Preparation method of porous frame type hollow microspheres based on microemulsion polymerization and enzyme catalytic application

Abstract

The application discloses a preparation method and enzyme catalysis application of porous framework hollow microspheres based on a microemulsion polymerization method, wherein the method is characterized in that the interaction of an oil-water interface in a microemulsion is regulated, a covalent organic framework (COFs) material is assembled in situ at the droplet interface, and therefore, the COFs nanoscale hollow microspheres with uniform morphology are obtained, and the method has good universality. The COFs nanoparticles prepared by the method can be used as biological carriers to efficiently load biological enzymes, and on the basis of retaining the original activity of the enzymes, the conformation and activity of the enzymes are ensured to be not affected by the outside world, the tolerance of the enzymes to bad environments is improved, the recycling stability of the biological enzymes is enhanced, and the method has great application potential in the field of immobilized enzyme catalysis.

Description

Technical Field

[0001] The present invention belongs to the technical field of nanomaterial preparation, and particularly relates to a preparation method of porous framework hollow microspheres based on a microemulsion polymerization method and enzyme catalysis application. Background Art

[0002] Due to their multifunctionality, topological structure, and connectivity diversity, COFs have become a powerful platform for structural design and customization of organic functional materials. In recent decades, COFs have attracted great interest in many fields, such as gas storage and separation, sensors, batteries, catalysis, etc.

[0003] Many properties of COFs are related to their internal chemical connections, porous structures, and nanoscale morphology. The traditional solvothermal method for synthesizing COFs requires high temperatures, vacuum operations, and long reaction times, which limits the morphology control and application scale of COFs. The hard template method and the template-free method are currently the most reported methods for controlling and regulating the morphology of COFs. However, the template-free method has high requirements for monomer properties and reaction conditions, which undoubtedly increases the difficulty of synthesizing highly crystalline COFs. The hard template method requires the selection of suitable template materials and ensures that the template can be removed without destroying the morphology of COFs. Relatively speaking, the soft template method is simple to operate, can effectively regulate the morphology of COFs, and can be easily removed by washing.

[0004] Typically, emulsion polymerization using surfactants as soft templates can control polymer morphology under mild conditions. However, this method requires that the structure-directed polymer must provide the driving force for micelle formation and that the precursors can cross-link to form a framework for the pore structure. The cross-linked material should have higher thermal stability than the soft template to facilitate the preservation of the original pore structure during template removal. Therefore, the following key issues need to be addressed: how to obtain COF nanocrystals through in situ polymerization in microemulsions using the soft template method while ensuring their good dispersion and morphological stability; and at the same time, explore the universality of this method, the optimal reaction conditions for the prepared materials, and their application value. Summary of the Invention

[0005] To address the deficiencies of the prior art, the present invention provides a method for preparing porous framework-based hollow microspheres using microemulsion polymerization and its application in enzyme catalysis. This method is simple and universal, resulting in nano-microspheres with controllable size, high crystallinity, and uniform morphology. Furthermore, bioenzymes can be in situ loaded into the hollow internal structure of COFs microspheres through embedding, imparting multifunctionality.

[0006] In order to achieve the above object, the technical solution of the present invention is:

[0007] A method for preparing porous framework-type hollow microspheres based on a microemulsion polymerization method comprises the following steps: using a surfactant as a soft template for COFs microemulsion polymerization; adding a nonionic lipophilic surfactant and an oil-soluble aldehyde or amine monomer to an organic solvent to prepare an organic phase; using an aqueous solution containing a water-soluble amine or aldehyde monomer and a hydrophilic surfactant as the aqueous phase; dripping the aqueous phase into the stirring organic phase to fully mix the oil and water phases to form an oil-in-water microemulsion; polymerizing under the action of an acid catalyst; precipitating with ethanol after the reaction; and generating a solid through an amine-aldehyde condensation reaction. The COFs-type hollow microspheres are obtained through post-treatment.

[0008] Furthermore, the input ratio of the lipophilic nonionic surfactant to the organic phase is 0.5 to 5 mg mL -1 The input ratio of hydrophilic surfactant to water phase is 0.5-4.5 mg·mL -1 .

