CN103980519B - A kind of preparation method of Magnetic Agarose sugar microsphere - Google Patents
A kind of preparation method of Magnetic Agarose sugar microsphere Download PDFInfo
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- 230000004913 activation Effects 0.000 claims abstract description 11
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Abstract
The invention discloses the preparation method of a kind of Magnetic Agarose sugar microsphere, including the preparation of the nonmagnetic microsphere of agarose, microsphere activation crosslinking that agarose is nonmagnetic, the preparation of microsphere ferric ion solutions that agarose is nonmagnetic and chemistry corotation prepare Magnetic Agarose sugar microsphere.The method of traditional agarose Coated magnetic particles is improved by the present invention, traditional coating directly be improved to indirect cladding process.Advantage of the invention is that, the Magnetic Agarose sugar microsphere prepared, intert because of inside and be coated with magnetic ferroferric oxide magnetic core particle, greatly the mechanical strength adding agarose of intensity, overcomes quality soft, and structure is easily destroyed, the defect that service life is short, magnetic particle possesses superparamagnetism simultaneously, and magnetic responsiveness is strong, can realize being rapidly separated under externally-applied magnetic field；Magnetic ball is compared with traditional method, and balling-up is improved, and spheroid is mellow and full, is difficult to caking of reuniting, good dispersion, even particle size distribution, and specific surface area increase makes isolated and purified rate improve.
Description
Technical field
The invention belongs to magnetic organic/inorganic composite material technical field, be specifically related to the preparation method of a kind of Magnetic Agarose sugar microsphere.
Background technology
What at present biomedicine field was conventional be used for carries out separating, the matrix of materials of purification can be divided into inorganic and organic two big classes.The medium of inorganic matrix mainly includes silica gel, cellular glass, hydroxyapatite etc., how as the inserts of high performance liquid chromatography.The medium of organic substrate is usually natural polysaccharide, synthetic high polymer etc., and it is spherical that the substrate that these materials are prepared as is usually pearl.The base ball warp of usual a kind of precursor structure is crossed chemical modification and can be derived and have difference in functionality group, different structure, different performance, different types of separating medium.As can be seen here, base ball is various separating medium the most basic, most important " raw material ".Owing to base ball has multiple premium properties, the most still parent of separating medium, it is also possible to as immobilized enzyme and the carrier etc. of catalyst, all obtain a wide range of applications in fields such as biological engineering, medicine preparation or even electronic information industry.Base ball can be as multifunctional material, and the most micron-sized microsphere is by wider application.
Agarose is a kind of linear, the water-soluble natural polysaccharide extracted from Sargassum, is by 1, the β-D-galactopyranose and the 3 of Isosorbide-5-Nitrae-connection that 3-connects, the polymer that 6-dehydration-α-L galactopyranose residues is alternately formed by connecting.Ball shape agarose gel has preferable isolation medium characteristic.Agarose gel, as substrate, has the following advantages: 1. rich in hydroxyl, after chemical activation, can the various aglucon of coupling；2. many empty network structure, beneficially macromolecular substances diffusions in bead, thus increase the effective density of aglucon；3. there is preferable hydrophilic so that biomolecule be prone near and with part effect, biomolecule will not be made to inactivate；4. neutral, non-specific adsorption ability is minimum.When buffer ionic strength >=0.05mol/L, to protein almost without non-specific adsorption.Therefore, it is a kind of good inert solid support of separating medium, it is often used as the substrate in affinity chromatography, ion exchange chromatography, hydrophobic exchange chromatography (HIC), reverse-phase chromatography, for separating, the biochemistry such as purification, such as, Shahab Lahootid et al. (Shahab Lahootid and Michael
V.Sefton, Effect of an immobilization matrix and
capsule permeability on thevariability of encapsulated HEK
Cells, Biomaterials.21 (2000) 987-995) describe the agarose as nuclear matter surrounded by hydroxyethyl methacrylate-methyl methacrylate copolymer shell, it is implanted in HEK cell.Research shows, agarose is conducive to implanting cytoactive and cell division and increment.Hiroyuki Hayashi et al. (Hiroyuki Hayashi, Kazutomo Inoue, TunAung et al., Application of a novel B cell line MIN6 to amesh
-reinforced polyvinyl alcohol hydrogel tube and three layeragarose
Microcapsules:An in vitro study, Cell Transplantation5 (1996) S56-S69) describe agarose purposes in three layers of gel micro-ball of preparation, described capsule is used to be implanted in B cell line MIN6.This research shows, the B cell line MIN6 of implantation has the insulin secretion speed exceeding twice than the most implanted MIN6.
