CN113680338B - Novel chromatographic material and preparation method thereof - Google Patents

Novel chromatographic material and preparation method thereof Download PDF

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CN113680338B
CN113680338B CN202110988910.5A CN202110988910A CN113680338B CN 113680338 B CN113680338 B CN 113680338B CN 202110988910 A CN202110988910 A CN 202110988910A CN 113680338 B CN113680338 B CN 113680338B
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CN113680338A (en
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周丽
汪群杰
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Tianjin Boyun Purification Equipment Material Technology Co ltd
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    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
    • B01J20/281—Sorbents specially adapted for preparative, analytical or investigative chromatography
    • B01J20/286—Phases chemically bonded to a substrate, e.g. to silica or to polymers
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Abstract

The invention relates to a novel chromatographic material and a preparation method thereof, wherein the material consists of a core-shell structure, an outer layer consists of porous shell hybrid silica gel, and the hybrid silica gel layer has corresponding chemical or physical surface modification. The preparation method of the novel chromatographic material comprises the following steps: firstly preparing a solid core, carrying out multilayer coating and surface hybridization of SiO 2 silica sol or one-step integral hybridization on the solid core, and finally carrying out chemical or physical surface modification on the hybridized silica gel. The invention combines the high-salt-resistant alkali-resistant hybrid shell silica gel with the natural polysaccharide coating process to synthesize the novel chromatographic separation and purification material, which not only overcomes the defects of soft matrix and fragility of the natural polysaccharide separation medium, but also overcomes the defects of intolerance to high salt and high pH of the silica gel matrix, thus realizing the combination of the excellent purification characteristic of the natural polysaccharide modified core-shell silica gel and the stability of the hybrid silica gel for the first time and realizing the combination of the advantages of soft gel and hard gel.

Description

Novel chromatographic material and preparation method thereof
Technical Field
The invention relates to the technical field of composite materials, in particular to the field of biological purification of biological macromolecules such as proteins, polypeptides, antibody nucleotides and the like, and in particular relates to a novel chromatographic material and a preparation method thereof.
Background
The biological innovative drugs are emerging industries in the pharmaceutical industry, and the antibody drugs, the polypeptide drugs and the nucleotide drugs have the characteristics of wide application, high safety, obvious curative effect and the like, and are widely applied to the prevention, diagnosis and treatment of diseases such as tumors, hepatitis, diabetes, AIDS and the like at present, thereby having wide development prospects. The biological separation technology for purifying biological macromolecules such as proteins, antibodies, polypeptides, nucleotides and the like needs to adapt to the characteristics of diversity and complexity of the biological macromolecules, and can be developed and perfected particularly in the aspects of molecular structure, physicochemical properties, biological activity and the like. The chromatographic technology is a key technology for separating and purifying biological macromolecules, has diversity and universality, and is suitable for the special properties of the biological macromolecules, the core of the development of the technology is a separating and purifying material, and no matter what chromatographic method is adopted, the main factor influencing the liquid chromatographic separation effect is the performance of a separating medium. In recent years, in order to meet the purification requirements of antibodies, polypeptides and nucleotide drugs, various biochemical separation media are appeared in the market.
The purifying material used for purifying the biological macromolecules is mainly agarose gel, sephadex and polyacrylamide gel matrix, and the matrix has the advantages of strong hydrophilicity, capability of separating and purifying in a wider pH range, high salt resistance, alkali resistance, weak acid resistance, organic solvent resistance and extremely low non-specific adsorption. The defects are that the matrix is softer, the strength is poor, the physical and mechanical temperature is relatively poor, the pressure resistance is poor, the matrix is easy to break, and the fast and efficient separation of experiments is difficult.
