JP2002350383A - Biosensor and method of manufacturing the same - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a biosensor having an arbitrary characteristic by forming a through hole 11, which is made up so as to penetrate a limited permeable film 8 formed on a reacting film 5 of the biosensor, so that the numerical aperture can be properly controlled using a lithography method, and to obtain a method of manufacturing the biosensor. SOLUTION: A working pole 2 and a counter pole 3 are formed on a dielectric substrate 1, and a selective permeable film 4 and an immobilized enzyme film 5 are in turn laminated. A projection 6 made of photoresist, which uses organic non-solvent as separating liquid, is formed on the immobilized enzyme film 5, and then a water non-permeable film 7 is formed. Next, the projection 6 is removed using the separating liquid, and the limited permeable film 8 is formed. Therefore, the biosensor having a controllable aperture and the method of manufacturing the biosensor are obtained.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a biosensor for quantifying a specific component in an aqueous solution and a method for manufacturing the biosensor. The present invention relates to an improvement of a restricted permeation membrane of a biosensor having the following.

[0002]

2. Description of the Related Art In recent years, measurement using a biosensor has been actively performed in the fields of medicine and analysis. Among the biosensors, a current detection type glucose sensor is well known. This glucose sensor is widely used for measuring a blood sugar level and a urine sugar level.

The most common type of glucose sensor utilizes a reaction in which glucose is converted to gluconolactone and hydrogen peroxide using glucose oxidase (GOX). That is, it reacts as in the following equation (1). Glucose + O ₂ → gluconolactone _{+ H 2 O 2 ··· (1} ) (C 6 H 12 O 6) GOX (C 6 H 10 O 6) The (1) acting the generated hydrogen peroxide formula electrode And the reaction amount is detected as a current value. The reaction at the electrode is (2)
It is as the formula. H ₂ O ₂ → O ₂ + 2e ⁻ + 2H ₂ ⁺ ... (2)

As is apparent from equations (1) and (2),
If O ₂ is present in excess of glucose, the glucose concentration in the solution can be quantified because the glucose concentration in the solution is proportional to the current. However, the O ₂ involved in the reaction
Is the dissolved oxygen in the sample, and the above reaction is saturated at a glucose concentration (blood sugar level) of about 40 mg / dl. 50-500mg / dl for practical blood glucose measurement,
The urine sugar level is required to be in the range of about 0 to 2000 mg / dl. Therefore, in order to extend the measurement range, 1) dilute the sample, 2) mount an electron acceptor that reacts instead of oxygen on the sensor, and 3) arrange a limited permeation membrane consisting of a porous membrane on the outer layer to reduce the permeation of glucose. Some measures have been taken such as limiting the relative O ₂ concentration.

Among them, 1) requires a diluent and a diluting device, and 2) has a problem that it can be used only once. On the other hand, 3) has the advantage that the mechanism is simple and can be used repeatedly. For this reason, various researches on a restricted permeable membrane have been conducted.

One example is disclosed in JP-A-9-304330. FIG. 8 shows a urine sugar measuring device using a biosensor, and FIG. 9 is a cross-sectional view of the biosensor taken along the line BB 'in FIG.

The urine sugar measuring device 110 shown in FIG. 8 detects sugar in urine using the action of an enzyme.
11, a sensor body 113 is detachably provided via a sensor cap 117, various electronic circuits are built in the measuring instrument main body 111, and a battery or the like can be inserted through a battery cover 112. On the surface of the main body 111, a display unit 114 such as an LCD for displaying a measured value of urine sugar is provided.

The sensor body 113 has the biosensor 100 built in the tip of the sensor holder 119, and when urine is applied to the biosensor 100, the sugar level in the urine is automatically displayed on the display unit 11.
4 will be displayed.

[0009] As shown in FIG. 9, the biosensor 100 has a working electrode 102 and a counter electrode 103 formed on an insulating substrate 101, on which a permselective membrane 104, an immobilized enzyme membrane 105,
Polycarbonate porous films 108 are sequentially laminated.
Here, the polycarbonate porous membrane acts as the restricted permeation membrane 108, and the measurement range of the blood glucose level and the like is 0 to 500 mg / d.
l.

