Magnetic molecularly imprinted material for ganoderma lucidum polysaccharide extraction and preparation method thereof

Abstract

This invention relates to the field of natural product separation and purification technology, and discloses a magnetic molecularly imprinted material for extracting Ganoderma lucidum polysaccharides and its preparation method. The magnetic molecularly imprinted material includes a magnetic metal-organic framework composite carrier and a molecularly imprinted polymer layer coated on the surface of the composite carrier. The magnetic molecularly imprinted material is prepared by polymerizing raw materials containing the following parts by weight in an aqueous phase and then eluting to remove Ganoderma lucidum polysaccharide template molecules: magnetic metal-organic framework composite carrier: 1.0-1.5 parts; Ganoderma lucidum polysaccharide template molecules: 0.02-0.05 parts; functional monomers: 0.02-0.06 parts. This invention enables the material to directly separate solid and liquid under the action of an external magnetic field after adsorbing Ganoderma lucidum polysaccharides, simplifying the separation operation steps and improving the separation efficiency. Simultaneously, it reduces the interference of monosaccharides, proteins, and other impurities in the extraction system, thereby improving the extraction purity of the target product.

Description

Technical Field

[0001] This invention relates to the field of natural product separation and purification technology, specifically to a magnetic molecular imprinting material for extracting Ganoderma lucidum polysaccharides and its preparation method. Background Technology

[0002] Ganoderma lucidum polysaccharides are the core components of Ganoderma lucidum with a variety of physiological activities and have a wide range of applications in food and medicine. At present, the main methods for obtaining Ganoderma lucidum polysaccharides are traditional extraction methods such as water extraction and alcohol precipitation and column chromatography. With the development of materials science, magnetic molecular imprinted materials are gradually being applied to the separation and extraction of target molecules in complex systems.

[0003] Magnetic molecularly imprinted materials are polymer layers containing pores formed on the surface of a magnetic substrate through polymerization. During the polymerization process, template molecules are combined with functional monomers, and after elution, pores matching the spatial structure and chemical sites of the template molecules are left behind. In actual separation operations, an external magnetic field can cause the material with adsorbed target molecules to be directly separated from the mixture. Metal-organic framework materials have a wide specific surface area and abundant pore structure, and are introduced into the adsorption system to provide a large number of adsorption sites.

[0004] Currently available Ganoderma lucidum polysaccharide separation materials suffer from several drawbacks. Firstly, some materials have limited internal binding sites, resulting in low overall adsorption capacity. Secondly, the separation process is cumbersome and inefficient. Thirdly, conventional adsorption materials lack specific recognition capabilities, easily adsorbing impurities such as monosaccharides and proteins during extraction, leading to low purity of the final extracted product. Fourthly, existing synthesis reactions often disrupt the hydration state of Ganoderma lucidum polysaccharides, causing the generated imprinted pores to mismatch with the polysaccharide molecular conformation in the actual extraction environment. Furthermore, existing materials exhibit high mass transfer resistance, and the molecular imprint layer's coating on the carrier surface is unstable, leading to structural damage after repeated use and a significant decrease in reusability.

[0005] Therefore, the purpose of this invention is to provide a magnetic molecular imprinting material for extracting Ganoderma lucidum polysaccharides and its preparation method, so as to overcome the shortcomings of the prior art. Summary of the Invention

[0006] To address the shortcomings of existing technologies, this invention provides a magnetic molecular imprinting material for Ganoderma lucidum polysaccharide extraction and its preparation method, which solves the problems of low adsorption capacity, poor separation efficiency, low extraction purity due to lack of specific recognition, high internal mass transfer resistance, and poor reusability of existing Ganoderma lucidum polysaccharide extraction and separation materials.

[0007] To achieve the above objectives, the present invention provides the following technical solution: In a first aspect, the present invention provides a magnetic molecular imprinting material for extracting Ganoderma lucidum polysaccharides, employing the following technical solution: A magnetic molecularly imprinted material for extracting Ganoderma lucidum polysaccharides includes a magnetic metal-organic framework composite carrier and a molecularly imprinted polymer layer coated on the surface of the composite carrier. The magnetic molecularly imprinted material is prepared by polymerizing raw materials comprising the following parts by weight in an aqueous phase and then eluting to remove the Ganoderma lucidum polysaccharide template molecules: Magnetic metal-organic framework composite carrier: 1.0–1.5 parts; Ganoderma lucidum polysaccharide template molecule: 0.02-0.05 parts; Functional monomer: 0.02–0.06 parts; Crosslinking agent: 0.1–0.18 parts; Initiator: 0.002–0.005 parts; Deionized water: 2.4–4.8 parts.

[0008] By adopting the above technical solution, and using a magnetic metal-organic framework composite carrier as the substrate, the magnetic separation characteristics of magnetic nanoparticles and the high specific surface area and porous structure of metal-organic framework materials are utilized. During the polymerization process, functional monomers and Ganoderma lucidum polysaccharide template molecules interact through hydrogen bonds to form a pre-assembly. The crosslinking agent fixes the pre-assembly in the polymer three-dimensional network. After the template molecules are washed away, specific recognition pores that match the spatial structure, size and chemical groups of Ganoderma lucidum polysaccharide are left in the polymer layer.

[0009] During the extraction of Ganoderma lucidum polysaccharides, the target molecules enter the pores and re-bond with the functional monomer residues via hydrogen bonds, achieving specific adsorption. After adsorption, solid-liquid separation is achieved by applying an external magnetic field. Furthermore, the porous structure of the support reduces the mass transfer resistance of the polymer layer, thereby improving the adsorption and elution rates of the material.

[0010] Preferably, the magnetic metal-organic framework composite carrier is prepared by reacting components comprising the following parts by weight: Amino-modified ferrite nanoparticles: 1–1.2 parts; Isoflurone diisocyanate: 10-50 parts; N,N-Dimethylformamide: 150-600 parts; Amino-metal-organic framework materials: 10–50 parts; Stannous isooctanoate: 0.2–0.8 parts.

[0011] By employing the above technical solution, isoflurane diisocyanate is used as a coupling agent, and its molecular structure contains two isocyanate groups with different reactivity. Under the catalysis of stannous isooctanoate, the isocyanate groups undergo nucleophilic addition reactions with the amino groups on the surface of amino-modified ferrite and amino groups on the surface of amino metal-organic framework materials, respectively, to generate urea bonds.

[0012] The specific reaction process is as follows: the isocyanate group reacts with the amino group to form a -NH-CO-NH- structure, thereby connecting the ferrite particles and the metal-organic framework material through stable covalent bonds. This chemical bonding method avoids component detachment from the composite carrier during use, maintaining the structural stability and magnetic response performance of the material.

[0013] Preferably, the ferrite core in the amino-modified ferrite nanoparticles is one of magnetite, cobalt ferrite, manganese ferrite, or zinc ferrite nanoparticles. The amino-metal-organic framework material is one of MIL-101-NH2, UiO-67-NH2, MIL-125-NH2, CAU-1-NH2, or NH2-MIL-88B.

[0014] By adopting the above technical solution, the selected ferrite core has a high saturation magnetization intensity, which ensures that the material can quickly achieve solid-liquid separation under an external magnetic field. The selected metal-organic framework material has free amino groups, which provide reaction sites for the grafting of isoflurane diisocyanate. At the same time, its porous framework restricts the shrinkage and deformation of the molecularly imprinted polymer layer during the polymerization process, maintaining the structural integrity of the imprinted pores.

[0015] Preferably, the functional monomer is one of methacrylic acid, acrylamide, 2-acrylamido-2-methylpropanesulfonic acid, or acrylic acid; The crosslinking agent is one of N,N'-methylenebisacrylamide, 1,3,5-triacryloylhexahydro-1,3,5-triazine, polyethylene glycol diacrylamide, or polyethylene glycol diacrylate. The initiator is one of the following: a mixture of potassium persulfate and sodium sulfite, azobisisobutyramidine hydrochloride, azobiscyanopentanoic acid, or a mixture of ammonium persulfate and sodium ascorbate.

[0016] By adopting the above technical solution, the selected functional monomer contains carboxyl, amide or sulfonic acid groups, which can form hydrogen bonds with the hydroxyl groups on the surface of Ganoderma lucidum polysaccharide molecules. The crosslinking agent contains multiple polymerizable double bonds, which form a crosslinked three-dimensional polymer skeleton during free radical polymerization, thus fixing the binding conformation of the functional monomer and the template molecule.

[0017] The water-soluble initiator undergoes homolytic cleavage at a set temperature to generate free radicals, which initiate the addition polymerization of double bonds in monomers and crosslinking agents. Since the polymerization process takes place in an aqueous environment, the hydrated conformation of Ganoderma lucidum polysaccharides is maintained, which enhances the ability of imprinted materials to recognize and adsorb target molecules in actual aqueous systems.

