Magnetic nanoscale enzyme microspheres, preparation method and application thereof

Abstract

The application discloses a kind of magnetic nano-enzyme microspheres and preparation method and application thereof, belong to the cross technical field of material, drug, rapid detection etc..The magnetic nano-enzyme microspheres take Fe3O4 microspheres as core, and platinum nanoparticles are in situ grown after being coated with dendritic mesoporous silica on the surface.The magnetic nano-enzyme microspheres of the application utilize magnetic response separation, high load content and enzyme catalytic amplification, greatly improve the detection sensitivity and signal strength, and are suitable for high-sensitivity detection of trace chloramphenicol and hexachlorobenzene.The qualitative detection cutoff value of chloramphenicol is 0.1 ng / mL, and the quantitative detection sensitivity is 0.0011 ng / mL.The quantitative detection sensitivity of hexachlorobenzene is 0.4 ng / mL.

Description

Technical Field

[0001] This invention relates to the interdisciplinary fields of materials, pharmaceuticals, and rapid detection, and particularly to a magnetic nanoenzyme microsphere, its preparation method, and its application. Background Technology

[0002] Chloramphenicol (CAP) is a broad-spectrum antibiotic that was once widely used in livestock and aquaculture. However, it has serious toxic side effects, leading to fatal risks such as aplastic anemia and bone marrow suppression. Therefore, most countries, including China, the European Union, and the United States, have banned its use in food animals and have established strict residue limits in animal-derived foods (such as honey, milk, aquatic products, and meat), typically requiring "not detectable." Developing rapid, sensitive, and accurate methods for detecting chloramphenicol residues is of great significance for ensuring food safety and meeting regulatory requirements.

[0003] Hexachlorobenzene (HCB) is a typical persistent organic pollutant (POP) and one of the first 12 typical POPs included in the Stockholm Convention's global control list. HCB was once widely used in agriculture as a seed dressing fungicide, but it is also a byproduct of chemical processes and an unintentional source of emissions from chlorination processes. HCB is persistent, bioaccumulative, and has long-distance migration characteristics; it is difficult to degrade in the environment and can accumulate and amplify in organisms through the food chain. The International Agency for Research on Cancer (IARC) of the World Health Organization classifies it as a Group 2B carcinogen. my country has banned the production, distribution, use, and import / export of HCB since May 17, 2009. Despite this, due to historical residues and unintentional industrial emissions, HCB pollution in environmental media such as soil and water remains a prominent problem, posing a long-term potential risk to the ecological environment and human health.

[0004] Currently, the detection of chloramphenicol and hexachlorobenzene residues mainly relies on instrumental analytical methods, such as high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS / MS) and gas chromatography (GC). These methods offer advantages such as high sensitivity and accuracy, but suffer from drawbacks including expensive equipment, complex operation, cumbersome pretreatment, and long detection cycles, making them unsuitable for the needs of grassroots testing units and rapid on-site screening. In contrast, immunoassay methods, with their advantages of high specificity, ease of operation, and low cost, have played a crucial role in the rapid screening of trace contaminants in food and environmental samples. Among them, lateral-flow immunoassay (LFIA) has become one of the preferred techniques for rapid on-site detection due to its ease of operation, intuitive results, and lack of complex equipment; enzyme-linked immunosorbent assay (ELISA), with its high throughput and quantitative analysis capabilities, is widely used in laboratory batch testing. In traditional LFIA, colloidal gold nanoparticles (AuNPs) are the most commonly used signal labeling probes. However, traditional AuNPs-LFIA has inherent limitations in practical applications, such as low sensitivity and significant matrix interference, making it difficult to meet the needs of trace detection. In traditional ELISA, the preparation and purification of enzyme-labeled antibodies are relatively cumbersome, and the stability of natural enzymes is poor, easily affected by environmental factors such as temperature and pH, which restricts the stability and practicality of the detection method.

[0005] In recent years, nanozymes, as a class of nanomaterials with natural enzyme catalytic activity, have attracted widespread attention in the field of immunoassay due to their advantages such as simple preparation, low cost, and excellent stability. By integrating magnetic nanomaterials with nanozymes into multifunctional composite probes, magnetic enrichment and separation of target analytes and enzymatic amplification of signals can be achieved simultaneously, significantly improving detection sensitivity and reducing matrix interference. For example, magnetic nanozymes with peroxidase-like activity can catalyze the colorimetric reaction of substrates (such as 3,3',5,5'-tetramethylbenzidine) in the presence of hydrogen peroxide, thereby achieving exponential signal amplification. In LFIA, magnetic nanozyme probes can reduce matrix interference in the sample through magnetic enrichment, while simultaneously enhancing the colorimetric signal using the catalytic activity of nanozymes, overcoming the sensitivity bottleneck of traditional AuNPs-LFIA. In ELISA, magnetic nanozymes, as novel labeled probes, can replace traditional natural enzymes, significantly improving the stability of the method, while simplifying the operation process through magnetic separation.

[0006] However, research on simultaneously applying magnetic nanozymes to the detection of chloramphenicol via lateral flow immunochromatography and hexachlorobenzene via ELISA is currently lacking. Both chloramphenicol and hexachlorobenzene are small molecule haptens, and their immunoassays require a competitive mode, placing higher demands on the sensitivity and specificity of the detection methods. Therefore, developing a novel immunoassay platform based on magnetic nanozymes to achieve highly sensitive, visualized, rapid lateral flow immunochromatographic detection of chloramphenicol and high-throughput, high-stability ELISA quantitative detection of hexachlorobenzene is of significant research importance and application value for improving food and environmental safety monitoring systems. Summary of the Invention

[0007] This invention aims to solve the key problems of insufficient sensitivity, weak anti-interference ability, and quantitative detection of hexachlorobenzene in existing rapid detection methods for chloramphenicol. It provides a magnetic nanozyme microsphere, its preparation method, and its application. A multifunctional composite microsphere probe with "magnetic response separation", "high loading content" and "nanozyme catalytic amplification" was successfully constructed and used for rapid detection of chloramphenicol LFIA and detection of hexachlorobenzene by ELISA.

[0008] In a first aspect, the present invention provides a magnetic nanoenzyme microsphere, wherein the magnetic nanoenzyme microsphere has Fe3O4 microspheres as its core, the surface of the Fe3O4 microspheres is coated with dendritic mesoporous silica, and the surface of the dendritic mesoporous silica is coated with polyethyleneimine and then loaded with platinum nanoparticles to form magnetic nanoenzyme microspheres Fe3O4@dSiO2 / Pt.

[0009] Furthermore, the average diameter of the Fe3O4 microspheres is 150 nm to 197 nm; the thickness of the dendritic mesoporous silica is 40 nm to 70 nm, and the pore size is 35 nm to 55 nm.

