A method for detecting tetracycline in meat products based on copper activation-upconversion-bismuth molybdate sensing system

Abstract

The present invention belongs to the field of antibiotic detection, and in particular to a method for detecting tetracycline in meat products based on a copper-activated upconversion bismuth molybdate sensing system. The steps are: preparing upconversion nanomaterials and rhodamine derivatives, dissolving the two in methanol to obtain two solutions and mixing them in proportion to construct a sensing system. At the same time, the present invention synthesizes a bismuth molybdate-bismuth sulfide complex by a solvent thermal method, effectively improving the decomposition efficiency of tetracycline and copper ion-tetracycline complex in the solution, and shortening the reaction time. By decomposing the copper ion-tetracycline complex and tetracycline, the remaining tetracycline in the complex and the solution can be avoided from continuing to react with the copper ion, further avoiding the interference of the subsequent reaction on the upconversion fluorescence signal, reducing the detection limit of the method, and improving the detection accuracy. In summary, the present invention provides a novel tetracycline detection method, which does not require expensive instruments, reduces the detection cost, can be used for the detection of actual samples, and has broad application prospects.

Description

Technical Field

[0001] The present invention belongs to the field of antibiotic detection, and in particular relates to a method for detecting tetracycline in meat products based on a copper activation-upconversion-bismuth molybdate sensing system. Background Art

[0002] In modern agriculture, the broad-spectrum antibiotic tetracycline is widely used in animal treatments because it effectively inhibits the growth of pathogens and treats animal diseases. However, due to the overuse of tetracycline by some livestock farmers, it has resulted in residues in many animal-derived foods. Tetracycline can bioaccumulate in the human body through the food chain, causing tooth yellowing, liver toxicity, gastrointestinal disturbances, and allergic reactions. It can also promote the development of bacterial resistance, thereby compromising drug efficacy.

[0003] Given its potential hazards, China's national standard GB 31650-2019 stipulates that the tetracycline content in pork, beef, and fish must not exceed 200 μg / kg. Traditional tetracycline detection methods include high-performance liquid chromatography, capillary electrophoresis, enzyme-linked immunosorbent assay, and liquid chromatography-mass spectrometry. Although these techniques have high accuracy and sensitivity for tetracycline, they still have limitations, such as being expensive, time-consuming, and requiring advanced laboratory equipment and well-trained staff. Improvements are still needed to reduce testing costs and simplify the testing process. Summary of the Invention

[0004] In view of the shortcomings of the existing technology, the purpose of the present invention is to provide a method for detecting tetracycline in meat products based on a copper activation-upconversion-bismuth molybdate sensing system, which overcomes the problems of complex operation and high detection cost in the existing detection technology.

[0005] To achieve the above object, the present invention provides the following solutions:

[0006] The present invention first provides a method for detecting tetracycline in meat products based on a copper activation-upconversion-bismuth molybdate sensing system, comprising the following steps:

[0007] Step 1: Preparation of upconversion fluorescent nanomaterials;

[0008] Yttrium chloride hexahydrate, ytterbium chloride hexahydrate, and erbium chloride hexahydrate were dissolved in methanol, oleic acid and 1-octadecene were added, nitrogen was introduced, and a first heating and stirring reaction was carried out under a nitrogen atmosphere. After the reaction was completed, the mixture was cooled to a certain temperature to obtain a solution, which was recorded as mixed solution A;

[0009] Ammonium fluoride and sodium hydroxide are dissolved in methanol and then mixed with mixed solution A. A second heating and stirring reaction is carried out. Nitrogen is introduced and a third heating and stirring reaction is carried out under a nitrogen atmosphere. After the reaction, the mixture is cooled to room temperature. The resulting solution is recorded as mixed solution B. Mixed solution B is washed with a mixture of ethanol and cyclohexane, and finally centrifuged and dried to obtain an upconversion fluorescent nanomaterial.

[0010] Step 2: Rhodamine B is dissolved in ethanol, and then hydrazine hydrate is added dropwise at room temperature. The resulting solution is stirred and recorded as mixture C. The solution obtained after the first heating and reflux in an air bath (the solution changes from dark purple to light orange) is recorded as mixture D; the mixture D is subjected to rotary evaporation to remove the solvent to obtain a solid product, hydrochloric acid is added to the solid product to obtain a red solution, the pH of the red solution is adjusted, and then the solution is centrifuged and dried to obtain rhodamine B hydrazine;

[0011] Step 3: dissolving rhodamine B hydrazine in ethanol, adding dihydroxybenzaldehyde and acetic acid, and heating the obtained mixed solution under a nitrogen atmosphere for a second time to reflux to obtain a reflux solution; then cooling and concentrating the reflux solution, and allowing it to stand at room temperature for a period of time to obtain pink crystals, which are then centrifuged and dried to obtain a rhodamine derivative;

[0012] Step 4:

[0013] (1) Weighing bismuth nitrate pentahydrate and adding it to dilute nitric acid, stirring it for the first time at room temperature; after stirring, adding ammonium molybdate tetrahydrate and stirring it for the second time, then adjusting the pH of the mixed solution with ammonia water, and then transferring the mixed solution to a hydrothermal reactor to carry out a hydrothermal reaction. After the reaction, naturally cooling to a certain temperature and then centrifuging to obtain a precipitate, the obtained precipitate is washed with ethanol and deionized water in sequence, and dried to obtain bismuth molybdate;

