170 results found
Gispert Contamina I, Hindley J, Pilkington C, et al., 2022, Stimuli-responsive vesicles as distributed artificial organelles for bacterial activation, Proceedings of the National Academy of Sciences of USA, Vol: 119, Pages: 1-10, ISSN: 0027-8424
Intercellular communication is a hallmark of living systems. As such, engineering artificial cells that possess this behavior has been at the heart of activities in bottom-up synthetic biology. Communication between artificial and living cells has potential to confer novel capabilities to living organisms that could be exploited in biomedicine and biotechnology. However, most current approaches rely on the exchange of chemical signals that cannot be externally controlled. Here, we report two types of remote-controlled vesicle-based artificial organelles that translate physical inputs into chemical messages that lead to bacterial activation. Upon light or temperature stimulation, artificial cell membranes are activated, releasing signaling molecules that induce protein expression in Escherichia coli. This distributed approach differs from established methods for engineering stimuli-responsive bacteria. Here, artificial cells (as opposed to bacterial cells themselves) are the design unit. Having stimuli-responsive elements compartmentalized in artificial cells has potential applications in therapeutics, tissue engineering, and bioremediation. It will underpin the design of hybrid living/nonliving systems where temporal control over population interactions can be exerted.
Allen ME, Hindley JW, Baxani DK, et al., 2022, Hydrogels as functional components in artificial cell systems, Nature Reviews Chemistry, Vol: 6, Pages: 562-578, ISSN: 2397-3358
Recent years have seen substantial efforts aimed at constructing artificial cells from various molecular components with the aim of mimicking the processes, behaviours and architectures found in biological systems. Artificial cell development ultimately aims to produce model constructs that progress our understanding of biology, as well as forming the basis for functional bio-inspired devices that can be used in fields such as therapeutic delivery, biosensing, cell therapy and bioremediation. Typically, artificial cells rely on a bilayer membrane chassis and have fluid aqueous interiors to mimic biological cells. However, a desire to more accurately replicate the gel-like properties of intracellular and extracellular biological environments has driven increasing efforts to build cell mimics based on hydrogels. This has enabled researchers to exploit some of the unique functional properties of hydrogels that have seen them deployed in fields such as tissue engineering, biomaterials and drug delivery. In this Review, we explore how hydrogels can be leveraged in the context of artificial cell development. We also discuss how hydrogels can potentially be incorporated within the next generation of artificial cells to engineer improved biological mimics and functional microsystems.
Zubaite G, Hindley JW, Ces O, et al., 2022, Dynamic reconfiguration of subcompartment architectures in artificial cells., ACS Nano, Vol: 16, ISSN: 1936-0851
Artificial cells are minimal structures constructed from biomolecular building blocks designed to mimic cellular processes, behaviors, and architectures. One near-ubiquitous feature of cellular life is the spatial organization of internal content. We know from biology that organization of content (including in membrane-bound organelles) is linked to cellular functions and that this feature is dynamic: the presence, location, and degree of compartmentalization changes over time. Vesicle-based artificial cells, however, are not currently able to mimic this fundamental cellular property. Here, we describe an artificial cell design strategy that addresses this technological bottleneck. We create a series of artificial cell architectures which possess multicompartment assemblies localized either on the inner or on the outer surface of the artificial cell membrane. Exploiting liquid-liquid phase separation, we can also engineer spatially segregated regions of condensed subcompartments attached to the cell surface, aligning with coexisting membrane domains. These structures can sense changes in environmental conditions and respond by reversibly transitioning from condensed multicompartment layers on the membrane surface to a dispersed state in the cell lumen, mimicking the dynamic compartmentalization found in biological cells. Likewise, we engineer exosome-like subcompartments that can be released to the environment. We can achieve this by using two types of triggers: chemical (addition of salts) and mechanical (by pulling membrane tethers using optical traps). These approaches allow us to control the compartmentalization state of artificial cells on population and single-cell levels.
