Constructing droplet interface bilayers from the ... - Wallace Lab

protocol 

Constructing droplet interface bilayers from the 

contact of aqueous droplets in oil 

Sebastian Leptihn 1,3 , Oliver K Castell 1 , Brid Cronin 1 , En-Hsin Lee 2 , Linda C M Gross 1 , David P Marshall 1 , 

James R Thompson 1,3 , Matthew Holden 2 & Mark I Wallace 1 

1 Department of Chemistry, Oxford University, Oxford, UK. 2 Department of Chemistry, University of Massachusetts, Amherst, Massachusetts, USA. 3 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.). Correspondence should be addressed to M.I.W. (mark.wallace@chem.ox.ac.uk). 

Published online 2 May 2013; doi:10.1038/nprot.2013.061 

© 2013 Nature America, Inc. All rights reserved. 

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 a-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

protocol 


Figure 1 | Images illustrating the formation of 

droplet-droplet bilayers and droplet-hydrogel 

bilayers. (a) Agarose is pipetted onto the ends 

of two Ag/AgCl electrodes and submerged in 

hexadecane, 200 nl of vesicle solution are 

pipetted onto the agarose, the droplets move 

down on the electrodes once a lipid monolayer 

has been formed, and the electrodes are 

manipulated to bring the droplets into contact 

and form a bilayer. (b) Agarose substrate is 

prepared by spin-coating 0.75% (wt/vol) agarose 

onto a glass coverslip and then assembling a 

PMMA device and filling it with rehydrating 

agarose, lipid in oil is added to the device and a 

monolayer assembles at the agarose oil interface, 

a droplet that has been prepared under lipid in 

oil is pipetted into the device, the droplet falls 

under gravity and contacts the surface to form a 

bilayer, and an Ag/AgCl electrode is inserted into 

the droplet using a micromanipulator. Fluorescent 

species at the bilayer can be investigated using 

optical microscopy. Scale bars, 200 µm. 

the two droplets. This is quicker and easier to achieve than conventional 

patch clamping, in which the careful manipulation of a 

micropipette to a cell or vesicle surface, followed by gentle suction, 

is required to form a gigaseal, which is not always successful. 

Supported lipid bilayers produced by fusing vesicles on a 

glass surface 1,11 or by using the Langmuir-Blodgett deposition 

method 52,53 have been widely used for optical imaging of artificial 

bilayers. However, considerable care must be taken to limit 

substrate interaction with the bilayer and also to produce homogenous 

defect-free bilayers 54 . In contrast, DIBs are inherently defect 

free, and single-particle tracking of labeled lipids in a DIB formed 

between a droplet and an agarose substrate shows diffusion coefficients 

similar to an unsupported bilayer 47 . GUVs provide another 

alternative model membrane that can be generated using gentle 

hydration 55 , electroformation 56 or other methods 18 . GUVs have 

the advantage of not requiring a substrate, but they require careful 

connection to a micropipette to access the vesicle interior. 

Other miniaturized technologies for the development of artificial 

lipid bilayers have recently been reviewed 29,39,57–60 . For highthroughput 

screening, DIBs are straightforward to parallelize, and 

their automated generation is possible. Networks of DIBs could 

a 

b 

Prepare electrodes Add droplets Droplets sag Bilayer forms 

Spin-coat hydrogel Add oil/lipids Add droplet Bilayer forms Add electrode 

also be used to create biological equivalents of electronic devices 21 . 

The evolution of the DIB technique is still ongoing, but the method 

has matured to a stage in which it represents a valuable alternative 

to other artificial lipid bilayer techniques because of its simplicity, 

robustness and versatility. 

Experimental design 

There are a number of different possible experimental configurations 

for producing a DIB. The primary difference lies in whether 

the bilayer is formed between two droplets or between a droplet 

and a hydrogel support. For both of these methods, bilayers can 

be formed using lipids present either in the oil phase or as vesicles 

within the aqueous phase. To highlight this flexibility, we have 

chosen to describe two examples of our method: droplet-droplet 

bilayers formed with lipids present as vesicles within the aqueous 

droplet and droplet-hydrogel bilayers formed from lipids 

present in the oil phase. The steps describing these two configurations 

are shown in Figure 1. Examples for two applications, 

measuring protein transport across DIBs and optical and electrical 

recordings from channels in DIBs, are described in Boxes 1 

and 2, respectively. 

xyz 

- V 

Box 1 | Measuring protein transport across DIB 

The transport of analytes from one droplet to another across the bilayer can be monitored. In this case, one can monitor the 

N-terminal domain of the anthrax lethal factor (LF N ) after translocation through the protective antigen (PA) pore 20 . 

