A Hydrogen Sulfide‐Activated NIR‐II Fluorescence/NIR‐I Photoacoustic Dual‐Ratiometric Nanoprobe With Unique Recognition Reaction for Precise Visual Diagnosis of Hepatitis Disease

Abstract Hydrogen sulfide (H 2 S) is a vital gaseous signaling molecule that plays a central role in various physiological and pathological processes. Given the complementary advantages of fluorescence (FL) and photoacoustic (PA) imaging, there is a growing demand for dual‐ratiometric probes that enable precise in vivo monitoring of H 2 S levels. In this study, the use of 2‐mercapto‐1,3,4‐thiadiazole (MTD) as a novel recognition group of H 2 S is presented for the first time, following conjugation with cyanine dyes to obtain a new PA probe Cy‐MTD. To achieve dual‐ratiometric imaging, Cy‐MTD is incorporated into down‐conversion nanoparticle (DCNP), resulting in the creation of a pioneering NIR‐II FL/NIR‐I PA dual‐ratiometric nanoprobe DCNP@Cy‐MTD. Cy‐MTD undergoes the blueshift in absorption from 840 to 670 nm after reaction with H 2 S, enabling NIR‐I ratiometric PA imaging of H 2 S by measuring the ratio of PA signal at 670 and 840 nm (PA 670 /PA 840 ). In addition, due to the strong absorption of Cy‐MTD ≈840 nm and the overlapping between the absorption spectrum of Cy‐MTD and 808 nm excitation band of DCNP, the 808 nm‐excited FL emission (F 1550 nm,808Ex ) of DCNP in DCNP@Cy‐MTD nanoprobe is quenched through the competitive absorption, while it is restored upon the interaction with H 2 S because of the blueshift in absorption of Cy‐MTD. Using the stable FL emission of DCNP under 980 nm excitation (F 1550 nm,980Ex ) as the reference signal, NIR‐II ratiometric FL imaging (F 1550 nm,808Ex /F 1550 nm,980Ex ) of H 2 S is achieved. The dual‐ratiometric response features of the DCNP@Cy‐MTD nanoprobe offer a significant advancement over traditional single‐signal or single‐modality imaging techniques. By providing enhanced accuracy and reliability, this probe allows for the diagnosis of hepatitis by monitoring the H 2 S, surpassing the capabilities of conventional histopathological methods, which provides a new way for more effective diagnostic strategies for liver diseases.