A method for the broad-spectrum detection of eugenol substances by ratio fluorescence immunoassay

Abstract

The application discloses a kind of broad-spectrum detection eugenol class of ratio fluorescent immunoassay method, it includes the following steps: S1.Synthesis red fluorescent bovine serum albumin coated gold nanoclusters: S2.The coating of microwell plate;S3.The establishment of standard curve;S4.Determination of eugenol class of substance in sample.The application carries out immunoassay based on the ratio change of the emission peak fluorescent intensity of two fluorescent materials, since the interference of two fluorescent materials is the same, the fluorescent intensity ratio of two fluorescent materials cancels out the interference of temperature, polarity, fluorophore concentration and other external factors to some extent, with self-contained internal standard effect, therefore with higher precision, signal-to-noise ratio and sensitivity.The method is more simple and fast than traditional instrument method, detection limit is as low as 0.50 pg / mL, can be used to determine eugenol, isoeugenol, eugenol methyl ether, isoeugenol methyl ether, eugenol acetate, acetyl isoeugenol 6 kinds of anesthetics in aquatic products, and has wide application prospect.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of fluorescence analysis, and particularly relates to a ratio fluorescence immunoassay method for detecting eugenol substances based on red fluorescent gold nanoclusters and o-diphenylamine. BACKGROUND

[0002] Eugenol compounds, mainly including eugenol and isoeugenol, methyl eugenol and methyl isoeugenol, and acetyl eugenol and acetyl isoeugenol, have been widely used in animal transportation since the early 1970s due to their strong anesthetic effect. Fresh aquatic products are added with eugenol in temporary water bodies during circulation and transportation to anesthetize fresh aquatic products, so that they can enter a state similar to dormancy to reduce physiological metabolism, effectively reduce fish damage rate and mortality, and thus increase economic benefits. However, studies have shown that eugenol may cause liver damage in mammals and even have potential carcinogenic effects. The National Toxicology Program (NTP) of the United States has shown through research on the toxic effects of eugenol compounds that rodents can develop cancer cells or potential carcinogens after eating eugenol for a period of time; the U.S. Food and Drug Administration (FDA) also prohibits eugenol compounds from being used as anesthetics and tranquilizers for fresh edible aquatic products; the report of the International Cancer Research Agency (IARC) shows that eugenol can increase the incidence of liver cancer in mice and is classified as the third type of carcinogen. Therefore, it is necessary to evaluate and monitor trace anesthetics to protect human health and control environmental pollution.

[0003] At present, the detection methods of eugenol mainly rely on instrument detection, including ultraviolet spectrophotometry, thin layer chromatography, capillary electrophoresis, gas chromatography, liquid chromatography, gas chromatography-tandem mass spectrometry, liquid chromatography-tandem mass spectrometry and other analysis methods. However, these methods still have some defects, such as low sensitivity of spectrophotometry, poor reproducibility of capillary electrophoresis, relatively complex pretreatment of chromatography and interference, which cannot meet the demand of rapid detection. Instrument analysis requires expensive detection equipment and relatively professional technical personnel for operation, which is limited for primary line detection.

[0004] ELISA method based on antigen-antibody specific binding reaction has the advantages of high throughput, sensitivity, simplicity, low cost, easy operation, good accuracy, etc., and is very suitable for on-site rapid screening and detection, and is concerned in rapid screening of food contaminants. There are relatively few eugenol immunoassay methods. Jie Chaonan et al. (Jie Chaonan, Li Qin, Han Gang, Liu Huan, Wu Lidong, Li Jincheng. Rapid detection of eugenol based on colloidal gold immunochromatography method by monoclonal antibody [J]. Journal of Food Safety and Quality Inspection, 2019, (20): 6938-6943) established a colloidal gold immunochromatography method for eugenol, which is rapid and low cost, and the detection limit is 2.0 mg / L. The current immunological analysis method for the detection of eugenol has relatively low sensitivity, and mainly determines the single compound of eugenol. There is no report on the method for simultaneously determining six kinds (three groups of isomers) of eugenol substances (eugenol and isoeugenol, methyl eugenol and methyl isoeugenol, ethyl acetate eugenol ester and acetyl isoeugenol). Moreover, the traditional ELISA has the disadvantage of insufficient sensitivity for complex samples such as aquatic products, and the sensitivity is low. Therefore, the introduction of fluorescent materials instead of traditional ELISA color developing agent can effectively improve the detection sensitivity. However, single fluorescence signal is easily affected by operation, environment, instrument and other factors, and small differences will cause great changes in fluorescence signal, which is not conducive to trace detection of samples. In order to overcome this problem, two fluorescence signals can be introduced for ratio determination. The two fluorescence emission peak intensities are basically the same in the external interference, have self-calibration effect, and can eliminate the error caused by external interference factors to a certain extent. Such system is more suitable for detection of aquatic products.

