Fluorescence / colorimetric dual-mode enterotoxin B detection method based on dual-enzyme cascade catalysis

Abstract

The invention belongs to the technical field of food safety detection, and particularly relates to a fluorescence / colorimetric bimodal enterotoxin B detection method based on bienzyme cascade catalysis. The preparation method comprises the following steps: firstly, preparing a BiO-NH2 and MOF-818 nano material; the preparation method comprises the following steps: firstly preparing a BiO-NH2-MOF-818 nano-material, then further preparing a BiO-NH2-MOF-818 composite nano-material based on the BiO-NH2 and MOF-818 nano-material, which has good fluorescence performance and can gradually catalyze substrates 3, 5-di-tert-butyl catechol and 3, 3 ', 5, 5'-tetramethyl benzidine to generate visible signals, double-enzyme cascade catalysis not only converts a single substrate into more signal molecules, reduces the loss of intermediate products, but also can catalyze the reaction of the signal molecules to generate visible signals, so that the preparation process is simplified, and the preparation cost is reduced. Detection signals are effectively amplified; and moreover, by reducing diffusion of the intermediate between enzymes, the utilization rate of the intermediate is effectively improved, so that the catalytic efficiency is further improved, the detection sensitivity of enterotoxin B is improved, and rapid detection of enterotoxin B in food is realized.

Description

Technical Field

[0001] The present invention belongs to the technical field of food safety detection, and in particular relates to a method for detecting enterotoxin B by fluorescence / colorimetry dual-modality based on dual-enzyme cascade catalysis. Background Art

[0002] Staphylococcus aureus is one of the most common foodborne pathogens and is considered to be the main cause of foodborne diseases around the world. Enterotoxins produced by Staphylococcus aureus are the main virulence factors that induce food poisoning, which can easily cause symptoms such as abdominal pain, stomach cramps and vomiting in humans. At present, 23 enterotoxins have been identified as different serological entities, among which enterotoxin B (SEB) is one of the most toxic types of staphylococcal food poisoning. Due to its compact tertiary structure, enterotoxin B has strong heat resistance and retains its virulent properties after high-temperature cooking. The lethal dose of enterotoxin B is 20 ng / kg, while a dose of <0.4 ng / kg will cause clinical symptoms. Therefore, rapid and sensitive detection of enterotoxin B in food samples is of great significance to reduce the risk of staphylococcal food poisoning and control food safety.

[0003] At present, the detection methods of enterotoxin B mainly include chromatography, molecular biology and immunology, and enzyme-linked immunosorbent assay (ELISA) has been developed for commercial kits. These methods show good performance in the detection of enterotoxin B, but are also restricted by their inherent shortcomings; such as time-consuming chromatography, false negative results of molecular biology methods, and high prices of immunology methods. In recent years, sensor detection methods have attracted much attention due to their advantages such as fast response and simple pretreatment. However, the existing sensors for the detection of enterotoxin B are still insufficient: on the one hand, the detection sensitivity of the sensor still needs to be improved; on the other hand, most nanosensors are single-mode detection for enterotoxin B, which only provides one output signal and is prone to false negative or false positive detection results. Therefore, there is an urgent need to establish a sensitive, rapid and efficient enterotoxin B detection method. Summary of the invention

[0004] In order to overcome the shortcomings of the existing methods, the present invention provides a method for detecting enterotoxin B by fluorescence / colorimetry based on dual enzyme cascade catalysis. First, a dual mimetic catalytic nanozyme BiO-NH 2 @MOF-818 has good fluorescence properties and can gradually catalyze the substrates 3,5-di-tert-butylcatechol and 3,3',5,5'-tetramethylbenzidine to produce visible signals, which can realize the rapid detection of enterotoxin B in food.

[0005] In order to achieve the above-mentioned object of the invention, the technical solution adopted by the present invention is as follows:

[0006] A method for detecting enterotoxin B by fluorescence / colorimetry dual-mode based on dual enzyme cascade catalysis, comprising the following steps:

[0007] S1. Dual mimetic catalytic nanozyme BiO-NH 2 @ Preparation of MOF-818 composite nanomaterials;

[0008] (1) Preparation of BiO-NH 2 Nanomaterials:

[0009] Firstly, bismuth nitrate pentahydrate is weighed and dissolved in a mixed solution consisting of N,N-dimethylformamide, glycerol and water, and the solution is completely dissolved by ultrasonication. Then, the solution is transferred to a reactor for heating reaction, cooled to room temperature after the reaction, and washed with ultrapure water and ethanol in sequence. After washing, the solution is centrifuged, the precipitate is collected, and vacuum dried to obtain BiO-COOH nanomaterials.

