Topographic SU-8 substrates for immobilization of three-dimensional nanotube-vesicle networks.
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Adhesiveness↗
Biomedical subjects
Owe Orwar
Publications and source records attributed to Owe Orwar.
Mechanical tweezer action by self-tightening knots in surfactant nanotubes.
Entanglements and trefoil knots on surfactant nanotubes in the liquid phase were produced by a combination of network self-organization and micromanipulation. The resulting knots are self-tightening, and the tightening is driven by minimization of surface free energy of the surfactant membrane material. The formation of the knot and the steady-state knot at quasi-equilibrium can be directly followed and localized by using fluorescence microscopy. Knots on nanotubes can be used as nanoscale mechanical tweezers for trapping and manipulation of single nano- and micrometer-sized high-aspect ratio objects. Furthermore, we demonstrate that by controlling the surface tension, objects captured by a knot can be transported along given trajectories defined by the nanotube axes.
Fluorescence↗
Biomimetic nanoscale reactors and networks.
Methods based on self-assembly, self-organization, and forced shape transformations to form synthetic or semisynthetic enclosed lipid bilayer structures with several properties similar to biological nanocompartments are reviewed. The procedures offer unconventional micro- and nanofabrication routes to yield complex soft-matter devices for a variety of applications for example, in physical chemistry and nanotechnology. In particular, we describe novel micromanipulation methods for producing fluid-state lipid bilayer networks of nanotubes and surface-immobilized vesicles with controlled geometry, topology, membrane composition, and interior contents. Mass transport in nanotubes and materials exchange, for example, between conjugated containers, can be controlled by creating a surface tension gradient that gives rise to a moving boundary or by induced shape transformations. The network devices can operate with extremely small volume elements and low mass, to the limit of single molecules and particles at a length scale where a continuum mechanics approximation may break down. Thus, we also describe some concepts of anomalous fluctuation-dominated kinetics and anomalous diffusive behaviours, including hindered transport, as they might become important in studying chemistry and transport phenomena in these confined systems. The networks are suitable for initiating and controlling chemical reactions in confined biomimetic compartments for rationalizing, for example, enzyme behaviors, as well as for applications in nanofluidics, bioanalytical devices, and to construct computational and complex sensor systems with operations building on chemical kinetics, coupled reactions and controlled mass transport.
Biological Transport↗
Stabilization of high-resistance seals in patch-clamp recordings by laminar flow.
The formation of a high-resistance electrical seal between a cell membrane and a glass micropipet tip is essential in patch-clamp experiments. We have studied the electrical properties and the mechanical stability of the seal using a microfluidic chip generating laminar flow in open volumes. We show that, by using fluid flow (1-10 mm/s) acting along the symmetry axis of the cell-pipet, seals of a higher mechanical stability with increased resistances can be achieved, allowing up to 100% longer recording times and over 40% decreased noise levels (Irms). These improved properties are beneficial for high-sensitivity patch-clamp recordings, in particular, in longtime studies of ion channel receptor systems that are relevant in biosensor applications of the technique. Furthermore, these observations support the combination of patch-clamp with microfluidic devices, for example, for rapid solution exchange around a single cell sensor for high-throughput electrophysiology and for highly resolved kinetic studies.
Journal Article↗
Amperometric detection of exocytosis in an artificial synapse.
A liposome model of exocytosis has been used to examine the nanometer environment of the space between the electrode and the membrane. First, this has been used to test models of coulometric efficiency for different size vesicles with 5- and 33-microm electrodes. The resulting model has a best fit that suggests that the liquid space is approximately 300 nm across the gap. Given this dimension, the volume of the electrode-membrane space is not large enough to accommodate the volume of larger vesicles in cells such as the mast cells of the beige mouse. Second, the model suggests that flow of solution from the exocytosis event is more important than diffusion. Flow from the finite vesicle volume past the electrode leads to less charge passed. Third, and finally, this system can be used to model transport in the synapse and so it is possible to examine the idea that transmitter flows in addition to diffusing from the synapse. This model should be useful in understanding and quantifying the escape of transmitter from synapses in vivo.
Animals↗
A nanofluidic switching device.
