Tetrahedral\nDNA-Based Ternary Recognition Ratiometric\nFluorescent Probes for Real-Time In Situ Resolving Lysosome Subpopulations\nin Living Cells via Cl<sup>–</sup>, Ca<sup>2+</sup>, and pH

Lysosomes are multifunctional organelles vital for cellular\nhomeostasis\nwith distinct subpopulations characterized by varying levels of Cl<sup>–</sup>, Ca<sup>2+</sup>, and H<sup>+</sup>. In situ visualization\nof these parameters is crucial for lysosomal research, yet developing\nprobes that can simultaneously detect multiple ions remains challenging.\nHerein, we developed a lysosome-targeting ternary recognition ratiometric\nfluorescent probe based on tetrahedral DNA nanostructures (TDNs) to\nanalyze lysosome subpopulations by Cl<sup>–</sup>, Ca<sup>2+</sup>, and pH. The TDN probe is assembled from four single-stranded DNAs,\neach end-modified with responsive fluorophores (Pr-Cl for Cl<sup>–</sup>, Pr-Ca for Ca<sup>2+</sup>, and Pr-pH for pH) or a reference fluorophore\n(Cy5). The fluorophores are integrated at the vertices of the rigid\nTDN to minimize mutual interference, and their fixed stoichiometry\nestablishes a robust ternary recognition ratiometric fluorescence\nsensor for in situ resolution of lysosome subpopulations in living\ncells. Accordingly, a rise in lysosome subpopulations 2/6 characterized\nby low [Cl<sup>–</sup>], medium/high [Ca<sup>2+</sup>], and\nhigh pH was observed in the Niemann–Pick disease model cells\nbut seldom observed in the control group. Conversely, there was a\nmarked decline in the fraction of subpopulations 1/4/5 characterized\nby high [Cl<sup>–</sup>], medium to low [Ca<sup>2+</sup>],\nand pH. These changes were substantially reversed upon treatment.\nThe probe holds great promise for studying lysosome subpopulations\nand the diagnosis and treatment of related diseases.