Nanoprotease, preparation, detection, testing method and device and application thereof

Abstract

The present disclosure relates to a kind of nano-enzyme and its preparation, detection, test method and device and application, it relates to nano-enzyme technical field.The preparation method of the present disclosure includes: respectively obtaining Mn (NO3) 2 stirring solution and aminoethanol stirring solution, the Mn (NO3) 2 stirring solution and the aminoethanol stirring solution are mixed, to obtain mixed solution;Under inert atmosphere, according to the set rate and third set time, the mixed solution is stirred, to obtain stirring mixed solution;According to the fourth set time, the stirring mixed solution is aged, to obtain corresponding suspension;The suspension is filtered, washed and dried, to obtain Mn3O4 oxidase.The present disclosure embodiment can realize the preparation, detection, test and application of nano-enzyme.

Description

TECHNICAL FIELD

[0001] The present disclosure relates to the technical field of nano-enzyme, in particular to a nano-enzyme, a preparation method, a detection method, a testing method, a device and an application thereof. BACKGROUND

[0002] Amino acids are important biomolecules in the human body, not only can assemble proteins, but also have important physiological significance as individuals. They are not only related to gene expression, but also are regulators of protein phosphorylation cascade. The content and metabolic state of amino acids in normal human blood and urine remain relatively stable, 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 transmission, protein biosynthesis, and phospholipid metabolism. Imbalance of cysteine levels can cause damage to various tissues. Therefore, quantitative detection of cysteine is of great significance for disease diagnosis and treatment.

[0003] At present, 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 professional personnel and large instruments, which are not suitable for routine analysis of cysteine. Colorimetric method reduces these limitations and adds advantages such as low cost, fast reaction, and easy-to-use instruments, but the low detection sensitivity of this method limits its wide application. Therefore, it is of great significance to develop a high-sensitivity cysteine detection method based on nano-enzyme.

[0004] Nano-enzyme is a kind of nano-material with enzyme-like catalytic properties, which has the advantages of convenient storage, simple preparation, and easy control, and has been widely used in biology, medicine, environment, food, and other fields. Current nano-enzyme materials are mostly based on peroxidase, and have low sensitivity. The catalytic process of peroxidase-like nano-enzyme requires the participation of hydrogen peroxide, which is unstable. The application of hydrogen peroxide in actual cysteine detection will greatly affect the accuracy of the detection. Therefore, it is extremely important to develop a simple nano-enzyme preparation method to obtain high-efficiency oxidase-like nano-enzyme. SUMMARY

[0005] The present disclosure provides a nano-enzyme, a preparation method, a detection method, a testing method, a device and an application thereof.

[0006] According to an aspect of the present disclosure, a nano-enzyme preparation method is provided, comprising:

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

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

[0009] aging the stirred mixed solution for a fourth set time to obtain a corresponding suspension;

[0010] performing filtration, washing, and drying processes on the suspension to obtain Mn3O4-type oxidase.

[0011] Preferably, before the Mn(NO3)2 stirring solution and the aminoethanol stirring solution are obtained, a first set volume and a first set concentration of a corresponding Mn(NO3)2 solution and a second set volume and a second set concentration of a corresponding aminoethanol solution are prepared; and 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.

[0012] Preferably, the first set volume and the second set volume correspond to the same value, and the second set concentration corresponds to twice the 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 is configured as 24 hours; and / or, the inert atmosphere is configured as an argon atmosphere.

[0015] Preferably, the method for performing filtration, washing, and drying processes on the suspension to obtain Mn3O4-type oxidase comprises: performing filtration and washing on the suspension, and drying the suspension after the filtration and washing at a first set temperature and for a fifth set time to obtain Mn3O4-type oxidase.

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

[0017] Preferably, the method for aging the stirred mixed solution for a fourth set time to obtain a corresponding suspension comprises: detecting a first color corresponding to the suspension in real time within the fourth set time; and if the first color becomes grayish brown, determining that the aging process of the stirred mixed solution is complete to obtain the corresponding suspension.

[0018] Preferably, the method for determining that the aging treatment of the mixed solution is completed if the color becomes grayish brown, comprising: if the color becomes grayish brown, obtaining a set delay time; after the set delay time, determining that the aging treatment of the mixed solution is completed; and / or the method for determining that the aging treatment of the mixed solution is completed if the first color of the suspension is grayish brown in real time, comprising: obtaining a suspension picture corresponding to the suspension by using a camera or a video camera; performing color extraction on the suspension picture; and if the extracted color is the same as the set grayish brown, determining that the aging treatment of the mixed solution is completed.

[0019] Preferably, the method for performing color extraction on the suspension picture, comprising: segmenting the suspended matter in the suspension picture by using a preset threshold or a preset segmentation model to obtain a suspended matter image; and then performing color extraction on the suspended matter image; and if the extracted first color is the same as the set grayish brown, determining that the aging treatment of the mixed solution is completed. The method for determining that the aging treatment of the mixed solution is completed if the extracted first color is the same as the set grayish brown, comprising: analyzing the first RGB value corresponding to the first three channels of the extracted first color; and if the first three-channel RGB value is the same as the three-channel RGB value corresponding to the grayish brown or if the first three-channel RGB value deviates from the three-channel RGB value corresponding to the grayish brown within a set range, determining that the aging treatment of the mixed solution is completed.

[0020] According to an aspect of the present disclosure, a nanoscale enzyme preparation device is provided, comprising:

[0021] The first mixing and stirring unit is configured to obtain an 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 configured to stir the mixed solution under an inert atmosphere at a set rate and for a third set time to obtain a mixed and stirred solution. The aging treatment unit is configured to age the mixed and stirred solution for a fourth set time to obtain a corresponding suspension. The filtration, washing and drying treatment unit is configured to perform filtration, washing and drying treatment on the suspension to obtain a Mn3O4 oxidase.

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

[0023] According to an aspect of the present disclosure, a nanoscale enzyme preparation device is provided, comprising: a computer readable storage medium having stored thereon computer program instructions, which, when executed by a processor, implement the nanoscale enzyme preparation method described above.

[0024] According to an aspect of the present disclosure, a nanoscale enzyme preparation device is provided, comprising: a computer program product comprising computer programs / instructions, which, when executed by a processor, implement the nanoscale enzyme preparation method described above.

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

[0026] According to an aspect of the present disclosure, a computer readable storage medium having stored thereon computer program instructions, which, when executed by a processor, implement the nanoscale enzyme preparation method described above.

[0027] According to an aspect of the present disclosure, a computer program product comprising computer programs / instructions, which, when executed by a processor, implement the nanoscale enzyme preparation method described above.

[0028] According to an aspect of the present disclosure, a nanoscale enzyme is provided, which is prepared by the nanoscale enzyme preparation method described above or by the nanoscale enzyme preparation device described above.

[0029] According to an aspect of the present disclosure, a nanoscale enzyme prepared by the nanoscale enzyme preparation method described above or by the nanoscale enzyme preparation device described above or the nanoscale enzyme described above, an activity detection and / or enzyme kinetics detection and / or oxidoenzyme activity mechanism detection method thereof is provided.

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

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

[0032] The oxidase-like enzyme activity mechanism detection method comprises: using electron paramagnetic resonance spectroscopy to capture a reaction intermediate in a color development process of catalyzing 3,3',5,5'-tetramethylbenzidine by using 5,5-dimethyl-1-oxypyrridine as a specific probe, and measuring a resonance spectrum; and determining the reaction intermediate based on the resonance spectrum.

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

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

[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 one of 5-12 times of the third set concentration.

[0037] Preferably, 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 comprises: obtaining Mn3O4 oxidase solution corresponding to a first different set concentration in a first set concentration range; determining different colors of the solution to be detected corresponding to the Mn3O4 oxidase solution corresponding to the first different set concentration; and configuring the concentration corresponding to the darkest color in the first different colors or the concentration corresponding to the adjacent color unchanged in the first different colors as the seventh set concentration.

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

[0039] Preferably, the method for determining the sixth set concentration comprises: obtaining 3,3',5,5'-tetramethylbenzidine and dimethyl sulfoxide solutions corresponding to a second different set concentration in a second set concentration range; preparing solutions to be detected corresponding to the 3,3',5,5'-tetramethylbenzidine and dimethyl sulfoxide solutions corresponding to the second different set concentration under the Mn3O4 oxidase solution of the seventh set concentration; placing the solutions to be detected corresponding to the second different set concentration at a second set temperature for a sixth set time, respectively, and then detecting a plurality of light absorption values of a reaction system corresponding to the active detection solution at a set wavelength by an enzyme label instrument; configuring the set concentration corresponding to the maximum peak value of the plurality of light absorption values corresponding to the second different set concentration as the sixth set concentration; or placing the solutions to be detected corresponding to the second different set concentration at a second set temperature for a sixth set time, respectively, and then determining a plurality of colors corresponding to the second different set concentration; configuring the set concentration corresponding to the darkest color or the adjacent color unchanged or the green color in the plurality of colors corresponding to the second different set concentration as the sixth set concentration.

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

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

[0042] Preferably, the method for determining the sixth set time, comprises: determining a plurality of colors of the solution to be detected at a plurality of set time points under a second set temperature; and configuring the set time point corresponding to the color tending to be stable in the plurality of colors as the sixth set time.

[0043] Preferably, the method for determining the second set temperature, comprises: placing the solution to be detected under a plurality of set temperatures respectively for a sixth set time, and then detecting a plurality of light absorption values of a reaction system corresponding to the solution to be detected at a set wavelength by an enzyme label instrument; and configuring the set temperature corresponding to any two light absorption values without difference in the plurality of light absorption values as the second set temperature.

[0044] Preferably, 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℃ or 25℃; and / or, the sixth set time is configured as 5 minutes; and / or, the set wavelength is configured as 652 nm.

[0045] Preferably, before the step of detecting the light absorption value of the reaction system corresponding to the solution to be detected by the enzyme label instrument, the method further comprises the steps of: sucking a fourth set volume of the solution to be detected; adding the fourth set volume of the solution to be detected into the hole plate; and detecting the light absorption value of the reaction system corresponding to the solution to be detected by the enzyme label instrument.

