Metal organic framework conjugated nanobody complex probe, and construction method and application thereof

Abstract

The application provides a metal organic framework combined nanobody composite probe and a construction method and application thereof, and comprises the following steps: (1) preparing a ZIF-8@HRP core; (2) preparing (ZIF-8@HRP) 2 Double-layer nanocomposite; (3) adding a fusion protein to prepare a metal organic framework combined nanobody composite probe. The metal organic framework combined nanobody composite probe provided by the application combines a high-load enzyme catalytic system and a fluorescently labeled nanobody, establishes an immunodetection platform with a double-signal output mode, and realizes high-sensitivity detection of peanut allergen Ara h 2.

Description

Technical Field

[0001] This invention belongs to the food field, and in particular relates to a metal-organic framework combined with nanoantibody composite probe, its construction method and application. Background Technology

[0002] Food allergies, an abnormal reaction of the human immune system to food proteins, have become a significant global public health issue. Epidemiological studies show that approximately 2-4% of adults and over 5% of children are affected by food allergies to varying degrees, and the incidence rate is on the rise. Currently, there is no cure, and strictly avoiding allergens remains the most effective protective measure. Therefore, establishing accurate and sensitive food allergen detection technologies is crucial for ensuring food safety. Peanuts are a common and highly allergenic food; severe allergic reactions can lead to anaphylactic shock and even death. Ara h2 is considered the most important and clinically relevant allergenic protein in peanuts. Belonging to the 2S albumin family, it has a stable structure, rich in α-helices and β-sheets, allowing it to maintain its stable structure during food processing, heat treatment, and digestion, thus exhibiting strong allergenic activity. Therefore, establishing highly sensitive detection methods for Ara h2 is of great significance for food allergy risk assessment.

[0003] Currently, methods for detecting food allergens mainly include polymerase chain reaction (PCR), liquid chromatography-mass spectrometry (LC-MS), biosensing technology, and immunoassay methods. Among these, enzyme-linked immunosorbent assay (ELISA) is widely used due to its simplicity, high sensitivity, and wide applicability, especially double-antibody sandwich ELISA, which is frequently used for the detection of Ara h2 in complex food matrices. However, traditional ELISA is highly dependent on antibody performance. Conventional monoclonal or polyclonal antibodies generally suffer from problems such as large molecular weight, insufficient stability, significant batch-to-batch variability, and limited ability to recognize occult epitopes, which limit their sensitivity, repeatability, and rapid detection applications.

[0004] In recent years, nanobodies (Nb), as single-domain antibodies derived from the variable region (VHH) of camel heavy chain antibodies, have attracted widespread attention in the field of bioassay due to their advantages such as small molecular weight, high structural stability, ease of genetic engineering expression, and ability to recognize cryptic antigenic epitopes. Compared with traditional antibodies, nanobodies are more suitable for constructing highly stable detection systems in complex environments and have good potential for engineering modification. However, the development of high-affinity nanobodies targeting Ara h 2 is still relatively limited, and their application in highly sensitive detection systems is still insufficient.

[0005] Furthermore, existing antibody-based detection methods still have shortcomings in signal amplification. Traditional enzyme-labeled systems (such as HRP) suffer from limited enzyme loading, limited catalytic efficiency, and insufficient stability, making them unsuitable for detecting low-concentration target analytes. Metal-organic frameworks (MOFs), especially ZIF-8, are widely used in enzyme immobilization and signal amplification system construction due to their high specific surface area, porous structure, and good biocompatibility. However, they still face limitations such as insufficient enzyme loading efficiency, unstable catalytic activity, and limited structural design, restricting their further application in highly sensitive detection. Summary of the Invention

[0006] In view of this, the present invention aims to overcome the defects in the prior art and propose a metal-organic framework combined with nanoantibody composite probe, its construction method and application, which is a novel detection system with both high molecular recognition capability and strong signal amplification capability, improving the detection sensitivity and anti-interference capability of food allergens such as Ara h2, thereby meeting the actual needs of rapid food safety detection and on-site detection.

