A method for specific detection of acrylamide based on upconversion solid-phase sensor

Abstract

This invention discloses a specific detection method for acrylamide based on an upconversion solid-phase sensor, relating to the field of food safety testing. The preparation steps of the specific detection system based on the upconversion solid-phase sensor include: first, modifying silicon nano-sized fluorescein isothiocyanate with an acrylamide aptamer; then, preparing upconversion nanomaterials, doping them with polydimethylsiloxane to cast a sensor film, and further modifying the sensor film with a complementary aptamer chain; finally, mixing the two to obtain the specific detection system. The fluorescence intensity signal of the specific detection system after adding acrylamide standard solutions of different concentrations is measured, and an acrylamide detection standard curve is established. This invention, by constructing an acrylamide content detection method based on an upconversion solid-phase sensor, achieves high specificity, sensitivity, and rapid real-time detection of acrylamide in food, with a wide concentration detection range and a low detection limit, showing good prospects for field application.

Description

Technical Field

[0001] This invention relates to the field of food safety testing, specifically to a method for the specific detection of acrylamide based on an upconversion solid-phase sensor. Background Technology

[0002] Acrylamide is a typical organic compound commonly used in industrial processes such as organic synthesis, papermaking, cosmetics, textiles, and wastewater treatment. Acrylamide has been proven to be toxic to the skin, organs, central nervous system, and even genes; in 1994, the International Agency for Research on Cancer (IARC) classified it as a Group 2A carcinogen. The main formation pathway of acrylamide involves the Maillard reaction between amino acids and reducing sugars at high temperatures.

[0003] Therefore, considering the potential toxicity of acrylamide, the effective identification of acrylamide in food is a key issue for food safety. Traditional detection methods include high-performance liquid chromatography (HPLC) and gas chromatography (GC), which have significant advantages in terms of accuracy and reliability. However, due to the high cost of equipment, slow analysis speed, and high personnel requirements, these methods are not suitable for widespread application in field testing. In recent years, fluorescence sensing technology has attracted much attention due to its simplicity and short processing time, but there is currently limited research on the detection of acrylamide using fluorescence sensing technology.

[0004] Chinese patent application CN112540064A discloses a specific detection system for upconversion nanomaterials-rhodamine B derivatives-glutathione prepared through controllable self-assembly, used for detecting acrylamide content in food samples. While this prior art claims a limit of detection (LOD) of 0.68 nM, the actual linear concentration range is 0.1–10000 μM. Therefore, this LOD far exceeds the linear concentration range, making the claimed LOD of 0.68 nM unattainable.

[0005] The invention patent with publication number CN113218927A discloses a fluorescent probe for the specific detection of acrylamide prepared by combining a metal-organic framework material with an acrylamide fluorescent aptamer chain modified with 6-carboxyfluorescein dye. The linear concentration detection range is 50nM to 10μM, and the detection limit is 15.6nM, which is still relatively high.

[0006] Furthermore, both of the aforementioned existing technologies use liquid phase sensors for detection, which involves a complicated preparation process and a long detection time. Summary of the Invention

[0007] To address the shortcomings of existing technologies, this invention provides a specific detection method for acrylamide based on an upconversion solid-phase sensor. The specific detection system is constructed using a polydimethylsiloxane film doped with aptamer-modified silicon nano-fluorescein isothiocyanate and aptamer-complementary chain-modified core-shell upconversion nanomaterials. This system is used for acrylamide content detection, thereby achieving rapid and sensitive on-site detection of acrylamide.

[0008] This invention provides a method for preparing an acrylamide-specific detection system based on an upconversion solid-phase sensor. The acrylamide-specific detection system based on the upconversion solid-phase sensor includes aptamer-modified silicon nanofiber isothiocyanate fluorescein and a polydimethylsiloxane film doped with a core-shell upconversion nanomaterial modified with an aptamer complementary chain.

[0009] The preparation method of aptamer-modified silicon nano-sized fluorescein isothiocyanate includes the following steps: preparing silicon nano-sized fluorescein isothiocyanate, and then modifying it with a nucleic acid aptamer that can bind acrylamide to prepare aptamer-modified silicon nano-sized fluorescein isothiocyanate.

[0010] The method for preparing a polydimethylsiloxane film doped with a core-shell upconversion nanomaterial modified with an aptamer complementary chain includes the following steps: preparing a polydimethylsiloxane film doped with a core-shell upconversion nanomaterial, and then modifying the polydimethylsiloxane film doped with a core-shell upconversion nanomaterial doped with a complementary chain that is complementary to the sequence of the nucleic acid aptamer, thereby preparing a polydimethylsiloxane film doped with a core-shell upconversion nanomaterial modified with an aptamer complementary chain.