[0014] (2) Weighing bismuth nitrate pentahydrate and adding it to deionized water, stirring it for the first time at room temperature; then adding sodium thiosulfate pentahydrate and stirring it for the second time, transferring the obtained mixed solution to a hydrothermal reactor, performing a hydrothermal reaction, cooling it naturally to room temperature, and obtaining a precipitate by centrifugation. The obtained precipitate is washed with ethanol and deionized water in sequence, and dried to obtain bismuth sulfide;

[0015] Step 5: taking the bismuth molybdate and bismuth sulfide obtained in step 4 and adding them to deionized water in a certain proportion, transferring the obtained mixed solution after ultrasonic treatment to a hydrothermal reactor for hydrothermal reaction; naturally cooling to room temperature and centrifuging to obtain a precipitate, and washing the obtained precipitate with ethanol and deionized water in sequence, and then drying to obtain a bismuth molybdate-bismuth sulfide complex;

[0016] Step 6: Dissolve the upconversion fluorescent nanomaterial prepared in step 1 and the rhodamine derivative prepared in step 3 in methanol respectively to obtain an upconversion fluorescent nanomethanol solution and a rhodamine derivative methanol solution, and mix the two in a certain proportion to obtain a mixed solution, which is the copper activation-upconversion-bismuth molybdate sensing system;

[0017] Step 7: Prepare tetracycline standard solutions of different concentrations;

[0018] First, the bismuth molybdate-bismuth sulfide complex obtained in step 5 is added to water and stirred evenly to obtain a bismuth molybdate-bismuth sulfide composite solution; a tetracycline standard solution and a copper sulfate solution are mixed in a certain proportion, shaken for a period of time, and then the bismuth molybdate-bismuth sulfide composite solution is added and irradiated with a xenon lamp for a period of time (to decompose the copper-tetracycline complex and tetracycline), and the precipitate is removed by centrifugation to obtain a test solution;

[0019] The copper activation-upconversion-bismuth molybdate sensing system obtained in step 6 is mixed with the test solution to obtain a mixed solution and the fluorescence intensity signal characteristic value of the mixed solution is detected. According to the fluorescence intensity signal characteristic values ​​corresponding to the test solutions containing different concentrations of tetracycline, a standard curve related to tetracycline concentration is drawn with the logarithm of the tetracycline standard solution concentration as the abscissa and the fluorescence intensity signal characteristic value as the ordinate;

[0020] Step 8: Detection of tetracycline in meat products;

[0021] (1) Preparation of sample solution;

[0022] (2) The sample solution and the copper sulfate solution are mixed in a certain proportion, shaken for a period of time, and a bismuth molybdate-bismuth sulfide composite solution is added. The mixture is irradiated with a xenon lamp for a period of time, and the precipitate is removed by centrifugation to obtain a test solution; the test solution is mixed with the copper activation-upconversion-bismuth molybdate sensing system in a certain proportion to obtain a mixed solution, and the fluorescence intensity signal characteristic value of the mixed solution is measured. According to the tetracycline detection standard curve obtained in step 5, the content of tetracycline in the meat product sample is calculated.

[0023] Furthermore, in step 1, the usage ratio of methanol, yttrium chloride hexahydrate, ytterbium chloride hexahydrate, erbium chloride hexahydrate, oleic acid and 1-octadecene is 6-10 mL: 236.6 mg: 77.5 mg: 7.6 mg: 6 mL: 15 mL;

[0024] The amount ratio of oleic acid to methanol, ammonium fluoride and sodium hydroxide in the mixed solution A is 6 mL:10 mL:0.1482 g:0.1 g; the temperature of the first heating and stirring reaction is 160-170° C., the stirring time is 25-40 min, and the stirring rate is 300-500 rpm.

[0025] Furthermore, in step one, the temperature of the second heating and stirring is 50-70°C, and the time is 70-100 min; the temperature of the third heating and stirring reaction is 290-300°C, and the stirring reaction time is 60-90 min; the stirring rates of the second heating and stirring and the third heating and stirring are 300-500 rpm; the volume ratio of the mixed solution B to the ethanol and cyclohexane mixed solution is 1:1, and the volume ratio of cyclohexane to ethanol is 1:1; the centrifugal rate is 8000 rpm, and the time is 6-8 min; the drying temperature is 40-60°C, and the time is 8-20 h.

[0026] Furthermore, in step 2, the ratio of rhodamine B: ethanol: hydrazine hydrate: hydrochloric acid is 1.2 g: 30 mL: 3 mL: 50 mL; the first heating reflux time is 100 to 150 min, and the temperature is 70 to 80° C.; the concentration of the hydrochloric acid is 1 mol / L; the solution used to adjust the pH of the red solution is sodium hydroxide with a concentration of 1 mol / L, and the pH is adjusted to 9 to 10; the centrifugal speed is 8000 rpm, and the time is 6 to 10 min; the drying temperature is 45 to 60° C., and the time is 12 to 15 h.

