CN112708172A - COF-loaded chitosan bionic thin film material and preparation and application thereof - Google Patents

Abstract

The invention relates to preparation of a chitosan loaded Covalent Organic Framework (COF) bionic thin film material. Specifically, COF powder is uniformly mixed into a chitosan film pre-polymerization solution, a layer of COF is uniformly loaded on the surface of the chitosan film after freeze drying is carried out, and the chitosan COF-loaded hierarchical porous film with special functions is prepared. The preparation method has the advantages of mild conditions, simplicity and low cost. The prepared film material can be applied to enrichment of glycosylated peptide segments, and efficient enrichment of the glycosylated peptide segments is realized; meanwhile, the copper ion adsorption material can also be applied to copper ion adsorption and has good adsorption capacity on copper ions.

Description

COF-loaded chitosan bionic thin film material and preparation and application thereof

Technical Field

The invention takes a chitosan bionic film as a carrier, loads a newly reported COF and prepares a COF-loaded chitosan film material. Specifically, the chitosan-loaded COF film is synthesized by two methods of uniformly mixing COF powder into a chitosan film pre-polymerization solution, freeze-drying the mixture and uniformly loading a layer of COF on the surface of the chitosan film, so that the chitosan film which originally does not have a mesoporous structure has a hierarchical pore structure of mesopores and macropores, the specific surface area of the chitosan film is remarkably increased, and the chitosan film is endowed with the capacity of absorbing copper ions.

Background

Hierarchical porous monoliths (mesoporous porous monoliths) typically have pore structures of more than two dimensions (micropores: <2 nm; mesopores: 2-50 nm; macropores: >50nm), and this particular hierarchical pore structure is advantageous for increasing the specific surface area of the monolith and improving the mass transfer process inside it (document 1.Feinle A. al "Sol-gel synthesis of monolithic materials with hierarchical pore space.," Chemical Society Reviews, 2016,45(12): 3377-3399.). The currently reported hierarchical pore monolithic materials mainly include silica gel monolithic materials, organic polymer monolithic materials, zeolite monolithic materials, carbon-based monolithic materials, Metal Organic Framework (MOF) monolithic materials and the like, and due to unique pore structures and physicochemical properties, the materials are widely applied to the fields of gas adsorption, energy storage, separation, catalysis and the like. Among these, Materials such as zeolite and MOF are generally powders having adjustable microporous or mesoporous structures, and the most common method for preparing them as monolithic Materials is the freeze-drying method (document 2.Zhang h.al "Aligned porous structures by direct free"; Advanced Materials, 2007,19(11):1529-1533.) (document 3.Zhang n.al "Fabrication of high pore biodizable monolithic stranded Materials by Carbon oxide and the same adsorption of metals"; Carbon, 49(3): 2011-837.) that a powdered material is first dispersed in a solvent, frozen into a bulk material at a low temperature, and then the solvent is removed by freeze-drying to obtain a multi-stage macroporous/microporous or mesoporous material. Compared with other multi-level hole integral materials, the regulation and control of two hole structures of the integral material prepared from the powder material are mutually independent, and the special multi-level hole integral material is easy to design according to the requirement.

