JP2007333571A - Cell electrophysiological sensor and method for measuring cell electrophysiological phenomenon using same - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To solve a problem that accurate decompression control is obstructed, if an air bubble exists, although a means sucking from a lower side of a flat plate is employed for making a cell retained and adhered closely to a through hole of the flat plate. <P>SOLUTION: The cell electrophysiological sensor includes a well 1 including a first through hole 5, a retention plate 2 including a second through hole 6 abutting on a lower part of the well 1, and a flowin port 9 and a flowout port 10 for liquid below the retention plate 2 on both ends, has a channel plate 3 including a first groove 11 abut thereon and has a thin plate 4 including a third through hole 7 abut on an inside of the second through hole 6. The cell electrophysiological sensor communicates to the second through hole 6 and includes a first projection part 8 on a part of the retention plate 2 between the second through hole 6 and the flowout port 10. <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

  The present invention relates to a cell electrophysiological sensor 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 cell electricity using the same. The present invention relates to a method for measuring physiological phenomena.

  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, the patch clamp method, on the other hand, requires an extremely high ability to insert a fine micropipette into a single cell with high accuracy in the measurement technique, which 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 Discloses a method for performing electrophysiological measurement of cells (see, for example, Patent Document 1).

  In addition, after sucking and positioning from the lower surface of the cell to the surface through which one channel penetrates, the pressure difference is increased to rupture a part of the lower surface of the cell, thereby measuring the cells contained in the liquid. The method of performing is disclosed (for example, refer to Patent Document 2).

As disclosed in these, 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 suction from the back side of the flat plate, etc. This method has the advantage that the cells are automatically attracted and can be easily retained.
Special table 2002-508516 gazette Japanese translation of PCT publication No. 2003-511699

  The main purpose of the cell electrophysiological sensor in the conventional configuration is to easily measure the electrophysiological phenomenon of cells without using a fine probe used in the conventional patch clamp method. It is necessary to keep the cells in close contact with the through holes (holes) formed in the part.

  However, in order to suck the lower part of the flat plate, the liquid needs to be confined in a sealed space under the flat plate. If air bubbles exist in the sealed space, the lower space is decompressed by suction. Even if the bubbles expand, high-precision decompression control may be hindered.

  The present invention solves the above-mentioned conventional problems, and reduces the amount of bubbles remaining in the sealed space when decompressing the sealed space, and the cell electrophysiological sensor capable of controlling the decompression with high accuracy and the cell using the same An object is to provide a method for measuring electrophysiological phenomena.

In order to solve the conventional problem, the present invention includes a well having a first through-hole,
A holding plate having a second through hole in contact with the lower side of the well and a flow path plate having a first groove having a liquid inlet and an outlet at both ends are provided below the holding plate. A cell electrophysiological sensor in contact with a thin plate having a third through hole inside the second through hole, the cell electrophysiological sensor communicating with the second through hole, the second through hole and the outlet A first protrusion is provided on a part of the holding plate between the two.

  The cell electrophysiological sensor of the present invention and the method for measuring a cell electrophysiological phenomenon using the sensor are highly accurate by reducing the amount of bubbles remaining in the sealed space when the pressure under the thin plate is reduced in order to hold the cells. Since a cell electrophysiological sensor that can perform a precise pressure reduction control can be realized, the electrophysiological phenomenon of cells can be measured with high accuracy.

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

  FIG. 1 is a cross-sectional view of a cell electrophysiological sensor according to Embodiment 1 of the present invention. 1 is a well made of resin, and a first through-hole 5 for storing intracellular fluid or extracellular fluid is formed in the well 1.

  It is sufficient that at least one first through hole 5 is formed, and a plurality of the first through holes 5 can be provided.

  A holding plate 2 having a second through hole 6 is in contact with the lower portion of the well 1, and at least one third hole is formed in the second through hole 6 of the holding plate 2. A sensor chip including a thin plate 4 having a flat surface with through holes 7 is set. This sensor chip is processed by processing a silicon substrate with a thickness of 20 μm and an opening diameter of the third through hole 7 of 1 to 3 μmφ. And the opening diameter of this 3rd through-hole 7 can be suitably selected with the magnitude | size of a cell.

  Further, a flow path plate 3 having at least one first groove 13 provided with an inlet 9 and an outlet 10 for allowing the liquid to flow in and out at both ends thereof is brought into contact with the lower side of the holding plate 2. A cell electrophysiological sensor is configured, and the subject cell is held in close contact with the upper surface of the third through-hole 7, and the electrophysiological phenomenon of the subject cell can be measured.