[0010] Subsequently, BiO-COOH nanomaterials and 2-aminoterephthalic acid were ultrasonically dissolved in a mixed solution consisting of N,N-dimethylformamide and methanol; the solution was then transferred to a reactor for heating reaction. After the reaction was completed, the reaction product was collected and washed with N,N-dimethylformamide, acetone and ethanol in turn, centrifuged after washing, and the precipitate was collected and vacuum dried to obtain BiO-NH 2 Nanomaterials;

[0011] (2) Synthesis of MOF-818 nanomaterials: First, zirconium oxychloride octahydrate, copper nitrate pentahydrate and 4-pyrazolecarboxylic acid were weighed, dissolved in N,N-dimethylformamide, and trifluoroacetic acid was added, mixed evenly, and a mixed solution was obtained; the mixed solution was transferred to a reactor for heating reaction, cooled to room temperature after the reaction was completed, centrifuged, and the precipitate was collected and vacuum dried to obtain MOF-818 nanomaterials;

[0012] (3) Preparation of BiO-NH by polyvinylpyrrolidone-assisted synthesis 2 @MOF-818 composite nanomaterial: First, the BiO-NH synthesized in step (1) 2 The nanomaterial and polyvinyl pyrrolidone are dissolved in N,N-dimethylformamide, stirred evenly and centrifuged, the precipitate is collected, and the precipitate is redissolved in N,N-dimethylformamide, and the MOF-818 nanomaterial prepared in step (2) is added, stirred evenly and centrifuged again, the precipitate is collected, and finally vacuum dried to obtain BiO-NH 2 @MOF-818 composite nanomaterials;

[0013] Preferably, in step S1 (1), the dosage relationship of bismuth nitrate pentahydrate, N,N-dimethylformamide, glycerol and water is 0.5-10 mmol: 2-20 mL: 5-50 mL: 1-20 mL; the dosage relationship of BiO-COOH nanomaterial, 2-aminoterephthalic acid, N,N-dimethylformamide and methanol is 0.05-2 mmol: 0.025-1 mmol: 5-40 mL: 1-10 mL; and the ultrasonic time is 5-10 min.

[0014] Preferably, in step S1 (1), the temperature of the heating reaction is 100-150°C, and the heating time is 12-72 hours; the centrifugal conditions are: the rotation speed is 5000-10000 rpm, and the centrifugal time is 2-20 minutes; the temperature of the vacuum drying is 30-70°C, and the time is 3-48 hours.

[0015] Preferably, in step S1 (2), the amount of zirconium oxychloride octahydrate, copper nitrate pentahydrate, 4-pyrazolecarboxylic acid, trifluoroacetic acid and N,N-dimethylformamide is in the following relationship: 0.1-1 mmol: 0.1-1 mmol: 0.2-2 mmol: 0.05-1 mL: 20 mL; the temperature of the heating reaction is 80-120° C., and the reaction time is 5-24 hours; the speed of the centrifugation is 5000-10000 rpm, and the centrifugation time is 5-30 minutes; the temperature of the vacuum drying is 30-70° C., and the time is 4-48 hours.

[0016] Preferably, in step S1 (3), BiO-NH 2 The dosage relationship of nanomaterials, polyvinyl pyrrolidone, N,N-dimethylformamide, N,N-dimethylformamide used for re-dissolution and MOF-818 nanomaterials is 10-200 mg: 2-50 mg: 2-50 mL: 2-50 mL: 10-500 mg; the stirring temperature is 15-50° C., and the stirring time is 6-72 hours; the centrifugal speed is 5000-10000 rpm, and the centrifugal time is 5-30 minutes; the vacuum drying temperature is 30-70° C., and the drying time is 4-48 hours.

