JP6619672B2 - Ion-permeable lipid bilayer membrane formation method and current measurement apparatus for ion-permeable lipid bilayer membrane formation - Google Patents

Description

  The present invention relates to a method for forming an ion permeable lipid bilayer membrane and a current measuring device for forming an ion permeable lipid bilayer membrane.

  Cells that make up living organisms, various organelles such as mitochondria, Golgi bodies, and endoplasmic reticulum, cell nuclei, etc. are covered with a biological membrane on the outside. This biological membrane is basically a lipid bilayer membrane. It is composed of Various proteins having physiological activity, that is, receptors, enzymes, and the like are retained on the lipid bilayer membrane in a form penetrating the lipid bilayer membrane. These transmembrane proteins play an important role in vivo. In particular, it has been found that various receptors present on cell membranes trigger various physiological reactions by binding to ligands present in the living body. For this reason, various ligands that enhance the function of receptors, inhibitors that inhibit the function of receptors, and the like are used as pharmaceuticals, and natural or artificial ligands and inhibitors that can be used as new pharmaceuticals have been studied. ing.

  In order to develop these transmembrane proteins, their ligands, inhibitors, etc., it is desired to perform various measurements in the same state as in the living body, that is, in a state where the transmembrane protein is held on the biological membrane. In drug discovery screening, etc., by using an automated device, a large number of lipid bilayers are formed simultaneously, and by using a number of formed lipid bilayers simultaneously, screening is performed, It is expected to increase the efficiency of screening. In order to cope with such automatic measurement, even when an automated device is used, the lipid bilayer membrane is formed with good reproducibility, and once formed, the lipid bilayer membrane is an automated device. It needs to exist stably without being destroyed during the movement and measurement process within.

  Patent Document 1 includes a step of adding a lipid solution that forms a lipid bilayer to each of two wells adjacent to each other through a partition wall having one or a plurality of through holes having a pore diameter of 500 nm to 500 μm; Adding water or an aqueous solution to each well to form droplets of water or an aqueous solution in the lipid solution; and allowing the well to stand in this state to form a lipid bilayer membrane in the through-hole portion. A method of forming a lipid bilayer, including a device suitable for this method is described.

  Patent Document 2 discusses the manufacturing method and configuration method described in Patent Document 1 for the purpose of making it possible to manufacture the instrument described in Patent Document 1 at a lower cost and increasing the efficiency of measurement. In addition, there is described an improved instrument that can be used for measurement again by exchanging only the partition wall by converting the partition wall into a cassette.

  Thus, by using the devices described in Patent Document 1 and Patent Document 2, it becomes possible to form a lipid bilayer without the need for special techniques, and a membrane protein is contained in the bilayer. Can be reconstructed with good reproducibility. Furthermore, Non-Patent Document 1 has the same configuration as that described in Patent Document 1, and even when 16 pairs of wells are provided, signals from all wells are confirmed, and ion channel measurement is performed efficiently. It is described that can be done.

JP 2012-81405 A Japanese Patent Laying-Open No. 2015-77559

R. Kawano et.al., Scientific Reports 3, Article number: 1995 (2013) doi: 10.1038 / srep01995

  When forming an ion permeable lipid bilayer membrane in which an ion channel is embedded in a lipid bilayer membrane, it can be confirmed that the ion channel has been formed by measuring the current passing through the ion channel. Since the current passing through the ion channel is on the order of 1 pA (picoampere) or 10 pA and is extremely weak, there is a problem that it is difficult to measure accurately due to noise.

  An object of this invention is to provide the method and apparatus which can measure the ion channel current of an ion permeable lipid bilayer membrane more correctly in ion permeable lipid bilayer membrane formation.

The method for forming an ion permeable lipid bilayer membrane according to the present invention includes a plate having at least a pair of wells on the upper surface and capable of holding a liquid therein, and the wells are arranged so that the pair of wells communicate with each other. Arranging on the electrode base so as to contact at least a pair of electrodes communicating with the inside of the well and the bottom surface of the plate, and at least a pair of contacts of the electrode base;
Using the urging means for urging the contact in the electrode direction and the fixing means for fixing the plate and the electrode base, the plate and the electrode base in a state where the electrode and the contact are in contact with each other. Fixing step to fix,
An injection step of forming a lipid bilayer membrane comprising an ion channel between the pair of wells;
A measurement step of applying a constant voltage between the pair of electrodes and measuring the current between the pair of electrodes using an ammeter;
Is provided.

