Method for rapidly and quantitatively detecting aflatoxin B1 in rice in magnetic control up-conversion fluorescence quenching mode

Abstract

The invention discloses a method for rapidly and quantitatively detecting aflatoxin B1 in rice in a magnetic control up-conversion fluorescence quenching mode, and belongs to the field of food safety detection. The detection method comprises the following steps: constructing an alendronate modified lanthanide up-conversion material donor, synthesizing and preparing magnetic gold nano Fe (at) AuNPs material particles, modifying and connecting Fe (at) AuNPs by utilizing a specific AFB1 aptamer, constructing a magnetic control up-conversion detection system, reacting by adopting a color developing reagent TMB to cause fluorescence quenching, and detecting the AFB1 in the magnetic control up-conversion detection system according to the characteristic that the specific aptamer is preferentially combined with the AFB1. Analyzing the linear correlation between the fluorescence intensity and the concentration content of the AFB1 based on the fluorescence inner filtration effect between the AFB1-aptamer and the up-conversion material in the supernatant after magnetic control separation; the alendronate modified upconversion detection system based on the fluorescence internal filtration effect established in the invention has stable quantitative detection range and detection limit, the detection range can be kept at 0.5-0.01 mg / kg, the detection limit can reach 0.1 mu g / mL, the adding standard recovery rate is 90.2-96.8%, and the content of AFB1 in rice can be specifically detected.

Description

Technical Field

[0001] The present invention belongs to the field of rapid detection of food hazards, and particularly relates to a method for rapid quantitative detection of aflatoxin B1 in paddy rice under a magnetically controlled upconversion fluorescence quenching mode. Background Art

[0002] There is a risk of mildew in the whole chain of paddy rice during circulation, storage, transportation, marketing, production and post-production. The growth of fungi may lead to the generation of harmful substances such as aflatoxin B1 (AFB1), which pose a serious threat to human health. Therefore, it is particularly necessary to ensure the safety of paddy rice, especially the detection of aflatoxin B1.

[0003] Currently, the detection methods for the content of aflatoxin B1 in grains mainly include high performance liquid chromatography (HPLC), gas chromatography (GC), and enzyme-linked immunosorbent assay (ELISA), etc. Although these traditional detection techniques have advantages in accuracy and sensitivity, they generally have problems such as long detection period, complex operation, and high cost, resulting in difficulty in meeting the rapid response requirements in the actual grain production and circulation links. In addition, the existing detection methods have high requirements for sample pretreatment and often require professional personnel to operate, further increasing the difficulty and time cost of detection. Therefore, it is urgent to improve the existing detection techniques to improve the detection efficiency and convenience. Summary of the Invention

[0004] In view of the above problems, the present application is based on a quantitative detection system for AFB1 in paddy rice under a magnetically controlled upconversion fluorescence quenching mode constructed by magnetic gold nanoparticles modified with aflatoxin B1 (AFB1) specific aptamer, water-soluble modified upconversion materials (upconversion luminescent materials), and 3,3',5,5'-tetramethylbenzidine, so as to shorten the detection time and improve the sensitivity and specificity of detection.

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

[0006] A method for rapid quantitative detection of aflatoxin B1 in paddy rice under a magnetically controlled upconversion fluorescence quenching mode, the specific steps are as follows:

[0007] Step 1: Extract AFB1 from paddy rice:

[0008] Weigh the sample paddy rice and place it in a methanol-aqueous solution, stir and extract, and then centrifuge; take the supernatant and filter it through a 0.22 μm pore size filter, and then add the filtrate to 2 times its volume of Tris-HCl buffer solution to obtain the sample extract for standby;

[0009] Step 2: Prepare a lanthanide upconversion solution:

[0010] Dissolve ytterbium(III) chloride hexahydrate, yttrium(III) chloride hexahydrate, erbium(III) chloride hexahydrate and gallium(III) chloride hexahydrate in methanol, then transfer to a beaker, and add oleic acid (C 18 H 34 O2) and 1-octadecene (C 18 H 36 ). Heat the mixture to 150 °C and react for 45 min to obtain a lanthanide upconversion solution;

[0011] Step 3: Prepare upconversion nanosolid powder:

