CN105170110A - Magnetic composite nanoparticle and preparation method thereof - Google Patents
Magnetic composite nanoparticle and preparation method thereof Download PDFInfo
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- CN105170110A CN105170110A CN201510254667.9A CN201510254667A CN105170110A CN 105170110 A CN105170110 A CN 105170110A CN 201510254667 A CN201510254667 A CN 201510254667A CN 105170110 A CN105170110 A CN 105170110A
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Abstract
The invention relates to a magnetic composite nanoparticle with selective adsorption to antibodies. The structural formula of the magnetic composite nanoparticle is shown as the specification. The method includes: taking an Fe3O4 nanoparticle with superparamagnetism as the core, using tetraethyl orthosilicate as the silicon source, and employing SiO2 or a polymer to coat the Fe3O4 magnetic nanoparticle, thus obtaining a core-shell structure Fe3O4SiO2 or Fe3O4 polymer magnetic composite particle; and bonding a silane coupling agent mercaptoethyl trimethoxy silane to the silica gel surface of the magnetic composite particle, and then carrying out mercapto-ene click reaction on product and double bond-containing vinylpyridine (MEP), thus obtaining the Fe3O4SiO2-S-MEP or Fe3O4 polymer-S-MEP magnetic composite nanoparticle adsorbent. The Fe3O4SiO2-S-MEP or Fe3O4 polymer-S-MEP magnetic composite nanoparticle is a novel bio-functional magnetic composite nano-material with selective adsorption to antibodies. The material has very good superparamagnetism, and can rapidly realize solid-liquid separation under the effect of an external magnetic field, also shows specificity to antibody adsorption, and can be used for rapid separation and preparation of antibodies.
Description
Technical field
The present invention relates to a kind of magnetic composite nanoparticles and preparation method thereof, relating to a kind of is specifically aglucon with vinylpyridine, antagonist have selective absorption composite magnetic nano particle and preparation method thereof.
Background technology
Antibody engineering technology is gradual perfection along with modern biotechnology development, has become the main force of biotechnology industry, especially in biotechnological pharmaceutics field, has occupied critical role.Antibody drug is with the curative effect of its high degree of specificity, and little to human body toxic and side effect, oneself is widely used in clinic diagnosis, is mainly used in improving body immunity and treatment malignant tumour etc.Because purposes is different, also there is very large difference in the source of antibody, as the blood plasma of antibody sources in Healthy People for improving body immunity; The antibody for the treatment of malignant tumour is then humanized monoclonal antibody, derives from hybridoma.The antibody being which kind of source all will through strict security and efficacy examination before being applied to; Purity as the application model also antagonist of ejection preparation is had higher requirement, and efficient separation purifying technique is the guarantee of antibody mass, is also the key link determining production cost.
The affinity chromatography that what applying frequency was more in antibody purification process is is aglucon with albumin A and Protein G.Albumin A and Protein G are the cell wall proteins of staphylococcus aureus, can with the Fc segment specific binding of IgG antibody-like.Because its antagonist has very high selective and be specifically designed to the separation and purification of antibody, be widely applied in the preparation of industrialized antibody (USPatent4230685).There are some defects in the method, as protein A affinity chromatography medium expensive, cost is high; Service life is short, lack effective cleaning method; Its loss of biological activity is easily caused in acidic elution condition and Reusability; Because aglucon belongs to protein, be easily stored in the protease hydrolytic in material liquid, thus reduce separating effect, and coming off of aglucon easily causes the pollution etc. of antibody product.These weak points limit its application in extensive antibody purification field.And the shortcoming that a-protein aglucon has, protein G and Protein L have equally, and the cost ratio a-protein of protein G also wants expensive in addition, is faced with the problem of large-scale application difficulty equally.
The affine method of immunity (IAC) is combined with carrier using antigen (or antibody) as part, composition Stationary liquid, for separating of the antibody (or antigen) of energy specific bond in sample.Because the specificity of the combination of antigen and antibody is very strong, immune affine method is the very effective method of separation antibody, and purity is very high.But antigen is quite expensive, service life is short, therefore is not suitable for commercial scale and produces in a large number.
Ion-exchange chromatography is used to the separation and purification of antibody as a kind of the most frequently used chromatographic technique.This chromatography media carries out under lower ionic strength (20-50mM buffer solution) the adsorption entails of protein under normal circumstances.Therefore, feed liquid generally needs desalination in advance or dilution, can increase operating procedure and time like this, affect production capacity.Because immunoglobulin (Ig) is hydrophobic mostly, and the hydrophobicity of different antibody is also different, so HC (HIC) is also used to the separation and purification of immunoglobulin (Ig).Because HIC uses the ammonium sulfate of high concentration as mobile phase, therefore, the sample after separation just can carry out next step lock out operation after must carrying out desalination.This not only can increase production cost, and the salt added likely can destroy the stability of target protein, the separating step of interfere with subsequent.But usually only adopt ion-exchange or HC to be difficult to single step purification and obtain highly purified antibody protein.
