CN116120580B - Ordered bicontinuous structure metal organic framework material SP-ZIF-8, preparation method and application thereof - Google Patents

Abstract

The invention discloses an ordered bicontinuous structure metal organic framework material SP-ZIF-8 and its preparation method and application, and relates to the field of biocatalysis technology. The preparation method comprises the following steps: self-assembling block copolymer polystyrene-b-polyethylene oxide into cubic phase microspheres of DP structure by a co-solvent method; using the cubic phase microspheres of DP structure as soft templates, reacting metal Zn source and 2-methylimidazole in the pores in a mixed solvent system, and after the reaction is completed, washing the template with a cleaning solvent to obtain cubic phase ZIF-8 microspheres of SP structure, named SP-ZIF-8. The present invention uses the amphiphilic biblock copolymer PS-b-PEO through a simple solution self-assembly method to obtain a bicontinuous cubic phase structure assembly in one step, which has good stability and large-sized mesopores; using the SP-ZIF-8 material as a carrier material for immobilized trypsin, a high loading of 325μg/mg is achieved; and the prepared trypsin@SP-ZIF-8 bioreactor has an amino acid coverage of 37% and a matching peptide number of 45 for BSA.

Description

Ordered bicontinuous structure metal organic framework material SP-ZIF-8 and its preparation method and application

Technical Field

The invention relates to the technical field of biocatalysis, and in particular to an ordered bicontinuous structure metal organic framework material SP-ZIF-8 and a preparation method and application thereof.

Background technique

Porous materials have very important application value in many fields such as biocatalysis, energy storage, gas adsorption, etc. It is well known that pore structure has a decisive influence on material properties, and achieving precise control of pore structure is crucial in the preparation of porous materials. Among them, the bicontinuous structure has a three-dimensional continuous and interconnected network and channels, which is conducive to material transport and exposure of active sites, making it have great application potential in the field of energy conversion. Bicontinuous structures can usually be divided into double gyroid structures (DG, space group ), double diamond structure (DD, space group ), double primitive structure (DP, space group ), a single gyroid structure (SG, space group I4 ₁ 32), a single diamond structure (SD, space group ), a single set of primitive structures (SP, space group ) six categories.

However, the artificial synthesis of bicontinuous structural materials is challenging and difficult to obtain through top-down (such as photolithography) or bottom-up (such as self-assembly) methods. Among them, the top-down method can realize the diversified design and large-scale production of bicontinuous structural materials, but it is difficult to achieve nano or submicron pore size and unit cell parameter regulation. The small molecule assembly templates commonly used in the bottom-up method have poor stability, small pore size (usually less than 2nm in diameter), and easy clogging, which limits the application of the replicated bicontinuous structural materials.

Solution self-assembly of block copolymers (BCPs) is an effective strategy to solve the above problems. It can be used as a soft template to prepare bicontinuous porous materials with adjustable pore size in the range of 5–500 nm. Compared with small molecule assemblies, its assemblies are more stable and the pore size control is more flexible. However, in the self-assembly morphology phase diagram, the phase region of the bicontinuous structure is narrow, and it is difficult to find suitable assembly conditions. Importantly, under thermodynamic drive, block copolymer assemblies usually tend to form a double-set network structure. The single-set structure that can be artificially prepared is currently limited to inorganic materials such as SiO ₂ , metal oxides (TiO ₂ , Nb ₂ O ₅ , CsTaWO ₆ ), and carbon. Among them, most of these materials are SG bicontinuous structure materials obtained by polymer templates prepared by solvent evaporation induced self-assembly (EISA) or bulk self-assembly methods. For example, He Rongming's team obtained a DG-structured BCP assembly through bulk self-assembly of linear triblock terpolymers PI-b-PS-b-PDLA or PI-b-PS-b-PLLA, and further selectively removed the polylactic acid block to obtain a polymer template with a mesoporous SG structure. Then, the open pores of the template were filled with a Ni source precursor by chemical plating to achieve replication, and after removing the template, SG-structured metal Ni was obtained, as shown in Figure 1 (Sci. Adv. 2020, 6, eabc3644).

However, in this method, the polymer block composition is relatively complex, and the synthetic conditions and operation requirements are relatively high. Although this method can obtain a single set of G structure materials, the polymer template preparation process is relatively complicated, which limits its further large-scale production and application.

