A dual-recognition fluorescence chip utilizing a substrate-borne DNA walker for precision profiling of small extracellular vesicles.
We report a fluorescence chip based on a membrane-anchored, bipedal DNAzyme walker assembled on small extracellular vesicles (sEVs) surfaces via catalytic hairpin assembly (CHA). Unlike previously reported single-arm or proximity-ligation-dependent walkers on nanoparticle supports, this CHA-triggered DNAzyme walker is designed with a bipedal architecture and operates directly on the sEVs membrane. In our design, CHA simultaneously generates two DNAzyme arms on the same sEVs, enabling faster substrate cleavage kinetics and superior signal amplification. The assay achieves a theoretical detection limit of 2.8 particles/μL, approximately 1.5-fold lower than a comparable single-arm architecture. Dual recognition of the sEVs lipid bilayer and surface EpCAM protein (using a cholesterol-modified substrate and an anti-EpCAM antibody) effectively eliminates false-positive signals from free EpCAM or soluble interferents. The entire reaction cascade is integrated onto a chip platform, improving reproducibility and point-of-care potential. The chip performs robustly in fetal bovine serum and clinical serum samples, with recovery rates of 99.1-103.8% and strong correlation with nanoparticle tracking analysis. By establishing a CHA-assisted, bipedal walking mechanism on biological membranes, this work opens a new paradigm for designing efficient DNA nanomachines on membrane-enveloped targets.