JP2005337734A - Ion channel analytical method by cis-trans liquid current method and ion channel analyzer used for the same - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a revolutional ion channel analytical method by a cis-trans liquid current method and an ion channel analyzer used for the same enabling to continue an intended ion channel analysis efficiently even when a membrane to be analyzed is damaged during an analyzing process by quickly repairing the membrane. <P>SOLUTION: The technical method adopts a coaxial tube-type cis-trans isomeric liquid tube comprising an insulating inner tube in which a micropore is formed at a predetermined position of the tube wall where a membrane to be analyzed is formed and a first liquid flow capillary channel is provided inside of the tube wall w, and an insulating outer tube which surrounds the insulating inner tube and in which a second liquid flow capillary channel is formed around the inner tube, an inner tube electrode which is provided near the micropore in the first liquid flow capillary channel, and an outer tube electrode which is provided near micropore in the second liquid flow capillary channel. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

  The present invention improves the ion channel analysis technique, more specifically, the flow performance of a cis-trans isomer liquid pipe having a coaxial tube structure composed of an insulating inner tube and an outer tube. By using this system, even if the analysis target membrane breaks during the analysis process, it is possible to quickly repair the target membrane and efficiently continue the intended ion channel analysis. The present invention relates to an ion channel analysis method using a cis-trans liquid flow method and an ion channel analysis apparatus used for the method.

As a method for elucidating the function of a membrane protein, an electrophysiological technique is used, and dynamic medical / biological information and single molecule information that cannot be obtained by other methods can be obtained. By the way, the conventional electrophysiological methods are roughly classified into a patch clamp method (target sample = cultured cells) and a lipid flat membrane method (target sample = membrane protein extracted biochemically). Here, the “patch clamp method” refers to the formation of a micro cell membrane in the electrode by stripping the cell membrane by sucking the inside of the electrode while pressing a glass electrode with a smooth opening at the tip of the cell. In this method, a single channel current flowing through an ion channel formed in the electrode is measured by applying a pulse current to the minute cell membrane (see Non-Patent Document 1 = FIG. 6 on page 19 and its explanatory text). On the other hand, the “lipid planar membrane method” is a method in which a small hole is made in the partition wall that partitions the inside of the container, a lipid planar membrane is stretched here, a membrane vesicle is added to the cis side, and the planar membrane is fused. This is a method in which a pulse voltage is applied from a silver-silver chloride electrode, and a single channel current flowing out from an ion channel due to the pulse voltage is measured (Non-patent Document 1 = FIG. 3 on page 44, and its description). See sentence). These “patch clamp method” and “lipid planar membrane method” both respond specifically and sensitively to a specific molecule at a low concentration of a protein present in a biological membrane, and the response can be performed with extremely high accuracy. It can be measured automatically, and the sensitivity is high enough to capture not only one molecule but also one photon. Realization of a dream sensor is expected, and various research and development have been attempted among researchers involved in the technical field.
Edited by Masahiro Sogabe, Narumi Ogiki, etc. (Kyoritsu Publishing Co., Ltd.) “AEON CHANNEL” on page 19, FIG. 6 and its description, and on page 44, FIG. 3 and its description

  However, these conventional methods have common problems and have limitations and restrictions on the scope of application. The biggest bottleneck pointed out as the cause is the mechanical vulnerability of the membrane (microcell membrane formed on the glass electrode in the case of the patch clamp method, or planar membrane in the case of the lipid planar membrane method). The “film” is easily damaged by the surrounding physical influence and cannot be stably measured.

  However, as a proposal to improve the fragility of such a “film”, an attempt to increase the mechanical strength of the film by using a reinforcing agent that modifies the hardness and elasticity of the film molecule is also made. However, the adverse effect that the biochemical properties of the “membrane” itself change and obstructs the acquisition of the desired medical / biological information or single molecule information is avoided. It was not possible.

  The present invention has been made in view of the above-described problems in the conventional means for electrophysiologically analyzing ion channels, and has a weak mechanical strength and delicate micro cell membranes and ion channel molecules. Under the assumption of the assumption that the built-in lipid planar membrane is damaged during the analysis process, the damaged “membrane (microcellular membrane, lipid planar membrane, etc.)” is immediately repaired and the desired ion is repaired. It is an object of the present invention to provide an innovative method and apparatus that enables efficient channel analysis to be continued.

  In addition, another object of the present invention is to enable electrical measurement by rapidly and efficiently substituting each of the electrolyte solutions existing on both sides with the ion channel molecule-incorporated lipid planar membrane as a boundary. It is to provide a practical method and apparatus.

  Another object of the present invention is that not only membrane proteins existing on the cell membrane surface, but also membrane proteins such as organelles and nuclei, and bacteria and virus membrane proteins cannot be processed by conventional patch clamp methods. An object of the present invention is to provide an electrophysiologically useful method and apparatus that can be used for the measurement and analysis of membrane protein molecules.

