JP5198369B2 - Current measurement device using artificial lipid bilayer membrane - Google Patents

Description

  The present invention relates to a microfluidic system for analyzing lipid bilayer membranes, specifically a planar lipid bilayer array device including a microdiverticulum with a novel reservoir connection and / or electrodes; The present invention relates to a microfluidic system including the device and an automatic analysis system for electrical characteristics of a lipid bilayer membrane using the planar lipid bilayer array device.

  In order to understand the functions of membrane proteins involved in substance transport and information transmission inside and outside biological membranes by single-molecule measurement and to apply them in engineering, artificial lipid bilayer membrane models incorporating isolated membrane proteins It is necessary to construct an experimental system. However, to perform such experiments, skilled techniques were required, the reproducibility was poor, and the throughput was low. The inventors of the present invention created a planar lipid bilayer array using MEMS technology, and provided portability, reduced analysis time, a small amount of necessary reagents, high reproducibility, etc. (patents) Reference 1).

  FIG. 1 shows a schematic diagram of one embodiment of a prior art planar lipid bilayer array microfluidic system 1 by the inventors of the present invention. The microfluidic system 1 includes a liquid feeding port 2, a main channel 3 arranged in parallel, a planar lipid bilayer array 5 including a plurality of micro diverticulas 4 that open on both side walls of the main channel 3, and a main channel 3. The liquid storage tank connection part 6 and the liquid storage tank 7 are included. The main flow path 3 from the liquid feeding port 2 branches in parallel. When the micro diverticulum 4 opens in one side wall in the planar lipid bilayer membrane array 5, the other side wall opposite to the opening does not open. The micro diverticulums 4 are provided alternately on the opposite side walls of the main channel along the main channel 3. A portion defined by the side wall of the main channel 3 and the adjacent micro diverticulum 4 provided on the side wall supports the main channel 3 and the top and bottom surfaces of the micro diverticulum 4 as columns. For this reason, the space | interval of the said top surface and a bottom face can be kept constant. In the liquid storage tank connection section 6, the main flow path 3 first communicates with a groove having a width of 6 times or more the width of the main flow path, and is connected to the liquid storage tank 7 while the width of the groove extends to 30 times or more of the width of the main flow path. To do. Hereinafter, the liquid storage tank connection part having the shape of the liquid storage tank connection part 6 is referred to as a “conventional liquid storage tank connection part”.

  In another embodiment of the prior art planar lipid bilayer array device 1, the microdiverticulum is provided alternately on the top and bottom surfaces of the main channel. The portion defined by the top surface or bottom surface of the main channel and the adjacent micro diverticulum provided on the top surface or bottom surface supports the space between the side walls of the main channel as a beam. Can be held.

  When forming a lipid bilayer membrane in the conventional lipid bilayer membrane array device of the prior art, the microfluidic system 1 is previously immersed and saturated in an aqueous solvent, and then a microsyringe, a pump and other liquid delivery devices Is connected to the liquid feeding port 2 through a tube. Air enters the first liquid composed of an aqueous solution, the second liquid composed of an oily solution in which lipids, which are components of the lipid bilayer membrane, and the third liquid composed of an aqueous solution from the liquid feeding device. Sequentially inject into the array device. Then, when the first liquid is first injected, the first liquid fills the main flow path and the minute diverticulum. When the second liquid is subsequently injected, the second liquid penetrates into the micro diverticulum while sweeping the first liquid in the main flow path, forms an interface with the first liquid left in the micro diverticulum, and Wet the wall of the minute diverticulum. Thereafter, when the third liquid is injected, the third liquid pushes away the second liquid in the main flow path, so that the second liquid in the micro diverticulum is suspended in the opening portion of the micro diverticulum and seals the opening. A film layer is formed. Since the oily solvent of the second liquid is absorbed from the wall of the microdiverticulum, the membrane layer of the second liquid gradually becomes thin, and finally the two lipid molecules are hydrophobic in a tail-to-tail manner. It becomes a lipid bilayer oriented so that atomic groups face each other. When a first liquid to which a purified membrane protein such as α-hemolysin and a fluorescent substance such as calcein are added is used, α-hemolysin is dissolved in the second liquid. When the second liquid membrane becomes a lipid bilayer membrane, α-hemolysin self-assembles to form a heptamer having transmembrane pores through which calcein can pass. Therefore, it can be detected that calcein flows out of the micro diverticulum into the main channel and the intensity of the fluorescence in the micro diverticulum is reduced, thereby forming a lipid bilayer.