[0027] Furthermore, in step three, the amount ratio of rhodamine B hydrazine, ethanol, dimethylbenzaldehyde, and acetic acid is 0.46 g: 20 mL: 0.16 g: 0.15-0.2 mL; the reaction temperature of the second heating reflux is 70-85 ° C, and the time is 3-3.5 h; the volume of the concentrated solution is 30-50% of the volume of the reflux solution, and the standing time is 12-24 h; the centrifugal rate is 8000 rpm, and the time is 6-10 min; the drying temperature is 50-60 ° C, and the time is 12-24 h.

[0028] Furthermore, in step 4 (1), the ratio of bismuth nitrate pentahydrate, dilute nitric acid, and ammonium molybdate tetrahydrate is 2 mmol:25 mL:2 mmol, wherein the concentration of dilute nitric acid is 1 mol / L; the first stirring and the second stirring time are both 30 to 40 minutes; the pH of the solution is adjusted to 7 with ammonia water; the hydrothermal reaction temperature is 150 to 160° C., and the time is 6 hours; and the cooling to a certain temperature is 20 to 35° C.

[0029] Furthermore, in step 4 (2), the ratio of bismuth nitrate pentahydrate, deionized water, and sodium thiosulfate is 0.5 mmol:40 mL; 0.75 mmol; the first and second stirring times are both 40 to 60 min; the temperature of the hydrothermal reaction is 160 to 170° C., and the time is 24 h.

[0030] Furthermore, in step five, the ratio of bismuth molybdate, bismuth sulfide, and deionized water is 1 g:0.3 g:30 ml; the high-temperature reaction temperature is 100-120° C., and the reaction time is 10 h.

[0031] Furthermore, in step six, the concentrations of the up-conversion fluorescent nano-methanol solution and the rhodamine derivative methanol solution are 0.25 mg / mL and 0.1 mg / mL, respectively; and the volume ratio of the two mixed is 2:1.

[0032] Furthermore, in step seven, the volume ratio of the tetracycline standard solution, the copper sulfate solution, and the bismuth molybdate-bismuth sulfide composite solution is 1:1:0.5; the concentration of the bismuth molybdate-bismuth sulfide composite solution is 1 g / L, the concentration of the copper sulfate solution is 32 mg / L, and the concentration range of the tetracycline standard solution is 50 to 50,000 μg / L; the shaking time is 1.5 to 2 hours; the xenon lamp irradiation time is 1 hour; the volume ratio of the copper activation-upconversion-bismuth molybdate sensing system to the measured liquid is 1:1; and the step of determining the characteristic value of the fluorescence intensity signal of the mixed solution is: exciting the mixed solution with excitation light of 980 nm wavelength, and recording the fluorescence intensity value at 540 nm of the fluorescence spectrum.

[0033] Furthermore, in step eight (1), the specific steps for preparing the sample solution are as follows: weigh a certain amount of sample, place it in a centrifuge tube, add perchloric acid solution A to homogenize for a period of time, shake evenly, and centrifuge to obtain the supernatant; add perchloric acid solution B to the residue in the centrifuge tube, repeat the operation once, and combine the supernatants; add n-hexane to the supernatant, shake and then centrifuge to remove the n-hexane, then add n-hexane again, shake and then centrifuge to remove the n-hexane, and the obtained solution is recorded as treatment solution A; wash the treatment solution A with deionized water using an ODS-C18 column to remove impurities, elute with methanol A, collect the eluate, blow dry with nitrogen, and then make up to volume with methanol B and filter with a filter membrane to obtain the sample solution;

[0034] The ratio of the sample, perchloric acid solution A, and perchloric acid solution B is 1 g: 2 mL: 1 mL; the mass fractions of perchloric acid solution A and perchloric acid solution B are both 0.5%; the homogenization time is 30 s, and the oscillation time on the oscillator is 3 min; the centrifugation speed is 4000 rpm for 10 min; the ratio of the sample to n-hexane A, n-hexane B, deionized water, methanol A, and methanol B is 5 g: 1 mL: 1 mL: 10 mL: 5 mL: 1 mL; the pore size of the filter membrane is 0.45 μm;

[0035] Furthermore, in step eight (2), the volume ratio of the sample solution, copper sulfate solution, and bismuth molybdate-bismuth sulfide composite solution is 1:1:0.5; the concentration of the bismuth molybdate-bismuth sulfide composite is 1 g / L, and the concentration of the copper sulfate solution is 32 mg / L; the shaking time is 1.5 to 2 hours; the xenon lamp irradiation time is 1 hour; and the volume ratio of the copper activation-upconversion-bismuth molybdate sensing system to the liquid to be tested is 1:1.

[0036] Note: Perchloric acid solution A and perchloric acid solution B described in the present invention are both perchloric acid solutions; methanol A and methanol B are both methanol; different capital letters are only used to distinguish the names.

