JPH10246717A - Small biosensor and its manufacture - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a sensor of superior response characteristic which can be mass-produced, by forming a cathode pattern and an anode pattern on a sheet from a water-repellent insulating film at both faces of the hydrophilic porous sheet, coating other parts than a pad part with an oxygen-passing membrane or the like, immobilizing a living body-related substance to a sensing part and letting an electrolyte permeate in the sheet. SOLUTION: A water-repellent insulating film 2 is formed at both faces of a hydrophilic porous sheet 1. A cathode pattern having a cathode 3 and a cathode pad 5 for the purpose of electric connection is formed at one face of the porous sheet 1, and an anode pattern having an anode 6 and an anode pad 7 for electric connection is formed at the other face. The one face except for the cathode pad 5 is coated with an oxygen-passing film 8. The other face except for the anode pad 7 is coated with the oxygen-passing film 8 or a plastic sheet. A chip is sliced from the obtained porous sheet 1. A side face of the chip is coated with the oxygen-passing membrane 8. A living body-related substance immobilization layer 12 having a living body-related substance immobilized at a sensing part is formed. An electrolyte is let to permeate in the porous sheet 1.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a biosensor using a Clark-type oxygen electrode, which can operate stably at normal temperature, as a transducer, and a method of manufacturing the same. More specifically, the present invention relates to a biosensor that can be mass-produced using inexpensive materials.

[0002]

2. Description of the Related Art An oxygen electrode can be used for manufacturing various biosensors by fixing a bio-related substance such as an enzyme or a microorganism on a sensitive portion thereof.
For example, if glucose oxidase is immobilized on the sensitive part, a sensor for measuring glucose can be formed, and can be used for a diabetes diagnostic measuring instrument or the like. In addition, if Trichosporon cutaneum, a kind of yeast, is fixed to the sensitive part, a sensor that measures the BOD value, which is one of the important indicators of river and other pollution, can be made. Since the Clark-type oxygen electrode has a sensitive part covered with a water-repellent gas-permeable membrane, a biosensor has an advantage that the influence of interfering substances is smaller than that of other types of sensors.

[0003] Oxygen electrodes used as transducers in biosensors can be used in many applications. However, since those currently on the market are basically made individually, they are typically about the size of a ballpoint pen and cost tens of thousands of yen. Therefore, when a biosensor is produced using the biosensor, it is difficult to dispose it after one or several uses. Further, in the biosensor, the life of the biological substance fixed to the sensitive part often becomes a problem. In the case of using the current oxygen electrode and a conventional transducer, this problem is solved by replacing the biomaterial-fixed membrane. However, such an operation is troublesome for the user, and it is desirable that the sensor can be disposable. It is desirable that the size of the sensor is small.

In order to solve such a problem, a semiconductor fine processing technology such as photolithography is used.
Attempts have been made to collectively form an oxygen electrode serving as a transducer on a silicon or glass substrate and miniaturize it. However, although this technology can reduce the price and mass production of oxygen electrodes to some extent, as long as ordinary semiconductor microfabrication technology is used, it is difficult to fabricate an electrode with a size of 100 yen or less per chip, for example. .

[0005]

SUMMARY OF THE INVENTION The present invention has been made in view of the above-mentioned problems of the conventional technology, and can be mass-produced in bulk using inexpensive materials, and is small in size and excellent in response. It is an object to provide a sensor and a method for manufacturing the sensor.

[0006]

Means for Solving the Problems The present inventors have found that the above problems can be solved by adopting a specific structure, and have completed the present invention.

That is, in the biosensor according to the first aspect of the present invention, a water-repellent insulating film is formed on a part of one surface of a hydrophilic porous sheet, and extends from the insulating film onto the porous sheet. A cathode pattern having a cathode and pads for the cathode to make electrical contact with the outside is formed, and a water-repellent insulating film is formed on a part of the opposite surface of the porous sheet. An anode pattern having an anode and a pad for the anode to make electrical contact with the outside is formed so as to extend from the membrane onto the porous sheet, and the chip side surface of the biosensor and the surface on which the cathode is formed are pad portions. , And the surface on which the anode is formed is covered with an oxygen permeable material or a plastic sheet leaving a pad portion. Part biological substance is fixed to a small biosensor, characterized in that the electrolyte in the porous sheet of hydrophilic are impregnated.

In the biosensor according to the second aspect of the present invention, a water-repellent insulating film is formed on a part of one surface of a hydrophilic porous sheet, and extends from the insulating film onto the porous sheet. A cathode pattern having a cathode and a pad for the cathode to make an electrical contact with the outside, and an anode pattern having an anode and a pad for the anode to make an electrical contact with the outside, and On the other side, a water-repellent insulating film is formed on the entire surface or covered with a plastic sheet, and the side surface of the chip of the biosensor and the surface on which the cathode and anode are formed are oxygen permeable except for the respective pad portions. The body is covered with a material, the bio-related substance is fixed to the sensitive part, and the electrolyte is impregnated in the hydrophilic porous sheet. A small biosensor characterized and.

[0009] The biosensor of the present invention minimizes the materials required for its manufacture. In the biosensor of the present invention, an inexpensive material such as paper can be used as a substrate, and screen printing or spraying can be applied to pattern formation of an insulating film or the like, and can be manufactured at low cost. First
In the biosensor according to the aspect, the cathode and the anode are formed on different surfaces, respectively, and in the biosensor according to the second aspect, the cathode and the anode are formed on the same surface.

Further, the present invention provides a differential biosensor having two basic structures of the above biosensor. That is, in the biosensor according to the third aspect of the present invention, a water-repellent insulating film is formed on a part of one surface of a hydrophilic porous sheet, and extends from the insulating film onto the porous sheet. A cathode pattern having a cathode and a pad for the cathode to make electrical contact with the outside is formed,
Further, a water-repellent insulating film is formed on a part of the surface on the opposite side of the porous sheet, and the anode and the anode make electrical contact with the outside so as to extend from the insulating film onto the porous sheet. An anode pattern having a pad is formed, the chip side surface of the biosensor and the surface on which the cathode is formed are covered with an oxygen-permeable material except for the pad portion, and the surface on which the anode is formed is oxygen-permeable while leaving the pad portion. Coated with conductive material or plastic sheet,
In the hydrophilic porous sheet, two structures impregnated with an electrolytic solution are arranged in parallel, one of the sensitive portions is provided with a bio-related substance fixing layer, and the other sensitive portion is exposed as it is. A differential small-sized biosensor characterized in that it is left alone or only the same carrier material on which the bio-related substance fixing layer is formed is fixed.

In the biosensor according to a fourth aspect of the present invention, a water-repellent insulating film is formed on a part of one surface of a hydrophilic porous sheet, and extends from the insulating film onto the porous sheet. Two cathode patterns each having a cathode and a pad for the cathode to make electrical contact with the outside are formed, and a water-repellent insulating film is formed on a part of the opposite surface of the porous sheet, An anode pattern having an anode and a pad for the anode to make electrical contact with the outside is formed so as to extend from the insulating film onto the porous sheet, and the chip side surface of the biosensor and the surface on which the cathode is formed are formed. Leaving the pad portion covered with an oxygen permeable material, and the anode formed surface covered with an oxygen permeable material or plastic sheet leaving the pad portion, The electrolytic solution was impregnated in the aqueous porous sheet, and one of the sensitive parts was formed with a bio-related substance fixed layer, and the other sensitive part was left as it was or the bio-related substance fixed layer was formed. A differential small-sized biosensor characterized in that only the same carrier material as described above is fixed.

