CuPH (at) KMO composite nano-enzyme material as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of nano-enzymes, and particularly relates to a CuPH (at) KMO composite nano-enzyme material as well as a preparation method and application thereof. Aiming at the problems of poor catalytic activity, complex steps, high raw material cost and low biocompatibility of nano-enzyme prepared by the existing method, the preparation method comprises the following steps: by taking terephthalic acid and histidine as dual-coordination copper-based nano-enzyme as a precursor, introducing manganese oxide in situ under a hydrothermal condition; and the flower-like nano mimic enzyme which is uniform in size and has high laccase-like catalytic characteristics at the same time is synthesized. The composite nano-enzyme provided by the invention can catalytically oxidize phenol amine organic compounds under the condition that the pH value is 3-9, can realize the detection of the content of ergothioneine in cosmetics and nutritional supplement capsules, and can be applied to the field of cosmetic and food safety monitoring.

Description

Technical Field

[0001] The present invention belongs to the technical field of nanozymes, and particularly relates to a CuPH@KMO composite nanozyme material, a preparation method thereof, and an application thereof. Background Art

[0002] Natural enzymes are effective biocatalysts and participate in almost all metabolic processes of organisms. Due to their high catalytic efficiency and high substrate selectivity, natural enzymes have broad application prospects in industrial production, medical research, bioanalysis and other fields. However, natural enzymes have disadvantages such as high preparation and purification costs, easy denaturation under conditions such as high temperature or extreme pH, and degradation by proteases. In recent years, researchers have been committed to developing "artificial enzymes" with enzymatic activity as substitutes for natural enzymes. In the research, nanozymes with the catalytic function of natural enzymes have attracted wide attention. Nanozymes are nanomaterials with the catalytic ability of natural enzymes and following the enzymatic properties of natural enzymes (such as catalytic kinetics). Nanozymes have excellent properties such as good stability, high catalytic activity, highly adjustable structure, and low cost, and are expected to be excellent substitutes for natural enzymes in the fields of chemical and biological sensing, biomedicine, etc. Therefore, it is of great significance to develop new nanozymes with simple preparation methods, low costs, high catalytic performance, and good stability.

[0003] So far, the developed nanozymes cover four categories of catalytic enzymes: oxidoreductases, hydrolases, isomerases, and lyases. At the present stage, 92.9% of the nanozymes exhibit oxidoreductase-like activities, mainly including peroxidase (POD), oxidase (OXD), catalase (CAT), and superoxide dismutase (SOD)-like activities. In terms of peroxidase-like nanozymes, it can catalyze chromogenic substrates (such as 3,3',5,5'-tetramethylbenzidine (TMB), o-phenylenediamine (OPD), 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS), etc.) in the presence of hydrogen peroxide to form colored oxidation products, thus providing a convenient, rapid, and intuitive colorimetric detection. However, we know that the use of unstable and destructive hydrogen peroxide (H2O2) often affects the reproducibility of the detection system. Therefore, it is extremely important to develop laccase that does not require nor produce hydrogen peroxide during the catalytic oxidation process. Laccase is an environmentally friendly multi-copper oxidase, and its catalytic center contains four copper ions, which can catalyze the oxidation of substrates such as monophenols, polyphenols, and amines through four-electron transfer. In addition, like any other natural enzyme, laccase has poor stability, high price, and difficult storage. To solve these inherent disadvantages, a variety of inorganic, organic, and hybrid materials that can mimic the active sites of laccase have been discovered. Although underdeveloped and still requiring a lot of attention, the use of laccase mimetic enzyme materials as green catalysts is becoming a new field in nanozyme research.

[0004] So far, most of the existing laccase mimetic enzymes are independent nanozymes with a single catalytic center, such as MnO2, CeO2, BH-Cu, etc. There are still significant problems in their catalytic activity and substrate selectivity. To solve the above problems, composite nanozymes have emerged. Composite is the process of combining nanozymes with other substances to achieve better performance or expand their application scope. Compared with traditional nanozymes, composite nanozymes have a high specific surface area and abundant catalytic active centers, which makes them perform well in catalytic reactions and can significantly improve the reaction rate and efficiency. In short, the composite of nanozymes is an effective way to achieve high-performance nanomaterials and can provide strong support for the development of fields such as chemistry and biomedicine. Summary of the Invention

[0005] Based on this, aiming at the problems of poor catalytic activity, complex preparation method, high raw material cost and low biocompatibility of nanozymes prepared by existing methods, the present invention provides a metal-based composite nanozyme (CuPH@KMO) with laccase activity synergy, its preparation method and application.