[0007] To achieve the above objectives, the technical solution of the present invention is implemented as follows: This invention provides a method for constructing a metal-organic framework-based nanobody composite probe, comprising the following steps: (1) Dissolve the cationic surfactant in deionized water, add imidazole organic ligands to it, add the labeled enzyme under stirring, then add zinc salt solution, stir evenly and let stand to obtain ZIF-8@HRP core; (2) Disperse the obtained ZIF-8@HRP nucleus in deionized water to obtain a ZIF-8@HRP nucleus suspension. Add an imidazole organic ligand to the suspension, add a labeled enzyme under stirring, and then add a zinc salt solution. After stirring evenly, let it stand to obtain (ZIF-8@HRP). 2 Bilayer nanocomposite; (3) The (ZIF-8@HRP) 2 The bilayer nanocomposite was dispersed in a buffer solution, activated, and then a fusion protein was added and incubated once at room temperature. Then, bovine serum albumin solution was added and incubated a second time at room temperature to obtain a bioconjugate. The bioconjugate was centrifuged, washed, and redispersed to obtain the metal-organic framework-bound nanoantibody composite probe.

[0008] Further, in step (1), the solid-liquid ratio of the cationic surfactant, imidazole organic ligand, labeled enzyme, and zinc salt solution is 0.30-0.40 g: 110-120 g: 0.15-0.25 mL: 0.20-0.30 mL; the labeled enzyme is HRP, and the concentration of the labeled enzyme is 4-6 mg / mL; the zinc salt solution is Zn(NO3)2•6H2O solution, and the concentration of the zinc salt solution is 25-35 mg / mL; the cationic surfactant is hexadecyltrimethylammonium bromide; and the imidazole organic ligand is 2-methylimidazole.

[0009] Furthermore, the settling time in step (1) is 2-4 hours.

[0010] Further, in step (2), the solid-liquid ratio of ZIF-8@HRP nuclear suspension, imidazole organic ligand, labeled enzyme, and zinc salt solution is 0.2-0.6 mL: 110-120 g: 0.15-0.25 mL: 0.20-0.30 mL; the labeled enzyme is HRP, and the concentration of the labeled enzyme is 4-6 mg / mL; the zinc salt solution is Zn(NO3)2•6H2O solution, and the concentration of the zinc salt solution is 25-35 mg / mL; the cationic surfactant is hexadecyltrimethylammonium bromide; and the imidazole organic ligand is 2-methylimidazole.

[0011] Furthermore, the settling time in step (2) is 0.5-2 hours; the (ZIF-8@HRP) 2 The particle size of the bilayer nanocomposite is 174-194 nm.

[0012] Furthermore, the buffer solution in step (3) is a PBS solution; the activation step in step (3) uses 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride; and the (ZIF-8@HRP) in step (3) 2 The solid-liquid ratio of the bilayer nanocomposite to the fusion protein and bovine serum albumin is 1-2 mg: 10-50 μg: 50-200 μL; the concentration of the bovine serum albumin solution in step (3) is 10% w / v.

[0013] Furthermore, the incubation time in step (3) is 1-3 hours; the incubation time in step (3) is 0.5-2 hours.

[0014] Furthermore, the fusion protein in step (3) is prepared by a method comprising the following steps: linking the reporter protein coding sequence and the nanobody coding sequence that specifically recognizes the target allergen into an expression vector to obtain a recombinant expression vector; transforming the recombinant expression vector into prokaryotic expression host cells; culturing and inducing expression; and separating and purifying the expression product to obtain the fusion protein; wherein the reporter protein coding sequence is an sfGFP fragment; and the nanobody coding sequence that specifically recognizes the target allergen is the amino acid sequence of the nanobody Nb82 that encodes the peanut allergen Ara h2.

[0015] The present invention also provides a metal-organic framework-bound nanoantibody composite probe prepared by the aforementioned preparation method.

[0016] This invention also provides an application of a metal-organic framework combined with a nanobody composite probe, characterized by: the application of the metal-organic framework combined with a nanobody composite probe in the detection of food allergens; and the application of the metal-organic framework combined with a nanobody composite probe in the detection of peanut allergen Ara h2.

