Lipid Bilayer Formation by Contacting Monolayers in a Microfluidic Device for Membrane Protein Analysis

Funakoshi, Kei; Suzuki, Hiroaki; Takeuchi, Shoji

doi:10.1021/ac0613479

Cited by 431 publications

(393 citation statements)

References 32 publications

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“…Since the maximum surface area for the lipid bilayer is the size of the aperture (100 m X 85 m), we obtain a specific membrane capacitance of at least 0.4 µF.cm -2 . Considering the lipid bilayer surface area measured with confocal microscopy, we estimate a specific membrane capacitance of ~ 0.5 µF.cm -2 , which is within capacitance values previously reported for DPhPC lipid membranes [29][30][31] , indicating little or no residual decane in the bilayer membrane, and remarkably less than for a bilayer at a droplet interface 30 .…”

Section: Electrical Measurements Of Lipid Bilayerssupporting

confidence: 85%

Stable Free-Standing Lipid Bilayer Membranes in Norland Optical Adhesive 81 Microchannels

et al. 2016

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ABSTRACT. We report a simple, cost-effective and reproducible method to form free-standing lipid bilayer membranes in microdevices made with Norland Optical Adhesive (NOA81).Surface treatment with either alkylsilane or fluoroalkylsilane enables the self-assembly of stable DPhPC and DOPC/DPPC membranes. Capacitance measurements are used to characterize the lipid bilayer and to follow its formation in real-time. With current recordings, we detect the insertion of single α-Hemolysin pores into the bilayer membrane, demonstrating the possibility of using this device for single-channel electrophysiology sensing applications. Optical transparency of the device and vertical position of the lipid bilayer with respect to the microscope focal plane, allows easy integration with other single-molecule techniques, such as optical tweezers. Therefore, this method to form long-lived lipid bilayers finds a wide range of applications, from sensing measurements to biophysical studies of lipid bilayers and associated proteins.

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Section: Electrical Measurements Of Lipid Bilayerssupporting

confidence: 85%

Stable Free-Standing Lipid Bilayer Membranes in Norland Optical Adhesive 81 Microchannels

et al. 2016

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“…[1][2][3] With phospholipid molecules as surfactants, droplet-droplet contact leads to the expulsion of the interdroplet oil film and the formation of an interdroplet lipid bilayer, 4,5 as illustrated in Fig. 1.…”

Section: Introductionmentioning

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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“…Recently, we demonstrated that submicrolitre aqueous droplets submerged in an apolar liquid containing lipid can be tightly connected by means of lipid bilayers [1][2][3][4][5] to form networks [4][5][6] . Droplet interface bilayers have been used for rapid screening of membrane proteins 7,8 and to form asymmetric bilayers with which to examine the fundamental properties of channels and pores 9 .…”

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confidence: 99%

Droplet networks with incorporated protein diodes show collective properties

et al. 2009

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Recently, we demonstrated that submicrolitre aqueous droplets submerged in an apolar liquid containing lipid can be tightly connected by means of lipid bilayers [1][2][3][4][5] to form networks [4][5][6] . Droplet interface bilayers have been used for rapid screening of membrane proteins 7,8 and to form asymmetric bilayers with which to examine the fundamental properties of channels and pores 9 . Networks, meanwhile, have been used to form microscale batteries and to detect light 4 . Here, we develop an engineered protein pore with diode-like properties that can be incorporated into droplet interface bilayers in droplet networks to form devices with electrical properties including those of a current limiter, a half-wave rectifier and a full-wave rectifier. The droplet approach, which uses unsophisticated components (oil, lipid, salt water and a simple pore), can therefore be used to create multidroplet networks with collective properties that cannot be produced by droplet pairs.To obtain directional ionic current flows in droplet networks ( Fig. 1), we constructed a diode-like pore from staphylococcal a-haemolysin (aHL). aHL forms a heptameric protein pore 10 that inserts vectorially into lipid bilayers 11 . The crystal structure of the pore reveals a 14-stranded transmembrane b barrel capped by an extramembraneous domain, which contains a roughly spherical cavity 10 ( Fig. 2a, left). The wild-type (WT) pore is a 'blank slate' for protein engineering with properties similar to those of an electrolyte-filled tube; it is weakly rectifying and weakly anion selective and gates only at extreme applied potentials of either polarity 12 .aHL has been modified by mutagenesis or targeted chemical modification to form pores with a wide range of properties [13][14][15][16] , but none has exhibited sufficient rectification for our purpose. We had, however, noticed that aHL pores with positively charged side chains projecting into the lumen of the transmembrane b barrel tended to gate (open and close) at negative potentials. Therefore, in an attempt to obtain a fully rectifying pore, we tested an extreme version of aHL in which seven residues were replaced with arginines (7R-aHL) to yield a heptameric pore in which 49 additional positively charged side chains were located within the barrel (Fig. 2a, right). In 1 M KCl, 25 mM Tris HCl at pH 8.0, 100 mM, in planar lipid bilayers, the 7R-aHL pore has a unitary conductance of 0.95 + 0.01 nS (þ50 mV, n ¼ 8). The conductance of the WT pore under the same conditions is similar (0.99 + 0.02 nS, n ¼ 4), which suggests, surprisingly, that the drastically altered 7R-aHL pore is properly formed. The current-voltage (I-V) characteristics of 7R-aHL in 1 M KCl, however, showed virtually complete current rectification (Fig. 2b,c). At positive applied potentials, 7R-aHL remained in an open form with a stable steady-state current and infrequent short-lived closures of less than 10 ms. By contrast, at negative applied potentials, the pore was closed, with occasional brief current spikes ascriba...

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Lipid Bilayer Formation by Contacting Monolayers in a Microfluidic Device for Membrane Protein Analysis

Cited by 431 publications

References 32 publications

Stable Free-Standing Lipid Bilayer Membranes in Norland Optical Adhesive 81 Microchannels

Stable Free-Standing Lipid Bilayer Membranes in Norland Optical Adhesive 81 Microchannels

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

Droplet networks with incorporated protein diodes show collective properties

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