MOF nano-enzyme sensor for rapidly detecting African swine fever virus and preparation method of MOF nano-enzyme sensor

Abstract

The invention discloses an MOF nano-enzyme sensor for rapidly detecting African swine fever virus and a preparation method of the MOF nano-enzyme sensor, and belongs to the technical field of nano-enzyme sensors. The preparation method comprises the following steps: mixing and stirring CuS, Cu-MOF and water to obtain CuS-coated Cu-MOF; the preparation method comprises the following steps: preparing CuS (at) Cu-MOF, mixing CuS (at) Cu-MOF, 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride, N-hydroxysuccinimide and water, carrying out first incubation, adding anti-ASFVp54, and carrying out second incubation to obtain the MOF nano-enzyme sensor. According to the invention, the MOF nano-enzyme sensor is prepared and has excellent peroxidase simulation activity; after the p54 antibody is coated, ASFV is specifically combined through biomolecular recognition, so that a wide concentration range and a low detection limit can be realized.

Description

Technical Field

[0001] The present invention belongs to the technical field of nanoenzyme sensors, and specifically relates to a MOF nanoenzyme sensor for rapidly detecting African swine fever virus and a preparation method thereof. Background Art

[0002] African swine fever (ASF) is an acute and highly lethal infectious disease of swine caused by the African swine fever virus (ASFV). While not contagious to humans, ASFV can be extremely lethal to domestic pigs and various wild boar species. Due to the lack of an effective ASFV vaccine and drug treatment, the epidemic has caused significant economic losses to the country and the livestock breeding industry, making the situation extremely serious. ASFV is a large double-stranded DNA virus with a genome of approximately 170-190 kbp encoding approximately 150-200 proteins. The p54 protein, a product of the ASFV gene E183L located within the viral envelope, is a key structural protein of ASFV and plays a crucial role in its rapid invasion and attachment to susceptible cells. Therefore, detecting the p54 protein and, by extension, the true virus is of great significance.

[0003] Currently, the most commonly used method for detecting p54 is enzyme-linked immunosorbent assay (ELISA), but this method is cumbersome to use and has low specificity and sensitivity. Therefore, it is necessary to establish a rapid and sensitive detection method for ASFV of p54 protein to achieve early detection and isolation of ASFV and reduce losses in the pig farming industry. Summary of the Invention

[0004] In view of this, the object of the present invention is to provide a MOF nanozyme sensor for rapid detection of African swine fever virus and a preparation method thereof.

[0005] In order to achieve the above object, the present invention provides the following technical solutions: The present invention provides a method for preparing a MOF nanozyme sensor for rapid detection of African swine fever virus, comprising the following steps: S1. CuS, Cu-MOF, and water were mixed and stirred to obtain CuS@Cu-MOF; S2. CuS@Cu-MOF, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC), N-hydroxysuccinimide (NHS), and water were mixed and incubated for the first time. Anti-ASFVp54 was then added for the second incubation to obtain the CuS@Cu-MOF-antibody.

[0006] Preferably, the ratio of CuS to Cu-MOF in S1 is 1:3.

[0007] Preferably, the preparation method of CuS is: mixing CuCl2·2H2O, PVP, sodium thiosulfate and water, and then heating to react, cooling, washing and drying to obtain CuS powder.

[0008] Preferably, the mass ratio of CuCl2·2H2O, PVP, sodium thiosulfate and water is (0.1-0.15):(0.2-0.3):(0.15-0.25):(15-18); more preferably, it is 0.1364:0.25:0.2:16.

[0009] Preferably, the heating reaction temperature is (95-100)°C and the time is (10-14) hours; more preferably, the heating reaction temperature is 100°C and the time is 12 hours.

[0010] Preferably, the preparation method of the Cu-MOF is: Polyvinyl pyrrolidone, N, N-dimethylformamide, ethanol and water are mixed and dispersed, and then H2BDC and CuCl2·2H2O are added in sequence and stirred to dissolve. Then triethylamine is added to obtain a homogeneous colloidal suspension and ultrasonic treatment is performed to obtain Cu-MOF.

