CN1316450A - Process for preparing magnetic sepharose microspheres by both-phase (oil and water) method - Google Patents
Process for preparing magnetic sepharose microspheres by both-phase (oil and water) method Download PDFInfo
- Publication number
- CN1316450A CN1316450A CN 01111575 CN01111575A CN1316450A CN 1316450 A CN1316450 A CN 1316450A CN 01111575 CN01111575 CN 01111575 CN 01111575 A CN01111575 A CN 01111575A CN 1316450 A CN1316450 A CN 1316450A
- Authority
- CN
- China
- Prior art keywords
- magnetic
- consumption
- under
- alloys
- phase
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Landscapes
- Soft Magnetic Materials (AREA)
- Immobilizing And Processing Of Enzymes And Microorganisms (AREA)
Abstract
A process for preparing magnetic sepharose gel microspheres by biphase (oil and water) method includes such steps as dispersing Nd-Fe-B particles in sepharose solution, stirring, suspending the mixture in salad oil mixed with span80 as emulsifier, cooling, solidification, cross-linking and reduction. Its advantages include high density up to 2.04 g/ml, uniform granularity (50-300 microns) and better effect on separating protein.
Description
The present invention relates to a kind of method of preparing magnetic sepharose microspheres by both-phase (oil and water), belong to magnetic-particle and be carried on microsphere preparation technology in the carrier.
In recent years, along with the development of recombinant DNA technology and cell-fusion techniques, the mass production of various biologically active substances such as cytokine, enzyme, antibody and nucleic acid etc. becomes possibility.The value added of these biologically active substances is high, but content is very low in stock liquid.Therefore, low-cost, high yield purifying purpose product and effectively to control objectionable impurities be the target that these biologic separation and purification processes are pursued.Because the diversity of biologic requires the new and effective and pointed isolation technique of research and development.Beginning the eighties in 20th century has the scholar magnetically stabilized fluidized bed to be used for the separation of biologic.Magnetically stabilized fluidized bed is a kind of integrated sepn process, the advantage that integrates fluidized-bed, fixed bed and magnetic separation technique, promptly can reduce unit operation steps such as centrifugal or filtration, velocity of separation is fast, operational condition is gentle, process efficiency is high, its lytic activity yield height, reduce the cost of separation and purification, has tempting DEVELOPMENT PROSPECT in bioseparation.The core of magnetically stabilized fluidized bed is the magnetic resolution medium, is applicable to that therefore the synthetic of magnetic resolution medium of magnetically stabilized fluidized bed is the key that the magnetically stabilized fluidized bed operation realizes.
At present, the shared ratio of ferromagnetic material is higher in the magnetic resolution medium commonly used, and some greatly reduces the adsorptive capacity of separating medium up to 40%, and this has seriously limited the development of magnetically stabilized fluidized bed in biological product separates.And, Fee publishes thesis in the 42nd volume of 1996 " AIChE Journal ", think that the optimum density of the magnetic resolution medium that is applicable to magnetically stabilized fluidized bed operation is between 1.6-1.9g/mL, but existing magnetic resolution density of medium is generally all less than 1.3g/mL.Therefore, the novel magnetic resolution medium with high-density, high-specific surface area, high-adsorption-capacity of research and development has caused various countries scientist's attention.For example Goetz etc. relates to the electrostatic interaction that utilizes between silica gel particle and nickel powder surface at the 37th curly hair table paper of 1991 " Biotechnology and Bioengineeing ", and silica gel is wrapped in around the nickel powder, has prepared the magnetic silica-gel carrier.But to adsorbing the wash-out of back silica-gel carrier, conditional request is harsh, so its use range is restricted.Natural macromolecular material owing to have is easy to the functional group of modifying in a large number, and good biocompatibility, the elution requirement gentleness.Therefore, utilize natural macromolecular material combine with magnetic substance the preparation magnetic microsphere cause investigator's concern especially.The profit two phase process because the microballoon regular shape of its preparation, simple to operate, equipment requirements is low, is widely used aspect preparing at microballoon, it is normally known in the prior art.For example Zundertlerf Miller-Shu Erte is that matrix prepares a kind of spherical polymer particles with the polyvinyl alcohol by the profit two phase process in Chinese patent CN 1198104A, has wherein prepared low density magnetic polyethylene alkoxide polymer by the encapsulation magnetic colloid.
