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.

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.

Magnetic Gold Nanoparticles with Idealized Coating for Enhanced Point-Of-Care Sensing.

Magnetic nanoparticles with hybrid sensing functions are in wide use for bioseparation, sensing, and in vivo imaging. Yet, nonspecific protein adsorption to the particle surface continues to present a technical challenge and diminishes the theoretical protein detection capabilities. Here, a magneto-plasmonic nanoparticle synthesis is developed that minimizes nonspecific protein adsorption. Building on the success of zwitterionic polymers, a highly stable and anergic nanomaterial, magnetic gold nanoparticles with idealized coating (MAGIC) is obtained with significantly lower serum protein adsorption compared to control nanoparticles coated with commonly used polymers (polyethylene glycol, polyethylenimine, or polyallylamine hydrochloride). MAGIC nanoparticles are able to sense specific bladder cancer biomarkers at low levels and in the presence of other proteins. This strategy may find wide spread applications for in vitro and in vivo sensing as well as isolations.

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.

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.

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.

Integrated Dual-Mode Chromatography to Enrich Extracellular Vesicles from Plasma.

Purifying extracellular vesicles (EVs) from complex biological fluids is a critical step in analyzing EVs molecularly. Plasma lipoprotein particles (LPPs) are a significant confounding factor as they outnumber EVs >104 -fold. Given their overlap in size, LPPs cannot be completely removed using standard size-exclusion chromatography. Density-based separation of LPPs can be applied but is impractical for routine use in clinical research and practice. Here a new separation approach, known as dual-mode chromatography (DMC), capable of enriching plasma EVs, and depleting LPPs is reported. DMC conveniently integrates two orthogonal separation steps in a single column device: i) size exclusion to remove high-density lipoproteins (HDLs) that are smaller than EVs; and ii) cation exchange to clear positively charged ApoB100-containing LPPs, mostly (very) low-density lipoproteins (V)LDLs, from negatively charged EVs. The strategy enables DMC to deplete most LPPs (>97% of HDLs and >99% of (V)LDLs) from human plasma, while retaining EVs (>30% of input). Furthermore, the two-in-one operation is fast (15 min per sample) and equipment-free. With abundant LPPs removed, DMC-prepared samples facilitate EV identification in imaging analyses and improve the accuracy for EV protein analysis.

Bead-Based Extracellular Vesicle Analysis Using Flow Cytometry.

Extracellular vesicles (EVs) represent promising circulating biomarkers for cancers, but their high-throughput analyses in clinical settings prove challenging due to lack of simple, fast, and robust EV assays. Here, a bead-based EV assay detected by flow cytometry is described, which integrates EV capture using microbeads with EV protein analyses by flow cytometry. The assay is fast (<4 h for 48 samples), robust, and compatible with conventional flow cytometry instruments for high-throughput EV analysis. With the method, a panel of pancreatic cancer biomarkers in EVs from plasma samples of pancreatic cancer patients is successfully analyzed. The assay is readily translatable to other biomarkers or cancer types and can be run with standard materials on conventional flow cytometers, making it highly flexible and adaptable to diverse research and clinical needs.

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.

Methods for Systematic Identification of Membrane Proteins for Specific Capture of Cancer-Derived Extracellular Vesicles.

Analysis of cancer-derived extracellular vesicles (EVs) in biofluids potentially provides a source of disease biomarkers. At present there is no procedure to systematically identify which antigens should be targeted to differentiate cancer-derived from normal host cell-derived EVs. Here, we propose a computational framework that integrates information about membrane proteins in tumors and normal tissues from databases: UniProt, The Cancer Genome Atlas, the Genotype-Tissue Expression Project, and the Human Protein Atlas. We developed two methods to assess capture of EVs from specific cell types. (1) We used palmitoylated fluorescent protein (palmtdTomato) to label tumor-derived EVs. Beads displaying antibodies of interest were incubated with conditioned medium from palmtdTomato-expressing cells. Bound EVs were quantified using flow cytometry. (2) We also showed that membrane-bound Gaussia luciferase allows the detection of cancer-derived EVs in blood of tumor-bearing animals. Our analytical and validation platform should be applicable to identify antigens on EVs from any tumor type.

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.

High-throughput intensity diffraction tomography with a computational microscope.

