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

Leptihn, Sebastian; Castell, Oliver K.; Cronin, Bríd; Lee, En-Hsin; Gross, Linda C. M.; Marshall, David P.; Thompson, James R.; Holden, Matthew A.; Wallace, Mark I.

doi:10.1038/nprot.2013.061

Cited by 131 publications

(165 citation statements)

References 63 publications

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“…To date, lipid bilayer-connected droplet networks have been constructed with two quite different methods: manual pipetting of a small number of microliter droplets into a bulk oil reservoir, 4,8 or microfluidic generation and positioning of nanoliter-to-picoliter droplets in microfluidic channels or chambers. [9][10][11] Microliter droplets-in-oil are large enough for wire electrodes to be inserted.…”

Section: Interdroplet Bilayer Arraysmentioning

confidence: 99%

Interdroplet bilayer arrays in millifluidic droplet traps from 3D-printed moulds

et al. 2014

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In droplet microfluidics, aqueous droplets are typically separated by an oil phase to ensure containment of molecules in individual droplets of nano-to-picoliter volume. An interesting variation of this method involves bringing two phospholipid-coated droplets into contact to form a lipid bilayer in-between the droplets. These interdroplet bilayers, created by manual pipetting of microliter droplets, have proved advantageous for the study of membrane transport phenomena, including ion channel electrophysiology.In this study, we adapted the droplet microfluidics methodology to achieve automated formation of interdroplet lipid bilayer arrays. We developed a 'millifluidic' chip for microliter droplet generation and droplet packing, which is cast from a 3D-printed mould. Droplets of 0.7-6.0 μL volume were packed as homogeneous or heterogeneous linear arrays of 2-9 droplets that were stable for at least six hours. The interdroplet bilayers had an area of up to 0.56 mm 2 , or an equivalent diameter of up to 850 μm, as determined from capacitance measurements. We observed osmotic water transfer over the bilayers as well as sequential bilayer lysis by the pore-forming toxin melittin. These millifluidic interdroplet bilayer arrays combine the ease of electrical and optical access of manually pipetted microdroplets with the automation and reproducibility of microfluidic technologies. Moreover, the 3D-printing based fabrication strategy enables the rapid implementation of alternative channel geometries, e.g. branched arrays, with a design-to-device time of just 24-48 hours.

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Section: Interdroplet Bilayer Arraysmentioning

confidence: 99%

Interdroplet bilayer arrays in millifluidic droplet traps from 3D-printed moulds

et al. 2014

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“…Once nanopores were formed, the dye in the liposomes rapidly (<1 min) diffused into the external solution. We note that the releases of molecules from liposomes is not synchronous, which may be a result of the fact the pores do not all form at the same time, 69,70 (Figures 4c and S11). In contrast, in the absence of membrane proteins, liposomes only exhibit slight decay in fluorescence due to photobleaching when observed at the same condition (Figures 4d-f).…”

mentioning

confidence: 98%

Monodisperse Uni- and Multicompartment Liposomes

Deng¹,

Yelleswarapu²,

Huck³

2016

J. Am. Chem. Soc.

223

228

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Liposomes are self-assembled phospholipid vesicles with great potential in fields ranging from targeted drug delivery to artificial cells. The formation of liposomes using microfluidic techniques has seen considerable progress, but the liposomes formation process itself has not been studied in great detail. As a result, high throughput, high-yielding routes to monodisperse liposomes with multiple compartments have not been demonstrated. Here, we report on a surfactant-assisted microfluidic route to uniform, single bilayer liposomes, ranging from 25 µm to 190 µm, and with or without multiple inner compartments. The key of our method is the precise control over the developing interfacial energies of complex W/O/W emulsion systems during liposome formation, which is achieved via an additional surfactant in the phase. The liposomes consist of single bilayers, as demonstrated by nanopore formation experiments and confocal fluoresce microscopy, and they can act as compartments for cell-free gene expression. The microfluidic technique can be expanded to create liposomes with a multitude of coupled compartments, opening routes to networks of multistep microreactors.There has been a significant interest in the use of liposomes, self-assembled phospholipid vesicles composed of bilayer membranes, in fields as diverse as targeted drug delivery, 1,2 membrane protein science, 3-5 bioreactors 6-8 and biosensors. 9,10 Cell-sized liposomes that encapsulate biomolecules and incorporate biological functions provide a versatile mimic of certain aspects of living cells, as exemplified by work showing RNA replication, [11][12][13] in vitro transcription and translation of gene networks, 6,14-16 and organization of cell division machinery in liposomes. [17][18][19][20][21][22][23][24]

