Nanocomposite enzyme materials and their preparation methods, nanocomposite enzyme detection antibodies, colorimetric immunosensors and their detection methods

Abstract

A nanocomposite enzyme material and its preparation method are disclosed, relating to the field of biodetection technology. This invention combines natural enzymes with nanozymes and loads them into a metal-organic framework (MOF) material, utilizing the MOF material to enhance its loading capacity, enzyme activity, and stability. Furthermore, embodiments of this invention also provide detection antibodies and colorimetric immunosensors prepared using the aforementioned nanocomposite enzyme material, as well as corresponding detection methods. Utilizing the unique properties of the nanocomposite enzyme material, the ability to recognize and detect antigens is effectively improved. Direct antigen recognition is achieved without the need for nucleic acid extraction and amplification, representing a reliable, inexpensive, highly sensitive, and high-throughput antigen detection method.

Description

Technical Field

[0001] This invention relates to the field of biodetection technology, and more specifically, to nanocomposite enzyme materials and their preparation methods, nanocomposite enzyme detection antibodies, colorimetric immunosensors and their detection methods. Background Technology

[0002] African swine fever virus (ASFV) is a highly virulent pathogen that causes a fatal hemorrhagic disease in domestic and wild pigs, resulting in a near 100% mortality rate. Currently, ASF is a serious epidemic in China and other Asian countries, causing catastrophic damage to the pork industry. To date, there is no effective vaccine or antiviral strategy. Although ASFV cannot infect humans, the disease's impact in Asia has already caused enormous economic losses, and there are no signs of it abating. In the absence of effective treatments and vaccines, early and accurate diagnosis is an effective alternative for rapidly controlling the epidemic.

[0003] However, there is currently no reliable, inexpensive, highly sensitive, and high-throughput method for ASFV detection. The gold standard for laboratory ASFV diagnosis is based on polymerase chain reaction (PCR). Although highly sensitive, PCR is too complex for point-of-care testing, requiring the cryopreservation of fragile enzymes and relying on bulky and expensive equipment. This hinders its widespread use in rural communities where infections occur. Enzyme-linked immunosorbent assay (ELISA), a technique for identifying viral antigens, is also widely used for ASFV detection. ELISA does not require expensive equipment or complex amplification processes. However, its sensitivity is low, and it requires temperature-sensitive reagents, which are fragile and difficult to transport and store. Therefore, ELISA must be combined with other molecular diagnostic methods to detect the pathogen in point-of-care settings. Developing a reliable, inexpensive, highly sensitive, and high-throughput method for ASFV detection is therefore essential. Summary of the Invention

[0004] The primary objective of this invention is to provide a nanocomposite enzyme material and its preparation method, which combines natural enzymes with nanozymes and loads them into a metal-organic framework material, thereby utilizing the metal-organic framework material to improve its loading capacity, enzyme activity, and stability.

[0005] The second objective of this invention is to provide a nanocomposite enzyme detection antibody, which utilizes metal-organic framework materials to improve enzyme activity and stability, and can also achieve rapid detection of antigens by specifically binding the loaded antibody to the antigen.

[0006] The third objective of this invention is to provide a colorimetric immunoassay sensor that utilizes the specific recognition of antigens by a first antibody and a second antibody to form a sandwich structure of "antibody-antigen-antibody" for rapid antigen recognition and detection.

[0007] The fourth objective of this invention is to provide a non-therapeutic method for detecting ASFV, which is simple and convenient to operate, requires no nucleic acid extraction and amplification, directly identifies ASFV particles, and achieves rapid detection of ASFV.

[0008] The embodiments of the present invention are implemented as follows:

[0009] A nanocomposite enzyme material, wherein the nanocomposite enzyme material is AuNPs@ZIF-8@Fe3O4@HRP, the nanocomposite enzyme material includes metal-organic framework material ZIF-8, Fe3O4 and HRP loaded inside ZIF-8, and AuNPs distributed on the surface of ZIF-8.

[0010] A method for preparing the above-mentioned nanocomposite enzyme material, comprising:

[0011] ZIF-8@Fe3O4@HRP and AuNPs were mixed at 0~10 o Mixing is performed at C.

[0012] A nanocomposite enzyme detection antibody is formed by loading a first antibody onto the surface of the aforementioned nanocomposite enzyme material.

[0013] A colorimetric immunosensor includes a second antibody and the aforementioned nanocomposite enzyme detection antibody, wherein the first antibody and the second antibody specifically bind to different epitopes of the same antigen.

[0014] A non-therapeutic method for detecting ASFV, employing the aforementioned colorimetric immunoassay sensor, comprising:

[0015] Add the second antibody to the ELISA plate for the first incubation;

[0016] After the first incubation, the cells were sealed with BSA.