[0011] Preferably, the volume mass ratio of polyvinylpyrrolidone, N,N-dimethylformamide, ethanol, water, H2BDC, CuCl2·2H2O, and triethylamine is (0.01-0.03) g:32 mL:2 mL:2 mL:(0.1-0.15) g:(0.1-0.15) g:800 μL; more preferably, it is 0.02 g:32 mL:2 mL:2 mL:0.1246 g:0.1193 g:800 μL.

[0012] Preferably, the ultrasonic time is 4-7 hours; more preferably, the ultrasonic time is 6 hours.

[0013] Preferably, the stirring time of S1 is 1-3 hours.

[0014] Nanozymes are a new class of functional nanomaterials that mimic the functions of natural enzymes and offer numerous advantages, such as robust catalytic activity, enhanced stability, and simple synthesis. Various nanomaterials have been employed as nanozymes, including noble metal nanoparticles, metal oxides, and metal-organic frameworks. Nanozymes are commonly used for electrochemical sensing, initiating catalytic reactions to enhance signals or acting as advanced catalysts to engineer electroactive probes.

[0015] Among various nanozymes, metal-organic framework (MOF) nanozymes possess structural predictability, large surface area, and tunable catalytic activity. Compared with single MOF structures, MOF-composite nanozymes with other materials have significant advantages in catalytic activity. Although these nanomolecules have better performance, the development of MOF heterojunctions with non-precious metal complexes has not received much attention. Therefore, it is necessary to construct MOF-based nanozymes with higher catalytic activity.

[0016] Copper sulfide (CuS) is a common p-type semiconductor material that acts as an electron acceptor and possesses excellent physical and chemical properties. The combined advantages of the CuS and MOF heterojunctions employed in this study include a conjugated structure that enables efficient charge transfer, a reduced charge recombination rate, and a metal center for redox reactions. Furthermore, MOFs serve as a scaffold for CuS, hindering its aggregation. Therefore, combining CuS with MOFs is an effective strategy for enhancing catalytic activity and improving detection sensitivity. The design and development of novel composite nanozymes can broaden the detection range and achieve lower detection limits.

[0017] Preferably, the mass ratio of CuS@Cu-MOF, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride, N-hydroxysuccinimide, and anti-ASFVp54 in S2 is (3-6):0.02:0.025:0.04; more preferably, it is 4:0.02:0.025:0.04.

[0018] Anti-ASFVp54 is an antibody against the P54 protein of African swine fever virus (ASFV). It is a core tool for ASF diagnosis and research. Its applications cover serological testing, viral mechanism research and vaccine evaluation.

[0019] Preferably, the temperature of the first incubation in S2 is 3-5°C, and the time is 10-20 minutes; the temperature of the second incubation is 3-5°C, and the time is 1.5-2.5 hours.

[0020] More preferably, the water used in S1-S2 is ultrapure water.

[0021] The present invention also provides a MOF nanozyme sensor for rapid detection of African swine fever virus, which is prepared by the above-mentioned preparation method.

[0022] Contains at least the following beneficial technical effects: The present invention prepares a composite material CuS@Cu-MOF nanozyme with excellent peroxidase mimicking activity. The functional CuS@Cu-MOF nanozyme can catalyze the oxidation of 3,3',5,5'-tetramethylbenzidine (TMB) in the presence of H2O2. When the oxidation product (OxTMB) is reduced to TMB, it can be electrochemically detected by cathode current. Experiments have shown that the integrated CuS accelerates the Cu (Ⅱ) / Cu (I) redox cycle and significantly improves the POD-like activity. After coating with p54 antibodies, it specifically binds to ASFV through biomolecular recognition. The nanozyme can be used as a signal probe (CuS@Cu-MOF@Ab) to generate an amplified electrochemical signal. The resulting peak current is related to the concentration of ASFV. 1×10 -10 -1×10 -6 TCID 50 / mL wide concentration range and 2.34×10 -15 TCID 50 This biosensor combines the advantages of MOFs and CuS to create a high-performance nanozyme capable of detecting trace amounts of ASFV, thus providing a powerful sensing platform for the development of innovative diagnostic assays in the veterinary field. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 This is a high-magnification image of CuS prepared in Example 1.

[0024] Figure 2 This is a high-magnification image of the Cu-MOF prepared in Example 1.

[0025] Figure 3 This is a high-magnification image of the CuS@Cu-MOF prepared in Example 1.

[0026] Figure 4 This is a high-magnification image of the CuS@Cu-MOF@Ab sensor prepared in Example 1.

