Ratiometrically pH-Insensitive Upconversion Nanoprobe: Toward Simultaneously Quantifying Organellar Calcium and...

Calcium and chloride levels are closely related to lysosome dysfunction. However, the simultaneous measurement of calcium (Ca²⁺) and chloride (Cl^-) in acidic subcellular organelles, which is conducive to a deep understanding of lysosome-related biological events, remains a challenge. In this study, we developed a pH-insensitive, ratiometric NIR nanoprobe for the simultaneous detection of Ca²⁺ and Cl^- in acidic lysosomes and determined the roles of the two ions in lysosome function. The upconversion nanoprobe with blue, green, and red emissions was modified with a Ca²⁺-sensitive dye (Rhod-5N) and Cl^--responsive fluorophore (10,10'-bis[3-carboxypropyl]-9,9'-biacridinium dinitrate, BAC). As a result of a dual-luminescence resonance energy transfer between upconversion nanoparticles (UCNPs) and Rhod-5N/BAC, the blue and green upconversion luminescence (UCL) of UCNPs were quenched and the red UCL was used as the reference signal. The ratiometric upconversion nanoprobe possesses a specific ability for the concurrent recognition of Ca²⁺ and Cl^- ions independent of the influence of the environmental pH. To locate the probe in the lysosome, dextran was further modified with upconversion nanoparticles. Then, the nanoprobe with a high spatial resolution was constructed for the simultaneous monitoring of Ca²⁺ and Cl^- in acidic lysosomes. Moreover, it was found that the reduction of lysosomal Cl^- affects the release of lysosomal Ca²⁺, which further blocks the activities of specific lysosomal enzymes. The ratiometric NIR nanoprobe has great potential for decoding and evaluating lysosomal diseases.