In recent years, the demand for biotechnology for producing products has been expanding, and higher demands have been put on media. The separation medium has a rigid or semi-rigid skeleton and good hydrophilicity. Although natural polysaccharides have higher hydrophilicity and good biological macromolecular compatibility, the skeleton structures of the natural polysaccharides are mostly soft matrixes, are easy to deform, cannot bear high flow velocity, and have low resolution and are easy to agglomerate. The separating material has certain mechanical strength, and can not deform in the chromatography process, and the increase of the mechanical strength can also lead the chromatography to be carried out in a higher pressure environment, thereby shortening the separation time. How to increase the strength of agarose/dextran-based gel chromatographic materials has become a technical pain point in the advancement of gel separation materials.
Disclosure of Invention
In order to solve the problems in the prior art, a novel chromatographic material, wherein,
(1) The core-shell structure is internally provided with a solid sphere, and the outer layer of the core-shell structure is provided with porous shell silica gel;
(2) The shell layer consists of hybrid silica gel, and the hybrid silica gel layer can be realized through surface hybrid modification or integral organic hybridization;
(3) The shell layer has corresponding chemical or physical surface modification.
Preferably, the chromatographic material has a particle size in the range of 2-100um, preferably 5-50um; pore diameter: 50-1000A, preferably 300-800A; the core-shell structure at least comprises 1 or more non-porous solid cores and a shell layer with nanoscale aperture, wherein the particle size of the solid cores is 1-90um, and the thickness of the shell layer is 0.1-50um; the hybrid silica gel layer contains silicon-carbon bonds and silicon-oxygen bonds at the same time; chemical modification of the shell surface includes monomers capable of forming chemical bonds with the silica gel hybrid layer, including, but not limited to, octadecyl trichlorosilane octane trichlorosilane, trimethylchlorosilane, methyltrimethoxysilane, trichloro-silicon ethyl benzenesulfonyl chlorosilane, aminopropyl trimethoxysilane, polar phenylsilane, cyano propyl dimethyl chlorosilane, ureido propyl trimethoxy silane, chiral amide silane, and the like, all silanes and other monomers that can be used in the art; further or multi-step coupling modification of other bondable monomers including but not limited to carboxyl natural polysaccharide after chemical modification of the shell surface; amino natural polysaccharide, proteinA, DEAE, CM, SP, phenyl, alkyl, carboxyl and other monomers.
In any of the above embodiments, it is preferred that the physical modification of the shell surface comprises a coating material applied to the silica gel hybrid layer, including but not limited to natural polysaccharides, cellulose, polyethylene glycol, and the like, which can be used for coating.
The invention also provides a preparation method of the novel chromatographic material, which comprises the following steps: firstly preparing a solid core, carrying out multilayer coating and surface hybridization or one-step integral hybridization on SiO 2 silica sol on the solid core, and finally carrying out chemical or physical surface modification on the hybridized silica gel.
Preferably, the chromatographic medium comprises at least 1 or more non-porous solid cores, and non-porous solid cores include, but are not limited to, solid silica, solid zirconium dioxide, solid peptide dioxide, ferroferric oxide, and the like. The SiO 2 content in the silica sol used for coating the shell layer is 5-30%, the diameter is 5-100nm, and the coating times of the shell layer are 5-30.
In any of the above embodiments, it is preferred that the surface hybridization modification is achieved by multi-layer inorganic/organic polycondensation of the silicon hydroxyl groups on the surface of the core-shell silica gel with the difunctional/trifunctional organosilanes, introducing alkyl chains into the siloxane network. The integral organic modification layer of the hybrid silica gel layer is realized by dispersing and coating the pre-synthesized polyorganosilane on the outer layer of the solid core; the monomer used for the hybridization surface modification is mono-functional and bi-functional/tri-functional organosilane with the structural formula including but not limited to ：R1R2SiX、R1R2SiX2、R1R2SiX3, R1 and R 2 containing 1 or 2 carbon atoms, and X can be Cl,OCH3,OC2H5,(CH3)2N,(CH3CH2) 2N,I,Br,CN,OOCH3,O(CO)CH3,, wherein the silicon hydroxyl on the surface of the core-shell silica gel is subjected to multi-layer inorganic/organic polycondensation with the bi-functional/tri-functional organosilane on the surface, and alkyl chains are introduced into the siloxane network structure. The number of inorganic/organic polycondensation layers is 1-15, i.e. the above steps are repeated 1-15 times.
