Investigation of on-line electrokinetic enrichment strategies for capillary electrophoresis of extracellular vesicles.

This work explores strategies for electrokinetic preconcentration of extracellular vesicles (EVs) that are potential source of biomarkers for different diseases. The first approach that led to successful preconcentration of EVs is based on large volume sample stacking (LVSS), allowing an enrichment factor of 7 for CE of EVs with long-end injection (using a capillary with an effective length of 50 cm). Attempts were also made to perform multiple cycles of LVSS, field amplified sample stacking (FASS) and field amplified sample injection (FASI), to improve EVs preconcentration performance. The focus was then put on development of capillary isotachophoresis under high ionic strengths (IS) for electrokinetic enrichment of slow migrating EVs having heterogeneous mobilities. This approach relies on the use of extremely high concentrations of the terminating electrolyte (TE) to slow down the mobility of TE co-ions, rendering them slower than those of EVs. The limit of detection for intact EVs using the developed ITP-UV method reached 8.3 × 10 ⁸ EVs/mL, allowing an enrichment of 25 folds and a linear calibration up to 4 × 10 ¹⁰ EVs/mL. The ITP-UV and ITP-LIF approaches were applied to provide the electrokinetic signature of EVs of bovine milk and human plasma as well as to visualize more specifically intravesicular fluorescently labelled EVs. The investigation of these strategies shredded light into the challenges still encountered with electrokinetic preconcentration and separation of heterogeneous EVs sub-populations which are discussed herein based on our results and other attempts reported in the literature.
Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
(Copyright © 2024. Published by Elsevier B.V.)