A lock-to-key, systematic strategy for room-temperature phosphorescence in cellular biosensing and tracking

Considering the self-fluorescence of biospecies, phosphorescence—as a time-resolved characteristic—has gained immense attention in cellular biosensing, including enzyme detection, cellular viscosity monitoring, and biochemical process tracking. These dynamic processes are precisely recorded in real time based on differences in wavelength and lifetime, observable with the naked eye. While the application of phosphorescence in cellular biosensing has enabled major advances, molecular design strategies and practical implementations remain less extensively explored. Here, we examine the modular approach to systematic strategies for room-temperature phosphorescence (RTP) probes. Various methods involving triplet-state stability, lifetime modulation, and rigid environment destruction have been employed to construct “turn-on” and “turn-off” RTP probes. We further discuss the applications of RTP probes in monitoring cellular ions, enzymes, microRNA, organelles, and bioimaging. Lastly, we explore potential applications, unresolved challenges, and future perspectives for RTP probes in depth.