Further, in a biosensor according to a fifth aspect of the present invention, a water-repellent insulating film is formed on a part of one surface of a hydrophilic porous sheet, and extends from the insulating film onto the porous sheet. A cathode pattern having a cathode and a pad for the cathode to make an electrical contact with the outside, and an anode pattern having an anode and a pad for the anode to make an electrical contact with the outside, and On the other side, a water-repellent insulating film is formed on the entire surface or covered with a plastic sheet, and the side surface of the chip of the biosensor and the surface on which the cathode and anode are formed are oxygen permeable except for the respective pad portions. Two structures each coated with a material and impregnated with an electrolytic solution in the hydrophilic porous sheet are arranged in parallel; A bio-related substance fixing layer is formed near one of the cathodes, and the vicinity of the other cathode is left as it is, or only the same carrier material as that on which the bio-related substance fixing layer is formed is fixed. This is a small differential biosensor.

Further, in the biosensor according to the sixth aspect of the present invention, a water-repellent insulating film is formed on a part of one surface of the hydrophilic porous sheet, and the insulating film is formed on the porous sheet. Two independent cathode patterns each having a cathode and a pad for the cathode to make an electrical contact with the outside are formed, and an anode pattern having a pad for the anode and the anode to make an electrical contact with the outside are formed. The surface opposite to the porous sheet is entirely coated with a water-repellent insulating film or covered with a plastic sheet, and the side surface of the chip of the biosensor and the surface on which the cathode and anode are formed are the respective surfaces. Covered with oxygen permeable material leaving the pad part,
The electrolyte is impregnated into the hydrophilic porous sheet, and a biomaterial-fixed layer is formed near one of the cathodes, and the other cathode is exposed as it is or the biomaterial-fixed layer is formed. A small differential biosensor characterized in that only the same carrier material is fixed.

[0014] The biosensors of the third and fifth aspects include:
Each of the differential biosensors has two basic structures of the biosensors of the first and second embodiments, and one of the biosensors does not have a biological substance fixed thereto. Thereby, the influence of the fluctuation of the dissolved oxygen concentration of the measurement solution can be reduced. 4th
The biosensors of the sixth and sixth aspects have the same anode as the biosensors of the third and fifth aspects, respectively.

In the present invention, the porous sheet is preferably paper. Preferably, both the cathode and the anode are silver. The cathode pattern may have a cathode lead wire between the cathode and the pad, and the cathode lead wire is sandwiched between two insulating films, and an oxygen permeable material covers the cathode lead wire. Is preferred.

[0016] The oxygen permeable material is preferably a silicone rubber. Further, it is preferable that a part of the anode is in contact with the electrolyte and the other part is covered with an insulating film.

Further, the bio-related substance-fixing layer is formed of yeast Tric.
It is preferably a layer to which hosporon cutaneum is fixed. In this case, it can be used as a BOD sensor. Further, the present invention provides a preferable method for manufacturing the biosensor.

That is, the manufacturing method according to the first aspect of the present invention is a method for manufacturing a small biosensor including the following steps. (1) A water-repellent insulating film is formed on both sides of a hydrophilic porous sheet so that a part of the porous sheet is exposed. (2) a cathode pattern having a cathode and a pad having a pad for making electrical contact with the outside on one side, and an anode and an anode being in electrical contact with the outside so as to extend from the insulating film onto the porous sheet; An anode pattern having a pad for taking the same is formed on the opposite surface. (3) The surface on which the cathode pattern is formed is covered with an oxygen-permeable material except for the pad portion, and the surface on which the anode pattern is formed is covered with an oxygen-permeable material or a plastic sheet except for the pad portion. . (4) Chips are cut out from the obtained porous sheet, and the side surfaces of the chips are covered with an oxygen-permeable material. (5) A bio-related substance is fixed to the sensitive part to form a bio-related substance fixing layer. (6) The electrolyte is impregnated into the porous sheet from a part of the chip.

The manufacturing method according to the second aspect of the present invention is a method for manufacturing a small-sized biosensor including the following steps. (1) A water-repellent insulating film is formed on both surfaces of a hydrophilic porous sheet from which a part of a boundary portion of a chip is cut out and removed so that a part of the porous sheet is exposed. (2) a cathode pattern having a cathode and a pad having a pad for making electrical contact with the outside on one side, and an anode and an anode being in electrical contact with the outside so as to extend from the insulating film onto the porous sheet; An anode pattern having a pad for taking the same is formed on the opposite surface. (3) The surface on which the cathode pattern is formed is covered with an oxygen-permeable material except for the pad portion, and the surface on which the anode pattern is formed is covered with a plastic sheet except for the pad portion. (4) Chips are cut out from the obtained porous sheet. (5) A bio-related substance is fixed to the sensitive part to form a bio-related substance fixing layer. (6) The electrolyte is impregnated into the porous sheet from a part of the chip.

Furthermore, a manufacturing method according to a third aspect of the present invention is a method for manufacturing a small-sized biosensor including the following steps. (1) A water-repellent insulating film is formed so that a part of the hydrophilic porous sheet is exposed. (2) a cathode pattern having a cathode and a pad for the cathode to make an electrical contact with the outside, and an anode and an anode making an electrical contact with the outside so as to extend from the insulating film onto the porous sheet. Pattern is formed on the surface on which the insulating film is formed. (3) The surface on which the cathode pattern and the anode pattern are formed is covered with an oxygen-permeable material except for the pad portion, and a water-repellent insulating film is formed on the entire surface on the opposite side, or a plastic sheet is used. Cover. (4) Chips are cut out from the obtained porous sheet, and the side surfaces of the chips are covered with an oxygen-permeable material. (5) A bio-related substance is fixed to the sensitive part to form a bio-related substance fixing layer. (6) The electrolyte is impregnated into the porous sheet from a part of the chip.

Furthermore, a manufacturing method according to a fourth aspect of the present invention is a method for manufacturing a small-sized biosensor including the following steps. (1) A water-repellent insulating film is formed on one surface of a hydrophilic porous sheet from which a part of a boundary portion of a chip is cut out and removed so that a part of the porous sheet is exposed. (2) a cathode pattern having a cathode and a pad for the cathode to make an electrical contact with the outside, and an anode and an anode making an electrical contact with the outside so as to extend from the insulating film onto the porous sheet. Pattern is formed on the surface on which the insulating film is formed. (3) The surface on which the cathode pattern and the anode pattern are formed is covered with an oxygen-permeable material except for the pad portion, and a water-repellent insulating film is formed on the entire surface on the opposite side, or a plastic sheet is used. Cover. (4) Chips are cut out from the obtained porous sheet. (5) A bio-related substance is fixed to the sensitive part to form a bio-related substance fixing layer. (6) The electrolyte is impregnated into the porous sheet from a part of the chip.

