Comparison of extracellular vesicle elution methods from aptamer-conjugated magnetic beads for downstream physical characterization and immunoassays

Aptamer-based affinity chromatography is a promising approach for the efficient capture and gentle release of extracellular vesicles (EVs) with preserved EV integrity; however, to date, there is no direct comparison of EV elution methods from the aptamers and evaluation of their effects on downstream EV analysis. This study compares the efficacy of two simple-to-implement elution methods using deoxyribonuclease (DNase) I and NaCl in releasing EVs from anti-CD63-aptamer-conjugated magnetic beads, focusing on downstream physical and protein analysis of the EVs. We used anti-CD63-aptamer-functionalized magnetic beads to capture EVs from ovarian cancer cell line OVCAR-3 culture media, subsequently eluting them using NaCl (0.5 or 1.0 M) or DNase I (50 or 100 U/mL). Dynamic light scattering and transmission electron microscopy (TEM) were used to assess elution efficiency and EV morphology, respectively, and showed intact EVs after elution. Enzyme-linked immunosorbent assay (ELISA) quantified CD9 and EGFR protein expression in the eluted EVs. Our results indicated that NaCl and DNase I can effectively elute EVs, maintaining their structural integrity as observed by TEM. 100 U/mL DNase I provided the highest elution efficiency of 88%, followed closely by 1.0 M NaCl with a comparable efficiency of 87%. With ELISA targeting two EV surface proteins, CD9 and EGFR, EVs eluted by 1.0 M NaCl demonstrated the highest signals for both proteins. Due to the high elution efficiency and low cost of NaCl, it is more suitable for immunoassay-based downstream analysis. This comparative study demonstrated the importance of selecting an appropriate elution method to optimize EV yield and maintain protein activity for downstream applications.