JP4425891B2 - 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, electrophysiological phenomena of a plurality of cells can be measured at a time, but there is a problem that when the number of cells to be measured is increased, the number of sensor chips becomes large and the structure becomes complicated. there were.

  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 sensor chip is configured such that a chip part made of silicon and a chip holding part made of glass are joined by glass welding.

  The cell electrophysiological sensor and the manufacturing method thereof according to the present invention, when a minute sensor chip is fixed to a holding plate, a sensor chip in which a chip part made of silicon and a chip holding part made of glass are joined by glass welding are attached to the holding plate Since it is held, it can improve the self-alignment property of the minute chip part and reduce the leakage current due to liquid leakage from the gap, so that it can measure with high accuracy and has excellent productivity A sensor and a manufacturing method thereof are realized.

(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 cross-sectional view for explaining a method for manufacturing a cell electrophysiological sensor.

  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 20 are introduced.

  A holding plate 2 made of resin having a second through hole 6 is in contact with the lower portion of the well 1, and at least one first through hole is formed in the second through hole 6 of the holding plate 2. A sensor in which a chip portion 4a made of silicon provided with a diaphragm 9 having three through holes 7 and a chip holding portion 4b made of glass having a hydrophilic property to hold the minute chip portion 4a are joined by glass welding. Chip 4 is set. The chip portion 4a can be formed in a minute and highly accurate shape by etching a silicon wafer using a semiconductor process. 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 chip portion 4a can be formed by processing in an integrated manner 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 chip portion 4a, it can be efficiently manufactured with high accuracy, and its production equipment is also easily available.

  However, the chip portion 4a made of such fine silicon has a problem from the viewpoint of handling and assembly.

  On the other hand, it is possible to realize a sensor structure and a manufacturing method capable of efficiently producing a cell electrophysiological sensor with high accuracy by fixing the chip part 4a to the holding plate 2 via a chip holding part 4b made of glass. it can. Detailed description thereof will be described later.

  A cell electrophysiological sensor is formed below the holding plate 2 by contacting a flow path plate 3 having cavities 8 for allowing liquid to flow in and out at both ends thereof, and a third through hole 7. 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. Then, the intracellular fluid 19 can be filled into the cavity 8 by filling the intracellular fluid 19 from the inlet 16 and suctioning from the outlet 17 using a suction pump or the like.

  In addition, the well 1, the holding plate 2 and the flow path plate 3 are conveniently made of resin, and more preferably thermoplastic resin. Thereby, these materials can obtain a highly homogeneous molded body with good productivity by using means such as injection molding. 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 having excellent hydrophilicity 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 polyethylene (PE), from the viewpoint of workability, production cost, and material availability. 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. Further, since these materials can transmit ultraviolet rays, they are effective when an ultraviolet curable adhesive is used.

  For the cell electrophysiological sensor having the above-described configuration, for example, when the holding plate 2 is integrally manufactured from a silicon substrate and the chip portion 4a is integrally formed on a part of the holding plate 2, the cost is increased and the yield is increased. When the defective chip portion 4a is present in part, the structure does not have repairability. On the other hand, if it is the structure of this invention, while producing the microchip part 4a efficiently collectively and selecting and using only the non-defective goods in it, a high-yield cell electrophysiological sensor can be realized, A configuration with repairability can be realized.

  Further, the diameter of the third through hole 7 is desirably 5 μm or less, and the through hole has an optimal shape for holding the cells 20. In this way, the sensor chip 4 including the chip portion 4 a and the chip holding portion 4 b and the holding plate 2 are separately manufactured, and the sensor chip 4 is fitted and joined to the second through hole 6 of the holding plate 2. Thus, a cell electrophysiological sensor can be produced efficiently. Furthermore, even when there is a defective sensor chip 4, it can be easily replaced.

  However, the chip portion 4a is difficult to handle because it has a small shape, and when the chip portion 4a is directly inserted into the second through-hole 6 formed in the holding plate 2, it may be static electricity, etc. Therefore, it was difficult to insert the tip part 4a into a predetermined position.

