CuCoNi-MOF aerogel nano-enzyme and preparation method and detection reagent thereof

Abstract

The invention provides a CuCoNi-MOF aerogel nano-enzyme, a preparation method thereof and a detection reagent, and belongs to the technical field of antibody detection.The preparation method of the aerogel nano-enzyme comprises the following steps that bivalent copper salt, bivalent cobalt salt and bivalent nickel salt are mixed, then a reducing agent is added to reduce bivalent metal ions, then a dimethylimidazole solution is added, and the CuCoNi-MOF aerogel nano-enzyme is obtained. And reacting at 25-35 DEG C for at least 1 hour, finally taking out the precipitate after the reaction, washing with deionized water, and freeze-drying to obtain the target product. The detection reagent provided by the invention is prepared from the aerogel nano-enzyme as a raw material. The aerogel nano enzyme has high activity and strong stability, and can form a detection reagent with stable properties with a protein biomarker, and meanwhile, the preparation method is simple and convenient, and does not need expensive instruments, so that the manufacturing cost can be reduced. The detection reagent provided by the invention has very high sensitivity and accuracy.

Description

Technical Field

[0001] The present invention belongs to the technical field of antibody detection, and specifically relates to a CuCoNi-MOF aerogel nanozyme, a preparation method thereof, and a detection reagent. Background Art

[0002] African swine fever (ASF) is a highly contagious disease of pigs caused by African swine fever virus (ASFV). ASF is characterized by high morbidity and high mortality, which can have a devastating impact on the pig farming industry. ASFV is a large double-stranded DNA virus belonging to the family Asfarviridae. Currently, there is no effective vaccine or treatment method. Therefore, prevention and control mainly rely on biosecurity measures, detection, and culling of infected pig herds. Enzyme-linked immunosorbent assay (ELISA) is a commonly used immunoassay method, which is not only highly sensitive but also simple to operate. However, due to the high cost and poor stability of natural enzymes, it is particularly important to find their substitutes for immunoassay. Nanozymes have properties similar to natural enzymes and are considered good substitutes for natural enzymes due to their low cost, good stability, and high accuracy.

[0003] Currently, it has been confirmed that a variety of metal-organic framework materials (MOF) have peroxidase-like properties, such as Ni-MOF, Co-MOF, and Fe-MOF. At the same time, researchers have also found that some metal-organic framework materials doped with different transition metals have better activity, such as FeCoNi-MOF, FeNi-MOF, etc. However, Fe also lacks in practical applications due to its poor stability. In addition, it has been found that aerogels often have a larger specific surface area, and the peroxidase-like activity of Cu-MOF aerogel is higher than that of Cu-MOF and Cu aerogel. Therefore, it is necessary to combine metal doping technology and aerogel technology to find peroxidase-like materials with more excellent performance. Summary of the Invention

[0004] To solve the above problems, the purpose of the present invention is to provide a CuCoNi-MOF aerogel nanozyme, a preparation method thereof, and a detection reagent.

[0005] To achieve the above objectives, the technical solution of the present invention is as follows:

[0006] A preparation method of a CuCoNi-MOF aerogel nanozyme, comprising the following steps:

[0007] S1. Mix soluble divalent copper salt, divalent cobalt salt, and divalent nickel salt evenly in deionized water according to a molar ratio of 0.75 - 0.85:0.05 - 0.15:0.05 - 0.15;

[0008] S2. Add a reducing agent to reduce the divalent metal ions to obtain an intermediate solution; the molar ratio of the reducing agent to the divalent metal ions is 2.5 to 3.5;

[0009] S3. Add the dimethylimidazole solution to the intermediate solution under stirring conditions, react at 25°C to 35°C for at least 1 hour, then take out the reacted precipitate, wash it with deionized water, and freeze-dry to obtain the target product; the molar ratio of the dimethylimidazole to the divalent metal ions is 0.3 to 0.4.

[0010] As a specific embodiment of the present invention, the divalent copper salt can be CuCl 2 , Cu(NO 3 ) 2 etc., the divalent cobalt salt can be CoCl 2 , Co(NO 3 ) 2 etc., the divalent nickel salt can be NiSO 4 , NiCl 2 etc. In order to disperse it sufficiently, the solution can be stirred or ultrasonically treated.

