Selection of a high-affinity DNA aptamer for puerarin and its application in fluorescence sensing

Puerarin (Pue), a naturally occurring isoflavone, is an important quality marker of Pueraria lobata, a widely used traditional Chinese medicinal material. Reliable determination of Pue is therefore relevant to both pharmaceutical quality control and studies of its biological activity. Here, a library-immobilized capture-SELEX protocol was used to isolate DNA sequences that recognize Pue. The predominant candidate, Pue-1, displayed an apparent dissociation constant (K_d) of 738 nM in a thioflavin T (ThT) fluorescence assay. An independent microscale thermophoresis (MST) measurement yielded a K_d of 111 nM. Pue-1 showed substantially weaker responses to the structurally related flavonoids tested, supporting its preferential recognition of Pue. A target-induced strand-displacement design was subsequently used to construct a turn-on fluorescent aptasensor, which exhibited preferential responses to Pue over the tested interferents. In 200-fold-diluted Pueraria root extract, the assay achieved a limit of detection of 29.5 nM, with spike recoveries ranging from 93.2% to 96.2%. These results demonstrate the suitability of Pue-1 as a recognition element for constructing a fluorescent sensing platform for Pue.