A method for the preparation of a solid phase biosensor and its use for the detection of nitrosamines

Abstract

The application belongs to the field of food safety detection, and particularly relates to a preparation method of a solid-phase biosensor and application of the solid-phase biosensor to nitrosamine detection; the steps are as follows: step one, preparation of up-conversion nanometer material and carboxyl modification on the surface; step two, preparation of bio-functionalized up-conversion nanometer material; step three, preparation of a bio-solid-phase biosensor convenient for on-site detection; the solid-phase biosensor is applied to rapid detection of nitrosamine, a standard curve is constructed by determination of the standard nitrosamine content, and detection of the nitrosamine content in food is realized; the application realizes rapid, economical and accurate detection of nitrosamine in food through preparation of a solid-phase fluorescence sensor; meanwhile, the designed solid-phase biosensor has the ability of on-site detection of nitrosamine, provides a method for real-time monitoring of nitrosamine in the food processing process, and has a good practical prospect.

Description

Technical Field

[0001] This invention belongs to the field of food safety testing, specifically relating to a method for preparing a solid-phase biosensor and its application in the detection of nitrosamines. Background Technology

[0002] Cured meat products satisfy people's pursuit of health while also meeting popular taste preferences, resulting in a high market share and promising future. However, several issues remain regarding their processing and consumption. The curing process typically relies on adding nitrites and nitrates to enhance color and inhibit bacterial growth. However, during processing, these nitrites and nitrates can react with proteins and biogenic amines, leading to the formation of carcinogenic nitrosamines, threatening public health. Most of these highly carcinogenic nitrosamines are volatile, with dimethylnitrosamine (DIMA) posing a greater threat and attracting more research from domestic and international scholars. Therefore, researching rapid and sensitive quantitative detection of DIMA during cured meat processing is crucial for meeting the needs of curing technology development and ensuring the rapid development of cured meat products in my country.

[0003] Domestic and international research has shown that the main methods for detecting volatile nitrosamines include analytical chemical chromatography and spectrophotometry. However, chromatographic methods are expensive and require professional operators, while spectrophotometry is susceptible to interference from other ions affecting nitrosamines. Therefore, it is essential to develop a rapid, highly sensitive, and easily on-site method for detecting nitrosamines. Summary of the Invention

[0004] To address the shortcomings of existing technologies, this invention provides a method for preparing a solid-phase biosensor and its application in detecting nitrosamines in food, thereby solving the problems of high detection costs, cumbersome detection steps, and unsuitability for on-site detection in the aforementioned existing technologies.

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

[0006] First, a method for preparing a solid-phase biosensor is provided:

[0007] Step 1: Preparation of water-soluble upconversion nanomaterials: Yttrium chloride hexahydrate, ytterbium chloride hexahydrate, and thulium chloride hexahydrate are dissolved in methanol A. Then, a certain proportion of oleic acid and 1-octadecene are added for the first heating and stirring. After stirring, the mixture is cooled to room temperature. Then, methanol B containing sodium hydroxide and ammonium fluoride is added for the second heating and stirring. After stirring, the mixture is cooled to room temperature. The precipitate is obtained by centrifugation, washed with a mixture of ultrapure water and ethanol, and dried to obtain oleic acid-coated upconversion nanomaterials for later use.

[0008] Weigh the dried oleic acid-coated upconversion nanomaterials and add them to toluene and chloroform for ultrasonic dissolution. Then add an ultrapure aqueous solution containing polyacrylic acid and stir for a third time. After stirring, centrifuge to collect the precipitate and wash it with ethanol to obtain a solid, which is the water-soluble upconversion nanomaterial. Finally, dissolve it in ultrapure water to obtain a water-soluble upconversion nanomaterial solution.

[0009] Preferably, in step one, the amounts of yttrium chloride hexahydrate, ytterbium chloride hexahydrate, thulium chloride hexahydrate, methanol A, oleic acid, 1-octadecene, sodium hydroxide, ammonium fluoride, and methanol B are in the following ratio: 120.6 mg: 38.8 mg: 0.7 mg: 10 mL: 4.0 mL: 9.0 mL: 50.0 mg: 74.1 mg: 10 mL; the amounts of the oleic acid-coated upconversion nanomaterial, toluene, chloroform, polyacrylic acid, and ultrapure water are in the following ratio: 50 mg: 6.0 mL: 4.0 mL: 300 mg: 20 mL; and the washing is performed using a mixture of ultrapure water and ethanol, wherein the volume ratio of ultrapure water to ethanol is 1:3.

[0010] Preferably, in step one, the first heating and stirring time is 20-30 min, and the heating temperature is 160℃; the second heating and stirring temperature is 295-305℃, and the stirring time is 1.0-1.5 h; the third stirring time is 48 h; the ultrasonic dissolution time is 10 min; the centrifugation conditions are: rotation speed of 8000-10000 rpm / min, time of 5-10 min; and the concentration of the water-soluble upconversion nanomaterial solution is 5 mg / mL.

