JP2006133001A - Cell electrophysiology measurement device - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To perform highly accurate measurement by improving the property of sealing between a hole and cells, to lower the cost of a chip, and to supply cells and chemicals with satisfactory operability, with respect to an electrophysiological measuring instrument. <P>SOLUTION: A member 102 of PDMS, for example, is used wherein the diameter of a patch hole 101 becomes smaller than an original diameter when thereon exerting negative pressure 202. Further, a micro flow-path chip 601 is formed in which a buffer liquid acts as a sheath flow 702 of chemicals, by supplying cells 104 onto the patch hole 101, and placing a buffer liquid supply flow path 602 and a chemical supply flow path 603 in the middle of a flow path of the patch hole 101. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

  The present invention relates to an apparatus for measuring electrophysiological signals of isolated cells and cultured cells.

  Due to the rapid increase in new drug candidate compounds in recent years, development of an apparatus for screening drugs using cell electrophysiological activity as an index is strongly desired. There is a potential difference (resting membrane potential) of several tens of mV inside and outside the cell membrane of living cells. In cells in which an arbitrary receptor is expressed on the cell surface in advance, when an added compound binds to the receptor, an ion channel existing in the membrane opens. As a result, the ion concentration inside and outside the membrane changes, and the membrane potential changes as a current flows through the membrane. In an apparatus for measuring an electrophysiological signal of a cell, protein quantification can be easily performed from a current change with respect to a fixed membrane potential. Therefore, electrophysiological information such as ion channels and transporters can be obtained directly with high accuracy. The results of these analyzes not only contribute to the design of clinical trial methods, but are an effective tool for the early selection of compounds that avoid human risk.

  As a conventional apparatus for measuring an electrophysiological signal of a cell, for example, there is a patch clamp method described in Non-Patent Document 1. In the patch clamp method, cells are aspirated with a glass pipette, the cells are brought into close contact with the tip of the glass pipette, and the cell membrane of cells in the area that is in close contact with the glass pipette is broken by additional suction pressure, and the pipette and intracellular Are at the same potential. After that, by detecting the potential of the electrode in the pipette as a difference from the potential of the reference electrode by using a detection means such as an amplifier, this is a method for detecting a potential change in the cell.

  On the other hand, for the purpose of high-speed chemical screening, particularly for the first screening (first candidate narrowing), since the quickness and convenience of measurement are more important, for example, the micropore disclosed in Patent Document 1 is used. There is an apparatus for measuring an electrophysiological signal using a flat plate chip. In an apparatus for measuring electrophysiological signals using a flat-plate chip having micropores, a biological sample such as a cell or tissue piece is placed on a flat plate electrode in a solution close to the biological salt concentration composition, and the potential of the electrode. The ion flow through the ion channel is detected by measuring the change. Since this method does not require an operation for bringing cells into close contact with a glass pipette, the electrophysiological activity of cells close to the state in a living body can be measured easily and quickly.

Japanese Patent Laid-Open No. 2004-12215

Essential cell biology, 5th edition, Nanedo, 2002, Japanese edition, translated by Keiko Nakamura et al., Pages 388-389

  However, in a conventional apparatus for measuring an electrophysiological signal using a flat plate chip having fine holes, the sealing performance between the fine holes and the cells is not always good. The sealing performance between the micropores and the cells becomes better as the diameter of the micropores is smaller. On the other hand, when the diameter of the micropore is reduced, the fluid resistance is increased, and the force for sucking the cell onto the micropore with a suction pump or the like is weakened. Thus, when the diameter of the micropore is reduced, the sealing performance between the micropore and the cell and the force for sucking the cell onto the micropore have a trade-off relationship. Seal performance is a factor that directly affects the accuracy of electrophysiological signals. For high-precision measurement, there was a problem of improving the sealing performance.

  Moreover, although the flat chip is usually disposable, silicon or glass has been used as the material of the conventional flat chip. In manufacturing such flat plate chips, photolithographic processes used in semiconductor manufacturing and ion beam drilling are utilized, and the number of processes is considerably large, the material cost is high, and the total cost is required unless they are produced in very large quantities. There was a problem of increasing the cost.

