WO2016129273A1

WO2016129273A1 - Method for manufacturing enzyme electrode, and enzyme electrode

Info

Publication number: WO2016129273A1
Application number: PCT/JP2016/000677
Authority: WO
Inventors: 松彦西澤; 雄大小川
Original assignee: 国立大学法人東北大学
Priority date: 2015-02-09
Filing date: 2016-02-09
Publication date: 2016-08-18
Also published as: JPWO2016129273A1

Abstract

A method for manufacturing an enzyme electrode characterized in that a substrate is imparted with a carbon material in the presence of an oxidoreductase whereby an enzyme electrode containing the carbon material and the oxidoreductase is obtained. An enzyme electrode characterized in containing a carbon material, an oxidoreductase, and at least one binding molecule selected from the group consisting of a binding molecule A for binding carbon materials to each other and a binding molecule B for binding the carbon material and the oxidoreductase to each other.

Description

Method for producing enzyme electrode and enzyme electrode

The present invention relates to a method for producing an enzyme electrode and an enzyme electrode.

An electrode (enzyme electrode) in which an enzyme is immobilized on an electrode material is known as a core component that determines the electrochemical performance of a biosensor or biobattery.
Biosensors are already used as indispensable tools for measurement in the medical, food, and environmental fields, and biocells (biofuel cells) are used in this field as an extremely safe power source that is friendly to living organisms and the environment. Future practical application is expected in the future.

As the enzyme of the enzyme electrode, an enzyme that catalyzes a redox reaction that accompanies exchange of electrons is usually used. These enzymes oxidize and reduce organic molecules, which are sensing targets in biosensors and fuel in biocells, and generate electric currents in the above devices, so that electrical information and information can be obtained from the presence and / or amount of organic molecules. It functions as a transducer that obtains energy.

On the other hand, as an electrode material for enzyme electrodes, nanocarbons such as carbon nanotubes (CNT) and fine carbon materials such as particulate carbon have been used in recent years (

Patent Documents

1 and 2 and Non-Patent Document 1). reference).

JP 2007-035437 A International Publication No. 2012/002290

However, the conventional enzyme electrode production method inevitably includes a plurality of steps, so that the overall process becomes complicated and extra production costs are generated (Patent Document 1). 2).
Moreover, in the enzyme electrode manufactured by the conventional method for manufacturing an enzyme electrode, it is difficult to obtain the uniformity of enzyme adsorption to the carbon material, and it is difficult to obtain the reproducibility of the performance of the enzyme electrode (Patent Document 1, 2), and there is room for improvement in that the carbon material such as CNT is likely to collapse or the enzyme leaks out and the structure of the enzyme electrode is low in stability (see Non-Patent Document 1).

Therefore, an object of the present invention is to produce an enzyme electrode simply and at low cost.

As a result of diligent research to solve the above problems, the present inventor solved the above problems by applying and drying a mixture of an oxidoreductase and a carbon material on an electrode substrate in a one-step process. The inventors have found that this is possible and have completed the present invention.

The gist of the present invention is as follows.
The method for producing an enzyme electrode of the present invention is characterized in that an enzyme electrode containing a carbon material and an oxidoreductase is obtained by applying the carbon material to a substrate under conditions where the oxidoreductase is present.
In the present specification, “oxidoreductase” refers to an enzyme that catalyzes an oxidation reaction and / or an enzyme that catalyzes a reduction reaction.

In the method for producing an enzyme electrode of the present invention, it is possible to include an electrode forming step of forming the enzyme electrode. Here, it is preferable to use a printing technique in the electrode forming step.

In the method for producing an enzyme electrode of the present invention, a suspension adjustment step of adjusting a suspension containing the carbon material and the oxidoreductase, and a suspension for applying the suspension to the substrate It is preferable that an application process is included.

Here, in the method for producing an enzyme electrode of the present invention, in addition to the oxidoreductase, a binding molecule A that binds the carbon materials and a binding molecule B that binds the carbon material and the oxidoreductase. It is preferable that the condition further includes at least one binding molecule selected from the group.
At this time, the binding molecule is preferably the binding molecule A, and more preferably the binding molecule A and the binding molecule B.

Here, it is preferable that the method for producing an enzyme electrode of the present invention includes a freeze-drying step in which trehalose is further present in addition to the oxidoreductase, and the enzyme electrode is freeze-dried.

The enzyme electrode of the present invention is selected from the group consisting of the carbon material, the oxidoreductase, a binding molecule A that binds the carbon materials, and a binding molecule B that binds the carbon material and the oxidoreductase. At least one binding molecule.
At this time, the binding molecule is preferably the binding molecule A, and more preferably the binding molecule A and the binding molecule B.

Here, the enzyme electrode of the present invention preferably further contains trehalose. The enzyme electrode of the present invention is preferably lyophilized.

In the method for producing an enzyme electrode of the present invention and the enzyme electrode of the present invention, the binding molecule A is at least one selected from the group consisting of a small molecule or polymer having an aromatic group, and a fluorine-substituted hydrocarbon. Preferably, it is at least one selected from the group consisting of polyvinylimidazole (PVI), poly (sodium 4-styrenesulfonate), and Nafion (registered trademark). The binding molecule B has a small molecule having an aromatic group and at least one selected from the group consisting of an N-hydroxysuccinimide ester group, an aldehyde group, an epoxy group, a maleimide group, a carbodiimide group, and a hydrazide group, or It is preferably a polymer, and more preferably at least one selected from the group consisting of 1-pyrenebutyric acid N-hydroxysuccinimide ester (PBSE) and N-benzoyloxysuccinimide.

According to the present invention, an enzyme electrode can be produced easily and at low cost.

