Composite material of nucleic acid aptamer modified magnetic graphene oxide as well as preparation and application of composite material

Abstract

The invention discloses a nucleic acid aptamer modified magnetic graphene oxide composite material, which is characterized in that Fe3O4 particles are loaded on the surface of graphene oxide in the composite material, a layer of close contact interface is also covered, and the interface is a coating structure formed by a nucleic acid aptamer and magnetic graphene oxide. According to the composite material disclosed by the invention, the magnetic graphene oxide synthesized by one step through a coprecipitation method is used as an adsorption material for dispersive solid-phase extraction after being modified by a ZEN aptamer, and is combined with HPLC-FLD for use, so that the defects that a traditional adsorbent is poor in selectivity, large in matrix effect and difficult to recycle are overcome; the detection requirements of trace zearalenone in various matrixes can be met.

Description

Technical Field

[0001] The present invention relates to the technical field of material analysis, and particularly relates to a composite material of nucleic acid aptamer modified magnetic graphene oxide, and its preparation and application. Background Art

[0002] Zearalenone (ZEN), also known as F-2 toxin, is a secondary metabolite produced by fungi of the genus Fusarium such as Fusarium graminearum. High levels of ZEN are often detected in grains such as corn, wheat, oats, and barley infected with Fusarium. ZEN has estrogenic and anabolic activities and can bind to estrogen receptors, leading to disruption of the body's hormone balance and subsequent reproductive system diseases. In addition, ZEN also has genotoxicity, hepatotoxicity, immunotoxicity, and hematotoxicity, etc. Also, due to its chemical and thermal stability, it is very difficult to degrade or remove during food processing. Therefore, it causes great harm to humans, livestock, and the environment. The International Agency for Research on Cancer has classified ZEN as a Group 3 carcinogen, and different countries and regions have also set strict regulations on the limit standards of ZEN. The national standard of China, "GB 2761-2017 Limits of Mycotoxins in Foods", stipulates that the residual limit of ZEN in wheat, corn and their products should be less than 60 μg / kg; the European Union stipulates that the residual limit of ZEN in all grains and products should be less than 75 μg / kg. However, due to the complex matrix of cereal samples themselves and the interference of other pollutants, a series of unstable factors often occur during the detection process. Therefore, developing a pretreatment method for highly selective extraction of zearalenone is of great significance for food and environmental safety.

[0003] At present, the sample pretreatment methods for zearalenone toxin in foods mainly include liquid-liquid extraction (LLE), solid-phase extraction (SPE), dispersive solid-phase extraction (DSPE), immunoaffinity column extraction (IAC), etc. Among these methods, immunoaffinity column extraction has good selectivity, but it is expensive and cannot be recycled. Liquid-liquid extraction consumes a large amount of solvent, is complicated to operate, and does not have the ability of selective extraction. Dispersive solid-phase extraction is considered to be one of the most efficient pretreatment methods at present because of its low solvent consumption, simple operation, and recyclability; however, there are also problems such as low adsorption capacity. Therefore, it is necessary to develop a highly selective dispersive solid-phase extraction adsorbent for the selective extraction and analysis of zearalenone in complex samples.

[0004] Magnetic solid-phase extraction (MSPE) combines functional magnetic nanoparticles with traditional SPE. The micron-sized or nano-sized particles used can be rapidly dispersed throughout the sample system, enabling full contact with the target substances, accelerating the adsorption process and adsorption efficiency. By applying an external magnetic field, the MNPs can be rapidly separated and collected from the liquid phase, and then the target substances are desorbed and analyzed from the adsorbent, and the analytical solution is taken for detection and analysis. Compared with traditional SPE, MSPE does not require packing adsorbents in an SPE column, the extraction device is simple, the functionalized MNPs have strong magnetism and are easily separated by an external magnetic field, avoiding cumbersome centrifugation and filtration operations and the adsorbent loss caused by multiple elutions. Avoiding the adsorbent loss caused by multiple elutions, at the same time, MSPE uses less organic solvents, has low pollution and is environmentally friendly during the process, meeting the concept of "green chemistry" advocated today.

