In contrast to common DNA walkers, multipedal DNA walkers exhibit larger walking area and faster walking kinetics and provide increased amplification efficiency. Consequently, they have received a considerable amount of attention in biosensing. However, most of them are synthesized by immobilizing multiple DNA walking strands on the surface of Au nanoparticles, which is tedious and time-consuming. Simple preparation of multipedal DNA walkers remains a challenge. Herein, we adopted a simple enzyme-free targettriggered catalytic hairpin assembly (CHA) circuit to synthesize a tripedal DNA walker. By walking on a DNA track-functionalized electrode, a sensitive electrochemiluminescence DNA nanomachine biosensor was constructed for sensing miRNA-21. The DNA walker was powered by toehold-mediated strand displacement; the whole process did not need the assistance of enzymes, thus avoiding tedious procedures and enzyme degradation under unfavorable environmental conditions. Specifically, a superior detection limit of 4 aM and a broad linear range of 10 aM to 1 pM were achieved. This CHA−tripedal DNA walker biosensor was then applied for the detection of miRNA-21 in human serum and showed high selectivity and excellent reproducibility, demonstrating its practical application in bioanalysis. In particular, the Y-shaped tripedal DNA walker comes from the DNA circuit, which makes the approach ideally suited for biosensing of small nucleic acid targets.

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