Droplet Interface Bilayers Research Articles

Crosslinking Strategies for the Microfluidic Production of Microgels.

This article provides a systematic review of the crosslinking strategies used to produce microgel particles in microfluidic chips. Various ionic crosslinking methods for the gelation of charged polymers are discussed, including external gelation via crosslinkers dissolved or dispersed in the oil phase; internal gelation methods using crosslinkers added to the dispersed phase in their non-active forms, such as chelating agents, photo-acid generators, sparingly soluble or slowly hydrolyzing compounds, and methods involving competitive ligand exchange; rapid mixing of polymer and crosslinking streams; and merging polymer and crosslinker droplets. Covalent crosslinking methods using enzymatic oxidation of modified biopolymers, photo-polymerization of crosslinkable monomers or polymers, and thiol-ene “click” reactions are also discussed, as well as methods based on the sol−gel transitions of stimuli responsive polymers triggered by pH or temperature change. In addition to homogeneous microgel particles, the production of structurally heterogeneous particles such as composite hydrogel particles entrapping droplet interface bilayers, core−shell particles, organoids, and Janus particles are also discussed. Microfluidics offers the ability to precisely tune the chemical composition, size, shape, surface morphology, and internal structure of microgels by bringing multiple fluid streams in contact in a highly controlled fashion using versatile channel geometries and flow configurations, and allowing for controlled crosslinking.

Chiral lipid bilayers are enantioselectively permeable.

Homochiral membrane bilayers organize biological functions in all domains of life. The membrane’s permeability–its key property–correlates with a molecule’s lipophilicity, but the role of the membrane’s rich and uniform stereochemistry as a permeability determinant is largely ignored in empirical and computational measurements. Here, we describe a new approach to measuring permeation using continuously generated microfluidic droplet interface bilayers (DIBs, 480/min) and benchmark this system by monitoring fluorescent dye DIB permeation over time. Permeation of non-fluorescent, alkyne-labeled molecules was measured using a fluorogenic click reaction. DIB transport measurements revealed enantioselective permeation of alkyne-labeled amino acids (Ala, Val, Phe, Pro) and dipeptides through a chiral phospholipid bilayer; the biological L enantiomers permeated faster than D (1.2–6-fold; Ala–Pro). Enantioselective permeation both poses a potentially unanticipated criterion for drug design and offers a kinetic mechanism for the abiotic emergence of homochirality via chiral transfer between sugars, amino acids, and lipids.

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.

Temperature-Controlled Assembly and Characterization of a Droplet Interface Bilayer.

The droplet interface bilayer (DIB) method for assembling lipid bilayers (i.e., DIBs) between lipid-coated aqueous droplets in oil offers key benefits versus other methods: DIBs are stable and often long-lasting, bilayer area can be reversibly tuned, leaflet asymmetry is readily controlled via droplet compositions, and tissue-like networks of bilayers can be obtained by adjoining many droplets. Forming DIBs requires spontaneous assembly of lipids into high density lipid monolayers at the surfaces of the droplets. While this occurs readily at room temperature for common synthetic lipids, a sufficient monolayer or stable bilayer fails to form at similar conditions for lipids with melting points above room temperature, including some cellular lipid extracts. This behavior has likely limited the compositions-and perhaps the biological relevance-of DIBs in model membrane studies. To address this problem, an experimental protocol is presented to carefully heat the oil reservoir hosting DIB droplets and characterize the effects of temperature on the lipid membrane. Specifically, this protocol shows how to use a thermally conductive aluminum fixture and resistive heating elements controlled by a feedback loop to prescribe elevated temperatures, which improves monolayer assembly and bilayer formation for a wider set of lipid types. Structural characteristics of the membrane, as well as the thermotropic phase transitions of the lipids comprising the bilayer, are quantified by measuring the changes in electrical capacitance of the DIB. Together, this procedure can aid in evaluating biophysical phenomena in model membranes over various temperatures, including determining an effective melting temperature (TM) for multi-component lipid mixtures. This capability will thus allow for closer replication of natural phase transitions in model membranes and encourage the formation and use of model membranes from a wider swath of membrane constituents, including those that better capture the heterogeneity of their cellular counterparts.

