JP4915293B2 - Cell electrophysiological sensor device and cell electrophysiological sensor using the same - Google Patents

Description

  The present invention relates to a cell electrophysiological sensor device and a cell electrophysiological sensor using the same.

  The patch clamp method in electrophysiology is known as a method for measuring an ion channel existing in a cell membrane, and various functions of the ion channel have been elucidated by this patch clamp method. And the action of ion channels is an important concern in cytology, which has also been applied to drug development.

  The cell electrophysiological sensor is an application of this patch clamp method, and is used, for example, in drug screening for detecting a reaction generated by a cell by a chemical substance.

  Here, as shown in FIG. 15, the conventional device for cell electrophysiology sensor includes a resin substrate as the base material 1 and a cell holding chip 2 fitted in the hole of the base material 1, and the cell holding chip 2. Has a conduction hole 3. And when this conduction hole 3 is very fine, it can form with high precision by dry-etching using silicon as the cell holding chip 2.

  The cell holding chip 2 and the substrate 1 are bonded with an adhesive 4. Here, since the cell holding chip 2 is very small, it is difficult to apply the adhesive 4 to the side surface of the cell holding chip 2, and the adhesive 4 was applied from the outside after being fitted into the hole of the base material 1.

  The conventional cell electrophysiological sensor includes the above-described device for cell electrophysiological sensor, electrolytic baths 5A and 5B arranged above and below the cell holding chip 2, and extracellular fluid and inside injected into these electrolytic baths 5A and 5B. Electrodes 6A and 6B for measuring the potential of an electrolytic solution such as a liquid are provided.

  This cell electrophysiological sensor captures the cell 7 at the opening of the conduction hole 3 and measures the potential difference between the electrolytic cells 5A and 5B, thereby changing the potential inside and outside the cell 7 when the cell 7 is active. Alternatively, a physicochemical change caused by the activity of the cell 7 can be measured.

In addition, the following patent document is mentioned as what discloses the example similar to the said cell electrophysiological sensor.
JP 2007-010430 A

  The conventional device for cell electrophysiology sensor has a problem that the measurement accuracy of the cell electrophysiology sensor is low.

  The reason is that the airtightness between the cell holding chip 2 and the substrate 1 was low.

  That is, conventionally, since the base material 1 and the cell holding chip 2 are joined by the adhesive 4, the adhesive strength is low. Further, since the cell holding chip 2 is very small, it is very difficult to uniformly apply the adhesive 4 to the side surface. Therefore, a gap is easily generated between the side surface of the cell holding chip 2 and the base material 1, and an electrical path is formed between the electrolytic cells 5A and 5B, resulting in a decrease in measurement accuracy.

  Accordingly, an object of the present invention is to improve the measurement accuracy of a cellular electrophysiological sensor.

  In order to achieve this object, according to the present invention, the base material is made of glass, and a hydrophilic film made of at least one of silicon oxide and silicon nitride is formed on the side surface of the cell holding chip. It was assumed that it was welded to the membrane.

  Thereby, this invention can improve the measurement precision of a cell electrophysiological sensor.

  The reason is that the airtightness between the side surface of the cell holding chip and the substrate is enhanced.

  That is, in the present invention, since the base material is glass and the side surface of the cell holding chip is hydrophilic, when the base material is heated, the glass component of the molten base material becomes familiar with the entire circumference of the side surface of the cell holding chip. Thereafter, the glass component of the base material is welded to the hydrophilic film on the side surface of the cell holding chip.

  Therefore, the gap between the side surface of the cell holding chip and the base material is reduced, the airtightness is increased, and as a result, the measurement accuracy of the cell electrophysiological sensor can be improved.

(Embodiment 1)
As shown in the cross-sectional view of FIG. 1, the cell electrophysiological sensor device according to Embodiment 1 of the present invention includes a resin substrate 8, a base material 9 inserted into a hole of the resin substrate 8, and the base material 9. And a cell holding chip 11 fitted in a through-hole 10 that connects the upper and lower surfaces. The base material 9 used in the present embodiment has a tube shape that covers the outer periphery of the side surface of the cell holding chip 11.

