Unlocking Pb²⁺ Sensing Potential in a DNA G‑Quadruplex via Loop Modification with Fluorescent Chalcone Surrogates

The ability of guanine (G)-rich DNA to bind toxic lead (Pb²⁺) ions within a G-quadruplex (GQ) motif is a leading DNA biosensor strategy. A major analytical hurdle for GQ detection of Pb²⁺ is competitive GQ templating by potassium (K⁺) ions. We employ the on-strand DNA synthesis of internal fluorescent chalcone surrogates within the 15-mer thrombin binding aptamer (TBA15) to address this challenge. Replacement of thymidine at the 3-position (T3) within TBA15 with an indole-4-hydroxy-indanone (Ind4HI) chalcone strongly decreases K⁺-GQ stability while enhancing Pb²⁺-GQ stability to increase Pb²⁺ binding specificity. The new T3-Ind4HI probe exhibits a 15-fold increase in fluorescence intensity upon binding of Pb²⁺ by the modified TBA15 and can detect 6.4 nM Pb²⁺ in the presence of 10 mM K⁺. Thus, replacement of the T3 residue of TBA15 with the new Ind4HI probe modulates metal ion affinity by native TBA15 to solve the analytical challenge posed by K⁺ in real water samples for detecting Pb²⁺ to meet regulatory guidelines by using a GQ biosensor.