique specificity in biomolecular recognition, as provided by surface-enhanced Raman spectroscopy (SERS). RESULTS: The SAA-SERS approach supported by machine learning data analysis allowed efficient identification of pathological Aß oligomers in the cerebrospinal fluid of patients with a clinical diagnosis of AD or mild cognitive impairment due to AD. CONCLUSIONS: Such analytical approach can be used to recognize disease features, thus allowing early stratification and selection of patients, which is fundamental in clinical treatments and pharmacological trials.

Raman Spectroscopy Techniques for the Investigation and Diagnosis of Alzheimer's Disease.

Alzheimer's disease (AD) is the most common neurodegenerative disorder, resulting in memory loss, cognitive decline, bodily function impairment, and finally death. The growing number of people suffering from AD increasingly urges the development of effective early diagnosis and monitoring techniques. Here, we review the most recent developments in the field of Raman-based techniques, which have shown a significant potential in identifying AD by detecting specific biomarkers in biological fluids, as well as in providing fundamental insights into key molecules involved in the disease progression or in the analysis of histological specimens of patients with AD. These techniques comprise spontaneous and resonant Raman spectroscopies, exploit plasmon- or fiber- enhanced effects, such as surface-, tip- or fiber- enhanced Raman spectroscopies, or involve non-linear techniques like coherent Raman scattering. The scientific efforts employed up to now as well as the rapid technological advancements in optical detection instruments (spectrometers, lasers, substrates for analysis, etc.) and the diffusion of advanced data processing methods suggest a leading role of Raman techniques in the perspective of a preclinical or clinical detection of AD.

Ion-exchanged glass microrods as hybrid SERS/fluorescence substrates for molecular beacon-based DNA detection.

Ion-exchange in molten nitrate salts containing metal ions (i.e. silver, copper, etc.) represents a well-established technique able to modify the chemical-physical properties of glass materials. It is widely used not only in the field of integrated optics (IO) but also, more recently, in plasmonics due to the possibility to induce the formation of metal nanoparticles in the glass matrix by an ad hoc thermal post-process. In this work, the application of this technology for the realisation of low-cost and stable surface-enhanced Raman scattering (SERS) active substrates, based on soda-lime glass microrods, is reported. The microrods, with a radius of a few tens of microns, were obtained by cutting the end of an ion-exchanged soda-lime fibre for a length less than 1 cm. As ion source, silver nitrate was selected due to the outstanding SERS properties of silver. The ion-exchange and thermal annealing post-process parameters were tuned to expose the embedded silver nanoparticles on the surface of the glass microrods, avoiding the use of any further chemical etching step. In order to test the combined SERS/fluorescence response of these substrates, labelled molecular beacons (MBs) were immobilised on their surface for deoxyribonucleic acid (DNA) detection. Our experiments confirm that target DNA is attached on the silver nanoparticles and its presence is revealed by both SERS and fluorescence measurements. These results pave the way towards the development of low-cost and stable hybrid fibres, in which SERS and fluorescence interrogation techniques are combined in the same optical device.

Cost Effective Silver Nanowire-Decorated Graphene Paper for Drop-On SERS Biodetection.

The use of SERS for real-world bioanalytical applications represents a concrete opportunity, which, however, is being largely delayed by the inadequacy of existing substrates used to collect SERS spectra. In particular, the main bottleneck is their poor usability, as in the case of unsupported noble metal colloidal nanoparticles or because of the need for complex or highly specialized fabrication procedures, especially in view of a large-scale commercial diffusion. In this work, we introduce a graphene paper-supported plasmonic substrate for biodetection as obtained by a simple and rapid aerosol deposition patterning of silver nanowires. This substrate is compatible with the analysis of small (2 µL) analyte drops, providing stable SERS signals at sub-millimolar concentration and a detection limit down to the nanogram level in the case of hemoglobin. The presence of a graphene underlayer assures an even surface distribution of SERS hotspots with improved stability of the SERS signal, the collection of well-resolved and intense SERS spectra, and an ultra-flat and photostable SERS background in comparison with other popular disposable supports.

Label-free SERS detection of proteins based on machine learning classification of chemo-structural determinants.

