A method for detecting tetracycline residues in animal-derived foods

Abstract

The present invention discloses a method for detecting tetracycline residues in animal-derived foods, belonging to the field of food safety analysis. By using the prepared spiropyran-modified covalent organic framework as the reference fluorescence, a composite nanofiber membrane doped with spiropyran-modified covalent organic framework and sodium dodecylbenzenesulfonate is prepared by electrospinning, and then a dynamic adsorption-solid phase sensor is constructed for the dynamic ratio fluorescence sensing and visual colorimetric sensing detection of tetracycline in animal-derived foods. Compared with the existing method of constructing a solid-phase sensor by drop coating or impregnation, the dynamic adsorption mode of the present invention selectively adsorbs tetracycline on the composite nanofiber membrane, and performs solid-phase sensing by forming a polystyrene-tetracycline-sodium dodecylbenzenesulfonate supramolecular complex, reducing the interference of impurities in complex samples and improving the sensitivity of tetracycline ratio fluorescence sensing and the refinement degree of colorimetric sensing.

Description

Technical Field

[0001] The present invention belongs to the field of food safety analysis, and particularly relates to a method for detecting tetracycline residues in animal-derived foods. Background Art

[0002] With the continuous development of current technology, tetracycline is a broad-spectrum antibiotic that is often used to treat infections caused by microorganisms and promote the growth of livestock. It has the advantages of remarkable effects and being cheap and easily available. Therefore, tetracycline is widely used in the livestock and poultry breeding industry; the abuse of tetracycline has led to its residues in animal-derived foods such as milk and meat. Long-term consumption of foods containing tetracycline residues is harmful to human health, such as poor development of bones and tooth enamel, allergic reactions, gastrointestinal diseases, and hepatotoxicity; to ensure food safety, the European Union, the World Health Organization, China, etc. have stipulated the maximum contamination limit of tetracycline in foods. Therefore, effectively detecting tetracycline residues in foods has important practical significance.

[0003] Solid-phase fluorescence / colorimetric sensing is a technology that immobilizes probes on solid carriers for the visual detection of target substances. Compared with liquid-phase sensing, solid-phase sensing has high stability and strong portability, so it has high practical application value; most of the currently reported fluorescence / colorimetric sensors for tetracycline can achieve sensitive liquid-phase fluorescence sensing. However, when the sensing system established in the liquid phase is directly used for solid-phase colorimetric sensing, the sensitivity is significantly reduced; the reported carriers such as filter paper, glass rods, films, or gloves selected for solid-phase colorimetric sensing have a small surface area and do not allow internal mass transfer. Therefore, the fluorescent probe can only be immobilized on the surface of the carrier at a low loading rate, and the interaction between the fluorescent probe and tetracycline is limited; in addition, the commonly reported probe immobilization methods such as impregnation or coating cannot ensure the stable loading of the probe, so the sensing performance cannot be improved; therefore, it is very necessary to select a suitable carrier to achieve the stable immobilization of the fluorescent probe and improve the sensitivity of solid-phase fluorescence / colorimetric sensing of tetracycline.

[0004] Compared with general solid carriers such as filter paper and films, nanofiber membranes have a higher specific surface area, which helps to achieve a higher probe loading rate and provides more sensing positions for the interaction between the probe and the target substance, helping to improve the detection sensitivity of solid-phase sensors; in addition, the high porosity on the surface of the nanofiber membrane and the network structure formed by the interconnected pores inside contribute to the diffusion and mass transfer of the target substance, thereby effectively shortening the response time and improving the detection speed of solid-phase sensors; before preparing the nanofiber membrane by electrospinning, doping the probe into the polymer solution, due to the rapid evaporation of the solvent, the probe can be quickly and effectively dispersed and embedded in the nanofiber membrane, thereby improving the loading stability and distribution uniformity of the probe and improving the sensing performance.

[0005] In addition to being a good carrier for immobilizing probes, the nanofiber membrane can also be modified with various functional groups to achieve selective adsorption of target substances, further improving the sensitivity of sensing. Therefore, a solid-phase sensor based on the nanofiber membrane can achieve highly sensitive visual fluorescence sensing detection of tetracycline. Summary of the Invention

[0006] Aiming at the deficiencies of the prior art, the purpose of the present invention is to provide a method for detecting tetracycline residues in animal-derived foods, preparing a composite nanofiber membrane as a novel solid-phase sensor, performing ratio fluorescence sensing and visual colorimetric detection after efficiently adsorbing tetracycline in animal-derived foods, and providing a reliable on-site screening method for improving food safety levels.

