Preparation method and enzyme catalysis application of porous framework-like hollow microspheres based on microemulsion polymerization method

Abstract

The invention discloses a preparation method and enzyme catalysis application of porous framework-like hollow microspheres based on a microemulsion polymerization method, and the method is characterized in that covalent organic frameworks (COFs) materials are assembled in situ at liquid drop interfaces by regulating and controlling the interaction of oil-water interfaces in microemulsion, so that the COFs nano-scale-like hollow microspheres with uniform morphology are obtained; and the method has good universality. The COFs nano-particles prepared by the method disclosed by the invention can be used as a biological carrier for efficiently loading a biological enzyme, on the basis of retaining the original activity of the enzyme, the conformation and activity of the enzyme are not influenced by the outside, the tolerance of the COFs nano-particles to a bad environment is improved, the recycling stability of the biological enzyme is enhanced, and the service life of the COFs nano-particles is prolonged. The method has huge application potential in the field of immobilized enzyme catalysis.

Description

Technical Field

[0001] The invention belongs to the technical field of nano material preparation, and specifically 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 the diversity of multifunctionality, topology, and connectivity, 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 adjusting 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 adjust the morphology of COFs, and can be easily removed by washing.

[0004] Generally, emulsion polymerization using surfactants as soft templates can control the morphology of polymers under mild conditions, but this method requires that the structure-guided polymer must provide the driving force for micelle formation, and the precursor can be cross-linked to form a framework of pore structure. The cross-linked material should have higher thermal stability than the soft template to maintain the original pore structure during template removal. Therefore, the following key issues need to be solved: how to obtain COFs nanocrystals by in situ polymerization in microemulsions through the soft template method and ensure their good dispersibility and morphological stability; at the same time, explore the universality of this method and the optimal reaction conditions and application value of the prepared materials. Summary of the invention

[0005] In order to solve the deficiencies of the prior art, the purpose of the present invention is to provide a method for preparing porous framework hollow microspheres based on microemulsion polymerization and enzyme catalysis application, the method is simple in steps, has universality, and the obtained nano-microspheres have controllable size, high crystallinity, and uniform morphology. In addition, the in-situ loading of biological enzymes can be carried out in the hollow internal structure of COFs microspheres by embedding method, giving them multifunctionality.

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

[0007] A preparation method of porous framework hollow microspheres based on microemulsion polymerization method, using surfactant as a soft template for COFs microemulsion polymerization, adding non-ionic lipophilic surfactant and oil-soluble aldehyde or amine monomers into an organic solvent to prepare an organic phase, using an aqueous solution containing water-soluble amine or aldehyde monomers and hydrophilic surfactant as an aqueous phase, dripping the aqueous phase into the stirring organic phase, making the oil and water phases fully mixed to form an oil-in-water microemulsion, polymerizing under the action of an acid catalyst, precipitating with ethanol after the reaction, and generating solids through amine-aldehyde condensation reaction, and obtaining COFs hollow microspheres 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 0.5~4.5mg·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 to extract with methanol and tetrahydrofuran for 12 hours respectively after filtration, and vacuum dry 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 enzymes in advance to an aqueous phase containing water-soluble monomers, and fixing the biological enzymes in the cavity of the COFs hollow microspheres by in situ encapsulation to obtain a biological enzyme@COFs composite catalyst, which is 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 carry out in-situ assembly of COFs monomers to obtain nano-sized quasi-hollow microspheres, and the method is simple to operate, mild in 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 load biological enzymes in situ inside the quasi-hollow microspheres to achieve spatial separation of the biological enzymes and acid catalysts. On the one hand, the activity of the enzyme can be effectively retained, and the initial activity is close to that of the free enzyme. The stable and diverse COFs shell can support the conformation of the enzyme from the influence of the external environment, and the high porosity of the COFs can ensure mass transfer in the system, so that the enzyme is fully in contact with the reaction substrate, and has great application potential in the field of immobilized enzyme catalysis. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] The accompanying drawings in the specification, 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;

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

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

[0023] Figure 4 This is a 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;

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

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

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

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

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

[0030] Fig.11 The environmental stability of the CALB@COFs prepared in Example 7 of the present invention for catalytic production of biodiesel;

