CN1304101C - Size-uniform agarose gel microball and its preparing method - Google Patents
Size-uniform agarose gel microball and its preparing method Download PDFInfo
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- CN1304101C CN1304101C CNB2004100000879A CN200410000087A CN1304101C CN 1304101 C CN1304101 C CN 1304101C CN B2004100000879 A CNB2004100000879 A CN B2004100000879A CN 200410000087 A CN200410000087 A CN 200410000087A CN 1304101 C CN1304101 C CN 1304101C
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Abstract
The present invention provides a separating sepharose gel medium with the advantages of uniform size, controllability, high hydrophilicity and high porosity, which is used for the separation, the purification, etc. of biologically active substances and is characterized in that (1) a certain amount of sepharose is dissolved in a certain amount of water under a heating condition to prepare a sepharose water solution with certain concentration, and then the water phase passes through a microporous film by pressure and then is pressed into an oil phase to obtain emulsion drops with uniform size; (2) the emulsion is transferred into a cooling device and then is cooled to below 15 DEG C under the conditions of program cooling and slowly mechanical agitation to solidify gel so as to obtain sepharose gel microspheres with uniform size. Under a preferable condition, the diameter distribution coefficient of the sepharose gel microspheres is controlled within 11%, and diameters can be freely controlled within 1 to 100 micrometers. The technology solves the problems of nonuniform particle diameters of sepharose gel microspheres prepared by a traditional agitating emulsification and poor separating effect caused by large pressure change during separation; the present invention uses the characteristic of uniformity of particle diameters to reach uniform column packing so as to stabilize pressure, enhance flow speed and enhance separating efficiency during separation.
Description
Technical field
The invention belongs to bionic biochemical separation field
Background technology
Ago-Gel is the natural polysaecharides chromatography media, just occur as far back as the sixties in 20th century, it has the numerous characteristics of perfect medium: high-hydrophilic, highly porous property, containing more activated hydroxyl, with large biological molecule non-specific adsorption does not take place, is most widely used up to now a kind of chromatography media
[1]Owing to contain more activated hydroxyl on the agarose, can insert different aglucons under certain conditions, medium as affinity chromatography, hydrophobic and ion-exchange chromatography, along with research and deepening continuously of using, Ago-Gel is not only as the medium of normal pressure and low pressure chromatography, and can in depress the operation and realize that chromatography rapidly and efficiently, separate object relate to water-soluble biochemical substances such as protein, nucleic acid, peptide class, carbohydrate
[2]-[6]Ago-Gel can also be as the living cells carrier, as Shahab Lahooti except as the separating medium
[7]Adopt the inner core materials embedding HEK cell of agarose Deng the people as ethoxy acryloyl methyl esters and methacryl methyl terpolymer microballoon, agarose can make cell evenly be disperseed on the one hand, help the diffusion of nutriment and metabolite, can reduce the concentration of microballoon film material on the other hand, increase the permeability of film, help enough nutriments and enter in the microballoon for the cell growth.The adding that studies show that agarose helps by the division growth of the maintenance of embedding cell activity and cell, experimental result show with do not have agarose to compare 14 days after cell proliferation for its twice many, this mainly is because Ago-Gel not only makes cell be uniformly dispersed, and provides a kind of effect of supporting substrate but also played to cell.Hiroyuki Hayashi
[8]Adopt agarose to prepare three layers of gel capsules Deng the people and be used for embedding B cell line MIN6, result of study shows with the MIN6 of not embedding to be compared, and secretion of insulin speed has increased more than 1 times.
Yet no matter be at present as separating medium or living cells carrier, the preparation method of Ago-Gel is very limited, mainly be that employing is ultrasonic, mechanical agitation, emulsification condensation method that emulsifying etc. are common and injection condensing method, adopt these methods in preparation emulsion process, because particle diameter heterogeneity, little emulsion droplet can be absorbed by big emulsion droplet, big emulsion droplet again can be destroyed because of the effect of shearing force simultaneously, this can cause very heterogeneity of the agarose gel microsphere particle diameter that finally makes, therefore in the washing and storage process of gel micro-ball, little gel beads is easily by big gel beads absorption, thus the formation condensation product.Generally speaking, after agarose gel microsphere preparation at present finishes, all need gel sieved and to be used as separating medium, this not only can cause raw-material waste, also can cause the raising of production cost,, will simplify procedures greatly if can prepare gel microspheres with uniform grain sizes, save raw material, reduce production costs.Even the agarose gel microsphere of conventional emulsification method preparation sieves, the homogeneity of its particle diameter is still not high, and this will bring many difficulties for practical application.Because the particle diameter heterogeneity, in the medium and small gel beads of separation process can gather space between the big gel beads in pillar, make separating pressure increase, can cause when serious separating and can't carry out.Therefore be necessary to study novel preparation method, prepare the uniform particle diameter agarose gel microsphere, so as to overcome the not enough of traditional preparation process method and the application that brings thus on defective.