[0007] The purpose of the present invention can be achieved through the following technical solutions:

[0008] A method for detecting tetracycline residues in animal-derived foods, comprising the following steps:

[0009] S1: Synthesize covalent organic frameworks by solvothermal method;

[0010] S2: Prepare spirooxazine-modified covalent organic frameworks;

[0011] S3: Prepare a composite nanofiber membrane;

[0012] S4: Prepare a dynamic adsorption-solid phase sensor;

[0013] S5: Obtain a sample extract by preliminary treatment of animal-derived foods;

[0014] S6: Pass the sample extract obtained in S5 through the dynamic adsorption-solid phase sensor under the action of gravity;

[0015] S7: Take out the composite nanofiber membrane in the dynamic adsorption-solid phase sensor and dry it;

[0016] S8: Perform quantitative analysis of fluorescence ratio sensing using a fluorescence microscope-fiber optic spectrometer;

[0017] S9: Qualitative visual colorimetric detection of tetracycline;

[0018] S10: Semi-quantitative colorimetric detection of tetracycline.

[0019] Further, the steps of preparing the covalent organic frameworks include:

[0020] 1) Using ethyl acetate as a solvent, prepare a solution of trimesoyl chloride with a mass concentration of 2-5% (w / v);

[0021] 2) Using ethyl acetate as a solvent, prepare a solution of p-phenylenediamine with a mass concentration of 1-3% (w / v);

[0022] 3) Under the conditions of an ice-water bath (0 °C) and magnetic stirring, the p-phenylenediamine solution was slowly added dropwise to the trimesoyl chloride solution within 1 - 2 h. After magnetic stirring at room temperature for 12 - 24 h, the reaction solution was added dropwise to distilled water with a volume ratio of 1:15 - 1:25 (v / v) within 5 - 10 min, ultrasonicated for 30 - 60 min, and then centrifuged. The upper layer solution was discarded, and the covalent organic framework was collected;

[0023] 4) The covalent organic framework was successively washed 3 - 5 times by continuous vortexing with ethanol and distilled water with a mass-to-volume ratio of 1:50 - 1:100 (w / v), centrifuged, and the upper layer solvent was discarded. The washed covalent organic framework was collected and placed in a vacuum drying oven at 40 - 60 °C for drying for 8 - 12 h to obtain a dry and clean covalent organic framework.

[0024] Further, the preparation steps of the spiropyran-modified covalent organic framework include:

[0025] 1) Using dichloromethane as a dispersion solvent, a covalent organic framework dispersion solution with a mass concentration of 1 - 3% (w / v) was prepared;

[0026] 2) 2 - 5% (w / v) spiropyran, 4 - 10% (w / v) 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, and 1 - 3% (w / v) 4-dimethylaminopyridine were successively added. After magnetic stirring at room temperature for 24 - 36 h, the reaction solution was centrifuged, and the upper layer solution was discarded to collect the spiropyran-modified covalent organic framework;

[0027] 3) The spiropyran-modified covalent organic framework was successively washed 3 - 5 times by continuous vortexing with ethanol and distilled water with a mass-to-volume ratio of 1:50 - 1:100 (w / v), centrifuged, and the upper layer solvent was discarded. The washed spiropyran-modified covalent organic framework was collected and placed in a vacuum drying oven at 40 - 60 °C for drying for 8 - 12 h to obtain a dry and clean spiropyran-modified covalent organic framework.

[0028] Further, the preparation steps of the composite nanofiber membrane include:

[0029] 1) Using N,N-dimethylformamide as a solvent, a mixed solution with a polystyrene mass concentration of 25 - 35% (w / v) and a sodium dodecylbenzenesulfonate mass concentration of 0.6 - 3% (w / v) was prepared, and continuously magnetically stirred at room temperature for 5 - 8 h to obtain a homogeneous solution;

[0030] 2) Using N,N-dimethylformamide as a dispersion solvent, a spiropyran-modified covalent organic framework dispersion solution with a mass concentration of 0.05 - 0.25% (w / v) was prepared. After ultrasonicating for 30 - 60 min, the dispersion solution was added to the above homogeneous solution and magnetically stirred overnight to obtain an electrospinning precursor solution;

[0031] 3) Place the spinning solution in a spraying container. Set the voltage of the high-voltage power supply of the high-voltage electrostatic field to 16 - 20 kV, adjust the distance from the nozzle to the aluminum foil receiving screen to 10 - 20 cm, and the solution propulsion speed to 1 - 3 mL / h. After collecting for 1 - 3 h, a composite nanofiber membrane is obtained.

[0032] Further, the animal-derived foods in S5 include eggs, milk, fish, shrimp, and internal organs.

[0033] Further, the step of preliminarily treating the milk into a sample extract is as follows: Add 5% trichloroacetic acid (V / V) solution to the milk sample, vortex for 0.5 min, and after centrifugation, take the supernatant.

