CN116983917A - Preparation method and application of mixed mode microsphere for biological sample impurity removal and purification - Google Patents
Preparation method and application of mixed mode microsphere for biological sample impurity removal and purification Download PDFInfo
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- PYWVYCXTNDRMGF-UHFFFAOYSA-N rhodamine B Chemical compound [Cl-].C=12C=CC(=[N+](CC)CC)C=C2OC2=CC(N(CC)CC)=CC=C2C=1C1=CC=CC=C1C(O)=O PYWVYCXTNDRMGF-UHFFFAOYSA-N 0.000 description 7
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01D—SEPARATION
- B01D15/00—Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
- B01J13/00—Colloid chemistry, e.g. the production of colloidal materials or their solutions, not otherwise provided for; Making microcapsules or microballoons
- B01J13/02—Making microcapsules or microballoons
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
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- B01J13/00—Colloid chemistry, e.g. the production of colloidal materials or their solutions, not otherwise provided for; Making microcapsules or microballoons
- B01J13/02—Making microcapsules or microballoons
- B01J13/06—Making microcapsules or microballoons by phase separation
- B01J13/14—Polymerisation; cross-linking
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- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
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- B01J13/00—Colloid chemistry, e.g. the production of colloidal materials or their solutions, not otherwise provided for; Making microcapsules or microballoons
- B01J13/02—Making microcapsules or microballoons
- B01J13/20—After-treatment of capsule walls, e.g. hardening
- B01J13/22—Coating
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
- G01N1/34—Purifying; Cleaning
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Abstract
The invention discloses a preparation method and application of mixed mode microspheres for biological sample impurity removal and purification; the mixed mode microsphere consists of an adsorption inner shell layer with hydrophobic and ionic interactions and a hydrophilic outer shell layer with a drainage effect, wherein the hydrophobic and ionic interactions of the inner shell layer are formed by bonding ionizable amino long-chain alkane and agarose microsphere resin, the outer shell layer is formed by inward dipping and outward deposition and crosslinking of polysaccharide such as agarose, dextran, cellulose and the like, the thickness and drainage limit of the drainage layer can be controlled by the porosity of the agarose microsphere resin, the concentration of polysaccharide and the crosslinking degree of the outer shell layer.
Description
Technical Field
The invention relates to the field of biological sample impurity removal and purification, in particular to a preparation method and application of mixed mode microspheres for biological sample impurity removal and purification.
Background
The rapid development and application of biotechnology have broken through many life difficulties, and separation and purification of biological samples occupy very large cost in the whole application process of biotechnology, and the functionality and cost of separation materials become the most concerned problems in the field of biological separation.
The technologies currently applied to biological separation mainly include differential centrifugation technology, membrane separation technology and microsphere chromatography technology, and the microsphere chromatography technology becomes the mainstream technology for purifying industrial biological samples in consideration of production scale and cost. The chromatographic medium used in the chromatographic technique is divided into a plurality of types, the material mechanism can be divided into a polymer microsphere mechanism and an agarose medium mechanism, the action principle can be further divided into affinity chromatography, ion chromatography, hydrophilic-hydrophobic chromatography, size exclusion chromatography and the like, and different materials and surface-bonded functional mechanisms can achieve a certain separation and purification function, such as protein A chromatographic medium affinity chromatography for antibody purification, antibody in biological samples, Q column strong anion exchange function for protein purification, positive-charge protein purification and the like.
The existing separation and purification technology mostly uses repeatedly and in combination chromatography media with different chromatography actions, for example, protein A chromatography media are often selected for affinity purification, then ion chromatography is carried out by using Q column, and if the purity of the sample does not reach the expected requirement, the strong hydrophobic column is considered again. The repeated or combined use of the chromatographic media of different chromatographic principles solves the aim of purifying and separating most biological samples, such as protein antibody purification, plasmid separation, nucleic acid purification, exosome separation and the like, but the repeated use of the chromatographic media of different functional principles also causes great increase in purification cost and process complexity.
For example, CN108883394a discloses a separation matrix for purifying a biomacromolecule comprising a plurality of particles having a core region and a shell region, the shell region being accessible to the biomacromolecule and the core region of the grafted functional polymer being inaccessible to the biomacromolecule by polymerization of the monomer residues. The invention has complex flow, uses dangerous bromine water to control microsphere activation and fire extinguishing areas through reaction rate, needs repeated activation and inactivation, has longer synthesis period and high production cost. The invention scheme does not apply the microsphere to the separation of macromolecular cell vesicles from biological samples.
CN113416235a establishes the surface chemistry of the shell and core layers by precisely controlling two key chemical reactions through proprietary surface modification patent technology. The core-shell structure composite mode chromatography medium can be widely applied to separation and purification of biological macromolecules such as proteins, antibodies, viruses, virus vectors, vaccines, DNA, RNA, plasmids and the like. The process of the invention also uses dangerous bromine water, establishes the surface chemistry of the shell layer and the core layer by precisely controlling two-step key chemical reactions, and adopts a monomer residue polymerization mode to bond functional molecules, thereby having complex process and higher cost. In addition, the invention develops the application of separating and purifying a large number of biological samples, and is not applied to separating macromolecular cell vesicles from biological samples.
