A colorimetric aptasensor fabricated with group-specific split aptamers and complex nanozyme for enrofloxacin and ciprofloxacin determination.

Developing simultaneous detection methods for multiple targets is crucial for the field of food analysis. Herein, enrofloxacin (ENR) and ciprofloxacin (CIP) were taken as model targets. For the first time, a strategy to generate group-specific split aptamers was established by revealing and splitting the critical binding domain, and the split aptamers were exploited to design a four-way DNA junction (4WJ) which could regulate the enzymatic activity of chitosan oligosaccharide (COS)-AuNPs nanozyme to develop a colorimetric aptasensor. A pair of split aptamers were obtained for ENR (K = 15.00 nM) and CIP (K = 4.870 nM). The mechanism of COS binding with double-stranded DNA in the 4WJ was elucidated. Under optimal conditions, the colorimetric aptasensor enabled a wide linear detection range of 1.4-1400 nM and a limit of detection (LOD) of 321.1 pM and 961.0 pM towards ENR and CIP, respectively, which exhibited excellent sensitivity, selectivity, and availability in detecting ENR/CIP in seafood. This study expands the general strategies for generating robust aptamers and nanozyme complex and provides a good reference for developing multi-target detection methods.