Droplet preparation: Ag/AgCl electrodes coated in agarose are immersed in a solution of lipid in hexadecane. A 200-nl droplet 

solution containing PA and LF N is added to one of the electrodes to form the ‘reservoir’ droplet. ‘Reservoir’ and ‘capture’ droplets are 

contacted and a bilayer is formed, followed by insertion of PA pores. Bilayers form with typically low failure rates ( < 5%). 

Cargo transport: A potential is applied across the bilayer using the electrodes. After the translocation of LF N , the droplets are 

separated. 

Readout: The reservoir droplet is discarded. A detection droplet is formed by adding 200 nl of bilayer buffer to a new agarose-coated 

electrode. A volume of 200 nl of diluted PA is added to the capture droplet, and a DIB is formed with the detection droplet. After PA 

insertion into the membrane, blockage of the ionic current through pores by cargo from the capture droplet is monitored at the 

single-pore level. 

nature protocols | VOL.8 NO.6 | 2013 | 1049

protocol 

Box 2 | Optical and electrical recording from channels in DIB 


Both single-channel electrophysiology and single-molecule fluorescence imaging can be performed simultaneously using DIBs. Here we 

describe the use of this protocol to study the pore-forming toxin α-hemolysin from S. aureus 25 . 

Protein sample preparation: A C-terminal single cysteine mutant of α-hemolysin monomer is recombinantly expressed in Escherichia 

coli before chemical labeling with Cy3b maleimide. 

Device preparation: Agarose is spin-coated onto a microscope coverslip and covered with a micromachined PMMA device. Channels 

within this device are filled with molten agarose and allowed to cool and gel. A solution of lipid in hexadecane is pipetted into wells 

present in the device and incubated for ~15 min. This device ensures that the agarose remains hydrated, and it also provides a well to 

contain the oil solution. 

Droplet preparation: Aqueous droplets of ~100 pM α-hemolysin are allowed to equilibrate for 15 min in a solution of lipid in 

hexadecane to form lipid monolayers at the oil-water interface. A droplet is pipetted into each well; the droplet sinks to the substrate 

agarose layer at the bottom of the well, and it then forms a bilayer. Bilayers form with typically low failure rates (< 5%). α-hemolysin 

assembles to form a heptameric pore in the bilayer. 

Electrophysiology: A 100-µm-diameter Ag/AgCl electrode is inserted into the droplet using a micromanipulator. By using a 

corresponding Ag/AgCl ground electrode in the agarose, ionic currents from single pores present in the bilayer can be recorded. 

Single-molecule fluorescence: For optical recordings, the device was placed on an inverted microscope; individual α-hemolysin 

pores can be detected diffusing in the bilayer by using and TIRF illumination and electron-multiplying charge-coupled 

device (CCD) detection. 

Limitations 

DIBs can be formed using a range of different lipid compositions 

44,46,49 with little variation in appearance (Fig. 2). The basic 

rule of thumb is that if a lipid or solvent composition can be used 

for a conventional lipid bilayer experiment, it can be easily adapted 

to the DIB technique. 

The major limitations that we have investigated include the 

following: 

• Lipid location. Bilayers form more rapidly 

when vesicles are present within the 

aqueous phase; however, for some experiments, 

the presence of vesicles within the 

droplet may be undesirable. 

• Lipid concentration. When the lipids are 

present in the oil phase, the bilayer formation 

rate is dependent on lipid concentration. 

Below a concentration of 

2 mg ml − 1 , there is typically insufficient 

lipid to form a monolayer, and droplets 

will immediately coalesce without bilayer 

formation. Above a concentration of 10 mg ml − 1 , bilayer formation 

times are markedly increased. 

• Detergent concentration. As with all bilayer systems, the presence 

of detergent will disrupt the bilayer and potentially compromise 

stability. We have found that 1,2-diphytanoyl-sn-glycero-3- 

phosphocholine (DPhPC) DIBs may tolerate up to 0.01% 

(wt/vol) n-decyl-β-d-maltopyranoside in the aqueous droplet. 

a b c 

d e f 

Figure 2 | Bright-field images of droplet 

bilayers formed under various lipid conditions. 

