Nano enzyme as well as preparation method and application thereof

Abstract

The invention provides a nano enzyme as well as a preparation method and application thereof. The preparation method of the nano enzyme comprises the following steps: adding a stabilizer into a preheated solvent, and carrying out ultrasonic treatment; then adding ferric salt and cobalt salt, and adding alkali to adjust the pH value of the solution; then transferring the solution into a reaction kettle for reaction; and after the reaction, washing and separating to obtain the product. The obtained nano-enzyme is appropriate and uniform in particle size and good in dispersity, the enzyme activity is as high as 9.56 U / mg, and the application of the nano-enzyme in immunochromatography detection and plant canker prevention and treatment is widened. When the immunochromatography detection card prepared from the nano enzyme is used for detecting human hemoglobin, the detection limit can reach 0.05 ng / mL and is at least 10 times lower than that of a commercially available kit. When the nano-enzyme suspension is used for preventing and treating the kiwifruit canker, the nano-enzyme suspension with the concentration larger than or equal to 0.1 mg / mL is used, and the plant canker can be well controlled.

Description

Technical Field

[0001] The present invention relates to the field of nanozyme preparation, and specifically relates to a nanozyme and a preparation method and application thereof. Background Art

[0002] Nanozymes are highly efficient catalysts made of nanomaterials. They are artificially improved on the structure and function of natural enzymes. Compared with natural enzymes, nanozymes have special advantages such as wider adaptability to reactants, higher stability, and easier to change and regulate. Nanozymes are mainly composed of nanomaterials (such as metal nanoparticles, magnetic nanoparticles, silica nanotubes, etc.) and functional molecules (such as oxidoreductase, peroxidase, cyclooxygenase, etc.).

[0003] The nanozymes currently prepared are mostly based on inorganic nanomaterials, which have the characteristics of stable enzymatic properties, easy surface regulation and green economy. However, they still have shortcomings in particle size uniformity, enzyme activity and surface morphology, which makes them easy to agglomerate and the enzyme activity is unstable. In the application of immunochromatographic tests, there are problems of low sensitivity and poor repeatability, and there are few results in the prevention and control of plant diseases and insect pests. Summary of the invention

[0004] In view of the shortcomings of the prior art, the technical problem to be solved by the present invention is how to obtain a nanozyme with suitable uniform particle size, good dispersibility, and high enzyme activity, so as to broaden its application in immunochromatographic detection and plant canker disease prevention and treatment. Based on this, the present invention provides a nanozyme and its preparation method and application.

[0005] In order to solve the above technical problems, the present invention provides the following technical solutions:

[0006] The present invention provides a method for preparing a nanozyme, comprising the following steps:

[0007] (1) adding a stabilizer to a preheated solvent, stirring until clear, and then performing ultrasonic treatment;

[0008] (2) After ultrasonic treatment, add iron salt and cobalt salt to step (1), stir until clear; then add alkali to adjust the pH of the solution, continue stirring until clear, and perform ultrasonic treatment again;

[0009] (3) After ultrasonication, the solution in step (2) is transferred to a reaction vessel for reaction;

[0010] (4) After the reaction, washing and separation are performed to obtain the product.

[0011] Preferably, in step (1), the stabilizer is a polymer compound, a surfactant or an amino acid dispersant; the solvent is pure water, ethanol, tetraethylene glycol, ethylene glycol or triethylene glycol;

[0012] The preheating temperature of the solvent is 40-70°C; the ultrasonic frequency is 30-40KHz, the ultrasonic power is 120-360W, and the ultrasonic time is 20-40min.

[0013] Preferably, in step (1), the stabilizer is polyvinyl pyrrolidone, polyvinyl alcohol, polyethylene glycol, polyacrylic acid, poly(4-styrenesulfonic acid-co-maleic acid) sodium salt, sodium lauryl sulfate, glycine, cysteine, hexadecyltrimethylammonium bromide, hexadecyltrimethylammonium chloride or sodium citrate;

[0014] The mass ratio of the stabilizer to the volume ratio of the solvent is (2-16) g: (10-50) mL.

[0015] Preferably, in step (2), the iron salt is a divalent iron salt or a trivalent iron salt; the cobalt salt is a divalent cobalt salt; and the base is sodium hydroxide, ammonia water or sodium acetate;

[0016] When ultrasonicating again, the ultrasonic frequency is 30-40KHz, the ultrasonic power is 120-360W, the ultrasonic time is 1-3h, and the ultrasonic temperature is 18-35°C.

[0017] Preferably, the iron salt is ferric chloride, ferrous chloride, ferrous sulfate, ferric nitrate or ferric acetylacetonate; the cobalt salt is cobalt chloride, cobalt bromide, cobalt iodide, cobalt sulfate or cobalt nitrate;

[0018] The molar ratio of Fe in the iron salt to Co in the cobalt salt is (1-4):1; the mass ratio of the stabilizer to the iron salt is (2-16):(1-2); and the pH of the solution is adjusted to 8-13 after adding alkali.

[0019] Preferably, in step (3), the reaction temperature is 120-250° C. and the reaction time is 5-24 h;

[0020] In step (4), washing is performed with water or an organic solvent; the separation method is centrifugation or external magnetic field adsorption; the organic solvent is acetone, tetrahydrofuran, ethanol or methanol; the centrifugal speed is 7000-9000rpm, and the centrifugal time is 5-10min.

[0021] The present invention provides a nanozyme, and the nanozyme is prepared by the preparation method.

[0022] The present invention provides an application of the nanozyme in preparing an immunochromatography reagent.

[0023] Preferably, in the immunochromatographic test card prepared by the nanozyme, the binding pad is a nanozyme dilution labeled with a mouse monoclonal antibody to human hemoglobin;

[0024] When testing samples, after the sample solution is chromatographed to the quality control line, add DAB colorimetric solution at the test line and observe the results after 5 minutes.

[0025] The present invention provides an application of the nanozyme in preventing and treating plant canker.

[0026] The technical solution of the present invention achieves the following beneficial technical effects:

[0027] The novel nanozyme prepared by the present invention completely overcomes the disadvantages of traditional natural enzymes, such as poor stability, high price, non-recyclability, complicated preparation process, and harsh storage conditions, and has the advantages of simple preparation, low raw material cost, and repeated recycling. Compared with traditional natural enzymes that need to be stored frozen, the novel nanozyme prepared by the present invention only needs to be stored at room temperature.

