| 1. |
- Agarwal, A., et al.
(author)
-
Control of the Release of Freely Diffusing Molecules in Single-Cell Electroporation
- 2009
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In: Analytical Chemistry. - : American Chemical Society (ACS). - 0003-2700 .- 1520-6882. ; 81:19, s. 8001-8008
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Journal article (peer-reviewed)abstract
- Single-cell electroporation using an electrolyte-filled capillary is an emerging technique for transient pore formation in adherent cells. Because adherent cells do not have a simple and consistent shape and because the electric field emanating from the tip of the capillary is inhomogeneous, the Schwan equation based on spherical cells in homogeneous electrical fields does not apply. We sought to determine experimental and cell parameters that influence the outcome of a single-cell electroporation experiment. A549 cells were exposed to the thiol-reactive dye Thioglo-1, leading to green fluorescence from intracellular thiol adducts. Electroporation causes a decrease with time of the intracellular fluorescence intensity of Thioglo-1-loaded cells from diffusive loss of thiol adducts. The transient curves thus obtained are well-described by a simple model originally developed by Puc et al. We find that the final fluorescence following electroporation is related to the capillary tip-to-cell distance and cell size (specifically, 2(A/pi)(1/2) where A is the area of the cell's image in pixels. This quantity is the diameter if the image is a circle). In separate experiments, the relationship obtained can be used to control the final fluorescence following electroporation by adjusting the tip-to-cell distance based on cell size. The relationship was applied successfully to A549 as Well as DU 145 and PC-3 cells. Finally, F-tests show that the variability in the final fluorescence (following electroporation) is decreased when the tip-to-cell distance is controlled according to the derived relationship in comparison to experiments in which the tip-cell distance is a constant irrespective of cell size.
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| 2. |
- Agarwal, A., et al.
(author)
-
Effect of cell size and shape on single-cell electroporation
- 2007
-
In: Analytical Chemistry. - : American Chemical Society (ACS). - 0003-2700 .- 1520-6882. ; 79:10, s. 3589-3596
-
Journal article (peer-reviewed)abstract
- Single-cell electroporation was performed using electrolyte-filled capillaries on fluorescently labeled A549 cells. Cells were exposed to brief pulses (50-300 ms) at various cell-capillary tip distances. Cell viability and electroporation success were measured. In order to understand the variability in single-cell electroporation, logistic regression was used to determine whether the probabilities of cell survival and electroporation depend on experimental conditions and cell properties. Both experimental conditions and cell properties (size and shape) have a significant effect on the outcome. Finite element simulations were used to compare bulk electroporation to single-cell electroporation in terms of cell size and shape. Cells are more readily permeabilized and are more likely to survive if they are large and hemispherical as opposed to small and ellipsoidal with a high aspect ratio. The dependence of the maximum transmembrane potential across the cell membrane on cell size is much weaker than it is for bulk electroporation. Observed survival probabilities are related to the calculated fraction of the cell's surface area that is electroporated. Observed success of electroporation is related to the maximum transmembrane potential achieved.
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| 3. |
- Agarwal, A., et al.
(author)
-
Simultaneous maximization of cell permeabilization and viability in single-cell electroporation using an electrolyte-filled capillary
- 2007
-
In: Analytical Chemistry. - : American Chemical Society (ACS). - 0003-2700 .- 1520-6882. ; 79:1, s. 161-167
-
Journal article (peer-reviewed)abstract
- A549 cells were briefly exposed to Thioglo-1, which converts thiols to fluorescent adducts. The fluorescent cells were exposed to short (50-300 ms) electric field pulses (500 V across a 15 cm capillary) created at the tip of an electrolyte-filled capillary. Fluorescence microscopy revealed varying degrees of cell permeabilization depending on the conditions. Longer pulses and a shorter cell-capillary tip distance led to a greater decrease in the cell's fluorescence. Live/dead (calcein AM and propidium iodide) testing revealed that a certain fraction of cells died. Longer pulses and shorter cell-capillary tip distances were more deadly. An optimum condition exists at a cell-capillary tip distance of 3.5-4.5 mu m and a pulse duration of 120-150 ms. At these conditions, > 90% of the cells are permeabilized and 80-90% survive.
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| 4. |
- Ahemaiti, Aikeremu, 1984, et al.
(author)
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A multifunctional pipette for localized drug administration to brain slices
- 2013
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In: Journal of Neuroscience Methods. - : Elsevier BV. - 0165-0270 .- 1872-678X. ; 219:2, s. 292-296
-
Journal article (peer-reviewed)abstract
- We have developed a superfusion method utilizing an open-volume microfluidic device for administration of pharmacologically active substances to selected areas in brain slices with high spatio-temporal resolution. The method consists of a hydrodynamically confined flow of the active chemical compound, which locally stimulates neurons in brain slices, applied in conjunction with electrophysiological recording techniques to analyze the response. The microfluidic device, which is a novel free-standing multifunctional pipette, allows diverse superfusion experiments, such as testing the effects of different concentrations of drugs or drug candidates on neurons in different cell layers with high positional accuracy, affecting only a small number of cells. We demonstrate herein the use of the method with electrophysiological recordings of pyramidal cells in hippocampal and prefrontal cortex brain slices from rats, determine the dependence of electric responses on the distance of the superfusion device from the recording site, document a multifold gain in solution exchange time as compared to whole slice perfusion, and show that the device is able to store and deliver up to four solutions in a series. Localized solution delivery by means of open-volume microfluidic technology also reduces reagent consumption and tissue culture expenses significantly, while allowing more data to be collected from a single tissue slice, thus reducing the number of laboratory animals to be sacrificed for a study. (C) 2013 Elsevier B.V. All rights reserved.
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| 5. |
- Ahemaiti, Aikeremu, 1984, et al.
(author)
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Spatial characterization of a multifunctional pipette for drug delivery in hippocampal brain slices
- 2015
-
In: Journal of Neuroscience Methods. - : Elsevier BV. - 0165-0270 .- 1872-678X. ; 241, s. 132-136
-
Journal article (peer-reviewed)abstract
- Background: Among the various fluidic control technologies, microfluidic devices are becoming powerful tools for pharmacological studies using brain slices, since these devices overcome traditional limitations of conventional submerged slice chambers, leading to better spatiotemporal control over delivery of drugs to specific regions in the slices. However, microfluidic devices are not yet fully optimized for such studies. New method: We have recently developed a multifunctional pipette (MFP), a free standing hydrodynamically confined microfluidic device, which provides improved spatiotemporal control over drug delivery to biological tissues. Results: We demonstrate herein the ability of the MFP to selectively perfuse one dendritic layer in the CA1 region of hippocampus with CNQX, an AMPA receptor antagonist, while not affecting the other layers in this region. Our experiments also illustrate the essential role of hydrodynamic confinement in sharpening the spatial selectivity in brain slice experiments. Concentration-response measurements revealed that the ability of the MFP to control local drug concentration is comparable with that of whole slice perfusion, while in comparison the required amounts of active compounds can be reduced by several orders of magnitude. Comparison with existing method: The multifunctional pipette is applied with an angle, which, compared to other hydrodynamically confined microfluidic devices, provides more accessible space for other probing and imaging techniques. Conclusions: Using the MFP it will be possible to study selected regions of brain slices, integrated with various imaging and probing techniques, without affecting the other parts of the slices.