[0018] Secondly, the present invention provides a method for preparing magnetic molecularly imprinted materials for extracting Ganoderma lucidum polysaccharides, using the following technical solution: A method for preparing a magnetic molecularly imprinted material for extracting Ganoderma lucidum polysaccharides includes the following steps: Ferrite nanoparticles were dispersed in a mixed solvent of anhydrous ethanol and deionized water. Sodium hydroxide aqueous solution was added to adjust the pH of the system to alkaline. Then, 3-aminopropyltriethoxysilane was slowly added and stirred for reaction. After the reaction was completed, the solid product was separated, washed and dried to obtain amino-modified ferrite. The obtained amino-modified ferrite was dispersed in anhydrous N,N-dimethylformamide, and isoflurane diisocyanate was added for the first stage reaction. Then, amino metal-organic framework material and stannous isooctanoate were added for the second stage reaction. After the reaction was completed, the solid phase was separated, washed and dried to obtain a magnetic metal-organic framework composite carrier. The obtained magnetic metal-organic framework composite carrier, Ganoderma lucidum polysaccharide template molecule, functional monomer, crosslinking agent, initiator and deionized water were sequentially added to a polymerization reaction flask equipped with a degassing device to form a polymerization reaction system. After deoxygenation treatment, aqueous phase polymerization was initiated under constant temperature conditions. After polymerization was completed, the reaction was diluted and quenched, and the solid phase was separated and dried to obtain a composite material coated with polymer precursor. The obtained composite material coated with polymer precursor was dispersed in deionized water to prepare a dispersion. High-temperature elution was carried out under heating and stirring conditions. After elution, magnetic separation was performed and the material was continuously washed with hot deionized water until no template molecules remained. After drying, the finished magnetic molecular imprinted material was obtained.

[0019] By employing the above technical solution, the preparation method utilizes surface imprinting technology to perform aqueous phase polymerization on the surface of a magnetic metal-organic framework composite carrier. The resulting molecularly imprinted layer coats the outside of the carrier, and the imprinted pores are distributed on the surface of the material. This surface distribution characteristic shortens the diffusion path of template molecules during adsorption and desorption, reduces mass transfer resistance, and improves the utilization rate of imprinted sites. The high-temperature elution step provides heat energy to disrupt the hydrogen bonds between Ganoderma lucidum polysaccharides and the polymer network, achieving effective removal of template molecules and ensuring the effective imprinting capacity of the material.

[0020] Preferably, when preparing amino-modified ferrite, the ferrite nanoparticles have a particle size of 50-500 nm and are subjected to ultrasonic dispersion treatment before use for 30-60 min.

[0021] By adopting the above technical solution, setting the particle size range of nanoparticles and pre-dispersing them ultrasonically, it is possible to break the van der Waals force aggregation between nanoparticles, increase their specific surface area in the mixed solvent, fully expose the surface hydroxyl groups, and improve the grafting density of subsequent silane coupling agents.

[0022] Preferably, in the preparation of amino-modified ferrite, the mass ratio of the ferrite, anhydrous ethanol and 3-aminopropyltriethoxysilane is 1:15-60:0.1-0.5. The pH range of the system should be adjusted to 8–10; The reaction temperature after adding 3-aminopropyltriethoxysilane is 50–80 °C, and the reaction time is 4–12 h.

[0023] By adopting the above technical solution, under alkaline conditions, the ethoxy group at the end of 3-aminopropyltriethoxysilane undergoes hydrolysis to generate silanol groups. Subsequently, the silanol groups undergo a condensation dehydration reaction with the hydroxyl groups on the ferrite surface to form stable Fe-O-Si covalent bonds. Controlling the reagent ratio and reaction temperature is beneficial to forming a silane coating layer on the particle surface and introducing surface amino groups, thereby reducing the phenomenon of silane molecules self-polymerizing to form gels.

[0024] Preferably, in the preparation of magnetic metal-organic framework composite supports, the temperature of the first stage reaction after the addition of isoflurane diisocyanate is 30-50°C and the time is 6-10 h. The temperature of the second stage reaction after adding the amino-metal-organic framework material and stannous isooctanoate is 60–90 °C, and the time is 2–6 h.

[0025] By adopting the above technical solution and employing a staged heating reaction strategy, the first stage of low-temperature reaction allows the isocyanate groups with smaller steric hindrance and higher reactivity in the isoflurane diisocyanate molecule to preferentially react with the amino groups on the ferrite surface; the second stage of heating, under the catalysis of stannous isooctanoate, promotes the reaction of the isocyanate groups with larger steric hindrance and lower reactivity in the molecule with the amino groups on the surface of the metal-organic framework material. This step-by-step control reduces the self-crosslinking phenomenon between metal-organic framework materials or ferrite particles, thus ensuring the core-shell dispersion structure of the composite material.

[0026] Preferably, in preparing the composite material coated with the polymer precursor, the oxygen removal treatment is specifically implemented as follows: Dissolved oxygen in the polymerization reaction system was removed by repeating the freezing, vacuuming, and thawing cycle three times. Each cycle includes the sequential steps of freezing, vacuuming, and thawing. The aqueous phase polymerization is carried out at a temperature of 60–80°C for a time of 12–22 hours.

[0027] By employing the above technical solution, dissolved oxygen in the system is eliminated through a freezing and vacuum cycle, thus preventing oxygen from inhibiting polymerization and terminating chains, ensuring the growth of polymer chains and the formation of cross-linked networks. The isothermal polymerization conditions ensure a balance between the polymerization rate and the conformational stability of the polysaccharide template molecules.

[0028] Preferably, during high-temperature elution, the solid content of the dispersion is configured to be 50–150 g / L; The high-temperature elution temperature is 60–90°C, and the stirring time is 2–6 hours.

[0029] By adopting the above technical solution, since the formation of hydrogen bonds is an exothermic process, high temperature conditions are conducive to the dissociation of hydrogen bonds. Setting the elution temperature between 60 and 90°C can provide sufficient internal energy to overcome the hydrogen bond binding between the polyhydroxyl groups of Ganoderma lucidum polysaccharide and the functional groups of the polymer, causing the template molecules to be released from the imprinted pores into the aqueous phase, thereby leaving specific recognition pores in the polymer layer that match the molecular structure of Ganoderma lucidum polysaccharide.

[0030] This invention provides a magnetic molecularly imprinted material for extracting Ganoderma lucidum polysaccharides and its preparation method. It has the following beneficial effects: 1. This invention uses a magnetic metal-organic framework composite carrier, which combines the high specific surface area and porous structure of metal-organic framework materials, increases the binding sites inside the material, thereby improving the overall adsorption capacity. At the same time, the composite carrier has magnetic response characteristics, which enables the material to directly separate solid and liquid under the action of an external magnetic field after adsorbing Ganoderma lucidum polysaccharides, simplifying the separation operation steps and improving the separation efficiency.

[0031] 2. This invention sets up a molecularly imprinted polymer layer on the surface of a magnetic metal-organic framework composite carrier. After aqueous polymerization, the template molecules of Ganoderma lucidum polysaccharides are eluted to remove them. This leaves imprinted pores in the polymer layer that match the structure and size of the Ganoderma lucidum polysaccharides. The imprinted layer can directionally recognize and adsorb Ganoderma lucidum polysaccharides, reducing the interference of other impurities such as monosaccharides and proteins in the extraction system and improving the extraction purity of the target product.

[0032] 3. This invention maintains the hydrated conformation of Ganoderma lucidum polysaccharide template molecules by setting the mass ratio of raw materials to carry out polymerization reaction in the aqueous phase, so that the generated molecularly imprinted pores are more consistent with the molecular state in the actual extraction environment. The ratio of functional monomers to crosslinking agents can ensure the stable coating of molecularly imprinted polymer layers on the surface of composite carriers, which not only reduces the mass transfer resistance inside the material, but also maintains the integrity of the material structure and improves the reusability of the material. Attached Figure Description

[0033] Figure 1 The graph shows the adsorption capacity of the magnetic molecularly imprinted material prepared in Example 1 of the present invention as a function of time. Figure 2 Magnetic response test diagram of the magnetic molecular imprinted material prepared in Example 1 of the present invention; Figure 3 VSM hysteresis loop diagram of the magnetic molecular imprinted material prepared in Example 1 of the present invention; Figure 4 This is a comparison chart of the specific surface area and pore parameters of the products at each stage of the embodiments and comparative examples of the present invention; Figure 5 These are characterization diagrams of the magnetic properties and separation parameters of the products at each stage of this invention; Figure 6 This is a comparison chart of the mass retention rates of the composite materials of the present invention under ultrasonic conditions in polar solvents. Figure 7 The graph shows the isothermal adsorption and kinetic parameters of Ganoderma lucidum polysaccharides by different adsorbent materials of the present invention. Figure 8 This is a comparison chart of the specific adsorption and extraction purity of each material in the mixed interference system of the present invention; Figure 9 This is a comparison chart of the stability of various materials in the present invention under multiple adsorption and desorption cycles. Detailed Implementation

[0034] The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0035] Preparation Examples 1-6: Preparation Example 1: This preparation example provides a method for preparing amino-modified Fe3O4 (iron tetroxide) nanoparticles, including the following steps: One part of Fe3O4 nanoparticles with a particle size of 50-500nm was dispersed in a mixed solvent prepared by anhydrous ethanol and deionized water at a mass ratio of 3:1, with the amount of anhydrous ethanol used controlled at 15 parts. After ultrasonic dispersion for 30 min, sodium hydroxide aqueous solution was added to adjust the pH of the system to 8, and then 0.1 parts of silane coupling agent KH550 (3-aminopropyltriethoxysilane) was slowly added, wherein the mass ratio of Fe3O4, anhydrous ethanol and KH550 was 1:15:0.1.

