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    Elani Y, Trantidou T, Wylie D, Dekker L, Polizzi K, Law R, Ces Oet al., 2018,

    Constructing vesicle-based artificial cells with embedded living cells as organelle-like modules

    , Scientific Reports, Vol: 8, ISSN: 2045-2322

    There is increasing interest in constructing artificial cells by functionalisinglipid vesicles with biological and synthetic machinery. Due to their reduced complexity and lack of evolved biochemical pathways, the capabilities of artificial cells are limitedin comparison to their biologicalcounterparts. We show that encapsulating living cells in vesicles provides a means for artificial cells to leverage cellular biochemistry, with the encapsulated cells serving organelle-like functions as living modules inside a larger syntheticcell assembly. Using microfluidic technologies to construct such hybrid systems, we demonstrate that the vesicle host and the encapsulated cell operate in concert. The external architecture of the vesicle shields the cell from toxic surroundings, whilethe cellacts as a bioreactor module that processes encapsulated feedstock which is further processedby a synthetic enzymatic cascadeco-encapsulated in the vesicle.

    Barlow NE, Bolognesi G, Haylock S, Flemming AJ, Brooks NJ, Barter LMC, Ces Oet al., 2017,

    Rheological Droplet Interface Bilayers (rheo-DIBs): Probing the Unstirred Water Layer Effect on Membrane Permeability via Spinning Disk Induced Shear Stress

    , Scientific Reports, Vol: 7, ISSN: 2045-2322

    A new rheological droplet interface bilayer (rheo-DIB) device is presented as a tool to apply shear stress on biological lipid membranes. Despite their exciting potential for affecting high-throughput membrane translocation studies, permeability assays conducted using DIBs have neglected the effect of the unstirred water layer (UWL). However as demonstrated in this study, neglecting this phenomenon can cause significant underestimates in membrane permeability measurements which in turn limits their ability to predict key processes such as drug translocation rates across lipid membranes. With the use of the rheo-DIB chip, the effective bilayer permeability can be modulated by applying shear stress to the droplet interfaces, inducing flow parallel to the DIB membranes. By analysing the relation between the effective membrane permeability and the applied stress, both the intrinsic membrane permeability and UWL thickness can be determined for the first time using this model membrane approach, thereby unlocking the potential of DIBs for undertaking diffusion assays. The results are also validated with numerical simulations.

    Thomas JM, Friddin MS, Ces O, Elani Yet al., 2017,

    Programming membrane permeability using integrated membrane pores and blockers as molecular regulators

    , Chemical Communications, Vol: 53, Pages: 12282-12285, ISSN: 1359-7345

    We report a bottom-up synthetic biology approach to engineering vesicles with programmable permeabilities. Exploiting the concentration-dependent relationship between constitutively active pores (alpha-hemolysin) and blockers allows blockers to behave as molecular regulators for tuning permeability, enabling us to systematically modulate cargo release kinetics without changing the lipid fabric of the system.

    de Bruin A, Friddin MS, Elani Y, Brooks N, Law R, Seddon J, Ces Oet al., 2017,

    A transparent 3D printed device for assembling droplet hydrogel bilayers (DHBs)

    , RSC Advances, Vol: 7, Pages: 47796-47800, ISSN: 2046-2069

    We report a new approach for assembling droplet hydrogel bilayers (DHBs) using a transparent 3D printed device. We characterise the transparency of our platform, confirm bilayer formation using electrical measurements and show that single-channel recordings can be obtained using our reusable rapid prototyped device. This method significantly reduces the cost and infrastructure required to develop devices for DHB assembly and downstream study.