[0011] Step 2, Biofunctionalization of Upconversion Nanomaterials:

[0012] Weigh out the water-soluble upconversion nanomaterial solution prepared in step one and add it to morpholine ethanesulfonic acid solution. Add N-hydroxythiosuccinimide and 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride to react and activate the carboxyl group. After the reaction, collect the precipitate by centrifugation and wash it with phosphate solution A. The washed precipitate is dispersed in phosphate solution B. Then add the amino-modified nitrosamine aptamer complementary chain and incubate. After incubation, centrifuge again. The precipitate is washed with ultrapure water and centrifuged again to obtain the upconversion nanomaterial modified with the nitrosamine aptamer complementary chain. Finally, add it to phosphate solution C to obtain the upconversion nanomaterial solution modified with the nitrosamine aptamer complementary chain.

[0013] Preferably, in step two, the amounts of the water-soluble upconversion nanomaterial solution, morpholine ethanesulfonic acid solution, N-hydroxythiosuccinimide, and 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride are in the following ratio: 1.0 mL: 1.0 mL: 1.0 mg: 0.5 mg; the amounts of the water-soluble upconversion nanomaterial solution, phosphate solution A, phosphate solution B, aminated nitrosamine aptamer complementary chain, and phosphate solution C are in the following ratio: 1.0 mL: 10 mL: 2.0 mL: 1.2 mL: 10 mL; the concentration of the morpholine ethanesulfonic acid solution is 1.0 mol / L, and the concentration of the aminated nitrosamine aptamer complementary chain is 0.1 mmol / L.

[0014] The mixing reaction was carried out at a carboxyl activation temperature of 4°C for 2 hours. The incubation was conducted on a shaker at a temperature of 37°C for 2 hours at a speed of 200 rpm / min. The centrifugation conditions were as follows: a speed of 6000-80000 rpm / min for 5-10 minutes. The concentration of the upconversion nanomaterial solution modified with the nitrosamine aptamer complementary chain was 0.5 mg / mL.

[0015] Step 3, Fabrication of solid-phase biosensors:

[0016] First, a 4-(4-dimethylaminoazo)benzoic acid-labeled nitrosamine aptamer solution was added to the upconversion nanomaterial solution modified with the complementary chain of nitrosamine aptamer prepared in step two. After mixing, the mixture was heated in a water bath for the first time. After the reaction, it was cooled to a certain temperature and stirred for a period of time. After stirring, the precipitate was collected by centrifugation. The precipitate was then washed with ultrapure water and redispersed in ultrapure water to obtain a biomolecule-modified upconversion nanomaterial solution.

[0017] The prepared biomolecule-modified upconversion nanomaterial solution was mixed with zein, and a certain amount of acetic acid and dimethyl sulfoxide were added and stirred for a period of time to prepare a spinning solution. The solid-phase biosensor was prepared by spinning using an electrospinning instrument under constant voltage, temperature, relative humidity and roller speed conditions.

[0018] Preferably, in step three, the ratio of the upconversion nanomaterial solution modified with the complementary chain of the nitrosamine aptamer to the nitrosamine aptamer solution labeled with 4-(4-dimethylaminoazo)benzoic acid is 5.0 mL: 300 μL.

[0019] The ratio of biomolecule-modified upconversion nanomaterial solution, zein, acetic acid, and dimethyl sulfoxide is 5.0 mL: 7.0 mg: 20 mL: 1.0 mL; the concentration of the 4-(4-dimethylaminoazo)benzoic acid-labeled nitrosamine aptamer solution is 0.1 mmol / L, and the concentration of the biomolecule-modified upconversion nanomaterial solution is 2.0 mg / mL.

[0020] Preferably, in step three, the temperature of the first heating reaction in the water bath is 95°C, and the reaction time is 3 min; after cooling, the temperature is 37°C, and the stirring time is 0.5 h; the voltage of electrospinning is 20 kV, the temperature is 37°C, the relative humidity is 50%, the roller speed is 0.6 mL / h, and the stirring time is 2 h.

[0021] This invention also provides an application of a solid-phase biosensor for the detection of nitrosamines in food, the steps of which are as follows:

[0022] (1) Establishment of standard curve for nitrosamine content: The solid-phase biosensor and standard solutions of nitrosamines of different concentrations were placed together in a closed reaction box. Different concentrations of nitrosamine corresponded to different solid-phase biosensors. After incubation at a certain temperature, the fluorescence intensity signal value of the solid-phase biosensor was detected. By fitting the fluorescence signal intensity with the corresponding nitrosamine concentration, a standard curve for nitrosamine detection was established.

[0023] (2) Detection of nitrosamine content in food samples:

[0024] The solid-phase biosensor and the sample to be tested are placed in a closed reaction box and incubated under certain temperature conditions. The fluorescence intensity signal value of the solid-phase biosensor is then detected. By substituting the obtained fluorescence signal value into the standard curve in step (1), the content of nitrosamines in the sample to be tested can be detected.

[0025] Preferably, the concentration range of the standard solution of nitrosamine in step (1) is 0.05 ng / mL to 500 ng / mL;

[0026] Preferably, the incubation time after the addition of nitrosamine in steps (1)-(2) is 3 min and the incubation temperature is 45℃; the detection of the fluorescence intensity signal value of the solid-phase biosensor is specifically: the fluorescence value of the solid-phase biosensor under the excitation of a 980nm exciter is measured as the signal characteristic value.

