JPWO2010021320A1 - Uniform diameter vesicle generating apparatus and generating method - Google Patents

Abstract

To develop a method for producing a large amount of a uniform monolayer bilayer vesicle having a size in the range of 1 μm to 100 μm and having a high encapsulation efficiency. The present invention provides an apparatus for producing a bilayer vesicle. The bilayer membrane vesicle production device of the present invention comprises a main channel, a plurality of micro diverticulas opening in the main channel, an atrioventricular chamber communicating with the micro diverticulum, and pulsation preventing means provided in the atrioventricular chamber. Contains multiple structures. The present invention provides a method for producing a bilayer vesicle. The method for producing a vesicle of the present invention comprises the steps of preparing the structure of the present invention, first and second aqueous solutions, and a phase separation fluid, and the phase separation fluid forms a phase separation fluid layer, The phase separation fluid layer is suspended on the side wall surface of the microdiverticulum, and the liquid flow of the second aqueous solution in the main channel shears the phase separation fluid layer, and The first aqueous solution surrounded by the phase separation fluid layer is bundled and cut into vesicles.

Description

  The present invention relates to a bilayer vesicle production apparatus and production method, and more specifically, a production apparatus using a microchannel of bilayer vesicles having an average size of 1 μm to 100 μm and a coefficient of variation of less than 5%, and It relates to a generation method.

  A vesicle consisting of a single layer of amphiphilic molecular membrane, fully encapsulated in the desired volume containing the desired polymer compound, as a microscopic scale chemical reactor, as a drug delivery vehicle, or for various environmental stimuli Use as a responding biosensor is expected. In particular, single-layer phospholipid vesicles having a uniform size in the range of 1 μm to 100 μm have almost the same dimensions as actual living cells, and are in great demand as a model system for complex functions of biological membranes. By creating artificial cells by combining the functions of the elements that make up living cells one by one, it becomes possible to understand and apply more advanced life phenomena and their molecular mechanisms.

However, in the method of adding an aqueous solution to a lipid film dried on a substrate, a uniform single-layer vesicle cannot be produced unless freezing and thawing, sonication or other treatment is performed. The treatment must be selected according to the characteristics of the liquid to be sealed, and is not a universal production method because of low sealing efficiency. The inverse emulsion method overcomes these problems, but the size of vesicles cannot be directly controlled, and mass production is difficult. For example, in the method reported in Non-Patent Document 1, vesicles having an average size of 4.12 μm to 62.4 μm were generated, and the variation in the size was about 10% to about 20% of the size of the vesicle. . In the method reported in Non-Patent Document 2, vesicles having an average size of 4.0 μm were generated, but the variation in size was 50%.
Tan, YC et al., J. Am. Chem. Soc., 128: 5656-5658 (2006). Dittrich, PS et al., Lab Chip, 6: 488-493 (2006)

  Therefore, it is necessary to develop a method for producing a large amount of a uniform single-layer bilayer vesicle having a size in the range of 1 μm to 100 μm and a high encapsulation efficiency.

  The present invention provides an apparatus for producing a bilayer vesicle. The bilayer membrane vesicle generating apparatus of the present invention comprises a main channel, a plurality of micro diverticulas opening in the main channel, an atrioventricular chamber communicating with the micro diverticulum, and pulsation preventing means provided in the atrioventricular chamber. Contains multiple structures.

  In the apparatus for generating a bilayer vesicle according to the present invention, the pulsation preventing means may be a communication path that connects the micro diverticulum and the atrioventricle.

  In the apparatus for generating a bilayer vesicle of the present invention, the structure may include a shearing means.

  In the apparatus for generating a bilayer vesicle of the present invention, a phase separation fluid reservoir is provided on the wall of the micro diverticulum, the phase separation fluid reservoir suspends a phase separation fluid layer, and the phase separation fluid layer comprises: The micro diverticulum may be sealed between the micro diverticulum opening and the atrioventricular chamber.

  In the apparatus for generating a bilayer vesicle according to the present invention, a plurality of phase separation fluid reservoirs are provided on a wall surface of the micro diverticulum, and the plurality of phase separation fluid reservoirs suspend a plurality of phase separation fluid layers, The layer of phase separation fluid may seal the microdiverticulum between the opening of the microdiverticulum and the chamber.

  In the bilayer vesicle production device of the present invention, the phase separation fluid reservoir may be an overhang extending from the side wall surface of the micro diverticulum into the micro diverticulum.

  In the apparatus for generating a bilayer vesicle according to the present invention, the atrioventricular chamber may be provided with a pressure generator.

  In the apparatus for generating a bilayer vesicle of the present invention, the pressure generating unit may generate pressure by expansion of microbubbles generated by laser light.

  In the apparatus for generating a bilayer vesicle according to the present invention, the pressure generating unit may generate pressure by expansion of microbubbles generated by electrolysis.

  In the apparatus for generating a bilayer vesicle according to the present invention, the pressure generating unit may generate pressure by expansion of microbubbles generated by an electric heater.

  In the apparatus for generating a bilayer vesicle of the present invention, the pressure generating unit may generate pressure by a microactuator.

  In the apparatus for generating a bilayer vesicle of the present invention, the atrioventricular chamber may be connected to an external pressure source.

  In the bilayer vesicle production device of the present invention, the external pressure source may be a syringe pump.

  In the apparatus for generating a bilayer vesicle of the present invention, the external pressure source may be a high-pressure gas cylinder.

  In the apparatus for generating a bilayer vesicle according to the present invention, the phase separation fluid passing through the main flow path may flow in one direction, and the shearing means may be provided on the downstream side of the opening of the micro diverticulum.

  In the apparatus for generating a bilayer vesicle according to the present invention, the liquid passing through the main flow path may flow in two directions, the forward direction and the reverse direction, and the shearing means may be provided on the downstream side of the flow in the two directions.

  In the apparatus for producing a bilayer vesicle according to the present invention, the atrioventricular chamber may be connected to an external supply source of an aqueous solution, and the aqueous solution may be supplied to the microdiverticulum via the communication channel.

  In the apparatus for generating a bilayer vesicle according to the present invention, the structure includes one or more aqueous solution flow paths, and the aqueous solution flow paths include at least two of the plurality of phase separation fluid reservoirs. There is a case where the opening of the wall of the micro diverticulum provided between the individual and the external source of each of the plurality of aqueous solutions are communicated.

  In the apparatus for generating a bilayer vesicle according to the present invention, the structure includes a sub-flow channel, and the sub-flow channel is connected to an external supply source of a phase separation fluid, and the phase separation fluid passes through the sub-flow channel There are cases where the phase separation fluid reservoir is supplied.

  In the apparatus for generating a bilayer vesicle of the present invention, the microdiverticulum may be arranged two-dimensionally.

  In the apparatus for generating a bilayer vesicle according to the present invention, the microdiverticulum may be arranged three-dimensionally.

  In the apparatus for producing a bilayer vesicle of the present invention, the phase separation fluid layer may form an interface with an aqueous solution.

  In the apparatus for producing a bilayer vesicle of the present invention, the phase separation fluid may be an organic solution containing amphiphilic molecules.

  In the apparatus for producing a bilayer vesicle of the present invention, the phase separation fluid may be a gas or an organic solution containing no amphiphilic molecules, and the aqueous solution may contain amphiphilic molecules.

  In the apparatus for producing a bilayer vesicle of the present invention, the structure is made of polydimethylsiloxane, and the organic solution may swell and deform polydimethylsiloxane.