Temperature-Controlled Assembly and Characterization of a Droplet Interface Bilayer

This protocol details the use of a feedback temperature-controlled heating system to promote lipid monolayer assembly and droplet interface bilayer formation for lipids with elevated melting temperatures, and capacitance measurements to characterize temperature-driven changes in the membrane.

Ibuprofen and the Phosphatidylcholine Bilayer: Membrane Water Permeability in the Presence and Absence of Cholesterol.

The interactions between drugs and cell membranes can modulate the structural and physical properties of membranes. The resultant perturbations of the membrane integrity may affect the conformation of the proteins inserted within the membrane, disturbing the membrane-hosted biological functions. In this study, the droplet interface bilayer (DIB), a model cell membrane, is used to examine the effects of ibuprofen, a nonsteroidal anti-inflammatory drug (NSAID), on transbilayer water permeability, which is a fundamental membrane biophysical property. Our results indicate that the presence of neutral ibuprofen (pH 3) increases the water permeability of the lipid membranes composed of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC). When cholesterol is present with the DOPC, however, the water permeability is not influenced by addition of ibuprofen, regardless of the cholesterol content in DOPC. Given the fact that cholesterol is generally considered to impact packing in the hydrocarbon chain regions, our findings suggest that a potential competition between opposing effects of ibuprofen molecules and cholesterol on the hydrocarbon core environment of the phospholipid assembly may influence the overall water transport phenomena. Results from confocal Raman microspectroscopy and interfacial tensiometry show that ibuprofen molecules induce substantial structural and dynamic changes in the DOPC lipid bilayer. These results, demonstrating that the presence of ibuprofen increases the water permeability of pure DOPC but not that of DOPC-cholesterol mixtures, provide insight into the differential effect of a representative NSAID on heterogeneous biological membranes, depending upon the local composition and structure, results which will signal increased understanding of the gastrointestinal damage and toxicity induced by these molecules.

Permeation Studies across Symmetric and Asymmetric Membranes in Microdroplet Arrays.

We investigated the permeation of molecules across lipid membranes on an open microfluidic platform. An array of droplet pairs was created by spotting aqueous droplets, dispersed in a lipid oil solution, onto a plate with cavities surrounded by a hydrophobic substrate. Droplets in two adjacent cavities come in contact and form an artificial lipid bilayer, called a droplet interface bilayer (DIB). The method allows for monitoring permeation of fluorescently tagged compounds from a donor droplet to an acceptor droplet. A mathematical model was applied to describe the kinetics and determine the permeation coefficient. We also demonstrate that permeation kinetics can be followed over a series of droplets, all connected via DIBs. Moreover, by changing the lipid oil composition after spotting donor droplets, we were able to create asymmetric membranes that we used to mimic the asymmetry of the cellular plasma membrane. Finally, we developed a protocol to separate and extract the droplets for label-free analysis of permeating compounds by liquid chromatography–mass spectrometry. Our versatile platform has the potential to become a new tool for the screening of drug membrane permeability in the future.

Droplet-Based Membranous Soft Materials.

Inspired by the structure and functionality of natural cellular tissues, droplet interface bilayer (DIB)-based materials strategically combine model membrane assembly techniques and droplet microfluidics. These structures have shown promising results in applications ranging from biological computing to chemical microrobots. This Feature Article briefly explores recent advances in the areas of construction, manipulation, and functionalization of DIB networks; discusses their unique mechanics; and focuses on the contributions of our lab in the advancement of this platform. We also reflect on some of the limitations facing DIB-based materials and how they might be addressed, highlighting promising applications made possible through the refinement of the material concept.

Surface energy and separation mechanics of droplet interface phospholipid bilayers.

