CN109865496A - Magnetic silica filler, magnetic affine filler and preparation method and purposes - Google Patents
Magnetic silica filler, magnetic affine filler and preparation method and purposes Download PDFInfo
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Abstract
The present invention relates to magnetic Nano material technical fields, and in particular to a kind of preparation method of magnetic silica filler.The preparation method is in situ to introduce Fe using porous polymer polymer microballoon as template3O4Magnetic nano-particle, and carry out the sol gel reaction of silicon source, then by magnetic Fe3O4In-situ oxidation is at γ-Fe2O3, magnetic silica filler magnetic silica microballoon is prepared after high-temperature calcination.The magnetic silica filler magnetic silica microballoon partial size and aperture that this method is prepared are controllable, and magnetic particle is γ-Fe2O3, and be dispersed in the skeleton structure of silica.Magnetic silica filler further is put to apply by the present invention, heparin modified by carrying out to magnetic silica filler, and the magnetic affine filler of preparation can be used for isolating and purifying Antithrombin III.
Description
Technical field
The present invention relates to magnetic Nano material technical fields, and in particular to a kind of preparation method of magnetic silica filler
And magnetic silica filler obtained, further relate to a kind of affine filler of magnetism and its system comprising the magnetic silica filler
Preparation Method and purposes.
Background technique
Porous silica has good specific surface area height, high mechanical strength, stability, good biocompatibility and easily modification etc.
Feature.Magnetic nano-particle can be transported from liquid phase separation or specific aim to privileged site under the action of external magnetic field, inhaled
Fufen is widely used from fields such as, chemical catalysis and targeted delivery of drugs, but due to its stability difference and it is easy to reunite the disadvantages of, application
It is subject to certain restrictions.Porous silica and magnetic nano-particle are be combined with each other and prepare composite material, these can be overcome
Disadvantage, while the advantages of porous silica and magnetic nano-particle, will be effectively bonded together, to have more wide
General practical value.Especially porous magnetic silicon dioxide microsphere can be used as emerging magnetic affinity chromatography separation filler, in life
Object field of medicaments has great application value.In general, filler used in affinity chromatography separation is the microballoon using polysaccharide as matrix,
There are bad mechanical strength, easily-deformable, separation, flow velocity is low etc. that disadvantages, porous magnetic silicon dioxide microsphere filler then solve this hardly possible
Topic.
The porous magnetic silicon dioxide microsphere prepared at present is mainly core-shell structure, and most important preparation method is, first
Magnetic nano particle daughter nucleus is prepared, then carries out silica sol-gel reaction on its surface, in the sol-gel process
Surfactant, which is added, can prepare porous magnetic silica.Deng et al. (J.Am.Chem.Soc.2008,130,28-
29) magnetic silicon dioxide composite microsphere of superparamagnetism is prepared, with Fe3O4Particle is core, in acid condition with orthosilicic acid four
Ethyl ester (TEOS) is that silicon source carries out sol gel, in Fe3O4Particle surface uniformly wraps up one layer of non-porous silicas, so as to
Prevent magnetic particle from reuniting.On this basis, with cetyl trimethylammonium bromide (CTAB) for soft template, TEOS is silicon
Source, ammonium hydroxide are alkali, carry out the sol gel of second step, that is, produce Fe3O4@nSiO2@CTAB/SiO2Complex microsphere.Lead to again
It crosses solvent-extracted mode and removes CTAB, mesoporous silicon oxide shell can be obtained, prepare the Fe of about 390nm3O4@nSiO2@
mSiO2Complex microsphere can be used to the Microcystin in rapidly and efficiently adsorbent solution.Gai et al.
(Adv.Funct.Mater.2010,20,1166-1172) prepares the magnetism of porous core-shell structure with two similar step packet silicon process
Earth silicon material, and fluorescent decoration is carried out to it, so that multifunctional material is prepared, the sustained release for ibuprofen pharmaceutical.