  In the cell electrophysiological sensor according to the first embodiment, the first protrusion 8 is formed at a position communicating with the second through hole 6 of the holding plate 2. The height of the first protrusion 8 is smaller than the depth of the first groove 13.

  With such a configuration, the depth of the first groove 13 formed in the flow path plate 3 becomes shallow in the portion where the first protrusion 8 is provided, and the fluid inside the first groove 13 flows. The flow path is narrowed.

  As a result, when liquid is introduced from the inlet 9 and discharged from the outlet 10, the pressure is limited to the outlet 9 side in the first groove 13 due to the restriction of the channel width by the first protrusion 8. It is relatively higher than the 10 side.

  Further, when the inflow port 9 is closed and the inside of the first groove 13 is a sealed space, if the outflow port 10 side is depressurized, the pressure is reduced at the outflow port 10 side due to the restriction of the flow path width by the first protrusion 8. Relatively low. In this way, the region in which the thin plate 4 and the second through hole 6 are formed always has a relatively higher pressure than the region of the outlet 10, so that the inlet 9 side, particularly the second through hole, is formed. Generation of bubbles in the vicinity of 6 can be suppressed. As described above, by providing the first protrusion 8, it is possible to suppress the generation of bubbles on the thin plate 4 side when the pressure is reduced by suction from the outlet 10 side using a suction pump or the like. As a result, a cell electrophysiological sensor capable of realizing highly accurate decompression control can be provided.

  It has also been found that the generation of bubbles can be further suppressed by providing such a first protrusion 8 adjacent to the second through hole 6. It is considered that this is because the ability to remove bubbles generated by instantaneously generating a larger decompression force is increased.

  Further, by providing a plurality of such first protrusions 8 on the outlet 10 side, fine pressure reduction control can be achieved.

  Next, a method for measuring a cell electrophysiological phenomenon using the cell electrophysiological sensor according to the first embodiment will be briefly described.

First, the well 1 is filled with intracellular fluid or extracellular fluid. Here, for example, in the case of mammalian muscle cells, the intracellular fluid is typically an electrolytic solution to which K ⁺ ions are added at about 155 mM, Na ⁺ ions at about 12 mM, and C 1 ⁻ ions at about 4.2 mM. The extracellular fluid is an electrolytic solution to which about 4 mM of K ⁺ ions, about 145 mM of Na ⁺ ions, and about 123 mM of C 1 ⁻ ions are added. In this state, between the first electrode (not shown) installed inside the well 1 and the second electrode (not shown) made of silver chloride or the like installed at an appropriate location in the first groove 13. Thus, a resistance value of about 100 kΩ to 10 MΩ can be observed. This is because the intracellular or extracellular fluid, which is an electrolytic solution, permeates the third through-hole 7 provided in the thin plate 4 of the cell electrophysiological sensor, and an electric circuit is formed between the electrodes. .

  Next, the cells are introduced into the first through-hole 5 using a micropipette or the like. At this time, the inner wall surface of the first through hole 5 is preferably tapered. This realizes a structure in which cells easily reach the vicinity of the third through-hole 7 formed in the thin plate 4 that is a sensor chip.

  Next, for example, when the inflow port 9 is closed and sucked from the outflow port 10, the cells are attracted to the third through hole 7, and can finally be held so as to close the third through hole 7. As a result, the electrical resistance between the well 1 and the first groove 13 becomes sufficiently high (this state is called a giga seal).

  In this state, when the potential inside and outside the cell changes due to the electrophysiological activity of the cell when a chemical solution or the like is added, even if there is a slight potential difference or current, there is a slight gap between the first electrode and the second electrode. By measuring the change in current or voltage in the cell, a highly accurate method for measuring the electrophysiological phenomenon of cells can be realized.

  Here, in order to measure quickly and with high accuracy, the cell is quickly moved onto the third through-hole 7 and then fixed to the third through-hole 7 with a certain sealing property (high giga shield). It is important to hold it while maintaining the property. In order to make this holding state constant, it is important to stably hold the cell on the third through-hole 7 after moving the cell quickly while finely controlling the suction pressure.

  On the other hand, if bubbles or the like are present in the vicinity of the second through-hole 6, the suction pressure becomes unstable, and it becomes difficult to maintain sufficient sealing performance necessary for measurement.

