Focusing ion funnel-assisted ambient electrospray enables high-density and uniform deposition of non-spherical gold nanoparticles for highly sensitive surface-enhanced Raman scattering.

Surface-enhanced Raman scattering (SERS) is a powerful technique for detecting trace amounts of analytes. However, the performance of SERS substrates depends on many variables including the enhancement factor, morphology, consistency, and interaction with target analytes. In this study, we investigated, for the first time, the use of electrospray deposition (ESD) combined with a novel ambient focusing DC ion funnel to deposit a high density of gold nanoparticles (AuNPs) to generate large-area, uniform substrates for highly sensitive SERS analysis. We found that the combination of ambient ion focusing with ESD facilitated high-density and intact deposition of non-spherical NPs. This also allowed us to take advantage of a polydisperse colloidal solution of AuNPs (consisting of nanospheres and nanorods), as confirmed by finite-difference time domain (FDTD) simulations. Our SERS substrate exhibited excellent capture capacity for model analyte molecules, namely 4-aminothiophenol (4-ATP) and Rhodamine 6G (R6G), with detection limits in the region of 10-11 M and a relative standard deviation of <6% over a large area (∼500 × 500 µm2). Additionally, we assessed the quantitative performance of our SERS substrate using the R6G probe molecule. The results demonstrated excellent linearity (R2 > 0.99) over a wide concentration range (10-4 M to 10-10 M) with a detection limit of 80 pM.

Biocompatible Inhibitor Based on Chitosan and Amphiphilic Peptide against Mutant Huntingtin Toxicity.

Huntington's disease (HD) is classified as a protein-misfolding disease correlated with the mutant Huntingtin (mHtt) protein with abnormally expanded polyglutamine (polyQ) domains. Because no effective drugs have yet been reported, attempts to develop better therapy to delay the age of onset are in urgent demand. In this study, an amphiphilic peptide consisting of negatively charged hexaglutamic acid and a stretch of decaglutamine (E6 Q10 ) was chemically synthesized as an inhibitor against polyQ and mHtt toxicity. It is found that E6 Q10 selfassembles into spherical vesicles, as shown by means of TEM, cryoelectron microscopy, and dynamic light scattering. Assembled E6 Q10 prevented the polyQ-rich peptide (KKWQ20 AKK) from forming amyloid fibrils. To enable the cell-penetration ability of E6 Q10 , the E6 Q10 ⋅chitosan complex was generated. It is demonstrated that the complex penetrates cells, interferes with the mHtt oligomerization and aggregation process, and prevents mHtt cytotoxicity. By combining positively charged chitosan and amphiphilic peptides with a negatively charge moiety, a new strategy is provided to develop biocompatible and biodegradable inhibitors against mHtt toxicity.

Navigating the Landscape of Tumor Extracellular Vesicle Heterogeneity.

The last decade has seen a rapid expansion of interest in extracellular vesicles (EVs) released by cells and proposed to mediate intercellular communication in physiological and pathological conditions. Considering that the genetic content of EVs reflects that of their respective parent cell, many researchers have proposed EVs as a source of biomarkers in various diseases. So far, the question of heterogeneity in given EV samples is rarely addressed at the experimental level. Because of their relatively small size, EVs are difficult to reliably isolate and detect within a given sample. Consequently, standardized protocols that have been optimized for accurate characterization of EVs are lacking despite recent advancements in the field. Continuous improvements in pre-analytical parameters permit more efficient assessment of EVs, however, methods to more objectively distinguish EVs from background, and to interpret multiple single-EV parameters are lacking. Here, we review EV heterogeneity according to their origin, mode of release, membrane composition, organelle and biochemical content, and other factors. In doing so, we also provide an overview of currently available and potentially applicable methods for single EV analysis. Finally, we examine the latest findings from experiments that have analyzed the issue at the single EV level and discuss potential implications.

Facile Coating Strategy to Functionalize Inorganic Nanoparticles for Biosensing.

The use of inorganic nanoparticles (NPs) for biosensing requires that they exhibit high colloidal stability under various physiological conditions. Here, we report on a general approach to render hydrophobic NPs into hydrophilic ones that are ready for bioconjugation. The method uses peglyated polymers conjugated with multiple dopamines, which results in multidentate coordination. As proof-of-concept, we applied the coating to stabilize ferrite and lanthanide NPs synthesized by thermal decomposition. Both polymer-coated NPs showed excellent water solubility and were stable at high salt concentrations under physiological conditions. We used these NPs as molecular-sensing agents to detect exosomes and bacterial nucleic acids.

