Self-protected circular DNAzyme for integrated enrichment and quantification of small extracellular vesicles.

Small extracellular vesicles (sEVs) hold immense potential for liquid biopsy given the wealth of biological information they carry. Currently, the clinical application of these methods is limited due to their low abundance and the complexities associated with traditional isolation techniques. To address this, we developed a strategy integrating cholesterol-mediated capture with a Self-Protected DNAzyme Walker for the rapid and simultaneous specific isolation and quantification of small extracellular vesicles (sEVs). Upon specific binding to CD63, the blocker strand is released, which activates the DNAzyme catalytic core, leading to substrate cleavage, which triggers the specific release of sEVs from magnetic beads and the generation of a fluorescent signal. Importantly, the circular DNA Shield design provides remarkable stability to the system by safeguarding the DNAzyme core from nuclease degradation. Furthermore, the cyclic cleavage mechanism allows for highly sensitive detection, achieving a limit of detection (LOD) as low as 361 particles per μL. In addition, by leveraging the lipid bilayer structure for sEV enrichment, this strategy effectively eliminates interference from free proteins. Furthermore, the clinical feasibility of this assay was validated by successfully distinguishing Stage I breast cancer patients from healthy individuals with high statistical significance (p < 0.001), highlighting its promise for early cancer diagnosis. This work presents a robust paradigm for sEV analysis and lays a solid foundation for their downstream biomedical applications.