CN116440874A - Cross-linked spore phenol adsorbent based on aptamer functionalized magnetic hydrophobic polymer and application thereof - Google Patents

Cross-linked spore phenol adsorbent based on aptamer functionalized magnetic hydrophobic polymer and application thereof Download PDF

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CN116440874A
CN116440874A CN202310457145.3A CN202310457145A CN116440874A CN 116440874 A CN116440874 A CN 116440874A CN 202310457145 A CN202310457145 A CN 202310457145A CN 116440874 A CN116440874 A CN 116440874A
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aoh
magnetic
hydrophobic polymer
aptamer
mhbpa
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栾云霞
赵丽萍
李成
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Beijing Academy of Agriculture and Forestry Sciences
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Beijing Academy of Agriculture and Forestry Sciences
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    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
    • B01J20/22—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof comprising organic material
    • B01J20/26—Synthetic macromolecular compounds
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D15/00—Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
    • B01D15/08—Selective adsorption, e.g. chromatography
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
    • B01J20/30—Processes for preparing, regenerating, or reactivating
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Abstract

The invention provides an crosslinked spore phenol adsorbent based on a nucleic acid aptamer functionalized magnetic hydrophobic polymer and application thereof, wherein the crosslinked spore phenol adsorbent is obtained by coupling a nucleic acid aptamer with a magnetic hydrophobic polymer, and the magnetic hydrophobic polymer is a copolymer of magnetic nanoparticles, methyl methacrylate and ethylene carbonate, wherein the nucleic acid aptamer can specifically identify AOH, the magnetic hydrophobic polymer is designed according to the molecular structure of the AOH, hydrophobic interaction with the AOH can be increased, and high-efficiency and high-specificity adsorption of the AOH can be realized under the synergistic effect of the nucleic acid aptamer and the magnetic hydrophobic polymer. The cross-linked spore phenol adsorbent based on the nucleic acid aptamer functionalized magnetic hydrophobic polymer provided by the invention can be used for high-efficiency and high-specificity adsorption of toxin target molecules with hydrophobicity on the premise of changing the bonded corresponding aptamer.

Description

Cross-linked spore phenol adsorbent based on aptamer functionalized magnetic hydrophobic polymer and application thereof
Technical Field
The invention belongs to the technical field of sample pretreatment, and particularly relates to a cross-linked spore phenol adsorbent based on a nucleic acid aptamer functionalized magnetic hydrophobic polymer.
Background
The alternaria phenol (AOH) is a main toxic metabolite of alternaria, and has strong teratogenicity, mutagenicity and genetic toxicity. HPLC-MS and other methods are traditional detection means of AOH. However, the content span range of the alternaria alternata in the actual sample is large, the sample matrix is complex and the interferents are many, which restrict the accuracy and precision of the analysis of the alternaria alternata in the actual sample. Therefore, for AOH, development of effective adsorption or enrichment materials and methods for separating and enriching the analyte and eliminating matrix interference has become a research hotspot for mycotoxin analysis in various practical samples in recent years.
Prior art 1: immunoaffinity columns (IACs) are a simplified sample extraction procedure commonly used in pretreatment of mycotoxin samples, with good recognition specificity.
Prior art 2: the molecular engram polymer (MIP) synthesized by taking target molecules as templates can imitate recognition sites of antibodies, is complementary with the target molecules in size, shape and functional group positions, and has potential of replacing IAC. Bondi (Food chem.2018; 243:357-364) and the like take 4-Vinylpyridine (VIPY) and methacrylamide (MAM) as functional monomers, ethylene glycol dimethacrylate (EDMA) as a cross-linking agent, and 3,8, 9-trihydroxy-6H-dibenzo [ B, D ] pyran-6-one (S2) as an AOH substitution template, so that MIP is prepared for selective adsorption of AOH in Food samples, and has better recovery rate.
Prior art 3: the aptamer refers to single-stranded DNA (ssDNA) or RNA obtained by in vitro screening by SELEX technology. The aptamer can be combined with a target with high specificity and high affinity, has a wide target range (including protein, polypeptide, small molecules, cells, bacteria and even tissues), can be synthesized by a chemical method, and has the characteristics of low cost, easiness in modification, good stability and the like. Once the aptamer sequence is determined, high purity, high reproducibility synthesis can be performed at lower cost. The aptamer is used as a chemical antibody, and plays an important role in specific recognition and adsorption of various targets due to the excellent property of the aptamer. The nucleic acid aptamer is used as a molecular recognition element, and Magnetic Nano Particles (MNPs) are combined as a carrier, so that the adsorption and separation of mycotoxins can be realized in a complex sample matrix.
Currently, there are few commercial IACs used for pretreatment of AOH samples, indicating that the development and preparation of highly specific and affinity antibodies to AOH is difficult.
The sample pretreatment technology based on MIP has complex preparation process, needs to consume template molecules and has no universality.
The nucleic acid aptamer functionalized MNPs are used as adsorbents, and enrichment and separation of target toxins can be realized by utilizing the characteristics of specific recognition of the nucleic acid aptamer and magnetic separation of the MNPs. However, MNPs have fewer functional groups on the surface and fewer interaction sites with the target, and thus cannot further improve the adsorption efficiency of MNPs functionalized with an aptamer as an adsorbent.
Disclosure of Invention
In order to solve the technical problem of low adsorption efficiency caused by fewer interaction sites with a target when the aptamer functionalized MNPs are used as an adsorbent, the invention provides an adsorbent based on the aptamer functionalized magnetic hydrophobic polymer.
In order to achieve the above purpose, the invention adopts the following technical scheme:
a magnetic cross-linked sporophenol adsorbent based on a nucleic acid aptamer functionalized magnetic hydrophobic polymer is prepared by coupling a nucleic acid aptamer with a magnetic hydrophobic polymer, wherein the magnetic hydrophobic polymer is a copolymer of magnetic nanoparticles, methyl methacrylate and ethylene carbonate, and the nucleic acid aptamer is used for combining cross-linked sporophenol (AOH).
A magnetic hydrophobic polymer material for aptamer functionalization, prepared by the following method: adding the magnetic nano particles into polyallylamine hydrochloride (PAHC) to carry out ultrasonic treatment for the first time, then adding polyvinylpyrrolidone (PVP) to carry out ultrasonic treatment for the second time, carrying out stirring reaction for the first time, washing with water, adding the magnetic nano particles and tetraethyl orthosilicate (TEOS) into absolute ethyl alcohol containing ammonia water, carrying out stirring reaction for the second time, and washing out excessive reactant with ethanol; then dispersing the mixture in ethanol, adding 3- (trimethoxysilyl) propyl Methacrylate (MPS), and stirring for the third time to synthesize MNPs@MPS;
MNPs@MPS reacts with VEC, MMA, AIBN and 1, 4-dioxane to synthesize the magnetic hydrophobic polymer material MHbP.
The magnetic hydrophobic polymer material as described above, preferably, the magnetic nanoparticle and the PAHC are in a mass ratio of 2:1, the mass ratio of the dosage of polyvinylpyrrolidone (PVP) to PAHC is 1:4, tetraethyl orthosilicate (TEOS) and 1.3% ammonia absolute ethanol with mass concentration are added according to the proportion of 1:100, and 3- (trimethoxy silicon based) propyl Methacrylate (MPS) and ethanol are added according to the proportion of 1:25.
