JP4742882B2 - Cell electrophysiological sensor and manufacturing method thereof - Google Patents

Description

  The present invention relates to a cell electrophysiological sensor for measuring a cell electrophysiological phenomenon such as an intracellular potential or an extracellular potential used for measuring a physicochemical change generated by a cell activity.

  Conventionally, the patch clamp method in electrophysiology is known as a method for measuring the ion channel function existing in the 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.

  However, on the other hand, the patch clamp method requires an extremely high ability to insert a fine micropipette into a single cell with high precision in the measurement technique, so it requires skilled workers and requires high throughput. Is not an appropriate method.

  For this reason, flat-type probes utilizing microfabrication techniques have been developed, which are suitable for automated systems that do not require the insertion of micropipettes for individual cells.

  For example, there is a hole in the carrier that separates the two regions, and an electric field is generated by the electrodes placed above and below this carrier to efficiently hold the cells in the hole, and electrical measurement is performed between the upper and lower electrodes Can be used for electrophysiological measurement of cells (see Patent Document 1), or a plate-like device is provided with a plurality of through-holes, and includes a continuous layer of cells adhered thereto. A technique for measuring is disclosed (see Patent Document 2).

  Also, a technique for measuring the electrophysiological phenomenon of cells by attracting cells to the microelements by reducing the pressure of the microelements provided inside the cavities provided on the substrate and the flow paths connected to the microelements (Patent Literature) 3), a technique for measuring a cell's electrophysiological phenomenon (see Patent Document 4) by providing a hole in the flat portion of the substrate and integrating the reference electrode and the measurement electrode on the substrate, and one channel penetrates. A method of measuring cells contained in a liquid by locating the surface by suction from the lower surface of the cell and then rupturing part of the lower surface of the cell by increasing the pressure difference (see Patent Document 5) ).

  In addition, an electrode is arranged in the first and second cavities, respectively, so that the object seals the orifice in use, thereby forming electrically insulated first and second cavities. An apparatus (see Patent Document 6) that performs electrical measurement of an object in a medium by changing the impedance between them is disclosed.

  In addition, the extracellular potential is measured by an extracellular potential measuring device provided with a cell holding means provided on a substrate, a measurement electrode for detecting an electric signal of the well, and a reference electrode. The technique (refer patent document 7) is disclosed.

A technique for measuring extracellular potential with high accuracy by maintaining HEK293 cells, a type of human cultured cell line, in a 2.5 μm hole provided inside the SiO ₂ membrane to ensure high adhesion (non-patented) Reference 1) discloses that the through hole made in the flat plate plays the same role as the tip hole in the glass pipette, and can record the electrophysiological phenomenon of the cell with high accuracy and from the back side of the flat plate. The cell is automatically attracted by a method such as suction, and has an advantage that the cell can be easily held.

Also disclosed is a device for detecting a change in the state of an object by sealing the object in an orifice and measuring the electrode impedance between the galvanically isolated cavities thereby, the liquid in which the object is placed A liquid supply means including a liquid supply capillary and a liquid suction capillary for quickly removing and replacing the environment is also disclosed (see Patent Document 8).
Special table 2002-508516 gazette JP 2002-518678 Gazette US Pat. No. 6,315,940 Special table 2003-511668 gazette Japanese translation of PCT publication No. 2003-511699 Japanese translation of PCT publication No. 2003-527581 International Publication No. 02/055653 Pamphlet Japanese translation of PCT publication No. 2003-527581 T.A. Sordel et al, Micro Total Analysis Systems 2004, P521-522 (2004)

  However, in the conventional configuration, although there is what is disclosed about a technique related to a cell holding unit of a cell electrophysiological sensor that measures a potential difference generated inside and outside the cell with high accuracy by holding cells with high adhesion, Little is disclosed about the configuration for realizing the efficient production of the cell electrophysiological sensor.

  The object of the present invention is to realize a cell electrophysiological sensor excellent in productivity and a method for producing the same.

  In order to solve the conventional problems, the present invention provides a thin plate provided with a first through hole, a support provided around the thin plate, and a frame substrate provided with a second through hole and an electrode pattern. A cell electrophysiological sensor comprising: a second body smaller than the outer shape of the support body, wherein the support body has a function of contracting the outer shape of the support body when the support body is installed inside the second through-hole. It is set as the structure fitted and installed in the inside of this through-hole.

  The cell electrophysiological sensor and the method for producing the same according to the present invention are particularly efficient because a sensor chip having a minute shape constituted by a thin plate and a support on a frame substrate can be easily mounted and can be reliably installed. A cell electrophysiological sensor that can be produced well and a method for manufacturing the same can be provided.

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

  1 is a top view of a cell electrophysiological sensor according to Embodiment 1 of the present invention, FIG. 2 is a sectional view thereof, and FIG. 3 is an exploded sectional view thereof. 4 is a top view of the sensor chip, FIG. 5 is a sectional view thereof, and FIG. 6 is a top view of another example of the sensor chip.

