Two-dimensional particle-oxidoreductase complex

Abstract

This two-dimensional particle-oxidoreductase complex includes a two dimensional particle having one or more layers. In the two-dimensional particle, each of the layers includes: a layer main body represented by the formula MmXn (in the formula, M is at least one metal belonging to groups 3, 4, 5, 6 and 7, X is a carbon atom, a nitrogen atom or a combination of these, n is 1-4, and m is greater than n and not more than 5); and a modification or terminal T that is present at the surface of the layer main body (T is at least one type selected from the group consisting of a hydroxyl group, a fluorine atom, a chlorine atom, an oxygen atom and a hydrogen atom). The two-dimensional particle has a structure in which an oxygen atom derived from a hydroxyl group that is the modification or terminal T is bonded to a phosphorus atom in a phosphonic acid having a saturated hydrocarbon group or an unsaturated hydrocarbon group. An oxidoreductase is immobilized on the phosphonic acid having a saturated hydrocarbon group or an unsaturated hydrocarbon group of the two-dimensional particle.

Description

Two-dimensional particle-oxidoreductase complex

[0001] The present disclosure relates to two-dimensional particle-oxidoreductase complexes.

[0002] In recent years, MXene has been attracting attention as a new material. MXene is a type of so-called two-dimensional material, and is a layered material having the form of one or more layers. ｍ X ｎ T s It is a layered compound represented by the following composition, and is obtained by etching the precursor MAX. Due to the etching of the MAX, modifications or terminations such as -OH, =O, and -F are generated on the surface of MXene. These modifications or terminations impart hydrophilic properties to MXene. Therefore, MXene has excellent water dispersion properties. MXene also exhibits excellent electrical conductivity.

[0003] The surface of MXene is rich in modified or terminated functional groups derived from the etchant used in precursor treatment. In recent years, ligand substitution, in which other ligands are substituted via hydroxyl groups by dehydration condensation or other methods, has attracted attention as it can impart various new functional groups not inherently present in MXene and thereby impart new properties.

[0004] For example, in Non-Patent Document 1, Ti was synthesized through coordination interactions with an aminosilane ligand spacer. ３ C ２ A method for covalently binding lipase to the surface of TX nanosheets has been reported. The immobilized lipase showed improved catalytic activity for the hydrolysis of p-nitrophenyl palmitate (pNPP) upon irradiation with near-infrared light. Furthermore, the immobilized lipase exhibited good pH and thermal stability, as well as reusability, which are important for the practical application of enzymes.

[0005] In Non-Patent Document 2, in consideration of the fact that enzymes are easily desorbed even when placed on the electrode, MXene-Ti ３ C ２It has been shown that a mediator-free biosensor with an oxidized surface can be fabricated by immobilizing an oxidized / reduced enzyme such as hemoglobin (Hb) on MXene in the presence of Nafion. ３ C ２ Surface immobilization has been shown to be an efficient method for developing a new class of highly sensitive, stable, and promising electrochemical biosensors. ３ C ２ It has been reported that the special structure and properties of promote the direct electron transfer of Hb, and that a redox current resulting from the direct electron transfer of Hb was confirmed.

[0006] Chaoying Ding et al., "Photothermal enhanced enzymatic activity of lipase covalently immobilized on functionalized Ti3C2TX nanosheets", Chemical Engineering Journal 2019, 378, 122205.Fen Wang et al., "An Organ-Like Titanium Carbide Material (MXene) with Multilayer Structure Encapsulating Hemoglobin for a Mediator-Free Biosensor", Journal of The Electrochemical Society, 162 (1) B16-B21 (2015).

[0007] In Non-Patent Document 1, substitution with a ligand having an amino group is performed, but the ligand-substituted MXene shown in Non-Patent Document 1 has the problem of low electron transfer efficiency. Non-Patent Document 2 also proposes a biosensor in which the oxidoreductase is immobilized using a polymer such as Nafion to prevent the oxidoreductase from being detached from MXene. However, this type of configuration has the problem of low electron transfer efficiency.

[0008] The present disclosure has been made in consideration of the above circumstances, and its purpose is to provide a two-dimensional particle-oxidoreductase complex in which the oxidoreductase is immobilized to MXene via phosphonic acid present on the surface of MXene in the two-dimensional particle, and which has high electron transfer efficiency between the oxidoreductase and MXene.

[0009] According to one aspect of the present disclosure, there is provided a two-dimensional particle comprising one or more layers, wherein the layers are of the following formula: M ｍ X ｎ (wherein M is at least one Group 3, 4, 5, 6 or 7 metal; X is a carbon atom, a nitrogen atom or a combination thereof; n is 1 or more and 4 or less; and m is greater than n and 5 or less), and a modified or terminal T (wherein T is at least one selected from the group consisting of a hydroxyl group, a fluorine atom, a chlorine atom, an oxygen atom and a hydrogen atom) present on the surface of the layer body, and the modified or terminal T has a structure in which an oxygen atom derived from the hydroxyl group is bonded to a phosphorus atom of a phosphonic acid, the phosphonic acid having a saturated or unsaturated hydrocarbon group, and an oxidoreductase is immobilized on the phosphonic acid.

[0010] According to the present disclosure, a two-dimensional particle-oxidoreductase complex can be provided in which an oxidoreductase is immobilized to MXene via phosphonic acid present on the surface of MXene in the two-dimensional particle, and which has high electron transfer efficiency between the oxidoreductase and MXene.

[0011] FIG. 1 is a schematic side view of a complex of ligand-substituted MXene and an enzyme using a silane coupling agent. FIG. 2 is another schematic side view of a complex of ligand-substituted MXene and an enzyme using a silane coupling agent. FIG. 3 is a schematic side view of a conventional complex of MXene and an enzyme in which an enzyme is captured in a polymer. FIG. 4 is a schematic cross-sectional view illustrating MXene constituting the two-dimensional particle of the present embodiment (or precursor particles used in producing the two-dimensional particle of the present embodiment), (a) showing single-layer MXene and (b) showing multi-layer (exemplarily two-layer) MXene. FIG. 5 is a schematic side view of a complex of two-dimensional particle and an enzyme of the present embodiment. FIG. 6 is another schematic side view of a complex of two-dimensional particle and an enzyme of the present embodiment. FIG. 7 is a diagram showing the results of electrochemical evaluation of an electrode of Example 1-1. FIG. 8 is a diagram showing the results of electrochemical evaluation of an electrode of Example 2-1. FIG. 9 is a diagram showing the results of electrochemical evaluation of an electrode of Comparative Example 2-2.

[0012] The present inventors have noticed that a complex between a two-dimensional particle in which the surface of MXene is modified with a compound having a nitrogen-containing functional group and an oxidoreductase (sometimes simply referred to as "enzyme" in this specification) has high electron transfer efficiency and is useful, for example, as an electrode, and have conducted extensive research to obtain this two-dimensional particle-oxidoreductase complex.

[0013] First, Non-Patent Document 1 utilizes the hydroxyl groups on MXene to study a substitution reaction using APTES as a ligand, which can be said to be a technology that adds amino groups to MXene. However, upon investigation of this technology, it was found that when this silane coupling agent is used as a ligand, a polymerization reaction occurs not only between MXene and the silane coupling agent, but also between the silane coupling agents themselves, resulting in the MXene being present in the form of a polysiloxane having Si-O-Si bonds. Furthermore, it was found that the presence of this polysiloxane on MXene poses the problem of not being able to achieve uniform and thin ligand substitution on MXene. Furthermore, when an enzyme is immobilized on conventional ligand-substituted MXene particles using this silane coupling agent, as shown schematically in Figure 1, a bulky polymeric ligand such as siloxane is present between MXene 10c and oxidoreductase 25, increasing the distance between MXene 10c and oxidoreductase 25. As a result, as shown schematically in FIG. 2, it is believed that conventional ligand-substituted MXene particles 21 prepared using a silane coupling agent are bulky, and the distance between MXene 10c and oxidoreductase 25 increases as indicated by the double arrow.

[0014] Furthermore, as in Non-Patent Document 2, when a polymer is used to immobilize the enzyme, as shown in Figure 3, the polymer 27 is bulky, and it is thought that the distance between MXene 10c and the oxidoreductase 25 increases as indicated by the double arrow.