[0046] Preferably, the method for detecting the activity of the Mn3O4-type oxidase based on the color of the solution to be detected comprises the steps of: detecting the second color of the solution to be detected in real time; and determining that the Mn3O4-type oxidase has activity if the second color changes to blue. Preferably, the method for detecting the second color of the solution to be detected in real time comprises the steps of: acquiring a picture of the solution to be detected by using a camera or a video camera; and extracting the second color of the solution to be detected from the picture of the solution to be detected. Preferably, 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 the steps of: acquiring a light absorption value corresponding to each wavelength in a set wavelength range including the set wavelength; plotting a 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; and determining that the Mn3O4-type oxidase has activity if the peak value is a maximum peak value. Preferably, the method for detecting the activity of the Mn3O4-type oxidase based on the color of the solution to be detected and the light absorption value of the solution to be detected at a set wavelength comprises the steps of: detecting the second color of the solution to be detected in real time; generating a first detection condition if the second color changes to blue; acquiring a light absorption value corresponding to each wavelength in a set wavelength range including the set wavelength; plotting a 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; generating a second detection condition if the peak value is a maximum peak value; and determining that the Mn3O4-type oxidase has activity if the first detection condition and the second detection condition are both satisfied.

[0047] Preferably, the activity verification of the Mn3O4-type oxidase comprises: obtaining a first to-be-detected solution and a second to-be-detected solution; the preparation method of the first to-be-detected solution and the second to-be-detected solution is the same as the preparation method of the to-be-detected solution; air and nitrogen are respectively introduced into the first to-be-detected solution and the second to-be-detected solution, and the first light absorption value and the second absorption value of the reaction system corresponding to the first to-be-detected solution and the second to-be-detected solution are respectively detected by the enzyme label instrument; the activity of the Mn3O4-type oxidase is determined 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 comprises: if there is a significant difference between the first light absorption value and the second absorption value, the Mn3O4-type oxidase has oxidase activity.

[0048] Preferably, the method for extracting the second color corresponding to the to-be-detected solution from the to-be-detected solution picture comprises: analyzing the second RGB value corresponding to the second three channels of the to-be-detected solution picture to obtain the second color corresponding to the to-be-detected solution. The method for determining that the second color changes to blue color comprises: 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 the blue color or if the RGB value corresponding to the second three channels is within a set range of the RGB value corresponding to the three channels of the blue color, it is determined that the second color changes to blue color.

[0049] Preferably, the enzyme kinetics detection method further comprises: determining the Michaelis constant and the maximum reaction rate of the enzyme kinetics parameter based on the speed curve of the plurality of reaction rates of the enzyme reaction and the different set concentrations in the third set concentration range.

[0050] Preferably, the enzyme kinetics detection method further comprises: taking the reciprocal of the different set concentrations of the 3,3',5,5'-tetramethylbenzidine in the third set concentration range and the corresponding plurality of reaction rates to obtain a plurality of first reciprocals corresponding to the different set concentrations in the third set concentration range and a plurality of second reciprocals corresponding to the plurality of reaction rates; and using a double-reciprocal equation to fit the plurality of first reciprocals and the corresponding plurality of second reciprocals to obtain a linear curve of the Michaelis constant of the enzyme kinetics parameter.

[0051] Preferably, the third set concentration range is configured as 0-120 μg / mL; and the different set concentrations in the third set concentration range are configured as any number of values in 0-120 μg / mL.

[0052] Preferably, the method further comprises: placing the solution to be detected at different set concentrations in the third set concentration range at the second set temperature for a sixth set time, respectively, and then detecting a plurality of light absorption values of the corresponding reaction system of the solution to be detected at different set concentrations in the third set concentration range at a set wavelength by using an enzyme label instrument.

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

[0054] Preferably, the method of 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 primary reaction intermediate in the reaction intermediate.

[0055] According to an aspect of the present disclosure, a nano-enzyme activity and / or enzyme kinetics and / or oxyenzyme-like activity mechanism detection device for the nano-enzyme obtained by the nano-enzyme preparation method described above or the nano-enzyme obtained by the nano-enzyme preparation device described above or the nano-enzyme corresponding to the nano-enzyme is provided.

[0056] The nanoscale enzyme activity detection device comprises: a first acquisition unit configured to acquire a third set concentration of Mn3O4 oxidase solution and a fourth set concentration of 3,3',5,5'-tetramethylbenzidine and dimethyl sulfoxide solution; a first liquid preparation unit configured to add 3,3',5,5'-tetramethylbenzidine and dimethyl sulfoxide solution corresponding to a sixth set concentration less than the fourth set concentration and Mn3O4 oxidase solution corresponding to a seventh set concentration less than the third set concentration to a third set volume, a fifth set concentration and a set pH value NaAc-HAc buffer solution to obtain a solution to be detected; a light absorption value determination unit configured to place the solution to be detected at a second set temperature for a sixth set time, and detect the light absorption value of the reaction system corresponding to the solution to be detected at a set wavelength by using an enzyme label instrument; and an activity detection unit configured to detect the activity of the Mn3O4 oxidase based on the color of the solution to be detected and / or the light absorption value at the set wavelength.

[0057] The nanoscale enzyme kinetics detection device comprises: a second acquisition unit configured to acquire a third set concentration of Mn3O4 oxidase solution and a third set concentration range of different set concentrations of 3,3',5,5'-tetramethylbenzidine and dimethyl sulfoxide solution; a second solution preparation unit configured to add less than the fourth set concentration of the sixth set concentration of 3,3',5,5'-tetramethylbenzidine and dimethyl sulfoxide solution and less than the third set concentration of the third set concentration range of different set concentrations of Mn3O4 oxidase solution in a third set volume, a fifth set concentration and a set pH value NaAc-HAc buffer to obtain a third set concentration range of different set concentrations of the to-be-detected solution; a first determination unit configured to determine a plurality of reaction rates corresponding to the third set concentration range of different set concentrations of 3,3',5,5'-tetramethylbenzidine in the to-be-detected solution; and a second determination unit configured to use the Michaelis equation to fit the third set concentration range of different set concentrations of 3,3',5,5'-tetramethylbenzidine and the corresponding plurality of reaction rates to determine a speed curve of the relationship between the plurality of reaction rates of the enzymatic reaction and the third set concentration range of different set concentrations; or, the nanoscale enzyme kinetics 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 nanoscale enzyme kinetics detection method; or, the nanoscale enzyme kinetics detection device comprises: a computer readable storage medium having computer program instructions stored thereon, the computer program instructions being executed by a processor to implement the above-mentioned nanoscale enzyme kinetics detection method; or, comprising: a computer program product comprising computer programs / instructions, which are executed by a processor to implement the above-mentioned nanoscale enzyme kinetics detection method.

[0058] The class oxidase activity mechanism detection device includes a resonance spectrum measurement unit configured to capture a reaction intermediate in a color development process of catalytic 3,3',5,5'-tetramethylbenzidine by using electron paramagnetic energy spectrum with 5,5-dimethyl-1-oxidized pyrroline as a specific probe to measure a resonance spectrum, and a third determination unit configured to determine the reaction intermediate based on the resonance spectrum.

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

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

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

[0062] According to an aspect of the present disclosure, there is provided a selective test method for a nanoenzyme prepared by the above-mentioned nanoenzyme preparation method or prepared by the above-mentioned nanoenzyme preparation device or the above-mentioned nanoenzyme, including: obtaining cysteine corresponding to an eighth set concentration, amino acids and / or ions corresponding to a ninth set concentration greater than the eighth set concentration; and determining selectivity of Mn3O4 class oxidase activity based on activity responses of the Mn3O4 class oxidase to the cysteine corresponding to the eighth set concentration and the amino acids and / or ions corresponding to the ninth set concentration.

[0063] According to an aspect of the present disclosure, a nanoscale enzyme prepared by the nanoscale enzyme preparation method described above, or a nanoscale enzyme prepared by the nanoscale enzyme preparation device described above, or a nanoscale enzyme selective testing device corresponding to the nanoscale enzyme described above, comprises:

[0064] The third acquisition unit is configured to acquire cysteine corresponding to an eighth set concentration, amino acids and / or ions corresponding to a ninth set concentration greater than the eighth set concentration; and the selective determination unit is configured to determine the selectivity of the Mn3O4-type oxidase based on the response of the cysteine corresponding to the eighth set concentration and the amino acids and / or ions corresponding to the ninth set concentration to the activity of the Mn3O4-type oxidase, respectively.

[0065] According to an aspect of the present disclosure, a nanoscale enzyme prepared by the preparation method described above, or a nanoscale enzyme prepared by the preparation device described above, or the nanoscale enzyme described above is applied to cysteine detection and / or cysteine detection in urine.

[0066] In the embodiments of the present disclosure, the present disclosure proposes a technical solution of a nanoscale enzyme, a preparation method, a detection method, a testing method, a device and an application, which overcomes the problems of the traditional method, such as complicated sample pre-treatment, expensive instruments and equipment, the need for professional technical personnel to operate, and high operating costs.

[0067] It should be understood that the foregoing general description and the following detailed description are only exemplary and explanatory, rather than limiting the present disclosure.

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

[0069] The accompanying drawings, which are incorporated into and form part of the specification, illustrate embodiments consistent with the present disclosure and, together with the specification, serve to explain the technical solutions of the present disclosure.

[0070] Figure 1 A flowchart of a nanoscale enzyme preparation method according to an embodiment of the present disclosure is shown;

[0071] Figure 2 UV-Vis absorption spectra under different systems according to an embodiment of the present disclosure are shown;

[0072] Figure 3 Absorbance (light absorption value) at 52 nm under air and nitrogen atmosphere according to an embodiment of the present disclosure is shown;

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

[0074] Figure 5 The reaction condition optimization results 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 Mn3O4 nanoscale enzyme oxidase kinetics parameters 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 The electron paramagnetic resonance spectrum according to the embodiment of the present disclosure is shown; wherein, Figure A: 1 the characteristic peak of O2, Figure B: the characteristic peak of O2· - , Figure C: the characteristic peak of ·OH;

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

[0078] Figure 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 under the concentration of 20 μg / mL TMB; Figure B is the linear fitting of Figure A; Figure C is the detection range of cysteine under the concentration of 110 μg / mL TMB; and Figure D is the linear fitting of Figure C;

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

[0080] Figure 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 explained in detail below with reference to the accompanying drawings. Like reference numerals in the drawings denote like elements or elements having a similar function. Although various aspects of the embodiments are illustrated in the drawings, the drawings are not necessarily drawn to scale unless specifically indicated.