At the weak point that exists in the existing magnetic resolution medium, the object of the present invention is to provide a kind of method of preparing magnetic sepharose microspheres by both-phase (oil and water), magnetic microsphere particle diameter with this method preparation is 50-300 μ m, density can have good effect to proteinic separation up to 2.04g/mL.
For achieving the above object, the present invention is realized by following technical proposals.Under 80-90 ℃, the Nd-Fe-B alloys particles dispersed in agarose solution, is suspended in this mixture in the salad oil that is mixed with emulsifying agent Span80 by stirring then, the Nd-Fe-B alloys particle is encapsulated in the agarose microbeads, cooling makes its curing rapidly.Then carry out crosslinked to it and reduction, obtain density and can reach 2.04g/mL magnetic agarose gel microsphere, it is characterized in that the Nd-Fe-B alloys grain diameter is 20-50 μ m, its density is 7.4g/mL; Wherein the consumption of salad oil be the water volume 8-12 doubly, aqueous phase agarose solution consumption be the Nd-Fe-B alloys particle volume 10-60 doubly, the add-on of emulsifying agent Span80 is 10-50g/L; The linking agent that adopts is an epoxy chloropropane, and under 0.3-0.7M NaOH condition, the concentration of epoxy chloropropane is 1-5% (volume), and reductive agent adopts sodium borohydride, and its consumption is 4-6g/L under 0.8-1.4M NaOH condition.
The consumption of above-mentioned salad oil is 9～10 times of water.
The agarose solution of above-mentioned aqueous phase is 15～30 times of Nd-Fe-B alloys particle volume.
The consumption of above-mentioned epoxy chloropropane is 2～4% (volumes) under 0.3～0.7M NaOH condition.
The consumption of above-mentioned sodium borohydride is 5g/L under 0.8～1.4M NaOH condition.
Below the present invention is described in detail.
Gordian technique of the present invention has 5 points: the one, and the Nd-Fe-B alloys particulate is selected.Nd-Fe-B alloys particulate density is 7.4g/mL, and the magnetic saturation height can be used for preparing high-density, microballoon that saturation magnetization is strong.Two of gordian technique is to determine to select for use the Nd-Fe-B alloys granular size.The particle diameter of material is the prerequisite of the magnetic microsphere of preparation appropriate particle size, and for the preparation particle diameter is the magnetic microsphere of 50-300 μ m, it is 20-50 μ m that the present invention has selected the Nd-Fe-B alloys particle grain size.Three of gordian technique is to adopt the profit two phase process to prepare magnetic microsphere.The present invention proposes in the salad oil that is mixed with emulsifying agent Span80 magnetic-particle to be carried out encapsulation reaction by the profit two phase process, and this helps the parcel of magnetic-particle complete, the even and anti-poly-property of raising of covering.Four of gordian technique is by regulating water agarose solution and neodymium iron boron particle volume ratio, can realizing the control to the magnetic microsphere density of preparation.Five of gordian technique is to pass through step process such as crosslinked and reduction in preparation process, and this helps improving the intensity of microballoon, thereby improves crushing resistance, and some chemical functional group that can improve microsphere surface simultaneously, helps modifying aglucon.
The magnetic agarose gel microsphere of the present invention preparation is compared with existing magnetic resolution medium, and its tangible advantage is, the easy economy of preparation process is not high to equipment requirements; Have stronger magnetic responsiveness, under magnetic field, can realize rapid separation; Active group is many, is easy to modify aglucon; By modifying aglucon, for example pigment aglucon, ion-exchange group etc. have higher loading capacity to protein, cell etc., and the elution requirement gentleness; Good biocompatibility, good hydrophilic property is with a wide range of applications at aspects such as the immobilization of enzyme, cellular segregation, DNA detection, protein purifications; Because microballoon has higher density, therefore can be used for magnetic and surely change multiple lock out operation such as bed and expanded bed.
Following example will give further instruction to method provided by the invention.