[0036] The reaction was carried out at 50℃ and a stirring rate of 150 r / min for 6 h. After the reaction was completed, the solid product was separated by a magnet and washed with deionized water until neutral. Finally, it was dried at 50℃ for 6 h to obtain amino-modified Fe3O4 nanoparticles.

[0037] Preparation Example 2: This preparation example provides a method for preparing amino-modified Fe3O4 nanoparticles, including the following steps: 1.5 parts of Fe3O4 nanoparticles with a particle size of 50-500 nm were dispersed in a mixed solvent prepared by anhydrous ethanol and deionized water at a mass ratio of 3:1, with the amount of anhydrous ethanol used controlled at 36 parts. After ultrasonic dispersion for 45 min, sodium hydroxide aqueous solution was added to adjust the pH of the system to 9.5, and then 0.48 parts of KH550 were slowly added, wherein the mass ratio of Fe3O4, anhydrous ethanol and KH550 was 1:24:0.32.

[0038] The reaction was carried out at 70℃ and a stirring rate of 250 r / min for 6 h. After the reaction was completed, the solid product was separated by a magnet and washed with deionized water until neutral. Finally, it was dried at 60℃ for 12 h to obtain amino-modified Fe3O4 nanoparticles.

[0039] Preparation Example 3: This preparation example provides a method for preparing amino-modified Fe3O4 nanoparticles, including the following steps: One part of Fe3O4 nanoparticles with a particle size of 50-500nm was dispersed in a mixed solvent prepared by anhydrous ethanol and deionized water at a mass ratio of 3:1, and the amount of anhydrous ethanol used was controlled to be 60 parts. After ultrasonic dispersion for 60 minutes, sodium hydroxide aqueous solution was added to adjust the pH of the system to 10, and then 0.5 parts of KH550 were slowly added, wherein the mass ratio of Fe3O4, anhydrous ethanol and KH550 was 1:60:0.5.

[0040] The reaction was carried out at 80℃ and a stirring rate of 300 r / min for 12 h. After the reaction was completed, the solid product was separated by a magnet and washed with deionized water until neutral. Finally, it was dried at 80℃ for 12 h to obtain amino-modified Fe3O4 nanoparticles.

[0041] Preparation Example 4: This preparation example provides a method for preparing a Fe3O4 and MIL-101-NH2 composite support layer, including the following steps: One part of the amino-modified Fe3O4 obtained in Preparation Example 1 was dispersed in 150 parts of anhydrous DMF (i.e., N,N-dimethylformamide), and 10 parts of IPDI (i.e., isoflurane diisocyanate) were added. The mixture was reacted at 30°C and a stirring rate of 150 r / min for 6 h. Subsequently, 10 parts of MIL-101-NH2 (as an amino-metal-organic framework material) and 0.2 parts of stannous isooctanoate were added to the above system. The mass ratio of amino-modified Fe3O4, IPDI, DMF, MIL-101-NH2 and stannous isooctanoate was 1:10:150:10:0.2.

[0042] The reaction was carried out at 60℃ and a stirring rate of 150 r / min for 2 h. After the reaction was completed, the solid product was separated by a magnet and washed with deionized water until neutral. Finally, it was dried at 50℃ for 6 h to obtain the Fe3O4 and MIL-101-NH2 composite support layer.

[0043] Preparation Example 5: This preparation example provides a method for preparing a Fe3O4 and MIL-101-NH2 composite support layer, including the following steps: 1.2 parts of the amino-modified Fe3O4 obtained in Preparation Example 2 were dispersed in 360 parts of anhydrous DMF. After adding 24 parts of IPDI, the mixture was reacted at 40°C with a stirring rate of 200 r / min for 8 h. Subsequently, 24 parts of MIL-101-NH2 and 0.72 parts of stannous isooctanoate were added to the above system. The mass ratio of amino-modified Fe3O4, IPDI, DMF, MIL-101-NH2 and stannous isooctanoate was 1:20:300:20:0.6.

[0044] The reaction was carried out at 75℃ and a stirring rate of 200 r / min for 4 h. After the reaction was completed, the solid product was separated by a magnet and washed with deionized water until neutral. Finally, it was dried at 80℃ for 10 h to obtain the Fe3O4 and MIL-101-NH2 composite support layer.

[0045] Preparation Example 6: This preparation example provides a method for preparing a Fe3O4 and MIL-101-NH2 composite support layer, including the following steps: One part of the amino-modified Fe3O4 obtained in Preparation Example 3 was dispersed in 600 parts of anhydrous DMF. After adding 50 parts of IPDI, the mixture was reacted at 50°C and a stirring rate of 300 r / min for 10 h. Subsequently, 50 parts of MIL-101-NH2 and 0.8 parts of stannous isooctanoate were added to the above system. The mass ratio of amino-modified Fe3O4, IPDI, DMF, MIL-101-NH2 and stannous isooctanoate was 1:50:600:50:0.8.

[0046] The reaction was carried out at 90℃ and a stirring rate of 300 r / min for 6 h. After the reaction was completed, the solid product was separated by a magnet and washed with deionized water until neutral. Finally, it was dried at 80℃ for 12 h to obtain the Fe3O4 and MIL-101-NH2 composite support layer.

[0047] Examples 1-6: Example 1: This example provides a method for preparing magnetic molecularly imprinted materials for Ganoderma lucidum polysaccharide extraction, including the following steps: (1) Modification of ferrite nanoparticles: 1.5 parts of Fe3O4 nanoparticles with a particle size of 70-80 nm were dispersed in a mixed solvent composed of 36 parts of anhydrous ethanol and 12 parts of deionized water. After ultrasonic dispersion for 45 min, sodium hydroxide aqueous solution was added to adjust the pH of the system to 9.5. Then, 0.48 parts of silane coupling agent KH550 were slowly added. At this time, the mass ratio of ferrite, anhydrous ethanol and KH550 in the system was 1:24:0.32. The reaction was carried out at 70 °C and mechanical stirring rate of 250 r / min for 6 h. Afterward, the solid phase was separated by an external magnet and washed with deionized water until neutral. Finally, it was dried at 60 °C for 12 h to obtain amino-modified Fe3O4.

[0048] (2) Preparation of ferrite and MOF (i.e., amino metal-organic framework material) composite carrier: 1.2 parts of amino-modified Fe3O4 were weighed and dispersed in 360 parts of anhydrous DMF (i.e., N,N-dimethylformamide), and 24 parts of IPDI (i.e., isoflurane diisocyanate) were added. The mixture was then reacted at 40°C and a stirring rate of 200 r / min for 8 h.

[0049] Subsequently, 24 parts of MIL-101-NH2 and 0.72 parts of stannous isooctanoate were added to the above system. At this point, the mass ratio of amino-modified Fe3O4, IPDI, DMF, MIL-101-NH2 and stannous isooctanoate in the system was 1:20:300:20:0.6. The reaction was continued at 75°C and a stirring rate of 200 r / min for 4 h. After the reaction was completed, the solid phase was separated by a magnet and washed with deionized water until neutral. The solid phase was dried at 80°C for 10 h to obtain a composite support bonded to Fe3O4 and MIL-101-NH2.

[0050] (3) Preparation of molecularly imprinted polymer layer coating: 1.2 parts of composite carrier, 0.03 parts of Ganoderma lucidum polysaccharide template molecule, 0.03 parts of methacrylic acid monomer (as functional monomer), 0.12 parts of N,N'-methylenebisacrylamide crosslinking agent, 0.002 parts of potassium persulfate, 0.002 parts of sodium sulfite (potassium persulfate and sodium sulfite mixed as initiator) and 4.5 parts of deionized water were added to the polymerization reaction flask equipped with a degassing device in sequence. Dissolved oxygen in the system was completely removed by three repeated freezing, vacuuming and thawing cycles. Then, aqueous phase polymerization was initiated at a constant temperature of 60°C for 18 h. After polymerization, 15 parts of deionized water were added to the reaction system for dilution and quenching reaction. The solid product was separated by a magnet and dried at 60°C for 12 h to obtain a composite material coated with polymer precursor.

