CN110665465A - Magnetic covalent organic framework material for glycopeptide enrichment and preparation method and application thereof - Google Patents

Abstract

The invention discloses a magnetic covalent organic framework material for glycopeptide enrichment, and a preparation method and application thereof₃O₄Nano magnetic ball coated on Fe₃O₄The preparation method comprises the following steps: preparation of Fe₃O₄/COFs nanoparticles, preparation of Fe₃O₄a/COF-GSH material; the magnetic covalent organic framework material is shown to be enriched in glycopeptideThe method has the advantages of high selectivity, strong binding capacity, high enrichment efficiency, good recovery efficiency and the like, has very important significance in the research of the glycosylation process of physiological behaviors, and has good application prospect.

Description

Magnetic covalent organic framework material for glycopeptide enrichment and preparation method and application thereof

technical field

The invention belongs to the technical field of biological materials, and relates to a magnetic covalent organic framework material, a preparation method thereof, and an application in the enrichment of endogenous glycopeptides.

Background technique

The existing magnetic covalent organic framework materials are composed of magnetic nanospheres and hydrophilic covalent organic frameworks (COFs) wrapped on the surface of the magnetic nanospheres. It has unique properties such as high porosity, highly ordered mesopores, abundant organic ligands, and structural stability. Therefore, magnetic covalent organic framework materials have attracted much attention in recent years and have been widely used in the field of biomedicine, especially protein or peptide separation, drug delivery, magnetic resonance imaging and so on.

At present, the application of magnetic covalent organic framework materials for the enrichment of glycopeptides basically adopts the method of hydrophilic interaction chromatography, that is, only the hydrophilicity of the covalent organic framework itself is used to enrich the glycopeptides. Xiangmin Zhang et al. disclosed a preparation method of a covalent organic framework modified magnetic graphene composite material, and gave the application of the composite material in glycopeptide enrichment, the covalent organic framework modified magnetic graphene composite material Graphene is used as the base material, then the surface of graphene is decorated with nano-magnetic balls, and finally a layer of 2,3,6,7,10,11-hexahydroxytriphenyl (HHTP) is modified on the surface of the graphene modified by the magnetic balls. ) and terephenyldiboronic acid (BDBA) as the covalent organic framework layer of the organic ligand, namely the magnetic graphene composite material modified by the covalent organic framework is obtained, and the magnetic composite material is based on the hydrophilic properties of the covalent organic framework. Enrichment of glycopeptides (Self-assembling covalent organic framework functionalized magneticgraphene hydrophilic biocomposites as an ultrasensitiv matrix for N-linked glycopeptide recognition, Nanoscale, 2017, 9, 10750-10756, Xiangmin Zhang et al.).

However, due to the limitation of hydrophilic properties of covalent organic framework materials and the lack of flexible functional modification, the above-mentioned covalent organic framework-based composite materials are not very ideal for the enrichment of glycopeptides.

SUMMARY OF THE INVENTION

The purpose of the present invention is to aim at the problems existing in the above-mentioned prior art, to provide a magnetic covalent organic framework material for glycopeptide enrichment and a preparation method thereof, so as to improve the hydrophilicity of the covalent organic framework material and achieve Efficient enrichment of glycopeptides.

The magnetic covalent organic framework material for glycopeptide enrichment according to the present invention is composed of Fe ₃ O ₄ nano-magnetic spheres, a covalent organic framework (COF) layer coated on the surface of Fe ₃ O ₄ nano-magnetic spheres, a grafted Glutathione (GSH) on a covalent organic framework layer; the covalent organic framework layer is composed of 1,3,5-tris(4-aminophenyl)benzene and 2,5-divinyl- 1,4-benzenedicarboxaldehyde is a honeycomb structure obtained by addition reaction according to the ratio of substance amount 1:1.5, and the glutathione is grafted on the vinyl group of the covalent organic framework layer.

The magnetic covalent organic framework material for glycopeptide enrichment of the present invention presents a complete spherical shape, with a uniform particle size and a narrow distribution, and the average particle size is about 240-300 nm. The nanoparticle with regular shape and uniform size It is more suitable for enrichment and separation of proteins and peptides. The magnetic covalent organic framework material uses superparamagnetic triiron tetroxide (Fe ₃ O ₄ nanometer magnetic balls) as the core, and has high magnetic saturation strength, so that it has good magnetic response performance to an external magnetic field; Fe ₃ O ₄ nanomagnetic spheres account for about 48% of the mass of the magnetic covalent organic framework material, so that the saturation magnetization of the magnetic covalent organic framework material reaches about 45 emu g ^-1 . The COF layer coated on the surface of Fe ₃ O ₄ nanomagnetic spheres has high porosity, highly ordered mesopores, and large specific surface area, which is beneficial for the enrichment application of glycopeptides. Two kinds of organic ligands, 1,3,5-tris(4-aminophenyl)benzene and 2,5-divinyl-1,4-benzenedicarboxaldehyde, are selected as the raw materials of the above COF layer. Among them, for the 2,5-divinyl-1,4-benzenedicarboxaldehyde organic ligand, on the one hand, it is one of the components for the synthesis of covalent organic frameworks, and at the same time, the remaining carbon-carbon double bonds can be used to further introduce high affinity Aqueous zwitterionic glutathione as reactive site. In the present invention, glutathione (GSH) is modified on the COF layer by a high-efficiency thioene click method, and GSH is a zwitterion, thereby greatly improving the hydrophilic performance of the magnetic covalent organic framework material based on COFs, overcoming The limitations of the existing COFs composites in terms of hydrophilic properties are overcome, and GSH can achieve high-efficiency enrichment of glycopeptides.