The magnetic hydrophobic polymer material as described above, preferably, the time of the first ultrasonic wave is 1 to 60 minutes, and the time of the second ultrasonic wave is 15 to 30 minutes; the first stirring time is 5-10 h, the second stirring time is 5-10 h, and the third stirring time is 10-24 h.
Further preferably, the time of the first ultrasonic wave is 60min, and the time of the second ultrasonic wave is 20min; the time of the first stirring is 10h, the time of the second stirring is 10h, and the time of the third stirring is 24h.
The magnetic polymer material as described above, preferably mnps@mps and VEC, MMA, AIBN and 1, 4-dioxane are carried out in a mass to volume ratio of 100:70 to 570:500:10:10, wherein the first four are all in mg by mass and the last one is carried out in mL by volume; the reaction to synthesize MHbP is carried out for 24h under the protection of nitrogen and the condition of 90 ℃ oil bath.
Further, most preferably, mnps@mps and VEC, MMA, AIBN and 1, 4-dioxane are performed in a mass to volume ratio of 100:285:500:10:10.
An crosslinked spore phenol adsorbent based on a nucleic acid aptamer functionalized magnetic hydrophobic polymer, the preparation method of the crosslinked spore phenol adsorbent comprises the following steps: and incubating the prepared magnetic hydrophobic polymer material MHbP with an AOH aptamer to obtain the nucleic acid aptamer functionalized magnetic hydrophobic polymer adsorbent, namely MHbPA.
Preferably, the reaction ratio of MHbP to AOH aptamer solution is 5.0mg: 320.0. Mu.L, AOH aptamer concentration of 10.0. Mu.M, incubation time of 24h, at room temperature.
Preferably, the nucleic acid sequence of the AOH aptamer is: 5' -NH 2 -(CH 2 ) 6 -GGC ACT CCA CGC ATA GGC ATA CTT AAC TAG TGT TCA AGT TAT CCT GTG CGT GGA TGT CC-3'。
Further, the preparation method of the alteromonol adsorbent as described above preferably comprises the following steps:
s1, preparation of Magnetic Nano Particles (MNPs): first 1.4g (5.0 mM) FeCl was added 3 ·6H 2 O is dissolved in 40mL of ethylene glycol, 3.6g of NaAC and 1.0g of polyethylene glycol 200 are added, after stirring is carried out for 30min, the obtained yellow viscous liquid is sealed in a stainless steel autoclave with a polytetrafluoroethylene lining, and the reaction is carried out for 8h at 200 ℃; after the reaction kettle is naturally cooled, collecting the generated MNPs by using a magnet, and washing off redundant reactants by using ethanol to obtain the MNPs;
S2, dispersing the MNPs in 50mL of water, adding 0.5g of PAHC, performing ultrasonic treatment for 1h, and washing off excessive reactants by using deionized water; adding 10.0mL of water and 40.0mL of PVP with the concentration of 50.0mg/mL, carrying out ultrasonic treatment for 20min, stirring at the speed of 600rpm at room temperature for 10h, and washing off excessive reactants with water; adding the product into 100mL of absolute ethanol containing 1.3% ammonia water, adding 1.0mL of TEOS, stirring at room temperature for 10h, and washing off excessive reactant with ethanol; dispersing the product in 100.0mL of ethanol, adding 4.0mL of MPS, stirring at room temperature for 24 hours, washing off excessive reactant with ethanol, and drying at 45 ℃ for 12 hours to obtain MNPs@MPS;
s3, preparing a magnetic hydrophobic polymer: 100.0mg MNPs@MPS, 285.0mg VEC,500.0mg MMA, 10.0mg AIBN and 10.0mL 1, 4-dioxane were added to the flask and reacted under nitrogen at 90℃in an oil bath for 24 hours; washing off superfluous reactants on the surface by using ethanol and water, and drying the obtained product at 45 ℃ for 12 hours to obtain MHbP;
s4, mixing 320.0 mu L of 10.0 mu M AOH aptamer with 5.0mg MHbP, and incubating for 24 hours at room temperature; after the reaction is completed, washing the mixture for three times by using a binding buffer solution, and removing redundant reactants to obtain MHbPA;
the binding buffer contains 10.0mM Tris-HCl,10.0mM NaCl,5.0mM MgCl 2 ，pAqueous solution of H7.3.
The application of the alternariol adsorbent or the alternariol adsorbent obtained by the preparation method in sample pretreatment for detecting AOH.
Further, the application is that the application of the cross-linked spore phenol adsorbent in the pretreatment of the sample is added, 5.0g of the sample to be detected is mixed with 10.0mL of water/acetonitrile (40/60, v/v) in a centrifuge tube, vortex oscillation is carried out for 10min, 1.0mL of the sample solution is mixed with 320 mu L of the cross-linked spore phenol adsorbent for incubation for 10min after the supernatant is filtered by a filter membrane, the MHbPA adsorbed with the AOH is washed three times by a binding buffer solution, then mixed with 0.8mL of 70% methanol (methanol/binding buffer solution, v/v, 70/30) for incubation for 10min to elute the adsorbed AOH, after magnetic separation, HPLC determination is carried out on the supernatant, and the eluted AOH content after the MHbPA adsorption is calculated, namely the AOH content after the sample to be detected is pretreated by the MHbPA.
The invention has the beneficial effects that:
the invention provides an AOH adsorbent based on a nucleic acid aptamer functionalized magnetic hydrophobic polymer, wherein the magnetic hydrophobic polymer is designed according to the molecular structure of AOH, has targeting property, and can increase hydrophobic interaction with the AOH. And such hydrophobic polymers are suitable for adsorption of other toxin-like target molecules that are hydrophobic.
The invention provides an AOH adsorbent based on a nucleic acid aptamer functionalized magnetic hydrophobic polymer, which takes the nucleic acid aptamer of the AOH as a specific recognition element, and ensures the specificity of adsorption and recognition on the basis that the magnetic hydrophobic polymer improves the adsorption capacity.
The invention provides an AOH adsorbent based on a nucleic acid aptamer functionalized magnetic hydrophobic polymer, and the constructed adsorbent can realize high-efficiency adsorption and desorption of the AOH within 10min, and the adsorption capacity can reach 187.6ng/mg; and has good selectivity and stability, and can be repeatedly used for more than 6 times.
Drawings
FIG. 1 is a schematic diagram of the preparation of MHbPA in example 1;
FIG. 2 is an infrared spectrum characterization of MHbP;
FIG. 3 shows XPS characterization results of MNPs, MHbP, MHbPA;
FIG. 4 is a VAM characterization result of MNPs, MHbP, MHbPA;
FIG. 5 is a TGA characterization result of MNPs, MHbP, MHbPA;
FIG. 6 is a graph showing the morphology of the transmission electron microscope with respect to the prepared material and particle size
FIG. 7 is a graph showing the effect of MHbP mass on adsorption efficiency;
FIG. 8 is an effect of hydrophobic monomer species on AOH adsorption efficiency;
FIG. 9 is a graph showing the effect of sample volume on AOH adsorption efficiency;
FIG. 10 is a graph showing the effect of different desorption solvents on desorption efficiency;
FIG. 11 is the effect of methanol ratio on desorption efficiency;
FIG. 12 is a graph showing the effect of different desorption times on desorption efficiency;
fig. 13 is the effect of different desorption volumes on desorption efficiency.
