The design of membrane-based constructs with multiple compartments is of increasing importance given their potential applications as microreactors, as artificial cells in synthetic-biology, as ...simplified cell models, and as drug delivery vehicles. The emergence of droplet microfluidics as a tool for their construction has allowed rapid scale-up in generation throughput, scale-down of size, and control over gross membrane architecture. This is true on several levels: size, level of compartmentalisation and connectivity of compartments can all be programmed to various degrees. This tutorial review explains and explores the reasons behind this. We discuss microfluidic strategies for the generation of a family of compartmentalised systems that have lipid membranes as the basic structural motifs, where droplets are either the fundamental building blocks, or are precursors to the membrane-bound compartments. We examine the key properties associated with these systems (including stability, yield, encapsulation efficiency), discuss relevant device fabrication technologies, and outline the technical challenges. In doing so, we critically review the state-of-play in this rapidly advancing field.
In this article we detail a robust high-throughput microfluidic platform capable of fabricating either symmetric or asymmetric giant unilamellar vesicles (GUVs) and characterise the mechanical ...properties of their membranes.
Giant unilamellar vesicles (GUVs) have a wide range of applications in biology and synthetic biology. As a result, new approaches for constructing GUVs using microfluidic techniques are emerging but ...there are still significant shortcomings in the control of fundamental vesicle structural parameters such as size, lamellarity, membrane composition and internal contents. We have developed a novel microfluidic platform to generate compositionally-controlled GUVs. Water-in-oil (W/O) droplets formed in a lipid-containing oil flow are transferred across an oil-water interface, facilitating the self-assembly of a phospholipid bilayer. In addition, for the first time we have studied the mechanical properties of the resultant lipid bilayers of the microfluidic GUVs. Using fluctuation analysis we were able to calculate the values for bending rigidity of giant vesicles assembled on chip and demonstrate that these correlate strongly with those of traditional low throughput strategies such as electroformation.
Phase separation in ternary model membranes is known to occur over a range of temperatures and compositions and can be induced by increasing hydrostatic pressure. We have used small angle X-ray ...scattering (SAXS) to study phase separation along pre-determined tie lines in dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC) and cholesterol (CHOL) mixtures. We can unequivocally distinguish the liquid ordered (Lo) and liquid disordered (Ld) phases in diffraction patterns from biphasic mixtures and compare their lateral compressibility. The variation of tie line endpoints with increasing hydrostatic pressure was determined, at atmospheric pressure and up to 100 MPa. We find an extension and shift of the tie lines towards the DOPC rich region of the phase diagram at increased pressure, this behaviour differs slightly from that reported for decreasing temperature.
Bicontinuous cubic structures offer enormous potential in applications ranging from protein crystallisation to drug delivery systems and have been observed in cellular membrane structures. One of the ...current bottlenecks in understanding and exploiting these structures is that cubic scaffolds produced in vitro are considerably smaller in size than those observed in biological systems, differing by almost an order of magnitude in some cases. We have addressed this technological bottleneck and developed a methodology capable of manufacturing highly swollen bicontinuous cubic membranes with length scales approaching those seen in vivo. Crucially, these cubic systems do not require the presence of proteins. We have generated highly swollen Im3m symmetry bicontinuous cubic phases with lattice parameters of up to 480 Å, composed of ternary mixtures of monoolein, cholesterol and negatively charged lipid (DOPS or DOPG) and we have been able to tune their lattice parameters. The swollen cubic phases are highly sensitive to both temperature and pressure; these structural changes are likely to be controlled by a fine balance between lipid headgroup repulsions and lateral pressure in the hydrocarbon chain region.
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).
Droplet interface bilayer (DIB) networks are emerging as a cornerstone technology for the bottom up construction of cell-like and tissue-like structures and bio-devices. They are an exciting and ...versatile model-membrane platform, seeing increasing use in the disciplines of synthetic biology, chemical biology, and membrane biophysics. DIBs are formed when lipid-coated water-in-oil droplets are brought together-oil is excluded from the interface, resulting in a bilayer. Perhaps the greatest feature of the DIB platform is the ability to generate bilayer networks by connecting multiple droplets together, which can in turn be used in applications ranging from tissue mimics, multicellular models, and bio-devices. For such applications, the construction and release of DIB networks of defined size and composition on-demand is crucial. We have developed a droplet-based microfluidic method for the generation of different sized DIB networks (300-1500 pl droplets) on-chip. We do this by employing a droplet-on-rails strategy where droplets are guided down designated paths of a chip with the aid of microfabricated grooves or "rails," and droplets of set sizes are selectively directed to specific rails using auxiliary flows. In this way we can uniquely produce parallel bilayer networks of defined sizes. By trapping several droplets in a rail, extended DIB networks containing up to 20 sequential bilayers could be constructed. The trapped DIB arrays can be composed of different lipid types and can be released on-demand and regenerated within seconds. We show that chemical signals can be propagated across the bio-network by transplanting enzymatic reaction cascades for inter-droplet communication.
We demonstrate that nanolitre-sized droplets are an effective tool in coupling two-dimensional separations in both time and space. Using a microfluidic droplet connector, chemically separated ...components can be segmented into nanolitre droplets. After oil filtering and droplet merging, these droplets are loaded into a second dimension for comprehensive separations.
Droplet interface
bilayers (DIBs) have become widely recognised as a robust platform for constructing model
membranes
and are emerging as a key technology for the bottom-up assembly of synthetic ...cell-like and
tissue-like structures. DIBs are formed when lipid-monolayer coated water droplets are brought together
inside a well of oil, which is excluded from the interface as the DIB forms. The unique
features of the system, compared to traditional approaches (e.g., supported lipid bilayers, black
lipid
membranes,
and liposomes), is
the ability to engineer multi-layered bilayer networks by connecting multiple
droplets
together in 3D, and the capability to impart bilayer asymmetry freely within these
droplet
architectures by supplying droplets with different lipids. Yet despite these achievements, one potential
limitation of the technology is that DIBs formed from biologically relevant components
have not been well studied. This could limit the reach of the platform to biological
systems where bilayer composition and asymmetry are understood to play a key role. Herein,
we address this issue by reporting the assembly of asymmetric DIBs designed to replicate
the plasma membrane compositions of three different plant species;
Arabidopsis thaliana, tobacco, and oats, by engineering vesicles with different amounts
of plant phospholipids, sterols and cerebrosides for the first time. We show that
vesicles made
from our plant lipid formulations are stable and can be used to assemble asymmetric
plant DIBs. We verify this using a bilayer permeation assay, from which we extract values
for absolute effective bilayer permeation and bilayer stability. Our results confirm that
stable DIBs can be assembled from our plant membrane mimics and could lead to new approaches for
assembling model systems to study membrane translocation and to screen new
agrochemicals in plants.
In this study, a novel droplet based microfluidic method for the generation of different sized droplet interface bilayers is reported. A microfluidic platform was designed, which allows the ...generation and packing of picoliter lipid coated water droplets. Droplets were generated by hydrodynamic focusing coupled with selective transport along grooves according to their size. A trapping structure at the end of the groove and a fine control of the flow pressures allowed for the droplets to be successfully trapped and aligned on demand. This technology facilitates the fine control of droplet size production as well as the generation of extended networks from a variety of lipids including 1,2-diphytanoyl-sn-glycero-3-phosphocholine and 1,2-dioleoyl-sn-glycero-3-phosphocholine in linear and non-linear configurations, which is vital to the application of Droplet Interface Bilayers to biological network construction on-chip.
PDF