Covalent organic framework materials (COFs) are a new porous organic polymer material developed in recent years, and have high crystallinity and ordered nano-pore structures. There have been many studies on the preparation and application of hydrazones linked with (document 4.Feng X.al "volatile organic frameworks". Chemical Society Reviews 2012,41(18):6010- & 2.) (document 5.Ding S.Y.al "volatile organic frameworks (COFs): from design to applications". Chemical Society Reviews, 2013,42(2):548- & 568.) (document 6.Cote A.P.al "ports, crystalloid, volatile organic frameworks". Science 2005,310(5751):1166- & 1170) and, in general, they require aromatic amines. However, COFs are generally in powder form under synthetic conditions and exhibit poor processability due to their difficulty in dissolving in common solvents. Therefore, shaping COF powders is a challenge. For example, in order to form COF into a film form, porous α -Al is first formed₂O₃A layer of aldehyde group is modified on the surface of a substrate, and then a COF film with the thickness of 4 μm is prepared on the substrate in situ (document 7.Lu H. Al "A novel 3D constant organic frame membrane growth on a porous alpha-Al)₂O₃substrate under silver conditions Chemical Communications 2015,51(85) 15562 and 15565. Of particular note, such COF membranes exhibit poor mass transport processes due to the lack of macropores. There is therefore a need for COF bulk materials with large pores, and there are few reports in the literature on such materials. In addition, many adsorbing materials prepared based on chitosan have been reported so far. For example, the green chelate fiber for adsorbing cadmium ions is prepared by chitosan such as Cui, and the adsorption capacity of the green chelate fiber for adsorbing cadmium ions reaches 47.65mg g^-1(document 8.Meng J. al "furniture synthesis of magnetic chips nano-no-particulate functions with N/O-relating groups for influencing the adaptation of U (VI) from a group solution". Journal of Materials Science, 2017,53(3): 2277-; the magnetic chitosan nano particles for treating the uranium-containing wastewater are prepared by Zhou and the like, and the adsorption capacity of the magnetic chitosan nano particles on uranium ions reaches 0.66mmol g^-1(document 9.Sheng L.al "A novel 3D synergistic organic frame membrane growth on a pore alpha-Al₂O₃substrate under solvent conditions Journal of radio and Nuclear Chemistry 2016,310, (3) 1361. 1371.). In view of the problems, the chapter loads one COF (TpODH) by two different methods on the basis of the chitosan film (CM) prepared in the chapter II, and two chitosan-supported COF films CM-COF-I and CM-COF-II with a hierarchical pore structure are prepared and applied to enrichment and heavy metal ion adsorption of glycosylated peptides.

Disclosure of Invention

The invention provides two methods for preparing a chitosan-loaded COF film based on a freeze-drying method. One method is to prepare the COF film loaded with chitosan by uniformly mixing COF powder in chitosan pre-polymerization liquid by ultrasound, freezing the mixture in liquid nitrogen and then carrying out a freeze-drying method. Secondly, placing the chitosan film in COF reaction liquid, heating by oil bath to enable COF to be polymerized on the surface of the chitosan film in situ, and thus preparing the chitosan-loaded COF film.

The technical scheme adopted by the invention is as follows:

two different chitosan-supported COF films CM-COF-I and CM-COF-II are synthesized through two ideas. One is to uniformly suspend the synthesized COF powder in a CM pre-polymerization solution, and finally embed the COF powder in a CM-COF-I film by a freeze drying method. And the other method is that the prepared CM film is immersed in COF reaction solution, amino on the surface of the CM reacts with aldehyde in the reaction solution, so that COF is subjected to in-situ polymerization on the surface of CM-COF-II, and the chitosan-loaded COF film is prepared.

The chitosan-loaded COF film material prepared by the invention can be applied to enrichment of glycosylated peptide segments, and the result shows that the material has good enrichment capacity on the glycosylated peptide segments by carrying out glycopeptide enrichment on IgG trypsin hydrolysate. The material can also be applied to copper ion adsorption, and the result shows that the material has good adsorption capacity on copper ions by adsorbing the copper ions in water.

The invention has the following beneficial effects and advantages:

according to the invention, the chitosan bionic film is used as a carrier, a newly reported COF is loaded, so that the CM film which does not have a mesoporous structure originally has a hierarchical pore structure, the specific surface area is obviously increased, and the capability of adsorbing copper ions is endowed for the CM film. As a combination of COF and an integral material, which is rarely reported at present, the CM-COF film has the advantages of convenient storage and use compared with powdery COF, simple preparation process and low cost and easily obtained raw materials. In addition, the CM-COF film retains the excellent enrichment capacity of the chitosan film on the glycosylated peptide section, so that the chitosan film becomes a novel multifunctional hierarchical porous film material

Drawings

FIG. 1 is a schematic diagram of the preparation of TpODH.

FIG. 2 is a schematic diagram of the preparation of CM-COF-I.

FIG. 3 is a schematic diagram of the preparation of CM-COF-II.

