JP2021082394A - Enzyme electrode and biosensor, and biofuel cell - Google Patents

Abstract

To provide an enzyme electrode with high safety, in which conductive carbon fillers with a high specific surface area, such as nanocarbon materials, have excellent dispersibility, and a biosensor and a biofuel cell in which the enzyme electrode is used.SOLUTION: An enzyme electrode is an enzyme electrode including a reagent part formed on a conductive substrate, the reagent part including an oxidoreductase enzyme, a conductive carbon filler, and a cellulose derivative.SELECTED DRAWING: Figure 1

Description

The present invention relates to enzyme electrodes and biosensors as well as biofuel cells.

Enzyme electrodes that use enzymatic reactions are used in biosensors and biofuel cells. For example, biosensors are used in various fields such as medical fields and clinical examination fields to measure substances to be detected in biological samples. As an example of a biosensor, a biosensor using an electrochemical measurement method is known. This biosensor forms a working electrode, a counter electrode, and a reference electrode on an insulating base material, and is in contact with these electrodes to contain an enzyme and an electron acceptor (hereinafter referred to as a mediator). Is forming. According to such a biosensor, in principle, various substances can be measured by selecting an enzyme whose substrate is the substance to be measured. For example, a glucose sensor that measures the glucose concentration in a sample solution by selecting glucose oxidase as an enzyme has been put into practical use.

In recent years, a conductive carbon filler having a high specific surface area such as a nanocarbon material has been used for an enzyme electrode from the viewpoint of achieving high sensitivity and high output by improving the specific surface area of the electrode. However, since the nanocarbon material is generally highly hydrophobic, it is difficult to disperse it in an aqueous system, and it is difficult to maintain the dispersed state because aggregation is likely to occur due to van der Waals force or the like. Therefore, when a nanocarbon material is used for the enzyme electrode, a method of preparing an electrode coating liquid by dispersing the nanocarbon material using an organic solvent (for example, Patent Documents 1 and 2, Non-Patent Documents 1 and 2) and an interface. A method of preparing an electrode coating liquid by dispersing a nanocarbon material in an aqueous solvent using an activator (for example, Non-Patent Documents 3 and 4) has been proposed.

International Publication No. 2005/0882888 Japanese Unexamined Patent Publication No. 2018-121545

Chemical Physics Letters 480 (2009) 123-126 Bioelectrochemistry 76 (2009) 10-13 Biosensors and Bioelectronics 74 (2015) 947-952 Journal of Power Sources 408 (2018) 1-6

However, in the case of an enzyme electrode produced by a conventional method, when the enzyme electrode is brought into direct contact with a measurement object such as food, a beverage, a cell culture solution, or a human body where contamination is a problem, an organic solvent or an interface is brought to the measurement object. There is a problem that the activator dissolves or adheres and contaminates the object to be measured. It is also possible to remove the organic solvent and the surfactant from the enzyme electrode by washing or the like, but with the removal, the nanocarbon material may reaggregate and the dispersibility may decrease.

Therefore, the present invention provides an enzyme electrode having excellent dispersibility and dispersion stability of a conductive carbon filler having a high specific surface area such as a nanocarbon material and having high safety, and a biosensor and a biofuel cell using the same. The purpose was to do.

In order to solve the above problems, the enzyme electrode of the present invention is an enzyme electrode having a reagent portion formed on a conductive substrate, and the reagent portion contains an oxidoreductase, a conductive carbon filler, and a cellulose derivative. It is characterized by including.

According to the present invention, it is possible to provide an enzyme electrode having excellent dispersibility and dispersion stability of a conductive carbon filler having a high specific surface area and high safety, and a biosensor and a biofuel cell using the enzyme electrode. Become.

It is a graph which shows the measurement result of Example 10 and Comparative Example 11 of this invention. It is a graph which shows the measurement result of Example 11 of this invention. It is a graph which shows the measurement result of Example 12 of this invention. It is a graph which shows the measurement result of the comparative example 12.

Hereinafter, embodiments of the present invention will be described in detail.
The enzyme electrode targeted by the present invention is not particularly limited as long as it is an electrode having a reagent portion formed on a conductive substrate, but the reagent portion contains an oxidoreductase, a conductive carbon filler, and a cellulose derivative. I'm out. In the present specification, dispersibility means that the conductive carbon filler is easily dispersed, and dispersion stability means that the dispersed state of the conductive carbon filler is maintained and reaggregation is difficult.

The shape of the conductive carbon filler used in the present invention is not particularly limited, and spherical, scaly, fibrous, porous and the like can be used. Further, those having an average particle size in the range of 10 nm to 20 μm measured by an electron microscope or the like can be used. The BET specific surface area of the conductive carbon filler is 10 m ² / g or more, preferably 30 m ² / g or more. Specific examples of the conductive carbon filler include carbon black, graphite powder, porous carbon, nanocarbon material and the like. Here, the porous carbon includes not only activated carbon powder or activated carbon fiber having mesopores of 2 to 50 nm, but also a carbon material having communication pores in which the mesopores are connected. Examples of carbon black include furnace black, thermal black, acetylene black, and ketjen black. Examples of graphite powder include pyrolysis graphite and spheroidal graphite. Examples of the nanocarbon material include single-walled carbon nanotubes, multi-walled carbon nanotubes, graphene, and fullerenes. Preferred are carbon black, porous carbon or nanocarbon materials.

Further, the cellulose derivative used in the present invention is a polymer in which a part of the hydroxyl group of cellulose is replaced with another substituent by an etherification reaction or an esterification reaction, and includes hydrophilic cellulose and non-hydrophilic cellulose. However, hydrophilic cellulose is preferable. Here, the hydrophilic cellulose is a cellulose derivative having a high affinity for water and having a property of being easily dissolved and miscible in an aqueous solvent and a polar solvent. Examples of the polar solvent include ethanol, 1-propanol, 2-propanol, formic acid, acetic acid, ethyl acetate, dimethyl sulfoxide and the like. Further, the aqueous solvent refers to a solvent mainly composed of water only. However, a trace amount of a polar solvent such as ethanol may be contained as long as the enzyme is not inactivated and safety is not a concern after drying. Examples of hydrophilic cellulose include hydroxyalkyl cellulose, carboxyalkyl cellulose, carboxyalkyl hydroxyalkyl cellulose, methyl cellulose and the like. Examples of hydroxyalkyl cellulose include hydroxyethyl cellulose, hydroxypropyl cellulose, hydroxypropyl methyl cellulose, hydroxyethyl methyl cellulose and the like. Moreover, as carboxyalkyl cellulose, carboxymethyl cellulose and the like can be mentioned. Moreover, as carboxyalkyl hydroxyalkyl cellulose, carboxymethyl hydroxyethyl cellulose and the like can be mentioned. Preferred cellulose derivatives are carboxymethyl cellulose or hydroxypropyl cellulose. Further, these cellulose derivatives may be used alone or in combination of two or more. Further, the cellulose derivative having a carboxy group may also contain a salt form such as an alkali metal salt. The non-hydrophilic cellulose is a cellulose having a low affinity for water and having a property of being difficult to dissolve and mix in an aqueous solvent and a polar solvent. For example, ethyl cellulose, propyl cellulose, butyl cellulose, acetyl cellulose and the like. Can be mentioned.

