JP2007278960A - Cell electric physiological sensor, and method of measuring cell electric physiological phenomenon using this - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a cell electric physiological sensor capable of accurately measuring an electric physiological phenomenon correspondingly to various cells, and to provide a method of measuring the cell electric physiological phenomenon using this. <P>SOLUTION: The sensor includes a thin plate 2 having a first through-hole 3, a holding plate 4 having a second through-hole 5, and a well 6 having an inner well 8. The thin plate 2 is held inside the second through-hole 5, the well 6 is arranged in the upper part of the second through-hole 5 and butted on the holding plate 4, and the inner well 8 is disposed to be included in the well 6. A third through-hole 7 is formed in a wall surface section, and one opening of the third through-hole 7 is opened toward the second through- hole 5 of the holding plate 4. <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

  The present invention relates to a cell electrophysiological sensor used for measuring a response of a cell to a drug and a method for measuring a cell electrophysiological phenomenon using the same.

  Conventionally, the patch clamp method in electrophysiology is known as a method of measuring ion channels existing in cell membranes using a glass micropipette with a fine tip, and this patch clamp method elucidates various functions of ion channels. It has been. 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 extremely high ability to insert a fine glass micropipette into a single cell with high accuracy in the measurement technique. It's not the right way if you need it.

  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, thereby efficiently holding the cell hole and performing electrical measurement between the upper and lower electrodes Discloses a technique that enables electrophysiological measurement, which is one of cell state quantity measurements (see, for example, Patent Document 1).

On the other hand, a microfluidic chamber suitable for flowing a solution containing a cell agent that can come into contact with cells continuously or intermittently, a living cell held in the microfluidic chamber, and a cell generated in the living cell A technique for measuring the effect of a cell agent flowing in the microfluidic chamber by preparing a means for measuring a mediated extracellular effect is disclosed (for example, see Patent Document 2). In particular, when this technique is used, a cell agent can be supplied to or discharged from the very periphery of the cell, which is effective when it is desired to quickly switch the solution environment around the cell.
Special table 2002-508516 gazette Japanese Patent No. 2993982

  However, in the conventional configuration, in order to quickly exchange the solution environment around the cells, the fluid chamber needs to be a closed space, and the fluidity, diffusion coefficient, concentration, etc. of the solution are adjusted according to each cell. In the case where it is desired to measure the electrophysiological change generated by the cells due to the solution under various conditions with high accuracy, it is difficult to change the size of the fluid chamber according to the size of the cells in the fixed closed space as described above.

  The present invention solves the above-described conventional problems, and is capable of measuring electrophysiological phenomena with high accuracy and efficiency even for various cells, and measurement of cell electrophysiological phenomena using the same. It is intended to provide a method.

  In order to solve the conventional problem, the present invention comprises a thin plate provided with a first through hole, a holding plate provided with a second through hole, and a well containing an inner well. An inner well that can hold a solution or a solution containing cells so as to be contained in the well is placed inside the through hole of the second well, and a well is disposed on the upper portion of the second through hole to contact the holding plate. A third through hole is provided in the wall surface of the inner well, and one opening of the third through hole is configured to open toward the second through hole of the holding plate. is there.

  The cell electrophysiological sensor of the present invention and the method for measuring a cell electrophysiological phenomenon using the same are held in the first through hole of the thin plate while having an open space above the desired inner well such as shape, dimensional accuracy, and material. A cell electrophysiological sensor capable of realizing highly accurate and efficient measurement by realizing a configuration capable of reliably supplying a drug to the vicinity of the various cells, and a It is possible to provide a method for measuring the cellular electrophysiological phenomenon used.

(Embodiment 1)
Hereinafter, a cell electrophysiological sensor according to Embodiment 1 of the present invention and a method for measuring a cell electrophysiological phenomenon using the same will be described with reference to the drawings.

  FIG. 1 is a perspective view of a cell electrophysiological sensor of the present invention, and FIG. 2 is a cross-sectional view taken along a line AA in FIG.

