A skin-inspired soft material with directional mechanosensation.

Lessons about artificial sensor design may be taken from evolutionarily perfected physiological systems. Mechanosensory cells in human skin are exquisitely sensitive to gentle touch and enable us to distinguish objects of different stiffnesses and textures. These cells are embedded in soft epidermal layers of gel-like consistency. Reproducing these mechanosensing capabilities in new soft materials may lead to the development of adaptive mechanosensors which will further enhance the abilities of engineered membrane-based structures with bioinspired sensing strategies. This strategy is explored here using droplet interface bilayers embedded within a thermoreversible organogel. The interface between two lipid-coated aqueous inclusions contained within a soft polymeric matrix forms a lipid bilayer resembling the lipid matrix of cell membranes. These interfaces are functionalized with bacterial mechanosensitive channels (V23T MscL) which convert membrane tension into changes in membrane conductance, mimicking mechanosensitive channel activation in mammalian mechanosensory cells. The distortion of encapsulated adhered droplets by cyclical external forces are first explored using a finite element composite model illustrating the directional propagation of mechanical disturbances imposed by a piston. The model predicts that the orientation of the droplet pair forming the membrane relative to the direction of the compression plays a role in the membrane response. The directional dependence of mechanosensitive channel activation in response to gel compression is confirmed experimentally and shows that purely compressive perturbations normal to the interface invoke different channel activities as compared to shearing displacement along a plane of the membrane. The developed system containing specially positioned pairs of droplets functionalized with bacterial mechanosensitive channels and embedded in a gel creates a skin-inspired soft material with a directional response to mechanical perturbation.

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

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.

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

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.

The voltage-dependence of MscL has dipolar and dielectric contributions and is governed by local intramembrane electric field.

Channels without canonical voltage sensors can be modulated by voltage acting on other domains. Here we show that besides protein dipoles, pore hydration can be affected by electric fields. In patches, both WT MscL and its V23T mutant show a decrease in the tension midpoint with hyperpolarization. The mutant exhibits a stronger parabolic dependence of transition energy on voltage, highly consistent with the favourable dielectric contribution from water filling the expanding pore. Purified V23T MscL in DPhPC droplet interface bilayers shows a similar voltage dependence. When reconstituted in an asymmetric DOPhPC/DPhPC bilayer carrying a permanent bias of ~130 mV due to a dipole potential difference between the interfaces, the channel behaved as if the local intramembrane electric field sets the tension threshold for gating rather than just the externally applied voltage. The data emphasize the roles of polarized water in the pore and interfacial lipid dipoles in channel gating thermodynamics.

Encapsulating Networks of Droplet Interface Bilayers in a Thermoreversible Organogel.

The development of membrane-based materials that exhibit the range and robustness of autonomic functions found in biological systems remains elusive. Droplet interface bilayers (DIBs) have been proposed as building blocks for such materials, owing to their simplicity, geometry, and capability for replicating cellular phenomena. Similar to how individual cells operate together to perform complex tasks and functions in tissues, networks of functionalized DIBs have been assembled in modular/scalable networks. Here we present the printing of different configurations of picoliter aqueous droplets in a bath of thermoreversible organogel consisting of hexadecane and SEBS triblock copolymers. The droplets are connected by means of lipid bilayers, creating a network of aqueous subcompartments capable of communicating and hosting various types of chemicals and biomolecules. Upon cooling, the encapsulating organogel solidifies to form self-supported liquid-in-gel, tissue-like materials that are robust and durable. To test the biomolecular networks, we functionalized the network with alamethicin peptides and alpha-hemolysin (αHL) channels. Both channels responded to external voltage inputs, indicating the assembly process does not damage the biomolecules. Moreover, we show that the membrane properties may be regulated through the deformation of the surrounding gel.

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. It has been discovered and rigorously studied using the patch-clamp technique for almost three decades. Its basic role of translating tension applied to the cell membrane into permeability response makes it a strong candidate to function as a mechanoelectrical transducer in artificial membrane-based biomolecular devices. Serving as building blocks to such devices, droplet interface bilayers (DIBs) can be used as a new platform for the incorporation and stimulation of MscL channels. Here, we describe a micropipette-based method to form DIBs and measure the activity of the incorporated MscL channels. This method consists of lipid-encased aqueous droplets anchored to the tips of two opposing (coaxially positioned) borosilicate glass micropipettes. When droplets are brought into contact, a lipid bilayer interface is formed. This technique offers control over the chemical composition and the size of each droplet, as well as the dimensions of the bilayer interface. Having one of the micropipettes attached to a harmonic piezoelectric actuator provides the ability to deliver a desired oscillatory stimulus. Through analysis of the shapes of the droplets during deformation, the tension created at the interface can be estimated. Using this technique, the first activity of MscL channels in a DIB system is reported. Besides MS channels, activities of other types of channels can be studied using this method, proving the multi-functionality of this platform. The method presented here enables the measurement of fundamental membrane properties, provides a greater control over the formation of symmetric and asymmetric membranes, and is an alternative way to stimulate and study mechanosensitive channels.

Activation of bacterial channel MscL in mechanically stimulated droplet interface bilayers.

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.

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

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.