Publications
106 results found
Ces O, Elani Y, Karamdad K, et al., 2016, Novel microfluidic technologies for the bottom-up construction of artificial cells
This talk will outline novel microfluidic strategies for biomembrane engineering that are capable of fabricating vesicles [1], droplet interface bilayer networks [2], multisomes [3] and artificial tissues [4] where parameters such as membrane asymmetry, membrane curvature, compartment connectivity and individual compartment contents can be controlled. Various bulk methods, such as extrusion, gentle hydration and electroformation, have been synonymous with the formation of lipid vesicles over recent years. However these strategies suffer from significant shortcomings associated with these processes including limited control of vesicle structural parameters such as size, lamellarity, membrane composition and internal contents. To address this technological bottleneck we have developed novel microfluidic platforms to form lipid vesicles in high-Throughput with full control over the composition of both the inner and outer leaflet of the membrane thereby enabling the manufacture of symmetric and asymmetric vesicles. This is achieved by manufacturing microfluidic channels with a step junction, produced by double-layer photolithography, which facilitates the transfer of a W/O emulsion across an oil-water phase boundary and the self-Assembly of a phospholipid bilayer. These platforms are being used to explore the role of asymmetry in biological systems [1] and study the engineering rules that regulate membrane mediated protein-protein interactions [5]. In addition, these technologies are enabling the construction of biological machines capable of acting as micro-reactors [6], environmental sensors and smart delivery vehicles [5] as well as complex multi-compartment artificial cells where the contents and connectivity of each compartment can be controlled. These compartments are separated by biological functional membranes that can facilitate transport between the compartments themselves and between the compartments and external environment. This approach has led to the deve
Carreras P, Elani Y, Law RV, et al., 2015, A microfluidic platform for size-dependent generation of droplet interface bilayer networks on rails, Biomicrofluidics, Vol: 9, ISSN: 1932-1058
Dropletinterface 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.
Barriga HMG, Law RV, Seddon JM, et al., 2015, The effect of hydrostatic pressure on model membrane domain composition and lateral compressibility, Physical Chemistry Chemical Physics, ISSN: 1463-9084
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.
Pommella A, Brooks NJ, Seddon JM, et al., 2015, Selective flow-induced vesicle rupture to sort by membrane mechanical properties, Scientific Reports, Vol: 5, ISSN: 2045-2322
Dent MR, López-Duarte I, Dickson CJ, et al., 2015, Imaging phase separation in model lipid membranes through the use of BODIPY based molecular rotors., Phys Chem Chem Phys, Vol: 17, Pages: 18393-18402
In order to fully understand the dynamics of processes within biological lipid membranes, it is necessary to possess an intimate knowledge of the physical state and ordering of lipids within the membrane. Here we report the use of three molecular rotors based on meso-substituted boron-dipyrrin (BODIPY) in combination with fluorescence lifetime spectroscopy to investigate the viscosity and phase behaviour of model lipid bilayers. In phase-separated giant unilamellar vesicles, we visualise both liquid-ordered (Lo) and liquid-disordered (Ld) phases using fluorescence lifetime imaging microscopy (FLIM), determining their associated viscosity values, and investigate the effect of composition on the viscosity of these phases. Additionally, we use molecular dynamics simulations to investigate the orientation of the BODIPY probes within the bilayer, as well as using molecular dynamics simulations and fluorescence correlation spectroscopy (FCS) to compare diffusion coefficients with those predicted from the fluorescence lifetimes of the probes.
Purushothaman S, Cicuta P, Ces O, et al., 2015, The Influence of high pressure on the bending rigidity of model membranes, Journal of Physical Chemistry B, Vol: 119, Pages: 9805-9810, ISSN: 1520-6106
Curvature is a fundamental lipid membrane property that influences many membrane-mediated biological processes and dynamic soft materials. One of the key parameters that determines the energetics of curvature change is the membrane bending rigidity. Understanding the intrinsic effect of pressure on membrane bending is critical to understanding the adaptation and structural behavior of bio-membranes in deep-sea organisms, as well as soft material processing. However, it has not previously been possible to measure the influence of high hydrostatic pressure on membrane bending energetics and this bottleneck has primarily been due to a lack of technology platforms for performing such measurements. We have developed a new high pressure microscopy cell which, combined with vesicle fluctuation analysis, has allowed us to make the first measurements of membrane bending rigidity as a function of pressure. Our results show a significant increase in bending rigidity at pressures up to 40 MPa. Above 40 MPa, the membrane mechanics become more complex. Corresponding small and wide angle X-ray diffraction shows an increase in density and thickness of the bilayer with increasing pressure which correlates with the micro-mechanical measurements and these results are consistent with recent theoretical predictions of the bending rigidity as a function of hydrocarbon chain density. This technology has the potential to transform our quantitative understanding of the role of pressure in soft material processing, the structural behavior of bio-membranes and the adaptation mechanisms employed by deep-sea organisms.
