Design and Optimization of a Silicon-Based Electrokinetic Microchip for Sensitive Detection of Small Extracellular Vesicles.

Detection of analytes using streaming current has previously been explored using both experimental approaches and theoretical analyses of such data. However, further developments are needed for establishing a viable microchip that can be exploited to deliver a sensitive, robust, and scalable biosensor device. In this study, we demonstrated the fabrication of such a device on silicon wafer using a scalable silicon microfabrication technology followed by characterization and optimization of this sensor for detection of small extracellular vesicles (sEVs) with sizes in the range of 30 to 200 nm, as determined by nanoparticle tracking analyses. We showed that the sensitivity of the devices, assessed by a common protein-ligand pair and sEVs, significantly outperforms previous approaches using the same principle. Two versions of the microchips, denoted as enclosed and removable-top microchips, were developed and compared, aiming to discern the importance of high-pressure measurement versus easier and better surface preparation capacity. A custom-built chip manifold allowing easy interfacing with standard microfluidic connections was also constructed. By investigating different electrical, fluidic, morphological, and fluorescence measurements, we show that while the enclosed microchip with its robust glass-silicon bonding can withstand higher pressure and thus generate higher streaming current, the removable-top configuration offers several practical benefits, including easy surface preparation, uniform probe conjugation, and improvement in the limit of detection (LoD). We further compared two common surface functionalization strategies and showed that the developed microchip can achieve both high sensitivity for membrane protein profiling and low LoD for detection of sEV detection. At the optimum working condition, we demonstrated that the microchip could detect sEVs reaching an LoD of 104 sEVs/mL (when captured by membrane-sensing peptide (MSP) probes), which is among the lowest in the so far reported microchip-based methods.

Analyses of single extracellular vesicles from non-small lung cancer cells to reveal effects of epidermal growth factor receptor inhibitor treatments.

Precision cancer medicine has changed the treatment landscape of non-small cell lung cancer (NSCLC) as illustrated by the introduction of tyrosine kinase inhibitors (TKIs) towards mutated epidermal growth factor receptor (EGFR). However, as responses to EGFR-TKIs are heterogenous among NSCLC patients, there is a need for ways to early monitor changes in treatment response in a non-invasive way e.g., in patient's blood samples. Recently, extracellular vesicles (EVs) have been identified as a source of tumor biomarkers which could improve on non-invasive liquid biopsy-based diagnosis of cancer. However, the heterogeneity in EVs is high. Putative biomarker candidates may be hidden in the differential expression of membrane proteins in a subset of EVs hard to identify using bulk techniques. Using a fluorescence-based approach, we demonstrate that a single-EV technique can detect alterations in EV surface protein profiles. We analyzed EVs isolated from an EGFR-mutant NSCLC cell line, which is refractory to EGFR-TKIs erlotinib and responsive to osimertinib, before and after treatment with these drugs and after cisplatin chemotherapy. We studied expression level of five proteins; two tetraspanins (CD9, CD81), and three markers of interest in lung cancer (EGFR, programmed death-ligand 1 (PD-L1), human epidermal growth factor receptor 2 (HER2)). The data reveal alterations induced by the osimertinib treatment compared to the other two treatments. These include the growth of the PD-L1/HER2-positive EV population, with the largest increase in vesicles exclusively expressing one of the two proteins. The expression level per EV decreased for these markers. On the other hand, both the TKIs had a similar effect on the EGFR-positive EV population.

Plasmon-Enhanced Fluorescence of Single Quantum Dots Immobilized in Optically Coupled Aluminum Nanoholes.

Fluorescence-based optical sensing techniques have continually been explored for single-molecule detection targeting myriad biomedical applications. Improving signal-to-noise ratio remains a prioritized effort to enable unambiguous detection at single-molecule level. Here, we report a systematic simulation-assisted optimization of plasmon-enhanced fluorescence of single quantum dots based on nanohole arrays in ultrathin aluminum films. The simulation is first calibrated by referring to the measured transmittance in nanohole arrays and subsequently used for guiding their design. With an optimized combination of nanohole diameter and depth, the variation of the square of simulated average volumetric electric field enhancement agrees excellently with that of experimental photoluminescence enhancement over a large range of nanohole periods. A maximum 5-fold photoluminescence enhancement is statistically achieved experimentally for the single quantum dots immobilized at the bottom of simulation-optimized nanoholes in comparison to those cast-deposited on bare glass substrate. Hence, boosting photoluminescence with optimized nanohole arrays holds promises for single-fluorophore-based biosensing.

Multi-marker profiling of extracellular vesicles using streaming current and sequential electrostatic labeling.