[0017] After sealing, the antigen is added for a second incubation;

[0018] After the second incubation, the nanocomposite enzyme detection antibody was added for a third incubation.

[0019] After the third incubation, a buffer solution was added, and TMB and H2O2 were used for color development.

[0020] The beneficial effects of the embodiments of the present invention are:

[0021] This invention provides a nanocomposite enzyme material and its preparation method, which combines natural enzymes with nanozymes and loads them into a metal-organic framework (MOF) material. The MOF material enhances the loading capacity, enzyme activity, and stability. Furthermore, this invention also provides detection antibodies and colorimetric immunosensors prepared using the aforementioned nanocomposite enzyme material, along with corresponding detection methods. Utilizing the unique properties of the nanocomposite enzyme material, the detection capability for antigens is effectively improved. Direct antigen recognition is achieved without the need for nucleic acid extraction and amplification, providing a reliable, inexpensive, highly sensitive, and high-throughput antigen detection method. Attached Figure Description

[0022] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as a limitation on the scope. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort.

[0023] Figure 1 This is a schematic diagram of the ASFV detection method provided in an embodiment of the present invention;

[0024] Figure 2 The scanning electron microscope (SEM) image (A) and transmission electron microscope (TEM) image (B) of ZIF-8 provided for embodiments of the present invention are shown.

[0025] Figure 3 SEM image (A) and TEM image (B) of Fe3O4 provided in the embodiments of the present invention;

[0026] Figure 4 SEM image (A) of ZIF-8@Fe3O4@HRP and TEM image (B) of the nanocomposite enzyme material provided in the embodiments of the present invention;

[0027] Figure 5 Infrared spectra (A) and Zeta potential results (B) of ZIF-8, Fe3O4, ZIF-8@Fe3O4 and ZIF-8@Fe3O4@HRP provided in the embodiments of the present invention;

[0028] Figure 6 X-ray diffraction results of ZIF-8, Fe3O4, and ZIF-8@Fe3O4@HRP provided in the embodiments of the present invention;

[0029] Figure 7 The above are the photoelectron spectroscopy (XPS) results of the nanocomposite enzyme material provided in the embodiments of the present invention;

[0030] Figure 8 The ultraviolet absorption spectra (A) and standard curves (B) of different concentrations of antigen in the non-therapeutic ASFV detection method provided in the embodiments of the present invention.

[0031] Figure 9 The specific detection results of the colorimetric immunoassay sensor provided in the embodiments of the present invention;

[0032] Figure 10 The results are optimizations of the ASFV detection method provided in the embodiments of the present invention.

[0033] Figure 11 The enzyme activity comparison results of ZIF-8@Fe3O4@HRP provided in the embodiments of the present invention;

[0034] Figure 12 The stability test results of ZIF-8@Fe3O4@HRP provided in the embodiments of the present invention;

[0035] Figure 13 The high-temperature resistance test results of ZIF-8@Fe3O4@HRP provided in the embodiments of the present invention;

[0036] Figure 14 The results of the low-temperature tolerance test of ZIF-8@Fe3O4@HRP provided in the embodiments of the present invention. Detailed Implementation

[0037] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased commercially.

[0038] The following is a detailed description of a nanocomposite enzyme material and its preparation method, a nanocomposite enzyme detection antibody, a colorimetric immunoassay sensor and its detection method according to embodiments of the present invention.

[0039] This invention provides a nanocomposite enzyme material, which is AuNPs@ZIF-8@Fe3O4@HRP. The nanocomposite enzyme material includes a metal-organic framework material ZIF-8, Fe3O4 and HRP loaded inside ZIF-8, and AuNPs distributed on the surface of ZIF-8.

[0040] This study synthesizes a nanozyme (Fe3O4) and a natural enzyme (HRP, horseradish peroxidase) within a metal-organic framework (ZIF-8). The combination of these two enzymes provides high enzyme activity, while the protection provided by the metal-organic framework ensures optimal enzyme loading and stability. Furthermore, the surface AuNPs (gold nanoparticles) can further bind antibodies, achieving specific binding to antigens.

[0041] This invention also provides a method for preparing the above-mentioned nanocomposite enzyme material, comprising:

[0042] ZIF-8@Fe3O4@HRP and AuNPs were mixed at 0~10 oC Mix them together.

[0043] Based on the mass of ZIF-8@Fe3O4@HRP, the molar amount of AuNPs is 0.5~5 mmol / g. That is, 0.5~5 mmol of AuNPs are required per gram of ZIF-8@Fe3O4@HRP to ensure optimal AuNP loading.

