JP2001021527A - Throw-away biosensor - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a throw-away biosensor capable of simplification of the operation at the time of measurement and shortening of the measuring time, and having a reactive reagent and a biocatalyst immobilized equally with excellent preservation stability. SOLUTION: This throw-away biosensor has an insulating substrate 3, two or three conductors fixed on the same plane of the substrate 3, a biocatalyst immobilizing layer 8 formed on one or more conductors among the conductors, a closed-system cell 6 fixed on the plane of the substrate 3 so as to form a sample liquid holding part 12 and having a sample liquid injection opening 4, and a reactive reagent immobilizing layer 7 formed on the inner surface of the cell 6 so as to be out of contact with the biocatalyst immobilizing layer 8. As the reactive reagent and the biocatalyst are equally immobilized with excellent preservation stability, the measurement operation of the sample liquid becomes simple and the measuring time can be shortened.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a disposable biosensor, and more particularly, to a disposable biosensor capable of simplifying a measurement operation in a sample liquid measurement and shortening a measurement time.

[0002]

2. Description of the Related Art Conventionally, biosensors have mainly adopted a method of continuously or repeatedly measuring a target chemical substance using a set of sensor elements. A biocatalyst is often immobilized on the sensor element. Therefore, the sensor element once used for the measurement is washed manually or by an automatic device, and the next measurement operation is started after the catalyst activity is restored.

On the other hand, in a field in which irregular or intermittent measurement that does not require continuous or regular repetitive measurement is required, measurement with one set of sensor elements is performed in consideration of storage and maintenance of sensor elements. A so-called one-time-use disposable biosensor has been developed. For example, a blood sugar level sensor in the medical field employs a disposable enzyme sensor element and realizes easy operability and maintenance-free, thereby enabling measurement at an individual level.

By the way, the method which is considered to be the easiest for the user in the measurement method of the disposable biosensor is to perform only the operation of dipping the sensor element in the liquid to be measured or dropping the liquid to be measured onto the sensor element. A method that can obtain the expected results after a certain period of time, specifically, requires complicated and time-consuming processing work such as addition of biocatalysts and preparation of reaction reagents that are performed as a pretreatment step of the measurement operation. And no way. Therefore, development of a biosensor in which all necessary biocatalysts and reaction reagents are quantitatively arranged in advance has been desired.

[0005] However, biocatalysts are usually unstable, and are often handled during storage, in that the enzyme may lose its activity during storage or may die if it is a microorganism or animal or plant cells. difficult. In particular, living organisms such as microorganisms, animal and plant cells react sensitively to drying, temperature changes, or coexistence with reaction reagents, and are liable to be rapidly inactivated or killed. Even in the case where deactivation or death does not occur, an operation for restoring the activity or a restoring time is often required until the original catalyst activity is exhibited.

Taking microbial cells as an example, it is technically possible to preserve microorganisms for a long period of time by freeze-drying or the like, but the substrate activity as a biocatalyst of the sensor is immediately restored at the time of measurement. It does not mean that a sufficient reaction can be obtained. In general, it takes one to two or more days for the microorganisms in a dry storage state to regain the original bioactive function. Therefore, in this case, it is desirable to store the microorganisms in an aqueous solution or in a wet state.

[0007] On the contrary, in the case of the reaction reagent, it is often possible to store it stably for a long time in the dry state. Therefore, in order to obtain a biosensor that can be measured with simple operations without complicated operations involved in measurement, a disposable biosensor in which both biocatalysts and reaction reagents having different properties described above are immobilized with good storage stability. The development of was desired.

[0008]

DISCLOSURE OF THE INVENTION The present invention has been made from the above-mentioned point of view, and enables both a reaction reagent and a biocatalyst to have a good storage stability so that the operation at the time of measurement can be simplified and the measurement time can be shortened. It is an object to provide an immobilized disposable biosensor.

[0009]

Means for Solving the Problems In order to solve the above problems, the present inventors dispose a biocatalyst and a reaction reagent separately in a disposable biosensor in consideration of their respective properties. Therefore, the present invention has the following configuration.

(1) an insulating substrate; two or three conductors fixed on the same plane of the substrate; a biocatalyst fixing layer provided on at least one of the conductors; A closed cell having a sample liquid inlet fixed so as to form a sample liquid holding portion on the flat surface, and a reaction reagent provided on the inner surface of the cell so as not to contact the biocatalyst immobilization layer. Disposable biosensor having an immobilization layer.

(2) a flat insulating substrate, two or three conductors fixed on the same plane of the substrate so as not to contact each other, and at least one of the two or three conductors A biocatalyst-immobilized layer provided, wherein a spacer is fixed on a surface of the substrate having a conductor and a biocatalyst-immobilized layer, and a sample liquid inlet is provided on the spacer. A cover having a reaction reagent immobilization layer on one side thereof is fixed so that the surface having the reaction reagent immobilization layer faces the surface of the substrate having the conductor and the biocatalyst immobilization layer, and the substrate A disposable biosensor comprising a sample liquid holding portion surrounded by the surface having the conductor and biocatalyst immobilization layer, the inner wall of the spacer, and the surface having the reaction reagent immobilization layer of the cover. (3) The disposable biosensor according to (2), wherein the cover further has an air hole.

(4) A flat insulating substrate, two or three conductors fixed on the same plane of the substrate so as not to contact each other, and at least one of the two or three conductors And a biocatalyst-immobilized layer provided, wherein a spacer having a sample liquid inlet and a reaction reagent are immobilized on the surface of the substrate having the conductor and the biocatalyst-immobilized layer. A mesh is laminated to form a sample liquid holding portion surrounded by the surface of the substrate having the conductor and the biocatalyst immobilization layer, the inner wall of the spacer, and the lower surface of the mesh on which the reaction reagent is immobilized. Disposable biosensor.

(5) The disposable biosensor according to any one of (1) to (4), wherein the biocatalyst-immobilized layer comprises a mesh on which the biocatalyst is immobilized.

(6) The reaction reagent is a buffer, a storage stabilizer,
The disposable biosensor according to (1) to (5), which is selected from a standard substance, a redox indicator, and a mediator for preparing a calibration curve.

(7) The disposable biosensor according to (1) to (6), wherein the biocatalyst is selected from microorganisms, animal cells and plant cells.