  According to the present invention, noise can be reduced and the ion channel current of the ion-permeable lipid bilayer membrane can be measured more accurately.

FIG. 3 is a top view illustrating a schematic configuration of a plate of the current measuring device according to the first embodiment. 2 is a cross-sectional view illustrating a schematic configuration of a plate of the current measuring device according to the first embodiment; It is a side view which shows schematic structure of the partition cassette of the electric current measurement apparatus concerning Embodiment 1. FIG. 1 is a cross-sectional view illustrating a schematic configuration of an electrode base of a current measuring device according to a first embodiment; 1 is a cross-sectional view illustrating a schematic configuration of a current measuring device according to a first embodiment. 1 is a cross-sectional view illustrating a schematic configuration of a current measuring device according to a first embodiment. 3 is a flowchart showing a procedure of an ion-permeable lipid bilayer formation method according to the first embodiment. It is a graph which shows an example of the current waveform before and behind reconstruction of αHL in Example 2. 6 is a diagram illustrating an example of a lead-out wiring on a bottom surface of a base plate in Comparative Example 1. FIG. It is the photograph which image | photographed the appearance of the connector with a cable in the comparative example 1. It is a graph which shows an example of the current waveform before and behind reconstruction of αHL in comparative example 2. It is sectional drawing which shows schematic structure of the electric current measurement apparatus concerning another embodiment.

Embodiment 1
Embodiments of the present invention will be described below with reference to the drawings. In each drawing, in order to obtain an understanding of the structure, each part is appropriately scaled up and down, and does not represent an accurate dimensional relationship.

  First, the current measuring device used for the ion permeable lipid bilayer membrane forming method will be described with reference to FIGS. The current measurement device according to the first embodiment includes a plate for forming a lipid bilayer membrane and an electrode base for outputting an ion channel current to the outside.

  First, each structure of the plate of the current measuring device according to the first embodiment will be described with reference to FIGS. 1 and 2. FIG. 1 is a top view illustrating a schematic configuration of a plate of the current measuring device according to the first embodiment. FIG. 2 is a cross-sectional view illustrating a schematic configuration of the plate of the current measuring device according to the first embodiment.

  1 and 2, the plate 110 includes a well 111, a well 112, an electrode 113, an electrode 114, and a partition wall cassette 120. The partition cassette 120 includes a partition 121, a pressing plate 122, and a pressing plate 123.

  The plate 110 is a plate having a plate shape having a main surface that forms the well 111 and the well 112 and a bottom surface that is substantially parallel to the main surface and connected to the electrode base. For example, the plate 110 is preferably a plastic base such as acrylic. Further, the plate 110 has a groove for inserting the partition cassette 120.

  The well 111 and the well 112 are holes that have openings on the main surface of the plate 110 and do not penetrate the plate 110. For example, the well 111 and the well 112 are formed by cutting a plastic base such as acrylic. In the well 111 and the well 112, an ion permeable lipid bilayer membrane in which the ion channel is embedded in the lipid bilayer membrane is formed.

  The well 111 and the well 112 are formed at positions where they communicate with each other at a part of the side surface. The well 111 and the well 112 are separated by the partition cassette 120 at a position where they communicate with each other at a part of the side surface.

  The electrode 113 is an electrode that electrically connects the bottom portion in the well 111 and the bottom surface of the plate 110. The electrode 113 is preferably made of metal. For example, the electrode 113 is formed by forming a through hole communicating with the bottom surface of the plate 110 at the bottom of the well 111 and filling the through hole with a silver / silver chloride paste.

  The electrode 114 is an electrode that electrically connects the bottom of the well 112 and the bottom of the plate 110. Like the electrode 113, the electrode 114 is preferably made of metal. For example, the electrode 114 is formed by forming a through hole communicating with the bottom surface of the plate 110 at the bottom of the well 112 and filling the through hole with a silver / silver chloride paste in the same manner as the electrode 113.

  Next, each structure of the partition cassette of the current measuring device of the current measuring device according to the first embodiment will be described with reference to FIGS. FIG. 3 is a side view illustrating a schematic configuration of the partition cassette of the current measuring device according to the first embodiment. In FIG. 3, the partition cassette 120 includes a partition 121, a pressing plate 122, and a pressing plate 123.