Vald é s-Sol í s et al. (Chem.Mater.2009,21,1806-1814) is more using being prepared based on the method that nanometer is cast
Hole magnetic silica composite material.Its method is first to prepare porous silica silicon materials (such as SBA-15), then by molysite
It deposits in the hole of silica, adds ethylene glycol and infiltration is entered to it.By sample lower 450 DEG C in nitrogen atmosphere after infiltration
2h is calcined, so that molysite is generated magnetic iron oxide, to prepare porous magnetic earth silicon material.Wherein magnetic nano-particle point
Cloth is reunited together on entire silica substrate material, and by part magnetic nano-particle.
The magnetic silica microballoon prepared at present has the disadvantages that mostly partial size only has several hundred rans, grain
Diameter and aperture are uncontrollable, and structure is mostly core-shell structure, and magnetic nano-particle is not the uniform bone for being dispersed in silica
In frame structure；Magnetic particle is mostly Fe3O4, it need to prepare in advance, it is cumbersome, and non-refractory, it is oxidizable, it is magnetic easily to disappear.Cause
This, prepares controllable, evenly dispersed, magnetic stability the magnetic silica filler magnetic silica microballoon of partial size and aperture,
It is the heavy difficult point of this field.
Summary of the invention
The object of the present invention is to overcome the problems of the prior art, provides a kind of magnetic silica filler, this is filled out
The magnetic particle of material is evenly dispersed, magnetic stability, and can be used for preparing has the affine filler of magnetism for isolating and purifying purposes.
Therefore, in a first aspect, the present invention provides a kind of preparation method of magnetic silica filler, this method include with
Lower step:
Sulfonated processing is carried out to porous polymer polymer microballoon；
By sulfonated porous polymer polymer microballoon and include Fe3+And Fe2+Source of iron contact to adsorb Fe3+And Fe2 +, Fe in the source of iron3+:Fe2+Ammonium hydroxide, in-situ preparation magnetic Fe is added after the completion of absorption in molar ratio=2:13O4/ polyphosphazene polymer
Close object microballoon；
Silicon source is added to the magnetic Fe3O4In/high molecular polymer microballoon, sol gel reaction is carried out, magnetic is obtained
Property Fe3O4/ high molecular polymer/SiO 2 composite microsphere；
With oxidant by the magnetic Fe3O4Magnetic Fe in/high molecular polymer/SiO 2 composite microsphere3O4Situ oxygen
It is melted into γ-Fe2O3, obtain γ-Fe2O3/ high molecular polymer/SiO 2 composite microsphere；
By the γ-Fe2O3/ high molecular polymer/SiO 2 composite microsphere carries out high-temperature calcination, removes as template
The high molecular polymer obtains the magnetic silica filler.
Specifically, which is polystyrene type or polyglycidyl acrylate class or it spreads out
The microballoon of biology.
Preferably, which is that polystyrene microsphere, poly (glycidyl methacrylate) are micro-
Ball, polystyrene/divinylbenzene microspheres, poly (glycidyl methacrylate)/ethylene glycol dimethacrylate microballoon.
Preferably, the partial size of the porous polymer polymer microballoon is from 3 μm to 50 μm, and aperture is from 2nm to 200nm.
For polystyrene/divinylbenzene microspheres and poly (glycidyl methacrylate)/ethyleneglycol dimethacrylate
The degree of cross linking of ester microsphere, divinylbenzene or ethylene glycol dimethacrylate is 5-80%, i.e. divinylbenzene monomer accounts for polyphenyl
Ethylene/divinylbenzene microspheres molar ratio is that 5-80% or ethylene glycol dimethacrylate monomer account for polymethylacrylic acid
Ethylene oxidic ester/ethylene glycol dimethacrylate microballoon molar ratio is 5-80%.
Specifically, the sulfonated processing concentrated sulfuric acid or sulphite carry out.Preferably, which is sulfurous acid
Sodium.