[0012] Cool the lanthanide upconversion solution obtained in Step 2 under nitrogen blowing and add it dropwise to a methanol solution containing ammonium fluoride and sodium hydroxide. Heat in a water bath and rapidly raise the temperature, and maintain for 60 min under magnetic stirring to synthesize nanoparticles with a particle size of 35 - 40 nm; Wash the nanoparticles several times with ethanol aqueous solution I (ethanol volume concentration is 25%) to wash away excess oleic acid, then centrifuge to remove the unreacted precursor solution, and finally dry under vacuum. The obtained solid powder is the upconversion nanoparticles, which are reserved for use;

[0013] Step 4: Water-soluble modification of upconversion nanomaterials:

[0014] Take 200 mg of the upconversion nanoparticles prepared in Step 3 and 50 mg of alendronic acid in 20 mL of an ethanol-chloroform solution (volume ratio of ethanol to chloroform is 2:5), ultrasonically dissolve, and rapidly stir and react. Adjust the pH to 2 - 3 with dilute hydrochloric acid, wash the mixture with ethanol aqueous solution II (ethanol volume concentration 50%), and dry under vacuum at 60 °C for 6 h to obtain water-soluble modified upconversion nanomaterials, which are reserved for use;

[0015] Step 5: Construct specific AFB1 aptamer:

[0016] Biotinylate the 5'-end of the aptamer shown in nucleotide sequence SEQ INNO.2, and modify the 3'-end with a thiol-modifying reagent to obtain a specific AFB1 aptamer with the nucleotide sequence shown in SEQ ID NO.1; Add a 20 mM PBS solution (phosphate buffer solution) to it to obtain an aptamer solution with a concentration of 50 mM;

[0017] Step 6: Prepare aptamer-magnetic Fe@AuNPs composite:

[0018] Mix 1 mL of chloroauric acid and 99 mL of deionized water, heat to boiling, add 1 mL (volume concentration 3%) of sodium citrate solution, and continuously stir for 30 min to obtain a gold nanoparticle colloidal solution (AuNPs), which is reserved for use;

[0019] Subsequently, polyethyleneimine (PEI)-modified magnetite was synthesized. The specific modification scheme was as follows: 100 mg of nano-magnetite was mixed with 75 mL of deionized water, and polyethyleneimine with a final concentration of 5 mg / mL was added thereto. After stirring for 2 h, centrifugation was performed, and drying was carried out at 70 °C to obtain polyethyleneimine (PEI)-modified magnetite. Then, the polyethyleneimine (PEI)-modified magnetite was dissolved in a 20 mM PBS solution to obtain a polyethyleneimine (PEI)-modified magnetite solution with a concentration of 1 g / m, which was reserved for use.

[0020] 10 mL of the synthesized polyethyleneimine (PEI)-modified magnetite was mixed with 30 mL of AuNPs prepared in the above steps, and ultrasonic treatment (ultrasonic treatment at 40 kHz for 1 h) was carried out to obtain a magnetic Fe@AuNPs solution; after the obtained magnetic Fe@AuNPs solution was subjected to magnetic separation, the solid magnetic Fe@AuNPs was redissolved in 10 mL of 20 mM PBS to obtain a purified magnetic Fe@AuNPs solution; 200 μL of the purified magnetic Fe@AuNPs solution was mixed with 200 μL of the AFB1 aptamer (50 mM) prepared in step 5. The aptamer was connected and modified to Fe@AuNPs through -SH to obtain an aptamer-magnetic Fe@AuNPs composite material, and 20 mM PBS was added to adjust the concentration of the ligand-magnetic Fe@AuNPs composite material to 0.5 mg / mL.

[0021] Step 7: Construct a magnetically controlled upconversion nanomaterial detection system:

[0022] The sample extract obtained in step 1 was used, and it was reacted with the aptamer-magnetic Fe@AuNPs composite material with a concentration of 0.5 mg / mL prepared in step 6 for 25 min. After magnetic separation, the supernatant was retained and mixed with 3,3',5,5'-tetramethylbenzidine (TMB) for reaction for 15 min. After completion, the water-soluble modified upconversion nanomaterial prepared in step 4 was added, and after mixing, the fluorescence signal intensity was measured. In this step, the volumes of the sample extract, aptamer-magnetic Fe@AuNPs composite material, TMB, and water-soluble modified upconversion nanomaterial added were the same, and the addition amount could be selected according to actual conditions. The addition amount was preferably 200 μL - 1.0 mL.

[0023] Meanwhile, with the concentration of AFB1 standards at concentration gradients of 0 (a blank sample obtained by mixing methanol and water in equal volumes), 50, 100, 250, and 500 ng / mL as the abscissa and the fluorescence signal intensity as the ordinate, a standard curve was constructed;

[0024] The fluorescence signal measured for the sample was brought into the above standard curve, and the AFB1 content in the sample was obtained.