We present a nanofluidic switching function which allows directed transport in lipid bilayer nanotubes ( approximately 100 nm in diameter) between any two or several nanotube-connected vesicle containers (5-10 mum radius) in a large network. Liquid is transported in the form of small vesicular structures (10-15-10-18 L), and the transport direction is controlled by a two-point perturbation technique, where the surface membrane tension is decreased in one container while it is increased in another container. When reaching a target container, the transported vesicular structures could also inject their material into it.
Journal Article↗
Nanofluidic networks based on surfactant membrane technology.
We explore possibilities to construct nanoscale analytical devices based on lipid membrane technology. As a step toward this goal, we present nanotube-vesicle networks with fluidic control, where the nanotube segments reside at, or very close (<2 microm) to optically transparent surfaces. These nanofluidic systems allow controlled transport as well as LIF detection of single nanoparticles. In the weak-adhesion regime, immobilized vesicles can be approximated as perfect spheres with nanotubes attached at half the height of the vesicle in the axial (z) dimension. In the strong-adhesion regime (relative contact area, Sr* approximately 0.3), nanotubes can be adsorbed to the surface with a distance to the interior of the nanotubes defined by the membrane thickness of approximately 5 nm. Strong surface adsorption restricts nanotube self-organization, enabling networks of nanotubes with arbitrary geometries. We demonstrate LIF detection of single nanoparticles (30-nm-diameter fluorescent beads) inside single nanotubes. Transport of nanoparticles was induced by a surface tension differential applied across nanotubes using a hydrodynamic injection protocol. Controlled transport in nanotubes together with LIF detection enables construction of nanoscale fluidic devices with potential to operate with single molecules. This opens up possibilities to construct analytical platforms with characteristic length scales and volume orders of magnitudes smaller than employed in traditional microfluidic devices.
Liposomes↗
Nanotube-vesicle networks with functionalized membranes and interiors.
We describe nanotube-vesicle networks with reconstituted membrane protein from cells and with interior activity defined by an injection of microparticles or molecular probes. The functionality of a membrane protein after reconstitution was verified by single-channel ion conductance measurements in excised inside-out patches from the vesicle membranes. The distribution of protein, determined by fluorescence detection, in the network membrane was homogeneous and could diffuse via a nanotube connecting two vesicles. We also show how injecting small unilamellar protein-containing vesicles can differentiate the contents of individual containers in a network. The combination of membrane activity and interior activity was demonstrated by ionophore-assisted accumulation, and internal Calcium Green-mediated detection, of Ca2+ within a single network container. This system can model a variety of biological functions and complex biological multicompartment structures and might serve as a platform for constructing complex sensor and computational devices.
Biomimetic Materials↗
Artificial cells: unique insights into exocytosis using liposomes and lipid nanotubes.
Exocytosis is the fundamental process underlying neuronal communication. This process involves fusion of a small neurotransmitter-containing vesicle with the plasma membrane of a cell to release minute amounts of transmitter molecules. Exocytosis is thought to go through an intermediate step involving formation of a small lipid nanotube or fusion pore, followed by expansion of the pore to the final stage of exocytosis. The process of exocytosis has been studied by various methods; however, when living cells are used it is difficult to discriminate between the molecular effects of membrane proteins relative to the mechanics of lipid-membrane-driven processes and to manipulate system parameters (e.g., membrane composition, pH, ion concentration, temperature, etc.). We describe the use of liposome-lipid nanotube networks to create an artificial cell model that undergoes the later stages of exocytosis. This model shows that membrane mechanics, without protein intervention, can drive expansion of the fusion pore to the final stage of exocytosis and can affect the rate of transmitter release through the fusion pore.
Animals↗
Electrophoretic separation and confocal laser-induced fluorescence detection at ultralow concentrations in constricted fused-silica capillaries.
Laser-induced fluorescence detection of labeled amino acids in a flowing stream at femtomolar (10(-15)M) concentrations was achieved by using a fused-silica capillary flow-cell comprising a constricted thin-walled detection region with inner diameters (IDs) ranging from 2 to 8 microm. The diameter of the constricted region was made to match a diffraction-limited focus of a uniphase transverse electromagnetic mode (TEM(00)) laser beam. Optimization of capillary dimensions and geometries (i.e., curvature, wall thickness, and outer-inner diameter ratio were performed in order to minimize cylindrical lensing of the focused laser beam. The fluorescence was collected in a confocal optical setup using a 1.3 numerical aperture (NA), 100x oil-immersion objective and a single-photon-counting avalanche diode (SPAD). Under conditions of fluid flow, the constriction in the capillary forces all analytes to traverse across the laser probe volume, resulting in a high sampling efficiency. Fluorescein isothiocyanate-labeled glutamate (FITC-Glu) was electrophoretically separated and detected in capillaries having an ID of 2 microm at the constricted region with detection limits of 250 fM (signal-to-noise ratio (S/N) = 3) in the injected solution.