[0016]

BEST MODE FOR CARRYING OUT THE INVENTION A disposable biosensor according to the present invention comprises an insulating substrate and a substrate fixed on the same plane of the substrate.
To three conductors, a biocatalyst fixing layer provided on at least one of the conductors, and a sample liquid inlet fixed to form a sample liquid holding part on the flat surface of the substrate And a reaction reagent-immobilized layer provided on the inner surface of the cell so as not to contact the biocatalyst-immobilized layer.

As a specific form of such a disposable biosensor of the present invention, a flat insulating substrate and two or three substrates fixed on the same plane of the substrate so as not to contact each other.
A biocatalyst-immobilizing layer provided on at least one of the two or three conductors, wherein the conductor and the biocatalyst-immobilizing layer of the substrate are provided. A spacer having a sample liquid inlet and a cover having a reaction reagent immobilization layer on one surface is provided on the spacer, and the surface having the reaction reagent immobilization layer is a conductor of the substrate. And fixed so as to face the surface having the biocatalyst immobilization layer, and surrounded by the surface of the substrate having the conductor and biocatalyst immobilization layer, the inner wall of the spacer, and the surface of the cover having the reaction reagent immobilization layer. A disposable biosensor (hereinafter, also referred to as a "disposable biosensor of the first embodiment of the present invention") characterized by forming a sample liquid holding portion, and a flat insulating substrate; Base Disposable type comprising: two or three conductors fixed on the same plane so as not to contact each other; and a biocatalyst fixing layer provided on at least one of the two or three conductors. A biosensor, wherein a spacer having a sample liquid inlet and a mesh on which a reaction reagent is immobilized are laminated on a surface of the substrate having a conductor and a biocatalyst immobilization layer, and the conductor and biocatalyst immobilization of the substrate are performed A disposable biosensor (hereinafter, referred to as a biosensor) characterized in that a sample liquid holding portion surrounded by a surface having a functionalized layer, an inner wall of a spacer, and a lower surface of a mesh on which a reaction reagent is immobilized is formed.
"Disposable biosensor of the second embodiment of the present invention"
And so on).

Hereinafter, the disposable biosensor of the first embodiment of the present invention will be described, and then the disposable biosensor of the second embodiment of the present invention will be described.

(1) Disposable Biosensor of the First Embodiment of the Present Invention The insulating substrate used for the disposable biosensor of the first embodiment of the present invention has a flat plate shape, and the biosensor can withstand use. The material is not particularly limited as long as the material and size can support the entire biosensor so as to have a sufficient strength.
Examples of the material constituting the insulating base material include resins such as polyester and glass epoxy resin, glass, and paper that has been surface-treated so that the sample solution does not penetrate.

In the disposable biosensor according to the first embodiment of the present invention, two or three conductors are fixed on the same plane of the substrate so as not to contact each other.
A biocatalyst fixing layer is provided on at least one of the three conductors.

In the case of two conductors, one is a conductor acting as a working electrode and the other is a conductor acting as a counter electrode when measuring a sample solution using the biosensor of the present invention. When the number of conductors is three, two are conductors serving as a working electrode and a counter electrode, and the other one is a conductor serving as a reference electrode. At least one conductor provided with the biocatalyst fixing layer is a conductor serving as a working electrode.

The working electrode is applied with a voltage when qualitatively and quantitatively measuring a substance to be measured in a sample using a biosensor, and electrically detects a change due to a biocatalytic reaction in the sample to be measured. The electrode and counter electrode are electrodes provided to generate a current between the working electrode and the reference electrode, and the reference electrode is an electrode serving as a reference for setting the potential of the working electrode. That is, each electrode plays a different role when used as a biosensor, but the configuration of the electrode itself is similar in that it is a conductor. Also, in general, the counter electrode and the reference electrode can be shared, so that
Also in the present invention, one conductor may have a role of both a counter electrode and a reference electrode as needed.

As the conductor, a conductor which is stable, has high conductivity, and is substantially harmless to a biocatalyst;
For example, platinum, gold, metal such as silver, or graphite,
And carbon materials such as carbon. Depending on the conductor used and the material and shape of the insulating substrate, the conductors are usually fixed on the same plane of the insulating substrate so as not to contact each other, for example, by a method such as vapor deposition, sputtering, or screen printing.

In the biosensor according to the first embodiment of the present invention, the biocatalyst fixing layer provided on at least one of the two or three conductors (working electrode) may be a biocatalyst in a normal method. Are fixed in layers.
When the biocatalyst is immobilized on the working electrode, usually, the biocatalyst is not immobilized on the conductor serving as the counter electrode or the reference electrode. However, in some cases, from the viewpoint of workability, all of the above two or three conductors are immobilized. The biocatalyst may be immobilized so as to cover the surface.

In the disposable biosensor according to the first embodiment of the present invention, a spacer is fixed on the surface of the substrate having the conductor and the biocatalyst fixing layer as described later. Therefore, the biocatalyst-immobilized layer is
Even when the biocatalyst fixing layer is provided on the insulating substrate so as to cover all the three conductors, it is usually preferable that the biocatalyst fixing layer does not reach the spacer fixing portion of the insulating substrate. Specifically, the spacer is formed on the insulating substrate by
Since it is often fixed to a portion near the outer edge of the insulating substrate so as to surround a range covering all three conductors, it is preferable to form a biocatalyst fixing layer inside the fixed portion.

Examples of the biocatalyst immobilized on the conductor include, but are not limited to, biocatalysts used in ordinary biosensors. Specific examples include glucose oxidase, glucoamylase, galactose oxidase, alcohol oxidase, tyrosinase, and the like. Catechol 1,
Enzymes such as 2-oxidase, pyruvate oxidase, uricase, amino acid oxidase, glutaminase, glutamate dehydrogenase, asparakinase, amine oxidase, cholesterol esterase, lipase, phospholipase, catalase, creatinase, phosphatase, urease; Gluconobacter, Pseudomonas, Methylomonas, Hansenula, Candida, Desulfovibrio, Thiobacillus, Clostridium, Methylococcus, Arthrobacter, Mycobacterium, Lactobacillus or Brevibacterium Prokaryotic microorganisms such as bacteria, actinomycetes,
Microorganisms such as eukaryotic microorganisms such as yeast belonging to the genus Trichosporon; organelles; antigens; antibodies;
Plant cells; animal tissues, plant tissues and the like.