[0005] The selection of the solid adsorbent in the MSPE method is an important factor determining the extraction efficiency, enrichment factor and service life of the adsorption material. To improve the selectivity of MSPE materials, a series of functionalized adsorbents based on "molecular recognition" have been developed, mainly including adsorption materials based on antibodies, molecular imprinting technology and aptamers. In recent years, many studies have applied aptamers as affinity ligands to MSPE and achieved good results. Aptamers are random oligonucleotide fragments screened by the systematic evolution of ligands by exponential enrichment (SELEX) technology in vitro, which can be DNA or RNA and can specifically bind to target substances. Aptamers can form various three-dimensional spatial structures through intramolecular interactions such as hydrogen bonds and base complementary pairing, including hairpin, bulge loop, pseudoknot and G-quadruplex structures. According to this three-dimensional spatial structure, aptamers can widely bind to targets, from metal ions to small molecules, macromolecular proteins, and even whole cells. Aptamers are also called "chemical antibodies", but different from antibodies, aptamers can be synthesized in large quantities in vitro, which gives aptamers a wider application space in the field of biochemical analysis.

[0006] The information disclosed in this background section is only intended to enhance the overall understanding of the present invention and should not be regarded as an admission or any form of suggestion that this information constitutes prior art already known to those of ordinary skill in the art. Summary of the Invention

[0007] The object of the present invention is to provide a composite material of nucleic acid aptamer modified magnetic graphene oxide, its preparation and application. The prepared composite material has good selective adsorption ability and good specificity for zearalenone. Using this composite material as an adsorbent for dispersive solid-phase extraction and applying it to the detection of zearalenone in grains, the detection sensitivity is high.

[0008] To achieve the above object, the present invention provides a composite material of nucleic acid aptamer modified magnetic graphene oxide, including: Fe3O4 particles are loaded on the surface of graphene oxide in the composite material, and a closely contacted interface is also covered, and this interface is a coating structure formed by nucleic acid aptamer and magnetic graphene oxide.

[0009] Preferably, in the above technical solution, for the composite material of nucleic acid aptamer modified magnetic graphene oxide, the bonding and immobilization amount of the nucleic acid aptamer is 1-5 μg / mg, the average size of Fe3O4 nanoparticles is 9-12 nm, and the dispersibility is good; the composite material of nucleic acid aptamer modified magnetic graphene oxide has high adsorption ability and magnetic separation performance, the saturation magnetization intensity (Ms) is 20-25 emu / g, and the adsorption capacity for zearalenone ZEN is 200-300 μg / g.

[0010] Preferably, in the above technical solution, the nucleic acid aptamer is ZEN aptamer 5'-NH2-(CH2)6-TCA TCTATC TAT GGT ACA TTA CTA TCT GTA ATG TGA-3'.

[0011] A preparation method of a composite material of nucleic acid aptamer modified magnetic graphene oxide. First, prepare graphene oxide GO, then use the co-precipitation method to synthesize magnetic graphene oxide GO / Fe3O4 in one step. Disperse GO / Fe3O4 in a buffer solution, add EDC and NHS to activate the surface activity of its carboxyl end, add an amino-modified aptamer and connect it to the material surface, incubate, and then separate to obtain the composite material of nucleic acid aptamer modified magnetic graphene oxide GO / Fe3O4 / Apt.

[0012] Preferably, in the above technical solution, graphene oxide GO is prepared by the Hummers method, including: stirring graphite powder, sodium nitrate and sulfuric acid in an ice-water bath, adding potassium permanganate and stirring at below 5 °C, then stirring at 30-40 °C, adding water, stirring at 90-100 °C, adding water and hydrogen peroxide and stirring, cooling to room temperature, centrifuging into a colloid, and dialyzing to neutrality to obtain graphene oxide GO.

[0013] Preferably, in the above technical solution, the method for one-step synthesis of magnetic graphene oxide GO / Fe3O4 by coprecipitation method includes: dispersing graphene oxide GO in water, adding FeCl3·6H2O and FeSO4·7H2O, heating to 40-50 °C, stirring and reacting under nitrogen protection, adding ammonia water during the reaction, washing to neutral after the reaction, drying, and grinding to obtain magnetic graphene oxide GO / Fe3O4.

[0014] Use of a composite material of nucleic acid aptamer modified magnetic graphene oxide as a dispersive solid phase extraction adsorbent material.