  In the prior art planar lipid bilayer membrane microfluidic system described above, the second fluid continues to be absorbed from the wall of the microdiverticulum once the lipid bilayer is formed. The bilayer ruptures over time. In order to extend the time from the formation of the lipid bilayer membrane to the rupture, that is, the lifetime, it is effective to increase the lipid concentration in the second liquid and to decrease the flow rate of the third liquid. However, when the lipid concentration in the second liquid is increased or the flow rate of the third liquid is decreased, the second liquid causes gelation and forms an embolus, and therefore the experiment can be continued only for several minutes at most.

  Moreover, in the above-described conventional planar lipid bilayer membrane microfluidic system, the volume in the micro diverticulum sealed by the lipid bilayer membrane is smaller than the size of the main channel. Therefore, even if the pressure applied to the liquid passing through the main flow path varies due to the liquid feeding, there is an advantage that the volume variation in the microrecess chamber is small and the lipid bilayer membrane is not easily ruptured. However, the conventional microdiverticulum has a disadvantage that the liquid in the microdiverticulum can be measured only by an optical method such as fluorescence. Since fluorescent dyes do not necessarily have high time resolution, they are not sufficient for analyzing the functions of membrane proteins involved in nerves and other higher life phenomena.

JP 2009-128206 A

  Therefore, it is necessary to develop a planar lipid bilayer array microfluidic system that can maintain a planar lipid bilayer for a long time without causing gelation even when a second liquid containing a high concentration of lipid is used. is there. Moreover, in order to perform a measurement with high time resolution on the function of the membrane protein incorporated in the lipid bilayer membrane, it is necessary to provide an electrode in the microdiverticulum.

  The present invention provides a planar lipid bilayer array device. The planar lipid bilayer array device of the present invention includes a planar lipid bilayer array including a main channel extending from a liquid delivery port, a micro diverticulum provided in the main channel, and the planar lipid bilayer array. A liquid storage tank connection section that communicates with the main flow path; a liquid storage tank connected to the liquid storage tank connection section; and a discharge port that communicates with the liquid storage tank. It consists of a plurality of discharge channels arranged in parallel with branches.

  In the planar lipid bilayer array device of the present invention, the width and depth of the discharge channel may be the same as the width and depth of the main channel.

  In the planar lipid bilayer array device of the present invention, the reservoir connection part may be composed of 18 discharge channels arranged in parallel, each of the two main channels being bifurcated eight times. is there.

  In the planar lipid bilayer array device of the present invention, the reservoir connection portion may include a sub-discharge channel that intersects and communicates with the discharge channel.

  The planar lipid bilayer array device of the present invention may include five auxiliary discharge channels per main channel.

  In the planar lipid bilayer array device of the present invention, the reservoir connection part may include a discharge promoting liquid channel for injecting a discharge promoting liquid into the discharge channel.

  The planar lipid bilayer array device of the present invention may include a main channel electrode electrically connected to the liquid in the main channel and a micro diverticulum electrode electrically connected to the liquid in the micro diverticulum. .

  In the planar lipid bilayer array device of the present invention, the microdiverticulum electrode may include a conductive gel exposed on a wall surface that defines the microdiverticulum.

  In the planar lipid bilayer array device of the present invention, the conductive gel may be an agarose gel.

  In the planar lipid bilayer array device of the present invention, the micro diverticulum electrode may include a conductor plate in contact with a wall surface that defines the micro diverticulum.

  In the planar lipid bilayer array device of the present invention, the conductor plate may be a gold plate.

  The present invention provides a microfluidic device comprising the planar lipid bilayer array device of the present invention, a liquid feeding device connected to a liquid feeding port of the device, and an analyzer for observing the planar lipid bilayer array device Provide a system.

  The present invention is connected to the planar lipid bilayer array device of the present invention, the liquid feeding device connected to the liquid feeding port of the device, and the discharge promoting liquid channel of the planar lipid bilayer array device. There is provided a microfluidic system including a liquid feeding device and an analyzer for observing the planar lipid bilayer membrane array device.

  The microfluidic system of the present invention includes a planar lipid bilayer array device including a main channel electrode electrically connected to a liquid in the main channel and a micro diverticulum electrode electrically connected to the liquid in the micro diverticulum. And a device for measuring electrical characteristics between the main channel electrode and the microdiverticulum electrode.

