External intervention can make quasi‐mechanical movements in enzyme‐free DNA nanomachine, suggesting the promise of developing novel biosensing systems. The superiority of the “signal‐on” strategies inspired researchers to fabricate sensitive and selective biosensors by combining the advantages of chemical amplification methods with DNA nanomachines. Herein, a smart enzyme‐free DNA nanomachine for biomolecule detection has been developed using DNA walker strategy, Click Chemistry, and electrochemically mediated surface‐initiated atom transfer radical polymerization. When the target biomarker is present, DNA walker (DW) could be unleashed which drives walking on the surface of the DNA‐coated Fe3O4/GCE. Then, this DW hybridized with Azide‐labeled DNA probe1 (H1) and subsequently substituted by DNA probe2 (H2) through walking process. Therefore, the loop of H1 was unlocked to form Azide group available at 3’ end. This procedure is repeated so that each released DW can open multiplex H1 strands, resulted in amplification of the signal. After successive walking, “Click reactions” carried out between Azide and 3‐Butynyl‐2‐bromoisobutyrate for initiating the radical polymerization reaction, which brought a great quantity of the electroactive labels on the electrode surface. The number of labels was quantified by square wave voltammetry which was proportional to the target biomarker concentration. In addition, this electroanalytical devicewas applied successfully for direct determination of biomolecules in real serum, and also showed wide dynamic range, excellent detection limit, storage stability and reproducibility. Thus, the proposed biosensor could be effectually utilized in biomedical research and clinical diagnosis.
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