It is a figure shown about the outline | summary of the manufacturing method of the enzyme electrode of embodiment of this invention. It is a figure which shows the result of the output performance measurement by the cyclic voltammetry of the enzyme electrode of Example 1. It is a figure which shows the result of the output performance measurement by the chronoamperometry of the enzyme electrode of Example 1. It is a figure which shows the result of the output performance measurement by the chronoamperometry of the enzyme electrode of Example 2, 3 (together with the result of Example 1). (A) is a figure which shows the result of the output performance measurement by cyclic voltammetry of the enzyme electrode of Example 4, (b) is the result of the output performance measurement by chronoamperometry of the enzyme electrode of Example 4. FIG. (A) is a figure which shows the result of the output performance measurement by cyclic voltammetry of the enzyme electrode of Example 5, (b) is the figure which shows the result of output performance measurement by the chronoamperometry of the enzyme electrode of Example 5. FIG. (A) is a figure which shows the result of the output performance measurement by the cyclic voltammetry of the enzyme electrode of Example 6, (b) shows the result of the output performance measurement by the chronoamperometry of the enzyme electrode of Example 6. FIG. (A) is a figure which shows the result of the output performance measurement by cyclic voltammetry of the enzyme electrode of Example 7, (b) is the result of the output performance measurement by chronoamperometry of the enzyme electrode of Example 7. FIG. (A) is a figure which shows the result of the output performance measurement by cyclic voltammetry of the enzyme electrode of Example 8, (b) is the result of the output performance measurement by chronoamperometry of the enzyme electrode of Example 8. FIG. (A) is a figure which shows the result of the output performance measurement by the cyclic voltammetry of the enzyme electrode of Example 9, (b) is the figure of the output performance measurement by the chronoamperometry of the enzyme electrode of Example 9. FIG. (A) is a figure which shows the result of the output performance measurement by the cyclic voltammetry of the enzyme electrode of Example 10, (b) is the figure which shows the result of the output performance measurement by the chronoamperometry of the enzyme electrode of Example 10. FIG.

Hereinafter, with reference to the drawings, a method for producing an enzyme electrode of the present invention and an embodiment of the enzyme electrode of the present invention will be described in detail.

(Method for producing enzyme electrode)
The method for producing an enzyme electrode according to an embodiment of the present invention (hereinafter, also referred to as “the production method of the present embodiment”) includes applying a carbon material to a substrate under conditions where an oxidoreductase is present. And obtaining an enzyme electrode containing a carbon material and an oxidoreductase.
FIG. 1 shows an outline of the manufacturing method of the present embodiment.

In the manufacturing method of the present embodiment, for example, a suspension containing a carbon material and an oxidoreductase is prepared, and this suspension is applied (described later), and a powder containing the carbon material and the oxidoreductase is prepared. The powder can be applied to the base material, or a paste-like sol containing a carbon material and an oxidoreductase can be prepared, and the sol can be applied to the base material.

Hereinafter, effects obtained by the present invention will be described.
As a conventional method for producing an enzyme electrode, a carbon material such as nanocarbon or particulate carbon is formed into a porous structure, and then an enzyme solution is applied to the porous structure to thereby apply the enzyme to the carbon material. Examples include a production method for adsorbing, a pretreatment step such as a degassing treatment or a surfactant treatment for carbon nanotubes, and then a production method for performing an enzyme inclusion step of immersing the pretreated carbon nanotubes in an enzyme solution. It is done.
However, since these conventional manufacturing methods necessarily include a plurality of steps, the entire process becomes complicated and extra manufacturing costs are generated.
According to the manufacturing method of the present embodiment, as described above, the application / drying of the mixture of the oxidoreductase and the carbon material on the electrode substrate can be performed in a single step, so that it is simple and low-cost. The enzyme electrode can be manufactured with

Also, in the above conventional method for producing an enzyme electrode, the shape of the electrode is naturally determined according to the shape of the carbon material forming the skeleton of the electrode. On the other hand, according to the manufacturing method of the present embodiment, it is possible to include a step of forming an enzyme electrode (described later). In this respect, it is different from the conventional enzyme electrode manufacturing method.

In the manufacturing method of this embodiment, as shown in FIG. 1, it is possible to include an electrode forming step of forming an enzyme electrode. In addition, in the manufacturing method of the enzyme electrode of this invention, it is not limited to this, The electrode shaping | molding process may not be included.

[[Electrode forming step]]
In this step, for example, an enzyme electrode can be formed by drafting a desired shape on the substrate and then applying the carbon material to the substrate in the presence of the enzyme according to the draft. In addition, for example, an enzyme electrode can be formed by forming a groove having a desired shape on a substrate and then applying a carbon material to the groove in the presence of an enzyme.
Furthermore, this process can also be performed using a printing technique. For example, an enzyme electrode is formed by applying a carbon material to a substrate so as to form a desired shape in the presence of an enzyme using a computer. be able to.

Hereinafter, the manufacturing method of this embodiment shown in FIG. 1 will be described in more detail.

Here, in the production method of the present embodiment, as shown in FIG. 1, in addition to the oxidoreductase, a binding molecule A that binds the carbon materials and a binding molecule B that binds the carbon material and the oxidoreductase include It is a condition that exists.

Both the binding molecule A and the binding molecule B exhibit the effect of stabilizing the electrode structure in the solution (described later).

Further, in the production method of the present embodiment, the following effects can be obtained particularly under the condition that the binding molecule B that binds the carbon material and the oxidoreductase exists.
As described above, the method of applying and drying a mixture of oxidoreductase and carbon material on an electrode substrate in a single step has great significance in the production of enzyme electrodes, but has been almost studied so far. There wasn't.
As the above method, it is conceivable to prepare a solution containing a fine carbon material and an enzyme.
Here, when a solution obtained by simply mixing these is applied to a substrate and dried, the surface of a fine carbon material such as a carbon nanotube is hydrophobic in the first place, so that the carbon material and the enzyme do not adsorb, Does not form an enzyme electrode. Based on this, it is conceivable to prepare a solution containing the carbon material and the enzyme adsorbed to each other in water. In order to adsorb the carbon material and the enzyme to each other, the carbon material is interfaced. In some cases, it is necessary to immerse the activator solution while applying ultrasonic waves. In this case, the enzyme is denatured and deactivated by the ultrasonic treatment.
If the binding molecule B is used in the production method of the present embodiment, the risk of inactivation of the oxidoreductase during production can be reduced, and the effect of the present invention of producing an enzyme electrode simply and at low cost can be achieved. Easy to obtain.

In the production method of the present embodiment shown in FIG. 1, both the binding molecule A and the binding molecule B are present. However, in the production method of the present invention, the binding molecule A and the binding molecule A are not limited to this. Conditions under which at least one binding molecule selected from the group consisting of the binding molecules B is present are preferable conditions, and it is preferable that the binding molecule A is present rather than the binding molecule B (described later).

