A kit for detecting eugenol residue and its application

Abstract

The invention discloses a compound. Based on the compound, a fluorescence polarization immunoassay of eugenol is established to detect the residual amount of eugenol in aquatic products. The detection range of the fluorescence polarization immunoassay for eugenol is 6.2-495.7 ng / mL, the detection limit is 1.7 ng / mL, and the sensitivity can reach 55.4 ng / mL. A kit for detecting the residual amount of eugenol is established based on the compound and the fluorescence polarization immunoassay. The method is simple to operate, has a large detection quantity, and is highly sensitive, and can be used for the rapid screening and determination of the residual amount of anesthetic eugenol in aquatic products.

Description

Technical Field

[0001] The present invention relates to the field of anesthetic detection and immunoassay technology, and in particular to a kit for detecting eugenol residues and an application thereof. Background Art

[0002] Eugenol, chemical name 4-allyl-2-methoxyphenol, is often used as an anesthetic for fish in the aquatic industry because of its good antibacterial and anesthetic effects on fresh aquatic products, can effectively reduce the injury rate and mortality of fish during transportation, and is cheap. However, the safety of eugenol is still controversial, and my country's agricultural administrative department has not yet included it in the "Aquaculture Drug Information Sheet". Studies have shown that eugenol residues may cause liver damage in mammals and even have potential carcinogenic effects. Its residues in food and its use in the collection, storage and transportation of aquatic products deserve attention.

[0003] The detection of eugenol currently mainly adopts the instrumental detection method, among which gas chromatography-mass spectrometry (GC-MS) and liquid chromatography-mass spectrometry (LC-MS) are the most widely used. However, the instrumental detection method requires expensive analytical equipment, and the method operation is complicated and requires specially trained personnel to operate. Its general application is greatly limited. Therefore, it is necessary to establish a simple, rapid and highly sensitive detection method for detecting eugenol in aquatic products. The detection limit of the eugenol detection method recorded in the prior art (CNKI: SUN: SPAJ.0.2019-20-036) is 2.0 mg / ml, which is not low enough and cannot accurately detect the residual eugenol in the substance.

[0004] Immunoassay detection has the advantages of high sensitivity, good specificity, rapidity and simplicity, and is very suitable for rapid on-site screening and detection. Among them, fluorescence polarization immunoassay is relatively special. It does not require multiple washing steps in the solid phase immunoassay process. The reaction system is completely in solution. It only requires mixing the sample to be tested, the fluorescent tracer and the specific antibody. The one-step reaction can quickly achieve the purpose of quantitative detection within a few minutes. Designing a fluorescent tracer with a reasonable structure is the core step in establishing the eugenol fluorescence polarization immunoassay. At present, there are no research reports on eugenol fluorescent tracers and related fluorescence polarization immunoassay methods at home and abroad. Summary of the invention

[0005] The purpose of the present invention is to overcome the above-mentioned deficiencies in the prior art and to provide a kit for detecting eugenol residues and its application.

[0006] The first object of the present invention is to provide a compound having a structural formula as shown in Formula I.

[0007] The second object of the present invention is to provide a method for preparing the above compound.

[0008] The third object of the present invention is to provide a eugenol fluorescence polarization immunoassay.

[0009] The fourth object of the present invention is to provide a kit for detecting the residual amount of eugenol.

[0010] In order to achieve the above object, the present invention is implemented by the following scheme:

[0011] A compound, the structural formula of the compound is as shown in formula (I),

[0012]

[0013] The method for preparing the compound having the structural formula as shown in Formula I comprises the following steps:

[0014] S1. Using eugenol and methyl acrylate as raw materials, 4-(4-hydroxy-3-methoxyphenyl)-but-2-enoic acid methyl ester is produced by olefin metathesis reaction;

[0015] S2. The 4-(4-hydroxy-3-methoxyphenyl)-but-2-enoic acid methyl ester generated in step S1 is subjected to a hydrolysis reaction to introduce a carboxyl group to generate 4-(4-hydroxy-3-methylaminophenyl)-but-2-enoic acid;

[0016] S3. The 4-(4-hydroxy-3-methylaminophenyl)-but-2-enoic acid generated in step S2 is coupled to ethylenediamine isothiocyanate fluorescein by an active ester method to generate a eugenol fluorescent tracer having a structure shown in formula I.