McCarthy NLC, Ces O, Law RV, et al., 2015, Separation of liquid domains in model membranes induced with high hydrostatic pressure., Chem Commun (Camb), Vol: 51, Pages: 8675-8678
We have imaged the formation of membrane microdomains immediately after their induction using a novel technology platform coupling high hydrostatic pressure to fluorescence microscopy. After formation, the ordered domains are small and highly dynamic. This will enhance links between model lipid assemblies and dynamic processes in cellular membranes.
Barriga HMG, Parsons ES, McCarthy NLC, et al., 2015, Pressure–Temperature Phase Behavior of Mixtures of Natural Sphingomyelin and Ceramide Extracts, Langmuir, Vol: 31, Pages: 3678-3686, ISSN: 0743-7463
Elani Y, Purushothaman S, Booth PJ, et al., 2015, Measurements of the effect of membrane asymmetry on the mechanical properties of lipid bilayers, Chemical Communications, Vol: 51, Pages: 6976-6979, ISSN: 1359-7345
<p>We detail an approach for constructing asymmetric membranes and characterising their mechanical properties, leading to the first measurement of the effect of asymmetry on lipid bilayer mechanics.</p>
Tang T-YD, Brooks NJ, Ces O, et al., 2015, Structural studies of the lamellar to bicontinuous gyroid cubic (Q<sub>II</sub><SUP>G</SUP>) phase transitions under limited hydration conditions, SOFT MATTER, Vol: 11, Pages: 1991-1997, ISSN: 1744-683X
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- Citations: 9
Tyler AII, Barriga HMG, Parsons ES, et al., 2015, Electrostatic swelling of bicontinuous cubic lipid phases, Soft Matter, Vol: 11, Pages: 3279-3286, ISSN: 1744-6848
Lipid bicontinuous cubic phases have attracted enormous interest as bio-compatible scaffolds for use in a wide range of applications including membrane protein crystallisation, drug delivery and biosensing. One of the major bottlenecks that has hindered exploitation of these structures is an inability to create targeted highly swollen bicontinuous cubic structures with large and tunable pore sizes. In contrast, cubic structures found in-vivo have periodicities approaching the micron scale. We have been able to engineer and control highly swollen bicontinuous cubic phases of spacegroup Im3m containing only lipids by a) increasing the bilayer stiffness by adding cholesterol and b) inducing electrostatic repulsion across the water channels by addition of anionic lipids to monoolein. By controlling the composition of the ternary mixtures we have been able to achieve lattice parameters up to 470 Å, which is 5 times that observed in pure monoolein and nearly twice the size of any lipidic cubic phase reported previously. These lattice parameters significantly exceed the predicted maximum swelling for bicontinuous cubic lipid structures, which suggest that thermal fluctuations should destroy such phases for lattice parameters larger than 300 Å.
Barriga HMG, Tyler AII, McCarthy NLC, et al., 2015, Temperature and pressure tuneable swollen bicontinuous cubic phases approaching nature's length scales, Soft Matter, Vol: 11, Pages: 600-607, ISSN: 1744-683X
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.
Casey D, Wylie D, Gallo J, et al., 2015, A novel, all-optical tool for controllable and non-destructive poration of cells with single-micron resolution, Bio-Optics: Design and Application 2015, Publisher: Optical Society of America
We demonstrate controllable poration within ≈1 µm regions of individual cells, mediated by a near-IR laser interacting with thin-layer amorphous silicon substrates. This technique will allow new experiments in single-cell biology, particularly in neuroscience.
Karamdad K, Law RV, Seddon JM, et al., 2014, Preparation and mechanical characterisation of giant unilamellar vesicles by a microfluidic method, Lab on a Chip, Vol: 15, Pages: 557-562, ISSN: 1473-0197
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.
Brooks NJ, Seddon JM, 2014, High Pressure X-ray Studies of Lipid Membranes and Lipid Phase Transitions, Zeitschrift für Physikalische Chemie, Vol: 228, Pages: 987-1004, ISSN: 0942-9352
Hydrostatic pressure has dramatic effects on biomembrane structure and stability and is a key thermodynamic parameter in the context of the biology of deep sea organisms. Furthermore, high-pressure and pressure-jump studies are very useful tools in biophysics and biotechnology, where they can be used to study the mechanism and kinetics of lipid phase transitions, biomolecular transforma- tions, and protein folding/unfolding. Here, we first give an overview of the tech- nology currently available for X-ray scattering studies of soft matter systems under pressure. We then illustrate the use of this technology to study a variety of lipid membrane systems.