  The microfluidic system of the present invention includes a planar lipid bilayer array device including a main channel electrode electrically connected to a liquid in the main channel and a micro diverticulum electrode electrically connected to the liquid in the micro diverticulum. A liquid-feeding device connected to the liquid-feeding port of the device, a liquid-feeding device connected to the discharge promoting liquid channel of the planar lipid bilayer array device, the main channel electrode and the microdiverticulum electrode And a device for measuring the electrical characteristics between them.

  In the microfluidic system of the present invention, the liquid feeding device may include a liquid feeding pump.

  In the microfluidic system of the present invention, the liquid feeding device may include a flow path switching switch.

  The present invention provides a method for analyzing lipid bilayer membranes. The method for analyzing a lipid bilayer membrane of the present invention includes (1) a planar lipid comprising a main channel electrode electrically connected to a liquid in the main channel and a micro diverticulum electrode electrically connected to the liquid in the micro diverticulum. A step of feeding a first liquid composed of an aqueous solution to the double membrane array device; and (2) measuring a current between the main flow path and the micro diverticulum, so that the first liquid is the micro diverticulum. And (3) the main flow channel, the micro diverticulum, and the step of sending a second liquid composed of an oily solution to which lipid has been added to the planar lipid bilayer array device. Current is measured to confirm that the second liquid has electrically insulated the minute diverticulum and the main flow path, and then the third liquid made of an aqueous solution is used as the planar lipid bilayer array device. And (4) measuring the current between the main flow path and the minute diverticulum, A step of detecting that the membrane layer of the second fluid sandwiched between the first fluid and the third fluid has formed a lipid bilayer; (5) a micro diverticulum in which the lipid bilayer is formed; and the main channel And a step of recording a measurement result of an electrical characteristic between.

  The present invention provides an automated analysis system for the electrical properties of lipid bilayer membranes. The automatic analysis system for the electrical characteristics of the lipid bilayer membrane of the present invention includes the microfluidic system of the present invention, a data storage system, and a control system, and executes the lipid bilayer membrane analysis method of the present invention.

  In the present invention, the lipid bilayer membrane refers to a membrane in which two lipid molecules are oriented so that the hydrophobic atomic groups face each other in a tail-to-tail manner. Examples of the lipid of the present invention include, but are not limited to, phospholipids such as phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, and sphingomyelin, and glycolipids such as cerebroside and ganglioside.

  A liquid feeding port, a main flow path, a micro diverticulum, a conducting conduit, a liquid storage tank, a discharge port, a discharge promoting liquid main flow path, and a discharge promoting liquid side flow path included in the planar lipid bilayer array device of the present invention are attached. However, the present invention is not limited to the shape shown in the drawings, and may have any shape. The reservoir connection part of the present invention may be connected to a planar lipid bilayer array of the prior art. Moreover, the planar lipid bilayer array provided with the electrode of the present invention may be connected to a conventional reservoir connection part.

  Even if the branch of the main flow path and the branch of the discharge flow path included in the liquid storage tank connection portion of the present invention are two branches, that is, a branch to two discharge flow paths, three or four The above branching into the discharge channel may be possible. Although the effect of the present invention has not been systematically verified, the liquid storage tank connection portion of the present invention has a second structure in which the growth of the gel is suppressed or the flow rate is reduced by the branching of the discharge flow path. There is a possibility that the flow path will not be clogged even if the concentration of lipid added to the second liquid is increased because the liquid solvent is easily absorbed by the base material of the planar lipid bilayer array device. There is.

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

  The planar lipid bilayer array device of the present invention is formed by using a mold having a three-dimensional shape, which is prepared by combining the conventional microfabrication technology and / or the imprint transfer method alone or in combination. It may be a product. The molded article may be made using materials including, but not limited to, plastics, ceramics and hydrogels, and composite materials thereof.

  The planar lipid bilayer array apparatus of the present invention sends an optical microscope for observing the formation of a planar lipid bilayer in the planar lipid bilayer array, a liquid passing through the main channel, and / or a liquid for promoting discharge. Analyze and record the electrical properties of the planar lipid bilayer membrane by connecting to a pump for liquid dispensing, for example, a syringe pump, a valve for switching liquids, and an electrode provided in the planar lipid bilayer membrane array For example, there may be an accompanying facility including, but not limited to, an electric circuit control device, a temperature control device, and a central processing unit for controlling these operations.

  The conductive gel that may be filled in the microdiverticulum electrode of the present invention and the current-carrying conduit includes agarose, polyacrylamide, methylcellulose, and other polymers that can be gelled at physiological temperatures, and aqueous solutions such as sodium salts and potassium salts. Including.