  The present invention provides a method for producing a bilayer vesicle. The method for producing a bilayer vesicle according to the present invention comprises a main channel, a plurality of micro diverticulas opening in the main channel, an atrioventricular chamber communicating with the micro diverticulum, and a pulsation preventing means provided in the atrioventricular chamber. A structure including the first and second aqueous solutions, and a phase separation fluid; and flowing the first aqueous solution through the main flow path of the structure to cause the first aqueous solution to flow into the micro diverticulum. Introducing the phase separation fluid into the main flow path of the structure, introducing the phase separation fluid into at least a part of the microdiverticulum, and introducing a second aqueous solution into the main flow path of the structure. Flowing a solution to remove the phase separation fluid in the main flow path, the phase separation fluid forming a phase separation fluid layer, the phase separation fluid layer being suspended on a side wall surface of the micro diverticulum; In the state where the phase separation fluid layer is suspended on the side wall surface of the micro diverticulum, By applying pressure to the first diverticulum, the first aqueous solution is swept into the micro diverticulum via the atrioventricular chamber, and the phase separation is performed so that the tip of the phase separation fluid layer enters the main flow path from the opening of the micro diverticulum. Stretching the fluid layer; and by flowing a second aqueous solution in the main flow channel shearing the phase separation fluid layer, the phase separation fluid layer and a first phase surrounded by the phase separation fluid layer. And the aqueous solution is cut together as a vesicle.

  The present invention provides a method for producing a bilayer vesicle. The method for producing a bilayer vesicle according to the present invention comprises a main channel, a plurality of micro diverticulas opening in the main channel, an atrioventricular chamber communicating with the micro diverticulum, and a pulsation preventing means provided in the atrioventricular chamber. A structure including a hydrophobic solvent, first and second aqueous solutions, and a phase separation fluid; and flowing the hydrophobic solvent through a main flow path of the structure, Introducing into the micro diverticulum; flowing a first aqueous solution into the main flow path of the structure; introducing the first aqueous solution into the micro diverticulum; and phase separation into the main flow path of the structure. Flowing a fluid to introduce the phase separation fluid into at least a part of the microdiverticulum; and flowing a second aqueous solution into the main flow path of the structure to remove the phase separation fluid in the main flow path And the phase separation fluid forms a phase separation fluid layer, In the state suspended from the side wall surface of the micro diverticulum, and in the state where the phase separation fluid layer is suspended from the side wall surface of the micro diverticulum, the hydrophobic solvent in the chamber is heated to generate and expand micro bubbles. By applying pressure, the first aqueous solution is swept into the micro diverticulum through the atrioventricle, and the phase separation fluid enters the main flow path from the opening of the micro diverticulum through the opening of the micro diverticulum. Stretching the layer, and the flow of the second aqueous solution in the main flow channel shears the phase separation fluid layer, whereby the phase separation fluid layer and the first surrounded by the phase separation fluid layer And the aqueous solution is cut into vesicles.

  In the method for producing a bilayer vesicle of the present invention, the phase separation fluid may be an organic solution containing amphiphilic molecules.

  In the method for producing a bilayer vesicle of the present invention, the first and second aqueous solutions may contain amphiphilic molecules, and the phase separation fluid may be a gas or an organic solution that does not contain amphiphilic molecules. .

  In the method for producing a bilayer vesicle of the present invention, the amphiphilic molecule contained in the first aqueous solution may have a composition different from that of the amphiphilic molecule contained in the second aqueous solution.

  In the method for producing a bilayer vesicle according to the present invention, the pulsation preventing means may be a communication path that connects the micro diverticulum and the atrioventricle.

  In the method for producing a bilayer vesicle according to the present invention, the structure includes a shearing unit, and the shearing unit may shear the phase separation fluid layer together with a liquid flow of the second aqueous solution in the main channel. .

  In the method for producing a bilayer vesicle according to the present invention, a phase separation fluid reservoir is provided on a wall surface of the micro diverticulum, the phase separation fluid reservoir suspends a phase separation fluid layer, and the phase separation fluid layer comprises: The micro diverticulum may be sealed between the opening of the micro diverticulum and the communication channel.

  In the method for producing a bilayer vesicle of the present invention, the phase separation fluid reservoir may be an overhang extending from the side wall surface of the micro diverticulum into the micro diverticulum.

  In the method for producing a bilayer vesicle of the present invention, applying pressure to the atrioventricular chamber may be performed by expansion of microbubbles generated by laser light.

  In the method for producing a bilayer vesicle of the present invention, applying pressure to the atrioventricular chamber may be performed by expansion of microbubbles generated by electrolysis.

  In the method for producing a bilayer vesicle of the present invention, applying pressure to the atrioventricular chamber may be performed by expansion of microbubbles generated by an electric heater.

  In the method for producing a bilayer vesicle according to the present invention, applying pressure to the atrioventricle may be performed by a microactuator.

  In the method for producing a bilayer vesicle of the present invention, applying pressure to the atrioventricle may be performed by being connected to an external pressure source.

  In the method for producing a bilayer vesicle of the present invention, the external pressure source may be a syringe pump.

  In the method for producing a bilayer vesicle according to the present invention, the external pressure source may be a high-pressure gas cylinder.

  In the method for producing a bilayer vesicle according to the present invention, the liquid passing through the main channel may flow in one direction, and the shearing means may be provided on the downstream side of the opening of the micro diverticulum.

  In the method for producing a bilayer vesicle according to the present invention, the liquid passing through the main channel may flow in two directions, the forward and reverse directions, and the shearing means may be provided on the downstream side of the two directions of flow.

  In the method for producing a bilayer vesicle according to the present invention, the atrioventricular chamber may be connected to an external supply source of an aqueous solution, and the aqueous solution may be supplied to the microdiverticulum via the communication channel.

  In the method for producing a bilayer vesicle according to the present invention, the structure includes a sub-flow channel, the sub-flow channel is connected to an external supply source of a phase separation fluid, and the phase separation fluid passes through the sub-flow channel. There are cases where the phase separation fluid reservoir is supplied.

  The present invention provides a method for producing a bilayer vesicle of N layers (N is an integer of 2 or 3 or more). In the method for producing an N-layer bilayer vesicle according to the present invention, a main channel, a plurality of micro diverticulas opening in the main channel, an atrioventricular chamber communicating with the micro diverticulum, and a pulsation prevention provided in the atrioventricular chamber A structure including means, a first to (N + 1) th aqueous solution, a first to an Nth phase separation fluid, and a first aqueous solution flowing in the main flow path of the structure Introducing the first aqueous solution into the micro diverticulum, and flowing the first phase separation fluid through the main flow path of the structure to introduce the first phase separation fluid into at least a part of the micro diverticulum. And a second aqueous solution is allowed to flow through the main flow path of the structure to remove the first phase separation fluid in the main flow path, and the first phase separation fluid is the first and second aqueous solutions. A sandwiched first phase separation fluid layer is formed, and the first phase separation fluid layer is located on the side of the micro diverticulum. And a m-th phase separation fluid (m is an integer from 2 to N-1) is allowed to flow through the main flow path of the structure, and the m-th phase separation fluid is allowed to flow through the microdivested chamber. Introducing the m + 1st aqueous solution into the main flow path of the structure, removing the mth phase separation fluid in the main flow path, and removing the mth phase separation fluid. Forming the mth phase separation fluid layer sandwiched between the mth and (m + 1) th aqueous solutions, the mth phase separation fluid layer being suspended on the side wall surface of the micro diverticulum, and the structure Flowing the Nth phase separation fluid through the body main flow path and introducing the Nth phase separation fluid into at least a part of the microdiverticulum; and the N + 1th aqueous solution in the main flow path of the structure To remove the Nth phase separation fluid in the main flow path, A separation fluid forms an Nth phase separation fluid layer sandwiched between the Nth and (N + 1) th aqueous solutions, the Nth phase separation fluid layer being suspended on the side wall surface of the micro diverticulum; In the state where the N phase-separated fluid layers are suspended on the side wall surface of the micro diverticulum, by applying pressure to the atrioventricular chamber, the first aqueous solution is pushed into the micro diverticulum through the atrioventricular chamber. Extending the N phase separation fluid layers so that the tips of the N phase separation fluid layers enter the main flow channel from the opening of the microdiverticulum, and the (N + 1) th aqueous phase in the main flow channel. The liquid flow of the solution shears the N phase separation fluid layers, thereby the N phase separation fluid layers and the first to Nth aqueous solutions surrounded by the N phase separation fluid layers. And the step of cutting as a vesicle. Here, the m-th phase separation fluid (m is an integer from 2 to N-1) flows through the main flow path of the structure, and the m-th phase separation fluid is introduced into at least a part of the micro diverticulum. Flowing the (m + 1) th aqueous solution through the main flow path of the structure to remove the mth phase separation fluid in the main flow path, so that the mth phase separation fluid is the mth and forming the mth phase separation fluid layer sandwiched between the m + 1st aqueous solution, and the mth phase separation fluid layer is suspended on the side wall surface of the microdiverticulum, the value of m being 2, 3, 4,..., N-1 is repeated N-2 times.

