Partition wall for forming lipid bilayer membrane and lipid bilayer membrane forming device including same, and method for forming lipid bilayer membrane using same

Abstract

Disclosed is a means capable of enhancing the efficiency of formation of a lipid bilayer membrane and the efficiency of introduction of a membrane protein to a lipid bilayer membrane compared with lipid bilayer membrane formation by a conventional droplet contact method. The partition wall for forming a lipid bilayer membrane has a through hole in which a planar lipid bilayer membrane is formed. The planar shape of the through hole has at least a linear portion, and the lipid bilayer membrane formed also has a linear portion. Also provided are: a lipid bilayer membrane formation device having the partition wall for forming a lipid bilayer membrane; a method for forming a lipid bilayer membrane using the device; and a lipid bilayer membrane formed in a through-hole portion of a partition wall, wherein the peripheral edge has a linear portion.

Description

Partition for forming lipid bilayer membrane, lipid bilayer membrane forming device including the same, and lipid bilayer membrane forming method using the same

[0001] The present invention relates to a partition for forming a lipid bilayer membrane, a lipid bilayer membrane-forming device including the partition, a lipid bilayer membrane-forming method using the partition, and the formed lipid bilayer membrane.

[0002] The cells that make up living organisms, as well as various intracellular organelles such as mitochondria, Golgi apparatus, and endoplasmic reticulum, and cell nuclei, are surrounded by a biological membrane, which is essentially composed of a lipid bilayer. Various physiologically active proteins, such as receptors and enzymes, are retained on the lipid bilayer and penetrate the membrane. These transmembrane proteins play important roles in living organisms. In particular, it has been found that various receptors present on the cell membrane trigger various physiological responses by binding to ligands present in the body. For this reason, various ligands that enhance receptor function and inhibitors that inhibit receptor function are used as pharmaceuticals, and natural or artificial ligands and inhibitors that can be used as new pharmaceuticals are also being researched.

[0003] It is also known that proteins held in lipid bilayer membranes can be used as sensors. For example, an odor sensor is formed by holding an olfactory receptor protein in a lipid bilayer membrane, or a sensor is known in which a specific binding substance that specifically binds to a test substance is contained in the droplet when the lipid bilayer membrane is formed by the droplet contact method, and an ion channel protein is held in the lipid bilayer membrane so that, in the presence of the test substance, the test substance binds to the specific binding substance and blocks the ion channel.

[0004] Conventionally, the droplet contact method has been widely used as a method for forming lipid bilayer membranes (see, for example, Patent Documents 1 to 4). The droplet contact method typically uses a device comprising a first well, a second well positioned adjacent to the first well, and a partition separating the first and second wells. The partition has through-holes through which a lipid bilayer membrane is formed. A lipid solution containing a lipid membrane-forming lipid is added to each of the first and second wells, and then an aqueous electrolyte solution is added to each well, forming a lipid bilayer membrane that blocks the through-holes in the partition. Membrane proteins and other components to be retained within the lipid bilayer membrane are typically contained in an aqueous solution. That is, proteins are introduced into the lipid bilayer membrane by adding lipid vesicles containing the membrane protein or purified membrane protein to an aqueous solution.

[0005] Japanese Patent Application Publication No. 2017-83210 Japanese Patent Application Publication No. 2019-072698 Japanese Patent Application Publication No. 2012-081405 Japanese Patent Application Publication No. 2019-022872

[0006] One-push membrane formation for iterative measurement of ion channel activity on arrayed chips, H. Mimura et al., 10.1109 / MEMS49605.2023.10052568. GTP-dependent regulation of myometrial KCa channels incorporated into lipid bilayers, L. Toro et al., J. Gen. Physiol., 96, 373-394 (1990). Yasunobu Okada (ed.), Latest Patch Clamp Experimental Techniques, Yoshioka Shoten (2011). One-Pot Species Release and Nanopore Detection in a Voltage-Stable Lipid Bilayer Platform, X. Kang et al., Nano Lett., 19, 9145-9153 (2019).

[0007] In the formation of lipid bilayer membranes by the droplet contact method, it goes without saying that high lipid bilayer formation efficiency (i.e., short time until the lipid bilayer membrane is formed) and high membrane protein introduction efficiency (i.e., short time until the membrane protein is introduced (also called reconstitution) into the lipid bilayer membrane) are advantageous for conducting experiments efficiently. In particular, with tiny lipid bilayer membranes (diameter 200 μm or less), this method has the advantage of a high signal-to-noise ratio and time resolution in the measurement results, but has the problem of low membrane protein introduction efficiency.

[0008] Therefore, the object of the present invention is to provide a means capable of increasing the efficiency of lipid bilayer membrane formation and the efficiency of membrane protein introduction into lipid bilayer membranes compared to lipid bilayer membrane formation by the conventional droplet contact method.

