Tandem SERS and MS/MS Profiling of Plasma Extracellular Vesicles for Early Ovarian Cancer Biomarker Discovery.

Extracellular vesicles (EVs) are vectors of biomolecular cargo that play essential roles in intercellular communication across a range of cells. Protein, lipid, and nucleic acid cargo harbored within EVs may serve as biomarkers at all stages of disease; however, the choice of methodology may challenge the specificity and reproducibility of discovery. To address these challenges, the integration of rigorous EV purification methods, cutting-edge spectroscopic technologies, and data analysis are critical to uncover diagnostic signatures of disease. Herein, we demonstrate an EV isolation and analysis pipeline using surface-enhanced Raman spectroscopy (SERS) and mass spectrometry (MS) techniques on plasma samples obtained from umbilical cord blood, healthy donor (HD) plasma, and plasma from women with early stage high-grade serous carcinoma (HGSC). Plasma EVs were purified by size exclusion chromatography and analyzed by surface-enhanced Raman spectroscopy (SERS), mass spectrometry (MS), and atomic force microscopy. After determining the fraction of highest EV purity, SERS and MS were used to characterize EVs from HDs, pooled donors with noncancerous gynecological ailments (n = 6), and donors with early stage [FIGO (I/II)] with HGSC. SERS spectra were subjected to different machine learning algorithms such as PCA, logistic regression, support vector machine, naïve Bayes, random forest, neural network, and k nearest neighbors to differentiate healthy, benign, and HGSC EVs. Collectively, we demonstrate a reproducible workflow with the potential to serve as a diagnostic platform for HGSC.

Colloidal Self-Assembly of Silver Nanoparticle Clusters for Optical Metasurfaces.

Optical metasurfaces are two-dimensional assemblies of nanoscale optical resonators and could constitute the next generation of ultrathin optical components. The development of methods to manufacture these nanostructures on a large scale is still a challenge, while most performance demonstrations were obtained with lithographically fabricated metasurfaces that are restricted to small scales. Self-assembly fabrication routes are promising alternatives and have been used to produce original nanoresonators. Reports of self-assembled metasurface fabrication, however, are still scarce. Here, we show that an emulsion-based formulation approach can be used both for the fabrication of complex colloidal resonators, presenting a strong interaction with light, in particular due to simultaneous magnetic and electric modes of resonance, and for their deposition in homogeneous films. This fabrication technique involves emulsification of an aqueous suspension of silver nanoparticles in an oil phase, followed by controlled drying of the emulsion, and produces silver colloidal clusters. We show that the drying process can be controlled in a liquid emulsion, producing a metafluid, as well as in a sedimented emulsion, producing a metasurface. The structural control of the synthesized colloidal clusters is demonstrated with electron microscopy and X-ray scattering techniques. Using a polarization-resolved multiangle light scattering setup in the visible wavelength range, we conduct a comprehensive angular and spectroscopic study of the optical resonant scattering of the nanoresonators in a metafluid and show that they present strong optical magnetic resonances and directional forward-scattering patterns, with scattering efficiencies of up to 4. The metasurfaces consist of homogeneous films, of variable surface density, of colloidal clusters that have the same extinction properties on the surface and in the fluid. This experimental approach allows for large-scale production of metasurfaces.

Silver nanoparticles inhibit nitrogen fixation in soybean (Glycine max) root nodules.

Antimicrobial silver nanoparticles (AgNPs) are popular in consumer and industrial products, leading to increasing concentrations in the environment. We tested whether exposure to AgNPs could be detrimental to a microbe, its host plant, and their symbiotic relationship. When subjected to 10 µg/mL AgNPs, growth of Bradyrhizobium japonicum USDA 110 was halted. Axenic nitrogen-fertilized Glycine max seedlings were unaffected by 2.5 µg/mL of 30 nm AgNPs, but growth was inhibited with the same dose of 16 nm AgNPs. With 2.5 µg/mL AgNPs, biomass of inoculated plants was 50% of the control. Bacteroids were not found in nodules on plants treated with 2.5 µg/mL AgNPs and plants given 0.5-2.5 µg/mL AgNPs had 40-65% decreased nitrogen fixation. In conclusion, AgNPs not only interfere with general plant and bacterial growth but also inhibit nodule development and bacterial nitrogen fixation. We should be mindful of not releasing AgNPs to the environment or to agricultural land.

