JP4425892B2 - 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, and a method for producing the same. is there.

  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.

  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 using microfabrication technology have been developed, and these do not require the insertion of a micropipette for each individual cell, and can automatically fix and measure cells simply by reducing the pressure. It is suitable as an automation system.

  For example, a technique is disclosed in which a plate-like device is provided with a plurality of through-holes and includes a continuous layer of cells adhered thereto, and a voltage-dependent ion channel activity is measured with an electrode (see Patent Document 1).

Further, an apparatus is disclosed in which an object seals an orifice during use, and an electrical measurement of an object in a medium is performed by a change in impedance between electrically insulated electrodes (see Patent Document 2).
JP 2002-518678 Gazette Japanese translation of PCT publication No. 2003-527581

  However, in the conventional technique, it is possible to measure the electrophysiological phenomenon of a plurality of cells at once, but as the number of cells to be measured increases, the number of sensor chips becomes larger and the structure becomes complicated. There was a problem of becoming.

  The present invention solves the above-described conventional problems, and realizes an efficient structure of a cell electrophysiological sensor excellent in productivity even when the number of cells to be measured is increased, and has a small leakage current. It is an object of the present invention to provide a cell electrophysiological sensor that can be measured with high accuracy in a state and a method for manufacturing the same.

  In order to solve the conventional problem, the present invention provides a well having a first through hole, a holding plate having a second through hole in contact with the lower part of the well, and a lower part of the holding plate. A flow path plate having a cavity having a liquid inlet and an outlet at both ends is contacted, and a sensor chip having a diaphragm having a third through hole is contacted inside the second through hole. In the cell electrophysiological sensor, the holding plate is made of glass having hydrophilicity, the sensor chip is made of silicon, and the sensor chip and the holding plate are joined by glass welding.

  The cell electrophysiological sensor of the present invention and the method for manufacturing the same are excellent in self-alignment when the sensor chip is inserted into the holding plate and are joined by glass welding, thereby reducing leakage current due to liquid leakage from the gap. Therefore, it is possible to realize a cell electrophysiological sensor that can measure with high accuracy and is excellent in productivity, and a manufacturing method thereof.

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

  1 is a cross-sectional view of a cell electrophysiological sensor according to Embodiment 1 of the present invention, and FIG. 2 is an enlarged cross-sectional view of a main part of FIG. FIG. 3 is a top view for explaining a method of fixing and holding the sensor chip by glass welding, and FIG. 4 is a sectional view thereof.

  In FIG. 1 and FIG. 2, reference numeral 1 denotes a well made of resin, and a first through hole 5 for storing the extracellular fluid 18 is formed in the well 1. The first through-hole 5 is formed with a tapered cross-sectional shape, so that it is efficient when a liquid such as an electrolytic solution or cells are introduced.

  In addition, a holding plate 2 made of hydrophilic glass provided with a second through hole 6 is in contact with the lower portion of the well 1. Inside the second through hole 6 of the holding plate 2, A sensor chip 4 made of silicon provided with a diaphragm 9 having at least one third through-hole 7 is set. The sensor chip 4 can be formed by etching a silicon wafer. For example, the cavity 10 is formed by etching a silicon wafer, and then the third through hole 7 is formed, whereby the thickness of the diaphragm 9 is 10 to 100 μm, and the opening diameter of the third through hole 7 is 1 to 1. The sensor chip 4 can be formed by being integrally processed using a fine processing technique such as a semiconductor process with a size of 3 μmφ. The opening diameter of the third through hole 7 can be appropriately selected depending on the size of the cell 20.

  Note that the order in which the cavity 10 or the third through-hole 7 is formed may be first.

  Thus, by using silicon as a constituent material of the sensor chip 4, it can be efficiently and accurately manufactured using a semiconductor process, and its production equipment is easily available.