[0027] Figure 5 XRD patterns of CuS, Cu-MOF, CuS@Cu-MOF and CuS@Cu-MOF@Ab prepared in Example 1.

[0028] Figure 6 XPS spectra of CuS@Cu-MOF and CuS@Cu-MOF@Ab prepared in Example 1.

[0029] Figure 7 This is the Fourier transform infrared spectrum of CuS@Cu-MOF and CuS@Cu-MOF@Ab prepared in Example 1.

[0030] Figure 8 This is the UV spectrum of the CuS, Cu-MOF and CuS@Cu-MOF prepared in Example 1 after the color development reaction.

[0031] Figure 9 This is the fluorescence intensity diagram of CuS, Cu-MOF and CuS@Cu-MOF prepared in Example 1.

[0032] Figure 10 These are the EPR spectra of CuS, Cu-MOF and CuS@Cu-MOF prepared in Example 1.

[0033] Figure 11 This is the current-concentration diagram when anti-ASFVAb solution is added during the preparation of the MOF nanozyme sensor in Example 1.

[0034] Figure 12 Screening diagram for the optimal incubation time for incubating antigens on the electrode of MOF nanozyme sensor.

[0035] Figure 13 The peak current of CuS@Cu-MOF-anti-ASFVAb detected each antigen protein near 0.3V.

[0036] Figure 14 The peak current diagram and logarithmic function diagram of the MOF nanozyme sensor in the present invention for different concentrations of African swine fever virus.

[0037] Figure 15 These are the current values ​​when the MOF nanozyme sensor of Example 1 captures African swine fever virus at different concentrations. DETAILED DESCRIPTION

[0038] Based on the embodiments of the present invention, all other embodiments obtained by persons of ordinary skill in the art without creative work shall fall within the scope of protection of the present invention. The present invention will be described below with reference to specific embodiments. It should be noted that these embodiments are merely illustrative and do not limit the present invention in any way.

[0039] Example 1 This embodiment provides a MOF nanozyme sensor for rapid detection of African swine fever virus: 1) Synthesis of CuS: 0.1364 g of CuCl2·2H2O, 0.25 g of PVP, and 0.2 g of sodium thiosulfate were dispersed sequentially in 16 ml of ultrapure water and stirred until completely dissolved. The mixture was then immediately transferred to a 20 ml microwave reaction vial and maintained at 100°C for 12 hours. At the end of the reaction, the vial was cooled to room temperature to yield a dark green precipitate. The product was washed three times with ultrapure water (1000 rpm for 30 minutes) and finally dried in a vacuum oven at 60°C for 12 hours to obtain a dark green powder.

[0040] 2) Synthesis of Cu-MOF: 0.02 g of polyvinyl pyrrolidone (PVP) was dispersed in a mixture of 32 mL of DMF (N,N-dimethylformamide), 2 mL of ethanol, and 2 mL of ultrapure water. 0.1246 g of H2BDC was then added and dissolved under a magnetic stirrer. 0.1193 g of CuCl2·2H2O was added and stirred until completely dissolved. A homogeneous colloidal suspension was obtained by rapidly injecting 800 μL of triethylamine into the system while stirring. The colloidal solution was then transferred to a sonicator and sonicated continuously for 6 hours (40 kHz) in the dark. The resulting light blue precipitate was collected, washed three times with ethanol by centrifugation, and stored in a desiccator at 60°C overnight to yield a blue powder, the Cu-MOF.

[0041] 3) Synthesis of CuS@Cu-MOF: CuS and Cu-MOF powders were dispersed in ultrapure water in a mass ratio of 1:3 and stirred for 2 hours to obtain the composite material CuS@Cu-MOF.

[0042] 4) Construction of MOF nanozyme sensors CuS@Cu-MOF was prepared into an aqueous solution (4.0 mL, 1.0 mg / mL), and EDC (25.0 μL, 0.8 mg / mL) and NHS (25.0 μL, 1.0 mg / mL) were added and incubated at 4°C for 10 min. Then, anti-ASFVp54 solution (200.0 μL, 0.2 mg / mL) was added and incubated at 4°C for 2 h to obtain a CuS@Cu-MOF-antibody solution, i.e., a composite MOF nanozyme sensor solution, which was recorded as MA solution.