[0028] As a new material, the nanozyme of the present invention has both the physical and chemical properties of the nanomaterial itself and the enzyme-like catalytic function, and has the advantages of both natural enzymes and artificial enzymes. Among them, the nanostructure not only gives the nanozyme an efficient catalytic function, but also makes the nanozyme more stable than the natural enzyme and easy to mass produce. Among them, according to the transmission electron microscopy analysis, the nanozyme of the present invention has the characteristics of small particle size and uniform distribution. The TEM particle size measured by nano measurer is 98.38±12.03nm; according to the enzyme activity determination, it can be seen that the enzyme activity of the nanozyme of the present invention can reach up to 9.56U / mg; according to the hydrated particle size analysis, it can be seen that the hydrated particle size of the nanozyme of the present invention is 136.53±6.00nm.

[0029] The nanozyme of the present invention can be applied to immunochromatographic technology, and the immunochromatographic detection card prepared by the nanozyme of the present invention can reach 0.05ng / mL when detecting human hemoglobin, which is at least 10 times lower than that of the commercially available test kit. This is because on the one hand, the immunochromatographic detection card prepared by the present invention adds a nanozyme catalytic color development system on the basis of the conventional detection card currently on the market. When the concentration of the test object is low, the antigen / antibody sandwich enrichment at the T line alone cannot achieve color development that can be recognized by the naked eye. By dripping DAB color development solution, the principle of nanozyme catalyzing DAB color development is used to achieve the amplification of the detection signal, thereby producing color development that can be recognized by the naked eye, and achieving the purpose of greatly improving the detection sensitivity; on the other hand, the high specific surface area and loose porous structure of the nanozyme facilitate the antigen and antibody in the system to be combined with the nanozyme as a carrier, making the detection card more suitable for the detection of trace antigens.

[0030] The nanozyme of the present invention can be used to prevent and treat kiwifruit canker. The nanozyme suspension with a concentration of ≥0.1 mg / mL can achieve good control of plant canker. Among them, the nanozyme of the present invention can exert POD and SOD activities, regulate the level of reactive oxygen (ROS), and improve the immunity of plants. The generation of ROS catalyzed by POD activity can destroy the active components (cell membrane, protein, nucleic acid and enzyme, etc.) of microorganisms, thereby killing a variety of pathogenic microorganisms including bacteria, fungi and viruses. In terms of antibacterial, nanozymes can also release metal ions, enhance bacterial autophagy, and kill bacteria through their own physical and chemical properties such as magnetism and photothermal conversion, combined with photothermal therapy and sonodynamic therapy and other physical and chemical pathways. In terms of antiviral infection, nanozymes can block the binding of viruses to host cells, deliver drugs to interfere with viral gene replication, and alleviate the inflammatory response caused by viral infection, effectively inhibiting viral infection. In addition, the nanozyme of the present invention has a moderate particle size and is evenly distributed, can be evenly dispersed in an aqueous solution system for a long time, can maintain an effective concentration for a long time during actual use, and is convenient for large-scale spraying. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 Linear fitting diagram of the specific enzyme activity determination of the nanozyme obtained in Example 1 of the present invention;

[0032] Figure 2 Linear fitting diagram of the specific enzyme activity determination of the nanozyme obtained in Example 2 of the present invention;

[0033] Figure 3 Linear fitting diagram of the specific enzyme activity determination of the nanozyme obtained in Example 3 of the present invention;

[0034] Figure 4 Linear fitting diagram of the specific enzyme activity determination of the nanozyme obtained in Example 4 of the present invention;

[0035] Figure 5 Linear fitting diagram of the specific enzyme activity determination of the nanozyme obtained in Example 5 of the present invention;

[0036] Figure 6 Linear fitting diagram of the specific enzyme activity determination of the nanozyme obtained in Example 6 of the present invention;

[0037] Figure 7 Linear fitting diagram of the specific enzyme activity determination of the nanozyme obtained in Example 7 of the present invention;

[0038] Figure 8 Linear fitting diagram of the specific enzyme activity determination of the nanozyme obtained in Example 8 of the present invention;

[0039] Fig. 9 Linear fitting diagram of the specific enzyme activity determination of the nanozyme obtained in Example 9 of the present invention;

[0040] Fig.10Linear fitting diagram of the specific enzyme activity determination of the nanozyme obtained in Example 10 of the present invention;

[0041] Fig.11 Linear fitting diagram of the specific enzyme activity determination of the nanozyme obtained in Example 11 of the present invention;

[0042] Fig.12 Linear fitting diagram of the specific enzyme activity determination of the nanozyme obtained in Example 12 of the present invention;

[0043] Fig.13 Transmission electron microscopy image of the nanozyme obtained in Example 1 of the present invention;

[0044] Fig.14 Transmission electron microscopy image of the nanozyme obtained in Example 2 of the present invention;

[0045] Fig.15 Transmission electron microscopy image of the nanozyme obtained in Example 3 of the present invention;

[0046] Fig.16 Transmission electron microscopy image of the nanozyme obtained in Example 4 of the present invention;

[0047] Fig.17 Transmission electron microscopy image of the nanozyme obtained in Example 5 of the present invention;

[0048] Fig.18 Transmission electron microscopy image of the nanozyme obtained in Example 6 of the present invention;

[0049] Fig.19 Transmission electron microscopy image of the nanozyme obtained in Example 7 of the present invention;

[0050] Fig. 20 Transmission electron microscopy image of the nanozyme obtained in Example 8 of the present invention;

[0051] Fig.21 Transmission electron microscopy image of the nanozyme obtained in Example 9 of the present invention;

[0052] Fig. 22 Transmission electron microscopy image of the nanozyme obtained in Example 10 of the present invention;

[0053] Fig.23 Transmission electron microscopy image of the nanozyme obtained in Example 11 of the present invention;

[0054] Fig.24 Transmission electron microscopy image of the nanozyme obtained in Example 12 of the present invention;

[0055] Fig.25 Hydration particle size diagram of the nanozyme obtained in Example 7 of the present invention;

[0056] Fig.26 The test results of the test card prepared by the nanozymes obtained in Example 7, Example 4, and Example 3 of the present invention on whole blood samples;

[0057] Fig. 27 The test results of the commercially available fecal occult blood test kit on 0.5 ng / mL whole blood samples;

[0058] Fig.28 The detection results of pure water samples using the detection cards prepared with the nanozymes obtained in Examples 7, 4 and 3 of the present invention.

[0059] Fig.29 A graph showing the results of the nanozyme obtained in Example 7 of the present invention preventing and treating kiwifruit canker. DETAILED DESCRIPTION

[0060] In order to make the purpose, technical scheme and technical effect of the invention of this application clearer, the technical scheme in the embodiment of this application will be clearly and completely described below in conjunction with the drawings in the embodiment of this application. Obviously, the described embodiment is a part of the embodiment of this application, not all the embodiments. Based on the embodiments in this application, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of this application.

