Enzyme electrode, electrochemical device, and method for detecting target substance

Abstract

The present disclosure provides an enzyme electrode that is advantageous from the viewpoint of causing a redox to occur which involves a target substance without replenishing an enzyme. An enzyme electrode (1a) according to the present disclosure is provided with: a conductive base material (10); an enzyme (21b); a metal organic structure (21a); and a fixing agent (22). The enzyme (21b) catalyzes the redox in which the target substance is involved. The fixing agent (22) fixes the enzyme (21b) and the metal organic structure (21a) to the base material (10).

Description

Enzyme electrode, electrochemical device, and method for detecting target substance

[0001] The present disclosure relates to an enzyme electrode, an electrochemical device, and a method for detecting a target substance.

[0002] Biosensors using oxidoreductases have been known. For example, Patent Document 1 describes a glucose sensor as a biosensor. According to the description, the glucose sensor is obtained as follows: an electrode system including at least a measuring electrode and a counter electrode is formed on an insulating substrate by a method such as screen printing. An enzyme reaction layer including a hydrophilic polymer, an oxidoreductase, and an electron mediator is formed on this electrode system.

[0003] In this glucose sensor, glucose oxidase is used as the oxidoreductase, and a metal complex or organic compound such as potassium ferricyanide, a ferrocene derivative, or a quinone derivative is used as the electron mediator. A buffer is added to the enzyme reaction layer as needed. When a sample solution containing a substrate is dropped onto the enzyme reaction layer of this biosensor, the enzyme reaction layer dissolves, causing a reaction between the enzyme and the substrate, resulting in the reduction of the electron mediator. After the enzyme reaction is complete, the substrate concentration in the sample solution can be determined from the current value required to electrochemically oxidize the reduced electron mediator.

[0004] Japanese Patent Application Laid-Open No. 2002-340839

[0005] The technology described in Patent Document 1 needs to be reconsidered from the viewpoint of generating redox reactions involving a target substance without supplementing an enzyme. Therefore, the present disclosure provides a technology that is advantageous from the viewpoint of generating redox reactions involving a target substance without supplementing an enzyme.

[0006] The present disclosure provides an enzyme electrode comprising: a conductive substrate; an enzyme that catalyzes an oxidation-reduction reaction involving a target substance; a metal organic framework; and an immobilizing agent that immobilizes the enzyme and the metal organic framework on the substrate.

[0007] The enzyme electrode of the present disclosure is advantageous in that it can cause oxidation-reduction involving a target substance without the need for supplemental enzymes.

[0008] FIG. 1 is a cross-sectional view schematically showing an example of an enzyme electrode according to the present embodiment. FIG. 2 is a cross-sectional view schematically showing another example of an enzyme electrode according to the present embodiment. FIG. 3 is a diagram schematically showing an example of an electrochemical device including an enzyme electrode according to the present embodiment. FIG. 4 is a flowchart showing an example of a method for detecting a target substance according to the present embodiment. FIG. 5 is a diagram schematically showing an example of a biosensor including an enzyme electrode according to the present embodiment. FIG. 6 is a diagram schematically showing another example of a biosensor including an enzyme electrode according to the present embodiment. FIG. 7 is a diagram schematically showing an example of a structure according to the present embodiment. FIG. 8 is a diagram showing the structure of Mil-100(Fe). FIG. 9 is a diagram schematically showing an example of a device according to the present embodiment. FIG. 10 is a graph showing the results of chronoamperometry measurement using the enzyme electrode according to Example 1. FIG. 11 is a graph showing the relationship between cortisol concentration and current value. FIG. 12A is a transmission image obtained by microscopic observation of the composite according to Example 1. FIG. 12B is a scattering image obtained by fluorescence microscopic observation of the composite according to Example 1. FIG. 12C is a fluorescent image obtained by fluorescence microscope observation of the composite according to Example 1. FIG. 13A is a transmission image obtained by microscope observation of a metal-organic framework (MOF). FIG. 13B is a scattering image obtained by fluorescence microscope observation of the MOF. FIG. 13C is a fluorescent image obtained by fluorescence microscope observation of the MOF. FIG. 14 is a graph showing the results of chronoamperometry measurement using the enzyme electrode according to Example 1. FIG. 15 is a graph showing the results of chronoamperometry measurement using the enzyme electrode according to Comparative Example 1. FIG. 16 is a graph showing the relationship between the reaction rate of the enzyme reaction and the initial cortisol concentration in the examples and comparative examples. FIG. 17 is a graph showing the Michaelis constant and maximum reaction rate of the enzyme reaction in the examples and comparative examples.

[0009] (Findings underlying the present disclosure) In the glucose sensor described in Patent Document 1, the target substance is sensed by oxidizing the reduced form of the electron mediator produced by an enzymatic reaction involving the target substance at an electrode. However, in measurements using this glucose sensor, the enzyme reaction layer dissolves, causing the enzyme to react with the substrate, and it is therefore understood that the enzyme must be added each time a measurement is performed. If the concentration of the target substance were to be measured continuously, it would be necessary to add a measurement reagent or the like multiple times. For this reason, it is practically difficult to perform permanent measurements using such a glucose sensor. It is difficult to apply such a glucose sensor to, for example, a wearable biosensor that measures the concentration of a biological substance contained in sweat. In this specification, a wearable biosensor generally refers to a biosensor that can be worn on the body.

[0010] For example, if an enzyme can be immobilized on an enzyme electrode, it is believed that oxidation-reduction involving a target substance can occur without the need for enzyme replenishment, making it possible to continuously measure the concentration of the target substance. However, the inventors' studies have revealed that immobilizing only an enzyme does not necessarily maintain high enzyme activity. After extensive trial and error, the inventors have newly discovered that immobilizing an enzyme and a metal-organic framework on an enzyme electrode makes it easier to maintain high activity of oxidation-reduction involving a target substance. Based on this new finding, the inventors have finally completed the enzyme electrode of the present disclosure.

[0011] Enzymes are proteins that function as catalysts in specific reactions with specific molecules. It is conceivable that enzyme properties, such as substrate specificity and reaction specificity, can be utilized in sensing. While enzymes can be used in sensing devices, their low stability of activity can hinder their continuous use. This poses a problem, for example, when using enzymes in wearable sensors. Wearable sensors generally refer to sensors that can be worn on the body.

[0012] Therefore, the present inventors have conducted extensive research into whether the stability of enzyme activity can be increased by conjugating an enzyme with a MOF. As a result, it has been found that, depending on the type of MOF, the stability of the enzyme activity may be low even when the enzyme is conjugated with a MOF. On the other hand, it has also been newly discovered that the activity of the enzyme can be stabilized by using a specific MOF in the conjugation of the enzyme with a MOF. Based on this new finding, the present inventors have finally completed the structure of the present disclosure.

[0013] (Embodiments of the Present Disclosure) Hereinafter, embodiments of the present disclosure will be described with reference to the drawings. Note that the embodiments described below are all comprehensive or specific examples. The numerical values, shapes, materials, components, the arrangement and connection of the components, steps, and the order of steps shown in the following embodiments are merely examples and are not intended to limit the scope of the claims. Furthermore, each figure is not necessarily an exact illustration. In each figure, substantially identical configurations are assigned the same reference numerals, and redundant explanations may be omitted or simplified. Furthermore, below, terms indicating the relationship between elements, such as "parallel" and "perpendicular," terms indicating the shape of elements, such as "rectangular," and numerical ranges do not only indicate strict meanings but also include substantially equivalent ranges, for example, differences of a few percent. Furthermore, below, detecting a target substance includes not only finding the target substance and confirming its presence, but also measuring the amount (e.g., number or concentration) or the range of that amount of the target substance. The embodiments shown as these comprehensive or specific examples may be realized using a system, an apparatus, an integrated circuit, a computer program, or a computer-readable recording medium such as a CD-ROM, or may be realized by any combination of a system, a method, an integrated circuit, a computer program, and a recording medium.

[0014] First Embodiment Hereinafter, a first embodiment will be specifically described with reference to FIGS. 1 to 6. FIG.

[0015] Fig. 1 is a cross-sectional view schematically illustrating an example of an enzyme electrode according to the present embodiment. As shown in Fig. 1, the enzyme electrode 1a includes a conductive substrate 10, an enzyme 21b, a metal-organic framework (MOF) 21a, and an immobilizing agent 22. The enzyme 21b catalyzes oxidation-reduction involving a target substance. The immobilizing agent 22 immobilizes the enzyme 21b and the MOF 21a to the substrate 10. For example, in the enzyme electrode 1a, an enzyme-containing layer 20 containing the enzyme 21b, the MOF 21a, and the immobilizing agent 22 is formed. The enzyme-containing layer 20 is formed along the surface of the substrate 10.

