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.

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.

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.

Decoration of silicon nanowires with silver nanoparticles for ultrasensitive surface enhanced Raman scattering.

Silicon nanowires (Si NWs), produced by the chemical etching technique, were decorated with silver nanoparticles (NPs) produced at room temperature by the pulsed laser deposition (PLD) technique. Silver NPs were obtained by means of nanosecond pulsed laser ablation of a target in the presence of a controlled Ar atmosphere. Two different laser pulse numbers and Si NWs having different lengths were used to change the NP number density on the Si NW surface. The resulting Ag NP morphologies were studied by scanning electron microscopy imaging. The results show that this industrially compatible technological approach allows the coverage of the Si NW walls with Ag NPs with a strong control of the NP size distribution and spatial arrangement. The obtained Ag NP decorated Si NWs are free from chemicals contamination and there is no need of post deposition high temperature processes. The optical properties of Si NW arrays were investigated by reflectance spectroscopy that showed the presence of a plasmon related absorption peak, whose position and width is dependent on the Ag NP surface morphology. Coupling the huge surface-to-volume ratio of Si NW arrays with the plasmonic properties of silver nanoparticles resulted in a 3D structure suitable for very sensitive surface enhanced Raman scattering (SERS) applications, as demonstrated by the detection of Rhodamine 6G in aqueous solution at a concentration level of 10(-8) M.

Exploring the Aß1-42 fibrillogenesis timeline by atomic force microscopy and surface enhanced Raman spectroscopy.

Introduction: Alzheimer's disease (AD) is a progressive debilitating neurological disorder representing the most common neurodegenerative disease worldwide. Although the exact pathogenic mechanisms of AD remain unresolved, the presence of extracellular amyloid-ß peptide 1-42 (Aß1-42) plaques in the parenchymal and cortical brain is considered one of the hallmarks of the disease. Methods: In this work, we investigated the Aß1-42 fibrillogenesis timeline up to 48 h of incubation, providing morphological and chemo-structural characterization of the main assemblies formed during the aggregation process of Aß1-42, by atomic force microscopy (AFM) and surface enhanced Raman spectroscopy (SERS), respectively. Results: AFM topography evidenced the presence of characteristic protofibrils at early-stages of aggregation, which form peculiar macromolecular networks over time. SERS allowed to track the progressive variation in the secondary structure of the aggregation species involved in the fibrillogenesis and to determine when the ß-sheet starts to prevail over the random coil conformation in the aggregation process. Discussion: Our research highlights the significance of investigating the early phases of fibrillogenesis to better understand the molecular pathophysiology of AD and identify potential therapeutic targets that may prevent or slow down the aggregation process.

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.

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.

Probing the Structure of Toxic Amyloid-ß Oligomers with Electron Spin Resonance and Molecular Modeling.

Structural models of the toxic species involved in the development of Alzheimer's disease are of utmost importance to understand the molecular mechanism and to describe early biomarkers of the disease. Among toxic species, soluble oligomers of amyloid-ß (Aß) peptides are particularly important, because they are responsible for spreading cell damages over brain regions, thus rapidly impairing brain functions. In this work we obtain structural information on a carefully prepared Aß(1-42) sample, representing a toxic state for cell cultures, by combining electron spin resonance spectroscopy and computational models. We exploited the binding of Cu2+ to Aß(1-42) and used copper as a probe for estimating Cu-Cu distances in the oligomers by applying double electron-electron resonance (DEER) pulse sequence. The DEER trace of this sample displays a unique feature that fits well with structural models of oligomers formed by Cu-cross-linked peptide dimers. Because Cu is bound to the Aß(1-42) N-terminus, for the first time structural constraints that are missing in reported studies are provided at physiological conditions for the Aß N-termini. These constraints suggest the Aß(1-42) dimer as the building block of soluble oligomers, thus changing the scenario for any kinetic model of Aß(1-42) aggregation.

Air Stable Nickel-Decorated Black Phosphorus and Its Room-Temperature Chemiresistive Gas Sensor Capabilities.

In the rapidly emerging field of layered two-dimensional functional materials, black phosphorus, the P-counterpart of graphene, is a potential candidate for various applications, e.g., nanoscale optoelectronics, rechargeable ion batteries, electrocatalysts, thermoelectrics, solar cells, and sensors. Black phosphorus has shown superior chemical sensing performance; in particular, it is selective for the detection of NO2, an environmental toxic gas, for which black phosphorus has highlighted high sensitivity at a ppb level. In this work, by applying a multiscale characterization approach, we demonstrated a stability and functionality improvement of nickel-decorated black phosphorus films for gas sensing prepared by a simple, reproducible, and affordable deposition technique. Furthermore, we studied the electrical behavior of these films once implemented as functional layers in gas sensors by exposing them to different gaseous compounds and under different relative humidity conditions. Finally, the influence on sensing performance of nickel nanoparticle dimensions and concentration correlated to the decoration technique and film thickness was investigated.

