Constructing droplet interface bilayers from the contact of aqueous droplets in oil

Nat Protoc. 2013 Jun;8(6):1048-57. doi: 10.1038/nprot.2013.061. Epub 2013 May 2.


We describe a protocol for forming an artificial lipid bilayer by contacting nanoliter aqueous droplets in an oil solution in the presence of phospholipids. A lipid monolayer forms at each oil-water interface, and when two such monolayers touch, a bilayer is created. Droplet interface bilayers (DIBs) are a simple way to generate stable bilayers suitable for single-channel electrophysiology and optical imaging from a wide variety of preparations, ranging from purified proteins to reconstituted eukaryotic cell membrane fragments. Examples include purified proteins from the α-hemolysin pore from Staphylococcus aureus, the anthrax toxin pore and the 1.2-MDa mouse mechanosensitive channel MmPiezo1. Ion channels and ionotropic receptors can also be reconstituted from membrane fragments without further purification. We describe two approaches for forming DIBs. In one approach, a lipid bilayer is created between two aqueous droplets submerged in oil. In the other approach, a membrane is formed between an aqueous droplet and an agarose hydrogel, which allows imaging in addition to electrical recordings. The protocol takes <30 min, including droplet generation, monolayer assembly and bilayer formation. In addition to the main protocol, we also describe the preparation of Ag/AgCl electrodes and sample preparation.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Alkanes
  • Animals
  • Antigens, Bacterial / chemistry
  • Bacterial Toxins / chemistry
  • Hemolysin Proteins / chemistry
  • Ion Channels / chemistry
  • Ion Channels / metabolism
  • Lipid Bilayers / chemical synthesis*
  • Lipid Bilayers / chemistry
  • Membranes, Artificial*
  • Mice
  • Nanostructures / chemistry*
  • Oils / chemistry*
  • Silver
  • Silver Compounds
  • Water / chemistry*


  • Alkanes
  • Antigens, Bacterial
  • Bacterial Toxins
  • Hemolysin Proteins
  • Ion Channels
  • Lipid Bilayers
  • Membranes, Artificial
  • Oils
  • Piezo1 protein, mouse
  • Silver Compounds
  • anthrax toxin
  • staphylococcal alpha-toxin
  • Water
  • Silver
  • n-hexadecane
  • heptadecane
  • silver chloride