[0006] The object of the present invention is achieved by the following technical solutions:

[0007] The first aspect of the present invention provides a preparation method of a CuPH@KMO composite nanozyme material, comprising the following steps:

[0008] Step 1: At room temperature, weigh histidine solid and dissolve it in a mixed solvent of N,N-dimethylformamide and ethanol with a volume ratio of 1:1. After stirring evenly, solution A is obtained; weigh copper nitrate solid and dissolve it in N,N-dimethylformamide solvent. Under continuous stirring, add terephthalic acid and polyvinylpyrrolidone K30 to obtain a uniform solution B.

[0009] Step 2: On a magnetic stirrer, slowly add solution B to solution A and continuously stir and react until a clear blue-green solution C is obtained.

[0010] Step 3: Transfer the obtained clear solution C into a hydrothermal reaction kettle with a polytetrafluoroethylene liner. Heat it in an oven at 110°C for 8 hours, and then slowly cool the reaction kettle to room temperature.

[0011] Step 4: Transfer the solution in the reaction kettle to a centrifuge tube, centrifuge it at 10000 r / min for 5 minutes, remove the upper clear liquid, collect the lower precipitate, rinse it repeatedly three times with ultrapure water and methanol solution, and place the precipitate in an oven at 50°C to dry for 24 hours to obtain the prepared precursor copper-based nanozyme.

[0012] Step 5: Weigh manganese sulfate monohydrate, potassium permanganate and polyvinylpyrrolidone K30 solids and dissolve them in 25 mL of ultrapure water, and continuously stir until they are evenly dispersed.

[0013] Step 6: Add the solid powder of the precursor copper-based nanozyme to the solution obtained in Step 5, and ultrasonically disperse it until it is uniform. Then transfer the mixed solution to a high-pressure reactor and react at 160 °C for 1 hour. After the reaction is completed, cool the reactor to room temperature, and centrifuge, wash, and dry the solution to obtain the CuPH@KMO composite nanozyme material.

[0014] Further, the molar ratio of metallic copper, terephthalic acid, and histidine in the solution C in Step 2 is 2:1:1.

[0015] Further, the mass ratio of copper nitrate trihydrate to polyvinylpyrrolidone K30 added to the solution B in Step 1 is 3:1; the mass ratio of manganese sulfate monohydrate to polyvinylpyrrolidone K30 added in Step 5 is 10:1.

[0016] Further, the molar ratio of manganese sulfate monohydrate to potassium permanganate solid added in Step 5 is 1:1.

[0017] Further, the addition amount of the precursor copper-based nanozyme in Step 5 is 10 - 30 mg.

[0018] The second aspect of the present invention provides a CuPH@KMO composite nanozyme synthesized by the above preparation method. This nanozyme has laccase-like catalytic activity and can catalytically oxidize phenolic amine organic compounds under the condition of pH 3 - 9.

[0019] The third aspect of the present invention provides an application of the CuPH@KMO composite nanozyme in the detection of ergothioneine. The detection methods are fluorescence and ultraviolet methods, which specifically include the following steps:

[0020] Step 1: Sequentially add resorcinol solution, dopamine solution, ergothioneine solutions with different concentrations, and CuPH@KMO nanozyme to a 2-(N-morpholino)ethanesulfonic acid monohydrate buffer solution with pH = 7. After diluting with deionized water, incubate at room temperature for 10 minutes, and respectively measure and record the fluorescence intensity at 478 nm and the ultraviolet absorbance at 420 nm, and draw a standard working curve.

[0021] Step 2: After pre-treating the actual sample, operate according to Step 1 to measure and record the fluorescence intensity at 478 nm and the ultraviolet absorbance at 420 nm, and obtain the ergothioneine content in the actual sample according to the working curve.

[0022] Further, the principle of the fluorescence and ultraviolet detection methods is: Ergothioneine with reducibility and chelating ability will inhibit the generation of fluorescein, resulting in a decrease in both the fluorescence at 478 nm and the ultraviolet absorption at 420 nm, thereby realizing the linear analysis of ergothioneine.

[0023] Furthermore, the actual samples are cosmetics and nutritional supplement capsules containing ergothioneine. The specific pre-treatment process is as follows: Dissolve the content of the nutritional supplement capsule in 1 mL of deionized water for standby, and filter the cosmetic essence through a 0.22-μm ultrafiltration membrane for standby.