  The cell holding chip 11 according to the present embodiment includes a thin plate 12 and a frame 13 formed on the upper surface side of the thin plate 12, and the thin plate 12 is formed with a conduction hole 14 that connects the upper and lower surfaces. Yes. In the present embodiment, the thickness of the thin plate 12 is 1 to 5 μm, and the diameter of the conduction hole 14 is 1 to 5 μm. This size is suitable for trapping cells 15 having a diameter of 10 to 20 μm. The frame 13 has a horizontal thickness of 150 μm, and the base material 9 has an inner diameter of 0.75 mm, an outer diameter of 1.5 mm, and a thickness of 0.375 mm.

  Further, the cell holding chip 11, that is, the thin plate 12 and the frame body 13 in the present embodiment are formed of silicon, and the base material 9 is formed of glass. Since the cell holding chip 11 is made of silicon, the conduction hole 14 on the order of μm can be formed with high accuracy by a dry etching technique.

  Further, a hydrophilic film 16 made of silicon dioxide is formed on the side surface of the cell holding chip 11, that is, on the side surfaces of the frame body 13 and the thin plate 12 so as to cover almost all of the side surfaces. Nine glass components are welded.

  In addition to silicon dioxide, the hydrophilic film 16 is preferably made of a material that is hydrophilic and can be generated by a reaction on the silicon surface, such as silicon oxide, silicon nitride, or a mixture of silicon oxide and silicon nitride. In particular, since silicon dioxide or the like can be covalently bonded to the molten glass, the bonding becomes stronger.

  Further, by forming the hydrophilic film 16 as uniformly as possible, the cell holding chip 11 can be stably bonded to the base material 9.

  Moreover, in this Embodiment, as shown in FIG. 1, said cell electrophysiological sensor device is used for a cell electrophysiological sensor.

  That is, this cell electrophysiological sensor includes a cell electrophysiological sensor device, electrolytic cells 17A and 17B provided above and below the cell holding chip 11, and electrodes 18A for measuring potentials in these electrolytic cells 17A and 17B, respectively. , 18B. A sealing material 19 was attached to the lower electrolytic cell 17B so as to seal the electrolytic cell 17B, and a pressure transmission tube 20 for sucking the liquid in the electrolytic cell 17B was installed.

  Next, a method for measuring the electrophysiological activity of a cell using the cell electrophysiological sensor in this embodiment will be described.

  First, an extracellular solution is filled in the upper electrolytic cell 17A shown in FIG. 1, and an intracellular solution is filled in the lower electrolytic cell 17B so as not to enter bubbles, and the electrodes 18A and 18B are brought into contact with the extracellular solution and intracellular solution, respectively. Let

Here, for example, in the case of mammalian muscle cells, the extracellular fluid 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. 10 is 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.

  In this state, a conduction resistance value of about 100 kΩ to 10 MΩ can be measured between the electrodes. This is because the extracellular fluid or the intracellular fluid permeates the conduction hole 14 and the two electrodes are conducted through the extracellular fluid and the intracellular fluid.

  Next, when the cells 15 are introduced from the upper side of the upper electrolytic cell 17A and the pressure is reduced by the pressure transmission tube 19, the cells 15 are attracted to the conductive holes 14, block the conductive holes 14, and the upper and lower electrolytic cells 17A, 17B. Is in a state of 1 GΩ or higher. In this state, when the potential inside and outside the cell changes due to the electrophysiological activity of the cell 15, even a slight potential difference or current can be measured. Here, at the time of this measurement, reducing the electrical path not passing through the cell 15 between the electrolytic cells 17A and 17B as much as possible contributes to improvement in measurement accuracy.

  A method for manufacturing the device for cellular electrophysiological sensor of the present embodiment will be described below.

  First, in the first step, a substrate 21 made of silicon as shown in FIG. 2 is prepared, and a first etching resist film 22 is formed in a predetermined pattern on the surface of the substrate 21. The substrate 21 is usually called a Si substrate and is generally available.