Further, the enzyme electrode of the present invention includes a reagent portion. The reagent portion means a portion other than the conductive substrate, and includes at least an oxidoreductase, a conductive carbon filler, and a cellulose derivative. The reagent section can be composed of one layer (single layer) or a plurality of laminated reagent layers.

When the reagent section is composed of one reagent layer, the reagent layer contains an oxidoreductase, a mediator, a conductive carbon filler and a cellulose derivative in the same layer (hereinafter, also referred to as a mixed reagent section). The mixed reagent portion contains 0.1 to 30 parts by weight, preferably 0.2 to 25 parts by weight, and more preferably 0.4 to 20 parts by weight of the cellulose derivative with respect to 1 part by weight of the conductive carbon filler. If the amount of the cellulose derivative is less than 0.1 parts by weight with respect to the conductive carbon filler, the dispersibility of the conductive carbon filler is not sufficient, and if it is more than 30 parts by weight, it becomes difficult for the conductive carbon fillers to come into contact with each other. This is because the conductivity is not sufficient.

When the reagent section is composed of a plurality of laminated reagent layers, the plurality of reagent layers include one or more first reagent layers and one or more second reagent layers, and the first reagent. The layer may contain a conductive carbon filler and a cellulose derivative, and the second reagent layer may contain an oxidoreductase. The first reagent layer and the second reagent layer can be alternately laminated (hereinafter, also referred to as a laminated reagent section). For example, as an example of the case where the reagent portion is composed of two layers, a first reagent layer (also referred to as a conductive carbon layer) containing a conductive carbon filler and a cellulose derivative is provided on the conductive substrate side, and the first reagent A second reagent layer containing an oxidoreductase can be laminated on the layer. Alternatively, a second reagent layer may be provided on the conductive substrate side, and the first reagent layer may be laminated on the second reagent layer. In the laminated reagent section, the conductive carbon layer contains 0.1 to 30 parts by weight, preferably 0.2 to 25 parts by weight, more preferably 0.4 parts by weight of the cellulose derivative with respect to 1 part by weight of the conductive carbon filler. Includes ~ 20 parts by weight.

Further, as an example of the case where the reagent portion is composed of three layers, a plurality of conductive carbon layers can be provided, and a first reagent layer, a second reagent layer, and a first reagent can be provided on the conductive substrate. The layers may be laminated in this order. Further, as another example, the second reagent layer, the first reagent layer, and the second reagent layer may be laminated in this order on the conductive substrate.

As the conductive substrate, a metal electrode such as gold, platinum, or silver, or a conductive portion formed of an insulating material can be used. The material used for the insulating base material is not particularly limited, but for example, polyethylene terephthalate, polycarbonate, polyimide, polyethylene, polypropylene, polystyrene, polyvinyl chloride, polyoxymethylene, monomer cast nylon, polybutylene terephthalate, methacrylic resin, ABS resin and the like. Resin material or glass material can be used. Polyethylene terephthalate, polycarbonate, and polyimide are preferable, and polyethylene terephthalate is more preferable. For the conductive portion on the insulating base material, for example, using carbon, gold, platinum, palladium or the like as a material, a conductive layer is formed by a sputtering method, a vapor deposition method, a screen printing method or the like, and then a laser trimming method is used. It can be formed by processing into a predetermined electrode pattern.

An example of the method for producing an enzyme electrode of the present invention will be described in detail below.
(1) When the reagent part has one layer A coating liquid containing a cellulose derivative, a conductive carbon filler and an oxidoreductase is prepared, and the coating liquid is applied to a conductive substrate to form the reagent part. Specifically, a conductive carbon filler is added to a cellulose derivative aqueous solution prepared to a predetermined concentration using an aqueous solvent so as to have a predetermined concentration, and a carbon dispersion is prepared by performing a vortex mixer or sonication. .. A carbon / oxidoreductase / mediator mixed solution (coating liquid) is prepared by mixing a separately prepared oxidoreductase / mediator solution with a carbon dispersion at a predetermined mixing ratio. This coating liquid is applied onto the conductive substrate a predetermined number of times and dried to form one reagent layer. Then, by immersing the conductive substrate having one reagent layer in the polymer solution for a protective film, pulling it up, and drying it a plurality of times, it is possible to obtain an enzyme electrode in which the reagent layer is covered with a protective film. it can. Here, the aqueous solvent refers to a solvent mainly composed of water, and if necessary, may contain a polar organic solvent as long as it does not inactivate the enzyme and does not impair safety after drying. It should be noted that the volume% of water is 70% or more, preferably 85% or more, and more preferably 95% or more when it is mainly composed of water. Examples of the polar organic solvent that can be used in the present invention include ethanol, 1-propanol, 2-propanol, formic acid, acetic acid, dimethyl sulfoxide and the like.
(2) When the reagent layer consists of two or more layers When the reagent part consists of two or more reagent layers, a step of preparing a carbon dispersion containing a cellulose derivative and a conductive carbon filler and a solution containing an oxidoreductase are prepared. The step of forming the reagent part includes the step of forming the first reagent layer using the carbon dispersion and the second reagent using the solution containing the oxidoreductase. An enzyme electrode can be produced by a production method having one or more steps for forming a layer. For example, when the reagent section is composed of two reagent layers, it can be produced by the following method. That is, a conductive carbon filler is added to a cellulose derivative aqueous solution prepared to a predetermined concentration using an aqueous solvent so as to have a predetermined concentration, and a vortex mixer or sonication is performed to prepare a carbon dispersion. This carbon dispersion is applied onto the conductive substrate a predetermined number of times and dried to form a first reagent layer (conductive carbon layer). Next, a separately prepared oxidoreductase / mediator solution is applied to the surface of the conductive carbon layer to form a second reagent layer, which is dried and dried to form two reagent layers (first reagent layer / second reagent). A reagent section consisting of layers) is formed. Then, by immersing the conductive substrate having the reagent part in the polymer solution for a protective film, pulling it up, and drying it a plurality of times, an enzyme electrode in which the reagent part is covered with a protective film can be obtained. When the reagent section is composed of three reagent layers, the carbon dispersion is applied a predetermined number of times on the second reagent layer above the two reagent layers to form the first reagent layer. By forming and drying, a reagent portion composed of three reagent layers (first reagent layer / second reagent layer / first reagent layer) is formed. Alternatively, the second reagent layer may be formed on a conductive substrate, and the first reagent layer may be laminated on the second reagent layer to form a reagent portion composed of two reagent layers. Further, a second reagent layer is formed on a conductive substrate, a first reagent layer is laminated on the first reagent layer, and a second reagent layer is further laminated on the second reagent layer, and a reagent composed of three reagent layers is laminated. The part may be formed.

