Immune probe for orderly immobilizing biological enzyme and antibody as well as preparation method and application of immune probe

Abstract

The invention discloses an immune probe for orderly immobilizing a biological enzyme and an antibody as well as a preparation method and application of the immune probe. The method comprises the following steps: S1, adding 2-methylimidazole and a metal salt solution into a polyvinyl pyrrolidone and cysteine mixed solution containing bio-enzyme, and aging at room temperature; after aging, centrifuging to obtain a precipitate, washing and dispersing to obtain a signal generating element suspension; s2, slowly dropwise adding an antibody solution into a 2-methylimidazole solution, then adding the signal generation element suspension prepared in the step S1, then adding a metal salt solution, and carrying out vortex oscillation; and finally, centrifugally collecting precipitates, washing and dispersing to obtain the immune probe integrating the dual functions of antibody recognition and signal generation. The method of the present invention shows an ordered, efficient strategy of synthetase-antibody conjugates that not only loads a large amount of enzymes to generate amplification signals, but also protects encapsulated proteins from denaturation conditions.

Description

Technical Field

[0001] The present invention relates to the technical fields of natural enzyme and antibody immobilization and biosensor technology, and particularly relates to an immune probe for orderly immobilizing bioenzymes and antibodies, a preparation method thereof, and an application thereof. Background Art

[0002] The enzyme-linked immunosorbent assay (ELISA) is currently the most common and technically mature immunoassay method, and has been widely used in the safety detection of food. This method is a method for qualitative or quantitative analysis by adsorbing an antigen or antibody on the surface of a solid-phase carrier, performing an antigen-antibody reaction with an enzyme-labeled antibody conjugate, and performing a color reaction after the enzyme acts on the substrate. In ELISA, the enzyme-antibody conjugate plays a crucial role in connecting the signal readout and the immunobinding reaction. However, in the synthesis process of the enzyme-antibody conjugate, complex covalent bonds and cumbersome purification processes often bring inevitable functional sacrifices, such as the inactivation of the antibody capture ability and enzyme activity, resulting in a relatively low detection limit in immunoassays. In addition, as a protein, the enzyme-antibody conjugate is extremely prone to conformational changes under harsh conditions (high temperature, extreme pH, organic solvents, etc.), resulting in loss of activity, which greatly hinders the industrial development of enzyme-linked immunosensors.

[0003] As a class of crystalline porous materials, metal-organic frameworks (MOFs) not only have high encapsulation efficiency, but also can protect biomolecules from the influence of denaturing conditions. These excellent properties bring bright prospects for the application of protein-MOF composites in the field of immunosensors. However, in current protein-MOF composites, usually single or multiple bioenzymes are integrated with MOFs, and a complex covalent binding is still required between the antibody and MOF, which inevitably reduces the antigen capture efficiency of the antibody. Recently, studies on simultaneously immobilizing bioenzymes and antibodies into MOFs as immunoassay markers have been reported. However, in this way of multi-protein disorderly mixed immobilization, there will be competitive coordination, which will hinder the best utilization of the respective advantages of bioenzymes and antibodies, resulting in low utilization rates. Therefore, it is necessary to study the optimal way of immobilizing bioenzymes and antibodies to meet the efficient utilization of the two proteins and the maintenance of stability, while improving the overall catalytic efficiency of bioenzymes and the recognition ability of antibodies. Summary of the Invention

[0004] Based on this, in view of the above technical problems, it is necessary to provide an immune probe for orderly immobilizing bioenzymes and antibodies, a preparation method thereof, and an application thereof.

[0005] In a first aspect, the present invention provides a preparation method for an immune probe for orderly immobilizing bioenzymes and antibodies, which comprises the following steps: S1. In a mixed solution of polyvinylpyrrolidone (PVP) and cysteine (Cys) containing bioenzymes, add 2-methylimidazole (2-mIM) and a metal salt solution, and age at room temperature; after aging is completed, centrifuge to obtain a precipitate, wash and redisperse it to obtain a signal generating element suspension; S2. Slowly drip the antibody solution into the 2-methylimidazole solution, then add the signal generating element suspension prepared in step S1, then add the metal salt solution, and perform vortex oscillation; finally, centrifuge to collect the precipitate, wash and redisperse it to obtain an immunoprobe integrating the dual functions of antibody recognition and signal generation.