  Furthermore, another object of the present invention is that the surface area of the lipid planar membrane can be made extremely small, the capacitance can be greatly reduced, the stray capacitance in the circuit is small, and the dielectric loss is also low. It is an object of the present invention to provide an ion channel analysis method and apparatus effective for test research that can accurately obtain highly reliable medical and biological information with extremely low noise.

The method means adopted by the inventor to achieve the above object will be described with reference to the accompanying drawings. That is, in the “ion channel analysis method by perfusion generation of a lipid planar membrane” provided by the present invention, a micropore 11a in which a lipid planar membrane is generated is opened at a predetermined position of the tube wall w, and the tube wall w is provided inside. Includes an insulating inner tube 11 having a capillary-shaped first liquid flow path T; and an insulating outer tube 12 surrounding the inner pipe 11 to form a second liquid flow path C around the inner pipe. For a cis-trans isomer liquid pipe 1 having a coaxial pipe structure composed of
The inner pipe 11 and the outer pipe 12 are each perfused with an electrolytic solution so that the first and second liquid flow paths T and C are filled with the electrolytic solution, and then the second liquid flow in the outer pipe 12 is filled. By sending a lipid solution into the channel C, a planar lipid membrane M is formed in the micropore 11a of the inner tube, and then a sample solution containing the ion channel molecule to be analyzed is fed to the first liquid channel T of the inner tube 11. The ion channel molecules are incorporated into the lipid planar membrane M by perfusion, and the one electrode 2 installed in the first and second liquid flow paths T and C in the vicinity of the ion channel lipid planar membrane Mb thus incorporated is formed. It is characterized in that a single channel current flowing between the electrodes 2 and 3 passing through the ion channel lipid planar membrane Mb is detected and measured by applying a pulse current.

In addition, another method means adopted by the inventor to achieve the above object is that a micropore 11a having a size capable of receiving a cell to be analyzed is established at a predetermined position on the tube wall w, and An insulating inner tube 11 having a capillary-shaped first liquid flow channel T inside the tube wall w; surrounding the inner tube 11 and forming a second liquid flow channel C around the inner tube For a cis-trans isomer liquid pipe 1 having a coaxial pipe structure including an insulating outer pipe 12,
Each of the inner tube 11 and the outer tube 12 is perfused with an electrolytic solution so that both the inner and outer tubes are filled with the electrolytic solution, and then the cell to be analyzed is placed in the second liquid channel or the first liquid channel. The cell Mb is received in the fine hole 11a of the inner tube by feeding the contained sample liquid, and then the first liquid flow path or the second liquid flow opposite to the flow path into which the sample liquid is fed. A negative pressure is instantaneously applied to the roadside to cleave the membrane patch on the negative pressure side of the cell Mb received in the micropore 11a, and thus the cell Mb of the micropore 11a in which the membrane patch is cleaved, By applying a pulse voltage to one of the electrodes installed in the first and second liquid flow paths T and C in the vicinity of the micropore 11a, it flows through the channel molecules on the cell membrane and flows between the two electrodes. It is characterized in that required data is obtained by detecting and measuring a single channel current.

On the other hand, the apparatus means adopted by the present inventor for achieving the above object will be described as follows with reference to the accompanying drawings.
That is, in the “ion channel analyzer” of the present invention, a micropore 11a in which a lipid planar membrane is formed is opened at a predetermined position on the tube wall w, and a capillary-shaped first liquid channel is formed inside the tube wall w. A cis in a coaxial tube structure including an insulating inner tube 11 having T and an insulating outer tube 12 surrounding the inner tube 11 and forming a second liquid flow path C around the inner tube 11 A trans isomer liquid pipe 1; an inner pipe electrode 2 disposed in the first liquid flow path T in the cis-trans isomer liquid pipe 1 in the vicinity of the fine hole 11a; and the second liquid flow The first and second liquid flow paths T and C formed by the inner and outer pipes 11 and 12 are adopted in the path C by adopting technical means such as the outer pipe electrode 3 disposed in the vicinity of the fine hole 11a. A state in which a lipid flat membrane Mb in which the ion channel molecules to be analyzed are incorporated is formed in the micropores 11a by being perfused with electrolyte However, it is characterized in that a single channel current passing through the lipid planar membrane Mb can be detected and measured by applying a pulse current having a constant peak value to either one of the inner and outer electrodes 2 and 3. .

