Plasmonic nano-aperture label-free imaging of single small extracellular vesicles for cancer detection.

BACKGROUND: Small extracellular vesicle (sEV) analysis can potentially improve cancer detection and diagnostics. However, this potential has been constrained by insufficient sensitivity, dynamic range, and the need for complex labeling. METHODS: In this study, we demonstrate the combination of PANORAMA and fluorescence imaging for single sEV analysis. The co-acquisition of PANORAMA and fluorescence images enables label-free visualization, enumeration, size determination, and enables detection of cargo microRNAs (miRs). RESULTS: An increased sEV count is observed in human plasma samples from patients with cancer, regardless of cancer type. The cargo miR-21 provides molecular specificity within the same sEV population at the single unit level, which pinpoints the sEVs subset of cancer origin. Using cancer cells-implanted animals, cancer-specific sEVs from 20 µl of plasma can be detected before tumors were palpable. The level plateaus between 5-15 absolute sEV count (ASC) per µl with tumors ≥8 mm3. In healthy human individuals (N = 106), the levels are on average 1.5 ASC/µl (+/- 0.95) without miR-21 expression. However, for stage I-III cancer patients (N = 205), nearly all (204 out of 205) have levels exceeding 3.5 ASC/µl with an average of 12.2 ASC/µl (±9.6), and a variable proportion of miR-21 labeling among different tumor types with 100% cancer specificity. Using a threshold of 3.5 ASC/µl to test a separate sample set in a blinded fashion yields accurate classification of healthy individuals from cancer patients. CONCLUSIONS: Our techniques and findings can impact the understanding of cancer biology and the development of new cancer detection and diagnostic technologies.

Small extracellular vesicles (sEVs) are tiny particles derived from cells that can be detected in bodily fluids such as blood. Detecting sEVs and analyzing their contents may potentially help us to diagnose disease, for example by observing differences in sEV numbers or contents in the blood of patients with cancer versus healthy people. Here, we combine two imaging methods ­ our previously developed method PANORAMA and imaging of fluorescence emitted by sEVs­to visualize and count sEVs, determine their size, and analyze their cargo. We observe differences in sEV numbers and cargo in samples taken from healthy people versus people with cancer and are able to differentiate these two populations based on our analysis of sEVs. With further testing, our approach may be a useful tool for cancer diagnosis and provide insights into the biology of cancer and sEVs.

Functional Plasmonic Microscope: Characterizing the Metabolic Activity of Single Cells via Sub-nm Membrane Fluctuations.

Metabolic abnormalities are at the center of many diseases, and the capability to film and quantify the metabolic activities of a single cell is important for understanding the heterogeneities in these abnormalities. In this paper, a functional plasmonic microscope (FPM) is used to image and measure metabolic activities without fluorescent labels at a single-cell level. The FPM can accurately image and quantify the subnanometer membrane fluctuations with a spatial resolution of 0.5 µm in real time. These active cell membrane fluctuations are caused by metabolic activities across the cell membrane. A three-dimensional (3D) morphology of the bottom cell membrane was imaged and reconstructed with FPM to illustrate the capability of the microscope for cell membrane characterization. Then, the subnanometer cell membrane fluctuations of single cells were imaged and quantified with the FPM using HeLa cells. Cell metabolic heterogeneity is analyzed based on membrane fluctuations of each individual cell that is exposed to similar environmental conditions. In addition, we demonstrated that the FPM could be used to evaluate the therapeutic responses of metabolic inhibitors (glycolysis pathway inhibitor STF 31) on a single-cell level. The result showed that the metabolic activities significantly decrease over time, but the nature of this response varies, depicting cell heterogeneity. A low-concentration dose showed a reduced fluctuation frequency with consistent fluctuation amplitudes, while the high-concentration dose showcased a decreasing trend in both cases. These results have demonstrated the capabilities of the functional plasmonic microscope to measure and quantify metabolic activities for drug discovery.

Progressive and instantaneous nature of lithium nucleation discovered by dynamic and operando imaging.

