JP2004069309A - Extracellular potential measuring device and its manufacturing method - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To solve the problem of a method for accurately achieving a shape is expected, wherein the size of an opening at a well side of a recess for retaining the cell of a specimen and the size of a through-hole side need to be set to two stages, when a plurality wells need to be arranged in a substrate with high density, in an extracellular potential measuring device. <P>SOLUTION: The substrate 1 comprises a a base 2; an intermediate layer 3 made of a material differing from the base 2; and the laminated structure of a thin plate 4 made of nearly the same material as the base. At least one well 5 is provided inside the base 2, and at least one through-hole 6 leading to the well 5 and the outside of the substrate 1 is provided inside the thin plate 4. <P>COPYRIGHT: (C)2004,JPO

Description

[0001]
TECHNICAL FIELD OF THE INVENTION
The present invention relates to an extracellular potential measurement device used for measuring extracellular potential or a physicochemical change occurring in the activity of a cell and a method for producing the same. Used.
[0002]
[Prior art]
Conventionally, drug screening using the electrical activity of cells as an index has been performed by a patch clamp method, a method using a fluorescent dye or a luminescence indicator.
[0003]
This patch clamp method is a method in which the transport of ions through a single channel protein molecule is electrically recorded by a microelectrode probe using a microportion (called a patch) of the cell membrane attached to the tip of a micropipette. This method is one of the few methods capable of examining the function of one protein molecule in real time (for example, Molecular Biology of Cells, Third Edition, Garland @ Publishing Inc., New York, 1994, Japanese Edition). (Translated by Keiko Nakamura et al., Pp. 181-182, 1995, Kyoikusha).
[0004]
There is also a method of measuring the electrical activity of a cell by monitoring the movement of ions in the cell using a fluorescent dye or a luminescence indicator that emits light in accordance with a change in the concentration of a specific ion.
[0005]
However, the patch clamp method requires a special technique for the preparation and operation of a micropipette, and it takes a lot of time to measure one sample. Therefore, the patch clamp method is not suitable for use in screening a large number of drug candidate compounds at high speed. In addition, although a method using a fluorescent dye or the like can screen a large amount of drug candidate compounds at high speed, a step of staining cells is required, and the measurement requires a high background due to the effect of the dye and decolorization with time. There is a disadvantage that the N ratio is poor.
[0006]
As an alternative, the methods disclosed in Japanese Patent Application Nos. 2001-001980 and 2001-170341 can obtain high-quality data equivalent to the data obtained by the patch clamp method, and use a fluorescent dye. A large amount of sample can be measured simply and at high speed as in the method. In these prior arts, at least one well having cell holding means provided on a substrate and a sensor means for detecting an electric signal in this well are used to measure extracellular potential or physicochemical changes generated by cells. A so-called extracellular potential measurement device is disclosed. The operation of the extracellular potential measurement device disclosed in the above prior art will now be described with reference to the drawings.
[0007]
FIG. 20 is a schematic cross-sectional view showing the well structure of the extracellular potential measurement device disclosed in the above prior art. A culture solution 48 is placed in a well 40, and a test cell 47 is provided on a substrate 42. Captured or held by the cell holding means. The cell holding means has a depression 41 formed in the substrate 42 and a through-hole 44 communicating with the depression 41 through an opening. Further, a measurement electrode 45 as a sensor means is disposed in the through hole 44, and the measurement electrode 45 is connected to a signal detection unit via a wiring.
[0008]
At the time of the measurement, the subject cell 47 is held in close contact with the recess 41 by means of a suction pump or the like from the through-hole 44 side. The electric signal generated by the activity of the subject cell 47 in this manner is detected by the measurement electrode 45 provided on the through hole 44 side without leaking to the culture solution 48 side in the well 40.
[0009]
Here, in order to hold the subject cell 47 in the recess 41, the well 40, which is a container for mixing the subject cell 47, the culture solution 48, and the drug that reacts with the subject cell 47, is required above the recess 41. . In the conventional example, the well 40 is provided separately from the substrate 42 and is set above the depression 41 as shown in FIG.
[0010]
Further, the size of the recess 41 holding the subject cell 47 is about 10 to 30 μm, the size of the through-hole 44 side is 1 to 5 μm in two stages, and the length of the through-hole 44 is , Usually about 10 μm. In order to accurately realize this shape, it is necessary to first dry-etch the depression 41 using the first mask and then dry-etch the through hole 44 using the second mask using two types of masks. There is.
[0011]
[Problems to be solved by the invention]
However, a particular problem in the extracellular potential measurement device as described above is that if the well 40, the through hole 44, and the substrate 42 having the depression 41 are separately provided, the well 40 and the substrate 42 need to be bonded to each other. And accurate alignment is also required. In particular, when it is necessary to arrange a plurality of wells 40 at a high density in the substrate 42, it is necessary to securely bond the wells so that the liquid does not leak out, in addition to the alignment, and the work becomes difficult. .