[0009] Furthermore, the volume ratio of the organic solvent to water is 15:0.5-8; the organic solvent is dichloromethane, toluene, xylene, n-hexane or cyclohexane.

[0010] Furthermore, the selected nonionic lipophilic surfactant is span85, span80, diglycerol dioleate or polyvinyl pyrrolidone, and the hydrophilic surfactant is hexadecylpyridinium bromide, polyethylene glycol or propylene glycol.

[0011] Furthermore, the aldehyde monomer is 1,3,5-triformylphloroglucinol, 2,5-dimethoxyterephthalaldehyde or 1,4-terephthalaldehyde, and the amine monomer is 1,4-p-phenylenediamine, ethidium bromide, melamine or 1,3,5-tris(4-aminophenyl)benzene.

[0012] Furthermore, the acid catalyst used is p-toluenesulfonic acid, benzoic acid or acetic acid.

[0013] Furthermore, the reaction time of the amine-aldehyde condensation reaction is 15-360 min, and the reaction temperature is 0-60°C.

[0014] Furthermore, the post-treatment process is as follows: after filtration, extraction with methanol and tetrahydrofuran for 12 hours respectively, and vacuum drying at 100° C. for 6 hours.

[0015] An application of porous framework hollow microspheres prepared by the above preparation method, using COFs hollow microspheres synthesized by microemulsion polymerization as immobilized enzyme carriers, adding a small amount of biological enzyme in advance to the aqueous phase containing water-soluble monomers, and immobilizing the biological enzyme in the cavity of the COFs hollow microspheres by in situ encapsulation to obtain a biological enzyme@COFs composite catalyst, which is then used for catalytic reactions.

[0016] Furthermore, the biological enzyme used is porcine pancreatic type II lipase PPL or Antarctic Candida lipase CALB.

[0017] The beneficial effects of the present invention are:

[0018] The present invention utilizes a microemulsion polymerization method to perform in-situ assembly of COFs monomers to produce nano-sized hollow-like microspheres. The method is simple to operate, requires mild conditions, and has good universality. The prepared COFs material has high crystallinity, uniform size, and stable morphology. As an application, the COFs nanoparticles prepared by the method of the present invention can be used to in-situ load biological enzymes inside the hollow-like microspheres, achieving spatial separation of the biological enzymes and acid catalysts. On the one hand, the activity of the enzymes can be effectively retained, with an initial activity close to that of the free enzymes. The stable and diverse COFs shell can support the conformation of the enzymes from the influence of the external environment. The high porosity of the COFs can ensure mass transfer in the system, allowing the enzymes to fully contact the reaction substrates. The COFs have great application potential in the field of immobilized enzyme catalysis. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] The accompanying drawings, which constitute a part of the present invention, are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute improper limitations on the present invention.

[0020] Figure 1 The SEM, TEM, and PXRD images of the COFs hollow nanospheres prepared in Example 1 of the present invention are shown;

[0021] Figure 2 The SEM, TEM, and PXRD images of the COFs hollow nanospheres prepared in Example 2 of the present invention are shown;

[0022] Figure 3 This is an SEM image of the COFs hollow nanospheres prepared in Example 3 of the present invention;

[0023] Figure 4 This is an SEM image of the COFs hollow nanospheres prepared in Example 4 of the present invention;

[0024] Figure 5 The SEM, TEM, and PXRD images of the COFs hollow nanospheres prepared in Example 5 of the present invention are shown;

[0025] Figure 6 The SEM and PXRD images of the COFs hollow nanospheres prepared in Example 6 of the present invention are shown;

[0026] Figure 7 These are the SEM, TEM, PXRD, and CLSM images of the COFs hollow nanospheres prepared in Example 7 of the present invention;

[0027] Figure 8 Schematic diagram of the catalytic process of PPL@COFs prepared in Example 7 of the present invention in a fixed bed;

[0028] Figure 9 Real-time catalytic performance monitoring of PPL@COFs prepared in Example 7 of the present invention in a fixed bed;

[0029] Figure 10 The cyclic stability of biodiesel produced by CALB@COFs and free enzyme catalysis prepared in Example 7 of the present invention;

[0030] Figure 11 The environmental stability of biodiesel produced by catalysis of CALB@COFs prepared in Example 7 of the present invention;

[0031] Figure 12 Gas chromatography for the production of biodiesel catalyzed by free CALB lipase;

[0032] Figure 13 Gas chromatography of the catalytic production of biodiesel by CALB@COFs prepared in Example 7 of the present invention. DETAILED DESCRIPTION

[0033] It should be noted that the following detailed descriptions are exemplary and intended to provide further explanation of the present invention. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which the present invention belongs.