[0005] There is no report on the broad-spectrum ratio fluorescence immunoassay method for eugenol substances. SUMMARY

[0006] In order to make up for the defects of the prior art, the present application aims to provide a ratio fluorescent immunoassay method for broad-spectrum detection of eugenol substances based on red fluorescent gold nanoclusters and o-phenylenediamine. By coating eugenol coating in a microplate, using eugenol monoclonal antibody (Anti-Eug Ab) as a recognition element, applying the specific binding reaction of antigen and antibody, constructing a competitive reaction mode, then washing away the unbound drug and antibody, then connecting the horseradish peroxidase labeled goat anti-mouse secondary antibody (Ab2-HRP) as a signal amplification element with the antibody, then washing away the unbound secondary antibody, connecting the HRP in the microplate to catalyze the subsequent addition of H2O2 (hydrogen peroxide) and OPD (o-phenylenediamine) to oxidize the generated fluorescent substance DAP (2,3-diaminophenazine), then adding red fluorescent bovine serum albumin coated gold nanoclusters (BSA-Au NCs) to the microplate, and the generated DAP will quench the fluorescence of BSA-Au NCs. The emission wavelength of BSA-Au NCs is 670 nm, and the emission wavelength of DAP is 570 nm. Therefore, due to the different concentrations of the drug, the competitive reaction of antigen and antibody makes the amount of HRP connected on the microplate different, resulting in different concentrations of DAP generated by enzyme catalytic oxidation, and finally the content of BSA-Au NCs and generated DAP that are not quenched is different, and finally the ratio of the fluorescence intensity at 570 nm and 670 nm wavelengths is calculated, which can achieve the purpose of broad-spectrum detection of eugenol substances. The method has the advantages of convenience, simple operation, high sensitivity, low detection limit, and is suitable for rapid detection of six kinds of eugenol substances in aquatic products, and has wide application prospect.

[0007] The above object of the present application is achieved by the following technical solutions.

[0008] A ratio fluorescent immunoassay method for broad-spectrum detection of eugenol substances comprises the following steps:

[0009] S1. Synthesis of red fluorescent bovine serum albumin coated gold nanoclusters: mix a certain concentration of chloroauric acid solution with an equal amount of bovine serum albumin solution, then add an appropriate amount of sodium hydroxide solution, adjust the pH of the solution to alkaline, heat and react overnight, and obtain a red fluorescent bovine serum albumin coated gold nanocluster aqueous solution (i.e. BSA-Au NCs solution) after the reaction is completed, store it in the refrigerator away from light, and dilute it by a certain multiple when used;

[0010] S2. Coating of the microplate: add eugenol coating to the microplate, stand in the refrigerator for a period of time, then wash away the coating liquid; dry the plate, add a certain volume of blocking solution, incubate for a period of time, then dry the plate, dry it in an oven, and store it for later use;

[0011] S3. Establishment of standard curve: different concentrations of eugenol substance standard solution and eugenol monoclonal antibody are added into the microwell plate obtained in step S2 at the same time, after incubation for a certain time, the plate is washed, goat anti-mouse secondary antibody labeled with horseradish peroxidase is added, the plate is washed after incubation for a certain time, H2O2 and OPD solution are added for incubation, then the BSA-Au NCs solution prepared in step S1 is added for incubation, under excitation at 360 nm, the fluorescence emission spectrum in the range of 400-750 nm is recorded; the fluorescence intensity ratio F 570 / F 670 is calculated, and a standard curve is drawn with eugenol substance concentration as the abscissa and F 570 / F 670 as the ordinate.