[0051] (4) Thermodynamic desorption of template molecules: One part of the composite material obtained in step (3) was uniformly dispersed in 10 parts of deionized water to prepare a dispersion with a solid content of 100 g / L. Then, it was eluted and stirred at 75°C and 250 r / min for 4 h. After the elution, it was separated by a magnet and washed with hot deionized water at 75°C until no template molecules remained in the filtrate. Finally, it was dried at 60°C for 12 h to obtain the finished magnetic molecular imprinted material.

[0052] The prepared magnetic molecularly imprinted materials are uniform in size, and the adsorption capacity of the magnetic molecularly imprinted materials changes over time as shown in the attached figure. Figure 1 As shown, the adsorption capacity of Ganoderma lucidum polysaccharides at room temperature and pressure is 115 mg / g; the magnetic responsiveness test diagram of the magnetic molecular imprinted material is attached. Figure 2 As shown, the magnetic separation time is 7 minutes, which is sufficient for effective separation; the VSM hysteresis loop diagram of the magnetic molecular imprinted material is attached. Figure 3 As shown in the figure, the hysteresis loop exhibits a typical S-shaped distribution and passes through the origin. There is no obvious coercivity or remanence, indicating that the prepared material possesses superparamagnetism and high saturation magnetization. This allows the material to achieve rapid solid-liquid separation when an external magnetic field is applied, and to quickly redisperse into the aqueous phase system after the magnetic field is removed. The material also shows good specificity recognition after being reused 8 times.

[0053] Example 2: This example provides a method for preparing magnetic molecularly imprinted materials for Ganoderma lucidum polysaccharide extraction, including the following steps: (1) Modification of ferrite nanoparticles: 1.5 parts of Fe3O4 nanoparticles with a particle size of 60-70 nm were dispersed in a mixed solvent consisting of 36 parts of anhydrous ethanol and 12 parts of deionized water. After ultrasonic dispersion for 60 min, sodium hydroxide aqueous solution was added to adjust the pH of the system to 10. Then, 0.57 parts of KH550 were slowly added. At this time, the mass ratio of ferrite, anhydrous ethanol and KH550 in the system was 1:24:0.38. The reaction was carried out at 80 °C and a stirring rate of 300 r / min for 4 h. Afterward, the solid phase was separated by a magnet and washed with deionized water until neutral. The solid phase was dried at 80 °C for 10 h to obtain amino-modified Fe3O4.

[0054] (2) Preparation of ferrite-MOF composite support: 1.2 parts of amino-modified Fe3O4 were dispersed in 300 parts of anhydrous DMF, and 30 parts of IPDI were added. The mixture was reacted at 50℃ and a stirring rate of 250 r / min for 6 h. Subsequently, 30 parts of UiO-67-NH2 (as an amino-metal-organic framework material) and 0.48 parts of stannous isooctanoate were added to the above system. The mass ratio of each component in the system was 1:25:250:25:0.4. The mixture was reacted at 90℃ and a stirring rate of 250 r / min for 2 h. After the reaction, the mixture was magnetically separated and washed until neutral. The mixture was then dried at 80℃ for 10 h to obtain the composite support bonded to Fe3O4 and UiO-67-NH2.

[0055] (3) Preparation of molecularly imprinted polymer layer coating: 1.2 parts of composite carrier, 0.03 parts of Ganoderma lucidum polysaccharide, 0.025 parts of acrylamide (as functional monomer), 0.12 parts of N,N'-methylenebisacrylamide (as crosslinking agent), 0.003 parts of azobisisobutyramidine hydrochloride (as initiator) and 4.3 parts of deionized water were added to the polymerization reaction flask. After three cycles of freezing, vacuuming and thawing to remove oxygen, polymerization was carried out at 80℃ for 12 h. After the reaction, the system was diluted with 18 parts of deionized water and magnetically separated. The composite material coated with polymer precursor was obtained by drying at 80℃ for 10 h.

[0056] (4) Thermodynamic desorption of template molecules: 1 part of the composite material obtained in step (3) was dispersed in 12.5 parts of deionized water to prepare a dispersion of 80 g / L. The mixture was stirred at 300 r / min at 90℃ for 2 h. After the mixture was stirred, it was magnetically separated and washed with 75℃ deionized water until the filtrate was free of template molecules. The mixture was dried at 80℃ for 10 h to obtain the magnetic molecular imprinted material.

[0057] Example 3: This example provides a method for preparing magnetic molecularly imprinted materials for Ganoderma lucidum polysaccharide extraction, including the following steps: (1) Modification of ferrite nanoparticles: The process parameters and reagent dosages are exactly the same as those in step (1) of Example 1, and amino-modified Fe3O4 is obtained.

[0058] (2) Preparation of ferrite and MOF composite support: The process parameters and reagent dosage are exactly the same as in step (2) of Example 1, and a composite support bonded to Fe3O4 and MIL-101-NH2 is obtained.

[0059] (3) Preparation of molecularly imprinted polymer layer coating: 1.5 parts of composite carrier, 0.05 parts of Ganoderma lucidum polysaccharide, 0.06 parts of 2-acrylamide-2-methylpropanesulfonic acid (as functional monomer), 0.18 parts of 1,3,5-triacryloylhexahydro-1,3,5-triazine (as crosslinking agent), 0.005 parts of azodicyanovalerate (as initiator) and 4.8 parts of deionized water were added to the polymerization bottle. After deoxygenation and circulation, polymerization was carried out at 65°C for 16 h. After the reaction, the mixture was diluted with 16 parts of deionized water and magnetically separated. The polymer precursor was dried at 80°C for 10 h.

[0060] (4) Thermodynamic desorption of template molecules: The process parameters and elution process are exactly the same as step (4) in Example 1, and finally magnetic molecular imprinted materials are obtained.

[0061] Example 4: This example provides a method for preparing magnetic molecularly imprinted materials for Ganoderma lucidum polysaccharide extraction, including the following steps: (1) Modification of ferrite nanoparticles: 1.5 parts of CoFe2O4 (cobalt ferrite) nanoparticles with a particle size of 80-90 nm were dispersed in a mixed solvent consisting of 36 parts of anhydrous ethanol and 12 parts of deionized water. After ultrasonic dispersion for 45 min, the pH was adjusted to 9, and 0.48 parts of KH550 were added. At this time, the mass ratio of the system was 1:24:0.32. The mixture was stirred at 250 r / min for 6 h at 70 °C, magnetically separated and washed until neutral, and dried at 60 °C for 14 h to obtain amino-modified CoFe2O4.

[0062] (2) Preparation of ferrite and MOF composite support: 1.2 parts of amino-modified CoFe2O4 were dispersed in 240 parts of anhydrous DMF, 12 parts of IPDI were added, and the mixture was stirred at 150 r / min for 10 h at 30 °C. Then, 12 parts of MIL-125-NH2 (as an amino metal-organic framework material) and 0.24 parts of stannous isooctanoate were added. The mass ratio of each component in the system was 1:10:200:10:0.2. The mixture was reacted at 150 r / min for 6 h at 60 °C. After magnetic separation and washing until neutral, the mixture was dried at 75 °C for 12 h to obtain the composite support of CoFe2O4 and MIL-125-NH2.

[0063] (3) Preparation of molecularly imprinted polymer layer coating: 1.2 parts of composite carrier, 0.03 parts of Ganoderma lucidum polysaccharide, 0.04 parts of acrylic acid (as functional monomer), 0.18 parts of N,N'-methylenebisacrylamide (as crosslinking agent), 0.004 parts of initiator composed of ammonium persulfate and sodium ascorbate and 4 parts of deionized water were added to the polymerization bottle. After deoxygenation, the mixture was polymerized at 70°C for 16 h. After the reaction, the system was diluted with 15 parts of water and magnetically separated. The mixture was dried at 60°C for 12 h to obtain the polymerization precursor.

[0064] (4) Thermodynamic desorption of template molecules: 1 part of the precursor obtained in step (3) was dispersed in 20 parts of deionized water to prepare a dispersion of 50 g / L. The mixture was stirred at 200 r / min for 6 h at 60 °C. Then it was washed with hot water at 80 °C until no template molecules remained. The mixture was dried at 60 °C for 12 h to obtain magnetic molecular imprinted material.

[0065] Example 5: This example provides a method for preparing magnetic molecularly imprinted materials for Ganoderma lucidum polysaccharide extraction, including the following steps: (1) Modification of ferrite nanoparticles: 1.5 parts of MnFe2O4 (manganese ferrite) nanoparticles with a particle size of 75-85 nm were dispersed in a mixed system of 36 parts of anhydrous ethanol and 12 parts of water. After ultrasonic dispersion for 45 min, the pH was adjusted to 9.2, and 0.45 parts of KH550 were added. The mass ratio of the system was 1:24:0.3. The mixture was stirred at 220 r / min for 5 h at 75 °C, washed, and dried at 60 °C for 14 h to obtain amino-modified MnFe2O4.

