A method for label-free detection of aflatoxin B1 in cereals based on intrinsic protein fluorescence

Abstract

This invention discloses a label-free method for detecting aflatoxin B1 in grains based on intrinsic protein fluorescence. The method utilizes the mechanism of gold nanoparticles enhancing the intrinsic fluorescence of an artificial antigen (aflatoxin B1-bovine serum albumin) on the surface of photonic crystal microspheres. Aflatoxin B1 monoclonal antibody is immobilized on the surface of the gold nanoparticles, and the artificial antigen aflatoxin B1-BSA is immobilized on the surface of the photonic crystal microspheres. After incubating aflatoxin B1 standard or sample extract with the gold nanoparticles and photonic crystal microspheres modified with the above-mentioned biomolecules, the fluorescence signal of the photonic crystal microspheres is detected. The aflatoxin B1 in the sample is qualitatively and quantitatively detected based on the fluorescence signal of the microspheres. This invention can sensitively and specifically detect aflatoxin B1 in samples, reducing detection costs.

Description

Technical Field

[0001] This invention pertains to the detection of aflatoxin B1, specifically relating to a method for detecting aflatoxin B1 in grains based on the intrinsic fluorescence of proteins without labeling. Background Technology

[0002] Aflatoxins (AFs) are a class of highly carcinogenic fungal toxins with serious harmful effects and stable physicochemical properties, and have been a major concern both domestically and internationally. They primarily enter the human and animal body through the diet, and excessive intake can lead to liver cancer. In addition, aflatoxins can cause growth disorders (such as stunted growth and emaciation). The International Agency for Research on Cancer (IARC) classifies aflatoxins B1 (AFB1), B2 (AFB2), G1 (AFG1), and G2 (AFG2) as Group 1 carcinogens. Furthermore, AFB1 has a relative molecular weight of 312.27 and is very stable to light and heat, only decomposing when heated to 280-300°C. Autoclaving for 2 hours reduces its toxicity by 25%-33%, and for 4 hours by 50%. Once AFB1 contaminates food or the traditional Chinese medicine supply chain, it can cause enormous losses. Currently, the most effective way to prevent AFB1 contamination of food or the traditional Chinese medicine supply chain is to develop efficient, sensitive, and low-cost rapid detection methods.

[0003] Currently, methods for detecting AFB1 in samples include thin-layer chromatography (TLC), high-performance liquid chromatography (HPLC), HPLC-mass spectrometry (HPLC-MS), immunoassay strips, and Raman spectroscopy. TLC has limited sensitivity, requires expensive equipment and skilled operators, involves complex sample preparation processes such as immunoaffinity column enrichment and purification, and sometimes derivatization, resulting in long detection times and high costs, making it unsuitable for rapid on-site detection. While rapid methods like immunoassay strips and Raman spectroscopy allow for rapid on-site detection of AFB1, most only offer semi-quantitative detection. Therefore, developing a method for detecting AFB1 that is highly specific, low-cost, and easy to operate is essential. Summary of the Invention

[0004] Purpose of the invention: To address the problems existing in the existing methods for detecting aflatoxin B1, this invention provides a method for detecting aflatoxin B1 in grains based on protein intrinsic fluorescence label-free detection. This invention can sensitively and specifically detect aflatoxin B1 in samples and reduce detection costs.

[0005] Technical Solution: To achieve the above objectives, the present invention provides a method for the label-free detection of aflatoxin B1 in grains based on intrinsic protein fluorescence, comprising the following steps:

[0006] Aflatoxin B1 monoclonal antibody was immobilized on the surface of gold nanoparticles, and artificial antigen aflatoxin B1-BSA was immobilized on the surface of photonic crystal microspheres. After aflatoxin B1 standard or sample extract was incubated with the above-mentioned gold nanoparticles and photonic crystal microspheres, the fluorescence signal of the photonic crystal microspheres was detected. The aflatoxin B1 in the sample was qualitatively and quantitatively detected based on the fluorescence signal of the microspheres.

[0007] The method utilizes gold nanoparticles with aflatoxin B1 monoclonal antibody immobilized on their surface to enhance the intrinsic fluorescence of the artificial antigen aflatoxin B1-BSA on the surface of photonic crystal microspheres for detection.

[0008] The photonic crystal microspheres are formed by self-assembly of silica emulsion and methyl silicone oil, wherein the silica emulsion is made of TEOS and ammonia.