[0017] S2. Acquire fluorescence signal and colorimetric signal;

[0018] (1) Preparation of enterotoxin B standard solution: Prepare n enterotoxin B standard solutions with different concentration gradients, with concentrations of C 1 , C 2 , C 3 , C 4 , ... C n-1 , C n , where n is a positive integer;

[0019] (2) Obtaining fluorescence signals: First, BiO-NH 2 @MOF-818 composite nanomaterials were dissolved in ultrapure water to obtain BiO-NH 2 @MOF-818 solution; then the enterotoxin B aptamer was mixed with BiO-NH 2 @MOF-818 solution was mixed and incubated for the first time; the enterotoxin B standard solution was added to the solution after the first incubation and incubated for the second time; then, 3,5-di-tert-butylcatechol solution and 3,3',5,5'-tetramethylbenzidine solution were added to the second incubation solution in sequence and incubated for the third time to obtain the test solution; the test solutions corresponding to the enterotoxin B standard solutions of different concentrations were recorded as D 1 , D 2 , D 3 , D 4 , ...D n-1 , D n Finally, the fluorescence intensity of the solution to be tested at the fluorescence emission peak is measured and recorded, which is denoted as F 1 , F 2 , F 3 , F 4 , ... F n-1 , F n , where n is a positive integer;

[0020] (3) Obtaining colorimetric signal:

[0021] After measuring the fluorescence signal of each solution to be tested, continue to measure and record the absorbance at the ultraviolet absorption peak, which is recorded as A 1 , A 2 , A 3 , A 4 , ... A n-1 , A n , where n is a positive integer;

[0022] Preferably, the concentration range of the enterotoxin B standard solution in step S2 (1) is 1×10 -9 ~100ng / mL.

[0023] Preferably, in step S2 (2), the enterotoxin B aptamer, BiO-NH 2 The dosage relationship of @MOF-818 solution, enterotoxin B standard solution, 3,5-di-tert-butylcatechol solution and 3,3',5,5'-tetramethylbenzidine solution is 0.005~1mL: 0.05~5mL: 0.1~2mL: 0.05~1mL: 0.05~1mL, where the concentration of enterotoxin B aptamer solution is 1nM~10μM, BiO-NH 2The concentration range of @MOF-818 solution is 10~1000μg / mL, the concentration range of 3,5-di-tert-butylcatechol solution is 0.05~5mmol / L, and the concentration range of 3,3',5,5'-tetramethylbenzidine solution is 0.1~5mmol / L.

[0024] Preferably, in step S2 (2), the first incubation time is 10 to 60 minutes; the second incubation time is 5 to 60 minutes; the third incubation time is 2 to 30 minutes; the incubation temperature is 2 to 40°C; when measuring and recording the fluorescence intensity at the fluorescence emission peak, the excitation wavelength is 300 to 400 nm, and the fluorescence emission peak is at 380 to 450 nm.

[0025] Preferably, in step S2 (3), the ultraviolet absorption peak is located at 330-450 nm.

[0026] S3. Establish a fluorescence / colorimetric rapid detection prediction model;

[0027] (1) Establish a rapid fluorescence detection prediction model:

[0028] A model is established using the concentration of the enterotoxin B standard solution in step S2 (1) and the corresponding fluorescence intensity in step S2 (2) to obtain a fluorescence rapid detection prediction model for enterotoxin B F=f(x), where F is the fluorescence intensity and x is the enterotoxin B concentration;

[0029] (2) Establish a colorimetric rapid detection prediction model:

[0030] The concentration of the enterotoxin B standard solution in step S2 (1) and the corresponding absorbance A in step S2 (3) are used to calculate the 1 , A 2 , A 3 , A 4 , ... A n-1 , A n Models were established respectively to obtain a colorimetric rapid detection prediction model Y=y(x) for enterotoxin B, where Y is the absorbance at the absorption peak and x is the concentration of enterotoxin B.

[0031] S4. Detect the content of enterotoxin B in the sample;

[0032] The sample to be tested is homogenized and then added with the extracting solution. After mixing and centrifugation, the supernatant is collected. Then, the fat extracting solution is added. After shaking and centrifugation, the fat layer is removed to obtain the sample solution to be tested.

[0033] The operation of step S2 is followed, except that the enterotoxin B standard solution is replaced by the sample test solution, and finally the fluorescence intensity and absorbance of the sample test solution are obtained and substituted into the fluorescence rapid detection prediction model and colorimetric rapid detection prediction model established in step S3, respectively, to realize the rapid detection of enterotoxin B by fluorescence and colorimetry.