[0027] Preferably, the sample to be tested in step (2) includes cured meat, ham, and sausage; the pretreatment method for the sample to be tested is to divide the sample into thin slices with a thickness of 0.2cm and a weight of 10g±0.2g.

[0028] The present invention discloses the following technical effects:

[0029] 1. This invention discloses a method for preparing a solid-phase biosensor and its application in the detection of nitrosamines. It explores a new method for fluorescence sensing detection of dimethyl nitrosamines during the processing of cured meat products. The main focus is on overcoming the challenge of controllable preparation of solid-phase sensors, and realizing rapid and real-time detection of volatile nitrosamines during the processing of cured meat products using solid-phase sensors.

[0030] 2. This invention uses a glutaraldehyde cross-linking method to bind the complementary sequence of an aptamer to an upconversion nanomaterial, and then uses a nitrosodimethylamine aptamer containing 4-(4-dimethylaminoazo)benzoic acid at the aptamer terminal to bind the complementary sequence of the aptamer through complementary base pairing. The upconversion nanomaterial acts as a donor for fluorescence resonance energy transfer, and 4-(4-dimethylaminoazo)benzoic acid acts as an acceptor for fluorescence energy.

[0031] 3. The specific detection system constructed in this invention exhibits a high fluorescence response to nitrosodimethylamine through optimized aptamer concentration and fluorescence resonance energy transfer between upconversion nanomaterials. By linking biorecognition molecules, it effectively overcomes interference from other homologous target compounds, improving detection sensitivity and specificity. This overcomes the shortcomings of traditional methods and safeguards public food health and safety.

[0032] 4. The solid-phase sensing detection method for nitrosamines established in this invention has a linear concentration range of fluorescence intensity signal characteristic values ​​of 0.05-500 ng / mL, exhibiting a wide linear detection range and a detection limit (LOD) of 0.017 ng / mL. The designed solid-phase sensing method can meet the high-sensitivity detection requirements of nitrosamine content in food, possesses good versatility, and provides a theoretical basis for real-time monitoring of nitrosamines in practical detection processes. Attached Figure Description

[0033] Figure 1 A schematic diagram illustrating the principle of constructing a solid-phase biosensor for nitrosamine detection.

[0034] Figure 2 The image shows a transmission electron microscope (TEM) image of the upconversion nanoparticles prepared in Example 1.

[0035] Figure 3 Scanning electron microscope image of the upconversion luminescent solid-phase biosensor prepared in Example 1.

[0036] Figure 4 The fluorescence standard curves established for detecting different concentrations of nitrosamines in Example 1 are shown below; where A is a fluorescence signal diagram of the sensor for detecting different concentrations of nitrosamines; and B is a standard curve established using the nitrosamine concentration and the characteristic value of the fluorescence intensity signal of the sensor at 450 nm.

[0037] Figure 5 For the specific analysis of solid-phase biosensors. 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 nitrosamines and zein used in this invention were purchased from Shanghai Maclean's Reagent Co., Ltd., and the nitrosamine aptamer complementary sequence solution and nitrosamine aptamer solution were purchased from Sangon Biotech (Shanghai) Co., Ltd.

[0043] Figure 1 A schematic diagram illustrating the principle of constructing a solid-phase biosensor for nitrosamine detection; detailed steps are provided in the embodiments.

[0044] Example 1:

[0045] Oleic acid-coated upconversion nanomaterials were prepared by high-temperature pyrolysis: 120.6 mg of yttrium chloride hexahydrate, 38.8 mg of ytterbium chloride hexahydrate, and 0.7 mg of thulium chloride hexahydrate were accurately weighed and ultrasonically dispersed in 10 mL of methanol. 4.0 mL of oleic acid and 9.0 mL of 1-octadecene were added, and the mixture was magnetically stirred at 160 °C for 30 min under argon atmosphere to obtain a transparent solution. After cooling to room temperature, a mixed solution containing 50 mg of sodium hydroxide and 74.1 mg of ammonium fluoride dissolved in 10 mL of methanol was added dropwise. The flask was sealed and heated at 70 °C for 40 min to evaporate the methanol. Then, the mixture was heated to 100 °C under an argon atmosphere and held for 10 min to remove excess methanol and air from the apparatus. The mixture was then magnetically stirred at 300 °C for 1 h. After cooling to room temperature, the upconversion nanoparticles were precipitated by centrifugation and washed three times with a 1:3 volume ratio of ultrapure water and ethanol. The precipitate was then dried to obtain pure oleic acid-coated upconversion nanoparticles.

[0046] To meet the requirements of biosensors, the synthesized oleic acid-coated upconversion nanoparticles should be modified into water-soluble upconversion nanoparticles. The procedure is as follows: Weigh 50.0 mg of oleic acid-coated upconversion nanoparticles, add 6.0 mL of toluene and 4.0 mL of chloroform, and sonicate for 10 min; then add 20.0 mL of ultrapure water containing 300.0 mg of polyacrylic acid and stir vigorously for 48 h. Next, centrifuge the resulting solution at 10000 rpm / min for 5 min, wash and remove excess polyacrylic acid to obtain water-soluble upconversion nanoparticles. Finally, dissolve the water-soluble upconversion nanoparticles in water to obtain a water-soluble upconversion nanomaterial solution with a concentration of 5 mg / mL.