FIG. 14 shows the results of the adsorption performance of MHbPA for different AOH concentrations;
FIG. 15 is a graph showing the fit of the adsorption behavior of MHbPA to AOH for different thermal adsorption models;
FIG. 16 shows the adsorption efficiency of MHbPA to AOH at various adsorption times;
FIG. 17 is a fit of different adsorption kinetics models to MHbPA adsorption AOH;
FIG. 18 shows the selectivity of MHbPA;
FIG. 19 is the stability of MHbPA;
FIG. 20 is a chart of the number of re-uses of MHbPA;
FIG. 21 shows the adsorption capacity results for AOH with different adsorbents.
Detailed Description
The invention aims to develop an adsorbent based on an aptamer functionalized magnetic hydrophobic polymer, solves the problem of low adsorption efficiency when the aptamer is independently used as an identification element through the synergistic interaction of the aptamer and the magnetic hydrophobic polymer, and realizes the rapid and efficient separation and enrichment of AOH in a complex matrix. Wherein the magnetic hydrophobic polymer (poly (methyl methacrylate-ethylene carbonate) @ MNPs, i.e., p (MMA-VEC) @ MNPs, abbreviated as MHbP) is designed according to the hydrophobicity of the AOH, and the amino-modified aptamer is coupled to the MHbP through the reaction between the aptamer and the VEC monomer to construct the aptamer functionalized magnetic hydrophobic polymer adsorbent, i.e., MHbPA. In the constructed adsorbent, the synergistic AOH can be rapidly and efficiently separated and enriched through the specific affinity of the aptamer and the hydrophobic interaction between the magnetic hydrophobic polymer and the AOH.
The invention relates to a design and synthesis of a targeting magnetic hydrophobic polymer: the magnetic hydrophobic polymer mainly comprises three parts: (1) magnetic nanoparticles containing double bonds; (2) providing a polymer monomer of an aptamer binding group; (3) a polymer monomer having hydrophobic properties.
Aiming at the structural characteristics of AOH, the invention prepares the targeting hydrophobic polymer, and increases the interaction with a target; on the basis, the specific recognition effect of the nucleic acid aptamer is combined, the nucleic acid aptamer functionalized magnetic hydrophobic polymer is constructed, and the rapid and efficient separation and enrichment of the AOH with the synergistic effect of the nucleic acid aptamer and the magnetic hydrophobic polymer are realized.
The following examples serve to further illustrate the invention but are not to be construed as limiting the invention. Modifications and substitutions made to the invention without departing from the spirit and nature of the invention are intended to be within the scope of the invention.
The technical means used in the examples are conventional means well known to those skilled in the art unless otherwise indicated. The nucleic acid aptamer of AOH in the following examples was obtained by screening the subject group, and the sequence thereof was: 5' -NH 2 -(CH 2 ) 6 GGC ACT CCA CGC ATA GGC ATA CTT AAC TAG TGT TCA AGT TAT CCT GTG CGT GGA TGT CC-3', synthesized by the company of Biotechnology Co., ltd,
Alternariol, alternaria Monomethyl Ether (AME), tenasconic acid (TeA), tenatoxin (TEN), and ochratoxin a (OTA) are available from peninsula pregangli bioengineering, inc.
Polyethylene glycol 200 is available from pichia pastoris (Shanghai, china); ethylene glycol, 1, 4-dioxane, 3- (trimethoxy silicon)Propyl Methacrylate (MPS), ferric chloride (FeCl) 3 ·6H 2 O) and Azobisisobutyronitrile (AIBN) and polyallylamine hydrochloride (PAHC) were purchased from aladine (Shanghai, china); methacrylate (MMA), ethyl Methacrylate (EMA), butyl Methacrylate (BMA), and polyvinylpyrrolidone (PVP) are available from enokay (beijing, china); ferrous chloride (FeCl) 3 ·6H 2 O) ammonia (NH) 3 ·H 2 O) and tetraethyl orthosilicate (TEOS) are available from beijing chemical company (beijing, china).
The buffers used were all composed of Tris-HCl,10.0mM NaCl,5.0mM MgCl at a concentration of 10.0mM 2 An aqueous solution having a pH of 7.3.
Example 1
The MHbP is synthesized by adopting a free radical polymerization method, MNPs@MPS, VEC and MMA are used as monomers, AIBN is used as an initiator, and the synthesis process is shown in figure 1. The specific operation is as follows:
(1) Synthesis of Fe by hydrothermal method 3 O 4 Magnetic Nanoparticles (MNPs) of (a): first 1.4g (5.0 mM) FeCl was added 3 ·6H 2 O was dissolved in 40mL of ethylene glycol, 3.6g of NaAC and 1.0g of polyethylene glycol 200 were added thereto, and after stirring for 30 minutes, the resulting yellow viscous liquid was sealed in a polytetrafluoroethylene-lined stainless steel autoclave and reacted at 200℃for 8 hours. After the reaction kettle is naturally cooled, collecting generated Fe by using a magnet 3 O 4 And washing off excessive reactant with ethanol, and storing the product MNPs in deionized water at 4 ℃ for standby.
(2) Mnps@mps was synthesized to introduce double bonds: dispersing all MNPs obtained in the steps in 50mL of water, adding 0.5g of PAHC, carrying out ultrasonic treatment for 1h, and washing off excessive reactants by using deionized water; adding 10.0mL of water and 40.0mL of PVP with the concentration of 50.0mg/mL into the mixture, carrying out ultrasonic treatment for 20min, stirring at room temperature for 10h (600 rpm), and washing off redundant reactants with water; adding the product into absolute ethanol (4.6 mL of ammonia water and 95.4mL of ethanol) containing ammonia water with volume concentration of 1.3%, adding 1.0mL of TEOS, stirring at room temperature for 10h, and washing off excessive reactant with ethanol; dispersing the product in 100.0mL of ethanol, adding 4.0mL of MPS, stirring at room temperature for 24h, washing off excessive reactant with ethanol, and drying at 45 ℃ for 12h for later use.
The above-mentioned first modifies the-C=C-bond on the MNPs, on the basis of which the polymer monomers VEC and MMA are polymerized onto the MNPs by free-radical polymerization. (3) synthesizing MHbP by a free radical polymerization method: 100.0mg MNPs@MPS, 285.0mg VEC,500.0mg MMA, 10.0mg AIBN and 10.0mL 1, 4-dioxane were added to the flask and reacted under nitrogen at 90℃in an oil bath for 24 hours. The excessive reactant on the surface is washed off by ethanol and water, and the obtained product is dried for 12 hours at 45 ℃ to obtain the magnetic hydrophobic polymer MHbP.
To verify whether the polymerization was successful, the MHbP was characterized by infrared spectroscopy (FT-IR), as shown in fig. 2. In MHbP, at 590.2cm -1 The peaks found here are characteristic peaks of Fe-O in MNPs, indicating that the polymerization process of the polymer has no significant effect on MNPs. In addition to the characteristic absorption peak of MNPs, at 1060.8cm -1 And 1793.7cm -1 Characteristic absorption peaks were found corresponding to asymmetric and symmetric stretching vibrations of-C-O-C-and-c=o-in VEC, respectively. Furthermore, at 1436.9 and 1728.3cm -1 at-CH 2 Bending vibrations and stretching vibrations of the carbonyl group, indicating the presence of MMA monomers in MHbP. FT-IR results showed that both functional monomers were successfully grafted onto MNPs.