The preparation method of the magnetic covalent organic framework material for glycopeptide enrichment of the present invention is mainly realized by the mechanism of epitaxial growth, and the surface of the magnetic sphere is wrapped with a layer of covalent covalent by the method of epitaxial growth under mild conditions. The organic framework layer (two ligands form a COF layer through covalent bonding), and then the surface of the covalent organic framework layer is modified with highly hydrophilic zwitterion glutathione by the method of efficient thioene click.

The preparation method of the magnetic covalent organic framework material for glycopeptide enrichment according to the present invention, the steps are as follows:

(1) Preparation of Fe ₃ O ₄ /COFs nanoparticles

Under ultrasonic conditions, Fe ₃ O ₄ nanomagnetic spheres, 1,3,5-tris(4-aminophenyl)benzene and 2,5-divinyl-1,4-benzenedicarboxaldehyde were mixed in dimethylmethylene The sulfone is mixed and dispersed uniformly to form a mixed solution; then, under the condition of ultrasonic, acetic acid is added dropwise to the above mixed solution to form a reaction system; then the obtained reaction system is left to incubate at room temperature for 10-30 minutes; The liquid is magnetically separated and the separated solid product is collected, the obtained solid product is washed to remove the unreacted material adsorbed on the surface of the solid product, and the covalent organic framework layer is coated with Fe ₃ O ₄ nanometer magnetic ball nanoparticles, referred to as Fe ₃ O ₄ /COFs nanoparticles; the mass ratio of the Fe ₃ O ₄ nano-magnetic spheres to 1,3,5-tris(4-aminophenyl)benzene is 1:(0.2～1),1,3,5 - the substance ratio of tris(4-aminophenyl)benzene to 2,5-divinyl-1,4-benzenedicarboxaldehyde is 1:1.5, the 1,3,5-tris(4-aminobenzene) The volume ratio of the sum of the mass of phenyl)benzene and 2,5-divinyl-1,4-benzenedicarboxaldehyde to acetic acid is (30-210): 1, the unit of mass is mg, and the volume of The unit is mL;

(2) Preparation of Fe ₃ O ₄ /COF-GSH magnetic covalent organic framework material

Azobisisobutyronitrile, glutathione and Fe ₃ O ₄ /COFs nanoparticles were sequentially added to the composite solvent to form a mixed solution, and then under nitrogen protection, the obtained mixed solution was subjected to mercaptoene click at 60-80 °C Reaction until the surface of Fe ₃ O ₄ /COFs nanoparticles turns yellow; after the completion of the mercaptoene click reaction, the obtained reaction solution is subjected to magnetic separation and the separated solid product is collected, and the obtained solid product is washed to remove unreacted materials adsorbed on the surface of the solid product After drying, a magnetic covalent organic framework material for glycopeptide enrichment, referred to as Fe ₃ O ₄ /COF-GSH material for short, is obtained; the composite solvent is uniformly obtained by mixing ethanol and deionized water in a volume ratio of 1:3, In the mixed solution, the mass ratio of Fe ₃ O ₄ /COFs nanoparticles to glutathione is 1:(0.3～1); the mass ratio of the glutathione to azobisisobutyronitrile is 1: (0.05～0.25).

In the above preparation method of magnetic covalent organic framework material for glycopeptide enrichment, in step (1), the Fe ₃ O ₄ nano-magnetic balls are mainly made of ferric chloride, sodium acetate and sodium citrate as raw materials, with The superparamagnetic iron tetroxide nanospheres with a particle size of about 200nm-250nm were synthesized by a hydrothermal method using ethylene glycol as a solvent; in addition, the particle size distribution of the magnetic spheres can be adjusted by adjusting the hydrothermal reaction time. The specific implementation of the preparation of Fe ₃ O ₄ nanomagnetic spheres can be obtained by referring to the conventional preparation methods disclosed in the prior art, see The design and synthesis of a hydrophilic core-shell-shell structured magnetic metal-organic framework as a novel immobilized metal ionaffinity platform for phosphoproteome research Chem. Commun., 2014, 50, 6228-6231, Chunhui Deng et al. and Ti ⁴⁺ -immobilized multilayer polysaccharide coated magnetic nanoparticles for highly selective enrichment of phosphopeptides J.Mater.Chem.B 2014,2,4473-4480, Hanfa Zou et al.

In the above preparation method of the magnetic covalent organic framework material for glycopeptide enrichment, in step (1), two ligands are prepared by addition reaction to obtain the COF layer, wherein acetic acid is used as a catalyst, Fe ₃ O ₄ nanomagnetic ball as a carrier. Under ultrasonic conditions, Fe ₃ O ₄ nanomagnetic spheres, 1,3,5-tris(4-aminophenyl)benzene and 2,5-divinyl-1,4-benzenedicarboxaldehyde were mixed and dispersed uniformly. Ultrasonic 5~10min is enough; the amount of dimethyl sulfoxide is as long as it can completely dissolve 1,3,5-tris(4-aminophenyl)benzene and 2,5-divinyl-1,4-benzenedicarboxaldehyde , and the Fe ₃ O ₄ nano-magnetic balls can be uniformly dispersed. Then, under ultrasonic conditions, add acetic acid dropwise to the mixed solution, and the dropwise addition time is generally controlled to be 5 to 15 minutes. After the dropwise addition of acetic acid, the obtained mixed solution is left to incubate at room temperature for 10-30 minutes, and the solid product in the obtained reaction solution is Fe ₃ O ₄ /COFs nanoparticles. Therefore, the reaction solution is subjected to solid-liquid separation and the solid product is washed to obtain Fe ₃ O ₄ /COFs nanoparticles. The purpose of washing is to remove the unreacted material adsorbed on the surface of the solid product. The washing method adopted in the present invention is: the isolated solid product is washed successively with anhydrous tetrahydrofuran, anhydrous methanol, ethanol, and deionized water. The lotion can be washed 3 to 5 times.

