A rapid detection method of nitrosamine based on metalloporphyrin upconversion fluorescence sensor

Abstract

The application belongs to the field of food safety detection, and particularly relates to a rapid detection method of nitrosamine based on a metal porphyrin upconversion fluorescence sensor. The steps are as follows: first, an oil coated upconversion nanomaterial and a tetraphenylporphyrin cobalt containing perchloric acid structure are prepared; then N-nitrosodiethylamine is introduced into the sensor to combine with the structured tetraphenylporphyrin cobalt to obtain the metal porphyrin upconversion fluorescence sensor; finally, a standard curve is constructed by determining the content of N-nitrosodiethylamine in a standard sample to realize the detection of the content of N-nitrosodiethylamine in food, which has the advantages of rapidness, economy and accuracy, a wide concentration detection range and a low detection limit; the application detects N-nitrosodiethylamine based on the sensor of the porphyrin material combined with the upconversion nanomaterial, proposes a rapid detection strategy of carcinogenic nitrosamine, and has a good practical prospect.

Description

Technical Field

[0001] This invention belongs to the field of food safety testing, specifically relating to a rapid detection method for nitrosamines based on a metalloporphyrin upconversion fluorescence sensor. Background Technology

[0002] Pickled foods have preservative properties and a long shelf life, resulting in widespread market demand. Pickled seafood is commonly found in coastal areas. Due to their unique flavor and texture, pickled foods are highly favored by consumers; however, the safety of pickled products remains a subject of considerable controversy. Pickled foods often contain nitrates and nitrites, which can react with proteins to form N-nitrosamines such as dimethylnitrosamine and pyrrolonitrosamine. Humans are a susceptible group to cancer caused by N-nitrosamines, and in regions where people habitually consume pickled seafood and cured meat products, the incidence of nasopharyngeal carcinoma and gastric cancer is very high. In 1978, the International Agency for Research on Cancer (IARC) classified N-nitrosamines as highly carcinogenic food contaminants, with N-nitrosodimethylamine and N-nitrosodiethylamine classified as Group 2A carcinogens—the most likely carcinogens—posing a significant threat to human health. Therefore, detecting nitrosamines in meat products is of paramount importance for ensuring public safety.

[0003] Currently, commonly used methods for detecting N-nitrosamines include gas chromatography, liquid chromatography, and liquid chromatography-mass spectrometry. However, these methods have some drawbacks, such as expensive equipment, high detection costs, unsuitability for on-site detection, and the need to use some toxic reagents. Therefore, for the automated detection of N-nitrosamines in pickled foods, it is essential to develop a rapid and portable method. Summary of the Invention

[0004] The purpose of this invention is to provide a rapid detection method for nitrosamines based on a metalloporphyrin upconversion fluorescence sensor, so as to solve the problems of high detection cost, cumbersome detection steps and slow detection speed in the prior art.

[0005] To achieve the above objectives, the present invention provides the following solution:

[0006] This invention first provides a method for preparing a metalloporphyrin-based upconversion fluorescence sensor, the steps of which are as follows:

[0007] Step 1: Yttrium chloride hexahydrate, ytterbium chloride hexahydrate, and thulium chloride hexahydrate are dissolved in methanol A. Oleic acid and 1-octadecene are then added. Under argon protection, the mixture is heated and stirred for the first time. After the reaction, it is cooled to room temperature to obtain a coolant. Ammonium fluoride, sodium hydroxide, and methanol B are mixed and added to the coolant. The mixture is heated to a certain temperature for the second reaction. After the reaction, argon gas is introduced, and the temperature is raised to a certain temperature for the third heating reaction. After the third heating reaction, under argon protection, the mixture is heated and stirred for the fourth time at a certain temperature. After the reaction, it is cooled to room temperature to obtain the reaction product. Finally, the reaction product is washed with a mixture of ultrapure water and ethanol, and then vacuum dried to obtain oleic acid-coated upconversion nanoparticles.

[0008] Step 2: Synthesize tetraphenylporphyrin cobalt material containing chlorate ions;

[0009] First, meso-tetraphenylporphyrin cobalt was dissolved in methanol A and sonicated until completely dissolved. Then, perchloric acid was added and stirred for the first time. After stirring, the mixture was dried under reduced pressure in a vacuum drying oven to remove the solvent. Methanol B and benzene A were added to the remaining solid and stirred for the second time until dissolved. Then, the mixture was refluxed in a water bath to obtain a crystalline product. Methanol C and benzene B were added dropwise to the crystalline product until completely dissolved to form a saturated solution. The solution was then cooled at a certain temperature. After purple-red crystals precipitated in the solution, anhydrous ethanol was added and stirred for the third time. After stirring and standing for a period of time, the final product was obtained by filtration and drying, which is the tetraphenylporphyrin cobalt material containing chlorate.

[0010] Step 3: Fabrication of the fluorescence sensor;

[0011] The upconversion nanomaterials prepared in step one are dissolved in dichloromethane A to obtain an upconversion nanosolution; then the tetraphenylporphyrin cobalt material containing chlorate prepared in step two is weighed and dissolved in dichloromethane B to obtain a tetraphenylporphyrin solution containing chlorate.