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| 6. |
- Ainla, Alar, 1982, et al.
(author)
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A Microfluidic Diluter Based on Pulse Width Flow Modulation
- 2009
-
In: Analytical Chemistry. - : American Chemical Society (ACS). - 0003-2700 .- 1520-6882. ; 81:13, s. 5549-5556
-
Journal article (peer-reviewed)abstract
- We demonstrate that pulse width flow modulation (PWFM) can be used to design fasts accurate, and precise multi-stage dilution modules for microfluidic devices. The PWFM stage unit presented here yields 10-fold dilution, but several PWFM stages can be connected in series to yield higher-order dilutions. We have combined two stages in a device thus capable of diluting up to 100-fold, and we have experimentally determined a set of rules that can be conveniently utilized to design multistage diluters. Microfabrication with resist-based molds yielded geometrical channel height variances of 7% (22.9(16) mu m) with corresponding hydraulic resistance variances of similar to 20%. Pulsing frequencies, channel lengths, and flow pressures can be chosen within a wide range to establish the desired diluter properties. Finally, we illustrate the benefits of on-chip dilution in an example application where we investigate the effect of the Ca2+ concentration on a phospholipid bilayer spreading from a membrane reservoir onto a SiO2 surface. This is one of many possible applications where flexible concentration control is desirable.
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| 7. |
- Ainla, Alar, 1982, et al.
(author)
-
A Microfluidic Pipette for Single-Cell Pharmacology
- 2010
-
In: Analytical Chemistry. - : American Chemical Society (ACS). - 0003-2700 .- 1520-6882. ; 82:11, s. 4529-4536
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Journal article (peer-reviewed)abstract
- We report on a free-standing microfluidic pipette made in poly(dimethylsiloxane) having a circulating liquid tip that generates a self-con-fining volume in front of the outlet channels. The method is flexible and scalable as the geometry and the size of the recirculation zone is defined by pressure, channel number, and geometry. The pipette is capable of carrying out a variety of complex fluid processing operations, such as mixing, multiplexing, or gradient generation at selected cells in cell and tissue cultures. Using an uptake assay, we show that it is possible to generate dose response curves in situ from adherent Chinese hamster ovary cells expressing proton-activated human transient receptor potential vanilloid (hTRPV1) receptors. Using confined superfusion and cell stimulation, we could activate hTRPV1 receptors in single cells, measure the response by a patch-clamp pipette, and induce membrane bleb formation by exposing selected groups of cells to formaldehyde/dithiothreitol-containing solutions, respectively. In short, the microfluidic pipette allows for complex, contamination-free multiple-compound delivery for pharmacological screening of intact adherent cells.
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| 8. |
- Ainla, Alar, 1982, et al.
(author)
-
A multi-purpose microfluidic pipette for single-cell analysis
- 2010
-
In: 14th International Conference on Miniaturized Systems for Chemistry and Life Sciences 2010, MicroTAS 2010; Groningen; Netherlands; 3 October 2010 through 7 October 2010. - 9781618390622 ; 2, s. 932-934
-
Conference paper (peer-reviewed)abstract
- We report a multi-purpose microfluidic pipette, with a recirculating liquid tip. This device, made in poly(dimethylsiloxane), enables contamination-free manipulation and chemical stimulation of selected single cells in cell collectives or tissue slices. The pipette is capable of carrying out a variety of complex fluid processing functionalities, such as mixing, multiplexing, or gradient generation. The concept is flexible and scalable as the geometry and the size of the recirculation zone is defined by pressure, channel number, and geometry. We have applied the pipette in a fluorescence uptake assay, electrophysiology studies and for chemical induction of membrane protrusion from biological cells.
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| 9. |
- Ainla, Alar, 1982, et al.
(author)
-
A multifunctional pipette
- 2012
-
In: Lab on a Chip - Miniaturisation for Chemistry and Biology. - : Royal Society of Chemistry (RSC). - 1473-0189 .- 1473-0197. ; 12:7, s. 1255-1261
-
Journal article (peer-reviewed)abstract
- Microfluidics has emerged as a powerful laboratory toolbox for biologists, allowing manipulation and analysis of processes at a cellular and sub-cellular level, through utilization of microfabricated features at size-scales relevant to that of a single cell. In the majority of microfluidic devices, sample processing and analysis occur within closed microchannels, imposing restrictions on sample preparation and use. We present an optimized non-contact open-volume microfluidic tool to overcome these and other restrictions, through the use of a hydrodynamically confined microflow pipette, serving as a multifunctional solution handling and dispensing tool. The geometries of the tool have been optimised for use in optical microscopy, with integrated solution reservoirs to reduce reagent use, contamination risks and cleaning requirements. Device performance was characterised using both epifluorescence and total internal reflection fluorescence (TIRF) microscopy, resulting in similar to 200 ms and similar to 130 ms exchange times at similar to 100 nm and similar to 30 mm distances to the surface respectively.
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| 10. |
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| 11. |
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| 12. |
- Bauer, Brigitte, 1978, et al.
(author)
-
Direct reconstitution of plasma membrane lipids and proteins in nanotube-vesicle networks
- 2006
-
In: Langmuir. - : American Chemical Society (ACS). - 1520-5827 .- 0743-7463. ; 22:22, s. 9329-9332
-
Journal article (peer-reviewed)abstract
- We demonstrate here that nanotube-vesicle networks can be constructed directly from plasma membranes of cultured cells. We used a combination of dithiothreitol (DTT) and formaldehyde to produce micron-sized plasma membrane vesicles that were subsequently shaped into networks using micromanipulation methods previously used on purely synthetic systems. Only a single cell is required to derive material sufficient to build a small network. This protocol covers the advantages of reconstitution in vesicles, such as full control over the solution environment, while keeping the proteins in their original surroundings with the proper orientation. Furthermore, control of membrane protein and lipid content in the networks is achievable by employing different cell types, for example, by overexpression of a desired protein or the use of specialized cell-types as sources for rare proteins and lipids. In general, the method provides simple accessibility for functional studies of plasma membrane constituents. Specifically, it provides a direct means to functionalize nanotube-vesicle networks with desired proteins and lipids for studies of transport activity both across membranes (protein-mediated) and across nanotubes (diffusion), and substrate conversion down to the single-molecule limit. Nanotube-vesicle networks can adopt different geometries and topologies and undergo shape changes at will, providing a flexible system for changing the physical and chemical environment around, for example, a membrane protein. Furthermore, the method offers unique possibilities for extracting membrane and protein material for nanotechnological sensor and analytical devices based on lipid membrane networks.
