JP5047878B2 - Microelectrode array device, manufacturing method thereof and bioassay method - Google Patents

Description

  The present invention relates to a microelectrode array device for measuring cell potential, a method for producing the same, and a bioassay method using the device.

  In recent years, with the development of genome planning and the progress of structural analysis of biomolecules, the movement to understand biological phenomena in terms of molecular biochemistry and apply them to industries has become active. On the other hand, in order to obtain biological information closer to in vivo, bioassay methods using cells are attracting attention. Through analysis using such cells, it is possible to obtain information on cell functions, cell-cell interactions, cell-chemical interaction, etc., such as life phenomena, production of useful substances, physiological activities of substances, New knowledge on toxicity, etc. can be obtained.

  Cell analysis methods can be obtained, for example, by modifying proteins and sugar chains present on the cell surface with fluorescently labeled antibodies, or introducing fluorescently labeled probes that specifically bind to the substance to be analyzed into the cells. Examples thereof include a method of analyzing a fluorescent image and a method of measuring intracellular calcium and pH by introducing an indicator such as a fluorescent reagent into the cell. However, in these methods, the labeling substance or indicator may cause damage to the cells due to stimulation or damage, and the analysis may not be performed with high accuracy. Therefore, as a technique for analyzing cells noninvasively, cells such as nerve cells, cardiomyocytes, and muscle cells whose action potential changes with life activity are cultured on a microelectrode, and the change in action potential at that time is measured. Methods have been proposed (see, for example, Patent Documents 1 and 2, Non-Patent Document 1). Changes in cell action potentials are caused by changes in the ion concentration inside and outside the cell membrane, and can be measured by measuring the changes in the ion concentration near the cell as an ionic current using an electrode. It can be detected.

On the other hand, in the conventional methods including the analysis methods as described above, a group of cells consisting of a plurality of cells is analyzed, and the average value of the data obtained by the analysis is as if it is a characteristic of each cell. Have been dealing with. However, in reality, cell cycles and circadian rhythms (circadian rhythms) are rarely synchronized in a cell group, and genes and proteins are expressed in different cycles for each cell. For this reason, the obtained analysis result is accompanied by the problem of fluctuations due to the handling of the cell group, and a highly accurate analysis result cannot be obtained. Therefore, in order to solve such problems, a technique such as synchronous culture has been developed and used. However, there has been a problem that cells of the same stage must be continuously supplied. Therefore, in order to solve such problems, a plurality of cells are individually cultured one by one, and a network of each cell is formed into a cell group, and information about each cell in the cell is obtained. Acquiring this information has increased the movement to conduct more accurate analysis.
As described above, a method for arbitrarily arranging cells to form a network has also been proposed. For example, a technique for controlling cell elongation and movement by culturing cells in a space having a barrier with a height of 10 μm or more is disclosed (for example, see Non-Patent Document 2). In addition, it has been reported that when a pattern is formed with a cell adhesive substance such as laminin, the projections of nerve cells extend along the pattern (see Non-Patent Document 3). A network can be constructed by arranging cells in a predetermined space.
JP-A-6-078889 Japanese Patent Laid-Open No. 11-187865 Jimbo Y. et al. , Tateno T., Robinson HPC. , Biophys. J76, 670-678 (1999) Stopak D.M. , Et al. Dev. Biol. , 90, 383-398 (1982) Letourneau P.M. C. Dev. Biol. , 66, 183-196 (1975)

The technique for culturing cells on a microelectrode and measuring the change in action potential at that time is useful as a technique for continuously analyzing cells for a long period of time. However, in the conventionally proposed technique, using a microelectrode array substrate, tissue fragments or cultured cells were measured, and average data on multiple cells was acquired and analyzed as described above. There was a problem that analysis was not possible with high accuracy. For example, in order to analyze a response to a drug or electrical stimulus with higher accuracy, it is necessary to analyze information for each cell. However, even if information is analyzed by culturing one cell alone, the analysis result does not reflect the influence of the interaction between cells, and the function as a tissue composed of a group of cells cannot be analyzed accurately.
Therefore, in order to perform analysis with high accuracy using cells, a technique for constructing a cell group in which cells interact with each other and analyzing the cells in the cell group for each cell is required. . Therefore, development of a new device that enables such analysis is strongly desired.

  The present invention has been made in view of the above circumstances, and provides a microelectrode array device capable of measuring the potential of each cell in a cell group, a production method thereof, and a bioassay method using the device. Is an issue.

To solve the above problem,
The invention according to claim 1 is a microelectrode array device for measuring a cell potential,
A microelectrode on the insulating substrate; wiring of the microelectrode; and an insulating film covering the insulating substrate so as to expose the microelectrode; and further attaching cells to a predetermined region on the insulating film. with a non-cell adhesive layer that inhibits are provided, said non without cell adhesion layer is provided, an insulating film and a region on the insulating substrate exposed in the vicinity thereof and said microelectrodes are a cell placement portion, the minute electrodes and / or the non-cell adhesive layer is microelectrode array device, wherein Rukoto such a material having optical transparency.
The invention according to claim 2 is characterized in that a plurality of the cell placement portions are connected by a cell connection portion in which the non-cell adhesion layer is not provided and the insulating film is exposed. 1. The microelectrode array device according to 1 .
The invention described in claim 3 is characterized in that the non-cell adhesion layer is formed by bonding a compound having a perfluoroalkyl group or a perfluoroalkyl ether group onto the insulating film. Item 3. The microelectrode array device according to Item 1 or 2 .
The invention according to claim 4 is the microelectrode array device according to claim 3 , wherein the compound is represented by the following general formula (1) or (2).

(In the formula, R ¹ represents a linear or branched perfluoroalkyl group or perfluoroalkyl ether group having ^{1 to} 16 carbon atoms; R ¹ ′ represents a linear or branched chain having ^{1 to} 16 carbon atoms; Represents a group in which one fluorine atom is removed from a perfluoroalkyl group or a perfluoroalkyl ether group; R ² is any one of groups represented by the following formulas (11) to (16), and a plurality of R ² may be the same or different; Y is a group represented by the formula “—NH—C (═O) —” or a carbonyl group, and a plurality of Y may be the same or different; Is an ethyleneoxy group in which one or more hydrogen atoms may be replaced by an alkyl group or alkyloxyalkyl group in which one hydrogen atom is substituted, and a plurality of Z may be the same or different from each other Even J and k are each independently 0 or 1, and a plurality of j or k may be the same or different from each other; l and m are each independently an integer of 0 or more, and a plurality of l or m may be the same or different from each other; when l is an integer of 2 or more, one Z may be the same or different from each other.

(In the formula, R ³ represents a hydroxyl group or an atom or group that can be substituted with a hydroxyl group; R ⁴ represents a hydrogen atom or a monovalent hydrocarbon group; n is 1, 2 or 3, and n is 2 or 3; And n R ³ may be the same or different from each other; when n is 1, the two R ⁴ may be the same or different from each other.)
Invention of claim 5, the compound represented by the general formula (1) or (2), according to claim 4, characterized by being represented by the following general formula (3) or (4) This is a microelectrode array device.

(In the formula, R ¹ represents a linear or branched perfluoroalkyl group or perfluoroalkyl ether group having ^{1 to} 16 carbon atoms; R ¹ ′ represents a linear or branched chain having ^{1 to} 16 carbon atoms; Represents a group in which one fluorine atom is removed from a perfluoroalkyl group or a perfluoroalkyl ether group; R ² is any one of groups represented by the following formulas (11) to (16), and a plurality of R ² may be the same or different; m is an integer of 0 or more, and a plurality of m may be the same or different.