[0011] As a specific embodiment of the present invention, the reducing agent can be KBH 4 or NaBH 4 .

[0012] A CuCoNi-MOF aerogel nanozyme is prepared by the above preparation method.

[0013] A detection reagent is prepared by a method comprising the following steps:

[0014] Dissolve the above-mentioned CuCoNi-MOF aerogel nanozyme in water to form a solution, then add the protein biomarker of a certain virus to the solution of the CuCoNi-MOF aerogel nanozyme and incubate; then add a standard protein solution and incubate; finally, centrifuge the above-mentioned mixed solution at 3°C to 5°C, and wash and dry the precipitate with deionized water to obtain the nanomaterial, which is the detection reagent for the virus.

[0015] As a specific embodiment of the present invention, the certain virus is African swine fever virus.

[0016] Beneficial effects:

[0017] (1) The preparation method of the CuCoNi-MOF aerogel nanozyme of the present invention is simple and convenient, and does not require expensive instruments, thus reducing the manufacturing cost.

[0018] (2) The CuCoNi-MOF aerogel nanozyme of the present invention has high enzyme activity and strong stability, and can form a detection reagent with stable properties with protein biomarkers. At the same time, it is doped with copper, and relatively inexpensive copper is used to replace other more expensive transition metals, thereby further reducing the cost.

[0019] (3) The detection reagent of the present invention has high sensitivity and accuracy. The response range of the colorimetric immunoassay for African swine fever antigen is 2.5 ng / mL to 1000 pg / mL, and the detection limit is 0.1884 ng / mL. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 Graph of the peroxidase-like activity test results of the aerogel nanozymes of each example and comparative example;

[0021] Figure 2 Graph of the peroxidase-like activity test results of the detection reagent prepared in Example 2 at different pH values;

[0022] Figure 3 Graph of the peroxidase-like activity test results of the detection reagent prepared from Cu-Co-Ni MOF aerogel nanozyme;

[0023] Figure 4 Absorption spectra corresponding to African swine fever antigen with different concentrations of Cu-Co-Ni MOF aerogel nanozyme;

[0024] Figure 5 Linear correlation curve graph of the absorbance at 652 nm and the concentration of African swine fever antigen. DETAILED DESCRIPTION OF THE INVENTION

[0025] The following will clearly and completely describe the specific embodiments of the present invention in conjunction with examples. Obviously, the described examples are only a part of the embodiments of the present invention, rather than all of the embodiments.

[0026] All drugs in the following examples are commercially available products.

[0027] Comparative Example 1 (Preparation of Cu, CoNi-MOF, Fe-Co-Ni-MOF, CrCoNi-MOF, AlCoNi-MOF, ZnCoNi-MOF aerogel nanozymes)

[0028] Put 3 mL of 0.1 mol / L CuCl 2 aqueous solution into a centrifuge tube, then sonicate for 3 min, and then quickly add NaBH 4The solution (9 ml, 0.1 mol / L) was vigorously stirred for 1 min and then allowed to stand for 30 min. The resulting precipitate was centrifuged and washed three times with deionized water (1000 rpm, 8 min), and then freeze-dried for 8 h to obtain Cu aerogel nanozyme.

[0029] 1.5 mL of 0.18 mol / L CoCl 2 aqueous solution and 1.5 mL of 0.02 mol / L NiSO 4 aqueous solution were placed in a centrifuge tube, then sonicated for 3 min, and then NaBH 4 solution (9 ml, 0.1 mol / L) was vigorously stirred for 1 min and allowed to stand for 30 min. Then, 1,2-dimethylimidazole solution (1 ml, 0.1 mol / L) after sonication for 3 min was added and stirred for 30 min, followed by standing for 20 min. The supernatant was aspirated, and the resulting precipitate was washed with deionized water and centrifuged three times (1000 rpm, 8 min), and then freeze-dried for 8 h to obtain CoNi-MOF aerogel nanozyme.

[0030] 1 mL of 0.24 mol / L Fe 2 (SO 4 ) 3 aqueous solution, 1 mL of 0.03 mol / L CoCl 2 aqueous solution and 1 mL of 0.03 mol / L NiSO 4 aqueous solution were placed in a centrifuge tube, then sonicated for 3 min, and then NaBH 4 solution (9 ml, 0.1 mol / L) was vigorously stirred for 1 min and allowed to stand for 30 min. Then, 1,2-dimethylimidazole solution (1 ml, 0.1 mol / L) after sonication for 3 min was added and stirred for 30 min, followed by standing for 20 min. The supernatant was aspirated, and the resulting precipitate was washed with deionized water and centrifuged three times (1000 rpm, 8 min), and then freeze-dried for 8 h to obtain FeCoNi-MOF aerogel nanozyme.

