A medium-entropy oxide nanofoam enzyme and its preparation method and application

Abstract

The present invention provides a medium-entropy oxide nanofoam enzyme and its preparation method and application, which belongs to the field of nanozyme technology. The preparation method of the medium-entropy oxide nanofoam enzyme comprises the following steps: nickel salt, iron salt, manganese salt, urea and molten salt 1 are mixed with a grinding aid and then ground to obtain a mixture A; the mixture A is added to a molten salt 2 in a molten state, and then cooled to room temperature, and finally washed and dried to obtain the medium-entropy oxide nanofoam enzyme. In the low-high temperature molten salt assisted calcination method of the present invention, the use of low-high temperature molten salt effectively inhibits the agglomeration of the entropy oxide in FeNiMnO4 during the preparation process, forming a nano-foam structure. The colorimetric sensor constructed based on the FeNiMnO4 medium-entropy oxide nanofoam enzyme exhibits excellent detection performance, and its detection limit (LOD) can be as low as 0.98mU / mL, while also having excellent stability.

Description

Technical Field

[0001] The present invention relates to the technical field of nanoenzymes, and in particular to a medium-entropy oxide nanofoam enzyme and a preparation method and application thereof. Background Art

[0002] Acetylcholinesterase (AChE) catalyzes the hydrolysis of the neurotransmitter acetylcholine (ATCh) into acetic acid and thiocholine (TCh). This enzyme, primarily found at the neuromuscular junction and in the cholinergic nervous system, terminates the excitatory effects of neurotransmitters on the postsynaptic membrane, ensuring the normal transmission of nerve signals within the body. It is a crucial hydrolase for maintaining cholinergic nerve impulses in the body. Clinically, measuring AChE activity is crucial for understanding the pathogenesis of neurodegenerative diseases such as Alzheimer's disease. Brain acetylcholine levels decline significantly with age, directly associated with cognitive impairment. Abnormal AChE activity accelerates acetylcholine degradation, further exacerbating the condition. Therefore, accurate measurement of AChE activity not only facilitates early diagnosis of Alzheimer's disease but also provides a dynamic monitoring indicator for evaluating drug efficacy.

[0003] In recent years, colorimetric sensing methods have been widely used for high-sensitivity detection of biomarkers due to their simple operation, high sensitivity and low cost. This method has strong anti-interference ability, is easy to operate and has low dependence on professional equipment, which will provide a simple alternative visual detection strategy for in vitro detection of AChE activity. However, the natural enzymes used in this detection technology are expensive to prepare and easily lose their activity, which limits their large-scale application. It is of great significance to develop efficient nanoenzymes to replace natural enzymes for the detection of AChE activity. For example, the Chinese invention patent document with authorization announcement number CN118287113B discloses a method for preparing sulfur-doped selenide heterojunction nanozymes and their application in the detection of acetylcholinesterase activity. The method first synthesizes NiO and FeO nanosheets, and then reacts with ligands under solvent thermal conditions to prepare FeO nanosheets. x Ni y DMBD-NS ultimately prepared a FeSe2 / NiSe2 heterojunction material through a selenization reaction under an inert atmosphere, which was used as a nanozyme to detect the activity of acetylcholinesterase. However, the preparation process involved in this patent is cumbersome, energy-intensive, inefficient, and costly, making it unsuitable for industrial production. Furthermore, the selenization process produces harmful gases, which is detrimental to environmental protection.

[0004] Metal oxides with peroxidase (POD)-like activity have become the most effective and promising candidates for replacing natural enzymes in colorimetric sensing applications due to their simple synthesis methods and low cost. However, due to insufficient catalytic performance, current results are unsatisfactory. To address these issues, existing research has mainly focused on modulating the POD-like properties of metal oxide nanozymes through material morphology and size regulation. However, less research has focused on the effect of increased entropy on the properties of metal oxide PODs. Intermediate entropy oxides (IDOs) have attracted widespread attention in recent years due to their unique structural characteristics, including the intermediate entropy effect, lattice distortion effect, slow diffusion effect, and "cocktail effect." These properties endow IDOs with excellent structural stability and nearly unlimited structural tunability. Therefore, increased entropy may be an effective means of modulating the POD-like properties of metal oxides. However, most IDOs are currently formed by calcining at high temperatures for long periods of time. For example, Chinese invention patent document CN113248255B discloses a high-temperature, thermally insulating fluorite-type IDO and its preparation method. The method first adds ammonia to the precursor solution. The stirred and dried sample is then heat-treated at 1100°C to 1500°C in an air atmosphere for 1 to 4 hours. After cooling, it is then kept at 1500°C to 1600°C for 16 to 60 hours to obtain a high-temperature, insulating fluorite-type intermediate entropy oxide. The intermediate entropy oxide prepared by this method requires long, high-temperature calcination, which consumes a lot of energy. In addition, the prepared material is prone to sintering, making it unsuitable for the preparation of nanozyme materials with high catalytic performance requirements. Therefore, developing a simple and effective method to prepare porous and loose intermediate entropy oxides as efficient nanozymes for AChE activity detection, thereby improving the detection sensitivity of nanozyme-based colorimetric sensing platforms, has important scientific significance and application value. Summary of the Invention