[0034] It should be noted that the terms used herein are only for describing specific embodiments and are not intended to limit the exemplary embodiments according to the present invention. As used herein, unless the context clearly indicates otherwise, the singular form is intended to include the plural form. In addition, it should be understood that when the terms "comprise" and / or "include" are used in this specification, they indicate the presence of features, steps, operations, devices, components and / or combinations thereof.

[0035] The microemulsion described in the present invention refers to an emulsion with a diameter of dispersed droplets between 5nm and 1000nm.

[0036] In view of the shortcomings of the existing covalent organic framework preparation, such as unsatisfactory size and crystallinity and complicated preparation process, in order to solve the above technical problems, the present invention proposes a preparation method of COFs-type hollow microspheres based on microemulsion polymerization and enzyme catalysis application.

[0037] The first typical embodiment of the present invention (Examples 1, 2, 3, 4, and 5) provides a method for preparing porous framework-type hollow microspheres based on microemulsion polymerization, using COFs monomers as synthetic raw materials, surfactants as templates, acids as efficient catalysts, and microemulsions as reaction systems. Nanoscale COFs-type hollow microspheres are prepared by in situ polymerization on the droplet surface, and the structure has high crystallinity and porosity.

[0038] In one or more examples of this embodiment, the microemulsion is a water-in-oil microemulsion system, which comprises: adding a nonionic lipophilic surfactant and an oil-soluble aldehyde or amine monomer to an organic solvent to form an organic phase; using an aqueous solution containing a water-soluble aldehyde or amine monomer and a hydrophilic surfactant as the aqueous phase; uniformly dripping the aqueous phase into the stirring oil phase to fully mix the oil and water phases to form a water-in-oil microemulsion; polymerizing under the action of an acid catalyst; and precipitating the solid formed by the amine-aldehyde condensation reaction with ethanol after the reaction. The organic phase is an organic substance that is immiscible with water, and the substance dissolved in the aqueous phase has poor solubility in the organic phase; when the aldehyde is an organic compound containing at least two aldehyde groups, the amine is an organic compound containing at least three primary amine groups; when the aldehyde is an organic compound containing at least three aldehyde groups, the amine is an organic compound containing at least two primary amine groups.

[0039] In this series of embodiments, the surfactant is selected to ensure the stability of the microemulsion and the rapid progress of the amine-aldehyde condensation reaction, wherein the hydrophilicity and lipophilicity of the surfactant are divided according to the hydrophilic-lipophilic balance value.

[0040] In this series of embodiments, the input ratio of the lipophilic nonionic surfactant to the organic phase is 0.5 to 5 mg·mL -1 The input ratio of hydrophilic surfactant to water phase is 0.5-4.5 mg·mL -1 .

[0041] In this series of embodiments, the volume ratio of the organic solvent to water is 15:0.5-8, and the organic solvent is dichloromethane.

[0042] In this series of embodiments, the molar ratio of aldehyde to amine is 3:2 or 2:3.

[0043] In this series of embodiments, the aldehyde is one of 1,4-terephthalaldehyde, 1,3,5-triformylphloroglucinol 2,5-dihydroxyterephthalaldehyde, and 2,5-dimethoxyterephthalaldehyde.

[0044] In this series of embodiments, the amine is one of 1,3,5-tris(4-aminophenyl)benzene, 1,4-p-phenylenediamine, and melamine.

[0045] In this series of embodiments, the hydrophilic surfactant used is cetylpyridinium bromide (CPB), and the lipophilic nonionic surfactant is span80.

[0046] In this embodiment, the acid catalyst is one of p-toluenesulfonic acid or benzoic acid.

