JP4720638B2 - Cell electrophysiological sensor and method for measuring cell electrophysiology using the same - Google Patents

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 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 reduced 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 above conventional problems, the present invention includes a well having a first through hole, made from the second holding plate having a through hole in contact with the lower side of the well, the second A cell electrophysiological sensor in which a thin plate having a third through-hole is contacted inside the through-hole, wherein a groove is provided below the holding plate along the flow direction of the flowing liquid. .

  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 enables accurate pressure reduction control can be realized, a highly accurate electrophysiological phenomenon of cells can be measured.

(Embodiment 1)
Hereinafter, the cell electrophysiological sensor 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, and FIG. 2 is a bottom view of a holding plate. 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 a through hole 7 is fixedly held. This sensor chip can be efficiently manufactured by processing a silicon substrate. For example, the thickness is 20 μm, and the opening diameter of the third through-hole 7 is made by etching with a dimension 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 11 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.

  As a configuration for suppressing and removing bubbles in the flow path of the first groove 11 and in the vicinity of the second through hole 6, a plurality of second grooves 12 are provided along the inlet 9 and the outlet 10. Each end face is in communication with the second through-hole 6.

  By adopting such a configuration, it is possible to suppress the generation of bubbles and quickly remove bubbles, and a cell electrophysiological sensor capable of measuring efficiently and with high accuracy and a cell electrophysiology using the same. A method for measuring a phenomenon can be realized.

  Next, the operation of the cell electrophysiological sensor having such a configuration will be described.

  Usually, the cell electrophysiological sensor before measurement is stored or packaged in a dry state. When measuring the electrophysiological phenomenon of a cell using this cell electrophysiological sensor, it is necessary to fill the inside of the flow path with a liquid as a step before entering the measurement. At this time, liquid such as pure water does not easily enter the valley of the second groove 12 at first, and a space where the gas-phase fluid can easily pass through the gap of the second groove 12 is formed. As a result, when the liquid flows from the inlet 9 toward the outlet 10, the generated gas-phase fluid bubbles pass through the gap in the formed second groove 12, and the liquid passes near the upper portion of the second groove 12. It turns out that it passes easily.

  As a result, the liquid and bubbles can flow quickly from the inlet 9 toward the outlet 10 without stagnation, so that all the bubbles inside the first groove 11 can be expelled to the outlet 10 side. . Thereafter, by filling a liquid such as a chemical solution necessary for measurement with pure water, it is possible to fill the first groove 11 with a liquid such as a chemical solution without leaving bubbles in the first groove 11.

  In this way, when cells are introduced in the absence of bubbles and sucked using, for example, a vacuum pump from the outlet 10, the cells introduced from the first through-hole 5 gradually fall by their own weight, and then The third through hole 7 can be held so as to be closed, and then a stable gigashield state can be held by suction with a stable pressure.

  For example, when the second through-hole 6 is formed in a shape of 50 μmφ, the width and depth of the second groove 12 is 50 μm, and the cross-sectional shape of the second groove 12 is etched so as to be a triangle. The effect can be exhibited by forming it by the method. Further, when the second through hole 6 has a dimensional shape of 200 to 1000 μmφ, a second groove 12 having a shape in which a plurality of triangular grooves having the above dimensions are arranged in parallel is formed. By doing so, the same effect can be exhibited.

  Thus, it can respond flexibly by appropriately selecting the shape and number of the second grooves 12 according to the size and shape of the opening diameter of the second through-hole 6. And it is important to determine these specifications from conditions that can suppress the generation of bubbles most.

  In addition, it turned out that the said phenomenon exhibits the effect remarkably, so that the space | interval of the 2nd groove | channel 12 is narrow and the trough of a processus | protrusion cuts into an acute angle. Therefore, it is more preferable to arrange a plurality of the second grooves 12 having small dimensions cut into acute angles in parallel.

  Accordingly, it is possible to provide a cell electrophysiological sensor capable of measuring with high accuracy by realizing more accurate decompression control and a method for measuring a cell electrophysiological phenomenon using the cell electrophysiological sensor.