  The present invention provides a single-layer bilayer vesicle having an average size in the range of 1 μm to 100 μm, a coefficient of variation of less than 5%, and the same composition of membrane and vesicle fluid.

  The present invention provides a bilayer vesicle produced by the bilayer vesicle production apparatus of the present invention.

  The present invention provides a single-layer bilayer vesicle produced by the bilayer vesicle production method of the present invention.

  The present invention provides an N-layer bilayer vesicle produced by the method for producing an N-layer bilayer vesicle of the present invention.

  The bilayer vesicle of the present invention may have the same membrane protein embedded therein.

  The present invention provides a pharmaceutical composition comprising the bilayer vesicle of the present invention.

  The present invention provides a cosmetic composition comprising the bilayer vesicle of the present invention.

  The present invention provides a food composition comprising the bilayer vesicle of the present invention.

  In the present invention, the vesicle refers to a bilayer membrane enclosing a fluid. A bimolecular membrane refers to a membrane composed of two amphiphilic molecules. Bilayer vesicles can be observed with an optical microscope and can be of any shape and size as long as they can be handled by microchannel technology, but they have a spherical or pancake shape. The vesicle is preferably a vesicle having a diameter of 1 μm to 100 μm and completely enclosed, and more preferably a vesicle having a diameter of 5 μm to 40 μm.

  In the present invention, the size of a vesicle means a distance from one point on the surface of a vesicle to another point farthest measured with an optical microscope calibrated with a micrometer for a substantially spherical vesicle. For pancake vesicles, it refers to the square root of the fluorescently labeled area of an image taken under a microscope.

  In the present invention, the phase separation fluid is an organic solution or a gas. When the phase separation fluid of the present invention is an organic solution, the organic solution contains amphiphilic molecules that are components of the bilayer membrane. When the phase separation fluid of the present invention is a gas, the first and second aqueous solutions of the present invention contain amphiphilic molecules. The amphiphilic molecules contained in the first and second aqueous solutions of the present invention may have the same composition or different compositions. The amphiphilic molecule of the present invention is a molecule that can be dissolved in both water and oil, and a molecule in which a hydrophilic atomic group and a hydrophobic atomic group coexist in the molecule. Amphiphilic molecules of the present invention include, for example, phospholipids such as phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, sphingomyelin, glycolipids such as cerebroside and ganglioside, long-chain alcohols, and long-chain carboxylic acids. Including, but not limited to, soaps and synthetic detergents.

  In the present invention, the hydrophobic solvent is caused to flow into the main flow path prior to the first aqueous solution and is introduced into the chamber from the micro diverticulum or directly from the source of the hydrophobic solvent connected to the chamber. May be placed in the chamber. The hydrophobic solvent may be a solvent having a boiling point lower than that of water, for example, a fluorocarbon.

  In the present invention, the first aqueous solution is encapsulated in vesicles with very high efficiency, as described in the examples below. Therefore, the first aqueous solution can be selected as a solution having the same composition as the desired vesicle solution. For example, an enzyme, a coenzyme, a substrate, a high energy phosphate compound, an electron donor, etc. constituting a transcription, translation, respiration, assimilation, catabolism, information transmission and other reaction systems can be selected alone or in any combination. . As shown in the following examples, the first is a cell-free expression system containing RNA polymerase and transcription factor necessary for transcription and translation, aminoacyl-tRNA and ribosome, and DNA encoding a transcription unit containing a promoter and a structural gene. May be contained in liquid. Fluorescent labeling is preferably performed to identify the generated vesicles.

  In some cases, a viscous polymer such as polyvinylpyrrolidone or sodium alginate is added to the first aqueous solution. In some cases, the polymer is a reversibly gelling polymer such as sodium alginate. Sodium alginate gels in the presence of calcium ions. The inside of the vesicle can be gelled by adding a factor that reversibly gels the polymer, such as calcium, to the second aqueous solution or the liquid outside the vesicle after the produced vesicle is recovered. . By removing the gelling factor from the vesicle external solution, the vesicle internal solution can be freely reversibly gelled. The gelling factor can be removed by, for example, dilution or addition of EDTA or GEDTA (EGTA), which is a specific chelating agent for calcium ions. When a viscous polymer or gel is present inside the vesicle, the chemical reaction system in the vesicle proceeds efficiently by molecular crowding or excluded volume effect. Further, in order to stably maintain the shape of the vesicle, it is useful when the generated vesicle is transported or stored for a long time.

  In the present invention, the organic solution is a component of the bilayer membrane, phospholipids such as phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, sphingomyelin, etc., glycolipids such as cerebroside, ganglioside, etc., cholesterol It is preferable to contain other compounds, membrane proteins, and organic solvents that can dissolve them, such as hexadecacene, squalene, and the like. When the structure of the present invention is made of polydimethylsiloxane, it is preferable to use hexadecacene as a solvent, which can be absorbed by polydimethylsiloxane and swelled and deformed.

  Since the second aqueous solution of the present invention is an external solution of the vesicle, in order to stably maintain the shape of the vesicle, it has a solution composition that is isotonic with the liquid in the vesicle, that is, the first aqueous solution. Is preferred. In addition, bovine serum albumin or polyvinylpyrrolidone may be added to prevent vesicles from being electrostatically adsorbed to the container or to stabilize the vesicles.

  Main flow path, micro diverticulum, pulsation prevention means, communication narrow path, atrioventricular chamber, shearing means, shearing section, fluid reservoir, overhanging section and sub-flow path included in the structure of the vesicle generating apparatus of the present invention, aqueous The connection between the external supply source of the solution and / or the external pressure source and the structure is not limited to the shape shown in the accompanying drawings, and may have any shape. The vesicle production method of the present invention uses not only the bimolecular film formation method disclosed in the present specification but also the bimolecular film formed by other methods including Japanese Patent Application No. 2005-220002. May be formed.

  The main flow path of the structure of the present invention flows so that an aqueous solution, an organic solution and / or a gas supplied from the outside of the structure are introduced into all the micro diverticulas inside the structure. Finally, it is discharged to a drain connected to the outside of the structure. The aqueous solution, organic solution, and / or gas that have flowed through the structure form a layer in the micro diverticulum in the order of flow through the main flow path.

  In the structure according to the present invention, the pulsation preventing means is such that an aqueous solution, an organic solution and / or a gas layer formed in a micro diverticulum is disturbed by an aqueous solution, an organic solution and / or a gas flowing in the main flow path. It is provided so that the layered structure is not destroyed. The pulsation preventing means of the present invention may be a communication path in which the connecting portion between the micro diverticulum and the atrioventricle is thinner than the opening size of the micro diverticulum. Alternatively, it is a valve provided in the chamber or a connection path from the chamber to the outside so that liquid can flow only in the direction from the main flow path to the chamber and is deformed when the pressure in the chamber increases. In some cases, the valve can push the liquid in the minute diverticulum toward the main channel. Further, it may be an orifice or any other pulsation prevention means known to those skilled in the art.