[0031] Fig.12 Gas chromatography for the preparation of biodiesel catalyzed by free CALB lipase;

[0032] Fig.13 Gas chromatography of the catalytic preparation 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 are intended to provide further explanation of the present invention. Unless otherwise specified, all technical and scientific terms used herein have the same meanings as those 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 exemplary embodiments according to the present invention. As used herein, unless the context clearly indicates otherwise, the singular form is also intended to include the plural form. In addition, it should be understood that when the terms "comprising" and / or "including" are used in this specification, it indicates 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 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 high-efficiency 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 implementation mode, the microemulsion is an oil-in-water microemulsion system, and the oil-in-water microemulsion system is: 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 aldehyde or amine monomer and a hydrophilic surfactant as the aqueous phase, and uniformly dropping the aqueous phase into the stirring oil phase, so that the oil and water phases are fully mixed to form an oil-in-water microemulsion, and polymerization occurs under the action of an acid catalyst. After the reaction is completed, ethanol is used to precipitate the solid generated by the amine-aldehyde condensation reaction. Among them, the organic phase is an organic substance that is not miscible 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 0.5~4.5mg·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 embodiments, the materials after the reaction are filtered, extracted, and dried to obtain purer covalent organic framework microspheres. The specific extraction method is: after the reaction, the solid is precipitated with ethanol, filtered, and extracted with methanol and tetrahydrofuran for 12 hours respectively. The drying conditions are: vacuum drying at 100°C for 6 hours.

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

[0050] This implementation method is to ultrasonically disperse COFs monomers in a single reaction solvent for polymerization, and the 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 implementation method.

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

[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 min.

[0054] The third embodiment of the present invention (Example 7) uses the COFs nano-sized hollow microspheres synthesized in the first embodiment as immobilized enzyme carriers to prepare composite biocatalysts. In the process of preparing nano-scale COFs hollow microspheres based on microemulsion polymerization, a small amount of bioenzyme is added to a phosphate buffered saline solution containing water-soluble COFs monomers, and then the input ratio of non-ionic surfactant to organic phase is adjusted to reduce the effect of organic solvent on bioenzyme activity during the reaction. The bioenzyme is fixed in the cavity pores of the COFs microspheres by in-situ encapsulation to obtain a bioenzyme@COFs composite catalyst.

[0055] This implementation method is similar to the first implementation method. A lipophilic nonionic surfactant and an oil-soluble monomer are added to an organic solvent to prepare an oil phase. A water-soluble monomer and a hydrophilic surfactant are dissolved in a phosphate buffered saline solution containing a small amount of porcine pancreatic type II lipase PPL as an aqueous phase. The aqueous phase is uniformly dripped into the stirring oil phase to fully mix the oil and water phases to form an oil-in-water microemulsion. An acid catalyst is added. During the polymerization process, the lipase can be in situ doped in the cavity of the COFs microspheres. After the reaction is completed, the solid is precipitated with ethanol, filtered and freeze-dried to obtain a composite biocatalyst PPL@COFs, and stored at 4°C. The porcine pancreatic type II lipase PPL is replaced with the Antarctic Candida lipase CALB, and CALB@COFs is prepared by the same method. The biological activity of the immobilized enzyme can be determined by the hydrolysis reaction 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 water 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 for 15 minutes at 40°C, stop the reaction, drop phenolphthalein 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 (U) of lipase activity is defined as the amount of lipase required to hydrolyze tributyrin to produce 1.0 μmol 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, and the specific method was to fill PPL@COFs in a packed bed reactor (with an inner diameter of 8 mm), install a 300-mesh sieve (volume of 4 mL) at both ends, prepare a reaction solution of 4-nitrobenzaldehyde and indole using 1,4-dioxane and water (1:4 in v / v) as solvent, and precipitate the reaction mixture 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 applied to the preparation of biodiesel. The specific method is to add soybean oil, anhydrous methanol, water content and a quantitative immobilized enzyme CALB@COFs into a screw-cap glass bottle, react at 40°C for 24 hours, centrifuge and take the supernatant after completion, 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 in conjunction with specific embodiments.

