Method for detecting aflatoxin in food products
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- SHANDONG DINGKE TESTING TECHNOLOGY CO LTD
- Filing Date
- 2026-05-28
- Publication Date
- 2026-06-26
Smart Images
Figure CN122282736A_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of aflatoxin detection technology, specifically relating to a method for detecting aflatoxin in grain products. Background Technology
[0002] Aflatoxin (especially aflatoxin B1, AFB1), a highly toxic secondary metabolite produced by fungi such as Aspergillus flavus during grain storage and processing, widely contaminates various grains and oil crops such as corn, peanuts, and rice. This toxin possesses strong carcinogenic, teratogenic, and hepatotoxic and nephrotoxic properties, posing a serious threat to human health and leading to significant losses in grain resources and economic damage. Therefore, developing a rapid, accurate, and sensitive detection technology for AFB1 in grains is a crucial requirement for ensuring food safety, protecting public health, and standardizing quality control in the grain industry.
[0003] Currently, the main methods for detecting AFB1 in grains include high-performance liquid chromatography (HPLC), liquid chromatography-mass spectrometry (LC-MS / MS), enzyme-linked immunosorbent assay (ELISA), fluorescence sensing, and electrochemical sensing. Among these, while HPLC and LC-MS / MS offer high detection accuracy and precise quantification, they rely on large, sophisticated instruments, have complex procedures, are costly, and time-consuming, making them unsuitable for rapid on-site screening and batch sample testing. ELISA is simple to operate and has a lower cost; however, it suffers from drawbacks such as antibody inactivation, poor specificity, and a high false-positive rate, and its sensitivity is insufficient for accurate quantification of trace AFB1.
[0004] In recent years, aptamer sensing technology based on nanomaterial modification has become a research hotspot in the field of rapid AFB1 detection. Aptamers possess high specificity and good stability, while nanomaterials have unique optical and electrochemical properties; the combination of the two provides a new approach for AFB1 detection. Existing technologies have constructed sensing systems based on CeO2, Fe-based nanomaterials, and SiO2-coated structures, aiming to improve detection performance by optimizing material properties. However, this field still faces many technical bottlenecks that urgently need to be overcome.
[0005] On the one hand, the limited conductivity and electron transfer efficiency of traditional nanomaterials result in insufficient detection sensitivity, making it difficult to accurately capture and quantify trace amounts of AFB1 (below ng / mL). On the other hand, grain extract systems are complex, containing various impurities such as proteins, polysaccharides, and oils. Existing nanomaterials exhibit poor dispersibility and stability, are easily affected by matrix interference, and have limited binding capacity with AFB1, leading to poor detection specificity and anti-interference performance. Furthermore, most existing sensing materials possess only a single signal response characteristic, are compatible with limited detection platforms, and lack sufficient salt and acid / alkali resistance in complex matrices, making them unsuitable for different grain pretreatment systems and limiting practical applications. In addition, most sensing materials lack recyclability, resulting in high costs per detection and failing to meet the practical needs of large-scale, routine detection in the grain industry.
[0006] Therefore, developing an AFB1 detection material that combines high sensitivity, high specificity, strong stability, and good adaptability to practical applications has become a key technical problem that urgently needs to be solved in this field. Summary of the Invention
[0007] The purpose of this invention is to provide a method for detecting aflatoxin in grain products, so as to solve the above-mentioned technical problems.
[0008] To achieve the above-mentioned technical objectives, the technical solution of the present invention is as follows:
[0009] The method for detecting aflatoxin in grain products includes the following steps:
[0010] S1: Mix the modified nanosensor composite solution with the sample solution to be tested, then bring the volume up with PBS buffer, vortex, and incubate at 37°C in the dark for 20 min. Measure the fluorescence intensity F of the sample solution to be tested. 样 ;
[0011] S2: Set up a sample blank. Mix the modified nanosensor composite solution with the sample blank solution, then bring the volume up with PBS buffer, vortex, and incubate at 37°C in the dark for 20 min. Measure the fluorescence intensity F of the sample blank. 样品空白 ;
[0012] S3: Calculate the actual fluorescence intensity F 测 = F 样 -F 样品空白 The actual fluorescence intensity F 测 Substituting into the standard curve regression equation, the AFB1 concentration c in the sample solution to be tested is calculated. 测 Based on this, the actual content of AFB1 in the grain samples was calculated;
[0013] The standard curve regression equation is: ΔF = 98.6c 测 +12.8;
[0014] The formula for calculating the actual content of AFB1 in grain samples is as follows: ;
[0015] Where X represents the actual content of AFB1 in the grain sample, and the unit is... c 测 V indicates the concentration of AFB1 in the sample solution to be tested, in ng / mL; 定容 The volume represents the final volume after pretreatment, in mL; D represents the dilution factor during the detection process, where 100 μL of the sample solution is diluted to 2 mL, which is a 20-fold dilution; m represents the mass of the grain sample, in g.
[0016] As a further improvement, the specific preparation process of the modified nanosensing composite liquid is as follows:
[0017] [C4(MIM)2]F2 was dissolved in Tris-HCl buffer and sonicated until completely dissolved to obtain the [C4(MIM)2]F2 modified solution;
[0018] The nanosensing complex was dispersed in [C4(MIM)2]F2 modification solution, incubated with shaking at 37°C for 1 h, centrifuged for 10 min, and washed 3 times to obtain the modified nanosensing complex. The modified nanosensing complex was diluted with PBS buffer and ultrasonically dispersed for 30 min to obtain the final product.
[0019] As a further improvement, the specific preparation process of the nanosensing composite is as follows:
[0020] Tb,Fe-CeO2@SiO2 powder was dispersed in anhydrous ethanol-deionized water mixture and sonicated for 30 min. γ-aminopropyltriethoxysilane was added and the mixture was stirred and reacted at 30 °C under N2 protection for 12 h. After the reaction was completed, the mixture was centrifuged and washed 3 times and then vacuum dried at 60 °C for 6 h to obtain aminated Tb,Fe-CeO2@SiO2.
[0021] Aminated Tb,Fe-CeO2@SiO2 was dispersed in MES buffer and ultrasonically dispersed evenly. EDC·HCl / NHS mixed activation reagent was added and activated by stirring in the dark at room temperature for 30 min. Carboxylated AFB1 nucleic acid aptamer stock solution was added and reacted in the dark at room temperature and under N2 protection for 24 h. After the reaction was completed, the mixture was centrifuged and washed 3 times to remove the supernatant. PBS buffer was then added and ultrasonically dispersed in an ice bath for 30-60 min, followed by centrifugation for 10-15 min to obtain the nucleic acid aptamer dispersion.
[0022] Add the nucleic acid aptamer dispersion to the AFB1 standard solution, bring the volume up to PBS buffer, incubate with shaking at room temperature for 30 min, centrifuge at 8000 rpm for 10 min, remove the supernatant, and collect the precipitate to obtain the final product.
[0023] As a further improvement, the specific preparation process of the Tb,Fe-CeO2@SiO2 powder is as follows:
[0024] Hexadecyltrimethylammonium bromide was added to a mixed solvent prepared from deionized water and anhydrous ethanol, and stirred at room temperature until completely dissolved to obtain a hexadecyltrimethylammonium bromide solution.
[0025] Under ultrasonic and stirring conditions, Tb,Fe-CeO2 dispersion was added dropwise to hexadecyltrimethylammonium bromide solution. After the addition was complete, ultrasonication was continued for 30 min to obtain a mixed system. Tetraethyl orthosilicate was mixed with anhydrous ethanol to prepare TEOS ethanol solution.
[0026] Under stirring at 300 rpm, TEOS ethanol solution was added dropwise to the mixture. After the addition was complete, the mixture was stirred at 300 rpm for 24 h at room temperature. Then, NH4NO3 buffer solution was added and stirred for 10 min. The mixture was then centrifuged and ultrasonically dispersed three times. After centrifugation, the supernatant was removed, the precipitate was collected, and the precipitate was vacuum dried at 40-60℃ for 6-12 h. Finally, the precipitate was ground to obtain the final product.