[0034] Further, the step of preliminarily treating the fish or shrimp into a sample extract is as follows: Add 5% trichloroacetic acid (V / V) solution to the fish or shrimp, homogenize, ultrasonically extract for 10 min, centrifuge and separate, and take out the supernatant.

[0035] Further, in S8, analyze the fluorescence spectrum of the composite nanofiber membrane by a fluorescence microscope - fiber optic spectrometer, and perform quantitative ratio fluorescence sensing detection of tetracycline according to the change in the fluorescence intensity ratio of the spiropyran - modified covalent organic framework and tetracycline.

[0036] Further, the qualitative visual colorimetric detection of tetracycline is to observe the color change of the composite nanofiber membrane before and after adsorbing tetracycline under the irradiation of a 365 nm ultraviolet lamp.

[0037] Further, in S10, after taking a photo of the composite nanofiber membrane in S5 with a smartphone, then use color analysis software to obtain the red, green, and blue (RGB) chromaticity values of the image, and perform semi - quantitative colorimetric detection of tetracycline using the green / red chromaticity ratio (G / R) of the image.

[0038] The beneficial effects of the present invention: The present invention provides a method for dynamic adsorption - solid - phase sensing detection of tetracycline residues in animal - derived foods. A composite nanofiber membrane of polystyrene doped with spiropyran - modified covalent organic framework and sodium dodecylbenzenesulfonate is prepared by electrospinning for dynamic ratio fluorescence sensing and visual colorimetric detection of tetracycline in various animal - derived foods such as eggs, milk, fish, shrimp, meat, and internal organs; compared with the existing method of constructing solid - phase sensing by drop - coating or impregnation, the novel dynamic solid - phase sensing mode has the following advantages and effects:

[0039] (1) High sensitivity of ratio fluorescence sensing:

[0040] The present invention uses the green fluorescence emitted by tetracycline at a wavelength of 550 nm as the recognition fluorescence signal. At the same time, the red fluorescence emitted by the spiropyran-modified covalent organic framework at a wavelength of 620 nm is used as the reference fluorescence signal. According to the linear relationship between the ratio of the fluorescence intensities of the two and the concentration of tetracycline, quantitative detection of tetracycline ratio fluorescence sensing is carried out. The composite nanofiber membrane is used as a solid-phase sensor. Polystyrene in its structure is a polymer with abundant π electrons. Using polystyrene as the backbone of the nanofiber membrane can form π-π interactions with tetracycline. In addition, the addition of sodium dodecylbenzenesulfonate can improve the hydrophilicity of the polystyrene nanofiber membrane, thereby promoting the full contact between tetracycline and the nanofiber membrane and making the interaction force between the two more likely to occur. At the same time, there is a hydrogen bond between the oxygen atom in the sulfonic acid group of sodium dodecylbenzenesulfonate and the hydroxyl group of tetracycline, further strengthening the interaction between the composite nanofiber membrane and tetracycline. The strong interaction between polystyrene, sodium dodecylbenzenesulfonate and tetracycline contributes to the formation of the polystyrene-tetracycline-sodium dodecylbenzenesulfonate supramolecular complex. The rigid structure of this supramolecular complex is significantly higher than that of tetracycline, and the absorption coefficient increases with the increase of the rigid structure. The non-radiative inactivation and quenching rate in the system decrease, and the quantum yield of tetracycline increases accordingly. Using the whole composite nanofiber membrane as a probe, while the sample dynamically adsorbs tetracycline through the sensor, the fluorescence performance of tetracycline is improved. Therefore, the fluorescence intensity of tetracycline at a wavelength of 550 nm increases, while the fluorescence intensity of the spiropyran-modified covalent organic framework at a wavelength of 620 nm remains stable. The increase in the ratio of the fluorescence intensities of the two is beneficial to improving the sensitivity of ratio fluorescence sensing.

[0041] (2) High degree of refinement in colorimetric sensing

[0042] Spiropyran is an important photochromic dye with good luminescence and color display properties. It can produce red fluorescence under ultraviolet light irradiation. The red fluorescence emitted by spiropyran and the green fluorescence emitted by tetracycline produce color superposition, thereby realizing visual colorimetric sensing of tetracycline. When the solid-phase colorimetric sensor of the present invention detects low-concentration tetracycline, an obvious color change from light rose red to orange-pink can be shown when the concentration changes twice. When the concentration of tetracycline is 50 nM, the color changes from the red system to the yellow system. The high degree of refinement in colorimetric sensing is closely related to the stable modification of spiropyran and the tunability of its fluorescence intensity. In the present invention, spiropyran is grafted onto the covalent organic framework by a covalent modification method. The large π-conjugated structure of the covalent organic framework interacts with the benzene ring of polystyrene to form π-π bonds, improving the stability of the sensor. At the same time, the adjustable reference fluorescence intensity can be achieved by adjusting the amount of the spiropyran-modified covalent organic framework added to the composite nanofiber membrane. Detailed implementation manners

[0043] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts belong to the scope of protection of the present invention.