[0006] In order to efficiently immobilize biomacromolecules to improve their stability, the present invention prepares a protein@MOFs composite material by a biomimetic mineralization process. First, relying on the strong binding between metal ion nodes and 2-methylimidazole, in the presence of polyvinylpyrrolidone and cysteine, bioenzymes are encapsulated in-situ to prepare signal generating elements (Enz@MOFs, Enz refers to bioenzymes). Subsequently, a metal salt solution is added to a mixed solution containing Enz@MOFs, 2-methylimidazole and antibody Ab2 to specifically prepare a MOFs-Ab2 coating, and an immunoprobe Enz@MOFs@MOFs-Ab2 integrating the dual functions of antibody recognition and signal generation is prepared. MOFs plays a protective role for biomacromolecules, and the enzyme immobilized therein shows better stability than natural enzymes in unsuitable and extreme environments. The orderly and hierarchical immobilization method in MOFs of the present invention effectively improves the utilization efficiency and stability of bioenzymes and antibodies.

[0007] Specifically, in step S1, the concentration of cysteine is 2-6 mg / mL; the concentration of 2-methylimidazole is 80-600 mM; the concentration of bioenzymes is 10-30 mg / mL. In step S2, the added concentration of the antibody is 5-20 mg / mL. Without cysteine, Enz@MOFs shows extremely low biological activity; too low or too high concentrations of bioenzymes or antibodies are not conducive to efficient loading in MOFs.

[0008] In steps S1 and S2, the metal salt is a transition metal salt, such as transition metal salt solutions of Zn salt, Co salt, Cu salt, Tb salt, etc.

[0009] In the second aspect, the present invention provides an immunoprobe prepared by the method of the first aspect above.

[0010] In the third aspect, the present invention provides an application of an immunoprobe in detecting isocarbophos. With the excellent catalytic activity and recognition ability of the immunoprobe Enz@MOFs@MOFs-Ab2 integrating the dual functions of antibody recognition and signal generation, an immunosensor is constructed to detect isocarbophos.

[0011] By means of the specific recognition reaction between antigen and antibody, the added isocarbophos standard competes with the antibody for the antigen on the solid-phase carrier to form an antigen-antibody complex. After binding with Enz@MOFs@MOFs-Ab2, NH3 is generated through urea decomposition, causing the phenol red solution to change from yellow to red. Finally, a standard curve of isocarbophos is established based on the absorbance value at 560 nm. For an isocarbophos solution with an unknown concentration, the absorbance value at 560 nm obtained by performing the same operation on it is substituted into this standard curve, and then the isocarbophos concentration of the unknown solution can be calculated, thereby realizing the detection of isocarbophos in food samples.

[0012] The immunosensor constructed based on Enz@MOFs@MOFs-Ab2 proposed by the present invention is used for the detection of isocarbophos, with a detection limit of 0.69 ng / mL and an IC 50 of 6.27 ng / mL, and a linear range of 1.56 - 25.25 ng / mL, providing new ideas and technical support for the on-site detection of isocarbophos in food.

[0013] Compared with the prior art, the present invention has the following beneficial effects: In the present invention, the functions of the signal amplification unit (biological enzyme Enz) and the biological recognition unit (antibody Ab2) are integrated with MOFs respectively through a mild biomimetic mineralization process to form Enz@MOFs@MOFs-Ab2, demonstrating an orderly and efficient strategy for synthesizing enzyme-antibody conjugates. The Enz@MOFs@MOFs-Ab2 prepared by the present invention not only loads a large amount of enzymes to generate amplified signals, but also protects the encapsulated proteins from denaturing conditions.

[0014] The present invention also constructs a rapid and sensitive method for detecting isocarbophos in food with Enz@MOFs@MOFs-Ab2 as the label. The detection limit of isocarbophos is 0.69 ng / mL, which is about 4 times lower than that of the conventional HRP-based ELISA. The method of the present invention shows high selectivity for isocarbophos, and acceptable accuracy and precision are obtained in the analysis of three samples, namely pitaya, pear and tea. This enzyme-antibody conjugation strategy is expected to replace the traditional enzyme-labeled antibody in immunoassay applications and expand the application of MOF materials in the field of immunosensors. Description of the Drawings

[0015] In order to more clearly illustrate the solutions in the present application or the prior art, the following will briefly introduce the drawings required for use in the description of the embodiments or the prior art. Obviously, the drawings in the following description are some embodiments of the present application. For those of ordinary skill in the art, other drawings can also be obtained based on these drawings without creative efforts.