[0047] In this series of embodiments, the aldehyde-amine condensation reaction time is 15 to 120 minutes, and the temperature is 25°C.

[0048] In this series of examples, the reaction material was filtered, extracted, and dried to obtain purer covalent organic framework microspheres. The specific extraction method was as follows: after the reaction, the solid was precipitated with ethanol, filtered, and extracted with methanol and tetrahydrofuran for 12 hours respectively. The drying conditions were: vacuum drying at 100°C for 6 hours.

[0049] The second embodiment of the present invention (Example 6) provides a method for preparing COFs materials in a homogeneous manner based on a one-pot process, which better explains the effect of microemulsion polymerization on the morphology and size of COFs in the first embodiment.

[0050] This embodiment is to ultrasonically disperse COFs monomers in a single reaction solvent for polymerization, and polymerization occurs under the action of an acid catalyst. After the amine-aldehyde condensation reaction is completed, the generated solid is filtered, washed, and dried to obtain a COFs material that is comparable to the first embodiment.

[0051] In this embodiment, the reaction solvent can be one of acetonitrile, dichloromethane, ethyl acetate, mesitylene, dioxane, and o-dichlorobenzene, or a mixture thereof.

[0052] In this embodiment, the acid catalyst is p-toluenesulfonic acid.

[0053] In this embodiment, the reaction temperature is 25° C. and the reaction time is 15 minutes.

[0054] The third embodiment of the present invention (Example 7) utilizes the COFs nanoscale hollow microspheres synthesized in the first embodiment as immobilized enzyme supports to prepare a composite biocatalyst. During the preparation of these nanoscale COFs hollow microspheres using a microemulsion polymerization method, a small amount of enzyme was added to a phosphate buffered saline solution containing water-soluble COFs monomers. The ratio of nonionic surfactant to organic phase was then adjusted to reduce the effect of the organic solvent on enzyme activity during the reaction. The enzyme was then immobilized within the pores of the COFs microspheres through in situ encapsulation, resulting in an enzyme@COFs composite catalyst.

[0055] This method is similar to the first. A lipophilic nonionic surfactant and an oil-soluble monomer are added to an organic solvent to form an oil phase. The water-soluble monomer and hydrophilic surfactant are dissolved in a phosphate-buffered saline solution containing a small amount of porcine pancreatic type II lipase (PPL) as the aqueous phase. The aqueous phase is then dripped into the stirring oil phase at a uniform rate to thoroughly mix the two phases, forming a water-in-oil microemulsion. An acid catalyst is then added. During the polymerization process, the lipase is in situ incorporated into the cavities of the COFs microspheres. After the reaction, the resulting solid is precipitated with ethanol, filtered, and freeze-dried to obtain the composite biocatalyst PPL@COFs, which is then stored at 4°C. The porcine pancreatic type II lipase (PPL) was replaced with the Candida antarctica lipase (CALB), and CALB@COFs were prepared using the same method. The bioactivity of the immobilized enzyme can be determined by the hydrolysis of tributyrin.

[0056] In this embodiment, the lipophilic nonionic surfactant used is span80, and the input ratio to the organic phase is 4.5 mg·mL -1 .

[0057] In this embodiment, the hydrophilic nonionic surfactant used is cetylpyridinium bromide (CPB), and the input ratio to the aqueous phase is 3 mg·mL -1 .

[0058] In this embodiment, the volume ratio of the organic solvent to water is 15:5.5.

[0059] In this embodiment, the amine is 1,4-p-phenylenediamine, and the aldehyde is 1,3,5-triformylphloroglucinol.

[0060] In this embodiment, the water phase drop rate is controlled to be 0.5 mL min -1 .

[0061] In this embodiment, the acid catalyst is benzoic acid.

[0062] In this embodiment, the reaction temperature is 25° C. and the reaction time is 30 min.

[0063] In this embodiment, for the determination of the hydrolysis activity of the immobilized enzyme, the specific method is to add 1.0 mL of tributyrin to 50 mL of phosphate buffer, add a quantitative amount of immobilized enzyme, vigorously stir at 40 ° C for 15 minutes, terminate the reaction, add phenolphthalein dropwise and titrate with NaOH solution to the reaction end point, and record the volume of NaOH solution consumed. The blank experiment is carried out under the same conditions without adding lipase. All experiments are performed three times. One unit of lipase activity (U) is defined as the amount of lipase required to hydrolyze tributyrin to produce 1.0 μmol of butyric acid per minute under the assay conditions.