[0005] In view of this, the present invention aims to provide a medium-entropy oxide nanofoam enzyme and its preparation method and application to solve the problems of low catalytic efficiency, high energy consumption of medium-entropy oxide, and easy sintering of metal oxide nanozymes prepared by traditional methods. The present invention uses a low-temperature molten salt-assisted calcination method to efficiently prepare medium-entropy oxide nanofoam enzyme. This material combines the medium-entropy effect and the cocktail effect, showing high intrinsic nanozyme catalytic activity.

[0006] In order to achieve the above object, the present invention provides the following technical solutions:

[0007] One of the technical solutions of the present invention is a method for preparing a medium-entropy oxide nanofoam enzyme, comprising the following steps:

[0008] Mixing nickel salt, iron salt, manganese salt, urea and molten salt 1 with a grinding aid and grinding the mixture to obtain a mixture A;

[0009] The mixture A is added to the molten salt 2 in a molten state, and then cooled to room temperature, and finally washed and dried to obtain the medium-entropy oxide nanofoam enzyme.

[0010] In the present invention, the nickel salt is nickel chloride hexahydrate or nickel sulfate hexahydrate, the iron salt is ferric chloride hexahydrate or ferric sulfate pentahydrate, and the manganese salt is manganese chloride tetrahydrate or manganese sulfate tetrahydrate.

[0011] In the present invention, the mass ratio of the nickel salt, the iron salt, the manganese salt and the urea is 1:1.125:0.825:3-8.

[0012] In the present invention, the molten salt 1 is a mixture of calcium chloride and sodium chloride; the molar ratio of the calcium chloride to the sodium chloride is 1:0.8-1.2; the mass ratio of the urea to the molten salt 1 is 3-8:7.5-20. In the present invention, the molten salt 1 is a low-temperature molten salt.

[0013] In the present invention, the grinding aid is ethanol. The present invention does not impose any particular limitation on the amount of ethanol used, and those skilled in the art can make routine adjustments to meet the grinding requirements.

[0014] In the present invention, the molten salt 2 is sodium chloride or potassium chloride. The molten salt 2 is a high-temperature molten salt. The amount of molten salt 2 is not particularly limited; it can be used in an amount sufficient for the molten salt calcination method. For example, the mass ratio of mixture A to molten salt 2 is 1:5-10. The molten salt 2 is prepared by calcining the molten salt 2 at a constant temperature of 800-1000°C until it is molten.

[0015] The second technical solution of the present invention is a medium-entropy oxide nanofoam enzyme prepared by the above preparation method.

[0016] The third technical solution of the present invention is an application of the above-mentioned medium-entropy oxide nanofoam enzyme in the detection of acetylcholinesterase activity.

[0017] The present invention discloses the following technical effects:

[0018] The present invention proposes a low-high temperature molten salt assisted calcination method. During the preparation process, the use of low-high temperature molten salt effectively inhibits the agglomeration of entropy oxides in FeNiMnO4 and forms a nano-foam structure.

[0019] The prepared medium-entropy oxide nanofoam enzyme has a rich pore structure, providing more reaction channels for the rapid adsorption and activation of substrate molecules and the desorption of products during the acetylcholinesterase activity detection process. Furthermore, the FeNiMnO4 medium-entropy oxide nanofoam enzyme exhibits both a medium-entropy effect and a cocktail effect, demonstrating high intrinsic nanozyme catalytic activity.