Further, among the disposable biosensors according to the first embodiment of the present invention in which the biocatalyst is immobilized, the biocatalysts such as living organisms such as microorganisms, animal cells, and plant cells are used.
In particular, it can be said that the configuration of the biosensor of the present invention is effective for a biosensor in which a biocatalyst that reacts sensitively to drying, temperature change, or coexistence with a reaction reagent during storage is immobilized.

When the biocatalyst is immobilized on the conductor, the biocatalyst may be immobilized by any method similar to the immobilization method used for immobilizing the biocatalyst at the time of manufacturing a normal biosensor. it can. Specifically, a biocatalyst is encapsulated and immobilized in a gel matrix such as alginate gel, carrageenan gel, agarose gel, curdlan gel, chitosan gel, or a photo-curable resin such as photo-crosslinkable polyvinyl alcohol, or a polymer. Examples of the method include immobilization by incorporating into a three-dimensional crosslinked structure such as acrylamide. Alternatively, the biosensor may be immobilized on a biocatalyst using glutaraldehyde or the like so as to be electrically connected to a conductor as an electrode on the surface of the substrate constituting the biosensor. Furthermore, it is also possible to fix the biocatalyst by combining these methods.

In order to immobilize the biocatalyst on the electrode substrate in a manner that the biocatalyst is contained in the various polymer structures, the biocatalyst is preferably mixed with each of the polymer raw material liquids as a suspension. The resulting material is applied to a desired range on the electrode substrate plane in an appropriate thickness by an appropriate method, and then solidified and cured by a method suitable for each polymer.

For fixation with glutaraldehyde, a suspension containing a biocatalyst is applied to a desired area on the flat surface of the electrode substrate by an appropriate method, and then exposed to vapor of an aqueous solution of glutaraldehyde having an appropriate concentration. Can immobilize the biocatalyst.

The thickness of the biocatalyst-immobilized layer in the biosensor of the first embodiment of the present invention is not particularly limited, but is preferably about 10 μm to 200 μm.

Among biocatalysts, those which do not like to be dried during storage, such as living organisms such as microorganisms, animal cells, plant cells, etc., are immobilized by a method in which the biocatalyst is not dried after immobilization. Immobilization method that can be stored in, for example,
Alginate gel, carrageenan gel, agarose gel,
An entrapment immobilization method using curdlan gel, photocrosslinkable polyvinyl alcohol, or the like is preferably used.

Further, in the biosensor according to the first embodiment of the present invention, a mesh on which a biocatalyst is immobilized (hereinafter sometimes referred to as “biocatalyst immobilized mesh”) is used as the biocatalyst immobilizing layer. It is also possible and preferred.

Such a biocatalyst-immobilized mesh is prepared, for example, by blending the above-mentioned biocatalyst, preferably as a suspension, with each of the above polymer raw material liquids on a mesh of an appropriate mesh size (hereinafter referred to as “biocatalyst”). An immobilized raw material) may be supplied in an appropriate amount, and the supplied biocatalyst immobilized raw material may be supplied using a squeegee having an appropriate size, for example, a squeegee including a rubber blade, a Teflon blade, a urethane blade, or the like. In the same manner as in the squeezing process in screen printing, the biocatalyst-immobilized raw material is stretched over the entire mesh so as to spread over the minute space inside the mesh, and then solidified and cured by a method suitable for each polymer. It can be produced by causing

As the material of the mesh used, for example, polyester, polyamide, cellulose, silk,
In addition, various natural / synthetic materials capable of forming fibers can be used. The thickness of the mesh is not particularly limited,
Preferably about 50 to 200 μm, more preferably about 70 to 200 μm.
About 150 μm. The mesh size and mesh shape are not particularly limited as long as the biocatalyst-immobilized raw material to be used can penetrate into a fine space inside the mesh. The mesh size depends on the type and concentration of the biocatalyst-immobilizing liquid used, but is generally about # 30 to # 30.
It is preferably about 200, and more preferably about # 80 to # 150.

The treatment for immobilizing the biocatalyst on the mesh as described above may be performed on the substrate after the mesh is fixed on the substrate in advance so as to cover at least the conductor serving as the working electrode. However, it is preferably performed at a different location than on the substrate. This is because if the biocatalyst-immobilized mesh is made in a different place from the substrate, it is possible to produce a uniform biocatalyst-immobilized mesh over a large area at one time, and cut it into the required size to obtain multiple meshes. This is because a plurality of biosensors with little variation among the biosensors can be efficiently manufactured by using the biosensors.

When such a biocatalyst-immobilized mesh is used as the biocatalyst-immobilized layer, the size and shape of the biocatalyst-immobilized mesh are adjusted so that at least the conductor serving as the working electrode is covered on the insulating substrate. Any size and shape that can be fixed to portions other than on the conductor may be used. Preferably, it has a size and a shape that can be fixed to a portion other than the conductors on the insulating substrate so as to cover all of the two or three conductors on the insulating substrate. Further, when using a biocatalyst immobilized mesh as the biocatalyst immobilization layer, the size and shape thereof, such as the fixed portion on the substrate of the spacer fixed on the insulating substrate as described later, such as, The shape and size may be used.

Specifically, the spacer is often fixed to a portion near the outer edge of the insulating substrate so as to surround the area covering all of the above-mentioned two or three conductors on the insulating substrate. Alternatively, the size of the biocatalyst-immobilized mesh may reach the outer edge of the spacer.

As a method of fixing the biocatalyst-fixed mesh or the mesh for biocatalyst fixation to the insulating base material,
For example, a method such as bonding with an adhesive such as water-resistant epoxy, a double-sided adhesive tape, or the like, and heat fusion may be used. The fixing portion of the mesh is on an insulating substrate other than the conductor.
For example, when the biocatalyst-fixed mesh is fixed so as to cover all of the two or three conductors, preferably, the outer peripheral portion of the insulating substrate in a range including all of the conductors is set as the fixing portion. Further, the biocatalyst-immobilized mesh fixing portion on the insulating substrate may be a portion that overlaps with the spacer fixing portion on the insulating substrate, and thus, the biocatalyst-immobilized mesh is firmly fixed on the insulating base material. It is preferable in doing.