  Furthermore, a cell electrophysiological sensor is configured below the holding plate 2 by contacting a flow path plate 3 having a cavity 8 for allowing liquid to flow in and out at both ends of the holding plate 2. The cell 20 is held in close contact with the upper surface of the cell 20 and the electrophysiological phenomenon of the cell 20 can be measured. The cavity 8 can be filled with the intracellular fluid 19 by suction from the outlet 17 using a suction pump or the like.

  Further, it is convenient from the viewpoint of productivity that the well 1 and the flow path plate 3 are made of resin, and it is particularly preferable to use a thermoplastic resin. Accordingly, these materials can be produced with high productivity by using means such as injection molding, and a highly homogeneous molded body can be obtained. More preferably, these thermoplastic resins are polycarbonate (PC), polyethylene (PE), olefin polymer, polymethyl methacrylate acetate (PMMA), or a combination thereof. These materials can be easily joined to the holding plate 2 made of glass by using an ultraviolet curable adhesive. More preferably, the thermoplastic resin is a cyclic olefin polymer, a linear olefin polymer, a cyclic olefin copolymer obtained by polymerizing them, or a thermoplastic resin made of polyethylene (PE). From the viewpoint of workability, production cost, and availability of materials. To preferred. In particular, the cyclic olefin copolymer is highly transparent and highly resistant to inorganic chemicals such as alkalis and acids, and is suitable for the production method or use environment of the present invention. Moreover, since these materials can transmit ultraviolet rays, they are effective when an ultraviolet curable adhesive is used.

  The diameter of the third through hole 7 is desirably 5 μm or less, and the through hole has an optimal size for holding the cells 20. As described above, the sensor chip 4 and the holding plate 2 are separately manufactured, and the sensor chip 4 is fitted into the second through hole 6 of the holding plate 2 to efficiently manufacture the cell electrophysiological sensor. Can do. In particular, even when there is a defective sensor chip 4, the sensor chip 4 can be easily replaced.

  On the other hand, for example, when the holding plate 2 is manufactured integrally from a silicon substrate, the cost is increased, the yield is deteriorated, and even if some of the sensor chips are defective, the repair property is also provided. It becomes the composition which does not.

  On the other hand, when the holding plate 2 is formed of resin, and the sensor chip 4 formed of silicon is directly inserted into the second through-hole 6 and fixed, the sensor chip 4 is difficult to handle because of its micro shape, When inserted into the minute second through-hole 6 formed in the holding plate 2, it was difficult to insert and fix the sensor chip 4 in a predetermined position due to the influence of static electricity or the like when performed in a dry state. .

  In order to overcome this problem, when the sensor chip 4 is immersed in pure water or the like and inserted into the second through-hole 6 in this state, the self-alignment property due to the surface tension of water of the sensor chip 4 is used. It was found that the work can be done very efficiently. Therefore, the sensor chip 4 manufactured by microfabrication is immersed in pure water in order to maintain hydrophilicity after cleaning by chemical treatment such as acid or alkali or physical treatment such as plasma or UV. It is preferable to keep it.

  The sensor chip 4 is taken out from the pure water just before being inserted into the second through hole 6 of the holding plate 2 and assembled, and a part of the sensor chip 4 is inserted into the entrance of the second through hole 6. .

  At this time, the surface of the sensor chip 4 and the inside of the cavity 10 are sufficiently filled with pure water, and the inner wall surface of the second through-hole 6 is formed using hydrophilic glass. Thus, the sensor chip 4 was found to be self-aligned uniformly at the position shown in FIG. 2 by the action of the surface tension of water.

  By utilizing this action, it is possible to easily arrange the sensor chips 4 uniformly. In particular, when producing a cell electrophysiological sensor having a plurality of sensor chips 4 in which the second through-holes 6 are arranged in a matrix, the sensor chip 4 is arranged inside the second through-hole 6 very efficiently. be able to.

  Note that there is no influence of self-alignment due to the direction of the sensor chip 4, and the sensor chip 4 can be self-aligned along one plane of the holding plate 2 even if the surface of the diaphragm 9 faces either the upper or lower surface. I know it.