  The shearing means included in the structure of the vesicle generator according to the present invention includes a tongue-shaped object (hereinafter referred to as “tongue-shaped”) including an organic solution layer deformed by a liquid flow in the main flow path and a first aqueous solution. It may be provided at a position away from the opening of the minute diverticulum, provided that the body can be in contact with the body. The shearing means refers to means for concentrating the shearing force that binds the tongue-like body together with the flow of the main flow path. The shearing unit may concentrate the shearing force by ultrasonic waves, electricity, or the like, in addition to the shearing part which is the physical structure of the structure of the present invention. The dimension of the shearing part and the distance from the opening are the properties of the shape and size of the opening, the viscoelasticity of the aqueous solution and organic solution used, the surface tension, and the side wall surface of the micro diverticulum that suspends the organic solution layer and the shearing part. Those skilled in the art can appropriately design according to the wetting characteristics and the like. The shearing part may be a ridge, protrusion, or base that is continuous with the wall surface of the main channel. A structure in which the shearing part is continuous with the opening of the micro diverticulum, for example, the narrowing of the opening of the micro diverticulum or the nozzle, or the downstream side of the tongue that has entered the main channel with the opening on the downstream side of the main channel extending into the main channel There is a case of a structure that supports. Alternatively, a structure such as a bridge, a beam, or a rod supported by protrusions, legs, or columns from the wall surface of one or more main flow paths may be used. The shearing part of the present invention may be provided downstream of the opening of the micro diverticulum with respect to the liquid flow in the main channel. The shearing part of the present invention may be provided on the wall surface of the main channel on the same side as the opening of the micro diverticulum, but may be provided on the wall surface of the main channel facing the opening of the micro diverticulum. In the present invention, the liquid passing through the main channel may flow in only one direction (forward direction), but may also flow in the direction opposite to the forward direction (reverse direction). In that case, one shearing portion of the present invention is provided on each side of the opening of the micro diverticulum along the longitudinal direction of the main flow path. The two shear portions may have different positions and / or shapes, and vesicles of different sizes can be generated by switching the direction of the liquid flow in the main flow path.

  The phase separation fluid, organic solution or gas of the present invention is flowed into the main flow path next to the first aqueous solution, and then introduced into each of the microdivertices by flowing the second aqueous solution through the main flow path. By providing an overhang or other phase separation fluid reservoir in the micro diverticulum near the opening of the micro diverticulum, a larger volume of organic solution can be suspended. The phase separation fluid reservoir of the present invention may be of any structure or shape, provided that a larger volume of organic solution or gas can adhere to the side wall surface of the microdiverticulum. For example, the side wall surface of the micro diverticulum may be subjected to a chemical surface treatment having a high affinity with an organic solution or gas, or the concave surface and / or the convex portion may be provided on the side wall surface to increase the surface area. There may be, and it is not limited to the overhang | projection part shown by FIG. The phase separation fluid reservoir of the present invention switches the functional state of the phase separation fluid reservoir by remotely controlling the affinity of the phase separation fluid of the present invention with the side wall of the microdiverticulum by optical, electrical or other techniques. Preferably it is possible.

  In another embodiment of the present invention, the hydrophobic solvent is flowed before flowing the first aqueous solution into the main flow path. The hydrophobic solvent enters the atrioventricle from the main channel through the micro diverticulum. The hydrophobic solvent is preferably a liquid having a low boiling point such as fluorocarbon. This is because the generation of heat necessary for generating and expanding microbubbles in the pressure generating section in the chamber can be minimized.

  The production of the vesicle of the present invention occurs when the tip of the tongue is pressed against the shearing means and is cut and bundled. Since the vesicle of the present invention is a monolayer bilayer vesicle as described in the following examples, the organic solution does not disappear immediately after the vesicle is separated from the tongue. However, the organic solution is consumed when a large amount of vesicles are produced. Therefore, in the present invention, a sub flow path may be provided to replenish the organic solution. The sub-flow path may form an organic solution flow path along the wall surface of the main flow path even after the second aqueous solution is flowed by surface-treating a part of the wall surface of the main flow path. In some cases, a three-dimensional flow path is provided separately from the path by a fine processing technique. The vesicle production | generation apparatus provided with the subchannel for replenishment of an organic solution can continue producing | generating the vesicle of the same quality stably for a long period of time. Similarly, the first aqueous solution of the present invention is also consumed when large amounts of vesicles are produced. Therefore, in the present invention, the first aqueous solution may be replenished from an external supply source. In an apparatus for producing an N-layer bilayer membrane vesicle having N phase separation fluid reservoirs, an m + 1th aqueous solution is placed between the mth phase separation fluid reservoir and the m + 1st phase separation fluid reservoir. An opening for replenishing from an external source may be provided. By remotely switching the functional state of each phase separation fluid reservoir, it is possible to control which layer is suspended by which phase separation fluid reservoir. Then, the aqueous solution between specific layers can be replenished from the outside. Thereby, the composition of the aqueous solution in the middle of each layer of the N-layer bilayer vesicle can be controlled.

  The vesicle generating device, structure, phase separation fluid, air, organic solution, and aqueous solution of the present invention are subjected to sterilization treatment if necessary, or manufactured with materials and manufacturing methods that are not biotoxic. Or prepared.

  The vesicle generating apparatus of the present invention includes an optical microscope for operating a laser beam and observing a vesicle generating process, a pump for feeding a liquid passing through a main channel, for example, a syringe pump, and a channel. It may be accompanied by auxiliary equipment including, but not limited to, a valve for switching, a controller for an electrical circuit for driving or controlling an actuator of the structure of the present invention, and a temperature controller.

  The structure of the present invention can be formed into a three-dimensional structure by removing a sacrificial layer by combining conventional microfabrication techniques such as photolithography and etching (EE Text Sensor Micromachine Engineering, edited by Hiroyuki Fujita, Ohm (2005)). As an alternative, there is a case where a stamp transfer method (Japanese Patent Application No. 2007-10867 and Onoe, H. et al., Journal of Micromechanics and Microengineering, 17: 1818-1827 (2007)) is used. In addition, a structure having a three-dimensional shape can be created by combining the conventional fine processing technique and the stamp transfer method.

  The structure of the present invention may be a molded product produced using a mold having a three-dimensional shape created by combining conventional fine processing techniques and / or the above-mentioned imprint transfer method alone or in combination. . The molded article may be made using materials including, but not limited to, plastics, ceramics and hydrogels, and composite materials thereof.

  In the present invention, “the micro diverticulum is two-dimensionally arranged” means that the main channel and the micro diverticulum are arranged on a single plane. For example, it refers to a case where a plurality of minute diverticulums are formed on a single glass substrate and arranged to face each other across the main channel.

  In the present invention, “the micro diverticulum is three-dimensionally arranged” means that there are two or more planes on which the main flow path and the micro diverticulum are arranged. For example, it refers to a case where the micro diverticulum is arranged so as to be rotationally symmetric with respect to the direction of the fluid flow in the main channel.

The schematic diagram of the structure of one embodiment of the vesicle production | generation apparatus of this invention. The schematic diagram which shows one embodiment of the vesicle production | generation method of this invention. The partial expansion schematic diagram which shows the mode of a deformation | transformation of the organic solution layer in the vicinity of opening of the micro diverticulum in one embodiment of the structure of the vesicle production | generation apparatus of this invention. Photomicrographs taken continuously under a fluorescence microscope of the production of fluorescently labeled vesicles. The fluorescence micrograph of the spherical vesicle produced | generated by one embodiment of the vesicle production | generation apparatus of this invention. The histogram of the size of the spherical vesicle produced | generated with one embodiment of the vesicle production | generation apparatus of this invention. The fluorescence micrograph of the pancake-like vesicle produced | generated with one embodiment of the vesicle production | generation apparatus of this invention. The histogram of the size of the pancake-like vesicle produced | generated with one embodiment of the vesicle production | generation apparatus of this invention. The graph which shows the relationship between the flow rate Q of a 1st aqueous solution, and the size of a vesicle. Graph showing the relationship between vesicle generation order and vesicle size The schematic diagram of the structure of one embodiment of the vesicle production | generation apparatus of this invention containing the subchannel connected to the external supply source of the organic solution. The microscope picture which shows the time-dependent change of the fluorescence intensity of the vesicle in which (alpha) -hemolysin was embedded. The graph which shows the time-dependent change of the fluorescence intensity of the vesicle in which (alpha) -hemolysin was embedded. The micrograph which shows the time-dependent change after the osmotic shock of the size of a vesicle. The graph which shows the time-dependent change after the osmotic shock of the size of a vesicle. Confocal micrographs before and after encapsulation of fluorescently labeled beads in vesicles. The micrograph which shows the time-dependent change of the fluorescence emission intensity by GFP in a vesicle. The graph which shows the time-dependent change of the fluorescence emission intensity by GFP in a vesicle. The schematic diagram of the structure of one embodiment of the vesicle production | generation apparatus of this invention which forms an amphiphilic molecular film between the 1st and 2nd aqueous solution using gas as a phase separation fluid.