[0061] In view of the technical problems existing in the prior art, the present invention provides a method for preparing a nanozyme, comprising the following steps:

[0062] (1) adding a stabilizer to a preheated solvent, stirring until clear, and then performing ultrasonic treatment;

[0063] (2) After ultrasonic treatment, add iron salt and cobalt salt to step (1), stir until clear; then add alkali to adjust the pH of the solution, continue stirring until clear, and perform ultrasonic treatment again;

[0064] (3) After ultrasonication, the solution in step (2) is transferred to a reaction vessel for reaction;

[0065] (4) After the reaction, washing and separation are performed to obtain the product.

[0066] In a preferred embodiment of the present invention, in step (1), the stabilizer is a polymer compound, a surfactant or an amino acid dispersant; the solvent is pure water, ethanol, tetraethylene glycol, ethylene glycol or triethylene glycol;

[0067] In a preferred embodiment of the present invention, in step (1), the stabilizer is polyvinyl pyrrolidone, polyvinyl alcohol, polyethylene glycol, polyacrylic acid, poly(4-styrenesulfonic acid-co-maleic acid) sodium salt, sodium lauryl sulfate, glycine, cysteine, hexadecyltrimethylammonium bromide, hexadecyltrimethylammonium chloride or sodium citrate;

[0068] In a preferred embodiment of the present invention, in step (1), the mass ratio of the stabilizer to the volume ratio of the solvent is (2-16) g:

[0069] (10-50) mL; slowly add the stabilizer into the solvent three times;

[0070] In a preferred embodiment of the present invention, in step (1), the mass ratio of the stabilizer to the volume ratio of the solvent is (7-8) g: 50 mL;

[0071] In a preferred embodiment of the present invention, in step (1), the preheating temperature of the solvent is 40-70°C; the ultrasonic frequency is 30-40KHz, the ultrasonic power is 120-360W, and the ultrasonic time is 20-40min;

[0072] In a preferred embodiment of the present invention, in step (2), the iron salt is a divalent iron salt or a trivalent iron salt; the cobalt salt is a divalent cobalt salt; the base is sodium hydroxide, ammonia water or sodium acetate;

[0073] In a preferred embodiment of the present invention, in step (2), the iron salt is ferric chloride, ferrous chloride, ferrous sulfate, ferric nitrate or ferric acetylacetonate; the cobalt salt is cobalt chloride, cobalt bromide, cobalt iodide, cobalt sulfate or cobalt nitrate;

[0074] In a preferred embodiment of the present invention, in step (2), the molar ratio of Fe in the iron salt to Co in the cobalt salt is (1-4):1; the mass ratio of the stabilizer to the iron salt is (2-16):(1-2); after adding the alkali, the pH of the solution is adjusted to 8-13;

[0075] In a preferred embodiment of the present invention, in step (2), the mass ratio of the stabilizer to the iron salt is (7-8):(1-2);

[0076] In a preferred embodiment of the present invention, in step (2), the ultrasonic frequency is 30-40KHz, the ultrasonic power is 120-360W, the ultrasonic time is 1-3h, and the ultrasonic temperature is 18-35°C;

[0077] In a preferred embodiment of the present invention, in step (3), the reaction temperature is 120-250°C (for example, 120°C, 140°C, 160°C, 180°C, 200°C, 220°C or 240°C), and the reaction time is 5-24h (for example, 5h, 10h, 12h, 15h, 20h or 24h); the volume of the reaction solution is 10%-90% of the volume of the cavity in the reactor;

[0078] In a preferred embodiment of the present invention, in step (3), the reaction temperature is 180° C. and the reaction time is 10 h;

[0079] In a preferred embodiment of the present invention, in step (4), washing is performed by water washing or organic solvent washing; the separation method is centrifugation or external magnetic field adsorption;

[0080] In a preferred embodiment of the present invention, in step (4), the organic solvent is acetone, tetrahydrofuran, ethanol or methanol; the centrifugal speed is 7000-9000 rpm, and the centrifugal time is 5-10 min;

[0081] In a preferred embodiment of the present invention, in step (4), the obtained product is in the form of a dispersion or a lyophilized powder and is stored at room temperature without being protected from light.

[0082] The present invention provides a nanozyme, and the nanozyme is prepared by the preparation method.

[0083] In a preferred embodiment of the present invention, the nanozyme is dispersed in pure water and stored.

[0084] The present invention provides an application of the nanozyme in preparing an immunochromatography reagent.

[0085] The present invention provides an application of the nanozyme in preparing a reagent for detecting human hemoglobin.

[0086] In a preferred embodiment of the present invention, in the immunochromatographic test card prepared by the nanozyme, the conjugate pad is a nanozyme dilution labeled with a mouse monoclonal antibody to human hemoglobin;

[0087] When testing samples, after the sample solution is chromatographed to the quality control line, add DAB colorimetric solution at the test line and observe the results after 5 minutes.

[0088] In a preferred embodiment of the present invention, the immunochromatographic detection card prepared using the nanozyme has a detection limit of 0.05 ng / mL when detecting human hemoglobin.

[0089] The present invention provides an application of the nanozyme in preventing and treating plant canker.

[0090] The present invention provides an application of the nanozyme in preventing and treating kiwifruit canker.

[0091] In a preferred embodiment of the present invention, when preventing and treating kiwifruit canker, the nanozyme is dispersed in pure water to form a suspension with a concentration of ≥0.1 mg / mL, which is then applied to the affected part of the plant.

[0092] The following is a detailed description of a nanozyme of the present invention and its preparation method and application through specific examples.

[0093] Example 1

[0094] This embodiment provides a method for preparing a nanozyme, comprising the following steps:

[0095] (1) 2.62 g of stabilizer (glycine) was slowly added to 50 mL of solvent (triethylene glycol) preheated at 60° C. in three portions, stirred until clear, and then subjected to ultrasonic treatment (ultrasonic frequency of 35 kHz, ultrasonic power of 240 W, and ultrasonic time of 30 min);

[0096] (2) After ultrasound, add FeCl to step (1) 3 6H 2 O (1.89 g, 0.0070 mol), CoCl 2 6H 2 O (0.84 g, 0.0035 mol), stirred until clear; then added 0.7 mL of ammonia water, adjusted the solution pH to 13, continued stirring until clear, and then performed ultrasonic treatment again (ultrasonic frequency of 35 kHz, ultrasonic power of 240 W, ultrasonic time of 2 h), adding crushed ice every 15-30 min during ultrasonic treatment to keep the ultrasonic temperature at a low temperature (18-35 ° C);

[0097] (3) After ultrasonication, the solution of step (2) was transferred to a hydrothermal reactor for reaction at a temperature of 180° C. for 10 h;

[0098] (4) After the reaction, the solid was washed with pure water for three times and centrifuged at 8000 rpm for 5 min. Finally, the solid was resuspended in pure water to obtain the product.