[0024] Compared with the prior art, the present invention has the following beneficial effects:

[0025] 1. The present invention uses copper nitrate trihydrate as the copper source and terephthalic acid and histidine as the two ligands to prepare the precursor metal nanozyme material (CuPH). Then, manganese oxide is successfully introduced onto the copper-based nanozyme by an in-situ synthesis method, thereby obtaining a composite laccase mimetic enzyme with a synergistic enhancement effect.

[0026] 2. The raw materials used in the method of the present invention are safe, easily available, low in price, simple in operation, and have low requirements for instruments and equipment, providing the possibility for large-scale industrial synthesis.

[0027] 3. The composite nanozyme provided by the present invention has uniform and stable properties and low toxicity, and has excellent laccase catalytic activity. It can catalyze the oxidation of phenolic amine organic compounds under the condition of pH 3-9, and has great application prospects in the fields of environmental monitoring, food safety monitoring, and bioanalysis. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for the description of the embodiments or the prior art. Obviously, the drawings in the following description are only the embodiments of the present invention. For those of ordinary skill in the art, other drawings can be obtained according to the provided drawings without creative efforts.

[0029] Figure 1 are the scanning electron microscope images and particle size distribution diagrams of the CuPH and CuPH@KMO-20 composite nanozymes prepared in Example 1 and Example 4;

[0030] Figure 2 is the elemental analysis diagram of the CuPH@KMO-20 composite nanozyme prepared in Example 4;

[0031] Figure 3 are the Fourier transform infrared spectra of the CuPH, KMO, and CuPH@KMO-20 composite nanozymes prepared in Example 1, Example 2, and Example 4;

[0032] Figure 4 are the thermogravimetric analysis curves of the CuPH, KMO, and CuPH@KMO-20 composite nanozymes prepared in Example 1, Example 2, and Example 4;

[0033] Figure 5Are the nitrogen adsorption - desorption isotherm curves of CuPH, KMO, and CuPH@KMO - 20 composite nanozymes prepared in Example 1, Example 2, and Example 4;

[0034] Figure 6 Are the ultraviolet absorption spectra of the oxidation of laccase substrates 2,4 - dichlorophenol (2,4 - DP) and 4 - aminophenazone (4 - AP) catalyzed by CuPH, KMO, and CuPH@KMO - 20 composite nanozymes prepared in Example 1, Example 2, and Example 4;

[0035] Figure 7 Is the comparison chart of the laccase catalytic activities of CuPH@KMO - 10, CuPH@KMO - 20, and CuPH@KMO - 30 composite nanozymes prepared in Example 3 - 5;

[0036] Figure 8 Are the catalytic activities of the nanozymes prepared in Example 1, Example 2, and Example 4 in the full pH range;

[0037] Figure 9 Are the fluorescence spectra and linear relationship charts for EGT detection;

[0038] Figure 10 Are the ultraviolet absorption spectra and linear relationship charts for EGT detection. Detailed implementation manners

[0039] The present invention will be further described in detail below with reference to the drawings and examples. The specific examples described here are only used to explain the present invention, rather than limiting the present invention. Without conflict, the embodiments and features in the embodiments of this application can be combined with each other. Additionally, it should be noted that for the convenience of description, only parts related to the present invention are shown in the drawings, rather than all structures. It should be pointed out that for those of ordinary skill in the art, without departing from the principle of the present invention, several modifications and improvements can also be made, which should also be regarded as within the protection scope of the present invention.

[0040] Example 1

[0041] This example provides a preparation method of a precursor copper - based nanozyme CuPH, and the steps are as follows:

[0042] Step 1: At room temperature, take 0.1 mmol of histidine compound and dissolve it in an 8 - mL beaker containing a mixed solution of N,N - dimethylformamide and ethanol (the volume ratio of DMF to EtOH is 1:1), and continuously stir with a glass rod to prepare a mixed solution A containing histidine;

[0043] At room temperature, 0.2 mmol of copper nitrate trihydrate was dissolved in a beaker containing 4 mL of N,N-dimethylformamide solvent. Under continuous stirring, 0.1 mmol of terephthalic acid and 16 mg of polyvinylpyrrolidone K30 (PVP) were added, and the mixture was continuously stirred with a glass rod to form a homogeneous mixed solution B.

