Preparation method of cobalt monatomic nano enzyme probe modified by polydopamine and detection device of cobalt monatomic nano enzyme probe modified by polydopamine

Abstract

The invention relates to a preparation method of a polydopamine modified cobalt monatomic nano-enzyme probe and a detection device thereof, and relates to the technical field of immunochromatography detection. The preparation method of the CoSAN (at) PDA probe comprises the following steps: S1, preparing CoSAN, and carrying out biocompatibility modification on the surface of the CoSAN by using polydopamine (PDA) to obtain CoSAN (at) PDA; and S2, combining the CoSAN (at) PDA with a tetracycline monoclonal antibody to form the probe. The technology can be used for rapid and sensitive detection of tetracycline residues in food and environmental samples, and has good repeatability and stability.

Description

Technical Field

[0001] The present application relates to the technical field of immunochromatographic detection, and more particularly to a method for preparing a polydopamine (PDA)-modified cobalt single atom nanozyme (CoSAN, Co Single Atom Nanozyme) probe and a detection device thereof. Background Art

[0002] Tetracycline (TC) is a type of antibiotic widely used in clinical medical treatment, animal husbandry and aquaculture. Due to its long-term and high-frequency use, the residues of tetracycline and its metabolites in food and the natural environment have been increasing year by year. These residues enter the human body through the ecological circulation system and the food chain, which not only greatly increases the risk of the spread of microbial resistance, but also poses a potential serious threat to human health.

[0003] The prevention and control of tetracycline pollution is currently carried out from three key dimensions: source control, food safety monitoring, and environmental sample testing. Among these prevention and control measures, efficient and portable on-site detection technology is the most critical and direct pollution control method, which can make accurate judgments on the pollution situation at the first time and provide a strong basis for subsequent control measures.

[0004] Immunoassay technology is based on the principle of specific binding between antigens and antibodies, and plays a vital role in the field of antibiotic residue monitoring and pollution prevention and control. The core of this technology is to use the highly specific recognition ability of antibodies for target antigens, and with the help of signal amplification mechanisms, to achieve accurate qualitative or quantitative detection of the target. Common immunoassay methods include enzyme-linked immunosorbent assay (ELISA), fluorescent immunoassay, chemiluminescent immunoassay, etc. However, these traditional immunoassay methods generally have problems such as cumbersome and complicated operating procedures, the need to be equipped with expensive professional testing equipment, and time-consuming sample pre-treatment. They are difficult to meet the actual needs of rapid on-site detection and are greatly restricted in practical applications.

[0005] Lateral flow immunochromatography (LFIA) is a paper-based detection platform based on antigen-antibody specific binding and nanoparticle labeling technology. It has significant advantages such as low cost, fast detection speed, and easy portability, and has certain application potential in the field of on-site detection. However, in terms of tetracycline detection, traditional LFIA has the problem of low sensitivity, and relies on expensive enzyme labels or precious metal nanomaterials as signal amplification labels during the detection process, which not only increases the detection cost, but also further limits its practical application scope. In addition, single-mode detection technology generally has the problems of insufficient sensitivity and limited anti-interference ability. When faced with complex samples, it is easily affected by background interference and it is difficult to meet the high-precision requirements of trace analysis.

[0006] In summary, the development of a multimodal immunochromatographic method based on highly catalytically active nanozymes for tetracycline detection has important practical significance and application value. Summary of the invention

[0007] The embodiment of the present application provides a method for preparing a CoSAN@PDA probe and a detection device thereof, aiming to solve the problem that the traditional immunochromatography method is insufficiently sensitive in tetracycline detection and cannot meet the detection requirements of trace residues. At the same time, the signal label cost is high and the detection range is limited, making it difficult to achieve economical and efficient detection.