  Therefore, when the tip portion 4a is immersed in pure water or the like and inserted into the opening of the tip holding portion 4b made of glass having excellent hydrophilicity in that state, the tip portion 4a is minute due to its affinity with glass having excellent hydrophilicity. It has been found that the chip portion 4a can be operated very efficiently by being well aligned at the position shown in FIG. 2 while self-aligning with the opening of the chip holding portion 4b.

  The chip portion 4a produced by micromachining is cleaned by chemical treatment such as acid or alkali, or physical treatment such as plasma or UV, and is then immersed in pure water in order to maintain hydrophilicity. It is preferable to keep it.

  Further, immediately before being inserted into the opening of the chip holding part 4b and assembling, the chip part 4a is taken out from pure water, and a part of the chip part 4a is inserted into the entrance of the opening of the chip holding part 4b. At this time, the surface of the chip part 4a and the inside of the cavity 10 are in a state where pure water is sufficiently adhered and filled, and the inner wall surface of the opening part of the chip holding part 4b is made of hydrophilic glass. The part 4a 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 the action of the surface tension of water, it is possible to easily dispose the tip portion 4a uniformly at a predetermined position of the tip holding portion 4b.

  In addition, there is no influence of self-alignment due to the direction of the chip part 4a, and the chip part 4a is self-aligned so as to be along one plane of the chip holding part 4b even if the surface of the diaphragm 9 faces up or down. I know.

  Moreover, in order to improve hydrophilicity, it is more preferable that the glass composition which comprises the chip | tip holding | maintenance part 4b contains silicon dioxide.

  Thereafter, in order to keep the tip portion 4a from moving from a predetermined position, the tip portion 4a and the tip holding portion 4b are fixed so as to be horizontal in the horizontal direction, and then placed in a heat treatment furnace or the like.

  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.

  And after drying the water adhering at about 100 degreeC, it heats to the predetermined temperature which glass welds, and joins the chip | tip part 4a and the chip | tip holding | maintenance part 4b by glass welding. Since the sensor chip 4 bonded by glass welding is excellent in sealing performance, it is possible to realize the sensor chip 4 having excellent reliability with little liquid leakage and the like, thereby realizing a cell electrophysiological sensor with high measurement accuracy. Can do.

  In particular, liquid leakage in the vicinity of the chip portion 4a has an important influence on the characteristics of the giga seal, and a structure that can reliably prevent liquid leakage on the outer wall surface of the chip portion 4a is very important as the structure of the cell electrophysiological sensor.

  Then, by setting the gap between the outer wall surface of the tip portion 4a and the inner wall surface of the opening portion of the chip holding portion 4b to be 300 μm or less, it is possible to surely perform welding and have a gap exceeding 300 μm. There arises a problem that the reliability of the welding and joining by the glass is lowered. Moreover, it is preferable to use borosilicate glass, aluminosilicate glass, or lead borosilicate glass as glass from a viewpoint of bondability, working temperature, and reliability.

  And it is important that the inner wall surface of the opening part of the chip | tip holding | maintenance part 4b in this Embodiment 1 has hydrophilicity. For this purpose, it is preferable to use, for example, borosilicate glass (Corning; # 7052, # 7056), aluminosilicate glass or lead borosilicate glass (Corning; # 8161) for the chip holding portion 4b. Furthermore, the melting point of the glass is important from the viewpoint of workability, and it is preferable that the temperature convenient for bonding with the chip portion 4a by glass welding is equal to or higher than the softening point of the glass, and more preferably 500 to 900. It is in the range of ° C. This is because if glass lower than 500 ° C. is used, the strength is insufficient, and if it exceeds 900 ° C., workability deteriorates.

  In order to impart hydrophilicity, the inner wall surface of the opening of the chip holding part 4b 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 the inner wall surface of the opening of the chip holding part 4b 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.

  Then, the sensor chip 4 prepared in advance in this way is inserted into the second through hole 6 of the holding plate 2 made of resin, and the sensor chip 4 is fixedly held by, for example, bonding with an adhesive or the like. can do. At this time, it is preferable from the viewpoint of workability to use an ultraviolet curable resin or the like as the adhesive.

