A rapid detection method of enrofloxacin based on fluorescent covalent organic framework magnetic beads

Abstract

The application discloses a kind of enrofloxacin rapid detection methods based on fluorescent covalent organic framework magnetic beads, belong to food safety detection field.The method uses the fluorescent covalent organic framework magnetic bead of coupling enrofloxacin monoclonal antibody, realizes detection in combination with immunochromatography test strip.Fluorescent covalent organic framework magnetic bead realizes target enrichment separation with inner core, shell fluorescent covalent organic framework material high-efficiency coupling antibody and exports fluorescence signal;Test strip is equipped with detection line (coated enrofloxacin artificial antigen) and quality control line (coated secondary antibody).By comparing the fluorescence intensity of detection line and quality control line, trace enrofloxacin in complex matrix can be qualitatively and quantitatively analyzed.The method is simple, rapid and sensitive, without large instrument, can be detected on site.

Description

Technical Field

[0001] This invention relates to the field of food quality and safety testing technology, specifically to a rapid detection method for enrofloxacin based on fluorescent covalent organic framework magnetic beads. Background Technology

[0002] Enrofloxacin (ENR) is a broad-spectrum fluoroquinolone antibiotic that inhibits bacterial DNA gyrase and topoisomerase IV, thereby blocking DNA replication and achieving a bactericidal effect. This drug has significant advantages such as a broad antibacterial spectrum, strong antibacterial activity, and strong tissue penetration, and is widely used in disease treatment in aquaculture and livestock farming. However, enrofloxacin is not completely metabolized in animals, and its residues can have potential toxic side effects on humans. Furthermore, long-term, high-dose use can induce bacterial resistance, seriously threatening human health. Therefore, establishing a rapid, sensitive, and reliable method for detecting enrofloxacin is crucial for ensuring food safety and standardizing drug use practices.

[0003] Immunochromatographic assay (ICA) has become an important tool for rapid food safety screening due to its outstanding advantages such as speed, low cost, portability, and ease of operation. Traditional ICA methods mainly use gold nanoparticles as signal labeling materials, but these materials have significant problems such as low sensitivity and limited quantitative ability, making it difficult to meet the needs of trace enrofloxacin detection.

[0004] In recent years, various fluorescent labeling materials have been integrated into ICA systems to improve detection sensitivity and quantification capabilities. However, existing fluorescent labeling materials generally suffer from drawbacks such as strong fluorescence bleaching properties, poor photostability, high toxicity, low fluorescence quantum yield, and cumbersome preparation processes, which seriously affect the detection performance of fluorescent ICA.

[0005] Furthermore, when using fluorescent ICA to detect trace amounts of enrofloxacin in food, sample dilution is usually required to reduce the matrix effect. This process simultaneously reduces the concentration of the target analyte, leading to decreased detection sensitivity and potentially false negative results. Therefore, developing novel enrichment and separation techniques that eliminate complex sample pretreatment steps (such as extraction, purification, washing, and centrifugation) to achieve rapid concentration of the target enrofloxacin, avoid sample loss, reduce the influence of the matrix effect, and thus improve detection sensitivity and shorten analysis time, has become crucial for the high-performance and industrialization of enrofloxacin detection technology. Summary of the Invention

[0006] (a) Technical problems to be solved

[0007] To overcome the shortcomings of existing enrofloxacin detection technologies, such as low sensitivity, limited quantitative capability, weak resistance to matrix interference, and cumbersome operation, this invention provides a rapid enrofloxacin detection method based on fluorescent covalent organic framework magnetic beads. This method utilizes a core material that combines magnetic separation and enrichment functions with efficient fluorescence signal output to achieve rapid enrichment and highly sensitive detection of trace enrofloxacin in complex matrices. It not only simplifies the detection process by omitting complex sample pretreatment and immunomagnetic bead elution steps, reducing operation time and sample loss, but also provides both qualitative and quantitative detection capabilities, improving the accuracy and reliability of the detection results. Furthermore, it eliminates the need for sophisticated and complex large-scale instruments, meeting the requirements for rapid on-site detection and expanding its application scenarios.

