CN115260538A - Preparation method of agarose gel microspheres with narrow particle size distribution - Google Patents

Preparation method of agarose gel microspheres with narrow particle size distribution Download PDF

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CN115260538A
CN115260538A CN202210561654.6A CN202210561654A CN115260538A CN 115260538 A CN115260538 A CN 115260538A CN 202210561654 A CN202210561654 A CN 202210561654A CN 115260538 A CN115260538 A CN 115260538A
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agarose
particle size
agarose gel
hpo
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徐偲
王晓钟
戴立言
陈英奇
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Quzhou Research Institute of Zhejiang University
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Quzhou Research Institute of Zhejiang University
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    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08J—WORKING-UP; GENERAL PROCESSES OF COMPOUNDING; AFTER-TREATMENT NOT COVERED BY SUBCLASSES C08B, C08C, C08F, C08G or C08H
    • C08J3/00—Processes of treating or compounding macromolecular substances
    • C08J3/12—Powdering or granulating
    • C08J3/16—Powdering or granulating by coagulating dispersions
    • 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
    • B01J13/06—Making microcapsules or microballoons by phase separation
    • B01J13/14—Polymerisation; cross-linking
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08J—WORKING-UP; GENERAL PROCESSES OF COMPOUNDING; AFTER-TREATMENT NOT COVERED BY SUBCLASSES C08B, C08C, C08F, C08G or C08H
    • C08J3/00—Processes of treating or compounding macromolecular substances
    • C08J3/02—Making solutions, dispersions, lattices or gels by other methods than by solution, emulsion or suspension polymerisation techniques
    • C08J3/03—Making solutions, dispersions, lattices or gels by other methods than by solution, emulsion or suspension polymerisation techniques in aqueous media
    • C08J3/075—Macromolecular gels
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08J—WORKING-UP; GENERAL PROCESSES OF COMPOUNDING; AFTER-TREATMENT NOT COVERED BY SUBCLASSES C08B, C08C, C08F, C08G or C08H
    • C08J2305/00—Characterised by the use of polysaccharides or of their derivatives not provided for in groups C08J2301/00 or C08J2303/00
    • C08J2305/12—Agar-agar; Derivatives thereof
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08J—WORKING-UP; GENERAL PROCESSES OF COMPOUNDING; AFTER-TREATMENT NOT COVERED BY SUBCLASSES C08B, C08C, C08F, C08G or C08H
    • C08J2405/00—Characterised by the use of polysaccharides or of their derivatives not provided for in groups C08J2401/00 or C08J2403/00
    • C08J2405/02—Dextran; Derivatives thereof

Abstract

The invention discloses a preparation method of agarose gel microspheres with narrow particle size distribution. The method uses dextran to pre-modify agarose, and adds Na2HPO4And the double-function emulsifier, reagent proportion regulation and an emulsification process are carried out, and the agarose gel microspheres with narrow particle size distribution are obtained through solidification and washing. The invention can prepare the agarose gel microspheres with the particle size concentrated between 50 and 160 mu m. The preparation method has the beneficial effects that: compared with a micro-membrane emulsification method and a high-pressure injection method, the method has the advantages of simple process, low requirement on equipment and easy amplification production; the reaction condition is mild, and the production period is short; agarose gelThe particle size distribution of the microspheres is concentrated; the gel microspheres with different particle sizes can be obtained by regulating and controlling the emulsification time, and the application range is wide.

Description

Preparation method of agarose gel microspheres with narrow particle size distribution
Technical Field
The invention relates to the field of downstream separation and purification of biomedicine, in particular to a preparation method of agarose gel microspheres with narrow particle size distribution.
Background
Agarose is a linear polysaccharide formed by alternating linkage of a plurality of galactose, and is mainly composed of alternating linkage of 1, 3-linked beta-D-galactose and 1, 4-linked 3, 6-lacto-L-galactose. Agarose has good hydrophilicity, almost has no charged groups, rarely causes denaturation to sensitive biological macromolecules, and is often used as a chromatography medium for separation and purification of biological medicines. The agarose gel microspheres have pentagonal macropores, and can allow diffusion of substances with large molecular weight, which is also an advantage of the agarose gel microspheres in application to separation and purification of biological macromolecules.
