CN114160110B - Pre-activated polysaccharide microsphere and preparation method and application thereof - Google Patents
Pre-activated polysaccharide microsphere and preparation method and application thereof Download PDFInfo
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- 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
- B01J20/28—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof characterised by their form or physical properties
- B01J20/28014—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof characterised by their form or physical properties characterised by their form
- B01J20/28016—Particle form
- B01J20/28021—Hollow particles, e.g. hollow spheres, microspheres or cenospheres
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- A—HUMAN NECESSITIES
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- A61M1/00—Suction or pumping devices for medical purposes; Devices for carrying-off, for treatment of, or for carrying-over, body-liquids; Drainage systems
- A61M1/36—Other treatment of blood in a by-pass of the natural circulatory system, e.g. temperature adaptation, irradiation ; Extra-corporeal blood circuits
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- B01D15/00—Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
- B01D15/08—Selective adsorption, e.g. chromatography
- B01D15/10—Selective adsorption, e.g. chromatography characterised by constructional or operational features
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- B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
- B01J20/22—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof comprising organic material
- B01J20/24—Naturally occurring macromolecular compounds, e.g. humic acids or their derivatives
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- B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
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- B01J2220/00—Aspects relating to sorbent materials
- B01J2220/40—Aspects relating to the composition of sorbent or filter aid materials
- B01J2220/48—Sorbents characterised by the starting material used for their preparation
- B01J2220/4812—Sorbents characterised by the starting material used for their preparation the starting material being of organic character
- B01J2220/4825—Polysaccharides or cellulose materials, e.g. starch, chitin, sawdust, wood, straw, cotton
Abstract
The invention provides a polysaccharide microsphere, wherein aldehyde groups are contained in the polysaccharide microsphere and on the surface of the polysaccharide microsphere; the polysaccharide comprises one or more of cellulose, agarose, dextran and chitosan. The polysaccharide microsphere prepared by the invention has high hydroformylation efficiency and higher aldehyde group content, and provides a favorable basis for the application of the subsequent polysaccharide microsphere. The polysaccharide microsphere prepared by pre-activation is prepared by dissolving polysaccharide, adding glutaraldehyde for pre-activation reaction, coupling active molecules by exposed aldehyde groups subsequently, mixing and emulsifying a pre-activated water phase solution and an oil phase containing a surfactant to form water-in-oil droplets, and cooling and curing to obtain the polysaccharide microsphere. In the invention, before the step of balling in the preparation stage, the preactivated polysaccharide molecular chain is firstly formylated to form microspheres, and the subsequent activation is not needed, so that ligands such as protein A and the like can be directly modified to be used for blood adsorption or used in the field of chromatographic separation media.
Description
Technical Field
The invention belongs to the field of polysaccharide microspheres, relates to a polysaccharide microsphere and a preparation method and application thereof, and particularly relates to a pre-activated polysaccharide microsphere and a preparation method and application thereof.
Background
The natural polysaccharide medium is the medium used for biochemical separation at the earliest time, and with the continuous development of the biological separation technology, the development of novel polysaccharide media such as chitosan, konjac glucomannan and the like is also continuous. The polysaccharide medium has the following characteristics: polysaccharides have abundant hydroxyl groups and thus are very hydrophilic. In many technical processes, such as renaturation and purification of proteins, it is necessary to work in a hydrophilic environment in order to ensure the structural and functional properties of the target substance. In addition, due to the existence of hydroxyl groups, the polysaccharide medium is easy to bond with functional groups, and various types of chromatographic separation media, such as anion and cation exchange, hydrophobic chromatography, affinity chromatography and the like, are derived. Because the surface of the polysaccharide medium only contains hydroxyl, the prepared polysaccharide microspheres also need to be further activated and applied to the fields of biological separation and blood adsorption.
In order to couple some bioactive substances, such as protein a, aldehyde groups need to be modified on the microspheres. Most of the aldehyde groups of the microspheres are generally aminated firstly with epoxy groups of the microspheres, and then the aldehyde groups are modified, such as CN113101900A. Such methods have long reaction paths and can result in excessive amounts of schiff bases being generated, thereby affecting the stability of the chromatographic medium. However, only the surface of the microsphere solid can be subjected to the direct aldehyde modification method, which is very inefficient.
Therefore, how to find a suitable method for preparing polysaccharide microspheres to solve the above problems in the prior art has become one of the focuses of great concern for many research and development manufacturers and first-line researchers.
Disclosure of Invention
In view of the above, the technical problem to be solved by the present invention is to provide a polysaccharide microsphere and a preparation method thereof, and particularly to a polysaccharide microsphere prepared by a pre-activation method.
The invention provides a polysaccharide microsphere, wherein aldehyde groups are contained in the polysaccharide microsphere and on the surface of the polysaccharide microsphere;
the polysaccharide comprises one or more of cellulose, agarose, dextran and chitosan.
Preferably, the particle size of the polysaccharide microsphere is 30-300 μm;
the polysaccharide microspheres have a porous structure;
the aperture of the polysaccharide microsphere is 10-100 nm;
the density of aldehyde groups in the polysaccharide microspheres is 20-200 mu mol/g;
the aldehyde groups are provided by glutaraldehyde.
