JP2009291135A - Cellular electrophysiological sensor - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To improve reliability in measurement by a cellular electrophysiological sensor by easily suppressing and removing bubbles. <P>SOLUTION: The cellular electrophysiological sensor includes a diaphragm 2 having at least one through-hole 1 and a frame 5 supporting the diaphragm 2 and having a cavity 6. The reliability of measurement by the cellular electrophysiological sensor is enhanced by using the diaphragm 2 having a laminar structure comprising a silicon layer 3 and a silicon dioxide layer 4 and making an indented form the surface of the silicon dioxide layer 4, thereby suppressing generation of bubbles and easily removing remaining bubbles. <P>COPYRIGHT: (C)2010,JPO&INPIT

Description

  The present invention relates to a cell electrophysiological sensor used for measuring a physicochemical change generated in an extracellular potential of a cell or a cell activity.

  As a conventional method for elucidating the function of an ion channel existing in a cell membrane by using the electrical activity of a cell as an index or screening (inspecting) a drug, a patch clamp method can be mentioned.

  In this patch clamp method, a small part of a cell membrane (referred to as a patch) is gently aspirated with the tip of the micropipette, and the membrane potential is obtained by fixing (clamping) the current across the patch using a microelectrode probe provided on the micropipette. Is measured under As a result, the state of opening and closing of one or a small number of ion channels present in the patch can be electrically recorded. This is one of the few methods that can examine the physiological function of cells in real time.

  However, the patch clamp method requires special techniques and skills to create and operate a micropipette, and it takes a lot of time to measure a single sample. Therefore, it is suitable for screening a large number of drug candidate compounds at high speed. Not.

  On the other hand, in recent years, a flat-plate microelectrode probe utilizing microfabrication technology has been developed, and this method is suitable for an automated system that does not require the insertion of a micropipette for each individual cell.

  For example, a plurality of through-holes are provided in a cell holding substrate, a subject cell is adhered to the opening of the through-hole, and a measurement electrode disposed below the through-hole is used to increase the potential-dependent ion channel activity of the subject cell. A technique for measuring is disclosed (for example, see Patent Document 1).

  Further, a 2.5 μm through hole is formed inside a silicon oxide cell holding substrate (membrane), and HEK293 cells, which are a type of human cultured cell line, are held in the through hole to provide high adhesion. Has been disclosed, and a technique for measuring the extracellular potential with high accuracy is disclosed (for example, see Non-Patent Document 1).

  Furthermore, as shown in FIG. 9, the conventional cell electrophysiological sensor 31 includes a cell holding substrate 32, a recess 33 formed on the upper surface of the cell holding substrate 32, and a lower portion of the cell holding substrate 32. A through hole 34 connected to the lower surface, a reference electrode 35 disposed above the cell holding substrate 32, and a measurement electrode 36 disposed inside the through hole 34 are provided. The measurement electrode 36 is connected to a signal detection unit via a wiring 37. The cell holding substrate 32 is disposed inside the well 38 (see, for example, Patent Document 2).

  Next, the operation method of the cell electrophysiological sensor 31 will be described below.

  First, the cells and the electrolytic solution 40 are injected into the well 38, and the cells are trapped (captured) by the concave portion 33 and held. The cells held in the recesses 33 are hereinafter referred to as subject cells 39.

At the time of measurement, the subject cell 39 is sucked from below the through hole 34 with a suction pump or the like, and is held in close contact with the opening of the through hole 34. That is, the through hole 34 plays the same role as the tip hole in the glass pipette. The functionality or pharmacological reaction of the ion channel of the subject cell 39 is analyzed by measuring the voltage or current before and after the reaction between the reference electrode 35 and the measurement electrode 36 and determining the potential difference inside and outside the cell. ing.
JP 2002-518678 Gazette International Publication No. 02/055653 Pamphlet T. Sordel et al, Micro Total Analysis Systems 2004, P521-522 (2004)

  However, the conventional cell electrophysiological sensor has a problem that an error occurs in the measured value of the potential difference between the reference electrode and the measurement electrode, and the reliability of the measurement by the cell electrophysiological sensor is lowered.

  This is because bubbles are likely to remain in the low hydrophilicity part of the inner wall surface of the flow path or on the cell holding substrate, and the resistance value of the bubbles is so large that the measured value varies depending on the presence or absence of the bubbles. .

