Magnetic rice husk carbon material, preparation method and application thereof, and sample pretreatment method and detection method of aflatoxin B1

Abstract

The invention belongs to the technical field of magnetic materials, and particularly relates to a magnetic rice husk carbon material and a preparation method and application thereof as well as a sample pretreatment method and a detection method of aflatoxin B1. The magnetic rice husk carbon material comprises Fe3O4 nano-particles and amorphous silicon dioxide loaded on the surfaces of the Fe3O4 nano-particles. The magnetic rice husk carbon material has the adsorption capacity of a carbon-based material and the separation characteristic of a magnetic material at the same time, the magnetic rice husk carbon material is used as a magnetic solid-phase extraction material to enrich and purify aflatoxin B1 in rice, the magnetic rice husk carbon material has superparamagnetism and excellent dispersity, and an adsorption material can be rapidly separated from a sample matrix. The magnetic rice husk carbon material is an efficient pretreatment enrichment purification material, the sample pretreatment process of the aflatoxin B1 is simple, the time is short, and the use of a large amount of organic reagents can be avoided.

Description

Technical Field

[0001] The present invention belongs to the technical field of magnetic materials, and particularly relates to a magnetic rice husk carbon material, a preparation method and application thereof, a sample pretreatment method and a detection method for aflatoxin B1. Background Art

[0002] Aflatoxin B1 is one of the pollutants affecting the quality and safety of rice. Aflatoxin B1 is easy to cause cancer, teratogenesis and mutagenesis, can induce bleeding, edema and immunosuppression, and even induce liver cancer, seriously threatening human life and health.

[0003] At present, there are various methods for determining aflatoxin B1, such as high performance liquid chromatography, ultra high performance liquid chromatography-mass spectrometry, etc. However, most of these methods require a time-consuming pretreatment process and cannot detect aflatoxin B1 quickly and sensitively. In order to improve the sensitivity and reduce the matrix effect, different types of solvents or adsorbents have been used in the pretreatment step, such as liquid-liquid extraction, solid phase extraction and immunoaffinity column adsorption. However, immunoaffinity column adsorption requires expensive antibodies, while liquid-liquid extraction and solid phase extraction consume a large amount of organic solvents and take a long time, which hinders their application in the quality screening of rice. Therefore, it is of great significance to develop a rapid and simple sample pretreatment method for aflatoxin B1. Summary of the Invention

[0004] In view of this, the purpose of the present invention is to provide a magnetic rice husk carbon material, a preparation method and application thereof, a sample pretreatment method and a detection method for aflatoxin B1. Using the magnetic rice husk carbon material of the present invention in the sample pretreatment process of aflatoxin B1, the method is simple and time-consuming.

[0005] The present invention provides a magnetic rice husk carbon material, including Fe3O4 nanoparticles and amorphous silica loaded on the surface of the Fe3O4 nanoparticles.

[0006] Preferably, the particle size of the magnetic rice husk carbon material is 200-400 nm.

[0007] The present invention also provides a preparation method of the magnetic rice husk carbon material according to the above technical solution, including the following steps:

[0008] Performing first calcination on rice husks to obtain a rice husk carbonization product; the main component of the rice husk carbonization product is amorphous silica;

[0009] Mixing and reacting the rice husk carbonization product, an alkali and water to obtain a silicate-containing solution;

[0010] Dropping the silicate-containing solution into a Fe3O4 dispersion, performing hydrothermal reaction under alkaline conditions, and performing second calcination on the obtained precipitate to obtain a magnetic rice husk carbon material.

[0011] Preferably, the temperature of the first calcination is 500-600 °C, and the heat preservation time is 6-10 h.

[0012] Preferably, the concentration of Fe3O4 in the Fe3O4 dispersion is 1-3 mg / mL, and the Fe3O4 dispersion also contains a surfactant.

[0013] Preferably, the alkaline condition of the hydrothermal reaction is pH = 10-12.

[0014] Preferably, the temperature of the hydrothermal reaction is 70-90 °C, and the time is 12-36 h.

[0015] The present invention also provides the application of the magnetic rice husk carbon material described in the above technical solution or the magnetic rice husk carbon material obtained by the above preparation method in the detection of aflatoxin B1.

[0016] The present invention also provides a sample pretreatment method for detecting aflatoxin B1, comprising the following steps:

[0017] Mix the sample to be tested with an aqueous methanol solution for extraction to obtain an extract:

[0018] Mix the extract with an adsorbent material for adsorption to obtain an adsorbed solid; the adsorbent material is the magnetic rice husk carbon material described in the above technical solution or the magnetic rice husk carbon material obtained by the above preparation method;

[0019] Elute the adsorbed solid to obtain a test solution.

[0020] The present invention also provides a detection method for aflatoxin B1, comprising the following steps:

[0021] Obtain a test solution according to the sample pretreatment method described in the above technical solution, perform time-resolved fluorescence immunochromatography on the test solution, and obtain the content of aflatoxin B1 in the sample to be tested according to the ratio of the obtained test line T value to the control line C value and a predetermined standard curve;

[0022] The predetermined standard curve is a linear curve of the aflatoxin B1 concentration and the ratio of the corresponding test line T value to the control line C value.

[0023] Compared with the prior art, the present invention has the following beneficial effects:

[0024] The present invention provides a magnetic rice husk carbon material, which includes Fe3O4 nanoparticles and amorphous silica loaded on the surface of the Fe3O4 nanoparticles. The present invention uses rice husk, a by-product in the rice processing process, as a raw material for high-value utilization. It is carbonized and magnetized to synthesize a magnetic rice husk carbon material (MRHC). The magnetic rice husk carbon material of the present invention simultaneously has the adsorption ability of carbon-based materials and the separation characteristics of magnetic materials. The Fe 3+ ions may form chelates with the carbonyl group of AFB1 for chemical adsorption, and at the same time, the pore structure in the material can perform physical adsorption. The magnetic rice husk carbon material is used as a magnetic solid-phase extraction material to enrich and purify aflatoxin B1 in rice. The magnetic rice husk carbon material has superparamagnetism and excellent dispersibility, and can quickly separate the adsorption material from the sample matrix. The magnetic rice husk carbon material is an efficient pretreatment enrichment and purification material. The sample pretreatment process of aflatoxin B1 is simple, time-consuming, and can avoid the use of a large amount of organic reagents.

