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By alternatively transforming microfluidic emulsions to double emulsions ( 12) or hydrogel beads equipped with polyelectrolyte shells ( 13), sorting in standard flow cytometers also becomes possible. The key technical module to make this possible is a microfluidic droplet sorter that has so far relied exclusively on fluorescent readouts (fluorescence-activated droplet sorting, FADS) ( 10, 11). Massive scale-down of assay volumes by compartmentalization of library members in water-in-oil emulsion droplets has recently led to the development of ultrahigh-throughput screening platforms that use small volumes (typically picoliters) and allow sorting of more than 10 6 variants per hour ( 7 – 9) (cf., schematic in Fig. Therefore, screening of large libraries of variants is essential and technologies to trawl sequence space efficiently are required. The AADS module makes the most widely used optical detection format amenable to screens of unprecedented size, paving the way for the implementation of chromogenic assays in droplet microfluidics workflows.ĭirected evolution has arguably become the most popular method to generate enzymes with improved and altered activities ( 1 – 5), but the success of this approach is dependent on exploring a maximum of combinatorial diversity ( 6). Fourteen hits showed increased activity (improved >4.5-fold in lysate k cat increased >2.7-fold), soluble protein expression levels (up 60%), and thermostability (T m, 12 ☌ higher). To demonstrate the utility of this module for protein engineering, two rounds of directed evolution were performed to improve the activity of phenylalanine dehydrogenase toward its native substrate. Sorting experiments showed that the AADS successfully enriched active variants up to 2,800-fold from an overwhelming majority of inactive ones at ∼100 Hz. The detection limit (10 μM in a coupled assay producing a formazan dye) enables accurate kinetic readouts sensitive enough to detect a minimum of 1,300 turnovers per enzyme molecule, expressed in a single cell, and released by lysis within a droplet. To validate this device, we implemented a miniaturized coupled assay for NAD +-dependent amino acid dehydrogenases. Using this module, microdroplets can be sorted based on absorbance readout at rates of up to 300 droplets per second (i.e., >1 million droplets per hour). Here we describe a highly efficient microfluidic absorbance-activated droplet sorter (AADS) that extends the range of assays amenable to this approach. Ultrahigh-throughput screening, in which members of enzyme libraries compartmentalized in water-in-oil emulsion droplets are assayed, has emerged as a powerful format for directed evolution and functional metagenomics but is currently limited to fluorescence readouts.