Target-promoted catalytic DNA molecular circuit network coupled with endonuclease amplification for sensitive and label-free electrochemical aptamer antibiotic assay

Sarafloxacin (SAR) is a common antibiotic that accumulates in animal tissues, which potentially poses health risks to humans via ingestion. Sensitive detection of SAR in animal-derived foods is therefore critical. Traditional SAR detection methods based on HPLC or antibody immunoassays encounter the limitations of cost, sensitivity and operational simplicity. In this study, with the design of a new DNAzyme/catalytic hairpin assembly (CHA) molecular circuit network coupled with endonuclease amplification, we developed an electrochemical aptamer (AP) biosensor with high sensitivity for label-free SAR detection in milk. The sensor functions by specific binding of SAR to APs, which release DNAzyme sequences to initiate the catalytic DNAzyme/CHA molecular circuit network amplification for forming many ssDNAs and dsDNA duplexes. These sequences further hybridize with hairpin signal probes on sensor electrode to create favorable nicking sites for endonuclease, which cleaves hairpin signal probes to liberate lots of G-quadruplex strands to complex with and capture hemin. Subsequent hemin reduction by electrochemistry thus results in dramatically enhanced currents to achieve label-free detection of SAR in linear range between 10 pM and 80 nM with detection limit of 1.62 pM. Our sensor also shows satisfactory reproducibility, repeatability and stability, demonstrates high selectivity for the target SAR against other interfering antibiotics, e.g., norfloxacin, penicillin and gentamicin and can realize detection of low concentrations of SAR in diluted complex milk and human serum samples, thereby underscoring its promises for convenient and sensitive assay of different trace antibiotics.