Droplet interface bilayers are a convenient model system to study the physio-chemical properties of phospholipid bilayers, the major component of the cell membrane. The mechanical response of these bilayers to various external mechanical stimuli is an active area of research because of its implications for cellular viability and the development of artificial cells. In this article, we characterize the separation mechanics of droplet interface bilayers under step strain using a combination of experiments and numerical modelling. Initially, we show that the bilayer surface energy can be obtained using principles of energy conservation. Subsequently, we subject the system to a step strain by separating the drops in a step-wise manner, and track the evolution of the bilayer contact angle and radius. The relaxation time of the bilayer contact angle and radius along with the decay magnitude of the bilayer radius were observed to increase with each separation step. By analysing the forces acting on the bilayer and the rate of separation, we show that the bilayer separates primarily through the peeling process with the dominant resistance to separation coming from viscous dissipation associated with corner flows. Finally, we explain the intrinsic features of the observed bilayer separation by means of a mathematical model comprising the Young-Laplace equation and an evolution equation. We believe that the reported experimental and numerical results extend the scientific understanding of lipid bilayer mechanics, and that the developed experimental and numerical tools offer a convenient platform to study the mechanics of other types of bilayers.

Efficient Lipid Bilayer Formation by Dipping Lipid-Loaded Microperforated Sheet in Aqueous Solution

This paper describes a method for a bilayer lipid membrane (BLM) formation using a perforated sheet along with an open chamber. Microscopic observation of the formed membrane showed a typical droplet interface bilayer. We proved that the formed membrane was a BLM based on electrical measurements of the membrane protein α-hemolysin, which produces nanopores in BLMs. Unlike the conventional approach for BLM formation based on the droplet contact method, this method provides aqueous surfaces with no organic solvent coating layer. Hence, this method is suitable for producing BLMs that facilitate the direct addition of chemicals into the aqueous phase.

The role of temperature in the formation of human-mimetic artificial cell membranes using droplet interface bilayers (DIBs).

Droplet interface bilayers (DIBs) have recently started to be used as human-mimetic artificial cell membranes. DIBs are bilayer sections created at the interface of two aqueous droplets, such that one droplet can be used as a donor compartment and the other as an acceptor compartment for the quantification of molecular transport across the artificial cell membrane. However, synthetic phospholipids are overwhelmingly used to create DIBs instead of naturally derived phospholipids, even though the diverse distribution of phospholipids in the latter is more biomimetic. We present the first systematic study of the role of temperature in DIB formation, which shows that the temperature at which DIBs are formed is a key parameter for the formation of DIBs using naturally derived phospholipids in a microfluidic platform. The phospholipids that are most abundant in mammalian cell membranes (phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylserine (PS), and phosphatidylinositol (PI)) only form DIBs when the temperature is above the phase transition temperature (Tm). Similarly, DIB formation usually only occurs above the highest Tm of a single phospholipid in a bespoke formulation. In addition, we show a new phenomenon wherein the DIB "melts" without disintegrating for bilayers formed predominantly of phospholipids that occupy cylindrical spaces. We also demonstrate differences in DIB formation rates as well as permeability of these biomimetic membranes. Given the difficulties associated with making DIBs using naturally derived phospholipids, we anticipate this work will illuminate the role of phospholipid phase transition in mono- and bilayer formation and lay the foundation for DIBs to be used as human-mimetic artificial cell membranes.

Activating mechanosensitive channels embedded in droplet interface bilayers using membrane asymmetry.

Droplet microcompartments linked by lipid bilayers show great promise in the construction of synthetic minimal tissues. Central to controlling the flow of information in these systems are membrane proteins, which can gate in response to specific stimuli in order to control the molecular flux between membrane separated compartments. This has been demonstrated with droplet interface bilayers (DIBs) using several different membrane proteins combined with electrical, mechanical, and/or chemical activators. Here we report the activation of the bacterial mechanosensitive channel of large conductance (MscL) in a dioleoylphosphatidylcholine:dioleoylphosphatidylglycerol DIB by controlling membrane asymmetry. We show using electrical measurements that the incorporation of lysophosphatidylcholine (LPC) into one of the bilayer leaflets triggers MscL gating in a concentration-dependent manner, with partial and full activation observed at 10 and 15 mol% LPC respectively. Our findings could inspire the design of new minimal tissues where flux pathways are dynamically defined by lipid composition.

Biomimetic artificial cells to model the effect of membrane asymmetry on chemoresistance.