Strutt R, Sheffield F, Barlow N, et al., 2022, UV-DIB: label-free permeability determination using droplet interface bilayers, Lab on a Chip: miniaturisation for chemistry, physics, biology, materials science and bioengineering, Vol: 22, Pages: 972-985, ISSN: 1473-0189
Simple diffusion of molecular entities through a phospholipid bilayer, is a phenomenon of great importance to the pharmaceutical and agricultural industries. Current model lipid systems to probe this typically only employ fluorescence as a readout, thus limiting the range of assessable chemical matter that can be studied. We report a new technology platform, the UV-DIB, which facilitates label free measurement of small molecule translocation rates. This is based upon the coupling of droplet interface bilayer technology with implemented fiber optics to facilitate analysis via ultraviolet spectroscopy, in custom designed PMMA wells. To improve on current DIB technology, the platform was designed to be reusable, with a high sampling rate and a limit of UV detection in the low μM regime. We demonstrate the use of our system to quantify passive diffusion in a reproducible and rapid manner where the system was validated by investigating multiple permeants of varying physicochemical properties across a range of lipid interfaces, each demonstrating differing kinetics. Our system permits the interrogation of structural dependence on the permeation rate of a given compound. We present this ability from two structural perspectives, that of the membrane, and the permeant. We observed a reduction in permeability between pure DOPC and DPhPC interfaces, concurring with literature and demonstrating our ability to study the effects of lipid composition on permeability. In relation to the effects of permeant structure, our device facilitated the rank ordering of various compounds from the xanthine class of compounds, where the structure of each permeant differed by a single group alteration. We found that DIBs were stable up to 5% DMSO, a molecule often used to aid solubilisation of pharmaceutical and agrochemical compounds. The ability of our device to rank-order compounds with such minor structural differences provides a level of precision that is rarely seen in current, industr
Abdel Aty H, Strutt R, Mcintyre N, et al., 2022, Machine learning platform for determining experimental lipid phase behaviour from small angle X-ray scattering patterns by pre-training on synthetic data, Digital Discovery, Vol: 1, Pages: 98-107
Lipid membranes are vital in a wide range of biological and biotechnical systems; they undepin functions from modulation of protein activity to drug uptake and delivery. Understanding the structure, interactions, self-assembly and phase behaviour of lipids is critical to developing a molecular undertanding of biological membrane mediated processes, establishing engineering approaches to biotechnical membrane application development. Small Angle X-ray Scattering (SAXS) is the de facto method used to analyse the structure of self-assembled lipid systems. The resultant diffraction patterns are however extremely difficult to assign automatically with researchers spending considerable time often analysing patterns ex situ from a beamline facility, reducing experimental capacity and optimisation. Furthermore, research projects will often focus on particular lipid compositions and thus would benefit significantly from a method which can be rapidly optimised for a range of samples of interest. We present a generalisable machine learning pipeline that is able to classify lipid phases based on their raw, experimental SAXS spectra, with >99% accuracy and an inference time of <60 ms, enabling high throughput on-site analysis. We achieved this through application of a synthetic data generation system, capable of building synthetic SAXS patterns from the underlying physics which dictate phase behaviour, and we also propose an extension of our system to synthetically generate co-existence phase spectra with known composition ratios. Pre-training our machine learning model on this synthetic data, and fine-tuning on experimental samples empowers the model in achieving state-of-the-art, rapid lipid phase classification, allowing researchers to be able to adapt their experiments on site if needed and hence massively accelerate high throughput lipid research.
Molisso S, Williams DR, Ces O, et al., 2021, Molecular interaction and partitioning in α-Keratin using 1H NMR Spin-Lattice (T1) relaxation times, Journal of the Royal Society Interface, Vol: 18, Pages: 1-8, ISSN: 1742-5662
The interactions between small molecules and keratins are poorly understood. In this paper an NMR method is presented to measure changes in the 1H T1 relaxation times of small molecules in human hair keratin to quantify their interaction with the fiber. Two populations of small molecule compounds were identified with distinct relaxation times, demonstrating the partitioning of the compounds into different keratin environments. The changes in relaxation time for solvent in hair compared to bulk solvent were shown to be related to the molecular weight, MW, and the partition coefficient, LogP, of the solvent investigated. Compounds with low molecular weights and high hydrophilicities had greater reductions in their T1 relaxation times and therefore experienced increased interactions with the hair fiber. The relative population sizes were also calculated. This is a significant step toward modelling the behavior of small molecules in keratinous materials and other large insoluble fibrous proteins.