Here, the protective film prevents or suppresses the leakage of the substance contained in the reagent portion to the outside of the protective film, and the substrate existing outside the protective film permeates into the protective film in which the reagent portion exists. It has a possible hole. Then, the protective film needs to be arranged so as to cover at least the reagent portion on the enzyme electrode. Further, when the enzyme electrode is used by inserting it into a living body, a medium, or a reagent for cell culture, the protective film covering the surface thereof generally has biocompatibility in which proteins and cells are not adsorbed or are difficult to be adsorbed. It is preferably formed of a polymer having such properties. Examples of the polymer capable of forming such a protective film include a copolymer of methyl methacrylate and hydroxyethyl methacrylate, a copolymer of butyl methacrylate and hydroxyethyl methacrylate, and poly (2-methacryloyloxyethyl phosphorylcholine-con-butyl methacrylate). ), Polyurethane and the like.

Further, the mediator means a redox substance that mediates electron transfer, and refers to a substance that is responsible for the transfer of electrons generated by the redox reaction of the substrate by the oxidoreductase at the enzyme electrode.

The substrate used in the present specification means a substance to be analyzed (also referred to as an analysis) when the enzyme electrode is used for a biosensor, and a fuel material when the enzyme electrode is used for a biofuel cell. means.

By using a cellulose derivative in the enzyme electrode of the present invention, the dispersibility and dispersion stability of the conductive carbon filler having a high specific surface area can be improved. The high specific surface area of the conductive carbon filler increases the effective area effective for the reaction and the amount of the enzyme carried, so that the current density of the enzyme electrode increases. This makes it possible to increase the output and sensitivity. Further, since the conductive carbon filler also functions as a supporting medium for enzymes and mediators, it is expected that the durability of the enzyme electrode will be improved. Further, since the enzyme electrode of the present invention does not contain an organic solvent or a surfactant, the enzyme electrode is in direct contact with a measurement object (subject) such as food, beverage, cell culture solution, or human body where contamination is a problem. Even if this is done, the object to be measured will not be contaminated. Cellulose derivatives are used as additives in foods and pharmaceuticals and are highly safe substances. The enzyme electrode of the present invention can be suitably used as an enzyme electrode for measuring a subject whose contamination is a problem, such as food, beverage, medium, cell culture reagent, blood product, and human body.

As an example of the enzyme electrode for contacting a subject, an electrode of a biosensor for biological monitoring, for example, an electrode of a biosensor for continuous glucose monitoring (abbreviated as CGM) can be mentioned. CGM can continuously measure the glucose concentration in the blood or interstitial fluid of the subject by a biosensor attached to the skin of the subject. Since the electrodes do not contain organic solvents or surfactants, it is possible to provide a highly safe biosensor for CGM. In addition, the electrodes of a biosensor for monitoring ketone bodies can be mentioned. The concentration of ketone bodies in the blood or interstitial fluid of the subject can be continuously measured, which can lead to the prevention of ketoacidosis in the subject. Since the electrode does not contain an organic solvent or a surfactant, a highly safe biosensor for ketone body monitoring can be provided. Further, the electrode of the biosensor for monitoring the medium in which the cell culture is performed can be mentioned. By continuously measuring glucose, lactic acid, glutamine, and glutamic acid with a biosensor inserted in the medium, it is possible to obtain information on the number of cells and cell metabolism. Since the electrodes do not contain organic solvents or surfactants, biosensors with very low cell toxicity can be provided.

(Biosensor)
The biosensor of the present invention can be constructed by using the enzyme electrode of the present invention. For example, the biosensor of the present invention has at least a pair of working and counter electrode, or at least one pair of working and counter electrode and reference electrode formed on an insulating substrate and is in contact with an enzyme. And a reagent layer containing media is formed.

The biosensor of the present invention includes amino acids such as hemoglobin, glycated hemoglobin, glutamine and glutamic acid, glycated amino acids such as fructosyl lysine and fructosylvaline, glycated peptides such as fructosylvalyl histidine, glycated albumins and sugars such as glucose. Amino acids such as vitamins such as vitamin C, alcohols, cholesterol, lysine, and ketone bodies (3-hydroxybutyric acid) can be detected.

Further, in the biosensor of the present invention, the reagent layer can contain both oxidoreductase and mediator, or only oxidoreductase.

The oxidoreductase used in the present invention includes an oxidase and a dehydrogenase. For example, glucose oxidase, lactate oxidase, cholesterol oxidase, bilirubin oxidase, glucose dehydrogenase, lactate dehydrogenase, amino acid oxidase, amino acid dehydrogenase, glutamate oxidase, glutamate dehydrogenase, fructosyl amino acid oxidase, fructosyl peptide oxidase, 3-hydroxybutyric acid dehydrogenase, alcohol oxidase. , Alcohol dehydrogenase and the like. These oxidoreductases are the amount of oxidoreductases that can be used to detect amino acids such as glucose, lactic acid, cholesterol, bilirubin, glutamine, and glutamic acid, glycated amino acids, or glycated peptides, ketone bodies (3-hydroxybutyric acid), and alcohol. Is, for example, per sensor or per measurement, for example, 0.01 to 100 U, preferably 0.05 to 10 U, and more preferably 0.1 to 5 U.

In addition, mediators include, but are not limited to, metal complexes (eg, osmium complex, ruthenium complex, iron complex, etc.), quinone compounds (eg, benzoquinone, naphthoquinone, phenanthrenquinone, phenanthrolinquinone, anthraquinone, and derivatives thereof). Etc.), phenazine compounds, viologen compounds, phenothiazine compounds, and phenol compounds. More specifically, potassium ferricyanide, hexaammine ruthenium, ferrocene, poly (1-vinylimidazole) -bis (bipyridine) chloroosmium, hydroquinone, 2-methyl-1,4-benzoquinone, 1,2-naphthoquinone-4-sulfon. Acid salt, 9,10-phenanthrenquinone-2-sulfonate, 9,10-phenanthrenquinone-2,7-disulfonate, 1,10-phenanthroline-5,6-dione, anthraquinone-2-sulfonate , Phenazine derivatives such as 1-methoxy-5-methylphenazinenium methylsulfate and 1-methoxy-5-ethylphenadinium ethylsulfate, methylviologen, benzylviologen, methyleneblue, methylenegreen, 2-aminophenol, 2-amino One or more selected from the group consisting of -4-methylphenol and 2,4-diaminophenol is used. Examples of the salt include, but are not limited to, sodium salt, potassium salt, calcium salt, magnesium salt, lithium salt and the like. The blending amount of the mediator is not particularly limited, and is, for example, 0.1 pmol to 1000 μmol, preferably 10 pmol to 500 μmol, and more preferably 500 pmol to 100 μmol per measurement or per sensor. ..