In any of the above schemes, preferably, the hybrid core-shell silica gel, amino or carboxyl silane is added into toluene solution for reflux reaction, filtration, toluene and methanol cleaning, vacuum drying, carboxylation or amination natural polysaccharide is added into ethanol, EDC/NHS catalyzes carboxyl to react with amino on the surface of the hybrid core-shell silica gel, stirring is carried out at room temperature in a dark place, filtration and cleaning are carried out, and the hybrid core-shell silica gel modified by natural polysaccharide is obtained.
In any of the above embodiments, preferably, the aminosilane includes, but is not limited to: amino-containing silanes such as gamma-aminopropyl triethoxysilane, aminopropyl trimethoxysilane, aminopropyl methyldiethoxysilane, aminopropyl methyldimethoxysilane, N-phenyl-gamma-aminopropyl trimethoxysilane, N-diethyl aminopropyl trimethoxysilane, N-dimethyl aminopropyl trimethoxysilane, N-beta- (aminoethyl) -aminopropyl triethoxysilane, gamma-diethylenetriaminopropyl methyldimethoxysilane, N-beta- (aminoethyl) -gamma-aminopropyl methyldimethoxysilane.
In any of the above embodiments, preferably, the carboxylated polysaccharide includes, but is not limited to: carboxyl natural polysaccharides such as carboxyl dextran, carboxyl-beta-cyclodextrin, carboxylated chitosan, and carboxyl agarose; the aminated polysaccharide is: amino natural polysaccharides such as aminodextran, amino-beta-cyclodextrin, aminated chitosan, and amino agarose.
In any of the above schemes, it is preferred that the novel chromatographic medium is suitable for extraction and separation of liquid or solution organic substances in the relevant industries of biological crude drugs, clinical, environmental, food, etc.
The novel chromatographic purification separation material is synthesized by combining the high-salt-resistant alkali-resistant hybrid shell silica gel with the natural polysaccharide coating process, overcomes the defects of soft matrix and fragility of the natural polysaccharide separation medium, overcomes the defects of intolerance to high salt and high pH of the silica gel matrix, realizes the combination of the excellent purification characteristic of the agarose modified core-shell silica gel and the stability of the hybrid silica gel for the first time, and realizes the combination of the advantages of soft gel and hard gel. The novel core-shell separation medium has a rigid framework, good hydraulic permeability, high flow rate and high load capacity, and the formed surface modification layer is also very thin, fast in mass transfer rate and narrow in peak shape. The patent perfectly realizes the fusion of the two technologies and develops a large-aperture core-shell type hybrid silica gel material; the second is to coat and crosslink the surface of the hybrid silica gel with but not limited to hydrophilic materials, y, reverse bonding phases, and further functionalize into the desired ion exchange functional groups or conjugated antibodies. Develop a novel chromatographic material.
Detailed Description
For a further understanding of the present invention, the present invention will be described in detail with reference to the following examples.
The invention provides a novel chromatographic material which consists of a core-shell structure, wherein the outer layer is made of porous shell hybrid silica gel, and the hybrid silica gel layer has corresponding chemical or physical surface modification.
Preferably, the preparation method of the novel chromatographic material comprises the following steps: firstly preparing a solid core, carrying out multilayer coating and surface hybridization or one-step integral hybridization on SiO 2 silica sol on the solid core, and finally carrying out chemical or physical surface modification on the hybridized silica gel. Wherein the particle size of the chromatographic medium ranges from 2 to 100um, preferably from 5 to 50um; pore diameter: 50-1000A, preferably 300-800A.