  The partition wall 121 has a plate shape having a through hole, and is a partition wall that separates the well 111 and the well 112. The partition wall 121 has one or a plurality of through holes. A lipid bilayer membrane can be formed in this through-hole. A current flows between the well 111 and the well 112 through the ion channel of the lipid bilayer membrane. For example, the partition wall 121 is a parylene (poly-p-xylylene) film having a thickness of 5 μm, and has a plurality of through holes having a diameter of 100 μm formed by photolithography.

  The pressing plate 122 and the pressing plate 123 are pressing plates that sandwich the partition wall 121. For example, the pressing plate 122 and the pressing plate 123 are preferably acrylic plates having a certain thickness. The pressing plate 122 and the pressing plate 123 have through holes that can expose both surfaces of the partition wall 121 in a state where both surfaces of the partition wall 121 are sandwiched. The through hole is formed at a position where the through hole formed in the partition wall 121 is exposed in a state where the pressing plate 122 and the pressing plate 123 sandwich both surfaces of the partition wall 121.

  The partition cassette 120 is formed by sandwiching both surfaces of the partition 121 between a pressing plate 122 and a pressing plate 123. The partition cassette 120 is mounted in the groove of the plate 110 so that the well 111 and the well 112 communicate with each other through the through-holes of the partition 121, the pressing plate 122, and the pressing plate 123.

  Next, each configuration of the electrode base of the current measuring device according to the first embodiment will be described with reference to FIG. FIG. 4 is a cross-sectional view illustrating a schematic configuration of the electrode base of the current measuring device according to the first embodiment.

  In FIG. 4, the electrode base 200 includes a base plate 201, a spacer 202, a movable electrode 203, and a movable electrode 204. The movable electrode 203 includes a contact 231, a shaft 232, an urging mechanism 233, a holder 234, and a connection terminal 235. Similarly, the movable electrode 204 includes a contact 241, a shaft 242, an urging mechanism 243, a holder 244, and a connection terminal 245.

  The base plate 201 has a plate shape and has a through hole that accommodates the movable electrode 203 and the movable electrode 204. The base plate 201 is preferably made of an insulating material and has sufficient strength to support the plate 110.

  The spacer 202 is a support that is fixed to the upper surface of the base plate 201 and supports the plate 110. In the spacer 202, it is desirable that the surface in contact with the plate 110 has a shape corresponding to the plate 110. For example, when the bottom surface of the plate 110 is planar, it is desirable that the surface of the spacer 202 that contacts the plate 110 is also planar. As with the base plate 201, the spacer 202 is preferably made of an insulating material and has sufficient strength to support the plate 110. The spacer 202 only needs to be a structure having a certain height in order to have a function of keeping the displacement amount of the movable electrode 203 and the movable electrode 204 in the Z-axis direction constant. For example, the spacer 202 is preferably a block shape, a column shape, or a cylindrical shape.

  The movable electrode 203 is an electrode that is accommodated in the through hole of the base plate 201 and is movable in the Z-axis direction. The movable electrode 203 is an electrode that is electrically connected to the electrode 113 by being in contact with the electrode 113 exposed on the bottom surface of the plate 110.

  Similar to the movable electrode 203, the movable electrode 204 is an electrode that is accommodated in the through hole of the base plate 201 and is movable in the Z-axis direction. The movable electrode 204 is an electrode that is electrically connected to the electrode 114 by being in contact with the electrode 114 exposed on the bottom surface of the plate 110.

  The contact 231 is a contact in contact with the electrode 113 exposed on the bottom surface of the plate 110. The contact 231 is preferably a material having electrical conductivity. Further, it is desirable that the contact surfaces of the contact 231 and the electrode 113 are flat.

  The shaft 232 is a rod-shaped conductor that connects the contact 231 at one end and connects the connection terminal 235 to the other end, and electrically connects the contact 231 and the connection terminal 235. The XY cross section of the shaft 232 may be any shape and size that can be accommodated in the through hole of the base plate 201. For example, the XY cross section of the shaft 232 is preferably a circular or square shape.

  The urging mechanism 233 is configured to urge the movable electrode 203 toward the plate 110 in the Z-axis direction. For example, the urging mechanism 233 is preferably formed of an elastic body. Specifically, the urging mechanism 233 is preferably configured by a coil spring. The inner diameter of the coil spring is larger than the outer diameter of the shaft 232, and the outer diameter of the coil spring is smaller than the outer diameter of the contact 231 and the outer diameter of the holder 234, so The contact 231 can be biased toward the plate 110 in the Z-axis direction.

