JP2020187049A - Insert well - Google Patents

Abstract

To provide a technique of doing an observation and an electric measurement at the same time on a biological sample held in an insert well.SOLUTION: An insert well 10 is arranged in a recess capable of storing liquid in a container. The insert well 10 includes: a bottom part 12 having a sample holding surface 122, the sample holding surface being provided with a hole for passage of liquid; a peripheral wall part 14 having a cylindrical inner surface 142, the cylindrical inner surface extending in a vertical direction D1 from the sample holding surface 122 of the bottom part 12; and an inner reference electrode 20 and an inner action electrode 30 located in the inner surface 142 and arranged along the inner surface 142.SELECTED DRAWING: Figure 1

Description

The present invention relates to a technique for electrically measuring a biological sample placed in an insert well.

In order to investigate the properties and states of biological samples such as cells or tissues, a technique for measuring the electrical resistance of the biological sample is known. For example, in transepithelial electrical resistance (TEER) measurement, a membrane for cell culture is arranged in a culture medium, and electrodes are arranged on one side and the other side of the membrane. Then, by measuring the electrical resistance between these electrodes, the electrical resistance of the cell group cultured on the membrane is measured (for example, Patent Document 1).

In Patent Document 1, a culture insertion dish (insert well) in which cultured cells are arranged at the bottom is inserted into a recess of the culture dish. Then, a pair of electrodes are inserted inside and outside the culture insertion dish, and the voltage applied between the pair of electrodes is measured to measure the resistance of the cultured cells.

Japanese Unexamined Patent Publication No. 2005-137307

However, in the case of the prior art, since the electrode is inserted inside the insert well, it may be difficult to observe the biological sample held at the bottom of the insert well due to the electrode or the shadow formed by the electrode. .. In addition, the four-terminal method may be applied in order to measure the electrical resistance with high accuracy. In this case, since the working electrode for applying the voltage and the reference electrode for measuring the voltage are inserted inside the insert well, the observation field of view is further narrowed. Therefore, improvement was required.

An object of the present invention is to provide a technique for performing both observation and electrical measurement of a biological sample held in an insert well.

In order to solve the above problems, the first aspect is an insert well arranged in a recess in a container in which a liquid can be stored, and has a bottom having a sample holding surface provided with a hole for passing the liquid, and the bottom. A peripheral wall portion having a tubular inner surface extending from the sample holding surface in the first direction, and an inner reference electrode and an inner working electrode provided on the inner surface and arranged along the inner surface are provided.

The second aspect is the insert well of the first aspect, wherein the inner reference electrode is connected to the first lead wire portion extending in the first direction along the inner surface and the first lead wire portion. It includes an inner reference portion extending in the circumferential direction around the axis parallel to the first direction along the inner surface.

The third aspect is the insert well of the second aspect, further including a collar portion protruding outward from the peripheral wall portion.

The fourth aspect is the insert well of the third aspect, in which the first lead wire portion of the inner reference electrode extends from the inner surface of the peripheral wall portion to the surface of the collar portion.

The fifth aspect is the insert well of the fourth aspect, in which the boundary portion between the surface of the collar portion and the inner surface of the peripheral wall portion is chamfered, and the first lead wire portion is chamfered. It extends through the boundary portion to the surface of the collar.

A sixth aspect is an insert well according to any one of the second to fifth aspects, wherein the inner reference portion has an annular shape.

A seventh aspect is an insert well of any one of the second to sixth aspects, wherein the inner working electrode has a second lead wire portion extending in the first direction along the inner surface and the second lead wire. It includes an inner working portion that is connected to the portion and extends in the circumferential direction along the inner surface.

The eighth aspect is the insert well of the seventh aspect, in which the inner reference portion is arranged closer to the sample holding surface than the inner working portion.

The ninth aspect is the insert well of the eighth aspect, in which the inner working portion includes a first inner working portion extending from the second conducting portion in one direction in the circumferential direction, and the inner working portion extending from the second conducting portion in the circumferential direction. The first lead wire portion includes a second inner working portion extending to the other side of the above, and the first conducting portion is arranged between the first inner working portion and the second inner working portion.

A tenth aspect is an insert well according to any one of the second to ninth aspects, further comprising an insulating film covering at least a part of the first lead wire portion.

The eleventh aspect is an insert well of any one of the first to tenth aspects, the outer reference electrode provided on the outer surface of the peripheral wall portion, and the outer reference electrode and the outer action arranged along the outer surface. Further provided with electrodes.

The twelfth aspect is the insert well of the eleventh aspect, wherein the outer reference electrode is connected to the third lead wire portion extending in the first direction along the outer surface and the third lead wire portion. It includes an outer reference portion extending in the circumferential direction around the axis along the first direction along the outer surface.

The thirteenth aspect is the insert well of the twelfth aspect, in which the outer working electrode is connected to the fourth lead wire portion extending in the first direction along the outer surface and the fourth lead wire portion. Includes an outer working portion extending in the circumferential direction along the outer surface.

According to the insert well of the first aspect, an inner working electrode for applying a voltage to the biological sample and an inner reference electrode for measuring the voltage applied to the biological sample are provided on the inner surface of the peripheral wall portion extending in the first direction. And, it is arranged along the inner surface. In this case, since the overlap of the inner working electrode and the inner reference electrode with respect to the sample holding surface in the first direction can be reduced, the sample arranged on the sample holding surface can be observed well. Therefore, it is possible to perform both observation and resistance value measurement for the biological sample in the insert well.

According to the insert well of the second aspect, when a voltage is applied to the biological sample, the current generated in the vicinity thereof can be detected with high sensitivity by the inner reference portion extending in the circumferential direction of the inner reference electrode. Therefore, since the voltage applied to the biological sample can be measured with high accuracy, the electrical resistance of the biological sample can be measured satisfactorily.