[0082] The word "exemplary" is used herein in the sense of being an example, illustration, or instance. Any embodiment described herein as "exemplary" is not necessarily to be construed as preferred or advantageous over other embodiments.

[0083] The term "and / or", as used herein, merely describes association between associated objects, and can indicate that three cases, such as A and / or B, can exist, that is, A exists alone, A and B exist together, and B exists alone. In addition, the term "at least one" herein indicates any one of a plurality or any combination of at least two of a plurality, for example, at least one of A, B, and C includes 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 specific embodiments below. Those skilled in the art should understand that the present disclosure can also be implemented without some specific details. In some examples, methods, means, elements and circuits well known to those skilled in the art are not described in detail, in order to highlight the main ideas of the present disclosure.

[0085] It can be understood that the above-mentioned various method embodiments of the present disclosure can be combined with each other to form combined embodiments without deviating from the principle logic. Due to the limited space, the present disclosure will not be described again.

[0086] In addition, the present disclosure also provides devices, electronic equipment, computer readable storage media, programs and applications corresponding to the nano-enzyme preparation, nano-enzyme detection, nano-enzyme selective test method. The above can be used to implement any one of the nano-enzyme preparation method, nano-enzyme detection method, nano-enzyme selective test method provided by the present disclosure, the corresponding technical solutions and description and see the corresponding description in the nano-enzyme preparation method, nano-enzyme detection method, nano-enzyme selective test method part, and will not be described again.

[0087] Figure 1 A flow chart of a nano-enzyme preparation method according to an embodiment of the present disclosure is shown as follows. Figure 1 As shown, the nano-enzyme preparation method comprises: step S101: obtaining Mn(NO3)2 stirring solution and 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 under an inert atmosphere at a set rate and for a third set time to obtain a stirred mixed solution; step S103: aging the stirred mixed solution for a fourth set time to obtain a corresponding suspension; step S104: performing suction filtration, washing and drying treatment on the suspension to obtain Mn3O4 oxidase. The method overcomes the problems of traditional methods, such as complicated sample pre-treatment, expensive equipment, the need for professional technical personnel to operate, and high running cost.

[0088] In the embodiments of the present disclosure, before the Mn(NO3)2 stirring solution and the aminoethanol stirring solution are obtained, a first set volume and a first set concentration of the Mn(NO3)2 solution and a second set volume and a second set concentration of the aminoethanol solution 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 the embodiments of the present disclosure, the first set volume and the second set volume correspond to the same value, and the second set concentration corresponds to twice the value of the first set concentration.

[0090] In the embodiments 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 the embodiments of the present disclosure, the third set time can be configured as 3 hours; and / or, the fourth set time is configured as 24 hours; and / or, the inert atmosphere is configured as an argon atmosphere.

[0092] In the embodiments of the present disclosure, the method for obtaining the Mn3O4 oxidase by performing the filtration, washing and drying processes on the suspension includes: performing the filtration and washing on the suspension, and performing the drying on the suspension after the filtration and washing at a first set temperature and for a fifth set time, to obtain the Mn3O4 oxidase.

[0093] In the embodiments of the present disclosure, the first set temperature is configured as 60 DEG C; and / or, the fifth set time is configured as 24 hours.

[0094] In the embodiments of the present disclosure, the method for aging the stirring mixed solution for the fourth set time to obtain the corresponding suspension includes: detecting the first color of the suspension in real time within the fourth set time; if the first color becomes grayish brown, it is determined that the aging treatment of the stirring mixed solution is completed to obtain the corresponding suspension. Wherein, if the color becomes grayish brown, it is determined that the aging treatment of the stirring mixed solution is completed, which includes: if the color becomes grayish brown, a set delay time is obtained; after the set delay time, it is determined that the aging treatment of the stirring mixed solution is completed; and / or, the method for detecting the first color of the suspension in real time, if the first color becomes grayish brown, it is determined that the aging treatment of the stirring mixed solution is completed, which includes: using a camera or a video camera to obtain a suspension picture corresponding to the suspension; color extraction is performed on the suspension picture; if the extracted color is the same as the set grayish brown, it is determined that the aging treatment of the stirring mixed solution is completed.

[0095] In the embodiments of the present disclosure, the method for color extraction of the suspension picture includes: using a preset threshold or a preset segmentation model to segment the suspended matter in the suspension picture 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 grayish brown, it is determined that the aging treatment of the stirring mixed solution is completed. Wherein, if the extracted first color is the same as the set grayish brown, it is determined that the aging treatment of the stirring mixed solution is completed, which includes: analyzing the first RGB value corresponding to the three channels of the extracted first color; if the RGB value corresponding to the first three channels is the same as the RGB value corresponding to the three channels of the grayish brown or if the RGB value corresponding to the first three channels is within a set range of the RGB value corresponding to the three channels of the grayish brown, it is determined that the aging treatment of the stirring mixed solution is completed.

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

[0097] In embodiments and other possible embodiments of the present disclosure, the first set volume and the first set concentration are respectively configured as 10 ml and 0.4 mM (millimolar), and the second set volume and the second set concentration are respectively configured as 10 ml and 0.8 mM (millimolar). The second set concentration corresponds to a value greater than that of the first set concentration. For example, the second set concentration corresponds to a value of 1.5-3 times that of the first set concentration. Specifically, the first set volume and the second set volume correspond to the same value, and the second set concentration corresponds to a value of twice that of the first set concentration.

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

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

[0100] The embodiments of the present disclosure further provide a method for detecting the activity of the nanoscale enzyme, the enzyme kinetics and / or the mechanism of the oxygens-like enzyme activity.

[0101] In the embodiments of the present disclosure, the activity of the nanoscale enzyme is detected by the following method: obtaining a third set concentration of Mn3O4 oxygens-like enzyme solution and a fourth set concentration of 3,3', 5,5'-tetramethylbenzidine and dimethyl sulfoxide solution; adding 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 Mn3O4 oxygens-like enzyme solution less than the third set concentration into a fifth set volume of NaAc-HAc buffer solution with a fifth set concentration and a set pH value to obtain a solution to be detected; placing the solution to be detected at a second set temperature for a sixth set time, and then detecting the light absorption value of the reaction system corresponding to the solution to be detected at a set wavelength by an enzyme label instrument; and detecting the activity of the Mn3O4 oxygens-like enzyme based on the color of the solution to be detected and / or the light absorption value at the set wavelength.

[0102] In the embodiments of the present disclosure, before the third set concentration of Mn3O4 oxygens-like enzyme solution is obtained, a first mass of powder sample of Mn3O4 oxygens-like enzyme is weighed, and the powder sample is dispersed in ultrapure water to prepare the third set concentration of Mn3O4 oxygens-like enzyme solution.

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

[0104] In the embodiments of the present disclosure, the third set concentration is configured as 1 mg / mL; and / or, the fourth set concentration is configured as 10 mg / mL; the fourth set concentration is any one of 5-12 times of the third set concentration; the sixth set concentration is configured as 25 μg / mL or 110 μg / mL; and the seventh set concentration is configured as 20 μg / mL.

[0105] In the embodiment of the present disclosure, the method for determining the seventh set concentration comprises: obtaining Mn3O4-type oxidase solutions corresponding to different first set concentrations in a first set concentration range; determining different colors of the solutions to be detected corresponding to the Mn3O4-type oxidase solutions corresponding to the different first set concentrations; and configuring the concentration corresponding to the deepest color in the different first colors or the concentration corresponding to the adjacent color unchanged in the different first colors as the seventh set concentration. The method for determining the different colors of the solutions to be detected corresponding to the Mn3O4-type oxidase solutions corresponding to the different set concentrations comprises: 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 third set concentration into a third set volume, a fifth set concentration, and a set pH value NaAc-HAc buffer, respectively, to obtain the solutions to be detected corresponding to the Mn3O4-type oxidase solutions corresponding to the different first set concentrations; and determining the different colors corresponding to the solutions to be detected corresponding to the Mn3O4-type oxidase solutions corresponding to the different first set concentrations, respectively.

[0106] In the embodiment of the present disclosure, the method for determining the sixth set concentration comprises: obtaining 3,3',5,5'-tetramethylbenzidine and dimethyl sulfoxide solutions corresponding to different second set concentrations in a second set concentration range; preparing solutions to be detected of the 3,3',5,5'-tetramethylbenzidine and dimethyl sulfoxide solutions corresponding to the different second set concentrations under the Mn3O4-type oxidase solutions corresponding to the seventh set concentration; placing the solutions to be detected corresponding to the different second set concentrations at a second set temperature for a sixth set time, respectively, and then detecting, by using an enzyme label instrument, a plurality of light absorption values of a reaction system corresponding to the solutions to be detected at a set wavelength; configuring the set concentration corresponding to the maximum peak value of the plurality of light absorption values corresponding to the different second set concentrations as the sixth set concentration; or placing the solutions to be detected corresponding to the different second set concentrations at a second set temperature for a sixth set time, respectively, and then determining a plurality of colors corresponding to the different second set concentrations; and configuring the set concentration corresponding to the deepest color in the plurality of colors corresponding to the different second set concentrations or the set concentration corresponding to the adjacent color unchanged or the set concentration corresponding to the green color as the sixth set concentration.