[0031] Similarly, by replacing Fe 2 (SO 4 ) 3 with ZnSO 4 , AlCl 3 , CrCl 3 , with the amounts of other substances remaining unchanged, following the above steps, CrCoNi-MOF aerogel, AlCoNi-MOF aerogel, and ZnCoNi-MOF aerogel nanozymes can be obtained.

[0032] Example 1 (Preparation of CuCoNi-MOF Aerogel Nanozyme)

[0033] 1 mL of 0.24 mol / L CuCl 2 aqueous solution, 1 mL of 0.03 mol / L CoCl 2 aqueous solution and 1 mL of 0.03 mol / L NiSO 4 aqueous solution were placed in a centrifuge tube, then ultrasonicated for 3 min, and then rapidly added with NaBH 4 solution (9 mL, 0.1 mol / L) and stirred vigorously for 1 min, then left standing for 30 min; then 1,2-dimethylimidazole solution (1 mL, 0.1 mol / L) after ultrasonicating for 3 min was added and stirred for 30 min, and then left standing for 20 min. The supernatant was aspirated, and the obtained precipitate was washed with deionized water and centrifuged three times (1000 rpm, 8 min), and then freeze-dried for 8 h to obtain CuCoNi-MOF aerogel nanozyme.

[0034] Example 2 (Preparation of African swine fever detection reagent)

[0035] 20 mg of the CuCoNi-MOF aerogel nanozyme prepared in Example 1 was dissolved in 10 mL of deionized water, and then 2 mL of the protein biomarker of African swine fever (200 μg / mL) was added, incubated at 4 °C for 2.5 h, and then 1 mL of standard protein solution (0.1 mg / mL) was added and incubated for 2 h. Then, the above mixed solution was centrifuged at 4 °C, and the separated solid was washed three times with deionized water and freeze-dried at -40 °C to obtain the African swine fever detection reagent.

[0036] Example 3 (Using the African swine fever detection reagent to detect the concentration of African swine fever antigen)

[0037] Take the coating solution (100 μL) containing 1 μg / mL African swine fever capture antibody in a 96-well plate and incubate overnight at 4 °C. At this time, the African swine fever capture antibody is fixed on the 96-well plate (solid phase). After washing 3 times with phosphate buffer solution (pH = 7), the excess African swine fever capture antibody not fixed on the solid phase is washed away, and then incubated with the standard protein solution (200 μL, 0.1 mg / mL) for 30 min. Then, wash 2 to 3 times with phosphate buffered saline to remove the excess standard protein solution.

[0038] Add African swine fever antigen to the 96-well plate and incubate for 30 min. The African swine fever antigen in the solution to be tested binds to the African swine fever capture antibody on the solid phase and is thus fixed on the solid phase, and then washed.

[0039] Add 100 μL (1 mg / mL) of the detection reagent prepared in Example 2, incubate at 37 °C for 30 min, and allow the African swine fever antigen in the solution to be tested to adsorb to the African swine fever antibody in the detection reagent, so that the detection reagent is solid-phase fixed through the antigen. Then, wash three times with deionized water to remove the unbound African swine fever detection reagent.

[0040] Finally, add 100 μL of acetic acid-sodium acetate solution (pH = 4), 50 μL of 500 mM H 2 O 2 , 50 μL of 100 mM / L 3,3',5,5'-tetramethylbenzidine (TMB) solution. After 3 min, measure the absorbance of the solution to be tested, and determine the concentration of African swine fever antigen based on the absorbance of the solution to be tested.

[0041] Test Example 1 (Peroxidase-like Activity Test)

[0042] In this test example, colorless 3,3',5,5'-tetramethylbenzidine (TMB) was used as the chromogenic substrate, and its oxidized state has a characteristic absorption at 652 nm.