[0009] The artificial antigen aflatoxin B1-BSA immobilized on the surface of the photonic crystal microspheres is obtained by modifying the photonic crystal microspheres with hydroxyl and epoxy groups, adding AFB1-BSA, and then reacting with a shaking reaction.

[0010] Preferably, the silica nanoparticles have a particle size of 320 nm, and the photonic crystal microspheres are prepared by microfluidic self-assembly with a size of 270 μm. The photonic crystal microspheres are placed in a centrifuge tube, and a piranha solution is prepared using concentrated sulfuric acid and hydrogen peroxide. The piranha solution is added to the centrifuge tube containing the photonic crystal microspheres, and the tube is placed in a decolorizing shaker for shaking at room temperature. After the reaction is complete, excess piranha solution is washed off the surface of the microspheres with double-distilled water, and the microspheres are dried in a forced-air drying oven until all moisture is evaporated. A toluene solution of diethoxymethyl[(3-epoxyethylenemethoxy)propyl]silane (GPTMS) was prepared using toluene solution. Different concentrations of GPTMS toluene solutions were added to centrifuge tubes containing hydroxylated photonic crystal microspheres. The tubes were placed in a shaking incubator and allowed to react. After the reaction, the microspheres were washed successively with toluene, anhydrous ethanol, and double-distilled water. After washing, the microspheres were dried in a forced-air drying oven until all moisture was evaporated and collected for later use. An AFB1-BSA solution was added to the collected microspheres, and the mixture was shaken, washed, blocked with BSA, and washed again to obtain the artificial antigen aflatoxin B1-BSA immobilized on the surface of the photonic crystal microspheres.

[0011] The gold nanoparticles are formed by reducing chloroauric acid with trisodium citrate.

[0012] The aflatoxin B1 monoclonal antibody immobilized on the surface of the gold nanoparticles is obtained by modifying a colloidal gold solution formed by reducing chloroauric acid with trisodium citrate, adding mercapto-polyethylene glycol-carboxyl solution and methoxy-polyethylene glycol-mercapto-sulfapy solution, activating the nanoparticles, adding the monoclonal antibody AFB1-ab, and then reacting with shaking.

[0013] Preferably, the nanoparticles are thiol-polyethylene glycol-carboxyl groups. After stirring, methoxy-polyethylene glycol-thiol groups are added to the nanoparticle solution and stirred again. The mixture is centrifuged to remove excess PEG, then EDC and NHS solutions are added. The mixture is placed in a decolorizing shaker and reacted at room temperature. After the reaction, it is washed with PB buffer to remove excess EDC and NHS solution. The colloidal gold solution obtained from the above reaction is then diluted with PB buffer to obtain a gold nanoparticle solution. The monoclonal antibody AFB1-ab is added to the above gold nanoparticle solution, and the mixture is shaken, centrifuged, washed, and the gold nanoparticles are suspended to obtain aflatoxin B1 monoclonal antibody immobilized on the surface of the gold nanoparticles.

[0014] The incubation is carried out at 35-37℃ for 90-100 minutes.

[0015] Preferably, the incubation is carried out at 37°C for 90 minutes.

[0016] The fluorescence signal of the photonic crystal microspheres was detected by measuring its fluorescence intensity using an inverted fluorescence microscope equipped with a fluorescence spectrometer and a CCD imaging system, recording the results, and analyzing them using OriginPro9 software.

[0017] The detection is performed by focusing under a 40× eyepiece, with the excitation wavelength in the range of 360-370nm.

[0018] This invention utilizes the mechanism of enhancing the intrinsic fluorescence of an artificial antigen (aflatoxin B1-bovine serum albumin) on the surface of photonic crystal microspheres using gold nanoparticles. Aflatoxin B1 monoclonal antibody is immobilized on the surface of the gold nanoparticles, and the artificial antigen aflatoxin B1-BSA is immobilized on the surface of the photonic crystal microspheres. When aflatoxin B1 standard or sample extract is incubated with the gold nanoparticles and photonic crystal microspheres modified with the above-mentioned biomolecules, the fluorescence signal of the photonic crystal microspheres is detected. Qualitative and quantitative detection of aflatoxin B1 in the sample is then performed based on the fluorescence signal of the microspheres. This invention can sensitively and specifically detect aflatoxin B1 in samples, reducing detection costs. This invention requires no labeling, is a one-step reaction, and is simple, rapid, sensitive, and low-cost for the detection of aflatoxin B1.