Preferably, the polysaccharide microsphere is obtained by pre-activating glutaraldehyde and grafting the glutaraldehyde on a polysaccharide molecular structure and then curing the glutaraldehyde into a sphere;
in the pre-activation grafting process, part of aldehyde group at one end of glutaraldehyde is grafted in a polysaccharide molecular structure;
the aldehyde group is an aldehyde active group;
the polysaccharide microspheres are polysaccharide microspheres with surface aldehyde groups and internal aldehyde groups;
the polysaccharide microspheres are solidified into spheres without an activation step;
the polysaccharide microspheres comprise polysaccharide microspheres for chromatographic separation media or blood adsorption.
The invention provides a preparation method of polysaccharide microspheres, which comprises the following steps:
1) Mixing a polysaccharide solution and glutaraldehyde, and performing pre-activation reaction to obtain a water phase solution;
2) Mixing the aqueous phase solution obtained in the step with an oil phase containing a surfactant again to obtain an emulsion;
3) And cooling and solidifying the emulsion obtained in the step to obtain the polysaccharide microspheres.
Preferably, the polysaccharide comprises one or more of cellulose, agarose, dextran and chitosan;
the viscosity value of the polysaccharide solution is 50-1000 mPa.s;
the polysaccharide solution is obtained by dissolving polysaccharide particles in water;
the glutaraldehyde comprises a glutaraldehyde solution;
the mass fraction of the glutaraldehyde solution is 5-10%;
the volume ratio of the glutaraldehyde solution to the polysaccharide solution is 1: (1-25).
Preferably, the mass fraction of the polysaccharide in the polysaccharide solution is 1-10%;
the mixing temperature is 85-95 ℃;
the time of the pre-activation reaction is 30-150 min;
the temperature of the preactivation reaction is 70-100 ℃;
the surfactant is specifically an emulsifier;
the surfactant comprises one or more of span 40, span 60, span80 and span 85.
Preferably, the oil phase comprises one or more of liquid paraffin, cyclohexane and toluene;
the mass content of the surfactant in the oil phase is 0.5-2%;
the volume ratio of the water phase to the oil phase is 1: (1-5);
the emulsion is a water-in-oil emulsion;
the temperature for cooling and curing is less than or equal to 45 ℃;
the time for cooling and solidifying is 5-30 min.
The invention provides a blood purification material, which comprises polysaccharide microspheres and a ligand connected with the polysaccharide microspheres;
the polysaccharide microsphere comprises the polysaccharide microsphere in any one of the technical schemes or the polysaccharide microsphere prepared by the preparation method in any one of the technical schemes.
The invention provides an adsorption column, which comprises a shell and a blood purification material arranged in the shell;
the blood purification material comprises the polysaccharide microsphere in any one of the technical schemes, the polysaccharide microsphere prepared by the preparation method in any one of the technical schemes, and the blood purification material in the technical scheme.
The invention also provides the polysaccharide microsphere prepared by the preparation method of any one of the above technical schemes, the blood purification material prepared by the preparation method of any one of the above technical schemes, and application of the adsorption column of the above technical schemes in the fields of bioseparation and preparation of blood adsorption devices.
The invention provides a polysaccharide microsphere, wherein aldehyde groups are contained in the polysaccharide microsphere and on the surface of the polysaccharide microsphere; the polysaccharide comprises one or more of cellulose, agarose, dextran and chitosan. Compared with the prior art, the invention aims at the hydroformylation of most of the existing polysaccharide microspheres, and the problems that the reaction path is long, the generated Schiff bases are excessive and the stability of a chromatography medium is influenced are caused because the epoxy groups of the microspheres are aminated firstly and then the aldehyde groups are modified. Other direct aldehyde group method, firstly solidified into ball, then activating reaction is carried out on the surface of microsphere, which has the defect of low efficiency because aldehyde reaction can only be carried out on the surface of microsphere solid.
The invention creatively provides a specific polysaccharide microsphere with a specific structure, wherein aldehyde groups are contained in the polysaccharide microsphere and on the surface of the polysaccharide microsphere, namely the polysaccharide microsphere can be subjected to aldehyde group conversion on the inner part and the surface of the polysaccharide microsphere at the same time. The polysaccharide microsphere prepared by the method has high hydroformylation efficiency and higher hydroformylation content, and provides a favorable basis for the application of the subsequent polysaccharide microsphere. The polysaccharide microsphere provided by the invention is prepared by pre-activation, firstly polysaccharide particles are dissolved, then glutaraldehyde is added for pre-activation reaction, exposed aldehyde groups can be coupled with active molecules subsequently, then the pre-activated water phase solution and an oil phase containing a surfactant are mixed and emulsified to form water-in-oil droplets, and the polysaccharide microsphere is obtained by cooling and curing. In the preparation process, before the step of balling the microspheres in the preparation stage, the polysaccharide molecular chains are pre-activated to be aldehyde-converted, and then the microspheres are formed, so that the polysaccharide microspheres with the surfaces and the interiors being aldehyde-converted are obtained. The invention can realize the preparation and activation of the microspheres at the same time. Through the control of experimental conditions, the surface and the interior of the prepared polysaccharide microsphere both contain unreacted aldehyde groups, so that the common aldehyde group formation inside and outside the microsphere is realized, subsequent activation is not required, and ligands such as protein A and the like can be directly modified for the field of blood adsorption.