[0044] Example 1:

[0045] This example is applied to the sensing detection of tetracycline residues in milk samples. The specific steps are as follows:

[0046] S1: Synthesize covalent organic frameworks by the solvothermal method;

[0047] First, use ethyl acetate as the solvent to prepare a 2% (w / v) solution of trimesoyl chloride; use ethyl acetate as the solvent to prepare a 1% (w / v) solution of p-phenylenediamine; then, under the conditions of an ice-water bath (0 °C) and magnetic stirring, slowly drop the p-phenylenediamine solution into the trimesoyl chloride solution within 1 h; after magnetic stirring at room temperature for 12 h, drop the reaction solution into distilled water with a volume ratio of 1:15 (v / v) within 5 min, ultrasonicate for 30 min and then centrifuge, discard the upper layer solution, and collect the covalent organic frameworks; finally, continuously vortex wash the covalent organic frameworks 3 times with ethanol and distilled water with a mass-volume ratio of 1:50 (w / v) in sequence, centrifuge, discard the upper layer solvent, collect the washed covalent organic frameworks, and place them in a vacuum drying oven at 40 °C for 8 h to obtain dry and clean covalent organic frameworks.

[0048] S2: Prepare spiro-pyran-modified covalent organic frameworks;

[0049] Use dichloromethane as the dispersion solvent to prepare a 1% (w / v) dispersion solution of covalent organic frameworks; sequentially add 2% (w / v) spiro-pyran, 4% (w / v) 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, and 1% (w / v) 4-dimethylaminopyridine, stir magnetically at room temperature for 24 h, centrifuge the reaction solution, discard the upper layer solution, and collect the spiro-pyran-modified covalent organic frameworks; finally, continuously vortex wash the spiro-pyran-modified covalent organic frameworks 3 times with ethanol and distilled water with a mass-volume ratio of 1:50 (w / v) in sequence, centrifuge, discard the upper layer solvent, collect the washed spiro-pyran-modified covalent organic frameworks, and place them in a vacuum drying oven at 40 °C for 8 h to obtain dry and clean spiro-pyran-modified covalent organic frameworks.

[0050] S3: Prepare a composite nanofiber membrane;

[0051] Using N-N dimethylformamide as the solvent, a mixed solution with a polystyrene mass concentration of 25% (w / v) and a sodium dodecylbenzenesulfonate mass concentration of 1% (w / v) was prepared, and a homogeneous solution was obtained by continuously magnetic stirring at room temperature for 5 h; Subsequently, using N-N dimethylformamide as the dispersion solvent, a dispersion solution of spiropyran-modified covalent organic framework with a mass concentration of 0.1% (w / v) was prepared. After ultrasonic treatment for 30 min, the dispersion solution was added to the above homogeneous solution, and magnetic stirring was carried out overnight to obtain an electrospinning precursor solution; Finally, the spinning solution was placed in a spraying container, the high-voltage power supply voltage of the high-voltage electrostatic field was set to 18 kV, the distance from the nozzle to the aluminum foil receiving screen was adjusted to 15 cm, the solution propulsion speed was 1 mL / h, and after collecting for 3 h, a composite nanofiber membrane was obtained.

[0052] S4: Prepare a dynamic adsorption-solid phase sensor;

[0053] The composite nanofiber membrane was filled in an empty column tube to prepare a dynamic adsorption-solid phase sensor.

[0054] S5: The milk sample was pretreated to obtain a sample extract;

[0055] Take 5 mL of milk sample, add 5 mL of 5% trichloroacetic acid (V / V) solution, vortex for 0.5 min, and after centrifugation, take the supernatant.

[0056] S6: Pass the sample extract obtained in S5 through the dynamic adsorption-solid phase sensor under the action of gravity;

[0057] The sample extract was passed through the composite nanofiber membrane in the dynamic adsorption-solid phase sensor under the action of gravity.

[0058] S7: Take out the composite nanofiber membrane in the dynamic adsorption-solid phase sensor and dry it;

[0059] Take out the composite nanofiber membrane and use a hair dryer to dry it quickly.

[0060] S8: Use a fluorescence microscope-fiber optic spectrometer for quantitative analysis of fluorescence ratio sensing;

[0061] Analyze the fluorescence spectrum of the composite nanofiber membrane by a fluorescence microscope-fiber optic spectrometer, and perform quantitative ratio fluorescence sensing detection of tetracycline according to the change in the fluorescence intensity ratio of spiropyran-modified covalent organic framework and tetracycline.