  On the other hand, in the conventional method in which the sensor chip 4 is inserted into the second through hole 6 in a dry state, the sensor chip 4 may be scattered due to static electricity or the like, and the workability is very good. In addition, the insertion position of the inserted sensor chip 4 also varies, and a lot of time is required for alignment.

  Moreover, in order to improve hydrophilicity, it is preferable that the glass composition which comprises the holding | maintenance plate 2 contains silicon dioxide.

  Thereafter, in order to keep the sensor chip 4 from moving from a predetermined position, the sensor chip 4 and the second through-hole 6 are fixed so as to be horizontal in the horizontal direction (in FIG. 1, rotated 90 degrees). And put it into a heat treatment furnace.

  And after drying the water adhering at about 100 degreeC, it heats to the predetermined temperature which glass welds, and the sensor chip 4 and the holding | maintenance plate 2 are joined by glass welding. Thereby, a highly productive cell electrophysiological sensor can be realized.

  Although an example using a heat treatment furnace has been described, welding can be performed by heating only the vicinity of the sensor chip 4 by local heating using a heater or the like, or local heating using a near infrared laser or the like.

  In addition, welding and joining can be reliably performed by setting the gap between the outer wall surface of the sensor chip 4 and the inner wall surface of the second through-hole 6 to 50 μm or less. If the gap exceeds 50 μm, there is a problem that the reliability of the welding and bonding by the glass is lowered.

  Furthermore, it is preferable to use, for example, borosilicate glass (Corning; # 7052, # 7056) or lead borosilicate glass (# 8161) as the holding plate 2 from the viewpoints of bondability, working temperature, and reliability.

  And it is important that the inner wall surface of the 2nd through-hole 6 in this Embodiment 1 has hydrophilicity.

  Moreover, it is preferable that the temperature convenient for joining the sensor chip 4 and the holding plate 2 is equal to or higher than the softening point of the glass, and more preferably in the range of 500 to 900 ° C. This is because if glass lower than 500 ° C. is used for the holding plate 2, the strength is insufficient, and if it exceeds 900 ° C., workability deteriorates.

  In order to impart hydrophilicity, the inner wall surface of the second through-hole 6 may be chemically or physically treated to increase the hydrophilicity. Alternatively, the effect can be further enhanced by forming a hydrophilic film or the like for further enhancing the hydrophilicity. Furthermore, as another method for imparting hydrophilicity, the removal of carbon compounds by oxygen plasma, the decomposition and removal of organic substances by ultraviolet irradiation, or the decomposition and removal of organic substances containing carbon atoms by wet treatment with sulfuric acid, hydrogen peroxide, etc. Is very effective and has excellent productivity.

  The hydrophilicity of at least the inner wall surface of the second through hole 6 is preferably 10 degrees or less in terms of contact angle. The contact angle refers to an angle formed between a droplet surface and a solid surface in a state where a droplet such as pure water is placed on the solid surface and is in equilibrium. And the measuring method can generally use the θ / 2 method. In this method, the contact angle can be obtained from the angle of the straight line connecting the left and right end points and the vertex of the droplet with respect to the solid surface. It is also possible to measure using a protractor or the like.

  In addition, as shown in FIG. 1, a first electrode 14 and a second electrode 15 are provided. These electrodes 14 and 15 are used for indicating an electrical index generated by an electrophysiological phenomenon of a cell, such as a potential and a current. Although it is for measuring, these shapes and materials are not particularly limited.

  Next, a method for measuring cell electrophysiological activity using the cell electrophysiological sensor of the present invention will be briefly described.

First, the extracellular fluid 18 is filled in the well 1 and the intracellular fluid 19 is sucked from the inlet 16 to the outlet 17 of the well 1 to fill the cavity 8. Here, the extracellular liquid 18 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. The intracellular liquid 19 is, for example, a mammalian muscle cell Typically, it is an electrolyte 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 this state, a conduction resistance value of about 100 kΩ to 10 MΩ can be observed between the first electrode 14 installed inside the well 1 and the second electrode 15 installed inside the cavity 8. This is because the intracellular fluid 19 or the extracellular fluid 18 penetrates and an electric circuit is formed between the first electrode 14 and the second electrode 15.