  Therefore, in order to realize highly accurate measurement, it is very important to use a cell electrophysiological sensor in which bubbles are not easily generated in the vicinity of the second through hole 6.

  FIG. 2 is a cross-sectional view for explaining the configuration of another example of the cellular electrophysiological sensor according to the first embodiment. As shown in FIG. The tip of the first protrusion 8 is made flat toward the outlet 10 side. As a result, the pressure on the inlet 9 side is relatively higher than that on the outlet 10 side as described above, so that the generation of bubbles in the vicinity of the second through-hole 6 holding the thin plate 4 is reduced. It has the advantage of being able to. Furthermore, by controlling the length of this flat shape, it is possible to realize optimal suction conditions and easily control the liquid flow into a laminar flow, so that the cell electrophysiological sensor can measure with higher accuracy. Can be realized.

  Next, the configuration of another example of the cell electrophysiological sensor according to the first embodiment will be described. FIG. 3 is a cross-sectional view for explaining the configuration of another example of the cell electrophysiological sensor according to the first embodiment. The first protrusion 8 is adjacent to the second through-hole 6 as shown in FIG. The plane of the opening 12 of the second through-hole 6 formed by the lower surface of the holding plate 2 and the lower surface of the first protrusion 8 is at least 0 degree with respect to the longitudinal line of the first groove 13. The opening 12 of the second through-hole 6 is formed toward the liquid inlet 9 side at the angle, and the second penetration is made so that the flat portion of the opening 12 and the thin plate 4 are horizontal. A thin plate 4 is installed inside the hole 6.

  By adopting such a configuration, compared to the configuration of the embodiment shown in FIG. 1, the liquid flows smoothly without generating a pool portion from the inlet 9 side to the outlet 10 side. The effect can be demonstrated.

  Further, since the flat surface of the thin plate 4 faces the inlet 9 side, the liquid sent from the inlet 9 side surely reaches the lower side of the thin plate 4, for example, the liquid inside the first groove 13. This is especially effective when you want to change the type.

(Embodiment 2)
Hereinafter, a cell electrophysiological sensor according to Embodiment 2 of the present invention will be described with reference to the drawings.

  As shown in FIG. 4, the cell electrophysiological sensor according to the second embodiment includes a well 1 having a first through hole 5 and a holding plate having a second through hole 6 below the well 1. 2, and a sensor chip made of a thin plate 4 having a flat surface having at least one third through hole 7 is fixedly held inside the second through hole 6.

  Further, below the holding plate 2, a flow path plate 3 having at least one first groove 13 provided with a liquid inlet 9 and an outlet 10 at both ends is abutted. Further, the second protrusion 11 is formed on the bottom surface of the flow path plate 3 so as to be on the outlet 10 side from below the second through hole 6.

  A cell electrophysiological sensor is configured to measure and measure the electrophysiological phenomenon of the subject cell by closely holding the subject cell in the third through-hole 7.

  In the cell electrophysiological sensor having the above-described configuration, in particular, by forming the second protrusion 11, the flow path through which the fluid inside the flow path plate 3 flows in the portion where the second protrusion 11 is formed. It is characterized by being narrowed.

  By adopting such a configuration, when the liquid is introduced from the inlet 9 and discharged from the outlet 10, the pressure of the passage is changed by the first groove 13 due to the restriction of the passage width by the second protrusion 11. The inlet 9 side is relatively higher than the outlet 10 side.

  Further, when the inflow port 9 is closed and the inside of the first groove 13 is a sealed space, if the outflow port 10 side is depressurized, the pressure is reduced due to the restriction of the channel width by the second protrusion 11. Is relatively low. As described above, it was found that the side where the thin plate 4 and the second through-hole 6 are provided always has a relatively high pressure, so that the generation of bubbles is reduced on the inlet 9 side. As a result, even if the pressure on the outlet 10 side is greatly reduced, it is possible to realize a cell electrophysiological sensor that does not generate bubbles on the thin plate 4 side and can be pressure-controlled with high accuracy.

  Moreover, as shown in FIG. 4, the cell electrophysiological sensor can be efficiently manufactured by adopting the configuration in which the second protrusion 11 of the flow path plate 3 is provided.

  First, as a normal method for producing the cell electrophysiological sensor in the present invention, first, the well 1 and the holding plate 2 are fusion-bonded using a thermoplastic resin or the like, and then the flow-path plate 3 is laminated and fused. It is efficient to produce a cellular electric sensor having a desired shape that is integrated by bonding. Here, if the first protrusion 8 is formed on the holding plate 2, inconvenience such as poor bonding may occur during the fusion bonding.