[0064] In this embodiment, the prepared composite biocatalyst PPL@COFs was applied to the general procedure of continuous flow cyclic addition. The specific method was as follows: PPL@COFs was filled in a packed bed reactor (with an inner diameter of 8 mm), 300-mesh sieves (volume 4 mL) were installed at both ends, and a reaction solution of 4-nitrobenzaldehyde and indole was prepared using 1,4-dioxane and water (1:4 in v / v) as solvents. The reaction mixture was stirred at 0.4 mL min -1 The effluent was collected regularly and analyzed by HPLC.

[0065] In this embodiment, the prepared composite biocatalyst CALB@COFs is used in the preparation of biodiesel. The specific method is to add soybean oil, anhydrous methanol, water content and a certain amount of immobilized enzyme CALB@COFs into a screw-capped glass bottle, react at 40°C for 24 hours, centrifuge and take the supernatant, and analyze the sample by gas chromatography.

[0066] In order to enable those skilled in the art to more clearly understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below with reference to specific embodiments.

[0067] Example 1:

[0068] 15mL of 1mg·mL -1 The dichloromethane solution of span 80 was added to a 50 mL round-bottom flask and stirred at 650 rpm. 1,3,5-triformylphloroglucinol (15.7 mg, 0.075 mmol) was added. 10 mg of hexadecylpyridinium bromide (CPB), 1,4-p-phenylenediamine (12 mg, 0.112 mmol) and 30 mg of p-toluenesulfonic acid (PTSA) were dissolved in 3.5 mL of deionized water by ultrasonication at 0.5 mL min. -1 The mixture was added dropwise to the dichloromethane solution at a rate of 1:1. The mixture was stirred at room temperature for 15 minutes. After the reaction was completed, 15 mL of ethanol was used to precipitate the solid. The solid orange powder was filtered and extracted with methanol and tetrahydrofuran for 12 hours respectively. The solid was dried under vacuum at 100 ° C for 6 hours. The obtained material was further characterized by SEM, TEM and PXRD. The results are shown in Figure 2. Figure 1 As shown (a) in the figure is a SEM image, b) is a TEM image, and c) is a PXRD image), the COFs material presents a regular spherical morphology, uniform size, an average diameter of about 300 nm, a hollow structure, and good crystallinity. Therefore, this experimental scheme was selected as the basic condition for the subsequent Example 6.

[0069] Example 2:

[0070] 15mL of 1mg·mL -1A dichloromethane solution of span 80 was added to a 50 mL round-bottom flask and stirred at 650 rpm. 1,4-terephthalaldehyde (15.1 mg, 0.112 mmol) was added. 10 mg of hexadecylpyridinium bromide (CPB), 1,3,5-tris(4-aminophenyl)benzene (26.4 mg, 0.075 mmol) and 30 mg of p-toluenesulfonic acid (PTSA) were dissolved in 3.5 mL of deionized water by ultrasonication at 0.5 mL min. -1 The mixture was added dropwise to the dichloromethane solution at a rate of 1:1. The mixture was stirred at room temperature for 15 minutes. After the reaction was completed, 15 mL of ethanol was used to precipitate the solid. The solid brown powder was filtered and extracted with methanol and tetrahydrofuran for 12 hours respectively. The solid was dried under vacuum at 100 ° C for 6 hours. The obtained material was further characterized by SEM, TEM and PXRD. The results are shown in Figure 2. Figure 2 As shown (a) is the SEM image, b) is the TEM image, and c) is the PXRD image), the COFs material has a smooth surface and regular structure, a spherical particle size of about 700 nm, and an obvious cavity structure can be observed under a transmission electron microscope, with good crystallinity.