    Trantidou T, Friddin M, Elani Y, Brooks NJ, Law RV, Seddon JM, Ces Oet al., 2017,

    Engineering compartmentalized biomimetic micro- and nanocontainers

    , ACS Nano, Vol: 11, Pages: 6549-6565, ISSN: 1936-086X

    Compartmentalization of biological content and function is a key architectural feature in biology, where membrane bound micro- and nanocompartments are used for performing a host of highly specialized and tightly regulated biological functions. The benefit of compartmentalization as a design principle is behind its ubiquity in cells and has led to it being a central engineering theme in construction of artificial cell-like systems. In this review, we discuss the attractions of designing compartmentalized membrane-bound constructs and review a range of biomimetic membrane architectures that span length scales, focusing on lipid-based structures but also addressing polymer-based and hybrid approaches. These include nested vesicles, multicompartment vesicles, large-scale vesicle networks, as well as droplet interface bilayers, and double-emulsion multiphase systems (multisomes). We outline key examples of how such structures have been functionalized with biological and synthetic machinery, for example, to manufacture and deliver drugs and metabolic compounds, to replicate intracellular signaling cascades, and to demonstrate collective behaviors as minimal tissue constructs. Particular emphasis is placed on the applications of these architectures and the state-of-the-art microfluidic engineering required to fabricate, functionalize, and precisely assemble them. Finally, we outline the future directions of these technologies and highlight how they could be applied to engineer the next generation of cell models, therapeutic agents, and microreactors, together with the diverse applications in the emerging field of bottom-up synthetic biology.

    Salehi-Reyhani A, Ces O, Elani Y, 2017,

    Artificial cell mimics as simplified models for the study of cell biology

    , Experimental Biology and Medicine, Vol: 242, Pages: 1309-1317, ISSN: 1535-3702

    Living cells are hugely complex chemical systems composed of a milieu of distinct chemical species (including DNA, proteins, lipids, and metabolites) interconnected with one another through a vast web of interactions: this complexity renders the study of cell biology in a quantitative and systematic manner a difficult task. There has been an increasing drive towards the utilization of artificial cells as cell mimics to alleviate this, a development that has been aided by recent advances in artificial cell construction. Cell mimics are simplified cell-like structures, composed from the bottom-up with precisely defined and tunable compositions. They allow specific facets of cell biology to be studied in isolation, in a simplified environment where control of variables can be achieved without interference from a living and responsive cell. This mini-review outlines the core principles of this approach and surveys recent key investigations that use cell mimics to address a wide range of biological questions. It will also place the field in the context of emerging trends, discuss the associated limitations, and outline future directions of the field.

    Trantidou T, Elani Y, Parsons E, Ces Oet al., 2017,

    Hydrophilic surface modification of PDMS for droplet microfluidics using a simple, quick, and robust method via PVA deposition

    , Microsystems and Nanoengineering, Vol: 3, ISSN: 2055-7434

    Polydimethylsiloxane (PDMS) is a dominant material in the fabrication of microfluidic devices to generate water-in-oil droplets, particularly lipid-stabilized droplets, because of its highly hydrophobic nature. However, its key property of hydrophobicity has hindered its use in the microfluidic generation of oil-in-water droplets, which requires channels to have hydrophilic surface properties. In this article, we developed, optimized, and characterized a method to produce PDMS with a hydrophilic surface via the deposition of polyvinyl alcohol following plasma treatment and demonstrated its suitability for droplet generation. The proposed method is simple, quick, effective, and low cost and is versatile with respect to surfactants, with droplets being successfully generated using both anionic surfactants and more biologically relevant phospholipids. This method also allows the device to be selectively patterned with both hydrophilic and hydrophobic regions, leading to the generation of double emulsions and inverted double emulsions.

    Barlow NE, Smpokou E, Friddin MS, Macey R, Gould I, Turnbull C, Flemming AJ, Brooks NJ, Ces O, Barter LMCet al., 2017,

    Engineering plant membranes using droplet interface bilayers

    , Biomicrofluidics, Vol: 11, ISSN: 1932-1058

    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.

    Barlow NE, Bolognesi G, Flemming AJ, Brooks N, Barter LMC, Ces Oet al., 2016,

    Multiplexed droplet Interface bilayer formation

    , Lab on a Chip, Vol: 16, Pages: 4653-4657, ISSN: 1473-0197

    We present a simple method for the multiplexed formation ofdroplet interface bilayers (DIBs) using a mechanically operatedlinear acrylic chamber array. To demonstrate the functionality ofthe chip design, a lipid membrane permeability assay is performed.We show that multiple, symmetric DIBs can be created andseparated using this robust low-cost approach.

