Explicit fluorescence sensing method for sensitive detection of Pb²⁺ ions based on DNA aptamer folding as a molecular...

Heavy metals can potentially create environmental and atmospheric pollution, significantly threatening human health. Lead is categorized as a hazardous heavy metal that can adversely affect the environment and human well-being. We present a novel method for detecting lead (Pb²⁺) ions utilizing crystal violet and thrombin binding aptamer (TBA), employing absorbance, fluorescence, and fluorescence anisotropy experiments. An increase in the absorbance peak of TBA at 300 nm and a significant decrease in the fluorescence intensity of the TBA-crystal violet complex with the addition of Pb²⁺ ions was observed. Fluorescence anisotropy experiments of the TBA-crystal violet complex in the presence of Pb²⁺ ions exhibited a decrease in the anisotropy values from 0.46 to 0.34. In the presence of Pb²⁺ ions, TBA exhibited a positive peak at 312 nm and a negative peak at 265 nm in the CD spectrum, indicating the formation of a G-quadruplex structure. While, in the presence of other metal ions, TBA adopted a random coil structure. A combination of absorption titration and kinetic measurements was employed to understand the role of Pb²⁺ ions in mediating the folding of G-quadruplex structures. A stopped-flow analysis revealed that the rate of G-quadruplex folding with Pb²⁺ ions was approximately 28 times faster than that of K⁺ ions. This method presents a rapid, accurate, and targeted approach for Pb²⁺ ions detection, with potential applications in environmental sample analysis. It can detect Pb²⁺ ions at concentrations as low as 1.18 nM (0.32 ppb), significantly lower than the WHO's drinking water limit of 72 nM.