A 2-pyridone-based ratiometric fluorescent probe for specific detection and imaging of singlet oxygen in live cells

Singlet oxygen (¹O₂) plays a crucial role in cancer chemotherapy and ROS biology, driving the need for highly specific probes to monitor its dynamics in real time. Herein, we developed the ratiometric fluorescent probe NAP-t-PY, utilizing a 2-pyridone recognition unit. The probe's 1-methyl-3-benzyl-2-pyridone moiety reacts specifically with ¹O₂via [4 + 2] cycloaddition, forming the endoperoxide NAP-t-PY-EP. This reaction attenuates intramolecular charge transfer (ICT), inducing a significant blue shift from 562 nm to 446 nm, thereby enabling ratiometric detection with a large Stokes shift (138 nm). Demonstrates high sensitivity (LOD = 0.12 μM), excellent selectivity and rapid response (<15 min) towards ¹O₂. Its excellent photostability and low cytotoxicity facilitated biological application, successfully enabling real-time live-cell imaging of PDT-induced endogenous ¹O₂ in both A549 lung cancer cells and NCM460 normal colon cells.