In order to solve the problems referred to above that a-protein Human serum protein medium exists, the smaller ligand adopting antagonist to have special affinity interaction replaces expensive a-protein affinity ligand.As will be the separation and purification of close sulphur chromatogram (Porathetal.FEBSLetters, 1985,185:306) for antibody of aglucon containing sulfuryl and mercaptoethanol.Dewatering electric charge inducing color chromatogram (hydrophobicchargeinductionchromatography, HCIC) be a kind of new technology (TongHF of separation and purification antibody, LinDQ, GaoD, etal.JChromatogrA, 2013,1285:88-96), HCIC, mainly through increasing the bond densities of hydrophobic grouping, selects suitable ionization aglucon simultaneously, making its neutral when higher pH, making albumen only rely on hydrophobic interaction when adsorbing.By regulating eluent pH value during desorption, make electric charge identical with protein surface on Stationary liquid surface band, thus rely on electrostatic repulsion to be got off by Protein elution.United States Patent (USP) (USPatent5,652,348; 5,719,269; 7,144,743) report and utilize heterocyclic ligand hydrophobic interaction novel separating medium carrying out adsorbing separation carrying out antibody and preparation method thereof.The chromatography media being wherein aglucon with mercaptoethyl pyridine has had PallBiosepara company merchandized handling.Chinese patent (CN101284224) report a kind of with mercaptopyridine and sulfuryl be aglucon, with the Expanded Bed Adsorption medium of cellulose/inorganic weighting agent complex microsphere separation antibody that is matrix and preparation method.Chinese patent (CN201210202165.8) reports one with hydrophilic gel microballoon for matrix, activated substrate is made in s-triazine activator, then two smaller ligand such as bonding 4-(2-hydroxyethyl)-pyridine, for separating of expansion bed separating medium and the preparation method of antibody.
Magnetic Nano material, as a kind of new function composite, has the features such as high adsorption capacity, modified surface, easily separated and good biocompatibility, has been widely used in the pretreatment process of the complex system such as biology, environment sample because of it.Relative to traditional chromatographic separation technology, can promptly carry out under the effect of magnetic composite microsphere outside magnetic field being separated, recovery and reuse.
In recent years, magnetic separation technique shows good advantage in protein separation.The magnetic responses of this adsorber particles can reach the object of depositing selective enrichment and separate targets thing in case at other SSs, direct separation and purification target protein from biological Raw samples is made to become possibility, such as, zymotic fluid, cell suspending liquid, blood plasma, milk, whey and plant extraction liquid etc.The application of magnetic separation technique obviates the loaded down with trivial details processing procedure of chromatography packed column, such as centrifugal, filtration, matrix separation etc., and, magnetic separation technique has its unique advantage, such as fast, gentle, applied widely, easy operation, can realize other technologies and is difficult to realize or irrealizable separation etc.
The Mercapto-Ethyl pyridine (MEP) antagonist with selective adsorption is bonded to the surface of magnetic composite microsphere as aglucon, the selective adsorption of the superparamagnetism of magnetic nano-particle and dewatering electric charge inducing color chromatogram aglucon MEP antagonist can be combined, preparing the magnetic composite nano material that a kind of antagonist has the new bio functionalization of selective absorption, is a kind of dewatering electric charge induction magnetic adsorbent.This magnetic composite microsphere contains an ionizable pyridine ring and a hydrophobic tail containing element sulphur, its pKa is 5.8, time under common physiological condition as pH7-8, pyridine ring is not charged, and MEP is combined by gentle hydrophobic interaction with antibody, after pH reduces, pyridine ring becomes positively charged lotus, now antibody also brings identical positive charge lower than its isoelectric point due to pH, and when electrostatic repulsion serious offense hydrophobic effect, antibody just elutes from medium.The existing good superparamagnetism of this magnetic composite microsphere, shows again antagonist absorption very high compatibility and selective, can be used for quick separating and the preparation of antibody.
Summary of the invention
Involved in the present invention take vinylpyridine as aglucon, and antagonist has the magnetic composite nanoparticles of selective absorption: with magnetic composite nanoparticles Fe
3o
4@SiO
2or Fe
3o
4@polymer is matrix, and aglucon is through the vinylpyridine of silane coupler mercapto ethyl trimethoxy silane by sulfydryl-alkene click-reaction coupling, and the structure of the magnetic composite nanoparticles of this experiment preparation consists of:
Aglucon is vinylpyridine, comprises 4-vinylpridine, 3-vinylpyridine, 2-vinylpyridine.
Described a kind of magnetic composite nanoparticles is a kind of is aglucon with vinylpyridine, and antagonist has the magnetic composite nanoparticles of selective absorption, and described magnetic composite nanoparticles is to have the Fe of superparamagnetism
3o
4nano particle is core, has coated SiO
2or polymer is the nucleocapsid structure of shell, Fe
3o
4@SiO
2or Fe
3o
4the particle diameter of@polymer magnetic composite nanoparticle is 250 ~ 1000nm, and described vinylpyridine comprises 4-vinylpridine, 3-vinylpyridine, 2-vinylpyridine.