[0020] A colorimetric sensor based on FeNiMnO4 entropy oxide nanofoam enzyme exhibits excellent detection performance, with a limit of detection (LOD) as low as 0.98 mU / mL and excellent stability. This innovation opens up a new technical path and solution for the rapid and accurate detection of acetylcholinesterase activity. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0022] Figure 1 is the powder X-ray diffraction pattern of FeNiMnO4-1 in Example 1;

[0023] Figure 2 The scanning electron micrograph and element distribution diagram of FeNiMnO4-1 of Example 1 are shown;

[0024] Figure 3 These are scanning electron microscopy images of the nanozymes of Comparative Example 1 and Comparative Example 2;

[0025] Figure 4 is the peroxidase-like activity of the samples of Example 1 and Comparative Examples 1-5;

[0026] Figure 5 The UV absorption spectra of the medium entropy oxide nanofoam enzyme at different acetylcholinesterase activities;

[0027] Figure 6 is the relationship between UV absorbance and acetylcholinesterase activity;

[0028] Figure 7 This is a graph showing the peroxidase-like activity of FeNiMnO4-1 after 9 cycles of detecting acetylcholinesterase activity in Example 1;

[0029] Figure 8 This is a scanning electron micrograph of Example 1 after FeNiMnO4-1 was used to detect acetylcholinesterase activity for 9 cycles. DETAILED DESCRIPTION

[0030] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0031] It should be understood that the terms described herein are intended only to describe particular embodiments and are not intended to limit the present invention. In addition, for numerical ranges herein, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. The intermediate value within any stated value or stated range, and each smaller range between any other stated value or intermediate value within the stated range, is also encompassed within the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded within the scope.

[0032] Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein may also be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of any conflict with any incorporated document, the contents of this specification shall prevail.

[0033] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments described herein without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from the description of the invention. The description and examples are intended to be exemplary only.

[0034] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0035] The present invention adopts the following methods to detect the activity of the medium entropy oxide nanofoam enzyme and the detection sensitivity of the medium entropy oxide nanofoam enzyme to the activity of acetylcholinesterase.

[0036] FeNiMnO4 entropy oxide nanofoam enzyme suspension (1 mg / mL), 3,3',5,5'-tetramethylbenzidine (TMB) (1 mM), and H2O2 (100 mM) solutions were prepared respectively in pure water. Then, 100 µL FeNiMnO4 entropy oxide nanofoam enzyme, 50 µL H2O2, and 100 µL TMB were mixed in 2750 µL sodium acetate-acetic acid (NaAc-Hac, pH = 4.5, 0.2 M) buffer solution. After incubation at room temperature for 20 min, the absorbance of the reaction solution was measured at 652 nm using a UV-visible spectrophotometer to evaluate the activity of FeNiMnO4 entropy oxide nanofoam enzyme.

[0037] 150µL of AChE (activity levels of 0, 0.5, 1, 2, 4, 6, 8, 10, 12, 16, and 20 mU / mL) was mixed with 90µL of 5mM ATCh, and the mixture was diluted to 300µL. After incubation at 37°C for 30 minutes, 50µL of 1mg / mL FeNiMnO4 entropy oxide nanofoam was added and incubated for another 1 minute to completely inhibit the activity of the FeNiMnO4 entropy oxide nanofoam. Subsequently, 2500µL of NaAc-Hac buffer (pH 4.5, 0.2M), 50µL of 100mM H2O2, and 100µL of 1mM TMB were added, and incubation was continued at 37°C for another 15 minutes. The absorbance was then recorded, and acetylcholinesterase activity was monitored.

[0038] Unless otherwise specified, the technical solutions described in the present invention are all conventional solutions in the field, and the reagents or raw materials used, unless otherwise specified, are purchased from commercial channels or have been disclosed.

[0039] In order to better understand the present invention, the content of the present invention is further illustrated below in conjunction with the examples, but the content of the present invention is not limited to the following examples.

[0040] Example 1

[0041] Step S1: 0.2 g of nickel chloride hexahydrate, 0.225 g of ferric chloride hexahydrate, 0.165 g of manganese chloride tetrahydrate, 1 g of urea, 2.5 g of a mixed molten salt of calcium chloride and sodium chloride (molar ratio 1:1), and 20 mL of ethanol were added to a mortar and ground until the liquid was completely evaporated to obtain a powdered solid A.