The understanding of lithium (Li) nucleation and growth is important to design better electrodes for high-performance batteries. However, the study of Li nucleation process is still limited because of the lack of imaging tools that can provide information of the entire dynamic process. We developed and used an operando reflection interference microscope (RIM) that enables real-time imaging and tracking the Li nucleation dynamics at a single nanoparticle level. This dynamic and operando imaging platform provides us with critical capabilities to continuously monitor and study the Li nucleation process. We find that the formation of initial Li nuclei is not at the exact same time point, and Li nucleation process shows the properties of both progressive and instantaneous nucleation. In addition, the RIM allows us to track the individual Li nucleus's growth and extract spatially resolved overpotential map. The nonuniform overpotential map indicates that the localized electrochemical environments substantially influence the Li nucleation.

Single-Exosome Counting and 3D, Subdiffraction Limit Localization Using Dynamic Plasmonic Nanoaperture Label-Free Imaging.

Blood-circulating exosomes as a disease biomarker have great potential in clinical applications as they contain molecular information about their parental cells. However, label-free characterization of exosomes is challenging due to their small size. Without labeling, exosomes are virtually indistinguishable from other entities of similar size. Over recent years, several techniques have been developed to overcome the existing challenges. This paper demonstrates a new label-free approach based on dynamic PlAsmonic NanO-apeRture lAbel-free iMAging (D-PANORAMA), a bright-field technique implemented on arrayed gold nanodisks on invisible substrates (AGNIS). PANORAMA provides high surface sensitivity and has been shown to count single 25 nm polystyrene beads (PSB) previously. Herein, we show that using the dynamic imaging mode, D-PANORAMA can yield 3-dimensional, sub-diffraction limited localization of individual 25 nm beads. Furthermore, we demonstrate D-PANORAMA's capability to size, count, and localize the 3-dimensional, sub-diffraction limited position of individual exosomes as they bind to the AGNIS surface. We emphasize the importance of both the in-plane and out-of-plane localization, which exploit the synergy of 2-dimensional imaging and the intensity contrast.

Exploring the synergy of radiative coupling and substrate undercut in arrayed gold nanodisks for economical, ultra-sensitive label-free biosensing.

We report radiatively coupled arrayed gold nanodisks on invisible substrate (AGNIS) as a cost-effective, high-performance platform for nanoplasmonic biosensing. By substrate undercut, the electric field distribution around the nanodisks has been restored to as if the nanodisks were surrounded by a single medium, thereby provides analyte accessibility to otherwise buried enhanced electric field. The AGNIS substrate has been fabricated by wafer-scale nanosphere lithography without the need for costly lithography. The LSPR blue-shifting behavior synergistically contributed by radiative coupling and substrate undercut have been investigated for the first time, which culminates in a remarkable refractive index sensitivity increase from 207 nm/RIU to 578 nm/RIU. The synergy also improves surface sensitivity to monolayer neutravidin-biotin binding from 7.4 nm to 20.3 nm with the limit of detection (LOD) of neutravidin at 50 fM, which is among the best label-free results reported to date on this specific surface binding reaction. As a potential cancer diagnostic application, extracellular vesicles such as exosomes excreted by cancer and normal cells were measured with a LOD within 112-600 (exosomes/µL), which would be sufficient in many clinical applications. Using CD9, CD63, and CD81 antibodies, label-free profiling has shown increased expression of all three surface antigens in cancer-derived exosomes. This work demonstrates, for the first time, strong synergy of arrayed radiative coupling and substrate undercut can enable economical, ultrasensitive biosensing in the visible light spectrum where high-quality, low-cost silicon detectors are readily available for point-of-care applications.

Nanoporous Metals: From Plasmonic Properties to Applications in Enhanced Spectroscopy and Photocatalysis.

The field of plasmonics is capable of enabling interesting applications in different wavelength ranges, spanning from the ultraviolet up to the infrared. The choice of plasmonic material and how the material is nanostructured has significant implications for ultimate performance of any plasmonic device. Artificially designed nanoporous metals (NPMs) have interesting material properties including large specific surface area, distinctive optical properties, high electrical conductivity, and reduced stiffness, implying their potentials for many applications. This paper reviews the wide range of available nanoporous metals (such as Au, Ag, Cu, Al, Mg, and Pt), mainly focusing on their properties as plasmonic materials. While extensive reports on the use and characterization of NPMs exist, a detailed discussion on their connection with surface plasmons and enhanced spectroscopies as well as photocatalysis is missing. Here, we report on different metals investigated, from the most used nanoporous gold to mixed metal compounds, and discuss each of these plasmonic materials' suitability for a range of structural design and applications. Finally, we discuss the potentials and limitations of the traditional and alternative plasmonic materials for applications in enhanced spectroscopy and photocatalysis.