[0012]
Furthermore, as described above, when using two types of masks, after performing dry etching using the first mask, misalignment of the mask occurs when performing dry etching using the second mask. I do.
[0013]
In addition, since two masks are prepared and photolithography is performed separately for each mask, it is troublesome in terms of manufacturing, which may lead to an increase in cost.
[0014]
[Means for Solving the Problems]
In order to solve the above-mentioned problem, the invention according to claim 1 of the present invention is characterized in that the substrate has a laminated structure of a base, an intermediate layer made of a material different from the base, and a thin plate made of the same material as the base. At least one or more wells for mixing a somatic cell, a culture solution and a drug, and at least one or more through-holes provided in the thin plate to the outside of the well and the substrate. It is an extracellular potential measurement device that measures chemical changes, and the well is provided in the base, so there is no need to provide a separate well, and the well and through hole are integrated into the substrate, making it extremely compact. An extracellular potential measurement device can be realized.
[0015]
The invention according to claim 2 of the present invention is the extracellular potential measurement device according to claim 1, wherein the device has a depression on the well side of the through hole, and the opening of the depression is larger than the opening of the through hole. In addition, the subject cells can be drawn into the through-hole side and securely held in the depression.
[0016]
The invention according to claim 3 of the present invention is the extracellular potential measurement device according to claim 2, wherein the inner wall of the recess is formed in a conical shape. Since it is possible to enter and only one subject cell can enter within the cone, an extracellular potential measuring device that can more reliably hold the subject cell can be realized.
[0017]
The invention according to claim 4 of the present invention is the extracellular potential measuring device according to claim 2, wherein the inner wall of the depression is formed into a hemispherical shape. Since the connection surface with the hemisphere has a hemispherical shape, the surface of the subject cell comes into more reliable contact with the connection surface with the through-hole, and an extracellular potential measurement device capable of ensuring the retention of the subject cell is provided. Can be realized.
[0018]
The invention according to claim 5 of the present invention is the extracellular potential measurement device according to claim 1, wherein the electrical resistivity of the intermediate layer is higher than the electrical resistivity of the thin plate, wherein the through-hole and the recess are formed from the thin plate side. When formed by dry etching, the etching ions accumulate in the intermediate layer having a large electric resistivity, so that the etching ions also dry-etch the thin plate near the intermediate layer. An extracellular potential measurement device having a manufacturing advantage that a depression can be formed can be realized.
[0019]
The invention according to claim 6 of the present invention is the extracellular potential measuring device according to claim 1, wherein the materials constituting the intermediate layer and the thin plate are made of materials having different etching rate ratios with respect to the same etching gas. When the through hole and the dent are formed by dry etching, the intermediate layer can be prevented from being dry-etched even after the etching reaches the intermediate layer, so that a more accurate formation of the through hole and the dent can be realized. Thus, an extracellular potential measuring device having an advantage in manufacturing can be realized.
[0020]
The invention according to claim 7 of the present invention is the extracellular potential measuring device according to claim 1, wherein the base and the thin plate are made of silicon, and the intermediate layer is made of silicon dioxide, wherein silicon and silicon dioxide are the same dry etching gas. , For example SF₆, CF₄, XeF₂This has a large difference in etching rate with respect to the above, and thus complements the effect of the invention according to claim 6 in that the through hole and the depression can be formed with high precision in the thin plate.
[0021]
The invention according to claim 8 of the present invention is the extracellular potential measuring device according to claim 2, wherein the opening of the depression is 10 to 100 μm and the opening of the through hole is 1 to 10 μm. If the size of the somatic cell is 10 μm, the size of the opening of the depression is 15 μm, and the size of the through hole is 5 μm, only one test cell can enter the depression, and Therefore, it is possible to realize an extracellular potential measurement device that is reliably held in the depression.
[0022]
The invention according to claim 9 of the present invention is characterized in that the substrate has a laminated structure of a base, an intermediate layer made of a material different from the base and a thin plate made of a material substantially the same as the base, and a test cell, a culture solution, At least one or more wells for mixing a drug and at least one or more through-holes provided in the thin plate to the outside of the well and the substrate are measured for an extracellular potential or a physicochemical change generated by a cell. A method of manufacturing an extracellular potential measurement device, the method comprising: providing the well by etching a base; providing the through-hole by dry etching using one type of mask on a thin plate; and forming an intermediate layer at the bottom of the well. Manufacturing of extracellular potential measurement device including the step of removing by dry etching and the step of forming detection electrode by thin film technology from the outside A method for manufacturing an extracellular potential measurement device that can easily form a recess wider than the size of a through-hole on the well side of the through-hole when forming the through-hole in a thin plate using only one type of mask. be able to.