[0015] Preferably, in the above technical solution, the composite material of nucleic acid aptamer modified magnetic graphene oxide is used for dispersive solid phase extraction to extract zearalenone from cereal samples for detecting zearalenone.

[0016] Preferably, in the above technical solution, the method for separating zearalenone using the composite material of nucleic acid aptamer modified magnetic graphene oxide includes the following steps:

[0017] (1) Wash the GO / Fe3O4 / Apt magnetic nanoparticles with PBS buffer solution, and magnetically separate and discard the supernatant.

[0018] (2) Prepare the sample loading solution to be measured, pipette the loading solution into the GO / Fe3O4 / Apt magnetic nanoparticles treated in step (1), place it on a shaker, carry out the adsorption reaction, and magnetically separate and discard the supernatant.

[0019] (3) Add water to the GO / Fe3O4 / Apt magnetic nanoparticles treated in step (2), and magnetically separate and discard the supernatant.

[0020] (4) Add the eluent, shake, carry out desorption, magnetically separate and collect the eluent, filter through a membrane, and separate zearalenone.

[0021] Preferably, in the above technical solution, the organic solvent of the eluent in step (4) is methanol, the elution ratio is 80% methanol-PBS buffer solution, the pH of the elution solvent is 7-7.5, and the elution time is 3-5 min.

[0022] Compared with the prior art, the present invention has the following beneficial effects:

[0023] (1) The composite material of nucleic acid aptamer modified magnetic graphene oxide of the present invention combines the aptamer with MSPE, providing a selective recognition site for the selective enrichment and extraction of trace zearalenone in actual samples.

[0024] (2) Preparation method of the composite material of the present invention: First, modified Hummers method is used to synthesize graphene oxide (GO), and magnetic nano-graphene oxide (GO / Fe3O4) is obtained by room-temperature synthesis. Then, with GO / Fe3O4 as the carrier, the ZEN aptamer with high affinity and specific recognition and binding ability to ZEN molecules is grafted to prepare the magnetic graphene oxide nanomaterial modified with ZEN aptamer (GO / Fe3O4 / Apt). The prepared GO / Fe3O4 / Apt nanomaterial has good selective adsorption ability and high specificity for zearalenone. Further, this GO / Fe3O4 / Apt nanomaterial is used as an adsorbent, and this adsorbent is combined with dispersive solid-phase extraction for highly selective extraction of zearalenone in actual grain samples.

[0025] (3) The composite material of nucleic acid aptamer-modified magnetic graphene oxide of the present invention: Magnetic graphene oxide is synthesized in one step by the co-precipitation method. After being modified with the ZEN aptamer, it is used as an adsorption material for dispersive solid-phase extraction and is combined with HPLC-FLD for successful detection of zearalenone in complex grain samples. The advantages of this method can be summarized as follows: 1) The prepared aptamer-functionalized magnetic graphene oxide nanomaterial (GO / Fe3O4 / Apt) has strong adsorption, and the adsorption capacity reaches 246.2 μg / g. 2) The dispersive solid-phase extraction material prepared based on GO / Fe3O4 / Apt has good specificity and selectivity for ZEN because the ZEN aptamer contains a G fragment, and the aptamer rich in the G fragment sequence has a strong affinity for the target molecule and can form stable secondary and tertiary structures. Moreover, the aptamer can also bind to the target through steric regulation, electrostatic interaction, hydrogen bond, van der Waals force, and π-π stacking interaction between aromatic groups. The ZEN aptamer not only binds tightly to ZEN but also has specificity for ZEN in the presence of other biotoxins (ochratoxin A, OTA; ochratoxin B, OTB). 3) It has good reusability, and the recovery rate of the target molecule is still more than 92% after being recycled 5 times. It overcomes the defects of traditional adsorbents such as poor selectivity, large matrix effect, and difficulty in recycling; it can meet the detection requirements of trace zearalenone in various matrices. Description of the Drawings

[0026] Figure 1 It is the schematic diagram of the composite material of nucleic acid aptamer-modified magnetic graphene oxide of the present invention for dispersive solid-phase extraction;

[0027] Figure 2 It is the FT-IR spectra of GO, GO / Fe3O4, and GO / Fe3O4 / Apt composite materials;