According to the insert well of the third aspect, the insert well can be easily arranged in the concave portion of the container by placing the collar portion on the peripheral edge of the concave portion of the container.

According to the insert well of the fourth aspect, since the first lead portion of the inner reference electrode extends from the inner surface of the peripheral wall portion to the surface of the collar portion, the inner reference electrode is connected to the circuit for resistance measurement on the surface of the collar portion. can do.

According to the insert well of the fifth aspect, the first lead wire portion of the inner reference electrode is chamfered on the surface of the collar portion and the inner surface and the boundary portion of the peripheral wall portion, and is arranged on the chamfered boundary portion. In this case, it is possible to reduce the possibility that the first lead wire portion is broken at the boundary portion thereof.

According to the insert well of the sixth aspect, the current generated in the vicinity of the voltage applied to the biological sample is sensitively detected by the annular inner reference portion arranged over the entire inner surface of the peripheral wall portion. Can be done. Therefore, since the voltage applied to the biological sample can be measured with high accuracy, the electrical resistance of the biological sample can be measured satisfactorily.

According to the insert well of the seventh aspect, since the inner working portion of the inner working electrode extends in the circumferential direction, when a voltage is applied between the inner working electrode and the electrode arranged outside the insert well, the circumferential direction An electric field can be generated in a wide range of. As a result, the electric field generated on the sample holding surface can be made uniform in the circumferential direction.

According to the insert well of the eighth aspect, the inner reference portion of the inner reference electrode is arranged closer to the sample holding surface than the inner working portion of the inner working electrode. In this case, the voltage applied between the inner working electrode and the working electrode arranged outside the insert well can be measured by the inner reference portion arranged in the current path.

According to the insert well of the ninth aspect, since the first and second inner working portions of the inner working electrode are provided so as not to overlap with the first conducting portion of the inner reference electrode, the inner reference electrode and the inner working electrode are separated from each other. It is possible to suppress direct conduction. Further, since each of the first and second inner working portions can be extended in the circumferential direction to the position where the first conducting wire portion is sandwiched, the electric field generated on the sample holding surface can be made uniform in the circumferential direction.

According to the insert well of the tenth aspect, since at least a part of the first lead wire portion in the inner reference electrode is covered with an insulating film, that portion can be insulated from the surroundings and can be protected.

According to the insert well of the eleventh aspect, the inner reference electrode and the inner working electrode are arranged in the insert well, and the outer reference electrode and the outer working electrode are arranged outside the insert well, respectively, across the sample holding surface on which the biological sample is placed. Will be done. In this case, since it is not necessary to arrange the pair of electrodes of the inner reference electrode and the pair of electrodes of the inner working electrode in the recess of the container in which the insert well is arranged, the electrical resistance of the biological sample can be easily increased. Can be measured.

According to the insert well of the twelfth aspect, the current generated in the vicinity of the voltage applied to the biological sample can be detected with high sensitivity by the outer reference portion having a length in the circumferential direction of the outer reference electrode. .. Therefore, since the voltage applied to the biological sample can be measured with high accuracy, the electrical resistance of the biological sample can be measured satisfactorily.

According to the insert well of the thirteenth aspect, since the outer working portion of the outer working electrode extends in the circumferential direction along the outer surface of the peripheral wall portion, an electric field can be generated in a wide range in the circumferential direction. In this case, the electric field generated on the sample holding surface can be made uniform as compared with the case where the electric field is applied to a narrow range in the circumferential direction.

It is a perspective view of the insert well of an embodiment. It is a cross-sectional view of the insert well, and is the AA arrow view of FIG. It is sectional drawing of the insert well, and is the BB arrow view of FIG. It is a cross-sectional view of the insert well, and is the CC arrow view of FIG. It is a side view of the insert well, and is the D arrow view of FIG. It is a side view of the insert well, and is the E arrow view of FIG. It is a side view of the insert well, and is the F arrow view of FIG. It is a top view of the insert well. It is a bottom view of the insert well. It is the schematic sectional drawing which shows the container in which the insert well was inserted. It is a schematic diagram which conceptually shows a resistance measurement circuit.

Hereinafter, embodiments of the present invention will be described with reference to the accompanying drawings. It should be noted that the components described in this embodiment are merely examples, and the scope of the present invention is not limited to them. In the drawings, the dimensions and numbers of each part may be exaggerated or simplified as necessary for easy understanding.

<1. Embodiment>
<Insert well 10>
FIG. 1 is a perspective view of the insert well 10 of the embodiment. FIG. 2 is a cross-sectional view of the insert well 10, and is a view taken along the line AA of FIG. FIG. 3 is a cross-sectional view of the insert well 10 and is a view taken along the line BB of FIG. FIG. 4 is a cross-sectional view of the insert well 10 and is a view taken along the line CC of FIG. FIG. 5 is a side view of the insert well 10 and is a view taken along the arrow D of FIG. FIG. 6 is a side view of the insert well 10 and is a view taken along the arrow E of FIG. FIG. 7 is a side view of the insert well 10, and is a view taken along the line F of FIG. FIG. 8 is a top view of the insert well 10. FIG. 9 is a bottom view of the insert well 10.

The insert well 10 is an instrument used together with a separate container such as a well plate when measuring the electrical resistance of a biological sample. As shown in FIG. 1, the insert well 10 includes a bottom portion 12, a peripheral wall portion 14, a flange portion 16, an inner reference electrode 20, an inner working electrode 30, an outer working electrode 40, and an outer working electrode 50. Hereinafter, each of these components will be described.