[0034] Preferably, the dosage relationship of the sample to be tested, the extract and the fat extract in step S4 is 2-100 g: 5-500 mL: 5-500 mL; the sample to be tested is starch, milk and dairy products, meat or eggs, the extract is PBS buffer, and the fat extract is n-heptane; the shaking time is 2-30 minutes, the centrifugal speed is 5000-10000 rpm, and the centrifugal time is 2-30 minutes.

[0035] Beneficial effects of the present invention:

[0036] (1) The dual-mimetic catalytic nanozyme prepared by the present invention has a high catalytic efficiency and can catalyze the substrates 3,5-di-tert-butylcatechol and 3,3',5,5'-tetramethylbenzidine in a short time and produce a significant signal change, thereby realizing the rapid detection of enterotoxin B.

[0037] (2) Dual-enzyme cascade catalysis not only converts a single substrate into more signal molecules, reduces the loss of intermediates, effectively amplifies the detection signal, realizes signal amplification, and improves detection sensitivity; it also effectively improves the utilization rate of intermediates by reducing the diffusion of intermediates between enzymes, thereby further improving the catalytic efficiency and improving the sensitivity of enterotoxin B detection.

[0038] (3) The dual-modal sensor constructed by the present invention combines the two detection modes of fluorescence and colorimetry, and can use the output signals of the two modes to mutually verify the detection results, thereby improving the accuracy and reliability of enterotoxin B detection.

[0039] (4) The enterotoxin B dual-enzyme cascade catalytic sensing detection method proposed in the present invention has a detection level of enterotoxin B that is lower than the national standard, meets daily detection needs, and can be used for rapid detection of enterotoxin B in a variety of food and agricultural products. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1 The dual-simulation catalytic nanozyme BiO-NH prepared in Example 1 2 @SEM image of MOF-818.

[0041] Figure 2 (a) is the fluorescence response of the dual-mimetic catalytic nanozyme to enterotoxin B in Example 1, and (b) is the colorimetric response of the dual-mimetic catalytic nanozyme to enterotoxin B. Specific implementation plan

[0042] The present invention is described in detail below through various embodiments; however, these embodiments are only used to illustrate the present invention and are not used to limit the scope of the present invention.

[0043] The enterotoxin B aptamer used in the present invention was purchased from Sangon Biotech (Shanghai) Co., Ltd.; meanwhile, the reagents used in the present invention can be obtained from conventional commercial sources unless otherwise specified.

[0044] Embodiment 1:

[0045] S1. Preparation of dual-mimic catalytic nanozymes

[0046] (1) Preparation of BiO-NH 2 Nanomaterials: First, weigh 2 mmol of bismuth nitrate pentahydrate and dissolve it in a mixed solution consisting of 10 mL of N, N-dimethylformamide, 20 mL of glycerol and 6 mL of water, and ultrasonicate for 5 minutes to completely dissolve it. Then transfer the above solution to a 50 mL stainless steel autoclave, heat at 120 ° C for 48 hours, and then cool to room temperature. Wash with ultrapure water and ethanol three times respectively, centrifuge (6000 rpm, 10 minutes), collect the precipitate, and dry it overnight under vacuum at 60 ° C to obtain BiO-COOH nanomaterials.

[0047] Subsequently, 0.4mmol BiO-COOH and 0.2mmol 2-aminoterephthalic acid were ultrasonically dissolved (5 minutes) in a mixed solution consisting of 16mL N,N-dimethylformamide and 4mL methanol. The solution was then transferred to a 50mL stainless steel autoclave and heated at 120°C for 48 hours. After the reaction was completed, the reaction product was collected and washed three times with N,N-dimethylformamide, acetone and ethanol in turn. Finally, centrifugation (6000 rpm, 10 minutes) was performed to collect the precipitate, which was then dried at 60°C under vacuum for 12 hours to obtain BiO-NH 2 Nanomaterials;

[0048] (2) Synthesis of MOF-818 nanomaterials: First, weigh 0.26mmol zirconium oxychloride octahydrate, 0.26mmol copper nitrate pentahydrate and 0.52mmol 4-pyrazole carboxylic acid, respectively, dissolve them in 20mL N,N-dimethylformamide solution, add 240μL trifluoroacetic acid and mix well. Transfer the above solution to a 50mL stainless steel autoclave and heat at 100℃ for 12 hours. After cooling to room temperature, centrifuge (8000 rpm, 15 minutes) and collect the precipitate. Finally, dry under vacuum at 60℃ for 12 hours to obtain MOF-818 nanomaterials.