Brooks NJ, 2014, Pressure effects on lipids and bio-membrane assemblies., IUCrJ, Vol: 1, Pages: 470-477, ISSN: 2052-2525
Membranes are amongst the most important biological structures; they maintain the fundamental integrity of cells, compartmentalize regions within them and play an active role in a wide range of cellular processes. Pressure can play a key role in probing the structure and dynamics of membrane assemblies, and is also critical to the biology and adaptation of deep-sea organisms. This article presents an overview of the effect of pressure on the mesostructure of lipid membranes, bilayer organization and lipid-protein assemblies. It also summarizes recent developments in high-pressure structural instrumentation suitable for experiments on membranes.
Carreras P, Elani Y, Law R, et al., 2014, A droplet trapping microfluidic device for the study of mass-transport across droplet interface bilayers, MicroTAS 2014
Carreras P, Law RV, Brooks NJ, et al., 2014, Microfluidic generation of droplet interface bilayer networks incorporating real-time size sorting in linear and non-linear configurations, Biomicrofluidics, Vol: 8, ISSN: 1932-1058
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.
Hamid HAA, Hashim R, Seddon JM, et al., 2014, Lyotropic phase behaviour and structural parameters of monosaccharide and disaccharide guerbet branched-chain β-D-glycosides, Advanced Materials Research, Vol: 895, Pages: 111-115, ISSN: 1022-6680
The phase behaviour and self-assembly structural parameters of a pair of monosaccharide and disaccharide Guerbet branched-chain β-D-glycosides, namely 2-octyldodecyl β-D-glucoside (β-Glc- C12C8) and 2-octyldodecyl β-D-maltoside (β-Mal- C12C8), have been studied by means of optical polarizing microscopy (OPM) and small-angle X-ray diffraction at room temperature (25°C). These compounds are sugar-based glycolipid surfactants having a total chain length of C20, and differ based on the increasing number of hydroxyl groups of the sugar headgroup (glucose and maltose). The repeat spacings obtained by X-ray diffraction as a function of water content have been used to determine the limiting hydration for the two glycosides. At room temperature, β-Glc-12C8 and β- Mal- C12C8 have limiting hydrations of 22 wt% and 25 wt%, corresponding to 8 - 10 and 10 - 12 water molecules per glycoside, respectively. At all water contents between 5 and 29 wt % water, these compounds adopt inverse hexagonal (HII) or fluid lamellar (Lα) phases. The structural parameters of these phases have been determined from the diffraction data, from the X-ray repeat spacings, densities and concentration of the glycosides. © (2014) Trans Tech Publications, Switzerland.
Karamdad K, Brooks N, Ces O, 2014, Microfluidic generation of asymmetric giant unilamellar vesicles, Pages: 339-341
We have developed a novel microfluidic platform to generate asymmetric giant unilamellar vesicles (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. We have confirmed the presence of unilameller lipid bilayers in each vesicle by incorporating fluorescently-tagged lipid into the membrane bilayer.
Cook AG, Martinez-Felipe A, Brooks NJ, et al., 2013, New insights into the transitional behaviour of methyl-6-O-(<i>n</i>-dodecanoyl)-α-D-glucopyranoside using variable temperature FTIR spectroscopy and X-ray diffraction, LIQUID CRYSTALS, Vol: 40, Pages: 1817-1827, ISSN: 0267-8292
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- Citations: 27
Zahid NI, Conn CE, Brooks NJ, et al., 2013, Investigation of the Effect of Sugar Stereochemistry on Biologically Relevant Lyotropic Phases from Branched-Chain Synthetic Glycolipids by Small-Angle X-Ray Scattering, Langmuir
Synthetic branched-chain glycolipids are suitable as model systems to understand biological cell membranes, particularly since certain natural lipids possess chain branching. Herein, four branched-chain glycopyranosides namely 2-hexyl-decyl-α-D-glucopyranoside (α-Glc-OC10C6), 2-hexyl-decyl-β-D-glucopyranoside (β-Glc-OC10C6), 2-hexyl-decyl-α-D-galactopyranoside (α-Gal-OC10C6) and 2-hexyl-decyl-β-D-galactopyranoside (β-Gal-OC10C6) with a total alkyl chain length of 16 carbon atoms have been synthesized and their phase behaviour studied. The partial binary phase diagrams of these non-ionic surfactants in water were investigated by optical polarizing microscopy (OPM) and small-angle X-ray scattering (SAXS). The introduction of chain branching in the hydrocarbon chain region is shown to result in the formation of inverse structures such as the inverse hexagonal and inverse bicontinuous cubic phases. Comparison of the four compounds showed that they exhibited different polymorphism, especially in the thermotropic state, due to contributions from anomeric and epimeric effects according to their stereochemistry. The neat compound of α-Glc-OC10C6 exhibited a lamellar (Lα) phase whereas dry α-Gal-OC10C6formed an inverse bicontinuous cubic Ia3d (QIIG) phase. Both β-anomers of glucoside and galactoside adopted the inverse hexagonal phase (HII) in the dry state. Generally, in the presence of water, all four glycolipids formed inverse bicontinuous cubic Ia3d (QIIG) and Pn3m (QIID) phases over a wide temperature and concentration range. The formation of inverse non-lamellar phases by these Guerbet branched-chain glycosides confirms their potential as materials for novel biotechnological applications such as drug-delivery and crystallization of membrane proteins.