  1 and 2, reference numeral 1 denotes a cell electrophysiological sensor, which includes a thin plate 2 made of silicon, a holding plate 4 made of an insulating material such as a resin, and a well 6 made of a material excellent in moldability such as a resin. The thin plate 2 is provided with at least one first through hole 3 having a diameter of 1 to 3 μm. The thin plate 2 is fixed inside the second through hole 5 of the holding plate 4.

  Furthermore, although the well 6 is in contact with the upper part of the holding plate 4, either the well 6 or the holding plate 4 is transparent to light in a specific wavelength region, and one of them is impermeable. preferable. As a result, the well 6 and the holding plate 4 can easily form a strong adhesive surface by irradiating a laser beam in a specific wavelength region from a transparent surface and thermally welding the joint surface.

  Further, an opening is provided in the well 6, and an inner well 8 is inserted and held along the inner wall surface of the opening. Further, the inner wall 8 of the inner well 8 has an inner surface extending from the upper surface to the lower surface. A third through hole 7 is provided toward the end, and one end communicates with a groove 10 provided in the lower portion of the well 6. In this way, by bringing the well 6 containing the inner well 8 into close contact with the holding plate 4, one opening of the third through hole 7 formed in the wall surface portion 9 of the inner well 8 is the second of the holding plate 4. The shape is open toward the second through hole 5 or the first through hole 3. The inner well 8 can be held in the opening of the well 6 by a bonding method such as press-fitting, an adhesive, or melt bonding. Further, the inner well 8 can be removed from the well 6.

  With the configuration as described above, the cell electrophysiological sensor 1 according to the first embodiment can easily inject a solution containing the cells 23 from the inner well 8 as described later. Furthermore, since another solution can be poured from the third through-hole 7, the solution inside the inner well 8 can be efficiently exchanged with another solution.

  In addition, since the shape of the inner well 8 is very small, it is easy to design and it is necessary to mold it with high dimensional accuracy. Therefore, only the inner well 8 is individually made of a resin material having excellent dimensional moldability. It is possible to mold other molded bodies (for example, well 6) with normal accuracy, and this can increase productivity.

  Next, a method for measuring a cell electrophysiological phenomenon using the cell electrophysiological sensor of Embodiment 1 will be described.

  3 to 7 are cross-sectional views for explaining the procedure for measuring the electrophysiological phenomenon of cells in the first embodiment. 8 to 10 are sectional views for explaining another example of the cell electrophysiological sensor, and FIGS. 11 to 13 are sectional views for explaining another example of the cell electrophysiological sensor.

  First, as shown in FIG. 3, when the first solution 24 is introduced from the upper part of the inner well 8 included in the well 6, as shown in FIG. 4, the first through hole 3 and the lower region of the holding plate 4 are placed. The first solution 24 penetrates, and the first solution 24 contacts the upper electrode 27 and the lower electrode 28 in each region. Since the first solution 24 is an electrolytic solution containing NaCl, a conduction resistance of about 1 to 10 MΩ is usually generated between the upper electrode 27 and the lower electrode 28.

  Note that a solution different from the first solution 24 (for example, a third solution 26 such as an electrolytic solution containing Kcl) may be placed in the lower region of the holding plate 4 in advance.

  On the other hand, the first solution 24 introduced from the inner well 8 slightly enters the side of the third through hole 7 provided in the inner well 8, but there is no particular problem.

  However, if bubbles remain in the vicinity of the first through-hole 3, the above-described conduction resistance may not be ensured. In particular, if air remains in the third through-hole 7, When another solution (for example, the second solution 25) is introduced from the third through hole 7, bubbles are generated in the vicinity of the first through hole 3. In order to prevent this, as shown in FIG. 7, when the first solution 24 is introduced from the inner well 8, the first bubble 24 is sucked from the third through-hole 7, so that the bubbles are first removed. Since the first solution 24 fills the inside of the third through hole 7 in a sufficient amount, another solution is poured into the third through hole 7 later. Even so, the generation of bubbles can be prevented.