[0044] Furthermore, during the reaction, ZIF-8@Fe3O4@HRP was first prepared into a suspension of 1-5 mg / mL, and ultrasonic treatment was used to ensure uniform dispersion of ZIF-8@Fe3O4@HRP. AuNPs were used in a solution with a concentration of approximately 0.2-0.3 mM. After the reaction was completed, the solution was magnetically washed three times before use.

[0045] Optionally, the preparation method of ZIF-8@Fe3O4@HRP includes:

[0046] Fe3O4, HRP, Zn(NO3)2·6H2O, and 2-methylimidazole were mixed at room temperature, centrifuged, and then freeze-dried. The mass ratio of Fe3O4 to HRP was 1:0.2~1. Preferably, the mass ratio of Fe3O4 to HRP was 1:0.5.

[0047] Fe3O4 is prepared by co-precipitation of Fe(III) and Fe(II) ions. Fe(III) can be obtained using FeCl3·6H2O, and Fe(II) can be obtained using FeSO4·7H2O, with a mass ratio of 2~5:1~3. The reaction requires vigorous stirring at 50~60℃ until complete dissolution, followed by heating to 70~80℃. oC Add trisodium citrate dropwise, and at 80-85°C... o The mixture was continuously stirred under nitrogen atmosphere. After the reaction was completed and cooled to room temperature, a strong magnet was immersed in the solution to separate the black precipitate. The obtained product was washed three times with deionized water and ethanol, and finally dried in a vacuum oven.

[0048] In preparing ZIF-8@Fe3O4@HRP, the Fe3O4 is first dispersed in water to a concentration of 5–15 mg / mL. This concentration is then mixed with HRP solution, Zn(NO3)2·6H2O solution, and 2-methylimidazole solution. The concentrations of the HRP solution, Zn(NO3)2·6H2O solution, and 2-methylimidazole solution are 5–15 mg / mL, 0.2–1 M, and 1–1.5 M, respectively. Based on the mass of Fe3O4, the amount of Zn(NO3)2·6H2O used is 0.015–0.025 mol, and the amount of 2-methylimidazole used is 0.6–0.7 mol.

[0049] Methods for preparing AuNPs include:

[0050] HAuCl4 and sodium citrate were mixed at 100~120°C. o The mixture was reacted at C for 20-40 min; the molar ratio of HAuCl4 to sodium citrate was 1:4-10.

[0051] In this reaction, HAuCl4 was used in a solution with a concentration of 20–30 mmol / L, and sodium citrate was used in a solution with a concentration of 10–15 mmol / L. The reaction was carried out at high temperature until the solution turned wine-red, then naturally cooled to room temperature, and then... o Stored under C.

[0052] This invention also provides a nanocomposite enzyme detection antibody, which is formed by loading a first antibody onto the surface of the aforementioned nanocomposite enzyme material.

[0053] The AuNPs on the surface of this nanocomposite enzyme material can effectively bind to the primary antibody, achieving a high loading rate. Optionally, the primary antibody is a nanobody that can specifically bind to ASFV, and the amino acid sequence of the primary antibody is SEQ NO. 1. Specifically, the amino acid sequence of the primary antibody is: LQESGGGSVQ AGGSLRLSCA VSGDRRARST DGRYCMAWFRQAPGKEREGV ANIYIGGGST YIADSVKGRF TMSHENAKNTLYLQMNSLKP EDTAMYYCAA GTAMVRRWLPSIRGYAARCT ALINDLYDSW GQGTQVTVSS AA

[0054] The preparation method of the antibody for detection using the nanocomposite enzyme includes:

[0055] The primary antibody was mixed with the nanocomposite enzyme material and incubated with shaking for 10–15 h. BSA (bovine serum albumin) was then added for blocking for 1–2 h, followed by magnetic washing for later use. The concentration of the primary antibody was 5–20 μg / mL, and the volume was 80–150 μL. The nanocomposite enzyme material was prepared using the above method, with a volume of 0.5–2 mL.

[0056] This invention also provides a colorimetric immunoassay sensor, comprising a second antibody and the nanocomposite enzyme detection antibody of claim 6 or 7, wherein the first antibody and the second antibody specifically bind to different epitopes of the same antigen. It utilizes the specific recognition of the antigen by the first and second antibodies to form an "antibody-antigen-antibody" sandwich structure for rapid antigen recognition and detection.

[0057] Furthermore, the second antibody specifically binds to ASFV, while the first and second antibodies specifically bind to different epitopes of ASFV. For example, the second antibody can be recombinant African swine fever p72 protein (product number: bs-41384R; English name: ASFV p72) produced by Beijing Bio-Sens Biotechnology Co., Ltd.