  In particular, bubbles generated in the periphery of the through hole have been a factor that greatly fluctuates the measured current or voltage detected by the measurement electrode.

  Therefore, an object of the present invention is to realize a cell electrophysiological sensor excellent in measurement reliability by suppressing the generation of bubbles around a through hole and efficiently removing bubbles.

  In order to solve the above-mentioned conventional problems, the present invention comprises a diaphragm having a two-layer structure having a through hole, and a frame that supports the diaphragm and has a cavity. The diaphragm includes a silicon layer and a silicon dioxide layer. The surface of the silicon dioxide layer has a concavo-convex shape.

  The cell electrophysiological sensor of the present invention suppresses the generation of bubbles in the periphery of the through-holes by making the surface of the diaphragm silicon dioxide having excellent hydrophilicity and making the surface concavo-convex. The sensor structure can be removed efficiently. As a result, bubbles attached to the surface of the diaphragm can be removed, and the reliability of the measurement by the cell electrophysiological sensor can be improved.

(Embodiment 1)
Hereinafter, the configuration of the cell electrophysiological sensor according to Embodiment 1 of the present invention will be described with reference to the drawings.

  FIG. 1 is a cross-sectional view of a cell electrophysiological sensor according to Embodiment 1 of the present invention, and FIG. 2 is a plan view of FIG. FIG. 3 is a cross-sectional view for explaining a method for measuring a cell electrophysiological phenomenon using the cell electrophysiological sensor of the first embodiment.

  1 and 2, the cell electrophysiological sensor according to the first embodiment is prepared in a batch by preparing a wafer having excellent availability and processability such as a silicon substrate and performing etching using a photolithographic technique. In addition, it is possible to efficiently produce a cell electrophysiological sensor having a minute shape with high dimensional accuracy.

  In particular, an SOI substrate having a stacked structure of silicon and silicon dioxide is preferably used. This SOI substrate is often used when manufacturing semiconductor devices, and is easily available, and can be easily manufactured by designating the thickness of silicon dioxide to a predetermined thickness.

  The basic configuration of the cell electrophysiological sensor in the first embodiment is to form a thin diaphragm 2 having at least one through hole 1, and the diaphragm 2 is composed of silicon 3 and silicon dioxide 4. It has a laminated structure. And the surface of the said silicon dioxide 4 has the uneven | corrugated | grooved part 4b, By providing this uneven | corrugated | grooved part 4b, the surface of the diaphragm 2 can improve hydrophilicity more, and the movement and removal of a bubble are performed easily. Can do.

  In addition, since the subject cell 9a for measurement is sucked and fixed in the through hole 1, it is preferable to provide a smooth portion 4a having a smooth surface in the vicinity of the through hole 1. The smoothness is preferably 1 nm or less in terms of centerline average roughness (Ra). As a result, the retainability and airtightness of the subject cell 9a can be kept high, and damage to the coating surface of the subject cell 9a can be prevented. Accordingly, it is preferable to provide the region where the smooth portion 4a is provided in the silicon dioxide 4 at the peripheral portion of the through hole 1 so as to be slightly larger than the size of the subject cell 9a.

  The cellular electrophysiological sensor according to the first embodiment is provided with a frame 5 for fixing and supporting the diaphragm 2 and for facilitating a fixed arrangement on a measuring device or the like. By using the frame 5 as silicon, By processing a commercially available SOI substrate having silicon dioxide 4 disposed in the middle, a cell electrophysiological sensor having excellent dimensional accuracy can be efficiently realized.

  Note that at least one through-hole 1 is sufficient, and when it is desired to measure a plurality of cells 9 simultaneously, a plurality of through-holes 1 can be formed. Thus, the S / N ratio can be increased by simultaneously measuring a plurality of cells 9 as a plurality of subject cells 9a.

  Further, a cavity 6 capable of storing a liquid or the like is formed inside the frame 5. The cavity 6 is for storing a liquid containing cells 9 and the like. The cell electrophysiological sensor having such a configuration fixes the cell 9 from the plurality of cells 9 floating in the liquid so as to close the through-hole 1 by means such as suction, and the subject cell 9a. It can be. Thereafter, the reaction of the subject cell 9a with respect to the drug solution or the like is measured with a connected electrode.