[0015] For example, when used as a transducer, heterogeneous materials different from MXene, such as nanoparticles, biomolecules, or organic molecules, are considered as receptors. It is generally known that in such composites of heterogeneous materials and MXene, the shorter the distance between MXene and the heterogeneous material, the easier electron transfer occurs. An electrode (MXene) in which an oxidoreductase is immobilized as the heterogeneous material is specifically called an enzyme electrode. The distance between the enzyme and MXene is also important in these enzyme electrodes; the shorter the distance, the more efficiently electrons generated by the enzyme can be transported to MXene (the electrode side). On the other hand, if the distance increases, the electron transfer efficiency decreases, and electrons cannot or do not transfer easily to the electrode side. For example, if the distance between MXene and the heterogeneous material exceeds approximately 15 Å, excited electrons generated on the heterogeneous material of the composite cannot or do not transfer easily to MXene. Therefore, as described in Non-Patent Documents 1 and 2, if bulky polysiloxanes or polymers are present on MXene and the distance between the enzyme and MXene is long, the efficiency of electron transfer between the enzyme and MXene will be very poor, making it difficult to use MXene in electronic devices that utilize its conductivity.

[0016] The present inventors therefore conducted extensive research to realize a complex of MXene and an oxidoreductase in which the distance between the enzyme and MXene is uniformly small. As a result, they discovered that a complex of a two-dimensional particle in which phosphonic acid is substituted as a ligand on MXene and an oxidoreductase would be sufficient.

[0017] (Embodiment 1: Two-dimensional particle-oxidoreductase complex) Hereinafter, a two-dimensional particle-oxidoreductase complex in one embodiment of the present invention will be described in detail, but the present disclosure is not limited to this embodiment.

[0018] The two-dimensional particle-oxidoreductase complex in this embodiment comprises a two-dimensional particle comprising one or more layers, wherein the layers are represented by the following formula: M ｍ X ｎ(wherein M is at least one Group 3, 4, 5, 6, or 7 metal; X is a carbon atom, a nitrogen atom, or a combination thereof; n is 1 or more and 4 or less; and m is greater than n and 5 or less), and a modified or terminal T (wherein T is at least one selected from the group consisting of a hydroxyl group, a fluorine atom, a chlorine atom, an oxygen atom, and a hydrogen atom) present on the surface of the layer body; and the modified or terminal T has a structure in which an oxygen atom derived from the hydroxyl group is bonded to a phosphorus atom of a phosphonic acid, the phosphonic acid having a saturated or unsaturated hydrocarbon group, and an oxidoreductase is immobilized on the phosphonic acid. This makes it possible to realize a two-dimensional particle-oxidoreductase complex with high electron transfer efficiency between the oxidoreductase and MXene. Furthermore, detachment of the oxidoreductase from the two-dimensional particle-oxidoreductase complex can be prevented.

[0019] In this specification, when an "atom" refers to a certain element, the oxidation number of the element is not limited to 0, but may be any number within the range of oxidation numbers that the element can take.

[0020] (Two-dimensional particle) The two-dimensional particle constituting the two-dimensional particle-oxidoreductase complex of this embodiment will be described first. The two-dimensional particle according to this embodiment includes a predetermined layer body and a modification or terminal T present on the surface of the layer body, and has a structure in which an oxygen atom derived from a hydroxyl group of the modification or terminal T is bonded to a phosphorus atom of a phosphonic acid having a saturated hydrocarbon group or an unsaturated hydrocarbon group.

[0021] (MXene Constituting Two-Dimensional Particles) MXene constituting the two-dimensional particles according to this embodiment (or precursor particles used to produce the two-dimensional particles of this embodiment) will be described.

[0022] The one or more layers may be understood as a layered compound and referred to as "M ｍ X ｎ T ｓ ", where s is any number, and conventionally, x or z may be used instead of s. Typically, n can be 1, 2, 3, or 4, but is not limited thereto.

[0023] In the above formula for MXene, M is preferably at least one selected from the group consisting of Ti, Zr, Hf, V, Nb, Ta, Cr, Mo, and Mn, more preferably at least one selected from the group consisting of Ti, V, Cr, and Mo. Most preferably, M contains Ti. M may be only Ti.

[0024] MXene is a compound represented by the formula: M ｍ X ｎ However, it is known that it can be expressed as follows: Sc ２ C, Ti ２ C, Ti ２ N, Zr ２ C, Zr ２ N, Hf ２ C, Hf ２ N.V. ２ C.V. ２ N, Nb ２ C, Ta ２ C, Cr ２ C, Cr ２ N, Mo ２ C, Mo １．３ C, Cr １．３ C, (Ti, V) ２ C, (Ti, Nb) ２ C.W. ２ C.W. １．３ C, Mo ２ N, Nb １．３ C, Mo １．３ Y ０．６ C (in the above formula, "1.3" and "0.6" mean approximately 1.3 (= 4 / 3) and approximately 0.6 (= 2 / 3), respectively), Ti ３ C ２ , Ti ３ N ２ , Ti ３ (CN), Zr ３ C ２ , (Ti, V) ３ C ２ , (Ti ２ Nb)C ２ , (Ti ２ Ta) C ２ , (Ti ２ Mn)C ２ , Hf ３ C ２、(Hf ２ V)C ２ 、(Hf ２ Mn)C ２ 、(V ２ Ti)C ２ 、(Cr ２ Ti)C ２ 、(Cr ２ V)C ２ 、(Cr ２ Nb)C ２ 、(Cr ２ Ta)C ２ 、(Mo ２ Sc)C ２ 、(Mo ２ Ti)C ２ 、(Mo ２ Zr)C ２ 、(Mo ２ Hf)C ２ 、(Mo ２ V)C ２ 、(Mo ２ Nb)C ２ 、(Mo ２ Ta)C ２ 、(W ２ Ti)C ２ 、(W ２ Zr)C ２ 、(W ２ Hf)C ２ 、 Ti ４ N ３ 、V ４ C ３ 、Nb ４ C ３ 、Ta ４ C ３ 、(Ti,Nb) ４ C ３ 、(Nb,Zr) ４ C ３ 、(Ti ２ Nb ２ )C ３ 、(Ti ２ Ta ２ )C ３ 、(V ２ Ti ２ )C ３ 、(V ２ Nb ２ )C ３ 、(V ２ Ta ２ )C ３ 、(Nb２ Ta ２ ) C ３ , (Cr ２ Ti ２ ) C ３ , (Cr ２ V ２ ) C ３ , (Cr ２ Nb ２ ) C ３ , (Cr ２ Ta ２ ) C ３ , (Mo ２ Ti ２ ) C ３ , (Mo ２ Zr ２ ) C ３ , (Mo ２ Hf ２ ) C ３ , (Mo ２ V ２ ) C ３ , (Mo ２ Nb ２ ) C ３ , (Mo ２ Ta ２ ) C ３ , (W ２ Ti ２ ) C ３ , (W ２ Zr ２ ) C ３ , (W ２ Hf ２ ) C ３ , (Mo ２．７ V １．３ ) C ３ (In the above formula, "2.7" and "1.3" mean approximately 2.7 (= 8 / 3) and approximately 1.3 (= 4 / 3), respectively.)

[0025] Typically, in the above formula, M is titanium or vanadium, and X is a carbon atom or a nitrogen atom. Preferably, the layer body is Ti ３ C ２ By using the above structure, it is possible to realize a two-dimensional particle-oxidoreductase complex with higher electron transfer efficiency. For example, the MAX phase is ３ AlC ２ and MXene is Ti ３ C ２ Tｓ (In other words, the layer body is Ti ３ C ２ wherein M is Ti, X is C, n is 2, and m is 3. ３ C ２ In this case, the MXene particles obtained have high dispersion stability in the dispersion medium and exhibit high electrical conductivity when formed into a film, which is preferable.

[0026] In this embodiment, MXene may contain a relatively small amount of A atoms derived from the precursor MAX phase, for example, 10% by mass or less of the original A atoms. The residual amount of A atoms is preferably 8% by mass or less, more preferably 6% by mass or less. However, even if the residual amount of A atoms exceeds 10% by mass, this may not be a problem depending on the application and use conditions of the two-dimensional particles.

