A Rational Design and Development of a Label-free Fluorescent Biosensor for the Simultaneous and Rapid Detection of Five Fluoroquinolones in Food

Fluoroquinolones (FQs) are veterinary drug residues in the food chain that threaten global food safety and human health and there is a great need for a rapid, sensitive and reliable monitoring method to better safeguard our food systems. In this study, we developed a high-performance aptamer via a recognition-mechanism-oriented truncation strategy, enabling rapid and sensitive detection of multiple FQs. The recognition mechanism between the aptamer and FQs were studied by the combination of computer-aided molecular simulation and experimental verifications. It was revealed that hydrophobicity of FQs critically governs aptamer-target interactions. Guided by these insights, the original sequence was truncated to a 40-nt aptamer, named as W-G1, which preserved key binding sites and structural stability. W-G1 with the characteristic G-quadruplex (G4) conformation demonstrated excellent affinity and stability for FQs. Exploiting the function of this conformation, a label-free fluorescent G4/thioflavine T biosensor was fabricated to detect FQs in chicken ham sausage within 30 minutes. The results showed that detection limits ranged from 1.32 ng/kg to 17.30 ng/kg that are lower than food safety regulation defined limits of 1.0 μg/kg (the lowest LOD of standard method for lomefloxacin), with recoveries of 81.17~115.94%. This mechanism-driven strategy enhances aptamer-based detection of FQs, offering improved performance, design flexibility, and reduced cost.