The preparation method of matrix microspheres has multiple at present, mainly has the different preparation technologies such as suspension polymerisation (including inverse suspension polymerization), emulsion polymerization, dispersin polymerization, seed swelling polymerization.The nearly more than ten years occur in that again film emulsion-suspension polymerization technique.Agarose is as important isolation medium, the most the most frequently used preparation method is that reverse microemulsion process will be heat-fused in aqueous phase by agarose, there is the organic facies of certain viscosity as oil phase, at high temperature make the agarose Aqueous dispersions of hot melt in organic facies, cooling down rapidly, agarose is made to condense beading ball, Chinese patent CN
1524878A has applied for agarose microbeads prepared by a kind of reverse microemulsion method technology for purifying gene engineering recombinant interferon.China Patent No. CN
1472002A has applied for that a kind of reverse microemulsion process prepares the technology of high flux agarose microbeads.The another kind of new preparation method risen in recent years is that reverse microemulsion method-film emulsion process prepares agarose microbeads, i.e. on the basis of reverse microemulsion method, by pressure change, agarose hot melt aqueous phase is entered cooling balling-up in organic facies by special pore size distribution film, thus it is uniform preferably to control microspherulite diameter.China Patent No. CN
101715364 B have applied for the method that reverse micro emulsion-film emulsion process prepares agarose polysaccharide ball.Chinese patent CN 20041000087.9 describes tradition microporous membrane and prepares the agarose sugar pearl with controllable grain size.
Agarose microbeads prepared by above-mentioned patent application, is merely able to be applied in various detached dowel reach isolated and purified purpose as inserts, be merely able to various chromatographic apparatuss with the use of, it is not possible to separately as substrate reach separate purpose.In chromatographic isolation and biomolecule purification process, whether separating medium can sustain high flow rate is an important restriction, and some problem being widely used in the agarose medium in bio-separation field relevant is known, agarose gel structure is to be formed by the interaction of hydrogen bond.At gel state; the hydrogen bond staggered by interchain is formed many empty network structure by polysaccharide chain; this gel that formed by the interaction of hydrogen bond has low mechanical strength; and the most it is not able to take the highest flow velocity; because granule is softer; usually there will be the phenomenon compressing or blocking chromatographic column when chromatography, cause flow velocity the slowest, the problems such as service life is short.Simultaneously because the restriction that instrument uses, add the complexity of operation, before entering post, sample has to pass through the pre-treatment of series of complex, such as need through operating procedures such as cell breakage, centrifugation, salt precipitation, dissolution precipitations, considerably increase isolated and purified difficulty and time, add that the problems such as chromatographic apparatus maintenance substantially increase the cost of operation, be not appropriate for large-scale industrial production.
In order to overcome the shortcoming that agarose gel mechanical strength is low, in recent years, there is relevant report, in agarose microbeads, introduce magnetisable material, be used as skeletal support with this, increase the mechanical strength of agarose, and instrument can be departed from, it is used alone by magnetic, to realize separating purpose.Such as, Dong Yusheng et al. is (with reference to Journal
OfNorthwestUniversity (NaturalScience Edition, Apr.
2001Vo.l 31 No. 2) reverse microemulsion method coated ferriferrous oxide prepares Magnetic Agarose sugar microsphere separation urokinase.Li Jiaxing et al. is (with reference to Chemical
Engineering Journal 172 (2011) 892 897) reverse microemulsion method coated ferriferrous oxide Adsorption of Radioactive nucleic etc..But the agarose microbeads of magnetic prepared by the method existence cladding is uneven, coating efficiency is low, difference spherical in shape, the shortcomings such as specific surface area is little, although improve the mechanical performance of agarose, but bring new defect, as low in isolated and purified worry simultaneously, magnetic responsiveness difference etc..