[0009] As a result of extensive research, the present inventors have discovered that by making the planar shape of the through-holes provided in the partition (also called "separator") used in lipid bilayer membrane formation by the droplet contact method non-circular, it is possible to form a lipid bilayer membrane with a straight edge, and that such a lipid bilayer membrane with a straight edge has a higher efficiency of lipid bilayer membrane formation and membrane protein introduction efficiency into the lipid bilayer membrane than conventional circular lipid bilayer membranes, thereby completing the present invention.

[0010] That is, the present invention provides the following: (1) A partition wall for forming a lipid bilayer membrane, having through-holes through which a planar lipid bilayer membrane is formed, wherein the planar shape of the through-holes has at least a linear portion, and the lipid bilayer membrane formed also has a linear portion. (2) The partition wall for forming a lipid bilayer membrane according to (1), which is a partition wall for forming a lipid bilayer membrane by a droplet contact method. (3) The partition wall for forming a lipid bilayer membrane according to (1) or (2), wherein the planar shape of the through-holes has a linear portion. (4) The partition wall for forming a lipid bilayer membrane according to any one of (1) to (3), wherein the planar shape of the lipid bilayer membrane to be formed is a polygon with six or fewer vertices. (5) The partition wall for forming a lipid bilayer membrane according to (4), wherein the polygon is a rectangle, a rhombus, an upward-facing triangle, or a downward-facing triangle. (6) A lipid bilayer membrane-forming partition according to any one of (1) to (3), wherein the planar shape of the through-hole is X-shaped, rectangular, cross-shaped, upward three-way radial, or downward three-way radial. (7) A lipid bilayer membrane-forming device comprising the lipid bilayer membrane-forming partition according to any one of (1) to (6). (8) The lipid bilayer membrane-forming device according to (7), comprising the lipid bilayer membrane-forming partition according to any one of (1) to (6) and at least one well provided in a substrate and separated by the partition. (9) A lipid bilayer membrane-forming method, comprising forming a lipid bilayer membrane having a linear portion that covers at least a part of the through-hole using the lipid bilayer membrane-forming device according to (7) or (8). (10) A lipid bilayer membrane formed in the through-hole portion of the partition, the lipid bilayer membrane having a linear periphery. (11) The lipid bilayer membrane according to (10), wherein the planar shape is a rhombus, a right-angled rectangle, an upward triangle, or a downward triangle. (12) Use of the partition wall according to any one of (1) to (6) for forming a lipid bilayer membrane. (13) Use of the partition wall according to any one of (1) to (6) for producing a lipid bilayer membrane-forming device.

[0011] By forming a lipid bilayer membrane by the droplet contact method using the lipid bilayer membrane forming diaphragm of the present invention, the efficiency of lipid bilayer membrane formation and the efficiency of membrane protein introduction into the lipid bilayer membrane can be improved compared to conventional droplet contact methods.

[0012] 1 is a diagram schematically illustrating a specific example of a substrate having a double-well chamber used in the method of the present invention.

[0034] FIG. 1 is a diagram illustrating the water or aqueous solution addition step in the method of the present invention.

[0035] FIG. 1 is a diagram schematically illustrating the shape of a through-hole in a partition wall prepared in the following example.

[0036] FIG. 1 is a diagram illustrating the experimental apparatus used in the following example, where (a) is a schematic perspective view of the entire apparatus, (b) is an enlarged view of the double-well chamber portion in (a), and (c) is a diagram of the apparatus actually prototyped and used.

[0037] FIG. 1 is a diagram illustrating the shapes of lipid bilayer membranes formed when non-circular and circular through-holes are used in the following example. The shapes of the lipid bilayer membranes formed depending on the shape of the through-hole are as follows: (a) upward three-way radiating type → upward triangle. (b) downward three-way radiating type → downward triangle. (c) cross → diamond. (d) X-shape → square. (e) asterisk → circle. (f) square (200 μm) → square. (g) circle (200 μm) → circle. To improve visibility, the shape of the lipid bilayer membrane is shown as a schematic diagram with dashed lines. This figure shows the lipid bilayer membrane formation efficiency measured in the Examples below when noncircular and circular through-holes are used. The tracing operation was performed 10 times at approximately 3-minute intervals (each set), and the number of times a lipid bilayer membrane was formed is shown. The graph shows the average and standard error of three sets of experiments. Squares (200 μm on each side) were used for the rectangular through-holes, and circles (200 μm in diameter) were used for the circular through-holes. This figure shows a comparison of the appearance rate of ion channel signals when noncircular through-holes were used, measured in the Examples below. The shapes of the planar lipid bilayer membranes formed by the through-holes used in the experiments are as follows: X-shaped → square, asterisk → circle, square (200 μm) → square. Ten experiments were performed, and the number of times an ion channel signal appeared is shown in the graph. This figure shows a comparison of the appearance rate of ion channel signals when noncircular through-holes of different sizes and shapes were used, measured in the Examples below. (a) Number of times a lipid bilayer membrane was formed. (b) Number of ion channel signal appearances. (c) Rate of ion channel signal appearance per number of lipid bilayer formations. The diameter of the circular through-holes and the length of one side of the square through-holes are indicated.The experiment involved 10 tracing runs, and the number of times a lipid bilayer was formed (a) and the number of times an ion channel signal appeared (b) were counted. The appearance rate of the ion channel signal per number of times a lipid bilayer was formed (c) was then calculated. The graph shows the average and standard error of four sets of experiments.

