A kind of preparation method of core/shell type magnetic particle product and application
Technical field
The invention belongs to magnetic and nano material preparation, relate to a kind of preparation method of core/shell type magnetic particle and the application in bio-separation thereof.
Technical background
Magnetic particle has wide application in biomedicine, cytology and the biotechnology field such as fixing of the separation of nucleic acid, protein and purifying, targeted drug, cell marking and sorting, biomolecular labeling and detection, immunoassay, enzyme.It is to have utilized the controlled characteristic of magnetic particle magnetic, and magnetic is controlled to be meant under the effect of magnetic particle outside magnetic field, moves and enrichment to the direction of appointment.Compare with separation method such as centrifugal process, the precipitation method, ion-exchange and chromatography commonly used; The magnetic of solid-liquid phase separates under the outside magnetic field effect; Have simple and quick, plurality of advantages such as separative efficiency is high, equipment needed thereby is simple, energy consumption is low, cost is few, pollution-free.Magnetic particle is as target medicine carrier, can be implemented in the enrichment of moving, fix a point of body interior orientation, thereby reach the purpose that improves result of treatment, reduces toxic and side effect, and having widely at aspects such as treatment tumour, cancers to have prospect.In animal, clinical testing, succeed in separating cancer cell and normal cell with magnetic particle, represented the application prospect that attracts people's attention.In the existing goods, mostly magnetic particle is the polymer microsphere that makes through investment, monomer copolymerization method or polymer microsphere swelling-take off swelling method etc., or the magnetic microsphere that is coated by polysaccharide molecule.Polymer microsphere is composited by magnetic grain and surface aggregate macromolecular material; The general more complicated of its synthesis technique; Regular meeting uses some poisonous organic solvent or polymerization single polymerization monomers in the synthesis technique; Molded microsphere surface is difficult to modify again, and this microballoon biocompatibility is relatively poor, only is adapted at in-vitro application; Though the microballoon biocompatibility that polysaccharide coats is better, the medicine that can be applied in the body is carried, and coating layer is unstable, the shortcoming that exists magnetic particle to come off easily, and these have all limited its range of application.
The preparation method of shell/caryogram magnetic particle reports a lot, but its kernel is the more weak Fe of magnetic
3O
4On ferrite.For example, patent ZL02109817.4 discloses a kind of preparation method of composite magnetic particle, and its kernel is that particle diameter is the superparamagnetism Fe of 5~8nm
3O
4Nano particle; Patent ZL03111351.6 also discloses magnetic core-shell particle with high-intensity magnetic field responding ability and preparation method thereof, and its kernel is the magnetic ferrites nano particles, and shell is a silicon dioxide.Its magnetic ferrites nano particle is to refer to Fe
3O
4Nano particle, γ-Fe
2O
3Nano particle or mix transition metal and the magnetic ferrites nano particle of compound (particle diameter is 3~12nm).The method for preparing this magnetic ferrites~silicon dixoide nucleocapsid structure particulate is: preparation magnetic ferrites nano particle~coating~organosilicon source, inorganic silicon source coats.Its magnetic ferrites nano particle adopts known coprecipitation preparation.It is with Na that above-mentioned inorganic silicon source coats
2SiO
3As the inorganic silicon source, washed magnetic ferrites nano particle directly is distributed to without any surface treatment contains SiO
2The aqueous solution in, stir and to carry out coated with silica first time, be coated on the thick silicon dioxide layer of magnetic ferrites nanoparticle surface formation 1~2nm through current inorganic silicon source.The organosilicon source is to be the silicon source with the tetraethoxysilane; With the alcohol and water mixture is solvent; Under the catalysis of alkali, carry out the coated with silica second time through the teos hydrolysis reaction, the particle diameter of the multilayer core/shell type magnetic particle that obtains is at 0.2~10 μ m; Nuclear/shell magnetic particle contains 20~1000 magnetic ferrites nano particles, and the quality percentage composition of magnetic ferrites nano particle is 10~60% in the particulate.Utilizing tilt-pour process to carry out pure water under the permanent magnetic field that permanent magnet through 0.05~0.15T in the preparation process provides cleans and removes impurity.The magnetic responsiveness ability of particulate is: the hydrosol of this core-shell particle of 10ml, under the externally-applied magnetic field of 0.05T, the particle enrichment time is in 1 minute.