[0042] Step S2: 30g of sodium chloride was added to a quartz crucible and calcined at 900°C in a muffle furnace until molten. The molten sodium chloride was then poured into a quartz crucible containing 5g of powdered solid A. After cooling to room temperature, the resulting solid powder was filtered through a Büchner funnel and rinsed with water and 0.5M dilute hydrochloric acid until the molten salt was completely removed. Finally, the remaining powder was placed in a forced air drying oven at 80°C for 24h to obtain a FeNiMnO4 entropy oxide nanofoam enzyme, named FeNiMnO4-1.

[0043] Example 2

[0044] The only difference from Example 1 is that in step S1, 1 g of urea, 2.5 g of a mixed molten salt of calcium chloride and sodium chloride (molar ratio 1:1), and 20 mL of ethanol are replaced with 1.6 g of urea, 4 g of a mixed molten salt of calcium chloride and sodium chloride (molar ratio 1:1), and 25 mL of ethanol, respectively. The remaining steps and parameters are the same as those in Example 1. The obtained FeNiMnO4 entropy oxide nanofoam enzyme is recorded as FeNiMnO4-2.

[0045] Example 3

[0046] The only difference from Example 1 is that in step S1, the calcium chloride-sodium chloride (molar ratio 1:1) mixed molten salt is replaced with calcium chloride-sodium chloride (molar ratio 1:1.2) mixed molten salt, and the remaining steps and parameters are the same as those in Example 1. The obtained FeNiMnO4 entropy oxide nanofoam enzyme is recorded as FeNiMnO4-3.

[0047] Example 4

[0048] The only difference from Example 1 is that 30 g of sodium chloride is replaced by 30 g of potassium chloride in step S2, and the remaining steps and parameters are the same as those in Example 1. The obtained FeNiMnO4 entropy oxide nanofoam enzyme is recorded as FeNiMnO4-4.

[0049] Example 5

[0050] The only difference from Example 1 is that the constant temperature calcination at 900°C in a muffle furnace to a molten state is changed to the constant temperature calcination at 850°C in a muffle furnace to a molten state, and the other steps and parameters are the same as Example 1. The obtained FeNiMnO4 entropy oxide nanofoam enzyme is recorded as FeNiMnO4-5.

[0051] Comparative Example 1

[0052] The only difference from Example 1 is that calcium chloride-sodium chloride low-temperature molten salt is not added in step S1, and the remaining steps and parameters are the same as those in Example 1. The obtained nanozyme is recorded as FeNiMnO4-D1.

[0053] Comparative Example 2

[0054] Step S1 is the same as step S1 in Example 1.

[0055] Step S2: 5 g of powdered solid A was added to a quartz crucible and calcined at 900°C in a muffle furnace for 30 minutes. After cooling to room temperature, the resulting solid powder was filtered through a Büchner funnel and rinsed with water and 0.5 M dilute hydrochloric acid until the molten salt was completely removed. Finally, the remaining powder was placed in a forced air drying oven at 80°C for 24 hours. The resulting nanozyme was designated FeNiMnO4-D2.

[0056] Comparative Example 3

[0057] The only difference from Example 1 is that in step S1, 0.2 g nickel chloride hexahydrate, 0.225 g ferric chloride hexahydrate, and 0.165 g manganese chloride tetrahydrate are replaced with 0.6 g nickel chloride hexahydrate, and the remaining steps and parameters are the same as in Example 1. The obtained nanozyme is recorded as NiO.

[0058] Comparative Example 4

[0059] The only difference from Example 1 is that in step S1, 0.2 g nickel chloride hexahydrate, 0.225 g ferric chloride hexahydrate, and 0.165 g manganese chloride tetrahydrate are replaced with 0.495 g manganese chloride tetrahydrate, and the remaining steps and parameters are the same as in Example 1. The obtained nanozyme is recorded as MnOx.

[0060] Comparative Example 5

[0061] The only difference from Example 1 is that in step S1, 0.2 g nickel chloride hexahydrate, 0.225 g ferric chloride hexahydrate, and 0.165 g manganese chloride tetrahydrate are replaced with 0.675 g ferric chloride hexahydrate, and the remaining steps and parameters are the same as in Example 1. The obtained nanozyme is recorded as FeOx.