DESCRIPTION OF SYMBOLS 1 Main flow path 2 Micro diverticulum 3 Communication channel 4 Atrioventricular chamber 5 Pressure generation part 6 Aluminum thin film 7 Laser beam 8 Fine bubble 9 Shear part 10 Overhang part 11 Subflow path 12 Amphiphile oriented on the surface of the first aqueous solution Molecule 13 Gas layer 14 Amphiphilic molecule oriented on the surface of the second aqueous solution

  Examples are shown below, but these are intended to illustrate embodiments and are not intended to limit the scope of the present invention.

1. Vesicle Generation The vesicle generation apparatus of one embodiment of the present invention includes a microfluidic device, the microfluidic device including one or more structures of the present invention. The structure is made of polydimethylsiloxane (PDMS) mounted on a glass surface, and a fine structure including a main flow path and a plurality of micro diverticulas opening in the main flow path is provided by fine processing. A pump and a channel switch are connected to the microfluidic device, and the type of liquid flowing in the main channel, the flow rate, and the flow method such as a uniform flow, a pulsating flow or other non-uniform flow can be freely set. Can be set. FIG. 1 is a schematic view of a structure of a vesicle generating apparatus according to one embodiment of the present invention. A micro diverticulum 2 is opened facing the main flow channel 1, and the micro diverticulum 2 communicates with the atrioventricular chamber 4 via the communication narrow path 3. The chamber 4 is provided with a pressure generator 5. In one embodiment of the present invention, the pressure generating unit 5 applies pressure for the expansion of the fine bubbles 8 generated by the laser light 7 focused on the aluminum thin film 6 arranged in a pattern on the bottom surface of the atrioventricular chamber 4. appear. The liquid is pushed out from the chamber 4 to the communication channel 3 by the pressure. However, since the communication channel 3 is narrow and long, a flow resistance is generated. Therefore, the communication channel 3 slows down the liquid flow from the atrioventricular chamber 4 toward the minute diverticulum 2. A shearing portion 9 is provided on the wall surface of the main channel on the downstream side of the opening of the micro diverticulum 2 with respect to the liquid flow in the main channel 1. In one embodiment of the present invention, the depth (dimension in the direction perpendicular to the plane of FIG. 1) mounted on the glass surface of the main channel, the microdiverticulum, and all other polydimethylsiloxane structures is 17 μm. The width of the opening to the wall surface of the main channel 1 of the micro diverticulum 2 is 20 μm. The width of the communication channel 3 is 6 μm, and the distance between the opening surface of the communication channel 3 to the minute diverticulum 2 and the opening surface of the communication channel 3 to the chamber 4, that is, the length of the communication channel 3 is 100 μm. The height of the shearing portion 9 from the main channel wall surface is 10 μm.

  FIG. 2 is a schematic view showing one embodiment of the vesicle production method of the present invention. First, the first aqueous solution, the water-insoluble organic solution containing amphiphilic molecules constituting the vesicle membrane, and the second aqueous solution are passed through the vesicle generator of the present invention in this order (FIG. 2A to FIG. 2). H). The first aqueous solution becomes a vesicle internal solution. The first aqueous solution flowing through the main channel enters the micro diverticulum from the opening of each micro diverticulum of the structure of the present invention, and fills the atrioventricular passage through the communicating narrow channel (FIG. 2A). Next, the organic solution is caused to flow through the main channel. Here, as the organic solution, for example, when an organic solution in which lipid is dissolved with hexadecane 17 is used, polydimethylsiloxane is not dissolved, but it has a property of getting wet on the surface of polydimethylsiloxane, and the organic solution is added to the polydimethylsiloxane resin. Is absorbed and the polydimethylsiloxane resin swells and deforms. Thereby, the said organic solution penetrate | invades into the inside of the micro diverticulum 2 of the structure of this invention (FIG. 2B). Since the organic solution is wetted on the polydimethylsiloxane surface, the organic solution layer is suspended on the opposite side wall surfaces of the micro diverticulum 2 to seal the micro diverticulum. Since the polydimethylsiloxane swells and deforms, the communication path is further narrowed, so that the organic solution does not enter the communication path. Finally, the organic solution in the main channel is washed away by flowing the second aqueous solution through the main channel. The organic solution phase-separates between the first and second aqueous solutions to form an organic solution layer separated by an interface (FIG. 2C). Thereafter, a pressure is generated in the pressure generating section in the chamber, and the first aqueous solution in the microdiverticulum pushes the organic solution layer toward the opening of the microdiverticulum. Since the pressure generated in the pressure generating part in the chamber is slowed down by the communication channel, the organic solution layer is suspended on the side wall surface of the micro diverticulum and the cross section is deformed convexly toward the outside of the micro diverticulum opening. (FIG. 2D). At this time, the layer becomes thin at the tip of the organic solution layer deformed into a convex shape. Since the second aqueous solution flows at a constant speed in the main channel (in FIG. 2, from left to right), when the tip of the organic solution layer further expands and enters the main channel from the opening of the microdiverticulum, The tip is pushed by the liquid flow in the main channel and bends downstream (right in FIG. 2) of the main channel (FIG. 2E). When the tip of the organic solution layer pushed by the first aqueous solution extends beyond the shearing means, the organic solution layer is pressed against the shearing means by the liquid flow in the main channel (FIG. 2F). And the front-end | tip of an organic solution layer is bundled and cut | disconnected (FIG. 2G). Thereafter, the tip of the organic solution layer is extended beyond the shearing means by the first aqueous solution, and the tip is successively bound and cut into vesicles (FIG. 2H). As a result, the vesicle containing the first aqueous solution flows through the second aqueous solution downstream of the main flow path.

  FIG. 3A shows a first aqueous solution (indicated as “Water” with thin hatching), an organic solution (indicated as “Oil”), and a second aqueous solution in the main channel of the vesicle generating apparatus of the present invention. (B) through (H) are the vicinity of the opening of the microdiverticulum in one embodiment of the structure of the vesicle generating device of the present invention. It is a partial expansion schematic diagram which shows the mode of a deformation | transformation of the organic solution layer in FIG. The first aqueous solution pushes air and enters the fine diverticulum and is introduced (FIG. 3B). After the organic solution is introduced into the fine diverticulum (Fig. C), a second aqueous solution enters the fine diverticulum and a layer of the organic solution is formed in the fine diverticulum (Fig. 3D). The molecule of the amphiphilic molecule dissolved in the organic solution layer is composed of a rod-like hydrophobic atomic group and a spherical hydrophilic atomic group, and is hydrophilic at the interface between the first and second aqueous solutions and the organic solution layer. The atomic groups are oriented so as to contact the aqueous solution (FIGS. 3C and D). A shearing portion 9 is provided on the wall surface of the main channel on the downstream side of the opening of the micro diverticulum with respect to the liquid flow in the main channel. In some cases, an overhanging portion 10 is provided on the peripheral edge of the opening where the micro diverticulum faces the main flow channel, and the wall surface of the diverticulum projects to narrow the inner diameter. Since the overhang 10 locally increases the surface area of the inner surface of the diverticulum, a larger volume of organic solution layer can be suspended over the opening of the microdiverticulum. As the organic solution layer stretches, it thins from the wall of the microdiverticulum and / or the periphery of the overhang to the tip of the stretch, and eventually all the amphiphilic molecules are converted into the first or second aqueous solution. A bimolecular film oriented so as to be in contact is formed (FIG. 3E). FIG. 4 is a photomicrograph obtained by adding a fluorescent dye to the first aqueous solution to fluorescently label vesicles and continuously photographing the generation of vesicles from the upper frame to the lower frame under a fluorescence microscope. As shown in FIG. 4, the tongue extending upward is pressed against the shearing means by the leftward flow of the main flow path, and the vesicles are constricted and generated. The fact that a single-layer bilayer film is actually formed in the film of the generated vesicle will be described in Example 3 below.

