A spiropyran with low pK_a for tracking DNA G-quadruplexes and revealing the dissipation of ΔΨm with senescence using an in-situ switching strategy

The in-situ fluorescence triggering of bioprobes only using endogenous bioforces is an ideal non-destructive real-time detection method, which is of particular interest to improve the accuracy of clinical diagnosis and treatment. We have recently reported a strategy of spiropyran in-situ switching triggered by endogenous biological forces in vivo to develop optical probes for this purpose. However, such probes, as with all spiropyrans, are sensitive to lysosomal acidity. We here present a spiropyran-based fluorescent probe TANG with low pK_a, which can recognize intranuclear DNA G4s in situ without the aid of exogenous light or chemicals and is as stable to lysosomal acidity due to a decreased pK_a value (4.3). Interestingly, despite the stability to lysosomal pH environment, the TANG spiropyran can be opened in situ by the negative membrane potential of extranuclear mitochondria (ΔΨm), causing a ratiometric change in fluorescence signals and providing the in-situ and real-time tracking of ΔΨm. Of note, ratiometric imaging using TANG indicates that ΔΨm decreases gradually with cellular senescence, which is to the best of our knowledge the first visualization of such mitochondrion-related aging processes using a ratiometric imaging approach.