Ratiometric Fluorescence Sensing System for Lead Ions Based on Self-Assembly of Bioprobes Triggered by Specific Pb²⁺–Peptide Interactions

Lead is one of the most toxic substances. However, there are few ratiometric fluorescent probes for sensing Pb 2+ in aqueous solution as well as living cells because specific ligands for Pb 2+ ions have not been well characterized. Considering the interactions between Pb 2+ and peptides, we developed ratiometric fluorescent probes for Pb 2+ based on the peptide receptor in two steps. First, we synthesized fluorescent probes ( 1 – 3 ) based on the tetrapeptide receptor (ECEE-NH 2 ) containing hard and soft ligands by conjugation with diverse fluorophores that showed excimer emission when they aggregated. After investigation of fluorescent responses to metal ions, benzothiazolyl-cyanovinylene was evaluated as an appropriate fluorophore for ratiometric detection of Pb 2+ . Next, we modified the peptide receptor to decrease the number of hard ligands and/or to replace Cys with disulfide bond and methylated Cys for improving selectivity and cell permeability. From this process, we developed two fluorescent probes ( 3 and 8 ) among the probes ( 1 – 8 ) that exhibited remarkable ratiometric sensing properties for Pb 2+ including high water solubility (≤2% DMF), visible light excitation, high sensitivity, selectivity for Pb 2+, low detection limits (<10 nM), and fast response (<6 min). The binding mode study revealed that specific Pb 2+ –peptide interactions of the probes caused nanosized aggregates in which the fluorophores of the probes came close each other, exhibiting excimer emission. In particular, 8 based on tetrapeptide bearing a disulfide bond and two carboxyl groups with a good permeability successfully quantified intracellular uptake of Pb 2+ in live cells through ratiometric fluorescent signals. The ratiometric sensing system based on specific metal–peptide interactions and excimer emission process could provide a valuable tool to quantify Pb 2+ in live cells and pure aqueous solutions.