Specifically, in the production method of the present embodiment, it is preferable to use a suspension containing a carbon material and an oxidoreductase. By using the suspension, the method for producing the enzyme electrode of the present invention can be easily carried out.
In particular, in the manufacturing method of this embodiment, as shown in FIG. 1, a suspension adjusting step for adjusting a suspension containing a carbon material and an oxidoreductase, and a suspension for applying the suspension to a substrate. A liquid coating process.

[[Suspension adjustment process]]
In this step, for example, a carbon material, an oxidoreductase, and optionally a binding molecule are added to water to prepare a suspension.
Suspension methods include stirring with a stirrer, pipetting with a pipette or syringe, stirring by shaking (shaking) a container containing the suspension, such as a mixer, and a suspension using a rod-like device. Stirring by stirring can be mentioned. At this time, the carbon material and the oxidoreductase can be brought into an environment suitable for the enzyme electrode to exhibit activity without substantially performing ultrasonic treatment on the suspension.

[[Suspension application process]]
In this step, the application may be performed by hand using a spatula or the like, or by using a printing technique by a machine.
Examples of printing technologies include a type of press that uses a printing machine to directly or indirectly apply a sheet to a plate and applies pressure to the plate to print, and a type that does not use a plate (inkjet printing, etc.). For example, a press is preferable because the suspension has a certain viscosity. Examples of the plate used in the press include a relief plate, a planographic plate, an intaglio plate, and a stencil plate (used for screen printing).

The carbon material content in the suspension may be 0.01 to 5 mg / mL, and preferably 0.1 to 1 mg / mL.
The content of the oxidoreductase in the suspension may be 0.05 to 50 mg / mL, and preferably 0.5 to 10 mg / mL.
The content of the binding molecule A in the suspension may be 0.005 to 5% by weight, and preferably 0.01 to 1% by weight.
The content of the binding molecule B in the suspension may be 1 to 100 mM, and preferably 2 to 200 mM.
The viscosity of the suspension is preferably 1 Pa · s or more, and preferably 20 Pa · s or more from the viewpoint of facilitating application to the substrate and facilitating application using a printing technique. Is more preferably 1000 Pa · s or less, more preferably 100 Pa · s or less.

In addition, in the manufacturing method of this embodiment including the suspension adjusting step and the suspension applying step, the electrode forming step described above is included in the suspension applying step as shown in FIG.

[Freeze drying process]
In the manufacturing method of the present embodiment, the enzyme electrode may optionally be freeze-dried (lyophilization step), for example, following the electrode forming step described above.
In this step, for example, the enzyme electrode can be impregnated with liquid nitrogen and then placed in a freeze dryer and dried under reduced pressure.

Here, in the manufacturing method of this embodiment, as shown in FIG. 1, in the above-mentioned suspension application | coating process, it is set as the conditions where trehalose exists in addition to an oxidoreductase.
If the enzyme electrode is freeze-dried in the presence of trehalose, an effect of preventing loss of catalytic activity during freezing and thawing of the oxidoreductase can be obtained (described later).
The trehalose content in the aforementioned suspension may be 1 to 30% by weight, preferably 5 to 20% by weight, for example, 10% by weight.

Hereinafter, each element used in the method for producing an enzyme electrode according to an embodiment of the present invention will be described in detail.

-Carbon material-
Examples of the carbon material used in the manufacturing method of the present embodiment include nanocarbons such as carbon nanotubes (hereinafter also referred to as CNT), graphene, and fullerene; particulate carbon such as ketjen black (activated carbon particles) and carbon fibers; and These mixtures etc. are mentioned. These may be used alone or in combination of two or more.

As the nanocarbon, CNT is preferable.
The CNT may be either a single-wall CNT (SWCNT) or a multi-wall CNT (MWCNT). Specific examples of SWCNT include CoMoCAT (registered trademark) (manufactured by South West Nano Technologies), and specific examples of MWCNT. An example is Baytubes (registered trademark) (manufactured by Bayer Material Science).
The CNT is preferably SWCNT from the viewpoint of improving the electrical conductivity of the enzyme electrode and increasing the specific surface area of the electrode substrate.

The average length of CNTs may be 50 nm or more, preferably 1 μm or more, and may be 2 mm or less from the viewpoint of giving high conductivity to the enzyme electrode and giving high mechanical strength to the electrode structure, The thickness is preferably 5 μm or less.
From the viewpoint of increasing the specific surface area of the electrode substrate and increasing the amount of enzyme that can be immobilized on the enzyme electrode, the average diameter of CNTs may be 0.5 nm or more, preferably 1 nm or more, and may be 1 μm or less. The thickness is preferably 200 nm or less.
From the viewpoint of increasing the amount of enzyme that can be immobilized on the enzyme electrode, the specific surface area of CNT is preferably 10 m ² / g or more, more preferably 600 m ² / g or more, and may be 2000 m ² / g or less. , 1000 m ² / g or less is preferable.

The SWCNT production method is not particularly limited, and includes a chemical vapor synthesis method (CVD method), an arc discharge method, a laser ablation method, and the like. In particular, from the viewpoint of relatively increasing the average length, The growth method is preferred.
The super-growth method is a method in which a raw material compound and a carrier gas are supplied onto a substrate having a catalyst layer for producing CNTs on the surface, and CNT is synthesized by chemical vapor deposition (CVD). In this method, the catalytic activity of the catalyst layer for producing CNTs is drastically improved by the presence of a small amount of oxidizing agent (see International Publication No. 2006/011655). In this method, the catalyst layer is formed on the surface of the base material by a wet process, and a gas mainly composed of acetylene is used as a raw material gas, and the units that perform the formation process, the growth process, and the cooling process are connected. By using a continuous CNT manufacturing apparatus, SWCNTs can be efficiently manufactured.
In the specification of the present application, SWCNT manufactured by the super-growth method is also particularly referred to as SGCNT.

As the particulate carbon, ketjen black is preferable.
The average particle size of the particulate carbon may be 10 nm or more, and may be 1 μm or less.

