Antibiotic-induced autocatalytic DNA circuit for enrofloxacin detection based on triple signal amplification strategy

Using hairpin DNA as the sensing probes and aptamer sequence as the recognition probe, we have successfully constructed an autocatalytic DNA circuit biosensor for enrofloxacin (ENR) detection with high sensitivity and selectivity. The antibiotic-induced release of the trigger DNA (T) can initiate the cross-hybridization of hairpin probes to form three-way DNA junction products, in which the originally split T fragments in hairpin probes can be pulled together to form the intact T sequence. Both the released T and the newly regenerated T can be reused to speed up the generation of the three-way DNA junction. In the products, intact Mg²⁺-dependent DNAzyme sequences will be generated at two arms of the DNA junctions, which can cleave the FAM and BHQ labeled substrate hairpin probe, leading to the release of another T. Through the triple signal amplification strategy in the autocatalytic DNA circuit, we can get an exponentially amplified signal for ENR detection. The DNA circuit biosensor is ultrasensitive with a detection limit of 25.8 fM. The sensing system is robust and has been applied to the detection of ENR in real fish and water samples with good reliability and accuracy. With the advantages of enzyme-free format, highly efficient signal amplification efficiency, and good robustness in complex samples, we anticipate that this biosensor will be a powerful tool for antibiotic residue monitoring in environmental and food samples.