[0016] Figure 1 (A) SEM image of UPC@MOFs prepared in Example 1; (B) Energy-dispersive spectroscopy image of UPC@MOFs prepared in Example 1. Figure 2 (A) km optimized for Cys concentration of UPC@MOFs prepared in Example 2; (B) km optimized for PVP concentration of UPC@MOFs prepared in Example 2; (C) km optimized for 2-mIM concentration of UPC@MOFs prepared in Example 2; (D) Enzyme loading optimized for urease concentration of UPC@MOFs prepared in Example 2. Figure 3 (A) SEM image of Urease@MOFs@MOFs-Ab2 prepared in Example 3; (B) Energy-dispersive spectroscopy image of Urease@MOFs@MOFs-Ab2 prepared in Example 3. Figure 4 Zeta potential diagram of Urease@MOFs@MOFs-Ab2 prepared in Example 3. Figure 5 Detection performance optimized for Ab2 concentration of Urease@MOFs@MOFs-Ab2 prepared in Example 4. Figure 6 (A) Detection effects of different Ab2 immobilization methods in Example 5; (B) Ab2 coupling efficiency of different Ab2 immobilization methods in Example 5. Figure 7 Confocal fluorescence image of Urease@MOFs@MOFs-Ab2 obtained in Example 6. Among them, FITC represents the confocal fluorescence image obtained by labeling urease with the green fluorescent dye fluorescein isothiocyanate (FITC), RhB represents the confocal fluorescence image obtained by labeling Ab2 with the red fluorescent dye rhodamine B (RhB), and Merge represents the image after merging the FITC and RhB confocal fluorescence images.

[0017] Figure 8 (A) Schematic diagram of the immunosensor for isocarbophos detection based on Urease@MOFs@MOFs-Ab2 in Example 7. In the figure, Antigen: Coated antigen; Anti-ICP mAbs: Monoclonal antibody against isocarbophos; Isocarbophos: Isocarbophos; Urea: Urea; NH3: Ammonia; Phenol red: Phenol red; Red product: Red product; (B) Standard curve for isocarbophos detection by the immunosensor based on Urease@MOFs@MOFs-Ab2 in Example 7; (C) Standard curve for isocarbophos detection by the traditional enzyme-linked immunosorbent assay method in Example 7.

[0018] Figure 9 (A) Specificity of the immunosensor based on Urease@MOFs@MOFs-Ab2 for detecting isocarbophos in Example 8; (B) Anti-interference ability of the immunosensor based on Urease@MOFs@MOFs-Ab2 for detecting isocarbophos in Example 8. Detailed implementation manners

[0019] In order to enable those skilled in the art to better understand the solution of the present invention, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0020] In order to enable those skilled in the art to better understand the solution of the present application, the technical solutions in the embodiments of the present application will be clearly and completely described below in conjunction with the accompanying drawings.

[0021] As described in the above technical background, the conjugation strategy of enzyme-antibody mainly relies on covalent binding. Although this method can achieve effective conjugation of enzyme-antibody, in the process of synthesizing enzyme-antibody conjugates, complex covalent bonds and cumbersome purification processes often bring inevitable functional sacrifices, and there is no reported research on hierarchically and orderly immobilizing enzymes and antibodies into MOFs as immunoassay markers.

[0022] Therefore, the present inventors conducted a large number of experiments and developed a method for preparing an immunosensor probe for orderly immobilizing bioenzymes and antibodies. By a mild biomimetic mineralization process, the functions of the signal amplification unit (bioenzyme) and the biorecognition unit (antibody) are integrated with MOFs (metal-organic frameworks) to form an Enz@MOFs@MOFs-Ab2 immunosensor probe, demonstrating an orderly and efficient strategy for synthesizing enzyme-antibody conjugates. The Enz@MOFs@MOFs-Ab2 prepared by the present invention not only loads a large amount of enzymes to generate amplified signals, but also protects the encapsulated proteins from denaturing conditions.

[0023] A method for preparing an immunosensor probe for orderly immobilizing bioenzymes and antibodies, which comprises the following steps: S1. In a mixed solution of polyvinylpyrrolidone (PVP) and cysteine (Cys) containing a bioenzyme, 2-methylimidazole (2-mIM) and a metal salt solution are added, and aging is carried out at room temperature; after aging is completed, the precipitate is obtained by centrifugation, and after washing and redispersion, a signal generation element suspension is obtained; S2. Slowly drip the antibody solution into the 2-methylimidazole solution, then add the signal generation element suspension prepared in step S1, and then add the metal salt solution, followed by vortex oscillation; finally, centrifuge to collect the precipitate, wash and redisperse it to obtain an immunoprobe integrating the dual functions of antibody recognition and signal generation.

[0024] The present invention also constructs an immunosensor based on Enz@MOFs@MOFs-Ab2 for detecting isocarbophos. The detection limit of this method for isocarbophos can be as low as 0.69 ng / mL, which is about 4 times lower than that of the conventional HRP-based ELISA, and can be used for the detection of isocarbophos in samples, showing broad application prospects.