[0025] The present invention uses rice husk, a by-product in the rice processing process, as a raw material to prepare a magnetic rice husk carbon material, which improves the bioavailability of rice husk, reduces resource waste, and has a high silicon content, which can provide an abundant silicon source in the synthesis of magnetic adsorption materials.

[0026] The present invention also provides a sample pretreatment method and a detection method for aflatoxin B1, which have the outstanding advantages of simple operation steps, short time consumption, and environmental friendliness. The present invention magnetizes the carbonized product of rice husk and prepares the carbonized product of rice husk into a magnetic solid-phase extraction adsorption material with a high specific surface area and rich active sites. The external magnetic field can play an important role in the pretreatment step of aflatoxin B1 detection. Combining time-resolved fluorescence immunochromatography for the detection of aflatoxin B1 in rice avoids the use of large-scale instruments, is simple, fast, efficient, and safe, and the entire extraction and detection process does not exceed 15 minutes. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] 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 in the embodiments. Obviously, the drawings in the following description are only some embodiments of the present invention. For those of ordinary skill in the art, other drawings can be obtained based on these drawings without creative efforts.

[0028] Figure 1 It is a schematic diagram of the synthesis and application of the magnetic rice husk carbon material of the present invention;

[0029] Figure 2 It is the magnetization curve of iron oxide (Fe3O4) and magnetic rice husk carbon material (MRHC) in Test Example 1;

[0030] Figure 3 Magnetic separation diagram in Test Example 1;

[0031] Figure 4 XRD diagram of the magnetic rice husk carbon material (MRHC) prepared in Example 1;

[0032] Figure 5 Infrared spectrum diagram of Fe3O4 and the magnetic rice husk carbon material (MRHC) prepared in Example 1;

[0033] Figure 6 Particle size distribution diagram of the magnetic rice husk carbon material (MRHC) prepared in Example 1;

[0034] Figure 7 Pore size distribution diagram of the magnetic rice husk carbon material (MRHC) prepared in Example 1;

[0035] Figure 8 Effect results of the volume fraction of methanol in the extraction solvent on the recovery rate of aflatoxin B1;

[0036] Figure 9 Effect results of the extraction time on the recovery rate of aflatoxin B1;

[0037] Figure 10 Effect results of the dosage of the adsorption material on the recovery rate of aflatoxin B1;

[0038] Figure 11 Effect results of the adsorption time on the recovery rate of aflatoxin B1;

[0039] Figure 12 Effect results of the elution volume on the recovery rate of aflatoxin B1;

[0040] Figure 13 Effect results of the elution time on the recovery rate of aflatoxin B1;

[0041] Figure 14 Correlation curve of the MSPE-TRFICA results and the IAC-UHPLC-MS / MS results. Detailed implementation mode

[0042] The present invention provides a magnetic rice husk carbon material, including Fe3O4 nanoparticles and amorphous silica supported on the surface of the Fe3O4 nanoparticles.

[0043] In the present invention, the particle size of the Fe3O4 nanoparticles is preferably 10 - 200 nm.

[0044] In the present invention, by magnetizing the carbonized product of rice husk, the rice husk charcoal is prepared into a magnetic solid-phase adsorption material with a high specific surface area and abundant active sites, which can play an important role in the sample pretreatment process for the detection of aflatoxin B1 by using an external magnetic field.

[0045] In the present invention, the magnetic rice husk charcoal material is preferably a red powder, and the particle size is preferably 200 - 400 nm.

[0046] The present invention also provides a preparation method of the magnetic rice husk charcoal material described in the above technical solution, including the following steps:

[0047] Perform the first calcination on the rice husk to obtain a rice husk carbonized product; the main component of the rice husk carbonized product is amorphous silica;

[0048] Mix and react the rice husk carbonized product, an alkali, and water to obtain a silicate-containing solution;

[0049] Drop the silicate-containing solution into the Fe3O4 dispersion, perform a hydrothermal reaction under alkaline conditions, and perform the second calcination on the obtained precipitate to obtain the magnetic rice husk charcoal material.

[0050] In the present invention, unless otherwise specified, the materials and equipment used are all commercially available products in the art.

[0051] In the present invention, the rice husk is subjected to the first calcination to obtain a rice husk carbonized product; the main component of the rice husk carbonized product is amorphous silica.

[0052] Rice husk is a by-product of the rice processing process, and most of it is discarded or burned on the cultivated land. The present invention uses rice husk to prepare the magnetic rice husk charcoal material, which can reduce resource waste and environmental pollution.

[0053] In the present invention, before the first calcination, it further includes: sequentially washing, pickling, and drying the rice husk;

[0054] The washing is preferably performed using distilled water, and the number of washing times is preferably 3 times; after the washing, first drying and grinding are preferably performed. The temperature of the first drying is preferably 50 - 60 °C, and the time is preferably 12 - 48 h, specifically 24 h; the present invention has no special requirements for the particle size after the grinding, as long as the sample is uniform;

[0055] The pickling is preferably: boiling the ground rice husk in hydrochloric acid; the boiling time is preferably 1 - 3 h, specifically 2 h; the concentration of the hydrochloric acid is preferably 2 mol / L, and the dosage ratio of the rice husk to the hydrochloric acid is preferably 100 g:500 mL. The function of the pickling is to remove the residual metal ions in the rice husk. The hydrochloric acid concentration and boiling time described in the present invention are conducive to the removal of metal ions in the rice husk, and the prepared material has a high adsorption efficiency for aflatoxin B1.