[0013] The lipid bilayer membrane formation method of the present invention can use a device similar to that used in known methods, except for the planar shape of the through-holes formed in the partition. The "partition" in the lipid bilayer membrane formation method is a plate with through-holes formed in it, and these partitions separate wells formed in the substrate, forming a lipid bilayer membrane that blocks these through-holes. Known methods for forming lipid bilayer membranes that cover the through-holes include the droplet contact method, the brush coating method, and the lamination method, and the present invention is applicable to any of these methods. Of these, the droplet contact method is preferred. Figure 1 is the same as Figure 1 of Patent Document 3, but the method of the present invention can also use a device with a structure basically similar to that of a known lipid bilayer membrane formation device used in the droplet contact method.

[0014] FIG. 1 is a schematic diagram of one specific example of a lipid bilayer membrane-forming device that can be preferably used in the method of the present invention. FIG. 1(a) is a plan view, and FIG. 1(b) is an end view of the cross section taken along line b-b' in FIG. 1(a). In this specific example, a first well 16 and a second well 14 are formed in a substrate 10, and the boundary between them is separated by a partition wall 12. This configuration is called a double-well chamber (DWC). The partition wall 12 has a through-hole 18 (see FIG. 1(b)). In the example shown in FIG. 1, the planar shape of the well is basically circular, with only the boundary where the two circles meet being linear. However, the shape of the well is not limited, and other shapes are also acceptable as long as they are separated by a partition wall having the through-hole. The size of the well is not particularly limited, and typically has a diameter of about 2 mm to 8 mm, particularly about 3 mm to 5 mm, and a depth of about 50% to 200%, particularly about 50% to 100% of the diameter. However, the method of the present invention can be carried out with wells larger or smaller than these ranges. The number of through-holes provided in the partition wall may be one or more. It is known that the efficiency of lipid bilayer membrane formation is increased when the through-holes are tapered in the thickness direction (i.e., the diameter of the through-holes is different on one surface of the partition wall than on the other surface), and this is also preferred in the present invention.

[0015] The partition wall of the present invention is characterized by the planar shape of the through-holes. Conventionally, the planar shape of the through-holes in the partition wall is all circular. Textbooks on the droplet contact method also state that the through-holes in the partition wall are circular. In the present invention, the planar shape of the through-holes is non-circular, and the lipid bilayer membrane formed has a straight portion at its periphery. This is significantly different from the conventional droplet contact method. In known droplet contact methods, the planar shape of the through-holes is always circular, and therefore the planar shape of the lipid bilayer membrane formed is also circular. A lipid bilayer membrane with a straight portion at its periphery has never been known before, and is provided for the first time by the present invention.

[0016] The planar shape of the through-hole is not particularly limited as long as it is non-circular and forms a lipid bilayer membrane with a straight portion at the periphery, but it is preferable that the planar shape of the through-hole have a straight portion. Note that the "planar shape" refers to the shape viewed from directly above when the partition is placed on a horizontal surface. The "planar shape" of the lipid bilayer membrane is similar, and refers to the shape viewed from directly above when the lipid bilayer membrane is placed on a horizontal surface, in other words, the shape of a lipid bilayer membrane formed in the vertical direction when viewed from the horizontal direction.

[0017] The planar shape of the lipid bilayer membrane to be formed is preferably a polygon with six or fewer vertices, preferably four or fewer vertices. Preferred examples of such polygons include right-angled quadrilaterals (squares and rectangles), rhombuses, and upward-pointing or downward-pointing triangles. The polygon is preferably a regular polygon.

[0018] Examples of the planar shape of the through-holes in the partitions that form such planar lipid bilayer membranes include X-shaped, rectangular, cross-shaped, upward three-way radiation, and downward three-way radiation. In the following description, "up" means vertically upward (i.e., the direction opposite to the direction of gravity), and "down" means vertically downward (i.e., the direction of gravity). In the following description, the numerical value indicating the size of a through-hole refers to the size of the smallest part of the through-hole when the through-hole tapers in the thickness direction.