In any of the above schemes, it is preferred that the novel chromatographic medium is suitable for extraction and separation of liquid or solution organic substances in the relevant industries of biological crude drugs, clinical, environmental, food, etc.
In any of the above embodiments, preferably, the chromatographic medium comprises at least 1 or more solid, non-porous cores, and the solid, non-porous cores may be solid silica, solid zirconia, solid peptide dioxide, or the like, solid materials that provide hydroxyl groups. The SiO2 content in the silica sol is 5-30%, the diameter is 5-100nm, and the coating times of the shell layer are 5-30.
Example 1
Preparation of Shell silica gel
(1) 50G of 3um solid silica gel spheres are heated to 90℃in 1000ml of 0.2M aqueous HF solution for 8 hours; filtered, 1000ml of pure water wash solution 2 times, 500ml of acetone 3 times, and vacuum dried for 12 hours.
(2) Mixing the dried solid silica gel, 500ml of aqueous nitric acid solution with pH of 3.0 and 250g of 1.0% aqueous polydimethyldiallyl ammonium chloride solution for 20 min, separating by a centrifuge, removing the supernatant, adding 300ml of deionized water for 3 times, and removing the supernatant. Then 500g of silica sol (SiO 2 content 15%, particle size 20 nm) with pH 3.5 was added and mixed for 20 minutes, the mixture was separated by a centrifuge, the supernatant was discarded, 300ml of deionized water was added and washed 3 times, and the supernatant was discarded.
(3) Repeating the step (2) for 10 times, and performing microscopic examination under a microscope to obtain a sphere with a diameter of 5.0um and uniformity.
(4) And (3) firing at a high temperature of 900 ℃ for 10 hours.
(5) 73G of the white solid after burning off is heated to 90℃in 1000ml of 0.2M aqueous HF for 8 hours; filtered, 1000ml of pure water wash solution 2 times, 500ml of acetone 3 times, and vacuum dried for 12 hours. Microscopic examination, 5.0um spherical, aperture 300A.
Example 2
Preparation of hybrid core-shell silica gel
10 G of 5um core-shell silica gel obtained in the above step, 4g of tris (dimethylamino) ethylsilane, 50 ml of toluene and reflux at about 110℃for 8 hours. The solid obtained is filtered and washed 3 times with 50 ml of toluene each time; washing with methanol 6 times, each time 50 ml; vacuum drying at 100deg.C for 8 hr. C content 3.14%
Repeating the above steps for 2 times, wherein the bonding coverage C content is 4.77%.
Example 3
Preparation of reversed-phase C18 hybridized core-shell silica gel
10 G of hybrid silica gel obtained in the preparation step of the hybrid core-shell silica gel, 10 g of octadecyl dimethyl chlorosilane, 4 g of triethylamine, 50 ml of toluene and reflux at about 110 ℃ for 6 hours. The solid obtained is filtered and washed 3 times with 50 ml of toluene each time; washing with methanol 6 times, each time 50 ml; vacuum drying at 100deg.C for 8 hr. The bonded silica gel, 5 g of trimethylchlorosilane, 2 g of triethylamine, 50 ml of toluene and reflux at about 110 ℃ for 2 hours. The solid obtained is filtered and washed 3 times with 50 ml of toluene each time; washing with methanol 6 times, each time 50 ml; vacuum drying at 100deg.C for 8 hr. 11 g of solid are obtained.
Example 4
Preparation of dextran hybridization core-shell silica gel
10 G of 5um hybrid core-shell silica gel obtained in the previous step, 4 g of aminopropyl triethoxysilane, 50ml of toluene and reflux at about 110 ℃ for 16 hours. Filtering the reacted sand core funnel, washing with toluene for 3 times, and 50 milliliters each time; washing with methanol 6 times, each time 50 ml; vacuum drying at 100deg.C for 8 hr.
