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ABSTRACT: Measuring protein expression in single cells is the basis of single cell proteomics. The sensitivity and dynamic range of a single cell immunoassay should ideally be such that proteins that are expressed between 1-10(6) copies per cell can be detected and counted. We have investigated the effect of miniaturizing antibody microarrays by reducing capture spot sizes from 100 μm to 15 μm using dip-pen nanolithography. We demonstrate that protocols developed for printing and passivating antibody capture spots using conventional pin-based contact printing can be directly transferred to dip-pen lithography whilst retaining the capture activity per unit area. Using a simple kinetic model, we highlight how the limit of detection and dynamic range of a sandwich immunoassay, respectively, increase and decrease when spot size is reduced. However, we show that reducing spot size is more effective than increasing assay chamber volume when seeking to multiplex such a microfluidic immunoassay. Although we make particular reference to single cell microfluidic immunoassays, the topics discussed here are applicable to capture assays in general.
Lab on a Chip 04/2013; · 5.67 Impact Factor
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ABSTRACT: Monoacylglycerol based lipids are highly important model membrane components and attractive candidates for drug encapsulation and as delivery agents. However, optimizing the properties of these lipids for applications requires a detailed understanding of the thermodynamic factors governing the self-assembled structures that they form. Here, we report on the effects of hydrostatic pressure, temperature, and water composition on the structural behavior and stability of inverse lyotropic liquid crystalline phases adopted by monolinolein (an unsaturated monoacylglycerol having cis-double bonds at carbon positions 9 and 12) under limited hydration conditions. Six pressure-temperature phase diagrams have been determined using small-angle X-ray diffraction at water contents between 15 wt % and 27 wt % water, in the range 10-40 °C and 1-3000 bar. The gyroid bicontinuous cubic (Q(II)(G)) phase is formed at low pressure and high temperatures, transforming to a fluid lamellar (L(α)) phase at high pressures and low temperature via a region of Q(II)(G)/L(α) coexistence. Pressure stabilizes the lamellar phase over the Q(II)(G) phase; at fixed pressure, increasing the water content causes the coexistence region to move to lower temperature. These trends are consistent throughout the hydration range studied. Moreover, at fixed temperature, increasing the water composition increases the pressure at which the Q(II)(G) to L(α) transition takes place. We discuss the qualitative effect of pressure, temperature, and water content on the stability of the Q(II)(G) phase.
Langmuir 08/2012; 28(36):13018-24. · 4.19 Impact Factor
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ABSTRACT: Droplet interface bilayer (DIB) networks have vast potential in the field of membrane biophysics, synthetic biology, and functional bio-electronics. However a technological bottleneck exists in network fabrication: existing methods are limited in terms of their automation, throughput, versatility, and ability to form well-defined 3-D networks. We have developed a series of novel and low-cost methodologies which address these limitations. The first involves building DIB networks around the contours of a microfluidic chip. The second uses flow rate and droplet size control to influence droplet packing geometries within a microfluidic chamber. The latter method enables the controlled formation of various 3-D network arrays consisting of thousands of interconnected symmetric and asymmetric lipid bilayers for the first time. Both approaches allow individual droplet position and composition to be controlled, paving the way for complex on-chip functional network synthesis.
Lab on a Chip 08/2012; 12(18):3514-20. · 5.67 Impact Factor
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ABSTRACT: Arylstibonates structurally resemble phosphotyrosine side chains in proteins and here we addressed the ability of such compounds to act as inhibitors of a panel of mammalian tyrosine and dual-specificity phosphatases. Two arylstibonates both possessing a carboxylate side chain were identified as potent inhibitors of the protein tyrosine phosphatase PTP-ß. In addition, they inhibited the dual-specificity, cell cycle regulatory phosphatases Cdc25a and Cdc25b with sub-micromolar potency. However, the Cdc25c phosphatase was not affected demonstrating that arylstibonates may be viable leads from which to develop isoform specific Cdc25 inhibitors.
Bioorganic & medicinal chemistry 05/2012; 20(14):4371-6. · 2.82 Impact Factor
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ABSTRACT: We present a top down separation platform for yeast ribosomal proteins using affinity chromatography and capillary electrophoresis which is designed to allow deposition of proteins onto a substrate. FLAG tagged ribosomes were affinity purified, and rRNA acid precipitation was performed on the ribosomes followed by capillary electrophoresis to separate the ribosomal proteins. Over 26 peaks were detected with excellent reproducibility (<0.5% RSD migration time). This is the first reported separation of eukaryotic ribosomal proteins using capillary electrophoresis. The two stages in this workflow, affinity chromatography and capillary electrophoresis, share the advantages that they are fast, flexible and have small sample requirements in comparison to more commonly used techniques. This method is a remarkably quick route from cell to separation that has the potential to be coupled to high throughput readout platforms for studies of the ribosomal proteome.
