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.