JP2007298350A - Cell electrophysiological sensor and cell electrophysiological phenomenon measuring method using this - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To materialize a cell electrophysiological sensor for suppressing the formation of air bubbles to the utmost and a cell electrophysiological phenomenon measuring method using this. <P>SOLUTION: This cell electrophysiological sensor comprises a thin sheet 1 having a first through hole 2, a holding plate 3 having a second through hole 4, and a flow-path plate 5 with a flow path formed thereon for causing a fluid to flow out of/into a lower part of the through hole 2. The thin sheet 1 is firmly fixed so as to close the through hole 4 while the flow-path plate 5 is firmly fixed to a lower part of the holding plate 3. A third through hole 6 is provided through the flow-path plate 5 coaxially with the through hole 4, and a hollow tube probe 7 is inserted through the through hole 6 from the exterior. <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 method for producing the same. is there.

  Conventionally, the patch clamp method in electrophysiology is known as a method for measuring ion channels existing in cell membranes, and various functions of ion channels have been elucidated by this patch clamp method. And the action of this ion channel is an important concern in cytology, which is also 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 It enables electrophysiological measurement of cells. This is because the through-hole made in the flat plate plays the same role as the tip hole in the glass pipette, and it can record the electrophysiological phenomenon of the cell with high accuracy, and the cell is automatically generated by a method such as suction from the back side of the flat plate. It has the advantage that it can be easily attracted and cells can be easily retained (see, for example, Patent Document 1).
Special table 2002-508516 gazette

  However, in the conventional configuration, when the electrophysiological phenomenon of a cell is measured, the cell exists in a solution, and this cell reacts extremely sensitively to the state of the solution, for example, the drug dissolution concentration, ion concentration, and the like. At this time, it is known that the state of bubbles remaining in the solution adversely affects the measurement result. For this reason, control of the flow of the solution is extremely important.

  In particular, in the process of measuring an ionic current flowing through a cell membrane, which is one of the electrophysiological phenomena of cells, there is often a need to exchange the type of liquid accumulated in each of the two regions. For this reason, there are a method of exchanging the liquid accumulated inside by a pipette, a method of confining the upper and lower areas of the flat plate with the flow path of the closed space, and replacing the solution inside the flow path of the closed space, etc. It was difficult to control the pressure inside the closed space, and there was a problem that measurement was unstable due to the generation of bubbles inside the flow path.

  An object of the present invention is to provide a cell electrophysiological sensor capable of measuring with high accuracy by suppressing the generation of bubbles as much as possible and a method for measuring a cell electrophysiological phenomenon using the cell electrophysiological sensor.

  In order to solve the above-described conventional problems, the present invention allows a fluid to flow into and out of a thin plate having a first through hole, a holding plate having a second through hole, and a lower portion of the first through hole. A flow path plate having a flow path for the cell electrophysiological sensor, wherein the thin plate is fixed so as to close the second through hole, and the flow path plate is fixed to a lower portion of a holding plate. The flow path plate is provided with a third through hole coaxially with the second through hole, the hollow tube probe is inserted from the outside through the third through hole, and the tip of the hollow tube probe is connected to the second through hole. The inside of the through hole is inserted while maintaining the flow path and the sealing property.

  The cell electrophysiological sensor of the present invention and the method for measuring a cell electrophysiological phenomenon using the sensor can rapidly exchange the solution in one region of the thin plate by allowing the solution to flow into and out of the flow path, and the third through hole. By inserting a hollow tube probe, the pressure control around one region of the thin plate can be controlled while suppressing the generation of bubbles as much as possible, so that cell electrophysiology can be measured efficiently and with high accuracy. A sensor and a method for measuring a cell electrophysiological phenomenon using the sensor can be provided.

(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 the cell electrophysiological sensor according to Embodiment 1 of the present invention, and shows the state of the sensor structure of the cell electrophysiological sensor at the time of measurement. In FIG. 1, reference numeral 1 denotes a thin plate made of silicon, silicon dioxide, glass or the like, and a first through hole 2 is provided so as to penetrate the upper and lower surfaces. Reference numeral 3 denotes a holding plate made of resin. The holding plate 3 is provided with a second through hole 4 penetrating vertically.

  The thin plate 1 is held inside the second through hole 4 so as to close the second through hole 4. As a result, the upper and lower spaces of the holding plate 3 communicate only through the first through hole 2.