Compared with the existing polysaccharide microspheres and preparation methods, the polysaccharide microspheres are pre-activated firstly, and then do not need to be activated again, which is equivalent to synchronously realizing the preparation and activation of the microspheres, and the obtained polysaccharide microspheres are the microspheres with active groups, so that the activation step is simplified, the reaction efficiency is improved, and the problems that the reaction is carried out on the solid surfaces of the microspheres in the existing activation process of the microspheres and the crosslinking rate is low are effectively solved; in addition, the invention carries out pre-activation in the preparation process of the polysaccharide microsphere, the dissolution process of the polysaccharide particles is a solution reaction, and the polysaccharide particles and glutaraldehyde are dissolved in water, thereby greatly improving the efficiency of pre-activation and crosslinking. Meanwhile, the traditional hydroformylation of the microspheres generally comprises amination of the microspheres and modification of aldehyde groups, and excessive Schiff bases are generated in the process, so that the stability of a chromatography medium is influenced. Therefore, the polysaccharide microsphere provided by the invention is expected to have wide application space in the fields of chromatographic separation media and blood adsorption.
Experimental results show that the polysaccharide microsphere which is pre-activated in the microsphere preparation process and is provided by the invention has the aldehyde group density of 120 mu mol/g determined by a hydroxylamine hydrochloride method, and compared with the traditional polysaccharide microsphere which is prepared firstly and then activated, the polysaccharide microsphere has the aldehyde group density of only 80 mu mol/g, and the content of aldehyde group active groups is greatly increased. And the density of aldehyde groups on the pre-activated polysaccharide microspheres is continuously increased along with the decrease of the content of the polysaccharide microspheres to reach 145 mu mol/g.
Drawings
FIG. 1 is a schematic diagram of a process for preparing polysaccharide microspheres by pre-activation according to the present invention;
FIG. 2 is a microphotograph of agarose microspheres prepared in example 1 of the present invention;
FIG. 3 is a distribution diagram of the particle size of agarose microspheres prepared in example 1 of the invention.
Detailed Description
For a further understanding of the invention, reference will now be made to the preferred embodiments of the invention by way of example, and it is to be understood that the description is intended to further illustrate features and advantages of the invention, and not to limit the scope of the claims.
All of the starting materials of the present invention, without particular limitation as to their source, may be purchased commercially or prepared according to conventional methods well known to those skilled in the art.
All raw materials of the invention are not particularly limited in purity, and the invention preferably adopts purity requirements which are conventional in the field of analytical purification or polysaccharide microsphere preparation.
All the raw materials, the marks and the acronyms thereof belong to the conventional marks and acronyms in the field, each mark and acronym is clear and definite in the field of related application, and the raw materials can be purchased from the market or prepared by a conventional method by the technical staff in the field according to the marks, the acronyms and the corresponding application.
The invention provides a polysaccharide microsphere, wherein aldehyde groups are contained in the polysaccharide microsphere and on the surface of the polysaccharide microsphere;
the polysaccharide comprises one or more of cellulose, agarose, dextran and chitosan.
In the present invention, the polysaccharide includes one or more of cellulose, agarose, dextran and chitosan, more preferably cellulose, agarose, dextran or chitosan.
In the present invention, the particle size of the polysaccharide microsphere is preferably 30 to 300. Mu.m, more preferably 45 to 165. Mu.m, and still more preferably 60 to 120. Mu.m.
In the present invention, the polysaccharide microsphere preferably has a porous structure.
In the present invention, the pore diameter of the polysaccharide microsphere is preferably 10 to 100nm, more preferably 15 to 70nm, and still more preferably 20 to 40nm.
In the invention, the density of aldehyde groups in the polysaccharide microspheres is preferably 20-200 [ mu ] mol/g, more preferably 50-190 [ mu ] mol/g, more preferably 100-190 [ mu ] mol/g, more preferably 110-180 [ mu ] mol/g, more preferably 120-160 [ mu ] mol/g.
In the present invention, the aldehyde group is preferably provided by glutaraldehyde.
In the invention, the polysaccharide microsphere is preferably obtained by pre-activating and grafting glutaraldehyde on a polysaccharide molecular structure and then curing to form a sphere.
In the present invention, the aldehyde group is preferably an aldehyde-based active group.
In the present invention, in the pre-activation grafting process, part of aldehyde groups at one end of glutaraldehyde are preferably grafted in the polysaccharide molecular structure. Specifically, the pre-activation process of the invention also comprises a cross-linking reaction, namely glutaraldehyde exists in two states, and a part of aldehyde groups at two ends of the glutaraldehyde are grafted in the molecular structure of the polysaccharide to form a cross-linked structure. And the other part of glutaraldehyde is aldehyde group grafted to the molecular structure of polysaccharide and the other part of aldehyde group becomes aldehyde group active group grafted to the molecular structure of polysaccharide. In particular, the invention adopts excessive glutaraldehyde, and better ensures the pre-activation process.