[0027] As a further improvement, the specific preparation process of the Tb,Fe-CeO2 dispersion is as follows:
[0028] Terbium nitrate hexahydrate, ferric nitrate nonahydrate, and polyvinylpyrrolidone were added sequentially to cerium nitrate hexahydrate and stirred until homogeneous. Sodium hydroxide solution was then added and mixed. Ethanol and ethylene glycol were added and stirred at room temperature for 2 hours. The mixture was then subjected to a solvothermal reaction at 160°C for 24 hours. After cooling to room temperature, the mixture was washed three times, vacuum dried at 80°C for 12 hours, calcined at 500°C for 2 hours, cooled to room temperature, and pulverized to obtain Tb,Fe-CeO2 nanoparticles.
[0029] Tb,Fe-CeO2 nanopowder was added to a mixed solvent of deionized water and anhydrous ethanol, stirred and pre-dispersed, and the pH of the system was adjusted to 10 with 30% concentrated ammonia. The mixture was stirred at 300 rpm for 15 min and then sonicated for 15 min to obtain the final product.
[0030] As a further improvement, the mass ratio of cerium nitrate hexahydrate, terbium nitrate hexahydrate, ferric nitrate nonahydrate, and polyvinylpyrrolidone is 21.5:1.1~2.2:0.9~1.8:1~2.
[0031] As a further improvement, the carboxylated AFB1 aptamer is a nucleotide sequence as shown in SEQ ID NO:1 with a carboxyl group modified at the 5' end.
[0032] As a further improvement, the specific preparation process of [C4(MIM)2]F2 is as follows:
[0033] 1-Methylimidazole and 1,4-dibromobutane were dissolved in anhydrous acetonitrile and refluxed and stirred at 80 °C under N2 protection for 48 h. After cooling to room temperature, the acetonitrile was removed by rotary evaporation. The mixture was washed three times with ethyl acetate, filtered, and dried under vacuum at 60 °C for 24 h to obtain [C4(MIM)2]Br2.
[0034] The strongly basic anion exchange resin was soaked in NaF solution for 24 hours to convert it into F. - Type 1 resin; wash with deionized water, filter, and collect F. - Type resin; dissolve [C4(MIM)2]Br2 in deionized water, add F - Type I resin, shaken and stirred at room temperature for 24 hours, then filtered to remove F - The resin was concentrated by rotary evaporation, and the filtrate was dried under vacuum at 60°C for 24 hours to obtain the final product.
[0035] As a further improvement, the mass ratio of [C4(MIM)2]F2 to the nanosensing composite is 0.1~0.15:0.3~0.6.
[0036] Due to the adoption of the above technical solution, the beneficial effects of the present invention are as follows:
[0037] The method for detecting aflatoxin in grain products provided by this invention demonstrates the beneficial effects of the modified nanosensor complex in aflatoxin detection in three dimensions: improved detection performance, enhanced specificity, and optimized adaptability for practical applications.
[0038] 1. The ionic liquid [C4(MIM)2]F2 can significantly improve the conductivity and electron transfer efficiency of the nanosensor composite. Combined with the enzyme-like catalytic activity of Tb,Fe-CeO2, it can significantly reduce the detection limit and improve the sensitivity, thus achieving the accurate quantification of trace AFB1 in grains.
[0039] 2. The SiO2 coating enhances the stability and dispersibility of Tb,Fe-CeO2, reduces the adsorption interference of impurities in the grain matrix, and, combined with the high specificity recognition function of the AFB1 aptamer, significantly reduces false positive results and greatly improves the accuracy and anti-interference ability of the detection results. At the same time, the modified nanosensor complex has both the fluorescence properties of Tb and the electrochemical activity of CeO2 / Fe, and has dual optical / electrochemical signal response characteristics. It can be adapted to a variety of detection platforms, is easy to operate and has a fast detection speed, and can meet the requirements of batch and rapid on-site screening of grain samples.
[0040] 3. The hydrophobicity and coordination of [C4(MIM)2]F2 not only enhances the binding ability of the nanosensor composite to AFB1 in the grain sample, but also improves the salt and acid / alkali resistance of the material in the complex system of grain extract. It can be adapted to the pretreatment system of different grains such as cereals and oilseeds, and has strong practicality. In addition, the designability of the nanocomposite structure also makes it reusable, which can effectively reduce the cost of a single detection and adapt to the large-scale and routine detection applications in the grain industry. Attached Figure Description
[0041] Figure 1 This is the standard curve for detecting AFB1 fluorescence in the modified nanosensor composite liquid in Example 1;
[0042] Figure 2 This is a specific detection diagram of the modified nanosensor composite. Detailed Implementation
[0043] The technical solution of the present invention will be clearly and completely described below with reference to specific embodiments. However, those skilled in the art will understand that the embodiments described below are some embodiments of the present invention, but not all embodiments, and are only used to illustrate the present invention, and should not be regarded as limiting the scope of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall be followed. Where the manufacturers of reagents or instruments are not specified, they are all conventional products that can be purchased commercially.
[0044] Example 1: A method for detecting aflatoxin in grain products, comprising the following steps:
[0045] S1. To 21.5g of cerium nitrate hexahydrate, add 1.6g of terbium nitrate hexahydrate, 1.4g of ferric nitrate nonahydrate, and 1.5g of polyvinylpyrrolidone in sequence, stir well, then add 100mL of sodium hydroxide solution to provide a strong alkaline environment, then add 30mL of ethanol and 30mL of ethylene glycol, stir at 300rpm for 2h at room temperature, then solvothermal reaction at 160℃ for 24h, cool to room temperature, wash three times with 70mL of deionized water and anhydrous ethanol respectively, vacuum dry at 80℃ for 12h, calcine at 500℃ for 2h by heating at 10℃ / min, cool to room temperature, and pulverize to obtain Tb,Fe-CeO2 nanoparticles.
[0046] S2. Add 1.6g of Tb,Fe-CeO2 nanopowder to a mixed solvent of 60mL deionized water and anhydrous ethanol at a volume ratio of 1:4. Pre-disperse the mixture by stirring at 300rpm. Adjust the pH of the system to 10 with 30% concentrated ammonia. Stir at 300rpm for 15min and then sonicate for 15min to obtain a Tb,Fe-CeO2 dispersion.
[0047] S3. Add 4.5 g of hexadecyltrimethylammonium bromide (CTAB) to a mixed solvent of 80 mL deionized water and 20 mL anhydrous ethanol. Stir at 300 rpm at room temperature until CTAB is completely dissolved to obtain a clear CTAB solution. Under continuous sonication and stirring at 300 rpm, add a Tb,Fe-CeO2 dispersion dropwise to the CTAB solution at a rate of 1.5 mL / min. After the addition is complete, sonicate for 30 min to obtain a mixed system. Mix 8.64 g of tetraethyl orthosilicate (TEOS) with 80 mL of anhydrous ethanol to prepare a TEOS ethanol solution. Under stirring at 300 rpm, add the TEOS ethanol solution dropwise to the mixed system at a rate of 0.75 mL / min. After the addition is complete, sonicate for 2 min and stir at 300 rpm at room temperature for 24 h. Then add 45 mL of NH4NO3 buffer solution and stir for 10 min. Perform three centrifugation-sonication dispersion purification cycles (centrifugation at 8000 rpm for 10 minutes each). After centrifugation for 1 minute, the precipitate was dispersed with deionized water and sonicated for 10 minutes for the first two centrifugations. After centrifugation for the third time, the precipitate was dispersed with anhydrous ethanol and sonicated for 10 minutes. The supernatant was discarded after each centrifugation. After centrifugation, the supernatant was discarded, and the bottom precipitate was collected. The precipitate was vacuum dried at 50℃ for 9 hours and ground to obtain Tb,Fe-CeO2@SiO2 powder.