[0008] (II) Technical Solution

[0009] A rapid detection method for enrofloxacin based on fluorescent covalent organic framework magnetic beads includes the following steps:

[0010] S1. Fluorescent covalent organic framework magnetic beads for conjugating monoclonal antibodies:

[0011] The fluorescent covalent organic framework magnetic beads are core-shell structured composite materials, with a superparamagnetic core and a fluorescent covalent organic framework material on the outer shell; the particle size of the fluorescent covalent organic framework magnetic beads is 20-300 nm; anti-enrofloxacin monoclonal antibody is coupled to the surface of the fluorescent covalent organic framework magnetic beads by electrostatic adsorption.

[0012] S2. Sample pretreatment:

[0013] Take the sample homogenate to be tested, add fluorescent covalent organic framework magnetic beads conjugated with monoclonal antibody and mix and incubate to obtain magnetic bead-enrofloxacin complex. The complex is rapidly separated from the sample matrix by applying an external magnetic field. After discarding the supernatant, the complex is reconstituted with buffer to obtain a reconstituted solution.

[0014] S3. Sample Testing:

[0015] The reconstituted solution of S2 was diluted with phosphate buffer and added dropwise to the sample well of the immunochromatographic test strip. With the help of capillary action, the complex moved along the chromatographic channel of the test strip and passed sequentially through the detection line T and the control line C, resulting in a specific immune reaction. The T line was coated with enrofloxacin artificial antigen that could competitively bind to the enrofloxacin monoclonal antibody on the fluorescent covalent organic framework magnetic beads, and the C line was coated with secondary antibody that could specifically bind to the monoclonal antibody on the fluorescent covalent organic framework magnetic beads.

[0016] S4. Signal Output and Result Determination:

[0017] Qualitative judgment: If the T line does not show fluorescence, it indicates that the concentration of enrofloxacin in the reconstituted solution exceeds the critical value; conversely, it indicates that the concentration of enrofloxacin in the reconstituted solution does not exceed the critical value.

[0018] Quantitative determination: The fluorescence intensity of the T and C lines is read by a fluorescence immunochromatographic analyzer, the T / C value is calculated, and the quantitative content of enrofloxacin in the reconstituted solution and the test sample is deduced based on the pre-plotted standard working curve of enrofloxacin concentration-T / C value.

[0019] According to a preferred embodiment of the present invention, in S1, the preparation method of fluorescent covalent organic framework magnetic beads is as follows: 1,3,6,8-tetra(4-formylphenyl)pyrene, terephthalic acid hydrazide, iron oxide nanoparticles, and a 1:1 volume ratio mixture of mesitylene / 1,4-dioxane are placed together in a reaction vessel for ultrasonic treatment. After adding acetic acid solution, the mixture is fully degassed, the reaction vessel is sealed, and the mixture is placed at 140-160°C for ≥72 hours. The precipitate is collected, washed sequentially with ethanol, tetrahydrofuran, and acetone, and then dried under vacuum to obtain fluorescent covalent organic framework magnetic beads.

[0020] The fluorescent covalent organic framework magnetic beads can generate fluorescence intensity signals in the wavelength range of 500-600nm under an excitation wavelength of 300-400nm; preferably, when the excitation wavelength is 365nm, the fluorescence intensity signal at 530nm is detected.

[0021] Preferably, in S1, the particle size of the fluorescent covalent organic framework magnetic beads is 50nm, 100nm, 150nm, 200nm or 250nm.

[0022] Preferably, the molar ratio of 1,3,6,8-tetra(4-formylphenyl)pyrene to phthalic acid hydrazide is 1:2, the mass ratio of 1,3,6,8-tetra(4-formylphenyl)pyrene to iron oxide nanoparticles is 1:1, the total mass of 1,3,6,8-tetra(4-formylphenyl)pyrene, phthalic acid hydrazide, and iron oxide nanoparticles to the volume ratio of the mesitylene / 1,4-dioxane mixture is 25-28 mg:1 mL, and the molar amount of added acetic acid is 10-20 times the total molar amount of 1,3,6,8-tetra(4-formylphenyl)pyrene and phthalic acid hydrazide.

[0023] According to a preferred embodiment of the present invention, in S1, the method for coupling anti-enrofloxacin monoclonal antibody onto the surface of fluorescent covalent organic framework magnetic beads is as follows:

[0024] Step 1, Prepare the working solution:

[0025] The working solution includes a coupling buffer, a washing buffer, and a blocking agent; the coupling buffer is a sterile aqueous solution containing 0.5-0.7 g / L borax and 0.8-1.0 g / L boric acid.

