Combining G-Quadruplex and Non-Quadruplex Aptamers with Distinct Thermodynamic Driving Forces for Highly Selective Pb ²⁺ Detection

Lead ions (Pb 2+ ) are important contaminants and have also emerged as valuable cofactors in nucleic acid chemistry. Both G-quadruplex (G4)-forming sequences and in vitro selected aptamers have been extensively explored for Pb 2+ binding and detection. Here, aptamer selection was conducted using a low Pb 2+ concentration (10 μM) and an extended incubation time (10 min), yielding two distinct families of Pb 2+ -binding aptamers. ITC revealed that the non-G-rich aptamer Pb-13 binds Pb 2+ through favorable enthalpic and entropic contributions. In contrast, the G-rich aptamer Pb-2-T forms a hybrid G-quadruplex and exhibits predominantly enthalpy-driven binding, whereas the classical G-quadruplex sequence T30695 binds Pb 2+ through an entropy-driven mechanism. A strand-displacement fluorescence sensor based on Pb-13 achieved a detection limit of 7.5 nM Pb 2+ . Thioflavin T (ThT) fluorescence further revealed opposite responses for the two aptamers, with Pb-13 exhibiting signal-off behavior and Pb-2-T displaying signal-on behavior. These complementary responses enabled selective and label-free detection of Pb 2+ . This work demonstrates that DNA can recognize Pb 2+ through multiple orthogonal structural and thermodynamic mechanisms, expanding our understanding of metal ion recognition and multidimensional sensing.