[0027] The MXene constituting the two-dimensional particles of this embodiment (or precursor particles used to produce the two-dimensional particles of this embodiment) will be described below with reference to Figure 4. Note that Figures 4(a) and 4(b) below do not show the substituents on the surface of the layer body that are derived from phosphonic acid having saturated or unsaturated hydrocarbon groups.

[0028] The MXene constituting the two-dimensional particles of this embodiment is an aggregate including one layer of MXene particles (hereinafter simply referred to as "MXene particles") 10a (single-layer MXene particles) as illustrated in Fig. 4(a). More specifically, the MXene particles 10a are ｍ X ｎ The layer body (M ｍ X ｎ The MXene layer 7a has a main layer 1a and modifications or terminations T3a, 5a present on the surface of the main layer 1a (more specifically, on at least one of the two opposing surfaces of each layer). ｍ X ｎ T ｓ ", where s is an arbitrary number.

[0029] The two-dimensional particles of this embodiment may contain one or more layers. Examples of MXene particles with multiple layers (multilayer MXene particles) include, but are not limited to, two-layer MXene particles 10b, as shown schematically in FIG. 4(b). 1b, 3b, 5b, and 7b in FIG. 4(b) are the same as 1a, 3a, 5a, and 7a in FIG. 4(a). Two adjacent MXene layers (e.g., 7a and 7b) in a multilayer MXene particle do not necessarily need to be completely separated and may be partially in contact. The MXene particle 10a may be a mixture of the single-layer MXene particle 10a and the multilayer MXene particle 10b, with the multilayer MXene particles 10b remaining unseparated.

[0030] Although not limiting this embodiment, the thickness of each layer (corresponding to the above-mentioned MXene layers 7a and 7b) contained in the MXene particle is, for example, 0.8 nm to 5 nm, particularly 0.8 nm to 3 nm (this may vary mainly depending on the number of M atomic layers contained in each layer). The interlayer distance (or gap dimension, shown as Δd in FIG. 4(b)) for each stack of the multilayer MXene particle that may be contained may be, for example, 0.8 nm to 10 nm, particularly 0.8 nm to 5 nm, more particularly about 1 nm, and the total number of layers may be 2 to 20,000.

[0031] The MXene constituting the two-dimensional particles of this embodiment is preferably a multilayer MXene particle with a small number of layers obtained by a delamination process. The term "small number of layers" refers to, for example, a stacked number of MXene layers of 10 or less, preferably 6 or less. Furthermore, the thickness of the multilayer MXene particle with a small number of layers in the stacking direction is preferably 15 nm or less, more preferably 10 nm or less. Hereinafter, these "multilayer MXene particles with a small number of layers" may be referred to as "small-layer MXene particles." Furthermore, single-layer MXene particles and small-layer MXene particles may be collectively referred to as "single-layer / small-layer MXene particles."

[0032] The MXene constituting the two-dimensional particles of this embodiment preferably contains a large amount of single-layered and few-layered MXene. By including a large amount of single-layered and few-layered MXene, the specific surface area of ​​MXene can be made larger than that of multi-layered MXene, which is thought to result in a larger contact area with the ligand during the substitution reaction and a higher ligand coverage. For example, the layered material particles of this embodiment have 10 or fewer MXene stacks and a thickness of 15 nm or less, preferably 10 nm or less, and the single-layered and few-layered MXene preferably accounts for 80 vol% or more, more preferably 90 vol% or more, and even more preferably 95 vol% or more of the total MXene. Furthermore, it is more preferable that the volume of single-layered MXene is greater than the volume of few-layered MXene. Because the true density of these MXenes does not vary significantly depending on their form, it can also be said that it is more preferable that the mass of single-layered MXene is greater than the mass of few-layered MXene. When these relationships are satisfied, the specific surface area can be further increased, resulting in, for example, improved dispersion stability and, for example, when the two-dimensional particles of this embodiment are used in electrodes, sufficient suppression of deterioration of conductivity over time. Most preferably, the MXene constituting the two-dimensional particles of this embodiment is formed solely of a single layer of MXene.

[0033] The two-dimensional particles according to this embodiment have a structure in which an oxygen atom derived from the hydroxyl group, which is the modified or terminal T, is bonded to the phosphorus atom of the phosphonic acid. In this specification, a phosphonic acid having a saturated hydrocarbon group or an unsaturated hydrocarbon group may be simply referred to as a "phosphonic acid." A phosphonic acid has a phosphorus oxoacid as a parent compound and has the general formula R-P(=O)X ２ (R is an organic group having a saturated hydrocarbon group or an unsaturated hydrocarbon group (which may further have a nitrogen-containing functional group, a sulfur-containing functional group, a hydroxyl group, or the like, as necessary), and at least one of the two Xs is an oxygen atom derived from the hydroxyl group that is the MXene modification or the terminal T).

[0034] The bond between the oxygen atom derived from the hydroxyl group of the modified or terminal T and the phosphorus atom may be, for example, a covalent bond or an ionic bond. Confirmation of the bond between the oxygen atom derived from the hydroxyl group of the main body and the phosphorus atom, i.e., whether phosphonic acid is substituted on MXene or whether an amino group (nitrogen atom) described below is present, can be confirmed using, for example, XRD, TG-DTA, XPS, solid-state NMR, TG-MS, or conductivity measurement.

[0035] There is no limitation on the length of the carbon chain of the saturated or unsaturated hydrocarbon group of the phosphonic acid. Examples include phosphonic acids having an alkyl group, such as methylphosphonic acid (C1), propylphosphonic acid (C3), hexylphosphonic acid (C6), octylphosphonic acid (C8), decylphosphonic acid (C10), and dodecylphosphonic acid (C12).

[0036] The phosphonic acid may further have a nitrogen-containing functional group, a sulfur-containing functional group, a hydroxyl group, etc., as necessary. In the case of the nitrogen-containing functional group, the unpaired electron on the nitrogen atom is important, so any functional group containing a nitrogen element in the molecule, such as pyridine, pyrrole, aniline, pyrrolidine, pyrimidine, a cyano group, a nitro group, an amino group, or an imino group, may be used. The sulfur-containing functional group may be, for example, a thiol group.

[0037] The nitrogen-containing functional group is preferably an amino group. This allows for the realization of a two-dimensional particle-oxidoreductase complex, such as an enzyme electrode, with high electron transfer efficiency. Examples of the phosphonic acid containing a saturated or unsaturated hydrocarbon group and an amino group include aminomethylphosphonic acid (C1) (aminophosphonic acid with an alkyl chain length of 1: hereinafter referred to as NH ２ -C1PA), aminopropylphosphonic acid (C3) (aminophosphonic acid with an alkyl chain length of 3: hereinafter referred to as NH ２ -C3PA), aminohexylphosphonic acid (C6) (aminophosphonic acid with an alkyl chain length of 6: hereinafter referred to as NH ２ -C6PA) can be used. The phosphonic acid is preferably aminomethylphosphonic acid (C1).

[0038] The bonded structure can be formed during the manufacturing process of the two-dimensional particles according to this embodiment by a dehydration condensation reaction between hydroxyl groups present on the surface of the MXene particle (precursor particle) layer and the phosphonic acid ligand. This reaction can also be described as a reaction in which hydroxyl groups (particularly hydrogen) present on the surface of the MXene particle (precursor particle) layer are replaced with phosphonic acid. This reaction is sometimes referred to as "ligand substitution." During the manufacturing process of the two-dimensional particles according to this embodiment, a substitution reaction of multiple hydroxyl groups present on the surface of the MXene particle (precursor particle) layer with the phosphonic acid proceeds, increasing the number of bonds between oxygen atoms derived from the hydroxyl groups on the MXene particle and the phosphorus atom. As a result, the amount of ligand present on the MXene can increase.

[0039] In the two-dimensional particles according to this embodiment, the phosphonic acids do not react with each other, and therefore bulky polysiloxanes or polymers, as in Non-Patent Documents 1 and 2, are not present on MXene, and the ligand can be distributed almost uniformly on MXene 10c, as shown schematically in Figure 5. As a result, even when an oxidoreductase is immobilized on the ligand-substituted MXene particles (two-dimensional particles), the distance between MXene 10c and oxidoreductase 25 is short, as indicated by the double arrow, as shown schematically in Figure 6, and it is believed that this increases the electron transfer efficiency.

