Time-resolved fluorescent probe for rapidly detecting ethoxyquin, immunochromatographic test strip, detection method and application

Abstract

The invention belongs to the technical field of food safety and rapid detection, and particularly relates to a time-resolved fluorescent probe for rapidly detecting ethoxyquin, an immunochromatography test strip, a detection method and application. The probe Eu-EQ-mAb is prepared by indirect coupling of europium fluorescent microspheres and ethoxyquin monoclonal antibodies, the particle size of the microspheres is 150-250 nm, and the emission peak is 610-625 nm. The test strip comprises a PVC (polyvinyl chloride) bottom plate, a sample pad, a nitrocellulose membrane coated with a detection line T and a quality control line C, and a water absorption pad, during detection, a sample and the probe are pre-reacted and then are subjected to chromatography, and a result is judged through a T / C value. During detection, after the Eu-EQ-mAb fluorescent probe and a sample are subjected to pre-reaction, immunochromatography is carried out, and a T / C value judgment result is read. The semi-quantitative detection limit of the method is 0.5-1 [mu] g / L, the detection sensitivity of the method in large yellow croakers, perches and pears reaches 5 [mu] g / kg, intra-batch and inter-batch variation coefficients are smaller than 10%, and the method has the advantages of being easy and convenient to operate, rapid in detection, high in sensitivity and the like and is suitable for on-site rapid screening of ethoxyquin residues in food.

Description

Technical Field

[0001] This invention relates to the field of food safety monitoring and immunoassay detection technology, and more specifically, to a time-resolved fluorescent probe, immunochromatographic test strip, detection method, and application for the rapid detection of ethoxyquinoline. Background Technology

[0002] Ethoxyquin (EQ) is an antioxidant commonly used in feed and aquatic product preservation, but it carries certain toxicological risks, and strict limits are set for its residues in food and feed in various countries. Current detection methods for ethoxyquin mainly rely on large-scale instruments such as high-performance liquid chromatography (HPLC) and liquid chromatography-mass spectrometry (LC-MS), which offer high sensitivity but involve complex sample pretreatment, high detection costs, and are not suitable for rapid on-site screening. Time-resolved fluorescent microspheres encapsulate a large number of lanthanide-containing fluorescent complexes within polystyrene microspheres, offering advantages such as large Stokes shift, long fluorescence lifetime, and low background interference. These methods can significantly improve the sensitivity of immunoassays and are suitable for integration with immunochromatography for rapid on-site detection. Therefore, it is necessary to develop a novel ethoxyquin immunochromatographic detection system based on time-resolved fluorescent microspheres to address the issue of insufficient sensitivity. Summary of the Invention

[0003] To overcome the shortcomings of existing technologies, this invention provides a time-resolved fluorescent probe, immunochromatographic test strip, detection method, and application for the rapid detection of ethoxyquinoline.

[0004] This invention is achieved through the following technical solution: a time-resolved fluorescent probe for rapid detection of ethoxyquinoline, wherein the time-resolved fluorescent probe is an ethoxyquinoline time-resolved fluorescent probe Eu-EQ-mAb, obtained by indirect coupling of a secondary fluorescent probe and an ethoxyquinoline monoclonal antibody EQ-mAb, wherein the secondary fluorescent probe is a solution of time-resolved fluorescent microspheres with europium ions as the luminescent center, wherein the average particle size of the time-resolved fluorescent microspheres is 150-250 nm, the polydispersity index (PDI) of the hydration particle size is ≤0.1, and it has a narrow-band emission spectrum with an emission peak located at 610-625 nm at an excitation wavelength of approximately 350 nm.

[0005] As a preferred embodiment, the preparation method of the time-resolved fluorescent probe includes: diluting 20 μL of secondary fluorescent probe to 100 μL with microsphere diluent, adding 0.25–10 μL of EQ-mAb with a concentration of 0.01 mg / mL, and mixing and reacting at room temperature for 1–10 min to obtain Eu-EQ-mAb.

[0006] As a preferred option, the amount of EQ-mAb added is 2 μL, and the reaction time is 5 min.

