Screening of Single-Stranded DNA Aptamer Specific for Florfenicol and Application in Detection of Food Safety

Abstract In this work, single-stranded DNA (ssDNA) aptamers specific to florfenicol (FF) having high binding affinity were prepared using magnetic-bead-based systematic evolution of ligands by exponential enrichment technique (MB-SELEX). After 10 rounds of MB-SELEX screening, aptamers that can simultaneously recognize FF and its metabolite FFA were obtained. The aptamer with the lowest dissociation constant (Kd) was truncated and optimized based on secondary structure analysis. The optimal aptamer selected was Apt-14t, with a length of 43 nt and its dissociation constant was 4.66 ± 0.75 nM, which was about 7 times higher than that of the full-length sequence. The potential binding sites and interactions with FF were demonstrated by molecular docking simulations. In addition, a colorimetric strategy for nanogold aptamers was constructed. The linear detection range of this method was 0.00128–500 ng/mL and the actual detection limit was 0.00128 ng/mL. Using this strategy to detect florfenicol in actual milk and eggs samples, the spiked recoveries were 88.9% − 123.1% and 84.0% − 112.2%, respectively and the relative standard deviation was less than 5.6%, showing high accuracy.