[0066] (2) Preparation of ferrite-MOF composite support: 1.2 parts of amino-modified MnFe2O4 were dispersed in 300 parts of anhydrous DMF, and 20 parts of IPDI were added. The mixture was stirred at 200 r / min for 9 h at 40 °C. Subsequently, 20 parts of CAU-1-NH2 (as an amino-metal-organic framework material) and 0.6 parts of stannous isooctanoate were added. The mass ratio of each component in the system was 1:16.7:250:16.7:0.5. The reaction was carried out at 70 °C for 5 h, and after washing, the mixture was dried at 85 °C for 10 h to obtain a composite support bonded to MnFe2O4 and CAU-1-NH2.

[0067] (3) Preparation of molecularly imprinted polymer layer coating: 1.0 part of composite carrier, 0.02 part of Ganoderma lucidum polysaccharide, 0.02 part of methacrylic acid (as functional monomer), 0.1 part of polyethylene glycol diacrylamide with a molecular weight of 200 (as crosslinking agent), 0.0024 part of azobisisobutyramidine hydrochloride (as initiator) and 2.4 part of deionized water were added to the polymerization bottle. After deoxygenation, the mixture was polymerized at 75°C for 22 h. 20 parts of water were added for dilution and separation. The mixture was dried at 60°C for 12 h to obtain the polymerization precursor.

[0068] (4) Thermodynamic desorption of template molecules: 1 part of the precursor obtained in step (3) was dispersed in 16.67 parts of deionized water to prepare a dispersion of 60 g / L. The dispersion was stirred at 200 r / min for 5 h at 70 °C. The mixture was washed with water at 80 °C until the filtrate was pure. The mixture was dried at 60 °C for 12 h to obtain the magnetic molecular imprinted material.

[0069] Example 6: This example provides a method for preparing magnetic molecularly imprinted materials for Ganoderma lucidum polysaccharide extraction, including the following steps: (1) Modification of ferrite nanoparticles: 1.5 parts of ZnFe2O4 (zinc ferrite) nanoparticles with a particle size of 85-90 nm were dispersed in a mixture of 36 parts of anhydrous ethanol and 12 parts of deionized water. After sonication, the pH was adjusted to 9.8, and 0.52 parts of KH550 were added. The mass ratio of the system was 1:24:0.35. The reaction was carried out at 280 r / min at 65 °C for 7 h. After washing, the mixture was dried at 55 °C for 18 h to obtain amino-modified ZnFe2O4.

[0070] (2) Preparation of ferrite and MOF composite support: 1.2 parts of amino-modified ZnFe2O4 were dispersed in 360 parts of anhydrous DMF, 30 parts of IPDI were added, and the reaction was carried out at 45℃ and 250 r / min for 7 h. Then, 30 parts of NH2-MIL-88B (as an amino metal-organic framework material) and 0.72 parts of stannous isooctanoate were added. The mass ratio of the system was 1:25:300:25:0.6. The reaction was carried out at 80℃ for 3 h. After washing, the mixture was dried at 80℃ for 10 h to obtain the composite support bonded to ZnFe2O4 and NH2-MIL-88B.

[0071] (3) Preparation of molecularly imprinted polymer layer coating: 1.2 parts of composite carrier, 0.03 parts of Ganoderma lucidum polysaccharide, 0.05 parts of 2-acrylamide-2-methylpropanesulfonic acid (as functional monomer), 0.15 parts of polyethylene glycol diacrylate with a molecular weight of 250 (as crosslinking agent), 0.004 parts of azodicyanovalerate (as initiator) and 4 parts of deionized water were added to the polymerization bottle. After deoxygenation, the mixture was polymerized at 65°C for 15 h, diluted and separated with 16 parts of water, and dried at 60°C for 12 h to obtain the polymerization precursor.

[0072] (4) Thermodynamic desorption of template molecules: 1 part of the precursor obtained in step (3) was dispersed in 6.67 parts of water to prepare a dispersion of 150 g / L. The mixture was stirred at 280 r / min for 4 h at 80 °C. After thorough washing with hot water at 80 °C, the mixture was dried at 60 °C for 12 h to obtain the magnetic molecular imprinted material.

[0073] Comparative Examples 1-2: Comparative Example 1: Compared with Example 1, the difference is that step (2) of coating preparation of MOF carrier layer is omitted, and amino-modified Fe3O4 is directly used for polymerization of molecular imprinted layer in step (3). The rest are the same.

[0074] Comparative Example 2: Compared with Example 1, the difference is that the IPDI connection modification step is not performed, and the Fe3O4 modified by KH550 is directly used for the coating of the MOF carrier layer. All other aspects are the same.

[0075] Test Examples 1-6: Test Example 1: Physical Adsorption Test of Specific Surface Area and Porosity (1) Extract 0.15 g of each of the amino-modified Fe3O4 obtained in step (1) of Example 1, the Fe3O4-MIL-101-NH2 composite carrier obtained in step (2), the final magnetic molecular imprinted material obtained in step (4), and the final product obtained in Comparative Example 1 and place them in glass sample tubes.

[0076] (2) Connect the sample tube to the degassing station of the fully automatic surface area and porosity analyzer, set the heating rate to 10℃ / min under vacuum conditions, raise the temperature to 120℃ and degas at a constant temperature for 10h to remove the moisture and air residues adsorbed on the surface and in the pores of the material.

[0077] (3) After degassing, the sample tube is transferred to the analysis station. At a liquid nitrogen temperature of 77K, high-purity nitrogen is used as the adsorbate for static volumetric testing. The relative pressure range is controlled between 0.01 and 0.99. The gas volume change during adsorption and desorption is recorded.

[0078] (4) After the experiment, the specific surface area of ​​each sample was calculated using the BET multi-point method model, the total pore volume and average pore size were calculated using the BJH model based on the desorption branch data, and the relevant physicochemical data were exported.

[0079] Table 1. Specific surface area and pore parameters of products at each stage in the examples and comparative examples

[0080] Conclusion: Based on Table 1 and Appendix Figure 4 The data shows that the specific surface area of ​​the amino-modified Fe3O4 in Example 1 is 42.15 m². 2 / g, pore volume is 0.112cm³ 3 The surface area was 1156.38 m² / g, with an average pore size of 15.42 nm, which is typical of inorganic nanoparticles. The measured porosity mostly originated from the gaps formed by particle packing. After preparing the composite support, the surface area reached 1156.38 m² / g. 2 / g, total pore volume increased to 0.845cm³. 3 / g, the average pore size decreased to 3.14nm. The MIL-101-NH2 material itself has micro-mesoporous structure characteristics. The changes in the above data indicate that the MOF material was grafted onto the ferrite surface, so that the composite carrier formed a porous framework structure.

[0081] In Example 1, the specific surface area of ​​the final magnetic molecularly imprinted material decreased to 345.67 m². 2 / g, total pore volume decreased to 0.382cm³. 3 / g, the average pore size increased to 6.51nm. The parameter decrease was caused by the polymerization reaction. The monomer and crosslinking agent reacted on the surface and inside the pores of the composite carrier. The generated molecularly imprinted polymer occupied part of the pore space. Since some smaller micropores were completely closed by the polymer, the testing instrument mainly detected the unfilled mesopores or macropores, which caused the calculated average pore size to increase.

[0082] According to Table 1 and Appendix Figure 4 The data shows that, due to the absence of MOF material in Comparative Example 1, the specific surface area of ​​the final product is 86.43 m². 2 / g, directly polymerized on the ferrite surface, the polymer can only be distributed on the outer surface of the particles. Due to the lack of support from the underlying porous structure, the overall specific surface area of ​​the material remains at a low level; Example 1 retained 345.67m². 2 The specific surface area of ​​ / g is much higher than that of Comparative Example 1, indicating that the MOF structure disperses the polymer on the surface. The expansion of the physical structure increases the actual contact area between the material and the liquid phase, allowing the imprinted sites to be dispersed in space. Combined with the adsorption requirements of macromolecules, the increased contact area is the physical basis for obtaining a high adsorption capacity when extracting Ganoderma lucidum polysaccharides in this example.

[0083] Test Example 2: Saturation Magnetization and Macroscopic Magnetic Deposition Test (1) Take 0.05 g of each of the unmodified Fe3O4 nanoparticles, the ferrite and MOF composite carrier of Example 1, the magnetic molecular imprinted material finally prepared in Example 1, and the magnetic molecular imprinted materials prepared in Examples 4 and 5, and pack them into the special sample box of the vibrating sample magnetometer (VSM) for compaction and fixation.

[0084] (2) At room temperature (298K), the external magnetic field strength of the vibrating sample magnetometer is set to -10000 Oe to +10000 Oe. The test program is started, the magnetization intensity change of each sample under the continuously changing magnetic field is recorded, the corresponding hysteresis loop data is obtained, and the saturation magnetization intensity (Ms) value of each sample is extracted.

[0085] (3) Weigh 0.1g of the products of each stage and the final materials of different embodiments, respectively, and add them to a glass bottle containing 10ml of deionized water. Disperse by ultrasonication for 15 minutes to prepare a uniform suspension with a concentration of 10g / L.