  Next, as shown in FIG. 5, the first solution 24 containing the cells 23 is put into the inner well 8, and then the lower portion of the holding plate 4 is decompressed as shown in FIG. And the cell 23 is drawn into the first through-hole 3. Here, the first solution 24 passes through the first through-hole 3, but the cell 23 cannot pass through the first through-hole 3 and is held near the opening of the first through-hole 3. . And if this cell 23 is hold | maintained so that the 1st through-hole 3 may be plugged with moderate adhesion, the 1st solution 24 will be electrically divided on the upper and lower sides of the holding | maintenance plate 4, and the upper electrode 27 and lower electrode Between 28, the resistance value is sufficiently high, for example, 100 MΩ, preferably 500 MΩ or more.

  Next, when a fixed voltage of, for example, about 10 mV is applied between the upper electrode 27 and the lower electrode 28, the potential applied to the membrane of the cell 23 (membrane potential) is fixed, and the current value at this time is measured, whereby the cell It is possible to measure the current flowing through the ion channel existing in the 23.

  The detailed procedure for measuring the current flowing through the ion channel is disclosed in detail in the conventional method such as the patch clamp method, and the description thereof is omitted here.

  Further, as another example of the configuration of the cell electrophysiological sensor, it can be introduced from the third through-hole 7 as a portion to which a solution containing cells is introduced as shown in FIG. Thereby, the cell 23 can be reliably introduced into the vicinity of the first through hole 3. Thereafter, when the lower portion of the holding plate 4 is decompressed as shown in FIG. 9, the first solution 24 and the cells 23 are drawn into the first through-hole 3, and the cells 23 can pass through the first through-hole 3. It cannot be held and is held near the opening of the first through hole 3. With this configuration, even with a small amount of cells 23, the cells 23 can be arranged and held so as to reliably block the first through-hole 3.

  Furthermore, as another example, the second solution 25 is introduced from the third through-hole 7 as shown in FIG. Here, since one opening part of the 3rd through-hole 7 is opening toward the 1st through-hole 3, the 1st solution 24 of the 1st through-hole 3 vicinity is a 2nd solution quickly. 25 can be exchanged.

  At this time, it is more preferable to form a plurality of third through holes 7. Accordingly, the second solution 25 is introduced from one third through-hole 7 (in the direction of the arrow), and suction (in the direction of the arrow) is performed from the other third through-hole 7, whereby the first solution Rapid solution exchange from 24 to the second solution 25 is possible. With this configuration, when the exchange of the solution is quickly performed in the upper region of the holding plate 4 while measuring the current flowing through the ion channel, the second solution 25 is not included in the first solution 24. By measuring the change in the ion channel current due to the drug component (for example, the channel inhibitor for a specific ion channel) contained in the pharmacological effect, the pharmacological effect can be determined quickly and accurately.

  Furthermore, as another example of the cellular electrophysiological sensor in the first embodiment, as shown in FIG. 11, the shape of the opening 13 below the inner well 8 is made smaller than the outer diameter of the thin plate 2, thereby The liquid (for example, the second solution 25) introduced from the through-hole 7 can promptly exchange the first solution 24 in the vicinity of the first through-hole 3 of the thin plate 2 more reliably.

  As still another example, when the thin plate 2 is held below the second through hole 5 of the holding plate 4 as shown in FIG. 12, the second through hole 5 is formed in the opening 13 below the inner well 8. By providing the projecting portion 11 projecting inwardly and downwardly, the solution in the vicinity of the first through-hole 3 can be quickly replaced more reliably.

  As still another example, as shown in FIG. 13, a frame body 12 is joined to the outer peripheral portion of the thin plate 2, and the frame body 12 is installed inside the second through-hole 5 and below the inner well 8. By providing the projecting portion 11 projecting inward and downward of the frame body 12 in the opening 13, the solution in the vicinity of the first through hole 3 can be more quickly exchanged more reliably.

