A detection method for acrylamide content based on an upconversion nanobiosensing system

Abstract

The present invention belongs to the technical field of food safety detection, and specifically relates to a method for detecting acrylamide content based on an upconversion and silver nanocluster specific system. The steps are as follows: First, prepare core-shell structured upconversion nanomaterials, and modify silica and amino groups on the surface of the core-shell upconversion nanomaterials; combine the surface-modified upconversion nanomaterials with DNA1, and then add an aptamer and DNA2 for binding to obtain a specific detection system; after adding acrylamide solutions with different concentrations, remove the detached aptamer and DNA2 strands; add silver nitrate and sodium borohydride again to synthesize silver nanoclusters using the remaining DNA2 as a template; finally, establish a standard curve with the fluorescence intensity signal in systems with different acrylamide concentrations as the ordinate and the logarithm of the acrylamide concentration as the abscissa; The present invention realizes the highly specific and sensitive detection of acrylamide in food, has a wide concentration detection range and a low detection limit, and has good application prospects.

Description

Technical Field

[0001] The present invention belongs to the technical field of food safety detection, and particularly relates to a method for detecting acrylamide content based on an upconversion and silver nanocluster specific system. Background Art

[0002] Acrylamide is a substance widely present in thermally processed foods, mainly produced through the Maillard reaction between asparagine and reducing sugars under high temperature and low humidity conditions. Many organs of the human body can absorb and accumulate acrylamide, and binding to hemoglobin, DNA or enzymes can cause harm. Studies have shown that acrylamide has genotoxicity, neurotoxicity, reproductive toxicity and potential carcinogenic effects. The World Health Organization stipulates that the limit of acrylamide in daily drinking water is 0.5 μg / L.

[0003] Traditional acrylamide detection methods, such as gas chromatography and high performance liquid chromatography, although having high accuracy and stability, have expensive instrument equipment and cumbersome operation steps, and cannot meet the rapid detection of acrylamide content. The present invention proposes a rapid and accurate method for detecting acrylamide content, overcoming the defects of high cost and slow speed of traditional methods, and improving the sensitivity and accuracy of mercury detection. Summary of the Invention

[0004] The purpose of the present invention is to solve the problems existing in the prior art, such as long detection time and high instrument cost, and provides a fluorescence nanosensor for quantitatively detecting acrylamide in food based on upconversion and silver nanoclusters, with the aid of the specific self-assembly characteristics of DNA, thereby realizing the rapid, sensitive and accurate detection of acrylamide in food.

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

[0006] The present invention provides a method for detecting acrylamide, comprising the following steps:

[0007] Step 1, preparation of core upconversion nanomaterials: Gadolinium chloride hexahydrate, ytterbium chloride hexahydrate, and thulium chloride hexahydrate are added to methanol A and dissolved by ultrasonic waves, and then oleic acid and 1-octadecene are added to form a mixed solution; under the protection of argon, a first heating and stirring reaction is carried out, and after the reaction, it is cooled to room temperature to obtain a cooling solution; ammonium fluoride, sodium hydroxide, and methanol B are mixed and then added to the cooling solution to carry out a first water bath reaction, and after the reaction, the temperature is raised to carry out a second water bath reaction; after the water bath reaction, a second heating and stirring reaction is carried out under the protection of argon, and after the reaction, it is cooled to room temperature to obtain a reaction product; after washing and vacuum drying, the core upconversion nanomaterials are obtained;

[0008] Step 2, Preparation of core-shell upconversion nanomaterials: Dissolve yttrium chloride hexahydrate in methanol C by ultrasonic treatment, and transfer it to a mixed solution of oleic acid and 1-octadecene; under the protection of argon, perform the first heating and stirring reaction, and after the reaction, cool to room temperature to obtain a cooling solution;

[0009] Dissolve the core upconversion nanomaterial obtained in Step 1 in cyclohexane, then mix it with ammonium fluoride, sodium hydroxide, and methanol D, add it to the cooling solution, and perform the first water bath reaction. After the reaction, raise the temperature to perform the second water bath reaction; after the water bath reaction, under the protection of argon, perform the second heating and stirring reaction, and cool to room temperature to obtain a reaction product; wash the reaction product and dry it under vacuum to obtain the core-shell upconversion nanomaterial;

[0010] Step 3, Functional modification of core-shell upconversion nanomaterials: Add the core-shell upconversion nanomaterials obtained in Step 2 to hydrochloric acid and disperse them by ultrasonic treatment; after washing, disperse them in ethanol, add deionized water and ammonia water, and perform the first stirring reaction under certain temperature conditions; after the reaction, add tetraethyl orthosilicate for the second stirring reaction; finally, add 3-aminopropyltriethoxysilane for the third stirring reaction to obtain a reaction product; after washing and drying under vacuum, obtain amino-functionalized core-shell upconversion nanomaterials;

[0011] Step 4, Core-shell upconversion nanomaterials functionalized with aptamer complementary strand 1 (DNA1): Dissolve the amino-functionalized core-shell upconversion nanomaterials obtained in Step 3 in phosphate buffer A; then add glutaraldehyde and stir the reaction; after centrifugal washing with phosphate buffer solution, dissolve it in phosphate buffer solution B, add avidin for the first incubation reaction; finally, add aptamer complementary strand 1 (DNA1) for the second incubation to obtain core-shell upconversion nanomaterials functionalized with DNA1 strand;

[0012] Step 5, Establishment of a specific detection system: Dissolve the DNA1-functionalized core-shell upconversion nanomaterials obtained in Step 4 in phosphate buffer, add acrylamide aptamer and aptamer complementary strand 2 (DNA2) for incubation to obtain a specific detection system for acrylamide;

[0013] Step 6. Establishment of acrylamide detection standard curve: Add acrylamide standard solutions with different concentrations to the specific detection system of acrylamide prepared in Step 5 to obtain detection solutions with different concentrations. One concentration of acrylamide standard solution corresponds to one specific detection system, and the two are in a one-to-one correspondence. Then, centrifuge the detection solutions with different concentrations to obtain precipitates, wash the precipitates with phosphate buffer solution, and redisperse the washed precipitates in phosphate buffer solution C. After the first reaction with silver nitrate solution, add sodium borohydride solution for the second reaction. After the reaction, measure the fluorescence intensity signal characteristic values of the specific detection systems added with acrylamide solutions with different concentrations, denoted as Y, and establish the relationship between acrylamide concentration (c) and fluorescence intensity signal characteristic values, that is, obtain the acrylamide detection standard curve.