Furthermore, in a conventional apparatus for measuring an electrophysiological signal using a flat plate chip having a fine hole, the method for supplying cells and chemicals is not always easy to operate. Particularly, in order to examine the influence of the concentration of a chemical solution to be administered to cells, it is necessary to prepare several concentrations of the chemical solution and administer the solution, and there is a problem that a supply system for cells and chemical solutions is not sufficiently prepared.
The present invention relates to an apparatus for measuring an electrophysiological signal using a flat chip having a micropore, to improve the sealing performance between the micropore and the cell, to reduce the cost of the chip, and to a cell or a chemical solution. The purpose of this system is to make the supply system of this product easy to operate.

  Therefore, in order to solve one of the above objects, the present invention includes a member having a hole smaller than the size of a cell, an electrode arranged so as to sandwich the member, and means for sucking a cell into the hole, A cell electrophysiology measuring apparatus comprising a member in which the diameter of the hole facing the cell side of the member becomes smaller than the original diameter when driving means for sucking cells into the hole is configured To do.

  In order to solve one of the above objects, the present invention uses polydimethylsiloxane (PDMS) as the member.

  Furthermore, in order to achieve one of the above objects, the present invention provides a chemical solution supply unit in the middle of the flow path to the buffer solution supply unit and the hole so that the buffer solution becomes a sheath flow of the chemical solution. A means for adjusting the amount of the chemical solution from the supply portion of the chemical solution was provided.

  As described above, according to the present invention, the sealing performance between the micropores and the cells is improved, the measurement is performed with high accuracy, the cost of the chip is reduced, and the supply of the cells and the chemicals is good. It becomes possible to make it a system.

  Hereinafter, the present invention will be described with reference to FIGS. FIG. 1 is a schematic diagram of the whole cell electrophysiology measuring apparatus of the present invention. A measurement chip 201 (see FIG. 2) configured by a flexible member 102 having a patch hole 101 smaller than the diameter of the cell 104 and a reinforcing member 103 that reinforces the flexible member 102 is installed in the measurement block 105. The measurement chip 201 is filled with the buffer solution 106, the reference electrode 107 is placed in the buffer solution 106, the electrode 108 is placed below the patch hole 101, and connected to the amplifier unit 109 to measure a weak current or voltage. It has become. The suction pump 110 is used when sucking one of the plurality of cells 104 in the buffer solution 106 into the patch hole 101. If the negative pressure 202 (see FIG. 2) is further applied after the cells 104 are sucked into the patch holes 101, the cell membrane of the cells 104 can be destroyed. In addition to the above, the cell electrophysiology measurement apparatus of the present invention is provided with a buffer solution supply unit 111, a cell supply unit 112, a drug solution supply unit 113, and a waste solution unit 114.

  FIG. 2 is a schematic diagram showing a change in the hole diameter of the measurement chip of FIG. The measurement chip 201 includes a flexible member 102 having a patch hole 101 having a diameter of several μm smaller than that of the cell 104 and a reinforcing member 103 that reinforces the flexible member 102. In this embodiment, the flexible member 101 is polydimethylsiloxane, which is a kind of silicon rubber: PDMS (width 1.5 mm × length 1.5 mm × height 0.5 mm, Young's modulus E = 7.5 × 105 Pa, Poisson's ratio ν = 0.49) The reinforcing member 103 is made of acrylic (width 1.5 mm × length 1.5 mm × height 0.5 mm, hole diameter φ1.0 mm, Young's modulus E = 3.0 × 109 Pa, Poisson's ratio ν = 0.23). FIG. 2A shows a state before applying a negative pressure by the suction pump 110, and the diameter φD0 of the patch hole 101 on the side facing the cell 104 is 5 μm. On the other hand, FIG. 2B shows a state in which a negative pressure is applied by the suction pump 110, and the patch hole 101 bends when sucked at 0.2 × 105 Pa, and the patch hole 101 on the side facing the cell 104. The diameter φD1 is about 4 μm. That is, the member 101 is made of a material in which the diameter (φD1) of the patch hole 101 facing the cell side becomes smaller than the original diameter (φD0) when the negative pressure 202 is applied. The sealing performance between the patch hole 101 and the cell 104 becomes better as the diameter of the patch hole 101 is smaller. On the other hand, when the diameter of the patch hole 101 is reduced, the resistance of the fluid flowing therethrough is increased, and the force for sucking the cell 104 onto the patch hole 101 by a suction pump or the like is in a trade-off relationship. However, as described above, when negative pressure is applied, the diameter of the patch hole 101 facing the cell 104 side gradually becomes smaller than the original diameter depending on the magnitude of the negative pressure. ) Or the like can be used to improve the sealing performance between the patch hole 101 and the cell 104. Furthermore, the cell 104 can be sucked onto the patch hole 101 without increasing the resistance of the fluid flowing through the patch hole 101 so much.