Embodiment one:
Under 90 ℃, be that the Nd-Fe-B alloys particles dispersed of 25-50 μ m is in the agarose solution of the 40g/L of 60mL with the particle diameter of 30g by ultra-sonic dispersion.This mixture is splashed in the 600mL organic phase that contains 10g/L Span80, keep 90 ℃ of temperature of reaction, control stirring velocity 1400rpm, behind the stirring 30min, cooling rapidly, controlled temperature is 15 ℃, and keeps stirring velocity 1400rpm, continues to stir 30min.Then, sedimentation magnetic microsphere under magnetic field, the microballoon under the sedimentation with using the distilled water repetitive scrubbing behind the acetone, are cleaned until organic phase earlier.Then, get the clean microballoon (deposition weight is 50g) of 50mL and mix, add NaBH with the 1mol/LNaOH of 50mL
4Make NaBH in the solution
4Concentration be 5g/L.In shaking bath, 25 ℃, under the 150rpm, reaction 30min.Then, add the epoxy chloropropane of 4mL, in shaking bath, 25 ℃, under the 150rpm, reaction 5h.Reaction finishes, and adds the distilled water repetitive scrubbing to neutral.Then, the 50mL microballoon is mixed with the 2mol/LNaOH of 50mL, add NaBH
4Make solution NaBH
4Concentration be 5g/L, boil.Reaction finishes, and adds the distilled water repetitive scrubbing to neutral, promptly obtains size distribution in 50-300 μ m magnetic microsphere.Obtain size distribution in 50-150 μ m by preferred screening, density be 1.875g/mL and Nd-Fe-B alloys granule content be 13% (volume) and size distribution in 150-300 μ m, density is that 2.04g/mL (volume) and Nd-Fe-B alloys granule content are 16% magnetic agarose microbeads.
Embodiment two:
Under 90 ℃, be that the Nd-Fe-B alloys particles dispersed of 25-50 μ m is in the agarose solution of the 50g/L of 70mL with the particle diameter of 8.75g by ultra-sonic dispersion.This mixture is splashed in the 700mL organic phase that contains 50g/L Span80, keep 90 ℃ of temperature of reaction, keep stirring velocity 1500rpm, behind the stirring 35min, cooling rapidly, controlled temperature is 15 ℃, and keeps stirring velocity 1500rpm, continues to stir 15min.Then, sedimentation magnetic microsphere under magnetic field, the microballoon under the sedimentation with using the distilled water repetitive scrubbing behind the acetone, are cleaned until organic phase earlier.Then, get the clean microballoon (deposition weight is 20g) of 20mL and mix, add NaBH with the 1mol/L NaOH of 60mL
4Make NaBH in the solution
4Concentration be 5g/L.In shaking bath, 25 ℃, under the 150rpm, reaction 30min.Then, add the epoxy chloropropane of 1.6mL, in shaking bath, 25 ℃, under the 150rpm, reaction 5h.Reaction finishes, and adds the distilled water repetitive scrubbing to neutral.Then, the 50mL microballoon is mixed with the 2mol/LNaOH of 50mL, add NaBH
4Make solution NaBH
4Concentration be 5g/L, boil.Reaction finishes, and adds the distilled water repetitive scrubbing to neutral, promptly obtains size distribution in 50-300 μ m magnetic microsphere.Obtain size distribution in 50-150 μ m by preferred screening, density be 1.26g/mL and Nd-Fe-B alloys granule content be 3.6% (volume) and size distribution in 150-300 μ m, density is that 1.19g/mL (volume) and Nd-Fe-B alloys granule content are 2.5% magnetic agarose microbeads.
Claims (2)
1. the method for a preparing magnetic sepharose microspheres by both-phase (oil and water), this method is under 80-90 ℃, with the Nd-Fe-B alloys particles dispersed in agarose solution, by stirring this mixture is suspended in the salad oil that is mixed with emulsifying agent Span80 then, the Nd-Fe-B alloys particle is encapsulated in the agarose microbeads, cooling makes its curing rapidly, then carry out crosslinked and reduction to it, obtain density and can reach 2.04g/mL magnetic agarose gel microsphere, it is characterized in that: the Nd-Fe-B alloys grain diameter is 20-50 μ m, and its density is 7.4g/mL; Wherein the consumption of salad oil be the water volume 8-12 doubly, aqueous phase agarose solution consumption be the Nd-Fe-B alloys particle volume 10-60 doubly, the add-on of emulsifying agent Span80 is 10-50g/L; The linking agent that adopts is an epoxy chloropropane, and under 0.3-0.7M NaOH condition, the concentration of epoxy chloropropane is 1-5% (volume), and reductive agent adopts sodium borohydride, and its consumption is 4-6g/L under 0.8-1.4M NaOH condition.