[0099] Example 2

[0100] This embodiment provides a method for preparing a nanozyme, comprising the following steps:

[0101] (1) 7.88 g of stabilizer (glycine) was slowly added to 50 mL of solvent (triethylene glycol) preheated at 60° C. in three portions, stirred until clear, and then subjected to ultrasonic treatment (ultrasonic frequency of 35 kHz, ultrasonic power of 240 W, and ultrasonic time of 30 min);

[0102] (2) After ultrasound, add FeCl to step (1) 3 6H 2 O (1.89 g, 0.0070 mol), CoCl 2 6H 2 O (0.84 g, 0.0035 mol), stirred until clear; then added 0.7 mL of ammonia water, adjusted the solution pH to 13, continued stirring until clear, and then performed ultrasonic treatment again (ultrasonic frequency of 35 kHz, ultrasonic power of 240 W, ultrasonic time of 2 h), adding crushed ice every 15-30 min during ultrasonic treatment to keep the ultrasonic temperature at a low temperature (18-35 ° C);

[0103] (3) After ultrasonication, the solution of step (2) was transferred to a hydrothermal reactor for reaction at a temperature of 180° C. for 5 h;

[0104] (4) After the reaction, the solid was washed with pure water for three times and centrifuged at 8000 rpm for 5 min. Finally, the solid was resuspended in pure water to obtain the product.

[0105] Example 3

[0106] This embodiment provides a method for preparing a nanozyme, comprising the following steps:

[0107] (1) 7.88 g of stabilizer (poly(4-styrenesulfonic acid-co-maleic acid) sodium salt) was slowly added to 50 mL of solvent (triethylene glycol) preheated at 60° C. in three portions, stirred until clear, and then subjected to ultrasonic treatment (ultrasonic frequency of 35 kHz, ultrasonic power of 240 W, and ultrasonic time of 30 min);

[0108] (2) After ultrasound, add FeCl to step (1) 3 6H 2 O (1.89 g, 0.0070 mol), CoCl 2 6H 2 O (0.84 g, 0.0035 mol), stirred until clear; then added 0.7 mL of ammonia water, adjusted the solution pH to 13, continued stirring until clear, and then performed ultrasonic treatment again (ultrasonic frequency of 35 kHz, ultrasonic power of 240 W, ultrasonic time of 2 h), adding crushed ice every 15-30 min during ultrasonic treatment to keep the ultrasonic temperature at a low temperature (18-35 ° C);

[0109] (3) After ultrasonication, the solution of step (2) was transferred to a hydrothermal reactor for reaction at a temperature of 180° C. for 10 h;

[0110] (4) After the reaction, the solid was washed with pure water for three times and centrifuged at 8000 rpm for 5 min. Finally, the solid was resuspended in pure water to obtain the product.

[0111] Example 4

[0112] This embodiment provides a method for preparing a nanozyme, comprising the following steps:

[0113] (1) 7.88 g of stabilizer (glycine) was slowly added to 50 mL of solvent (triethylene glycol) preheated at 60° C. in three portions, stirred until clear, and then subjected to ultrasonic treatment (ultrasonic frequency of 35 kHz, ultrasonic power of 240 W, and ultrasonic time of 30 min);

[0114] (2) After ultrasound, add FeCl to step (1)3 6H 2 O (1.89 g, 0.0070 mol), CoCl 2 6H 2 O (0.84 g, 0.0035 mol), stirred until clear; then added 0.7 mL of ammonia water, adjusted the solution pH to 13, continued stirring until clear, and then performed ultrasonic treatment again (ultrasonic frequency of 35 kHz, ultrasonic power of 240 W, ultrasonic time of 2 h), adding crushed ice every 15-30 min during ultrasonic treatment to keep the ultrasonic temperature at a low temperature (18-35 ° C);

[0115] (3) After ultrasonication, the solution of step (2) was transferred to a hydrothermal reactor for reaction at a temperature of 200° C. for 10 h;

[0116] (4) After the reaction, the product is washed with pure water, separated by magnetic adsorption, and then washed with pure water again to obtain the product.

[0117] Example 5

[0118] This embodiment provides a method for preparing a nanozyme, comprising the following steps:

[0119] (1) Slowly add 7.88 g of stabilizer (polyvinyl pyrrolidone / PVP K30) into 50 mL of solvent (triethylene glycol) preheated at 60 °C three times, stir until clear, and then perform ultrasonic treatment (ultrasonic frequency of 35 kHz, ultrasonic power of 240 W, and ultrasonic time of 30 min);

[0120] (2) After ultrasound, add FeCl to step (1) 3 6H 2 O (1.89 g, 0.0070 mol), CoCl 2 6H 2 O (0.84 g, 0.0035 mol), stirred until clear; then added 0.7 mL of ammonia water, adjusted the solution pH to 13, continued stirring until clear, and then performed ultrasonic treatment again (ultrasonic frequency of 35 kHz, ultrasonic power of 240 W, ultrasonic time of 2 h), adding crushed ice every 15-30 min during ultrasonic treatment to keep the ultrasonic temperature at a low temperature (18-35 ° C);

[0121] (3) After ultrasonication, the solution of step (2) was transferred to a hydrothermal reactor for reaction at a temperature of 120° C. for 10 h;

[0122] (4) After the reaction, the product is washed with pure water, separated by magnetic adsorption, and then washed with pure water again to obtain the product.

[0123] Example 6

[0124] This embodiment provides a method for preparing a nanozyme, comprising the following steps:

[0125] (1) Slowly add 7.88 g of stabilizer (polyvinyl alcohol / PVA) into 50 mL of solvent (triethylene glycol) preheated at 60°C three times, stir until clear, and then perform ultrasonic treatment (ultrasonic frequency of 35 kHz, ultrasonic power of 240 W, and ultrasonic time of 30 min);

[0126] (2) After ultrasound, add FeCl to step (1) 3 6H 2 O (1.89 g, 0.0070 mol), CoCl 2 6H 2 O (0.84 g, 0.0035 mol), stirred until clear; then added 0.7 mL of ammonia water, adjusted the solution pH to 13, continued stirring until clear, and then performed ultrasonic treatment again (ultrasonic frequency of 35 kHz, ultrasonic power of 240 W, ultrasonic time of 2 h), adding crushed ice every 15-30 min during ultrasonic treatment to keep the ultrasonic temperature at a low temperature (18-35 ° C);

[0127] (3) After ultrasonication, the solution of step (2) was transferred to a hydrothermal reactor for reaction at a temperature of 180° C. for 10 h;

[0128] (4) After the reaction, the solid was washed with pure water for three times and centrifuged at 8000 rpm for 5 min. Finally, the solid was resuspended in pure water to obtain the product.