[0062] Characterization and effect verification:

[0063] 1. Figure 1 The powder X-ray diffraction pattern of FeNiMnO4-1 of Example 1 is consistent with the standard card PDF#00-010-0325 of spinel oxide, indicating that Example 1 is a spinel medium entropy oxide.

[0064] 2. Figure 2 The scanning electron micrograph and element distribution diagram of FeNiMnO4-1 of Example 1 are presented. The results show that the FeNiMnO4-1 of Example 1 is a porous foam structure composed of accumulated nanoparticles, and that the three elements Fe, Ni, and Mn are evenly distributed within the nanoparticles, further confirming the medium entropy characteristics of Example 1.

[0065] 3. Figure 3 Scanning electron micrographs of Comparative Example 1 and Comparative Example 2 are shown. The results show that both Comparative Example 1 and Comparative Example 2 present a block structure at the micron level, indicating that the low-temperature molten salt and the high-temperature molten salt work together to promote the formation of a nano-foam structure, and both are indispensable. This is mainly because during the preparation of the sample, the high-temperature molten salt can quickly transfer heat to the powder solid A, causing the low-temperature molten salt in the powder solid A to melt rapidly. These molten low-temperature molten salts wrap around the metal-urea complex to prevent the aggregation of the metal-urea complex. At the same time, the dispersed metal urea complex decomposes under heat to produce a large amount of gas, which promotes the formation of foamed metal oxides. At the same time, the external molten salt fills the pores of the foamed metal oxide to inhibit the agglomeration of the metal oxides.

[0066] Table 1 further lists the N2 adsorption-desorption test results of Example 1 and Comparative Examples 1-5. It can be seen from the table that the specific surface areas of Example 1 and Comparative Examples 3-5 prepared by the low-high temperature molten salt method are in the range of 74.37-83.28 m 2 / g, which is much higher than that of Comparative Examples 1 and 2, and the pore size distribution range is wider than that of the comparative examples. This further shows that the low-temperature molten salt method can effectively prepare oxide nanofoams with rich pore structures. This rich pore structure is conducive to the exposure of nanozyme active sites, thereby improving the sensitivity and service life of nanozyme-based sensors.

[0067] Table 1 N2 adsorption-desorption test results of Example 1 and Comparative Examples 1-5

[0068]

[0069] 4. Use pure water to prepare FeNiMnO4 entropy oxide nanofoam enzyme suspension (1 mg / mL), TMB (1 mM), and H2O2 (100 mM) solutions, respectively. Then, 100 µL FeNiMnO4 entropy oxide nanofoam enzyme, 50 µL H2O2, and 100 µL TMB were mixed in 2750 µL sodium acetate-acetic acid (NaAc-Hac, pH = 4.5, 0.2 M) buffer solution. After incubation at room temperature for 20 minutes, the absorbance of the reaction solution was measured at 652 nm using a UV-visible spectrophotometer to evaluate the activity of FeNiMnO4 entropy oxide nanofoam enzyme.

[0070] Figure 4 The peroxidase-like activity of Example 1 and Comparative Examples 1-5 samples was compared. The order of strength of the absorption peak at 652nm represents the order of peroxidase-like activity, and the results are Example 1> Comparative Example 1> Comparative Example 5> Comparative Example 4> Comparative Example 2> Comparative Example 3. From the activity comparison between Example 1 and Comparative Example 3, Comparative Example 4, and Comparative Example 5, it can be seen that the activity of Example 1 of the medium entropy effect is significantly better than that of the single metal oxide, wherein the order of element activity is Fe>Mn>Ni. From the activity comparison between Example 1 and Comparative Example 1, Comparative Example 5, and Comparative Example 2, it can be seen that the foam-like structure plays a vital role in the activity of the nanozyme. It can be seen that the excellent activity of the nanozyme in Example 1 comes from the combined effect of its porous foam-like structure and the medium entropy effect.

[0071] 5. 150µL of AChE (activity levels of 0, 0.5, 1, 2, 4, 6, 8, 10, 12, 16, and 20 mU / mL) was mixed with 90µL of 5mM ATCh, and the mixture was diluted to 300µL. After incubation at 37°C for 30 minutes, 50µL of 1mg / mL FeNiMnO4 entropy oxide nanofoam was added and incubated for another 1 minute to completely inhibit the activity of the FeNiMnO4 entropy oxide nanofoam. Then, 2500µL of NaAc-Hac buffer (pH 4.5, 0.2M), 50µL of 100mM H2O2, and 100µL of 1mM TMB were added sequentially, and incubation was continued at 37°C for another 15 minutes. The absorbance was then recorded, and acetylcholinesterase activity was monitored.

