JP2001021523A - Immobilization method of biocatalyst - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide an immobilization method of biocatalyst, capable of executing, accurately and easily, quantitative immobilization of a biocatalyst on a biocatalyst electrode, and thereby capable of efficient production of the biocatalyst electrode on which the biocatalyst is immobilized quantitatively. SOLUTION: This immobilization method of a biocatalyst on a biocatalyst electrode having an insulating substrate, a conductor fixed on the insulating substrate, and the biocatalyst immobilized on the conductor includes a process for fixing a screen 3 having opening parts 3a, 3b, 3c or a mesh part on the insulating substrate so that the conductor is faced from the opening parts 3a, 3b, 3c or the mesh part, a process for supplying liquid including the biocatalyst and capable of being solidified by after-treatment onto the screen 3, a process for bringing a squeegee 6 into close contact with the screen 3 surface and for moving the squeegee 6 on the screen 3 so that the liquid is spread out on the whole of the opening parts 3a, 3b, 3c or the mesh part, and a process for immobilizing the liquid including the biocatalyst.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for immobilizing a biocatalyst on a biocatalyst electrode, and more particularly, to a biocatalyst capable of accurately and simply immobilizing a biocatalyst on a biocatalyst electrode. The method for immobilization of

[0002]

2. Description of the Related Art At present, biosensors that perform electrochemical measurement using biochemical reactions of biocatalysts (enzymes, microorganisms, organelles, antigens, antibodies, etc.) are used in the fields of environment, food inspection, medical diagnosis and the like. Widely used. In a biosensor, a biochemical reaction is performed on a biocatalyst electrode having a biocatalyst immobilized on the electrode. As a method for immobilizing a biocatalyst on this biocatalyst electrode,
A method of physically adsorbing directly or via a filter on the electrode surface, a method of chemically modifying the electrode surface to form a covalent bond, and a method of enclosing or cross-linking a polymer material and disposing it on the electrode have been proposed.

[0003] In particular, conventional biosensors often use a pair of biocatalyst electrodes to perform measurement continuously or a plurality of times. Therefore, the pair of biocatalyst electrodes is quantitatively applied to a certain substance to be measured. If the measurement can be performed in a short time, the concentration of the substance to be measured can be quantitatively predicted by a calibration curve created using the same biocatalyst electrode. Therefore, the correlation with another pair of similar biocatalyst electrodes having the same purpose, the difference in response value between the electrodes, and the like were not very important issues except in extreme cases.

[0004] That is, 2 is manufactured for the same purpose.
Even if the amount of immobilized biocatalyst of each pair of biocatalyst electrodes is different, and as a result, even if the response values to the same concentration of substrate are different, each response value is used independently, so each individual biocatalyst electrode is used. If the response value was linear, quantitative measurement was possible.

However, in recent years, when low-cost, maintenance-free, disposable biosensors are mass-produced, the measurement using the biocatalyst electrode is basically performed only once, and the biocatalyst required for preparing a calibration curve is required. Including electrodes, the possibility of using a plurality of biocatalyst electrodes for one measurement sample has emerged. In other words, the calibration curve preparation and sample measurement used in the continuous
The method of sequentially performing the measurement using a pair of biocatalyst electrodes is difficult to employ in a disposable biosensor whose measurement is basically performed in a short time. For this reason, conventional biosensors for continuous or repeated measurement cannot be ignored, but uniform response value characteristics between individual biosensors are particularly required for disposable biosensors, and biocatalysts are placed on the electrodes. It has become essential to develop a method for immobilizing biocatalysts that can produce a large amount of biocatalyst electrodes immobilized with good quantitativeness at low cost.

[0006]

DISCLOSURE OF THE INVENTION The present invention has been made in view of the above, and is a method for immobilizing a biocatalyst on a biocatalyst electrode, which can accurately and easily perform quantitative immobilization of the biocatalyst. Accordingly, it is an object of the present invention to provide a method for immobilizing a biocatalyst, which enables efficient production of a biocatalyst electrode in which the biocatalyst is immobilized with good quantitativeness.

[0007]

Means for Solving the Problems The inventors of the present invention have conducted intensive studies on a method for immobilizing a biocatalyst in order to solve the above-mentioned problems. The present inventors have found that the immobilization of a biocatalyst can be performed quantitatively and easily, and have completed the present invention. That is, the present invention is as follows.

(1) A method for immobilizing a biocatalyst on a biocatalyst electrode having an insulating substrate, a conductor fixed on the insulating substrate, and a biocatalyst fixed on the conductor, comprising: It is intended to provide a method comprising the steps of b), (c) and (d).

(A) fixing a screen having an opening or a mesh portion to an insulating substrate so that a conductor faces the opening or the mesh portion; and (b) a liquid containing a biocatalyst, which is solidified by a post-treatment. Supplying a possible liquid onto the screen, (c) bringing the squeegee into close contact with the screen surface, and moving it over the screen so that the liquid is stretched over the openings or meshes, (d) biocatalyst Solidifying a liquid containing

(2) The solidifiable liquid used in step (b) is a sodium alginate solution, and the solidification method in step (d) is performed by reacting the solution with a calcium salt solution. .