[0026] The washing buffer solution is a 2-(N-morpholino)ethanesulfonic acid buffer solution with a concentration of 0.002-0.003 g / mL and a pH of 5.5-6.0;

[0027] The sealing agent is a phosphate buffer solution containing 0.05 g / mL skim milk powder;

[0028] Step 2, conjugate with anti-enrofloxacin monoclonal antibody:

[0029] First, fluorescent covalent organic framework (FOB) magnetic beads are pretreated by soaking in conjugation buffer, followed by magnetic separation for later use. Anti-enrofloxacin monoclonal antibody and FOB magnetic beads are mixed at a mass ratio of 1:3 and placed in conjugation buffer. Conjugation is performed at 37℃±0.5℃ on a rotary apparatus for 30-60 min, followed by magnetic separation. The supernatant is discarded, and the mixture is washed three times with washing buffer. After washing, the FOB magnetic beads are blocked with a blocking agent for 1 h, followed by magnetic separation to obtain the fluorescent covalent organic framework magnetic beads containing the conjugated monoclonal antibody. The binding mechanism between the fluorescent covalent organic framework magnetic beads and the enrofloxacin monoclonal antibody is electrostatic adsorption. This method achieves highly efficient antibody conjugation and is a physical conjugation process, ensuring that antibody activity remains unaffected.

[0030] According to a preferred embodiment of the present invention, in S2, the sample to be tested is meat or fish.

[0031] According to a preferred embodiment of the present invention, the specific method in the sample pretreatment step of S2 is as follows: take 1-2g of the sample to be tested, homogenize it in a colloid mill, add it to a phosphate buffer solution with a pH of 7.4, add the fluorescent covalent organic framework magnetic beads of the conjugated monoclonal antibody, mix and incubate for 4-10min at a temperature of 37℃±0.5℃, incubate on a gyroscope at 10-20rpm, after incubation, perform magnetic separation, discard the supernatant, wash with phosphate buffer solution with a pH of 7.4, and reconstitute in 5mL of phosphate buffer solution with a pH of 7.4 to obtain a reconstituted solution.

[0032] According to a preferred embodiment of the present invention, in S3, the immunochromatographic test strip includes a base plate, on which filter paper, a sample pad, a nitrocellulose membrane and absorbent paper are sequentially overlapped. The nitrocellulose membrane is provided with a detection line (T line) and a control line (C line). The T line is coated with enrofloxacin artificial antigen, and the C line is coated with secondary antibody.

[0033] The sample pad, nitrocellulose membrane, and absorbent paper form a chromatography channel through capillary action. After the magnetic bead-enrofloxacin complex is added, the complex solution moves at a constant speed along the channel and reacts sequentially with the T-line artificial antigen and the C-line secondary antibody to complete competitive binding and efficacy verification.

[0034] According to a preferred embodiment of the present invention, in S4, during qualitative judgment, the results are observed with an ultraviolet lamp for qualitative analysis. If the T line does not show fluorescence, it indicates that the content of enrofloxacin in the reconstituted solution is higher than the minimum detection limit of 0.05 ng / mL; if the T line shows fluorescence, it indicates that the content of enrofloxacin in the reconstituted solution is lower than the minimum detection limit of 0.05 ng / mL.

[0035] According to a preferred embodiment of the present invention, in S4, during quantitative determination, after the reconstituted solution is added to the sample well of the immunochromatographic test strip for a period of time, the fluorescence intensity of the T line and C line is measured using a fluorescence immunochromatographic analyzer. The ratio of the fluorescence intensity of the T line and the C line is recorded as the T / C value. A standard working curve is plotted with the concentration values ​​of enrofloxacin standard solutions of different gradient concentrations on the x-axis and the T / C value on the y-axis to determine the enrofloxacin content in the reconstituted solution and the sample to be tested. The quantitative detection range is 3.25-560 μg / kg.