2. prepare the method for magnetic agarose gel microsphere according to the said a kind of profit two-phase of claim 1, it is characterized in that: the consumption of salad oil is 9～10 times of water; The agarose solution of aqueous phase is 15～30 times of Nd-Fe-B alloys particle volume; The consumption of epoxy chloropropane is 2～4% (volumes) under 0.3～0.7M NaOH condition; The consumption of sodium borohydride is 5g/L under the condition of 0.8～1.4MNaOH.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 01111575 CN1194029C (en) | 2001-03-24 | 2001-03-24 | Process for preparing magnetic sepharose microspheres by both-phase (oil and water) method |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 01111575 CN1194029C (en) | 2001-03-24 | 2001-03-24 | Process for preparing magnetic sepharose microspheres by both-phase (oil and water) method |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN1316450A true CN1316450A (en) | 2001-10-10 |
| CN1194029C CN1194029C (en) | 2005-03-23 |
Family
ID=4659125
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN 01111575 Expired - Fee Related CN1194029C (en) | 2001-03-24 | 2001-03-24 | Process for preparing magnetic sepharose microspheres by both-phase (oil and water) method |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN1194029C (en) |
Cited By (13)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1304101C (en) * | 2004-01-12 | 2007-03-14 | 中国科学院过程工程研究所 | Size-uniform agarose gel microball and its preparing method |
| CN100333828C (en) * | 2003-10-25 | 2007-08-29 | 玉环县海洋生物化学有限公司 | Biologic chromatography gelatin and producing texhnique |
| CN101036876B (en) * | 2007-01-25 | 2010-09-01 | 天津大学 | Method for preparing super-hole fibrin microsphere protein absorbing medium |
| CN1939582B (en) * | 2005-09-15 | 2010-12-15 | 米利波尔公司 | Method and apparatus for making porous agarose beads |
| CN101314648B (en) * | 2008-03-18 | 2010-12-22 | 天津大学 | Macromolecule blot gelose polymer microsphere and preparation method thereof |
| CN102488636A (en) * | 2011-12-22 | 2012-06-13 | 吴江市德佐日用化学品有限公司 | Hand sanitizer |
| CN102994489A (en) * | 2012-10-09 | 2013-03-27 | 山西农业大学 | Biotin-avidin system immobilized glucoamylase and preparation method thereof |
| CN103182199A (en) * | 2013-03-22 | 2013-07-03 | 西北大学 | Method for purifying polyphenols |
| CN103212377A (en) * | 2013-04-19 | 2013-07-24 | 哈尔滨益材新材料有限公司 | Preparation method of agarose immune magnetic microspheres and applications thereof |
| CN103769057A (en) * | 2012-10-25 | 2014-05-07 | 中国科学院过程工程研究所 | High-strength polysaccharide aerogel microsphere, and preparation method and application thereof |
| CN104593139A (en) * | 2015-01-21 | 2015-05-06 | 东北农业大学 | Method for carrying out enzymolysis on soybean by using gas-solid two-phase magnetically-stabilized fluidized bed |
| CN108056220A (en) * | 2017-11-22 | 2018-05-22 | 沂南帅克宠物用品有限公司 | It is a kind of that there is the pet food to clean the teeth and preparation method |
| CN110448737A (en) * | 2019-07-09 | 2019-11-15 | 复旦大学附属儿科医院 | Treatment magnetic gel and preparation method thereof is injected in vesicoureteric reflux |
-
2001
- 2001-03-24 CN CN 01111575 patent/CN1194029C/en not_active Expired - Fee Related
Cited By (16)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN100333828C (en) * | 2003-10-25 | 2007-08-29 | 玉环县海洋生物化学有限公司 | Biologic chromatography gelatin and producing texhnique |
| CN1304101C (en) * | 2004-01-12 | 2007-03-14 | 中国科学院过程工程研究所 | Size-uniform agarose gel microball and its preparing method |
| CN1939582B (en) * | 2005-09-15 | 2010-12-15 | 米利波尔公司 | Method and apparatus for making porous agarose beads |
| CN101036876B (en) * | 2007-01-25 | 2010-09-01 | 天津大学 | Method for preparing super-hole fibrin microsphere protein absorbing medium |
| CN101314648B (en) * | 2008-03-18 | 2010-12-22 | 天津大学 | Macromolecule blot gelose polymer microsphere and preparation method thereof |
| CN102488636A (en) * | 2011-12-22 | 2012-06-13 | 吴江市德佐日用化学品有限公司 | Hand sanitizer |
| CN102994489A (en) * | 2012-10-09 | 2013-03-27 | 山西农业大学 | Biotin-avidin system immobilized glucoamylase and preparation method thereof |
| CN102994489B (en) * | 2012-10-09 | 2014-01-08 | 山西农业大学 | Biotin-avidin system immobilized glucoamylase and preparation method thereof |