[0007] A time-resolved fluorescence immunochromatographic test strip for rapid detection of ethoxyquinoline includes a sample pad, a nitrocellulose membrane, and an absorbent pad sequentially attached to a base plate. The base plate is made of PVC. The nitrocellulose membrane has a detection line (T) and a control line (C). The T line is coated with ethoxyquinoline-coated antigen EQ-Ag, and the C line is coated with goat anti-mouse IgG antibody. The sample pad overlaps with one end of the nitrocellulose membrane, and the absorbent pad overlaps with the other end of the nitrocellulose membrane. EQ-Ag is obtained by coupling the ethoxyquinoline hapten with a carrier protein, wherein the carrier protein is bovine serum albumin or ovalbumin.

[0008] As a preferred embodiment, the preparation method of the time-resolved fluorescence immunochromatographic test strip includes: Step T1, Preparation of T / C line coating solution Dilute the EQ-Ag stock solution with a concentration of 4.0–5.0 mg / mL 10–45 times with T / C line diluent for later use; dilute the goat anti-mouse IgG antibody with the same diluent. Step T2, Preparation of the reaction membrane Nitrocellulose membrane (2) was laid flat on the coating area of ​​the base plate (6). EQ-Ag diluent was sprayed at a speed of 0.5-1.5 μL / cm using an XYZ three-dimensional gold sprayer to form T lines and goat anti-mouse IgG antibody was sprayed to form C lines. After spraying, the membrane was dried at 37 ℃ for 2 h. Step T3: Sample pad and absorbent pad treatment Cut the sample pad to the appropriate size; cut the absorbent pad and set it aside. Step T4: Assembly and Cutting Paste the sample pad, nitrocellulose membrane, and absorbent pad sequentially onto the base plate, with each adjacent component overlapping by 2 mm. Use an automatic strip cutter to cut the test strips into strips 2–4 mm wide, place them in a sealed bag with desiccant, and store them at room temperature away from light.

[0009] Furthermore, the T / C line diluent was PBS + 5% methanol, the stock solution concentration of EQ-Ag was 4.8 mg / mL, and it was diluted 15 times with the T / C line diluent during coating; the EQ-Ag diluent was sprayed at a speed of 1 μL / cm using an XYZ three-dimensional cross-cutting gold sprayer to form the T line; the width of the test strip was 3 mm.

[0010] A rapid detection method for ethoxyquinoline includes the following steps: Step S1: Pre-treat the sample to be tested to obtain the test solution; Step S2: Add 100 μL of the test solution to the microwell, add 5 μL of the time-resolved fluorescent probe Eu-EQ-mAb according to any one of claims 1 to 3, mix well and react at room temperature for 3 to 10 min; Step S3: Insert the time-resolved fluorescence immunochromatographic test strip of any one of claims 4 to 7 into the microwell, immerse the sample end of the test strip in the mixture, and perform the immunochromatographic reaction at room temperature for 5 to 15 minutes; Step S4: Take out the test strip and read the fluorescence signals of the detection line and control line under 365 nm ultraviolet light or immunofluorescence quantitative analyzer. Determine the presence and approximate concentration level of ethoxyquinoline in the sample based on the T / C value.

[0011] As a preferred embodiment, the chromatography time in step S3 is 10 min; when the sample is an ethoxyquinoline standard solution, a positive result is determined when the ethoxyquinoline concentration is ≥1 μg / L, and the semi-quantitative detection limit range is 0.5~1 μg / L.

[0012] As a preferred option, the sample to be tested is large yellow croaker, sea bass or pear. The pretreatment method is acetonitrile-n-hexane liquid-liquid extraction and the addition of antioxidants to obtain the sample test solution.

[0013] Application of the above-mentioned time-resolved fluorescent probe and the above-mentioned time-resolved fluorescent immunochromatographic test strip in the rapid qualitative or semi-quantitative detection of ethoxyquinoline residues in food.

[0014] This invention, employing the above technical solutions, offers the following advantages compared to existing technologies: Significantly improved sensitivity: Time-resolved fluorescent microspheres possess a high signal-to-noise ratio. The detection sensitivity of the ethoxyquinoline time-resolved fluorescent immunochromatographic test strip established in this invention is 5 μg / kg, approximately twice that of colloidal gold test strips. Simple labeling method: The secondary fluorescent probe is indirectly coupled to EQ-mAb, eliminating the need for chemical modification of the antibody or covalent coupling with the microspheres. This simplifies operation and facilitates large-scale preparation. Good repeatability: Intra-batch and inter-batch precision studies of EQ standard solutions at concentrations of 0, 5, and 10 μg / L showed coefficients of variation of less than 10%, indicating stable test strip preparation and reliable detection results. Suitable for rapid on-site screening: The method of this invention requires only a 5-minute pre-reaction in the microwells followed by 10 minutes of chromatography to complete the detection. The entire process does not rely on large instruments; only a quantitative immunofluorescence analyzer is needed for result reading, making it suitable for grassroots regulatory departments and enterprise self-inspection.

