Tetrahedral DNA-Based Ternary Recognition Ratiometric Fluorescent Probes for Real-Time In Situ Resolving Lysosome Subpopulations in Living Cells via Cl–, Ca2+, and pH

Lysosomes are multifunctional organelles vital for cellular homeostasis with distinct subpopulations characterized by varying levels of Cl^-, Ca²⁺, and H⁺. In situ visualization of these parameters is crucial for lysosomal research, yet developing probes that can simultaneously detect multiple ions remains challenging. Herein, we developed a lysosome-targeting ternary recognition ratiometric fluorescent probe based on tetrahedral DNA nanostructures (TDNs) to analyze lysosome subpopulations by Cl^-, Ca²⁺, and pH. The TDN probe is assembled from four single-stranded DNAs, each end-modified with responsive fluorophores (Pr-Cl for Cl^-, Pr-Ca for Ca²⁺, and Pr-pH for pH) or a reference fluorophore (Cy5). The fluorophores are integrated at the vertices of the rigid TDN to minimize mutual interference, and their fixed stoichiometry establishes a robust ternary recognition ratiometric fluorescence sensor for in situ resolution of lysosome subpopulations in living cells. Accordingly, a rise in lysosome subpopulations 2/6 characterized by low [Cl^-], medium/high [Ca²⁺], and high pH was observed in the Niemann-Pick disease model cells but seldom observed in the control group. Conversely, there was a marked decline in the fraction of subpopulations 1/4/5 characterized by high [Cl^-], medium to low [Ca²⁺], and pH. These changes were substantially reversed upon treatment. The probe holds great promise for studying lysosome subpopulations and the diagnosis and treatment of related diseases.