[0012] Then, a tetraphenylporphyrin cobalt solution containing chlorate and dichloromethane C were added to the upconversion nanosolution, and after ultrasonic treatment, a metalporphyrin-based upconversion fluorescent sensor was obtained.

[0013] Preferably, in step one, the ratio of yttrium chloride hexahydrate, ytterbium chloride hexahydrate, gadolinium chloride hexahydrate, methanol A, oleic acid, and 1-octadecene is 241.1 mg: 77.5 mg: 19.2 mg: 10 mL: 8.0 mL: 16.0 mL; and the ratio of methanol A to ammonium fluoride, sodium hydroxide, and methanol B in the coolant is 10 mL: 148.2 mg: 100 mg: 10 mL.

[0014] Preferably, in step one, the temperature of the first heating and stirring reaction is 150-170℃ and the time is 30-40 min; the temperature of the second reaction is 70-80℃ and the reaction time is 30 min; the third heating reaction is carried out under a 100℃ water bath for 10-15 min; and the temperature of the fourth heating and stirring reaction is 295-305℃ and the time is 60-70 min.

[0015] When washing the reaction products, the volume ratio of ultrapure water to ethanol is 1:1.

[0016] Preferably, in step two, the ratio of the amount of meso-tetraphenylporphyrin cobalt, methanol A, perchloric acid solution, methanol B, benzene A, methanol C, benzene B and anhydrous ethanol is 100mg:100mL:5mL:10mL:10mL:2mL:2mL:2mL; and the concentration of the perchloric acid is 0.72mol / L.

[0017] Preferably, in step two, the time for ultrasonication to complete dissolution is 5-10 minutes; the time for the first stirring reaction is 9-10 hours, and the rotation speed is 1500 rpm / min; the time for the second stirring reaction is 30 minutes, and the rotation speed is 500 rpm / min; the time for the third stirring reaction is 10-15 minutes, and the rotation speed is 500 rpm / min.

[0018] The vacuum drying oven has a temperature of 80℃ and a vacuum strength of -0.085 MPa; the reflux treatment in the water bath has a temperature of 35-40℃; the cooling temperature is 50℃ and the cooling time is 2 hours; the standing period is 2-3 hours.

[0019] Preferably, in step three, the concentration of the upconversion nanomaterial solution is 2.5 mg / mL, and the concentration of the tetraphenylporphyrin cobalt solution containing chlorate is 2 mg / mL; the volume ratio of the upconversion nanomaterial solution, the tetraphenylporphyrin cobalt solution containing chlorate, and dichloromethane C is 2:2:5; and the ultrasonic treatment time is 5-10 min.

[0020] In this context, dichloromethane A, dichloromethane B, and dichloromethane C are all dichloromethane, and benzene A and benzene B are both benzene. The different letters are only used to distinguish them in name.

[0021] The application of the metalloporphyrin-based upconversion fluorescence sensor prepared in this invention for detecting the content of N-nitrosodimethylamine in food, and its specific detection steps are as follows:

[0022] (1) Establishment of the standard curve for N-nitrosodimethylamine content:

[0023] Dichloromethane and N-nitrosodimethylamine standard solutions of different gradient concentrations were added to the prepared upconversion nanosensor. After incubation at a certain temperature for a period of time, a detection solution was obtained. Then, the fluorescence intensity signal characteristic value of the detection solution was measured, and a standard curve of N-nitrosodimethylamine concentration and its corresponding fluorescence intensity signal characteristic value was constructed.

[0024] (2) Detection of N-nitrosodimethylamine content in food samples: After food pretreatment, extract the sample liquid and then operate according to step (1). The difference is that the N-nitrosodimethylamine standard solution is replaced with the extracted sample liquid. Then measure the characteristic value of fluorescence intensity signal and input it into the standard curve of step (1) to calculate the content of N-nitrosodimethylamine in the food.

[0025] Preferably, the concentration of the N-nitrosodimethylamine standard solution in step (1) is 0.5-200 ng / mL; the ratio of the amounts of the upconversion nanosensor, dichloromethane, and N-nitrosodimethylamine standard solution is 4 mL: 5 mL: 100 μL; the incubation time is 2 min, and the incubation temperature is 30 °C; the step of determining the fluorescence intensity signal characteristic value of N-nitrosodimethylamine is as follows: the fluorescence value at 450 nm under 980 nm excitation light is used as the fluorescence intensity signal characteristic value.

[0026] Preferably, the food pretreatment steps in step (2) are as follows: weigh a certain amount of sample, add it to a centrifuge tube, and add n-hexane to remove oil; then add a certain amount of standard solution, sodium hydroxide solution and acetonitrile for homogenization, and perform vortex ultrasonic treatment; finally add an adsorbent, mix and centrifuge to obtain supernatant, and filter the supernatant with a filter membrane to obtain sample solution;

[0027] Preferably, the ratio of the meat product, n-hexane, standard solution, sodium hydroxide solution, acetonitrile, and adsorbent is 2g:5mL:50μL:4mL:6mL:50mg; the concentration of the sodium hydroxide solution is 1mmol / L; and the standard solution is an internal standard solution of N-nitrosodimethylamine with a concentration of 0.1μg / mL.