(In the formula, R ³ represents a hydroxyl group or an atom or group that can be substituted with a hydroxyl group; R ⁴ represents a hydrogen atom or a monovalent hydrocarbon group; n is 1, 2 or 3, and n is 2 or 3; And n R ³ may be the same or different from each other; when n is 1, the two R ⁴ may be the same or different from each other.)
Invention of Claim 6 is a manufacturing method of the microelectrode array device as described in any one of Claims 1-5 , Comprising: The process of forming a microelectrode and wiring of this microelectrode on an insulating substrate, A step of patterning an insulating film on the insulating substrate so as to expose the microelectrode; and a step of covering a surface on which the insulating film on the insulating substrate is patterned with a non-cell adhesion layer that inhibits cell adhesion; Patterning a photosensitive resin on the non-cell adhesive layer, removing a predetermined portion of the non-cell adhesive layer by etching using the photosensitive resin as a mask, and exposing the microelectrode and the insulating film in the vicinity thereof. to form the cell arrangement portion, further, to form a cell connecting portion insulating film is exposed in a predetermined region for connecting the cell arrangement portions, and patterning the photosensitive resin, the following In a method for producing a microelectrode array device characterized by having a step of removing the photosensitive resin.
The invention of claim 7 is a method for producing a microelectrode array device according to any one of claims 1 to 5 and forming a wiring microelectrodes and fine small electrodes on an insulating substrate Patterning an insulating film on the insulating substrate so as to expose the microelectrode, and covering the microelectrode and the insulating film in the vicinity thereof on the surface on which the insulating film on the insulating substrate is patterned. A step of patterning a photosensitive resin on the substrate, a step of coating a surface of the insulating substrate on which the photosensitive resin is patterned with a non-cell adhesion layer that inhibits cell adhesion, and a non-cell adhesion layer on the photosensitive resin. And a photosensitive resin coated with the non-cell adhesive layer to expose the microelectrode and the insulating film in the vicinity thereof to form a cell placement portion. Electric A method of manufacturing a polar array device.
The invention according to claim 8 is characterized in that the photosensitive resin is patterned so as to form, together with the cell placement portion, a cell connection portion in which an insulating film is exposed in a predetermined region that connects the cell placement portions. A method of manufacturing a microelectrode array device according to claim 7 .
The invention according to claim 9 is a bioassay method using the microelectrode array device according to any one of claims 1 to 5 , wherein a measurement target is placed in a cell arrangement portion on the microelectrode array device. A bioassay method comprising a step of arranging cells and measuring a potential of the cells with a microelectrode.

  According to the present invention, the potential can be measured for each cell in the cell group. As a result, the cells can be analyzed non-invasively with high accuracy, and can be applied to, for example, bioassays using various chemical substances.

<Microelectrode array device>
A microelectrode array device of the present invention (hereinafter sometimes abbreviated as device) is a microelectrode array device for measuring a cell potential, comprising a microelectrode on an insulating substrate, wiring of the microelectrode, An insulating film covering the insulating substrate so as to expose the microelectrode, and a non-cell adhesive layer is further provided in a predetermined region on the insulating film, and the non-cell adhesive layer is not provided, A region on the insulating substrate where the microelectrode and the insulating film in the vicinity thereof are exposed is a cell placement portion.
In the present invention, cells to be measured can be appropriately selected according to the purpose and are not particularly limited. And what can be proliferated by culture | cultivation is also suitable. Of these, animal cells are preferred, and specific examples of neuroblasts are cardiomyocytes, cardiomyocytes, hepatocytes, and fat cells. Differentiable cells such as embryonic stem cells (ES cells) and induced pluripotent stem cells (iPS cells) may also be used.

(First embodiment)
FIG. 1 is a diagram illustrating a device according to a first embodiment of the present invention, where (a) is a plan view and (b) is a perspective view.
An electrode 12 is patterned on the insulating substrate 11. The electrode 12 electrically connects the first connecting portion 12a serving as an electrical contact between the device 1 and a measuring instrument (not shown), the reference electrode 13 or the microelectrode for cell measurement, and the first connecting portion 12a. The second connection portion 12b to be connected to each other. A plurality of microelectrodes are arranged in the microelectrode arrangement portion 14 as will be described later.
An insulating film 15 is provided on the insulating substrate 11 so as to cover the second connection portion 12 b of the electrode 12, and the reference electrode 13 and the microelectrode arrangement portion 14 are further provided on the insulating film 15. A partition wall 16 is erected so as to enclose

The material of the insulating substrate 11 may be appropriately selected from known materials. Among these, transparent ones are preferable, and specific examples include glass and resin, and glass is particularly preferable.
What is necessary is just to select the thickness of the insulated substrate 11 and the magnitude | size of an upper surface suitably according to the objective. For example, if it is thickness, it is preferable that it is 0.1-10 mm normally, and it is more preferable that it is 0.3-1.5 mm. Within such a range, the workability of the insulating substrate 11 and the usability of the device 1 are particularly excellent.

What is necessary is just to select the material of the electrode 12 from a well-known thing suitably. For example, gold (Au), silver (Ag), chromium (Cr), platinum (Pt), and platinum black (PT-C) can be exemplified as materials usually used for electrodes. However, in the present invention, a material having optical transparency is preferable. Here, “having light transparency” means “having transparency at least for visible light”, and preferably has transparency equivalent to that of transparent glass or silicon dioxide (SiO ₂ ). By using an electrode made of a light-transmitting material, noise can be effectively reduced and analysis can be performed with higher sensitivity when a cell to be measured is optically analyzed using a microscope or the like. In particular, it is suitable for irradiating a laser under microscope observation and applying a technique usually called optical tweezers or applying light stimulation to a cell for analysis. Specific examples of such preferable materials include tin-doped indium oxide (hereinafter abbreviated as ITO), silver-doped ITO, indium / zinc oxide, indium / titanium oxide, aluminum-doped zinc oxide, gallium-doped zinc oxide, and cadmium. Examples include doped zinc oxide, antimony-doped tin oxide, fluorine-doped tin oxide, and niobium-doped titanium oxide.
The refractive index of the electrode 12 is preferably equivalent to transparent glass or silicon dioxide (SiO _2), it is preferably from 1.4 to 1.6. Since the electrode 12 has such preferable physical properties, cells can be optically analyzed with higher sensitivity.

The 1st connection part 12a is strip | belt shape among the electrodes 12, and is a connection part with a measuring device. The width is preferably set in consideration of the size of the device, and is usually preferably 0.1 to 2.0 mm. The second connection portion 12 b has a bent portion and has a band shape having a portion whose width decreases toward the microelectrode placement portion 14. Although the width is not particularly limited, it is usually preferable that the width is equal to or smaller than the width of the first connection portion 12a.
Here, although the strip-shaped thing is illustrated as the 1st connection part 12a and the 2nd connection part 12b, it is not limited to this, Other shapes may be sufficient. However, an elongated shape is preferable.
Moreover, it is preferable that the electrode 12 is the same thickness as the microelectrode described later.

The reference electrode 13 may be made of the same material as the electrode 12.
The reference electrode 13 is a substantially square shape, it is preferable sufficiently larger than the microelectrodes described later, usually is preferably ^{1.0 × 10 -8 ~1.0 × 10 -6} m 2 1.0 × 10 ^{−8 to} 2.5 × 10 ⁻⁷ m ² is more preferable.
Here, although the substantially square-shaped thing is illustrated as the reference electrode 13, it is not limited to this and another shape may be sufficient.
Moreover, it is preferable that the reference electrode 13 is the same thickness as the microelectrode described later.
Here, an example having four reference electrodes is shown, but the number of reference electrodes may be adjusted as appropriate according to the number of microelectrodes and the like.

The material of the insulating film 15 may be appropriately selected from known materials. Preferable examples include various resins and silicon oxide. A preferable example of the resin is a photosensitive resin such as a photoresist.
The size of the upper surface of the insulating film 15 may be set as appropriate according to the size of the insulating substrate 11. The thickness of the insulating film 15 can not only reliably electrically insulate, but also a thickness that does not give stress to the cells arranged in the cell arrangement portion, specifically, a thickness equal to or less than the height of the cells. It is preferable. From such a viewpoint, the thickness of the insulating film 15 is preferably 0.1 to 30 μm, and more preferably 0.5 to 5 μm.

As will be described later, the partition wall 16 is for holding the cell culture solution on a predetermined region including the reference electrode 13 and the microelectrode arrangement portion 14 of the device 1. By supplying the retained cell culture solution, the cells to be measured can survive. What is necessary is just to select a cell culture solution suitably according to the kind of cell of measurement object.
The partition wall 16 has a rectangular tube shape with a hollow portion in a plan view.
Moreover, the material of the partition wall 16 is preferably stable with respect to the retained cell culture solution, and for the same reason as that of the electrode 12, a material having optical transparency is preferable. A particularly preferable example is glass.
It is preferable to appropriately adjust the size of the surrounding surface surrounded by the partition wall 16 according to the arrangement form of the reference electrode 13 and the microelectrode arrangement portion 14. The height of the partition wall 16 may be adjusted as appropriate according to the amount of the cell culture solution to be held, and is not particularly limited.
The partition wall 16 is preferably disposed concentrically with the insulating substrate 11 as illustrated here.
The partition 16 is exemplified here as a rectangular tube, but is not limited thereto, and may have another shape such as a round tube, and can be appropriately selected.

  The microelectrode placement unit 14 includes a cell placement unit that places cells to be measured and a microelectrode that measures the potential of the placed cells. The microelectrode arrangement part 14 is arranged on the surface of the device 1 where the electrode 12 is provided, and is preferably arranged concentrically with the insulating substrate 11 as illustrated here.

