Microfluidic device and method for forming lipid bilayer membranes

Abstract

To provide a microfluidic device capable of operating fluid simply to form a lipid double membrane by distinguishing two flow paths into hydrophilic one and hydrophobic one, and a method for forming a lipid double membrane.SOLUTION: A microfluidic device 1 comprises: a first flow path 21 defined by a hydrophobic surface; a second flow path 22 defined by a hydrophilic surface, which extends parallel to the first flow path 21; and a connection path 23 that connects the first flow path 21 and the second flow path 22, where the second flow path 22 is filled with a first aqueous solution 31, the first flow path 21 is filled with a second aqueous solution 33, and the connection path 35 is formed with a lipid double membrane.SELECTED DRAWING: Figure 3

Description

[Technical Field]

[0001] The present invention relates to a microfluidic device and a method for forming a lipid bilayer membrane. [Background technology]

[0002] Cell membranes are primarily composed of lipid bilayers and membrane proteins, and have various functions, such as the selective permeation of ions and molecules. In recent years, research into the engineering applications of cell membranes has progressed, and nanopore sequencers and other devices have begun to be put to practical use. Meanwhile, microfluidics, which realizes various functions in microchannels, has led to advances in miniaturization, speedup, and device development in analysis. Microfluidic devices incorporating cell membranes within microchannels can be used to evaluate membrane properties and for applications such as nanopore sequencers, and the integration of cell membrane functions is expected to lead to the creation of new devices.

[0003] In the prior art, attempts have been made to apply lipid bilayer membrane synthesis methods that utilize the gas-liquid and liquid-liquid interfaces and the amphiphilic properties of lipid molecules to microchannels, and several channel shapes and fluid control methods have been proposed.

[0004] For example, Patent Document 1 relates to an apparatus and method for forming a lipid bilayer membrane. In this apparatus, a first container having oil on the surface of a substrate is provided, and multiple second containers are provided at the bottom of the first container, and the inner surface of the first container has a shape that converges toward the second container so that when an aqueous solution is added to the oil contained in the first container, droplets formed from the aqueous solution are guided to the second container, and the multiple second containers are arranged so that the droplets contained in the second containers come into contact with each other, and a lipid bilayer membrane is formed when the droplets contained in the second containers come into contact with each other through holes that penetrate walls separating adjacent second containers.

[0005] Non-Patent Document 1 discloses a device fabricated by providing microchambers consisting of multiple through-holes in a carbon-fluorine hydrophobic polymer on a glass slide and combining this with a spacer and a cover top. A lipid bilayer membrane is synthesized at the boundary between the microchannel and the microchamber by sequentially flowing an aqueous solution, an organic solvent containing lipid molecules, and an aqueous solution through access holes provided in the cover slip into a microchannel formed between the cover top, spacer, and microchamber.

[0006] Patent Document 2 discloses an apparatus for forming lipid planar membranes, which includes a first flow path and a second flow path, at least a portion of which is adjacent to at least a portion of the first flow path across a partition wall, and at least one small hole penetrating the partition wall that fluidly connects the first flow path and the second flow path. Thus, by filling one of the first and second flow paths with an aqueous solution and the other with an organic solution containing lipids, the aqueous solution and the organic solution can come into contact with each other through the small hole. Subsequently, by replacing the organic solution with the aqueous solution, a lipid planar membrane is formed in the small hole.

[0007] Patent Document 3 relates to a method for forming a lipid bilayer membrane in a microfluidic device including a main substrate having formed therein a first liquid delivery section, a second liquid delivery section, a first flow path filled with a first liquid injected from the first liquid delivery section, a second flow path filled with a second liquid injected from the second liquid delivery section, and an insertion section through which the first flow path and the second flow path are inserted, the method comprising: a first liquid injection step of injecting an oily solution in which lipids are dissolved from the first liquid delivery section and the second liquid delivery section to fill the first flow path, the second flow path, and the insertion section with the oily solution; and a second liquid injection step of injecting an aqueous solution from the first liquid delivery port and the second liquid delivery port to form a lipid bilayer membrane in the insertion section. [Prior art documents] [Patent documents]

[0008] [Patent Document 1] Patent No. 6877028 [Patent Document 2] Patent Publication No. 2005-98718 [Patent Document 3] Patent Publication No. 2014-30382 [Non-patent literature]

[0009] [Non-Patent Document 1] Watanabe et al., Nature Communications 5:4519,(2014) DOI: 10.1038 / ncomms5519 Summary of the Invention [Problem to be solved by the invention]

[0010] In the device of Patent Document 1, it is difficult to perform independent fluid manipulation on both sides of the lipid bilayer membrane, and its applications are limited.

[0011] In the device of Non-Patent Document 1, it is difficult to perform independent fluid manipulation on both sides of the lipid bilayer membrane constructed in the channel, and the device of Non-Patent Document 1 also has limited applications.

[0012] The device in Patent Document 2 requires the use of a pump to flow the liquid while maintaining a delicate pressure balance at the liquid-liquid interface, making fluid manipulation cumbersome. Furthermore, when the spatial size of the pores is less than a few microns, the influence of surface tension becomes dominant, making it difficult to maintain the liquid-liquid interface and form a lipid bilayer membrane, limiting its application to spaces of a few tens of microns. The diameter of the small holes ranges from several tens of microns to several hundred microns, but the production of lipid bilayer membranes in spaces smaller than this has not yet been realized.

[0013] Patent Document 3 is an improved version of the device by the same group of inventors as Patent Document 2, but it is still necessary to use a pump to flow the liquid while maintaining a delicate pressure balance at the liquid-liquid interface. Also, when the spatial size of the pores is less than a few μm, the influence of surface tension becomes dominant, making it difficult to maintain the liquid-liquid interface and form a lipid bilayer membrane, so its application is limited to spaces of a few tens of μm. In the examples, the area of ​​the lipid bilayer membrane formed is 200 μm 2 (10 μm × 20 μm), and it has not been possible to produce lipid bilayer membranes in smaller spaces.

[0014] The problem to be solved by the present invention is to provide a microfluidic device that can easily manipulate fluids and form lipid bilayer membranes by distinguishing between two flow paths, one hydrophilic and the other hydrophobic, and a method for forming lipid bilayer membranes. [Means for solving the problem]

[0015] As a result of extensive research to solve the above problems, the inventors discovered that by making the surfaces of the two flow channels of a microfluidic device hydrophilic and hydrophobic, and filling each channel with an aqueous solution and an organic solvent, it is possible to easily form a lipid bilayer membrane without having to maintain a delicate pressure balance at the liquid-liquid interface through complicated pressure control using an external pump, and thus completed the present invention.