Next, in the second step, as shown in FIG. 3, a gas that promotes etching (hereinafter referred to as a promotion gas) and a gas that suppresses (hereinafter referred to as a suppression gas) alternately from the opening of the first etching resist film 22. The conductive hole 14 is formed in the substrate 21 by introduction. As an etching method at this time, it is preferable to use a dry etching method using ICP plasma, for example, SF ₆ or CF ₄ is used as a promoting gas, and C ₄ F ₈ or CHF ₃ is used as a suppressing gas. Thus, the substrate 21 made of silicon is etched when the gas to be promoted is introduced, and a protective film is formed on the etched inner wall when the suppression gas is introduced. Therefore, the etching is performed by optimizing the combination of these etching gases. It progresses only under the opening of one etching resist film 22, and the conductive hole 14 can be etched into a substantially vertical shape.

  Thereafter, as a third step, the first etching resist film 22 is removed as shown in FIG.

  Next, as a fourth step, as shown in FIG. 5, a second etching resist film 23 is formed in a predetermined pattern on the surface of the substrate 21 (the surface opposite to the surface on which the conduction holes 14 are formed).

  Thereafter, as a fifth step, as shown in FIG. 6, a gas that promotes etching (promoting gas) and a gas that suppresses (suppressing gas) are alternately introduced from the opening of the second etching resist film 23. When 21 is etched, the cell holding chip 11 composed of the thin plate 12 and the frame body 13 can be formed. The etching method at this time is the same as the method for forming the conduction hole 14 described above.

  Next, as a sixth step, a hydrophilic film (16 shown in FIG. 1) made of silicon dioxide is formed on the side surface of the cell holding chip 11 by a method such as CVD, thermal oxidation, or vapor deposition.

  Thereafter, the cell holding chip 11 is inserted into the through hole 10 of the glass tube, which is the base material 9 shown in FIG. 1, and the outer peripheral surface of the cylindrical base material 9 is heated with a flame such as a gas burner. The glass component 9 is melted, and the hydrophilic film 16 on the inner peripheral surface side of the through hole 10 of the base material 9 and the outer peripheral surface of the cell holding chip 11 are directly bonded. The gas burner is used here because the base material 9 and the cell holding chip 11 can be locally heated and fused. The heating temperature at this time is preferably about 200 ° C to 1500 ° C. Moreover, in this Embodiment, since sufficient heating time was taken on high temperature conditions, the hydrophilic film | membrane 16 and the base material 9 of the cell holding chip 11 side surface can be covalently bonded. Thus, by bonding at least part of the hydrophilic film 16 to the base material 9, the adhesive strength between the cell holding chip 11 and the base material 9 can be further increased.

  And finally, in the present embodiment, an adhesive is applied to the lower end portion of the side surface of the base material 9 shown in FIG. 1, and the base material 9 is inserted into the hole of the resin substrate 8, thereby Forming.

  The effect in this Embodiment is demonstrated below.

  The cell electrophysiological sensor device of the present embodiment can improve the measurement accuracy of the cell electrophysiological sensor.

  The reason is that the airtightness between the side surface of the cell holding chip 11 and the substrate 9 is increased.

  That is, in the present embodiment, glass is used as the base material 9, and the hydrophilic film 16 made of silicon dioxide is formed on the side surface of the cell holding chip 11. When the base material 9 is heated here, the glass component of the melted base material 9 is welded to the side surface of the cell holding chip 11, but since the glass is hydrophilic, the hydrophilic film 16 is formed as described above. The glass component is well adapted to the side surface of the cell holding chip 11. And the glass component which became familiar to the side surface perimeter of the cell holding chip | tip 11 is firmly joined directly by thermally welding to the side surface of the cell holding chip | tip 11. FIG.

  Accordingly, the gap between the side surface of the cell holding chip 11 and the base material 9 can be reduced, the airtightness can be increased, and the leakage current can be suppressed. As a result, the measurement accuracy of the cell electrophysiological sensor can be improved. it can.

  In addition, although a glass plate etc. may be used as the base material 9, the direction using a glass tube is excellent in repair property. Further, since glass is less expensive than silicon, the size of the base material 9 can be increased, and the workability is improved as compared with the case where only the chip is fitted into the resin substrate 8.