[0043] Example 2 This embodiment provides a MOF nanozyme sensor for rapid detection of African swine fever virus: 1) Synthesis of CuS: 0.1 g of CuCl2·2H2O, 0.2 g of PVP, and 0.15 g of sodium thiosulfate were dispersed sequentially in 15 ml of ultrapure water and stirred until completely dissolved. The mixture was then immediately transferred to a 20 ml microwave reaction vial and maintained at 95°C for 10 h. At the end of the reaction, the vial was cooled to room temperature to yield a dark green precipitate. The product was washed three times with ultrapure water (1000 rpm for 30 min) and finally dried in a vacuum oven at 60°C for 12 h to obtain a dark green powder.

[0044] 2) Synthesis of Cu-MOF 0.01 g of PVP (polyvinyl pyrrolidone) was dispersed in a mixture of 32 mL of DMF (N,N-dimethylformamide), 2 mL of ethanol, and 2 mL of ultrapure water. 0.1 g of H2BDC was then added and dissolved under stirring on a magnetic stirrer. 0.1 g of CuCl2·2H2O was added and stirred until completely dissolved. A homogeneous colloidal suspension was obtained by rapidly injecting 800 μL of triethylamine into the system while stirring. The colloidal solution was then transferred to a sonicator and sonicated continuously for 4 hours (40 kHz) in the dark. The resulting light blue precipitate was collected, washed three times by centrifugation with ethanol, and stored in a desiccator at 60°C overnight to obtain a blue powder, the Cu-MOF.

[0045] 3) Synthesis of CuS@Cu-MOF CuS and Cu-MOF powders were dispersed in ultrapure water in a mass ratio of 1:3 and stirred for 2 hours to obtain the composite material CuS@Cu-MOF.

[0046] 4) Construction of MOF nanozyme sensors CuS@Cu-MOF was prepared into an aqueous solution (3.0 mL, 1.0 mg / mL), and EDC (25.0 μL, 0.8 mg / mL) and NHS (25.0 μL, 1.0 mg / mL) were added and incubated at 4°C for 10 min. Then, anti-ASFVp54 solution (200.0 μL, 0.2 mg / mL) was added and incubated at 4°C for 2 h to obtain a CuS@Cu-MOF-antibody solution, i.e., a composite MOF nanozyme sensor solution, which was recorded as MA solution.

[0047] Example 3 This embodiment provides a MOF nanozyme sensor for rapid detection of African swine fever virus: 1) Synthesis of CuS: 0.15 g of CuCl2·2H2O, 0.3 g of PVP, and 0.25 g of sodium thiosulfate were dispersed sequentially in 18 ml of ultrapure water and stirred until completely dissolved. The mixture was then immediately transferred to a 20 ml microwave reaction vial and maintained at 100°C for 14 hours. At the end of the reaction, the vial was cooled to room temperature to yield a dark green precipitate. The product was washed three times with ultrapure water (1000 rpm for 30 minutes) and finally dried in a vacuum oven at 60°C for 12 hours to obtain a dark green powder.

[0048] 2) Synthesis of Cu-MOF 0.03 g of polyvinyl pyrrolidone (PVP) was dispersed in a mixture of 32 mL of DMF (N,N-dimethylformamide), 2 mL of ethanol, and 2 mL of ultrapure water. 0.15 g of H2BDC was then added and dissolved under stirring on a magnetic stirrer. 0.15 g of CuCl2·2H2O was added and stirred until completely dissolved. A homogeneous colloidal suspension was obtained by rapidly injecting 800 μL of triethylamine into the system while stirring. The colloidal solution was then transferred to a sonicator and sonicated continuously for 7 hours (40 kHz) in the dark. The resulting light blue precipitate was collected, washed three times with ethanol by centrifugation, and stored in a desiccator at 60°C overnight to yield a blue powder, the Cu-MOF.

[0049] 3) Synthesis of CuS@Cu-MOF CuS and Cu-MOF powders were dispersed in ultrapure water in a mass ratio of 1:3 and stirred for 2 hours to obtain the composite material CuS@Cu-MOF.