In the above preparation method of the magnetic covalent organic framework material for glycopeptide enrichment, in step (2), the zwitterion glutathione is modified on the surface of the covalent organic framework layer by a high-efficiency thioene click method. The reaction needs to be carried out under anoxic conditions. Before the mixed solution obtained in the present invention reacts under nitrogen protection, deoxidation treatment is performed to remove oxygen in the mixed solution and above the mixed solution in the reactor. The specific treatment method is as follows: firstly, azobisisobutyronitrile, glutathione and Fe ₃ O ₄ /COFs nanoparticles are added to the composite solvent containing deionized water and ethanol in turn and ultrasonically mixed; then nitrogen purging is used. Deoxidize the mixed solution, and the deoxidation time is about 0.5-2h; then the oxygen in the reactor is removed by the cyclic operation mode of vacuum pumping and nitrogen passing, generally circulating 3 to 5 times. In each cycle, the reactor is first Evacuate the interior to a degree of vacuum not greater than 100Pa, and then pass nitrogen to normal pressure. In order to facilitate the later cleaning operation, the ultrasonic mixing vessel for adding azobisisobutyronitrile, glutathione and Fe ₃ O ₄ /COFs nanoparticles into the composite solvent in turn can be different from the reactor for the subsequent thioene click reaction. , after the mixed solution and the reactor are deoxidized respectively, then the deaerated mixed solution is transferred to the deoxidized reactor, and then reacted under the condition of magnetic stirring at 60-80 ℃ until the surface of the nanoparticles turns yellow, And the magnetic nanoparticles in the mixed solution are well dispersed and there is no wall sticking phenomenon, and the general reaction time is about 6-24h. The solid product in the obtained reaction solution is Fe ₃ O ₄ /COF-GSH material. Therefore, the solid-liquid separation of the reaction solution, the washing and drying of the solid product, and the Fe ₃ O ₄ /COF-GSH magnetic covalent organic framework material are obtained. The purpose of washing is to remove the unreacted material adsorbed on the surface of the solid product. The washing method adopted in the present invention is as follows: the separated solid product is washed with ethanol and deionized water in turn. Generally, each washing solution is washed 3 to 5 times. Can.

The present invention further provides the application of the magnetic covalent organic framework material in the enrichment of endogenous glycopeptides. The magnetic covalent organic framework material has a very good enrichment effect for glycopeptides in biological samples, especially in the process of enriching endogenous glycopeptides in human saliva, showing extremely excellent performance. It is very important in the process of behavioral protein glycosylation.

The operation of enriching glycopeptides in the magnetic covalent organic framework material of the present invention is as follows:

(1) Adsorption of glycopeptides: First, the glycosylated protein immunoglobulin G is digested into glycopeptides with trypsin, and diluted with buffer, then the magnetic covalent organic framework material of the present invention is added and mixed and stirred evenly, and then Shake with a shaker for 10-60 min at room temperature to enrich the glycopeptides on the surface of the magnetic covalent organic framework material, and then use magnetic separation under the action of an external magnetic field to separate the magnetic covalent organic framework material with glycopeptides on the surface from the solution. separated from

(2) Desorption of glycopeptides: adding the magnetic covalent organic framework material with glycopeptides adsorbed on the surface into the desorption solution to desorb the glycopeptides from the magnetic covalent organic framework material.

In the above-mentioned adsorption process for glycopeptides, the modified highly hydrophilic molecule GSH on the covalent organic framework utilizes the principle of hydrophilic interaction chromatography to realize the enrichment of glycopeptides. In the magnetic covalent organic framework material provided by the present invention, the COF layer is a highly ordered mesoporous material with a pore size of about 3.6 nm, which can block macromolecules (proteins, exosomes, etc.) Therefore, it is beneficial to the enrichment and application of endogenous glycopeptide peptides. By changing the components of the buffer, the adsorbed glycopeptides are detached from the surface of the magnetic covalent organic framework material, thereby realizing the capture and separation of the glycopeptides.

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

1. The magnetic covalent organic framework material provided by the present invention uses Fe ₃ O ₄ nano-magnetic spheres as the core, and introduces 1,3,5-tris(4-aminophenyl) benzene on the surface of Fe ₃ O ₄ nano-magnetic spheres. A covalent organic framework composed of 2,5-divinyl-1,4-benzenedicarboxaldehyde, using the remaining carbon-carbon double bond on 2,5-divinyl-1,4-benzenedicarboxaldehyde as the reaction site , the hydrophilic zwitterion GSH was modified by the thioene click reaction, the material not only exhibited good magnetic response properties, but also greatly improved the COF-based The hydrophilic properties of magnetic covalent organic framework materials show the advantages of high selectivity, strong binding ability, high enrichment efficiency, and good recovery efficiency in glycopeptide enrichment, which are very important in the study of physiological behavior glycosylation process. significance, and the application prospect is good.

2. In the magnetic covalent organic framework material provided by the present invention, a covalent organic framework is introduced on the surface of the Fe ₃ O ₄ nano-magnetic sphere, which has a high specific surface area and a highly ordered mesoporous structure, which makes the magnetic covalent organic framework material. It is very suitable for enrichment and separation of proteins or peptides, especially endogenous glycopeptides.