  Next, the cells 20 are introduced from the well 1 side. The sensor chip 4 may be disposed in the second through hole 6 so that the diaphragm 9 is closer to the first through hole 5 side. This selection should be optimally determined by the nature of the cell 20 being measured.

  Finally, when one of the inlet 16 or the outlet 17 of the well 1 is depressurized, the cell 20 is attracted to the third through-hole 7, and the cell 20 closes the third through-hole 7. Then, the electric resistance on the side of the cavity 8 is sufficiently high (GΩ seal). In this giga-seal state, when the potential inside and outside the cell changes due to the electrophysiological activity of the cell 20, highly accurate measurement is possible even with a slight potential difference or current.

  The manufacturing method of the cell electrophysiological sensor configured as described above will be described below.

  First, as shown in FIG. 3 and FIG. 4, the sensor chip 4 forms a diaphragm 9 from a silicon wafer or the like by using a semiconductor processing technique such as photolithography and dry etching. Hole 7 is formed. A large number of sensor chips 4 can be manufactured collectively by dividing into individual pieces.

  Thereafter, in order to increase the hydrophilicity as necessary, the silicon compound is formed using a thin film process such as thermal oxidation of silicon in an oxygen-mediated atmosphere or CVD or sputtering to increase the hydrophilicity. It is also possible. Thereafter, the sensor chip 4 is immersed in pure water and stored.

  On the other hand, for the holding plate 2, borosilicate glass having a thickness of 0.75 mm is prepared, and then the second through hole 6 is formed by drilling, for example, by etching.

  Thereafter, if necessary, the hydrophilicity of the inner wall surface of the second through hole 6 is confirmed, the holding plate 2 is held horizontally, the sensor chip 4 stored in the pure water is taken out, and the sensor chip 4 is taken out. A part of the sensor chip 4 is inserted from the upper surface of the holding plate 2 to the entrance of the second through-hole 6 in a state where the pure water is attached thereto. The inserted sensor chip 4 is allowed to stand by self-alignment at the position shown in FIGS. 3 and 4 by the interaction of the surface tension of pure water.

  It has been confirmed that self-alignment is performed in the same manner even when the vertical positional relationship is reversed in the direction in which the sensor chip 4 is inserted. It has also been confirmed that when the self-alignment position is inserted from below, self-alignment is performed on the lower surface side of the holding plate 2 in FIG.

  Thereafter, the holding plate 2 is rotated 90 degrees to fix the sensor chip 4 and the second through-hole 6 in a horizontal direction (arranged to support the sensor chip 4 on the inner wall surface of the second through-hole 6). ) This is to prevent the sensor chip 4 from moving easily because the surface tension of water disappears due to the absence of pure water.

  With such an arrangement, it is placed in a heat treatment furnace and heated. Optimum conditions can be set as appropriate for the heating method. In particular, heat treatment conditions for drying and heat treatment conditions for welding by glass are important. The heat treatment conditions for drying are preferably in the range of 80 to 120 ° C. And as a welding joining temperature at the time of using the said borosilicate glass (Corning; # 7052, # 7056), the conditions of 700-750 degreeC are preferable.

  And it is preferable to perform this drying process and welding joining process collectively. The movement of the sensor chip 4 can be suppressed by carrying out all at once.

  At this time, if the gap between the sensor chip 4 and the second through-hole 6 is 50 μm or less, it is confirmed that bonding by glass welding is possible. For example, as a result of performing the glass welding with the outer shape of the sensor chip 4 being 700 μm and the inner diameter of the second through hole 6 being 750 μm, it was possible to perform the bonding by the glass welding efficiently and reliably. The welding temperature at that time could be performed at 718 ° C. under heat treatment conditions of 10 seconds or less.