[0023]
According to a tenth aspect of the present invention, in the step of providing a through hole by dry etching using one type of mask on a thin plate, the thin plate is formed by dry etching from the outside, and the dry etching is performed at least by etching. 10. The method for producing an extracellular potential measuring device according to claim 9, wherein the method is performed using two types of gas: a gas for suppressing the etching and a gas for promoting the etching. Dry etching can be performed while controlling the rate of suppression and promotion of the extracellular potential, so that the extracellular potential measurement device can perform dry etching vertically so that the hole size of the through hole is at least the same size as the mask on the outer side Can be realized.
[0024]
According to an eleventh aspect of the present invention, in the dry etching for forming the through hole, the step of using a gas for suppressing the etching and the step of using a gas for promoting the etching are alternately repeated. The method of manufacturing an extracellular potential measuring device according to the above item, which complements the effect of the invention according to the claim 10, wherein the hole shape of the through hole can be dry-etched more vertically.
[0025]
The invention according to claim 12 of the present invention is characterized in that, in the step of providing a through hole, etching is performed such that the ratio of etching with a gas that promotes etching to a gas that suppresses etching increases as the etching proceeds. This is a method of manufacturing an extracellular potential measuring device, and as the ratio of gas that promotes dry etching increases as the process of forming the through hole progresses, the etching diameter of the well side of the through hole increases, and the depression easily forms on the well side. A method of manufacturing an extracellular potential measurement device that can be formed can be realized.
[0026]
According to a thirteenth aspect of the present invention, in the step of providing a through hole, the dry etching performed by repeating the step of promoting dry etching and the step of suppressing dry etching is stopped before reaching the intermediate layer made of silicon dioxide. 11. The method for manufacturing an extracellular potential measuring device according to claim 10, wherein dry etching is performed until the intermediate layer of silicon dioxide is reached only with a gas that promotes etching, and the dry etching conditions are changed before reaching the intermediate layer. Therefore, it is possible to realize a method of manufacturing an extracellular potential measurement device that can easily and continuously form the through-hole and the depression.
[0027]
The invention according to claim 14 of the present invention is characterized in that, in the step of using a gas for promoting dry etching and the step of using a gas for suppressing etching, an inductive coupling method using a coil is used as a means for decomposing the gas; The method for manufacturing an extracellular potential measuring device according to claim 10, wherein the step of promoting etching is performed by applying high frequency to the substrate, and the step of suppressing dry etching is performed without applying high frequency to the substrate. In addition, since a high frequency is applied to the substrate when promoting dry etching and is not applied when suppressing etching, a method for manufacturing an extracellular potential measuring device that can form a uniform protective film can be realized.
[0028]
In the invention according to claim 15 of the present invention, in the step of forming the through hole, the step of promoting dry etching and the step of suppressing dry etching are repeatedly performed, and dry etching is performed on the intermediate layer mainly composed of silicon dioxide. 11. The method for manufacturing an extracellular potential measuring device according to claim 10, wherein the thin plate is excessively dry-etched even after the etching reaches the intermediate layer. Therefore, in the dry etching, the etching spreads in the lateral direction near the intermediate layer. Therefore, it is possible to realize a method of manufacturing an extracellular potential measurement device that can easily form a depression on the well side simultaneously with the formation of the through hole.
[0029]
The invention according to claim 16 of the present invention is a method for manufacturing an extracellular measurement device in which a base and a thin plate are made of silicon and an intermediate layer is made of silicon dioxide, and is used when a through hole is formed in a lower layer. The gas that promotes etching is SF₆, CF₄, XeF₂Is a gas containing any one of the above, and the gas that suppresses etching is C₄F₈, CHF₃The method for manufacturing an extracellular potential measurement device according to claim 10, wherein a gas containing any one of the following is used: the outside of the through-hole is a vertical dry-etched shape having the same size as a mask, and a well is formed. The side can be a wider recess than this, and complements the effect of the invention according to claims 10 to 15.
[0030]
BEST MODE FOR CARRYING OUT THE INVENTION
Hereinafter, an extracellular potential measurement device and a method for manufacturing the same of the present invention will be described with reference to embodiments and drawings.
[0031]
(Embodiment 1)
The first to third aspects, the sixth to the twelfth, the fourteenth and the sixteenth aspects of the present invention will be described with reference to the first embodiment of the present invention and FIGS.
[0032]
FIG. 1 is a perspective view showing an embodiment of the extracellular potential measuring device according to Embodiment 1 of the present invention, and FIGS. 2 and 3 are sectional views thereof. 4 and 5 are enlarged sectional views of the essential parts. 6 to 13 are cross-sectional views for explaining a method for manufacturing the extracellular potential measuring device.