2. 2. Characterization of generated vesicles 2-1. Vesicle Size The size of the generated vesicle was measured by dissolving a fluorescent dye in the first aqueous solution and observing the vesicle flowing in the main channel with a fluorescence microscope calibrated with a micrometer. In the vesicle generating apparatus of one embodiment of the present invention described in Example 1, by adjusting the pressure of the first aqueous solution generated by the optical tweezers and the flow rate of the second aqueous solution flowing through the main flow path Vesicles with sizes from 5 μm to 40 μm were obtained. FIG. 5 is an example of a fluorescence micrograph of the generated spherical vesicles. FIG. 6 is a histogram of the size of 24 spherical vesicles. The horizontal axis represents the vesicle size (unit: μm), and the vertical axis represents the number of vesicles in each section having a size of about 0.5 μm. As shown in FIG. 6, the sizes of the generated vesicles were distributed almost symmetrically, the average size of the vesicles was 16.5 μm, and the coefficient of variation (cv) was 3.7%. The vesicles produced were stable and maintained a spherical shape for several days. Increasing the flow rate of the main channel increased the shearing force acting on the deformed organic solution layer, resulting in the formation of smaller sized spherical vesicles. When the flow velocity of the main channel was lowered, pancake or spheroid vesicles were generated (FIG. 7). FIG. 8 is a histogram of the size of 31 pancake-like vesicles. The sizes of the generated vesicles are distributed almost symmetrically, the average vesicle size is 28.0 μm, and the coefficient of variation (cv) is 4.4. %Met. When pancake-like vesicles were transferred into a wide volume solution, they became spherical. FIG. 9 shows the result of examining the influence on the size of the vesicle by changing the flow rate of the first aqueous solution flowing into the micro diverticulum from the communication channel. FIG. 9 is a graph plotting the flow velocity Q (unit: pL / sec) on the horizontal axis and the size of vesicles (unit: μm) on the vertical axis. The flow rate Q was obtained by dividing the volume of each vesicle by the time required to generate the vesicle. As shown in FIG. 9, since the correlation coefficient between the flow velocity Q and the vesicle size is as small as −0.058, it was concluded that the influence of the vesicle size is small even if the flow velocity Q is changed. This example demonstrates that the method and apparatus of the present invention can produce large amounts of uniform vesicles similar in size to typical cells.

  FIG. 10 is a graph in which the horizontal axis represents the number of vesicles generated in order of generation, and the vertical axis represents the size of the vesicle. From FIG. 10, it was recognized that the size of the vesicle generated later was larger and the variation in size was larger. This is because the concentration of the amphiphilic molecules in the organic solution decreases as the vesicles are formed, so that the surface tension at the tip of the tongue changes, and the transport of amphiphilic molecules to the tip of the tongue and // indicates that the orientation is non-uniform.

  In the embodiment shown in FIG. 11, a secondary channel 11 connected to an external source of organic solution is provided along the microdiverticulum. In this embodiment, since the organic solution is replenished via the sub-channel 11, a larger amount of vesicles can be generated uniformly. The filling of the organic solution into the sub-channel is preferably performed before flowing the organic solution into the main channel. Further, it is preferable that when the organic solution flows through the main channel, the organic solution is pushed out from the sub channel and the organic solution in the sub channel is continuous with the organic solution flowing in from the main channel. As will be described later, in the embodiment in which the first and second aqueous solutions are separated by the gas layer without using the organic solution, the amphiphilic molecule is added to the aqueous solution, and the gas is passed through the sub-flow channel 11. Replenish.

2-2. Membrane structure of the generated vesicle α-hemolysin derived from Staphylococcus aureus is embedded in a single bilayer membrane to form a through-hole that connects the inside and outside of the membrane. If the generated vesicle is made of a single-layer bilayer, α-hemolysin forms nanopores that penetrate the inside and outside of the membrane, so that the substance diffuses inside and outside the membrane, and a fluorescent dye is added to the solution inside the vesicle If so, the fluorescent dye will flow out of the vesicle and the fluorescence should disappear (FIG. 12A). Therefore, it was examined whether or not the generated vesicles cause diffusion of substances inside and outside the membrane by α-hemolysin. The fluorescent dye calcein was dissolved in the first aqueous solution serving as the vesicle internal solution, and α-hemolysin was dissolved together with the lipid in the organic solution serving as the vesicle membrane to produce vesicles. FIG. 12B is a graph showing the change over time of the fluorescence intensity of the generated vesicles. As shown in FIG. 12B, the fluorescence intensity of vesicles did not change for about 4 seconds after generation, but rapidly decreased after 5 seconds. This means that in the first 4 seconds, α-hemolysin self-assembled within the membrane and formed nanopores that penetrate the membrane, and then calcein in the vesicles flowed out of the vesicles through the nanopore. To do. That is, from the experimental results, it was suggested that the vesicles produced by the method and apparatus of the present invention are made of a single-layer bilayer. This example also demonstrated that the method and apparatus of the present invention can produce vesicles reconstituted with membrane proteins.

2-3. Membrane permeability and stability of vesicles One of the characteristics of biological membranes is that they have a semi-permeability that allows water to permeate but not sugar or salts. Therefore, the membrane permeability of vesicles produced by the method and apparatus of the present invention was examined. A vesicle produced using phosphate buffered saline (PBS) as a first aqueous solution was transferred into PBS containing 100 mM glucose, and the change in the size of the vesicle over time was measured. FIG. 13A is a series of photographs of changes in the size of one vesicle. FIG. 13B is a graph showing the change in the size of vesicles with time after the vesicle internal solution is PBS and vesicles having sizes of about 15 μm, about 20 μm, and about 25 μm are transferred to PBS containing 100 mM glucose and exposed to osmotic shock. It is. As shown in FIG. 6, the size of the vesicle decreased immediately after the start of measurement, and no change was observed after 1500 seconds (25 minutes). The vesicles were very stable and did not change in size after 1 week of production (not shown). The decrease in the size of the vesicle was linear, and the slope of the straight line was almost the same regardless of the size of the vesicle. This suggests that vesicles having the same membrane composition exhibit the same membrane permeation characteristics regardless of the size of the vesicles. This example proved that the vesicles produced by the method and apparatus of the present invention have a semi-permeability that permeates water but does not permeate glucose, similar to biological membranes. In addition, this example proved that the characteristics of the bilayer film can be measured in a vesicle state.

2-4. Control of Vesicle Liquid Composition In this example, it was examined whether the composition of the first aqueous solution and the liquid in the vesicle, particularly whether the polymer concentration was the same. A vesicle prepared using a high-concentration solution of fluorescently labeled bead beads having a particle size of 200 nm as the first aqueous solution was observed with a confocal microscope (FIG. 14). As a result of comparing the average number of bead balls per 5 μm square field of view in 20 frames photographed at 100 millisecond intervals, the number of bead balls per field observed inside 20 vesicles was as follows. The number of beads per field in the solution applied to the apparatus of the present invention as an aqueous solution was approximately the same. In this embodiment, in the production of vesicles by the method and apparatus of the present invention, the efficiency of encapsulation in the vesicle is very high, that is, when the vesicle is produced, the liquid in the vesicle is not enclosed but leaks out of the vesicle. It proved that the possibility of influx of vesicle fluid was very low.