[0017] Compared with the prior art, the present invention has the following advantages: The metal-organic framework combined with nanobody composite probe of the present invention establishes an immunoassay platform with dual signal output modes by combining a high-load enzyme catalytic system with fluorescently labeled nanobody, thereby achieving highly sensitive detection of peanut allergen Ara h2.

[0018] The method for preparing the metal-organic framework combined with nanobody composite probe of the present invention utilizes a multilayer metal-organic framework to embed enzymes to construct a high-load catalytic structure, and combines it with fluorescently labeled nanobody to form a composite nanoprobe with both molecular recognition and signal amplification functions. Based on this probe, a fluorescence-colorimetric dual-modal detection platform is established to achieve highly sensitive detection of peanut allergen Ara h2. Attached Figure Description

[0019] Figure 1 The (ZIF-8@HRP) described in this embodiment of the invention. 2 TEM and DLS characterization; Figure 2 The (ZIF-8@HRP) described in this embodiment of the invention. 2 EDS characterization; Figure 3 The (ZIF-8@HRP) described in this embodiment of the invention. 2 XRD characterization; Figure 4 The (ZIF-8@HRP) described in this embodiment of the invention. 2FT-IR characterization; Figure 5 The (ZIF-8@HRP) described in this embodiment of the invention. 2 XPS characterization; Figure 6 The (ZIF-8@HRP) described in this embodiment of the invention. 2 UV-vis absorption and Zeta potential; Figure 7 The (ZIF-8@HRP) described in this embodiment of the invention. 2 Characterization of HRP encapsulation amount; Figure 8 The ZIF-8@HRP and (ZIF-8@HRP) described in the embodiments of the present invention. 2 catalytic performance; Figure 9 The (ZIF-8@HRP) described in this embodiment of the invention. 2 Stability; Figure 10 The (ZIF-8@HRP) described in this embodiment of the invention. 2 Catalytic kinetics; Figure 11 This refers to the construction of recombinant fluorescent Nbs as described in the embodiments of the present invention; Figure 12 The (ZIF-8@HRP) described in this embodiment of the invention. 2 Assembly of @Nb probes; Figure 13 This invention optimizes the dual-mode detection method for peanut allergen Ara h2 protein described in the embodiments of the present invention. Figure 14 This invention relates to the construction of a standard curve and the validation of the method specificity for the dual-mode immunoassay method for peanut allergen Ara h2 protein described in this embodiment. Detailed Implementation

[0020] Unless otherwise defined, the technical terms used in the following embodiments have the same meanings as commonly understood by those skilled in the art. Unless otherwise specified, the experimental reagents used in the following embodiments are conventional biochemical reagents; and the experimental methods described are conventional methods.

[0021] The present invention will be described in detail below with reference to embodiments.