[0033]
1 to 3, a substrate 1 has a laminated structure of a base 2 made of silicon, an intermediate layer 3 made of silicon dioxide, and a thin plate 4 made of silicon. The base 2 is provided with a well 5 for storing a sample solution containing test cells and mixing the test cells 10 and a culture solution 11 thereof with a drug. Further, the thin plate 4 at the bottom of the well 5 is provided. A through hole 6 is provided. A recess 7 is provided on the well 5 side of the through hole 6, and the size of the opening on the well 5 side is larger than the size of the opening on the lower surface of the substrate 1.
[0034]
In addition, the size of the through-hole 6 and the depression 7 can be determined according to the size and properties of the subject cell 10. For example, if the size of the subject cell 10 is 10 μm, the size of the depression 7 is preferably 10 μm or more and 20 μm or less, and the size of the through hole 6 is preferably 5 μm or less.
[0035]
Further, in the first embodiment, the inner wall portion of the recess 7 has a conical shape with the well 5 side as the bottom surface.
[0036]
Next, an insulator 9 made of silicon dioxide is provided on silicon at least on the inner wall and the bottom surface of the well 5, the inner wall of the through hole 6, the inner wall of the depression 7, and the lower surface of the thin plate 4. Further, the inner wall of the through hole 6 is formed. On the outer side of the thin plate 4, a detection electrode 8 mainly composed of gold is provided on the insulator 9.
[0037]
Since the size of the cells is generally 5 to 20 μm, the opening of the depression 7 is preferably 10 to 100 μm, and the opening of the through hole 6 is preferably 1 to 10 μm.
[0038]
The extracellular potential measuring device having the above configuration can measure extracellular potential or physicochemical changes generated by cells by the following operation. This operation will be described with reference to the drawings.
[0039]
FIG. 3 is a cross-sectional view showing a state in which the test cell 10 and the culture solution 11 are put into the well 5 in the extracellular potential measuring device, and FIGS. It is a principal part expanded sectional view.
[0040]
As shown in FIG. 3, when the culture solution 11 and the test cell 10 are put into the well 5, the test cell 10 is suspended in the culture solution 11 for a while. Further, since the culture solution 11 is a liquid, it flows not only into the well 5 but also into the lower surface side of the well 5 after filling the depression 7 and the through hole 6. In this manner, the floating subject cell 10 is drawn into the depression 7 as shown in FIG. 4 by the pressure in the well 5. When the pressure of the retraction is insufficient, if the suction is performed from the through-hole 6 side by means of a suction pump or the like, the floating subject cells 10 can be more reliably suctioned to the dent 7 side.
[0041]
Next, the test subject cells 10 that have reached the depression 7 are held in the depression 7 as shown in FIG. When a drug (not shown) is administered to the culture solution 11 in the well 5 in such a state, the drug penetrates into the culture solution 11, and the subject cell 10 exchanges ions with the drug as shown in FIG. When the reaction is caused and activated, the electric signal generated in the through-hole 6 changes the potential of the culture solution 11 filled in the through-hole 6. This change in potential is taken out to the outside by the detection electrode 8 in contact with the culture solution 11.
[0042]
As described above, in the extracellular potential measuring device of the present invention, since the depression 7 is provided on the bottom surface of the well 5, it is not necessary to separately provide a well as in the conventional art. And the drug can be mixed directly. Further, since the well 5 and the recess 7 and the through-hole 6 provided on the bottom surface are integrated, the culture solution 11 can flow to the through-hole 6 side without inadvertently leaking out of the well 5. it can.
[0043]
Further, since an insulator 9 made of silicon dioxide is provided on the inner wall of the recess 7, the inner wall of the through hole 6, the lower surface of the thin plate 4, the bottom of the well 5, and the inner wall, the detection electrode 8 is electrically connected to the well 5 side. Electrically insulated. Furthermore, since the inner wall of the recess 7 has a conical shape with the well 5 as the bottom surface, the subject cells 10 can be drawn in without being blocked by the through-hole 6 and can be stably held. As an example, when the size of the subject cell 10 is 10 μm, the size of the well 7 on the well side, that is, the size of the conical bottom surface is set to 20 μm or less, so that two or more subject cells 10 At the same time, the penetration into the recess 7 is eliminated, and the subject cell 10 does not pass through the through-hole 6 by setting the size of the through-hole 6 to 5 μm or less.