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| 13. |
- Bauer, Brigitte, 1978, et al.
(author)
-
Proteomic Analysis of Plasma Membrane Vesicles
- 2009
-
In: Angewandte Chemie - International Edition. - : Wiley. - 1433-7851 .- 1521-3773. ; 48:9, s. 1656-1659
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Journal article (peer-reviewed)abstract
- (Figure Presented) A simple and scalable method is presented for harvesting, purification, and on-chip processing of mammalian plasma membrane vesicles (PMVs) optimized for downstream proteome analysis. After immobilization on a microfluidic flow-cell of PMVs, the embedded membrane proteins are proteolytically digested, and the peptides harvested and analyzed by LC-MS/MS. Over 93% of the detected proteins are plasma-membrane-derived. © 2009 Wiley-VCH Verlag GmbH & Co. KGaA.
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| 14. |
- Billerit, Celine, 1977, et al.
(author)
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Formation of giant unilamellar vesicles from spin-coated lipid films by localized IR heating
- 2012
-
In: Soft Matter. - : Royal Society of Chemistry (RSC). - 1744-6848 .- 1744-683X. ; 8:42, s. 10823-10826
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Journal article (peer-reviewed)abstract
- We report a novel method for the generation of GUVs (generate unilamellar vesicles) from spin-coated lipid films by means of localized heating. This technique enables GUV formation from both charged and neutral lipid species, as well as from a complex lipid mixture, in various ionic strength conditions. Encapsulation was possible during and after GUV formation.
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| 15. |
- Billerit, Celine, 1977, et al.
(author)
-
Heat-induced formation of single giant unilamellar vesicles
- 2011
-
In: Soft Matter. - : Royal Society of Chemistry (RSC). - 1744-6848 .- 1744-683X. ; 7:20, s. 9751-9757
-
Journal article (peer-reviewed)abstract
- Giant unilamellar vesicles (GUVs) are an excellent model system for the investigation of lipid membranes, the study of membrane proteins and ion channels in a biomimetic environment, and in the creation of artificial cells. Here, we describe a novel method for the preparation of GUVs from single multilamellar liposomes by means of directed infrared laser heating. Our method generates individual unilamellar vesicles at selected locations, not only from natural and artificial lipid mixtures containing negatively charged lipids, but also from preparations of single lipids, such as neutral phosphatidylethanolamine. The presented method provides a new efficient resource for giant vesicle research and offers an alternative to the electroformation and de/rehydration techniques.
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| 16. |
- Bridle, Helen, 1979, et al.
(author)
-
Automated control of local solution environments in open-volume microfluidics
- 2007
-
In: Analytical Chemistry. - : American Chemical Society (ACS). - 0003-2700 .- 1520-6882. ; 79:24, s. 9286-9293
-
Journal article (peer-reviewed)abstract
- We present an open-volume microfluidic system capable of on-fine modification of a patterned laminar flow by using programmable inlet valves. Each separate solution environment in the flow pattern can be independently exchanged between different preloaded input solutions where each exchange requires 20 s. The number of flow patterns that can be generated by one device is N-n, where N represents the number of valve inlets and n the number of microchannels in the microfluidic system. Furthermore, the system can be operated as a combinatorial mixer, in which mixture of the different input solutions can be obtained independently in each channel. Since the flow patterns are generated in an open volume, they are accessible to many different detection methods and types of probes, e.g., microelectrodes, cells, or cell fragments. This technology offers the possibility to adjust the flow pattern composition in response to an output from a probe. This is the first step toward creating an automated feedback device controlled by, for example, biological cells.
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| 17. |
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| 18. |
- Czolkos, Ilja, 1980, et al.
(author)
-
Controlled formation and mixing of two-dimensional fluids
- 2007
-
In: Nano Letters. - : American Chemical Society (ACS). - 1530-6992 .- 1530-6984. ; 7:7, s. 1980-1984
-
Journal article (peer-reviewed)abstract
- We introduce a novel technique for the controlled spreading and mixing of lipid monolayers from multilamellar precursors on surfaces covered by the hydrophobic epoxy resin SU-8. The lipid spreads as a monolayer as a result of the high surface tension between SU-8 and the aqueous environment. A micropatterned device with SU-8 lanes, injection pads, and mixing regions, surrounded by hydrophilic Au, was constructed to allow handling of lipid films and to achieve their mixing at controlled stoichiometry. Our findings offer a new approach to dynamic surface functionalization and decoration as well as surface-based catalysis and self-assembly.
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| 19. |
- Czolkos, Ilja, 1980, et al.
(author)
-
Flow control of thermotropic lipid monolayers
- 2011
-
In: Soft Matter. - : Royal Society of Chemistry (RSC). - 1744-6848 .- 1744-683X. ; 7:15, s. 6926-6933
-
Journal article (peer-reviewed)abstract
- There is an increasing interest in using liquid crystalline media as mobile phases in two-dimensional nanofluidic systems. Their small-scale, reduced dimensionality, and plentiful opportunities for functionalisation render such phases advantageous. However, flow control has been difficult to achieve, as the wetting processes which drive area expansion are not dynamically controllable. Here, we report on temperature-controlled monolayer spreading of 1,2-dielaidoyl-sn-glycero-3-phosphoethanolamine (DEPE) on the hydrophobic substrates SU-8, and Teflon AF (amorphous fluoropolymer). The gel/liquid phase transition of DEPE at T(c) similar to 38 degrees C is exploited to toggle spreading of a molecular lipid film on SU-8. We observed that on Teflon AF, DEPE monolayer spreading occurs even below T(c), and exhibits strongly accelerated spreading above the phase transition temperature. Our results demonstrate that switching DEPE monolayer spreading on and off, or, alternatively, switching between fast and slow area expansion, is a feasible approach towards establishing control over lipid film flow in two-dimensional fluidic systems. We also present a chip-based device integrating a patterned surface for 2D-microfluidics and on-chip heating.
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| 20. |
- Czolkos, Ilja, 1980, et al.