In the present invention, the polysaccharide microspheres are preferably polysaccharide microspheres that are surface-aldehyde-modified and interior-aldehyde-modified.
In the present invention, the polysaccharide microspheres are preferably cured into spheres without an activation step.
In the present invention, the polysaccharide microspheres preferably include polysaccharide microspheres for chromatographic separation media or blood adsorption.
The invention provides a preparation method of polysaccharide microspheres, which comprises the following steps:
1) Mixing a polysaccharide solution and glutaraldehyde, and performing pre-activation reaction to obtain a water phase solution;
2) Mixing the aqueous phase solution obtained in the step with an oil phase containing a surfactant again to obtain an emulsion;
3) And cooling and solidifying the emulsion obtained in the step to obtain the polysaccharide microspheres.
Firstly, mixing a polysaccharide solution and glutaraldehyde, and carrying out pre-activation reaction to obtain an aqueous phase solution.
In the present invention, the polysaccharide preferably comprises one or more of cellulose, agarose, dextran and chitosan, more preferably cellulose, agarose, dextran or chitosan.
In the present invention, the viscosity value of the polysaccharide solution is preferably 50 to 1000 mPas, more preferably 250 to 800 mPas, and still more preferably 450 to 600 mPas.
In the present invention, the polysaccharide solution is preferably obtained by dissolving polysaccharide particles in water. Specifically, the polysaccharide solution does not contain a solution of polysaccharide particles; more specifically, the polysaccharide solution is a clear solution having a certain viscosity.
In the present invention, the glutaraldehyde preferably includes a glutaraldehyde solution.
In the present invention, the glutaraldehyde may also be an acidic glutaraldehyde solution. Specifically, the pH value is less than or equal to 6.
In the present invention, the mass fraction of the glutaraldehyde solution is preferably 5% to 10%, more preferably 6% to 9%, and still more preferably 7% to 8%.
In the present invention, the mass fraction of the polysaccharide in the polysaccharide solution is preferably 1% to 10%, more preferably 3% to 8%, and still more preferably 5% to 6%.
In the present invention, the volume ratio of the glutaraldehyde solution to the polysaccharide solution is preferably 1: (1 to 25), more preferably 1: (6 to 20), more preferably 1: (11 to 15).
In the present invention, the time of the pre-activation reaction is preferably 30 to 150min, more preferably 50 to 130min, and still more preferably 70 to 110min.
In the present invention, the temperature of the pre-activation reaction is preferably 70 to 100 ℃, more preferably 75 to 95 ℃, and more preferably 80 to 90 ℃.
The invention mixes the water phase solution obtained in the above steps with the oil phase containing the surfactant again to obtain the emulsion.
In the present invention, the surfactant is particularly preferably an emulsifier.
In the present invention, the surfactant preferably comprises one or more of span 40, span 60, span80 and span 85, more preferably span 40, span 60, span80 or span 85.
In the present invention, the oil phase preferably includes one or more of liquid paraffin, cyclohexane, and toluene, and more preferably liquid paraffin, cyclohexane, or toluene.
In the present invention, the mass content of the surfactant in the oil phase is preferably 0.5% to 2%, more preferably 0.7% to 1.8%, and still more preferably 1.0% to 1.5%.
In the present invention, the volume ratio of the aqueous phase to the oil phase is preferably 1: (1 to 5), more preferably 1: (1.5 to 4.5), more preferably 1: (2 to 4), more preferably 1: (2.5-3.5).
In the present invention, the emulsion is preferably a water-in-oil emulsion.
Finally, cooling and solidifying the emulsion obtained in the step to obtain the polysaccharide microspheres.
In the present invention, the temperature for cooling and curing is preferably 45 ℃ or lower, more preferably 35 ℃ or lower, and still more preferably 25 ℃ or lower.
In the present invention, the time for cooling and curing is preferably 5 to 30min, more preferably 10 to 25min, and still more preferably 15 to 20min.
In the invention, the cooling solidification mode comprises that cold water is adopted to replace hot water for cooling solidification. Specifically, the hot water in the reaction device is discharged, and then cold water is added for replacement or cold water/ice blocks are directly added into the reaction device, so long as the temperature of the reaction device is quickly reduced to the room temperature.
The invention is a complete and refined integral preparation process, better ensures the specific structure of the polysaccharide microsphere, and further improves the activation performance of the polysaccharide microsphere, the preparation method of the polysaccharide microsphere can specifically comprise the following steps, and the preparation of the microsphere is completed while the hydroformylation is realized:
the method comprises the following steps: dispersing polysaccharide particles in water to obtain a dissolved polysaccharide solution;
step two: adding a glutaraldehyde solution into the polysaccharide solution for preactivation to obtain an aqueous phase solution;
step three: mixing the water phase solution in the step two with an oil phase containing a surfactant, and mechanically stirring to form a water-in-oil emulsion;
step four: and (4) cooling the emulsion in the third step, and solidifying to obtain the pre-activated polysaccharide microspheres.
Specifically, the polysaccharide may be one or a mixture of two or more of cellulose, agarose, dextran, and chitosan.