Described a kind of magnetic composite nanoparticles, has the character of dewatering electric charge induction solid-phase adsorbent, is a kind of dewatering electric charge induction magnetic adsorbent.Containing an ionizable pyridine ring and a hydrophobic tail containing element sulphur after ligand cou, time under common physiological condition as pH7-8, pyridine ring is not charged, MEP is combined by gentle hydrophobic interaction with antibody, after pH reduces, pyridine ring becomes positively charged lotus, and now antibody also brings identical positive charge lower than its isoelectric point due to pH, when electrostatic repulsion serious offense hydrophobic effect, antibody just elutes from medium.
Described a kind of magnetic composite nanoparticles, when carrying out specific adsorption, by the sample containing specific antibody, as blood, serum, blood plasma, ascites, cell culture fluid, emulsion, in the biological raw materials such as the monoclonal antibody that bioengineering is expressed or yolk, immunoglobulin (Ig) crude extract etc. mix according to a certain percentage with magnetic composite microsphere prepared by this method, absorption one hour of vibrating under room temperature is in case deposited at cushioning liquid, after specific antibody is combined with corresponding immunoglobulin (Ig), antibody and the foreign protein of non-specific binding is washed respectively off with cleaning fluid and eluent, finally adopt the immunoglobulin (Ig) of specific elution specific adsorption, obtain specific IgG or IgY.
1, take vinylpyridine as aglucon, antagonist have selective absorption the preparation method of magnetic composite microsphere:
(1) Fe
3o
4the preparation of nano particle
First hydro-thermal method is adopted, with FeCl
36H
2o is raw material, adds 4.0g polyethylene glycol PEG1000 and 14.4g sodium acetate, is transferred in the stainless steel cauldron of 50mLTeflon-lined after stirring and dissolving, to be packagedly put in temperature programmed control case 200 DEG C of reaction 12h, prepares size uniform and the Fe of favorable dispersibility
3o
4magnetic Nano magnetic fluid.Respectively with magnetic fluid prepared by the cyclic washing such as ethanol, water after cooling.Prepare size uniform and the Fe of favorable dispersibility
3o
4magnetic Nano material.Then dry 8h in the vacuum drying chamber of 60 DEG C is put in.Finally, by dried Fe
3o
4magnetic nanoparticle is scattered in absolute ethyl alcohol, gathers to prevent its magnetic.
(2) Fe
3o
4@SiO
2the preparation of magnetic composite microsphere
By the Fe of preparation in (1)
3o
4magnetic Nano material is core, take TEOS as the Fe of the nucleocapsid structure that silicon source adopts sol-gal process preparation size controlled
3o
4@SiO
2magnetic composite microsphere.Its preparation process is as follows: get the magnetic fluid of ethanol in proper amount dispersion (containing 700mgFe
3o
4), spend deionized water three times after Magneto separate to remove ethanol, then add the HCl solution 50mL of 0.1mol/L, ultrasonic 30min, with middle conjunction electric charge, subsequently, spend from water washing for several times, then proceed in the three-necked bottle of the cleaning of 500mL, add 4:1 absolute ethyl alcohol and deionized water, ultrasonic 10min, opens mechanical agitation, slowly adds 5mL28%NH
3h
2o, 30 DEG C are stirred 15min, more slowly add 4mLTEOS, after dropwising at 30 DEG C stirring reaction 8h.After reaction terminates, with absolute ethanol washing six times, Magneto separate, finally, by the product of gained vacuum drying 8h at 60 DEG C, for subsequent use, as shown in Figure 1.
(3) take vinylpyridine as the Fe of aglucon
3o
4@SiO
2the preparation of-S-MEP magnetic composite microsphere
With Fe
3o
4@SiO
2magnetic composite microsphere is matrix, weighs appropriate magnetic composite microsphere, adds solvent dry toluene, then adds appropriate 4-mercapto ethyl trimethoxy silane stirring and refluxing reaction 24h, the obtained Fe containing sulfydryl
3o
4@SiO
2magnetic composite microsphere.To the Fe containing sulfydryl after cleaning-drying
3o
4@SiO
2add dry toluene in magnetic composite microsphere, with azodiisobutyronitrile (AIBN) for initator, and slowly add appropriate 4-vinylpridine stirring reaction 30h, cleaning-drying obtains Fe
3o
4@SiO
2-S-MEP magnetic composite microsphere, as shown in Figure 5.
Fe prepared by the present invention
3o
4@SiO
2-S-MEP magnetic composite nanoparticles is a kind of dewatering electric charge induction magnetic adsorbent, be the aglucon MEP antagonist of the superparamagnetic of magnetic nano-particle and dewatering electric charge inducing color chromatogram is had specific absorption property combine, a kind of antagonist of preparation has the magnetic composite nano material of the new bio functionalization of selective absorption.The existing good superparamagnetism of this material, shows again the specificity of antagonist absorption, can be used for quick separating and the preparation of antibody.