  In the present embodiment, since the hydrophilic film 16 made of silicon dioxide exhibits electrical insulation, it is possible to suppress the formation of an electrical path at the interface between the side surface of the cell holding chip 11 and the base material 9. This contributes to further improvement in measurement accuracy. The thickness of the hydrophilic film 16 is proportional to the surface area of the side surface of the chip, but in the case of the present embodiment, electrical insulation can be improved by setting it to 0.1 μm or more. As the material of the hydrophilic film 16, silicon nitride or the like also shows electrical insulation, but silicon dioxide has a lower dielectric constant and thus has higher insulation and can improve measurement accuracy.

  The cell holding chip 11 may be a flat plate shape. However, when the thin plate 12 and the frame body 13 are formed as in the present embodiment, the frame body 13 even when the thin plate 12 is thin, that is, when the conduction hole 14 is shortened. Therefore, the bonding area with the base material 9 can be increased, and the adhesion strength between the cell holding chip 11 and the base material 9 can be increased. Note that the depth of the conduction hole 14 needs to be adjusted according to the size and nature of the cell 15 as the subject.

  Moreover, in this Embodiment, since the base material 9 was inserted in the resin substrate 8, the several cell holding | maintenance chip | tip 11 can be mounted on the one resin substrate 8, and it is suitable for a large amount of chemical | medical agent screening etc.

  In the present embodiment, a Si substrate (silicon) is used as the cell holding chip 11. However, as shown in FIG. 7, a substrate in which an etching stop layer 24 is provided between silicon may be used. Examples of the stop layer 24 include an SOI substrate using silicon dioxide having a slower etching rate than silicon. By disposing the etching stop layer 24 on the surface of the thin plate 12 and on the joint surface side with the frame 13, that is, at the interface between the thin plate 12 and the frame 13, the thickness of the thin plate 12 and the depth of the conduction hole 14 are reduced. It can be easily processed into a desired size.

Here, when silicon dioxide is used as the etching stop layer 24 as described above, it is preferable to use, for example, CF ₄ , Ar, etc. as a gas for promoting etching in the plasma etching process of silicon dioxide. Thereby, silicon dioxide can be preferentially etched, and unnecessary etching of silicon can be suppressed.

(Embodiment 2)
The difference between the present embodiment and the first embodiment is that the outer diameter of the thin plate 12 is formed to be smaller than the outer diameter of the frame body 13 as shown in FIG. This can be formed by adjusting the size and arrangement of the etching resist film when forming the thin plate 12 and when forming the frame 13 in the dry etching process. In the present embodiment, the hydrophilic film 16 is formed on the side surface of the frame body 13.

  As in the first embodiment, the thickness of the thin plate 12 in the present embodiment is about 1 μm to 5 μm, which is very thin compared to the thickness of the frame 13 (about 150 μm).

  In this Embodiment, since the thin plate 12 is located inside the frame 13, the stress which the thin plate 12 receives from the base material 9 can be reduced, and it can suppress that the thin plate 12 breaks. Such a structure is effective because the thickness of the thin plate 12 is reduced and the mechanical strength is reduced when the conduction hole 14 needs to be shortened.

  In addition, when the base material 9 and the cell holding chip 11 having different linear expansion coefficients are thermally welded as in the present embodiment, the thin plate 12 is attached to the frame body 13 because stress is easily applied by thermal shrinkage after molding. It is particularly effective to arrange it more inside.

  In the present embodiment, since the thin plate 12 is formed inside the frame body 13, a gap is formed on the outer periphery of the side surface of the thin plate 12. In the present embodiment, this gap is used as the glass reservoir 25, and the molten glass component 26 can be stored in the glass reservoir 25.

  Thereby, in this Embodiment, it can suppress that a glass component wraps around the thin plate 12 surface, and an unevenness | corrugation is made.

  In the present embodiment, since the glass component 26 attached to the glass reservoir 25 serves as a stopper and can hold the cell holding chip 11, the cell holding chip can be used even during or after the heat welding process. 11 can be prevented from sliding off the base material 9.

(Embodiment 3)
The difference between the present embodiment and the first embodiment is that, as shown in FIG. 9, the hydrophilic film 16 is inserted into the cell holding chip 11 through the cell holding surface (upper surface) of the thin plate 12 from the upper opening of the conduction hole 14. And the side surface of the cell holding chip 11 are covered with an integral hydrophilic film 16.