It is of great significance to realize the efficient utilization and application development of the advantages of the bicontinuous structure. As a natural biocatalyst, enzymes have irreplaceable advantages such as high catalytic efficiency, strong substrate specificity, mild reaction conditions, and environmental friendliness. They are widely used in biosensing, energy chemical industry, and environmental catalysis. However, free enzymes are sensitive to the external environment, have poor stability, high cost, and low reuse rate, which limits their industrial application. Immobilized enzyme technology came into being. It uses physical or chemical methods to combine enzymes with water-insoluble carriers, so that the enzymes are confined to a certain space for catalytic reactions. At the same time, the enzymes can be separated and recovered by centrifugation, sedimentation, or magnetic methods for reuse.

Metal-organic frameworks (MOFs) have become a new type of enzyme immobilization carrier in recent years due to their high specific surface area and pore volume, designable and controllable pore size and structure, and stability. However, MOFs immobilized enzymes also face some challenges, such as low enzyme loading rate and enzyme activity retention. For example, trypsin-catalyzed protein hydrolysis in traditional solutions is a time-consuming and inefficient process, and the large molecular size and stable tertiary structure of bovine serum albumin (BSA) make its complete enzymatic hydrolysis particularly difficult, which is not conducive to further research in proteomics. The mesoporous carrier materials reported so far have narrow and discontinuous pores, which to some extent limit the encapsulation of trypsin and the mass transfer of large-sized protein molecules inside the pores, and the enzymatic hydrolysis efficiency needs to be improved.

Although Gu Jinlou's research group prepared Ce-HMMOFs materials with columnar mesoporous structures (pore size of about 10 nm) for trypsin immobilization, as shown in Figure 2, a high enzyme loading of 304 μg/mg was achieved; cytochrome c (Cyt-c), myoglobin (MYG), and bovine serum albumin (BSA) standard proteins were used as model substrates, respectively, and their catalytic efficiency (of which the BSA enzymatic sequence coverage rate was 25%) was confirmed to be better than that of free enzyme (19%). (Angew. Chem. Int. Ed. 2020, 59, 14124–14128), however, its enzymatic efficiency for BSA still needs to be improved, and the amino acid coverage rate is only 25%.

Therefore, technicians in this field are committed to developing an ordered bicontinuous structure metal organic framework material for enzyme immobilization carrier to improve enzymatic hydrolysis efficiency and amino acid coverage.

Summary of the invention

In view of the above-mentioned defects of the prior art, the technical problem to be solved by the present invention is to provide an ordered bicontinuous structure metal organic framework material for enzyme immobilization carrier to improve the enzymatic hydrolysis efficiency and amino acid coverage.

To achieve the above object, the present invention provides a method for preparing an ordered bicontinuous structure metal organic framework material SP-ZIF-8 for enzyme immobilization carrier, comprising the following steps:

S1: self-assembly of block copolymer polystyrene-b-polyethylene oxide into cubic phase microspheres with DP structure by co-solvent method;

S2: Using DP-structured cubic microspheres as soft templates, metal Zn source and 2-methylimidazole were reacted in the pores in a mixed solvent system. After the reaction was completed, the template was washed away with a cleaning solvent to obtain SP-structured cubic ZIF-8 microspheres, named SP-ZIF-8.

In a preferred embodiment of the present invention, in step S1, preparing cubic microspheres with DP structure comprises the following steps:

S11: dissolving the block copolymer polystyrene-b-polyethylene oxide PS ₂₁₈ -b-PEO ₄₅ in the first mixed solvent, stirring at room temperature until the copolymer is completely dissolved, to obtain a first solution;

S12: adding deionized water dropwise to the first solution to obtain a second solution;

S13: dialyzing the obtained second dissolved solution in deionized water, then centrifuging and freeze-drying to obtain cubic phase microsphere powder with DP structure.

Furthermore, in step S11, the first mixed solvent consists of dioxane and N,N-dimethylformamide (DMF), and the volume ratio of dioxane to N,N-dimethylformamide (DMF) in the first mixed solvent is 92:8.

Furthermore, in step S12, 2 mL of deionized water is added dropwise at a rate of 1 mL·h ^-1 .

Furthermore, in step S13, the second dissolved solution is dialyzed in deionized water for 24 hours.