Accordingly, the following is a brief description of the method and apparatus of the present invention.
(1) Since the cis-trans isomer liquid pipe 1 having a coaxial pipe structure used in the present invention needs to be electrically insulating, it is common to use glass, ceramic, or synthetic resin as a material. Moreover, since it may be visually recognized from outside or irradiated with light, it is desirable to be transparent, and it is also desirable to have impact resistance, so that it is preferably flexible.
(2) The cis-trans isomer liquid pipe 1 used in the present invention is composed of an inner pipe 11 and an outer pipe 12. The inner pipe 11 and the outer pipe 12 surrounding the inner pipe 11 are both electrolytes. The inner tube 11 has an outer diameter of 2 to 0.5 mm and an inner diameter of 1.7 to 0.5 mm (pipe). Thickness of wall w = 0.2 ~ 0.5mm), outer diameter of outer pipe 12 is 8 ~ 3mm, inner diameter is 4 ~ 2.5mm (thickness of pipe wall w '= 4 ~ 0.5mm) is adopted The Incidentally, the inside of the inner tube 11 becomes a capillary-shaped first liquid channel T, while the gap between the inner tube 11 and the outer tube 12 becomes an annular second liquid channel C.
(3) In addition, a fine hole 11a is formed in the tube wall w of the inner pipe 11 at a place where it is easy to measure when the cis-trans isomer liquid pipe 1 is constructed. The smaller the through-hole diameter of the fine hole 11a, the better. In general, the diameter of the hole penetrating the center is 50 to 10 μm, and when the cell membrane is targeted, it is necessary to receive it on the micropore wall without passing one cell, and about 1 μm. It is necessary to be. In addition, it is advantageous for the formation of a planar lipid membrane if the periphery of the through-hole is a conical surface whose pore diameter gradually increases as it reaches the outer surface of the tube wall w. Incidentally, the through hole portion of the micro hole 11a formed in the inner tube 11 can be drilled by irradiating a well-known YAG gas laser, and as a processing method for forming a conical surface around it, the conical surface should be formed. There is a method in which masking (for example, polyethylene resin coating) is applied to other parts, and etching is performed with a known glass corrosive agent such as dilute hydrofluoric acid, hydrofluoric acid, xenon fluoride, etc. Is well known in the art.
(4) In the cis-trans isomer liquid pipe 1 configured as described above, it is necessary to perform hydrophobic treatment on at least the micropores 11a of the inner pipe 11 in which the lipid planar membrane is formed. As a hydrophobic treatment method applied in this case, the cis-trans isomer liquid tube is immersed in a well-known hydrophobic treatment solution such as a silicone-based water repellent or a fluorine-based water repellent, and then heated appropriately. The method of heat setting is taken. Thus, the inner and outer surfaces of the tube wall w in the inner tube 11 and the inner surface of the outer tube 12 are hydrophobized (in the case of this immersion method, the outer peripheral surface of the outer tube 12 is simultaneously hydrophobized. Not particularly necessary in the invention).
(5) An inner tube electrode 2 disposed in the first liquid channel T adjacent to the fine hole 11a, and an outer tube electrode disposed in the second liquid channel C adjacent to the minute hole 11a. As for 3, a metal material having good conductivity is preferably used, and a silver-silver chloride electrode is usually adopted.
(6) As a means for applying a pulse voltage to the inner tube electrode 2 or the outer tube electrode 3 described above, a known impulse generator 4 capable of arbitrarily setting and outputting a pulse voltage having a required peak value is adopted. As a means for detecting and measuring a single channel current flowing through the ion channel lipid planar membrane Mb to the inner tube electrode 2 or the outer tube electrode 3, a channel current measuring device 5 incorporating a patch clamp amplifier should be employed. Any of these impulse generators 4 and current measuring devices 5 can be used as they are generally used in the well-known patch clamp method and lipid planar membrane method.
(7) Then, in the “ion channel analyzer” of the present invention, a predetermined electrolyte or analysis target is provided to the first liquid channel T and the second liquid channel C of the cis-trans isomer liquid tube 1. As means for supplying and feeding ion channel sample liquid, cell-containing sample liquid, etc., the first syringe pump 6 is provided in the first liquid flow path T, and the second syringe pump 7 is provided in the second liquid flow path C. And the liquid can be ejected intermittently or continuously at an arbitrary pressure to perfuse the first and second liquid flow paths T and C, respectively.
(8) Further, in the present invention, there is provided a liquid supply means capable of causing a relative pressure difference to the pressure of the liquid perfused through the first liquid flow path T and the second liquid flow path C in the cis-trans isomer liquid pipe 1. If the arrangement is made so that a suction force can be generated from the flow path with high hydraulic pressure to the low flow path in the micropores 11a in the tube wall of the inner tube 11, the ion channel lipid planar membrane Mb is generated. Or can be done quickly and accurately when regenerating and repairing.

  In the method and apparatus of the present invention, a micropore for generating a lipid planar membrane is formed at a predetermined position on the tube wall, and an insulating inner wall having a capillary-shaped first liquid channel is formed inside the tube wall. A coaxial cis-trans isomerization electrolyte tube comprising a tube; and an insulating outer tube surrounding the inner tube and forming a second liquid flow path around the inner tube. Therefore, the necessary electrolyte solution, ion channel sample solution, and cell-containing sample solution are selectively delivered to the inner tube and the outer tube of the cis-trans isomerized electrolyte tube. By perfusing into the liquid flow path and the second liquid flow path, the ion channel lipid planar membrane to be analyzed is rapidly formed in the micropores, or the cells of the membrane patch dehiscence are rapidly received. In the course of ion channel analysis, the lipid planar membrane and the recipient cells of the micropores It is possible to continue the desired ion channel analysis efficiently immediately repaired even when damaged by factors.