[0008] In order to solve the above technical problems, the technical solution proposed in this application is:

[0009] The present application provides a method for preparing a CoSAN@PDA probe, comprising the following steps:

[0010] S1: preparing CoSAN, and modifying the surface of the CoSAN with polydopamine (PDA) for biocompatibility to obtain CoSAN@PDA;

[0011] S2: The CoSAN@PDA is combined with tetracycline monoclonal antibody to form a probe.

[0012] Furthermore, in S2:

[0013] CoSAN@PDA and monoclonal antibody were slowly stirred in buffer A at low temperature, BSA was added for blocking, and then centrifuged at low temperature and washed repeatedly with ultrapure water to obtain the bottom precipitate, which was dissolved in buffer B and dispersed evenly to obtain the probe solution.

[0014] Further, in S1, the preparation of CoSAN comprises the following steps:

[0015] (1) 7-14 parts of silicon dioxide, 15-25 parts of glucosamine, 1-7 parts of cobalt chloride hexahydrate, and 1-7 parts of zinc chloride are mixed in ultrapure water, and ultrasonicated for 20-40 minutes to disperse uniformly. The resulting mixed solution is frozen at -20°C for 4 hours and then overnight to obtain a lyophilized powder;

[0016] (2) grinding the powder obtained in step (1) and placing it in a tube furnace, burning it at 800-1000° C. for 1-4 hours with argon gas, and etching the obtained powder with 30% hydrofluoric acid for 10-14 hours;

[0017] (3) The etched solution is freeze-dried using a freeze dryer, and the resulting powder is ground to obtain CoSAN.

[0018] Further, in S1, modifying the surface of the CoSAN for biocompatibility with polydopamine (PDA) comprises the following steps:

[0019] Hydrogen peroxide was added dropwise to the CoSAN solution, and then a Tris-HCl solution containing dopamine hydrochloride was added dropwise to the CoSAN solution, and the mixture was stirred at room temperature for 1 h. After centrifugation, the mixture was washed with deionized water for several times and then resuspended in buffer C to obtain a CoSAN@PDA solution.

[0020] Furthermore, buffer A, buffer B or buffer C is at least one of Tris-hydrochloric acid buffer, phosphate buffer, sodium acetate buffer, sodium citrate buffer or borax buffer.

[0021] On the other hand, the present application also relates to a trimodal immunochromatographic detection device for detecting tetracycline based on CoSAN, characterized in that it comprises:

[0022] A test paper, comprising a sample pad, a nitrocellulose (NC) membrane and a water-absorbing pad, wherein the nitrocellulose (NC) membrane is sprayed with a test line and a control line formed by TC-OVA and GAM;

[0023] A container for implementing a competitive immune response, wherein the container can be filled with one of the CoSAN@PDA probes prepared above;

[0024] Detection equipment used to observe signal output, capable of capturing and identifying black signals, TMB colorimetric reactions, or luminol chemiluminescent reactions.

[0025] Furthermore, the test paper is inserted into the container filled with CoSAN@PDA probe solution, and the following three-mode signal outputs are included:

[0026] (1) After the test paper reaction is completed, the signal generated by the inherent black color of CoSAN@PDA on the T line and C line is used as an indicator. The detection equipment directly takes a photo and analyzes the gray value to calculate the tetracycline concentration of the sample;

[0027] (2) After the test paper reacts, add color developing solution to the T line and C line. After a period of reaction, a dark blue signal appears. The detection equipment takes a photo and analyzes the gray value to calculate the tetracycline concentration of the sample;

[0028] (3) After the test strip has reacted, add luminescent solution to the T line and the C line. After a period of reaction, use a chemiluminescence imaging system to record the luminescent signal of the test strip, take an image and analyze the signal. After the detection equipment measures the gray value, the tetracycline concentration of the sample can be obtained.

[0029] Furthermore, the color developing solution is H2O2 and precipitated TMB solution.

[0030] Furthermore, the luminescent solution is a solution of H2O2, luminol and 4-iodophenol.