Mora NL, Findlay HE, Brooks NJ, et al., 2021, The membrane transporter lactose permease increases lipid bilayer bending rigidity, BIOPHYSICAL JOURNAL, Vol: 120, Pages: 3787-3794, ISSN: 0006-3495
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Seligman H, Zaman S, Pitcher DS, et al., 2021, Reusable snorkel masks adapted as particulate respirators, PLoS One, Vol: 16, Pages: 1-11, ISSN: 1932-6203
ntroductionDuring viral pandemics, filtering facepiece (FFP) masks together with eye protection form the essential components of personal protective equipment (PPE) for healthcare workers. There remain concerns regarding insufficient global supply and imperfect protection offered by currently available PPE strategies. A range of full-face snorkel masks were adapted to accept high grade medical respiratory filters using bespoke-designed 3D-printed connectors. We compared the protection offered by the snorkel to that of standard PPE using a placebo-controlled respirator filtering test as well as a fluorescent droplet deposition experiment. Out of the 56 subjects tested, 42 (75%) passed filtering testing with the snorkel mask compared to 31 (55%) with a FFP3 respirator mask (p = 0.003). Amongst the 43 subjects who were not excluded following a placebo control, 85% passed filtering testing with the snorkel versus to 68% with a FFP3 mask (p = 0.008). Following front and lateral spray of fluorescence liquid particles, the snorkel mask also provided superior protection against droplet deposition within the subject’s face, when compared to a standard PPE combination of FFP3 masks and eye protection (3.19x108 versus 6.81x108 fluorescence units, p<0.001). The 3D printable adaptors are available for free download online at https://www.ImperialHackspace.com/COVID-19-Snorkel-Respirator-Project/.ConclusionFull-face snorkel masks adapted as particulate respirators performed better than a standard PPE combination of FFP3 mask and eye protection against aerosol inhalation and droplet deposition. This adaptation is therefore a promising PPE solution for healthcare workers during highly contagious viral outbreaks.
Zhang S, Contini C, Hindley J, et al., 2021, Engineering motile aqueous phase-separated droplets via liposome stabilisation, Nature Communications, Vol: 12, Pages: 1-11, ISSN: 2041-1723
There are increasing efforts to engineer functional compartments that mimic cellular behaviours from the bottom-up. One behaviour that is receiving particular attention is motility, due to its biotechnological potential and ubiquity in living systems. Many existing platforms make use of the Marangoni effect to achieve motion in water/oil (w/o) droplet systems. However, most of these systems are unsuitable for biological applications due to biocompatibility issues caused by the presence of oil phases. Here we report a biocompatible all aqueous (w/w) PEG/dextran Pickering-like emulsion system consisting of liposome-stabilised cell-sized droplets, where the stability can be easily tuned by adjusting liposome composition and concentration. We demonstrate that the compartments are capable of negative chemotaxis: these droplets can respond to a PEG/dextran polymer gradient through directional motion down to the gradient. The biocompatibility, motility and partitioning abilities of this droplet system offers new directions to pursue research in motion-related biological processes.
Strutt R, Hindley JW, Gregg J, et al., 2021, Activating mechanosensitive channels embedded in droplet interface bilayers using membrane asymmetry, Chemical Science, Vol: 12, Pages: 2138-2145, ISSN: 2041-6520
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.
