Amorphous MOF-mediated cascade antibody directed probe, and preparation method and application thereof

Abstract

The application relates to the technical field of immunodetection, in particular to a non-crystalline MOF-mediated cascade antibody directional probe and a preparation method and application thereof. The application specifically comprises the following steps: (1) mixing a second antibody solution with a coomassie brilliant blue solution to obtain a second antibody staining solution; (2) mixing a Zn(OAc)2.2H2O solution, an HmIM solution and the second antibody staining solution to obtain aR-ZIF-Ab2 nanocomposite material; and (3) mixing the aR-ZIF-Ab2 nanocomposite material with a first antibody and incubating to obtain aR-ZIF-Ab2-mAb1 cascade antibody directional probe. The CAbD strategy based on aMOF can be used as a promising antibody directional and probe construction means in immunochromatographic analysis, and provides a reference innovation direction for the design and development of an immunobiosensor.

Description

Technical Field

[0001] This invention relates to the field of immunoassay technology, and in particular to an amorphous MOF-mediated cascade antibody-directed probe, its preparation method, and its application. Background Technology

[0002] Lateral flow immunochromatography (LFIA) has wide applications in on-site detection for food safety, environmental monitoring, and medical diagnosis. Nanomaterials are a key component of traditional immunochromatographic techniques, and the effective binding of antibodies (Abs) to nanomaterials not only significantly affects the effectiveness of the formed probe but also greatly dominates the analytical performance of LFIA. In the immunochromatographic analysis of small molecule compounds, recent research has focused on the binding of nanomaterials and antibodies using different targeting techniques, such as chemically mediated, protein-mediated, and boric acid-mediated antibody targeting methods. Most of these targeting methods are based on binding to the Fc fragment of Abs, thereby exposing the Fab fragment of the antibody to achieve adequate antigen-antibody recognition. To a certain extent, these techniques help improve antigen-antibody binding efficiency, thus facilitating the establishment of subsequent immunological methods. However, due to steric hindrance and the occupancy of antibody active sites, some chemically mediated targeting operations may reduce antibody activity. Furthermore, traditional Ab targeting techniques often only focus on the targeting effect, neglecting the important parameter of Ab dosage.

[0003] In biosynthetic nanoscience, crystal formation is precisely controlled by a biomineralization process, in which biomolecules mediate highly ordered structures through nanocrystal nucleation and growth mechanisms. Metal-organic frameworks (MOFs) are excellent protein carriers, ensuring the bioactivity and stability of proteins. Therefore, the "protein-MOF" structure is the preferred structure for catalysts, biosensors, and delivery systems. To date, the most widely studied MOF for these applications is the zeolite imidazole salt framework-8 (ZIF-8). It is a Zn... 2+ The three-dimensional network structure composed of nodes, linked together by HmIM ligands, can be synthesized within minutes at room temperature in aqueous solution. This mild manufacturing technique explains the high activity retention of the loaded protein. Immunoglobulin G (IgG) antibodies, as a unique class of proteins, exhibit different surface chemistry in the Fab and Fc regions. Studies have demonstrated that the ZIF-C structure formed by local growth of the Fc region ensures that the Fab region protrudes from the surface of the MOF material, allowing the Fab region to freely bind its specific antigen. Specifically, Zn... 2+ The focus is on the negatively charged functional groups on the surface of the MOF structure (the Fc segment of IgG antibodies), rather than on the positively charged enrichment sites (the Fab segment). This study lays the foundation for the development of targeted antibody anchoring to MOFs. Summary of the Invention

[0004] The purpose of this invention is to provide a method for preparing amorphous MOF-mediated cascade antibody-directed probes, comprising the following steps:

[0005] (1) Mix the second antibody solution with Coomassie brilliant blue solution for staining to obtain the second antibody staining solution;

[0006] (2) The Zn(OAc)2·2H2O solution, HmIM solution and the second antibody staining solution were mixed and reacted to obtain aR-ZIF-Ab2 nanocomposite material;

[0007] (3) The aR-ZIF-Ab2 nanocomposite material was mixed with the first antibody and incubated to obtain the aR-ZIF-Ab2-mAb1 cascade antibody directional probe.