High heterogeneity in the membrane protein expression of small extracellular vesicles (sEVs) means that bulk methods relying on antibody-based capture for expression analysis have a drawback that each type of antibody may capture a different sub-population. An improved approach is to capture a representative sEV population, without any bias, and then perform a multiplexed protein expression analysis on this population. However, such a possibility has been largely limited to fluorescence-based methods. Here, we present a novel electrostatic labelling strategy and a microchip-based all-electric method for membrane protein analysis of sEVs. The method allows us to profile multiple surface proteins on the captured sEVs using alternating charge labels. It also permits the comparison of expression levels in different sEV-subtypes. The proof of concept was tested by capturing sEVs both non-specifically (unbiased) as well as via anti-CD9 capture probes (biased), and then profiling the expression levels of various surface proteins using the charge labelled antibodies. The method is the first of its kind, demonstrating an all-electrical and microchip based method that allows for unbiased analysis of sEV membrane protein expression, comparison of expression levels in different sEV subsets, and fractional estimation of different sEV sub-populations. These results were also validated in parallel using a single-sEV fluorescence technique.

Exploiting Electrostatic Interaction for Highly Sensitive Detection of Tumor-Derived Extracellular Vesicles by an Electrokinetic Sensor.

We present an approach to improve the detection sensitivity of a streaming current-based biosensor for membrane protein profiling of small extracellular vesicles (sEVs). The experimental approach, supported by theoretical investigation, exploits electrostatic charge contrast between the sensor surface and target analytes to enhance the detection sensitivity. We first demonstrate the feasibility of the approach using different chemical functionalization schemes to modulate the zeta potential of the sensor surface in a range -16.0 to -32.8 mV. Thereafter, we examine the sensitivity of the sensor surface across this range of zeta potential to determine the optimal functionalization scheme. The limit of detection (LOD) varied by 2 orders of magnitude across this range, reaching a value of 4.9 × 106 particles/mL for the best performing surface for CD9. We then used the optimized surface to profile CD9, EGFR, and PD-L1 surface proteins of sEVs derived from non-small cell lung cancer (NSCLC) cell-line H1975, before and after treatment with EGFR tyrosine kinase inhibitors, as well as sEVs derived from pleural effusion fluid of NSCLC adenocarcinoma patients. Our results show the feasibility to monitor CD9, EGFR, and PD-L1 expression on the sEV surface, illustrating a good prospect of the method for clinical application.

Multiplexed electrokinetic sensor for detection and therapy monitoring of extracellular vesicles from liquid biopsies of non-small-cell lung cancer patients.

Liquid biopsies based on extracellular vesicles (EVs) represent a promising tool for treatment monitoring of tumors, including non-small-cell lung cancers (NSCLC). In this study, we report on a multiplexed electrokinetic sensor for surface protein profiling of EVs from clinical samples. The method detects the difference in the streaming current generated by EV binding to the surface of a functionalized microcapillary, thereby estimating the expression level of a marker. Using multiple microchannels functionalized with different antibodies in a parallel fluidic connection, we first demonstrate the capacity for simultaneous detection of multiple surface markers in small EVs (sEVs) from NSCLC cells. To investigate the prospects of liquid biopsies based on EVs, we then apply the method to profile sEVs isolated from the pleural effusion (PE) fluids of five NSCLC patients with different genomic alterations (ALK, KRAS or EGFR) and applied treatments (chemotherapy, EGFR- or ALK-tyrosine kinase inhibitors). The vesicles were targeted against CD9, as well as EGFR and PD-L1, two treatment targets in NSCLC. The electrokinetic signals show detection of these markers on sEVs, highlighting distinct interpatient differences, e.g., increased EGFR levels in sEVs from a patient with EGFR mutation as compared to an ALK-fusion one. The sensors also detect differences in PD-L1 expressions. The analysis of sEVs from a patient prior and post ALK-TKI crizotinib treatment reveals significant increases in the expressions of some markers (EGFR and PD-L1). These results hold promise for the application of the method for tumor treatment monitoring based on sEVs from patient liquid biopsies.

Multiparametric Profiling of Single Nanoscale Extracellular Vesicles by Combined Atomic Force and Fluorescence Microscopy: Correlation and Heterogeneity in Their Molecular and Biophysical Features.