[0048] S4. Disperse 50 mg of Tb,Fe-CeO2@SiO2 powder in 50 mL of anhydrous ethanol-deionized water mixture (volume ratio 9:1), sonicate for 30 min to ensure uniform dispersion, add 225 μL of γ-aminopropyltriethoxysilane (APTES), and stir the reaction at 30 °C under N2 protection for 12 h. After the reaction is completed, wash the powder three times with anhydrous ethanol and deionized water at 8000 rpm for 10 min each time, and dry it under vacuum at 60 °C for 6 h to obtain aminated Tb,Fe-CeO2@SiO2.
[0049] S5. Prepare a 10 μmol / L stock solution of carboxylated AFB1 aptamer using 0.01 mol / L PBS (pH 7.4) buffer; prepare a 0.1 mol / L EDC·HCl / NHS mixed activating reagent (molar ratio of the two is 2:1). The carboxylated AFB1 aptamer is a nucleotide sequence with a carboxyl group modified at the 5' end, as shown in SEQ ID NO:1, specifically 5'-COOH-GTTGGGCACGTGTTGTCTCTCTGTGTCTCGTGCCCTTCGCTAGGCCC-3', where the sequence SEQ ID NO:1 is 5'-GTTGGGCACGTGTTGTCTCTCTCTGTGTCTCGTGCCCTTCGCTAGGCCC-3'.
[0050] S6. Disperse 35 mg of aminoated Tb,Fe-CeO2@SiO2 in 20 mL of 0.01 mol / L MES (pH 6.0) buffer, sonicate for 20 min to ensure uniform dispersion, add 1 mL of EDC·HCl / NHS mixed activation reagent, and activate by stirring at room temperature in the dark for 30 min. After activation, add 2.5 mL of 10 μmol / L carboxylated AFB1 nucleic acid aptamer stock solution, and react at room temperature and under N2 protection in the dark for 24 h with stirring. After the reaction, wash three times with 0.01 mol / L PBS (pH 7.4) buffer at 8000 rpm for 10 min each time, discard the supernatant, add 20 mL of 0.01 mol / L PBS buffer, sonicate for 45 min under ice bath conditions, and centrifuge at 9000 rpm for 13 min to obtain Tb,Fe-CeO2@SiO2@nucleic acid aptamer dispersion (nucleic acid aptamer dispersion).
[0051] S7. Take 100 μL of Tb,Fe-CeO2@SiO2@nucleic acid aptamer dispersion, add 20 ng / mL of AFB1 standard solution, and adjust the volume to 1 mL with PBS buffer. Incubate at room temperature with shaking for 30 min to allow the aptamer to fully bind with AFB1. Centrifuge at 8000 rpm for 10 min, discard the supernatant, collect the precipitate, and obtain the Tb,Fe-CeO2@SiO2@aptamer-AFB1 complex (nanosensing complex).
[0052] S8. Dissolve 1.642 g of 1-methylimidazole and 2.159 g of 1,4-dibromobutane in 30 mL of anhydrous acetonitrile. Reflux and stir at 80 °C under N2 protection for 48 h. Cool to room temperature, remove acetonitrile by rotary evaporation, wash three times with ethyl acetate, filter, and dry under vacuum at 60 °C for 24 h to obtain [C4(MIM)2]Br2. Take 10 g of strongly basic anion exchange resin (OH... - (Type), soaked in 50 mL of 1 mol / L NaF solution for 24 h, is converted to F. - Type resin, washed with deionized water F - Type-3 resin, until the effluent tests negative for F-ions using fluoride ion test paper. -Filter and collect F - For the type of resin, dissolve 1.901 g of [C4(MIM)2]Br2 in 20 mL of deionized water, and add F. - Type I resin, shaken and stirred at room temperature for 24 hours, then filtered to remove F - The resin was concentrated by rotary evaporation, and the filtrate was dried under vacuum at 60°C for 24 hours to obtain [C4(MIM)2]F2.
[0053] S9. Dissolve 0.125g of [C4(MIM)2]F2 in 15mL of 0.05mol / L Tris-HCl (pH 8.0) buffer and sonicate for 5min until completely dissolved to obtain the [C4(MIM)2]F2 modified solution; add 0.45g of... The Tb,Fe-CeO2@SiO2@aptor-AFB1 complex was dispersed in 5 mL of [C4(MIM)2]F2 modification solution and incubated with shaking at 37 °C for 1 h. This allowed the ionic liquid to adsorb onto the surface of the Tb,Fe-CeO2@SiO2@aptor-AFB1 complex via electrostatic and hydrophobic interactions. After incubation, the mixture was centrifuged at 10000 rpm for 10 min and washed three times with Tris-HCl buffer to remove unadsorbed free ionic liquid, yielding the [C4(MIM)2]F2-modified Tb,Fe-CeO2@SiO2@aptor-AFB1 complex (modified nanosensor complex). The [C4(MIM)2]F2-modified Tb,Fe-CeO2@SiO2@aptor-AFB1 complex was then subjected to 0.01 mol / L PBS. The modified nanosensor composite solution was obtained by diluting the buffer solution (pH 7.4) to a concentration of 1 mg / mL and ultrasonically dispersing it for 30 min.
[0054] Example 2: A method for detecting aflatoxin in grain products, comprising the following steps:
[0055] S1. To 21.5g of cerium nitrate hexahydrate, 1.1g of terbium nitrate hexahydrate, 0.9g of ferric nitrate nonahydrate, and 1g of polyvinylpyrrolidone were added sequentially and stirred until homogeneous. Then, 100mL of sodium hydroxide solution was added and mixed to provide a strong alkaline environment. Next, 30mL of ethanol and 30mL of ethylene glycol were added. The mixture was stirred at 300rpm for 2 hours at room temperature, and then solvothermal reacted at 160℃ for 24 hours. After cooling to room temperature, the mixture was washed three times with 70mL of deionized water and anhydrous ethanol, respectively. It was then vacuum dried at 80℃ for 12 hours, calcined at 500℃ at a rate of 10℃ / min for 2 hours, cooled to room temperature, and pulverized to obtain Tb,Fe-CeO2 nanoparticles.
[0056] S2. Add 1.2g of Tb,Fe-CeO2 nanopowder to a mixed solvent of 60mL deionized water and anhydrous ethanol at a volume ratio of 1:4. Stir at 300rpm to pre-disperse the mixture. Adjust the pH of the system to 10 with 30% concentrated ammonia. Stir at 300rpm for 15min and then sonicate for 15min to obtain a Tb,Fe-CeO2 dispersion.
[0057] S3. Add 3g of hexadecyltrimethylammonium bromide (CTAB) to a mixed solvent of 80mL deionized water and 20mL anhydrous ethanol. Stir at 300rpm at room temperature until CTAB is completely dissolved to obtain a clear CTAB solution. Under continuous sonication and stirring at 300rpm, add a Tb,Fe-CeO2 dispersion dropwise to the CTAB solution at a rate of 1mL / min. After the addition is complete, sonicate for 30min to obtain a mixed system. Mix 5.64g of tetraethyl orthosilicate (TEOS) with 80mL of anhydrous ethanol to prepare a TEOS ethanol solution. Under stirring at 300rpm, add the TEOS ethanol solution dropwise to the mixed system at a rate of 0.5mL / min. After the addition is complete, sonicate for 2min and stir at 300rpm at room temperature for 24h. Then add 40mL of NH4NO3 buffer solution and stir for 10min. Perform three centrifugation-sonication dispersion purification cycles (centrifugation at 8000rpm for 10 minutes each). After centrifugation for 1 minute, the precipitate was dispersed with deionized water and sonicated for 10 minutes for the first two centrifugations. After centrifugation for the third time, the precipitate was dispersed with anhydrous ethanol and sonicated for 10 minutes. The supernatant was discarded after each centrifugation. After centrifugation, the supernatant was discarded, and the bottom precipitate was collected. The precipitate was vacuum dried at 40℃ for 6 hours and ground to obtain Tb,Fe-CeO2@SiO2 powder.