3. The preparation method of the magnetic covalent organic framework material provided by the present invention firstly adopts an epitaxial growth method to wrap a COF layer on the surface of the Fe ₃ O ₄ nano-magnetic sphere, and then adopts a high-efficiency thioene click reaction to graft on the COF layer. The hydrophilic molecule GSH has simple operation and mild reaction conditions in the whole process, and the magnetic covalent organic framework material can be prepared in a short time, so it is easy to be popularized in the field of biomedicine.

Description of drawings

Fig. 1 is the flow chart of the preparation process of the magnetic covalent organic framework material for glycopeptide enrichment according to the present invention and the flow chart of the enrichment of glycopeptides, A is the preparation process flow chart of the magnetic covalent organic framework material, and B is the magnetic covalent organic framework material. Flow chart for the enrichment of glycopeptides by covalent organic framework materials.

2 is a characterization diagram of the magnetic nanomaterials prepared in the embodiment of the present invention, wherein A is the scanning electron microscope (SEM) image of the Fe ₃ O ₄ nanomagnetic spheres prepared in the embodiment 1, and B is the Fe ₃ O prepared in the embodiment 2. ₄ / The scanning electron microscope (SEM) image of the COFs nanoparticles, C is the scanning electron microscope (SEM) image of the Fe ₃ O _/ COF-GSH material prepared in Example 12, D is the Fe ₃ O ₄ nanomagnetic ball prepared in Example 1 The transmission electron microscope (TEM) image of the Fe ₃ O ₄ /COFs nanoparticles prepared in Example 2, E is the scanning electron microscope (TEM) image of the Fe ₃ O ₄ /COF-GSH material prepared in Example 12 (SEM) image.

3 is an X-ray diffraction pattern of the magnetic nanomaterial prepared in the embodiment of the present invention, wherein a corresponds to the Fe ₃ O ₄ nanomagnetic ball prepared in the embodiment 1, b corresponds to the Fe ₃ O ₄ /COFs nanoparticle prepared in the embodiment 2, c corresponds to the Fe ₃ O ₄ /COF-GSH material prepared in Example 12.

FIG. 4 is a characterization diagram of the elemental composition of the Fe ₃ O ₄ /COF-GSH material prepared in Example 12 of the present invention.

5 is the nitrogen adsorption-desorption curve and mesopore size distribution diagram of the magnetic nanomaterials prepared in Example 2 and Example 12 of the present invention, wherein A is the nitrogen adsorption of Fe ₃ O ₄ /COFs nanoparticles prepared in Example 2 -Desorption curve, B is the nitrogen adsorption-desorption curve of Fe ₃ O ₄ /COF-GSH material prepared in Example 12, C is the mesopore size distribution of Fe ₃ O ₄ /COFs nanoparticles prepared in Example 2 , D is the mesopore size distribution diagram of Fe ₃ O ₄ /COF-GSH material prepared in Example 12.

6 is a hysteresis loop spectrum of the magnetic nanomaterial prepared in the embodiment of the present invention in the range of -18000Oe to 18000Oe, wherein a corresponds to the Fe ₃ O ₄ nanomagnetic ball prepared in Example 1, and b corresponds to Fe prepared in Example 2 ₃ O ₄ /COFs nanoparticles, c corresponds to the Fe ₃ O ₄ /COF-GSH material prepared in Example 12.

7 is the MS spectrum of the immunoglobulin G digestion solution in Application Example 1 of the present invention, wherein A is the MS spectrum of the immunoglobulin G digestion solution without enrichment treatment, and B is the Fe ₃ O ₄ prepared in Example 12 / MS image of immunoglobulin G digested solution enriched with COF-GSH material.

Detailed ways

The technical solutions of the present invention will be clearly and completely described below through the embodiments and in conjunction with the accompanying drawings. Obviously, the described embodiments are only a part of the embodiments of the present invention, but not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative work fall within the protection scope of the present invention.

The structure of the magnetic covalent organic framework material for glycopeptide enrichment proposed by the present invention is shown in Figure 1. The magnetic covalent organic framework material is composed of Fe ₃ O ₄ nano-magnetic spheres, which are coated on Fe ₃ O ₄ nano-magnetic The COF intermediate layer on the spherical surface and the zwitterion GSH grafted on the COF layer are composed. The present invention prepares the magnetic covalent organic framework material based on the process flow shown in FIG. 1. As shown in FIG. 1A, firstly, Fe ₃ O ₄ nano-magnetic spheres are prepared by hydrothermal method; The surface of Fe ₃ O ₄ magnetic nanospheres is coated with a layer of COF intermediate layer to obtain magnetic nanoparticles (Fe ₃ O ₄ /COFs nanoparticles ₎ coated with COF layer _. The Fe ₃ O ₄ /COF-GSH material was prepared by grafting onto the surface of Fe ₃ O ₄ /COFs nanoparticles.

Theoretically, the ratio of two organic ligands, 1,3,5-tris(4-aminophenyl)benzene and 2,5-divinyl-1,4-benzenedicarboxaldehyde, is 1 according to the amount of substance: 1.5 The COF layer of honeycomb structure with six-membered ring as the basic unit as shown in Fig. 1A is obtained by addition reaction. Each six-membered ring is formed from six 1,3,5-tris(4-aminophenyl)benzenes and six 2,5-divinyl-1,4-benzenedicarboxaldehydes spaced by covalent bonds. The theoretical mesopore size of the COF layer of this honeycomb structure is about 3.9 nm. Each 2,5-divinyl-1,4-benzenedicarbaldehyde has two carbon-carbon double bonds as the reactive sites for the mercaptoene click reaction.