[0086] (4) Place the glass vial on the test platform and attach a neodymium iron boron square magnet with a surface magnetic field strength of 0.3T to its side wall. Start the stopwatch and observe the changes in the suspension. Record the time required from the start of directional particle movement in the suspension to the overall liquid phase returning to a transparent and clear state as the macroscopic magnetic separation time.

[0087] (5) After the separation process is completed, use a pipette to aspirate the supernatant 1 cm from the liquid surface, and use a UV-Vis spectrophotometer to measure the transmittance at a wavelength of 600 nm. Calculate the amount of suspended solids and the magnetic separation recovery rate of each material.

[0088] Table 2. Magnetic properties and separation test data of products at each stage and different core materials

[0089] Conclusion: Based on Table 2 and appendix Figure 5 The data shows that the saturation magnetization of unmodified Fe3O4 was 68.31 emu / g, and the liquid phase magnetic separation time was 12.4 seconds. During the preparation of the composite carrier in Example 1, the saturation magnetization decreased to 45.74 emu / g, and the corresponding magnetic separation time was extended to 28.1 seconds. The saturation magnetization of the final material in Example 1 further decreased to 32.46 emu / g, and the magnetic separation time was 45.8 seconds.

[0090] The stepwise change in parameters reflects the evolution of the material's mass composition. Saturation magnetization is the sum of magnetic moments per unit mass of material. After grafting non-magnetic KH550, IPDI, MIL-101-NH2, and polymer networks onto the surface of pure ferrite, the volume and total mass of the entire particle increase. Since the density of the outer coating layer is usually lower than that of inorganic metal oxides, the mass fraction of the inner magnetic core layer in the entire composite particle is diluted. When the instrument is tested, a fixed mass of sample is taken, in which the content of magnetic material actually participating in the magnetic field response has decreased compared to the pure core layer state, resulting in a decrease in the measured macroscopic saturation magnetization value.

[0091] The line graph reflects the physical relationship between separation time and magnetization intensity. Due to the weakening of magnetism per unit mass and the increase of the overall hydrodynamic radius of the particles, the magnetic attraction of the composite material in the liquid phase decreases while the fluid resistance increases, resulting in a longer directional sedimentation time. Comparing the data of Example 4 and Example 5, when CoFe2O4 or MnFe2O4 is used as the core layer, the final saturation magnetization intensity of the material is distributed between 28.92 and 35.18 emu / g. Combined with the line graph data on the Y-axis on the right, the magnetic separation time of the material does not exceed 60 seconds, and the separation recovery rate is maintained above 95%.

[0092] Experiments have shown that, within the component ratio defined in this invention, although the thick external non-magnetic MOF layer and imprinted polymer layer weaken the magnetic response index per unit mass, the residual magnetism retained by the composite material still meets the macroscopic sedimentation requirements under normal temperature and pressure. In the liquid phase extraction process, the separation of the solid phase adsorbent and the liquid phase extraction mother liquor can be achieved within 1 minute by applying a conventional magnetic field, which confirms that the rapid phase separation capability required for engineering applications is maintained while ensuring adsorption expansion.

[0093] Test Example 3: Quality Retention Rate Test under Harsh Environment (1) Accurately weigh 1.000 g of each of the dried magnetic molecular imprinted materials prepared in Examples 1, 2 and 3 and the final product of Comparative Example 2, and transfer them to polytetrafluoroethylene centrifuge tubes with a capacity of 50 mL.

[0094] (2) Add 30 mL of a mixed dispersion prepared by N,N-dimethylformamide and ultrapure water in a volume ratio of 1:1 to each centrifuge tube, and shake the solid in the tube to make it fully suspended.

[0095] (3) Fix the centrifuge tube in the water bath of the ultrasonic cleaner, set the water bath temperature to 25°C, turn on the ultrasonic generator with a power of 300W, and perform continuous mechanical cavitation oscillation treatment on the sample. The total treatment time is set to 120 minutes.

[0096] (4) When the ultrasonic treatment reaches the test nodes of 30 minutes, 60 minutes, 90 minutes and 120 minutes, temporarily turn off the ultrasonic equipment, take out the centrifuge tube and apply a static magnetic field of 0.3T to the outside of the tube wall for 5 minutes, so that the magnetic main composite material sinks to the tube wall.

[0097] (5) Use a pipette to transfer 20 mL of the upper suspension (containing non-magnetic MOF debris and polymer particles that have been physically destroyed) into an evaporating dish that has been dried to constant weight. Place the evaporating dish in a vacuum drying oven at 85°C to dry and evaporate the solvent, and weigh the dry weight of the remaining solid.

[0098] (6) The total mass of the detached material in the system is calculated by using the ratio of the sampling volume to the total solvent volume. The mass retention rate of the main body of the material under different ultrasonic time periods is calculated by subtracting the total mass of the detached material from the initial sample mass.

[0099] Table 3. Mass retention rate data of composite materials under strong polar solvents and ultrasonic cavitation treatment

[0100] Conclusion: Based on Table 3 and Appendix Figure 6It can be seen that the mass retention rate of the product of Comparative Example 2 decreased linearly with the extension of ultrasonic time. After 30 minutes of treatment, the retention rate was 85.34%, and when the treatment time reached 120 minutes, the value dropped to 61.27%. The loss of mass indicates that the non-magnetic MOF layer and the attached polymer network detached from the ferrite core surface and were suspended in the liquid phase. In the preparation process of Comparative Example 2, KH550 modified aminated ferrite was directly composited with MOF without the crosslinking process of IPDI. The two materials were mainly bonded by hydrogen bonds and van der Waals forces at the interface. Under the polar solvation effect of N,N-dimethylformamide, solvent molecules penetrated into the interface, and the mechanical shear force generated by ultrasonic waves overcame the weak intermolecular forces, resulting in the disintegration of the bonding structure between the composite layers.

[0101] Appendix Figure 6 The broken lines marked with three distinct colors—red, blue, and green—represent Example 1, Example 2, and Example 3, respectively. The three broken lines highly overlap in the high-order range of the graph, with only a slight decrease in numerical value. Table 3 and... Figure 6 Data shows that after 120 minutes of testing, the quality retention rates of the three embodiments were 96.88%, 96.15%, and 97.02%, respectively. The data proves that the structural stability of the material was substantially improved after the introduction of the IPDI process.

[0102] Test results verified that the embodiment utilizes the asymmetric reactivity of the isocyanate groups at both ends of the IPDI molecule to react the active secondary isocyanate groups with the amino groups on the ferrite surface at a lower temperature stage, and then, under higher temperature and catalysis, the remaining primary isocyanate groups are linked to the amino groups on the MOF surface. The stepwise polymerization method generates covalent urea bonds between the inorganic magnetic core layer and the organic framework layer. The bond energy of these covalent bonds is much higher than that of physical adsorption and hydrogen bonding, enabling them to resist the permeation and expansion of polar solvents and the hydrodynamic damage caused by ultrasonic cavitation. This improved structural stability ensures the service life of the magnetic molecularly imprinted material in actual industrial separation and high-temperature elution cycles.

[0103] Test Example 4: Comparison of Isothermal Adsorption Capacity and Adsorption Kinetics at Room Temperature and Pressure (1) Weigh out Ganoderma lucidum polysaccharide standard with a purity greater than 98%, and use deionized water to prepare a series of polysaccharide stock solutions with initial concentrations of 50 mg / L, 100 mg / L, 150 mg / L, 200 mg / L, 250 mg / L and 300 mg / L, and place them in glass containers for later use.

[0104] (2) Accurately weigh 20.0 mg of each of the magnetic molecular imprinted materials prepared in Examples 1, 3, 6 and Comparative Examples 1 and 2, and add them in parallel to each of the conical flasks containing 20 mL of the above-mentioned Ganoderma lucidum polysaccharide solutions of different concentrations.

[0105] (3) Place the conical flask in a constant temperature water bath shaker, set the water bath temperature to 25℃ and the shaking rate to 150r / min for isothermal adsorption experiments. For kinetic testing, select the experimental group with an initial concentration of 200mg / L. When the reaction proceeds to 5, 10, 20, 30, 45, 60, 90, 120 and 150 minutes, take 0.5mL of suspension from the system.

[0106] (4) Under the action of an external magnetic field, the suspension is separated into solid and liquid. The supernatant is taken and the absorbance in the solution is measured at a wavelength of 490 nm using a UV-Vis spectrophotometer using the phenol and sulfuric acid method. The concentration of the remaining Ganoderma lucidum polysaccharide in the liquid phase is calculated by using a standard curve.

[0107] (5) Calculate the material adsorption capacity data at each time point and when adsorption equilibrium is reached, extract the fitting rate constant of pseudo-second-order adsorption kinetics and the time required to reach adsorption equilibrium.