[0011] Preferably, fluorescein isothiocyanate is dissolved in a solvent with 3-aminopropyltriethoxysilane to obtain a concentrated solution. The concentrated solution is added to a microemulsion composed of cyclohexane, n-hexanol, Triton X-100 and deionized water. Then, tetraethyl orthosilicate, ammonia and 3-aminopropyltriethoxysilane are added in sequence to react and obtain silicon nanofiber fluorescein isothiocyanate.

[0012] The silicon nano-sized fluorescein isothiocyanate is cross-linked with glutaraldehyde and an acrylamide aptamer to obtain aptamer-modified silicon nano-sized fluorescein isothiocyanate.

[0013] Preferably, when preparing the polydimethylsiloxane film doped with the core-shell upconversion nanomaterial, the core upconversion nanomaterial is prepared first, and then the core-shell upconversion nanomaterial is prepared.

[0014] The core-shell upconversion nanomaterial and polydimethylsiloxane pregel are cured with a curing agent to obtain a polydimethylsiloxane film doped with core-shell upconversion nanomaterial.

[0015] The polydimethylsiloxane film doped with the core-shell upconversion nanomaterial was immersed in an activation solution containing sodium periodate, benzyl alcohol, acrylic acid, and deionized water, and irradiated with ultraviolet light. After activating the polydimethylsiloxane film doped with the core-shell upconversion nanomaterial with a solution of 1-ethyl-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide, the polydimethylsiloxane film doped with the core-shell upconversion nanomaterial was modified with a complementary strand that is complementary to the sequence of the nucleic acid aptamer to obtain a polydimethylsiloxane film modified with the complementary strand of the aptamer.

[0016] Preferably, the steps for obtaining the core upconversion nanomaterial include: dissolving and mixing gadolinium chloride, ytterbium chloride, thulium chloride with oleic acid and octadecene, and then heating and reacting them with ammonium fluoride under an inert gas atmosphere to obtain the core upconversion nanomaterial;

[0017] The steps for obtaining the core-shell upconversion nanomaterial include: dissolving yttrium chloride and terbium chloride and dissolving and mixing them with oleic acid and octadecene, and then heating and reacting them with the core upconversion nanomaterial and ammonium fluoride under an inert gas atmosphere to obtain the core-shell upconversion nanomaterial.

[0018] This invention provides an acrylamide-specific detection system based on an upconversion solid-phase sensor, comprising aptamer-modified silicon nanofibrillated isothiocyanate fluorescein and a polydimethylsiloxane film doped with a core-shell upconversion nanomaterial modified with an aptamer complementary chain.

[0019] The preparation method of aptamer-modified silicon nano-sized fluorescein isothiocyanate includes the following steps: preparing silicon nano-sized fluorescein isothiocyanate, and then modifying it with a nucleic acid aptamer that can bind acrylamide to prepare aptamer-modified silicon nano-sized fluorescein isothiocyanate.

[0020] A method for preparing a polydimethylsiloxane film doped with a core-shell upconversion nanomaterial modified with an aptamer complementary chain includes the following steps: preparing a polydimethylsiloxane film doped with a core-shell upconversion nanomaterial; then modifying the polydimethylsiloxane film doped with the core-shell upconversion nanomaterial using a complementary chain that is sequence-complementary to the nucleic acid aptamer; thereby preparing the polydimethylsiloxane film doped with a core-shell upconversion nanomaterial modified with an aptamer complementary chain.

[0021] Before use, the aptamer-modified silicon nano-fluorescein isothiocyanate was mixed and incubated with a polydimethylsiloxane film doped with a core-shell upconversion nanomaterial modified with the aptamer complementary chain, and then used to detect acrylamide.

[0022] This invention also provides an application of an acrylamide-specific detection system based on an upconversion solid-phase sensor in acrylamide detection.

[0023] This invention also provides a method for the specific detection of acrylamide based on an upconversion solid-phase sensor, using the aforementioned acrylamide specific detection system based on the upconversion solid-phase sensor.

[0024] Before detection, the aptamer-modified silicon nanoparticle fluorescein isothiocyanate was mixed and incubated with a polydimethylsiloxane film doped with a core-shell upconversion nanomaterial modified with the aptamer complementary chain, and then used to detect acrylamide.

[0025] During testing, the sample to be tested is crushed and dissolved, the supernatant is purified and added to the acrylamide-specific detection system after mixing and incubation. After the reaction, the polydimethylsiloxane film is taken out and its fluorescence intensity signal characteristic value is measured. The acrylamide content in the sample is calculated by using the acrylamide detection standard curve.