The agarose gel microsphere is a natural polysaccharide type biological chromatography filler which is porous, hydrophilic, free of charged groups and good in biocompatibility. And because the agarose gel microspheres are rich in polyhydroxy, derivative microspheres with different functional groups can be obtained through chemical bonding, so that the agarose gel microspheres can be applied to different types of chromatography processes. At present, the agarose gel microspheres are mainly used for separating and purifying macromolecular bioactive substances such as viruses, proteins, enzymes, polypeptides and the like, and are widely applied to the industries such as biomedicine, blood products, vaccines, cosmetics, health care products and the like.
Currently, agarose microspheres mainly used in chromatographic separation, such as Sepharose series, and a series of hydrophobic, affinity, ion exchange chromatographic microspheres prepared by coupling different ligands on the basis of the Sepharose series, have average particle sizes of about 90 μm, and particle size distribution ranges of generally 45-165 μm (Linnan. Large-particle size agarose microspheres prepared by conventional membrane emulsification methods, diss. Beijing: beijing university of chemical industry, 2009.). In recent years, researchers have done a lot of work on the preparation of agarose gel microspheres. In patent No. CN108905977A, a preparation method of agarose gel microspheres is reported, wherein agarose solid is used as a matrix, activated by a cross-linking agent and then reacted with sulfuric anhydride to obtain sulfuric anhydride agarose, and the sulfuric anhydride agarose is prepared into the agarose gel microspheres, wherein the size of the agarose gel microspheres is 10-1000 mu m. In patent No. CN111073061A, there is disclosed agarose gel microspheres prepared by a two-phase spheronization method, the particle size of which is concentrated in the range of 20 to 300. Mu.m. In addition, the patent with the publication number of CN1304101C discloses agarose gel microspheres prepared by a membrane emulsification method combined with a mechanical stirring method, wherein the size of the obtained agarose gel microspheres is 1-100 μm, the process flow is longer, and the requirement on equipment is higher.
In the actual preparation process of the agarose gel microspheres, the small emulsion drops are easily absorbed by the large emulsion drops, and the large emulsion drops generate the small emulsion drops under the action of shearing force, so the prepared agarose gel microspheres often have the problems of uneven particle size distribution, overlarge particle size distribution range and the like.
In general, it is still difficult to prepare sepharose microspheres with a particle size of 45-165 μm (the size range of sepharose microspheres mainly used in chromatographic separation) and narrow particle size distribution. Therefore, the development of the preparation process of the agarose gel microspheres with narrow particle size distribution and simple process is of great significance for promoting the healthy development of the biomedical industry in China, especially the downstream chromatographic separation and purification field.
Disclosure of Invention
Aiming at the problems of excessively wide particle size distribution, longer technological process for obtaining agarose gel microspheres with uniform particle size, higher equipment requirement and the like which are often generated in the preparation process of the agarose gel microspheres, the invention provides a preparation method of the agarose gel microspheres with narrow particle size distribution, and the proportion of microspheres with the particle size range of 50-160 mu m in the prepared agarose gel microspheres is not less than 85%.
The invention aims to overcome the defects of the prior art and provides the preparation method of the agarose gel microspheres, which is simple and convenient to synthesize, narrow in particle size distribution and easy for large-scale production.