  The holder 234 is a structure in which a hollow cylindrical lower portion that holds the shaft 232 and a hollow flat plate upper portion that supports the biasing mechanism 233 are connected. The lower part of the holder 234 can be accommodated in the through hole of the base plate 201 by making the outer diameter smaller than the through hole of the base plate 201. It is desirable that the shape of the outer diameter of the lower part of the holder 234 corresponds to the shape of the through hole of the base plate 201. Further, the holder 234 can hold the shaft 232 by making the inner diameter of the lower portion of the holder 234 larger than the outer diameter of the shaft 232 so that it can slide. It is desirable that the shape of the inner diameter of the lower portion of the holder 234 corresponds to the shape of the outer diameter of the shaft 232.

  The upper portion of the holder 234 has an outer diameter larger than the through hole of the base plate 201 and larger than the outer diameter of the coil spring, so that the holder 234 is kept at the height of the base plate 201 while The contact 231 of the movable electrode 203 can be used as a base point for biasing the plate 110 in the Z-axis direction.

  The connection terminal 235 is a terminal that is electrically connected to the outside. The connection terminal 235 is a substance having electrical conductivity.

  The contact 241, the shaft 242, the urging mechanism 243, the holder 244, and the connection terminal 245 have the same functions and configurations as the contact 231, the shaft 232, the urging mechanism 233, the holder 234, and the connection terminal 235, respectively.

  With the above configuration, the electrode base 200 can support the plate 110 and be electrically connected to the electrode exposed on the bottom surface of the plate 110.

  A current measuring device is configured by combining the plate 110 and the electrode base 200 described above. Next, the configuration of the current measuring device will be described.

  FIG. 5 is a cross-sectional view illustrating a schematic configuration of the current measuring device according to the first embodiment. In FIG. 5, the same components as those in FIGS. In FIG. 5, the current measuring device 100 includes a plate 110, an electrode base 200, a fixing mechanism 301, and a fixing mechanism 302.

  In the fixing mechanism 301 and the fixing mechanism 302, the contact 231 of the movable electrode 203 and the electrode 113 exposed on the bottom surface of the plate 110 are in contact, and the contact 241 of the movable electrode 204 and the electrode 114 exposed on the bottom surface of the plate 110 are in contact. Then, the plate 110 and the electrode base 200 are fixed. For example, the fixing mechanism 301 and the fixing mechanism 302 are configured by male screws. The fixing mechanism 301 and the fixing mechanism 302 are inserted into through holes 311 and 312 provided in the plate 110 and screwed with female screws 321 and 322 provided on the electrode base 200.

  As described above, the contact 231 of the movable electrode 203 is urged toward the plate 110 in the Z-axis direction by the urging mechanism 233. On the other hand, the electrode 113 in contact with the contact 231 is fixed to the bottom surface of the plate 110, and the plate 110 is fixed to the electrode base 200 by the fixing mechanism 301 and the fixing mechanism 302. Accordingly, the contact 231 of the movable electrode 203 presses and contacts the electrode 113 on the bottom surface of the plate 110 without being displaced in the Z-axis direction.

  As described above, according to the current measuring apparatus of the first embodiment, the biasing mechanism biases the contact of the movable electrode in the electrode direction of the plate, and the plate is fixed in a state where the electrode of the plate is in contact with the contact of the movable electrode. By fixing the plate and the electrode base by the mechanism, the contact of the movable electrode can be brought into contact with the electrode of the plate being pressed, and contact noise caused by imperfect contact can be reduced.

  Next, a configuration for reducing electromagnetic noise will be described. FIG. 6 is a cross-sectional view illustrating a schematic configuration of the current measuring device according to the first embodiment. In FIG. 6, the same components as those in FIGS.

  In FIG. 6, the current measuring device 100 includes a plate 110, an electrode base 200, a fixing mechanism 301, a fixing mechanism 302, a twisted pair cable 401, a connection terminal 402, a shield cable 403, and a housing 404. .

  The twisted pair cable 401 is a cable in which two electric wires are twisted in pairs. The twisted pair cable 401 connects one end of two pairs of electric wires to the connection terminal 235 and the connection terminal 245, respectively, and connects the other end of the two pairs of electric wires to the connection terminal 402. The twisted pair cable 401 transmits signals from a pair of electrodes 113 and 114 arranged in the pair of wells 111 and 112. Therefore, when the current measuring apparatus 100 has two pairs of electrodes arranged in two pairs of wells, signals from the two pairs of electrodes can be transmitted by providing the two twisted pair cables 401. That is, it is preferable that the current measuring apparatus 100 includes a number of twisted pair cables 401 corresponding to a plurality of pairs of electrodes.