Therefore, the invention aims to disclose a preparation method and application of mixed mode microspheres for biological sample impurity removal and purification; the mixed mode microsphere consists of an adsorption inner shell layer with hydrophobic and ionic interactions and a hydrophilic outer shell layer with a drainage effect, wherein the hydrophobic and ionic interactions of the inner shell layer are formed by bonding ionizable amino long-chain alkane and agarose microsphere resin, the outer shell layer is formed by inward dipping and outward deposition and crosslinking of polysaccharide such as agarose, dextran, cellulose and the like, the thickness and drainage limit of the drainage layer can be controlled by the porosity of the agarose microsphere resin, the concentration of polysaccharide and the crosslinking degree of the outer shell layer.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provides a preparation method and application of a mixed mode microsphere for biological sample impurity removal and purification; the mixed mode microsphere consists of an adsorption inner shell layer with hydrophobic and ionic interactions and a hydrophilic outer shell layer with a drainage effect, wherein the hydrophobic and ionic interactions of the inner shell layer are formed by bonding ionizable amino long-chain alkane and agarose microsphere resin, the outer shell layer is formed by inward dipping and outward deposition and crosslinking of polysaccharide such as agarose, dextran, cellulose and the like, the thickness and drainage limit of the drainage layer can be controlled by the porosity of the agarose microsphere resin, the concentration of polysaccharide and the crosslinking degree of the outer shell layer.
In order to achieve the technical effects, the following technical scheme is adopted:
a mixed mode microsphere for biological sample impurity removal and purification comprises an inner shell layer and an outer shell layer;
the inner shell layer adopts agarose microsphere resin as a base sphere core, and hydrophobic molecules capable of being ionized are grafted after the agarose microsphere resin is crosslinked and activated to form the agarose microsphere resin inner shell layer under the combined action of hydrophobic and ions;
the outer shell layer is formed by inwards dipping and outwards depositing a coated polysaccharide porous medium on the surface of the inner shell layer to form the outer shell layer, the polysaccharide porous medium can be inwards dipped and adsorbed in the pores of the outer shell layer of the agarose microsphere resin, and can be outwards deposited on the surface of the inner shell layer of the agarose microsphere resin to form a thin shell layer or a net-shaped branched structure, after inwards dipping and outwards depositing the coated polysaccharide porous medium, the outer shell layer is crosslinked, the formed outer shell layer can keep the hydrophilicity of the microsphere and resist biomolecules, and the pore diameter of the outer shell layer is smaller than that of the agarose microsphere resin-based spheres, so that the effect of protecting the agarose microsphere resin-based spheres and resist macromolecules from entering the agarose microsphere resin-based spheres is realized;
the shell layer consists of an inward dipping shell layer and an outward deposition shell layer, and the thickness of the inward dipping shell layer can be controlled by adjusting the pore diameter of the agarose microsphere resin-based spheres, the concentration of the polysaccharide porous medium and the crosslinking degree of the shell layer;
the inward immersed shell layer in the shell layer determines the exclusion limit of the mixed mode microsphere shell layer;
the thickness of the outward deposition shell layer is controlled by the adding amount of the polysaccharide when the shell layer coats the polysaccharide porous medium;
the hydrophobic molecules capable of ionization include primary, secondary or tertiary amines of 6-18 carbon long or branched chain, charged amine structures and hydrophobic long chains.
Further, the agarose microsphere resin is 2% -8% agarose microsphere, and the microsphere is grafted with hydrophobic molecules capable of being ionized after crosslinking and activation; the cross-linking agents are all mixtures of epichlorohydrin and sodium borohydride.
Further, the agarose microsphere resin is selected from 4% agarose microspheres, and the hydrophobic molecules capable of ionization are N-hexylamine, N-dimethylhexylamine or N-octylamine; the porous medium of polysaccharide is agarose, dextran, cyclodextrin, chitosan and cellulose, the shell layer is not bonded and not combined with any biological sample, the biological sample with larger size can be discharged, and only the biological sample with the discharge limit smaller than the shell layer can enter the interior.
Further, the agarose microsphere resin is selected from 3-6% agarose microsphere resin-based spheres, the mass concentration of the polysaccharide porous medium is 2% -8%, the thickness of the inward dipping shell layer is 1-10 mu m, and the exclusion limit is 20-50nm; the crosslinking control agent is one or more of dextran or hydroxypropyl cellulose.
Further, the outwardly deposited shell layer of the outer shell layer does not substantially form a shell layer, is presented in a network-like branched structure, exhibits better hydrophilicity, and simultaneously more effectively blocks and protects the biospecking components.
