JP2023046682A - Instrument for forming lipid bilayer membrane and regeneration method of lipid bilayer membrane therewith - Google Patents

Abstract

To provide a novel lipid bilayer membrane regeneration method having a higher regeneration rate of a lipid bilayer membrane than a conventional method and a lipid bilayer membrane forming instrument.SOLUTION: A lipid bilayer membrane forming instrument comprises a pair of wells having a bottom surface and adjacent to each other, and a separator that separates a boundary of these wells and has a throughhole in which a lipid bilayer membrane is formed. Further, gas ejection holes provided in the bottom surface of at least one well of the pair of wells, gas supply means for supplying gas to the gas ejection holes, and a control wall provided in the vicinity of the gas ejection hole and controlling a moving direction of bubbles to disturb for the gas ejected from the gas ejection holes to move in a direction leaving from the throughhole.SELECTED DRAWING: Figure 2

Description

TECHNICAL FIELD The present invention relates to a lipid bilayer membrane forming device for forming a lipid bilayer membrane by a droplet contact method and a method for regenerating a lipid bilayer membrane using the same.

Cells that make up living organisms, various organelles such as mitochondria, Golgi bodies, and endoplasmic reticulum present in cells, cell nuclei, etc. are covered with a biological membrane on the outside, and this biological membrane is basically a lipid bilayer membrane. consists of Various physiologically active proteins such as receptors and enzymes are retained on the lipid bilayer membrane in a form that penetrates the lipid bilayer membrane. These transmembrane proteins play important roles in vivo. In particular, various receptors present on cell membranes are known to trigger various physiological reactions by binding to ligands present in vivo. 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 being researched. ing.

It is also known to retain proteins in lipid bilayer membranes and use them as sensors. For example, an odorant sensor can be obtained by retaining an olfactory receptor protein in a lipid bilayer membrane, or specific binding properties that bind specifically to a test substance in a droplet when forming a lipid bilayer membrane by the droplet contact method. A sensor that contains a substance and retains an ion channel protein in the lipid bilayer membrane so that, when the test substance is present, the test substance binds to the specific binding substance to block the ion channel. is also known.

Conventionally, a droplet contact method is known as a widely used method for forming a lipid bilayer membrane (Patent Documents 1 to 4, etc.). In the droplet contact method, a device having a pair of wells and a partition wall (separator) separating the wells is usually used. The separator has through holes through which the lipid bilayer membrane is formed. A lipid solution containing a lipid membrane-forming lipid is added to each well, and then an aqueous electrolyte solution is added to each well, whereby a lipid bilayer membrane is formed in such a manner as to block the through-holes of the partition walls. Proteins and the like to be retained in the lipid bilayer membrane are usually contained in the aqueous solution.

However, lipid bilayer membranes have a structure in which lipid monomolecular membranes that are non-covalently bonded are laminated in two layers, so they are vulnerable to vibrations, etc., and are easily destroyed. have Conventionally, when the formed lipid bilayer membrane is destroyed, it has been necessary to redo the entire membrane formation process or redo a part of the membrane formation process from the beginning. In any case, there is a problem that the loss of human and time resources due to redoing the process is large. Technological developments have been made to make membrane breakage less likely, but it is not realistic to completely prevent membrane breakage.

The inventors of the present application previously found and announced that lipid bilayer membranes can be regenerated by introducing air bubbles into the well from the bottom of the well (Non-Patent Document 1). Even after the membrane is destroyed, the lipid and oil are present near the through-holes of the separator, and the lipid bilayer membrane is reformed by temporarily contacting the air bubbles with this. That is, the lipid bilayer membrane is formed in the process in which the air bubbles and the lipid monomolecular membrane formed at the interface of the aqueous solution trace the through-hole portion of the separator.

Japanese Patent Application Laid-Open No. 2017-83210 Japanese Patent Application Laid-Open No. 2019-072698 Japanese Patent Application Laid-Open No. 2012-081405 JP 2019-022872 A

I. Hashimoto et al., Proceedings of IEEE Micro Electro Mechanical Systems 2020, pp.1022-1023

The method described in Non-Patent Document 1 using air bubbles is an excellent method that can easily regenerate the lipid bilayer membrane, but the probability of regenerating the lipid bilayer membrane within 1 minute is only 31%. There is a problem.