[0025] An application of an immunoprobe in the detection of isocarbophos, and the specific application steps are as follows: (1) Prepare an immunosensor: Prepare an immunosensor based on the Enz@MOFs@MOFs-Ab2 immunoprobe; (2) Construct a standard curve: Configure standard solutions of isocarbophos with different concentrations, add them to an enzyme-linked immunosorbent assay (ELISA) plate coated with the isocarbophos coating antigen, and add an equal volume of the anti-isocarbophos monoclonal antibody; after the water bath reaction is complete, add the Enz@MOFs@MOFs-Ab2 immunoprobe obtained in step S2; after the water bath reaction is complete again, add the chromogenic solution, and after the color development is completed, measure the absorbance value with an ELISA reader to obtain a standard curve of the absorbance value versus the antigen concentration; (3) Determine isocarbophos in the sample: Replace the isocarbophos standard solution with an equal volume of the processed sample, measure the sample according to the method of the standard curve, and substitute it into the standard curve obtained in step S4 to obtain the concentration of isocarbophos in the sample.

[0026] With the specific recognition reaction between the antigen and the antibody, the added isocarbophos standard competes with the antibody for the antigen on the solid phase carrier to form an antigen-antibody complex, and then after binding with Enz@MOFs@MOFs-Ab2, NH3 is generated by urea decomposition, causing the phenol red solution to change from yellow to red. Finally, a standard curve of isocarbophos is established through the absorbance value at 560 nm. For an isocarbophos solution with an unknown concentration, the absorbance value at 560 nm obtained by performing the same operation on it is substituted into this standard curve, and the isocarbophos concentration of the unknown solution can be calculated, thus realizing the detection of isocarbophos in food samples.

[0027] The orderly and hierarchical immobilization method in MOFs proposed by the present invention effectively improves the utilization efficiency and stability of bioenzymes and antibodies. The immunosensor based on Enz@MOFs@MOFs-Ab2 is used for the detection of isocarbophos, and its detection limit is 0.69 ng / mL, IC 50It was 6.27 ng / mL, and the linear range was 1.56 - 25.25 ng / mL, providing new ideas and technical support for on-site detection of isocarbophos in foods.

[0028] The present invention will be described in detail below in conjunction with examples. The bio-enzyme is selected from urease, and the metal salt is selected from Zn(NO3)2·6H2O. However, it should be noted that the following examples are only the preferred embodiments of the present invention and do not limit the present invention. Unless otherwise specified in this article, the solution concentrations are all volume concentrations.

[0029] Example 1: Synthesis and characterization of UPC@MOFs After mixing 100 μL of urease solution (20 mg / mL), 200 μL of PVP solution (0.5 mg / mL), and 100 μL of Cys solution (4 mg / mL) in a 2 mL centrifuge tube and stirring for 30 seconds, 400 μL of 2-mIM (160 mmol / L) and 400 μL of Zn(NO3)2·6H2O solution (40 mmol / L) were added respectively, and aged at room temperature for 4 hours. Finally, the precipitate was collected by centrifugation at 10000 rpm for 10 min in a centrifuge, washed twice with ultrapure water, and dispersed in an appropriate amount of ultrapure water, and stored in a 4°C refrigerator for standby.

[0030] The results of scanning electron microscopy (SEM) showed the asymmetric spherical nanostructure of UPC@MOFs, and the diameter was about 600 nm ( Figure 1 Figure (A)). The energy dispersive spectroscopy (EDS) elemental mapping showed ( Figure 1 Figure (B)) that the elements Zn, C, N, O, and P were well distributed in the UPC@MOFs composite material.