[0056] The drying temperature is preferably 50 - 60°C, and the time is preferably 12 - 48 h, specifically it can be 24 h.

[0057] In the present invention, the temperature of the first calcination is preferably 500 - 600°C, specifically it can be 550°C, and the heat preservation time is preferably 6 - 10 h, specifically it can be 8 h. The rice husk carbonization product is a white powder, and its main component is amorphous silica (>98%). The Si-OH on the surface has relatively high reactivity, which is conducive to introducing active groups into the pore surface or framework for functionalization. The calcination temperature and time described in the present invention are conducive to the formation of a porous structure, which is beneficial to the improvement of the adsorption performance.

[0058] After obtaining the rice husk carbonization product, the present invention mixes and reacts the rice husk carbonization product, an alkali, and water to obtain a silicate-containing solution.

[0059] In the present invention, the alkali is preferably a strong base, and the strong base preferably includes one or more of alkali metal hydroxides and alkaline earth metal hydroxides, such as it can be one or more of sodium hydroxide, potassium hydroxide, and calcium hydroxide.

[0060] In the present invention, the mass ratio of the rice husk carbonization product to the alkali is preferably 4:4.5 - 5.5, specifically it can be 4:5. The amount of the alkali described in the present invention can convert the silica in the rice husk carbonization product into sodium silicate, and will not cause excessive alkaline substances to remain in the product.

[0061] In the present invention, the dosage ratio of the rice husk to water is preferably 100 g:200 - 300 mL, specifically it can be 100 g:250 mL.

[0062] In the present invention, the dosage ratio of the rice husk carbonization product to water is preferably 5 - 10 g:200 - 300 mL, specifically it can be 5.9 g:250 mL.

[0063] In the present invention, the temperature of the mixing reaction of the rice husk carbonization product, the alkali, and water is preferably 80°C. During the mixing reaction process, the silica in the rice husk carbonization product reacts with the alkali to form silicate.

[0064] After obtaining the silicate-containing solution, the present invention drops the silicate-containing solution into the Fe3O4 dispersion liquid, conducts a hydrothermal reaction under alkaline conditions, and conducts a second calcination on the obtained precipitate to obtain a magnetic rice husk carbon material.

[0065] In the present invention, the Fe3O4 in the Fe3O4 dispersion liquid is preferably a commercially available product or is prepared by a co-precipitation method. The present invention has no special requirements for the conditions of the co-precipitation method, and the conditions commonly used by those skilled in the art can be adopted.

[0066] In the present invention, the concentration of Fe3O4 in the Fe3O4 dispersion is preferably 1 to 3 mg / mL, specifically it can be 2 mg / mL; the particle size is preferably 10 to 200 nm.

[0067] In the present invention, the Fe3O4 dispersion preferably further contains a surfactant. The surfactant preferably includes cetyltrimethylammonium bromide (CTAB), sodium dodecyl sulfate or polyethyleneglycol octylphenyl ether, and more preferably cetyltrimethylammonium bromide. The surfactant of the present invention can form a micelle structure in an aqueous solution, which helps to stabilize and disperse the Fe3O4 magnetic particles, prevent agglomeration into large particles, and is conducive to the preparation of a magnetic adsorption material with small size and uniform dispersion; it can also form a thin coating layer on the surface of the Fe3O4 magnetic particles, which helps to protect the magnetic particles from being damaged and promotes the uniform deposition of silicate on the surface of the magnetic particles.

[0068] In the present invention, the concentration of the surfactant in the Fe3O4 dispersion is preferably 0.01 to 0.03 mol / L, specifically it can be 0.02 mol / L.

[0069] In the present invention, it is preferred to first mix the surfactant and water to obtain a surfactant solution, and then mix it with Fe3O4 for the second time to obtain an Fe3O4 dispersion; the second mixing method is preferably ultrasonic, and the ultrasonic time is preferably 30 to 60 min.

[0070] In the present invention, when the silicate-containing solution is dropped into the Fe3O4 dispersion, the temperature of the Fe3O4 dispersion is preferably 80 °C, the dropping rate is preferably 2 to 3 drops / second, stirring is preferably accompanied during the dropping, and stirring is preferably continued for 30 min after the dropping.

[0071] In the present invention, the volume ratio of the silicate-containing solution to the Fe3O4 dispersion is preferably 0.9 to 1.1:1.9 to 2.1, specifically it can be 1:2.

[0072] In the present invention, the mass ratio of the rice husk to Fe3O4 in the Fe3O4 dispersion is preferably 100:0.9 to 1.1, specifically it can be 100:1.

[0073] In the present invention, the mass ratio of Fe3O4 to the carbonized product of rice husk in the Fe3O4 dispersion is preferably 0.14 to 0.18:1, specifically 0.17:1 in the examples of the present invention. With the dosage of Fe3O4 of the present invention, the obtained magnetic rice husk carbon material has strong magnetism and good adsorption capacity.

[0074] In the present invention, the alkaline condition of the hydrothermal reaction is preferably a pH value of 10 - 12, specifically, it can be a pH value of 11. The present invention preferably adds a hydrochloric acid solution to adjust the pH value, and the concentration of the hydrochloric acid solution is preferably 1 mol / L. The pH value in the present invention is conducive to the loading of silicate on the surface of Fe3O4 particles, forming a stable adsorption structure.

[0075] In the present invention, the temperature of the hydrothermal reaction is preferably 70 - 90 °C, specifically, it can be 80 °C, and the time is preferably 12 - 36 h, specifically, it can be 24 h. With the reaction temperature in the present invention, the reaction rate of silicic acid or silicate with Fe3O4 particles is fast, and the surfactant therein acts as a template agent to promote the formation of the silicon-based material.