FIG. 2 is an enlarged view illustrating the microelectrode arrangement portion 14, (a) is a plan view, and (b) is a plan view further illustrating a part of (a).
In the microelectrode arrangement portion 14, a plurality of microelectrodes 17 having a substantially square shape are arranged. The insulating substrate 11 is covered with an insulating film 15 so that the plurality of microelectrodes 17 are exposed. Further, on the insulating film 15, there are a substantially circular region composed of the exposed microelectrode 17, a predetermined region surrounding the microelectrode 17, and a strip-shaped region connecting these substantially circular regions. A non-cell adhesion layer 19 is provided except for the non-stacked region 10. The substantially circular region is a cell placement portion 10a for placing cells to be measured, and the microelectrode 17 and the insulating film 15 are exposed. Moreover, the strip | belt-shaped area | region which connects adjacent cell arrangement | positioning parts 10a is the cell connection part 10b which connects the arrange | positioned cells, and the insulating film 15 is exposed.

The non-cell adhesion layer 19 includes a substance having non-cell adhesion (hereinafter abbreviated as a non-adhesive substance), and on the exposed surface (hereinafter abbreviated as a non-adhesion surface), the culture and non-culture periods In either case, cell adhesion is inhibited. The non-cell adhesive layer 19 can be formed by bonding a precursor of a non-adhesive substance on the insulating film. In other words, the precursor of the non-adhesive substance is a substance containing a functional group having a binding ability to the insulating film.
Specific examples of the non-cell adhesion layer 19 include liquid repellency. The term “liquid repellency” as used herein refers to “water repellency” or “oil repellency”.

The non-adhesive surface preferably has water repellency with a water contact angle of 100 ° or more, and preferably has oil repellency with a hexadecane contact angle of 30 ° or more. The magnitudes of water repellency and oil repellency can be adjusted by the kind of precursor of the non-adhesive substance.
As described above, since the non-adhesive surface has liquid repellency, the adsorption of biological material is suppressed on the non-adhesive surface, so that no scaffold is formed when cells are adsorbed. As a result, the measurement target cell is selectively adsorbed and fixed to the cell placement unit 10a in the device 1.

  In addition, the non-cell adhesive layer 19 is preferably light transmissive for the same reason as in the case of the electrode 12. In order to make the non-cell adhesive layer 19 light transmissive, a component such as a non-adhesive substance may be light transmissive.

In particular, the non-cell adhesive layer 19 is preferably made of a material having liquid repellency and light transmission.
As such a non-cell adhesive layer 19, more specifically, a non-adhesive substance containing a fluorine-containing compound having liquid repellency and light transmittance can be exemplified.

  As the precursor of the fluorine-containing compound, a compound having a perfluoroalkyl group or a perfluoroalkyl ether group at one end of the molecule is preferable, and a compound represented by the following general formula (1) or (2) is more preferable. (Hereinafter, abbreviated as compound (1) and compound (2), respectively).

(In the formula, R ¹ represents a linear or branched perfluoroalkyl group or perfluoroalkyl ether group having ^{1 to} 16 carbon atoms; R ¹ ′ represents a linear or branched chain having ^{1 to} 16 carbon atoms; Represents a group in which one fluorine atom is removed from a perfluoroalkyl group or a perfluoroalkyl ether group; R ² is any one of groups represented by the following formulas (11) to (16), and a plurality of R ² may be the same or different; Y is a group represented by the formula “—NH—C (═O) —” or a carbonyl group, and a plurality of Y may be the same or different; Is an ethyleneoxy group in which one or more hydrogen atoms may be replaced by an alkyl group or alkyloxyalkyl group in which one hydrogen atom is substituted, and a plurality of Z may be the same or different from each other Even J and k are each independently 0 or 1, and a plurality of j or k may be the same or different from each other; l and m are each independently an integer of 0 or more, and a plurality of l or m may be the same or different from each other; when l is an integer of 2 or more, one Z may be the same or different from each other.

(In the formula, R ³ represents a hydroxyl group or an atom or group that can be substituted with a hydroxyl group; R ⁴ represents a hydrogen atom or a monovalent hydrocarbon group; n is 1, 2 or 3, and n is 2 or 3; And n R ³ may be the same or different from each other; when n is 1, the two R ⁴ may be the same or different from each other.)

R ¹ is a linear or branched perfluoroalkyl group or perfluoroalkyl ether group having ¹ to 16 carbon atoms. Here, the “perfluoroalkyl group” refers to “a group in which all hydrogen atoms of the alkyl group are substituted with fluorine atoms”. The “perfluoroalkyl ether group” refers to “a monovalent group in which the perfluoroalkyl group is bonded to an oxygen atom”.
Examples of the alkyl in R ¹ include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, undecyl, dodecyl, tridecyl, Examples include tetradecyl, pentadecyl, and hexadecyl.
R ¹ preferably has 3 to 12 carbon atoms, and more preferably 6 to 10 carbon atoms.
R ¹ is preferably linear and is preferably a perfluoroalkyl group.

R ¹ ′ represents a group obtained by removing one fluorine atom from a linear or branched perfluoroalkyl group or perfluoroalkyl ether group having ^{1 to} 16 carbon atoms. Specifically, a divalent group in which one fluorine atom is removed from R ¹ can be exemplified. When R ¹ ′ has 2 or more carbon atoms, one fluorine atom bonded to the terminal carbon atom opposite to the carbon atom bonded to “Z” of R ¹ is removed. Divalent groups are preferred.

R ² is any of the groups represented by the formulas (11) to (16). Then, a plurality of R ² may be the same or different from each other, for example, Compound (2) R ² of both molecular ends in the may be the same or different from each other, the compound R ² in the compound (1) ( R ² in 2) may be the same as or different from each other.

R ³ represents a hydroxyl group or an atom or group that can be substituted with a hydroxyl group.
Preferable examples of the atom capable of substituting for the hydroxyl group of R ³ include halogen atoms such as fluorine atom, chlorine atom, bromine atom and iodine atom, and chlorine atom is particularly preferable.
Moreover, as a group which can be substituted by a hydroxyl group, a C1-C6 alkoxy group, an aryloxy group, an aralkyloxy group, and an acyloxy group are mentioned as a preferable thing.
Examples of the alkoxy group having 1 to 6 carbon atoms include methoxy group, ethoxy group, n-propoxy group, isopropoxy group, n-butoxy group, isobutoxy group, sec-butoxy group, tert-butoxy group, pentoxy group and hexyloxy. Examples are groups.
Examples of the aryloxy group include a phenoxy group and a naphthoxy group.
Examples of the aralkyloxy group include a benzyloxy group and a phenethyloxy group.
Examples of the acyloxy group include an acetoxy group, a propionyloxy group, a butyryloxy group, a valeryloxy group, a pivaloyloxy group, and a benzoyloxy group.
Among these, as R ³ , a chlorine atom, a methoxy group, and an ethoxy group are more preferable, and a chlorine atom is particularly preferable.

R ⁴ represents a hydrogen atom or a monovalent hydrocarbon group.
The monovalent hydrocarbon group for R ⁴ is not particularly limited, and may be a chain structure or a cyclic structure, and may be either saturated or unsaturated. In the case of a chain structure, either a straight chain or a branched chain may be used, and in the case of a cyclic structure, either a monocyclic structure or a polycyclic structure may be used.
Among these, preferred are an alkyl group having 1 to 6 carbon atoms, an aryl group, and an aralkyl group.
Examples of the alkyl group having 1 to 6 carbon atoms include methyl group, ethyl group, n-propyl group, isopropyl group, n-butyl group, isobutyl group, sec-butyl group, tert-butyl group, pentyl group and hexyl group. It can be illustrated.
Examples of the aryl group include a phenyl group and a naphthyl group.
Examples of the aralkyl group include a benzyl group and a phenethyl group.
Among these, as R ⁴ , a methyl group, an ethyl group, an n-propyl group, and an isopropyl group are more preferable, and a methyl group is particularly preferable.

Y is a group represented by the formula “—NH—C (═O) —” or a carbonyl group. That is, adjacent methylene groups and oxygen atoms are bonded as follows.
“— (CH ₂ ) _m — (NH—C (═O)) _j — (O) _k —”
_{"- (CH 2) m - (} C (= O)) j - (O) k - "
A plurality of Y may be the same as or different from each other. For example, Ys facing each other across “R ¹ ′” in the compound (2) may be the same as or different from each other. ) In Y) and Y in compound (2) may be the same as or different from each other.