    Chan CL, Bolognesi G, Bhandarkar A, Friddin M, Brooks NJ, Seddon J, Law R, Barter L, Ceset al., 2016,

    DROPLAY: laser writing of functional patterns within biological microdroplet displays

    , Lab on a Chip, Vol: 16, Pages: 4621-4627, ISSN: 1473-0197

    In this study, we introduce an optofluidic method for the rapid construction of large-area cell-sized droplet assemblieswith user-defined re-writable two-dimensional patterns of functional droplets. Light responsive water-in-oil dropletscapable of releasing fluorescent dye molecules upon exposure were generated and self-assembled into arrays in amicrofluidic device. This biological architecture was exploited by the scanning laser of a confocal microscope to ‘write’ userdefined patterns of differentiated (fluorescent) droplets in a network of originally undifferentiated (non-fluorescent)droplets. As a result, long lasting images were produced on a droplet fabric with droplets acting as pixels of a biologicalmonitor, which can be erased and re-written on-demand. Regio-specific light-induced droplet differentiation within a largepopulation of droplets provides a new paradigm for the rapid construction of bio-synthetic systems with potential as tissuemimics and biological display materials.

    Friddin MS, Bolognesi G, Elani Y, Brooks N, Law R, Seddon J, Neil M, ces Oet al., 2016,

    Optically assembled droplet interface bilayer (OptiDIB) networks from cell-sized microdroplets

    , Soft Matter, Vol: 12, Pages: 7731-7734, ISSN: 1744-6848

    We report a new platform technology to systematically assemble droplet interface bilayer (DIB) networks in user-defined 3D architectures from cell-sized droplets using optical tweezers. Our OptiDIB platform is the first demonstration of optical trapping to precisely construct 3D DIB networks, paving the way for the development of a new generation of modular bio-systems.

    Friddin MS, Bolognesi G, Elani Y, Brooks N, Law R, Seddon J, Neil M, Ces Oet al.,

    The optical assembly of bilayer networks from cell-sized droplets for synthetic biology

    , Systems and Synthetic Biology
    Elani Y, 2016,

    Construction of membrane-bound artificial cells using microfluidics: a new frontier in bottom-up synthetic biology

    , Biochemical Society Transactions, Vol: 44, Pages: 723-730, ISSN: 1470-8752

    The quest to construct artificial cells from the bottom-up using simple building blocks has received much attention over recent decades and is one of the grand challenges in synthetic biology. Cell mimics that are encapsulated by lipid membranes are a particularly powerful class of artificial cells due to their biocompatibility and the ability to reconstitute biological machinery within them. One of the key obstacles in the field centres on the following: how can membrane-based artificial cells be generated in a controlled way and in high-throughput? In particular, how can they be constructed to have precisely defined parameters including size, biomolecular composition and spatial organization? Microfluidic generation strategies have proved instrumental in addressing these questions. This article will outline some of the major principles underpinning membrane-based artificial cells and their construction using microfluidics, and will detail some recent landmarks that have been achieved.

    Friddin MS, Bolognesi G, Elani Y, Brooks N, Law R, Seddon J, Neil M, Ces Oet al.,

    Optical tweezers to assemble 2D and 3D droplet interface bilayer networks from cell-sized droplets

    , EMBL Microfluidics
    Poulos AS, Nania M, Lapham P, Miller RM, Smith AJ, Tantawy H, Caragay J, Gummel J, Ces O, Robles ESJ, Cabral JTet al., 2016,

    Microfluidic SAXS Study of Lamellar and Multilamellar Vesicle Phases of Linear Sodium Alkylbenzenesulfonate Surfactant with Intrinsic Isomeric Distribution