[0028] Figure 3It is the hysteresis loop diagram of the composite materials of GO / Fe3O4 and GO / Fe3O4 / Apt;

[0029] Figure 4 It is the bonding amount diagram of GO / Fe3O4 nanomaterials to the ZEN aptamer;

[0030] Figure 5 It is the TEM diagram of GO;

[0031] Figure 6 It is the TEM diagram of GO / Fe3O4 with 200 nm, Figure 5 The inset in it is the size distribution diagram of Fe3O4 nanoparticles;

[0032] Figure 7 It is the TEM diagram of GO / Fe3O4 with 50 nm,

[0033] Figure 8 It is the TEM diagram of GO / Fe3O4 / Apt;

[0034] Figure 9 It is the HPLC-FLD diagram of the blank sample (A) and the sample solution (B) added with 50 ng / mL ZEN;

[0035] Figure 10 It is the standard curve of ZEN concentration and peak area. Detailed implementation manners

[0036] The following combines the accompanying drawings to describe in detail the specific implementation manners of the present invention, but it should be understood that the protection scope of the present invention is not limited by the specific implementation manners.

[0037] Unless otherwise clearly stated, throughout the specification and claims, the term "comprising" or its variations such as "including" or "having" etc. will be understood to include the stated elements or components, without excluding other elements or other components.

[0038] As Figure 1 shown, a preparation method of a composite material of nucleic acid aptamer-modified magnetic graphene oxide, and a method thereof for dispersive solid-phase extraction. Figure 1 The dispersive solid-phase preparation process and the dispersive solid-phase extraction principle are given. First, graphene oxide (GO) is synthesized by the Hummers method, and magnetic nano-graphene oxide (Fe3O4 / GO) is obtained by the precipitation method. Then, using Fe3O4 / GO as the carrier, the ZEN aptamer with high affinity and specific recognition and binding ability to ZEN molecules is grafted to prepare the ZEN aptamer-modified magnetic graphene oxide nanomaterial (Fe3O4 / GO / Apt). Finally, the prepared Fe3O4 / GO / Apt nanomaterial is used as an adsorbent to develop a pre-treatment method for dispersive solid-phase extraction. Specifically as follows:

[0039] I. Preparation of a composite material of nucleic acid aptamer-modified magnetic graphene oxide

[0040] 1. Preparation of graphene oxide (GO)

[0041] Graphene oxide was synthesized by the Hummers method, which included: putting 2.0 g of graphite powder into a 250 mL beaker, adding 1.0 g of sodium nitrate and 46.0 mL of sulfuric acid (mass percentage 95 - 98%) and stirring well in an ice-water bath. Then 6.0 g of potassium permanganate was slowly added, and it was stirred at a temperature below 5 °C for 2 h. Then it was stirred at 35 °C for 60 min, and then 92 mL of water was added, and it was stirred at 95 °C for 15 min. The beaker was taken out of the water bath, and 100.0 mL of water and 8.0 mL of hydrogen peroxide (30% wt%) were added while it was still hot, and stirring was continued until it cooled to room temperature. Finally, the product was centrifuged into a colloid and dialyzed to neutrality. The prepared graphene oxide was denoted as GO.

[0042] 2. Preparation of magnetic graphene oxide (GO / Fe3O4) nanomaterials

[0043] GO / Fe3O4 nanomaterials were synthesized in one step by the co-precipitation method. 0.1 g of graphene oxide was dispersed in a 50 mL solution of FeCl3·6H2O (11 mmol) and FeSO4·7H2O (6 mmol), and magnetic stirring was carried out for 2 h under nitrogen protection. After the water bath temperature was raised to 45 °C, ammonia water (12 mL) was slowly dropped in, and the reaction was continued for 1 h. Finally, it was alternately washed with ultrapure water and absolute ethanol until it was nearly neutral, then dried in a vacuum drying oven at 60 °C for 12 h, and finally ground into granular form with an agate mortar, and then stored at 4 °C.