[0019] Beneficial effects: Compared with the prior art, the present invention has the following advantages:

[0020] (1) This method does not require fluorescent labeling for the detection of AFB1 toxin;

[0021] (2) The method of the present invention only requires one incubation step to detect the results, which is fast;

[0022] (3) Each sample in this invention requires only 2 μL of sample, resulting in low reagent consumption;

[0023] (4) The sensitivity of this invention for detecting AFB1 toxin reaches pg / g;

[0024] (5) Compared with other methods for detecting AFB1, the detection cost is low. Attached Figure Description

[0025] Figure 1 Roadmap for label-free detection of aflatoxin B1 in cereals using intrinsic fluorescence of proteins;

[0026] Figure 2 Morphology characterization and ultraviolet absorption spectrum of gold nanoparticles;

[0027] Figure 3 Surface properties of microspheres;

[0028] Figure 4 Fluorescence imaging was performed after different modifications to the surface of the microspheres.

[0029] Figure 5 The effect of different excitation wavelengths on fluorescence signal was investigated: a) excitation light 360-370 nm; b) excitation light 460-495 nm; c) excitation light 530-550 nm; d) comparison with glass microspheres of the same particle size.

[0030] Figure 6 The standard curve for detecting AFB1 is shown in Figure a, where a is the fluorescence spectrum of microspheres responding to different concentrations of AFB1; and b is the relationship between different concentrations of AFB1 and the fluorescence intensity of microspheres.

[0031] Figure 7 To detect AFB1 specificity;

[0032] Figure 8 The spiked recovery rate. Detailed Implementation

[0033] The present invention will be further described below with reference to specific embodiments and accompanying drawings.

[0034] Unless otherwise specified, the experimental methods described in the embodiments are conventional methods; unless otherwise specified, the reagents and materials are commercially available.

[0035] The mercapto-polyethylene glycol-carboxyl group was purchased from Xi'an Kaixin Biotechnology Co., Ltd., with an average molecular weight of 5000.

[0036] The methoxy-polyethylene glycol-mercapto group was purchased from Xi'an Kaixin Biotechnology Co., Ltd., with an average molecular weight of 5000.

[0037] The monoclonal antibodies AFB1-ab and AFB1-BSA were purchased from Shandong Binzhou Hongdu Biotechnology Co., Ltd.

[0038] In the embodiments and figures, SPCMs: photonic crystal microspheres; SPCMs+epoxy groups: epoxy groups on the surface of photonic crystal microspheres;

[0039] SPCMs+anti-AFB1: AFB1 antibody modification on the surface of photonic crystal microspheres;

[0040] SPCMs+AFB1-BSA: AFB1 artificial antigen modification on the surface of photonic crystal microspheres;

[0041] SPCMs+AFB1-BSA+anti-AFB1: Photonic crystal microspheres are modified with AFB1 artificial antigen and then with AFB1 antibody.

[0042] SPCMs+AuNPs-anti-AFB1: Gold nanoparticles modified with AFB1 antibody on the surface of photonic crystal microspheres;

[0043] SPCMs+AFB1-BSA+AuNPs-anti-AFB1: Photonic crystal microspheres modified with AFB1 artificial antigen followed by gold nanoparticle-AFB1 antibody modification.

[0044] Example 1

[0045] I. A method for preparing photonic crystal microspheres, comprising the following steps:

[0046] (1) Preparation of monodisperse silica microsphere emulsion

[0047] Table 1 Different Photonic Crystal Microsphere Configurations

[0048]

[0049] First, prepare two clean conical flasks. According to the proportions shown in Table 1, add ammonia, anhydrous ethanol, and water to one conical flask (labeled Solution A), and add TEOS and anhydrous ethanol to the other conical flask (labeled Solution B). Place Solution A on a magnetic stirrer and stir at a high speed of 1200 rpm while simultaneously adding Solution B. After 1-2 minutes, reduce the speed to 400 rpm, cover with plastic wrap to prevent ammonia evaporation, and continue stirring for approximately 6 hours. After the reaction is complete, a milky white emulsion will be obtained. Wash the emulsion 3-4 times each with anhydrous ethanol and double-distilled water until there is no ammonia odor. Finally, determine the solid content, ensuring it is around 15%, and store at room temperature for later use.