Specifically, which is tetraalkoxysilane.Preferably, which is tetraethyl orthosilicate.
Specifically, the magnetic Fe3O4/ high molecular polymer microballoon and the mass ratio of the silicon source are 1:0.5-15.
Specifically, the oxidant is ammonium ceric nitrate.
Specifically, which rises to 500-600 DEG C from room temperature with the speed of 1-10 DEG C/min, and at 500-600 DEG C
Lower holding 2-20h.
In second aspect, the present invention provides a kind of magnetic silica filler, which passes through
The preparation method of first aspect present invention is prepared, and is a kind of monodispersity, porous nanofiller.
In the third aspect, the present invention provides a kind of heparin modified affine filler of magnetism, and the heparin modified magnetism is affine
The magnetic silica filler and the heparin in conjunction with the magnetic silica filler that filler includes second aspect of the present invention.
In fourth aspect, the present invention provides the affine filler of magnetism of third aspect present invention, which mainly includes
Following steps:
Amination processing is carried out to the magnetic silica filler with 3- aminopropyl triethoxysilane, is obtained amidized
Magnetic silica filler；
It reacts the amidized magnetic silica filler in the presence of sodium cyanoborohydride with heparin sodium, obtains
The heparin modified affine filler of magnetism；
Preferably, the preparation method comprising the following specific steps
The magnetic silica filler is dried in vacuo, three ethoxy of 3- aminopropyl is added in dry toluene in ultrasonic disperse
Base silane, back flow reaction 10-24h obtain amidized magnetic silica filler through filtration washing and drying,
Heparin sodium is dissolved in the dipotassium hydrogen phosphate solution that concentration is 0.1-0.5mol/L, the amination after drying is added
Magnetic silica filler, ultrasonic disperse adds sodium cyanoborohydride, is heated to reflux 8-15h, through filtration washing and drains
Afterwards, sodium carbonate liquor is added, ultrasound mixes, and acetic anhydride is added, and 0 DEG C of reaction 20-40min adds acetic anhydride, reacts at room temperature
20-40min, filtering, is sufficiently washed with water, obtains the affine filler of the magnetism after dry.
In the 5th aspect, the affine filler of magnetism of present invention offer third aspect present invention or according to the present invention four directions
The affine filler of magnetism that the preparation method in face is prepared is used to isolate and purify the purposes of Antithrombin III.
Preferably, which is people's Antithrombin III in human plasma.
Beneficial effects of the present invention:
The preparation method of magnetic silica filler of the invention is drawn in situ using porous polymer polymer microballoon as template
Enter Fe3O4Magnetic nano-particle, and carry out the sol gel reaction of silicon source, then by magnetic Fe3O4In-situ oxidation is at γ-Fe2O3,
Magnetic silica filler magnetic silica microballoon is prepared after high-temperature calcination.The magnetic dioxy that this method is prepared
The magnetic particle of SiClx filler is γ-Fe2O3, and be dispersed in the skeleton structure of silica.
γ-Fe in magnetic nano-particle2O3Compared to Fe3O4It is advantageous in that, γ-Fe2O3It has been in oxidation state, has been kept
It will not be further oxided while magnetic, it is more stable.And magnetic Fe3O4It is unstable in air, it can be oxidized to nonmagnetic
α-Fe2O3.And magnetic particle γ-Fe2O3It is dispersed in the skeleton structure of silica, can guarantee that magnetic microsphere is filled out
Otherwise the homogeneity of material easily causes biggish differences between batches.
Moreover, the preparation method magnetic nano-particle introducing method of magnetic silica filler of the invention is simple, it is in situ
Synthesis, magnetic stability, high temperature resistant are not easy demagnetization and are not easy to leak.Also, the magnetic silica filler magnetism two being prepared
Silicon oxide microsphere is since with silicon dioxide skeleton, high mechanical strength solves the usually used polysaccharide of current affinity chromatography
Flexible glue mechanical strength is low, easily-deformable, the operation disadvantages such as flow velocity is low, and with monodispersity is good and partial size and aperture controllably excellent
Point.
