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 Pb2+ in aqueous solution as well as living cells because specific ligands for Pb2+ ions have not been well characterized. Considering the interactions between Pb2+ and peptides, we developed ratiometric fluorescent probes for Pb2+ based on the peptide receptor in two steps. First, we synthesized fluorescent probes (1–3) based on the tetrapeptide receptor (ECEE-NH2) 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 Pb2+. 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 Pb2+ including high water solubility (≤2% DMF), visible light excitation, high sensitivity, selectivity for Pb2+, low detection limits (<10 nM), and fast response (<6 min). The binding mode study revealed that specific Pb2+–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 Pb2+ 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 Pb2+ in live cells and pure aqueous solutions.