BMB reports 04/2012; 45(4):233-8. · 1.72 Impact Factor
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ABSTRACT: Mechanical properties of biological membranes are known to regulate membrane protein function. Despite this, current models of protein communication typically feature only direct protein-protein or protein-small molecule interactions. Here we show for the first time that, by harnessing nanoscale mechanical energy within biological membranes, it is possible to promote controlled communication between proteins. By coupling lipid-protein modules and matching their response to the mechanical properties of the membrane, we have shown that the action of phospholipase A(2) on acyl-based phospholipids triggers the opening of the mechanosensitive channel, MscL, by generating membrane asymmetry. Our findings confirm that the global physical properties of biological membranes can act as information pathways between proteins, a novel mechanism of membrane-mediated protein-protein communication that has important implications for (i) the underlying structure of signaling pathways, (ii) our understanding of in vivo communication networks, and (iii) the generation of building blocks for artificial protein networks.
Journal of the American Chemical Society 03/2012; 134(13):5746-9. · 9.91 Impact Factor
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ABSTRACT: The effect of hydrostatic pressure on lipid structure and dynamics is highly important as a tool in biophysics and bio-technology, and in the biology of deep sea organisms. Despite its importance, high hydrostatic pressure remains significantly less utilised than other thermodynamic variables such as temperature and chemical composition. Here, we give an overview of some of the theoretical aspects which determine lipid behaviour under pressure and the techniques and technology available to study these effects. We also summarise several recent experiments which highlight the information available from these approaches.
Chemistry and physics of lipids 02/2011; 164(2):89-98. · 2.15 Impact Factor
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Ali Salehi-Reyhani,
Joseph Kaplinsky,
Edward Burgin,
Miroslava Novakova,
Andrew J deMello,
Richard H Templer,
Peter Parker,
Mark A A Neil, Oscar Ces,
Paul French,
Keith R Willison,
David Klug
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ABSTRACT: We have developed a generic platform to undertake the analysis of protein copy number from single cells. The approach described here is 'all-optical' whereby single cells are manipulated into separate analysis chambers using an optical trap; single cells are lysed by a shock wave caused by laser-induced microcavitation, and the protein released from a single cell is measured by total internal reflection microscopy as it is bound to micro-printed antibody spots within the device. The platform was tested using GFP transfected cells and the relative precision of the measurement method was determined to be 88%. Single cell measurements were also made on a breast cancer cell line to measure the relative levels of unlabelled human tumour suppressor protein p53 using a chip incorporating an antibody sandwich assay format. These results suggest that this is a viable method for measuring relative protein levels in single cells.
Lab on a Chip 02/2011; 11(7):1256-61. · 5.67 Impact Factor
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ABSTRACT: A high pressure cell for small and wide-angle x-ray diffraction measurements of soft condensed matter samples has been developed, incorporating a fully automated pressure generating network. The system allows both static and pressure jump measurements in the range of 0.1-500 MPa. Pressure jumps can be performed as quickly as 5 ms, both with increasing and decreasing pressures. Pressure is generated by a motorized high pressure pump, and the system is controlled remotely via a graphical user interface to allow operation by a broad user base, many of whom may have little previous experience of high pressure technology. Samples are loaded through a dedicated port allowing the x-ray windows to remain in place throughout an experiment; this facilitates accurate subtraction of background scattering. The system has been designed specifically for use at beamline I22 at the Diamond Light Source, United Kingdom, and has been fully integrated with the I22 beamline control systems.
The Review of scientific instruments 06/2010; 81(6):064103. · 1.52 Impact Factor
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ABSTRACT: The effects of biological buffers on lipids have not been fully investigated because of the long-standing assumption that these buffers are too hydrophilic to substantially interact with the lipid membrane. We present evidence that for some buffers, this is not necessarily the case. Our research points toward a membrane softening effect caused by the buffer molecules interacting with the headgroup region of the lipid. Changes in the elastic properties of the membrane are known to control membrane protein behavior; this work serves as a warning for the design of assays utilizing model membranes in the presence of buffers.