[0017] More preferably, in step S1, the reaction solvent of the olefin metathesis reaction is dichloromethane, the reaction temperature is 70-80°C, the reaction time is 10-24h, and 1,3-bis(2,4,6-trimethylphenyl)-2-(imidazolidinyl) (dichlorobenzylidene) (tricyclohexylphosphine) ruthenium is used as a reaction catalyst in the reaction; the molar ratio of the eugenol, methyl acrylate and the reaction catalyst is 1:3.2-4:0.06-0.08.

[0018] More preferably, in step S2, the hydrolysis reaction uses sodium hydroxide and 4-(4-hydroxy-3-methoxyphenyl)-but-2-enoic acid methyl ester, the reaction solvent is ethanol, the reaction temperature is 70-80°C, and the reaction time is 5-12h; the molar ratio of the 4-(4-hydroxy-3-methoxyphenyl)-but-2-enoic acid methyl ester and sodium hydroxide is 1:2.8-5; after the hydrolysis reaction is completed, extraction is carried out with hydrochloric acid and ethyl acetate, and drying is carried out after extraction.

[0019] Further preferably, the drying after the extraction is: drying with anhydrous sodium sulfate.

[0020] More preferably, in step S3, the coupling agent of the active ester method is N,N'-dicyclohexylcarbodiimide and N-hydroxysuccinimide; the molar ratio of ethylenediamine isothiocyanate fluorescein, 4-(4-hydroxy-3-methylaminophenyl)-but-2-enoic acid and the coupling agent is 1:2.8~5:11.2~20.

[0021] More preferably, in step S3, the product obtained by the active ester method needs to be separated and purified using a preparative thin layer chromatography plate.

[0022] Further preferably, the developing solvent in the separation and purification process is a mixed solution with a volume ratio of chloroform:methanol=4:1.

[0023] Preferably, the preparation reaction formula of the compound is as shown below.

[0024]

[0025] A eugenol fluorescence polarization immunoassay, the fluorescence polarization immunoassay comprising the following steps:

[0026] S1. Adding eugenol standard solutions with concentrations ranging from 0.001 ng / mL to 10000 ng / mL, with a concentration gradient of not less than 8, to the fluorescent microwells of the fluorescent microwell plate, and then sequentially adding the compound according to claim 1 and the eugenol-specific antibody, mixing to obtain a mixed solution, and allowing the mixed solution to react fully;

[0027] S2. Use a multifunctional fluorescence polarization instrument to read the fluorescence polarization value of the mixed solution after sufficient reaction in the microplate;

[0028] S3. Establish the standard curve of eugenol fluorescence polarization immunoassay;

[0029] S4. Repeat steps S1 and S2, replace the eugenol standard solution in step S1 with the sample to be tested, and compare the measured fluorescence polarization value with the standard curve established in step S3 to obtain the eugenol content in the sample to be tested.

[0030] Preferably, the eugenol-specific antibody is obtained by immunizing an animal with Eul-4-Tbl-BSA, and the Eul-4-Tbl-BSA is obtained by coupling Eul-4-Tbl with a carrier protein BSA having a structural formula as shown in formula (II).

[0031]

[0032] Preferably, in step S1, the concentration of the eugenol fluorescent tracer is 1-5 nmol / L, the concentration of the eugenol-specific antibody is 0.5-5 μg / mL, and the sufficient reaction time is 1-15 min.

[0033] More preferably, in step S1, the volume ratio of the eugenol standard solution, the compound and the eugenol-specific antibody is 1-5:8-10:8-10.

[0034] Further preferably, in step S1, the volume ratio of the eugenol standard solution, the compound and the eugenol-specific antibody is 2:9:9.

[0035] Preferably, in step S3, the abscissa of the standard curve is the concentration of eugenol, and the ordinate is the fluorescence polarization signal intensity.

[0036] The present invention also claims a kit for detecting the residual amount of eugenol, which comprises: the above compound, a eugenol-specific antibody and a eugenol standard solution.

