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With the development of DNA nanotechnology, DNA has been widely used to construct a variety of nanomachines. Among them, a DNA walker is a unique nanomachine that can move continuously along a specific orbit to fulfill diverse functions. In this paper, a dual signal amplification electrochemical biosensor based on a DNA walker and DNA nanoflowers is constructed for high sensitivity detection of Staphylococcus aureus (S. aureus). Two groups of double-stranded DNA are modified on the surface of a gold electrode. The binding of S. aureus with its aptamer induces the disintegration of the long double strands and releases the DNA walker. With the help of exonuclease III (Exo III), the DNA walker moves along the electrode surface and continuously hydrolyzes the anchored short double strands. The introduction of a specially customized circular DNA and phi29 DNA polymerase initiates the rolling circle amplification (RCA) reaction. DNA nanoflowers are formed at high local concentration of DNA in the solution, which provide binding sites for electroactive methylene blue (MB) and thus produce intense signal. Under the best conditions, the current response is linearly related to the logarithm of the concentration of S. aureus ranging from 60 to 6 × 10 7 CFU/mL, and the detection limit is 9 CFU/mL. In addition, the proposed biosensor has achieved satisfactory results in the detection of actual water samples and diluted honey samples, which confirm the practicability of the biosensor and its application potential in environmental monitoring and food safety.

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