  In the method and apparatus of the present invention, since the cis-trans isomerized electrolyte tube having the coaxial tube structure is employed, the electrolyte solution existing on both sides with the ion channel lipid planar membrane or the patch-dehiscent cell as a boundary. Each can be quickly and efficiently exchanged individually and electrically, and electrical measurements are possible. Medical / biological information and single molecule information about specific ion channel lipid planar membranes and cells to be analyzed In addition to membrane proteins on the cell membrane surface, micropores established on the inner wall of the inner tube, membrane proteins such as organelles and nuclei, and bacteria and viruses Since ion channel lipid planar membranes derived from membrane proteins can be made freely, membrane protein molecules that could not be processed by the conventional patch clamp method can be measured and analyzed, for example, early onset of leukemia and cancer. It is intended range of use on the electrophysiological is extremely wide, such as.

  Furthermore, in the method and apparatus of the present invention, a capillary coaxial cis-trans isomerization electrolyte tube is adopted, and the pore size of the micropores established in the inner tube can be extremely reduced, so that the micropores are generated. The surface area of the ion channel lipid planar membrane can also be markedly reduced compared to the conventional patch clamp method and lipid planar membrane method, and the capacitance can be greatly reduced, and inner and outer tubes are used with highly insulating materials. Therefore, stray capacitance and dielectric loss in the electric circuit can be greatly reduced, and highly reliable medical / biological information can be obtained accurately with low noise.

  Hereinafter, the ion channel analysis method and apparatus according to the present invention will be described in more detail based on embodiments shown in the accompanying drawings.

  FIG. 1 and FIG. 2 are cross-sectional explanatory views showing the outline of an ion channel analyzer (Example) according to the present invention. What is indicated by reference numeral 1 in the figure is a cis-trans isomer liquid tube having a coaxial tube structure, and a flexible insulating inner tube 11 made of transparent quartz glass is provided in the center, and of these, The periphery of the tube 11 is surrounded by a transparent flexible quartz glass insulating outer tube 12 made of the same material as the inner tube, and a second wall is formed between the outer periphery of the inner wall of the inner tube 11 and the inner wall surface of the outer tube 12. A liquid flow path C is formed. Incidentally, the inside of the tube wall w of the inner tube 11 constitutes a capillary-shaped first liquid flow path T, and a predetermined position of the tube wall w (in FIG. 1, a position on the right side of the center of the inner tube 11). A micropore 11a in which a lipid planar membrane is to be formed is opened.

  The inner tube 11 in this embodiment has an outer diameter of 1 mm, an inner diameter of 0.7 mm, and a thickness of the tube wall w of 0.3 mm. The minimum hole diameter that penetrates the center of the microhole 11a is 30 μm in diameter. The maximum hole diameter on the outer surface side of the wall w is 86 μm, and the hole diameter from the maximum hole diameter to the minimum hole diameter gradually changes to form a cone shape. In this case, in this example, it is necessary to ensure adhesion stability of the generated lipid planar membrane on the pore wall surface of at least the cone-shaped micropores 11a, and since hydrophobic treatment is required, in use, The entire cis-trans isomer liquid tube 1 is immersed in a hydrophobic processing solution (trioctylsylane = 10% benzine diluent), defoamed and pulled up, and then heated with an infrared heater for about 15 seconds to be processed into a hit set.

  Next, reference numeral 2 in the figure denotes an inner tube electrode that is inserted and disposed in the first liquid flow channel T so as to be close to the fine hole 11a of the inner tube 11 in the cis-trans isomer liquid tube 1. Reference numeral 3 denotes an outer tube electrode inserted and disposed in the second liquid flow path C between the outer tube 12 and the inner tube 11 as close to the fine hole 11a. Since both the inner and outer electrodes 2 and 3 are required to have high conductivity, silver-silver chloride is used in this embodiment. The inner tube electrode 2 and the inner tube electrode 3 are respectively connected to conducting wires 21 and 23 made of a highly conductive metal, and are connected to an impulse generator and a channel current measuring device described later.

  On the other hand, what is indicated by reference numeral 4 in FIG. 1 is an impulse generator capable of arbitrarily setting and outputting a pulse voltage having a required peak value to the inner tube electrode 2 via the lead wire 21, and reference numeral 5 is a patch. This is a channel current measuring instrument with a built-in clamp amplifier. The channel current measuring device 5 is configured such that the impulse generator is interposed between the two electrolytes in the first liquid channel T and the second liquid channel C electrically insulated by the ion channel lipid planar membrane Mb of the micropore 11a. 4, the outer tube electrode 3 collects the current when the pulse voltage output by 4 is deformed by the action of the ion permeation / opening / closing mechanism unique to the ion channel lipid planar membrane and output as a unique single channel current. This is a circuit mechanism for receiving and inputting via the. In the present embodiment, the impulse generator 4 and the channel current measuring device 5 are housed and integrated in a case E, and are connected to the downstream side of the cis-trans isomer liquid pipe 1.