Beneficial effect of the present invention is embodied in:
1, in prior art, downstream produces the problem of immunoglobulin (Ig) operation cycle length, provides the magnetic substrate with specific adsorption, achieves quick separating.
2, in producing for downstream, the problem of Feedstock treating complexity, the needed raw material of this method single step purification is only simple centrifugal segregation fatty product, does not need to saltout except foreign protein.
3, produce the problem of albumin A affinity chromatography purifying immunoglobulin used for downstream, provide simple, economy, fast isolation and purification method.
4, produce the problem of the antibody purification easy in inactivation faced for downstream, this method adopts the agent of dewatering electric charge induced adsorption, provides a kind of antibody purification process of gentleness.
Accompanying drawing explanation
Fig. 1 is Fe
3o
4@SiO
2the preparation of magnetic composite microsphere
Fig. 2 is Fe
3o
4magnetic nano-particle SEM and Fe
3o
4@SiO
2magnetic composite microsphere and TEM figure
Fig. 3 is Fe
3o
4@SiO
2-S magnetic composite microsphere synthetic route chart
Fig. 4 is Fe
3o
4@SiO
2grafting MEP magnetic composite microsphere synthetic route chart
Fig. 5 is Fe
3o
4@SiO
2-S-MEP magnetic composite microsphere synthetic route chart
Fig. 6 is Fe
3o
4, Fe
3o
4@SiO
2, Fe
3o
4@SiO
2the hysteresis curve (298K) of-S-MEP particle
Fig. 7 is application Fe of the present invention
3o
4@SiO
2-S-MEP magnetic composite microsphere is to the Static Adsorption thermoisopleth of γ-gamma globulin and HSA
Fig. 8 is application Fe of the present invention
3o
4@SiO
2the comparison of-S-MEP magnetic coupling silicon ball absorption situation under different salt concentration conditions to γ-gamma globulin and HSA
Fig. 9 is application Fe of the present invention
3o
4@SiO
2the electrophoretogram of immunoglobulin (Ig) in-S-MEP magnetic composite microsphere separation and purification human serum, Lane1: standard I gG; Lane2: standard HSA; Lane3: serum former state; Lanes4-7: eluent (pH is respectively 6,5.5,5,4)
Figure 10 applies Fe of the present invention
3o
4@SiO
2immunoglobulin (Ig) electrophoretogram in-S-MEP magnetic composite microsphere separation and purification chicken with yolk, Lane1: egg yolk former state; Lanes2-4: eluent (pH is respectively 6,5.5,5); Lanes5-7: eluent (pH is respectively 4,3,2)
Detailed description of the invention
Below by way of example, the invention will be further described:
Embodiment 1:
(1) Fe
3o
4the preparation of nano particle
Hydro-thermal method is adopted to prepare size uniform and the Fe of favorable dispersibility
3o
4magnetic Nano material.It is roughly under process: in the three-necked bottle of dried and clean, add appropriate FeCl
36H
2o, then measure ethylene glycol and add as solvent in three-necked bottle, mechanical agitation, until when solution is yellow transparent, adds 4.0gPEG1000 wherein, 14.4g anhydrous sodium acetate, and mechanical agitation makes it to dissolve.Again above-mentioned solution is transferred in the stainless steel cauldron of Teflon-lined of 6 50mL, is packagedly put in temperature programmed control case 200 DEG C of reaction 12h, naturally cool to room temperature.The magnetic fluid of gained after cooling is poured out, with ethanol washing several (Magneto separate) after Magneto separate, use three water washings for several times (Magneto separate) again, finally again with absolute ethanol washing several, after Magneto separate, be put in dry 8h in the vacuum drying chamber of 60 DEG C.Finally, by dried Fe
3o
4magnetic nanoparticle is scattered in absolute ethyl alcohol, gathers to prevent its magnetic.
(2) Fe
3o
4@SiO
2the preparation of magnetic composite microsphere
By the Fe of preparation in (1)
3o
4magnetic Nano material is core, take TEOS as the Fe of the nucleocapsid structure that silicon source adopts sol-gal process preparation size controlled
3o
4@SiO
2magnetic composite microsphere, its preparation process is as follows: get the magnetic fluid of ethanol in proper amount dispersion (containing 700mgFe
3o
4), spend deionized water three times after Magneto separate to remove ethanol, then add the HCl solution 50mL of 0.1mol/L, ultrasonic 30min, with middle conjunction electric charge, subsequently, spend from water washing for several times, then proceed in the three-necked bottle of the cleaning of 500mL, add 4:1 absolute ethyl alcohol and deionized water, ultrasonic 10min, opens mechanical agitation, slowly adds 5mL28%NH
3h
2o, 30 DEG C are stirred 15min, more slowly add 4mLTEOS, after dropwising at 30 DEG C stirring reaction 8h.After reaction terminates, with absolute ethanol washing six times, Magneto separate, finally, by the product of gained vacuum drying 8h at 60 DEG C, for subsequent use.