[0108] Table 4. Equilibrium adsorption capacity and kinetic parameters of Ganoderma lucidum polysaccharides by different adsorbent materials

[0109] Conclusion: Based on Table 4 and Appendix Figure 7 It can be seen that the bar charts of Examples 1, 3, and 6 are at a relatively high level, with the equilibrium adsorption capacity concentrated in the range of 105.81 mg / g to 114.26 mg / g; at the same time, the solid line representing the equilibrium time is at a low level (46.5 to 54.2 minutes), and the dashed line representing the adsorption rate constant is at a high level. The combination of data shows that the materials of the examples can not only accommodate more target molecules at room temperature and pressure, but also have a faster mass transfer and diffusion process. In contrast, the adsorption capacity bar chart of Comparative Example 1 is significantly reduced to 64.15 mg / g, the solid line representing the equilibrium time climbs to 128.4 minutes, and the rate constant drops to the lowest point.

[0110] The contrasting data above confirms the surface area expansion and mass transfer promotion effects of the MOF support layer. Comparative Example 1 omits the MOF material coating step, and the initiator directly initiates monomer polymerization on the surface of a conventional inorganic ferrite. Due to the lack of porous structure support on the inorganic solid surface, the generated imprinted polymer network is mostly in a dense two-dimensional spread or locally aggregated state, limiting the total spatial distribution of effective adsorption sites, resulting in lower bar chart data. At the same time, the large molecular weight of Ganoderma lucidum polysaccharide causes it to encounter high hydrodynamic and steric hindrance when penetrating into the dense polymer layer, leading to a prolonged apparent adsorption time.

[0111] In the preparation of this example, a porous framework material was introduced to construct a mesoscopic layer. The structure provides a physical interface for the aqueous polymerization system to extend into three-dimensional space. The polymerization reaction occurs based on the channels and outer surface of the MOF. The cured material not only increases the total number of imprinted cavities, but the mesopores and macropores retained inside also reduce the liquid phase diffusion resistance, enabling polysaccharide molecules to quickly reach specific recognition sites, which is manifested as an increase in adsorption capacity and a shortening of the time to reach equilibrium.

[0112] According to Table 4 and Appendix Figure 7 It can be seen that the adsorption capacity corresponding to the bar chart of Comparative Example 2 is 76.82 mg / g, which is slightly higher than that of Comparative Example 1, but lower than that of all examples. Comparative Example 2 lacks the IPDI covalent bonding step. Under the physical force of continuous isothermal oscillation, the MOF layer and its attached polymer network, which are bound by non-covalent bonds, are peeled off from the ferrite surface. The detached material cannot be deposited with the core during the subsequent magnetic separation sampling process and remains suspended in the liquid phase or even extracted, resulting in a loss of the effective adsorption capacity per unit mass. The test results conversely confirm that the stepwise construction of covalent urea bonds using the asymmetric groups of IPDI is a necessary step to ensure the stability of the material pores and the availability of adsorption sites.

[0113] Test Example 5: Comparison Test of Specificity Identification and Product Purity in a Mixed Interference System (1) Prepare a mixed competitive adsorption simulation solution. Use deionized water as solvent. Mix Ganoderma lucidum polysaccharide standard, glucose (monosaccharide), maltodextrin (oligosaccharide) and bovine serum albumin (protein) in a mass ratio of 1:1:1:1 to prepare a mixed solution with a total organic matter concentration of 400 mg / L, wherein the concentration of each individual component is 100 mg / L.

[0114] (2) Weigh 25.0 mg of each of the magnetic molecular imprinted materials prepared in Example 1, Example 2, Example 4 and Comparative Example 1 and Comparative Example 2 respectively, and put them into a reaction bottle containing 20 mL of mixed simulated liquid. Place the reaction bottle in a water bath constant temperature shaker at 25°C and shake continuously at a rate of 180 r / min for 2 hours to make the adsorption mass transfer process of the solid and liquid phases reach thermodynamic equilibrium.

[0115] (3) The reaction system is separated into solid and liquid under the action of an external magnetic field, and the supernatant is poured off. 15 mL of room temperature deionized water is added to quickly rinse the retained solid material, shake for 1 minute and then magnetically separate again. This step is repeated twice to remove impurities that are non-specifically attached to the solid surface by physical van der Waals forces.

[0116] (4) Add 20 mL of hot deionized water at 75 °C to the washed solid material and perform high-temperature elution stirring at 200 r / min for 3 hours to promote the specific binding target molecules to overcome intermolecular forces and desorb from the imprinted cavity. Collect the eluent after magnetic separation.

[0117] (5) Quantitative analysis of the components in the eluent was performed using high performance liquid chromatography (HPLC). A differential refractive index detector was set up to determine the concentration of polysaccharides and oligosaccharides, and an ultraviolet detector was set up to determine the protein concentration. The specific adsorption amount of Ganoderma lucidum polysaccharides, the total non-specific adsorption amount of the three interfering substances, and the purity percentage of Ganoderma lucidum polysaccharides in the final extract were calculated respectively.

[0118] Table 5. Adsorption capacity distribution and extraction purity data of each material in the mixed system

[0119] Conclusion: Based on Table 5 and Appendix Figure 8 Data shows that in a mixed competitive solution containing monosaccharides, oligosaccharides, and proteins, the adsorption capacity of Ganoderma lucidum polysaccharides in Examples 1, 2, and 4 ranged from 68.91 to 75.14 mg / g. The light gray bar chart in the attached figure shows that the total adsorption of interfering substances in Examples 1, 2, and 4 did not exceed 8 mg / g, and the corresponding product purity was all above 90%. In Comparative Example 1, the adsorption capacity of Ganoderma lucidum polysaccharides decreased to 35.42 mg / g, the adsorption capacity of interfering substances increased to 26.85 mg / g, and the product purity decreased to 56.88%.

[0120] The data differences between the examples and Comparative Example 1 correspond to the differences in the spatial structure of the materials. Comparative Example 1 did not introduce an MOF layer, and the molecularly imprinted polymer was directly coated on the outer surface of the ferrite. In the mixed solution, large molecular proteins such as bovine serum albumin can easily contact and adhere to the polymer surface. Physical adhesion occupies the surface area of ​​the material and covers part of the imprinted cavity, resulting in a decrease in the adsorption of the target polysaccharide and an increase in the extraction ratio of impurities.

[0121] Examples 1, 2, and 4 use MOF materials with three-dimensional channels as a support for polymer growth. The pore structure of the MOF material generates size exclusion for large molecular impurities in the mixed solution. Large protein molecules are limited by their molecular diameter and have difficulty entering the interior of the MOF channels, and are mainly isolated outside the material. For low molecular weight sugars that can enter the channels, they cannot form effective hydrogen bonds because their molecular spatial configuration does not match the imprinted cavity reserved during polymerization. The size exclusion and spatial imprint recognition mechanisms work together to limit the adsorption of impurity molecules by the material and improve the purity of Ganoderma lucidum polysaccharides in the final hot eluent.

[0122] According to Table 5 and Appendix Figure 8 Data shows that the adsorption capacity of Ganoderma lucidum polysaccharides in Comparative Example 2 was 47.63 mg / g, and the product purity was 75.71%, both lower than those in the Example. Comparative Example 2 omitted the IPDI crosslinking process during preparation, resulting in a lack of covalent bonds between the material layers. During the 2-hour isothermal oscillation adsorption and subsequent high-temperature water bath elution, some of the outer layer structure with imprinted cavities detached. The destruction of structural integrity led to a reduction in the specific recognition sites of the material as a whole, affecting the separation and extraction effect. This indicates that enhancing the interlayer bonding force through chemical bonds is a physical prerequisite for maintaining the selective adsorption performance of the material.

[0123] Test Example 6: Stability Test of Multi-Round Adsorption-Desorption Cycle Regeneration (1) Prepare a 200 mg / L Ganoderma lucidum polysaccharide aqueous solution as the test stock solution. Weigh 30.0 mg of each of the magnetic molecular imprinted materials prepared in Examples 1, 3, 5 and Comparative Examples 1 and 2, and put them into a reaction container containing 25 mL of test stock solution.

[0124] (2) Place the reaction vessel in a water bath shaker at 25°C, set the shaking rate to 150 r / min, and continue the reaction for 2 hours to reach adsorption equilibrium.

[0125] (3) After the reaction is completed, solid and liquid are separated under an external magnetic field of 0.3T. The supernatant is extracted and the concentration of the remaining Ganoderma lucidum polysaccharide in the solution is determined at a wavelength of 490nm using a UV-Vis spectrophotometer. The adsorption capacity of the first cycle is calculated.

[0126] (4) Add 20 mL of deionized water at 80 °C to the retained solid adsorbent material, stir at a constant temperature of 200 r / min for 2 hours to desorb and elute, perform magnetic separation after elution, discard the eluent, add 10 mL of anhydrous ethanol to the solid material to wash once, and then dry under vacuum at 60 °C.

[0127] (5) The dried recovered material was put back into a freshly prepared 200 mg / L Ganoderma lucidum polysaccharide aqueous solution, and the above adsorption, determination, elution and drying steps were repeated to complete a total of 10 complete test cycles. The adsorption capacity of each material in the 1st, 3rd, 5th, 8th and 10th cycles was recorded, and the capacity retention rate was calculated by the ratio of the adsorption amount in the 10th cycle to the adsorption amount in the 1st cycle.