[0008] Preferably, the concentration of the second antibody solution in step (1) is 4–6 mg / mL. -1 The concentration of the Coomassie brilliant blue solution is 1.5–2.5 g / 100 mL; the volume ratio of the second antibody solution to the Coomassie brilliant blue solution is 2:3–7; and the staining time is 25–35 min.

[0009] Preferably, the concentration of the Zn(OAc)2·2H2O solution in step (2) is 0.4 to 0.6 mol / L; and the concentration of the HmIM solution is 1 to 2 mol / L.

[0010] Preferably, the volume ratio of the Zn(OAc)2·2H2O solution, HmIM solution, and second antibody staining solution in step (2) is 72-92:16-20:5-15.

[0011] Preferably, the reaction time in step (2) is 15 to 25 minutes; after the reaction is completed, the reaction solution is further separated and sealed, wherein the separation is centrifugal separation; the centrifugation speed is 4000 to 6000 rpm, the centrifugation time is 4 to 6 minutes, and the sealing time is 25 to 35 minutes.

[0012] Preferably, the volume-to-mass ratio of the aR-ZIF-Ab2 nanocomposite material to the first antibody in step (3) is 90-110 μL: 0.5-1.5 g; the first antibody is a monoclonal antibody against CPAOZ or NPSEM.

[0013] The present invention also provides an amorphous MOF-mediated cascade antibody-directed probe prepared by the aforementioned preparation method.

[0014] The present invention also provides a cascade antibody-directed probe obtained by the preparation method described above, or the application of the cascade antibody-directed probe in food detection.

[0015] Preferably, the cascaded antibody-directed probe is used to detect furazolidone metabolites or furazolidone metabolites in food.

[0016] Preferably, the method for detecting furazolidone metabolites or furazolidone metabolites in food is lateral flow immunochromatography; the food is fish or honey.

[0017] In this invention, Ab2 is a goat anti-mouse second antibody.

[0018] Compared with the prior art, the present invention has the following beneficial effects:

[0019] 1. This invention provides a cascade antibody targeting (CAbD) strategy, which, when applied to immunochromatographic analysis, demonstrates its potential in detecting nitrofuran metabolites. Since antibodies are proteins that can act as antigens and bind to secondary antibodies, constructing a targeting strategy mediated by the secondary antibody (Ab2) can significantly improve the activity of the detection antibody (mAb1) and reduce its consumption. Given the typically high difficulty and cost of screening detection antibodies, the establishment of this immunochromatographic method is of great significance.

[0020] Figure 1 A demonstrates the innovative principle of the CAbD strategy. First, goat anti-mouse secondary antibody (Ab2) is stained with Coomassie Brilliant Blue R-250. Then, the antibody-directed anchoring effect of ZIF is used to obtain the aR-ZIF-Ab2 signal tag. Finally, it is mixed with a monoclonal antibody against the target analyte (mAb1), and the mAb1 is effectively directed through the already anchored Ab2. This differs from direct-directed anchoring of mAb1 (…). Figure 1 C) and post-adsorbed mAb1 ( Figure 1 Compared to the fixed strategy of D), the CAbD strategy ( Figure 1 B) It can reduce the consumption of analyte mAb1 and speed up the construction time of the LFIA system, thereby improving the competitive efficiency of fixed antigen and free antigen against mAb1, and thus improving detection sensitivity.

[0021] Furthermore, the aR-ZIF-Ab2 signal tag is synthesized under mild conditions (room temperature) and uses a simple solvent (only water is required), thus having no effect on the activity of biomolecules (such as proteins, antibodies, etc.). To demonstrate the feasibility and innovation of this antibody-directed strategy, the CAbD strategy was applied to an immunochromatographic system for the simultaneous detection of furazolidone metabolites (SEM) and furazolidone metabolites (AOZ) in food samples, achieving satisfactory sensitivity and recovery.