On-line SERS detection of single bacterium using novel SERS nanoprobes and a microfluidic dielectrophoresis device.

The integration of novel surface-enhanced Raman scattering (SERS) nanoprobes and a microfluidic dielectrophoresis (DEP) device is developed for rapid on-line SERS detection of Salmonella enterica serotype Choleraesuis and Neisseria lactamica. The SERS nanoprobes are prepared by immobilization of specific antibody onto the surface of nanoaggregate-embedded beads (NAEBs), which are silica-coated, dye-induced aggregates of a small number of gold nanoparticles (AuNPs). Each NAEB gives highly enhanced Raman signals owing to the presence of well-defined plasmonic hot spots at junctions between AuNPs. Herein, the on-line SERS detection and accurate identification of suspended bacteria with a detection capability down to a single bacterium has been realized by the NAEB-DEP-Raman spectroscopy biosensing strategy. The practical detection limit with a measurement time of 10 min is estimated to be 70 CFU mL(-1) . In comparison with whole-cell enzyme-linked immunosorbent assay (ELISA), the SERS-nanoprobe-based biosensing method provides advantages of higher sensitivity and requiring lower amount of antibody in the assay (100-fold less). The total assay time including sample pretreatment is less than 2 h. Hence, this sensing strategy is promising for faster and effective on-line multiplex detection of single pathogenic bacterium by using different bioconjugated SERS nanoprobes.

Surface-enhanced Raman scattering of graphene with photo-assisted-synthesized gold nanoparticles.

This paper presents a convenient and reliable method to prepare gold nanoparticles (AuNPs) on graphene. Photo-assisted synthesis (PAS) was employed to grow AuNPs in AuCl(4)(-) electrolyte on graphene. The size of AuNPs could be as large as 130 nm. This optical method had a steady growth rate of AuNPs. The distribution of AuNPs was well controlled by focusing the laser for PAS. The minimum diameter of the distribution was approximately 1 µm. Surface-enhanced Raman scattering of graphene due to AuNPs was observed. Electrical fields near AuNPs calculated by the finite-difference time-domain algorithm ensured that the Raman enhancement was attributed to the localized surface plasmons of AuNPs.

Tapered optical fiber sensor based on localized surface plasmon resonance.

A tapered fiber localized surface plasmon resonance (LSPR) sensor is demonstrated for refractive index sensing and label-free biochemical detection. The sensing strategy relies on the interrogation of the transmission intensity change due to the evanescent field absorption of immobilized gold nanoparticles on the tapered fiber surface. The refractive index resolution based on the interrogation of transmission intensity change is calculated to be 3.2×10⁻5 RIU. The feasibility of DNP-functionalized tapered fiber LSPR sensor in monitoring anti-DNP antibody with different concentrations spiked in buffer is examined. Results suggest that the compact sensor can perform qualitative and quantitative biochemical detection in real-time and thus has potential to be used in biomolecular sensing applications.

Multiple resonance fiber-optic sensor with time division multiplexing for multianalyte detection.

A proof-of-concept multiwindow fiber-optic sensor utilizing multiple particle plasmon resonance (PPR) of silver nanoparticles and gold nanorods separately on two unclad portions of the fiber for multianalyte detection is demonstrated. The detection is based on intensity interrogation of multiple wavelengths by a single detector. Time division multiplexing is employed to modulate the illumination of dual-wavelength LEDs to induce PPRs for simultaneous real-time and label-free monitoring of two types of biomolecular interactions. Preliminary results reveal that a refractive index resolution of 9 ×10(-6) RIU is achieved. Moreover, the measured intensities of two windows independently respond to their respective binding events. The potential of the sensor architecture with multiple sensing windows for cascaded, higher throughput, and multianalyte biochemical detection can be expected.

Epstein-Barr viral product-containing exosomes promote fibrosis and nasopharyngeal carcinoma progression through activation of YAP1/FAPα signaling in fibroblasts.