[0040] (Oxidoreductase) The oxidoreductase in the two-dimensional particle-oxidoreductase complex is not limited. The oxidoreductase according to the present disclosure includes oxidoreductases and electron transfer proteins. The oxidoreductase and electron transfer protein are not limited to Cytochrome C from bovine heart (hereinafter abbreviated as Cyt. C) used in the examples, and any type of oxidoreductase or electron transfer protein may be used. For example, the cytochrome family, hemoglobin, and myoglobin having an electron transfer site such as heme may be used, or ferredoxin, NAD-dependent dehydrogenase, hydrogenase, coenzyme Q cytochrome C reductase, succinate dehydrogenase, and nitrogenase having an electron transfer site such as an Fe—S cluster may be used. Preferably, the oxidoreductase or electron transfer protein is one in which the distance from the electron transfer site in the oxidoreductase to the enzyme surface is within 15 Å, allowing direct electron transfer.

[0041] The "immobilization" mentioned above may be achieved by immobilizing the oxidoreductase via a modifying group, which is a ligand on MXene, and may include immobilization via a covalent bond as well as immobilization via one or more non-covalent bonds selected from the group consisting of electrostatic interactions, hydrophobic interactions, hydrogen bonds, and van der Waals forces. Immobilization via a covalent bond may be achieved by a covalent bond including an amide bond, as illustrated in FIG. 5 . Immobilization via a non-covalent bond may be achieved by immobilization via a non-covalent bond 29, such as a hydrogen bond, between the hydrogen atom or the like of a ligand (aminated phosphonic acid in FIG. 7 ) present on MXene 10c and the oxidoreductase 25, as shown in FIG. 7 .

[0042] The covalent bond may be one or more of an amide bond, an ester bond, a thioester bond, an ether bond, and a thioether bond. The covalent bond is preferably an amide bond. That is, from the viewpoint of realizing an enzyme electrode or the like with high electron transfer efficiency, it is preferable that the phosphonic acid and the oxidoreductase are immobilized via a covalent bond including an amide bond. The non-covalent bond is preferably one or more selected from the group consisting of electrostatic interaction, hydrophobic interaction, and hydrogen bond. Furthermore, "immobilization via a modification group on MXene" refers to a state in which all or a portion of the oxidoreductase remains on MXene even after the two-dimensional particle-oxidoreductase complex is washed with a buffer such as 1x PBS. The remaining oxidoreductase may be confirmed, for example, by an electrochemical method as described in the Examples below, or by observation using an electron microscope or a scanning probe microscope.

[0043] (Method for Producing a Two-Dimensional Particle-Oxidoreductase Conjugate) Hereinafter, a method for producing a two-dimensional particle-oxidoreductase conjugate in one embodiment of the present invention will be described in detail, but the present disclosure is not limited to this embodiment.

[0044] The method for producing a two-dimensional particle-oxidoreductase conjugate according to this embodiment includes the steps of: (a) providing a precursor particle comprising one or more layers, wherein the layers are represented by the following formula: M ｍ X ｎ(wherein M is at least one Group 3, 4, 5, 6, or 7 metal; X is a carbon atom, a nitrogen atom, or a combination thereof; n is 1 or more and 4 or less; and m is greater than n and 5 or less), and modified or terminal T (T is at least one selected from the group consisting of a hydroxyl group, a fluorine atom, a chlorine atom, an oxygen atom, and a hydrogen atom, and includes at least a hydroxyl group) present on the surface of the layer body; (b) preparing an aqueous dispersion containing the precursor particles and a solution containing a phosphonic acid having a saturated hydrocarbon group or an unsaturated hydrocarbon group; and (c) mixing and stirring the aqueous dispersion containing the precursor particles, the phosphonic acid solution, and an inorganic acid to obtain two-dimensional particles having a structure in which the oxygen atom derived from the hydroxyl group, which is the modified or terminal T, is bonded to the phosphorus atom of the phosphonic acid; and (d) immobilizing an oxidoreductase on the two-dimensional particles (ligand-substituted MXene). According to this method, the oxidoreductase is immobilized on MXene via the phosphonic acid present on the surface of MXene in the two-dimensional particle, thereby obtaining a two-dimensional particle-oxidoreductase complex with high electron transfer efficiency between the oxidoreductase and MXene.

[0045] Each step of the above-mentioned production method will be described in detail below. Step (a) Preparing predetermined MXene particles (precursor particles). The production of the MXene particles (precursor particles) is not limited, and they can be produced, for example, by the following method.

[0046] Etching of MAX Phases Particles of a given layered material (MXene particles, precursor particles) can be synthesized by selectively etching (removing and optionally layer-separating) A atoms (and optionally some of the M atoms) from a MAX phase. The MAX phase has the following formula: ｍ AX ｎ(wherein M, X, n and m are as defined above, A is at least one element of Groups 12, 13, 14, 15 and 16, and is usually an element of Group A, typically Group IIIA and Group IVA, and more particularly may contain at least one element selected from the group consisting of Al, Ga, In, Tl, Si, Ge, Sn, Pb, P, As, S and Cd, and is preferably Al), and M ｍ X ｎ (each X may have a crystal lattice in which it is located in an octahedral array of M) and a layer composed of A atoms is located between them. In the MAX phase, typically when m=n+1, one layer of X atoms is arranged between each of the n+1 layers of M atoms (these are collectively referred to as "M ｍ X ｎ The repeating unit has a layer of A atoms (also referred to as an "A atom layer") arranged as the next layer after the n+1th layer of M atoms, but is not limited thereto.

[0047] Etching is performed to remove at least some of the A atoms from the precursor using an etching solution. The etching solution is HF, H ３ P.O. ４ , HCl, HI and H ２ SO ４ The etching solution preferably contains at least one of HF (hydrofluoric acid) and H ３ P.O. ４ It is more preferable that the etching solution contains at least one of the following: (phosphoric acid). For example, etching can be performed by the so-called MILD method, in which HCl and LiF contained in the etching solution are reacted in the system to generate HF; however, it is preferable to perform etching using an etching solution containing HF (hydrofluoric acid), the so-called ACID method, or an etching solution containing phosphoric acid. These methods are preferable because they make it easier to obtain flake-shaped layered material particles (MXene particles) with a large planar area than the MILD method. Other etching conditions are not particularly limited, and known conditions can be adopted. As the etching solution, a mixture of the above acid and, for example, pure water as a solvent can be used. As the etching solution, a solution with an HF concentration of 1.5M or more and 14M or less, and H ３ P.O.４ concentration of 5.5M or more, HCl concentration of 6.0M or more, HI concentration of 5.0M or more, and H ２ SO ４ An etching solution having a concentration of at least one selected from the group consisting of 5.0 M or more can be used. In the etching of the A atoms, in addition to the A atoms, some of the M atoms may also be selectively etched in some cases. An example of the etched product obtained by the above etching is a slurry.

[0048] The MAX phase can be produced by a known method. For example, TiC powder, Ti powder, and Al powder are mixed in a ball mill, and the resulting mixed powder is sintered in an Ar atmosphere to obtain a sintered body (a block of the MAX phase). The sintered body is then pulverized with an end mill to obtain the powdered MAX phase for the next step.

[0049] The A atoms (and possibly some of the M atoms) are selectively etched (removed and possibly layer separated) from the MAX phase, thereby removing the A atom layer (and possibly some of the M atoms) to expose the M ｍ X ｎ The surface of the layer is modified with hydroxyl groups, fluorine atoms, chlorine atoms, oxygen atoms, hydrogen atoms, etc. present in the etching solution (usually, an aqueous solution of fluorine-containing acid is used, but this is not limited to this) to terminate the surface.

[0050] The etching solution may contain a metal compound containing a monovalent metal ion, and an intercalation treatment of the monovalent metal ion may be carried out simultaneously with the etching. Examples of metal compounds containing monovalent metal ions include those used in the intercalation treatment described below. The content of the metal compound containing monovalent metal ions in the etching solution is preferably 0.001% by mass or more. The content is more preferably 0.01% by mass or more, and even more preferably 0.1% by mass or more. On the other hand, from the viewpoint of dispersibility in the solution, the content of the metal compound containing monovalent metal ions in the etching solution is preferably 10% by mass or less, more preferably 1% by mass or less.