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“…DIBs are formed by the contact of two lipid monolayers; in this case, a monolayer formed at the interface between an aqueous droplet and a solution of phospholipids in oil, and another between a thin hydrogel film and the oil. DIBs are robust, long-lived, and defect-free, show unrestricted diffusion, form gigaohm resistance seals, and are compatible with high-resolution optical microscopy (24). .…”

mentioning

confidence: 99%

“…Here, we exploit the unique sensitivity of iSCAT to overcome the limitations in temporal resolution and sensitivity to image, track, and characterize lipid nanodomains without requiring any labels. We use droplet interface bilayers (DIBs) as an artificial membrane model (23,24) with phase-separated lipid mixtures (Fig. 1A).…”

mentioning

confidence: 99%

Dynamic label-free imaging of lipid nanodomains

Wit

Danial

Kukura

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

100

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Lipid rafts are submicron proteolipid domains thought to be responsible for membrane trafficking and signaling. Their small size and transient nature put an understanding of their dynamics beyond the reach of existing techniques, leading to much contention as to their exact role. Here, we exploit the differences in light scattering from lipid bilayer phases to achieve dynamic imaging of nanoscopic lipid domains without any labels. Using phase-separated droplet interface bilayers we resolve the diffusion of domains as small as 50 nm in radius and observe nanodomain formation, destruction, and dynamic coalescence with a domain lifetime of 220 ± 60 ms. Domain dynamics on this timescale suggests an important role in modulating membrane protein function.droplet interface bilayer | iSCAT | lipid nanodomains | label-free imaging | light scattering C ell membranes compartmentalize into lipid domains that enable the selective recruitment of specific proteins (1). These "lipid rafts" have been proposed to control many membrane processes including apical sorting, protein trafficking, and the clustering of proteins during signaling. The dynamic formation and destruction of lipid nanodomains are thought to provide the central mechanism to regulate this wide range of essential processes (2-4). Although many methods now provide strong evidence to support their existence in vivo (5), the combination of nanoscopic size and dynamics on millisecond timescales has placed the direct observation of their behavior beyond the scope of existing techniques. Consequently, although we know they exist, frustratingly little is known regarding their function and dynamics (6).Recent advances in fluorescence nanoscopy provide the only time-dependent information on the behavior of lipid nanodomains (7-9). Stimulated emission depletion-fluorescence correlation spectroscopy has shown cholesterol-mediated hindered nanoscale diffusion of single labeled sphingomyelin lipids that is consistent with the lipid raft hypothesis and transient binding of lipids (9). Superresolution fluorescence microscopy has also revealed protein clusters in cell membranes with 0.5-s temporal resolution (7). All of these experiments, however, are limited in temporal resolution by fluorescence, and must infer lipid nanodomains from the addition of fluorescent labels.Macroscopic phase separation in artificial lipid bilayers has been widely used to help understand the biological implications of domain formation. Different lipid phases can be visualized using fluorescence microscopy with labels that preferentially partition into a specific phase (10-12). This approach is successful for micrometer-sized domains but inevitably fails on the tens to few hundreds of nanometers scale due to limitations in phase specificity, the limited residence time of a label within a specific nanoscopic domain, and the achievable optical resolution (13). The fluorescent probe is itself an additional component that can perturb phase behavior, either directly or through photooxidation (14, 15). As ...

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

Cited by 131 publications

References 63 publications

Interdroplet bilayer arrays in millifluidic droplet traps from 3D-printed moulds

Interdroplet bilayer arrays in millifluidic droplet traps from 3D-printed moulds

Monodisperse Uni- and Multicompartment Liposomes

Dynamic label-free imaging of lipid nanodomains

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