FIG. 4 is a scanning electron micrograph of TpODH.

FIG. 5 is a scanning electron micrograph of CM-COF-I.

FIG. 6 is a scanning electron micrograph of CM-COF-II.

FIG. 7 shows nitrogen adsorption-desorption curves for TpODH, CM-COF-I and CM-COF-II.

FIG. 8 shows pore size distributions of TpODH, CM-COF-I and CM-COF-II.

FIG. 9 is an XRD spectrum of TpODH, CM-COF-I and CM-COF-II.

FIG. 10 is a MALDI-TOF mass spectrum of the glycopeptide enrichment and sugar cleavage with different acetonitrile concentrations in example 1.

FIG. 11 is a standard curve of copper ions in example 2.

Detailed Description

Example 1

Preparation of TpODH: the preparation process of TpODH is shown in fig. 1. To an ampoule, 18mg of ODH (0.15mmol) and 21mg of Tp (0.10mmol) were added, and 500. mu.L of anhydrous 1, 4-dioxane and 500. mu.L of mesitylene were added, and after sonication for 30min, 0.4mL of an aqueous acetic acid solution (6M) was added. And (3) rapidly freezing the ampoule bottle in liquid nitrogen, and after the ampoule bottle is completely frozen, pumping until the internal pressure is lower than 5Pa, and sealing the ampoule bottle by using a flame spray gun. The ampoule containing the reaction was placed in an oil bath and heated at 120 ℃ for 72 h. The resulting reddish brown solid was finally washed with DMF and THF and dried under vacuum at 100 ℃ overnight to give 30mg of dried COF powder.

Preparation of CM-COF-I: the preparation process of CM-COF-I is shown in FIG. 2. 30mg of chitosan is added into a centrifuge tube, then 3mL of 1% diluted acetic acid is added into the centrifuge tube, and the mixture is stirred until the chitosan is dissolved. 3mg of polyethylene glycol diglycidyl ether was added to the centrifuge tube as a crosslinking agent and sonicated for 30 min. Taking 30mg of TpODH, carrying out ultrasonic treatment for 30min, and then quickly pouring the obtained suspension into a copper mold. And (3) placing the copper mould on the surface of liquid nitrogen for 5min to completely freeze the uniform suspension in the mould. Then the copper mold is put into a vacuum freeze dryer for freeze drying for 24 h. And finally, putting the copper mold into an oven, heating at 80 ℃ for 12-24h to obtain a chitosan-supported COF film CM-COF-I, wherein the porosity of the obtained film is about 85%, and the COF accounts for 50% of the mass ratio of the material.

Preparation of CM-COF-II: the preparation process of CM-COF-II is shown in FIG. 3. 30mg of CM film was placed in an ampoule, and 18mg of ODH and 21mg of TP were added. Then adding 500. mu.L of anhydrous 1, 4-dioxane and 500. mu.L of mesitylene into the ampoule bottle, and carrying out ultrasonic treatment for 30 min. Immersing the obtained mixture into liquid nitrogen for quick freezing, pumping until the internal pressure is lower than 5Pa after the mixture is completely frozen, and sealing the ampoule bottle by using a flame spray gun. The ampoule containing the reaction was placed in an oil bath and heated at 120 ℃ for 72 h. And finally, washing the obtained reddish brown film with DMF and THF, standing at room temperature for 24h, and then drying in vacuum at 100 ℃ overnight to obtain 60mg of a dried CM-COF-II film, wherein the porosity of the obtained film is about 85%, and the COF accounts for 50% of the material by mass.

FIG. 4 is a scanning electron microscope image of TpODH, which shows that TpODH is a chrysanthemum-like crystal, and is formed by aggregating petal-like crystals having a length of about 2 μm, a width of 150 to 300nm, and a thickness of 20 to 40 nm.

FIG. 5 is a scanning electron micrograph of CM-COF-I, from which it can be seen that there are various loading modes of COF in CM-COF-I, including a full wrapping mode (4A) in which the surface is completely covered by chitosan, a similar transmembrane protein (4B), and a ring-holding mosaic mode (4C) embedded in the film and similar to the fixing gem of the ring-holding claw. It can be seen from the figure that there are different degrees of clogging of the surface of the COF.