According to the manufacturing method of the present invention, a large number of biosensor chips can be manufactured at once. In particular, according to the manufacturing methods of the second and fourth aspects, since the boundary portion between the chips of the porous sheet is cut out and removed in advance, when the chip is cut out from the porous sheet, the porous sheet has a pad in the cross section. Since the chip side is not exposed except for the part and the chip side surface is covered, the step of covering the chip side surface can be omitted.

When the biological substance is a microorganism, the microorganism is mixed with a photo-curable resin, applied to the sensitive part, and then irradiated with ultraviolet rays to cure the photo-curable resin. A fixed layer can be formed.

[0024] Preferably, the oxygen permeable material is a silicone rubber. When an applicable oxygen-permeable material such as silicone rubber is used as the oxygen-permeable material, the application of the oxygen-permeable material and the immobilization of a biological substance are performed as in the following production methods (a) to (d). The layers can be formed at the same time, and the step of preparing an independent bio-related substance fixed layer can be omitted.

(A) A method of manufacturing a small biosensor including the following steps. (1) A water-repellent insulating film is formed on both sides of a hydrophilic porous sheet so that a part of the porous sheet is exposed. (2) a cathode pattern having a cathode and a pad having a pad for making electrical contact with the outside on one side, and an anode and an anode being in electrical contact with the outside so as to extend from the insulating film onto the porous sheet; An anode pattern having a pad for removing the microorganism is formed on the opposite surface. At this time, the suspension of microorganisms is filtered through a membrane filter, and the microorganisms are adsorbed and fixed on the membrane filter. The surface on which the microorganisms are adsorbed and immobilized of the dried membrane filter is adhered to the oxygen-permeable material immediately after the application of the oxygen-permeable material and before the curing to form a bio-related substance fixing layer on the sensitive part. I do. (3) The surface on which the cathode pattern is formed is covered with an oxygen-permeable material except for the pad portion, and the surface on which the anode pattern is formed is covered with an oxygen-permeable material or a plastic sheet except for the pad portion. . (4) Chips are cut out from the porous sheet, and the side surfaces of the chips are covered with an oxygen-permeable material as necessary. (5) The electrolyte is impregnated into the porous sheet from a part of the chip.

(B) A method for manufacturing a small biosensor including the following steps. (1) A water-repellent insulating film is formed on both surfaces of a hydrophilic porous sheet from which a part of a boundary portion of a chip is cut out and removed so that a part of the porous sheet is exposed. (2) a cathode pattern having a cathode and a pad having a pad for making electrical contact with the outside on one side, and an anode and an anode being in electrical contact with the outside so as to extend from the insulating film onto the porous sheet; An anode pattern having a pad for taking the same is formed on the opposite surface. (3) The surface on which the cathode pattern is formed is covered with an oxygen-permeable material except for the pad portion, and the surface on which the anode pattern is formed is covered with a plastic sheet except for the pad portion. The suspension is filtered through a membrane filter, the microorganisms are adsorbed and fixed on the membrane filter, and the surface of the membrane filter obtained by naturally drying the adsorbed and fixed microorganisms is subjected to an oxygen-permeable material. Immediately after the application and before curing, a bio-related substance fixing layer is formed on the sensitive part by bonding to the oxygen-permeable material. (4) Cutting out chips from the porous sheet. (5) The electrolyte is impregnated into the porous sheet from a part of the chip.

(C) A method for manufacturing a small biosensor including the following steps. (1) A water-repellent insulating film is formed on one surface of a hydrophilic porous sheet so that a part of the porous sheet is exposed. (2) a cathode pattern having a cathode and a pad for the cathode to make an electrical contact with the outside, and an anode and an anode making an electrical contact with the outside so as to extend from the insulating film onto the porous sheet. Pattern is formed on the surface on which the insulating film is formed. (3) The surface on which the cathode pattern and the anode pattern are formed is covered with an oxygen-permeable material except for the pad portion, and a water-repellent insulating film is formed on the entire surface on the opposite side, or a plastic sheet is used. At that time, the suspension of microorganisms is filtered through a membrane filter, the microorganisms are adsorbed and fixed on the membrane filter, and then the microorganism filter is obtained by naturally drying the membrane filter to adsorb and fix the microorganisms. The surface is adhered to the oxygen-permeable material immediately after the application of the oxygen-permeable material and before the curing, so that a bio-related substance fixing layer is formed on the sensitive part. (4) Chips are cut out from the porous sheet, and the side surfaces of the chips are covered with an oxygen-permeable material. (5) The electrolyte is impregnated into the porous sheet from a part of the chip.

(D) A method for manufacturing a small biosensor including the following steps. (1) A water-repellent insulating film is formed on one surface of a hydrophilic porous sheet from which a part of a boundary portion of a chip is cut out and removed so that a part of the porous sheet is exposed. (2) a cathode pattern having a cathode and a pad for the cathode to make an electrical contact with the outside, and an anode and an anode making an electrical contact with the outside so as to extend from the insulating film onto the porous sheet. Pattern is formed on the surface on which the insulating film is formed. (3) The surface on which the cathode pattern and the anode pattern are formed is covered with an oxygen-permeable material except for the pad portion, and a water-repellent insulating film is formed on the entire surface on the opposite side, or a plastic sheet is used. At that time, the suspension of microorganisms is filtered through a membrane filter, the microorganisms are adsorbed and fixed on the membrane filter, and then the microorganism filter is obtained by naturally drying the membrane filter to adsorb and fix the microorganisms. The surface is adhered to the oxygen-permeable material immediately after the application of the oxygen-permeable material and before the curing, so that a bio-related substance fixing layer is formed on the sensitive part. (4) Cutting out chips from the porous sheet. (5) The electrolyte is impregnated into the porous sheet from a part of the chip.

[0029]

Embodiments of the present invention will be described below in detail. <1> Biosensor of the present invention The hydrophilic porous sheet 1 used in the present invention is used for impregnating and holding an electrolyte.
There is no particular limitation as long as an insulating film can be formed thereon with good adhesion, and the adhesion of the metal pattern to be the cathode and anode is good. Examples of such a porous sheet 1 include a filter paper and a nitrocellulose membrane filter. Preferably filter paper,
A commercially available product can be used.

The thickness of the porous sheet is usually from 100 to 3
The size is typically 0.2 to 0.5 cm × 1 to 2 cm, although the size varies depending on the application. The water-repellent insulating film 2 formed on the porous sheet 1 is usually composed of a water-repellent polymer, and the polymer used must satisfy the following conditions. The porous sheet 1 is not made water-repellent by penetrating into the porous sheet 1 such as paper. The adhesion of the metal formed on the insulating film 2 to the electrode pattern is practically sufficient.

Examples of such a polymer include polyimide and silicone rubber (oxygen permeable material). The insulating film 2 may be formed by applying the above polymer, or may be formed by bonding a sheet made of the above polymer with an adhesive or the like. An example of an adhesive applied to a sheet is a tape. When a tape is used, the following conditions must be satisfied in addition to the above conditions. The electrolyte permeates into the porous sheet and does not come off when the tape gets wet with water. This condition does not need to be fulfilled perfectly, but it must at least allow the user electrolyte to soak into the porous sheet and not peel off during use of the biosensor.