(3) The method according to (1), wherein the step (a) is a step of fixing a screen consisting only of the mesh portion to the insulating substrate so as to cover the entire conductor.

[0012]

BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, the present invention will be described in detail.
The method for immobilizing a biocatalyst of the present invention is a method for immobilizing a biocatalyst on a biocatalyst electrode having an insulating substrate, a conductor fixed on the insulating substrate, and a biocatalyst fixed on the conductor, The method includes (a) a screen fixing step, (b) a liquid supply step for biocatalyst immobilization, (c) a squeezing step, and (d) a biocatalyst immobilization step.

(A) Screen fixing step In the method for fixing a biocatalyst of the present invention, the biocatalyst is fixed on a conductor of an electrode substrate having a conductor fixed on an insulating substrate. Examples of the electrode substrate to which the immobilization method of the present invention is applied include, but are not particularly limited to, electrode substrates used in ordinary biosensors. Electrode substrate.

The conductor of the electrode substrate is a conductor which is stable, has high conductivity, and is substantially harmless to a biocatalyst, for example, a metal such as platinum, gold, silver or the like, graphite, carbon or the like. Carbon material. The insulating substrate is not particularly limited as long as it is a material and a shape that can support the entire electrode so that the biocatalyst electrode has strength enough to withstand use. Examples of the material include a resin such as polyester, glass, and a sample solution. Paper that has been subjected to a surface treatment so as not to penetrate. Depending on the conductor used and the material and shape of the insulating substrate, the conductor is usually fixed on the insulating substrate by, for example, a method such as vapor deposition, sputtering, or screen printing.

The number of conductors in the electrode substrate to which the method of the present invention is applied is one or more, and preferably one to three. If the number of conductors is 1,
The biocatalyst is immobilized on the conductor. When the number of conductors is two or more, the biocatalyst is immobilized on at least the conductor serving as the working electrode. In this case, the other conductors serve as a counter electrode, a reference electrode, and the like. However, the biocatalyst may be fixed on these conductors for reasons such as increasing work efficiency.

The screen used in the fixing method of the present invention has an opening or a mesh. The opening or the mesh portion is provided at a position corresponding to at least a conductor serving as a working electrode on the electrode substrate when the screen is fixed on the electrode substrate. When the opening or mesh portion is provided corresponding to only the conductor serving as the working electrode, the size and shape thereof are the same as the conductor serving as the working electrode, or include the entire conductor serving as the working electrode. In addition, a size and a shape including a part of the insulating substrate can be given.

When a conductor other than the working electrode is provided on the same plane of the electrode substrate, the arrangement of the openings or mesh portions in the screen may be performed only for the working electrode. May be carried out in a form including the conductor of (1). When an opening or a mesh is provided for a plurality of conductors, a plurality of openings or a mesh may be provided for each conductor as in the case of the working electrode, or one opening or a mesh may be provided. May be provided so as to cover all the conductors including a part of the insulating substrate.

The screen has such a size that the entire periphery of the opening or the mesh portion can be fixed to the insulating substrate, and further, when fixed, there is no portion protruding from the outer edge of the electrode substrate.
It is preferably a thin plate having a shape.

In the method of the present invention, the screen may be removed from the electrode substrate after immobilization of the biocatalyst, or the screen may be left on the substrate as a final product. The material of the screen is not particularly limited as long as it is substantially harmless to the biocatalyst when the screen is finally removed, and is stable to various compounds used for immobilizing the biocatalyst. When the screen is left as it is in the final product, the screen has, in addition to the above properties, a material having good adhesion to the insulating substrate and itself also insulating, for example, polyethylene terephthalate, polyethylene, polypropylene,
It is preferable to be made of plastic such as polycarbonate or glass.

When the screen has a mesh portion, the mesh portion and other portions (hereinafter, sometimes referred to as "screen main body") may be integrally formed of the same material, or a combination of different materials. May be configured. Examples of the material of the mesh portion include polyester, polyamide, cellulose, silk, and various other natural and synthetic materials capable of forming fibers. Further, the screen main body used in the present invention may be densely configured so as not to allow liquid to pass therethrough, or may be configured in a mesh shape. That is, the entire screen used in the present invention may be in a mesh shape. Also,
Preferably, the screen used in the method of the present invention includes a screen having an opening and other portions made of a dense material that is impermeable to liquid, and a screen having the entire screen formed in a mesh shape.

The thickness of the screen used in the method of the present invention is not particularly limited, but is preferably from 50 to 200.
μm, more preferably about 70 to 150 μm. The size and shape of the mesh of the mesh portion of the screen are not particularly limited as long as the size allows the liquid for immobilizing the biocatalyst to be used to pass therethrough. The mesh size depends on the type and concentration of the biocatalyst-immobilizing liquid used, but is generally preferably about # 30 to # 200, and more preferably about # 80 to # 150.

