Abstract
Near-infrared-I/II fluorescent proteins (NIR-I/II FPs) are crucial for in vivo imaging, yet the current NIR-I/II FPs face challenges including scarcity, the requirement for chromophore maturation, and limited emission wavelengths (typically < 800 nm). Here, we utilize synthetic protein-seeking NIR-II dyes as chromophores, which covalently bind to tag proteins (e.g., human serum albumin, HSA) through a site-specific nucleophilic substitution reaction, thereby creating proof-of-concept biomimetic NIR-II FPs. This chemogenic protein-seeking strategy can be accomplished under gentle physiological conditions without catalysis. Proteomics analysis identifies specific binding site (Cys 477 on DIII). NIR-II FPs significantly enhance chromophore brightness and photostability, while improving biocompatibility, allowing for high-performance NIR-II lymphography and angiography. This strategy is universal and applicable in creating a wide range of spectrally separated NIR-I/II FPs for real-time visualization of multiple biological events. Overall, this straightforward biomimetic approach holds the potential to transform fluorescent protein-based bioimaging and enables in-situ albumin targeting to create NIR-I/II FPs for deep-tissue imaging in live organisms.