Department of Chemistry, Oxford University, Oxford, UK.
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.
我们描述了一种通过在磷脂存在下接触油溶液中的纳升级水滴滴来形成人工脂质双层的方案。在每个油水界面形成一个脂质单层，当两个这样的单层接触时，就会形成一个双层。液滴界面双层（DIB）是一种简单的方法，可以从各种制剂中生成稳定的双层，适用于单通道电生理学和光学成像，这些制剂范围从纯蛋白到重组真核细胞膜片段。例子包括来自金黄色葡萄球菌的α-溶血素孔、炭疽毒素孔和 1.2-MDa 小鼠机械敏感通道 MmPiezo1 的纯蛋白。离子通道和离子型受体也可以从不进一步纯化的膜片段中重建。我们描述了两种形成 DIB 的方法。在一种方法中，在浸没在油中的两个水滴滴之间形成脂质双层。在另一种方法中，在水滴滴和琼脂糖水凝胶之间形成膜，这允许除了电记录之外的成像。该方案耗时<30 分钟，包括液滴生成、单层组装和双层形成。除了主要方案外，我们还描述了 Ag/AgCl 电极的制备和样品制备。