[0010]

However, the conventional method cited has the following problems. First, the film used as the restricted transmission membrane 108 is expensive. Since this film is produced by irradiating a neutron beam to the polycarbonate film and chemically etching a deteriorated portion changed by the neutron beam to form a predetermined through-hole, the film cannot be as inexpensive as a normal resin material. A second problem is that a step of attaching a film to each sensor is required. Such an operation requires more man-hours than a normal wafer film forming process, and it is particularly difficult to select an adhesive, which also increases costs. A third problem is that it is not possible to easily change the film parameters such as the diameter of the through-hole and the aperture ratio. Film production requires large-scale equipment and makes it difficult to produce many kinds and small quantities.

SUMMARY OF THE INVENTION The present invention has been made to solve the above-mentioned problems, and the problem to be solved by the present invention is to make it possible to easily set the aperture ratio of a restricted permeation membrane used in a biosensor by a simple process, Concentration is 0-5000m
It is intended to provide a biosensor that does not saturate to about g / dl and a method for manufacturing the biosensor.

[0012]

According to the first aspect of the present invention, a biomembrane having a permselective membrane 4 below a reaction layer (immobilized enzyme membrane) 5 and a restricted permeation membrane 8 outside the reaction layer 5 is provided. The sensor 10 includes a plurality of minute projections 6 formed of a photosensitive material using a non-organic solvent as a stripping liquid on a base layer of a restricted transmission film 8, and a plurality of projections 6 provided around the plurality of projections 6. A biosensor comprising a water-impermeable membrane 7, wherein the plurality of projections 6 are removed to form a restricted permeable membrane 8 having a plurality of small openings with the water-impermeable membrane remaining. It was done.

The invention according to claim 2 is a biosensor 10
Is a biosensor of the current detection type, wherein the biosensor according to claim 1 is provided.

The invention according to claim 3 is a biosensor 10
Is a glucose sensor, wherein the biosensor is a biosensor according to claim 1.

The invention according to claim 4 is a biosensor 10.
Is a glucose sensor comprising a hydrogen peroxide electrode and a glucose oxidase, which is a biosensor according to claim 1.

The invention according to claim 5 is a biosensor 10
Is a biosensor using an ion-sensitive field-effect transistor as a transducer.

According to a sixth aspect of the present invention, there is provided a method for manufacturing a biosensor having a permselective membrane below a reaction layer and a restricted permeation membrane outside the reaction layer, the method comprising: Forming a plurality of minute projections using a photosensitive material using a non-organic solvent as a removing liquid, forming a water-impermeable film around the projections, and removing the projections A method for producing a biosensor, characterized in that:

According to a seventh aspect of the present invention, there is provided the biosensor manufacturing method according to the sixth aspect, wherein the stripping solution for the photosensitive material is an aqueous sodium hydroxide solution.

The invention according to claim 8 is characterized in that the water-impermeable membrane has a thickness of 80%.
The method for producing a biosensor according to claim 6, wherein the resin is a resin curable at a temperature of not more than ° C.

According to a ninth aspect of the present invention, there is provided the method for manufacturing a biosensor according to the sixth aspect, wherein the water-impermeable membrane contains any one of silicone, polyurethane, polyacrylic acid and fluororesin as a main component. Things.

According to a tenth aspect of the present invention, there is provided a method of manufacturing a biosensor according to the sixth aspect, wherein the projection 6 has a columnar or conical shape.

The invention according to claim 11 is characterized in that, in the step of exposing the photosensitive material, overexposure or underexposure is performed, and the projection 6 is made thinner than when exposed under regular conditions. Item 7. A method for producing a biosensor according to Item 6.

According to a twelfth aspect of the present invention, there is provided the biosensor manufacturing method according to the sixth aspect, wherein the diameter of the projection 6 is 1 μm or less.

According to a thirteenth aspect of the present invention, there is provided the biosensor manufacturing method according to the sixth aspect, wherein the aperture ratio of the restricted permeable membrane 8 is 0.01 to 10%.

The invention according to claim 14 is the biosensor 1
7. A method for manufacturing a biosensor according to claim 6, wherein a plurality of 0s are simultaneously formed on the same substrate 14.

[0026]

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS Hereinafter, a biosensor and a method of manufacturing the biosensor according to the present invention will be described in detail with reference to FIGS.