[0049] (3) Synthesis of BiO-NH 2@MOF-818 composite nanomaterials: Preparation of BiO-NH by polyvinylpyrrolidone-assisted synthesis 2 @MOF-818 composite nanomaterials. First, 50 mg of synthesized BiO-NH 2 The nanomaterial and 10 mg of polyvinyl pyrrolidone were dissolved in 10 mL of N, N-dimethylformamide solution and stirred at room temperature for 12 hours. Centrifugation (6000 rpm, 10 minutes) was performed to remove unbound polyvinyl pyrrolidone. The precipitate was collected and redissolved in 20 mL of N, N-dimethylformamide solution, to which 100 mg of MOF-818 nanomaterial was added and stirred at room temperature for 12 hours. Centrifugation (8000 rpm, 15 minutes) was performed to collect the precipitate. Finally, the precipitate was dried under vacuum at 60 ° C for 12 hours to obtain BiO-NH 2 @MOF-818 composite nanomaterial is a dual-simulation catalytic nanozyme; its electron microscope image is as follows Figure 1 shown.

[0050] S2. Acquire fluorescence signal and colorimetric signal;

[0051] (1) Preparation of enterotoxin B standard solution: Prepare 8 enterotoxin B standard solutions with different concentration gradients, with concentrations of C 1 =1×10 -6 ng / mL, C 2 =1×10 -5 ng / mL, C 3 =1×10 -4 ng / mL, C 4 =1×10 -3 ng / mL, C 5 =1×10 - 2 ng / mL, C 6 =1×10 -1 ng / mL, C 7 =1ng / mL, C 8 =10 ng / mL;

[0052] (2) Obtaining fluorescence signals: First, 0.1 mL of enterotoxin B aptamer (100 nmol / L) was mixed with 1 mL of BiO-NH 2 @MOF-818 solution (50 μg / mL) was mixed and incubated at room temperature for 45 minutes; secondly, 0.5 mL of enterotoxin B standard solution of different concentrations was added thereto respectively and incubated at room temperature for 30 minutes; then, 0.2 mL of 3,5-di-tert-butylcatechol (1 mmol / L) and 0.2 mL of 3,3',5,5'-tetramethylbenzidine solution (1 mmol / L) were added to the above solution in sequence, mixed, and incubated at room temperature for 10 minutes to obtain the test solution D1 , D 2 , D 3 , D 4 , D 5 , D 6 , D 7 , D 8 Finally, the test solution was transferred to a quartz cuvette, and the cuvette was transferred to the sample pool of the fluorescence spectrometer, the excitation wavelength was set to 340 nm, and the fluorescence intensity of the fluorescence emission peak at 397 nm was measured and recorded as F 1 =521, F 2 =771, F 3 =976, F 4 =1190, F 5 =1445, F 6 =1628, F 7 =1898, F 8 =2208;

[0053] (3) Obtaining colorimetric signals: After measuring the fluorescence signal of each solution to be tested, transfer the solution in the quartz cuvette to a glass cuvette, and place the cuvette in a UV-visible spectrophotometer to measure and record the absorbance of the UV absorption peak at 373 nm as A. 1 =0.50, A 2 =0.77, A 3 =0.99, A 4 =1.18, A 5 =1.35, A 6 =1.50, A 7 =1.63, A 8 =1.73;

[0054] Figure 2 (a) is the fluorescence response of the dual-mimetic catalytic nanozyme to enterotoxin B, and (b) is the colorimetric response of the dual-mimetic catalytic nanozyme to enterotoxin B; where with SEB means containing enterotoxin B, and without SEB means not containing enterotoxin B.

[0055] S3. Establish a fluorescence / colorimetric rapid detection prediction model;

[0056] (1) Establishing a fluorescence rapid detection prediction model: Using the concentration C of the enterotoxin B standard solution described in step S2 (1) 1 , C 2 , C 3 , C 4 , C 5 , C 6 , C 7 , C 8The logarithm of -6, -5, -4, -3, -2, -1, 0, 1 and the fluorescence intensity F, F in step S2 (2) 2 , F 3 , F 4 , F 5 , F 6 , F 7 , F 8 A model was established to obtain a fluorescence rapid detection prediction model for enterotoxin B: F=7233.96log C+1914.59, where F is the fluorescence intensity at 527 nm and C is the logarithm of the enterotoxin B concentration.