A method for preparing mixed mode microspheres for biological sample impurity removal and purification, comprising the following steps:
step S1: crosslinking of agarose microsphere resin-based spheres: washing agarose microsphere resin-based spheres with pure water, draining, adding NaOH solution, mixing, stirring uniformly, adding epichlorohydrin and sodium borohydride, keeping stirring, and washing with pure water to obtain crosslinked agarose microsphere resin-based spheres;
step S2: activating agarose microsphere resin-based spheres: taking the crosslinked agarose microsphere resin-based spheres obtained in the step S1, draining, adding NaOH solution, mixing, uniformly stirring, adding epichlorohydrin and sodium borohydride, continuously keeping stirring, and cleaning with purified water to obtain activated crosslinked agarose microsphere resin-based spheres;
step S3: agarose microsphere resin-based spheres grafted with hydrophobic molecules capable of ionization: taking the activated agarose microsphere resin-based spheres obtained in the step S2, draining, adding NaOH solution, mixing, uniformly stirring, adding ionized hydrophobic molecules, continuously keeping stirring, and cleaning with purified water to obtain the ionized hydrophobic molecule grafted agarose microsphere resin-based spheres;
step S4: and (3) coating a shell layer: taking the ionized hydrophobic molecular grafted agarose microsphere resin-based spheres obtained in the step S3, draining, adding pure water and a polysaccharide porous medium, stirring, wherein the polysaccharide porous medium is one or more of agarose, dextran, cyclodextrin, chitosan and cellulose, heating until the polysaccharide porous medium is completely dissolved, keeping constant temperature stirring and soaking, stopping heating, keeping stirring and naturally cooling, cooling to room temperature, draining, and washing to obtain a coated product;
step S5: crosslinking of the outer shell layer: draining the product obtained in the step S4, adding NaOH solution, mixing, adding epichlorohydrin and sodium borohydride after stirring uniformly, continuously keeping stirring, and cleaning with purified water to obtain mixed die type microspheres;
the agarose microsphere resin is 2% -8% agarose microsphere;
the hydrophobic molecules capable of ionization include primary, secondary or tertiary amines of 6-18 carbon long or branched chain.
Further, in the step S1, the concentration of the NaOH solution is 0.5-10mol/L, the agarose microsphere resin-based spheres are washed 3-10 times by pure water, the mixture is stirred uniformly at 25-40 ℃ after being added with the NaOH solution, the epichlorohydrin and the sodium borohydride are added, the mixture is continuously kept at 25-50 ℃ and stirred for 1-4 hours, and then the mixture is washed 5-10 times by the pure water, wherein the mass ratio of the agarose microsphere resin-based spheres to the NaOH, the epichlorohydrin and the sodium borohydride is: 2-8:4-80:10-20:0.5-1; in the step S2, the concentration of the NaOH solution is 0.5-5mol/L, the NaOH solution is added for mixing, after the mixture is stirred uniformly at the temperature of 25-50 ℃, the epichlorohydrin and the sodium borohydride are added, the mixture is continuously kept at the temperature of 25-50 ℃ for stirring for 1-3 hours, and then the mixture is washed for 5-10 times by purified water, wherein the mass ratio of the crosslinked agarose microsphere resin-based spheres to the NaOH, the epichlorohydrin and the sodium borohydride is as follows: 2-8:3-60:20-40:0.15-0.3.
Further, in the step S3, the concentration of the NaOH solution is 0.1-1mol/L, the NaOH solution is added for mixing, after being stirred uniformly at the temperature of 25-40 ℃, the hydrophobic molecules capable of being ionized are added, after being stirred continuously at the temperature of 25-40 ℃ for 8-48 hours, purified water is used for cleaning for 5-10 times, and the mass ratio of the activated agarose microsphere resin-based spheres to the NaOH is as follows: 2-8:0.6-12:100-200.
Further, adding pure water in the step S4, raising the temperature to 60-110 ℃ and stirring; after the polysaccharide porous medium is dissolved, stirring for 0.5-2 hours at 60-75 ℃, stopping heating, and washing for 5-10 times; the mass ratio of the resin-based spheres of the hydrophobic molecular grafted agarose microsphere capable of being ionized to the dextran, the agarose and the purified water is 2-8:0.1-0.8:0.1-0.8:5-20 parts; the concentration of the NaOH solution in the step S5 is 0.5-10mol/L, the NaOH solution is added for mixing, after the mixture is stirred uniformly at the temperature of 25-40 ℃, the epichlorohydrin and the sodium borohydride are added, the mixture is continuously kept at the temperature of 25-40 ℃ for stirring for 1h-4h, and then purified water is used for cleaning for 5-10 times, wherein the mass ratio of the product obtained in the step S4, the NaOH, the epichlorohydrin and the sodium borohydride is 2-8:2-40:5-20:0.1-1; in the step S5, a crosslinking control agent may be added to control the degree of crosslinking before adding epichlorohydrin and sodium borohydride, where the crosslinking control agent is one or more of dextran and hydroxypropyl cellulose.
The mixed mode microsphere is applied to the impurity removal and purification of biological samples, and particularly to the impurity removal and purification field of exosome and delivery carrier purification.
The beneficial effects of the invention are as follows:
1. the invention provides a microsphere preparation technology, which realizes the comprehensive size exclusion chromatography, ion chromatography and hydrophilic-hydrophobic chromatography on microspheres without using strong toxic reagents, is used for the separation and purification of biological samples, and adopts an innovative microsphere structure and a synthesis scheme, and endows the microspheres with unique structures in a shell coating mode of inward impregnation and outward deposition, thereby realizing the biological application of complex system sample purification;
2. the invention provides a mixed mode chromatography scheme, which adopts a mixed mode microsphere chromatography medium to realize the separation and purification of biological samples in one step, and simplifies the flow and the separation and purification cost (including material cost and time cost) of the separation and purification of the biological samples; the workload of passing through 3 chromatographic columns is originally required, and the purpose of separation and purification can be achieved by adopting the mixed mode microsphere through 1 step of column purification, so that the purification flow and the purification time are greatly reduced; the cost of the chromatographic material is reduced from 3 parts to 1 part;
3. the invention is used as a new scheme for the rapid impurity removal and purification of specific biological samples, such as exosome purification and liposome delivery vehicle purification; the mixed mode microsphere can reject biological samples with the size of more than 20-50nm and adsorb biological impurities with the size of less than 20-50nm, and the mechanism can be directly applied to the purification of specific biological samples.