BACKGROUND: The progression of nasopharyngeal carcinoma (NPC) is profoundly affected by Epstein-Barr virus (EBV) infection. However, the role of EBV in the intercommunication between NPC and surrounding stromal cells has yet to be explored. METHODS: NPC biopsies were obtained for immunohistochemical (IHC) analyses. Clinical correlations between the expression of active YAP1/FAPα and the fibrotic response and between YAP1/FAPα and the density of cytotoxic CD8a+ T lymphocytes were determined. Survival times based on IHC scores were compared between groups using Kaplan-Meier survival and log-rank tests. Independent prognostic factors for metastasis/recurrence-free survival and overall survival were identified using univariate and multivariate Cox regression models. Fibroblasts were isolated from human nasopharyngeal biopsies. Exosomes were purified from culture supernatants of EBV+-positive NPC cells. The effects of EBV product-containing exosomes on fibroblast activation, fibrotic response, tumor growth, immune response, and correlations between the expression of featured genes were investigated using gel contraction assays, ELISAs, EdU incorporation assays, real-time impedance assays, RNA sequencing, immunostaining, 3D cancer spheroid coculture systems, and an NPC xenograft model. RESULTS: NPC patients who developed metastasis had significantly higher levels of active YAP1 and FAPα in their tumor stroma, which was further correlated with tumor fibrosis and poorer metastasis-free survival. Exosomes released from EBV+-NPC cells contained abundant FAPα protein and EBV-encoded latent membrane protein 1. Viral product-containing exosomes markedly enhanced the fibrotic response and tumor growth in a mouse xenograft model. IHC analyses of human NPC and NPC xenografts revealed positive correlations between levels of active YAP1 and FAPα, YAP1 and the fibrotic response, and FAPα and the fibrotic response. Mechanistic studies showed that treatment of fibroblasts with viral product-containing exosomes promoted the characteristics of cancer-associated fibroblasts by stimulating YAP1 signaling and the production of the immunosuppressive cytokines IL8, CCL2, and IL6. Inhibition of YAP1 activation markedly reversed these exosome-mediated protumoral effects, resulting in reduced contractility, inactivation of YAP1 signaling, and decreased production of immunosuppressive cytokines in fibroblasts. Furthermore, fibroblasts stimulated with these viral product-containing exosomes promoted NPC resistance to T cell-mediated cytotoxicity within tumor spheroids. In NPC tissues, a significant negative correlation was found between YAP1/FAPα and the density of CD8a+ T lymphocytes with a granzyme B signature. CONCLUSION: EBV orchestrates interactions with the host and surrounding stroma by stimulating the functions of YAP1 and FAPα in fibroblasts through exosome cargos to create a more immunosuppressive, proinvasive microenvironment.

Electrochemically fabricated self-aligned 2-D silver/alumina arrays as reliable SERS sensors.

A novel SERS sensor for adenine molecules is fabricated electrochemically using an ordered two-dimensional array of self-aligned silver nanoparticles encapsulated by alumina. Silver is electro-deposited on the interior surfaces at the bottom of nano-channels in a porous anodic aluminum oxide (AAO) film. After etching aluminum, the back-end alumina serves as a SERS substrate. SERS enhancement factor greater than 10(6) is measured by 532 nm illumination. It exhibits robust chemical stability and emits reproducible Raman signals from repetitive uses for eight weeks. The inexpensive mass production process makes this reliable, durable and sensitive plasmon based optical device promising for many applications.

Epstein-Barr Virus Induces Adipocyte Dedifferentiation to Modulate the Tumor Microenvironment.

The most frequent location of metastatic EBV+ nasopharyngeal carcinoma (NPC) is the bone marrow, an adipocyte-dominant region. Several EBV-associated lymphoepithelioma-like carcinoma (LELC) types also grow in the anatomical vicinity of fat tissues. Here we show that in an adipose tissue-rich tumor setting, EBV targets adipocytes and remodels the tumor microenvironment. Positive immunoreactivity for EBV-encoded early antigen D was detected in adipose tissue near tumor beds of bone marrow metastatic NPC. EBV was capable of infecting primary human adipocytes in vitro, triggering expression of multiple EBV-encoded mRNA and proteins. In infected adipocytes, lipolysis was stimulated through enhanced expression of lipases and the AMPK metabolic pathway. The EBV-mediated imbalance in energy homeostasis was further confirmed by increased release of free fatty acids, glycerol, and expression of proinflammatory adipokines. Clinically, enhanced serum levels of free fatty acids in patients with NPC correlated with poorer recurrence-free survival. EBV-induced delipidation stimulated dedifferentiation of adipocytes into fibroblast-like cells expressing higher levels of S100A4, a marker protein of cancer-associated fibroblasts (CAF). IHC analyses of bone marrow metastatic NPC and salivary LELC revealed similar structural changes of dedifferentiated adipocytes located at the boundaries of EBV+ tumors. S100A4 expression in adipose tissues near tumor beds correlated with fibrotic response, implying that CAFs in the tumor microenvironment are partially derived from EBV-induced dedifferentiated adipocytes. Our data suggest that adipose tissue serves as an EBV reservoir, where EBV orchestrates the interactions between adipose tissues and tumor cells by rearranging metabolic pathways to benefit virus persistence and to promote a protumorigenic microenvironment. SIGNIFICANCE: This study suggests that Epstein-Barr virus hijacks adipocyte lipid metabolism to create a tumor-promoting microenvironment from which reactivation and relapse of infection could potentially occur.