[0012] S4. Determination of eugenol substance concentration in sample: equal volume of treated sample is used to replace the eugenol substance standard solution, and the measured F 570 / F 670 is substituted into the standard curve obtained in step S3 to obtain the eugenol substance concentration in the sample.

[0013] As a preferred embodiment of the broad-spectrum eugenol substance detection ratio fluorescence immunoassay method provided by the application, the concentration of the chloroauric acid solution in step S1 is 10-20 mM, and the concentration of the bovine serum albumin solution is 50-60 mg / mL.

[0014] As a preferred embodiment of the broad-spectrum eugenol substance detection ratio fluorescence immunoassay method provided by the application, in step S1, the heating temperature of the chloroauric acid solution and the bovine serum albumin solution is 37°C, and the reaction is performed for 10-14 h.

[0015] As a preferred embodiment of the broad-spectrum eugenol substance detection ratio fluorescence immunoassay method provided by the application, in step S2, the volume of the eugenol coating precursor is 100 μL, and the standing time is 12-15 h.

[0016] As a preferred embodiment of the broad-spectrum eugenol substance detection ratio fluorescence immunoassay method provided by the application, in step S2, the volume of the blocking solution is 100-200 μL, and the blocking time is 1-3 h.

[0017] As a preferred embodiment of the broad-spectrum eugenol substance detection ratio fluorescence immunoassay method provided by the application, in step S3, the concentration of the eugenol monoclonal antibody is 1-5 μg / L, and the time is 30-40 min.

[0018] As a preferred embodiment of the ratio fluorescent immunoassay method for detecting eugenol substances provided by the present application, in step S3, the concentration of H2O2 and OPD solution is 0.5-5 mM, and the time is 30-40 min.

[0019] As a preferred embodiment of the ratio fluorescent immunoassay method for detecting eugenol substances provided by the present application, the eugenol substances are eugenol, isoeugenol, eugenol methyl ether, isoeugenol methyl ether, eugenol acetate, and acetyl isoeugenol.

[0020] As a preferred embodiment of the ratio fluorescent immunoassay method for detecting eugenol substances provided by the present application, the analysis method specifically comprises the following steps:

[0021] S11. Synthesis of red fluorescent bovine serum albumin-coated gold nanoclusters: 10 mL of chloroauric acid solution (10 mM) is mixed with 10 mL of bovine serum albumin solution (50 mg / mL), followed by the addition of 1 mL of sodium hydroxide solution (1 M) to adjust the pH of the solution to alkaline, and the solution is heated at 37°C for 12 h. After the reaction is completed, a red fluorescent bovine serum albumin-coated gold nanocluster aqueous solution is obtained, which is stored in a refrigerator at 4°C in the dark;

[0022] S12. Coating of 96-well microplates: 100 μL of eugenol coating stock is added to the microplate, which is coated in a refrigerator at 4°C for 12 h, followed by washing of the coating solution. The plate is dried by tapping, 120 μL of blocking solution is added for blocking for 3 h, the blocking solution is shaken off, and the plate is dried in an oven at 37°C for storage;

[0023] S13. Establishment of a standard curve: 50 μL of eugenol substance standard solution with different concentrations is added to the 96-well microplate obtained in step S12 at the same time as the addition of 50 μL of antibody. After incubation for 40 min, the plate is washed, 100 μL of 5000-fold diluted horseradish peroxidase-labeled goat anti-mouse secondary antibody is added, incubation is performed for 30 min, and then the plate is washed. 50 μL of 1 mM H2O2 and 1 mM OPD solution is added to each well, followed by the addition of 50 μL of BSA-Au NCs solution prepared in step S11. Incubation is performed for 30 min, and the fluorescence emission spectrum in the range of 400-750 nm is recorded under excitation at 360 nm. The fluorescence intensity ratio F 570 / F 670 is calculated. The standard curve is plotted with the concentration of eugenol substance as the abscissa and F 570 / F 670 as the ordinate.