  Accordingly, in the present embodiment, the airtightness between the base material 9 and the cell holding chip 11 is improved as in the first embodiment, and the hydrophilicity on the upper surface of the thin plate 12 is improved. Adhesion with the 14 opening is improved, and generation of bubbles in the vicinity of the conduction hole 14 can be suppressed, thereby improving measurement accuracy.

  In the present embodiment, since silicon dioxide is used as the hydrophilic film 16, the hydrophilic film 16 has electrical insulation. Therefore, the generation of capacitance in the electrical leak path can be suppressed by the insulating hydrophilic film 16, and the capacitance component on the current waveform can be suppressed. As a result, a minute change in current that flows when the cell 15 reacts can be detected with high accuracy.

  That is, as shown in FIG. 10, when the cell electrophysiological sensor of the present embodiment is represented by an equivalent circuit, the cell holding chip 11 is made of semiconductor silicon, and the electrolytic layer 17A is formed above and below the cell holding chip 11. Since 17B is arranged, a capacitive component is generated at the interface between the extracellular fluid and the intracellular fluid and the cell holding chip 11, and a resistance component is generated inside the cell holding chip 11, and the subject cell is formed in the opening of the conduction hole 14. Is in close contact, a resistance component is generated at the interface. Here, for example, if the capacitance component at the interface between the extracellular fluid and the cell holding chip 11 is large, the current response when a rectangular voltage is applied between the two electrodes becomes a distorted waveform with a large capacitance component, which is observed. It becomes difficult to understand the potential change through the cell. That is, a slight potential change due to the physicochemical reaction of the cell 15 cannot be measured with high accuracy if it is buried in the above-described capacitance component.

  In contrast, in the present embodiment, since the hydrophilic film 16 formed from the upper surface to the side surface of the cell holding chip 11 is insulative, the capacity component at the interface between the extracellular fluid and the cell holding chip 11 can be reduced. . Furthermore, by reducing one capacitance component, the combined capacitance value between the electrodes 18A and 18B disposed in the respective electrolytic cells 17A and 17B can be reduced. As a result, the generation of stray capacitance in the cell electrophysiological sensor can be suppressed, and only the potential change due to the cell reaction can be detected, so that highly accurate measurement is possible.

  When the insulating hydrophilic film 16 functions as an insulating film, the film thickness is preferably increased in proportion to the surface area of the cell holding chip 11, for example, when the diameter is 700 μm, the thickness is 0.1 μm or more. Is desirable. Thereby, the stray capacitance value can be suppressed to 100 pF or less, and highly accurate measurement can be performed.

  Further, as shown in FIG. 11, by forming an insulating hydrophilic film 16 on the lower surface side of the thin plate 12, it is possible to suppress the generation of bubbles and reduce the generation of stray capacitance. As shown in this embodiment, the formation of the hydrophilic film 16 on the upper surface side of the thin plate 12 effectively improves the stray capacitance in the electrical path that leads from the upper surface of the thin plate 12 to the electrolytic cell through the conduction hole 14. Can be reduced.

  Further, in this embodiment, the insulating hydrophilic film 16 is not formed on the surface of the thin plate 12 opposite to the cell holding surface, but it is formed on both the cell holding surface (upper surface) and the opposite surface (lower surface). May be. Further, it may be formed inside the conduction hole 14. For example, as shown in FIG. 12, an insulating hydrophilic film 16 may be formed on the entire surface of the cell holding chip 11. Thereby, generation | occurrence | production of a bubble and generation | occurrence | production of a floating capacity | capacitance can be suppressed effectively. Further, for example, even when the insulating hydrophilic film 16 on either the upper surface or the lower surface of the thin plate 12 is scratched and the silicon surface is exposed, the capacitance component can be reduced on the other surface.

  Furthermore, in the present embodiment, the side surfaces of the thin plate 12 and the frame body 13 are substantially flush, but the outer diameter of the thin plate 12 may be smaller than the outer diameter of the frame body 13 as shown in FIG. Thereby, damage to the thin plate 12 can be suppressed. When the hydrophilic film 16 is formed on the surface of the thin plate 12, the glass component easily goes around the surface of the thin plate 12, but the glass reservoir portion is arranged by arranging the thin plate 12 inside the frame body 13 as shown in FIG. 13. Since 25 is formed, the wraparound of the glass component to the surface of the thin plate 12 can be suppressed.