[0019] As specifically demonstrated in the examples below, when an X-shaped through-hole is used, a square lipid bilayer membrane is formed in the center of the X. Here, the "X-shape" preferably has lines extending from the center in all four directions at 90-degree angles, although variations of approximately ±20%, particularly approximately ±10%, from 90 degrees are acceptable. The "X-shape" may also consist solely of linear portions (i.e., grooves), or may have a circular central portion with linear portions extending radially in all directions from the central circular portion. The size of the X-shaped through-hole is not particularly limited as long as a square lipid bilayer membrane is formed, but the width of the linear portion is typically approximately 10 μm to 200 μm, preferably approximately 70 μm to 150 μm. The length of the linear portion is not particularly limited, but is typically approximately 100 μm to 500 μm, preferably approximately 200 μm to 300 μm. When a circular portion is provided in the center of the X-shape and straight portions extend radially from this circular portion, the diameter of this circle is usually about 50 μm to 800 μm, preferably about 200 μm to 700 μm. When a circular portion is provided, it is preferable to set the width of the straight portion so that an arc portion remains in the center of the X-shape.

[0020] When a rectangular through-hole is used, a rectangular lipid bilayer membrane is formed. The rectangular rectangle is preferably a square. In the case of a square, the length of one side is not particularly limited as long as a lipid bilayer membrane is formed, but is usually about 50 μm to 800 μm, preferably about 100 μm to 700 μm.

[0021] When a cross-shaped through-hole is used, a diamond-shaped lipid bilayer membrane is formed in the center of the cross. Here, the "cross" preferably has four straight lines extending from the center in all directions at 90-degree angles, although a deviation of about ±20%, particularly about ±10%, from 90 degrees is acceptable. The "cross" may also consist of only straight lines, or may have a circular central portion with straight lines extending radially in all directions from the central circular portion. The size of the cross-shaped through-hole is not particularly limited as long as a diamond-shaped lipid bilayer membrane is formed, but the width of the straight line portion is typically about 50 μm to 200 μm, preferably about 70 μm to 150 μm. The length of the straight line portion is not particularly limited, but is typically about 100 μm to 500 μm, preferably about 200 μm to 300 μm. When a circular portion is provided in the center of the cross shape and straight portions extend radially from this circular portion, the diameter of this circle is usually about 50 μm to 800 μm, preferably about 200 μm to 700 μm. When a circular portion is provided, it is preferable to set the width of the straight portion so that an arc portion remains in the center of the cross.

[0022] When an upward triangular through-hole is used, an upward triangular lipid bilayer membrane is formed. Here, the "upward triangular radiation" refers to a shape in which three straight lines extend radially from a single point, with one of the three straight lines extending directly upward. Here, the "upward triangular radiation" preferably has three lines extending radially from the center that are all at an angle of 120 degrees relative to each other, although a deviation of about ±20%, particularly about ±10%, from 120 degrees is acceptable. Furthermore, the "upward triangular radiation" may consist solely of straight lines, or may have a circular central portion from which straight lines extend radially in three directions. The size of the upward triangular radiation through-hole is not particularly limited as long as it allows the formation of an upward triangular lipid bilayer membrane, but the width of the straight lines is typically about 50 μm to 200 μm, preferably about 70 μm to 150 μm. The length of the straight line portion is not particularly limited, but is usually about 100 μm to 500 μm, preferably about 200 μm to 300 μm. When a circular portion is provided in the center of the upward three-way radiation type and the straight line portions extend radially from this circular portion, the diameter of this circle is usually about 50 μm to 800 μm, preferably about 200 μm to 700 μm. When a circular portion is provided, it is preferable to set the width of the straight line portion so that an arc portion remains in the center.

[0023] When a downward triangular radiating through-hole is used, a downward triangular lipid bilayer membrane is formed. Here, the "downward triangular radiating through-hole" refers to a shape in which three straight lines extend radially from a single point, with one of the three straight lines extending directly downward. Here, the "downward triangular radiating through-hole" preferably has three lines extending radially from the center that are all at an angle of 120 degrees, although a deviation of about ±20%, particularly about ±10%, from 120 degrees is acceptable. Furthermore, the "downward triangular radiating through-hole" may consist solely of straight lines, or may have a circular central portion from which straight lines extend radially in three directions. The size of the downward triangular radiating through-hole is not particularly limited as long as an upward triangular lipid bilayer membrane is formed; however, the width of the straight lines is typically about 50 μm to 200 μm, preferably about 70 μm to 150 μm. The length of the straight line portion is not particularly limited, but is usually about 100 μm to 500 μm, preferably about 200 μm to 300 μm. When a circular portion is provided in the center of the downward three-way radiation type and the straight line portions extend radially from this circular portion, the diameter of this circle is usually about 50 μm to 800 μm, preferably about 200 μm to 700 μm. When a circular portion is provided, it is preferable to set the width of the straight line portion so that an arc portion remains in the center.