[0058] S4. Disperse 50 mg of Tb,Fe-CeO2@SiO2 powder in 50 mL of anhydrous ethanol-deionized water mixture (volume ratio 9:1), sonicate for 30 min to ensure uniform dispersion, add 200 μL of γ-aminopropyltriethoxysilane (APTES), and stir the reaction at 30 °C under N2 protection for 12 h. After the reaction is completed, wash the powder three times with anhydrous ethanol and deionized water at 8000 rpm for 10 min each time, and dry it under vacuum at 60 °C for 6 h to obtain aminated Tb,Fe-CeO2@SiO2.
[0059] S5. Prepare a 10 μmol / L stock solution of carboxylated AFB1 aptamer using 0.01 mol / L PBS (pH 7.4) buffer; prepare a 0.1 mol / L EDC·HCl / NHS mixed activating reagent (molar ratio of the two is 2:1).
[0060] S6. Disperse 20 mg of aminoated Tb,Fe-CeO2@SiO2 in 20 mL of 0.01 mol / L MES (pH 6.0) buffer, sonicate for 20 min to ensure uniform dispersion, add 1 mL of EDC·HCl / NHS mixed activation reagent, and activate by stirring at room temperature in the dark for 30 min. After activation, add 2 mL of 10 μmol / L carboxylated AFB1 aptamer stock solution, and react at room temperature and under N2 protection in the dark for 24 h with stirring. After the reaction, wash three times with 0.01 mol / L PBS (pH 7.4) buffer at 8000 rpm for 10 min each time, discard the supernatant, add 20 mL of 0.01 mol / L PBS buffer, sonicate for 30 min under ice bath conditions, and centrifuge at 8000 rpm for 15 min to obtain Tb,Fe-CeO2@SiO2@aptamer dispersion.
[0061] S7. Take 100 μL of Tb,Fe-CeO2@SiO2@nucleic acid aptamer dispersion, add 20 ng / mL of AFB1 standard solution, and adjust the volume to 1 mL with PBS buffer. Incubate at room temperature with shaking for 30 min to allow the aptamer to fully bind with AFB1. Centrifuge at 8000 rpm for 10 min, discard the supernatant, collect the precipitate, and obtain the Tb,Fe-CeO2@SiO2@aptamer-AFB1 complex.
[0062] S8. Dissolve 1.642 g of 1-methylimidazole and 2.159 g of 1,4-dibromobutane in 30 mL of anhydrous acetonitrile. Reflux and stir at 80 °C under N2 protection for 48 h. Cool to room temperature, remove acetonitrile by rotary evaporation, wash three times with ethyl acetate, filter, and dry under vacuum at 60 °C for 24 h to obtain [C4(MIM)2]Br2. Take 10 g of strongly basic anion exchange resin (OH... - (Type), soaked in 50 mL of 1 mol / L NaF solution for 24 h, is converted to F. - Type resin; washing with deionized water F - Type-3 resin, until the effluent tests negative for F-ions using fluoride ion test paper. - Filter and collect F - Type resin; dissolve 1.901 g [C4(MIM)2]Br2 in 20 mL of deionized water, add F - Type I resin, shaken and stirred at room temperature for 24 hours, then filtered to remove F - The resin was concentrated by rotary evaporation, and the filtrate was dried under vacuum at 60°C for 24 hours to obtain [C4(MIM)2]F2.
[0063] S9. Dissolve 0.1g of [C4(MIM)2]F2 in 10mL of 0.05mol / L Tris-HCl (pH 8.0) buffer and sonicate for 5min until completely dissolved to obtain the [C4(MIM)2]F2 modified solution; dissolve 0.3g of [C4(MIM)2]F2 in [C4(MIM)2]F2 modified solution. The Tb,Fe-CeO2@SiO2@aptor-AFB1 complex was dispersed in 5 mL of [C4(MIM)2]F2 modification solution and incubated with shaking at 37 °C for 1 h. This allowed the ionic liquid to adsorb onto the surface of the Tb,Fe-CeO2@SiO2@aptor-AFB1 complex via electrostatic and hydrophobic interactions. After incubation, the mixture was centrifuged at 10,000 rpm for 10 min and washed three times with Tris-HCl buffer to remove unadsorbed free ionic liquid, yielding the [C4(MIM)2]F2-modified Tb,Fe-CeO2@SiO2@aptor-AFB1 complex. The [C4(MIM)2]F2-modified Tb,Fe-CeO2@SiO2@aptor-AFB1 complex was diluted to 1 mg / mL with 0.01 mol / L PBS buffer (pH 7.4) and ultrasonically dispersed for 30 min to obtain the modified nanosensor composite solution.
[0064] Example 3: A method for detecting aflatoxin in grain products, comprising the following steps:
[0065] S1. To 21.5g of cerium nitrate hexahydrate, add 2.2g of terbium nitrate hexahydrate, 1.8g of ferric nitrate nonahydrate, and 2g of polyvinylpyrrolidone in sequence, stir well, then add 100mL of sodium hydroxide solution to provide a strong alkaline environment, then add 30mL of ethanol and 30mL of ethylene glycol, stir at 300rpm for 2h at room temperature, then solvothermal reaction at 160℃ for 24h, cool to room temperature, wash three times with 70mL of deionized water and anhydrous ethanol respectively, vacuum dry at 80℃ for 12h, calcine at 500℃ for 2h by heating at 10℃ / min, cool to room temperature, and pulverize to obtain Tb,Fe-CeO2 nanoparticles.
[0066] S2. Add 2g of Tb,Fe-CeO2 nanopowder to a mixed solvent of 60mL deionized water and anhydrous ethanol at a volume ratio of 1:4. Stir at 300rpm to pre-disperse the mixture. Adjust the pH of the system to 10 with 30% concentrated ammonia. Stir at 300rpm for 15min and then sonicate for 15min to obtain a Tb,Fe-CeO2 dispersion.
[0067] S3. Add 6g of hexadecyltrimethylammonium bromide (CTAB) to a mixed solvent of 80mL deionized water and 20mL anhydrous ethanol. Stir at 300rpm at room temperature until CTAB is completely dissolved to obtain a clear CTAB solution. Under continuous sonication and stirring at 300rpm, add a Tb,Fe-CeO2 dispersion dropwise to the CTAB solution at a rate of 2mL / min. After the addition is complete, sonicate for 30min to obtain a mixed system. Mix 11.34g of tetraethyl orthosilicate (TEOS) with 80mL of anhydrous ethanol to prepare a TEOS ethanol solution. Under stirring at 300rpm, add the TEOS ethanol solution dropwise to the mixed system at a rate of 1mL / min. After the addition is complete, sonicate for 2min and stir at 300rpm at room temperature for 24h. Then add 50mL of NH4NO3 buffer solution and stir for 10min. Perform three centrifugation-sonication dispersion purification cycles (centrifugation at 8000rpm for 10 minutes each). After centrifugation for 1 minute, the precipitate was dispersed with deionized water and sonicated for 10 minutes for the first two centrifugations. After the third centrifugation, the precipitate was dispersed with anhydrous ethanol and sonicated for 10 minutes. The supernatant was discarded after each centrifugation. After centrifugation, the supernatant was discarded, and the bottom precipitate was collected. The precipitate was vacuum dried at 60℃ for 12 hours and ground to obtain Tb,Fe-CeO2@SiO2 powder.