[0051] (Water Washing) The etched product obtained by the etching is washed with water. By washing with water, the acid used in the etching can be sufficiently removed. The amount of water to be mixed with the etched product and the washing method are not particularly limited. For example, water may be added and the mixture may be stirred or centrifuged. Stirring methods include stirring using a hand shake, an automatic shaker, a shear mixer, a pot mill, or the like. The degree of stirring, such as the stirring speed and stirring time, may be adjusted depending on the amount and concentration of the material to be treated. The water washing may be performed one or more times. Preferably, the water washing is performed multiple times. For example, steps (i) to (iv) of (i) adding water (to the etched product or the remaining precipitate obtained in (iii) below), (ii) stirring, (iii) centrifuging the stirred product, and (iv) discarding the supernatant after centrifugation and recovering the remaining precipitate may be performed two or more times, for example, 15 or less times.

[0052] [MXene Layer Separation] The treated product (water-washed product) obtained by the water washing may be used to promote layer separation of MXene (delamination, separating multilayer MXene into single-layer MXene) by any appropriate post-treatment (e.g., ultrasonic treatment, hand shaking, automatic shaker, etc.). Since ultrasonic treatment can destroy MXene due to excessive shear force, if it is desired to obtain two-dimensional MXene particles with a larger aspect ratio (preferably single-layer MXene), it is preferable to apply an appropriate shear force using a hand shake or automatic shaker, etc. The intercalation treatment and delamination are described below.

[0053] (Intercalation Treatment) For example, a monovalent metal ion intercalation treatment may be performed, which includes a step of mixing the etched product obtained by the etching treatment with a metal compound containing a monovalent metal ion. Examples of the monovalent metal ion constituting the metal compound containing a monovalent metal ion include alkali metal ions such as lithium ions, sodium ions, and potassium ions, copper ions, silver ions, and gold ions. Examples of the metal compound containing a monovalent metal ion include ionic compounds in which the above metal ions are bonded to cations. Examples include iodides, phosphates, sulfide salts including sulfates, nitrates, acetates, and carboxylates of the above metal ions. As described above, lithium ions are preferred as the monovalent metal ion, and metal compounds containing lithium ions are preferred as metal compounds containing lithium ions, more preferably ionic compounds of lithium ions, and even more preferably one or more of iodides, phosphates, and sulfide salts of lithium ions. If lithium ions are used as the metal ion, it is thought that water hydrated with lithium ions has the most negative dielectric constant, making it easier to form a monolayer.

[0054] The content of the metal compound containing a monovalent metal ion in the formulation for intercalation treatment of a monovalent metal ion is preferably 0.001% by mass or more. The content is more preferably 0.01% by mass or more, and even more preferably 0.1% by mass or more. On the other hand, from the viewpoint of dispersibility in the solution, the content of the metal compound containing a monovalent metal ion is preferably 10% by mass or less, more preferably 1% by mass or less.

[0055] The specific method of intercalation is not particularly limited. For example, a metal compound containing a monovalent metal ion may be mixed with the MXene water medium clay, and the mixture may be stirred or left to stand. For example, stirring at room temperature may be used. Examples of the stirring method include a method using a stirring bar such as a stirrer, a method using a stirring blade, a method using a mixer, and a method using a centrifugal device. The stirring time can be set depending on the production scale of the two-dimensional particles, and may be set, for example, between 12 and 24 hours.

[0056] (Delamination) Delamination may be performed using the intercalation-treated product obtained by intercalation. For example, delamination may involve centrifuging the intercalation-treated product, discarding the supernatant, and then washing the remaining precipitate with water. The conditions for the delamination treatment are not particularly limited. The dispersion medium used for delamination is also not particularly limited, and examples include using one or more polar organic dispersion mediums and aqueous dispersion mediums. This process may be repeated at least once, preferably at least twice, but not more than 10 times, to obtain a supernatant containing single-layered and / or few-layered MXene as the delamination-treated product. Alternatively, the supernatant may be centrifuged, and the resulting supernatant may be discarded to obtain a single-layered and / or few-layered MXene-containing clay as the delamination-treated product. The resulting delamination-treated product may be used as an aqueous dispersion containing precursor particles.

[0057] The delamination-treated product, ie, the supernatant containing the single-layer / few-layer MXene and the single-layer / few-layer MXene-containing clay, may be subjected to removal of the dispersion medium to obtain dried precursor particles.

[0058] Step (b) Prepare an aqueous dispersion containing the precursor particles and a phosphonic acid-containing solution by dissolving phosphonic acid in, for example, pure water. The solution containing phosphonic acid (e.g., phosphonic acid having a nitrogen-containing functional group) can be prepared using not only pure water but also any of ethanol, 2-propanol, ethylene glycol, methanol, acetone, acetonitrile, DMF, NMF, DMSO, NMP, and the like, which are capable of dissolving phosphonic acid. One or more of these can be used.

[0059] The aqueous dispersion containing the precursor particles may be the single-layered or few-layered MXene-containing clay obtained by the delamination process, or the aqueous dispersion may be prepared by adjusting the water content. Alternatively, the aqueous dispersion may be prepared by dispersing the precursor particles (dried product) obtained by the above method in water. The proportion of the precursor particles contained in the aqueous dispersion containing the precursor particles may be, for example, in the range of 0.01% by mass to 1% by mass (solid content).

[0060] The reaction solution obtained by mixing the aqueous dispersion, the phosphonic acid-containing solution (e.g., the phosphonic acid-containing aqueous solution), and the inorganic acid such as hydrochloric acid, sulfuric acid, or phosphoric acid may have a pH of 4 or less. The pH is preferably in the range of 1 to 2.5.

[0061] Step (c): An aqueous dispersion containing precursor particles, the phosphonic acid-containing solution (e.g., an aqueous solution of phosphonic acid), and an inorganic acid are mixed and stirred to obtain two-dimensional particles having a structure in which oxygen atoms derived from the hydroxyl groups that are the modifying or terminal T are bonded to phosphorus atoms of the phosphonic acid.

[0062] The method for mixing and stirring the aqueous dispersion containing precursor particles and the phosphonic acid-containing solution is not particularly limited. The stirring can be performed, for example, for a period of 6 hours or more and 24 hours or less. According to this embodiment, the aqueous dispersion containing precursor particles and the phosphonic acid-containing solution are mixed and stirred, resulting in a reaction mode in which the precursor particles (MXene particles) and the ligand are contained in the same system, rather than a phase-separation type interfacial reaction as in Patent Document 1. Therefore, the contact frequency between the hydroxyl groups on MXene and the ligand is increased compared to the phase-separation type interfacial reaction, thereby accelerating the substitution reaction. As a result, the substitution rate of the ligand on the surface of the precursor particles (MXene particles) can be improved.

[0063] The proportion of phosphonic acid in the precursor particles is preferably high, and the molar ratio of precursor particles to phosphonic acid (preferably phosphonic acid having a nitrogen-containing functional group) is preferably 1: (3 or more and 50 or less). ３ C ２ (OH) ２ wherein the phosphonic acid is aminomethylphosphonic acid (NH ２-C1PA), the blending ratio may be precursor particles:aminomethylphosphonic acid (molar ratio) = 1: (3 to 20), and further may be precursor particles:aminomethylphosphonic acid (molar ratio) = 1: (6 to 10).

[0064] The method of ligand substitution is not limited to the above-mentioned method and may be any production method that can react with the functional group of MXene.

[0065] The obtained two-dimensional particles may be in the form of a two-dimensional particle-containing dispersion liquid dispersed in a dispersion medium. The two-dimensional particle-containing dispersion liquid contains two-dimensional particles that are ligand-substituted MXene particles, preferably aminated MXene particles (particularly, the layer body of MXene is Ti). ３ C ２ ) particles, and have excellent dispersion stability. The two-dimensional particles contained in the two-dimensional particle-containing dispersion are as described above in the (Two-dimensional Particles) section. The dispersion medium contained in the two-dimensional particle-containing dispersion according to this embodiment is not limited. As the dispersion medium, water (pure water), an organic dispersion medium compatible with water (solubility in water > 10% w / w), or an organic dispersion medium that is not compatible with water may be used. According to this embodiment, unlike Patent Document 1 and the like, dispersion is possible even in organic dispersion mediums compatible with water (solubility in water > 10% w / w), thereby providing a wider selection of organic dispersion mediums than conventional ones. Dispersion is also possible in alcohol dispersion mediums, which was not possible with conventional MXene, thereby reducing the energy consumption required for dispersion medium removal during film formation processes such as spray coating.