[0050] (2) Assembling photonic crystal microspheres: Prepare two syringes, one 5 mL syringe filled with the prepared emulsion and the other 50 mL syringe filled with methyl silicone oil. After fixing the syringes to the microfluidic injection pump, connect the two syringes using a three-way valve. The microfluidic flow rate of the oil phase is 10 mL / h, and the flow rate of the emulsion phase is 5 mL / h. Utilizing capillary action, the emulsion is cut off by the oil phase to form photonic crystal microspheres. Collect the prepared photonic crystal microspheres in a container filled with methyl silicone oil. Photonic crystal microspheres are prepared using the "water-in-oil" principle (see reference: Journal of Chromatography A. 2020, 1626, 461379 for details).

[0051] (3) Curing and drying: The photonic crystal microspheres collected in a container filled with methyl silicone oil are placed in a drying oven at 60°C and dried and cured until the moisture is completely evaporated.

[0052] (4) Hexane and ethanol cleaning: After the photonic crystal microspheres have been cured and dried, place them in a porcelain crucible. Recover the excess methyl silicone oil. First, clean the microspheres with hexane 3-4 times to remove excess oil from the surface of the microspheres. Then, clean them with ethanol 3-4 times to remove the residual hexane from the surface of the microspheres. After cleaning, place the crucible containing the microspheres in a drying oven to dry, or place it at room temperature to allow the ethanol to evaporate naturally.

[0053] (5) High-temperature calcination: At this time, the surface compressive strength of the microspheres is low and they are easy to break, which is not conducive to use. Therefore, the microspheres are placed in a tube furnace and the temperature is slowly raised to 700℃ and calcined at high temperature for about 3 hours. Then, the temperature is slowly lowered to room temperature to stabilize the surface structure of the photonic crystal microspheres.

[0054] II. Surface modification of photonic crystal microspheres and immobilization of artificial antigens:

[0055] (1) Modification of hydroxyl groups (-OH)

[0056] The photonic crystal microspheres obtained in step (I) were placed in a centrifuge tube. A piranha solution was prepared by mixing concentrated sulfuric acid (98% by mass) and hydrogen peroxide (30% by mass) in a volume ratio of 7:3. This piranha solution was added to the centrifuge tube containing the photonic crystal microspheres (10 μL / microsphere) for hydroxylation modification. The tube was then placed in a decolorizing shaker and reacted at 160 rpm for approximately 6 hours at room temperature. After the reaction, excess piranha solution was washed off the surface of the microspheres with double-distilled water. The microspheres were then placed in a forced-air drying oven and dried at 60°C until all moisture was evaporated. The dried microspheres were collected for later use.

[0057] (2) Modification of epoxy groups (-CH(O)CH-)

[0058] Prepare a 5% GPTMS toluene solution using toluene. Add the GPTMS toluene solution to a centrifuge tube containing photonic crystal microspheres hydroxylated in step (1) (10 μL per microsphere). Place the tube in a shaking incubator and shake at 160 rpm for about 6 hours at 60°C. After the reaction, wash the tube 3-4 times each with toluene, anhydrous ethanol, and double-distilled water. After washing, place the tube in a forced-air drying oven and dry at 60°C until all moisture is evaporated. Collect the tube for later use.

[0059] (3) Prepare 80 μg / mL AFB1-BSA and add it to a centrifuge tube containing photonic crystal microspheres obtained in step (2), 2 μL per microsphere. Shake at 160 rpm for 12 h at 4 °C. Wash three times with PB buffer (pH 7.4, the pH of which will be used for all subsequent PB buffers). Block with 1% BSA and wash three times with PB buffer. After washing, place in a drying oven and dry at 60 °C until the moisture is completely evaporated to obtain artificial antigen aflatoxin B1-BSA fixed on the surface of the photonic crystal microspheres. Collect for later use.

[0060] Example 2

[0061] Preparation of aflatoxin B1 monoclonal antibody immobilized on the surface of gold nanoparticles

[0062] (1) Preparation of colloidal gold solution

[0063] Place 50 mL of purified water in an Erlenmeyer flask and place it on a magnetic stirrer. Heat and stir simultaneously until the water boils. Add 0.375 mL of 1% trisodium citrate solution and 0.5 mL of 2% HAuCl4 solution to the Erlenmeyer flask. Heat until the colloidal gold turns a transparent purplish-red color. Stop heating and cool the colloidal gold solution to room temperature for later use.