[0036] (III) Beneficial Effects

[0037] This invention, through its innovative design of "dual-function integration of magnetic separation and fluorescence signal output," provides a breakthrough solution to the pain points of existing enrofloxacin detection technologies. Its technical effectiveness can be systematically summarized from three dimensions: core performance, operational practicality, and application adaptability, as detailed below:

[0038] (1) Achieving high-sensitivity capture of trace targets: The magnetic core of the fluorescent covalent organic framework magnetic beads has superparamagnetism, which can rapidly enrich trace enrofloxacin in the sample and avoid the decrease in sensitivity caused by sample dilution in traditional detection; at the same time, the outer shell fluorescent covalent organic framework material has good photostability and high quantum yield, which can efficiently amplify the fluorescence signal and achieve trace detection of as low as 0.05 ng / mL in complex matrices (such as fish and beef), completely solving the problem of false negatives.

[0039] (2) Accurately achieve both qualitative and quantitative objectives: Compared with the traditional colorimetric method, the fluorescence signal response is stronger, and qualitative judgment can be completed intuitively by using a UV lamp (whether the T-line fluorescence directly corresponds to whether the content exceeds the standard); relying on the ordered pore structure and stability of the fluorescent material, a stable T / C value standard curve can be constructed, and the quantitative range covers 3.25~560μg / kg, meeting the precise quantitative needs in different scenarios and making up for the deficiency of the weak quantitative ability of the traditional immunochromatographic method.

[0040] (3) Excellent anti-interference ability and detection stability: The magnetic separation step can directly separate the sample matrix and the target, eliminating complex pretreatment and greatly reducing the interference of impurities such as proteins and fats; at the same time, the immunomagnetic separation and immunochromatography are integrated, eliminating the steps of magnetic bead elution and fluorescent material spraying on the binding pad, avoiding sample loss and error during operation, and significantly improving the repeatability and reliability of the detection results.

[0041] (4) High coupling efficiency and stable antibody activity: Fluorescent covalent organic framework magnetic beads have the characteristics of large specific surface area and high density of functional sites. They achieve efficient coupling with antibodies through non-covalent interactions such as electrostatic adsorption, and the coupling effect is stable. Moreover, the physical coupling process does not destroy antibody activity, ensuring the specificity of the immune response, and no complicated chemical modification steps are required.

[0042] (5) Short detection cycle and few operation steps: The entire detection process does not require cumbersome pretreatment such as centrifugation, extraction, and purification. The sample and magnetic beads only need to be incubated for 5 minutes, and the chromatography reaction can be completed in 1 to 10 minutes. The whole process is short. The results can be interpreted by ultraviolet lamp (qualitative) or small fluorescence immunochromatographic analyzer (quantitative), without the need for professional operator training.

[0043] (6) Easy to operate, low cost, no need for large precision instruments: It gets rid of the dependence of traditional laboratory testing on large analytical instruments. The testing equipment is portable and can be directly applied to scenarios such as farms, food processing plants, and market supervision sites, realizing a closed loop of "on-site sampling - instant testing - rapid results".

[0044] This invention's detection method is adaptable to the detection of complex food matrices. Targeting the complex matrix characteristics of various food samples (such as fish and beef) including aquatic products and livestock, it utilizes a magnetic separation anti-interference design, allowing direct detection without special sample preparation. This adapts to the diverse needs of food safety testing and can meet the requirements of multiple scenarios. It can achieve rapid qualitative screening using ultraviolet light (e.g., rapid on-site screening of samples exceeding standards in market supervision) and precise quantitative analysis using standard curves (e.g., enterprise quality control, precise laboratory testing), balancing screening efficiency and detection accuracy. The preparation process of its fluorescent covalent organic framework magnetic beads is well-defined, the test strips can be mass-produced and sealed for storage, and the detection cost is lower than traditional fluorescence immunoassay technology, providing a solid industrial foundation for large-scale application. Attached Figure Description

[0045] Figure 1 This is a schematic diagram of the preparation process of fluorescent covalent organic framework magnetic beads coupled with monoclonal antibodies.

[0046] Figure 2 This is a schematic diagram of the immunomagnetic separation process of fluorescent covalent organic framework magnetic beads.

[0047] Figure 3 This is a graph showing the results of enrofloxacin detection using a fluorescent covalent organic framework magnetic bead immunoassay test strip (sample concentrations from left to right are 1.0, 0.5, 0.2, 0.1, 0.05, and 0 ng / mL). Detailed Implementation

[0048] To better explain and facilitate understanding of the present invention, the present invention will be described in detail below with reference to the accompanying drawings and specific embodiments.