[0076] In the present invention, the hydrothermal reaction is preferably carried out under stirring, and the rotation speed of the stirring is preferably 700 - 1100 rpm, specifically, it can be 1000 rpm.

[0077] In the present invention, before the second calcination, it preferably further includes: washing the obtained precipitate and drying; the washing is preferably water washing, and the number of water washing times is preferably 3 times; the drying temperature is preferably 50 - 60 °C, and the time is preferably 12 - 48 h.

[0078] In the present invention, the temperature of the second calcination is preferably 500 - 600 °C, specifically, it can be 550 °C, and the heat preservation time is preferably 1 - 3 h, specifically, it can be 2 h. The second calcination can remove the surfactant molecules.

[0079] The present invention also provides the application of the magnetic rice husk carbon material described in the above technical solution or the magnetic rice husk carbon material obtained by the above preparation method in detecting aflatoxin B1.

[0080] The present invention also provides a sample pretreatment method for detecting aflatoxin B1, including the following steps:

[0081] Mix the sample to be detected and an aqueous methanol solution for extraction to obtain an extract:

[0082] Mix the extract and an adsorption material for adsorption to obtain an adsorbed solid; the adsorption material is the magnetic rice husk carbon material described in the above technical solution or the magnetic rice husk carbon material obtained by the above preparation method;

[0083] Elute the adsorbed solid to obtain a solution to be detected.

[0084] The present invention mixes the sample to be detected and an aqueous methanol solution for extraction to obtain an extract.

[0085] In the present invention, the sample to be tested is rice, and the rice preferably includes one or more of white rice, glutinous rice, and brown rice; the sample to be tested is preferably pulverized before extraction, and the present invention has no special requirements for the pulverization method.

[0086] In the present invention, the volume fraction of methanol in the methanol aqueous solution is preferably 50% - 90%, and specifically can be 50%, 60%, 70%, 80%, or 90%.

[0087] In the present invention, the dosage ratio of the sample to be tested and the methanol aqueous solution is preferably 1 g: 0.5 - 1.5 mL, and specifically can be 1 g: 1 mL.

[0088] In the present invention, the extraction method is preferably vortex, and the rotation speed of the vortex is preferably 2000 - 2800 rpm. The extraction time is preferably 1 - 10 min, and specifically can be 1 min, 2 min, 3 min, 5 min, or 10 min.

[0089] In the present invention, after extraction, it preferably further includes: solid-liquid separation and dilution; the solid-liquid separation method is preferably centrifugation, the centrifugation speed is preferably 4000 rpm, and the time is preferably 2 min; the dilution multiple is preferably 10 times.

[0090] After obtaining the extract, the present invention mixes the extract and the adsorbent material for adsorption to obtain an adsorbed solid; the adsorbent material is the magnetic rice husk carbon material described in the above technical solution or the magnetic rice husk carbon material obtained by the above preparation method.

[0091] In the present invention, the adsorbent material is preferably activated before use, the activator used for activation is preferably dichloromethane, and the activation is preferably: vortex mixing the adsorbent material and dichloromethane, and discarding the dichloromethane; the time of the vortex mixing is preferably 2 min.

[0092] In the present invention, using dichloromethane as the activator, the recovery rate of aflatoxin B1 (AFB1) can be increased from 21.85% (without activation) to 80.27%, which is also significantly higher than the recovery rate (23.24%) when using methanol as the activator. After activation, impurities adsorbed on the magnetic rice husk carbon material (MRHC) can be removed, and at the same time, AFB1 can penetrate into the interior of the adsorbent material, improving the adsorption efficiency.

[0093] In the present invention, the dosage ratio of the sample to be tested and the adsorbent material is preferably 1 g: 2 - 20 mg, and specifically can be 1 g: 2 mg, 1 g: 5 mg, 1 g: 10 mg, 1 g: 15 mg, or 1 g: 20 mg.

[0094] In the present invention, the adsorption method is preferably vortex, and the rotational speed of the vortex is preferably 2000 - 2800 rpm. The adsorption time is preferably 0.5 - 4 min, specifically it can be 0.5 min, 1 min, 2 min, 3 min or 4 min. After the adsorption, aflatoxin B1 is adsorbed by the magnetic rice husk carbon material.

[0095] In the present invention, after the adsorption, it preferably further includes separation to obtain an adsorbed solid and a supernatant; the separation preferably utilizes an external magnetic field, and the external magnetic field preferably uses a 40*20*20 mm N52 magnet with a strength range of 6000 - 10000 gauss.

[0096] After obtaining the adsorbed solid, the present invention elutes the adsorbed solid to obtain a test solution.

[0097] In the present invention, the eluent used for elution is preferably methanol, and the dosage of the eluent and the adsorption material is preferably 0.5 - 4 mL:10 mg, specifically it can be 0.5 mL:10 mg, 1 mL:10 mg, 2 mL:10 mg, 3 mL:10 mg or 4 mL:10 mg.

[0098] In the present invention, the elution method is preferably vortex, and the rotational speed of the vortex is preferably 2000 - 2800 rpm. The elution time is preferably 0.5 - 4 min, specifically it can be 0.5 min, 1 min, 2 min, 3 min or 4 min.

[0099] In the present invention, after the elution, it preferably further includes separation. The obtained eluate is blown with nitrogen and re-dissolved; the separation preferably utilizes an external magnetic field, and the external magnetic field preferably uses a 40*20*20 mm N52 magnet with a strength range of 6000 - 10000 gauss; the re-dissolution preferably uses ultrapure water, and the volume of the ultrapure water is preferably 150 μL.