[0010] A second aspect of the present invention provides a method for preparing the above-mentioned magnetic nanoenzyme microspheres, comprising the following steps: Preparation of Fe3O4 microspheres: The Fe3O4 microspheres were prepared by a solvothermal method and dispersed in water; Preparation of Fe3O4@dSiO2: The heterogeneous microemulsion liquid phase method of Winsor III system was adopted. The dendritic mesoporous silica was grown on the surface of Fe3O4 microspheres by in-situ hydrolysis and condensation of tetraethyl orthosilicate to obtain Fe3O4@dSiO2, which was then dispersed in water. Preparation of Fe3O4@dSiO2-PEI: A polyethyleneimine solution and a Fe3O4@dSiO2 solution were mixed and subjected to ultrasonic and oscillation treatment to coat the Fe3O4@dSiO2 material surface with polyethyleneimine, thereby obtaining the Fe3O4@dSiO2-PEI, which was then dispersed in water; Preparation of magnetic nanoenzyme microspheres Fe3O4@dSiO2 / Pt: Pt nanoparticles were grown on the surface of Fe3O4@dSiO2-PEI by in-situ growth to obtain the magnetic nanoenzyme microspheres Fe3O4@dSiO2 / Pt.

[0011] Furthermore, the preparation method shall at least satisfy one of the following (1)-(4): (1) The preparation of Fe3O4 microspheres includes: dissolving ferric chloride hexahydrate and citrate in ethylene glycol solution, adding sodium acetate and stirring evenly, reacting at 190℃~210℃ for 9 h~11 h, and washing to obtain Fe3O4 precipitate; (2) The preparation of Fe3O4@dSiO2 includes: dissolving hexadecyltrimethylammonium bromide and urea in water, stirring, then adding the Fe3O4 microspheres and a cyclohexane solution containing n-pentanol and tetraethyl orthosilicate, stirring vigorously, and then refluxing the entire mixed solution in an oil bath at 65℃~75℃ for 15 h~17 h; after the reaction is completed, the precipitate is collected by magnetic separation and washed with ethanol, and then the precipitate is refluxed in an acetone solution at 75℃~85℃ to fully remove hexadecyltrimethylammonium bromide from the dendritic silica channels, and finally washing to obtain the Fe3O4@dSiO2; (3) The preparation of Fe3O4@dSiO2-PEI includes: taking Fe3O4@dSiO2 as a template and dispersing it in water, then adding polyethyleneimine, sonicating the mixed solution to homogenize it, and then shaking it in a shaker for 1 h to 2 h before washing it with water to obtain Fe3O4@dSiO2-PEI; (4) The preparation of magnetic nanoenzyme microspheres Fe3O4@dSiO2 / Pt includes: dispersing Fe3O4@dSiO2-PEI in water, then adding potassium chloroplatinate and polyvinylpyrrolidone, mechanically stirring evenly in an ice bath, adding sodium borohydride solution, and continuously stirring in an ice bath for 20 min to 100 min, obtaining the magnetic nanoenzyme microspheres Fe3O4@dSiO2 / Pt after magnetic separation and water washing.

[0012] Furthermore, the preparation method satisfies at least one of the following conditions: The mass concentration of the ferric chloride hexahydrate in the ethylene glycol solution is controlled to be 0.030 g / mL to 0.045 g / mL; The mass ratio of the Fe3O4 microspheres to the volume ratio of the tetraethyl orthosilicate is controlled to be 100 mg: (0.85~0.95) mL; The mass concentration of Fe3O4@dSiO2 in water is controlled to be 1.5 mg / mL to 2.5 mg / mL, and the mass ratio of Fe3O4@dSiO2 to polyethyleneimine is (1.5~2.5):1; The mass ratio of Fe3O4@dSiO2-PEI to potassium chloroplatinate is controlled to be 1:(0.5~2.5), and the molecular weight of polyvinylpyrrolidone is 10000~59000.

[0013] A third aspect of the present invention provides the application of the above-described magnetic nanoenzyme microspheres in the preparation of products for chloramphenicol detection or products for hexachlorobenzene detection.

[0014] In a fourth aspect, the present invention provides a chloramphenicol detection probe, which uses the above-mentioned magnetic nanoenzyme microspheres as a carrier, wherein streptavidin is adsorbed on the surface of the carrier, biotinylated chloramphenicol antibody is specifically bound to the streptavidin, and the non-specific binding sites on the surface of the carrier are blocked by bovine serum albumin.

[0015] A fifth aspect of the present invention provides an immunochromatographic test strip product based on magnetic nanozyme microsphere enhancement, comprising: Chloramphenicol detection probe, wherein the chloramphenicol detection probe is the chloramphenicol detection probe described above; The test strip includes a sample pad, a nitrocellulose membrane, an absorbent pad, and a base plate; wherein the sample pad, the nitrocellulose membrane, and the absorbent pad are disposed on the base plate, the sample pad and the absorbent pad are respectively disposed on both sides of the nitrocellulose membrane, and the nitrocellulose membrane has a detection line coated with chloramphenicol-bovine serum albumin conjugate and a control line coated with anti-IgG antibody; The instruction manual describes a method of use in which the sample to be tested is mixed with the chloramphenicol detection probe, purified by magnetic enrichment and magnetic separation, and then the resuspended sample solution is dropped onto the sample pad of the test strip for chromatographic detection.

[0016] In a sixth aspect, the present invention provides a hexachlorobenzene detection probe, which uses the above-mentioned magnetic nanoenzyme microspheres as a carrier, wherein hexachlorobenzene antibody is adsorbed on the surface of the carrier, and the non-specific binding sites on the surface of the carrier are blocked by bovine serum albumin.

[0017] A seventh aspect of the present invention provides an ELISA product based on magnetic nanoenzyme microspheres, comprising: A hexachlorobenzene detection probe, wherein the hexachlorobenzene detection probe is the hexachlorobenzene detection probe described above; A multi-well plate, wherein the antigen is coated with hexachlorobenzene and non-specific binding sites are blocked using bovine serum albumin.

[0018] Compared with the prior art, the present invention has at least the following beneficial effects: The magnetic nanoenzyme microspheres Fe3O4@dSiO2 / Pt provided by this invention have Fe3O4 microspheres as the core, with Pt platinum nanoparticles loaded onto the surface after being coated with dendritic mesoporous silica. Fe3O4 provides magnetic concentration and separation functions, while the dendritic mesoporous silica (dSiO2) has a large specific surface area and loading space, allowing a large number of Pt nanoparticles with superior enzyme catalytic activity to be modified onto the dSiO2 surface. Compared with other types of magnetic nanoenzymes (such as dSiO2 / Fe3O4 / Pt...), this invention... Compared with PEI and Fe-Au@Pt-D, the magnetic nanoenzyme microspheres of this invention possess high catalytic activity, high magnetic response, and good stability. The magnetic nanoenzyme microspheres Fe3O4@dSiO2 / Pt utilize their magnetic enrichment, magnetic separation, and enzyme catalysis functions to greatly improve detection sensitivity and signal intensity. For the qualitative detection of chloramphenicol using immunochromatography, the detection limit is 0.1 ng / mL, and the quantitative detection limit is 0.0011 ng / mL. For the ELISA detection of hexachlorobenzene, the detection limit is 0.4 ng / mL. Attached Figure Description

[0019] To more clearly illustrate the technical solutions in the embodiments of the present invention, the accompanying drawings used in the description of the embodiments will be briefly introduced below. Obviously, the accompanying drawings described below are only some embodiments recorded in the embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings.