The thickness of the insulating film 2 depends on the material constituting it, but is usually 50 to 100 μm. A cathode pattern having a cathode 3 and a pad 5 for the cathode 3 to make electrical contact with the outside extends from the insulating film 2 to the porous sheet 1. That is, it exists over both. The same applies to the anode 6.

The material constituting the cathode 3 and the anode 6 is conductive, and is not particularly limited as long as it does not react with the measurement solution or the like, but silver is preferably used. There is no particular limitation on the shapes of the cathode pattern and the anode pattern. When the cathode 3 and the anode 6 are formed on different surfaces, they can be simply formed over the entire surface without patterning. The term "pattern" as used herein includes such a shape formed on the entire surface. In any case, the portion that comes into contact with the porous sheet 1 (the electrolytic solution in the porous sheet) functions as the cathode 3 and the anode 6. Preferably, a portion of the lead wire 4 is provided between the cathode 3 and the pad 5, and the width of the portion of the cathode 3 and the portion of the lead wire 4 is formed thin (FIG. 1B). This width is usually 0.05 to 0.
2 mm.

When the cathode 3 and the anode 6 are formed on different surfaces (the biosensor according to the first embodiment), the dead space can be reduced, which is advantageous in terms of layout. Becomes simpler and easier to make. On the other hand, the cathode 3 and the anode 6 may be formed on the same surface (the biosensor of the second embodiment) (FIG. 2). In this case, the insulating layer on the back surface is formed on the entire surface so that the porous sheet is not exposed, or the entire surface is covered with a plastic sheet. Coating with a plastic sheet can be performed by bonding the sheet with an adhesive or the like. If necessary, an oxygen-permeable material may be further formed as an insulating film.

Here, the cathode lead wire 4 is insulated so as to be sandwiched between the insulating layer 2 and the oxygen permeable material 8. It is preferable to form another insulating film on the line 4 (FIG. 3).

The oxygen permeable material 8 is not particularly limited, but silicone rubber is preferably used. The thickness of the layer of the oxygen-permeable material 8 depends on its type, but usually the thickness on the cathode 3 is 50 to 200 μm.

The biological substance is a microorganism, an enzyme, or the like that can act on a measurement substance in a sample. These are appropriately selected depending on the use of the biosensor. The fixation of the bio-related substance to the sensitive part can be performed by a known method, for example, a microorganism is mixed with a photocurable resin, applied to the sensitive part,
It can be fixed by curing. Alternatively, the culture solution of the microorganism may be filtered through a membrane filter, dried, and the microorganism filter adsorbed and fixed on the membrane filter may be adhered to the sensitive part. The sensitive part means a portion of the oxygen permeable material 8 on the film near the cathode 3, and the effect of the action of the bio-related substance generated there is sufficient, so that the oxygen electrode as a transducer, more specifically, the cathode 3 Area. A reaction of the measurement substance in the sample occurs in the sensitive part, and the measurement substance is quantified.

The electrolyte is not particularly limited, and an appropriate one is used. For example, an aqueous solution of potassium chloride can be used.
In using the biosensor of the present invention, the anode 6
In contrast, a constant voltage of, for example, -1.0 V may be applied to the cathode 3. As a result, a reduction reaction of oxygen occurs on the surface of the cathode 3, and a current value proportional to the oxygen concentration can be obtained as a signal. On the anode side, the reaction mainly forming silver chloride proceeds, and a stable reference potential is obtained. When this biosensor is immersed in a buffer solution and stabilized, a certain amount of a sample solution is added.If the bio-related substance is a microorganism such as yeast, the measurement substance such as nutrients in the sample solution is utilized. At that time, respiratory activity is increased and oxygen consumption is increased. Since this change appears as a decrease in the current value, for example, the BOD value can be obtained using this as an index.

In the biosensor of the present invention, the portion where the cathode is formed has an arrangement of an oxygen-permeable membrane-cathode-electrolyte layer (a porous sheet containing an electrolytic solution), and oxygen first passes through the oxygen-permeable membrane. After entering the electrolyte layer,
It wraps around from the electrolyte layer side, reaches the cathode, and is reduced at the cathode. By adopting such a structure, it is possible to mass-produce a small-sized biosensor having excellent responsiveness using inexpensive materials.

Typically, the anode 6 is the cathode 3
Is formed without patterning on the entire surface on the side opposite to the surface on which is formed. However, the anode 3 is
If the area ratio is large, a silver chloride layer is not easily formed,
The anode potential may not be stable. In this case,
As shown in FIG. 4, only a part of the anode 6 may be exposed, and most of the anode 6 may be covered with the insulating film. The area of the exposed portion may be about the same as that of the cathode 3, but it is preferable that the width of A in FIG. 1B is sufficiently large so that the reaction on the anode side is not limited. The formation of silver chloride proceeds not only in the exposed portion of silver but also in the portion covered with the insulating film. Therefore, the advantages of shortening the stabilization time and extending the life can be achieved.

As a modified example of the biosensor, an actuated sensor can be manufactured. That is, two oxygen electrodes to be used as transducers are arranged in parallel, and a bio-related substance is fixed to one of the sensitive parts and a bio-related substance is not fixed to the other (the biosensor of the third embodiment) (FIG. 7). . Thereby, the influence of the fluctuation of the dissolved oxygen concentration of the measurement solution can be reduced. When a carrier such as a photo-curable resin is used for fixing, if the carrier layer is formed even on the side without a bio-related substance, the characteristics of the two sensitive parts are likely to be uniform, and the sensor The accuracy is improved. Also, instead of using two independent oxygen electrodes as transducers, one chip has two cathodes 3,
It is also possible to adopt a structure in which one anode 6 is shared (the biosensor of the fifth embodiment) (FIG. 8). Further, these cathode and anode can be formed on the same surface (fourth and sixth biosensors).

Next, a method of manufacturing the above-described biosensor of the present invention will be described.

<2> Manufacturing Method of the Present Invention First, the manufacturing method of the first embodiment of the present invention will be described step by step. Although one chip is shown in the figure for convenience of explanation, a plurality of chips are actually manufactured at once. Also,
1, 2, 6 (a), 7, and 8, the oxygen permeable membrane 8 is omitted for convenience of illustration.

(1) Formation of Insulating Film A water-repellent insulating film is formed on both surfaces of the hydrophilic porous sheet so that a part of the porous sheet is exposed.

As a method of forming an insulating film so that a part of the porous sheet is exposed, (a) a mask is formed on the porous sheet, a material for the insulating film is applied by spraying, and drying is performed. And (b) attaching a tape as an insulating film. The mask used in the case of the method (a) does not necessarily need to be a high-precision mask used for screen printing, and a mask obtained by cutting a part of a plastic sheet is sufficient. In the case of the method (b), the end of the tape becomes a step which is vertically steep in the pattern formation in the next step, and the pattern may not be formed sufficiently. In such a case, a pattern of a water-repellent polymer (for example, silicone rubber) may be formed only near the step.