Being a key player in intercellular communications, nanoscale extracellular vesicles (EVs) offer unique opportunities for both diagnostics and therapeutics. However, their cellular origin and functional identity remain elusive due to the high heterogeneity in their molecular and physical features. Here, for the first time, multiple EV parameters involving membrane protein composition, size and mechanical properties on single small EVs (sEVs) are simultaneously studied by combined fluorescence and atomic force microscopy. Furthermore, their correlation and heterogeneity in different cellular sources are investigated. The study, performed on sEVs derived from human embryonic kidney 293, cord blood mesenchymal stromal and human acute monocytic leukemia cell lines, identifies both common and cell line-specific sEV subpopulations bearing distinct distributions of the common tetraspanins (CD9, CD63, and CD81) and biophysical properties. Although the tetraspanin abundances of individual sEVs are independent of their sizes, the expression levels of CD9 and CD63 are strongly correlated. A sEV population co-expressing all the three tetraspanins in relatively high abundance, however, having average diameters of <100 nm and relatively low Young moduli, is also found in all cell lines. Such a multiparametric approach is expected to provide new insights regarding EV biology and functions, potentially deciphering unsolved questions in this field.

Electrokinetic sandwich assay and DNA mediated charge amplification for enhanced sensitivity and specificity.

An electrical immuno-sandwich assay utilizing an electrokinetic-based streaming current method for signal transduction is proposed. The method records the changes in streaming current, first when a target molecule binds to the capture probes immobilized on the inner surface of a silica micro-capillary, and then when the detection probes interact with the bound target molecules on the surface. The difference in signals in these two steps constitute the response of the assay, which offers better target selectivity and a linear concentration dependent response for a target concentration within the range 0.2-100 nM. The proof of concept is demonstrated by detecting different concentrations of Immunoglobulin G (IgG) in both phosphate buffered saline (PBS) and spiked in E. coli cell lysate. A superior target specificity for the sandwich assay compared to the corresponding direct assay is demonstrated along with a limit of detection of 90 pM in PBS. The prospect of improving the detection sensitivity was theoretically analysed, which indicated that the charge contrast between the target and the detection probe plays a crucial role in determining the signal. This aspect was then experimentally validated by modulating the zeta potential of the detection probe by conjugating negatively charged DNA oligonucleotides. The length of the conjugated DNA was varied from 5 to 30 nucleotides, altering the zeta potential of the detection probe from -9.3 ± 0.8 mV to -20.1 ± 0.9 mV. The measurements showed a clear and consistent enhancement of detection signal as a function of DNA lengths. The results presented here conclusively demonstrate the role of electric charge in detection sensitivity as well as the prospect for further improvement. The study therefore is a step forward in developing highly selective and sensitive electrokinetic assays for possible application in clinical investigations.

Comparison and optimization of nanoscale extracellular vesicle imaging by scanning electron microscopy for accurate size-based profiling and morphological analysis.

Nanosized extracellular vesicles (EVs) have been found to play a key role in intercellular communication, offering opportunities for both disease diagnostics and therapeutics. However, lying below the diffraction limit and also being highly heterogeneous in their size, morphology and abundance, these vesicles pose significant challenges for physical characterization. Here, we present a direct visual approach for their accurate morphological and size-based profiling by using scanning electron microscopy (SEM). To achieve that, we methodically examined various process steps and developed a protocol to improve the throughput, conformity and image quality while preserving the shape of EVs. The study was performed with small EVs (sEVs) isolated from a non-small-cell lung cancer (NSCLC) cell line as well as from human serum, and the results were compared with those obtained from nanoparticle tracking analysis (NTA). While the comparison of the sEV size distributions showed good agreement between the two methods for large sEVs (diameter > 70 nm), the microscopy based approach showed a better capacity for analyses of smaller vesicles, with higher sEV counts compared to NTA. In addition, we demonstrated the possibility of identifying non-EV particles based on size and morphological features. The study also showed process steps that can generate artifacts bearing resemblance with sEVs. The results therefore present a simple way to use a widely available microscopy tool for accurate and high throughput physical characterization of EVs.

Influence of molecular size and zeta potential in electrokinetic biosensing.

Electrokinetic principles such as streaming current and streaming potential are extensively used for surface characterization. Recently, they have also been used in biosensors, resulting in enhanced sensitivity and simpler device architecture. Theoretical models regarding streaming current/potential studies of particle-covered surfaces have identified features such as the particle size, shape and surface charge to influence the electrokinetic signals and consequently, the sensitivity and effective operational regime of the biosensor. By using a set of well-characterized proteins with varying size and net surface charge, this article experimentally verifies the theoretical predictions about their influence on the sensor signal. Increasing protein size was shown to enhance the signal when their net surface charge was either opposite to that of the sensor surface, or close to zero, in agreement with the theoretical predictions. However, the effect gradually saturated as the protein size exceeded the coulomb screening length of the electrolyte. In contrast, the proteins containing the same type of charge as the surface showed little or no difference, except that the signal inverted. The magnitude of the surface charge was also shown to influence the signal. The sensitivity of the technique for protein detection varied over two orders of magnitude, depending upon the size and surface charge. Furthermore, the capacity of the electrokinetic method for direct electrical detection of various proteins, including those carrying little or no net electric charges, is demonstrated.