Summary of the invention
In order to solve above-mentioned technical problem, the method for traditional agarose Coated magnetic particles is improved by the present invention, traditional coating directly be improved to indirect cladding process.The present invention adopts the following technical scheme that
The preparation method of a kind of Magnetic Agarose sugar microsphere, comprises the steps:
1. the preparation of the nonmagnetic microsphere of agarose
(1) providing the agarose solution containing additive as aqueous phase W, heating at a certain temperature makes agarose dissolve, and wherein agarose concentration in this solution is 0.1wt%-20wt%；
(2) offer and the organic solvent of aqueous phase W not phase mixing are as oil phase O, and dissolved emulsifier wherein, and wherein the emulsifier concentration in oil phase O is 0.1wt%-1.5wt%；
(3) oil phase O it is heated at 40 DEG C-90 DEG C and stirs under the mixing speed of 200-1000rpm, then agarose aqueous phase W being joined in oil phase O, keeping temperature and mixing speed to react a period of time acquisition W/O emulsion；The volume ratio of aqueous phase W and oil phase O is 1:1 ~ 1000；
(4) the W/O emulsion that in 5-10min, reduction step (3) obtains is to 15 ~ 40 DEG C, to form the nonmagnetic microsphere of agarose；
2. agarose nonmagnetic microsphere activation crosslinking
(1) preparation sodium hydroxide solution；
(2) the nonmagnetic microsphere of above-mentioned agarose and sodium borohydride are weighed according to the nonmagnetic microsphere of agarose with sodium borohydride mass ratio 1000 ~ 10:1, it is then added in sodium hydroxide solution, agarose is nonmagnetic microsphere and sodium borohydride mixture concentration 0.01 ~ 10g/ml in sodium hydroxide solution, react 10 ~ 45min under 20 ~ 30 DEG C and 300 ~ 600rpm；
(3) adding volume ratio in the solution of step (2) is epoxychloropropane and the dimethyl sulfoxide cross-linking agent mixed liquor of 1:1 ~ 100, and the liquor capacity of cross-linking agent mixed liquor and step (2) ratio for 1:1 ~ 100, reacts 5 ~ 20h under 20 ~ 30 DEG C and 300 ~ 600rpm；
(4) washing, removes unreacted reagent, obtains the nonmagnetic microsphere of agarose of activation crosslinking；
3. the preparation of agarose nonmagnetic microsphere ferric ion solutions
(1) preparation ferrous ion and ferric ion mixed solution；
(2) the nonmagnetic microsphere of agarose above-mentioned activation cross-linked joins in the mixed solution of step (1), and the concentration of agarose is nonmagnetic microsphere is 0.01-2g/ml, soaks 10-120h under 30-60 DEG C and 200-600rpm；
4. chemistry corotation prepares Magnetic Agarose sugar microsphere
(1) it is that ammonia is joined in agarose nonmagnetic microsphere ferric ion solutions by the ratio of 1 ~ 100:1 according to agarose nonmagnetic microsphere ferric ion solutions and ammonia volume ratio；Under 40-80 DEG C and 200-1000rpm, it is passed through nitrogen reaction, prepares ferroso-ferric oxide magnetic core；
(2) separating, washing under magnetic field, preserves, obtains Magnetic Agarose sugar microsphere.
Preferably, step 1.-(1) in, wherein the additive of aqueous phase W is sodium chloride, glucose, soft phospholipid or mannitol.
Preferably, step 1.-(2) in, one or more during wherein oil phase O is liquid paraffin, petroleum ether, olive oil, Oleum Gossypii semen, soybean oil, sunflower seed oil and some alkanes；The emulsifying agent added in oil phase O is glycerin ether polymer P O-500, NOFABLE SO-992 NOFABLE SO-902 Arlace
183, Cremophor RH40, sorbitan trioleate Span
85, dehydrated sorbitol mono-fatty acid ester Span
80 and sorbitan tristearate Span
One or more in 65.
Preferably, step 2.-(1) in naoh concentration be 0.1mol/L-2mol/L.
Preferably, step 3.-(1) in ferric ion solutions in the ratio of ferrous ion and the amount of the material of ferric ion be 1 ~ 10:1；Preparation water used by mixed solution is the water removing the oxygen wherein contained.