  1 and 2, the second groove 12 is provided before and after the second through hole 6 (the inlet 9 side and the outlet 10 side), but at least the inlet 9 and the second through hole are provided. The effect can be exerted by forming it between 6, and it is particularly effective to form it in the vicinity of the second through-hole 6. The reason is that bubbles are likely to be generated near the cell and in the vicinity of the second through hole 6.

  It is more effective to form the second groove 12 wider than at least the outer shape of the second through-hole 6.

  Note that pure water is used in the initial preparation stage in order to save liquid such as a chemical solution, and the same effect can be exhibited when a liquid such as a chemical solution is used directly.

  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 11. 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.

  Thereafter, 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 11 via the thin plate 4 is sufficiently high (this state is called a giga seal).

  In this state, when the internal and external potential of the cell changes due to the electrophysiological activity of the cell when a chemical solution or the like is added, even if a slight potential difference or current is present between the first electrode and the second electrode By measuring changes in current or voltage, it is possible to realize a highly accurate measurement method of the electrophysiological phenomenon of cells.

  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 retain the cells while maintaining the sex). In order to maintain this holding state constant, it is important to stably hold the cell on the third through-hole 7 after rapidly moving the cell 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 the generation of bubbles is small in the vicinity of the second through-hole 6 and the bubbles are easily removed.

(Embodiment 2)
Hereinafter, a cell electrophysiological sensor according to Embodiment 2 of the present invention will be described with reference to the drawings. FIG. 3 is a cross-sectional view of the cell electrophysiological sensor according to Embodiment 2 of the present invention, and FIG. 4 is a top view of the flow path plate 3.

  As shown in FIGS. 3 and 4, the cell electrophysiological sensor according to the second embodiment has a well 1 having a first through hole 5 and a second through hole 6 below the well 1. The sensor plate 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 11 having a liquid inlet 9 and an outlet 10 at both ends is abutted. Further, the second groove 12 is formed on the bottom surface of the flow path plate 3 so as to correspond to the position 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, by forming the second groove 12 so as to be disposed below the second through-hole 6, the same effect as in the first embodiment can be exhibited. It is possible to realize a cell electrophysiological sensor that can be used.

  In such a configuration, the liquid is less likely to enter the valleys of the plurality of second grooves 12, and a space in which the gas-phase fluid can easily pass between the second grooves 12 is formed.

  As a result, when the liquid flows from the inlet 9 toward the outlet 10, the gas phase fluid bubbles pass through the valley of the second groove 12, and the liquid passes near the upper part of the second groove 12. Thus, the liquid and bubbles flow from the inlet 9 toward the outlet 10 without stagnation. As a result, it is possible to expel all the bubbles in the first groove 11 to the outlet 10 side, and the liquid can be filled with a liquid such as a chemical solution or a culture solution without leaving the bubbles in the first groove 11. It becomes.

  Note that the above-described phenomenon can be more effective as the interval between the second grooves 12 is narrower and the valleys are cut into an acute angle.

  Moreover, as shown in FIG. 3 and FIG. 4, the cell electrophysiological sensor can be efficiently manufactured by providing the second groove 12 in the flow path plate 3.

  As a method for producing the cell electrophysiological sensor in the second embodiment, 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 fusion-bonded. Thus, it is efficient to produce a cellular electric sensor having a desired shape by integrating them. Here, when the second groove 12 is formed in the holding plate 2, the second groove 12 has an acute saw-shaped pointed shape when using a method of joining while applying pressure during the fusion joining. When it is processed, the tip may be damaged.

  In such a case, by forming the second groove 12 in 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. .

  In consideration of the above, when the second groove 12 is formed on both the lower surface side of the holding plate 2 and the upper surface side of the flow path plate 3, the bubbles can be removed more efficiently. .

  Next, a configuration of another example of the cell electrophysiological sensor according to the second embodiment will be described. FIG. 5 is a top view of a flow path plate 3 of another example of the cell electrophysiological sensor according to the second embodiment, and FIG. 6 is an enlarged view of a main part thereof.