Periodic Spiral Ripples on VS2 Flakes: A Tip-Enhanced Raman Investigation.

Using atmospheric-pressure chemical vapor deposition, we have synthesized vanadium disulfide (VS2) flakes with a metallic 1T phase that display nanoscale spiral surface ripples. To understand the origin of these chiral patterns in these transition metal dichalcogenides, tip-enhanced Raman spectroscopy and Kelvin probe force microscopies were jointly used to investigate their crystal structure, possible oxidation, and electronic properties, respectively. We found that the surface corrugation consists of small crystalline domains with distinct orientations. The change in local orientation is observed concomitantly with a spectral shift of the lattice modes of VS2 and results in the formation of grain boundaries between the domains with distinct orientation. Additionally, the periodic surface structure is modulating the work function of VS2 by 14 meV.

The Hidden Flower in WS2 Flakes: A Combined Nanomechanical and Tip-Enhanced Raman Exploration.

We report on tungsten disulfide (WS2) flakes grown by chemical vapor deposition (CVD), which exhibit a flower-like surface structure above the primary few-layer flake with a triangular shape. The fine structure is only revealed in the mechanical, chemical, and electronic properties of the flake but not in the topography. The origin of this structure is the peculiar one-step growth during the CVD process that permits to control the sulfur concentration at any time. A high concentration of S at the onset of the deposition process leads to a rapid growth of the flake, resulting in tungsten vacancies. Reducing the sulfur concentration toward the end of the growth slows down the reaction and leads to sulfur vacancies. These microscale domains were studied by confocal- and tip-enhanced Raman spectroscopy revealing their chemical composition with high spatial resolution. A strong quenching of the photoluminescence in the tungsten-vacancy domains is observed. Atomic force microscope measurements, performed in intermittent contact mode, force modulation mode (including lateral force mode), and PeakForce quantitative nanomechanics mode, show that the mechanical properties of these domains differ. Within the tungsten-vacancy domains, the adhesion force is reduced, while the friction force increased. Kelvin probe force microscopy measurements show that the electronic properties of the flakes are modulated by these domains. The combined nanomechanical and nanospectroscopy measurements provide detailed insights on the inhomogeneous surface properties of the single WS2 flake, further highlighting how its multidomain properties can be finely tuned using CVD.

Investigating the Performances of Wide-Field Raman Microscopy with Stochastic Optical Reconstruction Post-Processing.

Super-resolution fluorescence microscopy based on localization algorithms has tremendously impacted the field of imaging by improving the spatial resolution of optical measurements with specific blinking fluorophores and concomitant reduction of acquisition time. In vibrational spectroscopy and imaging, various methods have been developed to surpass the diffraction limit including near-field scattering methods, such as in tip-enhanced Raman and infrared spectroscopies. Although these scanning-probe techniques can provide exquisite spatial resolution, they often require long acquisition times and tedious fabrication of nano-scale scanning probes. Herein, stochastic optical reconstruction microscopy (STORM) protocol is applied on Raman measurements acquired using a wide-field home-built microscopy setup. We explore how the fluctuations of the Raman signal acquired over a series of time-lapse images at specific spectral ranges can be exploited with STORM processing, possibly revealing details with improved spatial resolution, under lower irradiance and with faster acquisition speed that cannot be achieved in point scanning mode over the same field of view. Samples studied here include patterned silicon, polystyrene microspheres on a silicon wafer, and graphene on a silicon/silicon dioxide substrate. The outcome presents an effective way to collect Raman images at selected spectral ranges with spatial resolutions of ∼200 nm over a large field of view under 532 nm excitation together with an acquisition speed improved by two orders of magnitude and under a significantly reduced irradiance compared to confocal laser scanning acquisition.

