Table 3.

The summary of EV analysis methods.

Methods	Benefits	Potential Drawbacks	Reference
Transmission Electron Microscopy	High‐resolution Minimal sample volume needed	Low throughput Sample preparation: laborious, risk of artifacts	[37, 200]
Scanning Electron Microscopy	Simpler sample preparation: less complex than TEM Minimal sample volume needed	Lower resolution: compared to TEM Risk of artifacts from sample dehydration. Might obscure/alter sample by conductive coating	[201]
Atomic Force Microscopy	Can provide 3D surface topography Probe the mechanical characteristics Analysis in physiological environments	Low throughput Expertise needed: complex use and data interpretation	[40, 202]
Nanoparticle Tracking Analysis	Provide both size distribution and particle concentration Real‐time visualization Better resolution for polydisperse samples	Difficulty detecting particles smaller than ≈30 nm More complex sample preparation	[203]
Dynamic Light Scattering	Require minimal steps before analysis Rapid determination of size distribution	No concentration data. Polydisperse challenge: difficult to accurately determine size in samples with a broad size distribution	[36, 204]
Tunable Resistive Pulse Sensing	Provide particle size and concentration Adjustable for different particle sizes	Particles or aggregates can block the pore Sensitive to changes in temperature, salt concentration, and other factors	[41, 205]
Western Blotting	Confirm the presence of specific EVs markers Can probe multiple proteins using different antibodies	Time‐consuming Not ideal for low‐abundance proteins	[42, 206]
Enzyme‐Linked Immunosorbent Assay	Provide quantitative data on protein concentrations Suitable for analyzing multiple samples simultaneously	Chance of antibodies detecting unintended targets Might not detect very low abundance proteins	[43, 207]
Mass Spectrometry	Broad profiling of proteins, lipids, and other molecules Can detect molecules present in low concentrations	Require advanced bioinformatics tools Need for rigorous sample preparation High setup and maintenance costs	[44, 208]
Polymerase Chain Reaction	Can detect low quantities of specific nucleic acid sequences Provide quantitative data	Sensitive to the purity of EV nucleic acid Risk of sample contamination leading to false positives	[45, 209]
Total Internal Reflection Fluorescence	High signal‐to‐noise ratio Enable real‐time observation of EVs interactions at the cell membrane or surface	Limited field of view Require precise setup alignment and calibration	[210]
Flow Cytometry	Can simultaneously measure multiple parameters (e.g., size and protein markers) Rapid data acquisition Provide information on individual EVs	Detection of smaller EVs can be challenging Non‐specific binding Require instruments with high sensitivity and resolution	[211]
Surface Plasmon Resonance	Capable of detecting low concentrations Label‐free detection	Highly sensitive to changes in refractive index Require careful preparation and optimization of the sensor chip surface	[46, 212]
Surface‐Enhanced Raman Scattering	Detect low‐abundance molecules Provide multiple targets simultaneously based on distinct Raman shifts	Reproducibility not ideal Background interference	[47, 213]