CuBi composite N and P doped MXene nano-enzyme, preparation method thereof, detection reagent and non-diagnostic purpose African swine fever detection method

Abstract

The invention discloses a CuBi composite N and P doped MXene nano-enzyme, a preparation method thereof, a detection reagent and a non-diagnostic purpose African swine fever detection method, and relates to the technical field of nano-enzymes. The preparation method of the nano-enzyme comprises the following steps: taking copper salt and bismuth salt to react under the action of a reducing agent, taking a solid phase after the reaction is finished, and freeze-drying to obtain the nano-enzyme, the preparation method comprises the following steps: dispersing Ti3C2MXene powder in water, adding ammonium dihydrogen phosphate, carrying out a reaction at normal temperature, continuously carrying out a hydrothermal reaction after the reaction is finished, taking a solid phase and carrying out freeze-drying after the reaction is finished, and taking the products of the first two steps, dispersing in water, reacting, and after the reaction is finished, taking the solid phase and freeze-drying to obtain the product. The detection reagent needs to be combined with a protein biomarker of a virus to be detected for incubation. According to the nano enzyme, the synthesis steps are convenient and rapid, and the raw materials are conventional commercial products in the field, low in price, simple and easy to obtain; the nano enzyme is relatively high in activity and relatively high in sensitivity; the detection reagent disclosed by the invention has relatively high sensitivity and accuracy.

Description

Technical Field

[0001] The present invention relates to the technical field of nanozymes, and specifically to a CuBi composite N, P-doped MXene nanozyme, a preparation method thereof, a detection reagent, and a method for detecting African swine fever for non-diagnostic purposes. Background Art

[0002] African swine fever (ASF) poses a major threat to the pig industry, not only because of its high mortality rate, but also because of the lack of effective vaccines and treatment methods. In addition, the outbreak of the disease may lead to significant economic and social losses, including trade restrictions, pork price fluctuations, etc.

[0003] Enzyme-linked immunosorbent assay (ELISA) is a commonly used immunoassay method, which is not only highly sensitive but also easy to operate. However, due to the high cost and poor stability of natural enzymes, it is particularly important to find substitutes for immunoassays. Nanozymes have properties similar to natural enzymes and are regarded as good substitutes for natural enzymes due to their low cost, good stability, and high accuracy. Nanozymes are usually used as signal probes, and MXene is used as one of the materials for nanozymes due to its high sensitivity and response speed, and its application is becoming more and more widespread. However, MXene is prone to stacking and aggregation under the interaction of strong van der Waals forces and hydrogen bonds, which greatly limits its application range. Summary of the Invention

[0004] To solve at least one of the above problems, the present invention provides a CuBi composite N, P-doped MXene nanozyme, a preparation method thereof, a detection reagent, and a method for detecting African swine fever for non-diagnostic purposes.

[0005] The technical solution of the present invention is as follows: A preparation method of a CuBi composite N, P-doped MXene nanozyme, comprising the following steps:

[0006] S1. Take a water-soluble copper salt and a water-soluble bismuth salt and dissolve them, then add a reducing agent and react. After the reaction is completed, take the solid phase and freeze-dry to obtain a CuBi metal aerogel; the reducing agent is one of KBH 4 and NaBH 4 The molar ratio of the copper salt, bismuth salt, and reducing agent is 3.6-5.0:0.04-1:20-25;

[0007] S2. Disperse Ti 3 C 2 MXene powder in water, then add ammonium dihydrogen phosphate and dissolve it. React under continuous stirring and at room temperature. After the reaction is completed, continue with a hydrothermal reaction. After the reaction is completed, take the solid phase and freeze-dry to obtain modified MXene powder; the Ti 3 C 2The addition amount of MXene powder is 30 to 50 parts by mass, and the addition amount of ammonium dihydrogen phosphate is 10,000 parts by mass;

[0008] S3. Take the CuBi metal aerogel of S1 and the modified MXene powder of S2 and disperse them in water, then stir and react for 20 to 30 h. After the reaction is completed, take the solid phase and freeze-dry it to obtain; wherein, for every 2 to 2.5×10 -3 mol of reducing agent in S1, it corresponds to 30 to 50 mg of Ti 3 C 2 MXene powder.