[0107] In the embodiment of the present disclosure, the method for preparing the second different concentration of Mn3O4 oxidase solution at the seventh set concentration, includes: adding the second different concentration of 3,3',5,5'-tetramethylbenzidine and dimethyl sulfoxide solution less than the fourth set concentration and the seventh set concentration of Mn3O4 oxidase solution less than the third set concentration into the third set volume and the fifth set concentration of NaAc-HAc buffer solution with a set pH value, to obtain the second different concentration of Mn3O4 oxidase solution 3,3',5,5'-tetramethylbenzidine and dimethyl sulfoxide solution corresponding to the second different concentration of 3,3',5,5'-tetramethylbenzidine and dimethyl sulfoxide solution corresponding to the second different concentration of the solution to be detected.

[0108] In the embodiment of the present disclosure, the method for determining the set pH value, includes: obtaining NaAc-HAc buffer solutions corresponding to different pH values in a set pH value range; determining a plurality of 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 in the plurality of color development values as the set pH value.

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

[0110] In the embodiment of the present disclosure, the method for determining the second set temperature, includes: placing the solution to be detected at a plurality of set temperatures respectively for a sixth set time, detecting a plurality of light absorption values of a reaction system corresponding to the solution to be detected at a set wavelength by an enzyme label instrument, and configuring the set temperature corresponding to any two light absorption values without difference in the plurality of light absorption values as the second set temperature.

[0111] In the embodiment of the present disclosure, 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; the set pH value is configured as 3.5; the second set temperature is configured as room temperature or 5°C or 25°C; the sixth set time is configured as 5 minutes; and / or, the set wavelength is configured as 652 nm.

[0112] In embodiments of the present disclosure, before the step of detecting the light absorption value of the reaction system corresponding to the solution to be detected by the enzyme label instrument at the set wavelength, the method further comprises: aspirating a fourth set volume of the solution to be detected, adding the fourth set volume of the solution to be detected into the well plate, and detecting the light absorption value of the reaction system corresponding to the solution to be detected by the enzyme label instrument at the set wavelength; wherein the fourth set volume is configured as 200 μL.

[0113] In embodiments 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 comprises: 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 has activity; and / or, the method for detecting the second color corresponding to the solution to be detected in real time comprises: acquiring a solution to be detected picture corresponding to the solution to be detected by using a camera or a video camera; performing color extraction on the solution to be detected picture 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 the set wavelength comprises: acquiring a light absorption value corresponding to each wavelength in a set wavelength range including the set wavelength; plotting a wavelength-light absorption value curve corresponding to the light absorption value corresponding to each wavelength 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, it is determined that the Mn3O4-type oxidase has activity; 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: detecting the second color corresponding to the solution to be detected in real time; if the second color changes to blue, a first detection condition is generated; acquiring a light absorption value corresponding to each wavelength in a set wavelength range including the set wavelength; plotting a wavelength-light absorption value curve corresponding to the light absorption value corresponding to each wavelength 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, a second detection condition is generated; if the first detection condition and the second detection condition are both satisfied, it is determined that the Mn3O4-type oxidase has activity.

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

[0115] Before the third set concentration of Mn3O4 oxidase solution is obtained, a first mass of Mn3O4 oxidase powder sample is weighed, the powder sample is dispersed in ultrapure water, and the third set concentration of Mn3O4 oxidase solution is prepared; before the fourth set concentration of 3,3',5,5'-tetramethylbenzidine and dimethyl sulfoxide solution is obtained, a second mass of 3,3',5,5'-tetramethylbenzidine powder is weighed, and the powder is dissolved in dimethyl sulfoxide to prepare the fourth set concentration of 3,3',5,5'-tetramethylbenzidine and dimethyl sulfoxide solution; the third set concentration is configured as 1 mg / mL. Wherein the fourth set concentration is configured as 10 mg / mL; the fourth set concentration is any one of 5-12 times of the third set concentration, for example, 5, 6, 7, 8, 9, 10, 11, 12 times.

[0116] In the embodiments of the present disclosure and other possible embodiments, the Mn3O4 oxidase activity detection includes the following steps: 1. First, a first mass of Mn3O4 oxidase powder sample is weighed and dispersed in ultrapure water to prepare a 1 mg / mL (milligrams per milliliter, concentration unit) Mn3O4 oxidase solution. A 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. 1 mL of NaAc-HAc buffer (pH 3.5, 0.1 M (moles, concentration unit)) is added to 110 μg / mL TMB solution (3,3',5,5'-tetramethylbenzidine and dimethyl sulfoxide solution) and 20 μg / mL Mn3O4 (Mn3O4 oxidase) solution to obtain a solution to be detected for activity. In addition, the NaAc-HAc buffer and the TMB solution are used as a control experiment to accurately determine the oxidase activity of Mn3O4. 3. After being placed at room temperature (for example, 25°C) for 5 minutes, 200 μL of the reaction solution (solution to be detected for activity) is added to a 96-well plate, and the light absorption value of the reaction system at 652 nm is detected by an enzyme marker.

[0117] In embodiments of this disclosure, the verification of the activity of the Mn3O4-type oxidase includes: obtaining a first test solution and a second test solution; wherein the preparation methods of the first test solution and the second test solution are the same as the preparation methods of the test solution; respectively purging the first test solution and the second test solution into an air atmosphere and a nitrogen atmosphere, and respectively using the microplate reader to detect the first light absorption value and the second absorbance value of the reaction system corresponding to the first test solution in an air atmosphere and the second test solution in a nitrogen atmosphere at a set wavelength; determining the activity of the Mn3O4-type oxidase based on the first light absorption value and the second absorbance value; and / or, the method for determining the activity of the Mn3O4-type oxidase based on the first light absorption value and the second absorbance value includes: if there is a significant difference between the first light absorption value and the second absorbance value, then it has oxidase-like activity.

[0118] In embodiments of this disclosure, the method for extracting colors from the image of the solution to be tested to obtain a second color corresponding to the solution includes: parsing the second RGB values ​​corresponding to the second and third channels of the image of the solution to be tested to obtain the second color corresponding to the solution. The method for determining that the second color changes to blue includes: obtaining the second RGB values ​​corresponding to the second color; if the second RGB values ​​are the same as the RGB values ​​corresponding to the three channels of blue, or if the deviation between the RGB values ​​corresponding to the second and third channels and the RGB values ​​corresponding to the three channels of blue is within a set range, then the second color change is determined to be blue.

[0119] Figure 2 The UV-Vis absorption spectra of different systems according to embodiments of this disclosure are shown. Detection results, such as... Figure 2 As shown, 3,3′,5,5′-tetramethylbenzidine (TMB) is oxidized to blue oxidized TMB (oxTMB), with a significant absorption peak at 652 nm, indicating that the nanomaterial possesses good oxidase-like activity. The table below shows... Figure 2 The corresponding original data (only a portion of the data is shown because it contains 251 rows).

[0120]

[0121]

[0122] In the embodiments and other possible embodiments of the present disclosure, aerobic (activity) verification: first add 20 μg / mL Mn304 (Mn304 oxidase-like enzyme) and 110 μg / mL TMB in 1 mL NaAc-HAc buffer, respectively, under air atmosphere and nitrogen atmosphere, and detect the absorbance at 652 nm using an enzyme marker.

[0123] Figure 3 The absorbance (light absorption value) at 52 nm under air and nitrogen atmosphere according to the embodiments of the present disclosure is shown. From Figure 3 It can be seen that when detected under nitrogen atmosphere, the absorbance at 652 nm is greatly weakened, which is significantly different from that under air, which shows that Mn304 nanoscale enzyme catalyzes TMB color development and needs the participation of oxygen, and shows that Mn304 has oxidase-like enzyme activity. The following table is Figure 3 the corresponding raw data.

[0124]

[0125] In the embodiments and other possible embodiments of the present disclosure, the optimization of experimental conditions includes the following steps: adding TMB and dimethyl sulfoxide solution and Mn304 (Mn304 oxidase-like enzyme) solution in NaAc-HAc buffer (pH 3.5, 0.1 M), and measuring the absorbance at 652 nm after 5 min of reaction. The concentration of nanoscale enzyme, TMB concentration, pH, and reaction time during the reaction, and the concentration of nanoscale enzyme and substrate TMB (TMB and dimethyl sulfoxide solution) are optimized.

[0126] First, the concentration of nanoscale enzyme (Mn304 oxidase-like enzyme) and substrate TMB is optimized. Figure 4 The results of the reaction system optimization according to the embodiments of the present disclosure are shown; wherein, Fig. A is the concentration of Mn304 nanoscale enzyme, and Fig. B is the substrate TMB, and the results are as shown in Figure 4 A, first, the concentration of nanoscale enzyme is optimized, when the final concentration of nanoscale enzyme is in the range of 5, 10, 20, 30, 40, 50 μg / mL, with the increase of the concentration of nanoscale enzyme, the color development trend gradually deepens, when the final concentration of Mn304 nanoscale enzyme is 20 μg / mL, the color development is best, so the best final concentration of nanoscale enzyme is 20 μg / mL; under this concentration, the effect of TMB concentration on the oxidase activity of Mn304 is explored, and TMB solution with a final concentration of 20-120 μg / mL is selected, and the reaction is carried out at 25°C (room temperature) for 5 min.

[0127] The results are as shown in Figure 4B, TMB at 25 μg / mL, the solution is green, indicating that the amount of TMB is between insufficient and sufficient; OD652 (light absorption value at 652 nm) value is the highest when TMB is 110 μg / mL, 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 A and Figure 4 B correspond to the original data as follows. Figure 4

[0128] Figure 4 A corresponds to the original data

[0129]

[0130]

[0131] Figure 4 B corresponds to the original data

[0132]

[0133] Figure 5 The reaction condition optimization results according to the embodiments of the present disclosure are shown; wherein A is pH, B is buffer concentration, C is reaction time, and D is reaction temperature. The results are shown in Figure 5 A, first, the pH of NaAc-HAc buffer is optimized, when the pH is in the range of 3 to 5, the color development is good, and the color development is best at pH 3.5, so the optimal pH is 3.5; the concentration of the reaction system will affect the catalytic ability of the nano-enzyme, so two commonly used concentrations of 0.1 and 0.2 M are selected for optimization to explore the best reaction system of the sensor. As shown in Figure 5 B, when the concentration of the reaction system has no significant difference, therefore, the concentration of 0.1 M is selected for subsequent experiments; when the color development time is optimized, as shown in Figure 5 C, with the increase of time, the color development gradually increases, and the color development tends to be stable at 5 min, and there is basically no difference compared with 20 min, considering the demand for rapid detection, combined with the experimental optimization results, therefore, 5 min is selected as the best reaction time; finally, the temperature of the reaction system is optimized, as shown in Figure 5 D, there is no difference between 5°C and 25°C, and with the increase of temperature, OD652 gradually decreases. Therefore, the experiment selects the normal temperature 25°C (room temperature) as the reaction temperature for subsequent experiments. Figure 5 A-D in the original data as follows.