Label-Free Surface Protein Profiling of Extracellular Vesicles by an Electrokinetic Sensor.

Small extracellular vesicles (sEVs) generated from the endolysosomal system, often referred to as exosomes, have attracted interest as a suitable biomarker for cancer diagnostics, as they carry valuable biological information and reflect their cells of origin. Herein, we propose a simple and inexpensive electrical method for label-free detection and profiling of sEVs in the size range of exosomes. The detection method is based on the electrokinetic principle, where the change in the streaming current is monitored as the surface markers of the sEVs interact with the affinity reagents immobilized on the inner surface of a silica microcapillary. As a proof-of-concept, we detected sEVs derived from the non-small-cell lung cancer (NSCLC) cell line H1975 for a set of representative surface markers, such as epidermal growth factor receptor (EGFR), CD9, and CD63. The detection sensitivity was estimated to be ∼175000 sEVs, which represents a sensor surface coverage of only 0.04%. We further validated the ability of the sensor to measure the expression level of a membrane protein by using sEVs displaying artificially altered expressions of EGFR and CD63, which were derived from NSCLC and human embryonic kidney (HEK) 293T cells, respectively. The analysis revealed that the changes in EGFR and CD63 expressions in sEVs can be detected with a sensitivity in the order of 10% and 3%, respectively, of their parental cell expressions. The method can be easily parallelized and combined with existing microfluidic-based EV isolation technologies, allowing for rapid detection and monitoring of sEVs for cancer diagnosis.

Electrokinetic effect for molecular recognition: A label-free approach for real-time biosensing.

We present a simple and inexpensive method for label-free detection of biomolecules. The method monitors the changes in streaming current in a fused silica capillary as target biomolecules bind to immobilized receptors on the inner surface of the capillary. To validate the concept, we show detection and time response of different protein-ligand and protein-protein systems: biotin-avidin and biotin-streptavidin, barstar-dibarnase and Z domain-immunoglobulin G (IgG). We show that specific binding of these biomolecules can be reliably monitored using a very simple setup. Using sequential injections of various proteins at a diverse concentration range and as well as diluted human serum we further investigate the capacity of the proposed technique to perform specific target detection from a complex sample. We also investigate the time for the signal to reach equilibrium and its dependence on analyte concentration and demonstrate that the current setup can be used to detect biomolecules at a concentration as low as 100pM without requiring any advanced device fabrication procedures. Finally, an analytical model based on diffusion theory has been presented to explain the dependence of the saturation time on the analyte concentration and capillary dimensions and how reducing length and inner diameter of the capillary is predicted to give faster detection and in practice also lower limit of detection.

Electrochemical reduction of O2 in 1-butyl-1-methylpyrrolidinium bis(trifluoromethanesulfonyl)imide ionic liquid containing Zn2+ cations: deposition of non-polar oriented ZnO nanocrystalline films.

The influence of the Zn(2+) concentration and temperature on the electrochemical reduction of O(2) in a solution of zinc bis(trifluoromethanesulfonyl)imide (Zn(TFSI)(2)) salt in 1-butyl-1-methylpyrrolidinium bis(trifluoromethanesulfonyl)imide (PYR(14)TFSI) ionic liquid is presented. ZnO nanocrystalline films were then electrodeposited, under enhanced O(2) reduction, at temperatures in the 75-150 °C range. Their morphology, chemical composition, structural and optical properties were analyzed. In contrast to the polar-oriented ZnO usually obtained from aqueous and conventional solvent based electrolytes, nanocrystalline films oriented along non-polar directions, (11 ̅10) and (11 ̅20), were obtained from this ionic liquid electrolyte. A significant content of carbon was detected in the films, pointing to the active participation and crucial effect of pyrrolidinium cation (and/or byproducts) during the electrodeposition. The films showed semiconducting behavior with an optical gap between 3.43 and 3.53 eV as measured by optical transmittance. Their room temperature photoluminescence spectra exhibited two different bands centered at ∼3.4 and ∼2.2 eV. The intensity ratio between both bands was found to depend on the deposition temperature. This work demonstrates the great potential of ionic liquids based electrolytes for the electrodeposition of ZnO nanocrystalline thin films with innovative microstructural and optoelectronic properties.