Summary of the invention
The invention provides the Ago-Gel separating medium of size homogeneous, controlled, high-hydrophilic and highly porous property, with separation and purification of being used for bioactivator etc.It is characterized in that (1) is dissolved in the certain density agarose solution of preparation in a certain amount of water with a certain amount of agarose under heating condition, this water is pressed in the oil phase by microporous barrier with pressure, obtain the emulsion droplet of size homogeneous; (2) this emulsion is transferred in the cooling device adopt be cooled under programmed cooling and the slow churned mechanically condition make below 15 ℃ the emulsion droplet gelling solidify the agarose gel microsphere of size homogeneous.Under optimal conditions, the diameter breadth coefficient of agarose gel microsphere is controlled in 11%, and diameter can freely be controlled in the 1-100 micron.
This technology has solved the agarose gel microsphere particle diameter heterogeneity of traditional stirring and emulsifying method preparation, pressure changes the problem of the inferior separating effect that causes greatly when separating.The present invention utilizes the homogeneity characteristic of particle diameter, and it is even to reach the dress post, thus make when separating pressure stability evenly, improve flow velocity, improve separative efficiency.
Agarose gel microsphere of size homogeneous provided by the invention and preparation method thereof can also bring down column effect:
(1) agarose gel microsphere provided by the invention is except as the biochemical separating medium, also can be used for as carriers such as living cells, genes, for the result of treatment in cell proliferation and the performance body provides favourable micro, effectively avoid the identification and the immunological rejection effect of body endolymph cell.
(2) agarose gel microsphere provided by the invention, because uniform particle diameter and controlled, use this gel micro-ball can effectively carry out relation research between macromolecular substances such as particle diameter and different proteins, nucleic acid and the separating effect thereof, thereby find out the gel micro-ball of the needed optimal particle diameter of different separate substances, if the size heterogeneity of gel micro-ball then can't be carried out the research of this respect.
(3) adopt preparation method of the present invention, can prepare the gel micro-ball that the conventional emulsification method is difficult to the small particle diameter of preparation easily, thereby realize the separation of the difficult bioactivator that separates.
(4), can prepare separating medium, the high specific gravity separating medium of magnetic in conjunction with the embedding techniques of method of the present invention and inorganic matters such as magnetic-particle in the past.In conjunction with method of the present invention and agarose method of modifying in the past, can prepare various separation agarose microbeads.
(5) preparation method provided by the invention, mild condition is expected to keep its biologically active and biological stability as living cells isoreactivity material carrier.
(6) gel micro-ball uniform particle diameter provided by the invention need not to sieve, simplify procedures greatly, and conservation, thus will reduce production costs greatly and follow-up separation and purification cost.
Description of drawings
The preparation flow of Fig. 1 agarose gel microsphere.
The light micrograph of emulsion droplet in the agarose emulsion of Fig. 2 embodiment 1 preparation.
The light micrograph of the agarose gel microsphere of Fig. 3 embodiment 1 preparation.
The light micrograph of emulsion droplet in the agarose emulsion of Fig. 4 comparative example 1 preparation.
The light micrograph of the agarose gel microsphere of Fig. 5 comparative example 1 preparation.
The light micrograph of emulsion droplet in the agarose emulsion of the different membrane aperture preparations of Fig. 6 embodiment 2 employings.
Graph of a relation between emulsion droplet particle diameter and the membrane aperture in the agarose emulsion of the different membrane aperture of Fig. 7 embodiment 2 preparations.
Fig. 8 embodiment 2 adopts the light micrograph of the agarose gel microsphere of different membrane aperture preparations.
Graph of a relation between the agarose gel microsphere particle diameter of the different membrane aperture of Fig. 9 embodiment 2 preparations and the membrane aperture.
