A dual-ratiometric strategy to design the NIR-IIb reference-based activatable nanoprobe for accurate nitric oxide detection

Fluorescence imaging offers a real-time, convenient method for detection nitric oxide (NO) in vivo, especially using the near-infrared IIb (NIR-IIb, 1500-1700 nm) region. However, traditional single-ratiometric fluorescent probes for NO detection suffer from signal distortion due to inherent limitations such as signal crosstalk and component separation. Herein, we proposed a dual-ratiometric strategy to ensure detection accuracy. The detection result was considered to be reliable upon the auxiliary ratio and practical ratio exhibiting a synchronous change. Based on this strategy, we fabricated a NO-responsive probe, comprising a conjugated polymer P-NH2 and a rare-earth doped nanocrystal NaYF4:Yb,Er,Ce@NaYF4:Yb,Nd@NaYF4. When interaction with NO, the probe generated two ratiometric signals: FNIR-I/FNIR-IIb and FNIR-II'/FNIR-IIb (NIR-I, 700-900 nm; NIR-II', 1000-1400 nm), where the latter acted as the practical ratio for its better imaging clarity. With an acetaminophen-induced liver injury model, the probe demonstrated the potential for precise ratiometric imaging of NO distribution in vivo. Particularly, ratio imaging revealed the intricate vascular structure of the abdomen, demonstrating the significant advantages of the NIR-IIb region. Our study offers an effective approach to reduce detection errors in the bioimaging applications and provides a powerful tool for basic biomedical research.