[0068] S4. Disperse 50 mg of Tb,Fe-CeO2@SiO2 powder in 50 mL of anhydrous ethanol-deionized water mixture (volume ratio 9:1), sonicate for 30 min to ensure uniform dispersion, add 250 μL of γ-aminopropyltriethoxysilane (APTES), and stir the reaction at 30 °C under N2 protection for 12 h. After the reaction is completed, wash the powder three times with anhydrous ethanol and deionized water at 8000 rpm for 10 min each time, and dry it under vacuum at 60 °C for 6 h to obtain aminated Tb,Fe-CeO2@SiO2.
[0069] S5. Prepare a 10 μmol / L stock solution of carboxylated AFB1 aptamer using 0.01 mol / L PBS (pH 7.4) buffer; prepare a 0.1 mol / L EDC·HCl / NHS mixed activating reagent (molar ratio of the two is 2:1).
[0070] S6. Disperse 50 mg of aminoated Tb,Fe-CeO2@SiO2 in 20 mL of 0.01 mol / L MES (pH 6.0) buffer, sonicate for 20 min to ensure uniform dispersion, add 1 mL of EDC·HCl / NHS mixed activation reagent, and activate by stirring at room temperature in the dark for 30 min. After activation, add 3 mL of 10 μmol / L carboxylated AFB1 aptamer stock solution, and react at room temperature and under N2 protection in the dark for 24 h with stirring. After the reaction, wash three times with 0.01 mol / L PBS (pH 7.4) buffer at 8000 rpm for 10 min each time, discard the supernatant, add 20 mL of 0.01 mol / L PBS buffer, sonicate for 60 min under ice bath conditions, and centrifuge at 10000 rpm for 10 min to obtain Tb,Fe-CeO2@SiO2@aptamer dispersion.
[0071] S7. Take 100 μL of Tb,Fe-CeO2@SiO2@nucleic acid aptamer dispersion, add 20 ng / mL of AFB1 standard solution, and adjust the volume to 1 mL with PBS buffer. Incubate at room temperature with shaking for 30 min to allow the aptamer to fully bind with AFB1. Centrifuge at 8000 rpm for 10 min, discard the supernatant, collect the precipitate, and obtain the Tb,Fe-CeO2@SiO2@aptamer-AFB1 complex.
[0072] S8. Dissolve 1.642 g of 1-methylimidazole and 2.159 g of 1,4-dibromobutane in 30 mL of anhydrous acetonitrile. Reflux and stir at 80 °C under N2 protection for 48 h. Cool to room temperature, remove acetonitrile by rotary evaporation, wash three times with ethyl acetate, filter, and dry under vacuum at 60 °C for 24 h to obtain [C4(MIM)2]Br2. Take 10 g of strongly basic anion exchange resin (OH... - (Type), soaked in 50 mL of 1 mol / L NaF solution for 24 h, is converted to F. - Type resin, washed with deionized water F - Type-3 resin, until the effluent tests negative for F-ions using fluoride ion test paper. - Filter and collect F - For the type of resin, dissolve 1.901 g of [C4(MIM)2]Br2 in 20 mL of deionized water, and add F. - Type I resin, shaken and stirred at room temperature for 24 hours, then filtered to remove F - The resin was concentrated by rotary evaporation, and the filtrate was dried under vacuum at 60°C for 24 hours to obtain [C4(MIM)2]F2.
[0073] S9. Dissolve 0.15g of [C4(MIM)2]F2 in 20mL of 0.05mol / L Tris-HCl (pH 8.0) buffer and sonicate for 5min until completely dissolved to obtain the [C4(MIM)2]F2 modified solution; add 0.6g... The Tb,Fe-CeO2@SiO2@aptor-AFB1 complex was dispersed in 5 mL of [C4(MIM)2]F2 modification solution and incubated with shaking at 37 °C for 1 h. This allowed the ionic liquid to adsorb onto the surface of the Tb,Fe-CeO2@SiO2@aptor-AFB1 complex via electrostatic and hydrophobic interactions. After incubation, the mixture was centrifuged at 10,000 rpm for 10 min and washed three times with Tris-HCl buffer to remove unadsorbed free ionic liquid, yielding the [C4(MIM)2]F2-modified Tb,Fe-CeO2@SiO2@aptor-AFB1 complex. The [C4(MIM)2]F2-modified Tb,Fe-CeO2@SiO2@aptor-AFB1 complex was diluted to 1 mg / mL with 0.01 mol / L PBS buffer (pH 7.4) and ultrasonically dispersed for 30 min to obtain the modified nanosensor composite solution.
[0074] Comparative Example 1: Compared with Example 1, Comparative Example 1 did not use [C4(MIM)2]F2 to modify the Tb,Fe-CeO2@SiO2@aptamer-AFB1 complex. The specific method is as follows:
[0075] S1. To 21.5g of cerium nitrate hexahydrate, add 1.6g of terbium nitrate hexahydrate, 1.4g of ferric nitrate nonahydrate, and 1.5g of polyvinylpyrrolidone in sequence, stir well, then add 100mL of sodium hydroxide solution to provide a strong alkaline environment, then add 30mL of ethanol and 30mL of ethylene glycol, stir at 300rpm for 2h at room temperature, then solvothermal reaction at 160℃ for 24h, cool to room temperature, wash three times with 70mL of deionized water and anhydrous ethanol respectively, vacuum dry at 80℃ for 12h, calcine at 500℃ for 2h by heating at 10℃ / min, cool to room temperature, and pulverize to obtain Tb,Fe-CeO2 nanoparticles.
[0076] S2. Add 1.6g of Tb,Fe-CeO2 nanopowder to a mixed solvent of 60mL deionized water and anhydrous ethanol at a volume ratio of 1:4. Pre-disperse the mixture by stirring at 300rpm. Adjust the pH of the system to 10 with 30% concentrated ammonia. Stir at 300rpm for 15min and then sonicate for 15min to obtain a Tb,Fe-CeO2 dispersion.
[0077] S3. Add 4.5 g of hexadecyltrimethylammonium bromide (CTAB) to a mixed solvent of 80 mL deionized water and 20 mL anhydrous ethanol. Stir at 300 rpm at room temperature until CTAB is completely dissolved to obtain a clear CTAB solution. Under continuous sonication and stirring at 300 rpm, add a Tb,Fe-CeO2 dispersion dropwise to the CTAB solution at a rate of 1.5 mL / min. After the addition is complete, sonicate for 30 min to obtain a mixed system. Mix 8.64 g of tetraethyl orthosilicate (TEOS) with 80 mL of anhydrous ethanol to prepare a TEOS ethanol solution. Under stirring at 300 rpm, add the TEOS ethanol solution dropwise to the mixed system at a rate of 0.75 mL / min. After the addition is complete, sonicate for 2 min and stir at 300 rpm at room temperature for 24 h. Then add 45 mL of NH4NO3 buffer solution and stir for 10 min. Perform three centrifugation-sonication dispersion purification cycles (centrifugation at 8000 rpm for 10 minutes each). After centrifugation for 1 minute, the precipitate was dispersed with deionized water and sonicated for 10 minutes for the first two centrifugations. After centrifugation for the third time, the precipitate was dispersed with anhydrous ethanol and sonicated for 10 minutes. The supernatant was discarded after each centrifugation. After centrifugation, the supernatant was discarded, and the bottom precipitate was collected. The precipitate was vacuum dried at 50℃ for 9 hours and ground to obtain Tb,Fe-CeO2@SiO2 powder.
[0078] S4. Disperse 50 mg of Tb,Fe-CeO2@SiO2 powder in 50 mL of anhydrous ethanol-deionized water mixture (volume ratio 9:1), sonicate for 30 min to ensure uniform dispersion, add 225 μL of γ-aminopropyltriethoxysilane (APTES), and stir the reaction at 30 °C under N2 protection for 12 h. After the reaction is completed, wash the powder three times with anhydrous ethanol and deionized water at 8000 rpm for 10 min each time, and dry it under vacuum at 60 °C for 6 h to obtain aminated Tb,Fe-CeO2@SiO2.