[0062] S9: Qualitative visual colorimetric detection of tetracycline;

[0063] Under the irradiation of a 365 nm ultraviolet lamp, observe the color change of the composite nanofiber membrane before and after adsorbing tetracycline to perform qualitative visual colorimetric detection of tetracycline.

[0064] S10: Semi - quantitative colorimetric detection of tetracycline;

[0065] After photographing the composite nanofiber membrane in S3 using a smartphone, the red, green, and blue (RGB) chromaticity values of the image are obtained using color analysis software, and the semi - quantitative colorimetric detection of tetracycline is carried out using the green / red chromaticity ratio (G / R) of the image.

[0066] The fluorescence sensing detection limit of tetracycline by the solid - phase sensing method based on the polystyrene composite nanofiber membrane is 2.14 nM, and the lowest detection concentration of colorimetric sensing is 20 nM.

[0067] Example 2:

[0068] This example is applied to the sensing detection of tetracycline residues in shrimp samples. The specific steps are as follows:

[0069] S1: Synthesize covalent organic frameworks by solvothermal method:

[0070] First, using ethyl acetate as the solvent, prepare a solution of trimesoyl chloride with a mass concentration of 3% (w / v); using ethyl acetate as the solvent, prepare a solution of p - phenylenediamine with a mass concentration of 2% (w / v). Subsequently, under the conditions of an ice - water bath (0 °C) and magnetic stirring, the p - phenylenediamine solution is slowly added dropwise to the trimesoyl chloride solution within 1 h. After magnetic stirring at room temperature for 18 h, the reaction solution is dropped into distilled water with a volume ratio of 1:20 (v / v) within 8 min, sonicated for 40 min, and then centrifuged. The upper layer solution is discarded, and the covalent organic frameworks are collected. Finally, the covalent organic frameworks are successively washed 4 times by continuous vortexing with ethanol and distilled water with a mass - to - volume ratio of 1:80 (w / v), centrifuged, the upper layer solvent is discarded, and the washed covalent organic frameworks are collected and placed in a vacuum drying oven at 50 °C for 10 h to obtain dry and clean covalent organic frameworks.

[0071] S2: Prepare spiropyran - modified covalent organic frameworks;

[0072] Using dichloromethane as the dispersion solvent, prepare a dispersion solution of covalent organic frameworks with a mass concentration of 2% (w / v). Successively add 3% (w / v) spiropyran, 6% (w / v) 1 - ethyl - (3 - dimethylaminopropyl) carbodiimide hydrochloride, and 2% (w / v) 4 - dimethylaminopyridine, and stir magnetically at room temperature for 30 h. The reaction solution is centrifuged, the upper layer solution is discarded, and the spiropyran - modified covalent organic frameworks are collected. Finally, the spiropyran - modified covalent organic frameworks are successively washed 4 times by continuous vortexing with ethanol and distilled water with a mass - to - volume ratio of 1:80 (w / v), centrifuged, the upper layer solvent is discarded, and the washed spiropyran - modified covalent organic frameworks are collected and placed in a vacuum drying oven at 50 °C for 10 h to obtain dry and clean spiropyran - modified covalent organic frameworks.

[0073] S3: Prepare a composite nanofiber membrane;

[0074] Using N,N-dimethylformamide as a solvent, prepare a mixed solution with a polystyrene mass concentration of 30% (w / v) and a sodium dodecylbenzenesulfonate mass concentration of 2% (w / v), and continuously magnetically stir at room temperature for 6 h to obtain a homogeneous solution; Subsequently, using N,N-dimethylformamide as a dispersion solvent, prepare a dispersion solution of spiropyran-modified covalent organic framework with a mass concentration of 0.15% (w / v). After ultrasonic treatment for 40 min, add the dispersion solution to the above homogeneous solution and magnetically stir overnight to obtain an electrospinning precursor solution; Finally, place the spinning solution in a spraying container, set the high-voltage power supply voltage of the high-voltage electrostatic field to 20 kV, adjust the distance from the nozzle to the aluminum foil receiving screen to 20 cm, and the solution propulsion speed to 2 mL / h. After collecting for 2 h, a composite nanofiber membrane is obtained.

[0075] S4: Prepare a dynamic adsorption-solid phase sensor:

[0076] Fill the composite nanofiber membrane in an empty column tube to prepare a dynamic adsorption-solid phase sensor.

[0077] S5: Obtain a sample extract by preliminary treatment of the shrimp meat sample;

[0078] Take 2 g of the shrimp meat sample, add 10 mL of 5% trichloroacetic acid (V / V) solution, homogenize, ultrasonically extract for 10 min, centrifuge, and take out the supernatant.

