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Related Experiment Video

Updated: Mar 12, 2026

Multi-enzyme Screening Using a High-throughput Genetic Enzyme Screening System
08:10

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Ultrahigh-throughput-directed enzyme evolution by absorbance-activated droplet sorting (AADS).

Fabrice Gielen1, Raphaelle Hours1, Stephane Emond1

  • 1Department of Biochemistry, University of Cambridge, Cambridge, CB2 1GA, United Kingdom.

Proceedings of the National Academy of Sciences of the United States of America
|November 9, 2016
PubMed
Summary

A new microfluidic absorbance-activated droplet sorter (AADS) enables high-throughput screening using color-based assays, expanding directed evolution capabilities. This technology allows for rapid sorting of enzyme variants, significantly advancing protein engineering and functional metagenomics.

Keywords:
directed evolutionemulsion dropletsmicrofluidicsprotein engineeringultrahigh-throughput

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Area of Science:

  • Biotechnology
  • Biochemistry
  • Microfluidics

Background:

  • Ultrahigh-throughput screening using droplet microfluidics is powerful for directed evolution and functional metagenomics.
  • Current methods are limited to fluorescence readouts, restricting assay compatibility.

Purpose of the Study:

  • To develop and validate a microfluidic absorbance-activated droplet sorter (AADS).
  • To extend droplet microfluidics screening to absorbance-based assays.
  • To enable high-throughput screening for enzyme engineering and functional metagenomics.

Main Methods:

  • Development of a microfluidic absorbance-activated droplet sorter (AADS).
  • Implementation of a miniaturized coupled assay for NAD+-dependent amino acid dehydrogenases.
  • Application of AADS for directed evolution of phenylalanine dehydrogenase.

Main Results:

  • The AADS sorts microdroplets based on absorbance at rates up to 300 droplets/second (>1 million/hour).
  • Detection limit of 10 μM with sensitive kinetic readouts (≥1,300 turnovers/molecule).
  • Enrichment of active enzyme variants up to 2,800-fold.
  • Directed evolution yielded phenylalanine dehydrogenase variants with >4.5-fold improved activity and enhanced stability.

Conclusions:

  • The AADS significantly expands the applicability of droplet microfluidics screening to chromogenic assays.
  • This technology facilitates high-throughput screening for protein engineering and functional metagenomics.
  • The AADS enables unprecedented screening sizes for enzyme discovery and optimization.