The light micrograph of emulsion droplet in the emulsion of the different agarose concentrations of Figure 10 embodiment 3 preparations.
Graph of a relation between emulsion droplet particle diameter and the agarose concentration in the agarose emulsion of the variable concentrations of Figure 11 embodiment 3 preparations.
The light micrograph of the gel micro-ball of the different agarose concentrations of Figure 12 embodiment 3 preparations.
The different agarose concentration gel micro-ball particle diameters of Figure 13 embodiment 3 preparations and the graph of a relation between the concentration.
Specific embodiments
The present invention includes agarose gel microsphere of size homogeneous and preparation method thereof.The preparation of agarose gel microsphere is by step preparation shown in Figure 1, and concrete grammar and step are described as follows:
1) preparation of w/o type emulsion
A certain amount of agarose, NaCl and other additive are joined in a certain amount of water, and under heating condition, fully dissolve as water; More than one oil soluble emulsifying agent is dissolved in oil-based liquid, and is heated to certain temperature as oil phase.Water is pressed into oil phase by hydrophobic porous film, obtains the w/o type emulsion of size homogeneous.
The concentration of agarose can be prepared as required, and needed temperature is also inequality during the agarose solution film emulsification of variable concentrations, can carry out choose reasonable as required; The water additive can comprise harmless water-soluble substanceses such as albumin, lecithin, glucose, sweet mellow wine.Oil phase is to be in a liquid state under the normal temperature, with the immiscible oily matter of water, can use atoleine and benzinum, olive oil, cotton seed oil, soya-bean oil, sunflower oil, other alkanes hydrocarbon etc., general selected oil phase boiling point is than higher, and volatility is better weak.Oil emulsifier must be dissolved in employed oily matter, can use polymer (as PO-500, PO-310), Crodaret, sorbitan trioleate (class of department 85), sorbitan monooleate (class of department 80), anhydrous sorbitol tristearate (class of department 65), the oleophylic-hydrophilic block copolymers of sorbitan sesquioleate (Arlacel83), glycerin ether etc.The concentration of emulsifying agent is 0.5-10wt% in the oil phase.
The volume ratio of water and oil phase is 1: 1-1: 1000.
2) preparation of agarose gel microsphere
With above-mentioned steps 1) resulting emulsion is transferred in the heat sink, slowly is being cooled to gelling below 15 ℃ and solidifies under mechanical agitation slowly, and washing, the gained gel micro-ball is stored in the distilled water.
Cooling rate was wanted slowly when gelling was solidified, and cooling extent adopts the mechanical agitation oar slowly to stir for being lower than 2 ℃/min during cooling, and revolution is 50-200rpm.
After end is solidified in gelling, the gained gel is washed, and will be stored in the distilled water under the gained gel normal temperature with benzinum, acetone, ethanol, distilled water successively.
Embodiment 1
With the aperture is that the hydrophobic film of 5.7 μ m places oil loving material to soak into, and makes pore membrane fully moistening to guarantee the complete spread apart of hydrophobic chain on the film.Accurately take by weighing a certain amount of agarose and a certain amount of NaCl joins in a certain amount of water, the concentration that makes agarose is 4%, and the concentration of NaCl is 0.9%, under heating condition it is fully dissolved, and makes solution keep certain temperature standby.Oil soluble emulsifying agent is joined in the atoleine of 200ml, be stirred to and dissolve fully and be heated to 50 ℃ as oil phase.Rapidly the water about 6.0g is transferred in the film emulsifier unit that is preheated to 50 ℃ in oil phase while hot, under constant pressure, the hydrophobic microporous membrane by the aperture homogeneous is pressed in the oil phase, obtains the w/o type emulsion; Emulsification finishes, and emulsion is transferred in the heat sink slowly cooling in air under the slow stirring of paddle, when reducing to room temperature, further lower the temperature with cold bath, in water-bath, add a small amount of ice then emulsion is cooled to below 15 ℃, the gelling of agarose emulsion droplet is solidified.At last the gained gel micro-ball is filtered, be kept in the distilled water successively with benzinum, acetone, ethanol and distilled water washing, and with it.The average grain diameter of emulsion droplet and gel micro-ball adopts the microscope that has scale to measure in the emulsion, survey 200 and average, wherein the average diameter of emulsion droplet is 20.55 μ m in the emulsion, and the CV value is 19.25%, the average diameter of gel micro-ball is 23.04 μ m in water, and the CV value is 18.46%.The optical microscope photograph of emulsion droplet and gel micro-ball is distinguished as shown in Figures 2 and 3, and particle diameter is homogeneous comparatively.