In the method of the present invention, the screen is fixed to the insulating base material such that the conductor on the insulating substrate faces the opening or the mesh portion. Here, when fixing the screen to the electrode substrate having several conductors other than the working electrode and finally removing the screen,
The screen body may be fixed to the insulating substrate so that the screen body covers the conductors other than the working electrode, but if the screen body is to be left as it is in the final product, if the screen body is densely configured, The screen is fixed to the insulating substrate so that the screen main body does not cover the conductors other than the working electrode.

When the screen is finally removed, the fixing portion is at least the entire periphery of the opening or the mesh portion. When the screen is left as it is in the final product, the opening or the mesh portion of the screen is removed. The whole surface of the part.

When the entire screen is formed in a mesh shape, the screen is fixed so as to cover the entire conductor. In this case, if the screen is to be finally removed, the fixing portion is the entire periphery of a portion corresponding to the insulating substrate including at least the conductor of the screen.
When the screen is left as it is on the final product, the fixing portion is the entire surface of the screen excluding the portion corresponding to the insulating substrate including at least the conductor.

As a method of fixing the screen to the insulating base material, when the screen is finally removed, a fixing method that allows easy removal operation is selected.
Also, if you leave the screen as it is in the final product,
A method of firmly fixing, for example, a fixing method such as adhesion with a water-resistant epoxy resin or an adhesive such as a double-sided adhesive tape, and heat fusion is selected.

(B) Biocatalyst Immobilization Liquid Supplying Step In the immobilization method of the present invention, the liquid containing the biocatalyst is placed on the screen fixed to the insulating base material by the step (a). A liquid that can be solidified by the treatment (hereinafter, also referred to as “biocatalyst fixing liquid”) is supplied.

The biocatalyst to which the immobilization method of the present invention can be applied includes, without particular limitation, biocatalysts used for ordinary biosensors. Specific examples include glucose oxidase, glucoamylase, galactose oxidase, and alcohol oxidase. , Tyrosinase, catechol 1,2-oxidase, pyruvate oxidase, uricase, amino acid oxidase, glutaminase, glutamate dehydrogenase, asparakinase, amine oxidase, cholesterol esterase, lipase, phospholipase, catalase, creatinase, enzymes such as phosphatase, urease; Bacillus, Acinetobacter, Gluconobacter, Pseudomonas, Methylomonas, Hansenula, Candida, Desulfobi Rio genus Thiobacillus spp., Clostridium, Methylococcus genus Ah solo genus,
Bacteria belonging to the genus Mycobacterium, Lactobacillus or Brevibacterium, prokaryotic microorganisms such as actinomycetes, and microorganisms such as eukaryotic microorganisms such as yeast belonging to the genus Trichosporon; organelles; antigens; antibodies, and the like.

In order to fix the biocatalyst on the conductor of the electrode substrate, in the present invention, these biocatalysts are supplied onto the screen in a form that is contained in a liquid that can be solidified by post-treatment.

The liquid that can be solidified by post-treatment is a liquid that can be solidified by post-treatment such as light irradiation, addition of a reagent, and drying, as long as it is substantially harmless to the biocatalyst to be used. There is no particular limitation. For example, a raw material solution of a polymer gel such as alginate gel, carrageenan gel, agarose gel, curdlan gel, chitosan gel, etc.
A raw material liquid of a photocurable resin such as photocrosslinkable polyvinyl alcohol, a raw material liquid of a three-dimensional crosslinked structure such as polyacrylamide, and the like can be given.

In order to make the above-mentioned biocatalysts contained in such various polymer raw material liquids, a method similar to the method used at the time of manufacturing a normal biosensor may be used. Here, the concentration of the biocatalyst-containing solution used is 200 to 5 from the viewpoint of workability.
The viscosity is preferably about 00 ps.

For example, when the biocatalyst is immobilized using a calcium alginate gel, a suspension containing a required amount of the biocatalyst may be added to an aqueous solution of about 0.5 to 3% of sodium alginate. Stir to produce a uniform mixture, and supply this mixture on the screen. The sodium alginate solution becomes a calcium alginate gel by being brought into contact with a calcium salt solution having an appropriate concentration in the step (d) described later. Further, the aqueous sodium alginate solution may contain salts such as sodium chloride and potassium chloride, buffers, and the like, in addition to sodium alginate. Further, the biocatalyst suspension and the like may contain salts such as sodium chloride and potassium chloride, a buffer, and the like, in addition to the biocatalyst.

In the method for immobilizing a biocatalyst of the present invention, a biocatalyst immobilizing liquid is supplied onto a screen.
The location where the biocatalyst fixing liquid is supplied is not particularly limited as long as it is on the screen. Here, in this specification, in the case of a screen having an opening, the upper part of the screen includes the upper part of the opening. Note that the phrase “on the opening” actually means on the insulating substrate including at least the conductor facing the opening.