[0044]
In this way, it is possible to realize a state where the subject cell 10 is securely held in the recess 7 during measurement. That is, since the culture solution 11 on the through-hole 6 side and the culture solution 11 on the well 5 side are in an electrically insulated state, the electric signal generated by the activity of the subject cell 10 is the culture solution on the well 5 side. 11 can be detected by the detection electrode 8 provided on the through-hole 6 side without leaking. The insulating layer 9 is necessary only when the signal generated near the through-hole 6 when the surface resistivity of the surface layer of silicon is low is small, and even if the signal leaks to the well 5 side, the measurement becomes impossible. Come.
[0045]
Therefore, if the surface resistivity of silicon itself is sufficiently high, sufficient electrical insulation can be realized just by holding the subject cell 10, and if the extracellular potential is large enough to cause little leakage The insulator 9 is not always necessary.
[0046]
Next, a method for manufacturing the extracellular potential measuring device of the present invention will be described with reference to FIGS.
[0047]
As shown in FIG. 6, the method for manufacturing the extracellular potential measuring device comprises preparing a substrate 1 comprising a base 2 made of silicon, an intermediate layer 3 made of silicon dioxide, and a thin plate 4 made of silicon. Then, a resist mask 12 is formed. The substrate 1 as described above is generally called an SOI substrate, and is often used when manufacturing a semiconductor device, and is easily available.
[0048]
Next, the through hole 6 is formed by dry etching until a predetermined etching depth of the thin plate 4 is reached. FIG. 7 is an enlarged view of a main part of the part A in FIG. The etching method at this time is optimally a dry etching method. In dry etching, it is important to use a gas that promotes etching and a gas that suppresses etching.
[0049]
The gas which promotes this etching is SF₆, CF₄, XeF₂Although these can be used, they have the effect of promoting the etching of silicon not only in the depth direction but also in the lateral direction.
[0050]
So CHF₃, C₄F₈, XeF₂By mixing a gas that suppresses etching such as CF, CF₂By forming the protective film made of the above polymer, the formation of the through hole 6 by dry etching can be advanced only below the resist mask 12 as shown in FIG.
[0051]
In addition, as another method of making the etching shape vertical, an almost vertical etching is performed by repeating a process of performing a small amount of etching with a gas that promotes etching and then forming a small amount of a protective film with a gas that suppresses etching. It can be shaped.
[0052]
In the experiment, SF was formed to form a through-hole 6 having a size of 5 μm.₆Is generated for 13 seconds while flowing 130 sccm, dry etching of about 1 μm is performed.₄F₈Is generated for 7 seconds while flowing 85 sccm to form a protective film of about 0.01 μm, and dry etching is repeated six times. As a result, a substantially vertical etching shape having a depth of about 7 μm can be obtained. did it. The protective film is formed not only on the wall surface of the through hole 6 but also on the bottom surface by the gas which suppresses the etching. However, the protective film formed on the bottom surface is a gas which promotes etching compared to the protective film formed on the wall surface. Can be easily removed, so that the etching can proceed only downward.
[0053]
Further, at the time of dry etching with a gas that promotes etching, a high frequency is applied to the substrate 1 by an inductive coupling method using an external coil to generate a negative bias voltage on the substrate 1, thereby increasing the positive ions SF in the plasma.₅ ⁺And CF₃ ⁺Collides against the substrate 1 so that the dry etching proceeds in a more vertical direction.
[0054]
Further, when suppressing the dry etching, no bias voltage is generated on the substrate 1 unless a high frequency is applied to the substrate 1, so that the CF which is a material of the protective film⁺Is no longer deflected, and a uniform protective film can be formed on the wall surface of the dry etching hole of the substrate 1.
[0055]
Further, as shown in FIG. 8, dry etching is performed until the intermediate layer 3 is reached by dry etching. What is important in this step is that when etching is performed by performing dry etching from the thin plate 4 side, as the etching proceeds, This means that the ratio of the gas that promotes the etching is gradually increased, or the etching time by the gas that promotes the etching is gradually increased. That is, in a process in which the process of promoting dry etching and the process of suppressing dry etching are repeated, the ratio of the process of promoting dry etching is gradually increased as dry etching proceeds.
[0056]
As a result, the shape of the etching is tapered so that the side of the well 5 is widened as shown in FIG. 8, and the through-hole 6 has an opening on the well 5 side smaller than an opening on the lower surface side of the thin plate 4. It can be formed to be large.
[0057]
In this dry etching step, since silicon dioxide is provided as the intermediate layer 3, when the dry etching of the through hole 6 is completed and reaches the intermediate layer 3, trouble does not occur even if the dry etching is not stopped immediately. do not do. This is because the etching gas does not immediately etch the intermediate layer 3.
[0058]
In particular, SF is used as a gas to promote etching.₆Is used, since this etching gas has a ratio of the etching rate of silicon to silicon dioxide of 10 or more, even if the dry etching of the through hole 6 is etched for a while after reaching the silicon dioxide, the etching gas is not in the middle of the silicon dioxide. Since the layer 3 is not easily removed, the recess 7 having high dimensional accuracy can be easily formed.