[0031] Furthermore, the test paper is inserted into the container filled with CoSAN@PDA probe solution, including: mixing the probe solution and buffer C in a well plate to obtain a mixed solution, then adding the sample extract to the mixed solution, vertically inserting the strip into the mixed solution, waiting for a certain period of time, selecting any mode, taking a photo to analyze the grayscale value, and measuring the tetracycline concentration in the sample.

[0032] Compared with the prior art, the preparation method of a polydopamine-modified cobalt single-atom nanozyme probe and its detection device in the present application have achieved the following beneficial technical effects:

[0033] The enzyme marker in the probe solution in this application is CoSAN@PDA with peroxidase activity. This marker can not only gather on the test line with its inherent black color to output signals, but also rely on the peroxide activity of CoSAN@PDA to convert hydrogen peroxide into active oxygen, which can not only convert the colorless precipitated TMB into a blue-purple complex, but also make luminol and 4-iodophenol produce chemiluminescence, thereby achieving dual amplification of color development signals and chemiluminescence signals, greatly improving the sensitivity and signal stability of the tetracycline test strip detection method. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] In order to more clearly illustrate the embodiments of the present application or the technical solutions in the prior art, a brief introduction will be given below to the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are some embodiments of the present application. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0035] Figure 1 Schematic diagram of the immunochromatographic principle of detecting tetracycline based on cobalt single-atom nanozyme of the present invention

[0036] Figure 2 This is a scanning electron microscope image of CoSAN@PDA according to an embodiment of the present invention. DETAILED DESCRIPTION

[0037] In order to make the purpose, technical solution and advantages of the embodiments of the present application clearer, the technical solution in the embodiments of the present application will be clearly and completely described below in conjunction with the drawings in the embodiments of the present application. Obviously, the described embodiments are part of the embodiments of the present application, not all of the embodiments. Based on the embodiments in the present application, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of this application.

[0038] The meanings of the terms in this application are explained as follows and used throughout this application: Cobalt single-atom nanozyme: CoSAN; Polydopamine: PDA; Cobalt single-atom nanozyme modified with polydopamine (PDA): CoSAN@PDA, see Figure 2 .

[0039] A method for preparing a CoSAN@PDA probe of the present application comprises the following steps:

[0040] S1: preparing CoSAN, and modifying the surface of the CoSAN with polydopamine (PDA) for biocompatibility to obtain CoSAN@PDA;

[0041] S2: The CoSAN@PDA is combined with tetracycline monoclonal antibody to form a probe.

[0042] In S2:

[0043] CoSAN@PDA and monoclonal antibody were slowly stirred in buffer A at low temperature, BSA was added for blocking, and then centrifuged at low temperature and washed repeatedly with ultrapure water to obtain the bottom precipitate, which was dissolved in buffer B and dispersed evenly to obtain the probe solution.

[0044] In this application, the probe solution consists of a substrate buffer, a tetracycline monoclonal antibody, and CoSAN@PDA with peroxidase activity. Nanozymes are a type of nanomaterial that can simulate the catalytic activity of natural enzymes, with the advantages of low cost, high stability, easy preparation and regulation. CoSAN@PDA combines with tetracycline monoclonal antibodies to form an efficient detection probe, and its peroxidase activity can catalyze hydrogen peroxide (H2O2) to generate reactive oxygen species with strong oxidizing effects. These reactive oxygen species can convert colorless TMB (3,3′,5,5′-tetramethylbenzidine) into a blue-purple complex for color signal amplification, and produce chemiluminescent signals when reacting with luminol and 4-iodophenol.

[0045] In S1, the preparation of CoSAN comprises the following steps:

[0046] (1) 7-14 parts of silicon dioxide, 15-25 parts of glucosamine, 1-7 parts of cobalt chloride hexahydrate, and 1-7 parts of zinc chloride are mixed in ultrapure water, and ultrasonicated for 20-40 minutes to disperse uniformly. The resulting mixed solution is frozen at -20°C for 4 hours and then overnight to obtain a lyophilized powder;

[0047] (2) grinding the powder obtained in step (1) and placing it in a tube furnace, burning it at 800-1000° C. for 1-4 hours with argon gas, and etching the obtained powder with 30% hydrofluoric acid for 10-14 hours;

[0048] (3) The etched solution is freeze-dried using a freeze dryer, and the resulting powder is ground to obtain CoSAN.