[0071] Example 3:

[0072] 15mL of 1mg·mL -1 The dichloromethane solution of span 80 was added to a 50 mL round-bottom flask and stirred at 650 rpm. 2,5-dimethoxyterephthalaldehyde (21.7 mg, 0.112 mmol) and 30 mg of benzoic acid were added. 10 mg of hexadecylpyridinium bromide (CPB) and melamine (9.5 mg, 0.075 mmol) were ultrasonically dissolved in 3.5 mL of deionized water and stirred at 0.5 mL min. -1 The mixture was added dropwise to the dichloromethane solution at a rate of 1:1. The mixture was stirred at room temperature for 15 minutes. After the reaction was completed, 15 mL of ethanol was used to precipitate the solid. The solid red powder was filtered and extracted with methanol and tetrahydrofuran for 12 hours respectively. The solid powder was vacuum dried at 100 ° C for 6 hours. The obtained material was further characterized by SEM. The results are shown in FIG. Figure 3 As shown, the particle size of the sphere is about 400 nm, and an obvious cavity structure can be observed under a scanning electron microscope.

[0073] Example 4:

[0074] According to the operation of Example 1, 15 mL of 1 mg·mL -1A dichloromethane solution of span 80 was added to a 50 mL round-bottom flask, and 1,3,5-triformylphloroglucinol (15.7 mg, 0.075 mmol) was added at different stirring speeds. 10 mg of hexadecylpyridinium bromide (CPB), 1,4-p-phenylenediamine (12 mg, 0.112 mmol), and 30 mg of p-toluenesulfonic acid (PTSA) were dissolved in different amounts of deionized water by ultrasonication at 0.5 mL min. -1 The solution was added dropwise to the dichloromethane solution at a rate of 1:1. After stirring at room temperature for different times, the solid was precipitated with 15 mL of ethanol after the reaction was completed, and extracted with methanol and tetrahydrofuran for 12 hours respectively, and dried under vacuum at 100 ° C for 6 hours. The specific operating conditions are shown in Table 1. The results are shown in Table 1. Figure 4 As shown in the figure, by regulating the reaction conditions, COFs-like hollow microspheres with different morphologies and sizes can be prepared.

[0075] Table 1 Summary of different operating conditions of Examples 1 and 4

[0076] Stirring speed (rpm) Deionized water dosage (mL) Reaction time (min) Example 1 650 3.5 15 Example 4-1 1500 3.5 15 Example 4-2 650 8 15 Example 4-3 650 3.5 120

[0077] Example 5:

[0078] 15mL of 1mg·mL -1 A dichloromethane solution of span 80 was added to a 50 mL round-bottom flask and stirred at 650 rpm. 2,5-dihydroxyterephthalaldehyde (18.7 mg, 0.112 mmol) was added. 10 mg of hexadecylpyridinium bromide (CPB), 1,3,5-tris(4-aminophenyl)benzene (26.4 mg, 0.075 mmol) and 30 mg of p-toluenesulfonic acid (PTSA) were dissolved in 3.5 mL of deionized water by ultrasonication at 0.5 mL min. -1 The mixture was added dropwise to the dichloromethane solution at a rate of 1:1. The mixture was stirred at room temperature for 15 minutes. After the reaction was completed, 15 mL of ethanol was used to precipitate the solid. The solid reddish-brown powder was filtered and extracted with methanol and tetrahydrofuran for 12 hours respectively. The solid was dried under vacuum at 100 ° C for 6 hours. The obtained material was further characterized by SEM, TEM and PXRD. The results are shown in Figure 2. Figure 5 As shown (a) is the SEM image, b) is the TEM image, and c) is the PXRD image), the COFs microspheres have a diameter of about 400 nm, are uniform in size, have groove-like protrusions on the surface, and have good crystallinity. However, transmission electron microscopy observed that the interior is a solid structure. Therefore, the carrier material required for subsequent in situ encapsulation of bioenzyme experiments cannot be prepared using amphiphilic 2,5-dihydroxyterephthalaldehyde as a monomer and 1,3,5-tris(4-aminophenyl)benzene as a monomer.