-Redox enzyme-
Examples of the enzyme that catalyzes the oxidation reaction used in the production method of the present embodiment include fructose dehydrogenase (D-Fructose Dehydrogenase, FDH), glucose oxidase (Glucose Oxydase, GOD), glucose dehydrogenase (Glucose Dehydrogenase, GDH), Examples include oxidoreductases such as oxidase, alcohol dehydrogenase, lactate oxidase, and lactate dehydrogenase. Fructose dehydrogenase (FDH) is preferred because it has resistance to pH and does not require a mediator. These enzymes are supported on a cathode (anode) in a biobattery including an enzyme electrode. These may be used alone or in combination of two or more.
Examples of the enzyme that catalyzes the reduction reaction used in the production method of this embodiment include bilirubin oxidase (Bilirubin Oxidase, BOD), laccase, Cu efflux oxidase (Cueo), ascorbate oxidase, and the like. From the standpoint of resistance to chloride ions and the like, bilirubin oxidase (BOD) is preferred. These enzymes are supported on the anode (cathode) in a biocell including an enzyme electrode. These may be used alone or in combination of two or more.

-Base material-
The base material used in the manufacturing method of the present embodiment is not particularly limited, and plastic, metal, carbon, ceramic, glass, paper, wood, and a molded body using these materials (for example, plate, cloth) , Sticks, strings, fibers, films, other solid materials, etc.). Moreover, what has previously arrange | positioned electrodes other than an enzyme electrode on the surface of the said base material is good also as a base material.

-Binding molecule-
As a binding molecule used in the production method of the present embodiment, at least one binding molecule selected from the group consisting of a binding molecule A that binds carbon materials to each other and a binding molecule B that binds a carbon material and an oxidoreductase. It is.
Both the binding molecule A and the binding molecule B exhibit an effect of stabilizing the structure of the electrode. For example, the structure of the electrode can be maintained even in a wet environment. As the binding molecule, it is preferable to use the binding molecule A rather than the binding molecule B from the viewpoint of efficiently obtaining the effect of the present invention. From the viewpoint of obtaining the effect of the present invention more efficiently, the binding molecule A and the binding molecule are used. More preferably, both B are used.

The binding molecule A that binds the carbon materials may be a substituted or unsubstituted saturated or unsaturated aliphatic or aromatic hydrocarbon, and a π-π interaction (π-π with the surface of the carbon material). A molecule that exhibits a binding force by stacking is preferred.
Specific examples of molecules exhibiting a binding force due to π-π interaction with the surface of a carbon material include aromatic groups (phenyl group, naphthyl group, pyrenyl group, phenanthrenyl group) that are likely to cause π-π interaction. And a small molecule or a polymer having an aromatic hydrocarbon group such as anthracenyl group; a heteroaromatic group such as imidazole group, pyrazole group, oxazole group, thiazole group, pyrazine group, quinoline and isoquinoline). Here, examples of the polymer include polyolefin and polyether. The weight average molecular weight of the polymer may be 500 to 500,000, and preferably 5000 to 50,000.
As the aliphatic hydrocarbon, a fluorine-substituted hydrocarbon is preferable, and examples thereof include Nafion (registered trademark).
More specifically, as binding molecule A, polyvinyl imidazole (PVI), poly (sodium 4-styrenesulfonate), and Nafion (registered trademark) are preferable from the viewpoint of water solubility and availability, and high output is obtained. From the viewpoint, PVI and poly (sodium 4-styrenesulfonate) are particularly preferable.
The binding molecule A has an effect of stabilizing the structure of the electrode.
These may be used alone or in combination of two or more.
In particular, PVI allows π-π interaction with CNTs in aqueous solution. More specifically, PVI is highly water-soluble and exhibits the effect of increasing the dispersibility in water by physical adsorption between the imidazole group and the carbon material. The effect of improving the adhesion of the electrode and stabilizing the electrode structure is also shown. Similarly, poly (sodium 4-styrenesulfonate) also has the effect of causing physical adsorption between the phenyl group and the carbon material and enhancing the adhesion between the carbon materials.

The binding molecule B that binds the carbon material and the oxidoreductase is a functional group (amino) present in the side chain of the amino acid constituting the enzyme while exhibiting a binding force due to π-π interaction with the surface of the carbon material. Group, hydroxyl group, carboxyl group, etc.), and is preferably a molecule that can be covalently bonded to an enzyme. Specifically, N-hydroxysuccinimide ester having the aforementioned aromatic group It is preferably a small molecule or a polymer having a group, an aldehyde group, an epoxy group, a maleimide group, a carbodiimide group, a hydrazide group, etc., and more specifically, 1-Pyrenebutyric acid N-hydroxysuccinimide ester (1-pyrenebutyric acid N -Hydroxysuccinimide ester) (PBSE), N-benzoyloxys Cincinimide, 4,4 ′-[(8,16-Dihydro-8,16-dioxodibenzo [a, j] perylene-2,10-diyl) dioxy] dibutylic acid di (N-succinimidyl ester) is preferred. From the viewpoint of influence, PBSE and N-benzoyloxysuccinimide are particularly preferable, and the binding molecule B also has an effect of stabilizing the structure of the electrode.
These may be used alone or in combination of two or more.
In particular, PBSE immobilizes the enzyme on the surface of the carbon material by reacting the N-hydroxysuccinimide ester group with the amino group of the enzyme while causing physical adsorption between the pyrenyl group and the carbon material. In N-benzoyloxysuccinimide, the phenyl group physically adsorbs to the carbon material, and the succinimide ester group immobilizes the enzyme on the surface of the carbon material.

In the production method of the present embodiment, the binding molecule A is an osmium complex (Os (bpy) ₂ Cl) (wherein bpy represents bipyridine) represented by the following formula instead of the aforementioned PVI. Modified PVI (PVI in which osmium is bonded at the 3-position of the imidazole part in a part of the side chain of PVI) (hereinafter also referred to as “PVI-Os”) can also be used. Note that m and n in the formula are not particularly limited, and may be, for example, m: n = 100: 1 to 1: 100.

The osmium complex represented by the above formula can function as a mediator with respect to an enzyme (for example, GOD) that requires a mediator (coenzyme) among the enzymes that catalyze the oxidation reaction described above. At this time, for example, electrons generated by GOD oxidizing glucose are transferred to the osmium complex portion, and the electrons are transferred to the carbon material.
Therefore, when PVI-Os is used as the binding molecule A, the range of enzymes that catalyze the oxidation reaction that can be used in the production method of the present embodiment can be expanded.

-water-
Examples of water include ultrapure water.