Heide C, Buldum G, Moya-Ramirez I, et al., 2021, Design, development and optimisation of a functional mammalian cell-free protein synthesis platform, Frontiers in Bioengineering and Biotechnology, Vol: 8, ISSN: 2296-4185
In this paper, we describe the stepwise development of a cell-free protein synthesis (CFPS) platform derived from cultured Chinese hamster ovary (CHO) cells. We provide a retrospective summary of the design challenges we faced, and the optimized methods developed for the cultivation of cells and the preparation of translationally active lysates. To overcome low yields, we developed procedures to supplement two accessory proteins, GADD34 and K3L, into the reaction to prevent deactivation of the translational machinery by phosphorylation. We compared different strategies for implementing these accessory proteins including two variants of the GADD34 protein to understand the potential trade-offs between yield and ease of implementation. Addition of the accessory proteins increased yield of turbo Green Fluorescent Protein (tGFP) by up to 100-fold depending on which workflow was used. Using our optimized protocols as a guideline, users can successfully develop their own functional CHO CFPS system, allowing for broader application of mammalian CFPS.
Potter M, Najer A, Kloeckner A, et al., 2020, Controlled dendrimersome nanoreactor system for localised hypochlorite-induced killing of bacteria, ACS Nano, Vol: 14, Pages: 17333-17353, ISSN: 1936-0851
Antibiotic resistance is a serious global health problem necessitating new bactericidal approaches such as nanomedicines. Dendrimersomes (DSs) have recently become a valuable alternative nanocarrier to polymersomes and liposomes due to their molecular definition and synthetic versatility. Despite this, their biomedical application is still in its infancy. Inspired by the localized antimicrobial function of neutrophil phagosomes and the versatility of DSs, a simple three-component DS-based nanoreactor with broad-spectrum bactericidal activity is presented. This was achieved by encapsulation of glucose oxidase (GOX) and myeloperoxidase (MPO) within DSs (GOX-MPO-DSs), self-assembled from an amphiphilic Janus dendrimer, that possesses a semipermeable membrane. By external addition of glucose to GOX-MPO-DS, the production of hypochlorite (−OCl), a highly potent antimicrobial, by the enzymatic cascade was demonstrated. This cascade nanoreactor yielded a potent bactericidal effect against two important multidrug resistant pathogens, Staphylococcus aureus (S. aureus) and Pseudomonas aeruginosa (P. aeruginosa), not observed for H2O2 producing nanoreactors, GOX-DS. The production of highly reactive species such as –OCl represents a harsh bactericidal approach that could also be cytotoxic to mammalian cells. This necessitates the development of strategies for activating –OCl production in a localized manner in response to a bacterial stimulus. One option of locally releasing sufficient amounts of substrate using a bacterial trigger (released toxins) was demonstrated with lipidic glucose-loaded giant unilamellar vesicles (GUVs), envisioning, e.g., implant surface modification with nanoreactors and GUVs for localized production of bactericidal agents in the presence of bacterial growth.
Contini C, Hindley J, Macdonald T, et al., 2020, Size dependency of gold nanoparticles interacting with model membranes, Communications Chemistry, Vol: 3, Pages: 1-12, ISSN: 2399-3669
The rapid development of nanotechnology has led to an increase in the number and variety of engineered nanomaterials in the environment. Gold nanoparticles (AuNPs) are an example of a commonly studied nanomaterial whose highly tailorable properties have generated significant interest through a wide range of research fields. In the present work, we characterise the AuNP-lipid membrane interaction by coupling qualitative data with quantitative measurements of the enthalpy change of interaction. We investigate the interactions between citrate-stabilised AuNPs ranging from 5 to 60 nm in diameter and large unilamellar vesicles acting as a model membrane system. Our results reveal the existence of two critical AuNP diameters which determine their fate when in contact with a lipid membrane. The results provide new insights into the size dependent interaction between AuNPs and lipid bilayers which is of direct relevance to nanotoxicology and to the design of NP vectors.