  Next, what is indicated by reference numeral 6 on the upstream side of the cis-trans isomer liquid pipe 1 in FIG. 1 is a first syringe pump connected to the first liquid flow path T in the inner pipe 11. The second syringe pump connected to the second liquid flow path C between the outer tube 12 and the inner tube 11 is designated by. Thus, the first syringe pump 6 injects the required electrolyte solution, the ion channel sample solution to be analyzed, or the sample solution containing the cells to be analyzed into the first solution channel T, and the first solution channel. On the other hand, the second syringe pump 7 is a mechanism for injecting a required electrolytic solution or qualitative solution and allowing it to perfuse into the second liquid flow path C.

  Further, on the downstream side of the cis-trans isomer liquid pipe 1, what is indicated by reference numeral 8 is a first for receiving and storing the electrolytic solution and the ion channel sample liquid flowing down through the first liquid flow path T. A liquid collector, indicated by reference numeral 9, is a second liquid collector that receives and accommodates the electrolytic solution and lipid solution flowing down through the second liquid flow path C. The electrolyte solution, the ion channel sample solution, the cell-containing sample solution, and the lipid solution perfused through the cis-trans isomer liquid tube 1 can be collected without leakage by the first and second collectors 8 and 9. .

  Further, in FIG. 1, what is indicated by reference numeral 10 is a workstation (Fujitsu: Celsius V 810), which is the impulse generator 4 and the channel current measuring device 5, and the first syringe pump 6 and the second syringe pump. 7 is connected.

  Then, the workstation 10 generates the impulse by transmitting a control signal to the impulse generator 4 arbitrarily or based on a predetermined program, such as the potential, peak value, pulse waveform, hertz number, etc. to be output from the impulse generator 4. Controls the pulse voltage output by the instrument. Further, a single channel current peculiar to each membrane Mb outputted according to each ion channel lipid planar membrane Mb of the micropore 11a is inputted to the channel current measuring device 5 incorporating the patch clamp amplifier. Each single channel current that is done will be sent to the workstation 10. The workstation 10 can record each of the input single channel currents on a recording medium (not shown), and can also display and monitor them on the display 10a.

[Example of use of the embodiment apparatus]
A specific procedure for performing ion channel analysis using the above-described embodiment apparatus of the present invention is as follows. In addition, the minimum hole diameter penetrating the center of the fine hole 11a in the inner tube 11 is 1 μm, and the maximum hole diameter on the outer surface side of the tube wall w is 30 μm.

(1) Cleaning and sterilizing treatment Sufficient cleaning and sterilizing treatment is required to use the apparatus of this embodiment. For the cis-trans isomer liquid pipe 1, the liquid pipe portion is removed and the inner and outer pipes 11 and 12 are placed inside. In a state where the air is removed, it is immersed in strong acid (concentrated nitric acid: concentrated sulfuric acid = 1: 3) for several days. Next, the cis-trans isomer liquid pipe 1 after the strong acid washing is thoroughly washed with fresh water and dried, and chloroform-methanol liquid (1: 1) is introduced into the inner and outer pipes 11 and 12 to sterilize. disinfect.

(2) Hydrophobization treatment around the micropores 11a The cis-trans isomer liquid tube 1 is immersed in trioctylsylane (10% benzine diluent) to attach the trioctylsylane to the walls of the liquid channels T and C of the inner and outer tubes 11 and 12. Then, heat and dry with a far-infrared heater from a position about 0.1 mm away to make the entire flow path T / C hydrophobic. At the same time, the hole wall surface of the fine hole 11a is also hydrophobized.

(3) Assembling of Example Apparatus The inner tube electrode 2 is inserted from the downstream end portion of the inner tube 11 of the cis-trans isomer liquid tube 1 to a position near the fine hole 11a, and the inner tube is fixed to the plastic holder. . Next, the outer tube electrode 3 is inserted from the downstream side of the outer tube 12 to the vicinity of the fine hole 11a, and the outer tube portion is also fixed to the plastic holder. The inner tube electrode 2 is connected to an impulse generator 4 built in the case E through a conducting wire 21, and the inner electrode 3 is connected to a channel current measuring device 5 built in the case E through a conducting wire 31. Connect. At this time, the downstream ends of the inner pipe 11 and the outer pipe are connected to the first liquid collector 8 and the second liquid collector 9, and the first liquid flow path T is connected to the first liquid collector 8 and the second liquid collector 9. The two-liquid channel C is communicated with the second liquid collector 9.