FIG. 1A is a plan view of a biosensor showing one embodiment of the present invention, and FIG. 1B is a sectional view taken along the line AA ′ of FIG.
It is a sectional arrow view. 1A and 1B, a substantially square working electrode 2 and a counter electrode 3 are formed on an insulating substrate 1 and are connected to substantially square electrode pads 12A and 12B. Next, the permselective film 4 is laminated. The permselective membrane 4 is provided for the purpose of excluding high molecular weight molecules only through H ₂ O _{2 to be} detected. Above this, an immobilized enzyme film 5 is formed. A projection 6 made of a photoresist using a non-organic solvent as a stripping liquid is formed thereon.

Here, the reason why a photoresist using a non-organic solvent as a stripping solution is used is as follows. Selective permeable membrane 4
For example, cellulose, an ion exchange resin or the like is often used. These materials generally dissolve in organic solvents. Therefore, if a photoresist using an organic solvent as a stripper is used, the permselective film 4 will be dissolved at the time of stripping.
Therefore, a photoresist using a non-organic solvent as a stripper must be used. As a material satisfying this requirement, there is a solder resist used for a printed wiring board and the like, which can be obtained at a low price.

A non-permeable membrane 7 is formed around the projection 6 of the permselective membrane 4. The non-permeable membrane 7 is desirably one that can be cured at room temperature. This is because many of the enzymes used in biosensors are deactivated at high temperatures and lose their catalytic function. The heat resistance temperature of GOX which is relatively resistant to heat is about 80 ° C. Suitable resins for the water-impermeable membrane 7 include silicone, polyurethane, polyacrylic acid, and fluororesin. After the water-impermeable membrane 7 has hardened, the projections 6 are removed with a stripping solution to remove
Is completed.

FIG. 1B shows a cross-sectional view of the biosensor 10 from which the single protrusion 6 has been removed. However, in actuality, the plan view of FIG. 3A and the cross-sectional view of FIG. ′ As shown in FIG.
4, working electrode 2 and counter electrode 3 and electrode pads 12A, 1
Selectively permeable membrane 4 on a plurality of biosensor elements composed of 2B
After integrally forming a plurality of biosensor elements in a state where the immobilized enzyme membrane 5 and the impermeable membrane 7 are sequentially formed, the single biosensor 10 is cut into a five-meshed board.
A single biosensor 10 is obtained as shown in FIGS.

FIGS. 2A to 2F show FIGS.
FIG. 3B is a schematic cross-sectional view illustrating a step of the biosensor 10 of FIG. Hereinafter, description will be made in the order of the steps. In FIG. 2A, a conductive film is formed on the insulating substrate 1 by vapor deposition or the like, and the working electrode 2 and the counter electrode 3 are formed by photolithography. FIG.
In (B), the permselective film 4 is formed on the entire surface by spin coating or the like. In FIG. 2C, an immobilized enzyme film 5 is formed on the entire surface by spin coating or the like, and a photoresist 9 using a non-organic solvent as a stripping solution is formed on the entire immobilized enzyme film 5, and then FIG. In ()), the photoresist 9 is exposed and developed.
A large number of minute projections 6 are formed. FIG. 2 (E)
Then apply the water-impermeable resin solution by spin coating, etc.
After drying, the water-impermeable film 7 is formed around the protrusion 6. In FIG. 2F, the protrusion 6 is removed using a stripping solution. In this manner, the restricted permeable membrane 8 having the minute through holes 11 and made of the water-impermeable material is formed.

One embodiment of the current detection type biosensor shown in the embodiment of FIGS. 1 to 3 will be described below.

Example 1 A glass plate was used as the insulating substrate 14 shown in FIGS.
The size of the biosensor element is 100 mm, and platinum (Pt) is vapor-deposited on the glass plate, and 100 pairs of biosensor elements in which the working electrode 2, the counter electrode 3, and the electrode pads 12A and 12B are patterned by a lift-off method or a milling method are formed. The conductive film may be gold (Au) or silver (Ag).

Next, an alcohol solution of an ion exchange resin (trademark, Nafion) is applied to the entire surface by spin coating and dried at room temperature to obtain a selective oxide film 4.

Next, a mixed solution of bovine serum albumin, glucose oxidase and glutaraldehyde is applied to the entire surface,
After drying at room temperature, an immobilized enzyme membrane 5 is formed.