[0037] Compared with the prior art, the present invention has the following beneficial effects:

[0038] The present invention provides a compound of the structural formula, the reaction raw materials of the compound are easily available, the operation process is simple, and the target product yield is high. Based on this compound, a fluorescence polarization immunoassay for eugenol is further established for detecting the residual amount of eugenol in aquatic products. The detection range of the fluorescence polarization immunoassay for eugenol is 6.2 to 495.7 ng / mL, the detection limit is 1.7 ng / mL, and the sensitivity can reach 55.4 ng / mL. A kit for detecting the residual amount of eugenol is established based on the compound and the fluorescence polarization immunoassay. The method is simple to operate, has a large detection quantity, and has a high detection sensitivity, and can be used for the rapid screening and determination of the anesthetic eugenol residues in aquatic products. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1 This is a high-resolution time-of-flight mass spectrometry identification diagram;

[0040] Figure 2 This is the standard curve of eugenol fluorescence polarization immunoassay. DETAILED DESCRIPTION

[0041] The present invention is further described in detail below in conjunction with the accompanying drawings and specific examples of the specification. The examples are only used to explain the present invention and are not used to limit the scope of the present invention. The test methods used in the following examples are conventional methods unless otherwise specified; the materials and reagents used are reagents and materials that can be obtained from commercial channels unless otherwise specified.

[0042] Example 1 Preparation of Eugenol Fluorescent Tracer

[0043] The reaction formula for the preparation of eugenol fluorescent tracer is shown below.

[0044]

[0045] 1. Experimental methods

[0046] 1.64 g of eugenol was weighed, and 30 mL of dichloromethane was used as a solvent to dissolve the eugenol to obtain a mixed solution 1. 100 mg of 1,3-bis(2,4,6-trimethylphenyl)-2-(imidazolidinyl)(dichlorobenzylidene)(tricyclohexylphosphine)ruthenium and 5.3 g of methyl acrylate were added to the mixed solution 1 in sequence. After mixing, the solution was placed under reflux and stirred at 80° C. for olefin metathesis reaction for 24 hours. After the olefin metathesis reaction was completed, the dichloromethane solvent was evaporated to obtain an intermediate: 4-(4-hydroxy-3-methoxyphenyl)-but-2-enoic acid methyl ester.

[0047] 1.5 g of 4-(4-hydroxy-3-methoxyphenyl)-but-2-enoic acid methyl ester was dissolved in 30 mL of ethanol as a solvent to obtain a mixed solution 2, 0.9 g of sodium hydroxide was added to the mixed solution 2, and the mixture was refluxed and stirred at 70° C. for hydrolysis reaction for 10 h. After the hydrolysis reaction was completed, the mixed solution 2 was subjected to reduced pressure distillation to remove ethanol, and 0.5 mol / L hydrochloric acid and ethyl acetate were added to the mixed solution 2 in sequence for extraction. After the extraction was completed, the mixture was washed with saturated brine and dried over anhydrous sodium sulfate, and 4-(4-hydroxy-3-methylaminophenyl)-but-2-enoic acid was obtained by concentration.

[0048] 8.3 mg (40 mmol) of 4-(4-hydroxy-3-methylaminophenyl)-but-2-enoic acid, 16 mg (80 mmol) of N, N'-dicyclohexylcarbodiimide and 10 mg (80 mmol) of N-hydroxysuccinimide were dissolved in 0.5 mL of N, N'-dimethylformamide, stirred at room temperature for 12 hours, and centrifuged after the reaction to collect the supernatant to obtain an activated ester supernatant. 4.5 mg (10 mmol) of ethylenediamine isothiocyanate fluorescein was added to the activated ester supernatant, stirred at room temperature for 4 hours to obtain the reaction product, i.e., eugenol fluorescent tracer.

[0049] The reaction product was separated and purified using a preparative thin layer chromatography plate, wherein the developing solvent was a mixed solution of chloroform and methanol in a volume ratio of 4:1. f = 0.1, 0.2, 0.6, 0.8 and 0.9 fluorescence bands, where R f It is a parameter that represents the migration position of a component and is defined as the ratio of the solute migration distance to the mobile phase migration distance.

[0050] Then, 1 mL of methanol was used to extract the substance on the fluorescent strip to obtain a yellow-green liquid, which was then subjected to activity testing, namely, specific binding with eugenol antibodies. The eugenol-specific antibodies were obtained by immunizing animals with Eul-4-Tbl-BSA, and the Eul-4-Tbl-BSA was obtained by coupling Eul-4-Tbl with a carrier protein BSA as shown in formula (II).

[0051]

[0052] Each R f The fluorescence band corresponds to the fluorescence polarization value (mP value) of the yellow-green liquid before and after binding to the antibody, and high-resolution time-of-flight mass spectrometry is performed on it at the same time.

