Nitroxide‐Mediated Fluorescence Quenching: Synthesis and Applications for the Detection of ROS in Biological Systems

ABSTRACT Profluorescent nitroxides (PFNs) have established themselves as powerful tools for monitoring oxidative processes and subcellular redox dynamics by reactive oxygen species (ROS). Their characteristic “off–on” behavior arises from the efficient, proximity‐driven quenching of covalently bound fluorophores by paramagnetic nitroxide radicals, after which restoration of fluorescence can be achieved by radical reduction or chemical conversion. Here, an overview of the design principles, synthesis strategies, and biological applications of nitroxide–fluorophore conjugates is provided, highlighting their functional roles in the detection of reactive oxygen species, with a particular focus on organelle‐specific probes for mitochondria, cell membranes, lipids, and DNA. Probes lacking specific organelle‐targeting motifs are also included. The chemical parameters, including the selection of fluorophores and nitroxides, the synthesis and stability of linkers, and strategies for controlling pH‐dependent fluorescence, are discussed. PFNs that can selectively detect specific ROS and demonstrate their broader applicability in vivo for monitoring disease‐related oxidative stress, antibacterial mechanisms, and collagen degradation are also introduced. Therefore, PFNs will continue in their expansion to be useful analytical toolkits for real‐time redox imaging and offer promising next‐generation organelle‐specific probes and theranostic applications.