[0049] Example 1

[0050] This embodiment describes a method for synthesizing fluorescent covalent organic framework magnetic beads (MCOFs). The synthesis steps are as follows: Figure 1 As shown, a mixture of 1,3,6,8-tetra(4-formylphenyl)pyrene (20 mg, 32 μmol), terephthalic acid hydrazide (12.43 mg, 64 μmol), iron oxide nanoparticles (20 mg), and mesitylene / 1,4-dioxane (1:1, 2.0 mL) was placed in a 15 mL heat-resistant glass tube, sonicated for 15 min, and 0.2 mL of acetic acid solution (6 M) was added. The tube was then rapidly frozen in a liquid nitrogen bath. Degassing was performed through three freeze-pump-melt cycles, and the tube was flame-sealed under vacuum. After being heated to room temperature, the sealed tube was placed in a 150 °C oven and allowed to stand for 3 days. The precipitate was collected by filtration, washed successively with ethanol, tetrahydrofuran, and acetone, and finally dried under vacuum at 80 °C for 12 h to obtain a yellow solid, namely fluorescent covalent organic framework magnetic beads (MCOFs).

[0051] Example 2

[0052] This embodiment describes the electrostatic adsorption method used to conjugate anti-enrofloxacin monoclonal antibody to fluorescent covalent organic framework magnetic beads prepared in Example 1. The steps are as follows:

[0053] Step 1, Prepare the working solution:

[0054] The working solution includes coupling buffer, washing buffer, and blocking agent;

[0055] The coupling buffer is prepared as follows: Add 3 mL of borax solution with a concentration of 19.07 g / L and 7 mL of boric acid solution with a concentration of 12.37 g / mL to a volumetric flask, and dilute with sterile distilled water to 100 mL.

[0056] The washing buffer solution is prepared as follows: Add 0.43 g of 2-(N-morpholino)ethanesulfonic acid buffer solution to a volumetric flask, dilute with sterile distilled water to 200 mL, and adjust the pH to 5.5-6.0;

[0057] The sealing agent is prepared by adding 1 mL of phosphate buffer solution to a sterile container, then adding 50 mg of skim milk powder, and gently vortexing until completely dissolved.

[0058] Step 2, conjugation of anti-enrofloxacin monoclonal antibody: Place 300 μL of conjugation buffer in a 2 mL centrifuge tube, add 0.5 mg of 50 nm fluorescent covalent organic framework magnetic beads, and magnetically separate for 3 min, discarding the supernatant. Add a mixture of 250 μg of anti-enrofloxacin monoclonal antibody and 0.75 mg of fluorescent covalent organic framework magnetic beads, place in 1 mL of conjugation buffer, and conjugate at 37 °C and 15 rpm for 45 min. Magnetically separate for 5 min, discarding the supernatant, and wash three times with 1 mL of washing buffer. Finally, add 1 mL of blocking agent, mix, and block for 1 h to obtain the fluorescent covalent organic framework magnetic beads conjugated with the monoclonal antibody. Figure 1 The MCOFs monoclonal antibody shown.

[0059] like Figure 1 As shown, the fluorescent covalent organic framework magnetic beads (MCOFs monoclonal antibodies) conjugated with monoclonal antibodies have a core-shell composite structure. The core is a superparamagnetic magnetic core, and the shell is a fluorescent covalent organic framework material. The surface is conjugated with anti-enrofloxacin monoclonal antibody.

[0060] Example 3

[0061] This embodiment describes the preparation method of enrofloxacin immunochromatographic test strips, which is briefly described below:

[0062] Enrofloxacin rabbit polyclonal antibody at a concentration of 1.5 mg / mL and rabbit anti-mouse secondary antibody at a concentration of 0.5 mg / mL were sprayed onto a nitrocellulose membrane to form a detection line (T line) and a control line (C line), respectively, with a spray volume of 0.5 μL / cm. The nitrocellulose membrane was vacuum dried overnight at 37°C. The sample pad, the dried nitrocellulose membrane, and absorbent paper were then sequentially pasted onto a PVC base plate, cut into 4 mm wide test strips, packaged, placed in a foil bag, sealed with desiccant, and stored in a desiccant container for later use.