[0016] The present invention encompasses the embodiments described below.

[0017] Item 1. A microfluidic device, a first flow path defined by a hydrophobic surface; a second flow path defined by a hydrophilic surface and extending in parallel with the first flow path; a connecting path that connects the first flow path and the second flow path, A microfluidic device, wherein the second flow path is filled with a first aqueous solution, and the first flow path is filled with a second aqueous solution, and a lipid bilayer membrane is formed in the connecting path.

[0018] Item 2. The microfluidic device according to Item 1, wherein the connecting path communicates with the first flow path at one end and communicates with the second flow path at the other end, the connecting path narrows in diameter from the one end toward the other end and from the other end toward the one end, and a lipid bilayer membrane is formed at the point of the connecting path where the diameter is smallest.

[0019] Item 3. A part of the first flow path extends toward the second flow path to form a first part of the connecting path, and a part of the second flow path extends toward the first flow path to form a second part of the connecting path, Connecting roads and the first part of Connecting roads The second portion of Connecting roads Item 3. The microfluidic device according to item 1 or 2, wherein a first portion of the connecting path has a hydrophobic surface and a second portion of the connecting path has a hydrophilic surface.

[0020] Item 4. The microfluidic device according to any one of Items 1 to 3, wherein the connecting path has a width of less than 100 μm.

[0021] Item 5. The microfluidic device according to any one of Items 1 to 4, wherein the first flow path and the second flow path extend linearly and parallel to each other on both sides of the portion connected to the connecting path in the longitudinal direction of each of the first flow path and the second flow path.

[0022] Item 6. The microfluidic device according to any one of Items 1 to 5, which is formed by joining a first substrate provided with the first flow channel, the second flow channel, and the connecting path, to a second substrate.

[0023] Item 7. A microfluidic device for forming a lipid bilayer membrane, a first flow path defined by a hydrophobic surface; a second flow path extending parallel to the first flow path and having a hydrophilic surface; a connecting path connecting the first flow path and the second flow path.

[0024] Item 8. A method for forming a lipid bilayer membrane, providing a microfluidic device including a first flow path defined by a hydrophobic surface, a second flow path having a hydrophilic surface and extending in parallel with the first flow path, and a connecting path connecting the first flow path and the second flow path; filling the first flow path of the microfluidic device with an organic solvent containing lipid molecules and filling the second flow path with a first aqueous solution, thereby contacting the organic solvent and the first aqueous solution in the connecting path; and replacing the organic solvent in the first flow path with a second aqueous solution, thereby forming a lipid bilayer membrane in the connecting path; A method comprising:

[0025] Item 9. The method according to Item 8, wherein the connecting path communicates with the first flow path at one end and communicates with the second flow path at the other end, the connecting path narrowing in diameter from the one end toward the other end and from the other end toward the one end, and the step of forming a lipid bilayer membrane on the connecting path includes forming a lipid bilayer membrane at a point on the connecting path where the diameter is smallest. [Effects of the Invention]

[0026] According to the microfluidic device and lipid bilayer membrane formation method of the present invention, it is not necessary to maintain a delicate pressure balance at the liquid-liquid interface of the solutions filling the two flow paths, and a lipid bilayer membrane can be formed with simple fluid manipulation. [Brief explanation of the drawings]

[0027] [Figure 1] FIG. 1 is an exploded perspective view of an embodiment of a microfluidic device. [Figure 2] (A) A schematic cross-sectional view of the microfluidic device according to the embodiment of Fig. 1. (B) A partially enlarged view of the boxed area indicated by the symbol A in Fig. 2(A). [Figure 3](A) A partially enlarged cross-sectional view of the area enclosed by the square indicated by the symbol B in Figure 2(B). (B) A partially enlarged plan view of the area surrounding the connecting path 13 of the microfluidic device 10 enclosed by the square dotted line indicated by the symbol C in Figure 3(A). [Figure 4] (A) Formation of a lipid monolayer membrane when the first channel is filled with an organic solvent and the second channel is filled with a first aqueous solution. (B) A schematic enlarged view of the area enclosed by the dotted square frame indicated by symbol D in Figure 4(A). (C) Formation of a lipid bilayer membrane when the first channel is filled with a second aqueous solvent and the second channel is filled with the first aqueous solution. (D) A schematic enlarged view of the area enclosed by the dotted square frame indicated by symbol E in Figure 4(C). [Figure 5] (A) A partially enlarged perspective view showing the flow and pressure of the organic solvent in the first channel and the first aqueous solution in the second channel of the microfluidic device in Figure 1. (B) A schematic diagram showing a longitudinal section taken along line 5B-5B in Figure 5(A) and the relationship between the pressure Porg applied from the organic solvent to the first aqueous solution, the pressure Paq applied from the first aqueous solution to the organic solvent, and the Laplace pressure. [Figure 6] Schematic diagram showing how a membrane protein is inserted into a lipid bilayer. [Figure 7] FIG. 10 is an enlarged partial cross-sectional view of another microfluidic device having multiple connections. [Figure 8] (A) Photograph of the state in which an organic solvent containing lipid molecules is flowed through the first flow path and a buffer solution is flowed through the second flow path, resulting in the formation of a monolayer lipid molecular membrane at the interface between the organic and aqueous phases. (B) Photograph of the state in which a lipid bilayer membrane is formed at the junction of the two flow paths after the organic solvent in the first flow path is replaced with a buffer solution. (C) Enlarged photograph of the area enclosed by the dotted line in Figure 8(B). [Figure 9] (A)-(D) Steps 1 to 4 are shown when water and an organic solvent are flowed through the microfluidic device of the comparative example. (E)-(F) are schematic diagrams showing the relationship between the pressures acting on the interfaces of the areas enclosed by the dotted squares in Figures 9(B)-(D). (H)-(K) are photographs of the flow paths of the microfluidic device corresponding to steps 1 to 4, respectively. DETAILED DESCRIPTION OF THE INVENTION

[0028] As used herein, a microfluidic device refers to a device having a channel or fluid chamber with at least one or more of the three dimensions (length, width, and depth) being less than 1 mm. A microchannel refers to a channel with at least one or more of the three dimensions (length, width, and depth) being less than 1 mm.