[0064] (2) Modifying groups

[0065] Prepare 10 μmol / L mercapto-polyethylene glycol-carboxyl solution and 50 μmol / L methoxy-polyethylene glycol-mercapto-metallic solution. Take 22.5 mL of the prepared colloidal gold solution, add 0.75 mL of mercapto-polyethylene glycol-carboxyl solution, place in a decolorizing shaker, and react at 160 rpm for about 20 min at room temperature. After the reaction, add 5 mL of methoxy-polyethylene glycol-mercapto-metallic solution, place in a decolorizing shaker, and react at 160 rpm for about 3 h at room temperature. After the reaction, centrifuge 3-4 times at high speed to remove residual PEG, collect the lower precipitate, and finally adjust the colloidal gold solution to 1 mL with PB buffer (pH 6.8) and collect for later use.

[0066] (3) Activating groups

[0067] Prepare 40 mg / mL EDC and 110 mg / mL NHS solutions using PB buffer (pH 7.4) as activators for carboxyl groups. Add 6 μL of EDC and 6 μL of NHS solution to 1 mL of the carboxyl-modified colloidal gold solution from step (2), place in a decolorizing shaker, and shake at 160 rpm for about 20 min at room temperature. After the reaction, wash 3-4 times with PB buffer (pH 7.4) to remove excess EDC and NHS solution, take the lower precipitate, and use PB buffer to adjust the volume of colloidal gold solution to 1 mL.

[0068] (4) Take 100 μL of 100 μg / mL monoclonal antibody AFB1-ab and add it to 150 μL of the colloidal gold solution obtained in step (3) above. Shake at 4°C for 2 h. After centrifugation at 10000g for 30 min, wash three times with PB buffer, take the solid precipitate, and resuspend the gold nanoparticles with 150 μL PB buffer to obtain aflatoxin B1 monoclonal antibody fixed on the surface of the gold nanoparticles.

[0069] The colloidal gold prepared in step (1) was dispersed in an ethanol solution. After dispersion, it was dropped onto a Cu mesh, and the ethanol was allowed to evaporate naturally. This operation was repeated several times, and the preparation of the colloidal gold was observed using a transmission electron microscope (TEM). See Figure 1 a. The colloidal gold particles are 38 nm in size; the UV spectrum of the nano-gold solution in step (1) is shown in [reference needed]. Figure 1 b indicates the size and uniformity of the gold nanoparticles.

[0070] Example 3

[0071] I. Construction of photonic crystal microspheres after immune response:

[0072] Take 6 photonic crystal microspheres (each with the AFB1-BSA solution modified in Example 1), add 150 μL of gold nanoparticles immobilized with aflatoxin B1 monoclonal antibody from Example 2, and place in a shaking incubator at 37°C and 160 rpm for 1.5 h to obtain immune-bound photonic crystal microspheres. After washing, extract the fluorescence signal on the surface of the microspheres using a fluorescence spectroscopy instrument under an inverted fluorescence microscope. Figure 2 .

[0073] II. Characterization of Photonic Crystal Microspheres

[0074] The photonic crystal microspheres obtained in step (i) of Example 1 were placed on a glass slide. A metallurgical microscope, adjusted to bright field, was used at 20x magnification to observe the color and brightness of the photonic crystal microspheres. Photographs were taken and analyzed. (See attached image.) Figure 3 a. The color of this microsphere is the characteristic color of photonic crystal microspheres.

[0075] The assembly of microspheres after immune binding in step (I) was observed using a scanning electron microscope, photographs were taken, and the results were analyzed. Figure 3 b, c, d. The microspheres are neatly arranged on the surface, and the colloidal gold is uniformly distributed.

[0076] III. Fluorescence Intensity of Photonic Crystal Microspheres at Different Stages

[0077] Unmodified photonic crystal microspheres obtained in step (i) of Example 1, epoxy-modified photonic crystal microspheres obtained in step (2) of Example 1, gold nanoparticles obtained in Example 2, and photonic crystal microspheres after the final immunoreaction in this example were placed on glass slides and their fluorescence intensity was observed using an inverted fluorescence microscope. The fluorescence intensity of the photonic crystal microspheres was detected using an inverted fluorescence microscope (Olympus, IX73, Japan) equipped with a fluorescence spectrometer (OceanOptics, QEPro, USA) and a CCD imaging system. The microspheres were placed on a glass slide and focused under a 40× eyepiece. The excitation wavelength was 360-370 nm, and the emission wavelength was 450 nm. See [link to relevant documentation]. Figure 4 Under near-ultraviolet excitation, the fluorescence imaging is blue only when AFB1-BSA appears, and the color is also the strongest.