Imaging the Electrochemical Impedance of Single Cells via Conductive Polymer Thin Film.

Many fundamentally important biological phenomena involve the cells to establish and break down the adhesive interactions with the substrate. Here, we report a novel optical method that could directly image the electrochemical impedance of cell-substrate interactions at the single cell level with conventional microscopes and cameras. A thin conductive polymer layer on top of the ITO substrate (poly(3,4-ethylenedioxythiophene) poly(styrenesulfonate), PEDOT:PSS) is used as the impedance imaging and sensing layer. A sinusoidal electrochemical potential is applied to the conductive polymer film, and the ion intercalation and transportation in the PEDOT:PSS layer will change the absorption spectrum of the polymer film. The attachment of the cells to the substrate will block and affect the ion doping and dedoping process, and therefore change the color of the polymer film. This process can be captured by any upright or inverted microscope with a simple camera. Utilizing this method, we have successfully imaged the impedance of single-cell attachment, observed the variations of cell-substrate interactions, and measured the impedance changes at different stages of the attachment process. This paper has proposed and successfully demonstrated a new strategy that translates the electrochemical impedance information to an optical signal that could be imaged and used to quantify the local responses. In addition, this method does not need any specially designed optical setup, which may lead to its broad applications in the clinics and biological research laboratories.

Directed Concentrating of Micro-/Nanoparticles via Near-Infrared Laser Generated Plasmonic Microbubbles.

Directed concentrating of micro- and nanoparticles via laser-generated plasmonic microbubbles in a liquid environment is an emerging technology. For effective heating, visible light has been primarily employed in existing demonstrations. In this paper, we demonstrate a new plasmonic platform based on nanoporous gold disk (NPGD) array. Thanks to the highly tunable localized surface plasmon resonance of the NPGD array, microbubbles of controlled size can be generated by near-infrared (NIR) light. Using NIR light provides several key advantages over visible light in less interference with standard microscopy and fluorescence imaging, preventing fluorescence photobleaching, less susceptible to absorption and scattering in turbid biological media, and much reduced photochemistry, phototoxicity, and so forth. The large surface-to-volume ratio of NPGD further facilitates the heat transfer from these gold nanoheaters to the surroundings. While the microbubble is formed, the surrounding liquid circulates and direct microparticles randomly dispersed in the liquid to the bottom NPGD surface, which can be made to yield a unique collection of 3D hollow dome microstructures with bubbles larger than 5 µm. Such capability can also be employed in concentrating suspended colloidal nanoparticles at desirable sites and with the preferred configuration enhancing the sensor performance. Specifically, the interaction among concentrated nanoparticles and their interactions with the underlying substrate have been investigated for the first time. These collections have been characterized using optical microscopy, scanning electron microscopy, hyperspectral localized surface plasmon resonance imaging, and hyperspectral Raman imaging. In addition to various micro- and nanoparticles, the plasmonic microbubbles are also shown to collect biological cells and extracellular nanovesicles such as exosomes. By using a spatial light modulator to project the laser in arbitrary patterns, parallel concentrating can be achieved to fabricate an array of clusters.

Plasmonic nano-aperture label-free imaging (PANORAMA).

Label-free optical imaging of nanoscale objects faces fundamental challenges. Techniques based on propagating surface plasmon resonance (SPR) and localized surface plasmon resonance (LSPR) have shown promises. However, challenges remain to achieve diffraction-limited resolution and better surface localization in SPR imaging. LSPR imaging with dark-field microscopy on metallic nanostructures suffers from low light throughput and insufficient imaging capacity. Here we show ultra-near-field index modulated PlAsmonic NanO-apeRture lAbel-free iMAging (PANORAMA) which uniquely relies on unscattered light to detect sub-100 nm dielectric nanoparticles. PANORAMA provides diffraction-limited resolution, higher surface sensitivity, and wide-field imaging with dense spatial sampling. Its system is identical to a standard bright-field microscope with a lamp and a camera - no laser or interferometry is needed. In a parallel fashion, PANORAMA can detect, count and size individual dielectric nanoparticles beyond 25 nm, and dynamically monitor their distance to the plasmonic surface at millisecond timescale.