[0044] Step 2: Place the prepared homogeneous mixed solution A on a magnetic stirrer. Under continuous stirring, slowly add the prepared homogeneous mixed solution B to the above system to form a mixed solution C. After stirring for 10 minutes, a clear blue-green solution was finally obtained, indicating that the coordination compound of Cu 2+ with terephthalic acid and histidine has been formed and is well dispersed in the solution.

[0045] Step 3: Transfer the above-obtained homogeneous and clear mixed solution C into a hydrothermal reaction kettle with a polytetrafluoroethylene liner. Heat it in an oven at 110 °C for 8 hours, and then slowly cool the reaction kettle to room temperature.

[0046] Step 4: Open the reaction kettle cooled to room temperature, transfer the prepared mixed solution of nanozyme to a centrifuge tube, place it in a centrifuge and centrifuge at 10000 r / min for 5 minutes. Remove the supernatant, collect the lower precipitate and wash it repeatedly three times with deionized water and methanol solution. Finally, collect the pure lower precipitate and transfer it to an oven at 50 °C to dry for 24 hours to obtain the precursor copper-based nanozyme (CuPH).

[0047] Example 2

[0048] This example provides a manganese oxide KMO nanozyme, and the steps are as follows:

[0049] Step 1: Add 0.5 mmol of manganese sulfate monohydrate, 0.5 mmol of potassium permanganate and 8.5 mg of polyvinylpyrrolidone K30 to 25 mL of deionized water in sequence, and continuously stir in a magnetic stirrer for 30 minutes. Finally, transfer the mixed solution to a high-pressure reaction kettle and react at a temperature of 160 °C for 1 hour.

[0050] Step 2: After the reaction is completed, open the reaction kettle cooled to room temperature, transfer the prepared mixed solution of the composite enzyme to a centrifuge tube, place it in a centrifuge and centrifuge at 10000 r / min for 5 minutes. Remove the supernatant, collect the lower precipitate and wash it repeatedly three times with deionized water and ethanol solution. Finally, collect the pure lower precipitate and transfer it to an oven at 50 °C to dry for 24 hours to obtain the prepared manganese oxide KMO.

[0051] Example 3

[0052] This example provides a preparation method of a CuPH@KMO-10 composite nanozyme, and the steps are as follows:

[0053] Step 1: Dissolve 0.5 mmol of manganese sulfate monohydrate, 0.5 mmol of potassium permanganate, and 8.5 mg of polyvinylpyrrolidone K30 solid in 25 mL of ultrapure water, and continuously stir in a magnetic stirrer for 30 minutes. Add 10 mg of the CuPH solid powder prepared in Example 1 to the above solution, and ultrasonicate for 10 minutes until the solid is uniformly dispersed. Finally, transfer the mixed solution to a high-pressure reaction kettle and react at 160 °C for 1 hour;

[0054] Step 2: After the reaction is completed, open the reaction kettle cooled to room temperature, transfer the prepared mixed solution of the composite enzyme to a centrifuge tube, place it in a centrifuge and centrifuge at 10000 r / min for 5 minutes, remove the supernatant, collect the lower precipitate, and rinse it three times with deionized water and ethanol solution. Finally, collect the pure lower precipitate and transfer it to an oven at 50 °C to dry for 24 hours to obtain the prepared composite nanozyme CuPH@KMO-10.

[0055] Example 4

[0056] This example provides a method for preparing a CuPH@KMO-20 composite nanozyme, and the steps are as follows:

[0057] Step 1: Dissolve 0.5 mmol of manganese sulfate monohydrate, 0.5 mmol of potassium permanganate, and 8.5 mg of polyvinylpyrrolidone K30 solid in 25 mL of ultrapure water, and continuously stir in a magnetic stirrer for 30 minutes. Add 20 mg of the CuPH solid powder prepared in Example 1 to the above solution, and ultrasonicate for 10 minutes until the solid is uniformly dispersed. Finally, transfer the mixed solution to a high-pressure reaction kettle and react at 160 °C for 1 hour;

[0058] Step 2: After the reaction is completed, open the reaction kettle cooled to room temperature, transfer the prepared mixed solution of the composite enzyme to a centrifuge tube, place it in a centrifuge and centrifuge at 10000 r / min for 5 minutes, remove the supernatant, collect the lower precipitate, and rinse it three times with deionized water and ethanol solution. Finally, collect the pure lower precipitate and transfer it to an oven at 50 °C to dry for 24 hours to obtain the prepared composite nanozyme CuPH@KMO-20.