[0063] Example 4

[0064] The fluorescent covalent organic framework magnetic beads (MCOFs monoclonal antibody) conjugated with monoclonal antibody prepared in Example 2 and the enrofloxacin immunochromatographic test strip in Example 3 were used to quantitatively detect enrofloxacin in fish meat. The preparation method is as follows:

[0065] (1) Sample preparation and enrichment:

[0066] See Figure 2 The procedure shown is for sample processing and enrichment. Take 2g of fish meat sample, homogenize it using a colloid mill, add 5mL of pH 7.4 phosphate buffer solution, then add 200μg of blocked MCOFs monoclonal antibody, and incubate at 37℃ and 20rpm for 5min; after magnetic separation for 1min, discard the supernatant. Figure 2This is a schematic diagram of the immunomagnetic separation process of fluorescent covalent organic framework magnetic beads. As shown in the figure, most of the enrofloxacin from the test sample has been captured by the MCOFs monoclonal antibody, and the interfering substances introduced by the matrix material in the test sample have been separated and removed. The sample is washed with phosphate buffer solution at pH 7.4 and reconstituted in 5 mL of phosphate buffer solution at pH 7.4 to obtain the reconstituted solution.

[0067] (2) Chromatographic detection: Take 50 μL of the above reconstituted solution, add 950 μL of pH7.4 phosphate buffer solution and mix well. Take 100 μL and add it to the sample well of the test strip. After 10 min, use a fluorescence immunochromatographic analyzer to read the fluorescence intensity of the T line and C line and the T / C value.

[0068] Result determination:

[0069] ① Qualitative analysis: When observed under a 365nm wavelength ultraviolet lamp, if the T line does not show fluorescence (emits fluorescence at 530nm), it indicates that the enrofloxacin content in the added sample is ≥0.05ng / mL; if the T line shows fluorescence, it indicates that the content in the added sample is <0.05ng / mL.

[0070] Figure 3 This image shows the results of an immunoassay test strip for enrofloxacin detection using fluorescent covalent organic framework magnetic beads. The concentrations of enrofloxacin in the added samples, from left to right, correspond to 1.0 ng / mL, 0.5 ng / mL, 0.2 ng / mL, 0.1 ng / mL, 0.05 ng / mL, and 0 ng / mL, respectively. When the concentration is ≥0.05 ng / mL, the T-line fluorescence gradually weakens with increasing enrofloxacin concentration in the added samples, while the T-line fluorescence signal is strongest in the blank control. This result indicates that the detection method of this invention has high detection sensitivity. Based on the concentration of enrofloxacin in the added samples, the amount of reconstituted sample, and the amount of the test sample, the enrofloxacin content in the test sample can be further calculated.

[0071] ② Quantitative Analysis: A series of enrofloxacin standard solutions with gradient concentrations were prepared in advance. Following the operation method of "chromatographic detection" in step (2) of this embodiment, the solutions were added to the sample wells of the test strip. After 10 minutes, the fluorescence intensity of the T line and C line and the T / C value were read using a fluorescence immunochromatographic analyzer. Then, a standard working curve was plotted with the concentration of the enrofloxacin standard solution as the abscissa and the T / C value as the ordinate. Using this standard working curve, the specific concentration of enrofloxacin in the reconstituted solution and the fish meat sample was calculated by comparing it with the T / C value of the actual sample to be tested.

[0072] Example 5

[0073] The fluorescent covalent organic framework magnetic beads conjugated with monoclonal antibodies prepared in Example 2 and the enrofloxacin immunochromatographic test strip from Example 3 were used to quantitatively detect enrofloxacin in beef. The preparation method is as follows:

[0074] (1) Sample processing and enrichment: Take 2g of beef sample, homogenize it with a colloid mill, add 5mL of pH 7.4 phosphate buffer solution, then add 200μg of blocked MCOFs monoclonal antibody, mix and incubate at 37℃ and 20rpm for 5min; after magnetic separation for 1min, discard the supernatant, wash with pH 7.4 phosphate buffer solution, and redissolve in 5mL of pH 7.4 phosphate buffer solution to obtain the redissolved solution;

[0075] (2) Chromatographic detection: Take 50 μL of the above reconstituted solution, add 950 μL of pH 7.4 phosphate buffer solution and mix well. Take 100 μL and add it to the sample well of the test strip. After 10 min, use a fluorescence immunochromatographic analyzer to read the fluorescence intensity of the T line and C line and the T / C value; Result interpretation:

[0076] ① Qualitative analysis: When observed under a 365nm wavelength ultraviolet lamp, if the T line does not show fluorescence (emits fluorescence at 530nm), it indicates that the enrofloxacin content in the added sample is ≥0.05ng / mL; if the T line shows fluorescence, it indicates that the content is <0.05ng / mL.