[0059]
Next, as shown in FIG. 9, after forming a resist mask 13 on the base 2 side by photolithography, the base 2 is etched by etching until reaching the intermediate layer 3 as shown in FIG. As an etching method at this time, dry etching using a gas that promotes etching and a gas that suppresses etching as described above is advantageous for arranging the wells 5 with high density, but arranging with such high density. If unnecessary, wet etching using TMAH or KOH can be used.
[0060]
Next, as shown in FIG. 11, the silicon dioxide intermediate layer 3 exposed on the bottom surface of the well 5 is removed by etching. The method at this time is not only wet etching with HF but also CF.₄Dry etching with gas or the like can be used.
[0061]
Thereafter, as shown in FIG. 12, after removing the resist mask 13, silicon dioxide is formed on the surface of the silicon constituting the base 2 and the thin plate 4 by a thermal oxidation method. Thereby, the insulating layer 9 made of silicon dioxide is formed on at least the inner wall and the bottom surface of the well 5, the inner wall of the recess 7, the inner wall of the through hole 6, and the lower surface of the thin plate 4.
[0062]
Next, as shown in FIG. 13, the detection electrode 8 is formed from the lower surface side of the thin plate 4 by vapor deposition or sputtering of gold. Thereby, the detection electrode 8 is formed not only on the lower surface of the thin plate 4 but also on the inner wall of the through hole 6. The material of the detection electrode 8 can be any electrode material that does not react with the culture solution 11, but it is particularly preferable to use gold, platinum, silver, silver chloride, aluminum, or the like. As to which electrode material is provided, an appropriate material should be selected depending on the type of the sample solution.
[0063]
As described above, by using the manufacturing method of the present invention, one photomask and one time from the side of the thin plate 4 can be formed in a state where the through hole 6 formed in the thin plate 4 is connected to the conical recess 7 on the well 5 side. The extracellular potential measurement device can be easily and accurately formed only by etching.
[0064]
(Embodiment 2)
The second and third aspects of the present invention will be described with reference to FIGS. FIG. 14 is a cross-sectional view for explaining the configuration of an extracellular potential measurement device and a method for manufacturing the same according to the second embodiment of the present invention, and FIGS. 15 and 16 are enlarged cross-sectional views of main parts thereof.
[0065]
Next, the configuration of the extracellular potential measuring device according to the second embodiment and a method for manufacturing the same will be described. The invention disclosed in the second embodiment has the same basic configuration as the extracellular potential measurement device disclosed in the first embodiment, and a description of the same configuration will be omitted, and only a different configuration will be described.
[0066]
FIG. 14 is a cross-sectional view of the extracellular potential measurement device according to the second embodiment, and is different in that the shape of the depression 21 formed in the thin plate 18 is hemispherical as shown in FIG. By forming the depression 21 in such a hemispherical shape, the subject cell 10 can be more closely held in the depression 21, so that the potential change of the culture solution 11 in the through hole 20 can be more easily captured. It is possible.
[0067]
Next, a method for manufacturing the extracellular potential measuring device will be described.
[0068]
The manufacturing method of the extracellular potential measuring device according to the second embodiment can be manufactured through substantially the same steps as the manufacturing method according to the first embodiment, except that a through hole 20 and a depression 21 are formed in the thin plate 18. Only the method will be described, and a method of manufacturing this part will be described with reference to the drawings.
[0069]
In this step, as shown in FIG. 15, in the state where the intermediate layer 17 and the thin plate 18 are laminated, a resist mask 24 is formed on the thin plate 18 side, and then a gas which promotes etching and etching is suppressed because the through hole 20 is formed. Dry etching is performed using a gas to be etched, and the etching is terminated when the etching reaches a predetermined depth. The predetermined depth is to stop before reaching the intermediate layer 17 made of silicon dioxide, and an optimum depth can be appropriately selected according to the size and shape of the depression 21.
[0070]
What is important at this time is that at the end of the etching, the supply of the gas for suppressing the etching is stopped, and the etching is performed.
[0071]
When dry etching is performed by repeating a process of forming a small amount of a protective film (not shown) with a gas that suppresses etching after performing a small amount of etching with a gas that promotes etching, the end of the etching accelerates the etching. It is important that the process be completed in the step of dry etching with the use of a gas. This is because the protective film formed by the gas for suppressing the etching is removed from the bottom surface of the etching.
[0072]
Next, as shown in FIG. 16, XeF₂Dry etching is performed by introducing a gas.
[0073]
By this operation, the dry etching proceeds from the bottom surface where silicon is exposed, and the etching proceeds so as to spread toward the well 19 side.
