EDTA-Masked Tl⁺ Detection Using a Pair of Non-G-Quadruplex Aptamers with Opposite Fluorescence Responses

Thallium (Tl) is a highly toxic trace heavy metal that poses significant environmental and ecological risks. Current analytical methods for its detection are limited to instrumentation. In this study, we employed a library-immobilized SELEX (systematic evolution of ligands by exponential enrichment) technique and isolated a pair of non-G-quadruplex DNA aptamers that can specifically recognize Tl⁺. Among them, the Tl-3 aptamer exhibited a dissociation constant (K_d) of 4.5 μM by a thioflavin T (ThT) fluorescence assay, where the fluorescence dropped upon adding Tl⁺. Using isothermal titration calorimetry (ITC), its K_d was determined to be 17.9 μM. Another aptamer, Tl-5, showed increased ThT fluorescence upon Tl⁺ addition, indicating a distinct structural feature. The K_d values for Tl-5 were 14.0 μM (ThT) and 34.2 μM (ITC). This pair of Tl⁺ aptamers made it possible to tell Tl⁺ apart from a few general G-quadruplex binding metals such as K⁺ and Pb²⁺. Taking advantage of the low affinity of Tl⁺ to EDTA, the responses of both aptamers to other metal ions were negligible using EDTA as a masking agent. The ThT-based label-free fluorescent detection of Tl⁺ lake water achieved limits of detection (LODs) of 0.8 and 1.6 μM using Tl-3 and Tl-5, respectively. This pair of aptamers holds great promise for the environmental analysis of Tl⁺.