After the magnetic polymer MHbP is obtained, the aptamer is coupled through the reaction of the modified amino group on the aptamer and the epoxy group in the VEC, and the nucleic acid aptamer functionalized magnetic hydrophobic polymer MHbPA is obtained, and the specific preparation method is as follows: 10.0. Mu.M AOH aptamer (320.0. Mu.L) was mixed with 5.0mg of MHbP prepared as described above and incubated for 24h at room temperature. After completion of the reaction, the reaction mixture was washed with binding buffer (10.0 mM Tris-HCl,10.0mM NaCl,5.0mM MgCl) 2 pH 7.3), washing for three times, removing excessive reactant on a magnetic rack under the washing joint, pouring out liquid to obtain MHbPA, and then storing in 320 mu L of binding buffer solution to obtain MHbPA with the concentration of 15.6mg/mL, and storing at the temperature of 4 ℃ for later use. When in use, the supernatant is removed for use after separation by a magnetic frame.
The preparation of MHbPA was confirmed by a series of characterization methods. First, elemental compositions of MNPs, MHbP, and MHbPA were obtained by X-ray diffraction (XPS), as shown in fig. 3. Fe2p and O1s peaks can be observed in the spectra of MNPs; in the MHbP spectra, in addition to the peaks of Fe2p and O1s, a peak of C1 was found, indicating that the polymerization of the polymer monomer introduced element C; in addition, an N1s peak was also observed in MHbPA, demonstrating that the aptamer was successfully coupled to MHbP.
Magnetic changes during MHbPA synthesis were detected using a Vibrating Sample Magnetometer (VSM), as shown in fig. 4. The magnetic response of MHbPA is related to the content of MNPs. In fig. 4, hysteresis regression curves of MNPs, MHbP and MHbPA are all in an S shape with origin symmetry, and have paramagnetism. The saturation magnetization of MNPs, MHbP and MHbPA were 68.7, 49.8 and 39.9emu/g, respectively. Although the saturation magnetization gradually decreases due to the polymerization of the monomer and the coupling of the aptamer, the retained magnetism is sufficient to achieve rapid separation under the action of an external magnetic field.
MNPs, MHbP and MHbPA were further validated using a thermogravimetric analyzer (TGA) and the results are shown in fig. 5: because the prepared materials all have certain residual moisture, the weight of the materials is slightly reduced at about 100 ℃; at 200-450 ℃, the weight of MHbP and MHbPA is significantly reduced (about 10%) due to degradation of the polymer; furthermore, MHbPA has about 4% more weight loss than MHbPA due to the binding of the aptamer.
The morphology of the above-prepared material and particle size was observed by Transmission Electron Microscopy (TEM), and as shown in fig. 6, all three were uniform spherical particles. The particle sizes of MNPs, MHbP and MHbPA are 196.8 + -7.0 nm, 214.9 + -2.0 nm and 218.4+ -5.1 nm, respectively. Compared with MNPs, the MHbP particle size is obviously increased, and the appearance level proves that the polymer is Fe 3 O 4 The surface of MNPs is polymerized to a certain degree; the particle size of MHbPA increases to a lesser extent than MHbPA, possibly due to the flexible structure of the aptamer.
Example 2
In order to realize the efficient adsorption of the MHbPA to the AOH, key factors such as the MHbP quality, the MHbP composition, the adsorbed sample volume and the like are examined.
The adsorption efficiency detection method comprises the following steps:
AOH adsorption and desorption: mixing 1.0mL of AOH solution with 320 mu L of MHbPA (separated by a magnetic frame and used after the supernatant is discarded) with the concentration of 15.6mg/mL prepared by the method of example 1 at room temperature for 10min, and taking the supernatant for liquid phase detection after magnetic separation, namely the content of the residual AOH after the absorption of the MHbPA; after washing the MHbPA adsorbed with the AOH with the binding buffer solution for three times, adding 0.8mL of 70% methanol (v/v, methanol/binding buffer solution) and mixing for 10min, and taking the supernatant after magnetic separation for liquid phase detection to obtain the eluted AOH content.
The high performance liquid chromatography detection method of the AOH comprises the following steps: the HPLC column was Venusil MP C18 (250X 4.6mm id, particle size 5 μm, bonna-Agela China). The HPLC detection conditions were: mobile phase A (water) -B (acetonitrile) is eluted with a combination gradient of 80% A (1 min), 80-40% A (3 min), 40% A (1 min), 40-80% A (1 min) and 80% A (1 min); the column temperature is 25 ℃; the flow rate is 1.0mL/min; the sample injection volume is 20 mu L; the detector is a fluorescence detector; the excitation wavelength was 258nm and the emission wavelength was 440nm.
Wherein the adsorption efficiency and recovery rate are calculated from the formula (1) and the formula (2), wherein A 0 For the peak area of AOH before adsorption, A 1 Peak area of supernatant after adsorption to MHbPA, a 2 Peak area of supernatant after MHbPA elution. Peak areas were all obtained by integration on liquid chromatography software.
Examining the MHbP quality:
10.0. Mu.M AOH aptamer (320.0. Mu.L) was mixed with 1.0, 3.0, 5.0, 7.0, 9.0, 11.0 and 13.0mg MHbP, respectively, and incubated for 24h at room temperature. After completion of the reaction, the reaction mixture was washed with binding buffer (10.0 mM Tris-HCl,10.0mM NaCl,5.0mM MgCl) 2 pH 7.3) and removing the redundant reactant to obtain the MHbPA with different MHbP dosage. 320. Mu.L of the above prepared solution was subjected to various concentrations of 15.6mg/mLMHbPA (used after separation by a magnetic rack and discarding the supernatant) was mixed with 1.0mL of 1.0 μg/mL AOH solution at room temperature for 10min, and after magnetic separation, the supernatant was taken for liquid phase detection. Liquid chromatography detection of 1.0. Mu.g/mL AOH (A 0 ) Peak area of supernatant after magnetic separation (A) 1 ) The adsorption efficiency is calculated by the formula (1), the adsorption efficiency is plotted on the abscissa with the MHbP mass as the ordinate, and the result is shown in fig. 7, i.e. the effect of the MHbP amount on the AOH adsorption efficiency. Experimental results indicate that MHbP mass has a significant impact on AOH adsorption efficiency, as MHbP is able to provide an aptamer binding group, thereby affecting the concentration of the aptamer bound on MHbPA. The result shows that the adsorption efficiency is improved along with the increase of the MHbP dosage; when the concentration of MHbP is 5.0mg, the adsorption efficiency of MHbPA to AOH is 92.1%; when the concentration of MHbP is 9.0mg, the adsorption efficiency of MHbPA to AOH is improved to 94.2%; the MHbPA mass (11.0 and 13.0 mg) was continuously increased, and the adsorption efficiency of MHbPA to AOH was kept stable. Considering that the adsorption efficiency of MHbPA is similar, the dosage difference between 5.0mg and 9.0mg is larger, and 5.0mg of MHbP is finally selected for the next study.
Examination of MHbP composition:
the design principle of MHbPA is combined, namely, the synergistic interaction of the specific affinity of the aptamer and the hydrophobic interaction of MHbP is used for realizing the high adsorption performance of MHbPA, and the composition of MHbP is examined.