Magnetic silica filler further is put to apply by the present invention, by carrying out liver to magnetic silica filler
Element modification, the magnetic affine filler of preparation, can be used for isolating and purifying the Antithrombin III in blood plasma, easy to operate, purification efficiency
Height improves blood plasma comprehensive utilization ratio.
Detailed description of the invention
Fig. 1 is the scanning electron microscope (SEM) photograph of magnetic silica filler prepared by the embodiment of the present invention 1；
Fig. 2 is the graph of pore diameter distribution of magnetic silica filler prepared by the embodiment of the present invention 1；
Fig. 3 is the B-H loop of magnetic silica filler prepared by the embodiment of the present invention 1；
Fig. 4 is the SDS-PAGE figure for the Antithrombin III that the embodiment of the present invention 4 isolates and purifies.
Specific embodiment
Below by specific embodiment and in conjunction with attached drawing, invention is further described in detail.
The present invention provides a kind of preparation method of magnetic silica filler, method includes the following steps:
Sulfonated processing is carried out to porous polymer polymer microballoon；
By sulfonated porous polymer polymer microballoon and include Fe3+And Fe2+Source of iron contact to adsorb Fe3+And Fe2 +, Fe in the source of iron3+:Fe2+Ammonium hydroxide, in-situ preparation magnetic Fe is added after the completion of absorption in molar ratio=2:13O4/ polyphosphazene polymer
Close object microballoon；
Silicon source is added to the magnetic Fe3O4In/high molecular polymer microballoon, sol gel reaction is carried out, magnetic is obtained
Property Fe3O4/ high molecular polymer/SiO 2 composite microsphere；
With oxidant by the magnetic Fe3O4Magnetic Fe in/high molecular polymer/SiO 2 composite microsphere3O4Situ oxygen
It is melted into γ-Fe2O3, obtain γ-Fe2O3/ high molecular polymer/SiO 2 composite microsphere；
By the γ-Fe2O3/ high molecular polymer/SiO 2 composite microsphere carries out high-temperature calcination, removes as template
The high molecular polymer obtains the magnetic silica filler.
The porous polymer polymer microballoon that the present invention uses be polystyrene type or polyglycidyl acrylate class or
The microballoon of its derivative.Specific preferred porous polymer polymer microballoon is polystyrene microsphere, polymethylacrylic acid shrink
Glycerol ester microsphere, polystyrene/divinylbenzene microspheres, poly (glycidyl methacrylate)/ethylene glycol dimethacrylate
Microballoon.
The porous polymer polymer microballoon that the present invention uses can be prepared by commercially available acquisition, or by polymerization reaction
It obtains.For example, polystyrene microsphere can be easy commercially available acquisition.In another example (" new hydrophobic chromatography is situated between the doctoral thesis of Wang Renwei
Preparation, modification and the application of the preparation and application of matter --- poly (glycidyl methacrylate) microballoon ") describe poly- methyl-prop
The preparation of olefin(e) acid glycidol ester microsphere；The doctoral thesis (" preparation of one divinylbenzene stephanoporate crosslinked microsphere of polystyrene of Li Lu
And the research of active matter adsorption sustained-release performance ") describe the preparation of one divinylbenzene of polystyrene；Polymethyl acid glycidyl
Ester/ethylene glycol dimethacrylate microballoon can refer to following documents: Samatya, S., Kabay, N., Tuncel,
A.Reactive&Functional Polymers,2010,70,555。
The packing material size that the preparation method of magnetic silica filler of the invention is prepared is at 3-10 μm, and aperture is at least
>=30nm, is suitable as chromatograph packing material, is especially suitable for the macromolecular substances such as protein isolate matter.