Optically Tunable Tin Oxide-Coated Hollow Gold-Silver Nanorattles for Use in Solar-Driven Applications.

Core@shell metal nanoparticles have emerged as promising photocatalysts because of their strong and tunable plasmonic properties; however, marked improvements in photocatalytic efficiency are needed if these materials are to be widely used in practical applications. Accordingly, the design of new and functional light-responsive nanostructures remains a central focus of nanomaterial research. To this end, we report the synthesis of nanorattles comprising hollow gold-silver nanoshells encapsulated within vacuous tin oxide shells of adjustable thicknesses (∼10 and ∼30 nm for the two examples prepared in this initial report). These composite nanorattles exhibited broad tunable optical extinctions ranging from ultraviolet to near-infrared spectral regions (i.e., 300-745 nm). Zeta potential measurements showed a large negative surface charge of approximately -35 mV, which afforded colloidal stability to the nanorattles in aqueous solution. We also characterized the nanorattles structurally and compositionally using scanning electron microscopy, transmission electron microscopy, and energy-dispersive X-ray spectroscopy. Futhermore, finite-difference time-domain simulation and photoluminescence properties of the composited nanoparticles were investigated. Collectively, these studies indicate that our tin oxide-coated hollow gold-silver nanorattles are promising candidates for use in solar-driven applications.

Magnetic Active Water Filter Membrane for Induced Heating to Remove Biofoulants.

Filter membrane processes are water purification methods that use a partially permeable membrane to separate contaminants from drinking water and wastewater. Although highly effective, they suffer from biofouling due to the aggregation of bacteria and contaminants from the filtrate, thus rendering the membrane unusable. Consequently, the membrane needs to be replaced on a regular basis, which interrupts filtration operation, reduces throughput, and increases production cost. To address this issue, we have developed a new method to remove biofoulants via induction heating on a modified membrane with magnetite (Fe3O4) magnetic nanoparticles (MNPs) coating. Under applied alternating magnetic field (AMF), the surface temperature of the MNPs coating reaches 180 °C with a heating rate of 1.03 °C/s, which disintegrates biofoulants generated by model bacteria (Bacillus subtilis) and by those present in environmental water samples collected from a local lake. The heating process is capable of cleaning biofoulants for several cycles without damaging the filtration function of the membrane. Furthermore, magnetic induction heating on the modified membrane allows uniform high-intensity heat generation on a large surface in only a few minutes using inexpensive MNPs, which can potentially be scaled up for industrial applications.

Single red blood cell analysis reveals elevated hemoglobin in poikilocytes.

Abnormally shaped red blood cells (RBCs), called poikilocytes, can cause anemia. At present, the biochemical abnormalities in poikilocytes are not well understood. Normal RBCs and poikilocytes were analyzed using whole-blood and single-cell methods. Poikilocytes were induced in rat blood by intragastrically administering titanium dioxide (TiO2) nanoparticles. Complete blood count and inductively coupled plasma mass spectrometry analyses were performed on whole-blood to measure average RBC morphology, blood hemoglobin (HGB), iron content, and other blood parameters. Follow-up confocal Raman spectroscopy was performed on single RBCs to analyze cell-type-specific HGB content. Two types of poikilocytes, acanthocytes and echinocytes, were observed in TiO2 blood samples, along with normal RBCs. Acanthocytes (diameter 7.7 ± 0.5 µm) and echinocytes (7.6 ± 0.6 µm) were microscopically larger (p < 0.05) than normal RBCs (6.6 ± 0.4 µm) found in control blood samples (no TiO2 administration). Similarly, mean corpuscular volume was higher (p < 0.05) in TiO2 whole-blood (70.70 ± 1.97 fl) than in control whole-blood (67.42 ± 2.03 fl). Poikilocytes also had higher HGB content. Mean corpuscular hemoglobin was higher (p < 0.05) in TiO2 whole-blood (21.84 ± 0.75 pg) than in control whole-blood (20.8 ± 0.32 pg). Iron content was higher (p < 0.001) in TiO2 whole-blood (697.0 ± 24.5 mg / l) than in control whole-blood (503.4 ± 38.5 mg / l), which supports elevated HGB as iron is found in HGB. HGB-associated Raman bands at 1637, 1585, and 1372 cm - 1 had higher (p < 0.001) amplitudes in acanthocytes and echinocytes than in RBCs from control blood and normal RBCs from TiO2 blood. Further, the 1585-cm - 1 band had a lower (p < 0.05) amplitude in normal RBCs from TiO2 versus control RBCs. This represents biochemical abnormalities in normal appearing RBCs. Overall, poikilocytes, especially acanthocytes, have elevated HGB.