[0079] S6: Pass the sample extract obtained in S5 through the dynamic adsorption-solid phase sensor under the action of gravity;

[0080] Pass the sample extract through the composite nanofiber membrane in the dynamic adsorption-solid phase sensor under the action of gravity.

[0081] S7: Take out the composite nanofiber membrane in the dynamic adsorption-solid phase sensor and dry it;

[0082] Take out the composite nanofiber membrane and use a hair dryer to quickly dry it.

[0083] S8: Perform quantitative analysis of fluorescence ratio sensing using a fluorescence microscope-fiber optic spectrometer;

[0084] Analyze the fluorescence spectrum of the composite nanofiber membrane by a fluorescence microscope-fiber optic spectrometer, and perform quantitative ratio fluorescence sensing detection of tetracycline according to the change in the fluorescence intensity ratio of the spiropyran-modified covalent organic framework and tetracycline.

[0085] S9: Qualitative visual colorimetric detection of tetracycline;

[0086] Under the irradiation of a 365 nm ultraviolet lamp, the color change of the composite nanofiber membrane before and after adsorbing tetracycline was observed to conduct qualitative visual colorimetric detection of tetracycline.

[0087] S10: Semi - quantitative colorimetric detection of tetracycline;

[0088] After photographing the composite nanofiber membrane in S3 using a smartphone, the red, green, and blue (RGB) chromaticity values of the image were obtained using color analysis software, and the semi - quantitative colorimetric detection of tetracycline was carried out using the green / red chromaticity ratio (G / R) of the image.

[0089] The fluorescence sensing detection limit of tetracycline by the solid - phase sensing method based on the polystyrene composite nanofiber membrane was 4.76 μg / kg, and the lowest detection concentration of colorimetric sensing was 44.45 μg / kg.

[0090] Example 3:

[0091] The example was applied to the sensing detection of tetracycline residues in fish samples. The specific steps were as follows:

[0092] S1: Synthesize covalent organic frameworks by the solvothermal method:

[0093] First, using ethyl acetate as the solvent, prepare a 5% (w / v) solution of trimesoyl chloride; using ethyl acetate as the solvent, prepare a 3% (w / v) solution of p - phenylenediamine. Subsequently, under the conditions of an ice - water bath (0 °C) and magnetic stirring, the p - phenylenediamine solution was slowly added dropwise to the trimesoyl chloride solution within 2 h. After magnetic stirring at room temperature for 24 h, the reaction solution was dropped into distilled water with a volume ratio of 1:25 (v / v) within 10 min, sonicated for 60 min, and then centrifuged. The upper layer solution was discarded, and the covalent organic frameworks were collected. Finally, the covalent organic frameworks were successively washed 5 times by continuous vortexing with ethanol and distilled water with a mass - volume ratio of 1:100 (w / v), centrifuged, the upper layer solvent was discarded, and the washed covalent organic frameworks were collected and placed in a vacuum drying oven at 60 °C for 12 h to obtain dry and clean covalent organic frameworks.

[0094] S2: Prepare spiropyran - modified covalent organic frameworks;

[0095] Using dichloromethane as the dispersion solvent, prepare a covalent organic framework dispersion solution with a mass concentration of 3% (w / v); sequentially add 5% (w / v) spiropyran, 10% (w / v) 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, and 3% (w / v) 4-dimethylaminopyridine, stir magnetically at room temperature for 36 h, centrifuge the reaction solution, discard the upper layer solution, and collect the spiropyran-modified covalent organic framework; finally, continuously vortex wash the spiropyran-modified covalent organic framework 5 times with ethanol and distilled water with a mass-volume ratio of 1:100 (w / v), centrifuge, discard the upper layer solvent, collect the washed spiropyran-modified covalent organic framework, and place it in a vacuum drying oven at 60 °C for drying for 12 h to obtain a dry and clean spiropyran-modified covalent organic framework.

[0096] S3: Prepare a composite nanofiber membrane;

[0097] Using N,N-dimethylformamide as the solvent, prepare a mixed solution with a mass concentration of 35% (w / v) of polystyrene and a mass concentration of 3% (w / v) of sodium dodecylbenzenesulfonate, and continuously stir magnetically at room temperature for 8 h to obtain a homogeneous solution; subsequently, using N,N-dimethylformamide as the dispersion solvent, prepare a spiropyran-modified covalent organic framework dispersion solution with a mass concentration of 0.25% (w / v), after ultrasonic treatment for 60 min, add the dispersion solution to the above homogeneous solution, stir magnetically overnight to obtain an electrospinning precursor solution; finally, place the spinning solution in a spraying container, set the high-voltage power supply voltage of the high-voltage electrostatic field to 16 kV, adjust the distance from the nozzle to the aluminum foil receiving screen to 10 cm, and the solution propulsion speed to 3 mL / h. After collecting for 3 h, a composite nanofiber membrane is obtained.