Mhbps having different hydrophobicity (MMA, EMA, BMA, respectively) were synthesized, and different mhbpas were prepared under the same operating conditions. Namely, in the step of synthesizing MHbP by the radical polymerization method in example 1, EMA and BMA are used for experiment to replace MMA respectively, and MHbP with different compositions is prepared. MHbPA of different composition mhbps were prepared as in example 1. 320. Mu.L of each of the different MHbPA (separated by a magnetic frame, used after discarding the supernatant) prepared above at a concentration of 15.6mg/mL was mixed with 1.0mL of 1.0. Mu.g/mL of AOH solution at room temperature for 10min, and after magnetic separation, the supernatant was taken for liquid phase detection. Liquid chromatography detection of 1.0. Mu.g/mL AOH (A 0 ) Peak area of supernatant after magnetic separation (A) 1 ) Calculating adsorption efficiency by the formula (1), wherein the adsorption efficiency is vertical sitting by taking the MHbP hydrophobic monomer type as an abscissaThe results are shown in FIG. 8, which shows the effect of different hydrophobic monomers on adsorption efficiency.
FIG. 8 illustrates that hydrophobicity has some effect on adsorption efficiency, with MMA being the highest adsorption efficiency when it is the hydrophobic monomer, and decreasing adsorption efficiency as hydrophobicity increases (EMA or MBA as monomer). The hydrophobic nature promotes to some extent hydrophobic interactions with AOH, whereas the strongly hydrophobic nature of the monomer may make it difficult for MHbPA to disperse uniformly in the sample solution, resulting in reduced adsorption efficiency. Therefore, MMA is preferable as the hydrophobic monomer.
Investigation of the monomer polymerization ratio:
the polymerization ratio of MMA and VEC in MHbP is an important parameter for the composition of MHbP. 7 MHbP were synthesized with different MMA and VEC mass ratios. Namely, in the step of synthesizing MHbP by the radical polymerization method in example 1, the experimental synthesis of MHbP1-7 was carried out using different amounts of VEC and MMA, respectively, and the specific amounts are shown in Table 1. 5.0mg of different MHbP was incubated with 320.0. Mu.L of aptamer solution (10.0. Mu.M) at room temperature for 24h, after the reaction was completed, with binding buffer (Tris-HCl, 10.0mM NaCl,5.0mM MgCl) 2 pH 7.3) and removing the redundant reactant to obtain the MHbPA with different monomer ratios. 320. Mu.L of each of the different MHbPA prepared above at a concentration of 15.6mg/mL (separated by a magnetic frame, used after discarding the supernatant) was mixed with 1.0mL of 1.0. Mu.g/mL of AOH solution at room temperature for 10min, and after magnetic separation, the supernatant was taken for liquid phase detection. Liquid chromatography detection of 1.0. Mu.g/mL AOH (A 0 ) Peak area of supernatant after magnetic separation (A) 1 ) The adsorption efficiency was calculated by the formula (1), and the results are shown in table 1, namely, the influence of different monomer polymer ratios on the adsorption efficiency.
TABLE 1 influence of different VEC and MMA usage on adsorption efficiency
In Table 1, the effect of VEC amount on adsorption efficiency was examined at a fixed MMA amount of 500.0mg, with the adsorption efficiency increasing from 89.1% to 96.9% as the VEC amount increased from 70.0mg to 285.0 mg; when the VEC amount was 570.0mg, the adsorption efficiency was lowered. The amount of VEC can influence the bonding amount of the aptamer, and the higher the VEC polymerization proportion is, the higher the aptamer fixed bonding concentration is, and the higher the adsorption efficiency is; however, when the VEC ratio is too high, polymerization of MMA is affected, and the synergistic effect may be impaired, and adsorption efficiency may be lowered. MMA is therefore preferred: the use level of the VEC is 500:70-570, and the most preferable is 500:285.
The effect of MMA usage was examined: the amount of VEC was kept constant (285.0 mg), and the adsorption efficiency was significantly affected by the amount of MMA, and when the amount of MMA was less than 500.0mg, the adsorption performance of MHbPA on AOH was poor. When the MMA content was increased to 500.0mg, the adsorption efficiency was highest. This suggests that the hydrophobic effect in MHbPA does favor adsorption of AOH.
Investigation of the adsorbed sample volume:
MHbPA was prepared as in example 1. 320. Mu.L of MHbPA prepared as described above at a concentration of 15.6mg/mL (used after separation by a magnetic frame and discarding the supernatant) was mixed with 0.5mL of 2.0. Mu.g/mL AOH, 1.0mL of 1.0. Mu.g/mL AOH, 2.0mL of 0.5. Mu.g/mL AOH, 3.0mL of 0.33. Mu.g/mL AOH and 4.0mL of 0.25. Mu.g/mL AOH, respectively, at room temperature for 10 minutes, and after magnetic separation, the supernatant was taken for liquid phase detection. Detection of Pre-adsorption AOH by liquid chromatography (A) 0 ) Peak area of supernatant after magnetic separation (A) 1 ) The adsorption efficiency is calculated by the formula (1), the AOH volume is taken as an abscissa, the adsorption efficiency is taken as an ordinate, and a graph is drawn, and the result is shown in fig. 9, namely, the influence of different sample volumes on the adsorption efficiency. When the sample volume was increased from 0.5mL to 1.0mL, the adsorption efficiency was increased from 82.9% to 96.9%, and when the sample volume was increased to 4.0mL, the adsorption efficiency was drastically decreased to 56.8%. To obtain better AOH adsorption performance, 1.0mL was chosen as the optimal sample volume.
Investigation of the desorption solvent:
MHbPA was prepared as in example 1. 320. Mu.L of MHbPA prepared above was concentrated at 15.6mg/mL, separated by a magnetic stand, the supernatant was discarded, and then mixed with 1.0mL of 1.0. Mu.g/mL AOH at room temperature for 10min, and after magnetic separation, the supernatant was collected. After washing the MHbPA adsorbed with AOH three times with a binding buffer, different eluting solvents were added respectively:mixing methanol, ethanol, acetonitrile, ultrapure water and 1.0M NaCl solution for 10min, magnetically separating, and taking supernatant for liquid phase detection to obtain the eluted AOH content. Detection of Pre-adsorption AOH Peak area by liquid chromatography (A 0 ) Peak area of supernatant after magnetic separation (A 1 ) And peak area of AOH eluted with different elution solvents (A 2 ) The recovery rate is calculated by the formula (2), the recovery rate is plotted on the abscissa with different adsorption solvents, and the result is shown in fig. 10, namely the influence of different elution solvents on the recovery rate. In fig. 10, the recovery of methanol is highest, 88.8%, and the remaining four solvents are below 60.0%, indicating that strong interaction of MHbPA with AOH is dramatically reduced after methanol addition. In order to further improve the recovery rate, the proportion of methanol in the desorption solvent was studied.