(2) Formation of a pattern of cathode and anode: A pattern extending from the insulating film onto the porous sheet is formed as follows.
A cathode and a cathode pattern having a pad for making electrical contact with the outside are formed on one surface, and an anode pattern having an anode and a pad for making the anode make electrical contact with the outside is formed on the opposite surface.

The pattern can be formed by various methods such as screen printing, vacuum deposition, and sputtering. However, from the viewpoint of cost, it is preferable to use a simple method such as screen printing.

(3) Formation of oxygen permeable film The surface on which the cathode pattern is formed is covered with an oxygen permeable material except for the pad portion, and the surface on which the anode pattern is formed is covered with the oxygen permeable material except for the pad portion. Cover with conductive material or plastic sheet.

When the oxygen permeable material is a silicone rubber, a method of applying the silicone rubber and baking it in an atmosphere saturated with steam at 80 ° C. for one hour can be exemplified. When forming the film, protect the pad part by applying an easily peelable tape, and after the silicone rubber is cured,
Peel off the tape on the pad. Alternatively, as in (1), a film is formed using a mask except for the pad portion.

The portion formed on the cathode by this coating functions as an oxygen permeable film. Since the function of the oxygen permeable membrane is not required on the surface on which the anode is formed, a plastic sheet may be bonded with an adhesive or the like instead of being coated with an oxygen permeable material.

The coating on the cathode side and the anode side may be performed first or simultaneously.

(4) Chip Cutting Chips are cut from the obtained porous sheet, and the side surfaces of the chips are covered with an oxygen-permeable material.

The oxygen electrodes formed collectively on the porous sheet are cut off with scissors or a cutter knife. At this time, since the porous sheet is exposed on the cut surface, the side surface is also covered with the same oxygen-permeable material as in (3) except for a part as the electrolyte inlet.

(5) Immobilization of bio-related substance A bio-related substance is immobilized on the sensitive part to form a bio-related substance fixing layer. For example, culturing microorganisms suitable for the analyte,
A method in which cells are separated by centrifugation, the separated cells are mixed with a carrier such as a photocurable resin, applied to a sensitive part (on an oxygen-permeable membrane near the cathode), and then cured by irradiating ultraviolet rays. Is preferably used. In the case of a BOD sensor,
For example, the yeast Trichosporon cutaneum can be used as a microorganism.

Note that this step may be performed collectively before the step (4).

(6) Injection of electrolyte An electrolyte is impregnated from a part of the chip into the porous sheet. Since the electrolyte injection port is provided, the electrolyte can be injected by immersing the entire chip in the electrolyte.

Through the above steps, the biosensor of the present invention can be obtained. Further, before forming the hydrophilic porous sheet on the water-repellent insulating film, a part of the boundary portion of the chip may be cut out and removed (the manufacturing method of the second embodiment) (FIG. 6). Normally, a portion that remains without being removed is made to be a pad portion, and this portion becomes an electrolyte solution injection port in an electrolyte solution injection step. Other steps are basically the same as the manufacturing method of the first embodiment, but in this case, in the step of cutting out the chips, when the chips are cut out, the porous sheet is not unnecessarily exposed to the cross section. This has the advantage that the step of coating the side surface of the chip can be omitted. When a plastic sheet is used, a sensor excellent in strength can be obtained.

In the case where the cathode and the anode are formed on the same surface, the method (1) to the method (1)
The step (3) may be performed as follows. (1) A hydrophilic porous sheet is used as it is or by cutting out and removing a part of a boundary part of a chip, and is repelled by exposing a part of the porous sheet to one surface of the porous sheet. An aqueous insulating film is formed. (2) a cathode pattern having a cathode and a pad for the cathode to make an electrical contact with the outside, and an anode and an anode making an electrical contact with the outside so as to extend from the insulating film onto the porous sheet. Pattern is formed on the surface on which the insulating film is formed. (3) The surface on which the cathode pattern and the anode pattern are formed is covered with an oxygen-permeable material except for the pad portion, and a water-repellent insulating film is formed on the entire surface on the opposite side, or a plastic sheet is used. Cover.

When the oxygen-permeable material is a coatable oxygen-permeable material such as silicone rubber, and the biological substance is a microorganism, the suspension of the microorganism is filtered through a membrane filter, and the microorganism-containing suspension is filtered. Is adsorbed and fixed on the membrane filter, and the surface of the membrane filter obtained by air drying is fixed on the oxygen permeable material immediately after the application of the oxygen permeable material and before curing. By bonding, a bio-related substance fixing layer can be formed on the sensitive part. In this case, mild conditions such as standing at room temperature are employed as the curing conditions for the coatable oxygen-permeable material such as silicone rubber. The membrane filter is appropriately selected according to the cell size of the microorganism to be used. In this case, there is no need to perform the step of fixing the biologically-related substance after the cutting.

When the coating or application of the oxygen-permeable material is performed in a plurality of steps, the oxygen-permeable material used in each step may be the same or different. The differential type biosensor can also be manufactured by applying the above manufacturing method.

[0061]

The present invention will be described below with reference to examples.
Note that a large number of small biosensors are collectively manufactured on one sheet, but for convenience, one (one chip) biosensor is shown in the drawing.

[0062]

Example 1 (1) Formation of Insulating Film Filter paper (manufactured by Whatman, Inc.) was used as the hydrophilic porous sheet 1.
Using a 180 μm thick mask with a mask on both sides to expose a portion of the filter paper, apply a water-repellent polymer (Universal, Plasticidip) by spraying and dry to obtain a thickness of approximately An insulating film 2 of 50 μm was formed on both sides of the filter paper (FIG. 1A).

(2) Formation of Cathode and Anode On the filter paper having the insulating film formed in (1), the cathode 3, the cathode lead wire 4 and the cathode pad 5 are extended so as to extend on the insulating film and the filter paper. A cathode pattern having portions and an anode pattern having portions of the anode 6 and the anode pad 7 were formed. The cathode pattern and the anode pattern were made of silver, and each was formed to a thickness of 1 μm by sputtering (FIG. 1).
(B)).

(3) Formation of Oxygen Permeation Film Silicone rubber (KE4895, manufactured by Shin-Etsu Chemical Co., Ltd.) was applied to the surface on which the cathode 3 was formed, and baked at 80 ° C. in an atmosphere saturated with water vapor for 1 hour. Then, similarly,
The same silicone rubber was applied to the surface on which the anode 6 was formed and baked. In the formation of each film, the pads 5 and 7, which are the contacts when connecting to the measuring instrument, were protected from sticking with silicone rubber by applying an easily peelable tape. After the silicone rubber on both sides was cured, the tape on the pad portion was peeled off (FIG. 1 (c)).

(4) Separation of Chip The oxygen electrode formed on the filter paper was cut off with scissors or a cutter knife. At this time, since the filter paper was exposed on the cut surface, the same silicone rubber as that used in (3) was applied to the side surface and cured, leaving the electrolytic solution inlet 9.

(5) Yeast culture The yeast Trichosporon cutaneum was cultured in Bacto YM broth at pH 7.0.
The cells were cultured in a medium (manufactured by DIFCO Laboratories) at 30 ° C. for 18 hours.