[0009] One embodiment of the present invention is that the copper salt is copper nitrate and the bismuth salt is bismuth nitrate.

[0010] One embodiment of the present invention is that in S1, the reaction time is 4 to 5 h; in S2, the normal temperature reaction time is 2 to 2.5 h, the hydrothermal reaction time is 10 to 15 h, and the hydrothermal reaction temperature is 120 to 150 °C.

[0011] One embodiment of the present invention is that in S1, the molar ratio of copper salt, bismuth salt and reducing agent is 3.6 to 4.9:0.4 to 0.5:20 to 25. When the molar ratio of the three is within this range, the prepared nanozyme has better effects.

[0012] Another object of the present invention is to disclose a CuBi composite N, P-doped MXene nanozyme prepared by any of the above methods.

[0013] Another object of the present invention is to disclose a detection reagent, and its preparation method includes the following steps: take the above-mentioned CuBi composite N, P-doped MXene nanozyme and disperse it in water to obtain a dispersion liquid, then add a protein biomarker of a certain virus to the dispersion liquid and incubate at 37 °C for 2 h to 2.5 h; then add a standard protein solution and incubate for 1.5 h to 2.5 h; finally, centrifuge the above mixed solution at 3 °C to 5 °C, take the solid phase and wash and dry it to obtain the detection reagent for this virus.

[0014] Another object of the present invention is to disclose a method for detecting African swine fever for non-diagnostic purposes, including the following steps:

[0015] S1. Take the coating solution of African swine fever capture antibody, fix it on the stationary phase, and then use the immobilized African swine fever capture antibody to adsorb the antigen in the test solution to fix the antigen on the stationary phase;

[0016] S2. Add the detection reagent of claim 5 into the stationary phase of S1 and incubate it to allow the African swine fever antigen to adsorb to the African swine fever antibody, thereby immobilizing the detection reagent on the stationary phase; wherein, the detection reagent is prepared from the protein biomarker of African swine fever.

[0017] S3. Add a buffer solution, hydrogen peroxide, and ABTS into the stationary phase of S2 and soak it, then detect its absorbance, and determine the concentration of African swine fever antigen in the test solution according to the magnitude of the absorbance.

[0018] The above detection method is a conventional method in the art, except that the detection reagent of the present invention is used.

[0019] One embodiment of the present invention is that in S4, the pH of the buffer solution is 3.5 - 4.5.

[0020] One embodiment of the present invention is that the chromogenic substrate is ABTS (2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)), and the oxidized state of this chromogenic substrate has an absorption characteristic peak at 652 nm.

[0021] Beneficial effects: The nanozyme of the present invention has a nanosheet structure, and its synthesis steps are convenient and fast. The raw materials used are all conventional commercial products in the art, which are cheap, simple, and easy to obtain; at the same time, this nanozyme has high activity and high sensitivity; the detection reagent of the present invention has high sensitivity and accuracy; based on the detection reagent of the present invention, when using colorimetric immunoassay to detect African swine fever, the response range of the antigen is 5 pg / mL - 2000 pg / mL, and the detection limit is 0.0452 pg / mL. Description of the Drawings

[0022] Figure 1 It is the X-ray photoelectron spectroscopy diagram of the product of Example 1;

[0023] Figure 2 It is the transmission electron microscope diagram of the product of Example 1;

[0024] Figure 3 It is the X-ray diffraction detection diagram of the product of Example 1;

[0025] Figure 4 It is the activity test diagram of the nanozymes prepared in different examples;

[0026] Figure 5 It is the absorbance test diagram under different African swine fever antigen concentration conditions;

[0027] Figure 6 It is the linear correlation fitting diagram of absorbance and African swine fever antigen concentration. Detailed Embodiments

[0028] Next, the specific embodiments of the present invention will be clearly and completely described in conjunction with examples and drawings. Obviously, the described examples are only a part of the embodiments of the present invention, rather than all of the embodiments.