[0028] The centrifugation speed was 12000 rpm / min, the time was 3 min, and the pore size of the filter membrane was 0.22 μm.

[0029] Preferably, the food includes smoked meat, ham, and sausage.

[0030] Beneficial effects:

[0031] 1. This invention discloses a rapid detection method for nitrosamines based on a metalloporphyrin upconversion fluorescence sensor. Upconversion nanomaterials are used as the fluorescence energy donor, and cobalt tetraphenylporphyrin is used as the energy acceptor carrier. After N-nitrosodimethylamine is added and binds to the energy acceptor, the ultraviolet absorption peak of the energy acceptor redshifts, resulting in a fluorescence internal rate effect with the fluorescence donor, leading to a decrease in the fluorescence intensity of the upconversion nanomaterials. The content of N-nitrosodimethylamine is inversely proportional to the change in fluorescence intensity. Therefore, a method combining upconversion nanomaterials and metalloporphyrin materials has been successfully constructed for the efficient detection of N-nitrosodimethylamine.

[0032] 2. The specific detection system constructed in this invention utilizes a novel quenching mechanism between novel nanomaterials and metalloporphyrins to prepare a novel fluorescent sensor for the rapid detection of N-nitrosodimethylamine. Compared with traditional methods such as chromatography and high-performance liquid chromatography, this method significantly improves detection efficiency. Furthermore, the introduction of fluorescence sensing provides a simple and convenient approach for the detection of nitrosamines, offering a strategic method for the on-site detection of the carcinogenic nitrosamine. In addition, the tetraphenylporphyrin cobalt containing a perchloric acid structure used exhibits specificity for the detection of N-nitrosodimethylamine, overcoming the shortcomings of traditional methods and achieving high sensitivity and high specificity for the detection of N-nitrosodimethylamine, which is crucial for ensuring food and environmental safety.

[0033] 3. This invention establishes a rapid detection method for nitrosamines based on a metalloporphyrin upconversion fluorescence sensor. The linear concentration range of its fluorescence intensity signal characteristic value is 0.5-200 ng / mL, exhibiting a wide linear detection range and a limit of detection (LOD) of 0.17 ng / mL. Using the constructed metalloporphyrin-integrated upconversion nanomaterial fluorescence sensor to detect N-nitrosodimethylamine, the detection results are far below the nationally stipulated detection limit. Therefore, the designed sensing method can meet the high-sensitivity detection requirements of N-nitrosodimethylamine in food, possesses good versatility, and offers higher sensitivity and shorter detection time compared to traditional methods, providing a new approach for on-site detection. Attached Figure Description

[0034] Figure 1 This is a schematic diagram illustrating the principle of rapid detection of N-nitrosodimethylamine based on quenching upconversion fluorescence using porphyrin materials.

[0035] Figure 2 The image shows a transmission electron microscope (TEM) image of the upconversion nanoparticles prepared in Example 1.

[0036] Figure 3Fluorescence standard curves were established to detect different concentrations of N-nitrosodimethylamine; where A is the fluorescence signal diagram of the sensor for detecting different concentrations of N-nitrosodimethylamine; and B is the standard curve established using the concentration of N-nitrosodimethylamine and the characteristic value of the fluorescence intensity signal of the sensor at 450 nm.

[0037] Figure 4 Analysis of the specificity and anti-interference properties of the sensor prepared in Example 1. Detailed Implementation

[0038] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0039] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Every smaller range between any stated value or intermediate value within a stated range, and any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0040] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0041] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be obvious to those skilled in the art. This application specification and embodiments are merely exemplary.

[0042] The N-nitrosodimethylamine, N-nitrosodiethylamine, N-nitrosodipropylamine, N-nitrosodiphenylamine, and N-nitrosopyrrolidine used in this invention were all purchased from Jiangsu Zhenjiang Dewei Chemical Co., Ltd.

[0043] Figure 1 This diagram illustrates the principle of fluorescence signal detection for N-nitrosodimethylamine in food. Detailed steps are described in the examples.

[0044] Example 1:

[0045] Step 1: Preparation of oleic acid-coated upconversion nanomaterials (UCNPs) using a high-temperature pyrolysis method:

[0046] 241.1 mg of yttrium chloride hexahydrate, 77.5 mg of ytterbium chloride hexahydrate, and 19.2 mg of thulium chloride hexahydrate were accurately weighed and ultrasonically dispersed in 10 mL of methanol. 8 mL of oleic acid and 16 mL of 1-octadecene were added, and the mixture was magnetically stirred at 150 °C for 30 min under argon atmosphere to obtain a transparent solution. After cooling to room temperature, a coolant was obtained. A mixed solution containing 100 mg of sodium hydroxide and 148.2 mg of ammonium fluoride (4.0 mmol) dissolved in 10 mL of methanol was added dropwise. The mixture was heated to 70 °C for 30 min to completely evaporate the methanol. Then, it was heated to 100 °C under an argon atmosphere and held for 15 min. Excess methanol and air were removed from the apparatus. The mixture was then magnetically stirred at 300 °C for 60 min. After cooling to room temperature, the upconversion nanoparticles were precipitated by centrifugation and washed three times with a 1:1 volume ratio of ultrapure water and ethanol. The precipitate was collected and dried to obtain pure oleic acid-coated upconversion nanoparticles. Figure 2 Transmission electron microscopy image of the prepared upconversion nanoparticles;

