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.

Large-Sized Nanocrystalline Ultrathin ß-Ga2O3 Membranes Fabricated by Surface Charge Lithography.

Large-sized 2D semiconductor materials have gained significant attention for their fascinating properties in various applications. In this work, we demonstrate the fabrication of nanoperforated ultrathin ß-Ga2O3 membranes of a nanoscale thickness. The technological route includes the fabrication of GaN membranes using the Surface Charge Lithography (SCL) approach and subsequent thermal treatment in air at 900 °C in order to obtain ß-Ga2O3 membranes. The as-grown GaN membranes were discovered to be completely transformed into ß-Ga2O3, with the morphology evolving from a smooth topography to a nanoperforated surface consisting of nanograin structures. The oxidation mechanism of the membrane was investigated under different annealing conditions followed by XPS, AFM, Raman and TEM analyses.

Low-Cost Synthesis of Silicon Quantum Dots with Near-Unity Internal Quantum Efficiency.

As a cost-effective batch synthesis method, Si quantum dots (QDs) with near-infrared photoluminescence, high quantum yield (>50% in polymer nanocomposite), and near-unity internal quantum efficiency were fabricated from an inexpensive commercial precursor (triethoxysilane, TES), using optimized annealing and etching processes. The optical properties of such QDs are similar to those prepared from state-of-the-art precursors (hydrogen silsesquioxane, HSQ) yet featuring an order of magnitude lower cost. To understand the effect of synthesis parameters on QD optical properties, we conducted a thorough comparison study between common solid precursors: TES, HSQ, and silicon monoxide (SiO), including chemical, structural, and optical characterizations. We found that the structural nonuniformity and abundance of oxide inherent to SiO limited the resultant QD performance, while for TES-derived QDs this drawback can be avoided. The presented low-cost synthetic approach would significantly favor applications requiring high loading of good-quality Si QDs, such as light conversion for photovoltaics.

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.

Wafer-scale fabrication of isolated luminescent silicon quantum dots using standard CMOS technology.

A wafer-scale fabrication method for isolated silicon quantum dots (Si QDs) using standard CMOS technology is presented. Reactive ion etching was performed on the device layer of a silicon-on-insulator wafer, creating nano-sized silicon islands. Subsequently, the wafer was annealed at 1100 °C for 1 h in an atmosphere of 5% H2 in Ar, forming a thin oxide passivating layer due to trace amounts of oxygen. Isolated Si QDs covering large areas (∼mm2) were revealed by photoluminescence (PL) measurements. The emission energies of such Si QDs can span over a broad range, from 1.3 to 2.0 eV and each dot is typically characterized by a single emission line at low temperatures. Most of the Si QDs exhibited a high degree of linear polarization along Si crystallographic directions [[Formula: see text]] and [[Formula: see text]]. In addition, system resolution-limited (250 µeV) PL linewidths (full width at half maximum) were measured for several Si QDs at 10 K, with no clear correlation between emission energy and polarization. The initial part of PL decays was measured at room temperature for such oxide-embedded Si QDs, approximately several microseconds long. By providing direct access to a broad size range of isolated Si QDs on a wafer, this technique paves the way for the future fabrication of photonic structures with Si QDs, which can potentially be used as single-photon sources with a long coherence length.

Wafer-level fabrication of individual solid-state nanopores for sensing single DNAs.

For biomolecule sensing purposes a solid-state nanopore platform based on silicon has certain advantages as compared to nanopores on other substrates such as graphene, silicon nitride, silicon oxide etc Capitalizing on the developed CMOS technology, nanopores on silicon are scalable without any requirement for additional processing, the devices are low cost and the process can be repeatable with a high yield. One of the essential requirements in biomolecule sensing is the ability of the nanopore to interact with the analyte. In this work, we present a method for processing high aspect ratio, single nanopores in the range of 10-30 nm in diameter and approximately 700 nm in length on a silicon-on-insulator (SOI) wafer. The presented method of manufacturing the high aspect ratio individual nanopores combines optical lithography and anisotropic KOH etching with a final electrochemical etching step to form the nanopores and is repeatable and can be processed in batches. We demonstrate electrical detection of dsDNA translocation, where the characteristic time of the process is in the millisecond range. We also analyse the translocation parameters and correlate the enhanced length of the nanopore to a longer translocation time as compared to other substrates.

The shell matters: one step synthesis of core-shell silicon nanoparticles with room temperature ultranarrow emission linewidth.

Here we present a one-step synthesis that provides silicon nanocrystals with a thin shell composed of a ceramic-like carbonyl based compound, embedded in a porous organosilicon film. The silicon nanocrystals were synthesised from hydrogen silsesquioxane molecules, modified with organic molecules containing carbonyl groups, which were annealed at 1000 °C in a slightly reducing 5% H2 : 95% Ar atmosphere. The organic character of the shell was preserved after annealing due to trapping of organic molecules inside the HSQ-derived oxide matrix that forms during the annealing. The individual silicon nanocrystals, studied by single dot spectroscopy, exhibited a significantly narrower emission peak at room temperature (lowest linewidth ∼ 17 meV) compared to silicon nanocrystals embedded in a silicon oxide shell (150 meV). Their emission linewidths are even significantly narrower than those of single CdSe quantum dots (>50 meV). It is hypothesized that the Si-core-thin shell structure of the nanoparticle is responsible for the unique optical properties. Its formation within one synthesis step opens new opportunities for silicon-based quantum dots. The luminescence from the produced nanocrystals covers a broad spectral range from 530-720 nm (1.7-2.3 eV) suggesting strong application potential for solar cells and LEDs, following the development of a suitable mass-fabrication protocol.

