Ultra-sensitive quantification and fluorescence-activated droplet sorting of UDG activity enabled by a novel fluorogenic strategy.

Uracil-DNA glycosylase (UDG) is a key enzyme in molecular diagnostics. However, the application of traditional methods is often limited by the operational complexity and safety concerns. Therefore, we developed an ultra-sensitive, convenient, safe detection method by engineering a novel UDG-splitting fluorogenic probe that enables absolute quantification within 30 min, without the need for radioactive materials, auxiliary enzymes, or additional probes. This method also features a wide dynamic range (2.5-20 mU/μL), excellent limits of detection, and high precision (RSD < 10%). Furthermore, the reliability of the method was cross-validated using multiple commercially available UDG enzymes. We further demonstrated the application of this system in fluorescence-activated droplet sorting (FADS), achieving high-throughput screening of UDG mutant libraries with significantly improved efficiency over conventional approaches. The thermal stability of the resulting mutant was up to 15 times higher than that of the wild type. Therefore, this work not only establishes a robust platform for ultrasensitive UDG activity analysis but also provides a powerful ultra-high-throughput screening tool to advance the molecular evolution of UDG.