In the biosensor according to the first embodiment of the present invention, a spacer is fixed on the surface of the substrate having the conductor and the biocatalyst fixing layer, and the spacer is further fixed on the spacer.
A cover having a sample liquid inlet and, if necessary, an air hole, and having a reaction reagent immobilization layer on one surface, and a surface having the reaction reagent immobilization layer having a conductor and a biocatalyst immobilization layer of the substrate. And is fixed so as to face.

In the biosensor of the first embodiment of the present invention, the spacer and the cover are laminated on the surface of the substrate having the conductor and the biocatalyst-immobilizing layer in this manner, whereby the conductor of the substrate is And a sample liquid holding portion surrounded by the surface having the biocatalyst fixing layer, the inner wall of the spacer, and the surface of the cover having the reaction reagent fixing layer.

The shape of the spacer is a frame shape having an opening, and the frame portion is fixed from the opening to the biocatalyst fixing layer fixed on the conductor serving as at least the working electrode; When there is a conductor on which the fixing layer is not formed, the conductor is fixed on the substrate so as to face all of the conductor.

The cover further fixed on the spacer has a sample liquid inlet, an air hole if necessary, and a reaction reagent immobilization layer on one side. The size, shape, position, and the like of the sample liquid inlet and the air holes provided as needed are not particularly limited. However, regarding the position,
These positions are such that they do not close when fixed on the spacer.

The material of the spacer may be any material that is stable to the sample solution, has good adhesion to the insulating substrate, and is insulative. Any material may be used as long as it is stable to various compounds used for immobilizing the reagent, has good adhesiveness to the spacer, and is insulating. Examples of such materials for the spacer and the cover commonly include plastics such as polyethylene terephthalate, polyethylene, polypropylene, and polycarbonate, glass, and the like. Further, the spacer and the cover used in the present invention may be integrally formed. The fixing of the spacer and the cover can be usually performed by bonding with the same adhesive as used for bonding the parts made of the above-mentioned materials, or by heat fusion.

Specific examples of the reaction reagent fixed in a layer on one surface of the cover include a buffer, a storage stabilizer, a standard substance for preparing a calibration curve, a redox indicator, a mediator, and the like. As these reaction reagents, those similar to the reaction reagents usually added to the sample solution when measuring the various sample solutions by the biosensor on which the various biocatalysts or the like are immobilized are not particularly limited.

More specifically, examples of the buffer include a composition for a Tris-HCl buffer, a composition for an acetate buffer, a composition for a phosphate buffer, a composition for a maleate buffer, and the like. Examples of the agent include sodium chloride, potassium chloride, calcium chloride, sodium alginate, sodium carboxymethylcellulose (CMC), agarose, pullulan, carrageenan and the like. In addition, as a standard substance for preparing a calibration curve, a substance that can be measured by the biocatalyst, for example, glucose, glutamic acid and the like can be mentioned.

The mediator promotes the transfer of electrons generated by the metabolism of various substances by the biocatalyst to the electrodes. Any mediator may be used as long as it promotes the transfer of electrons from the biocatalyst to the electrode. Specifically, 1-methoxy-5-methylphenazinium methylsulfonate (1-M-PMS), , 6-Dichloroindophenol (DCIP), 9-dimethylaminobenzo-α-phenazoxonium chloride, methylene blue, indigotrisulfonic acid, phenosafuranine,
Thionine, new methylene blue, 2,6-dichlorophenol, indophenol, Azure B, N, N,
N ', N'-tetramethyl-p-phenylenediamine dihydrochloride, resorufin, safranin, sodium anthraquinone β-sulfonate, dyes such as indigo carmine, riboflavin, L-ascorbic acid, flavin adenine dinucleotide, flavin mononucleotide, nicotine adenine Dinucleotide, lumichrome, ubiquinone, hydroquinone, 2,6-dichlorobenzoquinone, 2-methylbenzoquinone, 2,5-dihydroxybenzoquinone, 2-hydroxy-1,4-naphthoquinone, glutathione, peroxidase, cytochrome C,
Biological redox substances such as ferredoxin or derivatives thereof,
Other Fe-EDTA, Mn-EDTA, Zn-EDT
A, meso sulfate, 2,3,5,6-tetramethyl-p-phenylenediamine, potassium ferricyanide and the like.

The method for immobilizing the reaction reagent is not particularly limited as long as the reaction reagent can be immobilized in a state where the reaction reagent is uniformly dispersed in the reaction reagent immobilization layer. Therefore, as the immobilizing material for immobilizing the reaction reagent, a material that forms a matrix such as that used for immobilizing the biocatalyst may be used, or a material that does not form a matrix may be used. They may be used in combination. The immobilizing material for immobilizing the reaction reagent may be a material that dissolves in the sample solution together with the reaction reagent when the sample solution is measured, or may be a material that does not dissolve. As the immobilization material for immobilizing the reaction reagent, an immobilization material is used in which the reaction reagent immobilization layer obtained by using the immobilization material does not elute eluting components that impair the effects of the present invention.

Even when a reaction reagent-immobilized layer is formed by using a material that forms a matrix and does not dissolve in a sample solution as the above-mentioned immobilization material, the molecules of the reaction reagent are usually much larger than the matrix size. Since the sample solution is very small, if the sample solution comes into contact with the reaction reagent-immobilized layer, the reaction reagent can be sufficiently dissolved in the sample solution.

As a method for immobilizing a reaction reagent, specifically, a method similar to the above-mentioned method for immobilizing a biocatalyst, for example, an alginate gel, a carrageenan gel,
A method of encapsulating and immobilizing in a gel matrix such as agarose gel, curdlan gel, chitosan gel, or immobilizing by incorporating into a three-dimensional crosslinked structure such as a photocurable resin such as photocrosslinkable polyvinyl alcohol or polyacrylamide. Method and CM not used for immobilizing the biocatalyst
And a method of immobilizing with a water-soluble and viscous material such as C. Many of these immobilization methods include a step of adding a reaction reagent to a raw material of an immobilization material and mixing the reaction reagent. At this time, the reaction reagent may be added as it is or in the form of a solution.