  In such a case, by forming the second protrusion 11 on the flow path plate 3 as shown in the second embodiment, it is possible to prevent a reduction in work efficiency during assembly such as joining. it can.

  Next, in another example of the cell electrophysiological sensor of the second embodiment, a plurality of second protrusions 11 are formed along the direction of the inlet 9 and the outlet 10 as in the first embodiment. The same effect as in the first embodiment can be exhibited.

  Next, a configuration of another example of the cell electrophysiological sensor according to the second embodiment will be described. As shown in FIG. 5, it is found that the same effect as that of the cell electrophysiological sensor having the configuration shown in FIG. 2 of the first embodiment can be exhibited by making the tip of the second protrusion 11 flat. ing.

  As described above, by adopting such a configuration, the pressure on the inlet 9 side can be designed to be relatively higher than that on the outlet 10 side, and the second through hole 6 holding the thin plate 4 can be designed. It is possible to provide a cell electrophysiological sensor that can reduce the generation of bubbles in the vicinity of. When a cell electrophysiological sensor having such a configuration is used to measure a cell electrophysiological phenomenon, it can be efficiently and accurately measured.

  As described above, since the cell electrophysiological sensor and the cell electrophysiological measurement method using the same according to the present invention enable high-accuracy measurement, the present invention can be applied to a measuring instrument such as drug screening that performs pharmacological determination at high speed. Useful.

Sectional drawing of the cell electrophysiological sensor in Embodiment 1 of this invention Sectional view of another example of cellular electrophysiological sensor Cross-sectional view of another example of cell electrophysiological sensor Sectional drawing of the cell electrophysiological sensor in Embodiment 2 of this invention Sectional view of another example of cellular electrophysiological sensor

Explanation of symbols

DESCRIPTION OF SYMBOLS 1 Well 2 Holding plate 3 Flow path plate 4 Thin plate 5 1st through-hole 6 2nd through-hole 7 3rd through-hole 8 1st projection part 9 Inflow port 10 Outflow port 11 2nd projection part 12 Opening part 13 First groove

Claims (10)

A well having a first through hole, a holding plate having a second through hole in contact with the lower side of the well, and a liquid inlet and outlet at both ends below the holding plate. A cell electrophysiological sensor that abuts a flow path plate having one groove and abuts a thin plate having a third through hole inside the second through hole, A cell electrophysiological sensor that is in communication and has a first protrusion provided on a part of the holding plate between the second through hole and the outlet. The cell electrophysiological sensor according to claim 1, wherein the first protrusion is provided adjacent to the second through hole. The cell electrophysiological sensor according to claim 1, wherein a plurality of first protrusions are formed. The cell electrophysiological sensor according to claim 1, wherein the tip of the first protrusion is flat. The first protrusion is disposed adjacent to the second through hole, and the plane of the opening of the second through hole is disposed at an angle of 0 ° or more with respect to the longitudinal line of the first groove. 2. The cell electrophysiological sensor according to claim 1, wherein the opening is disposed toward the liquid inlet side, and the thin plate is disposed inside the opening so that the flat surface and the thin plate of the opening are horizontal. . A well having a first through hole, a holding plate having a second through hole in contact with the lower side of the well, and a liquid inlet and outlet at both ends below the holding plate. A cell electrophysiological sensor that abuts a flow path plate having one groove and a thin plate having a third through hole inside the second through hole, A cell electrophysiological sensor that is in communication and has a second protrusion provided on a part of the flow path plate between the second through hole and the outlet. The cell electrophysiological sensor according to claim 6, wherein the second protrusion is provided adjacent to the second through hole. The cell electrophysiological sensor according to claim 6, wherein a plurality of second protrusions are formed. The cell electrophysiological sensor according to claim 6, wherein the tip of the second protrusion is flat. A well having a first through hole, a holding plate having a second through hole in contact with the lower side of the well, and a liquid inlet and outlet at both ends below the holding plate. A flow path plate having a single groove, a thin plate having a third through hole in contact with the second through hole, communicating with the second through hole, and a second through hole. A method for measuring a cell electrophysiological phenomenon, in which a cell electrophysiological sensor using a cell electrophysiological sensor provided with a first protrusion on a part of the holding plate or the flow path plate between the gas outlet and the outlet is measured.

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