[0031] Example 2: Optimization of the synthesis conditions of UPC@MOFs (1) Optimization of Cys concentration: After mixing 100 μL of urease solution (20 mg / mL), 200 μL of PVP solution (0.5 mg / mL), and 100 μL of Cys solution (0 - 10 mg / mL) in a 2 mL centrifuge tube and stirring for 30 seconds, 400 μL of 2-mIM (160 mmol / L) and 400 μL of Zn(NO3)2·6H2O solution (40 mmol / L) were added respectively, and aged at room temperature for 4 hours. Finally, the precipitate was collected by centrifugation at 10000 rpm for 10 min in a centrifuge, washed twice with ultrapure water, and dispersed in an appropriate amount of ultrapure water, and stored in a 4°C refrigerator for standby; (2)Optimization of PVP concentration: After mixing 100 μL of urease solution (20 mg / mL), 200 μL of PVP solution (0 - 12 mg / mL), and 100 μL of Cys solution (4 mg / mL) in a 2 mL centrifuge tube and stirring for 30 seconds, 400 μL of 2-mIM (160 mmol / L) and 400 μL of Zn(NO3)2·6H2O solution (40 mmol / L) were added respectively, and then aged at room temperature for 4 hours. Finally, the precipitate was collected by centrifugation at 10000 rpm for 10 min, washed twice with ultrapure water, dispersed in an appropriate amount of ultrapure water, and stored in a 4°C refrigerator for standby; (3)Optimization of 2-mIM concentration: After mixing 100 μL of urease solution (20 mg / mL), 200 μL of PVP solution (0.5 mg / mL), and 100 μL of Cys solution (4 mg / mL) in a 2 mL centrifuge tube and stirring for 30 seconds, 400 μL of 2-mIM (80 - 800 mmol / L) and 400 μL of Zn(NO3)2·6H2O solution (40 mmol / L) were added respectively, and then aged at room temperature for 4 hours. Finally, the precipitate was collected by centrifugation at 10000 rpm for 10 min, washed twice with ultrapure water, dispersed in an appropriate amount of ultrapure water, and stored in a 4°C refrigerator for standby; (4)Optimization of urease concentration: After mixing 100 μL of urease solution (1 - 100 mg / mL), 200 μL of PVP solution (0.5 mg / mL), and 100 μL of Cys solution (4 mg / mL) in a 2 mL centrifuge tube and stirring for 30 seconds, 400 μL of 2-mIM (160 mmol / L) and 400 μL of Zn(NO3)2·6H2O solution (40 mmol / L) were added respectively, and then aged at room temperature for 4 hours. Finally, the precipitate was collected by centrifugation at 10000 rpm for 10 min, washed twice with ultrapure water, dispersed in an appropriate amount of ultrapure water, and stored in a 4°C refrigerator for standby; In 100 μL of the above-mentioned UPC@MOFs solution diluted to a certain multiple, 50 μL of urea solutions with different concentrations and 50 μL of phenol red aqueous solution were added respectively. The change in absorbance of enzyme-catalyzed substrates with different concentrations over time was measured, and the kinetic parameter Km was calculated. The smaller the Km, the better the affinity between UPC@MOFs and the substrate. Among them, the kinetic parameter Km was calculated by the Michaelis-Menten equation: 1 / V = (Km / Vmax × 1 / [S]) + 1 / Vmax, where [S] is the substrate concentration and V is the reaction rate at this substrate concentration. Figure 2(A)-(D) show that 4 mg / mL Cys, 0.5 mg / mL PVP, and 160 mmol / L 2-mIM show the minimum Km, and 20 mg / mL urease shows the highest enzyme loading. Preferably, the Cys concentration is selected from 2-8 mg / mL; the PVP concentration is selected from 0.2-2 mg / mL; the 2-mIM concentration is selected from 80-600 mmol / L; the urease concentration is selected from 10-30 mg / mL, and UPC@MOFs can all show good performance.

[0032] Figure 2 D shows that with the increase of urease concentration, the loading amount of urease shows a trend of first increasing and then decreasing, indicating that too high or too low urease concentration is not conducive to its efficient loading in MOFs.

[0033] Example 3: Synthesis and Characterization of Urease@MOFs@MOFs-Ab2 100 μL of a 10 mg / mL goat anti-mouse IgG (Ab2) solution was slowly added dropwise to 400 μL of a 160 mmol / L 2-mIM solution. Then, the above UPC@MOFs suspension was redissolved to 300 μL with ultrapure water and added to the pre-mixed Ab2 and 2-mIM. Next, 400 μL of a 40 mmol / L Zn(NO3)2·6H2O (40 mmol / L) was added and mixed for 30 seconds, followed by vortex oscillation at 25°C and 800 rpm for 30 min. Finally, the precipitate was collected by centrifugation at 8000 rpm for 5 min, washed twice with ultrapure water, and dispersed in an appropriate amount of ultrapure water and stored in a 4°C refrigerator for later use.

[0034] Scanning electron microscopy (SEM) results show ( Figure 3 (A)), the composite material Urease@MOFs@MOFs-Ab2 prepared by the two-step biomimetic mineralization process shows a typical rhombic dodecahedron shape with relatively uniform size (700 nm). According to the energy dispersive spectroscopy (EDS) elemental mapping ( Figure 3 (B)), the elements Zn, C, N, O, and P are well distributed in the Urease@MOFs@MOFs-Ab2 composite material.

[0035] Zeta potential test results show ( Figure 4PC@MOFs has a positive charge (+5.46 mV). Since urease is negatively charged, the potential value of UPC@MOFs is negative (-7.75 mV). Since urease is negatively charged, the potential value of UPC@MOFs is negative (-7.75 mV). Compared with UPC@MOFs, the potential of Urease@MOFs@MOFs-Ab2 (-13.33 mV) also changed significantly, indicating the effective connection between Ab2 and UPC@MOFs. Compared with UPC@MOFs, the potential of Urease@MOFs@MOFs-Ab2 (-13.33 mV) also changed significantly, indicating the effective connection between Ab2 and UPC@MOFs.