[0079] S5. Prepare a 10 μmol / L stock solution of carboxylated AFB1 aptamer using 0.01 mol / L PBS (pH 7.4) buffer; prepare a 0.1 mol / L EDC·HCl / NHS mixed activating reagent (molar ratio of the two is 2:1).
[0080] S6. Disperse 35 mg of aminoated Tb,Fe-CeO2@SiO2 in 20 mL of 0.01 mol / L MES (pH 6.0) buffer, sonicate for 20 min to ensure uniform dispersion, add 1 mL of EDC·HCl / NHS mixed activation reagent, and activate by stirring at room temperature in the dark for 30 min. After activation, add 2.5 mL of 10 μmol / L carboxylated AFB1 aptamer stock solution, and react at room temperature and under N2 protection in the dark for 24 h with stirring. After the reaction, wash three times with 0.01 mol / L PBS (pH 7.4) buffer at 8000 rpm for 10 min each time, discard the supernatant, add 20 mL of 0.01 mol / L PBS buffer, sonicate for 45 min under ice bath conditions, and centrifuge at 9000 rpm for 13 min to obtain Tb,Fe-CeO2@SiO2@aptamer dispersion.
[0081] S7. Take 100 μL of Tb,Fe-CeO2@SiO2@nucleic acid aptamer dispersion, add 20 ng / mL of AFB1 standard solution, and adjust the volume to 1 mL with PBS buffer. Incubate at room temperature with shaking for 30 min to allow the aptamer to fully bind with AFB1. Centrifuge at 8000 rpm for 10 min, discard the supernatant, collect the precipitate, and obtain the Tb,Fe-CeO2@SiO2@aptamer-AFB1 complex. Dilute the Tb,Fe-CeO2@SiO2@aptamer-AFB1 complex with 0.01 mol / L PBS buffer (pH 7.4) to a concentration of 1 mg / mL, and sonicate for 30 min to obtain the nanosensor composite solution.
[0082] Comparative Example 2: Compared with Example 1, Comparative Example 1 did not use [C4(MIM)2]F2 to modify the Tb,Fe-CeO2@SiO2@aptamer-AFB1 complex, nor did it graft nucleic acid aptamers. The specific method is as follows:
[0083] S1. To 21.5g of cerium nitrate hexahydrate, add 1.6g of terbium nitrate hexahydrate, 1.4g of ferric nitrate nonahydrate, and 1.5g of polyvinylpyrrolidone in sequence, stir well, then add 100mL of sodium hydroxide solution to provide a strong alkaline environment, then add 30mL of ethanol and 30mL of ethylene glycol, stir at 300rpm for 2h at room temperature, then solvothermal reaction at 160℃ for 24h, cool to room temperature, wash three times with 70mL of deionized water and anhydrous ethanol respectively, vacuum dry at 80℃ for 12h, calcine at 500℃ for 2h by heating at 10℃ / min, cool to room temperature, and pulverize to obtain Tb,Fe-CeO2 nanoparticles.
[0084] S2. Add 1.6g of Tb,Fe-CeO2 nanopowder to a mixed solvent of 60mL deionized water and anhydrous ethanol at a volume ratio of 1:4. Pre-disperse the mixture by stirring at 300rpm. Adjust the pH of the system to 10 with 30% concentrated ammonia. Stir at 300rpm for 15min and then sonicate for 15min to obtain a Tb,Fe-CeO2 dispersion.
[0085] S3. Add 4.5 g of hexadecyltrimethylammonium bromide (CTAB) to a mixed solvent of 80 mL deionized water and 20 mL anhydrous ethanol. Stir at 300 rpm at room temperature until CTAB is completely dissolved to obtain a clear CTAB solution. Under continuous sonication and stirring at 300 rpm, add a Tb,Fe-CeO2 dispersion dropwise to the CTAB solution at a rate of 1.5 mL / min. After the addition is complete, sonicate for 30 min to obtain a mixed system. Mix 8.64 g of tetraethyl orthosilicate (TEOS) with 80 mL of anhydrous ethanol to prepare a TEOS ethanol solution. Under stirring at 300 rpm, add the TEOS ethanol solution dropwise to the mixed system at a rate of 0.75 mL / min. After the addition is complete, sonicate for 2 min and stir at 300 rpm at room temperature for 24 h. Then add 45 mL of NH4NO3 buffer solution and stir for 10 min. Perform three centrifugation-sonication dispersion purification cycles (centrifugation at 8000 rpm for 10 minutes each). After centrifugation for 1 minute, the precipitate was dispersed with deionized water and sonicated for 10 minutes for the first two centrifugations. After centrifugation for the third time, the precipitate was dispersed with anhydrous ethanol and sonicated for 10 minutes. The supernatant was discarded after each centrifugation. After centrifugation, the supernatant was discarded, and the bottom precipitate was collected. The precipitate was vacuum dried at 50℃ for 9 hours and ground to obtain Tb,Fe-CeO2@SiO2 powder.
[0086] S4. Dilute the Tb,Fe-CeO2@SiO2 powder with 0.01mol / L PBS buffer (pH 7.4) to a concentration of 1mg / mL, and sonicate for 30min to obtain the Tb,Fe-CeO2@SiO2 composite solution.
[0087] Comparative Example 3: Tb-CeO2 nanoparticles were used directly to detect aflatoxin in grain products.
[0088] Tb-CeO2 powder was diluted to a concentration of 1 mg / mL with 0.01 mol / L PBS buffer (pH 7.4) and ultrasonically dispersed for 30 min to obtain Tb-CeO2 composite solution.
[0089] Standard curve regression equation
[0090] 100 μL of the composite solution from Example 1 and Comparative Examples 1-3 were mixed with 100 μL of AFB1 standard working solution at concentrations of 0, 0.01, 0.1, 1, 10, 50, and 100 ng / mL, respectively. The mixture was then brought to a final volume of 2 mL with PBS buffer (pH 7.4), and vortexed for 30 s to ensure homogeneity. The mixture was then incubated in a 37°C water bath for 20 min in the dark. The solutions were then placed in a fluorescence spectrophotometer with the following detection parameters: λex = 350 nm, λem = 545 nm, slit width 5 nm, and scan speed 120 nm / min. The fluorescence intensity F of each tube was measured, and the fluorescence intensity of the blank tube (0 ng / mL AFB1) was recorded as F0. A linear regression was performed with the AFB1 concentration c (ng / mL) as the abscissa and the fluorescence intensity difference as the ordinate. The regression equation for the standard curve of Example 1 was obtained as ΔF = 98.6c. 测 +12.8, correlation coefficient R 2 =0.9991, linear range is 0.01~100 ng / mL, method detection limit (LOD) = 0.03 ng / mL, limit of quantitation (LOQ) = 0.10 ng / mL, such as Figure 1 As shown in the figure; Table 1 shows the sensitivity tests of each group in Example 1 and Comparative Examples 1-3.
[0091] Table 1 Sensitivity test results for each group
[0092]
[0093] As shown in Table 1, the method used in Example 1 is significantly superior to Comparative Examples 1-3 in terms of sensitivity and linear range. Specifically, the standard curve regression equation of Example 1 has the largest slope, indicating that its fluorescence intensity response to changes in AFB1 concentration is the most sensitive; at the same time, it has the widest linear range, covering a concentration range from 0.01 to 100 ng / mL, meeting the detection requirements of aflatoxin at different concentration levels. In addition, the limit of detection and limit of quantitation of Example 1 are both low, at 0.03 ng / mL and 0.10 ng / mL, respectively, meaning that the method can detect lower concentrations of aflatoxin and the quantification is more accurate. In contrast, although Comparative Example 1 is comparable to Example 1 in terms of limit of detection, its linear range is narrower and its sensitivity is lower; the sensitivity, linear range, and limit of detection of Comparative Examples 2 and 3 are all inferior to those of Example 1, especially Comparative Example 3, which has the narrowest linear range and the highest limit of detection, indicating that the effect of directly using Tb-CeO2 nanopowder for detection is not ideal. Therefore, it can be concluded that the method used in Example 1 has higher sensitivity in the detection of aflatoxin in grain products.
