JP2007174990A - Cellular electrophysiological sensor array and method for producing the same - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a cellular electrophysiological sensor array capable of carrying out the measurement collectively by achieving an efficient structure of the cellular electrophysiological sensor in which the cellular electrophysiological sensors are arranged in an array shape; and to provide a method for producing the electrophysiological sensor array. <P>SOLUTION: The cellular electrophysiological sensor array is constituted so that the first through holes 6a and 6b are connected in the inside of the same groove 14 by connecting a well plate 1, a chip plate 2 having the first through holes 6a and 6b and electrodes 9 and 10, and a groove plate 3 having the groove 14, and a cellular electrophysiological sensor 4 have a structure incorporated in the first through holes 6a and 6b. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

  The present invention relates to a cell electrophysiological sensor array for measuring a cell electrophysiological phenomenon such as an intracellular potential or an extracellular potential used for measuring a physicochemical change generated by a cell activity, and a method for producing the same. It is.

  Conventionally, the patch clamp method in electrophysiology is known as a method for measuring the ion channel function existing in the cell membrane, and various functions of the ion channel have been elucidated by this patch clamp method. And the action of ion channels is an important concern in cytology, which has also been applied to drug development.

  However, on the other hand, the patch clamp method requires an extremely high ability to insert a fine micropipette into a single cell with high precision in the measurement technique, so it requires skilled workers and requires high throughput. Is not an appropriate method.

  For this reason, flat-type probes utilizing microfabrication techniques have been developed, which are suitable for automated systems that do not require the insertion of micropipettes for individual cells.

  For example, there is a hole in the carrier that separates the two regions, and an electric field is generated by the electrodes placed above and below this carrier to efficiently hold the cells in the hole, and electrical measurement is performed between the upper and lower electrodes Can be used for electrophysiological measurement of cells (see Patent Document 1), or a plate-like device is provided with a plurality of through-holes, and includes a continuous layer of cells adhered thereto. A technique for measuring is disclosed (see Patent Document 2).

  Also, a technique for measuring the electrophysiological phenomenon of cells by attracting cells to the microelements by reducing the pressure of the microelements provided inside the cavities provided on the substrate and the flow paths connected to the microelements (Patent Literature) 3), a technique for measuring a cell's electrophysiological phenomenon by providing a hole in a flat portion of the substrate and integrating the reference electrode and the measurement electrode on the substrate (see Patent Document 4), one channel penetrates A method of measuring cells contained in a liquid by locating the surface by suction from the lower surface of the cell and then rupturing part of the lower surface of the cell by increasing the pressure difference (see Patent Document 5) ).

  In addition, an electrode is arranged in the first and second cavities, respectively, so that the object seals the orifice in use, thereby forming electrically insulated first and second cavities. An apparatus (see Patent Document 6) that performs electrical measurement of an object in a medium by changing the impedance between them is disclosed.

  Further, the extracellular potential is measured by an extracellular potential measuring device including a well provided with a cell holding means provided on a substrate, a measurement electrode for detecting an electric signal of the well, and a reference electrode. The technique (refer patent document 7) is disclosed.

A technique for measuring extracellular potential with high accuracy by maintaining HEK293 cells, a type of human cultured cell line, in a 2.5 μm hole provided inside the SiO ₂ membrane to ensure high adhesion (non-patented) Reference 1) discloses that the through hole made in the flat plate plays the same role as the tip hole in the glass pipette, and can record the electrophysiological phenomenon of the cell with high accuracy and from the back side of the flat plate. The cell is automatically attracted by a method such as suction, and has an advantage that the cell can be easily held.