[0129] Example 7

[0130] This embodiment provides a method for preparing a nanozyme, comprising the following steps:

[0131] (1) 7.88 g of stabilizer (glycine) was slowly added to 50 mL of solvent (ethylene glycol) preheated at 60° C. in three portions, stirred until clear, and then subjected to ultrasonic treatment (ultrasonic frequency of 35 kHz, ultrasonic power of 240 W, and ultrasonic time of 30 min);

[0132] (2) After ultrasound, add FeCl to step (1) 3 6H 2 O (1.89 g, 0.0070 mol), CoCl 2 6H 2O (0.84 g, 0.0035 mol), stirred until clear; then added 0.7 mL of ammonia water, adjusted the solution pH to 13, continued stirring until clear, and then performed ultrasonic treatment again (ultrasonic frequency of 35 kHz, ultrasonic power of 240 W, ultrasonic time of 2 h), adding crushed ice every 15-30 min during ultrasonic treatment to keep the ultrasonic temperature at a low temperature (18-35 ° C);

[0133] (3) After ultrasonication, the solution of step (2) was transferred to a hydrothermal reactor for reaction at a temperature of 180° C. for 10 h;

[0134] (4) After the reaction, the solid was washed with pure water for three times and centrifuged at 8000 rpm for 5 min. Finally, the solid was resuspended in pure water to obtain the product.

[0135] Example 8

[0136] This embodiment provides a method for preparing a nanozyme, comprising the following steps:

[0137] (1) Slowly add 7.88 g of stabilizer (polyvinyl pyrrolidone / PVP K30) into 50 mL of solvent (triethylene glycol) preheated at 60 °C three times, stir until clear, and then perform ultrasonic treatment (ultrasonic frequency of 35 kHz, ultrasonic power of 240 W, and ultrasonic time of 30 min);

[0138] (2) After ultrasound, add FeCl to step (1) 3 6H 2 O (1.89 g, 0.0070 mol), CoCl 2 6H 2 O (0.84 g, 0.0035 mol), stirred until clear; then added 0.7 mL of ammonia water, adjusted the solution pH to 13, continued stirring until clear, and then performed ultrasonic treatment again (ultrasonic frequency of 35 kHz, ultrasonic power of 240 W, ultrasonic time of 2 h), adding crushed ice every 15-30 min during ultrasonic treatment to keep the ultrasonic temperature at a low temperature (18-35 ° C);

[0139] (3) After ultrasonication, the solution of step (2) was transferred to a hydrothermal reactor for reaction at a temperature of 180° C. for 10 h;

[0140] (4) After the reaction, the solid was washed with pure water for three times and centrifuged at 8000 rpm for 5 min. Finally, the solid was resuspended in pure water to obtain the product.

[0141] Example 9

[0142] This embodiment provides a method for preparing a nanozyme, comprising the following steps:

[0143] (1) 7.88 g of stabilizer (cysteine) was slowly added to 50 mL of solvent (triethylene glycol) preheated at 60° C. in three portions, stirred until clear, and then subjected to ultrasonic treatment (ultrasonic frequency of 35 kHz, ultrasonic power of 240 W, and ultrasonic time of 30 min);

[0144] (2) After ultrasound, add FeCl to step (1) 3 6H 2 O (1.89 g, 0.0070 mol), CoCl 2 6H 2 O (0.84 g, 0.0035 mol), stirred until clear; then added 0.7 mL of ammonia water, adjusted the solution pH to 13, continued stirring until clear, and then performed ultrasonic treatment again (ultrasonic frequency of 35 kHz, ultrasonic power of 240 W, ultrasonic time of 2 h), adding crushed ice every 15-30 min during ultrasonic treatment to keep the ultrasonic temperature at a low temperature (18-35 ° C);

[0145] (3) After ultrasonication, the solution of step (2) was transferred to a hydrothermal reactor for reaction at a temperature of 180° C. for 10 h;

[0146] (4) After the reaction, the solid was washed with pure water for three times and centrifuged at 8000 rpm for 5 min. Finally, the solid was resuspended in pure water to obtain the product.

[0147] Example 10

[0148] This embodiment provides a method for preparing a nanozyme, comprising the following steps:

[0149] (1) 7.88 g of stabilizer (glycine) was slowly added to 50 mL of solvent (triethylene glycol) preheated at 60° C. in three portions, stirred until clear, and then subjected to ultrasonic treatment (ultrasonic frequency of 35 kHz, ultrasonic power of 240 W, and ultrasonic time of 30 min);

[0150] (2) After ultrasound, add FeCl to step (1) 3 6H 2 O (1.89 g, 0.0070 mol), CoCl 2 6H 2 O (0.84 g, 0.0035 mol), stirred until clear; then added 0.7 mL of ammonia water, adjusted the solution pH to 13, continued stirring until clear, and then performed ultrasonic treatment again (ultrasonic frequency of 35 kHz, ultrasonic power of 200 W, ultrasonic time of 2 h), adding crushed ice every 15-30 min during ultrasonic treatment to keep the ultrasonic temperature at a low temperature (18-35 ° C);

[0151] (3) After ultrasonication, the solution of step (2) was transferred to a hydrothermal reactor for reaction at a temperature of 160° C. for 10 h;

[0152] (4) After the reaction, the solid was washed with pure water for three times and centrifuged at 8000 rpm for 5 min. Finally, the solid was resuspended in pure water to obtain the product.

[0153] Embodiment 11

[0154] This embodiment provides a method for preparing a nanozyme, comprising the following steps:

[0155] (1) Slowly add 7.88 g of stabilizer (cysteine) into 50 mL of solvent (pure water) preheated at 40° C. in three portions, stir until clear, and then perform ultrasonic treatment (ultrasonic frequency of 35 kHz, ultrasonic power of 240 W, and ultrasonic time of 30 min);

[0156] (2) After ultrasound, add FeCl to step (1) 3 6H 2 O (1.89 g, 0.0070 mol), CoCl 2 6H 2 O (0.84 g, 0.0035 mol), stirred until clear; ultrasonic treatment was performed again (ultrasonic frequency was 35 kHz, ultrasonic power was 240 W, and ultrasonic time was 30 min);

[0157] (3) After ultrasound, add 0.378 g NaBH 4 , react at 40℃ for 1h;

[0158] (4) After the reaction, the solid was washed with pure water for three times and centrifuged at 8000 rpm for 5 min. Finally, the solid was resuspended in pure water to obtain the product.