[0015] Additional aspects and advantages of the invention will become apparent in the following description or may be learned by practice of the invention. Attached Figure Description

[0016] The above and / or additional aspects and advantages of the present invention will become apparent and readily understood from the description of the embodiments taken in conjunction with the following drawings, in which: Figure 1 This is a schematic diagram of the test strip structure of the present invention; Figure 2 This is a diagram illustrating the determination of the test results of the test strip of the present invention. in, Figure 1 The correspondence between the reference numerals and components in the attached drawings is as follows: 1. Sample pad; 2. Nitrocellulose membrane; 3. Detection line; 4. Quality control line; 5. Absorbent pad; 6. Base plate. Detailed Implementation

[0017] To better understand the above-mentioned objectives, features, and advantages of the present invention, the present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments. It should be noted that, unless otherwise specified, the embodiments and features described in these embodiments can be combined with each other.

[0018] Many specific details are set forth in the following description in order to provide a full understanding of the invention. However, the invention may also be practiced in other ways different from those described herein, and therefore the scope of protection of the invention is not limited to the specific embodiments disclosed below.

[0019] This invention proposes a rapid time-resolved fluorescent probe for detecting ethoxyquinoline. The time-resolved fluorescent probe is an ethoxyquinoline time-resolved fluorescent probe Eu-EQ-mAb, obtained by indirect coupling of a secondary fluorescent probe and an ethoxyquinoline monoclonal antibody EQ-mAb. The secondary fluorescent probe is a solution of time-resolved fluorescent microspheres with europium ions as the luminescent center. The average particle size of the time-resolved fluorescent microspheres is 150–250 nm, the polydispersity index (PDI) of the hydration particle size is ≤0.1, and it has a narrow-band emission spectrum with an emission peak at 610–625 nm at an excitation wavelength of approximately 350 nm. The preparation method of the time-resolved fluorescent probe includes: diluting 20 μL of the secondary fluorescent probe to 100 μL with microsphere diluent, adding 0.25–10 μL of EQ-mAb at a concentration of 0.01 mg / mL, and mixing and reacting at room temperature for 1–10 min to obtain Eu-EQ-mAb. The amount of EQ-mAb added is 2 μL, and the reaction time is 5 min. The emission spectrum peak positions of Eu-EQ-mAb are basically consistent with those of the secondary fluorescent probe, with the maximum emission peak located at approximately 617 nm. Furthermore, the hydration particle size increases while the PDI remains less than 0.1.

[0020] A time-resolved fluorescence immunochromatographic test strip for rapid detection of ethoxyquinoline includes a sample pad 1, a nitrocellulose membrane 2, and an absorbent pad 5 sequentially attached to a base plate 6. The base plate 6 is made of rigid, non-absorbent PVC material. The nitrocellulose membrane 2 has a detection line T and a control line C. The T line is coated with ethoxyquinoline-coated antigen EQ-Ag, and the C line is coated with goat anti-mouse IgG antibody. The sample pad 1 overlaps with one end of the nitrocellulose membrane 2, and the absorbent pad 5 overlaps with the other end of the nitrocellulose membrane 2. The EQ-Ag is obtained by coupling the ethoxyquinoline hapten with a carrier protein, wherein the carrier protein is bovine serum albumin or ovalbumin.

[0021] The preparation methods for time-resolved fluorescence immunochromatographic test strips include: Step T1, Preparation of T / C line coating solution The EQ-Ag stock solution with a concentration of 4.8 mg / mL was diluted 15 times with T / C line diluent of PBS + 5% methanol for later use; the goat anti-mouse IgG antibody was diluted to an appropriate concentration with the same diluent. Step T2, Preparation of the reaction membrane Nitrocellulose membrane 2 was laid flat on the coating area of ​​substrate 6. EQ-Ag diluent was sprayed at a speed of 1 μL / cm using an XYZ three-dimensional gold sprayer to form T-lines, and goat anti-mouse IgG antibody was sprayed to form C-lines. After spraying, the membrane was dried at 37 ℃ for 2 h. Step T3: Sample pad and absorbent pad treatment Cut the sample pad to the appropriate size. If necessary, pre-impregnate it with a treatment solution containing protein blocking agent and buffer salt and then dry it. Cut the absorbent pad and set it aside. Step T4: Assembly and Cutting On the base plate 6, attach the sample pad 1, nitrocellulose membrane 2 and absorbent pad 5 in sequence, with each adjacent part overlapping by 2 mm; use an automatic strip cutter to cut the test strips into 3 mm wide strips, put them into a sealed bag with desiccant, and store them at room temperature away from light.

