Structure Switchable Single Fluorophore Biosensor to Measure Dissociation Constant in PAT Aptamer Tailoring

Abstract Aptamers, as synthetic oligonucleotide recognition elements, exhibit remarkable potential in biosensing applications with high specificity, chemical modifiability, and cost‐effectiveness. Current approaches remain fundamentally limited by their reliance on expensive instrumentation, complex operation, and labor‐intensive modification processes. Here, a structure‐switchable single‐fluorophore biosensor is developed integrating agarose‐immobilized targets with fluorescent light‐up extension primers for real‐time quantitative PCR (qPCR)‐based dissociation constant (K_d) determination. A 78‐mer patulin aptamer is used as a model for systematic tailoring. Aptamer M1 with the best affinity (K_d = 33.41nm), obtained by removing primer regions and terminal redundant bases, exhibits a 2.5‐fold increase in affinity compared to the 78 – mer. Method validation shows consistent trends, confirming reliability of the measurement platform and the efficacy of the aptamer engineering approach. Further, the molecular docking analysis identifies the central stem GC base pairs as the core interaction sites. In all, this study establishes a cost‐effective, aptamer affinity quantification platform based on standard qPCR, improveing the quantitative assessment of SELEX‐derived aptamers for functional element conversion, providing a robust technical framework for advancing aptamer applications in biosensing.