[0029] Example 1: Preparation of CuBi composite N, P-doped MXene nanozyme:

[0030] S1. Take 5 mL of water, add copper nitrate and bismuth nitrate in a molar ratio of 9:1, and make the total concentration of copper nitrate and bismuth nitrate 0.1 mol / L. Take 22 mL of water and add KBH 4 to prepare a reducing agent solution with a concentration of 0.1 mol / L. After mixing the two evenly, let it stand and react at room temperature for 4.5 h. After the reaction is completed, centrifuge to obtain the solid phase, wash it several times, and then freeze-dry to obtain CuBi metal aerogel;

[0031] S2. Take 37.5 mg of Ti 3 C 2 MXene powder and disperse it in water. Then add 10 g of diammonium hydrogen phosphate and stir and react at room temperature for 2.5 h. After the reaction is completed, continue to carry out hydrothermal reaction at 140 °C for 12 h. After the reaction is completed, centrifuge to obtain the solid phase and freeze-dry it to obtain.

[0032] S3. Uniformly disperse the products of S1 and S3 in water, then stir and react at room temperature for 24 h. After the reaction is completed, centrifuge to obtain the solid phase and freeze-dry it to obtain M1.

[0033] Example 2: Preparation of CuBi composite N, P-doped MXene nanozyme:

[0034] S1. Take 5 mL of water, add copper nitrate and bismuth nitrate in a molar ratio of 9:2, and make the total concentration of copper nitrate and bismuth nitrate 0.1 mol / L. Take 24 mL of water and add KBH 4 to prepare a reducing agent solution with a concentration of 0.1 mol / L. After mixing the two evenly, let it stand and react at room temperature for 4.5 h. After the reaction is completed, centrifuge to obtain the solid phase, wash it several times, and then freeze-dry to obtain CuBi metal aerogel;

[0035] S2. Take 37.5 mg of Ti 3 C 2 MXene powder and disperse it in water. Then add 10 g of diammonium hydrogen phosphate and stir and react at room temperature for 2.5 h. After the reaction is completed, continue to carry out hydrothermal reaction at 140 °C for 12 h. After the reaction is completed, centrifuge to obtain the solid phase and freeze-dry it to obtain.

[0036] S3. Uniformly disperse the products of S1 and S3 in water, then stir and react at room temperature for 24 h. After the reaction is completed, centrifuge to obtain the solid phase, and freeze-dry to obtain M2.

[0037] Example 3. Preparation of CuBi composite N, P-doped MXene nanozyme:

[0038] S1. Take 5 mL of water, add copper nitrate and bismuth nitrate in a molar ratio of 9:0.4, and make the total concentration of copper nitrate and bismuth nitrate 0.1 mol / L. Take 23 mL of water and add KBH 4 to prepare a reducing agent solution with a concentration of 0.1 mol / L. After mixing the two evenly, let them stand and react at room temperature for 4.5 h. After the reaction is completed, centrifuge to obtain the solid phase, wash it several times, and then freeze-dry to obtain CuBi metal aerogel;

[0039] S2. Take 37.5 mg of Ti 3 C 2 MXene powder and disperse it in water. Then add 10 g of diammonium hydrogen phosphate and stir and react at room temperature for 2.5 h. After the reaction is completed, continue the hydrothermal reaction at 140 °C for 12 h. After the reaction is completed, centrifuge to obtain the solid phase and freeze-dry to obtain it.

[0040] S3. Uniformly disperse the products of S1 and S3 in water, then stir and react at room temperature for 24 h. After the reaction is completed, centrifuge to obtain the solid phase, and freeze-dry to obtain M3.

[0041] Example 4. Preparation of CuBi composite N, P-doped MXene nanozyme:

[0042] S1. Take 5 mL of water, add copper nitrate and bismuth nitrate in a molar ratio of 9:0.1, and make the total concentration of copper nitrate and bismuth nitrate 0.1 mol / L. Take 21 mL of water and add KBH 4 to prepare a reducing agent solution with a concentration of 0.1 mol / L. After mixing the two evenly, let them stand and react at room temperature for 4.5 h. After the reaction is completed, centrifuge to obtain the solid phase, wash it several times, and then freeze-dry to obtain CuBi metal aerogel;

[0043] S2. Take 37.5 mg of Ti 3 C 2 MXene powder and disperse it in water. Then add 10 g of diammonium hydrogen phosphate and stir and react at room temperature for 2.5 h. After the reaction is completed, continue the hydrothermal reaction at 140 °C for 12 h. After the reaction is completed, centrifuge to obtain the solid phase and freeze-dry to obtain it.