Establishing standardized methods for a consistent analysis of spectral data remains a largely underexplored aspect in surface-enhanced Raman spectroscopy (SERS), particularly applied to biological and biomedical research. Here we propose an effective machine learning classification of protein species with closely resembled spectral profiles by a mixed data processing based on principal component analysis (PCA) applied to multipeak fitting on SERS spectra. This strategy simultaneously assures a successful discrimination of proteins and a thorough characterization of the chemostructural differences among them, ultimately opening up new routes for SERS evolution toward sensing applications and diagnostics of interest in life sciences.

Hollow core photonic crystal fiber-assisted Raman spectroscopy as a tool for the detection of Alzheimer's disease biomarkers.

SIGNIFICANCE: Alzheimer's disease (AD) is an irreversible and progressive disorder that damages brain cells and impairs the cognitive abilities of the affected. Developing a sensitive and cost-effective method to detect Alzheimer's biomarkers appears vital in both a diagnostic and therapeutic perspective. AIM: Our goal is to develop a sensitive and reliable tool for detection of amyloid ß (1-42) peptide (Aß42), a major AD biomarker, using fiber-enhanced Raman spectroscopy (FERS). APPROACH: A hollow core photonic crystal fiber (HCPCF) was integrated with a conventional Raman spectroscopic setup to perform FERS measurements. FERS was then coupled with surface-enhanced Raman spectroscopy (SERS) to further amplify the Raman signal thanks to a combined FERS-SERS assay. RESULTS: A minimum 20-fold enhancement of the Raman signal of Aß42 as compared to a conventional Raman spectroscopy scheme was observed using the HCPCF-based light delivery system. The signal was further boosted by decorating the fiber core with gold bipyramids generating an additional SERS effect, resulting in an overall 200 times amplification. CONCLUSIONS: The results demonstrate that the use of an HCPCF-based platform can provide sharp and intense Raman signals of Aß42, in turn paving the way toward the development of a sensitive label-free detection tool for early diagnosis of AD.

Nanoscopic insights into the surface conformation of neurotoxic amyloid ß oligomers.

Raman spectroscopy assisted by localized plasmon resonances generating effective hot spots at the gaps between intertwined silver nanowires is herein adopted to unravel characteristic molecular motifs on the surface of Aß42 misfolded oligomers that are critical in driving intermolecular interactions in neurodegeneration.

Biosensor surface functionalization by a simple photochemical immobilization of antibodies: experimental characterization by mass spectrometry and surface enhanced Raman spectroscopy.

Surface functionalization is a key step in biosensing since it is the basis of an effective analyte recognition. Among all the bioreceptors, antibodies (Abs) play a key role thanks to their superior specificity, although the available immobilization strategies suffer from several drawbacks. When gold is the interacting surface, the recently introduced Photochemical Immobilization Technique (PIT) has been shown to be a quick, easy-to-use and very effective method to tether Abs oriented upright by means of thiols produced via tryptophan mediated disulphide bridge reduction. Although the molecular mechanism of this process is quite well identified, the detailed morphology of the immobilized antibodies is still elusive due to inherent difficulties related to the microscopy imaging of Abs. The combination of Mass Spectrometry, Surface-Enhanced Raman Spectroscopy and Ellman's assay demonstrates that Abs irradiated under the conditions in which PIT is realized show only two effective disulphide bridges available for binding. They are located in the constant region of the immunoglobulin light chain so that the most likely position Ab assumes is side-on, i.e. with one Fab (i.e. the antigen binding portion of the antibody) exposed to the solution. This is not a limitation of the recognition efficiency in view of the intrinsic flexibility of the Ab structure, which makes the free Fab able to sway in the solution, a feature of great importance in many biosensing applications.

Seeding variability of different alpha synuclein strains in synucleinopathies.