[0026] More preferably, when obtaining the acrylamide detection standard curve, a gradient dilution is performed using a standard acrylamide solution of known concentration, and then detection is performed using the acrylamide-specific detection system based on the upconversion solid-phase sensor to obtain the characteristic values ​​of the detection fluorescence intensity signal of acrylamide at different known concentrations, and the acrylamide detection standard curve is obtained by linear fitting.

[0027] This invention provides an acrylamide-specific detection kit based on an upconversion solid-phase sensor, characterized in that it includes the acrylamide-specific detection system based on the upconversion solid-phase sensor.

[0028] The beneficial effects of this invention are:

[0029] (1) This invention discloses a method for detecting acrylamide content based on an upconversion solid-phase sensor, which is a solid-state luminescent sensor based on upconversion and nano-sized fluorescein isothiocyanate for detecting acrylamide. Upconversion nanoparticles are doped into a polydimethylsiloxane film as fluorescent donors and connected to the complementary chain of an acrylamide aptamer; nano-sized fluorescein isothiocyanate acts as a fluorescent acceptor and is connected to the acrylamide aptamer. In the absence of acrylamide, the aptamer and its complementary chain bind through base pairing, causing the fluorescent acceptor to bind to the donor, and upconversion luminescence is quenched through fluorescence resonance energy transfer; when acrylamide is present, the aptamer preferentially and specifically binds to acrylamide, the fluorescent acceptor detaches from the donor surface, and the donor fluorescence recovers. Therefore, a highly specific acrylamide concentration detection system is constructed by utilizing the change in fluorescence intensity of the solid-state sensor.

[0030] (2) The specific detection system constructed in this invention, specifically the upconversion nanomaterial-fluorescein isothiocyanate fluorescence detection system, employs an optimized polydimethylsiloxane solid detection membrane, which features high transmittance, low cost, and easy modification. Compared with liquid sensors, it can effectively simplify the detection process and is expected to provide a convenient and visible means for real-time analysis, promoting the application of on-site detection. The aptamer, as a recognition molecule, exhibits high specificity for the detection of acrylamide, enabling real-time detection of acrylamide content during processing, overcoming the shortcomings of traditional methods, and is crucial for ensuring food safety.

[0031] (3) The linear concentration range of the ratio of acrylamide concentration to fluorescence intensity signal established by this invention is 0.001 to 10 μM, which has a wide linear detection range and a detection limit (LOD) of 0.001 μM. It can meet the high sensitivity detection of acrylamide content, has good versatility and field application prospects, and has higher sensitivity and portability than traditional methods. Attached Figure Description

[0032] Figure 1 Figure 1 shows the characterization of the prepared nanomaterials; Figure A is a transmission electron microscope (TEM) image of nano-sized fluorescein isothiocyanate, Figure B is a TEM image of core-shell upconversion nanoparticles, and Figure C is a scanning electron microscope (SEM) image of the prepared polydimethylsiloxane film doped with core-shell upconversion nanoparticles.

[0033] Figure 2 The preparation process of polydimethylsiloxane thin films doped with core-shell upconversion nanoparticles is shown in Figure (a), where Figure (b) shows the morphology of the mixed adhesive at the beginning of casting, Figure (c) shows the morphology of the mixed adhesive during the intermediate state of vacuum degassing, Figure (d) shows the mold fixing method during curing, Figure (e) shows the initial morphology of the thin film, and Figure (f) shows the morphology of the thin film after loading nano-sized fluorescein.

[0034] Figure 3 This diagram illustrates the preparation process and detection principle of an acrylamide-specific detection system based on an upconversion solid-phase sensor. In the figure, CUCNPs represents the core upconversion nanomaterial, CSUCNPs represents the core-shell upconversion nanomaterial, Mould represents the fixed template, CSUCNPs@PDMS represents a polydimethylsiloxane film doped with core-shell upconversion nanomaterials, CSUCNPs@PDMS-FITC@SiO2 represents a polydimethylsiloxane film loaded with silicon nanofluorescein, AAm represents acrylamide, and Aptamer-FITC@SiO2 represents aptamer-modified silicon nanofibrillated fluorescein isothiocyanate.

[0035] Figure 4The diagram illustrates the principle of fluorescence quenching in thin films; Figure A shows the absorption spectrum and upconversion fluorescence emission spectrum of silicon nanofluorescein; Figure B shows the upconversion luminescence decay curves of the thin film before and after binding with silicon nanofluorescein.

[0036] Figure 5 The results show the fluorescence response of the thin film to different concentrations of acrylamide; Figure A shows the spectrum of the thin film at different acrylamide concentrations, and Figure B shows the standard curve of the fitting between acrylamide concentration and the characteristic value of the film fluorescence signal.