Z is an ethyleneoxy group in which one hydrogen atom is substituted for an alkyl group or an alkyloxyalkyl group in which one or more hydrogen atoms may be substituted with a fluorine atom. The terminal oxygen atom of the ethyleneoxy group is bonded to the adjacent R ¹ .
The number of hydrogen atoms that may be substituted in the ethyleneoxy group is preferably one. Further, the alkyl group or alkyloxyalkyl group in which one or more hydrogen atoms may be substituted with a fluorine atom is preferably linear or branched, and more preferably linear. . And it is preferable that carbon number is 1-16.
A plurality of Z may be the same as or different from each other. For example, in the compound (2), Zs facing each other across “R ¹ ′” may be the same as or different from each other. ) In Z) and Z in compound (2) may be the same or different.

j and k are each independently 0 or 1, and particularly preferably 0. Moreover, several j or k may mutually be same or different. For example, j relating to Y facing each other across “R ¹ ′” in compound (2) may be the same or different from each other, and j relating to Y in compound (1) may be different from that in compound (2). J for Y may be the same as or different from each other. The same applies to k.

l and m are each independently an integer of 0 or more.
L is particularly preferably 0.
m is preferably 0 to 3, more preferably 1 or 2, and particularly preferably 2.
A plurality of l or m may be the same as or different from each other. For example, in the compound (2), ^{1's related} to Z facing each other across “R ¹ ′” may be the same as or different from each other, and the 1's related to Z in the compound (1) and the 1 The l's corresponding to Z may be the same as or different from each other. The same applies to m.
Furthermore, when l is an integer greater than or equal to 2, 1 Z may mutually be same or different.

n is 1, 2 or 3, and when n is 2 or 3, n R ^{3 s} may be the same or different from each other, and when n is 1, two R ⁴ May be the same or different from each other.

  As a compound (1) or (2), what is represented by the following general formula (3) or (4) is mentioned as a more preferable thing.

(In the formula, R ¹ , R ¹ ′, R ² and m are the same as described above.)

In the general formula (1) or (2), a compound in which Z is represented by “— (CHR ⁵ —CHR ⁶ —O) _{1 ′} — (CHR ⁷ —CHR ⁸ —O) ₁ ″” It is mentioned as a more preferable thing.
Here, one of R ⁵ and R ⁶ represents a hydrogen atom, and the other represents one hydrogen atom of a methyl group in a linear or branched fluoroalkyl group or fluoroalkyl ether group having 1 to 16 carbon atoms. Represents a substituted group. Here, the “fluoroalkyl ether group” refers to “a monovalent group in which the linear or branched fluoroalkyl group having 1 to 16 carbon atoms is bonded to an oxygen atom”.
One of R ⁷ and R ⁸ represents a hydrogen atom, and the other represents a linear or branched alkyl group or alkyl ether group having 1 to 16 carbon atoms. Here, the “alkyl ether group” refers to “a monovalent group in which the linear or branched alkyl group having 1 to 16 carbon atoms is bonded to an oxygen atom”.

l ′ is an integer of 2 or more, preferably 2 to 20, and more preferably 5 to 10.
l ″ is an integer of 0 or more, preferably 0 to 20, and more preferably 0.

Examples of the fluoroalkyl group in which one hydrogen atom of the methyl group in R ⁵ and R ⁶ is substituted include CF ₃ —, CHF ₂ —, CClF ₂ —, C ₂ F ₅ —, CHF ₂ CF ₂ —, CClF. _{2 CF 2 -, (CF 3} ) 2 CF-, CF 3 CF 2 CF 2 -, (CHF 2) 2 CF -, (CF 3) (CHF 2) CF -, (CF 3) 2 CFCF 2 CF 2 - _{, (CF 3) 2 CF (} CF 2 CF 2) 2 -, CF 3 (CF 2) 3 -, CF 3 (CF 2) 5 -, CF 3 (CF 2) 7 -, CF 3 (CF 2) 9 _{-, CF 3 (CF 2)} 11 -, CF 3 (CF 2) 13 -, CF 3 (CF 2) 15 - it can be exemplified.
And in R ⁵ and R ^6, examples of the fluoroalkyl ether group in which one hydrogen atom is substituted methyl group, a monovalent group such fluoroalkyl groups linked to an oxygen atom can be exemplified.

Examples of the alkyl group for R ⁷ and R ⁸ include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, undecyl, Examples include dodecyl, tridecyl, tetradecyl, pentadecyl and hexadecyl.
The Examples of the alkyl ether groups in R ⁷ and R ^8, monovalent radicals the alkyl groups linked to an oxygen atom can be exemplified.

The non-cell adhesion layer 19 can be formed by bonding a precursor of a non-adhesive substance on the insulating film 15 as described above. A commercial product may be used as the precursor of the non-adhesive substance.
For example, when the insulating film 15 has a hydroxyl group, as the compound (1) or (2), R ² has a group represented by the formula (11), (12), or (16). It is preferable to use it.
Among these, the compound (1) or (2) having an organic silyl group represented by the formula (16) or the like is preferable because it is easy to obtain a commercial product. Examples of such commercially available products include CYTOP series (trademark, manufactured by Asahi Glass Co., Ltd.), MegaFuck (trademark, manufactured by Dainippon Ink Chemical Co., Ltd.), and Dick Guard (trademark, manufactured by Dainippon Ink Chemical Co., Ltd.). ), FPX-30G (trademark, manufactured by JSR Corporation), Novec EGC-1720 (trademark, manufactured by Sumitomo 3M Corporation), Patinal series (substance WR1, WR2, WR3) (trademark, manufactured by Merck Corporation), OPTOOL DSX ( Trademark, manufactured by Daikin Industries, Ltd.).

When R ² has a group other than the above formulas (11), (12) and (16), for example, a group represented by the above formula (13), (14) or (15) The insulating film may be treated so as to have a functional group capable of reacting with these groups.
For example, according to the method described in Chembiochem, 2,686-694 (2001) (Benters R. Et al.), A compound having a group represented by the formula (14) as R ² (that is, an amino group) (1) or (2) can be bonded to the insulating film.
Further, for example, 3-aminopropyltrimethoxysilane, 3-aminopropyltriethoxysilane, 3-aminopropyldiethoxymethylsilane, 3- (2-aminoethylamino) propyltrimethoxysilane, 3- (2-aminoethyl) If an organic silane compound having an amino group such as amino) propyltriethoxysilane or 3- (2-aminoethylamino) propyldimethoxymethylsilane is bonded to the surface of the insulating film, R ² is represented by the formula (13). The compound (1) or (2) having a group (that is, a carboxyl group) or a group represented by the formula (15) (that is, an epoxy group) can be bonded to the insulating film. The organosilane compound is easily available.

In the non-cell adhesion layer 19, the non-adhesive substance is composed of at least a part of a molecule, preferably one end of the molecule is bonded to a specific atom or functional group on the insulating film 15, while a portion having liquid repellency It is presumed that it is oriented so as to be exposed toward the outside of the device 1. For example, when the compound (1) is used as a precursor of a non-adhesive substance, the R ² is bonded to the insulating film, while the R ¹ is oriented so as to be exposed to the outside of the device 1. Further, when the compound (2) is used, it is presumed that one or two R ² bonds to the insulating film, while R ¹ ′ is oriented so as to be exposed to the outside of the device 1. .
Thus, it is presumed that the non-cell adhesion layer 19 develops non-adhesiveness to cells due to the orientation of the liquid-repellent sites. Therefore, by using a compound having a perfluoroalkyl group or a perfluoroalkyl ether group as in the compound (1) or (2), it is possible to develop a higher non-adhesive effect in the device 1.

  In addition, the non-cell adhesion layer 19 formed using the compound (1) or (2) is more transparent than a conventional layer containing polytetrafluoroethylene or the like, and is suitable for forming a thin single-layer thin film. It is excellent in fine workability such as patterning and removal. Since the transparency is excellent, when optically detecting a cell or a substance acting on the cell, noise can be greatly reduced, and highly sensitive analysis is possible. Moreover, since it is excellent in microfabrication, as will be described later, a large number of cell placement portions 10a and cell connection portions 10b having a small size can be formed on the insulation substrate 11, and the arrangement density of cells on the insulation substrate 11 can be increased. Analyzes with high efficiency.

The non-adhesive substance in the non-cell adhesive layer 19 may be one kind or two or more kinds. When two or more types are used in combination, the combination, ratio, and the like can be appropriately selected depending on the purpose.
The non-cell adhesion layer 19 may contain any component other than the non-adhesive substance as long as the effects of the present invention are not hindered.

The thickness of the non-cell adhesion layer 19 is preferably adjusted as appropriate according to the type of cells to be measured. Usually, it is preferably 5000 nm or less, more preferably 1000 nm or less, and particularly preferably 200 nm or less. In particular, when a monomolecular film of a non-adhesive substance is formed using the compound (1) to form a non-cell adhesion layer 19, a film having a thickness of 5 to 100 nm is suitably obtained.
If it is the thickness of the said range, the non-cell adhesion layer 19 is excellent also in workability.