[0029] 1. Microfluidic Device Construction FIG. 1 is an exploded perspective view of a microfluidic device 1 according to this embodiment. The microfluidic device 1 is constructed by bonding a first substrate 3 and a second substrate 4. The first substrate 3 and the second substrate 4 constitute a substrate 2. The first substrate 3 and the second substrate 4 may be formed from any material commonly used as a substrate for a microfluidic device, such as glass, silicon, plastic, or polydimethylsiloxane (PDMS). Glass, silicon, or polydimethylsiloxane (PDMS) is preferred in terms of ease of forming a hydrophobic surface 21S and a hydrophilic surface 22S, which will be described later. Silicon is not optically transparent, but is easier to microfabricate using MEMS technology than glass. Furthermore, in terms of allowing the internal structure of the substrate 2 to be viewed from the outside, it is preferred that one or both of the first substrate 3 and the second substrate 4 be made of a transparent material, such as glass, transparent plastic, or polydimethylsiloxane (PDMS).

[0030] The first substrate 3 is provided with grooves 100 that extend in the short direction from the center of the first substrate 3 in the longitudinal and short directions, branching twice before widening into eight separate linear grooves 101 to 108. Ends 101 to 108a of the linear grooves 101 to 108 that are farther from the center of the first substrate 3 in the longitudinal and short directions have the shape of a bottomed hole that is wider than the width of the linear portion of the linear grooves 101 to 108 that is closer to the base end than the ends 101 to 108a, and terminate within the first substrate 3.

[0031] In this embodiment, the linear grooves 101 and 104, the linear grooves 102 and 103, the linear grooves 105 and 108, and the linear grooves 106 and 107 are symmetrical to one another with respect to an imaginary center line P that passes through the centers of the two short sides of the first substrate 3 and extends along the longitudinal direction of the first substrate 3. Furthermore, the linear grooves 101 and 105, the linear grooves 102 and 106, the linear grooves 103 and 107, and the linear grooves 104 and 108 are symmetrical to one another with respect to an imaginary center line Q that passes through the centers of the two long sides of the first substrate 3 and extends along the short direction of the first substrate 3.

[0032] The second substrate 4 has four through holes 5a to 5h formed therein, which serve as liquid supply or drainage ports. When the facing surfaces of the first substrate 3 and the second substrate 4 are joined together so as to cover the groove 100 of the first substrate 3, the through holes 5a to 5h of the second substrate 4 are aligned with the ends 101 to 108a of the first substrate 3.

[0033] When the first substrate 3 and the second substrate 4 are bonded together, the three surfaces (bottom and two side surfaces) of the first substrate 3 that form the walls of the groove 100 and the surface (top surface) of the second substrate 4 that covers the groove 100 form a flow channel 10, which is a microchannel (Figure 2(A)).

[0034] Referring to FIG. 2(A), the flow path 10 has eight linear flow paths 11-18 defined by the walls of the first substrate 3 and the walls of the second substrate 4, which define eight separate linear grooves 101-108.

[0035] There are no particular limitations on the width, length, and depth of each of the linear channels 11 to 18. The width is, for example, 100 to 1000 μm. The length is, for example, 10 to 50 mm. The depth is, for example, 1 to 100 μm.

[0036] The positions of the linear flow channels 11 to 18 correspond to the linear grooves 101 to 108, respectively, and therefore, in this embodiment, the linear flow channels 11 and 14, the linear flow channels 12 and 13, the linear flow channels 15 and 18, and the linear flow channels 16 and 17 are symmetrical to one another with respect to the imaginary center line P. Furthermore, the linear flow channels 11 and 15, the linear flow channels 12 and 16, the linear flow channels 13 and 17, and the linear flow channels 14 and 18 are symmetrical to one another with respect to the imaginary center line Q.

[0037] The second substrate 4 has four through holes 5a to 5h formed therein, which serve as liquid supply or drainage ports. When the facing surfaces of the first substrate 3 and the second substrate 4 are joined together so as to cover the groove 100 of the first substrate 3, the through holes 5a to 5h of the second substrate 4 are aligned with the ends 101 to 108a of the first substrate 3.

[0038] 2(B), the linear flow channels 11 and 12 merge at the end opposite to the ends 11a and 12a to form a merger 19a, the linear flow channels 13 and 14 merge at the end opposite to the ends 13a and 14a to form a merger 19b, the linear flow channels 15 and 16 merge at the end opposite to the ends 15a and 16a to form a merger 19c, and the linear flow channels 17 and 18 merge at the end opposite to the ends 17a and 18a to form a merger 19d. Each of the mergers 19a, 19b, 19c, 19d tapers away from the side communicating with the linear flow channels 11 and 12, the linear flow channels 13 and 14, the linear flow channels 15 and 16, and the linear flow channels 17 and 18, respectively. Confluences 19a and 19c are connected to both ends of a substantially V-shaped first flow path 21. That is, if first flow path 21 is divided into a first portion 21a, a second portion 21b extending linearly in the middle, and a third portion 21c, confluence 19a is connected to first portion 21a, and confluence 19c is connected to third portion 21c. Confluences 19b and 19d are connected to both ends of a substantially V-shaped second flow path 22. That is, if second flow path 22 is divided into a first portion 22a, a second portion 22b extending linearly in the middle, and a third portion 22c, confluence 19b is connected to first portion 22a, and confluence 19d is connected to third portion 22c.

[0039] Fig. 3(A) is a partially enlarged perspective view of the microfluidic device 1 of the area enclosed by the square indicated by the symbol B in Fig. 2(B). The first flow channel 21 and the second flow channel 22 extend in parallel, and are connected to each other by a connecting path 23 therebetween so that fluids can pass between them.

[0040] When injecting a liquid into or discharging a liquid from the through-holes 5a to 5h in Fig. 1, a liquid supply tube 6 (Fig. 1) is connected to each of the through-holes 5a to 5h. Linear flow paths 11 and 12, linear flow paths 13 and 14, linear flow paths 15 and 16, and linear flow paths 17 and 18 are each paired, and in each pair of through-holes 5a and 5b, through-holes 5c and 5d, through-holes 5e and 5f, and through-holes 5g and 5h, one through-hole acts as an inlet for the liquid, while the other through-hole acts as an outlet.

[0041] For example, the liquid injected into through-hole 5a is discharged from through-hole 5b. The liquid injected into through-hole 5a passes through linear flow path 11 and junction 19a, and part of it passes through linear flow path 12 and is discharged from through-hole 5b, but part of it enters first flow path 21 from junction 19a and flows to junction 19c, then flows through linear flow path 15 and is discharged from through-hole 5e to the outside of substrate 2, and also flows through linear flow path 16 and is discharged from through-hole 5f to the outside of substrate 2.