The darkest area in each image is the tip of the 

Ag/AgCl electrode. (a) DPhPC in hexadecane. 

(b) DPhPC in tetradecane. (c) DPhPC in decane. 

(d) DPhPC:cholesterol (3:2) in hexadecane. 

(e) DPhPC:DOPG (9:1) in hexadecane. (f) DOPE: 

DOPG (4:1) in hexadecane. (g) DPhPC:DPPC: 

cholesterol (1:1:1) in hexadecane. (h) DPhPC/ 

DPhPC:DPPC:cholesterol (1:1:1) in hexadecane. 

(i) DOPC:DPPC:cholesterol (3:1:1)/DOPC in 

hexadecane. In the examples above, droplets appear 

as dark circles corresponding to the perimeter of the 

droplet. The bilayer can be seen as a white ring. The 

irregular out of focus black object in each image is 

the electrode with agarose anchor. Scale bar, 75 µm. 

g h i 

1050 | VOL.8 NO.6 | 2013 | nature protocols

protocol 

• Applied potential. As with other planar lipid bilayers, high 

applied potentials ( >150–200 mV) can cause electroporation 

and eventually rupture the bilayer. 

• Osmotic pressure. As with other bilayers, DIB integrity is influenced 

by the osmotic gradient across the bilayer. At higher osmolyte 

concentrations, droplets form lipid bilayers more readily and 

are more stable compared with droplets of a lower ionic strength. 

Although it has not been exhaustively explored, we have found 

that DIBs are able to withstand osmotic gradients of 50 atm. 

• Temperature. DIBs do not tolerate freezing of the oil (the 

freezing point of hexadecane is 18 °C). DIBs can be used at 

higher temperatures, and our experiments on planar supports 

show that bilayers are stable up to the melting temperature of 

the agarose. 

• Residual solvent. Capacitance measurements 44 show that the 

proportion of residual solvent present in the bilayer depends 

on the similarity between lipid chain length and solvent 

structure, as one would expect for a Montal-Mueller bilayer 

61 . We previously calculated that DPhPC DIBs formed 

in hexadecane contain 9.2% oil by volume 44 . The use of lipid 

solutions in heptadecane or squalene produce an essentially 

solvent-free bilayer 44 . 


MATERIALS 

REAGENTS 

CRITICAL See the Reagent Setup section for detailed preparation 

instructions. 

Agarose preparation 

• Agarose, low melt (Sigma-Aldrich, cat. no. A9414) 

Vesicle preparation 

• 1,2-Dipalmitoyl-sn-glycero-3-phosphocholine (DPPC, Avanti Polar Lipids, 

cat. no. 850355) 

• Chloroform (Sigma, cat. no. 650498) 

• Buffer solution: 100 mM KCl and 10 mM HEPES (pH 7.4, titrated 

with KOH) 

Lipid in hexadecane solution preparation 

• 1,2-Diphytanoyl-sn-glycero-3-phosphocholine (DPhPC; Avanti Polar 

Lipids, cat. no. 850356; Note that DPhPC can be replaced by other lipids or 

lipid combinations if desired) 

• Chloroform (Sigma, cat. no. 650498) 

• Hexadecane (Sigma, cat. no. H6703) 

Electrode preparation 

• Silver wire, 100 µm in diameter (Sigma, cat. no. 348783) 

• Silver wire, 1.5 mm in diameter (Sigma, cat. no. 348759) 

• Sodium hypochlorite solution (Sigma, cat. no. 425044) 

EQUIPMENT 

Experimental setting: droplet-droplet 

• Faraday cage 

• Oil bath, e.g., 1-ml Perspex chamber 

• Micromanipulators (NMN21, Narishige) 

• Patch-clamp amplifier (Axopatch 200B, Axon Instruments) 

Vesicle preparation 

• Vacuum desiccator 

• Mini-extruder (Avanti Polar Lipids, cat. no. 610000) 

• Nuclepore track-etched membrane, 0.1-µm pore size, 19-mm diameter 

(Whatman, cat. no. 800309) 