FIG. 6 is a scanning electron micrograph of CM-COF-II, from which it can be seen that CM-COF-II has an ordered pore structure, the microstructure is similar to honeycomb, the thickness of the pore wall is about 100nm, the pore diameter is between 20-100 μm, the COF in CM-COF-II uniformly covers the surface of the pore channel in the film, and the crystal shape is the same as that of the COF synthesized separately. This indicates that COF was successfully supported on chitosan films.

FIG. 7 is a nitrogen adsorption-desorption curve of CM, TpODH, CM-COF-I and CM-COF-II.

Table 1 shows specific surface areas of CM, TpODH, CM-COF-I and CM-COF-II. It can be seen from the table that the specific surface area of CM before COF loading is only 0.4m² g-¹The specific surface area of the COF was 298.1m² g-¹The specific surface area of CM-COF-I is smaller than that of CM-COF-II, which is caused by the fact that COF in CM-COF-I is coated with chitosan.

FIG. 8 is a graph showing pore size distributions of CM, TpODH, CM-COF-I and CM-COF-II. It can be seen from the figure that the pore size distribution of CM before COF loading is not uniform (fig. 8A). The pore diameter of COF is mainly distributed around 4nm (fig. 8B). Since the COF supported in the CM-COF-I film was coated with chitosan, no pores concentrated at around 4nm like the COF were present in the CM-COF-I-1 and CM-COF-I-2 (FIG. 8C, D). And CM-COF-II shows a large number of pores with the same size of COF around 4nm (FIG. 8E-I).

FIG. 9 is an XRD spectrum of TpODH, CM-COF-I and CM-COF-II. As shown in fig. 9, the COF shows a strong peak at 4.5 ° (± 0.2, 2 θ). CM-COF-I and CM-COF-II also show peaks around 4.5 deg., but the peak intensity of CM-COF-II is lower than COF due to different degrees of encapsulation by chitosan, while the peak intensity of CM-COF-I is the lowest, which indicates that the COF structure supported on the film is not damaged.

Example 2

3 portions of 1mg of CM-COF-II material prepared in example 1 were each taken in parallel and each enriched with buffer (volume ratio: ACN/H)₂O/TFA-83/16/1, 85/14/1,87/12/1, v/v/v) 3 rinses with 200 μ L each. Then 200 μ L of enrichment buffer containing 1 μ g IgG protein enzymolysis solution is added, and incubated in an oscillator at 25 deg.C for 30 min. Centrifuging the mixture at 2000rpm for 2min, discarding the solution, and eluting with three different enrichment buffers for 3 times with 200 μ L each time; the captured glycopeptides were then eluted 2 times for 10min at room temperature using 100. mu.L of eluent (ACN/H2O/TFA 30/69/1, v/v/v). Finally, the eluate will be analyzed by MALDI-TOF-MS. The results are shown in FIG. 10. When the IgG trypsin hydrolysate was directly analyzed by MALDI-TOF-MS, a large number of non-glycopeptide signals with high intensity were observed in FIG. 10A, and the glycopeptide signals were strongly suppressed. After enrichment, a significant amount of non-glycopeptide signal was still observed when ACN/H2O/TFA (85/14/1, v/v/v) was used as loading solution, but significantly reduced compared to the previous (FIG. 10B); when ACN/H2O/TFA (83/16/1, v/v/v) was used as the loading solution, it was found that the glycopeptide signal in fig. 10C was mainly higher in intensity, and the signal of non-glycopeptides was significantly reduced compared to the previous one, and 26 glycosylated peptide fragments were detected (table 2), indicating that the non-glycosylated peptide fragments were eluted and the glycosylated peptide fragments were better retained; the glycopeptide signal was significantly weaker when ACN/H2O/TFA (81/19/1, v/v/v) was used as the loading solution (FIG. 10D). The peptide fragment enriched with ACN/H2O/TFA (83/16/1, v/v/v) as the loading solution was deglycosylated with PNGaseF and analyzed by MALDI, and 2 strongly signaled deglycosylated peptide fragments were found in FIG. 10E, indicating thatCM-COF-II has excellent glycopeptide enrichment ability.