[0159] Example 12

[0160] This embodiment provides a method for preparing a nanozyme, comprising the following steps:

[0161] (1) Slowly add 7.88 g of stabilizer (glycine) into 50 mL of solvent (pure water) preheated at 40° C. in three portions, stir until clear, and then perform ultrasonic treatment (ultrasonic frequency of 35 kHz, ultrasonic power of 240 W, and ultrasonic time of 30 min);

[0162] (2) After ultrasound, add FeCl to step (1) 3 6H 2 O (1.89 g, 0.0070 mol), CoCl 2 6H 2O (0.84 g, 0.0035 mol), stirred until clear; ultrasonic treatment was performed again (ultrasonic frequency was 35 kHz, ultrasonic power was 240 W, and ultrasonic time was 30 min);

[0163] (3) After ultrasound, add 0.378 g NaBH 4 , react at 40℃ for 1h;

[0164] (4) After the reaction, the solid was washed with pure water for three times and centrifuged at 8000 rpm for 5 min. Finally, the solid was resuspended in pure water to obtain the product.

[0165] Performance measurement and analysis:

[0166] (1) Enzyme activity detection

[0167] The products obtained in Examples 1-12 of the present invention were respectively dispersed in pure water to obtain sample enzyme solutions to be tested, and the enzyme activity of the obtained nanozymes was detected according to the standard GB / T37966-2019 nanotechnology iron oxide nanoparticle peroxidase activity measurement method.

[0168] 2 mL of 0.2 M acetate buffer (pH 3.6), 100 μL of 30% (w / w) H 2 O 2 , 100 μL 10 mg / mL TMB DMSO solution were mixed with 100 μL enzyme solution of the test sample of different concentrations, and the absorbance value at 652 nm was measured at 37°C.

[0169] The peroxidase activity was calculated as follows: Where:

[0170] B nano : The number of nanozyme peroxidase activity units, unit is U;

[0171] V: total volume of reaction solution, in μL;

[0172] ε: molar absorption coefficient of TMB derivative, unit is 39000 mol -1 ·L·cm -1 ;

[0173] l: cuvette optical path, in cm;

[0174] ΔA / Δt: The rate of change of absorbance of reaction solutions of different concentrations over time, in cm -1 .

[0175] Then, according to the B corresponding to the enzyme solution of the sample with different concentrations nanoThe value was linearly fitted with the corresponding nanozyme mass, and the slope of the fitting line (k) was the measured peroxidase specific activity of the nanozyme, in U / mg. Figure 1-12 As shown in Table 1.

[0176] Table 1 Enzyme activity of nanozymes obtained in Examples 1-12 of the present invention

[0177] Group Enzyme activity (U / mg) Group Enzyme activity (U / mg) Group Enzyme activity (U / mg) Example 1 6.99 Example 5 5.30 Example 9 4.05 Example 2 5.63 Example 6 4.57 Example 10 5.90 Example 3 6.15 Example 7 9.56 Embodiment 11 5.62 Example 4 7.76 Example 8 5.77 Example 12 6.05

[0178] As shown in Table 1, the nanozyme prepared by using glycine as a stabilizer at 180°C for 10 h in Example 7 has the best enzyme activity, which is significantly higher than that of the nanozymes prepared by using other stabilizers. The main reason for this phenomenon may be that glycine contains both -COOH and -NH 2 , and has good thermal stability and tolerance to alkaline environments, so that in the process of nanozyme formation, it has better dispersibility and stability, and the particle size is more uniform, so that the activity of the nanozyme is maximized, which can be proved by TEM. 2 However, because it is sensitive to oxygen and unstable in an alkaline environment, it may decompose under high temperature and high pressure reaction conditions, resulting in low activity of the final prepared nanozyme. This can be proved by the fact that the enzyme activity is improved under the condition of 40°C without adding alkali in Example 11. Polyvinyl pyrrolidone and polyvinyl alcohol do not contain -COOH and -NH 2 And because of its film-forming property, a complete closed film will be formed on the surface of the nanozyme, causing the enzyme active site to be partially covered, making the enzyme activity slightly lower.

[0179] (2) Transmission electron microscopy analysis

[0180] Table 2 TEM particle size distribution of nanozymes obtained in Examples 1-12 of the present invention

[0181] Group <![CDATA[D min (nm)]]> <![CDATA[D max (nm)]]> Group <![CDATA[D min (nm)]]> <![CDATA[D max (nm)]]> Group <![CDATA[D min (nm)]]> <![CDATA[D max (nm)]]> Example 1 109.76 151.81 Example 5 65.39 153.70 Example 9 119.90 167.38 Example 2 103.49 165.95 Example 6 83.64 242.17 Example 10 85.58 242.68 Example 3 117.86 171.80 Example 7 77.87 108.99 Embodiment 11 57.75 132.89 Example 4 102.70 178.95 Example 8 102.59 181.96 Example 12 71.34 155.51

[0182] As shown in Table 2, the nanozyme prepared in Example 7 has the smallest particle size deviation and the best uniformity. The products obtained in Examples 1-12 were analyzed by transmission electron microscopy. The results are as follows: Figure 13-24 shown.

[0183] In the solvothermal reaction, the formation process of nanoparticles is divided into three stages. In the first stage (saturation), as the temperature rises, the solvent vaporizes, and the monomer concentration in the system gradually increases to a saturated state. No nanoparticles are generated in this process; in the second stage (nucleation), the monomer concentration is supersaturated, and after reaching a certain level, it begins to gradually precipitate nuclei. After that, the monomer concentration begins to decrease, and after reaching a certain level, nucleation stops; in the third stage (growth), further growth begins after nucleation. Among them, due to the difference in nucleation time, the second and third stages will proceed simultaneously.

[0184] Compared with the best embodiment 7, the types of stabilizers are screened in embodiments 3, 6, 8, and 9. As can be seen from the figure, the uniformity of the products obtained by the stabilizers used in embodiments 3, 6, 8, and 9 under the same conditions is quite different from that of embodiment 7. This is because the stabilizer can help the nucleus to disperse evenly during the growth process without forming aggregation, and the formed nanoparticles can achieve the purpose of increasing dispersibility through electrostatic repulsion due to the same electrical properties, while different stabilizers have different stabilizing effects due to their own unique physical and chemical properties (such as high temperature resistance, chemical bonding mode, whether they have active groups, etc.), which leads to different particle sizes and uniformity of nanoparticles.