[0049] In S1, the biocompatible modification of the surface of the CoSAN using polydopamine (PDA) comprises the following steps:

[0050] Hydrogen peroxide was added dropwise to the CoSAN solution, and then a Tris-HCl solution containing dopamine hydrochloride was added dropwise to the CoSAN solution, and the mixture was stirred at room temperature for 1 h. After centrifugation, the mixture was washed with deionized water for several times and then resuspended in buffer C to obtain a CoSAN@PDA solution.

[0051] The above-mentioned probe of the present application can be used in a trimodal immunochromatographic detection device for detecting tetracycline based on CoSAN, comprising:

[0052] A test paper, comprising a sample pad, a nitrocellulose (NC) membrane and a water-absorbing pad, wherein the nitrocellulose (NC) membrane is sprayed with a test line and a control line formed by TC-OVA and GAM;

[0053] A container for implementing a competitive immune response, wherein the container can be filled with the CoSAN@PDA probe prepared above;

[0054] Detection equipment used to observe signal output, capable of capturing and identifying black signals, TMB colorimetric reactions, or luminol chemiluminescent reactions.

[0055] Specifically, Figure 1 As shown, therein is shown a schematic diagram of the structure of an immunochromatographic test strip. The test strip consists of three main parts: a sample pad 201, a nitrocellulose membrane (NC membrane) 202, and an absorbent pad 203. The test antigen and the secondary antibody are sprayed on the NC membrane to form a test line (T line) 204 and a control line (C line) 205, respectively. The test line consists of a tetracycline antigen and a primary antibody, while the control line consists of a secondary antibody, in order to verify the reliability of the detection process. The backboard card 206 of the test strip preferably uses a PVC board, which not only provides good mechanical strength, but also ensures the stability of the test strip, so that it can maintain consistent performance in different detection environments.

[0056] The device can be used for a trimodal immunochromatographic method for detecting tetracycline based on cobalt single-atom nanozymes. In this method, the CoSAN@PDA probe is enriched in the test line (T line) and the control line (C line) through the specific binding of antigen and antibody to form a visual detection signal. Combining three detection modes (naked eye observation, color enhancement and chemiluminescence enhancement), the detection system can achieve highly sensitive and rapid detection of tetracycline residues.

[0057] In the immunochromatographic detection device of the present application, glucosamine is used as a nitrogen source during the synthesis of CoSAN@PDA nanozyme. Glucosamine as a nitrogen source helps to enhance the biocompatibility of the nanozyme and reduce its toxicity in biological systems, making the method more suitable for the detection of biological samples such as milk, honey, aquatic products, etc.

[0058] In the immunochromatographic detection device of the present application, Tris-hydrochloric acid buffer is used as the buffer type in the preparation of CoSAN@PDA probe. Tris-hydrochloric acid buffer can provide a stable pH environment, protect antibody activity, ensure efficient immune response, and is suitable for the detection of various complex samples containing tetracycline, such as soil, water, animal tissue, etc.

[0059] In the immunochromatographic detection device of the present application, the specific pH value of the buffer in the probe preparation is 7.4. pH 7.4 is the pH value of the human physiological environment, which can ensure the stability and activity of the probe in human sample detection and is suitable for residual detection of tetracycline-related drugs in the medical field, such as blood, urine, etc.

[0060] In the immunochromatographic detection device of the present application, the immunochromatographic reaction is carried out in a well plate container. The use of the well plate container can process multiple samples at the same time, improve the detection efficiency, and is suitable for large-scale food and environmental sample screening, such as food processing plants, farms, water quality monitoring stations, etc.