[0074]
At this time, since a protective film (not shown) formed by a gas that suppresses etching in the previous step is formed on the side wall of the through hole 20, XeF is formed.₂Is not dry etched. In this way, the depression 21 can be formed in a hemispherical shape as shown in FIG.
[0075]
Thereafter, similarly to the first embodiment, an extracellular potential measurement device can be manufactured through the steps shown in FIGS. 9 to 13.
[0076]
(Embodiment 3)
The third and fifth aspects of the present invention will be described with reference to FIGS. FIG. 17 is a cross-sectional view for explaining a method of manufacturing the extracellular potential measuring device according to the third embodiment of the present invention, and FIGS. 18 and 19 are enlarged cross-sectional views of the main part.
[0077]
In the third embodiment, another manufacturing method for forming the through holes 6 and 20 and the recesses 7 and 21 disclosed in the first and second embodiments will be disclosed.
[0078]
The only difference from the manufacturing method described in the first and second embodiments is the method of forming the through-hole 30 in the thin plate 26. The method of forming this portion will be described with reference to the drawings.
[0079]
FIG. 17 shows a state in which an intermediate layer 25 made of silicon dioxide and a thin plate 26 made of silicon are stacked, and a resist mask 27 is formed on the thin plate 26 side. This shows a state where dry etching has been performed for formation. In this state, the etching is performed excessively until the etching reaches the intermediate layer 25.
[0080]
Then, even after the dry etching reaches the intermediate layer 25, the etching is further continued. At this time, the intermediate layer 25 is an insulator, and the thin plate 26 made of silicon or the like has lower insulation resistance than the intermediate layer 25. If the above-described etching is continued excessively in such a configuration, the surface of the intermediate layer 25 is subjected to dry etching with a gas that promotes etching, as shown in FIG.₅ ⁺) Stays, and the etching ions 29 supplied from the plasma are repelled in the direction of the arrow shown in FIG.
[0081]
As a result, if the dry etching is excessively performed in such a configuration, the dry etching is intensively performed near the side wall of the intermediate layer 25, and as shown in FIG. Can be formed.
[0082]
In the experiment, a 10 μm depression 31 could be formed by excessively performing the dry etching of the through-hole 30 having a diameter of 3 μm even after reaching the intermediate layer 25.
[0083]
【The invention's effect】
As described above, according to the present invention, the substrate has a base and a laminated structure of an intermediate layer made of a material different from the base and a thin plate made of the same material as the base, and a through hole is provided in the well and the thin plate in the base, This through-hole is provided with a separate well by realizing a device for measuring extracellular potential or a physicochemical change generated by a cell and a method of manufacturing the same, in which the opening on the well side has a recess larger than the outer side. There is no need to provide an extremely compact extracellular potential measurement device in which wells and through holes are integrally formed in a substrate.
[0084]
In the case of manufacturing the above device, there is no need to perform photolithography on two types of masks from the well side, and even if the substrate has irregularities such as a well-provided substrate, a spray that uniformly coats the resist on the irregular surface. Provided is a manufacturing method in which extremely high-precision through holes and depressions are provided on the bottom of a well without requiring expensive equipment such as a coating apparatus or a projection or a stepper capable of exposing a high-precision pattern without contact between a photomask and a substrate. be able to.
[Brief description of the drawings]
FIG. 1 is a perspective view of an extracellular potential measurement device according to a first embodiment of the present invention.
FIG. 2A is a sectional view of the same, and FIG.
FIG. 3 is a sectional view showing the operation of the extracellular potential measuring device.
FIG. 4 is an enlarged sectional view of a main part of the same.
FIG. 5 is an enlarged sectional view of a main part of the same.
FIG. 6 is a cross-sectional view for explaining the method for manufacturing the extracellular potential measurement device according to the first embodiment of the present invention.
FIG. 7 is an enlarged sectional view of a main part of the same.
FIG. 8 is an enlarged sectional view of a main part of the same.
FIG. 9 is a sectional view of the same.
FIG. 10 is a sectional view of the same.
FIG. 11 is a sectional view of the same.
FIG. 12 is a sectional view of the same.
FIG. 13 is a sectional view of the same.
FIG. 14 is a cross-sectional view of the extracellular potential measurement device according to the second embodiment of the present invention.
FIG. 15 is an enlarged sectional view of a main part for explaining a method of manufacturing the extracellular potential measuring device.
FIG. 16 is an enlarged sectional view of a main part of the same.
FIG. 17 is an enlarged sectional view of a main part for describing a method of manufacturing the extracellular potential measuring device according to the third embodiment of the present invention.
FIG. 18 is an enlarged cross-sectional view of the main part.
FIG. 19 is an enlarged sectional view of a main part of the same.
