Table 2.

Techniques used for detection and isolation of extracellular vesicles

S. No.	Techniques for detection and isolation of EVs	Sub-types of detection and isolation techniques of EVs	Advantages	Disadvantages	Refs.
1	Filtration-based techniques for EV isolation.	Centrifugal ultrafiltration, Tangential flow filtration, Exodisc, ExoTIC (exosome total isolation chip), Integrated double-filtration microfluidic device, Hydrostatic dialysis	High purity, Fast, Scalable, Simple	Time-consuming, Low purity, lower EV yield, reduced sample recovery, Protein contamination	606–612
2	Flow field-flow fractionation-based techniques	Immunoaffinity chromatography - asymmetrical flow field-flow fractionation (IAC-AsFlFFF), Frit-inlet AsFlFFF	Avoiding yield loss, reducing potential EV’s integrity damage, and Scalable	The AF4 process requires high expertize to operate and customize; tangential flow filtration is often not used as an EV purification method	606,613–616
3	Size-based/ Density-Based /Centrifugation technique	Size-exclusion chromatography, Differential ultracentrifugation, CUC: cushioned-density (ultra)centrifugation, DGUC: density gradient (ultra)centrifugation, Sucrose density gradient centrifuge, Iodixanol density gradient ultracentrifugation	Low cost, Low risk of pollution Fast, Scalable, Simple, Easily automated, and integrated with diagnosis	Protein contamination; sample volume limited, Low extraction volume; Extensive laboratory equipment requirements Time-consuming, operator and equipment-sensitive process, purity depending on the optimization based on starting sample type, rotor used, and the applied g-forces	606,617–620
4	Ion-exchange based techniques	Anion-exchange chromatography, Anion exchange, Nickel-based isolation, Cation- and anion-exchange chromatography	Require shorter isolation time, higher purity	Ion-exchange methods in EV research are limited to cell culture but face challenges in complex biological matrices like blood and plasma due to high amounts of charged biomolecules	621–624
5	Electrophoresis and dielectrophoresis (DEP) based techniques	Alternating current electrokinetic, microarray chip device, Agarose gel electrophoresis, Capillary electrophoresis, Capillary zone electrophoresis, Direct current–insulator-based dielectrophoresis, Electrophoresis with dialysis, On-chip immunoelectrophoresis, On-chip microcapillary electrophoresis		The electric field has the potential to influence the properties of exosomes	625–627
6	Affinity-based EV isolation and separation techniques.	Magnetic beads, Silica nano spring, Agarose resin, Polymeric monolithic disks, Agarose gel column, Immunoaffinity Enrichment, Immunocapture, Enzyme-Linked Immunosorbent Assay (ELISA)	High purity to isolate specific EVs subtypes	The affinity approach to EV removal is limited by factors like beads’ binding capacity, antigen exposure, epitope stability, antibody affinity, acidic elution buffers, and high costs	606,628,629
7	Methods utilizing the change in EV solubility and aggregation	Precipitation with Hydrophilic Polymers, Precipitation with Protamine, EV Precipitation with Sodium Acetate, Precipitation of Proteins with Organic Solvent (PROSPR)	The process is quick, easy, and scalable; it doesn’t damage electric vehicles (EVs), and it doesn’t need any special isolation equipment.	The sample may be contaminated with proteins, complexes, lipoproteins, nucleoproteins, viral particles, and biopolymers, potentially affecting further analysis, long process, gel filtration is required, PROSPR technique is inferior to gel chromatography, acetone can disrupt the functionality of vesicular membranes	630–632
8	EV isolation methods utilizing interactions	Antibodies to EVs receptors, Phosphatidylserine-Binding proteins, Heparin modified sorbents, Binding of heat shock proteins, lectins.	Low cost, simple, high purity, preservation of functional integrity, readily reversible bonding, does not require complex equipment.	Obstacles include detachment, intact vesicle analysis, nonspecific binding, initial purification and concentration requirements, high selectivity, cost, and antibody availability	632–638
9	Microfluidics	Microfluidics-based immunoaffinity capture, acoustofluidics, membrane-filtration microfluidics, viscoelastic flows or nanowire traps, Viscoelasticity-based microfluidic system, λ-DNA mediated viscoelastic microfluidic system, Electroosmotic flow-driven DLD pillar array.	Low cost, Fast, Simple, Easily automated and integrated with a diagnosis	Requires a specific level of expertize, not suitable for preparative purposes (e.g., therapeutic applications), low sample volume might be a limitation, need additional equipment, high cost	606,639,640
10	Precipitation based isolation	Commercial kits for polymer precipitation, polymer precipitates EV, Urine Exosome RNA Isolation Kit, Total Exosome Isolation Solution, and RIBO™ Exosome Isolation Reagent	User-friendly, cheap, simple, and does not require complex equipment.	Costly for high sample sizes, post-cleanup is required for downstream applications due to polymer and protein contamination.	606,641
11	Other techniques used for the detection and isolation of EVs	nano-sized deterministic lateral displacement, Oscillatory viscoelastic, NanoDLD pillar array, Electroosmotic flow-driven DLD pillar array, nanoplasmon-enhanced scattering assay, ExoSearch Chip, Acoustic Nanofilter, Facile PEG-based isolation, Nanoparticle tracking analysis, Contact-Free Sorting, Two-phase isolation, KeepEX, Label-Free SERS to Identify EV, using carboxyl group-functionalized iron oxide nanoparticles, Matrix-assisted laser desorption ionization time-of-flight mass spectrometry	Higher yield, DLD is a nondestructive method that enables rapid, continuous, single-particle sorting in a continuous flow, without particle labeling, using small sample volumes.	Need more equipments, a lengthy process, Limitation on the number of samples that may be processed simultaneously (to six samples).	642–652