Nanometer enzyme, preparation method, detection method, test method, device and application thereof

Abstract

The invention relates to a nano-enzyme, a preparation method, a detection method, a test method, a device and application thereof, and relates to the technical field of nano-enzymes. The preparation method comprises the following steps: respectively obtaining a Mn (NO3) 2 stirring solution and an aminoethanol stirring solution, and mixing the Mn (NO3) 2 stirring solution and the aminoethanol stirring solution to obtain a mixed solution; stirring the mixed solution in an inert atmosphere according to a set rate and a third set time to obtain a stirred mixed solution; performing aging treatment on the stirred and mixed solution according to a fourth set time to obtain a corresponding suspension; and carrying out suction filtration, washing and drying treatment on the suspension to obtain the Mn3O4 oxidase. According to the embodiment of the invention, preparation, detection, testing and application of the nano-enzyme can be realized.

Description

Technical Field

[0001] The present disclosure relates to the field of nanozyme technology, and in particular to a nanozyme and a preparation, detection, testing method, device and application thereof. Background Art

[0002] Amino acids are important biological molecules in the human body. They can not only assemble proteins, but also have important physiological significance when they exist alone. They are not only related to gene expression, but also regulators of protein phosphorylation cascade reactions. Amino acids in normal human blood and urine maintain relatively stable content and metabolic state, and their intake and excretion should be balanced. Cysteine ​​is an important essential amino acid that participates in many physiological processes in the human body, including signal transduction, protein biosynthesis, phospholipid metabolism, etc. Imbalance in cysteine ​​levels can lead to damage to various tissues. Therefore, quantitative detection of cysteine ​​is of great significance for the diagnosis and treatment of diseases.

[0003] Currently, fluorescence spectroscopy, chemiluminescence, and chromatography have been used to detect cysteine, but they have limitations such as high cost, complex sample pretreatment, and the need for professionals and large instruments during the operation process, and are not suitable for routine analysis of cysteine. The colorimetric method reduces these limitations and adds advantages such as low cost, fast reaction, and easy-to-operate instruments, but the low detection sensitivity of this method limits its widespread application. Therefore, it is of great significance to develop a highly sensitive cysteine ​​detection method based on nanozymes.

[0004] Nanozyme is a nanomaterial with enzyme-like catalytic properties. It has the advantages of convenient storage, simple preparation, and easy control. It has been widely used in biology, medicine, environment, food and other fields. Most of the current nanozyme materials are based on peroxidase, and the sensitivity is low. Since the catalytic process of peroxidase-like nanozymes requires the participation of hydrogen peroxide, and hydrogen peroxide is unstable, its application in actual cysteine ​​detection will greatly affect the accuracy of the detection. Therefore, it is extremely important to develop a simple nanozyme preparation method to obtain efficient oxidase-like nanozymes. Summary of the invention

[0005] The present invention discloses a nanozyme and a technical solution for its preparation, detection, testing method, device and application.

[0006] According to one aspect of the present disclosure, there is provided a method for preparing a nanozyme, comprising:

[0007] Obtaining a Mn(NO3)2 stirring solution and an aminoethanol stirring solution respectively, and mixing the Mn(NO3)2 stirring solution and the aminoethanol stirring solution to obtain a mixed solution;

[0008] Under an inert atmosphere, stirring the mixed solution at a set rate and for a third set time to obtain a stirred mixed solution;

[0009] Performing an aging treatment on the stirred mixed solution according to a fourth set time to obtain a corresponding suspension;

[0010] The suspension is filtered, washed and dried to obtain the Mn3O4 oxidase.

[0011] Preferably, before obtaining the Mn(NO3)2 stirring solution and the aminoethanol stirring solution, a Mn(NO3)2 solution corresponding to a first set volume and a first set concentration and an aminoethanol solution corresponding to a second set volume and a second set concentration are prepared respectively; under the inert atmosphere, the Mn(NO3)2 solution and the aminoethanol solution are stirred for a first set time and a second time respectively to obtain the corresponding Mn(NO3)2 stirring solution and aminoethanol stirring solution.

[0012] Preferably, the first set volume and the second set volume have the same numerical value, and the second set concentration has a numerical value that is twice the numerical value of the first set concentration.

[0013] Preferably, the first set volume and the first set concentration are configured as 10 ml and 0.4 mM respectively, and the second set volume and the second set concentration are configured as 10 ml and 0.8 mM respectively.

[0014] Preferably, the third set time can be configured as 3 hours; and / or, the fourth set time can be configured as 24 hours; and / or, the inert atmosphere can be configured as an argon atmosphere.

[0015] Preferably, the method of filtering, washing and drying the suspension to obtain a Mn3O4-type oxidase comprises: filtering and washing the suspension, and drying the filtered and washed suspension at a first set temperature and a fifth set time to obtain a Mn3O4-type oxidase.

[0016] Preferably, the first set temperature is configured to be 60° C.; and / or the fifth set time is configured to be 24 hours.

[0017] Preferably, the method of performing aging treatment on the stirred mixed solution according to a fourth set time to obtain a corresponding suspension includes: detecting a first color corresponding to the suspension in real time within the fourth set time; if the first color turns into gray-brown, it is determined that the aging treatment of the stirred mixed solution is completed and the corresponding suspension is obtained.

[0018] Preferably, the method for determining that the aging treatment of the stirred mixed solution is completed if the color turns into gray-brown includes: if the color turns into gray-brown, obtaining a set delay time; after the set delay time, determining that the aging treatment of the stirred mixed solution is completed; and / or, the method for real-time detection of the first color corresponding to the suspension, if the first color turns into gray-brown, determining that the aging treatment of the stirred mixed solution is completed, includes: using a camera or a video camera to obtain a suspension image corresponding to the suspension; performing color extraction on the suspension image; if the extracted color is the same as the set gray-brown, determining that the aging treatment of the stirred mixed solution is completed.

[0019] Preferably, the method for color extraction of the suspension image comprises: using a preset threshold or a preset segmentation model to segment the suspended matter in the suspension image to obtain a suspended matter image; further, performing color extraction on the suspended matter image; if the extracted first color is the same as the set gray-brown color, it is determined that the aging treatment of the stirred mixed solution is completed. Wherein, the method for determining that the aging treatment of the stirred mixed solution is completed if the extracted first color is the same as the set gray-brown color comprises: parsing the first RGB values ​​corresponding to the three channels corresponding to the extracted first color; if the RGB values ​​corresponding to the first three channels are the same as the RGB values ​​corresponding to the three channels corresponding to the gray-brown color or if the deviation between the RGB values ​​corresponding to the first three channels and the RGB values ​​corresponding to the three channels corresponding to the gray-brown color is within a set range, it is determined that the aging treatment of the stirred mixed solution is completed.

[0020] According to one aspect of the present disclosure, there is provided a nanozyme preparation device, comprising:

[0021] The first mixing and stirring unit is used to obtain a Mn(NO3)2 stirring solution and an aminoethanol stirring solution respectively, and mix the Mn(NO3)2 stirring solution and the aminoethanol stirring solution to obtain a mixed solution; the second mixing and stirring unit is used to stir the mixed solution at a set rate and a third set time under an inert atmosphere to obtain a stirred mixed solution; the aging treatment unit is used to perform an aging treatment on the stirred mixed solution according to a fourth set time to obtain a corresponding suspension; the filtration, washing and drying treatment unit is used to perform a filtration, washing and drying treatment on the suspension to obtain a Mn3O4-type oxidase.

[0022] According to one aspect of the present disclosure, a nanozyme preparation device is provided, comprising: a processor; a memory for storing instructions executable by the processor; wherein the processor is configured to call the instructions stored in the memory to execute the above-mentioned nanozyme preparation method.

[0023] According to one aspect of the present disclosure, a nanozyme preparation device is provided, comprising: a computer-readable storage medium having computer program instructions stored thereon, wherein the computer program instructions implement the above-mentioned nanozyme preparation method when executed by a processor.

[0024] According to one aspect of the present disclosure, there is provided a nanozyme preparation device, comprising: a computer program product, comprising a computer program / instructions, which implement the above-mentioned nanozyme preparation method when executed by a processor.

[0025] According to one aspect of the present disclosure, an electronic device is provided, comprising: a processor; and a memory for storing instructions executable by the processor; wherein the processor is configured to execute the above-mentioned nanozyme preparation method.

[0026] According to one aspect of the present disclosure, a computer-readable storage medium is provided, on which computer program instructions are stored, and when the computer program instructions are executed by a processor, the above-mentioned nanozyme preparation method is implemented.

[0027] According to one aspect of the present disclosure, a computer program product is provided, comprising a computer program / instruction, which implements the above-mentioned nanozyme preparation method when executed by a processor.

[0028] According to one aspect of the present disclosure, a nanozyme is provided, which is prepared using the nanozyme preparation method as described above or the nanozyme preparation device as described above.

[0029] According to one aspect of the present disclosure, there is provided a method for detecting the activity and / or enzyme kinetics and / or oxidase-like activity mechanism of a nanozyme obtained by the nanozyme preparation method as described above or a nanozyme prepared by the nanozyme preparation device as described above or a nanozyme as described above;

[0030] Among them, the activity detection of the nanozyme includes: obtaining a Mn3O4-type oxidase solution with a third set concentration and a 3,3',5,5'-tetramethylbenzidine and dimethyl sulfoxide solution with a fourth set concentration; adding a 3,3',5,5'-tetramethylbenzidine and dimethyl sulfoxide solution corresponding to a sixth set concentration less than the fourth set concentration and a Mn3O4-type oxidase solution corresponding to a seventh set concentration less than the third set concentration to a NaAc-HAc buffer solution with a third set volume, a fifth set concentration and a set pH value to obtain a solution to be detected; after placing the solution to be detected for a sixth set time at a second set temperature, detecting the light absorption value of the reaction system corresponding to the solution to be detected by an enzyme marker at a set wavelength; detecting the activity of the Mn3O4-type oxidase based on the color corresponding to the solution to be detected and / or the light absorption value at the set wavelength; and / or,

[0031] Wherein, the enzyme kinetics detection method includes: obtaining a Mn3O4-type oxidase solution of a third set concentration and 3,3',5,5'-tetramethylbenzidine and dimethyl sulfoxide solutions of different set concentrations within the third set concentration range; adding a 3,3',5,5'-tetramethylbenzidine and dimethyl sulfoxide solution corresponding to a sixth set concentration less than the fourth set concentration and a Mn3O4-type oxidase solution corresponding to different set concentrations within the third set concentration range less than the third set concentration to a NaAc-HAc buffer solution of a third set volume, a fifth set concentration and a set pH value, to obtain a solution to be detected with different set concentrations within the third set concentration range; respectively determining a plurality of reaction rates corresponding to different set concentrations of 3,3',5,5'-tetramethylbenzidine within the third set concentration range in the solution to be detected; using the Michaelis-Menten equation, fitting the different set concentrations of 3,3',5,5'-tetramethylbenzidine within the third set concentration range and the corresponding plurality of reaction rates, to determine a velocity curve of the relationship between the plurality of reaction rates of the enzymatic reaction and the different set concentrations within the third set concentration range; and / or,

[0032] Among them, the method for detecting the mechanism of oxidase activity includes: using electron paramagnetic energy spectrum, using 5,5-dimethyl-1-pyrroline oxide as a specific probe to capture the reaction intermediates in the color development process of catalyzing 3,3',5,5'-tetramethylbenzidine, and measuring the resonance spectrum; based on the resonance spectrum, determining the reaction intermediates.

[0033] Preferably, before obtaining the Mn3O4-type oxidase solution of the third set concentration, a first mass of a powder sample of the Mn3O4-type oxidase is weighed, and the powder sample is dispersed in ultrapure water to prepare the Mn3O4-type oxidase solution of the third set concentration.

[0034] Preferably, before obtaining the 3,3',5,5'-tetramethylbenzidine and dimethyl sulfoxide solution of the fourth set concentration, a second mass of 3,3',5,5'-tetramethylbenzidine powder is weighed and the powder is dissolved in dimethyl sulfoxide to prepare the 3,3',5,5'-tetramethylbenzidine and dimethyl sulfoxide solution of the fourth set concentration.

[0035] Preferably, the third set concentration is configured as 1 mg / mL; and / or the fourth set concentration is configured as 10 mg / mL.

[0036] Preferably, the fourth set concentration is any value between 5 and 12 times the third set concentration.

[0037] Preferably, the sixth set concentration is configured to 25 μg / mL or 110 μg / mL; and / or, the seventh set concentration is configured to 20 μg / mL; and / or, the method for determining the seventh set concentration includes: obtaining a Mn3O4-type oxidase solution corresponding to a first different set concentration within a first set concentration range; determining the different colors of the solution to be detected corresponding to the Mn3O4-type oxidase solution corresponding to the first different set concentrations; and configuring the concentration corresponding to the darkest color among the first different colors or the concentration corresponding to the adjacent colors among the first different colors that remain unchanged as the seventh set concentration.

[0038] Preferably, the method for determining the different colors of the solution to be detected corresponding to the Mn3O4-type oxidase solution corresponding to the different set concentrations includes: adding 3,3',5,5'-tetramethylbenzidine and dimethyl sulfoxide solutions corresponding to a sixth set concentration less than the fourth set concentration and Mn3O4-type oxidase solutions corresponding to different set concentrations less than the three set concentrations to the third set volume, fifth set concentration and set pH value NaAc-HAc buffer solution, respectively, to obtain the solution to be detected corresponding to the Mn3O4-type oxidase solution of the first different set concentration; and respectively determine the different colors of the solution to be detected corresponding to the Mn3O4-type oxidase solution of the first different set concentration.

[0039] Preferably, the method for determining the sixth set concentration includes: obtaining 3,3',5,5'-tetramethylbenzidine and dimethyl sulfoxide solutions corresponding to the second different set concentrations within the second set concentration range; preparing the test solutions of 3,3',5,5'-tetramethylbenzidine and dimethyl sulfoxide solutions corresponding to the second different set concentrations under the Mn3O4-type oxidase solution of the seventh set concentration; at the second set temperature, after placing the test solutions corresponding to the second different set concentrations for a sixth set time, detecting multiple light absorption values ​​of the reaction system corresponding to the solution to be detected for activity at a set wavelength by using an enzyme reader; configuring the set concentration corresponding to the maximum peak value of the multiple light absorption values ​​corresponding to the second different set concentrations as the sixth set concentration; or, at the second set temperature, after placing the test solutions corresponding to the second different set concentrations for a sixth set time, determining multiple colors corresponding to the second different set concentrations; configuring the set concentration corresponding to the darkest color among the multiple colors corresponding to the second different set concentrations, or the color that remains unchanged due to the adjacent colors, or the color that is green as the sixth set concentration.