Specifically, the viscosity of the polysaccharide solution may be 50 to 1000 mPas.
Specifically, the mass fraction of the glutaraldehyde solution in the second step may be 5% to 10%. Wherein the pH may be less than 6.
Specifically, the reaction time of the polysaccharide solution and the glutaraldehyde solution can be 30-150 min, and the reaction temperature can be 70-100 ℃.
Specifically, the volume ratio of the glutaraldehyde solution to the polysaccharide solution is 1: (1-25).
Specifically, the oil phase is one or a mixture of more than two of liquid paraffin, cyclohexane and toluene.
Specifically, the surfactant is preferably a surfactant emulsifier, and may be one or a mixture of more than two of span 40, span 60, span80 and span 85.
Specifically, when the preparation of the microsphere is completed, the activation is already completed, and aldehyde groups are modified on the surface and inside of the microsphere.
Specifically, the hot water in the water bath kettle is discharged, and then cold water is added for replacement or cold water/ice blocks are directly added into the water bath kettle, and the temperature of the water bath kettle is quickly reduced to the room temperature.
The method comprises the steps of mixing a polysaccharide solution with an acidic glutaraldehyde solution at 85-95 ℃, and taking the solution obtained by reaction as a water phase; then mixing the water phase solution with oil phase containing surfactant, and mechanically stirring to form water-in-oil emulsion; and finally, cooling the emulsion, solidifying into balls, and washing to obtain the pre-activated polysaccharide microspheres.
Referring to fig. 1, fig. 1 is a schematic diagram of a process flow for preparing polysaccharide microspheres by pre-activation according to the present invention.
The invention is a complete and detailed integral technical scheme, is better convenient for the subsequent application of the pre-activated polysaccharide microspheres prepared by the invention, and also provides a blood purification material, wherein the blood purification material comprises polysaccharide microspheres and a ligand connected with the polysaccharide microspheres;
the polysaccharide microsphere comprises the polysaccharide microsphere in any one of the technical schemes or the polysaccharide microsphere prepared by the preparation method in any one of the technical schemes.
The invention is a complete and detailed integral technical scheme, is better convenient for the subsequent application of the pre-activated polysaccharide microspheres prepared by the invention, and also provides an adsorption column, wherein the adsorption column comprises a shell and a blood purification material arranged in the shell;
the blood purification material comprises the polysaccharide microsphere in any one of the technical schemes, the polysaccharide microsphere prepared by the preparation method in any one of the technical schemes, and the blood purification material in the technical scheme.
The invention also provides the polysaccharide microsphere prepared by the preparation method of any one of the above technical schemes, the blood purification material prepared by the preparation method of any one of the above technical schemes, and application of the adsorption column of the above technical schemes in the fields of bioseparation and preparation of blood adsorption devices.
The steps of the invention provide a pre-activated polysaccharide microsphere and a preparation method and application thereof. The polysaccharide microsphere is a specific polysaccharide microsphere with a specific structure, and both the interior and the surface of the polysaccharide microsphere contain aldehyde groups, namely the polysaccharide microsphere can be subjected to aldehyde group conversion simultaneously. The polysaccharide microsphere prepared by the method has high hydroformylation efficiency and higher hydroformylation content, and provides a favorable basis for the application of the subsequent polysaccharide microsphere.
The polysaccharide microsphere provided by the invention is prepared by pre-activation, firstly polysaccharide particles are dissolved, then glutaraldehyde is added for pre-activation reaction, exposed aldehyde groups can be coupled with active molecules subsequently, then the pre-activated water phase solution and an oil phase containing a surfactant are mixed and emulsified to form water-in-oil droplets, and the polysaccharide microsphere is obtained by cooling and curing. In the preparation process, before the microsphere is formed into a sphere in the preparation stage, the polysaccharide molecular chain is pre-activated to be formylated and then the microsphere is formed, so that the polysaccharide microsphere with the surface and the inner part being formylated is obtained. The invention can realize the preparation and activation of the microsphere at the same time. Through the control of experimental conditions, the surface and the interior of the prepared polysaccharide microsphere both contain unreacted aldehyde groups, so that the common aldehyde group formation inside and outside the microsphere is realized, subsequent activation is not required, and ligands such as protein A and the like can be directly modified for the field of blood adsorption.
Compared with the existing polysaccharide microspheres and preparation methods, the polysaccharide microspheres are pre-activated firstly, and then do not need to be activated again, which is equivalent to synchronously realizing the preparation and activation of the microspheres, and the obtained polysaccharide microspheres are the microspheres with active groups, so that the activation step is simplified, the reaction efficiency is improved, and the problems that the microspheres are reacted with an activating agent in the activation process of the existing microspheres, the reaction is carried out on the solid surfaces of the microspheres, and the crosslinking rate is low are effectively solved; in addition, the invention carries out pre-activation in the preparation process of the polysaccharide microsphere, the dissolution process of the polysaccharide particles is a solution reaction, and the polysaccharide particles and glutaraldehyde are dissolved in water, thereby greatly improving the efficiency of pre-activation and crosslinking. Meanwhile, the traditional hydroformylation of the microspheres generally comprises amination of the microspheres and modification of aldehyde groups, and excessive Schiff bases are generated in the process, so that the stability of a chromatography medium is influenced. Therefore, the polysaccharide microsphere provided by the invention is expected to have wide application space in the fields of chromatographic separation media and blood adsorption.