[0014] Step 7. Detection of acrylamide content in food samples: Crush the food samples, add deionized water, shake and perform ultrasonic treatment. Then, purify the supernatant through a solid-phase extraction column, add the purified supernatant to the specific detection system of acrylamide, measure the fluorescence intensity signal characteristic values of the specific detection system, and calculate the acrylamide content in the food samples through the acrylamide detection standard curve constructed in Step 6.

[0015] Preferably, in Step 1, the dosage ratio of gadolinium chloride hexahydrate, ytterbium chloride hexahydrate, thulium chloride hexahydrate, and methanol A is 0.07434 g: 0.07593 g: 0.0015 g: 4 mL; the ultrasonic dissolution time is 10 min; for the first heating and stirring reaction, the temperature is 150 - 170 °C and the time is 30 min; for the second heating and stirring reaction, the temperature is 290 - 310 °C and the time is 1 h; the volume ratio of methanol A, oleic acid, and 1-octadecene is 4 mL: 4 mL: 7 mL; the dosage ratio of methanol A, ammonium fluoride, sodium hydroxide, and methanol B is 4 mL: 0.06 g: 0.04 g: 4 mL; for the first water bath reaction, the temperature is 40 - 60 °C and the water bath reaction time is 40 min, and for the second water bath reaction, the temperature is 100 °C and the water bath reaction time is 10 min.

[0016] Preferably, in step two, the dosage ratio of yttrium chloride hexahydrate to methanol C is 0.1214 g: 4 mL; the ultrasonic dissolution time is 10 - 15 min; the temperature of the first heating and stirring reaction is 150 - 170 °C, and the time is 30 min; the temperature of the second heating and stirring reaction is 290 - 310 °C, and the time is 1 h; the dosage ratio of methanol C, oleic acid, 1-octadecene, core upconversion nanomaterials, cyclohexane, ammonium fluoride, sodium hydroxide, and methanol D is 4 mL: 4 mL: 7 mL: 50 mg: 5 mL: 0.06 g: 0.04 g: 4 mL; the temperature of the first water bath reaction is 40 - 60 °C, the water bath reaction time is 40 min, the temperature of the second water bath reaction is 60 - 80 °C, and the water bath reaction time is 30 min.

[0017] Preferably, in step three, the dosage ratio of the core-shell upconversion nanomaterials, hydrochloric acid, ethanol, deionized water, ammonia water, tetraethyl orthosilicate, and 3-aminopropyltriethoxysilane is 20 mg: 1 mL: 60 mL: 10 mL: 2.5 mL: 0.01 - 0.05 mL: 0.03 - 0.08 mL; the concentration of hydrochloric acid is 0.1 M; the ultrasonic dispersion time is 10 - 15 min; the volume concentration of ammonia water is 25%; the temperature of the first stirring is 65 °C, and the stirring time is 10 min; the temperature of the second stirring is 65 °C, and the stirring time is 2 - 6 h; the temperature of the third stirring is 65 °C, and the stirring time is 1 - 3 h.

[0018] Preferably, in step four, the dosage ratio of the amino-functionalized core-shell upconversion nanomaterials, phosphate buffer A, glutaraldehyde, phosphate buffer solution B, avidin, and DNA1 is 20 mg: 10 mL: 2.5 mL: 10 mL: 0.1 mL: 0.1 mL; the stirring reaction time is 2 h; the concentrations of phosphate buffer A and phosphate buffer B are both 10 mM, and the pH values are both 7.4; the volume concentration of glutaraldehyde is 25%; the concentration of avidin is 1 mg / mL.

[0019] Preferably, the concentration of DNA1 in step four is 60 nM, the solvent is phosphate buffer, and the sequence of DNA1 is 5’-Biotin-TTTTTTTTTT AAT CGC TGA ATG AAA TCC ACG TGG CCA CAC ACT CAT CCA CCACCC GGG TGG GGT GGG GTG GGG-3’; the temperature of the first incubation reaction is 37 °C, and the incubation time is 12 h; the temperature of the second incubation reaction is 37 °C, and the incubation time is 2 - 24 h.

[0020] Preferably, in step five, the dosage ratio of the DNA1-functionalized core-shell upconversion nanomaterial, phosphate buffer, acrylamide aptamer, and DNA2 is 20 mg: 10 mL: 0.1 mL: 0.1 mL; the incubation temperature is 37 °C, and the incubation time is 10 - 60 min.

[0021] Preferably, in step five, the concentration of the acrylamide aptamer is 60 nM, the solvent is phosphate buffer, and the aptamer sequence is 5’-CAG TCC AGG ACA GAT TCG CGA GTG GTC GTG GTG AGG TGC GTG TATGGG TGG TGG ATG AGT GTG TGG CCA CGT GGA TTT CAT TCA GCG ATT-3’; the concentration of DNA2 is 60 nM, the solvent is phosphate buffer, and the DNA2 sequence is 5’-CCC TTA ATC CCC ATA CAC GCA CCT CACCAC GAC CAC TCG CGA ATC TGT CCT GGA CTG-3’.

[0022] Preferably, in step six, the dosage ratio of the acrylamide specific detection system, acrylamide standard solution, phosphate buffer C, silver nitrate solution, and sodium borohydride solution is 0.5 mL: 0.15 mL: 0.65 mL: 0.016 mL: 0.016 mL; the acrylamide detection time is 5 - 60 min; the first reaction condition is to stand still at 4 °C for 15 min; the second reaction condition is to shake for 30 - 40 s; the concentration of silver nitrate is 1.2 μM; the concentration of sodium borohydride is 1.2 μM; the concentration of phosphate buffer D is 10 mM, and the pH is 7.4; the concentration of the acrylamide standard solution is 0.001 - 100 μM;

[0023] Measuring the fluorescence intensity signal characteristic value Y of the detection solution specifically refers to measuring the fluorescence intensity value at 450 nm under the excitation of 980 nm excitation light; the acrylamide detection standard curve refers to the standard curve that constructs the relationship between the fluorescence intensity signal characteristic value of the specific detection system and the acrylamide concentration, that is, Y = alog(c) + b, where a and b are constants; Y: fluorescence intensity characteristic value; c: acrylamide concentration.