[0016] In the enzyme electrode 1a, the enzyme 21b and the MOF 21a are immobilized on the substrate 10 by the immobilizing agent 22. This allows the enzyme 21b to remain on the enzyme electrode 1a even when a sample containing a target substance comes into contact with the enzyme electrode 1a, making it easier to cause oxidation-reduction involving the target substance without supplementing the enzyme. In addition, the activity of the enzyme 21b is more likely to be maintained at a high level than when the enzyme electrode does not contain a MOF.

[0017] The enzyme 21b and the MOF 21a form, for example, a complex 21. In the complex 21, for example, a part of the enzyme 21b is arranged so as to be embedded in a part of the MOF 21a. In the complex 21, the enzyme 21b may be adsorbed to the MOF 21a, or the MOF 21a may be adsorbed to the enzyme 21b. Alternatively, the enzyme 21b and the MOF 21a may integrally form the complex 21 due to an interaction between a part of the enzyme 21b and the MOF 21a.

[0018] The complex 21 is produced by, for example, a coprecipitation method or an adsorption method. According to the coprecipitation method, the MOF 21a is synthesized in a solution containing the enzyme 21b to obtain the complex 21. According to the adsorption method, the enzyme 21b is adsorbed onto the MOF 21a synthesized in advance to obtain the complex 21.

[0019] The target substance involved in the oxidation-reduction catalyzed by the enzyme 21b is not limited to a specific substance. The target substance is, for example, a substance that is oxidized or reduced by the catalytic action of the enzyme 21b, and is a substance that is the target of detection in detection using the enzyme electrode 1a. The target substance may also be called an analyte or a detection target. The target substance is, for example, a biological substance such as glucose, corticosteroids, and hormones. These biological substances may be contained in body fluids.

[0020] The enzyme 21b is not limited to a specific enzyme as long as it catalyzes oxidation-reduction involving a target substance. The enzyme 21b is appropriately selected depending on the target substance. The enzyme 21b is, for example, a protein that can selectively react with the target substance. The enzyme electrode 1a may contain only one type of enzyme 21b, or may contain two or more types of enzymes 21b. The enzyme 21b is preferably an enzyme that causes oxidation-reduction involving the target substance as a substrate. The enzyme 21b preferably acts specifically on the target substance. The enzyme 21b is preferably an oxidoreductase that causes charge transfer in oxidation-reduction involving the target substance. The enzyme 21b may be, for example, an oxidase or dehydrogenase that uses the target substance as a substrate. Examples of oxidoreductases include glucose oxidase and glucose dehydrogenase when the target substance is glucose, and lactate oxidase and lactate dehydrogenase when the target substance is lactic acid. Other examples of oxidoreductases include cholesterol esterase and cholesterol oxidase when the target substance is cholesterol, and alcohol oxidase and alcohol dehydrogenase when the target substance is alcohol. Further examples of oxidoreductases include bilirubin oxidase when the target substance is bilirubin, and 11β-hydroxysteroid dehydrogenase when the target substance is cortisol. Further examples of oxidoreductases include creatinase and sarcosine oxidase when the target substance is creatine. Further examples of oxidoreductases include urate oxidase when the target substance is uric acid. Further examples of oxidoreductases include fructose dehydrogenase when the target substance is fructose. Further examples of oxidoreductases include mannitol dehydrogenase when the target substance is mannitol.

[0021] The oxidoreductase, which is the enzyme 21b, may undergo an enzymatic reaction with the participation of a coenzyme. In other words, the enzyme 21b may be coenzyme-dependent. The coenzyme-dependence of the enzyme 21b is not limited to a specific coenzyme. When the enzyme 21b is dependent on a specific coenzyme, the enzyme electrode 1a may contain that specific coenzyme. Examples of specific coenzymes are nicotinamide adenine dinucleotide (NAD), nicotinamide adenine dinucleotide phosphate (NADP), pyrroloquinoline quinone (PQQ), and flavin adenine dinucleotide (FAD). The coenzyme contained in the enzyme electrode 1a is preferably FAD or PQQ. In this case, the coenzyme is bound to the protein, which is the enzyme 21b. Alternatively, the coenzyme may constitute a part of that protein. Therefore, in an electrochemical device including the enzyme electrode 1a or in a measurement using the enzyme electrode 1a, it is not necessary to add a coenzyme separately from the enzyme 21b, which tends to simplify the manufacture of the electrochemical device and the measurement using the enzyme electrode 1a.

[0022] The coenzyme contained in the enzyme electrode 1a may be NAD or NADP. These coenzymes can function without binding to the protein enzyme 21b. These coenzymes can be added separately from the enzyme 21b. Using NAD and NADP can complicate the fabrication of the electrochemical device and the measurement using the enzyme electrode 1a compared to using FAD and PQQ. However, the enzyme 21b may be dependent on NAD or NADP, and the enzyme electrode 1a may contain NAD or NADP. The enzyme 21b may be an FAD-dependent oxidase or a dehydrogenase that is NAD-dependent, NADP-dependent, or PQQ-dependent.

[0023] In the enzyme electrode 1a, for example, the target substance is oxidized by the enzyme 21b. In this case, the coenzyme can be converted to a reduced form. The reduced coenzyme is oxidized in the substrate 10. This converts the reduced coenzyme to an oxidized form, and a current is generated in the substrate 10 as the coenzyme is oxidized. By measuring this current, the concentration of the target substance, which is the substrate, can be determined. Alternatively, the target substance is reduced by the enzyme 21b. In this case, the coenzyme can be converted to an oxidized form. The oxidized coenzyme is reduced in the substrate 10. This converts the oxidized coenzyme to a reduced form, and a current is generated in the substrate 10 as the coenzyme is reduced. By measuring this current, the concentration of the target substance, which is the substrate, can be determined.

[0024] The content of the enzyme 21b contained in the enzyme electrode 1a is not limited to a specific value, and is adjusted, for example, so as to enable detection of a target substance.

[0025] MOF21a has a structure in which metal ions and organic molecules are bonded. Combining a MOF with an enzyme can increase the stability of the enzyme. For example, the following literature describes the relationship between MOFs and enzymes: Weibin Liang, Peter Wied, Francesco Carraro, Christopher J. Sumby, Bernd Nidetzky, Chia-Kuang Tsung, Paolo Falcaro, and Christian J. Doonan, "Metal-Organic Framework-Based Enzyme Biocomposites", Chem. Rev. 2021, 121, 3, 1077-1129(2021) Shan Liang, Xiao-Ling Wu, Jun Xiong, Min-Hua Zong, Wen-Yong Lou, "Metal-organic frameworks as novel matrices for efficient enzyme immobilization: An update review", Coordination Chemistry Reviews,Volume 406(2020) These documents do not consider immobilizing MOFs and enzymes on conductive substrates. If enzymes are immobilized on conductive substrates using immobilizing agents other than MOFs, it seems that there is no need to use MOFs. However, as described above, the inventors' investigations have revealed that when only an enzyme is immobilized using an immobilizing agent other than a MOF, the activity of the enzyme is not necessarily maintained at a high level. In the enzyme electrode 1a, the enzyme 21b and the MOF 21a are immobilized on the substrate 10. This makes it easier for the activity of the enzyme 21b to be maintained at a high level compared to when the enzyme electrode does not contain a MOF.