[0047] Step 2: The synthesis method of tetraphenylporphyrin cobalt is as follows:

[0048] First, 100 mg of racemic tetraphenylporphyrin cobalt was ultrasonically dissolved in 100 mL of methanol solution. 5 mL of 0.72 mol / L perchloric acid was added and stirred for 10 h until completely dissolved. The solvent was then removed under reduced pressure in a vacuum drying oven. The remaining solid was recrystallized from methanol and benzene to obtain purplish-red crystals. The specific recrystallization steps are as follows: 2.0 g of the crude product was accurately weighed and added to a round-bottom flask containing 10 mL each of methanol and benzene. The flask was heated to 38 °C in a water bath under reflux. Then, 2 mL each of methanol and benzene were added dropwise until the precipitate was completely dissolved, forming a saturated solution. Finally, the solution was cooled at 50 °C for 2 h, and purplish-red crystals precipitated. Anhydrous ethanol was then added and stirred. After stirring, the solution was allowed to stand for 2 h, filtered, and dried to obtain the final product, which is the tetraphenylporphyrin cobalt material containing chlorate.

[0049] Step 3: Preparation of a fluorescent sensor for the detection of N-nitrosodimethylamine: Dissolve the upconversion nanomaterials prepared in Step 1 in dichloromethane to obtain an upconversion nanosolution (UCNPs solution) with a concentration of 2.5 mg / mL; then weigh the tetraphenylporphyrin cobalt material containing chlorate prepared in Step 2 and dissolve it in dichloromethane to obtain a tetraphenylporphyrin solution containing chlorate with a concentration of 2 mg / mL.

[0050] Then, 2 mL of a 2.5 mg / mL UCNPs solution was added to the test tube, followed by 2 mL of a 2 mg / mL tetraphenylporphyrin cobalt solution containing chlorate. Then, 5 mL of dichloromethane was added to the test tube using a pipette, and the mixture was sonicated for 5 minutes to mix thoroughly. Finally, an upconversion nanosensor containing porphyrin material for detecting N-nitrosodimethylamine was obtained.

[0051] The fluorescent nanosensor prepared based on this invention is used to detect the content of N-nitrosodimethylamine in food;

[0052] (1) Establishment of the standard curve for the fluorescence detection of N-nitrosodimethylamine:

[0053] The prepared fluorescence sensor was added to 5 mL of dichloromethane solution; then, N-nitrosodimethylamine standard solutions of different concentrations were added to obtain detection solutions of different concentrations. After incubation, the fluorescence intensity signal characteristic values ​​of the detection solutions were measured. Figure 3 A); A standard curve for the detection of N-nitrosodimethylamine content was established by linear fitting of the concentration and fluorescence intensity signal characteristics. Figure 3 B) Specifically: Prepare N-nitrosodimethylamine solutions of different concentrations (0.5, 5, 20, 40, 70, 90, 120, 150 and 200 ng / mL), add 100 μL of each solution to the specific detection system, and collect fluorescence signal characteristic values ​​after incubation at 30℃ for 2 min.

[0054] The step of determining the fluorescence intensity signal characteristic value of the detection solution is as follows: the fluorescence intensity value at 450nm under 980nm excitation light is the fluorescence intensity signal characteristic value of the detection solution.

[0055] Figure 3 A standard curve was established for the fluorescence detection of different concentrations of N-nitrosodimethylamine; from Figure 3 It can be seen that the fluorescence signal at 450 nm continuously decreases with the increase of N-nitrosodimethylamine concentration; the standard curve for N-nitrosodimethylamine content detection was obtained by linear fitting: y = -22.24x + 7098.69; where y is the characteristic value of the fluorescence intensity signal, and x is the concentration of N-nitrosodimethylamine (N-nitrosodimethylamine). Figure 3 B), Correlation coefficient R 2 =0.9905, the limit of detection (LOD) is 0.17 ng / mL, and the linear range is 0.5-200 ng / mL.

[0056] (2) Detection of N-nitrosodimethylamine content in smoked meat:

[0057] Weigh 2.0g of smoked meat sample and add it to a 50mL centrifuge tube. Then add 5mL of n-hexane to remove the grease. Next, add 50μL of standard solution to the mixture, and then add 4mL of sodium hydroxide solution and acetonitrile for homogenization, vortexing, and ultrasonic dispersion. Transfer the solution to a centrifuge tube containing 50mg of adsorbent. After centrifugation, retain the supernatant and filter it using a filter membrane. Take 500μL of the filtrate into the sensor for detection. After incubation for 2min, measure the fluorescence signal characteristic value and input it into the standard curve obtained in step (1). Calculate that the content of N-nitrosodimethylamine in the smoked meat is 1.7ng / g.

[0058] The content of N-nitrosodimethylamine in the sample was detected by gas chromatography-mass spectrometry, a national standard method, and the content of N-nitrosodimethylamine was found to be 1.67 ng / g. The calculated P value showed that there was no significant difference between the designed fluorescence sensing method and the national standard method.