(author)
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High-Resolution Micropatterned Teflon AF Substrates for Biocompatible Nanofluidic Devices
- 2012
-
In: Langmuir. - : American Chemical Society (ACS). - 1520-5827 .- 0743-7463. ; 28:6, s. 3200-3205
-
Journal article (peer-reviewed)abstract
- We describe a general photolithography-based process for the microfabrication of surface-supported Teflon AF structures. Teflon AF patterns primarily benefit from superior optical properties such as very low autofluorescence and a low refractive index. The process ensures that the Teflon AF patterns remain strongly hydrophobic in order to allow rapid lipid monolayer spreading and generates a characteristic edge morphology which assists directed cell growth along the structured surfaces. We provide application examples, demonstrating the well-controlled mixing of lipid films on Teflon AF structures and showing how the patterned surfaces can be used as biocompatible growth-directing substrates for cell culture. Chinese hamster ovary (CHO) cells develop in a guided fashion along the sides of the microstructures, selectively avoiding to grow over the patterned areas.
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| 21. |
- Czolkos, Ilja, 1980, et al.
(author)
-
Molecular phospholipid films on solid supports
- 2011
-
In: Soft Matter. - : Royal Society of Chemistry (RSC). - 1744-6848 .- 1744-683X. ; 7:10, s. 4562-4576
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Journal article (peer-reviewed)abstract
- Phospholipid membranes are versatile structures for mimicking biological surfaces. Bilayer and monolayer membranes can be formed on solid supports, leading to enhanced stability and accessibility of the biomimetic molecular film. This has facilitated functional studies of membrane proteins and aided the development of membrane-based applications in, for example, biosensing, self-assembled reaction kinetics and catalysis. Assembly and preparation of lipid films on supporting surfaces is a challenging engineering task with the goal of fabricating mechanically, chemically and thermodynamically stable lipid membranes. In this review, the current state of the art of molecularly thin lipid layer fabrication is presented with an emphasis on support materials, film formation mechanisms, characterisation methods, and applications.
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| 22. |
- Czolkos, Ilja, 1980, et al.
(author)
-
Platform for Controlled Supramolecular Nano-Assembly
- 2009
-
In: Nano Letters. - : American Chemical Society (ACS). - 1530-6992 .- 1530-6984. ; 9:6, s. 2482-2486
-
Journal article (peer-reviewed)abstract
- We here present a two-dimensional (2D) micro/nano-fluidic technique where reactant-doped liquid−crystal films spread and mix on micro- and nanopatterned substrates. Surface-supported phospholipid monolayers are individually doped with complementary DNA molecules which hybridize when these lipid films mix. Using lipid films to convey reactants reduces the dimensionality of traditional 3D chemistry to 2D, and possibly to 1D by confining the lipid film to nanometer-sized lanes. The hybridization event was observed by FRET using single-molecule-sensitive confocal fluorescence detection. We could successfully detect hybridization in lipid streams on 250 nm wide lanes. Our results show that the number and density of reactants as well as sequence of reactant addition can be controlled within confined liquid crystal films, providing a platform for nanochemistry with potential for kinetic control.
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| 23. |
- Davidson, M., et al.
(author)
-
Fluid mixing in growing microscale vesicles conjugated by surfactant nanotubes
- 2005
-
In: Journal of the American Chemical Society. - : American Chemical Society (ACS). - 1520-5126 .- 0002-7863. ; 127:4, s. 1251-1257
-
Journal article (peer-reviewed)abstract
- This work addresses novel means for controlled mixing and reaction initiation in biomimetic confined compartments having volume elements in the range of 10-12 to 10-15 L. The method is based on mixing fluids using a two-site injection scheme into growing surfactant vesicles. A solid-state injection needle is inserted into a micrometer-sized vesicle (radius 5-25 μm), and by pulling on the needle, we create a nanoscale surfactant channel connecting injection needle and the vesicle. Injection of a solvent A from the needle into the nanotube results in the formation of a growing daughter vesicle at the tip of the needle in which mixing takes place. The growth of the daughter vesicle requires a flow of surfactants in the nanotube that generates a flow of solvent B inside the nanotube which is counterdirectional to the pressure-injected solvent. The volume ratio ψ between solvent A and B inside the mixing vesicle was analyzed and found to depend only on geometrical quantities. The majority of fluid injected to the growing daughter vesicle comes from the pressure-based injection, and for a micrometer-sized vesicle it dominates. For the formation of one daughter vesicle (conjugated with a 100-nm radius tube) expanded from 1 to 200 μm in radius, the mixing ratios cover almost 3 orders of magnitude. We show that the system can be expanded to linear strings of nanotube-conjugated vesicles that display exponential dilution. Mixing ratios spanning 6 orders of magnitude were obtained in strings of three nanotube-conjugated micrometer-sized daughter vesicles.
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| 24. |
- Davidson, M., et al.
(author)
-
Nanotube-vesicle networks with functionalized membranes and interiors
- 2003
-
In: Journal of the American Chemical Society. - 0002-7863 .- 1520-5126. ; 125:2, s. 374-8
-
Journal article (peer-reviewed)abstract
- 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.
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| 25. |
- Dommersnes, Paul Gunnar, 1971, et al.
(author)
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Marangoni transport in lipid nanotubes
- 2005
-
In: Europhysics Letters. - : IOP Publishing. - 0295-5075 .- 1286-4854. ; 70:2, s. 271-277
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Journal article (peer-reviewed)abstract
- We give a simple picture of transient and stationary transport in lipid nanotubes connecting two vesicles, when a difference of membrane tension is imposed at time t = 0, either by pressing one vesicle with a micro-fiber, or by adding a surplus of membrane lipid. The net result is a transport of membrane from the tense towards the floppy vesicle. In the early stage, the tube remains cylindrical, and the gradient of surface tension gives rise to two opposite flows of the internal liquid: a Marangoni flow towards the direction of high tension, and a Poiseuille flow (induced by Laplace pressures) in the opposite direction. At longer time, the tube reaches a stationary state, where curvature and Laplace pressure are balanced. Marangoni flows dominate for giant vesicles, where Laplace pressure is negligible.
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| 26. |
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| 27. |
- Erkan, Yavuz, 1982, et al.