Zhang S, Contini C, Hindley J, et al., 2020, Engineering motile aqueous phase-separated droplets via liposome stabilisation, Publisher: Research Square Platform LLC
<jats:title>Abstract</jats:title> <jats:p>There are increasing efforts to engineer functional compartments that mimic aspects of cellular behaviour in a drive to construct an artificial cell from the bottom-up. One behaviour that is receiving particular attention is motility, due to its biotechnological potential and the fact that movement of discrete cells is a ubiquitous feature of living systems. Many existing platforms make use of the Marangoni effect to achieve motion in water/oil (w/o) droplet systems. However, most of these systems are unsuitable for biological applications due to issues with biocompatibility caused by the presence of oil phases. Here we report a biocompatible all aqueous (w/w) PEG/dextran Pickering-like emulsion system consisting of liposome-stabilized cell-sized droplets, where the stability can be easily tuned by adjusting liposome composition and concentration. We demonstrate that the compartments are capable of negative chemotaxis: if water is introduced into the emulsion system, these droplets can respond through directional motion away from PEG in the continuous phase and down to the polymer gradient with a velocity change proportional to the rearrangement of liposome stabilisers in the PEG/dextran interface. The biocompatibility, motility and partitioning abilities of this novel droplet system offers new directions to pursue research in motion-related biological processes.</jats:p>
Haylock S, Friddin M, Hindley J, et al., 2020, Membrane protein mediated bilayer communication in networks of droplet interface bilayers, Communications Chemistry, Vol: 3, ISSN: 2399-3669
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.
Hindley JW, Law RV, Ces O, 2020, Membrane functionalization in artificial cell engineering, SN Applied Sciences, Vol: 2, ISSN: 2523-3963
Bottom-up synthetic biology aims to construct mimics of cellular structure and behaviour known as artificial cells from a small number of molecular components. The development of this nascent field has coupled new insights in molecular biology with large translational potential for application in fields such as drug delivery and biosensing. Multiple approaches have been applied to create cell mimics, with many efforts focusing on phospholipid-based systems. This mini-review focuses on different approaches to incorporating molecular motifs as tools for lipid membrane functionalization in artificial cell construction. Such motifs range from synthetic chemical functional groups to components from extant biology that can be arranged in a ‘plug-and-play’ approach which is hard to replicate in living systems. Rationally designed artificial cells possess the promise of complex biomimetic behaviour from minimal, highly engineered chemical networks.
Barriga H, Ces O, Law R, et al., 2019, Engineering swollen cubosomes using cholesterol and anionic lipids, Langmuir: the ACS journal of surfaces and colloids, Vol: 35, Pages: 16521-16527, ISSN: 0743-7463
Dispersions of non-lamellar lipid membrane assemblies are gaining increasing interest for drug delivery and protein therapeutic application. A key bottleneck has been the lack of rational design rules for these systems linking different lipid species and conditions to defined lattice parameters and structures. We have developed robust methods to form cubosomes (nanoparticles with a porous internal structure) with water channel diameters of up to 171 Å which are over 4 times larger than archetypal cubosome structures. The water channel diameter can be tuned via the incorporation of cholesterol and the charged lipids DOPA, DOPG or DOPS. We have found that large molecules can be incorporated into the porous cubosome structure and these molecules can interact with the internal cubosome membrane. This offers huge potential for accessible encapsulation and protection of biomolecules, and development of confined interfacial reaction environments.
Hindley JW, Zheleva DG, Elani Y, et al., 2019, Building a synthetic mechanosensitive signaling pathway in compartmentalized artificial cells, Proceedings of the National Academy of Sciences, Vol: 116, Pages: 16711-16716, ISSN: 0027-8424
To date reconstitution of one of the fundamental methods of cell communication, the signaling pathway, has been unaddressed in the bottom-up construction of artificial cells (ACs). Such developments are needed to increase the functionality and biomimicry of ACs, accelerating their translation and application in biotechnology. Here we report the construction of a de novo synthetic signaling pathway in microscale nested vesicles. Vesicle cell models respond to external calcium signals through activation of an intracellular interaction between phospholipase A2 and a mechanosensitive channel present in the internal membranes, triggering content mixing between compartments and controlling cell fluorescence. Emulsion-based approaches to AC construction are therefore shown to be ideal for the quick design and testing of new signaling networks and can readily include synthetic molecules difficult to introduce to biological cells. This work represents a foundation for the engineering of multi-compartment-spanning designer pathways that can be utilised to control downstream events inside an artificial cell, leading to the assembly of micromachines capable of sensing and responding to changes in their local environment.