-Trehalose-
Trehalose used in the production method of the present embodiment is a kind of non-reducing disaccharide in which two glucose molecules are α, α-1, 1 linked, and has extremely high hydrophilicity.
Trehalose is widely present in nature and is not only used as an energy source in organisms such as algae, bacteria, fungi, yeasts, insects, and invertebrates, but also stress from the outside world (dry, frozen) , High osmotic pressure, heat, etc.).
For example, by impregnating a high-concentration (1M) trehalose solution with an enzyme electrode containing an oxidoreductase and then freeze-drying the enzyme electrode, the higher-order structure is destroyed when the oxidoreductase is frozen and thawed. Therefore, loss of catalytic activity can be prevented. The hydroxyl group of trehalose is responsible for hydrogen bonds with the enzyme instead of water molecules during water sublimation, preventing the destruction of the higher-order structure of the enzyme, and the trehalose molecules concentrated during the water sublimation process This is probably because the enzyme was protected.

(Enzyme electrode)
The enzyme electrode according to an embodiment of the present invention (hereinafter also referred to as “enzyme electrode of the present embodiment”) includes a carbon material, an oxidoreductase, a binding molecule A that binds the carbon materials, and the carbon material and the oxidation. And at least one binding molecule selected from the group consisting of binding molecules B that bind to a reductase.

The enzyme electrode of the present embodiment may be manufactured by the method for manufacturing an enzyme electrode according to the embodiment of the present invention.

The enzyme electrode of the present embodiment includes a base material used in the manufacturing method of the above-described present embodiment, but the enzyme electrode of the present invention is not limited to this, and does not include the base material. Also good.

As an enzyme electrode manufactured by a conventional enzyme electrode manufacturing method, a carbon material such as nanocarbon or particulate carbon is formed into a porous structure, and then an enzyme solution is applied to the porous structure. The enzyme electrode manufactured by the manufacturing method which adsorb | sucks an enzyme to a carbon material by this is mentioned.
However, this enzyme electrode has a tendency that it is difficult to obtain the uniformity of enzyme adsorption to the carbon material, and it is difficult to obtain the reproducibility of the performance of the enzyme electrode.
According to the enzyme electrode manufactured by the manufacturing method of the present embodiment, it is possible to sufficiently spread the enzyme solution in the gaps between the carbon materials during the manufacturing, so that the enzyme can be uniformly adsorbed on the carbon material. The performance of the enzyme electrode can be obtained with good reproducibility.

In addition, as an enzyme electrode manufactured by a conventional enzyme electrode manufacturing method, a pellet shape manufactured by a manufacturing method in which a mixture obtained by mixing enzyme powder and CNT powder is pressed (pressed) under pressure. The enzyme electrode is also included.
However, this enzyme electrode has a high risk of collapse of a carbon material such as CNT and leakage of the enzyme, and the stability of the structure of the enzyme electrode tends to be low.
According to the enzyme electrode manufactured by the manufacturing method of the present embodiment, the stability of the structure of the enzyme electrode is achieved by the action of the binding molecules existing between the carbon materials and / or between the carbon material and the oxidoreductase. Can be increased.

The details of the carbon material, oxidoreductase, binding molecule A, and binding molecule B constituting the enzyme electrode of the present embodiment may be as described in the method for manufacturing the enzyme electrode of the present embodiment.

As the content of each element in the enzyme electrode of the present embodiment in a dry state, the content of the carbon material may be 0.1 to 10% by weight, with the whole enzyme electrode being 100% by weight, and 0.5 to 5% by weight. % Is preferred.
The content of the oxidoreductase may be 0.1 to 20% by weight, preferably 1 to 10% by weight, with the whole enzyme electrode being 100% by weight.
The content of the binding molecule A may be 0.1 to 10% by weight, preferably 0.5 to 5% by weight, with the whole enzyme electrode being 100% by weight.
The content of the binding molecule B may be 0.1 to 20% by weight with 100% by weight of the whole enzyme electrode, and preferably 1 to 10% by weight.

The enzyme electrode of this embodiment is preferably lyophilized, and the water content of the enzyme electrode of this embodiment in the lyophilized state may be 10% by weight or less, with the whole enzyme electrode being 100% by weight. It is preferably not more than wt%, and preferably not more than 1 wt%.

As described above, the enzyme electrode of the present embodiment preferably further contains trehalose.
In this case, the trehalose content in the enzyme electrode of the present embodiment in a dry state may be 10 to 98% by weight, preferably 70 to 95% by weight, with the whole enzyme electrode being 100% by weight.

Enzyme electrodes used in biosensors and biocells (biofuel cells) are usually disposable, and therefore need to be produced inexpensively with a small number of processes. Advantages in terms of production efficiency and cost obtained by the method for producing an enzyme electrode of the present embodiment are great. In particular, since the enzyme electrode manufacturing method of the present embodiment can be applied to printing technology, it can be integrated with the manufacturing process of printable electronics that is currently progressing rapidly. This will greatly contribute to the expansion of usage.
The enzyme electrode of the present embodiment can be suitably used as, for example, a core part of a biosensor or a bio battery in the medical, food, and environmental fields.

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

(Example 1)
A. Production of enzyme electrode A-1. Electrode material SGCNT (average length: 200 μm, provided by AIST Nanotube Research Center) was used as the carbon material.
As an oxidoreductase, fructose dehydrogenase (D-fructose dehydrogenase (FDH), derived from Gluconobacter, EC number: 1.1.99.11, 20 U / mg, manufactured by Toyobo Co., Ltd.), an enzyme that catalyzes an oxidation reaction. Using.
A ceramic plate-like member was used as the substrate.
As the binding molecule A, synthesized polyvinyl imidazole (PVI) (molecular weight: about 60000) was used.
As the binding molecule B, 1-pyrenebutyric acid N-hydroxysuccinimide ester (PBSE) (product number: 457078, manufactured by Sigma Aldrich) was used.
As water, ultrapure water was used.
As trehalose, 1 trehalose dihydrate (product number: 204-18451, manufactured by Wako Pure Chemical Industries, Ltd.) was used.

The buffer solution used in this example was prepared by the following procedure.
50 mM citrate-phosphate buffer (McIVaine buffer solution, MAC buffer, pH 5.0): 0.1 M citrate solution and 0.2 M Na ₂ HPO ₄ solution were prepared using deionized water, 0 A 50 mM MAC buffer was obtained by mixing 242.5 mL of 0.1 M citric acid solution and 257.5 mL of 0.2 M Na ₂ HPO ₄ solution.
50 mM phosphate buffer solution (PBS buffer, pH 7.0): 0.1M KH ₂ PO ₄ solution is prepared using deionized water, and 14.5 mL of 2M NaOH solution is added to 500 mL of this solution. Thereafter, the solution was diluted to 1000 mL with distilled water to obtain 50 mM PBS buffer.
In the following, these buffers were appropriately diluted and used.

