A Proximity-Induced pH-Responsive DNA Switch for Reversible Capture and Release of Extracellular Vesicles

Extracellular vesicles (EV) serve as critical mediators in physiological and pathological processes, holding great promise for cancer diagnosis, real-time monitoring, and prognostic applications. However, their small size and low density present considerable challenges in achieving efficient, specific, and mild isolation, which currently hinders widespread clinical translation. Here, we report a proximity-induced pH-responsive DNA switch (PPS) that enables reversible EV capture via Hoogsteen triplex formation. Specifically, dual-aptamer probes targeting EpCAM and CD63 on EV exploit membrane fluidity to form proximity-induced duplex helix, which assemble into Hoogsteen triplex helix with magnetic bead-conjugated strands at mildly acidic conditions (pH6.5) for EV capture. Crucially, simply adjusting the pH to neutral (pH7.4) triggers triplex dissociation, releasing intact EV without using chemical denaturants and preserving vesicle integrity for high-purity isolation. Under optimized conditions, this method achieves 79.4% and 72.6% EV purification efficiency in PBS and complex biological matrices, respectively, within 1 h. Furthermore, its modular aptamer design enables rapid adaptation to diverse EV subpopulations through simple probe substitution, without modifying the core framework, thereby reducing cost and complexity for broad applications. Meanwhile, due to the gentle capture-release process, the structural integrity and bioactivity of EV are well preserved, as demonstrated by wound-healing and cellular uptake assays. These advantageous features─rapid processing, high specificity, mild operation, and preservation of EV activity─indicate that the PPS strategy is a robust, nondestructive method for EV isolation. It thus holds significant potential for further application in diverse EV-related research fields such as disease diagnosis and drug delivery.