In the preparation method of magnetic silica filler of the invention, sulfonated processing is to repair polymer microsphere
Sulfonate radical is adornd into, sulfonate radical is negatively charged, adsorbable Fe2+And Fe3+, so that ammonium hydroxide, which is added, produces magnetic Fe3O4, introduce magnetic
Nanoparticle adds TEOS, carries out sol gel reaction, then can carry out silica on magnetic macromolecular microsphere surface
Cladding, and silica is the base portion of last filler.Following in-situ oxidation again, by unstable magnetic Fe3O4It is oxidized to
Stable magnetic γ-Fe2O3。
Magnetic silica filler also is put to apply by the present invention, by with 3- aminopropyl triethoxysilane to magnetism two
Cilicon oxide filler carry out amination processing, obtain amidized magnetic silica filler, and make this amidized magnetic two
Cilicon oxide filler is reacted in the presence of sodium cyanoborohydride with heparin sodium, and the heparin modified affine filler of magnetism is obtained.It should
Magnetic affine filler can be used for isolating and purifying Antithrombin III (AT-III).
AT-III is the important factor for participating in physiological blood clotting mechanism and controlling blood coagulation, and molecular weight 58kD is that blood is solidifying
Gu the major inhibitors of system are the weights for causing thrombus and pulmonary embolism to maintain the normal circulation AT-III of body blood to lack
Want reason, disease incidence 1/500-1/5000.AT-III is 100-300 μ g/ml, human body in normal adults Plasma
Interior AT-III level is lower than the 70% of normal level, and the risk of thrombosis can be made to increase.AT-III can be used for treating AT-III
The patient of shortage, such as congenital and acquired Antithrombin III lack, and can be used for treating congenital antithrombin III shortage and draw
The thrombus risen, the illnesss such as dispersivity blood vessel internal haemorrhage, septicemia, wound, tumour, thromboembolism and complicated pregnancy.
The present invention isolates and purifies the Antithrombin III in blood plasma, easy to operate, purification efficiency using magnetic affine filler
Height improves blood plasma comprehensive utilization ratio.
The present invention is given an example below by way of non-limiting embodiment.
Embodiment 1: the preparation of magnetic silica filler
Weigh the magnetic silica filler poly (glycidyl methacrylate)/glycol dinitrate of 7.42 μm of partial sizes of 20g
Base acrylate (PGMA/EGDMA) microballoon, 20g sodium sulfite is added in 500ml water in ultrasonic disperse, mechanical at 160rpm
It is stirred to react for 24 hours.It filters, is washed respectively 3 times with water and ethyl alcohol after reaction, it is micro- to obtain sulfonated porous PGMA/EGDMA
Ball, it is spare in 50 DEG C of oven dryings.
Weigh 1.645g FeCl2·4H2O and 4.34g FeCl3·6H2O is dissolved in 20ml water, is vacuumized, with obtained
To Fe2+:Fe3+Molar ratio is the source of iron solution of 1:2, N2Under save backup.By the above-mentioned 7.42 sulfonated μ being prepared of 0.5g
The source of iron solution that 0.5ml has been prepared is added in 20ml water in the porous PGMA/EGDMA microballoon ultrasonic disperse of m, after adsorbing 12h, directly
The ammonium hydroxide that 28 weight % of 1ml is added into solution is connect, mechanical stirring 3h generates magnetic Fe3O4/ porous PGMA/EGDMA microballoon.
Continue the ammonium hydroxide of addition 28 weight % of 2ml in above-mentioned reaction solution, 10ml water and 80ml ethyl alcohol is added, surpasses
Sound dispersion microsphere.3g TEOS is dissolved into 10ml ethyl alcohol, the solution of TEOS is slowly dropped in reaction solution with peristaltic pump,
It is reacted at room temperature for 24 hours after being added dropwise.It filters after reaction, is washed respectively 3 times with water and ethyl alcohol, obtain magnetic Fe3O4/ porous
PGMA/EGDMA/ SiO 2 composite microsphere.