10×-Enhanced Heterogeneous Nanocatalysis on a Nanoporous Gold Disk Array with High-Density Hot Spots.

Certain noble metal nanostructures as heterogeneous photocatalysts have drawn significant attention in the recent past because of their unique optical properties which lead to the excitation of localized surface plasmon resonance (LSPR). The LSPR concentrates electromagnetic fields to the surfaces and its relaxation processes can convert photon energy to energetic charge carriers or heat, which can be subsequently harvested to enhance surface catalysis. Here, we report the catalytic performance of a novel plasmonic nanostructure, disk-shaped nanoporous gold (NPG) nanoparticles or simply NPG disks, using a well-tested reduction pathway of resazurin to resorufin. We show that the catalytic reaction rate of NPG disks is enhanced by 10-fold upon external light illumination because of the excitation of LSPR. The plasmon-enhanced catalytic reaction follows a linear-to-superlinear transition in the rate dependence on the input light power. In addition, the light input results in a room temperature reaction rate equivalent to that of an ambient temperature of 70 °C. Together, the results support that hot charge carriers play the dominant role in the enhancement.

Investigation of Thermal Properties of Graphene-Coated Membranes by Laser Irradiation to Remove Biofoulants.

In the present study, we take advantage of the high thermal conductivity of graphene nanomaterials to develop a filter that can be easily cleaned via laser irradiation after biofouling occurs. In this investigation, the intensity of the laser beam and the amount of graphene used for membrane coating were investigated with Bacillus subtilis to achieve the most efficient removal of biofoulants. Thermographic measurements of glass microfiber filters coated with 500 µg of graphene showed an increase in temperature of about 328 ± 9 °C in about 6 s when the filters were irradiated with a 21.6 W/cm-2 laser intensity, which allowed successful removal of biofoulants. The thermal cleaning was effective for at least four filtrations without impacting the subsequent microbial removals, which were of ∼5 log for each filtration step followed by laser irradiation. Additionally, the permeability of the coated filters only dropped from 17.8 to 15.9 L/m2s after the laser cleaning procedure. The cleaning procedure was validated by using bayou water with a complex composition of biofoulants. Graphene-coated membranes coupled with laser irradiation afford a very fast and nonhazardous approach to clean biofoulants on graphene-coated membranes.

Symmetry Breaking-Induced Plasmonic Mode Splitting in Coupled Gold-Silver Alloy Nanodisk Array for Ultrasensitive RGB Colorimetric Biosensing.

We report the first observation of symmetry breaking-induced mode splitting in coupled gold-silver alloy nanodisk array (ANA). According to the plasmonic hybridization picture, the original localized surface plasmon resonance (LSPR) of individual nanodisk is split into a pair of high and low energy modes when placed in between a superstrate and a substrate. Although well studied in single silver nanoparticles, the high energy mode has been largely suppressed in gold nanoparticles, which nevertheless are more chemically robust and have superior environmental stability. Herein, we show that the high energy mode can be partially restored and precisely engineered to ∼540 nm for silver-rich alloy nanodisk which has excellent environmental stability. However, peak broadening and red-shifting occur due to plasmonic dephasing when the nanodisk diameter increases. We next demonstrate that a far-field coupled ANA fabricated by low-cost nanosphere lithography can fully restore the high energy mode with electric field concentration extended into the superstrate, thereby imparting greater sensitivity to local refractive index changes. The high energy mode at 540 nm is of key importance for color change detection using low-cost RGB cameras/human vision and broadband light sources (e.g., the sun). The index sensitivity of ANA is the highest among existing plasmonic arrays (particles or holes) within a similar resonance wavelength region. We demonstrate colorimetric detection of sub-nanomolar and sub-monolayer biotin-streptavidin surface binding with a smartphone camera and a white light lamp. The high performance yet low-cost fabrication and detection technology could potentially result in affordable point-of-care biosensing technologies.