[0024] S14. Determination of the concentration of eugenol substances in a sample: equal volumes of treated sample are used to replace the eugenol substance standard solution, and the steps in step S13 are followed. The measured F 570 / F 670The standard curve obtained in the step S3 is substituted to obtain the concentration of eugenol substances in the sample.

[0025] Compared with the prior art, the present application has the beneficial effects that:

[0026] By coating eugenol coating in a microplate, using eugenol monoclonal antibody (Anti-Eug Ab) as a recognition element, applying the specific binding reaction of antigen and antibody, constructing a competitive reaction mode, then washing away the unbound drug and antibody, then connecting the horseradish peroxidase labeled goat anti-mouse secondary antibody (Ab2-HRP) as a signal amplification element with the antibody, then washing away the unbound secondary antibody, connecting the HRP catalyzed H2O2 (hydrogen peroxide) and OPD (o-phenylenediamine) oxidation to generate fluorescent substance DAP (2,3-diaminophenazine) which is subsequently added to the red fluorescent bovine serum albumin coated gold nanoclusters (BSA-Au NCs), and the generated DAP quenches the fluorescence of BSA-Au NCs. The emission wavelength of BSA-Au NCs is 670nm, and the emission wavelength of DAP is 570nm. Therefore, due to the different concentrations of drugs, the competitive reaction of antigen and antibody makes the amount of HRP connected on the microplate different, resulting in different concentrations of DAP generated by enzyme catalyzed oxidation, and finally the content of BSA-Au NCs and generated DAP which is not quenched is different, and finally the fluorescence intensity ratio at 570nm and 670nm wavelengths can achieve the purpose of broad-spectrum detection of eugenol substances.

[0027] The present application is based on the ratio change of the fluorescence intensity of the emission peaks of two fluorescent materials for immunological analysis. Since the two fluorescent materials are subjected to the same external interference, the fluorescence intensity ratio of the two fluorescent materials to some extent offsets the interference of external factors such as temperature, polarity and fluorophore concentration, has a self-contained internal standard effect, and therefore has higher precision, signal-to-noise ratio and sensitivity. Compared with the traditional instrument method, the method is simpler and faster, and the detection limit is as low as 0.50 pg / mL, which can be used for determining six anesthetics of eugenol, isoeugenol, eugenol methyl ether, isoeugenol methyl ether, eugenol acetate and acetyl isoeugenol in aquatic products, and has a wide application prospect. BRIEF DESCRIPTION OF DRAWINGS

[0028] Figure 1 It is a schematic diagram of the ratio fluorescence immunoassay method for broad-spectrum detection of eugenol substances based on red fluorescent gold nanoclusters and o-phenylenediamine.

[0029] Figure 2 It is a transmission electron microscope image of the red fluorescent gold nanoclusters prepared in step S11 of the present application.

[0030] Figure 3 It is a fluorescence spectrum diagram of the red fluorescent gold nanoclusters prepared in step S11 of the present application.

[0031] Figure 4 To detect the standard curve of eugenol, isoeugenol, methyleugenol, isomethyleugenol, eugenol acetate and acetyl isoeugenol 6 kinds of anesthetics. DETAILED DESCRIPTION

[0032] As described in the above technical background, the detection of eugenol substances at present mainly relies on large instrument method, and there is still no immunological analysis method combined with fluorescence for the detection of eugenol substances.