[0022] Therefore, the CAbD strategy based on aMOF proposed in this invention can serve as a promising method for antibody targeting and probe construction in immunochromatographic analysis, providing a reference for the design and development of immunobiosensors.

[0023] 2. The CAbD strategy provided by this invention innovatively integrates three elements: staining goat anti-mouse secondary antibody (Ab2) with Coomassie Brilliant Blue R-250, directional anchoring of the secondary antibody (Ab2) with an amorphous metal-organic framework (aMOF), and further directional orientation of the detection antibody (mAb1) using the already directionally anchored Ab2. aMOF has a dual function of antibody orientation and signal display. Due to the difference in surface charge properties between the Fc and Fab fragments in immunoglobulin G, excess R-250-stained Ab2 is anchored within an imidazole salt framework (ZIF), forming a colorimetric tag with a significant signal intensity. Compared to the direct directional anchoring or post-adsorption of mAb1 by ZIF, this strategy reduces mAb1 consumption, better preserves mAb1 activity and binding efficiency, and shortens the construction time of the immunochromatographic system. As expected, the CAbD-based immunochromatographic system exhibited satisfactory performance, with visualization sensitivities of 0.22 ng / mL for nitrofurazone and 0.18 ng / mL for furazolidone metabolites. -1 Since the first antibody targeting in the CAbD strategy is Ab2, and this goat anti-mouse secondary antibody can bind to all murine antibodies, this aMOF-based cascade antibody targeting strategy can serve as a universal, non-invasive antibody targeting method, especially with significant advantages in ultrasensitive multiplex immunoassays for food safety monitoring. Attached Figure Description

[0024] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on the provided drawings without creative effort.

[0025] Figure 1 This is a schematic diagram of the probe preparation process; where A represents a schematic diagram of the probe preparation process based on CAbD, B represents the LFIA principle based on CAbD, C represents LFIA based on direct orientation of mAb1, and D represents LFA based on post-adsorption of mAb1.

[0026] Figure 2 Comparison of key parameters in probe preparation for different antibody targeting strategies.

[0027] Figure 3Analysis results of AOZ and SEM standard solutions based on the CAbD directional scheme LFIA.

[0028] Figure 4 Analysis results of AOZ standard solution based on direct orientation scheme (A) and post-adsorption orientation scheme (B) LFIA.

[0029] Figure 5 The results of standard addition detection of AOZ and SEM in fish and honey samples based on the CAbD directional protocol LFIA. Detailed Implementation

[0030] This invention provides a method for preparing amorphous MOF-mediated cascade antibody-directed probes, comprising the following steps:

[0031] (1) Mix the second antibody solution with Coomassie brilliant blue solution for staining to obtain the second antibody staining solution;

[0032] (2) The Zn(OAc)2·2H2O solution, HmIM solution and the second antibody staining solution were mixed and reacted to obtain aR-ZIF-Ab2 nanocomposite material;

[0033] (3) The aR-ZIF-Ab2 nanocomposite material was mixed with the first antibody and incubated to obtain the aR-ZIF-Ab2-mAb1 cascade antibody directional probe.

[0034] In this invention, the concentration of the Coomassie Brilliant Blue solution in step (1) is 1.5–2.5 g / 100 mL; preferably 1.7–2.3 g / 100 mL; more preferably 1.9–2.1 g / 100 mL; and even more preferably 2 g / 100 mL.

[0035] In this invention, the Coomassie Brilliant Blue is preferably Coomassie Brilliant Blue R-250.

[0036] In this invention, the concentration of the second antibody solution in step (1) is 4–6 mg / mL. -1 Preferably 5 mg·mL -1 .

[0037] In this invention, the volume ratio of the second antibody solution to the Coomassie brilliant blue solution in step (1) is 2:3 to 7; preferably 2:4 to 6; and more preferably 2:5.

[0038] In this invention, the staining in step (1) involves mixing the second antibody solution with Coomassie brilliant blue solution and allowing it to stand for 25 to 35 minutes; preferably 27 to 33 minutes; more preferably 29 to 31 minutes; and even more preferably 30 minutes.