[0024] Among the through-holes with linear portions at the periphery described above, X-shaped, cross-shaped, upward three-way radial, and downward three-way radial through-holes form a lipid bilayer in their center, but not in the linear portions (grooves), leaving them as through-holes. However, when a lipid bilayer is formed in the center, the linear portions (grooves) are filled with an organic solvent (e.g., n-decane) in which lipids are dispersed, and ions in the liquid cannot pass through the organic solvent layer in which lipids are dispersed, as with a lipid bilayer. Therefore, the reconstitution of ion channel proteins can be measured in the same way as when a conventional circular lipid bilayer is formed.

[0025] Except for the use of the lipid bilayer membrane-forming partition wall of the present invention described above, the lipid bilayer membrane formation method of the present invention is similar to the well-known droplet contact method. A lipid bilayer membrane can be formed in the portion of the through-hole 18 by the well-known droplet contact method using a device similar to the well-known lipid bilayer membrane-forming device shown in Figure 1 , except that the partition wall 12 of the lipid bilayer membrane-forming device described based on Figure 1 has the planar through-hole 18 described above.

[0026] Briefly, as described in Patent Document 3, for example, a lipid bilayer-forming lipid solution is added to each of the two wells 14 and 16, and the lipid solution is filled into each well. Here, the lipid bilayer-forming lipid may be a well-known phospholipid used in preparing liposomes. To mimic reactions in biological membranes, a lipid that is the same as or similar to biological membranes is preferred. Preferred examples of phospholipids that have been widely used in this field include diphytanoyl phosphatidylcholine (DPhPC), dipalmitoyl phosphatidylcholine (dipalmytoyl phosphatidylcholine), palmitoyl oleoyl phosphatidylcholine (1-Palmitoyl 2-Oleoyl phosphatidylcholine (POPC)), and dioleoyl phosphatidylcholine (DOPC). Many of these are commercially available, and commercially available products can be preferably used.

[0027] The concentration of phospholipids in the solution used to form the lipid bilayer membrane is not particularly limited as long as it is a concentration that allows the formation of a lipid bilayer membrane, but is usually about 5 g / L to 30 g / L, preferably about 10 g / L to 20 g / L. The solvent for the phospholipid solution is not particularly limited, but is preferably an organic solvent, and more preferably an aliphatic hydrocarbon solvent such as n-decane.

[0028] Next, water or an aqueous solution is added to each well to form droplets of water or an aqueous solution in the lipid solution. This process is shown schematically in FIG. 2. FIG. 2 shows how a micropipette 22 is inserted into the lipid solution 20 filled in each well to form droplets 24 of water or an aqueous solution. FIG. 2 also shows a schematic diagram of a droplet 24 being formed in one well and another droplet 24 being formed in the other well. The liquid forming the droplets in this process may be pure water or an aqueous solution such as an aqueous buffer solution (a buffer solution whose primary solvent is water). The amount of water or an aqueous solution added to each well is not particularly limited, but from the perspective of efficiently forming a lipid bilayer membrane, it is preferably about 1 to 25 times the volume of the lipid solution filled in each well, and more preferably about 5 to 10 times.

[0029] The lipid bilayer membranes of the present invention are suitable for use in various assays, such as examining the properties and function of proteins retained in the lipid bilayer membrane, screening for ligands that bind to the protein and alter its physiological activity, and examining their properties. Therefore, the lipid bilayer membrane preferably contains a protein, particularly a transmembrane protein that functions in vivo while retained in a biological membrane. Proteins retained in the lipid bilayer membrane can include various receptors and enzymes, including, but not limited to, peptide proteins such as α-hemolysin, gramicidin, and alamethicin, various ion channels, and ABC transporter proteins. When the protein is water-soluble, an aqueous solution of the protein is preferably used as the aqueous solution, and an aqueous solution containing the protein in an aqueous buffer is even more preferable. The protein concentration in these solutions is not particularly limited and can be selected as appropriate, but is typically approximately 1 nM to 1 mM, preferably approximately 0.1 μM to 10 μM. The protein may be contained in the aqueous solution added to either one of the two wells, or it may be contained in the aqueous solutions added to both wells. Furthermore, when the protein to be held in the lipid bilayer membrane is lipophilic, proteoliposomes expressing the membrane protein can be used by fusing them with the lipid membrane in the device.