[0020] Figure 1 Scanning electron microscope (SEM), transmission electron microscope (TEM), and EDS elemental distribution maps of Fe3O4, Fe3O4@dSiO2, and Fe3O4@dSiO2 / Pt provided in Embodiment 1 of the present invention; wherein, Figure 1 a is a diagram showing the preparation steps. Figure 1 b- Figure 1 Images d are SEM images of Fe3O4, Fe3O4@dSiO2, and Fe3O4@dSiO2 / Pt, respectively. Figure 1 e- Figure 1 g are TEM images of Fe3O4, Fe3O4@dSiO2, and Fe3O4@dSiO2 / Pt, respectively. Figure 1 h1 is a STEM image of Fe3O4@dSiO2 / Pt. Figure 1 h2- Figure 1 h8 is the EDS elemental distribution diagram of Fe3O4@dSiO2 / Pt.

[0021] Figure 2SEM images of Fe3O4 prepared under different amounts of ferric chloride hexahydrate provided in Examples 2 and 1 of the present invention, and SEM images of Fe3O4@dSiO2 prepared under different tetraethyl orthosilicate feeding conditions provided in Examples 3 and 1 of the present invention; wherein Figure 2 a- Figure 2 The corresponding amounts of ferric chloride hexahydrate for c are 0.972g, 1.134g, and 1.296g, respectively. Figure 2 d~ Figure 2 The corresponding amounts of tetraethyl orthosilicate for f are 0.75 mL, 0.85 mL, and 0.95 mL, respectively.

[0022] Figure 3 Example 4 of this invention provides surface potential diagrams, hydration particle size, and polydispersity index diagrams of Fe3O4@dSiO2 / Pt using PVP with molecular weights of 10,000, 24,000, 40,000, or 59,000 as stabilizers, showing the effect of reaction time. Figure 3 a- Figure 3 d corresponds to the surface potential diagrams of Fe3O4@dSiO2 / Pt when the molecular weight of PVP is 10000, 24000, 40000, and 59000 respectively; Figure 3 e- Figure 3 h corresponds to the hydrated particle size and polydispersity index of Fe3O4@dSiO2 / Pt when the molecular weight of PVP is 10000, 24000, 40000, and 59000, respectively.

[0023] Figure 4 Example 5 of this invention uses PVP with molecular weights of 10,000, 24,000, 40,000, or 59,000 as a stabilizer to prepare Fe3O4@dSiO2 / Pt with increasing synthesis time. K m ( Figure 4 a) and K cat ( Figure 4 b).

[0024] Figure 5 The magnetic capture efficiency of Fe3O4@dSiO2 / Pt in PBS buffer as a function of separation time, as provided in Example 6 of this invention ( Figure 5 a) and its magnetic capture efficiency after incubation in PBS for different times ( Figure 5 b).

[0025] Figure 6 The surface potential of Fe3O4@dSiO2 / Pt and the prepared probe provided in Example 7 of the present invention ( Figure 6 a) and hydrated particle size diagram ( Figure 6 b).

[0026] Figure 7 The diagram shows the process of magnetic separation, magnetic concentration, and detection on a test strip using a chloramphenicol detection probe, as provided in Embodiment 8 of the present invention; wherein, the reference numerals are 1, sample pad, 2, nitrocellulose membrane, 3, absorbent pad, and 4, base plate.

[0027] Figure 8 The chloramphenicol detection probe provided in Example 9 of this invention was incubated at chloramphenicol concentrations of 0 ng / mL, 0.00156 ng / mL, 0.00313 ng / mL, 0.00625 ng / mL, 0.0125 ng / mL, 0.025 ng / mL, 0.05 ng / mL, 0.1 ng / mL, 0.2 ng / mL, 0.4 ng / mL, and 0.8 ng / mL. The results were then applied to a test strip. A photograph was taken with a mobile phone, and the grayscale value of the T line on the test strip was extracted. The relationship between the grayscale value and the chloramphenicol concentration was then analyzed. Figure 8 a); After adding the catalytic colorimetric solution, take a photo with a mobile phone and identify the gray value of the T line on the test strip. Then, analyze the relationship between the gray value and the concentration of chloramphenicol. Figure 8 b).

[0028] Figure 9 This is a schematic diagram illustrating the preparation of the hexachlorobenzene detection probe and the detection of hexachlorobenzene on a 96-well plate, as provided in Example 10 of the present invention.

[0029] Figure 10 The relationship between absorbance and hexachlorobenzene concentration was described in Example 10 of this invention, where a hexachlorobenzene detection probe was incubated at hexachlorobenzene concentrations of 0 ng / mL, 0.4 ng / mL, 0.8 ng / mL, 1.6 ng / mL, 3.2 ng / mL, 6.4 ng / mL, 12.8 ng / mL, 25.6 ng / mL, 51.2 ng / mL, 102.4 ng / mL, and 204.8 ng / mL, followed by colorimetric development of 3,3',5,5'-tetramethylbenzidine and detection on a microplate reader. Detailed Implementation

[0030] To better understand the above technical solutions, the technical solutions of the embodiments of this application will be described in detail below with reference to the accompanying drawings and specific embodiments. It should be understood that the embodiments of this application and the specific features in the embodiments are detailed descriptions of the technical solutions of the embodiments of this application, rather than limitations on the technical solutions of this application. In the absence of conflict, the embodiments of this application and the technical features in the embodiments can be combined with each other.

[0031] In a first aspect, the present invention provides a magnetic nanoenzyme microsphere, wherein the magnetic nanoenzyme microsphere has Fe3O4 microspheres as its core, the surface of the Fe3O4 microspheres is coated with dendritic mesoporous silica, and the surface of the dendritic mesoporous silica is coated with polyethyleneimine and then loaded with platinum nanoparticles to form magnetic nanoenzyme microspheres Fe3O4@dSiO2 / Pt.

[0032] The Fe3O4 microspheres have an average diameter of 150 nm to 197 nm; the dendritic mesoporous silica has a thickness of 40 nm to 70 nm and a pore size of 35 nm to 55 nm.

[0033] Understandably, given the limited surface space of Fe3O4 supports, it is difficult to load large quantities of nanozymes, and the enzyme-like catalytic activity of nanozymes is highly dependent on surface-active catalytic sites. Therefore, the catalytic activity of the internal nanozyme building blocks may be inhibited. Silica, with its large and open mesopores, due to its large pore size, high specific surface area, good hydrophilicity, and ease of modification, can serve as an excellent catalyst support for firmly immobilizing nanozyme building blocks and effectively preventing random leakage.