Also disclosed is a device for detecting a change in the state of an object by sealing the object in an orifice and measuring the electrode impedance between the galvanically isolated cavities thereby, the liquid in which the object is placed Also disclosed is a liquid supply means having a liquid supply capillary and a liquid suction capillary in order to quickly remove and replace the environment (see Patent Document 8).
Special table 2002-508516 gazette JP 2002-518678 Gazette US Pat. No. 6,315,940 Special table 2003-511668 gazette Japanese translation of PCT publication No. 2003-511699 Japanese translation of PCT publication No. 2003-527581 International Publication No. 02/055653 Pamphlet Japanese translation of PCT publication No. 2003-527581 T.A. Sordel et al, Micro Total Analysis Systems 2004, P521-522 (2004)

  However, the main purpose of the conventional cell electrophysiological sensor is to easily measure the electrophysiological phenomenon of the cell without using the glass fine probe used in the conventional patch clamp method. The measurement procedure can be simplified by this, but there is almost no description regarding the structure that can perform many measurement processes at once, and an efficient structure is not known.

  The present invention solves the above-described conventional problems, and realizes an efficient cellular electrophysiological sensor structure in which cellular electrophysiological sensors are arranged in an array, and a cell electrophysiological sensor array that can be collectively measured and its A manufacturing method is realized.

  In order to solve the above-described conventional problems, the present invention provides a well plate having a plurality of wells, a chip plate having a plurality of first through holes and two types of electrodes, and a groove having a plurality of grooves. A plurality of first through holes are connected to each other in the same groove, and the second through hole is formed inside. A cell electrophysiological sensor having a thin plate is embedded in the first through hole.

  The cell electrophysiological sensor array and the manufacturing method thereof according to the present invention enable efficient measurement all at once in use and realize a manufacturing method excellent in mass productivity.

(Embodiment 1)
Hereinafter, the cell electrophysiological sensor array and the manufacturing method thereof according to Embodiment 1 of the present invention will be described with reference to the drawings.

  1 is a cross-sectional view for explaining the structure of a cell electrophysiological sensor array according to Embodiment 1 of the present invention, FIG. 2 is an enlarged cross-sectional view of a portion A in FIG. 1, and FIGS. 3 and 4 are in FIG. It is an expanded sectional view of the B section.

  1 to 4, the cell electrophysiological sensor array according to Embodiment 1 of the present invention includes a well plate 1 having a plurality of wells 5a, 5b, 5c, 5d, and 5e, and a plurality of first through holes 6a, 6b, a plurality of inflow / outflow holes 7a, 7b and one electrode take-out hole 8 are arranged on a straight line at a predetermined pitch, and the first electrode 9 and the second electrode 10 are arranged on one side thereof. A chip plate 2 provided on the side and a groove plate 3 having a linear groove 14, and a cell electrophysiological sensor 4 is embedded in the first through holes 6 a and 6 b, and one end of the second electrode 10 is formed. Is embedded in the electrode extraction hole 8. The first through holes 6a and 6b, the inflow / outflow holes 7a and 7b, and the electrode extraction hole 8 are disposed inside the openings on the lower surfaces of the wells 5c, 5d, 5a, 5e, and 5b, respectively. Further, the first through holes 6a and 6b, the outflow / inflow holes 7a and 7b, and the lower surface of the electrode lead-out hole 8 are arranged in a straight line. The well plate 1, the chip plate 2 and the groove plate 3 are joined to each other, whereby the first through holes 6 a and 6 b are connected by the groove 14 of the groove plate 3. That is, the lower surface of the cell electrophysiological sensor 4 is partitioned from the outside as a region constituted by the grooves 14.

  Here, the first electrode 9 and the second electrode 10 are preferably composed of electrodes including at least one selected from electrode materials consisting of chromium, titanium, copper, gold, platinum, silver and silver chloride. Further, the two types of electrodes 9 and 10 are preferably formed on the same surface of the chip plate 2.

  In particular, it has been confirmed that copper adheres to the thermoplastic resin described later with good adhesion, and silver and silver chloride can reduce electrical contact resistance with the electrolyte solution described later.

  Preferably, an electrode paste 11, which is a conductive adhesive in which silver and silver chloride are mixed in a fine particle state, is embedded in the electrode extraction hole 8 so as to be connected to one end of the second electrode 10. As a result, the electrode extraction hole 8 can be reliably closed, and the groove 14 is blocked from the outside.