[0079] Example 6:

[0080] 1,3,5-triformylphloroglucinol (15.7 mg, 0.075 mmol) and 1,4-p-phenylenediamine (12 mg, 0.112 mmol) were ultrasonically dispersed in 15 mL of dichloromethane, 30 mg of p-toluenesulfonic acid catalyst was added, and after stirring at 650 rpm for 15 min at room temperature, an orange-red solid powder was obtained by filtration. The powder was extracted with methanol and tetrahydrofuran for 12 h, respectively, and dried in vacuum at 100 ° C for 6 h. The obtained material was further characterized by SEM and PXRD. The results are shown in FIG. Figure 6 As shown in the figure (a) is the SEM image, b) is the PXRD pattern), the COFs prepared by the one-pot method by the condensation of 1,3,5-triformylphloroglucinol and 1,4-p-phenylenediamine amine aldehyde have a block structure, which is completely different from the morphology obtained in Example 1. This further proves that the microemulsion polymerization method has a certain ability to regulate the morphology and size of COFs.

[0081] Example 7:

[0082] 15mL of 4.5mg·mL -1 The dichloromethane solution of span80 was added to a 50 mL round-bottom flask and stirred at 650 rpm. 1,3,5-triformylphloroglucinol (15.7 mg, 0.075 mmol) and 30 mg of benzoic acid were added. 16.5 mg of hexadecylpyridinium bromide (CPB) and 1,4-p-phenylenediamine (12 mg, 0.112 mmol) were ultrasonically dissolved in 5.5 mL of phosphate buffered saline containing porcine pancreatic type II lipase PPL (saturated ~ 66 mg / g) and stirred at 0.5 mL min. -1 The mixture was added dropwise to the above dichloromethane solution at a rate of 1:1. Stir at room temperature for 30 minutes. After the reaction was completed, 15 mL of ethanol was used to precipitate the solid, which was filtered and freeze-dried to obtain a solid orange-red powder of PPL@COFs. The composite biocatalyst CALB@COFs was prepared by the same method after replacing the porcine pancreatic type II lipase PPL with a phosphate buffered saline solution of Antarctic Candida lipase CALB (saturated ~76 mg / g). The obtained material was further characterized by SEM, TEM, PXRD and CLSM. The results are shown in Figure 2. Figure 7 As shown in the figure (a) is the SEM image, b) is the TEM image, c) is the PXRD image, and d) is the CLSM image), the original morphology of COFs was not changed after the lipase was in situ doped inside the COFs. The TEM image showed a hollow structure similar to that in Example 1. The CLSM image showed that the addition of lipase caused the color inside the microspheres to change, proving that the biological enzyme was successfully embedded inside the COFs microspheres.

[0083] The activity of the immobilized lipase was determined by the hydrolysis reaction of tributyrin. 1.0 mL of tributyrin was added to 50 mL of phosphate buffer (25 mM, pH 5.0), followed by 10 mg of immobilized enzyme. The reaction was vigorously stirred at 40°C for 15 min. After terminating the reaction, phenolphthalein was added dropwise and the immobilized enzyme was treated with 0.1 mol·L -1 The NaOH solution was titrated to the endpoint of the reaction, and the volume of NaOH solution consumed was recorded. A blank experiment was performed under the same conditions without the addition of lipase. All experiments were performed three times. One unit of lipase activity (U) is defined as the amount of lipase required to hydrolyze tributyrin to produce 1.0 μmol butyric acid per minute under the assay conditions. The calculation formula is as follows:

[0084]

[0085] Where V is the volume of NaOH solution consumed after adding lipase (mL); V0 is the volume of NaOH solution consumed in the blank control (mL); N is the concentration of the NaOH solution (mol / L); T is the reaction time (min); and M is the amount of lipase used (mL or mg). Multiple measurements revealed that the hydrolytic activity of the immobilized PPL@COFs enzyme was 4033 U / g, approaching the initial activity of the free enzyme (4337 U / g). The enzyme@COFs catalyst prepared using the method of this invention effectively retains the enzyme's intrinsic catalytic activity.