[0077] ② Quantitative analysis: A series of enrofloxacin standard solutions with gradient concentrations were prepared in advance. Following the operation method of step (2) above in this embodiment, the solutions were added to the sample wells of the test strip. After 10 minutes, the fluorescence intensity of the T line and C line and the T / C value were read using a fluorescence immunochromatographic analyzer. Then, a standard working curve was plotted with the concentration of the enrofloxacin standard solution as the abscissa and the T / C value as the ordinate. Using this standard working curve, the specific concentration of enrofloxacin in the reconstituted solution and beef sample was calculated by comparing it with the T / C value of the actual sample to be tested.

[0078] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features. These modifications or substitutions, or combinations of technical features in the above embodiments that do not conflict with each other, can be made in accordance with the manner described in the embodiments. These modifications, substitutions or combinations do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A rapid detection method for enrofloxacin based on fluorescent covalent organic framework magnetic beads, characterized in that, Includes the following steps: S1. Fluorescent covalent organic framework magnetic beads for conjugating monoclonal antibodies: The fluorescent covalent organic framework magnetic beads are core-shell structured composite materials, with a superparamagnetic core and a fluorescent covalent organic framework material on the outer shell; the particle size of the fluorescent covalent organic framework magnetic beads is 20-300 nm; anti-enrofloxacin monoclonal antibody is coupled to the surface of the fluorescent covalent organic framework magnetic beads by electrostatic adsorption. The preparation method of fluorescent covalent organic framework magnetic beads is as follows: 1,3,6,8-tetra(4-formylphenyl)pyrene, terephthalic acid hydrazide, iron oxide nanoparticles and a 1:1 volume ratio mixture of mesitylene / 1,4-dioxane are placed together in a reaction vessel and subjected to ultrasonic treatment. After adding acetic acid solution, the mixture is fully degassed, the reaction vessel is sealed, and the mixture is placed at 140-160℃ and allowed to stand for ≥72h. The precipitate is collected, washed successively with ethanol, tetrahydrofuran and acetone, and dried under vacuum to obtain fluorescent covalent organic framework magnetic beads. The method for conjugating anti-enrofloxacin monoclonal antibodies to the surface of fluorescent covalent organic framework magnetic beads is as follows: Step 1, Prepare the working solution: The working solution includes a coupling buffer, a washing buffer, and a blocking agent; the coupling buffer is a sterile aqueous solution containing 0.5-0.7 g / L borax and 0.8-1.0 g / L boric acid. The washing buffer solution is a 2-(N-morpholino)ethanesulfonic acid buffer solution with a concentration of 0.002-0.003 g / mL and a pH of 5.5-6.0; The sealing agent is a phosphate buffer solution containing 0.05 g / mL skim milk powder; Step 2, conjugate with anti-enrofloxacin monoclonal antibody: First, the fluorescent covalent organic framework magnetic beads were pretreated by soaking in conjugation buffer, followed by magnetic separation for later use. Anti-enrofloxacin monoclonal antibody and covalent organic framework magnetic beads were mixed at a mass ratio of 1:3 and placed in conjugation buffer. The mixture was conjugated at 37℃±0.5℃ on a rotary apparatus for 30-60 minutes, followed by magnetic separation. The supernatant was discarded, and the mixture was washed three times with washing buffer. After washing, the fluorescent covalent organic framework magnetic beads were blocked with blocking agent for 1 hour, followed by magnetic separation to obtain the fluorescent covalent organic framework magnetic beads containing the conjugated monoclonal antibody. S2. Sample pretreatment: Take the sample homogenate to be tested, add fluorescent covalent organic framework magnetic beads conjugated with monoclonal antibody and mix and incubate to obtain magnetic bead-enrofloxacin complex. The complex is rapidly separated from the sample matrix by applying an external magnetic field. After discarding the supernatant, the complex is reconstituted with buffer to obtain a reconstituted solution. S3. Sample Testing: The reconstituted solution of S2 was diluted with phosphate buffer and added dropwise to the sample well of the immunochromatographic test strip. With the help of capillary action, the complex moved along the chromatographic channel of the test strip and passed sequentially through the detection line T and the control line C, resulting in a specific immune reaction. The T line was coated with enrofloxacin artificial antigen that could competitively bind to the enrofloxacin monoclonal antibody on the fluorescent covalent organic framework magnetic beads, and the C line was coated with secondary antibody that could specifically bind to the monoclonal antibody on the fluorescent covalent organic framework magnetic beads. S4. Signal Output and Result Determination: Qualitative judgment: If the T line does not show fluorescence, it indicates that the concentration of enrofloxacin in the reconstituted solution exceeds the critical value; Conversely, it indicates that the concentration of enrofloxacin in the reconstituted solution has not exceeded the critical value; Quantitative determination: The fluorescence intensity of the T and C lines is read by a fluorescence immunochromatographic analyzer, the T / C value is calculated, and the quantitative content of enrofloxacin in the reconstituted solution and the test sample is deduced based on the pre-plotted standard working curve of enrofloxacin concentration-T / C value.