[0037] Compared with the existing technology, the present invention has the following beneficial effects:

[0038] (1) The present invention discloses a copper activation-upconversion-bismuth molybdate sensing system for detecting tetracycline in meat products. Since tetracycline contains electron donor groups, it can react with Cu 2+ The present invention combines to form a stable tetracycline-Cu complex. The invention utilizes the complexation reaction between tetracycline and copper ions. A copper ion solution is added to the liquid to be tested. The content of residual copper ions in the solution is detected, thereby reflecting the content of tetracycline in the substance to be tested, amplifying the detection signal, improving the detection sensitivity, and reducing the detection limit.

[0039] (2) The rhodamine derivatives used in the present invention can specifically react with copper ions to produce changes in ultraviolet absorption intensity. The reaction formula for the reaction of rhodamine derivatives with copper ions is:

[0040]

[0041] Rhodamine derivatives and Cu 2+ After binding, the lactam ring opens, the solution turns pink, and its UV absorbance increases. The compound produced by this reaction has a large absorption peak in the 450-600nm range, overlapping with the fluorescence peak of the upconversion nanoparticles at 540nm. Therefore, this compound can quench the fluorescence of the upconversion nanoparticles through the fluorescence inner filter effect. The degree of quenching varies with the residual copper ion concentration in the solution. By measuring the intensity of the solution's fluorescence signal, the tetracycline content in the analyte can be accurately inferred. Higher tetracycline concentrations in the test solution promote the complexation of copper ions with tetracycline. Lower residual copper ion concentrations in the solution reduce the amount of compound produced after reaction with the rhodamine derivative, resulting in a lower degree of quenching of the upconversion nanomaterial. The reaction is very rapid and operates under mild conditions. Within a certain concentration range, the tetracycline content in the test solution can be roughly determined by visually observing the color change. This method eliminates the need for complex procedures, shortens detection time, and improves detection efficiency.

[0042] (3) The present invention synthesizes a bismuth molybdate-bismuth sulfide complex by a solvothermal method. Precisely controlling the mass ratio of bismuth molybdate to bismuth sulfide to be 1:0.3 can effectively improve the decomposition efficiency of tetracycline and the copper ion-tetracycline complex in the solution and shorten the reaction time. By decomposing the copper ion-tetracycline complex and tetracycline, the complex and the remaining tetracycline in the solution can be prevented from continuing to react with the copper ion, further preventing the interference of subsequent reactions on the upconversion fluorescence signal, reducing the detection limit of the method, and improving the detection accuracy.

[0043] (4) The rhodamine derivative prepared by the present invention is combined with the upconversion nanomaterial to detect tetracycline in actual samples. The detection probe is prepared by precisely controlling the mixing of 0.1 mg / mL rhodamine derivative methanol solution and 0.25 mg / mL upconversion nanomethanol solution in a volume ratio of 1:2, which can improve the sensitivity and reliability of the detection. The method proposed by the present invention is simple to operate, low in cost, and can be used for the detection of actual samples. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] Figure 1 This is a transmission electron microscope image of upconversion fluorescent nanomaterials.

[0045] Figure 2 This is the fluorescence spectrum of the upconversion fluorescent nanomaterial.

[0046] Figure 3 This is the UV absorption spectrum of the methanol solution of rhodamine derivative before and after adding copper sulfate solution.

[0047] Figure 4 The fluorescence standard curve of tetracycline at different concentrations is shown in Figure 2. DETAILED DESCRIPTION

[0048] Exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0049] It should be understood that the terms described herein are intended only to describe particular embodiments and are not intended to limit the present invention. In addition, for numerical ranges herein, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Each smaller range between any intermediate value within a stated value or stated range and any other stated value or intermediate value within the stated range is also encompassed by the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded within the scope.

[0050] Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein may also be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of any conflict with any incorporated document, the contents of this specification shall prevail.

[0051] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0052] Example 1:

[0053] In order to further verify the detection effect of the detection method prepared by the present invention on the detection of tetracycline in meat products, the present invention takes the detection of tetracycline in pork as an example, and the specific operation steps are as follows:

[0054] (1) 236.6 mg of yttrium chloride hexahydrate, 77.5 mg of ytterbium chloride hexahydrate, and 7.6 mg of erbium chloride hexahydrate were dissolved in 6 mL of methanol, 6 mL of oleic acid and 15 mL of 1-octadecene were added, nitrogen was introduced, and the mixture was heated to 160° C. and stirred for 30 min at a stirring rate of 400 rpm under a nitrogen atmosphere. After the reaction was completed, the mixture was cooled to room temperature to obtain a mixed solution A; 0.1482 g of ammonium fluoride and 0.1 g of sodium hydroxide were dissolved in 10 mL of methanol, mixed with the mixed solution A, heated to 70° C. and stirred for 90 min, and then nitrogen was introduced, heated to 300° C. and maintained for 80 min under a nitrogen atmosphere, and cooled to room temperature to obtain a mixed solution B; the mixed solution B was washed with a mixed solution of ethanol and cyclohexane, collected by centrifugation, and dried to obtain an upconversion fluorescent nanomaterial ( Figure 1 , Figure 2 );