[0022] Example 1 (ZIF-8@HRP) 2 Synthesis and Characterization (ZIF-8@HRP) 2The bilayer nanocomposite was synthesized at room temperature via a two-step epitaxial overgrowth method. First, hexadecyltrimethylammonium bromide (CTAB, 0.35 mg) was dissolved in 1.75 mL of deionized water, 2-methylimidazole (113.5 mg) was added, and the mixture was stirred at 500 rpm for 4 minutes. Then, HRP (0.2 mL, 5 mg / mL) was added, followed by Zn(NO3)2•6H2O (0.25 mL, 29.2 mg / mL) after 1 minute. The mixture was stirred at 500 rpm for 5 minutes, then allowed to stand at room temperature for 3 hours to form the first layer of ZIF-8@HRP cores. The product was collected by centrifugation (12,000 rpm, 10 minutes), washed twice with deionized water, and redispersed in 1 mL of deionized water. For the second layer, 0.5 mL of the ZIF-8@HRP core suspension was taken, 2-methylimidazole (113.5 mg) was added, and the mixture was stirred at 500 rpm for 4 minutes. Then, HRP (0.2 mL, 5 mg / mL) was added, followed by Zn(NO3)2•6H2O (0.25 mL, 29.2 mg / mL) after 1 minute. The mixture was stirred at 500 rpm for 5 minutes, then allowed to stand for 1 hour. The final product was denoted as (ZIF-8@HRP). 2 Bilayer nanocomposite. Transmission electron microscopy (TEM) reveals a uniform nanocubic morphology. Figure 1 The average size is 184 ± 10 nm, consistent with the hydrodynamic diameter obtained by dynamic light scattering (DLS). Figure 1 High-angle annular dark-field scanning transmission electron microscopy (HAADF-STEM) combined with energy-dispersive X-ray spectroscopy (EDS) elemental mapping confirmed the uniform distribution of C, N, O, Zn, P, S, and Fe in the nanocubic structure, indicating the successful construction of a hierarchical (ZIF-8@HRP) structure. 2 structure( Figure 2 (ZIF-8@HRP) 2 The powder X-ray diffraction (XRD) pattern matches the simulated ZIF-8 diffraction peaks very well, indicating that the crystal framework of ZIF-8 was well preserved after protein encapsulation and secondary growth. Figure 3 Fourier transform infrared (FT-IR) spectroscopy further confirmed the successful formation of this framework, at 2927 cm⁻¹. -1 The peak at 1581 cm⁻¹ represents the C-H bond stretching vibration of the 2-methylimidazolium ligand. -1 and 1383 cm -1 The characteristic bands at the locations are attributed to the C=N and CN tensile vibrations of the imidazole ring, respectively, confirming the integrity of the ZIF-8 structure. Figure 4X-ray photoelectron spectroscopy (XPS) further identified the presence of Zn, C, N, and O elements, consistent with the successful formation of the MOF-based hybrid system. Figure 5 In summary, these results confirm the design of (ZIF-8@HRP). 2 The architecture was successfully manufactured. From ZIF-8@HRP to (ZIF-8@HRP) 2 The UV-vis absorption intensity and Zeta potential gradually increase, consistent with the layer-by-layer growth of the MOF shell. Figure 6 The enhanced optical response further supports the increased loading of HRP during the secondary encapsulation process, indicating efficient immobilization of biomolecules within the layered framework. Thermogravimetric analysis (TGA) was further used to quantify the HRP loading. (ZIF-8@HRP) 2 The HRP loading content in the medium was 442 μg / mg, confirming the high encapsulation efficiency of the multi-shell MOF system. Figure 7 ).

[0023] Example 2 (ZIF-8@HRP) 2 catalytic performance and stability To evaluate its signal amplification capability, the H2O2-mediated oxidation of 3,3',5,5'-tetramethylbenzidine (TMB) was used as a model reaction for ZIF-8@HRP and (ZIF-8@HRP). 2 The peroxidase-like activity was evaluated. Compared with ZIF-8@HRP, (ZIF-8@HRP) 2 The catalytic performance was significantly improved. The catalytic activity of both structures increased with increasing concentration. (ZIF-8@HRP) 2 It consistently exhibited excellent performance. To quantitatively elucidate the catalytic kinetics, the Michaelis-Menten parameters, including the maximum reaction rate (Vmax) and the Michaelis-Menten constant (Km), were determined by varying the concentrations of TMB and H2O2. (ZIF-8@HRP) 2 The Km values ​​for TMB and H2O2 were 0.9048 mM and 9.767 mM, respectively, demonstrating good substrate affinity and efficient catalytic transformation, highlighting its signal amplification potential on the ELISA platform. Figure 8 ).