By above-mentioned experimental technique, the Fe of the controlled nucleocapsid structure of size can be carried out
3o
4@SiO
2the preparation of magnetic composite microsphere, the size tunable of this composite nano-microsphere is built in 50-1000nm.This invents the Fe adopted
3o
4nano particle diameter is 200nm, Fe
3o
4@SiO
2nano particle diameter is as 300-400nm, and building-up process and particle diameter test result are as depicted in figs. 1 and 2.
(3) take 4-vinylpyridine as the preparation of adsorbing medium of ligand separation antibody
With Fe
3o
4@SiO
2magnetic composite microsphere is matrix, and selecting the magnetic nano-particle of optimum grain-diameter and carry out modification (magnetic composite microsphere of about 200nm is selected in this practice) to it, take MEP as the Fe that aglucon prepares that antagonist has specific adsorption effect
3o
4@SiO
2-S-MEP magnetic composite microsphere, preparation process is as follows: first weigh appropriate silicone dioxide magnetic microsphere in the 100mL three-necked bottle of a drying, add 50mL dry toluene wherein, add 0.1g4-mercapto ethyl trimethoxy silane wherein, be warming up to 90 DEG C, under return stirring, react 24h.After completion of the reaction, use methyl alcohol and water washing three times respectively, be then put in dry in 50 DEG C of baking ovens stand-by.Then be put in a 100mL three-necked bottle by above-mentioned product, add 50mL dry toluene wherein, 0.01g azodiisobutyronitrile (AIBN) also slowly adds 1.0mL4-vinylpyridine.Be warming up to 60 DEG C, under agitation react 30h.After reaction terminates, use water and methanol wash respectively for several times, be then put in 50 DEG C of vacuum drying chambers and dry.Retained product is stand-by.
Fe prepared by the present invention
3o
4@SiO
2-S-MEP magnetic composite microsphere is for form by the coupling of 4-mercapto ethyl trimethoxy silane, introduce-S-atom, by sulfydryl-alkene click-reaction, 4-vinylpridine is fixed on magnetic substrate, under the condition that the present invention applies, sulphur atom and vinylpyridine are by interacting, can obtain the magnetic composite microsphere of contraposition coupling, result as shown in Figure 5.
Embodiment 2:
The preparation of the adsorbing medium being ligand separation antibody with 3-vinylpyridine
With Fe
3o
4@SiO
2magnetic composite microsphere is matrix, and selecting the magnetic nano-particle of optimum grain-diameter and carry out modification (selecting the magnetic composite microsphere of about 200nm) to it, take MEP as the Fe that aglucon prepares that antagonist has specific adsorption effect
3o
4@SiO
2-MEP magnetic composite microsphere, preparation process is as follows: first weigh appropriate silicone dioxide magnetic microsphere in the 100mL three-necked bottle of a drying, add 50mL dry toluene wherein, add 0.1g4-mercapto ethyl trimethoxy silane wherein, be warming up to 90 DEG C, under return stirring, react 24h.After completion of the reaction, use methyl alcohol and water washing three times respectively, be then put in dry in 50 DEG C of baking ovens stand-by.Then be put in a 100mL three-necked bottle by above-mentioned product, add 50mL dry toluene wherein, 0.01g azodiisobutyronitrile (AIBN) also slowly adds 1.0mL3-vinylpyridine.Be warming up to 60 DEG C, under agitation react 30h.After reaction terminates, use water and methanol wash respectively for several times, be then put in 50 DEG C of vacuum drying chambers and dry.Retained product is stand-by.
Between the Fe of position
3o
4@SiO
2-S-MEP magnetic composite microsphere is for form by the coupling of 4-mercapto ethyl trimethoxy silane, introduce-S-atom, by sulfydryl-alkene click-reaction, 3-vinylpyridine is fixed on magnetic substrate, under applied condition, sulphur atom and 3-vinylpyridine interact, and obtain a magnetic composite microsphere for digit pair connection.
Embodiment 3
The preparation of the adsorbing medium being ligand separation antibody with 2-vinylpyridine
With Fe
3o
4@SiO
2magnetic composite microsphere is matrix, and selecting the magnetic nano-particle of optimum grain-diameter and carry out modification (selecting the magnetic composite microsphere of about 200nm) to it, take MEP as the Fe that aglucon prepares that antagonist has specific adsorption effect
3o
4@SiO
2-MEP magnetic composite microsphere, preparation process is as follows: first weigh appropriate silicone dioxide magnetic microsphere in the 100mL three-necked bottle of a drying, add 50mL dry toluene wherein, add 0.1g4-mercapto ethyl trimethoxy silane wherein, be warming up to 90 DEG C, under return stirring, react 24h.After completion of the reaction, use methyl alcohol and water washing three times respectively, be then put in dry in 50 DEG C of baking ovens stand-by.Then be put in a 100mL three-necked bottle by above-mentioned product, add 50mL dry toluene wherein, 0.01g azodiisobutyronitrile (AIBN) also slowly adds 1.0mL2-vinylpyridine.Be warming up to 60 DEG C, under agitation react 30h.After reaction terminates, use water and methanol wash respectively for several times, be then put in 50 DEG C of vacuum drying chambers and dry.Retained product is stand-by.
