Generation of specific aptamers for constructing CoOOH nanozymes-based colorimetric aptasensor for detection of fleroxacin in milk

The misuse or abuse of fleroxacin (FLE) in livestock and aquaculture can result in elevated levels of FLE in animal-derived foods, posing significant health risks to humans. Detection of FLE has been relying on instrumental analysis methods that need expensive equipment and longer analysis time. Herein we report the first isolation of two specificity aptamers against FLE (FLE-9 and FLE-51) by capture-SELEX, characterized them well and explored the binding mechanism. Furthermore, FLE-9 was selected to construct a CoOOH@Apt based biosensor. The catalytic activity of the biosensor was evaluated using the Michaelis equation, revealing a Vmax of 4.87×10−8 M s−1 and a Km of 0.11 mM. These results suggest that the affinity of CoOOH@Apt is higher than that of CoOOH nanozymes. In the present of FLE, the aptamer FLE-9 specifically binds to FLE, distancing itself from CoOOH nanozymes and thereby reducing the catalytic activity of the biosensor. Finally, the label-free colorimetric aptasensor was successfully applied to detect FLE, with a detection limit of 4.26 ng/mL. The recovery rates ranged from 96.70 % to 101.70 %, with a relative standard deviation (RSD) below 3.02 %. This study presents promising probes and a novel strategy for the determination of FLE in milk samples, contributing to improved food safety monitoring.