[0043] In a 96-well microplate, add 20 μL (1 mg / mL) of the CuCoNi-MOF aerogel nanozyme of Example 1, the Cu aerogel nanozyme of Comparative Example 1, CoNi-MOF aerogel nanozyme, FeCoNi-MOF aerogel nanozyme, CrCoNi-MOF aerogel nanozyme, AlCoNi-MOF aerogel nanozyme, and ZnCoNi-MOF aerogel nanozyme to 7 wells respectively. Each comparative example is 20 μL, 1 mg / mL. Number the 7 wells from 1 to 7 in sequence. Then, simultaneously add acetic acid-sodium acetate buffer (180 μL, pH = 4) to these 7 wells, and then simultaneously add H 2 O 2 (50 μL, 500 mM) to them. Subsequently, simultaneously add TMB (50 μL, 100 mM / L) to the wells and react for 3 min. Place it in an enzyme-linked immunosorbent assay (ELISA) reader and observe the absorbance value at 652 nm. The test results are as Figure 1 shown.

[0044] During the experiment, it was found that the FeCoNi-MOF aerogel nanozyme showed an obvious color change under static conditions, which might be caused by its rapid oxidation. To determine the stability of each aerogel nanozyme, after each aerogel nanozyme was prepared, it was placed for different times (5 min, 30 min, 1 d), and then its absorbance value was measured. The test results are shown in Table 1.

[0045] Table 1 Absorbance values of each aerogel nanozyme at different times

[0046]

[0047] As can be seen from Table 1, the absorbance of the FeCoNi-MOF aerogel nanozyme changes rapidly, and its performance is unstable, so it is not suitable as a test strip. According to the absorbance change rate at 1d to determine the stability of each aerogel nanozyme (the larger the change rate, the worse the stability), the stability ranking is as follows: Cu-Co-NiMOF aerogel nanozyme > CoNi-MOF aerogel nanozyme > ZnCoNi-MOF aerogel > Cu aerogel nanozyme > CrCoNi-MOF aerogel nanozyme > AlCoNi-MOF aerogel nanozyme > FeCoNi-MOF aerogel nanozyme. The Cu-Co-NiMOF aerogel nanozyme has the smallest change rate, only 1.9%, and it is an ideal test strip material.

[0048] Test Example 2 (Single-factor experiment for optimizing the reaction pH value)

[0049] Select 6 wells in a 96-well microplate and add the detection reagent prepared in Example 2 (100 μL, 1 mg / mL). Then, sequentially add 100 μL of acetic acid-sodium acetate buffer solution with different pH values, and then sequentially add H 2 O 2 (50 μL, 500 mM). Subsequently, sequentially add TMB (50 μL, 100 mM / L) and react for 3 min. Place it in an enzyme-labeling instrument and observe the absorbance value at 652 nm. The test results are as Figure 2 shown.

[0050] As Figure 2 can be seen, its peroxidase-like activity is the highest at pH 4.

[0051] Test Example 4 (Single-factor experiment for optimizing the nanozyme concentration)

[0052] Dissolve different masses of the CuCoNi-MOF aerogel nanozyme prepared in Example 1 in 10 mL of deionized water respectively. Then, add 2 mL of the protein biomarker of African swine fever (200 μg / mL) respectively, incubate at 37 °C for 2.5 h, and then add 1 mL of the standard protein solution (0.1 mg / mL) respectively and incubate for 2 h. Then, centrifuge and separate the respective mixed solutions at 4 °C, wash three times with deionized water, and freeze-dry at -40 °C to obtain the African swine fever detection reagents with different nanozyme concentrations for peroxidase-like activity testing. The test results are as Figure 3 shown.

[0053] As Figure 3 can be seen, by detecting the absorbance at 652 nm with an enzyme-labeling instrument, it can be known that the absorbance of the detection reagent itself increases almost linearly with the increase of the nanozyme concentration and tends to be stable at about 2 mg / mL.

[0054] Test Example 5 (Actual Detection Limit Test)

[0055] To investigate the analytical performance of the designed enzyme-linked immunosorbent assay, ultraviolet absorption spectra of different African swine fever antigen concentrations (concentration range: 2.5 ng / mL - 1000 ng / mL) were collected under optimized conditions (i.e., the CuCoNi-MOF aerogel nanozyme added during the preparation of the detection reagent was 2 mg / ml and the pH during the peroxidase-like activity test was 4). As Figure 4 shown, as the concentration of African swine fever antigen increased, the ultraviolet absorption spectrum gradually increased, showing positive feedback. As Figure 5 shown, within the range of 2.5 ng / mL - 1000 ng / mL, the maximum value of the ultraviolet absorption spectrum (652 nm) showed a good linear relationship, and the corresponding linear calibration curve was Y = 0.52399X + 0.03186 (R2 = 0.9783), and the detection limit (LOD) was 0.1884 ng / mL (calculated by 3σ / S, σ = 0.002).

