Loop-mediated isothermal amplification (LAMP), a DNA amplification technique valued for its speed, specificity, sensitivity, and simplicity, is increasingly employed in disease diagnostics and genetically modified organism detection. At the heart of this method lies the Bst DNA polymerase, whose enzymatic properties—such as strand displacement activity, thermostability, and catalytic efficiency—are critical to the success of isothermal amplification, typically conducted at 60–65°C.

A research group led by Ma Fuqiang from the Suzhou Institute of Biomedical Engineering and Technology (SIBET) of the Chinese Academy of Sciences, has long been committed to enzyme molecular modification and ultra-high-through-put enzyme screening technologies, and has established a Fluorescence-Activated Droplet Sorting (FADS) platform. Based on droplet microfluidics technology, this platform can screen large mutant libraries at the single-cell level, with a daily screening throughput of over 10 million mutants.

Using this platform, the researchers designed an ultra-high-throughput screening protocol for Bst DNA polymerase base-d on fluorescence-activated single-cell microfluidics. 

For the first time, this method was applied to the directed evolution of Bst polymerase, resulting in the identification of mutant enzymes with significantly enhanced activity. When integrated into LAMP assays, these mutants reduced the reaction's peak-emergence time and demonstrated improved thermal stability, allowing for extended enzyme storage.

As a result, it achieved a leap from the wild-type enzyme to commercially valuable enzymes, fully demonstrating the great potential of FADS technology in the efficient evolution of molecular enzymes.

The FADS Ultra-High-Throughput Enzyme Screening Platform (Image by MA Fuqiang's group)

Schematic Diagram of FADS-Based Directed Evolution of Bst DNA Polymerase (Image by MA Fuqiang's group)