ArticleAnalytical chemistry2014
One in a million: flow cytometric sorting of single cell-lysate assays in monodisperse picolitre double emulsion droplets for directed evolution.
Article in Analytical chemistry, 2014. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 87 papers.
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Who cites it
87 citing papers in PubMed.
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- Real-time optical spectroscopy forNanoscale advances · 2026Article
- Design and Synthesis of Fluorosurfactants for Microfluidic Droplet-Based Bioapplications.Polymer science & technology (Washington, D.C.) · 2026Review
- Microfluidic-Assisted Evolution of a Robust NADChemSusChem · 2025Article
- A Microfluidics-Based Ultrahigh-Throughput Screening Unveils Diverse Ketoreductases Relevant to Pharmaceutical Synthesis.Analytical chemistry · 2025Article
- Continuous FACS Sorting of Double Emulsion Picoreactors with a 3D-Printed Vertical Mixer.Analytical chemistry · 2025Article
- Artificial Intelligence-Empowered Automated Double Emulsion Droplet Library Generation.Small (Weinheim an der Bergstrasse, Germany) · 2025Article
- Fluorogenic, Subsingle-Turnover Monitoring of Enzymatic Reactions Involving NAD(P)H Provides a Generalized Platform for Directed Ultrahigh-Throughput Evolution of Biocatalysts in Microdroplets.Journal of the American Chemical Society · 2025Article
- Continuous FACS sorting of double emulsion picoreactors with a 3D printed vertical mixer.bioRxiv : the preprint server for biology · 2025Article
- From specialization to broad adoption: Key trends in droplet microfluidic innovations enhancing accessibility to non-experts.Biomicrofluidics · 2025Article
- Cell-Free Gene Expression: Methods and Applications.Chemical reviews · 2025Review
- A Comprehensive Review on Bioactive Molecules and Advanced Microorganism Management Technologies.Current issues in molecular biology · 2024Review
- Passive Droplet Microfluidic Platform for High-Throughput Screening of Microbial Proteolytic Activity.Analytical chemistry · 2024Article
- Robust Double Emulsions for Multicolor Fluorescence-Activated Cell Sorting.Analytical chemistry · 2024Article
- Controlling phase separations and reactions in trapped microfluidic droplets.Scientific reports · 2024Article
- Droplet Microfluidics for High-Throughput Screening and Directed Evolution of Biomolecules.Micromachines · 2024Review
- Carbohydrate-active enzyme (CAZyme) discovery and engineeringRSC chemical biology · 2024Review
- Ultrahigh Throughput Evolution of Tryptophan Synthase in Droplets via an Aptamer Sensor.ACS catalysis · 2024Article
- Tailored Fluorosurfactants through Controlled/Living Radical Polymerization for Highly Stable Microfluidic Droplet Generation.Angewandte Chemie (International ed. in English) · 2024Article
- Versatile Product Detection via Coupled Assays for Ultrahigh-Throughput Screening of Carbohydrate-Active Enzymes in Microfluidic Droplets.ACS catalysis · 2023Article
27 more citing papers are in PubMed but not listed here.
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Authors and funding
6 authors.
Funding
Abstract
Directed evolution relies on iterative cycles of randomization and selection. The outcome of an artificial evolution experiment is crucially dependent on (i) the numbers of variants that can be screened and (ii) the quality of the assessment of each clone that forms the basis for selection. Compartmentalization of screening assays in water-in-oil emulsion droplets provides an opportunity to screen vast numbers of individual assays with good signal quality. Microfluidic systems have been developed to make and sort droplets, but the operator skill required precludes their ready implementation in nonspecialist settings. We now establish a protocol for the creation of monodisperse double-emulsion droplets in two steps in microfluidic devices with different surface characteristics (first hydrophobic, then hydrophilic). The resulting double-emulsion droplets are suitable for quantitative analysis and sorting in a commercial flow cytometer. The power of this approach is demonstrated in a series of enrichment experiments, culminating in the successful recovery of catalytically active clones from a sea of 1 000 000-fold as many low-activity variants. The modular workflow allows integration of additional steps: the encapsulated lysate assay reactions can be stopped by heat inactivation (enabling ready control of selection stringency), the droplet size can be contracted (to concentrate its contents), and storage (at -80 °C) is possible for discontinuous workflows. The control that can be thus exerted on screening conditions will facilitate exploitation of the potential of protein libraries compartmentalized in droplets in a straightforward protocol that can be readily implemented and used by protein engineers.
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