Experimental setting: droplet-hydrogel 

• Faraday cage 

• Micromanipulator 

• Patch-clamp amplifier 

• Inverted microscope 

Lipid in hexadecane preparation 

• Vacuum desiccator 

Poly(methyl methacrylate) device and droplet incubation chamber 

• Extruded sheet poly(methyl methacrylate) (PMMA, 4-mm thickness 

(RS, cat. no. 824-519) 

• Subtractive computer numerical control (CNC) milling machine 

(e.g., Roland Modela MDX-40A) 

• CAD software (e.g., SolidWorks) 

Surface preparation 

• Microscope coverslips (borosilicate glass, 24 mm × 40 mm, thickness no. 1 

(0.13–0.16 mm); Gerhard Menzel, cat. no. BB024040A1) 

• Plasma cleaner (Diener Electronic, Femto) 

• Spin coater (3,000 r.p.m.; Laurell, cat. no. WS-650MZ-23NPP/LITE) 

• Adhesive tape (e.g., Scotch, 3M) 

Device assembly 

• Hot plate capable of 35 °C 

• Dry heat block capable of 90 °C 

REAGENT SETUP 

Droplet-droplet vesicle preparation Prepare a 50 mg ml − 1 lipid stock by dissolving 

DPPC in chloroform. Take 29 µl of this stock in a glass vial. While 

rotating the vial gently, dry the lipid solution under a stream of nitrogen until 

the solvent is removed and a lipid film remains. Place the vial in a vacuum desiccator 

for at least 30 min to remove any residual solvent. Resuspend the lipid 

in 1 ml of buffer and vortex the lipid for 60 s to obtain a cloudy 2 mM lipid 

suspension. Extrude the solution with 21 passes through a membrane filter 

(e.g., Avanti mini-extruder with Whatman-GE Nuclepore track-etched 

membranes). As illustrated in Figure 2, other lipids and lipid ratios can be 

prepared using this method. Vesicle solutions can be stored at 4 °C for 1 week. 

Droplet-droplet protein preparation (optional) This protocol can be 

adapted to incorporate membrane proteins into the DIB. Several strategies 

are possible, depending on the nature of the users’ protein sample. Standard 

protocols exist for preparation of the samples used in these procedures. For 

example, membrane proteins can be first reconstituted into lipid vesicles 62,63 , 

detergent-solubilized proteins can also be used 64 and proteins can also be 

synthesized in the droplet 20 . 

Droplet-droplet agarose preparation Dissolve 30 mg of agarose in 1 ml of 

desired experimental buffer to produce a 3% (wt/vol) solution. This can be 

stored for several weeks at room temperature (22 °C). 

Droplet-droplet electrode preparation Briefly melt one end of a 20-mm 

length of 100-µm-diameter silver wire over a flame to create a silver ball 

of ~250 µm in diameter. Immerse the ball end of the wire in 20% (wt/vol) 

sodium hypochlorite solution for 30 min. A gray color indicates that a 

AgCl coating has formed. Two Ag/AgCl electrodes should be prepared. 

Solder them to insulated cable and fit appropriate connectors for head 

stage (e.g., Axon Axopatch 200B). These can be used for up to 2 weeks 

before recoating. 

Droplet-hydrogel agarose preparation Dissolve 75 mg of agarose in 10 ml 

of deionized water to produce a 0.75% (wt/vol) solution. Dissolve 325 mg of 

agarose in 10 ml of the desired experimental buffer to produce 3.25% (wt/vol) 

solution. These can be stored for at least 2 weeks at room temperature. 

Droplet-hydrogel: lipid in hexadecane solution preparation Prepare a 

50 mg ml − 1 lipid stock by dissolving DPhPC in chloroform. Take 380 µl of 

this stock in a glass vial. Swirl the vial rapidly under nitrogen stream until 

the solvent is removed and a lipid film is formed. Place the vial in vacuum 

desiccator for at least 30 min to remove the solvent completely. Add 2 ml of 

hexadecane and vortex the vial to produce a 9.5 mg ml − 1 solution. Lipid in 

chloroform solution can be kept at least 1 month in a sealed vial at − 20 °C. 

Lipid in hexadecane is stable at room temperature. 