  By adopting such a configuration, it is possible to suppress the generation of bubbles in the intracellular liquid 15 flowing through the groove 14 by completely closing the electrode extraction hole 8, and through the intracellular liquid 15 and the electrode paste 11. Since it can be electrically connected to the second electrode 10, forming the electrodes 9 and 10 on one surface facilitates contact with a probe and the like and realizes a structure with excellent productivity.

  Further, as shown in FIG. 2, since the electrode paste 11 which is a conductive adhesive is exposed on the groove 14 side, the potential state of the electrolytic solution accumulated in the groove 14 can be measured.

  Preferably, the two types of electrodes, that is, the first electrode 9 and the second electrode 10 are formed only on one surface of the chip plate 2 as in the first embodiment. As a result, all the lead-out patterns of the electrodes are embedded between the well plate 1 and the chip plate 2, which is effective for protecting the electrodes. Furthermore, since these electrodes are formed on the same surface, there is an advantage that connection to an external device becomes easy.

  The well plate 1, the chip plate 2, and the groove plate 3 are all formed of a thermoplastic resin. And as these thermoplastic resins, it is preferable to use any one of polycarbonate resin, polyethylene resin, olefin polymer, polymethyl methacrylate resin, or a combination thereof. Furthermore, it is more preferable to include at least one resin selected from a material consisting of a cyclic olefin polymer, a linear olefin polymer, a cyclic olefin copolymer copolymerized with these, and polyethylene.

  Further, as shown in FIG. 4, a second through hole 12 is formed in the thin plate 13 of the cell electrophysiological sensor 4, and the second through hole 12 is extremely small and has a diameter of 5 μm or less. Yes.

  Next, a method for measuring the electrophysiological phenomenon of a cell using the cell electrophysiological sensor array having the configuration as described above will be described.

  5 to 7 are cross-sectional views showing examples of use of the cell electrophysiological sensor array of the present invention, and FIG. 8 is an enlarged cross-sectional view of a main part of FIG.

  First, as shown in FIG. 5, the intracellular fluid 15 is introduced from the outflow / inflow hole 7a, and the inside of the groove 14 is filled with the intracellular fluid 15 until the intracellular liquid 15 reaches the outflow / inflow hole 7b. Here, in the case of mammalian muscle cells, the intracellular fluid 15 is typically an electrolytic solution to which K + ions are added at about 155 mM, Na + ions at about 12 mM, and Cl- ions are added at about 4.2 mM. The external solution 16 is an electrolytic solution to which about 4 mM of K + ions, about 145 mM of Na + ions, and about 123 mM of Cl − ions are added.

  Next, when the electrical resistance between the first electrode 9 and the second electrode 10 is measured in a state where the extracellular fluid 16 is introduced into the wells 5c and 5d as shown in FIG. 6, it is about 100 kΩ to 10 MΩ. The resistance value can be observed.

  This is because the intracellular fluid 15 or the extracellular fluid 16 which is an electrolytic solution penetrates into the second through-hole 12 provided in the thin plate 13 of the cell electrophysiological sensor 4, and the extracellular fluid 16 is inside the wells 5 c and 5 d. This is because the intracellular fluid 15 is in contact with the electrode paste 11 that is a conductive adhesive in the electrode extraction hole 8 inside the groove 14 to form an electric circuit.

  Next, as shown in FIG. 7, when the cells 17 are introduced into the wells 5 c and 5 d and suctioned from the outflow / inflow holes 7 a or 7 b, the cells 17 in the wells 5 c and 5 d together with the extracellular fluid 16 It is attracted to the second through hole 12.

  Then, as shown in FIG. 8, when the cells 17 are in close contact with each other so as to completely block the second through-hole 12, the electrical resistance between the extracellular fluid 16 and the intracellular fluid 15 increases, and is usually 100 MΩ or more, preferably 1 GΩ or more. The resistance value becomes. As described above, in a state where the electrical resistance is increased, even if the number of ions flowing into or out of the cell 17 is small, the movement of ions flowing inside and outside the cell 17 can be measured with high accuracy. . This is the measurement of the ion channel, which is an electrophysiological phenomenon of the cell 17.