  In particular, the feature of the configuration of the cell electrophysiological sensor in the first embodiment is that the diaphragm 2 has a laminated structure of silicon 3 and silicon dioxide 4, and the exposed surface of the silicon dioxide 4 has an uneven shape. . By adopting such a structure, the silicon dioxide 4 having excellent hydrophilicity is used as an exposed surface, and the exposed surface is formed into an uneven shape, whereby the hydrophilicity is further enhanced and the generation of bubbles on the surface of the diaphragm 2 is suppressed. Therefore, it is possible to realize a cell electrophysiological sensor that enables highly accurate measurement.

  Here, when a liquid in which droplet-like cells 9 are dispersed using a dispenser or the like is injected into the cavity 6 on the surface of the silicon dioxide 4 having the concavo-convex shape, the liquid is spherical by surface tension. It is necessary to dispense a predetermined amount of liquid into the cavity 6 via an automatic dispenser or the like as it is. In order to measure the electrochemical change of the subject cell 9a stably and accurately, it is important to fill the liquid around the through hole 1 with no bubbles remaining (FIG. 3). reference). In order to realize this, the surface of the diaphragm 2 has a surface structure with excellent hydrophilicity as shown in FIG. 1, so that the dropped liquid droplets quickly spread from the tip of the concavo-convex portion 4b. Thus, it is possible to realize the diaphragm 2 that can efficiently suppress the generation of bubbles and remove the bubbles.

  Here, as for the surface roughness of the uneven | corrugated | grooved part 4b which is an exposed surface of the silicon dioxide 4, Ra> = 5nm is preferable, More preferably, Ra: 5-200nm. It is because hydrophilicity will fall when Ra is less than 5 nm, and workability will fall when it exceeds 200 nm. It is preferable to use an etching technique for the uneven portion 4b from the viewpoint of productivity.

  Using the cell electrophysiological sensor having such a configuration, a liquid is filled in a state where no bubbles remain, for example, an extracellular fluid 10 in which cells 9 are dispersed inside the cavity 6 is stored, and one cell therein By trapping 9 on the through-hole 1 by suction means or the like, it is possible to obtain a subject cell 9a that can be measured with high accuracy.

  On the other hand, from the viewpoint of suppressing and removing the generation of bubbles, it is preferable that the exposed surface of the diaphragm 2 is composed entirely of silicon dioxide 4 composed of the concavo-convex portions 4b, but the subject cell 9a sucked into the through hole 1 is a thin film The thin film of the subject cell 9a may be damaged by the protrusions of the silicon dioxide 4.

  In order to prevent this, it is preferable that the surface of the peripheral portion of the through hole 1 has a smooth shape.

  Further, the subject cell 9a is sucked and fixed so as to block the through-hole 1, and the electrical change in the two regions separated by the through-hole 1 is measured, so that the hermeticity of the subject cell 9a in the through-hole 1 is measured. Is an important factor. In order to keep the airtightness high and uniform, the silicon dioxide 4 at the peripheral edge of the through-hole 1 is preferably composed of a smooth portion 4a. As a result, the electrical change of the subject cell 9a can be measured while stably maintaining the airtightness. Here, since the size of the cell 9 is several μm to 50 μm, it is preferable that the surface of the smooth silicon dioxide 4 is slightly larger than the size of the cell 9.

  On the other hand, in the sensor structure in which the silicon dioxide 4 having the concavo-convex portion 4b is not provided, when the liquid is dispensed using the micropipette, the droplet formed at the tip of the micropipette is dropped as it is. The problem was that bubbles such as air that existed in the cavity 6 could not be removed well and remained on the surface of the diaphragm 2 as bubbles.

  If the bubbles remain in the vicinity of the through-hole 1, a minute change in current or voltage, which is an electrochemical change of the subject cell 9a fixed above the through-hole 1 using suction means, is increased. It was difficult to measure accurately.

  In addition, in order to stably measure the electrophysiological phenomenon of the cell, the size of the through hole 1 is slightly smaller than the size of the subject cell 9a, and the subject cell is closed so as to close the through hole 1. It is preferable that 9a has a shape in which the subject cell 9a can be fixed by means such as suction, and the diameter of the opening of the through hole 1 is 3 μm.