Droplet-hydrogel protein preparation (optional) Membrane proteins can 

also be incorporated into a droplet-hydrogel DIB by following the suggested 

sample preparation steps outlined above. In addition, proteins can be 

introduced into the lipid bilayer from the agarose support: for detergentsolubilized 

proteins, samples are diluted to detergent concentrations that 


protocol 

do not disrupt the bilayer (for n-decyl-β-d-maltopyranoside ~0.01%, 

usually 1:1,000, for single-channel recordings up to 1:1,000,000). Membrane 

fragments can also be used 27,65 . For proteins in native cell membranes, 

membrane fragments or vesicles, samples are diluted (usually 1:1,000, but 

up to 1:1,000,000 for single-channel recordings). Before device assembly, 

the sample is mixed with the low-melting substrate agarose, or pipetted 

onto the spin-coated hydrogel substrate layer. 

Droplet-hydrogel electrode preparation For the ground electrode, cut a 

10-mm length of 1.5-mm silver wire. Solder it to 18-gauge insulated copper 

wire with an appropriate connector for the head stage. For the droplet electrode, 

cut a 20-mm length of 100-µm-diameter silver wire and solder it to 

insulated cable with connector for the head stage. Immerse silver portions 

of 1.5-mm electrode in 20% (wt/vol) sodium hypochlorite overnight, and, 

also, immerse the 100-µm electrode for 1 h. A gray color indicates that an 

AgCl coating has been formed. Electrodes need to be checked regularly for 

the uniformity of the Ag/AgCl coating and need to be recoated if required. 

As a general guideline, we are able to use electrodes for about 2 weeks 

before recoating. 

EQUIPMENT SETUP 

Droplet-hydrogel PMMA device manufacture This is a reusable device in which 

droplet hydrogel DIBs can be formed; it can be machined by CNC micromachining. 

An array of 1-mm-diameter holes machined in PMMA acts as chambers for 

the formation of individual DIBs. On the underside of the PMMA substrate, a 

microfluidic channel surrounding each well is machined (Fig. 3a–c). 

Droplet-hydrogel incubation chamber manufacture Prepare a chamber 

for droplet incubation by micromilling recesses of 43 mm × 27 mm × 

5.5 mm into a planar PMMA substrate (Fig. 3d). 

Droplet-hydrogel coverslip surface preparation Plasma-clean 

microscope coverslips for 7 min at 95 W with a 0.5-bar 

oxygen stream. 


PROCEDURE 

1| Depending on the desired application, DIBs may be generated in option A, a droplet-droplet configuration, or option B, 

a droplet-hydrogel format. Both approaches share a number of common steps, including coating the electrodes with agarose 

and mounting the Faraday cage. 

(A) Droplet-droplet bilayers ● TIMING 8 min 

(i) Preparation of oil bath chamber. Fill up an oil bath (e.g., a 1-ml Perspex chamber) with hexadecane and place this 

chamber, together with two micromanipulators and the patch-clamp amplifier head stage, in a metal enclosure to act 

as a Faraday cage. 

(ii) Preparation of agarose-coated electrodes (Fig. 1a). Pipette 200 nl of 3% (wt/vol) agarose at 90 °C onto the end of two 

coated electrodes. A stereomicroscope may assist with this process. On cooling, agarose gelation generates an agarose 

droplet at the end of each electrode. 

(iii) To avoid dehydration of the agarose, submerge each droplet in the hexadecane-containing oil chamber immediately 

after gelation of the agarose. Attach the impaled electrode to the micromanipulator to support the agarose droplet 

within the chamber. 

(iv) Application of the lipid solution to the agarose-coated electrode (Fig. 1a). By using a stereomicroscope to view the 

droplets, pipette 200 nl of pre-prepared vesicle solution onto each agarose droplet immersed in the oil by placing the 

pipette tip close to the supported agarose droplet. The vesicle droplets will adhere to the suspended hydrogel. The 

droplet solution can contain proteoliposomes or detergent-solubilized proteins if incorporation of membrane proteins 

is required. 

(v) Incubate the droplets (Fig. 1a). Wait for ~2 min to allow the formation of a self-assembled lipid monolayer. It is 

important to give the droplets time to form a defect-free lipid monolayer; otherwise, droplets tend to burst and the 

procedure will fail. On doing so, reduction of the interfacial tension results in the droplet sagging from the electrode, 

indicating that the droplets are ready to form a DIB. 

CRITICAL STEP Allow time for monolayer formation or droplets will fuse when they contact. 