[0100] The present invention also provides a detection method for aflatoxin B1, comprising the following steps:

[0101] Obtain a test solution according to the sample pretreatment method described in the above technical solution, perform time-resolved fluorescence immunochromatography on the test solution, and obtain the content of aflatoxin B1 in the test sample according to the ratio of the obtained test line T value to the control line C value and a predetermined standard curve;

[0102] The predetermined standard curve is a linear curve of the aflatoxin B1 concentration versus the ratio of the corresponding test line T value to the control line C value.

[0103] In the present invention, the time-resolved fluorescence immunochromatography preferably includes: adding 50 μL of the test solution into a reaction micro-well and mixing it with the fluorescence immuno-probe in the well. After incubating at 37 °C for 5 min, insert the aflatoxin B1 fluorescence immunoassay strip, wait for 5 min, and detect using a dry fluorescence immunoassay analyzer at an excitation wavelength of 365 nm and an emission wavelength of 615 nm to obtain the ratio of the test line T value to the control line C value.

[0104] The present invention uses rice husk as a raw material to prepare a new magnetic solid-phase extraction material for aflatoxin B1, and establishes a simple, rapid, sensitive, and economical detection method for aflatoxin B1.

[0105] To further illustrate the present invention, the following will describe in detail the magnetic rice husk carbon material provided by the present invention, its preparation method and application, the sample pretreatment method and detection method for aflatoxin B1 with reference to the accompanying drawings and embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0106] The following is carried out according to Figure 1 the synthesis and application schematic diagram of the magnetic rice husk carbon material shown to prepare and apply the magnetic rice husk carbon material.

[0107] Example 1 Preparation of magnetic rice husk carbon material

[0108] First, wash 100 g of rice husk three times in distilled water, and then dry it at 50 °C for 24 h. After grinding, boil it in 500 mL of hydrochloric acid (2 mol / L) solution for 2 h, wash the precipitate with distilled water, dry it at 50 °C for 24 h, and then calcine the dried sample at 550 °C for 6 h to obtain the rice husk carbonization product (5.9 g). After weighing, mix it with sodium hydroxide in a ratio of 4:5 (w / w) and dissolve it thoroughly in 250 mL of distilled water at 80 °C to form a sodium silicate solution.

[0109] Subsequently, prepare magnetic Fe3O4 particles by the co-precipitation method:

[0110] Place 150 mL of deionized water and 2 mL of N2H4·H2O into a 250 mL three-necked round-bottom flask equipped with a mechanical stirrer, and stir for 30 min to remove oxygen. Subsequently, introduce aqueous solutions of Fe 3+ / Fe 2+ with a molar ratio maintained at 1.75:1 of FeCl3 and FeSO4 into the flask. Immediately afterwards, quickly inject 8 mL of ammonia water (25%, v / v) into the mixture under vigorous stirring. The resulting solution is kept at 80 °C for another 30 min. After this step, filter the precipitate and wash it 10 times with deionized water and absolute ethanol respectively. The precipitate is then dried under vacuum for 24 h, and then ground into a powder with a particle size of 150 - 200 nm, with an average particle size of 182 nm.

[0111] Magnetic Fe3O4 particles (200 mg) were dispersed in 100 mL of hexadecyltrimethylammonium bromide (CTAB) solution (0.02 mol / L) and ultrasonically treated for 30 min. The obtained suspension was heated to 80 ° C under mechanical stirring, and then the sodium silicate solution (50 mL) obtained in the above step was slowly added (2-3 drops / second). After further stirring for 30 min, 1 mol / L hydrochloric acid solution was added dropwise to the mixture, the pH value was adjusted to 11, and then stirred (1000 rpm) for 2 h, and then water bathed at 80 ° C for 24 h. The formed precipitate was washed 3 times with distilled water, then dried at 50 ° C for 12 h, and finally calcined at 550 ° C for 2 h. The red powder finally obtained was a magnetic rice husk carbon material (658 mg).

[0112] Application Example 1: Detection of Aflatoxin B1 in White Rice

[0113] 2 g of crushed white rice sample was placed in a 15 mL centrifuge tube, AFB1 was added to the white rice until the AFB1 concentration was 2, 5, and 10 μg / kg, respectively, 2 mL of 70% volume fraction methanol-water solution was added, and high-speed vortex (2000 rpm) was mixed for 3 min. After centrifugation at 4000 rpm for 2 min, 1 mL of supernatant extract was transferred to a 15 mL centrifuge tube and diluted to 10 mL with ultrapure water to obtain the diluted sample extract for standby use.

[0114] 10 mg of the magnetic rice husk carbon material prepared in Example 1 was placed in a 15 mL centrifuge tube, and then 2 mL of dichloromethane was added for vortex activation for 2 min. After discarding the dichloromethane, 10 mL of the diluted sample extract was added to the centrifuge tube. After vigorous vortexing (2000 rpm) for 1 min, the magnetic rice husk carbon material adsorbed aflatoxin B1, and then separated by an external magnetic field (using a 40*20*20 mm N52 magnet) and the supernatant was discarded, 0.5 mL of methanol was added for vortex oscillation for 1 min, separated by an external magnetic field (using a 40*20*20 mm N52 magnet), eluted with liquid nitrogen and blown to dryness, added 150 μL of ultrapure water for re-dissolution, and detected by TRFICA.

[0115] Time-resolved fluorescence immunochromatographic analysis (TRFICA) includes: taking 50 μL of the test solution and adding it to the reaction microwell, and fully mixing it with the fluorescent immunoprobe in the well. After incubation at 37°C for 5 minutes, insert the aflatoxin B1 fluorescent immunoassay strip, wait for 5 minutes, and use a dry fluorescent immunoassay analyzer to detect at an excitation wavelength of 365nm and an emission wavelength of 615nm, record the ratio of the test line (T line) and the quality control line (C line), and use the T / C value to establish a calibration curve for quantitative analysis.