When a screen having an opening is used in the present invention, it is preferable that the liquid for immobilizing a biocatalyst is supplied onto the opening. In this case, the amount of the biocatalyst-fixing liquid supplied onto the screen is determined by an extension surface extending from the surface of the screen main body to the opening and an inner wall constituting the opening of the screen (hereinafter referred to as “opening inner wall”). And at least the same amount as the space filling the space formed by the surface of the insulating substrate including at least the conductor facing the opening, and the capacity is 1.0 to 2.0 times the space volume. Is preferred.

When a screen having a mesh portion is used in the present invention, the biocatalyst-immobilizing liquid is preferably supplied near the boundary between the mesh portion and the screen body. The amount of the biocatalyst-fixing liquid supplied onto the screen is determined by the mesh portion of the screen when there is no gap between the surface of the mesh portion facing the insulating substrate and the surface of the insulating substrate including at least the conductor facing the mesh portion. It is sufficient that the amount is at least the same as the amount that fills all the small spaces inside. Further, when there is a gap between the surface of the mesh portion facing the insulating substrate and the surface of the insulating substrate including at least the conductor facing the mesh portion, the amount that fills the gap and the amount that fills all the small spaces inside the mesh portion. And at least the same amount. Also in these cases, it is preferable that the amount of the supplied biocatalyst fixing liquid is 1.0 to 2.0 times the total volume of the space.

Further, when the entire screen is formed in a mesh shape, the supply position of the biocatalyst fixing liquid is not particularly limited. The supply amount of the biocatalyst fixing liquid is the same as in the case where the screen having the mesh portion is used.

The method for supplying the liquid for fixing the biocatalyst onto the screen in the fixing method of the present invention is not particularly limited. For example, a fixed amount liquid supply device such as a piston cylinder or a pipette can be used. The biocatalyst-immobilizing liquid may be supplied at a single point, or may be supplied in a small amount at a plurality of points so that the following squeezing step is performed smoothly.

(C) Squeegeeing Step In the squeegeeing step, the squeegee is moved on the screen in a state in which the squeegee is in close contact with the screen surface, so that the biocatalyst fixing liquid supplied on the screen is opened or meshed. This is the step of stretching the entire part.

This step can be performed in the same manner as the squeezing step in ordinary screen printing. In other words, in this step, the metal mask in the screen printing technology using a metal mask or a mesh screen, the mesh screen was replaced with a screen having openings or mesh portions, and the ink in the screen printing was replaced with a biocatalyst fixing liquid. In this state, it can be considered as a step of performing a squeezing step in screen printing.

As the squeegee used in the fixing method of the present invention, a squeegee similar to that used in ordinary screen printing, specifically, a rubber blade, a Teflon blade, a urethane blade or the like can be used. The size of the squeegee is preferably such that when the squeegee is brought into close contact with the screen surface at an appropriate position, both ends of the squeegee reach both ends of the screen.

When a screen having an opening is used in the present invention, the squeegee is moved by an extension surface extending from the surface of the screen main body to the opening, the inner wall of the opening, It is performed in such a manner that a space formed by the surface of the insulating substrate including at least the conductor facing the opening is filled. Here, in the step (b), when the biocatalyst fixing liquid having a volume equal to or larger than the space volume is supplied, the surplus liquid is discharged out of the electrode substrate, and the screen surface after squeezing becomes flat. So that
Move the squeegee.

When a screen having a mesh portion is used in the present invention, the liquid for fixing a biocatalyst is
In such a manner that all the small spaces inside the mesh part of the screen are filled, and if there is a gap between the surface of the mesh part facing the insulating substrate and the surface of the insulating substrate including at least the conductor facing the mesh part, The squeegee is moved in such a way that all the small spaces inside the mesh part are filled and the gaps are filled. Also in this case, if excess biocatalyst fixing liquid is supplied,
The excess is discharged out of the electrode substrate, and the squeegee is moved so that the screen surface after squeezing becomes flat.

Even when the entire screen is formed in a mesh shape, the squeezing step can be performed in the same manner as described above.

(D) Biocatalyst Immobilization Step The electrode substrate in which the opening or mesh portion of the screen is filled with the biocatalyst immobilization liquid in the above step (c) is then subjected to a treatment for solidifying the liquid. Is done. The solidification treatment differs depending on the type of the polymer material for immobilization to be used, and a treatment corresponding to each material, for example, a solidification treatment such as a gelling treatment, a cross-linking treatment, and a covalent bonding treatment is performed.

For example, when a biocatalyst is immobilized by a calcium alginate gel and a biocatalyst-containing sodium alginate solution is used as the biocatalyst fixing liquid, the electrode substrate after the step (c) is used. By immersing the electrode in a calcium salt solution such as a calcium chloride solution at an appropriate temperature for an appropriate time, a biocatalyst-immobilized electrode can be obtained.

The biocatalyst-immobilized electrode that has been thus immobilized can be stored in a dry state or in a solution depending on the characteristics of the biocatalyst. In the method of the present invention, if necessary, a step of removing the screen after the step (d) may be provided.