[0044] S3. Uniformly disperse the products of S1 and S3 in water, then stir and react at room temperature for 24 h. After the reaction is completed, centrifuge to obtain the solid phase, and freeze-dry to obtain M4.

[0045] Comparative Example 1, Preparation of Cu composite N, P-doped MXene nanozyme:

[0046] S1. Take 5 mL of water, add copper nitrate to prepare a solution with a concentration of 0.1 mol / L, take 23 mL of water and add KBH 4 to prepare a reducing agent solution with a concentration of 0.1 mol / L. After mixing the two evenly, let it stand and react at room temperature for 4.5 h. After the reaction is completed, centrifuge to obtain the solid phase, wash it several times, and then freeze-dry to obtain CuBi metal aerogel;

[0047] S2. Take 37.5 mg of Ti 3 C 2 MXene powder and disperse it in water. Then add 10 g of diammonium hydrogen phosphate and stir and react at room temperature for 2.5 h. After the reaction is completed, continue to carry out hydrothermal reaction at 140 °C for 12 h. After the reaction is completed, centrifuge to obtain the solid phase and then freeze-dry to obtain it.

[0048] S3. Uniformly disperse the products of S1 and S3 in water, then stir and react at room temperature for 24 h. After the reaction is completed, centrifuge to obtain the solid phase and freeze-dry to obtain D1.

[0049] Example 5, Preparation of African swine fever detection reagent:

[0050] Take CuBi composite N, P-doped MXene nanozyme, add water to make it uniformly dispersed to form a dispersion. Then add 2 mL of the protein biomarker of African swine fever (200 μg / mL) to the dispersion and incubate at 4 °C for 2.5 h. Then add 1 mL of standard protein solution (0.1 mg / mL) and incubate for 2 h. Then, centrifuge and separate the above mixed solution at 4 °C, wash the separated solid three times with deionized water, and then freeze-dry to obtain it.

[0051] Example 6, Detection of African swine fever antigen concentration:

[0052] S1. Take the coating solution (100 μL) containing 1 μg / mL of African swine fever capture antibody in a 96-well plate (stationary phase) and incubate overnight at 4 °C. At this time, the African swine fever capture antibody is fixed on the 96-well plate; after washing 3 times with phosphate buffer solution (pH = 7), wash away the excess African swine fever capture antibody that is not fixed on the 96-well plate; then incubate with the standard protein solution (200 μL, 0.1 mg / mL) for 30 min. Then, wash with phosphate buffered saline two to three times to remove the excess standard protein solution; add the test solution to the 96-well plate and incubate for 30 min. The African swine fever antigen in the test solution binds to the African swine fever capture antibody on the 96-well plate and is thus fixed by the 96-well plate. Then wash several times with phosphate buffered saline.

[0053] S2. Add 100 μL (1 mg / mL) of the detection reagent prepared in Example 4 into a 96-well plate, incubate at 37 °C for 30 min, and then wash the 96-well plate several times with water;

[0054] S3. Add 100 μL of acetic acid-sodium acetate solution (pH = 4), 50 μL of 18 mM H 2 O 2 , and 50 μL of 0.0014 mg / mL ABTS (2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)) solution into the 96-well plate. After maintaining at room temperature for 3 min, take the solution in the 96-well plate and perform absorbance detection, and determine the African swine fever antigen concentration in the test solution according to the absorbance.

[0055] To further illustrate the effects of the products prepared by the present invention, specific test examples are used to illustrate it below.

[0056] 1. Characterization

[0057] Take the Cu 9 Bi 1 composite N, P-doped MXene nanozyme M1 prepared in Example 1, and perform X-ray photoelectron spectroscopy, transmission electron microscopy and X-ray diffraction detection on it. The final results are as Figures 1 to 3 shown.