FIG. 20 is a schematic cross-sectional view of a conventional extracellular potential measuring device.
[Explanation of symbols]
1 substrate
2 base
3 Middle class
4mm thin plate
5 well
6mm penetration
7 hollow
8 detection electrode
9 insulator
10 Subject cell
11 culture solution
12 resist mask
13 resist mask
15mm board
16 base
17 middle class
18mm thin plate
19 well
20mm penetration
21 hollow
22 detection electrode
23 insulator
24 resist mask
25 middle class
26mm thin plate
27 resist mask
28 ° etching ion
29 etching ion
30mm penetration
31 hollow

Claims (16)

The substrate has a base and an intermediate layer made of a material different from the base and a laminated structure of a thin plate made of the same material as the base, and at least one or more wells for mixing a test cell, a culture solution, and a drug in the base, An extracellular potential measuring device for measuring an extracellular potential or a physicochemical change generated by a cell, provided with at least one or more through-holes communicating with the outside of the well and the substrate in a thin plate. 2. The extracellular potential measuring device according to claim 1, wherein the through-hole has a recess on the well side, and the opening of the recess is larger than the opening of the through-hole. The extracellular potential measuring device according to claim 2, wherein the inner wall of the depression has a conical shape. 3. The extracellular potential measuring device according to claim 2, wherein the inner wall of the depression has a hemispherical shape. The extracellular potential measuring device according to claim 1, wherein the electric resistivity of the intermediate layer is higher than the electric resistivity of the thin plate. 2. The extracellular potential measuring device according to claim 1, wherein the materials constituting the intermediate layer and the thin plate are made of materials having different etching rate ratios to the same etching gas. The extracellular potential measuring device according to claim 1, wherein the base and the thin plate are made of silicon, and the intermediate layer is made of silicon dioxide. The extracellular potential measuring device according to claim 2, wherein the opening of the depression is 10 to 100 m, and the opening of the through hole is 1 to 10 m. The substrate has a base and an intermediate layer made of a material different from the base and a laminated structure of a thin plate made of the same material as the base, and at least one or more wells for mixing a test cell, a culture solution, and a drug in the base, A method for manufacturing an extracellular potential measurement device for measuring extracellular potential or physicochemical change generated by cells provided with at least one or more through-holes communicating with the outside of the well and the substrate in a thin plate, comprising: Forming the through-hole by dry etching using one type of mask on a thin plate; removing the intermediate layer at the bottom of the well by dry etching; A method for producing an extracellular potential measurement device, comprising a step of forming a detection electrode by a technique. In the step of providing a through hole by dry etching using one type of mask in a thin plate, the thin plate is formed by dry etching from the outer side, and this dry etching is at least two types of a gas that suppresses etching and a gas that promotes etching. The method for producing an extracellular potential measurement device according to claim 9, wherein the method is carried out by using. The method for producing an extracellular potential measurement device according to claim 10, wherein in the dry etching for forming the through-hole, a step of using a gas for suppressing etching and a step of using a gas for promoting etching are alternately repeated. The method for manufacturing an extracellular potential measurement device according to claim 10, wherein in the step of providing the through-hole, the etching is performed such that the ratio of dry etching using a gas that promotes etching and a gas that suppresses etching increases as the etching proceeds. In the step of providing a through-hole, dry etching performed by repeating the step of accelerating etching and the step of suppressing etching is stopped before reaching the intermediate layer made of silicon dioxide, and then the intermediate layer made of silicon dioxide is etched only with a gas that promotes etching. The method for manufacturing an extracellular potential measurement device according to claim 10, wherein dry etching is performed until the layer reaches the layer. In the step of using a gas that promotes etching and the step of using a gas that suppresses etching, an inductive coupling method using a coil is used as a means for decomposing the gas, and a high frequency is applied to the substrate in the step of promoting dry etching. The method for manufacturing an extracellular potential measuring device according to claim 10, wherein the step of suppressing the dry etching is performed without applying high frequency to the substrate. In the step of forming a through hole, a step of accelerating dry etching and a step of suppressing dry etching are repeatedly performed to dry-etch a thin plate excessively even after the dry etching reaches an intermediate layer mainly composed of silicon dioxide. Item 11. The method for producing an extracellular potential measurement device according to item 10. A method of manufacturing an extracellular measurement device in which a base and a thin plate are made of silicon and an intermediate layer is made of silicon dioxide, wherein a gas for promoting etching is SF ₆ , a gas used when forming a through hole in a lower layer. The extracellular potential measurement according to claim 10, wherein the gas is a gas containing any one of CF ₄ and XeF ₂ , and the gas for suppressing etching is any one of C ₄ F ₈ and CHF ₃ or a gas containing these. Device manufacturing method.

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