[0036] Example 4: Optimization of the synthesis conditions of Urease@MOFs@MOFs-Ab2 100 μL of an Ab2 solution with a concentration of 1 - 20 mg / mL was slowly added dropwise to 400 μL of a 2-mIM solution with a concentration of 160 mmol / L. Then, the above UPC@MOFs suspension was redissolved in 300 μL of ultrapure water and added to the pre-mixed Ab2 and 2-mIM. Next, 400 μL of Zn(NO3)2·6H2O with a concentration of 40 mmol / L (40 mmol / L) was added and mixed for 30 seconds, and then vortexed at 25°C and 800 rpm for 30 min. Finally, the precipitate was collected by centrifugation at 8000 rpm for 5 min, washed twice with ultrapure water, and dispersed in an appropriate amount of ultrapure water and stored in a refrigerator at 4°C for later use.

[0037] The detection performance of Urease@MOFs@MOFs-Ab2 prepared with different Ab2 concentrations was determined by ic-ELISA method. The experimental results showed that ( Figure 5 ) when the composite material was diluted to the same concentration for use, 10 mg / mL showed the best detection performance. Among them, the Ab2 concentration showed good detection performance in the range of 5 - 20 mg / mL.

[0038] Example 5: Performance evaluation of immunoprobes prepared by different Ab2 immobilization methods Surface adsorption: The above UPC@MOFs suspension was redissolved in 1000 μL of ultrapure water, and then 100 μL of Ab2 solution with a concentration of 10 mg / mL was slowly added dropwise to the Urease@MOFs suspension. It was vortexed at 800 rpm at 25 °C for 1 h, 100 μL of 10% BSA was added and kept for 30 min. Finally, the precipitate was collected by centrifugation at 8000 rpm for 5 min, washed twice with ultrapure water, and dispersed in an appropriate amount of ultrapure water and stored in a refrigerator at 4 °C for later use.

[0039] Ovalent attachment: 100 μL of EDC solution (2 mg / mL) and 100 μL of NHS solution (4 mg / mL) were added to 800 μL of UPC@MOFs suspension. After vortexing at 800 rpm at 25 °C for 30 min, it was centrifuged at 8000 rpm for 5 min and dispersed in 1000 μL of ultrapure water. 100 μL of Ab2 solution containing 10 mg / mL was added to the centrifuge tube. After vortexing at 800 rpm at 25 °C for 1 h, it was placed at 4 °C and vortexed at 800 rpm. After 12 h, 100 μL of 10% BSA was added to the system and vortexed at 800 rpm at 25 °C for 30 min. Finally, the precipitate was collected by centrifugation at 8000 rpm for 5 min, washed twice with ultrapure water, and dispersed in an appropriate amount of ultrapure water and stored in a refrigerator at 4 °C for later use.

[0040] In-situ encapsulation: 100 μL of Ab2 solution with a concentration of 10 mg / mL was slowly added dropwise to 400 μL of 2-mIM solution with a concentration of 160 mmol / L. Then the above UPC@MOFs suspension was redissolved in 300 μL of ultrapure water and added to the pre-mixed Ab2 and 2-mIM. Then 400 μL of Zn(NO3)2·6H2O (40 mmol / L) was added and mixed for 30 s, and it was vortexed at 800 rpm at 25 °C for 30 min. Finally, the precipitate was collected by centrifugation at 8000 rpm for 5 min, washed twice with ultrapure water, and dispersed in an appropriate amount of ultrapure water and stored in a refrigerator at 4 °C for later use.

[0041] The detection performance of the three Ab2 immobilization methods was determined by ic-ELISA. The experimental results showed that ( Figure 6((A)), when the composite materials are diluted to the same concentration for use, in-situ encapsulation shows the most excellent detection performance. Further, the coupling efficiencies of the three Ab2 immobilization methods are determined. As Figure 6 (B) shows, the antibody coupling efficiencies of surface adsorption, covalent attachment, and in-situ encapsulation are 52.99%, 42.14%, and 83.28%, respectively. In-situ encapsulation exhibits superior antibody coupling efficiency, about 1.6 times that of surface adsorption and about 2 times that of covalent attachment. Among them, the coupling efficiency = (absorbance value of labeled Ab2 / absorbance value of total Ab2) × 100. Further, in the in-situ encapsulation method, if the suspension and metal salt are added to the pre-mixed solution simultaneously, it is not conducive to the uniform distribution of Ab2 on MOFs-Ab2.

[0042] Example 6: Characterization of the spatial position distribution of urease and antibody in the Urease@MOFs@MOFs-Ab2 structure To determine the distribution of urease and Ab2 in the nanostructure, Urease@MOFs@MOFs-Ab2 was synthesized using fluorescein isothiocyanate (FITC)-labeled urease (FITC-Urease) and rhodamine B-labeled Ab2 (RhB-Ab2) as components. The preparation methods of FITC-Urease and RhB-Ab2 refer to the literature Control of structure topology and spatial distribution of biomacromolecules in protein@ZIF-8 biocomposites. Chem. Mater. 2018, 30, 1069-1077.