We present a microfluidic platform that enables the formation of bespoke asymmetric droplet interface bilayers (DIBs) as artificial cell models from naturally-derived lipids. We use them to perform pharmacokinetic assays to quantify how lipid asymmetry affects the permeability of the chemotherapy drug doxorubicin. Previous attempts to model bilayer asymmetry with DIBs have relied on the use of synthetic lipids to achieve asymmetry. Use of natural lipids serves to increase the biomimetic nature of these artificial cells, showcasing the next step towards forming a true artificial cell membrane in vitro. Here we use our microfluidic platform to form biomimetic, asymmetric and symmetric DIBs, with their asymmetry quantified through their life-mimicking degree of curvature. We subsequently examine permeability of these membranes to doxorubicin, and reveal measurable differences in its pharmacokinetics induced by membrane asymmetry, highlighting another factor that potentially contributes to chemoresistance in some forms of cancer.

Single-molecule imaging of pore-forming toxin dynamics in droplet interface bilayers.

Single-channel recording from pore-forming toxins (PFTs) provides a clear and direct molecular readout of toxin action. However to complete any mechanistic understanding of PFT behavior, this functional kinetic readout must be linked to the underlying changes in toxin structure, binding, conformation, or stoichiometry. Here we review how single-molecule imaging methods might be used to further our understanding of PFTs, and provide detailed practical guidance on the use of droplet interface bilayers as a method capable of examining both single-molecule fluorescence and single-channel electrical signals from PFTs.

Microfluidic platform enables tailored translocation and reaction cascades in nanoliter droplet networks

In the field of bottom-up synthetic biology, lipid membranes are the scaffold to create minimal cells and mimic reactions and processes at or across the membrane. In this context, we employ here a versatile microfluidic platform that enables precise positioning of nanoliter droplets with user-specified lipid compositions and in a defined pattern. Adjacent droplets make contact and form a droplet interface bilayer to simulate cellular membranes. Translocation of molecules across membranes are tailored by the addition of alpha-hemolysin to selected droplets. Moreover, we developed a protocol to analyze the translocation of non-fluorescent molecules between droplets with mass spectrometry. Our method is capable of automated formation of one- and two-dimensional droplet networks, which we demonstrated by connecting droplets containing different compound and enzyme solutions to perform translocation experiments and a multistep enzymatic cascade reaction across the droplet network. Our platform opens doors for creating complex artificial systems for bottom-up synthetic biology.

Bioluminescent detection of isothermal DNA amplification in microfluidic generated droplets and artificial cells

Microfluidic droplet generation affords precise, low volume, high throughput opportunities for molecular diagnostics. Isothermal DNA amplification with bioluminescent detection is a fast, low-cost, highly specific molecular diagnostic technique that is triggerable by temperature. Combining loop-mediated isothermal nucleic acid amplification (LAMP) and bioluminescent assay in real time (BART), with droplet microfluidics, should enable high-throughput, low copy, sequence-specific DNA detection by simple light emission. Stable, uniform LAMP–BART droplets are generated with low cost equipment. The composition and scale of these droplets are controllable and the bioluminescent output during DNA amplification can be imaged and quantified. Furthermore these droplets are readily incorporated into encapsulated droplet interface bilayers (eDIBs), or artificial cells, and the bioluminescence tracked in real time for accurate quantification off chip. Microfluidic LAMP–BART droplets with high stability and uniformity of scale coupled with high throughput and low cost generation are suited to digital DNA quantification at low template concentrations and volumes, where multiple measurement partitions are required. The triggerable reaction in the core of eDIBs can be used to study the interrelationship of the droplets with the environment and also used for more complex chemical processing via a self-contained network of droplets, paving the way for smart soft-matter diagnostics.

Continuous and Rapid Solution Exchange in a Lipid Bilayer Perfusion System Based on Droplet-Interface Bilayer.