[0026] MOF 21a is not limited to a specific MOF as long as it can be used with enzyme 21b. The metal ion contained in MOF 21a is not limited to a specific metal ion. The metal ion may be an ion of at least one metal selected from metals in groups 1 to 16 of the IUPAC periodic table of elements. The metal may include actinides and lanthanides. Examples of metal ions include Li + , Na + , K. + , Rb + , Be 2+ , Mg 2+ , Ca 2+ , Sr 2+ , Ba 2+ , Sc 3+ , Y 3+ , Ti 4+ , Zr 4+ , Hf 4+ , V 5+ , V 4+ , V 3+ , V 2+ , Nb 3+ , Nb 5+ , Ta 5+ , Cr 6+ , Cr 3+ , Mo 6+ , Mo 3+ , W 6+ , W 3+ , Mn 4+ , Mn 3+ , Mn 2+ , Re 7+ , Re 2+ , Fe 3+ , Fe 2+ , Ru 4+ , Ru 3+ , Ru 2+ , Os 3+ , Os 2+ , Co 3+ , Co 2+ , Rh 3+ , Rh 2+ , Rh + , Ir 4+ , Ir 2+ , Ir + , Ni 2+ , Pd 4+ , Pd 2+ , Pt 4+ , Pt 2+ , Cu 2+, Cu + , Ag + , Au + , Zn 2+ , Cd 2+ , Hg 2+ , B 3+ , Al 3+ , Ga 3+ , In 3+ , T.I. 3+ , Si 4+ , Si 2+ , Ge 4+ , Ge 2+ , Sn 4+ , Sn 2+ , Pb 4+ , Pb 2+ , As 5+ , As 3+ , Sb 5+ , Sb 3+ , Bi 5+ , Bi 3+ , La 3+ , Ce 3+ , Ce 4+ , Pr 3+ , Pr 4+ , Nd 3+ , Sm 3+ , Sm 2+ , Eu 3+ , Eu 2+ , Gd 3+ , Tb 3+ , Tb 4+ , Dy 3+ , Ho 3+ , Er 3+ , Tm 3+ , Tm 2+ , Yb 3+ , Yb 2+ , Lu 3+ , Th 4+ , U 6+ , U 5+ , U 4+ , and U 3+ The MOF 21a may contain only one type of metal ion, or may contain multiple types of metal ions.

[0027] The MOF 21a contains ions of at least one metal selected from the group consisting of iron, zinc, aluminum, and copper. In this case, the stability of the enzyme 21b in the enzyme electrode 1a is likely to be further improved. In addition, the enzyme electrode 1a can be easily manufactured at low cost.

[0028] The MOF 21a preferably contains iron ions, which can improve the stability of the enzyme 21b in the enzyme electrode 1a and facilitate the production of the enzyme electrode 1a at low cost.

[0029] The organic molecule contained in MOF21a is not limited to a specific organic molecule. Examples of the organic molecule include 4,4',4''-[benzene-1,3,5-triyl-tris(ethyne-2,1-diyl)]tribenzoate, biphenyl-4,4'-dicarboxylate, 4,4',4''-[benzene-1,3,5-triyl-tris(benzene-4,1-diyl)]tribenzoate, 1,3,5-benzenetribenzoate, 1,4-benzenedicarboxylate, benzene-1,3,5-tris(1H-tetrazole), 1,3,5-benzenetricarboxylic acid, terephthalic acid, imidazole, and benzimidazolate. , 2-nitroimidazole, 2-methylimidazole, 2-ethylimidazole, 5-chlorobenzimidazole, purine, fumaric acid, α-cyclodextrin, β-cyclodextrin, γ-cyclodextrin, 1,4-bis(1-imidazolyl)benzene, 4,4'-bispyridyl, 1,4-diazabicyclo[2.2.2]octane, 2-amino-1,4-benzenedicarboxylate, 2-amino-1,4-benzenedicarboxylic acid, 4,4'-azobenzenedicarboxylate, 4,4'-azobenzenedicarboxylic acid, azobenzenedicarboxylic acid, Aniline-2,4,6-tribenzoate, Aniline-2,4,6-tribenzoic acid, Biphenyl-4,4'-dicarboxylic acid, 1,1'-biphenyl-2,2',6,6'-tetracarboxylate, 1,1'-biphenyl-2,2',6,6'-tetracarboxylic acid, 2,2'-bipyridyl-5,5'-dicarboxylate, 2,2'-bipyridyl-5,5'-dicarboxylic acid, 1,3,5-tris(4-carboxyphenyl)benzene, 1,3,5-tris(4-carboxylatephenyl)benzene, 1,3,5-benzenetricarbo xylate, 2,5-dihydroxy-1,4-benzenedicarboxylate, 2,5-dihydroxy-1,4-benzenedicarboxylic acid, 2,5-dimethoxy-1,4-benzenedicarboxylate, 2,5-dimethoxy-1,4-benzenedicarboxylic acid, 1,4-naphthalenedicarboxylate, 1,4-naphthalenedicarboxylic acid, 1,3-naphthalenedicarboxylate, 1,3-naphthalenedicarboxylic acid, 1,7-naphthalenedicarboxylate, 1,7-naphthalenedicarboxylic acid, 2,6-naphthalenedicarboxylate, 2,6-Naphthalenedicarboxylic acid, 1,5-naphthalenedicarboxylate, 1,5-naphthalenedicarboxylic acid, 2,7-naphthalenedicarboxylate, 2,7-naphthalenedicarboxylic acid, 4,4',4''-nitrilotrisbenzoate, 4,4',4''-nitrilotrisbenzoic acid, 2,4,6-tris(2,5-dicarboxyphenylamino)-1,3,5-triazine, 2,4,6-tris(2,5-dicarboxylatephenylamino)-1,3,5-triazine, 1,3,6,8-tetrakis(4-carboxyphenyl)pyrene, 1, 3,6,8-tetrakis(4-carboxylatephenyl)pyrene, 1,2,4,5-tetrakis(4-carboxylatephenyl)benzene, 1,2,4,5-tetrakis(4-carboxylatephenyl)benzene, 5,10,15,20-tetrakis(4-carboxylatephenyl)porphyrin, 5,10,15,20-tetrakis(4-carboxylatephenyl)porphyrin, adenine, adeninate, fumarate, 1,2,4,5-benzenetetracarboxylate, 1,2,4,5-benzenetetracarboxylic acid, 1,3,5-benzene tribenzoic acid, 3-amino-1,5-benzenedicarboxylic acid, 3-amino-1,5-benzenedicarboxylate, 1,3-benzenedicarboxylic acid, 1,3-benzenedicarboxylate, 4,4',4''-[benzene-1,3,5-triyl-tris(ethyne-2,1-diyl)]tribenzoic acid, 4,4',4''-[benzene-1,3,5-triyl-tris(benzene-4,1-diyl)]tribenzoic acid, oxalic acid, oxalate, fumaric acid, fumarate, maleic acid, maleate, trans,trans-muconic acid, trans,tr ans-muconate, cis,trans-muconic acid, cis,trans-muconate, cis,cis-muconic acid, cis,cis-muconate, pyrazole, 2,5-dimethylpyrazole, 1,2,4-triazole, 3,5-dimethyl-1,2,4-triazole, pyrazine, 2,5-dimethylpyrazine, hexamethylenetetraamine, nicotinic acid, nicotinate, isonicotinic acid, isonicotinate, 4-(3,5-dimethyl-1H-pyrazole)-benzoic acid, 2,5-furandicarboxylic acid, 2,5-furandicarboxylate, 3,5-dimethyl-4-carboxypyrazole, 3,5-dimethyl-4-carboxylate pyrazole, and 4-(3,5-dimethyl-1H-pyrazol-4-yl)-benzoic acid, 4-(3,5-dimethyl-1H-pyrazol-4-yl)-benzoate. MOF21a may contain only one type of organic molecule or multiple types of organic molecules.

[0030] The organic molecule contained in the MOF 21a has, for example, a benzene ring, which tends to further increase the stability of the enzyme 21b in the enzyme electrode 1a.

[0031] The organic molecule having a benzene ring contained in the MOF 21a is preferably trimesic acid, which is likely to further increase the stability of the enzyme 21b in the enzyme electrode 1a.

[0032] MOF 21a may be a lanthanide (Ln)-containing MOF, such as Er(bdc), Dy(bdc), Tb(bpdc), Gd(bpdc), Tb(bpydc), Tb(bdc), Eu(bdc), Gd(bdc), and Ln(bpedc), where bdc is 1,4-benzenedicarboxylate, bpdc is 4,4'-biphenyldicarboxylate, bpydc is 2,2'-bipyridine-5,5'-dicarboxylate, and bpedc is biphenylethene-4,4'-dicarboxylate. MOF 21a may be selected from mixed-component metal-organic frameworks known as MC-MOFs. MC-MOFs have structures characterized by more than one type of organic ligand and / or metal. MC-MOFs can be obtained by using different organic ligands and / or metals in the solution combination of metal-organic framework precursors and biomolecules, or by post-synthetic substitution of the organic ligands and / or metal species of the metal-organic framework.