[0037] Figure 6 Figure A shows the performance evaluation results of the upconversion detection thin film; Figure B shows the molecular structure models of the target and interfering components; Figure B shows the results of the selectivity and anti-interference tests. Detailed Implementation

[0038] Example 1

[0039] A method for preparing an acrylamide-specific detection system based on an upconversion solid-phase sensor, the specific preparation steps are as follows:

[0040] I. Preparation of aptamer-modified silicon nanoparticles of fluorescein isothiocyanate.

[0041] (1) Preparation of silicon nano-sized fluorescein isothiocyanate: 0.5 mg of fluorescein isothiocyanate was mixed with 0.05 mL of 3-aminopropyltriethoxysilane and 2.5 mL of ethanol and purged with nitrogen for 40 min to obtain a concentrated solution; 1.5 mL of cyclohexane, 1.6 mL of n-hexanol, 1.77 mL of Triton X-100 and 0.48 mL of deionized water were stirred for 20 min to form a microemulsion; 0.4 mL of the concentrated solution was added to the above microemulsion, and after stirring for 15 min again, 0.05 mL of tetraethyl orthosilicate was added, and after stirring for 30 min, 0.06 mL of ammonia water with a volume concentration of 30% was added, and after stirring for 24 h again; then 0.05 mL of 3-aminopropyltriethoxysilane was added and stirred for 24 h; finally, the solution was washed with ethanol and deionized water respectively to obtain silicon nano-sized fluorescein isothiocyanate.

[0042] Figure 1 A is a transmission electron microscope image of the prepared nano-sized fluorescein isothiocyanate. As can be seen from the image, fluorescein isothiocyanate was successfully nano-sized with uniform particle size, a diameter of about 40 nm, and good dispersibility.

[0043] (2) Preparation of aptamer-modified silicon nano-fluorescein isothiocyanate: 10 mg of silicon nano-fluorescein isothiocyanate obtained in step (1) was dissolved in 5 mL of phosphate buffer, and 1.25 mL of 25% glutaraldehyde was added and stirred for 2 h. After centrifugation and washing, the nanomaterial was dissolved again in 5 mL of phosphate buffer to obtain nano-fluorescein isothiocyanate solution. 0.1 mL of acrylamide aptamer solution (60 nM) was added. The aptamer sequence was 5'-NH2-CAG TCC AGG ACA GATTCG CGA GTG GTC GTG GTG AGG TGC GTG TAT GGG TGG TGG ATG AGT GTG TGG CCA CGTGGA TTT CAT TCAGCG ATT-3'. After incubation at 37℃ for 12 h, the aptamer-modified silicon nano-fluorescein isothiocyanate was obtained by washing.

[0044] II. Preparation of polydimethylsiloxane thin films doped with aptamer complementary chain modified core-shell upconversion nanomaterials.

[0045] (1) Preparation of core upconversion nanomaterials: 0.07434 g gadolinium chloride hexahydrate, 0.07593 g ytterbium chloride hexahydrate and 0.00150 g thulium chloride hexahydrate were accurately weighed, dissolved in 4 mL methanol by sonication for 10 min, and then transferred to a three-necked flask. 4 mL oleic acid and 7 mL octadecene were added. Under the protection of argon, the mixture was heated to 160 °C for the first time and magnetically stirred for 30 min, and then cooled to room temperature to obtain a coolant. 4 mL of methanol solution containing 0.06 g ammonium fluoride and 0.04 g sodium hydroxide was added dropwise to the coolant. The mixture was then reacted in a water bath at 50 °C for the first time for 40 min, and in a water bath at 70 °C for the second time for 30 min to allow the methanol in the solution to completely evaporate. Then, under the protection of argon, the mixture was heated to 300 °C for the second time and magnetically stirred for 1 h. After cooling to room temperature, the reaction product was obtained. Finally, the reaction product was washed with a mixed solution of ethanol and cyclohexane (volume ratio of 1:2) and vacuum dried to obtain the core upconversion nanomaterials.

[0046] (2) Preparation of core-shell upconversion nanomaterials: 0.12140 g of yttrium chloride hexahydrate and 0.00746 g of terbium chloride hexahydrate were added to 4 mL of methanol and sonicated for 10 min. The mixture was then transferred to a three-necked flask, and 4 mL of oleic acid and 7 mL of octadecene were added. Under argon protection, the mixture was heated to 160 °C for the first time and magnetically stirred for 30 min. After cooling to room temperature, a coolant was obtained. 4 mL of methanol solution containing 0.06 g of ammonium fluoride and 0.04 g of sodium hydroxide and 50 mg of core-shell upconversion nanomaterials were added to the core-shell upconversion nanomaterials. 5 mL of cyclohexane solution of the nanomaterial (obtained in step (1)) was added dropwise to the cooling liquid. Then, the reaction was carried out in a water bath at 50°C for 40 min for the first time, and in a water bath at 100°C for 10 min for the second time, so that the methanol in the solution could be completely evaporated. Then, under argon protection, the reaction was heated to 300°C for the second time and magnetically stirred for 1 h. After cooling to room temperature, the reaction product was obtained. Finally, the reaction product was purified with a mixed solution of ethanol and cyclohexane (volume ratio of 1:2) and vacuum dried to obtain the core-shell upconversion nanomaterial.

