Single-wavelength-excited fluorogenic nanoprobe for accurate real-time ratiometric analysis of broad pH fluctuations in mitophagy

Mitophagy has a critical role in maintaining cellular homeostasis through acidic lysosomes engulfing excess or impaired mitochondria, thereby pH fluctuation is one of the most significant indicators for tracking mitophagy. Then such precise pH tracking demands the fluorogenic probe that has tailored contemporaneous features, including mitochondrial-specificity, excellent biocompatibility, wide pH-sensitive range of 8.0-4.0, and especially quantitative ability. However, available molecular probes cannot simultaneously meet all the requirements since it is extremely difficult to integrate multiple functionalities into a single molecule. To fully address this issue, we herein integrate two fluorogenic pH sensitive units, a mitochondria-specific block, cell-penetrating facilitator, and biocompatible segments into an elegant silica nano scaffold, which greatly ensures the applicability for real-time tracking of pH fluctuations in mitophagy. Most significantly, at a single wavelength excitation, the integrated pH-sensitive units have spectra-distinguishable fluorescence towards alkaline and acidic pH in a broad range that covers mitochondrial and lysosomal pH, thus enabling a ratiometric analysis of pH variations during the whole mitophagy. This work also provides constructive insights into the fabrication of advanced fluorescent nanoprobes for diverse biomedical applications.