[0067] Embodiment 1:

[0068] 15mL of 1mg·mL -1 The dichloromethane solution of span 80 was added to a 50 mL round-bottom flask, stirred at 650 rpm, and 1,3,5-triformylphloroglucinol (15.7 mg, 0.075 mmol) was added. 10 mg of hexadecylpyridinium bromide (CPB) and 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 1.5 ℃. The mixture was stirred at room temperature for 15 min. After the reaction was completed, 15 mL of ethanol was used to precipitate the solid. The solid orange-red powder was filtered and extracted with methanol and tetrahydrofuran for 12 h respectively. The solid was dried under vacuum at 100 °C for 6 h. The obtained material was further characterized by SEM, TEM and PXRD. The results are shown in Figure 1 As shown (a) is the SEM image, b) is the TEM image, and c) is the PXRD image), the COFs material presents a regular spherical morphology, with uniform size, an average diameter of about 300 nm, a hollow structure, and good crystallinity. Therefore, this experimental scheme is selected as the basic condition for the subsequent Example 6.

[0069] Embodiment 2:

[0070] 15mL of 1mg·mL -1The dichloromethane solution of span 80 was added to a 50 mL round-bottom flask, stirred at 650 rpm, and 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 100 ℃. The mixture was stirred at room temperature for 15 min. After the reaction was completed, 15 mL of ethanol was used to precipitate the solid. The solid brown powder was filtered out and extracted with methanol and tetrahydrofuran for 12 h respectively. The solid was dried under vacuum at 100 °C for 6 h. The obtained material was further characterized by SEM, TEM and PXRD. The results are shown in 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 a regular structure. The spherical particle size is about 700nm, and an obvious cavity structure can be observed under a transmission electron microscope, with good crystallinity.

[0071] Embodiment 3:

[0072] 15mL of 1mg·mL -1 The dichloromethane solution of span 80 was added to a 50 mL round-bottom flask, stirred at 650 rpm, and 2,5-dimethoxyterephthalaldehyde (21.7 mg, 0.112 mmol) and 30 mg 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 100 ℃. The mixture was stirred at room temperature for 15 min. 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 h respectively. The solid powder was dried under vacuum at 100 °C for 6 h. The obtained material was further characterized by SEM. The results are shown in 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] Embodiment 4:

[0074] According to the operation of Example 1, 15 mL of 1 mg mL -1The 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) and 1,4-p-phenylenediamine (12 mg, 0.112 mmol) and 30 mg of p-toluenesulfonic acid (PTSA) were ultrasonically dissolved in different amounts of deionized water at 0.5 mL min. -1 After stirring at room temperature for different time periods, the solid was precipitated with 15 mL of ethanol, extracted with methanol and tetrahydrofuran for 12 h, and dried under vacuum at 100 °C for 6 h. The specific operating conditions are shown in Table 1. Figure 4 As shown, 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] Embodiment 5:

[0078] 15mL of 1mg·mL -1 The dichloromethane solution of span 80 was added to a 50 mL round-bottom flask, stirred at 650 rpm, and 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 min. After the reaction was completed, 15 mL of ethanol was used to precipitate the solid. The solid red-brown powder was filtered and extracted with methanol and tetrahydrofuran for 12 h, respectively, and dried under vacuum at 100 °C for 6 h. The obtained material was further characterized by SEM, TEM and PXRD. The results are shown in 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 revealed that the interior is a solid structure. Therefore, the carrier material required for the subsequent in-situ encapsulation of bioenzyme experiments cannot be prepared using amphiphilic 2,5-dihydroxyterephthalaldehyde as a monomer and 1,3,5-tri(4-aminophenyl)benzene as a monomer.

[0079] Embodiment 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, which 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 Figure 6 As shown in the figure (a) is the SEM image, b) is the PXRD image), the COFs prepared by the one-pot method through 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] Embodiment 7:

[0082] 15mL 4.5mg·mL -1 The dichloromethane solution of span80 was added to a 50 mL round-bottom flask, stirred at 650 rpm, and 1,3,5-triformylphloroglucinol (15.7 mg, 0.075 mmol) and 30 mg 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 the mixture was stirred at 0.5 mL min. -1 Add it dropwise to the above dichloromethane solution at a rate of 1.5 ℃. Stir at room temperature for 30 minutes. After the reaction is completed, use 15 mL of ethanol to precipitate the solid, filter and freeze-dry to obtain PPL@COFs solid orange-red powder. Replace porcine pancreatic type II lipase PPL with a phosphate buffered saline solution of Antarctic Candida lipase CALB (saturated ~76 mg / g) and use the same method to prepare the composite biocatalyst CALB@COFs. The obtained material was further characterized by SEM, TEM, PXRD and CLSM. The results are as follows Figure 7 As shown (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 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 mixture was stirred vigorously at 40 °C for 15 min. After the reaction was terminated, phenolphthalein was added dropwise and 0.1 mol·L -1 The NaOH solution was titrated to the end point of the reaction, and the volume of NaOH solution consumed was recorded. The blank experiment was performed in the same manner without adding 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, and the calculation formula is as follows:

[0084]

[0085] Wherein, V is the volume of NaOH solution consumed after adding lipase (mL); V0 is the volume of NaOH solution consumed by the blank control (mL); N is the concentration of NaOH solution (mol / L); T is the reaction time (min); M is the amount of lipase (mL or mg). After multiple measurements, the hydrolysis activity of the immobilized enzyme PPL@COFs is 4033U / g, which is close to the initial activity of the free enzyme (4337U / g). The bioenzyme@COFs catalyst prepared by the method of the present invention can effectively retain the intrinsic catalytic activity of the enzyme.

[0086] Application 1:

[0087] Using PPL@COFs as heterogeneous catalyst, 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 catalyst leakage. 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 after a period of time, and the yield was determined by HPLC. After multiple experiments, the yield was stabilized at 40%, showing good stability. Fig. 9 shown.

[0088] Application 2:

[0089]

[0090] In a 10mL screw-cap glass bottle, add 0.65g soybean oil and a certain amount of anhydrous methanol, with an oil-to-alcohol molar ratio of 1:3, 15% water content and 30mg immobilized enzyme CALB@COFs, add methanol every 8h, stir continuously at 300r / min at 40℃ for 24h, centrifuge at 4000r / min for 10min after the reaction, and take the supernatant for gas chromatography analysis. After multiple experiments, the highest fatty acid methyl ester conversion rate of 87% was obtained for synthesized biodiesel, which has a catalytic performance far exceeding that of free enzymes. After 5 cycles, the conversion rate remained stable and had good stability. The results are as follows Fig.10 , 11 , 12, and 13.

[0091] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope 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. Nonionic lipophilic surfactants and oil-soluble aldehydes or amine monomers are added to organic solvents to prepare an organic phase. An aqueous solution containing water-soluble amines or aldehyde monomers and hydrophilic surfactants is used as the aqueous phase. The aqueous phase is dropped 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 is completed, ethanol precipitation is used. The amine-aldehyde condensation reaction generates a solid, and COFs hollow microspheres are obtained after post-treatment.

2. The method for preparing porous framework hollow microspheres based on microemulsion polymerization as claimed in claim 1, characterized in that: 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 0.5~4.5mg·mL -1 .

3. The method for preparing porous framework hollow microspheres based on microemulsion polymerization as claimed in claim 1, characterized in that: 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.

4. The method for preparing porous framework hollow microspheres based on microemulsion polymerization as claimed in claim 1, characterized in that: 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.

5. The method for preparing porous framework hollow microspheres based on microemulsion polymerization as claimed in claim 1, characterized in that: 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.

6. The method for preparing porous framework hollow microspheres based on microemulsion polymerization as claimed in claim 1, characterized in that: The acid catalyst used is p-toluenesulfonic acid, benzoic acid or acetic acid.

7. The method for preparing porous framework hollow microspheres based on microemulsion polymerization as claimed in claim 1, characterized in that: The reaction time of the amine-aldehyde condensation reaction is 15-360 minutes, and the reaction temperature is 0-60°C.

8. The method for preparing porous framework hollow microspheres based on microemulsion polymerization as claimed in claim 1, characterized in that: The post-treatment process is to extract with methanol and tetrahydrofuran for 12 hours respectively after filtration, and vacuum dry at 100°C for 6 hours.

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

10. The use according to claim 9, characterized in that The biological enzyme used is porcine pancreatic type II lipase PPL or Antarctic Candida lipase CALB.

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