Characterization of ovarian cancer-derived extracellular vesicles by surface-enhanced Raman spectroscopy.

Ovarian cancer is the most lethal gynecological malignancy, owing to the fact that most cases are diagnosed at a late stage. To improve prognosis and reduce mortality, we must develop methods for the early diagnosis of ovarian cancer. A step towards early and non-invasive cancer diagnosis is through the utilization of extracellular vesicles (EVs), which are nanoscale, membrane-bound vesicles that contain proteins and genetic material reflective of their parent cell. Thus, EVs secreted by cancer cells can be thought of as cancer biomarkers. In this paper, we present gold nanohole arrays for the capture of ovarian cancer (OvCa)-derived EVs and their characterization by surface-enhanced Raman spectroscopy (SERS). For the first time, we have characterized EVs isolated from two established OvCa cell lines (OV-90, OVCAR3), two primary OvCa cell lines (EOC6, EOC18), and one human immortalized ovarian surface epithelial cell line (hIOSE) by SERS. We subsequently determined their main compositional differences by principal component analysis and were able to discriminate the groups by a logistic regression-based machine learning method with ∼99% accuracy, sensitivity, and specificity. The results presented here are a great step towards quick, facile, and non-invasive cancer diagnosis.

Correction: Extending nanoscale patterning with multipolar surface plasmon resonances.

Correction for 'Extending nanoscale patterning with multipolar surface plasmon resonances' by Issam Kherbouche et al., Nanoscale, 2021, 13, 11051-11057, DOI: .

Characterization of extracellular vesicles derived from mesenchymal stromal cells by surface-enhanced Raman spectroscopy.

Extracellular vesicles (EVs) are secreted by all cells into bodily fluids and play an important role in intercellular communication through the transfer of proteins and RNA. There is evidence that EVs specifically released from mesenchymal stromal cells (MSCs) are potent cell-free regenerative agents. However, for MSC EVs to be used in therapeutic practices, there must be a standardized and reproducible method for their characterization. The detection and characterization of EVs are a challenge due to their nanoscale size as well as their molecular heterogeneity. To address this challenge, we have fabricated gold nanohole arrays of varying sizes and shapes by electron beam lithography. These platforms have the dual purpose of trapping single EVs and enhancing their vibrational signature in surface-enhanced Raman spectroscopy (SERS). In this paper, we report SERS spectra for MSC EVs derived from pancreatic tissue (Panc-MSC) and bone marrow (BM-MSC). Using principal component analysis (PCA), we determined that the main compositional differences between these two groups are found at 1236, 761, and 1528 cm-1, corresponding to amide III, tryptophan, and an in-plane -C=C- vibration, respectively. We additionally explored several machine learning approaches to distinguish between BM- and Panc-MSC EVs and achieved 89 % accuracy, 89 % sensitivity, and 88 % specificity using logistic regression.

Extending nanoscale patterning with multipolar surface plasmon resonances.

Plasmonic excitation of metallic nanoparticles can trigger chemical reactions at the nanoscale. Such optical effects can also be employed to selectively and locally graft photopolymer layers at the nanostructure surface, and, when combined with a surface functionalization agent, new pathways can be explored to modify the surface of a plasmonic nanoparticle. Among these approaches, diazonium salt chemistry is seen as an attractive strategy due to the high photoinduced reactivity of these salts. In this work, we demonstrate that it is possible to trigger the site-selective grafting of aryl films derived from diazonium salts on distinct nano-localized area of single gold nanotriangles, by taking advantage of their multipolar localized surface plasmon modes. It is shown the aryl film will preferentially graft in areas where the electric field enhancement is maximum, independently of the considered excited surface plasmon mode. These experimental findings are in very good qualitative agreement with the calculations of the local electric field, using the finite-difference time-domain (FDTD) method. We believe that this plasmonic-based approach will not only pave a new way for the spatially controlled surface functionalization of plasmonic nanoparticles, but also provide a general strategy to attach distinct molecules to hot spot regions on a single nanoparticle, opening promising prospects in sensing and multiplexing, and optically nano-scale patterning of functional groups.