  Thus, when the size of the cell 9 is about 5 to 50 μm, in order to hold the cell 9 and the through hole 1 with high adhesion, the diameter of the opening of the through hole 1 is set to 3 μm or less. This is because it is desirable.

  The optimum size of the opening of the through hole 1 can be determined by the shape and properties of the subject cell 9a to be measured.

  And it is preferable that the thickness of the diaphragm 2 shall be 10-300 micrometers from viewpoints, such as workability and mechanical strength.

  Moreover, it is preferable from a viewpoint of productivity that the internal diameter of the cavity 6 shall be 100 micrometers-2.0 mm. When the thickness is smaller than 100 μm, it becomes difficult to dispense liquid, and a large amount of bubbles remain. On the other hand, if it exceeds 2 mm, the productivity of the sensor is lowered and it is not suitable for measuring a liquid containing a small amount of cells 9.

  Moreover, it is preferable from the viewpoint of mechanical strength and handling that the diaphragm 2 is configured to support the diaphragm 2 and have a cavity 6 for storing liquid.

  Next, a method for measuring the electrophysiological activity generated when the cell 9 is activated using the cell electrophysiological sensor having the above-described configuration will be described in more detail with reference to the drawings. FIG. 3 is a schematic cross-sectional view of the measuring device when the cellular electrophysiological sensor is set in the measuring device.

  As shown in FIG. 3, the cell electrophysiological sensor is set inside a partition plate 14 provided inside a container 17 made of an insulator such as plastic. A well 15 for storing a liquid is disposed in the upper layer portion of the partition plate 14.

  The partition plate 14 and the well 15 are preferably made of resin or the like from the viewpoints of productivity, workability, dimensional accuracy, and the like.

  In addition, an opening is provided inside the partition plate 14, and the cell electrophysiological sensor is formed with the diaphragm 2 on the lower surface side inside the opening, and joined without gaps so that liquid leakage does not occur in the opening. The space inside the container 17 is divided into two regions with the diaphragm 2 as a boundary. In the upper and lower regions partitioned by the partition plate 14, a liquid such as the extracellular fluid 10 or the culture fluid and the intracellular fluid 11 are placed. Or liquids, such as a chemical | medical solution, will be stored, respectively. The movement of the liquid above and below these subject cells 9a is performed only through the through hole 1. In addition, a channel 16 is formed on the lower surface side of the partition plate 14 using a part of the container 17, and the intracellular solution 11 or the drug solution is provided inside the channel 16 by using a liquid feeding means such as a micropump. It is possible to fill and remove such liquids.

  Further, a reference electrode 12 composed of a silver / silver chloride electrode or the like is disposed in the extracellular solution 10 on the upper side of the partition plate 14, and a silver / silver chloride electrode is placed in the intracellular solution 11 on the lower side of the partition plate 14. The measurement electrode 13 comprised by these is arrange | positioned.

  The reference electrode 12 and the measurement electrode 13 may be interchanged. The reference electrode 12 and the measurement electrode 13 can be selected from materials consisting of chromium, titanium, copper, gold, platinum, silver, and silver chloride. Further, the reference electrode 12 and the measurement electrode 13 may be needle-shaped microelectrode probes.

  Next, with the cell potential measuring device as described above prepared, the cells 9 dispersed in a liquid such as the extracellular fluid 10 to be measured are dispensed from the upper side of the container 17 using a dispenser. Then, by using a suction pump or the like to control the suction force so that the lower side of the partition plate 14 becomes a low pressure, a predetermined pressure difference is generated between the upper and lower sides of the partition plate 14. At this time, one cell 9 is attracted and held by the opening of the through-hole 1 to form a subject cell 9a.

  And if this pressure difference is maintained, sufficient adhesiveness will be ensured and it will have an electrical resistance value between the extracellular fluid 10 and the intracellular fluid 11 which are inside.

  By controlling the suction pressure of the subject cell 9a, a part of the coating of the subject cell 9a is broken, and the intracellular fluid 11 is injected from the lower surface side of the through hole 1 of the subject cell 9a. A part of the subject cell 9a can be brought into contact with the intracellular fluid 11. As a result, the electrochemical change of the subject cell 9a can be measured.