    , Langmuir, Vol: 32, Pages: 5852-5861, ISSN: 1520-5827

    The structure and flow behavior of a concentrated aqueous solution (45 wt %) of the ubiquitous linear sodium alkylbenzenesulfonate (NaLAS) surfactant is investigated by microfluidic small-angle X-ray scattering (SAXS) at 70 °C. NaLAS is an intrinsically complex mixture of over 20 surfactant molecules, presenting coexisting micellar (L1) and lamellar (Lα) phases. Novel microfluidic devices were fabricated to ensure pressure and thermal resistance, ability to handle viscous fluids, and low SAXS background. Polarized light optical microscopy showed that the NaLAS solution exhibits wall slip in microchannels, with velocity profiles approaching plug flow. Microfluidic SAXS demonstrated the structural spatial heterogeneity of the system with a characteristic length scale of 50 nL. Using a statistical flow–SAXS analysis, we identified the micellar phase and multiple coexisting lamellar phases with a continuous distribution of d spacings between 37.5 and 39.5 Å. Additionally, we showed that the orientation of NaLAS lamellar phases is strongly affected by a single microfluidic constriction. The bilayers align parallel to the velocity field upon entering a constriction and perpendicular to it upon exiting. On the other hand, multilamellar vesicle phases are not affected under the same flow conditions. Our results demonstrate that despite the compositional complexity inherent to NaLAS, microfluidic SAXS can rigorously elucidate its structure and flow response.

    Elani Y, Solvas XC, Edel JB, Law RV, Ces Oet al., 2016,

    Microfluidic generation of encapsulated droplet interface bilayer networks (multisomes) and their use as cell-like reactors.

    , Chemical Communications, Vol: 52, Pages: 5961-5964, ISSN: 1364-548X

    Compartmentalised structures based on droplet interface bilayers (DIBs), including multisomes and compartmentalised vesicles, are seen by many as the next generation of biomimetic soft matter devices. Herein, we outline a microfluidic approach for the construction of miniaturised multisomes of pL volumes in high-throughput and demonstrate their potential as vehicles for in situ chemical synthesis.

    Miller RM, Poulos AS, Robles ESJ, Brooks NJ, Ces O, Cabral JTet al., 2016,

    Isothermal Crystallization Kinetics of Sodium Dodecyl Sulfate–Water Micellar Solutions

    , Crystal Growth & Design, Vol: 16, Pages: 3379-3388, ISSN: 1528-7505

    The crystallization mechanisms and kinetics of micellar sodium dodecyl sulfate (SDS) solutions in water, under isothermal conditions, were investigated experimentally by a combination of reflection optical microscopy (OM), differential scanning calorimetry (DSC), and attenuated total reflection Fourier transform infrared spectroscopy (ATR-FTIR). The rates of nucleation and growth were estimated from OM and DSC across temperatures ranging from 20 to −6 °C for 20% SDS-H2O, as well as for 10 and 30% SDS-H2O at representative temperatures of 6, 2, and −2 °C. A decrease in temperature increased both nucleation and growth rates, and the combined effect of the two processes on the morphology was quantified via both OM and ATR-FTIR. Needles, corresponding to the hemihydrate polymorph, become the dominant crystal form at ≤ −2 °C, while platelets, the monohydrate, predominate at higher temperatures. Above 8 °C, crystallization was only observed if seeded from crystals generated at lower temperatures. Our results provide quantitative and morphological insight into the crystallization of ubiquitous micellar SDS solutions and its phase stability below room temperature.

    Friddin MS, Bolognesi G, Elani Y, Brooks NJ, Law RV, Seddon JM, Neil MAA, Ces Oet al., 2016,

    Light-driven drag and drop assembly of micron-scale bilayer networks for synthetic biology

    , Pages: 545-546

    We have developed a new method to assemble single- or multi-layered networks of droplet interface bilayers (DIBs) from cell-sized droplets using a single beam optical trap (optical tweezers). The novelty of our approach is the ability to directly trap the microdroplets with the laser and manipulate them in 3D to construct DIB networks of user-defined architectures. Our method does not require a complex optical setup, is versatile, contactless, benefits from both high spatial and temporal resolution, and could set a new paradigm for the assembly of smart, synthetic biosystems.

    Carreras P, Elani Y, Law RV, Brooks NJ, Seddon JM, Ces Oet 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, Ces O, Brooks NJet 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.