[0044] 3. Preparation of a composite material of nucleic acid aptamer-modified magnetic graphene oxide (GO / Fe3O4 / Apt)

[0045] 6 mg of magnetic graphene oxide nanomaterials were added to 1.0 mL of an EDC / NHS mixture (concentrations were 16 mmol / L and 4 mmol / L respectively). After reacting for 30 min, 0.4 mL of a 32.7 μg / mL nucleic acid aptamer solution was added, and it was incubated at 37 °C for 2 h to obtain the GO / Fe3O4 / Apt composite material.

[0046] After magnetic separation of the obtained GO / Fe3O4 / Apt composite material, it was rinsed with 1.0 mL of Tris-HCl buffer solution. Finally, the reaction solution and the rinsing solution were mixed, and the volume was fixed to 5 mL with Tris-HCl buffer solution. 40 μL of Oligreen fluorescent dye was added under dark conditions to react with the remaining ZEN aptamer in the buffer solution for 10 min, and the fluorescence spectrum of the solution was measured. The fluorescence intensity value at 521 nm was recorded, and then the binding amount of the GO / Fe3O4 nanomaterial to the ZEN aptamer was calculated through the fluorescence intensity.

[0047] Among them, the aptamer is the ZEN aptamer, and the nucleotide sequence is: 5'-NH2-(CH2)6-TCA TCT ATC TAT GGTACA TTA CTA TCT GTA ATG TGA-3'.

[0048] As Figure 2 shown, Figure 2 are the FT-IR spectra of GO, GO / Fe3O4, and GO / Fe3O4 / Apt composite materials. For GO, there is a broad absorption peak caused by the stretching of —OH at 3159~3402 cm -1 , stretching vibration peaks of the C=O and C=C groups are respectively at 1728 cm -1 and 1624 cm-1, and stretching vibration peaks of O—H, C—O—C, and C—O are respectively at 1394 cm -1 , 1256 cm -1 , and 1065 cm -1 . This indicates that GO contains many oxygen-containing groups. In contrast, for GO / Fe3O4, the stretching vibration peaks corresponding to —OH, C=O, C=C, and O—H are respectively at 3159~3402 cm -1 , 1715 cm -1 , 1624 cm -1 , and 1394 cm -1 , which is consistent with the characteristic absorption peaks of GO. The Fe—O characteristic absorption peak of Fe3O4 appears at 587 cm -1 . These phenomena indicate the successful synthesis of GO / Fe3O4. Compared with GO / Fe3O4, some new characteristic peaks appear in the infrared spectrum of the GO / Fe3O4 / Apt composite material. The peak values at 1715 cm -1 and 1538 cm -1 correspond to the stretching vibrations of C=O and C—N in the amide respectively. The absorption band appearing at 1282 cm -1 belongs to amide Ш. The results show that the aptamer is successfully bound to GO / Fe3O4.

[0049] At 300 K, the magnetic properties of GO / Fe3O4 and GO / Fe3O4 / Apt composites were measured using a VSM. As Figure 3 shown, the hysteresis loops of the samples are S-shaped curves, passing through the origin, with no coercivity and remanence, indicating that the GO / Fe3O4 and GO / Fe3O4 / Apt composites exhibit superparamagnetism at 300 K. The saturation magnetization (Ms) of the GO / Fe3O4 / Apt composite is 23.5 emu / g, slightly lower than the 32.8 emu / g saturation magnetization of GO / Fe3O4. This indicates that the prepared GO / Fe3O4 / Apt composite has good magnetic separation performance.

[0050] Figure 4 Figure shows the binding amount of ZEN aptamer to the GO / Fe3O4 nanomaterial. GO / Fe3O4 / Apt was prepared using 0.02, 0.05, 0.1, 0.2, 0.4, and 0.6 mL of 32.7 μg / mL ZEN aptamer respectively. The experimental results are as Figure 4 shown. As the amount of aptamer increases, the binding amount on the surface of GO / Fe3O4 increases accordingly. When the amount of nucleic acid aptamer is 0.4 mL (13.08 μg), the aptamer binding reaches saturation, and the maximum binding amount is approximately 2.18 μg / mg.