  The conductor plate that may be used for the micro diverticulum electrode and the main channel electrode of the present invention and the current-carrying conduit is a metal such as gold, silver, or copper, a conductive resin, a conductive composite material, or other conductors of the present invention. The affixed to the base material of the planar lipid bilayer array device. The conductor plate is processed by etching, vapor deposition, or other processing techniques known to those skilled in the art. As the conductor used for the conductor plate electrode, gold, silver and silver chloride are preferable.

  In the micro diverticulum electrode of the present invention, the conductor plate is placed on the bottom surface of the micro diverticulum and is sandwiched between the bottom surface of the micro diverticulum and the wall surface of the micro diverticulum or embedded in the top surface or the bottom surface of the micro diverticulum. In some cases, it may be exposed to the current-carrying conduit on the back side of the wall across the wall of the minute diverticulum.

  The main channel electrode of the present invention may be installed anywhere in the planar lipid bilayer array device of the present invention, but it is installed immediately after the main channel extends from the liquid feeding port and first branches into two. Is preferred. The main flow path electrode of the present invention may be a conductor plate electrode or a conductive gel, but the main flow path electrode of the present invention may be crossed across the bottom surfaces of the two branched main flow paths. In some cases, the conductor plate is placed.

  The electrical characteristics between the main channel electrode and the microdiverticulum electrode of the present invention include, but are not limited to, current, potential difference, resistance, impedance, dielectric constant, frequency addition change, frequency attenuation, frequency phase change, and the like. . Since the planar lipid bilayer array device of the present invention is equipped with electrodes, it can perform analysis with higher time resolution than the conventional planar lipid bilayer array device.

  The first liquid and the third liquid of the present invention are composed of an aqueous solution and may contain any component on the condition that the formation of the planar lipid bilayer membrane of the present invention is not affected. The first fluid may contain a membrane protein that is incorporated into the planar lipid bilayer. When the membrane protein is involved in the transport of a specific ion such as an ion channel, it is desirable that the first liquid and the third liquid have a salt composition suitable for detecting the fluctuation of the ion.

  The second liquid of the present invention is a component of a planar lipid bilayer membrane, such as phospholipids such as phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, sphingomyelin, and glycolipids such as cerebroside, ganglioside, etc. And cholesterol and other compounds, and an organic solvent capable of dissolving them, such as hexadecane, squalene, and the like are preferable. When the structure of the present invention is made of polydimethylsiloxane, it is preferable to use hexadecane that can be absorbed and swelled by polydimethylsiloxane as a solvent.

The schematic diagram of one embodiment of the planar lipid bilayer array apparatus 1 of a prior art. The schematic diagram of one embodiment of the manifold storage tank connection part of this invention. The schematic diagram of another embodiment of the manifold storage tank connection part of this invention. The schematic diagram of another embodiment of the manifold storage tank connection part of this invention. The schematic diagram of another embodiment of the manifold storage tank connection part of this invention. (A) A perspective view of one embodiment of a microdiverticulum comprising a conductive gel electrode of the present invention and (B) a conductor plate electrode, and (C) one embodiment of a microdiverticulum comprising a conductor plate electrode. Micrograph. The graph which shows the relationship between the lipid gelation time in a plane lipid bilayer membrane apparatus provided with a different reservoir connection part, and the lipid concentration of a 2nd liquid. The graph which recorded the time-dependent change of the electric current between the main flow paths in the case of using an agarose electrode (A) and a gold pattern electrode (B) on the minute diverticulum side. The graph which recorded the time-dependent change of the electric current when ionophore by alpha hemolysin is formed in a lipid bilayer membrane in another plane lipid bilayer membrane array device of the present invention using a gold plate electrode on the side of a micro diverticulum.

DESCRIPTION OF SYMBOLS 1 Microfluidic system 2 Liquid supply port 3 Main flow path 4 Micro diverticulum 5 Planar lipid bilayer membrane 6 Reservoir connection part 7 Reservoir 8 Discharge port 9, 90, 91, 92, 93, 94, 95, 96, 97, 98 Discharge flow path 10 Sub discharge flow path 11 Discharge promotion liquid main flow path 12 Discharge promotion liquid side flow path 13 Discharge promotion liquid side flow path 14 Conducting conduit 15 Agarose gel 16 Gold plate electrode

  The embodiments of the present invention described below are for illustrative purposes only and are not intended to limit the technical scope of the present invention. The technical scope of the present invention is limited only by the appended claims.