[0030] When the droplets are formed in the lipid solution, the lipid solution is compressed and pressed against the partition wall, where the lipid solution layer and the aqueous layer come into contact with each other under a certain amount of pressure. At this time, a lipid bilayer membrane is formed in the through-holes of the partition wall. The lipid bilayer membrane is formed by leaving the water or aqueous solution droplets in the formed state for typically 3 minutes to 1 hour, blocking the through-holes of the partition wall.

[0031] When the protein is an ion channel-forming protein, such as α-hemolysin or alamethicin, if the protein is properly retained in the lipid bilayer, the retained protein portion forms an ion channel, establishing electrical continuity between the two wells. Therefore, by applying a predetermined voltage between the two wells and measuring the current flowing through the lipid bilayer, it is possible to confirm that a lipid bilayer has been formed in which the protein is properly retained in the lipid bilayer. Furthermore, by applying various substances to the protein, it is possible to screen for substances that change the protein structure and make the ion channel easier to close or open. Such substances may exert some physiological activity and become candidates for pharmaceuticals. Therefore, measuring the current between the two wells is often desirable. As specifically described in the Examples below, the DWC used in the method of the present invention has electrodes placed on the upper surface of the base plate that forms the bottom of each well, and these electrodes can be connected to the outside of the wells, making it easy to wire each well.

[0032] The present invention will be specifically described below based on examples, although the present invention is not limited to the following examples.

[0033] 1. Fabrication of partition walls (separators) with non-circular through holes (1) Design and fabrication of separators with non-circular through holes Separators with non-circular through holes were fabricated (Fig. 3). Specifically, separators with non-circular through holes were fabricated (Fig. 3(a), (△; a through groove of 250 μm length and 100 μm width extending from the center of a 200 μm circular hole toward the apex of an equilateral triangle)), a downward three-way radial type (Fig. 3(b), (▽; a through groove of 250 μm length and 100 μm width extending from the center of a 200 μm circular hole toward the apex of an inverted equilateral triangle)), a cross type (Fig. 3(c), +; a through groove of 250 μm length and 100 μm width extending from the center of a 200 μm circular hole toward the top, bottom, left, and right), an X type (Fig. 3(d), X; a through groove of 250 μm length and 100 μm width extending from the center of a 200 μm circular hole toward the apex of a square), and an asterisk type. (Fig. 3(e), *; a separator with a through-hole of 250 μm length and 100 μm width in the up / down / left / right and square vertex directions from the center of a 200 μm circular hole) (Note that, as will be described later, the asterisk type is not an example of the present invention because a circular lipid bilayer membrane is formed in the asterisk type), and a square (Fig. 3(f), □; a separator with a square through-hole. A conventional circular (○) type was also fabricated for comparison. These separators were fabricated using an NC precision machining center with a thickness of 0.3 mm. The through-holes were fabricated by cutting a 300 mm acrylic plate. The through-holes were tapered on one side, with a large diameter of 1.2 mm and a small diameter of 200 μm. To investigate the relationship between the size of the through-hole and the efficiency of introducing ion channels into the lipid bilayer membrane, square through-holes with side lengths of 141 μm, 200 μm, 283 μm, and 495 μm were fabricated, and circular through-holes with small diameters of 200 μm, 400 μm, and 700 μm were fabricated. For this reason, these were distinguished by their lengths.

[0034] (2) Integration into a Trace Device To evaluate its performance, the fabricated separator was attached to a single-trace device consisting of a pair of double wells (Figure 4). This device has a double-well chip at the bottom and a trace module at the top. The trace module has a microplate and is set on the guide post of the double-well chip. The guide post is equipped with a spring that supports the vertical movement of the trace module. For electrophysiological measurements, a pair of 1-mm-diameter silver-silver chloride electrodes was placed at the bottom of the double well. The well containing the working electrode was designated R (Recording), and the well containing the reference electrode was designated G (Ground). To accurately maintain the distance from the microplate, the separator was attached with the small-diameter side of the tapered through-hole facing R and fixed with adhesive. Tracing refers to the process of tracing the diaphragm with the trace microplate, which is performed by moving the trace microplate up and down while in contact with the diaphragm. When tracing is performed, if a lipid bilayer membrane is formed in the through-hole, this lipid bilayer membrane is destroyed and reset to its original state, and even if the lipid bilayer membrane is in the process of being formed, it is reset to its original state. Therefore, in the experiment below, tracing is performed every 3 minutes, which means that the lipid bilayer membrane formation is reset every 3 minutes.

[0035] 2. Relationship between the shape of the through-holes and the shape of the planar lipid bilayer membrane formed (1) Shape of the through-holes To investigate the shape of the lipid bilayer membrane formed by using noncircular through-holes, we performed microscopic observations using a tracing device equipped with a separator with noncircular through-holes.