[0050] 4) Construction of MOF nanozyme sensors CuS@Cu-MOF was prepared into an aqueous solution (6.0 mL, 1.0 mg / mL), and EDC (25.0 μL, 0.8 mg / mL) and NHS (25.0 μL, 1.0 mg / mL) were added and incubated at 4°C for 10 min. Then, anti-ASFVp54 solution (200.0 μL, 0.2 mg / mL) was added and incubated at 4°C for 2 h to obtain a CuS@Cu-MOF-antibody solution, i.e., a composite MOF nanozyme sensor solution, which was recorded as MA solution.

[0051] Test Example 1 (1) Characterization of MOF nanozyme sensors Figure 1 A is a low-magnification image of the CuS sensor prepared in Example 1 of the present invention, and B is an enlarged partial image. Figure 2 This is a high-magnification image of Cu-MOF. Figure 3 This is a high-magnification image of the CuS@Cu-MOF sensor. Figure 4 This is the enlarged image of CuS@Cu-MOF after binding to p54 monoclonal antibody; Figure 3 and Figure 4 It can be seen that the African swine fever antibody protein anti-ASFVp54 in the nanozyme sensor is modified on the surface of the composite nanozyme CuS@Cu-MOF, making its surface rough.

[0052] Figure 5 XRD patterns of CuS, Cu-MOF, CuS@Cu-MOF and CuS@Cu-MOF@Ab (CuS@Cu-MOF-antibody) prepared in Example 1 of the present invention. Figure 5 It can be seen that in the Cu-MOF pattern, diffraction peaks were observed at 8.34°, 15.48°, 16.7°, and 26.32°, corresponding to the

[001] ,

[200] ,

[201] , and

[220] planes, respectively. These peaks were also detected in the pattern of the CuS@Cu-MOF hybrid, indicating that the crystal structure of CuS remained intact after loading onto the Cu-MOF. In addition, new characteristic peaks at 27.68°, 29.28°, 31.79°, 32.85°, 47.94°, 52.72°, and 59.35° were observed in the CuS@Cu-MOF hybrid, corresponding to the

[101] ,

[102] ,

[103] ,

[006] ,

[110] ,

[108] , and

[116] planes of CuS. The crystal structure of CuS@Cu-MOF@Ab is similar to that of CuS@Cu-MOF. The results showed that the crystallinity of CuS@Cu-MOF was retained after modification with anti-ASFVp54.

[0053] Figure 6 XPS spectra of CuS@Cu-MOF and CuS@Cu-MOF@Ab prepared in Example 1 of the present invention; Figure 7 This is the Fourier transform infrared spectrum of CuS@Cu-MOF and CuS@Cu-MOF@Ab prepared in Example 1 of the present invention. Figure 6 The S 2p peak intensity of CuS@Cu-MOF@Ab increases. Figure 7 The amide bond of CuS@Cu-MOF@Ab vibrates and stretches, which verifies that the African swine fever antibody protein has been modified on CuS@Cu-MOF, and the MOF nanozyme sensor has been successfully prepared. Figure 7 The black vertical lines in the middle represent the peak positions 1651 (left) and 1570 (right).

[0054] Test Example 2 (2) Detection of catalytic activity of MOF nanozyme sensors The POD activity of CuS@Cu-MOF in the MOF nanozyme sensor was evaluated by a typical binding color development reaction. The binding color development mechanism was as follows: in the presence of H2O2 (30.0 mM, 100.0 μL), colorless TMB (30.0 mM, 100.0 μL) could be oxidized by MOF nanozyme to generate Ox-TMB; CuS and Cu-MOF were selected as controls, and H2O2 / TMB solution containing H2O2 and TMB and TMB solution were selected as blanks.

[0055] Figure 8 This is the UV spectrum of CuS, Cu-MOF, CuS@Cu-MOF and blank solution after the color reaction. Figure 8 It can be seen that there is no absorbance in the combined chromogenic solutions of TMB and H2O2 / TMB blank without the addition of MOF nanozyme, while the combined chromogenic solutions after adding CuS, Cu-MOF and CuS@Cu-MOF all produce obvious absorbance at 652nm, and the absorbance of the combined chromogenic solution of CuS@Cu-MOF is higher, verifying the effectiveness of the catalytic detection of the composite material CuS@Cu-MOF.

[0056] Test Example 3 (3) Study on the catalytic performance of MOF nanozyme sensors ① The catalytic performance of three different MOF nanozymes was evaluated by examining the amount of hydroxyl radicals produced by CuS, Cu-MOF and CuS@Cu-MOF in the H2O2 / TMB system at pH 4.5.