[0047] Figure 1 B is a transmission electron microscope image of the prepared core-shell upconversion nanoparticles. As can be seen from the image, the core-shell upconversion nanoparticles have a uniform particle size of about 45 nm and good dispersibility.

[0048] (3) Preparation of polydimethylsiloxane film doped with core-shell upconversion nanomaterials: 25 mg of core-shell upconversion nanomaterials obtained in step (2) were added to 10 g of polydimethylsiloxane pregel. The mixture was stirred for 24 h to mix evenly. Then, an active silane-based organosilicon curing agent (Dow Corning SYLGARD 184, USA) was added and stirred for 20 min to obtain a mixed adhesive. The mixed adhesive was poured into a template, degassed under vacuum, and compacted with a cover plate. After curing at 90 °C for 1.5 h, the film was demolded to obtain polydimethylsiloxane film doped with core-shell upconversion nanomaterials.

[0049] Figure 1 C is a scanning electron microscope image of the polydimethylsiloxane film doped with core-shell upconversion nanoparticles. As can be seen from the image, the polydimethylsiloxane film is smooth and the core-shell upconversion nanomaterials are uniformly distributed inside it. Figure 2 This describes the degassing process for polydimethylsiloxane film molding. The mold is a 5×5 metal groove mold, with unit dimensions of 1cm×1cm. Figure 2 As can be seen, the mixed adhesive is initially a liquid containing a large number of air bubbles. As degassing proceeds, the number of air bubbles gradually decreases until they disappear. Figure 2 AC is the degassing process for polydimethylsiloxane films. As vacuum degassing proceeds, the air bubbles in the adhesive gradually decrease in size until they disappear. Further encapsulation is then performed, followed by curing in an oven. Figure 2 D).

[0050] (4) Preparation of polydimethylsiloxane film modified with aptamer complementary chain: 0.01 g sodium periodate, 0.5 g benzyl alcohol, 10 g acrylic acid and 89.5 g deionized water were mixed evenly to prepare an activation solution. The polydimethylsiloxane film doped with core-shell upconversion nanomaterials obtained in step (3) was completely immersed in the solution and irradiated with ultraviolet light for 2.5 h. After the irradiation, the film was washed with distilled water at 70 °C. Then the film was transferred to 1 mL of 2-(N-morpholino)ethanesulfonic acid buffer (10 mM, pH = 6.0), and 0.5 mL of 1-ethyl-(3-dimethylaminopropyl)carbodiimide solution with a concentration of 2 mg / mL and 0.5 mL of N-hydroxysuccinimide solution with a concentration of 1 mg / mL were added for activation reaction. After washing with phosphate buffer, 0.1 mL of aptamer complementary chain solution (60 nM) was added. The aptamer complementary chain is 5'-NH2-AAT CGC TGA ATG AAA TCC ACG TGG CCA CAC ACT CATCCA CCA CCC ATA CAC GCA CCT CAC CAC GAC CAC TCG CGA ATC TGT CCT GGA CTG-3' was incubated at 37°C for 12 h to obtain a polydimethylsiloxane film modified with complementary chains.

[0051] III. Establishment of a specific detection system.

[0052] The 0.6 mL aptamer-modified silicon nanofiber isothiocyanate fluorescein solution (2 mg / mL) prepared in the above steps and one aptamer complementary chain modified polydimethylsiloxane film were dispersed in 2 mL phosphate buffer. After incubation, the solution and film formed a specific detection system. Figure 2 E and Figure 2 F represents the morphology of the polydimethylsiloxane film modified with aptamer complementary chain before and after loading nano-fluorescein. The film is orange after loading, indicating that the aptamer-modified silicon nano-fluorescein isothiocyanate was successfully bound to the film surface.