[0040] Preferably, the method for preparing the test solutions of 3,3',5,5'-tetramethylbenzidine and dimethyl sulfoxide solutions corresponding to the second different set concentrations under the Mn3O4-type oxidase solution of the seventh set concentration, comprises: adding 3,3',5,5'-tetramethylbenzidine and dimethyl sulfoxide solutions corresponding to the second different concentrations less than the fourth set concentration and Mn3O4-type oxidase solutions corresponding to the seventh set concentration less than the third set concentration to NaAc-HAc buffer of the third set volume, the fifth set concentration and the set pH value, respectively, to obtain the test solutions corresponding to the Mn3O4-type oxidase solutions 3,3',5,5'-tetramethylbenzidine and dimethyl sulfoxide solutions of the second different set concentrations; and respectively determining the different colors corresponding to the test solutions corresponding to the 3,3',5,5'-tetramethylbenzidine and dimethyl sulfoxide solutions of the second different set concentrations.

[0041] Preferably, the method for determining the set pH value comprises: obtaining NaAc-HAc buffer solutions corresponding to different pH values ​​within the set pH value range; determining a plurality of color development values ​​corresponding to the NaAc-HAc buffer solutions with different pH values; configuring the pH value corresponding to the best color development among the plurality of color development values ​​as the set pH value; and / or,

[0042] Preferably, the method for determining the sixth set time includes: determining multiple colors corresponding to the solution to be detected at multiple set time points at the second set temperature; and configuring the set time point corresponding to the color tending to be stable among the multiple colors as the sixth set time.

[0043] Preferably, the method for determining the second set temperature includes: after placing the solution to be detected for a sixth set time at multiple set temperatures, detecting multiple light absorption values ​​of the reaction system corresponding to the solution to be detected for activity at a set wavelength by an enzyme marker; and configuring the set temperature corresponding to any two indifferent light absorption values ​​among the multiple light absorption values ​​as the second set temperature.

[0044] Preferably, the third set volume is configured to 1 mL; and / or the fifth set concentration is configured to 0.1 M or 0.2 M; and / or the set pH value is configured to 3.5; and / or the second set temperature is configured to room temperature or 5°C or 25°C; and / or the sixth set time is configured to 5 minutes; and / or the set wavelength is configured to 652 nm.

[0045] Preferably, before detecting the light absorption value of the reaction system corresponding to the solution to be detected for activity at a set wavelength by using an enzyme-labeled instrument, it also includes: aspirating a fourth set volume of the solution to be detected, adding the fourth set volume of the solution to be detected into a well plate, and detecting the light absorption value of the reaction system corresponding to the solution to be detected for activity at a set wavelength by using an enzyme-labeled instrument; and / or, the fourth set volume is configured to be 200 μL.

[0046] Preferably, the method for detecting the activity of the Mn3O4-type oxidase based on the color corresponding to the solution to be detected comprises: detecting the second color corresponding to the solution to be detected in real time; if the second color changes to blue, determining that the Mn3O4-type oxidase is active; and / or, the method for detecting the second color corresponding to the solution to be detected in real time comprises: obtaining a picture of the solution to be detected corresponding to the solution to be detected using a camera or a video camera; performing color extraction on the picture of the solution to be detected to obtain the second color corresponding to the solution to be detected; or, the method for detecting the activity of the Mn3O4-type oxidase based on the light absorption value of the solution to be detected at a set wavelength comprises: obtaining the light absorption value corresponding to each wavelength within a set wavelength range including the set wavelength; drawing a corresponding wavelength-light absorption value curve based on the light absorption value corresponding to each wavelength; and extracting the light absorption value of the solution to be detected based on the wavelength-light absorption value curve. value curve, determine the peak value corresponding to the light absorption value at the set wavelength; if the peak value is the maximum peak value, determine that the Mn3O4-type oxidase is active; or, the method for detecting the activity of the Mn3O4-type oxidase based on the color corresponding to the solution to be detected and the light absorption value at the set wavelength, comprises: real-time detection of a second color corresponding to the solution to be detected; if the second color changes to blue, generate a first detection condition; obtain the light absorption value corresponding to each wavelength within the set wavelength range including the set wavelength; draw the corresponding wavelength-light absorption value curve based on the light absorption value corresponding to each wavelength; based on the wavelength-light absorption value curve, determine the peak value corresponding to the light absorption value at the set wavelength; if the peak value is the maximum peak value, generate a second detection condition; if the first detection condition and the second detection condition are met at the same time, determine that the Mn3O4-type oxidase is active.

[0047] Preferably, the verification of the activity of the Mn3O4-type oxidase includes: obtaining a first solution to be detected and a second solution to be detected; wherein the preparation method of the first solution to be detected and the second solution to be detected is the same as the preparation method of the solution to be detected; respectively introducing an air atmosphere and a nitrogen atmosphere into the first solution to be detected and the second solution to be detected, and respectively using the microplate reader to detect the first light absorption value and the second absorption value of the reaction system corresponding to the first solution to be detected introduced into the air atmosphere and the second solution to be detected introduced into the nitrogen atmosphere at a set wavelength; determining the activity of the Mn3O4-type oxidase based on the first light absorption value and the second absorption value; and / or, the method for determining the activity of the Mn3O4-type oxidase based on the first light absorption value and the second absorption value includes: if there is a significant difference between the first light absorption value and the second absorption value, then there is oxidase-like activity.

[0048] Preferably, the method of extracting the color of the image of the solution to be detected to obtain the second color corresponding to the solution to be detected includes: parsing the second RGB value corresponding to the second three channels of the image of the solution to be detected to obtain the second color corresponding to the solution to be detected. Among them, the method of judging that the second color changes to blue includes: obtaining the second RGB value corresponding to the second color; if the second RGB value is the same as the RGB value corresponding to the three channels of blue or if the deviation of the RGB value corresponding to the second three channels and the RGB value corresponding to the three channels of blue is within a set range, then it is determined that the second color changes to blue.

[0049] Preferably, the enzyme kinetics detection method further comprises: determining the Michaelis constant and the maximum reaction rate of the enzyme kinetics parameters based on a velocity curve of the relationship between multiple reaction rates of the enzymatic reaction and different set concentrations within the third set concentration range.

[0050] Preferably, the enzyme kinetics detection method further includes: taking the reciprocals of different set concentrations within the third set concentration range of the 3,3',5,5'-tetramethylbenzidine and their corresponding multiple reaction rates, respectively, to obtain multiple first reciprocals corresponding to different set concentrations within the third set concentration range and multiple second reciprocals corresponding to the multiple reaction rates; using a double reciprocal equation, fitting the multiple first reciprocals and their corresponding multiple second reciprocals to obtain a linearization curve of the Michaelis constant of the enzyme kinetic parameters.

[0051] Preferably, the third set concentration range is configured as 0-120 μg / mL; wherein the different set concentrations within the third set concentration range are configured as any multiple values ​​of 0-120 μg / mL.

[0052] Preferably, it also includes: at the second set temperature, after placing the solutions to be detected with different set concentrations within the third set concentration range for a sixth set time, detecting multiple light absorption values ​​of the reaction system corresponding to the solutions to be detected with different set concentrations within the third set concentration range at the set wavelength by using an enzyme marker.

[0053] Preferably, the method for determining the reaction intermediate based on the resonance spectrum comprises: determining different free radicals having characteristic peaks based on the resonance spectrum; and determining the different free radicals as the reaction intermediates.

[0054] Preferably, the method for determining the reaction intermediate based on the resonance spectrum further comprises: determining the free radical corresponding to the maximum value among the values ​​corresponding to the characteristic peaks of the different free radicals as the most important reaction intermediate among the reaction intermediates.

[0055] According to one aspect of the present disclosure, there is provided a device for detecting the nanozyme activity and / or enzyme kinetics and / or oxidase-like activity mechanism of a nanozyme obtained by the nanozyme preparation method as described above, a nanozyme prepared by the nanozyme preparation device as described above, or a nanozyme corresponding to the nanozyme as described above.

[0056] Among them, the nanozyme activity detection device includes: a first acquisition unit, used to obtain a Mn3O4-type oxidase solution with a third set concentration and a 3,3',5,5'-tetramethylbenzidine and dimethyl sulfoxide solution with a fourth set concentration; a first liquid preparation unit, used to add a 3,3',5,5'-tetramethylbenzidine and dimethyl sulfoxide solution corresponding to a sixth set concentration less than the fourth set concentration and a Mn3O4-type oxidase solution corresponding to a seventh set concentration less than the third set concentration to a NaAc-HAc buffer solution with a third set volume, a fifth set concentration and a set pH value to obtain a solution to be detected; a light absorption value determination unit, used to place the solution to be detected for a sixth set time at a second set temperature, and then detect the light absorption value of the reaction system corresponding to the solution to be detected at a set wavelength through an enzyme marker; activity detection A unit for detecting the activity of the Mn3O4-type oxidase based on the color corresponding to the solution to be detected and / or the light absorption value at a set wavelength; or, wherein the nanozyme activity detection device comprises: a processor; a memory for storing processor executable instructions; wherein the processor is configured to call the instructions stored in the memory to execute the above-mentioned nanozyme activity detection method; or, wherein the nanozyme activity detection device comprises: a computer-readable storage medium having computer program instructions stored thereon, and the computer program instructions implement the above-mentioned nanozyme activity detection method when executed by the processor; or, wherein the nanozyme activity detection device comprises: a computer program product, comprising a computer program / instruction, which implements the above-mentioned nanozyme activity detection method when executed by the processor.

[0057] Among them, the nanozyme kinetic detection device includes: a second acquisition unit, used to obtain a Mn3O4-type oxidase solution of a third set concentration and 3,3',5,5'-tetramethylbenzidine and dimethyl sulfoxide solutions of different set concentrations within the third set concentration range; a second liquid preparation unit, used to add 3,3',5,5'-tetramethylbenzidine and dimethyl sulfoxide solutions corresponding to a sixth set concentration less than the fourth set concentration and Mn3O4-type oxidase solutions corresponding to different set concentrations within the third set concentration range less than the three set concentrations to a NaAc-HAc buffer solution of a third set volume, a fifth set concentration and a set pH value, to obtain a solution to be detected with different set concentrations within the third set concentration range; a first determination unit, used to respectively determine multiple reaction rates corresponding to 3,3',5,5'-tetramethylbenzidine of different set concentrations within the third set concentration range in the solution to be detected; and a second determination unit, used to Using the Michaelis equation, the different set concentrations within the third set concentration range of the 3,3',5,5'-tetramethylbenzidine and their corresponding multiple reaction rates are fitted to determine the speed curve of the relationship between the multiple reaction rates of the enzymatic reaction and the different set concentrations within the third set concentration range; or, wherein the nanozyme kinetic detection device comprises: a processor; a memory for storing processor executable instructions; wherein the processor is configured to call the instructions stored in the memory to execute the above-mentioned nanozyme kinetic detection method; or, wherein the nanozyme kinetic detection device comprises: a computer-readable storage medium having computer program instructions stored thereon, and the computer program instructions implement the above-mentioned nanozyme kinetic detection method when executed by the processor; or, comprising: a computer program product, comprising a computer program / instruction, which implements the above-mentioned nanozyme kinetic detection method when executed by the processor.

[0058] Wherein, the oxidase-like activity mechanism detection device comprises: a resonance spectrum measurement unit, which is used to use electron paramagnetic energy spectrum to capture the reaction intermediates of catalyzing 3,3′,5,5′-tetramethylbenzidine in the color development process with 5,5-dimethyl-1-pyrroline oxide as a specific probe to measure the resonance spectrum; a third determination unit, which determines the reaction intermediates based on the resonance spectrum; or, wherein, the oxidase-like activity mechanism detection device comprises: a processor; a memory for storing processor executable instructions; wherein the processor is configured to call the instructions stored in the memory to execute the above-mentioned oxidase-like activity mechanism detection method; or, wherein, the oxidase-like activity mechanism detection device comprises: a computer-readable storage medium on which computer program instructions are stored, and the computer program instructions implement the above-mentioned oxidase-like activity mechanism detection method when executed by the processor; or, wherein, the oxidase-like activity mechanism detection device comprises: a computer program product, including a computer program / instruction, and the computer program / instruction implements the above-mentioned oxidase-like activity mechanism detection method when executed by the processor.

[0059] According to one aspect of the present disclosure, an electronic device is provided, comprising: a processor; a memory for storing instructions executable by the processor; wherein the processor is configured to perform one or more of the above-mentioned activity detection and / or enzyme kinetics detection and / or oxidase-like activity mechanism detection methods.

[0060] According to one aspect of the present disclosure, a computer-readable storage medium is provided, on which computer program instructions are stored, and when the computer program instructions are executed by a processor, one or more of the above-mentioned activity detection and / or enzyme kinetics detection and / or oxidase-like activity mechanism detection methods are implemented.

[0061] According to one aspect of the present disclosure, there is provided a computer program product, comprising a computer program / instruction, which, when executed by a processor, implements one or more of the above-mentioned activity detection and / or enzyme kinetics detection and / or oxidase-like activity mechanism detection methods.

[0062] According to one aspect of the present disclosure, there is provided a nanozyme obtained by the nanozyme preparation method as described above, or a nanozyme prepared by the nanozyme preparation device as described above, or a nanozyme as described above, and a method for testing its selectivity, comprising: obtaining cysteine ​​corresponding to an eighth set concentration, and amino acids and / or ions corresponding to a ninth set concentration greater than the eighth set concentration; determining the selectivity of the Mn3O4-type oxidase activity based on the activity responses of cysteine ​​corresponding to the eighth set concentration and amino acids and / or ions corresponding to the ninth set concentration to the Mn3O4-type oxidase, respectively.

[0063] According to one aspect of the present disclosure, a nanozyme obtained by the above-mentioned nanozyme preparation method or a nanozyme prepared by the above-mentioned nanozyme preparation device or a nanozyme selectivity testing device corresponding to the above-mentioned nanozyme is provided, comprising:

[0064] The third acquisition unit is used to obtain cysteine ​​corresponding to the eighth set concentration and amino acids and / or ions corresponding to the ninth set concentration greater than the eighth set concentration; the selectivity determination unit is used to determine the selectivity of the Mn3O4-type oxidase activity based on the activity response of cysteine ​​corresponding to the eighth set concentration and amino acids and / or ions corresponding to the ninth set concentration to the Mn3O4-type oxidase.