Experimental results show that the polysaccharide microsphere which is pre-activated in the microsphere preparation process and is provided by the invention has the aldehyde group density of 120 mu mol/g determined by a hydroxylamine hydrochloride method, and compared with the traditional polysaccharide microsphere which is prepared firstly and then activated, the polysaccharide microsphere has the aldehyde group density of only 80 mu mol/g, and the content of aldehyde group active groups is greatly increased. And the density of aldehyde groups on the pre-activated polysaccharide microspheres is continuously increased along with the decrease of the content of the polysaccharide microspheres to reach 145 mu mol/g.
For further illustration of the present invention, the following will describe in detail a polysaccharide microsphere and its preparation method and application in conjunction with the following examples, but it should be understood that these examples are implemented on the premise of the technical solution of the present invention, and the detailed embodiments and specific procedures are given, only for further illustration of the features and advantages of the present invention, not for limitation of the claims of the present invention, and the scope of the present invention is not limited to the following examples.
Example 1
Preparation of Pre-activated agarose microspheres at 6% Mass concentration
(1) Preparation of agarose solution
6g of the agarose dry powder was dispersed in 100mL of water and heated at 95 ℃ for 30min to obtain a dissolved agarose solution. After it was dissolved, the viscosity was measured to 286 mpa-S with an NDJ-5S viscometer.
(2) Preparation of preactivated agarose solutions
Adding 6% glutaraldehyde solution into the agarose solution obtained in the step (1), and uniformly mixing, wherein the ratio of the glutaraldehyde solution to the agarose solution is 1: 10. heating at 95 deg.C for 60min to obtain preactivated agarose solution as water phase.
(3) Preparation of preactivated agarose microspheres
Preheating 200mL of liquid paraffin (containing 3g of span 80) at 90 ℃ for 30min to serve as an oil phase, quickly adding the water phase obtained in the step (2) into the oil phase, and mechanically stirring at the rotating speed of 500rpm for 60min to form a water-in-oil emulsion. And replacing hot water with cold water, cooling, solidifying and cleaning to obtain the preactivated agarose microspheres.
The agarose microspheres prepared in example 1 of the invention were characterized.
Referring to FIG. 2, FIG. 2 is a microphotograph of agarose microspheres prepared in example 1 of the present invention.
As shown in FIG. 2, the morphology of the preactivated agarose microspheres is observed by an XSP-8C optical microscope, and the morphology of the preactivated agarose microspheres prepared by the invention is regular and round.
Referring to FIG. 3, FIG. 3 is a graph showing the particle size distribution of agarose microspheres prepared in example 1 of the present invention.
As shown in FIG. 3, the particle size distribution of the agarose microspheres prepared by the invention is 45-165 μm, and the average particle size is 90 μm, which is determined by analyzing the particle size of the agarose microspheres by an LS-609 laser particle size analyzer.
The performance of the pre-activated agarose microspheres prepared in example 1 of the invention was tested.
The density of aldehyde groups of the preactivated agarose microspheres prepared in example 1 of the present invention was 120. Mu. Mol/g, as determined by hydroxylamine hydrochloride. The polysaccharide microsphere is prepared under the parallel condition by adopting the conventional process of firstly preparing the microsphere and then activating, and the aldehyde group density of the polysaccharide microsphere is only 80 mu mol/g and is far lower than that of the preactivated microsphere prepared by the invention.
Example 2
Preparation of preactivated agarose microspheres at 4% mass concentration
Similar to example 1, the difference is the agarose mass concentration.
(1) Preparation of agarose solution
4g of dry agarose powder was dispersed in 100mL of water and heated at 95 ℃ for 30min to obtain a dissolved agarose solution. After it had dissolved, the viscosity number was measured to be 210 mPa.s using an NDJ-5S viscometer.
(2) Preparation of Pre-activated agarose solutions
Adding 4% glutaraldehyde solution into the agarose solution obtained in the step (1), and uniformly mixing, wherein the ratio of the glutaraldehyde solution to the agarose solution is 1:12. heating at 95 deg.C for 60min to obtain preactivated agarose solution as aqueous phase.
(3) Preparation of preactivated agarose microspheres
200mL of liquid paraffin (containing 3g of span 80) was preheated at 90 ℃ for 30min to serve as an oil phase, and the water phase obtained in step (2) was rapidly added to the oil phase and mechanically stirred at 500rpm for 60min to form a water-in-oil emulsion. And replacing hot water with cold water, cooling, solidifying and cleaning to obtain the preactivated agarose microspheres.
The performance of the pre-activated agarose microspheres prepared in example 2 of the invention was tested.
The aldehyde group density of the preactivated agarose microspheres prepared in example 2 of the invention was 145. Mu. Mol/g, as determined by the hydroxylamine hydrochloride method. The polysaccharide microsphere is prepared under the parallel condition by adopting the conventional process of firstly preparing the microsphere and then activating, and the aldehyde group density of the polysaccharide microsphere is only 80 mu mol/g and is far lower than that of the preactivated microsphere prepared by the invention.