[0053] The preparation method and detection principle of the acrylamide-specific detection system based on the upconversion solid-phase sensor are as follows: Figure 3 As shown, in the specific detection system, the aptamer and its complementary chain bind through base pairing, and the binding of the fluorescent acceptor and donor quenches the upconversion membrane's luminescence. When acrylamide is added, the aptamer preferentially and specifically binds to acrylamide, causing the nano-sized fluorescein to detach from the membrane surface, thus restoring the membrane's fluorescence. The principle of upconversion fluorescence quenching of the membrane is illustrated in the diagram below. Figure 4 As shown, the absorption peak of silicon nanostructured fluorescein isothiocyanate completely overlaps with the upconversion emission peak at 450 nm, thus quenching the upconversion emission peak. Figure 4As shown in B, the presence of silicon nanofiber shortens the upconversion fluorescence lifetime of the film from 693.79 μs to 300.27 μs, indicating that the energy transfer caused fluorescence quenching.

[0054] Example 2

[0055] The specific preparation steps for establishing a standard curve for acrylamide detection are as follows:

[0056] (1) Determination of fluorescence intensity signal characteristic values ​​of acrylamide standard solutions: Eight specific detection systems, each consisting of 2.6 mL of solution and one film, were prepared. 0.4 mL of acrylamide standard solutions with concentrations of 0.001 μM, 0.01 μM, 0.1 μM, 1 μM, 5 μM, 10 μM, 50 μM, and 100 μM were added to each system, resulting in different concentrations of test solutions. Each concentration of acrylamide solution corresponded to one specific detection system, with a one-to-one correspondence. The fluorescence intensity signal characteristic value Y of the specific detection systems with different concentrations of acrylamide solution was then measured. Figure 4 As shown in Figure A, the fluorescence intensity of the specific detection system is highest at a wavelength of 450 nm. Therefore, the fluorescence intensity value at 450 nm was measured under excitation light with a wavelength of 980 nm. Figure 5 A represents the fluorescence signal of the detection system at different acrylamide concentrations. As can be seen from the figure, the fluorescence intensity of the detection system increases with the increase of acrylamide concentration.

[0057] (2) Establishing a standard curve for acrylamide detection: Establish the relationship between the logarithm of acrylamide concentration (c) and the characteristic value Y of the fluorescence intensity signal. The standard curve for acrylamide detection is obtained through linear fitting, as shown below. Figure 5 As shown in Figure B, the logarithm of acrylamide concentration and the characteristic value of fluorescence intensity signal exhibit a linear relationship in the concentration range of 0.001–10 μM, but deviate from the linear curve in the range of 10–100 μM. Therefore, the linear equation obtained in the linear range of 0.001–10 μM is Y = 5090.6log(c) + 15886, with a coefficient of determination R0. 2 =0.9975, and the detection limit is calculated to be 0.001 μM according to the formula LOD = 3σ / K (where σ is the standard deviation of 10 blank experiments and K is the slope of the standard curve).

[0058] Example 3

[0059] Solid-phase sensor for the specificity and anti-interference detection of acrylamide.

[0060] L-Asparagine (Asn), methacrylamide (MA), methacrylic acid (MAa), acrylic acid (Aa), and propionic acid (PA) were used as interfering substances. Figure 6A) Acrylamide (AAm) was used at a concentration 100 times higher than that used in the method of the present invention to evaluate its selectivity. The results are as follows: Figure 6 As shown in B, only the target acrylamide can cause a significant change in the fluorescence signal, and high concentrations of interfering substances do not cause a significant change in the fluorescence detection signal. Therefore, the detection method constructed in this invention has high specificity and high sensitivity to acrylamide.

[0061] Acrylamide was further added to each interfering substance system to evaluate its anti-interference ability; the results are as follows: Figure 6 As shown in Figure B, the addition of acrylamide caused a significant change in the signal value, indicating that the constructed solid-phase sensor has high specificity and anti-interference ability for acrylamide.

[0062] Example 4

[0063] Detection of acrylamide content in food samples.

[0064] A 1g sample of commercially available potato chips was crushed, 1mL of deionized water was added, and the mixture was shaken and sonicated for 40min. The supernatant was then purified using a solid-phase extraction column, and 0.4mL of the supernatant was added to the specific detection system constructed in Example 1, which consisted of 2.6mL of detection solution and one membrane. The fluorescence intensity signal characteristic value of the specific detection system was measured. The acrylamide content in the food sample was calculated using the acrylamide detection standard curve established in Example 2.

[0065] Detection Example 1

[0066] High-performance liquid chromatography (HPLC) is commonly used in standards for detecting acrylamide content in food samples. To further evaluate the accuracy and practical feasibility of the method described in this invention, the results of detecting acrylamide content using this method were compared with those using standard methods. Three potato chip samples with randomly added unknown concentrations of acrylamide were analyzed using the constructed acrylamide fluorescence detection method. After measuring the fluorescence intensity, the fluorescence values ​​were substituted into the standard curve equation to calculate the acrylamide content. Simultaneously, HPLC was used to detect the same samples, and the acrylamide content in the potato chip samples was calculated. The significant differences between the HPLC detection results and the fluorescence detection results were compared.