[0134] Figure 5 The original data of A

[0135]

[0136] ​Figure 5 Raw data of B

[0137]

[0138] Figure 5 Raw data of C

[0139]

[0140] Figure 5 Raw data of D

[0141]

[0142] In an embodiment of the present disclosure, the enzyme kinetics detection method comprises: obtaining a third set concentration of Mn3O4 oxidase solution and a third set concentration range of different set concentrations of 3,3',5,5'-tetramethylbenzidine and dimethyl sulfoxide solution; adding less than the fourth set concentration of the sixth set concentration of 3,3',5,5'-tetramethylbenzidine and dimethyl sulfoxide solution and less than the third set concentration of the third set concentration range of different set concentrations of Mn3O4 oxidase solution in a third set volume, a fifth set concentration and a set pH value NaAc-HAc buffer solution to obtain a third set concentration range of different set concentrations of the to-be-detected solution; determining a plurality of reaction rates corresponding to the third set concentration range of different set concentrations of 3,3',5,5'-tetramethylbenzidine in the to-be-detected solution respectively; using the Michaelis equation, fitting the third set concentration range of different set concentrations of 3,3',5,5'-tetramethylbenzidine and the corresponding plurality of reaction rates to determine the velocity curve of the plurality of reaction rates of the enzyme reaction and the third set concentration range of different set concentrations.

[0143] In an embodiment of the present disclosure, the enzyme kinetics detection method further comprises: determining the Michaelis constant and the maximum reaction rate of the enzyme kinetics parameter based on the velocity curve of the plurality of reaction rates of the enzyme reaction and the third set concentration range of different set concentrations.

[0144] In an embodiment of the present disclosure, the enzyme kinetics detection method further comprises: respectively taking the reciprocal of the third set concentration range of different set concentrations of 3,3',5,5'-tetramethylbenzidine and the corresponding plurality of reaction rates to obtain a plurality of first reciprocals corresponding to the third set concentration range of different set concentrations and a plurality of second reciprocals corresponding to the plurality of reaction rates; using the double-reciprocal equation, fitting the plurality of first reciprocals and the corresponding plurality of second reciprocals to obtain a linearization curve of the Michaelis constant of the enzyme kinetics parameter.

[0145] In embodiments of the present disclosure, the third set concentration range is configured as 0-120 pg / mL; wherein different set concentrations in the third set concentration range are configured as any multiple values in 0-120 pg / mL.

[0146] In embodiments of the present disclosure, further comprising: placing the different set concentrations of the solution to be detected in the third set concentration range at the second set temperature for a sixth set time, respectively, and then detecting a plurality of light absorption values of the reaction system corresponding to the different set concentrations of the solution to be detected in the third set concentration range at a set wavelength by an enzyme label instrument.

[0147] In embodiments of the present disclosure and other possible embodiments, enzyme kinetics detection includes the following steps: 1, 20 pg / mL Mn304 (Mn304 oxidase) is added to 1 mL NaAc-HAc buffer (pH 3.5, 0.1 M), and 0-120 pg / mL (third set concentration range) of TMB is added, respectively. 3, After placing at room temperature for 5 minutes, 200 pL of reaction solution is taken and added to a 96-well plate, and the light absorption value of the reaction system at 652 nm is detected by an enzyme label instrument. 3, a plot of substrate concentration (different set concentrations of 3,3',5,5'-tetramethylbenzidine in the third set concentration range of the solution to be detected) versus reaction rate is drawn.

[0148] Figure 6 Mn304 nanoscale enzyme oxidase kinetics parameters according to embodiments of the present disclosure are shown. Figure 6 A is the Michaelis-Menten equation of Mn304 (different set concentrations of TMB corresponding to the substrate); Figure 6 B is the Lineweaver-Burk curve. As Figure 6 , the Michaelis-Menten equation (Michaelis equation) is used to fit the enzyme kinetics parameters Km and Vmax of the nanoscale enzyme (Mn304 oxidase), and the results are as shown in Figure 6 A, the Michaelis constant Km of Mn304 (Mn304 oxidase) is 0.1896 mM, and the maximum reaction rate is 0.2834 pmol / s. Based on Figure 6 the data in A, the enzyme kinetics parameters of the two nanoscale enzymes are fitted by the Michaelis-Menten equation.

[0149] By Figure 6 It can be seen that by using the Michaelis equation, different C TMB and the corresponding V establish the A graph; by using the double-reciprocal equation different 1 / C TMB corresponding to 1 / V establishes the B graph. Wherein, C TMBThe concentration of TMB is represented by V, and the reaction rate is represented by V. The following table is the original data corresponding to A and B in the formula. Figure 6 The original data corresponding to A and B in the formula.

[0150] Figure 6 A original data

[0151]

[0152]

[0153] Figure 7 B original data

[0154]

[0155] In an embodiment of the present disclosure, the oxido-like enzyme activity mechanism detection method comprises: using electron paramagnetic resonance spectroscopy, using 5,5-dimethyl-1-oxypyrridine as a specific probe to capture the reaction intermediate in the catalysis of 3,3',5,5'-tetramethylbenzidine in the color development process, and measuring the resonance spectrum; and determining the reaction intermediate based on the resonance spectrum.

[0156] In an embodiment of the present disclosure, the method for determining the reaction intermediate based on the resonance spectrum comprises: determining different free radicals with characteristic peaks based on the resonance spectrum; and determining the different free radicals as the reaction intermediate. In the method for determining the reaction intermediate based on the resonance spectrum, the method further comprises: determining the free radical corresponding to the maximum value in the values corresponding to the characteristic peaks of the different free radicals as the main reaction intermediate in the reaction intermediate.

[0157] In an embodiment of the present disclosure and other possible embodiments, electron paramagnetic resonance spectroscopy is used to capture the reaction intermediate in the catalysis of TMB color development process by using 5,5-dimethyl-1-oxypyrridine (DMPO) as a specific probe, and the resonance spectrum is measured. The characteristic peaks are analyzed to determine the existence of the reaction intermediate.

[0158] Figure 7 The electron paramagnetic resonance spectrum according to an embodiment of the present disclosure is shown in the figure; wherein, figure A: 1 The characteristic peak of O2, figure B: O2· - The characteristic peak of ·OH, figure C: The characteristic peak of ·OH. The results are as follows Figure 7 A, corresponding free radical 1 The characteristic peak of O2 is 1:1:1, and the molar concentration is 1.926e-07; Figure 7 B is six peaks of four large and two small, corresponding to the free radical O2· - The characteristic peak of ·OH, and the molar concentration is 1.287e-06; Figure 8C corresponds to the characteristic peak of the free radical ·OH 1:2:2:1, the molar concentration is 3.201e-07. The above results confirm the existence of three kinds of free radicals, and the molar concentration of O2· - is the highest, so O2· - is the main reaction intermediate.

[0159] The embodiment of the disclosure also proposes a selective test method of the nanoscale enzyme prepared by the nanoscale enzyme preparation method or prepared by the nanoscale enzyme preparation device or the nanoscale enzyme, comprising: obtaining cysteine corresponding to an eighth set concentration, amino acids and / or ions corresponding to a ninth set concentration greater than the eighth set concentration; and determining the selectivity of the Mn3O4 oxidase activity based on the response of the Mn3O4 oxidase activity to the cysteine corresponding to the eighth set concentration and the amino acids and / or ions corresponding to the ninth set concentration, respectively.

[0160] In the embodiment and other possible embodiments of the disclosure, the selectivity test is an important performance indicator for measuring the detection specificity of the nanoscale enzyme, which determines whether the nanoscale enzyme can be used for target molecule detection. By using common amino acids, ions and other substances to explore the substances affecting the activity of Mn3O4 oxidase, the disclosure selects a plurality of common amino acids, glucose, Na + , K + The selectivity test of the nanoscale enzyme (Mn3O4 oxidase) is performed, and the concentration of all substances to be detected is 100 μM, which is 5-10 times the concentration of cysteine. Figure 8 The detection specificity of cysteine according to the embodiment of the disclosure is shown, as Figure 8 It can be seen from the figure that cysteine can significantly inhibit the activity of Mn3O4 oxidase, and has almost no response to the remaining substances. It is proved that the constructed Mn3O4 nanoscale enzyme has specificity for the detection of cysteine. Figure 9 The corresponding raw data are as follows.

[0161]

[0162] The disclosure also proposes a nanoscale enzyme prepared by the preparation method, or a nanoscale enzyme prepared by the preparation device, or the nanoscale enzyme in the application of cysteine detection and / or in the application of cysteine detection in urine.