[0072] Given the key role of acetylcholinesterase as an enzyme in the nervous system, the development of a biosensor for rapid and sensitive detection of acetylcholinesterase activity is of great significance. The medium-entropy oxide nanofoam enzyme FeNiMnO4 was used to establish an acetylcholinesterase activity sensing platform due to its excellent peroxidase (POD)-like activity. Because the active site of the nanozyme is easily poisoned by the product generated by the hydrolysis of acetylcholine catalyzed by acetylcholinesterase. To ensure the feasibility of this detection method, such as Figure 5 As shown in the figure, under different acetylcholinesterase concentrations, the ultraviolet absorption spectrum of the medium entropy oxide nanofoam enzyme FeNiMnO4-1 system prepared in Example 1 shows that as the acetylcholinesterase concentration of the solution increases from 0 to 20 mU / mL, the absorbance value of the solution decreases significantly, indicating that the activity of the nanozyme is inhibited by the activity of acetylcholinesterase. Therefore, the decrease in absorbance can directly reflect the activity of acetylcholinesterase. The absorbance data was converted according to the Lambert-Beer law to obtain Figure 6 The relationship between UV absorption value and acetylcholinesterase activity. In the range of 0-8mU / mL, UV absorption value and acetylcholinesterase activity showed a good linear relationship (see Figure 6 Illustration), the linear equation is: y=0.0907x+0.0920 (R 2 =0.993). Therefore, the detection limit (LOD) of the medium-entropy oxide nanofoam enzyme FeNiMnO4-1 material is as low as 0.98 mU / mL, indicating that the colorimetric sensing method based on this material has excellent detection sensitivity for acetylcholinesterase activity.

[0073] 6. Figure 7 This is a graph of the peroxidase-like activity after 9 cycles of detecting acetylcholinesterase activity in Example 1. It can be seen from the graph that Example 1 has excellent enzyme activity stability, and the peroxidase-like activity after 9 cycles can still maintain about 90% of the initial activity.

[0074] 7. Figure 8 This is a scanning electron microscope image of Example 1 after 9 cycles of detecting acetylcholinesterase activity. The results show that Example 1 still maintains a nano-foam structure after 9 cycles of detecting acetylcholinesterase activity, indicating that Example 1 has excellent structural stability, which is conducive to the stability of its enzyme activity.

[0075] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. An application of a medium entropy oxide nanofoam enzyme in the detection of acetylcholinesterase activity, characterized in that: The preparation method of the medium-entropy oxide nanofoam enzyme comprises the following steps: Mixing nickel salt, iron salt, manganese salt, urea and molten salt 1 with a grinding aid and grinding the mixture to obtain a mixture A; The mixture A is added to the molten salt 2, and then cooled to room temperature, and finally washed and dried to obtain the medium entropy oxide nanofoam enzyme; The molten salt 1 is a mixture of calcium chloride and sodium chloride; the molar ratio of calcium chloride to sodium chloride is 1:0.8-1.2; The molten salt 2 is sodium chloride or potassium chloride.

2. The use of the medium entropy oxide nanofoam enzyme in the detection of acetylcholinesterase activity according to claim 1, characterized in that: The nickel salt is nickel chloride hexahydrate or nickel sulfate hexahydrate, the iron salt is ferric chloride hexahydrate or ferric sulfate pentahydrate, and the manganese salt is manganese chloride tetrahydrate or manganese sulfate tetrahydrate.

3. The use of the medium entropy oxide nanofoam enzyme in the detection of acetylcholinesterase activity according to claim 2, characterized in that: The mass ratio of the nickel salt, iron salt, manganese salt and urea is 1:1.125:0.825:3-8.

4. The use of the medium entropy oxide nanofoam enzyme in acetylcholinesterase activity detection according to claim 1, characterized in that: The mass ratio of the urea to the molten salt 1 is 3-8:7.5-20.

5. The use of the medium entropy oxide nanofoam enzyme in acetylcholinesterase activity detection according to claim 1, characterized in that: The grinding aid is ethanol.

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