It weighs 0.06g ammonium ceric nitrate to be dissolved in 4ml acetone, acquired solution is added to the magnetic Fe of above-mentioned preparation3O4/ more
In the PGMA/EGDMA/ SiO 2 composite microsphere of hole, it is ultrasonically treated 3h, makes magnetic Fe3O4It is transformed into magnetic γ-Fe2O3, obtain
Magnetic γ-Fe2O3/ porous PGMA/EGDMA/ SiO 2 composite microsphere, then this complex microsphere is filtered, it washs, it is dry.
By magnetism γ-Fe obtained above2O3/ porous PGMA/EGDMA/ SiO 2 composite microsphere is placed in Muffle furnace,
550 DEG C of high-temperature calcination 6h are risen to the speed of 2 DEG C/min, remove porous PGMA/EGDMA template, obtained magnetic silica is filled out
Material.
The scanning electron microscope (SEM) photograph of the magnetic silica filler that the present embodiment is prepared as shown in Figure 1, graph of pore diameter distribution such as
Shown in Fig. 2, B-H loop is as shown in Figure 3.
Embodiment 2: the preparation of magnetic silica filler
Magnetic silica filler polystyrene/divinylbenzene (PS/DVB) microballoon of 4.54 μm of partial sizes of 20g is weighed, is surpassed
Sound is scattered in 500ml water, and 20g sodium sulfite is added, and mechanic whirl-nett reaction is for 24 hours at 160rpm.It filters, uses after reaction
Water and ethyl alcohol wash 3 times respectively, obtain sulfonated porous PS/DVB microballoon, spare in 50 DEG C of oven dryings.
Weigh 1.645g FeCl2·4H2O and 4.34g FeCl3·6H2O is dissolved in 20ml water, is vacuumized, with obtained
To Fe2+:Fe3+Molar ratio is the source of iron solution of 1:2, N2Under save backup.By the above-mentioned 4.54 sulfonated μ being prepared of 0.5g
The source of iron solution that 0.125ml has been prepared is added in 20ml water in the porous PS/DVB microballoon ultrasonic disperse of m, after adsorbing 12h, directly
The ammonium hydroxide of 28 weight % of 1ml is added into solution, mechanical stirring 3h generates magnetic Fe3O4/ porous PS/DVB microballoon.
Continue the ammonium hydroxide of addition 28 weight % of 2ml in above-mentioned reaction solution, 10ml water and 80ml ethyl alcohol is added, surpasses
Sound dispersion microsphere.2g tetraethyl orthosilicate (TEOS) is dissolved into 10ml ethyl alcohol, the solution of TEOS is slowly added dropwise with peristaltic pump
Into reaction solution, reacted at room temperature for 24 hours after being added dropwise.It filters after reaction, is washed 3 times, obtained respectively with water and ethyl alcohol
Magnetic Fe3O4/ porous PS/DVB/ SiO 2 composite microsphere.
It weighs 0.045g ammonium ceric nitrate to be dissolved in 4ml acetone, acquired solution is added to the magnetic Fe of above-mentioned preparation3O4/ more
In the PS/DVB/ SiO 2 composite microsphere of hole, it is ultrasonically treated 3h, makes magnetic Fe3O4It is transformed into magnetic γ-Fe2O3, obtain magnetism
γ-Fe2O3/ porous PS/DVB/ SiO 2 composite microsphere, then this complex microsphere is filtered, it washs, it is dry.
By magnetism γ-Fe obtained above2O3/ porous PS/DVB/ SiO 2 composite microsphere is placed in Muffle furnace, with 2
DEG C/speed of min rises to 600 DEG C of high-temperature calcination 6h, porous PS/DVB template is removed, magnetic silica filler is made.
Embodiment 3: the preparation of magnetic silica filler
The present embodiment replaces embodiment using poly (glycidyl methacrylate) microballoon (PGMA) microballoon of 6.5 μm of partial sizes
1 polystyrene/divinylbenzene (PS/DVB) microballoon, remaining is same as Example 1.