[0116] The test results are shown in Table 1. The sample recovery rate was 88.1% - 109.4%, and the intra-day and inter-day precisions were both no more than 10.7%.

[0117] Table 1 Recovery rate and precision of AFB1 in white rice

[0118]

[0119] Application Example 2 Detection of Aflatoxin B1 in Glutinous Rice in Actual Samples

[0120] Take the pulverized glutinous rice sample. The pretreatment method and test method are the same as those in Application Example 1. The test results are shown in Table 2. The sample recovery rate was 85.2% - 94.6%, and the intra-day and inter-day precisions were both no more than 11.5%.

[0121] Table 2 Recovery rate and precision of AFB1 in glutinous rice

[0122]

[0123] Application Example 3 Detection of Aflatoxin B1 in Brown Rice in Actual Samples

[0124] Take the pulverized brown rice sample. The pretreatment method and test method are the same as those in Application Example 1. The test results are shown in Table 3. The sample recovery rate was 90.6% - 98.7%, and the intra-day and inter-day precisions were both no more than 11.3%.

[0125] Table 3 Recovery rate and precision of AFB1 in brown rice

[0126]

[0127] Sensitivity and Standard Curve

[0128] Mix equal amounts of white rice, glutinous rice, and brown rice without aflatoxin B1 to obtain a blank mixed sample. Add different gradient concentrations of aflatoxin B1 to the blank mixed sample. Establish a quantitative standard curve with the concentration of aflatoxin B1 on the x-axis and the T / C value on the y-axis. Use the standard curve to quantify 20 blank matrix samples, and calculate the visual detection limit (LOD). The quantification limit (LOQ) is 3 × LOD. The results are shown in Table 4:

[0129] Table 4 Linear range, calibration equation, correlation coefficient, detection limit (LOD), and quantification limit (LOQ) of AFB1

[0130]

[0131] Test Example 1 Magnetic Intensity Test

[0132] The magnetic properties of the material prepared in Example 1 were analyzed by a vibrating magnetometer (VSM) test. Magnetic separation test: 30 mg of the magnetic rice husk carbon material was placed in 30 mL of water and stirred to disperse it evenly. Then, an external magnet was placed, and it was observed whether the magnetic rice husk carbon material could be separated from the water and adsorbed on the container wall.

[0133] Figure 2 are the magnetization curves of Fe3O4 and the magnetic rice husk carbon material (MRHC). The absence of a hysteresis loop in the magnetic profiles of Fe3O4 and MRHC indicates that they have superparamagnetism. Among them, the saturation magnetization intensity of Fe3O4 is 49.32 emu / g, while that of MRHC is 16.32 emu / g, indicating a slight decrease in the saturation magnetization intensity. However, MRHC retains sufficient magnetization intensity to be rapidly isolated from the aqueous medium by an external magnetic field. Figure 3 The magnetic separation diagram of also confirms this observation. The magnetic rice husk carbon material is separated from the water and adsorbed on the container wall.

[0134] Test Example 2 XRD and infrared spectra, particle size distribution and pore size distribution

[0135] X-ray diffraction (XRD) was used for phase structure analysis, infrared spectroscopy (IR) was used to determine the functional groups of the material, the particle size distribution was tested by a laser particle size analyzer, and the pore size distribution was tested by BET.

[0136] Figure 4 is the XRD pattern of the magnetic rice husk carbon material (MRHC) prepared in Example 1. A broad diffraction peak was observed at 2θ = 15° - 30°, indicating the presence of an amorphous silica structure in MRHC. In addition, the diffraction peaks at 2θ angles of 30.26°, 35.60°, 43.30°, 53.80°, 57.42° and 62.88° correspond to the (220), (310), (400), (422), (511) and (440) planes of Fe3O4, respectively. This result indicates the successful combination of Fe3O4 nanoparticles and RHC (rice husk carbon) after hydrothermal treatment.

[0137] Figure 5 are the infrared spectra of Fe3O4 and the magnetic rice husk carbon material (MRHC) prepared in Example 1. The FT-IR spectrum of MRHC shows many absorption peaks (3431, 1630, 1089, 962, 799, 467 and 560 cm -1 ). The main absorption peaks shown by MRHC at 3431 and 1630 cm -1 mainly come from the stretching and bending modes of the O-H bond vibration. At (1089, 799 and 467 cm -1) The spectral features observed at [x] cm⁻¹ correspond to the stretching, bending, and rocking motions of Si-O, while the peak at 962 cm⁻¹ corresponds to the bending vibration of Si-OH. In addition, the MRHC vibration spectrum at 560 cm⁻¹ and the Fe₃O₄ vibration spectrum at 580 cm⁻¹ are both related to the stretching vibration mode of Fe-O, indicating that Fe-O has successfully combined with RHC to form MRHC, which is consistent with the XRD results. -1 The peak at [x] cm⁻¹ corresponds to the bending vibration of Si-OH. -1 In addition, the MRHC vibration spectrum at 560 cm⁻¹ and the Fe₃O₄ vibration spectrum at 580 cm⁻¹ -1 are both related to the stretching vibration mode of Fe-O, indicating that Fe-O has successfully combined with RHC to form MRHC, which is consistent with the XRD results.

[0138] Figure 6 Figure [x] is the particle size distribution diagram of the magnetic rice husk carbon material (MRHC) prepared in Example 1. Figure 7 Figure [x] is the pore size distribution diagram of the magnetic rice husk carbon material (MRHC) prepared in Example 1. The size of MRHC particles varies between 255.0 nm and 396.06 nm, with an obvious peak observed at 341.99 nm, accounting for 43.98% of the entire particle distribution, and the average diameter is approximately 337.60 nm. The pore size of MRHC is 3.41 nm, which is larger than the molecular size of AFB1 (0.853 × 1.175 × 1.484 nm). Therefore, AFB1 can freely enter the porous channels of MRHC.