Supramaniam P, Ces O, Salehi-Reyhani A, 2019, Microfluidics for artificial life: techniques for bottom-up synthetic biology, Micromachines, Vol: 10, Pages: 1-27, ISSN: 2072-666X
Synthetic biology is a rapidly growing multidisciplinary branch of science that exploits the advancement of molecular and cellular biology. Conventional modification of pre-existing cells is referred to as the top-down approach. Bottom-up synthetic biology is an emerging complementary branch that seeks to construct artificial cells from natural or synthetic components. One of the aims in bottom-up synthetic biology is to construct or mimic the complex pathways present in living cells. The recent, and rapidly growing, application of microfluidics in the field is driven by the central tenet of the bottom-up approach—the pursuit of controllably generating artificial cells with precisely defined parameters, in terms of molecular and geometrical composition. In this review we survey conventional methods of artificial cell synthesis and their limitations. We proceed to show how microfluidic approaches have been pivotal in overcoming these limitations and ushering in a new generation of complexity that may be imbued in artificial cells and the milieu of applications that result.
Khan H, Seddon JM, Law RV, et al., 2019, Effect of glycerol with sodium chloride on the Krafft point of sodium dodecyl sulfate using surface tension, Journal of Colloid and Interface Science, Vol: 538, Pages: 75-82, ISSN: 0021-9797
The effect of glycerol with sodium chloride (NaCl) on the phase behaviour of sodium dodecyl sulfate (SDS) near the Krafft point was studied by surface tension analysis using the pendant drop method. The critical micelle concentration (CMC) and Krafft Temperature (TK) of SDS in water: glycerol mixtures, across the full composition range, and in NaCl solutions within 0.005–0.1 M were obtained. The pendant drop method successfully allowed us to determine the Krafft point of SDS in high glycerol systems where other traditional methods (e.g. conductivity) have been ineffective. Overall the addition of glycerol increases the CMC and the TK, thus shifting the Krafft point of SDS to higher temperatures (increasing crystallisation temperatures) and higher SDS content in the presence of glycerol, which is interpreted as a result of the reduction in solvent polarity which opposes micellization. The addition of NaCl to the SDS – water-glycerol systems brings the CMC back down, while having no significant effect on the TK. Our results establish a robust route for tuning the Krafft point of model surfactant SDS by adjusting solvent quality and salt content.
Friddin MS, Elani Y, Trantidou T, et al., 2019, New directions for artificial cells using rapid prototyped biosystems, Analytical Chemistry, Vol: 91, Pages: 4921-4928, ISSN: 0003-2700
Microfluidics has been shown to be capable of generating a range of single- and multi- compartment vesicles and bilayer delineated droplets that can be assembled in 2D and 3D. These model systems are becoming increasingly recognized as powerful biomimetic constructs for assembling tissue models, engineering therapeutic delivery systems and for screening drugs. One bottleneck in developing this technology is the time, expertise and equipment required for device fabrication. This has led to interest across the microfluidics community in using rapid prototyping to engineer microfluidic devices from Computer Aided Design (CAD) drawings. We highlight how this rapid prototyping revolution is transforming the fabrication of microfluidic devices for bottom-up synthetic biology. We provide an outline of the current landscape and present how advances in the field may give rise to the next generation of multifunctional biodevices, particularly with Industry 4.0 on the horizon. Successfully developing this technology and making it open-source could pave the way for a new generation of citizen-led science, fueling the possibility that the next multi-billion dollar start-up could emerge from an attic or a basement.
Ces O, Elani Y, 2019, Community building in synthetic biology., Experimental biology and medicine (Maywood, N.J.), Vol: 244, Pages: 281-282, ISSN: 0037-9727
Friddin M, Bolognesi G, Salehi-Reyhani A, et al., 2019, Direct manipulation of liquid ordered lipid membrane domains using optical traps, Communications Chemistry, Vol: 2, Pages: 1-7, ISSN: 2399-3669
Multicomponent lipid bilayers can give rise to coexisting liquid domains that are thought to influence a host of cellular activities. There currently exists no method to directly manipulate such domains, hampering our understanding of their significance. Here we report a system that allows individual liquid ordered domains that exist in a liquid disordered matrix to be directly manipulated using optical tweezers. This allows us to drag domains across the membrane surface of giant vesicles that are adhered to a glass surface, enabling domain location to be defined with spatiotemporal control. We can also use the laser to select individual vesicles in a population to undergo mixing/demixing by locally heating the membrane through the miscibility transition, demonstrating a further layer of control. This technology has potential as a tool to shed light on domain biophysics, on their role in biology, and in sculpting membrane assemblies with user-defined membrane patterning.