[0024] Preferably, in step seven, the dosage ratio of the food sample and deionized water is 1 g: 1 mL; the dosage ratio of the acrylamide specific detection system and the purified supernatant is 0.5 mL: 0.15 mL; the ultrasonic time is 30 - 60 min.

[0025] The methanol A, methanol B, methanol C, and methanol D used in the present invention are all methanol, and the letters A, B, C, and D are only for naming differences; the phosphate buffer A, phosphate buffer B, and phosphate buffer C used in the present invention are all phosphate buffers (pH = 7.4), and the letters A, B, and C are only for naming differences.

[0026] Compared with the existing detection technologies, the beneficial effects of the present invention are as follows:

[0027] 1. The present invention discloses a fluorescence detection method for acrylamide in food, which is a detection method for the acrylamide content in food based on an upconversion-silver nanocluster specific system. Specifically, upconversion nanoparticles are used as a fluorescence donor, and silver nanoclusters are used as a fluorescence acceptor. An acrylamide detection system is constructed through aptamers and DNA assembly, which has high specificity and anti-interference ability, and realizes highly sensitive, low-cost, and specific detection of acrylamide in food.

[0028] 2. The specific detection system constructed by the present invention, specifically an optimized designed hybrid detection system of upconversion nanomaterials-silver nanoclusters, has a strong fluorescence response to acrylamide, can effectively eliminate background fluorescence and the interference of other molecules, has high specificity for the detection of acrylamide, overcomes the deficiencies of traditional methods, and is crucial for ensuring food safety.

[0029] 3. The linear concentration range of the acrylamide concentration and the fluorescence intensity signal characteristic value established by the present invention is 0.001 - 100 μM, which has a wide linear detection range. The detection limit LOD is 0.011 μM, which can meet the highly sensitive detection of acrylamide content in food, has good versatility, and is more accurate and sensitive than traditional methods. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required to be used in the embodiments. Obviously, the following described drawings are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0031] Figure 1 It is a transmission electron microscope image of the core-shell upconversion nanoparticles prepared in Example 1.

[0032] Figure 2 It is a transmission electron microscope image of the functionalized core-shell upconversion nanoparticles prepared in Example 1.

[0033] Figure 3 It is a transmission electron microscope image of the silver nanoclusters prepared in Example 1.

[0034] Figure 4Fluorescence signals of the upconversion nanomaterial-silver nanocluster hybrid system at different acrylamide concentrations in Example 1.

[0035] Figure 5 Detection standard curve of acrylamide for the upconversion nanomaterial-silver nanocluster hybrid system in Example 1. Detailed implementation manners

[0036] To make the objectives, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly described below. Obviously, the described embodiments are some but not all of the embodiments of the present invention. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0037] The following further elaborates in detail the detailed implementation manners of the present invention with reference to the accompanying drawings of the specification.

[0038] Example 1:

[0039] The present invention discloses a method for detecting the acrylamide content in food based on an upconversion and silver nanocluster specific system. The specific steps are as follows:

[0040] Step 1, preparation of the core upconversion nanomaterial: Accurately weigh 0.07434 g of gadolinium chloride hexahydrate, 0.07593 g of ytterbium chloride hexahydrate, and 0.0015 g of thulium chloride hexahydrate. Dissolve them in 4 mL of methanol by ultrasonic treatment for 10 min, transfer to a three-necked flask, and add 4 mL of oleic acid and 7 mL of octadecene. Under the protection of argon, heat to 160 °C for the first time, stir magnetically for 30 min, and cool to room temperature. Gradually add 4 mL of a methanol solution containing 0.06 g of ammonium fluoride and 0.04 g of sodium hydroxide dropwise to the coolant, then react at 50 °C in a water bath for 40 min for the first time and at 100 °C in a water bath for 10 min for the second time to completely volatilize the methanol in the solution. Then, under the protection of argon, heat to 300 °C for the second time, stir magnetically and react for 1 h, and cool to room temperature to obtain the reaction product. Finally, wash the reaction product with a mixed solution of ethanol and cyclohexane (volume ratio 1:2), and vacuum dry to obtain the core upconversion nanomaterial.

[0041] Figure 1 Transmission electron microscope image of the core upconversion nanoparticles prepared in Example 1 of the present invention. The electron microscope image shows that the core upconversion nanoparticles are monodispersed with a diameter of about 18 nm.

[0042] Step 2, Preparation of core-shell upconversion nanomaterials: Add 0.1214 g of yttrium chloride hexahydrate to 4 mL of methanol and ultrasonically dissolve for 10 min. Transfer it to a three-necked flask, and add 4 mL of oleic acid and 7 mL of octadecene. Under the protection of argon, heat to 160 °C for the first time, stir magnetically for 30 min, and then cool to room temperature. Gradually add 4 mL of a methanol solution containing 0.06 g of ammonium fluoride and 0.04 g of sodium hydroxide dropwise to the coolant, and then react in a water bath at 50 °C for 40 min for the first time and react in a water bath at 70 °C for 30 min for the second time to completely volatilize the methanol in the solution. Then, under the protection of argon, heat to 300 °C for the second time, stir magnetically for 1 h, and cool to room temperature to obtain the reaction product. Finally, wash the reaction product with a mixed solution of ethanol and cyclohexane (volume ratio 1:2), and vacuum dry to obtain the core-shell upconversion nanomaterials;

[0043] Figure 2 Figure 4 is the transmission electron microscopy image of the core-shell upconversion nanoparticles prepared in Example 1 of the present invention. As can be seen from the figure, the diameter of the core-shell upconversion nanoparticles increases to about 50 nm and the dispersion is good.