<Bottom 12>
The bottom portion 12 has a sample holding surface 122 provided with a hole for passing a liquid. The bottom portion 12 is a portion that covers the opening below the peripheral wall portion 14, which will be described later. The bottom portion 12 is a member provided with a large number of fine through holes, and for example, a film-like (porous film-like) member having cell adhesion is suitable. In this example, the plan view shape of the bottom portion 12 is circular, but is not limited to this, and may be any shape such as an elliptical shape or a polygonal shape. Further, the bottom portion 12 has a flat plate shape having a uniform thickness, but the thickness may not be uniform. Further, although the sample holding surface 122 is substantially flat, it may have irregularities.

<Peripheral wall 14>
The peripheral wall portion 14 has a tubular inner surface 142 and an outer surface 144 extending from the sample holding surface 122 of the bottom portion 12 in the vertical direction D1 (first direction) perpendicular to the sample holding surface 122. Here, the inner surface 142 and the outer surface 144 have a cylindrical shape centered on the axis AX1 parallel to the vertical direction D1 corresponding to the shape (circular shape) of the bottom portion 12. It is not essential that the inner surface 142 and the outer surface 144 of the peripheral wall portion 14 extend parallel to the vertical direction D1, and may extend diagonally with respect to the vertical direction D1. Further, in this example, the inner diameter and the outer diameter of the peripheral wall portion 14 are constant in the vertical direction D1, but may be different in the vertical direction D1.

<Tsuba 16>
The flange portion 16 is a portion that protrudes outward (that is, in a direction away from the axis AX1) from the peripheral wall portion 14. The collar portion 16 has an upper surface 162 (front surface) and a lower surface 164 (back surface). The collar portion 16 has an annular shape corresponding to the shape of the inner surface 142 of the peripheral wall portion 14, and here, the collar portion 16 has an annular shape. It is not essential that the flange portion 16 has an annular shape. For example, the collar portion 16 may be composed of a plurality of projecting portions that radiate outward from the upper end portion of the peripheral wall portion 14.

The peripheral wall portion 14 and the flange portion 16 are made of, for example, a material having high optical transparency (polyethylene terephthalate, polycarbonate, etc.). Further, the peripheral wall portion 14 and the flange portion 16 may be integrally molded, or each may be composed of individual members. The peripheral wall portion 14 and the flange portion 16 are insulators.

<Inside reference electrode 20>
The inner reference electrode 20 is provided on the inner surface 142 of the peripheral wall portion 14, and is arranged along the inner surface 142. That is, the inner reference electrode 20 has a shape corresponding to the shape of the inner surface 142. The inner reference electrode 20 has a first lead wire portion 22 and an inner reference portion 24, and the first lead wire portion 22 and the inner reference portion 24 are in direct contact with the inner surface 142.

The first lead wire portion 22 extends along the inner surface 142 of the peripheral wall portion 14 in parallel with the vertical direction D1. Further, the first lead wire portion 22 extends from the inner surface 142 to the upper surface 162 of the flange portion 16, and here extends from the inner edge portion to the outer edge portion of the upper surface 162 (see FIG. 8). The inner reference electrode 20 is connected to a resistance measurement circuit 80 (see FIG. 11), which will be described later, via a portion of a first lead wire portion 22 arranged on the upper surface 162.

The inner reference portion 24 is connected to the lower end portion of the first lead wire portion 22. Further, the inner reference portion 24 extends along the inner surface 142 of the peripheral wall portion 14 in the circumferential direction D2 around the axis AX1 along the vertical direction D1. The inner reference portion 24 has an annular shape (here, an annular shape) along the inner surface 142.

The inner reference electrode 20 may be, for example, a thin film-like portion provided by depositing metal on the inner surface 142 of the peripheral wall portion 14 and the upper surface 162 (surface) of the flange portion 16, or may be conductive. It may be a plate-shaped or rod-shaped conductive member having. In the latter case, the conductive member may be fixed to the inner surface 142 or the upper surface 162 via an adhesive or the like. The inner working electrode 30, the outer reference electrode 40, and the outer working electrode 50, which will be described later, may be provided on the surfaces of the peripheral wall portion 14 and the flange portion 16 in the same manner as the inner reference electrode 20.

<Inner working electrode 30>
Like the inner reference electrode 20, the inner working electrode 30 is provided on the inner surface 142 of the peripheral wall portion 14, and is arranged along the inner surface 142. That is, the inner working electrode 30 has a shape corresponding to the shape of the inner surface 142. The inner working electrode 30 has a second conducting wire portion 32 and an inner working portion 34, and the second conducting wire portion 32 and the inner working portion 34 are in direct contact with the inner surface 142.

The second lead wire portion 32 extends in the vertical direction D1 along the inner surface 142 of the peripheral wall portion 14. Further, the second lead wire portion 32 extends from the inner surface 142 to the upper surface 162 of the flange portion 16, and here extends from the inner edge portion to the outer edge portion of the upper surface 162 (see FIG. 8). The inner working electrode 30 is connected to a resistance measurement circuit 80 (see FIG. 11), which will be described later, via a portion of a second lead wire portion 32 arranged on the upper surface 162.

The inner working portion 34 extends in the circumferential direction D2 along the inner surface 142 of the peripheral wall portion 14. As shown in FIGS. 2 to 4, on the inner surface 142, the lower end of the first lead wire portion 22 of the inner reference electrode 20 is closer to the sample holding surface 122 than the lower end portion of the second lead wire portion 32 of the inner working electrode 30. It is located on the near side (lower side). The inner reference portion 24 connected to the lower end of the first lead wire portion 22 is arranged closer to the sample holding surface 122 (lower side) than the inner action portion 34 connected to the lower end portion of the second lead wire portion 32. Has been done.