[0066] As the organic dispersion medium, for example, one or more selected from the group consisting of ethanol, 2-propanol, ethylene glycol, methanol, acetone, acetonitrile, DMF, NMF, DMSO, and NMP can be used. Examples of organic dispersion media that are not compatible with water include 1-hexanol, chloroform, and toluene. The dispersion medium is preferably one or more selected from the group consisting of pure water, ethanol, and 2-propanol. Two-dimensional particle-containing dispersions using these dispersion media have high dispersion stability of the two-dimensional particles.

[0067] Step (d): Immobilizing an oxidoreductase on the two-dimensional particles (ligand-substituted MXene). Immobilization methods include covalently linking two-dimensional particles (e.g., MXene to which amino groups have been added by ligand substitution) and an oxidoreductase. For example, aminated MXene is used as the ligand-substituted MXene, and an amide bond is formed by dehydration condensation between the amino group on the aminated MXene and the carboxyl group on Cytochrome C. In this case, the reagent used may be any amide bond-forming reagent that forms an amide bond through a dehydration condensation reaction, and is not limited to N-hydroxy sulfosuccinimide or 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride, as shown in the Examples below. Furthermore, the method for forming a non-covalent bond between the two-dimensional particles and an oxidoreductase is not limited. For example, as shown in the Examples below, it can be formed by dropping and drying ligand-substituted MXene (aminated MXene), then dropping an oxidoreductase dissolved in a buffer solution, and allowing it to dry naturally.

[0068] The method for producing a two-dimensional particle-oxidoreductase conjugate according to this embodiment may include the above steps in the order listed, and other steps are not particularly limited. For example, after step (c), the method may include a step of washing the ligand-substituted MXene particles obtained by the reaction.

[0069] The present disclosure will be described in more detail below with reference to examples. The present disclosure is not limited to the following examples, and appropriate modifications can be made within the scope of the above-mentioned and below-mentioned aims, and all such modifications are within the technical scope of the present disclosure. For example, the present disclosure is not limited to the preparation methods described below, and any preparation method that can modify MXene or react with the terminal functional group can be used.

[0070] Example 1 In Example 1, the electron transfer efficiency was evaluated for an example in which the phosphonic acid of ligand-substituted MXene was covalently bonded to an oxidoreductase.

[0071] [Example 1-1] In this example, NH was used as a ligand (phosphonic acid having a saturated hydrocarbon group or an unsaturated hydrocarbon group and a nitrogen-containing functional group, a surface modifier). ２ Aminophosphonic acid-substituted MXene particles were prepared as two-dimensional particles (ligand-substituted MXene particles) using -C1PA (aminophosphonic acid with an alkyl chain length of 1 (C1)). There is no limit to the alkyl chain length, and in addition to aminomethylphosphonic acid, for example, aminopropylphosphonic acid (C3) or aminohexylphosphonic acid (C6) can also be used. Furthermore, the ligand is not limited to aminophosphonic acid. When a nitrogen-containing functional group is present, the unpaired electron on the nitrogen atom is important, so any phosphonic acid containing a nitrogen element within the molecule will suffice. If the raw materials are changed, the weight added may change, taking into account the molecular weight of each.

[0072] (Preparation of MXene particles (precursor particles)) First, the following steps were carried out in order: (1) preparation of precursor (MAX), (2) etching of precursor, (3) cleaning after etching, (4) Li intercalation, and (5) delamination, as detailed below, to obtain precursor particles (single-layer / few-layer MXene-containing samples, MXene particles) to be subjected to treatments such as ligand substitution.

[0073] [1] Preparation of Precursor (MAX) TiC powder, Ti powder, and Al powder (all manufactured by Kojundo Chemical Laboratory Co., Ltd.) were mixed in a molar ratio of 2:1:1 in a ball mill containing zirconia balls for 24 hours. The resulting mixed powder was fired at 1350°C for 2 hours in an Ar atmosphere. The fired body (block-shaped MAX) was then crushed with an end mill to a maximum size of 40 μm or less. This resulted in the preparation of TiC powder as a precursor (powdered MAX). ３ AlC ２ particles were obtained.

[0074] [2] Etching of precursor (MAX) Ti prepared by the above method ３ AlC ２ Using particles (powder), etching was performed under the following etching conditions to remove Ti ３ AlC ２ A solid-liquid mixture (slurry) containing solid components derived from the powder was obtained. (Etching conditions) Precursor: Ti３ AlC ２ (Sieved through a 45 μm mesh) Etching solution composition: 49% HF 6 mL H ２ O 18 mL HCl (12 M) 36 mL Precursor input amount: 3.0 g Etching container: 100 mL Eye Boy Etching temperature: 35°C Etching time: 24 h Stirrer rotation speed: 400 rpm

[0075] [3] Post-etching cleaning The above slurry was divided into two parts and placed in two 50 mL centrifuge tubes. Then, after centrifuging at 3500 G for 5 minutes using a centrifuge, the supernatant was discarded. Thereafter, (i) 40 mL of pure water was added to the remaining precipitate in each centrifuge tube, (ii) centrifugation was again carried out at 3500 G for 5 minutes, and (iii) the supernatant was separated and removed. This procedure from (i) to (iii) was repeated 11 times. After the final centrifugation, the supernatant was discarded, and Ti ３ AlC ２ T ｘ - Water medium clay was obtained.

[0076] [4] Li intercalation Ti prepared by the above method ３ AlC ２ T ｘ The clay was subjected to Li intercalation using LiCl as a Li-containing compound under the following conditions: the clay was stirred at 20°C to 25°C for 12 hours. (Li intercalation conditions) ３ AlC ２ T ｘ - Water medium clay (MXene after washing): solid content 0.75 g LiCl: 0.75 g Intercalation container: 100 mL Eye Boy Temperature: 20 ° C or higher and 25 ° C or lower (room temperature) Time: 12 hours Stirrer rotation speed: 800 rpm

[0077] [5] Delamination The slurry obtained by Li intercalation was placed in a 50 mL centrifuge tube and centrifuged at 3500 G using a centrifuge. The supernatant was then discarded. Next, (i) 40 mL of pure water was added to the remaining precipitate, followed by stirring for 15 minutes on a shaker. (ii) The mixture was centrifuged at 3500 G. (iii) The supernatant was recovered as a single-layer / sparse-layer MXene-containing solution. These steps (i) to (iii) were repeated four times to obtain a single-layer / sparse-layer MXene-containing supernatant. Furthermore, this supernatant was centrifuged at 4300 G for 2 hours using a centrifuge. The supernatant was then discarded, and the remaining precipitate, a single-layer / sparse-layer MXene-containing clay (hereinafter simply referred to as MXene clay), was obtained as a clay containing MXene particles (precursor particles).

[0078] In this example, clay (MXene clay) containing MXene particles (precursor particles) obtained in the preparation of the MXene particles (precursor particles) described above was used to carry out the following steps, which are described in detail below: (1) synthesis of ligand-substituted MXene particles, (2) washing of the ligand-substituted MXene particles, (3) preparation of a dispersion containing ligand-substituted MXene particles, (4) fabrication of an MXene-enzyme electrode, and (5) electrochemical evaluation of the MXene-enzyme electrode. In this example, ligand-substituted MXene particles refer to aminated MXene particles modified with amino groups. Each of these steps is described below.

[0079] (1) Synthesis of Ligand-Substituted MXene Particles MXene clay was dispersed in pure water to prepare an aqueous dispersion containing MXene particles, with the MXene solids concentration adjusted to 1 mg / mL. 20 mL of this aqueous dispersion containing MXene particles was prepared and transferred to a 50 mL vial containing a stirrer. NH ２ 73.5 mg (0.660 mmol) of -C1PA (manufactured by Aldrich) was weighed and dissolved in 1.32 mL of pure water. ２ - As long as C1PA dissolves, the water is not limited to pure water.

[0080] 10 μL of hydrochloric acid (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) and NH ２1.32 mL of -C1PA was added, and the mixture was stirred at a room temperature of 20 to 25°C using a magnetic stirrer at a stirring speed of 800 rpm for 24 hours.