[0025] Preferably, for the paddy sample in Step 1: 1 g of paddy is finely pulverized and sieved through a 40-mesh sieve. The methanol-aqueous solution is 4 mL of a 70% (by volume) methanol-aqueous solution. The stirring extraction reaction time is 30 min. The centrifugation conditions are 4 °C, 12,000 rpm, and 10 min. The dilution ratio of the filtrate is that 1.5 mL of the supernatant is diluted in 3 mL of Tris-Hcl.

[0026] Preferably, in Step 2, for the preparation of the lanthanide upconversion solution: 0.065 g of ytterbium(III) chloride hexahydrate, 0.117 g of yttrium(III) chloride hexahydrate, 0.006 g of erbium(III) chloride hexahydrate, and 0.091 g of gallium(III) chloride hexahydrate are added to 4 mL of methanol (pure methanol). The addition amounts of oleic acid and 1-octadecene are 6 mL and 14 mL, respectively.

[0027] Preferably, in Step 3, for the preparation of the upconversion nanosolid powder: The methanol solution containing ammonium fluoride and sodium hydroxide mentioned above means that 0.4446 g of ammonium fluoride and 0.3 g of sodium hydroxide are added to 35 mL of methanol. After heating in a water bath at 70 °C for 45 min, it is rapidly (heating rate > 20 °C / min) heated to 300 °C. The ethanol-aqueous solution I mentioned means a solution with an ethanol volume concentration of 25%, and the added volume is 75 mL. The centrifugation conditions are centrifugation at 6000 r / min for 10 min. The vacuum drying conditions are continuous drying at 60 °C for 24 h.

[0028] Preferably, in Step 4, in the ethanol-chloroform solution, the volume ratio of ethanol to chloroform is 2:5, and in the ethanol-aqueous solution II, the volume ratio of ethanol to water is 1:1.

[0029] Preferably, in Step 5, for the construction of the specific AFB1 aptamer: ssDNA as shown in SEQ ID NO.2 is selected, its base number is 50, the corresponding number of PCR amplification cycles is set to 5 times, and the amount of streptavidin-labeled horseradish peroxidase added to every 10 μM of ssDNA is 0.5 U.

[0030] Preferably, in Step 7, for the construction of the magnetically controlled upconversion nanomaterial detection system: The volume ratio of the Fe@AuNPs composite material to TMB is 1:1, and the volume ratio of the Fe@AuNPs composite material to the upconversion nanomaterial is 1:1. After adding 0.5 mg / mL of the water-soluble modified upconversion nanomaterial and reacting for 10 min, the fluorescence signal intensity is measured under 980 nm fluorescence excitation.

[0031] In view of the problem of huge time and labor costs in the traditional chemical quantitative detection process, the present invention realizes the purpose of rapid detection of AFB1 in rice through an aptamer-modified magnetic gold nanoparticle-alendronate-modified upconversion nanomaterial-3,3',5,5'-tetramethylbenzidine reaction system. Based on the characteristic that AFB1 preferentially binds to the specific aptamer, and then based on the technical principle of reducing the fluorescence quenching effect, compared with the prior art, the method of the present application has the following beneficial effects:

[0032] (1) Based on the specificity of AFB1 and the AFB1 aptamer, and based on the fluorescence inner filter effect between horseradish peroxidase and the upconversion material, the linear relationship between the fluorescence intensity and the AFB1 concentration is analyzed; this method can quickly identify and quantitatively analyze aflatoxin B1 in the sample, avoiding the long detection experiment process in the traditional detection method.

[0033] (2) The specific detection system constructed by the present invention shows a high transferability for the detection of AFB1 in rice, can eliminate the interference of other toxins including vomitoxin and ochratoxin, eliminate the phenomenon of weak background fluorescence signal, and overcome the deficiencies of the traditional detection method.

[0034] (3) The established linear detection range of AFB1 in the present invention is 0.5 μg - 0.01 mg / kg, the corresponding detection limit LOD is 0.1 μg / kg, and the spiked recovery rate is between 90.2% and 96.8%. Comparing with the enzyme-linked immunosorbent assay for the determination of aflatoxin B1 in feed GB / T17480-2008, the lowest detection limit is 0.5 μg / kg and the quantification limit is 2 μg / kg. This method can realize the rapid detection of aflatoxin B1 in rice from the original detection time of 2 - 3 hours to 0.2 - 0.5 hours on the premise of the same detection accuracy as the national standard method.