Next, as shown in FIG. 2C, a photoresist 9 in which a stripping solution is a non-organic solvent is applied, and this photoresist 9 is exposed and developed to form a projection 9 as shown in FIG. To form

The pattern of the mask for exposing and developing the photoresist 9 is a circle having a diameter of 1 μm and a square of 10 μm.
The pieces were placed on the entire surface, that is, at a density of 10 ⁸ / cm ² . A 1% aqueous solution of sodium carbonate (Na ₂ CO ₃ ) was used as a developer. Next, a 1 μm-thick silicone resin solution was applied to the entire surface by spin coating. Next, the protrusion 6 was removed using a 4% aqueous solution of sodium hydroxide (NaOH). In this way, a restricted permeable membrane 8 having a large number of through holes 6 having a diameter of 1 μm was formed. In photolithography using ultraviolet light, the limit of patterning is about 0.1 μm and 1 μm
It is relatively easy to make a pattern. The aperture ratio of the thus obtained restricted transmission film 8 is less than 1%. Since the measurement range of urine sugar and the like is inversely proportional to the aperture ratio, in this case, the measurement range is designed to be about 100 times the original. Next, the insulating substrate 14 was divided to obtain 100 glucose sensors 10.

The glucose sensor 10 was evaluated using a potentiostat (constant voltage stabilized power supply). In the evaluation using a phosphate buffer containing glucose, a linear calibration curve was obtained in a glucose concentration range of 0 to 5000 mg / dl. Next, serum containing 500 mg / dl glucose was repeatedly measured 10 times, and the coefficient of variation (standard deviation / mean) was 0.3%. Then 2000mg /
When urine containing dl glucose was similarly measured, the coefficient of variation was 10%, and the magnitude of the output current was reduced by 10% in 10 measurements.

In the first embodiment, the silicone resin is used as the water-impermeable membrane 7. However, any resin that can be cured at 80 ° C. or lower may be used. For example, a polyurethane resin, a polyacrylic acid resin, a fluororesin, or the like may be used. I can do it.

The aperture ratio of the through-hole 11 formed on the restricted transmission film 8 will be described with reference to FIGS.
FIG. 4A schematically shows a through hole 11 on a working electrode 2 formed in an insulating substrate 1 used in a biosensor 10 of another embodiment described later. The aperture ratio may be controlled. Here, the aperture ratio indicates the ratio of the circular area of the working electrode to the total area of all the through holes.
FIG. 4B shows the saturation characteristic curve when the output current obtained between the working electrode 2 and the counter electrode 3 of the current detection type biosensor is plotted on the vertical axis, and the glucose concentration is plotted on the horizontal axis 7.
The saturation characteristic curve 22 in FIG. 4 (B) is a saturation characteristic curve when the restricted permeation film 8 is not provided, and 23 is a saturation characteristic curve when the polycarbonate porous film shown in FIG. In the first embodiment of the present invention, the aperture ratio is 1%.
The diameter of the through hole 11 of the restricted transmission film 8 and the design of the aperture ratio are appropriately selected by selecting the pattern of the mask used at the time of exposing and developing the photoresist 9 and setting the diameter to 1 μm.
m or less (not including 0), when the opening ratio is selected from 0.01 to 10%, the saturation point of the glucose concentration is set to 5 at the time of blood glucose measurement.
0-500 mg / dl, and 0-20 when measuring urinary sugar level
A characteristic saturation curve 25 having a linear calibration curve can be easily and freely obtained up to 00 mg / dl (characteristic saturation curve 24) or a glucose concentration of 5000 mg / dl or more.

FIGS. 5A and 5B show another embodiment of the present invention. The difference from FIGS. 1A and 1B is that
A semicircular (2 mmφ) working electrode 2 patterned on the insulating substrate 1 with t, Ag, Au, or the like so as to surround the working electrode 2 from the left and right, a semilunar counter electrode 3, a reference electrode (reference electrode) 13, and an electrode pad 12 A, 12B and 12C are provided.
5 (A) and 5 (B), the reference electrode 13 is also used as the counter electrode 3, but in FIGS. 5 (A) and 5 (B), an independent reference electrode 13 is provided to improve measurement accuracy. It is.

The selective permeable membrane 4, the immobilized enzyme membrane 5, the water-impermeable membrane 7, and the restricted permeable membrane 8 composed of the pores 11 coated on the working electrode 2, the counter electrode 3 and the reference electrode 13 are shown in FIG. ,
It is configured similarly to (B).