[0036] (2) Experimental Method and Conditions: 1) The silver-silver chloride electrodes of the double-well chip were connected to a patch-clamp amplifier. 2) 1.5 μL of phospholipid (1,2-diphytanoyl-sn-glycero-3-phosphocholine, DPhPC, concentration 20 mg / mL) dispersed in organic solvent (decane) was added to the right well of the double-well chip. 3) 25 μL of solution (10 mM Tris / MOPS, pH 7.4, 240 mM KCl, 10 mM Potassium Methanesulfonate, 2 mM CaCl2) was added to both wells of the double-well chip. 4) The tracing module was placed on the guide post, pressed down with a finger, and then released. The spring force returned the tracing module to its original position, moving it up and down (referred to as the tracing operation). By repeating this process, the microplate was moved up and down near the small-diameter side of the separator's through-holes, and lipid bilayer formation was attempted using noncircular through-holes. 5) The formation of lipid bilayers was observed under a microscope from the side of the double-well chip. A triangular pulse (±50 mV, 1 Hz) was applied using a patch clamp amplifier. 6) The trace module was moved up and down approximately every 3 minutes over a 30-minute period, and the number of times lipid bilayers formed was counted. This allowed for a comparison of the efficiency of lipid bilayer formation when using noncircular through-holes. Lipid bilayers were identified by their characteristic contours.

[0037] (3) Results Figure 5 shows microscopic images of lipid bilayers formed using noncircular and circular through-holes. It was confirmed that the lipid bilayers formed had the following shapes depending on the shape of the through-holes used (through-hole shape → lipid bilayer shape): upward triangular radiation → upward triangle, downward triangular radiation → downward triangle, cross → diamond, X → square, asterisk → circle, square → square, circle → circle. It was confirmed that the use of noncircular through-holes also allowed the formation of lipid bilayers with straight segments other than circles.

[0038] Figure 6 shows the results of counting the number of times membranes were formed for each through-hole shape when tracing was performed 10 times at 3-minute intervals. The graph shows the average value and standard error for three sets of experiments. The formation efficiency of lipid bilayer membranes tended to be higher when noncircular through-holes were used compared to when circular through-holes were used. The formation efficiency of X-shaped and asterisk-shaped through-holes was particularly high. This confirmed that one of the advantages of using noncircular through-holes is the improved formation efficiency of lipid bilayer membranes.

[0039] 3. Relationship between Planar Lipid Bilayer Shape and Signal Appearance Rate (1) Comparison of Ion Channel Signal Appearance Rates Using Square and Circular Lipid Bilayers We investigated whether the shape of the lipid bilayer formed using noncircular through-holes affects the efficiency of membrane protein incorporation into the lipid bilayer. The noncircular through-holes compared were X-shaped, asterisk-shaped, and square (200 μm). As mentioned above, the X-shaped, asterisk-shaped, and square-shaped lipid bilayers form square, round, and square, respectively. The ion channel signal appearance rates (the efficiency of membrane protein incorporation into the lipid bilayer) were compared by electrically measuring ion channel activity using the noncircular through-holes that formed these specific lipid bilayer shapes.

[0040] (2) Experimental Method and Conditions: 1) to 4) were performed in the same manner as in "2. Relationship between Through-hole Shape and Planar Lipid Bilayer Membrane Shape" above. 5) 0.5 μL of a sample containing ion channels (a membrane extract fraction prepared from porcine uterus by a known method (Non-Patent Document 2), 1 mg / mL protein concentration, suspended in 5 mM PIPES / NaOH, pH 6.8, 100 mM KCl, and 300 mM sucrose) was added to the right-side well. 6) Electrical measurements were performed by recording current values ​​for 30 minutes under a constant applied voltage (+30 mV). During measurements, the trace module was moved up and down approximately every 3 minutes to promote membrane reformation and the appearance of ion channel signals. 7) Following the above procedure, 10 experiments using each through-hole shape were performed, and the number of experiments in which ion channel signals appeared was counted.

[0041] Results The experimental results are shown in Figure 7. The number of times that ion channel signals appeared in experiments using X-shaped, asterisk-shaped, and square noncircular through-holes was 5, 1, and 5 times, respectively, out of 10 experiments. This indicates that the rectangular lipid bilayer membranes formed with X-shaped and square noncircular through-holes have a higher appearance rate of ion channel signals than circular lipid bilayer membranes formed with asterisk-shaped through-holes.