(6) Separation of cells The cultured yeast was centrifuged at 1500 rpm for 15 minutes.
The plate was washed twice with a phosphate buffer (100 mM, pH 7.0) and centrifuged again.

(7) Fixation of yeast The yeast obtained in (6), a photo-curable resin (manufactured by Kansai Paint, ENT-3400) and a phosphate buffer were mixed at a weight ratio of 1: 1: 1. Was applied so as to cover it, and the resin was cured by irradiating ultraviolet rays for 3 minutes under a nitrogen atmosphere to fix yeast (FIG. 1 (d)). The obtained yeast fixed layer was immersed in a phosphate buffer at room temperature overnight.

(8) Injection of Electrolyte Solution The entire chip obtained in the steps up to (7) is immersed in an electrolyte solution (aqueous potassium chloride solution), and the electrolytic solution is injected from the portion where the filter paper is exposed, that is, from the electrolyte solution inlet 9. The liquid was soaked.

By the above steps, a BOD sensor was obtained.

[0071]

Example 2 (1) Formation of insulating film Filter paper (manufactured by Whatman, Inc.) was used as hydrophilic porous sheet 1.
A polyimide tape (Kapton film tape No. 5413 (trade name, manufactured by Sumitomo 3M), a thickness of 70 μm) as an insulating film 2 was attached to both surfaces thereof, and the two layers were attached.

(2) Formation of Cathode and Anode In the same manner as in Example 1, a silver cathode pattern and an anode pattern were formed on the insulating film 2 formed in (1).

(3) Culture of yeast and fixation by adsorption The yeast was cultured in the same manner as in Example 1, and 1 ml of the culture was filtered through a membrane filter (manufactured by Millipore, VMWP09025, pore size 0.5 μm). The plate was washed three times with a phosphate buffer (pH 7.0). Next, it was sufficiently dried and fixed by adsorption.

(4) Formation of oxygen-permeable membrane and immobilization of yeast Silicone rubber (KE4895, manufactured by Shin-Etsu Chemical Co., Ltd.) was applied to the surface on which the cathode 3 was formed, and immediately thereafter, the membrane filter obtained in (3) was applied appropriately. The yeast was fixed in a fixed shape, and the surface on which the yeast was adsorbed and fixed was stuck on silicone rubber to fix the yeast. The curing of the silicone rubber was performed at room temperature in an atmosphere saturated with water vapor. Silicone rubber was also applied to the surface on which the anode 6 was formed and cured, except that the membrane filter was not attached. In addition,
In forming each of the films, the portion of the pad for making a contact for immediate connection was protected in the same manner as in Example 1 so that silicone rubber did not adhere.

(5) Separation of Chip The oxygen electrode formed on the filter paper was cut off with scissors or a cutter knife. As in Example 1, the same silicone rubber as in (4) was also applied to the side surface except for the electrolyte injection port 9.

(6) Injection of Electrolyte The procedure was the same as in Example 1. FIG. 5 shows the response when the sensitive part of the small BOD sensor prepared by this method was immersed in a 0.1 M phosphate buffer and a standard sample was added.

[0077]

Example 3 (1) Formation of Insulating Film, Cathode and Anode In the same manner as in Example 1, an insulating layer 2 was formed on filter paper, and a cathode pattern and an anode pattern were formed thereon. However, a filter paper cut out from the vicinity of the chip boundary other than the vicinity of the pad was used (FIG. 6A).

(2) Adhesion of Plastic Sheet An adhesive (epoxy adhesive) 11 is spin-coated on the entire surface of a polyester sheet (thickness: 100 μm) 10 having a shape exposing a portion of the anode pad 7, and immediately (1) The obtained filter paper was bonded to the surface on which the cathode 3 was not formed.

(3) Formation of Oxygen-permeable Membrane and Fixation of Yeast In the same manner as in Example 2, silicone rubber to be an oxygen-permeable membrane was applied to the surface on which the cathode 3 was formed, except for the pad 5. The yeast was fixed in the same manner as in Example 2.

(4) Separation of Chip The oxygen electrode formed on the filter paper was cut off with scissors or a cutter knife. The boundary portion other than the chip pad portion has a laminated structure of polyester sheet-adhesive-silicone rubber, so that an external aqueous solution is not soaked, so it is not necessary to apply silicone rubber to the side surface. No (FIG. 6 (b)).

(5) Injection of Electrolyte The procedure was the same as in Example 2. The electrolyte was injected from the portion of the pad where the filter paper was exposed.

[0082]

Example 4 The same as Example 1 except that two oxygen electrodes serving as transducers were arranged in parallel, one was fixed with yeast, and the other was formed with a fixed layer using only a photocurable resin and a phosphate buffer. To produce a differential sensor.

[0083]

Embodiment 5 A differential sensor was prepared in the same manner as in Embodiment 2 except that two oxygen electrodes serving as transducers were arranged in parallel, one of which was fixed with yeast and the other was a membrane filter which did not adsorb and fix yeast. Make it.

[0084]

As described above, according to the present invention,
A small-sized biosensor which can be mass-produced using inexpensive materials and has excellent responsiveness, and a method for manufacturing the same are provided.

[Brief description of the drawings]

FIG. 1 shows the structure and manufacturing method of a small biosensor according to Examples 1 and 2.

FIG. 2 shows an example of a layout when a cathode and an anode are formed on the same surface.

FIG. 3 is a cross-sectional view of an embodiment in which an insulating layer for a cathode lead wire is added.

FIG. 4 is a cross-sectional view of an embodiment in which a part of the anode is exposed and most of the anode is covered with an insulating film.

FIG. 5 shows how a small biosensor according to the second embodiment responds.

FIG. 6 shows a structure and a manufacturing method of a small-sized biosensor according to a third embodiment. (A) is a plan view, and (b) is a cross-sectional view along the line aa 'in (a).

FIG. 7 shows an example of a structure of a differential biosensor.

FIG. 8 shows an example of a structure in which a common anode is used in a differential biosensor.

[Explanation of symbols]

 DESCRIPTION OF SYMBOLS 1 Porous sheet 2 Insulating film 3 Cathode 4 Cathode lead wire 5 Cathode pad 6 Anode 7 Anode pad 8 Oxygen permeable membrane 9 Electrolyte injection port 10 Plastic sheet 11 Adhesive 12 Bio-related substance fixed layer

 ──────────────────────────────────────────────────続 き Continuing on the front page (72) Inventor Masao Karube 1-3-16 Higashi-Arima, Miyamae-ku, Kawasaki-shi, Kanagawa (72) Inventor Hiroaki Suzuki 3-6-6 Takezono, Tsukuba-shi, Ibaraki 305-103

Claims (22)