[0058] This invention also provides a method for detecting ASFV for non-therapeutic purposes, which employs the above-mentioned colorimetric immunoassay sensor, comprising:

[0059] S1. Add the second antibody to the ELISA plate for the first incubation;

[0060] S2. After the first incubation is completed, seal with BSA;

[0061] S3. After sealing, add antigen for a second incubation;

[0062] S4. After the second incubation, add the nanocomposite enzyme detection antibody and continue the third incubation;

[0063] S5. After the third incubation, add buffer solution and perform color development using TMB and H2O2.

[0064] Furthermore, the first incubation is at 0~10 o The incubation was carried out at C for 10–20 h. After incubation, the sample was washed with PBST (phosphate buffer) at pH 7.2 and then blocked with BSA for 30–60 min.

[0065] The second incubation lasted 40-90 minutes. After incubation, the sample was washed with PBST at pH 7.2, and then nanocomposite enzyme was added to detect the antibody.

[0066] The third incubation lasts 40–90 minutes. After incubation, a buffer solution is added. The buffer solution here is an acetate buffer solution with a pH of 5–6, preferably pH 5.5. The color development time is 2–10 minutes.

[0067] After color development, qualitative detection can be performed by visual comparison. Alternatively, the absorbance of different concentrations of antigen at OD625nm can be measured, and a working curve can be plotted to achieve quantitative detection.

[0068] The detection principle of this detection method is as follows: Figure 1 As shown, the second antibody is first adsorbed onto the surface of the ELISA plate and then blocked with BSA. The second antibody specifically recognizes the ASFV antigen. Subsequently, the nanocomposite enzyme detection antibody specifically recognizes the ASFV antigen, forming an antibody-antigen-antibody sandwich structure. After the colorimetric reaction, it can be observed with the naked eye or using UV-Vis spectroscopy. It should be noted that... Figure 1 For ease of illustration, the names of some substances have been simplified. For example, in the figure, Nb2 represents the first antibody; Au@MOF represents the nanocomposite enzyme material; Au@MOF@Nb2 represents the nanocomposite enzyme detection antibody; Ab1 represents the second antibody; Ab1@ASFV represents the combination of the second antibody and ASFV antigen; and Ab1@ASFV@Nb2 represents the sandwich structure formed by the combination of the second antibody, ASFV antigen, and nanocomposite enzyme detection antibody.

[0069] The features and performance of the present invention will be further described in detail below with reference to embodiments.

[0070] Example 1

[0071] This embodiment provides a ZIF-8@Fe3O4@HRP preparation method including:

[0072] S1. Dissolve 3.25 g FeCl3·6H2O and 1.67 g FeSO4·7H2O in 50 mL of deionized water, and then... o Stir vigorously at C for 30 minutes. After complete dissolution, heat the mixture to 75°C. o C, and ammonia solution (6.25 mL, 25% w / w) was added dropwise while stirring continuously for 60 min. Subsequently, 1.5 M trisodium citrate (6.25 mL) was added, and the solution was heated to 85°C. o The solution was stirred continuously under nitrogen atmosphere for 90 min and then cooled to room temperature. A strong magnet was then immersed in the solution for 5 min to separate the black precipitate. The resulting product was washed three times with deionized water and ethanol, and finally cooled at 60 °C. o The solid Fe3O4 was obtained by drying in a vacuum oven at C for 6 h.

[0073] S2. Prepare a Fe3O4 suspension by suspending 20 mg of the above solid Fe3O4 in 2 mL of deionized water and sonicating for 15 min to ensure uniform dispersion. Prepare 1 mL of a 10 mg / mL horseradish peroxidase (HRP) solution beforehand. Then, mix 2 mL of the Fe3O4 suspension, 1 mL of the HRP solution, 1 mL of a 0.4 M Zn(NO3)2·6H2O MOF solution, and 10 mL of a 1.25 M 2-methylimidazole solution at room temperature for 30 min. Centrifuge the mixture at 6000 rpm for 10 min and wash the precipitate three times with deionized water.

[0074] Figures 2-4 SEM and TEM images of ZIF-8, Fe3O4, and ZIF-8@Fe3O4@HRP are shown respectively. Figure 2 As can be seen, ZIF-8 has a dodecahedral framework structure, composed of... Figure 3 It can be seen that the Fe3O4 nanoparticles are uniform and exhibit a nano-flower-like structure. Figure 4 -A shows that ZIF-8 is uniformly coated on HRP and Fe3O4 nanoparticles, and the material is well dispersed.