[0057] (2) Establishing a colorimetric rapid detection prediction model: Using the enterotoxin B standard solution C described in step S2 (1) 1 , C 2 , C 3 , C 4 , C 5 , C 6 , C 7 , C 8 The logarithm of -6, -5, -4, -3, -2, -1, 0, 1 and the absorbance A in step S2 (3) 1 , A 2 , A 3 , A 4 , A 5 , A 6 , A 7 , A 8 A model was established to obtain a colorimetric rapid detection prediction model for enterotoxin B: A=0.14log C+1.11, where A is the absorbance at 373 nm and C is the enterotoxin B concentration.

[0058] S4. Using chicken as a sample, detecting the content of enterotoxin B in the chicken sample;

[0059] (1) Sample pretreatment: Take a chicken sample and divide it into 5 portions, each with 10 g, and record them as S 1 , S 2 , S 3 , S 4 , S 5 After homogenization, add 15 mL of PBS buffer to each sample, shake and mix for 15 minutes, and centrifuge (5000 rpm, 10 minutes). Take 10 mL of supernatant, transfer to another centrifuge tube, add 10 mL of n-heptane, shake and mix for 5 minutes, centrifuge (5000 rpm, 10 minutes), completely remove the n-heptane on the upper layer, and collect the lower clear liquid, which is the sample solution to be tested, and the corresponding record is Y 1 , Y 2 , Y 3 , Y 4 , Y5 .

[0060] (2) According to the method of obtaining fluorescence signal in step S2 (2), the fluorescence intensity of each sample test liquid is measured to obtain a fluorescence response signal, which is substituted into the fluorescence rapid detection prediction model established in step S4 (1), and the fluorescence rapid prediction concentration value of each sample is calculated; according to the method of obtaining colorimetric signal in step S2 (3), the absorbance of each sample test liquid is measured, which is substituted into the colorimetric rapid detection prediction model established in step S4 (2), and the colorimetric rapid prediction concentration value of each sample is calculated; the results are shown in Table 1.

[0061] Table 1 Results of detection of enterotoxin B in chicken samples using dual enzyme cascade catalytic fluorescence / colorimetric dual-modal sensor

[0062]

[0063] It can be seen from Table 1 that the relative errors of the detection results of enterotoxin B by the fluorescence method and the colorimetric method and the results of the liquid chromatography-mass spectrometry method are less than 5%, indicating that the fluorescence colorimetric dual-modal sensing detection method based on dual-enzyme cascade catalysis described in the present invention can be used for the detection of enterotoxin B in chicken.

[0064] 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, a person skilled in the art should understand that the present invention can still be modified or replaced by equivalents; and all technical solutions and improvements thereof that do not depart from the spirit and scope of the present invention should be included in the scope of the claims of the present invention.

