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

Hydrogen sulfide (H₂S) 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 H₂S levels. In this study, the use of 2-mercapto-1,3,4-thiadiazole (MTD) as a novel recognition group of H₂S 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 H₂S, enabling NIR-I ratiometric PA imaging of H₂S by measuring the ratio of PA signal at 670 and 840 nm (PA₆₇₀/PA₈₄₀). 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 (F₁₅₅₀ _nm,808Ex) of DCNP in DCNP@Cy-MTD nanoprobe is quenched through the competitive absorption, while it is restored upon the interaction with H₂S because of the blueshift in absorption of Cy-MTD. Using the stable FL emission of DCNP under 980 nm excitation (F₁₅₅₀ _nm,980Ex) as the reference signal, NIR-II ratiometric FL imaging (F₁₅₅₀ _nm,808Ex/F₁₅₅₀ _nm,980Ex) of H₂S 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 H₂S, surpassing the capabilities of conventional histopathological methods, which provides a new way for more effective diagnostic strategies for liver diseases.