[0024] Considering that probe stability is crucial for the reliability and repeatability of the assay, we systematically studied (ZIF-8@HRP) using free HRP as a control. 2 pH stability, thermal stability, and long-term storage stability. (ZIF-8@HRP) 2It maintains stable catalytic activity under slightly acidic conditions (pH 4-6). When the pH increases from 7 to 10, (ZIF-8@HRP)... 2 The remaining activity remained at 85.2%, while free HRP decreased to 25.4%, indicating that encapsulation within the ZIF-8 framework effectively mitigated pH-induced inactivation. Thermal stability was further assessed by monitoring TMB oxidation over a temperature range of 20–90 °C. Figure 9 (ZIF-8@HRP) 2 The catalytic activity of the enzyme decreased from 100% to 69.6%, while that of free HRP decreased to 41.2%, indicating that MOF encapsulation significantly enhanced the enzyme's robustness at high temperatures. Long-term stability was assessed by monitoring catalytic activity after storage at 4 °C for 15 days. (ZIF-8@HRP) 2 The test showed negligible loss of activity, confirming excellent storage stability. In summary, these results indicate that (ZIF-8@HRP) exhibits... 2 It possesses excellent catalytic efficiency and enhanced resistance to environmental stress, making it a reliable signal amplification probe for ELISA applications. Figure 10 ).

[0025] Example 3 Construction of recombinant fluorescent Nbs To construct a bifunctional recognition probe, the PCR-amplified sfGFP fragment and the sequence of the nanobody Nb82 encoding the peanut allergen Ara h2 (amino acid sequence shown in SEQ ID NO.1) were integrated into the pHEN6c vector using a one-step seamless cloning method, and a 6x His tag was fused. The recombinant plasmid was transformed into *E. coli* WK6 competent cells, plated on LB agar containing 100 μg / mL ampicillin, and incubated overnight at 37°C. Positive clones were confirmed by colony PCR, and the recombinant plasmid was extracted. After overnight culture, 5 mL of the bacterial culture was transferred to 500 mL of TB medium (containing 2.5 mL of 20% glucose, 100 μg / mL ampicillin, and 500 μL of 2 M MgCl2), and cultured at 37°C with shaking at 220 rpm until OD200. 600The concentration was approximately 0.6. IPTG was added to a final concentration of 1 mM, and expression was induced overnight at 28°C with shaking. Cells were then purified using the following method: lysis buffer containing 50 mM Tris-HCl (pH 8.0), 300 mM NaCl, 10 mM imidazole, 1 mM PMSF, and 1 mg / mL lysozyme was sonicated; the Ni-NTA column was washed sequentially with buffers containing 20, 50, and 100 mM imidazole (pH 7.4), followed by elution with 300 mM imidazole. Verification was performed by colony PCR and nucleotide sequencing. The eluted protein was dialyzed against PBS (pH 7.4), and protein concentration was determined using a NanoDrop spectrophotometer. The purified product was analyzed by SDS-PAGE and Western blot. The fusion protein purified by sonication and imidazole gradient elution showed an expected molecular weight of ~45 kDa and a soluble product concentration of 5 mg / L on SDS-PAGE and Western blot. The structural and functional integrity of the sfGFP domain was confirmed by its bright green appearance under ambient conditions and the characteristic photoluminescence maximum at 530 nm (λex = 488 nm). Crucially, the gene fusion strategy significantly enhanced target binding affinity. ELISA evaluation showed that the epigenetic binding affinity of Nb82-sfGFP-H6 (21.91 nM) was 3-fold greater than that of the Nb82 monomer (65.50 nM). Figure 11 ). Example 4 (ZIF-8@HRP) 2 Assembly of @Nb probes To assemble the target nanobioprobe, the lyophilized (ZIF-8@HRP) was first... 2 The nanocomposite material was uniformly dispersed in 10 mM PBS (pH 7.4). Surface activation was initiated by adding EDC-HCl (63 μg), followed by the introduction of the Nb-GFP fusion protein. The reaction mixture was incubated with continuous gentle stirring (450 rpm) at room temperature for 2 hours to promote covalent coupling. Subsequently, bovine serum albumin (BSA) solution (50 μL, 10% w / v) was added to passivate residual active sites, followed by incubation for another 2 hours. The resulting bioconjugate was separated by centrifugation at 14000 rpm for 10 min and subjected to two rigorous washes with deionized water to remove unbound reagents. Purified (ZIF-8@HRP) 2 The @Nb probe was finally redispersed in PBS (10 mM, pH 7.4) and stored at 4 °C prior to analytical applications. The high specific surface area of ​​the MOF composite facilitated dense interfacial loading of fluorescent nanobody via electrostatic adsorption. Therefore, the fully assembled (ZIF-8@HRP) 2The @Nb probe exhibited strong green emission under 488nm excitation, similar to the bare (ZIF-8@HRP) probe. 2 The precursor's non-luminescent white appearance contrasts sharply with its appearance. Successful electrostatic complexation was systematically confirmed by a corresponding change in the Zeta potential, and also compared to the original (ZIF-8@HRP). 2 Compared to the previous model, it maintained almost the same catalytic activity. After quantitative optimization, the ideal Nb loading was determined to be 30 μg by BCA assay. The designed nanobioprobe exhibited excellent interfacial robustness and maintained excellent catalytic stability during the 15-day evaluation period. Figure 12 ).