  This ultraviolet curable resin can improve the workability and provide a highly sealing joint surface by providing the glass of the holding plate 2 and the chip holding portion 4b with the property of transmitting ultraviolet rays. This is because the ultraviolet curable resin is hydrophilic, and the outer wall surface of the chip holding portion 4b made of glass has hydrophilicity, so that the permeability of the ultraviolet curable resin into the gap between the bonding surfaces is increased. Bonding reliability can be improved.

  On the other hand, in the method of inserting the chip part 4a directly into the second through hole 6 formed in the holding plate 2 in a dry state, the chip part 4a may be scattered by static electricity or the like. The workability is not so good, the insertion positions of the inserted chip portions 4a vary, and a lot of time is required for alignment. Further, for example, when inserted into the second through-hole 6 of the holding plate 2 and then joined using the adhesive 21, the adhesive or the like hardly permeates the joining interface, and liquid leakage or the like There was a bonding failure.

  Next, as shown in FIG. 1, the holding plate 2 is provided with a first electrode 14 and a second electrode 15, and these electrodes 14 and 15 are electrical indicators generated by the electrophysiological phenomenon of the cell 20, For example, for measuring potential, current, etc., the shape and material are not particularly limited.

  Next, a method for measuring the electrophysiological activity of the cell 20 using the cell electrophysiological sensor of the present invention will be briefly described.

First, the extracellular fluid 18 is stored 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 decompressed, 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, the chip portion 4a forms a diaphragm 9 from a silicon wafer or the like by using a semiconductor processing technique such as photolithography and dry etching, and then a third through hole 7 is formed in the diaphragm 9. Form. A large number of chip portions 4a can be manufactured in a lump by separating the chip portions 4a 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, it is preferable to store the chip portion 4a by immersing it in pure water.

  On the other hand, the tip holding part 4b prepares a glass tube made of borosilicate glass whose inner diameter is slightly larger than the outer shape of the tip part 4a, and then confirms the hydrophilicity of the inner wall surface of the opening of the glass tube as necessary. Then, the tip portion 4a stored in the pure water is taken out, and a portion of the tip portion 4a is inserted into the entrance of the opening of the glass tube with the pure water attached to the tip portion 4a. The inserted tip portion 4a is allowed to stand on the upper surface portion of the opening of the glass tube by self-alignment by the interaction of the surface tension of pure water.

  It is confirmed that the self-alignment is performed in the same manner even when the upper and lower positional relationships of the diaphragm 9 are reversed as the direction in which the chip portion 4a is inserted.

  In addition, when the self-alignment position is inserted from the bottom of the glass tube, self-alignment is confirmed on the lower surface side of the glass tube.

  Thereafter, the glass tube is cut into predetermined pieces and separated into pieces. A plurality of singulated chips can be manufactured by repeating such operations.

  The glass tube may be cut in advance to a predetermined size, the chip holding part 4b is produced and aligned, and the chip part 4a is inserted into the aligned chip holding part 4b by the above method. Is possible.

  Such an individualized chip is fixed in a direction in which the chip portion 4a and the opening of the glass tube overlap in the horizontal direction (arranged so that the inner wall surface of the opening of the glass tube supports the outer wall surface of the chip portion 4a). . This is to prevent the tip portion 4a from moving easily due to the absence of pure water and the absence of surface tension of water 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.

  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.

  And it is preferable to perform this drying process and the joining process by heat welding collectively. This is for preventing the positional deviation between the chip part 4a and the chip holding part 4b by carrying out collectively. At this time, if the gap between the tip portion 4a and the opening of the glass tube is 300 μm or less, it is confirmed that bonding by glass welding is possible. For example, as a result of glass welding with the outer diameter of the tip portion 4a being 700 μm, the inner diameter of the opening of the glass tube being 1.0 mm, and the outer diameter being 1.50 mm, it was possible to efficiently perform bonding by glass welding. The welding temperature at that time was 718 ° C., and the welding time was 10 seconds or less. The outer diameter of the glass tube may be a shape that can be fixedly held on the holding plate 2.