    Findlay H, Purushothaman S, Ces O, Booth Pet al., 2015,

    Secondary transporter structure and function in synthetic lipid bilayer systems

    , 29th Annual Symposium of the Protein-Society, Publisher: WILEY-BLACKWELL, Pages: 50-51, ISSN: 0961-8368
    McDonald C, Jovanovic G, Ces O, Buck Met al., 2015,

    Membrane Stored Curvature Elastic Stress Modulates Recruitment of Maintenance Proteins PspA and Vipp1

    , mBio, Vol: 6, Pages: e01188-15-e01188-15, ISSN: 2161-2129

    Phage shock protein A (PspA), which is responsible for maintaining inner membrane integrity under stress in enterobacteria, and vesicle-inducting protein in plastids 1 (Vipp1), which functions for membrane maintenance and thylakoid biogenesis in cyanobacteria and plants, are similar peripheral membrane-binding proteins. Their homologous N-terminal amphipathic helices are required for membrane binding; however, the membrane features recognized and required for expressing their functionalities have remained largely uncharacterized. Rigorously controlled, in vitro methodologies with lipid vesicles and purified proteins were used in this study and provided the first biochemical and biophysical characterizations of membrane binding by PspA and Vipp1. Both proteins are found to sense stored curvature elastic (SCE) stress and anionic lipids within the membrane. PspA has an enhanced sensitivity for SCE stress and a higher affinity for the membrane than Vipp1. These variations in binding may be crucial for some of the proteins’ differing roles in vivo. Assays probing the transcriptional regulatory function of PspA in the presence of vesicles showed that a relief of transcription inhibition occurs in an SCE stress-specific manner. This in vitro recapitulation of membrane stress-dependent transcription control suggests that the Psp response may be mounted in vivo when a cell’s inner membrane experiences increased SCE stress.

    Yeh JS-M, Sennoga CA, McConnell E, Eckersley R, Tang M-X, Nourshargh S, Seddon JM, Haskard DO, Nihoyannopoulos Pet al., 2015,


    , ULTRASOUND IN MEDICINE AND BIOLOGY, Vol: 41, Pages: 2478-2496, ISSN: 0301-5629
    Ja'afar F, Leow CH, Garbin V, Sennoga CA, Tang M-X, Seddon JMet al., 2015,

    Surface Charge Measurement of SonoVue, Definity and Optison: A Comparison of Laser Doppler Electrophoresis and Micro-Electrophoresis

    , Ultrasound in Medicine and Biology, Vol: 41, Pages: 2990-3000, ISSN: 0301-5629

    Microbubble (MB) contrast-enhanced ultrasonography is a promising tool for targeted molecular imaging. It is important to determine the MB surface charge accurately as it affects the MB interactions with cell membranes. In this article, we report the surface charge measurement of SonoVue, Definity and Optison. We compare the performance of the widely used laser Doppler electrophoresis with an in-house micro-electrophoresis system. By optically tracking MB electrophoretic velocity in a microchannel, we determined the zeta potentials of MB samples. Using micro-electrophoresis, we obtained zeta potential values for SonoVue, Definity and Optison of −28.3, −4.2 and −9.5 mV, with relative standard deviations of 5%, 48% and 8%, respectively. In comparison, laser Doppler electrophoresis gave −8.7, +0.7 and +15.8 mV with relative standard deviations of 330%, 29,000% and 130%, respectively. We found that the reliability of laser Doppler electrophoresis is compromised by MB buoyancy. Micro-electrophoresis determined zeta potential values with a 10-fold improvement in relative standard deviation.

    Pommella A, Brooks NJ, Seddon JM, Garbin Vet al., 2015,

    Selective flow-induced vesicle rupture to sort by membrane mechanical properties

    , Scientific Reports, Vol: 5, ISSN: 2045-2322
    Gaffney PRJ, ces O, arduin A, 2015,

    Regulation of PLCβ2 by the electrostatic and mechanical properties of lipid bilayers

    , Scientific Reports, Vol: 5, ISSN: 2045-2322

    Phosphoinositide-specific phospholipase C (PLC) is an important family of enzymes constituting a junction between phosphoinositide lipid signaling and the trans-membrane signal transduction processes that are crucial to many living cells. However, the regulatory mechanism of PLC is not yet understood in detail. To address this issue, activity studies were carried out using lipid vesicles in a model system that was specifically designed to study protein-protein and lipid-protein interactions in concert. Evidence was found for a direct interaction between PLC and the GTPases that mediate phospholipase activation. Furthermore, for the first time, the relationships between PLC activity and substrate presentation in lipid vesicles of various sizes, as well as lipid composition and membrane mechanical properties, were analyzed. PLC activity was found to depend upon the electrostatic potential and the stored curvature elastic stress of the lipid membranes.