[0039] In this invention, the concentration of the Zn(OAc)2·2H2O solution in step (2) is 0.4 to 0.6 mol / L, preferably 0.5 mol / L.

[0040] In this invention, the concentration of the HmIM solution in step (2) is 1-2 mol / L; preferably 1.5 mol / L.

[0041] In this invention, the volume ratio of the Zn(OAc)2·2H2O solution, HmIM solution and the second antibody staining solution in step (2) is 72-92:16-20:5-15; preferably 76-88:17-19:7-13; further preferably 80-84:18:9-11; and more preferably 82:18:10.

[0042] In this invention, the reaction time in step (2) is 15 to 25 minutes; preferably 17 to 23 minutes; more preferably 19 to 21 minutes; and more preferably 20 minutes.

[0043] In this invention, after the reaction in step (2) is completed, the process of separating and sealing the solution after the reaction is also included. The separation is centrifugal separation to remove unreacted ions.

[0044] In this invention, the centrifugation speed is 4000-6000 rpm and the centrifugation time is 4-6 min; preferably, the centrifugation speed is 5000 rpm and the centrifugation time is 5 min.

[0045] In this invention, the sealing is achieved by resuspending the centrifuged precipitate in a 1-3% BSA solution, preferably a 2% BSA solution, and then sealing it; the sealing time is 25-35 min; preferably 27-33 min; more preferably 29-31 min; and even more preferably 30 min.

[0046] In this invention, after the sealing and binding process, a washing step with deionized water is included to remove excess CBB R-250. The deionized water washing is preferably performed three times until the supernatant becomes clear.

[0047] In this invention, the volume-to-mass ratio of the aR-ZIF-Ab2 nanocomposite material to the first antibody in step (3) is 90-110 μL: 0.5-1.5 g; preferably 94-106 μL: 1 g; more preferably 98-102 μL: 1 g; and even more preferably 100 μL: 1 g.

[0048] In this invention, the first antibody in step (3) is a monoclonal antibody against CPAOZ or NPSEM.

[0049] This invention also provides an amorphous MOF-mediated cascade antibody targeting probe prepared by the aforementioned method. The probe is preferably stored at 4°C for later use.

[0050] The present invention also provides a cascade antibody-directed probe obtained by the preparation method described above, or the application of the cascade antibody-directed probe in food detection.

[0051] In this invention, the cascaded antibody-directed probe is used to detect furazolidone metabolites or furazolidone metabolites in food.

[0052] In this invention, the method for detecting furazolidone metabolites or furazolidone metabolites in food is lateral flow immunochromatography; the food is fish or honey; preferably fish; more preferably pomfret.

[0053] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0054] Example 1

[0055] A method for preparing an amorphous MOF-mediated cascade antibody-directed probe, comprising the following steps:

[0056] (1) Dispense 4 mg / mL of the solution separately. -1 The second antibody solutions of CPAOZ and NPSEM were thoroughly mixed with Coomassie Brilliant Blue solution at a volume ratio of 2:7 and allowed to stand for 25 minutes to obtain two antigen Ab2 staining solutions.

[0057] (2) Mix 0.4M Zn(OAc)2·2H2O solution, 1M HmIM solution and Ab2 staining solutions of the two antigens prepared in step (1) at a volume ratio of 72:16:1 and react for 15 min. Then centrifuge (4000 rpm for 4 min) to obtain a precipitate, remove unreacted ions, then resuspend the precipitate with 1.5% BSA and block for 25 min. Wash the blocked precipitate twice with deionized water until the supernatant is clear to obtain aR-ZIF-Ab2 of the two antigens.

[0058] (3) The two aR-ZIF-Ab2 obtained in step (2) correspond to their respective anti-CPAOZ and NPSEM monoclonal antibodies (mAb1). AOZ mAb1 SEM The mixtures were incubated at volume ratios of 90:0.5 and 90:0.5 to obtain aR-ZIF-Ab2-mAb1(aR-ZIF-Ab2-mAb1). AOZ and aR-ZIF-Ab2-mAb1 SEMStore at 4°C for further use.