Investigation of the methanol ratio of the desorption solvent:
MHbPA was prepared as in example 1. 320. Mu.L of MHbPA prepared above was concentrated at 15.6mg/mL, separated by a magnetic stand, the supernatant was discarded, and then mixed with 1.0mL of 1.0. Mu.g/mL AOH at room temperature for 10min, and after magnetic separation, the supernatant was collected. After washing the MHbPA adsorbed with AOH three times with the binding buffer, 0.8mL of methanol of different proportions was added as an eluting solvent (methanol and binding buffer mixed): 50% methanol, 70% methanol, 80% methanol, 90% methanol and 100% methanol are mixed for 10min, after magnetic separation, the supernatant is taken for liquid phase detection, namely the AOH content eluted when different methanol ratios are used as eluting solvents. Detection of Pre-adsorption AOH Peak area by liquid chromatography (A 0 ) Peak area of supernatant after magnetic separation (A 1 ) And peak area of AOH eluted with different elution solvents (A 2 ) The recovery rate is calculated by the formula (2), the methanol ratio is taken as an abscissa, the recovery rate is taken as an ordinate, and a graph is drawn, and the result is shown in fig. 11, namely, the influence of different methanol ratios on the recovery rate. The recovery rate is the lowest when the methanol proportion is 50%; the recovery rate was 93.4% at the highest when the methanol ratio was 70%. Thus, a mixture of 30% binding buffer and 70% methanol was used as desorption solvent.
Investigation of desorption time:
according toExample 1 MHbPA was prepared. 320. Mu.L of MHbPA prepared above was concentrated at 15.6mg/mL, separated by a magnetic frame, the supernatant was discarded, and then mixed with 1.0mL of 1.0. Mu.g/mL AOH at room temperature for 10min, respectively, and after magnetic separation, the supernatant was collected. After the MHbPA adsorbed with the AOH is washed three times by using a binding buffer solution, 0.8mL of 70% methanol is added as an eluting solvent to be respectively mixed for 10min, 20 min, 30 min, 60min, 120 min, 240 min and 360min, and after magnetic separation, the supernatant is taken for liquid phase detection, namely the content of the AOH eluted when different methanol ratios are used as the eluting solvents. Detection of Pre-adsorption AOH Peak area by liquid chromatography (A 0 ) Peak area of supernatant after magnetic separation (A 1 ) And peak areas of AOH eluted with different elution times (A 2 ) The recovery rate was calculated by the formula (2), the elution time was taken as the abscissa, the recovery rate was taken as the ordinate, and a graph was drawn, and the result was shown in fig. 12, namely, the effect of different elution times on the recovery rate. The recovery rate is obviously increased in 0-10min, and the recovery rate is not obviously increased along with the continuous increase of the desorption time, which indicates that the desorption action of MHbPA can be completed in a shorter desorption time, the pretreatment time of an actual sample is greatly reduced, the elution time is preferably 10-60 min, and in order to rapidly realize the pretreatment process of an AOH sample, 10min is selected as the optimal desorption time of MHbPA.
Investigation of desorption volume:
MHbPA was prepared as in example 1. 320. Mu.L of MHbPA prepared above was concentrated at 15.6mg/mL, separated by a magnetic frame, the supernatant was discarded, and then mixed with 1.0mL of 1.0. Mu.g/mL AOH at room temperature for 10min, respectively, and after magnetic separation, the supernatant was collected. After the MHbPA adsorbed with the AOH is washed three times by using a binding buffer solution, 0.2, 0.4, 0.6, 0.8 and 1.0mL of 70% methanol are respectively added as eluting solvents to be mixed for 10min, after magnetic separation, the supernatant is taken to carry out liquid phase detection, and the AOH content eluted by the eluting solvents with different volumes is obtained. Detection of Pre-adsorption AOH Peak area by liquid chromatography (A 0 ) Peak area of supernatant after magnetic separation (A 1 ) And peak areas of AOH eluted with different elution times (A 2 ) Calculating recovery rate (0.8 is changed into corresponding elution volume) by the formula (2), plotting the elution volume as abscissa and the recovery rate as ordinateThe graph shows the results in FIG. 13, namely the effect of different elution volumes on recovery. Experimental results show that the recovery rate gradually increases along with the increase of the desorption volume, and the maximum value is reached when the desorption volume is 0.8mL, so that the desorption volume of 0.8mL is selected, and the enrichment effect is 1.25 times.
Example 3
Isothermal adsorption experiments:
MHbPA was prepared as in example 1. 320. Mu.L of MHbPA prepared above at a concentration of 15.6mg/mL was mixed with 1.0mL 0.5,0.6,0.8,1.0,2.0 and 5.0. Mu.g/mLAOH, respectively, at room temperature for 10min, and after magnetic separation, the supernatant was collected. Detection of Pre-adsorption AOH Peak area by liquid chromatography (A 0 ) Peak area of supernatant after magnetic separation (A 1 ) Adsorption efficiency is calculated by formula (1), and adsorption capacity Q is calculated by formula (3). The graph is drawn with the AOH concentration as the abscissa and the adsorption efficiency and adsorption capacity as the ordinate, and the result is shown in FIG. 14, namely the adsorption isotherm of MHbPA. In FIG. 14, as the AOH concentration increases, the adsorption efficiency gradually decreases, and the adsorption capacity gradually increases, with a maximum value of 187.6ng/mg.
Fig. 15 is obtained by fitting the obtained data to Langmuir adsorption model and Freundlich adsorption model by using the origin software through the formula (4) and the formula (5). In the Langmuir adsorption model, the saturation adsorption capacity of MHbPA is 190ng/mg; the fitting result of the Freundlich adsorption model is poor (R 2 0.697). Therefore, the adsorption type of MHbPA is closer to Langmuir model, indicating that the adsorption of MHbPA to AOH is monolayer adsorption.
Wherein C is 0 To the initial concentration of AOH before adsorption, C e The concentration of AOH after adsorption, i.e., the residual concentration (mg/L), V is the AOH volume, m is the amount of MHbPA (mg), Q m Saturated adsorption quantity (mg/g), K L Is Langmuir constant (L/mg), K F The adsorption constant (mg/g) was Freundlich, and n was a constant. Adsorption kinetics:
MHbPA was prepared as in example 1. 320. Mu.L of MHbPA prepared above at a concentration of 15.6mg/mL and 1.0mL of 1.0. Mu.g/mLAOH were mixed at room temperature for 0.5, 1, 3, 5, 10, 20, 60 and 120min, respectively, and after magnetic separation, the supernatant was collected. Detection of Pre-adsorption AOH Peak area by liquid chromatography (A 0 ) Peak area of supernatant after magnetic separation (A 1 ) The adsorption efficiency was calculated by the formula (1). The adsorption efficiency is plotted on the abscissa and the adsorption efficiency is plotted on the ordinate, and the results are shown in fig. 16, namely the adsorption kinetics of MHbPA. In fig. 16, from 0.5min to 10min, the adsorption efficiency was significantly improved and reached a plateau after 10 min.
The adsorption kinetics are calculated and fitted by equation (6) and equation (7) using origin software to the obtained data according to pseudo first order kinetics model and pseudo second order kinetics model, resulting in fig. 17. The adsorption of MHbPA to AOH is more in accordance with a pseudo-secondary adsorption kinetics model, and a pseudo-primary adsorption kinetics model R 2 Poor (0.164), the adsorption reaches equilibrium within 10min, the equilibrium adsorption amount is 186.0ng/mg, and the equilibrium adsorption amount is consistent with the experimental result.
ln(Q e -Q t )＝ln Q e -K 1 t (6)
Q e In terms of adsorption amount (mg/g) at adsorption equilibrium, Q t The adsorption quantity (mg/g) at any time t (min), K 1 Is the equilibrium constant of the quasi-first order dynamics (1/min), K 2 Is the equilibrium constant (g/mg.min) of the quasi-second order kinetics.