Further, the mediator is preferably a polymerized mediator bonded to a polymer from the viewpoint of durability of the sensor and suppression of outflow to the outside of the sensor. The polymer to which the mediator binds is a homopolymer, a random copolymer, a block copolymer, or a polymer to which they are bonded or mixed. The weight average molecular weight of the polymer is 10,000 or more, preferably 50,000 or more, more preferably 100,000 or more, and the upper limit of the weight average molecular weight is less than 10,000,000, preferably 1,000. Less than 000. The polymer is not particularly limited, but a polymer in which a plurality of atoms selected from at least one of a carbon atom, a nitrogen atom, an oxygen atom and a sulfur atom are bonded in a chain to form a main chain. Can be mentioned. Specific examples thereof include natural polymers such as proteins, polypeptides and polynucleotides, and synthetic polymers such as polyamino acids, polyimines, polyallyl compounds, poly (meth) acrylates, polyalkylene oxides, and copolymers thereof. Can be mentioned. Examples of polyamino acids include poly (L-glutamic acid) and poly (L-lysine). Moreover, as polyimine, polyalkyleneimine such as polyethyleneimine and polypropyleneimine can be mentioned. Further, examples of the polyallyl compound include polyallylamine and polydiallylamine. Further, examples of the polyalkylene oxide include polyethylene oxide and polypropylene oxide. The entire polymerized mediator is preferably hydrophilic, and the polymer to which the mediator is bound is preferably hydrophilic.

As the polymerized mediator, one in which the mediator and the polymer are bonded via a covalent bond can be used. As the covalent bond, for example, those described in JP-A-2003-514924 can be used, and specific examples thereof include ether bond, thioether bond, ester bond, urethane bond, amide bond and the like. it can. The covalent bond may be formed from the reactive groups originally possessed by the monomer forming the polymer, or may be formed from the reactive groups possessed by the linker separately introduced into the mediator or the polymer. As a specific example of the polymerized mediator, for example, a phenazine compound is used as the mediator, poly (L-lysine) is used as the polymer, and the phenazine compound and poly (L-lysine) are bonded via an amide bond. Can be mentioned.

In the biosensor of the present invention, high-sensitivity detection is possible by using an enzyme electrode capable of increasing the current density. In addition, since the enzyme electrode does not contain an organic solvent or a surfactant, even if the biosensor is brought into direct contact with the object to be measured, the object to be measured will not be contaminated, so that a highly safe biosensor can be used. Can be provided.

As an example of the biosensor, a biosensor for biological monitoring, for example, a biosensor for continuous glucose monitoring (abbreviated as CGM) can be mentioned. As described above, since the electrode does not contain an organic solvent or a surfactant, a highly safe biosensor for CGM can be provided.

(Bio fuel cell)
The biofuel cell of the present invention uses the enzyme electrode of the present invention for at least one of a positive electrode and a negative electrode. For example, when an enzyme electrode containing an oxidase is used for the negative electrode, when a saccharide is used as a fuel, the saccharide supplied to the negative electrode is oxidized to generate electrons and protons, and the electrons are carried to the positive electrode through an external circuit. At the positive electrode, protons, electrons, and oxygen react to produce water. If necessary, an electrolyte as a proton conductor may be used.

In the biofuel cell of the present invention, high output can be achieved by using an enzyme electrode capable of increasing the current density. Further, since the enzyme electrode does not contain an organic solvent or a surfactant, it does not contaminate the fuel, so that a highly safe biofuel cell can be provided.

Hereinafter, the present invention will be described in more detail with reference to Examples, but the present invention is not limited to the following Examples.

<Material>
The materials used in Examples and Comparative Examples are as follows. The abbreviations of the materials were appropriately marked and used.

(Conductive carbon filler)
・ Ketjen Black (EC-300J: manufactured by Lion Specialty Chemicals Co., Ltd.)
DBP oil absorption (cm ³ / 100g): 360 (9g method)
BET specific surface area (m ² / g): 800
Volatile content (%): 0.5
pH: 9.0
Ash content (%): 0.05
Primary particle size (nm): 39.5
・ Acetylene carbon black (100% compression: manufactured by Strem Chemicals)
Specific surface area (m ² / g): 80
Average particle size: 0.042 micron, porous carbon (Knobel MJ (4) 010: manufactured by Toyo Tanso Co., Ltd.)
BET specific surface area (m ² / g): 1100
Design mesopore diameter (nm): 10
Total pore volume (mL / g): 2.0
Micropore volume (mL / g): 0.4
Bulk density (g / mL): 0.10.
Here, the Knobel MJ (4) 010 has a communication hole to which the meso holes are connected.
-Graphene (graphene, nanoplatelet (900394): manufactured by Sigma-Aldrich)
Specific surface area (m ² / g): 300
Average particle size: less than 2 microns Bulk density (g / cm ³ ): 0.2-0.4
-Carbon nanotubes (Carbon nanotube, few-walled (900788): manufactured by Sigma-Aldrich)
Diameter x length: 2.5 to 3 nm x 2 to 6 μm
G / D ratio: ≧ 10 (Raman)
Median number of layers: 2-3 (TEM)
BET specific surface area (m ² / g): 700 or more ^{Bulk density (g / cm 3} ): 0.05 (ASTMD7481)

(Cellulose derivative)
-Carboxymethyl cellulose (cellogen F-7A: manufactured by Dai-ichi Kogyo Seiyaku Co., Ltd.) (hereinafter abbreviated as CMC)
2% aqueous solution viscosity (mPa · s / 25 ° C): 12-18
Degree of etherification: 0.70 to 0.80
-Hydroxypropyl cellulose (NISSO HPC L: manufactured by Nippon Soda) (hereinafter abbreviated as HPC L)
2% aqueous solution viscosity (mPa · s / 20 ° C): 6-10
-Hydroxypropyl cellulose (NISSO HPC H: manufactured by Nippon Soda) (hereinafter abbreviated as HPC H)
2% aqueous solution viscosity (mPa · s / 20 ° C): 1000-4000
-Methyl cellulose (SM-4000: manufactured by Shin-Etsu Chemical Co., Ltd.) (hereinafter abbreviated as MC (S))
Degree of Substitution (Average number of hydroxyl groups substituted with methoxy groups per glucose ring unit of cellulose: 1.8
-Methyl cellulose (M0290: manufactured by Tokyo Chemical Industry Co., Ltd.) (hereinafter abbreviated as MC (T))
2% aqueous solution viscosity (mPa · s / 20 ° C.): 13-18
-Hydroxyethyl methyl cellulose (SEB-04T: manufactured by Shin-Etsu Chemical Co., Ltd.) (hereinafter abbreviated as HEMC)
Degree of Substitution (Average number of hydroxyl groups substituted with methoxy groups per glucose ring unit of cellulose: 1.5
Substitution number of moles (average number of moles of hydroxyethoxy groups added per glucose ring of cellulose): 0.20
-Hydroxypropyl methylcellulose (09963: manufactured by Sigma-Aldrich) (hereinafter abbreviated as HPMC)
2% aqueous solution viscosity (mPa · s / 25 ° C): ~ 15