[0059] Example 2:

[0060] Step 1: Preparation of oleic acid-coated upconversion nanomaterials using a high-temperature pyrolysis method:

[0061] 241.1 mg of yttrium chloride hexahydrate, 77.5 mg of ytterbium chloride hexahydrate, and 19.2 mg of thulium chloride hexahydrate were accurately weighed and ultrasonically dispersed in 10 mL of methanol. 8 mL of oleic acid and 16 mL of 1-octadecene were added, and the mixture was magnetically stirred at 150 °C for 30 min under argon atmosphere to obtain a transparent solution. After cooling to room temperature, a mixed solution containing 100 mg of sodium hydroxide and 148.2 mg of ammonium fluoride (4.0 mmol) dissolved in 10 mL of methanol was added dropwise. The mixture was heated to 70 °C for 30 min to completely evaporate the methanol. Then, it was heated to 100 °C under an argon atmosphere and held for 10-15 min to remove excess methanol and air from the apparatus. The mixture was then magnetically stirred at 300 °C for 60 min. After cooling to room temperature, the upconversion nanoparticles were precipitated by centrifugation and washed three times with a 1:1 volume ratio of ultrapure water and ethanol. The precipitate was dried to obtain pure oleic acid-coated upconversion nanoparticles. Figure 2 Transmission electron microscopy image of the prepared upconversion nanoparticles;

[0062] Step 2: The synthesis method of tetraphenylporphyrin cobalt is as follows:

[0063] First, 100 mg of racemic tetraphenylporphyrin cobalt was ultrasonically dissolved in 100 mL of methanol solution. 5 mL of 0.72 mol / L perchloric acid was added and stirred for 10 h until completely dissolved. Then, the solvent was completely removed in a vacuum drying oven. The remaining solid was recrystallized from methanol and benzene to obtain purplish-red crystals. The specific recrystallization steps are as follows: 2.0 g of the crude product was accurately weighed and added to a round-bottom flask containing 10 mL each of methanol and benzene. The flask was heated to 38 °C in a water bath under reflux. Then, 2 mL each of methanol and benzene were added dropwise until the precipitate was completely dissolved, forming a saturated solution. Finally, the solution was cooled at 50 °C for 2 h, and purplish-red crystals precipitated. Anhydrous ethanol was then added and stirred. After stirring, the solution was allowed to stand for 2 h, filtered, and dried to obtain the final product, which is the tetraphenylporphyrin cobalt material containing chlorate.

[0064] Step 3: Preparation of a fluorescent sensor for the detection of N-nitrosodimethylamine:

[0065] The upconversion nanomaterials prepared in step one were dissolved in dichloromethane to obtain an upconversion nanosolution (UCNPs solution) with a concentration of 2.5 mg / mL; then the tetraphenylporphyrin cobalt material containing chlorate prepared in step two was weighed and dissolved in dichloromethane to obtain a tetraphenylporphyrin solution containing chlorate with a concentration of 2 mg / mL.

[0066] Then, 2 mL of a 2.5 mg / mL UCNPs solution was added to the test tube, followed by 2 mL of a 2 mg / mL tetraphenylporphyrin cobalt solution containing chlorate. Then, 5 mL of dichloromethane was added to the test tube using a pipette, and the mixture was sonicated for 5 minutes to mix thoroughly. Finally, an upconversion nanosensor containing porphyrin material for detecting N-nitrosodimethylamine was obtained.

[0067] The fluorescent nanosensor prepared based on this invention is used to detect the content of N-nitrosodimethylamine in food;

[0068] (1) Establishment of the standard curve for the fluorescence detection of N-nitrosodimethylamine:

[0069] The prepared fluorescence sensor was added to 5 mL of dichloromethane solution; then, N-nitrosodimethylamine standard solutions of different concentrations were added to obtain detection solutions of different concentrations. After incubation, the fluorescence intensity signal characteristic values ​​of the detection solutions were measured. Figure 3 A); A standard curve for the detection of N-nitrosodimethylamine content was established by linear fitting of the concentration and fluorescence intensity signal characteristics. Figure 3B) Specifically, N-nitrosodimethylamine solutions of different concentrations (0.5, 5, 20, 40, 70, 90, 120, 150, and 200 ng / mL) were prepared, and 100 μL of each solution was added to the specific detection system. After incubation at 30°C for 2 min, fluorescence signal characteristic values ​​were collected.

[0070] The step of determining the fluorescence intensity signal characteristic value of the detection solution is as follows: the fluorescence intensity value at 450nm under 980nm excitation light is the fluorescence intensity signal characteristic value of the detection solution.

[0071] Figure 3 A standard curve was established for the fluorescence detection of different concentrations of N-nitrosodimethylamine; from Figure 3 It can be seen that the fluorescence signal at 450 nm continuously decreases with the increase of N-nitrosodimethylamine concentration; the standard curve for N-nitrosodimethylamine content detection was obtained by linear fitting: y = -22.24x + 7098.69; where y is the characteristic value of the fluorescence intensity signal, and x is the concentration of N-nitrosodimethylamine (N-nitrosodimethylamine). Figure 3 B), Correlation coefficient R 2 =0.9905, the limit of detection (LOD) is 0.17 ng / mL, and the linear range is 0.5-200 ng / mL.