Ultrafiltration and Injection of Islet Regenerative Stimuli Secreted by Pancreatic Mesenchymal Stromal Cells.

The secretome of mesenchymal stromal cells (MSCs) is enriched for biotherapeutic effectors contained within and independent of extracellular vesicles (EVs) that may support tissue regeneration as an injectable agent. We have demonstrated that the intrapancreatic injection of concentrated conditioned media (CM) produced by bone marrow MSC supports islet regeneration and restored glycemic control in hyperglycemic mice, ultimately providing a platform to elucidate components of the MSC secretome. Herein, we extend these findings using human pancreas-derived MSC (Panc-MSC) as "biofactories" to enrich for tissue regenerative stimuli housed within distinct compartments of the secretome. Specifically, we utilized 100 kDa ultrafiltration as a simple method to debulk protein mass and to enrich for EVs while concentrating the MSC secretome into an injectable volume for preclinical assessments in murine models of blood vessel and islet regeneration. EV enrichment (EV+) was validated using nanoscale flow cytometry and atomic force microscopy, in addition to the detection of classical EV markers CD9, CD81, and CD63 using label-free mass spectrometry. EV+ CM was predominately enriched with mediators of wound healing and epithelial-to-mesenchymal transition that supported functional regeneration in mesenchymal and nonmesenchymal tissues. For example, EV+ CM supported human microvascular endothelial cell tubule formation in vitro and enhanced the recovery of blood perfusion following intramuscular injection in nonobese diabetic/severe combined immunodeficiency mice with unilateral hind limb ischemia. Furthermore, EV+ CM increased islet number and ß cell mass, elevated circulating insulin, and improved glycemic control following intrapancreatic injection in streptozotocin-treated mice. Collectively, this study provides foundational evidence that Panc-MSC, readily propagated from the subculture of human islets, may be utilized for regenerative medicine applications.

Three-color plasmon-mediated reduction of diazonium salts over metasurfaces.

Surface plasmon-mediated chemical reactions are of great interest for a variety of applications ranging from micro- and nanoscale device fabrication to chemical reactions of societal interest for hydrogen production or carbon reduction. In this work, a crosshair-like nanostructure is investigated due to its ability to induce local enhancement of the local electromagnetic field at three distinct wavelengths corresponding to three plasmon resonances. The structures are irradiated in the presence of a solution containing diazonium salts at wavelengths that match the resonance positions at 532 nm, 632.8 nm, and 800 nm. The resulting grafting shows polarization and wavelength-dependent growth patterns at the nanoscale. The plasmon-mediated reactions over arrays of the crosshair structures are further investigated using scanning electron microscopy and supported by finite domain time domain modelling revealing wavelength and polarization specific reactions. Such an approach enables nanoscale molecular printing using light source opening multiplexing applications where different analytes can be grafted under distinct opto-geometric conditions.

Deciphering tip-enhanced Raman imaging of carbon nanotubes with deep learning neural networks.

Recent release of open-source machine learning libraries presents opportunities to unify machine learning with nanoscale research, thus improving effectiveness of research methods and characterization protocols. This paper outlines and demonstrates the effectiveness of such a synergy with artificial neural networks to provide for an accelerated and enhanced characterization of individual carbon nanotubes deposited over a surface. Our algorithms provide a rapid diagnosis and analysis of tip-enhanced Raman spectroscopy mappings and the results show an improved spectral assignment of spectral features and spatial contrast of the collected images. Using several examples, we demonstrate the robustness and versatility of our deep learning neural network models. We highlight the use of machine learning and data science in tandem with tip-enhanced Raman spectroscopy technique enables a fast and accurate understanding of experimental data, thus leading to a powerful and comprehensive imaging analysis applied to spectroscopic measurements.