[0075] Figure 5 -A shows the infrared comparison images of ZIF-8, Fe3O4, ZIF-8@Fe3O4, and ZIF-8@Fe3O4@HRP. The images show that the ZIF-8@Fe3O4@HRP surface absorbs water (at 3446 cm⁻¹). -1 Stretching vibration of CN single bond (1178 cm) -1 ), and Fe-O functional groups (576 cm ), -1 The corresponding new absorption band is significantly enhanced. -C=O (1642 cm⁻¹) was observed in ZIF-8 and ZIF-8@Fe₃O₄@HRP composites. -1 The characteristic peaks of HRP and Fe3O4 indicate that HRP and Fe3O4 were successfully composited onto ZIF-8.

[0076] Figure 5-B shows the Zeta potential test results of ZIF-8, Fe3O4, ZIF-8@Fe3O4, and ZIF-8@Fe3O4@HRP. In the figure, a represents ZIF-8, b represents Fe3O4, c represents ZIF-8@Fe3O4, and d represents ZIF-8@Fe3O4@HRP. It can be seen that the average Zeta potential of ZIF-8 is 24.83 mV, that of Fe3O4 is -26.47 mV, that of ZIF-8@Fe3O4 is 14.13 mV, and that of ZIF-8@Fe3O4@HRP is 0.23 mV. The decrease in charge is due to the negative charge of HRP at pH=7, indicating the successful synthesis of the ZIF-8@Fe3O4@HRP nanocomposite material.

[0077] Figure 6 The X-ray diffraction results of ZIF-8, Fe3O4, and ZIF-8@Fe3O4@HRP are shown. As can be seen from the figures, Fe3O4 exhibits diffraction patterns at 36.21 nm. o There is a sharp peak at 36.21, while ZIF-8@Fe3O4@HRP has a peak at 36.21. o This diffraction peak also appeared at [location missing]. This proves that the encapsulation of Fe3O4 and HRP did not cause any change in the morphology of ZIF-8. The ZIF-8@Fe3O4@HRP composite material has been successfully synthesized.

[0078] Example 2

[0079] This embodiment provides a nanocomposite enzyme material, the preparation method of which includes:

[0080] S1. Add 1.07 mL of HAuCl4 solution with a concentration of 23.46 mmol / L to 100 mL of deionized water, and incubate at 110 °C. o Heat and stir at C for 5 min, add 10 ml of 14.53 mmol / L sodium citrate solution, reflux for 20-40 min until the solution turns wine red, cool naturally to room temperature to obtain AuNPs solution, and then... o Stored under C.

[0081] S2. Prepare a 1 mg / mL ZIF-8@Fe3O4@HRP suspension, disperse by sonication, and add 10 mL to 50 mL of AuNPs solution. o C. Stir for 12 h, wash three times with magnetic suction, and resuspend to 10 mL to obtain a solution of nanocomposite enzyme material for later use.

[0082] Figure 4-B shows a TEM image of the nanocomposite enzyme material, which clearly shows that AuNPs particles are uniformly distributed on the surface of ZIF-8@Fe3O4@HRP.

[0083] Figure 7 The XPS results of the nanocomposite enzyme material are shown. The figure shows that due to Zn... 2p Au 4f Fe 2p and O 1s The binding energies peaked at 1020, 81.3, 707, and 528 eV, respectively, proving the presence of Zn, Au, Fe, and O in the nanocomposite material, indicating the successful synthesis of the nanocomposite enzyme material.

[0084] Example 3

[0085] This embodiment provides a nanocomposite enzyme detection antibody, the preparation method of which includes:

[0086] Mix 100 μL of the 10 μg / mL first antibody with 1 mL of the nanocomposite enzyme material prepared in Example 2, shake and incubate for 12 h, add 100 μL of 1 mg / mL BSA for blocking for 1 h, wash with magnetic adsorption to obtain the nanocomposite enzyme detection antibody for later use.

[0087] Example 4

[0088] This embodiment provides a colorimetric immunosensor, comprising a second antibody and a nanocomposite enzyme detection antibody prepared in Example 5. The first antibody is a nanobody, and its amino acid sequence is SEQ NO.1. The second antibody is recombinant African swine fever p72 protein (product number: bs-41384R; English name: ASFV p72) produced by Beijing Bio-Sens Biotechnology Co., Ltd.

[0089] This embodiment also provides a method for detecting ASFV for non-therapeutic purposes, which uses the above-mentioned colorimetric immunoassay sensor, and the specific steps are as follows:

[0090] S1. Add 50 μL of a 1 μg / mL secondary antibody that specifically recognizes ASFV to the ELISA plate. o Incubate at C for 12 hours;

[0091] S2. After incubation, wash three times with 100 μL of 0.1 mol / L PBST at pH 7.2, and block with 50 μL of 1 mg / mL BSA for 30 min.