[0033] Therefore, the purpose of the present application is to provide a ratio fluorescence immunoassay method for detecting eugenol substances based on red fluorescence gold nanoclusters and o-phenylenediamine. By coating eugenol coating in a microplate, using eugenol monoclonal antibody (Anti-Eug Ab) as a recognition element, applying the specific binding reaction of antigen and antibody, constructing a competitive reaction mode, then washing away the unbound drug and antibody, then connecting the horseradish peroxidase labeled goat anti-mouse secondary antibody (Ab2-HRP) as a signal amplification element with the antibody, then washing away the unbound secondary antibody, connecting the HRP catalyzed H2O2 (hydrogen peroxide) and OPD (o-phenylenediamine) added subsequently to generate fluorescent substance DAP (2,3-diaminophenazine), then adding red fluorescent bovine serum albumin coated gold nanoclusters (BSA-Au NCs) to it, and the generated DAP will quench the fluorescence of BSA-Au NCs. The emission wavelength of BSA-Au NCs is 670 nm, and the emission wavelength of DAP is 570 nm. Therefore, due to the different concentrations of drugs, the competitive reaction of antigen and antibody makes the amount of HRP connected on the microplate different, resulting in different concentrations of DAP generated by enzyme catalytic oxidation, and finally the content of BSA-Au NCs and generated DAP that are not quenched is different, and finally the calculation of the fluorescence intensity ratio at 570 nm and 670 nm wavelengths can achieve the purpose of broad-spectrum detection of eugenol substances. The method has the advantages of convenience, simple operation, high sensitivity and low detection limit, and is suitable for rapid detection of 6 kinds of eugenol substances in aquatic products, and has wide application prospect.

[0034] The technical solutions of the present application will be further described in detail in combination with specific embodiments.

[0035] Unless otherwise specified, the reagents, methods and instruments used in the present application are conventional reagents, methods and instruments in the technical field.

[0036] Unless otherwise specified, the reagents and materials used in the following examples are commercially available

[0037] Wherein, the unit "M" referred to in the present application represents the amount concentration of the substance, which can also be expressed as mol / L, and common ones are nM (nmol / L), μM (umol / L), mM (mmol / L), M (mol / L).

[0038] Example 1

[0039] The present embodiment provides a ratio fluorescence immunoassay method for detecting eugenol substances in a broad spectrum, which specifically comprises the following steps:

[0040] Step S11. Synthesis of red fluorescent bovine serum albumin coated gold nanoclusters: 10 mL of chloroauric acid solution (10 mM) is mixed with 10 mL of bovine serum albumin solution (50 mg / mL), followed by adding 1 mL of sodium hydroxide solution (1 M), adjusting the pH of the solution to alkaline, and heating the reaction at 37°C for 12 h. After the reaction is completed, a red fluorescent bovine serum albumin coated gold nanocluster aqueous solution is obtained, which is stored in a refrigerator at 4°C in the dark;

[0041] From Figure 2 It can be seen that the prepared red fluorescent bovine serum albumin coated gold nanoclusters are circular particles with a particle size of 5.1 nm. The optimal excitation wavelength of the red fluorescent gold nanoclusters is 360 nm, and the optimal emission wavelength is at 670 nm Figure 3 ).

[0042] Step S12. Coating of 96-well microplate: 100 μL of eugenol coating stock is added to the microplate, which is coated in a refrigerator at 4°C for 12 h, followed by washing away the coating liquid. The plate is dried, 120 μL of blocking solution is added for blocking for 3 h, the blocking solution is shaken off, and the plate is dried in a 37°C oven for storage;

[0043] Step S13. Establishment of standard curve: 50 μL of different concentrations of eugenol substance standard solution and 50 μL of eugenol monoclonal antibody are simultaneously added to the 96-well microplate obtained in step S12, incubated for 40 min, and then the plate is washed. 100 μL of 5000-fold diluted horseradish peroxidase labeled goat anti-mouse secondary antibody is added, incubated for 30 min, and then the plate is washed. 50 μL of 1 mM H2O2 and 1 mM OPD solution is added to each well, followed by adding 50 μL of BSA-Au NCs solution prepared in step S11, incubating for 30 min, and recording the fluorescence emission spectrum in the range of 400-750 nm under excitation at 360 nm. The fluorescence intensity ratio F 570 / F 670 is calculated. The standard curve is plotted with the concentration of eugenol substance as the abscissa and F 570 / F 670 as the ordinate.