[0061] In the immunochromatographic detection device of the present application, the test line is composed of tetracycline antigen and primary antibody, and the control line is composed of secondary antibody. This design can ensure the accuracy and repeatability of the test results, and is suitable for scientific research institutions, testing laboratories, food processing companies and other occasions with strict requirements on the test results.

[0062] In the immunochromatographic detection device of the present application, the test strip has a detection range of 0.01 to 100 ppb when detecting tetracycline. The wide detection range enables the method to meet the needs of different scenarios, from extremely low concentration environmental monitoring to high concentration drug residue detection, and can provide accurate results.

[0063] In the immunochromatographic detection device of the present application, the generation of black signal is generated by the redox reaction catalyzed by CoSAN@PDA. The generation of black signal is not interfered by the color of the sample and is suitable for the detection of food and environmental samples of various colors, such as dark beverages, soil, water, etc.

[0064] Preferably, in the immunochromatographic detection device, the detection equipment includes a spectrum analyzer and image recognition software. The spectrum analyzer can accurately measure the signal intensity, and the image recognition software can automatically analyze the test results to reduce human errors, and is suitable for high-throughput detection and automated detection systems, such as food safety testing centers, environmental monitoring stations, etc.

[0065] Example

[0066] Reagents required in the examples: cobalt chloride hexahydrate, zinc chloride, silicon dioxide, D-glucosamine hydrochloride, from Shanghai Solebao Co., Ltd.; hydrofluoric acid, phosphate buffer, acetate buffer, Tris-hydrochloric acid buffer, boric acid, borax, sodium citrate, hydrogen peroxide, 4-iodophenol, luminol and Tween-20, dimethyl sulfoxide from Aladdin Reagent Company. 3,3',5,5'-tetramethylbenzidine from Sigma Reagent Company.

[0067] This embodiment provides a method for preparing CoSAN nanozyme, which is as follows:

[0068] 2.25 g of silicon dioxide, 2.0 g of D-glucosamine hydrochloride and 0.272 g of zinc chloride were dissolved in deionized water, 0.24 g of cobalt chloride hexahydrate was added, the resulting solution was freeze-dried for 12 hours, and the solid was ground to obtain a powder. The powder was pyrolyzed at 900°C for 3 hours at a heating rate of 3°C / min in an argon atmosphere. The powder was etched with 10% hydrofluoric acid for 12 hours and vacuum dried at 65°C overnight to obtain CoSAN.

[0069] Embodiment 2:

[0070] This embodiment provides a method for preparing a CoSAN@PDA probe, which is as follows:

[0071] CoSAN was dissolved in deionized water and ultrasonicated to obtain a 1 mg / mL dispersion solution. Dopamine hydrochloride was dissolved in Tris-HCl to obtain a 5 mg / mL DH-Tris solution. Hydrogen peroxide was diluted to 10%, and 10 uL of hydrogen peroxide dilution was added. After reacting for 5 minutes, 15 uL of DH-Tris solution was added, stirred at 25°C for 1 hour, and centrifuged at 8000 rpm for 30 minutes after stirring. The obtained precipitate was washed 3 times by centrifugation in deionized water and resuspended in a borate buffer with a pH of 8 to obtain CoSAN@PDA. 1 mL of CoSAN@PDA solution (2.0 mg / mL) was mixed with 1 mL of borate buffer (pH = 8.0), and an anti-tetracycline monoclonal antibody solution (8.3 mg / mL) was slowly added dropwise. The mixture was slowly stirred at 4°C for 4 hours, centrifuged at 8000 rpm for 25 minutes, and the precipitate was dissolved in a PBS buffer containing 0.2% Tween-20 and 1.5% (m / v) BSA to obtain the CoSAN@PDA probe.