A-2. Preparation of enzyme electrode and lyophilization 25 mM MAC buffer (pH 5.0) and PVI (final concentration (the final concentration refers to the concentration in the dispersion solution at the time of application to the substrate. The same applies hereinafter). : 0.1 wt%), 1 trehalose dihydrate (final concentration: 10.0 wt%), SGCNT (final concentration: 1 mg / mL) in this order, and sonicating the solution for 10 minutes By doing so, SGCNT was dispersed in the solution.
Next, 1 M PBSE DMSO solution was added to the dispersion solution so that the final concentration of PBSE was 20 mM, and this solution was mixed by 50 tappings. Thereafter, the mixed solution was allowed to stand for 1 hour to cause physical adsorption between the pyrenyl group of PBSE and SGCNT, and SGCNT was modified with PBSE.
Thereafter, a 100 mg / mL FDH solution was prepared using a 50 mM MAC buffer. This FDH solution was added to the dispersion solution so that the final concentration of FDH was 5 mg / mL, and this solution was mixed by 50 tappings. Thereafter, the mixed solution was allowed to stand for 1 hour, and the N-hydroxysuccinimide ester group of PBSE was reacted with the amino group of the enzyme to immobilize the enzyme on the SGCNT surface.
On the base material, 5 μL (about 5.5 mg) of the above dispersion solution was applied so as to have a desired shape.
Thereafter, the plastic plate containing SGCNT and FDH was allowed to stand at room temperature for 10 minutes to remove excess water, thereby making it semi-dry.
Thus, an enzyme electrode for negative electrode (anode) containing SGCNT and an enzyme (FDH) that catalyzes the oxidation reaction was produced.

Then, the plastic plate is flash-frozen by immersing it in liquid nitrogen for about 5 minutes, and then 5 hours or more using a freeze dryer (product number: ALPHA 2-4 LSC, manufactured by CHRiST (Kubota Corporation)). Dried.
Thus, a freeze-dried enzyme electrode was obtained. The weight of the enzyme electrode was about 0.56 mg.

B. Output Performance Measurement The output performance was evaluated using a three-electrode electrochemical measurement system (product number: 730C electroanalyzer, manufactured by BAS) for the produced electrode.
Specifically, cyclic voltammetry (CV) under the conditions of sweep potential: −0.2 to 0.6 V, sweep speed: 10 mV / s, and chrono under the condition that the potential is fixed at 0.5 V. Amperometry (evaluation of stability of electrode activity) was performed.
Ag / AgCl (saturated KCl) was used for the reference electrode, and a platinum electrode was used for the counter electrode. Moreover, the 0.2M fructose solution prepared using 100 mM MAC buffer was used as a measurement solution. As a control, 100 mM MAC buffer not containing fructose was used.
All of the above measurements were performed while stirring the solution. The measurement was performed twice for Example 1 and the control.
As the area (cm ² ) for determining the current density in both measurements, the area (one side only) (calculated as the area of a circle with a diameter of 4 mm) of the plate-like member that is the substrate of the enzyme electrode was used.

The CV results of Example 1 are shown in FIG. 2 (Example 1 is indicated by a solid line, and control is indicated by a broken line). The horizontal axis represents the sweep potential (V), and the vertical axis represents the current density (mA / cm ² ).
As shown in FIG. 2, in the case of Example 1, a maximum current density of 1.5 mA / cm ² (at a sweep potential of 0.6 V) was observed. This value is comparable to the value described in the previous report. On the other hand, in the case of control, the current density was approximately 0 mA / cm ² .

The results of chronoamperometry of Example 1 are shown in FIG. The elapsed time (hour) is shown on the horizontal axis, and the current density (mA / cm ² ) is shown on the vertical axis.
An increase in current density was observed for a few minutes immediately after immersing the enzyme electrode in the measurement solution. This seems to indicate that the enzyme involved in the reaction increased as the dry enzyme electrode was wetted with the fructose solution. Thereafter, although the current density gradually decreased, it kept 1 mA / cm ² or more (80% or more of the initial current) even after 10 hours. This is probably because the binding molecule prevented the enzyme from leaking out of the electrode and the structure of the enzyme electrode was stable.

(Example 2, Example 3)
The influence of the presence or absence of binding molecules PVI and PBSE on the output performance of the enzyme electrode was examined.

(Example 2)
Instead of using both PVI and PBSE as binding molecules, an enzyme electrode was prepared and the output performance was measured by chronoamperometry, as in Example 1, except that only PVI was used.
(Example 3)
Instead of using both PVI and PBSE as binding molecules, an enzyme electrode was prepared and the output performance was measured by chronoamperometry, as in Example 1, except that neither PVI nor PBSE was used. did.

The results of Examples 2 and 3 are shown in FIG. The elapsed time (hour) is shown on the horizontal axis, and the current density (mA / cm ² ) is shown on the vertical axis. In FIG. 4, the results of Example 1 are also shown again. Example 1 is indicated by a solid line, Example 2 is indicated by an alternate long and short dash line, and Example 3 is indicated by an intersection.
In the case of Example 2, the collapse of the structure of the enzyme electrode is not observed by visual observation, and it is considered that the binding between the CNTs is maintained. However, as shown in FIG. 4, the activity of the enzyme electrode is smaller than that in Example 1, and the degree of decrease in activity over time is also large. In the case of Example 2, it is considered that a large amount of enzyme leaked because CNT and FDH were not immobilized on the carbon material surface.
In Example 3, immediately after the enzyme electrode was immersed in the measurement solution, it was observed that the dry enzyme electrode was wetted with the fructose solution, and an increase in current density was observed as shown in FIG. . However, as shown in FIG. 4, no current was observed in about 1 minute thereafter. In the case of Example 3, it was found by visual observation that the structure of the enzyme electrode collapsed as the fructose solution was wetted, and CNTs and the like were dissipated in the solution.

(Examples 4 to 6)
The output performance of the enzyme electrode when a material other than SGCNT was used as the carbon material was examined.
The measurement was performed twice for each of Examples 4 to 6 and each control.