Carboxylated dextran 20g, ethanol 100ml, EDC10ml 10mg/ml, stirring in dark for 5h, NHS 20ml 10mg/ml, stirring in dark for 16h. Adding amino hybridized core-shell silica gel, stirring at room temperature for 24h, filtering, washing with ethanol for three times, each time with 50ml, and washing with water for three times, each time with 50ml. Finally obtaining the glucan hybridization core-shell silica gel.
The novel chromatographic material can realize the purification preparation of polypeptide, antibody and nucleic acid by utilizing the coupling technology of natural polysaccharide.
Example 5
Preparation of DEAE-dextran hybrid core-shell silica gel and purification of gingko galactose binding protein
10Ml of dextran hybridization core-shell silica gel was taken, transferred into a 250ml conical flask, 20ml of DEAE-HCl of a certain concentration was added, and another 20ml of NaOH solution of a certain concentration was put into another conical flask. Sealing, preheating in a constant temperature shaking table, and pouring NaOH into the bottle 1 after 10 min. Sealing and starting the shaking table. After the established reaction time, taking a bottle 1, cooling with water, and cleaning a medium in a sand core funnel with distilled water to obtain DEAE-dextran hybridization core-shell silica gel with DEAE and diethylaminoethyl.
Taking the DEAE-dextran hybridization core-shell microsphere, washing for a plurality of times by distilled water, washing for 2 times by phosphate buffer (0.01 mol/L, PH 7.2), and loading into a column. Fully balancing a chromatographic column with 5 column volumes of PBS, fully dialyzing the same PBS with the prepared crude protein extract, centrifugally collecting supernatant, using an automatic constant flow pump to perform column loading, continuously balancing the column with phosphate buffer until the penetrating peak is reduced to a base line, and continuously eluting with eluent (0.01 mol/LPBS, PH7.2 and 0.25 mol/LNaCl); collecting the eluting peak liquid, and taking part of the sample for freeze drying electrophoresis detection.
Example 6
Carboxydextran hybridization material covalently coupled protein a protein and purification of IgG antibodies
20Mg of EDC and 2mg of NHS were dissolved in MES (pH=5.0), added to the carboxydextran hybridization material washed 3 times with PBS, shaken for 1.5 hours at room temperature, centrifuged, and the supernatant was discarded. 1mg of biological protein molecule ProteinA is dissolved in 2ml of PBS, added into the centrifuged carboxyl dextran hybridization material, vibrated for 4 hours at room temperature, added with 1M glycine for reaction for 1 hour, washed for 3 times by PBS after the reaction, and stored in a storage solution. The prepared protein A coupled glucan hybrid material is obtained.
1Mg of ProteinA is added into a new centrifuge tube, washed 3 times with 500mg of PBS, centrifuged, supernatant is removed, 500uLPBS and 50uL of plasma are added, vortexed, placed at room temperature for 30min, centrifuged, the upper layer is removed, PBST is added again, vortexed, the upper layer is removed, repeated twice, 50-100uL of 0.1M sodium citrate is added, vortexed and oscillated, the supernatant is dissociated at room temperature for 2-3min, centrifuged, and transferred into a prepared new centrifuge tube of 1M Tris-HcL.