As a result of intensive research to solve the above problems, the inventors of the present application have found that by providing a control wall adjacent to the gas discharge hole provided on the bottom surface of the well for controlling the moving direction of bubbles generated in the gas discharge hole, , found that the probability of regenerating a lipid bilayer membrane within 1 minute can be significantly increased, and completed the present invention.

That is, the present invention provides the following.
(1) A lipid bilayer membrane comprising a pair of wells adjacent to each other and having a bottom surface, and a separator separating the boundaries of these wells and having through holes in which the lipid bilayer membrane is formed. A forming instrument,
a gas ejection hole provided in the bottom surface of at least one of the pair of wells;
gas supply means for supplying gas to the gas discharge holes;
a control wall provided in the vicinity of the gas ejection hole for controlling the moving direction of the air bubble, which prevents the air bubble ejected from the gas ejection hole from moving in a direction away from the through hole. Membrane forming device.
(2) The device for forming a lipid bilayer membrane according to (1), wherein the control wall is provided adjacent to the gas discharge hole and opened at a central angle θ toward the separator when viewed from above.
(3) The device for forming a lipid bilayer membrane according to (2), wherein the central angle θ is 30° to 180°.
(4) The device for forming a lipid bilayer membrane according to (3), wherein the central angle θ is 60° to 120°.
(5) The device for forming a lipid bilayer membrane according to any one of (1) to (4), wherein the control wall has a height of 0.2 mm to 2 mm.
(6) The device for forming a lipid bilayer membrane according to any one of (1) to (5), wherein the distance from the separator to the center of the gas discharge hole is 0.2 mm to 1 mm when viewed from above.
(7) The device for forming a lipid bilayer membrane according to any one of (1) to (6), wherein the gas discharge hole has a diameter of 0.2 mm to 1 mm.
(8) The device for forming a lipid bilayer membrane according to any one of (1) to (7), wherein the distance from the center position of the through-hole to the bottom surface is 0.2 mm to 2 mm.
(9) The device for forming a lipid bilayer membrane according to any one of (1) to (8), wherein the control wall is composed of side walls of plates laminated on the bottom surface.
(10) Using the lipid bilayer membrane forming device according to any one of (1) to (9), when the lipid bilayer membrane formed in the through-hole is broken by a droplet contact method, the gas A method for regenerating a lipid bilayer membrane, comprising exhaling a gas from an ejection hole and bringing the ejected air bubbles into contact with the through-hole to regenerate the lipid bilayer membrane in the through-hole.
(11) The method according to (10), wherein the air bubble interrupts contact between the droplets in the pair of wells through the through-holes, and the droplet breakup time is 1 to 10 seconds.
(12) The method according to (10) or (11), wherein the speed at which the gas is discharged from the gas discharge holes is 20 μL/min to 200 μL/min.

INDUSTRIAL APPLICABILITY According to the present invention, a destroyed lipid bilayer membrane can be easily regenerated with high probability.

1 is a schematic diagram of a known device for forming a lipid bilayer membrane; FIG. (a) is a plan view, and (b) is an end elevation view taken along line bb' of (a). 1 is a schematic cross-sectional view of a preferred specific example of the present invention; FIG. FIG. 3 is a schematic plan view of the vicinity of the gas ejection holes 30 of a preferred specific example of the present invention, viewed from above. 1 is a schematic diagram of a device for a preliminary experiment produced in Reference Example 1 below. FIG. (a) is a schematic plan view, and (b) is a schematic cross-sectional view. 1 is a schematic perspective view of a device for a preliminary experiment produced in Reference Example 1 below. FIG. FIG. 4 is a diagram showing changes in current value when bubbles are ejected after lipid bilayer membrane disruption, obtained in the following examples. FIG. 10 is a diagram showing the relationship between the ejection speed of air and the breakup time of droplets due to air bubbles, obtained in the following examples. FIG. 4 is a diagram showing the relationship between the air ejection speed and the reformation rate of lipid bilayer membranes, obtained in the following examples.

Preferred embodiments of the present invention will now be described with reference to the drawings.

FIG. 1 is a schematic diagram of a known device for forming a lipid bilayer film described in Patent Document 3. FIG. The lipid bilayer membrane formation device of the present invention also has the same basic structure as the known lipid bilayer membrane formation device as shown in FIG. 1 except for the novel features described later.