[0185] Compared with the best embodiment 7, embodiments 1, 2, 4 and 10 respectively make corresponding changes to the dosage of the same stabilizer (glycine), reaction time and reaction temperature. For example, in embodiment 1, the insufficient amount of stabilizer causes the nuclei formed during the reaction to aggregate, and the regrowth after aggregation will lead to an increase in particle size, and the difference in the degree of aggregation will further lead to poor uniformity of the final nanozyme particle size; in embodiment 2, the reaction time is shortened. Due to the difference in the "nucleation" time, shortening the reaction time will lead to insufficient growth of the later nucleated particles, resulting in a large difference in particle size. In the figure, the minimum particle size is about 103.49nm, and the maximum particle size is about 165.95nm, with a large deviation; in embodiments 4 and 10, the reaction temperature is changed. Since triethylene glycol has a high boiling point, the solution is at a lower temperature (embodiment 10). The vaporization process of the agent becomes slow, and an effective high-pressure environment cannot be formed to provide sufficient energy for "nucleation", so that the change of the monomer concentration in the system is relatively slow, which greatly prolongs the time of the nucleation stage, so that the first nucleated has grown to a certain extent, while the growth of the later nucleated is insufficient. In the figure, the minimum particle size is about 85.58nm, and the maximum particle size is about 242.68nm, which can also be verified from Examples 5 and 8; and too high a temperature (Example 4) will damage the crystal structure of the nanoparticles, so that the enzyme active site is destroyed. Although the difference cannot be intuitively seen from the morphology, the obvious reduction in enzyme activity can illustrate this point.

[0186] In Example 11 and Example 12, NaBH 4Reduction under low temperature conditions has the advantages of low reaction temperature and short reaction time compared to the solvothermal method. However, the solvothermal method is in a state of high temperature and high pressure during the reaction process, which makes the crystallization of nanoparticles more uniform and the dispersion better. Therefore, the activity of nanozymes prepared by the solvothermal method is generally higher than that of NaBH 4 The nanozyme prepared by reduction was shown in Figure 1, but the activity of the nanozyme prepared in Example 11 was higher than that in Example 9, which may be mainly due to the instability of cysteine ​​in an alkaline environment.

[0187] (3) Hydration particle size analysis

[0188] The product obtained in Example 7 of the present invention was subjected to hydration particle size analysis. The results are as follows Fig.25 .

[0189] according to Fig.25 It can be seen that the particle size distribution of the nanozyme prepared in Example 7 is relatively uniform, and its hydrated particle size is 136.53±6.00nm, which is slightly increased compared to 98.38±12.03nm measured by TEM. This is a normal phenomenon because DLS measures the state of particles in solution, including the hydration layer formed by the interaction between particles and solvent molecules.

[0190] Application Example 1

[0191] Preparation of nanozyme dilution: Disperse the nanozymes obtained in Example 7, Example 4, and Example 3 with higher enzyme activity in water to obtain a nanozyme dilution with a concentration of 0.5 mg / mL for later use;

[0192] Based on immunochromatographic technology, the nanozymes obtained in Examples 7, 4 and 3 of the present invention were used to prepare immunochromatographic detection cards, and human hemoglobin was detected. The preparation method of the detection card is as follows: the absorbent pad and the nitrocellulose membrane are overlapped by 2 mm and sequentially pasted on the PCV bottom plate to make a test paper; the quality control line goat anti-rabbit IgG secondary antibody and human hemoglobin (Hb) mouse monoclonal antibody (capture antibody) were diluted to 1 mg / mL with pH7.4, 10mM phosphate buffer (PB), and the antibody was coated on the nitrocellulose membrane of the test paper using a film stripper, and a nanozyme dilution labeled with 1 mg / mL human hemoglobin mouse monoclonal antibody (labeled antibody) was added to the conjugate pad, and placed in a 37°C oven for drying for 30 min, and then the test paper was cut into 4 mm wide test strips using a strip cutter, and a desiccant was added to seal for standby use. Among them, the detection area is the NC membrane reaction area coated with human hemoglobin (Hb) mouse monoclonal antibody (capture antibody), which serves as the T line, and the quality control area is coated with goat anti-rabbit IgG secondary antibody, which serves as the C line.

[0193] (1) Whole blood sample testing:

[0194] Whole blood samples with concentrations of 0.05 ng / mL, 0.5 ng / mL, 5 ng / mL, 50 ng / mL, 100 ng / mL, and 500 ng / mL were used as test objects, and the immunochromatographic detection cards prepared using the nanozymes obtained in Example 7, Example 4, and Example 3 were used to measure 85 μL of whole blood samples. After adding the sample, when the sample solution was chromatographed to the C line, a drop of 3,3-diaminobenzidine (DAB) colorimetric solution was added to the T line, and the results were observed after 5 minutes. The results are as follows: Fig.26 As shown (where Fig.26 Among them, number one is embodiment 7, number two is embodiment 4, and number three is embodiment 3).

[0195] DAB colorimetric solution: DAB 50 mg + 0.05 mol / L Tris buffer (pH 7.6) 100 mL + 30% H 2 O 2 40μL.

[0196] according to Fig.26 It can be seen that for whole blood samples of different concentrations, the immunochromatographic detection cards prepared by the nanozymes obtained in Example 7, Example 4, and Example 3 are all positive; among them, the color development for the whole blood sample of 0.05ng / mL is slightly weaker, but still visible to the naked eye. It can be seen that the detection limit of the immunochromatographic detection card prepared by the nanozyme obtained in the present invention can reach 0.05ng / mL. In addition, using the commercially available fecal occult blood detection kit (colloidal gold method) as a control, the whole blood sample with the above concentration of 0.5ng / mL was tested according to the operating method in its instruction manual, and the results were observed after 5 minutes. The results are as follows Fig. 27 As shown. Fig. 27 It can be seen that the T line does not show color and the test is negative.

[0197] By comparison, it can be seen that the detection limit of the immunochromatographic detection card prepared by the nanozyme obtained in the embodiment of the present invention is reduced by more than 10 times compared with the conventional detection reagents available on the market.

[0198] (2) Pure water sample testing:

[0199] The immunochromatographic detection cards prepared by the nanozymes obtained in Example 7, Example 4, and Example 3 were used to measure 85 μL of purified water. The results were observed after 5 minutes. Fig. 22 As shown (where Fig.28 Among them, number one is embodiment 7, number two is embodiment 4, and number three is embodiment 3).