The reaction reagent-immobilized layer obtained by each of the above-mentioned immobilization methods is usually in a wet state. In the biosensor of the first embodiment of the present invention, the reaction reagent-immobilized layer may be kept in a wet state. From the viewpoint that the reaction reagent can be stably stored, it is preferable that the wet reaction reagent-immobilized layer is dried by an appropriate drying treatment. Examples of the drying method include general methods, for example, drying by heating, vacuum drying, and the like.

The thickness of the reaction reagent-immobilized layer in the biosensor of the first embodiment of the present invention is not particularly limited, but is preferably 5 μm in a dry state.
The thickness is about m to 100 μm.

The reaction reagent-immobilized layer may be formed at any position on at least one surface of the cover as long as it has at least a sample liquid inlet and an air hole, excluding the air hole. Preferably, it is formed in a portion other than the spacer fixing portion. As an example of a preferable arrangement of the reaction reagent immobilization layer, one formed on the entire surface of one surface of the cover excluding the spacer immobilization site and the sample liquid inlet and, if having air holes, excluding the air holes.

Further, in the biosensor of the first embodiment of the present invention, a mesh on which a reaction reagent is immobilized (hereinafter also referred to as a “reaction reagent immobilization mesh”) is used as the reaction reagent immobilization layer. It is also possible. The production of the reaction reagent-immobilized mesh can be performed, for example, by the same method as the production of the biocatalyst-immobilized mesh except that a reaction reagent is used instead of the biocatalyst in the production of the biocatalyst-immobilized mesh. Further, it is also possible to immobilize the reaction reagent on the mesh with a water-soluble and viscous material such as CMC which is not used for immobilizing the biocatalyst. Also in this case, it is possible to add a drying treatment similar to the above at the end, and it is preferable to perform the drying treatment from the viewpoint of the storage stability of the reaction reagent as described above.

The fixing of the reaction reagent-immobilized mesh on one side of the cover can be performed using the same adhesive or the like as used for fixing the biocatalyst-immobilized mesh to the insulating substrate. However, point fixation may be used. Also, like the biocatalyst-immobilized mesh, the reaction reagent-immobilized mesh can be sized to reach the spacer-immobilized portion on one surface of the cover, and the immobilized portion can be positioned to overlap the spacer-immobilized portion. It is.

The reaction reagent immobilized on one surface of the cover in this manner is dissolved in the sample liquid to be injected into and held in the sample liquid holding part when measuring the sample liquid due to the structure of the biosensor of the present invention. It is.

The amount of the reaction reagent immobilized on one surface of the cover is appropriately selected depending on the type of the reaction reagent and the amount of the sample solution held in the sample solution holding section. Specifically, the amount of the reaction reagent in the sample solution held in the sample solution holding unit is usually such that the concentration becomes the same as the concentration at which the reaction reagent is added to the sample solution when measuring the sample solution. The reaction reagent is fixed.

In the biosensor according to the first embodiment of the present invention, when a standard substance for preparing a calibration curve is immobilized as the reaction reagent, measurement is performed in place of the sample liquid in the sample holder. A buffer or the like containing no target substance is injected and used as a biosensor for measuring a standard substance.

Hereinafter, a specific example of the disposable biosensor according to the first embodiment of the present invention will be described with reference to FIGS. FIG. 1 is a diagram showing a front view of an insulating substrate to which a conductor used for an example of a disposable biosensor according to the first embodiment of the present invention is fixed. FIG. 2 is a front view showing an example of the disposable biosensor according to the first embodiment of the present invention. FIG. 3 is a sectional view taken along line XX ′ of an example of the disposable biosensor according to the first embodiment of the present invention shown in FIG. 2, and FIG. 4 is an exploded view thereof.

As shown in FIG. 1, the flat insulating substrate 3
A working electrode 1 and a counter electrode 2 having the same area are formed substantially in parallel on the same plane. In this example, the counter electrode 2 also functions as a reference electrode.

As shown in FIGS. 2 to 4, in the biosensor 20 of this embodiment, the spacer and the cover are integrally formed. This is hereinafter referred to as cell 6. The biocatalyst fixing layer 8 covers the working electrode 1 and the counter electrode 2 on the plane of the insulating substrate 3 and is formed on the entire surface except the cell 6 fixing portion.

For example, when the biocatalyst is immobilized using a calcium alginate gel, a suspension containing the required amount of the biocatalyst is added to an aqueous solution of about 1 to 3% of sodium alginate, and the mixture is stirred well. Then, a uniform mixture is prepared, and an appropriate amount of the mixture is applied to the entire surface of the insulating substrate excluding the cell 6 fixing portion. The aqueous sodium alginate solution may contain sodium chloride, potassium chloride, various buffers, and the like, in addition to sodium alginate.
Further, the biocatalyst suspension and the like may contain sodium chloride, potassium chloride, various buffers, and the like, in addition to the biocatalyst.

Next, the whole of the electrode substrate coated with the mixed solution is immersed in a calcium salt solution having an appropriate concentration to immobilize the mixed solution as a calcium alginate gel containing a biocatalyst.

Here, the biocatalyst-immobilized layer 8 is located only in the above range.
In order to form the cells, a method may be adopted in which the cell fixing portion is masked and then the fixing process is performed, and after the fixing is completed, the cell fixing portion is removed.

The cell 6 has a portion 6a corresponding to its cover.
The substrate has a sample liquid inlet 4 and an air hole 5, and a reaction reagent fixing layer 7 is provided on a surface of the substrate facing the biocatalyst fixing layer when fixed on the substrate. The reaction reagent-immobilized layer 7 is formed over the entire area of the surface of the substrate facing the biocatalyst-immobilized layer except for the sample liquid inlet 4 and the air hole 5. The reaction reagent-immobilized layer 7 is immobilized by the same method as the biocatalyst-immobilized layer 8, for example, by being contained in a calcium alginate gel. The reaction reagent immobilization layer 7
It is preferable to perform a drying treatment after the immobilization from the viewpoint of storage stability of the reaction reagent. As the drying treatment in that case, for example, sufficient drying with warm air at about 30 to 80 ° C. or the like can be mentioned.

