A near-infrared ratiometric fluorescence probe for mitochondrial H₂O₂ imaging in biosystems and water samples

Hydrogen peroxide (H₂O₂) is a crucial signaling molecule involved in various physiological and pathological processes. Mitochondria are the primary site of endogenous H₂O₂ generation, and its dysregulation is closely linked to diseases such as cancer and neurodegenerative disorders. Therefore, developing robust tools for real-time, accurate, and in-situ H₂O₂ detection is of great significance. Herein, we report a novel near-infrared (NIR) ratiometric fluorescent probe, NIR1, designed for the selective imaging of mitochondrial H₂O₂. The probe utilizes a hemicyanine scaffold and a boronate ester as the specific H₂O₂ recognition group. Upon reaction with H₂O₂, NIR1 exhibits a distinct ratiometric fluorescence signal change (F_{715 nm}/F_{675 nm}) in the NIR region, accompanied by a visible color shift from blue to light green. This probe demonstrates high sensitivity (detection limit: 15.8 nM), excellent selectivity, and a fast response time (40 min). Crucially, NIR1 shows outstanding mitochondrial targeting capability (Pearson coefficient: 0.9792) and good biocompatibility. It has been successfully applied to monitor both exogenous and endogenous H₂O₂ fluctuations in living cells via confocal microscopy. Furthermore, NIR1 is effective for the quantitative determination of H₂O₂ in various real water samples with satisfactory recoveries. This work provides a promising NIR ratiometric tool for investigating H₂O₂-related physiological and pathological events at the subcellular level.