  Thereafter, a wiring pattern is formed by a thin film technique, a plating technique, etc., and further, a first electrode 14 and a second electrode 15 are formed by a technique such as dispensing or screen printing using a mixture of Ag and AgCl. It should be noted that the process sequence of forming holes by etching or the like and the electrode forming process may be different.

  Next, the well 1 and the flow path plate 3 can be manufactured by injection molding using an acrylic resin or the like to have a shape as shown in FIG.

  Next, the holding plate 2 and the well 1 are joined. As a joining method, joining with an ultraviolet curable adhesive is preferable.

  Thereafter, the flow path plate 3 is joined, and a cell electrophysiological sensor as shown in FIG. 1 can be produced.

  Note that the holding plate 2 and the flow path plate 3 can be joined to the holding plate 2 at the same time, and any one of the methods can be appropriately employed.

  The adhesive is preferably an ultraviolet curable adhesive, and the holding plate 2 is made of glass that transmits ultraviolet light, so that the ultraviolet rays are irradiated from any direction, and an ultraviolet curable resin is used. It becomes possible to bond, the adhesive can be reliably cured at the time of ultraviolet irradiation, and a structure with little liquid leakage is realized by reliably bonding the holding plate 2 to the well 1 or the flow path plate 3. Thus, a cell electrophysiological sensor capable of reliably measuring the cell 20 can be realized.

  The cell electrophysiological sensor and the method for producing the same of the present invention are useful for measuring the electrophysiological phenomenon of a cell that can efficiently measure a plurality of cells collectively.

Sectional drawing of the cell electrophysiological sensor in Embodiment 1 of this invention Enlarged sectional view of the main part Top view for explaining a method for fixing and holding the sensor chip Sectional view

Explanation of symbols

DESCRIPTION OF SYMBOLS 1 Well 2 Holding plate 3 Flow path plate 4 Sensor chip 5 1st through-hole 6 2nd through-hole 7 3rd through-hole 8 Cavity 9 Diaphragm 14 1st electrode 15 2nd electrode 16 Inlet 17 Outlet 18 Extracellular fluid 19 Intracellular fluid 20 cells

Claims (9)

A well having a first through hole and a holding plate having a second through hole in contact with the lower side of the well; and a third through hole is provided inside the second through hole. the sensor chip a contact cell electrophysiological sensor, the holding plate and glass, the sensor chip is a silicon, the cell electrophysiological sensor the sensor chip and the retaining plate are joined by glass fusing. The cell electrophysiological sensor according to claim 1, wherein the glass is glass containing silicon dioxide. The cell electrophysiological sensor according to claim 1, wherein the glass has a softening point of 500 to 900 ° C. The cell electrophysiological sensor according to claim 2, wherein the glass is borosilicate glass, aluminosilicate glass, or lead borosilicate glass. The cell electrophysiological sensor according to claim 1, wherein the glass is glass that transmits ultraviolet rays. The cell electrophysiological sensor according to claim 1, wherein a contact angle between glass and water is 10 degrees or less. The cell electrophysiological sensor according to claim 1, wherein the well is selected from a material comprising a cyclic olefin polymer, a linear olefin polymer, a cyclic olefin copolymer obtained by copolymerization thereof, or polyethylene. Cell electrophysiological sensor according to claim 1, the junction between the well and the glass are joined by ultraviolet curing resin. A well having a first through hole, and a holding plate made of glass having a second through hole in contact with the lower side of the well; and a third through hole in the second through hole a method of manufacturing a contact with fine胞電vapor physiological sensor of the sensor chip made of silicon and a step of forming a well having said first through hole, the holding plate having a second through hole Forming a sensor chip having the third through hole , wetting the surface of the sensor chip with water, and inserting the sensor chip into the second through hole to perform self-alignment. process and method for producing a cell electrophysiological sensor that includes a step of bonding the glass fusing and the holding plate and the sensor chip by heat treatment for.

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