[0043] After mixing 100 μL of FITC-Urease solution (2 mg / mL), 200 μL of PVP solution (0.5 mg / mL), and 100 μL of Cys solution (4 mg / mL) in a 2 mL centrifuge tube and stirring for 30 seconds, 400 μL of 2-mIM (160 mmol / L) and 400 μL of Zn(NO3)2·6H2O solution (40 mmol / L) were added respectively, and aged at room temperature for 4 hours. Then, the precipitate was collected by centrifugation at 10000 rpm for 10 min, washed twice with ultrapure water, and dispersed in 300 μL of ultrapure water. Next, 100 μL of RhB-Ab2 solution with a concentration of 10 mg / mL was slowly added dropwise to 400 μL of 2-mIM solution with a concentration of 160 mmol / L. Then, the above UPC@MOFs suspension was redissolved to 300 μL with ultrapure water, added to the pre-mixed Ab2 and 2-mIM, and then 400 μL of Zn(NO3)2·6H2O (40 mmol / L) with a concentration of 40 mmol / L was added and mixed for 30 seconds, and vortexed at 800 rpm at 25°C for 30 min. Finally, the precipitate was collected by centrifugation at 8000 rpm for 5 min, washed twice with ultrapure water, and dispersed in an appropriate amount of ultrapure water and stored in a refrigerator at 4°C for later use. The obtained sample was tested using a fluorescence confocal microscope to determine the distribution positions of the two proteins (urease and Ab2) in MOFs.

[0044] As Figure 7 shown, where FITC represents the fluorescence confocal image obtained by labeling urease with the green fluorescent dye fluorescein isothiocyanate (FITC), RhB represents the fluorescence confocal image obtained by labeling Ab2 with the red fluorescent dye rhodamine B (RhB), and Merge represents the image after merging the FITC and RhB fluorescence confocal images. The results show that the distribution of the two proteins (urease and Ab2) in MOFs is that urease is distributed in the inner layer and Ab2 is distributed in the outer layer, realizing the ordered hierarchical distribution of enzyme-antibody.

[0045] Example 7: Construction of an immunosensor based on Urease@MOFs@MOFs-Ab2 and detection of isocarbophos First, coat the antigen on the microplate in advance. When isocarbophos is present, the coated antigen and isocarbophos will competitively bind to the anti-isocarbophos monoclonal antibody. The amount of the coated antigen-antibody immune complex formed on the well is inversely proportional to the isocarbophos concentration. After binding to Urease@MOFs@MOFs-Ab2, a signal pathway is generated, and then Urea and phenol red solution are added as colorimetric substrates for signal amplification. Urease on the composite material catalyzes the hydrolysis of Urea to produce ammonia, significantly increasing the pH value of the solution, and then causing an obvious color change in the pH indicator (phenol red solution), with an increase in absorbance at 560 nm. According to the indirect competition mode, the higher the concentration of isocarbophos, the less the amount of the bound Urease@MOFs@MOFs-Ab2 probe, and the yellower the color. Therefore, the concentration of isocarbophos is negatively correlated with the absorbance at 560 nm. With the concentration of the isocarbophos standard as the abscissa and B / B0 as the ordinate, a standard curve is plotted through a four-parameter nonlinear equation. The traditional enzyme-linked immunosorbent assay method uses commercially available HRP-labeled Ab2, and the chromogenic substrate is TMB.

[0046] Figure 8 (A)shows the schematic diagram for the detection of isocarbophos based on Urease@MOFs@MOFs-Ab2. As Figure 8 shown in (B), the standard curve of isocarbophos gives IC 50 = 6.27 ng / mL, with a linear range of 1.56 - 25.25 ng / mL, IC 10 = 0.69 ng / mL, and R 2 = 0.99. The detection limit of this method for isocarbophos can be as low as 0.69 ng / mL, which is about 4 times lower than that of the conventional HRP-based ELISA Figure 8 (C), and it can be used for the detection of isocarbophos in samples, showing broad application prospects.

[0047] Example 8: Construction of an immunosensor based on Urease@MOFs@MOFs-Ab2 and evaluation of the detection performance for isocarbophos The excellent specificity and anti-interference ability of Urease@MOFs@MOFs-Ab2 are beneficial for establishing an accurate and reliable immunosensor. By introducing other organophosphorus pesticides (profenofos, bromophos, monocrotophos, dimethoate, methamidophos, dichlorvos, and triazophos) for comparative determination, the results are as Figure 9 shown in (A). When adding 100-fold equivalent concentrations of other pesticides, the inhibition rate (IE%) is all below 10%. When adding isocarbophos, the IE% value is as high as about 58%. In addition, the influence of adding other common substances (such as ions, amino acids, and proteins, etc.) on the immunosensing system is relatively small Figure 9((B)), indicating that the sensor has excellent anti-interference performance. The above results show that the method established in the present invention has good specificity and anti-interference ability for the detection of isocarbophos. Among them, the inhibition rate = (absorbance value without isocarbophos - absorbance value with isocarbophos) ÷ (absorbance value without isocarbophos - absorbance value without isocarbophos and anti-isocarbophos antibody) × 100%.