[0055] (2) 1.2 g of rhodamine B was dissolved in 30 mL of ethanol, and then 3 mL of hydrazine hydrate was added dropwise at room temperature and stirred vigorously to obtain a mixture C. The stirred mixture C was heated to reflux at 80°C in an air bath for 120 min, and the solution changed from dark purple to light orange. It was then cooled and the solvent was removed by rotary evaporation to obtain solid A. 50 mL of 1 mol / L hydrochloric acid was added to solid A, and then 1 mol / L sodium hydroxide was slowly added to adjust the pH of the solution to 9. Subsequently, it was centrifuged at 8000 rpm for 8 minutes, washed with deionized water, and dried at 50°C for 12 h to obtain rhodamine B hydrazine;

[0056] (3) 0.46 g of rhodamine B hydrazine was dissolved in 20 mL of ethanol, 0.16 g of dihydroxybenzaldehyde and 0.2 mL of acetic acid were added, and the mixture was heated under reflux at 80°C for 3 h under a nitrogen atmosphere. The resulting solution was cooled and concentrated to 8 mL, and allowed to stand at room temperature. After standing for 15 h, the rhodamine derivative was obtained. Finally, the rhodamine derivative was collected after centrifugation at 8000 rpm for 8 min and dried at 50°C for 20 h ( Figure 3 );

[0057] (4) Weigh 2 mmol of bismuth nitrate pentahydrate and add it to 25 mL of 1 mol / L dilute nitric acid, stirring for 30 minutes; add 2 mmol of ammonium molybdate tetrahydrate to the above solution and continue stirring for 30 minutes. Then adjust the pH of the solution to 7.0 with ammonia water, transfer the solution to a hydrothermal reactor, and place it in a 160°C oven to react for 6 hours. After cooling naturally to 30°C, centrifuge to obtain a precipitate, wash the precipitate with ethanol and deionized water, dry it in an oven at 60°C, and dry it in an oven to obtain bismuth molybdate.

[0058] Weigh 0.5 mmol of bismuth nitrate pentahydrate and add it to 40 mL of deionized water, stirring for 40 minutes. Then, add 0.75 mmol of sodium thiosulfate pentahydrate and stir for 40 minutes. Transfer the resulting solution to a hydrothermal reactor and react for 24 hours. After cooling naturally, centrifuge to obtain a precipitate. Wash the precipitate with ethanol and deionized water, dry it in an oven at 60°C, and dry it in an oven to obtain bismuth sulfide.

[0059] (5) 1 g of bismuth molybdate and 0.3 g of bismuth sulfide were added to 30 mL of deionized water and ultrasonicated for 20 min. The resulting solution was transferred to a hydrothermal reactor and placed in an oven at 100°C for 10 h. After natural cooling, the precipitate was obtained by centrifugation. The precipitate was washed with ethanol and deionized water and dried in an oven to obtain a bismuth molybdate-bismuth sulfide complex;

[0060] (6) dissolving the upconversion fluorescent nanomaterial and the rhodamine derivative in methanol respectively to obtain a 0.25 mg / mL upconversion fluorescent nanomethanol solution and a 0.1 mg / mL rhodamine derivative methanol solution, and mixing the two in a volume ratio of 2:1 to obtain a detection probe (i.e., a copper activation-upconversion-bismuth molybdate sensing system);

[0061] (7) Prepare tetracycline standard solutions with concentrations of 50, 100, 1000, 5000, 10000, and 50000 μg / L, respectively. Mix 500 μL of tetracycline standard solutions of different concentrations with 500 μL of 32 mg / L copper sulfate solution, shake for 1.5 h, then add 250 μL of 1 g / L bismuth molybdate-bismuth sulfide complex, irradiate with a xenon lamp for 1 h, and centrifuge to remove the precipitate to obtain the test solution. Then, take 500 μL of the test solution and mix it with 500 μL of the detection probe, detect the fluorescence intensity signal characteristic value of the mixed solution, and draw a tetracycline concentration-related standard curve (based on the fluorescence intensity signal characteristic value corresponding to the test solution containing different concentrations of tetracycline, with the logarithm of the tetracycline standard solution concentration as the horizontal axis and the fluorescence intensity signal characteristic value as the vertical axis) Figure 4 ). Tetracycline can be complexed with copper ions, converting the tetracycline concentration into a copper ion concentration signal, which is beneficial to signal amplification and facilitates quantitative detection. The synthesized bismuth molybdate-bismuth sulfide complex can decompose the copper ion-tetracycline complex and tetracycline under xenon lamp irradiation, avoiding the complex and the remaining tetracycline in the solution to continue to react with copper ions, and further avoiding the interference of subsequent reactions on the upconversion fluorescence signal. Therefore, this method has a lower detection limit and higher detection accuracy. Bismuth molybdate is composed of MoO6 perovskite layer and (Bi2O2) 2+ It has a layered structure, a unique valence band structure, and excellent thermal stability, enabling it to degrade organic pollutants under visible light. Bismuth molybdate-bismuth sulfide, synthesized using a solvothermal method, effectively improves the degradation efficiency of copper ion-tetracycline complexes and tetracycline, shortening the reaction time. Furthermore, since copper ions can bind to rhodamine derivatives, the lactam ring in the rhodamine derivatives opens, increasing UV absorbance. The resulting complex effectively quenches the fluorescence of the upconversion nanoparticles. The degree of fluorescence quenching is monitored to reflect the tetracycline concentration: the more intense the fluorescence quenching, the higher the residual copper ion concentration in the solution and the lower the concentration of the tetracycline standard solution. The rapid and sensitive reaction between copper ions and rhodamine derivatives facilitates shorter detection times. Furthermore, the reaction is highly specific, effectively eliminating interference from other ions and improving detection accuracy.