OBJECTIVES: Currently, the exact reasons why different α-synucleinopathies exhibit variable pathologies and phenotypes are still unknown. A potential explanation may be the existence of distinctive α-synuclein conformers or strains. Here, we intend to analyze the seeding activity of dementia with Lewy bodies (DLB) and Parkinson's disease (PD) brain-derived α-synuclein seeds by real-time quaking-induced conversion (RT-QuIC) and to investigate the structure and morphology of the α-synuclein aggregates generated by RT-QuIC. METHODS: A misfolded α-synuclein-enriched brain fraction from frontal cortex and substantia nigra pars compacta tissue, isolated by several filtration and centrifugation steps, was subjected to α-synuclein/RT-QuIC analysis. Our study included neuropathologically well-characterized cases with DLB, PD, and controls (Ctrl). Biochemical and morphological analyses of RT-QuIC products were conducted by western blot, dot blot analysis, Raman spectroscopy, atomic force microscopy, and transmission electron microscopy. RESULTS: Independently from the brain region, we observed different seeding kinetics of α-synuclein in the RT-QuIC in patients with DLB compared to PD and Ctrl. Biochemical characterization of the RT-QuIC product indicated the generation of a proteinase K-resistant and fibrillary α-synuclein species in DLB-seeded reactions, whereas PD and control seeds failed in the conversion of wild-type α-synuclein substrate. INTERPRETATION: Structural variances of α-synuclein seeding kinetics and products in DLB and PD indicated, for the first time, the existence of different α-synuclein strains in these groups. Therefore, our study contributes to a better understanding of the clinical heterogeneity among α-synucleinopathies, offers an opportunity for a specific diagnosis, and opens new avenues for the future development of strain-specific therapies. Ann Neurol 2019;85:691-703.

Nanoscale Discrimination between Toxic and Nontoxic Protein Misfolded Oligomers with Tip-Enhanced Raman Spectroscopy.

Highly toxic protein misfolded oligomers associated with neurological disorders such as Alzheimer's and Parkinson's diseases are nowadays considered primarily responsible for promoting synaptic failure and neuronal death. Unraveling the relationship between structure and neurotoxicity of protein oligomers appears pivotal in understanding the causes of the pathological process, as well as in designing novel diagnostic and therapeutic strategies tuned toward the earliest and presymptomatic stages of the disease. Here, it is benefited from tip-enhanced Raman spectroscopy (TERS) as a surface-sensitive tool with spatial resolution on the nanoscale, to inspect the spatial organization and surface character of individual protein oligomers from two samples formed by the same polypeptide sequence and different toxicity levels. TERS provides direct assignment of specific amino acid residues that are exposed to a large extent on the surface of toxic species and buried in nontoxic oligomers. These residues, thanks to their outward disposition, might represent structural factors driving the pathogenic behavior exhibited by protein misfolded oligomers, including affecting cell membrane integrity and specific signaling pathways in neurodegenerative conditions.

Triggering molecular assembly at the mesoscale for advanced Raman detection of proteins in liquid.

An advanced optofluidic system for protein detection based on Raman signal amplification via dewetting and molecular gathering within temporary mesoscale assemblies is presented. The evaporation of a microliter volume of protein solution deposited in a circular microwell precisely follows an outward-receding geometry. Herein the combination of liquid withdrawal with intermolecular interactions induces the formation of self-assembled molecular domains at the solid-liquid interface. Through proper control of the evaporation rate, amplitude of the assemblies and time for spectral collection at the liquid edge are extensively raised, resulting in a local enhancement and refinement of the Raman response, respectively. Further signal amplification is obtained by taking advantage of the intense local electromagnetic fields generated upon adding a plasmonic coating to the microwell. Major advantages of this optofluidic method lie in the obtainment of high-quality, high-sensitivity Raman spectra with detection limit down to sub-micromolar values. Peculiarly, the assembled proteins in the liquid edge region maintain their native-like state without displaying spectral changes usually occurring when dried drop deposits are considered.

Site-Selective Surface-Enhanced Raman Detection of Proteins.

Strategies for protein detection via surface-enhanced Raman spectroscopy (SERS) currently exploit the formation of randomly generated hot spots at the interfaces of metal colloidal nanoparticles, which are clustered together by intrusive chemical or physical processes in the presence of the target biomolecule. We propose a different approach based on selective and quantitative gathering of protein molecules at regular hot spots generated on the corners of individual silver nanocubes in aqueous medium at physiological pH. Here, the protein, while keeping its native configuration, experiences an intense local E-field, which boosts SERS efficiency and detection sensitivity. Uncontrolled signal fluctuations caused by variable molecular adsorption to different particle areas or inside clustered nanoparticles are circumvented. Advanced electron microscopy analyses and computational simulations outline a strategy relying on a site-selective mechanism with superior Raman signal enhancement, which offers the perspective of highly controlled and reproducible routine SERS detection of proteins.