Example 4
Example 1 except that single-walled carbon nanotubes (average length: 1 μm, Single-Walled Carbon Nanotube (SWCNT), product number: 704121, manufactured by South West Nano Technologies) were used instead of SGCNT as the carbon material. In the same manner as described above, an enzyme electrode was prepared, and the output performance was measured by cyclic voltammetry and chronoamperometry.
(Example 5)
In the case of Example 1 except that multi-walled carbon nanotubes (average length: 5 μm, Multi-Walled Carbon Nanotube (MWCNT), product number: C70P, manufactured by Bayer Material Science) were used instead of SGCNT as the carbon material. Similarly, an enzyme electrode was prepared, and the output performance was measured by cyclic voltammetry and chronoamperometry.
(Example 6)
An enzyme electrode was prepared in the same manner as in Example 1 except that Ketjenblack (Ketjenblack (KB), product number: EC-600JD, manufactured by Ketjen Black International) was used instead of SGCNT as the carbon material. The output performance was measured by cyclic voltammetry and chronoamperometry.

The results of cyclic voltammetry and chronoamperometry in Examples 4 to 6 are shown in FIG. 5 (a), FIG. 5 (b) to FIG. 7 (a), and FIG. 7 (b), respectively. Each of Examples 4 to 6 is indicated by a solid line, and only the CV result of Example 5 is indicated by a broken line for control). In each figure, (a) shows the sweep potential (V) on the horizontal axis, the current density (mA / cm ² ) on the vertical axis, and (b) shows the elapsed time (hours) on the horizontal axis. The current density (mA / cm ² ) is shown on the vertical axis.
First, in the case of Example 4 (SWCNT) and Example 5 (MWCNT), a current density of about 1 mA / cm ^{2 at} maximum (at a sweep potential of 0.6 V) was observed (FIGS. 5A and 6). (See (a)). Further, the current density was maintained at 0.6 mA / cm ² or more even after 10 hours from the immersion of the enzyme electrode in the measurement solution, and the effect of the binding molecule was clearly observed (FIG. 5 (b) and (Refer FIG.6 (b)).
Moreover, in the case of Example 6 (KB), the collapse of the structure of the enzyme electrode was not observed by visual observation, indicating the effect of the binding molecule. However, in the case of Example 6, from the value of the current density and the state of the passage of time, the activity of the enzyme electrode is maintained to some extent even after 10 hours from the immersion of the enzyme electrode in the measurement solution. It was found that the structural stability was inferior to that of the CNTs of Examples 4 and 5 (see FIGS. 7A and 7B).

However, in any of Examples 4 to 6, in the case of the control without using the binding molecule, it was observed that the structure of the enzyme electrode collapsed as soon as the enzyme electrode was immersed in the measurement solution (not shown). ). In this respect, in any of Examples 4 to 6, it was confirmed that the effect of the binding molecule was confirmed since it had activity even after 10 hours.

The effect of the type of carbon material on the performance of the enzyme electrode can be discussed from the shape and crystallinity of the carbon material.
In Example 4 (SGCNT) in which the most excellent performance was obtained, since the average length is extremely long as 200 μm and the aspect ratio is high, a structure in which the carbon materials are entangled with each other is formed, and the binding molecule is It is considered that the effect can be exhibited effectively. On the other hand, in Example 5 using CNT having a relatively short average length and Example 6 using fine carbon particles, it is considered that bonding between carbon materials is relatively difficult to occur.
Furthermore, since the binding molecules (PVI and PBSE) bind to the surface of the carbon material, particularly to the graphite structure, by the π-π interaction, the effect of the binding molecule is hardly exhibited in KB which is a carbon material having low crystallinity. it is conceivable that.

(Example 7)
Instead of using PVI as the binding molecule A, an enzyme electrode was prepared in the same manner as in Example 1 except that poly (sodium 4-styrenesulfonate) (product number: 243051, manufactured by Sigma Aldrich) was used. The output performance was measured by cyclic voltammetry and chronoamperometry.
The results of cyclic voltammetry and chronoamperometry of Example 7 are shown in FIG. 8 (a) and FIG. 8 (b), respectively (Example 7 is indicated by a solid line. Note that only the CV result is controlled. Is indicated by a broken line). In (a), the horizontal axis indicates the sweep potential (V), the vertical axis indicates the current density (mA / cm ² ), and in (b), the horizontal axis indicates the elapsed time (hours), and the vertical axis indicates the current. The density (mA / cm ² ) is indicated.
In the case of Example 7, the maximum current density was 0.12 mA / cm ² , and although the activity of the enzyme electrode was lower than that of Example 1, it was shown to function as an electrode. Moreover, even after 10 hours, the current density was maintained at 0.07 mA / cm ² or more, indicating that the durability of the enzyme electrode can be maintained.

(Example 8)
An enzyme electrode was prepared in the same manner as in Example 1 except that Nafion (registered trademark) (product number: 527122, Nafion perfluorinate resin solution 20 wt%) was used instead of PVI as the binding molecule A. Electrodes were prepared, and output performance was measured by cyclic voltammetry and chronoamperometry.
The results of cyclic voltammetry and chronoamperometry of Example 8 are shown in FIG. 9 (a) and FIG. 9 (b), respectively (Example 8 is indicated by a solid line. Note that only the CV result is controlled. Is indicated by a broken line). In (a), the horizontal axis indicates the sweep potential (V), the vertical axis indicates the current density (mA / cm ² ), and in (b), the horizontal axis indicates the elapsed time (hours), and the vertical axis indicates the current. The density (mA / cm ² ) is indicated.
In the case of Example 8, the maximum current density was 0.18 mA / cm ² , and although the activity of the enzyme electrode was lower than that of Example 1, it was shown to function as an electrode. Moreover, even after 10 hours, the current density was maintained at 0.15 mA / cm ² or more, indicating that the durability of the enzyme electrode can be maintained.