[0044] Step 3, Functional modification of core-shell upconversion nanomaterials: Add 20 mg of the core-shell upconversion nanomaterials obtained in Step 2 to 1 mL of hydrochloric acid (0.1 M), and ultrasonically disperse for 15 min. After washing with ethanol, disperse it again in 60 mL of ethanol, add 10 mL of deionized water and 2.5 mL of ammonia water (25%), and stir and react at 65 °C for 10 min. Add 0.03 mL of tetraethyl orthosilicate, stir and react for 4 h. Add 0.05 mL of 3-aminopropyltriethoxysilane, stir and react for 2 h to obtain the reaction product. Wash the reaction product and vacuum dry to obtain the amino-functionalized core-shell upconversion nanomaterials;

[0045] Figure 3 Figure 5 is the transmission electron microscopy image of the functionalized core-shell upconversion nanoparticles prepared in Example 1. It can be seen that the core-shell upconversion nanoparticles are uniformly coated with a silica shell about 10 nm thick, indicating the successful amino-functionalization of the upconversion nanoparticles.

[0046] Step 4, Aptamer-complementary DNA1 strand 1 (DNA1)-functionalized core-shell upconversion nanomaterials: Dissolve 20 mg of the amino-functionalized core-shell upconversion nanomaterials obtained in Step 3 in 10 mL of phosphate buffer (10 mM, pH = 7.4); then add 2.5 mL of glutaraldehyde (25%), and stir for 2 h; after washing with phosphate buffer solution, dissolve it in 10 mL of phosphate buffer solution (10 mM, pH = 7.4), add 0.1 mL of avidin (1 mg / mL), and incubate at 37 °C for 12 h; finally, add 0.1 mL of DNA1 (60 nM), and the DNA1 strand is 5’-Biotin-TTTTTTTTTT AAT CGC TGA ATG AAA TCC ACG TGG CCA CACACT CAT CCA CCA CCC GGG TGG GGT GGG GTG GGG-3’, and incubate at 37 °C for 6 h to obtain DNA1 strand-functionalized core-shell upconversion nanomaterials;

[0047] Step 5, Establishment of a specific detection system: Dissolve 20 mg of the DNA1 strand-functionalized core-shell upconversion nanomaterials obtained in Step 4 in 10 mL of phosphate buffer, add 0.1 mL of acrylamide aptamer (60 nM), and the aptamer is 5’-CAG TCC AGG ACA GAT TCG CGA GTG GTC GTG GTG AGG TGC GTG TAT GGG TGG TGG ATGAGT GTG TGG CCA CGT GGA TTT CAT TCA GCG ATT-3’, add 0.1 mL of DNA2 strand (60 nM), and DNA2 is 5’-CCC TTA ATC CCC ATA CAC GCA CCT CAC CAC GAC CAC TCG CGA ATC TGT CCT GGACTG-3’, and incubate at 37 °C for 20 min to obtain a specific detection system for acrylamide.

[0048] Step 6, Establishment of an acrylamide detection standard curve: Add acrylamide standard solutions with different concentrations to 0.65 mL of the specific detection system prepared in Step 5, and incubate for 10 min to obtain detection solutions with different concentrations. One concentration of acrylamide solution corresponds to one specific detection system, and the two are in a one-to-one correspondence; after incubating the core-shell upconversion nanomaterials with acrylamide, wash them with phosphate buffer solution, redisperse them in 0.65 mL of phosphate buffer solution, add 0.16 mL of silver nitrate solution (1.2 μM), let it stand at 4 °C for 15 min, and then add 0.16 mL of sodium borohydride solution (1.2 μM) and shake violently for 30 s to synthesize silver nanoclusters, as Figure 4The diameter of the synthesized silver nanoclusters is about 4 nm; then, under the excitation of 980 nm excitation light, the characteristic value Y of the fluorescence intensity signal of the specific detection system with acrylamide solutions of different concentrations added is measured, the relationship between acrylamide concentration (c) and the characteristic value Y of the fluorescence intensity signal is established, and the acrylamide detection standard curve is obtained, as Figure 5 shown as Y = 886.15 log(c) + 5956.6, the coefficient of determination R 2 = 0.9749, the detection limit is 0.011 μM, and the linear range is 0.001 - 100 μM.

[0049] Step 7: Detection of acrylamide content in food samples: Crush 1 g of food samples, add 1 mL of deionized water, shake and ultrasonically treat for 40 min; then purify the supernatant and add it to the specific detection system, measure the characteristic value of the fluorescence intensity signal of the specific detection system, and calculate the acrylamide content in the food samples through the acrylamide detection standard curve constructed in Step 6.

[0050] Example 2:

[0051] The present invention discloses a method for detecting acrylamide content in food based on an upconversion and silver nanocluster specific system, and the specific steps are as follows:

[0052] Step 1, preparation of core upconversion nanomaterials: Accurately weigh 0.07434 g of gadolinium chloride hexahydrate, 0.07593 g of ytterbium chloride hexahydrate, and 0.0015 g of thulium chloride hexahydrate, ultrasonically dissolve them in 4 mL of methanol for 10 min, transfer them to a three-necked flask, and add 4 mL of oleic acid and 7 mL of octadecene; under the protection of argon, heat to 150 °C for the first time, stir magnetically for 30 min, and cool to room temperature; gradually add 4 mL of a methanol solution of 0.06 g of ammonium fluoride and 0.04 g of sodium hydroxide dropwise to the coolant, then react in a water bath at 40 °C for 40 min for the first time and react in a water bath at 100 °C for 10 min for the second time to completely volatilize the methanol in the solution; then, under the protection of argon, heat to 290 °C for the second time, stir magnetically and react for 1 h, and cool to room temperature to obtain the reaction product; finally, wash the reaction product with a mixed solution of ethanol and cyclohexane (volume ratio 1:2), and vacuum dry to obtain the core upconversion nanomaterials.