(Material for manufacturing protective film)
The following materials were used to prepare the protective film.
-Poly (ter. Butyl methacrylate-b-4-vinylpyridine) (manufactured by Polymer Source) (hereinafter abbreviated as tBuMA4VP)
Number average molecular weight Mn: Poly (ter. Butyl methacrylate) 80,000
Poly (4-vinyl pyridine) 77,000
Mw / Mn: 1.15
-Random copolymer of polypropylene glycol methyl ether-styrene-4-vinylpyridine (manufactured by Polymer Source) (hereinafter abbreviated as PGMAS4VP)
Polypropylene glycol methyl ether: Styrene: 4-vinylpyridine = 24:16:60
Number average molecular weight Mn: 52,000
Weight average molecular weight Mw: 94,000
Mw / Mn: 1.8
-Polyethylene glycol dimethyl ether (manufactured by Sigma-Aldrich) (hereinafter abbreviated as PEGDGE)
Number average molecular weight Mn: ~ 1,000
2- [4- (2-Hyrodoxyethyl) -1-piperazinyl] ethanesulfonic acid (manufactured by Dojin Chemical Co., Ltd.) (hereinafter abbreviated as HEPES)

Example 1
In this example, Ketjen Black (EC-300J: manufactured by Lion Specialty Chemicals Co., Ltd.) was used as the conductive carbon filler, and the effect of the cellulose derivative on the dispersibility of Ketjen Black was investigated.

(experimental method)
Aqueous solutions of the following 6 types of cellulose derivatives were prepared using Milli-Q water.
-Carboxymethyl cellulose (cellogen F-7A)
-Hydroxypropyl cellulose (NISSO HPC L)
-Hydroxypropyl cellulose (NISSO HPC H)
-Hydroxyethyl methyl cellulose (SEB-04T)
Methyl cellulose (M0290)
Hydroxypropyl Methyl Cellulose (09963)

The cellulose derivative was adjusted to an aqueous solution having a predetermined concentration, and the adjusted aqueous solution was filtered in advance with a filter having a pore size of 0.8 μm (25CS080AS manufactured by Advantech) for HEMC and MC (T). Next, Ketjen black was added to each prepared aqueous solution of the cellulose derivative so that the concentration was 1.5 mg / mL. Then, the mixture was stirred with a vortex mixer (VORTEX GENIE2) for about 30 seconds, and then treated with an ultrasonic bath for 15 minutes. When HEMC was used, the treatment was performed with an ultrasonic homogenizer for about 30 seconds instead of the ultrasonic bath. Next, after stirring again with a vortex mixer for 30 seconds, the dispersion treatment liquid was filtered through a filter having a pore size of 0.8 μm. 100 μL of the filtrate (hereinafter referred to as sample solution) was put into a microplate, and the absorption spectrum of the sample solution was measured with a plate reader. Here, as the microplate, UV-Star (registered trademark) 96Well F-Boden manufactured by Greiner Bio One Co., Ltd. was used. Further, as the plate reader, infinite (registered trademark) M200 Pro manufactured by Tecan Co., Ltd. was used.

(result)
Table 1 shows the absorbance values of each sample solution at a wavelength of 760 nm. In addition, the case of dispersing with an ultrasonic bath using only Milli-Q water without containing a cellulose derivative was designated as Comparative Example 1, and the case of dispersing with an ultrasonic homogenizer was designated as Comparative Example 2. The absorbance of each sample solution in Table 1 is a value after background correction obtained by subtracting the absorbance of each cellulose derivative aqueous solution or milliQ water only at a wavelength of 760 nm.

It can be seen that when the cellulose derivative is added, the absorbance at 760 nm is higher than that in the case of using only milliQ water. In addition, when the cellulose derivative was added visually, the sample solution was almost transparent when only Milli-Q water was used, whereas the color of the sample solution was blacker. This indicates that, as compared with the case of using only Milli-Q water, a large amount of Ketjen black was present in the filtrate even after the filtration treatment, and the dispersibility of Ketjen black was improved.

Example 2
In this example, the same procedure as in Example 1 was carried out except that acetylene carbon black (100% compressed: manufactured by Strem Chemicals) was used as the conductive carbon filler. The results are shown in Table 2.

Also in this example, it can be seen that when the cellulose derivative is added, the absorbance at 760 nm is higher than that in the case of using only milliQ water. In addition, when the cellulose derivative was added visually, the sample solution was almost transparent when only Milli-Q water was used, whereas the color of the sample solution was blacker. This indicates that a large amount of acetylene carbon black was present in the filtrate even after the filtration treatment as compared with the case of using only Milli-Q water, and the dispersibility of acetylene carbon black was improved.

Example 3
In this example, graphene (graphene, nanoplatelet (900394): manufactured by Sigma-Aldrich) was used as the conductive carbon filler and added so as to have a concentration of 3.0 mg / mL, instead of the ultrasonic bath. The procedure was the same as in Example 1 except that the treatment was performed with an ultrasonic homogenizer for about 30 seconds. The results are shown in Table 3.

Also in this example, it can be seen that when the cellulose derivative is added, the absorbance at 760 nm is higher than that in the case of using only milliQ water. In addition, when the cellulose derivative was added visually, the color of the sample solution was blacker than that in the case of using only Milli-Q water. This indicates that graphene was abundantly present in the filtrate even after the filtration treatment as compared with the case of using only Milli-Q water, and the dispersibility of graphene was improved.

Example 4
In this example, carbon nanotubes (Carbon nanotube, few-walled (900788): manufactured by Sigma Aldrich Co., Ltd.) were used as the conductive carbon filler and added to the aqueous solution of the cellulose derivative so as to have a concentration of 6.0 mg / mL. The same procedure as in Example 1 was carried out except that the treatment was performed with an ultrasonic homogenizer for about 30 seconds instead of the ultrasonic bath. The results are shown in Table 4.

Also in this example, it can be seen that when the cellulose derivative is added, the absorbance at 760 nm is higher than that in the case of using only milliQ water. In addition, when the cellulose derivative was added visually, the sample solution was almost transparent when only Milli-Q water was used, whereas the color of the sample solution was blacker. This indicates that, as compared with the case of using only Milli-Q water, a large amount of carbon nanotubes were present in the filtrate even after the filtration treatment, and the dispersibility of the carbon nanotubes was improved.