[0034] The magnetic nanoenzyme microspheres Fe3O4@dSiO2 / Pt provided in this invention have Fe3O4 microspheres as the core, with dendritic mesoporous silica grown in situ on the surface, followed by the growth of Pt platinum nanoparticles. Fe3O4 provides magnetic concentration and separation functions, while the dendritic mesoporous silica has a large specific surface area and loading space, allowing a large number of Pt nanoparticles with superior enzyme catalytic activity to be modified onto the dSiO2 surface. Utilizing its magnetic enrichment, magnetic separation, and enzyme catalytic functions, the magnetic nanoenzyme microspheres Fe3O4@dSiO2 / Pt significantly improve detection sensitivity and signal intensity, achieving a cutoff value as low as 0.1 ng / mL and a detection limit as low as 0.0011 ng / mL for immunochromatographic detection of chloramphenicol.

[0035] A second aspect of the present invention provides a method for preparing the above-mentioned magnetic nanoenzyme microspheres, comprising the following steps: Preparation of Fe3O4 microspheres: Fe3O4 microspheres were prepared by a solvothermal method and dispersed in water; Preparation of Fe3O4@dSiO2: The heterogeneous microemulsion liquid phase method of Winsor III system was adopted. Dendritic mesoporous silica was grown on the surface of Fe3O4 microspheres by in-situ hydrolysis and condensation of tetraethyl orthosilicate to obtain Fe3O4@dSiO2, which was then dispersed in water. Preparation of Fe3O4@dSiO2-PEI: Polyethyleneimine solution and Fe3O4@dSiO2 solution were mixed and subjected to ultrasonic and oscillation treatment to coat the surface of Fe3O4@dSiO2 material with polyethyleneimine, thus obtaining Fe3O4@dSiO2-PEI, which was then dispersed in water; Preparation of magnetic nanoenzyme microspheres Fe3O4@dSiO2 / Pt: Pt nanoparticles were grown on the surface of Fe3O4@dSiO2-PEI by in-situ growth to obtain magnetic nanoenzyme microspheres Fe3O4@dSiO2 / Pt.

[0036] In this embodiment of the invention, a solvothermal method was used to synthesize Fe3O4. This method utilizes a high-temperature, high-pressure, and sealed environment to provide the high temperature and reducing atmosphere required for crystal growth, thereby significantly improving the crystallinity and saturation magnetization of Fe3O4 and preventing its oxidation. Simultaneously, by flexibly controlling the solvent, surfactant, and reaction parameters, the particle size, morphology, and surface functionalization of the nanoparticles can be precisely controlled, achieving a narrow particle size distribution and good monodispersity. Furthermore, this method is relatively simple, has good batch-to-batch reproducibility, and overcomes the limitations of traditional wet chemical synthesis methods in terms of weak magnetic response, easy agglomeration, and uncontrollable morphology. The in-situ growth of dendritic mesoporous silicon employs a heterogeneous microemulsion liquid phase method based on the Winsor III system, providing a unique water-oil interface network where the silicon source hydrolyzes and deposits, ultimately forming radial channels radiating from the center outwards. By simply adjusting the water-oil-surfactant ratio or co-solvent, the product structure can be precisely controlled from ordinary mesoporous to dendritic folds, and even the spacing between folds can be controlled. This achieves in-situ, direct, and uniform coating of the core material, as well as wide-range and broad-size control of pore size. The advantages of modifying amino groups on the dSiO2 surface with polyethyleneimine via electrostatic adsorption are: modification in aqueous solution, mild operating conditions, and environmental safety; polyethyleneimine is a cationic polymer rich in amino groups with extremely high amino density; in aqueous solution, positively charged PEI can be directly adsorbed onto the negatively charged silica surface through electrostatic interaction, making it simple and easy to control. The advantages of growing Pt nanoparticles on the Fe3O4@dSiO2-PEI surface through in-situ growth are: utilizing the strong reducing properties of sodium borohydride to achieve small size control, utilizing the steric hindrance of PVP to achieve high dispersion and stability, and achieving a strong coupling interface through in-situ growth. The resulting composite material typically exhibits: ultra-small particle size, high loading, high dispersion, and excellent catalytic stability.

[0037] In some embodiments, the preparation of Fe3O4 microspheres includes: Ferric chloride hexahydrate and citrate were dissolved in ethylene glycol solution, sodium acetate was added and stirred evenly, and the reaction was carried out at 190℃~210℃ for 9 h~11 h. After washing, Fe3O4 precipitate was obtained. The mass concentration of ferric chloride hexahydrate in the ethylene glycol solution was controlled to be 0.030 g / mL to 0.045 g / mL.

[0038] Specifically, the preparation method of Fe3O4 microspheres achieves synergistic optimization of product particle size, dispersibility, and magnetism by precisely controlling the precursor concentration, solvent system, and reaction conditions. Ethylene glycol, at high temperatures (190℃~210℃), acts not only as a high-boiling-point solvent but also as a reducing agent. Sodium acetate provides an alkaline environment, promoting the hydrolysis and nucleation of iron ions. Citrate ions adsorb onto the surface during particle growth, acting as a capping agent and dispersant. The optimized concentration range (0.030 g / mL~0.045 g / mL) controls the number of nuclei and balances particle size and magnetism. Within this range, particles with moderate size (e.g., around 150 nm~300 nm, assembled from secondary nanocrystals), strong magnetic response, and good monodispersity are typically obtained. 190℃~210℃ is a typical range where ethylene glycol exhibits good reducing performance while maintaining system stability. 9 h~11 h ensures sufficient crystal growth and maturation.

[0039] In some embodiments, the preparation of Fe3O4@dSiO2 includes: Hexadecyltrimethylammonium bromide and urea were dissolved in water and stirred. Then, Fe3O4 microspheres and a cyclohexane solution containing n-pentanol and tetraethyl orthosilicate were added. After vigorous stirring, the entire mixture was refluxed in an oil bath at 65℃~75℃ for 15h~17h. After the reaction was completed, the precipitate was collected by magnetic separation and washed with ethanol. Then, the precipitate was refluxed in an acetone solution at 75℃~85℃ to fully remove hexadecyltrimethylammonium bromide from the dendritic silica channels. Finally, Fe3O4@dSiO2 was obtained after washing. The mass ratio of Fe3O4 microspheres to tetraethyl orthosilicate was controlled to be 100 mg: (0.85~0.95) mL.

[0040] Specifically, the advantages of the Fe3O4@dSiO2 preparation method are: (1) Clear core-shell structure: Monodisperse Fe3O4 cores are uniformly encapsulated. (2) Unique shell structure: Dendritic / wrinkled mesoporous shell with open and accessible pores, which is beneficial for subsequent modification and loading. (3) Strong magnetic responsiveness: Low-temperature template removal avoids magnetic oxidation. (4) Clean pores: CTAB is completely removed, resulting in high specific surface area and large adsorption capacity. (5) Good repeatability: Specific ratio and temperature parameters provide a good process window.

[0041] In some embodiments, the preparation of Fe3O4@dSiO2-PEI includes: Fe3O4@dSiO2 was used as a template and dispersed in water. Polyethyleneimine was then added, and the mixture was homogenized by sonication. After shaking in a shaker for 1-2 hours, the mixture was washed with water to obtain Fe3O4@dSiO2-PEI. The mass concentration of Fe3O4@dSiO2 in water was controlled to be 1.5 mg / mL to 2.5 mg / mL, and the mass ratio of Fe3O4@dSiO2 to polyethyleneimine was (1.5~2.5):1.