[0059] Example 2

[0060] A method for preparing an amorphous MOF-mediated cascade antibody-directed probe, comprising the following steps:

[0061] (1) Dispense 6 mg / mL of the solution separately. -1 The second antibody Ab2 solution of CPAOZ and NPSEM was thoroughly mixed with Coomassie Brilliant Blue solution at a volume ratio of 2:3 and allowed to stand for 35 min to obtain two antigen Ab2 staining solutions.

[0062] (2) Mix 0.6MZn(OAc)2·2H2O solution, 2MMmI solution and Ab2 staining solutions of the two antigens prepared in step (1) at a volume ratio of 92:20:10 and react for 25 min. Then centrifuge (6000 rpm for 6 min) to obtain a precipitate, remove unreacted ions, then resuspend the precipitate with 2.5% BSA and block for 35 min. Wash the blocked precipitate with deionized water 4 times until the supernatant is clear to obtain aR-ZIF-Ab2 of the two antigens.

[0063] (3) The two aR-ZIF-Ab2 obtained in step (2) correspond to their respective anti-CPAOZ and NPSEM monoclonal antibodies (mAb1). AOZ mAb1 SEM The mixture was incubated at a ratio of 110 μL:1.5 μg and 110 μL:1.5 μg to obtain aR-ZIF-Ab2-mAb1(aR-ZIF-Ab2-mAb1). AOZ and aR-ZIF-Ab2-mAb1 SEM Store at 4°C for further use.

[0064] Example 3

[0065] A method for preparing an amorphous MOF-mediated cascade antibody-directed probe, comprising the following steps:

[0066] (1) 5 mg / mL -1 Goat anti-mouse AOZ and SEM secondary antibody Ab2 solution were thoroughly mixed with 2g / 100mL Coomassie brilliant blue solution at a volume ratio of 2:5 and allowed to stand for 30min to obtain the stained Ab2 solution.

[0067] (2) Mix 0.5M Zn(OAc)2·2H2O solution, 1.5M HmIM solution and Ab2 staining solutions of the two antigens prepared in step (1) at a volume ratio of 82:18:7 and react for 20 min. Then centrifuge (5000 rpm for 5 min) to obtain a precipitate, remove unreacted ions, then resuspend the precipitate with 2% BSA and block for 30 min. Wash the blocked precipitate with deionized water 3 times until the supernatant is clear to obtain aR-ZIF-Ab2 of the two antigens.

[0068] (3) The two aR-ZIF-Ab2 obtained in step (2) correspond to their respective anti-CPAOZ and NPSEM monoclonal antibodies (mAb1). AOZ mAb1 SEM The mixture was incubated at volume ratios of 100 μL:1 μg and 100 μL:1.3 μg to obtain aR-ZIF-Ab2-mAb1(aR-ZIF-Ab2-mAb1). AOZ and aR-ZIF-Ab2-mAb1 SEM Store at 4°C for further use.

[0069] Comparative Example 1

[0070] Preparation of probe (aR-ZIF-mAb1) for direct targeted detection of antibodies based on ZIF-8

[0071] The other methods are exactly the same as in Example 3, except that Ab2 in step (1) of Example 3 is replaced with mAb1. AOZ ), to obtain aR-ZIF-mAb1 AOZ .

[0072] Comparative Example 2

[0073] Preparation of probe (aR-ZIF@mAb1) based on post-adsorption detection antibody

[0074] (1) Loading Coomassie Brilliant Blue R-250 onto ZIF: Mix 10 μL of 2% (g / 100 mL) Coomassie Brilliant Blue R-250 solution, 82 μL of 0.5 mol / L Zn(OAc)₂·2H₂O and 18 μL of 1.5 mol / L HmIM and react for 20 min. After centrifugation (5000 rpm, 10 min) to remove the supernatant, add 2% BSA to block for 30 min. Then wash the solution with deionized water until the supernatant becomes colorless, and resuspend the precipitate in deionized water to 1 mL.

