Table 2.

Summary of exosome characterization techniques.

Methods	Instrument	Description
Morphological	SEM	Provides three-dimensional surface information.
	TEM	Superior image resolution and can be used with immunogold labeling to provide molecular characterization.
	cryo-EM	Enables analysis of EVs morphology without extensive processing.
	AFM	Provide information on both surface topology and local material properties.
Size	NTA( Nanoparticle tracking analysis )	Tracks individual vesicle scattering over time, as they diffuse and scatter under light illumination then it could determine vesicle concentration and size distribution. Analysis of size and concentration. Fast and easy. Low specificity for same size particles.
	DLS	Measures bulk scattered light from EVs as the vesicles undergo continuous Brownian motion. The dynamic information on the vesicles is derived from an autocorrelation of the scattered intensity and could be used to determine vesicle size. As the original size distribution measured by DLS is intensity-weighted, the data is dominated by large vesicles. It is affected by the color, electrical, magnetic and other physical and chemical properties of the measured substance, and is very sensitive to dust and impurities.
	TRPS (Tunable resistive pulse sensing)	Two fluidic reservoirs, each connected to an electrode, are separated by a membrane with a pore. The ionic current between reservoirs is then measured. When a EV passes through the pore, it blocks the current flow, leading to a transient current decrease.
	SEA (Single EV analysis)	EVs are biotinylated and captured on a flat surface coated with neutravidin (Av). EVs are then stained with fluorescent antibodies and imaged. Subsequently, fluorophores are quenched, and the staining process is repeated for a different set of markers.
	SP-IRIS	The coherent light formed by the substrate and the particle is imaged, and the size of the nanoparticles is directly calculated by the brightness after imaging. Sp-iris technology has the advantages of high precision, high sensitivity and single exosome fluorescence imaging.
Surface marker	Flow cytometry -Small particle flow cytometry	Highly sensitive flow cytometry instrument, termed vesicle flow cytometry. Fluorescent intensity from liposomes, labeled with di-8-ANEPPS, were calibrated for the vesicle diameter. Analysis of size, count and surface protein expression. Commonly found in facilities. Size limitation of conventional flow cytometers
	Western blot	Conventional EV protein analysis. EV protein lysate is separated by SDS−PAGE, before being transferred over to a membrane for immunoblotting of specific EV protein targets (e.g., CD81, TSG101, CD9 and CD63). Analysis of protein components. Technique commonly performed in several laboratories. Time consuming processing.
	ELISA (Enzyme-linked immuno- sorbent assay)	In the specific “sandwich” configuration, vesicles or lysates could be applied to a solid support that has been pretreated with an immobilized capturing antibody. Captured vesicle targets are then exposed to a detecting target antibody.
New Technologies for Analysis of EVs	Micronuclear magnetic resonance	(a) Assay schematics to maximize magnetic nanoparticle (NMP) binding onto EVs. A two-step bio- orthogonal click chemistry was used to label EVs with MNPs. (b) The microfluidic system for on-chip detection of circulating EVs is designed to detect MNP-targeted vesicles, concentrate MNP-tagged vesicles (while removing unbound MNPs), and provide in-line NMR detection.
	Surface plasmon resonance	(a) The nPLEX sensing is based on transmission SPR through periodic nanohole arrays. The hole diameter is 200 nm with a periodicity of 450 nm. The structure was patterned in a gold film (200 nm thick) deposited on a glass substrate. (b) Finite-difference time- domain simulation shows the enhanced electromagnetic fields tightly confined near a periodic nanohole surface. The field distribution overlaps with the size of EVs captured onto the sensing surface, maximizing the detection sensitivity. (c) The sensing array can be integrated with multichannel microfluidics for independent and parallel analyses. (d) Assay schematic of changes in transmission spectra showing EV detection. The gold surface is prefunctionalized by a layer of polyethylene glycol (PEG), and antibody conjugation and specific EV binding were monitored by transmission spectral shifts as measured by sensor. (e) In comparison to gold standard methods, the nPLEX assay demonstrated excellent sensitivity, more sensitive than Western blotting and chemiluminescence ELISA, respectively. (f) Correlation between nPLEX and ELISA measurements. The marker protein expression level was determined by normalizing the marker signal with that of anti-CD63, which accounted for variation in exosomal counts across samples.
	Electrochemical detection	EVs are captured on magnetic beads directly in plasma and labeled with HRP enzyme for electrochemical detection. The magnetic beads are coated with antibodies against CD63(an enriched surface marker in exosomes).
	ExoScreen technology	This proximity assay requires two types of immunobeads: (1) donor beads, which are excited at 680 nm to release singlet oxygen, and (2) acceptor beads, which can be only excited by the released singlet oxygen when they are situated within 200 nm away from the donor beads. (b) Assay workflow. Biological samples are first treated with biotinylated antibodies and acceptor beads conjugated with a second antibody. Streptavidin-coated donor beads were then added to complete the proximity assay for data acquisition. (c) Correlation between ExoScreen measurements for CD9 positive EVs, CD63 positive EVs, or CD63/CD9 double-positive EVs and EV protein concentration in a dilution series. The addition of biotinylated antibodies and acceptor beads conjugated antibodies is denoted “bCD9/aCD9” or “bCD63/aCD63”.