It can be seen from the above analysis that the surface of the Fe ₃ O ₄ /COFs nanoparticles provided by the present invention can be modified with more zwitterion GSH, thereby greatly improving the hydrophilic properties of the magnetic covalent organic framework material.

Example ₁ Preparation of superparamagnetic _Fe3O4 nanomagnetic balls by hydrothermal method

The specific preparation process of the Fe ₃ O ₄ nano-magnetic balls adopted in the following examples is: adding raw materials 2.43g FeCl ₃ ·6H ₂ O, 3.6g NaAc (sodium acetate) and 0.4g Na ₃ CT (sodium citrate) to a In a reaction kettle with 60 mL of ethylene glycol, magnetic stirring for 1 h to mix the above raw materials uniformly; then remove the stirrer, heat the reaction kettle to 200 ° C, and react for 12 hours; then cool the reaction kettle to room temperature, and magnetically conduct the reaction liquid. Separate and collect the separated solid product; then wash the solid product with ethanol and deionized water in sequence (five times for each washing solution) to obtain Fe ₃ O ₄ nanomagnetic spheres.

_{The Fe3O4} nanomagnetic spheres obtained by this method can be uniformly dispersed in water to form a stable superparamagnetic nanoparticle suspension. The DLS (Dynamic Light Scattering, dynamic light scattering) analysis of the obtained Fe ₃ O ₄ nanomagnetic spheres shows that the Fe ₃ O ₄ magnetic spheres have a particle size of about 220 nm.

By adjusting the hydrothermal reaction time from 10 to 16 h, the particle size of the obtained Fe ₃ O ₄ nanomagnetic spheres can be adjusted to be between 200 and 300 nm.

Example 2-Example 8 Preparation of Fe ₃ O ₄ /COFs nanoparticles (select 220nm Fe ₃ O ₄ nano-magnetic balls)

Weigh the raw materials according to Table 1, and prepare Fe ₃ O ₄ /COFs nanoparticles according to the following operation process in combination with the process parameters given in Table 1:

Fe ₃ O ₄ nanomagnetic spheres, 1,3,5-tris(4-aminophenyl)benzene (Tab) and 2,5-divinyl-1,4-benzenedicarboxaldehyde (Dva) were combined with two organic compounds. The two organic ligands were completely dissolved under ultrasonic conditions, and the Fe ₃ O ₄ nano-magnetic spheres were completely dispersed to obtain a uniformly dispersed mixed solution. Under ultrasonic conditions, acetic acid was added dropwise to the obtained mixed solution, and after the dropwise addition, the entire reaction system was left to incubate at room temperature. Finally, the obtained reaction solution is subjected to magnetic separation and the separated solid product is collected. The obtained solid product is washed with anhydrous tetrahydrofuran, anhydrous methanol, ethanol and deionized water in turn (each washing solution is washed three times) to obtain Fe ₃ O ₄ /COFs nanoparticles. DLS (Dynamic Light Scattering, dynamic light scattering) analysis of the obtained Fe ₃ O ₄ /COFs nanoparticles showed that the obtained Fe ₃ O ₄ /COFs nanoparticles had a particle size of about 250-670 nm, as shown in Table 1.

Table 1 Raw materials and their ratios and process parameters for preparing Fe ₃ O ₄ /COFs nanoparticles

As can be seen from Table ₁ , the thickness of the COF interlayer and the morphology _of the nanocomposite can be adjusted by adjusting the ratio of the two organic ligands, the ultrasonic time, and the incubation time. Considering factors such as the coating firmness of the COF intermediate layer, the size, the morphology of the nanocomposite material, and the saving of medicines, the Fe ₃ O ₄ /COFs nanoparticles of 270 nm prepared by the method described in Example 2 are now selected for the following examples. Preparation of Fe ₃ O ₄ /COF-GSH Magnetic Covalent Organic Framework Materials.

Example 9-Example 16 Preparation of Fe ₃ O ₄ /COF-GSH materials (Fe ₃ O ₄ /COFs nanoparticles of 270 nm were selected)

The following examples 9-16 adopt the reaction method of mercaptoene click, and the reaction device used is a two-necked flask reaction vessel heated in an oil bath and magnetically stirred.

Weigh the raw materials according to Table 2, and prepare Fe ₃ O ₄ /COF-GSH material according to the following operation process in combination with the process parameters given in Table 2:

Azobisisobutyronitrile, glutathione and Fe ₃ O ₄ /COFs nanoparticles were sequentially added to a compound solvent containing 20 mL (the compound was obtained by uniformly mixing ethanol and deionized water according to a volume ratio of 1:3), and ultrasonic waves were used. Treatment, the Fe ₃ O ₄ /COFs nanoparticles are completely dispersed, and the azobisisobutyronitrile and glutathione are completely dissolved. Then, the mixed solution was deoxygenated by nitrogen purging for 1 h; and then the branch-mouth bottle as the reactor was deoxygenated by the cyclic operation mode of vacuuming and passing nitrogen (the number of cycles was 3). Subsequently, under the protection of nitrogen, the mixed solution after deoxygenation is added to the branch-neck flask that removes oxygen in advance, and then the reaction temperature described in Table 2 is obtained by the oil bath, and the reaction temperature described in Table 2 is obtained under magnetic stirring, The reaction time completes the thioene click reaction. Finally, the obtained reaction solution is magnetically separated and the separated solid product is collected. The obtained solid product is washed with ethanol and deionized water in turn (each washing solution is washed three times) to obtain Fe ₃ O ₄ /COF-GSH material. The obtained Fe ₃ O ₄ /COF-GSH material was analyzed by DLS (Dynamic Light Scattering, dynamic light scattering) to investigate its dispersibility in water: the more successful GSH modification was, the better the dispersion of Fe ₃ O ₄ /COF-GSH material in water. The better the performance, the smaller the PDI value of the DLS test.