Example 3 shows that the type of adsorption of MHbPA to AOH is closer to the Langmuir model, demonstrating that the adsorption of MHbPA to AOH is monolayer adsorption; kinetic adsorption is more in line with a pseudo-secondary adsorption kinetic model. The maximum adsorption capacity is 187.6ng/mg, the optimal adsorption time is 10min, and the high-efficiency adsorption of the AOH can be realized in a short time.
Example 4
MHbPA was prepared as in example 1, the adsorption procedure was performed as in example 2, and the selectivity of the adsorbent MHbPA was evaluated by exchanging the target for 5 mycotoxins such as AME, teA, TEN, OTA and DON. 1.0. Mu.g/mL of different mycotoxins were incubated with 320. Mu.L of MHbPA at a concentration of 15.6mg/mL, respectively, and after 10min the supernatants were magnetically separated and assayed by HPLC.
Calculation of adsorption capacity Q by equation (3) in fig. 18, the adsorption capacity for AME is high (this is related to the selectivity of aptamer) because AME is structurally similar to AOH, and in addition, the adsorption capacity for hydrophobic small molecule mycotoxins such as TEN, OTA, etc. is between 30 and 40ng/mL (adsorption capacity for teas is 3.6 ng/mg), the adsorption capacity is high, indicating that the hydrophobic interaction between polymer and hydrophobic molecule actually promotes adsorption. But the adsorption capacity is far lower than MHbPA, and the synergistic effect of the polymer and the aptamer proves that the adsorption performance is enhanced, and the aptamer also ensures the recognition specificity. The MHbPA has better selectivity and higher adsorption capacity to AOH as an adsorbent.
MHbPA was prepared as in example 1, and after storing in a binding buffer for 4 weeks, the adsorption process was performed as in example 2, and the stability of MHbPA was examined by calculating the adsorption efficiency as in formula (1). In fig. 19, MHbPA has an adsorption efficiency of AOH maintained at 80% or more after 4 weeks of storage, showing good stability. In fig. 20, after the MHbPA is reused 6 times, the adsorption efficiency is still maintained at 80% or more, and good reusability is exhibited.
Example 5
To evaluate the feasibility of the MHbPA adsorbent to adsorb AOH in actual samples, it was applied to AOH recognition and adsorption in three AOH-labeled wheat flour samples. 5.0g wheat flour was mixed with 10.0mL water/acetonitrile (40/60, v/v) in a centrifuge tube, vortexed for 10min, the supernatant was filtered through a filter membrane and added with different concentrations of AOH, and the mixture was diluted 10-fold with binding buffer. The final AOH addition concentrations were 250.0ng/mL,375.0ng/mL and 500.0ng/mL, respectively. Three wheat flour samples with AOH of different concentrations were mixed with 320. Mu.L of 15.6mg/mL MHbPA prepared as in example 1 and incubated for 10min, after magnetic separation, the supernatant was taken, after washing the MHbPA with AOH adsorbed three times with binding buffer, incubation was performed for 10min with 0.8mL of 70% methanol (methanol/binding buffer, v/v, 70/30) to elute the adsorbed AOH, after magnetic separation, the supernatant was taken for HPLC measurement, and the content of the eluted AOH after adsorption of MHbPA was obtained.
According to the liquid chromatography detection method, AOH markers (10 ng/mL, 50ng/mL, 100ng/mL, 500ng/mL, 1000ng/mL, 2000 ng/mL) of different concentrations were detected, and the peak areas of AOH of different concentrations were integrated on a liquid chromatography workstation. Linear fitting was performed with AOH concentration on the abscissa and peak area on the ordinate to obtain a linear equation of y=0.13X-0.52 (R 2 =0.990), each set of experiments was assayed in triplicate.
The peak area (Y value) of AOH after elution was brought into AOH liquid chromatography linear equation y=0.13X-0.52 to obtain the concentration (X value) of AOH after elution. Since the volume of the eluent was 0.8mL, the final eluent concentration was multiplied by 0.8 based on the eluent concentration to obtain 1mL sample AOH, and the standard recovery rate was calculated according to formula (8), and the results are shown in table 2. In Table 2, the recovery of MHbPA to AOH in the actual wheat sample ranged between 81.4-105.9%, indicating that it still had a good adsorption capacity in the actual sample.
Wherein C is 0 For the AOH addition concentration, C 2 Is the concentration after AOH elution.
TABLE 2 recovery of AOH from actual labeled wheat samples by MHbPA
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Example 6
The method for measuring the content of AOH in the sample to be measured comprises the following steps: 5.0g of the sample to be tested was mixed with 10.0mL of water/acetonitrile (40/60, v/v) in a centrifuge tube, vortexed for 10min, the supernatant was filtered through a filter membrane, and the mixture was diluted 10-fold with binding buffer. 1.0mL of the sample solution was mixed with 320. Mu.L of MHbPA prepared in example 1 at a concentration of 15.6mg/mL (separated by a magnetic frame, used after discarding the supernatant) and incubated for 10min, after magnetic separation, the MHbPA adsorbed with AOH was washed three times with a binding buffer, and then mixed with 0.8mL of 70% methanol (methanol/binding buffer, v/v, 70/30) and incubated for 10min to elute the adsorbed AOH, after magnetic separation, the supernatant was taken for HPLC measurement, and the content of the AOH eluted after adsorption of the MHbPA was obtained. The concentration of AOH after elution (X value) was obtained by carrying the peak area of AOH after elution (Y value) into AOH liquid chromatography linear equation Y=0.13X-0.52 in triplicate for each set of experiments. Because the volume of the eluent is 0.8mL, the eluent is multiplied by 0.8 based on the concentration of the eluent to obtain the final elution concentration of the AOH of the 1mL sample, and the value is enlarged by 10 times based on the final elution concentration of the AOH of the 1mL sample, so that the AOH content of the sample to be detected is obtained.
Comparative example 1
MNPs were prepared as in example 1 and MNPs (MNP-Apt) with appropriate ligands bonded thereto were prepared as in the preparation of MHbPA, i.e.MHbP was replaced with MNPs during the preparation of MHbPA.
MMA monomer during the preparation was removed to prepare MNP@pVEC in the same manner as in example 1. And preparing MNP@pVEC-Apt according to the MHbPA preparation method, namely replacing the MHbP with MNP@pVEC in the MHbPA preparation process.
MHbP was prepared as in example 1.
MHbPA was prepared as in example 1.
The four materials prepared above were used as adsorbents respectively, AOH adsorption was performed according to the procedure of example 2, adsorption efficiency was calculated according to formula (3), and a histogram was drawn with different adsorbents as abscissa and adsorption efficiency as ordinate, and the result is shown in fig. 21. Compared with MNP-Apt, MNP@pVEC-Apt shows better adsorption capacity, and polymerization of VEC on MNPs provides more aptamer binding groups. Compared with MHbP, the adsorption capacity of MHbPA is higher and is 4 times that of MHbP. In addition, the adsorption capacity of MHbPA to AOH is also obviously higher than MNP@pVEC-Apt. The results indicate that higher adsorption capacities cannot be obtained by either aptamer affinity with AOH or polymer hydrophobic with AOH alone. Enhanced adsorption can be achieved only under the synergistic effect of the affinity of the aptamer and the hydrophobic effect of the polymer.