[0042] Alternatively, through hole 5b may be used as an injection hole and through hole 5a as a discharge hole. In this case, the liquid injected into through hole 5b passes through linear flow path 12 and confluence 19a, and part of it passes through linear flow path 11 and is discharged from through hole 5a, while part of it enters first flow path 21 from confluence 19a and flows to confluence 19c, then flows through linear flow path 15 and is discharged outside substrate 2 from through hole 5e, and also flows through linear flow path 16 and is discharged outside substrate 2 from through hole 5f.

[0043] The liquid injected into through hole 5c passes through linear flow path 13 and confluence 19b, and part of it passes through linear flow path 14 and is discharged from through hole 5d, but part of it enters second flow path 22 from confluence 19b and flows to confluence 19d, then flows through linear flow path 17 and is discharged outside substrate 2 through through hole 5g, and also flows through linear flow path 18 and is discharged outside substrate 2 through through hole 5f.

[0044] Alternatively, through hole 5d may be used as an injection hole and through hole 5c as a discharge hole. In this case, the liquid injected into through hole 5d passes through linear flow path 14 and confluence 19b, and part of it passes through linear flow path 113 and is discharged from through hole 5c, while part of it enters second flow path 22 from confluence 19b, flows to confluence 19d, flows through linear flow path 17 and is discharged outside substrate 2 from through hole 5g, and also flows through linear flow path 18 and is discharged outside substrate 2 from through hole 5f.

[0045] Instead of discharging the liquid injected into through hole 5a or through hole 5b from through hole 5e or through hole 5f, the liquid may be injected from through hole 5e or through hole 5f and then discharged from through hole 5a or through hole 5b.

[0046] For example, liquid injected into through-hole 5e passes through linear flow path 15 and confluence 19c, and part of it passes through linear flow path 16 and is discharged from through-hole 5f, while part of it enters first flow path 21 from confluence 19c and flows to confluence 19a, then flows through linear flow path 11 and is discharged outside of substrate 2 from through-hole 5a, and also flows through linear flow path 12 and is discharged outside of substrate 2 from through-hole 5b.

[0047] Alternatively, through hole 5f may be used as an injection hole and through hole 5e as a discharge hole. In this case, the liquid injected into through hole 5f passes through linear flow path 16 and confluence 19c, and part of it passes through linear flow path 15 and is discharged from through hole 5e, while part of it enters first flow path 21 from confluence 19c and flows to confluence 19a, then flows through linear flow path 11 and is discharged outside substrate 2 from through hole 5a, and also flows through linear flow path 12 and is discharged outside substrate 2 from through hole 5b.

[0048] The liquid injected into through hole 5g passes through linear flow path 17 and confluence 19d, and part of it passes through linear flow path 18 and is discharged from through hole 5h, but part of it enters second flow path 22 from confluence 19d and flows to confluence 19b, then flows through linear flow path 13 and is discharged outside substrate 2 from through hole 5c, and also flows through linear flow path 14 and is discharged outside substrate 2 from through hole 5d.

[0049] Alternatively, through hole 5h may be used as an injection hole and through hole 5g as a discharge hole. In this case, the liquid injected into through hole 5h passes through linear flow path 18 and confluence 19d, and part of it passes through linear flow path 17 and is discharged from through hole 5g, while part of it enters second flow path 22 from confluence 19d and flows to confluence 19b, then flows through linear flow path 13 and is discharged outside substrate 2 from through hole 5c, and also flows through linear flow path 14 and is discharged outside substrate 2 from through hole 5d.

[0050] By this procedure, the first flow path 21 can be filled with the first liquid, and the first liquid filling the first flow path 21 can be replaced with a different second liquid. Furthermore, if necessary, the second flow path 22 can be filled with the first liquid, and the first liquid filling the second flow path 22 can be replaced or switched with a different second liquid. The inside of the connection path 23 between the first flow path 21 and the second flow path 22 can also be filled with the first liquid at each of the first portion 23a on the side communicating with the first flow path 21 and the second portion 23b on the side communicating with the second flow path 22, and can then be replaced with a different second liquid.

[0051] The flow channel 10 of the microfluidic device 1 of this embodiment is symmetrical with respect to both the imaginary center lines P and Q in the longitudinal and lateral directions, so the operability of the microfluidic device 1 remains the same regardless of the dominant hand of the operator. Furthermore, there is no distinction between the rear and front sides when using the microfluidic device 1, which is advantageous.

[0052] In this embodiment, the bottom surface and two side surfaces of the first flow channel 21, and the top surface of the second substrate 4 that defines the first flow channel 21, have hydrophobic surfaces 21S. Therefore, the first flow channel 21 is defined by the hydrophobic surfaces 21S. Furthermore, the bottom surface and two side surfaces of the second flow channel 22, and the top surface of the second flow channel 22 that defines the second substrate 4, have hydrophilic surfaces 22S. Therefore, the second flow channel 22 that extends in parallel with the first flow channel 21 is defined by the hydrophilic surfaces 22S.

[0053] Preferably, the linear flow paths 11 and 12, the confluence 19a, the first flow path 21, the confluence 19c, and the linear flow paths 15 and 16 all have hydrophobic surfaces, and the linear flow paths 13 and 14, the confluence 19b, the second flow path 22, the confluence 19d, and the linear flow paths 17 and 18 all have hydrophilic surfaces.

[0054] The hydrophobic surface 21S of the first flow channel 21 may be any hydrophobic surface, for example, a hydrophobic surface formed by silane coupling, which substitutes hydrophilic groups such as hydroxyl groups present on the surface of the first substrate 3 (or both the first substrate 3 and the second substrate 4) with hydrophobic groups. Examples of silane coupling agents having hydrophobic groups include trimethylsilane (TMS) and hexamethyldisilazane.

[0055] The hydrophilic surface 22S of the second flow path 22 may be any hydrophilic surface, and may be the hydrophilic surface of the first substrate 3 (or both the first substrate 3 and the second substrate 4) used as is, or the surface of the first substrate 3 (or both the first substrate 3 and the second substrate 4) made of transparent silicon such as PDMS that has been made hydrophilic by plasma treatment, surface treatment using fluorine gas, etc.

[0056] 3(B) is a partially enlarged plan view of the connection channel 23 of the microfluidic device 1 enclosed by the dotted square frame indicated by the symbol C in FIG. 3(A). In this embodiment, the connection channel 23 communicates with the first flow channel 21 at one end and with the second flow channel 22 at the other end, and the diameter of the connection channel 23 decreases from one end to the other end and also decreases from the other end to the one end. As will be described later, a lipid bilayer membrane is formed at a point 23X in the connection channel 23 where the diameter is smallest.