Each of the cell placement portions 10a has a substantially circular shape with a diameter D. On the insulating substrate 11, a total of 16 cell arrangement portions 10a are arranged at equal intervals in two directions orthogonal to each other, and one microelectrode 17 is arranged for each cell arrangement portion 10a.
On the other hand, all of the cell connecting portions 10b are band-like in width. Each cell connection part is preferably arranged so as to connect each cell arrangement part to be connected with the shortest distance, and in FIG. 2, it overlaps with a line connecting substantially the center parts of the cell arrangement parts to be connected. The cell connection part 10b is arrange | positioned. The connection distance S corresponds to the center-to-center distance between the cell placement units 10a to be connected.
That is, the cell arrangement part 10a is arranged in a grid shape so as to constitute the apex, and the cell connection part 10b constitutes a side.

What is necessary is just to adjust the area of the cell arrangement | positioning part 10a suitably according to the kind and number of cells to arrange | position, and other purposes. For example, when the number of cells to be arranged is one per cell arrangement part, the area of the cell arrangement part is usually 2.0 × 10 ^{−11 to} 4.0 × 10 ⁻⁸ m ^2. Preferably, it is 1.8 × 10 ^{−10 to} 2.3 × 10 ⁻⁸ m ² . In order to obtain such a preferable area, the diameter D of the cell placement portion 10a may be adjusted to correspond to this area, preferably 5 to 225 μm, more preferably 15 to 150 μm. A smaller diameter D is preferable in that the number of bonding surfaces 131 on the substrate 1 can be increased.
The width W and the connection distance S of the cell connection part 10b may be appropriately adjusted according to the type and number of cells to be arranged and other purposes. Similarly to the above, when the number of cells to be arranged is one per cell arrangement portion 10a, the width W is usually preferably 30 μm or less, and more preferably 8 μm or less. The connection distance S varies depending on the cell, but is usually preferably adjusted according to the diameter D, preferably D + 5 to D + 350 μm, and more preferably D + 20 to D + 250 μm.

Here, an example is shown in which 16 cell placement portions 10a are placed on the microelectrode placement portion 14, but the number of cell placement portions takes into account the size of the insulating substrate, the number of cells used for measurement, and the like. And may be adjusted as appropriate. And the number of cell arrangement | positioning parts is preferable at the point which can use a device for various analysis.
Moreover, although the substantially circular shape is illustrated as a cell arrangement | positioning part, it is not limited to this, Other shapes, such as polygonal shape, may be sufficient.
As shown here, the shape and size of the cell placement portions are not necessarily the same as shown here, but the same is preferable in that the device can be easily manufactured.
In addition, here, an example is shown in which the cell arrangement portions are arranged at equal intervals in two directions orthogonal to each other on the insulating substrate, but the arrangement direction and interval are not limited to this, and can be appropriately selected according to the purpose. .

Here, although the strip-shaped thing is illustrated as a cell connection part, it is not limited to this, Other shapes may be sufficient. However, an elongated shape is preferable.
In addition, the number of cell connection portions may be adjusted as appropriate according to the number of cell placement portions and the purpose.
As shown here, the shape and size of the cell connecting portion are not necessarily the same, but the same is preferable in that the device can be easily manufactured.
In addition, here, an example is shown in which the cell placement portion is arranged in a grid shape so that the cell connection portion constitutes the apex, and the cell connection portion is configured as a side. You may arrange | position so that it may overlap, and may arrange | position so that it may overlap only on a diagonal line, and it does not need to be any of these.

The material of the microelectrode 17 may be appropriately selected from known materials, and the same material as the electrode 12 can be exemplified. For the same reason as in the case of the electrode 12, a material having optical transparency is preferable.
The size of the microelectrode 17 is preferably adjusted as appropriate according to the type of cell to be measured, but usually the area of the electrode surface is 1.0 × 10 ^{−11 to} 5.0 × 10 ⁻⁹ m ^2. It is preferable that it is 3.0 * 10 < ^-11 > -2.0 * 10 < ^-9 > m < ² >. The thickness of the microelectrode 17 is preferably 50 to 1000 nm, and more preferably 100 to 400 nm.

Here, an example is shown in which 16 microelectrodes 17 are arranged in the microelectrode arrangement portion 14, but the number of microelectrodes may be adjusted as appropriate in consideration of the number of cells used for measurement and the like. For example, the cell potential can be measured even if only one microelectrode is provided in the microelectrode arrangement part, but if a plurality of microelectrodes are provided, the potential of a plurality of cells can be measured simultaneously, For example, various analyzes such as the function of specific cells in the cell group network and the interaction between cells become possible.
Further, here, an example is shown in which one microelectrode 17 is disposed per cell placement portion 10a, but a plurality of microelectrodes may be placed per cell placement portion. In the case where a plurality of ion channel molecules are arranged, for example, ion channel molecules that selectively permeate ions into and out of the cell by measuring and comparing the potential at a plurality of locations (for example, near the nucleus and other portions) per cell. More advanced analysis is possible, such as obtaining information about the distribution.
In addition, the microelectrodes do not necessarily have to be arranged in all the cell arrangement portions, and may be appropriately selected according to the purpose. In the case where a cell arrangement part where no microelectrode is arranged is provided, for example, it is suitable for analysis using a heterogeneous cell in which the cell potential does not change, or for reducing the load on the analysis apparatus by reducing the total number of microelectrodes. .

On the other hand, although the substantially square shape is illustrated here as the microelectrode 17, it is not limited to this, Other shapes, such as other polygonal shape and circular shape, may be sufficient.
As shown here, the shapes of the microelectrodes are not necessarily the same as shown here, but the same is preferable in that the device can be easily manufactured.
The microelectrode 17 may not necessarily be surrounded by the exposed insulating film 15. For example, a part of the peripheral edge may be in contact with the non-cell adhesive layer 19. However, the microelectrode 17 is preferably surrounded by the exposed insulating film 15 because the cells can be stably arranged by the cell arrangement portion. And it is preferable that the microelectrode 17 is arrange | positioned concentrically with the cell arrangement | positioning part 10a so that it may illustrate here.

FIG. 3 is a longitudinal sectional view taken along line III-III in FIG.
The wiring 18 has a band shape, and electrically connects the second connecting portion 12b of the electrode 12 and the microelectrode 17. Therefore, normally, the number of electrodes 12, wirings 18, and microelectrodes 17 is the same. With such a configuration, the microelectrode 17 is electrically connected to an external measuring instrument (not shown) to which the device 1 is connected.
The wiring 18 preferably has the same thickness as the microelectrode 17 in consideration of forming the wiring 18 simultaneously with the microelectrode 17 as described in the device manufacturing method described later.
The width of the wiring 18 may be appropriately adjusted in consideration of the size of the microelectrode 17 and the like, but is usually preferably 2 to 70 μm, and more preferably 3 to 20 μm.
Here, a strip-shaped wiring is illustrated as an example, but the present invention is not limited to this and may have other shapes. However, an elongated shape is preferable.

  The cell to be measured is arranged in the cell arrangement unit 10 a so as to be in contact with the microelectrode 17. At this time, the whole cell may be accommodated in the cell placement portion 10a, that is, the recess where the microelectrode 17 on the insulating substrate 11 is exposed, or a part of the cell may not be accommodated in the recess. May be. This is because the arrangement position of the whole cell is fixed by the non-cell adhesion layer 19 surrounding the cell arrangement portion 10a even if the whole cell is not accommodated in the recess.

  In the present invention, it is preferable that at least one of the non-cell adhesive layer 19 and the microelectrode 17 has light transmittance, and it is more preferable that both the non-cell adhesive layer 19 and the microelectrode 17 have light transmittance.

  Up to this point, the device in which the wirings of the microelectrodes are all covered with an insulating film is exemplified as the device. However, for example, a part of the wiring may be exposed without being covered with the insulating film. However, in order to suppress the deterioration of the wiring, the fewer the exposed portions of the wiring, the better.

  In the present invention, the cell linking part may not necessarily be provided, but by providing the cell linking part as exemplified here, the cells can be easily bonded to each other at the linking part. A network is built and a cell group is formed. As a result, the potential of each cell in the cell group can be measured, and the function of the cell can be analyzed with high accuracy. In addition, it is possible to acquire highly accurate information regarding the interaction between cells, the interaction between cells and chemical substances, and the like.

(Second embodiment)
FIG. 4 is a plan view illustrating a device according to the second embodiment of the invention.
The device 2 exemplified here is different from the first embodiment in the form of the cell arrangement portion and the partition wall, and the same components as those in the first embodiment are denoted by the same reference numerals, and the detailed description thereof is as follows. Omitted.