It will be appreciated by those skilled in the art that the novel chromatographic material and method of making the same of the present invention includes any combination of the parts of the summary of the invention and the detailed description of the invention described above, and is limited in scope and does not constitute a complete description of the various aspects of these combinations for the sake of brevity. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims (2)

1. A method for purifying a ginkgo galactose binding protein, comprising:
1) Preparation of shell silica gel:
① 50g of 3um solid silica gel spheres are heated to 90℃in 1000ml of 0.2M aqueous HF solution for 8 hours; filtering, washing with 1000ml pure water 2 times, washing with 500ml acetone 3 times, and vacuum drying for 12 hr;
② Mixing the dried solid silica gel, 500ml of nitric acid aqueous solution with pH of 3.0 and 250g of 1.0% polydimethyldiallyl ammonium chloride aqueous solution for 20 minutes, separating by a centrifugal machine, discarding the supernatant, adding 300ml of deionized water for 3 times, discarding the supernatant, adding 500g of silica sol with pH of 3.5, wherein the SiO 2 content of the silica sol is 15%, the particle size is 20nm, mixing for 20 minutes, separating by the centrifugal machine, discarding the supernatant, adding 300ml of deionized water for 3 times, discarding the supernatant;
③ Repeating the steps ② times, and performing microscopic examination under a microscope to obtain a sphere shape with a size of 5.0um and uniformity;
④ High-temperature firing is carried out at 900 ℃ for 10 hours;
⑤ 73g of the white solid after burning off is heated to 90℃in 1000ml of 0.2M aqueous HF for 8 hours; filtering, washing with 1000ml pure water 2 times, washing with 500ml acetone 3 times, vacuum drying for 12 hr, and performing microscopic examination to obtain 5.0um sphere with aperture of 300A;
2) Preparing hybridized core-shell silica gel:
10 g of 5um core-shell silica gel, 4g of tris (dimethylamino) ethylsilane, 50ml of toluene and reflux at 110 ℃ for 8 hours; the solid obtained is filtered and washed 3 times with 50ml of toluene each time; washing with methanol 6 times, each time 50 ml; vacuum drying at 100deg.C for 8 hr; c content 3.14%;
repeating the steps for 2 times, wherein the bonding coverage C content is 4.77%;
3) Preparing glucan hybridization core-shell silica gel:
10 g of 5um hybridized core-shell silica gel obtained in the previous step, 4g of aminopropyl triethoxysilane, 50 ml of toluene and reflux at 110 ℃ for 16 hours; filtering the reacted sand core funnel, washing with toluene for 3 times, and 50 milliliters each time; washing with methanol 6 times, each time 50 ml; vacuum drying at 100deg.C for 8 hr;
Carboxylated glucan 20g, ethanol 100ml, EDC10ml 10mg/ml, stirring for 5h in dark, NHS 20ml 10mg/ml, stirring for 16h in dark; adding amino hybridized core-shell silica gel, stirring at room temperature for 24 hours, filtering, washing with ethanol for three times, each time with 50ml, and washing with water for three times, each time with 50ml, and finally obtaining glucan hybridized core-shell silica gel;
4) Preparation of DEAE-dextran hybrid core-shell silica gel and purification of ginkgo galactose binding proteins:
Transferring 10ml of dextran hybridization core-shell silica gel into a 250ml conical flask, adding 20ml of DEAE-HCl with certain concentration, and placing 20ml of NaOH solution with certain concentration into another conical flask; sealing, preheating in a constant temperature shaking table, and pouring NaOH into the bottle 1 after 10 min; sealing, starting a shaking table, taking a bottle 1 after a set reaction time, cooling with water, and cleaning a medium in a sand core funnel with distilled water to obtain DEAE-dextran hybridization core-shell silica gel with DEAE and diethylaminoethyl;
Taking the DEAE-dextran hybridization core-shell microsphere, washing for a plurality of times by distilled water, washing for 2 times by 0.01mol/L phosphate buffer with pH of 7.2, and loading into a column; fully balancing a chromatographic column by using PBS with 5 column volumes in advance, fully dialyzing the same PBS by using the prepared crude protein extract, centrifugally collecting supernatant, using an automatic constant flow pump to perform column loading, continuously balancing the column by using phosphate buffer until the penetrating peak is reduced to a base line, and continuously eluting by using eluent which is 0.01mol/L PBS and has the pH of 7.2 and contains 0.25mol/L NaCl; collecting the eluting peak liquid, and taking part of the sample for freeze drying electrophoresis detection.