[0065] According to one aspect of the present disclosure, there is provided a nanozyme prepared by the preparation method as described above; or, a nanozyme prepared by the preparation device as described above; or, the application of the nanozyme as described above in cysteine ​​detection and / or in cysteine ​​detection in urine.

[0066] In the embodiments of the present disclosure, the present disclosure proposes a nanozyme and its preparation method, detection method, test method, device and application technical solution, which overcomes the shortcomings of traditional methods such as cumbersome sample pre-processing, expensive instruments and equipment, requirement for professional technicians to operate, and high operating costs.

[0067] It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the disclosure.

[0068] Further features and aspects of the present disclosure will become apparent from the following detailed description of exemplary embodiments with reference to the attached drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0069] The drawings herein are incorporated into the specification and constitute a part of the specification. These drawings illustrate embodiments consistent with the present disclosure and are used to illustrate the technical solutions of the present disclosure together with the specification.

[0070] Figure 1 A flow chart showing a method for preparing a nanozyme according to an embodiment of the present disclosure;

[0071] Figure 2 Showing the UV-visible absorption spectra under different systems according to the embodiments of the present disclosure;

[0072] Figure 3 The absorbance (light absorption value) at 52 nm under air and nitrogen atmospheres according to the embodiments of the present disclosure is shown;

[0073] Figure 4The optimization results of the reaction system according to the embodiment of the present disclosure are shown; wherein, Figure A is the concentration of Mn3O4 nanozyme, and Figure B is the substrate TMB;

[0074] Figure 5 The results of reaction condition optimization according to the embodiment of the present disclosure are shown; wherein A is pH, B is buffer concentration, C is reaction time, and D is reaction temperature;

[0075] Figure 6 The kinetic parameters of the Mn3O4 nanozyme oxidase according to the embodiment of the present disclosure are shown; wherein A is the Michaelis-Menten equation of Mn3O4 (different set concentrations of TMB corresponding to the substrate); B is the Lineweaver-Burk curve;

[0076] Figure 7 An electron paramagnetic resonance spectrum according to an embodiment of the present disclosure is shown; wherein, Figure A: 1 Characteristic peak of O2, Figure B: O2· - Characteristic peaks of, Figure C: characteristic peaks of OH;

[0077] Figure 8 The detection specificity of cysteine ​​according to the embodiments of the present disclosure is shown;

[0078] Fig. 9 The detection sensitivity of cysteine ​​according to the embodiment of the present disclosure is shown; wherein, Figure A is the detection range of cysteine ​​at a TMB concentration of 20 μg / mL; Figure B is the linear fit of Figure A; Figure C is the detection range of cysteine ​​at a TMB concentration of 110 μg / mL; Figure D is the linear fit of Figure C;

[0079] Fig.10 is a block diagram of an electronic device 800 according to an exemplary embodiment;

[0080] Fig.11 is a block diagram of an electronic device 1900 according to an exemplary embodiment. DETAILED DESCRIPTION

[0081] Various exemplary embodiments, features and aspects of the present disclosure will be described in detail below with reference to the accompanying drawings. The same reference numerals in the accompanying drawings represent elements with the same or similar functions. Although various aspects of the embodiments are shown in the accompanying drawings, the drawings are not necessarily drawn to scale unless otherwise specified.

[0082] The word “exemplary” is used exclusively herein to mean “serving as an example, example, or illustration.” Any embodiment described herein as “exemplary” is not necessarily to be construed as preferred or advantageous over other embodiments.

[0083] The term "and / or" herein is only a description of the association relationship of the associated objects, indicating that there may be three relationships. For example, A and / or B can represent: A exists alone, A and B exist at the same time, and B exists alone. In addition, the term "at least one" herein represents any combination of at least two of any one or more of a plurality of. For example, including at least one of A, B, and C can represent including any one or more elements selected from the set consisting of A, B, and C.

[0084] In addition, in order to better illustrate the present disclosure, numerous specific details are given in the following specific embodiments. It should be understood by those skilled in the art that the present disclosure can also be implemented without certain specific details. In some examples, methods, means, components and circuits well known to those skilled in the art are not described in detail in order to highlight the subject matter of the present disclosure.

[0085] It can be understood that the above-mentioned various method embodiments mentioned in the present disclosure can be combined with each other to form a combined embodiment without violating the principle logic. Due to space limitations, the present disclosure will not go into details.

[0086] In addition, the present disclosure also provides devices, electronic devices, computer-readable storage media, programs and applications corresponding to the nanozyme preparation, nanozyme detection and nanozyme selectivity testing methods, all of which can be used to implement any nanozyme preparation method, nanozyme detection method and nanozyme selectivity testing method provided by the present disclosure. The corresponding technical solutions and descriptions refer to the corresponding records in the nanozyme preparation method, nanozyme detection method and nanozyme selectivity testing method sections, which will not be repeated here.

[0087] Figure 1 A flow chart of a method for preparing a nanozyme according to an embodiment of the present disclosure is shown, as shown in FIG. Figure 1 As shown, the method for preparing nanoenzyme comprises: step S101: obtaining a Mn(NO3)2 stirring solution and an aminoethanol stirring solution respectively, mixing the Mn(NO3)2 stirring solution and the aminoethanol stirring solution to obtain a mixed solution; step S102: stirring the mixed solution at a set rate and a third set time under an inert atmosphere to obtain a stirred mixed solution; step S103: aging the stirred mixed solution at a fourth set time to obtain a corresponding suspension; step S104: filtering, washing and drying the suspension to obtain a Mn3O4 oxidase. The method overcomes the shortcomings of the traditional method, such as cumbersome sample pre-processing, expensive instruments and equipment, the need for professional technicians to operate, and high operating costs.

[0088] In the embodiments of the present disclosure, before obtaining the Mn(NO3)2 stirring solution and the aminoethanol stirring solution, a Mn(NO3)2 solution corresponding to a first set volume and a first set concentration and an aminoethanol solution corresponding to a second set volume and a second set concentration are prepared respectively; under the inert atmosphere, the Mn(NO3)2 solution and the aminoethanol solution are stirred for a first set time and a second time respectively to obtain the corresponding Mn(NO3)2 stirring solution and the aminoethanol stirring solution.

[0089] In an embodiment of the present disclosure, the first set volume and the second set volume have the same numerical value, and the second set concentration has a numerical value that is twice the numerical value of the first set concentration.

[0090] In an embodiment of the present disclosure, the first set volume and the first set concentration are configured as 10 ml and 0.4 mM, respectively, and the second set volume and the second set concentration are configured as 10 ml and 0.8 mM, respectively.

[0091] In an embodiment of the present disclosure, the third setting time may be configured as 3 hours; and / or, the fourth setting time may be configured as 24 hours; and / or, the inert atmosphere may be configured as an argon atmosphere.

[0092] In an embodiment of the present disclosure, the method of filtering, washing and drying the suspension to obtain a Mn3O4-type oxidase comprises: filtering and washing the suspension, and drying the filtered and washed suspension at a first set temperature and a fifth set time to obtain a Mn3O4-type oxidase.

[0093] In an embodiment of the present disclosure, the first set temperature is configured to be 60° C.; and / or the fifth set time is configured to be 24 hours.

[0094] In an embodiment of the present disclosure, the method of performing an aging treatment on the stirred mixed solution according to a fourth set time to obtain a corresponding suspension includes: within the fourth set time, detecting the first color corresponding to the suspension in real time; if the first color changes to gray-brown, determining that the aging treatment of the stirred mixed solution is completed to obtain the corresponding suspension. Wherein, the method of determining that the aging treatment of the stirred mixed solution is completed if the color changes to gray-brown includes: if the color changes to gray-brown, obtaining a set delay time; after the set delay time, determining that the aging treatment of the stirred mixed solution is completed; and / or, the method of detecting the first color corresponding to the suspension in real time, and determining that the aging treatment of the stirred mixed solution is completed if the first color changes to gray-brown, includes: using a camera or a video camera to obtain a suspension picture corresponding to the suspension; performing color extraction on the suspension picture; if the extracted color is the same as the set gray-brown, determining that the aging treatment of the stirred mixed solution is completed.

[0095] Wherein, in an embodiment of the present disclosure, the method for color extraction of the suspension image comprises: using a preset threshold or a preset segmentation model to segment the suspended matter in the suspension image to obtain a suspended matter image; then, color extraction is performed on the suspended matter image; if the extracted first color is the same as the set gray-brown color, it is determined that the aging treatment of the stirred mixed solution is completed. Wherein, if the extracted first color is the same as the set gray-brown color, the method for determining that the aging treatment of the stirred mixed solution is completed comprises: parsing the first RGB values ​​corresponding to the three channels corresponding to the extracted first color; if the RGB values ​​corresponding to the first three channels are the same as the RGB values ​​corresponding to the three channels corresponding to the gray-brown color or if the deviation between the RGB values ​​corresponding to the first three channels and the RGB values ​​corresponding to the three channels corresponding to the gray-brown color is within a set range, it is determined that the aging treatment of the stirred mixed solution is completed.

[0096] In the embodiments of the present disclosure and other possible embodiments, the method for preparing the nanozyme includes: preparing a Mn(NO3)2 solution corresponding to a first set volume and a first set concentration and an aminoethanol solution corresponding to a second set volume and a second set concentration, respectively; stirring the Mn(NO3)2 solution and the aminoethanol solution for a first set time and a second time, respectively, under an inert atmosphere to obtain a corresponding Mn(NO3)2 stirred solution and an aminoethanol stirred solution; mixing the Mn(NO3)2 stirred solution and the aminoethanol stirred solution to obtain a mixed solution; stirring the mixed solution at a set rate and a third set time under the inert atmosphere to obtain a stirred mixed solution; aging the stirred mixed solution according to a fourth set time to obtain a corresponding suspension; filtering and washing the suspension, and drying the filtered and washed suspension at a first set temperature and for a fifth set time to obtain a Mn3O4-type oxidase.

[0097] Among them, in the embodiments of the present disclosure and other possible embodiments, the first set volume and the first set concentration are configured to 10 ml and 0.4 mM (millimolar), respectively; the second set volume and the second set concentration are configured to 10 ml and 0.8 mM (millimolar), respectively. The numerical value corresponding to the second set concentration is greater than the numerical value corresponding to the first set concentration. For example, the numerical value corresponding to the second set concentration is 1.5-3 times the numerical value corresponding to the first set concentration. Specifically, the numerical values ​​corresponding to the first set volume and the second set volume are the same, and the numerical value corresponding to the second set concentration is twice the numerical value corresponding to the first set concentration.

[0098] Among them, in the embodiments of the present disclosure and other possible embodiments, the inert atmosphere is configured as an argon atmosphere, the first set time and the second time are respectively configured to 30 minutes; the third set time can be configured to 3 hours; the fourth set time is configured to 24 hours; the first set temperature is configured to 60°C; and the fifth set time is configured to 24 hours.

[0099] In the embodiments of the present disclosure and other possible embodiments, 10 ml of 0.4 mM Mn(NO3)2 solution and 10 ml of 0.8 mM aminoethanol (AE) solution were stirred for 30 minutes under an argon atmosphere, and then the two solutions were mixed. The mixed solution was stirred at a constant speed for 3 hours under an argon atmosphere, and then aged for 24 hours to form a gray-brown suspension. The obtained suspension was filtered, washed several times, and vacuum dried at 60°C for 24 hours to obtain a powder sample corresponding to the Mn3O4 oxidase.

[0100] The disclosed embodiments also propose a method for detecting the activity and / or enzyme kinetics and / or oxidase-like activity mechanism of a nanozyme obtained by the nanozyme preparation method as described above, or a nanozyme prepared by the nanozyme preparation device as described above, or a nanozyme as described above.

[0101] Among them, in an embodiment of the present disclosure, the activity detection of the nanozyme includes: obtaining a Mn3O4-type oxidase solution of a third set concentration and a 3,3',5,5'-tetramethylbenzidine and dimethyl sulfoxide solution of a fourth set concentration; adding a 3,3',5,5'-tetramethylbenzidine and dimethyl sulfoxide solution corresponding to a sixth set concentration less than the fourth set concentration and a Mn3O4-type oxidase solution corresponding to a seventh set concentration less than the third set concentration to a NaAc-HAc buffer solution of a third set volume, a fifth set concentration and a set pH value to obtain a solution to be detected; after placing the solution to be detected for a sixth set time at a second set temperature, detecting the light absorption value of the reaction system corresponding to the solution to be detected for activity at a set wavelength by an enzyme marker; detecting the activity of the Mn3O4-type oxidase based on the color corresponding to the solution to be detected and / or the light absorption value at the set wavelength.

[0102] In an embodiment of the present disclosure, before obtaining the Mn3O4-type oxidase solution of the third set concentration, a first mass of a powder sample of the Mn3O4-type oxidase is weighed, and the powder sample is dispersed in ultrapure water to prepare the Mn3O4-type oxidase solution of the third set concentration.

[0103] In an embodiment of the present disclosure, before obtaining the 3,3',5,5'-tetramethylbenzidine and dimethyl sulfoxide solution of the fourth set concentration, a second mass of 3,3',5,5'-tetramethylbenzidine powder is weighed, and the powder is dissolved in dimethyl sulfoxide to prepare the 3,3',5,5'-tetramethylbenzidine and dimethyl sulfoxide solution of the fourth set concentration.

[0104] In an embodiment of the present disclosure, the third set concentration is configured to 1 mg / mL; and / or, the fourth set concentration is configured to 10 mg / mL; the fourth set concentration is any value between 5 and 12 times the third set concentration; the sixth set concentration is configured to 25 μg / mL or 110 μg / mL; the seventh set concentration is configured to 20 μg / mL.

[0105] In an embodiment of the present disclosure, the method for determining the seventh set concentration includes: obtaining a Mn3O4-type oxidase solution corresponding to a first different set concentration within a first set concentration range; determining different colors of a solution to be detected corresponding to the Mn3O4-type oxidase solution corresponding to the first different set concentration; configuring the concentration corresponding to the darkest color in the first different colors or the concentration corresponding to the adjacent colors in the first different colors remaining unchanged as the seventh set concentration. Wherein, the method for determining different colors of a solution to be detected corresponding to the Mn3O4-type oxidase solution corresponding to the different set concentrations includes: respectively adding 3,3',5,5'-tetramethylbenzidine and dimethyl sulfoxide solution corresponding to a sixth set concentration less than the fourth set concentration and Mn3O4-type oxidase solutions corresponding to different set concentrations less than the three set concentrations to the third set volume, fifth set concentration and set pH value NaAc-HAc buffer to obtain the solution to be detected corresponding to the Mn3O4-type oxidase solution of the first different set concentration; respectively determining different colors of the solution to be detected corresponding to the Mn3O4-type oxidase solution of the first different set concentration.