In this embodiment, the cell 6 is provided at the portion 6b corresponding to the spacer on the insulating substrate 3 at the working electrode 1
And on a plane having the counter electrode 2 and the biocatalyst fixing layer 8,
It is fixed using a double-sided adhesive tape or the like. by this,
The surface of the substrate having the working electrode 1 and the counter electrode 2 and the biocatalyst fixing layer 8 and the inner wall of the spacer 6b of the cell 6 and the reaction reagent fixing layer 7 of the cell cover 6a
The sample liquid holding part 12 surrounded by the surface having the above is formed.

The disposable biosensor of the present invention is
When not used immediately after the production, it is preferable to store the sample liquid inlet by plugging it, and further, if it has an air hole, by plugging the air hole. The stopper is removed when the biosensor is used.

In the measurement of the sample solution, the disposable biosensor of the first embodiment of the present invention differs from the ordinary disposable biosensor in that the sample solution uses a buffer, a storage stabilizer, and a redox indicator. There is no need to add a mediator or the like. By connecting the biosensor of the present invention to a measuring instrument, injecting an untreated sample liquid from the sample liquid inlet to the sample liquid holding portion so as to fill the sample liquid holding section, and holding in that state, the sample liquid in the sample liquid The reaction reagent elutes from the reaction reagent-immobilized layer. Thereafter, by applying a potential to the working electrode, the working electrode detects a biocatalytic reaction that is performed on the substance to be measured in the sample liquid holding unit, and by measuring the current flowing between the working electrode and the counter electrode, The concentration of the target substance in the sample solution injected into the sample solution holding section is measured.

In the biosensor according to the first embodiment of the present invention, the fixed reaction reagent dissolves into the sample solution when measuring the sample solution as described above. Can not be used again in the state. That is, it is a disposable biosensor. But,
In order to use again, only the cover is detachably fixed, the cover is removed after use, and after performing washing etc. as necessary, a cover similar to the state before use is used instead of the cover after use. It is also conceivable to use the method by fixing. In the case of using an expensive biocatalyst, this method may be effective, but since it takes time to replace the cover and the like, it is contrary to the simplification of the measurement operation which is the object of the present invention. The biosensor of the present invention is preferably used as a disposable type.

Further, for example, a biosensor 2 according to the first embodiment of the present invention, in which a standard substance (a substance to be measured in a sample solution) for preparing a calibration curve is immobilized in two amounts together with a mediator and a buffer. Measurement of a standard substance by injecting pure water using a set of biosensors in which one biosensor of the first embodiment of the present invention, in which a mediator and a buffer are immobilized, is fixed on the same support. By simultaneously or sequentially performing the measurement of the sample liquid by injecting the untreated sample liquid, the measurement operation of the concentration of the measurement target substance in the sample liquid can be performed very easily.

(2) Disposable Biosensor of the Second Embodiment of the Present Invention The disposable biosensor of the second embodiment of the present invention
In the disposable biosensor of the first embodiment of the present invention, the reaction reagent-immobilized mesh is fixed on the spacer in place of the cover, and the sample liquid inlet is eliminated by not using the cover. Therefore, instead of this, a sample liquid inlet is provided in the spacer, and the other configuration is exactly the same as that of the disposable biosensor of the first embodiment of the present invention.

The reaction reagent-immobilized mesh can be the same as that described in the disposable biosensor of the first embodiment of the present invention. Also,
The size, shape, and position of the sample liquid inlet provided in the spacer are not particularly limited, but are provided in such a size, shape, and position that the sample liquid can be easily injected and the sample liquid does not leak from the sample liquid during measurement. Preferably.

A specific example of the disposable biosensor according to the second embodiment of the present invention will be described below with reference to FIGS. FIG. 5 is a diagram illustrating a front view of an insulating substrate to which a conductor used in an example of a disposable biosensor according to a second embodiment of the present invention is fixed. FIG. 6 shows a second embodiment of the present invention.
FIG. 6A is a diagram showing a spacer used in an example of the disposable biosensor according to the embodiment, FIG. 6A is a front view, and FIG.
FIG. 7B is a cross-sectional view taken along line YY ′ of FIG. FIG. 7 is a front view illustrating an example of a disposable biosensor according to the second embodiment of the present invention. FIG. 8 is an exploded cross-sectional view taken along line ZZ ′ of an example of the disposable biosensor according to the second embodiment of the present invention shown in FIG.

As shown in FIG. 5, the flat insulating substrate 3
A working electrode 1 and a counter electrode 2 having the same area are formed substantially in parallel on the same plane. In this example, the counter electrode 2 also functions as a reference electrode.

As shown in FIG. 6, the spacer 9
It has a sample liquid inlet 4 and an opening corresponding to a range including all of the working electrode 1 and the counter electrode 2 on the substrate plane. As shown in FIGS. 7 and 8, the biocatalyst-fixed mesh 11 is fixed with a double-sided adhesive tape or the like so as to cover a range including all of the working electrode 1 and the counter electrode 2 of the insulating substrate 3. The size of the biocatalyst-immobilized mesh 11 is the same as the size of the spacer 9,
The fixing portion on the insulating substrate 3 is a portion that substantially overlaps with the spacer body laminated from above.

The spacer 9 is provided on the biocatalyst-fixed mesh 11 fixed on the insulating substrate 3, with an opening in a range including all of the working electrode 1 and the counter electrode 2 on the substrate plane below the biocatalyst-fixed mesh. Correspondingly, it is fixed with a double-sided adhesive tape or the like. The fixing site is the entire spacer main body in the spacer.

The biocatalyst-immobilized mesh 11 is, for example,
A biocatalyst immobilized on a mesh of polyester or the like using calcium alginate gel is used.
When the biocatalyst is immobilized on the mesh by calcium alginate gel, a suspension containing the required amount of the biocatalyst is added to an aqueous solution of about 1 to 3% of sodium alginate, and the mixture is stirred well. Is prepared and an appropriate amount of the mixed solution is supplied onto the mesh. Using a squeegee, this mixed solution was stretched over the entire mesh so that the mixed solution spread over the fine space inside the mesh in the same manner as in the squeezing step in screen printing, and then the mixed solution was filled. By immersing the entire mesh in a calcium salt solution having an appropriate concentration, the mixture is immobilized as a calcium alginate gel containing a biocatalyst.