    Dent MR, López-Duarte I, Dickson CJ, Geoghegan ND, Cooper JM, Gould IR, Krams R, Bull JA, Brooks NJ, Kuimova MKet 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.

    Yeh JS-M, Sennoga CA, McConnell E, Eckersley R, Tang M-X, Nourshargh S, Seddon JM, Haskard DO, Nihoyannopoulos Pet al., 2015,

    A targeting microbubble for ultrasound molecular imaging

    , PLoS ONE, Vol: 10, ISSN: 1932-6203
    Purushothaman S, Cicuta P, Ces O, Brooks NJet 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, Seddon JM, Brooks NJet 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.

    Elani Y, Law RV, Ces O, 2015,

    Vesicle-based artificial cells: recent developments and prospects for drug delivery

    , THERAPEUTIC DELIVERY, Vol: 6, Pages: 541-543, ISSN: 2041-5990
    Barriga HMG, Parsons ES, McCarthy NLC, Ces O, Seddon JM, Law RV, Brooks NJet al., 2015,

    Pressure–Temperature Phase Behavior of Mixtures of Natural Sphingomyelin and Ceramide Extracts

    , Langmuir, Vol: 31, Pages: 3678-3686, ISSN: 0743-7463
    Barriga HMG, Bazin R, Templer RH, Law RV, Ces Oet al., 2015,

    Buffer-Induced Swelling and Vesicle Budding in Binary Lipid Mixtures of Dioleoylphosphatidylcholine:Dioleoylphosphatidylethanolamine and Dioleoylphosphatidylcholine:Lysophosphatidylcholine Using Small-Angle X-ray Scattering and P-31 Static NMR

    , LANGMUIR, Vol: 31, Pages: 2979-2987, ISSN: 0743-7463
    Tang T-YD, Brooks NJ, Ces O, Seddon JM, Templer RHet al., 2015,

    Structural studies of the lamellar to bicontinuous gyroid cubic (Q(II)(G)) phase transitions under limited hydration conditions

    , SOFT MATTER, Vol: 11, Pages: 1991-1997, ISSN: 1744-683X
    Tyler AII, Barriga HMG, Parsons ES, McCarthy, Ces, Law RV, Seddon JM, Brooks NJet 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.

    Salehi-Reyhani A, Gesellchen F, Mampallil D, Wilson R, Reboud J, Ces O, Willison KR, Cooper JM, Klug DRet al., 2015,

    Chemical-Free Lysis and Fractionation of Cells by Use of Surface Acoustic Waves for Sensitive Protein Assays

    , ANALYTICAL CHEMISTRY, Vol: 87, Pages: 2161-2169, ISSN: 0003-2700
    Elani Y, Law R, Ces O,

    Vesicle-based artificial cells: recent developments and prospects for drug delivery

    , Therapeutic delivery, ISSN: 2041-6008
    Barriga HMG, Tyler AII, McCarthy NLC, Parsons ES, Ces O, Law RV, Seddon JM, Brooks NJet 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
    Bolognesi G, Hargreaves A, Ward AD, Kirby AK, Neil M, Bain CD, Ces Oet al., 2015,

    Microfluidic generation and optical manipulation of ultra-low interfacial tension droplets

    , Conference on Integrated Photonics - Materials, Devices, and Applications III, Publisher: SPIE-INT SOC OPTICAL ENGINEERING, ISSN: 0277-786X
    Elani Y, Law RV, Ces O, 2015,

    Protein synthesis in artificial cells: using compartmentalisation for spatial organisation in vesicle bioreactors