Example 9
An enzyme electrode was prepared in the same manner as in Example 1 except that N-benzoyloxysuccinimide (product number: 774324, manufactured by Sigma Aldrich) was used instead of PBSE as the binding molecule B. The output performance was measured by cyclic voltammetry and chronoamperometry.
The results of cyclic voltammetry and chronoamperometry of Example 9 are shown in FIG. 10 (a) and FIG. 10 (b), respectively (Example 9 is indicated by a solid line. Note that only the CV result is controlled. Is indicated by a broken line). In (a), the horizontal axis indicates the sweep potential (V), the vertical axis indicates the current density (mA / cm ² ), and in (b), the horizontal axis indicates the elapsed time (hours), and the vertical axis indicates the current. The density (mA / cm ² ) is indicated.
In the case of Example 9, the maximum current density was 1.5 mA / cm ² , indicating that the activity of the enzyme electrode was as high as in Example 1.
Moreover, even after 10 hours, the current density was maintained at 0.7 mA / cm ² or more, indicating that the durability of the enzyme electrode can be maintained.

(Example 10)
A case where a binding molecule having a mediator part was used was examined.
The measurement was performed twice for Example 10 and the control.

Instead of PVI as a binding molecule, synthesized PVI-Os (molecular weight: about 120,000, m: n = 5: 1) was used, glucose oxidase (GOD, EC number: 1.1.3) instead of FDH. .4, 240 U / mg, manufactured by Toyobo Co., Ltd.), an enzyme electrode was prepared in the same manner as in Example 1, and the output performance was measured by cyclic voltammetry and chronoamperometry.
In Example 10, 0.2 M glucose prepared using 50 mM PBS was used as a measurement solution. As a control, 100 mM MAC buffer containing no glucose was used.
The results of cyclic voltammetry and chronoamperometry in Example 10 are shown in FIGS. 11 (a) and 11 (b) (Example 10 is indicated by a solid line. Only the CV result is indicated by a broken line for control). Show). In FIG. 11 (a), the horizontal axis indicates the sweep potential (V), the vertical axis indicates the current density (mA / cm ² ), and in FIG. 11 (b), the horizontal axis indicates the elapsed time (hours). The vertical axis represents current density (mA / cm ² ).
As shown in FIG. 11A, in Example 10, a maximum current density of 0.25 mA / cm ² (at a sweep potential of 0.6 V) was observed.
Further, as shown in FIG. 11 (b), also in Example 10, as in Example 1, an increase in current density was observed for several minutes immediately after the enzyme electrode was immersed in the measurement solution. Although the density gradually decreased, about 80% of the initial current density was maintained even after 10 hours.
From this result, it was shown that PVI-Os can exert the function of a mediator of GOD in addition to the function as the binding molecule A.

According to the present invention, an enzyme electrode can be produced easily and at low cost.
The enzyme electrode of the present invention produced by the production method of the present invention can be suitably used, for example, as a core part of a biosensor or a bio battery (biofuel cell) in the medical, food and environmental fields.

Claims

A method for producing an enzyme electrode, characterized in that an enzyme electrode containing a carbon material and an oxidoreductase is obtained by applying a carbon material to a substrate under conditions where the oxidoreductase is present.
The method for producing an enzyme electrode according to claim 1, comprising an electrode forming step of forming the enzyme electrode.
The method for producing an enzyme electrode according to claim 2, wherein a printing technique is used in the electrode forming step.
A suspension adjusting step of adjusting a suspension containing the carbon material and the oxidoreductase;
The method for producing an enzyme electrode according to any one of claims 1 to 3, further comprising a suspension application step of applying the suspension to the substrate.
In addition to the oxidoreductase, there is further at least one binding molecule selected from the group consisting of a binding molecule A that binds the carbon materials and a binding molecule B that binds the carbon material and the oxidoreductase. The method for producing an enzyme electrode according to any one of Claims 1 to 4, which is a condition for conducting the enzyme electrode treatment.
The method for producing an enzyme electrode according to claim 5, wherein the binding molecule is the binding molecule A.
The method for producing an enzyme electrode according to claim 5, wherein the binding molecules are the binding molecule A and the binding molecule B.
The enzyme according to any one of claims 5 to 7, wherein the binding molecule A is at least one selected from the group consisting of a small molecule or a polymer having an aromatic group, and a fluorine-substituted hydrocarbon. Electrode manufacturing method.
The enzyme electrode production according to claim 8, wherein the binding molecule A is at least one selected from the group consisting of polyvinylimidazole (PVI), poly (sodium 4-styrenesulfonate), and Nafion (registered trademark). Method.
The binding molecule B has a small molecule having an aromatic group and at least one selected from the group consisting of an N-hydroxysuccinimide ester group, an aldehyde group, an epoxy group, a maleimide group, a carbodiimide group, and a hydrazide group, or The method for producing an enzyme electrode according to claim 5 or 7, which is a polymer.
The method for producing an enzyme electrode according to claim 10, wherein the binding molecule B is at least one selected from the group consisting of 1-pyrenebutyric acid N-hydroxysuccinimide ester (PBSE) and N-benzoyloxysuccinimide.
In addition to the oxidoreductase, a condition where trehalose is further present,
Freeze-drying the enzyme electrode, comprising a freeze-drying step;
The method for producing an enzyme electrode according to any one of claims 1 to 11.
At least one binding molecule selected from the group consisting of the carbon material, the oxidoreductase, a binding molecule A that binds the carbon materials, and a binding molecule B that binds the carbon material and the oxidoreductase. And an enzyme electrode.
The enzyme electrode according to claim 13, wherein the binding molecule is the binding molecule A.
The enzyme electrode according to claim 13, wherein the binding molecules are the binding molecule A and the binding molecule B.
The enzyme according to any one of claims 13 to 16, wherein the binding molecule A is at least one selected from the group consisting of a small molecule or a polymer having an aromatic group, and a fluorine-substituted hydrocarbon. electrode.
The enzyme electrode according to claim 16, wherein the binding molecule A is at least one selected from the group consisting of polyvinylimidazole (PVI), poly (sodium 4-styrenesulfonate), and Nafion (registered trademark).
The binding molecule B has a small molecule having an aromatic group and at least one selected from the group consisting of an N-hydroxysuccinimide ester group, an aldehyde group, an epoxy group, a maleimide group, a carbodiimide group, and a hydrazide group, or The enzyme electrode according to claim 13 or 15, which is a polymer.
The enzyme electrode according to claim 18, wherein the binding molecule B is at least one selected from the group consisting of 1-pyrenebutyric acid N-hydroxysuccinimide ester (PBSE) and N-benzoyloxysuccinimide.
The enzyme electrode according to any one of claims 13 to 19, further comprising trehalose.
The enzyme electrode according to claim 20, which is freeze-dried.