  Here, as shown in FIG. 5 and FIG. 6, the configuration of the third groove 13 having a plurality of acute-shaped concave portions in the shape of the inner wall on the side wall side of the first groove 11 formed in the flow path plate 3. It is said. At this time, the second groove 12 is formed on the upper surface or the lower surface of the first groove 11, and the third groove 13 communicates with the second groove 12. This configuration is realized by making the width of the second groove 12 the same as the width of the first groove 11 and forming the third groove 13 on the side wall surface of the first groove 11. can do.

  In such a configuration, the liquid layer fluid is less likely to enter the sharp concave tip formed in the third groove 13, and becomes a passage for the gas phase fluid of bubbles generated on the side wall. Thereby, bubbles, which are gas phase fluids generated near the side wall of the first groove 11, move through the third groove 13, then move to the second groove 12, and then quickly stagnate from the outlet 10. Combined with the structure described in the other embodiments so far, there is an effect that bubbles are not left inside the first groove 11 and are finally easily and efficiently filled with liquid. It can be demonstrated.

  As described above, by adopting such a configuration, the pressure on the inlet 9 side can be designed relatively accurately and higher than that on the outlet 10 side, and the second penetration holding the thin plate 4 can be achieved. A cell electrophysiological sensor capable of reducing the generation of bubbles in the vicinity of the hole 6 can be provided. And when measuring the electrophysiological phenomenon of a cell using the cell electrophysiological sensor which has such a structure, the measuring method of the cell electrophysiological phenomenon which can be measured efficiently with high precision can be provided.

  As described above, the cell electrophysiological sensor according to the present invention and the method for measuring a cell electrophysiological phenomenon using the sensor enable measurement with high accuracy. Useful for.

Sectional drawing of the cell electrophysiological sensor in Embodiment 1 of this invention Bottom view of the holding plate Sectional drawing of the cell electrophysiological sensor in Embodiment 2 of this invention Top view of the same channel plate Top view of flow path plate of another example Enlarged view of the main part

Explanation of symbols

1 well 2 holding plate 3 flow path plate 4 thin plate 5 first through hole 6 second through hole 7 third through hole 9 inflow port 10 outflow port 11 first groove 12 second groove 13 third groove

Claims (11)

A thin plate comprising a well having a first through-hole and a holding plate having a second through-hole abutting below the well, and having a third through-hole inside the second through-hole A cell electrophysiological sensor in which a groove is provided on the lower surface of the holding plate along the flow direction of the flowing liquid. The cell electrophysiological sensor according to claim 1, wherein the width of the groove is larger than the width of the second through hole. The cell electrophysiological sensor according to claim 1, wherein a plurality of the grooves are formed. The cell electrophysiological sensor according to claim 3, wherein a width of the plurality of grooves is larger than a width of the second through hole. A flow path plate having a first groove is abutted below the holding plate, the groove provided in the holding plate is a second groove, and the width of the second groove is the same as the width of the first groove. The cell electrophysiological sensor according to claim 1, wherein a third groove is provided on a side wall surface of the first groove. A well having a first through-hole, a holding plate having a second through-hole in contact with the lower side of the well, a liquid inlet and an outlet at the both ends, and a second plate are provided below the holding plate. A cell electrophysiological sensor in which a flow path plate having one groove is contacted and a thin plate having a third through hole is contacted inside the second through hole, the flow direction of the liquid being A cell electrophysiological sensor in which a second groove is provided in a part of the flow path plate. The cell electrophysiological sensor according to claim 6, wherein the width of the second groove is larger than the width of the second through hole. The cell electrophysiological sensor according to claim 6, wherein a plurality of second grooves are formed. The cell electrophysiological sensor according to claim 8, wherein the width of the plurality of second grooves is larger than the width of the second through hole. The cell electrophysiological sensor according to claim 6, wherein the width of the second groove is the same as the width of the first groove, and a third groove is provided on the side wall surface of the first groove. A well having a first through-hole, a holding plate having a second through-hole in contact with the lower side of the well, a liquid inlet and an outlet at the both ends, and a second plate are provided 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 method for measuring a cell electrophysiological phenomenon, wherein a cell electrophysiological sensor using a cell electrophysiological sensor in which a second groove is provided in a part of a holding plate or a flow path plate is measured.

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