FIG. 1(a) is a plan view, and FIG. 1(b) is an end view taken along line bb' in FIG. 1(a). In this embodiment, a first well 16 and a second well 14 are formed in substrate 10 and separated at their boundaries by separator 12 . Such a configuration is called a double well chamber (DWC) or double well device. The separator 12 is provided with a through hole 18 (see (b) of FIG. 1). In the example shown in FIG. 1, the planar shape of the well is basically circular, and only the boundary portion where two circles are in contact is linear. Other shapes are acceptable as long as they are separated by partitions having The size of the well is not particularly limited, and the diameter is usually about 2 mm to 8 mm, particularly about 3 mm to 5 mm, and the depth is usually about 50% to 200%, especially 50% to 100% of the diameter. , it is possible to practice the method of the present invention with more or less than this range. The number of through-holes 18 provided in the separator 12 may be one or plural.

FIG. 2 shows a schematic cross-sectional view of a preferred specific example of the lipid bilayer membrane-forming device of the present invention. The diagram on the left side of FIG. 2 is similar to FIG. 1(b) described above. is attached. The drawing on the right side of FIG. 2 is a partially enlarged view of the vicinity of the through hole 18 in the drawing on the left side.

In FIG. 2, reference number 20 indicates droplets formed in first well 16 and reference number 22 indicates droplets formed in second well 14 . A gas discharge hole 30 is provided in the bottom surface of one of the wells (the second well 14 in the illustrated example) (see the enlarged view on the right side of FIG. 2). A microchannel 32 is connected to the gas discharge hole 30 , and a pump 34 is connected to the other end of the microchannel 32 . A gas supplied from the pump 34 is discharged into the second well 14 from the gas discharge holes 30 through the microchannel 32 . Reference number 36 in the enlarged view on the right side of FIG. 2 is a lipid monolayer (lipid monolayer). The distance from the separator 12 to the center of the gas discharge hole is not particularly limited, but is usually about 0.2 mm to 1 mm, preferably about 0.3 mm to 0.7 mm when viewed from above (see FIG. 3, which will be described later). Also, the diameter of the gas discharge hole 30 is not particularly limited, but is usually about 0.2 mm to 1 mm, preferably about 0.3 mm to 0.7 mm. The distance from the center of the through hole 18 to the bottom surface of the second well 14 (reference symbol h in the enlarged view of FIG. 2) is usually about 0.2 mm to 2 mm, preferably about 0.3 mm to 1 mm. be. It should be noted that, for the sake of clarity, FIG. 2 does not show the control wall, which is an important feature of the invention and will be described later.

FIG. 3 is a schematic plan view of the vicinity of the gas ejection holes 30 of one preferred embodiment of the present invention, viewed from above. A control wall 38 is provided in the vicinity of the gas discharge hole 30 to control the moving direction of the air bubbles, which prevents the air bubbles discharged from the gas discharge hole 30 from moving in the direction away from the through hole 18 . A control wall 38 is a wall provided on the bottom surface of the second well 14 . In the illustrated embodiment, a control wall 38 is provided adjacent the gas discharge hole 30 and opens at a central angle θ toward the separator 12 when viewed from above. As specifically described in the examples below, the central angle θ is preferably 30° to 180° (when the control wall is 180°, the control wall is straight when viewed from above), especially 60° to 120°. is preferred. When the central angle θ is within this range, the probability of forming a lipid bilayer within 1 minute after gas ejection increases. It is preferable that the control wall 38 is provided symmetrically with respect to the separator 12 (that is, the central angle θ is bisected by a perpendicular line drawn from the center of the gas discharge hole 30 to the separator 12), but there may be some deviation (for example, , the central angle θ is divided into 6:4 to 4:6 by the perpendicular line) is no problem. Further, it is preferable that the through hole 18 is positioned right above the vertical line, but a slight deviation (for example, a deviation of about 0.4 mm or less in the horizontal direction in FIG. 3) is not a problem. Although the control wall 38 may be provided on the bottom surface of the second well 14, the control wall 38 may be formed by side walls of plates stacked on the bottom surface. This method is preferable because the control wall 38 can be formed simply by stacking plates each having one end face formed in the desired shape of the control wall 38 described above, and the manufacturing process is simple. In this case, the plate forms a step on the bottom surface of the well, and the end face (side wall) of this step becomes the control wall 38 .