[0163] Figure 9Detection sensitivity of cysteine according to an embodiment of the present disclosure is shown; wherein Figure A is the detection range of cysteine at a TMB concentration of 20 pg / mL; Figure B is the linear fitting of Figure A; Figure C is the detection range of cysteine at a TMB concentration of 110 pg / mL; and Figure D is the linear fitting of Figure C. In the embodiments of the present disclosure and other possible embodiments, the application of Mn304 nanoscale enzyme in the detection of cysteine includes the following steps: (1) Preparation of cysteine solution: First, a certain amount of cysteine powder is weighed and dissolved in ultrapure water to obtain a cysteine solution with a concentration of 1 mM. (2) 25 or 110 pg / mL TMB and 20 pg / mL Mn304 are added to 1 mL NaAc-HAc buffer (pH 3.5, 0.1 M). (3) Light absorption detection of different concentrations of cysteine: In step 2, the cysteine solution is added to make its final concentration 0, 0.5, 1, 2, 4, 8, 10, 14, 16, 18, 20, 25, 30, 35, 40, 42, 45 pM, respectively. After 10 minutes of room temperature standing, 200 pL of the reaction solution is taken and added to a 96-well plate, and the light absorption value of the reaction system at 652 nm is detected by an enzyme marker. (4) Establishing a linear relationship: The concentration C of added cysteine is taken as the abscissa, and the light absorption value A is taken as the ordinate to establish a standard linear regression curve, as shown in Figure 9 Figure B, and the linear regression equation is: A = -0.01419(C) + 0.01221 (correlation coefficient R 2 = 0.9901), and the minimum detection limit of cysteine can reach 0.02636 pM.

[0164] The results are shown in Figure 9 Figure A, when 25 pg / mL TMB is added to the reaction system, the TMB usage is between insufficient and sufficient, and after the addition of cysteine, the OD652 shows a trend of first rising and then falling. The concentration C of added cysteine is taken as the abscissa, and the light absorption change value A is taken as the ordinate to establish a standard linear regression curve, as shown in Figure 9 Figure B, and the linear regression equation is: A = -0.01419(C) + 0.01221 (correlation coefficient R 2 = 0.9901), and the minimum detection limit of cysteine can reach 0.02636 pM, and the linear detection range is 0.5-45 pM.

[0165] When 110 pg / mL TMB is added to the reaction system, the TMB usage is sufficient, and after the addition of cysteine, the OD652 shows a gradually decreasing trend, as shown in Figure 9 Figure C. The concentration C of added cysteine is taken as the abscissa, and the light absorption change value A is taken as the ordinate to establish a standard linear regression curve, as shown in Figure 9 Figure D, and the linear regression equation is: A = 0.02534(C) + 0.0526 (correlation coefficient R2 The lowest detection limit of cysteine can reach 0.23 μM, and the linear detection range is 2-60 μM. Figure 9 The original data corresponding to A-D are as follows.

[0166] Figure 9 Original data of A

[0167]

[0168]

[0169] Figure 9 Original data of B

[0170]

[0171] Figure 10 Original data of C

[0172]

[0173] Figure 10 Original data of D

[0174]

[0175] After comparing the above results, it is found that when the TMB usage in the reaction system is between insufficient and sufficient, that is, TMB is added to 25 μg / mL, the lowest detection limit of cysteine can reach 0.02636 μM, which is nearly 10 times higher than that when the TMB usage is sufficient, that is, TMB is added to 110 μg / mL, and the linear detection range is larger.

[0176] The execution subject of the nanocatalyst preparation method, the nanocatalyst detection method, and the nanocatalyst selectivity test method can be a nanocatalyst preparation device, a nanocatalyst activity detection device, and a nanocatalyst selectivity test device. For example, the nanocatalyst preparation method, the nanocatalyst detection method, and the nanocatalyst selectivity test method can be executed by a terminal device or a server or other processing device. 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, and the like. In some possible implementation manners, the nanocatalyst preparation method, the nanocatalyst detection method, and the nanocatalyst selectivity test method can be realized by a processor calling computer-readable instructions stored in a memory.

[0177] It can be understood by those skilled in the art that, in the above-mentioned preparation of nanoscale enzyme, nanoscale enzyme detection, and nanoscale enzyme selective test method of the specific embodiment, the writing order of each step does not mean a strict execution order and does not constitute any limitation on the implementation process, and the specific execution order of each step should be determined according to its function and possible internal logic.

[0178] The embodiment of the present disclosure further provides a nanoscale enzyme preparation device, comprising: a first mixing and stirring unit, configured 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, configured to stir the mixed solution under an inert atmosphere at a set rate and for a third set time to obtain a stirred mixed solution; an aging treatment unit, configured to age the stirred mixed solution for a fourth set time to obtain a corresponding suspension; and a filtration, washing and drying treatment unit, configured to perform filtration, washing and drying treatment on the suspension to obtain a Mn3O4 oxidase-like enzyme.

[0179] The embodiment of the present disclosure further provides a nanoscale enzyme preparation device, comprising: a processor; a memory for storing processor-executable instructions; wherein the processor is configured to invoke the instructions stored in the memory to execute the above-mentioned nanoscale enzyme preparation method.

[0180] The embodiment of the present disclosure further provides a nanoscale enzyme preparation device, comprising: a computer-readable storage medium having computer program instructions stored thereon, wherein the computer program instructions are executed by a processor to implement the above-mentioned nanoscale enzyme preparation method.

[0181] The embodiment of the present disclosure further provides a nanoscale enzyme preparation device, comprising: a computer program product comprising computer programs / instructions, wherein the computer programs / instructions are executed by a processor to implement the above-mentioned nanoscale enzyme preparation method.

[0182] The embodiment of the present disclosure further provides a nanoscale enzyme or a nanoscale enzyme prepared by using the above-mentioned nanoscale enzyme preparation device or a nanoscale enzyme activity and / or enzyme kinetics and / or oxidase-like enzyme activity mechanism detection device corresponding to the above-mentioned nanoscale enzyme.

[0183] In the embodiments of the present disclosure, the nano-enzyme activity detection device comprises: a first acquisition unit configured to acquire a third set concentration of Mn3O4 oxidase solution and a fourth set concentration of 3,3', 5,5'-tetramethylbenzidine and dimethyl sulfoxide solution; a first solution preparation unit configured to add 3,3', 5,5'-tetramethylbenzidine and dimethyl sulfoxide solution corresponding to a sixth set concentration less than the fourth set concentration and Mn3O4 oxidase solution corresponding to a seventh set concentration less than the third set concentration to a third set volume, a fifth set concentration and a set pH value NaAc-HAc buffer solution to obtain a solution to be detected; a light absorption value determination unit configured to place the solution to be detected at a second set temperature for a sixth set time, and then detect the light absorption value of the reaction system corresponding to the solution to be detected at a set wavelength by using an enzyme label instrument; and an activity detection unit configured to detect the activity of the Mn3O4 oxidase based on the color of the solution to be detected and / or the light absorption value at the set wavelength.

[0184] In the embodiments of the present disclosure, the nano-enzyme activity detection device comprises: a processor; a memory configured to store processor-executable instructions; wherein the processor is configured to invoke the instructions stored in the memory to execute the above-mentioned nano-enzyme activity detection method.

[0185] In the embodiments of the present disclosure, the nano-enzyme activity detection device comprises: a computer program product comprising computer programs / instructions, which, when executed by a processor, implement the above-mentioned nano-enzyme activity detection method.

[0186] In the embodiments of the present disclosure, the nano-enzyme activity detection device comprises: a computer readable storage medium having computer program instructions stored thereon, wherein the computer program instructions, when executed by a processor, implement the above-mentioned nano-enzyme activity detection method.

[0187] In the embodiment of the present disclosure, the nanoscale enzyme kinetics detection device comprises: a second acquisition unit configured to acquire a third set concentration of Mn3O4 oxidase solution and a third set concentration range of different set concentrations of 3,3',5,5'-tetramethylbenzidine and dimethyl sulfoxide solution; a second solution preparation unit configured to add 3,3',5,5'-tetramethylbenzidine and dimethyl sulfoxide solution corresponding to a sixth set concentration less than the fourth set concentration and Mn3O4 oxidase solution corresponding to a third set concentration less than the third set concentration in the third set volume, the fifth set concentration and the set pH value NaAc-HAc buffer solution to obtain a third set concentration range of different set concentrations of the to-be-detected solution; a first determination unit configured to determine a plurality of reaction rates corresponding to the third set concentration range of different set concentrations of 3,3',5,5'-tetramethylbenzidine in the to-be-detected solution; and a second determination unit configured to use the Michaelis equation to fit the third set concentration range of different set concentrations of 3,3',5,5'-tetramethylbenzidine and the corresponding plurality of reaction rates to determine a speed curve of the plurality of reaction rates of the enzymatic reaction and the third set concentration range of different set concentrations.

[0188] In the embodiment of the present disclosure, the nanoscale enzyme kinetics 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 nanoscale enzyme kinetics detection method.

[0189] In the embodiment of the present disclosure, the nanoscale enzyme kinetics detection device comprises: a computer readable storage medium having computer program instructions stored thereon, wherein the computer program instructions are executed by a processor to implement the above-mentioned nanoscale enzyme kinetics detection method.

[0190] In the embodiment of the present disclosure, the nanoscale enzyme kinetics detection device comprises: a computer program product comprising computer programs / instructions, wherein the computer programs / instructions are executed by a processor to implement the above-mentioned nanoscale enzyme kinetics detection method.

[0191] In the embodiments of the present disclosure, the class oxidase activity mechanism detection device includes: a resonance spectrum measurement unit configured to use electron paramagnetic energy spectrum to capture a reaction intermediate in a color development process of catalytic 3,3',5,5'-tetramethylbenzidine by using 5,5-dimethyl-1-oxidized pyrroline as a specific probe, and measure a resonance spectrum; and a third determination unit configured to determine the reaction intermediate based on the resonance spectrum; or, includes: a processor; a memory configured to store processor-executable instructions; wherein the processor is configured to invoke the instructions stored in the memory to perform the class oxidase activity mechanism detection method described above; or, includes: a computer readable storage medium having computer program instructions stored thereon, wherein the computer program instructions are executed by a processor to implement the class oxidase activity mechanism detection method described above; or, includes: a computer program product including computer programs / instructions, wherein the computer programs / instructions are executed by a processor to implement the class oxidase activity mechanism detection method described above.

[0192] The present disclosure further provides a nano-enzyme selective test device corresponding to the nano-enzyme prepared by the nano-enzyme preparation method or the nano-enzyme preparation device described above, including: a third acquisition unit configured to acquire cysteine corresponding to an eighth set concentration, amino acids and / or ions corresponding to a ninth set concentration greater than the eighth set concentration; and a selectivity determination unit configured to determine selectivity of Mn3O4 class oxidase activity based on responses of the Mn3O4 class oxidase to the cysteine corresponding to the eighth set concentration and the amino acids and / or ions corresponding to the ninth set concentration, respectively.