In the device 2, the number of electrodes 12 and reference electrodes 13 that are patterned on the insulating substrate 11 is different from that of the first embodiment in accordance with the arrangement form of the microelectrodes in the microelectrode arrangement part 24.
Further, the partition wall 26 has a round cylindrical shape with a hollow portion in a plan view, and is otherwise the same as the partition wall 16 in the first embodiment.

FIG. 5 is an enlarged view illustrating the microelectrode arrangement portion 24, (a) is a plan view, and (b) is a plan view further illustrating a part of the plan view of (a). .
In the microelectrode placement portion 24, a plurality of microelectrodes 27 having a substantially circular shape are placed. The microelectrode 27 is the same as the microelectrode 17 in the first embodiment except that the shape is different.
Then, an insulating film 15 is coated on the insulating substrate 11 so that the plurality of microelectrodes 27 are exposed. Furthermore, a substantially circular cell arrangement portion 20a composed of the exposed microelectrode 27 and a predetermined region surrounding the microelectrode 27, and a band-shaped cell connection portion 20b connecting these cell arrangement portions 20a. A non-cell adhesion layer 19 is provided on the insulating film 15 except for the non-stacked region 20 made of.

FIG. 6 is a longitudinal sectional view taken along line IV-IV in FIG.
The wiring 28 electrically connects the second connecting portion 12b of the electrode 12 and the microelectrode 27. Therefore, normally, the number of the electrodes 12, the wirings 28, and the microelectrodes 27 is the same. With such a configuration, the microelectrode 27 is electrically connected to an external measuring instrument (not shown) to which the device 1 is connected.
The wiring 28 is the same as the wiring 18 in the first embodiment, except that the number differs depending on the arrangement form of the microelectrodes 27.

Each of the cell placement portions 20a has a substantially circular shape with a diameter D ′. On the insulating substrate 11, a total of ten cell arrangement portions 20a are arranged at equal intervals in two directions orthogonal to each other, and the number of arrangement of the microelectrodes 27 per cell arrangement portion 20a is 0 or 1. It is.
On the other hand, each of the cell connecting portions 20b has a band shape having a width W ′. Each cell connection part is preferably arranged so as to connect each cell arrangement part to be connected with the shortest distance, and in FIG. 5, the cell connection parts overlap with a line connecting the substantially central parts of the cell arrangement parts to be connected. The cell connecting portion 20b is disposed in the cell. The connection distance S ′ corresponds to the center-to-center distance between the cell placement portions 20a to be connected.
The area of the cell placement portion 20a is the same as that of the cell placement portion 10a in the first embodiment. The sizes of D ′, W ′, and S ′ are the same as D, W, and S in the first embodiment.

Since the device of the present invention is covered with a non-cell adhesion layer in the microelectrode placement portion except for the cell placement portion and the cell connection portion, the cell is placed in a region other than the cell placement portion when the measurement target cell is applied. Even if it adheres, it can be easily removed. And a cell can be stably hold | maintained to a cell arrangement | positioning part.
In particular, the non-cell adhesion layer formed using the compound (1) or (2) is suitable for making a thin single-layer thin film, has excellent microfabrication properties, and has a cell arrangement part and a cell connection part. It can be formed with higher definition and higher accuracy than before, and the processing accuracy is not impaired. Therefore, since the arrangement density of cells can be increased, measurement can be performed with high efficiency. Further, when optically detecting a cell or a substance acting on the cell with excellent transparency, noise can be greatly reduced. Therefore, since the S / N ratio at the time of analysis is significantly improved as compared with the conventional case, analysis can be performed with higher accuracy.

By using the device of the present invention, the function of each cell in the cell group can be analyzed with high accuracy, and various analyzes and substance production using the function can be performed with high efficiency. For example, it is possible to objectively analyze the influence of cells under various environments using community effects between different types of cells. More specifically, for example, a bioassay can be performed using various chemical substances, and can be applied to a toxicity test or the like. As a result, for example, the effect of the administration of pharmaceuticals and chemical substances on the living body can be evaluated by comparing specific numerical values, compared to the subjective expression of good / bad physical condition. .
And when performing these analysis and substance production, the method similar to the case of using the conventional biochip, device, etc. may be applied except using the device of the present invention.

<Method for manufacturing microelectrode array device>
The device of the present invention can be manufactured as follows.
(Manufacturing method 1)
FIGS. 7-9 is a figure which illustrates the manufacturing process of the device of this invention illustrated to FIGS. 1-3. In the following, the microelectrode placement portion 14 will be described in particular with reference to the drawings.
First, the electrode 12, the reference electrode 13, the microelectrode 17 and the wiring 18 are deposited on the entire surface of one surface of the insulating substrate 11, and then patterned to form the electrode 12, the reference electrode 13, and FIG. As shown in a) and (b), the microelectrode 17 and the wiring 18 are formed, respectively. Fig.7 (a) is a top view of the microelectrode arrangement | positioning part 14, (b) is a longitudinal cross-sectional view in the II line | wire of (a). The film forming method may be appropriately selected from known methods, but sputtering is preferred. The patterning method after film formation may be appropriately selected from known methods, but wet etching or lift-off processing is preferable.

  Next, in the predetermined region on the insulating substrate 11 on which the second connection portion 12b, the reference electrode 13, the microelectrode 17 and the wiring 18 of the electrode 12 are formed, the reference electrode 13 and FIGS. As shown in b), the insulating film 15 is patterned so that the microelectrodes 17 are exposed. 8A is a plan view of the microelectrode placement portion 14, and FIG. 8B is a longitudinal sectional view taken along line II-II in FIG. The patterning method of the insulating film 15 may be appropriately selected from known methods according to the material of the insulating film 15. For example, when a photosensitive resin is used as the insulating film, patterning may be performed by photolithography. When a silicon oxide film is used as the insulating film, a silicon oxide film is formed by a plasma CVD method or the like, and then a photosensitive resin such as a photoresist is laminated on the silicon oxide film, which is then used for photolithography. Then, the silicon oxide film may be patterned so that the reference electrode 13 and the microelectrode 17 are exposed by etching using the patterned photosensitive resin as a mask. Etching at this time is preferably wet etching.

  Next, the surface of the insulating substrate 11 on which the insulating film 15 is patterned, specifically, the insulating film 15, the reference electrode 13, and the microelectrode 17 are covered with a precursor of a non-adhesive substance, and the non-cell adhesive layer 19 is covered. Form. Examples of the coating method at this time include a dip coating method, a vacuum deposition method, a CVD (chemical vapor deposition) method, and a plasma polymerization method. For example, in the case of the dip coating method, it is preferable to prepare a coating treatment solution containing a precursor of a non-adhesive substance, apply this, and remove the solvent component in the treatment solution by drying after coating. . The thickness of the non-cell adhesion layer 19 can be adjusted by the molecular size and amount of the precursor.

  Next, the insulating substrate 11 on which the non-cell adhesion layer 19 is formed may be washed and dried. For washing, an alcohol such as methanol or ethanol may be used, and when the coating solution contains a fluorinated solvent, a fluorinated solvent may be used.

Next, a region on the non-cell adhesion layer 19 other than the region corresponding to the reference electrode 13, the cell arrangement portion 10a, and the cell connection portion 10b is covered with a photosensitive resin by photolithography, and the non-cell adhesion layer is used with the resin as a mask. 19 is etched. Any etching method may be used at this time, but dry etching is preferable, and more specifically, etching using plasma generated from a kind of gas selected from the group consisting of oxygen, argon and chlorine is preferable. Further, etching may be performed by ultraviolet light or ozone irradiation.
By etching, the non-cell adhesion layer 19 not masked with the photosensitive resin is removed, and the non-cell adhesion layer 19 is patterned. As a result, the reference electrode 13 and the microelectrode 17 are exposed, and the insulating film 15 is exposed in a region corresponding to the cell placement portion 10a and the cell connection portion 10b.

  Next, by removing all the photosensitive resin remaining on the insulating substrate 11, the reference electrode 13 is exposed and, as shown in FIGS. 9 (a) and 9 (b), non-cell adhesion is performed on the microelectrode arrangement portion 14. The layer 19 is patterned to obtain the insulating substrate 11 on which the cell arrangement part 10a and the cell connection part 10b are formed. Fig.9 (a) is a top view of the microelectrode arrangement | positioning part 14, (b) is a longitudinal cross-sectional view in the III-III line of (a).

  Next, the device 1 is obtained by fixing the partition wall 16 in a predetermined region on the insulating film 15 so as to surround the reference electrode 13 and the microelectrode arrangement portion 14. The partition wall 16 is preferably fixed using an adhesive. The adhesive may be appropriately selected from known materials depending on the material of the insulating film 15 and the partition wall 16.