[0053] Step 2: Preparation of core-shell upconversion nanomaterials: Add 0.1214 g of yttrium chloride hexahydrate to 4 mL of methanol and ultrasonically dissolve for 10 min. Transfer it to a three-necked flask, and add 4 mL of oleic acid and 7 mL of octadecene. Under the protection of argon, heat to 150 °C for the first time, stir magnetically for 30 min, and then cool to room temperature. Gradually add 4 mL of a methanol solution containing 0.06 g of ammonium fluoride and 0.04 g of sodium hydroxide to the coolant, and then react in a water bath at 40 °C for 40 min for the first time and react in a water bath at 60 °C for 30 min for the second time to completely volatilize the methanol in the solution. Then, under the protection of argon, heat to 290 °C for the second time, stir magnetically for 1 h, and cool to room temperature to obtain the reaction product. Finally, wash the reaction product with a mixed solution of ethanol and cyclohexane (volume ratio 1:2), and vacuum dry to obtain the core-shell upconversion nanomaterials.

[0054] Step 3, Functional modification of core-shell upconversion nanomaterials: Add 20 mg of the core-shell upconversion nanomaterials obtained in Step 2 to 1 mL of hydrochloric acid (0.1 M), and ultrasonically disperse for 15 min. After washing with ethanol, disperse it again in 60 mL of ethanol, add 10 mL of deionized water and 2.5 mL of ammonia water (25%), and stir and react at 65 °C for 10 min. Add 0.01 mL of tetraethyl orthosilicate, stir and react for 2 h. Add 0.03 mL of 3-aminopropyltriethoxysilane, stir and react for 1 h to obtain the reaction product. Wash the reaction product and vacuum dry to obtain amino-functionalized core-shell upconversion nanomaterials;

[0055] Step 4, Core-shell upconversion nanomaterials functionalized with aptamer complementary DNA1 strand 1 (DNA1): Dissolve 20 mg of the amino-functionalized core-shell upconversion nanomaterials obtained in Step 3 in 10 mL of phosphate buffer (10 mM, pH = 7.4); then add 2.5 mL of glutaraldehyde (25%), and stir and react for 2 h. After washing with phosphate buffer solution, dissolve it in 10 mL of phosphate buffer solution (10 mM, pH = 7.4), add 0.1 mL of avidin (1 mg / mL), and incubate and react at 37 °C for 12 h; finally add 0.1 mL of DNA1 (60 nM), and the DNA1 strand is 5’-Biotin-TTTTTTTTTT AAT CGC TGA ATG AAA TCC ACG TGG CCA CACACT CAT CCA CCA CCC GGG TGG GGT GGG GTG GGG-3’, and incubate at 37 °C for 2 h to obtain DNA1 strand-functionalized core-shell upconversion nanomaterials;

[0056] Step 5. Establishment of the specific detection system: Dissolve 20 mg of the core-shell upconversion nanomaterials functionalized with DNA1 strand obtained in Step 4 in 10 mL of phosphate buffer, add 0.1 mL of acrylamide aptamer (60 nM), and the aptamer is 5’-CAG TCC AGG ACA GAT TCG CGA GTG GTC GTG GTG AGG TGC GTG TAT GGG TGG TGG ATGAGT GTG TGG CCA CGT GGA TTT CAT TCA GCG ATT-3’, add 0.1 mL of DNA2 strand (60 nM), and DNA2 is 5’-CCC TTA ATC CCC ATA CAC GCA CCT CAC CAC GAC CAC TCG CGA ATC TGT CCT GGACTG-3’. Incubate at 37 °C for 10 min to obtain the specific detection system for acrylamide.

[0057] Step 6. Establishment of the acrylamide detection standard curve: Add acrylamide standard solutions with different concentrations to 0.65 mL of the specific detection system prepared in Step 5, and incubate for 5 min to obtain detection solutions with different concentrations. One concentration of acrylamide solution corresponds to one specific detection system, and the two are in a one-to-one correspondence relationship; after incubating the core-shell upconversion nanomaterials with acrylamide, wash them with phosphate buffer, and then redisperse them in 0.65 mL of phosphate buffer. Add 0.16 mL of silver nitrate solution (1.2 μM), let it stand and react at 4 °C for 15 min, and then add 0.16 mL of sodium borohydride solution (1.2 μM) and shake vigorously for 30 s to synthesize silver nanoclusters; then, under the excitation of 980 nm excitation light, measure the characteristic value Y of the fluorescence intensity signal at 450 nm of the specific detection system added with acrylamide solutions with different concentrations, establish the relationship between acrylamide concentration (c) and the characteristic value Y of the fluorescence intensity signal, and obtain the acrylamide detection standard curve Y = 782.26log(c) + 6074.5, the determination coefficient R 2 = 0.9802, the detection limit is 0.013 μM, and the linear range is 0.001 - 100 μM.

[0058] Step 7. Detection of acrylamide content in food samples: Crush 1 g of food samples, add 1 mL of deionized water, shake and ultrasonically treat for 30 min; then purify the supernatant and add it to the specific detection system, measure the characteristic value of the fluorescence intensity signal of the specific detection system, and calculate the acrylamide content in the food samples through the acrylamide detection standard curve constructed in Step 6.

[0059] Example 3:

[0060] The present invention discloses a method for detecting acrylamide content in food based on an upconversion and silver nanocluster specific system. The specific steps are as follows:

[0061] Step 1, preparation of core upconversion nanomaterials: Accurately weigh 0.07434 g of gadolinium chloride hexahydrate, 0.07593 g of ytterbium chloride hexahydrate, and 0.0015 g of thulium chloride hexahydrate. Dissolve them in 4 mL of methanol by ultrasonic treatment for 10 min, transfer to a three-necked flask, and add 4 mL of oleic acid and 7 mL of octadecene. Under the protection of argon, heat to 170 °C for the first time, stir magnetically for 30 min, and then cool to room temperature. Gradually add 4 mL of a methanol solution containing 0.06 g of ammonium fluoride and 0.04 g of sodium hydroxide dropwise to the coolant, and then react in a water bath at 60 °C for 40 min for the first time and at 100 °C for 10 min for the second time to completely volatilize the methanol in the solution. Then, under the protection of argon, heat to 310 °C for the second time, stir magnetically for 1 h, and cool to room temperature to obtain the reaction product. Finally, wash the reaction product with a mixed solution of ethanol and cyclohexane (volume ratio 1:2), and dry it under vacuum to obtain the core upconversion nanomaterials.

