Development of an iridium complex fluorescent probe for FGF21 protein labeling and tracking

Fluorescent or luminescent labeling of biomolecules, as biosensors, with high sensitive and spatiotemporal resolution enable it an outstanding imaging technique for detecting and tracking biomolecular dynamics in many areas of life sciences and biomedical research. Ir(III) complex IrCN with solvent ligands could selectively recognize His via covalent attachment to the His imidazole group, and serve as a reaction-based "turn-on" fluorescent probe for the detection of His and His containing proteins. Fibroblast growth factor 21 (FGF21) was an essential glucose and lipid metabolic regulator and a promising therapeutic target for metabolic disorder syndromes. In this study, a non-emissive cyclometalated Ir(III) solvent complex IrCN was synthesized for FGF21 protein labeling. Binding test showed that the optimal binding ratio of IrCN and FGF21 protein was 1:100 (W/W). The binding between IrCN and FGF21 protein was very rapid, and the reaction could be completed in 10 min. IrCN probe no longer bound to other proteins after it specifically bound to the FGF21 protein. Biocompatibility studies shown that IrCN exhibited low cytotoxicity and tissue toxicity when the concentration was not higher than 50 μg/mL. Whereas, high concentration of IrCN caused organ-specific toxicity, with notable effects observed in both the spleen and skeletal muscle. Cell imaging experiments showed that revealed that unbound IrCN exhibits significant potential as a versatile cytoplasmic labeling agent, while its protein-conjugated form demonstrates effective protein tracing capabilities in cellular systems. Functional validation experiments by quantitative analysis of FGF21-mediated downstream pathway markers demonstrated that IrCN labeling preserves the native biological activity of FGF21 protein. This study demonstrated that IrCN served as a highly sensitive and stable probe for cell imaging and protein fluorescent labeling applications, which established a solid foundation for further exploration of its potential applications in diverse areas of biomolecular research, particularly in protein tracking and live-cell imaging studies.