Example 5
In this example, porous carbon (Knobel MJ (4) 010: manufactured by Toyo Carbon Co., Ltd.) was used as the conductive carbon filler and added to the aqueous cellulose derivative solution so as to have a concentration of 6.0 mg / mL, and an ultrasonic bath was used. Instead, the treatment was carried out in the same manner as in Example 1 except that the treatment was performed with an ultrasonic homogenizer for about 30 seconds. The results are shown in Table 5.

Also in this example, it can be seen that when the cellulose derivative is added, the absorbance at 760 nm is higher than that in the case of using only milliQ water. In addition, when the cellulose derivative was added visually, the sample solution was almost transparent when only Milli-Q water was used, whereas the color of the sample solution was blacker. This indicates that, as compared with the case of using only Milli-Q water, a large amount of porous carbon was present in the filtrate even after the filtration treatment, and the dispersibility of the porous carbon was improved.

Example 6
In this example, Ketjen Black (EC-300J: manufactured by Lion Specialty Chemicals Co., Ltd.) was used as the conductive carbon filler, and the effect of the cellulose derivative on the dispersibility stability of Ketjen Black was investigated.

(experimental method)
Aqueous solutions of the following three types of cellulose derivatives were prepared using Milli-Q water.
-Carboxymethyl cellulose (cellogen F-7A)
-Hydroxypropyl cellulose (NISSO HPC L)
-Hydroxypropyl cellulose (NISSO HPC H)

Ketjen black was added to the prepared aqueous solution of the cellulose derivative so that the concentration was 1 mg / mL. Then, after stirring with a vortex mixer for about 30 seconds, it was treated with an ultrasonic bath for 15 minutes. Next, after stirring again with a vortex mixer for 30 seconds, the dispersion treatment liquid was centrifuged at 2000 G for 5 minutes using a centrifuge (Kubota 7780). The supernatant of the centrifuged sample was taken and diluted 4-fold with Milli-Q water. 100 μL of the supernatant diluted solution (hereinafter referred to as sample solution) was put into a microplate, and the absorption spectrum of the sample solution was measured with a plate reader. The results are shown in Table 6. The absorbance of each sample solution in Table 6 is a value after background correction obtained by subtracting the absorbance of each cellulose derivative aqueous solution or milliQ water only at a wavelength of 760 nm.

It can be seen that when the cellulose derivative is added, the absorbance at 760 nm is higher than that in the case of using only milliQ water. In addition, when the cellulose derivative was added visually, the color of the sample solution was blacker than that in the case of using only Milli-Q water. This indicates that, as compared with the case of using only Milli-Q water, a large amount of Ketjen black was present in the supernatant liquid even after the centrifugation treatment, and the dispersion stability of Ketjen black was improved.

Example 7
In this example, HEMC and MC (S) were used as the cellulose derivative, and the prepared aqueous solution of the cellulose derivative was filtered through a filter having a pore size of 0.8 μm, and then Ketjen black was added so that the concentration became 3 mg / mL. The same procedure as in Example 6 was carried out except that the sample solution was treated with an ultrasonic homogenizer instead of the ultrasonic bath and a 10-fold diluted supernatant solution was used as the sample solution. The results are shown in Table 7.

Also in this example, a large amount of Ketjen black is present in the supernatant, indicating that the dispersion stability of Ketjen black is improved.

Example 8
In this example, the same procedure as in Example 6 was carried out except that acetylene carbon black (100% compressed: manufactured by Strem Chemicals) was used as the conductive carbon filler. The results are shown in Table 8.

Also in this example, it can be seen that when the cellulose derivative is added, the absorbance at 760 nm is higher than that in the case of using only milliQ water. In addition, when the cellulose derivative was added visually, the color of the sample solution was blacker than that in the case of using only Milli-Q water. This indicates that, as compared with the case of using only Milli-Q water, a large amount of acetylene carbon black was present in the supernatant liquid even after the centrifugation treatment, and the dispersion stability of the acetylene carbon black was improved.

Example 9
In this example, porous carbon (Knobel MJ (4) 010: manufactured by Toyo Tanso Co., Ltd.) was used as the conductive carbon filler and added so as to have a concentration of 3 mg / mL. The treatment was carried out in the same manner as in Example 6 except that the treatment was performed with an ultrasonic homogenizer for about 30 seconds. The results are shown in Table 9.

Also in this example, it can be seen that when the cellulose derivative is added, the absorbance at 760 nm is higher than that in the case of using only milliQ water. In addition, when the cellulose derivative was added visually, the color of the sample solution was blacker than that in the case of using only Milli-Q water. This indicates that, as compared with the case of using only Milli-Q water, a large amount of porous carbon was present in the supernatant liquid even after the centrifugation treatment, and the dispersion stability of the porous carbon was improved.

Example 10
In this embodiment, an enzyme electrode (also referred to as a mixed enzyme electrode) having a mixed reagent portion composed of one layer of a reagent layer containing a conductive carbon filler, an oxidoreductase and a mediator is prepared on the surface of the gold electrode. , The responsiveness of the enzyme electrode was evaluated.

(Preparation of carbon dispersion)
As the conductive carbon filler, Ketjen Black (EC-300J: manufactured by Lion Specialty Chemicals Co., Ltd.) was used. As the cellulose derivative, hydroxypropyl cellulose (NISSO HPC L: manufactured by Nippon Soda Co., Ltd.) was used. HPC L was dissolved in milliQ water at a concentration of 2.5 mg / mL, and Ketjenblack was added to the solution at a concentration of 4 mg / mL. Then, it was treated with an ultrasonic homogenizer for about 10 minutes, and allowed to stand for 3 days or more to obtain a carbon dispersion liquid. Then, the ultrasonic treatment was performed again for about 5 minutes before use.

(Synthesis of polymerized mediator)
As the polymerized mediator, a polymerized mediator (hereinafter, abbreviated as PLL-PEG25-Ph) in which phenazine was bound to poly (L-lysine) via a covalent bond was synthesized and used.

As the phenazine compound, the phenazine compound shown in [Chemical Formula 1] below was used. Further, PLL-PEG25-Ph can be expressed by the following general formula of [Chemical formula 2]. X in the formula is the introduction rate of the phenazine compound, and can usually be in the range of 0 <X ≦ 1.

[Chemical 1]

[Chemical 2]

Weigh 2.5 mg of the phenazine compound (manufactured by Tokyo Chemical Industry Co., Ltd.) shown in [Chemical Formula 1], and add 200 mM sodium chloride (NaCl) to 10 mM sodium phosphate buffer (hereinafter abbreviated as PB) (pH 7.4). A phenazine-containing solution was prepared by dissolving in 167 μL of the added solution. On the other hand, as the polymer, 1.07 mg of poly (L-lysine) hydrochloride (PLKC800 manufactured by Alamanda Polymers) was weighed and dissolved in 1033 μL of a solution containing NaCl in PB to prepare a polymer-containing solution. The phenazine-containing liquid and the polymer-containing liquid were mixed and reacted at room temperature for 4 hours with stirring. Here, the molar ratio of the phenazine compound to the poly (L-lysine) hydrochloride at the time of preparation (phenazine compound / poly (L-lysine) hydrochloride) is the number of moles in terms of monomer for the poly (L-lysine) hydrochloride. Is about (18/100).