[0026] Example 5: Condition optimization of the "fluorescence-colorimetric" dual-readout Nb immunoassay method for Ara h2 Another nanobody for peanut Ara h2 allergy, Nb154 (amino acid sequence shown in SEQ ID NO.2), was used as a capture antibody, with (ZIF-8@HRP) 2 Using Nb as the detection probe, we systematically optimized the probe amount, incubation time, and color development time. The specific steps are as follows: Nb154 was coated onto an ELISA plate and incubated overnight at 4 °C. The next day, the coating solution was discarded, and the plate was washed three times with PBST. 5% BSA was added to each well, and the plate was blocked at room temperature for 2 hours, followed by three more washes with PBST. After antigen incubation, different concentrations of probe were added, different incubation times were achieved, and 100 μL of TMB substrate was added to each well for different color development times. The absorbance was measured at 652 nm. The signal-to-noise ratio (OD) of different concentration combinations (experimental group OD) was compared. 450 / Control group OD 450 Based on the absolute signal intensity, the amount of detection probe, incubation time, and color development time were determined. The optimal probe volume was finally determined to be 10 μL to ensure robustness and a stable signal-to-noise ratio. Kinetic optimization further showed that a 60-minute probe incubation, combined with a 5-minute rapid catalytic reading, produced saturated and highly reproducible signal intensity. Figure 13 ).

[0027] Example 6: Establishment of the standard curve for the "fluorescence-colorimetric" dual-readout Nb immunoassay method for Ara h2 Based on optimal detection conditions, a fluorescence-colorimetric dual-readout Nb immunoELISA method was established and used to plot the standard curve for peanut allergen Ara h2 protein. Purified Ara h2 antigen was serially diluted and detected under optimized experimental conditions. The logarithm of antigen concentration was plotted on the x-axis, corresponding to the OD values. 652 nm value or OD 530A dose-response curve was plotted using fluorescence values ​​at nm as the ordinate. The linear detection range of the method was determined based on this curve. The limit of detection (LOD) was calculated by adding three standard deviations (Meanblank + 3SDblank) to the average absorbance of the blank wells, and the limit of quantitation (LOQ) was determined by adding ten standard deviations (Meanblank + 10SDblank) to the average absorbance of the blank wells. In colorimetric mode, the detection range was 0.06–250 ng / mL, with an LOD of 0.0056 ng / mL; in fluorescence mode, the detection range was 0.03–125 ng / mL, with an LOD of 0.01 ng / mL. The dual-mode immunosensing method exhibited strict specificity for a group of non-homologous dietary antigens (OVO, OVA, BLG, 7S and 11S globulins, and shellfish extracts), with negligible observed cross-reactivity. Figure 14 ).

[0028] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for constructing a metal-organic framework combined with a nanobody composite probe, characterized in that: Includes the following steps: (1) Dissolve the cationic surfactant in deionized water, add imidazole organic ligands to it, add the labeled enzyme under stirring, then add zinc salt solution, stir evenly and let stand to obtain ZIF-8@HRP core; (2) Disperse the obtained ZIF-8@HRP nucleus in deionized water to obtain a ZIF-8@HRP nucleus suspension. Add an imidazole organic ligand to the suspension, add a labeled enzyme under stirring, and then add a zinc salt solution. After stirring evenly, let it stand to obtain (ZIF-8@HRP). 2 Bilayer nanocomposite; (3) The (ZIF-8@HRP) 2 The bilayer nanocomposite was dispersed in a buffer solution, activated, and then a fusion protein was added and incubated once at room temperature. Then, bovine serum albumin solution was added and incubated a second time at room temperature to obtain a bioconjugate. The bioconjugate was centrifuged, washed, and redispersed to obtain the metal-organic framework-bound nanoantibody composite probe.