[0035] (4) By constructing a specific AFB1 detection system, the present invention realizes the high-specificity and high-sensitivity detection of the AFB1 content in rice. The detection steps are reasonable, the operation is simple, and it has a wide detection linear range and a low detection limit, so that this method has good practical value. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for use in the embodiments. Obviously, the following described drawings are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0037] Figure 1 It is the transmission electron microscope image after the water-soluble modification of the upconversion material in Example 1;

[0038] Figure 2 It is the transmission electron microscopy image of the gold nanoparticles synthesized by the chloroauric acid redox method in Example 3;

[0039] Figure 3 It is the transmission electron microscopy image of the PEI-modified nano-ferroferric oxide in Example 3;

[0040] Figure 4 It is the transmission electron microscopy image of the Fe@AuNPs nanoparticles prepared in Example 3;

[0041] Figure 5 It is the fluorescence signal standard curve in the magnetically controlled upconversion fluorescence quenching mode in Example 4;

[0042] Figure 6 It is the specific verification result of the magnetically controlled upconversion fluorescence quenching mode in Example 4. Detailed implementation manners

[0043] To make the objectives, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly described below. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all of the embodiments; based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention.

[0044] The following further describes the detailed implementation manners of the present invention with reference to the accompanying drawings of the specification.

[0045] The reagents involved in the embodiments are all of analytical grade. The fluorescence detection instrument used in the embodiments is purchased from Zolix HVC1800 (High Voltage Power Supply) DCS200PC (Photon Counting) Omni-200i (Monochromator / Spectrograph).

[0046] The preparation method of the PBS buffer solution in the embodiments is as follows: Beyotime 10×PBS buffer solution (sterile, pH 7.4), product number ST476, and after dilution by 50 times, a 20 mM PBS buffer solution is obtained.

[0047] Example 1: Preparation and characterization of water-soluble upconversion materials

[0048] In this example, the preparation process of the water-soluble upconversion material includes:

[0049] Dissolve 0.065 g of ytterbium(III) chloride hexahydrate, 0.117 g of yttrium(III) chloride hexahydrate, 0.006 g of erbium(III) chloride hexahydrate and 0.091 g of gallium(III) chloride hexahydrate in 4 mL of a methanol-aqueous solution with a volume concentration of 70%. Then add 6 mL of oleic acid and 14 mL of 1-octadecene, mix and heat, and react at 150 °C for 45 min to obtain a lanthanide upconversion solution;

[0050] Subsequently, cool the reaction solution under nitrogen blowing, and gradually add dropwise a methanol solution containing ammonium fluoride and sodium hydroxide (dissolve 0.4446 g of ammonium fluoride and 0.3 g of sodium hydroxide in 35 mL of methanol); heat in a 70 °C water bath for 45 min, then quickly (heating rate > 20 °C / min) raise the temperature to 300 °C, and react under magnetic stirring for 60 min to complete the synthesis of nanoparticles. The synthesized nanoparticles have a particle size of 35 - 40 nm. Then wash the particles 3 times with 75 mL of an ethanol-aqueous solution with a volume concentration of 25%, centrifuge at 6000 r / min for 10 min to remove unreacted precursors, and finally dry in vacuo at 60 °C. The obtained solid powder is the upconversion nanoparticles.

[0051] To make the material water-soluble, dissolve 200 mg of the prepared upconversion nanoparticles and 50 mg of alendronic acid in 20 mL of an ethanol-chloroform solution (volume ratio of ethanol to chloroform is 2:5), and perform ultrasonic treatment (40 kHz for 1 hour) to dissolve, and rapidly stir and react at 1500 rpm for 1 h. Then adjust the pH to 3 with 1 mol of dilute hydrochloric acid solution, wash the particles 3 times with an ethanol-aqueous solution (ethanol and water are mixed in equal volumes), and then dry in vacuo at 60 °C for 6 h to obtain a water-soluble modified upconversion nanomaterial.

[0052] The above preparation process was characterized by techniques such as fluorescence spectroscopy, transmission electron microscopy, and X-ray diffraction to evaluate the optical properties and structure of the material. The results of electron microscopy detection are as Figure 1 shown, indicating that the water-soluble modified upconversion nanomaterial prepared in this example changes the original oil-soluble characteristics. After alendronic acid modification, it can be uniformly dispersed in the aqueous phase and can provide a signal switch for the later entire Aspergillus flavus water-soluble system.