  Since the cell electrophysiological sensor array according to the first embodiment includes a plurality of cell electrophysiological sensors 4 that can measure the ion channel, efficient measurement can be performed simultaneously.

  In the description of the first embodiment, only two cell electrophysiological sensors 4 have been described. However, more cell electrophysiological sensors 4 may be provided, preferably 96 in 12 × 8 rows, or 24 × 16 rows of 384 or 48 × 32 rows of 1536 wells 5 are provided, and the cell electrophysiological sensor 4 is preferably disposed therein. FIGS. 9, 10 and 11 are an exploded perspective view, a top view and a cross-sectional view showing an example of a cell electrophysiological sensor array using a well plate 1 in which 384 wells 5 in 24 × 16 rows are arranged. In FIG. 9, the wells 5 are arranged in 384 arrays of 24 × 16 rows, and among these, the cell electrophysiological sensor 4 has column numbers E, F, G, H, I, J as shown in FIG. , K, and L, row number 3 to 22 and a total of 160 wells 5 are arranged, row number 1 and row number 24 well 5 are connected to inflow / outflow holes 7a and 7b, row number 2 or row number 23 The well 5 is connected to the electrode extraction hole 8.

  Thus, by arranging the cell electrophysiological sensors 4 in a lattice shape, the grooves 14 through which the intracellular fluid 15 flows can be arranged efficiently in a straight line. Furthermore, since the array of the wells 5 is an array generally used in many other related apparatuses (for example, an assay process for producing a plurality of types of drugs, a dispensing process for separating cells, etc.), the versatility that can be used by a dispensing robot is also provided. Have. This is an important factor for efficiently cooperating with related processes for screening drugs.

  As described above, with the configuration shown in the first embodiment, a plurality of cellular electrophysiological sensors 4, wells 5 that can administer and accumulate extracellular fluid 16, stimulating drugs, and cells 17, and intracellular fluid 15 flow in. The groove 14 to be formed, the first electrode 9 placed in the extracellular fluid 16, the electrode paste 11 and the second electrode 10 connected to the intracellular fluid 15 are integrated, and all on the same surface. By configuring the above, it becomes possible to perform a probe for measurement on the upper surface, thereby realizing a cell electrophysiological sensor array capable of performing efficient measurement all at once.

  In this manner, by forming the second electrode 10 on the same surface of the chip plate 2 and forming the electrode pattern on one surface together, a highly productive cell electrophysiological sensor array can be realized.

  The cell electrophysiological sensor 4 is made of an inorganic material such as silicon or glass. Usually, an expensive device is required to accurately process the thin plate 13 and the second through hole 12 in these inorganic materials. The cell electrophysiology sensor 4 is expensive if manufactured with this structure, but the cell electrophysiology that requires high-precision processing in the chip plate 2 formed of an inexpensive thermoplastic resin as in the present invention. By embedding the sensor 4 in the first through holes 6a and 6b, even if a plurality of cell electrophysiological sensors 4 are configured, an inexpensive cell electrophysiological sensor array can be provided.

  Further, by selecting a resin material as shown in the present invention, the well plate 1, the chip plate 2 and the groove plate 3 may be formed by heat welding or solvent welding which can be firmly bonded to each other without using an adhesive. It has the manufacturing advantage of being able to.

  Next, a method for producing the cell electrophysiological sensor array of the present invention will be described with reference to the drawings.

  12-15 is sectional drawing which shows a mode that the cell electrophysiological sensor array in Embodiment 1 of this invention is manufactured.

  First, as shown in FIG. 12, the well plate 1 is formed so that the well 5 is configured in a predetermined arrangement. Usually, in this process, it is efficient to use injection molding when a resin material is used, and a desired shape is obtained by injecting the thermoplastic resin while heating.