The technical scheme adopted by the invention for realizing the aim is as follows:
a preparation method of agarose gel microspheres with narrow particle size distribution comprises the following steps:
(1) Adding agarose into deionized water, stirring and heating until agarose is completely dissolved, adding dextran and Na under continuous stirring after clear sol is formed2HPO4Continuously stirring for 5-30 min at the stirring speed of 100-200 rpm to obtain solution A;
the agarose, the deionized water, the dextran, the Na2HPO4The proportion of (A) is 0.2-3g;
the glucan is added in the form of aqueous solution, and the mass percent of the glucan in the aqueous solution of the glucan is 0.1-5%;
the Na is2HPO4Adding in the form of an aqueous solution, na2HPO4Na in aqueous solution2HPO4Has a mass concentration of 0.05-0.5 g mL-1；
(2) Adding an emulsifier into the oil phase, heating to 60-120 ℃, and continuously stirring for 20-30 min at the stirring speed of 250-350 rpm to obtain a solution B;
the emulsifier is a combination of Span80 and Tween80, wherein the mass ratio of Span80 to Tween80 is 1-10;
the volume ratio of the oil phase to the deionized water is 10-100;
in the solution B, the mass concentration of the emulsifier is 1-10%;
(3) Adding the solution A obtained in the step (1) into the solution B obtained in the step (2), continuously stirring at the stirring speed of 100-200 rpm, and emulsifying for a certain time to obtain a corresponding product;
(4) And (4) cooling and solidifying the product obtained in the step (3), standing and layering, removing an upper oil layer, and removing an oil phase in a lower product by using a detergent to obtain the agarose gel microspheres with narrow particle size distribution.
Preferably, in step (1), the agarose, the deionized water, the dextran, the Na2HPO4The proportion of (A) is 0.6-2g.
Preferably, in the step (2), the oil phase is one or more of toluene, n-octane, liquid paraffin and n-heptane, and is further preferably toluene.
Preferably, in the step (2), the volume ratio of the oil phase to the deionized water is 60-80.
Preferably, in the step (2), the mass concentration of the emulsifier in the solution B is 2-5%.
Preferably, in the step (2), the mass ratio of Span80 to Tween80 in the emulsifier is 2-6.
Preferably, in the step (3), the time for emulsification is 10 to 60min.
Preferably, in step (3), during the emulsification, sampling is immediately carried out to observe under an optical microscope, and emulsification is stopped after a desired gel microsphere size is obtained.
Preferably, in step (4):
the detergent is one or more of ethanol, water, acetone and n-hexane; further preferred is a combination of ethanol and water, wherein the volume fraction of ethanol is 10%.
The invention adopts an improved two-phase balling method to prepare the agarose gel microspheres, and in order to effectively regulate and control the particle size distribution of the agarose gel microspheres, the invention has the improvement that:
(a) The agarose sol is pre-modified by using a glucan solution, and mutual collision among agarose molecules is effectively reduced through cross-linking of glucan molecules and agarose molecules, so that excessive cross-linking among the agarose molecules is effectively avoided;
(b) Using Na2HPO4Effectively improving the ionic environment of the agarose-glucan mixed solution and promoting the cross-linking process of two molecules;
(c) In the emulsification process, the mass ratio of 1-10: 1, span80 and Tween80 are used as emulsifiers, the emulsifier combination is a bifunctional emulsifier, wherein the hydrophilic-lipophilic balance value of Span80 is 4.3, the hydrophilic-lipophilic balance value of Tween80 is 15.0, the stability of the obtained emulsion is good under the condition of the mass ratio, and the stirring speed is set to be 100-200 rpm, so that excessive small emulsion drops caused by the shearing force action of large emulsion drops in the stirring process are effectively avoided;
(d) Adjusting agarose, deionized water, dextran, and Na to make the particle size distribution of the prepared agarose gel microspheres more concentrated2HPO4Oil phase and proportion of bifunctional emulsifier in the oil phase. In the ratioDextran, na2HPO4The modification effect of the double-function emulsifier and the emulsification effect of the double-function emulsifier can achieve good synergistic effect, ensure the stability of emulsion and facilitate the preparation of agarose gel microspheres with narrow particle size distribution. Example 1 is an example under preferable conditions, and the prepared agarose gel microspheres have a concentrated particle size distribution, wherein the proportion of microspheres having a particle size of 50 to 160 μm is 96% or more.
Compared with the prior art, the invention has the beneficial effects that at least:
1. compared with a membrane emulsification method and a high-pressure injection method, the preparation method provided by the invention has the advantages of simple process, low requirement on equipment and easiness in large-scale industrial production.
2. The preparation method provided by the invention has the advantages of mild reaction conditions, short production period and less environmental pollution.