[0042] Specifically, the core of the Fe3O4@dSiO2-PEI preparation method is to utilize an electrostatic self-assembly process to efficiently and gently load polyethyleneimine onto the surface of a pre-fabricated Fe3O4@dSiO2 core-shell structure. Specific parameters (concentration, ratio, and time) have been optimized to achieve uniform coating of the PEI layer, prevent particle aggregation, and maximize the surface amino function.

[0043] In some embodiments, the preparation of magnetic nanoenzyme microspheres Fe3O4@dSiO2 / Pt includes: Fe3O4@dSiO2-PEI was dispersed in water, then potassium chloroplatinate and polyvinylpyrrolidone were added. After mechanical stirring in an ice bath until homogeneous, sodium borohydride solution was added and stirred continuously in an ice bath for 20 min to 100 min. After magnetic separation and washing with water, magnetic nanoenzyme microspheres Fe3O4@dSiO2 / Pt were obtained. The mass ratio of Fe3O4@dSiO2-PEI to potassium chloroplatinate is controlled at 1:(0.5~2.5), the molecular weight of polyvinylpyrrolidone is 10000~59000; preferably, the molecular weight of polyvinylpyrrolidone is 40000, and the preferred reaction time is 60 min.

[0044] Specifically, the advantages of the magnetic nanoenzyme microsphere Fe3O4@dSiO2 / Pt preparation method are as follows: (1) The platinum nanoparticles are ultra-small and uniform in size: thanks to the synergistic effect of ice bath and PVP, the size of platinum nanoparticles is usually <5 nm. (2) Excellent dispersibility: the steric hindrance of PVP and the anchoring effect of PEI together prevent the migration and aggregation of platinum on the inner and outer surfaces of the mesoporous structure. (3) Strong bonding: the chemical anchoring of PEI ensures that the platinum nanoparticles are not easily detached even after multiple ultrasonic washings or catalytic cycles. (4) Accessible active sites: PVP with a molecular weight of 40,000 is preferred, which provides protection while ensuring that the substrate molecules can contact the platinum surface and maintain high enzyme-like catalytic activity. (5) Stable structure: all reactions are carried out in a low-temperature aqueous phase, which completely preserves the pore structure of dendritic mesoporous silica and the magnetism of Fe3O4.

[0045] A third aspect of this invention provides the application of the above-described magnetic nanozyme microspheres in the preparation of products for chloramphenicol detection or for hexachlorobenzene detection. The chloramphenicol detection products include, but are not limited to, lateral flow immunoassay strips; the hexachlorobenzene detection products include, but are not limited to, ELISA.

[0046] In a fourth aspect of the present invention, a chloramphenicol detection probe is provided, which uses the above-mentioned magnetic nanoenzyme microspheres as a carrier, with streptavidin adsorbed on the surface of the carrier, biotinylated chloramphenicol antibody specifically binding to the streptavidin, and the non-specific binding sites on the surface of the carrier being blocked by bovine serum albumin.

[0047] The preparation method of the above-mentioned chloramphenicol detection probe includes the following steps: Magnetic nanozyme microspheres were ultrasonically dispersed in a phosphate buffer solution with a pH of 5.0–8.0, and streptavidin solution was added. The mixture was then shaken and incubated to allow streptavidin to adsorb onto the surface of the magnetic nanozyme microspheres. After magnetic separation and washing, the microspheres were resuspended in phosphate buffer solution at pH 6.5-7.5, biotinylated chloramphenicol antibody was added, and the mixture was incubated at room temperature with shaking. The probe was blocked by adding bovine serum albumin solution. After the reaction was completed, the probe was washed and resuspended in PBST buffer to obtain the chloramphenicol detection probe. The mass ratio of streptavidin to magnetic nanozyme microspheres is (0.02~0.2):1; the mass ratio of biotinylated chloramphenicol antibody to magnetic nanozyme microspheres is (0.01~0.2):1; and the mass concentration of bovine serum albumin solution is 1%~15%. Preferably, the mass ratio of streptavidin to magnetic nanozyme microspheres is 0.1:1; the mass ratio of biotinylated chloramphenicol antibody to magnetic nanozyme microspheres is 0.05:1; and the mass concentration of bovine serum albumin solution is 10%.

[0048] A fifth aspect of this invention provides an immunochromatographic test strip product enhanced with magnetic nanozyme microspheres, comprising: the aforementioned chloramphenicol detection probe, test strip, and instructions; wherein, Test strips, such as Figure 7 As shown, the test strip includes a sample pad 1, a nitrocellulose membrane 2, an absorbent pad 3, and a base plate 4. The sample pad 1, the nitrocellulose membrane 2, and the absorbent pad 3 are disposed on the base plate 4. The sample pad 1 and the absorbent pad 3 are respectively disposed on both sides of the nitrocellulose membrane 2. The nitrocellulose membrane 2 has a detection line T (CAP-BSA) and a control line C (goat anti-mouse IgG) drawn on it. Optionally, the base plate 4 is a polyvinyl chloride base plate.

[0049] The instruction manual states that when using this immunochromatographic test strip, the solution to be tested should be mixed evenly with the above-mentioned chloramphenicol detection probe, and then placed on the sample pad after passing through magnetic enrichment, magnetic separation, and redispersion steps.

[0050] The process of detecting chloramphenicol using a chloramphenicol detection probe, including magnetic separation, magnetic concentration, and detection on a test strip, is as follows: Figure 7As shown, the test sample containing chloramphenicol is mixed with the chloramphenicol detection probe and incubated, and then separated and enriched under the action of a magnetic field; the supernatant is discarded, and the magnetic bead precipitate is resuspended in a buffer solution to obtain a purified sample solution; the sample solution is dropped onto the sample pad 1 of the test strip for chromatography; the qualitative or quantitative detection of chloramphenicol is achieved by reading the signal intensity at the T line.

[0051] In a sixth aspect of the present invention, a hexachlorobenzene detection probe is provided, which uses the above-mentioned magnetic nanoenzyme microspheres as a carrier, the surface of the carrier is adsorbed with hexachlorobenzene antibody, and the non-specific binding sites on the surface of the carrier are blocked by bovine serum albumin.

[0052] A seventh aspect of the present invention provides a method for preparing the above-described hexachlorobenzene detection probe, comprising the following steps: Magnetic nanozyme microspheres were ultrasonically dispersed in phosphate buffer solution at pH 5.0–8.0. Streptavidin solution was added, and the mixture was shaken and incubated to allow streptavidin to adsorb onto the surface of the magnetic nanozyme microspheres. After magnetic separation and washing, the microspheres were resuspended in phosphate buffer solution at pH 6.5–7.5, and biotinylated hexachlorobenzene antibody was added. The mixture was then shaken and incubated at room temperature. Bovine serum albumin solution was added for blocking. After the reaction was completed, the microspheres were washed and resuspended in PBST buffer to obtain the hexachlorobenzene detection probe. The mass ratio of biotinylated hexachlorobenzene antibody to magnetic nanoenzyme microspheres is (0.01~0.2):1; the mass concentration of bovine serum albumin solution is 1%~15%. Preferably, the mass ratio of hexachlorobenzene antibody to magnetic nanoenzyme microspheres is 0.02:1; the mass concentration of bovine serum albumin solution is 10%.