[0022] A rapid detection method for ethoxyquinoline, characterized by comprising the following steps: Step S1: The sample to be tested is pretreated to obtain the test solution; the sample to be tested is large yellow croaker, sea bass or pear. The pretreatment method is acetonitrile-n-hexane liquid-liquid extraction and antioxidant addition. The detection sensitivity of the test strip for ethoxyquinoline in the obtained sample test solution is 5 μg / kg.

[0023] Step S2: Add 100 μL of the test solution to the microwell, add 5 μL of the time-resolved fluorescent probe Eu-EQ-mAb, mix well, and react at room temperature for 3 to 10 min. Step S3: Insert the time-resolved fluorescence immunochromatographic test strip into the microwell, immerse the sample end of the test strip in the mixture, and perform the immunochromatographic reaction at room temperature for 5–15 min; the chromatography time is 10 min; when the sample is an ethoxyquinoline standard solution, a positive result is determined when the ethoxyquinoline concentration is ≥1 μg / L, and the semi-quantitative detection limit range is 0.5–1 μg / L.

[0024] Step S4: Take out the test strip and read the fluorescence signals of the detection line and control line under 365 nm ultraviolet light or immunofluorescence quantitative analyzer. Determine the presence and approximate concentration level of ethoxyquinoline in the sample based on the T / C value.

[0025] Application of a time-resolved fluorescent probe and a time-resolved fluorescent immunochromatographic test strip in the rapid qualitative or semi-quantitative detection of ethoxyquinoline residues in food.

[0026] The following is combined Figures 1 to 2 The present invention provides a detailed description of the time-resolved fluorescent probe, immunochromatographic test strip, detection method, and application for the rapid detection of ethoxyquinoline.

[0027] Example 1 Preparation and characterization of the ethoxyquinoline time-resolved fluorescent probe Eu-EQ-mAb: Europium time-resolved fluorescent microspheres with a particle size of 200 nm were used as a secondary fluorescent probe and diluted with the manufacturer's provided microsphere diluent. The time-resolved fluorescent microspheres appear milky white under sunlight and exhibit red fluorescence under 365 nm ultraviolet light, with a maximum emission wavelength of approximately 617 nm and a relatively narrow full width at half maximum (FWHM) emission spectrum.

[0028] The ethoxyquinoline monoclonal antibody EQ-mAb is a mouse-derived monoclonal antibody prepared by conventional methods in the field, and it has specific recognition ability for EQ-Ag.

[0029] The steps for preparing the ethoxyquinoline time-resolved fluorescent probe are as follows: (1) Take 20 μL of secondary fluorescent probe and dilute it to 100 μL with microsphere diluent; (2) Add different volumes of 0.01 mg / mL EQ-mAb (0.25, 0.5, 1, 2, 5, 10 μL) to the above solution, mix and react at room temperature for 5 min to obtain Eu-EQ-mAb with different antibody labeling amounts; Example 2 Preparation of ethoxyquinoline time-resolved fluorescence immunochromatographic test strips (1) Preparation of T / C line coating solution Dilute the EQ-Ag stock solution (4.8 mg / mL) with T / C line diluent (PBS + 5% methanol) 10, 15, 30, and 45 times respectively for later use; dilute the goat anti-mouse IgG antibody with the same diluent to the appropriate concentration.

[0030] (2) Preparation of reaction membrane Nitrocellulose membrane was laid flat on the coating area of ​​PVC board. EQ-Ag diluent was sprayed at a speed of 1 μL / cm using an XYZ three-dimensional scribing and gold spraying instrument to form T-lines, and goat anti-mouse IgG antibody was sprayed to form C-lines. After spraying, the membrane was dried at 37 ℃ for 2 h.

