A novel mitochondrial-targeted fluorescent probe for ratiometric imaging of nitric oxide in cells and zebrafish

Nitric oxide (NO) is a key signaling molecule that regulates energy metabolism, apoptosis, and antioxidant balance within mitochondria. It is closely associated with the development of cardiovascular diseases, neurodegenerative diseases, and cancer. Therefore, developing fluorescent probes capable of accurately detecting NO levels in mitochondria is essential for understanding disease mechanisms and clinical diagnostics. In this study, we developed a novel fluorescent probe based on the isophorone fluorophore. This probe achieves high sensitivity and specific ratiometric detection of NO in mitochondria by regulating the intramolecular charge transfer (ICT) effect. The probe emits red fluorescence before reacting with NO, and the addition of NO triggers an amine- NO addition reaction that inhibits the ICT effect, resulting in a color change to yellow-green fluorescence. This ratiometric fluorescence response provides a new method for quantitatively detecting NO. Additionally, the probe has a significant Stokes shift and good ratiometric wavelength separation, enhancing detection accuracy. It localizes explicitly to mitochondria, directly reflecting changes in mitochondrial NO concentration. Experiments in HeLa cells and zebrafish models have demonstrated the potential application of the probe in diagnosing and studying NO-related diseases. This provides new strategies and tools for researching the biological functions of NO and the early diagnosis of related diseases.