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

Lysosomes are multifunctional organelles vital for cellular homeostasis with distinct subpopulations characterized by varying levels of Cl, Ca, 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, Ca, 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 Ca, 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 [Ca], 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 [Ca], 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.