  On the other hand, as shown in FIG. 13, the two liquid inflow / outflow holes 7a and 7b, the two first through holes 6a and 6b, and the electrode take-out hole 8 are arranged in a straight line, and on the same surface. The chip plate 2 on which the first electrode 9 and the second electrode 10 are formed is produced. In addition, since the formation method of these electrodes 9 and 10 and the formation method of 1st through-hole 6a, 6b are not the structural requirements of this invention, detailed description is abbreviate | omitted.

  Here, the chip plate 2 is preferably made of the same thermoplastic resin material as the well plate 1, and more effectively, polycarbonate resin (PC), polyethylene resin (PE), olefin polymer, polymethyl methacrylate acetate resin ( PMMA), or a combination thereof. These resin materials have high solid solubility between materials, and can be easily bonded to each other. Further, as will be described later, it is easy to impart transparency to the material.

  With these configurations, the chip plate 2 can be easily attached to the well plate 1 in the next step as shown in FIG. At this time, as a method of attaching, it is possible to easily fix the bonding surface of the resin material by irradiating the bonding surface of the resin material with a laser beam. And in order to implement | achieve this method, at least one resin material has transparency with respect to a laser beam, and also two resin materials must be the same, or should be solid-solved mutually. Is desirable. It is preferably selected from a cyclic olefin polymer, a linear olefin polymer, a cyclic olefin copolymer obtained by polymerizing them, or a resin material made of polyethylene. In this case, these resin materials not only have high adhesion when bonded to each other, but especially in the case of cyclic olefin copolymers, the heat resistance is improved, and the chemical resistance to acids and alkalis is also increased. Has advantages.

  At this time, in the first embodiment, the first electrode 9 and the second electrode 10 are formed on one surface of the chip plate 2. The electrodes 9 and 10 are embedded between the well plate 1 and the chip plate 2, and the surfaces of these electrodes 9 and 10 can be protected without touching the liquid except where necessary.

  Next, as shown in FIG. 15, an electrode paste 11 made of silver / silver chloride is embedded in the electrode extraction hole 8 and connected to one end of the second electrode 10.

  Next, as shown in FIG. 16, the cell electrophysiological sensor 4 is embedded in the first through holes 6a and 6b, and the periphery is sealed with an adhesive 18 (see FIG. 8).

  Next, the cell electrophysiological sensor array shown in FIG. 18 is obtained by bonding the groove plate 3 formed with the grooves 14 as shown in FIG. 17 by the same means as the method of bonding the well plate 1 and the chip plate 2 together. . Therefore, it is desirable that the material of the groove plate 3 is the same material as the chip plate 2 or a resin material that is solid-dissolved therewith. Incidentally, the method of forming the groove 14 in the groove plate 3 can be produced by injection molding in the same manner as the well plate 1.

  In addition to the method described above, the well plate 1, the chip plate 2 and the groove plate 3 can be bonded together by applying a nonpolar organic solvent to the bonding surface.

  For example, when the resin material is a cyclic olefin copolymer, the resin material can be dissolved and fixed by applying a solvent typically containing toluene as a nonpolar organic solvent. In this case, heat is not generated on the bonding surface, which is effective when it is necessary to embed an electronic circuit such as a heat-sensitive electrode in the bonding surface.

  As described above, the cell electrophysiological sensor array in which the plurality of cell electrophysiological sensors 4 are efficiently arranged can be produced by the manufacturing method as described above.

  As described above, the cell electrophysiological sensor array and the method for producing the same according to the present invention can be used in a measuring instrument for drug screening and the like that can perform pharmacological determination at high speed because it can simultaneously perform measurement processing of a plurality of cell electric characteristics. It is done.