Comparative example 1 (mechanical mixing method)
Adopt the prescription identical with example 1, accurately take by weighing a certain amount of agarose and a certain amount of NaCl joins in a certain amount of water, the concentration that makes agarose is 4%, and the concentration of NaCl is 0.9%, under heating condition, it is fully dissolved, and make the certain temperature of solution maintenance standby.Oil soluble emulsifying agent is joined in the atoleine of 200ml, be stirred to and dissolve fully and be heated to 50 ℃ as oil phase.Rapidly the water about 6.0g is transferred to while hot in oil phase, magnetic agitation 1000rpm, 30min obtains the w/o type emulsion; Emulsification finishes, and emulsion is transferred in the heat sink slowly cooling in air under the slow stirring of paddle, when reducing to room temperature, further lower the temperature with cold bath, in water-bath, add a small amount of ice then emulsion is cooled to below 15 ℃, the agarose emulsion droplet is solidified.At last the gained gel micro-ball is filtered, be kept in the distilled water successively with benzinum, acetone, ethanol and distilled water washing, and with it.The average grain diameter of emulsion droplet and gel micro-ball adopts the microscope that has scale to measure in the emulsion, survey 200 and average, wherein the average diameter of emulsion droplet is 18.47 μ m in the emulsion, and the CV value is 39.37%, the average diameter of the gel micro-ball in the water is 28.08 μ m, and the CV value is 31.16%.The optical microscope photograph of emulsion droplet and gel micro-ball is distinguished as shown in Figure 4 and Figure 5, and particle diameter is heterogeneity very.
The hydrophobic film that the aperture is respectively 4.7 μ m, 5.7 μ m, 13 μ m, 19.6 μ m places oil loving material to soak into, and makes pore membrane fully moistening to guarantee the complete spread apart of hydrophobic chain on the film.Accurately take by weighing a certain amount of agarose and a certain amount of NaCl joins in a certain amount of water, the concentration that makes agarose is 4%, and the concentration of NaCl is 0.9%, under heating condition it is fully dissolved, and makes solution keep certain temperature standby.Oil soluble emulsifying agent is joined in the atoleine of 200ml, be stirred to and dissolve fully and be heated to 50 ℃ as oil phase.Rapidly the water about 6.0g is transferred in the film emulsifier unit that is preheated to 50 ℃ in oil phase while hot, respectively under constant pressure, the hydrophobic microporous membrane by the aperture homogeneous is pressed in the oil phase, obtains the w/o type emulsion; Emulsification finishes, and emulsion is transferred in the heat sink slowly cooling in air under the slow stirring of paddle, when reducing to room temperature, further lower the temperature with cold bath, in water-bath, add a small amount of ice then emulsion is cooled to below 15 ℃, the agarose emulsion droplet is solidified.With the gained gel filtration, be kept in the distilled water at last successively with benzinum, acetone, ethanol and distilled water washing, and with it.The average grain diameter of emulsion droplet and gel micro-ball adopts the microscope that has scale to measure, surveying 200 averages, wherein the average diameter of emulsion droplet is respectively 16.03 μ m, 20.55 μ m, 42.76 μ m, 61.88 μ m in the emulsion, and the average diameter of gel micro-ball is respectively 17.90 μ m, 23.04 μ m, 50.70 μ m, 62.37 μ m in the water.The optical microscope photograph of emulsion droplet and gel micro-ball is respectively as Fig. 6 and shown in Figure 8, relation between emulsion droplet particle diameter and the membrane aperture as shown in Figure 7, relation between gel micro-ball particle diameter and the membrane aperture as shown in Figure 9, by the aperture of emulsion droplet particle diameter and gel micro-ball particle diameter and film is linear as can be seen on the figure, the particle diameter of emulsion droplet and gel micro-ball approximately is 3 times of membrane aperture.