2. The rapid detection method for enrofloxacin based on fluorescent covalent organic framework magnetic beads according to claim 1, characterized in that: In S1, the molar ratio of 1,3,6,8-tetra(4-formylphenyl)pyrene to phthalic acid hydrazide is 1:2, and the mass ratio of 1,3,6,8-tetra(4-formylphenyl)pyrene to iron oxide nanoparticles is 1:1; the total mass of 1,3,6,8-tetra(4-formylphenyl)pyrene, phthalic acid hydrazide, and iron oxide nanoparticles to the volume ratio of the mesitylene / 1,4-dioxane mixture is 25-28 mg:1 mL, and the molar amount of added acetic acid is 10-20 times the total molar amount of 1,3,6,8-tetra(4-formylphenyl)pyrene and phthalic acid hydrazide.

3. The rapid detection method for enrofloxacin based on fluorescent covalent organic framework magnetic beads according to claim 1, characterized in that: In S2, the sample to be tested is meat or fish.

4. The rapid detection method for enrofloxacin based on fluorescent covalent organic framework magnetic beads according to claim 1, characterized in that, The specific method for the sample pretreatment step in S2 is as follows: Take 1-2g of the sample to be tested, homogenize it in a colloid mill, add it to a phosphate buffer solution with a pH of 7.4, add the fluorescent covalent organic framework magnetic beads of the conjugated monoclonal antibody, mix and incubate for 4-10 min at a temperature of 37℃±0.5℃ on a gyroscope at 10-20 rpm. After incubation, perform magnetic separation, discard the supernatant, wash with phosphate buffer solution with a pH of 7.4, and reconstitute in 5mL of phosphate buffer solution with a pH of 7.4 to obtain the reconstituted solution.

5. The rapid detection method for enrofloxacin based on fluorescent covalent organic framework magnetic beads according to claim 1, characterized in that: In S3, the immunochromatographic test strip includes a base plate, on which filter paper, a sample pad, a nitrocellulose membrane, and absorbent paper are sequentially stacked. The nitrocellulose membrane is provided with a detection line (T line) and a control line (C line). The T line is coated with enrofloxacin artificial antigen, and the C line is coated with secondary antibody.

6. The rapid detection method for enrofloxacin based on fluorescent covalent organic framework magnetic beads according to claim 1, characterized in that: In S4, for qualitative judgment, the results are observed under a UV lamp for qualitative analysis. If the T line does not show fluorescence, it indicates that the content of enrofloxacin in the reconstituted solution is higher than the lower limit of detection (0.05 ng / mL); if the T line shows fluorescence, it indicates that the content of enrofloxacin in the reconstituted solution is lower than the lower limit of detection (0.05 ng / mL).

7. The rapid detection method for enrofloxacin based on fluorescent covalent organic framework magnetic beads according to claim 1, characterized in that: In S4, during quantitative determination, after the reconstituted solution is added to the sample well of the immunochromatographic test strip for a period of time, the fluorescence intensity of the T line and C line is measured using a fluorescence immunochromatographic analyzer. The ratio of the fluorescence intensity of the T line and C line is recorded as the T / C value. A standard working curve is plotted with the concentration values ​​of enrofloxacin standard solutions of different gradient concentrations on the x-axis and the T / C value on the y-axis to determine the enrofloxacin content in the reconstituted solution and the sample to be tested.

Images