Conclusions
These developments will promote the translational value of the dual-mode separation - a fast, equipment-free, and non-biased way for EV isolation from plasma samples.
Methods
We built an enhanced dual-mode chromatography (eDMC) device which performed i) size-exclusion to remove particles smaller than EVs and LDLs and ii) cation-exchange in an acidic elution to retain LDLs longer than EVs. The performance of the eDMC, in comparison to size-exclusion only, was evaluated by analyzing the yield and purity of the isolated EVs.
Results
By measuring and modeling zeta potentials at different buffer pH, we estimated surface charge densities of EVs (-6.2 mC/m2) and LDLs (-3.6 mC/m2), revealing that EVs are more negatively charged than LDLs. Furthermore, the charge difference between EVs and LDLs was maximal at a weak acidic condition (pH = 6.4). By applying these findings, we optimized eDMC operation to enrich EVs directly from plasma, depleting >99.8% of LPPs within 30 min. Minimizing LDL contamination improved analytical signals in EV molecular assays, including single vesicle imaging, bulk protein measurements, and mRNA detection. Conclusions: These developments will promote the translational value of the dual-mode separation - a fast, equipment-free, and non-biased way for EV isolation from plasma samples.