Trantidou T, Friddin M, Gan KB, et al., 2018, Mask-free laser lithography for rapid and low-cost microfluidic device fabrication, Analytical Chemistry, Vol: 90, Pages: 13915-13921, ISSN: 0003-2700
Microfluidics has become recognized as a powerful platform technology associated with a constantly increasing array of applications across the life sciences. This surge of interest over recent years has led to an increased demand for microfluidic chips, resulting in more time being spent in the cleanroom fabricating devices using soft lithography—a slow and expensive process that requires extensive materials, training and significant engineering resources. This bottleneck limits platform complexity as a byproduct of lengthy delays between device iterations and affects the time spent developing the final application. To address this problem, we report a new, rapid, and economical approach to microfluidic device fabrication using dry resist films to laminate laser cut sheets of acrylic. We term our method laser lithography and show that our technique can be used to engineer 200 μm width channels for assembling droplet generators capable of generating monodisperse water droplets in oil and micromixers designed to sustain chemical reactions. Our devices offer high transparency, negligible device to device variation, and low X-ray background scattering, demonstrating their suitability for real-time X-ray-based characterization applications. Our approach also requires minimal materials and apparatus, is cleanroom free, and at a cost of around $1.00 per chip could significantly democratize device fabrication, thereby increasing the interdisciplinary accessibility of microfluidics.
Miller RM, Cabral J, Robles E, et al., 2018, Crystallisation of sodium dodecyl sulfate–water micellar solutions with structurally similar additives: counterion variation, CrystEngComm, Vol: 20, Pages: 6834-6843, ISSN: 1466-8033
The effects of a series of structurally similar sodium dodecyl sulfate (SDS) additives on the crystallisation of SDS–water micellar solutions were investigated using a combination of differential scanning calorimetry, dynamic light scattering, optical microscopy and inductively coupled plasma optical emission spectroscopy. Seven different counterions were chosen from groups 1 and 2 of the periodic table to replace the sodium on SDS: LDS, (SDS), KDS, RbDS, CsDS, Mg(DS)2, Ca(DS)2 and Sr(DS)2. Two representative temperature profileswere employed – linear cooling ramps at rate of 0.5 °C min−1 to determine near-equilibrium kinetics and transitions and isothermal holds at 6 °C to elucidate morphological changes. Crystallisation of the reference solution 20% SDS–H2O with 0.25, 1.0 and 2.5% additive was generally promoted or inhibited even at the lowest concentrations. Melting points however remained largely unchanged, suggesting that the additives predominantly had a kinetic rather than thermodynamic effect. ICP-OES measurements for the solutions containing 1% additive indicated that most of the additives were integrated into the SDS crystals which was reflected by morphological changes, including the formation of hexagonal and oval shaped crystals. Our results both quantify and provide a morphological insight into the effect of a series of additives on the crystallisation of micellar SDS solutions, which can readily form due to preferential Na exchange.
Barlow N, Kusumaatmaja H, Salehi-Reyhani A, et al., 2018, Measuring bilayer surface energy and curvature in asymmetric droplet interface bilayers, Journal of the Royal Society Interface, Vol: 15, ISSN: 1742-5662
For the past decade, droplet interface bilayers (DIBs) have had an increased prevalence in biomolecular and biophysical literature. However, much of the underlying physics of these platforms is poorly characterized. To further our understanding of these structures, lipid membrane tension on DIB membranes is measured by analysing the equilibrium shape of asymmetric DIBs. To this end, the morphology of DIBs is explored for the first time using confocal laser scanning fluorescence microscopy. The experimental results confirm that, in accordance with theory, the bilayer interface of a volume-asymmetric DIB is curved towards the smaller droplet and a lipid-asymmetric DIB is curved towards the droplet with the higher monolayer surface tension. Moreover, the DIB shape can be exploited to measure complex bilayer surface energies. In this study, the bilayer surface energy of DIBs composed of lipid mixtures of phosphatidylgylcerol (PG) and phosphatidylcholine are shown to increase linearly with PG concentrations up to 25%. The assumption that DIB bilayer area can be geometrically approximated as a spherical cap base is also tested, and it is discovered that the bilayer curvature is negligible for most practical symmetric or asymmetric DIB systems with respect to bilayer area.