  In addition, after preparing a glass substrate and forming openings in a matrix shape with predetermined dimensions of the glass substrate, the chip portion 4a is inserted into the opening of the glass substrate by the above-described method and bonded by thermal welding. It is also possible to manufacture the sensor chip 4 by cutting the glass substrate into pieces by cutting or etching.

  The sensor chip 4 manufactured in this way is fixed to the inside of the second through hole 6 of the holding plate 2 made of a thermoplastic resin, which has been molded in advance into a predetermined size and shape by injection molding, for example, with an adhesive 21. Hold.

  Thereafter, a wiring pattern is formed by a thin film technique, a plating technique, etc., and a first electrode 14 and a second electrode 15 are part of the holding plate 2 by a method such as dispensing or screen printing using a mixture of Ag and AgCl. To form.

  It should be noted that the process sequence of fixing and holding the sensor chip 4 and forming the electrodes 14 and 15 may be different.

  Next, the well 1 and the flow path plate 3 can be manufactured by molding using a die by injection molding or the like using acrylic resin or the like, and can be configured as shown in FIG. First, the holding plate 2 and the well 1 are joined. As the bonding method, thermal welding bonding with a laser or bonding with an ultraviolet curable adhesive by ultraviolet irradiation is preferable.

  Next, the flow path plate 3 is joined by the same method as described above, and a cell electrophysiological sensor as shown in FIG. 1 can be produced.

  In addition, the well 1 and the flow path plate 3 can be simultaneously bonded to the holding plate 2 at the same time, and either method can be appropriately employed.

  Moreover, it is preferable to use an ultraviolet curable adhesive as the adhesive used for the bonding, and any direction can be obtained by making the well 1, the holding plate 2, and the flow path plate 3 a resin that transmits ultraviolet light. It is possible to irradiate with ultraviolet rays and bond using an ultraviolet curable resin, and the adhesive can be reliably cured when irradiated with ultraviolet rays. The holding plate 2 and the well 1 or the flow path plate 3 By efficiently joining, a structure with little liquid leakage can be realized, and thereby a cell electrophysiological sensor capable of reliably measuring the cell 20 can be realized.

  As described above, the cell electrophysiological sensor and the method for producing the same according to the present invention are useful for measuring the electrophysiological phenomenon of cells capable of efficiently measuring 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 Sectional drawing for demonstrating the manufacturing method

Explanation of symbols

DESCRIPTION OF SYMBOLS 1 Well 2 Holding plate 3 Flow path plate 4 Sensor chip 4a Chip part 4b Chip holding part 5 1st through-hole 6 2nd through-hole 7 3rd through-hole 8 Cavity 9 Diaphragm 10 Cavity 14 1st electrode 15 1st Second electrode 16 Inlet 17 Outlet 18 Extracellular fluid 19 Intracellular fluid 20 Cell 21 Adhesive

Claims (11)

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. A cell electrophysiological sensor in contact with a sensor chip, the sensor chip comprising a chip part made of silicon and a chip holding part made of glass, wherein the cell part is joined by glass welding. Physiological sensor. 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, the holding plate and the flow path plate are selected from a material comprising a cyclic olefin polymer, a linear olefin polymer, a cyclic olefin copolymer obtained by copolymerizing these, or polyethylene. The cell electrophysiological sensor according to claim 1, wherein the well, the holding plate, and the flow path plate are joined by heat welding. The cell electrophysiological sensor according to claim 1, wherein the well, the holding plate, and the flow path plate are joined with an ultraviolet curable resin. The cell electrophysiological sensor according to claim 1, wherein the sensor chip and the holding plate are joined with an ultraviolet curable resin. 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 and a chip holding portion made of glass which holds the tip portion and the tip portion of silicon a contact with fine胞電gas producing method of the sanitary sensor, the wells with the first through hole A step of forming, a step of forming a holding plate having the second through-hole, a step of forming a third through-hole in the tip portion, a step of wetting the surface of the tip portion with water, and a chip by heat treatment forming a sensor chip parts and the chip holding portion are bonded by glass fusing, cell electrophysiological sensor that includes a step of bonding the sensor chip inserted to the inside of the second through hole of the holding plate Manufacturing method.

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