[0086] Application 1:

[0087] Using PPL@COFs as heterogeneous catalyst, a continuous flow cycloaddition reaction was carried out on a packed bed with a size of 100 mm × 8 mm, as shown in Figure 8 As shown. Filters were installed at both ends of the packed bed to prevent the catalyst from leaking. Then, 4-nitrobenzaldehyde (3.35 mmol) and indole (6.4 mmol) were dissolved in 50 mL of a mixed solvent (40 mL of water and 10 mL of 1,4-dioxane) and stirred at 0.4 mL min. -1 The flow rate was introduced into the reactor. Without changing the catalyst, the flow reaction was carried out at 50°C. The progress of the reaction was monitored in real time every time, and the yield was determined by HPLC. After multiple experiments, the yield was stable at 40%, showing good stability. Figure 9 shown.

[0088] Application 2:

[0089]

[0090] In a 10mL screw-cap glass bottle, 0.65g of soybean oil and a certain amount of anhydrous methanol were added, with an oil-to-methanol molar ratio of 1:3, a water content of 15%, and 30mg of immobilized enzyme CALB@COFs. Methanol was added every 8 hours, and stirring was continued at 300r / min at 40°C for 24 hours. After the reaction, centrifugation was performed at 4000r / min for 10 minutes. The supernatant was collected and analyzed by gas chromatography. After multiple experiments, the highest fatty acid methyl ester conversion rate of 87% was achieved in biodiesel synthesis, which has a catalytic performance far exceeding that of free enzyme. After 5 cycles of use, the conversion rate remained stable, showing good stability. The results are as follows Figure 10 、 11 , 12, and 13.

[0091] The foregoing description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Those skilled in the art will readily appreciate that various modifications and variations of the present invention are possible. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present invention are intended to be within the scope of protection of the present invention.

Claims

1. A method for preparing porous framework hollow microspheres based on microemulsion polymerization, characterized in that: Surfactants are used as soft templates for COFs microemulsion polymerization. Non-ionic lipophilic surfactant Span80 and oil-soluble aldehyde or amine monomers are added to an organic solvent to prepare an organic phase. An aqueous solution containing water-soluble amine or aldehyde monomers and a hydrophilic surfactant, cetylpyridinium bromide, is used as the aqueous phase. The aqueous phase is dripped into the stirring organic phase to fully mix the oil and water phases to form an oil-in-water microemulsion. Polymerization occurs under the action of an acid catalyst. After the reaction, ethanol precipitation is used to generate a solid through an amine-aldehyde condensation reaction. After post-treatment, COFs hollow microspheres are obtained. The input ratio of lipophilic nonionic surfactant to organic phase is 0.5-5 mg·mL -1 The input ratio of hydrophilic surfactant to water phase is 0.5-4.5 mg·mL -1 ; The volume ratio of the organic solvent to water is 15:0.5-8; the organic solvent is dichloromethane, toluene, xylene, n-hexane or cyclohexane; The aldehyde monomer is 1,3,5-triformylphloroglucinol, 2,5-dimethoxyterephthalaldehyde or 1,4-terephthalaldehyde, and the amine monomer is 1,4-p-phenylenediamine, ethidium bromide, melamine or 1,3,5-tris(4-aminophenyl)benzene.

2. The method for preparing porous framework hollow microspheres based on microemulsion polymerization according to claim 1, wherein: The acid catalyst used is p-toluenesulfonic acid, benzoic acid or acetic acid.

3. The method for preparing porous framework hollow microspheres based on microemulsion polymerization according to claim 1, wherein: The reaction time of the amine-aldehyde condensation reaction is 15-360 min, and the reaction temperature is 0-60°C.

4. The method for preparing porous framework hollow microspheres based on microemulsion polymerization according to claim 1, wherein: The post-treatment process was as follows: filtration, extraction with methanol and tetrahydrofuran for 12 h respectively, and vacuum drying at 100 °C for 6 h.

5. An application of porous framework hollow microspheres prepared by the preparation method according to any one of claims 1 to 4, characterized in that: COFs-type hollow microspheres synthesized by microemulsion polymerization are used as immobilized enzyme carriers. A small amount of biological enzyme is pre-added to the aqueous phase containing water-soluble monomers. The biological enzyme is immobilized in the cavity of the COFs-type hollow microspheres through in situ encapsulation to obtain a biological enzyme@COFs composite catalyst, which is then used for catalytic reactions.

6. The use according to claim 5, characterized in that The biological enzyme used is porcine pancreatic type II lipase PPL or Antarctic Candida lipase CALB.

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