(author)
-
Direct immobilization of cholesteryl-TEG-modified oligonucleotides onto hydrophobic SU-8 surfaces
- 2007
-
In: Langmuir. - : American Chemical Society (ACS). - 1520-5827 .- 0743-7463. ; 23:10, s. 5259-5263
-
Journal article (peer-reviewed)abstract
- We introduce a rapid, simple one-step procedure for the high-yield immobilization of cholesteryl- tetraethyleneglycol-modified oligonucleotides ( chol- DNA) at hydrophobic sites made of SU-8 photoresist. Topographic structures of SU-8 were microfabricated on microscope glass coverslips sputtered with a Ti/Au layer. Upon application, chol-DNA adsorbed to the SU-8 structures from solution, leaving the surrounding gold surface free of chol- DNA. chol-DNA immobilization is complete within 15 min and yields a surface coverage in the range of 20- 95 pmol/ cm(2), which corresponds to a film density of 10(12)- 10(13) molecules/cm(2). chol-DNA immobilization is stable and can be sustained despite rinsing, drying, dry storage for several hours, and rehydration of chips. Furthermore, complementary DNA in solution hybridizes efficiently to immobilized chol-DNA. We introduce a rapid, simple one-step procedure for the high-yield immobilization of cholesteryl-tetraethyleneglycol-modified oligonucleotides (chol-DNA) at hydrophobic sites made of SU-8 photoresist. Topographic structures of SU-8 were microfabricated on microscope glass coverslips sputtered with a Ti/Au layer. Upon application, chol-DNA adsorbed to the SU-8 structures from solution, leaving the surrounding gold surface free of chol-DNA. chol-DNA immobilization is complete within 15 min and yields a surface coverage in the range of 20-95 pmol/cm(2), which corresponds to a film density of 10(12)-10(13) molecules/cm(2). chol-DNA immobilization is stable and can be sustained despite rinsing, drying, dry storage for several hours, and rehydration of chips. Furthermore, complementary DNA in solution hybridizes efficiently to immobilized chol-DNA.
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| 28. |
- Granfeldt, D., et al.
(author)
-
Controlling desensitized states in ligand-receptor interaction studies with cyclic scanning patch-clamp Protocols
- 2006
-
In: Analytical Chemistry. - : American Chemical Society (ACS). - 0003-2700 .- 1520-6882. ; 78:23, s. 7947-7953
-
Journal article (peer-reviewed)abstract
- Ligand-gated ion channels are important control elements in regulation of cellular activities, and increasing evidence demonstrates their role as therapeutic targets. The receptors display complex desensitization kinetics, occurring on vastly different time scales. This is not only important in biology and pharmacology but might also be of technological significance since populations of receptors under microfluidic control can function analogously to DRAM memory circuits. Using a novel microfluidic method, and computer modeling of the receptor state distributions, we here demonstrate that GABA(A) receptor populations can be controlled to display high or low EC50 values, depending on input function (i.e., the exact pattern of agonist application). The sensitivity of the receptors can be tuned up to 40-fold (beta-alanine) by the particular agonist exposure pattern. By combining patch-clamp experiments with computer modeling of receptor state distributions, we can control the assembly of receptors in desensitized states. The technique described can be used as an analytical tool to study the effect of desensitization on the activity of ion channel effectors. We describe the differential blocking effect of the competitive antagonist bicuculline on the high- and low-EC50 GABA(A) receptor preparations and conclude that the inhibition is dramatically dependent on how the different desensitized states are populated. Furthermore, we show that both GABA and beta-alanine, two agonists with different affinity but similar efficacy, induce the same type of desensitization behavior and memory effects in GABA(A) receptors.
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| 29. |
- Gözen, Irep, 1980, et al.
(author)
-
Calcium-ion-controlled nanoparticle-induced tubulation in supported flat phospholipid vesicles
- 2011
-
In: Soft Matter. - : Royal Society of Chemistry (RSC). - 1744-6848 .- 1744-683X. ; 7:20, s. 9706-9713
-
Journal article (peer-reviewed)abstract
- Biological nanotubes, often referred to as tunneling nanotubes, fulfill important functions within the cell, e.g. by supplying cell components, conducting signals and transporting virus particles and bacteria. Many functions are still insufficiently understood, which has placed these nanostructures in the focus of recent investigation. We report here on our observations of transient tubulation in nanoparticle-containing, supported flat giant unilamellar vesicles (FGUVs). The encapsulation of nanoparticles in FGUVs in conjunction with low (1-4 mM) Ca(2+) in the ambient buffer solution resulted in transient tube formation. Tubes extended from the FGUV up to a length of several hundred micrometres and exhibited, on some occasions, vesicle encapsulation. The findings represent an interesting confirmation of several reported theoretical and practical models of tube formation in biological or biomimetic systems.
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| 30. |
- Gözen, Irep, 1980, et al.
(author)
-
Evidence for membrane flow through pores in stacked phospholipid membranes
- 2012
-
In: Soft Matter. - : Royal Society of Chemistry (RSC). - 1744-6848 .- 1744-683X. ; 8:23, s. 6220-6225
-
Journal article (peer-reviewed)abstract
- We present evidence for an inter-bilayer transport mode of lipid molecules in solid-supported double bilayer membranes via membrane defects. The observation of avalanche rupturing in isolated membrane areas of flat giant unilamellar vesicles, i.e., double bilayers, in the distal (with respect to the solid support) bilayer revealed migration of the membrane material between the stacked bilayers, which we can explain by the presence of defects. The presence of the interconnections was indirectly observed by monitoring the area development of the isolated membrane patches during rupturing. These new findings can help to better describe the dynamic processes in biological structures, featuring stacks of phospholipid bilayers, such as mitochondrial shape dynamics.
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| 31. |
- Gözen, Irep, 1980, et al.
(author)
-
Fractal avalanche ruptures in biological membranes
- 2010
-
In: Nature Materials. - 1476-4660 .- 1476-1122. ; 9:11, s. 908-912
-
Journal article (peer-reviewed)abstract
- Bilayer membranes envelope cells as well as organelles, and constitute the most ubiquitous biological material found in all branches of the phylogenetic tree. Cell membrane rupture is an important biological process, and substantial rupture rates are found in skeletal and cardiac muscle cells under a mechanical load(1). Rupture can also be induced by processes such as cell death(2), and active cell membrane repair mechanisms are essential to preserve cell integrity(3). Pore formation in cell membranes is also at the heart of many biomedical applications such as in drug, gene and short interfering RNA delivery(4). Membrane rupture dynamics has been studied in bilayer vesicles under tensile stress(5-8), which consistently produce circular pores(5,6). We observed very different rupture mechanics in bilayer membranes spreading on solid supports: in one instance fingering instabilities were seen resulting in floral-like pores and in another, the rupture proceeded in a series of rapid avalanches causing fractal membrane fragmentation. The intermittent character of rupture evolution and the broad distribution in avalanche sizes is consistent with crackling-noise dynamics(9). Such noisy dynamics appear in fracture of solid disordered materials(10), in dislocation avalanches in plastic deformations(11) and domain wall magnetization avalanches(12). We also observed similar fractal rupture mechanics in spreading cell membranes.