2-5. In this example, it was examined whether or not a chemical reaction proceeds in a vesicle by a substrate and an enzyme enclosed in the vesicle. FIG. 15A shows a mixed solution (RTS100, E. coli HY kit, Roche Diagnostics Inc., catalog number: 3186148) containing a cell-free expression system of E. coli and a GFP expression construct as a first aqueous solution, Fluorescence micrographs by GFP of vesicles produced using a reaction replenisher containing ribonucleotides and amino acids as a second aqueous solution (taken after 0, 1, 2, 5 and 15 hours after vesicle production from the left, respectively) FIG. 15B is a graph showing the change over time in the emission intensity. The horizontal axis represents time (unit: time), and the vertical axis represents relative fluorescence intensity. As shown in FIG. 15B, the fluorescence by GFP increased immediately after vesicle generation, and reached a plateau after 5 hours. As a control experiment, when no DNA was added, no vesicle fluorescence was observed. In this example, among the vesicles produced by the method and apparatus of the present invention, it is possible to carry out complex chemical reactions that synthesize mRNA and protein by consuming ATP, transcription from DNA and translation of GFP. That is, the vesicle of the present invention proved useful as a bioreactor for producing artificial cells.

  Depending on the use of the vesicle, it may be desirable not to use an organic solution. In such a case, another embodiment of the present invention is employed in which the organic solution is replaced with a gas and the amphiphilic molecule is supplied by being dissolved in the first and / or second aqueous solution. In this embodiment, as shown in the schematic diagram on the left side of FIG. 16, the amphiphilic molecules 12 oriented on the surface of the first aqueous solution are oriented on the surface of the second aqueous solution with the gas layer 13 therebetween. Facing the amphiphilic molecule 14. The surfaces of the first and second aqueous solutions are in contact with each other at the tip. At this time, the hydrophobic portions of the amphiphilic molecules oriented outside the respective aqueous solutions are bonded to each other, so that the first and second aqueous solutions are bonded. The surface of the solution is sealed with a bilayer. Therefore, also in this embodiment, a tongue-like body similar to the embodiment using the organic solution is formed (FIG. 16, right side).

  As described above, the bilayer vesicle of the present invention is a vesicle having the same dimensions as a living cell and having a very uniform size. In the vesicle, a complex chemical reaction such as transcription and translation is allowed to proceed, The same selective permeability as the membrane can be reproduced. Therefore, the bilayer vesicle production | generation apparatus and production | generation method of this invention have great significance as an important bridgehead for future artificial cell preparation.

Claims (47)