1. Configuration of planar lipid bilayer array device of the present invention The planar lipid bilayer array device of the present invention comprises a liquid feeding port, a main channel extending from the liquid feeding port, and a plurality of openings opened on both side walls of the main channel. A planar lipid bilayer membrane including a micro diverticulum, a multi-layered reservoir connecting portion of the main channel, and a reservoir, wherein the main channel and at least a part of the micro diverticulum are provided with electrodes. . The liquid feeding port of the planar lipid bilayer array device of the present invention is the same as the liquid feeding port of the conventional planar lipid bilayer array device.

  Referring to FIG. 2, in one embodiment of the multi-purpose reservoir connection part of the planar lipid bilayer array apparatus of the present invention, one main flow channel is provided downstream of the planar lipid bilayer array (not shown). 3 branches into two discharge channels 9. Of the two discharge channels, one discharge channel 91 not adjacent to another discharge channel further branches a new discharge channel 92. In this way, the outermost discharge flow path branches into two one after another and branches into new discharge flow paths 93, 94, 95, 96, 97, and 98. All the discharge flow paths 9 are arranged in parallel and communicate with the liquid storage tank 7. The barrier between the discharge flow channel and the adjacent discharge flow channel branched from the discharge flow channel is continuous until it is once branched and then connected to the liquid storage tank. That is, all the discharge flow paths branch a new discharge flow path only once before reaching the liquid storage tank. The new discharge channel is arranged so as to be adjacent only to the discharge channel before branching. That is, the new discharge flow path produced by the branch is disposed outside the discharge flow path before the branch. In this way, the manifold type reservoir connection part of the present embodiment has a fan-like shape in which the discharge channel 9 is branched into two from the main channel 3 one after another. The discharge channel 9 may have the same width and depth as the main channel 3.

  A discharge promoting liquid main flow path 11 is provided to supply the discharge promoting liquid to the manifold storage tank connection portion of this embodiment. The discharge promotion liquid main flow path 11 extends from a liquid supply port (not shown) of the discharge promotion liquid and reaches a branch point from the main flow path 3 to the discharge flow path 9. The discharge promoting liquid side channels 12 and 13 branched from the discharge promoting liquid main channel reach the branch point of the discharge channel close to the liquid storage tank. The main flow path, the discharge flow path, and the discharge promotion liquid main flow path may have the same width and depth. The discharge promoting liquid side flow path has the same depth as the discharge promoting liquid main flow path, but the discharge promoting liquid side flow path reaching the branch point closer to the liquid storage tank may have a narrower width. For example, the width of the discharge promoting liquid side channel reaching the branch point closer to the liquid storage tank may be two thirds, one second, or one third of the width of the discharge promoting liquid main channel.

  Referring to FIG. 3, in another embodiment of the multi-part reservoir connection part of the planar lipid bilayer array device of the present invention, the discharge channel is successively discharged from the main channel 3 downstream of the planar lipid bilayer array. 9 repeats the bifurcation, but the barrier between the discharge flow channel and the discharge flow channel that is branched from the discharge flow channel and is adjacent to the discharge flow channel is branched at least once before being connected to the liquid storage tank. In some cases, the secondary discharge flow path intersects. In other words, the discharge flow channel and the discharge flow channel adjacent to the discharge flow channel communicate with each other through the sub discharge flow channel 10 at least at one point from when the flow is branched to communication with the liquid storage tank. In other words, in the storage tank connecting portion, the sub-discharge channel 10 passes through the barrier between the two discharge channels 9 and intersects and communicates with the adjacent discharge channel. The sub-discharge flow path may cross and communicate with three or more adjacent discharge flow paths. The main channel, the discharge channel, and the sub-discharge channel may have the same width and depth.

  A discharge promoting liquid main flow path 11 is provided to supply the discharge promoting liquid to the manifold storage tank connection portion of this embodiment. The discharge promotion liquid main flow path 11 extends from a liquid supply port (not shown) of the discharge promotion liquid and reaches a branch point from the main flow path 3 to the discharge flow path 9. The discharge promoting liquid side flow path 12 branched from the discharge promoting liquid main flow path reaches the branch point of the discharge flow path close to the liquid storage tank. The main flow path, the discharge flow path, and the discharge promotion liquid main flow path may have the same width and depth. The discharge promoting liquid side flow path has the same depth as the discharge promoting liquid main flow path, but the discharge promoting liquid side flow path reaching the branch point closer to the liquid storage tank may have a narrower width. For example, the width of the discharge promoting liquid side channel reaching the branch point closer to the liquid storage tank may be two thirds or one half of the width of the discharge promoting liquid main channel.