2. A method for purifying an IgG antibody, comprising:
1) Preparation of shell silica gel:
① 50g of 3um solid silica gel spheres are heated to 90℃in 1000ml of 0.2M aqueous HF solution for 8 hours; filtering, washing with 1000ml pure water 2 times, washing with 500ml acetone 3 times, and vacuum drying for 12 hr;
② Mixing the dried solid silica gel, 500ml of nitric acid aqueous solution with pH of 3.0 and 250g of 1.0% polydimethyldiallyl ammonium chloride aqueous solution for 20 minutes, separating by a centrifugal machine, discarding the supernatant, adding 300ml of deionized water for 3 times, discarding the supernatant, adding 500g of silica sol with pH of 3.5 for mixing for 20 minutes, separating by a centrifugal machine, discarding the supernatant, adding 300ml of deionized water for 3 times, and discarding the supernatant;
③ Repeating the steps ② times, and performing microscopic examination under a microscope to obtain a sphere shape with a size of 5.0um and uniformity;
④ High-temperature firing is carried out at 900 ℃ for 10 hours;
⑤ 73g of the white solid after burning off is heated to 90℃in 1000ml of 0.2M aqueous HF for 8 hours; filtering, washing with 1000ml pure water 2 times, washing with 500ml acetone 3 times, vacuum drying for 12 hr, and performing microscopic examination to obtain 5.0um sphere with aperture of 300A;
2) Preparing hybridized core-shell silica gel:
10 g of 5um core-shell silica gel, 4g of tris (dimethylamino) ethylsilane, 50ml of toluene and reflux at 110 ℃ for 8 hours; the solid obtained is filtered and washed 3 times with 50ml of toluene each time; washing with methanol 6 times, each time 50 ml; vacuum drying at 100deg.C for 8 hr; c content 3.14%;
repeating the steps for 2 times, wherein the bonding coverage C content is 4.77%;
3) Preparing glucan hybridization core-shell silica gel:
10 g of 5um hybridized core-shell silica gel obtained in the previous step, 4g of aminopropyl triethoxysilane, 50 ml of toluene and reflux at 110 ℃ for 16 hours; filtering the reacted sand core funnel, washing with toluene for 3 times, and 50 milliliters each time; washing with methanol 6 times, each time 50 ml; vacuum drying at 100deg.C for 8 hr;
Carboxylated glucan 20g, ethanol 100ml, EDC10ml 10mg/ml, stirring for 5h in dark, NHS 20ml 10mg/ml, stirring for 16h in dark; adding amino hybridized core-shell silica gel, stirring at room temperature for 24 hours, filtering, washing with ethanol for three times, each time with 50ml, and washing with water for three times, each time with 50ml, and finally obtaining glucan hybridized core-shell silica gel;
4) Preparation of carboxydextran hybridization material covalently coupled protein a protein and purification of IgG antibodies:
Taking 20mg of EDC and 2mg of NHS, dissolving in MES with pH=5.0, adding into a carboxyl glucan hybridization material washed 3 times with PBS, oscillating for 1.5 hours at room temperature, centrifuging, and removing the supernatant; 1mg of biological protein molecule ProteinA is dissolved in 2ml of PBS, added into the centrifuged carboxyl dextran hybridization material, vibrated for 4 hours at room temperature, then added with 1M glycine for reaction for 1 hour, washed for 3 times by PBS after the reaction, and stored in a storage solution, thus obtaining the prepared ProteinA coupled dextran hybridization material;
1mg of ProteinA is added into a new centrifuge tube, washed 3 times with 500mg of PBS, centrifuged, supernatant is removed, 500uLPBS and 50uL of plasma are added, vortexed, placed at room temperature for 30min, centrifuged, the upper layer is removed, PBST is added again, vortexed, the upper layer is removed, repeated twice, 50-100uL of 0.1M sodium citrate is added, vortexed and oscillated, the supernatant is dissociated at room temperature for 2-3min, centrifuged, and transferred into a prepared new centrifuge tube of 1M Tris-HcL.
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