(vi) DIB formation (Fig. 1a). By using the stereomicroscope to view the droplets, move the micromanipulator(s) to carefully 

bring the two droplets into contact. It is crucial for the formation of a bilayer to move the droplets so that they 

‘touch’, but do not push them too close together as this may result in droplet fusion and failure of the procedure. 

Close the lid of the metal enclosure to shield it from external electrical noise. Bilayer formation generally occurs within 

1 or 2 min of contacting the droplets. Progression of the bilayer formation process may be monitored by capacitance 

measurement. To accelerate bilayer formation, a potential of 50 mV can be applied between the electrodes. 

CRITICAL STEP Droplets must be repositioned carefully or they will fuse when they contact. 

? TROUBLESHOOTING 

(vii) Adjust bilayer size (optional). Micromanipulation of the droplet positions can be used to adjust bilayer size, if required. 

(B) Droplet-hydrogel bilayers ● TIMING 30 min 

(i) Spin-coat an agarose layer onto a glass coverslip (Fig. 1b). Apply adhesive tape (e.g., Scotch tape, 3M) to the perimeter 

of a plasma-cleaned coverslip to restrict the area to be coated. 

(ii) Spin-coat 140 µl of agarose solution (0.75% wt/vol, 90 °C, 4,000 r.p.m., 30 s) onto the coverslip to provide the 

hydrogel support for bilayer formation. These conditions produce a layer sufficiently thin to permit total internal 

reflection fluorescence (TIRF) imaging of the bilayer (i.e., < 300 nm). The thickness of the agarose layer may be 

modulated by altering the agarose concentration and/or spin speed. To ensure uniform coverage, care should be taken 

to ensure that the coverslip is suitably attached to the spin coater and does not distort under the applied vacuum. 


protocol 


a 

Figure 3 | Device construction for droplethydrogel 

DIBs. (a,b) Subtractive micromilling 

(Modela MDX-40A, Roland DG) was used to 

fabricate a device in which the droplet interface 

2.10 

bilayers were formed. An array of 1-mm-diameter 

holes machined in PMMA act as chambers for the 

formation of individual DIBs. On the underside 

of the PMMA substrate, a microfluidic channel 

surrounding each well is machined. This allows 

sealing of the device with molten agarose 

c 

d 

to prevent leakage of lipid and oil, and also 

maintains hydration of the spin-coated hydrogel 

1 

layer upon which the DIB forms. (c) Image 

2 

of the device in situ: (1) The ground Ag/AgCl 

electrode connecting to the substrate gel. (2) 

The 100-µm Ag/AgCl electrode used for electrical 

3 

measurements inside droplets. (3) The DHB 

device attached to the bottom of the Faraday 

cage. White tape is used to seal the agarose 

channel. Black tape is used to mount the 

device on the microscope. (d) Droplet incubation chamber. A chamber for droplet incubation was prepared by micromilling 43 mm × 27 mm × 3.5 mm recesses 

into a planar PMMA substrate. 

27 

(iii) Remove the tape to leave an agarose-free border. This prevents electrical leakage from the underside of the device. 

(iv) Add membrane fragments or proteoliposomes to agarose (optional). If protein incorporation is desired, this sample 

(typically 1–20 µl) can be pipetted onto the agarose surface. 

(v) Assemble the device. The purpose of this step is both to introduce a reservoir of buffer in contact with the spin-coated 

agarose layer in order to maintain hydration and to create a leak-free seal around each droplet-containing well in the 

device. The bottom of the device is constructed such that a coverslip fits flush into the device to aid in making a seal 

(Fig. 3). Position the device on top of the agarose-coated face of the coverslip and place it on a hot plate heated to 35 °C. 


(vi) Fill the agarose reservoir. By using a 200-µl pipette, fill the underside cavity of the device with agarose (3.25% wt/vol 

in an appropriate experimental buffer) via the connecting fill-holes on the upper side of the PMMA device (Fig. 3). 

(vii) Place the ground Ag/AgCl electrode into the fill-hole before the agarose sets. 

(viii) Cover the agarose fill-holes on the upper side of the device with adhesive tape to prevent dehydration. 

(ix) Transfer to a microscope. Place the device within a Faraday cage mounted on an inverted microscope. 