[0200] according to Fig.28 It can be seen that the T line in the test card corresponding to Example 4 is obviously colored, and a false positive is detected; the T line in the test card corresponding to Example 3 is slightly colored, and a false positive is also detected; and the T line in the test card corresponding to Example 7 is not colored, which is normal.

[0201] In summary, the nanozyme prepared in Example 7 of the present invention has a detection limit at least 10 times lower than that of the commercially available fecal occult blood detection kit (colloidal gold method), and there is no false positive. The commercially available kit cannot effectively detect 0.5 ng / mL whole blood samples, while the nanozyme prepared in the present invention can also be effectively detected at 0.05 ng / mL whole blood samples, which greatly improves the sensitivity of the detection and has more accurate detection results.

[0202] Application Example 2

[0203] The peak period of kiwifruit canker is from the end of February to the beginning of March each year. As the temperature rises, the kiwifruit skin ulcers and flossing caused by the canker will decrease in plants that are in the final stage of infection. However, the pathogen has already invaded the branches and trunks, gradually causing some branches or the entire kiwifruit tree to wither and die.

[0204] This experiment adopted a smearing method to conduct a prevention and control experiment. The experiment was conducted on kiwifruit plants in the same garden that were suffering from ulcer disease and were in the final stage of ulcer disease infection. The nanozyme obtained in Example 7 of the present invention was dispersed in pure water, and the reagents with concentrations of 1.0 mg / mL, 0.5 mg / mL, 0.1 mg / mL, 0.05 mg / mL, and 0.01 mg / mL were diluted respectively, and directly applied to the kiwifruit ulcer affected area, and reapplied once every 15 days. The status of the experimental plants at 0 days, 15 days, and 30 days was recorded, and the ulcer disease plants treated with only pure water were used for comparison as a blank control group. The experimental results are as follows: Fig.29 shown.

[0205] The experimental results show that after 15 days of treatment with different concentrations of nanozymes, only the 1 mg / mL treatment group showed obvious signs of improvement in the erosion wounds of the diseased plants compared with the control group. After re-application at 15 days, 0.5 mg / mL and 0.1 mg / mL showed signs of epidermal recovery and improvement after 30 days compared with the control group, but the other concentration groups did not have obvious epidermal recovery effects. In general, the diseased plants treated with nanozymes no longer showed large-scale erosion and dieback, and the scope of ulcer disease was basically controlled. From the observation, the epidermal healing and regeneration phenomenon was more obvious in the diseased parts of the plants smeared with nanozyme suspension. Therefore, the nanozyme of the present invention is dispersed in pure water and configured into a suspension with a concentration greater than or equal to 0.1 mg / mL, which can achieve a good prevention and treatment effect on ulcer disease.

[0206] Obviously, the above embodiments are merely examples for the purpose of clear explanation, and are not intended to limit the implementation methods. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation methods here. The obvious changes or modifications derived therefrom are still within the scope of protection of the claims of this patent application.

Claims

1. A method for preparing a nanozyme, It is characterized in that The following steps are involved: (1) adding a stabilizer to a preheated solvent, stirring until clear, and then performing ultrasonic treatment; (2) After ultrasonic treatment, add iron salt and cobalt salt to step (1), stir until clear; then add alkali to adjust the pH of the solution, continue stirring until clear, and then perform ultrasonic treatment again; (3) After ultrasonication, the solution in step (2) is transferred to a reaction vessel for reaction; (4) After the reaction, washing and separation are performed to obtain the product.

2. The preparation method according to claim 1, It is characterized in that In step (1), the stabilizer is a polymer compound, a surfactant or an amino acid dispersant; the solvent is pure water, ethanol, tetraethylene glycol, ethylene glycol or triethylene glycol; The preheating temperature of the solvent is 40-70°C; the ultrasonic frequency is 30-40KHz, the ultrasonic power is 120-360W, and the ultrasonic time is 20-40min.

3. The preparation method according to claim 1, It is characterized in that In step (1), the stabilizer is polyvinyl pyrrolidone, polyvinyl alcohol, polyethylene glycol, polyacrylic acid, poly(4-styrenesulfonic acid-co-maleic acid) sodium salt, sodium lauryl sulfate, glycine, cysteine, hexadecyltrimethylammonium bromide, hexadecyltrimethylammonium chloride or sodium citrate; The mass ratio of the stabilizer to the volume ratio of the solvent is (2-16) g: (10-50) mL.

4. The preparation method according to claim 1, It is characterized in that In step (2), the iron salt is a divalent iron salt or a trivalent iron salt; the cobalt salt is a divalent cobalt salt; and the base is sodium hydroxide, ammonia water or sodium acetate; When ultrasonication is performed again, the ultrasonic frequency is 30-40KHz, the ultrasonic power is 120-360W, the ultrasonic time is 1-3h, and the ultrasonic temperature is 18-35°C.

5. The preparation method according to claim 4, It is characterized in that In step (2), the iron salt is ferric chloride, ferrous chloride, ferrous sulfate, ferric nitrate or ferric acetylacetonate; the cobalt salt is cobalt chloride, cobalt bromide, cobalt iodide, cobalt sulfate or cobalt nitrate; The molar ratio of Fe in the iron salt to Co in the cobalt salt is (1-4):1; the mass ratio of the stabilizer to the iron salt is (2-16):(1-2); and the pH of the solution is adjusted to 8-13 after adding alkali.

6. The preparation method according to claim 1, It is characterized in that In step (3), the reaction temperature is 120-250° C. and the reaction time is 5-24 h; In step (4), washing is performed with water or an organic solvent; the separation method is centrifugation or external magnetic field adsorption; the organic solvent is acetone, tetrahydrofuran, ethanol or methanol; the centrifugal speed is 7000-9000rpm, and the centrifugal time is 5-10min.

7. A nanozyme, It is characterized in that The nanozyme is prepared by the preparation method according to any one of claims 1 to 6.

8. Use of the nanozyme as claimed in claim 7 in the preparation of immunochromatography reagents.

9. The use according to claim 8, It is characterized in that In the immunochromatographic test card prepared by the nanozyme, the conjugate pad is a nanozyme dilution labeled with a mouse monoclonal antibody to human hemoglobin; When testing samples, after the sample solution is chromatographed to the quality control line, add DAB colorimetric solution at the test line and observe the results after 5 minutes.

10. Use of the nanozyme as claimed in claim 7 in preventing and treating plant ulcer disease.

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