  The connection terminal 402 is a terminal connected to the twisted pair cable 401 and the shield cable 403. The connection terminal 402 connects the two pairs of wires of the twisted pair cable 401 to the core wire of the shielded cable 403, respectively.

  The shielded cable 403 is a cable having a core wire and a shielded cable. For example, the shielded cable 403 has two core wires, and each core wire is connected to two pairs of electric wires of the twisted pair cable 401 via the connection terminal 402.

  The housing 404 is a case that accommodates the plate 110, the electrode base 200, the fixing mechanism 301, the fixing mechanism 302, and the twisted pair cable 401 therein. The housing 404 functions as an electromagnetic shield that blocks electromagnetic waves from the outside by being composed entirely of a conductive substance or a composite containing a conductive substance. The housing 404 is preferably made of, for example, a resin coated with a conductive metal such as aluminum or a conductive material. The housing 404 is preferably grounded. Further, the housing 404 may be provided with an openable / closable lid for exchanging the plate 110 and injecting the solution into the plate 110.

  As described above, according to the current measuring device of the first embodiment, the pair of electrodes arranged in the well is connected by the twisted pair cable via the electrode-based movable electrode, so that the magnetic flux penetrating the twisted pair cable is used. Since the directions of the generated currents are reversed between adjacent ones, they cancel each other, and the influence of the external magnetic flux on the signal transmitted by the twisted pair cable is reduced.

  In addition, according to the current measuring device of the first embodiment, the current measurement is performed by housing the plate, the electrode base, and the twisted pair cable in the casing that is entirely composed of a conductive substance or a composite containing a conductive substance. Electromagnetic waves from outside the device can be reduced by electromagnetic shielding.

  In addition, according to the current measuring apparatus of the first embodiment, by grounding the electromagnetically shielded casing, the energy of the electromagnetic wave absorbed by the electromagnetic shield can be released to the ground side, and the potential of the casing is set to the ground potential. Therefore, it is possible to prevent the occurrence of noise due to a change in the potential of the casing.

  Next, an ion permeable lipid bilayer membrane forming method will be described. FIG. 7 is a flowchart of a procedure of the ion-permeable lipid bilayer formation method according to the first embodiment.

  First, in step S501, the plate 110 is disposed on the electrode base 200 so that the pair of electrodes 113 and 114 exposed on the lower surface of the plate 110 and the pair of contacts 231 and 241 provided on the electrode base 200 are brought into contact with each other. Then, the process proceeds to step S502.

  In step S <b> 502, the electrodes 113 and 114 and the contacts 231 and 241 are connected by using biasing mechanisms 233 and 243 that bias the contacts 231 and 241 in the electrode direction and a fixing mechanism that fixes the plate 110 and the electrode base 200. In the state of contact, the plate 110 and the electrode base 200 are fixed, and the process proceeds to step S503.

  In step S503, a solution (oil system or water system) as a raw material is injected into the wells 111 and 112, a lipid bilayer is formed between both wells, and the process proceeds to step S504. Specifically, in step S503, a lipid bilayer-forming lipid solution is injected, and a solution containing at least one of a protein, a peptide, and an organometallic complex that forms an ion channel (preferably a buffer solution) is added to both wells. Or a buffer solution is injected into one well and the other well to form a lipid bilayer, and the process proceeds to step S504.

  In step S504, a constant voltage is applied between the pair of electrodes 113 and 114, the current between the pair of electrodes 113 and 114 is measured using an ammeter, and the process proceeds to step S505.

In step S505, it is determined whether or not an ion channel is formed by determining whether or not the measured current is equal to or greater than a predetermined threshold value. If the measured current is greater than or equal to the predetermined threshold, the process proceeds to step S506. If the measured current is less than the predetermined threshold, the process returns to step S504.
In step S506, it is determined that an ion channel has been formed, and the process returns to step S504.

  The completion of the ion-permeable lipid bilayer membrane can be confirmed by the above measurement, and the ion-permeable lipid bilayer membrane can be used for the measurement.

  Thus, according to the ion-permeable lipid bilayer formation method of Embodiment 1, the contact point of the movable electrode is biased in the direction of the plate electrode by the biasing mechanism, and the contact point between the electrode of the plate and the movable electrode is contacted. In this state, the plate and the electrode base are fixed by the fixing mechanism, so that the contact point of the movable electrode can be brought into contact with the electrode of the plate and the contact noise due to the imperfection of the contact is reduced. Therefore, it can be accurately determined whether or not an ion channel is formed.