The inner working portion 34 extends from the lower end portion of the second conducting wire portion 32 to both sides in the circumferential direction D2. Specifically, the inner working portion 34 extends from the lower end portion of the second conducting wire portion 32 to one of the circumferential directions D2, and from the lower end portion of the second conducting wire portion 32 to the other side of the circumferential direction D2. It has a second inner working portion 344 that extends. It is not essential that the inner working portion 34 extends from the lower end of the second conducting wire portion 32 to both sides in the circumferential direction D2, and may extend to only one side in the circumferential direction D2.

As shown in FIGS. 1 and 3, the tip portions of the first and second inner working portions 342 and 344 extend to positions close to the first conducting wire portion 22 of the inner reference electrode 20. As a result, the first lead wire portion 22 is arranged between the tip portion of the first inner working portion 342 and the tip portion of the second inner working portion 344. As described above, the inner working portion 34 has an arc shape rather than a perfect circular ring shape.

<Outside reference electrode 40>
The outer reference electrode 40 is provided on the outer surface 144 of the peripheral wall portion 14, and is arranged along the outer surface 144. That is, the outer reference electrode 40 has a shape corresponding to the shape of the outer surface 144. The outer reference electrode 40 has a third lead wire portion 42 and an outer reference portion 44, and the third lead wire portion 42 and the outer reference portion 44 are in direct contact with the outer surface 144.

The third lead wire portion 42 is provided on the outer surface 144 and extends in the vertical direction D1. Further, the second lead wire portion 32 extends from the outer surface 144 to the lower surface 164 of the collar portion 16, and here extends from the inner edge portion to the outer edge portion of the lower surface 164 (see FIG. 9). The outer reference electrode 40 is connected to a resistance measurement circuit 80 (see FIG. 11), which will be described later, via a portion of a second lead wire portion 32 arranged on the lower surface 164.

The outer reference portion 44 is connected to the lower end portion of the third lead wire portion 42, and extends in the circumferential direction D2 along the outer surface 144 of the peripheral wall portion 14. The outer reference portion 44 has an annular shape (here, an annular shape) along the outer surface 144.

<Outer working electrode 50>
The outer working electrode 50 is provided on the outer surface 144 of the peripheral wall portion 14, and is arranged along the outer surface 144. That is, the outer working electrode 50 has a shape corresponding to the shape of the outer surface 144. The outer working electrode 50 has a fourth conducting wire portion 52 and an outer working portion 54, and the fourth conducting wire portion 52 and the outer working portion 54 are in direct contact with the outer surface 144.

The fourth lead wire portion 52 extends in the vertical direction D1 along the outer surface 144 of the peripheral wall portion 14. Further, the fourth conducting wire portion 52 extends from the outer surface 144 to the lower surface 164 of the collar portion 16, and here extends from the inner edge portion to the outer edge portion of the lower surface 164 (see FIG. 9). The outer working electrode 50 is connected to a resistance measurement circuit 80 (see FIG. 11) described later via a portion of a fourth conducting wire portion 52 arranged on the lower surface 164.

The outer working portion 54 is connected to the lower end portion of the fourth conducting wire portion 52 and extends in the circumferential direction D2 along the outer surface 144 of the peripheral wall portion 14. As shown in FIGS. 5 to 7, on the outer surface 144, the lower end portion of the third lead wire portion 42 of the outer reference electrode 40 is closer to the sample holding surface 122 than the lower end portion of the fourth lead wire portion 52 of the outer working electrode 50. It is located on the near side (lower side). The outer reference portion 44 connected to the lower end of the third lead wire portion 42 is arranged closer to the sample holding surface 122 (lower side) than the outer action portion 54 connected to the lower end portion of the fourth lead wire portion 52. Has been done.

The outer working portion 54 extends from the lower end portion of the fourth conducting wire portion 52 to both sides in the circumferential direction D2. Specifically, the outer working portion 54 extends from the lower end portion of the fourth conducting wire portion 52 to one of the circumferential directions D2, and from the lower end portion of the fourth conducting wire portion 52 to the other in the circumferential direction D2. It has a second outer working portion 544 that extends. It is not essential that the outer working portion 54 extends from the lower end of the fourth conducting wire portion 52 to both sides in the circumferential direction D2, and may extend to only one side in the circumferential direction D2.

As shown in FIGS. 1 and 6, the tip portions of the first and second outer working portions 542 and 544 each extend to a position close to the third conducting wire portion 42 of the outer reference electrode 40. As a result, the third conducting wire portion 42 is arranged between the tip portion of the first outer working portion 542 and the tip portion of the second outer working portion 544. As described above, the outer working portion 54 has an arc shape rather than a perfect circular ring shape.

As shown in FIG. 1, the inner reference portion 24 and the outer reference portion 44 are provided at the same height in the vertical direction D1 and have overlaps with each other in the horizontal direction orthogonal to the vertical direction D1. The inner working portion 34 and the outer working portion 54 are provided at the same height in the vertical direction D1 and have overlaps with each other in the horizontal direction. The inner reference portion 24 and the outer reference portion 44 may be provided at different heights from each other. Further, the inner working portion 34 and the outer working portion 54 may also be provided at different heights from each other.

The vertical width (width in the vertical direction D1) of the inner reference portion 24 and the outer reference portion 44 is not particularly limited, but may be, for example, the same size. The vertical widths of the inner working portion 34 and the outer working portion 54 are not particularly limited, but may be, for example, the same size. Further, from the viewpoint of increasing the current detection sensitivity, the vertical width of the inner reference portion 24 and the outer reference portion 44 may be larger than the vertical width of the inner action portion 34 or the outer action portion 54.