[0033] MOF21a may be a zinc imidazolate framework (ZIF). ZIFs are a subclass of metal-organic frameworks that are particularly suitable for biological applications due to (i) their long-term stability under physiological conditions, (ii) the pH-responsive nature of the metal-organic ligand bond, and (iii) negligible cytotoxicity. Due to property (ii), ZIFs can be used as triggers for pH-triggered drug delivery applications. Furthermore, ZIFs can be synthesized in water and are chemically stable in water for extended periods, even at high temperatures. For example, ZIFs are chemically stable for several weeks near the boiling point of water. The stability of ZIFs in water makes them desirable matrices for hosting biomolecules for use in biological environments.

[0034] ZIFs have four-coordinated transition metal ions connected by organic imidazolate ligands. As a result, ZIFs can be formed as three-dimensional porous solids. Examples of four-coordinated transition metal ions are Fe, Co, Cu, and Zn. ZIFs, like zeolites, have very high thermal and chemical stability. By selecting ZIF precursors, many different ZIF topologies can be synthesized depending on the type of biomolecule. Therefore, MOF 21a can be a carboxylate-based MOF, a heterocyclic azolate-based MOF, or a metal cyanide MOF. MOF 21a can be, for example, one commonly known in the art.Examples of MOF21a are CD-MOF-1, CD-MOF-2, CD-MOF-3, CPM-13, FJI-1, FMOF-1, HKUST-1, IRMOF-1, IRMOF-2, IRMOF-3, IRMOF-6, IRM OF-8, IRMOF-9, IRMOF-13, IRMOF-20, JUC-48, JUC-62, MIL-101, MIL-100, MIL-125, MIL-53, MIL-88, MOF-5, MOF-74, MOF-1 77, MOF-210, MOF-200, MOF-205, MOF-505, MOROF-2, MOROF-1, NOTT-100, NOTT-101, NOTT-102, NOTT-103, NOTT-105, NOTT -106, NOTT-107, NOTT-109, NOTT-110, NOTT-111, NOTT-112, NOTT-113, NOTT-114, NOTT-140, NU-100, rho-ZMOF, PCN-6, PC N-6', PCN9, PCN10, PCN12, PCN12', PCN14, PCN16, PCN-17, PCN-21, PCN46, PCN66, PCN68, PMOF-2 (Cu), PMOF-3, SNU-5, SNU -15', SNU-21S, SNU-21H, SNU-50, SNU-77H, UiO-66, UiO-67, soc-MOF, sod-ZMOF, TUDMOF-1, UMCM-2, UMCM-150, UTSA-20, Z IF-2, ZIF-3, ZIF-4, ZIF-8, ZIF-9, ZIF-10, ZIF-11, ZIF-12, ZIF-14, ZIF-20, ZIF-21, ZIF-23, ZIF-60, ZIF-61, ZIF-62, ZI F-64, ZIF-65, ZIF-67, ZIF-68, ZIF-69, ZIF-70, ZIF-71, ZIF -72, ZIF-73, ZIF-74, ZIF-75, ZIF-76, ZIF-77, and ZIF-90. MIL-88 includes the MIL-88A, MIL-88B, MIL-88C, and MIL-88D series. The enzyme electrode 1a may contain only one type of MOF 21a, or may contain multiple types of MOFs 21a.

[0035] The substrate 10 is not limited to a specific material as long as it is conductive. The substrate 10 may contain a metal or carbon. The substrate 10 may be configured as a metal electrode or a carbon electrode. The carbon electrode is, for example, a glassy carbon electrode. The substrate 10 may perform the function of receiving or donating electrons due to, for example, oxidation-reduction involving a target substance.

[0036] The immobilizing agent 22 is not limited to a specific immobilizing agent as long as it can immobilize the enzyme 21b and the MOF 21a to the substrate 10. In this specification, immobilization may include the enzyme 21b and the MOF 21a remaining in the vicinity of the substrate 10. The immobilizing agent 22 may immobilize the enzyme 21b and the MOF 21a, which are separated from each other, to the substrate. In other words, the enzyme-containing layer 20 may include a portion where the enzyme 21b is dispersed alone, or a portion where the MOF 21a is dispersed alone.

[0037] The immobilizing agent 22 includes, for example, a polymer. This polymer, for example, keeps the enzyme 21b in the vicinity of the substrate 10. In this case, oxidation-reduction involving the target substance is more likely to occur without supplementation of the enzyme.

[0038] The polymer used as the immobilizing agent 22 is not limited to a specific polymer. The polymer may include, for example, at least one selected from the group consisting of nylon, collagen, gelatin, cellulose, and chitosan. In this case, the enzyme 21b is likely to be immobilized on the substrate in a desired state.

[0039] The immobilizing agent 22 may be an agent that binds the enzyme 21b to the substrate 10 through a chemical bond. This chemical bond is not limited to a specific chemical bond. This chemical bond includes, for example, hydrogen bonds and intermolecular bonds. For example, the immobilizing agent 22 may be an agent that can form an avidin-biotin complex between the substrate 10 and the enzyme 21b. The immobilizing agent 22 may be an agent that immobilizes the MOF 21a to the substrate 10.

[0040] 1 , the enzyme electrode 1a further includes, for example, a mediator 23. The mediator 23 performs the function of donating and receiving electrons generated by oxidation-reduction of the target substance. The enzyme 21b, for example, donates electrons generated by oxidation-reduction of the target substance to the mediator 23. The enzyme 21b donates electrons to the mediator 23 in an amount corresponding to the concentration of the target substance or the reaction amount of the target substance.

[0041] The mediator 23 is not limited to a specific mediator as long as it can donate and accept electrons generated by the oxidation-reduction of the target substance. Examples of the mediator 23 are NAD, NADP, polyethylenedioxythiophene, and methyl viologen. The mediator 23 is disposed in contact with the substrate 10 in the enzyme-containing layer 20, for example. The mediator 23 may be dispersed throughout the enzyme-containing layer 20.

[0042] As shown in Fig. 1, the enzyme electrode 1a includes, for example, a conductive material 24. The conductive material 24 is mixed with an immobilizing agent 22. Such a configuration tends to increase the sensitivity of target substance detection using the enzyme electrode 1a. Examples of the conductive material 24 include carbon materials such as carbon black and carbon nanotubes. The conductive material 24 may contain a metal.

[0043] Fig. 2 is a cross-sectional view schematically showing another example of the enzyme electrode according to the present embodiment. The enzyme electrode 1b shown in Fig. 2 has the same configuration as the enzyme electrode 1a, except for the parts that will be particularly described. The same reference numerals are used to designate the components of the enzyme electrode 1b that are the same as or correspond to the components of the enzyme electrode 1a, and detailed descriptions thereof will be omitted. The description of the enzyme electrode 1a also applies to the enzyme electrode 1b, unless technically inconsistent.

[0044] As shown in FIG. 2 , the enzyme electrode 1b further includes, for example, an intermediate layer 30. The intermediate layer 30 is disposed between the substrate 10 and the enzyme-containing layer 20 in the thickness direction. The intermediate layer 30 is, for example, a conductive layer. With this configuration, the enzyme-containing layer 20 is less likely to peel off from the enzyme electrode 1b, and the enzyme electrode 1b can exhibit high durability. The intermediate layer 30 may include, for example, a conductive polymer such as poly(3,4-ethylenedioxythiophene / poly(4-styrenesulfonic acid)) (PEDOT / PSS) or a carbon material such as graphene. The intermediate layer 30 may also include a mediator 23.

[0045] As described above, a target substance can be detected using the enzyme electrode 1a. Fig. 3 is a diagram schematically illustrating an example of an electrochemical device including the enzyme electrode 1a. As shown in Fig. 3, the electrochemical device 2 includes, in addition to the enzyme electrode 1a, a reference electrode 4a and an auxiliary electrode 4b, for example. The electrochemical device 2 detects the target substance according to, for example, a three-point electrode method. The enzyme electrode 1a functions as a working electrode.

[0046] Examples of the reference electrode 4a include a calomel electrode, a silver-silver chloride electrode, a hydrogen electrode, a mercury-mercury oxide electrode, a mercury-mercury sulfide electrode, a silver-silver ion electrode, a quasi-reference electrode, and a redox system reference electrode. Examples of the auxiliary electrode 4b include a glassy carbon electrode and a platinum electrode.

[0047] 4 is a flowchart showing an example of a method for detecting a target substance according to this embodiment. This method includes, for example, the following steps (i), (ii), and (iii): (i) supplying a target substance to the electrochemical device 2; (ii) bringing the target substance into contact with the enzyme electrode 1a to cause an oxidation-reduction reaction involving the target substance; and (iii) determining the concentration of the target substance based on the magnitude of the current generated at the enzyme electrode 1a by the oxidation-reduction reaction involving the target substance.