  Next, examples of current measurement using the current measuring device according to the present invention will be described based on examples.

(Example 1) Production of current detection unit (current measuring device 100) 1-1.
By cutting, a pair of wells 111 and 112 having a diameter of 4 mm and a depth of 3 mm were formed on one surface of an acrylic plate having a thickness of 4 mm. The distance between the centers of the pair of wells 111 and 112 was 3.5 mm. And the plate 110 was formed by providing the through-hole of diameter 1mm which penetrates an acrylic board in the center of the bottom part of the well 111,112.

1-2.
Electrodes 113 and 114 were formed by filling the through holes at the bottom of the wells 111 and 112 with a silver / silver chloride paste, respectively, and drying and solidifying. In addition, the electrodes 113 and 114 are slightly convex from the bottom of the plate 110 by being filled with an appropriate amount of paste in order to make complete contact with the contacts 231 and 241 of the electrode base 200 facing each other at the bottom of the plate 110. Bumps are formed.

1-3.
A groove having a width of 0.5 mm, a length of 4.4 mm, and a depth of 3.3 mm is provided in a contact portion between the well 111 and the well 112, and a 5 μm-thick parylene sandwiched between the pressing plates 122 and 123 is provided in the groove. A partition wall 121 was disposed. The partition wall 121 was provided with eleven round holes with a diameter of 100 μm by oxygen plasma etching.

1-4.
A predetermined number of holes were formed by drilling holes with a predetermined diameter in an acrylic plate having a thickness of 5 mm. Then, movable electrodes 203 and 204 that expand and contract via biasing mechanisms 233 and 243 were inserted into the holes, and an electrode base 200 was produced. The metal electrode used for the movable electrodes 203 and 204 had a diameter of about 1.6 mm and a length of about 28 mm.

1-5.
The electrode base 200 was placed in an aluminum case 404, and the connection terminals 402 and the connection terminals 235 and 245 attached to the case 404 were connected. A twisted pair cable was used for connection.

1-6.
The electrode base 200 and the plate 110 were fixed with male screws (fixing mechanisms 301 and 302) to complete the current detection unit (current measurement device 100).

(Example 2) Measurement of membrane protein ion channel current 2-1.
Commercially available phospholipid DPhPC (diphytanoylphosphatidylcholine) was dissolved in n-decane (normal decane) to prepare a solution of 20 mg / ml DPhPC.

2-2.
5 μL of this solution was dropped into each of the wells 111 and 112 of the plate 110 described in Example 1. A preferable dropping amount is 5 μL, but the dropping amount may be appropriately selected within a range of 3 to 10 μL.

2-3.
Subsequently, 20 μL of potassium phosphate buffer (KCl 1M, PB 10 mM, pH = 7.4) is dropped into one well 111, and 3 nM αHL (alpha-hemolysin) is mixed into the other well 112. Was dripped. A preferable dropping amount is 20 μL, but the dropping amount may be appropriately selected between 15 to 30 μL. Further, the preferable mixing amount of αHL is 3 nM, but the mixing amount may be selected in the range of 0.1 to 300 nM depending on the purpose.

2-4.
A micro current measuring amplifier (JET16ch) manufactured by Tecella is connected to the current measuring device 100 described in the first embodiment, and a clamp voltage of 50 mV is applied between the electrode 113 of the well 111 and the electrode 114 of the well 112, The current between the well 111 and the well 112 was measured. The gain of the amplifier was 10 GOhm (1 pA = 10 mV), and a low-pass filter with a sampling rate of 5 kHz and 1 kHz was inserted.

2-5.
When left in this state for a while, a lipid bilayer membrane is formed. Next, ion channel current is observed by the reconstruction of αHL. FIG. 8 is a graph showing an example of a current waveform before and after the reconstruction of αHL in the second embodiment. In FIG. 8, the vertical axis represents the current value, and the horizontal axis represents time.