Tight-binding calculations of the optical properties of Si nanocrystals in a SiO2 matrix.

We develop an empirical tight binding approach for the modeling of the electronic states and optical properties of Si nanocrystals embedded in a SiO2 matrix. To simulate the wide band gap SiO2 matrix we use the virtual crystal approximation. The tight-binding parameters of the material with the diamond crystal lattice are fitted to the band structure of ß-cristobalite. This model of the SiO2 matrix allows us to reproduce the band structure of real Si nanocrystals embedded in a SiO2 matrix. In this model, we compute the absorption spectra of the system. The calculations are in an excellent agreement with experimental data. We find that an important part of the high-energy absorption is defined by the spatially indirect, but direct in k-space transitions between holes inside the nanocrystal and electrons in the matrix.

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.

Non-stationary analysis of molecule capture and translocation in nanopore arrays.

Analytical formulas for the ON- and OFF-time distributions as well as for the autocorrelation function were derived for the case of single molecule translocation through nanopore arrays. The obtained time-dependent expressions describe very well experimentally recorded statistics of DNA translocations through an array of solid state nanopores, which allows us to extract molecule and system related physical parameters from the experimental traces. The necessity of non-stationary analysis as opposite to the steady-state approximation has been vindicated for the molecule capture process, where different time-dependent regimes were identified. A long tail in the distribution of translocation times has been rationalized invoking Markov jumps, where a possible sequential ordering of events was elucidated through autocorrelation function analysis.

Photodegradation of Organometal Hybrid Perovskite Nanocrystals: Clarifying the Role of Oxygen by Single-Dot Photoluminescence.

Photostability has been a major issue for perovskite materials. Understanding the photodegradation mechanism and suppressing it are of central importance for applications. By investigating single-dot photoluminescence spectra and the lifetime of MAPbX3 (MA = CH3NH3+, X = Br, I) nanocrystals with quantum confinement under different conditions, we identified two separate pathways in the photodegradation process. The first is the oxygen-assisted light-induced etching process (photochemistry). The second is the light-driven slow charge-trapping process (photophysics), taking place even in oxygen-free environment. We clarified the role of oxygen in the photodegradation process and show how the photoinduced etching can be successfully suppressed by OSTE polymer, preventing an oxygen-assisted reaction.

Thermophoresis-Controlled Size-Dependent DNA Translocation through an Array of Nanopores.

Large arrays of nanopores can be used for high-throughput biomolecule translocation with applications toward size discrimination and sorting at the single-molecule level. In this paper, we propose to discriminate DNA length by the capture rate of the molecules to an array of relatively large nanopores (50-130 nm) by introducing a thermal gradient by laser illumination in front of the pores balancing the force from an external electric field. Nanopore arrays defined by photolithography were batch processed using standard silicon technology in combination with electrochemical etching. Parallel translocation of single, fluorophore-labeled dsDNA strands is recorded by imaging the array with a fast CMOS camera. The experimental data show that the capture rates of DNA molecules decrease with increasing DNA length due to the thermophoretic effect of the molecules. It is shown that the translocation can be completely turned off for the longer molecule using an appropriate bias, thus allowing a size discrimination of the DNA translocation through the nanopores. A derived analytical model correctly predicts the observed capture rate. Our results demonstrate that by combining a thermal and a potential gradient at the nanopores, such large nanopore arrays can potentially be used as a low-cost, high-throughput platform for molecule sensing and sorting.

Light-Converting Polymer/Si Nanocrystal Composites with Stable 60-70% Quantum Efficiency and Their Glass Laminates.

Thiol-ene polymer/Si nanocrystal bulk hybrids were synthesized from alkyl-passivated Si nanocrystal (Si NC) toluene solutions. Radicals in the polymer provided a copassivation of "dark" Si NCs, making them optically active and leading to a substantial ensemble quantum yield increase. Optical stability over several months was confirmed. The presented materials exhibit the highest photoluminescence quantum yield (∼65%) of any solid-state Si NC hybrid reported to date. The broad tunability of thiol-ene polymer reactivity provides facile glass integration, as demonstrated by a laminated structure. This, together with extremely fast polymerization, makes the demonstrated hybrid material a promising candidate for light converting applications.

Influence of swift heavy ion irradiation on the photoluminescence of Si-nanoparticles and defects in SiO2.

The influence of swift heavy ion (SHI) irradiation on the photoluminescence (PL) of silicon nanoparticles (SiNPs) and defects in SiO2-film is investigated. SiNPs were formed by implantation of 70 keV Si+ and subsequent thermal annealing to produce optically active SiNPs and to remove implantation-induced defects. Seven different ion species with energy between 3-36 MeV and fluence from 1011-1014 cm-2 were employed for irradiation of the implanted samples prior to the thermal annealing. Induced changes in defect and SiNP PL were characterized and correlated with the specific energy loss of the employed SHIs. We find that SHI irradiation, performed before the thermal annealing process, affects both defect and SiNP PL. The change of defect and SiNP PL due to SHI irradiation is found to show a threshold-like behaviour with respect to the electronic stopping power, where a decrease in defect PL and an anticorrelated increase in SiNP PL after the subsequent thermal annealing are observed for electronic stopping exceeding 3-5 keV nm-1. PL intensities are also compared as a function of total energy deposition and nuclear energy loss. The observed effects can be explained by ion track formation as well as a different type of annealing mechanisms active for SHI irradiation compared to the thermal annealing.