[0053] An eighth aspect of the present invention provides an ELISA product based on magnetic nanoenzyme microspheres, wherein the magnetic nanoenzyme microspheres described above are used as a carrier, the surface of the carrier is adsorbed with biotinylated hexachlorobenzene antibody, and the non-specific binding sites on the surface of the carrier are blocked by bovine serum albumin.

[0054] Example 1: A method for preparing magnetic nanoenzyme microspheres See Figure 1 A method for preparing magnetic nanoenzyme microspheres includes the following steps: (1) Preparation of Fe3O4 microspheres: First, 1.134 g FeCl3·6H2O and 0.318 g sodium citrate dihydrate were dissolved in 30 mL of ethylene glycol solution. Then, 1.8 g sodium acetate was added and stirred for 30 minutes. Finally, the mixed solution was placed in a 50 mL reactor and reacted at 200 °C for 10 h. After the reactor cooled naturally, the obtained Fe3O4 precipitate was washed three times with ethanol and water respectively and stored in an aqueous solution for later use.

[0055] (2) Preparation of Fe3O4@dSiO2: 3g of hexadecyltrimethylammonium bromide and 0.9g of urea were dissolved in 45 mL of water and stirred mechanically. Then, 100 mg of Fe3O4 and 45 mL of cyclohexane solution containing 0.85 mL of n-pentanol and 0.85 mL of tetraethyl orthosilicate (TEOS) were added. After vigorous stirring for 1 h, the entire mixture was refluxed in an oil bath at 70 °C for 16 h. After the reaction was completed, the precipitate was collected by magnetic separation and washed three times with ethanol. Then, the precipitate was refluxed in acetone solution at 80 °C for 48 h to fully remove hexadecyltrimethylammonium bromide from the dendritic silica channels. The final Fe3O4@dSiO2 was washed twice each with ethanol and aqueous solution and stored in 40 mL of aqueous solution for later use.

[0056] (3) Preparation of Fe3O4@dSiO2 encapsulated with polyethyleneimine (Fe3O4@dSiO2-PEI): 40 mg of the above Fe3O4@dSiO2 template was dispersed in 20 mL of aqueous solution, followed by the addition of 20 mg of polyethyleneimine (PEI) with a molecular weight of 1800. The mixture was homogenized by ultrasonic assistance. After shaking in a shaker for 1.5 hours, the mixture was washed three times with water, and the resulting Fe3O4@dSiO2-PEI microspheres were redispersed in 20 mL of aqueous solution.

[0057] (4) Preparation of Fe3O4@dSiO2 / Pt: 3.75 mL of Fe3O4@dSiO2-PEI solution was subjected to magnetic separation to remove the supernatant and uniformly dispersed in 9 mL of ultrapure water. Then, 0.75 mL of K2PtCl4 (20.8 mg / mL) and 1 mL of 15 mg / mL PVP with a molecular weight of 40,000 were added to the above solution and mechanically stirred in an ice bath for 10 min. 0.75 mL of NaBH4 (29.12 mg / mL) was added to the above solution and stirred continuously in an ice bath for 1 h. Fe3O4@dSiO2 / Pt was obtained by magnetic separation, the supernatant was removed, and the solution was washed twice with water and then redispersed in 4 mL of ultrapure water.

[0058] The scanning electron microscopy and transmission electron microscopy characterization results of Fe3O4, Fe3O4@dSiO2 and Fe3O4@dSiO2 / Pt obtained in Example 1 are as follows: Figure 1 This indicates that the magnetic nanoenzyme microspheres were successfully prepared.

[0059] Example 2 The difference between Example 2 and Example 1 is that the amount of FeCl3·6H2O used in step (1) is set to 0.972g or 1.296g respectively, and the rest is the same as in Example 1.

[0060] Scanning electron microscopy results as follows Figure 2 As shown, the diameter of the prepared Fe3O4 increases from 150 nm to 197 nm as the amount of FeCl3·6H2O increases. Here, in preferred Example 1, when the amount of FeCl3·6H2O used is 1.134 g, the prepared Fe3O4 has an average diameter of approximately 180 nm. Figure 2 b) Used for subsequent in-situ growth of dendritic mesoporous silicon.

[0061] Example 3 The difference between Example 3 and Example 1 is that the amount of TEOS used in step (2) is set to 0.75 mL or 0.95 mL respectively, and the rest is the same as in Example 1.

[0062] Scanning electron microscopy results as follows Figure 2 As shown, this indicates that with the increase of TEOS feed amount, 0.75 mL TEOS ( Figure 2 d) The silicon source is insufficient under these conditions, making it difficult to generate dSiO2. Example 1 used 0.85 mL of TEOS (… Figure 2 Under conditions e), dSiO2 with a thickness of approximately 50 nm and a pore size of approximately 50 nm is produced. 0.95 mL TEOS ( Figure 2 f) To produce dSiO2 with a thickness of approximately 65 nm and a pore size of approximately 35 nm. Here, the amount of TEOS is preferably 0.85 mL.

[0063] Example 4 Example 4 investigated the surface potential, hydrated particle size, and polydispersity index of Fe3O4@dSiO2 / Pt under the following conditions: the molecular weight of PVP in step (4) of Example 1 was set to 10000, 24000, 40000, or 59000, and NaBH4 was added. The reaction time was set to 20 min, 40 min, 60 min, 80 min, or 100 min in an ice bath. The remaining preparation steps were the same as in Example 1.

[0064] like Figure 3 As shown, the surface potential of Fe3O4@dSiO2 / Pt gradually decreases and tends to stabilize with increasing reaction time, while the hydrated particle size and polydispersity index gradually increase. Here, a reaction time of 60 min is preferred.

[0065] Example 5 Example 5 uses the magnetic nanozyme microspheres prepared in Example 4 as signal tags. The enzyme activity of the magnetic nanozyme microspheres is measured using acetate / sodium acetate buffer, 3,3',5,5'-tetramethylbenzidine, and hydrogen peroxide to better reflect the nanozyme activity. Michaelis constant. K m and catalytic constant KcatIt is a characteristic physical quantity of enzyme activity.

[0066] like Figure 4 As shown, when the reaction time is 60 min, Fe3O4@dSiO2 / Pt prepared using PVP with a molecular weight of 40000 as a stabilizer exhibits relatively low [resistance / lowering]. K m and the highest Kcat Here, a molecular weight of 40,000 is preferred for PVP.

[0067] Example 6 Example 6 examines the magnetic capture efficiency of Fe3O4@dSiO2 / Pt prepared in Example 1 in PBS buffer, considering the increase in magnetic capture efficiency with separation time and the magnetic capture efficiency after incubation in PBS for different times. Figure 5 As shown in Figure a, under an applied magnetic field, its capture efficiency gradually increases with time, approaching 100% after two minutes, verifying its rapid magnetic separation capability; furthermore, Figure 5 b indicates that Fe3O4@dSiO2 / Pt maintains a stable magnetic capture efficiency during the incubation period of 0~12 hours.