In another preferred embodiment of the present invention, in step S2, preparing cubic ZIF-8 microspheres with SP structure comprises the following steps:

S21: Using DP-structured cubic microspheres as templates, dispersing them in the second mixed solvent, then adding 2-methylimidazole and stirring to diffuse them into the pores, then adding _ZnBr2 to react, and stirring until the reaction is complete;

S21: The product was washed with ethanol and THF respectively to remove the template, and then dried in vacuum at 60 °C to obtain SP-structured cubic ZIF-8 microspheres.

The present invention also provides an ordered bicontinuous structure metal organic framework material SP-ZIF-8 for enzyme immobilization carrier prepared by the above method.

The present invention also provides an application of the metal organic framework material SP-ZIF-8 as described above in trypsin immobilization and a biocatalytic reactor.

The present invention also provides a method for preparing a ZIF-8 biocatalytic reactor trypsin@SP-ZIF-8 for immobilizing trypsin, characterized in that trypsin is loaded into the metal organic framework material SP-ZIF-8 as described in claim 7 by a mixing and stirring method.

Preferably, the method specifically comprises the following steps:

Trypsin and SP-ZIF-8 were dispersed in a HEPES buffer solution and stirred at 25°C for 1 hour. The solid was washed and collected by centrifugation to obtain trypsin@SP-ZIF-8, which was then dispersed in _{an NH4HCO3} buffer solution to obtain a trypsin@SP-ZIF-8 bioreactor.

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

The present invention uses an amphiphilic diblock copolymer PS-b-PEO through a simple solution self-assembly method to obtain a bicontinuous cubic phase structure assembly in one step, which has good stability and large-sized mesopores;

The present invention uses the prepared bicontinuous cubic phase structure assembly as a soft template and successfully prepares an ordered single set of bicontinuous cubic phase structure SP-ZIF-8 material for the first time;

The present invention uses SP-ZIF-8 material as a carrier material for immobilizing trypsin, achieving a high loading amount of 325 μg/mg;

The trypsin@SP-ZIF-8 bioreactor prepared by the present invention can achieve complete enzymatic hydrolysis of bovine serum albumin BSA in 30 minutes, and LC-MS confirms 37% amino acid coverage and 45 matching peptide segments. The three-dimensionally connected double continuous pores shorten the diffusion distance between BSA and trypsin, which can promote their full contact. The large mesopores (~65nm) of SP-ZIF-8 can load large-sized BSA molecules, so that the catalytic process can proceed smoothly in the pores.

The trypsin@SP-ZIF-8 reactor prepared by the present invention has excellent reusability and can still maintain a relatively high catalytic efficiency (amino acid coverage of 33%) after 5 cycles.

The concept, specific structure and technical effects of the present invention will be further described below in conjunction with the accompanying drawings to fully understand the purpose, characteristics and effects of the present invention.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 is a 3D schematic diagram of the PI block network disclosed in the background technology of the present invention and a SEM image of the Ni network obtained after replicating the polylactic acid block; (A, B) simulation images of PI-b-PS-b-PLLA and PI-b-PS-b-PDLA respectively; (C, D) 3D reconstruction images of electron tomography and (E, F) FESEM images of Ni metal after replicating;

FIG2 is a schematic diagram of the trypsin immobilization and protein digestion process disclosed in the background art of the present invention;

Fig. 3 is a schematic diagram of the principle of the present invention;

FIG4 is a morphology and structural characterization diagram of a DP-structured cubic phase assembly (PCs) of a preferred embodiment of the present invention: (a, b) SEM diagram; (c) SAXS diagram;

FIG5 is a morphology and structural characterization diagram of a cubic phase ZIF-8 (SP-ZIF-8) of a SP structure according to a preferred embodiment of the present invention: (a, b) SEM diagram; (c) SAXS diagram;

6 is a growth mechanism diagram of a cubic phase ZIF-8 (SP-ZIF-8) of a SP structure according to a preferred embodiment of the present invention;

FIG. 7 is a schematic diagram of the immobilization of trypsin on SP-ZIF-8 and the BSA protein digestion process according to a preferred embodiment of the present invention.

Detailed ways

The following describes several preferred embodiments of the present invention with reference to the drawings in the specification, so that the technical content is clearer and easier to understand. The present invention can be embodied in many different forms of embodiments, and the protection scope of the present invention is not limited to the embodiments mentioned in the text.

In the drawings, components with the same structure are indicated by the same numerical reference numerals, and components with similar structures or functions are indicated by similar numerical reference numerals. The size and thickness of each component shown in the drawings are arbitrarily shown, and the present invention does not limit the size and thickness of each component. In order to make the illustration clearer, the thickness of the components is appropriately exaggerated in some places in the drawings.