[0057] Since the walls of the first substrate 3 are continuous between the first flow path 21, the second flow path 22, and the connecting path 23, it can also be considered that a part of the first flow path 21 extends toward the second flow path 22 to form the first portion 23a of the connecting path 23, and a part of the second flow path extends toward the first flow path 21 to form the second portion 23b of the connecting path 23, and that the first portion 23a of the connecting path 23 and the second portion 23b of the connecting path 23 are connected to form the entire length of the connecting path 23. In this embodiment, the first portion 23a of the connecting path 23 has a hydrophobic surface 23aS, and the second portion 23b of the connecting path 23 has a hydrophilic surface 23bS, on either side of the point 23X where the diameter of the connecting path 23 is smallest.

[0058] As shown in FIG. 3(B), the widths W1 and W2 of the first flow path 21 and the second flow path 22 are preferably 1 to 100 μm and may be as small as 10 μm or less or less than 10 μm. The lengths of the linear second portions 21b and 22b of the first flow path 21 and the second flow path 22 are not particularly limited, but are preferably 10 to 1000 μm, but are not limited thereto. The depths of the first flow path 21 and the second flow path 22 are preferably 1 to 100 μm and may be as small as 1 μm or less. The width W3 of the connecting path 23 is preferably 1 to 100 μm and may be as small as 1 μm or less. The depth of the connecting path 23 is preferably 1 to 100 μm and may be as small as 1 μm or less. In this embodiment, the first flow path 21 has a hydrophobic surface 21S and the second flow path 22 has a hydrophilic surface 22S, so that a lipid bilayer membrane can be formed even in a small flow path. In a particularly preferred embodiment, the widths W1 and W2 of the first flow channel 21 and the second flow channel 22 are 10 μm or less or less than 10 μm, the lengths of the linear portions 11b and 11b are 10 to 100 μm, the depths of the first flow channel 21 and the second flow channel 22 are 10 μm or less, and the width W3 of the connecting channel 23 is 1 to 10 μm, and the depth of the connecting channel 23 is 10 μm or less. According to the microfluidic device of this embodiment, the spatial size of the connecting channel 23 that forms the lipid bilayer membrane can be further miniaturized.

[0059] Returning to FIG. 3(A), when forming a lipid bilayer membrane in the connecting channel 23, both the first channel 21 and the second channel 22 are initially filled with an aqueous solution. In this case, since it is sufficient that the first channel 21, the second channel 22, and the connecting channel 23 are entirely filled with the aqueous solution, the aqueous solution may be introduced through any of the through-holes 5a to 5h. For example, the aqueous solution may be introduced into the first channel 21 from the direction of the open arrow in the upper left of the drawing, and into the second channel 22 from the direction of the open arrow in the lower left of the drawing. Alternatively, the aqueous solution may be introduced into the first channel 21 from the direction of the open arrow in the upper left of the drawing, and into the second channel 22 from the direction of the black arrow in the lower right of the drawing.

[0060] As shown in FIG. 4(A), the aqueous solution in the first channel 21 is replaced with an organic solvent 32 containing lipid molecules, as will be explained in more detail in the method of forming a lipid bilayer membrane.

[0061] Lipids are components that form lipid bilayer membranes and have a hydrophilic group (hydrophilic atomic group) and a hydrophobic group (hydrophobic atomic group). As lipids, one or more types are appropriately selected from, for example, phospholipids, glycolipids, cholesterol, or other compounds depending on the lipid bilayer membrane to be formed. Phospholipids include phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, sphingomyelin, etc. Glycolipids include cerebrosides, gangliosides, etc.

[0062] The organic solvent 32 for dissolving or dispersing the lipids is selected from various organic solvents, such as chloroform, decane, dodecane, hexadecane, squalene, and the like.

[0063] Aqueous solutions (including the first and second aqueous solutions described below) include water or aqueous solutions containing water-soluble substances, such as, but not limited to, saline, phosphate buffer, acetate buffer, citrate buffer, citrate phosphate buffer, Tris buffer, Tris phosphate buffer, etc. Water-soluble substances include, but are not limited to, acids, bases, salts, etc.

[0064] 2. Microfluidic Device Fabrication Method Next, a method for manufacturing the microdevice of this embodiment will be described.

[0065] First, a groove 100 is provided in the first substrate 3. A known method used to form a channel in a substrate of a microfluidic device can be used to form the groove 100 in the first substrate 3, such as etching. After covering the area other than the groove 100 with a mask, etching can be performed to form the groove 100 defined by the walls of the substrate 3.

[0066] Next, the first substrate 3 and the second substrate 4 are bonded together to form the substrate 2 having the flow path 10 therein. For bonding the first substrate 3 and the second substrate 4, it is preferable to use a known direct bonding technique such as a low-temperature bonding method in which the substrate surfaces are activated by oxygen plasma irradiation, a heat fusion method, fusion bonding, hydrofluoric acid bonding, or anodic bonding.

[0067] Next, a hydrophobic surface 21S is formed on the first flow channel 21. As described above, the hydrophobic surface 21S of the first flow channel 21 is formed by a hydrophobic treatment using a silane coupling agent or the like.

[0068] Next, a hydrophilic surface 22S is provided on the second flow path 22. A known method can be used for the hydrophilic treatment to provide the hydrophilic surface 22S on the second flow path 22. As described above, for example, the surface of a hydrophilic substrate may be used as is, or the surface of a transparent silicon substrate such as PDMS may be hydrophilized by surface treatment using plasma, fluorine gas, or the like. A preferred example is a method in which the first substrate 3 is a glass substrate, the first flow path 21, the second flow path 22, and the connecting path 23 are hydrophobized by applying a silane coupling agent, and then a base such as sodium hydroxide is passed through the second flow path 22 to remove the silane coupling agent, while avoiding passing the base such as sodium hydroxide through the first flow path 21, thereby making the second flow path 22 hydrophilic.

[0069] In this manner, a microfluidic device 1 is completed, which is a microfluidic device including a first flow channel 21 having a hydrophobic surface 21S, a second flow channel 22 having a hydrophilic surface 22S that extends parallel to the first flow channel 21, and a connecting channel 23 that connects the first flow channel 21 and the second flow channel 22. Such a microfluidic device 1 can be used to form a lipid bilayer membrane.

[0070] 3. Method for forming lipid bilayer membrane Next, a method for forming a lipid bilayer membrane using the microfluidic device 1 of this embodiment will be described.