Table 2 Raw materials and their proportions and process parameters for preparing Fe ₃ O ₄ /COF-GSH materials

It can be seen from Table 2 that the best GSH modification effect can be achieved by adjusting the amount of zwitterion GSH and catalyst AIBN, reaction temperature and reaction time. Considering factors such as modification effect, saving reaction time and saving raw materials, the scheme of Example 12 is now selected to prepare Fe ₃ O ₄ /COF-GSH material to study the structural characteristics, properties and enrichment of glycopeptides.

Structure Characterization

In order to explore whether COFs were successfully compounded on Fe ₃ O ₄ nanomagnetic spheres, the Fe ₃ O ₄ nanomagnetic spheres prepared in Example 1, the Fe 3 O 4 /COFs nanoparticles prepared in Example 2, and the Fe ₃ O ₄ /COFs nanoparticles prepared in Example 12 were tested The morphology, size and microstructure of the ₃ O ₄ /COF-GSH magnetic covalent organic framework material were characterized, as shown in Figures 2 to 5.

The Fe ₃ O ₄ /COFs nanoparticles prepared in Example 2 and the Fe ₃ O ₄ /COF-GSH magnetic covalent organic framework material prepared in Example 12 were morphologically characterized by scanning electron microscopy (SEM) and transmission electron microscopy (TEM). Analysis, the results are shown in Figure 2. It can be seen from Figure 2 that the prepared Fe ₃ O ₄ /COFs nanoparticles and Fe ₃ O ₄ /COF-GSH materials are spherical with uniform size and regular morphology. In addition, it can be seen from Figure 2E that the surface of the nanoparticles presents a rough network structure, indicating that the COFs layer has been successfully wrapped on the surface of the Fe ₃ O ₄ nanomagnetic spheres; from Figure 2F, it can be seen that the Fe ₃ O ₄ /COF-GSH material is closely related to Fe 3 O 4 . The morphology of ₃ O ₄ /COFs nanoparticles is almost the same, and the surface modification of zwitterion GSH has no obvious effect on the morphology of Fe ₃ O ₄ /COFs nanoparticles.

The Fe ₃ O ₄ nanomagnetic balls prepared in Example 1, the Fe ₃ O ₄ /COFs nanoparticles prepared in Example 2, and the Fe ₃ O ₄ /COF-GSH materials prepared in Example 12 were subjected to X-ray diffraction analysis, and the analysis results were As shown in Figure 3. It can be seen from Figure 3 that all three materials have characteristic peaks consistent with the standard Fe ₃ O ₄ diffraction peaks, indicating that the Fe ₃ O ₄ /COF-GSH material retains the crystal structure of Fe ₃ O ₄ nanomagnetic spheres. At the same time, Fe ₃ O ₄ /COF-GSH material has a new diffraction peak at 2.7°, which is the characteristic diffraction peak of covalent organic framework, indicating that the covalent organic framework was successfully synthesized on the surface of magnetic nanoparticles.

X-ray energy spectrum analysis (EDX) was performed on the Fe ₃ O ₄ /COF-GSH magnetic covalent organic framework material prepared in Example 12, and the analysis results are shown in FIG. 4 and Table 3. As can be seen from Figure 4 and Table 3, in the Fe ₃ O ₄ /COF-GSH magnetic covalent organic framework material, the atomic percentage of C element is 55.97%, the atomic percentage of N element is 15.52%, the atomic percentage of O element is 23.28%, and the atomic percentage of S element is 55.97%. The element atomic percentage is 2.01%, and the Fe atomic percentage is 3.22%. It can be seen from the above test results that there are S element and high content of N element in Fe ₃ O ₄ /COF-GSH material, which further proves that the COFs layer and zwitterion GSH have been successfully formed and modified on the surface of Fe ₃ O ₄ nanomagnetic spheres , high content of GSH is beneficial to the enrichment of glycopeptides.

Table 3 Atomic percentage of each element obtained in EDX analysis

Element Weight% Atomic% C 44.63 55.97 N 14.43 15.52 o 24.72 23.28 S 4.29 2.01 Fe 11.92 3.22 Total 100 100

The Fe ₃ O ₄ /COFs nanoparticles and Fe ₃ O ₄ /COF-GSH materials prepared in Example ₂ and Example ₁₂ were subjected to N adsorption/desorption tests. The test results are shown in Figure 5. The desorption isotherm curve shows that the magnetic covalent organic framework nanocomposite has a large specific surface area and porosity, and a narrow pore size distribution, which is about 3.6 nm (similar to the theoretical analysis results), indicating that it has a highly ordered mesostructure. The covalent organic framework of the porous material was successfully synthesized on the surface of the magnetic sphere, which is beneficial for the enrichment of peptides.

In summary, the shell layer in Fe ₃ O ₄ /COF-GSH material has a covalent organic framework structure, and this structure does not significantly affect the magnetocrystalline structure of the composite nanomaterials. This unique COF shell layer will be beneficial to Applications in glycopeptide capture and isolation.

