The elastic properties of nanoscale extracellular vesicles (EVs) are believed to influence their cellular interactions, thus having a profound implication in intercellular communication. However, accurate quantification of their elastic modulus is challenging due to their nanoscale dimensions and their fluid-like lipid bilayer. We show that the previous attempts to develop atomic force microscopy-based protocol are flawed as they lack theoretical underpinning as well as ignore important contributions arising from the surface adhesion forces and membrane fluctuations. We develop a protocol comprising a theoretical framework, experimental technique, and statistical approach to accurately quantify the bending and elastic modulus of EVs. The method reveals that membrane fluctuations play a dominant role even for a single EV. The method is then applied to EVs derived from human embryonic kidney cells and their genetically engineered classes altering the tetraspanin expression. The data show a large spread; the area modulus is in the range of 4 to 19 mN/m and the bending modulus is in the range of 15 to 33 kBT, respectively. Surprisingly, data for a single EV, revealed by repeated measurements, also show a spread that is attributed to their compositionally heterogeneous fluid membrane and thermal effects. Our protocol uncovers the influence of membrane protein alterations on the elastic modulus of EVs.

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.

Fluorescence-based single-molecule detection has been widely investigated and applied in biosensing and bioimaging due to its ultrahigh sensitivity and specificity. However, bulky and expensive commercial fluorescence microscopes are usually required. The Stokes shift property of most commonly used fluorophores requires optical sets such as dichroic mirrors and specific filters in the optical pathway before a photodetector to eliminate excitation and scattering lights from the fluorescence signals. The fluorescence signal collected by an objective is further unavoidably attenuated, and the optical resolution is diffraction-limited. Herein, a proof of concept of a lab-on-a-chip compatible molecular sensor is shown by integrating upconversion nanoparticles (UCNPs) and amorphous hydrogen-doped InGaZnO (InGaZnO:H) thin-film phototransistor (IGZO:H TFTs) aiming to alleviate those issues. Upon illumination with a 980 nm infrared light, the phototransistor shows no photocurrent without UCNPs but yields a high photocurrent with UV-visible fluorescent light emitted from the UCNPs. The molecular detection is enabled by further involving the Förster resonance energy transfer (FRET) mechanism, with the UCNPs as donors. The photocurrent falls back to its original low level when biotinylated gold nanoparticles are added to selectively bind and quench the UCNPs via biotin-streptavidin coupling. Each UCNP shows an estimated photocurrent-to-dark current ratio of 10³ and each biotinylated gold nanoparticle causes at least 1 order of magnitude decrease of the photocurrent. Our integrated setup presents a promising platform for further development toward an optoelectronic biosensor capable of single-molecule detection.

Extracellular vesicles (EVs) are nanoparticles encapsulated with a lipid bilayer, and they constitute an excellent source of biomarkers for multiple diseases. However, the heterogeneity in their molecular compositions constitutes a major challenge for their recognition and profiling, thereby limiting their application as an effective biomarker. A single-EV analysis technique is crucial to both the discovery and the detection of EV subpopulations that carry disease-specific signatures. Herein, a plasmonic nanohole array is designed for capturing single EVs and subsequently performing fluorescence detection of their membrane proteins by exploiting plasmonic amplification of the fluorescence signal. Unlike other reported methods, our design relies on an exclusive detection of single EVs captured inside nanoholes, thus allowing us to study only plasmonic effects and avoid other metal-induced phenomena while leveraging on the proximity of emitters to the plasmonic hotspots. The method is optimized through numerical simulations and verified by a combination of atomic force, scanning electron microscopy, and fluorescence microscopy. Fluorescence enhancement is then estimated by measuring the CD9 expression of small EVs derived from the human embryonic kidney (HEK293) cell line and carefully considering the spatial distribution of emission and excitation intensities. Fluorescence intensities of immunostained EVs show a moderate overall enhancement of intensity and follow the intensity trend predicted by simulation for nanohole arrays with different nanohole periods. Moreover, the number of observed EVs in the best-performing nanohole array increases by more than 12 times compared with EVs immobilized on a reference substrate, uncovering a vast amount of weakly fluorescent EVs that would remain undetected with the regular fluorescent method. Our nanohole array provides a basis for a future platform of single-EV analyses, also promising to capture the signature arising from low-expressing proteins.

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 tech-nique 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.

A microfabricated biosensor based on the streaming current method is presented in this work. The microfabricated sensor consists of a silicon microchannel, which is enclosed with a glass capping to form a closed microchannel. The depth of the microchannel is approximately 10 µm in width and length varying from 50 to 100 µm. The silicon is etched using deep reactive ion etching (DRIE) to form a microchannel. For the capping of the channel, a glass wafer of type Borofloat is used and anodically bonded to the silicon wafer to form a closed microchannel. The microchannel is then functionalized to be specific to certain biomarkers which can be a potential biomarker for cancer, for example. The method used for detection is called the streaming current method. In this method, fluid is flown through the microchannel with high pressure close to six bars. The surface of the silicon is oxidized, which has a zeta potential of approximately 2.7. Depending on the type of fluid the charge concentration varies. By having a pressure in the channel, the charges get distributed as an anode and cathode at the inlet and outlet electrodes of the microfluidic channels. At a fixed potential, a streaming current is observed, which is proportional to the charge accumulated. The difference between the streaming current with and without the biomarker is correlated to the concentration. Hence, a biosensor based on the streaming current method can be realized, which could be used for potential cancer biomarker detection.

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.

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.

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.