[0092] S3. After blocking, wash three times with 100 μL of 0.1 mol / L PBST at pH 7.2, and incubate with 50 μL of ASFV at different concentrations for 1 h.

[0093] S4. After incubation, wash the plate three times with 100 μL of 0.1 mol / L PBST (pH=7.2), add 50 μL of the nanocomposite enzyme detection antibody prepared in Example 3, incubate for 1 h, and then wash the plate.

[0094] S5. Add 200 μL of acetic acid buffer solution with pH=5.5, and add 50 μL of 10 mM TMB and 50 μL of 10 mM H2O2 for color development for 5 min. Measure the absorbance at OD652nm and plot the working curve.

[0095] Figure 8 -A shows the absorbance changes of different antigen concentrations. It can be seen that as the ASFV concentration increases, the absorbance signal detected at 652 nm increases, indicating a good linear relationship within the ASFV concentration range of 0.0005–2000 ng / mL. Based on the absorption peak changes at 652 nm using a UV spectrophotometer, the absorption peak values ​​for each concentration were taken for data analysis. A standard curve was constructed with the logarithm of the virus concentration as the X-axis and the absorbance value as the Y-axis. Figure 8 -B), the standard curve equation obtained is

[0096] Y = 0.0548 log[C] + 0.6322 (R) 2 = 0.9930)

[0097] In the formula, Y is the absorbance and C is the ASFV concentration (unit: ng / mL).

[0098] Experimental Example 1

[0099] This experiment tested the specificity of the colorimetric immunosensor using the detection method described in Example 4, replacing ASFV with bovine serum albumin (BSA), lysozyme, and Escherichia coli, respectively. E. coli Staphylococcus aureus ( S. aureus Na + Mg 2+ K + Ca 2+ Zn 2+ Cl - Porcine reproductive and respiratory syndrome virus (PRRSV), porcine epidemic diarrhea virus (PEDV), etc., test results are as follows Figure 9 As shown.

[0100] Depend on Figure 9 It can be seen that with the addition of BSA, lysozyme, E. coli , S. aureus Na + Mg 2+ K + Ca 2+ Zn 2+ Cl - No signal response was detected after PRRSV and PEDV. However, when ASFV standard was used or added to the mixture of the above samples, the colorimetric immunoassay sensor showed a significant signal response. This indicates that the sensor can specifically detect ASFV.

[0101] Experimental Example 2

[0102] This experimental example screens the conditions for ASFV detection methods, including the following steps:

[0103] 1) Optimal antigen incubation time

[0104] The detection method of Example 4 was used, with the ASFV antigen concentration fixed at 100 ng / mL and the volume at 50 μL. The incubation time in step S3 was set to 5 min, 10 min, 20 min, 30 min, 40 min, 50 min, 60 min, 70 min, 80 min, and 90 min, respectively. The final colorimetric results were compared. Figure 10 -A). As shown in the figure, the absorbance increases with the increase of incubation time, but no significant increase is observed after 60 min. Therefore, 60 min is selected as the optimal incubation time for the antigen.

[0105] 2) Detection of optimal antibody incubation time using nanocomposite enzymes

[0106] The detection method of Example 4 was used, with the ASFV antigen concentration fixed at 100 ng / mL and the volume at 50 μL. The incubation time in step S4 was set to 5 min, 10 min, 20 min, 30 min, 40 min, 50 min, 60 min, 70 min, 80 min, and 90 min, respectively. The final colorimetric results were compared. Figure 10 -B). As shown in the figure, the absorbance increases with the increase of incubation time, but no significant increase is observed after 50 min. Therefore, 50 min is selected as the optimal incubation time for the nanocomposite enzyme to detect the antibody.

[0107] 3) The optimal pH value of the colorimetric reaction buffer

[0108] The detection method of Example 4 was used, with the ASFV antigen concentration fixed at 100 ng / mL and the volume at 50 μL. The pH of the acetate buffer solution in step S5 was set to 3.5, 4, 4.5, 5, 5.5, 6, 6.5, and 7, respectively. The final colorimetric results were compared. Figure 10 -C). As shown in the graph, the absorbance first increases and then decreases with increasing pH, reaching its maximum at pH=5.5. Therefore, pH=5.5 is chosen as the optimal pH value for the acetate buffer solution in the colorimetric reaction.

[0109] 4) Optimal reaction time for colorimetric reaction

[0110] The detection method of Example 4 was used, with the ASFV antigen concentration fixed at 100 ng / mL and the volume at 50 μL. The colorimetric reaction time in step S5 was set to 0.5 min, 1 min, 2 min, 3 min, 4 min, 5 min, 6 min, and 7 min, respectively. The final colorimetric results were compared. Figure 10 -D). As can be seen from the figure, the absorbance increases with the increase of the color development reaction time, but decreases after 5 minutes. Therefore, 5 minutes is selected as the optimal reaction time for the color development reaction.