  In addition, the upstream plastic part is connected to the upstream end of the inner pipe 11 of the cis-trans isomerate liquid pipe 1 via the upstream plastic holder and the upstream end part of the first syringe pump 6 and the outer pipe 12. Connect the second syringe pump 7 through the holder.

  Then, the impulse generator 4, the channel current measuring device 5, the first syringe pump 6, and the second syringe pump 7 are connected to the interface adapter of the workstation 10.

  Thus, based on a control program installed in the workstation 10, a predetermined pulse voltage / command is applied between the inner tube electrode 2 and the outer tube electrode 3 and is input to the channel current measuring device 5. One channel current is detected and measured and recorded on the recording medium of the workstation 10. In this case, it is useful to measure each of the electrolyte solutions existing on both sides of the first liquid flow channel T and the second liquid flow channel C with the ion channel lipid planar membrane Mb as a boundary. Medical and biological information and single molecule information can be collected very dynamically.

(4) Ion channel analysis process First, the workstation 10 is operated to drive the first syringe pump 6 and the second syringe pump 7, and the inner tube 11 and the outer tube 12 in the cis-trans isomer liquid tube 1 An electrolyte solution (1M NaCl solution) is poured into the first liquid channel T and the second liquid channel C to be filled with the electrolyte solution (flow rate: 0.5 ml / min).

  Next, the impulse generator 4 is set to a voltage fixing mode by the workstation 10 and a pulse voltage having a constant peak value is repeatedly applied between the inner tube electrode 2 and the outer tube electrode 3 to thereby obtain electric resistance and electric capacity. Are continuously monitored on the display 10a. In this case, the standard of electric resistance is 500Ω, and the standard of electric capacity is 1 pF (stray capacity).

  Subsequently, a phospholipid solution (phosphatidylcholine 20 mg / ml hexadecane) is supplied to the second syringe pump 7, the workstation 10 is operated to drive the syringe pump 7, and the outer tube 12 and the inner tube are driven. 50 μl of the same solution is poured into the second fluid channel C between In this case, if the fluid pressure of the phospholipid solution in the second fluid channel C is set slightly higher than the fluid pressure in the first fluid channel T, a suction force is generated in the fine holes 11a. The flat lipid membrane M is easily generated. In this state, the display 10a monitors the amount of increase in electric resistance and electric capacity as a guide to check whether or not the lipid planar membrane M is formed in the micropores 11a. In this case, the standard for the formation of the lipid planar membrane M is an electric resistance of 300 GΩ and an electric capacity of 10 pF. If sufficient characteristics are not exhibited even when a lipid flat membrane is formed, the workstation 10 is operated to drive the first and second syringe pumps 6 and 7, and the first liquid channel T and the second liquid flow are driven. The insufficient lipid planar membrane is broken by generating a large pressure difference with the path C, and the same operation is repeated until a sufficient and appropriate lipid planar membrane M is formed by performing the above-described operation again.

  When the lipid planar membrane M necessary for the micropores 11a is generated, channel molecules are incorporated into the lipid planar membrane M. The channel molecules that can be incorporated in the present invention include not only membrane proteins on the cell membrane surface, but also membrane proteins such as organelles and nuclei, and all ion channel molecules derived from bacterial and viral membrane proteins. It is included.

In this analysis, analysis processing is performed according to the following procedure.
(1) First, blood is collected from a blood vessel of a patient suspected of having leukemia using a syringe, and the collected blood is centrifuged to prepare a sample solution containing only white blood cells.
(2) Next, the prepared sample solution is poured into the second liquid channel C between the outer tube 12 and the inner tube 11 via the second syringe pump 7. At this time, if the liquid pressure of the sample liquid in the second liquid flow path C is set to be slightly higher than the first liquid flow path T, the white blood cell Mb to be analyzed is placed on the cone-shaped wall of the micropore 11a. It is easily sucked along and received.
(3) Thus, when the white blood cell Mb is received in the fine hole 11a, the negative pressure (1 mH ₂ 0) is applied to the white blood cell exposed to the first fluid channel T by the suction drive of the first syringe pump 6. Is applied instantaneously (0.1 sec) to cleave only the membrane patch portion subjected to negative pressure, and the whole cell recording state is set to the workstation 10.
(4) Next, a pulse voltage (200 mV) is applied to the inner tube electrode 2 by the impulse generator 4. Since leukocytes affected with leukemia depolarize the membrane potential in response to potassium ions K ⁺ , the single channel current detected at the outer tube electrode 3 is measured. This single channel current will be recorded at the workstation 10 via the channel current meter 5. (5) According to research results that the inventor has independently discovered through many years of clinical examination, leukemia can be diagnosed when the channel current pattern indicates a HERG potassium channel. Incidentally, the present inventor changed the voltage applied to the white blood cells from −80 mV to +50 mV, maintained the time level of 500 ms, and again measured the current value after 10 ms after returning to −80 mV. If the value is large, leukemia is diagnosed.