[0139] Test Example 3 Optimization of the magnetic solid-phase extraction process (MSPE)

[0140] Using blank white rice samples spiked with AFB1 at a concentration of 5 μg / kg, the effects of different conditions (extraction solvent, extraction time, amount of adsorption material, adsorption time, elution volume, elution time) on MSPE were studied. To ensure the accuracy and optimization of these key parameters, all experiments were repeated three times and the average values were calculated.

[0141] Test procedure: Place 2 g of crushed white rice samples in a 15 mL centrifuge tube, add 2 mL of 70% (v / v) methanol-aqueous solution (extraction solvent), and mix by high-speed vortexing (2000 rpm) for 3 min (extraction time). After centrifuging at 4000 rpm for 2 min, transfer 1 mL of the supernatant extract to a 15 mL centrifuge tube and dilute it to 10 mL with ultrapure water to obtain the diluted sample extract for use.

[0142] Place 10 mg (adsorbent dosage) of magnetic rice husk carbon material in a 15 mL centrifuge tube, then add 2 mL of dichloromethane and vortex for activation for 2 min. After discarding the dichloromethane, add 10 mL of diluted sample extract to the centrifuge tube. After mixing vigorously by vortex (2000 rpm) for 1 min (adsorption time), the magnetic rice husk carbon material adsorbs aflatoxin B1, then separate it with an external magnetic field and discard the supernatant. Add 1 mL (elution volume) of methanol and vortex for 1 min (elution time), separate it with an external magnetic field, blow the eluate to dry with nitrogen, and add 150 μL of ultrapure water for reconstitution to obtain the test solution.

[0143] 1) Investigate the effect of methanol - aqueous solution with volume fractions from 50% to 90% on the recovery rate. As Figure 8 shown, when the methanol concentration increases to 70%, the recovery rate of AFB1 gradually increases. The polarity of the extraction agent may be an important factor in increasing the quantity of the analyte. Therefore, the optimal choice of this invention is 70% volume fraction of methanol - aqueous solution as the extraction solvent (at this time, the recovery rate of AFB1 is 90.78%).

[0144] 2) Investigate the effect of vortex extraction time from 1 min to 10 min on the recovery rate. From Figure 9 it can be seen that within the first 3 min of extraction, the recovery rate of AFB1 gradually increases, while then extending the extraction time from 3 min to 10 min, the recovery rate remains almost unchanged. This is because within 1 min to 3 min, due to the insufficient mixing of the extraction agent and the test solution, AFB1 is not fully extracted, and the recovery rate gradually increases with the extension of the vortex time; while when the vortex time reaches 3 min, the mass transfer process basically reaches equilibrium, and the recovery rate almost remains unchanged. Therefore, 3 min is determined as the optimal extraction time (at this time, the recovery rate of AFB1 is 91.65%).

[0145] 3) Investigate the effect of the adsorbent dosage from 2 mg to 20 mg on the recovery rate. As Figure 10 shown, the recovery rate of AFB1 increases significantly between 2 and 10 mg, while it fluctuates slightly between 10 and 20 mg. Fast adsorption can be achieved with less adsorbent because the average diameter of MRHC particles is very small, about 337.60 nm, and MRHC particles have a large specific surface area, which can promote the affinity interaction for effective adsorption. In the MSPE procedure, the dosage of the adsorbent is crucial because its dispersibility can quickly adsorb the target substance. The results show that 10 mg of the adsorbent is sufficient to extract and purify the analyte in white rice samples (at this time, the recovery rate of AFB1 is 90.92%).

[0146] 4) Investigate the effect of adsorption time from 0.5 min to 4 min on the recovery rate. The results are as Figure 11As shown, it indicates that the mass transfer process between the magnetic adsorption material and the analyte can complete adsorption within 1 min (at this time, the recovery rate of AFB1 is 91.82%). The reason why the magnetic extraction process can be completed quickly is that they have superparamagnetism and excellent dispersibility.

[0147] 5) Investigate the effect of the elution volume from 0.1 mL to 2 mL on the recovery rate. The results are as Figure 12 shown. 0.5 mL of methanol is an effective eluent for AFB1 extracted from MRHC. When more methanol is used, the recovery rate basically remains the same. Therefore, the optimal elution solvent volume is set at 0.5 mL (at this time, the recovery rate of AFB1 is 93.32%).

[0148] 6) Investigate the effect of the elution time from 0.5 min to 4 min on the recovery rate. The results are as Figure 13 shown. As the elution time increases from 0.5 min to 1 min, the recovery rate of the analyte also increases. When the elution time is further extended, the recovery rate of the analyte almost remains unchanged. Therefore, the optimal elution time is set at 1 min (at this time, the recovery rate of AFB1 is 93.57%).

[0149] Therefore, the present invention determines the optimal conditions: the extraction solvent is 70% methanol aqueous solution, the extraction time is 3 min, the dosage of the adsorption material is 10 mg, the adsorption time is 1 min, the eluent volume is 0.5 mL, and the elution time is 1 min.

[0150] Comparison of Test Example 4 with the confirmatory detection method

[0151] To verify the accuracy of the magnetic solid-phase extraction-time-resolved immunochromatography (MSPE-TRFICA) method, it is compared with the IAC-UHPLC-MS / MS detection method in the national food safety standard "GB5009.22-2016 Aflatoxins B and G in Foods".

[0152] The pretreatment process of the IAC-UHPLC-MS / MS detection method is as follows:

[0153] Extraction: Weigh 5 g of the test sample (accurate to 0.01 g) into a 50 mL centrifuge tube, add methanol-aqueous solution (volume ratio 70 + 30), vortex and mix evenly, place it in a shaker and shake for 20 min, centrifuge at 6000 r / min for 10 min, and take the supernatant for standby.