Drawings
FIG. 1 is a photograph of a mixed mode agarose microsphere 1 according to an embodiment of the invention;
FIG. 2 is a photograph of a mixed mode agarose microsphere 2 according to an embodiment of the invention;
FIG. 3 is a photograph of a mixed mode agarose microsphere 3 according to an embodiment of the invention;
FIG. 4 is a photograph showing adsorption of rhodamine B by the mixed mode agarose microsphere 2 in the embodiment of the invention;
FIG. 5 is a photograph showing adsorption of 30nm aqueous iron by the mixed mode agarose microsphere 2 according to the example of the present invention;
FIG. 6 is a photograph showing adsorption of mixed mode agarose microspheres 2 immersed in 40nm aqueous iron for 10min in an embodiment of the invention;
FIG. 7 is a photograph showing adsorption of a 40nm aqueous iron impregnation for 20 minutes by a mixed mode agarose microsphere 2 according to an embodiment of the invention;
FIG. 8 is a photograph showing adsorption of 50nm aqueous phase iron by mixed mode agarose microsphere 2 in the example of the present invention;
FIG. 9 is a photograph showing adsorption of 60nm aqueous phase iron by the mixed mode agarose microsphere 2 according to the embodiment of the invention;
FIG. 10 is a photograph showing adsorption of 70nm aqueous phase iron by mixed mode agarose microsphere 2 in the example of the present invention;
FIG. 11 is a photograph showing adsorption of 100nm aqueous phase iron by the mixed mode agarose microsphere 2 in the example of the present invention.
Detailed Description
The present invention will be described in further detail with reference to the following examples in order to make the objects, technical solutions and advantages of the present invention more apparent. It should be understood that the specific embodiments described herein are for purposes of illustration only and are not intended to limit the scope of the invention.
Example 1: the inner shell layer is as follows: 3% agarose microsphere resin-based sphere+n-hexylamine, and the coating shell layer is as follows: agarose with a mass concentration of 4% and dextran with a mass concentration of 4%
3% agarose microsphere resin-based sphere cross-linking: 100mL (3 g dry powder) of 3% agarose microsphere resin-based spheres are taken, washed 3 times by pure water, drained and added with 200mL of 1M NaOH solution to be mixed, after being stirred uniformly at a medium speed at 37 ℃, 20mL of epichlorohydrin and 1.0g of sodium borohydride are added, after being kept at the medium speed at 37 ℃ to be stirred continuously for 2.5 hours, the resin-based spheres are washed 10 times by the pure water, and the crosslinked 3% agarose microsphere resin-based spheres are obtained;
cross-linked 3% agarose microsphere resin-based sphere activation: taking 100mL of crosslinked 3% agarose microsphere resin-based spheres, draining, adding 150mL of 1M NaOH solution, mixing, stirring uniformly at a medium speed at 37 ℃, adding 40mL of epichlorohydrin and 0.3g of sodium borohydride, continuously keeping stirring for 2 hours at a medium speed at 37 ℃, and washing with purified water for 10 times to obtain activated crosslinked 3% agarose microsphere resin-based spheres;
grafting n-hexylamine: taking 100mL of activated crosslinked 3% agarose microsphere resin matrix, draining, adding 150mL of 1MNaOH solution, mixing, stirring uniformly at a medium speed at 37 ℃, adding 200mL of n-hexylamine, continuously keeping stirring for 24 hours at a medium speed at 37 ℃, and washing with purified water for 10 times to obtain the n-hexylamine grafted 3% agarose microsphere resin.
And (3) coating a shell layer: taking 100mL of n-hexylamine grafted 3% agarose microsphere resin, draining, adding 10mL of pure water, 0.4g of glucan and 0.4g of agarose, setting the temperature of an oil bath to 110 ℃, stirring at a medium speed, reducing the temperature of the oil bath to 60 ℃ after the solution boils, keeping the temperature of the oil bath at the medium speed, stirring for 1h at the medium speed, stopping heating, keeping the stirring at the medium speed, naturally cooling, cooling to room temperature, draining, and washing for 10 times.
Crosslinking of the outer shell layer: the coated microspheres were drained, 100mL of 1M NaOH solution was added and mixed, after stirring uniformly at a moderate speed at 37 ℃,10 mL of epichlorohydrin and 0.5g of sodium borohydride were added, stirring was continued at a moderate speed at 37 ℃ for 2.5 hours, and then the mixture was washed with purified water 10 times to obtain mixed mode agarose microspheres 1 (inner shell: 3% agarose microsphere resin-based spheres+n-hexylamine; outer shell: agarose microspheres with a mass concentration of 4% and dextran microspheres with a mass concentration of 4%).
The outer shell layer of the resulting mixed mode agarose microsphere 1 had a thickness of 7 μm and a exclusion limit of 50nm. As shown in FIG. 1, a photograph of a mixed mode agarose microsphere 1 is shown.