  Thus, the above-described method for diagnosing leukemia by the method of the present invention performs diagnosis in 15 minutes in total, 10 minutes for centrifugation of blood and leukocytes, and 5 minutes for ion channel analysis by cis-trans liquid flow treatment. In addition, since the white blood cells to be analyzed can be obtained by a normal blood sampling method, the pain of the subject is extremely light. On the other hand, the diagnosis of leukemia that is generally performed at present is very painful for the test subject because the white blood cells must be collected from the bone marrow rather than from peripheral blood, and at least 1 in the entire course of the diagnosis. Time is needed. As described above, the present invention brings about an epoch-making utility that is unprecedented even when it is applied only to the diagnosis of leukemia.

  The embodiments of the present invention specifically disclosed in the present specification are as described above. However, the present invention is not limited to the above-described embodiments, and various modifications can be made within the scope of the claims. For example, as shown in FIG. 3, it is possible to design a plurality of cis-trans liquid tubes 1 to be arranged in parallel so that many types of ion channel lipid planar membranes can be analyzed in parallel. It is clear that it belongs to the technical scope of the present invention.

  As is clear from the above description, the ion channel analysis method and apparatus provided by the present invention are structurally simple, but the cell itself, membrane proteins on the cell membrane surface, organelles, nuclei, etc. Highly accurate analysis data can be acquired using ion channel molecules derived from membrane proteins, and even membrane proteins of bacteria and viruses, and it can contribute greatly to medical and biological research and development, Furthermore, since it can also be utilized as a biosensor technology, its industrial utility value is extremely large.

FIG. 1 is an explanatory cross-sectional view showing an outline of an ion channel analyzer (Example) according to the present invention. FIG. 2 is a partially enlarged cross-sectional view showing the AA portion of FIG. 1 in an enlarged manner so that the structure of the cis-trans isomer liquid pipe and the form of micropores in the apparatus of this embodiment can be understood. FIG. 3 is a conceptual diagram showing a modified embodiment in which the apparatus of the present invention is configured in multiple types.

Explanation of symbols

1 cis-trans liquid tube
11 Insulating inner pipe
11a Micropore
12 Insulating outer tube 2 Inner tube electrode 3 Outer tube electrode 4 Impulse generator 5 Channel current measuring device 6 First syringe pump 7 Second syringe pump 8 First collector 9 Second collector 10 Workstation
10a Display C Second liquid flow path M Lipid flat film Mb Ion channel lipid flat film T First liquid flow path w Inner tube wall

Claims (13)