Amino Acids↗
Single-cell electroporation.
Electroporation is a widely used method for the introduction of polar and charged agents such as dyes, drugs, DNA, RNA, proteins, peptides, and amino acids into cells. Traditionally, electroporation is performed with large electrodes in a batch mode for treatment of a large number of cells in suspension. Recently, microelectrodes that can produce extremely localized electric fields, such as solid carbon fiber microelectrodes, electrolyte-filled capillaries and micropipettes as well as chip-based microfabricated electrode arrays, have proven useful to electroporate single cells and subcellular structures. Single-cell electroporation opens up a new window of opportunities in manipulating the genetic, metabolic, and synthetic contents of single targeted cells in tissue slices, cell cultures, in microfluidic channels or at specific loci on a chip-based device.
Cell Membrane Permeability↗
A cell-based bar code reader for high-throughput screening of ion channel-ligand interactions.
This paper presents a microfluidics-patch clamp platform for performing high-throughput screening and rapid characterization of weak-affinity ion channel-ligand interactions. This platform integrates a microfluidic chip consisting of multiple channels entering an open volume with standard patch clamp equipment. The microfluidic chip is placed on a motorized scanning stage and the method relies on the ability to scan rapidly, on the order of milliseconds, a patch-clamped cell across discrete zones of different solutions created in the open volume. Under ideal conditions, this method has the capacity to obtain kinetically resolved patch clamp measurements and dose-response curves of up to 10(3) ligand solutions in a single day.
Animals↗
Formation of geometrically complex lipid nanotube-vesicle networks of higher-order topologies.
We present a microelectrofusion method for construction of fluid-state lipid bilayer networks of high geometrical complexity up to fully connected networks with genus = 3 topology. Within networks, self-organizing branching nanotube architectures could be produced where intersections spontaneously arrange themselves into three-way junctions with an angle of 120 degrees between each nanotube. Formation of branching nanotube networks appears to follow a minimum-bending energy algorithm that solves for pathway minimization. It is also demonstrated that materials can be injected into specific containers within a network by nanotube-mediated transport of satellite vesicles having defined contents. Using a combination of microelectrofusion, spontaneous nanotube pattern formation, and satellite-vesicle injection, complex networks of containers and nanotubes can be produced for a range of applications in, for example, nanofluidics and artificial cell design. In addition, this electrofusion method allows integration of biological cells into lipid nanotube-vesicle networks.
Lipids↗
Functional screening of intracellular proteins in single cells and in patterned cell arrays using electroporation.
A tool for detection and characterization of intracellular enzyme-substrate and receptor-ligand interactions inside the cytoplasm of single targeted cells or small confined groups of cells is presented. Fluorogenic enzyme substrates and receptor ligands were rapidly delivered by electroosmosis and internalized by electroporation in cells using an electrolyte-filled capillary (EFC) biased at a high voltage. Specifically, alkaline phosphatase and proteases were detected in single NG108-15 cells using fluorescein diphosphate and casein BODIPY FL, respectively. The intracellular 1,4,5-inositol triphosphate (IP3) and ryanodine receptors were detected after EFC introduction of the selective receptor agonists IP3 and cyclic adenosine diphosphate ribose (cADPr), respectively. Receptor activation in both cases resulted in increased cytosolic concentrations of free calcium ions that were measured using the calcium-ion-selective probe, fluo-3. The effect of cADPr could be blocked by coadministration of the ryanodine receptor antagonist ruthenium red. Furthermore, electroporation of a plurality of cells grown in microwell structures (100 x 100 x 45 microm) molded in PDMS is demonstrated. The methods and systems described using an EFC for electroporation and delivery of protein markers, ligands, and substrates might be useful in high-throughput screening of intracellular targets, with applications in proteomics and phenotype profiling, as well as in drug discovery.
Alkaline Phosphatase↗