[0062] (8) Tetracycline detection;

[0063] Weigh 5g of pork sample, place it in a centrifuge tube, add 10mL of perchloric acid solution and homogenize for 30s, shake for 3min, and centrifuge to obtain the supernatant. Add 5mL of perchloric acid solution to the residue in the centrifuge tube, repeat the operation once, and combine the supernatants. Add 1mL of n-hexane, shake and centrifuge at 4000 rpm for 10min, remove the n-hexane, add 1mL of n-hexane, shake and centrifuge, remove the n-hexane, and obtain treatment liquid A. Use ODS-C18 column to add 10mL of deionized water to wash away impurities in treatment liquid A, add 5mL of methanol to elute, collect the eluate, blow dry with nitrogen, add 1mL of methanol, and filter with a 0.45μm filter membrane to obtain the sample liquid;

[0064] Take 500 μL of sample solution, add 500 μL of 32 mg / L copper sulfate solution, shake for 1.5 hours, then add 250 μL of 1 g / L bismuth molybdate-bismuth sulfide composite solution, irradiate with a xenon lamp for 1 hour, and centrifuge to remove the precipitate to obtain the test solution; take 500 μL of the test solution and add 500 μL of copper activation-upconversion-bismuth molybdate sensing system, excite the mixed solution with 980 nm wavelength excitation light, measure the fluorescence intensity signal characteristic value of the obtained solution, and calculate the tetracycline content in the meat product sample based on the tetracycline detection standard curve obtained in step 5.

[0065] Three pork samples (respectively designated as sample A, sample B, and sample C) were selected, and the tetracycline content in the pork was determined using the method and steps described in Example 1 of the present invention. The results were verified using the national standard method. The determination results are shown in Table 1. It can be seen that the method of the present invention has good accuracy in actual samples and has good application prospects.

[0066] Table 1 Results of the tetracycline content in pork samples detected by the method of the present invention and the national standard method

[0067]

[0068] Note: The above embodiments are only used to illustrate the present invention and are not intended to limit the technical solutions described in the present invention. Therefore, although this specification has described the present invention in detail with reference to the above embodiments, it should be understood by those skilled in the art that the present invention may still be modified or replaced by equivalents. All technical solutions and improvements that do not depart from the spirit and scope of the present invention should be included within the scope of the claims of the present invention.