  Further, when the surface of the polydimethylsiloxane (PDMS) measurement chip 201 having the patch hole 101 is subjected to a process of improving the wettability of the surface by irradiating the plasma, the adhesion between the patch hole 101 and the cell 104 is improved. Get better. As a result, the sealing performance is improved, and the electrophysiological signal can be measured with high accuracy.

  The hole shape of the measuring chip 201 in FIG. 2 may be a mortar shape in which the diameter of the hole facing the cell side is larger than the diameter of the hole on the back surface side as shown in the measuring chip 301 in FIG. In the case of this shape, the cell 104 can be stably adsorbed to the patch hole 101.

  Next, FIG. 4 shows an example of a method for manufacturing a polydimethylsiloxane (PDMS) measuring chip 201 having patch holes 101. First, as shown in FIG. 4A, the glass tube 401 is fixed to the micromanipulator 402 and is arranged so that the tip of the glass tube 401 is in contact with the surface of the container 403. Thereafter, as shown in FIG. 4 (B), liquid polydimethylsiloxane (PDMS) 404 containing a curing agent is poured and waiting for curing. After curing, as shown in FIG. 4C, when the glass tube 401 is removed, patch holes 405 can be formed.

  As described above, the use of the polydimethylsiloxane (PDMS) measuring chip 201 having the patch holes 405 reduces the number of manufacturing processes and material costs compared to the conventional silicon or glass chip. The total cost can be kept low.

  FIG. 5 is a diagram showing a microchannel structure for supplying cells onto the patch holes in the measurement chip of FIG. A flow path is formed between the cell supply part 112 and the waste liquid part 114 through the patch hole 101. The cell supply part 112 is in the upstream part of the patch hole 101, and there is a cell supply flow path 503 having a flow path width of about 300 μm in the middle. The waste liquid part 114 is in the downstream part of the patch hole 101, and there is a waste liquid flow path 504 having a flow path width of about 600 μm in the middle. In this embodiment, a contraction part 502 is provided in the middle of the flow path to the cell supply part 112 and the patch hole 101, and corresponds to the radius of the cell 104 from the center of the patch hole 101 on the downstream side of the patch hole 101. In the position, a plurality of coughs 501 are installed at an interval narrower than the diameter of the cell 104. By installing such a microchannel structure, the cells 104 can be efficiently supplied to the patch holes 101.

  The microchannel measurement chip 601 in FIG. 6 has a buffer solution supply channel 602 and a chemical solution supply channel 603 installed in the microchannel (cell supply channel 503 and waste solution channel 504) in FIG. It is a schematic diagram of the measurement chip | tip which connected the microchannel which mixes a chemical | medical solution.

  FIG. 7 is a schematic view showing a state of mixing the buffer solution and the chemical solution in the measurement chip 601 of FIG. A chemical solution supply channel 603 with a channel width of about 300 μm is installed in the middle of the buffer solution supply channel 602 and the patch hole 101 with a channel width of about 600 μm, and the buffer solution becomes a sheath flow 702 of the chemical solution. It is the feature that it was made into such a shape. Since the buffer solution and the chemical solution flow through the micro-order flow path, the molecular diffusion is fast and they are completely mixed with each other in the mixing region 701. The concentration of the chemical solution can be changed as appropriate by controlling the flow rate flowing from the chemical solution supply unit 113 to the chemical solution supply channel 603.

  As described above, by installing the microchannel structure as described above, it becomes possible to improve the operability of the cell or drug solution supply system without using a pipettor.

