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  • Journal article
    Chatzimichail S, Supramaniam P, Ces O, Salehi-Reyhani Set 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.

  • Journal article
    Bolognesi G, Friddin MS, Salehi-Reyhani S, Barlow N, Brooks NJ, Ces O, Elani Yet al., 2018,

    Sculpting and fusing biomimetic vesicle networks using optical tweezers

    , Nature Communications, Vol: 9, Pages: 1-11, ISSN: 2041-1723

    Constructing higher-order vesicle assemblies has discipline-spanning potential from responsive soft-matter materials to artificial cell networks in synthetic biology. This potential is ultimately derived from the ability to compartmentalise and order chemical species in space. To unlock such applications, spatial organisation of vesicles in relation to one another must be controlled, and techniques to deliver cargo to compartments developed. Herein, we use optical tweezers to assemble, reconfigure and dismantle networks of cell-sized vesicles that, in different experimental scenarios, we engineer to exhibit several interesting properties. Vesicles are connected through double-bilayer junctions formed via electrostatically controlled adhesion. Chemically distinct vesicles are linked across length scales, from several nanometres to hundreds of micrometres, by axon-like tethers. In the former regime, patterning membranes with proteins and nanoparticles facilitates material exchange between compartments and enables laser-triggered vesicle merging. This allows us to mix and dilute content, and to initiate protein expression by delivering biomolecular reaction components.

  • Journal article
    Karamdad K, Hindley J, Friddin MS, Bolognesi G, Law RV, Brooks NJ, Ces O, Elani Yet al., 2018,

    Engineering thermoresponsive phase separated vesicles formed via emulsion phase transfer as a content-release platform

    , Chemical Science, Vol: 9, Pages: 4851-4858, ISSN: 2041-6520

    Giant unilamellar vesicles (GUVs) are a well-established tool for the study of membrane biophysics and are increasingly used as artificial cell models and functional units in biotechnology. This trend is driven by the development of emulsion-based generation methods such as Emulsion Phase Transfer (EPT), which facilitates the encapsulation of almost any water-soluble compounds (including biomolecules) regardless of size or charge, is compatible with droplet microfluidics, and allows GUVs with asymmetric bilayers to be assembled. However, the ability to control the composition of membranes formed via EPT remains an open question; this is key as composition gives rise to an array of biophysical phenomena which can be used to add functionality to membranes. Here, we evaluate the use of GUVs constructed via this method as a platform for phase behaviour studies and take advantage of composition-dependent features to engineer thermally-responsive GUVs. For the first time, we generate ternary GUVs (DOPC/DPPC/cholesterol) using EPT, and by compensating for the lower cholesterol incorporation efficiencies, show that these possess the full range of phase behaviour displayed by electroformed GUVs. As a demonstration of the fine control afforded by this approach, we demonstrate release of dye and peptide cargo when ternary GUVs are heated through the immiscibility transition temperature, and show that release temperature can be tuned by changing vesicle composition. We show that GUVs can be individually addressed and release triggered using a laser beam. Our findings validate EPT as a suitable method for generating phase separated vesicles and provide a valuable proof-of-concept for engineering content release functionality into individually addressable vesicles, which could have a host of applications in the development of smart synthetic biosystems.

  • Journal article
    Loftus C, Saeed M, Davis D, Dunlop IEet al., 2018,

    Activation of human Natural Killer cells by graphene oxide-templated antibody nanoclusters

    , Nano Letters: a journal dedicated to nanoscience and nanotechnology, Vol: 18, Pages: 3282-3289, ISSN: 1530-6984

    An emerging new paradigm is that immune cell activation is controlled by transient interactions between supramolecular assemblies of receptors and ligands. Current immunotherapy biologic pharmaceuticals that activate or desensitize NK cells are, however, individual molecules that do not replicate this nanoscale organization of proteins. Here, we use nanoscale graphene oxide (NGO) as a template to generate soluble nanoscale clusters of Natural Killer cell-activating antibodies. We control nanocluster size and molecular number to mimic reported values for cell surface proteins. These NGO-templated molecular nanoclusters, used to stimulate NK cells via the CD16 receptor, successfully induced cellular activation, indicated by degranulation of cytolytic granules and IFN-γ secretion. Importantly, activation significantly exceeded that induced by the same antibodies applied as a solution of individual molecules. These results demonstrate that future immunotherapies could be enhanced by assembling immunomodulatory drugs into nanoclusters and establish NGO-templating as a candidate technology.