Embodiment 4: the preparation of magnetic silica filler
The present embodiment replaces polystyrene/divinylbenzene of embodiment 1 using polystyrene (PS) microballoon of 5.2 μm of partial sizes
(PS/DVB) microballoon, remaining is same as Example 1.
Embodiment 5: the preparation of magnetic affine filler
Magnetic silica filler 20g prepared by the embodiment 1 after weighing vacuum drying, ultrasonic disperse are anhydrous in 200ml
In toluene.3- aminopropyl triethoxysilane 5ml is added, back flow reaction is for 24 hours.Reactant is filtered, is distinguished with toluene and ethyl alcohol
Washing 3 times, it is dry in baking oven after draining, obtain amination magnetic silica filler.
It weighs 500mg heparin sodium to be dissolved in 50ml 0.2mol/L dipotassium hydrogen phosphate solution, the amination magnetic after drying is added
Property silica filler 12g, ultrasonic disperse.200mg sodium cyanoborohydride is added, 10h is heated to reflux.Through filtration washing and pumping
After dry, 2ml 0.2mol/L sodium carbonate liquor is added, ultrasound mixes.2ml acetic anhydride is added, 0 DEG C of reaction 30min adds 2ml
Acetic anhydride reacts at room temperature 30min.Filtering is sufficiently washed with water to neutrality, and magnetic affine filler is obtained after dry.
Embodiment 6: Antithrombin III is isolated and purified with magnetic affine filler
Human blood product cold ethanol production technology Cohn method component FIV-1 precipitating 50g is weighed, 500ml 20mM is dissolved in
In Tris-HCl buffer.After precipitating has sufficiently dissolved, the affine filler 5g of magnetism prepared by embodiment 3, adsorption reaction is added
24h.Reaction terminates, and is separated filler and reaction solution with magnet.20mM Tris-HCl+1M is added into the filler after absorption
NaCl solution impregnates 2h, removes the impurity protein adsorbed on filler, then separated filler from solution with magnet.With
20mM Tris-HCl+1M NaCl solution is washed filler 3 times repeatedly, completely removes impurity protein.20mM is added into filler again
Tris-HCl+3M NaCl impregnates 2h, makes one Antithrombin III and elute.Filler is separated from solution with magnet,
Obtain people's Antithrombin III solution of high-purity.
The people's Antithrombin III isolated and purified out is subjected to SDS-PAGE detection.Prepare 15% separation gel and 5% it is dense
Contracting glue, is fitted into electrophoresis tank, and electrophoretic buffer is added.Sample-loading buffer is added in sample, and 100 DEG C are boiled 5min.Sample after boiling
Product sequentially add in gel duct, plug in, and voltage is set as 80V, run 3.5h.After electrophoresis, by gel from glass
It is slowly stripped down on plate, dyes 3h using coomassie brilliant blue staining liquid, then decolourized overnight with destainer.Gel after decoloration is used
Gel imager is scanned, as shown in the SDS-PAGE figure of Fig. 4.It is normalized to calculated purity to peak area is carried out in duct,
The Antithrombin III purity being purified into is 91%.As it can be seen that the affine filler of magnetism that the present invention is prepared can drip separation very well
Purification of Human Antithrombin III.
Use above specific example is expounded the present invention, is merely used to help understand the present invention, not to
The limitation present invention.The design of those skilled in the art according to the present invention can also be made and several simply push away
It drills, deform or replaces.These are deduced, deformation or alternative are also fallen into scope of the presently claimed invention.