[0062] Step 2: Preparation of core-shell upconversion nanomaterials: Add 0.1214 g of yttrium chloride hexahydrate to 4 mL of methanol and dissolve it by ultrasonic treatment for 10 min. Transfer to a three-necked flask, and add 4 mL of oleic acid and 7 mL of octadecene. Under the protection of argon, heat to 170 °C for the first time, stir magnetically for 30 min, and then cool to room temperature. Gradually add 4 mL of a methanol solution containing 0.06 g of ammonium fluoride and 0.04 g of sodium hydroxide dropwise to the coolant, and then react in a water bath at 60 °C for 40 min for the first time and at 80 °C for 30 min for the second time to completely volatilize the methanol in the solution. Then, under the protection of argon, heat to 310 °C for the second time, stir magnetically for 1 h, and cool to room temperature to obtain the reaction product. Finally, wash the reaction product with a mixed solution of ethanol and cyclohexane (volume ratio 1:2), and dry it under vacuum to obtain the core-shell upconversion nanomaterials.

[0063] Step 3, functional modification of core-shell upconversion nanomaterials: Add 20 mg of the core-shell upconversion nanomaterials obtained in Step 2 to 1 mL of hydrochloric acid (0.1 M), and disperse them by ultrasonic treatment for 15 min. After washing with ethanol, disperse them again in 60 mL of ethanol, add 10 mL of deionized water and 2.5 mL of ammonia water (25%), and react by stirring at 65 °C for 10 min. Add 0.05 mL of tetraethyl orthosilicate, and stir and react for 6 h. Add 0.08 mL of 3-aminopropyltriethoxysilane, and stir and react for 3 h to obtain the reaction product. Wash the reaction product, and dry it under vacuum to obtain the amino-functionalized core-shell upconversion nanomaterials;

[0064] Step 4, Aptamer-complementary DNA1 strand 1 (DNA1)-functionalized core-shell upconversion nanomaterials: Dissolve 20 mg of the amino-functionalized core-shell upconversion nanomaterials obtained in Step 3 in 10 mL of phosphate buffer (10 mM, pH = 7.4); then add 2.5 mL of glutaraldehyde (25%), and stir and react for 2 h; after washing with phosphate buffer solution, dissolve it in 10 mL of phosphate buffer solution (10 mM, pH = 7.4), add 0.1 mL of avidin (1 mg / mL), and incubate and react at 37 °C for 12 h; finally, add 0.1 mL of DNA1 (60 nM), and the DNA1 strand is 5’-Biotin-TTTTTTTTTT AAT CGC TGA ATG AAA TCC ACG TGG CCA CACACT CAT CCA CCA CCC GGG TGG GGT GGG GTG GGG-3’, and incubate at 37 °C for 24 h to obtain DNA1 strand-functionalized core-shell upconversion nanomaterials;

[0065] Step 5, Establishment of a specific detection system: Dissolve 20 mg of the DNA1 strand-functionalized core-shell upconversion nanomaterials obtained in Step 4 in 10 mL of phosphate buffer, add 0.1 mL of acrylamide aptamer (60 nM), and the aptamer is 5’-CAG TCC AGG ACA GAT TCG CGA GTG GTC GTG GTG AGG TGC GTG TAT GGG TGG TGG ATGAGT GTG TGG CCA CGT GGA TTT CAT TCA GCG ATT-3’, add 0.1 mL of DNA2 strand (60 nM), and DNA2 is 5’-CCC TTA ATC CCC ATA CAC GCA CCT CAC CAC GAC CAC TCG CGA ATC TGT CCT GGACTG-3’, and incubate at 37 °C for 60 min to obtain a specific detection system for acrylamide.

[0066] Step 6. Establishment of acrylamide detection standard curve: Add acrylamide standard solutions with different concentrations to 0.65 mL of the specific detection system prepared in Step 5, incubate for 60 min to obtain detection solutions with different concentrations. One concentration of acrylamide solution corresponds to one specific detection system, and the two are in a one-to-one correspondence. Wash the core-shell upconversion nanomaterials after acrylamide incubation with phosphate buffer, redisperse them in 0.65 mL of phosphate buffer, add 0.16 mL of silver nitrate solution (1.2 μM), let it stand and react at 4°C for 15 min, then add 0.16 mL of sodium borohydride solution (1.2 μM) and shake vigorously for 30 s to synthesize silver nanoclusters. Then, under the excitation of 980 nm excitation light, measure the characteristic value Y of the fluorescence intensity signal at 450 nm of the specific detection system added with acrylamide solutions with different concentrations, establish the relationship between acrylamide concentration (c) and the characteristic value Y of the fluorescence intensity signal, obtain the acrylamide detection standard curve, Y = 7146.15 log(c) + 5893.7, and the determination coefficient R 2 = 0.9702, the detection limit is 0.014 μM, and the linear range is 0.001 - 100 μM.

[0067] Step 7: Detection of acrylamide content in food samples: Crush 1 g of food samples, add 1 mL of deionized water, shake and ultrasonically treat for 60 min. Then purify the supernatant and add it to the specific detection system, measure the characteristic value of the fluorescence intensity signal of the specific detection system, and calculate the acrylamide content in the food samples through the acrylamide detection standard curve constructed in Step 6.

[0068] Detection: Since Example 1 is the optimal example, the method and steps described in Example 1 are adopted in the present invention to determine the acrylamide content in 4 food standard samples. Sample 1 is potato chips, Sample 2 is cookies, Sample 3 is coffee, and Sample 4 is bread. As shown in Table 1 of the measurement results, it can be seen that there is no significant difference between the detection results of the method of the present invention and the standard high-performance liquid chromatography method, indicating that the method of the present invention can detect the acrylamide content in food with high precision.