[0094] Detection of aflatoxin B1
[0095] Take a grain sample to be tested, remove impurities, crush it, and pass it through an 80-mesh sieve for later use.
[0096] Add 20 mL of methanol-water solution (volume ratio 7:3) to 5 g of pulverized grain sample, vortex for 1 min, and sonicate for 30 min, vortexing once every 10 min to ensure thorough extraction; add 2 g of anhydrous sodium sulfate and 5 mL of n-hexane, vortex for 30 s, centrifuge at 0-4℃ and 8000-8500 rpm for 10 min, collect the lower aqueous phase, and use an activated C18 solid-phase extraction column (activated with 5 mL of methanol for 5 min, then with 5 mL of deionized water for 5 min) at a flow rate of 1 mL / min. First, rinse the column with 5 mL of deionized water (to remove water-soluble impurities), then elute AFB1 with 3 mL of methanol. Collect the eluent, place it in a 40℃ water bath and blow it dry with N2, and make up to 5 mL with 0.01 mol / L PBS buffer (pH 7.4), vortex to dissolve, and filter through a 0.22 μm aqueous filter membrane to obtain the sample solution to be tested.
[0097] Mix 100 μL of the 1 mg / mL composite solution from Examples 1-3 and Comparative Examples 1-3 with 100 μL of the sample solution to be tested. Make up to 2 mL with PBS buffer (pH 7.4), vortex for 30 s, incubate at 37°C in the dark for 20 min, and place in a fluorescence spectrophotometer. Set the detection parameters as follows: λex = 350 nm, λem = 545 nm, slit width 5 nm, scan speed 120 nm / min. Measure the fluorescence intensity F of the sample tube. 样 ;
[0098] Prepare a sample blank (take 5g of grain sample without AFB1, prepare the sample blank solution in the same way as the sample solution to be tested, mix 100μL of the sample blank solution with 100μL of the 1mg / mL composite solution from Examples 1-3 and Comparative Examples 1-3, and then bring the volume to 2mL with PBS buffer, vortex for 30s, and incubate at 37℃ in the dark for 20min). Place the sample blank in a fluorescence spectrophotometer and set the detection parameters: λex=350nm, λem=545nm, slit width 5nm, scan speed 120nm / min, and measure the fluorescence intensity F of the sample blank tube. 样品空白Method blank (20 mL methanol-water solution, volume ratio 7:3, vortexed for 1 min, ultrasonically extracted for 30 min, vortexed once every 10 min, added 2 g anhydrous sodium sulfate and 5 mL n-hexane, vortexed for 30 s, centrifuged at 0-4℃ and 8000-8500 rpm for 10 min, collected the lower aqueous phase, and used an activated C18 solid-phase extraction column with a flow rate controlled at 1 mL / min. First, rinsed the column with 5 mL deionized water, then added 3 mL methanol, collected the eluent, and dried it in a 40℃ water bath with N2. Then, used 0.01 mol / L... The PBS buffer was brought to a final volume of 5 mL, vortexed to dissolve, and filtered through a 0.22 μm aqueous filter membrane to obtain the method blank solution. 100 μL of the method blank solution was taken and mixed with 100 μL of the composite solution according to the detection procedure. The volume was brought to a final volume of 2 mL with PBS, vortexed for 30 s, and incubated at 37°C in the dark for 20 min. The solution was then placed in a fluorescence spectrophotometer, and the detection parameters were set as follows: λex = 350 nm, λem = 545 nm, slit width 5 nm, and scan speed 120 nm / min. The fluorescence intensity F of the method blank was measured. 方法空白 Eliminate matrix and reagent interference and calculate the actual fluorescence intensity F. 测 = F 样 -F 样品空白 Three parallel samples were prepared for each sample. The actual fluorescence intensity F of the sample was substituted into the regression equation of the standard curve to calculate the AFB1 concentration c in the sample solution. 测 (ng / mL), and based on this, the actual content of AFB1 in the grain sample was calculated.
[0099] The formula for calculating the actual content of AFB1 in a grain sample is:
[0100] AFB1 content in grains
[0101] Where: c 测 —The concentration of AFB1 in the sample solution to be tested (unit: ng / mL), substituted into F by the standard curve regression equation. 测 Calculated.
[0102] V 定容 —The final volume of the pretreatment (unit: mL), here it is 5 mL.
[0103] D – Dilution factor during the detection process, which is 20 here (because 100μL of the sample solution to be tested is brought to a final volume of 2mL, which is a 20-fold dilution).
[0104] m — the mass of the grain sample (unit: g), here it is 5g.
[0105] Since 1 μg / kg = 1 ng / g, the AFB1 content in grains... c测 ×20.
[0106] Specific detection
[0107] When detecting AFB1, the specificity of the [C4(MIM)2]F2-modified Tb,Fe-CeO2@SiO2@aptamer-AFB1 complex for AFB1 directly affects the accuracy of the detection method when multiple toxins are present in the detection environment. If the [C4(MIM)2]F2-modified Tb,Fe-CeO2@SiO2@aptamer-AFB1 complex also responds to other toxins, especially structurally similar fungal toxins, then during the experiment, the signal of non-target fungal toxins may be misinterpreted as an AFB1 signal, leading to an increase in false positive results. To verify whether the [C4(MIM)2]F2-modified Tb,Fe-CeO2@SiO2@aptamer-AFB1 complex has the ability to specifically recognize AFB1, the [C4(MIM)2]F2-modified Tb,Fe-CeO2@SiO2@aptamer-AFB1 complex was used to test for AFB1, aflatoxin G1 (AFG1), and aflatoxin G2 (AFG2) to determine whether the [C4(MIM)2]F2-modified Tb,Fe-CeO2@SiO2@aptamer-AFB1 complex has the ability to specifically recognize AFB1. Under optimal reaction conditions, AFB1, AFG1, AFG2, and phosphate-buffered saline (PBS) were added separately to three portions of [C4(MIM)2]F2 modified Tb,Fe-CeO2@SiO2@aptor-AFB1 complexes. The concentration of each toxin was set to 20 ng / mL. After centrifugation, the fluorescence intensity of the supernatant was detected.
[0108] Test results as follows Figure 2 As shown, the fluorescence signal is extremely significant in the presence of AFB1. The supernatant with only AFB1 added exhibits a strong fluorescence intensity, while the supernatant with other fungal toxins added shows very weak fluorescence intensity. This indicates that the [C4(MIM)2]F2-modified Tb,Fe-CeO2@SiO2@aptamer-AFB1 complex constructed in this study has a specific recognition ability for AFB1 and can accurately identify AFB1.
[0109] In summary, this invention develops a fluorescence detection method based on a [C4(MIM)2]F2-modified Tb,Fe-CeO2@SiO2@aptamer-AFB1 complex for the high-sensitivity and specificity detection of aflatoxin B1 (AFB1) in grain products. This method combines the high specificity of nucleic acid aptamers with the excellent fluorescence properties of Tb,Fe-CeO2@SiO2 nanomaterials. Modification with [C4(MIM)2]F2 further enhances the stability and fluorescence response of the Tb,Fe-CeO2@SiO2@aptamer-AFB1 complex. Experimental results show that the [C4(MIM)2]F2-modified Tb,Fe-CeO2@SiO2@aptamer-AFB1 complex exhibits extremely high sensitivity for AFB1, with a standard curve regression equation of ΔF = 98.6c. 测 The method exhibits a linear range of +12.8, covering 0.01–100 ng / mL, with a detection limit as low as 0.03 ng / mL and a quantitation limit of 0.10 ng / mL. Furthermore, specificity assays confirmed that the [C4(MIM)2]F2-modified Tb,Fe-CeO2@SiO2@aptamer-AFB1 complex accurately identifies AFB1 without interference from other structurally similar mycotoxins, effectively avoiding false positives. In addition, the method is simple to operate, with relatively straightforward sample pretreatment steps, making it suitable for rapid detection of aflatoxin B1 in actual grain samples.