    , PHYSICAL CHEMISTRY CHEMICAL PHYSICS, Vol: 17, Pages: 15534-15537, ISSN: 1463-9076
    Elani Y, Purushothaman S, Booth PJ, Seddon JM, Brooks NJ, Law RV, Ces Oet 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
    Furse S, Mak L, Tate EW, Templer RH, Ces O, Woscholski R, Gaffney PRJet al., 2015,

    Synthesis of unsaturated phosphatidylinositol 4-phosphates and the effects of substrate unsaturation on SopB phosphatase activity

    , ORGANIC & BIOMOLECULAR CHEMISTRY, Vol: 13, Pages: 2001-2011, ISSN: 1477-0520
    Zhang Y, Lervik A, Seddon J, Bresme Fet al., 2015,

    A coarse-grained molecular dynamics investigation of the phase behavior of DPPC/cholesterol mixtures

    , CHEMISTRY AND PHYSICS OF LIPIDS, Vol: 185, Pages: 88-98, ISSN: 0009-3084
    Tyler AII, Clarke JA, Seddon JM, Law RVet al., 2015,

    Solid state NMR of lipid model membranes.

    , Methods Mol Biol, Vol: 1232, Pages: 227-253

    In this chapter we describe the use of solid state nuclear magnetic spectroscopy to study the structure of lyotropic phases and lipid model membranes and show its ability to probe, site specifically, at a sub-Ångstrom resolution. Here, we demonstrate the immense versatility of the technique and its ability to provide information on the different liquid crystalline phases present. A multinuclear for example (31)P, (1)H, and (13)C approach is able to elucidate both the structure and dynamics over a wide variety of timescales. This coupled with a non-perturbing label (2)H is able to provide information such as the order parameters for a wide variety of different liquid phases.

    Casey D, Wylie D, Gallo J, Dent M, Salehi-Reyhani A, Wilson R, Brooks N, Long N, Willison K, Klug D, Neil M, Neale S, Cooper J, Ces Oet 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.

    Tyler AII, Law RV, Seddon JM, 2015,

    X-ray diffraction of lipid model membranes.

    , Methods Mol Biol, Vol: 1232, Pages: 199-225

    In this chapter the use of X-ray diffraction to study the structure of lyotropic phases and lipid model membranes is described. Determination of the phase symmetry and lattice parameters from small-angle X-ray scattering (SAXS), and of the nature of the hydrocarbon chain packing from wide-angle X-ray scattering (WAXS), are discussed. Methods by which the sign of the interfacial curvature of non-lamellar phases may be determined are then presented. Finally, the calculation of electron density profiles from the intensities of the observed Bragg peaks is described, for the lamellar phase and for the inverse hexagonal phase.

    Bolognesi G, Hargreaves A, Ward AD, Kirby AK, Bain CD, Ces Oet al., 2014,

    Microfluidic generation of monodisperse ultra-low interfacial tension oil droplets in water

    , RSC Advances, Vol: 5, Pages: 8114-8121, ISSN: 2046-2069

    We present a novel microfluidic approach for the generation of monodisperse oil droplets in water with interfacial tensions of the order of 1 μN m−1. Using an oil-in-water emulsion containing the surfactant aerosol OT, heptane, water and sodium chloride under conditions close to the microemulsion phase transition, we actively controlled the surface tension at the liquid–liquid interface within the microfluidic device in order to produce monodisperse droplets. These droplets exhibited high levels of stability with respect to rupture and coalescence rates. Confirmation that the resultant emulsions were in the ultra-low tension regime was determined using real space detection of thermally-induced capillary waves at the droplet interface.

    Karamdad K, Law RV, Seddon JM, Brooks NJ, Ces Oet al., 2014,

    Preparation and mechanical characterisation of giant unilamellar vesicles by a microfluidic method

    , Lab on a Chip, 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.

    Salehi-Reyhani A, Burgin E, Ces O, Willison KR, Klug DRet al., 2014,

    Addressable droplet microarrays for single cell protein analysis

    , ANALYST, Vol: 139, Pages: 5367-5374, ISSN: 0003-2654

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