The aqueous sodium alginate solution may contain sodium chloride, potassium chloride, various buffers and the like in addition to sodium alginate. Further, the biocatalyst suspension and the like may contain sodium chloride, potassium chloride, various buffers, and the like, in addition to the biocatalyst.

In the biosensor 20 of this embodiment, the reaction reagent fixing mesh 10 is further fixed on the spacer 9 with a double-sided adhesive tape or the like. by this,
Biocatalyst-immobilized mesh 11 fixed on the substrate 3
A sample liquid holding portion is formed which is surrounded by the surface, the inner wall of the spacer, and the lower surface of the mesh 10 on which the reaction reagent is immobilized.
The sample holder communicates with the outside of the biosensor 20 via the sample inlet 4 provided in the spacer 9.

The preparation of the reaction reagent-immobilized mesh 10 is carried out in the same manner as that of the biocatalyst-immobilized mesh 11 except that a reaction reagent is used instead of the biocatalyst in the preparation of the biocatalyst-immobilized mesh 11. it can. However, in producing the reaction reagent-immobilized mesh 10,
Finally, it is preferable to add a drying treatment similar to the above. For example, as a drying treatment when the reaction reagent is fixed using a calcium alginate gel, drying with hot air at about 30 to 80 ° C. and the like can be mentioned.

The method of using the disposable biosensor of the second embodiment of the present invention can be the same as that of the above-described disposable biosensor of the first embodiment of the present invention.

Further, the disposable biosensor of the present invention is not limited to the first and second embodiments, and in any other form, the method of use has been described for the two embodiments. Can be the same as By using the disposable biosensor of the present invention as described above, the measurement operation of the concentration of the substance to be measured in the sample liquid can be performed very easily, and the measurement time can be reduced.

[0083]

Embodiments of the present invention will be described below.

[0084]

EXAMPLE 1 (1) Production of Biosensor A working electrode 1 of the same area made of a carbon material and a working electrode 1 made of a carbon material were placed on the same plane of an insulating substrate 3 (15 × 35 mm) made of glass epoxy resin as shown in FIG. The counter electrode (also used as a reference electrode) 2 (3 × 7 mm) was fixed so as not to contact each other. Next, the same volume of microorganism (Pseudomonas fluorescens (Pseudomo
nas Fluorescens)), a suspension (suspension medium; distilled water) was added thereto to prepare a mixed solution, and a certain amount of the obtained mixed solution was further transferred onto the working electrode 1 and the counter electrode 2 of the substrate 3. After dropping the liquid on the substrate and spreading it over the entire substrate except for at least the portion where the cells are fixed, the entire substrate is
The mixture was gelled by immersion in a 4% aqueous solution of calcium chloride in a refrigerator at 4 ° C. for 30 minutes to obtain a wet immobilized microorganism electrode.

On the other hand, a 3% aqueous sodium alginate solution and 0.1 M sodium chloride and 40
A predetermined amount of a mixed solution obtained by uniformly mixing an aqueous solution containing mM mM ferricyanide was dropped on the inner bottom surface of the cell having one side closed, and the entire solution was spread over the entire bottom surface.
The mixture was gelled by immersing it in a 4% aqueous solution of calcium chloride in a refrigerator at 4 ° C. for 30 minutes. Then change this to 40
The solution was dried for 2 hours using a drier at a temperature of 0 ° C. to remove water, thereby obtaining an immobilized reaction reagent cell in a dry state.

The immobilized microbial electrode thus prepared and the immobilized reaction reagent cell in a dry state are adhered to each other with a double-sided adhesive tape in the same manner as shown in FIGS. Liquid holder 12 formed on the inner wall of the cell including the surface of the cell and the surface of the immobilized microorganism layer on the insulating substrate
Was obtained. A sample solution inlet 4 and an air hole 5 are provided at the bottom of the cell 6 and are plugged when the biosensor is stored. However, the stopper is removed and opened just before the measurement of the sample solution concentration, and the sample solution is injected. The liquid is introduced into the sample liquid holding unit 12 from the inlet 4.

(2) Evaluation of Biosensor The disposable biosensor of the present invention was evaluated by performing BOD measurement of an organic substance mixed solution (artificial drainage) using nine of the disposable biosensors obtained above. .

As an organic mixed solution, artificial sewage having a composition shown in the following Table 1, which is adopted in a report of OECD (Economic Cooperation and Development Organization), has a BOD ₅ value (5-day method, JI
(SK102, modified by Winkler sodium azide) 0 mgO / L, 70 mgO / L, 140 mgO / L
The solution prepared so that it may be contained at a concentration of 1% was used.

[0089]

[Table 1]

In the measurement of the above three kinds of organic substance mixed solutions having BOD concentrations, the measurement was carried out by the following method using three biosensors for each kind. The disposable biosensor obtained above is connected to a potentiostat, and the organic substance mixed solution is injected into the sample holding portion of the disposable biosensor so that the sample holding portion is filled with the measurement sample. After reacting for 1 minute, a potential of +400 mV was applied to the working electrode by a potentiostat for 1 minute.
The voltage was applied for 0 seconds, and after 5 seconds, the current flowing between the working electrode and the counter electrode was measured.

FIG. 9 shows the results. The correlation coefficient (R
² ) As shown by 0.9817, the BO of the solution used
Very good correlation to the current value measured by the D ₅ value and the disposable biosensors were obtained. The equation shown in FIG. 9 is an equation representing a regression line obtained by the least square method.

From the results, it can be seen that if the sample solution is measured using the biosensor of the present invention, accurate measurement can be performed without adding any pretreatment to the sample solution.

[0093]

Example 2 In the same manner as in Example 1 above, a mixed solution of a sodium alginate aqueous solution and a Pseudomonas fluorescens suspension, and a sodium alginate aqueous solution,
Mixtures of aqueous solutions each containing 0.1 M sodium chloride and 40 mM potassium ferricyanide were prepared. These two types of mixed liquids are used for screen printing in # 200
Tetron (trade name, polyester) mesh was separately imprinted with a squeegee so as to be uniform in the stitches. After that, two tetron meshes each filled with a stitch are separately immersed in a 2% aqueous solution of calcium chloride in a refrigerator at 4 ° C. for 30 minutes to obtain a mixed solution in the stitches. The gel was formed to obtain a microorganism-immobilized mesh and a non-dried reaction reagent-immobilized mesh (unfinished product). After completion of the gelation, the reaction reagent-immobilized mesh was dried with a dryer at 40 ° C. for 2 hours to remove water, thereby obtaining a finished product.