In FIGS. 1A and 1B, (1) and (2)
According to the reaction formula, glucose as a substrate is GOX.
Although the amount of O ₂ consumed by reacting with the enzyme is measured by current conversion, it is also possible to use an ion-sensitive transistor (ISFET) to be described later or the like. As the enzyme, alcohol dehydrogenase other than glucose can be used. Penicillinase and the like can be used. That is, the biosensor and the biosensor manufacturing method of the present invention can be applied to a biosensor other than the glucose sensor.

Further, another embodiment of the biosensor and the method of manufacturing the biosensor according to the present invention will be described with reference to FIGS. 6 (A) and 6 (B).

6A and 6B, the configuration is the same as that of the biosensor of FIGS. 1 and 2 except for the formation of the projection 6. FIG. After forming the immobilized enzyme film 5, a photoresist 9 using a non-organic solvent as a stripping solution is applied to the entire surface. At this time, it is desirable that the thickness of the resist be thinner than in the case of FIG. Next, as shown in FIG. 6A, the resist 9 is exposed from the light source 14 using a predetermined mask 16. When a negative resist is used, the exposure amount at this time is reduced by several tens of percent from the normal condition. Next, the projection 6 formed by developing the photoresist 9 is formed. By doing so, the mask 1
Thus, the protrusion 6 having a diameter smaller than that of the pattern 17 of FIG.

FIG. 6A shows the pattern 1 on the mask 16.
Assuming that the diameter of 7 is 1 μm, a columnar projection 6 of −1 μm of 1 μm is formed.

Conversely, when a positive resist is used as shown in FIG.
Similar results are obtained. That is, if the diameter of the pattern 17 of the transmission part on the mask 16 is 1 μm, the projection 6 having a diameter smaller than the diameter of the transmission part can be obtained by over-exposure. In the case of FIG. 6B, the protrusion 6 is formed in a conical shape.

Example 2 In the same manner as in Example 1, up to the immobilized enzyme membrane 5 was formed. Next, a negative photoresist 9 was applied with a thickness of 0.5 μm. The mask pattern was the same as in Example 1. Next, the exposure amount was set to 20
The photoresist 9 was exposed at a reduced percentage. When this was developed, a protrusion 6 having a diameter of 0.7 μm was obtained. Then, a 0.5 μm thick silicone solution is used as a water-impermeable membrane 7.
And dried at room temperature. Next, 4% N
The protrusion 6 was removed using an aOH aqueous solution.

The glucose sensor 10 was evaluated using a potentiostat. In the evaluation using a phosphate buffer containing glucose, the glucose concentration was 0 to 8000 mg.
A linear calibration curve was obtained in the range of / dl. Then 500
When serum containing mg / dl glucose was repeatedly measured 10 times, the coefficient of variation was 0.3%. Then 20
When urine containing 00 mg / dl glucose was similarly measured, the coefficient of variation was 5%, and no systematic decrease in output current was observed. The reason that the reproducibility was better than that of Example 1 is that the contaminants (proteins and the like) penetrated into the immobilized enzyme membrane 5 due to the smaller pore size of the perforated hole 11 in the restricted permeation membrane 8. It seems to have been prevented. It is understood that the mask may be changed to further reduce the hole diameter of the through hole 11 in order to further increase the accuracy.

FIG. 7 shows an insulating substrate 1 as an ISFET (Ion Sens) as a chemical sensor to which the manufacturing method of the present invention can be applied.
FIG. 1 is a schematic side sectional view of a biosensor 10A which is a field effect transistor (FET) 2 as a negative FET.
1 is a permselective membrane 4, an immobilized enzyme membrane 5, and a water-impermeable membrane 7 in which a permeation hole 11 is formed in place of a gate provided between a source 18 and a drain 19 on an insulating substrate 20 such as silicon. 8 are sequentially formed.

The ISFET is different from the current detection type biosensor 10 in that the electrochemical potential change is detected by the FET 21.
When the sample (aqueous solution) is immersed in the biosensor 10, the potential of the surface of the FET 21 changes via the ion-sensitive layer such as the immobilized enzyme membrane 5. Utilizing the fact that it is proportional to the value, the hydrogen ion concentration (PH) is measured.

In this case, the restricted permeable membrane 8 is also
Thus, a biosensor and a method for manufacturing a biosensor can be obtained in which the opening ratio of the through hole 11 formed in the biosensor can be easily controlled by photolithography using an inexpensive photosensitive material.

[0053]

As described above, according to the present invention, a biosensor having a wide measurement range can be obtained easily and inexpensively.
In addition, since the pore size and the aperture ratio of the restricted permeable membrane can be freely designed, it is possible to manufacture a biosensor and a biosensor having arbitrary characteristics without increasing the cost.

