multifunctional-micropipette-based-method-for-incorporation

Multifunctional, Micropipette-based Method for Incorporation And Stimulation of Bacterial Mechanosensitive Ion Channels in Droplet Interface Bilayers

MscL, a large conductance mechanosensitive channel (MSC), is a ubiquitous osmolyte release valve that helps bacteria survive abrupt hypo-osmotic shocks. ...

University of Georgia

A new method called the regulated attachment method (RAM) for reproducibly forming lipid bilayers within flexible substrates has been developed that enables precise control over the size of the bilayer. This technique uses a deformable flexible substrate to open and close an aperture that subdivides aqueous volumes submersed in an organic solvent. Phospholipids incorporated as vesicles in the aqueous phase self-assemble at the oil/water interface to form lipid monolayers that encapsulate each aqueous volume. Controlled attachment of opposing lipid monolayers is achieved by regulating the dimensions of the aperture in the substrate that separates the adjacent aqueous volumes. In this manner, the size of a lipid bilayer formed within a flexible substrate is a function of the substrate and aperture dimensions, and not determined by the sizes or shapes of the aqueous volumes. Lipid bilayers formed within the prototype flexible substrate exhibit DC resistances consistently higher than 10 GOmega and can survive 20-30x changes in area without rupture. Furthermore, RAM permits lipid bilayers to be completely unzipped after thinning by applying sufficient force to fully close the dividing aperture and even allows the introduction of species, such as alamethicin channels, into preformed lipid bilayers via controlled injection through an intersecting channel within the substrate. Controlling the size of the interface through indirect interactions with the supporting substrate offers a new platform for assembling durable lipid bilayers. We envision that this technology can be scaled to higher dimensions consisting of multiple apertures required for creating aqueous networks partitioned by functional lipid bilayers and to smaller length scales to produce very small lipid bilayers capable of hosting single proteins.

Regulated attachment method for reconstituting lipid bilayers of prescribed size within flexible substrates.

Physically-encapsulated droplet interface bilayers are formed by confining aqueous droplets encased in lipid monolayers within connected compartments of a solid substrate. Each droplet resides within an individual compartment and is positioned on a fixed electrode built into the solid substrate. Full encapsulation of the network is achieved with a solid cap that inserts into the substrate to form a closed volume. Encapsulated networks provide increased portability over unencapsulated networks by limiting droplet movement and through the integration of fixed electrodes into the supporting fixture. The formation of encapsulated droplet interface bilayers constructed from diphytanoyl phosphocoline (DPhPC) phospholipids is confirmed with electrical impedance spectroscopy, and cyclic voltammetry is used to measure the effect of alamethicin channels incorporated into the resulting lipid bilayers. The durability of the networks is quantified using a mechanical shaker to oscillate the bilayer in a direction transverse to the plane of the membrane and the results show that single droplet interface bilayers can withstand 1-10g of acceleration prior to bilayer failure. Observed failure modes include both droplet separation and bilayer rupturing, where the geometry of the supporting substrate and the presence of integrated electrodes are key contributors. Physically-encapsulated DIBs can be shaken, moved, and inverted without bilayer failure, enabling the creation of a new class of lab-on-chip devices.

Physical encapsulation of droplet interface bilayers for durable, portable biomolecular networks.

Solidified biomolecular networks that incorporate liquid-supported lipid bilayers are constructed by attaching lipid-encased, water-swollen hydrogels contained in oil. Poly(ethylene glycol) dimethacrylate (PEG-DMA) and a free-radical photoinitiator are added to an aqueous lipid vesicle solution such that exposure to ultraviolet light results in solidification of neighboring aqueous volumes. Bilayer formation can occur both prior to photopolymerization with the aqueous mixture in the liquid state and after solidification by using the regulated attachment method (RAM) to attach the aqueous volumes contained within a flexible substrate. In addition, photopolymerization of the hydrogels can be performed in a separate mold prior to placement in the supporting substrate. Membranes formed across a wide range of hydrogel concentrations [0-80% (w/v); MW=1000 g/mol PEG-DMA] exhibit high electrical resistances (1-10 GΩ), which enable single-channel recordings of alamethicin channels and show significant durability and longevity. We demonstrate that just as liquid phases can be detached and reattached using RAM, reconfiguration of solid aqueous phases is also possible. The results presented herein demonstrate a step toward constructing nearly solid-state biomolecular materials that retain fluid interfaces for driving molecular assembly. This work also introduces the use of three-dimensional printing to rapidly prototype a molding template used to fabricate polyurethane substrates and to shape individual hydrogels.

Bilayer formation between lipid-encased hydrogels contained in solid substrates.

MscL, a stretch-activated channel, saves bacteria experiencing hypo-osmotic shocks from lysis. Its high conductance and controllable activation makes it a strong candidate to serve as a transducer in stimuli-responsive biomolecular materials. Droplet interface bilayers (DIBs), flexible insulating scaffolds for such materials, can be used as a new platform for incorporation and activation of MscL. Here, we report the first reconstitution and activation of the low-threshold V23T mutant of MscL in a DIB as a response to axial compressions of the droplets. Gating occurs near maximum compression of both droplets where tension in the membrane is maximal. The observed 0.1-3 nS conductance levels correspond to the V23T-MscL sub-conductive and fully open states recorded in native bacterial membranes or liposomes. Geometrical analysis of droplets during compression indicates that both contact angle and total area of the water-oil interfaces contribute to the generation of tension in the bilayer. The measured expansion of the interfaces by 2.5% is predicted to generate a 4-6 mN/m tension in the bilayer, just sufficient for gating. This work clarifies the principles of interconversion between bulk and surface forces in the DIB, facilitates the measurements of fundamental membrane properties, and improves our understanding of MscL response to membrane tension.

Article	Year
Regulated attachment method for reconstituting lipid bilayers of prescribed size within flexible substrates. Analytical chemistry\| PubMed ID: 20058855	2010
Physical encapsulation of droplet interface bilayers for durable, portable biomolecular networks. Lab on a chip\| PubMed ID: 20221558	2010
Bilayer formation between lipid-encased hydrogels contained in solid substrates. ACS applied materials & interfaces\| PubMed ID: 21067200	2010
Activation of bacterial channel MscL in mechanically stimulated droplet interface bilayers. Scientific reports\| PubMed ID: 26348441	2015

Donald J. Leo

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