Construction of a fluorescent platform for sensitive small extracellular vesicles analysis based on sequential self-priming mediated DNAzyme catalyzed DNA walker

Small extracellular vesicles (sEVs) have emerged as highly promising diagnostic biomarkers for various diseases, including osteonecrosis of the femoral head and multiple malignancies. However, there remains a critical need for rapid, sensitive, and practical platforms capable of on-site sEVs detection. In this study, we developed a fluorescent bio-sensing platform for sensitive sEVs analysis based on a sequential self-priming DNAzyme-catalyzed DNA walker system. The biosensor incorporates a dumbbell-shaped probe, constructed by hybridizing two stem-loop DNA structures immobilized on a solid surface. This design enables precise target recognition and initiates a self-primed DNA polymerization process, which is further coupled with endonuclease-assisted strand extension and displacement. By integrating multiple signal amplification strategies, the platform achieves a broad detection range and an exceptionally low detection limit of 4.1 particles/μL. A key innovative aspect of our design is that the DNAzyme component of the DNA walker is not pre-assembled; rather, it is generated specifically in the presence of target sEVs. This target-induced activation mechanism substantially reduces background signal and effectively minimizes false-positive outcomes. Consequently, the biosensor maintains excellent detection stability, even within complex sample matrices. Given its high sensitivity and operational robustness, the proposed method represents a versatile bioassay platform. Its applicability can be readily extended to other analytes by incorporating specific aptamers into the probe design, thereby significantly broadening its potential in the field of bio-sensing. • We developed a fluorescent biosensing platform for sensitive sEVs analysis. • The platform is developed based on a sequential self-priming DNAzyme-catalyzed DNA walker system. • The integration of multiple signal amplification results in a broad detection range and a low LOD of 4.1 particles/μL. • Given its high sensitivity, the proposed method holds great potential as a versatile bioassay platform.