Magnetic Agarose sugar microsphere balling-up prepared by the present invention is round and smooth, good dispersion, do not produce reunion and its be the superparamagnetic nanoparticle that magnetic responsiveness is strong.Advantage of the invention is that, the Magnetic Agarose sugar microsphere prepared, interts because of inside and is coated with magnetic ferroferric oxide magnetic core particle, the mechanical strength adding agarose of very big intensity, overcome quality soft, structure is easily destroyed, the defect that service life is short, and magnetic particle possesses superparamagnetism simultaneously, magnetic responsiveness is strong, can realize under externally-applied magnetic field being rapidly separated, depart from chromatographic apparatus, independent use is to avoid the pre-treatment of complexity；Magnetic ball is compared with traditional method, and balling-up is improved, and spheroid is mellow and full, is difficult to caking of reuniting, good dispersion, even particle size distribution, and specific surface area increase makes isolated and purified rate improve.Magnetisable material clad ratio is improved significantly, up to more than 90%.
Accompanying drawing explanation
Fig. 1 is the Magnetic Agarose sugar microsphere optical microphotograph that the present invention prepares；
Fig. 2 is the nonmagnetic agarose microbeads optical microscope photograph that the present invention prepares；
Fig. 3 is what the present invention prepared Magnetic Agarose sugar microsphere photomacrograph, photo under wherein (a) is dispersity；B () is rapidly separated photo for (a) under magnetic field.
Detailed description of the invention
Below in conjunction with the accompanying drawings and the present invention is described in further details by embodiment.
1. the preparation method of the nonmagnetic microsphere of a kind of agarose of the present invention, comprises the steps:
(1) providing the agarose solution containing one or more additives as aqueous phase W, heating at a certain temperature makes agarose dissolve.
(2) provide with aqueous phase can not one or more organic solvents of phase mixing as oil phase O, and dissolve one or more emulsifying agents wherein.Under steady temperature, constant rotational speed, heat and stir oil phase O, according to a certain percentage aqueous phase W is joined in oil phase O, and under certain rotating speed mechanic whirl-nett reaction certain time obtain W/O emulsion.
(3) very fast reduction W/O emulsion drips to uniform temperature, forms agarose microbeads.
More specific explanation is, in step (1), it is provided that agarose solution there is a preset concentration, should be containing aqueous solution of agarose using as aqueous phase W in the present invention, this agarose solution, it will at the organic facies droplet that middle formation is aqueous mutually that water is immiscible.Agarose concentration in this solution is 0.1wt%-20wt%.Wherein the additive of aqueous phase can include harmless water-soluble substances, such as, sodium chloride, glucose, soft phospholipid, mannitol etc..
The oil phase provided in step (2) is mutual exclusive in water, it it is at room temperature the organic solvent of liquid, and need to have certain viscosity, the preferably organic reagent of high boiling point, low volatility, as, liquid paraffin, petroleum ether, olive oil, Oleum Gossypii semen, soybean oil, sunflower seed oil and some alkanes.Oil phase can be one can also be several with a certain proportion of mixture.Oil phase needs add one or more blended emulsifiers, this emulsifying agent must molten can be dissolved in this oil phase, such as, glycerin ether polymer (PO-500), NOFABLE SO-992 NOFABLE SO-902 (Arlace 183), Cremophor RH40, sorbitan trioleate (Span 85), dehydrated sorbitol mono-fatty acid ester (Span
80) or sorbitan tristearate (Span 65).Emulsifier concentration in oil phase is 0.1wt%-1.5wt%, and aqueous phase compares 1:1-1:1000 with oil phase volume.
The operation temperature of step (3) and (4) is above room temperature, and temperature required fusing point with Sepharose material and agarose aqueous solution content are relevant, and temperature range controls 40 DEG C-90 DEG C.Mechanical agitation speed is relevant with oil phase composition, controls at 200-1000rpm.
It is relevant with Sepharose material and content that step (5) reduces temperature, need to be reduced to 15-40 DEG C.
2. agarose microbeads activation crosslinking, including following step.
(1) certain density sodium hydroxide solution is prepared
(2) take the above-mentioned microsphere of certain mass, add sodium borohydride and the sodium hydroxide solution of certain volume of certain mass, constant temperature constant speed reaction certain time.