Trantidou T, Dekker L, Polizzi K, et al., 2018, Functionalizing cell-mimetic giant vesicles with encapsulated bacterial biosensors, Interface Focus, Vol: 8, ISSN: 2042-8901
The design of vesicle microsystems as artificial cells (bottom-up synthetic biology) has traditionally relied on the incorporation of molecular components to impart functionality. These cell mimics have reduced capabilities compared with their engineered biological counterparts (top-down synthetic biology), as they lack the powerful metabolic and regulatory pathways associated with living systems. There is increasing scope for using whole intact cellular components as functional modules within artificial cells, as a route to increase the capabilities of artificial cells. In this feasibility study, we design and embed genetically engineered microbes (Escherichia coli) in a vesicle-based cell mimic and use them as biosensing modules for real-time monitoring of lactate in the external environment. Using this conceptual framework, the functionality of other microbial devices can be conferred into vesicle microsystems in the future, bridging the gap between bottom-up and top-down synthetic biology.
Chatzimichail S, Supramaniam P, Ces O, et al., 2018, Micropatterning of planar metal electrodes by vacuum filling microfluidic channel geometries, Scientific Reports, Vol: 8, ISSN: 2045-2322
We present a simple, facile method to micropattern planar metal electrodes defined by the geometry of a microfluidic channel network template. By introducing aqueous solutions of metal into reversibly adhered PDMS devices by desiccation instead of flow, we are able to produce difficult to pattern “dead end” or discontinuous features with ease. We characterize electrodes fabricated using this method and perform electrical lysis of mammalian cancer cells and demonstrate their use as part of an antibody capture assay for GFP. Cell lysis in microwell arrays is achieved using the electrodes and the protein released is detected using an antibody microarray. We show how the template channels used as part of the workflow for patterning the electrodes may be produced using photolithography-free methods, such as laser micromachining and PDMS master moulding, and demonstrate how the use of an immiscible phase may be employed to create electrode spacings on the order of 25 – 50 μm, that overcome the current resolution limits of such methods. This work demonstrates how the rapid prototyping of electrodes for use in total analysis systems can be achieved on the bench with little or no need for centralized facilities.
Trantidou T, Friddin M, Salehi-Reyhani S, et al., 2018, Droplet microfluidics for the construction of compartmentalised model membranes, Lab on a Chip, Vol: 18, Pages: 2488-2509, ISSN: 1473-0189
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.
Chatzimichail S, Supramaniam P, Ces O, et al., 2018, Counting proteins in single cells with addressable droplet microarrays, Jove-Journal of Visualized Experiments, Vol: 137, ISSN: 1940-087X
Often cellular behaviour and cellular responses are analysed at the population level where the responses of many cells are pooled together as an average result masking the rich single cell behaviour within a complex population. Single cell protein detection and quantification technologies have made a remarkable impact in recent years. Here we describe a practical and flexible single cell analysis platform based on addressable droplet microarrays. This study describes how the absolute copy numbers of target proteins may be measured with single cell resolution. The tumour suppressor p53 is the most commonly mutated gene in human cancer, with more than 50% of total cancer cases exhibiting a non-healthy p53 expression pattern. The protocol describes steps to create 10nL droplets within which single human cancer cells are isolated and the copy number of p53 protein is measured with single molecule resolution to precisely determine the variability in expression. The method may be applied to any cell type including primary material to determine the absolute copy number of any target proteins of interest.
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