[0081] (2) Washing of Ligand-Substituted MXene Particles After stirring, the sample solution was transferred to a 50 mL centrifuge tube and centrifuged at room temperature, 8000 rpm, and for 8 minutes, after which the supernatant was discarded. Next, 20 mL of pure water was added to the precipitate to redisperse it. This procedure of adding pure water to the precipitate, centrifuging, and discarding the supernatant was repeated two more times (a total of three times) to remove unreacted NH ２ -C1PA was removed.

[0082] (3) Preparation of a dispersion containing ligand-substituted MXene particles After discarding the supernatant resulting from the third centrifugation in (2) above, 20 mL of pure water was added as a dispersion medium to the precipitate, and the precipitate was dispersed to prepare a dispersion of ligand-substituted MXene particles in pure water. Note that there are no limitations on the dispersion medium used to disperse the ligand-substituted MXene particles, and any dispersion medium other than those listed above can be used. In this example, pure water was used as the dispersion medium, but this is not a limitation.

[0083] The aminophosphonic acid-substituted MXene prepared in this example was analyzed by XPS (Quantes, manufactured by ULVAC-PHI, Inc.), solid-state NMR (NMR AVAVCE400, measurement conditions: 9.4 T magnetic field, ３１ P measurement and １ Using 1H measurement, it was confirmed that phosphonic acid was substituted on MXene and that it had an amino group (nitrogen atom).

[0084] (4) Preparation of MXene-enzyme electrode: 10 μL of the approximately 1 mg / mL ligand-substituted MXene dispersion prepared in (3) above was dropped onto a 3 mm diameter glassy carbon electrode (BAS: 002012) and allowed to dry naturally. Before use, this glassy carbon electrode was polished with 1.0 μm outer diameter diamond particles and 0.05 μm outer diameter alumina particles, and then polished and cleaned by ultrasonic treatment in pure water. After the ligand-substituted MXene was dripped and dried, 30 μL of a solution containing 5 mg / mL Cytochrome C from bovine heart (dissolved in 1x PBS, pH 7.4), 10 mM N-hydroxy sulfosuccinimide aqueous solution (Wako: 087-09371), and 10 mM 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride aqueous solution (Thermo Fisher: 22981) was dripped onto the electrode and allowed to dry naturally. This resulted in the production of an electrode (MXene-enzyme electrode, two-dimensional particle-oxidoreductase complex) in which the amino group on the ligand-substituted MXene (aminated MXene) and the carboxyl group on the Cytochrome C formed an amide bond through dehydration condensation. It should be noted that other dehydration condensation agents may be used in addition to the above-mentioned reagents as long as they can immobilize the amino groups on Cytochrome C and MXene.

[0085] (5) Electrochemical Evaluation of the MXene-Enzyme Electrode The electrochemical characteristics of the MXene-enzyme electrode prepared in (4) above were measured using a potentiostat (Hokuto Denko: 1280Z). 10 mL of 1x PBS was added as a measurement buffer to a cell containing a stir bar, and a three-electrode system was constructed using the MXene-enzyme electrode prepared in (4) above as the working electrode, a platinum wire as the counter electrode, and Ag / AgCl (3M NaCl) as the reference electrode.

[0086] N ２ After degassing by bubbling (50 cc / min) into the measurement buffer, chronoamperometry was carried out under the following conditions: After the start of chronoamperometry measurement, the charging current settled to around 0 A, and then 3% H ２ O ２50 μL of the aqueous solution was dropped into the above system as an oxidizing agent, and the stirring bar was rotated using a magnetic stirrer for 1 s to introduce H into the solution. ２ O ２ The current corresponding to the oxidation-reduction of the enzyme was confirmed by diffusing Ag. The measurement results are shown in Figure 8. (Measurement conditions) Measurement potential: -0.2 V (vs Ag / AgCl) Measurement time: 600 s

[0087] The assumptions of this experiment are as follows. Cytochrome C immobilized on aminated MXene on the working electrode is applied with a potential of -0.2 V, and there is a redox-capable site called heme iron (heme iron) in Cytochrome C, which can take on the states of divalent and trivalent iron ions. The redox potential of the iron ions is approximately 0 V (Bull. Chem. Soc. Jpn. 2021, 94, 433.). Therefore, during chronoamperometry measurement, Cytochrome C exists in a state where heme iron is a divalent reduced form. If this state is maintained, no current flows through the working electrode, and the current value is approximately 0 A. A 3% H ２ O ２ By adding the aqueous solution, the cheme iron in Cytochrome C is oxidized from divalent to trivalent. However, because the electrode is applied with a voltage of -0.2 V, electrons are immediately supplied from the electrode to Cytochrome C through aminated MXene, which is thought to cause a negative current to flow (because a potentiostat considers a current flowing from the working electrode to the counter electrode to be positive).

[0088] Chronoamperometry of the MXene-enzyme electrode prepared by the above method showed that 3% H ２ O ２ After adding the aqueous solution as an oxidizing agent to the system, the current was -24.3 μA / cm ２ The current response was confirmed.

[0089] Comparative Example 1-1 In Comparative Example 1-1, a ligand-substituted MXene film was prepared using the same silane coupling agent as in Non-Patent Document 1. Specifically, in (1) of Example 1-1, aminated MXene was synthesized using a silane coupling agent (APTES) instead of aminophosphonic acid as the ligand, and the MXene-enzyme electrode was prepared and electrochemically evaluated using the same procedures as in Example 1-1. Non-Patent Document 1 was referenced for the ligand substitution procedure.

[0090] Chronoamperometry of the MXene-enzyme electrode prepared by the above method showed that 3% H ２ O ２ After adding the aqueous solution as an oxidizing agent to the system, the ２ The current response was confirmed.

[0091] From the results of Example 1-1 and Comparative Example 1-1 above, it can be seen that in ligand substitution using a silane coupling agent (APTES), the polymerization reaction between APTES and the ligand substitution reaction with MXene occur in competition with each other, resulting in the ligand being present on MXene in the form of a bulky polymer such as siloxane. As a result, the presence of the bulky polymer likely increased the distance between MXene and Cytochrome C, resulting in a decrease in electron transfer efficiency. On the other hand, in this example, phosphonic acid was used as the ligand rather than a silane coupling agent. Compared to silane coupling agents, phosphonic acid is a very stable reagent that does not self-polymerize and only reacts with functional groups on MXene, allowing for uniform ligand substitution. As a result, the distance between MXene and the enzyme could be shortened, improving electron transfer efficiency. As a result, the oxidoreductase could be uniformly immobilized on MXene. Furthermore, the distance between MXene and the redox enzyme (Cytochrome C) was shortened, resulting in improved electron transfer efficiency (increased current value). Comparing the above results, the enzyme electrode of this example had a current value four times higher than that of the comparative example, and a response speed 16 times faster than that of the conventional electrode.

[0092] Example 2 In Example 2, the electron transfer efficiency was evaluated for an example in which the phosphonic acid of ligand-substituted MXene was non-covalently bonded to an oxidoreductase.

[0093] [Example 2-1] In this example, steps (1) to (3) of the procedure from (1) to (5) in Example 1-1 were carried out in the same manner as in Example 1-1. Steps (4) to (5) are described in detail below.

[0094] (4) Preparation of MXene-enzyme electrode Approximately 1 mg / mL of the ligand-substituted MXene dispersion prepared in Example 1-1 (3) was dropped in 10 μL onto a φ3 mm glassy carbon electrode (BAS: 002012) and allowed to dry naturally. This glassy carbon electrode was polished with diamond particles with an outer diameter of 1.0 μm and alumina particles with an outer diameter of 0.05 μm, and then polished and cleaned by ultrasonic treatment with pure water. After the ligand-substituted MXene was dropped and dried, 10 μL of 5 mg / mL Cytochrome C from bovine heart (dissolved in 1x PBS at pH 7.4) was dropped and allowed to dry naturally to prepare an electrode.