(3) in mentioned reagent, add dimethyl sulfoxide and the epoxychloropropane of certain volume, constant temperature constant speed reaction certain time.
Concrete body explanation, the sodium hydroxide provided in step (1) is used for activating microsphere surface group, and its concentration is in scope 0.1mol/L-2mol/L.
Agarose microbeads and sodium borohydride mass ratio 1000:1-10:1 in step (2).Agarose microbeads and sodium borohydride mixture concentration 0.01-10g/ml in sodium hydroxide solution.
Step (3) epoxychloropropane, dimethyl sulfoxide volume range 1:1-1:100.Cross-linking agent mixed liquor and microsphere sodium hydroxide mixeding liquid volume are than scope, 1:1-1:100.
3. prepared by the agarose microbeads Han iron ion
(1) a certain proportion of ferrous ion and ferric ion solution are prepared.
(2) microsphere after above-mentioned activation crosslinking presses finite concentration preparation with ferric ion solutions, and constant temperature, constant speed soak certain time.
Describe in detail, in the ferric ion solutions in step (1), ferrous ion and the thing mass ratio 1:1-10:1 of ferric ion, prepare the water used by solution and should be deoxidized water, remove oxygen therein, in order to avoid oxidation of divalent iron ion.
Microsphere ferric ion solutions concentration 0.01-2g/ml in step (2), temperature is relevant with agarose microbeads, and at 30-60 DEG C, speed controlling, at 200-600rpm, is immersed in 10-120h.
4. chemistry corotation prepares magnetic core
(1) the microsphere ferric ion solutions of the above-mentioned preparation of certain mass, adds ammonia by a certain percentage, controls solution alkaline condition.Constant temperature constant speed, is passed through nitrogen reaction, prepares ferroso-ferric oxide magnetic core.
(2) magnetic field separation washing, preserves, obtains Magnetic Agarose sugar microsphere.
It is expanded on further, microsphere ferric ion solutions and ammonia volume ratio 1:1-100:1 in step (1).Temperature, mixing speed are relevant with microsphere itself, control at 40-80 DEG C, rotating speed 200-1000rpm.
Embodiment one:
Prepared by the most nonmagnetic agarose microbeads:
(1) 2.0g agarose microbeads adds 100ml ultra-pure water, 100 DEG C of heating, prepares agarose aqueous phase W.
(2) liquid paraffin 180ml, petroleum ether 40ml, adds 8.0g emulsifying agent Span 80, fully dispersed prepares oil phase O.
(3) oil phase O heated at constant temperature 80 DEG C, mechanical agitation 400rpm, rapidly aqueous phase is moved into, react 3h.
(4) ice-water bath is cooled to rapidly 15 DEG C.
The activation crosslinking of the most nonmagnetic agarose microbeads:
(1) taking microsphere 5g, add NaOH7.5ml, the sodium borohydride 0.01g of 0.8mol/L, 25 DEG C, 400rpm shakes 30min.
(2) dimethyl sulfoxide 7.5ml, epoxychloropropane 3.75ml are added to above-mentioned solution.30 DEG C of constant temperature 400rpm react 20h.
(3) washing, removes unreacted reagent.
3. prepared by agarose ferric ion solutions
(1) 4.32gFeCl is taken3 ,15.92gFeCl2 ,It is dissolved in 200ml deoxidized water, prepares ferric ion solutions.
(2), during 30g microsphere is dissolved in above-mentioned ferric ion solutions, 45 DEG C, 90h is soaked in 400rpm concussion.
4. prepared by ferroso-ferric oxide magnetic core
(1) microsphere after the above-mentioned immersion of 15g adds 150ml deoxidized water.At 60 DEG C, adding 40ml ammonia, stirring is passed through nitrogen, reacts 3h.
(2) magnetic field separation, dehydrated alcohol, water wash.
(3) during microsphere is saved in 20% ethanol solution.4 DEG C of preservations.