The results show that the MHbPA and the AOH prepared by the invention have stronger adsorption performance.
Comparative example 2
The results of comparing the performance of the adsorbent prepared by the invention with the adsorption effect of different mycotoxins reported in the literature are shown in Table 3.
TABLE 3 comparison of adsorption effects of different adsorbents on different mycotoxins
The comparison result shows that the MHbPA prepared by the invention has the advantages of high efficiency and high speed on the adsorption of AOH.

Claims (10)

1. The magnetic cross-linked sporophenol adsorbent based on the nucleic acid aptamer functionalized magnetic hydrophobic polymer is characterized by being obtained by coupling a nucleic acid aptamer with a magnetic hydrophobic polymer, wherein the magnetic hydrophobic polymer is a copolymer of magnetic nanoparticles, methyl methacrylate and ethylene carbonate, and the nucleic acid aptamer is used for specifically binding cross-linked sporophenol.
2. A magnetic hydrophobic polymer material for aptamer functionalization, characterized in that it is prepared by the following method: adding the magnetic nano particles into polyallylamine hydrochloride, performing ultrasonic treatment for the first time, washing, adding polyvinylpyrrolidone solution, performing ultrasonic treatment for the second time, stirring for the first time, washing with water, adding the magnetic nano particles and tetraethyl orthosilicate into absolute ethyl alcohol containing ammonia water, stirring for the second time, and washing out excessive reactants with the ethyl alcohol; then dispersing the mixture in ethanol, adding 3- (trimethoxysilyl) propyl methacrylate, and stirring for the third time to synthesize MNPs@MPS;
MNPs@MPS reacts with VEC, MMA, AIBN and 1, 4-dioxane to synthesize the magnetic polymer material MHbP.
3. The magnetic hydrophobic polymer material of claim 2, wherein the magnetic nanoparticle to PAHC mass ratio is 2:1, the mass ratio of the dosage of polyvinylpyrrolidone to PAHC is 1:4, the ratio of tetraethyl orthosilicate to 1.3 percent ammonia absolute ethanol with volume concentration is 1:100, and the ratio of 3- (trimethoxy silicon based) propyl methacrylate to ethanol is 1:25.
4. The magnetic hydrophobic polymer material of claim 2, wherein the time of the first ultrasonic wave is 1 to 60 minutes and the time of the second ultrasonic wave is 15 to 30 minutes; the first stirring time is 5-10 h, the second stirring time is 5-10 h, and the third stirring time is 10-24 h.
5. The magnetic hydrophobic polymer material of claim 2, wherein mnps@mps and VEC, MMA, AIBN and 1, 4-dioxane are carried out in a mass to volume ratio of 100:70 to 570:500:10:10, wherein the first four are all in mg by mass and the last one is carried out in mL by volume; the reaction to synthesize MHbP is carried out for 24h under the protection of nitrogen and the condition of 90 ℃ oil bath.
6. An aptamer functionalized magnetic hydrophobic polymer-based cross-linked spore phenol adsorbent, which is characterized in that the preparation method comprises the following steps: incubating the magnetic hydrophobic polymer material of any of claims 2-5 with an AOH aptamer to obtain a aptamer functionalized magnetic hydrophobic polymer adsorbent, MHbPA.
7. The cross-linked spore phenol adsorbent as claimed in claim 6, characterized in that the reaction ratio of MHbP to AOH aptamer solution is 5.0mg: 320.0. Mu.L, AOH aptamer concentration of 10.0. Mu.M, incubation time of 24h, at room temperature.
8. The cross-linked spore phenol sorbent according to claim 6, wherein the AOH aptamer has a nucleic acid sequence of: 5' -NH 2 -(CH 2 ) 6 -GGC ACT CCA CGC ATA GGC ATA CTT AAC TAG TGT TCA AGT TAT CCT GTG CGT GGA TGT CC-3'。
9. The preparation method of the cross-linked spore phenol adsorbent based on the aptamer functionalized magnetic hydrophobic polymer is characterized by comprising the following steps of:
s1, preparation of Magnetic Nano Particles (MNPs): first 1.4g (5.0 mM) FeCl was added 3 ·6H 2 O is dissolved in 40mL of ethylene glycol, 3.6g of NaAC and 1.0g of polyethylene glycol 200 are added, after stirring is carried out for 30min, the obtained yellow viscous liquid is sealed in a stainless steel autoclave with a polytetrafluoroethylene lining, and the reaction is carried out for 8h at 200 ℃; after the reaction kettle is naturally cooled, collecting the generated MNPs by using a magnet, and washing off redundant reactants by using ethanol to obtain the MNPs;
S2, dispersing the MNPs in 50mL of water, adding 0.5g of PAHC, performing ultrasonic treatment for 1h, and washing off excessive reactants by using deionized water; adding 10.0mL of water and 40.0mL of PVP with the concentration of 50.0mg/mL, carrying out ultrasonic treatment for 20min, stirring at the speed of 600rpm at room temperature for 10h, and washing off excessive reactants with water; adding the product into 100mL of absolute ethanol containing 1.3% ammonia water, adding 1.0mL of TEOS, stirring at room temperature for 10h, and washing off excessive reactant with ethanol; dispersing the product in 100.0mL of ethanol, adding 4.0mL of MPS, stirring at room temperature for 24 hours, washing off excessive reactant with ethanol, and drying at 45 ℃ for 12 hours to obtain MNPs@MPS;
s3, preparing a magnetic hydrophobic polymer: 100.0mg MNPs@MPS, 285.0mg VEC,500.0mg MMA, 10.0mg AIBN and 10.0mL 1, 4-dioxane were added to the flask and reacted under nitrogen at 90℃in an oil bath for 24 hours; washing off superfluous reactants on the surface by using ethanol and water, and drying the obtained product at 45 ℃ for 12 hours to obtain MHbP;
s4, mixing 320.0 mu L of 10.0 mu M AOH aptamer with 5.0mg MHbP, and incubating for 24 hours at room temperature; after the reaction is completed, washing the mixture for three times by using a binding buffer solution, and removing redundant reactants to obtain MHbPA;
the binding buffer contains 10.0mM Tris-HCl,10.0mM NaCl,5.0mM MgCl 2 An aqueous solution at pH 7.3.
10. Use of the alternariol adsorbent according to any one of claims 6-8 or obtainable by the process of claim 9 in sample pretreatment for AOH detection.
CN202310457145.3A 2023-04-25 2023-04-25 Cross-linked spore phenol adsorbent based on aptamer functionalized magnetic hydrophobic polymer and application thereof Pending CN116440874A (en)

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN117607464A (en) * 2024-01-19 2024-02-27 北京市农林科学院 Immediate detection method of progesterone by using aptamer sensor based on portable glucometer

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN117607464A (en) * 2024-01-19 2024-02-27 北京市农林科学院 Immediate detection method of progesterone by using aptamer sensor based on portable glucometer
CN117607464B (en) * 2024-01-19 2024-04-05 北京市农林科学院 Immediate detection method of progesterone by using aptamer sensor based on portable glucometer

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