[Brief description of the drawings]

FIG. 1 is a schematic plan view and a cross-sectional view illustrating an embodiment of the present invention.

FIG. 2 is a schematic cross-sectional view schematically showing the process of FIG.

FIGS. 3A and 3B are a plan view and a schematic cross-sectional view illustrating a configuration in which a biosensor of the present invention is obtained at the same time.

FIG. 4 is an explanatory diagram of an aperture ratio on a working electrode of a biosensor used in the present invention and an explanatory diagram of a saturation characteristic curve showing a relationship between a glucose concentration and a detection output current of the biosensor.

FIG. 5 is a front view and a schematic sectional view showing another embodiment of the present invention.

FIG. 6 is a sectional side view of a main part showing a method for producing a restricted permeable membrane of the biosensor of the present invention.

FIG. 7 is a schematic sectional view showing another embodiment of the biosensor of the present invention.

FIG. 8 is an external view of a conventional urine sugar measuring device.

FIG. 9 is a schematic sectional view showing an example of a conventional biosensor.

[Explanation of symbols]

1,14,20 ‥‥ insulating substrate, 2,102 ‥‥ working electrode, 3,103 ‥‥ counter electrode, 4,104 ‥‥ selective permeable membrane,
5,105 ‥‥ immobilized enzyme membrane, 6 ‥‥ protrusion, 7 ‥‥ impermeable membrane, 8 ‥‥ restricted permeability membrane, 9 ‥‥ photoresist,
11 ‥‥ through-hole, 12A, 12B, 12C ‥‥ electrode pad, 13 ‥‥ reference electrode, 21 ‥‥ FET, 108 ‥‥ polycarbonate porous membrane

 ──────────────────────────────────────────────────の Continued on the front page (72) Inventor Satoru Ikeda 2-15-12 Nakane, Meguro-ku, Tokyo Inside Tama Electric Industry Co., Ltd.

Claims (14)

[Claims] 1. A biosensor having a permselective membrane below a reaction layer and a restricted permeation membrane outside the reaction layer, wherein a non-organic solvent is stripped on an underlayer of the restricted permeation membrane. A plurality of minute projections provided by a photosensitive material, and a water-impermeable film provided around the plurality of projections. The water-impermeable film is formed by removing the plurality of projections. A biosensor comprising the above-described restricted permeation membrane having a plurality of small openings except for the above. 2. The biosensor according to claim 1, wherein the biosensor is a current detection type biosensor. 3. The biosensor according to claim 1, wherein said biosensor is a glucose sensor. 4. The biosensor according to claim 1, wherein said biosensor is a glucose sensor comprising a hydrogen peroxide electrode and glucose oxidase. 5. The biosensor according to claim 1, wherein said biosensor is a biosensor using an ion-sensitive field-effect transistor as a transducer. 6. A method for producing a biosensor having a permselective membrane below a reaction layer and a restricted permeation membrane outside the reaction layer, wherein a non-organic solvent is provided on an underlayer of the restricted permeation membrane. Forming a plurality of minute projections using a photosensitive material that uses as a stripping liquid; forming a water-impermeable film around the projections; and removing the projections. A method for producing a biosensor. 7. The method for manufacturing a biosensor according to claim 6, wherein the stripping solution for the photosensitive material is an aqueous solution of sodium hydroxide. 8. The method according to claim 6, wherein the water-impermeable membrane is a resin curable at 80 ° C. or lower. 9. The method for manufacturing a biosensor according to claim 6, wherein the water-impermeable membrane contains any of silicone, polyurethane, polyacrylic acid, and fluororesin as a main component. 10. The method for manufacturing a biosensor according to claim 6, wherein the protrusion has a columnar shape or a conical shape. 11. The method according to claim 6, wherein, in the step of exposing the photosensitive material, overexposure or underexposure is performed so that the protrusion is thinner than when exposed under normal conditions. Manufacturing method of biosensor. 12. The method according to claim 6, wherein the diameter of the protrusion is 1 μm or less. 13. An aperture ratio of the restricted permeation membrane of 0.01 to 0.01.
The method for producing a biosensor according to claim 6, wherein the content is 10%. 14. The method of manufacturing a biosensor according to claim 6, wherein a plurality of said biosensors are simultaneously produced on the same substrate.