Microfluidic droplet platform for ultrahigh-throughput single-cell screening of biodiversity

Proc Natl Acad Sci U S A. 2017 Mar 7;114(10):2550-2555. doi: 10.1073/pnas.1621226114. Epub 2017 Feb 15.

Abstract

Ultrahigh-throughput screening (uHTS) techniques can identify unique functionality from millions of variants. To mimic the natural selection mechanisms that occur by compartmentalization in vivo, we developed a technique based on single-cell encapsulation in droplets of a monodisperse microfluidic double water-in-oil-in-water emulsion (MDE). Biocompatible MDE enables in-droplet cultivation of different living species. The combination of droplet-generating machinery with FACS followed by next-generation sequencing and liquid chromatography-mass spectrometry analysis of the secretomes of encapsulated organisms yielded detailed genotype/phenotype descriptions. This platform was probed with uHTS for biocatalysts anchored to yeast with enrichment close to the theoretically calculated limit and cell-to-cell interactions. MDE-FACS allowed the identification of human butyrylcholinesterase mutants that undergo self-reactivation after inhibition by the organophosphorus agent paraoxon. The versatility of the platform allowed the identification of bacteria, including slow-growing oral microbiota species that suppress the growth of a common pathogen, Staphylococcus aureus, and predicted which genera were associated with inhibitory activity.

Keywords: Staphylococcus aureus; butyrylcholinesterase; cell–cell interactions; microfluidic encapsulation; ultrahigh-throughput screening.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Antibiosis
  • Biodiversity
  • Butyrylcholinesterase / chemistry*
  • Cell Communication
  • Emulsions
  • Flow Cytometry
  • Genotype
  • High-Throughput Nucleotide Sequencing
  • High-Throughput Screening Assays / instrumentation*
  • Humans
  • Microfluidic Analytical Techniques / instrumentation
  • Microfluidic Analytical Techniques / methods*
  • Oils, Volatile / chemistry
  • Paraoxon / chemistry*
  • Phenotype
  • Single-Cell Analysis / instrumentation*
  • Staphylococcus aureus / drug effects
  • Staphylococcus aureus / growth & development
  • Water / chemistry

Substances

  • Emulsions
  • Oils, Volatile
  • Water
  • Butyrylcholinesterase
  • Paraoxon