[0071] First, a microfluidic device 1 is provided, which includes a first flow path 21 having a hydrophobic surface 21S, a second flow path 22 having a hydrophilic surface 22S extending parallel to the first flow path 21, and a connecting path 23 connecting the first flow path and the second flow path.

[0072] First, the liquid supply tube 6 is connected to the through-holes 5a, 5b, 5c, and 5d of the microfluidic device 1. The first aqueous solution 31 is caused to flow into the flow path 10 from the through-hole 5a and to be discharged from the through-hole 5b, and the first aqueous solution 31 is caused to flow into the flow path 10 from the through-hole 5d and to be discharged from the through-hole 5c. As a result, the first flow path 21, the second flow path 22, and the connecting path 23 are filled with the first aqueous solution 31.

[0073] As long as the first flow path 21, the second flow path 22 and the connecting path 23 are filled with the first aqueous solution 31, the first aqueous solution 31 may be flowed into the flow path 10 through any of the through holes 5a to 5h, and the first aqueous solution 31 may be discharged from the flow path 10 through any of the through holes 5a to 5h.

[0074] Next, the organic solvent 32 is introduced into the flow path 10 through the through-hole 5a and discharged through the through-hole 5b, thereby causing the organic solvent 32 to flow from one end of the first flow path 21 to the other. Then, as shown in FIG. 4(A), the first aqueous solution 31 is expelled from the first flow path 21, and the first flow path 21 is filled with the organic solvent 32. At this time, the first aqueous solution 31 is caused to flow into the flow path 10 through the through-hole 5d, maintaining the second flow path 22 filled with the first aqueous solution 31. As shown by the arrows in FIG. 4(A), the direction in which the organic solvent 32 flows through the first flow path 21 and the direction in which the first aqueous solution 31 flows through the second flow path 22 are the same (from left to right in the figure).

[0075] By filling the first flow path 21 and the first portion 23a of the connecting path 23 on the first flow path 21 side with an organic solvent 32, and filling the second flow path 22 and the second portion 23b of the connecting path 23 on the second flow path 22 side with a first aqueous solution 31, the organic solvent 32 and the first aqueous solution 31 come into contact in the connecting path 23 and form an interface. As shown in FIG. 4(B), at the interface between the first aqueous solution 31 and the organic solvent 32 present in the connecting path 23, the hydrophilic groups of some of the lipids in the organic solvent are aligned toward the first aqueous solution 31, and the hydrophobic groups of some of the lipids in the same organic solvent are aligned toward the organic solvent 32, forming a lipid monolayer membrane 34. The lipid monolayer membrane 34 is also referred to as a lipid monolayer membrane.

[0076] Next, the second aqueous solution 33 is injected through one of the through-holes 5a, 5b, 5e, or 5f, and the second aqueous solution 33 flows from one end of the first flow path 21 to the other. As a result, as shown in FIG. 4(C), the organic solvent 32 is expelled from the first flow path 21, and the first flow path 21 is filled with the second aqueous solution 33. That is, the organic solvent 32 is swept away by the second aqueous solution 33 and replaced with the second aqueous solution 33. Even during this process, the second flow path 22 remains filled with the first aqueous solution 31. In FIG. 4(C), the flow direction of the second aqueous solution 33 through the first flow path 21 and the flow direction of the first aqueous solution 31 through the second flow path 22 are shown as being the same (from left to right in the figure), but the flow direction of the second aqueous solution 33 through the first flow path 21 may be opposite to the flow direction of the first aqueous solution 31 through the second flow path 22.

[0077] The second aqueous solution 33 may be water or an aqueous solution containing a water-soluble substance, such as, but not limited to, saline, phosphate buffer, acetate buffer, citrate buffer, citrate phosphate buffer, Tris buffer, Tris phosphate buffer, etc. Examples of water-soluble substances include, but are not limited to, acids, bases, salts, etc. The second aqueous solution may be the same as or different from the first aqueous solution 31.

[0078] As shown in Figure 4(D), when the organic solution 32 is replaced with the second aqueous solution 33, in addition to the lipid monolayer 34 arranged at the connection portion, the hydrophobic groups of the lipids in the organic solution 32 are arranged and further arranged so as to face the hydrophobic groups of the monolayer lipid membrane, forming a lipid bilayer 35. In other words, the lipid bilayer 35 has a structure in which two lipid molecules in two lipid monolayers are oriented so that their hydrophobic groups face each other. The lipid bilayer 35 is formed at the point 23X (see Figure 3(B)) where the diameter of the connecting path 23 is smallest.

[0079] According to the method for forming a lipid bilayer membrane using the microfluidic device 1 of this embodiment described above, the second flow path 22 is filled with the first aqueous solution 31, and no organic solvent flows in before the lipid bilayer membrane 35 is formed. This prevents mixing of the organic solvent and the aqueous solution, which is advantageous for the formation of the lipid bilayer membrane 35.

[0080] Although the present applicant does not wish to restrict the present invention to any particular hypothesis or theory, it is believed that the reason lipid bilayer membranes 35 can be stably synthesized using the microfluidic device 1 of this embodiment is due to the effect of Laplace pressure caused by the hydrophobic and hydrophilic coating of the two flow paths 21, 22. As shown in FIGS. 5(A) to 5(C), when organic solvent 32 is flowing into first flow path 21, Laplace pressure is applied to organic solvent 32 at the interface between organic solvent 32 and first aqueous solution 31 in connecting path 23 (FIG. 5(B)). When first aqueous solution 31 is flowing into second flow path 22, Laplace pressure is applied to first aqueous solution 31 (FIG. 5(C)). The interfacial Laplace pressure, which is due to surface tension, is inversely proportional to the spatial size. Therefore, the smaller the connecting path 23, the greater the Laplace pressure that maintains the interface, enabling more stable fluid manipulation. This is believed to enable lipid bilayer membrane synthesis not only in flow paths of 10 μm or larger, as in the past, but also in extremely small flow paths of several μm or less, which was previously difficult.

[0081] The above explanation based on Laplace pressure is a theory, and even if a microfluidic device and a lipid bilayer membrane formation method do not completely follow this theory, they are included within the technical scope of the present invention as long as they satisfy the requirements stipulated in the present invention.