[0072] (2) Detection of N-nitrosodimethylamine content in ham:

[0073] Weigh 2.0g of ham sample and add it to a 50mL centrifuge tube. Then add 5mL of n-hexane to remove the grease. Next, add 50μL of standard solution to the mixture, and then add 4mL of sodium hydroxide solution and acetonitrile for homogenization, vortexing, and ultrasonic dispersion. Transfer the solution to a centrifuge tube containing 50mg of adsorbent. After centrifugation, retain the supernatant and filter it using a filter membrane. Take 500μL of the filtrate into the sensor for detection. After incubation for 2min, measure the fluorescence signal characteristic value and input it into the standard curve obtained in step (1). Calculate that the content of N-nitrosodimethylamine in the ham is 2.0ng / g, while the detection value of the national standard method is 2.07ng / g. After calculating the P value, it can be concluded that there is no significant difference between the designed fluorescence sensing method and the national standard method.

[0074] Example 3:

[0075] Step 1: Preparation of oleic acid-coated upconversion nanomaterials using a high-temperature pyrolysis method:

[0076] 241.1 mg of yttrium chloride hexahydrate, 77.5 mg of ytterbium chloride hexahydrate, and 19.2 mg of thulium chloride hexahydrate were accurately weighed and ultrasonically dispersed in 10 mL of methanol. 8 mL of oleic acid and 16 mL of 1-octadecene were added, and the mixture was magnetically stirred at 150 °C for 30 min under argon atmosphere to obtain a transparent solution. After cooling to room temperature, a mixed solution containing 100 mg of sodium hydroxide and 148.2 mg of ammonium fluoride (4.0 mmol) dissolved in 10 mL of methanol was added dropwise. The mixture was heated to 70 °C for 30 min to completely evaporate the methanol. Then, it was heated to 100 °C under an argon atmosphere and held for 10-15 min to remove excess methanol and air from the apparatus. The mixture was then magnetically stirred at 300 °C for 60 min. After cooling to room temperature, the upconversion nanoparticles were precipitated by centrifugation and washed three times with a 1:1 volume ratio of ultrapure water and ethanol. The precipitate was dried to obtain pure oleic acid-coated upconversion nanoparticles. Figure 2 Transmission electron microscopy image of the prepared upconversion nanoparticles;

[0077] Step 2: The synthesis method of tetraphenylporphyrin cobalt is as follows:

[0078] First, 100 mg of racemic tetraphenylporphyrin cobalt was ultrasonically dissolved in 100 mL of methanol solution. 5 mL of 0.72 mol / L perchloric acid was added and stirred for 10 h until completely dissolved. Then, the solvent was completely removed in a vacuum drying oven. The remaining solid was recrystallized from methanol and benzene to obtain purplish-red crystals. The specific recrystallization steps are as follows: 2.0 g of the crude product was accurately weighed and added to a round-bottom flask containing 10 mL each of methanol and benzene. The flask was heated to 38 °C in a water bath under reflux. Then, 2 mL each of methanol and benzene were added dropwise until the precipitate was completely dissolved, forming a saturated solution. Finally, the solution was cooled at 50 °C for 2 h, and purplish-red crystals precipitated. Anhydrous ethanol was then added and stirred. After stirring, the solution was allowed to stand for 2 h, filtered, and dried to obtain the final product, which is the tetraphenylporphyrin cobalt material containing chlorate.

[0079] Step 3: Preparation of a fluorescent sensor for the detection of N-nitrosodimethylamine:

[0080] The upconversion nanomaterials prepared in step one were dissolved in dichloromethane to obtain an upconversion nanosolution (UCNPs solution) with a concentration of 2.5 mg / mL; then the tetraphenylporphyrin cobalt material containing chlorate prepared in step two was weighed and dissolved in dichloromethane to obtain a tetraphenylporphyrin solution containing chlorate with a concentration of 2 mg / mL.

[0081] Then, 2 mL of a 2.5 mg / mL UCNPs solution was added to the test tube, followed by 2 mL of a 2 mg / mL tetraphenylporphyrin cobalt solution containing chlorate. Then, 5 mL of dichloromethane was added to the test tube using a pipette, and the mixture was sonicated for 5 minutes to mix thoroughly. Finally, an upconversion nanosensor containing porphyrin material for detecting N-nitrosodimethylamine was obtained.

[0082] The fluorescent nanosensor prepared based on this invention is used to detect the content of N-nitrosodimethylamine in food;

[0083] (1) Establishment of the standard curve for the fluorescence detection of N-nitrosodimethylamine:

[0084] The prepared fluorescence sensor was added to 5 mL of dichloromethane; then, N-nitrosodimethylamine standard solutions of different concentrations were added to obtain detection solutions of different concentrations. After incubation, the fluorescence intensity signal characteristic values ​​of the detection solutions were measured. Figure 3 A); A standard curve for the detection of N-nitrosodimethylamine content was established by linear fitting of the concentration and fluorescence intensity signal characteristics. Figure 3 B) Specifically, N-nitrosodimethylamine solutions of different concentrations (0.5, 5, 20, 40, 70, 90, 120, 150, and 200 ng / mL) were prepared, and 100 μL of each solution was added to the specific detection system. After incubation at 30°C for 2 min, fluorescence signal characteristic values ​​were collected.