Example 3

511.44mg of CuCl were weighed out₂·H₂And O is subjected to constant volume in a 500mL volumetric flask. 10mg of CM-COF-II prepared in example 1 was put into a 15mL centrifuge tube, 10mL of the above copper chloride solution was added, and the reaction was stirred at room temperature for 24 hours. Dissolving another 1g of starch in 10mL of cold water, adding 90mL of boiling water, dissolving into a transparent colloid, and standing overnight for later use. 1.068g of NH were taken₄Cl was dissolved in 40mL of water and 1.44mL of NH was added₃·H₂O, adjusting the pH to 9.25 to obtain NH₄Cl-NH₃·H₂And (4) O buffer solution. 500mg of sodium diethyldithiocarbamate was weighed out of the dark and dissolved in a 50mL brown volumetric flask to constant volume. Preparing standard solutions with copper ion concentrations of 0.5, 1.0, 1.5, 2.0 and 2.5. mu.g mL-1, adding into 25mL volumetric flasks, respectively, adding 2mL buffer solution, and adding 1mL 10g L^-1Starch, and finally adding 0.2. mu.L of copper reagent and fixing the volume. Standard curves were drawn by uv analysis. Taking 164 mu L of copper chloride solution adsorbed by the chitosan-loaded COF film, respectively adding a buffer solution, starch and a copper reagent according to the previous steps, and comparing the volume with a standard curve through ultraviolet analysis after constant volume. Calculated that the maximum adsorption capacity of CM-COF-II to copper ions can reach 131mg g^-1。

FIG. 11 is a standard curve for copper ions.

Table 3 shows the adsorption amount of copper ions by CM-COF-II.

TABLE 1

TABLE 2

TABLE 3

The invention relates to preparation of a chitosan loaded Covalent Organic Framework (COF) bionic thin film material. The preparation method has the advantages of mild conditions, simplicity and low cost. The prepared film material can be applied to enrichment of glycosylated peptide segments, and efficient enrichment of the glycosylated peptide segments is realized; meanwhile, the copper ion adsorption material can also be applied to copper ion adsorption and has good adsorption capacity on copper ions.

Claims (8)

1. A COF-loaded chitosan bionic thin film material is characterized in that: the method comprises the following steps of (1) cross-linking chitosan and polyethylene glycol diglycidyl ether by using a freeze-drying method to prepare a film with the thickness of about 1-2 mm, and loading COF on the surface of the film, so that the COF with mesopores of about 3-4 nm is loaded on the surface of a 1-20 mu m honeycomb macroporous pore channel in the chitosan film and the surface of the chitosan film, thereby forming a hierarchical porous film material; the porosity is 80-90%, and the COF accounts for 10-50% of the total mass.

2. A preparation method of a COF-loaded chitosan biomimetic thin film material is characterized by comprising the following steps:

ultrasonically and uniformly mixing COF powder containing a plurality of imino groups into acetic acid solution of chitosan and polyethylene glycol diglycidyl ether, and preparing a COF-loaded chitosan film by one step through a freeze-drying method;

or, firstly, preparing a chitosan film by adopting chitosan and polyethylene glycol diglycidyl ether, dissolving a functional monomer containing two imino groups and two amino groups and a functional monomer containing three phenolic hydroxyl groups into anhydrous 1, 4-dioxane and mesitylene, adding a chitosan film and an initiator, sealing a reactor, and heating in an oil bath to polymerize COF (chip on film) in situ on the surface of the chitosan film to prepare the COF-loaded chitosan film;

the COF containing a plurality of imino groups is TpODH, the functional monomer containing two imino groups and two amino groups is oxalic acid dihydrazide (ODH), the functional monomer containing three phenolic hydroxyl groups is trialdehyde phloroglucinol (Tp), and the initiator is acetic acid.