Because of the high sensitivity of lipid bilayers to external pressure fluctuations, a major challenge in functional studies of biological pores or ion channels is the difficulty in exchanging solutions rapidly while maintaining the stability of the lipid bilayer in a model membrane. Here we describe a droplet-interface bilayer-based perfusion system that has been routinely used in our research and is currently the most robust and stable perfusion system that provides prompt solution exchange surrounding a lipid bilayer. In this model membrane system, solutions can be completely exchanged within 1-2s to obtain prompt responses of a lipid bilayer or membrane pores to the membrane environments. Also, our system is stable enough to sustain continuous perfusions up to at least dozens of minutes. To demonstrate, we show that acidification-induced protein channel insertion,substrate binding to protein channels, and pH gradient-driven protein translocation of anthrax toxin can be sequentially initiated by continuous perfusions in our system. Moreover, by rapidly switching the solutions, the protein translocation based on ratchet mechanisms can be paused and reinitiated iteratively in our system. Overall, this perfusion system provides a controllable and reliable solution exchange platform for investigations of pores and translocations on lipid bilayers.

Penetratin translocation mechanism through asymmetric droplet interface bilayers

Penetratin is a cell penetrating peptide (CPP) that can enter cells by direct translocation through the plasma membrane. The molecular mechanism of this translocation still remains poorly understood. Here we provide insights on this mechanism by studying the direct translocation of the peptide across model membranes based on Droplet Interface Bilayers (DIBs), which are bilayers at the interface between two adhering aqueous-in-oil droplets. We first showed with symmetric bilayers made of a mix of 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-(1′-rac-glycerol) (POPG) and 1,2-dioleoyl-sn-glycero-3-phosphocholine (POPC) that the translocation of penetratin required the presence of at least 40% of POPG on both leaflets. Interestingly when replacing POPG with another anionic lipid 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-l-serine (POPS), translocation was inefficient. To elucidate the lipid partners required at each step of the CPP translocation process, we then investigated the crossing of asymmetric bilayers. We found that POPG on the proximal leaflet and POPS on the distal leaflet allowed penetratin translocation. Translocation was not observed when POPS was on the proximal leaflet and POPG on the distal leaflet or if POPS on the distal leaflet was replaced with POPC. These observations led us to propose a three-step translocation mechanism: (i) peptide recruitment by anionic lipids, (ii) formation of a transient peptide-lipid structure leading to the initiation of translocation which required specifically POPG on the proximal leaflet, (iii) termination of the translocation process favored by a driving force provided by anionic lipids in the distal leaflet.

Flowing droplet interface bilayers: A microfluidic tool to control droplet trajectories and to study mechanical properties of unsupported lipid bilayers.

We introduce the concept of Flowing Droplet Interface Bilayers (FDIBs) that are made of two droplets maintained in contact due to the presence of an adhesive lipidic surfactant. This system is similar to a flowing dumbbell made of two droplets interconnected by a lipid bilayer and driven by an external flow. Interestingly, such a dumbbell does not show a straight flow trajectory, but it oscillates between the sidewalls while moving along the microchannel. The origin of this unusual motion is hydrodynamic interactions, as demonstrated by analytical calculations and micro particle image velocimentry (µPiV) measurements. The hydrodynamic motion appears to be highly sensitive to the mechanical properties of the lipid bilayer connecting the two droplets (FDIB). Thus, droplet trajectories can be controlled by tuning the lipid bilayer composition, which enables in turn investigating mechanical properties of free-standing lipid bilayers.

Membrane protein mediated bilayer communication in networks of droplet interface bilayers

Droplet interface bilayers (DIBs) are model membranes formed between lipid monolayer-encased water droplets in oil. Compared to conventional methods, one of the most unique properties of DIBs is that they can be connected together to generate multi-layered ‘tissue-like’ networks, however introducing communication pathways between these compartments typically relies on water-soluble pores that are unable to gate. Here, we show that network connectivity can instead be achieved using a water-insoluble membrane protein by successfully reconstituting a chemically activatable mutant of the mechanosensitive channel MscL into a network of DIBs. Moreover, we also show how the small molecule activator can diffuse through an open channel and across the neighbouring droplet to activate MscL present in an adjacent bilayer. This demonstration of membrane protein mediated bilayer communication could prove key toward developing the next generation of responsive bilayer networks capable of defining information flow inside a minimal tissue.