[0053] Example 2: Construction of specific AFB1 aptamer

[0054] To obtain an ssDNA sequence highly specific for AFB1, in this example, the SELEX method was used to specifically screen AFB1 from a gene library, and the obtained aptamer was further purified. All aptamers used were purified by HPLC, and the obtained aptamer ssDNA nucleotides are shown in SEQ ID NO.2.

[0055] The above HPLC purification method is a conventional method in the art. In this example, the method disclosed in the literature "Optimisation of denaturing ion pair reversed phase HPLC for the purification of ssDNA in SELEX" (https: / / www.sciencedirect.com / science / article / pii / S0021967324000724?via%3Dihu b) was referred to.

[0056] The 5'-end of the target aptamer sequence was biotinylated as follows: 1 μg of single-stranded DNA with a length of 47 nt (SEQ ID NO. 2: 5'-GTTGGGCACGTGTTGTCTCTCTGTGTCT-3') was mixed with 10 U of T4 PNK enzyme (Thermo Scientific TM , catalog number EK0032), 1 mM biotinylated ATP (Biotin-7-ATP, CAS 343988-25-6), 10 mM ATP, and 2 μL of 10× reaction buffer (500 mM Tris-HCl, 100 mM MgCl2, 50 mM DTT, pH 7.6) in 20 μL of nuclease-free water, and incubated at 37 °C for 30 minutes. Subsequently, the enzyme activity was inactivated by heating at 75 °C for 10 minutes. The reaction product was purified by ethanol precipitation: 2.5 volumes of pre-cooled ethanol and 0.1 volume of 3 M sodium acetate (pH 5.2) were added, and after sedimentation at -20 °C for 30 minutes, centrifuged at 12,000×g for 15 minutes, washed twice with 70% ethanol, and finally dissolved in 20 μL of TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8.0), and the DNA recovery rate was ≥90%. To verify the modification efficiency, 10 μL of the purified DNA was incubated with streptavidin magnetic beads (Dynabeads TM M-270) in a binding buffer containing 2 M NaCl for 15 minutes. After magnetic separation, the unbound DNA in the supernatant was detected by Qubit TM fluorescence quantitative detection, and the biotin labeling efficiency was calculated to be ≥92%; further mass spectrometry analysis showed that the molecular weight of the modified DNA increased by 244.3 Da, with a deviation from the theoretical value (244.31 Da) of ≤0.05%, proving that the modification was successful.

[0057] Modify the 3'-end of the target aptamer sequence (SEQ ID NO.2) with a thiol group. The specific implementation steps are as follows: During the synthesis process, select a 3'-thiol modifier reagent (3’Thiol Modifier C6 S-S, catalog number 10-1937-95). This reagent is chemically coupled to the 3'-end of the DNA strand through phosphoramidite in the last step of solid-phase synthesis to form a stable disulfide bond (-S-S-) intermediate. After synthesis, deprotect by ammonia removal, and purify using reverse HPLC (mobile phase: 0.1M TEAA buffer / acetonitrile gradient, C18 column). Collect the main peak and lyophilize to obtain a DNA product with a disulfide bond at the 3'-end. To reduce the disulfide bond to a free thiol group (-SH), dissolve the DNA in Tris-HCl buffer (pH 8.0, concentration 1μg / μL) containing 50mM DTT (dithiothreitol), incubate at 37°C for 1 hour, then remove DTT through a desalting column. The final product is dissolved in TE buffer without EDTA (pH 8.0) and stored in the dark at -80°C. Verify the modified molecular weight by mass spectrometry, and the deviation between the measured value and the theoretical value (increase of 136.2Da, corresponding to C3-S-S modification) is ≤0.1%; further quantify the free thiol group using Ellman's reagent (5,5’-dithiobis-2-nitrobenzoic acid, DTNB): React 1μg of the modified DNA with 0.1mM DTNB in a pH 8.0 buffer at room temperature for 10 minutes. Absorbance detection at 412nm shows that the thiol concentration is 0.95±0.05nmol / μg DNA, and the labeling efficiency is ≥90%, proving successful modification.

[0058] The nucleotide sequence of the modified aptamer ssDNA is shown as SEQ ID NO.1 in Table 1. Add 20mM pbs solution to the modified ssDNA to obtain an aptamer solution with a concentration of 50mM.