[0056] In addition, the detection of African swine fever antigen in porcine serum samples was similar to the above procedure, except that diluted porcine serum samples were used instead of pure African swine fever antigen. For the detection of actual samples, after diluting the serum samples to an appropriate concentration, they were detected by the above method.

[0057] To verify the accuracy of the clinical African swine fever antigen detection method proposed in this study, we measured the African swine fever antigen levels in four serum samples and compared the results with those obtained from the hospital on this basis. First, the serum was diluted to the linear range according to the above method, and then directly detected with the African swine fever detection reagent. As shown in Table 2, the recovery rate was 90% - 105%, indicating that the method we proposed is applicable to the clinical analysis of African swine fever antigen.

[0058] Table 2 Detection Results of African Swine Fever Antigen

[0059] Sample Detected Concentration (ng / ml) Actual Concentration (ng / ml) Recovery Rate (%) 1 10.6 10.1 104 2 21.2 22 96 3 159 176 90 4 530 501 105

[0060] Similarly, according to the method for determining the CuCoNi-MOF aerogel nanozyme, the detection limits of the aerogel nanozymes in each comparative example were determined, and the results are shown in Table 3.

[0061] Table 3 Comparison Table of Detection Limits for Detecting African Swine Fever with Different Materials

[0062]

[0063]

[0064] As can be seen from Table 3, the CuCoNiMOF aerogel nanozyme has a relatively excellent detection limit. The present invention constructs a highly sensitive and highly selective sensing platform, and verifies the practicability of the sensing system by detecting African swine fever virus antigen.

[0065] The present invention has been disclosed in the preferred embodiments above. However, those skilled in the art should understand that these embodiments are only used to describe the present invention and should not be construed as limiting the scope of the present invention. Without departing from the principle of the present invention, further improvements can be made, and these improvements should also be included within the protection scope of the present invention.

Claims

1. A method for preparing CuCoNi-MOF aerogel nanozyme, characterized in that: The steps include: S1. Mix soluble divalent copper salt, divalent cobalt salt and divalent nickel salt in deionized water at a molar ratio of 0.75-0.85:0.05-0.15:0.05-0.15; S2, adding a reducing agent to reduce the divalent metal ions to obtain an intermediate solution; the molar ratio of the reducing agent to the divalent metal ions is 2.5 to 3.5; S3. Add the dimethylimidazole solution to the intermediate solution under stirring conditions, react at 25°C to 35°C for at least 1 hour, take out the precipitate after the reaction, wash it with deionized water, and freeze-dry it to obtain an aerogel nanozyme; the molar ratio of the dimethylimidazole to the divalent metal ion is 0.3 to 0.

4.

2. The method for preparing a CuCoNi-MOF aerogel nanozyme according to claim 1, characterized in that: The reducing agent is KBH4 or NaBH4.

3. The method for preparing a CuCoNi-MOF aerogel nanozyme according to claim 1, characterized in that: The divalent copper salt is CuCl2 or Cu(NO3)2, the divalent cobalt salt is CoCl2 or Co(NO3)2, and the divalent nickel salt is NiSO4 or NiCl2.

4. A CuCoNi-MOF aerogel nanozyme, characterized in that: The aerogel nanozyme is prepared by the preparation method of the CuCoNi-MOF aerogel nanozyme according to any one of claims 1 to 3.

5. A detection reagent, characterized in that: The method is prepared by the following steps: B1. Prepare the CuCoNi-MOF aerogel nanozyme according to claim 4 into an aqueous solution; B2. Add a protein biomarker of a certain virus to the aqueous solution prepared in step B1 and incubate; then add a standard protein solution and incubate; B3. Centrifuge, wash and dry the solution after incubation in step B2 at 3°C ​​to 5°C to obtain a virus detection reagent.

6. A detection reagent according to claim 4, characterized in that, The virus is African swine fever virus.

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