Next, a method for regenerating a lipid bilayer membrane using the device for forming a lipid bilayer membrane of the present invention will be described.

First, a lipid bilayer membrane that closes the through-hole 18 is formed by the conventional droplet contact method described in Patent Documents 1-4. That is, each well 14, 16 is filled with a lipid liquid. An aqueous solution is then added to one well. Then, a lipid monolayer, which is a lipid monomolecular layer, is spontaneously formed at the interface between the aqueous solution and the lipid liquid in such a manner as to block the through-holes 18 . Then add the aqueous solution to the other well. As a result, a lipid monolayer membrane is formed between the aqueous solution and the lipid liquid on the other side in such a manner as to close the through-hole 18, and as a result, a lipid bilayer membrane that closes the through-hole 18 is formed.

The lipid liquid and electrolyte aqueous solution used can be the same as the lipid liquid and electrolyte aqueous solution used in the conventional droplet contact method. That is, the phospholipids constituting the lipid bilayer membrane include, for example, diphytanoyl phosphatidylcholine (DPhPC), dipalmytoyl phosphatidylcholine, dioleoyl phosphatidylcholine (Dioleoyl phosphatidylcholine). phosphatidylcholine, DOPC) and the like can be mentioned as preferable examples. These lipid molecules are commercially available in reagent grade. An aliphatic hydrocarbon solvent such as n-decane can be used as the organic solvent that dissolves the phospholipid. Although the concentration of lipid molecules in the organic solvent is not particularly limited, it is usually 5 mg/mL or higher, preferably 20 mg/mL or higher for stable lipid bilayer membrane formation. As the aqueous electrolyte solution, an aqueous buffer such as a phosphate buffer containing a salt such as potassium chloride is usually used.

Lipid bilayer membranes are used to investigate the properties and functions of proteins in a state retained by the lipid bilayer membrane, to screen ligands that bind to the proteins and change their physiological activities, and to examine their properties. Since the lipid bilayer membrane is preferably used for various measurements, it is preferable that the lipid bilayer membrane contains a protein, and in particular, a transmembrane protein that functions in vivo while being retained by the biological membrane is preferable. Examples of proteins retained in lipid bilayer membranes include various receptors and enzymes, examples of which include peptide proteins such as α-hemolysin, gramicidin and alamethicin, various ion channels, and ABC transporter proteins. However, it is not limited to these. When the protein is water-soluble, it is preferable to use an aqueous solution of the protein as the aqueous solution, and it is more preferable to use an aqueous solution containing the protein in an aqueous buffer. The protein concentration in these solutions is not particularly limited and can be selected as appropriate, but is usually about 0.1 nM to 10 nM, preferably about 0.5 nM to 2 nM. The protein may be contained in the aqueous solution added to either one of the two wells, but may be contained in the aqueous solution added to both wells. Also, the aqueous solutions added to the two wells may have the same composition or different compositions.

When utilizing the formed lipid bilayer membrane, it is usual to measure the value of the current passing through the channel protein or the like retained in the lipid bilayer membrane, that is, the value of the current flowing through the lipid bilayer membrane. done. This is usually done by connecting an amplifier circuit for current measurement to the electrode (not shown) provided at the bottom of each well. Such circuits are the same as those used in conventional droplet contact methods and are well known and are described, for example, in US Pat.

The method of forming a lipid bilayer membrane by the droplet contact method described above is the same as the conventional method and well known. The lipid bilayer membrane regeneration method of the present invention is carried out after the lipid bilayer membrane formed as described above is destroyed by some cause (vibration or spontaneous destruction may occur). When the formed lipid bilayer membrane is destroyed, the gas is sent from the pump 34 and discharged from the gas discharge hole 30 . As the gas, it is most convenient and inexpensive to use air, but if desired, it is possible to use a gas other than air, such as an inert gas such as nitrogen gas or argon gas. Ejecting the gas creates air bubbles within the droplet. By bringing these bubbles into contact with the through-holes 18 , the lipid bilayer membrane is formed again in such a manner as to block the through-holes 18 . Even after the lipid bilayer membrane is destroyed, the lipid and oil remain in the vicinity of the through-hole 18, and the lipid bilayer membrane is re-formed by temporarily contacting the bubbles with the lipid and oil. That is, the lipid bilayer membrane is formed in the process in which the air bubbles and the lipid monomolecular membrane formed at the interface of the aqueous solution trace the through-hole portion of the separator. Note that regeneration of a lipid bilayer membrane by air bubbles itself is known (Non-Patent Document 1).