A micropore in which a lipid planar membrane is formed at a predetermined position of the tube wall, and an insulating inner tube having a capillary-shaped first liquid channel inside the tube wall; For a cis-trans isomer liquid pipe having a coaxial pipe structure including an insulating outer pipe that forms a second liquid flow path around the inner pipe,
Each of the inner tube and the outer tube is perfused with an electrolyte solution so that both the inner and outer tubes are filled with the electrolyte solution, and then a lipid solution is fed into the second liquid channel of the outer tube to thereby supply the inner tube. A lipid flat membrane is generated in the micropores, and then the sample liquid containing the ion channel molecule to be analyzed is perfused into the first liquid flow path of the inner tube to incorporate the ion channel molecule into the lipid flat membrane, thus A single voltage that flows between the two electrodes through the ion channel lipid planar membrane by applying a pulse voltage to one of the electrodes installed in the first and second liquid flow paths in the vicinity of the ion channel lipid planar membrane formed in an integrated manner. An ion channel analysis method using a cis-trans liquid flow system, characterized in that a channel current is detected and measured.   When the planar lipid membrane formed in the micropores of the inner tube is broken, the lipid solution is again fed into the second liquid channel of the outer tube to generate the lipid planar membrane in the micropores of the inner tube, The cis-trans liquid flow system according to claim 1, wherein a sample liquid containing an ion channel molecule to be analyzed is perfused into the first liquid flow path, and the ion channel molecule is incorporated into the lipid planar membrane. Ion channel analysis method.   A method of using a cis-trans isomer liquid tube having a coaxial glass tube structure made of a transparent glass capable of transmitting ultraviolet rays as an insulating inner tube and an insulating outer tube. After forming a lipid planar membrane and incorporating a membrane protein into the lipid planar membrane as an ion channel molecule to be analyzed, the membrane plane incorporating the membrane protein is irradiated with ultraviolet rays to polymerize the membrane plane in two dimensions. The ion channel analysis method according to the cis-trans liquid flow method according to claim 1 or 2. An insulative inner tube having a microscopic hole of a size capable of receiving a cell to be analyzed at a predetermined position on the tube wall and having a capillary-shaped first liquid flow path inside the tube wall; A cis-trans isomer liquid pipe having a coaxial pipe structure including an insulating outer pipe that surrounds the inner pipe and forms a second liquid flow path around the inner pipe.
Each of the inner tube and the outer tube is perfused with an electrolytic solution so that both the inner and outer tubes are filled with the electrolytic solution, and then the analysis target cell is contained in the second liquid channel or the first liquid channel. By feeding the sample liquid, the cells are received in the micropores of the inner tube, and then, on the side of the first liquid channel or the second liquid channel on the side opposite to the channel through which the sample liquid is fed, The membrane patch on the negative pressure side of the cells received in the micropores is cleaved by applying negative pressure, and the cells in the micropores thus cleaved by the membrane patch are in close proximity to the micropores. A feature of detecting and measuring a single channel current flowing between both electrodes through a channel molecule on the cell membrane by applying a pulse voltage to one electrode installed in the second liquid flow path An ion channel analysis method using a cis-trans liquid flow method.   The micropores in the inner wall of the inner tube are perforated to a pore size of about 1 μm, and the sample liquid containing white blood cells is fed into the second liquid channel or the first liquid channel of the cis-trans isomer liquid tube The white blood cells are received in the micropores of the inner tube, and then a negative pressure is instantaneously applied to the first liquid channel or the second liquid channel side opposite to the channel through which the sample solution is sent. The membrane patch on the negative pressure side in the white blood cells received in the micropores was cleaved, and thus the white blood cells in the micropores in which the membrane patch was cleaved were placed in the first and second liquid flow paths. By applying a pulse voltage to one of the electrodes, a single channel current that passes through the channel molecules on the cell membrane and flows between the two electrodes is detected and measured, and electrical data relating to the signs of leukemia are obtained. A cis-trans solution according to claim 4 Ion channel analysis method by the system.   The electrolyte solution to be perfused into the first liquid flow path of the inner tube and the second electrolyte flow path of the outer tube uses a plurality of types of electrolyte solutions having different compositions. The ion channel analysis method by a cis-trans liquid flow system according to any one of the above.   A relative hydraulic pressure difference is provided between the first liquid channel and the second liquid channel, and a fine hole in the tube wall of the inner tube is caused to generate a suction force from a high fluid pressure channel to a low fluid channel. The ion channel analysis method according to any one of claims 1 to 6, characterized by a cis-trans liquid flow method.   A micropore in which a lipid membrane is formed is opened at a predetermined position on the tube wall, and an inner tube having a capillary-shaped first liquid flow path is enclosed in the tube wall, and the inner tube is surrounded by the inner tube. A cis-trans isomer liquid pipe having a coaxial pipe structure including an insulating outer pipe that forms a second liquid flow path around the pipe; and in the first liquid flow path in the cis-trans isomer liquid pipe An inner tube electrode disposed in the vicinity of the micropore; an outer tube electrode disposed in the second liquid channel in the vicinity of the microhole; the inner tube and the outer tube The electrolyte solution is perfused and filled into the first and second liquid flow paths formed by the above and a planar lipid membrane incorporating the ion channel molecules to be analyzed is generated in the micropores, or in the micropores. In a state where the cell to be analyzed is received with the membrane patch being cleaved, a constant wave is applied to one of the inner and outer electrodes. By applying a pulse voltage value, cis and wherein the detection of measurable constructed of a single channel current through the planar lipid bilayer - ion channel analyzer of the transformer liquid flow method.   The fine hole formed in the tube wall of the insulating inner tube has a cone shape in which the diameter is small on the first liquid channel side and the diameter gradually increases toward the second liquid channel side. 9. A cis-trans liquid flow type ion channel analyzer according to claim 8.   10. The cis-trans liquid flow system according to claim 8 or 9, wherein at least an inner tube of a cis-trans isomer liquid pipe having a coaxial tube structure is subjected to a hydrophobic treatment on a cone-shaped hole wall surface of a fine hole. Ion channel analyzer.   On the inflow side of the cis-trans isomerary liquid pipe, a first syringe pump for injecting a required liquid into the first liquid flow path of the inner pipe and perfusing the first liquid flow path, and a second liquid flow path of the outer pipe A second syringe pump for injecting and perfusing the required liquid to the cis-trans isomer liquid pipe, while receiving the liquid perfusing the first liquid flow path and receiving it. The first liquid collector connected to the second liquid collector containing the liquid perfused through the second liquid flow path, and the inner tube electrode disposed in the first liquid flow path is a pulse. 11. An outer tube electrode connected to an impulse generator for generating a voltage and disposed in the second liquid flow path is connected to a channel current measuring device. A cis-trans liquid flow type ion channel analyzer as described.   The cis-trans liquid flow type ion channel analyzer according to any one of claims 8 to 11, wherein the inner and outer tubes of the cis-trans isomer liquid tube are made of transparent and flexible quartz glass. .   The ion channel of the cis-trans liquid flow system according to any one of claims 8 to 12, comprising at least two cis-trans isomer liquid pipes comprising an insulating inner pipe and an insulating outer pipe. Analysis device.

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