  In the above embodiment, the frame body 13 is arranged on the upper surface of the thin plate 12, but only the thin plate 12 may be used, and the frame body 13 may be arranged on the lower surface of the thin plate 12 as shown in FIG. 14. In the above embodiment, the conduction hole 14 is linear. However, when it is desired to reduce the aspect ratio of the conduction hole 14, a recess is formed on the surface of the thin plate 12, and then a hole is formed from the deepest part of the recess. Also good. In this case, it becomes easy to trap cells by making the concave portion hemispherical in particular.

  The cell electrophysiological sensor of the present invention is useful in performing cell electrophysiology measurement with high accuracy.

Sectional drawing of the cell electrophysiological sensor in Embodiment 1 of this invention Sectional drawing which shows the manufacturing process of the device for cell electrophysiological sensors in Embodiment 1 of this invention. Sectional view Sectional view Sectional view Sectional view Sectional drawing of the cell electrophysiological sensor of another example in Embodiment 1 of this invention Sectional drawing of the cell electrophysiological sensor in Embodiment 2 of this invention Sectional drawing of the cell electrophysiological sensor in Embodiment 3 of this invention Sectional drawing which shows the equivalent circuit of the cell electrophysiological sensor in Embodiment 3 of this invention Sectional drawing of the cell electrophysiological sensor of another example in Embodiment 3 of this invention Sectional view Sectional view Sectional drawing of the cell electrophysiological sensor in one embodiment of this invention Sectional view of a conventional cellular electrophysiological sensor

Explanation of symbols

DESCRIPTION OF SYMBOLS 8 Resin board | substrate 9 Base material 10 Through-hole 11 Cell holding chip 12 Thin plate 13 Frame 14 Conduction hole 15 Cell 16 Hydrophilic film 17A, 17B Electrolyzer 18A, 18B Electrode 19 Sealing material 20 Pressure transmission tube 21 Substrate 22 First etching resist Film 23 Second etching resist film 24 Etching stop layer 25 Glass reservoir 26 Glass component

Claims (9)

A cell holding chip having a conduction hole;
A substrate covering the side surface of the cell holding chip,
In the cell electrophysiological sensor device in which the main component of the cell holding chip is silicon,
The substrate is formed of glass;
A hydrophilic film made of at least one of silicon oxide and silicon nitride is formed on the entire surface of the cell holding chip,
The cell electrophysiological sensor device , wherein the substrate and the side surface of the cell holding chip are welded via the hydrophilic film . The cell electrophysiological sensor device according to claim 1, wherein at least a part of the hydrophilic film is covalently bonded to the base material. The hydrophilic film has electrical insulation,
The cell electrophysiological sensor device according to claim 1, wherein the device is also formed on a cell holding surface side of the cell holding chip and from an opening of the conduction hole to a side surface of the cell holding chip. The cell holding chip is
A thin plate having the conduction hole;
The cell electrophysiological sensor device according to any one of claims 1 to 3, further comprising a frame formed on an upper surface or a lower surface side of the thin plate. The outer diameter of the thin plate is
The cell electrophysiological sensor device according to claim 4, wherein the device is smaller than an outer diameter of the frame body. On the outer periphery of the side surface of the thin plate,
The cell electrophysiological sensor device according to claim 5, wherein a glass reservoir is formed. The substrate is cylindrical,
The cell electrophysiological sensor device according to any one of claims 1 to 6, wherein the base material is inserted into a hole provided in the substrate. Cell holding chip
The cell electrophysiological sensor device according to any one of claims 4 to 7, further comprising an etching stop layer between the thin plate and the frame. A device for cellular electrophysiological sensor according to any one of claims 1 to 8,
A cell electrophysiological sensor comprising an electrolytic cell provided above and below the cell holding chip of the device for cell electrophysiological sensor, and an electrode for measuring a potential in these electrolytic cells.

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