Claims

1. A method for detecting enterotoxin B by fluorescence / colorimetry dual-mode based on dual enzyme cascade catalysis, characterized in that: The following steps are involved: S1. Preparation of dual-mimic catalytic nanozyme BiO-NH2@MOF-818 composite nanomaterials; (1) First, weigh bismuth nitrate pentahydrate, dissolve it in a mixed solution consisting of N,N-dimethylformamide, glycerol and water, and completely dissolve it by ultrasonication. Then, transfer the solution to a reactor for heating reaction, cool it to room temperature after the reaction, and wash it with ultrapure water and ethanol in sequence. After washing, centrifuge it, collect the precipitate, and vacuum dry it to obtain BiO-COOH nanomaterials; Subsequently, BiO-COOH nanomaterials and 2-aminoterephthalic acid were ultrasonically dissolved in a mixed solution consisting of N,N-dimethylformamide and methanol; The solution is then transferred to a reactor for heating reaction. After the reaction is completed, the reaction product is collected and washed with N,N-dimethylformamide, acetone and ethanol in sequence. After washing, the product is centrifuged, the precipitate is collected, and vacuum dried to obtain BiO-NH2 nanomaterials. (2) Weighing zirconium oxychloride octahydrate, copper nitrate pentahydrate and 4-pyrazolecarboxylic acid respectively, dissolving them in N,N-dimethylformamide, adding trifluoroacetic acid, and mixing them evenly to obtain a mixed solution; transferring the mixed solution to a reactor for heating reaction, cooling to room temperature after the reaction, centrifuging, collecting the precipitate, and vacuum drying to obtain MOF-818 nanomaterials; (3) dissolving the BiO-NH2 nanomaterial and polyvinyl pyrrolidone synthesized in step (1) in N,N-dimethylformamide, stirring evenly and centrifuging, collecting the precipitate, redissolving it in N,N-dimethylformamide, adding the MOF-818 nanomaterial prepared in step (2), stirring evenly and centrifuging again, collecting the precipitate, and finally vacuum drying to obtain the BiO-NH2@MOF-818 composite nanomaterial; S2. Acquire fluorescence signal and colorimetric signal; (1) Preparation of enterotoxin B standard solution: Prepare n enterotoxin B standard solutions with different concentration gradients, with concentrations of C1, C2, C3, C4, ... C n-1 , C n , where n is a positive integer; (2) First, the BiO-NH2@MOF-818 composite nanomaterial is dissolved in ultrapure water to obtain a BiO-NH2@MOF-818 solution; then, the enterotoxin B aptamer is mixed with the BiO-NH2@MOF-818 solution and incubated for the first time; and the enterotoxin B standard solution is added to the solution after the first incubation and incubated for the second time; Subsequently, 3,5-di-tert-butylcatechol solution and 3,3',5,5'-tetramethylbenzidine solution were added to the second incubation solution in sequence, and the third incubation was performed to obtain the test solution; the test solutions corresponding to the enterotoxin B standard solutions with different concentrations were recorded as D1, D2, D3, D4, ... D n-1 , D n Finally, the fluorescence intensity of the solution to be tested at the fluorescence emission peak is measured and recorded, which are recorded as F1, F2, F3, F4, ... F n-1 , F n , where n is a positive integer; (3) After measuring the fluorescence signal of each solution to be tested, continue to measure and record the absorbance at the ultraviolet absorption peak, which is recorded as A1, A2, A3, A4, ... A in sequence. n-1 , A n , where n is a positive integer; S3. Establish a fluorescence / colorimetric rapid detection prediction model; (1) Establish a rapid fluorescence detection prediction model: A model is established using the concentration of the enterotoxin B standard solution in step S2 (1) and the corresponding fluorescence intensity in step S2 (2) to obtain a fluorescence rapid detection prediction model for enterotoxin B F=f(x), where F is the fluorescence intensity and x is the enterotoxin B concentration; (2) Establish a colorimetric rapid detection prediction model: The concentration of the enterotoxin B standard solution in step S2 (1) and the corresponding absorbances A1, A2, A3, A4, ... A in step S2 (3) are used to calculate the concentration of the enterotoxin B standard solution in step S2 (1) and the corresponding absorbances A1, A2, A3, A4, ... A n-1 , A n Models were established respectively to obtain a colorimetric rapid detection prediction model Y=y(x) for enterotoxin B, where Y is the absorbance at the absorption peak and x is the concentration of enterotoxin B; S4. Detect the content of enterotoxin B in the sample; The sample to be tested is homogenized and then added with the extracting solution. After mixing and centrifugation, the supernatant is collected. Then, the fat extracting solution is added. After shaking and centrifugation, the fat layer is removed to obtain the sample solution to be tested. The operation of step S2 is followed, except that the enterotoxin B standard solution is replaced by the sample test solution, and finally the fluorescence intensity and absorbance of the sample test solution are obtained and substituted into the fluorescence rapid detection prediction model and colorimetric rapid detection prediction model established in step S3, respectively, to realize the rapid detection of enterotoxin B by fluorescence and colorimetry.

2. The method for detecting enterotoxin B by fluorescence / colorimetry based on dual enzyme cascade catalysis according to claim 1, characterized in that: In step S1 (1), the amount of bismuth nitrate pentahydrate, N,N-dimethylformamide, glycerol and water is in the following relationship: 0.5-10 mmol: 2-20 mL: 5-50 mL: 1-20 mL; the amount of BiO-COOH nanomaterial, 2-aminoterephthalic acid, N,N-dimethylformamide and methanol is in the following relationship: 0.05-2 mmol: 0.025-1 mmol: 5-40 mL: 1-10 mL; and the ultrasonication time is 5-10 min.