[0031] (3) Treatment of sample pads and absorbent pads Cut the sample pads to the appropriate size. If necessary, pre-impregnate them with a treatment solution containing protein blocking agent and buffer salts and then dry them. Cut the absorbent pads and set them aside.

[0032] (4) Assembly and cutting Attach the sample pad, reaction membrane, and absorbent pad sequentially to the PVC base plate, with each adjacent component overlapping by approximately 2 mm. Use an automatic strip cutter to cut the strips into test strips approximately 3 mm wide, place them in a sealed bag with desiccant, and store them at room temperature away from light.

[0033] Example 3 Test strip performance evaluation (1) Detection limit Prepare EQ standard solutions at concentrations of 0, 0.1, 0.2, 0.5, 1, 2, 5, and 10 μg / L. Using a micropipette, add 100 μL of the sample solution to the sample pad of the test strip vertically. Start timing once the liquid flows. React for 10 minutes and then determine the result. Negative (-): The T line is darker than or the same as the C line, indicating that the concentration of ethoxyquinoline in the sample is below the detection limit. Positive (+): The T line is lighter than the C line or the T line is not visible, indicating that the concentration of ethoxyquinoline in the sample is equal to or higher than the detection limit. Invalid: No C line appears, indicating incorrect operation or that the test strip has deteriorated and become ineffective.

[0034] (2) Precision Three concentration levels of 0, 5, and 10 μg / L were selected to examine the intra-batch repeatability of test strips from the same batch and the inter-batch repeatability of test strips from different batches. The coefficient of variation (CV) of the T / C value was calculated. The results were all less than 10%, indicating that the test strips had good precision.

[0035] (3) Actual sample testing Blank samples and spiked samples at concentrations of 1, 2, 5, 8, 10, and 15 μg / kg were tested for ethoxyquinoline in large yellow croaker, sea bass, and pear. The results showed that the detection sensitivity for ethoxyquinoline in all three food samples was 5 μg / kg, significantly lower than the relevant limits, making it suitable for rapid on-site screening of ethoxyquinoline residues in food.

[0036] In the description of this invention, the term "a plurality of" refers to two or more. Unless otherwise explicitly defined, the terms "upper," "lower," etc., indicate the orientation or positional relationship based on the orientation or positional relationship shown in the accompanying drawings, and are only for the convenience of describing the invention and simplifying the description, and do not indicate or imply that the device or element referred to must have a specific orientation, or be constructed and operated in a specific orientation, and therefore should not be construed as a limitation of the invention. The terms "connection," "installation," "fixing," etc., should be interpreted broadly. For example, "connection" can be a fixed connection, a detachable connection, or an integral connection; it can be a direct connection or an indirect connection through an intermediate medium. For those skilled in the art, the specific meaning of the above terms in this invention can be understood according to the specific circumstances.

[0037] In the description of this specification, the terms "one embodiment," "some embodiments," "specific embodiment," etc., refer to a specific feature, structure, material, or characteristic described in connection with that embodiment or example, which is included in at least one embodiment or example of the present invention. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples.

[0038] The above are merely preferred embodiments of the present invention and are not intended to limit the present invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A time-resolved fluorescence probe for rapid detection of ethoxyquin, characterized in that The time-resolved fluorescence probe is an ethoxyquinoline time-resolved fluorescence probe Eu-EQ-mAb, which is indirectly coupled between a secondary fluorescence probe and an ethoxyquinoline monoclonal antibody EQ-mAb, the secondary fluorescence probe is a time-resolved fluorescence microsphere solution with europium ions as a luminescent center, the average particle size of the time-resolved fluorescence microsphere is 150-250 nm, the hydrated particle size polydispersity index PDI is less than or equal to 0.1, and the time-resolved fluorescence microsphere has a narrow-band emission spectrum with an emission peak at 610-625 nm at an excitation wavelength of about 350 nm. 2.The time-resolved fluorescence probe for rapid detection of ethoxyquin according to claim 1, characterized in that, The preparation method of the time-resolved fluorescence probe comprises: diluting 20 μL of the secondary fluorescence probe with a microsphere diluent to 100 μL, adding 0.25-10 μL of EQ-mAb with a concentration of 0.01 mg / mL, and mixing at room temperature for 1-10 min to obtain Eu-EQ-mAb. 3.The time-resolved fluorescence probe for rapid detection of ethoxyquin according to claim 1, characterized in that, The addition amount of the EQ-mAb is 2 μL, and the reaction time is 5 min.