On the other hand, as shown in FIG. 5, a working electrode 1 made of a carbon material and a counter electrode (also used as a reference electrode) of the same area are formed on the same plane of an insulating substrate 3 (15 × 35 mm) made of glass epoxy resin. 2 (3 × 7 mm) were fixed so as not to contact each other.

The microbe-immobilized mesh and the reagent-immobilized mesh obtained above were cut into appropriate sizes, and FIG.
As shown in FIG. 8, first, the mesh 11 for immobilizing microorganisms is fixed on the surface of the insulating substrate to which the working electrode 1 and the counter electrode 2 are fixed, using a double-sided adhesive tape. Spacer 9 having an opening and a sample liquid inlet 4 and a reaction reagent-immobilized mesh 1
0 was adhered and laminated with a double-sided adhesive tape to obtain a disposable biosensor 20 having a sample liquid holding portion formed on the surface of the microorganism-immobilized mesh, the inner wall of the spacer, and the lower surface of the reaction reagent-immobilized mesh.

It is preferable that the biosensor 20 obtained in this embodiment, like the biosensor obtained in the first embodiment, be plugged in the sample liquid inlet 4 of the spacer 9 during storage. Immediately before the start of the measurement of the concentration of the sample liquid, the stopper is removed to open the sample liquid inlet 4, from which the sample liquid is introduced into the sample liquid holding unit.

[0097]

According to the disposable biosensor of the present invention, both the reaction reagent and the biocatalyst are immobilized with good storage stability. By using the disposable biosensor of the present invention, the measurement operation of the sample liquid is simple and the measurement time can be shortened.

[Brief description of the drawings]

FIG. 1 is a diagram illustrating a front view of an insulating substrate to which a conductor used in an example of a disposable biosensor according to a first embodiment of the present invention is fixed.

FIG. 2 is a front view illustrating an example of a disposable biosensor according to the first embodiment of the present invention.

FIG. 3 is a diagram showing a cross-sectional view taken along line XX ′ of an example of the disposable biosensor according to the first embodiment of the present invention shown in FIG. 2;

FIG. 4 is an exploded sectional view taken along line XX ′ of an example of the disposable biosensor according to the first embodiment of the present invention shown in FIG. 2;

FIG. 5 is a diagram illustrating a front view of an insulating substrate to which a conductor used in an example of a disposable biosensor according to a second embodiment of the present invention is fixed.

FIG. 6 is a diagram illustrating a spacer used in an example of a disposable biosensor according to a second embodiment of the present invention.
FIG. 7A is a front view, and FIG. 6B is a cross-sectional view taken along line YY ′ of FIG.

FIG. 7 is a front view illustrating an example of a disposable biosensor according to a second embodiment of the present invention.

FIG. 8 is an exploded cross-sectional view taken along the line ZZ ′ of an example of the disposable biosensor according to the second embodiment of the present invention shown in FIG. 7;

FIG. 9 is a diagram showing evaluation results of the disposable biosensor obtained in Example 1.

[Explanation of symbols]

 1. Working electrode 2. 2. Counter electrode (also used as reference electrode) Insulating substrate 4. Sample liquid inlet 5. Air hole 6. 6. Cell (Integrally molded product of spacer and cover) 7. Reagent immobilization layer 8. Biocatalyst immobilization layer Spacer 10. 10. Mesh on which reaction reagent is immobilized Mesh with immobilized biocatalyst Sample liquid holding unit 20. Biosensor

 ──────────────────────────────────────────────────続 き Continuing on the front page (72) Inventor Junko Hoshita 5-4-171 Higashi, Hanyu-shi, Saitama Prefecture Akebono Brake Central Research Institute Co., Ltd. (72) Akira Yoshida 5-4-71 Higashi, Hanyu-shi, Saitama Akebono Brake Central Research Institute

Claims (7)

[Claims] 1. An insulating substrate, two or three conductors fixed on the same plane of the substrate, a biocatalyst fixing layer provided on at least one of the conductors, A closed cell having a sample liquid inlet fixed to form a sample liquid holding portion on the flat surface, and a reaction reagent immobilization provided on the inner surface of the cell so as not to contact the biocatalyst immobilization layer Disposable biosensor having an activated layer. 2. A flat insulating substrate, two or three conductors fixed on the same plane of the substrate so as not to contact each other, and provided on at least one of the two or three conductors. A biocatalyst-immobilized layer, and a disposable biosensor comprising a conductor and a biocatalyst-immobilized layer of the substrate, a spacer fixed on a surface of the biosensor, and a sample liquid inlet on the spacer. Having a cover having a reaction reagent immobilization layer on one side, and fixing so that the surface having the reaction reagent immobilization layer faces the surface of the substrate having the conductor and biocatalyst immobilization layer, A disposable biosensor, comprising: a sample liquid holding portion surrounded by a surface having a conductor and a biocatalyst immobilization layer, an inner wall of a spacer, and a surface having a reaction reagent immobilization layer of a cover. 3. The disposable biosensor according to claim 2, wherein the cover further has an air hole. 4. A flat insulating substrate, two or three conductors fixed on the same plane of the substrate so as not to contact each other, and provided on at least one of the two or three conductors. A biocatalyst-immobilized layer, comprising: a mesh having a spacer having a sample liquid inlet and a reaction reagent immobilized on a surface of the substrate having a conductor and a biocatalyst-immobilized layer. And disposing a sample liquid holding portion surrounded by the surface of the substrate having the conductor and the biocatalyst immobilization layer, the inner wall of the spacer, and the lower surface of the mesh on which the reaction reagent is immobilized. Type biosensor. 5. The disposable biosensor according to claim 1, wherein the biocatalyst-immobilized layer comprises a mesh on which the biocatalyst is immobilized. 6. The disposable biosensor according to claim 1, wherein the reaction reagent is selected from a buffer, a storage stabilizer, a standard substance for preparing a calibration curve, a redox indicator, and a mediator. . 7. The disposable biosensor according to claim 1, wherein the biocatalyst is selected from a microorganism, an animal cell, and a plant cell.