[0048] To further evaluate the reliability of the immunosensor, the spiked recovery rate of the method for isocarbophos was investigated. The actual sample solutions of pitaya, pear and tea after pretreatment were used to prepare isocarbophos solutions with concentrations of 0, 10, 20 and 30 ng / mL. This solution was used to replace the isocarbophos solutions with different concentrations used to construct the standard curve, and the remaining operations were the same. The absorbance values at 560 nm finally obtained were substituted into the standard curve, so that the concentration of isocarbophos in the added solution could be calculated.

[0049] The results are shown in Table 1. The actual sample solutions (pitaya, pear and tea) containing different concentrations of isocarbophos were analyzed 3 times. The spiked recovery rates were between 92.4–110.6%, and the coefficient of variation was less than 4.92% (n = 3). The results show that the immunosensor based on Urease@MOFs@MOFs-Ab2 has good accuracy and precision in detecting isocarbophos residues in complex biological samples. Among them, the spiked recovery rate = (measured value of spiked sample - measured value of blank sample) ÷ spiked amount × 100%. These satisfactory results prove the feasibility of using the immunosensor constructed with Urease@MOFs@MOFs-Ab2 for complex biological matrices, and can directly detect trace amounts of isocarbophos residues in foods.

[0050] Table 1: Detection of isocarbophos in actual samples using the immunosensor prepared by the present invention (n = 3) Note: ND: Not detected, not detected.

[0051] It should be noted that in this article, relational terms such as first and second are only used to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Moreover, the term "comprising", "including" or any other variant thereof is intended to cover non-exclusive inclusion, so that a process, method, article or device comprising a series of elements not only includes those elements, but also includes other elements not expressly listed, or also includes elements inherent to such process, method, article or device.

[0052] As described above, it is only the preferred specific embodiment of the present invention, but the protection scope of the present invention is not limited thereto. Any person skilled in the art within the technical scope disclosed by the present invention, according to the technical solution and inventive concept of the present invention, making equivalent substitutions or changes, shall be covered by the protection scope of the present invention.

[0053] Obviously, the embodiments described above are only a part of the embodiments of the present application, rather than all of the embodiments. The preferred embodiments of the present application are given in the drawings, but do not limit the patent scope of the present application. The present application can be implemented in many different forms. On the contrary, the purpose of providing these embodiments is to make the understanding of the disclosed content of the present application more thorough and comprehensive. Although the present application has been described in detail with reference to the foregoing embodiments, for those skilled in the art, they can still modify the technical solutions described in the foregoing specific embodiments, or make equivalent substitutions for some of the technical features. Any equivalent structure made by using the content of the specification and drawings of the present application, directly or indirectly applied in other related technical fields, shall be within the scope of the patent protection of the present application by the same token.

Claims

1. A method for preparing an immunoprobe for orderly immobilizing bioenzymes and antibodies, characterized in that, It includes the following steps: S1. Add 2-methylimidazole and a metal salt solution to a mixed solution of polyvinylpyrrolidone and cysteine containing a biological enzyme, and age at room temperature; After aging, centrifuge to obtain a precipitate, wash and redisperse it to obtain a signal generating element suspension; S2. Slowly drop the antibody solution into the 2-methylimidazole solution, then add the signal generating element suspension prepared in step S1, then add the metal salt solution, and perform vortex oscillation; finally, centrifuge to collect the precipitate, wash and redisperse it to obtain an immunoprobe integrating the dual functions of antibody recognition and signal generation.

2. The method according to claim 1, wherein In step S1, the concentration of cysteine is 2-6 mg / mL; the concentration of 2-methylimidazole is 80-600 mM; the concentration of the biological enzyme is 10-30 mg / mL.

3. The method according to claim 1, characterized in that, In step S2, the added concentration of the antibody is 5-20 mg / mL.

4. The method according to claim 1, characterized in that, In steps S1 and S2, the metal salt is a transition metal salt.

5. The method according to claim 1, wherein The concentration of the metal salt is 20-60 mmol / L.

6. The method according to claim 1, characterized in that In step S2, stir and mix before performing vortex oscillation.

7. The method according to claim 1, wherein In step S2, the vortex oscillation is specifically performed at 25 °C and 800 rpm for 30 min.

8. An immune probe, characterized in that, It is prepared by the method according to any one of claims 1-7.

9. The immunoprobe according to claim 8, wherein The biological enzyme is urease.

10. Use of the immunoprobe according to claim 9 for detecting isocarbophos.

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