The Fe at ortho position
3o
4@SiO
2-S-MEP magnetic composite microsphere is for form by the coupling of 4-mercapto ethyl trimethoxy silane, introduce-S-atom, by sulfydryl-alkene click-reaction, 2-vinylpyridine is fixed on magnetic substrate, under applied condition, sulphur atom and MEP are by interacting, and obtain the magnetic composite microsphere of ortho position coupling.
Embodiment 4:
To Fe prepared by application the present invention
3o
4@SiO
2-S-MEP magnetic composite microsphere has carried out magnetic property test.In the present invention, our homogeneous Fe that adopted sol-gal process to prepare
3o
4nano particle, and it is carried out coated, prepare Fe
3o
4@SiO
2microballoon and Fe
3o
4@SiO
2-S-MEP magnetic composite microsphere.Its nanometer character is tested under 298K respectively by superconductive quantum interference magnetic measurement system (SQUID), and Fig. 6 is Fe
3o
4, Fe
3o
4@SiO
2microballoon and Fe
3o
4@SiO
2the hysteresis curve of-S-MEP magnetic composite microsphere, the external magnetic field of applying is from-25000Oe to 25000Oe.Sample is under the effect of externally-applied magnetic field, and intensity of magnetization when reaching capacity reduces gradually along with the increase of coated thickness, Fe
3o
4the intensity of magnetization for being about 66.48emu/g, Fe
3o
4@SiO
2the intensity of magnetization be about about 61.25emu/g, and Fe
3o
4@SiO
2the intensity of magnetization of@MEP is about 58.13emu/g.
Fe prepared by application the present invention
3o
4@SiO
2-S-MEP magnetic composite microsphere is under the effect of externally-applied magnetic field, realize quick separating, this process can complete within one minute, illustrated that the complex microsphere that we synthesize has good superparamagnetism, can by the method for Magnetic Isolation by magnetic-particle and solution quick separating.
Embodiment 5
Apply Fe of the present invention
3o
4@SiO
2-S-MEP magnetic composite microsphere is to the Static Adsorption thermoisopleth of γ-gamma globulin and HSA
Adsorptive selectivity evaluates the indispensable factor of performance of the adsorbent.In serum, there is a variety of albumen.It mainly comprises three kinds: albumin, globulin and fibrinogen, because fibrinogen content is less and laboratory condition is limited, does not study in the method.We are to the major protein existed in serum, and immunoglobulin (Ig) and albuminous absorption property are studied, and have inquired into Fe of the present invention
3o
4@SiO
2the adsorption selectivity of the antagonist of-S-MEP magnetic composite microsphere.
First, the Fe of two parts of bonding MEP is accurately taken
3o
4@SiO
2-S-MEP magnetic composite microsphere 10mg, is put in respectively by it in two 5mL centrifuge tubes, draws 200 μ L γ-gamma globulin and HSA standard liquid (10mg/mL) is injected in two centrifuge tubes respectively, then uses PBS (20mmol/LKH
2pO
4, pH is 7.4,0.5mol/LNaCl) and solution dilution is to 4mL.At 25 DEG C, slowly shake absorption, and carry out Magneto separate every 30min and draw a certain amount of supernatant to carry out protein content mensuration at 595nm place.
As can be seen from experimental result: MEP magnetic adsorbent can specific adsorbed target albumen γ-gamma globulin, and does not substantially adsorb foreign protein HSA.Under normal circumstances, HCIC aglucon mainly comprises hydrophobic forces, specific effect power, Coulomb repulsion, Van der Waals force and Hyarogen-bonding etc. to the active force of target protein, with optimal conditions, MEP is very weak to the hydrophobic forces of HSA, substantially do not adsorbed, IgG is due to its strong hydrophobic forces and specific adhesion, and under adsorption conditions, active force is still very strong, can be adsorbed in a large number.Thus, with optimal conditions, the magnetic substrate of this experiment preparation can realize the object of specific target acquisition albumen in the serum of simulation, and result as shown in Figure 7.
Embodiment 6
Apply Fe of the present invention
3o
4@SiO
2the comparison of-S-MEP magnetic coupling silicon ball absorption situation under different salt concentration conditions to γ-gamma globulin and HSA
When protein retains in desirable dewatering electric charge induced adsorption agent, should be only relevant with the hydrophobic forces between protein and adsorbent, but actually, often there is departing from various degree, this abnormal behaviour may be that the character having ion-exchange due to the agent of dewatering electric charge induced adsorption causes, the agent of dewatering electric charge induced adsorption also has faint Van der Waals force except hydrophobic effect, the existence such as hydrogen bond and electrostatic force, although HCIC has salt-independent, but in order to avoid these active forces are on the impact of hydrophobic forces, when this measuring NaCl concentration is from 0.5moL/L to 3.0mol/L, salinity is on the impact of adsorption equilibrium.