[0047] Biofunctionalization of upconversion nanomaterials: 1.0 mL of upconversion nanoparticle solution was added to a centrifuge tube containing 1.0 mL of morpholine ethanesulfonic acid solution (concentration 1.0 mol / L). Then, N-hydroxythiosuccinimide (1.0 mg) and 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride (0.5 mg) were added to the mixture and mixed at 4 °C for 2 h to activate the carboxyl groups on the surface of the nanoparticles. The mixed solution was then centrifuged and washed with phosphate buffer at 6000 rpm / min for 5 min. The precipitate was collected and dispersed in 2.0 mL of phosphate buffer, followed by the addition of 1.2 mL of amino-modified nitrosodimethylamine aptamer complementary chain. The mixture was incubated at 37 °C for 2 h on a shaker at 200 rpm / min. After incubation, the mixture was centrifuged again to obtain upconversion nanomaterials modified with nitrosamine aptamer complementary chains. These nanomaterials were then added to phosphate buffer to obtain a solution of upconversion nanomaterials modified with nitrosamine aptamer complementary chains at a concentration of 0.5 mg / mL.

[0048] First, 5.0 mL of upconversion nanomaterial solution modified with nitrosamine aptamer complementary chain was transferred to a reaction tube, followed by the addition of 300.0 μL of 4-(4-dimethylaminoazo)benzoic acid-labeled nitrosamine aptamer solution to obtain a mixed solution. Then, the mixed solution was heated to 95 °C in a water bath and held for 3.0 min, followed by cooling to 37 °C and stirring for 0.5 h. The precipitate was collected by centrifugation and then washed three times with ultrapure water to obtain biomolecule-modified upconversion nanomaterials. These nanomaterials were then redispersed in ultrapure water to obtain a biomolecule-modified upconversion nanomaterial solution with a concentration of 2.0 mg / mL.

[0049] Figure 2 Transmission electron microscopy image of the biofunctionalized upconversion nanoparticles prepared in Example 1; from Figure 2 It can be seen that the prepared biofunctionalized upconversion nanoparticles have standard morphology and good dispersibility.

[0050] More importantly, zein and upconversion nanomaterials modified with biorecognition molecules are combined to form a membrane. The specific steps are as follows:

[0051] A spinning solution was prepared by dispersing a biomolecule-modified upconversion nanomaterial solution (5.0 mL) and zein (7.0 g) in a mixed solution of acetic acid (20.0 mL) and dimethyl sulfoxide (1.0 mL), and rapidly stirring until electrospinning began. The obtained spinning solution was loaded into a 5.0 mL syringe and placed on the worktable of an electrospinning machine under constant voltage, temperature, humidity, and roller speed (20 kV, 35 °C, 50% relative humidity, 0.6 mL / h). The spun membrane was a zein-upconversion nanomaterial membrane, which is a solid-phase biosensor.

[0052] Figure 3 The image shows a scanning electron microscope (SEM) image of the solid-phase biosensor prepared in Example 1. Figure 3 As can be seen, the prepared solid-phase biosensor has a uniform internal structure.

[0053] Detection of nitrosamine content in cured pork:

[0054] (1) Establishment of the standard curve for fluorescence detection of nitrosodimethylamine:

[0055] Eleven solid-phase biosensors were placed in sealed reaction chambers with corresponding standard solutions of nitrosamine at concentrations of 0.05, 0.1, 0.5, 1.0, 5.0, 10, 50, 100, 150, 200, and 500 ng / mL. After incubation at 45°C for 3 min, the fluorescence intensity signal values ​​on the solid-phase biosensors were measured. Figure 4A); A standard curve for the detection of nitrosodimethylamine content was established by linear fitting of the characteristic values ​​of nitrosamine concentration and fluorescence intensity signals. Figure 4 B);

[0056] 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.

[0057] Figure 4 A standard curve was established for the fluorescence detection of different concentrations of nitrosodimethylamine; from Figure 4 It can be seen that the fluorescence intensity at 450 nm gradually increases with the increase of nitrosodimethylamine concentration, indicating a positive correlation between nitrosodimethylamine concentration and increased fluorescence intensity. A good linear regression equation was obtained through linear fitting: y = 937.9x + 2629.5, with a correlation coefficient R0. 2 The value is 0.9867, with a range of 0.05 ng / mL to 100 ng / mL, which meets the requirements for the detection of nitrosodimethylamine. Here, y represents the upconversion fluorescence intensity at 450 nm and x represents the logarithmic concentration of nitrosodimethylamine.

[0058] (2) Detection of nitrosamine content in cured meat:

[0059] First, the cured meat was pretreated by cutting it into 0.2cm pieces with a weight of about 10g to ensure the accuracy of nitrosodimethylamine detection. Different concentrations of nitrosodimethylamine (0.5μg / kg, 2.5μg / kg, and 5.0μg / kg) were added to the surface of the cured meat samples. After incubation at 45℃ for 3 minutes, the fluorescence intensity signal value on the solid-phase biosensor was detected. The fluorescence signal characteristic value was detected by the solid-phase biosensor method, and the standard curve obtained in step (1) was substituted to calculate the spiked recovery rate. Statistical processing using a t-test showed that the solid-phase biosensor has good practicality. See Table 1 for details.