  • Journal article
    Craven G, Affron D, Allen C, Matthies S, Greener J, Morgan R, Tate E, Armstrong A, Mann Det al., 2018,

    High-throughput kinetic analysis for target-directed covalent ligand discovery

    , Angewandte Chemie International Edition, Vol: 57, Pages: 5257-5261, ISSN: 1433-7851

    Cysteine-­reactive small molecules are used as chemical probes of biological systems and as medicines. Identifying high-­quality covalent ligands requires comprehensive kinetic analysis to distinguish selective binders from pan-­reactive compounds. Here we describe quantitative irreversible tethering(qIT), a general method for screening cysteine-­reactive small moleculesbased upon the maximization of kinetic selectivity. We apply this method prospectively to discover covalent fragments that target the clinically important cell cycle regulator Cdk2. Crystal structures of the inhibitor complexes validate the approach and guide further optimization. The power of this technique is highlighted by the identification of a Cdk2-­selective allosteric (type IV) kinase inhibitor whose novel mode-­of-­action could be exploited therapeutically.

  • Journal article
    Cadinu P, Campolo G, Pud S, Yang W, Edel J, Dekker C, Ivanov Aet al., 2018,

    Double barrel nanopores as a new tool for controlling single-molecule transport

    , Nano Letters, Vol: 18, Pages: 2738-2745, ISSN: 1530-6984

    The ability to control the motion of single biomolecules is key to improving a wide range of biophysical and diagnostic applications. Solid-state nanopores are a promising tool capable of solving this task. However, molecular control and the possibility of slow readouts of long polymer molecules are still limited due to fast analyte transport and low signal-to-noise ratios. Here, we report on a novel approach of actively controlling analyte transport by using a double-nanopore architecture where two nanopores are separated by only a ∼ 20 nm gap. The nanopores can be addressed individually, allowing for two unique modes of operation: (i) pore-to-pore transfer, which can be controlled at near 100% efficiency, and (ii) DNA molecules bridging between the two nanopores, which enables detection with an enhanced temporal resolution (e.g., an increase of more than 2 orders of magnitude in the dwell time) without compromising the signal quality. The simplicity of fabrication and operation of the double-barrel architecture opens a wide range of applications for high-resolution readout of biological molecules.

  • Conference paper
    Wang S, Gould I, 2018,

    All-atom molecular dynamics of an ion-channel formed by viral protein U in lipid bilayers

    , 255th National Meeting and Exposition of the American-Chemical-Society (ACS) - Nexus of Food, Energy, and Water, Publisher: AMER CHEMICAL SOC, ISSN: 0065-7727
  • Journal article
    Hindley JW, Elani Y, McGilvery CM, Ali S, Bevan CL, Law R, Ces Oet al., 2018,

    Light-triggered enzymatic reactions in nested vesicle reactors

    , Nature Communications, Vol: 9, Pages: 1-6, ISSN: 2041-1723

    Cell-sized vesicles have tremendous potential both as miniaturised pL reaction vessels and in bottom-up synthetic biology as chassis for artificial cells. In both these areas the introduction of light-responsive modules affords increased functionality, for example, to initiate enzymatic reactions in the vesicle interior with spatiotemporal control. Here we report a system composed of nested vesicles where the inner compartments act as phototransducers, responding to ultraviolet irradiation through diacetylene polymerisation-induced pore formation to initiate enzymatic reactions. The controlled release and hydrolysis of a fluorogenic β-galactosidase substrate in the external compartment is demonstrated, where the rate of reaction can be modulated by varying ultraviolet exposure time. Such cell-like nested microreactor structures could be utilised in fields from biocatalysis through to drug delivery.

  • Journal article
    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, Pages: 1-8, ISSN: 2045-2322

    There is increasing interest in constructing artificial cells by functionalising lipid vesicles with biological and synthetic machinery. Due to their reduced complexity and lack of evolved biochemical pathways, the capabilities of artificial cells are limited in comparison to their biological counterparts. 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 synthetic cell assembly. Using microfluidic technologies to construct such hybrid cellular bionic 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, while the cell acts as a bioreactor module that processes encapsulated feedstock which is further processed by a synthetic enzymatic metabolism co-encapsulated in the vesicle.

  • Journal article
    Kim Y, Warren S, Favero F, Stone J, Clegg J, Neil M, Paterson C, Knight J, French P, Dunsby CWet al., 2018,

    Semi-random multicore fibre design for adaptive multiphoton endoscopy

    , Optics Express, Vol: 26, Pages: 3661-3673, ISSN: 1094-4087

    This paper reports the development, modelling and application of a semi-random multicore fibre (MCF) design for adaptive multiphoton endoscopy. The MCF was constructed from 55 sub-units, each comprising 7 single mode cores, in a hexagonally close-packed lattice where each sub-unit had a random angular orientation. The resulting fibre had 385 single mode cores and was double-clad for proximal detection of multiphoton excited fluorescence. The random orientation of each sub-unit in the fibre reduces the symmetry of the positions of the cores in the MCF, reducing the intensity of higher diffracted orders away from the central focal spot formed at the distal tip of the fibre and increasing the maximum size of object that can be imaged. The performance of the MCF was demonstrated by imaging fluorescently labelled beads with both distal and proximal fluorescence detection and pollen grains with distal fluorescence detection. We estimate that the number of independent resolution elements in the final image – measured as the half-maximum area of the two-photon point spread function divided by the area imaged – to be ~3200.

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