Example 1

A method for preparing an ordered bicontinuous structure metal organic framework material SP-ZIF-8 for enzyme immobilization carrier, as shown in FIG3 , comprises the following steps:

Block copolymer polystyrene-b-polyethylene oxide (PS-b-PEO) was self-assembled into cubic microspheres with DP structure and average diameter of 2.6±0.8μm by co-solvent method. Using it as soft template, metal Zn source (ZnBr ₂ ) and 2-methylimidazole reacted in the pores in a water/methanol mixed solvent system, and then the template was washed away by solvent to obtain cubic ZIF-8 microspheres with SP structure.

Specifically, the preparation method of DP structure cubic phase assembly (PCs) template and SP structure cubic phase ZIF-8 (SP-ZIF-8) is:

(1) Preparation of DP-structured cubic phase assembly (PCs) template: First, 20 mg of PS ₂₁₈ -b-PEO ₄₅ was dissolved in 2 mL of dioxane/DMF (v/v 92:8) mixed solvent and stirred at room temperature for several hours until the copolymer was completely dissolved. Subsequently, 2 mL of deionized water was added dropwise to the above solution at a rate of 1 mL·h ^-1 . The obtained solution was dialyzed with a large amount of deionized water for 24 hours, centrifuged several times, and freeze-dried to obtain a bicontinuous cubic phase assembly template powder, as shown in Figure 4.

(2) Preparation of SP-structured cubic ZIF-8 (SP-ZIF-8): 10 mg of the DP-structured assembly template was dispersed in 15 mL of a methanol/water (v/v 1/2) mixed solvent, 2-methylimidazole was added and stirred for several hours to diffuse into the pores, and then ZnBr ₂ was added to react. The reaction was completed by stirring at 25°C for 1 hour. Finally, the product was washed with ethanol and THF respectively and the template was removed. After vacuum drying at 60°C, the SP-ZIF-8 product was obtained, as shown in Figure 5.

As shown in Figure 6, the ZIF-8 precursor nucleated in the polymer template and grew as a "seed", forming porous polyhedral particles with an average size of 550±70nm in 25 minutes. The particle size of the small particles grew to 640±65nm in 30 minutes, and at the same time they connected to each other to form larger spheres. This process confirmed the "growth" and "fusion" process of ZIF-8 particles under the restriction of the template. SP-ZIF-8 microspheres can be prepared precisely and controllably by controlling different reaction times, revealing the growth mechanism of SP-ZIF-8 "seed-growth-fusion", and realizing the controllable and precise preparation of a single set of dual-continuous cubic phase structure ZIF-8.

Example 2

A method for preparing a ZIF-8 biocatalytic reactor for immobilized trypsin, trypsin@SP-ZIF-8, comprises the following steps: loading trypsin into SP-ZIF-8 material by a simple mixing and stirring method. As shown in FIG7 , specifically, 5 mg of trypsin and 10 mg of SP-ZIF-8 are weighed and dispersed in 5 mL of HEPES buffer solution and stirred at 25° C. for 1 hour, followed by washing and centrifugation to collect the solid, thereby obtaining trypsin@SP-ZIF-8, which is then dispersed in 1 mL of 25 mM NH ₄ HCO ₃ buffer solution, thereby obtaining a trypsin@SP-ZIF-8 bioreactor.

SP-ZIF-8 was applied to the immobilization carrier material of trypsin, and a high loading of 325 μg/mg could be achieved. This was mainly attributed to the coordination or hydrogen bonding between ZIF-8 and trypsin, and the encapsulation effect of the bicontinuous pores on trypsin. The trypsin@SP-ZIF-8 reactor could achieve complete enzymatic hydrolysis of bovine serum albumin in 30 minutes, and LC-MS confirmed 37% amino acid coverage and 45 matching peptides. The three-dimensionally connected bicontinuous pores shortened the diffusion distance between BSA and trypsin, promoting their full contact. The large mesopores (~65 nm) of SP-ZIF-8 can load large-sized BSA molecules, allowing the catalytic process to proceed smoothly in the pores.