[0048] 4, in step S11, a target substance is supplied to the electrochemical device 2. The method for supplying the target substance to the electrochemical device 2 is not limited to a specific method as long as the target substance comes into contact with the enzyme electrode 1a. For example, a sample containing the target substance is supplied to the inside of a container in which the enzyme electrode 1a, the reference electrode 4a, and the auxiliary electrode 4b are arranged. The target substance may be supplied through a flow path extending over the surface of the enzyme electrode 1a, or the sample containing the target substance may be dripped toward the enzyme electrode 1a.

[0049] In step S12, the target substance comes into contact with the enzyme electrode 1a, and an oxidation-reduction reaction involving the target substance occurs. Next, in step S13, the current value generated at the enzyme electrode 1a due to the oxidation-reduction reaction is obtained. For example, in the electrochemical device 2, the enzyme electrode 1a, which is the working electrode, is polarized to a constant potential relative to the reference electrode 4a using a potentiostat (not shown). At this time, a current resulting from the oxidation-reduction reaction involving the target substance is generated between the enzyme electrode 1a, which is the working electrode, and the auxiliary electrode 4b. For example, the change over time in the current at the enzyme electrode 1a, which is the working electrode, is measured, and chronoamperometry measurement is performed.

[0050] Next, in step S14, the concentration of the target substance is determined based on the acquired current value. For example, if the enzyme electrode 1a or the sample containing the target substance contains a mediator, when the target substance is oxidized or reduced by the enzyme 21b, the mediator is reduced or oxidized. This reduced or oxidized mediator is oxidized or reduced on the substrate 10 of the enzyme electrode 1a, which is the working electrode. This can be used to quantify the target substance in the sample. The current value obtained by chronoamperometry measurement tends to increase as the concentration of the target substance increases. Therefore, the concentration of the target substance can be detected by measuring the current at the substrate 10. The concentration of the target substance may be detected from the change or integrated amount of the acquired current value. For example, the concentration of the target substance can be determined by referring to a table or function that corresponds the change or integrated amount of the current value of the enzyme electrode 1a to the concentration of the target substance, which has been previously prepared by chronoamperometry measurement using a sample with a known concentration of the target substance.

[0051] Fig. 5 is a schematic diagram showing an example of a biosensor equipped with an enzyme electrode according to the present embodiment. As shown in Fig. 5, the biosensor 3a includes, in addition to the enzyme electrode 1a, an ammeter 7a and a signal processor 7b, for example. In the biosensor 3a, the ammeter 7a measures the current at the enzyme electrode 1a. The ammeter 7a is, for example, a potentiostat or a galvanostat. The signal processor 7b processes a signal indicating the measurement result of the current at the enzyme electrode 1a and performs calculations for detecting the target substance. The signal processor 7b includes, for example, an A / D conversion circuit and a digital signal processor (DSP).

[0052] In the biosensor 3a, the target substance is oxidized or reduced by the enzyme 21b contained in the enzyme electrode 1a. For example, the coenzyme or mediator 23, which is reduced or oxidized as a result of the oxidation or reduction of the target substance, is oxidized or reduced at the enzyme electrode 1a, and electrons are transferred. The ammeter 7a measures the current generated by this electron transfer. The signal processor 7b determines the concentration of the target substance based on the current value measured by the ammeter 7a. The concentration of the target substance may be determined based on the integrated amount of the current value or the amount of change in the current value.

[0053] Fig. 6 is a diagram schematically illustrating another example of a biosensor equipped with an enzyme electrode according to this embodiment. The biosensor 3b shown in Fig. 6 has the same configuration as the biosensor 3a, except for the parts that will be particularly described. The components of the biosensor 3b that are the same as or correspond to the components of the biosensor 3a are designated by the same reference numerals, and detailed description thereof will be omitted. The description of the biosensor 3a also applies to the biosensor 3b, unless technically inconsistent.

[0054] As shown in FIG. 6 , the biosensor 3b includes, in addition to the enzyme electrode 1a, for example, an ammeter 7a, a signal processor 7b, a supply mechanism 8, and a recording medium 9. The biosensor 3b is configured as, for example, a wearable sensor. The supply mechanism 8 is a mechanism capable of supplying a target substance toward the enzyme electrode 1a. The supply mechanism 8 supplies the target substance toward the enzyme electrode 1a using, for example, a pump, a moisture absorbent, or capillary action. The recording medium 9 is connected to the signal processor 7b by wire or wirelessly. Data indicating the concentration of the target substance determined by the signal processor 7b is transferred to the recording medium 9 by wire or wireless communication and recorded therein. The biosensor 3b may be configured as a stationary sensor.

[0055] (Other Embodiments) The enzyme electrode, the electrochemical device, and the method for detecting a target substance have been described based on the embodiments. However, the present disclosure is not limited to these embodiments. As long as they do not deviate from the gist of the present disclosure, various modifications that a person skilled in the art can conceive of to the embodiments and other embodiments formed by combining some of the components of the embodiments are included in the scope of the present disclosure.

[0056] Second Embodiment Hereinafter, a second embodiment will be specifically described with reference to FIGS.

[0057] FIG. 7 is a schematic diagram illustrating an example of a structure according to the present embodiment. As shown in FIG. 7, the structure 100a includes a metal-organic framework (MOF) 110 and an enzyme 120. In the structure 100a, the MOF 110 and the enzyme 120 are hybridized. The MOF 110 has a window size of 0.5 nm or more. With this configuration, the activity of the enzyme 120 is likely to be stabilized. Although the reason for this is unclear, it is thought that when the MOF 110 has a window size of 0.5 nm or more, the movement of substances is less likely to be hindered when the substrate reacts in the structure 100a due to the catalytic action of the enzyme 120, and the three-dimensional structure of the MOF 110 stabilizes the structure of the enzyme 120, making it easier to stabilize the activity of the enzyme 120. The window size of a MOF is the size of the opening between the interior and exterior of the MOF, and is the diameter of a circle inscribed in that opening. The window size of the MOF can be determined, for example, based on the results of a computer simulation of the structure of the MOF.

[0058] The method for evaluating the activity of the enzyme 120 is not limited to a specific method and may be a general method. For example, the activity of the enzyme 120 can be evaluated by a method for determining the reaction rate of an enzymatic reaction involving the enzyme 120. The activity of the enzyme 120 can be evaluated based on the amount of decrease in substrate concentration accompanying the enzymatic reaction involving the enzyme 120 or the amount of increase in product concentration accompanying the enzymatic reaction involving the enzyme 120. The amount of decrease in substrate concentration or the amount of increase in product concentration can be determined based on measurements such as predetermined optical measurements and electrochemical measurements.

[0059] The method for evaluating the stability of the activity of the enzyme 120 is not limited to a specific method and may be a general method. For example, the evaluation may be based on the rate of decrease in the activity of the enzyme 120 during storage of the structure 100a or the rate of decrease in the activity of the enzyme 120 in an accelerated test.

[0060] In the structure 100a, the enzyme 120 may be adsorbed to the MOF 110, or the MOF 110 may be adsorbed to the enzyme 120. At least a part of the enzyme 120 may be disposed inside the MOF 110. The enzyme 120 may be encapsulated in the MOF 110.

[0061] The window size of the MOF 110 is, for example, 2.0 nm or less.

[0062] In the structure 100a, the MOF 110 is not limited to a specific MOF as long as it has a window size of 0.5 nm or more. The MOF 110 contains, for example, Mil-100(Fe). In this case, the three-dimensional structure of the enzyme 120 is more likely to be maintained, and the activity of the enzyme 120 is more likely to be stabilized. Figure 8 is a diagram showing the structure of Mil-100(Fe). As shown in Figure 8, the window size w of Mil-100(Fe) is 0.84 nm. Furthermore, the cage size c of Mil-100(Fe) is 2.3 nm. For example, the following document describes a method for preparing Mil-100(Fe), but does not describe the complexation of Mil-100(Fe) with an enzyme or the enhancement of enzyme stability by the complexation of Mil-100(Fe) with an enzyme. Weifeng Li, Tao Zhang, “Room-temperature synthesis of MIL-100(Fe) and its adsorption fluoride removal from water”, Colloids and Surfaces A: Physicochemical and Engineering Aspects, 624(2021), 1-13. MIL-100(Fe) is also known as Iron(III) 1,3,5-benzenetricarboxylate hydrate porous (F-free MIL-100(Fe), AF-100(Fe)) and Iron trimesate. 3+ Mil-100 (Fe) is composed of Fe ions and trimesic acid. 2+ is oxidized by an oxidizing agent 3+ It may also be synthesized by oxidizing it to

[0063] The MOF 110 may be Mil-100(Fe), MOF5, or MOF74. The structure 100a may include only a specific type of MOF 110, or may include multiple types of MOFs 110.