[0044] From Figure 4It can be seen that, with the increase of eugenol concentration, the amount of monoclonal antibody connected to the microplate is reduced due to the competition of the antibody and the drug, and then the amount of goat anti-mouse labeled with horseradish peroxidase is reduced, and the content of DAP generated by H2O2 oxidizing OPD catalyzed by the enzyme is reduced, and the content of BSA-Au NCs and the generated DAP is different, which is specifically manifested as F 570 / F 670 value is reduced, and F 570 / F 670 is negatively correlated with the concentration of eugenol. The detection limit is as low as 0.50 pg / mL, which meets the determination of six anesthetics of eugenol, isoeugenol, eugenol methyl ether, isoeugenol methyl ether, eugenol acetate, and acetyl isoeugenol in aquatic products.

[0045] Step S14. Determination of the concentration of eugenol substances in the sample: replace the eugenol substance standard solution with an equal volume of the treated sample, and operate according to the steps of step S13, and then F 570 / F 670 is measured, and the standard curve obtained in step S3 is substituted to obtain the concentration of eugenol substances in the sample.

[0046] For actual sample fish and shrimp: take fish and shrimp samples added at levels of 10 ng / g, 50 ng / g, 100 ng / g, and 1000 ng / g, 5000 ng / g, and 10000 ng / g, and determine the six anesthetics of eugenol, isoeugenol, eugenol methyl ether, isoeugenol methyl ether, acetyl isoeugenol, and eugenol acetate according to the steps described in step S13. The recovery rate is calculated according to the formula: recovery rate (%) = (determined value after addition-blank value) / addition amount x 100%. The results are shown in Tables 1 and 2. The recovery rate of this addition is between 82.9% and 111.9%, and the coefficient of variation is less than 7.9%, indicating that the method for determining eugenol substances has good accuracy and stability.

[0047] Table 1 Addition recovery results of six eugenol substances in fish samples

[0048]

[0049] Table 2 Addition recovery results of six eugenol substances in shrimp samples

[0050]

[0051]

[0052] Example 2

[0053] The present embodiment provides a ratio fluorescent immunoassay method for detecting eugenol substances in a broad spectrum, which specifically comprises the following steps:

[0054] Step S21. Synthesis of red fluorescent bovine serum albumin coated gold nanoclusters: 10 mL of chloroauric acid solution (15 mM) was mixed with 10 mL of bovine serum albumin solution (60 mg / mL), followed by the addition of 1 mL of sodium hydroxide solution (1 M), the pH of the solution was adjusted to alkaline, and the reaction was heated at 37°C for 10 h. After the reaction was completed, a red fluorescent bovine serum albumin coated gold nanoclusters aqueous solution was obtained, which was stored in a refrigerator at 4°C in the dark;

[0055] Step S22. Coating of 96-well microplate: 100 μL of eugenol coating stock was added to the microplate, which was coated in a refrigerator at 4°C for 12 h, followed by washing away the coating solution. The plate was dried, 100 μL of blocking solution was added and blocked for 2 h, the blocking solution was shaken off, and the plate was dried in an oven at 37°C. The plate was stored for later use;

[0056] Step S23. Establishment of standard curve: 50 μL of different concentrations of eugenol standard solution was added to the 96-well microplate obtained in step S22, and 50 μL of eugenol monoclonal antibody was added at the same time. After incubation for 40 min, the plate was washed, 100 μL of 5000-fold diluted horseradish peroxidase labeled goat anti-mouse secondary antibody was added, and incubated for 30 min. Then, 50 μL of 0.5 mM H2O2 and 0.5 mM OPD solution was added to each well, followed by the addition of 50 μL of BSA-Au NCs solution prepared in step S21. After incubation for 40 min, the fluorescence emission spectrum in the range of 400-750 nm was recorded under excitation at 360 nm. The fluorescence intensity ratio F 570 / F 670 was calculated. 570 670 The standard curve was plotted with eugenol concentration as the abscissa and F 570 / F 670 as the ordinate.

