Size photometry and fluorescence imaging of immobilized immersed extracellular vesicles.

Immunofluorescence analysis of individual extracellular vesicles (EVs) in common fluorescence microscopes is gaining popularity due to its accessibility and high fluorescence sensitivity; however, EV number and size are only measurable using fluorescent stains requiring extensive sample manipulations. Here we introduce highly sensitive label-free EV size photometry (SP) based on interferometric scattering (iSCAT) imaging of immersed EVs immobilized on a glass coverslip. We implement SP on a common inverted epifluorescence microscope with LED illumination and a simple 50:50 beamsplitter, permitting seamless integration of SP with fluorescence imaging (SPFI). We present a high-throughput SPFI workflow recording >10,000 EVs in 7 min over ten 88 × 88 µm2 fields of view, pre- and post-incubation imaging to suppress background, along with automated image alignment, aberration correction, spot detection and EV sizing. We achieve an EV sizing range from 37 to ∼220 nm in diameter with a dual 440 and 740 nm SP illumination scheme, and suggest that this range can be extended by more advanced image analysis or additional hardware customization. We benchmark SP to flow cytometry using calibrated silica nanoparticles and demonstrate superior, label-free sensitivity. We showcase SPFI's potential for EV analysis by experimentally distinguishing surface and volumetric EV dyes, observing the deformation of EVs adsorbed to a surface, and by uncovering distinct subpopulations in <100 nm-in-diameter EVs with fluorescently tagged membrane proteins.

Effect of Sample Preprocessing and Size-Based Extraction Methods on the Physical and Molecular Profiles of Extracellular Vesicles.

Extracellular vesicles (EVs) are nanometric lipid vesicles that shuttle cargo between cells. Their analysis could shed light on health and disease conditions, but EVs must first be preserved, extracted, and often preconcentrated. Here we first compare plasma preservation agents, and second, using both plasma and cell supernatant, four EV extraction methods, including (i) ultracentrifugation (UC), (ii) size-exclusion chromatography (SEC), (iii) centrifugal filtration (LoDF), and (iv) accousto-sorting (AcS). We benchmarked them by characterizing the integrity, size distribution, concentration, purity, and expression profiles for nine proteins of EVs, as well as the overall throughput, time-to-result, and cost. We found that the difference between ethylenediaminetetraacetic acid (EDTA) and citrate anticoagulants varies with the extraction method. In our hands, ultracentrifugation produced a high yield of EVs with low contamination; SEC is low-cost, fast, and easy to implement, but the purity of EVs is lower; LoDF and AcS are both compatible with process automation, small volume requirement, and rapid processing times. When using plasma, LoDF was susceptible to clogging and sample contamination, while AcS featured high purity but a lower yield of extraction. Analysis of protein profiles suggests that the extraction methods extract different subpopulations of EVs. Our study highlights the strengths and weaknesses of sample preprocessing methods, and the variability in concentration, purity, and EV expression profiles of the extracted EVs. Preanalytical parameters such as collection or preprocessing protocols must be considered as part of the entire process in order to address EV diversity and their use as clinically actionable indicators.

nELISA: A high-throughput, high-plex platform enables quantitative profiling of the secretome.

We present the nELISA, a high-throughput, high-fidelity, and high-plex protein profiling platform. DNA oligonucleotides are used to pre-assemble antibody pairs on spectrally encoded microparticles and perform displacement-mediated detection. Spatial separation between non-cognate antibodies prevents the rise of reagent-driven cross-reactivity, while read-out is performed cost-efficiently and at high-throughput using flow cytometry. We assembled an inflammatory panel of 191 targets that were multiplexed without cross-reactivity or impact on performance vs 1-plex signals, with sensitivities as low as 0.1pg/mL and measurements spanning 7 orders of magnitude. We then performed a large-scale secretome perturbation screen of peripheral blood mononuclear cells (PBMCs), with cytokines as both perturbagens and read-outs, measuring 7,392 samples and generating ~1.5M protein datapoints in under a week, a significant advance in throughput compared to other highly multiplexed immunoassays. We uncovered 447 significant cytokine responses, including multiple putatively novel ones, that were conserved across donors and stimulation conditions. We also validated the nELISA's use in phenotypic screening, and propose its application to drug discovery.

Extracellular Vesicle Antibody Microarray for Multiplexed Inner and Outer Protein Analysis.

Proteins are found both outside and inside of extracellular vesicles (EVs) and govern the properties and functions of EVs, while also constituting a signature of the cell of origin and of biological function and disease. Outer proteins on EVs can be directly bound by antibodies to either enrich EVs, or probe the expression of a protein on EVs, including in a combinatorial manner. However, co-profiling of inner proteins remains challenging. Here, we present the high-throughput, multiplexed analysis of EV inner and outer proteins (EVPio). We describe the optimization of fixation and heat-induced protein epitope retrieval for EVs, along with oligo-barcoded antibodies and branched DNA signal amplification for sensitive, multiplexed, and high-throughput assays. We captured four subpopulations of EVs from colorectal cancer (CRC) cell lines HT29 and SW403 based on EpCAM, CD9, CD63, and CD81 expression, and quantified the co-expression of eight outer [integrins (ITGs) and tetraspanins] and four inner (heat shock, endosomal, and inner leaflet) proteins. The differences in co-expression patterns were consistent with the literature and known biological function. In conclusion, EVPio analysis can simultaneously detect multiple inner and outer proteins in EVs immobilized on a surface, opening the way to extensive combinatorial protein profiles for both discovery and clinical translation.

Microfluidic Capillaric Circuit for Rapid and Facile Bacteria Detection.

Urinary tract infections (UTI) are one of the most common bacterial infections and would greatly benefit from a rapid point-of-care diagnostic test. Although significant progress has been made in developing microfluidic systems for nucleic acid and whole bacteria immunoassay tests, their practical application is limited by complex protocols, bulky peripherals, and slow operation. Here we present a microfluidic capillaric circuit (CC) optimized for rapid and automated detection of bacteria in urine. Molds for CCs were constructed using previously established design rules, then 3D-printed and replicated into poly(dimethylsiloxane). CCs autonomously and sequentially performed all liquid delivery steps required for the assay. For efficient bacteria capture, on-the-spot packing of antibody-functionalized microbeads was completed in <20 s followed by autonomous sequential delivery of 100 µL of bacteria sample, biotinylated detection antibodies, fluorescent streptavidin conjugate, and wash buffer for a total volume ≈115 µL. The assay was completed in <7 min. Fluorescence images of the microbead column revealed captured bacteria as bright spots that were easily counted manually or using an automated script for user-independent assay readout. The limit of detection of E. coli in synthetic urine was 1.2 × 102 colony-forming-units per mL (CFU/mL), which is well below the clinical diagnostic criterion (>105 CFU/mL) for UTI. The self-powered, peripheral-free CC presented here has potential for use in rapid point-of-care UTI screening.