Table 2.

Comparison of different EV isolation and enrichment methods

Platform	Working principle	Advantages	Limitations
Conventional methods
Ultracentrifugation methods60, 61	Differential centrifugal force depending on particle density and size	Gold standard Large-scale EV preparations Little technical required	Time consuming Low recovery and purity Inability to isolate molecular subtypes (specificity)
DGUC62, 63	UC in density gradient matrix	Increased purity	Lengthy process Low throughput Lower yield
Size-based separation (ultrafiltration, SEC)62, 63	Differential transport based on size or molecular weight	Facile and user-friendly High yield	Prone to clogging Lack of specificity Pressure-induced damage
Field Flow Fractionation65	Differential flow transport under a perpendicular field	Gentle and rapid separation Efficient analyte recovery	Low resolution Low scalability
Precipitation59, 66	Polymeric additives induced precipitation.	Simple workflow Minimal equipment requirement Scalable sample preparation	Lack of specificity Low purity and recovery Hard-to-remove additives may affect subsequent assays and applications.
Immunoaffinity-based methods67-69	Capture of EVs using specific antibodies to target surface proteins.	Specificity for molecularly defined subtypes High purity Scalable sample preparation	High cost for large-scale isolation Prone to nonspecific binding Availability of specific antibodies
Microfluidics-based methods
Microfluidic Filtering70-73	Nanofiltration using porous materials or membranes on chip	Suitable for small-volume samples High throughput High size selectivity	Prone to clogging Lack of specificity Pressure-induced damage
Deterministic lateral displacement76, 77	Asymmetric bifurcation of laminar flow by micro-/nanoscale post arrays	Fast sorting High size resolution Amenable to automation	Complex device fabrication Low throughput Lack of specificity
Viscoelastic flow sorting80, 81	Size-dependent distribution across the flow of a viscoelastic fluid	Contact-free and label-free Simple chip design No need of external fields	Limited throughput Lack of specificity
Diffusiophoretic trapping83	Balanced particle and fluidic transport induced by a salt gradient and the nanochannel geometry	No need of electric field High enrichment rate Single measurements of size, concentration, and surface charge	Complex device design and fabrication Limited capacity for large-volume samples Purified samples required
Immunomagnetic isolation84-86	Magnetic capture of EVs using specific antibodies to target surface proteins.	Relatively simple process High specificity and purity for molecularly defined subtypes	High cost and low capacity Prone to nonspecific binding Availability of specific antibodies
Micro-/nano-structure-based isolation87-91	Combination of multiple factors (immunoaffinity, size, charge, et al.)	Greatly enhanced isolation efficiency	Complex device fabrication Limited capacity for large-scale processing
Acoustofluidic technology103, 104	Mechanical property-dependent acoustic force on particles induced by ultrasound waves	Fast, high-resolution sorting of intact EVs Contact-free and label-free No effects to EV properties	Complex device fabrication Lack of specificity Limited purity
Dielectrophoretic separation106-108	Displacement of dielectric particles by an electric field gradient.	Label-free and contact-free Fast enrichment of dielectric particles Improve specificity for immunoaffinity capture	Complex device design and fabrication Prone to influence of sample matrix and surface charges of EVs.
Thermophoretic enrichment111-113	Size-dependent particle transport driven by a thermal gradient.	Homogeneous process Low cost, non-destructive enrichment with raw samples.	Complex instrumentation Limited capacity for processing large volumes Difficulty in analyte recovery