[0059] Table 1 Aptamer base sequence table

[0060]

[0061] Example 3 Synthesis of aptamer-magnetic Fe@AuNPs composite

[0062] The synthesis process of magnetic Fe@AuNPs in this example is divided into two steps: First, synthesize AuNPs using the chloroauric acid redox method. The specific synthesis scheme is to mix 1mL of chloroauric acid and 99mL of deionized water, heat to boiling, add 1mL (volume concentration 3%) of sodium citrate solution, and continuously stir for 30 minutes to obtain a colloidal solution of gold nanoparticles (AuNPs) for standby. The transmission electron microscopy image of the obtained AuNPs is as shown in Figure 2 shown, Figure 2 where a and b are electron microscopy images at different magnifications.

[0063] Subsequently, polyethyleneimine (PEI)-modified iron oxide was synthesized. The specific modification scheme was as follows: 100 mg of nano iron oxide was mixed with 75 mL of deionized water, and polyethyleneimine with a final concentration of 5 mg / mL was added thereto. After stirring for 2 h, centrifugation was performed, and drying was carried out at 70 °C for 8 h. Covalent bonding was carried out on the polyethyleneimine (PEI) nano iron oxide powder to obtain polyethyleneimine (PEI)-modified iron oxide, and its transmission electron micrograph is as shown in Figure 3 shown. Then, the polyethyleneimine (PEI)-modified iron oxide was dissolved in a 20 mM PBS solution to obtain a polyethyleneimine (PEI)-modified iron oxide solution with a concentration of 1 g / m for standby;

[0064] 10 mL of the polyethyleneimine (PEI)-modified iron oxide solution was taken and mixed with 30 mL of AuNPs, and ultrasonic treatment (40 kHz) was performed for 1 hour to obtain a magnetic Fe@AuNPs solution; after the obtained magnetic Fe@AuNPs solution was magnetically separated, the solid magnetic Fe@AuNPs was redissolved in 10 mL of 20 mM PBS to obtain a purified magnetic Fe@AuNPs solution, and the synthesis of the target magnetic Fe@AuNPs was completed, and its transmission electron microscope is as shown in Figure 4 shown. By careful observation Figure 3 it can be found that the irregular serrated edges in the figure gradually changed to a more regular form in Figure 4 after binding with gold nanoparticles. This change indicates the effective binding and morphological improvement of the particles during the synthesis process, laying a foundation for subsequent application in the detection of aflatoxin B1.

[0065] Preparation of aptamer-magnetic Fe@AuNPs composite material: 200 μL of the prepared purified magnetic Fe@AuNPs solution was taken and 200 μL of the aptamer solution with a concentration of 50 mM prepared in Example 2 was added to obtain an aptamer-magnetic Fe@AuNPs composite material.

[0066] Example 4 Detection and analysis of AFB1 in paddy rice under the mode of magnetically controlled fluorescence quenching

[0067] On the basis of Example 3, gradient concentration AFB1 standard solutions were added to the obtained 0.5 mg / mL aptamer-magnetic Fe@AuNPs composite material, specifically 0 ng / mL (methanol and water were mixed in equal volumes, blank sample), 50 ng / mL, 100 ng / mL, 250 ng / mL, 500 ng / ml (the standard solution was diluted with a methanol aqueous solution, and the methanol aqueous solution was obtained by mixing methanol and water in equal volumes). The fluorescence spectrophotometer was used to observe the different degrees of fluorescence signal quenching, and then the fluorescence signal standard curve was plotted, as shown in Figure 5 shown.

[0068] The specific scheme is as follows: Take 200 μL of the aptamer-magnetic Fe@AuNPs composite material prepared in Example 3 and mix it with 200 μL of AFB1 standard solutions with different concentrations, and react for 25 min. After magnetic separation, take the supernatant; then add 200 μL of TMB chromogenic solution to the supernatant, react for 15 min, then add 200 μL of the water-soluble modified upconversion nanomaterial prepared in Example 1 and react for 10 min. Finally, measure the fluorescence intensity under a 980 nm excitation light source, and form a standard curve as shown in Figure 5 Y = -2.661X + 2040, where Y represents the fluorescence intensity signal and X represents the AFB1 concentration.

[0069] Perform AFB1 extraction treatment on the rice samples, and detect them using the same method as the above scheme.