3. The method for detecting enterotoxin B by fluorescence / colorimetry based on dual enzyme cascade catalysis according to claim 1, characterized in that: In step S1 (1), the temperature of the heating reaction is 100-150° C., and the heating time is 12-72 hours; the centrifugal conditions are: the rotation speed is 5000-10000 rpm, and the centrifugal time is 2-20 minutes; the temperature of the vacuum drying is 30-70° C., and the time is 3-48 hours.

4. The method for detecting enterotoxin B by fluorescence / colorimetry based on dual enzyme cascade catalysis according to claim 1, characterized in that: In step S1 (2), the amount of zirconium oxychloride octahydrate, copper nitrate pentahydrate, 4-pyrazolecarboxylic acid, trifluoroacetic acid and N,N-dimethylformamide is in the following relationship: 0.1-1 mmol: 0.1-1 mmol: 0.2-2 mmol: 0.05~1mL:20mL; the temperature of the heating reaction is 80~120℃, and the reaction time is 5~24 hours; the speed of the centrifugation is 5000~10000 rpm, and the centrifugation time is 5~30 minutes; the temperature of the vacuum drying is 30~70℃, and the time is 4~48 hours.

5. The method for detecting enterotoxin B by fluorescence / colorimetry based on dual enzyme cascade catalysis according to claim 1, characterized in that: In step S1 (3), the amount of BiO-NH2 nanomaterial, polyvinyl pyrrolidone, N,N-dimethylformamide, N,N-dimethylformamide used for re-dissolution and MOF-818 nanomaterial is in the following relationship: 10-200 mg: 2-50 mg: 2-50 mL: 2-50 mL: 10-500 mg; the stirring temperature is 15-50°C, and the stirring time is 6-72 hours; the centrifugal speed is 5000-10000 rpm, and the centrifugal time is 5-30 minutes; the vacuum drying temperature is 30-70°C, and the drying time is 4-48 hours.

6. The method for detecting enterotoxin B by fluorescence / colorimetry based on dual enzyme cascade catalysis according to claim 1, characterized in that: The concentration range of the enterotoxin B standard solution in step S2 (1) is 1×10 -9 ~100ng / mL.

7. The method for detecting enterotoxin B by fluorescence / colorimetry based on dual enzyme cascade catalysis according to claim 1, characterized in that: In step S2 (2), the dosage of the enterotoxin B aptamer, BiO-NH2@MOF-818 solution, enterotoxin B standard solution, 3,5-di-tert-butylcatechol solution and 3,3',5,5'-tetramethylbenzidine solution is 0.005-1 mL: 0.05-5 mL: 0.1-2 mL: 0.05-1 mL: 0.05~1mL, among which the concentration of enterotoxin B aptamer solution is 1nM~10μM, the concentration range of BiO-NH2@MOF-818 solution is 10~1000μg / mL, the concentration range of 3,5-di-tert-butylcatechol solution is 0.05~5mmol / L, and the concentration range of 3,3',5,5'-tetramethylbenzidine solution is 0.1~5mmol / L.

8. The method for detecting enterotoxin B by fluorescence / colorimetry based on dual enzyme cascade catalysis according to claim 1, characterized in that: In step S2 (2), the first incubation time is 10 to 60 minutes; the second incubation time is 5 to 60 minutes; the third incubation time is 2 to 30 minutes; the incubation temperature is 2 to 40°C; when measuring and recording the fluorescence intensity at the fluorescence emission peak, the excitation wavelength is 300 to 400 nm, and the fluorescence emission peak is at 380 to 450 nm.

9. The method for detecting enterotoxin B by fluorescence / colorimetry based on dual enzyme cascade catalysis according to claim 1, characterized in that: In step S2 (3), the ultraviolet absorption peak is located at 330-450 nm.

10. The method for detecting enterotoxin B by fluorescence / colorimetry based on dual enzyme cascade catalysis according to claim 1, characterized in that: The dosage relationship of the sample to be tested, the extract and the fat extract in step S4 is 2-100 g: 5-500 mL: 5-500 mL; the sample to be tested is starch, milk and dairy products, meat or eggs, the extract is PBS buffer, and the fat extract is n-heptane; the shaking time is 2-30 minutes, the centrifugal speed is 5000-10000 rpm, and the centrifugal time is 2-30 minutes.

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