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.

Fractal Silver Dendrites as 3D SERS Platform for Highly Sensitive Detection of Biomolecules in Hydration Conditions.

In this paper, we report on the realization of a highly sensitive and low cost 3D surface-enhanced Raman scattering (SERS) platform. The structural features of the Ag dendrite network that characterize the SERS material were exploited, attesting a remarked self-similarity and scale invariance over a broad range of length scales that are typical of fractal systems. Additional structural and optical investigations confirmed the purity of the metal network, which was characterized by low oxygen contamination and by broad optical resonances introduced by the fractal behavior. The SERS performances of the 3D fractal Ag dendrites were tested for the detection of lysozyme as probe molecule, attesting an enhancement factor of ~2.4 × 106. Experimental results assessed the dendrite material as a suitable SERS detection platform for biomolecules investigations in hydration 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.

Low cost tips for tip-enhanced Raman spectroscopy fabricated by two-step electrochemical etching of 125 µm diameter gold wires.

Tip-enhanced Raman spectroscopy (TERS) has become a well-applied technique for nanospectroscopy, allowing for single molecule sensitivity with sub-nanometer spatial resolution. The demand for efficient, reproducible and cost-effective probes for TERS is increasing. Here we report on a new electrochemical etching protocol to fabricate TERS tips starting from 125 µm diameter gold wires in a reproducible way. The process is reliable (50% of the tips have radius of curvature <35 nm, 66% <80 nm), fast (less than 2 min) and 2.5 times cheaper than the etching of standard 250 µm diameter wires. The TERS performance of the tips is tested on dyes, pigments and biomolecules and enhancement factors higher than 105 are observed. TERS mapping with a spatial resolution of 5 nm is demonstrated.

Optical Aggregation of Gold Nanoparticles for SERS Detection of Proteins and Toxins in Liquid Environment: Towards Ultrasensitive and Selective Detection.

Optical forces are used to aggregate plasmonic nanoparticles and create SERS-active hot spots in liquid. When biomolecules are added to the nanoparticles, high sensitivity SERS detection can be accomplished. Here, we pursue studies on Bovine Serum Albumin (BSA) detection, investigating the BSA-nanorod aggregations in a range from 100 µM to 50 nM by combining light scattering, plasmon resonance and SERS, and correlating the SERS signal with the concentration. Experimental data are fitted with a simple model describing the optical aggregation process. We show that BSA-nanorod complexes can be optically printed on non-functionalized glass surfaces, designing custom patterns stable with time. Furthermore, we demonstrate that this methodology can be used to detect catalase and hemoglobin, two Raman resonant biomolecules, at concentrations of 10 nM and 1 pM, respectively, i.e., well beyond the limit of detection of BSA. Finally, we show that nanorods functionalized with specific aptamers can be used to capture and detect Ochratoxin A, a fungal toxin found in food commodities and wine. This experiment represents the first step towards the addition of molecular specificity to this novel biosensor strategy.

Structural differences between toxic and nontoxic HypF-N oligomers.

We have studied two misfolded oligomeric forms of the protein HypF-N, which show similar morphologies but very different toxicities. We measured over 80 intermolecular distance-dependent parameters for each oligomer type using FRET, in conjunction with solution- and solid-state NMR and other biophysical techniques. The results indicate that the formation of a highly organised hydrogen bonded core in the toxic oligomers results in the exposure of a larger number of hydrophobic residues than in the nontoxic species, causing the former to form aberrant interactions with cellular components.

SERS detection of Biomolecules at Physiological pH via aggregation of Gold Nanorods mediated by Optical Forces and Plasmonic Heating.

Strategies for in-liquid molecular detection via Surface Enhanced Raman Scattering (SERS) are currently based on chemically-driven aggregation or optical trapping of metal nanoparticles in presence of the target molecules. Such strategies allow the formation of SERS-active clusters that efficiently embed the molecule at the "hot spots" of the nanoparticles and enhance its Raman scattering by orders of magnitude. Here we report on a novel scheme that exploits the radiation pressure to locally push gold nanorods and induce their aggregation in buffered solutions of biomolecules, achieving biomolecular SERS detection at almost neutral pH. The sensor is applied to detect non-resonant amino acids and proteins, namely Phenylalanine (Phe), Bovine Serum Albumin (BSA) and Lysozyme (Lys), reaching detection limits in the µg/mL range. Being a chemical free and contactless technique, our methodology is easy to implement, fast to operate, needs small sample volumes and has potential for integration in microfluidic circuits for biomarkers detection.