[0067] Table 1 Comparison of acrylamide content in food standard samples detected by the method of this invention and the standard method (unit: μM)

[0068]

[0069] As shown in Table 1, the detection results of the two methods are quite similar. The test results show that the P-value is 0.56, which is much greater than 0.05, indicating that there is no significant difference between the constructed acrylamide fluorescence detection method and the high-performance liquid chromatography method. All these results demonstrate that the acrylamide detection method proposed in this study has excellent accuracy and practicality in actual sample detection.

Claims

1. A method for preparing an acrylamide-specific detection system based on an upconversion solid-phase sensor, characterized by, The acrylamide-specific detection system based on an upconversion solid-phase sensor includes aptamer-modified silicon nano-fluorescein isothiocyanate and a polydimethylsiloxane film doped with a core-shell upconversion nanomaterial modified with an aptamer complementary chain. The preparation method of aptamer-modified silicon nano-sized fluorescein isothiocyanate includes the following steps: preparing silicon nano-sized fluorescein isothiocyanate, and then modifying it with a nucleic acid aptamer that can bind acrylamide to prepare aptamer-modified silicon nano-sized fluorescein isothiocyanate. The method for preparing a polydimethylsiloxane film doped with a core-shell upconversion nanomaterial modified with an aptamer complementary chain includes the following steps: preparing a polydimethylsiloxane film doped with a core-shell upconversion nanomaterial, and then modifying the polydimethylsiloxane film doped with a core-shell upconversion nanomaterial doped with a complementary chain that is complementary to the sequence of the nucleic acid aptamer, thereby preparing a polydimethylsiloxane film doped with a core-shell upconversion nanomaterial modified with an aptamer complementary chain.

2. The method for preparing the acrylamide-specific detection system based on an upconversion solid-phase sensor according to claim 1, characterized in that, Fluorescein isothiocyanate was dissolved in a solvent to obtain a concentrated solution. The concentrated solution was added to a microemulsion composed of cyclohexane, n-hexanol, Triton X-100 and deionized water. Then, tetraethyl orthosilicate, ammonia and 3-aminopropyltriethoxysilane were added in sequence to react and obtain silicon nanofiber fluorescein isothiocyanate. The silicon nano-sized fluorescein isothiocyanate is cross-linked with glutaraldehyde and an acrylamide aptamer to obtain aptamer-modified silicon nano-sized fluorescein isothiocyanate.

3. The method for preparing the acrylamide-specific detection system based on an upconversion solid-phase sensor according to claim 1, characterized in that, When preparing the polydimethylsiloxane film doped with the core-shell upconversion nanomaterial, the core upconversion nanomaterial is prepared first, and then the core-shell upconversion nanomaterial is prepared. The core-shell upconversion nanomaterial and polydimethylsiloxane pregel are cured with a curing agent to obtain a polydimethylsiloxane film doped with core-shell upconversion nanomaterial. The polydimethylsiloxane film doped with the core-shell upconversion nanomaterial was immersed in an activation solution containing sodium periodate, benzyl alcohol, acrylic acid, and deionized water, and irradiated with ultraviolet light. After activating the polydimethylsiloxane film doped with the core-shell upconversion nanomaterial with a solution of 1-ethyl-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide, the polydimethylsiloxane film doped with the core-shell upconversion nanomaterial was modified with a complementary strand that is complementary to the sequence of the nucleic acid aptamer to obtain a polydimethylsiloxane film doped with the core-shell upconversion nanomaterial modified with the complementary strand.

4. The method for preparing the acrylamide-specific detection system based on an upconversion solid-phase sensor according to claim 3, characterized in that, The steps for obtaining the core upconversion nanomaterial include: dissolving and mixing gadolinium chloride, ytterbium chloride, thulium chloride with oleic acid and octadecene, and then heating and reacting them with ammonium fluoride under an inert gas atmosphere to obtain the core upconversion nanomaterial. The preparation steps of the core-shell upconversion nanomaterial include: dissolving yttrium chloride and terbium chloride and dissolving and mixing them with oleic acid and octadecene, and then heating and reacting them with the core upconversion nanomaterial and ammonium fluoride under inert gas protection to obtain the core-shell upconversion nanomaterial.