  FIG. 8 is a schematic diagram of a system in which the measurement chip 601 in FIG. 6 is installed in the measurement block 801. By using a plurality of joints 802, the buffer solution supply unit 111 and the buffer solution supply channel 602, the cell supply unit 112 and the cell supply channel 503, the drug solution supply unit 113 and the drug solution supply channel 603, and the waste solution unit 114 are used as a waste solution flow. The path 504 is connected to configure the cell electrophysiology measuring apparatus shown in FIG. 1 so that the measurement can be performed.

  FIG. 9 is a schematic diagram when eight systems 601 in which the measurement chips of FIG. 8 are installed in a measurement block are arranged in a circle. A cell supply unit 901, a chemical solution supply diagram (not shown), and a buffer solution supply unit (not shown) are arranged in the central portion, and eight cells in the radial direction are arranged in parallel with the chip, so that cells, drug solutions, and buffer solutions are arranged. It is made the structure which can supply.

  So far, the embodiment has been described in which measurement is performed without taking time after the cell 104 is arranged on the patch hole 101. Hereinafter, a method will be described in which the cells 104 are sucked into the patch holes 101 of the member and then cultured in a culture container for a certain period of time, and then the member is installed in the apparatus and measurement is performed.

  FIG. 10 is a schematic diagram of a method for measuring after cells are previously attached to the patch holes on the measurement chip of FIG. The measuring chip described in FIG. 1 is removable. FIG. 10A shows a step of sucking one cell 1001 out of the plurality of cells 104 in the buffer solution into the patch hole 101. In FIG. 10B, after removing the measurement chip from the cell electrophysiology measurement apparatus, the measurement chip 201 in a state where the cell 1001 is on the patch hole 101 is placed in the cell culture container 1002 for a certain time (about several hours). In this step, the cells 1001 are sufficiently adhered onto the patch holes 101. FIG. 10C shows a process of measuring a weak current or voltage after placing the measurement chip 201 with the cell 1001 sufficiently adhered on the patch hole 101 on the measurement block 105 and applying a negative pressure 202. It is. By using such a method, since the patch hole 101 and the cell are sufficiently attached, the sealing performance between the patch hole 101 and the cell is further improved, and a highly accurate measurement result can be obtained. .

FIG. 11 is a schematic diagram when the measurement chip of FIG. 10 is multi-channeled and cells are attached to the patch holes in advance. FIG. 12 is a schematic diagram of the AA cross section of FIG. Using the syringe 1202, 16 cells 1102 are simultaneously sucked into each hole (not shown) of the multi-channel measurement chip 1101, and then, after being placed in a culture container for a certain period of time in the same manner as described above, measurement is performed. Do. The cells arranged in each hole may all be the same cell 1102, or may contain cells 1103 in which different ion channels are expressed.
The multi-channel measurement chip 1101 taken out from the culture vessel is installed in the multi-channel measurement block 1201 so that measurement can be performed. The suction pump 1202 applies a negative pressure to the plurality of cells 1102, and then supplies a chemical solution from the chemical solution supply unit 1203 to each measurement unit. Each measurement unit has a reference electrode 1204 and an electrode 1205, and is connected to the amplifier unit 1207 via a switch 1206. By switching the switch 1206 as appropriate, multi-channel measurement can be performed.

The present invention can be applied to an apparatus for screening drugs using electrophysiological activity of cells as an index.

The schematic diagram of the whole electrophysiology measuring device of the cell of the present invention. The schematic diagram which showed the mode of the change of the hole diameter of the measurement chip | tip of FIG. The schematic diagram at the time of making the hole shape of the measurement chip | tip of FIG. 1 into a mortar shape. An example of the manufacturing method of the measurement chip made from polydimethylsiloxane (PDMS) of FIG. The microchannel structure which supplies a cell on a patch hole in the measurement chip | tip of FIG. FIG. 6 is a schematic diagram of a measurement chip in which a microchannel for mixing a buffer solution and a chemical solution is connected to the microchannel in FIG. 5. The schematic diagram which showed the mode of mixing of a buffer solution and a chemical | medical solution in the measuring chip of FIG. The schematic diagram of the system which installed the measurement chip | tip of FIG. 6 in the measurement block. The schematic diagram at the time of arranging the system which installed the measurement chip | tip of FIG. 8 in the measurement block on the circumference of eight pieces. The schematic diagram of the method of measuring, after making a cell adhere beforehand on a patch hole to the measurement chip | tip of FIG. The schematic diagram at the time of making the measurement chip | tip of FIG. 10 multichannel, and making a cell adhere beforehand on a patch hole. The schematic diagram of the AA cross section of FIG.