[0111] Experimental Example 3

[0112] This experiment tested the enzyme activity of ZIF-8@Fe3O4@HRP. The specific steps included:

[0113] H₂O (as a blank control), ZIF-8, Fe₃O₄, HRP, ZIF-8@Fe₃O₄, ZIF-8@HRP, Fe₃O₄@HRP, and ZIF-8@Fe₃O₄@HRP were selected as samples for enzyme activity investigation. 100 μL of the above sample (0.1 mg / mL), 300 μL of pH 5.5 acetate buffer, 50 μL of 10 mM TMB (3,3',5,5'-tetramethylbenzidine), and 50 μL of 10 mM H₂O₂ were weighed respectively for color development, and the results were measured using a UV spectrophotometer. The test results are shown below. Figure 11 As shown.

[0114] in, Figure 11 -A shows the UV spectra of each sample. As can be seen from the figure, there are significant differences in the absorption peak at 652 nm among the various samples. Comparing the absorption peaks of each sample at 652 nm yields... Figure 11-B. It can be seen that the blank control and ZIF-8 showed almost no absorption. The absorbance of ZIF-8@Fe3O4 and ZIF-8@HRP loaded with metal framework materials was significantly increased compared to unloaded Fe3O4 and HRP, indicating that the application of metal framework materials has a positive effect on enzyme activity. The enzyme activity of the combined Fe3O4@HRP sample was also significantly increased compared to Fe3O4 and HRP alone, indicating that the synergistic effect of the two enzymes does exist. The ZIF-8@Fe3O4@HRP sample, obtained by embedding the two enzymes in metal framework material, showed the most significant enzyme activity, far exceeding that of the other samples.

[0115] Test Example 4

[0116] This experiment tested the stability of ZIF-8@Fe3O4@HRP. The specific steps included:

[0117] The synthesized ZIF-8@Fe3O4@HRP was resuspended in 10 mL of 0.1 mg / mL liquid and placed at 4°C. o Store at C. Enzyme activity was measured on days 1, 2, 3, 4, 5, 6, 7, 15, 30, 60, 90, 120, 150, and 180. For each test, 100 μL of 0.1 mg / mL ZIF-8@Fe3O4@HRP, 300 μL of pH 5.5 acetate buffer, 50 μL of 10 mM TMB (3,3',5,5'-tetramethylbenzidine), and 50 μL of 10 mM H2O2 were used for color development, and the results were measured using a UV spectrophotometer. The test results are shown below. Figure 12 As shown.

[0118] Depend on Figure 12 It can be seen that the enzyme activity of ZIF-8@Fe3O4@HRP remained almost unchanged during the 120-day storage period, and only slightly decreased after 180 days of storage, which fully demonstrates that ZIF-8@Fe3O4@HRP has high stability.

[0119] Experimental Example 5

[0120] This experiment tested the high-temperature resistance of ZIF-8@Fe3O4@HRP. The specific steps included:

[0121] Equal amounts of ZIF-8@Fe3O4@HRP and HRP were placed in 30 °C. o C, 40 o C, 50 o C, 60 o C, 70 o C, 80 o C, 90 oC, 100 o Enzyme activity was measured after 10 min at C. 100 μL of 0.1 mg / mL ZIF-8@Fe3O4@HRP or HRP, 300 μL of pH 5.5 acetate buffer, 50 μL of 10 mM TMB (3,3',5,5'-tetramethylbenzidine), and 50 μL of 10 mM H2O2 were used for color development. The results were measured using a UV spectrophotometer, and the remaining enzyme activity was calculated. The test results are shown below. Figure 13 As shown.

[0122] It should be noted that, in order to more clearly demonstrate enzyme activity, Figure 13 The ordinate represents the relative value of enzyme activity, i.e., 30. o The enzyme activity at temperature C is 100%. Calculate the relative values ​​of enzyme activity at each temperature. As can be seen from the graph, after 60... o After high temperature (C), HRP retained only 26.7% of its activity, while ZIF-8@Fe3O4@HRP retained 91.36% of its activity, even at 100°C. o After high-temperature treatment, ZIF-8@Fe3O4@HRP still retained 68.66% of its enzyme activity, while HRP was almost completely inactivated. This fully demonstrates that the metal framework material provides better protection for the enzyme embedded within it, reducing the impact of high temperatures.