<Curved surface 146,148>
The boundary portion between the upper end portion of the peripheral wall portion 14 and the inner edge portion of the flange portion 16 is chamfered. Here, the boundary portion between the upper surface 162 of the collar portion 16 and the inner surface 142 of the peripheral wall portion 14 is connected by a curved surface 146 by chamfering (see FIG. 2). Further, the boundary portion between the lower surface 164 of the flange portion 16 and the outer surface 144 of the peripheral wall portion 14 is also connected by a curved surface 148. The first lead wire portion 22 of the inner reference electrode 20 and the second lead wire portion 32 of the inner working electrode 30 are arranged along the curved surface 146, respectively. In this case, it is possible to reduce the possibility that the first and second conducting wire portions 22 and 32 will be disconnected at the boundary portion between the inner surface 142 and the upper surface 162. Further, the third lead wire portion 42 of the outer reference electrode 40 and the fourth lead wire portion 52 of the outer working electrode 50 are arranged along the curved surface 148, respectively. In this case, it is possible to reduce the possibility that the third and fourth conducting wire portions 42 and 52 are disconnected at the boundary portion between the outer surface 144 and the lower surface 164. The boundary between the peripheral wall portion 14 and the flange portion 16 may be a flat inclined surface instead of curved surfaces 146 and 148.

<Insulating film 60>
The first lead wire portion 22 of the inner reference electrode 20 is preferably covered with an insulating film 60. The insulating film 60 is composed of, for example, an insulating resin as a main component. In addition, all (that is, the portion arranged from the flange portion 16 to the peripheral wall portion 14) of the first lead wire portion 22 may be covered with the insulating film 60, or only a part thereof is covered with the insulating film 60. It may be covered. In the latter case, for example, of the first conducting wire portion 22, only the portion arranged on the inner surface 142 of the peripheral wall portion 14 is covered with the insulating film 60, and the portion arranged on the upper surface 162 of the flange portion 16 is It may be exposed to the outside.

By covering at least a part of the first conducting wire portion 22 with the insulating film 60, that portion can be electrically insulated from the surroundings. Further, since the insulating film 60 can protect the first conducting wire portion 22, the possibility that the first conducting wire portion 22 is broken can be reduced.

For example, in the first lead wire portion 22, the portion arranged on the peripheral wall portion 14 is a portion that comes into contact with the liquid when measuring the electric resistance. Therefore, when this portion is covered with the insulating film 60, the influence of the first lead wire portion 22 on the electrical measurement of the biological sample 9 can be reduced. Further, in this case, as shown in FIG. 3, the first conducting wire portion 22 can be insulated from the inner working portion 34 (specifically, the first and second inner working portions 342 and 344) of the inner working electrode 30. it can. Therefore, the inner working electrode 30 and the inner reference electrode 20 can be well insulated. Therefore, the resistance of the biological sample 9 can be measured satisfactorily.

As with the first wire portion 22, each of the second to fourth wire portions 32, 42, and 52 may be partially or wholly covered with the insulating film 60.

<Container 70>
FIG. 10 is a schematic cross-sectional view showing a container 70 into which the insert well 10 is inserted. When the resistance of the biological sample 9 is measured using the insert well 10, the insert well 10 is inserted into the recess 72 of the container 70 as shown in FIG. Then, the biological sample 9 is arranged on the sample holding surface 122 of the insert well 10. For example, when the biological sample 9 is a cultured cell, the cultured cell is cultured on the sample holding surface 122.

The inner diameter of the recess 72 is larger than the outer diameter of the peripheral wall portion 14 of the insert well 10, and the depth of the recess 72 is larger than the length of the peripheral wall portion 14 in the vertical direction D1. Further, since the inner diameter of the recess 72 is smaller than that of the flange 16 of the insert well 10, the lower surface 164 of the flange 16 is supported by the peripheral edge 74 of the recess 72. As a result, the insert well 10 is arranged inside the recess 72. Specifically, the lower surface of the bottom portion 12 (the surface opposite to the sample holding surface 122) is arranged at a position away from the bottom surface of the recess 72, and the outer surface 144 of the peripheral wall portion 14 is the inner wall surface of the recess 72 (the recess 72). It is arranged at a position away from the wall surface having a length in the depth direction of.

A conductive liquid (for example, a culture solution) for measuring resistance is stored inside the recess 72 and inside the insert well 10. The liquid in the insert well 10 can move into the recess 72 (ie, outside the insert well 10) through a plurality of holes provided in the bottom 12. Similarly, the liquid in the recess 72 can also move into the insert well 10. In the insert well 10, the liquid is stored at least to a depth at which the inner reference portion 24 of the inner reference electrode 20 and the inner working portion 34 of the inner working electrode 30 are completely immersed. Further, outside the insert well 10, the liquid is stored in the recess 72 at least to a depth at which the outer reference portion 44 of the outer reference electrode 40 and the outer working portion 54 of the outer working electrode 50 are immersed.

Two conductive portions 76 having conductivity are provided on the upper surface of the peripheral portion 74 of the container 70. The conductive portion 76 is a metal film and is provided linearly. Each of the conductive portions 76 comes into contact with the third conductor portion 42 and the fourth conductor portion 52 provided on the lower surface 164 of the flange portion 16. As a result, the outer reference electrode 40 and the outer working electrode 50 are physically and electrically connected to the resistance measurement circuit 80 (specifically, the conducting wires 85 and 87) described later via the respective conductive portions 76.

Further, as shown in FIG. 10, the first lead wire portion 22 of the inner reference electrode 20 and the second lead wire portion 32 of the inner working electrode 30 are physically and physically attached to the resistance measurement circuit 80 on the upper surface 162 of the flange portion 16. It is electrically connected. Specifically, the first lead wire portion 22 is connected to the lead wire 87, and the second lead wire portion 32 is connected to the lead wire 85. The container 70 may be provided with two conductive portions for connecting the first and second conducting wire portions 22 and 32 to the resistance measurement circuit 80, respectively. Such two conductive portions are located at positions corresponding to the two conductive portions, the first and second conducting portions 22, 32, for example, on the back surface of the lid material that closes the recess 72 of the container 70 and the upper opening of the insert well 10. It may be provided in. In this case, by attaching the lid material to the container 70, the two conductive portions can be brought into contact with the first and second conductor portions 22, 32, respectively.