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| 32. |
- Gözen, Irep, 1980, et al.
(author)
-
Repair of large area pores in supported double bilayers
- 2013
-
In: Soft Matter. - : Royal Society of Chemistry (RSC). - 1744-6848 .- 1744-683X. ; 9:10, s. 2787-2792
-
Journal article (peer-reviewed)abstract
- We describe an experimental system where we can generate, and subsequently close, multiple large membrane ruptures in supported double bilayers. We show in this study for the first time that large membrane pores (similar to 10-150 mu m in size) in flat phospholipid vesicles can be reduced in size or completely closed by a pore edge tension driven area reduction mechanism. We can dynamically control the membrane tension of a flat giant unilamellar vesicle and its interplay with the surface adhesion to a solid support. Adhesion to the support surface causes increased membrane tension, which eventually relaxes by the formation of several pores in the membrane. We show that the tension propagation time tau(max) is exceptionally long in this system, which allows for simultaneous opening of multiple pores. The pores can be stabilized by Ca2+-mediated pinning sites in the interior of the flat giant unilamellar vesicle. After pore formation followed by pinning, we depleted Ca2+ ions resulting in removal of pinning and relaxation of membrane tension. This allows the pore to close, driven by the pore edge tension.
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| 33. |
- Hannestad, Jonas, 1981, et al.
(author)
-
Kinetics of Diffusion-Mediated DNA Hybridization in Lipid Monolayer Films Determined by Single-Molecule Fluorescence Spectroscopy
- 2013
-
In: ACS Nano. - : American Chemical Society (ACS). - 1936-086X .- 1936-0851. ; 7:1, s. 308-315
-
Journal article (peer-reviewed)abstract
- We use single-molecule fluorescence microscopy to monitor individual hybridization reactions between membrane-anchored DNA strands, occurring in nanofluidic lipid monolayer films deposited on Teflon AF substrates. The DNA molecules are labeled with different fluorescent dyes, which make it possible to simultaneously monitor the movements of two different molecular species, thus enabling tracking of both reactants and products. We employ lattice diffusion simulations to determine reaction probabilities upon interaction. The observed hybridization rate of the 40-mer DNA was more than 2-fold higher than that of the 20-mer DNA. Since the lateral diffusion coefficient of the two different constructs is nearly identical, the effective molecule radius determines the overall kinetics. This implies that when two DNA molecules approach each other, hydrogen bonding takes place distal from the place where the DNA is anchored to the surface. Strand closure then propagates bidirectionally through a zipper-like mechanism, eventually bringing the lipid anchors together. Comparison with hybridization rates for corresponding DNA sequences in solution reveals that hybridization rates are lower for the lipid-anchored strands and that the dependence on strand length is stronger.
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| 34. |
- Hurtig, Johan, 1974, et al.
(author)
-
Electrophoretic transport in surfactant nanotube networks wired on microfabricated substrates
- 2006
-
In: Analytical Chemistry. - : American Chemical Society (ACS). - 0003-2700 .- 1520-6882. ; 78:15, s. 5281-5288
-
Journal article (peer-reviewed)abstract
- Nanofluidic devices are rapidly emerging as tools uniquely suited to transport and interrogate single molecules. We present a simple method to rapidly obtain compact surfactant nanotube networks of controlled geometry and length. The nanotubes, 100- 300 nm in diameter, are pulled from lipid vesicles using a micropipet technique, with multilamellar vesicles serving as reservoirs of surfactant material. In a second step, the nanotubes are wired around microfabricated SU-8 pillars. In contrast to unrestrained surfactant networks that minimize their surface free energy by minimizing nanotube path length, the technique presented here can produce nanotube networks of arbitrary geometries. For example, nanotubes can be mounted directly on support pillars, and long stretches of nanotubes can be arranged in zigzag patterns with turn angles of 180 degrees. The system is demonstrated to support electrophoretic transport of colloidal particles contained in the nanotubes down to the limit of single particles. We show that electrophoretic migration velocity is linearly dependent on the applied field strength and that a local narrowing of the nanotube diameter results from adhesion and bending around SU-8 pillars. The method presented here can aid in the fabrication of fully integrated and multiplexed nanofluidic devices that can operate with single molecules.
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| 35. |
- Hurtig, Johan, 1974, et al.
(author)
-
Injection and Transport of Bacteria in Nanotube-Vesicle Network
- 2008
-
In: Soft Matter. - : Royal Society of Chemistry (RSC). - 1744-6848 .- 1744-683X. ; 4:7, s. 1515-1520
-
Journal article (peer-reviewed)abstract
- Microinjection of bacteria (the MG1655 strain of E. coli.) into unilamellar lipid vesicles contained in surface-immobilized nanotube-vesicle networks is demonstrated. Injected baceria can not escape from one vesicle to another as the size of interconnecting nanotubes is too small (~200 nm in diameter) to allow for entry. Bacteria can, however, be moved from one vesicle to another by using Marangoni flows. Thus, single or several species can be transferred to a neighboring vesicle at will. The technique offers new possibilities for live matter functionalization into synthetic host networks, and may provide a means of studying the effect of compartmentalization and chemical species on a single bacterium. Thus, it may serve as an experimental platform to study how vesicle-encapsulated bacteria evade destruction in macrophages or how bacteria surf along thin membrane nanotubes toward connected macrophage cell bodies
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| 36. |
- Hurtig, Johan, 1974, et al.
(author)
-
Topographic SU 8 Substrates for Immobilization of Three-Dimensional Nanotube-Vesicle Networks
- 2004
-
In: Langmuir. - : American Chemical Society (ACS). - 1520-5827 .- 0743-7463. ; 20:13, s. 5637-5641
-
Journal article (peer-reviewed)abstract
- A method to create three-dimensional compact liposome networks, adhered to topographic substrates fabricated in the epoxy polymer SU-8 was described. The polymeric photoresist SU-8 is a highly suitable material for soybean lipid vesicle immobilization displaying sufficient contact potential for secure anchoring of liposomes and minimal lipid spreading. The material allows fluid-state lipid membrane structures to retain their structural integrity for long time periods. The use of the construction techniques increases the compactness of lipid nanotube networks as the tube density from a single vesicle is increased when access to the off-equatorial area is provided.
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| 37. |
- Jansson, Erik, 1984, et al.
(author)
-
Effect of cholesterol depletion on the pore dilation of TRPV1
- 2013
-
In: Molecular Pain. - : SAGE Publications. - 1744-8069. ; 9:1
-
Journal article (peer-reviewed)abstract
- The TRPV1 ion channel is expressed in nociceptors, where pharmacological modulation of its function may offer a means of alleviating pain and neurogenic inflammation processes in the human body. The aim of this study was to investigate the effects of cholesterol depletion of the cell on ion-permeability of the TRPV1 ion channel. The ion-permeability properties of TRPV1 were assessed using whole-cell patch-clamp and YO-PRO uptake rate studies on a Chinese hamster ovary (CHO) cell line expressing this ion channel. Prolonged capsaicin-induced activation of TRPV1 with N-methyl-D-glucamine (NMDG) as the sole extracellular cation, generated a biphasic current which included an initial outward current followed by an inward current. Similarly, prolonged proton-activation (pH 5.5) of TRPV1 under hypocalcemic conditions also generated a biphasic current including a fast initial current peak followed by a larger second one. Patch-clamp recordings of reversal potentials of TRPV1 revealed an increase of the ion-permeability for NMDG during prolonged activation of this ion channel under hypocalcemic conditions. Our findings show that cholesterol depletion inhibited both the second current, and the increase in ion-permeability of the TRPV1 channel, resulting from sustained agonist-activation with capsaicin and protons (pH 5.5). These results were confirmed with YO-PRO uptake rate studies using laser scanning confocal microscopy, where cholesterol depletion was found to decrease TRPV1 mediated uptake rates of YO-PRO. Hence, these results propose a novel mechanism by which cellular cholesterol depletion modulates the function of TRPV1, which may constitute a novel approach for treatment of neurogenic pain.
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| 38. |
- Jansson, Erik, 1984, et al.
(author)
-
Microfluidic Flow Cell for Sequential Digestion of Immobilized Proteoliposomes
- 2012
-
In: Analytical Chemistry. - : American Chemical Society (ACS). - 0003-2700 .- 1520-6882. ; 84:13, s. 5582-5588
-
Journal article (peer-reviewed)abstract
- We have developed a microfluidic flow cell where stepwise enzymatic digestion is performed on immobilized proteoliposomes and the resulting cleaved peptides are analyzed with liquid chromatography–tandem mass spectrometry (LC–MS/MS). The flow cell channels consist of two parallel gold surfaces mounted face to face with a thin spacer and feature an inlet and an outlet port. Proteoliposomes (50–150 nm in diameter) obtained from red blood cells (RBC), or Chinese hamster ovary (CHO) cells, were immobilized on the inside of the flow cell channel, thus forming a stationary phase of proteoliposomes. The rate of proteoliposome immobilization was determined using a quartz crystal microbalance with dissipation monitoring (QCM-D) which showed that 95% of the proteoliposomes bind within 5 min. The flow cell was found to bind a maximum of 1 μg proteoliposomes/cm2, and a minimum proteoliposome concentration required for saturation of the flow cell was determined to be 500 μg/mL. Atomic force microscopy (AFM) studies showed an even distribution of immobilized proteoliposomes on the surface. The liquid encapsulated between the surfaces has a large surface-to-volume ratio, providing rapid material transfer rates between the liquid phase and the stationary phase. We characterized the hydrodynamic properties of the flow cell, and the force acting on the proteoliposomes during flow cell operation was estimated to be in the range of 0.1–1 pN, too small to cause any proteoliposome deformation or rupture. A sequential proteolytic protocol, repeatedly exposing proteoliposomes to a digestive enzyme, trypsin, was developed and compared with a single-digest protocol. The sequential protocol was found to detect 65% more unique membrane-associated protein (p
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| 39. |
- Jesorka, Aldo, 1967, et al.
(author)
-
COMPLEX NANOTUBE-LIPOSOME NETWORKS
- 2009
-
In: Methods in Enzymology. - 1557-7988 .- 0076-6879. ; 464:C, s. 309-325
-
Journal article (peer-reviewed)abstract
- Surfactant nanotube-vesicle networks (NVN) belong to the smallest artificial devices known to date for performing controlled chemical operations with enzymes. Newly established means for transport of chemical reactants between containers, as well as advancements in initiation and control of chemical reactions in such systems have opened pathways to new devices with a resolution down to the single-molecule level. Here, we summarize the fabrication and functionalization of complex nanotube-liposome networks for such devices, and discuss related aspects of their application for studying chemical kinetics and materials transport phenomena in ultrasmall-scale bio-mimetic environments.
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| 40. |
- Jesorka, Aldo, 1967, et al.
(author)
-
Controlling Chemistry in Dynamic Nanoscale Systems
- 2010
-
In: Springer Series in Chemical Physics. - Berlin, Heidelberg : Springer Berlin Heidelberg. - 0172-6218. - 9783642025969 ; 96, s. 449-468
-
Conference paper (other academic/artistic)abstract
- The biological cell, the fundamental building block of the living world, is a complex maze of compartmentalized biochemical reactors that embed tens of thousands of chemical reactions running in parallel. Several, if not all, reactors are systematically interconnected by a web of nanofluidic transporters, such as nanotubes, vesicles, and membrane pores with ever-changing shapes and structures [1]. To initiate, terminate, or control chemical reactions, small-scale poly-/pleiomorphic systems undergo rapid and violent shape changes with energy barriers close to kBT , where, due to the small dimensions, diffusional mixing of reactants is rapid. The geometry, i.e. volume, and shape changes can be utilized to control both kinetic and thermodynamic properties of the system. This is in sharp contrast to the man-made macroscopic bioreactors, in which mixing of reactants is aided by mechanical means, such as stirring or sonication, under the assumption that reactions take place in volumes that do not change over time. Such reaction volumes are compact, like a sphere, a cube, or a cylinder, and do not provide for variation of shape. Ordinarily, reaction rates, mechanisms, and thermodynamic properties of chemical reactions in condensed media are based on these assumptions. A number of important questions and challenges arise from these facts. For example, how will we achieve fundamental understanding of how reactor shape affects chemistry on the nanoscale, how do we develop appropriate and powerful experimental model systems, and last but not least what impact will this knowledge have on the design and function of nanotechnological devices with new operation modes derived from natural principles.
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| 41. |
- Jesorka, Aldo, 1967, et al.