[0064] The enzyme 120 is not limited to a specific enzyme. The structure 100a may include only a specific type of enzyme 120, or may include multiple types of enzymes 120. The enzyme 120 is, for example, an oxidoreductase. The oxidoreductase may be an oxidase or a dehydrogenase. When the substrate is glucose, examples of the oxidoreductase are glucose oxidase and glucose dehydrogenase. When the substrate is lactic acid, examples of the oxidoreductase are lactate oxidase and lactate dehydrogenase. When the substrate is cholesterol, examples of the oxidoreductase are cholesterol esterase and cholesterol oxidase. When the substrate is alcohol, an example of the oxidoreductase is alcohol oxidase. When the substrate is bilirubin, an example of the oxidoreductase is bilirubin oxidase. When the substrate is cortisol, an example of the oxidoreductase is 11β-hydroxysteroid dehydrogenase.

[0065] The enzyme 120 may or may not be coenzyme-dependent. For example, if the enzyme 120 is an oxidoreductase, the oxidoreductase may be one that exhibits activity in response to a coenzyme. Even in such a case, the activity of the enzyme 120 is likely to be stabilized by the structure 100a.

[0066] The enzyme 120 may be an enzyme dependent on a coenzyme, such as nicotinamide adenine dinucleotide (NAD), nicotinamide adenine dinucleotide phosphate (NADP), pyrroloquinoline quinone (PQQ), or flavin adenine dinucleotide (FAD). The enzyme 120 may be a coenzyme-dependent oxidoreductase, such as an FAD-dependent oxidase, an NAD-dependent dehydrogenase, a PQQ-dependent dehydrogenase, or an FAD-dependent dehydrogenase. For example, when the enzyme 120 is an 11β-hydroxysteroid dehydrogenase (11β-HSD), NADP may act as a coenzyme.

[0067] The content of the enzyme 120 in the structure 100a is not limited to a specific value. For example, when the structure 100a is used to detect a target substance such as a substrate, the structure 100a contains the enzyme 120 in an amount that enables detection of the target substance. The content of the enzyme 120 in the structure 100a is, for example, 0.0001% or more and 5.0% or less by mass.

[0068] The method for producing the structure 100a is not limited to a specific method. The structure 100a can be produced by, for example, a coprecipitation method or a surface modification method. In the coprecipitation method, the MOF 110 is produced in a solution containing the enzyme 120, and the structure 100a is obtained by collecting the precipitate. In the surface modification method, the structure 100a is obtained by adsorbing the MOF 110 to the enzyme 120 by chemical adsorption or physical adsorption.

[0069] FIG. 9 is a diagram schematically illustrating an example of a device according to the present embodiment. As shown in FIG. 9, device 300 includes structure 100a. Device 300 detects target substance 500 based on at least one selected from the group consisting of an optical change and an electrochemical change caused by an enzymatic reaction involving enzyme 120 in structure 100a. In this manner, device 300 can detect target substance 500 through the action of enzyme 120. In addition, since the activity of enzyme 120 tends to be stable in structure 100a, device 300 can be used to stably detect target substance 500.

[0070] 9 , a target substance 500 reacts with an enzyme reaction involving the enzyme 120 to produce a product 500a. The device 300 further includes, for example, a detector 200. The detector 200 detects at least one selected from the group consisting of an optical change and an electrochemical change caused by the enzyme reaction involving the enzyme 120. A signal indicating the detection result in the detector 200 is processed by a signal processor (not shown), and calculations are performed for detecting the target substance 500. The signal processor includes, for example, an A / D conversion circuit and a digital signal processor (DSP).

[0071] The detector 200 is not limited to a specific detector, as long as it detects at least one change selected from the group consisting of an optical change and an electrochemical change caused by an enzyme reaction. When the detector 200 detects an optical change caused by an enzyme reaction, the detector 200 detects the target substance by, for example, measuring the absorbance at the maximum absorption wavelength of the product 500a. In this case, the wavelength of light at which the absorbance is measured may be changed using a reducing dye. When the detector 200 detects an electrochemical change caused by an enzyme reaction, the detector 200 includes, for example, an electrode and detects the target substance by measuring the voltage between the electrode and a reference potential. For example, if the enzyme 120 is NAD-dependent alcohol dehydrogenase and the substrate is ethanol, the target substance ethanol can be detected by oxidizing NADH produced in the reaction of ethanol at an electrode and measuring the oxidation voltage generated by the oxidation of NADH. In this case, a mediator may be used to change the oxidation voltage.

[0072] (Other Embodiments) The structures and devices have been described based on the embodiments. However, the present disclosure is not limited to these embodiments. As long as they do not deviate from the gist of the present disclosure, various modifications that a person skilled in the art can conceive of to the embodiments and other forms formed by combining some of the components of the embodiments are included in the scope of the present disclosure.

[0073] (Additional Note) From the above description, the following techniques are disclosed.

[0074] (Technology 1) An enzyme electrode comprising: a conductive substrate; an enzyme that catalyzes oxidation-reduction involving a target substance; a metal organic framework; and an immobilizing agent that immobilizes the enzyme and the metal organic framework to the substrate.

[0075] (Technology 2) The enzyme electrode according to Technology 1, wherein the enzyme and the metal organic framework form a complex.

[0076] (Technology 3) The enzyme electrode according to Technology 1, wherein the immobilizing agent includes a polymer.

[0077] (Technology 4) The enzyme electrode according to Technology 3, wherein the polymer includes at least one selected from the group consisting of nylon, collagen, gelatin, cellulose, and chitosan.

[0078] (Technology 5) The enzyme electrode according to any one of Technologies 1 to 4, wherein the immobilizing agent binds the enzyme to the substrate by chemical bonding.

[0079] (Technology 6) The enzyme electrode according to any one of Technologies 1 to 5, wherein the enzyme includes an oxidoreductase that causes charge transfer in oxidation-reduction involving the target substance.

[0080] (Technology 7) The enzyme electrode according to Technology 6, wherein the oxidoreductase causes an enzymatic reaction with the participation of a coenzyme.

[0081] (Technology 8) The enzyme electrode according to any one of technologies 1 to 7, wherein the enzyme includes 11β-hydroxysteroid dehydrogenase.

[0082] (Technology 9) The enzyme electrode according to any one of Techniques 1 to 8, wherein the metal organic framework contains ions of at least one metal selected from the group consisting of iron, zinc, aluminum, and copper.

[0083] (Technology 10) The enzyme electrode according to Technology 9, wherein the metal organic framework contains iron ions.

[0084] (Technology 11) The enzyme electrode according to any one of Techniques 1 to 10, wherein the metal organic framework includes an organic molecule having a benzene ring.

[0085] (Technology 12) The enzyme electrode according to Technology 11, wherein the organic molecule is trimesic acid.

[0086] (Technology 13) The enzyme electrode according to any one of Technologies 1 to 12, further comprising a mediator that donates and accepts electrons generated by oxidation-reduction involving the target substance.

[0087] (Technology 14) The enzyme electrode according to any one of technologies 1 to 13, further comprising a conductive substance mixed with the immobilizing agent.

[0088] (Technology 15) An electrochemical device comprising the enzyme electrode according to any one of technologies 1 to 14.

[0089] (Technology 16) A method for detecting a target substance, comprising: supplying a target substance to the electrochemical device according to Technology 15; bringing the target substance into contact with the enzyme electrode to cause an oxidation-reduction reaction involving the target substance; and determining the concentration of the target substance based on the magnitude of a current generated at the enzyme electrode by the oxidation-reduction reaction.

[0090] (Technology 17) A structure comprising: a metal organic framework; and an enzyme, wherein the metal organic framework and the enzyme are complexed, and the metal organic framework has a window size of 0.5 nm or more.

[0091] (Technology 18) The structure according to Technology 17, wherein the metal organic framework contains Mil-100(Fe).

[0092] (Technology 19) The structure according to Technology 17 or 18, wherein the enzyme is an oxidoreductase.