FIG. 11 is a schematic diagram conceptually showing the resistance measurement circuit 80. The resistance measuring circuit 80 is a device (transepithelial electrical resistance measuring device) for measuring the electrical resistance of the biological sample 9 by the so-called four-terminal method. Specifically, the resistance measurement circuit 80 includes a power supply device 81 and a voltmeter 83. The power supply device 81 is an AC voltage source. The output terminals on both sides of the power supply device 81 are connected to the inner working electrode 30 and the outer working electrode 50 of the insert well 10 via the lead wire 85, respectively. Further, the input terminals on both sides of the voltmeter 83 are connected to the inner reference electrode 20 and the outer reference electrode 40 of the insert well 10 via the lead wire 87, respectively.

In FIG. 11, the resistance Rm corresponds to the electrical resistance of the bottom 12 of the insert well 10 and the biological sample 9 (eg, cultured cells) held on the sample holding surface 122 of the bottom 12. Hereinafter, the bottom portion 12 and the biological sample 9 are collectively referred to as a “biological sample portion”. The resistance Rr1 corresponds to the electrical resistance of the liquid (for example, the culture solution) between the inner reference electrode 20 and the biological sample portion, and the resistance Rr2 corresponds to the electrical resistance of the liquid between the outer reference electrode 40 and the biological sample portion. Corresponds to electrical resistance. The resistance Rw1 corresponds to the electrical resistance of the liquid between the inner working electrode 30 and the biological sample portion, and the resistance Rw2 corresponds to the electrical resistance of the liquid between the outer working electrode 50 and the biological sample portion.

The resistors Rr1, Rr2, Rw1, Rw2 are measured in advance as controls. Further, the resistance Rm of the biological sample portion in the absence of the biological sample 9 is also measured in advance as a control. When measuring the resistance of the biological sample 9, first, a potential is applied between the inner working electrode 30 and the outer working electrode 50 by driving the power supply device 81. At the same time, the voltmeter 83 measures the voltage between the inner reference electrode 20 and the outer reference electrode 40. Then, the resistance value of the resistance Rm of the biological sample portion is calculated from the measured value of the voltage by a computer (not shown) or a manual calculation. The resistance value of the biological sample 9 is calculated by appropriately subtracting the resistance value when there is no biological sample 9 measured as a control from this resistance value.

<Effect>
In the present embodiment, the inner reference electrode 20 and the inner working electrode 30 are provided on the inner surface 142 of the peripheral wall portion 14 and are arranged along the inner surface 142. In this case, the inner working electrode 30 can apply a voltage to the biological sample 9 arranged at the bottom 12, and the inner reference electrode 20 can measure the voltage applied to the biological sample.

Further, since the inner reference electrode 20 and the inner working electrode 30 are provided on the inner surface 142 of the peripheral wall portion 14, the region where these electrodes 20 and 30 overlap the sample holding surface 122 in the vertical direction D1 is defined as the sample holding surface. It can be limited to the peripheral edge of 122. That is, since the overlapping portion of the inner reference electrode 20 and the inner working electrode 30 in the insert well 10 and the sample holding surface 122 in the vertical direction D1 can be reduced, the biological sample 9 held on the sample holding surface 122 can be moved in the vertical direction. It can be observed well from D1. Therefore, the biological sample 9 in the insert well 10 can be observed and electrically measured at the same time. An upright microscope, an inverted microscope, or the like can be applied as a means for observing the biological sample 9 in the insert well 10.

When the insert well 10 is inserted into the recess 72 of the container 70, the inner reference electrode 20 and the inner working electrode 30 are placed in the insert well 10 with the outer reference electrode 40 sandwiching the sample holding surface 122 on which the biological sample 9 is arranged. And the outer working electrode 50 is arranged outside the insert well 10, respectively. In this case, since it is not necessary to separately arrange the paired electrode of the inner reference electrode 20 and the paired electrode of the inner working electrode 30 in the recess 72 of the container 70 in which the insert well 10 is arranged, the biological sample 9 The electrical resistance of the can be easily measured. It is not essential that the insert well 10 is provided with the outer reference electrode 40 and the outer working electrode 50. When the insert well 10 is not provided with the outer reference electrode 40 and the outer working electrode 50, the paired electrode of the inner reference electrode 20 and the paired electrode of the inner working electrode 30 are placed in the recess 72 of the container 70, etc. It may be provided in.

Further, since the insert well 10 is provided with the flange portion 16, the insert well 10 can be easily arranged in the recess 72 by placing the collar portion 16 on the peripheral edge portion 74 of the recess 72 of the container 70. can do.

As shown in FIGS. 2 to 4, since the inner reference portion 24 of the inner reference electrode 20 extends in the circumferential direction D2, the current generated in the vicinity of the biological sample 9 when an electric field is applied is applied in the circumferential direction. It can be detected with high sensitivity over a wide range of D2. Therefore, since the voltage applied to the biological sample 9 can be measured with high accuracy, the resistance of the biological sample 9 can be measured satisfactorily.

Further, since the inner reference portion 24 has an annular shape extending along the inner surface 142 over the entire circumferential direction D2, it is possible to detect the current generated in the vicinity of the biological sample 9 with high sensitivity when a voltage is applied to the biological sample 9. In this case, since the voltage applied to the biological sample 9 can be measured with high accuracy, the resistance of the biological sample 9 can be measured satisfactorily.

As shown in FIGS. 2 to 4, the inner reference portion 24 of the inner reference electrode 20 is arranged closer to the sample holding surface 122 than the inner working portion 34 of the inner working electrode 30. In this case, the current generated between the inner working portion 34 and the working electrode (here, the outer working electrode 50) arranged outside the insert well 10 is detected by the inner reference portion 24 arranged in the current path. can do.