  As another embodiment of the present invention, there is the embodiment of FIGS. 4 and 5 in which the discharge promotion liquid main flow path and the discharge promotion liquid side flow path are removed from the embodiment shown in FIGS.

  With reference to FIG. 6, the electrode of the micro diverticulum of the present invention will be described. At least a part of the micro diverticulum of the planar lipid bilayer array of the present invention has an electrode for electrically connecting the liquid inside the micro diverticulum and the outside of the planar lipid bilayer array apparatus of the present invention. Provided. FIG. 6A illustrates an agarose electrode. One of the wall surfaces defining the micro diverticulum 4 is in communication with a current-carrying conduit 14, which is connected to an external electrode (not shown) of the planar lipid bilayer array device by a conductive gel, a conductor plate or other conductor. ) And electrically connected. At least a part of the current-carrying conduit 14 is filled with the agarose gel 15, and the end surface of the agarose gel 15 becomes a wall surface that defines the minute diverticulum 4.

  6B and 6C, the gold pattern electrode will be described. The gold plate electrode 16 is exposed on the bottom surface of the micro diverticulum 4, and the gold plate electrode 15 extends through the base material of the wall surface of the micro diverticulum 4 and is exposed inside the energizing conduit 14 separated by the wall surface. Thereby, the inside of the micro diverticulum 4 and the conducting conduit 14 are electrically connected.

  The planar lipid bilayer array device of this example is made of polydimethylsiloxane (PDMS) mounted on a glass surface, and has a liquid feeding port, a main channel extending from the liquid feeding port, and an opening in the main channel. And a micro structure including a plurality of micro diverticulas, a discharge channel branched from the main channel, and a current-conducting conduit. When a gold pattern electrode is provided, gold may be coated on the glass surface, and a PDMS structure may be mounted on the glass surface. The main flow path electrode of the present embodiment is a gold plate placed across both bottom surfaces of the bifurcated main flow path. The planar lipid bilayer array device is connected to a liquid feed pump and a flow path changeover switch, and the type of liquid flowing in the main flow path, the flow velocity, whether it is a uniform flow, a pulsating flow or other non-uniform flow It is possible to set freely how to flow.

  The dimensions of the planar lipid bilayer array device of the present invention were as follows. The bottom surface of the main channel was a glass surface, the main channel was 60 μm wide and 7 μm deep, and the minute diverticulum was 17 μm wide, 22 μm deep, and 7 μm deep. In the conventional storage tank connecting portion shown in FIG. 1, the width of the groove communicating with the main flow path is 400 μm, and the groove communicates with the liquid storage tank 1 mm away from the end face of the groove where the main flow path communicates with the groove. The width expands. The width of the liquid storage tank was 2 mm, and a circular discharge port having a diameter of 1.5 mm was provided on the bottom surface of the liquid storage tank. In the first manifold type storage tank connecting portion shown in FIG. 2 (hereinafter referred to as “manifold type (1)”), the discharge channel branched from the main channel has a width of 60 μm, a depth of 7 μm, and 1 mm. Branching eight times in the middle, nine discharge flow paths branch for one main flow path and communicate with the liquid storage tank in parallel. The liquid storage tank had a width and a depth of 3 mm, respectively, and a circular discharge port having a diameter of 1.5 mm was provided on the bottom surface of the liquid storage tank. In the second manifold type storage tank connection part (hereinafter referred to as “manifold type (2)”) shown in FIG. 3, the discharge channel branched from the main channel has a width of 60 μm, a depth of 7 μm, and 1 mm. Branching 8 times in between, nine discharge flow paths branch for one main flow path and communicate with the liquid storage tank. Five sub-discharge passages communicate with the discharge passage between the discharge passages. The liquid storage tank was 3 mm in width and depth, and a circular discharge port having a diameter of 1.5 mm was provided on the bottom surface of the liquid storage tank. In the following experiments, each planar lipid bilayer array device was immersed in water for 12 hours or more in advance to saturate the base polydimethylsiloxane and then used.