  Including a plurality of structures including a main channel, a plurality of micro diverticulums opening in the main channel, an atrioventricular chamber communicating with the micro diverticulum, and a pulsation preventing means provided in the atrioventricle, An apparatus for generating bilayer vesicles with amphiphilic molecules.   2. The apparatus for generating a bilayer vesicle according to claim 1, wherein the pulsation preventing means is a communication channel that connects the micro diverticulum and the atrioventricle.   The apparatus for generating a bilayer vesicle according to claim 1, wherein the structure includes a shearing unit.   A wall of the micro diverticulum is provided with a phase separation fluid reservoir, the phase separation fluid reservoir suspends a phase separation fluid layer, and the phase separation fluid layer is located between the opening of the micro diverticulum and the atrioventricular chamber. The apparatus for producing a bilayer vesicle according to claim 1, wherein the minute diverticulum is sealed with a.   A plurality of phase separation fluid reservoirs are provided on a wall surface of the micro diverticulum, the plurality of phase separation fluid reservoirs suspend a plurality of phase separation fluid layers, and the plurality of phase separation fluid layers are disposed on the micro diverticulum. 4. The apparatus for producing a bilayer vesicle according to claim 1, wherein the micro diverticulum is sealed between an opening and the atrioventricular chamber.   The bilayer vesicle generator according to claim 4 or 5, wherein the phase separation fluid reservoir is an overhanging portion extending from a side wall surface of the micro diverticulum into the micro diverticulum.   The apparatus for generating a bilayer vesicle according to claim 1, wherein a pressure generating unit is provided in the atrioventricular chamber.   The apparatus for generating a bilayer vesicle according to claim 7, wherein the pressure generation unit generates pressure by expansion of microbubbles generated by laser light.   The apparatus for generating a bilayer vesicle according to claim 7, wherein the pressure generator generates pressure by expansion of microbubbles generated by electrolysis.   The apparatus for generating a bilayer vesicle according to claim 7, wherein the pressure generating unit generates pressure by expansion of microbubbles generated by an electric heater.   The apparatus for generating a bilayer vesicle according to claim 7, wherein the pressure generating unit generates pressure by a microactuator.   The apparatus for generating a bilayer vesicle according to any one of claims 1 to 6, wherein the atrioventricle is connected to an external pressure source.   The apparatus for generating a bilayer vesicle according to claim 12, wherein the external pressure source is a syringe pump.   13. The apparatus for generating a bilayer vesicle according to claim 12, wherein the external pressure source is a high-pressure gas cylinder.   The bilayer membrane vesicle according to any one of claims 3 to 14, wherein the fluid passing through the main channel flows in one direction, and the shearing means is provided downstream of the opening of the microdiverticulum. Generator.   15. The fluid passing through the main flow path flows in two forward and reverse directions, and the shearing means is provided on the downstream side of each of the two flow directions. Bilayer vesicle generator.   17. The atrioventricular chamber is connected to an external supply source of an aqueous solution, and the aqueous solution is supplied to the micro diverticulum via the communication channel. Molecular membrane vesicle generator.   The structure includes one or two or more aqueous solution channels, and the aqueous solution channel is a wall surface of the micro diverticulum provided between at least two of the plurality of phase separation fluid reservoirs. The apparatus for generating a bilayer membrane vesicle according to any one of claims 5 to 17, wherein the opening of each of the plurality of aqueous solutions is in communication with an external source of each of the plurality of aqueous solutions.   The structure includes a sub-channel, the sub-channel is connected to an external supply source of a phase separation fluid, and the phase separation fluid is supplied to the phase separation fluid reservoir via the sub-channel. An apparatus for producing a bilayer vesicle according to any one of claims 4 to 18.   The apparatus for generating a bilayer vesicle according to any one of claims 1 to 19, wherein the minute diverticulum is two-dimensionally arranged.   The apparatus for generating a bilayer vesicle according to any one of claims 1 to 19, wherein the minute diverticulum is arranged three-dimensionally.   The apparatus for producing a bilayer vesicle according to any one of claims 4 to 21, wherein the phase separation fluid layer forms an interface with an aqueous solution.   The apparatus for generating a bilayer vesicle according to any one of claims 4 to 22, wherein the phase separation fluid is an organic solution containing amphiphilic molecules.   The bilayer vesicle according to claim 22, wherein the phase separation fluid is a gas or an organic solution free of amphiphilic molecules, and the aqueous solution contains amphiphilic molecules. apparatus.   25. The apparatus for generating a bilayer vesicle according to claim 1, wherein the structure is made of polydimethylsiloxane, and the organic solution swells and deforms polydimethylsiloxane. A method for producing a bilayer vesicle,
A structure including a main flow channel, a plurality of micro diverticulums opening in the main flow channel, an atrioventricular chamber communicating with the micro diverticulum, and pulsation preventing means provided in the atrioventricular chamber; Providing a solution and a phase separation fluid;
Flowing a first aqueous solution through the main flow path of the structure to introduce the first aqueous solution into the microdiverticulum;
Flowing the phase separation fluid through the main flow path of the structure and introducing the phase separation fluid into at least a portion of the microdiverticulum;
A second aqueous solution is allowed to flow in the main flow path of the structure to remove the phase separation fluid in the main flow path, the phase separation fluid forms a phase separation fluid layer, and the phase separation fluid layer is the micro separation layer. A step suspended on the side wall of the diverticulum;
In a state where the phase separation fluid layer is suspended on the side wall surface of the micro diverticulum, by applying pressure to the atrioventricular chamber, the first aqueous solution is pushed into the micro diverticulum through the atrioventricular chamber, and the phase Stretching the phase separation fluid layer such that the tip of the separation fluid layer enters the main flow path from the opening of the microdiverticulum;
The liquid flow of the second aqueous solution in the main channel shears the phase separation fluid layer, thereby binding the phase separation fluid layer and the first aqueous solution surrounded by the phase separation fluid layer. A method for producing a bilayer vesicle, comprising the step of cutting the vesicle. A method for producing a bilayer vesicle,
A structure including a main channel, a plurality of micro diverticulums opening in the main channel, an atrioventricular chamber communicating with the micro diverticulum, a pulsation preventing means provided in the atrioventricular chamber, a hydrophobic solvent, And providing a second aqueous solution and a phase separation fluid;
Flowing the hydrophobic solvent through the main flow path of the structure and introducing the hydrophobic solvent into the microdiverticulum;
Flowing a first aqueous solution through the main flow path of the structure to introduce the first aqueous solution into the microdiverticulum;
Flowing the phase separation fluid through the main flow path of the structure and introducing the phase separation fluid into at least a portion of the microdiverticulum;
A second aqueous solution is allowed to flow in the main flow path of the structure to remove the phase separation fluid in the main flow path, the phase separation fluid forms a phase separation fluid layer, and the phase separation fluid layer is the micro separation layer. A step suspended on the side wall of the diverticulum;
In the state where the phase separation fluid layer is suspended on the side wall surface of the micro diverticulum, the hydrophobic solvent in the chamber is heated and pressure is generated by the generation and expansion of micro bubbles, thereby passing the chamber through the chamber. Extending the phase separation fluid layer such that a first aqueous solution is swept into the micro diverticulum and the tip of the phase separation fluid layer enters the main channel from the opening of the micro diverticulum;
The liquid flow of the second aqueous solution in the main channel shears the phase separation fluid layer, thereby binding the phase separation fluid layer and the first aqueous solution surrounded by the phase separation fluid layer. A method for producing a bilayer vesicle, comprising the step of cutting the vesicle.   The method for producing a bilayer vesicle according to claim 26 or 27, wherein the phase separation fluid is an organic solution containing amphiphilic molecules.   28. The first and second aqueous solutions include amphiphilic molecules, and the phase separation fluid is a gas or an organic solution that does not include amphiphilic molecules. Of producing bilayer vesicles.   30. The method for producing a bilayer vesicle according to claim 29, wherein the amphiphilic molecule contained in the first aqueous solution has a composition different from that of the amphiphilic molecule contained in the second aqueous solution.   The method for producing a bilayer vesicle according to any one of claims 26 to 30, wherein the pulsation preventing means is a communication channel that connects the micro diverticulum and the atrioventricle.   32. The structure according to any one of claims 26 to 31, wherein the structure includes a shearing means, and the shearing means shears the phase separation fluid layer together with a liquid flow of the second aqueous solution in the main flow path. A method for producing the bilayer vesicle according to claim.   A wall of the micro diverticulum is provided with a phase separation fluid reservoir, the phase separation fluid reservoir suspends a layer of the phase separation fluid, and the phase separation fluid layer is formed between the opening of the micro diverticulum and the communication channel. 33. The method for producing a bilayer vesicle according to any one of claims 26 to 32, wherein the microdiverticulum is sealed in between.   34. The method of generating a bilayer vesicle according to claim 33, wherein the phase separation fluid reservoir is an overhang extending from a side wall surface of the micro diverticulum into the micro diverticulum.   35. The method for producing a bilayer vesicle according to claim 26, wherein the pressure is applied to the atrioventricle by the expansion of microbubbles generated by laser light.   35. The method for producing a bilayer vesicle according to claim 26, wherein the application of pressure to the atrioventricle is performed by expansion of microbubbles generated by electrolysis.   35. The method for producing a bilayer vesicle according to claim 26, wherein the pressure is applied to the atrioventricle by expansion of microbubbles generated by an electric heater.   The method for producing a bilayer vesicle according to any one of claims 26 and 28-34, wherein the pressure is applied to the atrioventricle by a microactuator.   The method for producing a bilayer vesicle according to any one of claims 26 and 28-34, wherein the pressure is applied to the atrioventricle by being connected to an external pressure source. .   40. The method of generating a bilayer vesicle according to claim 39, wherein the external pressure source is a syringe pump.   40. The method of generating a bilayer vesicle according to claim 39, wherein the external pressure source is a high-pressure gas cylinder.   The bilayer membrane vesicle according to any one of claims 32 to 41, wherein the liquid passing through the main channel flows in one direction, and the shearing means is provided downstream of the opening of the microdiverticulum. Generation method.   The liquid passing through the main flow path flows in two directions, forward and reverse, and the shearing means is provided on the downstream side of the flow in the two directions, respectively. Method for producing bilayer vesicles.   44. The apparatus according to claim 26, wherein the atrioventricular chamber is connected to an external supply source of an aqueous solution, and the aqueous solution is supplied to the micro diverticulum via the communication channel. Method for producing molecular membrane vesicles.   The structure includes a sub-channel, the sub-channel is connected to an external supply source of a phase separation fluid, and the phase separation fluid is supplied to the phase separation fluid reservoir via the sub-channel. The method for producing a bilayer vesicle according to any one of claims 32 to 44. A method for producing a bilayer vesicle of N layers (N is an integer of 2 or 3 or more),
A structure including a main flow channel, a plurality of micro diverticulums opening in the main flow channel, an atrioventricular chamber communicating with the micro diverticulum, and a pulsation preventing means provided in the atrioventricular chamber, and first to (N + 1) th aqueous Providing a solution and first to Nth phase separation fluids;
Flowing a first aqueous solution through the main flow path of the structure to introduce the first aqueous solution into the microdiverticulum;
Flowing a first phase separation fluid through the main flow path of the structure and introducing the first phase separation fluid into at least a portion of the microdiverticulum;
A second aqueous solution is caused to flow through the main flow path of the structure to remove the first phase separation fluid in the main flow path, and the first phase separation fluid is sandwiched between the first and second aqueous solutions. Forming a first phase separation fluid layer, the first phase separation fluid layer being suspended on the side wall surface of the microdiverticulum;
Flowing an m-th (m is an integer from 2 to N-1) phase separation fluid through the main flow path of the structure, and introducing the m-th phase separation fluid into at least a part of the microdiverticulum; ,
The m + 1st aqueous solution is allowed to flow through the main flow path of the structure to remove the mth phase separation fluid in the main flow path, and the mth phase separation fluid is the mth and m + 1th aqueous solutions. Forming an mth phase separation fluid layer sandwiched between solutions, the mth phase separation fluid layer being suspended on a side wall surface of the microdiverticulum;
Flowing an Nth phase separation fluid through the main flow path of the structure and introducing the Nth phase separation fluid into at least a portion of the microdiverticulum;
The N + 1th aqueous solution is allowed to flow through the main flow path of the structure to remove the Nth phase separation fluid in the main flow path, and the phase separation fluid is sandwiched between the Nth and N + 1th aqueous solutions. Forming an Nth phase separation fluid layer, wherein the Nth phase separation fluid layer is suspended on the side wall surface of the microdiverticulum;
In the state where the N phase separation fluid layers are suspended on the side wall surface of the micro diverticulum, the first aqueous solution is pushed into the micro diverticulum through the atrioventricle by applying pressure to the atrioventricular chamber. Elongating the N phase separation fluid layers such that the tips of the N phase separation fluid layers enter the main flow path from the opening of the microdiverticulum,
The liquid flow of the (N + 1) th aqueous solution in the main flow path is surrounded by the N phase separation fluid layers and the N phase separation fluid layers by shearing the N phase separation fluid layers. And a method of producing an N-layer bilayer vesicle, wherein the first to Nth aqueous solutions are bundled together and cut into vesicles.   The structure includes N phase separation fluid reservoirs provided on a wall surface of the micro diverticulum, and less than N aqueous solution flow paths, and the aqueous solution flow paths include the N phase separation fluids. The communication device according to claim 46, characterized in that an opening in the wall of the microdiverticulum provided between at least two of the reservoirs communicates with an external source of each of the less than N aqueous solutions. Method for producing N-layer bilayer vesicles.