[0123] Experimental Example 6

[0124] This experiment tested the low-temperature tolerance of ZIF-8@Fe3O4@HRP. The specific steps included:

[0125] Equal amounts of ZIF-8@Fe3O4@HRP and HRP were repeatedly freeze-thawed in liquid nitrogen 20 times before enzyme activity assays were performed. 100 μL of 0.1 mg / mL ZIF-8@Fe3O4@HRP or HRP, 300 μL of pH 5.5 acetate buffer, 50 μL of 10 mM TMB (3,3',5,5'-tetramethylbenzidine), and 50 μL of 10 mM H2O2 were used for color development, and the results were measured using a UV spectrophotometer. The remaining enzyme activity was then calculated. The test results are shown below. Figure 14 As shown.

[0126] Similarly, Figure 14The ordinate of the graph represents the relative value of enzyme activity, with the enzyme activity before liquid nitrogen freeze-thaw cycles set at 100%. As shown in the graph, the enzyme activity of HRP continuously decreased during repeated freeze-thaw cycles, reaching only 9.72% after 20 cycles. While the enzyme activity of ZIF-8@Fe3O4@HRP also showed an overall downward trend, the decrease was accompanied by fluctuations, and the magnitude of the decrease was significantly less than that of HRP. ZIF-8, acting as a framework material, loaded and immobilized the HRP, thus protecting it.

[0127] In summary, this invention provides a nanocomposite enzyme material and its preparation method, which combines natural enzymes with nanozymes and loads them into a metal-organic framework (MOF) material. The MOF material enhances the loading capacity, enzyme activity, and stability. Furthermore, this invention also provides detection antibodies and colorimetric immunosensors prepared using the aforementioned nanocomposite enzyme material, along with corresponding detection methods. Utilizing the unique properties of the nanocomposite enzyme material, the detection capability for antigens is effectively improved. Direct antigen recognition is achieved without the need for nucleic acid extraction and amplification, making it a reliable, inexpensive, highly sensitive, and high-throughput antigen detection method.

[0128] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A detection antibody loaded with a nanocomposite enzyme, characterized in that, The detection antibody loaded on the nanocomposite enzyme is obtained by loading a first antibody onto the surface of the nanocomposite enzyme material; the amino acid sequence of the first antibody is shown in SEQ ID NO.

1. The first antibody specifically binds to African swine fever virus; The nanocomposite enzyme material is AuNPs@ZIF-8@Fe3O4@HRP, which includes a metal-organic framework material ZIF-8, Fe3O4 and HRP loaded inside the ZIF-8, and AuNPs distributed on the surface of the ZIF-8. The preparation method of nanocomposite enzyme materials includes the following steps: ZIF-8@Fe3O4@HRP was mixed with AuNPs at 0~10℃.

2. The detection antibody loaded with the nanocomposite enzyme according to claim 1, characterized in that, Based on the mass of ZIF-8@Fe3O4@HRP, the molar amount of AuNPs is 0.5~5 mmol / g.

3. The detection antibody loaded with the nanocomposite enzyme according to claim 2, characterized in that, The preparation method of the ZIF-8@Fe3O4@HRP includes: The Fe3O4, HRP, Zn(NO3)2·6H2O, and 2-methylimidazole were mixed at room temperature, centrifuged, and then freeze-dried; the mass ratio of Fe3O4 to HRP was 1:0.2~1.

4. The detection antibody loaded with the nanocomposite enzyme according to claim 3, characterized in that, The preparation method of the AuNPs includes: mixing and reacting HAuCl4 and sodium citrate at 100~120℃ for 20~40 min; the molar ratio of HAuCl4 and sodium citrate is 1:4~10; The increase in absorbance signal detected at 652 nm indicates a good linear relationship within the ASFV concentration range of 0.0005 to 2000 ng / mL.

5. A colorimetric immunosensor, characterized in that, It includes a second antibody and a detection antibody loaded with a nanocomposite enzyme as described in claim 1, wherein the first antibody and the second antibody specifically bind to different epitopes of the same antigen.

6. The colorimetric immunoassay sensor according to claim 5, characterized in that, The second antibody specifically binds to African swine fever virus, and the first antibody and the second antibody specifically bind to different epitopes of African swine fever virus.

7. A method for detecting African swine fever virus for purposes other than disease diagnosis and treatment, characterized in that, The colorimetric immunosensor according to claim 5 or 6 comprises: Add the second antibody to the ELISA plate for the first incubation; After the first incubation, the cells were sealed with BSA. After sealing, the antigen is added for a second incubation; After the second incubation, the detection antibody loaded with the nanocomposite enzyme is added for a third incubation. After the third incubation, a buffer solution was added, and TMB and H2O2 were used for color development.

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