An integrated magneto-electrochemical device for the rapid profiling of tumour extracellular vesicles from blood plasma.

Assays for cancer diagnosis via the analysis of biomarkers on circulating extracellular vesicles (EVs) typically have lengthy sample workups, limited throughput or insufficient sensitivity, or do not use clinically validated biomarkers. Here we report the development and performance of a 96-well assay that integrates the enrichment of EVs by antibody-coated magnetic beads and the electrochemical detection, in less than one hour of total assay time, of EV-bound proteins after enzymatic amplification. By using the assay with a combination of antibodies for clinically relevant tumour biomarkers (EGFR, EpCAM, CD24 and GPA33) of colorectal cancer (CRC), we classified plasma samples from 102 patients with CRC and 40 non-CRC controls with accuracies of more than 96%, prospectively assessed a cohort of 90 patients, for whom the burden of tumour EVs was predictive of five-year disease-free survival, and longitudinally analysed plasma from 11 patients, for whom the EV burden declined after surgery and increased on relapse. Rapid assays for the detection of combinations of tumour biomarkers in plasma EVs may aid cancer detection and patient monitoring.

Second harmonic generation imaging - a new method for unraveling molecular information of starch.

We present a new method, second harmonic generation (SHG) imaging for the study of starch structure. SHG imaging can provide the structural organization and molecular orientation information of bio-tissues without centrosymmetry. In recent years, SHG has proven its capability in the study of crystallized bio-molecules such as collagen and myosin. Starch, the most important food source and a promising future energy candidate, has, for a decade, been shown to exhibit strong SHG response. By comparing SHG intensity from different starch species, we first identified that the SHG-active molecule is amylopectin, which accounts for the crystallinity in starch granules. With the aid of SHG polarization anisotropy, we extracted the complete χ((2)) tensor of amylopectin, which reflects the underlying molecular details. Through χ((2)) tensor analysis, three-dimensional orientation and packing symmetry of amylopectin are determined. The helical angle of the double-helix in amylopectin is also deduced from the tensor, and the value corresponds well to previous X-ray studies, further verifying amylopectin as SHG source. It is noteworthy that the nm-sized structure of amylopectin inside a starch granule can be determined by this far-field optical method with 1-µm excitation wavelength. Since SHG is a relatively new tool for plant research, a detailed understanding of SHG in starch structure will be useful for future high-resolution imaging and quantitative analyses for food/energy applications.

Plasmon-induced optical switching of electrical conductivity in porous anodic aluminum oxide films encapsulated with silver nanoparticle arrays.

We report on plasmon induced optical switching of electrical conductivity in two-dimensional (2D) arrays of silver (Ag) nanoparticles encapsulated inside nanochannels of porous anodic aluminum oxide (AAO) films. The reversible switching of photoconductivity greatly enhanced by an array of closely spaced Ag nanoparticles which are isolated from each other and from the ambient by thin aluminum oxide barrier layers are attributed to the improved electron transport due to the localized surface plasmon resonance and coupling among Ag nanoparticles. The photoconductivity is proportional to the power, and strongly dependent on the wavelength of light illumination. With Ag nanoparticles being isolated from the ambient environments by a thin layer of aluminum oxide barrier layer of controlled thickness in nanometers to tens of nanometers, deterioration of silver nanoparticles caused by environments is minimized. The electrochemically fabricated nanostructured Ag/AAO is inexpensive and promising for applications to integrated plasmonic circuits and sensors.

Direct near-field optical imaging of plasmonic resonances in metal nanoparticle pairs.

In this paper we investigate the near-field optical behavior of plasmon coupling in gold nanoparticle pairs. In particular, by performing series measurements through a fiber-collection mode near-field scanning optical microscope (NSOM), we directly observed the localized electromagnetic (EM) field distribution between two nanospheres is sensitively depended on the incident polarization and interparticle distance. The qualitative near-field observation and quantitative analysis facilitate more understanding of localized hot spots in surface-enhanced Raman scattering (SERS), and nano-applications in selectively controlling the spatial distribution of localized surface plasmon (SP) modes on a fabricated nanostructure by adjusting the polarization direction.

Broadband tunable optical parametric amplification from a single 50 MHz ultrafast fiber laser.