  1 to 3, reference numeral 1 denotes a thin plate made of a substrate having excellent workability such as silicon, and at least one first through hole 2 is formed inside, and silicon or the like is formed around the thin plate 1. The support 4 made of a material excellent in workability is integrated by a joining means. Reference numeral 5 denotes a frame substrate having excellent moldability such as a resin. A second through hole 6 is formed in the frame substrate 5, and a thin plate is formed inside the second through hole 6. A sensor chip comprising 1 and a support 4 is mounted.

  The frame substrate 5 is provided with a first electrode 7 and a second electrode 8 so as to face each other with the sensor chip as a center, and between the regions partitioned by the thin plate 1 by these electrodes 7 and 8. The potential and current can be measured.

  The first electrode 7 is connected to the first electrode pattern 12, and the second electrode 8 is a second electrode provided on the upper surface of the frame substrate 5 via the through-hole electrode 11 of the frame substrate 5. It is connected to the pattern 13. In addition, in order to store a culture solution or a chemical solution in the upper and lower spaces of the thin plate 1, a first container 9 and a second container 10 are joined to each other.

  In the cell electrophysiological sensor having the above-described configuration, the inner diameter of the second through-hole 6 is designed to be slightly smaller than the outer shapes of the thin plate 1 and the support 4 before the assembly shown in FIG. As shown in FIG. 4, a plurality of slits 3 are formed at one end of the support 4 toward the other end of the support 4 provided with the thin plate 1. With the structure of the support body 4 provided with the plurality of slits 3 as described above, when the support body 4 and the thin plate 1 are fitted into the second through-hole 6 as shown in FIGS. Since the plurality of slits 3 can be contracted along the shape of the second through hole 6, the slit 3 can be fixed by securely fitting into the second through hole 6 smaller than the outer shape of the support 4. Yes.

  Since such a cell electrophysiological sensor generally has a very small cell size of several μm to several tens of microns, the shape of the sensor chip used therefor also has a dimensional shape of the μm level. Therefore, there is a great demand for a cellular electrophysiological sensor that can be easily manufactured while maintaining high dimensional accuracy. Therefore, the configuration as in the first embodiment is desired. It is possible to provide a cell electrophysiological sensor that can meet the above requirements and a method for manufacturing the same.

  Further, as shown in FIG. 4, the shape of the slit 3 is formed radially toward the center of the support 4. Accordingly, the slit 3 can be easily processed and formed, and the support 4 can contract the slit 3 while having an appropriate strength.

  Further, as shown in FIG. 5, the depth of the slit 3 formed in the sensor chip is preferably smaller than the thickness of the support 4. As a result, the spring property can be enhanced while maintaining the mechanical strength. For example, when the thickness of the support 4 is about 500 μm, the depth of the slit 3 is preferably 200 to 400 μm from the viewpoint of productivity. Furthermore, it is preferable that the width of the slit 3 is sufficiently smaller than the inner diameter of the support 4. This can increase the productivity advantage. For example, when the inner diameter of the support 4 is about 500 μm, the width of the slit 3 is preferably 50 μm or less from the viewpoint of the holding strength of the sensor chip. With such a structure, the support 4 can deform and contract the slit 3 while having an appropriate spring strength, and can also provide efficient productivity in the manufacturing method described later.

  In addition, as shown in FIG. 6, the slit 3 is formed in a spiral shape toward the center of the support body 4, so that the sensor chip is rotated in the spiral direction to the inside of the second through hole 6. When inserting and fitting, the support body 4 can be easily fitted while the slit 3 is contracted.

  Next, FIG. 7 is an exploded sectional view of another example of the cell electrophysiological sensor, and FIG. 8 is a sectional view thereof.

  As shown in FIGS. 7 and 8, the inner diameter of the second through hole 6 is tapered so as to decrease toward the slit 3 side of the support 4, and a thin plate is formed inside the second through hole 6. 1 and the support 4 are mounted. Thereby, in addition to the support 4 being easily held and closely attached to the inside of the second through-hole 6, there is a manufacturing advantage that the support 4 is easily put in automatically.

  Next, the manufacturing method of the cell electrophysiological sensor in Embodiment 1 of this invention is demonstrated using drawing. 9-15 is sectional drawing for demonstrating the manufacturing method of the cell electrophysiological sensor in Embodiment 1 of this invention.

  First, as shown in FIG. 9, a resist mask 15 is formed on the surface of the silicon substrate 14 by a normal photolithography process.

Next, as shown in FIG. 10, the first through hole 2 is formed by etching. The shape of the first through hole 2 can be changed depending on the type of etching. For example, a gas that promotes etching such as SF ₆ and CF ₄ and a gas that suppresses etching such as C ₄ F ₈ and CHF ₃ are mixed. Etching is performed vertically by performing dry etching.

For example, as shown in FIG. 11, only a gas that promotes etching, such as SF ₆ or XeF _2, can be used to form a tapered shape or a round shape, or a combination thereof can be used as shown in FIG. . By forming the first through-hole 2 having such a shape, a sensor chip having high cell retention and sealing properties can be produced in accordance with the size, shape and cell surface characteristics of the cell.