  Next, in order to measure this electrical change, the reference electrode 12 is disposed so as to be connected to the extracellular fluid 10, and the measurement electrode 13 that is connected to the intracellular fluid 11 is disposed. By measuring the current or voltage change with the electrode 13, the electrochemical change of the subject cell 9a can be measured and detected.

  For example, when a stimulus such as a chemical compound such as a drug is applied to the subject cell 9a, the subject cell 9a exhibits an electrophysiological response. As a result, for example, a voltage is applied between the reference electrode 12 and the measurement electrode 13. It can be observed as an electrical response such as current.

Here, for example, in the case of mammalian muscle cells, the extracellular fluid 10 is typically an electrolytic solution to which about 4 mM of K ⁺ ions, about 145 mM of Na ⁺ ions, and about 123 mM of Cl ⁻ ions are added, For example, in the case of mammalian muscle cells, the intracellular solution 11 is typically an electrolytic solution to which K ⁺ ions are added at about 155 mM, Na ⁺ ions at about 12 mM, and Cl ⁻ ions at about 4.2 mM.

  In addition, the thing of a different composition may be used for the extracellular fluid 10 and the intracellular fluid 11 like this Embodiment 1, and you may use the same thing.

  When the well 15 is filled with the extracellular fluid 10, a resistance value of about 100 kΩ to 10 MΩ is generated between the reference electrode 12 installed in the well 15 and the measurement electrode 13 installed in the flow channel 16. Can be observed. This is because the electrolytic solution penetrates through the through hole 1 provided in the cell electrophysiological sensor, and an electric circuit is formed between the electrodes 12 and 13.

  Next, for example, one of the channels 16 is sealed, and the pressure is reduced from the other, so that the cells 9 are attracted to the through-holes 1. The electrical resistance between the cavity 6 filled with 10 and the flow path 16 filled with a liquid such as the intracellular fluid 11 is sufficiently high.

  Then, by further reducing the pressure or introducing a chemical solution having an action of dissolving the outer wall of the subject cell 9a, such as nystatin, into the flow channel 16, a minute hole is formed in the subject cell 9a.

  Thereafter, chemical stimulation or physical stimulation is applied to the subject cell 9a. Examples of chemical stimuli include chemicals, poisons, and physical stimuli include mechanical mutation, light, heat, electricity, and electromagnetic waves.

  When the subject cell 9a actively responds to these stimuli, the subject cell 9a releases or absorbs various ions through ion channels in the cell membrane. Then, an ionic current passing through the subject cell 9a is generated, and the potential gradient inside and outside the subject cell 9a changes. Therefore, this change is caused by the voltage or current between the reference electrode 12 and the measurement electrode 13 before and after the reaction. Can be detected by measuring.

  At this time, if bubbles exist in the vicinity of the through-hole 1, the measured value of the current / voltage detected by the measurement electrode 13 fluctuates due to the increase of the resistance value. However, the cell electrophysiology having the configuration as shown in FIG. Cell electrophysiological sensor with improved measurement reliability because it has a sensor structure that can suppress the generation of residual bubbles by using the sensor and can quickly remove the remaining bubbles. Can be realized.

  Next, the manufacturing method of the cell electrophysiological sensor in this Embodiment 1 is demonstrated using drawing. 4-8 is sectional drawing of the process for demonstrating a manufacturing method.

  First, as shown in FIG. 4, an SOI substrate including a silicon layer 20 having a thickness of about 20 μm, a silicon dioxide layer 21 having a thickness of about 2 μm, and a silicon layer 22 having a thickness of 400 to 500 μm is prepared, and the through hole 1 is formed. A resist mask (not shown) is formed on one surface of the silicon layer 20 to be formed. At this time, the shape of the mask hole of the resist mask is designed to be substantially the same as the desired shape of the through hole 1. In the first embodiment, since the minimum opening diameter of the through hole 1 is 3 μm, the opening diameter of the mask hole is also 3 μm.

