Technique | Principle | Advantages | Limitations |
---|---|---|---|
Ultracentrifugation | Sedimentation velocity | High purity; Widely used. | Lengthy process; Low yield; Require specialized equipment; Contamination of soluble proteins to vesicular material resulting in low specificity. |
Ultrafiltration | Size-based (membrane filters with specified molecular weight cut-offs) | High purity; Easily applied. | Contamination of same-sized vesicles resulting in low specificity; Breakage or deformation of the vesicles can occur due to pressure. |
Size exclusion chromatography | Size-based exclusion (EVs are eluted in earlier fractions based on their exclusion from pores within the stationary phase) | Relatively high yield; Easily applied. | Contamination of same-sized vesicles resulting in low specificity; Co-isolation of certain lipoproteins along with EVs which may require sequential techniques for separation. |
Precipitation methods | Â | Â | Â |
Polyethylene glycol | Precipitation | High yield and recovery; Popular; Easily applied. | Low specificity due to the presence of residual contaminants. |
Commercialized reagent kits (Examples: â—¦ ExoQuick PLUS â—¦ ExoQuick-TC PLUS) | Precipitation | High yield and recovery; Easily applied; Ability to precipitate EVs from a wide range of biofluids with reduced carryover contaminants; Relatively short processing time; Compatibility for use in downstream applications. | Relatively low specificity, but recent developments have reduced carryover contaminants; High costs. |
Immunoaffinity-based assays | Â | Â | Â |
Magneto-immunocapture | Antibody-coated magnetic particles | High specificity; Selective enrichment for EV populations (mainly small EVs). | Marker dependent; High costs. |
Microfluidic immunochips (Examples: ◦ ExoChip: CD63 antibody-coated microfluidic chip. ◦ ExoDIF and Exo-ID chip: immunoaffinity-based microfluidics combined with filtration approaches. ◦ Sub-ExoProfile chip: a microfluidic nanodevice with three self-assembled 3D nanopillars immobilized with capture antibodies for CD81, EpCAM, and Her2). ◦ EVs on demand chip (EVOD) and EV Click Chip: Click chemistry purification system integrating multimarker antibody cocktails for small EVs. | Antibody-coated microchannel chip (selective capture of small EV molecules) | High specificity; Time-saving procedures; Small consumption of samples and reagents; Increased efficiency in specifically trapping and separating small EVs; High compatibility for high-throughput analysis. | Marker dependent; High costs. |
Others | Â | Â | Â |
Size-based microfluidic chips (Examples: â—¦ ExoTIC â—¦ EXODUS | Physical properties (Nanoporous membrane for isolating small EVs of certain size range) | Time-saving procedures; Small consumption of samples and reagents; Increased efficiency in separating small EVs; High compatibility for high-throughput analysis. | Relatively low yield; Blockage of membrane pores can limit the continuous separation of small EVs. Recent developments with faster systems (such as those integrating periodic negative pressure oscillations and acoustofluidic streaming in EXODUS) are reducing these limitations. |
Acoustic-based microfluidic chips | Physical properties (ultrasonic waves with forces to separate particles) | Rapid; Continuous and efficient separation of particles and EVs; Non-contact; Application to a wide range of particles and vesicles allowing EV sorting. | Design and manipulation of bioparticles and submicrometer particles for deterministic sorting. |
Lipid nanoprobes | Lipid layer labelling and magnetic enrichment | Rapid; Efficient isolation of nanoscale EVs. | Low specificity; Contamination of other phospholipid membrane vesicles can occur. |
Peptide affinity assays (Examples: â—¦ Vn96: can bind heat shock proteins on EV surface) | Synthetic peptide binds molecules on the EV surface | Rapid; Inexpensive; High efficiency in isolating EVs from a minimum volume of liquid biopsy; Feasibility of simultaneous analyses of proteins, small RNAs and DNA. | Affinity for both EVs and circulating cell free DNA. The synthetic peptide must be removed from the sample prior to proteomic analysis. |
Aptamer-based assays | Short oligonucleotides that can bind with high affinity to specific molecules targeting specific markers | Rapid; Binding targets with a higher affinity and better biosensing in comparison to antibodies; Capture of specific cancer biomarkers allowing enrichment of tumor-derived exosomes; Less expensive than antibodies. | Risk of degradation by nuclease activity; Limited reproducibility for small EV proteins in clinical setting; Need to develop standardized selection and modification strategies for highly performing aptamers. |