Example 2: the inner shell layer is as follows: 4% agarose microsphere resin-based sphere +N, N-dimethylhexylamine, the cladding crust layer is: agarose with a mass concentration of 4% and dextran with a mass concentration of 4%
4% agarose microsphere resin-based sphere cross-linking: 100mL (4 g dry powder) of 4% agarose microsphere resin-based spheres are taken, washed 3 times by pure water, drained and added with 200mL of 1M NaOH solution to be mixed, after being stirred uniformly at a medium speed at 37 ℃, 20mL of epichlorohydrin and 1.0g of sodium borohydride are added, after being kept at the medium speed at 37 ℃ to be stirred continuously for 2.5 hours, the resin-based spheres are washed 10 times by the pure water, and the crosslinked 4% agarose microsphere resin-based spheres are obtained;
cross-linking 4% agarose microsphere resin-based sphere activation: taking 100mL of crosslinked 4% agarose microsphere resin-based spheres, draining, adding 150mL of 1M NaOH solution, mixing, stirring uniformly at a medium speed at 37 ℃, adding 40mL of epichlorohydrin and 0.3g of sodium borohydride, continuously keeping stirring for 2 hours at a medium speed at 37 ℃, and washing with purified water for 10 times to obtain activated crosslinked 4% agarose microsphere resin-based spheres;
grafting N, N-dimethylhexylamine: 100mL of activated crosslinked 4% agarose microsphere resin matrix is drained, 150mL of 1MNaOH solution is added for mixing, 200mLN, N-dimethylhexylamine is added after uniform stirring at a medium speed of 37 ℃, after continuous stirring at a medium speed of 37 ℃ for 24 hours, purified water is used for cleaning for 10 times, and the N, N-dimethylhexylamine grafted 4% agarose microsphere resin is obtained.
And (3) coating a shell layer: taking 100mLN, draining the N-dimethylhexylamine grafted 4% agarose microsphere resin, adding 10mL of pure water, 0.4g of glucan and 0.4g of agarose, setting the temperature of an oil bath to 110 ℃, stirring at a medium speed, reducing the temperature of the oil bath to 60 ℃ after the solution is boiled, keeping the temperature of the 60 ℃ and stirring at the medium speed for 1h, stopping heating, keeping the stirring at the medium speed, naturally cooling, cooling to room temperature, draining, and washing for 10 times.
Crosslinking of the outer shell layer: the coated microspheres were drained, mixed with 0.4g dextran (adjustment crosslinking) and 100mL1M NaOH solution, stirred at medium speed at 37℃uniformly, then 10mL epichlorohydrin and 0.5g sodium borohydride were added, and after continuing to keep stirring at medium speed at 37℃for 2.5 hours, the mixture was washed with purified water 10 times to obtain mixed mode agarose microspheres 2 (inner shell: 4% agarose microsphere resin-based spheres+N, N-dimethylhexylamine, outer shell: agarose with mass concentration of 4% and dextran microsphere with mass concentration of 4%).
The outer shell layer thickness of the resulting mixed mode agarose microsphere 2 was 5 μm and the exclusion limit was 40nm. As shown in FIG. 2, a photograph of the mixed-mode agarose microsphere 2 was obtained.
Example 3: the inner shell layer is as follows: 6% agarose microsphere resin-based sphere +N, N-dimethylhexylamine, the cladding crust layer is: agarose with a mass concentration of 4% and cellulose with a mass concentration of 4%
6% agarose microsphere resin-based sphere cross-linking: 100mL (6 g of dry powder) of 6% agarose microsphere resin-based spheres are taken, washed 3 times by pure water, drained and added with 200mL of 1MNaOH solution to be mixed, after being stirred uniformly at a medium speed at 37 ℃, 20mL of epichlorohydrin and 1.0g of sodium borohydride are added, after being stirred continuously at the medium speed at 37 ℃ for 2.5 hours, the mixture is washed 10 times by the pure water, and the crosslinked 6% agarose microsphere resin-based spheres are obtained;
cross-linking 6% agarose microsphere resin-based sphere activation: taking 100mL of crosslinked 6% agarose microsphere resin-based spheres, draining, adding 150mL of 1M NaOH solution, mixing, stirring uniformly at a medium speed at 37 ℃, adding 40mL of epichlorohydrin and 0.3g of sodium borohydride, continuously keeping stirring for 2 hours at a medium speed at 37 ℃, and washing with purified water for 10 times to obtain activated crosslinked 6% agarose microsphere resin-based spheres;
grafting N, N-dimethylhexylamine: 100mL of activated crosslinked 6% agarose microsphere resin matrix is drained, 150mL of 1MNaOH solution is added for mixing, 200mLN, N-dimethylhexylamine is added after uniform stirring at a medium speed of 37 ℃, after continuous stirring at a medium speed of 37 ℃ for 24 hours, purified water is used for cleaning for 10 times, and the N, N-dimethylhexylamine grafted 6% agarose microsphere resin is obtained.
And (3) coating a shell layer: taking 100mLN, draining the 6% agarose microsphere resin grafted by N-dimethylhexylamine, adding 10mL of pure water, 0.8g of cellulose and 0.8g of agarose, setting the temperature of an oil bath to 110 ℃, stirring at a medium speed, reducing the temperature of the oil bath to 60 ℃ after the solution is boiled, keeping the temperature of the 60 ℃ and stirring at the medium speed for 1h, stopping heating, keeping the stirring at the medium speed, naturally cooling, cooling to room temperature, draining, and washing for 10 times.