  Reference numeral 5 denotes a flow path plate made of a resin material or the like. The flow path plate 5 is brought into contact with the lower surface of the holding plate 3 to form a flow path 12a therein. It communicates with the outside through the inflow hole 10 and the outflow hole 11.

  Further, the flow path plate 5 is provided with a third through hole 6, and the third through hole 6 is configured so that a hollow tube probe 7 can be inserted and installed.

  A first measurement electrode 8 is formed inside the hollow tube probe 7, and a second measurement electrode 13 is formed above the thin plate 1. The measurement electrodes 8 and 13 are in contact with the second solution 16 or the first solution 15 accumulated in the respective regions. As a result, a potential difference, current, resistance value, etc. generated between the two regions can be measured.

  At the time of measurement, the tip of the hollow tube probe 7 is placed in close contact with the opening of the second through-hole 4 to maintain the flow path and the sealing property.

  As a result, the second solution 16 accumulated in the hollow tube probe 7 does not leak to the flow path 12a side and is electrically insulated, so that electricity generated in the upper and lower regions of the thin plate 1 is maintained. Chemical changes can be measured with high accuracy. Here, the electrochemical change that occurs in the upper and lower regions is a cell membrane that is generated by allowing the subject cell 18 to permeate various ions into and out of the cell by holding the subject cell 18 in the first through-hole 2. Including current, cell membrane potential and the like.

  In addition, in order to insert closely into the opening of the second through hole 4 of the hollow tube probe 7, a slit is provided on the inner wall surface at the tip of the hollow tube probe 7 as shown in FIG. While making it easy to insert into the second through-hole 4, it is possible to maintain airtightness with higher sealing performance.

  In such a configuration, it is possible to easily introduce the liquid inside the flow path 12a into the hollow tube probe 7 by loosening the degree of adhesion when the hollow tube probe 7 is installed. . That is, the second solution 16 that has flowed and accumulated in the flow path 12a through the inflow hole 10 and the outflow hole 11 removes the hollow tube probe 7 from the installation position of the second through-hole 4 as shown in FIG. By forming a gap, the first solution 15 accumulated in the hollow tube probe 7 can be pushed out and the second solution 16 can be introduced into the inside.

  At this time, the suction system 9 is preferably connected to the base side of the hollow tube probe 7, whereby the liquid in the flow path 12 a can be more easily introduced into the hollow tube probe 7.

  If the hollow tube probe 7 is brought into close contact with the second through-hole 4 again, the liquid inside the hollow tube probe 7 can be separated from the flow path 12a side while maintaining the sealing property. Therefore, it can also be electrically insulated, and electrochemical changes generated in the upper and lower regions of the thin plate 1 can be measured with high accuracy.

  As described above, by using the above-described configuration, it is possible to realize a cell electrophysiological sensor that makes it easy to freely change the type of solution to a liquid such as a culture solution, a chemical solution, or a reagent.

  Since the solution can be exchanged by the configuration and method as described above, it is not necessary to once extract the liquid inside the hollow tube probe 7, and the generation of bubbles can be efficiently suppressed. Can do.

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

  FIG. 4 shows a state before the hollow tube probe 7 is inserted into the cell electrophysiological sensor according to Embodiment 1 of the present invention. Before the hollow tube probe 7 is inserted, the third through-hole 6 is closed with a film 14 so that the liquid does not leak even if the liquid flows in and accumulates in the flow path 12a. .

  First, as shown in FIG. 5, the first solution 15 is introduced into the upper region and the lower region of the thin plate 1. For example, the first solution 15 is an extracellular fluid.

  Next, the hollow tube probe 7 is inserted into the third through-hole 6 as shown in FIG. At this time, the film 14 is squeezed. However, if the material of the film 14 is made of a highly elastic material such as polyethylene or silicon rubber, the first solution 15 leaks to the outside even when squeezed. It is convenient because it does not come out.

  Then, as shown in FIG. 7, the first solution 15 can be quickly introduced into the hollow tube probe 7 by suction from the suction system 9 on the base side of the hollow tube probe 7.

  In order to prevent bubbles from remaining in the vicinity of the second through hole 4 or in the hollow tube probe 7, the first solution 15 is poured from the inflow hole 10 toward the outflow hole 11 or the hollow tube probe 7. It is extremely effective to keep it flowing.