[0059] Example 5

[0060] This example provides a method for preparing a CuPH@KMO-30 composite nanozyme, and the steps are as follows:

[0061] Step 1: Dissolve 0.5 mmol of manganese sulfate monohydrate, 0.5 mmol of potassium permanganate, and 8.5 mg of polyvinylpyrrolidone K30 solid in 25 mL of ultrapure water, and continuously stir in a magnetic stirrer for 30 minutes. Add 30 mg of the CuPH solid powder prepared in Example 1 to the above solution, sonicate for 10 minutes until the solid is evenly dispersed, and finally transfer the mixed solution to a high-pressure reaction kettle and react at 160 °C for 1 hour;

[0062] Step 2: After the reaction is completed, open the reaction kettle cooled to room temperature, transfer the prepared mixed solution of the composite enzyme to a centrifuge tube, place it in a centrifuge and centrifuge at 10000 r / min for 5 minutes, remove the supernatant, collect the lower precipitate, and rinse it three times with deionized water and ethanol solution. Finally, collect the pure lower precipitate, transfer it to an oven at 50 °C and dry it for 24 hours to obtain the prepared composite nanozyme CuPH@KMO-30.

[0063] Example 6

[0064] This example provides the laccase-like activity of the nanozymes prepared according to Examples 1-5:

[0065] 2,4-Dichlorophenol and 4-aminoantipyrine can be oxidized by natural laccase to form red quinone imine substances with a strong absorption peak at 510 nm. The amount of the generated red quinone imine is proportional to the absorbance at 510 nm and the laccase activity. Therefore, the strength of the laccase activity of the nanozyme can be studied according to the intensity of the absorbance at 510 nm.

[0066] This example selects 2,4-dichlorophenol and 4-aminoantipyrine, the substrates of natural laccase, as the oxidation substrates, and studies the laccase-like catalytic activities of five nanomaterials, CuPH, KMO, CuPH@KMO-10, CuPH@KMO-20, and CuPH@KMO-30, prepared in Examples 1-5. The specific steps are as follows:

[0067] Add 60 μL of 1 mg / mL CuPH, KMO, CuPH@KMO-10, CuPH@KMO-20, and CuPH@KMO-30 nanozymes to 300 μL of 50 mM 2-(N-morpholino)ethanesulfonic acid monohydrate (MES) buffer solution (pH = 6) respectively, and then add 100 μL of 1 mg / mL 4-aminoantipyrine and 100 μL of 1 mg / mL 2,4-dichlorophenol solution. Finally, add 440 μL of deionized water to the mixture. After reacting at room temperature for 20 minutes, scan the ultraviolet absorption spectrum in the range of 300-700 nm using an ultraviolet spectrophotometer.

[0068] Example 7

[0069] This example provides a study on the catalytic pH range of CuPH, KMO, and CuPH@KMO-20 nanozymes with laccase activity. The specific steps are as follows:

[0070] Add 60 μL of 1 mg / mL CuPH, KMO, or CuPH@KMO-20 nanozyme to 300 μL of 2-(N-morpholino)ethanesulfonic acid monohydrate (MES) buffer solutions with different pH values (pH = 3, 4, 5, 6, 7, 8, 9), and then add 100 μL of 1 mg / mL 4-aminoantipyrine and 100 μL of 1 mg / mL 2,4-dichlorophenol solution. Finally, dilute the reaction system to 1 mL with deionized water. After reacting at room temperature for 20 minutes, record the ultraviolet absorbance value at 510 nm.

[0071] Example 8

[0072] This example provides an ergothioneine sensing method based on CuPH@KMO-20 nanozyme:

[0073] Sequentially add 150 μL of 10 mM resorcinol (RS) solution, different concentrations of ergothioneine (EGT) solutions (0, 2, 5, 11, 25, 45, 70, 115, 185, 230, 290, 330, 400 μg / mL), 20 μL of 10 mM dopamine (DA) solution, and 50 μL of 1 mg / mL CuPH@KMO nanozyme to a centrifuge tube containing 300 μL of 2-(N-morpholino)ethanesulfonic acid monohydrate (MES) buffer solution (pH = 7), and dilute with deionized water to ensure that the total volume of the system is 1 mL. After incubating at 25 °C for 10 minutes, record the fluorescence intensity at 478 nm respectively to construct a standard working curve.