[Claims] 1. A water-repellent insulating film is formed on a part of one surface of a hydrophilic porous sheet, and the cathode and the cathode are electrically connected to the outside so as to extend from the insulating film onto the porous sheet. A cathode pattern having a pad for removing the same, a water-repellent insulating film is formed on a part of the surface on the opposite side of the porous sheet, and extends from the insulating film onto the porous sheet. An anode pattern having an anode and a pad for the anode to make electrical contact with the outside is formed, and the chip side surface of the biosensor and the surface on which the cathode is formed are covered with an oxygen-permeable material except for a pad portion. The surface on which is formed is covered with an oxygen-permeable material or a plastic sheet leaving a pad portion, and a bio-related substance is fixed on the sensitive portion, and the hydrophilic porous material A small biosensor characterized in that an electrolyte is impregnated in a sheet. 2. A water-repellent insulating film is formed on a part of one surface of a hydrophilic porous sheet, and the cathode and the cathode are in electrical contact with the outside so as to extend from the insulating film onto the porous sheet. A cathode pattern having a pad for taking an electrical contact with the outside and an anode pattern having a pad for taking an electrical contact with the anode and the outside are formed, and the opposite surface of the porous sheet is entirely provided with a water-repellent insulating film. Is formed or covered with a plastic sheet, the chip side surface of the biosensor and the surface on which the cathode and anode are formed are covered with an oxygen-permeable material except for the respective pad portions, and a bio-related substance is applied to the sensitive portion. A small-sized biosensor fixed, wherein an electrolyte is impregnated in the hydrophilic porous sheet. 3. A water-repellent insulating film is formed on a part of one surface of a hydrophilic porous sheet, and the cathode and the cathode are electrically connected to the outside so as to extend from the insulating film onto the porous sheet. A cathode pattern having a pad for removing the same, a water-repellent insulating film is formed on a part of the surface on the opposite side of the porous sheet, and extends from the insulating film onto the porous sheet. An anode pattern having an anode and a pad for the anode to make electrical contact with the outside is formed, and the chip side surface of the biosensor and the surface on which the cathode is formed are covered with an oxygen-permeable material except for a pad portion. The surface on which was formed was covered with an oxygen-permeable material or a plastic sheet except for a pad portion, and the electrolyte was impregnated in the hydrophilic porous sheet. Two structures are arranged in parallel. One of the sensitive parts is provided with a bio-related substance fixing layer, and the other sensitive part is left as it is or the bio-related substance fixing layer is formed. A small differential biosensor characterized in that only the same carrier material as above is fixed. 4. A water-repellent insulating film is formed on a part of one surface of a hydrophilic porous sheet, and the cathode and the cathode are electrically connected to the outside so as to extend from the insulating film onto the porous sheet. Two cathode patterns each having a pad for removing the water, a water-repellent insulating film is formed on a part of the opposite surface of the porous sheet, and extends from the insulating film onto the porous sheet. An anode pattern having an anode and a pad for the anode to make electrical contact with the outside is formed, and the chip side surface of the biosensor and the surface on which the cathode is formed are coated with an oxygen-permeable material except for a pad portion. The surface on which the anode is formed is covered with an oxygen-permeable material or a plastic sheet except for a pad portion, and the electrolyte is impregnated in the hydrophilic porous sheet. In this case, a bio-related substance fixing layer is formed on one of the sensitive parts, and the other sensitive part is exposed as it is or only the same carrier material as that on which the bio-related substance fixing layer is formed is fixed. Differential small biosensor. 5. A water-repellent insulating film is formed on a part of one surface of a hydrophilic porous sheet, and the cathode and the cathode are electrically connected to the outside so as to extend from the insulating film onto the porous sheet. A cathode pattern having a pad for taking an electrical contact with the outside and an anode pattern having a pad for taking an electrical contact with the anode and the outside are formed, and the opposite surface of the porous sheet is entirely provided with a water-repellent insulating film. Is formed or coated with a plastic sheet, and the chip side surface of the biosensor and the formed surface of the cathode and anode are coated with an oxygen-permeable material except for respective pad portions, and the hydrophilic porous sheet is formed. There are two structures in which the electrolyte is impregnated.
It is arranged in parallel with each other, forming a bio-related substance fixed layer near one cathode, and leaving the other cathode vicinity as it is, or as the same carrier material as forming the bio-related substance fixed layer A small differential biosensor characterized in that only one is fixed. 6. A water-repellent insulating film is formed on a part of one surface of a hydrophilic porous sheet, and the cathode and the cathode are electrically connected to the outside so as to extend from the insulating film onto the porous sheet. Independently, two cathode patterns each having a pad for taking an electrode, an anode and an anode pattern having a pad for the anode to make an electrical contact with the outside are formed, and the opposite surface of the porous sheet is entirely covered. A water-repellent insulating film is formed or covered with a plastic sheet, and the chip side surface of the biosensor and the surface on which the cathode and anode are formed are covered with an oxygen-permeable material except for respective pad portions. The electrolyte is impregnated into the hydrophilic porous sheet, and a biomaterial-fixed layer is formed near one cathode, and near the other cathode. Should be exposed as it is,
A small differential biosensor characterized in that only the same carrier material on which the biomaterial-fixed layer is formed is fixed. 7. The sheet according to claim 1, wherein said porous sheet is paper.
7. The small-sized biosensor according to any one of 6. 8. The small biosensor according to claim 1, wherein both the cathode and the anode are silver. 9. The cathode pattern has a cathode lead between the cathode and the pad, the cathode lead being sandwiched between two layers of insulating films, and further having an oxygen permeable material thereon. The small biosensor according to any one of claims 1 to 8, wherein 10. The small biosensor according to claim 1, wherein the oxygen-permeable material is a silicone rubber. 11. The anode according to claim 1, wherein a part of the anode is in contact with the electrolyte and the other part is covered with an insulating film.
11. The small biosensor according to any one of items 10 to 10. 12. The bio-related substance-fixed layer is formed of yeast Tric.
A layer to which hosporon cutaneum is fixed.
The small biosensor according to any one of the above. 13. A method for manufacturing a small biosensor comprising the following steps. (1) A water-repellent insulating film is formed on both sides of a hydrophilic porous sheet so that a part of the porous sheet is exposed. (2) a cathode pattern having a cathode and a pad having a pad for making electrical contact with the outside on one side, and an anode and an anode being in electrical contact with the outside so as to extend from the insulating film onto the porous sheet; An anode pattern having a pad for taking the same is formed on the opposite surface. (3) The surface on which the cathode pattern is formed is covered with an oxygen-permeable material except for the pad portion, and the surface on which the anode pattern is formed is covered with an oxygen-permeable material or a plastic sheet except for the pad portion. . (4) Chips are cut out from the obtained porous sheet, and the side surfaces of the chips are covered with an oxygen-permeable material. (5) A bio-related substance is fixed to the sensitive part to form a bio-related substance fixing layer. (6) The electrolyte is impregnated into the porous sheet from a part of the chip. 14. A method for manufacturing a small biosensor comprising the following steps. (1) A water-repellent insulating film is formed on both surfaces of a hydrophilic porous sheet from which a part of a boundary portion of a chip is cut out and removed so that a part of the porous sheet is exposed. (2) a cathode pattern having a cathode and a pad having a pad for making electrical contact with the outside on one side, and an anode and an anode being in electrical contact