[0060] Table 1: Detection and recovery rate of dimethylnitrosamine in cured meat samples

[0061]

[0062] RSD: Relative Standard Deviation

[0063] Example 2:

[0064] Oleic acid-coated upconversion nanomaterials were prepared by high-temperature pyrolysis: 120.6 mg of yttrium chloride hexahydrate, 38.8 mg of ytterbium chloride hexahydrate, and 0.7 mg of thulium chloride hexahydrate were accurately weighed and ultrasonically dispersed in 10 mL of methanol. 4.0 mL of oleic acid and 9.0 mL of 1-octadecene were added, and the mixture was magnetically stirred at 160 °C for 30 min under argon atmosphere to obtain a transparent solution. After cooling to room temperature, a mixed solution containing 50 mg of sodium hydroxide and 74.1 mg of ammonium fluoride dissolved in 10 mL of methanol was added dropwise. The flask was then sealed. Methanol was evaporated by heating at 70℃ for 40 min, then heated to 100℃ under an argon atmosphere and held for 10 min to remove excess methanol and air from the apparatus. The mixture was then magnetically stirred at 300℃ for 1 h, cooled to room temperature, and centrifuged to obtain upconversion nanoparticle precipitate. This precipitate was washed three times with a 1:3 volume ratio of ultrapure water and ethanol, and dried to obtain pure oleic acid-coated upconversion nanoparticles. To meet the requirements of biosensors, the synthesized oleic acid-coated upconversion nanoparticles should be modified into water-soluble UCNPs. In short, 50.0 mg of oleic acid-coated upconversion nanoparticles were weighed, and 6.0 mL of toluene and 4.0 mL of chloroform were added, followed by sonication for 10 min. Then, 20.0 mL of ultrapure water containing 300.0 mg of polyacrylic acid was added, and the mixture was stirred vigorously for 48 h. The resulting solution was then centrifuged at 10000 rpm for 5 min, washed to remove excess polyacrylic acid, and finally, the water-soluble upconversion nanoparticles were dissolved in 10.0 mL of water for further use.

[0065] Biofunctionalization of upconversion nanomaterials: 1.0 mL of upconversion nanoparticles were added to a centrifuge tube containing 1.0 mL of morpholine ethanesulfonic acid solution. N-hydroxythiosuccinimide (1.0 mg) and 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride (0.5 mg) were then added to the mixture, and the mixture was stirred at 4 °C for 2 h to activate the carboxyl groups on the nanoparticle surface. The mixture was then centrifuged and washed with phosphate buffer at 6000 rpm / min, and then dispersed in 2.0 mL of phosphate buffer. Subsequently, 1.2 mL of amino-modified nitrosodimethylamine aptamer complementary chain was added, and the mixture was incubated at 37 °C for 2 h on a shaker at a speed of 200 rpm / min. After incubation, the mixture was washed with ultrapure water and centrifuged again to obtain upconversion nanomaterials modified with nitrosodimethylamine aptamer complementary chains. These nanomaterials were then stored in 10 mL of phosphate buffer solution to obtain a solution of upconversion nanomaterials modified with nitrosodimethylamine aptamer complementary chains.

[0066] First, 5.0 mL of upconversion nanomaterial solution modified with complementary chains of nitrosodimethylamine aptamers was transferred to a reaction tube, followed by the addition of 300.0 μL of 4-(4-dimethylaminoazo)benzoic acid-labeled nitrosamine aptamer solution. The mixture was then heated to 95 °C in a water bath and held for 3.0 min before cooling. After stirring at 37 °C for 0.5 h, the mixture was washed three times to obtain functional upconversion nanomaterials, which were then redispersed in ultrapure water to obtain a biomolecularly modified upconversion nanomaterial solution with a concentration of 2.0 mg / mL.

[0067] A membrane was formed by combining zein and upconversion nanomaterials modified with biorecognition molecules. The specific steps are as follows: A spinning solution was prepared by dispersing a solution of biomolecule-modified upconversion nanomaterials (5.0 mL) and zein (7.0 g) in a mixed solution of acetic acid (20.0 mL) and dimethyl sulfoxide (1.0 mL), and rapidly stirring until electrospinning began. The obtained spinning solution was loaded into a 5.0 mL syringe and placed on the worktable of an electrospinning machine under constant voltage, temperature, humidity, and roller speed (20 kV, 35 °C, 50% relative humidity, 0.6 mL / h). The obtained membrane is a zein-upconversion nanomaterial membrane, i.e., a solid-phase biosensor.

[0068] Detection of nitrosodimethylamine content in ham:

[0069] (1) Establishment of the standard curve for fluorescence detection of nitrosodimethylamine:

[0070] The method is the same as in Example 1, using a linear regression equation of y = 937.9x + 2629.5, and a correlation coefficient R0. 2 The value is 0.9867, with a range of 0.05 ng / mL to 100 ng / mL, which meets the requirements for the detection of nitrosodimethylamine; where y represents the upconversion fluorescence intensity at 450 nm and x represents the logarithmic concentration of nitrosodimethylamine.