Crosslinking of the outer shell layer: the coated microspheres were drained, mixed with 0.4g of hydroxypropyl cellulose (adjusting crosslinking) and 100mL of 1M NaOH solution, stirred uniformly at a medium speed at 37 ℃, then 10mL of epichlorohydrin and 0.5g of sodium borohydride were added, stirred continuously at a medium speed at 37 ℃ for 2.5 hours, and then washed with purified water 10 times to obtain mixed mode agarose microspheres 3 (inner shell: 6% agarose microsphere resin-based spheres+N, N-dimethylhexylamine, outer shell: agarose with a mass concentration of 4% and cellulose with a mass concentration of 4%).
The outer shell layer thickness of the resulting mixed mode agarose microsphere 3 was 2 μm and the exclusion limit was 20nm. As shown in FIG. 3, a photograph of the mixed-mode agarose microsphere 3 was obtained.
Example 4:
evaluation of mixed mode agarose microsphere 2 prepared in example 2:
1. rhodamine B impregnation of mixed mode agarose microsphere 2:
rhodamine B impregnation: 2mL of mixed mode agarose microspheres 2 were taken, placed in a 10mL centrifuge tube, 2mL of 1mg/mL rhodamine B solution was added, and the mixture was incubated for 5min with spin.
The effect of the mixed mode agarose microsphere 2 on adsorbing the fat-soluble rhodamine B was observed by a microscope, and as shown in fig. 4, rhodamine B molecules were substantially all captured and adsorbed by the interior of the microsphere, and rhodamine B molecules were substantially absent from the solution.
2. Adsorption of 30nm aqueous phase iron (oil phase iron to aqueous phase iron) by mixed mode agarose microsphere 2
30nm aqueous iron impregnation: 1mL of mixed mode agarose microsphere 2 is taken, filled into a 10mL centrifuge tube, 1mL of 1mg/mL30nm oil phase iron solution is added, and the mixture is rotated and incubated for 10min.
The effect of the mixed mode agarose microsphere 2 on adsorbing 30nm aqueous phase iron was observed by a microscope, and as shown in fig. 5, 30nm aqueous phase iron converted from oil phase iron can enter the interior of the microsphere and be captured by the microsphere.
3. Adsorption of 40nm aqueous phase iron (oil phase iron to aqueous phase iron) by mixed mode agarose microsphere 2
40nm aqueous iron impregnation: taking 1mL of mixed mode agarose microsphere 2 respectively, loading into a 10mL centrifuge tube, adding 1mL of 1mg/mL30nm oil phase iron solution, and rotating and incubating for 10min and 20min respectively.
The effect of the mixed mode agarose microsphere 2 on adsorbing 40nm aqueous phase iron was observed by microscopy, and as shown in fig. 6-7, the 40nm aqueous phase iron converted from oil phase iron required sufficient impregnation time (20 min for small amount of particles to enter the interior of the microsphere) to enter the microsphere.
4. Mixed mode agarose microsphere 2 pairs of 50nmFe 3 O 4 Impregnation adsorption of nanoparticles
50nmFe 3 O 4 Nanoparticle impregnation: 1mL of mixed mode agarose microsphere 2 was taken, placed in a 10mL centrifuge tube, and 1mL of 1mg/mL50nmFe was added 3 O 4 The nanoparticle solution was spin incubated for 20min.
The mixed die type microsphere 2 is observed to absorb 50nmFe by a microscope 3 O 4 The effect of the nanoparticle is shown in FIG. 8, 50nmFe 3 O 4 Cannot enter the interior of the microsphere and is discharged outside the microsphere.
5. Mixed mode agarose microsphere 2 pairs of 60nmFe 3 O 4 Impregnation adsorption of nanoparticles
60nmFe 3 O 4 Nanoparticle impregnation: 1mL of mixed mode agarose microsphere 2 was taken, placed in a 10mL centrifuge tube, and 1mL of 1mg/mL60nmFe was added 3 O 4 The nanoparticle solution was spin incubated for 30min.
The mixed die type microsphere 2 is observed to absorb 60nmFe by a microscope 3 O 4 The effect of the nanoparticle is, as shown in FIG. 9, 60nmFe 3 O 4 Cannot enter the interior of the microsphere and is discharged outside the microsphere.
6. Mixed mode agarose microsphere 2 pairs of 70nmFe 3 O 4 Impregnation adsorption of nanoparticles
70nmFe 3 O 4 Nanoparticle impregnation: 1mL of mixed mode agarose microsphere 2 was taken, placed in a 10mL centrifuge tube, and 1mL of 1mg/mL70nmFe was added 3 O 4 The nanoparticle solution was spin incubated for 30min.
The mixed die type microsphere 2 is observed to absorb 70nmFe through a microscope 3 O 4 The effect of the nanoparticle is, as shown in FIG. 10, 70nmFe 3 O 4 Cannot enter the interior of the microsphere and is discharged outside the microsphere.
7. Mixed mode agarose microsphere 2 pairs of 100nmFe 3 O 4 Impregnation adsorption of nanoparticles
100nmFe 3 O 4 Nanoparticle impregnation: 1mL of mixed mode agarose microsphere 2 was taken, placed in a 10mL centrifuge tube, and 1mL of 1mg/mL100nmFe was added 3 O 4 The nanoparticle solution was spin incubated for 30min.