[0068] Example 7: Preparation method of chloramphenicol or hexachlorobenzene detection probe The preparation method of chloramphenicol or hexachlorobenzene detection probe includes: ultrasonically dispersing 2 mg Fe3O4@dSiO2 / Pt obtained in Example 1 in 1 mL PB (pH 5.5, 10 mM) buffer solution, then adding 0.1 mg streptavidin and shaking in a shaker for 2 h, washing twice with PB (pH 5.5, 10 mM) solution after shaking and redispersing in 1 mL PB (pH 7.0, 10 mM) buffer solution, then adding 0.05 mg biotinylated chloramphenicol antibody or biotinylated hexachlorobenzene antibody, shaking at room temperature for 0.5 h, and then adding 200 µL 10% bovine serum albumin (BSA) for blocking for 0.5 h; after the reaction, washing twice with PB and resuspending in PBST (pH 7.4, 10 mM) buffer solution to obtain chloramphenicol detection probe or hexachlorobenzene detection probe.

[0069] like Figure 6 As shown, after Fe3O4@dSiO2 / Pt was modified into a probe, the surface potential increased from -21.9 mV to -5.7 mV, and the hydrated particle size increased from 409.2 nm to 541.2 nm, indicating that a chloramphenicol detection probe was successfully prepared.

[0070] Example 8: Preparation method of chloramphenicol competitive immunochromatographic test strip The preparation method of chloramphenicol competitive immunochromatographic test strips includes the following steps: (1) The untreated sample pad was soaked in a 0.02 mol / L PB solution (pH=7.4) containing 1% BSA, 2% sucrose and 2% Tween 20 for 1 min. After soaking, the sample pad was removed from the solution and dried in an oven at 37°C for 12 hours.

[0071] (2) The T-line coating solution was a chloramphenicol-bovine serum albumin conjugate dispersion (0.3 mg / mL), and the C-line coating solution was a PBS solution of goat anti-mouse IgG (2 mg / mL). The T-line and C-line coating solutions were sprayed onto the nitrocellulose membrane at a rate of 0.75 μL / cm to serve as the T-line and C-line, respectively. The sample pad and nitrocellulose membrane were then dried overnight at 37°C.

[0072] (3) Assemble the sample pad, nitrocellulose membrane and absorbent pad onto the polyvinyl chloride base plate with a 2 mm overlap, cut into strips 3.2 mm wide, and store in a desiccator.

[0073] like Figure 7 As shown, from left to right on the base plate 4 are sample pad 1, nitrocellulose membrane 2, and absorbent pad 3.

[0074] Example 9: Plotting standard photographs and standard curves of chloramphenicol test strips Chloramphenicol standards were prepared using 20 mM pH 7.4 PBS solution to obtain chloramphenicol concentrations of 0 ng / mL, 0.00156 ng / mL, 0.00313 ng / mL, 0.00625 ng / mL, 0.0125 ng / mL, 0.025 ng / mL, 0.05 ng / mL, 0.1 ng / mL, 0.2 ng / mL, 0.4 ng / mL, and 0.8 ng / mL for test strip detection. The chloramphenicol detection probe prepared in Example 7 was then added to 1 mL of the standard solution and incubated for 5 min, followed by magnetic separation for 10 min. The supernatant was then discarded, and the precipitate was redispersed in 90 μL of 10 mM PBS solution to obtain a sample solution. For detection, 80 μL of the sample solution was added to the sample pad. After 10 min, a photograph was taken using a smartphone. Subsequently, 2.5 μL of catalytic colorimetric solution (a mixed solution of N,N-dimethylformamide / water in a volume ratio of 1 / 1, containing 80 mM 3,3',5,5'-tetramethylbenzidine, 2 M hydrogen peroxide, and 50 mM pH 3.8 acetic acid / sodium acetate) was added dropwise to the T line and C line respectively.

[0075] like Figure 8As shown, before catalytic color development, the cutoff value of the test strip was 0.1 ng / mL; a linear relationship existed in the range of 0.00156 ng / mL to 0.025 ng / mL, and the limit of detection (LOD) was 0.0042 ng / mL. After catalytic color development, the cutoff value was 0.4 ng / mL; a linear relationship existed in the range of 0 ng / mL to 0.025 ng / mL, and the limit of detection (LOD) was 0.0011 ng / mL.

[0076] Example 10: Detection of hexachlorobenzene by ELISA and plotting of the standard curve like Figure 9 As shown, the detection of hexachlorobenzene on a 96-well plate includes the following steps: (1) Pretreatment of microplate: Add 100 μL of 1% polyethyleneimine (PEI) solution to each well of the 96-well plate and let it stand at room temperature (20℃~25℃) for 1 hour; discard the solution and wash twice with 20 mM pH7.4 phosphate buffer (PB); then add 100 μL of acetate-sodium acetate buffer containing 1% glutaraldehyde to each well and let it stand at room temperature for 1 hour; then wash twice with the same PB buffer.

[0077] (2) Immobilization of the coated antigen: Add 100 μL of coating solution to each well after the above treatment. The coating solution is 20 mM PB buffer (pH 7.4) containing 5 μg hexachlorobenzene coated antigen. Place the 96-well plate in a 4°C refrigerator for 12 hours to allow the antigen to be fully adsorbed. Discard the coating solution and wash 3 times with PB buffer.

[0078] (3) Blocking: Add 150 μL of 5% bovine serum albumin (BSA) solution to each well and let stand at room temperature for 2 hours; discard the blocking solution and wash 3 times with PBST washing buffer (20 mM PB, pH 7.4, containing 1% Tween-20 by volume).

[0079] (4) Sample addition and competitive reaction: Take 5 μL of the pre-prepared hexachlorobenzene detection probe (the preparation process of the hexachlorobenzene detection probe is as follows) Figure 9 As shown in the figure, the solution was added dropwise to each well, and each well contained a standard solution of hexachlorobenzene at different concentrations (prepared with an aqueous solution containing 20% ​​methanol by volume, with a hexachlorobenzene concentration range of 0~204.8 ng / mL); and incubated at room temperature for 30 minutes.

[0080] (5) Washing: Discard the reaction solution and wash 3 times with PBST washing solution.

[0081] (6) Colorimetric reaction: Add 100 μL of substrate buffer (containing 40 mM acetate-sodium acetate buffer and 20 mM hydrogen peroxide) and 100 μL of 5 mM 3,3',5,5'-tetramethylbenzidine solution to each well in sequence, and catalyze color development at room temperature for 10 minutes.

[0082] (7) Absorbance determination: The absorbance value of each well at a wavelength of 652 nm was measured using an enzyme-linked immunosorbent assay (ELISA) reader, and the content of hexachlorobenzene in the sample was calculated based on the standard curve.

[0083] like Figure 10 As shown, after catalytic color development, a non-linear curve relationship exists in the range of 0.4 ng / mL to 204.8 ng / mL, and the limit of detection (LOD) is 0.4 ng / mL.