[0071] (2) Detection of nitrosamine content in ham:

[0072] To ensure the accuracy of nitrosodimethylamine detection, a ham sample of approximately 10g and 0.2cm thickness was cut. Different concentrations of nitrosodimethylamine (0.5μg / kg, 2.5μg / kg, and 5.0μg / kg) were added to the sample surface. After incubation at 45℃ for 3 minutes, the fluorescence intensity signal on the solid-phase biosensor was detected. The signal was then input into the standard curve obtained in step (1), and the spiked recovery rate was calculated. Statistical analysis using a t-test showed that the solid-phase biosensor has good practicality. See Table 2 for details.

[0073] Table 2: Spiking recovery rate of dimethylnitrosamine in ham samples

[0074]

[0075] RSD: Relative Standard Deviation

[0076] Example 3:

[0077] Oleic acid-coated upconversion nanomaterials were prepared by high-temperature pyrolysis: 120.6 mg of yttrium chloride hexahydrate, 38.8 mg of ytterbium chloride hexahydrate, and 0.7 mg of thulium chloride hexahydrate were accurately weighed and ultrasonically dispersed in 10 mL of methanol. 4.0 mL of oleic acid and 9.0 mL of 1-octadecene were added, and the mixture was magnetically stirred at 160 °C for 30 min under argon atmosphere to obtain a transparent solution. After cooling to room temperature, a mixed solution containing 50 mg of sodium hydroxide and 74.1 mg of ammonium fluoride dissolved in 10 mL of methanol was added dropwise. The flask was then sealed. Methanol was evaporated by heating at 70℃ for 40 min, then heated to 100℃ under an argon atmosphere and held for 10 min to remove excess methanol and air from the apparatus. The mixture was then magnetically stirred at 300℃ for 1 h, cooled to room temperature, and centrifuged to obtain upconversion nanoparticle precipitate. This precipitate was washed three times with a 1:3 volume ratio of ultrapure water and ethanol, and dried to obtain pure oleic acid-coated upconversion nanoparticles. To meet the requirements of biosensors, the synthesized oleic acid-coated upconversion nanoparticles should be modified into water-soluble UCNPs. In short, 50.0 mg of oleic acid-coated upconversion nanoparticles were weighed, and 6.0 mL of toluene and 4.0 mL of chloroform were added, followed by sonication for 10 min. Then, 20.0 mL of ultrapure water containing 300.0 mg of polyacrylic acid was added, and the mixture was stirred vigorously for 48 h. The resulting solution was then centrifuged at 10000 rpm for 5 min, washed to remove excess polyacrylic acid, and finally, the water-soluble upconversion nanoparticles were dissolved in 10.0 mL of water for further use.

[0078] Biofunctionalization of upconversion nanomaterials: 1.0 mL of upconversion nanoparticles were added to a centrifuge tube containing 1.0 mL of morpholine ethanesulfonic acid solution. N-hydroxythiosuccinimide (1.0 mg) and 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride (0.5 mg) were then added to the mixture, and the mixture was stirred at 4 °C for 2 h to activate the carboxyl groups on the nanoparticle surface. The mixture was then centrifuged and washed with phosphate buffer at 6000 rpm / min, and then dispersed in 2.0 mL of phosphate buffer. Subsequently, 1.2 mL of an amino-modified nitrosodimethylamine aptamer complementary chain was added, and the mixture was incubated at 37 °C for 2 h on a shaker at 200 rpm / min. After incubation, the mixture was washed with ultrapure water and stored in 10 mL of phosphate buffer to obtain an upconversion nanomaterial solution modified with a nitrosodimethylamine aptamer complementary chain.

[0079] First, 5.0 mL of upconversion nanomaterial solution modified with the complementary chain of nitrosodimethylamine aptamer was transferred to a reaction tube, followed by the addition of 300.0 μL of 4-(4-dimethylaminoazo)benzoic acid-labeled nitrosamine aptamer solution. The mixture was then heated to 95 °C in a water bath and held for 3.0 min, followed by cooling. After stirring at 37 °C for 0.5 h, the mixture was washed three times to obtain functional upconversion nanomaterials, which were then redispersed in ultrapure water to obtain a biomolecularly modified upconversion nanomaterial solution with a concentration of 2.0 mg / mL.

[0080] A membrane was formed by combining zein and upconversion nanomaterials modified with biorecognition molecules. The specific steps are as follows: A spinning solution was prepared by dispersing a solution of biomolecule-modified upconversion nanomaterials (5.0 mL) and zein (7.0 g) in a mixed solution of acetic acid (20.0 mL) and dimethyl sulfoxide (1.0 mL), and rapidly stirring until electrospinning began. The obtained spinning solution was loaded into a 5.0 mL syringe and placed on the worktable of an electrospinning machine under constant voltage, temperature, humidity, and roller speed (20 kV, 35 °C, 50% relative humidity, 0.6 mL / h). The obtained membrane is a zein-upconversion nanomaterial membrane, i.e., a solid-phase biosensor.

