Table 2.

Detection methods of EVs with their advantages and limitations.

Detection Methods	Principles of Detection	Advantages	Limitations
Dynamic light scattering [173]	Measuring EV size distribution	Accurate, reliable, and repeatable particle size analysis in very short time; Size measurement of molecules with MW < 1000Da; very low sample volume	Low refractive index of vesicles makes problematic to distinguish MVs from polydispersed and size heterogeneous samples
Nanoparticle Tracking Analysis [174]	Quantification of nanoscale particles in liquid suspension moving under Brownian motion	Detection of single vesicles with a diameter ≤50nm	Only semi-quantification; Inaccurate with size heterogeneous samples and particle aggregates; Considerable intra-assay count variability
Electron microscopy	Measuring the size and morphology of EVs	Direct assessment of morphology and size; small sample amount	Time consuming; size and morphology modifications during sample preparation
Flow cytometry [175,176]	EV characterization with fluorescent antibodies EV counting	Quantitative and qualitative characterization of EVs using specific markers	Detection limit of flow cytometers (>100 nm, Nonspecific: swarming effect, detection of protein/antibody aggregates
ELISA/ Western Blot [177]	EV characterization and quantification using specific antibodies	Standard immunological methods; specific characterization of EV protein markers	Time consuming; possible detection of non-EV proteins; nonspecific information on EV concentration/size/distribution
Surface plasmon resonance [178]	Label-free detection of ligand binding to target receptors immobilized on a sensing surface	Measures the total mass of EVs, including proteins, lipids, and nucleotides; small sample volumes	Inadequate quality control and normalization across study groups;
Atomic force microscopy [179]	EV three-dimensional topography	Fast; small sample amount	Size and morphology modifications due to sample dehydration