Take the Fe of four parts of 10mg bonding MEP respectively
3o
4@SiO
2@MEP magnetic composite microsphere, and be put in four 2mL centrifuge tubes respectively, then, respectively to adding 100 μ L γ-gamma globulin standard liquid (10mg/mL) in four centrifuge tubes, the concentration of NaCl in the phosphate buffer of 20mmol/LpH=7.4 is adjusted to 0.5mol/L respectively, 1.0mol/L, 2.0mol/L, 3.0mol/L, the PBS solution adding different N aCl concentration is respectively divided in four centrifuge tubes to 2mL, at being placed in shaking table 25 DEG C, Slow Isothermal shakes 3.5h, then Magneto separate is carried out, leave and take each absorption supernatant in 4 DEG C of Refrigerator stores, reference liquid is nonprotein plain buffer, under 595nm, the absorbance after each system adsorbed proteins is measured with ultraviolet-spectrophotometer, same γ-the gamma globulin of method of testing of HSA.
As can be seen from accompanying drawing 8, when NaCl concentration fades to 3.0mol/L from 0.5mol/L, the adsorbance of γ-gamma globulin and HSA also increases thereupon, γ-gamma globulin changes and not obvious in 0.5mol/L to 3.0mol/L scope, and increasing degree is little, but when salinity increases, the adsorbance of HSA obviously increases, when concentration is increased to 3.0mol/L, adsorbance almost reaches 39.8 μ g/mg, and it may the mechanism of action be: due to novel medium Fe
3o
4@SiO
2matrix is nano-scale magnetic microballoon, and make it have high aglucon and modify density, like this, namely the aglucon of dewatering electric charge induced adsorption agent and target protein realize larger adsorption capacity by hydrophobic effect and specific effect under low salt concn; And for foreign protein HSA due to its weak hydrophobicity and nonspecific action, aglucon does not adsorb substantially to it, when salinity continues to increase, there is hydrophobic effect between the aglucon of dewatering electric charge induction character and albumen also strengthen gradually, until reach maximal absorptive capacity, and after salinity continues increase, the hydrophobic grouping of albumen also comes out gradually, hydrophobic forces between albumen and aglucon is also strengthened gradually, and dissociation constant also decreases.As can be seen from experimental result, have the target protein of strong-hydrophobicity and specific adsorption effect, salinity change is little on its impact, and this is consistent with the salt-independent characteristic of dewatering electric charge induced adsorption agent to a certain extent.
Embodiment 7
With the Fe that mercaptoethyl pyridine is aglucon
3o
4@SiO
2-S-MEP magnetic composite microsphere is to the selective absorption of Immunoglobulin in Serum (IgG)
Accurately take appropriate Fe
3o
4@SiO
2-S-MEP magnetic composite microsphere, adds appropriate serum, then is diluted to 7mL with equilibrium liquid.Then be put in shaking table, at room temperature slowly 1h is adsorbed in concussion.Then carry out Magneto separate, retain supernatant in 4 DEG C of refrigerators; Then ultrasonic cleaning 2min is carried out to magnetic silicon ball, Magneto separate with 7mL equilibrium liquid, retain cleaning fluid, be put in 4 DEG C of refrigerators.Then in centrifuge tube, add the eluent of the different pH of 7mL, then place in shaking table and shake wash-out 2h, Magneto separate, retain eluent stand-by in 4 DEG C of refrigerators.Finally, SDS-PAGE polyacrylamide gel electrophoresis is utilized to carry out electrophoretic analysis to the fluid of inclining of the Magneto separate in above steps.
Fig. 9 is Fe
3o
4@SiO
2-S-MEP magnetic composite microsphere to the separation and purification electrophoretogram of IgG in serum, Lane1: standard I gG; Lane2: standard HSA; Lane3: serum former state; Lanes4-7: eluent (pH is respectively 6,5.5,5,4), as can be seen from Figure 8, containing a large amount of foreign protein in serum former state, is mainly HSA, through Fe
3o
4@SiO
2after the absorption of-S-MEP magnetic composite microsphere, the elution through different pH can be found out, this Fe
3o
4@SiO
2-S-MEP magnetic composite microsphere can carry out selective absorption to the IgG in serum, and impurity protein such as HAS etc. is not adsorbed, and adopts Fe of the present invention
3o
4@SiO
2-S-MEP magnetic composite microsphere can realize step separation and concentration and a purifying to the IgG in serum, and purity is greater than 95%.
Embodiment 8
With the Fe that mercaptoethyl pyridine is aglucon
3o
4@SiO
2-MEP magnetic composite microsphere is to the selective absorption of immunoglobulin (Ig) in chicken with yolk (IgY)
Adopt the processing method in example 7, use Fe
3o
4@SiO
2-S-MEP magnetic composite microsphere carries out selective absorption to immunoglobulin (Ig) in chicken with yolk (IgY).Figure 10 is Fe
3o
4@SiO
2-S-MEP magnetic composite microsphere is to the separation and purification electrophoretogram of IgY in ovum gallinaceum.As can be seen from Figure 10, when pH is down to 5, do not have target protein IgY to be washed out, along with pH reduction magnetic microsphere surface and protein surface start with positive charge, hydrophobic interaction weakens gradually and electrostatic repulsion strengthens gradually, is eluted gradually by the albumen adsorbed.After pH is reduced to 4, in eluent, comprise target protein IgY, and foreign protein is little.Target protein IgY a large amount of after pH is down to 4 and adds a small amount of urea is eluted.