Magnetic performance test

Model BHV-525 vibration samples were used for Fe ₃ O ₄ nanomagnetic balls prepared in Example 1, Fe ₃ O ₄ /COFs nanoparticles prepared in Example 2, and Fe ₃ O ₄ /COF-GSH materials prepared in Example 12 The magnetometer (VSM) conducts magnetic tests in the range of -18000Oe to 18000Oe, and the hysteresis loop obtained from the test is shown in Figure 6. It can be seen from Figure 6 that the hysteresis loops of all samples pass through the origin without remanence and coercivity, indicating that Fe ₃ O ₄ nanomagnetic balls, Fe ₃ O ₄ /COFs nanoparticles, Fe ₃ O ₄ / All COF-GSH materials are superparamagnetic, and the saturation magnetization of Fe ₃ O ₄ /COF-GSH material reaches about 45emug ^-1 .

Application example

The present invention further provides the application of the above Fe ₃ O ₄ /COF-GSH material in the enrichment of glycopeptides, the capture and separation process of the Fe ₃ O ₄ /COF-GSH material for glycopeptides, as shown in FIG. Fe ₃ O ₄ /COF-GSH material is added to the sample to be treated, and then captured in a shaker. The time can be adjusted according to the sample size. After the capture process, the solid product is separated by magnetic separation, and then used The desorbed buffer desorbs the magnetic spheres with glycopeptides adsorbed on the surface, thereby obtaining a buffer containing glycopeptides. The obtained buffer solution containing glycopeptides can be analyzed by MS (Mass Spectrometry, mass spectrometry) to further determine the enrichment effect of Fe ₃ O ₄ /COF-GSH material on glycopeptides.

Application example 1 Enrichment of glycopeptides in the digested solution of glycosylated protein immunoglobulin G

Dissolve 2 mg of immunoglobulin G in 1 ml of 50Mm NH ₄ HCO ₃ buffer with a pH of 8.2, add 50 μg of trypsin, and digest at 37 ° C for 16 h; then use the first buffer (volume concentration of 90% Aqueous acetonitrile solution containing 1% trifluoroacetic acid by volume (ie, 90% ACN-H ₂ O containing 1% TFA) was diluted to a concentration of 10 ^-7 M to obtain an immunoglobulin G digestion solution. Take 1 mg of Fe ₃ O ₄ /COF-GSH material prepared in Example 12 and add it to 200 μl of immunoglobulin G digestion solution sample, then incubate at room temperature for 45 min on a shaker at 150-200 rpm; then wash with the first buffer for 3 The non-specifically adsorbed polypeptides were removed from the surface of the magnetic covalent organic framework material by pass (200 μl each time); finally, the magnetic covalent organic framework material with adsorbed glycopeptides was added to 10 μl of the second buffer (30% acetonitrile by volume). In an aqueous solution containing 0.1% trifluoroacetic acid by volume, that is, 30% ACN-H ₂ O containing 0.1% TFA), desorption was carried out for 30 min under vigorous shaking on a shaker at 800-1200 rpm, and magnetic co-polymers were separated by magnetic separation. The valence organic framework material is obtained to obtain a desorption solution. Then, 1 μl of the desorption solution and 1 μl of the unenriched immunoglobulin G digestion solution were taken for mass spectrometry analysis, and the analysis results are shown in FIG. 7 .

As can be seen from Figure 7, the immunoglobulin G digestion solution without enrichment treatment only obtained 5 glycopeptide signals by mass spectrometry, and the signals were very low, and the entire MS image was basically the signal of heteropeptides [see Figure 7]; After enrichment with Fe ₃ O ₄ /COF-GSH material, 35 glycopeptide signals can be detected on the entire mass spectrum, and the entire MS map is the signal peak of glycopeptides [see Figure 7, dots The labeled peaks are all characteristic peaks of glycopeptides]. The above analysis results show that the Fe ₃ O ₄ /COF-GSH material of the present invention can enrich glycopeptides well, and has good selectivity and high efficiency at the same time.

Application example 2 Enrichment of endogenous glycopeptides in human saliva

Take 10 μl of healthy human saliva and add it to 200 μl of the first buffer (90% acetonitrile aqueous solution by volume, which contains 1% volume concentration of trifluoroacetic acid, that is, 90% ACN-H ₂ O contains 1% TFA) . Take 1 mg of Fe ₃ O ₄ /COF-GSH material prepared in Example 12 and add it to the above sample, then incubate at room temperature for 45 min under the condition of a shaker at 150-200 rpm; then wash with the first buffer 3 times (200 μl each time) The non-specifically adsorbed polypeptides were removed from the surface of the magnetic covalent organic framework material; finally, the magnetic covalent organic framework material with adsorbed glycopeptides was added to 60 μl of the second buffer (30% volume concentration of acetonitrile aqueous solution, which contained volume concentration is 0.1% trifluoroacetic acid, that is, 30% ACN-H ₂ O containing 0.1% TFA), desorbed for 30 min under vigorous shaking of the shaker at 800-1200 rpm, and separated the magnetic covalent organic framework material by magnetic separation to obtain Desorption solution. The adsorption liquid was then used for LC-MS-MS analysis, the results are shown in Table 4, a total of 322 endogenous glycopeptides could be detected.

Table 5 shows the comparison of the Fe ₃ O ₄ /COF-GSH material provided by the present invention with the reported material for enriching endogenous glycopeptides in human saliva. It can be seen from Table 5 that, compared with the reported materials, the Fe ₃ O ₄ /COF-GSH material provided by the present invention can enrich the most endogenous glycopeptides in human saliva.

In conclusion, the Fe ₃ O ₄ /COF-GSH magnetic covalent organic framework material of the present invention can achieve excellent enrichment performance for glycopeptides and endogenous glycopeptides in human saliva.