A series of operations of the biocatalyst fixing method of the present invention described above can be said to be an application of a screen printing technique using a metal mask or a mesh screen. The screen having an opening or a mesh portion in the biocatalyst fixing method of the present invention is equivalent to a metal mask or a mesh screen in a screen printing technique,
The biocatalyst fixing liquid corresponds to the ink in screen printing. However, with normal screen printing technology,
The metal mask or the mesh screen is removed from the printed substrate after printing, whereas in the fixing method of the present invention, the screen having the opening or the mesh portion is finally adhered to the substrate in the initial step, for example. Differs in that it may not be removed.

In the method for immobilizing a biocatalyst of the present invention, by using such a screen printing method, a highly quantitative biocatalyst electrode for a biosensor can be made uniform and uniform in a short time. At the same time, it enables mass production. This is particularly advantageous in the manufacture of biocatalyst electrodes for disposable sensors, where multiple biocatalyst electrodes are required for one measurement.

The method for immobilizing a biocatalyst of the present invention comprises the steps of:
Compared to a method in which a polymer material for immobilizing a biocatalyst is simply dropped on an electrode substrate and stretched, the method is excellent in that quantitative fixation can be performed accurately. Furthermore, when a screen having a mesh portion is used in the method of the present invention and the screen is left as it is in the final product, the space inside the mesh is also filled with the immobilized polymer material. Can be very firmly fixed.
For this reason, it is possible to prevent the polymer material immobilizing the biocatalyst from peeling off from the electrode surface due to swelling of the polymer gel or the like, and as a result, it is possible to produce an immobilized electrode with good reproducibility. it can.

The method for immobilizing a biocatalyst of the present invention will be described more specifically for each type of screen used. First,
An example of a fixing method using a screen having an opening will be described with reference to FIGS.

(1) Example of Fixing Method Using Screen Having Opening FIG. 1 shows an electrode substrate 4 and a screen 3 having an opening in an example of the fixing method of the present invention using a screen having an opening. FIG. 2 is a front view of the electrode substrate 4 to which the screen 3 is fixed. FIG.
It is a figure which shows the liquid supply process for biocatalyst fixation in this example, and a squeezing process. FIG. 3I is a front view of the electrode substrate 4 to which the screen 3 shown in FIG. 2 is fixed.
It is sectional drawing in the -X 'line. FIG. 3 (ii) is a diagram showing a state where the biocatalyst fixing liquid 5 is supplied to the opening 3a, and FIGS. 3 (iii) and 3 (iv) are diagrams showing a squeezing process.

As shown in FIG. 1, the electrode substrate 4 used in this example
The working electrode 2a, the counter electrode 2b and the reference electrode 2c made of a conductor such as a carbon material are formed on the same plane of the insulating substrate 1. The screen 3 is a working electrode 2 of the electrode substrate.
a, openings 3a, 3b, and 3c having the same area as the size of each electrode are provided at positions corresponding to the counter electrode 2b and the reference electrode 2c. The screen body 3d is made of a liquid-impermeable insulating material,
For example, polyethylene terephthalate, polyethylene,
It is formed of plastic such as polypropylene or polycarbonate, glass, or the like.

In this embodiment, as shown in FIG. 2, the screen 3 is fixed so that each electrode faces each of the openings 3a, 3b and 3c corresponding to the working electrode 2a, the counter electrode 2b and the reference electrode 2c. . In this example, since the screen 3 is left on the electrode substrate even after the biocatalyst is fixed, the screen is fixed firmly using an adhesive or the like. Specifically, the fixing method is as follows.
An adhesive is applied to the entire surface on one side of d, and this surface is pressed against an insulating substrate. When the material of the insulating base is polyethylene terephthalate and the material of the screen is polyester, examples of the adhesive used include a water-resistant epoxy resin and a double-sided adhesive tape.

FIGS. 3 (i) to 3 (iv) show the steps of supplying the biocatalyst-immobilizing liquid 5 onto the screen and extending the liquid over the entire screen opening. FIG.
(I) is a cross-sectional view taken along line XX ′ of the front view of the electrode substrate 4 to which the screen 3 shown in FIG. 2 is fixed. In this example, the biocatalyst is located on the working electrode 2a where the opening 3a faces. The immobilizing liquid 5 is supplied. Normally, the biocatalyst is not fixed on the counter electrode 2b and the reference electrode 2c. However, in some cases, in terms of workability, the reference electrode 2c on which the opening 3b faces on the counter electrode 2b facing the opening 3b as described above. The liquid 5 for immobilizing a biocatalyst may be supplied to the upper part.

As the biocatalyst fixing liquid 5, the same liquid as described above can be used. In this example, as shown in FIG. 3 (ii), the amount of the biocatalyst fixing liquid 5 supplied onto the screen is determined by the extension surface extending from the surface of the screen main body 3d onto the opening 3a and the opening The amount is slightly larger than the amount filling the space formed by the inner wall 3a and the surface of the working electrode 2a facing the opening 3a.