[0058] Figure 1 is the X-ray photoelectron spectroscopy diagram. As can be seen from Figure 1 , the nanozyme contains elements such as N and P, indicating successful doping.

[0059] Figure 2 is the transmission electron microscopy diagram. As can be seen from Figure 2 , M1 has a flaky structure.

[0060] Figure 3 is the X-ray diffraction detection diagram. Among them, we tested M1, MXene and modified MXene. As can be seen from Figure 3 , the (002) peak is significantly broadened, which is due to the formation of more vacancies and defects after heteroatom doping into MXene. At the same time, the change in the MXene layer spacing (d-spacing) can be judged by the displacement of the (002) peak. It is speculated that the larger layer spacing in Ti3C2-N,P is due to the structural distortion introduced by doping nitrogen and phosphorus atoms in the matrix, while the Cu 9 Bi 1 composite N, P-doped MXene also shows a displacement, which is presumably also due to the doping of Cu 9 Bi 1 on the basis of Ti3C2-N,P.

[0061] 2. Activity test:

[0062] Take the CuBi composite N, P-doped MXene nanozyme prepared in Examples 1-4 and Comparative Example 1, prepare the African swine fever detection reagent according to the method of Example 5, and detect its absorbance value at 652 nm according to the following method: sequentially add CuBi composite N, P-doped MXene nanozyme (20 ul, 2 mg / ml), acetic acid-sodium acetate buffer solution (20 ul, pH = 4), H 2 O 2 (50 ul, 18 mM) and ABTS (50 ul, 0.0014 mg / ml), then react at room temperature for 3 min. After the reaction, take the reaction solution and observe its absorbance value at 652 nm in an enzyme-labeling instrument; at the same time, take MXene to replace the CuBi composite N, P-doped MXene nanozyme as a blank. The final results are as Figure 4 shown.

[0063] It can be seen from Figure 4 that, compared with the Cu composite N, P-doped MXene nanozyme and MXene, the CuBi composite N, P-doped MXene nanozyme prepared in the examples of the present invention has higher activity.

[0064] 3. Detection limit test

[0065] Take the CuBi composite N, P-doped MXene nanozyme prepared in Example 1, prepare the African swine fever detection reagent according to the method of Example 5, and perform ultraviolet absorption spectrum detection on the test solutions with different African swine fever antigen concentrations (5 pg / mL - 2000 pg / mL) according to the method of Example 6. The final results are as Figure 5 and 6 shown.

[0066] It can be seen from Figure 5 that as the concentration of African swine fever antigen increases, its ultraviolet absorption spectrum gradually rises, and the two are positively correlated.

[0067] Based on Figure 5 the detection results, taking the logarithm of the African swine fever antigen concentration as the abscissa and the absorbance value as the ordinate to plot Figure 6 , it can be seen from Figure 6 that the two show a linear relationship and have a good correlation, and its R 2 reaches 0.99714; at the same time, according to Figure 6 it can be calculated that its detection limit is 0.0452 pg / mL.

[0068] Meanwhile, the African swine fever detection reagent prepared in the embodiment of the present invention was compared with the African swine fever detection reagent of the prior art, and Table 1 was obtained. It can be seen from Table 1 that the detection limit of the African swine fever detection reagent in the embodiment of the present invention is much lower than that of the existing detection reagent.

[0069] Table 1 Comparison table of detection limits of different African swine fever detection reagents

[0070]

[0071] 4. Clinical detection of African swine fever antigen

[0072] Take a pig serum sample, dilute it, and detect the African swine fever antigen concentration according to the method of Example 6. Among them, the African swine fever detection reagent used is prepared from the CuBi composite N, P-doped MXene nanozyme of Example 1, and the detection results are compared with the results of the existing detection method (enzyme-linked immunosorbent assay) in the hospital as the actual results. The final results are shown in Table 2.

[0073] Table 2 Pig serum detection result table

[0074]

[0075]

[0076] It can be seen from Table 2 that the detection reagent and detection method in the embodiment of the present invention have a high recovery rate and can meet the actual use requirements.