[0078] By changing the excitation wavelength of the inverted microscope, the fluorescence spectra of the inverted fluorescence microscope and fluorescence spectrometer were recorded respectively. Figure 5 The fluorescence spectrum of ac is strongest when AFB1-BSA and gold nanoparticle-labeled antibody appear on the surface of the microspheres under near-ultraviolet excitation at only 365 nm.

[0079] Example 4

[0080] Creating the AFB1 standard curve

[0081] (1) Immune binding reaction

[0082] AFB1 was prepared to concentrations of 0.01, 0.1, 1, 10, and 100 ng / mL using PB buffer (pH 6.8). Five portions of photonic crystal microspheres (6 microspheres / portion) with modified AFB1-BSA solution from Example 1 were taken, and 150 μL of aflatoxin B1 monoclonal antibody immobilized on the surface of gold nanoparticles from Example 2 and the above-mentioned AFB1 solutions of different concentrations (25 μL / microsphere) were added respectively. The mixtures were placed in a shaking incubator and shaken at 160 rpm for 1.5 h at 37 °C.

[0083] (2) Result determination

[0084] After the reaction, the microspheres were washed 3-4 times with PB buffer. The fluorescence spectra were recorded using an inverted fluorescence microscope and a fluorescence spectrometer. The maximum fluorescence value was recorded under near-UV excitation at 365 nm. The results were analyzed using OriginPro9 software. (See attached image for details.) Figure 6 Linear equations such as Figure 6 As shown, the detection range is 0.1-10 ng / mL, and the limit of detection is 0.025 ng / mL.

[0085] Example 5

[0086] AFB1 specificity analysis

[0087] AFB1, AFG1, and AFG2 were prepared as standards with concentrations of 0.01, 0.1, 1, 10, and 100 ng / mL using PB buffer. The specificity of this method for structural analogs was then tested, following the same detection method as in Example 3. Results are shown below. Figure 7 It exhibits good detection specificity for AFB1 within the linear detection range and can separate different mycotoxins and structural analogs.

[0088] Example 6

[0089] Comparison of glass spheres and photonic crystal microspheres

[0090] Equal amounts of glass spheres and photonic crystal microspheres were placed in centrifuge tubes, modified with epoxy groups, and then conjugated with toxin antigens. The fluorescence signals of both types of microspheres conjugated with the toxin antigens were measured, and the results were recorded. Data were analyzed using OriginPro9 software. The modification process for the glass microspheres and photonic crystal microspheres was the same; only the microspheres were different. The results are as follows: Figure 5 As shown in d, this demonstrates that photonic crystal microspheres and gold nanoparticles significantly enhance the fluorescence signal of the microspheres.

[0091] Example 7

[0092] Determination of spiked recovery

[0093] 1. Sample pretreatment

[0094] Purchase corn, rice, and wheat from a supermarket, grind them into powder using a traditional Chinese medicine grinder, and then sift the powder through a 100-mesh sieve. Weigh out three portions (5g each) of rice, wheat, and corn, placing each portion into a 200mL Erlenmeyer flask with a cork stopper. Prepare a 100ng / mL AFB1-methanol solution using methanol, adding 25, 250, and 2500 μL of AFB1-methanol solution to each sample respectively, then dilute to 10mL with methanol. Gently shake the Erlenmeyer flask to mix thoroughly, and place it in a fume hood until the methanol evaporates naturally.

[0095] 2. Sample extraction

[0096] After the methanol has completely evaporated, prepare an 80% methanol extract. Take 25 mL of the extract and weigh 1 g of NaCl, then add it to each of the conical flasks from step (1). Gently shake the flasks to mix thoroughly, then homogenize at high speed for 60 seconds. Next, place the flasks in a shaker at 25°C and extract for 30 minutes at 180 rpm. Afterward, filter the liquid using Whatman No. 4 filter paper, and then filter the filtered liquid a second time through a 0.45 μm filter membrane. The filtered liquid is the sample to be tested.

[0097] Recovery rate determination

[0098] Take 25 μL of the filtered liquid, dry it, and then dilute the AFB1 sample solution to 0.1, 1, and 10 ng / mL using PB buffer, respectively. Replace the AFB1 standard in Example 3 with AFB1 sample solutions of 0.1, 1, and 10 ng / mL, and follow the same conditions and steps as in Example 3, and determine the recovery rate.