3. The agarose gel microspheres prepared by the preparation method provided by the invention have concentrated particle size distribution, and the agarose gel microspheres prepared in the embodiment 1 have concentrated particle size distribution within 50-160 mu m, and the content of the agarose gel microspheres is more than 96%. The particle size of the agarose gel microspheres mainly used in chromatographic separation is 50-160 mu m, so the method provided by the invention greatly reduces the screening cost of the agarose gel microspheres.
4. The agarose gel microspheres prepared by the preparation method provided by the invention have the advantages that the particle size is easy to regulate, the emulsification time can be regulated according to different target particle sizes to obtain gel microsphere products with different particle sizes, and the application range is wide.
Drawings
FIG. 1 is an optical microscope photograph of the Sepharose beads obtained in example 1;
FIG. 2 is a graph showing the distribution of the particle size of the Sepharose beads obtained in example 1.
Detailed Description
The invention is further described with reference to the following drawings and specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. The experimental procedures, in which specific conditions are not noted in the following examples, are generally carried out under conventional conditions or conditions recommended by the manufacturers.
Example 1
In a 100mL three-necked flask, 0.85g of agarose and 20mL of water were added, and the mixture was warmed to 105 ℃ with sufficient stirring until the agarose was completely dissolved and a clear and transparent sol was formed. At this point, the temperature was reduced to 80 ℃ and 2mL of a 0.2% dextran solution and 2mL of 0.1gmL were slowly added dropwise with stirring-1Na of (2)2HPO4The solution was stirred at 150rpm for 20min, and the mixture was designated as solution A.
In a 250mL three-necked flask, 70mL of toluene, 2.25g of Span80 and 0.75g of Tween80 emulsifier were sequentially added, the temperature was set at 70 ℃, the stirring rate was 300rpm, and the solution was stirred for 30min, and the solution was labeled as solution B.
And then, quickly pouring the solution A into the solution B, continuously and fully stirring at the stirring speed of 150rpm, sampling to observe under an optical microscope when emulsifying for 30min, and quickly cooling and curing the gel microspheres when the gel microspheres reach the ideal size. Standing for layering, removing upper oil phase, washing lower layer to obtain target product, and storing in water.
FIG. 1 is an optical microscopic image of the Sepharose beads obtained in example 1, in which the particle size distribution is uniform; FIG. 2 is a graph showing a distribution of particle sizes of the agarose gel beads obtained in example 1, wherein the distribution of particle sizes is narrow, and the proportion of the agarose gel beads having a particle size of 50 to 160 μm is more than 96%.
Example 2
In a 100mL three-necked flask, 2.45g of agarose and 20mL of water were added, and the temperature was raised to 105 ℃ with thorough stirring until the agarose was completely dissolved and a clear and transparent sol was formed. At this point, the temperature was reduced to 80 ℃ and, with continued stirring, 3mL of 1.0% dextran solution and 6mL of 0.1gmL were slowly added dropwise-1Na (b) of2HPO4The solution was stirred at 150rpm for 20min, and the mixture was designated as solution A.
In a 250mL three-necked flask, 50mL of toluene, 4.00g of Span80 and 0.50g of Tween80 emulsifier were sequentially added, the temperature was set at 70 ℃, the stirring rate was 300rpm, and the solution was stirred for 30min, and the solution was labeled as solution B.
And then, quickly pouring the solution A into the solution B, continuously and fully stirring at the stirring speed of 150rpm, emulsifying for 40min, sampling to an optical microscope for observation, and quickly cooling and curing the gel microspheres to reach the ideal size. Standing for layering, removing upper oil phase, washing lower layer to obtain target product, and storing in water. The particle size of the agarose gel microspheres obtained by example 2 is concentrated in the range of 20 to 250 μm, wherein the proportion of microspheres having a particle size range of 50 to 160 μm is 85% or more.
Comparative example 1
Comparative example 1 differs from example 1 only in the amount of dextran used.