[0084] It will be readily understood by those skilled in the art that the above-described advantageous methods can be freely combined and superimposed without conflict. The above are merely preferred embodiments of this application and are not intended to limit this application. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of this application should be included within the protection scope of this application. The above are merely preferred embodiments of this application. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the technical principles of this application, and these improvements and modifications should also be considered within the protection scope of this application.

Claims

1. A magnetic nanoenzyme microsphere, characterized in that, The magnetic nanoenzyme microspheres have Fe3O4 microspheres as the core, and the surface of the Fe3O4 microspheres is coated with dendritic mesoporous silica. The surface of the dendritic mesoporous silica is then coated with polyethyleneimine and loaded with platinum nanoparticles to form magnetic nanoenzyme microspheres Fe3O4@dSiO2 / Pt.

2. The magnetic nanoenzyme microspheres according to claim 1, characterized in that, The Fe3O4 microspheres have an average diameter of 150 nm to 197 nm; the dendritic mesoporous silica has a thickness of 40 nm to 70 nm and a pore size of 35 nm to 55 nm.

3. A method for preparing magnetic nanoenzyme microspheres according to claim 1 or 2, characterized in that, Includes the following steps: Preparation of Fe3O4 microspheres: The Fe3O4 microspheres were prepared by a solvothermal method and dispersed in water; Preparation of Fe3O4@dSiO2: The heterogeneous microemulsion liquid phase method of Winsor III system was adopted. The dendritic mesoporous silica was grown on the surface of Fe3O4 microspheres by in-situ hydrolysis and condensation of tetraethyl orthosilicate to obtain Fe3O4@dSiO2, which was then dispersed in water. Preparation of Fe3O4@dSiO2-PEI: A polyethyleneimine solution and a Fe3O4@dSiO2 solution were mixed and subjected to ultrasonic and oscillation treatment to coat the Fe3O4@dSiO2 material surface with polyethyleneimine, thereby obtaining the Fe3O4@dSiO2-PEI, which was then dispersed in water; Preparation of magnetic nanoenzyme microspheres Fe3O4@dSiO2 / Pt: Pt nanoparticles were grown on the surface of Fe3O4@dSiO2-PEI by in-situ growth to obtain the magnetic nanoenzyme microspheres Fe3O4@dSiO2 / Pt.

4. The preparation method according to claim 3, characterized in that, The preparation method shall satisfy at least one of the following (1)-(4): (1) The preparation of Fe3O4 microspheres includes: dissolving ferric chloride hexahydrate and citrate in ethylene glycol solution, adding sodium acetate and stirring evenly, reacting at 190℃~210℃ for 9 h~11 h, and washing to obtain Fe3O4 precipitate; (2) The preparation of Fe3O4@dSiO2 includes: dissolving hexadecyltrimethylammonium bromide and urea in water, stirring, then adding the Fe3O4 microspheres and a cyclohexane solution containing n-pentanol and tetraethyl orthosilicate, stirring vigorously, and then refluxing the entire mixed solution in an oil bath at 65℃~75℃ for 15 h~17 h; after the reaction is completed, the precipitate is collected by magnetic separation and washed with ethanol, and then the precipitate is refluxed in an acetone solution at 75℃~85℃ to fully remove hexadecyltrimethylammonium bromide from the dendritic silica channels, and finally washing to obtain the Fe3O4@dSiO2; (3) The preparation of Fe3O4@dSiO2-PEI includes: taking Fe3O4@dSiO2 as a template and dispersing it in water, then adding polyethyleneimine, sonicating the mixed solution to homogenize it, and then shaking it in a shaker for 1 h to 2 h before washing it with water to obtain Fe3O4@dSiO2-PEI; (4) The preparation of magnetic nanoenzyme microspheres Fe3O4@dSiO2 / Pt includes: dispersing Fe3O4@dSiO2-PEI in water, then adding potassium chloroplatinate and polyvinylpyrrolidone, mechanically stirring evenly in an ice bath, adding sodium borohydride solution, and continuously stirring in an ice bath for 20 min to 100 min, obtaining the magnetic nanoenzyme microspheres Fe3O4@dSiO2 / Pt after magnetic separation and water washing.

5. The preparation method according to claim 4, characterized in that, The preparation method shall satisfy at least one of the following conditions: The mass concentration of the ferric chloride hexahydrate in the ethylene glycol solution is controlled to be 0.030 g / mL to 0.045 g / mL; The mass ratio of the Fe3O4 microspheres to the volume ratio of the tetraethyl orthosilicate is controlled to be 100 mg: (0.85~0.95) mL; The mass concentration of Fe3O4@dSiO2 in water is controlled to be 1.5 mg / mL to 2.5 mg / mL, and the mass ratio of Fe3O4@dSiO2 to polyethyleneimine is (1.5~2.5):1; The mass ratio of Fe3O4@dSiO2-PEI to potassium chloroplatinate is controlled to be 1:(0.5~2.5), and the molecular weight of polyvinylpyrrolidone is 10000~59000.

6. The use of the magnetic nanoenzyme microspheres according to claim 1 or 2 in the preparation of products for chloramphenicol detection or products for hexachlorobenzene detection.

7. A chloramphenicol detection probe, characterized in that, It uses the magnetic nanoenzyme microspheres as described in claim 1 or 2 as a carrier, wherein streptavidin is adsorbed on the surface of the carrier, and biotinylated chloramphenicol antibody is specifically bound to the streptavidin, and the non-specific binding sites on the surface of the carrier are blocked by bovine serum albumin.

8. An immunochromatographic test strip product enhanced with magnetic nanoenzyme microspheres, characterized in that, include: A chloramphenicol detection probe, wherein the chloramphenicol detection probe is the chloramphenicol detection probe according to claim 7; The test strip includes a sample pad (1), a nitrocellulose membrane (2), an absorbent pad (3), and a base plate (4); wherein the sample pad (1), the nitrocellulose membrane (2), and the absorbent pad (3) are disposed on the base plate (4), the sample pad (1) and the absorbent pad (3) are respectively disposed on both sides of the nitrocellulose membrane (2), and the nitrocellulose membrane (2) has a detection line coated with chloramphenicol-bovine serum albumin conjugate and a control line with anti-IgG antibody. The instruction manual describes the method of use as follows: mix the sample to be tested with the chloramphenicol detection probe, and after magnetic enrichment and magnetic separation purification, add the resuspended sample solution to the sample pad (1) of the test strip for chromatographic detection.

9. A hexachlorobenzene detection probe, characterized in that, It uses the magnetic nanoenzyme microspheres as described in claim 1 or 2 as a carrier, wherein the surface of the carrier is adsorbed with hexachlorobenzene antibody, and the non-specific binding sites on the surface of the carrier are blocked by bovine serum albumin.

10. An ELISA product based on magnetic nanoenzyme microspheres, characterized in that, include: A hexachlorobenzene detection probe, wherein the hexachlorobenzene detection probe is the hexachlorobenzene detection probe according to claim 9; A multi-well plate, wherein the antigen is coated with hexachlorobenzene and non-specific binding sites are blocked using bovine serum albumin.

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