Capture‐SELEX of DNA Aptamers for Highly Selective Binding of Folate

Folate (vitamin B9) is an essential micronutrient for human health, particularly during pregnancy. Aptamers have emerged as attractive recognition elements for small-molecule detection, as demonstrated by numerous natural riboswitches. Although an RNA aptamer for folate has been reported, the development of a DNA aptamer is highly desirable for biosensing applications due to its superior chemical stability and ease of modification. Herein, we employed a library-immobilization-based selection strategy to isolate a family of high-affinity DNA aptamers for folate. Isothermal titration calorimetry (ITC) measurements yielded a dissociation constant of 640 nM. Based on the selected aptamer (FA1), we developed a signal-on, strand-displacement fluorescent biosensor by hybridizing a 5'-FAM-labeled aptamer with a short quencher-labeled complementary strand. The sensor achieved a limit of detection (LOD) of 8.5 nM in selection buffer and 31 nM in 10% serum. Selectivity studies demonstrated negligible responses to structurally related compounds, including guanine, adenine, theophylline, xanthine, and caffeine. Tetrahydrofolate generated a measurable signal only at elevated concentrations; however, ITC confirmed no direct binding, indicating that the observed response likely arises from its oxidation to folate. These results establish a robust DNA aptamer for folate for both fundamental studies and applied contexts, such as clinical monitoring and food fortification quality control.