  When the inner well 8 is enclosed in the well 6 as in the first embodiment, the formation of the third through hole 7 can be easily changed to various configurations. That is, in all the examples shown in FIGS. 10 to 13, the shape of the well 6 is the same, and only the inner well 8 may be changed and installed.

  14 is a perspective view of the inner well 8 of this example. FIG. 15 is a perspective view of the well plate by installing the inner well 8 inside the well 6, as shown in FIG. The inner wells 8 having different shapes can be relatively easily aligned.

  Further, if an inner well 8 having a different shape is provided for a plurality of wells 6, a well array having a plurality of shapes can be easily manufactured.

  As described above, the cell electrophysiological sensor and the cell electrophysiological measurement method using the same according to the present invention can measure changes in the electrophysiological phenomenon of cells with high accuracy and efficiency. Used as a measuring instrument for chemical screening.

The perspective view of the cell electrophysiological sensor in Embodiment 1 of this invention Sectional drawing in the AA part of the same FIG. Sectional drawing for demonstrating the measuring method of the electrophysiological phenomenon of a cell using the sensor Cross section Cross section Cross section Cross section Sectional view of another example of cellular electrophysiological sensor Cross section Cross section Cross-sectional view of another example of cell electrophysiological sensor Cross-sectional view of another example of cell electrophysiological sensor Cross-sectional view of another example of cell electrophysiological sensor Perspective view of the inner well Perspective view of the well plate

Explanation of symbols

DESCRIPTION OF SYMBOLS 1 Cell electrophysiological sensor 2 Thin plate 3 1st through-hole 4 Holding plate 5 2nd through-hole 6 Well 7 3rd through-hole 8 Inner well 9 Wall surface part 10 Groove 11 Projection part 12 Frame body 13 Opening part 23 Cell 24 First solution 25 Second solution 26 Third solution 27 Upper electrode 28 Lower electrode

Claims (7)

A cell electrophysiological sensor comprising a thin plate provided with at least one first through-hole, a holding plate provided with a second through-hole, and a well containing an inner well, wherein the thin plate is attached to the second through-hole. An inner well that can store a solution or a solution containing cells so as to be contained inside the well is disposed in contact with the holding plate by placing a well above the second through hole. A cell electrophysiological sensor in which a third through hole is provided in the wall surface of the inner well, and one opening of the third through hole opens toward the second through hole of the holding plate. The cell electrophysiological sensor according to claim 1, wherein the shape of the opening on the holding plate side of the wall surface of the inner well is smaller than the outer diameter of the thin plate. The cell electrophysiological sensor according to claim 1, wherein a projecting portion projecting into the inside of the second through hole is provided in an opening portion on the holding plate side of the wall surface portion of the inner well. A frame body portion is provided on the outer peripheral portion of the thin plate, the frame body portion is held in the second opening of the holding plate so that the frame body portion is located on the inner well side, and the protruding portion of the inner well is disposed on the frame body portion. The cell electrophysiological sensor according to claim 3, which is inserted into a cavity constituted by A thin plate provided with at least one first through hole, a holding plate provided with a second through hole, and a well containing an inner well. The thin plate is held inside the second through hole. A well is disposed above the second through-hole and is in contact with the holding plate, and an inner well is provided that can accumulate a solution or a solution containing cells so as to be contained inside the well. A step of preparing a cell electrophysiological sensor in which one opening of the third through-hole opens toward the second through-hole of the holding plate; and inside the inner well A method for measuring a cell electrophysiological phenomenon, comprising at least a step of accumulating a first solution and a step of introducing a second solution into the inside of a third through hole. The method for measuring a cell electrophysiological phenomenon according to claim 5, wherein the first solution inside the inner well is replaced with a second solution. The method for measuring a cell electrophysiological phenomenon according to claim 6, wherein cells are contained in the second solution.

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