Thereafter, etching is performed from the surface of the silicon layer 20 by using a dry etching technique to form a recess 23 that becomes a part of the through hole 1. At this time, when the recess 23 is formed with high accuracy, it is preferable to perform dry etching processing using alternately an etching gas such as SF ₆ that promotes etching and a gas that suppresses etching. Gases for suppressing this etching include CHF ₃ , C ₄ F _{8 and the} like. When a gas for suppressing this etching is sprayed on the etched wall surface, a protective film that is a polymer of CF ₂ is formed on the surface. The concave portion 23 can be formed substantially perpendicularly from the deepest portion of the concave portion 23 forming the through hole 1 toward the exposed surface of the silicon layer 20. When SF ₆ or the like that promotes silicon etching is used as the dry etching gas, the silicon dioxide layer 21 becomes an etching stop layer due to the difference in etching grade. By using such an etching technique, the depth of the through hole 1 can be formed in a predetermined shape with high accuracy, and a manufacturing method that can be easily manufactured even if it is a minute shape can be obtained.

Next, as shown in FIG. 5, dry etching is performed from the recess 23 to the lower surface of the silicon dioxide layer 21, thereby forming a via hole that becomes the through hole 1. At this time, the through-hole 1 can be formed by etching the silicon dioxide layer 21 from the upper surface of the SOI substrate using a gas such as CF ₄ . Thereafter, the resist mask is removed.

Next, after forming a resist mask (not shown) on the exposed surface of the silicon layer 22, etching similar to the above is performed using an etching gas such as SF ₆ that promotes etching from the silicon layer 22 to the silicon dioxide layer 21. By etching using the method, the frame 5 and the cavity 6 can be formed as shown in FIG. Also at this time, since the silicon dioxide layer 21 becomes an etching stop layer, the frame 5 and the cavity 6 can be manufactured with high accuracy.

  In the manufacturing method, it is possible to manufacture the sensor at once using an SOI substrate. When the resist mask for each manufacturing process is devised, a large number of cell electrophysiological sensors are manufactured at once. can do.

Next, as shown in FIG. 7, a resist mask (not shown) is formed at a peripheral portion of the through hole 1 on the cavity 6 side of the silicon dioxide layer 21 so as to have a predetermined dimension. Here, a resist mask having a circular shape of 20 μmφ was formed. The resist mask can be formed using a dispenser or an ink jet method. After the resist mask is formed, an etching process for forming irregularities on the exposed surface of the silicon dioxide layer 21 is performed. As the etching gas used for this, the surface of the silicon dioxide layer 21 exposed by performing plasma etching using a mixed gas of CHF ₃ and Ar gas can be processed to form the uneven portion 4b. At this time, the surface roughness of the concavo-convex portion 4b can be controlled by changing the plasma generation condition. By controlling this, the surface roughness of the concavo-convex portion 4b is formed in the range of Ra ≧ 5 to 100 nm. For each sample, its hydrophilicity and the state of bubbles are confirmed for each surface roughness, and it has a surface property that can efficiently suppress and remove bubbles in the above range. Was confirmed.

  Moreover, the smooth part 4a can be formed in the peripheral part of the through-hole 1 by removing a resist mask after uneven | corrugated processing. At this time, the size of the smooth portion 4a is preferably changed according to the size of the cell to be measured, and is preferably designed to be slightly larger than the size of the cell. Thus, a part of the subject cell 9a comes into contact with a part of the smooth portion 4a. If the surface has a smoothness of 1 nm (Ra) or less, the skin of the subject cell 9a is damaged. A cell electrophysiological sensor capable of exhibiting high sealing performance (G sealing performance) due to the fact that adhesion does not enter and that adhesion is enhanced when sucked through the through-hole 1, and can be measured with high accuracy. be able to.

  In addition, as a method of forming the uneven portion 4b, a sand blast method, a chemical etching method such as HF, or the like can be used as appropriate.

  Next, the configuration of another example of the cell electrophysiological sensor will be described with reference to FIG.

  The cellular electrophysiological sensor shown in FIG. 8 differs greatly from the configuration of the cellular electrophysiological sensor shown in FIG. 1 in that a silicon dioxide film 25 is formed on the inner wall surface of the through hole 1 and the inner wall surface of the frame 5. It is that. This makes it possible to realize a cell electrophysiological sensor that can more efficiently suppress and remove bubbles. This is because the surface of silicon that comes into contact with the liquid is covered with a silicon dioxide film 25 to enhance the hydrophilicity, thereby suppressing the generation of bubbles from the respective wall surfaces, and reducing the residual of bubbles while reducing the residual of bubbles. It can be filled with a liquid. In particular, at least the inner wall surface of the through hole 1 and the inner wall surface of the frame 5 are preferably covered with the silicon dioxide film 25. The reason is that it is a portion that is easily affected by bubbles when the subject cell 9a is measured.