ZnO hierarchical nanostructures: simple solvothermal synthesis and growth mechanism.

Hierarchical nano/micro structures of ZnO have been fabricated by solvothermal approach on sol-gel derived ZnO thin films. Paintbrush like nano/micro rod assembly, double-sided brush and windmill type architectures of ZnO are obtained when the ZnO thin film coated substrates were treated solvothermally in water at pH 10. Aligned nanorods are obtained at pH approximately 13.5 in water. In ethylenediamine-water solvent divergent micro/nanorod assemblies such as hemispherical dandelion, rice plant type bush of ZnO are obtained. Increase in the percentage of ethyelendiamine resulted in the formation of smaller assemblies of relatively thin nanorods. Initial slow reaction caused by the slow increase of the temperature inside the reaction medium and the different growth kinetics of the ZnO crystals are supposed to be the reason behind the architectural assemblies of the ZnO crystals.

Direct synthesis of ZnO nanowire arrays on Zn foil by a simple thermal evaporation process.

ZnO nanowire arrays were synthesized on zinc foil by a simple thermal evaporation process at relatively low temperature. Morphology and size controlled synthesis of the ZnO nanostructures was achieved by variation of the synthesis temperature, reaction time and the surface roughness of the substrate. A gas-solid and self-catalytic liquid-solid mechanism is proposed for the growth of nanowires at different temperatures. High-resolution transmission electron microscopy (HRTEM) showed that the as-grown nanowires were of single crystal hexagonal wurtzite structure, growing along the [101] direction. Photoluminescence exhibited strong UV emission at ∼382 nm and a broad green emission at ∼513 nm with 325 nm excitation. Raman spectroscopy revealed a phonon confinement effect when compared with results from bulk ZnO. The nanowire arrays also exhibited a field emission property.

Growth of ZnO nanocrystals by a solvothermal technique and their photoluminescence properties.

ZnO nanocrystals with various morphologies such as nanorod arrays, flower like assemblies, spherical particles, hexagonal cones, and self assembled microstructures were prepared by a solvothermal approach. It was observed that morphology of the ZnO nanostructures were very much solvent dependent in solvothermal approach. Water, ethylenediamine, and ethylene glycol-water mixture favors the formation of nanorods. Flower like assemblies of ZnO were produced in benzene. Spherical as well as cone like nanoparticles and their assemblies were produced in ethylene glycol. The ZnO nanostructures were characterized by X-ray diffraction, scanning and transmission electron microscopy, and photoluminescence studies.

Simple solvothermal route to synthesize ZnO nanosheets, nanonails, and well-aligned nanorod arrays.

ZnO nanosheets, nanonails, and well-aligned nanorods were fabricated on Zn foils by a solvothermal approach using ethanol as the solvent. A lower synthesis temperature and a shorter time period favor the formation of nanosheets. By optimizing the synthesis temperature and time period, ZnO nanonails with a hexagonal cap and a long stem could be produced. A higher temperature was not favorable to produce uniform and smooth nanorods. Well-aligned ZnO nanorod arrays were produced with diameters within 100-250 nm and lengths up to approximately 6 microm when NaOH was added to the solvent. By optimizing the reaction parameters, the morphology, size, and orientation of the nanoforms could be tailored. The ZnO nanorods exhibit an excitonic strong UV emission and a defect-related broad green emission at room temperature. The defect-related green emission band decreased with the improvement of the degree of alignment of the nanorods.

Surfactant-assisted route to synthesize well-aligned ZnO nanorod arrays on sol-gel-derived ZnO thin films.

Anisotropic growth of ZnO nanorod arrays on ZnO thin films was achieved at a temperature of 90 degrees C by a surfactant-assisted soft chemical approach with control over size and orientation. ZnO thin films with c-axis preferred orientation had been achieved by the sol-gel technique. Lengths, diameters, and the degree of alignment of the ZnO nanorods were controlled by changing the experimental parameters. It was observed that the surfactant was essential to restrict the lateral growth of the nanorods, whereas the pH level of the reaction medium controlled the length of the nanorods. On the other hand, the orientation of the nanorods depended on the crystalline orientation of the film as well as the pH of the reaction medium. Room-temperature photoluminescence studies revealed that the ZnO nanorods with the best alignment exhibited the best emission property. The ZnO nanorods exhibited a strong UV emission peak at approximately 3.22 eV, ascribed to the band-edge emission. The field emission studies of the well-aligned nanorod arrays exhibited a low turn-on field of 1.7 V/microm to get an emission current density of 0.1 microA/cm(2).