[0051] Figures 5 - 8 Figures show the TEM images of GO, GO / Fe3O4, and GO / Fe3O4 / Apt. It can be Figure 5 seen that GO has a transparent and very thin large lamellar structure with obvious wrinkles, and in some parts, the GO layers can be seen stacked together. Figure 6 In, Fe3O4 nanoparticles are aggregated on the surface of graphene oxide flakes. The average size of these particles is 11.2 nm. Figure 7 In, Fe3O4 is interspersed irregularly among the GO lamellae, and Fe3O4 has a polyhedral crystal structure. Figure 8 In, in addition to being loaded with a large number of Fe3O4 particles on the surface of GO, a closely contacted interface is also covered, which is the coating structure formed by the aptamer and GO / Fe3O4.

[0052] II. Method for using the composite material GO / Fe3O4 / Apt modified with nucleic acid aptamer magnetic graphene oxide for dispersive solid-phase extraction

[0053] 1. Preparation of solutions

[0054] (1) Preparation of Tris-HCl (10 mM, pH 7.4) buffer: Weigh 1.21 g of tris(hydroxymethyl)aminomethane and dissolve it in 800 mL of water. Then adjust the pH to 7.4 with HCl and make up the volume to 1000 mL.

[0055] (2) Preparation of PBS buffer: Weigh 8 g of NaCl, 0.2 g of KCl, 3.63 g of Na2HPO4·12H2O, and 0.24 g of KH2PO4, dissolve them in 800 mL of water, adjust the pH to 7.4 with HCl, and make up the volume to 1000 mL.

[0056] (3) Preparation of 10% methanol-PBS buffer: The preparation of 10% methanol-PBS buffer is to add 10 mL of methanol to 90 mL of PBS (10 mM, pH 7.4) buffer, that is, the volume ratio of methanol to PBS buffer is mixed at 1:9.

[0057] (4) Preparation of 80% methanol-PBS buffer: The preparation of 80% methanol-PBS buffer is to add 80 mL of methanol to 20 mL of PBS (10 mM, pH 7.4) buffer, that is, the volume ratio of methanol to PBS buffer is mixed at 8:2.

[0058] 2. Establishment of the separation method of the composite material GO / Fe3O4 / Apt modified with nucleic acid aptamer

[0059] (1) Wash the prepared GO / Fe3O4 / Apt magnetic nanoparticles three times with PBS buffer, and discard the supernatant after magnetic washing and separation.

[0060] (2) Prepare the ZEN standard solution with the prepared 10% methanol-PBS buffer, place the sample solution in the above-mentioned treated GO / Fe3O4 / Apt magnetic nanoparticles, place it on a shaker, and carry out the adsorption reaction at 150 rpm, then perform magnetic separation and discard the supernatant. Among them, the pH of the sample solution is 5.0, and the extraction time is 5 min.

[0061] (3) Add 10 mL of ultrapure water to the above-mentioned GO / Fe3O4 / Apt magnetic nanoparticles adsorbed with ZEN, perform magnetic separation and discard the supernatant;

[0062] (4) Add the eluent, vortex it on a vortex mixer for desorption, perform magnetic separation to collect the eluent, and filter it through a 0.22 μm organic filter membrane, then load it into a sample vial for HPLC-FLD detection. Among them, the elution organic solvent is methanol, the elution ratio is 80% methanol-PBS buffer, the pH of the elution solvent is 7.4, and the elution time is 3 min.

[0063] 3. Making the standard curve

[0064] Preparation of standard solution: Use ZEN standard sample and methanol to prepare a stock solution with a concentration of 100 μg / mL. Then, take a certain amount of the stock solution and add it to 10 mM PBS buffer solution with pH = 7.4, and make up the volume to obtain a series of ZEN standard solutions with different concentrations to be measured. Under the optimized experimental conditions, after desorbing different concentrations of ZEN standard solutions by dispersive solid-phase extraction, pass through a 0.22 μm organic filter membrane and detect by HPLC-FLD. Figure 10 It can be seen from 2 that in the range of 8.0 - 1000.0 ng / mL, the concentration of ZEN has a linear relationship with the peak area, the correlation coefficient is 0.9991, and the linear equation is: y = 2013.1x + 1070.6 (R 2 = 0.9991), and the detection limit (S / N = 3) is 1.51 ng / mL.

[0065] As Figure 9 shown in (A - B), Figure 9 the HPLC-FLD chromatograms of blank sample (A) and sample solution (B) added with 50 ng / mL ZEN are given. There is no peak between 3 - 4 min in the sample blank and the baseline is stable. The peak time of ZEN is about 3.48 min, indicating that this detection method is applicable and there is no background interference.