[0081] Detection of nitrosodimethylamine content in sausages:

[0082] (1) Establishment of the standard curve for fluorescence detection of nitrosodimethylamine:

[0083] The method is the same as in Example 1, using a linear regression equation of y = 937.9x + 2629.5, and a correlation coefficient R0. 2 The value is 0.9867, with a range of 0.05 ng / mL to 100 ng / mL, which meets the requirements for the detection of nitrosodimethylamine; where y represents the upconversion fluorescence intensity at 450 nm and x represents the logarithmic concentration of nitrosodimethylamine.

[0084] (2) Detection of nitrosamine content in sausages:

[0085] To ensure the accuracy of nitrosodimethylamine detection, sausage samples with a thickness of 0.2 cm and a weight of approximately 10 g were cut. Different concentrations of nitrosodimethylamine (0.5 μg / kg, 2.5 μg / kg, and 5.0 μg / kg) were added to the sample surface. After incubation at 45℃ for 3 min, the fluorescence intensity signal value on the solid-phase biosensor was detected. The result was substituted into the standard curve obtained in step (1), and the spiked recovery rate was calculated. Statistical analysis using a t-test showed that the solid-phase biosensor has good practicality. See Table 3 for details.

[0086] Table 3: Spiking recovery rate of dimethylnitrosamine in sausage samples

[0087]

[0088] RSD: Relative Standard Deviation

[0089] Example 4:

[0090] Specificity of the detection method: In order to evaluate the specificity of the constructed solid-phase biosensor for the detection of nitrosamines, this invention selects other structural analogs, including nitrosodimethylamine, nitrosodiethylamine, nitrosodipropylamine, nitrosopyrrolidine, and nitrosodiphenylamine, as interfering ions to further study the fluorescence characteristics of the solid-phase biosensor.

[0091] The results are as follows Figure 5 As shown, the fluorescence response efficiency of the prepared sensor to nitrosamine is significantly higher than that of other ions, indicating that the constructed solid-phase biosensor has good specificity.

[0092] 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 preparing a solid-phase biosensor, characterized in that, Includes the following steps: Step 1: Preparation of water-soluble upconversion nanomaterials: Yttrium chloride hexahydrate, ytterbium chloride hexahydrate, and thulium chloride hexahydrate are dissolved in methanol A. Then, a certain proportion of oleic acid and 1-octadecene are added for the first heating and stirring. After stirring, the mixture is cooled to room temperature. Then, methanol B containing sodium hydroxide and ammonium fluoride is added for the second heating and stirring. After stirring, the mixture is cooled to room temperature. The precipitate is obtained by centrifugation, washed with a mixture of ultrapure water and ethanol, and dried to obtain oleic acid-coated upconversion nanomaterials for later use. Weigh the dried oleic acid-coated upconversion nanomaterials and add them to toluene and chloroform for ultrasonic dissolution. Then add an ultrapure aqueous solution containing polyacrylic acid and stir for a third time. After stirring, centrifuge to collect the precipitate and wash it with ethanol to obtain a solid, which is the water-soluble upconversion nanomaterial. Finally, dissolve it in ultrapure water to obtain a water-soluble upconversion nanomaterial solution. Step 2, Biofunctionalization of Upconversion Nanomaterials: Weigh out the water-soluble upconversion nanomaterial solution prepared in step one and add it to morpholine ethanesulfonic acid solution. Add N-hydroxythiosuccinimide and 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride to react and activate the carboxyl group. After the reaction, centrifuge to collect the precipitate and wash it with phosphate solution A. The washed precipitate is dispersed in phosphate solution B. Then add the amino-modified nitrosamine aptamer complementary chain and incubate. After incubation, centrifuge again. The precipitate is washed with ultrapure water and centrifuged again to obtain the upconversion nanomaterial modified with the nitrosamine aptamer complementary chain. Finally, add it to phosphate solution C to obtain the upconversion nanomaterial solution modified with the nitrosamine aptamer complementary chain. Step 3, Fabrication of solid-phase biosensors: First, a 4-(4-dimethylaminoazo)benzoic acid-labeled nitrosamine aptamer solution was added to the upconversion nanomaterial solution modified with the complementary chain of nitrosamine aptamer prepared in step two. After mixing, the mixture was heated in a water bath for the first time. After the reaction, it was cooled to a certain temperature and stirred for a period of time. After stirring, the precipitate was collected by centrifugation. The precipitate was then washed with ultrapure water and redispersed in ultrapure water to obtain a biomolecule-modified upconversion nanomaterial solution. The prepared biomolecule-modified upconversion nanomaterial solution was mixed with zein, and a certain amount of acetic acid and dimethyl sulfoxide were added and stirred for a period of time to prepare a spinning solution. The solid-phase biosensor was prepared by spinning using an electrospinning instrument under constant voltage, temperature, relative humidity and roller speed conditions.

