A single-molecule platform for photo-triggered peroxynitrite release with built-in fluorescence calibration

Peroxynitrite (ONOO^-) is central to both physiological signaling and diverse pathological processes. Its dual nature underscores the need for precise tools to investigate its spatiotemporal dynamics and biological functions. However, the controlled generation and real-time tracking of ONOO^- remain challenging due to its short half-life and high reactivity. Current small-molecule ONOO^- donors often suffer from limitations such as slow release, low efficiency, and off-target effects. To overcome these challenges, here we report a new class of photo-triggered ONOO^- donors (O-PND and Si-PND) based on a single rhodamine-derived scaffold, enabling precise ONOO^- release with built-in fluorescence calibration. These molecular tools facilitate efficient ONOO^- generation under blue light irradiation, as confirmed in PBS and live cells, and exhibit excellent cell membrane permeability. Upon intracellular activation, O-PND and Si-PND induced a marked increase in oxidative stress. However, further studies reveal that the rapid transient ONOO^- burst in RAW264.7 cells was insufficient to significantly modulate macrophage polarization. Collectively, these robust self-reporting ONOO^- donors provide a powerful single-molecule platform for investigating ONOO^--mediated biological mechanisms with spatiotemporal precision.