High-Fidelity NADH Imaging with a Mitochondria-Targeted Probe: Specific Interrogation of Oxidative Stress in...

Abnormal accumulation of NADH in pneumonia and tumor microenvironments is a key pathological event driven by oxidative stress. It is very important to make a fluorescent probe that can accurately detect NADH and has high sensitivity and selectivity. Our research introduces a novel mitochondria-targeted probe, Mito-NQ, through a stepwise design strategy: screening N-methylquinoxaline salt as the optimal recognition group, extending the emission wavelength, and anchoring mitochondria with triphenylphosphine (TPP) for precise mitochondrial localization. Mechanistic studies revealed that the N-methylquinoxaline unit conferred exceptional selectivity by enabling electron transfer specifically with NADH while excluding interference from structural analogs (NAD⁺/NADPH) and biothiols. The Mito-NQ probe can accurately detected endogenous and exogenous NADH. In LPS-induced inflammatory cells, NADH levels increased 2.0-fold and were restored to approximately 1.32-2.63-fold after treatment with NAC, GSH, and dexamethasone (Dex). These dynamic changes are related to the situation that NADH accumulation directly reflects the intensity of oxidative stress and also to the situation that antioxidant/anti-inflammatory intervention can restore redox homeostasis by rebalancing mitochondrial NADH metabolism. Spray imaging of dissected lung tissues from an LPS-induced pneumonia mouse model revealed 4.0-fold NADH enrichment in alveolar regions (reduced to 2.8-5.7-fold after NAC/Dex treatment). Moreover, in A549 xenograft tumors, the tumor-to-adjacent tissue signal ratio reached 3.0, and three-dimensional imaging further revealed a spatial NADH gradient in the tumor core region. This probe provides a reliable visualization tool for studying oxidative stress mechanisms and evaluating drug efficacy for lung-related diseases.