2. The method for preparing a solid-phase biosensor according to claim 1, characterized in that, The dosage relationships of yttrium chloride hexahydrate, ytterbium chloride hexahydrate, thulium chloride hexahydrate, methanol A, oleic acid, 1-octadecene, sodium hydroxide, ammonium fluoride, and methanol B in step one are 120.6 mg: 38.8 mg: 0.7 mg: 10 mL: 4.0 mL: 9.0 mL: 50.0 mg: 74.1 mg: 10 mL; the dosage relationships of the oleic acid-coated upconversion nanomaterial, toluene, chloroform, polyacrylic acid, and ultrapure aqueous solution containing polyacrylic acid are 50 mg: 6.0 mL: 4.0 mL: 300 mg: 20 mL; the washing is performed using a mixture of ultrapure water and ethanol, wherein the volume ratio of ultrapure water to ethanol is 1:

3.

3. The method for preparing a solid-phase biosensor according to claim 1, characterized in that, In step one, the first heating and stirring time is 20-30 min, and the heating temperature is 160 ℃; the second heating and stirring temperature is 295-305 ℃, and the stirring time is 1.0-1.5 h; the third stirring time is 48 h; the ultrasonic dissolution time is 10 min; the centrifugation conditions are: rotation speed of 8000 rpm / min, time of 5-10 min; and the concentration of the water-soluble upconversion nanomaterial solution is 5 mg / mL.

4. The method for preparing a solid-phase biosensor according to claim 1, characterized in that, In step two, the amounts of the water-soluble upconversion nanomaterial solution, morpholine ethanesulfonic acid solution, N-hydroxythiosuccinimide, and 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride are in the following ratio: 1.0 mL: 1.0 mL: 1.0 mg: 0.5 mg; the amounts of the water-soluble upconversion nanomaterial solution, phosphate solution A, phosphate solution B, amino-modified nitrosamine aptamer complementary chain, and phosphate solution C are in the following ratio: 1.0 mL: 10 mL: 2.0 mL: 1.2 mL: 10 mL; the concentration of the morpholine ethanesulfonic acid solution is 1.0 mol / L, and the concentration of the amino-modified nitrosamine aptamer complementary chain is 0.1 mmol / L. The mixing reaction was carried out at a carboxyl activation temperature of 4 °C for 2 h. The incubation was conducted on a shaker at a temperature of 37 °C for 2 h at a speed of 200 rpm / min. The centrifugation conditions were as follows: speed of 6000-80000 rpm / min for 5-10 min. The concentration of the upconversion nanomaterial solution modified with the complementary chain of the nitrosamine aptamer was 0.5 mg / mL.

5. A method for preparing a solid-phase biosensor according to claim 1, characterized in that, In step three, the ratio of the upconversion nanomaterial solution modified with the complementary chain of nitrosamine aptamer to the nitrosamine aptamer solution labeled with 4-(4-dimethylaminoazo)benzoic acid is 5.0 mL: 300 mL. The ratio of biomolecule-modified upconversion nanomaterial solution, zein, acetic acid, and dimethyl sulfoxide is 5.0 mL: 7.0 mg: 20 mL: 1.0 mL; the concentration of the 4-(4-dimethylaminoazo)benzoic acid-labeled nitrosamine aptamer solution is 0.1 mmol / L, and the concentration of the biomolecule-modified upconversion nanomaterial solution is 2.0 mg / mL.

6. A method for preparing a solid-phase biosensor according to claim 1, characterized in that, In step three, the temperature of the first heating reaction in the water bath is 95 ℃, and the reaction time is 3 min; after cooling, the temperature is 37 ℃, and the stirring time is 0.5 h; the voltage of electrospinning is 20 kV, the temperature is 37 ℃, the relative humidity is 50%, the roller speed is 0.6 mL / h, and the stirring time is 2 h.

7. The use of the solid-phase biosensor prepared according to any one of claims 1-6 for the detection of nitrosamines in food, characterized in that, The specific testing steps are as follows: (1) Establishment of standard curve for nitrosamine content: The solid-phase biosensor and standard solutions of nitrosamines of different concentrations were placed together in a closed reaction box. Different concentrations of nitrosamine corresponded to different solid-phase biosensors. After incubation at a certain temperature, the fluorescence intensity signal value of the solid-phase biosensor was detected. By fitting the fluorescence signal intensity with the corresponding nitrosamine concentration, a standard curve for nitrosamine detection was established. (2) Detection of nitrosamine content in food samples: The solid-phase biosensor and the sample to be tested are placed in a closed reaction box and incubated under certain temperature conditions. The fluorescence intensity signal value of the solid-phase biosensor is then detected. By substituting the obtained fluorescence signal value into the standard curve in step (1), the content of nitrosamines in the sample to be tested can be detected.

8. The use according to claim 7, characterized in that, In step (1), the concentration range of the standard solution of nitrosamine is 0.05 ng / mL to 500 ng / mL.

9. The use according to claim 7, characterized in that, In steps (1)-(2), the incubation time after the addition of nitrosamine is 3 min and the incubation temperature is 45 ℃. The specific method for detecting the fluorescence intensity signal value of the solid-phase biosensor is to measure the fluorescence value of the solid-phase biosensor under the excitation of a 980 nm exciter as the signal characteristic value.

10. The use according to claim 7, characterized in that, The sample to be tested in step (2) includes cured meat, ham or sausage; the pretreatment method for the sample to be tested is to divide the sample into thin slices with a thickness of 0.2 cm and a weight of 10 g ± 0.2 g.

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