Then, the reaction solution was subjected to gel filtration chromatography using a PD-10 column (manufactured by GE Healthcare) using a solution prepared by adding NaCl to PB as an elution buffer. The solution after gel filtration was subjected to ultrafiltration using a centrifugal ultrafiltration filter (Amicon Ultra-450k; manufactured by Merck Millipore).

The obtained PLL-PEG25-Ph was adjusted to a predetermined concentration by adding 100 μL of the solution to a microplate and measuring the absorption spectrum with a plate reader. Here, as the microplate, UV-Star (registered trademark) 96Well F-Boden manufactured by Greiner Bio One Co., Ltd. was used. Further, as the plate reader, infinite (registered trademark) M200 Pro manufactured by Tecan Co., Ltd. was used.

(Preparation of carbon / oxidoreductase / mediator mixed solution)
Sodium phosphate buffer (pH 6.5) prepared with disodium hydrogen phosphate (manufactured by Wako Pure Chemical Industries, Ltd.) and sodium dihydrogen phosphate (manufactured by Wako Pure Chemical Industries, Ltd.), glucose dehydrogenase (FAD-dependent) ( BBI International's GDH GLD1), glutaaldehyde 25% solution (manufactured by Wako Pure Chemical Industries, Ltd.) and the prepared PLL-PEG25-Ph, carbon dispersion reagents are mixed so as to have the composition shown in Table 10 below. , Reacted for about 1 hour.

(Making an enzyme electrode)
0.5 μL of a carbon / enzyme / mediator mixed solution was applied onto the gold electrode and dried for about 15 minutes. Further, coating and drying were repeated twice, and the carbon / oxidoreductase / mediator mixed solution was applied three times in total. Then, it was dried for 2 hours to obtain a mixed enzyme electrode containing carbon, oxidoreductase, and mediator.

Comparative Example 11
An enzyme electrode was prepared in the same manner as in Example 10 except that the composition not containing the conductive carbon filler shown in Table 11 below was used, and the responsiveness of the enzyme electrode was evaluated.

(Enzyme / mediator solution)

(Evaluation of responsiveness of enzyme electrodes)
A three-electrode type using the prepared enzyme electrode as the working electrode, a gold electrode as the counter electrode, and an Ag / AgCl electrode (saturated KCl) (manufactured by BAS) as the reference electrode, and a potential made by BAS. Using a stat, the time change of the current was measured by the amperometric method in phosphate buffered physiological saline. Specifically, glucose was added so that the theoretical values were 50, 150, 300, 500, 700 mg / dL every 100 seconds from 150 seconds after the start of measurement, and the current response value was continuously measured. The current value of each glucose is the measured value of 5 points 5 seconds before, 10 seconds before, 15 seconds before, 20 seconds before, and 25 seconds before the next glucose addition after the glucose to be measured is added. Calculated from the average value. The final current value is the average value of the measured values at 5 points 80 seconds, 85 seconds, 90 seconds, 95 seconds, and 100 seconds after adding the glucose solution so that the glucose concentration becomes 700 mg / dL. Calculated from. The current value of each glucose concentration is the current value after background correction obtained by subtracting the current value of the glucose concentration of 0 mg / dL.

(result)
FIG. 1 is a graph showing the relationship between the current value and the glucose concentration in the mixed enzyme electrode of Example 10 and the enzyme electrode of Comparative Example 11 not containing the conductive carbon filler. Table 12 summarizes the measurement results at each glucose concentration. The results in FIGS. 1 and 12 are the average values of the three measured values of each enzyme electrode.

In both Example 10 and Comparative Example 11, an increase in the current value depending on the glucose concentration could be confirmed. In addition, Example 10 showed a higher current response than Comparative Example 11. This indicates that the presence of the conductive carbon filler increased the effective area effective for the reaction, resulting in an increase in the current density of the enzyme electrode.

Example 11
In this embodiment, an enzyme electrode (laminated enzyme electrode) in which a first reagent layer (conductive carbon layer) and a second reagent layer (layer containing an oxidoreductase and a mediator) are laminated on the surface of a gold electrode. (Also known as) was prepared and the electrode durability was evaluated.

(Preparation of polymer solution for protective film)
Poly (ter. Butyl methacrylate-b-4-vinylpyridine), a random copolymer of polypropylene glycol methyl ether-styrene-4-vinylpyridine, and polyethylene glycol dimethyl ether dissolved in ethanol were added to 2- [4- (2-). It was mixed with a HEPES buffer (pH 8.0) prepared using hirodoxyethyl) -1-piperazinyl] ethanesulfonic acid so as to have the composition shown in Table 13 below.

(Making an enzyme electrode)
0.29 μL of the carbon dispersion used in Example 10 was applied onto the gold electrode and dried for about 15 minutes. Further, coating and drying were repeated twice, and the carbon dispersion was applied three times in total. After the final coating, it was dried for about 90 minutes to form a conductive carbon layer. Next, 0.5 μL of the redox enzyme / mediator solution after the 1-hour reaction used in Comparative Example 11 was applied to the conductive carbon layer and dried for about 15 minutes. Further, coating and drying were repeated twice, and the solution was applied three times in total. Then, it was dried for 2 hours to form a reagent layer, and a laminated enzyme electrode in which a conductive carbon layer and a layer containing an oxidoreductase were laminated on a gold electrode was obtained. The laminated enzyme electrode was immersed in a polymer solution for a protective film, pulled up, dried for 8 minutes, and then immersed again. This operation was repeated 12 times in total and dried for 1 day to form a protective film and obtain an enzyme electrode.

Example 12
In this embodiment, an enzyme electrode having a mixed reagent portion composed of a single reagent layer containing a conductive carbon filler, an oxidoreductase and a mediator is prepared on the surface of the gold electrode, and the durability of the enzyme electrode is evaluated. went.

(Making an enzyme electrode)
A protective film was formed on the mixed enzyme electrode prepared in the same manner as in Example 10 by the same method as in Example 11 to obtain an enzyme electrode.

Comparative Example 12
An enzyme electrode containing no conductive carbon filler having a protective film formed by the same method as in Example 11 was prepared on the enzyme electrode prepared by the same method as in Comparative Example 11, and the durability of the enzyme electrode was evaluated. ..