[0106] In an embodiment of the present disclosure, the method for determining the sixth set concentration includes: obtaining 3,3',5,5'-tetramethylbenzidine and dimethyl sulfoxide solutions corresponding to the second different set concentrations within the second set concentration range; preparing the test solutions of 3,3',5,5'-tetramethylbenzidine and dimethyl sulfoxide solutions corresponding to the second different set concentrations under the Mn3O4 type oxidase solution of the seventh set concentration; at the second set temperature, after placing the test solutions corresponding to the second different set concentrations for a sixth set time, detecting multiple light absorption values ​​of the reaction system corresponding to the solution to be detected for activity at a set wavelength by an ELISA reader; configuring the set concentration corresponding to the maximum peak value of the multiple light absorption values ​​corresponding to the second different set concentrations as the sixth set concentration; or, at the second set temperature, after placing the test solutions corresponding to the second different set concentrations for a sixth set time, determining multiple colors corresponding to the second different set concentrations; configuring the set concentration corresponding to the darkest color among the multiple colors corresponding to the second different set concentrations, or the adjacent colors remain unchanged, or the color is green as the sixth set concentration.

[0107] In an embodiment of the present disclosure, the method for preparing the test solutions of 3,3',5,5'-tetramethylbenzidine and dimethyl sulfoxide solutions corresponding to the second different set concentrations under the seventh set concentration of Mn3O4-type oxidase solution, comprises: adding 3,3',5,5'-tetramethylbenzidine and dimethyl sulfoxide solutions corresponding to the second different concentrations less than the fourth set concentration and Mn3O4-type oxidase solutions corresponding to the seventh set concentration less than the third set concentration to the third set volume, fifth set concentration and set pH value NaAc-HAc buffer to obtain the test solutions corresponding to the Mn3O4-type oxidase solutions 3,3',5,5'-tetramethylbenzidine and dimethyl sulfoxide solutions of the second different set concentrations; and determining the different colors corresponding to the test solutions corresponding to the 3,3',5,5'-tetramethylbenzidine and dimethyl sulfoxide solutions of the second different set concentrations.

[0108] In an embodiment of the present disclosure, the method for determining the set pH value includes: obtaining NaAc-HAc buffer solutions corresponding to different pH values ​​within the set pH value range; determining multiple color development values ​​corresponding to the NaAc-HAc buffer solutions with different pH values; and configuring the pH value corresponding to the best color development among the multiple color development values ​​as the set pH value.

[0109] In an embodiment of the present disclosure, the method for determining the sixth set time includes: determining, at a second set temperature, multiple colors corresponding to the solution to be detected at multiple set time points; and configuring the set time point corresponding to a color tending to be stable among the multiple colors as the sixth set time.

[0110] In an embodiment of the present disclosure, the method for determining the second set temperature includes: after placing the solution to be detected for a sixth set time at multiple set temperatures, detecting multiple light absorption values ​​of the reaction system corresponding to the solution to be detected for activity at a set wavelength by an ELISA instrument; and configuring the set temperature corresponding to any two indifferent light absorption values ​​among the multiple light absorption values ​​as the second set temperature.

[0111] In the embodiments of the present disclosure, the third set volume is configured to 1 mL; and / or the fifth set concentration is configured to 0.1 M or 0.2 M; the set pH value is configured to 3.5; the second set temperature is configured to room temperature or 5°C or 25°C; the sixth set time is configured to 5 minutes; and / or the set wavelength is configured to 652 nm.

[0112] In an embodiment of the present disclosure, before detecting the light absorption value of the reaction system corresponding to the solution to be detected for activity at a set wavelength by using an ELISA reader, it also includes: aspirating a fourth set volume of the solution to be detected, adding the fourth set volume of the solution to be detected into a well plate, and detecting the light absorption value of the reaction system corresponding to the solution to be detected for activity at a set wavelength by using an ELISA reader; wherein the fourth set volume is configured to be 200 μL.

[0113] In an embodiment of the present disclosure, the method for detecting the activity of the Mn3O4-type oxidase based on the color corresponding to the solution to be detected includes: detecting the second color corresponding to the solution to be detected in real time; if the second color changes to blue, it is determined that the Mn3O4-type oxidase is active; and / or, the method for detecting the second color corresponding to the solution to be detected in real time includes: obtaining a picture of the solution to be detected corresponding to the solution to be detected using a camera or a video camera; performing color extraction on the picture of the solution to be detected to obtain the second color corresponding to the solution to be detected; or, the method for detecting the activity of the Mn3O4-type oxidase based on the light absorption value of the solution to be detected at a set wavelength includes: obtaining the light absorption value corresponding to each wavelength within a set wavelength range including the set wavelength; drawing a corresponding wavelength-light absorption value curve based on the light absorption value corresponding to each wavelength; and drawing a color curve based on the wavelength-light absorption value curve. light absorption value curve, determine the peak value corresponding to the light absorption value at the set wavelength; if the peak value is the maximum peak value, determine that the Mn3O4-type oxidase is active; or, the method for detecting the activity of the Mn3O4-type oxidase based on the color corresponding to the solution to be detected and the light absorption value at the set wavelength, comprising: real-time detection of a second color corresponding to the solution to be detected; if the second color changes to blue, generate a first detection condition; obtain the light absorption value corresponding to each wavelength within the set wavelength range including the set wavelength; based on the light absorption value corresponding to each wavelength, draw the corresponding wavelength-light absorption value curve; based on the wavelength-light absorption value curve, determine the peak value corresponding to the light absorption value at the set wavelength; if the peak value is the maximum peak value, generate a second detection condition; if the first detection condition and the second detection condition are met at the same time, determine that the Mn3O4-type oxidase is active.

[0114] In the embodiment of the present disclosure and other possible embodiments, a Mn3O4-based oxidase solution of a third set concentration and a 3,3',5,5'-tetramethylbenzidine and dimethyl sulfoxide solution of a fourth set concentration are obtained;

[0115] Before obtaining the Mn3O4 oxidase solution of the third set concentration, weigh a first mass of a powder sample of the Mn3O4 oxidase, disperse the powder sample in ultrapure water, and prepare the Mn3O4 oxidase solution of the third set concentration; before obtaining the 3,3',5,5'-tetramethylbenzidine and dimethyl sulfoxide solution of the fourth set concentration, weigh a second mass of 3,3',5,5'-tetramethylbenzidine powder, dissolve the powder in dimethyl sulfoxide, and prepare the 3,3',5,5'-tetramethylbenzidine and dimethyl sulfoxide solution of the fourth set concentration; the third set concentration is configured to be 1 mg / mL. The fourth set concentration is configured to be 10 mg / mL; the fourth set concentration is any value of 5-12 times the third set concentration, such as 5, 6, 7, 8, 9, 10, 11, 12 times.

[0116] In the embodiments of the present disclosure and other possible embodiments, the activity detection of Mn3O4-type oxidases includes the following steps: 1. First, weigh a first mass of a powder sample of a Mn3O4-type oxidase and disperse it in ultrapure water to prepare a 1 mg / mL (milligrams per milliliter, concentration unit) Mn3O4-type oxidase solution. The second mass of TMB powder is dissolved in DMSO (dimethyl sulfoxide) to prepare a 10 mg / mL 3,3',5,5'-tetramethylbenzidine and dimethyl sulfoxide solution. 2. Add 110 μg / mL TMB solution (3,3',5,5'-tetramethylbenzidine and dimethyl sulfoxide solution) and 20 μg / mL Mn3O4 (Mn3O4-type oxidase) solution to 1 mL NaAc-HAc buffer (pH 3.5, 0.1 M (molar, concentration unit)) to obtain a solution to be tested for activity. In addition, the NaAc-HAc buffer and TMB solution are used as control experiments to accurately determine the oxidase activity of Mn3O4. 3. After standing at room temperature (eg, 25°C) for 5 minutes, 200 μL of the reaction solution (solution to be detected for activity) was pipetted into a 96-well plate, and the light absorption value of the reaction system at 652 nm was detected by an ELISA instrument.

[0117] In an embodiment of the present disclosure, verification of the activity of the Mn3O4-type oxidase includes: obtaining a first solution to be detected and a second solution to be detected; wherein the preparation method of the first solution to be detected and the second solution to be detected is the same as the preparation method of the solution to be detected; respectively introducing an air atmosphere and a nitrogen atmosphere into the first solution to be detected and the second solution to be detected, and respectively using the ELISA instrument to detect the first light absorption value and the second absorption value of the reaction system corresponding to the first solution to be detected introduced into the air atmosphere and the second solution to be detected introduced into the nitrogen atmosphere at a set wavelength; determining the activity of the Mn3O4-type oxidase based on the first light absorption value and the second absorption value; and / or, the method for determining the activity of the Mn3O4-type oxidase based on the first light absorption value and the second absorption value includes: if there is a significant difference between the first light absorption value and the second absorption value, then there is oxidase-like activity.

[0118] In an embodiment of the present disclosure, the method of extracting the color of the image of the solution to be detected to obtain the second color corresponding to the solution to be detected includes: parsing the second RGB value corresponding to the second three channels of the image of the solution to be detected to obtain the second color corresponding to the solution to be detected. The method of judging that the second color changes to blue includes: obtaining the second RGB value corresponding to the second color; if the second RGB value is the same as the RGB value corresponding to the three channels of blue or if the deviation of the RGB value corresponding to the second three channels and the RGB value corresponding to the three channels of blue is within a set range, then it is determined that the second color changes to blue.

[0119] Figure 2 The UV-visible absorption spectra of different systems according to the embodiments of the present disclosure are shown. Figure 2 As shown in the table below, 3,3′,5,5′-tetramethylbenzidine (TMB) is oxidized to blue oxidized TMB (oxTMB), and has an obvious absorption peak at 652nm, indicating that the nanomaterial has good oxidase-like activity. Figure 2 The corresponding original data (since the data contains 251 rows, only part of the data is intercepted for display).

[0120] Wavelength (nm) TMB <![CDATA[Mn3O4]]> <![CDATA[TMB+Mn3O4]]> 300 2.6808 0.7167 2.5108 302 2.5994 0.6336 2.3299 304 2.4795 0.5802 2.156 306 2.3148 0.5425 1.9803 308 2.1171 0.5149 1.8092 310 1.8959 0.493 1.6419 312 1.7076 0.4788 1.513 314 1.5238 0.4668 1.3962 316 1.3429 0.4552 1.2849 318 1.1503 0.4416 1.171 320 0.9933 0.4293 1.0816 322 0.8488 0.4171 1.0031 324 0.7292 0.4068 0.942 326 0.6264 0.3976 0.894 328 0.5518 0.3905 0.8644 330 0.4838 0.3828 0.8422 332 0.4234 0.3738 0.8277 334 0.3701 0.3638 0.8229 336 0.3336 0.3556 0.8284 338 0.3044 0.3481 0.8434 340 0.2806 0.3413 0.8685 342 0.2601 0.3352 0.9103 344 0.2462 0.3306 0.9587 346 0.236 0.3268 1.0131 348 0.2272 0.3231 1.0831 … … … …

[0121] In the embodiments of the present disclosure and other possible embodiments, aerobicity (activity) verification: first, 20 μg / mL Mn3O4 (Mn3O4-type oxidase) and 110 μg / mL TMB were added to 1 mL NaAc-HAc buffer, and air atmosphere and nitrogen atmosphere were introduced respectively, and the absorbance at 652 nm was detected using an ELISA reader.

[0122] Figure 3The absorbance (light absorption value) at 52 nm in air and nitrogen atmosphere according to the embodiment of the present disclosure is shown. Figure 3 It can be seen that when the detection is carried out under nitrogen atmosphere, the absorbance at 652nm is greatly weakened, which is significantly different from the detection under air. This shows that the Mn3O4 nanozyme needs oxygen to catalyze the color development of TMB, indicating that Mn3O4 has oxidase-like activity. Figure 3 The corresponding original data.

[0123]

[0124] In the embodiments of the present disclosure and other possible embodiments, the optimization of experimental conditions includes the following steps: TMB and dimethyl sulfoxide solution and Mn3O4 (Mn3O4 oxidase) solution are added to NaAc-HAc buffer (pH 3.5, 0.1M), and the absorbance at 652nm is measured after 5 minutes of reaction. The nanozyme concentration, TMB concentration, pH and reaction time during the reaction, as well as the concentrations of nanozyme and substrate TMB (TMB and dimethyl sulfoxide solution) are optimized respectively.

[0125] First, the concentrations of nanozyme (Mn3O4-type oxidase) and substrate TMB were optimized. Figure 4 The results of the reaction system optimization according to the embodiment of the present disclosure are shown; wherein, Figure A is the concentration of Mn3O4 nanozyme, Figure B is the substrate TMB, and the results are as follows Figure 4 As shown in A, the nanozyme concentration was first optimized. When the final concentration of the nanozyme was in the range of 5, 10, 20, 30, 40, and 50 μg / mL, the color development trend gradually deepened with the increase of the nanozyme concentration. When the final concentration of the Mn3O4 nanozyme was 20 μg / mL, the color development was best, so the optimal final concentration of the nanozyme was 20 μg / mL. At this concentration, the effect of TMB concentration on the oxidase activity of Mn3O4 was investigated, and a TMB solution with a final concentration of 20-120 μg / mL was selected for reaction at 25°C (room temperature) for 5 minutes.

[0126] The results are as follows Figure 4 B, When TMB is 25 μg / mL, the solution is green, indicating that the amount of TMB used is between insufficient and sufficient; when TMB is 110 μg / mL, the OD652 (light absorption value at 652 nanometers) value is the highest, indicating that the amount of TMB is sufficient. Therefore, the final concentration of TMB is selected as 25 or 110 μg / mL for subsequent experiments. Figure 4 In Figure 4 A and Figure 4 The original data corresponding to B is as follows.