Claims (10)
1. a kind of preparation method of magnetic silica filler, which is characterized in that the described method comprises the following steps:
Sulfonated processing is carried out to porous polymer polymer microballoon；
By sulfonated porous polymer polymer microballoon and include Fe3+And Fe2+Source of iron contact to adsorb Fe3+And Fe2+, institute
State the Fe in source of iron3+:Fe2+Ammonium hydroxide, in-situ preparation magnetic Fe is added after the completion of absorption in molar ratio=2:13O4/ high molecular polymerization
Object microballoon；
Silicon source is added to the magnetic Fe3O4In/high molecular polymer microballoon, sol gel reaction is carried out, magnetism is obtained
Fe3O4/ high molecular polymer/SiO 2 composite microsphere；
With oxidant by the magnetic Fe3O4Magnetic Fe in/high molecular polymer/SiO 2 composite microsphere3O4In-situ oxidation
At γ-Fe2O3, obtain γ-Fe2O3/ high molecular polymer/SiO 2 composite microsphere；
By the γ-Fe2O3/ high molecular polymer/SiO 2 composite microsphere carries out high-temperature calcination, removes the institute as template
High molecular polymer is stated, the magnetic silica filler is obtained.
2. preparation method according to claim 1, which is characterized in that the porous polymer polymer microballoon is polyphenyl second
The microballoon of alkenes or polyglycidyl acrylate class or derivatives thereof；Preferably, the porous polymer polymer microballoon is
Polystyrene microsphere, poly (glycidyl methacrylate) microballoon, polystyrene/divinylbenzene microspheres, polymethylacrylic acid contracting
Water glyceride/ethylene glycol dimethacrylate microballoon；Preferably, the partial size of the porous polymer polymer microballoon is from 3 μm
To 50 μm, aperture is from 2nm to 200nm.
3. preparation method according to claim 1, which is characterized in that sulfonated the processing concentrated sulfuric acid or the sulphite
It carries out；Preferably, which is sodium sulfite.
4. preparation method according to claim 1, which is characterized in that the silicon source is tetraalkoxysilane, preferably former silicon
Sour tetra-ethyl ester；The magnetic Fe3O4/ high molecular polymer microballoon and the mass ratio of the silicon source are 1:0.5-15.
5. preparation method according to claim 1, which is characterized in that the oxidant is ammonium ceric nitrate.
6. preparation method according to claim 1, which is characterized in that the high-temperature calcination with the speed of 1-10 DEG C/min from
Room temperature rises to 500-600 DEG C, and keeps 2-20h at 500-600 DEG C.
7. a kind of magnetic silica filler, which is characterized in that the magnetic silica filler passes through according to claim 1-6
Any one of described in preparation method be prepared.
8. a kind of heparin modified affine filler of magnetism, which is characterized in that the heparin modified affine filler of magnetism includes root
According to magnetic silica filler as claimed in claim 7 and the heparin in conjunction with the magnetic silica filler.
9. the preparation method of the affine filler of magnetism according to claim 8, which is characterized in that the preparation method include with
Lower step:
Amination processing is carried out to the magnetic silica filler with 3- aminopropyl triethoxysilane, obtains amidized magnetic
Property silica filler；
It reacts the amidized magnetic silica filler in the presence of sodium cyanoborohydride with heparin sodium, obtains institute
State the heparin modified affine filler of magnetism；
Preferably, the preparation method comprising the following specific steps
The magnetic silica filler is dried in vacuo, 3- aminopropyl-triethoxy is added in dry toluene in ultrasonic disperse
Silane, back flow reaction 10-24h obtain amidized magnetic silica filler through filtration washing and drying,
Heparin sodium is dissolved in the dipotassium hydrogen phosphate solution that concentration is 0.1-0.5mol/L, it is described amidized after drying is added
Magnetic silica filler, ultrasonic disperse add sodium cyanoborohydride, are heated to reflux 8-15h, through filtration washing and drain
Afterwards, sodium carbonate liquor is added, ultrasound mixes, and acetic anhydride is added, and 0 DEG C of reaction 20-40min adds acetic anhydride, reacts at room temperature
20-40min, filtering, is sufficiently washed with water, obtains the affine filler of magnetism after dry.
10. the affine filler of magnetism according to claim 8 or preparation method according to claim 9 are prepared
The affine filler of magnetism be used to isolate and purify the purposes of Antithrombin III.
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