The speed at which the gas is discharged from the gas discharge holes 30 is not particularly limited, but is preferably about 20 μL/min to 200 μL/min. By setting the gas ejection speed within this range, the probability of regenerating the lipid bilayer membrane within 1 minute can be increased. Also, the droplet breakup time during which contact between the droplets in the pair of wells through the through-holes 18 is interrupted by air bubbles is not particularly limited, but is preferably about 1 second to 10 seconds. By setting the droplet breakup time within this range, the probability of regeneration of the lipid bilayer membrane within 1 minute can be increased. Note that the droplet breakup time depends on the gas ejection speed, various dimensions of the device (for example, the diameter of the gas ejection hole 30, the distance from the separator 12 to the center of the gas ejection hole 30, and the position of the center of the through hole 18). to the bottom surface, etc.) can be adjusted within the preferred range described above.

According to the regeneration method of the present invention, the lipid bilayer membrane is regenerated within 1 minute with a high probability (up to 97% as exemplified in the examples below). In addition, since it is possible to judge from the current value everything from lipid bilayer membrane disruption to re-formation, automation by electronic control can be expected. Air bubble ejection can also be solved with a simple pump and air bubbles in each well, and it is considered possible to develop a parallel membrane array device.

EXAMPLES The present invention will be specifically described below based on examples. However, the present invention is not limited to the following examples.

Reference Example 1 Optimization of Central Angle θ In order to optimize the central angle θ of the control wall 38 described above, the following preliminary experiment was conducted.

1. Device A device having a stepped bottom surface as shown in FIGS. 4 and 5 was fabricated. FIG. 4(a) is a schematic plan view of the manufactured device, and FIG. 4(b) is a schematic cross-sectional view. FIG. 5 is a schematic exploded perspective view of the device. The device has a three-layer structure of a well portion 40 having a square bottom surface of 5 mm on each side, a bottom portion 42 having a stepped portion with gas ejection holes 30, and a microchannel portion 44 for ejecting gas. As shown in FIG. 4A, the stepped shape of the bottom portion 42 is 30°, 60°, 90°, 120°, θ = 30°, 60°, 90°, 120°, with the angle (θ) as a parameter, starting from the air bubble introduction hole. We designed 7 types of devices with 150°, 180°, and stepless shapes. The height of the steps was 0.5 mm. The end face (side wall) of this step functions as a control wall. The three-layer part was made by cutting an acrylic plate using an NC precision machine. The parts were bonded together by thermocompression bonding. A glass plate was attached to the side of the well with an adhesive for observation.

2. Experimental method/conditions
1) 6 μL of organic solvent (n-decane) in which phospholipid (20 mg/mL DPhPC (1,2-diphytanoyl-sn-glycero-3-phosphocholine)) was dispersed was dropped into the well.
2) 44 μL of a buffer solution (1 M KCl, 10 mM phosphate buffer, pH 7.4) was added dropwise to the wells and allowed to stand for 5 minutes.
3) Air is discharged from a gas-tight syringe installed in a syringe pump (Pump elite 11 (trade name), manufactured by Harvard Apparatus), and bubbles are released from the gas discharge hole 30 provided on the bottom of the well through the microchannel 32 at the bottom of the device. caused The inflow rate of air was 30 μL/min. Air discharge was stopped when the air bubbles separated from the gas discharge holes 30 due to buoyancy. This was done once every minute and repeated 10 times.
4) A video of the introduction of air bubbles was taken from above with a digital microscope (VHX-1000, KEYENCE). We observed the guiding direction of bubbles with respect to the angle of the step shape.
5) Plotted the center point of the bubble every second from the time the bubble was introduced until it left. At this time, the center point of the bubble introduction hole was defined as the origin, the direction of θ/2 as the guide direction was defined as y, and the perpendicular direction was defined as x.

3. Results The experimental results are shown in Table 1 below. When the step angle (θ) was 90°, the bubble swayed to the side the shortest and advanced the most in the guiding direction. Based on this result, experiments were conducted with the central angle θ set to 90° in the following examples.