Claims (5)
1. a preparation method for Magnetic Agarose sugar microsphere, comprises the steps: that the preparation (1) of the 1. nonmagnetic microsphere of agarose provides containing additive
Agarose solution as aqueous phase W, at a certain temperature heating make agarose dissolve, wherein agarose concentration in this solution is
0.1wt%～20wt%；(2) offer and the organic solvent of aqueous phase W not phase mixing are as oil phase O, and dissolved emulsifier wherein, wherein oil phase
Emulsifier concentration in O is 0.1wt%～1.5wt%；(3) oil phase O is heated to 40 DEG C～90 DEG C and 200～1000rpm stirring speed
The lower stirring of degree, then joins in oil phase O by agarose aqueous phase W, keeps temperature and mixing speed to react a period of time acquisition W/O emulsion；Water
Phase W is 1:1～1000 with the volume ratio of oil phase O；The W/O emulsion that in (4) 5～10min, reduction step (3) obtains is to 15～40 DEG C, with shape
Become the nonmagnetic microsphere of agarose；2. agarose nonmagnetic microsphere activation crosslinking (1) preparation sodium hydroxide solution；(2) according to agarose without magnetic
Property microsphere and sodium borohydride mass ratio 1000～10:1 weigh the nonmagnetic microsphere of above-mentioned agarose and sodium borohydride, be then added in sodium hydroxide solution,
Agarose is nonmagnetic microsphere and sodium borohydride mixture concentration 0.01～10g/ml in sodium hydroxide solution, at 20～30 DEG C and 300～600rpm
Lower reaction 10～45min；(3) in the solution of step (2), epoxychloropropane and the dimethyl sulfoxide cross-linking agent that volume ratio is 1:1～100 is added
Mixed liquor, the liquor capacity of cross-linking agent mixed liquor and step (2) ratio for 1:1～100, reacts 5～20h under 20～30 DEG C and 300～600rpm；
(4) washing, removes unreacted reagent, obtains the nonmagnetic microsphere of agarose of activation crosslinking；3. agarose nonmagnetic microsphere ferric ion solutions
Preparation (1) preparation ferrous ion and ferric ion mixed solution；(2) the nonmagnetic microsphere of agarose above-mentioned activation cross-linked joins step
Suddenly in the mixed solution of (1), the concentration of agarose is nonmagnetic microsphere is 0.01～2g/ml, soaks 10～120h under 30～60 DEG C and 200～600rpm；
4. chemistry corotation prepares Magnetic Agarose sugar microsphere (1) according to the ratio that agarose nonmagnetic microsphere ferric ion solutions and ammonia volume ratio are 1～100:1
Ammonia is joined in agarose nonmagnetic microsphere ferric ion solutions；Under 40～80 DEG C and 200～1000rpm, it is passed through nitrogen reaction, prepares
Ferroso-ferric oxide magnetic core；(2) separating, washing under magnetic field, preserves, obtains Magnetic Agarose sugar microsphere.
The preparation method of Magnetic Agarose sugar microsphere the most according to claim 1, it is characterised in that: step 1.-(1) in, wherein aqueous phase W
Additive is sodium chloride, glucose, soft phospholipid or mannitol.
The preparation method of Magnetic Agarose sugar microsphere the most according to claim 1, it is characterised in that: step 1.-(2) in, wherein oil phase O is
One or more in liquid paraffin, petroleum ether, olive oil, Oleum Gossypii semen, soybean oil, sunflower seed oil；The emulsifying agent added in oil phase O is sweet
Oil ether polymer PO-500, NOFABLE SO-992 NOFABLE SO-902 Arlacel 83, Cremophor RH40, sorbitan trioleate
Span 85, dehydrated sorbitol mono-fatty acid ester Span 80 and sorbitan tristearate Span 65 and in one or more.
The preparation method of Magnetic Agarose sugar microsphere the most according to claim 1, it is characterised in that: step 2.-(1) in naoh concentration be
0.1mol/L-2mol/L。
The preparation method of Magnetic Agarose sugar microsphere the most according to claim 1, it is characterised in that: step 3.-(1) in ferric ion solutions in two
Valency iron ion is 1～10:1 with the ratio of the amount of the material of ferric ion；Preparation water used by mixed solution is the water removing the oxygen wherein contained.
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| CN114950375B (en) * | 2022-06-02 | 2024-04-30 | 杭州隆基生物技术有限公司 | Streptavidin agarose magnetic beads, preparation method and application thereof |
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