Localized Detection of Superoxide Generation and Monitoring of Photosensitizer Performance in Hypoxic Tumors by an In Situ Precipitating Fluorescent Probe

Type I photodynamic therapy (PDT), generating superoxide anion radicals (O₂^•-), presents a potent strategy against tumor resistance by virtue of its low oxygen dependence, ensuring efficacy in hypoxic tumors where traditional Type II PDT is limited. However, accurate O₂^•- detection with high spatiotemporal and subcellular resolution remains a critical unmet need. Herein, we report the rational design and synthesis of HPQ-CF₃, a novel fluorescent probe. Upon selective reaction with O₂^•-, HPQ-CF₃'s trifluoromethanesulfonate group departs, unmasking a hydroxyl group to generate the highly fluorescent HPQ-OH, a fluorophore incorporating the HPQ moiety. Crucially, the inherent low aqueous solubility of HPQ-OH leads to its in situ precipitation, enabling precise spatial localization of O₂^•- generation sites. HPQ-CF₃ exhibits high specificity, a rapid response (<300 s), a remarkable ∼200-fold fluorescence enhancement, a low limit of detection (LOD, 0.13 μM), and excellent linearity. HPQ-CF₃ demonstrated significantly superior performance over the commercial probe DHR123 for intracellular O₂^•- detection; its unique precipitation-based mechanism inherently minimizes background fluorescence while anchoring the signal at its origin, affording a substantially enhanced signal-to-noise ratio and markedly improved localization accuracy. Furthermore, HPQ-CF₃ successfully monitored O₂^•- production in complex biological settings, including solid tumors. HPQ-CF₃ is anticipated to be an invaluable tool for investigating O₂^•--related pathophysiology and advancing Type I photosensitizer development.