(x) Form a monolayer on the agarose surface (Fig. 1b). Pipette the lipid in hexadecane solution (9.5 mg ml − 1 ) into each 

well of the assembled device. 

(xi) Incubate the droplets (Fig. 1b). Fill the droplet incubation chamber with the lipid in hexadecane solution (9.5 mg ml − 1 ) 

and pipette approximately three 200-nl droplets into each groove in the chamber. The droplet solution can 

contain proteoliposomes or detergent-solubilized proteins, if the incorporation of membrane proteins via the droplet 

is required. 

(xii) Wait. Allow 20 min until monolayer assembly is complete. 

CRITICAL STEP Allow time for monolayer formation or droplets will fuse with the hydrogel. 

(xiii) Transfer droplets to the device. Pipette individual droplets from the incubation chamber to each of the 12 wells present 

in the device. 

(xiv) DIB formation (Fig. 1b). Bilayers form spontaneously as the droplets touch down on the surface. 


(xv) Insert the electrode (optional) (Fig. 1b). By using the micromanipulator, insert the agarose-coated electrode into the 

droplet by contacting the electrode with the droplet. This is observed as a slight deformation of the droplet. The electrode 

does not need to be forced into the droplet. After ~30 s, the droplet will spontaneously move onto the electrode. 

Connect both the electrodes to a patch-clamp amplifier to make conventional electrical recordings of ionic current. 


(xvi) Imaging the bilayer (optional) (Fig. 1b). The DIB is now formed on the agarose surface and positioned on an inverted 

microscope. Optical microscopy can then be used to image the bilayer, including TIRF imaging. 


43 

b 

4.20 

3.50 

0.90 

0.50 

ø0.50 

ø2 

3.50 

0.20 


protocol 


Troubleshooting advice can be found in Table 1. 

Table 1 | Troubleshooting table. 

Step Problem Possible reason Solution 

1A(vi), 1B(xiv) 

Droplets fuse shortly after 

bilayer formation 

Osmotic imbalance 

Check the osmotic balance of 

buffers 

Other amphiphiles are present. This may be 

diagnosed before fusion by the growth of 

unusually large bilayers; a result of reduced 

interfacial tension 

Check reagents for the presence of 

excess of detergent 


Droplets do not form 

bilayers 

Other lipid structures prevent monolayer contact 

1B(v) Agarose fills the wells Agarose solution is not sufficiently viscous. 

Agarose gelation is influenced by pH and ionic 

strength 

1B(xiv) 

1B(xv, xvi) 

Droplets fuse on contact 

with agarose 

Droplets shrink or grow 

over time 

Poor seal between device and coverslip surface 

One or both monolayers are not formed 

Other amphiphiles are present 

Osmotic imbalance or partitioning of water into 

the oil phase 

Decrease lipid concentration. 

Reduce incubation time in Step 

1A(v) or Step 1B(xii) 

Increase the agarose concentration 

Reassemble the device 

Increase lipid concentration by 

10%. Increase incubation time in 

Step 1B(xi) in 5-min increments 

Check reagents for the presence of 

detergent 

Check the osmotic balance of 

buffers 

1B(xv) Electrical noise Movement of droplet Reduce vibration 

External interference 

Droplet is touching the edge of the well 

Check whether the Faraday cage is 

grounded 

Move the droplet into the center of 

the well 

Current drift Evaporation; electrode corrosion Check whether the device is sealed 

and the agarose is properly hydrated 

1B(xvi) 

Cannot image bilayer using 

TIRF excitation 

Agarose layer is too thick 

Adjust the agarose concentration 

and spin-coater parameters 

● TIMING 

Reagent Setup 

Vesicle preparation: 1 h 

Agarose preparation: 5 min 

Electrode preparation: 30 min 

Lipid in hexadecane solution preparation: 45 min 

Electrode preparation: 18 h 

Equipment Setup 

Surface preparation: 10 min 

Procedural Steps 

Step 1A, droplet-droplet bilayers: 8 min 

Step 1A(i–iv), preparation: 5 min 

Step A(v), incubate monolayers: 2 min 

Step 1A(vi,vii), bilayer formation: 1 min 

Step 1B, droplet-hydrogel bilayers: 30 min 


protocol 

Figure 4 | Optical and electrical measurement of 

droplets. (a) Capacitance measured during bilayer 

formation by the application of a triangular 

potential. The bilayer again corresponds to 

the white circle at the center of the image 

within the droplet. The droplet edge appears 

as a darker ring. (b) Stepwise changes in ionic 

current corresponding to individual insertions 

of S. aureus α-hemolysin protein pores into the 

bilayer prepared as described in ref. 21. (Buffer: 