Embodiment 3
With the aperture is that the hydrophobic film of 4.7 μ m places oil loving material to soak into, and makes pore membrane fully moistening to guarantee the complete spread apart of hydrophobic chain on the film.Accurately taking by weighing a certain amount of agarose and a certain amount of NaCl joins in a certain amount of water, make the concentration of agarose be respectively 2%, 4%, 6% and 8%, the concentration of NaCl is 0.9%, under heating condition it is fully dissolved, and makes the certain temperature of solution maintenance standby.Oil soluble emulsifying agent is joined in the atoleine of 200ml, be stirred to and dissolve fully and be heated to uniform temperature as oil phase.Rapidly the water about 6.0g is transferred to while hot and is preheated in oil phase in the temperature required film emulsifier unit, respectively under constant pressure, the hydrophobic microporous membrane by the aperture homogeneous is pressed in the oil phase, obtains the w/o type emulsion; Emulsification finishes, and emulsion is transferred in the heat sink slowly cooling in air under the slow stirring of paddle, when reducing to room temperature, further lower the temperature with cold bath, in water-bath, add a small amount of ice then emulsion is cooled to below 15 ℃, the Ago-Gel gelling is solidified.With the gained gel filtration, be kept in the distilled water at last successively with benzinum, acetone, ethanol and distilled water washing, and with it.The average grain diameter of emulsion droplet and gel micro-ball adopts the microscope that has scale to measure, surveying 200 averages, the average grain diameter of emulsion droplet is respectively 17.45 μ m, 16.03 μ m, 16.47 μ m and 16.79 μ m, the CV value is respectively 12.29%, 19.18%, 14.96% and 19.29%, the average grain diameter of gel micro-ball is respectively 18.27 μ m, 17.90 μ m, 16.28 μ m and 17.04 μ m, and the CV value is respectively 17.67%, 12.59%, 13.75% and 16.25%.The optical microscope photograph of emulsion droplet and gel micro-ball is respectively as Figure 10 and shown in Figure 12, and the agarose emulsion droplet of variable concentrations and the relation between gel micro-ball particle diameter and the concentration are shown in Figure 11 and 13.Emulsion droplet and gel micro-ball diameter are not subjected to the influence of agarose concentration substantially.
List of references
[1] Wen Shaohong, Su Zhiguo, the epoxychloropropane cross-linked agarose gel is not delivered.
[2]Stellan?Hjertén，The?preparation?of?agarose?spheres?for?chromatography?of?molecules?and?particles，Biochimica?etBiophysica?Acta，79(1964)393-398.
[3]A.M.Egorov，A.KH.Vakhabov，V.YA.Chernyak，Isolation?of?agarose?and?granulation?of?agar?and?agarose?gel，Journal?ofChromatography，46(1970)143-148.
[4]Per-Erik?Gustavsson，Per-Olof?Larsson，Continuous?superporous?agarose?beds?for?chromatography?and?electrophoresis，Journal?of?Chromatography?A，832(1999)29-39.
[5]Ingo?Gottschalk，Per-Erik?Gustavsson，Bo?Ersson，Per?Lundahl，Improved?lectin-mediated?immobilization?of?human?redblood?cells?in?superporous?agarose?beads，Journal?of?Chromatography?B，784(2003)203-208.
[6] Zhang Wei, Zhang Jiaoqiang, Shao Wei, gold is super, Guo Lian, chemical industry is used in the preparation of Ago-Gel and chemical modification research, and 2003, the 32nd volume, the 1st phase, 24-27.
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Claims (8)
1, a kind of preparation method of agarose gel microsphere of size homogeneous is characterized in that (1) water (W) is certain density agarose solution; (2) oil phase (O) be dissolved oil soluble emulsifying agent and with the immiscible oily matter of water; (3) water is pressed into oil phase with pressure by microporous barrier at a certain temperature, obtains the w/o type emulsion droplet of size homogeneous; (4) the w/o type emulsion of size homogeneous is made below programmed cooling to 15 under the mechanical agitation slowly ℃ gelling solidify agarose gel microsphere.
2, the preparation method of the agarose gel microsphere of a kind of size homogeneous as claimed in claim 1 is characterized in that, the concentration range of agarose solution is 0.1wt%-20.0wt%
3, the preparation method of the agarose gel microsphere of a kind of size homogeneous as claimed in claim 2 is characterized in that, the concentration range of agarose solution is 1.0-10.0wt%.
4, the preparation method of the agarose gel microsphere of a kind of size homogeneous as claimed in claim 1 is characterized in that, water is 1 with the volume ratio of outer oil phase: 1-1: 1000.