[0066] Figure 9 The standard curve of ZEN concentration and peak area is given. In the range of 8.0 - 1000.0 ng / mL, the linear equation is: y = 2013.1x + 1070.6 (R 2 = 0.9991), and the detection limit (S / N = 3) is 1.51 ng / mL.

[0067] 4. Evaluation of repeatability, stability and specificity of the method

[0068] The precision and accuracy of this method were evaluated by performing 5 repeated extractions on 100 ng / mL ZEN solution. The relative standard deviation (RSD) within a day was determined by extracting 5 samples in parallel within one day. The sample solutions prepared continuously for 5 days were analyzed to obtain the RSD between days. The results showed that the RSD within a day and between days were 3.61% and 5.28% respectively. The selectivity of the adsorbent was investigated by mixing ZEN with other toxins (ochratoxin A, ochratoxin B) using the MSPE method. After MSPE treatment, the peak area of ZEN increased significantly, indicating effective enrichment. However, the peak areas of OTA and OTB did not increase significantly. The results showed that the prepared GO / Fe3O4 / Apt adsorbent has good specificity and selectivity for ZEN.

[0069] III. Sample testing

[0070] The samples to be tested are corn and wheat. Referring to the standard method (GB 5009.209-2016), the dried corn and wheat samples (purchased from the local market) are crushed and mixed. 2 g of each is accurately weighed, 10.0 mL of extraction solution (acetonitrile + water 9:1) is added, and vortex extraction is carried out for 2 min. Then about 1 g of sodium chloride is added and vortex extraction is carried out for 1 min. Centrifugation is carried out at 5000 r / min for 5 min. The supernatant is transferred to a clean glass bottle and dried at 50 °C under a nitrogen stream. The dried sample is redissolved with methanol-water (10 / 90, v / v). Finally, the sample solution is subjected to dispersive solid-phase extraction prepared with GO / Fe3O4 / Apt magnetic nanoparticles and a 0.22 μm organic filter membrane to obtain the test solution. After detection by HPLC-FLD, the peak area of the test solution is obtained, and then it is substituted into the linear equation y = 2013.1x + 1070.6. According to the measured peak area, the corresponding ZEN content is calculated from the standard curve, and its spike recovery rate is calculated. The results are shown in Table 1:

[0071] Table 1 Determination results of ZEN spike recovery in corn and wheat samples

[0072]

[0073]

[0074] In summary, the magnetic graphene oxide synthesized in one step by the coprecipitation method in the present invention is modified with a ZEN aptamer and used as an adsorption material for dispersive solid-phase extraction, which is successfully applied to the detection of zearalenone in complex cereal samples in combination with HPLC-FLD.

[0075] The advantages of this method can be summarized as follows: (1) The prepared aptamer-functionalized magnetic graphene oxide nanomaterial (GO / Fe3O4 / Apt) has strong adsorption, and the adsorption capacity reaches 246.2 μg / g; (2) The dispersive solid-phase extraction material prepared based on GO / Fe3O4 / Apt has good specificity and selectivity for ZEN because the aptamer rich in G fragment sequence has a strong affinity for the target molecule and can form stable secondary and tertiary structures. Moreover, the aptamer can also bind to the target through spatial regulation, electrostatic interaction, hydrogen bond, van der Waals force, and π-π stacking interaction between aromatic groups; the ZEN aptamer not only binds tightly to ZEN, but also has specificity for ZEN in the presence of other mycotoxins (OTA, OTB); (3) It has good reusability, and the recovery rate of the target molecule is still more than 92% after being recycled 5 times. It overcomes the defects of traditional adsorbents such as poor selectivity, large matrix effect, and difficulty in recycling; it can meet the detection requirements of trace zearalenone in various matrices.

[0076] The foregoing description of specific exemplary embodiments of the invention is for purposes of illustration and exemplification. These descriptions are not intended to limit the invention to the precise forms disclosed, and it is apparent that, according to the above teachings, many modifications and variations are possible. The purpose of selecting and describing the exemplary embodiments is to explain the specific principles of the invention and its practical applications, so that those skilled in the art can implement and utilize the various different exemplary embodiments of the invention, as well as various different selections and modifications. The scope of the invention is intended to be defined by the claims and their equivalents.