[0127] Figure 4 The original data corresponding to A

[0128]

[0129] Figure 4 The original data corresponding to B

[0130]

[0131] Figure 5 The results of reaction condition optimization according to the embodiment of the present disclosure are shown in Figure 1, wherein A is pH, B is buffer concentration, C is reaction time, and D is reaction temperature. Figure 5 As shown in A, the pH of NaAc-HAc buffer was first optimized. When the pH range was between 3 and 5, the color development was good, and the color development was best at pH 3.5, so the optimal pH was 3.5; the concentration of the reaction system would affect the catalytic ability of the nanozyme, so two commonly used concentrations of 0.1 and 0.2 M were selected for optimization to explore the optimal reaction system of the sensor. Figure 5 B. When there is no significant difference in the concentration of the reaction system, a concentration of 0.1 M is selected for subsequent experiments; when optimizing the color development time, Figure 5 C. As time goes by, the color development gradually increases. The color development has stabilized at 5 minutes, and there is basically no difference compared with 20 minutes. Considering the need for rapid detection and combined with the experimental optimization results, 5 minutes is selected as the optimal reaction time. Finally, the temperature of the reaction system is optimized, such as Figure 5 D. There is no difference between 5℃ and 25℃. As the temperature increases, OD652 gradually decreases. Therefore, the experiment selects room temperature 25℃ as the subsequent experimental reaction temperature. Figure 5 The original data corresponding to AD in is as follows.

[0132] Figure 5 The original data of A

[0133]

[0134] Figure 5 The original data of B

[0135]

[0136]

[0137] Figure 5 The original data of C

[0138]

[0139] Figure 5 The original data of D

[0140]

[0141] In the embodiments of the present disclosure, the enzyme kinetics detection method includes: obtaining a Mn3O4-type oxidase solution of a third set concentration and 3,3',5,5'-tetramethylbenzidine and dimethyl sulfoxide solutions of different set concentrations within the third set concentration range; adding 3,3',5,5'-tetramethylbenzidine and dimethyl sulfoxide solutions corresponding to a sixth set concentration less than the fourth set concentration and different set concentrations within the third set concentration range less than the third set concentration to a NaAc-HAc buffer solution of a third set volume, a fifth set concentration and a set pH value. A corresponding Mn3O4-type oxidase solution is obtained to obtain solutions to be tested with different set concentrations within the third set concentration range; a plurality of reaction rates corresponding to different set concentrations of 3,3',5,5'-tetramethylbenzidine within the third set concentration range in the solution to be tested are determined respectively; and the different set concentrations of 3,3',5,5'-tetramethylbenzidine within the third set concentration range and their corresponding multiple reaction rates are fitted using the Michaelis-Menten equation to determine a speed curve of the relationship between the multiple reaction rates of the enzymatic reaction and the different set concentrations within the third set concentration range.

[0142] In an embodiment of the present disclosure, the enzyme kinetics detection method further includes: determining the Michaelis constant and the maximum reaction rate of the enzyme kinetic parameters based on a velocity curve of the relationship between multiple reaction rates of the enzymatic reaction and different set concentrations within the third set concentration range.

[0143] In an embodiment of the present disclosure, the enzyme kinetics detection method also includes: taking the reciprocals of different set concentrations within the third set concentration range of the 3,3',5,5'-tetramethylbenzidine and their corresponding multiple reaction rates, respectively, to obtain multiple first reciprocals corresponding to different set concentrations within the third set concentration range and multiple second reciprocals corresponding to the multiple reaction rates; using a double reciprocal equation, fitting the multiple first reciprocals and their corresponding multiple second reciprocals to obtain a linearization curve of the Michaelis constant of the enzyme kinetic parameters.

[0144] In an embodiment of the present disclosure, the third set concentration range is configured as 0-120 μg / mL; wherein, the different set concentrations within the third set concentration range are configured as any multiple values ​​of 0-120 μg / mL.

[0145] In an embodiment of the present disclosure, it also includes: at a second set temperature, after the solutions to be detected with different set concentrations within the third set concentration range are placed for a sixth set time, multiple light absorption values ​​of the reaction system corresponding to the solutions to be detected with different set concentrations within the third set concentration range at a set wavelength are detected by an ELISA instrument.

[0146] In the embodiments of the present disclosure and other possible embodiments, the enzyme kinetics detection includes the following steps: 1. Add 20 μg / mL Mn3O4 (Mn3O4-type oxidase) to 1 mL NaAc-HAc buffer (pH 3.5, 0.1 M), and add 0-120 μg / mL (third set concentration range) of TMB. 3. After standing at room temperature for 5 minutes, draw 200 μL of the reaction solution and add it to a 96-well plate, and use an ELISA reader to detect the light absorption value of the reaction system at 652 nm. 3. Draw an image of the substrate concentration (different set concentrations within the third set concentration range corresponding to 3,3',5,5'-tetramethylbenzidine in the solution to be detected) versus the reaction rate.

[0147] Figure 6 The kinetic parameters of the Mn3O4 nanozyme-like oxidase according to an embodiment of the present disclosure are shown. Figure 6 A is the Michaelis-Menten equation of Mn3O4 (different set concentrations of TMB corresponding to the substrate); Figure 6 B is the Lineweaver-Burk curve. Figure 6 The Michaelis-Menten equation was used to fit the enzyme kinetic parameters of nanozyme (Mn3O4-type oxidase), including the Michaelis constant (Km) and the maximum reaction rate (Vmax). Figure 6 As shown in A, the Michaelis constant Km of Mn3O4 (Mn3O4 oxidase) is 0.1896mM, and the maximum reaction rate is 0.2834μmol / s. Figure 6 The data of A were used to fit the enzyme kinetic parameters of the two nanozymes using the Michaelis-Menten equation.

[0148] pass Figure 6 It can be seen that using the Michaelis equation, by different C TMB and its corresponding V to establish the A graph; using the double reciprocal equation different 1 / C TMB The corresponding 1 / V establishes the B graph. Among them, C TMB represents the concentration of TMB, and V represents the reaction rate. Figure 6 The original data corresponding to A and B in .

[0149] Figure 6 AOriginal data

[0150]

[0151] Figure 6 B. Original data

[0152]

[0153] In the embodiments of the present disclosure, the method for detecting the mechanism of oxidase activity includes: utilizing electron paramagnetic energy spectroscopy and using 5,5-dimethyl-1-pyrroline oxide as a specific probe to capture the reaction intermediates of catalyzing 3,3′,5,5′-tetramethylbenzidine in the color development process, and measuring a resonance spectrum; and determining the reaction intermediates based on the resonance spectrum.

[0154] In an embodiment of the present disclosure, the method for determining the reaction intermediate based on the resonance spectrum includes: determining different free radicals with characteristic peaks based on the resonance spectrum; and determining the different free radicals as the reaction intermediates. Wherein, the method for determining the reaction intermediate based on the resonance spectrum also includes: determining the free radical corresponding to the maximum value among the values ​​corresponding to the characteristic peaks of the different free radicals as the most important reaction intermediate among the reaction intermediates.

[0155] In the embodiments of the present disclosure and other possible embodiments, electron paramagnetic spectroscopy is used to capture reaction intermediates in the catalytic TMB color development process using 5,5-dimethyl-1-pyrroline oxide (DMPO) as a specific probe, and the resonance spectrum is measured and its characteristic peaks are analyzed to determine the presence of the reaction intermediates.

[0156] Figure 7 An electron paramagnetic resonance spectrum according to an embodiment of the present disclosure is shown; wherein, Figure A: 1 Characteristic peak of O2, Figure B: O2· - The characteristic peak of Figure C: the characteristic peak of OH. Figure 7 A, corresponding to free radicals 1 O2 characteristic peak 1:1:1, molar concentration 1.926e-07; Figure 7 B is six peaks with four large peaks and two small peaks, corresponding to the free radical O2· - The characteristic peak of , the molar concentration is 1.287e-06; Figure 7 C corresponds to the characteristic peak of free radical ·OH 1:2:2:1, and the molar concentration is 3.201e-07. The above results confirm the existence of three free radicals, and O2· - The molar concentration of O2· - It is the main reaction intermediate.

[0157] The embodiments of the present disclosure also propose a nanozyme obtained by the nanozyme preparation method as described above, or a nanozyme prepared by the nanozyme preparation device as described above, or a nanozyme as described above, and a selectivity testing method thereof, comprising: obtaining cysteine ​​corresponding to an eighth set concentration, and amino acids and / or ions corresponding to a ninth set concentration greater than the eighth set concentration; determining the selectivity of the Mn3O4-type oxidase activity based on the activity responses of cysteine ​​corresponding to the eighth set concentration and amino acids and / or ions corresponding to the ninth set concentration to the Mn3O4-type oxidase, respectively.

[0158] In the embodiments of the present disclosure and other possible embodiments, selectivity testing is an important performance indicator to measure the specificity of nanozyme detection, which determines whether the nanozyme can be used for target molecule detection. Common amino acids, ions, etc. are used to explore the substances that affect the activity of Mn3O4-type oxidases. This disclosure selects a variety of common amino acids, glucose, Na + , K + The nanozymes (Mn3O4-type oxidases) were selectively tested, and the concentration of all the test substances was 100 μM, which is 5-10 times the concentration of cysteine. Figure 8 The detection specificity of cysteine ​​according to the embodiment of the present disclosure is shown as follows: Figure 8 From the figure, it can be seen that cysteine ​​can significantly inhibit the oxidase-like activity of Mn3O4, and has almost no response to other substances. This shows that the constructed Mn3O4 nanozyme has specificity for the detection of cysteine. Figure 8 The corresponding original data are as follows.

[0159]

[0160] The present disclosure also proposes a nanozyme prepared by the preparation method as described above; or, a nanozyme prepared by the preparation device as described above; or, the application of the nanozyme as described above in cysteine ​​detection and / or the application of cysteine ​​detection in urine.

[0161] Fig. 9The detection sensitivity of cysteine ​​according to the embodiment of the present disclosure is shown; wherein, Figure A is the detection range of cysteine ​​at a TMB concentration of 20 μg / mL; Figure B is the linear fit of Figure A; Figure C is the detection range of cysteine ​​at a TMB concentration of 110 μg / mL; Figure D is the linear fit of Figure C. In the embodiment of the present disclosure and other possible embodiments, the cysteine ​​detection application of Mn3O4 nanozyme includes the following steps: (1) Preparation of cysteine ​​solution: First, weigh a specific amount of cysteine ​​powder and dissolve it in ultrapure water to obtain a cysteine ​​solution with a concentration of 1 mM. (2) Add 25 or 110 μg / mL TMB and 20 μg / mL Mn3O4 to 1 mL NaAc-HAc buffer (pH 3.5, 0.1 M). (3) Absorption detection of different concentrations of cysteine: In step 2, cysteine ​​solution was added to make the final concentrations 0, 0.5, 1, 2, 4, 8, 10, 14, 16, 18, 20, 25, 30, 35, 40, 42, 45 μM, respectively. After standing at room temperature for 10 minutes, 200 μL of the reaction solution was added to a 96-well plate, and the absorbance value of the reaction system at 652 nm was detected by an ELISA instrument; (4) Establishing a linear relationship: A standard linear regression curve was established with the added cysteine ​​concentration C as the horizontal axis and the absorbance value A as the vertical axis, such as Fig. 9 B and 9D, the linear regression equation is: A = -0.01419 (C) + 0.01221 (correlation coefficient R 2 =0.9901), the minimum detection limit of cysteine ​​can reach 0.02636μM.

[0162] The results are as follows Fig. 9 As shown in A, when 25 μg / mL TMB was added to the reaction system, the amount of TMB used was between insufficient and sufficient. After the addition of cysteine, OD652 showed a trend of first increasing and then decreasing. A standard linear regression curve was established with the added cysteine ​​concentration C as the horizontal axis and the light absorption change value A as the vertical axis, as shown in Fig. 9 B, its linear regression equation is: A = -0.01419 (C) + 0.01221 (correlation coefficient R 2 =0.9901), the minimum detection limit of cysteine ​​can reach 0.02636μM, and the linear detection range is 0.5-45μM.

[0163] When 110 μg / mL TMB was added to the reaction system, the amount of TMB used was sufficient, and after the addition of cysteine, OD652 showed a gradual downward trend, such as Fig. 9 C. A standard linear regression curve was established with the added cysteine ​​concentration C as the horizontal axis and the light absorption change value A as the vertical axis, such as Fig. 9 D, its linear regression equation is: A = 0.02534 (C) + 0.0526 (correlation coefficient R2 =0.9881), the minimum detection limit of cysteine ​​can reach 0.23 μM, and the linear detection range is 2-60 μM. Fig. 9 The original data corresponding to AD are as follows.

[0164] Figure 7 The original data of A

[0165]

[0166] Figure 7 The original data of B

[0167]

[0168]

[0169] Figure 7 The original data of C

[0170]

[0171] Figure 7 The original data of D

[0172]

[0173]

[0174] After comparing the above results, it was found that when the amount of TMB used in the reaction system was between insufficient and sufficient, that is, when TMB was added at 25 μg / mL, the minimum detection limit of cysteine ​​could reach 0.02636 μM, which was nearly 10 times higher than when the amount of TMB used was sufficient, that is, when TMB was added at 110 μg / mL, and the linear detection range span was larger.

[0175] The execution subject of the nanozyme preparation, nanozyme detection, and nanozyme selectivity testing method can be a nanozyme preparation device, a nanozyme activity detection device, and a nanozyme selectivity testing device. For example, the nanozyme preparation method, the nanozyme detection method, and the nanozyme selectivity testing method can be executed by a terminal device or a server or other processing device, wherein the terminal device can be a user equipment (UE), a mobile device, a user terminal, a terminal, a cellular phone, a cordless phone, a personal digital assistant (PDA), a handheld device, a computing device, a vehicle-mounted device, a wearable device, etc. In some possible implementations, the nanozyme preparation method, the nanozyme detection method, and the nanozyme selectivity testing method can be implemented by a processor calling a computer-readable instruction stored in a memory.

[0176] Those skilled in the art can understand that in the above-mentioned nanozyme preparation, nanozyme detection, and nanozyme selectivity testing methods of the specific embodiments, the writing order of each step does not mean a strict execution order and does not constitute any limitation on the implementation process. The specific execution order of each step should be determined by its function and possible internal logic.

[0177] The disclosed embodiment also proposes a nanozyme preparation device, comprising: a first mixing and stirring unit, used to obtain a Mn(NO3)2 stirring solution and an aminoethanol stirring solution respectively, and mix the Mn(NO3)2 stirring solution and the aminoethanol stirring solution to obtain a mixed solution; a second mixing and stirring unit, used to stir the mixed solution at a set rate and a third set time under an inert atmosphere to obtain a stirred mixed solution; an aging treatment unit, used to perform an aging treatment on the stirred mixed solution according to a fourth set time to obtain a corresponding suspension; a filtration, washing and drying treatment unit, used to perform a filtration, washing and drying treatment on the suspension to obtain a Mn3O4-type oxidase.