Biochemistry 11/2009; 48(47):11149-51. · 3.42 Impact Factor
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ABSTRACT: The field of drug-membrane interactions is one that spans a wide range of scientific disciplines, from synthetic chemistry, through biophysics to pharmacology. Cell membranes are complex dynamic systems whose structures can be affected by drug molecules and in turn can affect the pharmacological properties of the drugs being administered. In this tutorial review we aim to provide a guide for those new to the area of drug-membrane interactions and present an introduction to areas of this topic which need to be considered. We address the lipid composition and structure of the cell membrane and comment on the physical forces present in the membrane which may impact on drug interactions. We outline methods by which drugs may cross or bind to this membrane, including the well understood passive and active transport pathways. We present a range of techniques which may be used to study the interactions of drugs with membranes both in vitro and in vivo and discuss the advantages and disadvantages of these techniques and highlight new methods being developed to further this field.
Chemical Society Reviews 10/2009; 38(9):2509-19. · 28.76 Impact Factor
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ABSTRACT: We recently introduced a novel platform based upon optically trapped lipid coated oil droplets (Smart Droplet Microtools-SDMs) that were able to form membrane tethers upon fusion with the plasma membrane of single cells. Material transfer from the plasma membrane to the droplet via the tether was seen to occur. Here we present a customised version of the SDM approach based upon detergent coated droplets deployed within a microfluidic format. These droplets are able to differentially solubilise the plasma membrane of single cells with spatial selectivity and without forming membrane tethers. The microfluidic format facilitates separation of the target cells from the bulk SDM population and from downstream analysis modules. Material transfer from the cell to the SDM was monitored by tracking membrane localized EGFP.
Lab on a Chip 05/2009; 9(8):1096-101. · 5.67 Impact Factor
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ABSTRACT: In this paper, we report on a novel osmotic cell, developed to simultaneously subject a sample to osmotic stress and measure structural changes by small angle x-ray diffraction. The osmotic cell offers many advantages over more conventional methods of osmotically stressing soft materials to measure their structural response. In particular, a full osmotic analysis can be performed with a single small sample (25 microl). This reduces sample handling and the associated systematic errors, as well as enabling tight control and monitoring of the thermodynamic environment during osmosis, thereby increasing measurement precision. The cell design enables control of osmotic pressure to +/-0.04 bar over a pressure range of 1-100 bar, and temperature control to +/-0.05 degrees C. Under these conditions, the lattice spacing in lyotropic structures was resolved to better than +/-0.005 A. Using the osmotic cell, we demonstrate good agreement with previous conventional measurements on the energy of dehydrating the fluid lamellar phase of dioleoylphosphatidylcholine in water.
The Review of scientific instruments 04/2009; 80(3):035107. · 1.52 Impact Factor
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ABSTRACT: This chapter describes a method for the preparation of giant unilamellar vesicles containing phosphatidylinositol 4,5-bisphosphate that are larger than 20 microm in size. The phospholipids composition of the vesicular membrane is such that fluid lamellar and liquid-ordered or gel phases are formed and separate within the confines of one vesicle. It outlines the preparation of a protein fluorescent label, pleckstrin homology domain from phospholipase C-delta 1, that binds specifically to phosphatidylinositol 4,5-bisphosphate. Using fluorescence microscopy, the presence and spatial position of this phosphorylated phosphatidylinositol lipid on the lipid membrane have been located with the pleckstrin homology domain. We show that phosphatidylinositol 4,5-bisphosphate and the phospholipase C-delta 1 pleckstrin homology domain are located to the fluid phase of the vesicle membrane. This approach can therefore show how membrane physical properties can affect enzyme binding to phosphatidylinositol 4,5-bisphosphate and thus further the understanding of important membrane processes such as endocytosis.
Methods in molecular biology (Clifton, N.J.) 02/2009; 462:135-44.
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ABSTRACT: Lipids that are found in cell membranes form a variety of self-assembled phases in the presence of water. Many of these structures are liquid-crystalline with structural motifs mirrored in cells and organelles and can be exploited in the delivery of drugs and genes. We report the discovery of a lyotropic liquid crystalline phase based on a 3-D hexagonal close-packed arrangement of inverse micelles, of space group P6(3)/mmc. This is the first new inverse lyotropic liquid-crystalline phase to be reported for two decades and is the only known lyotropic phase whose structure consists of a close packing of identical inverse micelles.