[0085] The step of determining the fluorescence intensity signal characteristic value of the detection solution is as follows: the fluorescence intensity value at 450nm under 980nm excitation light is the fluorescence intensity signal characteristic value of the detection solution.

[0086] Figure 3 A standard curve was established for the fluorescence detection of different concentrations of N-nitrosodimethylamine; from Figure 3 It can be seen that the fluorescence signal at 450 nm continuously decreases with the increase of N-nitrosodimethylamine concentration; the standard curve for N-nitrosodimethylamine content detection was obtained by linear fitting: y = -22.24x + 7098.69; where y is the characteristic value of the fluorescence intensity signal, and x is the concentration of N-nitrosodimethylamine (N-nitrosodimethylamine). Figure 3 B), Correlation coefficient R 2 =0.9905, the limit of detection (LOD) is 0.17 ng / mL, and the linear range is 0.5-200 ng / mL.

[0087] (2) Detection of N-nitrosodimethylamine content in sausages:

[0088] Weigh 2.0g of sausage sample and add it to a 50mL centrifuge tube. Then add 5mL of n-hexane to remove the grease. Next, add 50μL of standard solution to the mixture, and then add 4mL of sodium hydroxide solution and acetonitrile for homogenization, vortexing, and ultrasonic dispersion. Transfer the solution to a centrifuge tube containing 50mg of adsorbent. After centrifugation, retain the supernatant and filter it using a filter membrane. Take 500μL of the filtrate into the sensor for detection. After incubation for 2min, measure the fluorescence signal characteristic value and input it into the standard curve obtained in step (1). Calculate that the content of N-nitrosodimethylamine in the sausage is 2.4ng / g, while the detection value of the national standard method is 2.23ng / g. After calculating the P value, it can be concluded that there is no significant difference between the designed fluorescence sensing method and the national standard method.

[0089] Specificity of the detection method: In order to evaluate the specificity of the constructed fluorescent sensor for the detection of N-nitrosodimethylamine, other nitrosamines, including N-nitrosodiethylamine, N-nitrosodipropylamine, N-nitrosodiphenylamine, and N-nitrosopyrrolidine, were selected as interfering substances to further study the fluorescence characteristics of the constructed fluorescent sensor, taking the sensor prepared in Example 1 as an example.

[0090] The results are as follows Figure 4 As shown, the fluorescence response efficiency of the prepared sensor to N-nitrosodimethylamine is significantly higher than that of other types of nitrosamines, indicating that the constructed fluorescence sensor has good specificity.

[0091] 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 the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that modifications or equivalent substitutions can still be made to the present invention. All technical solutions and improvements that do not depart from the spirit and scope of the present invention should be covered within the scope of the claims of the present invention.

Claims

1. A method for fabricating a metalloporphyrin-based upconversion fluorescence sensor, characterized in that, Includes the following steps: Step 1: Yttrium chloride hexahydrate, ytterbium chloride hexahydrate, and thulium chloride hexahydrate are dissolved in methanol A. Oleic acid and 1-octadecene are then added. Under argon protection, the mixture is heated and stirred for the first time. After the reaction, it is cooled to room temperature to obtain a coolant. Ammonium fluoride, sodium hydroxide, and methanol B are mixed and added to the coolant. The mixture is heated to a certain temperature for the second reaction. After the reaction, argon gas is introduced, and the temperature is raised to a certain temperature for the third heating reaction. After the third heating reaction, under argon protection, the mixture is heated and stirred for the fourth time at a certain temperature. After the reaction, it is cooled to room temperature to obtain the reaction product. Finally, the reaction product is washed with a mixture of ultrapure water and ethanol, and then vacuum dried to obtain oleic acid-coated upconversion nanoparticles. The ratio of yttrium chloride hexahydrate, ytterbium chloride hexahydrate, gadolinium chloride hexahydrate, methanol A, oleic acid, and 1-octadecene is 241.1 mg: 77.5 mg: 19.2 mg: 10 mL: 8.0 mL: 16.0 mL; the ratio of methanol A to ammonium fluoride, sodium hydroxide, and methanol B in the coolant is 10 mL: 148.2 mg: 100 mg: 10 mL. The first heating and stirring reaction is carried out at a temperature of 150-170 ℃ for 30-40 min; the second reaction is carried out at a temperature of 70-80 ℃ for 30 min; the third heating reaction is carried out in a 100 ℃ water bath for 10-15 min; and the fourth heating and stirring reaction is carried out at a temperature of 295-305 ℃ for 60-70 min. Step two: First, meso-tetraphenylporphyrin cobalt is dissolved in methanol A and sonicated until completely dissolved. Then, perchloric acid is added and stirred for the first time. After stirring, the mixture is dried under reduced pressure in a vacuum drying oven to remove the solvent. Methanol B and benzene A are added to the remaining solid and stirred for the second time until dissolved. Then, the mixture is refluxed in a water bath to obtain a crystalline product. Methanol C and benzene B are added dropwise to the crystalline product until completely dissolved to form a saturated solution. Then, the solution is cooled at a certain temperature. After purple-red crystals precipitate in the solution, anhydrous ethanol is added and stirred for the third time. After stirring and standing for a period of time, the final product is obtained by filtration and drying, which is the tetraphenylporphyrin cobalt material containing chlorate. Step 3: Dissolve the upconversion nanomaterial prepared in Step 1 in dichloromethane A to obtain an upconversion nanosolution; then weigh the tetraphenylporphyrin cobalt material containing chlorate prepared in Step 2 and dissolve it in dichloromethane B to obtain a tetraphenylporphyrin solution containing chlorate. Then, a tetraphenylporphyrin cobalt solution containing chlorate and dichloromethane C were added to the upconversion nanosolution, and after ultrasonic treatment, a metalporphyrin-based upconversion fluorescent sensor was obtained. The concentration of the upconversion nanomaterial solution is 2.5 mg / mL, and the concentration of the tetraphenylporphyrin cobalt solution containing chlorate is 2 mg / mL; the volume ratio of the upconversion nanomaterial solution, the tetraphenylporphyrin cobalt solution containing chlorate, and dichloromethane C is 2:2:5; the ultrasonic treatment time is 5-10 min.