As shown in FIGS. 3 and 4, since the inner working portion 34 (specifically, the first and second inner working portions 342 and 344) of the inner working electrode 30 extends in the circumferential direction D2, the inner working electrode When a voltage (AC voltage) is applied between the 30 and an electrode (for example, the outer working electrode 50) arranged outside the insert well 10, an electric field can be generated in a wide range in the circumferential direction D2. As a result, the electric field generated on the sample holding surface 122 can be made uniform in the circumferential direction D2.

Further, by bringing the inner working portion 34 closer to an annular shape, an electric field can be generated from almost the entire circumference of the circumferential direction D2 along the inner surface 142 of the peripheral wall portion 14. As a result, the electric field generated on the sample holding surface 122 can be brought close to the uniform.

Further, as shown in FIG. 3 and the like, the inner working portion 34 (specifically, the first and second inner working portions 342 and 344) of the inner working electrode 30 does not overlap with the first conducting wire portion 22, so refer to the inside. It is possible to suppress the direct conduction between the electrode 20 and the inner working electrode 30. Further, by extending the tip portions of the first and second inner working portions 342 and 344 in the circumferential direction D2 to the positions where the first conducting wire portion 22 is sandwiched, the electric field generated on the sample holding surface 122 is transmitted with respect to the circumferential direction D2. It can be made evenly. When the insulating film 60 is provided on the first conducting wire portion 22, the inner working portion 34 may be arranged so as to be overlapped on the first conducting wire portion 22 (specifically, on the insulating film 60). .. In this case, the inner working portion 34 may be made circular by connecting the tip portions of the first and second inner working portions 342 and 344.

As shown in FIG. 3 and the like, the first and second inner working parts 342 and 344 of the inner working part 34 extend parallel to the sample holding surface 122, and hold the sample from each part of the inner working part 34. The distance to the center of the surface 122 is constant. By making the inner working portion 34 parallel to the sample holding surface 122 in this way, the electric field applied to the biological sample 9 arranged on the sample holding surface 122 can be uniformly approached in the circumferential direction D2. ..

As shown in FIG. 1, the first and second lead wire portions 22 and 32 are arranged at positions facing each other with the shaft AX1 interposed therebetween. In this case, since the first and second conducting wire portions 22 and 32 can be separated from each other, it is possible to prevent them from being directly conducted. It is not essential that the first and second lead wire portions 22 and 32 are arranged so as to face each other. For example, they may be arranged close to the circumferential direction D2.

As shown in FIGS. 5 to 7, since the outer reference portion 44 of the outer reference electrode 40 extends in the circumferential direction D2 along the outer surface 144 of the peripheral wall portion 14, in the vicinity thereof when a voltage is applied to the biological sample 9. The generated current can be detected with high sensitivity over a wide range in the circumferential direction D2. Therefore, since the voltage applied to the biological sample 9 can be measured with high accuracy, the resistance of the biological sample 9 can be measured satisfactorily.

Further, when the outer reference portion 44 has an annular shape extending along the outer surface 144 over the entire circumferential direction D2, the current generated in the vicinity of the biological sample 9 when a voltage is applied can be detected with high sensitivity. In this case, since the voltage applied to the biological sample 9 can be measured with high accuracy, the resistance of the biological sample 9 can be measured satisfactorily.

As shown in FIG. 6, since the outer working portion 54 (specifically, the first and second outer working portions 542,544) of the outer working electrode 50 extends in the circumferential direction D2 along the outer surface 144 of the peripheral wall portion 14. , An electric field can be applied over a wide range in the circumferential direction D2. In this case, the electric field generated on the sample holding surface 122 can be made uniform as compared with the case where the electric field is applied to a narrow range in the circumferential direction D2.

Further, by bringing the outer working portion 54 closer to an annular shape, an electric field can be generated from the inner working portion 34 of the inner working electrode 30 to almost the entire circumference of the circumferential direction D2 along the outer surface 144 of the peripheral wall portion 14. As a result, the electric field generated on the sample holding surface 122 can be brought close to the uniform.

Further, since the outer working portion 54 does not overlap with the third conducting wire portion 42, it is possible to suppress the direct conduction between the outer working electrode 50 and the outer reference electrode 40. Further, by extending the tip portions of the first and second outer working portions 542 and 544 in the circumferential direction D2 to the positions where the third conducting wire portion 42 is sandwiched, the electric field generated on the sample holding surface 122 is generated with respect to the circumferential direction D2. It can be made evenly. When the insulating film 60 is provided on the third conducting wire portion 42, the outer working portion 54 may be arranged so as to be overlapped on the third conducting wire portion 42 (specifically, on the insulating film 60). In this case, the outer working portion 54 may be made circular by connecting the tip portions of the first and second outer working portions 542 and 544, respectively.

As shown in FIGS. 5 to 7, the outer reference portion 44 of the outer reference electrode 40 is arranged closer to the sample holding surface 122 than the outer working portion 54 of the outer working electrode 50. In this case, the current generated between the outer working portion 54 and the inner working portion 34 of the inner working electrode 30 arranged in the insert well 10 is detected by the outer reference portion 44 arranged in the current path. Can be done.

As shown in FIGS. 6 and 7, the first and second outer working parts 542,544 of the outer working part 54 extend parallel to the sample holding surface 122, and from each part of the outer working part 54, The distance to the center of the sample holding surface 122 is constant. By making the outer working portion 54 parallel to the sample holding surface 122 in this way, the electric field applied to the biological sample 9 arranged on the sample holding surface 122 can be uniformly brought close to the circumferential direction D2. ..