We have demonstrated a 0.7 microm - 1.9 microm wavelength-tunable light source based on a single-pass optical parametric amplification (OPA) in a multiperiod magnesium oxide-doped periodically poled lithium niobate crystal. The OPA pump was a frequency-doubled ultrafast ytterbium-doped fiber oscillator, and the residual 1040 nm laser power after frequency doubling was recycled to generate a supercontinuum seeding source. Compared with conventional OPAs, this system is free from timing jitter between the pump laser and the seeding source. Over 50% conversion efficiency was obtained with 10 nJ pump energy. Combined with a 50 MHz repetition rate, this versatile source is ideal for biomedical and spectroscopic applications.

Compact and Filter-Free Luminescence Biosensor for Mobile in Vitro Diagnoses.

We report a sensitive and versatile biosensing approach, LUCID (luminescence compact in vitro diagnostics), for quantitative molecular and cellular analyses. LUCID uses upconversion nanoparticles (UCNPs) as luminescent reporters in mutually exclusive photoexcitation and read-out sequences implemented on a smartphone. The strategy improves imaging signal-to-noise ratios, eliminating interference from excitation sources and minimizing autofluorescence, and thus enables filterless imaging. Here we developed a miniaturized detection system and optimized UCNPs for the system and biological applications. Nanoparticle luminescence lifetime was extended by controlling particle structure and composition. When tested with a range of biological targets, LUCID achieved high detection sensitivity (0.5 pM for protein and 0.1 pM for nucleic acids), differentiated bacterial samples, and allowed profiling of cells. In proof-of-concept clinical use, LUCID demonstrated effective screening of cancer cells in cervical brushing specimens, identifying patients at high risk for malignancy. These results suggest that LUCID could serve as a broadly applicable and inexpensive diagnostic platform.

The phase-response effect of size-dependent optical enhancement in a single nanoparticle.

We demonstrate detailed simulations and experiments of near-field phase-response in a single silver nanoparticle. The plasmon-photon interaction is directly observed in the vicinity of silver nanoparticles through a near-field scanning optical microscope (NSOM). Our results manifest the correlation of phase-response and size-dependent optical enhancement. Detailed interference behaviors between optical excitation and plasmon mediated re-radiation are revealed on a single particle basis. This observation facilitates nano-applications in controlling the spatial distribution of surface plasmon (SP) modes by means of nanostructures.

Integrated Magneto-Chemical Sensor For On-Site Food Allergen Detection.

Adverse food reactions, including food allergies, food sensitivities, and autoimmune reaction (e.g., celiac disease) affect 5-15% of the population and remain a considerable public health problem requiring stringent food avoidance and epinephrine availability for emergency events. Avoiding problematic foods is practically difficult, given current reliance on prepared foods and out-of-home meals. In response, we developed a portable, point-of-use detection technology, termed integrated exogenous antigen testing (iEAT). The system consists of a disposable antigen extraction device coupled with an electronic keychain reader for rapid sensing and communication. We optimized the prototype iEAT system to detect five major food antigens in peanuts, hazelnuts, wheat, milk, and eggs. Antigen extraction and detection with iEAT requires <10 min and achieves high-detection sensitivities (e.g., 0.1 mg/kg for gluten, lower than regulatory limits of 20 mg/kg). When testing under restaurant conditions, we were able to detect hidden food antigens such as gluten within "gluten-free" food items. The small size and rapid, simple testing of the iEAT system should help not only consumers but also other key stakeholders such as clinicians, food industries, and regulators to enhance food safety.

Multiparametric plasma EV profiling facilitates diagnosis of pancreatic malignancy.

Pancreatic ductal adenocarcinoma (PDAC) is usually detected late in the disease process. Clinical workup through imaging and tissue biopsies is often complex and expensive due to a paucity of reliable biomarkers. We used an advanced multiplexed plasmonic assay to analyze circulating tumor-derived extracellular vesicles (tEVs) in more than 100 clinical populations. Using EV-based protein marker profiling, we identified a signature of five markers (PDACEV signature) for PDAC detection. In our prospective cohort, the accuracy for the PDACEV signature was 84% [95% confidence interval (CI), 69 to 93%] but only 63 to 72% for single-marker screening. One of the best markers, GPC1 alone, had a sensitivity of 82% (CI, 60 to 95%) and a specificity of 52% (CI, 30 to 74%), whereas the PDACEV signature showed a sensitivity of 86% (CI, 65 to 97%) and a specificity of 81% (CI, 58 to 95%). The PDACEV signature of tEVs offered higher sensitivity, specificity, and accuracy than the existing serum marker (CA 19-9) or single-tEV marker analyses. This approach should improve the diagnosis of pancreatic cancer.