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

Nature Protocols volume 8, pages 1048–1057 (2013)Cite this article

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Abstract

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.

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Figure 1: Images illustrating the formation of droplet-droplet bilayers and droplet-hydrogel bilayers.
Figure 2: Bright-field images of droplet bilayers formed under various lipid conditions.
Figure 3: Device construction for droplet-hydrogel DIBs.
Figure 4: Optical and electrical measurement of droplets.
Figure 5: Stability and lifetime data from 300-nl droplet hydrogel bilayers of aqueous buffer (Tris-HCl pH 8.0, 0.5 M NaCl) formed in a range of concentrations of DPhPC lipid (32 droplets at each lipid concentration).

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Acknowledgements

We thank H. Bayley for his comments. This work was funded by the Biotechnology and Biological Sciences Research Council (BBSRC). M.H. is funded by the US National Science Foundation (NSF; CAREER award no. 1253565). M.I.W. is funded by the European Research Council (ERC). B.C. is an Engineering and Physical Sciences Research Council (EPSRC) Life Sciences Interface (LSI) Postdoctoral Fellow.

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Author notes

  1. Sebastian Leptihn & James R Thompson

    Present address: Present addresses: Institute for Microbiology and Molecular Biology, University of Hohenheim, Stuttgart, Germany (S.L.); Mork Family Department of Chemical Engineering and Materials and Molecular Biology, University of Southern California, Los Angeles, California, USA (J.R.T.).,

Authors and Affiliations

  1. Department of Chemistry, Oxford University, Oxford, UK

    Sebastian Leptihn, Oliver K Castell, Brid Cronin, Linda C M Gross, David P Marshall, James R Thompson & Mark I Wallace

  2. Department of Chemistry, University of Massachusetts, Amherst, Massachusetts, USA

    En-Hsin Lee & Matthew Holden

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Contributions

All authors contributed equally to this work. M.H. and E.-H.L. conducted the experiments on droplet-droplet DIBs. L.C.M.G. and S.L. conducted the experiments on droplet-hydrogel DIBs. B.C., S.L. and M.I.W. prepared the figures. J.R.T. helped develop the protocol. S.L., D.P.M., O.K.C., M.H. and M.I.W. wrote the main paper. All authors discussed the results and commented on the manuscript at all stages.

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Correspondence to Mark I Wallace.

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Leptihn, S., Castell, O., Cronin, B. et al. Constructing droplet interface bilayers from the contact of aqueous droplets in oil. Nat Protoc 8, 1048–1057 (2013). https://doi.org/10.1038/nprot.2013.061

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