[0070] The steps for extracting aflatoxin B1 from rice are as follows: Weigh the test rice samples (purchased from the market), inoculate with Aspergillus flavus and store for 7 d, then select 1 g of the mildewed particles, finely pulverize them and pass through a 40-mesh sieve, place them in 4 mL of a methanol-aqueous solution (volume concentration 70%) and stir for extraction for 30 min, and then centrifuge at 4 °C and 12,000 rpm for 10 min. Take the supernatant and filter it through a 0.22 μm needle filter, and take 1.5 mL of the filtered supernatant and dilute it to 3 mL of Tris-Hcl buffer to obtain a sample extract.

[0071] Take 200 μL of the aptamer-magnetic Fe@AuNPs composite material and mix it with 200 μL of the above sample extract, react for 25 min, then perform magnetic separation, and take the supernatant; add 200 μL of TMB chromogenic solution to the supernatant, react for 15 min, then add 200 μL of the water-soluble modified upconversion nanomaterial prepared in Example 1 and react for 10 min. Finally, measure the fluorescence intensity under a 980 nm excitation light source to obtain the fluorescence signal intensity, and obtain the final concentration result through the standard curve.

[0072] In addition, in the specificity test, add other toxins with the same concentration that may exist in rice, including the common ochratoxin A (OTA), ochratoxin B (OTB), and deoxynivalenol (DON) in the detection of AFB1 in rice. The corresponding detected fluorescence signal results are as shown in Figure 6 The results show that in the experiment carried out in this example, only the AFB1 group showed a significant decrease in fluorescence intensity, indicating that the detection method proposed in this application has high specificity.

Claims

1. A method for rapid quantitative detection of aflatoxin B1 in paddy rice under the mode of magnetically controlled up-conversion fluorescence quenching, characterized in that, The specific steps are as follows: 1) Extract AFB1 from paddy rice: Weigh the test paddy rice and place it in an aqueous methanol solution. Stir for extraction and then centrifuge. Take the supernatant and filter it through a 0.22 μm pore size filter. Then add the filtrate to 2 times its volume of Tris-HCl buffer solution to obtain the sample extract for standby; 2) Dissolve ytterbium(III) chloride hexahydrate, yttrium(III) chloride hexahydrate, erbium(III) chloride hexahydrate and gallium(III) chloride hexahydrate in methanol. Then add oleic acid and 1-octadecene, heat to 150 °C and react for 45 min to obtain a lanthanide upconversion solution; 3) After the lanthanide upconversion solution obtained in step 2) is cooled, dropwise add it to a methanol solution containing ammonium fluoride and sodium hydroxide, heat for reaction to obtain nanoparticles; wash with ethanol aqueous solution I, then centrifuge and dry to obtain upconversion nanoparticles; 4) Take 200 mg of the upconversion nanoparticles prepared in step 3) and 50 mg of alendronic acid and dissolve them in 20 mL of ethanol-chloroform solution, stir for reaction; then adjust the pH to 2-3, wash with ethanol aqueous solution II, and dry to obtain a water-soluble modified upconversion nanomaterial for standby; in the ethanol-chloroform solution, the volume ratio of ethanol to chloroform is 2:5; 5) Biotinylate the 5' end of the aptamer with the nucleotide sequence shown in SEQ IN NO.2, and modify the 3' end with a thiol-modifying reagent to obtain the AFB1 aptamer with the nucleotide sequence shown in SEQ ID NO.1; add a 20 mM PBS solution to obtain an aptamer solution with a concentration of 50 mM for standby; 6) Mix 100 mg of nano-ferroferric oxide with 75 mL of deionized water, add polyethyleneimine with a final concentration of 5 mg / mL, stir for reaction and then centrifuge. Take the precipitate and dry it to obtain PEI-modified ferroferric oxide; dissolve it in a 20 mM PBS solution to obtain a PEI-modified ferroferric oxide solution with a concentration of 1 g / m; Mix the PEI-modified ferroferric oxide solution with a gold nanoparticle colloidal solution for reaction to obtain a magnetic Fe@AuNPs solution. After magnetic separation, take the solid and redissolve it in 10 ml of 20 mM PBS solution to obtain a purified magnetic Fe@AuNPs solution; the volume ratios of the added PEI-modified ferroferric oxide solution, gold nanoparticle colloidal solution, and PBS solution are 1:3:1 in sequence; Mix the purified magnetic Fe@AuNPs solution and an equal volume of the aptamer solution prepared in step 5) to obtain a complex, and adjust the concentration of the complex to 0.5 mg / mL with a 20 mM PBS solution, which is the aptamer-magnetic Fe@AuNPs composite material for standby; 7) Add the aptamer-magnetic Fe@AuNPs composite material prepared in step 6) to the sample extract obtained in step 1) for reaction. After magnetic separation, take the supernatant, add TMB and mix for reaction; after the reaction is completed, add the water-soluble modified upconversion nanomaterial prepared in step 4), mix well and then measure the fluorescence signal intensity; Meanwhile, with the concentrations of the AFB1 standard at concentration gradients of blank sample, 50, 100, 250, and 500 ng / mL as the abscissa and the fluorescence signal intensity as the ordinate, a standard curve was constructed; the blank sample was obtained by mixing methanol and water in equal volumes; The fluorescence signal measured from the sample was substituted into the above standard curve to obtain the AFB1 content in the sample; The volumes of the sample extraction solution, aptamer-magnetic Fe@AuNPs composite material, TMB, and water-soluble modified upconversion nanomaterial added were the same.