  Next, after forming a resist mask 18 on the back surface of the silicon substrate 14 as shown in FIG. 13, cavities 17 and slits 3 are formed by etching as shown in FIG. Here, the thin plate 1 and the support 4 are manufactured by processing from one silicon substrate. However, after the thin plate 1 and the support 4 are manufactured in separate steps, they can be manufactured by joining them. It is. As to which of these manufacturing methods is adopted, an optimal manufacturing method can be selected by appropriately selecting from the viewpoint of sensor structure and productivity.

  At this time, as shown in FIG. 15, the resist mask 18 viewed from above has an opening for forming the cavity 17 and an opening for forming the slit 19. The width of the slit 19 is the opening of the cavity 17. The shape is sufficiently smaller. As a result, when the silicon substrate 14 is etched, a difference in etching rate occurs between the slit 19 and the cavity 17, and even if the cavity 17 is etched to a predetermined depth, the depth of the slit 19 is not the same depth. As a result, since the slit 19 does not reach the thin plate 1, the slit 19 can be easily processed by etching. The slit 19 has a function of contracting the support 4 and can also maintain the strength of the support 4.

  As an etching method at this time, dry etching performed by mixing a gas for promoting etching and a gas for suppressing etching is preferable. As a result, the difference between the etching rates of the slit 19 and the cavity 17 can be increased, and there is a manufacturing advantage that production can be performed under high-efficiency conditions.

  Next, after the support body 4 is inserted and fitted into the second through-hole 6 of the frame substrate 5, the first container 9 and the second container made of a polymer material excellent in moldability are used. A cell electrophysiological sensor can be produced by bonding 10.

  In addition, although the external shape of the support body 4 is larger than the internal diameter of the 2nd through-hole 6, since the support body 4 is provided with the slit 19, it can shrink | contract moderately and is sufficient, without destroying. It can be fixedly installed while maintaining strength.

  As described above, the cell electrophysiological sensor and the manufacturing method thereof according to the present invention realize a sensor structure excellent in productivity and a manufacturing method thereof capable of electrically detecting a physicochemical change emitted by a cell. Therefore, it is useful as a device used in a drug screening apparatus for measuring the reaction of an ion channel to a drug at high speed.

Top view of cellular electrophysiological sensor according to Embodiment 1 of the present invention Sectional view Exploded sectional view Expanded top view of the main part Sectional view Main part enlarged top view of another example Exploded sectional view of another example of cellular electrophysiological sensor Sectional view Sectional drawing for demonstrating the manufacturing method of the same cell electrophysiological sensor Sectional view Sectional view Sectional view Sectional view Sectional view Top view

Explanation of symbols

DESCRIPTION OF SYMBOLS 1 Thin plate 2 1st through-hole 3 Slit 4 Support body 5 Frame body board 6 2nd through-hole 7 1st electrode 8 2nd electrode 9 1st container 10 2nd container 11 Through-hole electrode 12 1st Electrode pattern 13 Second electrode pattern 14 Silicon substrate 15 Resist mask 17 Cavity 18 Resist mask 19 Slit

Claims (8)

A thin plate provided with a first through hole, a support provided around the thin plate, and a frame substrate provided with a second through hole. A cell electrophysiological sensor for measuring an electrophysiological phenomenon of a cell with an electrode , wherein when the support is installed in the second through-hole, the outer shape of the support contracts. As a function of contracting the size of the outer shape of the support body, the cell electricity provided with a plurality of slits in the support body and fitted and installed in a second through-hole smaller than the outer shape of the support body Physiological sensor. The cell electrophysiological sensor according to claim 1 , wherein the depth of the slit is smaller than the thickness of the support. The cell electrophysiological sensor according to claim 1 , wherein the slit has a radial shape toward the center of the support. The cell electrophysiological sensor according to claim 1 , wherein the slit has a spiral shape toward the center of the support. The cell electrophysiological sensor according to claim 1 , wherein the width of the slit is smaller than the inner diameter of the support. The cell electrophysiological sensor according to claim 1 , wherein the inner diameter of the second through hole is tapered. The cell electrophysiological sensor according to claim 1, wherein an electrode pattern is provided on the frame substrate. A thin plate provided with a first through hole, a support provided around the thin plate, and a frame substrate provided with a second through hole. A cell electrophysiological sensor for measuring an electrophysiological phenomenon of a cell with an electrode , wherein when the support is installed in the second through-hole, the outer shape of the support contracts. A method for manufacturing a cell electrophysiological sensor provided and fitted in a second through-hole smaller than the outer shape of the support, the depth of the support being smaller than the thickness of the support And a cell having a step of providing a slit whose width is sufficiently smaller than the inner diameter of the support, and this step is performed using dry etching using a gas for promoting etching and a gas for suppressing etching. Manufacture of electrophysiological sensors Law.

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