(Manufacturing method 2)
The device of the present invention can also be manufactured by the following method.
In the same manner as in the manufacturing method 1, the insulating film 15 is formed in a predetermined region on the insulating substrate 11 so that the reference electrode 13 and the microelectrode 17 are exposed as shown in FIGS. Pattern.
Next, on the surface of the insulating substrate 11 on which the insulating film 15 is patterned, the reference electrode 13, the microelectrode 17, the insulating film 15 in the area surrounding the microelectrode 17, and the areas surrounding the microelectrode 17 are The insulating film 15 in a predetermined region to be connected is covered with the photosensitive resin 9 by photolithography and patterned. Here, the “region surrounding the microelectrode 17” is a region corresponding to the cell placement portion 10a, and the “predetermined region connecting the regions surrounding the microelectrode 17” is the cell connecting portion. This is an area corresponding to 10b.
Next, the surface on which the photosensitive resin 9 on the insulating substrate 11 is patterned, specifically, the coated photosensitive resin 9 and the insulating film 15 are covered with a precursor of a non-adhesive substance, and FIG. As shown in (b), the non-cell adhesion layer 19 is formed. FIG. 10A is a plan view of the microelectrode placement portion 14, and FIG. 10B is a longitudinal sectional view taken along line VV in FIG. The method for coating the precursor of the non-adhesive substance may be the same as in the manufacturing method 1.
Next, the non-cell adhesive layer 19 on the photosensitive resin 9 is removed together with the photosensitive resin 9 covered with the non-cell adhesive layer 19. Thereby, the reference electrode 13 and the microelectrode 17 are exposed, and the insulating film 15 is exposed in a region corresponding to the cell placement portion 10a and the cell connection portion 10b. As a result, as in the case of the manufacturing method 1, as shown in FIGS. 9A and 9B, the insulating substrate 11 in which the non-cell adhesive layer 19 is patterned on the microelectrode arrangement portion 14 is obtained.
Next, the device 1 is obtained by fixing the partition wall 16 so as to surround the reference electrode 13 and the microelectrode arrangement portion 14 in a predetermined region on the insulating film 15 by a method similar to the manufacturing method 1.

  Here, although the manufacturing method of the device 1 according to the first embodiment of the present invention illustrated in FIGS. 1 to 3 has been described, for example, the device 2 and the like illustrated in FIGS. It goes without saying that devices can be manufactured as well. For example, when it is desired to form the cell placement portion and / or the cell connection portion in different shapes, the photosensitive resin used for removing the non-cell adhesion layer may be patterned so as to become a desired one. When the cell connection portion is unnecessary, the photosensitive resin may be patterned so that the reference electrode is exposed and only the cell placement portion is formed.

Hereinafter, the present invention will be described in more detail with reference to specific examples. However, the present invention is not limited to the following examples.
Example 1
The device illustrated in FIGS. 1 to 3 was manufactured by the manufacturing method 1.
That is, ITO was deposited on the entire surface of one side of a glass substrate (thickness: 0.5 mm) of Pyrex (registered trademark) (Code 7059, manufactured by Corning International Co., Ltd.) by sputtering so as to have a thickness of 200 nm. By patterning by wet etching, an electrode, a reference electrode, a microelectrode, and a wiring were formed.
Of the electrodes, the first connecting portion was formed into a strip shape having a width of 0.8 mm, and the second connecting portion was formed into a strip shape having a width reduced within a range of 0.8 to 0.01 mm. Further, the reference electrode was in a square shape with a side of 0.2 mm. Further, the microelectrode was in the shape of a square having a side of 10 μm, and the wiring was in the form of a strip having a width of 7.5 μm.
Next, a photoresist as an insulating film is patterned in a predetermined region on the glass substrate by photolithography so as to have a thickness of 1.2 μm, and OPTOOL DSX (trademark, Daikin Industries, Ltd.) is formed on the patterning surface, the reference electrode, and the microelectrode. Dip coating). And after drying overnight, it heated at 100 degreeC for 1 hour, and the non-cell adhesion layer was formed by wash | cleaning using Novec HFE (trademark, Sumitomo 3M Co., Ltd.) which is a fluorine-type solvent.
Next, a predetermined region on the non-cell adhesion layer was coated with a photoresist by photolithography, and the non-cell adhesion layer was etched with oxygen plasma for 5 minutes using the resin as a mask to pattern the non-cell adhesion layer.
Next, all the photoresist on the insulating substrate was removed to form a cell placement portion and a cell connection portion. The diameter D of the cell arrangement part was 25 μm, the width W of the cell connection part was 1 μm, and the connection distance S was 50 μm. The thickness of the non-cell adhesion layer was 12 nm.
Next, a rectangular tube-shaped partition wall made of a glass plate having a thickness of 0.7 mm, a height of 2.0 mm, and a hollow portion having a square of 2.5 mm on a side in plan view is formed on the insulating film. The device of the present invention was obtained by bonding and fixing to the region.

Using this device, the action potential of rat cardiomyocytes was measured according to the following procedure.
First, a collagen solution obtained by dissolving Cell Matrix Type III (manufactured by Nitta Gelatin Co., Ltd.) in a dilute hydrochloric acid aqueous solution with a pH of 3 so as to have a concentration of 0.3 mg / mL is applied as a culture solution in the partition wall for 10 minutes. After standing, it was washed with a dilute hydrochloric acid aqueous solution of pH 3.
Next, rat myocardial primary cultured cells (produced by Hokkaido System Science Co., Ltd.) are applied to the septum to which the collagen solution is applied, and the cells are placed one by one in the cell placement part using optical tweezers using an infrared laser. Placed and adsorbed.
Since both the microelectrode and the non-cell adhesion layer are transparent, the cell arrangement portion can be clearly seen. As a result, the cells can be easily arranged on the cell arrangement portion without being arranged on the non-cell adhesion layer. Furthermore, as shown in FIG. 11, it was confirmed that the cells were connected to each other at the connecting portion, and the pulsation was synchronized. FIG. 11 is a plan view schematically showing the microelectrode placement portion 14 at this time, and is a cell in which the reference numeral 110 is placed. FIG. 12 shows the measurement result of the action potential of one cell arranged at this time. In FIG. 12, the horizontal axis represents time, and the vertical axis represents potential (V). As shown in FIG. 12 (a), a potential change accompanying the pulsation of the cells in the culture solution was confirmed every 0.44 seconds. Next, when epinephrine, which is known to have a pharmacological effect for accelerating the pulsation of the myocardium, was added to the culture solution in the septum, the potential change was 0.20 seconds as shown in FIG. 12 (b). It was confirmed that the heartbeat beats faster.
From the above, it was confirmed that the action potential of a cell can be measured with high accuracy for each single cell.

(Example 2)
OPTOOL DSX (TM, manufactured by Daikin Industries, Ltd.) in place of, _CF 3 concentration is _{0.02mol / L - (CF 2)} 7 - (CH 2) Novec HFE (trademark of 2 -SiCl _3, Sumitomo stock A device was obtained in the same manner as in Example 1 except that the solution was used.
When the action potential of the rat myocardial primary cultured cells was measured by the same method as in Example 1 using the obtained device, the same result as in Example 1 was obtained.

(Example 3)
The device illustrated in FIGS. 4 to 6 was manufactured by the manufacturing method 2.
That is, a chromium thin film was formed to a thickness of 100 nm by sputtering on the entire surface of one side of a glass substrate (thickness: 0.5 mm) of Pyrex (registered trademark) (code 7059, manufactured by Corning International Co., Ltd.). Further, a platinum thin film having a thickness of 200 nm was formed by sputtering and patterned by lift-off processing to form an electrode, a reference electrode, a microelectrode, and a wiring.
Of the electrodes, the first connection portion was in the form of a strip having a width of 1 mm, and the second connection portion was in the form of a strip having a width reduced in the range of 1 to 0.015 mm. Further, the reference electrode was in a square shape with a side of 0.3 mm. Further, the microelectrodes were in a square shape with a side of 30 μm, and the wirings were in a strip shape with a width of 15 μm.
Next, a silicon dioxide film having a thickness of 500 nm is formed on the obtained insulating substrate by plasma CVD using tetraethoxysilane as a deposition source, and a photoresist is patterned on the silicon dioxide film by photolithography. The silicon oxide film was patterned so that the reference electrode and the microelectrode were exposed by performing wet etching with hydrofluoric acid using this as a mask.
Next, a predetermined region on the insulating substrate is covered with a photoresist by photolithography, and further, a vacuum evaporation method using WR1 Partial (trade name, manufactured by Merck Co., Ltd.) as an evaporation source is applied on the coated photoresist and the insulating film. Then, the non-adhesive material precursor was coated to form a non-cell adhesion layer. At this time, the temperature of the vapor deposition source was 360 ° C. to 450 ° C., and vapor deposition was performed at a vacuum degree of 10 ⁻³ Pa for 30 seconds.
Subsequently, the non-cell adhesion layer on the photoresist was removed using acetone together with the photoresist coated with the non-cell adhesion layer, thereby forming a cell arrangement portion and a cell connection portion. The diameter D ′ of the cell arrangement part was 30 μm, the width W ′ of the cell connection part was 0.75 μm, and the connection distance S ′ was 100 μm.
Next, a circular cylindrical partition wall made of a glass plate having a thickness of 0.7 mm, a height of 2.5 mm, and a hollow portion having a diameter of 3 mm in plan view is formed in a predetermined region on the insulating film. The device of the present invention was obtained by bonding and fixing.