Comparison with example 1 shows that the aldehyde group density on the pre-activated polysaccharide microspheres increases further as the content of polysaccharide microspheres decreases.
The foregoing detailed description of a pre-activated polysaccharide microsphere provided in accordance with the present invention, as well as methods for making and using the same, and the specific examples used herein to describe the principles and embodiments of the present invention, are presented solely to aid in the understanding of the methods and their underlying concepts, including the best mode, and to enable any person skilled in the art to practice the invention, including making and using any devices or systems and performing any incorporated methods. It should be noted that, for those skilled in the art, without departing from the principle of the present invention, it is possible to make various improvements and modifications to the present invention, and those improvements and modifications also fall within the scope of the claims of the present invention. The patentable scope of the invention is defined by the claims, and may include other examples that occur to those skilled in the art. Such other embodiments are intended to be within the scope of the claims if they have structural elements that do not differ from the literal language of the claims, or if they include equivalent structural elements with insubstantial differences from the literal languages of the claims.
Claims (10)
1. Polysaccharide microspheres are characterized in that the interior and the surface of the polysaccharide microspheres contain aldehyde groups;
the polysaccharide is agarose;
the preparation method of the polysaccharide microsphere comprises the following steps:
1) Mixing a polysaccharide solution and a glutaraldehyde solution, and carrying out pre-activation reaction to obtain an aqueous phase solution;
2) Mixing the aqueous phase solution obtained in the step with an oil phase containing a surfactant again to obtain an emulsion;
3) And cooling and solidifying the emulsion obtained in the step to obtain the polysaccharide microspheres.
2. The polysaccharide microsphere according to claim 1, wherein the particle size of the polysaccharide microsphere is 30 to 300 μm;
the polysaccharide microspheres have a porous structure;
the aperture of the polysaccharide microsphere is 10 to 100nm;
the density of aldehyde groups in the polysaccharide microspheres is 20 to 200 mu mol/g;
the aldehyde groups are provided by glutaraldehyde.
3. The polysaccharide microsphere of claim 1, wherein the polysaccharide microsphere is obtained by pre-activating glutaraldehyde and grafting the glutaraldehyde onto a polysaccharide molecular structure, and then curing the glutaraldehyde into a sphere;
in the pre-activation grafting process, part of aldehyde group at one end of glutaraldehyde is grafted in a polysaccharide molecular structure;
the aldehyde group is an aldehyde group active group;
the polysaccharide microspheres are polysaccharide microspheres with surface aldehyde groups and internal aldehyde groups;
the polysaccharide microspheres are solidified into spheres without an activation step;
the polysaccharide microspheres comprise polysaccharide microspheres for chromatographic separation media or blood adsorption.
4. The preparation method of the polysaccharide microsphere is characterized by comprising the following steps:
1) Mixing a polysaccharide solution and a glutaraldehyde solution, and carrying out pre-activation reaction to obtain an aqueous phase solution;
the mass fraction of the glutaraldehyde solution is 5% -10%;
the volume ratio of the glutaraldehyde solution to the polysaccharide solution is 1: (1 to 25);
the mass fraction of polysaccharide in the polysaccharide solution is 1% -10%;
the mixing temperature is 85 to 95 ℃;
the time of the preactivation reaction is 30 to 150min;
the temperature of the preactivation reaction is 70 to 100 ℃;
the polysaccharide is agarose;
2) Mixing the aqueous phase solution obtained in the step with an oil phase containing a surfactant again to obtain an emulsion;
3) And cooling and solidifying the emulsion obtained in the step to obtain the polysaccharide microspheres.
5. The method according to claim 4, wherein the polysaccharide solution has a viscosity of 50 to 1000mPas;
the polysaccharide solution is obtained by dissolving polysaccharide particles in water.
6. The method according to claim 4, wherein the surfactant is specifically an emulsifier;
the surfactant comprises one or more of span 40, span 60, span80 and span 85.
7. The method according to claim 4, wherein the oil phase comprises one or more of liquid paraffin, cyclohexane, and toluene;
the mass content of the surfactant in the oil phase is 0.5-2%;
the volume ratio of the water phase to the oil phase is 1: (1 to 5);
the emulsion is a water-in-oil emulsion;
the temperature for cooling and curing is less than or equal to 45 ℃;
the time for cooling and curing is 5 to 30min.
8. A blood purification material, which is characterized by comprising polysaccharide microspheres and a ligand connected with the polysaccharide microspheres;
the polysaccharide microsphere comprises the polysaccharide microsphere of any one of claims 1 to 3 or the polysaccharide microsphere prepared by the preparation method of any one of claims 4 to 7.
9. An adsorption column is characterized by comprising a shell and a blood purification material arranged inside the shell;
the blood purification material comprises the polysaccharide microsphere as defined in any one of claims 1 to 3, the polysaccharide microsphere prepared by the preparation method as defined in any one of claims 4 to 7, or the blood purification material as defined in claim 8.