Effect of Diversified Reservoir Connection on Lipid Gelation In the following examples, Calcein (C0875-5G, Dulbecco phosphate buffered saline (hereinafter referred to as “DPBS”, Invitrogen Corporation) was used as the first solution. Sigma Aldrich Japan Co., Ltd.) was added at 50 μM. As the second liquid, phosphatidylcholine (DPhPc, 850356C, Avanti Polar Lipids INC, Funakoshi Co., Ltd.) was added to hexadecane in an amount of 5-20 mg / mL. DPBS was used as the third liquid. Unless otherwise specified in the following examples, the flow rate of the first liquid was 1 μL / min, the flow rate of the second liquid was 0.3-0.6 μL / min, and the flow rate of the third liquid was 0.15 μL / min at the maximum. It was.

  In both the manifold type (1) and the manifold type (2) storage tank connection portions, no discharge promoting liquid was allowed to flow in the discharge promoting liquid main flow path and the discharge promoting liquid side flow path. Then, the second liquid slightly entered the discharge promotion liquid main flow path and the discharge promotion liquid side flow path from the discharge flow path, and the liquid flow stopped.

  FIG. 7 shows the relationship between the lipid gelation time and the lipid concentration of the second liquid in a conventional lipid bilayer membrane array apparatus having a multi-layer (1) or multi-layer (2) reservoir connection part. It is a graph to show. In a planar lipid bilayer membrane array device equipped with a conventional reservoir connection part, gelation occurs in 5 minutes when the lipid concentration of the second liquid is 5 mg / mL, and gelation occurs in 3 minutes when it reaches 10 mg / mL. I woke up. At a concentration of 20 mg / mL represented by *, gelation occurred in less than 3 minutes. On the other hand, the multi-types (1) and (2) did not gel in 10 minutes even when the lipid concentration was 20 mg / mL.

Current Measurement of Planar Lipid Bilayer Array FIG. 8 is a graph recording changes with time of the current between the main flow path when an agarose electrode (A) and a gold pattern electrode (B) are used on the minute diverticulum side. DPBS was used as the first liquid and the third liquid, and hexadecane added with 10 mg / mL phosphatidylcholine hexadecane was used as the second liquid. The flow rate of the first solution was 1 μL / min, the flow rate of the second solution was 0.3-0.6 μL / min, and the flow rate of the third solution was not stationary but could not be measured very slowly. When the second liquid is injected into the planar lipid bilayer membrane array apparatus of the present invention, the current between the main flow path and the microdiverticulum suddenly decreases and a nearly complete insulating state is obtained. Thereafter, when the third liquid is injected, the second liquid in the main channel is pushed away into the third liquid. The second liquid left in the opening of the minute diverticulum forms a film layer sandwiched between the first liquid and the third liquid. During this time, almost no current flows between the main flow path and the minute diverticulum, and the almost complete insulation state continues. However, when hexadecane, which is the solvent in the second liquid, is absorbed by the polydimethylsiloxane, which is the base material, the film layer of the second liquid becomes thinner, and this increases the current between the main flow path and the microdiverticulum. At the beginning, a plateau was reached when the lipid bilayer was formed. As described above, it was possible to measure the membrane permeation current corresponding to the shape of the artificial membrane observed with a microscope using either an agarose electrode or a gold plate electrode.

FIG. 9 shows the current between the main flow path and the microdiverticulum when an ionophore of α-hemolysin is formed on the lipid bilayer in another planar lipid bilayer array device of the present invention using a gold plate electrode on the side of the microdiverticulum. It is the graph which recorded change with time of. DPBS to which 5 μg / mL α-hemolysin (H9395-0.5 mg, Sigma-Aldrich Japan Co., Ltd.) is added is the first solution, hexadecane to which 10 mg / mL phosphatidylcholine hexadecane is added is the second solution, and DPBS is the third solution. Using. The flow rate of each solution was the same as in the experiment of FIG. After injecting the third solution, the amount of current immediately increased by 0.82 pA. Here, since the applied voltage was 60 mV, the resistance value was 73.2 gigaohms. The surface area of the membrane formed in one minute diverticulum is 119 μm ² , and when the thickness is 10 nm, the specific resistance is calculated to be 0.871 gigaohm · m. This value is calculated by the patch clamp method. It belongs to a numerical range that is used as an indicator of sealing. The state sealed with the lipid bilayer membrane is called a gigaohm seal.

  In FIG. 9, after the gigaohm seal state was established, a rapid current increase of about 8 pA was observed twice. This indicates that α-hemolysin incorporated from the first liquid into the lipid bilayer forms a heptamer and a micropore penetrating the membrane is formed. Therefore, the planar lipid bilayer array device of the present invention electrically confirms the formation of a gigaohm-sealed artificial lipid bilayer membrane and the formation of transmembrane micropores by a 7-mer that self-assembles α-hemolysin. We were able to.