FIGS. 3 (iii) and 3 (iv) show that the squeegee 6 is pressed against the screen 3 and moved in a certain direction so that the biocatalyst fixing liquid 5 is
d shows that the space formed by the extended surface extending from the surface of the opening 3a onto the opening 3a, the inner wall of the opening 3a, and the surface of the working electrode 2a facing the opening 3a is sufficiently filled, and the excess liquid 7 is scraped off. Have been. In this example, the squeegee moves from the X ′ side of the screen toward the X side.
The movement of the squeegee may be performed once in the above-mentioned direction, but may be performed several times in the same direction as necessary, and one to several reciprocating movements are also possible. Further, movement in a direction different from the XX ′ direction, for example, a direction perpendicular to the XX ′ direction is also possible.

In FIG. 3 (iv), after the surplus biocatalyst fixing liquid 5 is completely removed from the screen, the biocatalyst fixing liquid that fills the space is solidified. After solidification of the biocatalyst fixing liquid, the screen on the electrode substrate is used as a biocatalyst electrode while being left as it is.
The screen may be removed, but in that case, it is preferable to fix the screen by an easy-to-remove method.

Next, an example of a fixing method using a screen formed entirely in a mesh shape will be described with reference to FIGS.

(2) An example of an immobilization method using a screen entirely formed in a mesh shape FIG. 5 shows an electrode in an example of an immobilization method of the present invention using a screen entirely formed in a mesh shape. Substrate 4,
FIG. 6 is a diagram illustrating the bonding member 8 and the mesh screen 3, and FIG. 6 is a diagram illustrating the electrode substrate 4 to which the mesh screen 3 is fixed via the bonding member 8. FIG. 6 (A)
6B is a front view, and FIG. 6B is an exploded cross-sectional view of the electrode substrate 4 shown in FIG.

FIG. 7 shows a biocatalyst fixing liquid supply step and a squeezing step in this example. FIG. 7 (i) is a cross-sectional view taken along the line ZZ ′ of the front view of the electrode substrate 4 to which the mesh screen 3 shown in FIG. 6 (A) is fixed. It is a figure which shows the state in which was supplied. FIGS. 7 (ii) and 7 (iii) are views showing a squeezing process.

As shown in FIG. 5, the electrode substrate 4 used in this example
The working electrode 2a and the counter electrode 2b made of a conductor such as a carbon material are formed on the same plane of the insulating substrate 1. The material of the mesh screen, the size of the mesh, and the like are as described above. The bonding member 8 for fixing the mesh screen 3 to the electrode substrate 4 includes the working electrode 2
a and the counter electrode 2b are formed in a shape surrounding the periphery including both of them. When the material of the insulating base material is polyethylene terephthalate and the material of the mesh screen is polyester, a double-sided adhesive tape,
A water-resistant epoxy resin or the like may be used.

In this embodiment, as shown in FIG. 6, the mesh screen 3 is attached to the working electrode 2 via the adhesive member 8.
and is fixed to the electrode substrate 4 so as to cover the electrode a and the counter electrode 2b. 7 (i) to 7 (iii) show that the biocatalyst-immobilizing liquid 5 is supplied onto the mesh screen, and the liquid fills the entire internal space of the mesh screen.
It is a figure which shows the process of extending the said liquid over the whole screen so that it may fill the space which consists of the inner surface of a mesh screen, the inner wall of an adhesive member, and the electrode substrate surface.

FIG. 7I is a front view of the electrode substrate 4 to which the mesh screen 3 shown in FIG. 6A is fixed.
It is sectional drawing in the Z 'line, and is a figure which shows the state in which the liquid 5 for biocatalyst fixation was supplied on the screen.

As the biocatalyst-immobilizing liquid 5, the same liquid as described above can be used. In this example, the amount of the biocatalyst fixing liquid 5 supplied on the screen fills the entire internal space of the mesh screen 3 and the inner surface of the mesh screen,
The amount is slightly larger than the amount filling the space formed by the inner wall of the adhesive member and the surface of the electrode substrate.

FIGS. 7 (ii) and 7 (iii) show that the squeegee 6 is pressed against the surface of the mesh screen 3 and moved in a certain direction, so that the biocatalyst-immobilizing liquid 5 allows the mesh screen 3 to move. While filling all the internal space of the mesh screen, the inner surface of the mesh screen, the inner wall of the adhesive member and the space formed by the electrode substrate surface,
The manner in which the excess liquid 7 is scraped out is shown.
In this example, the squeegee moves from the Z side of the screen toward the Z ′ side. The movement of the squeegee may be performed once in the above-mentioned direction, but may be performed several times in the same direction as necessary, and one to several reciprocating movements are also possible. Further, movement in a direction different from the ZZ 'direction, for example, a direction perpendicular to the ZZ' direction is also possible.