10 mM Tris-HCl, 0.5 M NaCl, pH 8.0. Applied 

potential: + 100 mV). Scale bar, 200 µm. 

a 

100 pF 

200 ms 

No bilayer 

Bilayer 

b 

50 pA 

5 s 

Step 1B(i–ix), assemble device: 7 min 

Step 1B(x–xiii), incubate monolayers: 20 min 

Step 1B(xiv), bilayer formation: 1 min 

Step 1B(xv,xvi), insert electrodes and image: 2 min 


ANTICIPATED RESULTS 

With this protocol, one should be able to create DIBs. This protocol provides procedures for making both droplet-droplet and 

droplet-hydrogel variants of DIBs. 

Bilayer imaging 

Typical example images obtained during the steps of this protocol are shown in Figure 1. A range of representative droplethydrogel 

bilayers from a range of different lipid compositions is shown in Figure 3. Little obvious difference in appearance is 

seen as the lipid composition changes. Bilayers incorporating proteins show no visible differences. 

Electrical recording 

A typical electrical response from a DIB is shown in Figure 4. Figure 4a shows the capacitate response of the system during 

bilayer formation. Capacitance is measured by monitoring the square-wave current output upon application of a triangularwave 

input voltage 44 . The trace indicates an increase in capacitance as the bilayer forms. 

The most definitive measure of successful bilayer formation 

is the recording of ionic currents through a single ion 

a 

100 

channel or pore embedded in the bilayer. For these integral 

membrane proteins to function, they must span a single 

lipid bilayer and elicit a characteristic current. Figure 4b 

shows one example of this and demonstrates successive 

insertion events of the pore-forming toxin α-hemolysin from 

S. aureus. This protein was produced by following the 

procedures used in our previously published work 25 . Each 

stepwise increase in current corresponds to an individual 

α-hemolysin pore inserting into the bilayer. 

b 

80 

60 

40 

20 

40 

Bilayer formation 

Data showing the variation in bilayer stability and 

formation rate are presented in Figure 5 for a DPhPC in 

Immediate rupture (%) 

Formation time (min) 

30 

20 

10 

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). 

Lipid solutions were prepared from a single stock of DPhPC in hexadecane. 

Droplets were equilibrated in lipid hexadecane for 5 min before imaging. 

(a) The proportion of droplets that burst immediately is shown. (b) For 

those droplets that do not immediately rupture, the time from droplet 

addition to a well in the device to bilayer formation increases with lipid 

concentration. Devices were prepared ~10 min before droplet addition. 

Bilayers do not form without lipid (×). (c) Bilayer lifetime increases 

with lipid concentration. The experiment was terminated at 10 h for all 

remaining droplets (dotted line). Error bars indicate ± 1 s.d. 

c 

Bilayer lifetime (min) 

0 

1,000 

100 

10 

1 

0.1 

0 2 4 6 8 10 

Lipid concentration (mg ml –1 ) 


protocol 

hexadecane DIB formed between a droplet and an agarose surface. Similar trends are observed with other lipid mixtures. 

Lipid concentration is important for bilayer formation and droplet stability. Too-low concentration will result in insufficiently 

assembled monolayers with subsequent droplet rupture (Fig. 5a). The other extreme, too-high lipid concentrations, results in 

increased viscosity of the solution and potentially in the formation of multilamellar lipid layers. As a result, the formation of 

a lipid bilayer is delayed or does not occur at all (Fig. 5b). Bilayer lifetime also correlates with lipid concentration (Fig. 5c); 

to obtain a stable droplet, a concentration of >5 mg ml − 1 should be used. If care is taken to prevent evaporation of the oil 

and to ensure the agarose remains hydrated, droplet bilayers appear to be stable indefinitely. Once formed, DIBs are stable for 

many weeks, given conditions that prevent dehydration of the droplet. 


Acknowledgments 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. 

AUTHOR 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. 

COMPETING FINANCIAL INTERESTS The authors declare no competing financial 

interests. 

Reprints and permissions information is available online at http://www.nature. 

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