[0098] S4: Prepare a dynamic adsorption-solid phase sensor:

[0099] Fill the composite nanofiber membrane in an empty column tube to prepare a dynamic adsorption-solid phase sensor.

[0100] S5: The shrimp meat sample is preliminarily processed to obtain a sample extract;

[0101] Take 2 g of the fish meat sample, add 10 mL of 5% trichloroacetic acid (V / V) solution, homogenize, ultrasonically extract for 10 min, centrifuge, and take the supernatant.

[0102] S6: Pass the sample extract obtained in S5 through the dynamic adsorption-solid phase sensor under the action of gravity;

[0103] Pass the sample extract through the composite nanofiber membrane in the dynamic adsorption-solid phase sensor under the action of gravity.

[0104] S7: Take out the composite nanofiber membrane in the dynamic adsorption-solid phase sensor and dry it;

[0105] Take out the composite nanofiber membrane and use a hair dryer to dry it quickly.

[0106] S8: Conduct quantitative analysis of fluorescence ratio sensing using a fluorescence microscope - optical fiber spectrometer;

[0107] Analyze the fluorescence spectrum of the composite nanofiber membrane by a fluorescence microscope - optical fiber spectrometer, and conduct quantitative ratio fluorescence sensing detection of tetracycline according to the change in the fluorescence intensity ratio of spiropyran - modified covalent organic framework and tetracycline.

[0108] S9: Qualitative visual colorimetric detection of tetracycline;

[0109] Under the irradiation of a 365 - nm ultraviolet lamp, observe the color change of the composite nanofiber membrane before and after adsorbing tetracycline to conduct qualitative visual colorimetric detection of tetracycline.

[0110] S10: Semi - quantitative colorimetric detection of tetracycline;

[0111] After taking a photo of the composite nanofiber membrane in S5 using a smartphone, obtain the red, green, and blue (RGB) chromaticity values of the image using color analysis software, and conduct semi - quantitative colorimetric detection of tetracycline using the green / red chromaticity ratio (G / R) of the image.

[0112] The fluorescence sensing detection limit of tetracycline by the solid - phase sensing method based on the polystyrene composite nanofiber membrane is 4.76 μg / kg, and the lowest detection concentration of colorimetric sensing is 44.45 μg / kg.

[0113] In the description of this specification, the descriptions referring to terms such as "one embodiment", "example", "specific example", etc. mean that the specific features, structures, materials, or characteristics described in connection with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representations of the above - mentioned terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials, or characteristics described can be combined in a suitable manner in any one or more embodiments or examples.

[0114] The above shows and describes the basic principles, main features, and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited by the above - mentioned embodiments. The above - mentioned embodiments and the descriptions in the specification only illustrate the principles of the present invention. Without departing from the spirit and scope of the present invention, the present invention will have various changes and improvements, and these changes and improvements all fall within the scope of the present invention claimed.

Claims

1. A method for detecting tetracycline residues in animal-derived foods, characterized in that, It includes the following steps: S1: Synthesize covalent organic frameworks by solvothermal method; S2: Prepare spiropyran-modified covalent organic frameworks; S3: Prepare composite nanofiber membranes; S4: Fill the composite nanofiber membranes into empty column tubes to prepare dynamic adsorption-solid phase sensors; S5: Obtain sample extracts from animal-derived foods through preliminary treatment; S6: Pass the sample extracts obtained in S5 through the dynamic adsorption-solid phase sensor under the action of gravity; S7: Take out the composite nanofiber membranes in the dynamic adsorption-solid phase sensor and dry them; S8: Perform quantitative analysis of fluorescence ratio sensing using a fluorescence microscope-fiber optic spectrometer; S9: Qualitative visual colorimetric detection of tetracycline; S10: Semi-quantitative colorimetric detection of tetracycline; The preparation steps of the composite nanofiber membranes include: Based on the spiropyran-modified covalent organic frameworks, obtain an electrospinning precursor solution, place it in a spraying container, and obtain the composite nanofiber membranes.