[0074] Sequentially add 150 μL of 10 mM resorcinol (RS) solution, different concentrations of ergothioneine (EGT) solutions (0, 5, 11, 25, 45, 70, 115, 150, 185, 230, 280 μg / mL), 20 μL of 10 mM dopamine (DA) solution, and 50 μL of 1 mg / mL CuPH@KMO nanozyme to a centrifuge tube containing 300 μL of 2-(N-morpholino)ethanesulfonic acid monohydrate (MES) buffer solution (pH = 7), and dilute with deionized water to ensure that the total volume of the system is 1 mL. After incubating at 25 °C for 10 minutes, record the ultraviolet absorbance at 420 nm respectively to construct a standard working curve.

[0075] Example 9

[0076] This example provides the detection of the content of ergothioneine (EGT) in cosmetics and nutritional supplement capsules. The specific steps are as follows:

[0077] Select cosmetics and nutritional supplement capsules containing EGT as the actual samples for EGT analysis. First, completely dissolve the obtained content of the supplement capsules in 1 mL of deionized water for later use. Filter the cosmetic essence through a 0.22 μm ultrafiltration membrane for later use. Then, add different concentrations of EGT to the processed actual samples respectively. After that, operate according to the steps of EGT analysis. That is, add 150 μL of 10 mM RS solution, different concentrations of EGT solution, 20 μL of 10 mM DA solution, and 50 μL of 1 mg / mL CuPH@KMO nanozyme into a centrifuge tube containing 300 μL of MES buffer solution (pH = 7) respectively. After incubating at 25 °C for 10 minutes, measure the fluorescence intensity at 478 nm and the ultraviolet absorbance at 420 nm respectively.

[0078] Table 1 Detection of EGT Content in Cosmetics and Nutritional Supplement Capsules

[0079]

[0080] According to Table 1, 48.5 μg / mL of EGT was detected by the fluorescence mode in the supplement capsules, and 49.2 μg / mL of EGT was detected by the ultraviolet mode, which is not much different from the marked addition amount of 50 μg / mL in the capsules. Moreover, the recovery rate of EGT obtained by the standard addition method in the fluorescence mode is between 98.73 - 103.71%, and the RSD is less than 4.11%. The recovery rate of EGT obtained by the standard addition method in the ultraviolet mode is between 95.22 - 102.02%, and the RSD is less than 3.87%. Further, the recovery rate of EGT in the cosmetic essence obtained by the standard addition method in the fluorescence mode is between 98.97 - 103.26%, and the RSD is less than 3.24%. The recovery rate of EGT in the cosmetic essence obtained by the standard addition method in the ultraviolet mode is between 99.55 - 102.05%, and the RSD is less than 4.17%.

[0081] Figure 1 For the physical morphology of the CuPH@KMO-20 composite nanozyme obtained by the steps of Example 1 and 4, according to the attached Figure 1 The results of A scanning electron microscopy show that CuPH synthesized with the ratio of terephthalic acid to histidine of 1:1 presents smooth-surfaced granular particles. And according to Figure 1 the particle size distribution in B, its diameter is mostly distributed around 500 nm, and it is a kind of nano-particle with uniform size. As Figure 1 shown in C and 1D, after in-situ growth of manganese oxide, the scanning electron microscopy image of the composite material CuPH@KMO-20 presents a complete flower-like lamellar structure, and the overall size is roughly distributed around 600 - 700 nm.

[0082] Figure 2Element distribution map of the CuPH@KMO-20 composite nanozyme obtained according to the steps of Examples 1 and 4. The results show that the CuPH@KMO-20 composite nanozyme contains five elements including carbon, nitrogen, oxygen, copper, and manganese, indicating that the composite nanozyme not only includes the copper-based nanozyme with dual ligands but also contains in-situ synthesized manganese oxide.

[0083] Figure 3 Fourier transform infrared spectroscopy characterization of the CuPH, KMO, and CuPH@KMO-20 composite nanozymes prepared in Examples 1, 2, and 4. In the spectrum of CuPH, the absorption peaks at 1672 cm -1 and 1143 cm -1 correspond to the stretching vibration absorption peaks of C=O and C-O in the carboxyl group, respectively. The irregular absorption band at 1450 - 1600 cm -1 is related to the skeletal vibration of the benzene ring. The above results indicate the successful coordination of terephthalic acid with Cu 2+ The vibration peak at 1265 cm -1 corresponds to the stretching vibration of the aromatic amine C-N bond. In addition, the vibration peak at 828 cm -1 corresponds to the stretching vibration of the aromatic amine N-H bond, indicating the successful coordination of histidine in the CuPH nanozyme, which proves the successful preparation of the CuPH nanozyme. In the infrared spectrum of CuPH@KMO-20, it not only contains the infrared vibration peaks of CuPH but also the vibration peak of the typical Mn-O bond at 516.8 cm -1 in the manganese oxide KMO, which proves the successful preparation of the CuPH@KMO-20 composite nanozyme.

