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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Rapid purification and multiparametric characterization of circulating small extracellular vesicles utilizing a label-free lab-on-a-chip device
Nano-scale extracellular vesicles are lipid-bilayer delimited particles that are naturally secreted by all cells and have emerged as valuable biomarkers for a wide range of diseases. Efficient isolation of small extracellular vesicles while maintaining yield and purity is crucial to harvest their potential in diagnostic, prognostic, and therapeutic applications. Most conventional methods of isolation suffer from significant shortcomings, including low purity or yield, long duration, need for large sample volumes, specialized equipment, trained personnel, and high costs. To address some of these challenges, our group has reported a novel insulator-based dielectrophoretic device for rapid isolation of small extracellular vesicles from biofluids and cell culture media based on their size and dielectric properties. In this study, we report a comprehensive characterization of small extracellular vesicles isolated from cancer-patients’ biofluids at a twofold enrichment using the device. The three-fold characterization that was performed using conventional flow cytometry, advanced imaging flow cytometry, and microRNA sequencing indicated high yield and purity of the isolated small extracellular vesicles. The device thus offers an efficient platform for rapid isolation while maintaining biomolecular integrity.
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- Award ID(s):
- 2046037
- PAR ID:
- 10479678
- Publisher / Repository:
- natureportfolio
- Date Published:
- Journal Name:
- Scientific Reports
- Volume:
- 13
- Issue:
- 1
- ISSN:
- 2045-2322
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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