  The present invention can be used in fields such as cell function research, cell-based bioassay and substance production.

It is a figure which illustrates the device concerning a first embodiment of the present invention, (a) is a top view and (b) is a perspective view. It is a figure which expands and illustrates the microelectrode arrangement | positioning part on the device which concerns on 1st embodiment of this invention, (a) is a top view, (b) further expands and illustrates a part of (a). FIG. It is a longitudinal cross-sectional view in the III-III line of Fig.2 (a). It is a top view which illustrates the device concerning a second embodiment of the present invention. It is a figure which expands and illustrates the microelectrode arrangement | positioning part on the device which concerns on 2nd embodiment of this invention, (a) is a top view, (b) further expands and illustrates a part of (a). FIG. It is a longitudinal cross-sectional view in the IV-IV line of Fig.5 (a). It is a figure which illustrates the manufacturing process of the device which concerns on 1st embodiment of this invention, (a) is a top view of a microelectrode arrangement | positioning part, (b) is a longitudinal cross-sectional view in the II line of (a). is there. It is a figure which illustrates the manufacturing process of the device which concerns on 1st embodiment of this invention, (a) is a top view of a microelectrode arrangement | positioning part, (b) is a longitudinal cross-sectional view in the II-II line of (a). is there. It is a figure which illustrates the manufacturing process of the device which concerns on 1st embodiment of this invention, (a) is a top view of a microelectrode arrangement | positioning part, (b) is a longitudinal cross-sectional view in the III-III line of (a). is there. It is a figure which shows the other example of the manufacturing process of the device which concerns on 1st embodiment of this invention, (a) is a top view of a microelectrode arrangement | positioning part, (b) is a longitudinal section in the VV line of (a). FIG. 3 is a plan view schematically showing the arrangement state of cells in the microelectrode arrangement portion in Example 1. FIG. It is a measurement result of the action potential of the cell in Example 1, (a) is a measurement result before epinephrine addition, (b) is a measurement result after epinephrine addition.

Explanation of symbols

DESCRIPTION OF SYMBOLS 1, 2 ... Microelectrode array device, 11 ... Insulating substrate, 17, 27 ... Microelectrode, 18, 28 ... Wiring, 15 ... Insulating film, 19 ... Non-cell adhesion layer DESCRIPTION OF SYMBOLS 10,20 ... Non-stacking area | region, 10a, 20a ... Cell arrangement | positioning part, 10b, 20b ... Cell connection part, 9 ... Photosensitive resin, 14, 24 ... Microelectrode arrangement | positioning part

Claims (9)

A microelectrode array device for measuring cell potential,
A microelectrode on the insulating substrate, wiring of the microelectrode, and an insulating film covering the insulating substrate so as to expose the microelectrode;
An insulating substrate in which a non-cell adhesive layer that inhibits cell adhesion is further provided in a predetermined region on the insulating film, and the non-cell adhesive layer is not provided, and the microelectrode and the insulating film in the vicinity thereof are exposed. The upper area is the cell placement area ,
The microelectrodes and / or the microelectrode array device non-cell adhesive layer and wherein the Rukoto such a material having optical transparency. 2. The microelectrode array device according to claim 1 , wherein a plurality of the cell arrangement portions are connected by a cell connection portion in which the non-cell adhesion layer is not provided and the insulating film is exposed. The microelectrode according to claim 1 or 2 , wherein the non-cell adhesion layer is formed by bonding a compound having a perfluoroalkyl group or a perfluoroalkyl ether group onto the insulating film. Array device. The microelectrode array device according to claim 3 , wherein the compound is represented by the following general formula (1) or (2).

(In the formula, R ¹ represents a linear or branched perfluoroalkyl group or perfluoroalkyl ether group having ^{1 to} 16 carbon atoms; R ¹ ′ represents a linear or branched chain having ^{1 to} 16 carbon atoms; Represents a group in which one fluorine atom is removed from a perfluoroalkyl group or a perfluoroalkyl ether group; R ² is any one of groups represented by the following formulas (11) to (16), and a plurality of R ² may be the same or different; Y is a group represented by the formula “—NH—C (═O) —” or a carbonyl group, and a plurality of Y may be the same or different; Is an ethyleneoxy group in which one or more hydrogen atoms may be replaced by an alkyl group or alkyloxyalkyl group in which one hydrogen atom is substituted, and a plurality of Z may be the same or different from each other Even J and k are each independently 0 or 1, and a plurality of j or k may be the same or different from each other; l and m are each independently an integer of 0 or more, and a plurality of l or m may be the same or different from each other; when l is an integer of 2 or more, one Z may be the same or different from each other.

(In the formula, R ³ represents a hydroxyl group or an atom or group that can be substituted with a hydroxyl group; R ⁴ represents a hydrogen atom or a monovalent hydrocarbon group; n is 1, 2 or 3, and n is 2 or 3; And n R ³ may be the same or different from each other; when n is 1, the two R ⁴ may be the same or different from each other.) The microelectrode array device according to claim 4 , wherein the compound represented by the general formula (1) or (2) is represented by the following general formula (3) or (4).

(Wherein R1 represents a linear or branched perfluoroalkyl group or perfluoroalkyl ether group having 1 to 16 carbon atoms; R1 ′ represents a linear or branched chain group having 1 to 16 carbon atoms; A group obtained by removing one fluorine atom from a perfluoroalkyl group or a perfluoroalkyl ether group; R2 is any one of groups represented by the following formulas (11) to (16), and a plurality of R2s are the same as each other However, m may be different; m is an integer of 0 or more, and a plurality of m may be the same or different from each other.)

(In the formula, R ³ represents a hydroxyl group or an atom or group that can be substituted with a hydroxyl group; R ⁴ represents a hydrogen atom or a monovalent hydrocarbon group; n is 1, 2 or 3, and n is 2 or 3; And n R ³ may be the same or different from each other; when n is 1, the two R ⁴ may be the same or different from each other.) It is a manufacturing method of the microelectrode array device according to any one of claims 1 to 5 ,
Forming a microelectrode and wiring of the microelectrode on an insulating substrate;
Patterning an insulating film on the insulating substrate to expose the microelectrodes;
Coating the surface on which the insulating film on the insulating substrate is patterned with a non-cell adhesion layer that inhibits cell adhesion;
A photosensitive resin is patterned on the non-cell adhesive layer, a predetermined portion of the non-cell adhesive layer is removed by etching using the photosensitive resin as a mask, and the microelectrode and the insulating film in the vicinity thereof are exposed to form a cell. In addition to forming the placement portion, the photosensitive resin is patterned so as to form a cell connection portion in which an insulating film is exposed in a predetermined region where the cell placement portions are connected to each other , and then the photosensitive resin is formed. Removing, and
A method for producing a microelectrode array device, comprising: It is a manufacturing method of the microelectrode array device according to any one of claims 1 to 5 ,
Forming a microelectrode and wiring of the microelectrode on an insulating substrate;
Patterning an insulating film on the insulating substrate to expose the microelectrodes;
Patterning a photosensitive resin so as to cover the microelectrode and the insulating film in the vicinity thereof on the surface on which the insulating film on the insulating substrate is patterned;
Coating the surface on which the photosensitive resin on the insulating substrate is patterned with a non-cell adhesion layer that inhibits cell adhesion;
Removing the non-cell adhesion layer on the photosensitive resin together with the photosensitive resin coated with the non-cell adhesion layer to expose the microelectrode and the insulating film in the vicinity thereof to form a cell arrangement portion When,
A method for producing a microelectrode array device, comprising: Together with the cell arrangement portion, further, claim 7, characterized in that to form the cell connecting portion where the insulating film is exposed in a predetermined region for connecting the cell arrangement portions, patterning the photosensitive resin A manufacturing method of the microelectrode array device described in 1. A bioassay method using the microelectrode array device according to any one of claims 1 to 5 ,
A bioassay method comprising a step of placing a measurement target cell on a cell placement portion on the microelectrode array device and measuring the potential of the cell with the microelectrode.

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