GSK3787-Loaded Poly(Ester Amide) Particles for Intra-Articular Drug Delivery.

Osteoarthritis (OA) is a debilitating joint disorder affecting more than 240 million people. There is no disease modifying therapeutic, and drugs that are used to alleviate OA symptoms result in side effects. Recent research indicates that inhibition of peroxisome proliferator-activated receptor δ (PPARδ) in cartilage may attenuate the development or progression of OA. PPARδ antagonists such as GSK3787 exist, but would benefit from delivery to joints to avoid side effects. Described here is the loading of GSK3787 into poly(ester amide) (PEA) particles. The particles contained 8 wt.% drug and had mean diameters of about 600 nm. Differential scanning calorimetry indicated the drug was in crystalline domains in the particles. Atomic force microscopy was used to measure the Young's moduli of individual particles as 2.8 MPa. In vitro drug release studies showed 11% GSK3787 was released over 30 days. Studies in immature murine articular cartilage (IMAC) cells indicated low toxicity from the drug, empty particles, and drug-loaded particles and that the particles were not taken up by the cells. Ex vivo studies on murine joints showed that the particles could be injected into the joint space and resided there for at least 7 days. Overall, these results indicate that GSK3787-loaded PEA particles warrant further investigation as a delivery system for potential OA therapy.

Investigation of Au SAMs Photoclick Derivatization by PM-IRRAS.

In this work, we present a clean one-step process for modifying headgroups of self-assembled monolayers (SAMs) on gold using photo-enabled click chemistry. A thiolated, cyclopropenone-caged strained alkyne precursor was first functionalized onto a flat gold substrate through self-assembly. Exposure of the cyclopropenone SAM to UVA light initiated the efficient photochemical decarbonylation of the cyclopropenone moiety, revealing the strained alkyne capable of undergoing the interfacial strain-promoted alkyne-azide cycloaddition (SPAAC). Irradiated SAMs were derivatized with a series of model azides with varied hydrophobicity to demonstrate the generality of this chemical system for the modification and fine-tuning of the surface chemistry on gold substrates. SAMs were characterized at each step with polarization-modulation infrared reflection-absorption spectroscopy (PM-IRRAS) to confirm the successful functionalization and reactivity. Furthermore, to showcase the compatibility of this approach with biochemical applications, cyclopropenone SAMs were irradiated and modified with azide-bearing cell adhesion peptides to promote human fibroblast cell adhesion, and then imaged by live-cell fluorescence microscopy. Thus, the "photoclick" methodology reported here represents an improved, versatile, catalyst-free protocol that allows for a high degree of control over the modification of material surfaces, with applicability in materials science as well as biochemistry.

Probing mid-infrared plasmon resonances in extended radial fractal structures.

Infrared (IR) antennas made of metallic nanostructures are widely tunable from the near- to the far-IR range. They can be utilized for a variety of applications such as light harvesting and photonic filters, and their structural linear or circular anisotropy can be exploited to further enhance the sensitivity of spectroscopic measurements. Here gold dendritic fractal structures that were optimized to exhibit multiple resonances in the mid-IR range were characterized using a scattering-type scanning near-field optical IR microscope. The spatially resolved IR maps associated with the individual modes serve as a basis to understand the mode evolution between each fractal generation.

Optoelectronic, Aggregation, and Redox Properties of Double-Rotor Boron Difluoride Hydrazone Dyes.

