Superoxide (O₂^·−) is the primary reactive oxygen species in mammal cells. Detecting superoxide is crucial for understanding redox signaling but remains challenging. Herein, we introduce a class of activity-based sensing probes. The probes utilize 1,2,4,5-tetrazine as a superoxide-responsive trigger, which can be modularly tethered to various fluorophores to tune probe sensitivity and emission color. These probes afford ultra-specific and ultra-fluorogenic responses towards superoxide, and enable multiplexed imaging of various cellular superoxide levels in an organelle-resolved way. Notably, the probes reveal the aberrant superoxide generation in the pathology of myocardial ischemia/reperfusion injury, and facilitate the establishment of a high-content screening pipeline for mediators of superoxide homeostasis. One such identified mediator, coprostanone, is shown to effectively ameliorating oxidative stress-induced injury in mice with myocardial ischemia/reperfusion injury. Collectively, these results showcase the potential of 1,2,4,5-tetrazine-tethered probes as versatile tools to monitor superoxide in a range of pathophysiological settings.

超氧化物（O ₂ ^·−) 是哺乳动物细胞中的主要活性氧。检测超氧化物对于理解氧化还原信号至关重要，但仍然具有挑战性。在此，我们介绍了一类基于活动的传感探针。探针利用 1,2,4,5-四嗪作为超氧化物响应触发器，可以模块化地拴在各种荧光团上以调节探针灵敏度和发射颜色。这些探针对超氧化物提供超特异性和超荧光反应，并能够以细胞器分辨的方式对各种细胞超氧化物水平进行多重成像。值得注意的是，探针揭示了心肌缺血/再灌注损伤病理学中的异常超氧化物生成，并促进了超氧化物稳态介质的高含量筛选管道的建立。一种这样的确定的调解员，coprostanone，显示可有效改善小鼠心肌缺血/再灌注损伤的氧化应激损伤。总的来说，这些结果展示了 1,2,4,5-四嗪栓系探针作为在一系列病理生理环境中监测超氧化物的多功能工具的潜力。