Claims

1. A composite material of a nucleic acid aptamer modified magnetic graphene oxide, characterized in that: include: The surface of graphene oxide in the composite material is loaded with Fe3O4 particles and is also covered with a layer of closely contacted interface, which is a coating structure formed by nucleic acid aptamers and magnetic graphene oxide.

2. The composite material of nucleic acid aptamer modified magnetic graphene oxide according to claim 1, characterized in that: The bonding and solidification capacity of the nucleic acid aptamer is 1-5 μg / mg, the average size of the Fe3O4 nanoparticles is 9-12 nm, and the dispersibility is good; the composite material of the nucleic acid aptamer modified magnetic graphene oxide has efficient adsorption capacity and magnetic separation performance, the saturation magnetization intensity Ms is 20-25 emu / g, and the adsorption capacity for zearalenone is 200-300 μg / g.

3. The composite material of nucleic acid aptamer modified magnetic graphene oxide according to claim 1, characterized in that, The nucleic acid aptamer is a ZEN aptamer 5'-NH2-(CH2)6-TCA TCT ATC TAT GGT ACA TTA CTA TCT GTAATG TGA-3'.

4. A method for preparing a composite material of a nucleic acid aptamer modified with magnetic graphene oxide as claimed in any one of claims 1 to 3, characterized in that: First, graphene oxide GO is prepared, and then magnetic graphene oxide GO / Fe3O4 is synthesized in one step by a co-precipitation method. GO / Fe3O4 is dispersed in a buffer solution, and EDC and NHS are added to activate the carboxyl end surface. An amino-modified aptamer is added to connect to the material surface, incubated, and then separated to obtain a composite material of magnetic graphene oxide modified with a nucleic acid aptamer GO / Fe3O4 / Apt.

5. The method according to claim 4, characterized in that The Hummers method is used to prepare graphene oxide GO, including: stirring graphite powder, sodium nitrate and sulfuric acid in an ice water bath, adding potassium permanganate and stirring at below 5°C, then stirring at 30-40°C, adding water, stirring at 90-100°C, adding water and hydrogen peroxide and stirring, cooling to room temperature, centrifuging into colloid, dialyzing into neutral, and obtaining graphene oxide GO.

6. The method according to claim 4, characterized in that The method for synthesizing magnetic graphene oxide GO / Fe3O4 in one step by a co-precipitation method comprises: dispersing graphene oxide GO in a mixed solution containing FeCl3·6H2O and FeSO4·7H2O, heating to 40-50°C, stirring and reacting under nitrogen protection, adding ammonia water during the reaction, washing to neutrality after the reaction, drying, and grinding to obtain magnetic graphene oxide GO / Fe3O4.

7. Use of the composite material of nucleic acid aptamer modified magnetic graphene oxide as claimed in claim 1 as a dispersed solid phase extraction adsorption material.

8. The use according to claim 7, characterized in that The composite material of nucleic acid aptamer modified magnetic graphene oxide was used for dispersed solid phase extraction to extract zearalenone in grain samples for the detection of zearalenone.

9. The use according to claim 8, characterized in that The method for separating zearalenone using a composite material of a nucleic acid aptamer modified with magnetic graphene oxide comprises the following steps: (1) Wash the GO / Fe3O4 / Apt magnetic nanoparticles with PBS buffer, perform magnetic separation and discard the supernatant; (2) placing the prepared sample solution in the GO / Fe3O4 / Apt magnetic nanoparticles treated in step (1), shaking, performing adsorption reaction, and then magnetically separating and discarding the supernatant; (3) adding ultrapure water to the GO / Fe3O4 / Apt magnetic nanoparticles after adsorption of zearalenone in step (2), and then performing magnetic separation and discarding the supernatant; (4) adding an eluent, vortexing, desorbing, collecting the eluent by magnetic separation, filtering the membrane, and separating the zearalenone.

10. The use according to claim 9, characterized in that In step (4), the organic solvent of the eluent is methanol, the elution ratio is 80% methanol-PBS buffer, the pH of the elution solvent is 7-7.5, and the elution time is 3-5 min.

Images