  Next, as shown in FIG. 8, the hollow tube probe 7 is abutted against the inside of the second through hole 4 sufficiently. At this time, as another example, it is preferable to make the tip of the hollow tube probe 7 into a tapered shape as shown in FIG. And the external shape of the front-end | tip of the hollow tube probe 7 is made smaller than the 2nd through-hole 4, is made smaller than the 3rd through-hole 6, and it goes to the 3rd through-hole 6 from the 2nd through-hole 4. It is preferable to have a tapered shape that increases. As a result, the insertion can be facilitated and the sealing performance can be easily enhanced.

  In the state shown in FIG. 8, the upper and lower regions of the thin plate 1, the inside of the first through hole 2, and the inside of the hollow tube probe 7 are all filled with the first solution 15, and the first measurement is performed. A predetermined resistance value, typically 1 MΩ to 10 MΩ, is observed between the electrode 8 and the second measurement electrode 13.

  Next, as shown in FIG. 9, the subject cell 18 is put into the upper region of the thin plate 1. At this time, the subject cell 18 is suspended in the first solution 15 in the upper region of the thin plate 1, and thereafter, suction is performed from the suction system 9 connected to the hollow tube probe 7 as shown in FIG. When this is done, the subject cell 18 is drawn into the first through-hole 2.

  However, since the size of the subject cell 18 is larger than the first through hole 2, it cannot pass through the first through hole 2 and is held on the surface of the opening of the first through hole 2. In this state, when the adhesion force of the subject cell 18 to the opening of the first through hole 2 is sufficiently high, the resistance value observed between the first measurement electrode 8 and the second measurement electrode 13. Is 100 MΩ or more, preferably 1 GΩ or more.

As described above, when the subject cell 18 is held in the first through-hole 2 with a sufficiently high degree of adhesion, ions such as Na ⁺ , Ca ²⁺ , and K ⁺ flow in and out of the subject cell 18. Can be measured with high accuracy as changes in the current value and voltage value observed between the first measurement electrode 8 and the second measurement electrode 13. .

  When measuring the resistance value, current, and voltage, if it is necessary to change the type of solution in the upper and lower regions of the thin plate 1, as shown in FIG. After separating from the through-hole 4, the second solution 16 is introduced into the flow path 12 a to replace the inside of the flow path 12 a and the inside of the hollow tube probe 7. It is possible to perform suction while closely contacting the through hole 4. In this way, the above process can be performed as needed even after the subject cells 18 are in close contact with each other and the resistance value becomes sufficiently high. For example, the third solution is further formed in the lower region of the thin plate 1 and the hollow tube probe 7. It is also possible to substitute (not shown).

  As described above, the type of solution accumulated in the upper and lower regions partitioned by the thin plate 1 and the subject cell 18 held by the thin plate 1 can be easily determined by the configuration of the cell electrophysiological sensor in the first embodiment. Provided is a cell electrophysiological sensor capable of more efficiently and accurately measuring an electrophysiological phenomenon, which is a physicochemical phenomenon emitted by a cell, and a method for measuring a cell electrophysiological phenomenon using the same be able to.

(Embodiment 2)
Next, a cell electrophysiological sensor according to Embodiment 2 of the present invention and a cell electrophysiological measurement method using the same will be described with reference to the drawings.

  In particular, the second embodiment is different from the first embodiment in that a plurality of second through holes 4 are provided in the holding plate 3 as shown in FIG. 12, and the thin plate 1 is held and fixed to each of them. The lower side of the plurality of thin plates 1 has a common flow path 12b, an inflow hole 10, and an outflow hole 11. Note that the upper side of the thin plate 1 may be individually partitioned by a partition wall 19.

  Then, a third through hole 6 is provided on the flow path plate 5 coaxially with the second through hole 4 so as to correspond to the position of each thin plate 1, and is hollow from the lower part of the third through hole 6. A tube probe 7 can be inserted.

  Moreover, the first measurement electrode 8 and the second measurement electrode 13 for measurement are provided for each, and the electrophysiological phenomenon of the subject cell 18 held on each thin plate 1 can be individually measured.

  In order to insert a plurality of such hollow tube probes 7 together, the first measurement electrode 8 which is a measurement electrode is attached to the inner wall surface of the hollow tube probe 7 by a method such as bonding or embedding. By fixing, the simplification of the measurement operation can be improved.