[0069] Table 1 Results of detecting acrylamide content in food standard samples by the method of the present invention and the standard method (unit, μM)

[0070]

[0071] t = 0.55 < t 0.05(3) = 3.182, P > 0.05

[0072] When the constructed detection method is used to detect other structurally similar standard solutions, such as L-asparagine, methacrylamide, methacrylic acid, acrylic acid, acetic acid, and propionic acid, etc., the fluorescence signal of the system will not change. Only when acrylamide solution is added to the system will the fluorescence signal value of the system change significantly. And in the anti-interference experiment of adding acrylamide to the specific detection system in the presence of the above-mentioned analogs, adding acrylamide will cause a significant change in the fluorescence signal value. The results show that the constructed specific detection system is hardly affected by other coexisting analogs when detecting acrylamide aldehyde, indicating that the constructed detection method has high specificity and high selectivity for acrylamide.

[0073] Note: The above embodiments are only used to illustrate the present invention and do not limit the technical solutions described in the present invention; therefore, although this specification has described the present invention in detail with reference to the above embodiments, those of ordinary skill in the art should understand that the present invention can still be modified or equivalently replaced; and all technical solutions and their improvements that do not depart from the spirit and scope of the present invention shall be covered by the claims of the present invention.

Claims

1. A detection method for acrylamide content based on an upconversion nanobiosensing system, characterized in that, it comprises the following steps: Step 1, Gadolinium chloride hexahydrate, ytterbium chloride hexahydrate, and thulium chloride hexahydrate are added to methanol A and dissolved by ultrasonic treatment. Then oleic acid and 1-octadecene are added to form a mixed solution. Under the protection of argon, the first heating and stirring reaction is carried out. After the reaction, it is cooled to room temperature to obtain a coolant. After mixing ammonium fluoride, sodium hydroxide, and methanol B, they are added to the coolant for the first water bath reaction. After the reaction, the temperature is raised for the second water bath reaction. After the water bath reaction, the second heating and stirring reaction is carried out under the protection of argon. After the reaction, it is cooled to room temperature to obtain a reaction product. After washing and vacuum drying, the core upconversion nanomaterial is obtained; Step 2, Yttrium chloride hexahydrate is added to methanol C and dissolved by ultrasonic treatment, and then transferred to a mixed solution of oleic acid and 1-octadecene. Under the protection of argon, the first heating and stirring reaction is carried out. After the reaction, it is cooled to room temperature to obtain a coolant; The core upconversion nanomaterial obtained in Step 1 is dissolved in cyclohexane, and then mixed with ammonium fluoride, sodium hydroxide, and methanol D and added to the coolant for the first water bath reaction. After the reaction, the temperature is raised for the second water bath reaction. After the water bath reaction, the second heating and stirring reaction is carried out under the protection of argon and cooled to room temperature to obtain a reaction product. The reaction product is washed and vacuum dried to obtain a core-shell upconversion nanomaterial; Step 3, The core-shell upconversion nanomaterial obtained in Step 2 is added to hydrochloric acid and dispersed by ultrasonic treatment. After washing, it is dispersed in ethanol, deionized water and ammonia water are added, and the first stirring reaction is carried out at a certain temperature. After the reaction, tetraethyl orthosilicate is added for the second stirring reaction. Finally, 3-aminopropyltriethoxysilane is added for the third stirring reaction to obtain a reaction product. After washing and vacuum drying, an amino-functionalized core-shell upconversion nanomaterial is obtained; Step 4, The amino-functionalized core-shell upconversion nanomaterial obtained in Step 3 is dissolved in phosphate buffer A. Then glutaraldehyde is added and stirred. After centrifugal washing with phosphate buffer solution, it is dissolved in phosphate buffer solution B, and avidin is added for the first incubation reaction. Finally, the complementary strand 1 of the aptamer is added for the second incubation to obtain a DNA1-strand-functionalized core-shell upconversion nanomaterial; Step 5, Establishment of a specific detection system: The DNA1-strand-functionalized core-shell upconversion nanomaterial obtained in Step 4 is dissolved in phosphate buffer, and acrylamide aptamer and complementary strand 2 of the aptamer are added for incubation to obtain a specific detection system for acrylamide; Step 6. Establishment of acrylamide detection standard curve: Add acrylamide standard solutions with different concentrations into the specific detection system of acrylamide prepared in Step 5 to obtain detection solutions with different concentrations. One concentration of acrylamide standard solution corresponds to one specific detection system, and the two are in a one-to-one correspondence relationship. Then, centrifuge the detection solutions with different concentrations to obtain precipitates, wash the precipitates with phosphate buffer solution, redisperse the washed precipitates in phosphate buffer solution C, add silver nitrate solution for the first reaction, and then add sodium borohydride solution for the second reaction. After the reaction, measure the fluorescence intensity signal characteristic values of the specific detection systems added with acrylamide solutions with different concentrations, denoted as Y. Establish the relationship between acrylamide concentration and fluorescence intensity signal characteristic values, that is, obtain the acrylamide detection standard curve. Step 7. Detection of acrylamide content in food samples: Crush the food samples, add deionized water, shake and perform ultrasonic treatment. Then, purify the supernatant through a solid-phase extraction column, add it to the specific detection system, measure the fluorescence intensity signal characteristic values of the specific detection system, and calculate the acrylamide content in the food samples through the acrylamide detection standard curve constructed in Step 6.

2. A method for detecting acrylamide content based on an upconversion nanobiosensing system according to claim 1, characterized in that, In Step 1, the dosage ratio of gadolinium chloride hexahydrate, ytterbium chloride hexahydrate, thulium chloride hexahydrate, and methanol A is 0.07434 g: 0.07593 g: 0.0015 g: 4 mL; the ultrasonic dissolution time is 10 min; the temperature of the first heating and stirring reaction is 150 - 170 °C, and the time is 30 min; the temperature of the second heating and stirring reaction is 290 - 310 °C, and the time is 1 h; the volume ratio of methanol A, oleic acid, and 1-octadecene is 4 mL: 4 mL: 7 mL; the dosage ratio of methanol A, ammonium fluoride, sodium hydroxide, and methanol B is 4 mL: 0.06 g: 0.04 g: 4 mL; the temperature of the first water bath reaction is 40 - 60 °C, the water bath reaction time is 40 min, the temperature of the second water bath reaction is 100 °C, and the water bath reaction time is 10 min.