[0110] The specific embodiments of the present invention described above do not constitute a limitation on the scope of protection of the present invention. Any other corresponding changes and modifications made in accordance with the technical concept of the present invention should be included within the scope of protection of the claims of the present invention.
Claims
1. A method for detecting aflatoxin in grain products, characterized in that, Includes the following steps: S1: Mix the modified nanosensor composite solution with the sample solution to be tested, then bring the volume up with PBS buffer, vortex, and incubate at 37°C in the dark for 20 min. Measure the fluorescence intensity F of the sample solution to be tested. 样 ; S2: Set up a sample blank. Mix the modified nanosensor composite solution with the sample blank solution, then bring the volume up with PBS buffer, vortex, and incubate at 37°C in the dark for 20 min. Measure the fluorescence intensity F of the sample blank. 样品空白 ; S3: Calculate the actual fluorescence intensity F 测 = F 样 -F 样品空白 The actual fluorescence intensity F 测 Substituting into the standard curve regression equation, the AFB1 concentration c in the sample solution to be tested is calculated. 测 Based on this, the actual content of AFB1 in the grain samples was calculated; The standard curve regression equation is: ΔF = 98.6c 测 +12.8; The formula for calculating the actual content of AFB1 in grain samples is as follows: ; Where X represents the actual content of AFB1 in the grain sample, and the unit is... c 测 V indicates the concentration of AFB1 in the sample solution to be tested, in ng / mL; 定容 The volume represents the final volume after pretreatment, in mL; D represents the dilution factor during the detection process, where 100 μL of the sample solution is diluted to 2 mL, which is a 20-fold dilution; m represents the mass of the grain sample, in g.
2. The method for detecting aflatoxin in grain products according to claim 1, characterized in that, The specific preparation process of the modified nanosensing composite liquid is as follows: [C4(MIM)2]F2 was dissolved in Tris-HCl buffer and sonicated until completely dissolved to obtain the [C4(MIM)2]F2 modified solution; The nanosensing complex was dispersed in [C4(MIM)2]F2 modification solution, incubated with shaking at 37°C for 1 h, centrifuged for 10 min, and washed 3 times to obtain the modified nanosensing complex. The modified nanosensing complex was diluted with PBS buffer and ultrasonically dispersed for 30 min to obtain the final product.
3. The method for detecting aflatoxin in grain products according to claim 2, characterized in that, The specific preparation process of the nanosensing composite is as follows: Tb,Fe-CeO2@SiO2 powder was dispersed in anhydrous ethanol-deionized water mixture and sonicated for 30 min. γ-aminopropyltriethoxysilane was added and the mixture was stirred and reacted at 30 °C under N2 protection for 12 h. After the reaction was completed, the mixture was centrifuged and washed 3 times and then vacuum dried at 60 °C for 6 h to obtain aminated Tb,Fe-CeO2@SiO2. Aminated Tb,Fe-CeO2@SiO2 was dispersed in MES buffer and ultrasonically dispersed evenly. EDC·HCl / NHS mixed activation reagent was added and activated by stirring in the dark at room temperature for 30 min. Carboxylated AFB1 nucleic acid aptamer stock solution was added and reacted in the dark at room temperature and under N2 protection for 24 h. After the reaction was completed, the mixture was centrifuged and washed 3 times to remove the supernatant. PBS buffer was then added and ultrasonically dispersed in an ice bath for 30-60 min, followed by centrifugation for 10-15 min to obtain the nucleic acid aptamer dispersion. Add the nucleic acid aptamer dispersion to the AFB1 standard solution, bring the volume up to PBS buffer, incubate with shaking at room temperature for 30 min, centrifuge at 8000 rpm for 10 min, remove the supernatant, and collect the precipitate to obtain the final product.
4. The method for detecting aflatoxin in grain products according to claim 3, characterized in that, The specific preparation process of the Tb,Fe-CeO2@SiO2 powder is as follows: Hexadecyltrimethylammonium bromide was added to a mixed solvent prepared from deionized water and anhydrous ethanol, and stirred at room temperature until completely dissolved to obtain a hexadecyltrimethylammonium bromide solution. Under ultrasonic and stirring conditions, Tb,Fe-CeO2 dispersion was added dropwise to hexadecyltrimethylammonium bromide solution. After the addition was complete, ultrasonication was continued for 30 min to obtain a mixed system. Tetraethyl orthosilicate was mixed with anhydrous ethanol to prepare TEOS ethanol solution. Under stirring at 300 rpm, TEOS ethanol solution was added dropwise to the mixture. After the addition was complete, the mixture was stirred at 300 rpm for 24 h at room temperature. Then, NH4NO3 buffer solution was added and stirred for 10 min. The mixture was then centrifuged and ultrasonically dispersed three times. After centrifugation, the supernatant was removed, the precipitate was collected, and the precipitate was vacuum dried at 40-60℃ for 6-12 h. Finally, the precipitate was ground to obtain the final product.
5. The method for detecting aflatoxin in grain products according to claim 4, characterized in that, The specific preparation process of the Tb,Fe-CeO2 dispersion is as follows: Terbium nitrate hexahydrate, ferric nitrate nonahydrate, and polyvinylpyrrolidone were added sequentially to cerium nitrate hexahydrate and stirred until homogeneous. Sodium hydroxide solution was then added and mixed. Ethanol and ethylene glycol were added and stirred at room temperature for 2 hours. The mixture was then subjected to a solvothermal reaction at 160°C for 24 hours. After cooling to room temperature, the mixture was washed three times, vacuum dried at 80°C for 12 hours, calcined at 500°C for 2 hours, cooled to room temperature, and pulverized to obtain Tb,Fe-CeO2 nanoparticles. Tb,Fe-CeO2 nanopowder was added to a mixed solvent of deionized water and anhydrous ethanol, stirred and pre-dispersed, and the pH of the system was adjusted to 10 with 30% concentrated ammonia. The mixture was stirred at 300 rpm for 15 min and then sonicated for 15 min to obtain the final product.
6. The method for detecting aflatoxin in grain products according to claim 5, characterized in that, The mass ratio of cerium nitrate hexahydrate, terbium nitrate hexahydrate, ferric nitrate nonahydrate, and polyvinylpyrrolidone is 21.5:1.1~2.2:0.9~1.8:1~2.
7. The method for detecting aflatoxin in grain products according to claim 3, characterized in that, The carboxylated AFB1 aptamer is a nucleotide sequence as shown in SEQ ID NO:1, with a carboxyl group modified at the 5' end.
8. The method for detecting aflatoxin in grain products according to claim 2, characterized in that, The specific preparation process of [C4(MIM)2]F2 is as follows: 1-Methylimidazole and 1,4-dibromobutane were dissolved in anhydrous acetonitrile and refluxed and stirred at 80 °C under N2 protection for 48 h. After cooling to room temperature, the acetonitrile was removed by rotary evaporation. The mixture was washed three times with ethyl acetate, filtered, and dried under vacuum at 60 °C for 24 h to obtain [C4(MIM)2]Br2. The strongly basic anion exchange resin was soaked in NaF solution for 24 hours to convert it into F. - Type 1 resin; wash with deionized water, filter, and collect F. - Type resin; dissolve [C4(MIM)2]Br2 in deionized water, add F - Type I resin, shaken and stirred at room temperature for 24 hours, then filtered to remove F - The resin was concentrated by rotary evaporation, and the filtrate was dried under vacuum at 60°C for 24 hours to obtain the final product.
9. The method for detecting aflatoxin in grain products according to claim 2, characterized in that, The mass ratio of [C4(MIM)2]F2 to the nanosensing composite is 0.1~0.15:0.3~0.6.