[0077] The above is only a preferred embodiment of the present invention, and it does not impose any form of limitation on the present invention. Although the present invention has been disclosed above with a preferred embodiment, it is not intended to limit the present invention. Any person skilled in the art can make some changes or modifications to the above-disclosed technical content to be equivalent embodiments without departing from the technical solution of the present invention. However, any simple modification, equivalent change and modification made to the above embodiments based on the technical essence of the present invention still fall within the scope of the technical solution of the present invention.

Claims

1. A method for preparing CuBi composite N, P doped MXene nanozyme, characterized in that: The following steps are involved: S1, taking a water-soluble copper salt and a water-soluble bismuth salt and dissolving them, then adding a reducing agent and reacting, after the reaction is completed, taking a solid phase and freeze-drying to obtain a CuBi metal aerogel; the reducing agent is one of KBH4 and NaBH4, and the molar ratio of the copper salt, the bismuth salt and the reducing agent is 3.6-5.0:0.04-1:20-25; S2, dispersing Ti3C2 MXene powder in water, then adding ammonium dihydrogen phosphate and dissolving it, reacting under continuous stirring and room temperature conditions, and after the reaction is completed, continuing the hydrothermal reaction, and after the reaction is completed, taking the solid phase and freeze-drying it to obtain the modified MXene powder; the amount of the Ti3C2 MXene powder added is 30 to 50 parts by mass, and the amount of the ammonium dihydrogen phosphate added is 10,000 parts by mass; S3, take the CuBi metal aerogel of S1 and the modified MXene powder of S2 and disperse them in water, then stir and react for 20 to 30 hours. After the reaction is completed, take the solid phase and freeze-dry it; wherein, every 2 to 2.5×10 -3 mol of reducing agent, corresponding to 30-50 mg of Ti3C2 MXene powder in S2.

2. The method according to claim 1, characterized in that The copper salt is copper nitrate, and the bismuth salt is bismuth nitrate.

3. The method according to claim 1, characterized in that In S1, the reaction time is 4 to 5 hours; in S2, the reaction time at room temperature is 2 to 2.5 hours, the hydrothermal reaction time is 10 to 15 hours, and the hydrothermal reaction temperature is 120 to 150°C.

4. The method according to claim 1, characterized in that: In S1, the molar ratio of copper salt, bismuth salt and reducing agent is 3.6-4.9:0.4-0.5:20-25.

5. A CuBi composite N, P-doped MXene nanozyme prepared by the method described in any one of claims 1 to 4.

6. A detection reagent, characterized in that: The preparation method comprises the following steps: taking the CuBi composite N, P doped MXene nanozyme as described in claim 5 and dispersing it in water to obtain a dispersion, then adding a protein biomarker of a virus to the dispersion and incubating it at 37°C for 2h to 2.5h; then adding a standard protein solution and incubating it for 1.5h to 2.5h; finally, centrifuging the mixed solution at 3°C ​​to 5°C, taking the solid phase, washing it, and drying it to obtain a detection reagent for the virus.

7. A method for detecting African swine fever for non-diagnostic purposes, characterized in that: The following steps are involved: S1. Take the coating solution of African swine fever capture antibody and fix it on the stationary phase, and then use the fixed African swine fever capture antibody to adsorb the antigen in the test solution to fix the antigen on the stationary phase; S2. Adding the detection reagent of claim 6 into the stationary phase of S1 and incubating, so that the African swine fever antigen and the African swine fever antibody are adsorbed, and then the detection reagent is fixed on the stationary phase; wherein the detection reagent is prepared with a protein biomarker of African swine fever; S3. Add buffer solution, hydrogen peroxide and chromogenic substrate to the stationary phase of S2 and soak it, then detect its absorbance and determine the concentration of African swine fever antigen in the test solution based on the absorbance.

8. The method according to claim 7, characterized in that In S3, the pH of the buffer solution is 3.5-4.

5.

9. The method according to claim 7, characterized in that: The chromogenic substrate is ABTS.

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