Droplet Interface Bilayer Formation Research Articles

Investigating the effect of phospholipids on droplet formation and surface property evolution in microfluidic devices for droplet interface bilayer (DIB) formation.

Despite growing interest in droplet microfluidic methods for droplet interface bilayer (DIB) formation, there is a dearth of information regarding how phospholipids impact device function. Limited characterization has been carried out for phospholipids, either computationally (in silico) or experimentally (in situ) in polydimethylsiloxane (PDMS) microfluidic devices, despite recent work providing a better understanding of how other surfactants behave in microfluidic systems. Hence, microfluidic device design for DIB applications relies heavily on trial and error, with many assumptions made about the impact of phospholipids on droplet formation and surface properties. Here, we examine the effects of phospholipids on interfacial tension, droplet formation, wetting, and hence device longevity, using DPhPC as the most widely used lipid for DIB formation. We use a customized COMSOL in silico model in comparison with in situ experimental data to establish that the stabilization of droplet formation seen when the lipid is dosed in the aqueous phase (lipid-in) or in the oil phase (lipid-out) is directly dependent on the effects of lipids on the device surface properties, rather than on how fast they coat the droplet. Furthermore, we establish a means to visually characterize surface property evolution in the presence of lipids and explore rates of device failure in the absence of lipid, lipid-out, and lipid-in. This first exploration of the effects of lipids on device function may serve to inform the design of microfluidic devices for DIB formation as well as to troubleshoot causes of device failure during microfluidic DIB experiments.

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.

Passive and parallel microfluidic formation of droplet interface bilayers (DIBs) for measurement of leakage of small molecules through artificial phospholipid membranes

We present a passive microfluidic system for easy and rapid generation of Droplet Interface Bilayer pairs, each formed with two aqueous nanoliter droplets comprising controlled chemical composition. The system allows for rapid screening to quantify leakage of small molecules through artificial phospholipid bilayers. The droplets are generated, diluted and stored in-situ on the microfluidic chip. Our device comprises microfluidic Meter&Store (M&S) modules – hydrodynamic traps, which enable hard-wired operations on small – ca. 9 nL in volume – aqueous droplets including splitting, merging and derailing the droplets to side storage wells. Consequently, the droplets are locked and positioned next to each other and form bilayers at the point of contact between the nanoliter aqueous compartments. Additionally, a set of trap modules provides the possibility for in situ preparation of dilutions of a sample. We demonstrated the basic capacity of the trap-based system for formation of an array of 12 (controllably different) lipid bilayers in less than 5 min. Thanks to the small volumes of the droplets, the system is capable for monitoring the transport across the artificial membrane within a relatively short interval – opening the possibility to use dyes and alleviating the difficulty of parallel electrophysiological measurements. We use this functionality to prepare on-chip dilutions of small molecules and determine their permeation rates through the bilayer. Finally, we used the M&S system and calcium sensitive dyes to quantify the ion flux through the model α-hemolysin (αHL) nanopores.

Evaporation-induced monolayer compression improves droplet interface bilayer formation using unsaturated lipids.

In this article, we report on a new experimental methodology to enable reliable formation of droplet interface bilayer (DIB) model membranes with two types of unsaturated lipids that have proven difficult for creating stable DIBs. Through the implementation of a simple evaporation technique to condition the spontaneously assembled lipid monolayer around each droplet, we increased the success rates of DIB formation for two distinct unsaturated lipids, namely 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), from less than 10% to near 100%. Separately, using a pendant drop tensiometer, we learned that: (a) DOPC and POPC monolayers do not spontaneously assemble into their tightest possible configurations at an oil-water interface, and (b) reducing the surface area of a water droplet coated with a partially packed monolayer leads to a more tightly packed monolayer with an interfacial tension lower than that achieved by spontaneous assembly alone. We also estimated from Langmuir compression isotherms obtained for both lipids that the brief droplet evaporation procedure prior to DIB formation resulted in a 6%-16% reduction in area per lipid for DOPC and POPC, respectively. Finally, the increased success rates of formation for DOPC and POPC DIBs enabled quantitative characterization of unsaturated lipid membrane properties including electrical resistance, rupture potential, and specific capacitance.

Broadly Accessible Assays with Signal Amplification across Lipid Bilayers

Over the past decade, droplet interface bilayers (DIBs) have emerged as a robust platform for performing studies across lipid membranes with the potential for high-throughput applications. Here we explore the potential of DIBs for detecting the presence of analytes in a heterogeneous sample without the need for an external power source. We discuss the formation of DIBs by a broadly accessible and straightforward pipetting step leading to stable DIBs between two aqueous reservoirs with a proton gradient across the bilayer interface. When a pore-forming peptide is present, the pH of the reservoirs change, which can be detected by the unaided eye based on the color change of a pH sensitive dye. This approach takes advantage of bio-inspired amplification in the sense that one ion channel, after analyte-triggered activation, facilitates the flux of thousands of ions across the DIB. We describe our attempts to impede H+/OH- flux using known pore blockers in order to detect alterations in the rate of color change in the presence of these molecules. We also discuss the possibility of using pore-forming peptides engineered to alter their function in the presence of a specific analyte in order to detect this analyte within a sample. Our ultimate goal is to develop an assay to screen samples of biofluids in a broadly-accessible and robust manner that can be adapted to various analytes and biomarkers.

Formation of droplet interface bilayers in a Teflon tube.

Droplet-interface bilayers (DIBs) have applications in disciplines ranging from biology to computing. We present a method for forming them manually using a Teflon tube attached to a syringe pump; this method is simple enough it should be accessible to those without expertise in microfluidics. It exploits the properties of interfaces between three immiscible liquids, and uses fluid flow through the tube to pack together drops coated with lipid monolayers to create bilayers at points of contact. It is used to create functional nanopores in DIBs composed of phosphocholine using the protein α-hemolysin (αHL), to demonstrate osmotically-driven mass transfer of fluid across surfactant-based DIBs, and to create arrays of DIBs. The approach is scalable, and thousands of DIBs can be prepared using a robot in one hour; therefore, it is feasible to use it for high throughput applications.

A microfluidic approach for high-throughput droplet interface bilayer (DIB) formation

We present a simple, automated method for high-throughput formation of droplet interface bilayers (DIBs) in a microfluidic device. We can form complex DIB networks that are able to fill predefined three dimensional architectures. Moreover, we demonstrate the flexibility of the system by using a variety of lipids including 1,2-diphytanoyl-sn-glycero-3-phosphocholine (DPhPC) and 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC).