Patent Application
20150321152
The most important class of two-dimensional fluids are biological membranes. Biomembranes consist of lipids and are the key components of all living organisms, defining walls of cells and organelles inside them, which allows separating different chemical cellular processes from each other. In order to fulfill transport and control requirements of life, these membranes host numerous membrane proteins, which act as gateways responsible for uptake of nutrients and transmitting chemical signals from and to the surrounding, to name a few examples. Besides membrane proteins, other micro- and nano-scale objects, ranging from vesicles, bacteria and viruses to synthetic nanoparticles, are of interest under the aspect of binding or associating to biological membranes.
This invention provides a method and a system to locally create, modify and use two-dimensional fluid membrane structures on a surface.
One aspect of the invention provides a method for fabrication of a membrane on a surface. The method includes: providing a surface interfacing two environments, wherein one of the environments is a liquid; providing a flow-recirculating fluidic device having channel exits in the liquid environment in proximity of the surface; and locally delivering one or more processing solutions. The one or more processing sources include one or more membrane sources adapted and configured to form a membrane on the surface.
This aspect of this invention can have a variety of embodiments. In one embodiment, the flow-recirculating fluidic device is a microfluidic device. The channel exits can be positioned at a distance of about 10 μm to about 100 μm from the surface.
The method can further include controlling a positioning device to facilitate translation of the channel exits relative to the surface. The positioning device can be adapted and configured to translate the channel exits to create a two-dimensional fluid membrane having a geometry defined by a translation path. The positioning device can be a micromanipulator. The positioning device can be a scanning stage. The positioning device can include an electronic controller adapted and configured to control speed and trajectory of translation.
The processing solution can be switched between two or more solutions. Two or more different processing solutions can be delivered simultaneously. One of the processing solutions can be selected from the group consisting of: a chemical conjugation agent addressing a functional group in the membrane, a membrane soluble dye, and a fixation agent. One of the processing solutions can be a detergent adapted and configured to remove a portion of the membrane or its components from the surface.
A gel can be formed near the surface and the gel adapted and configured to remove the membrane from the surface.
Multiple membrane sources can be used to create the membrane with variable composition. Translation and switching between membrane sources can be synchronized, thereby creating spatially heterogeneous membrane geometries. Pulse width flow modulation can be used to create membranes with continuously variable composition.
The membrane can be a surfactant multilayer, a surfactant single layer, a surfactant double layer, a double lipid bilayer, a single lipid bilayer, a lipid monolayer. The membrane can contain additional components.
One of the processing solutions can be adapted and configured to modify the membrane locally.
The membrane may be formed only when the membrane source is in direct contact with the surface. The membrane can be formed through fusion of the membrane source into an existing membrane. The membrane can be spatially heterogeneous in composition, and components of the membrane can be transported in the membrane by two-dimensional diffusion.
The processing solution can be used to remove the membrane or parts of the membrane locally from the surface. Removed membrane components can be collected for analysis.
The surface can be a solid-liquid interface. The surface can include one or more selected from the group consisting of: glass, metal, plastic, rubber, silicon, and oxides.
The surface can be a gel-liquid interface. The surface can contain a pattern. The pattern can have different chemical or physical properties than non-patterned regions of the surface. The membrane can be deposited from the membrane source selectively on the pattern. The membrane may propagate exclusively on the pattern.
Transport properties in the membrane can vary depending on location on the pattern.
The membrane source can comprise surfactant micelles.
The membrane source can comprise surfactant vesicles. The membrane source can comprises membrane extracted from biological cells. The vesicles can be small unilamellar vesicles. The vesicles can be multilamellar vesicles.
The method can further comprise applying a field or gradient along some part of the membrane. The field can be a hydrodynamic flow field sufficient to impart a shear stress on the membrane. The field can be an electric field. The field can be a magnetic field. The gradient can be a thermal gradient. The gradient can be a chemical gradient. The field or gradient can cause the membrane to propagate along the surface. The field or gradient can cause one or more of the membrane components to migrate in the membrane. Migration can cause separation of two or more of the membrane components. The field or gradient can change direction along the membrane. The direction of the field can change at a stable stagnation point for membrane attached components. The stable stagnation point can be used to accumulate or trap membrane-attached components.
The method can further include heating the membrane. Heat can be applied to a local region of the membrane. Heating can be applied globally. Heating can cause a change in membrane fluidity. Heating can cause a change in membrane adhesion strength.
The method can further comprise analyzing the membrane. The analyzing step can utilize a sensor, electrochemical sensing, microscopy, spectroscopy, and/or total internal reflection.
The surface can be a liquid-liquid interface. The surface can be a liquid-gas interface. The liquid can include water. The channel cross-sectional dimensions can be between about 10 μm and about 100 μm.
The modification can be chemical or biological and can be selected from the group consisting of: a conjugation reaction, a cleavage reaction, dissociation, formation or breakage of covalent bond or coordination bond, nucleic acid hybridization, antigen-antibody recognition, and ion pairing. One or more additional components can be selected from the group consisting of: proteins, nanoparticles, microspheres, virus particles, vesicles, cell, bacterial cells, surfactant molecules, lipid molecules and non-lipid molecules.
Another aspect of the invention provides a method of fabricating a membrane. The method includes: introducing a fluidic device into a volume of confining liquid such that one or more outlet ports of the fluidic device are positioned below an outer surface of the confining liquid; and dispensing one or more surfactant-containing liquids from the fluidic device into the confining liquid such that the one or more surfactant-containing liquids are hydrodynamically confined between the confining liquid and a substrate below the confining liquid, thereby fabricating a membrane.
This aspect of the invention can have a variety of embodiments. The fluidic device can be a flow-recirculating fluidic device. The flow-recirculating fluidic device can include at least one inlet port.
The confining liquid can be water or an aqueous solution.
The one or more surfactant-containing liquids can include a suspension of vesicles or micelles. The vesicles can rupture upon contact with the substrate in order to form a membrane.
The surface can be a solid substrate. The substrate can include glass. The substrate can bear a pattern. The pattern can have different chemical or physical properties than non-patterned regions of the substrate. The substrate can include one or more selected from the group consisting: metal, plastic, rubber, and silicone.
The substrate can include a liquid. The substrate can be more viscous than the confining liquid. The substrate can be a gel. The substrate can be a hydrogel.
The method can further include translating the fluidic device about the substrate while dispensing the one or more surfactant-containing liquids.
The method can further include dispensing one or more processing solutions. The one or more processing solutions can comprise a chemical conjugation agent addressing a functional group in the membrane. The one or more processing solutions can comprise a membrane-soluble dye. The one or more processing solutions can comprise an antibody. The one or more processing solutions can comprise a detergent.
The processing solution can be dispensed in a sequential manner relative to the one or more surfactant-containing liquids. The processing solution can be dispensed simultaneously with the one or more surfactant-containing liquids. The surfactant-containing liquids can be dispensed using pulse-width modulation so that the membrane will have a variable composition.
The method can further comprise removing a portion of the membrane.
The membrane can be a surfactant multilayer membrane, a surfactant single layer membrane, a double lipid bilayer membrane, a single lipid bilayer membrane, or a lipid monolayer membrane.
The method can further include depositing one or more additional components into the membrane.
The one or more additional components can include one or more selected from the group consisting of: a protein, a nanoparticle, a microsphere, a virus particle, a vesicle, a cell, a bacterial cell, a surfactant molecule, a lipid molecule, and a non-lipid molecule.
The method can further include modifying a local region of the membrane. The modifying step can include one or more selected from the group consisting of: an additive reaction, a cleavage reaction, and a dissociation reaction.
Unless defined otherwise, all technical and scientific terms used herein have the meaning commonly understood by a person skilled in the art to which this invention belongs. The following references provide one of skill with a general definition of many of the terms used in this invention: Singleton et al., Dictionary of Microbiology and Molecular Biology (2nd ed. 1994); The Cambridge Dictionary of Science and Technology (Walker ed., 1988); The Glossary of Genetics, 5th Ed., R. Rieger et al. (eds.), Springer Verlag (1991); and Hale & Marham, The Harper Collins Dictionary of Biology (1991). As used herein, the following terms have the meanings ascribed to them below, unless specified otherwise.
In this disclosure, “comprises,” “comprising,” “containing” and “having” and the like can have the meaning ascribed to them in U.S. patent law and can mean “includes,” “including,” and the like; “consisting essentially of” or “consists essentially” likewise has the meaning ascribed in U.S. patent law and the term is open-ended, allowing for the presence of more than that which is recited so long as basic or novel characteristics of that which is recited is not changed by the presence of more than that which is recited, but excludes prior art embodiments.
The term “microfabrication” is meant to refer to a set of techniques used for fabrication of micro- or nanostructures. In certain preferred embodiments, microfabrication includes, but is not limited only to, the following techniques: photolithography, electron beam lithography, laser ablation, direct optical writing, thin film deposition (spin-coating, spray coating, chemical vapor deposition, physical vapor deposition, sputtering), thin film removal (development, dry etching, wet etching), replica molding (soft lithography), embossing, forming or bonding.
The term “microchannel” is meant to refer to a tube with nano- or microscopic cross-section. In certain preferred embodiments, a microchannel or channel has a size in the range of 0.1-200 μm. In other preferred embodiments of the present invention, microchannels are fabricated into microfluidic devices by means of microfabrication.
The term “macrochannel” is meant to refer to a tube of size larger than a microchannel (>200 μm)
The term “channel” is meant to refer to either a microchannel or a macrochannel.
The term “fluidic device” is meant to refer to a device which is used to handle and move fluids. A microfluidic device is a fluidic device. A capillary is a fluidic device.
The term “microfluidic device” is meant to refer to the microfabricated device comprising microchannels or circuits of microchannels, which are used to handle and move fluids. Preferably, microfluidic devices may include components like junctions, reservoirs, valves, pumps, mixers, filters, chromatographic columns, electrodes, waveguides, sensors, etc. Microfluidic devices can be made of polymer (e.g., PDMS, PMMA, PTFE, PE, epoxy resins, thermosetting polymers), amorphous (e.g., glass), crystalline (e.g., silicon, silicon dioxide) or metallic (e.g., Al, Cu, Au, Ag, alloys) materials. In certain preferred embodiments, a microfluidic device may contain composite materials or may be a composite material. The microfluidic pipette is a microfluidic device.
The term “membrane” is meant to refer to a molecular film with two-dimensional fluidity.
The term “object of interest” is meant to refer to the material entity to be studied, investigated, transported, positioned, separated, or otherwise influenced or modified by means of the invention.
The term “membrane attached” is meant to refer to a relationship between the membrane and the object of interest, which is characterized by a covalent or non-covalent binding or anchoring interaction.
The term “membrane embedded” is meant to refer to a relationship between the membrane and the object of interest, which is characterized by the object of interest being located within the physical boundaries of the membrane.
The term “reservoir” is meant to refer to the liquid volume that encapsulates the membrane.
The term “channel exit” is meant to refer to an open end of a channel that leads into the open volume.
The term “flow-recirculating fluidic device” is meant to refer to a fluidic device that features outflow from the device and aspiration back into the device, such that fluid leaving the device is fully or partially returned into the device. An example of a flow-recirculating device includes two closely-spaced capillaries.
The term “flow-recirculating microfluidic device” is meant to refer to a microfluidic device that features outflow from the device and aspiration back into device, such that fluid leaving the device is fully or partially returned into the device.
The term “mobility” is meant to refer to a parameter of the object relating its velocity to applied force.
The term “processing solution” is meant to refer to a solution which is delivered by the flow-recirculation device to the surface. Examples of functions of processing solution are membrane fabrication, removal, and functionalization.
The term “membrane source” is meant to refer to the processing solution used to fabricate the membrane.
Artificially created lipid membranes are versatile structures for mimicking biomembranes. Lipid membranes can be formed on solid supports, leading to enhanced stability of the molecular film. This enables functional studies of membrane properties, membrane-associated molecules and membrane proteins, and aids the development of applications such as biosensing, 2D chemical reactions and catalysis.
Aspects of this disclosure describes a method to fabricate, modify, remove and utilize a two-dimensional fluid membrane (later referred to as “membrane”) on a surface. The key aspect of the method is the localized assembly of a mesoscale membrane from precursors on a surface and its subsequent manipulation, using an open volume microfluidic device for lipid delivery. This membrane assembly and manipulation method is especially favorable for the convenient and reproducible preparation of molecular films of desired composition and size on a solid support. Compared to other supported membrane preparation methods, this method is more versatile and reproducible.
This disclosure describes a method and a system to fabricate, modify, remove and utilize a two-dimensional fluid membrane on a surface.
The invention comprises a surface 0101 covered by a liquid 0102, and two or more channels 0103, 0104, which are part of a “fluidic device” that is capable of producing a hydrodynamically confined flow 0105 near the surface 01010. The channels of the device can be translated relative to the surface 0101 along or more axes 0106 such that the hydrodynamically-confined flow exposes desired areas of the surface 01010. The system also includes one or more processing solutions, delivered though one or more of the channels to the surface, and confined hydrodynamically in a volume between the outlet of the device and the surface. One or more of the processing solutions contain a “membrane source”, which when brought into contact with the surface forms a two-dimensional fluid membrane (“membrane”) 0107. The membrane 0107 has two-dimensional fluidity, meaning that the building blocks of the membrane 0107 as well as the object attached to it can freely migrate within the membrane 0107.
Two or more processing solutions (0201, 0202) are supplied to the flow-recirculating device (
One embodiment of a flow-recirculating fluidic device is the multifunctional pipette described in International Publication Nos. WO 2011/067670 and WO 2012/153192 and Ainla, et al., Lab Chip 2012, DOI: 10.1039/C2LC20906C). This device allows valveless switching of up to 4 different processing solutions, which are provided from reservoirs inside the device. Other embodiments can comprise metal, glass or plastic capillaries, which are fabricated so that they can be brought sufficiently close to the solid surface and can be brought or are sufficiently close to each other.
External or internal reservoirs can be used for storage and delivery of processing solutions. In one embodiment, the channels are in the size range of 20 to 40 μm, positioned between 1-50 μm above the surface, and 5-50 μm separated from each other. The invention can comprise larger channels, wherein the channel separation scales with channel size. In one embodiment, the flow of processing solution through the recirculation zone can be driven by pressure and vacuum, or by electrical fields.
One or more processing solutions contain a membrane source, which in a preferred embodiment comprises small unilamellar vesicles. Other types of surfactant assemblies can also serve as membrane source. Nonlimiting examples of membrane sources are liposomes, phospholiposomes and niosomes. One or more membrane sources are simultaneously or sequentially recirculated by the flow-recirculating fluidic device, such that they are brought in contact with the surface. Upon contact, the membrane source adheres to the surface, and is there transformed into a membrane. H. Schonherr, J. M. Johnson, P. Lenz, C. W. Frank, S. G. Boxer, Vesicle adsorption and lipid bilayer formation on glass studied by atomic force microscopy, Langmuir 20 (2004) 11600-11606. Different types of membrane can be fabricated. In one embodiment, the fabricated membrane is a monolayer (0904) (
The time of exposure of the surface by the membrane source determines the coverage of the surface, and the fluidic properties of the formed membrane. Short exposure times prevent the formation of a coherent membrane that consistently covers the entire exposed surface. If the surface is insufficiently covered by the membrane source, the membrane is not coherent over the exposed area, and transport within the membrane is not possible. In one embodiment, the time of exposure of the surface is chosen to allow sufficient coverage for two-dimensional transport. In another embodiment, the time of exposure is chosen to provide insufficient coverage for two-dimensional transport.
In one example, membrane source is deposited onto the surface such that the surface area exposed by the flow recirculation is fully covered. In this case deposition stops upon full coverage of the exposed surface area (“nonspreading deposition”) (
An example of nonspreading deposition is shown in
Different membrane sources are either simultaneously, or sequentially supplied. By this means a membrane of desired composition can be fabricated. One example of simultaneous application is on-device mixing prior to supplying the membrane sources to the flow recirculation, another example is mixing on the surface after individual membrane sources have been supplied to the recirculation zone. One example of sequential application is pulse-width-modulation-like flow switching inside the flow-recirculating fluidic device. Another example of sequential application is the insertion of membrane source into a membrane by means of “spreading deposition”.
In one embodiment, multiple individual membrane deposits of well-defined and individually different composition can be fabricated on selected regions on a surface, using a different membrane source on each selected region. Here, the composition of the membrane source is defined prior to deposition.
In the example section (Example 3), sequential deposition (multiplexing) of two differently fluorescently labeled POPC membrane sources is demonstrated.
In one embodiment, membranes can be deposited in different geometries. If the surface is translated relative to the flow-recirculating fluidic device, extended areas on the surface can be covered with the membrane. The shape and geometry of the membrane area deposited depends on the trajectory, speed, and sequence of the translations. In one embodiment, the time of deposition is chosen such that the membrane is coherently covering the whole deposition area and has two-dimensional fluidity over the entire deposition area.
In one embodiment, the invention comprises a positioning device that allows translation of the device relative to the surface. Examples of positioning devices are micromanipulators and scanning stages. In a preferred embodiment, the positioning device features electronic control. In another preferred embodiment the setup comprises a control unit, which allows defining the speed and trajectory of the translation.
In another preferred embodiment, the control unit can also determine the flow rates in one or more channel(s). In one embodiment, the composition of the membrane is the same over the entire deposited membrane geometry. In another embodiment, the composition of the membrane differs over the entire deposited membrane geometry, creating a spatially heterogeneous geometry with respect to membrane composition.
In one embodiment, different deposited geometries are overlapping and are fluidically connected (0308).
In one embodiment, membranes of different composition are deposited in overlapping geometries in such a way, that the geometries are fluidically connected. Membrane components can cross over from one geometry to the other by means of diffusion. Example 6 demonstrates the fabrication and diffusional exchange between overlapping membrane geometries.
In one embodiment, a chemical reactant that dissolves or decomposes the deposited membrane is recirculated on a selected area on deposited membrane geometry, such that the deposited membrane is disassembled and removed from the surface in the exposed area. In another embodiment, new membrane is deposited onto the surface from which the membrane was removed, re-connecting the separated membrane geometries. In another embodiment, new membrane is deposited onto the surface from which the membrane was removed, connecting either geometry with a different membrane geometry in the vicinity. This allows the separation, repair and reconfiguration of deposited membrane geometries, as well as the establishment of reconfigurable membrane networks. In another embodiment, the membrane material that was removed from the surface is collected in an external or on-chip reservoir and used for membrane post-processing or chemical analysis.
In one embodiment, a deposited membrane geometry is functionalized with a chemical or biological reagent.
In one embodiment, the modification involves an additive reaction, where a reagent is coupled to the membrane. In one embodiment, the modification involves a cleavage reaction, or dissociation.
Examples of chemical or biological reagents are proteins, peptides, sugars, lipids, DNA, enzymes, ions, ligands, and small organic molecules. In one embodiment, global modification of a deposited membrane is performed by adding the reagent to the liquid. In another embodiment, modification is performed using a conventional fluidic device such as a glass needle. In another embodiment, local modification is performed by means of the processing solutions of the flow-recirculating fluidic device. In one aspect, the processing solution contains a chemical conjugation agent addressing a functional group available in the membrane.
In one aspect, the processing solution contains a functional molecule coupled to a chemical conjugation agent addressing a functional group available in the membrane.
In another aspect, the processing solution contains a functional molecule addressing a chemical conjugation group available in the membrane. In another aspect, the processing solution contains a functional molecule addressing a complexing group available in the membrane. In another aspect, the processing solution contains a functional molecule addressing a receptor available in the membrane. In another aspect, the processing solution contains a functional molecule addressing a ligand available in the membrane. In one embodiment, the processing solution contains a membrane soluble dye. In one embodiment, the processing solution contains an antibody. The processing solution can contain a detergent. In another aspect, the processing solution causes morphological change of the membrane attached objects (
In one aspect, the processing solution causes fixation or immobilization of the membrane or membrane components. In one aspect, the fixation is caused by antibody binding. In one aspect, the fixation is caused by receptor-ligand binding. In one aspect, the fixation is caused by gel formation near the membrane (
Membrane functionalization can be used in combination with the diverse functionalities of a microfluidic device. In one embodiment, the flow-recirculating fluidic device is a microfluidic device. In another embodiment, the flow-recirculating fluidic device is connected to a microfluidic device. Examples of microfluidic functionalities are the delivery of single chemical or biochemical solutions to the membrane or membrane-attached object, the processing and subsequent delivery of multiple chemical or biochemical solutions to the membrane or membrane-attached object, where processing comprises mixing, dilution, switching and temperature regulation.
Further examples of microfluidic functionalities are the processing of aspirated fluid. The aspirated fluid can comprise membrane, membrane components, membrane-attached objects, and fragments or products of chemical reactions involving membrane or membrane-attached objects. Examples of aspirated fluid comprise DNA, proteins, peptides, lipids, sugars, ions, and ligands. Microfluidic processing functionalities for aspirated fluid can comprise sensing, partitioning, division into aliquots, concentration, dilution, chemical modification, digestion, fractionation, separation, and detection. In some embodiments, the aspirated fluid can be transferred to external processing devices.
Example 8 demonstrates sequential membrane functionalization.
In one embodiment, the membranes are deposited on a patterned surface. The feature size of a pattern can be between 10 nm and the size scale of the entire surface. Patterns can be regions that are physically or chemically different from the remainder of the surface.
Chemical patterns can have a different surface chemistry or different material than the remainder of the surface. Physical patterns can have differences in surface morphology, such as roughness. Examples of chemical patterns are protein coatings, photoresist or polymer coatings, hydrogel coatings, self-assembled monolayers, or deposited thin films. Examples of physical patterns are regular or irregular arrays of particles or pillars, surface roughness resulting from polishing, etching, or sputtering. Examples of methods to produce patterns on surfaces are localized treatment with chemicals, lithography, plasma treatment, coating, physical and chemical deposition, wet and dry etching, chemical and physical etching.
In another aspect, the patterns are defined by the geometry of the features. Examples of geometric patterns are patches, lanes, and interconnected combinations of patches and lanes of variable sizescales from 10 nm to the size scale of the surface. Examples of methods to define geometries are lithography, engraving, embossing, direct writing techniques, and physical masks.
Patterns of different type and size scale can coexist on the same surface.
Example 10 demonstrates controlled lipid flow in patterned 2D channels. In
In one embodiment, a membrane can be exposed to a field or a gradient. The field or gradient acts on membrane or its components causing migration of membrane or membrane components or causing modification of the membrane. Examples of fields are hydrodynamic flow field, electrical field, magnetic fields, and electromagnetic fields. Examples of gradients are surface tension gradient, thermal gradient, solution composition gradient, and surface chemical gradient. The field or gradient can be applied during or after membrane deposition, or both. The source of the field (0808) or gradient can be part of the fluidic device, or can be part of the surface (
In one embodiment, the field or gradient changes polarity or direction. In a preferred embodiment, the lateral component of the field or gradient changes polarity or direction. The point of polarity or direction change (0812) can be a stable stagnation point for membrane components or membrane attached components (0811), which are migrating in the field or gradient (
In one embodiment, a field is applied that features a stagnation point in one or both of its lateral components. The stagnation point, in which the field changes polarity, is located in the area that is covered by the membrane. In one embodiment, the membrane components interact with the field, causing a stable stagnation point for these membrane components. In one embodiment, the stable stagnation point is used to concentrate or accumulate membrane components. In this embodiment, a static field source is translated across the membrane, translating the stable stagnation point. Membrane components or membrane-attached objects migrate in the plane of the membrane together with the stagnation point.
The invention can be utilized to confine, trap, accumulate, position, move, transport, separate and extract objects which are attached to or embedded in a membrane. In one aspect, the invention comprises a method to control the local concentration of membrane attached objects. This control of the concentration means accumulation of the membrane attached objects in the vicinity of the stagnation point.
To produce a stagnation point, a field is generated by a field source near the membrane. If projected to the membrane plane, then the field lines 2004 are converging towards a point (stagnation/sink point) near the field source 2003. The field exerts a force, which is moving the objects towards the stagnation point. Since the objects are membrane-attached, they cannot follow the field towards the field source, and become stably trapped in the stagnation point. While trapped, the objects are governed by two opposing processes, 2D diffusion in the membrane, which would broaden their spatial distribution in the membrane, and field confinement, which is pulling them towards the stagnation point, and keeping them in it. This balance can be adjusted by changing the field strength, or force, around the stagnation point. By changing the field strength, or by moving the field source perpendicularly towards and away from the membrane, the force on the objects is adjusted. The adjustment allows control of the balance, and determines the concentration distribution of the membrane-attached objects around the stagnation point. The maximum achievable concentration has a limit, due to the crowding effects, which are more significant in 2D space than in 3D.
By deliberately moving the stagnation point 2005, the membrane-attached objects follow it, and are thus transported between desired areas, or regions, on the membrane. If the objects are located outside of the stagnation point, they experience a restoring force pulling them towards it. The magnitude of the force depends on the spatial offset of the object from the stagnation point. At small offsets, the force will increase with offset distance, which provides a negative feedback. However, the force has a maximum 2010, after which it will drop rapidly 2008 with distance from the stagnation point 2009.
The force (2013) further depends on the field strength (2004) and size and shape of the membrane-attached object (2011). On the other hand, the transported object experiences viscous drag (2012), which is generated by the membrane and depends on the anchoring. The size, shape and anchoring contribute to a “mobility factor” (μ), which determines the relation (ν=μΦ) between transport velocity (ν) of the object and the field or gradient strength (Φ) around it. These are material properties of the membrane and the membrane-attached object.
Positioning of the stagnation point can be achieved by scanning the field source or changing the field strength in two or more field sources (
In some embodiments, the scanning rate or the force are varied during the separation to sequentially separate objects with different mobility factors μ from each other (2006, 2007).
In some aspects, the invention comprises an additional means for local delivery of materials into the vicinity of the stagnation point. In some aspects, the material can be membrane material for formation of membrane (
In one aspect, the method is used in combination with a means for fixation of the membrane (
Example 9 demonstrates hydrodynamic vesicle trapping.
In one aspect, the control of the concentration is used to concentrate membrane-bound biomolecules, such as nuclear-receptors, or G-protein coupled receptors. In a related embodiment, the invention is used to concentrate membrane proteins in a membrane area so that they assume an ordered structure or crystallize.
The invention can be applied where positioning of a membrane-bound objects close to a sensor or probe is desired. Exemplary embodiments of such sensors or probes are chemical or optical sensors, or electrodes. In one aspect, trapping is used to exert a force onto a membrane-bound object, to measure forces acting on membrane-bound objects, to measure interactions between membrane-bound objects, or to measure interactions between membrane-bound objects and non-membrane-bound objects. An exemplary embodiment is the monitoring of allosteric interactions between membrane proteins (
The invention can be used in combination with detection methods and devices. Such detectors can be utilized to analyze chemical or physical modifications or structural changes of the membrane, or material released from the membrane. In one embodiment, the system is coupled with an analytical detection mechanism. In one embodiment, the detection mechanism is electrical. In one embodiment, the detection is optical. In one embodiment, the detection mechanism is electrochemical. In one embodiment, the detection mechanism is mechanical. In one embodiment, the optical detection mechanism is microscopy-based. The invention can be applied in environments including an optical microscope. Optical microscopes can be both upright and inverted light microscopes. Examples of optical microscopes include fluorescence, epi-fluorescence, confocal, or TIRF microscopes.
In one aspect, the invention includes temperature control in the vicinity of the membrane. In one aspect, the temperature control is global, wherein the temperature of the entire membrane is changed. In another aspect, the temperature control is locally applied to a selected membrane region. In one embodiment, temperature control is used to change the fluidity of the deposited membrane. In one aspect, the combination of temperature control and the phase transition of surfactants in the membrane is used for on/off-switching of transport and diffusion of membrane-attached objects. An exemplary embodiment uses thermotropic lipids as membrane source.
In one embodiment, temperature control is used to change the composition of the membrane. In one aspect, the composition change of the membrane comprises phase transition and phase separation. In another aspect, the composition change of the membrane comprises lipid raft formation. In one embodiment, temperature control is used to modify the chemical reactivity of membrane components. In one embodiment, temperature control is used to modify the chemical reactivity of processing solutions. In one embodiment, temperature control is used to cause morphological changes of the membrane, or disintegrate the membrane.
In one embodiment, temperature control is achieved by means of heating. Examples of heat sources are resistive heaters, Peltier elements, radiative heaters, continuous wave or pulsed laser heaters, and convective heaters. In one preferred embodiment, the temperature control is achieved with a resistive heater under the membrane (
In one embodiment, the temperature control is used to establish a temperature gradient over the membrane or over a local area of the membrane. In one embodiment, the thermal gradient is used to transport membrane material by means of thermomigration or thermo diffusion. In one embodiment, temperature control is used in the vicinity of a stagnation point created by a field (
In some preferred embodiments, the waveguide can be attached to the field source. In some embodiments, the waveguide is an optical fiber. In some other embodiments, the waveguide is microfabricated into the flow-recirculating fluidic device (2203/2209). In another embodiment, the radiation can be provided through a microscope objective 2211 (
Non-limiting examples of the invention are presented herein.
Aspiration control was achieved my means of a microfluidic device described in Ainla, et al. A multifunctional pipette, Lab Chip 2012 coupled to a pressure controller. The microfluidic device has the following properties and dimensions. Channel size: 30 μm×30 μm, channel-channel separation at the tip: 20 μm, channel-bottom separation at the tip: 20 μm, solution reservoirs: 35 μL, flow conductance of supply channels: 53 nL/(s*bar), outflow: 3.2 nL/s, inflow (from 2 channels): 10.6 nL/s, ratio (outflow/inflow): 0.3. The device has the capability to switch in a valveless fashion between four different solutions.
Micropositioning was implemented using manual water hydraulic micromanipulators (Narishige MH-5, Japan) or electronic computer controllable micromanipulators (Scientifica PatchStar, UK). The micromanipulators allow positioning of the pipette and bringing the tip into proximity of the desired objects of interest inside the reservoir.
The experimental setup comprised the multifunctional pipette, a laser scanning confocal microscope Leica IRE2 (Leica Microsystems GmbH, Wetzlar, Germany) equipped with Leica TCS SP2 confocal scanner with AOBS™ and Ar/ArKr and HeNe lasers to provide excitation wavelength 488, 594 and 633 nm. Objectives used were HC PL APO CS 20×0.70 UV and HCX PL APO CS 40×1.25 OIL UV. The sample position was controlled by a scanning stage SCAN IM 120×100 (Märzhäuser Wetzlar GmbH & Co. KG, Wetzlar, Germany), equipped with a CORVUS™ stage controller (Märzhäuser). Both scanning stage and pipette control unit were connected to a PC computer via USB port. Custom software, written in Microsoft Visual C++ (.NET), allowed simultaneous control of stage position and pipette control unit, through which the liquid composition and deposition spot size were controlled. The pipette was held and positioned in the beginning of an experiment by a 3-axis water hydraulic micromanipulator Narishige MH-5 (Japan). During the experiment, the pipette tip was positioned about ˜10-20 μm above the surface, so that materials could be delivered to the surface, while avoiding direct contact, which would damage the lipid film.
Circular microscope cover glasses #1.5 (Menzel-Gläser, 47 mm diameter) were obtained from Thermo Scientific (Sweden). Before use, the glass surfaces were cleaned in the MC2 process laboratory at Chalmers University of Technology. First, the slides were immersed in freshly prepared piranha solution (3:1 v/v mixture of concentrated H2SO4 and 30% H2O2, heated to 100-110° C.) for 10 min, followed by rinsing with deionixed water and blow drying with nitrogen. Thereafter, the glass slices were mounted to a WILLCO WELLS™ dish frame using a dedicated double sided tape and assembly kit (Willco Wells B. V., Amsterdam, Netherlands) and stored in a sealed plastic bag until use.
The cleaned cover glasses were coated with ˜2 μm high SU-8 patterns using the procedure provided by Microchem Corporation. SU-8 2002 (Microchem Corp, Massachusetts, USA) was spin-coated at 3000 rpm for 30 s, followed by soft baking for 2 min at 95° C. on a hot-plate. The SU-8 film was exposed with a dose of 120 mJ/cm2 on a Karl-Süss™ contact mask aligner MA6 (G-line, 5-6 mW/cm2), using the “Low-Vac” mode with a bright-field chromium mask. The substrates were then post-exposure-baked for 2 min at 95° C. on a hot-plate. Thereafter, the SU-8 was developed in SU-8 Developer (Microchem) for 1 min using two sequential bathes, rinsed by spraying with clean developer, and blow dried with nitrogen, yielding a SU-8 coated cover glass where the channels are formed by the exposed glass. The surfaces were plasma cleaned briefly in a Plasma Therm BatchTop RIE (50 W, 250 mTorr, 1 min) plasma chamber, and hard baked for 10 min at 200° C. on a hot plate with slow heating and cooling to prevent crack formation. The so-prepared glass slides were mounted to dish frames like the plain glass slides described in the previous section.
The following vesicle compositions were used:
POPC-488: POPC 99%, ATTO88-DOPE 1%;
POPC-655: POPC 99%, ATTO655-DOPE 1%;
POPC-B: POPC 99%, Biotin-PE 1%;
POPC-488B: POPC 98%, ATTO488-DOPE 1%, Biotin-PE 1%;
POPC-655B: POPC 98%, ATTO655-DOPE 1%, Biotin-PE 1%; and
DOTAP-655: PC 49%, DOTAP 50%, ATTO655-DOPE 1%.
1-Palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), Soy L-α-phosphatidylcholine (PC), 1,2-dioleoyl-3-trimethyl-ammonium-propane (DOTAP), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(cap biotinyl) (Biotin-PE) were obtained from Avanti Polar Lipids (USA). ATTO 488 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (ATTO 488-DOPE) and ATTO 655 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (ATTO 655-DOPE) were provided by ATTO-TEC GmbH (Germany).
For each recipe, a designated amount (see above) of lipids and lipid conjugates in chloroform were mixed and diluted with chloroform to a total concentration of 10 mg/ml. 300 μl of this solution was placed in a 10 ml round bottom flask, and the chloroform was removed in a rotary evaporator at reduced pressure (−80 kPa) over a period of 6 hours. The dry lipid film at the walls of the flask was rehydrated with 3 ml of PBS buffer containing 5 mM Trisma Base (Sigma Aldrich), 30 mM K3PO4 (Sigma Aldrich), 30 mM KH2PO4 (Sigma Aldrich), 3 mM MgSO4*7H2O (Merck), and 0.5 mM Na2EDTA (Sigma Aldrich). The pH was adjusted to 7.4 with H3PO4 (Sigma Aldrich). The rehydrated lipid cake was placed in the fridge (4° C.) overnight. In the final step, the lipid cake was sonicated at 120 W/35 kHz (Bandelin Sonorex, Germany) at room temperature for 15-30 s, to induce the formation of giant vesicles of varying, mainly multiple lamellarity.
Small unilamellar vesicles were prepared on the day the experiments were conducted. 100 μl of the desired vesicle stock solutions were diluted (1:10) with TRIS buffer [125 mM NaCl (Sigma Aldrich), 10 mM TRIS (VWR), 1 mM Na2EDTA (Sigma Aldrich), adjusted to pH=7.4 and sonicated using a Sonics & Materials Vibra Cell™ High Intensity Ultrasonic Liquid Processor (Model 501, CIAB, Chemical Instruments AB, Sweden)] at 15° C. for 10 minutes. The sonicated samples were subsequently ultra-centrifuged at 40,000 rpm at 15° C. for 30 minutes to separate multilamellar aggregates and tip debris (Beckman TL-100 Ultracentrifuge, USA). The small unilamellar vesicles in the supernatant were transferred to a separate tube.
Triton X detergent was obtained from Sigma Aldrich and diluted 1:10 with TRIS buffer.
Antibodies were obtained from Agrisera (Sweden). 0.2 mg of goat anti-biotin antibody conjugated to DyLight 650 (“Goat anti-biotin”) was dissolved in 1 ml of 10 mM TRIS buffer. 0.2 mg of donkey anti-Goat IgG antibody conjugated to DyLight 594 (“Anti-goat”) was dissolved in 1 ml of 10 mM TRIS buffer. All antibody solutions were prepared instantly prior to the experiments, at room temperature.
1 mg powder of ATTO 488-biotin (Sigma Aldrich, Mo., USA) was diluted with 5 ml of HEPES buffer (10 mM HEPES, 100 mM NaCl, pH=7.4 adjusted with NaOH) to a final concentration of 0.2 mg biotin/ml HEPES.
8 μl of NEUTRAVIDIN®-coated latex particles (NEUTRAVIDIN® Labeled Microspheres, 0.2 μm, Yellow-Green Fluorescent (505/515), 1% solids, Invitrogen (Life Technologies); CA, USA) was diluted with 392 μl of HEPES buffer (10 mM HEPES, 100 mM NaCl, pH=7.4). The diluted solution is sonicated for 15 minutes (sonication frequency: 35 kHz, sonication power: 30/120 W, Bandelin Sonorex, Germany) and filtered through a PVDF (Hydrophilic polyvinylidene fluoride) membrane (Acrodisc LC syringe filter with effective filtration area of 13 mm with 0.2 μm pore size, PALL Life Sciences; NY, USA).
The flow-recirculating microfluidic device was loaded with POPC-488 as membrane source. The device was positioned and the flow of lipid vesicle suspension (POPC-488) was switched-on for 160 s (
The flow-recirculating microfluidic device was loaded with DOTAP-655 as membrane source. The device was positioned and the flow of lipid vesicle suspension (DOTAP-655) was switched-on for 600 s (
Switching between different membrane sources was used to create membranes with a variable composition. The flow-recirculating microfluidic device was loaded with two different membrane sources (POPC-488, POPC-655). The pipette was positioned and the flow of lipid vesicle suspension was started. Pulse width modulation was used to multiplex the flow according to the desired composition. The composition was changed over time in steps of 10%.
The flow-recirculating microfluidic device was loaded with two types of lipid vesicles (POPC-488 and DOTAP-655). Thereafter, the pipette was positioned and the flow of POPC-488 vesicle suspension was switched on for 60 s to deposit a POPC spot. Thereafter, the flow was switched off and the pressure conditions were set such that the outflow rate would be about half the previous rate, to ensure that the size of the hydrodynamically confined flow (HCF) volume is reduced and the DOTAP is deposited within the boundaries of the already existing POPC film. Thereafter, DOTAP-655 deposition was switched on. The DOTAP membrane source fused into the previously formed membrane, resulting in an increase in the patch size due to spreading.
The flow-recirculating microfluidic device was loaded with POPC-655 as a membrane source. The device was positioned and the flow of membrane source (POPC-655) was switched-on. Three written membrane geometries (heart, stickman, and smile, as shown in
Multicomponent surface writing of two partially overlapping membrane patches is depicted in
The flow-recirculating microfluidic device was loaded with three solutions: two types of membrane source (POPC-488 and POPC-655) and a solution of dilute Triton-X. Two parallel lanes of 150 μm length were written (
The flow-recirculating microfluidic device was loaded with four solutions: two types of membrane source (POPC-488B and POPC-B) and two antibody (primary goat anti biotin and secondary anti goat) solutions. The device was positioned and a 200 μm lane was written with a step size of 10 μm and a deposition time of 5 s, such that first 100 μm were composed of POPC-488B, followed by 100 μm of POPC-B (
The flow-recirculating microfluidic device was loaded with two different membrane sources (POPC-488, DOTAP-655). This experiment was started by depositing POPC membrane source onto a DOTAP membrane (
Flow-recirculating microfluidic device was loaded with two different membrane sources (POPC-488, DOTAP-655) and positioned onto the SU-8 patterned area (
The surface was approached with the flow-recirculating microfluidic device. NEUTRAVIDIN®-coated particles were injected through the outflow channel of the device, followed by a resting time of ˜10 seconds. Subsequently, biotin solution was injected through the same channel after internal switching between the two solutions into the vicinity of the lipid membrane patch. This biotin blocking of the remaining free binding sites of avidin prevents the encapsulation of the latex particles by the biotinylated lipid membrane. After adjustment of the flow parameters of the pipette, accumulation of particles in a confined area around the stagnation point is observed on the lipid patch. The particles migrate on the surface of the membrane, following the movement of the stagnation point created by the pipette.
Conductive indium tin oxide (ITO) coated cover glass slides were coated with 80 nm of DUPONT® TEFLON® AF amorphous fluoropolymer by spin-coating the coated surfaces and were e-beam patterned, using a JEOL JBX9300 e-beam lithography system, an acceleration voltage of 100 kV, and an exposure dose of 1000 μC/cm2. The exposed pattern defined the edge of the area on which a lipid monolayer is able to spread. The patterned cover glass-slides were submerged to aqueous TRIS buffer under a confocal microscope (as described in the previous example). The flow-recirculating microfluidic device was loaded with DOTAP-655 as membrane source. The device was positioned on the top of deposition area encompassed by the exposed spreading barrier. The flow of lipid vesicle suspension was switched on for 20 min (
The entire contents of all patents, published patent applications, and other references cited herein are hereby expressly incorporated by herein in their entireties by reference.
The functions of several elements may, in alternative embodiments, be carried out by fewer elements, or a single element. Similarly, in some embodiments, any functional embodiment may perform fewer, or different, operations than those described with respect to the illustrated embodiments. Also, functional elements shown as distinct for purposes of illustration may be incorporated within other functional elements separated in different hardware or distributed in a particular implementation.
While certain embodiments according to the invention have been described, the invention is not limited to just the described embodiments. Various changes and/or modifications can be made to any of the described embodiments without departing from the spirit or scope of the invention. Also, various combinations of elements, steps, features, and/or aspects of the described embodiments are possible and contemplated even if such combinations are not expressly identified herein.
This application is a continuation under 35 U.S.C. §120 of International Application No. PCT/IB2014/001089, filed Jan. 19, 2014, which claims the benefit of priority to U.S. Provisional Patent Application Ser. No. 61/754,554, filed Jan. 19, 2013. The entire contents of each of these applications are hereby incorporated by reference herein.
| Number | Date | Country | |
|---|---|---|---|
| 61754554 | Jan 2013 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | PCT/IB2014/001089 | Jan 2014 | US |
| Child | 14801878 | US |