2. The detection method for tetracycline residues in animal-derived foods according to claim 1, characterized in that, The steps for preparing the covalent organic frameworks include: 1) Using ethyl acetate as a solvent, prepare a trimesoyl chloride solution with a mass concentration of 2-5%; 2) Using ethyl acetate as a solvent, prepare a p-phenylenediamine solution with a mass concentration of 1-3%; 3) Under the conditions of an ice-water bath and magnetic stirring, slowly drop the p-phenylenediamine solution into the trimesoyl chloride solution within 1-2 h. After magnetic stirring at room temperature for 12-24 h, drop the reaction solution into distilled water with a volume ratio of 1:15-1:25 within 5-10 min, ultrasonicate for 30-60 min, then centrifuge, discard the upper layer solution, and collect the covalent organic frameworks; 4) Continuously vortex wash the covalent organic frameworks 3-5 times successively with ethanol and distilled water with a mass-to-volume ratio of 1:50-1:100, then centrifuge, discard the upper layer solvent, collect the washed covalent organic frameworks, and place them in a vacuum drying oven at 40-60 °C for drying for 8-12 h to obtain dry and clean covalent organic frameworks.

3. The detection method of tetracycline residues in animal-derived foods according to claim 1, characterized in that, The preparation steps of the spiropyran-modified covalent organic frameworks include: 1) Using dichloromethane as a dispersion solvent, prepare a covalent organic framework dispersion solution with a mass concentration of 1-3%; 2) Add 2-5% w / v spiropyran, 4-10% w / v 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, and 1-3% w / v 4-dimethylaminopyridine in sequence. After magnetic stirring at room temperature for 24-36 h, centrifuge the reaction solution, discard the upper layer solution, and collect the spiropyran-modified covalent organic frameworks; 3) Continuously vortex wash the spiropyran-modified covalent organic frameworks 3-5 times successively with ethanol and distilled water with a mass-to-volume ratio of 1:50-1:100, centrifuge, discard the upper layer solvent, collect the washed spiropyran-modified covalent organic frameworks, and place them in a vacuum drying oven at 40-60 °C for drying for 8-12 h to obtain dry and clean spiropyran-modified covalent organic frameworks.

4. The detection method for tetracycline residues in animal-derived foods according to claim 1, characterized in that, The preparation steps of the composite nanofiber membranes include: 1) Using N,N-dimethylformamide as a solvent, prepare a mixed solution with a mass concentration of polystyrene of 25-35% and a mass concentration of sodium dodecylbenzenesulfonate of 0.6-3%, and continuously magnetically stir at room temperature for 5-8 h to obtain a homogeneous solution; 2) Using N-N dimethylformamide as the dispersion solvent, prepare a dispersion solution of spiropyran-modified covalent organic framework with a mass concentration of 0.05 - 0.25%. After ultrasonic treatment for 30 - 60 min, add the dispersion solution to the above homogeneous solution and stir magnetically overnight to obtain an electrospinning precursor solution; 3) Place the spinning solution in a spraying container. Set the voltage of the high-voltage power supply of the high-voltage electrostatic field to 16 - 20 kV, adjust the distance from the nozzle to the aluminum foil receiving screen to 10 - 20 cm, and the solution feeding speed to 1 - 3 mL / h. After collecting for 1 - 3 h, obtain a composite nanofiber membrane.

5. The detection method of tetracycline residues in animal-derived foods according to claim 1, wherein The animal-derived foods in S5 include eggs, milk, fish, shrimp, and internal organs.

6. The detection method for tetracycline residues in animal-derived foods according to claim 5, characterized in that, The steps for preliminarily treating milk into a sample extract are as follows: Add 5% V / V trichloroacetic acid solution to the milk sample, vortex for 0.5 min, and after centrifugation, take the supernatant.

7. The detection method for tetracycline residues in animal-derived foods according to claim 5, characterized in that, The steps for preliminarily treating fish or shrimp into a sample extract are as follows: Add 5% V / V trichloroacetic acid solution to the fish or shrimp, homogenize, ultrasonically extract for 10 min, centrifuge and separate, and take out the supernatant.

8. The detection method for tetracycline residues in animal-derived foods according to claim 1, wherein In S8, analyze the fluorescence spectrum of the composite nanofiber membrane by a fluorescence microscope - optical fiber spectrometer, and perform quantitative ratio fluorescence sensing detection of tetracycline according to the change in the fluorescence intensity ratio of spiropyran-modified covalent organic framework and tetracycline.

9. The detection method for tetracycline residues in animal-derived foods according to claim 1, characterized in that The qualitative visual colorimetric detection of the tetracycline is to observe the color change of the composite nanofiber membrane before and after adsorbing tetracycline under the irradiation of a 365 nm ultraviolet lamp.

10. The detection method for tetracycline residues in animal-derived foods according to claim 1, characterized in that, In S10, after taking a photo of the composite nanofiber membrane in S5 using a smartphone, then use color analysis software to obtain the red, green, and blue color values of the image, and use the green / red color ratio of the image for semi-quantitative colorimetric detection of tetracycline.