Sectional view of the cell electrophysiological sensor array of the present invention Enlarged sectional view of the main part Enlarged sectional view of the main part Enlarged sectional view of the main part Sectional drawing explaining the usage method of the cell sensor array of this invention Cross section Cross section Enlarged sectional view of the main part The exploded perspective view which shows an example of the cell electrophysiological sensor array of this invention Top view Cross section Sectional drawing for demonstrating the manufacturing method of the cell sensor array of this invention Cross section Cross section Cross section Cross section Cross section Cross section

Explanation of symbols

DESCRIPTION OF SYMBOLS 1 Well plate 2 Chip plate 3 Groove plate 4 Cell electrophysiological sensor 5, 5a, 5b, 5c, 5d, 5e Well 6a, 6b First through-hole 7a, 7b Outflow / inflow hole 8 Electrode extraction hole 9 First electrode 10 Second electrode 11 Electrode paste 12 Second through-hole 13 Thin plate 14 Groove 15 Intracellular fluid 16 Extracellular fluid 17 Cell 18 Adhesive

Claims (12)

A well plate in which wells having wall surfaces made of one curved surface having two openings at least at both ends are arranged and arranged;
At least two outflow / inflow holes are provided on the lower surface of the opening of the well, and a plurality of first through holes and at least one electrode extraction hole are regularly arranged between the at least two outflow / inflow holes, and at least A chip plate with two types of electrodes on one side;
A groove plate having at least one linear groove on the bottom surface of the outflow / inflow hole, the first through hole and the electrode extraction hole of the chip plate;
A cell electrophysiology in which an outflow / inflow hole, a first through hole, and an electrode extraction hole are connected by the groove by joining the chip plate and the groove plate, and a thin plate having at least one second through hole is provided. A cell electrophysiological sensor array in which a sensor chip is embedded in the first through hole. The cell electrophysiological sensor array according to claim 1, wherein the well plate, the chip plate, and the groove plate are made of a thermoplastic resin. The cell electrophysiological sensor array according to claim 2, wherein the thermoplastic resin is a thermoplastic resin containing any one of polycarbonate resin, polyethylene resin, olefin polymer resin, and polymethyl methacrylate resin. The cell electrophysiological sensor array according to claim 2, wherein the thermoplastic resin is a thermoplastic resin containing any one of polycarbonate resin, polyethylene resin, olefin polymer resin, and polymethyl methacrylate resin. 2. The cell according to claim 1, wherein the array of wells arranged in the well plate is a grid array of any one of 96 × 12 × 8 format, 384 × 24 × 16 format, and 1536 × 48 × 32 format. Electrophysiological sensor array. The cell electrophysiological sensor array according to claim 1, wherein the two types of electrodes arranged on the chip plate are at least one selected from an electrode material group consisting of chromium, titanium, copper, gold, platinum, silver and silver chloride. . The cell electrophysiological sensor array according to claim 1, wherein two types of electrodes are arranged on the same surface of the chip plate. 8. The cell electrophysiological sensor array according to claim 7, wherein one of the two types of electrodes is filled with a conductive adhesive inside the electrode take-out hole to seal the electrode take-out hole. The cell electrophysiological sensor array according to claim 8, wherein the conductive adhesive is an electrode in which fine particles of silver and silver chloride are mixed. Preparing a well plate made of a thermoplastic resin in which wells provided with wall surfaces made of one curved surface having two openings at both ends are arranged at a constant interval;
At least two liquid outflow / inflow holes are formed on the lower surface of the well opening, and a plurality of first through holes and at least one electrode extraction hole are regularly arranged between the at least two outflow / inflow holes. And a step of preparing a chip plate made of a thermoplastic resin having at least one surface with two kinds of electrodes;
Preparing a groove plate made of a thermoplastic resin having at least one linear groove on the lower surface of the outflow / inflow hole, the first through hole and the electrode extraction hole of the chip plate;
Bonding the well plate and the chip plate by thermal welding or solvent welding;
A step of sealing a cell electrophysiological sensor provided with a thin plate having a second through-hole inside the first through-hole so that there is no gap between the cell electrophysiological sensor and the chip plate When,
A method of manufacturing a cell electrophysiological sensor array comprising a step of bonding the chip plate and the groove plate by heat welding or solvent welding so that the first through holes are connected by the same groove. The method for producing a cell electrophysiological sensor array according to claim 10, wherein thermal welding is performed by irradiating the bonding surface with a laser beam to perform thermal welding. The method for producing a cell electrophysiological sensor array according to claim 10, wherein solvent welding is applied to a bonding surface with a nonpolar organic solvent to perform solvent welding.

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