[0128] Table 6. Adsorption capacity and retention rate of each material in multiple adsorption-desorption cycles.

[0129] Conclusion: Based on Table 6 and Appendix Figure 9The data shows that after 10 complete cycles of adsorption and 80°C hot elution, the adsorption performance of the materials in the examples and comparative examples showed varying degrees of decline. Figure 9 The dark gray and light gray bar charts on the left visually reflect the difference in absolute adsorption capacity between the initial and final states of each group of materials, while the corresponding black line on the right reflects the overall capacity retention rate.

[0130] According to Table 6 and Appendix Figure 9 The data shows that the adsorption capacities of Examples 1, 3, and 5 during the first cycle ranged from 106.15 mg / g to 113.84 mg / g. After 10 cycles, the light gray bar chart showed that the adsorption capacity remained above 96.47 mg / g. The retention rate lines representing these three examples in the attached figure were all in the high range above 90%. The adsorption capacity of Comparative Example 1 decreased from 63.87 mg / g to 40.25 mg / g in the 10th cycle, with a retention rate of 63.01%. Comparative Example 2 showed the most significant decline, with an initial adsorption capacity of 75.93 mg / g, which was reduced to only 22.84 mg / g after the 10th cycle, resulting in a capacity retention rate as low as 30.08%.

[0131] The difference in decay rate mentioned above verifies the necessity of the covalent cross-linking process between the MOF support layer and IPDI in this invention. The structure of Comparative Example 1 lacks the MOF support layer, and the molecularly imprinted polymer is directly covered on the surface of the inorganic ferrite. During multiple cycles, the material repeatedly undergoes room temperature aqueous phase expansion and high temperature elution shrinkage. Due to the lack of rigid framework, the single polymer network will undergo structural relaxation under the influence of thermodynamic deformation. The imprinted cavity originally customized for the molecular size of Ganoderma lucidum polysaccharide will undergo spatial misalignment or deformation. The change in the conformation of the specific recognition site leads to an irreversible decrease in the effective adsorption capacity.

[0132] In the preparation of this example, a polymer network is constructed within or on the surface of a rigid MOF crystal framework. The three-dimensional channels of the MOF provide a physical spatial confinement effect. During repeated temperature alternation and solvent replacement, the support of the bottom MOF restricts the large-scale displacement of the outer polymer molecular chains. The thermodynamic deformation of the polymer network is constrained within a limited space, maintaining the stability of the imprinted cavity size and functional group arrangement, thereby retaining a high target binding capacity after multiple cycles.

[0133] The retention rate of Comparative Example 2 plummeted to an extremely low level, revealing the defects in the interfacial bonding of the materials. Comparative Example 2 used physical methods to bond the MOF, polymer, and ferrite, but lacked the covalent urea bonds established by IPDI. Long-term mechanical oscillation, fluid shear force, and thermal stress exceeded the threshold of hydrogen bonding and van der Waals forces, causing the outer structure to peel off from the magnetic core over a large area. This part of the material with adsorption sites was lost with the waste liquid during the magnetic separation process, resulting in a substantial loss of the adsorption capacity of the remaining solid. The test results confirmed that covalent bonding is a key technical feature for maintaining the structural integrity of composite magnetic adsorbents in industrial-grade regeneration cycles.

Claims

1. A magnetic molecular imprinting material for extracting Ganoderma lucidum polysaccharides, characterized in that, The magnetic molecularly imprinted material includes a magnetic metal-organic framework composite carrier and a molecularly imprinted polymer layer coated on the surface of the composite carrier. The magnetic molecularly imprinted material is prepared by polymerizing raw materials comprising the following parts by weight in an aqueous phase and then eluting to remove the Ganoderma lucidum polysaccharide template molecules: Magnetic metal-organic framework composite carrier: 1.0–1.5 parts; Ganoderma lucidum polysaccharide template molecule: 0.02-0.05 parts; Functional monomer: 0.02–0.06 parts; Crosslinking agent: 0.1–0.18 parts; Initiator: 0.002–0.005 parts; Deionized water: 2.4–4.8 parts.

2. The magnetic molecular imprinted material according to claim 1, characterized in that, The magnetic metal-organic framework composite carrier is prepared by reacting components comprising the following parts by mass: Amino-modified ferrite nanoparticles: 1–1.2 parts; Isoflurone diisocyanate: 10-50 parts; N,N-Dimethylformamide: 150-600 parts; Amino-metal-organic framework materials: 10–50 parts; Stannous isooctanoate: 0.2–0.8 parts.

3. The magnetic molecular imprinted material according to claim 2, characterized in that, The ferrite core in the amino-modified ferrite nanoparticles is one of magnetite, cobalt ferrite, manganese ferrite or zinc ferrite nanoparticles. The amino-metal-organic framework material is one of MIL-101-NH2, UiO-67-NH2, MIL-125-NH2, CAU-1-NH2, or NH2-MIL-88B.

4. The magnetic molecular imprinted material according to claim 1, characterized in that, The functional monomer is one of methacrylic acid, acrylamide, 2-acrylamide-2-methylpropanesulfonic acid, or acrylic acid. The crosslinking agent is one of N,N'-methylenebisacrylamide, 1,3,5-triacryloylhexahydro-1,3,5-triazine, polyethylene glycol diacrylamide, or polyethylene glycol diacrylate. The initiator is one of the following: a mixture of potassium persulfate and sodium sulfite, azobisisobutyramidine hydrochloride, azobiscyanopentanoic acid, or a mixture of ammonium persulfate and sodium ascorbate.

5. A method for preparing a magnetic molecularly imprinted material for extracting Ganoderma lucidum polysaccharides, used to prepare the magnetic molecularly imprinted material as described in any one of claims 1-4, characterized in that, Includes the following steps: Ferrite nanoparticles were dispersed in a mixed solvent of anhydrous ethanol and deionized water. Sodium hydroxide aqueous solution was added to adjust the pH of the system to alkaline. Then, 3-aminopropyltriethoxysilane was slowly added and stirred for reaction. After the reaction was completed, the solid product was separated, washed and dried to obtain amino-modified ferrite. The obtained amino-modified ferrite was dispersed in anhydrous N,N-dimethylformamide, and isoflurane diisocyanate was added for the first stage reaction. Then, amino metal-organic framework material and stannous isooctanoate were added for the second stage reaction. After the reaction was completed, the solid phase was separated, washed and dried to obtain a magnetic metal-organic framework composite carrier. The obtained magnetic metal-organic framework composite carrier, Ganoderma lucidum polysaccharide template molecule, functional monomer, crosslinking agent, initiator and deionized water were sequentially added to a polymerization reaction flask equipped with a degassing device to form a polymerization reaction system. After deoxygenation treatment, aqueous phase polymerization was initiated under constant temperature conditions. After polymerization was completed, the reaction was diluted and quenched, and the solid phase was separated and dried to obtain a composite material coated with polymer precursor. The obtained composite material coated with polymer precursor was dispersed in deionized water to prepare a dispersion. High-temperature elution was carried out under heating and stirring conditions. After elution, magnetic separation was performed and the material was continuously washed with hot deionized water until no template molecules remained. After drying, the finished magnetic molecular imprinted material was obtained.

6. The preparation method according to claim 5, characterized in that, When preparing amino-modified ferrite, the ferrite nanoparticles have a particle size of 50-500 nm and are subjected to ultrasonic dispersion treatment before use for 30-60 min.

7. The preparation method according to claim 5, characterized in that, In preparing amino-modified ferrite, the mass ratio of the ferrite, anhydrous ethanol, and 3-aminopropyltriethoxysilane is 1:(15-60):(0.1-0.5). The pH range of the system should be adjusted to 8–10; The reaction temperature after adding 3-aminopropyltriethoxysilane is 50–80 °C, and the reaction time is 4–12 h.

8. The preparation method according to claim 5, characterized in that, In the preparation of magnetic metal-organic framework composite supports, the temperature of the first stage reaction after the addition of isoflurone diisocyanate is 30-50℃ and the time is 6-10h. The temperature of the second stage reaction after adding the amino-metal-organic framework material and stannous isooctanoate is 60–90 °C, and the time is 2–6 h.

9. The preparation method according to claim 5, characterized in that, In preparing composite materials coated with polymer precursors, the specific implementation method of the deoxygenation treatment is as follows: Dissolved oxygen in the polymerization reaction system is removed by repeating the freezing, vacuuming, and thawing cycle three times. Each cycle includes the sequential steps of freezing, vacuuming, and thawing. The aqueous phase polymerization is carried out at a temperature of 60–80°C for a time of 12–22 hours.

10. The preparation method according to claim 5, characterized in that, When performing high-temperature elution, the solid content of the dispersion is configured to be 50-150 g / L; The high-temperature elution temperature is 60–90°C, and the stirring time is 2–6 hours.

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