[0053] 2. Experimental results

[0054] The multifunctional fluorescence polarization instrument reads each R f The corresponding fluorescence polarization value data of the yellow-green liquid before and after binding to the antibody are shown in Table 1.

[0055] Table 1 Fluorescence polarization results

[0056] <![CDATA[R f Value]]> mP value (before binding) mP value (after combination) 0.1 152 155 0.2 128 141 0.6 135 150 0.8 140 308 0.9 133 158

[0057] From the results in Table 1, we can see that R f The fluorescence band at R = 0.8 can specifically bind to the eugenol antibody and give a strong fluorescence polarization signal (308mP), while the other R f The fluorescence polarization signal of the fluorescent band at the value is not much different before and after binding to eugenol antibody.

[0058] High resolution time-of-flight mass spectrometry identification Figure 1 As shown, R f = 0.8 fluorescence MS (EI+) m / z: 656.1527, corresponding to the target fluorescent tracer molecule C 34 H 29 N 3 O 9 The quasi-molecular ion peak of S is the desired eugenol fluorescent tracer.

[0059] Example 2 Establishment of Eugenol Fluorescence Polarization Immunoassay

[0060] 1. Experimental methods

[0061] To the fluorescent microwells of the fluorescent microwell plate, a standard solution of eugenol with a concentration ranging from 0.001 ng / mL to 10000 ng / mL, with a concentration gradient of no less than 8, was sequentially added, followed by the eugenol fluorescent tracer prepared in Example 1 with a concentration of 2 nmol / L and 1 μg / mL of eugenol-specific antibody. The eugenol-specific antibody was obtained by immunizing an animal with Eul-4-Tbl-BSA, wherein the Eul-4-Tbl-BSA was obtained by coupling the Eul-4-Tbl with a carrier protein BSA as shown in the structural formula (II),

[0062]

[0063] The volume ratio of eugenol standard solution, compound 1 and eugenol-specific antibody was 2:9:9. The mixed solution was obtained after mixing. The mixed solution was fully reacted at room temperature for 5 minutes.

[0064] After the reaction is complete, the fluorescence polarization x =480nm, E m =535 nm) to read the fluorescence polarization value of the mixture in the fluorescence microplate.

[0065] The four-parameter logarithmic function in OriginPro 8.5 software was used for data analysis, in which the abscissa was the logarithmic value of the concentration of the eugenol standard solution and the ordinate was the measured fluorescence polarization value, and the standard curve of the eugenol fluorescence polarization immunoassay was drawn.

[0066] 2. Experimental results

[0067] The standard curve of eugenol fluorescence polarization immunoassay is shown in the figure below. Figure 2 The results showed that the linear detection range of the established eugenol fluorescence polarization immunoassay reached 6.2-495.7 ng / mL, the detection limit (LOD) was 1.7 ng / mL, and the sensitivity (IC 50 ) can reach 55.4ng / mL.

[0068] It shows that the detection effect of the eugenol fluorescence polarization immunoassay established in this example is very good and can well meet the detection sensitivity requirements. In addition, this method uses a high-throughput fluorescence microplate and can detect 96 samples at the same time, which has higher detection efficiency. The detection limit of eugenol by the method described in this example is 1.7 ng / mL, which is much better than the detection limit of 2.0 mg / mL of the method recorded in the prior art.

[0069] Example 3 Rapid Detection of Anesthetic Eugenol Residues in Aquatic Products

[0070] 1. Experimental methods

[0071] In this embodiment, seabass, mandarin fish and prawns were selected as aquatic samples. 2 μg / kg, 20 μg / kg and 200 μg / kg of eugenol standard solution were added to seabass, mandarin fish and prawns without eugenol, respectively, to obtain seabass, mandarin fish and prawns used in the experiment.