The Fe of existing patent (ZL03111351.6)
3O
4The coating layer of-silicon dioxide core/shell type magnetic particle is a silicon dioxide; Thereby the existing excellent biological compatibility of particulate; Better chemical stability; Can also carry out various finishinges through different silylating reagents, the microparticle surfaces after modifying can carry wears the various easy active groups that are connected with biomaterial.But the nuclear of this based fine particles more weak ferrite that is magnetic causes the responding ability in this external magnetic field of based fine particles on the weak side, need apply the enrichment that high-intensity external magnetic field could realize this particulate.In addition, also can reach enhancing particulate magnetic response ability, but the specific surface decline that the mean particle dia increase causes can cause surface-active reduction, thereby can directly reduce separative efficiency through the number (causing particulate to increase) that increases nanoparticle in the nuclear.Therefore, need under the condition that keeps particle size appropriateness (preferably being in the nanoscale category), further increase particulate is only the best to the magnetic response ability of externally-applied magnetic field selection.
Summary of the invention
Technical problem to be solved by this invention is to overcome above-mentioned weak point, and research can improve the magnetosensitive sensitivity of particulate, improves the responding ability of particulate to external magnetic field, makes kernel magnetic nano-particle safety with stable.
The present invention provides a kind of goods of core/shell type magnetic particle.
Of the present invention is the core/shell type magnetic particle of nuclear with the metallic nano crystal, and its kernel is the mixture of nano metallic nickel granular material or nano metallic nickel granular material and magnetic oxide nano particle, and shell is a silica.The mixture of said nano metallic nickel granular material or nano metallic nickel granular material and magnetic oxide nano particle refers to Fe, Co, Ni or its alloy, Fe
3O
4, γ-Fe
2O
3, Co
3O
4, Co
2O
3Or Fe
(1-x)M
xFe
2O
4The nano particle of (M=Fe, Co, Ni, Zn, two valency transition metal ionss such as Mn), their particle diameters have superparamagnetism below 10nm.Said reducing agent is meant hydrogen (H
2), hydrazine hydrate (H
4N
2.H
2O), potassium borohydride (KBH
4), sodium borohydride (NaBH
4) or its mixture in any one.Described particulate has coenocytism, and particle diameter sees that through transmission electron microscope the number of the magnetic nanometer particles of kernel in the silica core/shell type magnetic particle is 5~500 at 0.05~5 μ m.
Another object of the present invention provides a kind of preparation method of core/shell type magnetic particle product.
The inventive method is at first coating skim eyed structure silica (shell) outside magnetic metal oxide nano particle (nuclear); To examine whole or in part with reducing agent that in-situ reducing becomes nano metallic nickel granular material then, finally obtain that silica coats, be the core/shell type magnetic particle of kernel with the mixture of nano metallic nickel granular material or nano metallic nickel granular material and few part magnetic oxide nano particle.The result of in-situ reducing not only makes the high-load of magnetisable material in the nuclear be able to keep, and owing to the more weak metal oxide of magnetic in the nuclear has become the stronger metal of magnetic whole or most ofly, has stronger responding ability thereby make magnetic check external magnetic field.Thicken coating through further silica, the compactness and the chemical stability of silica shell are further enhanced, and have strengthened the protection of internal nuclear magnetism metal greatly, and the kernel magnetic metal is able to avoid the influence of extraneous air and the state that is in safety and stability.Through the individual layer for the third time of microparticle surfaces being coated (modification), make the surface possess the required functional group of various application, thereby can adapt to various application demands widely again with the silylating reagent of various functional groups.