As shown in FIG. 1, the third and fourth lead wire portions 42 and 52 are arranged at positions facing each other with the shaft AX1 interposed therebetween. In this case, since the third and fourth conducting wire portions 42 and 52 can be separated from each other, it is possible to prevent them from being directly conducted. It is not essential that the third and fourth lead wire portions 42 and 52 are arranged so as to face each other, and they may be arranged close to the circumferential direction D2.

<2. Modification example>
Although the embodiments have been described above, the present invention is not limited to the above, and various modifications can be made.

For example, in the above embodiment, it is not essential that the first to fourth lead wire portions 22, 32, 42, 52 extend in parallel with the vertical direction D1, for example, in both the vertical direction D1 and the circumferential direction D2. It may extend in the synthetic direction in which the components are combined.

It is not essential that the inner reference portion 24, the inner working portion 34, the outer reference portion 44 and the outer acting portion 54 extend parallel to the circumferential direction D2, for example, components in both the vertical direction D1 and the circumferential direction D2. It may extend in the combined synthetic direction.

As shown in FIGS. 1, 2 and 5, the first and third lead wire portions 22, 42 are in the horizontal direction (thickness direction of the peripheral wall portion 14) in the peripheral wall portion 14 and in the vertical direction in the flange portion 16. There is an overlap with D1, but these are not essential. For example, the first and third lead wire portions 22, 42 may be arranged in the flange portion 16 so as not to overlap in the vertical direction D1. In this case, when the first and third conducting wire portions 22 and 42 are connected to the resistance measurement circuit 80, the possibility that the first and third conducting wire portions 22 and 42 are directly conductive can be reduced. The second and fourth conductive portions 32 and 52 also have an overlap in the horizontal direction in the peripheral wall portion 14 and in the vertical direction D1 in the flange portion 16, but these are not essential.

Further, a plurality of insert wells 10 shown in FIG. 1 may be arranged in a one-dimensional or two-dimensional direction, and the collar portions 16 may be connected to each other. In this case, by holding the biological sample 9 in each of the plurality of insert wells 10, it is possible to collectively perform electrical measurement on the plurality of biological samples 9.

Although the present invention has been described in detail, the above description is exemplary in all aspects and the invention is not limited thereto. It is understood that a myriad of variations not illustrated can be envisioned without departing from the scope of the invention. The configurations described in the above embodiments and the modifications can be appropriately combined or omitted as long as they do not conflict with each other.

10 Insert well 12 Bottom 122 Sample holding surface 14 Peripheral wall 142 Inner surface 144 Outer surface 146,148 Curved surface (boundary)
16 collar 162 upper surface (surface)
20 Inner reference electrode 22 1st lead wire part 24 Inner reference part 30 Inner working electrode 32 2nd lead wire part 34 Inner working part 342 1st inner working part 344 2nd inner working part 40 Outer reference electrode 42 3rd lead wire part 44 Outer reference Part 50 External working electrode 52 4th conducting wire 54 Outer working part 542 1st outer working part 544 2nd outer working part 60 Insulation film 70 Container 72 Recess 80 Resistance measurement circuit 9 Biological sample AX1 axis D1 Vertical direction (1st direction)
D2 circumferential direction

Claims (13)

An insert well that is placed in a recess in the container where the liquid can be stored.
A bottom with a sample holding surface provided with holes for liquid to pass through,
A peripheral wall portion having a tubular inner surface extending in the first direction from the sample holding surface of the bottom portion,
An inner reference electrode and an inner working electrode provided on the inner surface and arranged along the inner surface,
With insert well. The insert well of claim 1
The inner reference electrode is
A first conducting wire portion extending in the first direction along the inner surface,
An inner reference portion that is connected to the first lead wire portion and extends in the circumferential direction around the axis parallel to the first direction along the inner surface.
Including, insert well. The insert well of claim 2.
A collar that protrudes outward from the peripheral wall,
Further equipped with insert wells. The insert well of claim 3
An insert well in which the first lead wire portion of the inner reference electrode extends from the inner surface of the peripheral wall portion to the surface of the collar portion. The insert well of claim 4
The boundary portion between the surface of the collar portion and the inner surface of the peripheral wall portion is chamfered.
An insert well in which the first lead wire portion extends through the chamfered boundary portion to the surface of the collar portion. The insert well according to any one of claims 2 to 5.
An insert well in which the inner reference portion has an annular shape. The insert well according to any one of claims 2 to 6.
The inner working electrode is
A second conducting wire portion extending in the first direction along the inner surface,
An inner working portion that is connected to the second conducting wire portion and extends in the circumferential direction along the inner surface,
Including, insert well. The insert well of claim 7.
An insert well in which the inner reference portion is arranged closer to the sample holding surface than the inner working portion. The insert well of claim 8.
The inner working part is
A first inner working portion extending from the second conducting wire portion in one direction in the circumferential direction,
A second inner working portion extending from the second conducting portion to the other in the circumferential direction,
Including
An insert well in which the first lead wire portion is arranged between the first inner working portion and the second inner working portion. The insert well according to any one of claims 2 to 9.
An insulating film that covers at least a part of the first lead wire portion,
Further equipped with insert wells. The insert well according to any one of claims 1 to 10.
An outer reference electrode and an outer working electrode provided on the outer surface of the peripheral wall portion and arranged along the outer surface.
Further equipped with insert wells. The insert well of claim 11.
The outer reference electrode is
A third conducting wire portion extending in the first direction along the outer surface,
An outer reference portion that is connected to the third lead wire portion and extends in the circumferential direction around the axis along the outer surface and along the first direction.
Including, insert well. The insert well of claim 12.
The outer working electrode is
A fourth conducting wire portion extending in the first direction along the outer surface,
An outer working portion that is connected to the fourth conducting wire portion and extends in the circumferential direction along the outer surface,
Including, insert well.

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