| CN103769057B (en) * | 2012-10-25 | 2016-07-13 | 中国科学院过程工程研究所 | A kind of high intensity polysaccharide gel micro-ball, preparation method and its usage |
| CN103769057A (en) * | 2012-10-25 | 2014-05-07 | 中国科学院过程工程研究所 | High-strength polysaccharide aerogel microsphere, and preparation method and application thereof |
| CN103182199A (en) * | 2013-03-22 | 2013-07-03 | 西北大学 | Method for purifying polyphenols |
| CN103182199B (en) * | 2013-03-22 | 2014-12-24 | 西北大学 | Method for purifying polyphenols |
| CN103212377A (en) * | 2013-04-19 | 2013-07-24 | 哈尔滨益材新材料有限公司 | Preparation method of agarose immune magnetic microspheres and applications thereof |
| CN104593139A (en) * | 2015-01-21 | 2015-05-06 | 东北农业大学 | Method for carrying out enzymolysis on soybean by using gas-solid two-phase magnetically-stabilized fluidized bed |
| CN108056220A (en) * | 2017-11-22 | 2018-05-22 | 沂南帅克宠物用品有限公司 | It is a kind of that there is the pet food to clean the teeth and preparation method |
| CN110448737A (en) * | 2019-07-09 | 2019-11-15 | 复旦大学附属儿科医院 | Treatment magnetic gel and preparation method thereof is injected in vesicoureteric reflux |
Also Published As
| Publication number | Publication date |
|---|---|
| CN1194029C (en) | 2005-03-23 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN1194029C (en) | Process for preparing magnetic sepharose microspheres by both-phase (oil and water) method | |
| CN101256864B (en) | Superparamagnetism mesoporous silicon dioxide composite ball and preparing method thereof | |
| Xie et al. | Immobilized lipase on core–shell structured Fe3O4–MCM-41 nanocomposites as a magnetically recyclable biocatalyst for interesterification of soybean oil and lard | |
| CN101549270B (en) | Preparation method of magnetic polymer inorganic composite micro-sphere | |
| Shao et al. | Effective adsorption and separation of lysozyme with PAA-modified Fe3O4@ silica core/shell microspheres | |
| Zhou et al. | Preparation of uniform-sized agarose beads by microporous membrane emulsification technique | |
| EP0538467A1 (en) | Process and means for down stream processing | |
| CN101090018A (en) | Silica-magnetic composite micropartical and its preparation method | |
| CN106745317A (en) | One-step method prepares method and its application of porous ferroferric oxide magnetic Nano microsphere | |
| CN102489273B (en) | Preparation method of magnetic beads of dendritic molecular imprinting polymer on surface of estrogenic nano silica gel | |
| CN1947848A (en) | Functional magnetic separating rod and its making method | |
| CN1194811C (en) | Macropore high-capacity agarose gel media preparing method | |
| EP1306128A1 (en) | Sorptive composite materials | |
| CN1203916C (en) | Ferromagnetic multiporous silica gel microsphere and its preparation method | |
| Dunlop et al. | Magnetic separation in biotechnology | |
| EP2035106B1 (en) | Highly porous magnetic temporary fixed bed | |
| CN1206023C (en) | Method for preparing high density core material coated with thin shell medium of agarose gel | |
| Altıntaş et al. | Monosize magnetic hydrophobic beads for lysozyme purification under magnetic field | |
| Gu et al. | Preparation and preliminary evaluation of macroporous magnetic agarose particles for bioseparation | |
| CN115504515B (en) | Magnetic nano gamma-Fe2O3@Al2O3Magnetic response type nano material as well as preparation method and application thereof | |
| Tai et al. | Synthesis of core/shell magnetic porous microspheres for lipase immobilization | |
| Xue et al. | Characterization of PVA-based magnetic affinity support for protein adsorption | |
| CN113633624B (en) | Preparation method of alginate-magnetic chitosan microspheres with core-shell structures | |
| Altıntaş et al. | Synthesis and characterization of monosize magnetic poly (glycidyl methacrylate) beads | |
| Lu et al. | Synthesis and characterization of magnetic polymer microspheres with a core–shell structure |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| C19 | Lapse of patent right due to non-payment of the annual fee | ||
| CF01 | Termination of patent right due to non-payment of annual fee |