2. The rapid quantitative detection method of aflatoxin B1 in paddy under the magnetically controlled up-conversion fluorescence quenching mode according to claim 1, wherein, In step 1), the test sample of paddy rice was obtained by crushing paddy rice and passing it through a 40-mesh sieve; the methanol aqueous solution was a 70% (v / v) methanol aqueous solution; the stirring extraction reaction time was 30 min.

3. The rapid quantitative detection method of aflatoxin B1 in paddy under the magnetically controlled up-conversion fluorescence quenching mode according to claim 1, wherein, In step 2), the mass ratio of ytterbium(III) chloride hexahydrate, yttrium(III) chloride hexahydrate, erbium(III) chloride hexahydrate, and gallium(III) chloride hexahydrate was 0.065:0.117:0.006:0.091 in sequence; the volume ratio of methanol, oleic acid, and 1-octadecene added was 4:6:14 in sequence; the mass-volume ratio of ytterbium(III) chloride hexahydrate to methanol added was 0.065:4, and the unit of the mass-volume ratio was g / ml.

4. The rapid quantitative detection method of aflatoxin B1 in paddy rice in the magnetron upconversion fluorescence quenching mode according to claim 1, characterized in that, In step 3), the method for preparing the methanol solution containing ammonium fluoride and sodium hydroxide was as follows: 0.4446 g of ammonium fluoride and 0.3 g of sodium hydroxide were added to 35 mL of methanol; the heating reaction meant heating to 70 °C for reaction for 45 min, and then raising the temperature to 300 °C for reaction for 60 min, with the heating rate > 20 °C / min.

5. The method for rapid quantitative detection of aflatoxin B1 in paddy rice under the magnetically controlled up-conversion fluorescence quenching mode according to claim 1, wherein In step 3), the particle size of the nanoparticles was 35 - 40 nm.

6. The rapid quantitative detection method of aflatoxin B1 in paddy under the magnetically controlled up-conversion fluorescence quenching mode according to claim 1, wherein, In step 4), the volume ratio of ethanol to chloroform in the ethanol-chloroform solution was 2:

5.

7. The rapid quantitative detection method of aflatoxin B1 in paddy under the magnetic control up-conversion fluorescence quenching mode according to claim 1, wherein, In the ethanol aqueous solution I, the volume concentration of ethanol was 25%; in the ethanol aqueous solution II, the volume concentration of ethanol was 50%.

8. The method for rapid quantitative detection of aflatoxin B1 in paddy rice in the magnetically controlled up-conversion fluorescence quenching mode according to claim 1, wherein, In step 6), the reaction time between the sample extraction solution and the aptamer-magnetic Fe@AuNPs composite material was 25 min, and the reaction time between the supernatant and TMB was 15 min.

9. The method for rapid quantitative detection of aflatoxin B1 in paddy rice under the magnetic control up-conversion fluorescence quenching mode according to claim 1, characterized in that, In step 6), the gold nanoparticle colloidal solution was prepared by the following method: 1 mL of chloroauric acid and 99 mL of deionized water were mixed, heated to boiling, and then 1 mL of a 3% (v / v) sodium citrate solution was added, and after continuous stirring for 30 min, the gold nanoparticle colloidal solution was obtained.

10. The method for rapid quantitative detection of aflatoxin B1 in rice in the magnetically controlled up-conversion fluorescence quenching mode according to claim 1, wherein, In step 7), the measurement of the fluorescence signal intensity meant measuring the fluorescence signal intensity under 980 nm fluorescence excitation.

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