[0099]

[0100] See results Figure 8 The spiked recoveries ranged from 84% to 101%.

Claims

1. A method for non-labeled detection of aflatoxin B1 in cereals based on intrinsic fluorescence of proteins, characterized in that, Comprising the following steps: The surface of the nano gold particles is fixed with aflatoxin B1 monoclonal antibody, the surface of the photonic crystal microspheres is fixed with artificial antigen aflatoxin B1-BSA, after the aflatoxin B1 standard or sample extract is incubated with the above nano gold particles and photonic crystal microspheres, the fluorescence signal of the photonic crystal microspheres is detected under the excitation light wavelength of 360-370nm, and the fluorescence signal of the microspheres is used for qualitative or quantitative detection of aflatoxin B1 in the sample; The photonic crystal microspheres are self-assembled by a silica emulsion and methyl silicone oil, and the silica emulsion is prepared from TEOS and ammonia water; The surface of the photonic crystal microspheres fixed with artificial antigen aflatoxin B1-BSA is obtained after the hydroxyl and epoxy groups of the photonic crystal microspheres are modified, and AFB1-BSA is added and oscillation reaction is carried out; Preparation of the surface of the nano gold particles fixed with aflatoxin B1 monoclonal antibody: (1) Preparation of colloidal gold solution Take 50mL of pure water and heat and stir, until the water boils, take 0.375mL of 1% trisodium citrate solution and 0.5mL of 2% HAuCl4 solution together, heat to transparent purple red, stop heating, cool the colloidal gold solution to room temperature, and wait for use; (2) Modification of groups Prepare 10 μmol / L thiol-polyethylene glycol-carboxyl solution and 50 μmol / L methoxy-polyethylene glycol-thiol solution, take 22.5mL of prepared colloidal gold solution, first add 0.75mL of thiol-polyethylene glycol-carboxyl solution, oscillate at room temperature at a speed of 160rpm for 20min, after the reaction is completed, add 5mL of methoxy-polyethylene glycol-thiol solution, oscillate at room temperature at a speed of 160rpm for 3h, after the reaction is completed, centrifuge 3-4 times to remove residual PEG, take the lower layer precipitate, and finally use PB buffer solution with pH 6.8 to make the colloidal gold solution to 1mL, and collect for use; (3) Activation of groups Use PB buffer solution with pH 7.4 to prepare 40 mg / mL EDC and 110 mg / mL NHS solutions respectively, which are used as activators for activating carboxyl groups, add 6 μL of EDC and 6 μL of NHS solution respectively to 1mL of colloidal gold solution with modified carboxyl groups in step (2), oscillate at room temperature at a speed of 160rpm for about 20min, after the reaction is completed, wash 3-4 times with PB buffer solution with pH 7.4 to remove excess EDC and NHS solution, take the lower layer precipitate, and use PB buffer solution to make the colloidal gold solution to 1mL; (4) Take 100 μL, 100 μg / mL monoclonal antibody AFB1-ab and add it to 150 μL of colloidal gold solution obtained in step (3) above, oscillate on a 4 °C shaking table for 2h, centrifuge at 10000g for 30min, wash 3 times with PB buffer solution, take the solid precipitate, and resuspend the nano gold particles with 150 μL of PB buffer solution to obtain nano gold particles with surface-fixed aflatoxin B1 monoclonal antibody. The gold nanoparticles have a size of 38 nm, the incubation is incubation at 35-37 ℃ for 90-100 min, only 2 μL of each sample is needed, and the method uses gold nanoparticles with surface-fixed aflatoxin B1 monoclonal antibody to enhance the inherent fluorescence of the surface artificial antigen aflatoxin B1-BSA of the photonic crystal microspheres to realize detection.

2. The method for non-labeled detection of aflatoxin B1 in cereals based on intrinsic fluorescence of proteins according to claim 1, characterized in that, The fluorescence signal of the detected photonic crystal microspheres is determined by using an inverted fluorescence microscope configured with a fluorescence spectrometer and a CCD imaging system, the results are recorded, and OriginPro9 software is used for analysis.

3. The method for non-labeled detection of aflatoxin B1 in cereals based on intrinsic fluorescence of proteins according to claim 2, characterized in that, The detection is focused under a 40x objective lens, and the excitation light wavelength is 360-370 nm.

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