with the outside so as to extend from the insulating film onto the porous sheet; An anode pattern having a pad for taking the same is formed on the opposite surface. (3) The surface on which the cathode pattern is formed is covered with an oxygen-permeable material except for the pad portion, and the surface on which the anode pattern is formed is covered with a plastic sheet except for the pad portion. (4) Chips are cut out from the obtained porous sheet. (5) A bio-related substance is fixed to the sensitive part to form a bio-related substance fixing layer. (6) The electrolyte is impregnated into the porous sheet from a part of the chip. 15. A method for manufacturing a small biosensor comprising the following steps. (1) A water-repellent insulating film is formed on one surface of a hydrophilic porous sheet so that a part of the porous sheet is exposed. (2) a cathode pattern having a cathode and a pad for the cathode to make an electrical contact with the outside, and an anode and an anode making an electrical contact with the outside so as to extend from the insulating film onto the porous sheet. Pattern is formed on the surface on which the insulating film is formed. (3) The surface on which the cathode pattern and the anode pattern are formed is covered with an oxygen-permeable material except for the pad portion, and a water-repellent insulating film is formed on the entire surface on the opposite side, or a plastic sheet is used. Cover. (4) Chips are cut out from the obtained porous sheet, and the side surfaces of the chips are covered with an oxygen-permeable material. (5) A bio-related substance is fixed to the sensitive part to form a bio-related substance fixing layer. (6) The electrolyte is impregnated into the porous sheet from a part of the chip. 16. A method for manufacturing a small biosensor comprising the following steps. (1) A water-repellent insulating film is formed on one surface of a hydrophilic porous sheet from which a part of a boundary portion of a chip is cut out and removed so that a part of the porous sheet is exposed. (2) a cathode pattern having a cathode and a pad for the cathode to make an electrical contact with the outside, and an anode and an anode making an electrical contact with the outside so as to extend from the insulating film onto the porous sheet. Pattern is formed on the surface on which the insulating film is formed. (3) The surface on which the cathode pattern and the anode pattern are formed is covered with an oxygen-permeable material except for the pad portion, and a water-repellent insulating film is formed on the entire surface on the opposite side, or a plastic sheet is used. Cover. (4) Chips are cut out from the obtained porous sheet. (5) A bio-related substance is fixed to the sensitive part to form a bio-related substance fixing layer. (6) The electrolyte is impregnated into the porous sheet from a part of the chip. 17. The living body-related substance is a microorganism, and the microorganism is mixed with a photocurable resin, applied to the sensitive portion, and then irradiated with ultraviolet light to cure the photocurable resin. 14. A substance fixing layer is formed.
The production method according to any one of items 16 to 16, wherein 18. A method for manufacturing a small biosensor including the following steps. (1) A water-repellent insulating film is formed on both sides of a hydrophilic porous sheet so that a part of the porous sheet is exposed. (2) a cathode pattern having a cathode and a pad having a pad for making electrical contact with the outside on one side, and an anode and an anode being in electrical contact with the outside so as to extend from the insulating film onto the porous sheet; An anode pattern having a pad for removing the microorganism is formed on the opposite surface. At this time, the suspension of microorganisms is filtered through a membrane filter, and the microorganisms are adsorbed and fixed on the membrane filter. The surface on which the microorganisms are adsorbed and immobilized of the dried membrane filter is adhered to the oxygen-permeable material immediately after the application of the oxygen-permeable material and before the curing to form a bio-related substance fixing layer on the sensitive part. I do. (3) The surface on which the cathode pattern is formed is covered with an oxygen-permeable material except for the pad portion, and the surface on which the anode pattern is formed is covered with an oxygen-permeable material or a plastic sheet except for the pad portion. . (4) Chips are cut out from the porous sheet, and the side surfaces of the chips are covered with an oxygen-permeable material as necessary. (5) The electrolyte is impregnated into the porous sheet from a part of the chip. 19. A method for manufacturing a small biosensor comprising the following steps. (1) A water-repellent insulating film is formed on both surfaces of a hydrophilic porous sheet from which a part of a boundary portion of a chip is cut out and removed so that a part of the porous sheet is exposed. (2) a cathode pattern having a cathode and a pad having a pad for making electrical contact with the outside on one side, and an anode and an anode being in electrical contact with the outside so as to extend from the insulating film onto the porous sheet; An anode pattern having a pad for taking the same is formed on the opposite surface. (3) The surface on which the cathode pattern is formed is covered with an oxygen-permeable material except for the pad portion, and the surface on which the anode pattern is formed is covered with a plastic sheet except for the pad portion. The suspension is filtered through a membrane filter, the microorganisms are adsorbed and fixed on the membrane filter, and the surface of the membrane filter obtained by naturally drying the adsorbed and fixed microorganisms is subjected to an oxygen-permeable material. Immediately after the application and before curing, a bio-related substance fixing layer is formed on the sensitive part by bonding to the oxygen-permeable material. (4) Cutting out chips from the porous sheet. (5) The electrolyte is impregnated into the porous sheet from a part of the chip. 20. A method for manufacturing a small biosensor comprising the following steps. (1) A water-repellent insulating film is formed on one side of a hydrophilic porous sheet so that a part of the porous sheet is exposed. (2) a cathode pattern having a cathode and a pad for the cathode to make an electrical contact with the outside so as to extend from the insulating film onto the porous sheet; and an anode and the anode making an electrical contact with the outside. Pattern is formed on the surface on which the insulating film is formed. (3) The surface on which the cathode pattern and the anode pattern are formed is covered with an oxygen-permeable material except for the pad portion, and a water-repellent insulating film is formed on the entire surface on the opposite side, or a plastic sheet is used. At that time, the suspension of microorganisms is filtered through a membrane filter, the microorganisms are adsorbed and fixed on the membrane filter, and the microorganism filter is obtained by naturally drying the membrane filter. The surface is adhered to the oxygen-permeable material immediately after the application of the oxygen-permeable material and before the curing, so that a bio-related substance fixing layer is formed on the sensitive part. (4) Chips are cut out from the porous sheet, and the side surfaces of the chips are covered with an oxygen-permeable material. (5) The electrolyte is impregnated into the porous sheet from a part of the chip. 21. A method for manufacturing a small biosensor comprising the following steps. (1) A water-repellent insulating film is formed on one surface of a hydrophilic porous sheet from which a part of a boundary portion of a chip is cut out and removed so that a part of the porous sheet is exposed. (2) a cathode pattern having a cathode and a pad for the cathode to make an electrical contact with the outside, and an anode and an anode making an electrical contact with the outside so as to extend from the insulating film onto the porous sheet. Pattern is formed on the surface on which the insulating film is formed. (3) The surface on which the cathode pattern and the anode pattern are formed is covered with an oxygen-permeable material except for the pad portion, and a water-repellent insulating film is formed on the entire surface on the opposite side, or a plastic sheet is used. At that time, the suspension of microorganisms is filtered through a membrane filter, the microorganisms are adsorbed and fixed on the membrane filter, and then the microorganism filter is obtained by naturally drying the membrane filter to adsorb and fix the microorganisms. The surface is adhered to the oxygen-permeable material immediately after the application of the oxygen-permeable material and before the curing, so that a bio-related substance fixing layer is formed on the sensitive part. (4) Cutting out chips from the porous sheet. (5) The electrolyte is impregnated into the porous sheet from a part of the chip. 22. The method according to claim 13, wherein said oxygen-permeable material is silicone rubber.