(author)
-
Controlling the internal structure of giant unilamellar vesicles by means of reversible temperature dependent sol-gel transition of internalized poly(N-isopropyl acrylamide)
- 2005
-
In: Langmuir. - : American Chemical Society (ACS). - 1520-5827 .- 0743-7463. ; 21:4, s. 1230-1237
-
Journal article (peer-reviewed)abstract
- In this work, we present preparation and basic applications of lipid-bilayer-enclosed picoliter volumes (microcontainers) of solutions of poly(N-isopropylacrylamide) (PNIPAAm). Giant unilamellar vesicles (GUVs) were prepared from phospholipids using a standard swelling procedure and subsequently surface immobilized. Clear, slightly viscous solutions of PNIPAAm of varying concentration in aqueous buffer were directly pressure-microinjected into the GUVs, using a submicrometer-sized, pointed capillary. The GUV was subjected to changing temperature over a 21-40 °C range. The typical phase transition of the polymeric material upon heating and cooling across the lower critical solution temperature was followed using optical microscopy and shown to be reversible over multiple sequential heating/cooling cycles without compromising the integrity of the GUV membrane. Fluorescent, carboxylic acid modified 200 nm latex beads, co-injected with the PNIPAAm solution, were temperature-reversibly immobilized during the phase transition, practically freezing the Brownian motion of the entrapped particles in the volume. Furthermore, a co-injected water soluble fluorescent polysaccharide - dye conjugate was shown not to migrate from the aqueous phase into the hydrophobic polymer part upon heating, whereas the fluorescent beads were completely but reversibly immobilized in the hydrophobic domains of dense polymer agglomerates. The system reported here provides a feasible method for the reversible stabilization and solidification of GUV interior volumes, e.g., as a micrometer-sized model system for controlled drug release.
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| 42. |
- Jesorka, Aldo, 1967, et al.
(author)
-
Generation of phospholipid vesicle-nanotube networks and transport of molecules therein
- 2011
-
In: Nature Protocols. - : Springer Science and Business Media LLC. - 1754-2189 .- 1750-2799. ; 6:6, s. 791-805
-
Journal article (peer-reviewed)abstract
- We describe micromanipulation and microinjection procedures for the fabrication of soft-matter networks consisting of lipid bilayer nanotubes and surface-immobilized vesicles. These biomimetic membrane systems feature unique structural flexibility and expandability and, unlike solid-state microfluidic and nanofluidic devices prepared by top-down fabrication, they allow network designs with dynamic control over individual containers and interconnecting conduits. The fabrication is founded on self-assembly of phospholipid molecules, followed by micromanipulation operations, such as membrane electroporation and microinjection, to effect shape transformations of the membrane and create a series of interconnected compartments. Size and geometry of the network can be chosen according to its desired function. Membrane composition is controlled mainly during the self-assembly step, whereas the interior contents of individual containers is defined through a sequence of microneedle injections. Networks cannot be fabricated with other currently available methods of giant unilamellar vesicle preparation (large unilamellar vesicle fusion or electroformation). Described in detail are also three transport modes, which are suitable for moving water-soluble or membrane-bound small molecules, polymers, DNA, proteins and nanoparticles within the networks. The fabrication protocol requires similar to 90 min, provided all necessary preparations are made in advance. The transport studies require an additional 60-120 min, depending on the transport regime.
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| 43. |
- Jesorka, Aldo, 1967, et al.
(author)
-
Liposomes: Technologies and Analytical Applications
- 2008
-
In: Annual Review of Analytical Chemistry. - 1936-1327 .- 1936-1335. ; 1, s. 801-832
-
Journal article (peer-reviewed)abstract
- Liposomes are structurally and functionally some of the most versatile supramolecular assemblies in existence. Since the beginning of active research on lipid vesicles in 1965, the field has progressed enormously and applications are well established in several areas, such as drug and gene delivery. In the analytical sciences, liposomes serve a dual purpose: Either they are analytes, typically in quality-assessment procedures of liposome preparations, or they are functional components in a variety of new analytical systems. Liposome immunoassays, for example, benefit greatly from the amplification provided by encapsulated markers, and nanotube-interconnected liposome networks have emerged as ultrasmall-scale analytical devices. This review provides information about new developments in some of the most actively researched liposome-related topics
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| 44. |
- Jesorka, Aldo, 1967, et al.
(author)
-
NANOFLUIDICS Neither shaken nor stirred
- 2012
-
In: Nature Nanotechnology. - : Springer Science and Business Media LLC. - 1748-3387 .- 1748-3395. ; 7:1, s. 6-7
-
Journal article (peer-reviewed)
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| 45. |
|
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| 46. |
- Karlsson, A., et al.
(author)
-
Controlled initiation of enzymatic reactions in micrometer-sized biomimetic compartments
- 2005
-
In: Journal of Physical Chemistry B. - 1520-6106 .- 1520-5207. ; 109:4, s. 1609-17
-
Journal article (peer-reviewed)abstract
- We present a technique to initiate chemical reactions involving few reactants inside micrometer-scale biomimetic vesicles (10(-12) to 10(-15) L) integral to three-dimensional surfactant networks. The shape of these networks is under dynamic control, allowing for transfer and mixing of two or several reactants at will. Specifically, two nanotube-connected vesicles were filled with reactants (substrate and enzyme, respectively) by microinjection. Initially, the vesicles are far apart and any diffusive mixing (on relevant experimental time scales) between the contents of the separated vesicles is hindered because of the narrow diameter and long axial extension of the nanotube. To initiate a reaction, the vesicles were brought close together, the nanotube was consumed by the vesicles and at a critical distance, the nanotube-vesicle junctions were dilated leading to formation of one spherical reactor, and hence mixing of the contents. We demonstrate the concept using a model enzymatic reaction, which yields a fluorescent product (two-step hydrolysis of fluorescein diphosphate by alkaline phosphatase), where product formation was measured as a function of time using a FRAP fluorescence microscopy protocol. By comparing the enzymatic activity with bulk measurements, the enzyme concentration inside the vesicle could be determined. Reactions could be followed for systems having as few as approximately 15 enzyme molecules confined to a reactor vesicle. To describe the experiments we use a simple diffusion-controlled reaction model and solve it using a survival probability approach. The agreement with experiment is qualitative, but the model describes the trends well. It is shown that the model correctly predicts (i) single-exponential decay after a few seconds, and (ii) that the substrate decay constant depends on the number of enzymes and geometry of reaction container. The numerical correction factor Lambda is introduced in order to ensure semiquantitative agreement between experiment and theory. It was shown that this numerical factor depends weakly on vesicle radius and number of enzymes, thus it is sufficient to determine this factor only once in a single calibration measurement.
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| 47. |
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| 48. |
|
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| 49. |
- Karlsson, A., et al.
(author)
-
Nanofluidic networks based on surfactant membrane technology
- 2003
-
In: Analytical Chemistry. - 0003-2700 .- 1520-6882. ; 75:11, s. 2529-37
-
Journal article (peer-reviewed)abstract
- 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.
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| 50. |
- Karlsson, A., et al.
(author)
-
Nanofluidic networks based on surfactant membrane technology
- 2003
-
In: Analytical Chemistry. - 0003-2700 .- 1520-6882. ; 75:11, s. 2529-37
-
Journal article (peer-reviewed)abstract
- 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 (
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