10. The polysaccharide microsphere according to any one of claims 1 to 3, the polysaccharide microsphere prepared by the preparation method according to any one of claims 4 to 7, the blood purification material according to claim 8, and the application of the adsorption column according to claim 9 in the fields of bioseparation and preparation of blood adsorption devices.
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Citations (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4732811A (en) * | 1982-02-28 | 1988-03-22 | Yeda Research And Development Company, Ltd. | Agarose-polyaldehyde beads and their biological application affinity chromatography, hemoperfusion, cell separation, etc. |
| US4861705A (en) * | 1983-01-31 | 1989-08-29 | Yeda Research And Development Company, Ltd. | Method for removing components of biological fluids |
| CN102000550A (en) * | 2010-10-20 | 2011-04-06 | 广州康盛生物科技有限公司 | Synthetic method used for preparing adsorbent for clearing pathogenic antibody by oxidizing periodate |
| CN106423079A (en) * | 2016-10-17 | 2017-02-22 | 江苏大学 | Preparation method and application of chitosan-microsphere immunoaffinity adsorbent |
| CN109897202A (en) * | 2019-03-15 | 2019-06-18 | 中科森辉微球技术（苏州）有限公司 | Big partial size agarose microbeads and preparation method thereof |
| WO2020154038A1 (en) * | 2018-12-05 | 2020-07-30 | Cytosorbents Corporation | Crosslinked polysaccharide based absorbents for removal of anti-a and/or anti-b antibodies from human plasma and whole blood |
| CN111718427A (en) * | 2019-03-20 | 2020-09-29 | 中国科学院化学研究所 | Dialdehyde cellulose with high aldehyde group content and preparation method and application thereof |
| CN112547029A (en) * | 2020-12-21 | 2021-03-26 | 有研资源环境技术研究院(北京)有限公司 | Microsphere composite material for arsenic-containing water body and soil heavy metal restoration and preparation method thereof |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0427715A1 (en) * | 1988-06-20 | 1991-05-22 | E.I. Du Pont De Nemours And Company | Biocompatible, substance-specific reagents for treating physiological fluids |
| US10046985B2 (en) * | 2016-02-11 | 2018-08-14 | King Fahd University Of Petroleum And Minerals | Method for removing cationic dyes from an aqueous solution using an adsorbent |
-
2021
- 2021-11-30 CN CN202111466689.3A patent/CN114160110B/en active Active
Patent Citations (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4732811A (en) * | 1982-02-28 | 1988-03-22 | Yeda Research And Development Company, Ltd. | Agarose-polyaldehyde beads and their biological application affinity chromatography, hemoperfusion, cell separation, etc. |
| US4861705A (en) * | 1983-01-31 | 1989-08-29 | Yeda Research And Development Company, Ltd. | Method for removing components of biological fluids |
| CN102000550A (en) * | 2010-10-20 | 2011-04-06 | 广州康盛生物科技有限公司 | Synthetic method used for preparing adsorbent for clearing pathogenic antibody by oxidizing periodate |
| CN106423079A (en) * | 2016-10-17 | 2017-02-22 | 江苏大学 | Preparation method and application of chitosan-microsphere immunoaffinity adsorbent |
| WO2020154038A1 (en) * | 2018-12-05 | 2020-07-30 | Cytosorbents Corporation | Crosslinked polysaccharide based absorbents for removal of anti-a and/or anti-b antibodies from human plasma and whole blood |
| CN113166233A (en) * | 2018-12-05 | 2021-07-23 | 西托索尔本茨公司 | Cross-linked polysaccharide-based absorbents for removing anti-A and/or anti-B antibodies from human plasma and whole blood |
| CN109897202A (en) * | 2019-03-15 | 2019-06-18 | 中科森辉微球技术（苏州）有限公司 | Big partial size agarose microbeads and preparation method thereof |
| CN111718427A (en) * | 2019-03-20 | 2020-09-29 | 中国科学院化学研究所 | Dialdehyde cellulose with high aldehyde group content and preparation method and application thereof |
| CN112547029A (en) * | 2020-12-21 | 2021-03-26 | 有研资源环境技术研究院(北京)有限公司 | Microsphere composite material for arsenic-containing water body and soil heavy metal restoration and preparation method thereof |
Non-Patent Citations (6)
| Title |
|---|
| Co-immobilized enzymes in magnetic chitosan beads for improved hydrolysis of macromolecular substrates under a time-varying magnetic field;Yang, K et al.;《Journal Of Biotechnology》;20100720;119-127 * |
| Fe3O4磁性复合功能微球制备及蛋白分离和光催化应用研究;侯雪梅;《中国博士学位论文全文数据库》;20190115;B014-26 * |
| Novel effective immunoadsorbents based on agarose-polyaldehyde microsphere beads: Synthesis and affinity chromatography;S.Margel、M.Offarim;《Analytical Biochemistry》;19830201;342-350 * |
| 微米级壳聚糖微球的制备;陈宇等;《大连工业大学学报》;20120515;203-206 * |
| 细菌纤维素/胶原蛋白多孔微球的制备及性能;张雯等;《陕西科技大学学报》;20190109;18-24+58 * |
| 血液净化用内毒素亲和吸附剂的制备研究;李永桂等;《广东化工》;20170630;96-98 * |
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