Carbon Monoxide: A Second Biomarker to Couple with Viscosity for the Construction of "Dual-Locked" Near-Infrared Fluorescent Probes for Accurately Diagnosing Non-Alcoholic Fatty Liver Disease.

Nonalcoholic fatty liver disease (NAFLD) can progress to cirrhosis and liver cancer if left untreated. Therefore, it is of great importance to develop useful tools for the noninvasive and accurate diagnosis of NAFLD. Increased microenvironmental viscosity was considered as a biomarker of NAFLD, but the occurrence of increased viscosity in other liver diseases highly reduces the diagnosis accuracy of NAFLD by a single detection of viscosity. Hence, it is very necessary to seek a second biomarker of NAFLD. It has been innovatively proposed that the overexpressed heme oxygenase-1 enzyme in NAFLD would produce abnormally high concentrations of CO in hepatocytes and that CO could serve as a potential biomarker. In this work, we screened nine lactam Changsha dyes (HCO-1-HCO-9) with delicate structures to obtain near-infrared (NIR), metal-free, and "dual-locked" fluorescent probes for the simultaneous detection of CO and viscosity. Changsha dyes with a 2-pyridinyl hydrazone substituent could sense CO, and the 5-position substituents on the 2-pyridinyl moiety had a great electron effect on the reaction rate. The double bond in these dyes served as the sensing group for viscosity. Probe HCO-9 was utilized for precise diagnosis of NAFLD by simultaneous detection of CO and viscosity. Upon reacting with CO in a high-viscosity microenvironment, strong fluorescence at 745 nm of probe HCO-9 was turned on with NIR excitation at 700 nm. Probe HCO-9 was proven to be an effective tool for imaging CO and viscosity. Due to the advantages of NIR absorption and low toxicity, probe HCO-9 was successfully applied to image NAFLD in a mouse model.