[0057] Figure 9 This is a fluorescence intensity diagram of CuS, Cu-MOF, and CuS@Cu-MOF prepared in Example 1 of the present invention. Figure 10 The EPR images of CuS, Cu-MOF and CuS@Cu-MOF prepared in Example 1 of the present invention are shown in Figure 1. Figure 9 It can be seen that the hydroxyl radical production produced by CuS@Cu-MOF catalysis is the highest. Figure 10 The above results were also verified.

[0058] Test Example 4 (4) Optimization of preparation conditions for MOF nanozyme sensors The optimal concentration of anti-ASFVAb solution added during the preparation of MOF nanozyme sensor was determined as follows: CuS@Cu-MOF aqueous solution (10 μL, 2.0 mg / mL) was modified on the surface of gold electrode, and after infrared drying, it was fixed with 0.5% naphthol as glue. Then, EDC (5.0 μL, 0.8 mg / mL) and NHS (5.0 μL, The Au electrode-CuS@Cu-MOF-anti-ASFVAb was prepared by adding 6.0 μL of anti-ASFVAb solution (1.0 mg / mL) at a concentration of 2 μg / mL, 4 μg / mL, 6 μg / mL, 8 μg / mL, and 10 μg / mL, respectively, and incubated at 4°C for 10 min. ASFVP-54Ab protein (6.0 μL, 5.0 μg / mL) was then added dropwise to the electrode surface and incubated at 4°C for 20 min. The electrode was placed in a mixture of H2O2 (500 μL, 20 mM), acetate buffer (9.0 mL, pH 4.5), and TMB (500 μL, 20 mM) for DPV testing. The results showed a characteristic peak near 0.3 V. The relationship between the current value (μA) and the concentration of the anti-ASFVAb solution was obtained, thereby obtaining the optimal anti-ASFVAb solution concentration.

[0059] Figure 11 This is the current-concentration diagram of adding anti-ASFVAb solution during the preparation process of the MOF nanozyme sensor of Example 1 of the present invention. Figure 11 As can be seen, as the anti-ASFVAb concentration increased from 2 μg / mL to 6 μg / mL, the current value of the MOF nanozyme sensor at 0.3V dropped sharply. Subsequently, as the anti-ASFVAb concentration increased, the current value tended to level off. This indicates that as the anti-ASFVAb concentration increased, more and more anti-ASFVAb covered the CuS@Cu-MOF surface, resulting in a shielding effect. This shielded some metal active sites, but it was unable to fully cover the entire CuS@Cu-MOF surface. Therefore, to ensure the high efficiency of the MOF enzyme biosensor, the optimal concentration of anti-ASFVAb was 6 μg / mL. The same operation method was used to optimize the antigen incubation time, and the final incubation time of the antigen on the electrode was 20, 30, 40, 50, and 60 minutes, respectively. Figure 12 The results showed that 40 min was the optimal incubation time.

[0060] Test Example 5 Study on the specificity and sensitivity of MOF nanozyme sensor for detecting influenza B virus ① Specificity Specificity is a key factor in detecting the target with high sensitivity and avoiding false positives.

[0061] In the present invention, the same concentrations of P72, P30, A104R, and C129R (5 μg / mL) were selected as control solutions to study the specificity of CuS@Cu-MOF-anti-ASFVAb detection.

[0062] Figure 13 The peak current of each antigen protein near 0.3V was detected by CuS@Cu-MOF-anti-ASFVAb. The results showed that at the same concentration of each protein, only the electrochemical signal of p54 decreased significantly, while no obvious changes were observed for P72, P30, A104R, and C129R. This is because the antibody modified on the sensor surface can only recognize and bind to the P54 antigen. These results indicate that the CuS@Cu-MOF-based immunosensor platform has excellent selectivity for P54 in complex matrices.

[0063] ② Sensitivity 6 μL of serum containing varying concentrations of African swine fever virus (ASFVAb) was added to the Au electrode-CuS@Cu-MOF-anti-ASFVAb and incubated for 20 minutes (0-3723.34 ng / mL). The electrode was then placed in 500 μL of 20 mM H₂O₂, 9 mL of acetate buffer (pH 4.5), and 500 μL of 20 mM TMB. The DPV peak current was then measured using the DPV test function on the electrochemical workstation.