Of the present invention is that the technology of preparing of core/shell type magnetic particle of nuclear comprises with the metallic nano crystal: preparation magnetic oxide nano particle, the coating of organosilicon source, electronation, inorganic silicon source coat, with 5 technical processs such as silylating reagent modification of various functional groups.Concrete steps are following:
(1) utilizes the preparation method of patent (200410073680.6) to prepare the magnetic oxide nano particle, be i.e. after the mixed reaction of molysite and aqueous slkali, add entry and stir, accomplish the crystallization reaction of particle; Again crystalline particle is passed through external magnetic field or centrifugation, clean impurity with distilled water, the particle diameter of the magnetic oxide nano particle that makes is at 3~10nm;
(2) the magnetic oxide nano particle after will washing directly is dispersed in the mixed solvent of alcohol and water; Under alkali condition; Carry out the silica coating first time through esters of silicon acis (methyl silicate, tetraethoxysilane, butyl silicate etc.) hydrolysis, the surface of the magnetic oxide nano particle that process organosilicon source coats has the silica of meso-hole structure to coat thin layer, and the thickness of coating layer can be regulated through regulating and control organosilyl amount; Through external magnetic field or centrifugation, clean impurity with distilled water;
(3) the magnetic oxide Nanoparticles Hydrosol that will accomplish silica coating for the first time is with hydrogen (H
2), hydrazine hydrate (H
4N
2.H
2O), potassium borohydride (KBH
4), sodium borohydride (NaBH
4) or its mixture in any one reducing agent reduce; Because the silicon oxide layer on surface has meso-hole structure, reducing agent sees through the silica mesh and gets into kernel, and the magnetic oxide nano particle is under the effect of reducing agent; The all or part of magnetic metal simple substance nano particle that is reduced into; Obtain coating for the first time with silica, metallic nano crystal is the core/shell type magnetic compound particles of nuclear, through external magnetic field or centrifugation, cleans impurity with distilled water;
(4) will accomplish the magnetic compound particles that silica coats with reduction reaction for the first time and be dispersed in SiO
2Water-soluble glue in stir; With 0.5mol/L acetic acid, hydrochloric acid, sulfuric acid; Regulator solution pH value to 6~10 mixed solution 2~20 hours, and the silica once more of accomplishing magnetic compound particles coats; The silica shell that makes the core/shell type magnetic particle is enough fine and close abundant, can secluding air with the outer medium of nuclear, avoid metal nanoparticle by the oxidation of the institute of the oxidizing substance in air or the solution; Through external magnetic field or centrifugation, clean impurity with distilled water; Obtaining particle diameter is the core/shell type magnetic particle that the silica of nuclear coats with the metallic nano crystal at 0.05~5 mu m range.
(5) step (4) core/shell type magnetic particle carries out finishing with the silylating reagent that has groups such as amino, sulfydryl, epoxy radicals, ester group, alkylene, obtains the silica that the surface has different functional groups and coats, is the core/shell type magnetic particle of nuclear with the metallic nano crystal.
Wherein said nano metallic nickel granular material is meant nanometer Fe particle, nano Co particle, nanometer Ni particle or Nanoalloy particle, and the magnetic oxide nano particle is meant nanometer Fe
3O
4Particle, nanometer γ-Fe
2O
3Particle, nano Co
3O
4Particle, nano Co
2O
3Or Fe
(1-x)M
xFe
2O
4Particle etc.
In the above-mentioned cladding process; When the said organosilicon of step (2) source coats; The concentration that is dispersed in magnetic oxide nano particle in the mixed solvent of alcohol and water is 0.01~0.15mol/L; Organosilyl concentration is 0.01~1.0mol/L, and organosilicon can be methyl silicate, tetraethoxysilane or butyl silicate; Alcohol in the mixed solvent of alcohol and water can be methyl alcohol, ethanol, ethylene glycol, propyl alcohol, propane diols, glycerine, isopropyl alcohol or butanols, the pure volumn concentration 40%~98% in the mixed solvent, the volumn concentration 2%~60% of water; In above-mentioned solution, add mass percent concentration and be 25%~28% concentrated ammonia liquor, the concentration of ammoniacal liquor in reaction system is 0.2~1.5mol/L, then stirring reaction 2~20 hours under the speed of 100~2000rpm.In the organosilicon source coated, organosilyl adding can be dropwise to add, and also can disposablely add, and can also add in batches, and the time interval of adjacent twice adding is 1~5 hour in batches, after organosilicon all adds, was continuing reaction 3~20 hours.Through controlling organosilyl addition, can regulate the thickness of the silicon oxide layer that coats for the first time.