[0082] When an aqueous solution containing a membrane protein 36 is used as the second aqueous solution 33 filling the first flow path 21, the membrane protein 36 is inserted into the lipid bilayer membrane 35 from the first flow path 21 side, as shown in Figure 6. On the other hand, when an aqueous solution containing a membrane protein 36 is used as the first aqueous solution 31 filling the second flow path 22, the membrane protein 36 is inserted into the lipid bilayer membrane 1 from the second flow path 22 side. Thus, according to the method for forming a lipid bilayer membrane of this embodiment, the direction in which the membrane protein 36 can be inserted into the lipid bilayer membrane 35 is not limited, and further new compounds can be inserted into the lipid bilayer membrane 35, making it possible to form a lipid bilayer membrane with higher functionality.

[0083] The membrane protein 36 may be any membrane protein capable of being inserted into a lipid bilayer. Membrane proteins include integral membrane proteins that reside within the lipid bilayer and peripheral membrane proteins that are bound to the lipid bilayer or integral membrane proteins by non-covalent forces such as hydrophobic interactions and electrostatic interactions. Integral membrane proteins include, but are not limited to, transmembrane proteins.

[0084] The effects of the microfluidic device 1 according to the embodiment of the present invention and the method for forming a lipid bilayer membrane using the same will be described. The parallel-running first flow path 21 is provided with a hydrophobic surface 21S, and the second flow path 22 is provided with a hydrophilic surface 22S, so that the interface between the organic solvent 32 flowing through the first flow path 21 and the first aqueous solution 31 flowing through the second flow path 22 is maintained stable.

[0085] Therefore, a lipid bilayer membrane can be easily formed without using an external pressure pump to precisely control the pressure of the organic solvent 32 in the first flow path 21 and the pressure of the first aqueous solution 31 in the second flow path 22. Furthermore, the spatial size of the connecting path 23 where the lipid bilayer membrane is formed can be further reduced to the order of several μm. Since the parallel first flow path 21 is provided with a hydrophobic surface 21S and the second flow path 22 is provided with a hydrophilic surface 22S, it is only necessary to flow the second aqueous solution 33 through the second flow path 22, and there is no need to exchange liquids in the second flow path 22 to form a lipid bilayer membrane. Therefore, no solution mixing occurs between the second aqueous solution 33 and the organic solvent, and the lipid bilayer membrane 35 can be easily formed. The diameter of the connection path 23 is tapered from one end to the other end, and from the other end to the one end. Therefore, the lipid bilayer membrane is formed at the point 23X where the diameter of the connection path 23 is smallest, and the lipid bilayer membrane 35 can be easily formed.

[0086] Although the present invention has been described above by taking specific embodiments as examples, the present invention is not limited to these and the following modifications are possible.

[0087] 1-4, all of the surfaces defining the first flow path 21 over the entire length thereof are hydrophobic, and all of the surfaces defining the second flow path 22 over the entire length thereof are hydrophilic. However, the entire length and entire area of ​​the walls defining the first flow path 21 and the second flow path 22 do not have to be defined by hydrophobic and hydrophilic surfaces. For example, as shown in FIG. 3, a part of the first portion 21a, a part of the second portion 21b, and a part of the third portion 21c of the first flow path 21 around the connecting path 13 may be defined by hydrophobic surfaces, and a part of the first portion 22a, a part of the second portion 22b, and a part of the third portion 22c of the second flow path 22 may be defined by hydrophilic surfaces. Alternatively, it is sufficient that the second portion 21b of the first flow path 21 is defined by a hydrophobic surface and the second portion 22b of the second flow path 22 of the second flow path 21 is defined by a hydrophilic surface, or it is also possible that only the second portion 21b of the first flow path 21 is defined by a hydrophobic surface and only the second portion 22b of the second flow path 22 of the second flow path 21 is formed by a hydrophilic surface. Not all of the four surfaces defining the first flow path 21 need to be hydrophobic surfaces, for example, two side surfaces and the bottom surface may be hydrophobic surfaces. Also, not all of the four surfaces defining the second flow path 22 need to be hydrophobic surfaces, for example, two side surfaces and the bottom surface may be hydrophobic surfaces. As shown in FIG. 7, the connection channel 23 of the microfluidic device 1 may be a plurality of connection channels 23 that connect the first channel 21 and the second channel 22 at a plurality of locations.

[0088] According to this configuration, a lipid bilayer membrane is formed in each of the plurality of connection paths 23, so that a plurality of lipid bilayer membranes can be tested simultaneously. As long as a lipid bilayer membrane can be formed in the microfluidic device 1, the linear channels 11 and 14, the linear channels 12 and 13, the linear channels 15 and 18, and the linear channels 16 and 17 may be asymmetric with respect to the imaginary center line P. Furthermore, the linear channels 11 and 15, the linear channels 12 and 16, the linear channels 13 and 17, and the linear channels 14 and 18 may be asymmetric with respect to the imaginary center line Q. According to the above-described embodiment, a lipid bilayer membrane can be formed without precise control of the delivery of the organic solvent and aqueous solution to the microfluidic device 1 using a pump or the like. However, optionally, the delivery tube may be further connected to a pressure pump to more precisely control the delivery and discharge of the organic solvent and aqueous solution to the microfluidic device 1.

[0089] The microfluidic device 1 and lipid bilayer membrane formation method according to an embodiment of the present invention solve the problems inherent in conventional artificial lipid bilayer membrane synthesis methods and are therefore expected to be applicable to a variety of fields. For example, by embedding membrane protein nanopores in lipid bilayer membranes and verifying in situ sequencing, they could be applied to blood microRNA analysis devices. Conventional methods involve extracting blood microRNA, amplifying it via PCR, and analyzing the base sequence using a next-generation sequencer. However, due to microRNA loss during sample pretreatment and biased PCR amplification, it has been difficult to obtain a microRNA profile that reflects the living body. The application of the present invention makes it possible, for the first time, to pretreat microRNA without loss using microfluidics and sequence it directly without PCR amplification, fundamentally resolving the problems inherent in conventional methods. This could lead to the discovery of biomarkers for cancer, immune diseases, and other conditions, as well as the realization of liquid biopsies using blood as a sample.