[0064] Figure 14 The peak current diagram and logarithmic function diagram of the MOF nanozyme sensor used for different concentrations of African swine fever virus in the present invention are shown in FIG. Figure 14 (A) is the peak current diagram, Figure 14 (B) is the logarithmic function graph, from Figure 14 It can be seen that the characteristic peak current value of Ox-TMB at 0.3V decreases with the increase of African swine fever virus concentration. Therefore, there is a good linear relationship between the characteristic peak current of Ox-TMB at 0.3V and the logarithmic function of African swine fever virus concentration (I=0.649lgC+1.44), r=0.976, and the calculated LOD detection limit is as low as 2.34×10 -15 TCID 50 / mL.

[0065] ③ Comparison of sensitivity between ELISA and electrochemical detection In order to evaluate the sensitivity of the detection method of the present invention, ELISA detection and fitting of influenza B virus were carried out, and the results are as follows: Figure 15 shown.

[0066] like Figure 15The solid black line in the middle shows the current values ​​recorded when capturing different concentrations of African swine fever virus. The change in current value is linearly negatively correlated with the log value of the virus concentration. The linear equation is I = 0.649 logC + 1.44 (r = 0.976), and the detection limit is 2.34×10 -15 TCID 50 / mL. As shown by the red solid line in Figure 15, the absorbance decreases with increasing virus concentration, and its linear equation is A = -0.0679 - 0.2173lgC (r = 0.999). The detection limit is 4.29×10 -10 TCID 50 According to these results, the detection limit of the sensor in the present invention is lower than that of the commercial ELISA kit.

[0067] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. A method for preparing a MOF nanozyme sensor for rapid detection of African swine fever virus, characterized in that: The following steps are involved: S1. CuS, Cu-MOF, and water were mixed and stirred to obtain CuS@Cu-MOF; S2. CuS@Cu-MOF, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride, N-hydroxysuccinimide, and water were mixed and incubated for the first time, and then anti-ASFVp54 was added for the second incubation to obtain CuS@Cu-MOF-antibody, i.e., MOF nanozyme sensor.

2. The preparation method according to claim 1, characterized in that The mass ratio of CuS to Cu-MOF in the S1 is 1:

3.

3. The preparation method according to claim 2, characterized in that The preparation method of CuS comprises the following steps: mixing CuCl2·2H2O, PVP, sodium thiosulfate and water, heating for reaction, cooling, washing and drying to obtain CuS powder.

4. The preparation method according to claim 3, characterized in that The mass ratio of the CuCl2·2H2O, PVP, sodium thiosulfate and water is (0.1-0.15): (0.2-0.3): (0.15-0.25): (15-18).

5. The preparation method according to claim 3, characterized in that The temperature of the heating reaction is (95-100) °C and the time is (10-14) h.

6. The preparation method according to claim 2, characterized in that The preparation method of the Cu-MOF is: Polyvinyl pyrrolidone, N, N-dimethylformamide, ethanol and water are mixed and dispersed, and then H2BDC and CuCl2·2H2O are added in sequence and stirred to dissolve. Then triethylamine is added to obtain a homogeneous colloidal suspension and ultrasonic treatment is performed to obtain Cu-MOF.

7. The preparation method according to claim 6, characterized in that The volume mass ratio of the polyvinyl pyrrolidone, N,N-dimethylformamide, ethanol, water, H2BDC, CuCl2·2H2O, and triethylamine is (0.01-0.03) g:32 mL:2 mL:2 mL:(0.1-0.15) g:(0.1-0.15) g:800 μL.

8. The preparation method according to claim 1, characterized in that The mass ratio of CuS@Cu-MOF, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride, N-hydroxysuccinimide, and anti-ASFVp54 in the S2 is (3-6):0.02:0.025:0.

04.

9. The preparation method according to claim 1, characterized in that The first incubation temperature in S2 is 3-5°C and the time is 10-20 minutes; the second incubation temperature is 3-5°C and the time is 1.5-2.5 hours.

10. A MOF nanozyme sensor for rapid detection of African swine fever virus, characterized in that: The invention is prepared by the preparation method according to any one of claims 1 to 9.

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