Rapid isolation of extracellular vesicles from diverse biofluid matrices via capillary-channeled polymer fiber solid-phase extraction micropipette tips

Extracellular vesicles (EVs) play essential roles in biological systems based on their ability to carry genetic and protein cargos, intercede in cellular communication and serve as vectors in intercellular transport. As such, EVs are species of increasing focus from the points of view of fundamental biochemistry, clinical diagnostics, and therapeutics delivery. Of particular interest are 30-200 nm EVs called exosomes, which have demonstrated high potential for use in diagnostic and targeted delivery applications. The ability to collect exosomes from patient biofluid samples would allow for comprehensive yet remote diagnoses to be performed. While several exosome isolation methods are in common use, they generally produce low recoveries, whose purities are compromised by concomitant inclusion of lipoproteins, host cell proteins, and protein aggregates. Those methods often work on lengthy timescales (multiple hours) and result in very low throughput. In this study, capillary-channeled polymer (C-CP) fiber micropipette tips were employed in a hydrophobic interaction chromatography (HIC) solid-phase extraction (SPE) workflow. Demonstrated is the isolation of exosomes from human urine, saliva, cervical mucus, serum, and goat milk matrices. This method allows for quick (<15 min) and low-cost (<$1 per tip) isolations at sample volume and time scales relevant for clinical applications. The tip isolation was evaluated using absorbance (scattering) detection, nanoparticle tracking analysis (NTA), and transmission electron microscopy (TEM). Exosome purity was assessed by Bradford assay, based on the removal of free proteins. An enzyme-linked immunosorbent assay (ELISA) to the CD81 tetraspanin protein was used to confirm the presence of the known exosomal-biomarker on the vesicles.