5. The application of an acrylamide-specific detection system based on an upconversion solid-phase sensor in acrylamide detection, characterized in that, The acrylamide-specific detection system based on upconversion solid-phase sensors includes aptamer-modified silicon nano-fluorescein isothiocyanate and polydimethylsiloxane films doped with core-shell upconversion nanomaterials modified with aptamer complementary chains. The preparation method of aptamer-modified silicon nano-sized fluorescein isothiocyanate includes the following steps: preparing silicon nano-sized fluorescein isothiocyanate, and then modifying it with a nucleic acid aptamer that can bind acrylamide to prepare aptamer-modified silicon nano-sized fluorescein isothiocyanate. A method for preparing a polydimethylsiloxane film doped with a core-shell upconversion nanomaterial modified with an aptamer complementary chain includes the following steps: preparing a polydimethylsiloxane film doped with a core-shell upconversion nanomaterial; then modifying the polydimethylsiloxane film doped with the core-shell upconversion nanomaterial using a complementary chain that is sequence-complementary to the nucleic acid aptamer; thereby preparing the polydimethylsiloxane film doped with a core-shell upconversion nanomaterial modified with an aptamer complementary chain. Before use, the aptamer-modified silicon nano-fluorescein isothiocyanate was mixed and incubated with a polydimethylsiloxane film doped with a core-shell upconversion nanomaterial modified with the aptamer complementary chain, and then used to detect acrylamide.

6. A method for specific detection of acrylamide based on upconversion solid phase sensor, characterized in that, An acrylamide-specific detection system based on an upconversion solid-phase sensor was used. The acrylamide-specific detection system based on an upconversion solid-phase sensor includes aptamer-modified silicon nano-fluorescein isothiocyanate and polydimethylsiloxane film doped with core-shell upconversion nanomaterials modified with aptamer complementary chains. The preparation method of aptamer-modified silicon nano-sized fluorescein isothiocyanate includes the following steps: preparing silicon nano-sized fluorescein isothiocyanate, and then modifying it with a nucleic acid aptamer that can bind acrylamide to prepare aptamer-modified silicon nano-sized fluorescein isothiocyanate. A method for preparing a polydimethylsiloxane film doped with a core-shell upconversion nanomaterial modified with an aptamer complementary chain includes the following steps: preparing a polydimethylsiloxane film doped with a core-shell upconversion nanomaterial; then modifying the polydimethylsiloxane film doped with the core-shell upconversion nanomaterial using a complementary chain that is sequence-complementary to the nucleic acid aptamer; thereby preparing the polydimethylsiloxane film doped with a core-shell upconversion nanomaterial modified with an aptamer complementary chain. Before detection, the aptamer-modified silicon nanoparticle fluorescein isothiocyanate was mixed and incubated with a polydimethylsiloxane film doped with a core-shell upconversion nanomaterial modified with the aptamer complementary chain, and then used to detect acrylamide. During testing, the sample to be tested is crushed and dissolved, the supernatant is purified and added to the acrylamide-specific detection system after mixing and incubation. After the reaction, the polydimethylsiloxane film is taken out and its fluorescence intensity signal characteristic value is measured. The acrylamide content in the sample is calculated by using the acrylamide detection standard curve.

7. The method of claim 6, wherein the acrylamide specific detection method is based on an upconversion solid-phase sensor. To obtain the acrylamide detection standard curve, a standard solution of acrylamide with a known concentration was used for gradient dilution, and then the acrylamide-specific detection system based on the upconversion solid-phase sensor was used for detection to obtain the fluorescence intensity signal characteristic values ​​of acrylamide at different known concentrations. The acrylamide detection standard curve was obtained by linear fitting.

8. A specific assay kit for acrylamide based on upconversion solid phase sensor, characterized by, This includes an acrylamide-specific detection system based on an upconversion solid-phase sensor, which includes aptamer-modified silicon nanofiber isothiocyanate fluorescein and polydimethylsiloxane films doped with core-shell upconversion nanomaterials modified with aptamer complementary chains. The preparation method of aptamer-modified silicon nano-sized fluorescein isothiocyanate includes the following steps: preparing silicon nano-sized fluorescein isothiocyanate, and then modifying it with a nucleic acid aptamer that can bind acrylamide to prepare aptamer-modified silicon nano-sized fluorescein isothiocyanate. A method for preparing a polydimethylsiloxane film doped with a core-shell upconversion nanomaterial modified with an aptamer complementary chain includes the following steps: preparing a polydimethylsiloxane film doped with a core-shell upconversion nanomaterial; then modifying the polydimethylsiloxane film doped with the core-shell upconversion nanomaterial using a complementary chain that is sequence-complementary to the nucleic acid aptamer; thereby preparing the polydimethylsiloxane film doped with a core-shell upconversion nanomaterial modified with an aptamer complementary chain. Before use, the aptamer-modified silicon nano-fluorescein isothiocyanate was mixed and incubated with a polydimethylsiloxane film doped with a core-shell upconversion nanomaterial modified with the aptamer complementary chain, and then used to detect acrylamide.

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