Confocal Absorbance‐Activated Droplet Sorting (cAADS) for Enzyme Engineering

Abstract Directed evolution relies on iterative cycles of variant generation, screening, and selection to identify enzyme variants with improved activities. Droplet‐based microfluidics accelerates this process by enabling rapid screening of enzyme variants in water‐in‐oil emulsions acting as picoliter‐scale microcompartments. In fluorescence‐activated droplet sorting (FADS), single E.coli cells are screened using a fluorogenic substrate at high throughput (≈2 kHz). However, fluorogenic assays for enzymatic systems are limited, while absorbance‐based detection assays represent a larger spectrum. At the micron scale, light absorption is weak, and scattering induced by droplet interfacial curvature further decreases detection sensitivity. Measurements are therefore performed at a cost of increasing droplet sizes or acquisition times, which limits throughput to <1 kHz. Here, this challenge is addressed with a confocal Absorbance‐Activated Droplet Sorting (cAADS) system. The platform achieves sensitive absorbance measurements at ultrahigh throughput (5.4 kHz) from droplets as small as 10 pL, and sorting of 50 pL droplets at frequencies up to 2.6 kHz. The cAADS methodology is demonstrated by enrichment of active Bilirubin Oxidase (BOD) variants, with a sorting efficiency of 99%. Its versatility and potential for absorbance‐based microfluidic screening in enzyme engineering are also demonstrated using a different enzyme: Glucose Oxidase.