2. The method for fabricating a metalloporphyrin-based upconversion fluorescence sensor according to claim 1, characterized in that, In step one, the volume ratio of ultrapure water to ethanol is 1:1 when washing the reaction product.

3. The method for preparing a metalloporphyrin-based upconversion fluorescence sensor according to claim 1, characterized in that, In step two, the ratio of the amounts of meso-tetraphenylporphyrin cobalt, methanol A, perchloric acid solution, methanol B, benzene A, methanol C, benzene B and anhydrous ethanol is 100 mg: 100 mL: 5 mL: 10 mL: 10 mL: 2 mL: 2 mL: 2 mL; and the concentration of the perchloric acid is 0.72 mol / L.

4. The method for fabricating a metalloporphyrin-based upconversion fluorescence sensor according to claim 1, characterized in that, In step two, the time for complete sonication is 5-10 min; the time for the first stirring reaction is 9-10 h, and the rotation speed is 1500 rpm / min; the time for the second stirring reaction is 30 min, and the rotation speed is 500 rpm / min; the time for the third stirring reaction is 10-15 min, and the rotation speed is 500 rpm / min. The vacuum drying oven has a temperature of 80℃ and a vacuum strength of -0.085 MPa; the reflux treatment temperature in the water bath is 35-40℃; the cooling temperature is 50℃ and the cooling time is 2 hours; and the standing time is 2-3 hours.

5. The use of the metalloporphyrin-based upconversion fluorescence sensor prepared according to any one of claims 1-4 for detecting the content of N-nitrosodimethylamine in meat products, characterized in that, The specific testing steps are as follows: (1) Establishment of the standard curve for N-nitrosodimethylamine content: Dichloromethane and N-nitrosodimethylamine standard solutions of different gradient concentrations were added to the prepared upconversion nanosensor. After incubation at a certain temperature for a period of time, a detection solution was obtained. Then, the fluorescence intensity signal characteristic value of the detection solution was measured, and a standard curve of N-nitrosodimethylamine concentration and its corresponding fluorescence intensity signal characteristic value was constructed. (2) Detection of N-nitrosodimethylamine content in food samples: After food pretreatment, extract the sample liquid and then operate according to step (1). The difference is that the N-nitrosodimethylamine standard solution is replaced with the extracted sample liquid. Then measure the fluorescence intensity signal characteristic value and input it into the standard curve of step (1) to calculate the N-nitrosodimethylamine content in the food.

6. The use according to claim 5, characterized in that, The concentration of the N-nitrosodimethylamine standard solution in step (1) is 0.5-200 ng / mL; the ratio of the amount of the upconversion nanosensor, dichloromethane and N-nitrosodimethylamine standard solution is 4 mL: 5 mL: 100 mL; the incubation time is 2 min and the incubation temperature is 30 ℃; the fluorescence intensity signal characteristic value determination step of N-nitrosodimethylamine is as follows: the fluorescence value at 450 nm under 980 nm excitation light is used as the fluorescence intensity signal characteristic value.

7. The use according to claim 5, characterized in that, The food pretreatment steps in step (2) are as follows: Weigh a certain amount of sample, add it to a centrifuge tube, and add n-hexane to remove oil; then add a certain amount of standard solution, sodium hydroxide solution and acetonitrile for homogenization, and perform vortex ultrasonic treatment; finally add an adsorbent, mix and centrifuge to obtain supernatant, and filter the supernatant with a filter membrane to obtain sample solution. The ratio of sample, n-hexane, standard solution, sodium hydroxide solution, acetonitrile, and adsorbent was 2 g: 5 mL: 50 mL: 4 mL: 6 mL: 50 mg; the concentration of sodium hydroxide solution was 1 mmol / L; the standard solution was an internal standard solution of N-nitrosodimethylamine with a concentration of 0.1 mg / mL; the centrifugation speed was 12000 rpm / min for 3 min; and the pore size of the filter membrane was 0.22 mm.

8. The use according to claim 5, characterized in that, The food items mentioned include smoked meat, ham, and sausage.

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