[0178] The disclosed embodiment also proposes a nanozyme preparation device, comprising: a processor; a memory for storing instructions executable by the processor; wherein the processor is configured to call the instructions stored in the memory to execute the above-mentioned nanozyme preparation method.

[0179] The disclosed embodiment also proposes a nanozyme preparation device, comprising: a computer-readable storage medium on which computer program instructions are stored, and the computer program instructions implement the above-mentioned nanozyme preparation method when executed by a processor.

[0180] The disclosed embodiment also proposes a nanozyme preparation device, including: a computer program product, including a computer program / instruction, which implements the above-mentioned nanozyme preparation method when executed by a processor.

[0181] The disclosed embodiments also propose a device for detecting the nanozyme activity and / or enzyme kinetics and / or oxidase-like activity mechanism of a nanozyme obtained by the nanozyme preparation method as described above, or a nanozyme prepared by the nanozyme preparation device as described above, or a nanozyme corresponding to the nanozyme as described above.

[0182] Among them, in an embodiment of the present disclosure, the nanoenzyme activity detection device includes: a first acquisition unit, used to obtain a Mn3O4-type oxidase solution of a third set concentration and a 3,3',5,5'-tetramethylbenzidine and dimethyl sulfoxide solution of a fourth set concentration; a first liquid preparation unit, used to add a 3,3',5,5'-tetramethylbenzidine and dimethyl sulfoxide solution corresponding to a sixth set concentration less than the fourth set concentration and a Mn3O4-type oxidase solution corresponding to a seventh set concentration less than the third set concentration to a NaAc-HAc buffer solution of a third set volume, a fifth set concentration and a set pH value to obtain a solution to be detected; a light absorption value determination unit, used to place the solution to be detected for a sixth set time at a second set temperature, and then detect the light absorption value of the reaction system corresponding to the solution to be detected at a set wavelength through an enzyme marker; an activity detection unit, used to detect the activity of the Mn3O4-type oxidase based on the color corresponding to the solution to be detected and / or the light absorption value at the set wavelength.

[0183] In an embodiment of the present disclosure, the nanozyme activity detection device comprises: a processor; a memory for storing instructions executable by the processor; wherein the processor is configured to call the instructions stored in the memory to execute the above-mentioned nanozyme activity detection method.

[0184] Among them, in an embodiment of the present disclosure, the nanozyme activity detection device includes: a computer program product, including a computer program / instruction, which implements the above-mentioned nanozyme activity detection method when executed by a processor.

[0185] Among them, in an embodiment of the present disclosure, the nanozyme activity detection device includes: a computer-readable storage medium on which computer program instructions are stored, and the computer program instructions implement the above-mentioned nanozyme activity detection method when executed by a processor.

[0186] Among them, in the embodiment of the present disclosure, the nanozyme kinetics detection device includes: a second acquisition unit, which is used to obtain a Mn3O4-type oxidase solution of a third set concentration and 3,3',5,5'-tetramethylbenzidine and dimethyl sulfoxide solutions of different set concentrations within the third set concentration range; a second liquid preparation unit, which is used to add 3,3',5,5'-tetramethylbenzidine and dimethyl sulfoxide solutions corresponding to a sixth set concentration less than the fourth set concentration and different set concentrations within the third set concentration range less than the three set concentrations into a NaAc-HAc buffer solution of a third set volume, a fifth set concentration and a set pH value. A Mn3O4-type oxidase solution corresponding to the degree is obtained to obtain a solution to be detected with different set concentrations within the third set concentration range; a first determining unit is used to respectively determine a plurality of reaction rates corresponding to different set concentrations of 3,3',5,5'-tetramethylbenzidine within the third set concentration range in the solution to be detected; a second determining unit is used to fit the different set concentrations of 3,3',5,5'-tetramethylbenzidine within the third set concentration range and the corresponding plurality of reaction rates using the Michaelis-Menten equation, and determine a speed curve of the relationship between the plurality of reaction rates of the enzymatic reaction and the different set concentrations within the third set concentration range.

[0187] Among them, in an embodiment of the present disclosure, the nanozyme kinetic detection device includes: a processor; a memory for storing processor executable instructions; wherein the processor is configured to call the instructions stored in the memory to execute the above-mentioned nanozyme kinetic detection method.

[0188] Among them, in an embodiment of the present disclosure, the nanozyme kinetic detection device includes: a computer-readable storage medium on which computer program instructions are stored, and the computer program instructions implement the above-mentioned nanozyme kinetic detection method when executed by a processor.

[0189] Among them, in an embodiment of the present disclosure, the nanozyme kinetic detection device includes: a computer program product, including a computer program / instruction, which implements the above-mentioned nanozyme kinetic detection method when executed by a processor.

[0190] Among them, in the embodiments of the present disclosure, the oxidase-like activity mechanism detection device includes: a resonance spectrum measurement unit, which is used to use electron paramagnetic energy spectrum to capture the reaction intermediates of catalyzing 3,3′,5,5′-tetramethylbenzidine in the color development process with 5,5-dimethyl-1-pyrroline oxide as a specific probe to measure the resonance spectrum; a third determination unit, based on the resonance spectrum, determines the reaction intermediates; or, includes: a processor; a memory for storing processor executable instructions; wherein the processor is configured to call the instructions stored in the memory to execute the above-mentioned oxidase-like activity mechanism detection method; or, includes: a computer-readable storage medium on which computer program instructions are stored, and when the computer program instructions are executed by the processor, the above-mentioned oxidase-like activity mechanism detection method is implemented; or, includes: a computer program product, including a computer program / instruction, and when the computer program / instruction is executed by the processor, the above-mentioned oxidase-like activity mechanism detection method is implemented.

[0191] The disclosed embodiments also propose a nanozyme obtained by the nanozyme preparation method as described above, or a nanozyme prepared by the nanozyme preparation device as described above, or a nanozyme selectivity testing device corresponding to the nanozyme as described above, comprising: a third acquisition unit, used to obtain cysteine ​​corresponding to an eighth set concentration, and amino acids and / or ions corresponding to a ninth set concentration greater than the eighth set concentration; a selectivity determination unit, used to determine the selectivity of the Mn3O4-type oxidase activity based on the activity responses of cysteine ​​corresponding to the eighth set concentration and amino acids and / or ions corresponding to the ninth set concentration to the Mn3O4-type oxidase, respectively.

[0192] The disclosed embodiments also propose the application of the nanozyme obtained by the nanozyme preparation method as described above, or the nanozyme prepared by the nanozyme preparation device as described above, or the nanozyme corresponding to the nanozyme as described above in cysteine ​​detection and / or in cysteine ​​detection in urine.

[0193] Based on the above, the purpose of the present invention is to provide a preparation of a sheet-like Mn3O4-like oxidase and on this basis innovate a homocysteine ​​detection method to solve the problems raised in the above-mentioned background technology. The detection principle of the present invention is: the Mn3O4 nanozyme with an oxidase-like enzyme can oxidize 3,3',5,5'-tetramethylbenzidine (TMB) to develop color and generate oxTMB. When the substrate TMB is sufficient, the Mn3O4 nanozyme can completely oxidize TMB, and each TMB loses one electron to form a blue product. When the substrate TMB is insufficient, the Mn3O4 nanozyme can oxidize part of the TMB, and each oxidized TMB will lose two electrons and polymerize to produce a yellow product. When the concentration of the substrate TMB is between the two, the substrate TMB will lose one or two electrons, and the color may appear green, which is the result of a mixture of blue and yellow.

[0194] Cysteine ​​contains sulfhydryl groups, which inhibit the activity of Mn3O4-type oxidases and can reduce blue, yellow or green oxTMB to colorless and transparent TMB. Green oxTMB indicates that there may be a mixture of yellow oxTMB and blue oxTMB oxidation forms at the same time. After adding cysteine, due to its reducing properties, both yellow and blue oxTMB are reduced. Yellow oxTMB obtains an electron and is converted into blue oxTMB, while blue oxTMB is reduced and gradually loses its blue color after obtaining an electron. In this process, since blue oxTMB has a maximum absorption peak at 652 nanometers (this is the property of oxTMB itself, this substance has a maximum absorption peak at 652 nanometers, which is the characteristic peak of this substance, and is a feature different from other substances), when cysteine ​​is added, the absorption peak will appear enhanced, then weakened and finally disappeared. The present invention utilizes this principle, the principle that cysteine ​​reduces green oxTMB to colorless and transparent TMB, and realizes the highly sensitive detection of cysteine ​​by Mn3O4 nanozyme by colorimetric detection of the absorbance value at 652nm.

[0195] At the same time, the present invention provides the following inferred mechanism of the oxidase-like activity of Mn3O4: Mn3O4 can catalyze the dissolved oxygen adsorbed on its surface to produce O2 - , O2 - It is extremely unstable in aqueous solution and will react quickly to produce H2O2 and 1 O2, H2O2 and O2 - The reaction generates ·OH. In addition, Mn3O4 can also directly catalyze H2O2 to generate O2 by a Fenton-like reaction. - and OH.

[0196] Compared with the prior art, the beneficial effects of the present invention are as follows: 1. The preparation method of the Mn3O4 oxidase is simpler, and when applied to cysteine ​​detection, the detection time is shorter, with a total time of about 10 minutes. 2. Compared with traditional fluorescence methods, spectroscopy, chromatography and other methods, the colorimetric method used by the Mn3O4 oxidase has the characteristics of low cost, easy operation, short time and no sample processing, which greatly overcomes the shortcomings of the traditional method of cumbersome sample pre-processing, expensive instruments and equipment, requiring professional technicians to operate, and high operating costs. 3. The sheet-like Mn3O4 oxidase is the first to achieve high-sensitivity detection of cysteine ​​content by regulating the amount of substrate TMB used in the cysteine ​​detection method and adjusting the electron transfer in the reaction system. The detection limit is as low as 0.02636μM, and the detection linear range is 0.5-45μM. Compared with the detection system with sufficient TMB usage currently used, the detection limit is increased by nearly ten times, and the linear detection range is wider. 4. Compared with nanomaterials with peroxidase-like activity that can be used to detect cysteine, the preparation and application of the Mn3O4-type oxidase in cysteine ​​detection does not require the participation of unstable hydrogen peroxide and has higher detection accuracy.

[0197] In some embodiments, the functions or modules included in the device provided in the embodiments of the present disclosure can be used to execute the nanozyme preparation method, nanozyme detection method, and nanozyme selectivity testing method described in the above method embodiments. The specific implementation can refer to the description of the above nanozyme preparation method, nanozyme detection method, and nanozyme selectivity testing method embodiments. For the sake of brevity, they will not be repeated here.

[0198] The present disclosure also provides a computer-readable storage medium on which computer program instructions are stored, and when the computer program instructions are executed by a processor, one or more of the above-mentioned nanozyme preparation, nanozyme detection, and nanozyme selectivity test methods are implemented. The computer-readable storage medium can be a non-volatile computer-readable storage medium.

[0199] The disclosed embodiment also proposes an electronic device, comprising: a processor; a memory for storing instructions executable by the processor; wherein the processor is configured to perform one or more of the above-mentioned nanozyme preparation, nanozyme detection, and nanozyme selectivity test methods. The electronic device can be provided as a terminal, a server, or other forms of equipment.

[0200] The disclosed embodiments also provide a computer program product, including a computer program / instruction, which, when executed by a processor, implements one or more of the above-mentioned nanozyme preparation, nanozyme detection, and nanozyme selectivity testing methods.

[0201] Fig.108 is a block diagram of an electronic device 800 according to an exemplary embodiment. For example, the electronic device 800 may be a mobile phone, a computer, a digital broadcast terminal, a messaging device, a game console, a tablet device, a medical device, a fitness device, a personal digital assistant, or the like.

[0202] Reference Fig.10 , the electronic device 800 may include one or more of the following components: a processing component 802 , a memory 804 , a power component 806 , a multimedia component 808 , an audio component 810 , an input / output (I / O) interface 812 , a sensor component 814 , and a communication component 816 .

[0203] The processing component 802 generally controls the overall operation of the electronic device 800, such as operations associated with display, phone calls, data communications, camera operations, and recording operations. The processing component 802 may include one or more processors 820 to execute instructions to complete all or part of the steps of the above-mentioned method. In addition, the processing component 802 may include one or more modules to facilitate the interaction between the processing component 802 and other components. For example, the processing component 802 may include a multimedia module to facilitate the interaction between the multimedia component 808 and the processing component 802.

[0204] The memory 804 is configured to store various types of data to support operations on the electronic device 800. Examples of such data include instructions for any application or method operating on the electronic device 800, contact data, phone book data, messages, pictures, videos, etc. The memory 804 can be implemented by any type of volatile or non-volatile storage device or a combination thereof, such as static random access memory (SRAM), electrically erasable programmable read-only memory (EEPROM), erasable programmable read-only memory (EPROM), programmable read-only memory (PROM), read-only memory (ROM), magnetic memory, flash memory, magnetic disk or optical disk.

[0205] The power supply component 806 provides power to the various components of the electronic device 800. The power supply component 806 may include a power management system, one or more power supplies, and other components associated with generating, managing, and distributing power to the electronic device 800.

[0206] The multimedia component 808 includes a screen that provides an output interface between the electronic device 800 and the user. In some embodiments, the screen may include a liquid crystal display (LCD) and a touch panel (TP). If the screen includes a touch panel, the screen may be implemented as a touch screen to receive input signals from the user. The touch panel includes one or more touch sensors to sense touch, slide, and gestures on the touch panel. The touch sensor may not only sense the boundaries of the touch or slide action, but also detect the duration and pressure associated with the touch or slide operation. In some embodiments, the multimedia component 808 includes a front camera and / or a rear camera. When the electronic device 800 is in an operating mode, such as a shooting mode or a video mode, the front camera and / or the rear camera may receive external multimedia data. Each front camera and rear camera may be a fixed optical lens system or have a focal length and optical zoom capability.

[0207] The audio component 810 is configured to output and / or input audio signals. For example, the audio component 810 includes a microphone (MIC), and when the electronic device 800 is in an operating mode, such as a call mode, a recording mode, and a voice recognition mode, the microphone is configured to receive an external audio signal. The received audio signal can be further stored in the memory 804 or sent via the communication component 816. In some embodiments, the audio component 810 also includes a speaker for outputting audio signals.

[0208] I / O interface 812 provides an interface between processing component 802 and peripheral interface modules, such as keyboards, click wheels, buttons, etc. These buttons may include but are not limited to: home button, volume button, start button, and lock button.