Claims (5)
1. a magnetic composite nanoparticles, is characterized in that, described magnetic composite nanoparticles is Fe
3o
4@SiO
2-S-MEP (MEP is vinylpyridine) or Fe
3o
4@polymer-S-MEP, with SiO
2or the magnetic composite nanoparticles Fe of polymer overmold
3o
4@SiO
2or Fe
3o
4@polymer is matrix, and aglucon is that its structure consists of through the vinylpyridine of silane coupler mercapto ethyl trimethoxy silane by sulfydryl-alkene click-reaction coupling:
2. a kind of magnetic composite nanoparticles according to claim 1, it is characterized in that, described a kind of magnetic composite nanoparticles is a kind of is aglucon with vinylpyridine, antagonist has the magnetic composite nanoparticles of selective absorption, and described magnetic composite nanoparticles is to have the Fe of superparamagnetism
3o
4nano particle is core, coated SiO
2or polymer is the nucleocapsid structure of shell, Fe
3o
4@SiO
2or Fe
3o
4@polymer magnetic composite nanoparticle, particle diameter is 50 ~ 1000nm, and described vinylpyridine comprises 4-vinylpridine, 3-vinylpyridine, 2-vinylpyridine.
3. a kind of magnetic composite nanoparticles according to claim 1, it is characterized in that, described magnetic composite nanoparticles has the character of dewatering electric charge induction solid-phase adsorbent, it is a kind of dewatering electric charge induction magnetic adsorbent, aglucon contains an ionizable pyridine ring and a hydrophobic tail containing element sulphur, when pH value is 7-8, pyridine ring is not charged, MEP is combined by gentle hydrophobic interaction with antibody, after pH reduces, pyridine ring becomes positively charged lotus, now antibody also brings identical positive charge lower than its isoelectric point due to pH, when electrostatic repulsion serious offense hydrophobic effect, antibody just elutes from medium.
4. a kind of magnetic composite nanoparticles according to claim 1, it is characterized in that, when carrying out specific adsorption, by the sample containing specific antibody, as blood, serum, blood plasma, ascites, cell culture fluid, emulsion, in the biological raw materials such as the monoclonal antibody that bioengineering is expressed or yolk, immunoglobulin (Ig) crude extract etc. mix with magnetic composite microsphere prepared by this method, absorption one hour of vibrating under room temperature is in case deposited at cushioning liquid, after specific antibody is combined with corresponding immunoglobulin (Ig), antibody and the foreign protein of non-specific binding is washed respectively off with cleaning fluid and eluent, finally adopt the immunoglobulin (Ig) of specific elution specific adsorption, obtain specific IgG or IgY.
5. a preparation method for magnetic composite nanoparticles, is characterized in that, comprises the following steps:
Step one: adopt SiO
2or polymer is to Fe
3o
4magnetic nano-particle carries out coated, preparation Fe
3o
4@SiO
2or Fe
3o
4@polymer magnetic composite nanoparticle; With Fe
3o
4magnetic nano-particle is core, the Fe of the nucleocapsid structure adopting sol-gal process preparation size controlled for silicon source with ethyl orthosilicate TEOS
3o
4@SiO
2magnetic compound particles, or with Fe
3o
4magnetic nano-particle is core, take polymer as shell, preparation Fe
3o
4@polymer magnetic compound particle;
Step 2: Silica Surface silane coupler mercapto ethyl trimethoxy silane being bonded to composite magnetic nano particle, obtains the Fe of surface with sulfydryl
3o
4@SiO
2magnetic composite nanoparticles; With Fe
3o
4@SiO
2composite magnetic particle is matrix, is solvent with toluene, adds 0.1g4-mercapto ethyl trimethoxy silane and stirs, be heated to backflow, and obtained surface is containing the Fe of sulfydryl
3o
4@SiO
2magnetic composite microsphere or surface contain the Fe of sulfydryl
3o
4@polymer magnetic composite nanoparticle;
Step 3: prepare Fe based on sulfydryl-alkene click-reaction
3o
4@SiO
2-S-MEP or Fe
3o
4@polymer-S-MEP magnetic composite nanoparticles; The sulfydryl of composite nanoparticle Surface Creation in toluene with azodiisobutyronitrile (AIBN) for initator, slowly add 1mL4-vinylpyridine stirring reaction 30h, carry out sulfydryl-alkene clicking chemistry with double bond containing vinylpyridine (MEP) to react, prepare the Fe that antagonist has selective absorption
3o
4@SiO
2-S-MEP or Fe
3o
4@polymer-S-MEP magnetic composite nanoparticles adsorbent.
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