[0057] Step S24. Determination of eugenol concentration in sample: equal volume of treated sample was used to replace the eugenol standard solution, and the steps in step S23 were followed. The measured F 570 / F 670 was substituted into the standard curve obtained in step S3 to obtain the eugenol concentration in the sample.

[0058] Example 3

[0059] The present embodiment provides a broad-spectrum detection of eugenol ratio fluorescence immunoassay method, which specifically comprises the following steps:

[0060] Step S31. Synthesis of red fluorescent bovine serum albumin coated gold nanoclusters: 10 mL of chloroauric acid solution (20 mM) was mixed with 10 mL of bovine serum albumin solution (55 mg / mL), followed by the addition of 1 mL of sodium hydroxide solution (1 M), the pH of the solution was adjusted to alkaline, and the solution was heated at 37°C for 14 h. After the reaction was completed, a red fluorescent bovine serum albumin coated gold nanocluster aqueous solution was obtained, which was stored in a refrigerator at 4°C in the dark;

[0061] Step S32. Coating of 96-well microplate: 100 μL of eugenol coating stock was added to the microplate, which was coated in a refrigerator at 4°C for 15 h, followed by washing of the coating solution, and the plate was dried by tapping. 120 μL of blocking solution was added and blocked for 3 h, and the blocking solution was shaken dry. The plate was stored in a 37°C oven for storage.

[0062] Step S33. Establishment of standard curve: 50 μL of eugenol standard solution of different concentrations was added to the 96-well microplate obtained in step S32, and 50 μL of eugenol monoclonal antibody was added at the same time. After incubation for 40 min, the plate was washed, 100 μL of 5000-fold diluted horseradish peroxidase labeled goat anti-mouse secondary antibody was added, and incubation was performed for 30 min. Then, 50 μL of 5 mM H2O2 and 5 mM OPD solution was added to each well, followed by the addition of 50 μL of BSA-Au NCs solution prepared in step S31. After incubation for 35 min, the fluorescence emission spectrum in the range of 400-750 nm was recorded under excitation at 360 nm. The fluorescence intensity ratio F 570 / F 670 was calculated. 570 670 The standard curve was plotted with the concentration of eugenol as the abscissa and F 570 / F 670 as the ordinate.

[0063] Step S34. Determination of the concentration of eugenol in the sample: equal volume of the treated sample was used to replace the eugenol standard solution, and the steps in step S33 were followed. The measured F 570 / F 670 was substituted into the standard curve obtained in step S3 to obtain the concentration of eugenol in the sample.

[0064] The above examples only express the embodiments of the present application, which are described in detail and specifically, but should not be understood as limiting the scope of the present application. Any technical solution obtained by equivalent substitution or equivalent transformation should fall within the scope of the present application.