[0072] Take the edible muscle part of the sea bass, make it into a uniform minced meat in a homogenizer, accurately weigh 2.00g and put it into a 50mL plastic centrifuge tube, add 1mL of distilled water to the centrifuge tube, vortex disperse for 1min, then add 9mL of acetonitrile and vortex extract for 5min, centrifuge at 5000r / min for 5min, take 5mL of supernatant after centrifugation and load it onto the PRiME HLB column for solid phase extraction, collect the effluent and blow it with nitrogen to less than 1mL in a 45°C water bath, then dissolve it with 50mmol / L, pH 8.0 borate buffer and dilute it to 5mL to obtain the sample to be tested. The sample to be tested was tested according to the method described in Example 2, and the specific detection steps are as follows:

[0073] Add 20 μL of the sample to be tested to the fluorescent microplate, and then add 90 μL of the 2 nmol / L eugenol fluorescent tracer prepared in Example 1 and 90 μL of the 1 μg / mL eugenol-specific antibody in sequence, mix well and react at room temperature for 5 minutes.

[0074] After the reaction is complete, the fluorescence polarization x =480nm, E m =535nm) to read the fluorescence polarization value of the sample to be tested in the fluorescence microplate, and calculate the eugenol content in the sample using the eugenol fluorescence polarization immunoassay standard curve established in Example 2.

[0075] The mandarin fish and shrimp samples were treated in the same way to measure the fluorescence polarization values.

[0076] 2. Experimental results

[0077] The fluorescence polarization detection results of seabass, mandarin fish and shrimp samples are shown in Table 1.

[0078] Table 1 Fluorescence polarization detection results of aquatic samples

[0079]

[0080]

[0081] The results showed that in different aquatic samples, the recovery rate of eugenol content determined by the eugenol fluorescence polarization immunoassay method established in Example 2 of the present invention was between 80.6% and 104.8%, and the relative standard deviation (RSD) was between 5.5% and 14.1%, indicating that the detection results of this method are accurate and reliable and can be used for rapid screening and determination of eugenol residues in aquatic products.

[0082] Example 4 Specific detection of eugenol by fluorescence polarization immunoassay

[0083] 1. Experimental methods

[0084] The analogs selected are isoeugenol and methyl eugenol, compounds with similar structures to eugenol, and tricaine, propofol and benzocaine, compounds with similar functions to eugenol, and the above analogs are used for specific detection of eugenol by fluorescence polarization immunoassay. The method steps are consistent with the method steps of Example 2. The competitive inhibition curves of the analogs are drawn in turn by fluorescence polarization immunoassay to obtain the IC values ​​of the structural analogs. 50 , and the cross-reactivity rate (CR) with eugenol was calculated by the formula:

[0085] CR=(IC 50 (Eugenol) / IC 50 (Structural analogues))×100%

[0086] 2. Experimental results

[0087] IC of eugenol and its analogs 50 The values ​​and cross-reaction rates are shown in Table 2.

[0088] Table 2 IC of eugenol and its analogs 50 Value and cross-reaction rate

[0089]

[0090]

[0091] The results show that the eugenol fluorescence polarization immunoassay established in the present invention has basically no cross-reaction to the structural analogs of eugenol, isoeugenol and methyleugenol, and the functional analogs of tricaine, propofol and benzocaine, indicating that this method is not affected by the structural analogs of eugenol and can only detect the content of eugenol.

[0092] Example 5 A kit for detecting eugenol

[0093] 1. Kit Composition

[0094] 1. A eugenol fluorescent tracer working solution containing the eugenol fluorescent tracer prepared in Example 1;

[0095] 2. Eugenol standard solution;

[0096] 3. Sample diluent;

[0097] 4. Fluorescence microplate;

[0098] 5. Eugenol-specific antibody working solution obtained by immunizing animals with Eul-4-Tbl-BSA.

[0099] 2. How to use the kit

[0100] The sample to be tested was added to the fluorescent microplate of the kit, and the eugenol fluorescent tracer working solution and antibody working solution were added in sequence for full reaction. x =480nm, E m =535 nm) Read the fluorescence polarization value of the sample to be tested in the kit, and calculate the eugenol content in the sample using the eugenol fluorescence polarization immunoassay standard curve established in Example 2.

[0101] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit the protection scope of the present invention. For ordinary technicians in this field, other different forms of changes or modifications can be made based on the above descriptions and ideas. It is not necessary and impossible to list all the implementation methods here. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the claims of the present invention.