Explanation of symbols

DESCRIPTION OF SYMBOLS 101: Patch hole 102: Deflection member 103: Reinforcement member 104: Cell 105: Measurement block 106: Buffer liquid 107: Reference electrode 108: Electrode 109: Amplifier unit 110: Suction pump 111: Buffer liquid supply part 112: Cell supply part 113 : Chemical solution supply unit 114: Waste liquid unit 201: Measuring chip 202: Negative pressure 301: Deflection member having a mortar-shaped hole 401: Glass tube 402: Micromanipulator 403: Container 404: Polydimethylsiloxane (PDMS)
405: Patch hole 501: Cough 502: Shrinkage portion 503: Cell supply flow path 504: Waste liquid flow path 601: Measurement chip for micro flow path 602: Buffer liquid supply flow path 603: Chemical liquid supply flow path 701: Buffer liquid and chemical liquid 702: sheath flow 801: microchannel measurement block 802: joint 901: cell supply unit 1001: cell 1002: cell culture vessel 1101: multichannel measurement chip 1102: cell 1103: cell 1201: multichannel measurement Block 1202: Suction pump 1203: Chemical solution supply unit 1204: Reference electrode
1205: Electrode 1206: Switch 1207: Amplifier unit

Claims (9)

A cell comprising a member having a hole smaller than the size of the cell, an electrode arranged so as to sandwich the member, and means for sucking the cell into the hole, and measuring the cell in a state in which the cell is sucked into the hole In the electrophysiological measurement device according to claim 1, wherein a member is used in which the diameter of the hole facing the cell side of the member is smaller than the original diameter when the means for sucking the cell into the hole is driven. Cell electrophysiology measurement device. 2. The cell electrophysiology measurement apparatus according to claim 1, wherein polydimethylsiloxane (PDMS) is used as the member. 3. The cell electrophysiology measurement apparatus according to claim 2, wherein a surface irradiated with plasma is used as a surface for sucking the cells of the member. 4. The cell electrophysiology measurement apparatus according to claim 1, wherein the hole has a mortar shape in which the diameter of the hole facing the cell side is larger than the diameter of the hole on the back surface side. Cell electrophysiology measurement device. The cell electrophysiology measurement device according to any one of claims 1 to 4, further comprising a flow path in which a cell supply unit is installed in an upstream part of the hole of the member and a waste liquid part is installed in a downstream part, and the cell supply unit and the A constricted portion is provided in the middle of the flow path to the hole, and a plurality of coughs are installed on the downstream side of the hole at a position corresponding to the radius of the cell from the center of the hole at intervals smaller than the cell diameter. An electrophysiological measurement device for cells characterized by the above. 6. The cell electrophysiology measurement apparatus according to claim 5, wherein a buffer solution supply unit is installed upstream of the hole of the member, and a chemical solution is provided in the middle of the flow path to the buffer solution supply unit and the hole. A cell electrophysiological measurement device characterized in that a flow path is formed so that a buffer solution becomes a sheath flow of a chemical solution. The cell electrophysiology measurement device according to claim 6, further comprising means for adjusting the amount of the chemical solution from the chemical solution supply unit. A cell comprising a member having a hole smaller than the size of the cell, an electrode arranged so as to sandwich the member, and means for sucking the cell into the hole, and measuring the cell in a state where the cell is sucked into the hole In the method of using the electrophysiological measurement device, after the cells are sucked into the holes of the member, the member is detached from the electrophysiological measurement device and cultured in a culture container for a certain period of time, and then the member is installed in the device. And a method of using a cell electrophysiology measuring apparatus characterized by performing measurement. The method of using the cell electrophysiological measurement device according to claim 8, wherein after sucking a plurality of cells into the plurality of holes of the member, the member is removed from the electrophysiological measurement device and transferred to a culture vessel, A method of using a cell electrophysiology measuring apparatus, comprising: culturing in a culture container for a certain period of time, and then installing the member in the apparatus to measure a plurality of cells.

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