Claims

1. A method for detecting tetracycline in meat products based on a copper activation-upconversion-bismuth molybdate sensing system, characterized in that: The following steps are involved: Step 1: Preparation of upconversion fluorescent nanomaterials; Yttrium chloride hexahydrate, ytterbium chloride hexahydrate, and erbium chloride hexahydrate were dissolved in methanol, oleic acid and 1-octadecene were added, nitrogen was introduced, and a first heating and stirring reaction was carried out under a nitrogen atmosphere. After the reaction was completed, the mixture was cooled to a certain temperature to obtain a solution, which was recorded as mixed solution A; Ammonium fluoride and sodium hydroxide are dissolved in methanol and then mixed with mixed solution A. A second heating and stirring reaction is carried out. Nitrogen is introduced and a third heating and stirring reaction is carried out under a nitrogen atmosphere. After the reaction, the mixture is cooled to room temperature. The resulting solution is recorded as mixed solution B. Mixed solution B is washed with a mixture of ethanol and cyclohexane, and finally centrifuged and dried to obtain an upconversion fluorescent nanomaterial. Step 2: Rhodamine B is dissolved in ethanol, and then hydrazine hydrate is added dropwise at room temperature. The resulting solution is stirred and recorded as mixture C. The solution obtained after the first heating and reflux in an air bath is recorded as mixture D; the solvent is removed from mixture D by rotary evaporation to obtain a solid product, hydrochloric acid is added to the solid product to obtain a red solution, the pH of the red solution is adjusted, and then the solution is centrifuged and dried to obtain rhodamine B hydrazine; Step 3: dissolving rhodamine B hydrazine in ethanol, adding dihydroxybenzaldehyde and acetic acid, and heating the obtained mixed solution under a nitrogen atmosphere for a second time to reflux to obtain a reflux solution; then cooling and concentrating the reflux solution, and allowing it to stand at room temperature for a period of time to obtain pink crystals, which are then centrifuged and dried to obtain a rhodamine derivative; Step 4: (1) Weighing bismuth nitrate pentahydrate and adding it to dilute nitric acid, and stirring it for the first time at room temperature; after stirring, adding ammonium molybdate tetrahydrate and stirring it for the second time, then adjusting the pH of the mixed solution with ammonia water, and then transferring the mixed solution to a hydrothermal reactor to carry out a hydrothermal reaction. After the reaction, naturally cooling to a certain temperature and then centrifuging to obtain a precipitate, the obtained precipitate is washed with ethanol and deionized water in sequence, and dried to obtain bismuth molybdate; (2) Weighing bismuth nitrate pentahydrate and adding it to deionized water, stirring it for the first time at room temperature; then adding sodium thiosulfate pentahydrate and stirring it for the second time, transferring the obtained mixed solution to a hydrothermal reactor, performing a hydrothermal reaction, cooling it naturally to room temperature, and obtaining a precipitate by centrifugation. The obtained precipitate is washed with ethanol and deionized water in sequence, and dried to obtain bismuth sulfide; Step 5: taking the bismuth molybdate and bismuth sulfide obtained in step 4 and adding them to deionized water in a certain proportion, transferring the obtained mixed solution after ultrasonic treatment to a hydrothermal reactor for hydrothermal reaction; naturally cooling to room temperature and centrifuging to obtain a precipitate, and washing the obtained precipitate with ethanol and deionized water in sequence, and then drying to obtain a bismuth molybdate-bismuth sulfide complex; Step 6: Dissolve the upconversion fluorescent nanomaterial prepared in step 1 and the rhodamine derivative prepared in step 3 in methanol respectively to obtain an upconversion fluorescent nanomethanol solution and a rhodamine derivative methanol solution, and mix the two in a certain proportion to obtain a mixed solution, which is the copper activation-upconversion-bismuth molybdate sensing system; Step 7: Prepare tetracycline standard solutions of different concentrations. First, the bismuth molybdate-bismuth sulfide complex obtained in step 5 is added to water and stirred evenly to obtain a bismuth molybdate-bismuth sulfide composite solution; a tetracycline standard solution and a copper sulfate solution are mixed in a certain proportion, shaken for a period of time, and then the bismuth molybdate-bismuth sulfide composite solution is added and irradiated with a xenon lamp for a period of time, and the precipitate is removed by centrifugation to obtain a test solution; The copper activation-upconversion-bismuth molybdate sensing system obtained in step 6 is mixed with the test solution to obtain a mixed solution and the fluorescence intensity signal characteristic value of the mixed solution is detected. According to the fluorescence intensity signal characteristic values ​​corresponding to the test solutions containing different concentrations of tetracycline, a standard curve related to tetracycline concentration is drawn with the logarithm of the tetracycline standard solution concentration as the abscissa and the fluorescence intensity signal characteristic value as the ordinate; Step 8: Detection of tetracycline in meat products; (1) Preparation of sample solution; (2) The sample solution and copper sulfate solution are mixed in a certain proportion, shaken for a period of time, bismuth molybdate-bismuth sulfide composite solution is added, irradiated with a xenon lamp for a period of time, and centrifuged to remove the precipitate to obtain the test solution; the test solution is mixed with the copper activation-upconversion-bismuth molybdate sensing system in a certain proportion to obtain a mixed solution, the fluorescence intensity signal characteristic value of the mixed solution is measured, and the tetracycline content in the meat product sample is calculated based on the tetracycline detection standard curve obtained in step 5.

2. The method for detecting tetracycline in meat products based on the copper activation-upconversion-bismuth molybdate sensing system according to claim 1, characterized in that: In step 1, the usage ratio of methanol, yttrium chloride hexahydrate, ytterbium chloride hexahydrate, erbium chloride hexahydrate, oleic acid and 1-octadecene is 6-10 mL:236.6 mg:77.5 mg:7.6 mg:6 mL:15 mL; The ratio of oleic acid to methanol, ammonium fluoride, and sodium hydroxide in the mixed solution A is 6 mL: 10 mL: 0.1482 g: 0.1 g; the temperature of the first heating and stirring reaction is 160-170° C., the stirring time is 25-40 min, and the stirring rate is 300-500 rpm; The second heating and stirring temperature is 50-70°C, and the time is 70-100 min; the third heating and stirring reaction temperature is 290-300°C, and the stirring reaction time is 60-90 min; the stirring rate of the second heating and stirring and the third heating and stirring is 300-500 rpm; the volume ratio of the mixed solution B to the ethanol and cyclohexane mixed solution is 1:1, and the volume ratio of cyclohexane to ethanol is 1:1; the centrifugal rate is 8000 rpm, and the time is 6-8 min; the drying temperature is 40-60°C, and the time is 8-20 h.

3. The method for detecting tetracycline in meat products based on the copper activation-upconversion-bismuth molybdate sensing system according to claim 1, characterized in that: In step 2, the ratio of rhodamine B: ethanol: hydrazine hydrate: hydrochloric acid is 1.2 g: 30 mL: 3 mL: 50 mL; the first heating reflux time is 100-150 min, and the temperature is 70-80 ° C; the concentration of the hydrochloric acid is 1 mol / L; the solution used to adjust the pH of the red solution is sodium hydroxide with a concentration of 1 mol / L, and the pH is adjusted to 9-10; the centrifugal rate is 8000 rpm, and the time is 6-10 min; the drying temperature is 45-60 ° C, and the time is 12-15 h.