Claims

1. A eugenol fluorescence polarization immunoassay, characterized in that: The following steps are involved: S1. adding eugenol standard solutions with concentrations ranging from 0.001 ng / mL to 10000 ng / mL, with a concentration gradient of not less than 8, to the fluorescent microwells of the fluorescent microwell plate, and then adding the compound described in formula (I) and the eugenol-specific antibody, respectively, mixing to obtain a mixed solution, and reacting fully; Formula (I); S2. Use a multifunctional fluorescence polarization instrument to read the fluorescence polarization value of the mixed solution after sufficient reaction in the microplate; S3. Establishing the standard curve of eugenol fluorescence polarization immunoassay; S4. Repeat step S1 and step S2, replace the eugenol standard solution in step S1 with the sample to be tested, and compare the measured fluorescence polarization value with the standard curve established in step S3 to obtain the eugenol content in the sample to be tested; The eugenol-specific antibody in step S1 is obtained by immunizing an animal with Eul-4-Tbl-BSA, and the Eul-4-Tbl-BSA is obtained by coupling Eul-4-Tbl with a carrier protein BSA as shown in formula (II). Formula (II).

2. The eugenol fluorescence polarization immunoassay according to claim 1, characterized in that: The concentration of the compound in step S1 is 1-5 nmol / L, the concentration of the eugenol-specific antibody is 0.5-5 μg / mL, and the time for sufficient reaction is 1-15 min.

3. The eugenol fluorescence polarization immunoassay according to claim 1, characterized in that: The horizontal axis of the standard curve in step S3 is the concentration of eugenol, and the vertical axis is the fluorescence polarization signal intensity.

4. The eugenol fluorescence polarization immunoassay according to claim 1, characterized in that: The preparation method of the compound represented by formula (I) comprises the following steps: S1. Using eugenol and methyl acrylate as raw materials, 4-(4-hydroxy-3-methoxyphenyl)-but-2-enoic acid methyl ester was prepared by olefin metathesis reaction; S2. introducing a carboxyl group into the 4-(4-hydroxy-3-methoxyphenyl)-but-2-enoic acid methyl ester produced in step S1 through a hydrolysis reaction to produce 4-(4-hydroxy-3-methylaminophenyl)-but-2-enoic acid; S3. The 4-(4-hydroxy-3-methylaminophenyl)-but-2-enoic acid generated in step S2 is coupled to ethylenediamine isothiocyanate fluorescein by an active ester method to generate a eugenol fluorescent tracer having a structure shown in formula I.

5. The eugenol fluorescence polarization immunoassay according to claim 4, characterized in that: In step S1 of the preparation method, the reaction solvent of the olefin metathesis reaction is dichloromethane, the reaction temperature is 70-80°C, the reaction time is 10-24 h, and 1,3-bis(2,4,6-trimethylphenyl)-2-(imidazolidinyl) (dichlorobenzylidene) (tricyclohexylphosphine) ruthenium is used as a reaction catalyst in the reaction; the molar ratio of the eugenol, methyl acrylate and the reaction catalyst is 1:3.2-4:0.06-0.

08.

6. The eugenol fluorescence polarization immunoassay according to claim 4, characterized in that: In step S2 of the preparation method, the hydrolysis reaction uses sodium hydroxide and 4-(4-hydroxy-3-methoxyphenyl)-but-2-enoic acid methyl ester, the reaction solvent is ethanol, the reaction temperature is 70-80°C, and the reaction time is 5-12 hours; the molar ratio of the 4-(4-hydroxy-3-methoxyphenyl)-but-2-enoic acid methyl ester and sodium hydroxide is 1:2.8-5; after the hydrolysis reaction is completed, extraction is carried out with hydrochloric acid and ethyl acetate, and drying is carried out after extraction.

7. The eugenol fluorescence polarization immunoassay according to claim 4, characterized in that: In step S3 of the preparation method, the coupling agents of the active ester method are N,N'-dicyclohexylcarbodiimide and N-hydroxysuccinimide; the molar ratio of ethylenediamine isothiocyanate fluorescein, 4-(4-hydroxy-3-methylaminophenyl)-but-2-enoic acid and the coupling agent is 1:2.8~5:11.2~20.

8. The kit for detecting residual eugenol by fluorescence polarization immunoassay of eugenol according to claim 1, characterized in that: The kit comprises: the compound of formula (I) as described in claim 1, a eugenol-specific antibody and a eugenol standard solution; The eugenol-specific antibody is obtained by immunizing an animal with Eul-4-Tbl-BSA, and the Eul-4-Tbl-BSA is obtained by coupling Eul-4-Tbl with a carrier protein BSA as shown in formula (II). Formula (II).

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