4. A time-resolved fluorescence immunochromatographic test strip for rapid detection of ethoxyquin, characterized by, The time-resolved fluorescence immunoassay test strip comprises a sample pad (1), a nitrocellulose membrane (2) and a water absorption pad (5) attached on a bottom plate (6) in sequence, the bottom plate (6) is made of PVC, the nitrocellulose membrane (2) is provided with a detection line T and a quality control line C, the T line is coated with an ethoxyquinoline coated antigen EQ-Ag, and the C line is coated with a goat anti-mouse IgG antibody; one end of the sample pad (1) is overlapped with the nitrocellulose membrane (2), and the other end of the water absorption pad (5) is overlapped with the nitrocellulose membrane (2); the EQ-Ag is obtained by coupling an ethoxyquinoline hapten with a carrier protein, and the carrier protein is bovine serum albumin or egg white albumin. 5.The time-resolved fluorescence immunochromatographic test strip for rapid detection of ethoxyquin according to claim 4, characterized in that, The preparation method of the time-resolved fluorescence immunoassay test strip comprises: Step T1, preparation of T / C line coating solution The EQ-Ag stock solution with a concentration of 4.0-5.0 mg / mL is diluted by 10-45 times with a T / C line diluent for standby; the goat anti-mouse IgG antibody is diluted with the same diluent; Step T2, preparation of reaction membrane The nitrocellulose membrane (2) is laid on the coating area of the bottom plate (6), the EQ-Ag diluent is sprayed at a speed of 0.5-1.5 μL / cm by using an XYZ three-dimensional membrane drawing and gold spraying instrument to form the T line, and the goat anti-mouse IgG antibody is sprayed to form the C line, and after spraying, it is placed at 37 ℃ for drying for 2 h; Step T3, sample pad and water absorption pad treatment The sample pad is cut into appropriate size; the water absorption pad is cut and standby; Step T4, assembly and cutting The sample pad (2), the nitrocellulose membrane (2) and the water absorption pad (5) are pasted on the bottom plate (6) in sequence, and each adjacent part is overlapped by 2 mm; an automatic cutting machine is used to cut into a test strip with a width of 2-4 mm, which is put into a sealed bag with a desiccant and stored at room temperature in the dark. 6.The time-resolved fluorescence immunochromatographic test strip for rapid detection of ethoxyquin according to claim 5, characterized in that, The T / C line diluent is PBS+5% methanol, the stock solution concentration of the EQ-Ag is 4.8 mg / mL, and the T / C line diluent is diluted by 15 times when coating; the XYZ three-dimensional membrane drawing and gold spraying instrument is used to spray the EQ-Ag diluent at a speed of 1 μL / cm to form the T line; the width of the test strip is 3 mm.

7. A detection method for rapid detection of ethoxyquin, characterized by, Specifically comprising the following steps: Step S1, obtaining a to-be-tested solution by pretreating a to-be-tested sample; Step S2, add 100 μL of the sample solution into the microwell, add 5 μL of the time-resolved fluorescent probe of any one of claims 1-3, mix and react at room temperature for 3-10 min; Step S3, insert the time-resolved fluorescent immunochromatographic test strip of any one of claims 4-7 into the microwell, immerse the sample end of the test strip into the mixed solution, and perform immunochromatographic reaction at room temperature for 5-15 min; Step S4, take out the test strip, read the fluorescent signals of the detection line and the quality control line on a 365 nm ultraviolet light or an immunofluorescence quantitative analyzer, and determine the presence and approximate concentration level of ethoxyquin in the sample according to the T / C value. 8.The method according to claim 7, wherein, In the step S3, the chromatographic time is 10 min; when the sample is an ethoxyquin standard solution, the sample is determined as positive when the ethoxyquin concentration is ≥1 μg / L, and the semi-quantitative detection limit range is 0.5-1 μg / L. 9.The method of claim 7, wherein the ethoxyquin is detected in less than 10 minutes. The sample to be tested is large yellow croaker, sea bass or pear, and the sample to be tested is obtained after pretreatment by using the pretreatment method of acetonitrile-n-hexane liquid-liquid extraction and adding an antioxidant.

10. Use of the time-resolved fluorescent probe of any one of claims 1-3 and the time-resolved fluorescent immunochromatographic test strip of any one of claims 4-6 for rapid qualitative or semi-quantitative detection of ethoxyquin residues in food.

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