[0042] 4. Relationship between the appearance rate of ion channel signals and the shape and size of the lipid bilayer membrane (1) Comparison of the appearance rate of ion channel signals using square and circular through-holes of different sizes To investigate whether the high uptake efficiency of membrane proteins in square lipid bilayer membranes is dependent on the size of the through-holes, circular and square through-holes of different sizes were fabricated. Square through-holes were used to form square lipid bilayer membranes. The sizes of the through-holes used in the experiment were three round types, each with a diameter and area of ​​200 μm (31,400 μm 2 ), 400μm (125,600μm 2 ), 700μm (384,650 μm 2 ) On the other hand, the length of one side and area of ​​the three square types are 141 μm (19,881 μm 2 ), 283μm (80,089μm 2 ), 495μm (245,025μm 2 Electrical measurements were performed using these through-holes of different shapes and sizes, and the occurrence rates of ion channel signals were compared.

[0043] (2) Experimental methods and conditions 1) to 6) were performed in the same manner as in "3. Relationship between the shape of the planar lipid bilayer membrane and the rate of signal appearance" above. 7) During electrical measurements, the double-well chip was observed under a microscope from the side to confirm the formation of a lipid bilayer membrane. 8) For each through-hole, the tracing operation (moving the tracing module up and down approximately every 3 minutes) was performed 10 times, and the number of times a lipid bilayer membrane was formed and the number of times an ion channel signal appeared were counted. The experiment was performed four times each.

[0044] (3) Results Figure 8 shows the experimental results. Because the lipid bilayer formation efficiency differs between circular and square through-holes, we calculated (a) the number of lipid bilayers formed per 10 tracing operations, (b) the number of ion channel signal appearances per 10 tracing operations, and (c) the ion channel signal appearance rate per lipid bilayer formation. The graph shows the average values ​​and standard errors for four sets of experiments. We confirmed that both the number of lipid bilayers formed and the number of ion channel signal appearances increased with increasing pore size for both circular and square through-holes. The ion channel signal appearance rate per lipid bilayer formation showed a similar trend. Notably, square lipid bilayers showed a higher ion channel signal appearance rate than circular ones, regardless of through-hole size.

[0045] Square lipid bilayers have corners (small corners) and straight edges that are not found in round lipid bilayers. In these regions, the local lipid arrangement and density may be different. These local changes may have promoted the ion channel's integration by altering the fluidity and tension of the lipid bilayer. Therefore, the through-hole structure that forms corners and straight edges in the lipid bilayer is thought to be important for efficient incorporation of membrane proteins into the lipid bilayer.

[0046] REFERENCE SIGNS LIST 10 Substrate 12 Partition 14 Second well 16 First well 18 Through-hole 20 Lipid solution 22 Micropipette 24 Droplet of water or aqueous solution

Claims

1. A partition for forming a lipid bilayer membrane, having a through-hole through which a planar lipid bilayer membrane is formed, wherein the planar shape of the through-hole has at least a straight portion, and the lipid bilayer membrane formed also has a straight portion.

2. The partition for forming a lipid bilayer membrane according to claim 1, wherein the partition for forming a lipid bilayer membrane is a partition for forming a lipid bilayer membrane by a droplet contact method.

3. The partition for forming a lipid bilayer membrane according to claim 1 or 2, wherein the planar shape of the through-hole has a straight portion.

4. The partition for forming a lipid bilayer membrane according to claim 1 or 2, wherein the planar shape of the lipid bilayer membrane to be formed is a polygon having six or less vertices.

5. The partition for forming a lipid bilayer membrane according to claim 4, wherein the polygon is a rectangle, a rhombus, an upward triangle or a downward triangle.

6. The partition for forming a lipid bilayer membrane according to claim 1 or 2, wherein the planar shape of the through-hole is X-shaped, rectangular, cross-shaped, upward three-way radial, or downward three-way radial.

7. A lipid bilayer membrane forming device comprising the lipid bilayer membrane forming partition according to claim 1 or 2.

8. A lipid bilayer membrane forming device according to claim 7, comprising a lipid bilayer membrane forming partition according to claim 1 or 2, and at least one well provided in a substrate and separated by the partition.

9. A method for forming a lipid bilayer membrane, comprising forming a lipid bilayer membrane having a straight portion that covers at least a portion of the through-hole, using the lipid bilayer membrane forming device according to claim 7.

10. A lipid bilayer membrane formed in the through-hole portion of a partition, the lipid bilayer membrane having a straight edge portion.

11. The lipid bilayer membrane according to claim 10, wherein the planar shape is a rhombus, a right-angled rectangle, an upward-facing triangle, or a downward-facing triangle.

12. Use of the partition according to any one of claims 1 to 6 for forming a lipid bilayer membrane.

13. Use of the partition according to any one of claims 1 to 6 for producing a lipid bilayer forming device.

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