In a 100mL three-necked flask, 0.85g of agarose and 20mL of water were added, and the mixture was warmed to 105 ℃ with sufficient stirring until the agarose was completely dissolved and a clear and transparent sol was formed. At this time, the temperature was reduced to 80 ℃ and 2mL of 6% dextran solution and 2mL of 0.1g mL were slowly added dropwise with stirring-1Na of (2)2HPO4The solution was stirred at 150rpm for 20min, and the mixture was designated as solution A.
In a 250mL three-necked flask, 70mL of toluene, 2.25g of Span80 and 0.75g of Twen 80 emulsifier were sequentially added, the temperature was set at 70 ℃, the stirring rate was 300rpm, and the mixture was stirred for 30min, and the solution was designated as liquid B.
And then, quickly pouring the solution A into the solution B, continuously and fully stirring at the stirring speed of 150rpm, and quickly cooling and solidifying when emulsifying for 30 min. Standing for layering, removing upper oil phase, washing lower layer to obtain target product, and storing in water. The agarose gel microspheres obtained by comparative example 1 had a particle size of 20 to 550 μm.
Comparative example 2
Comparative example 2 differs from example 1 only in Na2HPO4The amount of (A) to (B).
In a 100mL three-necked flask, 0.85g of agarose and 20mL of water were added, and the mixture was warmed to 105 ℃ with sufficient stirring until the agarose was completely dissolved and a clear and transparent sol was formed. At this time, the temperature was reduced to 80 ℃ and 2mL of a 0.2% dextran solution and 2mL of 0.6gmL were slowly added dropwise with stirring-1Na (b) of2HPO4Stirring the solution at a mechanical stirring speed of 150rpm for 20min, and recording the mixed solution as solution A。
In a 250mL three-necked flask, 70mL of toluene, 2.25g of Span80 and 0.75g of Tween80 emulsifier were sequentially added, the temperature was set at 70 ℃, the stirring rate was 300rpm, and the solution was stirred for 30min, and the solution was labeled as solution B.
And then, quickly pouring the solution A into the solution B, continuously and fully stirring at the stirring speed of 150rpm, and quickly cooling and solidifying when emulsifying for 30 min. Standing for layering, removing upper oil phase, washing lower layer to obtain target product, and storing in water. The agarose gel microspheres obtained by comparative example 2 had a particle size of 30 to 600. Mu.m.
Comparative example 3
The comparative example 3 differs from example 1 only in the ratio of Span80 and Tween80 in the emulsifier.
In a 100mL three-necked flask, 0.85g of agarose and 20mL of water were added, and the mixture was warmed to 105 ℃ with sufficient stirring until the agarose was completely dissolved and a clear and transparent sol was formed. At this point, the temperature was reduced to 80 ℃ and 2mL of a 0.2% dextran solution and 2mL of 0.1gmL were slowly added dropwise with stirring-1Na (b) of2HPO4The solution was stirred at 150rpm for 20min, and the mixture was designated as solution A.
In a 250mL three-necked flask, 70mL of toluene, 2.8g of Span80 and 0.2g of Tween80 as an emulsifier were sequentially charged, the temperature was set at 70 ℃, the stirring rate was 300rpm, and the mixture was stirred for 30min, and the solution was designated as solution B.
And then, quickly pouring the solution A into the solution B, continuously and fully stirring at the stirring speed of 150rpm, and quickly cooling and solidifying when emulsifying for 30 min. Standing for layering, removing the upper oil phase, washing the lower layer to obtain the target product, and storing in water. The agarose gel microspheres obtained by comparative example 3 were too dispersed in particle size, and the particle size was 10 to 750 μm.
Comparative example 4
Comparative example 4 differs from example 1 only in the amount of toluene used.
In a 100mL three-necked flask, 0.85g agarose and 20mL water were added, and the temperature was raised to 105 ℃ with thorough stirring until the agarose was completely dissolved and a clear and transparent sol was formed. Cooling to 80 deg.C, and stirringThen, 2mL of a 0.2% dextran solution and 2mL of 0.1gmL were slowly added dropwise-1Na (b) of2HPO4The solution was stirred at 150rpm for 20min, and the mixture was designated as solution A.
