Abstract
The pH value and viscosity in the lysosomal microenvironment play a crucial role in maintaining cellular homeostasis, and their abnormalities are closely related to various diseases. However, most of the existing fluorescent probes only respond to a single parameter, such as pH or viscosity, making it difficult to achieve highly specific imaging. Therefore, in this study, a novel "dual-lock" activated fluorescent probe BT-CZ was designed and synthesized based on the "AND gate" logic. This probe only turns on green fluorescence when both acidic conditions and high viscosity are present, effectively avoiding false positive signals caused by single environmental fluctuations. In vitro experiments demonstrated that BT-CZ exhibits sensitivity to viscosity and high selectivity under acidic conditions. Cell imaging and orthogonal pharmacological experiments confirmed that it can accurately target lysosomes and exhibit "dual-lock" activation behavior. Using the EBSS starvation-induced autophagy model, BT-CZ successfully monitored the coordinated alterations in lysosomal acidity and viscosity and could distinguish between functional autophagy and autophagic flux blockage. Notably, BT-CZ demonstrated effectiveness in the real-time in-situ diagnosis of tumor tissues with almost no background signal in normal organs.