In FIG. 7 (iii), after the surplus biocatalyst fixing liquid 5 is completely removed from the screen, the biocatalyst fixing liquid that fills the space is solidified. The screen on the electrode substrate may be removed after the liquid for fixing the biocatalyst is solidified, but may be left as it is and used as a biocatalyst electrode. For a mesh screen,
In particular, to keep the biocatalyst firmly immobilized,
Preferably, the screen is not removed.

[0066]

Embodiments of the present invention will be described below.

[0067]

Embodiment 1 An opening is formed on an electrode substrate having a working electrode, a counter electrode, and a reference electrode formed on the same plane of an insulating substrate as shown in FIG. Microorganisms (Pseudomonas fluorescens (Pseudomona
Fluorescens)) was immobilized to prepare a microorganism-immobilized electrode and evaluated.

(1) Preparation of Microorganism-Immobilized Electrode (i) Fixing of Screen An electrode substrate having a working electrode 2a, a counter electrode 2b and a reference electrode 2c made of a carbon material formed on the same plane of an insulating substrate 1 made of polyethylene terephthalate. 4 (see FIG. 1), a polyester screen 3 having openings 3a, 3b, 3c at portions corresponding to the working electrode 2a, the counter electrode 2b, and the reference electrode 2c is fixed using an adhesive (double-sided adhesive tape). (See FIG. 2).

(Ii) Immobilization of microorganisms A 3% aqueous sodium alginate solution in the same volume of a microorganism (Pseudomonas fluorescens) suspension (OD (Optica
l (Density (turbidity)) = 30, a suspending medium; 0.1M aqueous sodium chloride solution), and uniformly mix the mixed solution with the electrodes (2a, 2b, 3b) in the openings (3a, 3b, 3c). 2
c) 20 μl was dropped on each. Figure 3 shows the state before dropping.
As shown in (i), the state after dropping is shown in FIG.
As shown in (ii). By pressing and moving the squeegee 6 on the screen 3, an extended surface extending from the surface of the screen main body 3 d onto the openings (3 a, 3 b, 3 c) with the mixed liquid 5 supplied on the screen,
The excess 7 was scraped while sufficiently filling the space formed by the inner walls of the openings (3a, 3b, 3c) and the surfaces of the electrodes (2a, 2b, 2c) facing the openings (FIGS. 3 (iii) and (i)).
v)).

Thereafter, the electrode substrate obtained in a refrigerator at 5 ° C. was immersed in a 2% calcium chloride solution for 30 minutes to gel the mixed solution filled in the space to obtain a microorganism-immobilized electrode.

(2) Evaluation of Microorganism-Immobilized Electrode A BOD ₅ value (5-day method, JIS K102, modified by Winkler sodium azide method) of 35 mgO / L and 70%
A phosphate buffer (0.1 M), sodium chloride (0.1 M), potassium ferricyanide (40 mM) was added to each of the mgO / L organic substance mixed solution (artificial drainage) so that the final concentration became the value in parentheses. The resulting microorganism-immobilized electrode was evaluated using the reaction solution to which was added.

First, the microorganism-immobilized electrode obtained above was immersed in the reaction solution, and the microorganism-immobilized electrode was connected to a constant potential circuit. At the time of measurement, a potential of +400 mV was applied to the working electrode, and a current flowing between the working electrode and the counter electrode was measured 10 minutes after the potential was applied. A total of ten microorganism-immobilized electrodes were used, and five electrodes were measured for each of the above two types of solutions. FIG. 4 shows the results. The measurement error was calculated and found to be ± 5% or less for both solutions, indicating that the biocatalyst immobilization method of the present invention can produce a microorganism-immobilized electrode with good reproducibility.

[0073]

Embodiment 2 A working electrode and a counter electrode made of a carbon material similar to those shown in FIG. 5 are formed on the same plane of an insulating substrate by using a mesh screen as follows. The microorganisms (Pseudomonas Fluorescen) are covered so as to cover the two electrodes.
s)) was immobilized to prepare a microorganism-immobilized electrode and evaluated.

(1) Preparation of Microorganism-Immobilized Electrode (i) Fixing of Mesh Screen An electrode substrate 4 having a working electrode 2a made of carbon material and a counter electrode 2b formed on the same plane of an insulating substrate 1 made of polyethylene terephthalate. A double-sided adhesive tape 8 is applied so as to surround a portion occupying about half the area of the substrate including the working electrode 2a and the counter electrode 2b, and a # 200 tetron (trade name, polyester )
A screen 3 made of a mesh was attached (see FIGS. 5 and 6). As shown in FIG. 6, the mesh screen 3 is fixed to the electrode substrate 4 via the double-sided adhesive tape 8 so as to cover the working electrode 2a and the counter electrode 2b.