The mixed mode agarose microsphere 2 is observed to adsorb 100nmFe by a microscope 3 O 4 The effect of the nanoparticles, as shown in FIG. 11, was 100nmFe 3 O 4 Cannot enter the interior of the microsphere and is discharged outside the microsphere.
Example 5: purification of 40-150 μm exosome solution
Mixed mode microsphere column packing: 2mL of mixed mode agarose microsphere 2 is taken and filled into 10mL of pre-packed column, the column is vertically placed, and the column is washed 3 times by purified water after all the preservation solution in the column flows out.
Crude and pure exosome solution of 40-150 μm: centrifuging the exosome expression sample with the particle size of 40-150 μm to remove cell disruption impurities and large-particle-size impurities, and filtering with a 0.22 μm filter membrane.
Removing impurities from exosomes of 40-150 μm, purifying: passing the crude and pure exosome sample through a mixed mode microsphere 2 purification column, and collecting flow passing through exosome at the bottom. The recovery rate of exosomes of 40-150 μm is 95%.
The synthesized mixed mode microsphere 2 is applied to impurity removal and purification of the exosomes of 40-150 mu m, and can rapidly adsorb small molecular impurities from the crude pure exosome solution, while the exosomes of 40-150 mu m cannot be adsorbed by the microsphere, so that the rapid impurity removal and purification of the exosomes of 40-150 mu m are realized.
In summary, the invention discloses a preparation method and application of mixed mode microspheres for biological sample impurity removal and purification; the mixed mode microsphere consists of an adsorption inner shell layer with hydrophobic and ionic interactions and a hydrophilic outer shell layer with a drainage effect, wherein the hydrophobic and ionic interactions of the inner shell layer are formed by bonding ionizable amino long-chain alkane and agarose microsphere resin, the outer shell layer is formed by inward dipping and outward deposition and crosslinking of polysaccharide such as agarose, dextran, cellulose and the like, the thickness and drainage limit of the drainage layer can be controlled by the porosity of the agarose microsphere resin, the concentration of polysaccharide and the crosslinking degree of the outer shell layer.
So far, those skilled in the art will recognize that while embodiments of the present invention have been shown and described in detail herein, many other variations or modifications that are in accordance with the principles of the present invention may be directly ascertained or inferred from the present disclosure without departing from the spirit and scope of the present invention. Accordingly, the scope of the present invention should be understood and deemed to cover all such other variations or modifications.
Claims (10)
1. A mixed mode microsphere for purification of biological samples, wherein the mixed mode microsphere is composed of an inner shell layer and an outer shell layer;
the inner shell layer adopts agarose microsphere resin as a base sphere core, and hydrophobic molecules capable of being ionized are grafted after the agarose microsphere resin is crosslinked and activated to form the agarose microsphere resin inner shell layer under the combined action of hydrophobic and ions;
the outer shell layer is formed by inwards dipping the surface of the inner shell layer and outwards depositing a coated polysaccharide porous medium, a thin shell layer or a net-shaped branched structure can be formed by outwards depositing the surface of the outer layer of the agarose microsphere resin inner shell layer, after inwards dipping and outwards depositing the coated polysaccharide porous medium, a crosslinking control agent and a crosslinking agent are added to crosslink the outer shell layer, and the pore diameter of the outer shell layer is smaller than that of the agarose microsphere resin matrix spheres;
the shell layer consists of an inward dipping shell layer and an outward deposition shell layer;
the inward immersed shell layer in the shell layer determines the exclusion limit of the mixed mode microsphere shell layer;
the thickness of the outward deposition shell layer is controlled by the adding amount of the polysaccharide when the shell layer coats the polysaccharide porous medium;
the hydrophobic molecules capable of ionization include primary, secondary or tertiary amines of 6-18 carbon long or branched chain, charged amine structures and hydrophobic long chains.
2. The mixed mode microsphere for purification of biological samples according to claim 1, wherein the agarose microsphere resin is 2% -8% agarose microsphere, and the microsphere is grafted with hydrophobic molecules capable of ionization after crosslinking and activation; the cross-linking agents are all mixtures of epichlorohydrin and sodium borohydride.
3. A mixed mode microsphere for purification of biological samples according to claim 1, wherein the agarose microsphere resin is 4% agarose microsphere, and the hydrophobic molecules capable of ionization are N-hexylamine, N-dimethylhexylamine or N-octylamine; the porous medium for the inward dipping and outward depositing of the outer shell layer is one or more of agarose, dextran, cyclodextrin, chitosan and cellulose.
4. The mixed mode microsphere for biological sample impurity removal and purification according to claim 1, wherein 3-6% agarose microsphere resin-based spheres are selected as agarose microsphere resin, the mass concentration of the polysaccharide porous medium is 2% -8%, the thickness of the inward impregnation shell layer is 1-10 μm, and the exclusion limit is 20-50nm; the crosslinking control agent is one or more of dextran or hydroxypropyl cellulose.
5. A mixed mode microsphere for use in purification of biological samples according to claim 1, wherein the outward deposited shell layer of said outer shell layer is in the form of a network branched structure.