3. A method for preparing a film material according to claim 2, wherein:

the operation can be carried out in either of two ways,

the method comprises the following steps:

1) the prepared TpODH has a specific surface area of 200-300 m²The pore diameter is 3-4 nm, and the porosity is 70% -80%;

2) adding 100-200 mg of chitosan into a reaction container;

3) adding 50-200 mL of dilute acetic acid with the mass concentration of 1% -5% into the reaction container in the step 2) as a solvent;

4) adding 10-20 mg of polyethylene glycol diglycidyl ether into the solution obtained in the step 3) as a crosslinking agent;

5) carrying out ultrasonic treatment on the solution obtained in the step 4) for 30-60 min;

6) adding 10-50 mL of the solution obtained in the step 5) into a centrifuge tube;

7) adding 10-100 mg of TpODH obtained in the step 1) into the centrifugal tube obtained in the step 6);

8) carrying out ultrasonic treatment on the suspension obtained in the step 7) for 30-60 min;

9) pouring the suspension liquid obtained in the step 8) into a mould quickly;

10) placing the mould in the step 9) on the surface of liquid nitrogen for 5-10 min to completely freeze the uniform suspension in the mould;

11) putting the mould in the step 10) into a vacuum freeze dryer for freeze drying for 24-48 h;

12) putting the die in the step 11) into an oven, and heating at 60-80 ℃ for 12-24h to obtain a chitosan-loaded COF film;

or, method 2:

1) preparing and obtaining a chitosan film;

2) adding 20-50 mg of the chitosan film obtained in the step 1) into an ampoule bottle;

3) adding 10-20 mg of oxalic dihydrazide and 20-40 mg of trialdehyde phloroglucinol into the ampoule bottle in the step 2);

4) adding 500-1000 mu L of anhydrous 1, 4-dioxane and 500-1000 mu L of mesitylene into the ampoule bottle in the step 3) as a solvent;

5) carrying out ultrasonic treatment on the solution obtained in the step 4) for 30-60 min;

6) adding an initiator acetic acid aqueous solution (6-10 mol L) into the solution obtained in the step 5)^-1)400～800μL；

7) Placing the ampoule bottle in the step 6) in liquid nitrogen for 1-2 min to completely freeze the solution;

8) vacuumizing the ampoule bottle in the step 7), and sealing the bottle opening by flame;

9) placing the ampoule bottle in the step 8) in an oil bath for reaction at 100-120 ℃ for 48-96 h;

10) and (3) sequentially and respectively washing the product obtained in the ampoule bottle in the step 9) with mesitylene (DMF) and Tetrahydrofuran (THF), and drying to obtain the chitosan-loaded COF film.

4. The production method according to claim 3, characterized in that:

preparation of TpODH: adding 10-20 mg of oxalic acid dihydrazide and 20-40 mg of trialdehyde phloroglucinol into an ampoule bottle, adding 500-1000 mu L of anhydrous 1, 4-dioxane and 500-1000 mu L of mesitylene, performing ultrasonic treatment for 30-60 min, and adding 400-800 mu L of acetic acid aqueous solution (6-10 mol L)^-1)；

Rapidly freezing the ampoule bottle in liquid nitrogen, vacuumizing after complete freezing, and sealing the ampoule bottle by using a flame spray gun; placing the ampoule bottle containing the reactant in an oil bath pan, and heating for 48-96 h at 100-120 ℃; finally, the resulting reddish brown solid was washed with DMF and THF in order and dried under vacuum at 100 ℃ overnight to give dry COF powder TpODH.

5. The production method according to claim 3, characterized in that: the preparation process of the chitosan film comprises the steps of 4) ultrasonic treatment for 30-60 min, 6) freezing for 1-2 min, and 8) heating for 48-96 h.

6. A chitosan-supported COF film material prepared by the preparation method of any one of claims 1 to 5.

7. The film material of claim 6 can be used as an adsorbent for enrichment of glycosylated peptide fragments and/or copper ion adsorption.

8. The film material according to claim 7 can be used as an adsorbent for enriching glycosylated peptide segments in an immunoglobulin G (IgG) enzymolysis solution or adsorbing copper ions in water.

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