[0209] The sensor assembly 814 includes one or more sensors for providing various aspects of status assessment for the electronic device 800. For example, the sensor assembly 814 can detect the open / closed state of the electronic device 800, the relative positioning of the components, such as the display and keypad of the electronic device 800, and the sensor assembly 814 can also detect the position change of the electronic device 800 or a component of the electronic device 800, the presence or absence of contact between the user and the electronic device 800, the orientation or acceleration / deceleration of the electronic device 800, and the temperature change of the electronic device 800. The sensor assembly 814 may include a proximity sensor configured to detect the presence of a nearby object without any physical contact. The sensor assembly 814 may also include an optical sensor, such as a CMOS or CCD image sensor, for use in imaging applications. In some embodiments, the sensor assembly 814 may also include an acceleration sensor, a gyroscope sensor, a magnetic sensor, a pressure sensor, or a temperature sensor.

[0210] The communication component 816 is configured to facilitate wired or wireless communication between the electronic device 800 and other devices. The electronic device 800 can access a wireless network based on a communication standard, such as WiFi, 2G or 3G, or a combination thereof. In an exemplary embodiment, the communication component 816 receives a broadcast signal or broadcast-related information from an external broadcast management system via a broadcast channel. In an exemplary embodiment, the communication component 816 also includes a near field communication (NFC) module to facilitate short-range communication. For example, the NFC module can be implemented based on radio frequency identification (RFID) technology, infrared data association (IrDA) technology, ultra-wideband (UWB) technology, Bluetooth (BT) technology and other technologies.

[0211] In an exemplary embodiment, the electronic device 800 may be implemented by one or more application-specific integrated circuits (ASICs), digital signal processors (DSPs), digital signal processing devices (DSPDs), programmable logic devices (PLDs), field programmable gate arrays (FPGAs), controllers, microcontrollers, microprocessors, or other electronic components to perform the above methods.

[0212] In an exemplary embodiment, a non-volatile computer-readable storage medium is also provided, such as a memory 804 including computer program instructions, which can be executed by a processor 820 of an electronic device 800 to perform the above method.

[0213] Fig.11 1 is a block diagram of an electronic device 1900 according to an exemplary embodiment. For example, the electronic device 1900 may be provided as a server. Fig.11 , the electronic device 1900 includes a processing component 1922, which further includes one or more processors, and a memory resource represented by a memory 1932 for storing instructions executable by the processing component 1922, such as an application. The application stored in the memory 1932 may include one or more modules, each corresponding to a set of instructions. In addition, the processing component 1922 is configured to execute instructions to perform the above method.

[0214] The electronic device 1900 may also include a power supply component 1926 configured to perform power management of the electronic device 1900, a wired or wireless network interface 1950 configured to connect the electronic device 1900 to a network, and an input / output (I / O) interface 1958. The electronic device 1900 may operate based on an operating system stored in the memory 1932, such as Windows Server™, Mac OS X™, Unix™, Linux™, FreeBSD™ or the like.

[0215] In an exemplary embodiment, a non-volatile computer-readable storage medium is also provided, such as a memory 1932 including computer program instructions, which can be executed by the processing component 1922 of the electronic device 1900 to perform the above method.

[0216] The present disclosure may be a system, a method and / or a computer program product. The computer program product may include a computer-readable storage medium carrying computer-readable program instructions for causing a processor to implement various aspects of the present disclosure.

[0217] A computer-readable storage medium may be a tangible device that can hold and store instructions used by an instruction execution device. A computer-readable storage medium may be, for example, but not limited to, an electrical storage device, a magnetic storage device, an optical storage device, an electromagnetic storage device, a semiconductor storage device, or any suitable combination of the foregoing. More specific examples of computer-readable storage media (a non-exhaustive list) include: a portable computer disk, a hard disk, a random access memory (RAM), a read-only memory (ROM), an erasable programmable read-only memory (EPROM or flash memory), a static random access memory (SRAM), a portable compact disk read-only memory (CD-ROM), a digital versatile disk (DVD), a memory stick, a floppy disk, a mechanical encoding device, such as a punch card or a raised structure in a groove on which instructions are stored, and any suitable combination of the foregoing. As used herein, a computer-readable storage medium is not to be interpreted as a transient signal per se, such as a radio wave or other freely propagating electromagnetic wave, an electromagnetic wave propagating through a waveguide or other transmission medium (e.g., a light pulse through a fiber optic cable), or an electrical signal transmitted through a wire.

[0218] The computer-readable program instructions described herein can be downloaded from a computer-readable storage medium to each computing / processing device, or downloaded to an external computer or external storage device via a network, such as the Internet, a local area network, a wide area network, and / or a wireless network. The network can include copper transmission cables, optical fiber transmissions, wireless transmissions, routers, firewalls, switches, gateway computers, and / or edge servers. The network adapter card or network interface in each computing / processing device receives the computer-readable program instructions from the network and forwards the computer-readable program instructions for storage in the computer-readable storage medium in each computing / processing device.

[0219] The computer program instructions for performing the operation of the present disclosure may be assembly instructions, instruction set architecture (ISA) instructions, machine instructions, machine-related instructions, microcode, firmware instructions, state setting data, or source code or object code written in any combination of one or more programming languages, including object-oriented programming languages, such as Smalltalk, C++, etc., and conventional procedural programming languages, such as "C" language or similar programming languages. Computer-readable program instructions may be executed completely on a user's computer, partially on a user's computer, as an independent software package, partially on a user's computer, partially on a remote computer, or completely on a remote computer or server. In the case of a remote computer, the remote computer may be connected to the user's computer via any type of network, including a local area network (LAN) or a wide area network (WAN), or may be connected to an external computer (e.g., using an Internet service provider to connect via the Internet). In some embodiments, an electronic circuit, such as a programmable logic circuit, a field programmable gate array (FPGA), or a programmable logic array (PLA), may be customized by utilizing the state information of the computer-readable program instructions, and the electronic circuit may execute the computer-readable program instructions, thereby realizing various aspects of the present disclosure.

[0220] Various aspects of the present disclosure are described herein with reference to the flowcharts and / or block diagrams of the methods, devices (systems) and computer program products according to the embodiments of the present disclosure. It should be understood that each box in the flowchart and / or block diagram and the combination of each box in the flowchart and / or block diagram can be implemented by computer-readable program instructions.

[0221] These computer-readable program instructions can be provided to a processor of a general-purpose computer, a special-purpose computer, or other programmable data processing device, thereby producing a machine, so that when these instructions are executed by the processor of the computer or other programmable data processing device, a device that implements the functions / actions specified in one or more boxes in the flowchart and / or block diagram is generated. These computer-readable program instructions can also be stored in a computer-readable storage medium, and these instructions cause the computer, programmable data processing device, and / or other equipment to work in a specific manner, so that the computer-readable medium storing the instructions includes a manufactured product, which includes instructions for implementing various aspects of the functions / actions specified in one or more boxes in the flowchart and / or block diagram.

[0222] Computer-readable program instructions may also be loaded onto a computer, other programmable data processing apparatus, or other device so that a series of operating steps are performed on the computer, other programmable data processing apparatus, or other device to produce a computer-implemented process, thereby causing the instructions executed on the computer, other programmable data processing apparatus, or other device to implement the functions / actions specified in one or more boxes in the flowchart and / or block diagram.

[0223] The flow chart and block diagram in the accompanying drawings show the possible architecture, function and operation of the system, method and computer program product according to multiple embodiments of the present disclosure. In this regard, each square box in the flow chart or block diagram can represent a part of a module, program segment or instruction, and a part of the module, program segment or instruction includes one or more executable instructions for realizing the specified logical function. In some alternative implementations, the function marked in the square box can also occur in a sequence different from that marked in the accompanying drawings. For example, two continuous square boxes can actually be executed substantially in parallel, and they can sometimes be executed in reverse order, depending on the functions involved. It should also be noted that each square box in the block diagram and / or flow chart, and the combination of the square boxes in the block diagram and / or flow chart can be implemented with a dedicated hardware-based system that performs the specified function or action, or can be implemented with a combination of special hardware and computer instructions.

[0224] The embodiments of the present disclosure have been described above, and the above description is exemplary, not exhaustive, and is not limited to the disclosed embodiments. Many modifications and variations will be apparent to those of ordinary skill in the art without departing from the scope and spirit of the described embodiments. The selection of terms used herein is intended to best explain the principles of the embodiments, practical applications, or technical improvements to the technology in the market, or to enable other persons of ordinary skill in the art to understand the embodiments disclosed herein.

Claims

1. A method for preparing nanozyme, It is characterized in that include: Get Mn(NO 3 ) 2 Stirring the solution and aminoethanol stirring the solution, the Mn(NO 3 ) 2 The stirring solution and the aminoethanol stirring solution are mixed to obtain a mixed solution; Under an inert atmosphere, stirring the mixed solution at a set rate and for a third set time to obtain a stirred mixed solution; Performing an aging treatment on the stirred mixed solution according to a fourth set time to obtain a corresponding suspension; The suspension is filtered, washed and dried to obtain Mn 3 O 4 Oxidase-like enzymes.

2. The method for preparing the nanozyme according to claim 1, It is characterized in that In the acquisition of Mn(NO 3 ) 2 Before stirring the solution and the aminoethanol stirring solution, prepare the first set volume and the first set concentration of Mn(NO 3 ) 2 solution and an aminoethanol solution corresponding to a second set volume and a second set concentration; under the inert atmosphere, the Mn(NO 3 ) 2 The solution and the aminoethanol solution are stirred for a first set time and a second time to obtain the corresponding Mn(NO 3 ) 2 stirring solution and aminoethanol stirring solution; and / or, The first set volume and the second set volume have the same numerical value, and the second set concentration has a numerical value that is twice the numerical value of the first set concentration. and / or, The first set volume and the first set concentration are configured as 10 ml and 0.4 mM respectively, and the second set volume and the second set concentration are configured as 10 ml and 0.8 mM respectively; and / or, The third setting time may be configured as 3 hours; and / or, the fourth setting time may be configured as 24 hours; and / or, the inert atmosphere may be configured as an argon atmosphere; and / or, The suspension is filtered, washed and dried to obtain Mn 3 O 4 The method comprises: filtering and washing the suspension, and drying the filtered and washed suspension at a first set temperature and a fifth set time to obtain Mn 3 O 4 Oxidase-like enzymes; and / or, The first set temperature is configured as 60°C; and / or, the fifth set time is configured as 24 hours; and / or, The method of performing an aging treatment on the stirred mixed solution according to a fourth set time to obtain a corresponding suspension includes: within the fourth set time, real-time detection of a first color corresponding to the suspension; if the first color changes to gray-brown, determining that the aging treatment of the stirred mixed solution is completed and the corresponding suspension is obtained.

3. A nanozyme preparation device, It is characterized in that include: The first mixing and stirring unit is used to obtain Mn(NO 3 ) 2 Stirring the solution and aminoethanol stirring the solution, the Mn(NO 3 ) 2 The stirring solution and the aminoethanol stirring solution are mixed to obtain a mixed solution; the second mixing and stirring unit is used to stir the mixed solution at a set rate and a third set time under an inert atmosphere to obtain a stirred mixed solution; An aging treatment unit, used for performing an aging treatment on the stirred mixed solution according to a fourth set time to obtain a corresponding suspension; The filtration, washing and drying treatment unit is used to filter, wash and dry the suspension to obtain Mn 3 O 4 Oxidase-like enzyme; or, comprising: a processor; a memory for storing processor-executable instructions; wherein the processor is configured to call the instructions stored in the memory to execute the nanozyme preparation method described in any one of claims 1 to 2; or, comprising: a computer-readable storage medium having computer program instructions stored thereon, wherein the computer program instructions, when executed by the processor, implement the nanozyme preparation method described in any one of claims 1 to 2; or, comprising: a computer program product, comprising a computer program / instructions, wherein the computer program / instructions, when executed by the processor, implement the nanozyme preparation method described in any one of claims 1 to 2.

4. A nanozyme, It is characterized in that The nanozyme is prepared by using the nanozyme preparation method according to any one of claims 1-2 or the nanozyme preparation device according to claim 3.

5. A method for detecting the activity and / or enzyme kinetics and / or oxidase-like activity mechanism of a nanozyme obtained by the method for preparing a nanozyme according to any one of claims 1 to 2, or a nanozyme prepared by the nanozyme preparation device according to claim 3, or a nanozyme according to claim 4, It is characterized in that The activity detection of the nanozyme comprises: obtaining a third set concentration of Mn 3 O 4 oxidase solution and a fourth set concentration of 3,3',5,5'-tetramethylbenzidine and dimethyl sulfoxide solution; add a sixth set concentration of 3,3',5,5'-tetramethylbenzidine and dimethyl sulfoxide solution less than the fourth set concentration and a seventh set concentration of Mn corresponding to the third set concentration to the third set volume, fifth set concentration and set pH value NaAc-HAc buffer. 3 O 4 The oxidase-like solution is prepared to obtain a solution to be detected; after the solution to be detected is placed for a sixth set time at a second set temperature, the light absorption value of the reaction system corresponding to the solution to be detected for activity is detected by an ELISA instrument at a set wavelength; the Mn is detected based on the color corresponding to the solution to be detected and / or the light absorption value at the set wavelength. 3 O 4 oxidase-like activity; and / or, The enzyme kinetics detection method comprises: obtaining a third set concentration of Mn 3 O 4 oxidase solution and 3,3',5,5'-tetramethylbenzidine and dimethyl sulfoxide solutions of different set concentrations within the third set concentration range; 3,3',5,5'-tetramethylbenzidine and dimethyl sulfoxide solutions corresponding to a sixth set concentration less than the fourth set concentration and Mn corresponding to different set concentrations within the third set concentration range less than the third set concentration are added to the NaAc-HAc buffer solution of the third set volume, the fifth set concentration and the set pH value. 3 O 4 oxidase-like solution, obtaining solutions to be tested with different set concentrations within the third set concentration range; respectively determining multiple reaction rates corresponding to different set concentrations of 3,3',5,5'-tetramethylbenzidine within the third set concentration range in the solution to be tested; using the Michaelis-Menten equation, fitting the different set concentrations of 3,3',5,5'-tetramethylbenzidine within the third set concentration range and the corresponding multiple reaction rates, and determining the speed curve of the relationship between the multiple reaction rates of the enzymatic reaction and the different set concentrations within the third set concentration range; and / or, The method for detecting the mechanism of oxidase-like activity comprises: utilizing electron paramagnetic energy spectrum, taking 5,5-dimethyl-1-pyrroline oxide as a specific probe to capture the reaction intermediates in the color development process of catalyzing 3,3′,5,5′-tetramethylbenzidine, and measuring a resonance spectrum; and determining the reaction intermediates based on the resonance spectrum.