[0095] (5) Electrochemical Evaluation of MXene-Enzyme Electrode The electrochemical characteristics of the MXene-enzyme electrode prepared in (4) above were measured using a potentiostat (1280Z manufactured by Hokuto Denko). Cyclic voltammetry (hereinafter abbreviated as CV) was carried out under the following conditions using 10 mL of 1x PBS as the measurement buffer, a platinum wire as the counter electrode, and Ag / AgCl (3M NaCl) as the reference electrode. Before the measurement, N ２ (50 cc / min) was bubbled into the measurement buffer to degas the solution. (Measurement conditions) Measurement potential range: -0.3 to +0.14 V (vs Ag / AgCl) Sweep rate: 10 mV / s Number of cycles: 3

[0096] The measurement results are shown in Figure 9. From the measurement results, the oxidation-reduction current derived from Cytochrome C of the MXene-enzyme electrode prepared by the above method was 28.6 μA / cm ２ It should be noted that the current density is a faradaic current value derived from the enzyme, and is a current value obtained by dividing the charging current derived from the ligand-substituted MXene.

[0097] Comparative Example 2-1: This comparative example was carried out in the same manner as in Example 2-1, except that the ligand-substituted MXene used in Example 2-1 was replaced with ligand-unsubstituted MXene (at a concentration of 1 mg / mL). Specifically, the MXene clay obtained in [5] delamination in Example 1-1 was dispersed in pure water to prepare an MXene particle-containing aqueous dispersion at a MXene solids concentration of 1 mg / mL. This dispersion was used to prepare the MXene-enzyme electrode (4).

[0098] When the CV of the MXene-enzyme electrode prepared by the above method was measured, almost no redox current derived from Cytochrome C was observed.

[0099] Comparative Example 2-2 This comparative example was carried out in the same manner as in Example 2-1, except that the ligand-substituted MXene used in Example 2-1 was replaced with ligand-unsubstituted MXene (concentration: 1 mg / mL), and (4) the MXene-enzyme electrode was prepared. Details of the preparation of the modified (4) MXene-enzyme electrode are given below. Note that this comparative example corresponds to the conditions described in Non-Patent Document 2.

[0100] (4) Preparation of MXene-enzyme electrode The MXene clay obtained in [5] delamination in Example 1-1 was dispersed in pure water to prepare an MXene solids concentration of 1 mg / mL. 10 μL of this MXene particle-containing aqueous dispersion (ligand-unsubstituted MXene) was dropped onto a φ3 mm glassy carbon electrode (BAS: 002012) and allowed to air dry. Before use, this glassy carbon electrode was polished with 1.0 μm outer diameter diamond particles and 0.05 μm alumina particles, followed by polishing and cleaning with ultrasonic treatment in pure water. After the MXene was dripped and dried, a total of 10 μL of a mixed solution prepared by adding 10 μL of 5 wt % Nafion (manufactured by Aldrich: 527084) to 400 μL of 5 mg / mL Cytochrome C from bovine heart (dissolved in 1× PBS, pH 7.4) was dripped onto the electrode and allowed to dry naturally, thereby preparing an electrode.

[0101] The CV measurement results of the MXene-enzyme electrode prepared by the above method are shown in Figure 10. From the measurement results, the oxidation-reduction current derived from Cytochrome C of the electrode prepared by the above method was 10.0 μA / cm ２ It should be noted that this current density is also a faradaic current value derived from the enzyme, and is the current value obtained by dividing the charging current derived from the ligand-substituted MXene.

[0102] The results of Example 2-1, Comparative Example 2-1, and Comparative Example 2-2 demonstrate that while conventional enzyme immobilization on MXene required polymers such as Nafion, the use of MXene with ligand substitution by phosphonic acid enabled enzyme immobilization through ligand-mediated noncovalent bonds, typically electrostatic interactions, hydrophobic interactions, and hydrogen bonds, without the need for polymers such as Nafion. Specifically, this example uses aminophosphonic acid as the preferred ligand, and provides amino groups as the preferred surface functional groups on MXene, enabling enzyme immobilization on MXene through electrostatic and hydrophobic interactions between the amino group and the enzyme. More specifically, the amino groups contained in the ligand assume a positive charge upon protonation, and because the amino groups are capable of hydrogen bonding, they can form electrostatic interactions with negative charges (e.g., carboxyl groups) on the enzyme surface and hydrogen bonds with oxygen (O) atoms present on the enzyme surface. These factors are believed to be the factors that enabled this enzyme immobilization.

[0103] This application claims priority from Japanese Patent Application No. 2024-030291, which is incorporated herein by reference.

[0104] The two-dimensional particle-oxidation / reduction enzyme complex of the present disclosure can be used for any suitable application, and can be particularly preferably used for high-yield production of useful substances such as drugs, for example, in electrodes such as biological signal sensing electrodes and enzyme electrodes, biosensors, batteries, and even pharmaceuticals.

[0105] The disclosure of the present specification may include the following aspects. <1> The two-dimensional particle includes one or more layers, wherein the layer is represented by the following formula: M ｍ X ｎ (wherein M is at least one metal of Group 3, 4, 5, 6 or 7; X is a carbon atom, a nitrogen atom or a combination thereof; n is 1 or more and 4 or less; and m is greater than n and 5 or less), and a modified or terminal T (wherein T is at least one selected from the group consisting of a hydroxyl group, a fluorine atom, a chlorine atom, an oxygen atom and a hydrogen atom) present on the surface of the layer body, and the modified or terminal T has a structure in which an oxygen atom derived from the hydroxyl group is bonded to a phosphorus atom of a phosphonic acid, the phosphonic acid having a saturated or unsaturated hydrocarbon group, and an oxidoreductase is immobilized on the phosphonic acid. <2> A two-dimensional particle-oxidoreductase complex, wherein the layer body is Ti ３ C ２ <3> The two-dimensional particle-oxidoreductase complex according to <1> or <2>, wherein the phosphonic acid has a nitrogen-containing functional group. <4> The two-dimensional particle-oxidoreductase complex according to <3>, wherein an oxidoreductase is immobilized to the phosphonic acid having an amino group as the nitrogen-containing functional group via a covalent bond including an amide bond. <5> The two-dimensional particle-oxidoreductase complex according to <3>, wherein an oxidoreductase is immobilized to the phosphonic acid having an amino group as the nitrogen-containing functional group via a non-covalent bond including one or more selected from the group consisting of electrostatic interaction, hydrophobic interaction, and hydrogen bond. <6> The two-dimensional particle-oxidoreductase complex according to any one of <1> to <5>, which is used in a biological signal sensing electrode, a biosensor, or a pharmaceutical.

[0106] 1a, 1b Layer body (M ｍ X ｎLayer) 3a, 5a, 3b, 5b Modified or terminal T 7a, 7b MXene layer 10a, 10b, 10c MXene particle 21 Conventional ligand 23 Ligand of the present embodiment 25 Oxidoreductase 27 Polymer 29 Non-covalent bond

Claims

1. A two-dimensional particle comprising one or more layers, wherein said layers are of the following formula: M ｍ X ｎ (wherein M is at least one metal of Group 3, 4, 5, 6 or 7; X is a carbon atom, a nitrogen atom or a combination thereof; n is 1 or more and 4 or less; and m is greater than n and 5 or less), and a modified or terminal T (wherein T is at least one selected from the group consisting of a hydroxyl group, a fluorine atom, a chlorine atom, an oxygen atom and a hydrogen atom) present on the surface of the layer body, wherein the modified or terminal T has a structure in which an oxygen atom derived from the hydroxyl group is bonded to a phosphorus atom of a phosphonic acid, the phosphonic acid having a saturated or unsaturated hydrocarbon group, and an oxidoreductase is immobilized on the phosphonic acid.

2. The layer body is Ti ３ C ２ The two-dimensional particle-oxidoreductase complex according to claim 1, 3. The two-dimensional particle-oxidoreductase complex according to claim 1 or 2, wherein the phosphonic acid has a nitrogen-containing functional group.

4. The two-dimensional particle-oxidoreductase complex according to claim 3, wherein an oxidoreductase is immobilized to a phosphonic acid having an amino group as the nitrogen-containing functional group via a covalent bond including an amide bond.

5. The two-dimensional particle-oxidoreductase complex according to claim 3, wherein an oxidoreductase is immobilized to the phosphonic acid having an amino group as the nitrogen-containing functional group by a non-covalent bond comprising one or more selected from the group consisting of electrostatic interaction, hydrophobic interaction, and hydrogen bond.

6. The two-dimensional particle-oxidoreductase complex according to any one of claims 1 to 5, which is used in a biological signal sensing electrode, a biosensor, or a pharmaceutical.

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