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ABSTRACT Exosomes, a subset of extracellular vesicles (EVs) ranging in size from 30 to 150 nm, are of significant interest for biomedical applications such as diagnostic testing and therapeutics delivery. Biofluids, including urine, blood, and saliva, contain exosomes that carry biomarkers reflective of their host cells. However, isolation of EVs is often a challenge due to their size range, low density, and high hydrophobicity. Isolations can involve long separation times (ultracentrifugation) or result in impure eluates (size exclusion chromatography, polymer‐based precipitation). As an alternative to these methods, this study evaluates the first use of nylon‐6 capillary‐channeled polymer (C‐CP) fiber columns to separate EVs from human urine via a step‐gradient hydrophobic interaction chromatography method. Different from previous efforts using polyester fiber columns for EV separations, nylon‐6 shows potential for increased isolation efficiency, including somewhat higher column loading capacity and more gentle EV elution solvent strength. The efficacy of this approach to EV separation has been determined by scanning electron and transmission microscopy, nanoparticle flow cytometry (NanoFCM), and Bradford protein assays. Electron microscopy showed isolated vesicles of the expected morphology. Nanoparticle flow cytometry determined particle densities of eluates yielding up to 5 × 108particles mL−1, a typical distribution of vesicle sizes in the eluate (60–100 nm), and immunoconfirmation using fluorescent anti‐CD81 antibodies. Bradford assays confirmed that protein concentrations in the EV eluate were significantly reduced (approx. sevenfold) from raw urine. Overall, this approach provides a low‐cost and time‐efficient (< 20 min) column separation to yield urinary EVs of the high purities required for downstream applications, including diagnostic testing and therapeutics.
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Electroformed Inverse‐Opal Nanostructures for Surface‐Marker‐Specific Isolation of Extracellular Vesicles Directly from Complex Media
Abstract Extracellular vesicles (EVs) – nanoscale membranous particles that carry multiple proteins and nucleic acid cargoes from their mother cells of origin into circulation – have enormous potential as biomarkers. However, devices appropriately scaled to the nanoscale to match the size of EVs (30–200 nm) have orders of magnitude too low throughput to process clinical samples (1012EVs mL−1in serum). To address this challenge, we develop a novel approach that incorporates billions of nanomagnetic sorters that act in parallel to precisely isolate sparse EVs based on immunomagnetic labeling directly from clinical samples at flow rates billions of times greater than that of a single nanofluidic device. To fabricate these chips, the ferromagnetic metals are electro‐deposited into a self‐assembled microlattice, achieving >109nanoscale magnetophoretic sorting devices in a 3D postage stamp‐sized lattice with >70x magnetic traps and >20x enrichment of magnetic nanoparticles versus our previous work. The immunomagnetically labeled EVs are isolated and achieve a ≈100% increase in yield as well as increased purity compared to conventional methods. Building on the proof‐of‐concept demonstrations in this manuscript, this new approach has the potential to enhance the future clinical translation of EV biomarkers by enabling rapid, sensitive, and specific isolation of EV subpopulations from clinical samples.
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- Award ID(s):
- 2344714
- PAR ID:
- 10419155
- Publisher / Repository:
- Wiley Blackwell (John Wiley & Sons)
- Date Published:
- Journal Name:
- Advanced Materials Technologies
- Volume:
- 8
- Issue:
- 9
- ISSN:
- 2365-709X
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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