We develop the chemistry of boron difluoride hydrazone dyes (BODIHYs) bearing two aryl substituents and explore their properties. The low-energy absorption bands (λmax =427-464 nm) of these dyes depend on the nature of the N-aryl groups appended to the BODIHY framework. Electron-donating and extended π-conjugated groups cause a redshift, whereas electron-withdrawing groups result in a blueshift. The title compounds were weakly photoluminescent in solution and strongly photoluminescent as thin films (λPL =525-578 nm) with quantum yields of up to 18 % and lifetimes of 1.1-1.7 ns, consistent with the dominant radiative decay through fluorescence. Addition of water to THF solutions of the BODIHYs studied causes molecular aggregation which restricts intramolecular motion and thereby enhances photoluminescence. The observed photoluminescence of BODIHY thin films is likely facilitated by a similar molecular packing effect. Finally, cyclic voltammetry studies confirmed that BODIHY derivatives bearing para-substituted N-aryl groups could be reversibly oxidized (Eox1 =0.62-1.02 V vs. Fc/Fc+ ) to their radical cation forms. Chemical oxidation studies confirmed that para-substituents at the N-aryl groups are required to circumvent radical decomposition pathways. Our findings provide new opportunities and guiding principles for the design of sought-after multifunctional boron difluoride complexes that are photoluminescent in the solid state.

Advancements in fractal plasmonics: structures, optical properties, and applications.

The structural characteristics of plasmonic nanostructures directly influence their plasmonic properties, and therefore, their potential role in applications ranging from sensing and catalysis to light- and energy-harvesting. For a structure to be compatible with a selected application, it is critical to accurately tune the plasmonic properties over a specific spectral range. Fabricating structures that meet these stringent requirements remains a significant challenge as plasmon resonances are generally narrow with respect to the considered selected spectral range. Adapted from their well-established role in GHz applications, plasmonic fractal structures have emerged as architectures of interest due to their ability to support multiple tunable resonances over broad spectral domains. Here, we review the advancements that have been made in the growing field of fractal plasmonics. Iterative and space-filling geometries that can be prepared by advanced nanofabrication techniques, notably electron-beam lithography, are presented along with the optical properties of such structures and metasurfaces. The distributions of electromagnetic enhancement for some of these fractals is shown, along with how the resonances can be mapped experimentally. This review also explores how fractal structures can be used for applications in solar cell and plasmon-based sensing applications. Finally, the future areas of physical and analytical science that could benefit from fractal plasmonics are discussed.

Analytical Considerations in Nanoscale Flow Cytometry of Extracellular Vesicles to Achieve Data Linearity.

BACKGROUND: Platelet microparticles (PMPs) and their abundance in the blood are a prognostic biomarker in thrombotic disorders and cancer. Nanoscale flow cytometry (nFC) is ideal for high-throughput analysis of PMPs but these clinical assays have not been developed previously. OBJECTIVE: This article demonstrates that nFC is a suitable technology to enumerate PMPs present in plasma samples in a clinical setting. MATERIALS AND METHODS: nFC was performed using the Apogee A50-Micro instrument. Instrument settings and acquisition parameters were developed with the use of fluorescent beads and plasma samples. Sample preparation and handling was also optimized. RESULTS: nFC allows for linear detection of particles between approximately 200 and 1,000 nm based on calibration beads and was dependent on dilution factor and flow rate. Linearity in event analysis as samples became more diluted was lost when events approximately 100 nm were gated while linearity was maintained despite dilution of sample in events larger than 200 nm in diameter. Higher flow rates lead to an under-estimation of events analysed per microlitre of analyte and this was more pronounced when plasma samples were not diluted more than 1/20×. CONCLUSION: nFC offers multi-parametric analysis of PMPs when optimal calibration of acquisition and sample processing settings is performed. Analysis of plasmas from metastatic prostate cancer patients and leukaemia patients revealed that PMP levels were larger than 100 nm and were equally abundant in patients that responded to or failed androgen deprivation therapy or between patients representing different stages of leukaemia.