[0075] (2) Adsorb mAb1 onto the stained ZIF: Take 100 μL of the above resuspension and adsorb it onto the detection antibody mAb1. AOZMix 8 μL thoroughly, let stand for 2 hours, and then centrifuge to 100 μL to remove unbound mAb1.

[0076] Experimental Example 1

[0077] Take several 50mL centrifuge tubes, and fill each tube with 4mL of ultrapure water. Then, add different concentrations of AOZ (0, 0.045, 0.09, 0.18, 0.22, 0.26, 0.3, 0.35, 0.45, and 0.55 ng / mL) to these tubes respectively. -1 ) or SEM (0, 0.05, 0.1, 0.15, 0.2, 0.25, 0.3, 0.35, 0.45, 0.5 ng mL) -1 The standard solutions were prepared and thoroughly vortexed at room temperature. Next, 0.5 mL of 1.0 M HCl was added to both groups. For SEM derivatization, 100 mL of 40 mM 2-NBA dissolved in DMF was added to the SEM group, and for AOZ derivatization, 100 mL of 40 mM 4-CBA dissolved in DMSO was added to the AOZ group. These mixtures were thoroughly mixed by vortexing and shaken at 37 °C for 16 hours. After derivatization, 5 mL of 0.1 M Na₂HPO₄ solution, 0.4 mL of 1.0 M NaOH solution, and 6 mL of ethyl acetate were added to both groups sequentially. After thorough vortexing, the mixtures were centrifuged at 5000 rpm for 10 minutes, and the upper organic phase was extracted into 15 mL centrifuge tubes. The extracted ethyl acetate supernatant was dried over N₂ at 45 °C. Finally, add 2 mL of a mixed solution (hexane: 0.1 M PBS, V:V = 1:1) to dissolve the contents, then transfer the solution to a 2 mL centrifuge tube and centrifuge at 12000 rpm for 10 minutes. After centrifugation, store the bottom aqueous phase at 4°C for later use.

[0078] 1. Comparison of different antibody fixation strategies

[0079] Preparation of immersion immunochromatographic test strips: These strips consist of three parts: a nitrocellulose membrane (NC membrane), a sample pad, and an absorbent pad. The immunochromatographic test strips are prepared by orderly assembling the NC membrane, sample pad, and absorbent pad on a PVC plate.

[0080] Specifically, the sample pad was soaked in a blocking buffer containing 2% BSA, 1% PVP-K30, and 0.5% Tween 20, using 2 mg / mL... -1 Goat anti-mouse IgG PBS solution (10 mM, pH 7.4), 0.35 mg / mL -1 CPSEM-BSAPBS solution, 0.25 mg / mL -1 The CPAOZ-BSAPBS solution was prepared at 0.5 μL / cm³. -1The sample was sprayed onto the NC membrane at a certain rate to serve as the control line (C line), detection line 1 (T1), and detection line 2 (T2). It was then dried at 37°C for 5 hours. After drying, each component was adhered to a PVC board (connection order: NC membrane, sample pad, absorbent pad), with an overlap of approximately 2.5 mm between adjacent components. Then, it was cut into 3 mm wide test strips using a strip cutter. (In this invention, the test strips for the dual-detection section have two detection lines: SEM and AOZ. In the comparative experiment, this invention uses AOZ as an example (the operation for SEM and AOZ is exactly the same), illustrating the advantages of the Ab2 fixation method in terms of antibody dosage and performance compared to direct AOZ antibody targeting and random AOZ antibody adsorption.)

[0081] A novel probe, aR-ZIF-Ab2-mAb1, was prepared using a CAbD cascade antibody targeting strategy. For ease of comparison, it was labeled Probe-C0 (the probe prepared in Example 3). To demonstrate the superiority of the CAbD strategy, the anti-AOZ monoclonal antibody (mAb1) was directly targeted in the same manner as CAbD to form the aR-ZIF-mAb1 nanoprobe, which served as comparative probe 1 and was labeled Probe-C1 (the probe prepared in Comparative Example 1). An aR-ZIF@mAb1 probe was prepared by first staining ZIF and then adsorbing mAb1; this probe served as comparative probe 2 and was labeled Probe-C2 (the probe prepared in Comparative Example 2).