Comparative Example 1 Production of Current Detection Unit 1-1.
A plate 110 was produced in the same manner as described in Examples 1-1 to 1-3 in Example 1. The base plate 601 of the plate 110 is made of acrylic having a thickness of 4 mm, and has a structure in which 8 pairs are arranged in two rows each, for a total of 16 pairs. In the first comparative example, unlike the method described in the first embodiment, a lead wiring made of silver paste is provided on the bottom surface of the base plate. FIG. 9 is a diagram illustrating an example of the lead wiring on the bottom surface of the base plate in the first comparative example. A screen printing method was used to apply the silver paste to a predetermined position. In FIG. 9, portions 602 to 605 are designed so that the printed pattern acts as a contact point in contact with the electrode on the connector side.

1-2.
In a Faraday cage that connects two sets of connectors with cables (Tecella 8-channel electrode holder: Terrapin) to the plate 110 described in 1-1 of Comparative Example 1, and shields the plate 110 and the electrode holder from external electric field fluctuations. And completed the current detection device. 10 is a photograph of the appearance of the connector with cable in Comparative Example 1. FIG.

(Comparative Example 2) Detection of membrane protein ion channel current 2-1.
The current detection unit described in 1-1 of Comparative Example 1 was connected to a microcurrent measurement amplifier similar to that in Example 2-4, and a lipid solution and potassium phosphate were contained in the well in the same manner as described in Example 2. A buffer solution and a buffer solution mixed with αHL were sequentially added dropwise. The measurement conditions were the same as described in Example 2.

2-2.
By leaving it in this state for a while, a lipid bilayer membrane was formed, and then the ion channel current accompanying the reconstitution of αHL was measured. FIG. 11 shows an example of current waveforms before and after the αHL reconstruction. FIG. 11 is a graph illustrating an example of a current waveform before and after reconstruction of αHL in Comparative Example 2.

3. Comparison of noise level with the device described in Example 3-1.
The noise levels of the current measuring device described in Example 1 and the current measuring device described in Comparative Example 1 based on the present invention were compared.

3-2.
The noise values are obtained by measuring the effective value of the noise current for 5 to 10 seconds in a no-signal state before αHL is reconstructed three times each and performing an averaging process. The results are shown in Table 1.

  When Example 1 is compared with Comparative Example 1, in Example 1, the noise at the time of no signal is less than 1 pA, which is about 1/5 of the noise of the comparative example.

  In the current detection chip of the comparative example, routing wiring is printed for connection with the connector, and a loop is inevitably formed by this wiring portion. The magnitude of the induced electromotive force due to the time variation of the magnetic field from the outside is proportional to the area of the interlaced loop. Although it is not a metal casing in the comparative example, since the current detection device is housed in the Faraday cage, it is considered that the noise resistance against electric field fluctuations is almost equivalent to the device shown in the example. Therefore, the area of the loop formed by the routing wiring is larger in Comparative Example 1 than in Example 1, and it is considered that noise induced from this loop portion is generated.

  On the other hand, in Example 1, since the direction of the current generated by the magnetic flux penetrating the twisted pair cable is reversed between adjacent ones by connecting with the twisted pair cable, the magnetic fluxes from the outside cancel each other. Affects the signal transmitted by the twisted pair cable.

  As described above, according to the ion permeable lipid bilayer membrane formation method and the ion permeable lipid bilayer membrane formation method of the present invention, it is possible to measure an ion channel current of 1 pA order, which has been difficult in the past. Therefore, it can be applied to ion channel current measurement of membrane proteins, which could not be measured conventionally.

  Note that the present invention is not limited to the above-described embodiment, and can be changed as appropriate without departing from the spirit of the present invention. For example, although an example using a pair of wells and electrodes has been described in the above embodiment, the number of wells and electrodes is not particularly limited, and a pair or more of wells and electrodes may be provided.

  Moreover, in the said embodiment, although the screw is shown as an example of the fixing mechanisms 301 and 302 which fix the plate 110 and the electrode base 200, a fixing mechanism is not limited to a screw and generally fixes a panel. Push rivets, fasteners and the like can also be used. In addition, a friction pin or the like can be used instead of a screw because of the relationship with the spring pressure of the contact.

  Further, the fixing mechanisms 301 and 302 may be L-shaped brackets. FIG. 12 is a cross-sectional view illustrating a schematic configuration of a current measuring device according to another embodiment. 12, the same components as those in FIGS. 1 to 6 are denoted by the same reference numerals, and description thereof is omitted. In FIG. 12, the fixing mechanism 301 includes an L-shaped bracket 701 and a rotation shaft 702. Similarly, the fixing mechanism 302 includes an L-shaped bracket 703 and a rotation shaft 704.