6. The method for detecting nanozyme activity and / or enzyme kinetics and / or oxidase-like activity mechanism according to claim 5, It is characterized in that In the step of obtaining the third set concentration of Mn 3 O 4 Before preparing the oxidase solution, weigh the first mass of Mn 3 O 4 The powder sample of the oxidase-like enzyme is dispersed in ultrapure water to prepare the third set concentration of Mn 3 O 4 an oxidase-like solution; and / or, Before obtaining the 3,3',5,5'-tetramethylbenzidine and dimethyl sulfoxide solution of the fourth set concentration, weigh a second mass of 3,3',5,5'-tetramethylbenzidine powder, dissolve the powder in dimethyl sulfoxide, and prepare the 3,3',5,5'-tetramethylbenzidine and dimethyl sulfoxide solution of the fourth set concentration; and / or The third set concentration is configured as 1 mg / mL; and / or, the fourth set concentration is configured as 10 mg / mL; and / or, the sixth set concentration is configured as 25 μg / mL or 110 μg / mL; and / or, the seventh set concentration is configured as 20 μg / mL; and / or, the method for determining the seventh set concentration includes: obtaining the Mn corresponding to the first different set concentrations within the first set concentration range; 3 O 4 Determine the Mn corresponding to the first different set concentrations 3 O 4 different colors of the solution to be detected corresponding to the oxidase-like solution; configuring the concentration corresponding to the darkest color among the first different colors or the adjacent colors among the first different colors remaining unchanged as the seventh set concentration; and / or, The method for determining the sixth set concentration includes: obtaining a 3,3',5,5'-tetramethylbenzidine and dimethyl sulfoxide solution corresponding to a second different set concentration within the second set concentration range; 3 O 4 Under the oxidase-like solution, prepare the test solutions of 3,3',5,5'-tetramethylbenzidine and dimethyl sulfoxide solution corresponding to the second different set concentrations respectively; at the second set temperature, the test solutions corresponding to the second different set concentrations are placed for a sixth set time, and then the multiple light absorption values ​​of the reaction system corresponding to the solution to be detected for activity are detected by an ELISA instrument at a set wavelength; the set concentration corresponding to the maximum peak value of the multiple light absorption values ​​corresponding to the second different set concentrations is configured as the sixth set concentration; or, at the second set temperature, the test solutions corresponding to the second different set concentrations are placed for a sixth set time, and then the multiple colors corresponding to the second different set concentrations are determined; the set concentration corresponding to the darkest color or the unchanged adjacent colors or the green color among the multiple colors corresponding to the second different set concentrations is configured as the sixth set concentration; and / or, The method for determining the set pH value comprises: obtaining NaAc-HAc buffer solutions corresponding to different pH values ​​within the set pH value range; determining a plurality of color development values ​​corresponding to the NaAc-HAc buffer solutions with different pH values; configuring the pH value corresponding to the best color development among the plurality of color development values ​​as the set pH value; and / or, The method for determining the sixth set time comprises: determining a plurality of colors corresponding to the solution to be detected at a plurality of set time points at a second set temperature; configuring a set time point corresponding to a color tending to be stable among the plurality of colors as the sixth set time; and / or, The method for determining the second set temperature comprises: after placing the solution to be detected for a sixth set time at multiple set temperatures, detecting multiple light absorption values ​​of the reaction system corresponding to the solution to be detected for activity at a set wavelength by using an ELISA instrument; configuring the set temperature corresponding to any two indifferent light absorption values ​​among the multiple light absorption values ​​as the second set temperature; and / or, The third set volume is configured as 1 mL; and / or, the fifth set concentration is configured as 0.1 M or 0.2 M; and / or, the set pH value is configured as 3.5; and / or, the second set temperature is configured as room temperature or 5°C or 25°C; and / or, the sixth set time is configured as 5 minutes; and / or, the set wavelength is configured as 652 nm; and / or, Before detecting the light absorption value of the reaction system corresponding to the solution to be detected by the microplate reader at the set wavelength, the method further includes: absorbing the solution to be detected by a fourth set volume, adding the solution to be detected by the fourth set volume to the well plate, and detecting the light absorption value of the reaction system corresponding to the solution to be detected by the microplate reader at the set wavelength; and / or, the fourth set volume is configured to be 200 μL; and / or, The Mn is detected based on the color corresponding to the solution to be detected 3 O 4 The method for detecting the activity of a class of oxidases comprises: detecting in real time the second color corresponding to the solution to be detected; if the second color changes to blue, determining that the Mn 3 O 4 The oxidase-like enzyme is active; or, The Mn is detected based on the light absorption value of the solution to be detected at a set wavelength. 3 O 4 The method for measuring the activity of an oxidase-like enzyme comprises: obtaining a light absorption value corresponding to each wavelength within a set wavelength range including a set wavelength; drawing a corresponding wavelength-light absorption value curve based on the light absorption value corresponding to each wavelength; determining a peak value corresponding to the light absorption value at the set wavelength based on the wavelength-light absorption value curve; if the peak value is a maximum peak value, determining the Mn 3 O 4 The oxidase-like enzyme is active; or, The Mn is detected based on the color corresponding to the solution to be detected and the light absorption value at a set wavelength. 3 O 4 The method for detecting the activity of a type oxidase comprises: detecting the second color corresponding to the solution to be detected in real time; if the second color changes to blue, generating a first detection condition; obtaining the light absorption value corresponding to each wavelength within a set wavelength range including the set wavelength; drawing a corresponding wavelength-light absorption value curve based on the light absorption value corresponding to each wavelength; determining the peak value corresponding to the light absorption value at the set wavelength based on the wavelength-light absorption value curve; if the peak value is the maximum peak value, generating a second detection condition; if both the first detection condition and the second detection condition are satisfied, determining the Mn 3 O 4 The oxidase-like enzyme is active; and / or, For the Mn 3 O 4 The activity verification of the oxidase-like enzyme comprises: obtaining a first solution to be detected and a second solution to be detected; introducing an air atmosphere and a nitrogen atmosphere into the first solution to be detected and the second solution to be detected respectively, and using the microplate reader to detect the first light absorption value and the second absorption value of the reaction system corresponding to the first solution to be detected introduced into the air atmosphere and the second solution to be detected introduced into the nitrogen atmosphere at a set wavelength; determining the Mn based on the first light absorption value and the second absorption value. 3 O 4 and / or, determining the Mn based on the first light absorption value and the second absorption value. 3 O 4 The method for detecting the activity of an oxidase-like enzyme comprises: if there is a significant difference between the first light absorption value and the second absorption value, then there is an oxidase-like activity; and / or, The enzyme kinetics detection method further includes: determining the Michaelis constant and the maximum reaction rate of the enzyme kinetic parameters based on the velocity curve of the relationship between the multiple reaction rates of the enzymatic reaction and different set concentrations within the third set concentration range; and / or, The enzyme kinetics detection method further includes: taking the reciprocals of different set concentrations within the third set concentration range of the 3,3',5,5'-tetramethylbenzidine and the corresponding multiple reaction rates, respectively, to obtain multiple first reciprocals corresponding to the different set concentrations within the third set concentration range and multiple second reciprocals corresponding to the multiple reaction rates; fitting the multiple first reciprocals and the corresponding multiple second reciprocals using a double reciprocal equation to obtain a linearization curve of the Michaelis constant of the enzyme kinetics parameter; and / or, The third set concentration range is configured as 0-120 μg / mL; wherein the different set concentrations within the third set concentration range are configured as any multiple values ​​of 0-120 μg / mL; and / or, It also includes: after placing the solutions to be detected with different set concentrations within the third set concentration range for a sixth set time at the second set temperature, detecting multiple light absorption values ​​of the reaction system corresponding to the solutions to be detected with different set concentrations within the third set concentration range at a set wavelength by using an ELISA instrument; and / or, The method for determining the reaction intermediate based on the resonance spectrum includes: determining different free radicals with characteristic peaks based on the resonance spectrum; and determining the different free radicals as the reaction intermediates; and / or, The method for determining the reaction intermediate based on the resonance spectrum further includes: determining the free radical corresponding to the maximum value among the values ​​corresponding to the characteristic peaks of the different free radicals as the most important reaction intermediate among the reaction intermediates.

7. A device for detecting nanozyme activity and / or enzyme kinetics and / or oxidase-like activity mechanism corresponding to the nanozyme obtained by the nanozyme preparation method according to any one of claims 1 to 2, or the nanozyme prepared by the nanozyme preparation device according to claim 3, or the nanozyme according to claim 4, It is characterized in that The nanoenzyme activity detection device comprises: a first acquisition unit for acquiring a third set concentration of Mn 3 O 4 The first liquid preparation unit is used to add the 3,3',5,5'-tetramethylbenzidine and dimethyl sulfoxide solution corresponding to the sixth set concentration less than the fourth set concentration and the Mn corresponding to the seventh set concentration less than the third set concentration to the NaAc-HAc buffer solution of the third set volume, the fifth set concentration and the set pH value. 3 O 4 a light absorption value determining unit for detecting the light absorption value of the reaction system corresponding to the solution to be detected for activity at a set wavelength by using an ELISA instrument after placing the solution to be detected for a sixth set time at a second set temperature; an activity detection unit for detecting the Mn based on the color corresponding to the solution to be detected and / or the light absorption value at the set wavelength. 3 O 4 oxidase-like activity; or, comprising: a processor; a memory for storing processor executable instructions; wherein the processor is configured to call the instructions stored in the memory to execute the nanozyme activity detection method described in any one of claims 5 to 6; or, comprising: a computer-readable storage medium having computer program instructions stored thereon, wherein the computer program instructions are executed by the processor to implement the nanozyme activity detection method described in any one of claims 5 to 6; or, comprising: a computer program product, comprising a computer program / instruction, wherein the computer program / instruction is executed by the processor to implement the nanozyme activity detection method described in any one of claims 5 to 6; and / or, The nanozyme kinetics detection device comprises: a second acquisition unit for acquiring a third set concentration of Mn 3 O 4 The invention relates to an oxidase-like solution and 3,3',5,5'-tetramethylbenzidine and dimethyl sulfoxide solutions with different set concentrations within a third set concentration range; a second liquid preparation unit is used to add 3,3',5,5'-tetramethylbenzidine and dimethyl sulfoxide solutions corresponding to a sixth set concentration less than the fourth set concentration and Mn corresponding to different set concentrations within the third set concentration range less than the third set concentration to the NaAc-HAc buffer solution with a third set volume, a fifth set concentration and a set pH value. 3 O 4 A method for preparing an oxidase-like solution to obtain a solution to be detected with different set concentrations within the third set concentration range; a first determining unit, used to respectively determine a plurality of reaction rates corresponding to different set concentrations of 3,3',5,5'-tetramethylbenzidine within the third set concentration range in the solution to be detected; a second determining unit, used to fit the different set concentrations of 3,3',5,5'-tetramethylbenzidine within the third set concentration range and the corresponding plurality of reaction rates using the Michaelis-Menten equation to determine a velocity curve of the relationship between the plurality of reaction rates of the enzymatic reaction and the different set concentrations within the third set concentration range; or, comprising: a processor; A memory storing processor executable instructions; wherein the processor is configured to call the instructions stored in the memory to execute the nanozyme kinetic detection method described in any one of claims 5 to 6; or, comprising: a computer-readable storage medium having computer program instructions stored thereon, wherein the computer program instructions, when executed by the processor, implement the nanozyme kinetic detection method described in any one of claims 5 to 6; or, comprising: a computer program product, comprising a computer program / instructions, wherein the computer program / instructions, when executed by the processor, implement the nanozyme kinetic detection method described in any one of claims 5 to 6; and / or, The device for detecting the mechanism of oxidase activity comprises: a resonance spectrum measurement unit for capturing the reaction intermediates of catalyzing 3,3′,5,5′-tetramethylbenzidine in the color development process by using electron paramagnetic energy spectrum and taking 5,5-dimethyl-1-pyrroline oxide as a specific probe to obtain a resonance spectrum; a third determination unit for determining the reaction intermediates based on the resonance spectrum; or, comprising: a processor; a memory for storing processor executable instructions; wherein the processor is configured to call the instructions stored in the memory to execute the method for detecting the mechanism of oxidase activity as described in any one of claims 5 to 6; or, comprising: a computer-readable storage medium having computer program instructions stored thereon, wherein the computer program instructions implement the method for detecting the mechanism of oxidase activity as described in any one of claims 5 to 6 when executed by the processor; or, comprising: a computer program product comprising a computer program / instruction, wherein the computer program / instruction implements the method for detecting the mechanism of oxidase activity as described in any one of claims 5 to 6 when executed by the processor.

8. A nanozyme obtained by the nanozyme preparation method according to any one of claims 1 to 2, or a nanozyme prepared by the nanozyme preparation device according to claim 3, or a nanozyme according to claim 4, and a selectivity test method thereof, It is characterized in that include: Obtaining cysteine ​​corresponding to the eighth set concentration and amino acids and / or ions corresponding to a ninth set concentration greater than the eighth set concentration; and pairing Mn based on the cysteine ​​corresponding to the eighth set concentration and the amino acids and / or ions corresponding to the ninth set concentration. 3 O 4 The activity response of the oxidase-like enzyme was determined by 3 O 4 Selectivity of oxidase-like activity.

9. A nanozyme obtained by the nanozyme preparation method according to any one of claims 1 to 2, or a nanozyme prepared by the nanozyme preparation device according to claim 3, or a nanozyme selectivity testing device corresponding to the nanozyme according to claim 4, It is characterized in that include: A third acquisition unit is used to acquire cysteine ​​corresponding to an eighth set concentration, and amino acids and / or ions corresponding to a ninth set concentration greater than the eighth set concentration; The selectivity determination unit is used to respectively determine the cysteine ​​corresponding to the eighth set concentration and the amino acid and / or ion pair Mn corresponding to the ninth set concentration. 3 O 4 The activity response of the oxidase-like enzyme was determined by 3 O 4 Selectivity of oxidase-like activity.

10. Application of the nanozyme prepared by the preparation method according to any one of claims 1 to 2, or the nanozyme prepared by the preparation device according to claim 3, or the nanozyme according to claim 4 in cysteine ​​detection and / or in cysteine ​​detection in urine.

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