Example 1
1. Preparation of Lipid Bilayer Membrane Forming Instrument A double well device having a step (control wall 38) and a gas discharge hole 30 shown in FIGS. 2 and 3 was prepared. This device consists of a double-well chip that forms a lipid bilayer and a BNC connector for connecting to an amplifier. The double-well chip has a three-layer structure of a double-well portion having a pair of figure-eight-shaped wells forming a lipid bilayer membrane, a bottom portion having gas ejection holes and steps, and a microchannel portion. In the double well section, an acrylic separator (75 μm thick) with a through-hole of 400 μm diameter was inserted at the interface between the two wells. As shown in FIG. 3, the step shape of the bottom portion was set to have a center angle θ of 90° and a height of 0.5 mm starting from the gas discharge hole 30 having a diameter of 0.5 mm. It was designed so that the center of the gas discharge hole was positioned 0.45 mm from the separator. A channel (depth of 0.5 mm) for supplying air was provided in the microchannel part and connected to the gas discharge hole so that the air could be sent. The three-layer part was made by cutting an acrylic plate using an NC precision machine. The parts were bonded together by thermocompression bonding. A pair of silver electrodes (working electrode, reference electrode) with a diameter of 1 mm were embedded in the bottom of the well for electrophysiological measurement of the lipid bilayer membrane. Electrodes were connected to BNC connectors.

2. Control of contact time between bubbles and through-holes by gas discharge speed
(1) Experimental method and conditions
1) 2.4 μL of organic solvent (n-decane) in which phospholipid (20 mg/mL DPhPC) was dispersed was added dropwise to each well, followed by buffer solution (40 nM alpha-hemolysin, 1 M KCl, 10 mM phosphoric acid 25 μL of buffer solution, pH 7.4) was added dropwise. By this operation, a lipid bilayer membrane was formed in the through-holes of the separator.
2) In order to reform the lipid bilayer membrane, we applied a physical stimulus to the device to destroy the lipid bilayer membrane.
3) Air was sent from a syringe pump, and air bubbles were generated from the gas discharge holes provided on the bottom of the device through the microchannel at the bottom of the device. There were four conditions for air ejection: 30 µL/min, 100 µL/min, 200 µL/min, and 400 µL/min.
4) Based on the relationship between the state of the through-holes in the separator and the current value described below, the breakup time of droplets due to air bubbles was observed and analyzed from changes in the current value. The applied voltage was 50 mV. A well-known configuration described in Patent Document 3 and the like was adopted for the circuit and electrodes for current measurement.
5) Observation and analysis were performed by repeating the rupture of the lipid bilayer and ejection of air bubbles 10 times every minute.

(2) Results
(i) Relationship between state of through-holes in separator and current value FIG. 6 shows how the current value changes when air bubbles are ejected after the lipid bilayer membrane is broken.
1) Breakage of lipid bilayer membrane: Since two water droplets are fused at the through-hole, the electrical resistance is very small and the current value overflows.
2) Droplet breakup due to air bubbles: Due to the presence of air bubbles in the through-holes, the electrical resistance increases and the current value becomes zero.
3) Detachment of bubbles: Electrical noise generated when bubbles are detached from the gas discharge port is observed. If the droplets are kept separated by the through-hole, the current value remains 0. On the other hand, if the droplets merge after the bubbles are detached, the current value overflows.
4) Reformation of lipid bilayer membranes: Alpha-hemolysin reconstitutes into lipid bilayer membranes and forms nanometer-sized pores. The reconstitution of this nanopore shows a step-like current increase of about 50 pA under the experimental conditions. Reformation of the lipid bilayer membrane was judged based on the observation of this step-like current signal after the droplets were separated from each other in 3) above. If not observed within 1 minute, it was determined that the organic solvent was excessive and the lipid bilayer membrane could not be formed.

(ii) FIG. 7 shows the relationship between the ejection speed of bubbles and the breakup time of droplets due to bubbles. Based on this result and the relationship between the bubble ejection speed and the lipid bilayer regeneration success rate, which will be described later, the lipid bilayer membrane regeneration success rate is high when the droplet fragmentation time is about 1 to 10 seconds. Recognize.