3. A method for detecting acrylamide content based on an upconversion nanobiosensing system according to claim 1, characterized in that, In Step 2, the dosage ratio of yttrium chloride hexahydrate to Methanol C is 0.1214 g : 4 mL; the time for ultrasonic dissolution is 10 - 15 min; the temperature for the first heating and stirring reaction is 150 - 170 °C, and the time is 30 min; the temperature for the second heating and stirring reaction is 290 - 310 °C, and the time is 1 h; the dosage ratio of Methanol C, oleic acid, 1-octadecene, core upconversion nanomaterials, cyclohexane, ammonium fluoride, sodium hydroxide, and Methanol D is 4 mL : 4 mL : 7 mL : 50 mg : 5 mL : 0.06 g : 0.04 g : 4 mL; the temperature for the first water bath reaction is 40 - 60 °C, the water bath reaction time is 40 min, the temperature for the second water bath reaction is 60 - 80 °C, and the water bath reaction time is 30 min.

4. A method for detecting acrylamide content based on an upconversion nano-biosensing system according to claim 1, characterized in that, In Step 3, the dosage ratio of the core-shell upconversion nanomaterials, hydrochloric acid, ethanol, deionized water, ammonia water, tetraethyl orthosilicate, and 3-aminopropyltriethoxysilane is 20 mg : 1 mL : 60 mL : 10 mL : 2.5 mL : 0.01 - 0.05 mL : 0.03 - 0.08 mL; the concentration of hydrochloric acid is 0.1 M; the ultrasonic dispersion time is 10 - 15 min; the volume concentration of ammonia water is 25%; the temperature for the first stirring is 65 °C, and the stirring time is 10 min; the temperature for the second stirring is 65 °C, and the stirring time is 2 - 6 h; the temperature for the third stirring is 65 °C, and the stirring time is 1 - 3 h.

5. A method for detecting acrylamide content based on an upconversion nano-biosensing system according to claim 1, characterized in that, In Step 4, the dosage ratio of the amino-functionalized core-shell upconversion nanomaterials, phosphate buffer A, glutaraldehyde, phosphate buffer solution B, avidin, and DNA1 is 20 mg : 10 mL : 2.5 mL : 10 mL : 0.1 mL : 0.1 mL; the stirring reaction time is 2 h; the concentrations of phosphate buffer A and phosphate buffer B are both 10 mM, and the pH values are both 7.4; the volume concentration of glutaraldehyde is 25%; the concentration of avidin is 1 mg / mL.

6. A method for detecting acrylamide content based on an upconversion nano-biosensing system according to claim 1, characterized in that, In Step 4, the concentration of DNA1 is 60 nM, the solvent is phosphate buffer, and the sequence of the aptamer complementary strand 1 is 5’-Biotin-TTTTTTTTTT AAT CGC TGA ATG AAA TCC ACG TGG CCA CAC ACTCAT CCA CCA CCC GGG TGG GGT GGG GTG GGG-3’; the temperature for the first incubation reaction is 37 °C, and the incubation time is 12 h; the temperature for the second incubation reaction is 37 °C, and the incubation time is 2 - 24 h.

7. A method for detecting acrylamide content based on an upconversion nanobiosensing system according to claim 1, characterized in that, in step five, the dosage ratio of the core-shell upconversion nanomaterial functionalized with the aptamer complementary strand 1, phosphate buffer, acrylamide aptamer and aptamer complementary strand 2 is 20 mg: 10 mL: 0.1 mL: 0.1 mL; the incubation temperature is 37 °C, and the incubation time is 10 - 60 min.

8. A method for detecting acrylamide content based on an upconversion nanobiosensing system according to claim 1, characterized in that, in step five, the concentration of the acrylamide aptamer is 60 nM, the solvent is phosphate buffer, and the aptamer sequence is 5’-CAG TCC AGG ACA GAT TCG CGA GTG GTC GTG GTG AGG TGC GTG TATGGG TGG TGG ATG AGT GTG TGG CCA CGT GGA TTT CAT TCA GCG ATT-3’; the concentration of the aptamer complementary strand 2 is 60 nM, the solvent is phosphate buffer, and the aptamer complementary strand 2 sequence is 5’-CCC TTA ATC CCCATA CAC GCA CCT CAC CAC GAC CAC TCG CGA ATC TGT CCT GGA CTG -3’.

9. A method for detecting acrylamide content based on an upconversion nanobiosensing system according to claim 1, characterized in that, in step six, the dosage ratio of the acrylamide specific detection system, acrylamide standard solution, phosphate buffer C, silver nitrate solution and sodium borohydride solution is 0.5 mL: 0.15 mL: 0.65 mL: 0.016 mL: 0.016 mL; the acrylamide detection time is 5 - 60 min; the first reaction condition is to stand still at 4 °C for 15 min; the second reaction condition is to shake for 30 - 40 s; the concentration of silver nitrate is 1.2 μM; the concentration of sodium borohydride is 1.2 μM; the concentration of phosphate buffer D is 10 mM, pH is 7.4; the concentration of the acrylamide standard solution is 0.001 - 100 μM; Measure the fluorescence intensity signal characteristic value Y, specifically measure the fluorescence intensity value at 450 nm under the excitation of 980 nm excitation light; the acrylamide detection standard curve refers to the standard curve that constructs the relationship between the fluorescence intensity signal characteristic value of the specific detection system and the acrylamide concentration, that is, Y = alog(c) + b, where a and b are constants; Y: fluorescence intensity characteristic value; c: acrylamide concentration.

10. A method for detecting acrylamide content based on an upconversion nanobiosensing system according to claim 1, characterized in that, In Step 7, the dosage ratio of the food sample to deionized water is 1 g:1 mL; the dosage ratio of the specific detection system for acrylamide to the purified supernatant is 0.5 mL:0.15 mL; the ultrasonic time is 30 - 60 min.

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