[0084] Figure 4 Thermogravimetric analysis (TGA) curves of CuPH, KMO, and CuPH@KMO-20 synthesized in Examples 1, 2, and 4. The results show that due to the influence of the ligand, CuPH loses 10% of its weight at 257 °C, and the manganese oxide KMO still maintains more than 90% of its original weight at 600 °C. Due to the combination of CuPH and the manganese oxide, the thermodynamic performance of CuPH@KMO-20 is greatly improved, resulting in its losing 10% of its original weight at 456 °C.

[0085] Figure 5 Nitrogen adsorption-desorption isotherm curves of CuPH, KMO, and CuPH@KMO-20 synthesized in Examples 1, 2, and 4. By fitting with the Brunauer-Emmett-Teller method, the specific surface areas (BET) of CuPH, KMO, and CuPH@KMO-20 are 28.11, 84.28, and 171.09 m 2 / g. The results show that the composite CuPH@KMO-20 has the best specific surface area, which also provides a theoretical basis for the synergistic effect of enhanced laccase activity.

[0086] Figure 6 Investigation of the laccase catalytic activities of CuPH, KMO, and CuPH@KMO-20 synthesized in Examples 1, 2, and 4. It can be seen that there is no absorption peak at 510 nm for individual 2,4-dichlorophenol and 4-aminoantipyrine. Only when 2,4-dichlorophenol, 4-aminoantipyrine, and CuPH, KMO, or CuPH@KMO-20 coexist, an obvious ultraviolet absorption peak appears at 510 nm. Among them, the absorbance value of CuPH@KMO-20 is the largest and is greater than the sum of the absorbance values of CuPH and KMO. This indicates that among the three nanozymes, CuPH, KMO, and CuPH@KMO-20, the composite CuPH@KMO-20 has the strongest laccase-like catalytic activity due to the synergistic effect.

[0087] Figure 7 The absorbance values of the quinoneimine substances generated by the oxidation of 2,4-dichlorophenol and 4-aminoantipyrine catalyzed by the composite nanozymes prepared with different precursor doping amounts were compared at 510 nm. The results show that the magnitude of the absorbance value at 510 nm continuously increases with the increase in the mass of the doped precursor CuPH and reaches the maximum value at 20 mg, indicating that CuPH@KMO-20 synthesized by doping 20 mg of the precursor CuPH has the best laccase catalytic effect. In summary, the nanozyme synthesized by doping 20 mg of the precursor CuPH has a uniform and complete morphology and the optimal laccase catalytic effect.

[0088] Figure 8 For the catalytic activities of the nanozymes prepared in Example 1, Example 2, and Example 4 under different pH conditions. As Figure 8 shown, CuPH has more excellent catalytic activity under alkaline conditions, while KMO has more excellent catalytic activity under acidic conditions. The reaction pH range of the CuPH@KMO composite nanozyme is significantly broadened and can cover any pH from acidic to alkaline. This greatly increases the applicable range and application environment of CuPH@KMO.

[0089] Figure 9 For the fluorescence spectrogram and linear relationship diagram of EGT detection. When the concentration range of EGT is 2 - 330 μg / mL, the fluorescence intensity at 478 nm is also linearly correlated with the concentration of EGT, and the fitting equation is F = -15.1122C EGT +6210, R 2 = 0.9933, and its detection limit is 0.33 μg / mL.

[0090] Figure 10 UV absorption spectrum and linear relationship diagram for EGT detection. When the concentration range of EGT is 5 - 230 μg / mL, the UV absorbance at 420 nm shows a linear correlation with the concentration of EGT, and the fitting equation is A = -0.0026C EGT + 0.9539, R 2 = 0.9960, and its detection limit is 1.32 μg / mL.

[0091] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications made without departing from the spirit and principle of the present invention shall be equivalent replacement methods and are all included in the protection scope of the present invention.

