A Sensitive Aptamer Fluorescence Anisotropy Sensor for Cd²⁺ Using Affinity-Enhanced Aptamers with Phosphorothioate Modification

Rapid and sensitive detection of heavy metal cadmium ions (Cd²⁺) is of great significance to food safety and environmental monitoring, as Cd²⁺ contamination and exposure cause serious health risk. In this study we demonstrated an aptamer-based fluorescence anisotropy (FA) sensor for Cd²⁺ with a single tetramethylrhodamine (TMR)-labeled 15-mer Cd²⁺ binding aptamer (CBA15), integrating the strengths of aptamers as affinity recognition elements for preparation, stability, and modification, and the advantages of FA for signaling in terms of sensitivity, simplicity, reproducibility, and high throughput. In this sensor, the Cd²⁺-binding-induced aptamer structure change provoked significant alteration of FA responses. To acquire better sensing performance, we further introduced single phosphorothioate (PS) modification of CBA15 at a specific phosphate backbone position, to enhance aptamer affinity by possible strong interaction between sulfur and Cd²⁺. The aptamer with PS modification at the third guanine (G) nucleotide (CBA15-G3S) had four times higher affinity than CBA15. Using as an aptamer probe CBA15-G3S with a TMR label at the 12th T, we achieved sensitive selective FA detection of Cd²⁺, with a detection limit of 6.1 nM Cd²⁺. This aptamer-based FA sensor works in a direct format for detection without need for labeling Cd²⁺, overcoming the limitations of traditional competitive immuno-FA assay using antibodies and fluorescently labeled Cd²⁺. This FA method enabled the detection of Cd²⁺ in real water samples, showing broad application potential.