5, the preparation method of the agarose gel microsphere of a kind of size homogeneous as claimed in claim 4 is characterized in that, water is 1 with the volume ratio of outer oil phase: 5-1: 100.
6, the preparation method of the agarose gel microsphere of a kind of size homogeneous as claimed in claim 1, it is characterized in that, water is to be pressed in the oil phase by microporous barrier with pressure under temperature is higher than 45 ℃ condition, the agarose solution of variable concentrations carries out the needed temperature of film emulsification and also is not quite similar, and the big more needed temperature of concentration is high more.
7, the preparation method of the agarose gel microsphere of a kind of size homogeneous as claimed in claim 1, it is characterized in that, the gelling under programmed cooling and mechanical agitation condition of the agarose emulsion of size homogeneous is solidified, cooling and mechanical agitation all want slow, cooling rate is lower than 2 ℃/min, mixing speed is cooled to and promptly gets agarose gel microsphere below 15 ℃ about 100rpm.
8, the preparation method of the agarose gel microsphere of a kind of size homogeneous as claimed in claim 1 is characterized in that, employed microporous barrier surface is a hydrophobicity.
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| SG131015A1 (en) * | 2005-09-15 | 2007-04-26 | Millipore Corp | Method and apparatus for making porous agarose beads |
| CN101293191B (en) * | 2007-04-25 | 2011-11-09 | 中国科学院过程工程研究所 | Agarose gelatin microsphere preparation method |
| ES2630803T3 (en) * | 2007-12-03 | 2017-08-24 | Patheon Holdings I B.V. | Method to produce pearls |
| CN101486799B (en) * | 2008-01-16 | 2012-05-23 | 中国科学院过程工程研究所 | Agarose gel microsphere and preparation thereof |
| CN101314648B (en) * | 2008-03-18 | 2010-12-22 | 天津大学 | Macromolecule blot gelose polymer microsphere and preparation method thereof |
| CN103831066B (en) * | 2012-11-27 | 2015-12-02 | 山东鼎欣生物科技有限公司 | Microsphere preparation technology is analysed by CM agar basic unit |
| CN103923355B (en) * | 2014-04-09 | 2016-03-09 | 无锡百运纳米科技有限公司 | A kind of preparation method of agarose-hydroxyapatite composite microspheres |
| CN104826605B (en) * | 2015-04-24 | 2017-09-29 | 广州极泰生物科技有限公司 | One kind prepares highly cross-linked uniform particle size Ago-Gel medium using ultrasonic inverse suspension method |
| CN105170042B (en) * | 2015-09-17 | 2017-10-20 | 湖州师范学院 | A kind of method of the antibody separation based on magnetic microsphere |
| CN105504334B (en) * | 2015-12-30 | 2019-07-23 | 浙江工业大学 | A kind of agarose crystalline substance gel matrix particle and preparation method thereof with super large hole |
| CN105777942B (en) * | 2016-03-19 | 2017-12-22 | 绿麒（厦门）海洋生物科技有限公司 | The agarose gel microsphere production system and its production technology of uniform particle diameter |
| CN112266427B (en) * | 2020-11-06 | 2022-04-19 | 内蒙古科技大学 | Hydrophobic agarose aerogel microspheres and preparation method thereof |
| CN114191848B (en) * | 2021-12-06 | 2022-12-30 | 武汉瑞法医疗器械有限公司 | Method for cleaning agarose microspheres |
| CN114702732B (en) * | 2022-06-07 | 2022-08-26 | 江苏集萃智能液晶科技有限公司 | Polymer particle with double-size pore channel and preparation method thereof |
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| US4839111A (en) * | 1987-02-02 | 1989-06-13 | The University Of Tennessee Research Corporation | Preparation of solid core liposomes |
| CN1316450A (en) * | 2001-03-24 | 2001-10-10 | 天津大学 | Process for preparing magnetic sepharose microspheres by both-phase (oil and water) method |
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| US4839111A (en) * | 1987-02-02 | 1989-06-13 | The University Of Tennessee Research Corporation | Preparation of solid core liposomes |
| CN1316450A (en) * | 2001-03-24 | 2001-10-10 | 天津大学 | Process for preparing magnetic sepharose microspheres by both-phase (oil and water) method |
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