We demonstrate a motion-free intensity diffraction tomography technique that enables the direct inversion of 3D phase and absorption from intensity-only measurements for weakly scattering samples. We derive a novel linear forward model featuring slice-wise phase and absorption transfer functions using angled illumination. This new framework facilitates flexible and efficient data acquisition, enabling arbitrary sampling of the illumination angles. The reconstruction algorithm performs 3D synthetic aperture using a robust computation and memory efficient slice-wise deconvolution to achieve resolution up to the incoherent limit. We demonstrate our technique with thick biological samples having both sparse 3D structures and dense cell clusters. We further investigate the limitation of our technique when imaging strongly scattering samples. Imaging performance and the influence of multiple scattering is evaluated using a 3D sample consisting of stacked phase and absorption resolution targets. This computational microscopy system is directly built on a standard commercial microscope with a simple LED array source add-on, and promises broad applications by leveraging the ubiquitous microscopy platforms with minimal hardware modifications.

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.

Integrated Kidney Exosome Analysis for the Detection of Kidney Transplant Rejection.

Kidney transplant patients require life-long surveillance to detect allograft rejection. Repeated biopsy, albeit the clinical gold standard, is an invasive procedure with the risk of complications and comparatively high cost. Conversely, serum creatinine or urinary proteins are noninvasive alternatives but are late markers with low specificity. We report a urine-based platform to detect kidney transplant rejection. Termed iKEA (integrated kidney exosome analysis), the approach detects extracellular vesicles (EVs) released by immune cells into urine; we reasoned that T cells, attacking kidney allografts, would shed EVs, which in turn can be used as a surrogate marker for inflammation. We optimized iKEA to detect T-cell-derived EVs and implemented a portable sensing system. When applied to clinical urine samples, iKEA revealed high level of CD3-positive EVs in kidney rejection patients and achieved high detection accuracy (91.1%). Fast, noninvasive, and cost-effective, iKEA could offer new opportunities in managing transplant recipients, perhaps even in a home setting.

Direct detection of orchid viruses using nanorod-based fiber optic particle plasmon resonance immunosensor.

A fiber optic particle plasmon resonance (FOPPR) immunosensor is developed for label-free detection of orchid viruses that use gold nanorods (AuNRs) as the sensing material. The AuNRs are employed to create a near-infrared sensing window to solve the color interference problem of sample matrix for direct sensing of target analyte. This work cannot be achieved using gold nanospheres (AuNSs) because the signal of sample color absorption largely overlaps the signal of molecular recognition events in the visible spectrum, making the signal interpretation much more difficult. The AuNRs are immobilized on the unclad fiber core surface, and functionalized by antibodies which can specifically recognize the corresponding Cymbidium mosaic virus (CymMV) or Odontoglossum ringspot virus (ORSV) for rapid viral infection diagnosis. The refractive index resolution of the AuNR-FOPPR sensor is estimated to be 8×10(-6) RIU. The limits of detection (LODs) for CymMV and ORSV in leaf saps are 48 and 42 pg/mL, respectively, which are better than the LODs of 1200 pg/mL for both viruses obtained by enzyme-linked immunosorbent assay (ELISA). Exploiting the AuNR-FOPPR sensing strategy not only solves the color interference problem encountered by using AuNSs, but provides faster analysis, better reproducibility, and lower detection limit than ELISA. The sensor can distinguish between healthy and infected orchids in 10 min, and can further provide the quantitative analysis of infection level. It is potentially applicable to the quality control of orchid cultivation industry, but not limited to this, especially for creating special spectral sensing window for particular samples.

Probing substrate influence on graphene by analyzing Raman lineshapes.

We provide a new approach to identify the substrate influence on graphene surface. Distinguishing the substrate influences or the doping effects of charged impurities on graphene can be realized by optically probing the graphene surfaces, included the suspended and supported graphene. In this work, the line scan of Raman spectroscopy was performed across the graphene surface on the ordered square hole. Then, the bandwidths of G-band and 2D-band were fitted into the Voigt profile, a convolution of Gaussian and Lorentzian profiles. The bandwidths of Lorentzian parts were kept as constant whether it is the suspended and supported graphene. For the Gaussian part, the suspended graphene exhibits much greater Gaussian bandwidths than those of the supported graphene. It reveals that the doping effect on supported graphene is stronger than that of suspended graphene. Compared with the previous studies, we also used the peak positions of G bands, and I2D/IG ratios to confirm that our method really works. For the suspended graphene, the peak positions of G band are downshifted with respect to supported graphene, and the I2D/IG ratios of suspended graphene are larger than those of supported graphene. With data fitting into Voigt profile, one can find out the information behind the lineshapes.