Journal of the American Chemical Society 02/2009; 131(5):1678-9. · 9.91 Impact Factor
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ABSTRACT: We show that we can manipulate the stability of a metastable gel phase, either to enhance its transitory nature or to "lock" it in. Using simple additives such as salt and fatty alcohol we were able to examine both the long-range effect, acting between charged bilayers, and short-range effects on the metastability. We found that the addition of salt to the cationic surfactant diethanolamine ester dimethyl ammonium chloride destabilized the gel phase, and at high concentrations it was able to decrease the length of time taken for the gel phase to revert to a hydrated solid "coagel" phase by an order of magnitude. The growth of the coagel phase was also found to be affected by increasing salt concentration, changing from needle-like (1D) to spherical growth. In contrast to the marked destabilization of the gel phase by salt, the addition of 1-octadecanol was found to prolong the lifetime of the gel phase almost indefinitely by disrupting the short-range packing between the surfactant molecules. This suggests that counterion binding plays a major role in the stability of metastable lamellar gel phases.
The Journal of Physical Chemistry B 02/2009; 113(7):1948-53. · 3.70 Impact Factor
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ABSTRACT: This chapter describes a method for the preparation of giant unilamellar vesicles containing phosphatidylinositol 4,5-bisphosphate
that are larger than 20 μm in size. The phospholipids composition of the vesicular membrane is such that fluid lamellar and
liquid-ordered or gel phases are formed and separate within the confines of one vesicle. It outlines the preparation of a
protein fluorescent label, pleckstrin homology domain from phospholipase C-delta 1, that binds specifically to phosphatidylinositol
4,5-bisphosphate. Using fluorescence microscopy, the presence and spatial position of this phosphorylated phosphatidylinositol
lipid on the lipid membrane have been located with the pleckstrin homology domain. We show that phosphatidylinositol 4,5-bisphosphate
and the phospholipase C-delta 1 pleckstrin homology domain are located to the fluid phase of the vesicle membrane. This approach
can therefore show how membrane physical properties can affect enzyme binding to phosphatidylinositol 4,5-bisphosphate and
thus further the understanding of important membrane processes such as endocytosis.
KeywordsGiant unilamellar vesicles–phosphatidylinositol 4,5-bisphosphate–phospholipase C-delta 1–PH domain
12/2008: pages 135-144;
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ABSTRACT: The lyotropic phase behaviour of two analogues of dioctadecyl dimethylammonium chloride was investigated. Both the inclusion of ester groups and subsequent minor structural rearrangement of the interfacial region of the surfactant were found to increase the chain melting temperature, although the overall phase behaviour remained similar for both compounds. Both of the two analogues were found to underswell, due to the formation of multi-lamellar vesicles. We also found that the inclusion of these ester linkages substantially reduced the metastability of the 'gel phase' in which the surfactants usually reside, accelerating the rate of collapse to a coagel state. This occurred via a nucleation-growth mechanism, where the growth was found to be one-dimensional, i.e. needle-like.
Journal of Colloid and Interface Science 12/2008; 331(2):463-9. · 3.07 Impact Factor
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ABSTRACT: For this special issue of J. R. Soc. Interface we present an overview of the driving forces behind technological advances in the field of single-cell analysis. These range from increasing our understanding of cellular heterogeneity through to the study of rare cells, areas of research that cannot be tackled effectively using current high-throughput population-based averaging techniques.
Journal of The Royal Society Interface 10/2008; 5 Suppl 2:S111-2. · 4.40 Impact Factor
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Peter M P Lanigan,
Karen Chan,
Tanya Ninkovic,
Richard H Templer,
P M W French,
A J de Mello,
K R Willison,
P J Parker,
M A A Neil, Oscar Ces,
D R Klug
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ABSTRACT: We present a platform for the spatially selective sampling of the plasma membrane of single cells. Optically trapped lipid-coated oil droplets (smart droplet microtools, SDMs), typically 0.5-5 microm in size, composed of a hexadecane hydrocarbon core and fusogenic lipid outer coating (mixture of 1,2-dioleoyl-phosphatidylethanolamine and 1,2-dioleoyl-sn-glycero-3-phosphatidylcholine) were brought into controlled contact with target colon cancer cells leading to the formation of connecting membrane tethers. Material transfer from the cell to the SDM across the membrane tether was monitored by tracking membrane-localized enhanced green fluorescent protein.
Journal of The Royal Society Interface 10/2008; 5 Suppl 2:S161-8. · 4.40 Impact Factor