[0193] The present disclosure further provides a nano-enzyme selective test device corresponding to the nano-enzyme prepared by the nano-enzyme preparation method or the nano-enzyme preparation device described above, including: a third acquisition unit configured to acquire cysteine corresponding to an eighth set concentration, amino acids and / or ions corresponding to a ninth set concentration greater than the eighth set concentration; and a selectivity determination unit configured to determine selectivity of Mn3O4 class oxidase activity based on responses of the Mn3O4 class oxidase to the cysteine corresponding to the eighth set concentration and the amino acids and / or ions corresponding to the ninth set concentration, respectively.

[0194] Based on the above, the purpose of the present application is to provide a preparation method of sheet-like Mn3O4 oxidase and to innovate the homocysteine detection method based on the preparation method, so as to solve the problems in the background art. The detection principle of the present application is as follows: the Mn3O4 nanoscale enzyme with oxidase activity can oxidize 3,3',5,5'-tetramethylbenzidine (TMB) to generate oxTMB. When the substrate TMB is sufficient, the Mn3O4 nanoscale enzyme can completely oxidize TMB, each TMB loses one electron to form a blue product. When the substrate TMB is insufficient, the Mn3O4 nanoscale enzyme can oxidize part of TMB, each oxidized TMB loses two electrons and polymerizes to produce a yellow product. When the concentration of the substrate TMB is between the two, the substrate TMB loses one or two electrons, and the color may appear green, which is the result of the mixture of blue and yellow.

[0195] Cysteine contains a thiol group, which inhibits the activity of Mn3O4 oxidase, and can reduce blue, yellow or green oxTMB to colorless transparent TMB. Green oxTMB indicates that there may be a mixture of yellow oxTMB and blue oxTMB oxidation forms. After adding cysteine, both yellow and blue oxTMB are reduced due to its reducing property. Yellow oxTMB gets one electron to turn into blue oxTMB, while blue oxTMB gradually loses blue color after being reduced by one electron. In this process, since blue oxTMB has a maximum absorption peak at 652 nm (this is the property of oxTMB itself, which has a maximum absorption peak at 652 nm, which is the characteristic peak of this substance and is different from other substances), when cysteine is added, the absorption peak will appear enhanced, then weakened and finally disappeared. The present application utilizes this principle, and the principle that cysteine reduces green oxTMB to colorless transparent TMB, to realize high-sensitivity detection of cysteine by Mn3O4 nanoscale enzyme through colorimetric detection of the absorbance at 652 nm.

[0196] Meanwhile, the present application provides a speculated mechanism of the oxidase activity of Mn3O4 as follows: Mn3O4 can catalyze the dissolved oxygen adsorbed on its surface to generate O2 - , O2 - is extremely unstable in aqueous solution and will rapidly react to generate H2O2 and 1 O2, H2O2 reacts with O2 - to generate ·OH, in addition, Mn3O4 can also directly catalyze H2O2 to generate O2 - and ·OH by Fenton-like reaction.

[0197] Compared with the prior art, the beneficial effects of the application are: 1. The preparation method of the Mn3O4 oxidase is simpler, and when applied to cysteine detection, the detection time is shorter, and the total time is about 10 minutes. 2. Compared with traditional fluorescence method, spectrum method, chromatography method and the like, the Mn3O4 oxidase uses the colorimetric method, which has the characteristics of low cost, easy operation, short time and no need to process the sample, and greatly overcomes the shortcomings of the traditional method, such as complicated sample pre-treatment, expensive instrument equipment, the need for professional technical personnel to operate, high operation cost and the like. 3. The flaky Mn3O4 oxidase is used in the cysteine detection method for the first time, the use amount of the substrate TMB is adjusted, the electron transfer in the reaction system is adjusted, the high-sensitivity detection of the content of cysteine is realized, the detection limit is as low as 0.02636 muM, and the linear detection range is 0.5-45 muM. Compared with the detection system using sufficient TMB, the detection limit is increased by nearly ten times, and the linear detection range is wider. 4. The preparation of the Mn3O4 oxidase and the application in the detection of cysteine do not need the participation of unstable hydrogen peroxide compared with the nano material with peroxidase-like activity that can be used for detecting cysteine, and the detection accuracy is higher.

[0198] In some embodiments, the device provided by the embodiments of the present disclosure has functions or contains modules that can be used to perform the nano-enzyme preparation method, nano-enzyme detection method and nano-enzyme selectivity test method described in the foregoing method embodiment descriptions. For brevity, the specific implementation will not be described here again.

[0199] The embodiments of the present disclosure also provide a computer-readable storage medium having computer program instructions stored thereon, wherein the computer program instructions are executed by a processor to implement one or more of the nano-enzyme preparation method, nano-enzyme detection method and nano-enzyme selectivity test method. The computer-readable storage medium can be a non-volatile computer-readable storage medium.

[0200] The embodiments of the present disclosure also provide an electronic device, including: a processor; a memory for storing processor-executable instructions; wherein the processor is configured to implement one or more of the nano-enzyme preparation method, nano-enzyme detection method and nano-enzyme selectivity test method. The electronic device can be provided as a terminal, a server or other forms of devices.

[0201] The embodiments of the present disclosure also provide a computer program product, including a computer program / instruction, which, when executed by a processor, implements one or more of the nano-enzyme preparation method, nano-enzyme detection method and nano-enzyme selectivity test method.

[0202] Figure 11is a block diagram of an electronic device 800 according to an exemplary embodiment. The electronic device 800 can be, for example, a terminal such as a mobile phone, a computer, a digital broadcasting terminal, a messaging device, a game console, a tablet device, a medical device, a fitness device, a personal digital assistant, etc.

[0203] Referring to Figure 11 The electronic device 800 can include one or more of the following components: a processing component 802, a memory 804, a power supply 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.

[0204] The processing component 802 usually controls overall operations of the electronic device 800, such as operations associated with displaying, making a phone call, data communication, camera operation and recording operation. The processing component 802 can include one or more processors 820 to execute instructions to complete all or part of steps of the above-described methods. In addition, the processing component 802 can include one or more modules to facilitate interaction between the processing component 802 and other components. For example, the processing component 802 can include a multimedia module to facilitate the interaction between the multimedia component 808 and the processing component 802.

[0205] The memory 804 is configured to store various types of data to support operations of the electronic device 800. Examples of these data include instructions for any application or method operating on the electronic device 800, contact data, phonebook data, messages, pictures, videos, etc. The memory 804 can be implemented by any type of volatile or non-volatile storage devices 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 storage, flash memory, magnetic disk or optical disk.

[0206] The power supply component 806 supplies electric power for the various components of the electronic device 800. The power supply component 806 can include a power supply management system, one or more power supplies, and other components associated with generating, managing and distributing electric power for the electronic device 800.

[0207] The multimedia component 808 includes a screen to provide an output interface between the electronic device 800 and a user. In some embodiments, the screen can include a liquid crystal display (LCD) and a touch panel (TP). If the screen includes a touch panel, the screen can be implemented as a touch screen to receive an input signal from a user. The touch panel includes one or more touch sensors to sense a touch, a slide, and a gesture on the touch panel. The touch sensor can not only sense a boundary of a touching or a sliding action, but also detect duration and intensity of the touching or sliding action. 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 camera mode or a video mode, the front camera and / or the rear camera can receive external multimedia data. Each of the front camera and the rear camera can be a fixed optical lens system or have a focal length and optical zooming capability.

[0208] The audio component 810 is configured to output and / or input an audio signal. For example, the audio component 810 includes a microphone (MIC) to receive an external audio signal when the electronic device 800 is in an operating mode, such as a call mode, a recording mode, and a voice recognition mode. The received audio signal can be further stored in the memory 804 or transmitted via the communication component 816. In some embodiments, the audio component 810 further includes a speaker to output an audio signal.

[0209] The I / O interface 812 provides an interface for the processing component 802 and peripheral interface modules, such as a keypad, a click wheel, buttons, and the like. The buttons can include, but are not limited to, a home button, a volume button, a start button, and a lock button.

[0210] The sensor component 814 includes one or more sensors to provide various state assessments for the electronic device 800. For example, the sensor component 814 can detect an open / closed position of the electronic device 800, relative positioning of components, such as a display and a keypad of the electronic device 800, a change in position of the electronic device 800 or a component of the electronic device 800, presence or absence of user contact with the electronic device 800, orientation or acceleration / deceleration of the electronic device 800, and a temperature change of the electronic device 800. The sensor component 814 can include a proximity sensor configured to detect presence of a nearby object without any physical touch. The sensor component 814 can further include a light sensor such as a CMOS or CCD image sensor for use in an imaging application. In some embodiments, the sensor component 814 can further include an acceleration sensor, a gyroscope sensor, a magnetic sensor, a pressure sensor, or a temperature sensor.

[0211] 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 further 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) techniques, infrared data association (IrDA) techniques, ultra-wideband (UWB) techniques, Bluetooth (BT) techniques, and other techniques.

[0212] In an exemplary embodiment, the electronic device 800 can be implemented with 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, micro-controllers, microprocessors, or other electronic elements, for performing the above-described methods.

[0213] In an exemplary embodiment, a non-transitory computer-readable storage medium, such as the memory 804 including computer program instructions, is also provided, which can be executed by the processor 820 of the electronic device 800 to complete the above-described methods.

[0214] ​ is a block diagram of an electronic device 1900 according to an exemplary embodiment. For example, the electronic device 1900 can be provided as a server. Referring to ​ , 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, such as application programs, executable by the processing component 1922. The application programs stored in the memory 1932 can include one or more than one module each corresponding to a set of instructions. In addition, the processing component 1922 is configured to execute the instructions to perform the above-described methods.

[0215] The electronic device 1900 can 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 can operate based on an operating system stored in the memory 1932, such as Windows Server™, Mac OS X™, Unix™, Linux™, FreeBSD™, or the like.