  In order to fabricate such a cell electrophysiological sensor, the surface of the exposed silicon is thermally oxidized at a temperature of 700 ° C. or higher and in the presence of oxygen to thermally oxidize the surface of the silicon, thereby forming a silicon dioxide film. 25 can be formed. It is also effective to perform heat treatment by adding water vapor.

  And the film of the silicon dioxide film 25 by this thermal oxidation is preferably 5 nm or more. If the thickness is less than 5 nm, it is difficult to obtain a uniform thermal oxide film with few defects.

  And in order to maintain the hydrophilicity of the surface of this cell electrophysiological sensor, it is preferable to make it the clean surface which removed the carbon molecule adhering to the surface. After cleaning, the cleanliness of the surface can be maintained by storing the sensor immediately inside a container filled with pure water. Further, the hydrophilicity at this time is preferably 10 ° or less in terms of contact angle. This contact angle can be measured by applying pure water droplets from which ions have been removed to the surface of the object to be measured.

  Thus, it is more preferable that all surfaces such as the inner wall surface of the cavity 6, the exposed surface of the diaphragm 2, and the inner wall surface of the through hole 1 are in contact with each other. Thereby, the generation of bubbles can be further suppressed by increasing the wettability with the liquid for various measurements. Thereby, the wettability between the wall of the cell electrophysiological sensor and a liquid such as a chemical solution can be improved, and the liquid can be quickly filled into the through hole 1 from the cavity 6 and there is no bubble around the through hole 1. A cell electrophysiological sensor that can be filled in a state can be realized.

  This realizes a cell electrophysiological sensor that can suppress the generation of bubbles and efficiently remove the remaining bubbles.

  Since the cellular electrophysiological sensor of the present invention can easily suppress the generation of bubbles and remove the remaining bubbles, it is possible to improve the reliability of the measurement of the cell electrophysiological sensor. Therefore, the present invention has great applicability as a cellular electrophysiological sensor in the medical field where high-precision measurement is required.

Sectional drawing of the cell electrophysiological sensor in Embodiment 1 of this invention Plan view Sectional drawing for demonstrating the method to measure the electrophysiological phenomenon of the cell Process sectional drawing for demonstrating the manufacturing method of the same cell electrophysiological sensor Sectional view Sectional view Sectional view Sectional view of another example of cellular electrophysiological sensor Sectional view of a conventional extracellular potential measurement sensor

Explanation of symbols

DESCRIPTION OF SYMBOLS 1 Through-hole 2 Diaphragm 3 Silicon 4 Silicon dioxide 4a Smooth part 4b Uneven part 5 Frame 6 Cavity 9 Cell 9a Test cell 10 Extracellular fluid 11 Intracellular fluid 12 Reference electrode 13 Measurement electrode 14 Partition plate 15 Well 16 Channel 17 Container 20 Silicon layer 21 Silicon dioxide layer 22 Silicon layer 23 Recess 25 Silicon dioxide film

Claims (5)

A cellular electrophysiological sensor comprising a diaphragm having at least one through-hole and a frame supporting the diaphragm and having a cavity, wherein the diaphragm has a laminated structure of a silicon layer and a silicon dioxide layer, and the silicon dioxide A cell electrophysiological sensor having an irregular surface on the surface of the layer. The cellular electrophysiological sensor according to claim 1, wherein the surface roughness of the uneven shape of the silicon dioxide layer is 5 nm or more in terms of centerline average roughness (Ra). The cell electrophysiological sensor according to claim 1, wherein the surface of the silicon dioxide layer at the periphery of the through hole is smoothed. The cell electrophysiological sensor according to claim 3, wherein a region of the silicon dioxide layer at a peripheral portion of the through hole having a smooth surface is made larger than a size of the cell. The cell electrophysiological sensor according to claim 1, wherein at least the inner wall surfaces of the cavity and the through hole are covered with a silicon dioxide film.