Claims (19)

  A planar lipid bilayer array including a main channel extending from the liquid delivery port, and a micro diverticulum provided in the main channel, and a reservoir connection part communicating with the main channel extending from the planar lipid bilayer array A liquid storage tank connected to the liquid storage tank connection section and a discharge port communicating with the liquid storage tank. The liquid storage tank connection section includes a plurality of parallel arrangements in which the main flow path is branched. A planar lipid bilayer array device characterized by comprising a discharge channel.   The planar lipid bilayer array device according to claim 1, wherein the width and depth of the discharge channel are the same as the width and depth of the main channel.   The said liquid storage tank connection part consists of 18 discharge flow paths which are arranged in parallel with each of the two main flow paths being bifurcated 8 times, respectively. Planar lipid bilayer array device.   The planar lipid bilayer array device according to any one of claims 1 to 3, wherein the reservoir connection part includes a sub-discharge channel that intersects and communicates with the discharge channel.   5. The planar lipid bilayer array device according to claim 4, wherein each of the main channels includes five sub-discharge channels.   The plane according to any one of claims 1 to 5, wherein the storage tank connection portion includes a discharge promotion liquid channel for injecting a discharge promotion liquid into the discharge channel. Lipid bilayer array device.   The main flow path electrode that is electrically connected to the liquid in the main flow path, and the micro diverticulum electrode that is electrically connected to the liquid in the micro diverticulum are included. The planar lipid bilayer array device described in 1.   The planar lipid bilayer array device according to claim 7, wherein the micro diverticulum electrode includes a conductive gel exposed on a wall surface defining the micro diverticulum.   The planar lipid bilayer array device according to claim 8, wherein the conductive gel is an agarose gel.   The planar lipid bilayer array device according to claim 8, wherein the micro diverticulum electrode includes a conductor plate in contact with a wall surface defining the micro diverticulum.   The planar lipid bilayer array device according to claim 10, wherein the conductor plate is a gold plate.   The planar lipid bilayer array device according to any one of claims 1 to 6, a liquid feeding device connected to a liquid feeding port of the device, and an analyzer for observing the planar lipid bilayer array device And a microfluidic system.   The planar lipid bilayer array device according to claim 6, a liquid feeding device connected to a liquid feeding port of the device, and a feeding channel connected to a discharge flow path of the planar lipid bilayer array device. A microfluidic system comprising a liquid device and an analyzer for observing the planar lipid bilayer array device.   The planar lipid bilayer array device according to any one of claims 7 to 11, a liquid feeding device connected to a liquid feeding port of the device, and an analyzer for observing the planar lipid bilayer array device And a device for measuring electrical characteristics between the main flow path electrode and the microdiverticulum electrode.   15. The microfluidic system according to claim 14, further comprising a liquid feeding device connected to a liquid flow path for discharge promotion of the planar lipid bilayer array device.   The microfluidic system according to any one of claims 12 to 15, wherein the liquid feeding device includes a liquid feeding pump.   The microfluidic system according to claim 16, wherein the liquid feeding device includes a flow path switching switch. A method for analyzing a lipid bilayer membrane,
(1) a step of feeding a first liquid comprising an aqueous solution to the planar lipid bilayer array device according to any one of claims 7 to 11, and
(2) After measuring the current between the main flow path and the micro diverticulum and confirming that the first liquid is filled in the micro diverticulum, the second is made of an oily solution to which lipid has been added. Feeding a liquid to the planar lipid bilayer array device;
(3) The current between the main flow path and the micro diverticulum is measured, and after confirming that the second liquid electrically insulates the micro diverticulum and the main flow path, it is made of an aqueous solution. Feeding a third liquid to the planar lipid bilayer array device;
(4) Measure current between the main flow path and the micro diverticulum to detect that the membrane layer of the second fluid sandwiched between the first fluid and the third fluid has formed a lipid bilayer. Steps,
(5) A method for analyzing a lipid bilayer, comprising a step of recording a measurement result of electrical characteristics between a micro diverticulum in which a lipid bilayer membrane is formed and the main channel.   18. A lipid bilayer membrane comprising the microfluidic system according to claim 17, a data storage system, and a control system, wherein the lipid bilayer membrane analysis method according to claim 18 is executed. System for automatic analysis of physical characteristics.