A proximity cleavage assisted DNAzyme catalyzed loading DNA walker system for accurate and sensitive small extracellular vesicle analysis.

The significant biological functions and clinical importance of small extracellular vesicles (sEVs) in lung malignancies make them crucial diagnostic markers. However, the current detection methods for cancer related sEVs typically recognize single molecules on the surface of sEVs, which lack enough specificity. We developed a proximity cleavage-assisted DNAzyme-catalyzed loading DNA walker system for the precise investigation of sEVs. The target sEVs served as a loading medium by utilizing a cholesterol substrate and recognition probe to attach to the lipid bilayer and EpCAM on the surface, thereby initiating the proximity ligation-triggered DNAzyme-catalyzed DNA walker. The DNAzyme-catalyzed DNA walker enables the simultaneous detection of the lipid bilayer and EpCAM, thereby conferring high specificity to the technique. The DNAzyme-catalyzed DNA walker on the surface of sEVs and the signal generation mediated by the cleavage of free DNAzymes constitute the signal generation system. The proposed approach demonstrated a low detection limit of 2.5 particles per μL based on this design. The proposed method offers a novel approach for the sensitive detection of sEVs and other membrane structural molecules, indicating promising potential for disease diagnosis.