[0093] (Technology 20) The structure according to Technology 19, wherein the oxidoreductase exhibits activity in the presence of a coenzyme.

[0094] (Technology 21) The structure according to Technology 20, wherein the oxidoreductase comprises 11β-hydroxysteroid dehydrogenase.

[0095] (Technology 22) A device comprising the structure according to any one of technologies 17 to 21, which detects a target substance based on at least one selected from the group consisting of an optical change and an electrochemical change caused by an enzymatic reaction involving the enzyme of the structure.

[0096] The present disclosure will be described in more detail below with reference to examples, but the present disclosure is not limited to the following examples.

[0097] Example 1 Mil-100(Fe) was synthesized by coprecipitation using a solution containing iron ions, trimesic acid, and 11β-hydroxysteroid dehydrogenase type 1 (11b-HSD1), and Mil-100(Fe) and 11b-HSD1 were complexed to obtain the complex 11b-HSD1@Mil-100(Fe) according to Example 1.

[0098] An intermediate layer and an enzyme-containing layer were laminated in this order on a glassy carbon electrode. The intermediate layer was prepared by dropping 1.0 μL of graphene / PEDOT:PSS onto the surface of the glassy carbon electrode and drying the resulting solution. Graphene / PEDOT:PSS is a graphene-containing polyethylenedioxythiophene:polystyrenesulfonic acid solution with a concentration of 2.0 mg / mL. The graphene concentration in the graphene / PEDOT:PSS was 1.0 mg / mL. The enzyme-containing layer was prepared by dropping 1.5 μL of a chitosan and carbon nanotube (CNT) solution containing the 11b-HSD1@Mil-100(Fe) complex onto the intermediate layer and then drying the solution. The concentrations of 11b-HSD1, chitosan, and CNT in this solution were 13 mg / mL, 0.02 mg / mL, and 2.0 mg / mL, respectively. An acetic acid solution was used to suspend 11b-HSD1@Mil-100(Fe). Chitosan is a biocompatible gel that can be applied directly to the skin, and the addition of CNTs reduced the solution's viscosity and improved electron transfer efficiency. After the solution dried, the chitosan was crosslinked using 10 μg / mL sodium polyphosphate to reinforce the enzyme-containing layer. In this way, the enzyme electrode of Example 1 was obtained.

[0099] Chronoamperometry measurements were performed according to the three-point electrode method using the enzyme electrode of Example 1, an Ag / AgCl electrode, and a platinum electrode as the working electrode, reference electrode, and auxiliary electrode, respectively. The potential of the working electrode relative to the reference electrode was adjusted to 0.4 V. A measurement buffer containing 50 mM Tris-HCl and 500 mM NaCl was used. The pH of this measurement buffer was 7.0. The initial concentration of the coenzyme NADP in the measurement buffer was adjusted to 5.0 mM. A 10 mM cortisol solution was added dropwise in amounts of 50 μL, 100 μL, 200 μL, and 400 μL every 100 seconds, and the change in current at the working electrode was measured. The results are shown in FIG. 10.

[0100] In Figure 10, arrows (a), (b), (c), and (d) indicate the dripping of the cortisol solution. As shown in Figure 10, an increase in current value was confirmed each time the cortisol solution was dripped. This is understood to be due to the current generated when the cortisol contained in the dripped cortisol solution was oxidized by the enzyme 11b-HSD1 contained in the enzyme electrode of Example 1, and the reduced coenzyme NADPH produced at the same time was oxidized by the enzyme electrode. This suggests that the enzyme electrode of Example 1 can cause oxidation-reduction involving a target substance without enzyme supplementation, allowing continuous detection of the target substance. The difference in current value patterns between (a) and (d) is expected to be due to the reaction being unstable immediately after the cortisol solution was dripped.

[0101] Figure 11 is a graph showing the relationship between cortisol concentration and current value. This graph shows the average current value and its standard deviation from 10 seconds after the instillation of the cortisol solution to 30 seconds after the instillation in the chronoamperometric measurement described above. Figure 11 suggests that the concentration of the target substance can be estimated from the current value. It is expected that by miniaturizing and increasing the concentration of the enzyme, it will be possible to detect lower concentrations of the target substance.

[0102] Samples for fluorescence microscopy were prepared from Mil-100(Fe) and the complex 11b-HSD1@Mil-100(Fe) of Example 1. In preparing these samples, the enzyme 11b-HSD1 was modified with a fluorophore. Figure 12A shows a transmission image obtained by microscopy of the complex 11b-HSD1@Mil-100(Fe) of Example 1. Figure 12B shows a scattering image obtained by fluorescence microscopy of the complex of Example 1. Figure 12C shows a fluorescence image obtained by fluorescence microscopy of the complex of Example 1. Figure 13A shows a transmission image obtained by microscopy of the MOF Mil-100(Fe). Figure 13B shows a scattering image obtained by fluorescence microscopy of Mil-100(Fe). Figure 13C shows a fluorescence image obtained by fluorescence microscopy of Mil-100(Fe). For these fluorescence microscopic observations, a biological microscope BX43F manufactured by Olympus Corporation was used. The wavelength λ of the excitation light used for these fluorescence microscopic observations was ex The fluorescence image was obtained using a low-pass filter that transmits light in the wavelength range of 590 nm or more.

[0103] Comparing Figures 12A to 12C with Figures 13A to 13C, it is clear that the enzyme is distributed throughout the complex 11b-HSD1@Mil-100(Fe), suggesting that the MOF Mil-100(Fe) and the enzyme 11b-HSD1 form the complex 11b-HSD1@Mil-100(Fe).

[0104] Comparative Example 1 An enzyme electrode according to Comparative Example 1 was produced in the same manner as in Example 1, except that the enzyme 11b-HSD1 was used instead of the complex 11b-HSD1@Mil-100(Fe).

[0105] Chronoamperometry measurements were performed according to the three-point electrode method using the enzyme electrode of Example 1, an Ag / AgCl electrode, and a platinum electrode as the working electrode, reference electrode, and auxiliary electrode, respectively. The potential of the working electrode relative to the reference electrode was adjusted to 0.4 V. A measurement buffer containing 50 mM Tris-HCl and 500 mM NaCl was used. The pH of this measurement buffer was 7.5. The initial concentration of the coenzyme NADP in the measurement buffer was adjusted to 1.0 mM. The enzyme electrode of Example 1 was immersed in a 0.1 mM cortisol solution for 30 minutes, and then chronoamperometry measurements were performed. Similarly, chronoamperometry measurements were performed using the enzyme electrode of Example 1 that was not immersed in the cortisol solution. FIG. 14 is a graph showing the results of chronoamperometry measurements using the enzyme electrode of Example 1. In FIG. 14, (Ia) shows the results when the enzyme electrode according to Example 1 was immersed in the cortisol solution for 30 minutes, and (Ib) shows the results when the enzyme electrode according to Example 1 was not immersed in the cortisol solution.

[0106] Chronoamperometry measurements were performed in the same manner as in Example 1, except that the enzyme electrode of Comparative Example 1 was used instead of the enzyme electrode of Example 1. The enzyme electrode of Comparative Example 1 was immersed in a 0.1 mM cortisol solution for 30 minutes, and then chronoamperometry measurements were performed. Meanwhile, chronoamperometry measurements were performed in the same manner using the enzyme electrode of Comparative Example 1 that was not immersed in the cortisol solution. Figure 15 is a graph showing the results of chronoamperometry measurements using the enzyme electrode of Comparative Example 1. (IIa) shows the results when the enzyme electrode of Example 1 was immersed in the cortisol solution for 30 minutes, and (IIb) shows the results when the enzyme electrode of Example 1 was not immersed in the cortisol solution.

[0107] As shown in Figure 14, the current value measured when the enzyme electrode according to Example 1 was immersed in a cortisol solution was larger than the current value measured when the enzyme electrode was not immersed in a cortisol solution. On the other hand, as shown in Figure 15, the current value measured when the enzyme electrode according to Comparative Example 1 was immersed in a cortisol solution was not significantly different from the current value measured when the enzyme electrode was not immersed in a cortisol solution. This suggests that high enzyme activity can be easily maintained by immobilizing a complex of an MOF and an enzyme on a conductive substrate using an immobilizing agent.