Claims

1. A ratiometric immunoassay method for broad-spectrum detection of eugenol compounds, characterized in that, It includes the following steps: S1. Synthesis of gold nanoclusters coated with bovine serum albumin: A certain concentration of chloroauric acid solution was mixed with an equal volume of bovine serum albumin solution, and an appropriate amount of sodium hydroxide solution was added to adjust the pH of the solution to alkaline. The reaction was heated overnight. After the reaction was completed, an aqueous solution of gold nanoclusters coated with red fluorescent bovine serum albumin was obtained. The solution was stored in a refrigerator protected from light and diluted before use. S2. Coating of microplates: Add eugenol coating agent to microplates, let stand in the refrigerator for a period of time, wash off the coating solution; pat the plate dry, add a certain volume of blocking solution, incubate for a period of time, pat the plate dry, dry in an oven, and store for later use. S3. Establishment of standard curve: Eugenol standard solutions of different concentrations and eugenol monoclonal antibody were added simultaneously to the microplate obtained in step S2. After incubation for a certain period of time, the plate was washed, horseradish peroxidase-labeled goat anti-mouse secondary antibody was added, and after incubation for a period of time, the plate was washed again. H2O2 and OPD solution were added and incubated. Then, the gold nanoclusters coated with red fluorescent bovine serum albumin prepared in step S1 were added and incubated. The fluorescence emission spectrum in the range of 400-750 nm was recorded under 360 nm excitation. Calculate the fluorescence intensity ratio F 570 / F 670 Plotting the concentration of eugenol compounds on the x-axis, F 570 / F 670 Plot a standard curve for the ordinate; S4. Determination of eugenol concentration in the sample: Replace the eugenol standard solution with an equal volume of the pre-treated sample, and follow the steps in S3. Measure the concentration of F... 570 / F 670 Substitute the standard curve obtained in step S3 into the sample to obtain the concentration of eugenol compounds in the sample. In step S1, the concentration of chloroauric acid solution is 10-20 mM, and the concentration of bovine serum albumin solution is 50-60 mg / mL. In step S1, the heating temperature of the chloroauric acid solution and the bovine serum albumin solution is 37°C, and the reaction time is 10–14 h. In step S2, the eugenol coating is 100 μL in its original volume and left to stand for 12–15 h. In step S2, the sealing solution needs to be 100-200 μL, and the sealing time is 1-3 hours. In step S3, the concentration of eugenol monoclonal antibody is 1–5 μg / L, and the incubation time is 30–40 min. In step S3, the concentrations of H2O2 and OPD solution are 0.5mM to 5mM, and the incubation time is 30 to 40 minutes.

2. The ratiometric fluorescence immunoassay method for broad-spectrum detection of eugenol compounds according to claim 1, characterized in that, The eugenol compounds are eugenol, isoeugenol, eugenol methyl ether, isoeugenol methyl ether, eugenol acetate, and acetyl isoeugenol.

3. The ratiometric fluorescence immunoassay method for broad-spectrum detection of eugenol compounds according to claim 1, characterized in that, In step S1, the refrigerator temperature for storing items away from light is set to 4°C.

4. The ratiometric fluorescence immunoassay method for broad-spectrum detection of eugenol compounds according to claim 1, characterized in that, Specifically, the following steps are included: S11. Synthesis of gold nanoclusters coated with red fluorescent bovine serum albumin: 10 mL of 10 mM chloroauric acid solution was mixed with 10 mL of 50 mg / mL bovine serum albumin solution, and then 1 mL of 1 M sodium hydroxide solution was added to adjust the pH of the solution to alkaline. The reaction was heated at 37 °C for 12 h. After the reaction was completed, an aqueous solution of gold nanoclusters coated with red fluorescent bovine serum albumin, namely BSA-Au NCs solution, was obtained and stored in a refrigerator at 4 °C in the dark. S12. Coating of 96-well microplates: Add 100 μL of eugenol coating agent to the microplate and coat it at 4°C for 12 h. Then wash off the coating solution, pat the plate dry, add 120 μL of blocking solution and block for 3 h. Shake off the blocking solution, dry it at 37°C and store it for later use. S13. Establishment of standard curve: Add 50 μL of eugenol standard solutions of different concentrations and 50 μL of antibody to the 96-well microplate obtained in step S12. After incubation for 40 min, wash the plate and add 100 μL of horseradish peroxidase-labeled goat anti-mouse secondary antibody diluted 5000 times. After incubation for 30 min, wash the plate and add 50 μL each of 1 mM H2O2 and 1 mM OPD solution to each well. Then add 50 μL of BSA-Au NCs solution prepared in step S11 and incubate for 30 min. Record the fluorescence emission spectrum in the range of 400–750 nm under 360 nm excitation. Calculate the fluorescence intensity ratio F 570 / F 670 Plotting the concentration of eugenol compounds on the x-axis, F 570 / F 670 Plot a standard curve for the ordinate; S14. Determination of eugenol concentration in the sample: Replace the eugenol standard solution with an equal volume of the pre-treated sample, and follow the steps in S13. Measure the concentration of F... 570 / F 670 Substitute the standard curve obtained in step S13 into the standard curve to obtain the concentration of eugenol in the sample.

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