Controlled Veiling of Silver Nanocubes with Graphene Oxide for Improved Surface-Enhanced Raman Scattering Detection.

Hybrid graphene oxide (GO)/metal nanocomposites have been recently proposed as novel surface-enhanced Raman scattering (SERS) substrates. Despite an increasing interest in these systems, standardization in their fabrication process is still lacking but urgently required to support their use for real-life applications. In this work we investigate how the assembly of GO should be conducted to control adsorption geometry and optical properties at the interface with plasmonic nanostructures as monolayer assemblies of silver nanocubes, by tuning main experimental parameters including GO concentration and self-assembly time. We finally identified the experimental conditions for building up a close-fitting soft dressing of the plasmonic surface, which shows optimal characteristics for flexible and reliable SERS detection.

Concave gold nanocube assemblies as nanotraps for surface-enhanced Raman scattering-based detection of proteins.

SERS detection of proteins is typically performed by using labeling agents with stable and high Raman scattering cross sections. This is a valuable approach for trace detection and quantification of a target protein but is unsuitable for inspecting its inherent structural and functional properties. On the other hand, direct SERS of proteins has been mainly devoted to the study of short peptides and aminoacid sequences or of prosthetic groups with intense Raman signals, which is of scarce interest for a thorough characterization of most proteins. Here we try to overcome these limitations by setting-up an effective platform for the structural SERS analysis of proteins. The platform consists of an extended bidimensional array of gold concave nanocubes (CNCs) supported on a PDMS film. CNCs are closely-packed through face-face and face-corner interactions generating a monolayered arrangement featuring well distributed nanoholes. Here the protein homogeneously experiences an E-field enhancement outward from the metal surfaces surrounding it, which causes a large number of vibrations to be contemporarily amplified. The proposed platform provides stable and detailed SERS spectra and confers rapidity and reproducibility to the analysis.

Thermal transitions of fibrillar collagen unveiled by second-harmonic generation microscopy of corneal stroma.

The thermal transitions of fibrillar collagen are investigated with second-harmonic generation polarization anisotropy microscopy. Second-harmonic generation images and polarization anisotropy profiles of corneal stroma heated in the 35-80°C range are analyzed by means of a theoretical model that is suitable to probe principal intramolecular and interfibrillar parameters of immediate physiological interest. Our results depict the tissue modification with temperature as the interplay of three destructuration stages at different hierarchical levels of collagen assembly including its tertiary structure and interfibrillar alignment, thus supporting and extending previous findings. This method holds the promise of a quantitative inspection of fundamental biophysical and biochemical processes and may find future applications in real-time and postsurgical functional imaging of collagen-rich tissues subjected to thermal treatments.

Hybrid nanocomposite films for laser-activated tissue bonding.

We report new advancements in the biomedical exploitation of plasmonic nanoparticles as an effective platform for the photothermal repair of biological tissue. Chitosan films are loaded with gold nanorods with intense optical absorption in the "therapeutic window" of deepest light penetration through the body, and then activated by near infrared laser excitation to give adhesion with adjacent connective tissues. The adhesion consists of 0.07 mm(2) welds of ~20 kPa tensile strength at the film/tissue interface, which are obtained by administration of pulses with duration in the hundreds of millisecond timescale from a diode laser at ~130 J cm(-2). We investigate the adhesive effect as a function of pulse power and duration and identify an optimal operative window to achieve effective and reproducible welds with minimal detrimental superheating. These results may prove valuable to standardize laser bonding techniques and meet current needs for new knowledge which is urged by the penetration of nanotechnology into biomedical optics.

In-situ visualization, monitoring and analysis of electric field domain reversal process in ferroelectric crystals by digital holography.

In-situ monitoring of domain reversal in congruent lithium niobate by a digital holographic technique is described. While the ferroelectric polarization is reversed by electric field poling, the two-dimensional distribution of the phase shift, due mainly to the linear electro-optic and piezoelectric effects, is measured and visualized. Digital holography is used to reconstruct both amplitude and phase of the wavefield transmitted by the sample to reveal the phase shift induced by adjacent reversed domains during the poling. The resulting movies of both amplitude and phase maps, for in-situ visualization of domain pattern formation, are shown. The possibility of using the technique as tool for monitoring in real-time the periodic poling of patterned samples is discussed.