[0108] Example 1A: 1.0 mL of a 0.90 mg / mL and 1.0 mL 11β-HSD1 solution was added dropwise to a 20 millimolar (mM) and 5.0 milliliter (mL) aqueous solution of sodium trimesate. Next, 5.0 mL of a 20 mM and 5.0 mL aqueous solution of iron(II) sulfate was added, and 1.0 mL of a 5.0 mass% and 1.0 mL aniline solution was added dropwise to obtain a mixed solution. Next, the mixed solution was stirred in a refrigerator, and a reaction to produce the structure of Example 1A, in which Mil-100(Fe) and 11β-HSD1 were complexed, was carried out for 24 hours. Next, this structure was washed with ultrapure water. Next, this structure was suspended in a buffer solution to obtain a suspension of Example 1A. This suspension was used to evaluate the activity of 11β-HSD1. The supernatant of the mixed solution after the reaction was quantitatively analyzed for protein according to the Bradford method, and the protein concentration was below the detection limit. Therefore, it is considered that most of the added 11β-HSD1 solution was composited with Mil-100(Fe). It is considered that the content of 11β-HSD1 in the structure of Example 1A was 0.01 mass%.

[0109] Comparative Example 1A A suspension according to Comparative Example 1A was obtained in the same manner as in Example 1A, except that 11β-HSD1 was used instead of the structure according to Example 1A.

[0110] Comparative Example 2A: 1.0 mL of 0.90 mg / mL and 1.0 mL of 11β-HSD1 solution was added dropwise to 1200 mM and 9.0 mL of 2-methyltriazole aqueous solution. Next, 400 mM and 9.0 mL of zinc sulfate aqueous solution were added to obtain a mixed solution. Next, the mixed solution was stirred in a refrigerator, and a reaction to prepare the structure according to Comparative Example 2A, in which ZIF-8 and 11β-HSD1 were complexed, was carried out for 24 hours. This structure was then washed with ultrapure water. This structure was then suspended in a buffer solution to obtain the suspension according to Comparative Example 2A. This suspension was used to evaluate the activity of 11β-HSD1. Protein quantitative analysis of the supernatant of the mixed solution after the reaction was performed according to the Bradford method, but the protein concentration was below the detection limit. Therefore, it is believed that most of the added 11β-HSD1 was complexed with ZIF-8. The window size of ZIF-8 is 0.31 nm. The content of 11β-HSD1 in the structure according to Comparative Example 2A is thought to have been 0.1 mass %.

[0111] (Evaluation of Enzyme Activity) For the buffer solutions of Example 1A, Comparative Example 1A, and Comparative Example 2A, buffer solutions that had been heated at 40°C for 5 minutes and buffer solutions that had not been subjected to such heating treatment were prepared. The concentration of 11β-HSD1 in each buffer solution was estimated to be 0.2 mg / mL. Samples for enzyme activity evaluation were prepared with or without the addition of cortisol so that the cortisol concentrations in these buffer solutions were 0 μM, 5 μM, 10 μM, 20 μM, 40 μM, 50 μM, 80 μM, or 100 μM. To these samples, the coenzyme NADP was added at a concentration of 1.0 mM, WST-3 was added at a concentration of 1.0 mM, and 1-Methoxy PES was added at a concentration of 0.1 mM. The reaction rate of the enzymatic reaction in which cortisol was the substrate was calculated based on the change in cortisol concentration in the sample to which cortisol was added. 11β-HSD1 acts as a catalyst for the dehydrogenation of cortisol through the action of the coenzyme NADP. As a result of the dehydrogenation of cortisol, NADP is converted to reduced NADP (NADPH). WST-3 is reduced by reduced NADP (NADPH) with 1-Methoxy PES acting as an electron carrier to produce yellow formazan. The maximum absorption wavelength of WST-3 formazan is λ max is 433 nm, and the absorption maximum wavelength λ max The absorbance of formazan in this sample is proportional to the concentration of NADPH. Therefore, the concentration of cortisol can be determined based on a calibration curve prepared in advance from the absorbance of the sample at a wavelength of 433 nm, and the reaction rate of the enzymatic reaction in which cortisol is the substrate can be calculated. In this way, the reaction rate of the enzymatic reaction in which cortisol is the substrate for each sample was calculated. The results are shown in Figure 16.

[0112] 16 is a graph showing the relationship between the reaction rate of the enzyme reaction and the initial cortisol concentration in the examples and comparative examples. As shown in FIG. 16, it can be seen that in Example 1A, the higher the initial cortisol concentration, the faster the reaction rate. Therefore, it can be seen that an enzyme reaction in which cortisol is a substrate occurs using the structure of Example 1A. By fitting the following formula (1) to the graph of FIG. 16, the Michaelis constant K of the enzyme can be calculated for the buffer solutions of Example 1A, Comparative Example 1A, and Comparative Example 2A.m and maximum reaction rate V max was determined. The results are shown in Figure 17. In equation (1), v is the reaction rate and [S] is the substrate concentration.

[0113] v = V max [S] / (K m + [S]) Equation (1) Figure 17 is a graph showing the Michaelis constant and maximum reaction rate of the enzyme reaction in the examples and comparative examples. According to Figure 17, the activity of 11β-HSD1 in Comparative Example 1A was reduced by about 90% by heat treatment, and the activity of 11β-HSD1 in Comparative Example 2A was reduced by about 70% by heat treatment. On the other hand, it can be seen that the activity of 11β-HSD1 in Example 1A was maintained even after heat treatment. Comparison of Example 1A with Comparative Examples 1A and 2A suggests that the activity of the enzyme is more likely to be stabilized by using a structure in which an MOF having a window size of 0.5 nm or more is combined with an enzyme.

[0114] The enzyme electrode of the present disclosure can be used to continuously detect a target substance that serves as a substrate for an oxidoreductase. The enzyme electrode of the present disclosure can be used, for example, in a detection device that detects a target substance such as cortisol. In addition, the enzyme electrode of the present disclosure can be applied to continuous measurement of biological substances contained in body fluids or to wearable sensors. In addition, the enzyme electrode of the present disclosure can be used for electrochemical sensing of substances in the environment.

[0115] 1a, 1b Enzyme electrode 2 Electrochemical device 10 Substrate 21 Composite 21a MOF 21b Enzyme 22 Immobilizing agent 23 Mediator 24 Conductive material 30 Intermediate layer 100a Structure 110 MOF 120 Enzyme 300 Device 500 Target substance

Claims

1. An enzyme electrode comprising: a conductive substrate; an enzyme that catalyzes an oxidation-reduction reaction involving a target substance; a metal-organic framework; and an immobilizing agent that immobilizes the enzyme and the metal-organic framework to the substrate.

2. The enzyme electrode according to claim 1, wherein the enzyme and the metal-organic framework form a complex.

3. The enzyme electrode according to claim 1, wherein the immobilizing agent comprises a polymer.

4. The enzyme electrode according to claim 3, wherein the polymer comprises at least one selected from the group consisting of nylon, collagen, gelatin, cellulose, and chitosan.

5. The enzyme electrode according to claim 1, wherein the immobilizing agent binds the enzyme to the substrate by a chemical bond.

6. The enzyme electrode according to claim 1, wherein the enzyme comprises an oxidoreductase that causes charge transfer in an oxidation-reduction reaction involving the target substance.

7. The enzyme electrode according to claim 6, wherein the oxidoreductase causes an enzyme reaction with the participation of a coenzyme.

8. The enzyme electrode according to claim 1, wherein the enzyme comprises 11β-hydroxysteroid dehydrogenase.

9. The enzyme electrode according to claim 1, wherein the metal organic framework contains ions of at least one metal selected from the group consisting of iron, zinc, aluminum, and copper.

10. The enzyme electrode according to claim 9, wherein the metal-organic framework contains iron ions.

11. The enzyme electrode according to claim 1, wherein the metal-organic framework comprises an organic molecule having a benzene ring.

12. The enzyme electrode according to claim 11, wherein the organic molecule is trimesic acid.

13. The enzyme electrode according to claim 1, further comprising a mediator that receives electrons generated by oxidation-reduction involving the target substance.

14. The enzyme electrode according to claim 1, further comprising a conductive material mixed with the immobilizing agent.

15. An electrochemical device comprising the enzyme electrode according to any one of claims 1 to 14.

16. A method for detecting a target substance, comprising: supplying a target substance to the electrochemical device described in claim 15; bringing the target substance into contact with the enzyme electrode to cause an oxidation-reduction reaction involving the target substance; and determining the concentration of the target substance based on the magnitude of the current generated at the enzyme electrode by the oxidation-reduction reaction.

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