3. Success rate of lipid bilayer reformation
(1) Experimental method and conditions The following experiment was conducted using the double well device prepared above.
1) 2.4 μL of organic solvent (n-decane) in which phospholipid (20 mg/mL DPhPC) was dispersed was added dropwise to each well, followed by buffer solution (40 nM alpha-hemolysin, 1 M KCl, 10 mM phosphoric acid 25 μL of buffer solution, pH 7.4) was added dropwise. By this operation, a lipid bilayer membrane was formed in the through-holes of the separator.
2) In order to reform the lipid bilayer membrane, we applied a physical stimulus to the device to destroy the lipid bilayer membrane.
3) Air was sent from a syringe pump, and air bubbles were generated from the gas discharge holes provided on the bottom of the device through the microchannel at the bottom of the device. There were 4 air discharge conditions of 30 μL/min, 100 μL/min, 200 μL/min, and 400 μL/min.
4) Based on the relationship between the state of the through-holes of the separator and the current value, changes in the current value indicate the destruction of the lipid bilayer membrane in the through-holes, the fragmentation of the droplet by bubbles, the detachment of the bubbles, and the lipid bilayer membrane. We observed and analyzed the reformation of The applied voltage was 50 mV. A well-known configuration described in Patent Document 3 and the like was adopted for the circuit and electrodes for current measurement.
5) The rupture of the lipid bilayer membrane and ejection of air bubbles were repeated 30 times every minute, and observation and analysis were performed.

(2) Results FIG. 8 shows the relationship between the air ejection speed and the lipid bilayer membrane reformation rate. It was found that the highest reformation rate of 97% was obtained at air ejection rates of 30 and 100 μL/min.

10 substrate 12 separator 14 second well 16 first well 18 through hole 20 droplet 22 droplet 30 gas discharge hole 32 microchannel 34 pump 36 lipid monolayer (single film)
38 control wall 40 well portion 42 bottom portion 44 microchannel portion h distance from the center of the through-hole 18 to the bottom surface of the second well 14 θ central angle

Claims (12)

A lipid bilayer membrane forming device comprising a pair of wells adjacent to each other and having a bottom surface, and a separator separating the boundaries of these wells, the separator having through holes in which a lipid bilayer membrane is formed. There is
a gas ejection hole provided in the bottom surface of at least one of the pair of wells;
gas supply means for supplying gas to the gas discharge holes;
a control wall provided in the vicinity of the gas ejection hole for controlling the moving direction of the air bubble, which prevents the air bubble ejected from the gas ejection hole from moving in a direction away from the through hole. Membrane forming device. 2. The device for forming a lipid bilayer membrane according to claim 1, wherein said control wall is provided adjacent to said gas discharge hole and opens at a central angle θ toward said separator when viewed from above. The device for forming a lipid bilayer membrane according to claim 2, wherein the central angle θ is 30° to 180°. The device for forming a lipid bilayer membrane according to claim 3, wherein the central angle θ is 60° to 120°. The device for forming a lipid bilayer membrane according to any one of claims 1 to 4, wherein the control wall has a height of 0.2 mm to 2 mm. The device for forming a lipid bilayer membrane according to any one of claims 1 to 5, wherein the distance from the separator to the center of the gas discharge hole is 0.2 mm to 1 mm when viewed from above. The device for forming a lipid bilayer membrane according to any one of claims 1 to 6, wherein the gas discharge hole has a diameter of 0.2 mm to 1 mm. The device for forming a lipid bilayer membrane according to any one of claims 1 to 7, wherein the distance from the center position of said through-hole to said bottom surface is 0.2 mm to 2 mm. The device for forming a lipid bilayer membrane according to any one of claims 1 to 8, wherein the control wall is constituted by side walls of plates laminated on the bottom surface. Using the lipid bilayer membrane forming device according to any one of claims 1 to 9, when the lipid bilayer membrane formed in the through-hole is broken by a droplet contact method, A method for regenerating a lipid bilayer membrane, comprising discharging a gas and bringing the discharged bubbles into contact with the through-hole to regenerate the lipid bilayer membrane in the through-hole. 11. The method according to claim 10, wherein the droplet breakup time during which contact between the droplets in the pair of wells through the through-hole is interrupted by the bubble is 1 to 10 seconds. 12. The method according to claim 10, wherein the speed at which the gas is discharged from the gas discharge holes is 20 μL/min to 200 μL/min.

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