4. The method for detecting tetracycline in meat products based on the copper activation-upconversion-bismuth molybdate sensing system according to claim 1, characterized in that: In step three, the amount ratio of rhodamine B hydrazine, ethanol, dimethylbenzaldehyde, and acetic acid is 0.46 g: 20 mL: 0.16 g: 0.15~0.2 mL; the reaction temperature of the second heating reflux is 70~85 ° C, and the time is 3~3.5 h; the volume of the concentrated solution is 30~50% of the volume of the reflux solution, and the standing time is 12~24 h; the centrifugal rate is 8000 rpm, and the time is 6~10 min; the drying temperature is 50~60 ° C, and the time is 12~24 h.

5. The method for detecting tetracycline in meat products based on the copper activation-upconversion-bismuth molybdate sensing system according to claim 1, characterized in that: In step 4 (1), the ratio of bismuth nitrate pentahydrate, dilute nitric acid, and ammonium molybdate tetrahydrate is 2 mmol:25 mL:2 mmol, wherein the concentration of dilute nitric acid is 1 mol / L; the first stirring and the second stirring time are both 30-40 min; the pH of the solution is adjusted to 7 with ammonia water; the hydrothermal reaction temperature is 150-160°C, and the time is 6 h; the cooling to a certain temperature is 20-35°C; in step 4 (2), the ratio of bismuth nitrate pentahydrate, deionized water, and sodium thiosulfate is 0.5 mmol:40 mL; 0.75 mmol; the first and second stirring times are both 40-60 min; the temperature of the hydrothermal reaction is 160-170°C, and the time is 24 h.

6. The method for detecting tetracycline in meat products based on the copper activation-upconversion-bismuth molybdate sensing system according to claim 1, characterized in that: In step 5, the ratio of bismuth molybdate, bismuth sulfide, and deionized water is 1 g:0.3 g:30 ml; the temperature of the hydrothermal reaction is 100-120° C., and the time is 10 h.

7. The method for detecting tetracycline in meat products based on the copper activation-upconversion-bismuth molybdate sensing system according to claim 1, characterized in that: In step six, the concentrations of the upconversion fluorescent nanoparticle methanol solution and the rhodamine derivative methanol solution are 0.25 mg / mL and 0.1 mg / mL, respectively; and the volume ratio of the two mixtures is 2:

1.

8. The method for detecting tetracycline in meat products based on the copper activation-upconversion-bismuth molybdate sensing system according to claim 1, characterized in that: In step seven, the volume ratio of the tetracycline standard solution, the copper sulfate solution, and the bismuth molybdate-bismuth sulfide composite solution is 1:1:0.5; the concentration of the bismuth molybdate-bismuth sulfide composite solution is 1 g / L, the concentration of the copper sulfate solution is 32 mg / L, and the concentration range of the tetracycline standard solution is 50~50000 μg / L; the shaking time is 1.5~2 h; the xenon lamp irradiation time is 1 h; the volume ratio of the copper activation-upconversion-bismuth molybdate sensing system to the test solution is 1:1; the step of detecting the fluorescence intensity signal characteristic value of the mixed solution is: exciting the mixed solution with excitation light of 980 nm wavelength, and recording the fluorescence intensity value at 540 nm of the fluorescence spectrum.

9. The method for detecting tetracycline in meat products based on a copper activation-upconversion-bismuth molybdate sensing system according to claim 1, characterized in that: In step eight (1), the specific steps for preparing the sample solution are as follows: weigh a certain amount of sample, place it in a centrifuge tube, add perchloric acid solution A to homogenize for a period of time, shake evenly, and centrifuge to obtain the supernatant; add perchloric acid solution B to the residue in the centrifuge tube, repeat the operation once, and combine the supernatants; add n-hexane to the supernatant, shake and then centrifuge to remove the n-hexane, then add n-hexane again, shake and then centrifuge to remove the n-hexane, and the obtained solution is recorded as treatment solution A; wash the treatment solution A with deionized water using an ODS-C18 column to remove impurities, elute with methanol A, collect the eluate, blow dry with nitrogen, and then make up to volume with methanol B and filter with a filter membrane to obtain the sample solution; The ratio of the sample, perchloric acid solution A, and perchloric acid solution B is 1 g:2 mL:1 mL; the mass fractions of perchloric acid solution A and perchloric acid solution B are both 0.5%; the homogenization time is 30 s, and the oscillation time on the oscillator is 3 min; the centrifugal speed is 4000 rpm, and the time is 10 min; the ratio of the sample to n-hexane A, n-hexane B, deionized water, methanol A, and methanol B is 5 g:1 mL:1 mL:10 mL:5 mL:1 mL; and the pore size of the filter membrane is 0.45 µm.

10. The method for detecting tetracycline in meat products based on the copper activation-upconversion-bismuth molybdate sensing system according to claim 1, characterized in that: In step eight (2), the volume ratio of the sample solution, copper sulfate solution, and bismuth molybdate-bismuth sulfide composite solution is 1:1:0.5; the concentration of the bismuth molybdate-bismuth sulfide composite is 1 g / L, and the concentration of the copper sulfate solution is 32 mg / L; the shaking time is 1.5 to 2 h; the xenon lamp irradiation time is 1 h; and the volume ratio of the copper activation-upconversion-bismuth molybdate sensing system to the measured solution is 1:1.

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