(Evaluation of durability of enzyme electrodes)
A three-electrode type using the prepared enzyme electrode as the working electrode, a gold electrode as the counter electrode, and an Ag / AgCl electrode (saturated KCl) (manufactured by BAS) as the reference electrode, and a potential made by BAS. Using a stat, the time change of the current was measured by the amperometric method in phosphate buffered physiological saline. Specifically, glucose was added so as to have a theoretical value of 100, 300, 500, 700 mg / dL every 500 seconds from 1000 seconds after the start of measurement, and the current response value was continuously measured. After the measurement, the electrode was stored in phosphate buffered saline at 37 ° C., stored for 7 days, and then the same measurement was performed. The current value of each glucose is the measured value of 5 points 5 seconds before, 10 seconds before, 15 seconds before, 20 seconds before, and 25 seconds before the next glucose addition after the glucose to be measured is added. Calculated from the average value. The final current value is the average value of the measured values at 5 points after adding the glucose solution so that the glucose concentration becomes 700 mg / dL, 480 seconds, 485 seconds, 490 seconds, 495 seconds, and 500 seconds. Calculated from. The current value of each glucose concentration is the current value after background correction obtained by subtracting the current value of the glucose concentration of 0 mg / dL.

(result)
FIG. 2 is a graph showing the relationship between the current value and the glucose concentration in the laminated enzyme electrode of Example 11 on the first day (immediately after production) and after 7 days. Further, FIG. 3 is a graph showing the relationship between the current value and the glucose concentration in the mixed enzyme electrode of Example 12 on the first day and after 7 days. Further, FIG. 4 is a graph showing the relationship between the current value and the glucose concentration on the first day and after 7 days in the enzyme electrode containing no conductive carbon filler of Comparative Example 12. Table 14 shows the current values on the first day and after 7 days and the current retention rate (relative to the current value on the first day of the current value on the 7th day) in Examples 11 and 12 and Comparative Example 12 when the glucose concentration was 700 mg / dL. Ratio) is shown. Further, the results of Examples 11 and 12 are the average values of the measured values of each of the three enzyme electrodes, and the results of Comparative Example 12 are the average values of the measured values of the two enzyme electrodes.

In all of Examples 11 and 12 and Comparative Example 12, an increase in the current value depending on the glucose concentration could be confirmed. In addition, Examples 11 and 12 had a higher current retention rate after 7 days and were excellent in durability as compared with Comparative Example 12. Further, as compared with Example 11, Example 12 had a higher current retention rate after 7 days and was excellent in durability.

The present invention can provide a highly sensitive and highly safe biosensor using not only an enzyme but also a biomolecule other than the enzyme, for example, an antibody, a peptide, or DNA.

Claims (22)

An enzyme electrode containing a reagent portion formed on a conductive substrate, wherein the reagent portion is an enzyme electrode containing an oxidoreductase, a conductive carbon filler, and a cellulose derivative. The enzyme electrode according to claim 1, wherein the cellulose derivative is hydrophilic. The enzyme electrode according to claim 1 or 2, wherein the reagent section further contains a mediator. The enzyme electrode according to claim 3, wherein the mediator is bound to a polymer. The enzyme electrode according to any one of claims 1 to 4, wherein the reagent portion contains 0.1 to 30 parts by weight of the cellulose derivative with respect to 1 part by weight of the conductive carbon filler. The enzyme electrode according to any one of claims 1 to 5, wherein the conductive carbon filler is carbon black, graphite powder, porous carbon or nanocarbon. The invention according to any one of claims 1 to 6, wherein the cellulose derivative is at least one selected from the group consisting of methyl cellulose, carboxymethyl cellulose, hydroxyethyl cellulose, hydroxypropyl cellulose, hydroxyethyl methyl cellulose, and hydroxypropyl methyl cellulose. Enzyme electrode. The enzyme electrode according to any one of claims 1 to 7, wherein the reagent portion is covered with a protective film. The one according to any one of claims 1 to 8, wherein the reagent section comprises one reagent layer, and the one-layer reagent layer contains the oxidoreductase, the conductive carbon filler, and the cellulose derivative. Enzyme electrode. The reagent section is composed of a plurality of laminated reagent layers, and the plurality of reagent layers include one or more first reagent layers and one or more second reagent layers, and the first reagent layer. The enzyme electrode according to any one of claims 1 to 8, wherein the second reagent layer contains the conductive carbon filler and the cellulose derivative, and the second reagent layer contains the oxidoreductase. The enzyme electrode according to claim 10, wherein one layer of the first reagent layer and one layer of the second reagent layer are laminated in this order on the conductive substrate. A claim that one layer of the first reagent layer, one layer of the second reagent layer, and one layer of the first reagent layer are laminated in this order on the conductive substrate. Item 10. The enzyme electrode according to item 10. The enzyme electrode according to claim 10, wherein one layer of the second reagent layer and one layer of the first reagent layer are laminated in this order on the conductive substrate. The enzyme electrode according to any one of claims 1 to 13, wherein the substrate is one selected from the group consisting of glucose, lactic acid, ketone bodies, glutamic acid, and glutamine. The oxidoreductase is one selected from the group consisting of glucose oxidase, glucose dehydrogenase, lactate oxidase, lactate dehydrogenase, ketone body dehydrogenase, glutamate oxidase, and glutamate dehydrogenase, any one of claims 1 to 14. The enzyme electrode according to. The enzyme electrode according to any one of claims 1 to 15, wherein the object to be measured is food, a beverage, a medium, a reagent for cell culture, a blood product, or a human body. A method for producing an enzyme electrode having a reagent portion composed of a plurality of reagent layers laminated on a conductive substrate.
It includes a step of preparing a carbon dispersion containing a cellulose derivative and a conductive carbon filler, a step of preparing a solution containing an oxidoreductase, and a step of forming a reagent portion.
The step of forming the reagent portion includes a step of forming a first reagent layer using the carbon dispersion and a step of forming a second reagent layer using the solution containing the oxidoreductase, respectively. A method for producing an enzyme electrode, which has more than one time. A method for producing an enzyme electrode having a reagent portion formed on a conductive substrate.
A method for producing an enzyme electrode, which comprises preparing a coating solution containing a cellulose derivative, a conductive carbon filler, and an oxidoreductase, and applying the coating solution to a conductive substrate to form the reagent portion. The production method according to claim 17, wherein the carbon dispersion liquid contains 0.1 to 30 parts by weight of the cellulose derivative with respect to 1 part by weight of the conductive carbon filler. The production method according to claim 18, wherein the coating liquid contains 0.1 to 30 parts by weight of the cellulose derivative with respect to 1 part by weight of the conductive carbon filler. A biosensor having at least one pair of working electrode and counter electrode and a reference electrode for detecting or quantifying an analysis, wherein the enzyme electrode according to any one of claims 1 to 16 is attached to the working electrode. Biosensor to be used. A biofuel cell having a positive electrode and a negative electrode, wherein the enzyme electrode according to any one of claims 1 to 15 is used for at least one of the positive electrode and the negative electrode.

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