  Also in the second embodiment, the lower region of the thin plate 1 and the solution inside the hollow tube probe 7 can be exchanged as in the first embodiment. Since it is common, it is possible to replace the same solution with the inside of each hollow tube probe 7 and the hollow tube probe 7 is sufficiently adhered to the inside of the third through-hole 6 at the time of measurement. In this case, the solutions in the hollow tube probes 7 are electrically blocked from each other, so that the influence of the measurement in each region on the other regions can be eliminated.

  On the other hand, since the flow path 12b, the inflow hole 10 and the outflow hole 11 are common, even when the number of installed thin plates 1 is increased, the occupied area of the flow path 12b, the inflow hole 10 and the outflow hole 11 is reduced. Therefore, it is effective for miniaturization and simplification of the cell electrophysiological sensor.

  As described above, the cell electrophysiological sensor according to the present invention and the cell electrophysiological measurement method using the same can efficiently measure the cell electrophysiological phenomenon. Useful for screening systems.

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

Explanation of symbols

DESCRIPTION OF SYMBOLS 1 Thin plate 2 1st through-hole 3 Holding plate 4 2nd through-hole 5 Flow path plate 6 3rd through-hole 7 Hollow tube probe 8 1st measuring electrode 9 Suction system 10 Inflow hole 11 Outflow hole 12a, 12b Channel 13 Second measurement electrode 14 Film 15 First solution 16 Second solution 18 Subject cell 19 Septum

Claims (10)

A thin plate having a first through hole, a holding plate having a second through hole, and a flow path plate having a flow path for allowing fluid to flow into and out of the lower portion of the first through hole. And a cell electrophysiological sensor in which the thin plate is fixed so as to close the second through hole, and the flow path plate is fixed to a lower portion of the holding plate, wherein the flow path plate has a third through hole. Is provided coaxially with the second through hole, a hollow tube probe is inserted from the outside through the third through hole, and the tip of the hollow tube probe is inserted into the second through hole in the flow path. And cell electrophysiological sensor inserted with sealing performance. The cell electrophysiological sensor according to claim 1, wherein the outer shape of the hollow tube probe is made smaller than that of the third through hole, and the tip thereof is inserted into the second through hole while maintaining the flow path and the sealing property. . The outer shape of the tip of the hollow tube probe is made smaller than the second through hole, smaller than the third through hole, and tapered so as to become larger from the second through hole toward the third through hole. The cell electrophysiological sensor according to claim 2. The cell electrophysiological sensor according to claim 1, wherein the third through hole is sealed with a sealable material before the hollow tube probe is inserted. The cell electrophysiological sensor according to claim 1, wherein a slit is provided on the inner wall surface at the tip of the hollow tube probe. The cell electrophysiological sensor according to claim 1, wherein a thin plate is inserted into the second through-hole and fixed so as to be flush with the plane of the holding plate to close the second through-hole. The cell electrophysiological sensor according to claim 1, wherein an electrode is provided on the inner wall surface of the hollow tube of the hollow tube probe. The cell electrophysiological sensor according to claim 1, wherein the cell electrophysiological sensor is connected to means for sucking the inside of the hollow tube of the hollow tube probe. The cell electrophysiological sensor according to claim 1, wherein a plurality of thin plates are held inside a holding plate, and the plurality of thin plates are connected to a common flow path. A thin plate having a first through hole, a holding plate having a second through hole, and a flow path plate having a flow path for allowing fluid to flow into and out of the lower portion of the first through hole. The thin plate is fixed so as to close the second through hole, the flow path plate is fixed to a lower portion of the holding plate, and the third through hole is connected to the flow path plate with the second through hole. Provided on the same axis, a hollow tube probe is inserted from the outside through this third through-hole, and the tip of this hollow tube probe is inserted inside the second through-hole while maintaining the flow path and sealing performance A first step of preparing a cellular electrophysiological sensor,
A second step of flowing a fluid into the upper part of the thin plate and the flow path;
A third step of forming the third through hole by inserting the hollow tube probe into a part of the flow path plate; and inserting the tip of the hollow tube probe into the second through hole. A fourth step;
Measuring a cell electrophysiological phenomenon in which a cell is held in the opening of the first through-hole and includes a fifth step of measuring a potential, current, or resistance between the upper and lower regions of the thin plate of the holding plate Method.

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