Table 4 Fe ₃ O ₄ @COF-GSH magnetic covalent organic framework materials

Details of endogenous glycopeptides enriched from human saliva

Table 5 Performance comparison of Fe ₃ O ₄ /COF-GSH magnetic covalent organic framework material and reported materials for enriching endogenous glycopeptides in human saliva

Claims (10)

1. a magnetic covalent organic framework material for glycopeptide enrichment is characterized in that by Fe ₃ O ₄ nanometer magnetic spheres, the covalent organic framework layer coated on the surface of Fe ₃ O ₄ nanometer magnetic spheres, grafted glutathione on a covalent organic framework layer; the covalent organic framework layer is composed of 1,3,5-tris(4-aminophenyl)benzene and 2,5-divinyl-1,4 - A honeycomb structure obtained by addition reaction of phthalaldehyde in a substance ratio of 1:1.5, the glutathione is grafted on the vinyl group of the covalent organic framework layer. 2 . The magnetic covalent organic framework material for glycopeptide enrichment according to claim 1 , wherein the shape is spherical particles and the average particle size is 240-300 nm. 3 . 3. the preparation method of the magnetic covalent organic framework material that is used for glycopeptide enrichment described in claim 1 or 2, is characterized in that step is as follows: (1) Preparation of Fe ₃ O ₄ /COFs nanoparticles Under ultrasonic conditions, Fe ₃ O ₄ nanomagnetic spheres, 1,3,5-tris(4-aminophenyl)benzene and 2,5-divinyl-1,4-benzenedicarboxaldehyde were mixed in dimethylmethylene The sulfone is mixed and dispersed uniformly to form a mixed solution; then, under ultrasonic conditions, acetic acid is added dropwise to the above mixed solution to form a reaction system; then the obtained reaction system is left to incubate at room temperature for 10 to 30 minutes, and the obtained reaction solution is treated after the incubation. Perform magnetic separation and collect the separated solid product, wash the obtained solid product to remove the unreacted material adsorbed on the surface of the solid product, and obtain a covalent organic framework layer coated Fe ₃ O ₄ nanometer magnetic ball nanoparticles, referred to as Fe ₃ O ₄ /COFs nanoparticles; the mass ratio of the Fe ₃ O ₄ nano-magnetic spheres to 1,3,5-tris(4-aminophenyl)benzene is 1:(0.2～1),1,3,5- The substance amount ratio of tris(4-aminophenyl)benzene to 2,5-divinyl-1,4-benzenedicarboxaldehyde is 1:1.5, the 1,3,5-tris(4-aminobenzene) The volume ratio of the sum of the mass of benzene and 2,5-divinyl-1,4-benzenedicarboxaldehyde to acetic acid is (30-210): 1, the unit of mass is mg, and the unit of volume is mL; (2) Preparation of Fe ₃ O ₄ /COF-GSH material Azobisisobutyronitrile, glutathione and Fe ₃ O ₄ /COFs nanoparticles were sequentially added to the composite solvent to form a mixed solution, and then under nitrogen protection, the obtained mixed solution was subjected to mercaptoene click at 60-80 °C Reaction until the surface of Fe ₃ O ₄ /COFs nanoparticles turns yellow; after the completion of the mercaptoene click reaction, the obtained reaction solution is subjected to magnetic separation and the separated solid product is collected, and the obtained solid product is washed to remove unreacted materials adsorbed on the surface of the solid product After drying, a magnetic covalent organic framework material for glycopeptide enrichment, referred to as Fe ₃ O ₄ /COF-GSH material for short, is obtained; the composite solvent is uniformly obtained by mixing ethanol and deionized water in a volume ratio of 1:3, In the mixed solution, the mass ratio of Fe ₃ O ₄ /COFs nanoparticles to glutathione is 1:(0.3～1); the mass ratio of the glutathione to azobisisobutyronitrile is 1: (0.05～0.25). 4 . The method for preparing a magnetic covalent organic framework material for glycopeptide enrichment according to claim 3 , wherein in step (1), the dropwise addition time of acetic acid is 5-15 min. 5 . 5. the preparation method of the magnetic covalent organic framework material for glycopeptide enrichment according to claim 3 or 4, is characterized in that in step (2), before gained mixed solution carries out mercaptoene click reaction under nitrogen protection, A deoxygenation treatment is performed first to remove oxygen in the mixed solution and above the mixed solution in the reactor. 6. The preparation method of the magnetic covalent organic framework material for glycopeptide enrichment according to claim 5, characterized in that the deoxygenation treatment operation is: firstly adopting nitrogen purging mode to deoxygenate the mixed solution; The oxygen above the mixed liquid in the reactor was removed by vacuum-nitrogen circulation. 7. the preparation method of the magnetic covalent organic framework material that is used for glycopeptide enrichment according to claim 3 or 4 is characterized in that the solid product in step (1) is successively used anhydrous tetrahydrofuran, anhydrous methanol, ethanol, Deionized water is used for washing, and the solid product in step (2) is washed with ethanol and deionized water in sequence. 8. the preparation method of the magnetic covalent organic framework material for glycopeptide enrichment according to claim 5 is characterized in that the solid product in step (1) is successively used anhydrous tetrahydrofuran, anhydrous methanol, ethanol, deionized Wash with water, and the solid product in step (2) is washed with ethanol and deionized water in sequence. 9. the preparation method of the magnetic covalent organic framework material for glycopeptide enrichment according to claim 6 is characterized in that the solid product in step (1) is successively used anhydrous tetrahydrofuran, anhydrous methanol, ethanol, deionized Wash with water, and the solid product in step (2) is washed with ethanol and deionized water in sequence. 10. The application of the magnetic covalent organic framework material of claim 1 or 2 in the enrichment of endogenous glycopeptides.

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