Purushothaman S, Gauthe BLLE, Brooks NJ, et al., 2013, Automated laboratory based X-ray beamline with multi-capillary sample chamber, REVIEW OF SCIENTIFIC INSTRUMENTS, Vol: 84, ISSN: 0034-6748
Zahid IN, Conn CE, Brooks NJ, et al., 2013, Effects of sugar stereochemistry on lyotropic mesophases of branched-chain synthetic glycolipids, 9th European-Biophysical-Societies-Association Congress, Publisher: SPRINGER, Pages: S132-S132, ISSN: 0175-7571
Tyler AII, Shearman GC, Parsons ES, et al., 2013, Tuning curvature in inverse micellar and bicontinuous cubic phases, 9th European-Biophysical-Societies-Association Congress, Publisher: SPRINGER, Pages: S140-S140, ISSN: 0175-7571
Johnson S, Brooks NJ, Smith RAG, et al., 2013, Structural basis for recognition of the pore-forming toxin intermedilysin by human complement receptor CD59, Cell Reports, Vol: 3
Pore-forming proteins containing the structurally conserved membrane attack complex/perforin fold play an important role in immunity and host-pathogen interactions. Intermedilysin (ILY) is an archetypal member of a cholesterol-dependent cytolysin subclass that hijacks the complement receptor CD59 to make cytotoxic pores in human cells. ILY directly competes for the membrane attack complex binding-site on CD59, rendering cells susceptible to complement lysis. To understand how these bacterial pores form in lipid bilayers and the role CD59 plays in complement regulation, we determined the crystal structure of human CD59 bound to ILY. Here we show the ILY-CD59 complex at 3.5 Å resolution and identify two interfaces mediating this hostpathogen interaction. An ILY-derived peptide based on the binding-site inhibits pore formation in a CD59-containing liposome model system. These data provide insight into how CD59 coordinates ILY monomers, nucleating an early prepore state, and suggest a potential mechanism of inhibition for the complement terminal pathway.
Tang TYD, Brooks NJ, Jeworrek C, et al., 2012, Hydrostatic Pressure Effects on the Lamellar to Gyroid Cubic Phase Transition of Monolinolein at Limited Hydration, Langmuir
Cook AG, Wardell JL, Brooks NJ, et al., 2012, Non-symmetric liquid crystal dimer containing a carbohydrate-based moiety, Carbohydrate Research, Vol: 360, Pages: 78-83
The synthesis and characterisation of a novel non-symmetric liquid crystal dimer, 1-[3-O-(D-glucopyranos-3-yl)]-8-[(4-methoxyazobenzene-40-oxy)]octane is reported. This exhibits glassy behaviour and a highly interdigitated smectic A phase in which the aromatic and alkyl structural fragments overlap. Variable temperature infrared spectroscopy reveals that the strength and extent of hydrogen bonding within the system does not show a marked change at either the glass transition or at the smectic A-isotropic transition. This observation indicates that the smectic A-isotropic transition is driven by changes in the van der Waals interactions between the molecules while hydrogen bonded aggregates persist into the isotropic phase.
Shaw KP, Brooks NJ, Clarke JA, et al., 2012, Pressure – temperature phase behaviour of natural sphingomyelin extracts, Soft Matter, Vol: 8, Pages: 1070-1078
Sphingomyelin is the only sphingolipid occurring naturally in mammalian cells and can form up to 50% of the total phospholipid content of the myelin sheath which surrounds nerves. Having predominantly long, saturated acyl chains, it has a relatively high chain melting temperature and has been strongly associated with formation of lipid microdomains. Here, the lyotropic phase behaviour of sphingomyelin from three different natural sources (bovine brain, egg yolk and milk) in excess water is studied as a function of temperature and pressure by small- and wide-angle X-ray scattering, and solid state NMR. The different hydrocarbon chain length distributions of the three lipid extracts results in significant differences in their gel phase structure; both the bovine brain and egg yolk sphingomyelins can form a ripple gel phase but milk sphingomyelin forms an interdigitated gel phase due to the high degree of chain mismatch in its longer hydrocarbon chain components.
Furse S, Brooks NJ, Seddon AM, et al., 2012, Lipid membrane curvature induced by distearoyl phosphatidylinositol 4-phosphate, Soft Matter
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