[0090] The present invention will be described in more detail below with reference to examples, but the present invention is not limited to these examples. [Example]

[0091] Example First, a microfluidic device with the configuration shown in Figure 1 was constructed. Specifically, a groove 100 was fabricated on a quartz substrate using electron beam lithography and dry etching, and then the groove 100 was covered by bonding to another substrate to create a device equipped with a channel 10 consisting of through-holes 5a-5h, linear channels 11-18, merging channels 19a-19d, a first channel 21, a second channel 22, and a connecting channel 23. Next, the entire channel was hydrophobically modified by vapor-phase modification using a silane coupling agent (trimethylsilane: TMS). Each linear channel 11-18 had a width of 100 μm and a depth of 26 μm. After the hydrophobic modification, sodium hydroxide was passed through the second channel 22 to remove the TMS and modify the surface to a hydrophilic one, thereby creating two parallel microchannels: the first channel 210 with a hydrophobic surface and the second channel 22 with a hydrophilic surface. The widths W1 and W2 of the first flow channel 22 and the second flow channel 22 were 8 μm and the depth was 4 μm. The width W3 of the connection channel 23 between the two flow channels 21 and 22 was 2 μm.

[0092] Next, an organic solvent containing lipid molecules was passed through the hydrophobic flow path of the first flow path 21, and ion-exchanged water was passed through the second flow path 22, forming a monolayer lipid molecular membrane at the interface between the organic and aqueous phases (Figure 8(A)). The lipid molecules used were a 14:1 mixture of 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) and 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) fluorescently labeled with ATTO488 (ATOO-TEC), and were dissolved in chloroform at 4 mg / mL.

[0093] Next, the organic solvent in the first flow channel 21 was replaced with water, and a lipid bilayer membrane was formed on the connecting channel 23 between the two flow channels 21 and 22 (FIGS. 8(B) and (C)).

[0094] In this way, by making the two parallel microchannels 21 and 22 hydrophobic and hydrophilic, respectively, the Laplace pressure resulting from surface tension acts in the direction of maintaining the interface, thereby stably maintaining the interface between the organic and aqueous phases, enabling the formation of parallel two-phase flows between the organic and aqueous phases and the fluid manipulation of replacing the organic phase with the aqueous phase to be performed simply and reliably, resulting in the successful synthesis of a lipid bilayer membrane. By utilizing microchannels of a few micrometers or less, it is possible to achieve miniaturization and high sensitivity by an order of magnitude compared to conventional lab-on-a-chip.

[0095] Comparative Example We conducted a verification experiment for lipid bilayer membrane synthesis using a microchannel 10 whose entire surface was hydrophobically modified (the first channel 21 and second channel 22 were 2 μm wide and 2 μm deep, and the connecting channel 23 was 4 μm wide). In previous studies, we investigated a method for compensating for the surface tension that prevails in a microspace by applying pressure from the outlet side of the second channel (hereafter referred to as back pressure). First, in step 1, both the first channel 21 and the second channel 22 were filled with water (Figures 9(A) and (H)). Then, in step 2, the water was replaced with an organic solvent, and a liquid-liquid interface between the organic solvent and the aqueous solution could be maintained with the same pressure on the first channel 21 and the second channel 22 (Figures 9(B), (E), and (I)). However, when the organic solvent in the first flow channel 21 was replaced with the second aqueous solution in step 3 (Figures 9(C), (F), and (J)), the Laplace pressure disappeared at the liquid-liquid interface between the aqueous solutions, and the back pressure prevented the pressure balance from being maintained at the liquid-liquid interface, resulting in failure of the fluid manipulation for lipid bilayer membrane synthesis (Figures 9(D), (G), and (K)).

[0096] As described above, it was difficult to simultaneously maintain the liquid-liquid interface between the organic solvent and the aqueous solution and perform the replacement operation while controlling the fluid driving pressure while taking into account the Laplace pressure within the flow channel 10 whose entire surface was hydrophobically modified. [Explanation of symbols]

[0097] 1...microfluidic device, 3...first substrate, 4...second substrate, 21...first flow path, 21S...hydrophobic surface, 22...second flow path, 22S...hydrophilic surface, 23...connecting path, 23a...first part of connecting path, 23X...smallest diameter part of connecting path, 23b...second part of connecting path, 31...first aqueous solution, 32...organic solvent, 33...second aqueous solution, 35...lipid bilayer membrane.

Claims

1. 1. A microfluidic device comprising: a first flow path defined by a hydrophobic surface; a second flow path defined by a hydrophilic surface and extending in parallel with the first flow path; a connecting path connecting the first flow path and the second flow path, A microfluidic device, wherein the second flow path is filled with a first aqueous solution, and the first flow path is filled with a second aqueous solution, and a lipid bilayer membrane is formed in the connecting path.

2. 2. The microfluidic device according to claim 1, wherein the connecting path is connected to the first flow path at one end and to the second flow path at the other end, the connecting path narrows in diameter from the one end to the other end and from the other end to the one end, and a lipid bilayer membrane is formed at the point where the diameter of the connecting path is smallest.

3. 3. The microfluidic device of claim 1, wherein a portion of the first flow path extends toward the second flow path to form a first portion of the connecting path, a portion of the second flow path extends toward the first flow path to form a second portion of the connecting path, the first portion of the connecting path and the second portion of the connecting path are connected to form the entire length of the connecting path, and the first portion of the connecting path has a hydrophobic surface and the second portion of the connecting path has a hydrophilic surface.

4. The microfluidic device according to any one of claims 1 to 3, wherein the width of the connecting path is less than 100 µm.

5. The microfluidic device according to any one of claims 1 to 4, wherein the first flow path and the second flow path extend linearly and parallel to each other on both sides of the portion connected to the connecting path in the longitudinal direction of each of the first flow path and the second flow path.

6. The microfluidic device according to any one of claims 1 to 5, comprising a first substrate provided with the first flow path, the second flow path, and the connecting path, and a second substrate bonded together.

7. A microfluidic device for forming a lipid bilayer membrane, comprising: a first flow path defined by a hydrophobic surface; a second flow path extending parallel to the first flow path and having a hydrophilic surface; a connecting path connecting the first flow path and the second flow path.

8. A method for forming a lipid bilayer membrane, comprising: providing a microfluidic device including a first flow path defined by a hydrophobic surface, a second flow path having a hydrophilic surface and extending in parallel with the first flow path, and a connecting path connecting the first flow path and the second flow path; filling the first flow path of the microfluidic device with an organic solvent containing lipid molecules and filling the second flow path with a first aqueous solution, thereby contacting the organic solvent and the first aqueous solution in the connecting path; and replacing the organic solvent in the first flow path with a second aqueous solution, thereby forming a lipid bilayer membrane in the connecting path; A method comprising:

9. The method according to claim 8, wherein the connecting path communicates with the first flow path at one end and communicates with the second flow path at the other end, the connecting path narrowing in diameter from the one end to the other end and from the other end to the one end, and the step of forming a lipid bilayer membrane in the connecting path includes forming a lipid bilayer membrane at a point in the connecting path where the diameter is smallest.

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