[0216] In example embodiments, a non-transitory computer-readable storage medium, e.g., memory 1932 including computer program instructions, is also provided that can be executed by processing component 1922 of electronic device 1900 to implement the above-described methods.

[0217] The present disclosure can be a system, a method, and / or a computer program product. The computer program product can include a computer readable storage medium (or media) having computer readable program instructions thereon for causing a processor to carry out aspects of the present disclosure.

[0218] The computer readable storage medium can be a tangible device that can retain and store instructions for use by an instruction execution device. The computer readable storage medium can be, for example, but is not limited to, an electronic 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. A non-exhaustive list of more specific examples of the computer readable storage medium includes the following: a portable computer diskette, 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 disc read-only memory (CD-ROM), a digital versatile disk (DVD), a memory stick, a floppy disk, a mechanically encoded device such as punch-cards or punched tape, a

[0219] The computer readable program instructions described herein can be downloaded to respective computing / processing devices from a computer readable storage medium or to an external computer or external storage device via a network, for example, the Internet, a local area network, a wide area network and / or a wireless network. The network can comprise copper transmission cables, optical transmission fibers, wireless transmission, routers, firewalls, switches, gateway computers and / or edge servers. A network adapter card or network interface in each computing / processing device receives computer readable program instructions from the network and forwards the computer readable program instructions for storage in a computer readable storage medium within the respective computing / processing device.

[0220] Computer readable program instructions for carrying out operations of the present disclosure can be assembler instructions, instruction-set-architecture (ISA) instructions, machine instructions, machine dependent instructions, microcode, firmware instructions, state-setting data, or either source code or object code written in any combination of one or more programming languages, including an object oriented programming language such as Smalltalk, C++ or the like and conventional procedural programming languages, such as the "C" programming language or similar programming languages. The computer readable program instructions can execute entirely on the user's computer, partly on the user's computer, as a stand-alone software package, partly on the user's computer and partly on a remote computer or entirely on the remote computer or server. In the latter scenario, the remote computer can be connected to the user's computer through any type of network, including a local area network (LAN) or a wide area network (WAN), or the connection can be made to an external computer (for example, through the Internet using an Internet Service Provider). In some embodiments, electronic circuitry including, for example, programmable logic circuitry, field-programmable gate array (FPGA), or programmable logic array (PLA) can execute the computer readable program instructions by utilizing state information of the computer readable program instructions to personalize the electronic circuitry, in order to perform aspects of the present disclosure.

[0221] The computer readable program instructions can also be loaded onto a computer, other programmable data processing apparatus, or other device to cause a series of operational steps to be performed on the computer, other programmable apparatus or other device to produce a computer implemented process such that the instructions which execute on the computer or other programmable apparatus provide processes for implementing the functions / acts specified in the flowchart and / or block diagram block or blocks.

[0222] These computer readable program instructions can be provided to a processor of a general purpose computer, special purpose computer, or other programmable data processing apparatus to produce a machine, such that the instructions, which execute via the processor of the computer or other programmable data processing apparatus, create means for implementing the functions / acts specified in the flowchart and / or block diagram block or blocks. These computer readable program instructions can also be stored in a computer readable storage medium that can include random access memory (RAM), read only memory (ROM), electrically erasable programmable read only memory (EEPROM), flash memory or other data storage device. When the computer readable program instructions are loaded into the computer and other programmable data processing apparatus, a series of operational steps are implemented that provide processes such that the computer or other programmable apparatus provide processes for implementing the functions / acts specified in the flowchart and / or block diagram block or blocks.

[0223] The computer readable program instructions can also be loaded onto a computer, other programmable data processing apparatus, or other device to cause a series of operational steps to be performed on the computer, other programmable data processing apparatus or other device to produce a computer implemented process such that the instructions which execute on the computer, other programmable data processing apparatus, or other device implement the functions / acts specified in the flowchart and / or block diagram block or blocks.

[0224] The flow diagrams and the block diagrams in the drawings are presented to illustrate the architecture, functionality, and operation of possible implementations of systems, methods and computer program products according to various embodiments of the present disclosure. In this regard, each block in the flow diagrams and the block diagrams can represent a module, segment, or portion of instructions, which comprises one or more executable instructions for implementing the specified logic functions. In some alternative implementations, the functions noted in the blocks can occur out of the order noted in the figures. For example, two blocks shown in succession may, in fact, be executed substantially concurrently, or the blocks can sometimes be executed in the reverse order, depending upon the functionality involved. It will also be noted that each block of the block diagrams and / or flow diagrams, and combinations thereof, can be implemented by special purpose hardware-based systems that perform the specified functions or acts, or combinations of special purpose hardware and

[0225] Embodiments of the present disclosure have been described above, and the description is intended to be illustrative of the embodiments and not restrictive. Many modifications and variations of the described embodiments are possible and are within the scope of the disclosure. The selection of the terms to be used in the description is not intended to limit the scope of the embodiments described herein, but rather to best describe the principles of the embodiments in the context of the specific application.

Claims

1. A method for preparing a nano-enzyme, characterized in that, The method comprises the following steps: obtaining a Mn(NO3)2 stirring solution with a first set volume and a first set concentration of 0.4 mM, and an aminoethanol stirring solution with a second set volume and a second set concentration of 0.8 mM, wherein the second set volume is the same as the first set volume; mixing the Mn(NO3)2 stirring solution and the aminoethanol stirring solution to obtain a mixed solution; stirring the mixed solution under an inert atmosphere at a set rate and for a third set time of 3 hours to obtain a stirred mixed solution; aging the stirred mixed solution for a fourth set time of 24 hours to obtain a corresponding suspension; performing filtration, washing and drying on the suspension to obtain Mn3O4 oxidase.

2. The nanoscale enzyme production method of claim 1, wherein, Before the step of obtaining the Mn(NO3)2 stirring solution with the first set volume and the first set concentration of 0.4 mM and the aminoethanol stirring solution with the second set volume and the second set concentration of 0.8 mM, the method comprises the following steps: preparing the Mn(NO3)2 solution with the first set volume and the first set concentration and the aminoethanol solution with the second set volume and the second set concentration; stirring the Mn(NO3)2 solution and the aminoethanol solution under the inert atmosphere for a first set time and a second time to obtain the Mn(NO3)2 stirring solution and the aminoethanol stirring solution.

3. The method for preparing nanozymes according to any one of claims 1 or 2, characterized in that, The first set volume and the second set volume are respectively 10 ml.

4. The nanoscale enzyme production method according to any one of claims 1 or 2, wherein, The inert atmosphere is an argon atmosphere.

5. The method for preparing nanozymes according to any one of claims 3, characterized in that, The inert atmosphere is an argon atmosphere.

6. The nanoscale enzyme preparation method according to any one of claims 1 or 2 or 5, wherein, The step of performing filtration, washing and drying on the suspension to obtain the Mn3O4 oxidase comprises the following steps: performing filtration and washing on the suspension; performing drying on the suspension after the filtration and washing at a first set temperature and for a fifth set time to obtain the Mn3O4 oxidase.

7. The nanoscale enzyme production method of claim 3, wherein the step of introducing the first and second nucleic acid sequences into the host cell is performed by transfection, transformation, transduction, electroporation, or viral infection. The step of performing filtration, washing and drying on the suspension to obtain the Mn3O4 oxidase comprises the following steps: performing filtration and washing on the suspension; performing drying on the suspension after the filtration and washing at a first set temperature and for a fifth set time to obtain the Mn3O4 oxidase.

8. The nanoscale enzyme production method of claim 4, wherein, The step of performing filtration, washing and drying on the suspension to obtain the Mn3O4 oxidase comprises the following steps: performing filtration and washing on the suspension; performing drying on the suspension after the filtration and washing at a first set temperature and for a fifth set time to obtain the Mn3O4 oxidase.

9. The nanoscale enzyme production method of claim 6, wherein, The first set temperature is 60°C.

10. The nanoscale enzyme preparation method according to any one of claims 7 or 8, wherein, The first set temperature is 60°C.

11. The nanoscale enzyme production method of claim 6, wherein, The fifth set time is 24 hours.

12. The nanoscale enzyme preparation method according to any one of claims 7-9, wherein, The fifth set time is 24 hours.

13. The nanoscale enzyme production method of claim 10, wherein, The fifth set time is 24 hours.

14. The nanoscale enzyme production method of claim 6, wherein, The step of aging the stirred mixed solution for the fourth set time to obtain the corresponding suspension comprises the following steps: real-time detecting a first color of the suspension within the fourth set time; if the first color becomes grayish brown, it is determined that the aging of the stirred mixed solution is completed, and the corresponding suspension is obtained.

15. The nanoscale enzyme preparation method according to any one of claims 7-9, 11, 13, wherein, The aging treatment of the stirring mixed solution according to the fourth setting time obtains corresponding suspension, including: In the fourth setting time, the first color corresponding to the suspension is detected in real time; If the first color becomes grayish brown, it is determined that the aging treatment of the stirring mixed solution is completed, and the corresponding suspension is obtained.

16. The nanoscale enzyme production method of claim 10, wherein, The aging treatment of the stirring mixed solution according to the fourth setting time obtains corresponding suspension, including: In the fourth setting time, the first color corresponding to the suspension is detected in real time; If the first color becomes grayish brown, it is determined that the aging treatment of the stirring mixed solution is completed, and the corresponding suspension is obtained.

17. The nanoscale enzyme production method of claim 12, wherein, The aging treatment of the stirring mixed solution according to the fourth setting time obtains corresponding suspension, including: In the fourth setting time, the first color corresponding to the suspension is detected in real time; If the first color becomes grayish brown, it is determined that the aging treatment of the stirring mixed solution is completed, and the corresponding suspension is obtained.

18. A nano-enzyme, characterized in that, The nano-enzyme is prepared by using the nano-enzyme preparation method in any one of claims 1-17.

19. The nano-enzyme prepared by using the preparation method in any one of claims 1-17 is applied in the detection of cysteine.

20. The nano-enzyme prepared by using the preparation method in any one of claims 1-17 is applied in the detection of cysteine in urine.

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