[0082] Probes formed using three different antibody immobilization strategies were applied to the CPAOZ immunochromatographic analysis system, and the conditions for each system were optimized. The optimized experimental conditions for the probe obtained in Example 3 were: aR-ZIF-Ab2 concentration, mAb1 dosage, and probe dosage. The optimized conditions for the probes obtained in Comparative Examples 1 and 2 were: mAbAOZ dosage and probe dosage.

[0083] Under optimal conditions, such as Figure 2 As shown ( Figure 2 In the CAbD-based cascade antibody targeting strategy, Probe-C0 represents the consumption of each antibody and the probe construction time. Probe-C1 corresponds to directional mAbAOZ, and Probe-C2 corresponds to randomly adsorbed mAbAOZ. In CAbD-based LFIA, direct directional anchoring LFIA, and post-adsorption LFIA, the consumption of mAb1 per test strip is 0.07 μg, 0.30 μg, and 1.11 μg, respectively. Therefore, the mAb1 consumption of CPAOZ in the direct directional anchoring and post-adsorption strategies is 4.28 times and 15.85 times that in the CAbD strategy, respectively. More importantly, convenience and efficiency are also considered important evaluation indicators in the construction of LFIA systems. Detection time mainly depends on the synthesis time of the detection probe. Figure 2As shown, for immunochromatographic assay systems, CAbD-based LFIA requires only one mAb1 labeling process (80 min) because the pre-anchored Ab2 can bind to any murine antibody, regardless of whether there are one or more target analytes. In contrast, the direct-directed anchoring method requires a probe construction time that increases exponentially with the number of target analytes; for example, with two target analytes, it requires 160 min (80 min × 2). The post-adsorption-directed method is even more time-consuming, requiring another labeling step after signal labeling preparation, totaling 290 min. For competitive LFIA, the amount of mAb1 used is directly related to its sensitivity.

[0084] Probes formed by three antibody immobilization strategies were applied to CPAOZ and NPSEM immunochromatographic analysis systems, respectively.

[0085] The optimal detection conditions are: for SEM detection, the aR-ZIF-Ab2 concentration is 12 mg (100 μL). -1 The amount of mAbSEM added to 100 μL of aR-ZIF-Ab2 was 1.25 μg, and the amount of probeSEM was 9 μL. When detecting AOZ, the concentration of aR-ZIF-Ab2 was 18 mg (100 μL). -1 The amount of mAbAOZ added was 1 μg, and the amount of probeAOZ added was 7 μL.

[0086] Under optimal conditions, the visual limit of detection (vLOD) for AOZ using the CAbD strategy-based LFIA is 0.18 ng / mL. (T1 is the SEM detection line, i.e., TSEM; T2 is the AOZ detection line, i.e., TAOZ) -1 The concentration of the test strip with the disappearing line was 0.55 ng / mL. -1 ( Figure 3 LFIA based on CAbD strategy. In the direct-directed anchoring strategy, the visual detection limit for AOZ is 0.2 ng / mL. -1 The concentration at which the test strip disappears is 0.7 ng / mL. -1 ( Figure 4 A) In the post-adsorption strategy, the vLOD for AOZ is 0.40 ng / mL. -1 The concentration of the disinfectant was 1.2 ng / mL. -1 ( Figure 4 B). In colorimetric analysis of test strips, the concentration at which the line disappears represents the analytical sensitivity. Therefore, the LFIA sensitivity based on CAbD is 1.3 times and 2.2 times that based on direct directional anchoring LFIA and post-adsorption LFIA, respectively.

[0087] Experiment Example 2

[0088] Application of furacilin and furazolidone metabolites in food samples

[0089] To verify the application performance of the CAbD antibody targeting strategy, fish and honey were selected as food matrices (processed as in Experiment 1, using the probe obtained by the method in Example 3), and 0.3 μg / kg of each were added to them. -1 AOZ and SEM standard solutions.