[0072] Embodiment 3:

[0073] This embodiment provides a method for preparing a test strip, which is as follows:

[0074] Dilute TC-OVA and GAM to appropriate concentrations, evenly distribute them on the test line and control line of the NC membrane with a sprayer, package at 40℃, and dry after 2 hours. Apply the sample pad treatment liquid evenly on the glass fiber membrane and air-dry at room temperature and humidity less than 60%. Using PVC board as the substrate, assemble each part into strips in sequence, cut the test strips into single strips with a cutter, and store at 4℃. Use a cutter to divide the strips into single test strips and store at 4℃.

[0075] Embodiment 3:

[0076] This embodiment provides a signal-free enhanced immunochromatography method for detecting tetracycline based on cobalt single-atom nanozymes:

[0077] In a 96-well plate, add 20 μL of CoSAN@PDA-mAb solution to 90 μL of PBS buffer, then add 40 μL of the sample to be tested, mix for 2 minutes, insert the test paper into the liquid, wait for 3 minutes for gray lines to appear on the test paper, take a picture for grayscale analysis, and obtain the tetracycline concentration in the sample.

[0078] Embodiment 4:

[0079] This embodiment provides a color-enhanced immunochromatographic method for detecting tetracycline based on cobalt single-atom nanozymes:

[0080] In a 96-well plate, 50 μL of CoSAN@PDA-mAb solution was added to 200 μL PBS buffer, and then 50 μL of the sample to be tested was added, mixed for 5 minutes, and the test paper was inserted into the liquid. After waiting for 5 minutes, a gray line appeared on the test paper, and 10 μL of colorimetric solution (acetate buffer, pH = 4, containing 1mM precipitated TMB and 1mM H2O2) was added to the test line to enhance the color development. After 1 minute, an enhanced signal was observed due to peroxidase activity, and a photo was taken and the gray value was analyzed to determine the tetracycline concentration of the sample.

[0081] Embodiment 5:

[0082] This embodiment provides a chemiluminescence enhanced immunochromatography method for detecting tetracycline based on cobalt single-atom nanozymes:

[0083] In a 96-well plate, add 40 μL of CoSAN@PDA-mAb solution to 150 μL of PBS buffer, then add 30 μL of the sample to be tested, mix for 6 minutes, insert the test paper into the liquid, wait for 10 minutes for gray lines to appear on the test paper, and drop 20 μL of the luminescent mixture (consisting of 20 μL 100mM luminol, 50 μL 50mM p-iodophenol, 1mL Tris-HCl buffer, and 1 μL 30% hydrogen peroxide) on the test line and control line of the test paper. After incubation in the dark for 1 minute, the luminescent signal of the test paper is recorded with a chemiluminescence imaging system, and the image is captured or received using a smartphone and the information is directly analyzed. The tetracycline concentration of the sample can be obtained by measuring the grayscale value.

[0084] Through the above detailed description, it can be seen that the immunochromatographic detection method and device of the present application have significant technical advantages. First, by using CoSAN@PDA nanozyme, the sensitivity and accuracy of the detection are improved, ensuring the reliability of food safety and environmental monitoring. Secondly, the design of multimodal signal output enhances the stability of the test results and makes them unaffected by external environmental factors. In addition, the method is easy to operate, suitable for rapid on-site detection, and greatly improves the efficiency of detection.

[0085] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present application, rather than to limit it. Although the present application has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present application.

Claims

1. A method for preparing a CoSAN@PDA probe, characterized in that: The steps include: S1: preparing CoSAN, and modifying the surface of the CoSAN with polydopamine (PDA) for biocompatibility to obtain CoSAN@PDA; S2: The CoSAN@PDA is combined with tetracycline monoclonal antibody to form a probe.

2. The method for preparing the CoSAN@PDA probe according to claim 1, characterized in that: In S2: CoSAN@PDA and monoclonal antibody were slowly stirred in buffer A at low temperature, BSA was added for blocking, and then centrifuged at low temperature and washed repeatedly with ultrapure water to obtain the bottom precipitate, which was dissolved in buffer B and dispersed evenly to obtain the probe solution.