Bovine serum albumin was used as a substrate to evaluate the high catalytic efficiency of the bioreactor. Specifically: 40 μL of the dispersion of trypsin@SP-ZIF-8 was added to an NH ₄ HCO ₃ buffer solution containing 0.5 nmol/μL bovine serum albumin (BSA), and incubated in a 37°C constant temperature shaker for 30 minutes. Trypsin@SP-ZIF-8 was then recovered by centrifugation, and the supernatant was the digestion product solution, which was desalted and then subjected to mass spectrometry testing. The digestion product at 30 minutes characterized by liquid phase mass spectrometry had a sequence coverage of 37% and a matching number of 45 peptides, both of which were better than free trypsin (sequence coverage of 30% and a matching number of 31 peptides).

The preferred specific embodiments of the present invention are described in detail above. It should be understood that ordinary technicians in the field can make many modifications and changes based on the concept of the present invention without creative work. Therefore, all technical solutions that can be obtained by technicians in the technical field based on the concept of the present invention through logical analysis, reasoning or limited experiments on the basis of the prior art should be within the scope of protection determined by the claims.

Claims (8)

1. A method for preparing an ordered bicontinuous structure metal organic framework material SP-ZIF-8 for enzyme immobilization carrier, characterized in that it comprises the following steps: S1: self-assembling block copolymer polystyrene-b-polyethylene oxide into cubic microspheres of DP structure by co-solvent method; the DP structure is a double-set primitive structure; S2: Using DP-structured cubic microspheres as soft templates, a metal Zn source and 2-methylimidazole are reacted in the pores in a mixed solvent system. After the reaction is completed, the template is washed away with a cleaning solvent to obtain SP-structured cubic ZIF-8 microspheres, named SP-ZIF-8. The SP structure is a single set of primitive structures. In the step S1, preparing cubic microspheres with DP structure comprises the following steps: S11: dissolving the block copolymer polystyrene-b-polyethylene oxide PS ₂₁₈ -b-PEO ₄₅ in the first mixed solvent, stirring at room temperature until the copolymer is completely dissolved, to obtain a first solution; S12: adding deionized water dropwise to the first solution to obtain a second solution; S13: dialyzing the obtained second dissolved solution in deionized water, then centrifuging and freeze-drying to obtain a cubic phase microsphere powder of DP structure; In the step S2, preparing cubic ZIF-8 microspheres with SP structure comprises the following steps: S21: Using DP-structured cubic microspheres as templates, dispersing them in the second mixed solvent, then adding 2-methylimidazole and stirring to diffuse them into the pores, then adding _ZnBr2 to react, and stirring until the reaction is complete; S21: The product was washed with ethanol and THF respectively to remove the template, and then vacuum dried at 60°C to obtain cubic ZIF-8 microspheres with SP structure. 2. The method for preparing the ordered bicontinuous structure metal-organic framework material SP-ZIF-8 for enzyme immobilization carrier according to claim 1, characterized in that, in step S11, the first mixed solvent consists of dioxane and N,N-dimethylformamide, and the volume ratio of dioxane to N,N-dimethylformamide in the first mixed solvent is 92:8. 3. The method for preparing the ordered bicontinuous structure metal-organic framework material SP-ZIF-8 for enzyme immobilization carrier according to claim 1, characterized in that in step S12, 2 mL of deionized water is added dropwise at a speed of 1 mL·h ^-1 . 4. The method for preparing the ordered bicontinuous structure metal-organic framework material SP-ZIF-8 for enzyme immobilization carrier according to claim 1, characterized in that in step S13, the second dissolving solution is dialyzed in deionized water for 24 hours. 5. An ordered bicontinuous structure metal-organic framework material SP-ZIF-8 for enzyme immobilization carrier prepared by the method described in any one of claims 1 to 4. 6. Use of the metal organic framework material SP-ZIF-8 as claimed in claim 5 in trypsin immobilization and biocatalytic reactor. 7. A method for preparing a ZIF-8 biocatalytic reactor for immobilizing trypsin, trypsin@SP-ZIF-8, characterized in that trypsin is loaded into the metal organic framework material SP-ZIF-8 as described in claim 5 by a mixing and stirring method. 8. The method according to claim 7, characterized in that it specifically comprises the following steps: Trypsin and SP-ZIF-8 were dispersed in a HEPES buffer solution and stirred at 25°C for 1 hour. The solid was washed and collected by centrifugation to obtain trypsin@SP-ZIF-8, which was then dispersed in _{an NH4HCO3} buffer solution to obtain a trypsin@SP-ZIF-8 bioreactor.