[0090] like Figure 5 As shown, for SEM detection, the limit of detection for visualization in fish samples is 0.1 μg·kg. -1 The concentration at which SEM eliminated the test strip was 0.35 μg / kg. -1 The limit of detection for SEM visualization in honey samples was 0.05 μg / kg. -1 The concentration for SEM sterilization was also 0.5 μg / kg. -1 Furthermore, at concentrations of 0.15, 0.2, and 0.3 μg·kg⁻¹, [further details needed]. -1 At the specified concentrations, the recoveries of SEM in fish were 101.51%, 109.77%, and 91.05%, respectively, while those in honey were 88.18%, 96.17%, and 81.52%, respectively. The coefficients of variation (CV) for both were less than 9.71% (range 2.31%–9.71%). For AOZ detection, the limit of detection (LOD) in fish samples was 0.15 μg / kg. -1 The AOZ concentration for eliminating the test strip is 0.5 μg / kg. -1 The visual detection limit of AOZ in honey samples was 0.07 μg·kg⁻¹. -1 The concentration of AOZ at the elimination line is also 0.5 μg / kg. -1 Furthermore, at concentrations of 0.15, 0.2, and 0.3 μg·kg⁻¹, [further details needed]. -1 At the specified concentrations, the recoveries of AOZ in fish were 85.06%, 104.5%, and 101.15%, respectively, while those in honey were 87.53%, 118.82%, and 84.56%, respectively, with coefficients of variation (CV) less than 8.86% (1.78%–8.86%). The slight differences in detection limits across different samples are largely due to the different food matrices in which the target analyte is contained. Therefore, the amorphous MOF-mediated cascade antibody-directed LFIA proposed in this invention can be applied to complex food matrices, exhibiting good applicability and accuracy.

[0091] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for preparing an amorphous MOF mediated cascade antibody directed probe, comprising: The method comprises the following steps: (1) mixing and dyeing the second antibody solution with the coomassie brilliant blue solution to obtain a second antibody dyeing solution; (2) mixing and reacting the Zn(OAc)2·2H2O solution, the HmIM solution and the second antibody dyeing solution to obtain aR-ZIF-Ab2 nanocomposite; (3) mixing and incubating the aR-ZIF-Ab2 nanocomposite with the first antibody to obtain aR-ZIF-Ab2-mAb1 cascade antibody directional probe; The concentration of the second antibody solution in step (1) is 4-6 mg / mL -1 ; the concentration of the Coomassie brilliant blue solution is 1.5-2.5 g / 100 mL; the volume ratio of the second antibody solution to the Coomassie brilliant blue solution is 2:3-7; and the staining time is 25-35 min. In step (2), the concentration of the Zn(OAc)2·2H2O solution is 0.4-0.6 mol / L; and the concentration of the HmIM solution is 1-2 mol / L. In step (2), the volume ratio of the Zn(OAc)2·2H2O solution, the HmIM solution and the second antibody dyeing solution is 72-92:16-20:5-15. In step (2), the reaction time is 15-25 min; after the reaction, the solution after the reaction is separated and sealed, the separation is centrifugal separation, the centrifugal speed is 4000-6000 rpm, the centrifugal time is 4-6 min, and the sealing time is 25-35 min. In step (3), the volume / mass ratio of the aR-ZIF-Ab2 nanocomposite to the first antibody is 90-110 μL:0.5-1.5 g; and the first antibody is a monoclonal antibody against CPAOZ or NPSEM.

2. The non-crystalline MOF-mediated cascade antibody directional probe prepared by the preparation method of claim 1.

3. The application of the cascade antibody directional probe of claim 2 in food detection.

4. Use according to claim 3, characterized in that, The application of the cascade antibody directional probe in detecting furanocillin metabolites or furazolidone metabolites in food.

5. Use according to claim 4, characterized in that, The method for detecting furanocillin metabolites or furazolidone metabolites in food is lateral flow immunochromatography technology; and the food is fish or honey.

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