[0154] Immunoaffinity column pretreatment: Accurately pipette 4 mL of the supernatant, add 23 mL of PBS containing 1% Triton X-100 (or Tween-20), and mix well. Restore the immunoaffinity column stored at low temperature to room temperature. After the original liquid in the immunoaffinity column has drained completely, transfer the above sample solution into a 50 mL syringe barrel, adjust the dropping speed, and control the sample solution to drop steadily at a speed of 1 mL / min to 3 mL / min. After the sample solution has dropped completely, add 2 × 10 mL of water into the syringe barrel and wash the immunoaffinity column at a stable flow rate. After the water has dropped completely, dry the affinity column with a vacuum pump. Disconnect from the vacuum system, place a 10 mL graduated test tube under the affinity column, remove the 50 mL syringe barrel, add 2 × 1 mL of methanol to elute the affinity column, control the dropping speed at 1 mL / min to 3 mL / min, then dry the affinity column with a vacuum pump, and collect all the eluate into the test tube. Slowly blow the eluate to near dryness with nitrogen at 50 °C, add 1.0 mL of the initial mobile phase, vortex for 30 s to dissolve the residue, filter through a 0.22 μm filter membrane, and collect the filtrate into an injection vial for injection preparation.

[0155] Chromatographic conditions: The chromatographic separation temperature is 40 °C, and a Hypesil Gold C18 chromatographic column (100 mm × 2.1 mm, 3 μm) is used. The mobile phase system consists of A (aqueous solution of 5 mmol / L ammonium acetate) and B (a mixture of methanol and acetonitrile with a volume ratio of 1:1). Isocratic elution is carried out with 30% (v / v) mobile phase B for 5 min. The mobile phase flow rate is 300 μL / min, and the injection volume is 1 μL.

[0156] Mass spectrometry conditions: Multiple reaction monitoring (MRM); positive ion mode scanning with an electrospray ionization source (ESI); nebulizer gas flow rate of 3.0 L / min; drying gas flow rate of 10.0 L / min; heating gas flow rate of 10.0 L / min; heating block temperature of 400 °C; DL temperature of 250 °C; dwell time of 33 ms.

[0157] Two methods were used to quantitatively analyze white rice samples with an AFB1 concentration of 2 - 10 μg / kg, and the test results are shown in Table 5:

[0158] Table 5 Comparison of MSPE-TRFICA and IAC-UHPLC-MS / MS results (n = 3)

[0159]

[0160]

[0161] Taking the results of MSPE-TRFICA as the abscissa and the results of IAC-UHPLC-MS / MS as the ordinate, a correlation curve was made, as shown in Figure 14As shown. When the correlation coefficient is close to 1, it indicates a strong positive correlation between the two variables. Generally speaking, the correlation coefficient of the standard curve should not be lower than 0.98. Figure 14 The correlation coefficient in Figure 14 is 0.9801, indicating that the measurement results of the two methods are in good agreement.

[0162] Although the above embodiments have described the present invention in detail, they are only a part of the embodiments of the present invention, rather than all embodiments. People can also obtain other embodiments according to the embodiments of the present invention without creative work, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A magnetic rice husk carbon material, characterized in that, It includes Fe3O4 nanoparticles and amorphous silica supported on the surface of the Fe3O4 nanoparticles.

2. The magnetic rice husk carbon material according to claim 1, wherein, The particle size of the magnetic rice husk carbon material is 200 - 400 nm.

3. The preparation method of the magnetic rice husk carbon material according to claim 1 or 2, characterized in that, It includes the following steps: Perform the first calcination on rice husks to obtain a rice husk carbonization product; the main component of the rice husk carbonization product is amorphous silica; Mix and react the rice husk carbonization product, alkali, and water to obtain a silicate-containing solution; Drop the silicate-containing solution into the Fe3O4 dispersion, carry out a hydrothermal reaction under alkaline conditions, and perform the second calcination on the obtained precipitate to obtain the magnetic rice husk carbon material.

4. The preparation method according to claim 3, characterized in that The temperature of the first calcination is 500 - 600 °C, and the heat preservation time is 6 - 10 h.

5. The preparation method according to claim 3, characterized in that, The concentration of Fe3O4 in the Fe3O4 dispersion is 1 - 3 mg / mL, and the Fe3O4 dispersion also contains a surfactant.

6. The preparation method according to claim 3, characterized in that, The alkaline condition of the hydrothermal reaction is pH value = 10 - 12.

7. The preparation method according to claim 3 or 6, characterized in that, The temperature of the hydrothermal reaction is 70 - 90 °C, and the time is 12 - 36 h.

8. Use of the magnetic rice husk carbon material according to claim 1 or 2 or the magnetic rice husk carbon material obtained by the preparation method according to any one of claims 3 - 7 in detecting aflatoxin B1.

9. A sample pretreatment method for detecting aflatoxin B1, characterized in that, It includes the following steps: Mix the sample to be tested and an aqueous methanol solution for extraction to obtain an extract: Mix the extract and an adsorbent material for adsorption to obtain an adsorbed solid; the adsorbent material is the magnetic rice husk carbon material according to claim 1 or 2 or the magnetic rice husk carbon material obtained by the preparation method according to any one of claims 3 - 7; Elute the adsorbed solid to obtain a test solution.

10. A detection method for aflatoxin B1, characterized in that, It includes the following steps: Obtain a test solution according to the sample pretreatment method according to claim 9, perform time-resolved fluorescence immunochromatography on the test solution, and obtain the content of aflatoxin B1 in the sample to be tested according to the ratio of the obtained test line T value to the control line C value and a predetermined standard curve; The predetermined standard curve is a linear curve of the aflatoxin B1 concentration and the ratio of the corresponding test line T value to the control line C value.

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