Smartphone Nanocolorimetry for On-Demand Lead Detection and Quantitation in Drinking Water.

Lead ions (Pb2+) contamination in drinking water, a major source of lead poisoning to the general population, is typically detected by bulky and costly laboratory analytical instrument. A mobile analytical device for rapid Pb2+ sensing is a growing demand. Herein, we report smartphone nanocolorimetry (SNC) as a new technique to detect and quantify dissolved Pb2+ in drinking water. Specifically, we have employed a single-step sedimentation approach by mixing a controlled quantity of chromate ion (CrO42-) to react with Pb2+ containing solutions to form highly insoluble lead chromate (PbCrO4) nanoparticles as vivid yellow precipitates. This is followed by microscopic color detection and intensity quantitation at nanoscale level using dark-field smartphone microscopy. The sum of the intensity of yellow pixels bears a highly reproducible relationship with Pb2+ concentration between 1.37 and 175 ppb in deionized water and 5-175 ppb in city tap water. In contrast to traditional colorimetric techniques analyzing bulk color changes, SNC achieves unparalleled sensitivity by combining nanocolorimetry with dark-field microscopy and mobilized the metal ions detection by integrating the detection into the smartphone microscope platform. SNC is rapid and low-cost and has the potential to enable individual citizens to examine Pb2+ content in drinking water on-demand in virtually any environmental setting.

Integrated Nanogap Platform for Sub-Volt Dielectrophoretic Trapping and Real-Time Raman Imaging of Biological Nanoparticles.

A rapid, label-free, and broadly applicable chemical analysis platform for nanovesicles and subcellular components is highly desirable for diagnostic assays. We demonstrate an integrated nanogap plasmonic sensing platform that combines subvolt dielectrophoresis (DEP) trapping, gold nanoparticles (AuNPs), and a lineated illumination scheme for real-time, surface-enhanced Raman spectroscopy (SERS) imaging of biological nanoparticles. Our system is capable of isolating suspended sub-100 nm vesicles and imaging the Raman spectra of their cargo within seconds, 100 times faster than conventional point-scan Raman systems. Bare AuNPs are spiked into solution and simultaneously trapped with the nanovesicles along the gap to boost local optical fields. In addition, our platform offers simultaneous and delay-free spatial and temporal multiplexing functionality. These nanogap devices can be mass-produced via atomic layer lithography and provide a practical platform for high-speed SERS analysis of biological nanoparticles.

Photothermal generation of programmable microbubble array on nanoporous gold disks.

We present a novel technique to generate microbubbles photothermally by continuous-wave laser irradiation of nanoporous gold disk (NPGD) array covered microfluidic channels. When a single laser spot is focused on the NPGDs, a microbubble can be generated with controlled size by adjusting the laser power. The dynamics of both bubble growth and shrinkage are studied. Using computer-generated holography on a spatial light modulator (SLM), simultaneous generation of multiple microbubbles at arbitrary locations with independent control is demonstrated. A potential application of flow manipulation is demonstrated using a microfluidic X-shaped junction. The advantages of this technique are flexible bubble generation locations, long bubble lifetimes, no need for light-adsorbing dyes, high controllability over bubble size, and relatively lower power consumption.

Compressed sensing hyperspectral imaging in the 0.9-2.5 µm shortwave infrared wavelength range using a digital micromirror device and InGaAs linear array detector.

A hyperspectral imaging system based on compressed sensing has been developed to image in the 0.9-2.5 µm shortwave infrared wavelengths. With a programmable digital micromirror device utilized as spatial light modulator, we have successfully performed spectrally resolved image reconstruction with a 256-element InGaAs linear array detector without traditional raster scanning or a push-broom mechanism by a compressed sensing (CS) single-pixel camera approach. The chemical sensitivity of the imaging sensor to near-infrared (NIR) overtone signatures of hydrocarbons was demonstrated using hydrocarbon and ink patterns on glass, showing spectral selectivity for the chemical components. Compared to point-by-point raster scanning, we show that the CS scheme can effectively accelerate image acquisition with lower but reasonable quality.