  The L-shaped bracket 701 is provided on the electrode base 200 so that one end of the L-shaped bracket 701 can be freely rotated by the rotation shaft 702. Similarly, the L-shaped bracket 703 is also provided on the electrode base 200 so that one end of the L-shaped bracket 703 can be freely rotated by the rotation shaft 704.

  Then, after placing the plate 110 on the electrode base 200, the L-shaped brackets 701 and 703 are rotated so that one side of the L-shaped brackets 701 and 703 is positioned on the plate 110, thereby the electrode base 200 and the plate 110. May be fixed.

  The partition 121 is not necessarily provided, and an ion-permeable lipid bilayer can be formed as long as the well 111 and the well 112 communicate with each other. Further, the size of the diameter of the through hole of the partition wall 121 is not particularly limited. Further, the number of through holes of the partition wall 121 is not particularly limited.

100 Current measuring device 110 Plate 111, 112 Well 113, 114 Electrode 120 Bulkhead cassette 121 Bulkhead 122, 123 Holding plate 200 Electrode base 201 Base plate 202 Spacer 203, 204 Movable electrodes 231, 241 Contact 232, 242 Shaft 233, 243 Energizing Mechanisms 234, 244 Holders 235, 245 Connection terminals 301, 302 Fixing mechanisms 311, 312 Through holes 321, 322 Female screw 401 Twisted pair cable 402 Connection terminal 403 Shield cable 404 Cases 701, 703 L-shaped brackets 702, 704 Rotating shaft

Claims (8)

A plate having at least a pair of wells on the upper surface capable of holding a liquid therein and having the wells arranged to communicate between the pair of wells is disposed on an electrode base, and the inside of the well and the well An arrangement step of contacting at least a pair of electrodes communicating with the bottom surface of the plate and at least a pair of contacts of the electrode base;
Using the urging means for urging the contact in the electrode direction and the fixing means for fixing the plate and the electrode base, the plate and the electrode base in a state where the electrode and the contact are in contact with each other. Fixing step to fix,
An injection step of forming a lipid bilayer membrane comprising an ion channel between the pair of wells;
A measurement step of applying a constant voltage between the pair of electrodes and measuring the current between the pair of electrodes using an ammeter;
An ion-permeable lipid bilayer formation method comprising :
At least the plate and the electrode base are housed in a housing;
The housing has a resin coated with a metal conductor or conductive material,
A connection line between the at least one pair of contacts and the ammeter in the casing is a twisted pair cable, the connection line outside the casing is a shielded cable, and a shield of the shielded cable is grounded. Ion-permeable lipid bilayer formation method . The method for forming an ion-permeable lipid bilayer according to claim 1 , wherein the metal conductor or the conductive substance is grounded. If the current measured at the measuring step is greater than a predetermined threshold value, the ion-permeable lipid bilayer according to claim 1 or 2 comprising a determination step with the lipid bilayer membrane and embedded ion channel is formed Film forming method. The method of forming an ion-permeable lipid bilayer according to any one of claims 1 to 3 , wherein at least a part of a connection line between the at least one pair of contacts and the ammeter is a twisted pair cable. The method of forming an ion-permeable lipid bilayer according to any one of claims 1 to 4 , wherein the ion-permeable lipid bilayer is a substance in which an ion channel is embedded in a lipid bilayer. A plate having at least a pair of wells on the upper surface capable of holding a liquid therein, the wells are arranged so that the pair of wells communicate with each other, and each well has an electrode communicating with the inside of the well and the bottom of the plate. The plate;
An electrode base having at least a pair of contacts in contact with the electrode;
An ammeter that applies a constant voltage between the at least one pair of electrodes and measures current;
A biasing means for biasing the contact in the electrode direction;
A fixing means for fixing the plate and the electrode base in a state where the electrode and the contact are in contact;
A current measuring device for forming an ion-permeable lipid bilayer comprising :
A housing for storing at least the plate and the electrode base;
The housing has a resin coated with a metal conductor or conductive material,
Before Kikatami body, said at least one pair of contacts, the connection line between the current meter is a twisted pair cable, wherein the connecting lines in the housing outer is a shielded cable, the shield of the shielded cable is grounded Current measurement device for ion permeable lipid bilayer formation . The current measuring apparatus for forming an ion-permeable lipid bilayer according to claim 6 , wherein the metal conductor or the conductive substance is grounded. The current measuring device for forming an ion-permeable lipid bilayer according to claim 6 or 7 , wherein at least a part of a connection line between the at least one pair of contacts and the ammeter is a twisted pair cable.