A near-infrared probe for the real-time detection of lysosomal pH in living cells under "wash free" conditions

Lysosomal pH is an important indicator for the physiological state of eukaryotic cells. The real-time detection of intracellular lysosomal pH is critical for understanding and studying many physiological and pathological processes of cells. Herein, we designed and synthesized a series of novel pH sensors, namely W1, W2 and W3. By comparing the spectroscopic properties of the three molecules and their ability to target lysosomes in living cells, a specific probe W1 was selected for the quantitative analysis of lysosomal pH changes in live cells. W1 shows a fast, sensitive and highly selective red fluorescence response to an acidic pH value. The pK(a) value of W1 is 5.84, and the fluorescence intensity ratios of I-743/I-680 under acidic conditions show a good linear relationship with the pH value. In addition, W1 shows a 100-fold difference in fluorescence from an extracellular environment to an intracellular environment, allowing it to be used as a "wash free" staining probe to visualize the pH change of lysosomes. W1 was further applied to detect the changes of lysosomal pH during apoptosis and mitophagy. Thus, W1 is expected to be a potentially useful tool for monitoring the changes of lysosomal pH in cell-related physiological or pathological states.