3. The method for preparing the CoSAN@PDA probe according to claim 1, characterized in that: In S1, the preparation of CoSAN comprises the following steps: (1) 7-14 parts of silicon dioxide, 15-25 parts of glucosamine, 1-7 parts of cobalt chloride hexahydrate, and 1-7 parts of zinc chloride are mixed in ultrapure water, and ultrasonicated for 20-40 minutes to disperse uniformly. The resulting mixed solution is frozen at -20°C for 4 hours and then overnight to obtain a lyophilized powder; (2) grinding the powder obtained in step (1) and placing it in a tube furnace, burning it at 800-1000° C. for 1-4 hours with argon gas, and etching the obtained powder with 30% hydrofluoric acid for 10-14 hours; (3) The etched solution is freeze-dried using a freeze dryer, and the resulting powder is ground to obtain CoSAN.

4. The method for preparing the CoSAN@PDA probe according to claim 1, characterized in that: In S1, the biocompatible modification of the surface of the CoSAN using polydopamine (PDA) comprises the following steps: Hydrogen peroxide was added dropwise to the CoSAN solution, and then a Tris-HCl solution containing dopamine hydrochloride was added dropwise to the CoSAN solution, and the mixture was stirred at room temperature for 1 h. After centrifugation, the mixture was washed with deionized water for several times and then resuspended in buffer C to obtain a CoSAN@PDA solution.

5. The method for preparing the CoSAN@PDA probe according to claim 2 or 4, characterized in that: Buffer A, buffer B or buffer C is at least one of Tris-hydrochloric acid buffer, phosphate buffer, sodium acetate buffer, sodium citrate buffer or borax buffer.

6. A trimodal immunochromatographic detection device for detecting tetracycline based on CoSAN, characterized in that: include: A test paper, comprising a sample pad, a nitrocellulose (NC) membrane and a water-absorbing pad, wherein the nitrocellulose (NC) membrane is sprayed with a test line and a control line formed by TC-OVA and GAM; A container for implementing a competitive immune response, wherein the container can be filled with the CoSAN@PDA probe prepared according to any one of claims 1 to 4; Detection equipment used to observe signal output, capable of capturing and identifying black signals, TMB colorimetric reactions, or luminol chemiluminescent reactions.

7. A trimodal immunochromatographic detection device for detecting tetracycline based on CoSAN according to claim 6, characterized in that: The test paper is inserted into the container filled with CoSAN@PDA probe solution, and includes the following three-mode signal output: (1) After the test paper reaction is completed, the signal generated by the inherent black color of CoSAN@PDA on the T line and C line is used as an indicator. The detection equipment directly takes a photo and analyzes the gray value to calculate the tetracycline concentration of the sample; (2) After the test paper reacts, add color developing solution to the T line and C line. After a period of reaction, a dark blue signal appears. The detection equipment takes a photo and analyzes the gray value to calculate the tetracycline concentration of the sample; (3) After the test strip has reacted, add the luminescent reaction solution to the T line and the C line. After a period of reaction, use the chemiluminescence imaging system to record the luminescent signal of the test strip. After setting the parameters to capture the image, the detection equipment measures the gray value to obtain the tetracycline concentration of the sample.

8. A trimodal immunochromatographic detection device for detecting tetracycline based on CoSAN according to claim 7, characterized in that: The color developing solution is H2O2 and precipitated TMB solution.

9. A trimodal immunochromatographic detection device for detecting tetracycline based on CoSAN according to claim 7, characterized in that: The luminescent solution is a solution of H2O2, luminol and 4-iodophenol.

10. A trimodal immunochromatographic detection device for detecting tetracycline based on CoSAN according to claim 7, characterized in that: The test paper is inserted into the container filled with CoSAN@PDA probe solution, comprising: mixing the probe solution and buffer C in a well plate to obtain a mixed solution, then adding the sample extract to the mixed solution, vertically inserting the strip into the mixed solution, waiting for a certain period of time, selecting any mode, taking pictures and analyzing the grayscale value, and measuring the tetracycline concentration in the sample.

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