Claims

1. A method for preparing a CuPH@KMO composite nanozyme material, characterized in that: The following steps are involved: Step 1: Under room temperature, weigh solid histidine and dissolve it in a mixed solvent of N,N-dimethylformamide and ethanol in a volume ratio of 1:1, and stir to obtain solution A; weigh solid copper nitrate trihydrate and dissolve it in N,N-dimethylformamide solvent, and add terephthalic acid and polyvinylpyrrolidone K30 under continuous stirring to obtain a uniform solution B; Step 2: On a magnetic stirrer, slowly add solution B to solution A and continue stirring to obtain a clear blue-green solution C. Step 3: Transfer the solution C obtained above into a hydrothermal reactor with a polytetrafluoroethylene liner, heat it in an oven at 110° C. for 8 hours, and then slowly cool the reactor to room temperature; Step 4: Transfer the solution in the reaction kettle to a centrifuge tube, centrifuge at 10000r / min for 5 minutes, remove the supernatant and collect the lower precipitate, rinse it three times with ultrapure water and methanol solution, and dry the precipitate in a 50°C oven for 24 hours to obtain the prepared precursor copper-based nanozyme; Step 5: Dissolve monohydrated manganese sulfate, potassium permanganate and polyvinylpyrrolidone K30 solids in 25 mL of ultrapure water and stir until uniformly dispersed; Step 6: Add the precursor copper-based nanozyme solid powder to the solution obtained in step 5, ultrasonicate until uniformly dispersed, then transfer the mixed solution to a high-pressure reactor and react at 160°C for 1 hour. After the reaction is completed, cool the reactor to room temperature, centrifuge, wash, and dry the solution to obtain the CuPH@KMO composite nanozyme material.

2. The method for preparing a CuPH@KMO composite nanozyme material according to claim 1, characterized in that: The molar ratio of metallic copper, terephthalic acid and histidine in the solution C in step 2 is 2:1:

1.

3. The method for preparing a CuPH@KMO composite nanozyme material according to claim 1, characterized in that: In the step 1, copper nitrate trihydrate and polyvinyl pyrrolidone K30 are added to solution B in a mass ratio of 3:1; The mass ratio of manganese sulfate monohydrate to polyvinyl pyrrolidone K30 added in step 5 is 10:

1.

4. The method for preparing a CuPH@KMO composite nanozyme material according to claim 1, characterized in that: The molar ratio of manganese sulfate monohydrate and potassium permanganate solid added in step 5 is 1:

1.

5. The method for preparing a CuPH@KMO composite nanozyme material according to claim 1, characterized in that: In step 5, the amount of the precursor copper-based nanozyme added is 10 to 30 mg.

6. A CuPH@KMO composite nanozyme prepared by the preparation method as described in any one of claims 1 to 5.

7. The CuPH@KMO composite nanozyme according to claim 6, characterized in that: The composite nanozyme has laccase-like catalytic activity and can catalyze the oxidation of phenolamine organic compounds under pH conditions of 3 to 9.

8. The application of the CuPH@KMO composite nanozyme in thioneine detection according to claim 6, characterized in that, The detection method is a fluorescence and ultraviolet method, which specifically includes the following steps: Step 1, in the 2-(N-morpholine)ethanesulfonic acid monohydrate buffer solution of pH=7, resorcinol solution, dopamine solution, thioneine solution of different concentrations and CuPH@KMO nanozyme were added successively, diluted with deionized water, incubated at room temperature for 10 minutes, and the fluorescence intensity at 478nm and the ultraviolet absorbance at 420nm were tested and recorded respectively, and a standard working curve was drawn; Step 2, after actual sample is carried out pre-treatment, test and record the fluorescence intensity at 478nm and the ultraviolet absorbance at 420nm by step 1 operation, obtain the thioneine content in actual sample according to working curve.

9. the application of a kind of CuPH@KMO composite nanozyme according to claim 8 in thioneine detection, the principle of described fluorescence and ultraviolet detection method is: thioneine with reducibility and chelating ability can suppress the generation of fluorescein, so that the fluorescence at 478nm and the ultraviolet absorption at 420nm are both reduced, thereby realizing the linear analysis of thioneine.

10. According to the application of CuPH@KMO composite nanozyme in thioneine detection according to claim 8, it is characterized in that, The actual samples are cosmetics and nutritional supplement capsules containing ergothioneine, and the pretreatment process is specifically as follows: dissolving the contents of the nutritional supplement capsules in 1 mL of deionized water for later use, and filtering the cosmetic essence through a 0.22 μm ultrafiltration membrane for later use.

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