6. A method for preparing mixed mode microspheres for purification of biological samples, the method comprising the steps of:
step S1: crosslinking of agarose microsphere resin-based spheres: washing agarose microsphere resin-based spheres with pure water, draining, adding NaOH solution, mixing, stirring uniformly, adding epichlorohydrin and sodium borohydride, keeping stirring, and washing with pure water to obtain crosslinked agarose microsphere resin-based spheres;
step S2: activating agarose microsphere resin-based spheres: taking the crosslinked agarose microsphere resin-based spheres obtained in the step S1, draining, adding NaOH solution, mixing, uniformly stirring, adding epichlorohydrin and sodium borohydride, continuously keeping stirring, and cleaning with purified water to obtain activated crosslinked agarose microsphere resin-based spheres;
step S3: agarose microsphere resin-based spheres grafted with hydrophobic molecules capable of ionization: taking the activated agarose microsphere resin-based spheres obtained in the step S2, draining, adding NaOH solution, mixing, uniformly stirring, adding ionized hydrophobic molecules, continuously keeping stirring, and cleaning with purified water to obtain the ionized hydrophobic molecule grafted agarose microsphere resin-based spheres;
step S4: and (3) coating a shell layer: taking the ionized hydrophobic molecular grafted agarose microsphere resin-based spheres obtained in the step S3, draining, adding pure water and a polysaccharide porous medium, stirring, wherein the polysaccharide porous medium is one or more of agarose, dextran, cyclodextrin, chitosan and cellulose, heating until the polysaccharide porous medium is completely dissolved, keeping constant temperature stirring and soaking, stopping heating, keeping stirring and naturally cooling, cooling to room temperature, draining, and washing to obtain a coated product;
step S5: crosslinking of the outer shell layer: draining the product obtained in the step S4, adding NaOH solution, mixing, adding epichlorohydrin and sodium borohydride after stirring uniformly, continuously keeping stirring, and cleaning with purified water to obtain mixed die type microspheres;
the agarose microsphere resin is 2% -8% agarose microsphere;
the hydrophobic molecules capable of ionization include primary, secondary or tertiary amines of 6-18 carbon long or branched chain.
7. The method for preparing mixed mode microspheres for biological sample impurity removal and purification according to claim 6, wherein in the step S1, naOH solution concentration is 0.5-10mol/L, the agarose microsphere resin-based spheres are washed 3-10 times by pure water, after adding NaOH solution for mixing, stirring uniformly at 25-40 ℃, adding epichlorohydrin and sodium borohydride, continuing to keep stirring at 25-50 ℃ for 1-4 hours, and then washing 5-10 times by pure water, wherein the mass ratio of the agarose microsphere resin-based spheres to the NaOH, the epichlorohydrin and the sodium borohydride is: 2-8:4-80:10-20:0.5-1; in the step S2, the concentration of the NaOH solution is 0.5-5mol/L, the NaOH solution is added for mixing, after the mixture is stirred uniformly at the temperature of 25-50 ℃, the epichlorohydrin and the sodium borohydride are added, the mixture is continuously kept at the temperature of 25-50 ℃ for stirring for 1-3 hours, and then the mixture is washed for 5-10 times by purified water, wherein the mass ratio of the crosslinked agarose microsphere resin-based spheres to the NaOH, the epichlorohydrin and the sodium borohydride is as follows: 2-8:3-60:20-40:0.15-0.3.
8. The method for preparing mixed mode microspheres for purification of biological samples according to claim 6, wherein the concentration of NaOH solution in step S3 is 0.1-1mol/L, the NaOH solution is added and mixed, hydrophobic molecules capable of ionization are added after stirring uniformly at 25-40 ℃, stirring is continued for 8-48 hours at 25-40 ℃, purified water is used for washing 5-10 times, and the mass ratio of the activated agarose microsphere resin-based spheres, naOH, hydrophobic molecules capable of ionization is: 2-8:0.6-12:100-200.
9. The method for preparing mixed mode microspheres for purification of biological samples as claimed in claim 6, wherein pure water is added in the step S4, and the temperature is raised to 60-110 ℃ and stirred; after the polysaccharide porous medium is dissolved, stirring for 0.5-2 hours at 60-75 ℃, stopping heating, and washing for 5-10 times; the mass ratio of the resin-based spheres of the hydrophobic molecular grafted agarose microsphere capable of being ionized to the dextran, the agarose and the purified water is 2-8:0.1-0.8:0.1-0.8:5-20 parts; the concentration of the NaOH solution in the step S5 is 0.5-10mol/L, the NaOH solution is added for mixing, after the mixture is stirred uniformly at the temperature of 25-40 ℃, the epichlorohydrin and the sodium borohydride are added, the mixture is continuously kept at the temperature of 25-40 ℃ for stirring for 1h-4h, and then purified water is used for cleaning for 5-10 times, wherein the mass ratio of the product obtained in the step S4, the NaOH, the epichlorohydrin and the sodium borohydride is 2-8:2-40:5-20:0.1-1; and in the step S5, before the epichlorohydrin and the sodium borohydride are added, adding a crosslinking control agent for controlling the crosslinking degree, wherein the crosslinking control agent is one or more of glucan or hydroxypropyl cellulose.
10. An application method of mixed mode microspheres for biological sample impurity removal and purification, which is characterized in that the mixed mode microspheres according to any one of claims 1-5 or the mixed mode microspheres prepared by the preparation method according to any one of claims 6-9 are applied to the field of impurity removal and purification of biological samples, in particular to the field of impurity removal and purification of exosomes and delivery carriers.
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