(Ii) Immobilization of microorganisms A suspension of microorganisms (Pseudomonas fluorescens) in the same volume in a 3% aqueous sodium alginate solution (OD = 30,
A suspension liquid (0.1 M sodium chloride aqueous solution) was added to prepare a mixed liquid that was uniformly mixed, and the obtained mixed liquid was dropped on a mesh screen (see FIG. 7 (i)). Next, a squeegee 6 is pressed against the surface of the mesh screen, and the liquid fills the entire inner space of the mesh screen with the mixed liquid 5, and the inner surface of the mesh screen, the inner wall of the double-sided adhesive tape 8, and the electrode substrate The excess 7 of the dropped mixture was scraped off by moving on a screen so that the space consisting of the surface was sufficiently filled (see FIGS. 7 (ii) and (iii)).

Thereafter, the electrode substrate obtained in a refrigerator at 5 ° C. was immersed in a 2% calcium chloride solution for 30 minutes to gel the mixed solution filled in the space to obtain a microorganism-immobilized electrode.

(2) Evaluation of Microorganism-Immobilized Electrode Using the microorganism-immobilized electrode obtained above, the response to an organic substance-containing solution was examined. The organic substance-containing solution was converted to a BOD ₅ value of 0, 35, 70, 175,
Potassium ferricyanide was added to a 350 mg O / L solution (artificial drainage) so that the final concentration was 50 mM, and used. The microorganism-immobilized electrode was immersed in the organic substance-containing solution, and a reaction was performed for 10 minutes. During the reaction, a constant potential of +400 mV was applied to the working electrode side of the microorganism-immobilized electrode by a potentiostat, and the current flowing between the working electrode and the counter electrode at the end of the reaction for 10 minutes was measured. FIG. 8 shows the result. From this result, BOD ₅ =
Current value in a range of 0~350mgO / L and BOD ₅
Very linear correlation with the value (correlation coefficient; 0.998)
6) was obtained. The equation shown in FIG. 8 is an equation representing a regression line obtained by the least square method.

[0078]

According to the method for immobilizing a biocatalyst of the present invention, quantitative immobilization of a biocatalyst on a biocatalyst electrode can be performed by:
It can be performed accurately and easily. Furthermore, this enables efficient production of a biocatalyst electrode in which the biocatalyst is immobilized with good quantitativeness.

[Brief description of the drawings]

FIG. 1 is a diagram showing a front view of an electrode substrate and a screen having an opening in an example of a fixing method of the present invention.

FIG. 2 is a diagram showing a front view of an electrode substrate with a screen fixed in an example of the fixing method of the present invention.

FIG. 3 is a diagram showing a biocatalyst immobilizing liquid supply step and a squeezing step in an example of the immobilization method of the present invention.

FIG. 4 is a view showing evaluation results of the microorganism-immobilized electrode obtained in Example 1.

FIG. 5 is a diagram showing a front view of an electrode substrate, an adhesive member, and a mesh screen in another example of the fixing method of the present invention.

FIG. 6 is a view schematically showing an electrode substrate in a state where a mesh screen is fixed via an adhesive member in another example of the fixing method of the present invention. (A) is a front view,
(B) is an exploded sectional view as viewed from the Y direction.

FIG. 7 is a diagram showing a biocatalyst immobilizing liquid supply step and a squeezing step in another example of the immobilization method of the present invention.

FIG. 8 is a diagram showing evaluation results of the microorganism-immobilized electrode obtained in Example 2.

[Explanation of symbols]

1. 1. Insulating substrate 2. Conductor (2a; working electrode, 2b; counter electrode, 2c; reference electrode) 3. Screen (3a, 3b, 3c; opening, 3d; main body) Electrode substrate 5. 5. Liquid for immobilizing biocatalyst Squeegee 7. 7. Excess biocatalyst-immobilizing liquid Adhesive member

 ──────────────────────────────────────────────────続 き Continuing on the front page (72) Inventor Seiko Yano 5-4-71 Higashi, Hanyu-shi, Saitama Prefecture Akebono Brake Central Research Laboratory Co., Ltd. (72) Inventor Scott McNeven 5-4-171 Higashi, 5-yu Higashi, Hanyu-shi, Saitama Akebono Brake Central Technical Research Institute

Claims (3)

[Claims] 1. A method for immobilizing a biocatalyst on a biocatalyst electrode comprising an insulating substrate, a conductor fixed on the insulating substrate, and a biocatalyst fixed on the conductor, comprising the following steps (a) and (b): ), (C) and (d): (a) fixing a screen having an opening or a mesh portion to an insulating substrate so that a conductor faces through the opening or the mesh portion; Supplying a liquid containing a catalyst, which can be solidified by post-treatment, onto the screen, (c) bringing a squeegee into close contact with the screen surface and stretching the liquid over the entire opening or mesh part; The process of moving on the screen,
(D) a step of solidifying the liquid containing the biocatalyst; 2. The method according to claim 1, wherein the solidifiable liquid used in the step (b) is a sodium alginate solution, and the solidification method in the step (d) is performed by reacting a calcium salt solution with the solution. . 3. The method according to claim 1, wherein the step (a) is a step of fixing a screen comprising only the mesh portion to the insulating substrate so as to cover the entire conductor.