From DNA Aptamer Selection to Label-Free Fluorescent Biosensor for Chebulinic Acid

Abstract Chebulinic acid is a bioactive hydrolyzable tannin of increasing interest in natural product research, quality control, and pharmaceutical studies. In this work, four candidate DNA aptamers were identified using a restriction endonuclease-assisted SELEX (REase-SELEX) strategy, and Aptamer 1 was selected based on its enrichment level, predicted structural stability, thermal stability, and structural characteristics. A label-free fluorescent aptasensor was developed using thioflavin T (ThT) as the signal reporter. Structural prediction indicated that the selected aptamer was a G-rich sequence containing multiple stem-loop motifs and potential G-quadruplex-forming regions. Molecular docking further suggested possible binding modes between the aptamer and chebulinic acid involving multiple noncovalent interactions. The binding affinity was further validated by microscale thermophoresis and a gold nanoparticle-based colorimetric assay, yielding dissociation constants of 4.112 and 2.770 μM, respectively. Under optimized conditions, the proposed aptasensor exhibited a good linear response toward chebulinic acid in the range of 0.1-50 μM, with a regression equation of Y = -1.260X + 248.6 and an R2 of 0.9872. The limit of detection (LOD) was 8.29 nM. In addition, the sensor showed good selectivity and satisfactory recovery in a compound blank matrix and 10% fetal bovine serum (FBS), with recoveries of 94.3%-102.9%. These results demonstrate that the proposed platform provides a simple, low-cost, and sensitive strategy for chebulinic acid detection.