Cooperation of CRISPR/Cas12a and exonuclease III-assisted cascade cycling amplification for ultrasensitive electrochemical detection of ciprofloxacin

Antibiotic residues have been a serious public health concern worldwide, while sensitive and reliable detection of antibiotic residues is significant to control antibiotic contamination, ensure food safety, and safeguard human health. Herein, an ultrasensitive electrochemical biosensor is engineered for the detection of ciprofloxacin (CIP) based on the cooperation of CRISPR/Cas12a and exonuclease III (Exo III)-assisted cascade cycling amplification. The presence of CIP induces the conformational change of DNA probes and further triggers Exo III to catalyze the cascade cycling amplification, enabling propagation and ongoing accumulation of DNA fragments which act as the target strands to activate the trans-cleavage activity of CRISPR/Cas12a. Consequently, the activated CRISPR/Cas12a initiates its trans-cleavage activity to swiftly cleave the signal probes on the surface of electrode, bringing about remarkable change of electrochemical signal and eventually realizing the ultrasensitive detection of CIP. The exceptional enzymatic cycle amplification of Exo III incorporated with the superior trans-cleavage activity of CRISPR/Cas12a synergistically facilitates considerable improvement of analytical performance, resulting in a limit of detection as low as 0.022 ng mL^-1. Benefiting from the effective amplification capacity, high fidelity and programmability of the designed detection system, the biosensor shows good precision and specificity along with robust stability for CIP detection. Moreover, the proposed electrochemical biosensor dispensing with complicated probe construction is label-free and convenient-operated, which contributes to the credible application for CIP detection in real food samples with satisfactory results, indicating promising practicability in food safety monitoring.