In a 250mL three-necked flask, 130mL of toluene, 2.25g of Span80 and 0.75g of Tween80 emulsifier were sequentially added, the temperature was set at 70 ℃, the stirring rate was 300rpm, and the solution was stirred for 30min, and the solution was labeled as solution B.
And then, quickly pouring the solution A into the solution B, continuously and fully stirring at the stirring speed of 150rpm, and quickly cooling and solidifying when emulsifying for 30 min. Standing for layering, removing the upper oil phase, washing the lower layer to obtain the target product, and storing in water. The agarose gel microspheres obtained by comparative example 4 had a particle size of 30 to 600. Mu.m.
Furthermore, it should be understood that various changes and modifications can be made by one skilled in the art after reading the above description of the present invention, and equivalents also fall within the scope of the invention as defined by the appended claims.

Claims (9)

1. The preparation method of the agarose gel microspheres with narrow particle size distribution is characterized by comprising the following steps:
(1) Adding agarose into deionized water, stirring, heating until agarose is completely dissolved, adding dextran and Na under continuous stirring after clear sol is formed2HPO4Continuously stirring for 5-30 min at the stirring speed of 100-200 rpm to obtain solution A;
the agarose, the deionized water, the dextran, the Na2HPO4The proportion of (A) is 0.2-3g;
the glucan is added in the form of aqueous solution, and the mass percent of the glucan in the aqueous solution of the glucan is 0.1-5%;
the Na is2HPO4Adding in the form of an aqueous solution, na2HPO4Na in aqueous solution2HPO4Has a mass concentration of 0.05-0.5 g mL-1；
(2) Adding an emulsifier into the oil phase, heating to 60-120 ℃, and continuously stirring for 20-30 min at the stirring speed of 250-350 rpm to obtain a solution B;
the emulsifier is a combination of Span80 and Tween80, wherein the mass ratio of Span80 to Tween80 is 1-10;
the volume ratio of the oil phase to the deionized water is 10-100;
in the solution B, the mass concentration of the emulsifier is 1-10%;
(3) Adding the solution A obtained in the step (1) into the solution B obtained in the step (2), continuously stirring at the stirring speed of 100-200 rpm, and emulsifying for a certain time to obtain a corresponding product;
(4) And (4) cooling and solidifying the product obtained in the step (3), standing and layering, removing an upper oil layer, and removing an oil phase in a lower product by using a detergent to obtain the agarose gel microspheres with narrow particle size distribution.
2. The production method according to claim 1, wherein in step (1):
the agarose, the deionized water, the dextran, the Na2HPO4The proportion of (A) is 0.6-2g.
3. The preparation method according to claim 1, wherein in the step (2), the oil phase is one or more of toluene, n-octane, liquid paraffin, and n-heptane.
4. The method according to claim 1, wherein in the step (2), the volume ratio of the oil phase to the deionized water is 60 to 80.
5. The method according to claim 1, wherein in the step (2), the emulsifier is present in the solution B at a concentration of 2 to 5% by mass.
6. The preparation method according to claim 1, wherein in the step (2), the mass ratio of Span80 to Tween80 in the emulsifier is 2-6.
7. The method according to claim 1, wherein in the step (3), the time for emulsification is 10 to 60min.
8. The method according to claim 1, wherein in the step (3), during the emulsification, a sample is immediately taken to be observed under an optical microscope, and when a desired size of the gel microsphere is obtained, the emulsification is stopped.
9. The preparation method according to claim 1, wherein in the step (4), the detergent is one or more of ethanol, water, acetone and n-hexane.
CN202210561654.6A 2022-05-23 2022-05-23 Preparation method of agarose gel microspheres with narrow particle size distribution Pending CN115260538A (en)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN115739050A (en) * 2022-12-05 2023-03-07 浙江大学衢州研究院 Preparation method of crosslinked agarose microspheres

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN115739050A (en) * 2022-12-05 2023-03-07 浙江大学衢州研究院 Preparation method of crosslinked agarose microspheres
CN115739050B (en) * 2022-12-05 2024-05-03 浙江大学衢州研究院 Preparation method of crosslinked agarose microsphere

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