2. The method for constructing a metal-organic framework combined with a nanobody composite probe according to claim 1, characterized in that: In step (1), the solid-liquid ratio of the cationic surfactant, imidazole organic ligand, labeled enzyme, and zinc salt solution is 0.30-0.40 g: 110-120 g: 0.15-0.25 mL: 0.20-0.30 mL; the labeled enzyme is HRP, and the concentration of the labeled enzyme is 4-6 mg / mL; the zinc salt solution is Zn(NO3)2•6H2O solution, and the concentration of the zinc salt solution is 25-35 mg / mL; the cationic surfactant is hexadecyltrimethylammonium bromide; and the imidazole organic ligand is 2-methylimidazole.

3. The method for constructing a metal-organic framework combined with a nanobody composite probe according to claim 1, characterized in that: The settling time in step (1) is 2-4 hours.

4. The method for constructing a metal-organic framework combined with a nanobody composite probe according to claim 1, characterized in that: In step (2), the solid-liquid ratio of ZIF-8@HRP nuclear suspension, imidazole organic ligand, labeled enzyme, and zinc salt solution is 0.2-0.6 mL: 110-120 g: 0.15-0.25 mL: 0.20-0.30 mL; the labeled enzyme is HRP, and the concentration of the labeled enzyme is 4-6 mg / mL; the zinc salt solution is Zn(NO3)2•6H2O solution, and the concentration of the zinc salt solution is 25-35 mg / mL; the cationic surfactant is hexadecyltrimethylammonium bromide; and the imidazole organic ligand is 2-methylimidazole.

5. The method for constructing a metal-organic framework combined with a nanobody composite probe according to claim 1, characterized in that: The settling time in step (2) is 0.5-2 hours; the (ZIF-8@HRP) 2 The particle size of the bilayer nanocomposite is 174-194 nm.

6. The method for constructing a metal-organic framework combined with a nanobody composite probe according to claim 1, characterized in that: The buffer solution in step (3) is PBS solution; the activation step in step (3) uses 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride; the (ZIF-8@HRP) in step (3) 2 The solid-liquid ratio of the bilayer nanocomposite to the fusion protein and bovine serum albumin is 1-2 mg: 10-50 μg: 50-200 μL; the concentration of the bovine serum albumin solution in step (3) is 10% w / v.

7. The method for constructing a metal-organic framework combined with a nanobody composite probe according to claim 1, characterized in that: The incubation time in step (3) is 1-3 hours; the incubation time in step (3) is 0.5-2 hours.

8. The method for constructing a metal-organic framework combined with a nanobody composite probe according to claim 1, characterized in that: The fusion protein in step (3) is prepared by a method comprising the following steps: linking the reporter protein coding sequence and the nanobody coding sequence that specifically recognizes the target allergen into an expression vector to obtain a recombinant expression vector; transforming the recombinant expression vector into prokaryotic expression host cells; culturing and inducing expression; and separating and purifying the expression product to obtain the fusion protein; wherein the reporter protein coding sequence is an sfGFP fragment; and the nanobody coding sequence that specifically recognizes the target allergen is the amino acid sequence of the nanobody Nb82 encoding the peanut allergen Ara h2.

9. A metal-organic framework-bound nanoantibody composite probe prepared by the preparation method according to any one of claims 1-8.

10. An application of the metal-organic framework combined with nanoantibody composite probe as described in claim 9, characterized in that: The application of the metal-organic framework combined with nanobody composite probe in the detection of food allergens; the application of the metal-organic framework combined with nanobody composite probe in the detection of peanut allergen Ara h 2.

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