Step (3) is said when carrying out electronation, and the mol ratio of the consumption of reducing agent and magnetic oxide nano particle is 0.1~5, and the reduction reaction time is 0.5~10 hour.When the inorganic silicon source coats, SiO
2Concentration be 0.05~3mol/L, be adjusted between 6~10 with the pH value of 0.5mol/L hydrochloric acid reaction solution, mixing speed is to react 2~20 hours under the condition of 100~2000rpm.The modification of silylating reagent can also can be carried out in above-mentioned alcohol in the aqueous solution, and the volume percent content of silylating reagent is 0.1%~10%.
The good dispersion of core-shell type magnetic particulate in water of silica double-coated described in the invention, inner core materials magnetic is strong; The silica shell structurre is fine and close, so chemical stability is high, and oxidation resistance is strong; Do not have exposed magnetic particle, good leak integrity is arranged.
Another purpose of the present invention provides the application of a kind of core/shell type magnetic particle product in bio-separation.
The core/shell type magnetic particle of the present invention preparation joined in the solution that contains nucleic acid mix; Combine liquid through adding; Under the outside magnetic field, isolate nucleic acid-magnetic particle compound, using volume fraction again is that 70% ethanol cleans nucleic acid-magnetic particle compound; The last pH value of using again is that 7~9 TE buffer solution or aqua sterilisa elute nucleic acid, realizes purpose quick, high efficiency separation nucleic acid.
Utilization has the silane coupler of groups such as amino, sulfydryl, epoxy radicals silicon oxide surface is modified; Obtain having the surface of functional groups such as amino, sulfydryl, epoxy radicals, be prone to be connected with drug molecule with various bioactivators such as protein, antigen, antibody, enzymes.Be specially adapted to separating and purifying of biomaterial and pharmaceutical products, and can be used as the carrier of biomolecule and drug molecule.
Embodiment
Embodiment 1: the preparation of core-shell type magnetic particulate kernel
With 0.1mol/L FeCl
2With 0.2mol/L FeCl
3Mixed with concentrated ammonia liquor, react after 10 minutes, adds 100 times of amounts, 10 ℃ water, stirring is 3 hours under 5000rpm, the crystallization reaction completion of particle; Under 10000rpm,, promptly obtain the Fe that particle diameter is 2~12nm after cleaning with the crystalline particle centrifugation
3O
4Nano particle.Get 2.3 gram Fe
3O
4It is in the mixed solvent of 25%~28% concentrated ammonia liquor that nano particle is dispersed in by 500ml ethanol, 10ml water, 15ml mass percent concentration; At room temperature; Methyl silicate with 500rpm mixing speed dropping 0.1mol/L reacted 12 hours, isolated black solid with the permanent magnet of 0.1T; Solid matter is with distilled water washing 3~5 times, and accomplishing for the first time, silica coats.The magnetic composite that the first time, silica coated is dispersed in the water, at room temperature adds 4 gram NaBH
4Carry out reduction reaction, separate with the permanent magnet of 0.1T, the gained solid fully washs with distilled water, promptly gets magnetic Fe nano particle and Fe
3O
4Nano particle is mixed into the composite material of the silica coating of kernel.
Embodiment 2: the preparation of core-shell type magnetic particulate kernel
With 0.2mol/L FeCl
2With 0.4mol/L FeCl
3Mixed with 2mol/L NaOH, react after 25 minutes, adds 100 times of amounts, 15 ℃ water, stirring is 1 hour under 10000rpm, the crystallization reaction completion of particle; Through applying magnetic field crystalline particle is separated, promptly obtain the Fe that particle diameter is 2~12nm after use distilled water is cleaned
3O
4Nano particle.Get 3.5 gram Fe
3O
4It is in the mixed solvent of 25%~28% concentrated ammonia liquor that nano particle is dispersed in by 1000ml isopropyl alcohol, 20ml water, 40ml mass percent concentration; At room temperature; Tetraethoxysilane with 800rpm mixing speed dropping 0.6mol/L reacted 20 hours, isolated black solid with the permanent magnet of 0.1T; Solid matter is with distilled water washing 3~5 times, and accomplishing for the first time, silica coats.The magnetic composite that the first time, silica coated is dispersed in the water, at room temperature adds 8 gram KBH
4Carry out reduction reaction, separate with the permanent magnet of 0.05T, the gained solid fully washs with distilled water, promptly gets magnetic Fe nano particle and Fe
3O
4Nano particle is mixed into the composite material of the silica coating of kernel.
Embodiment 3: the preparation of core-shell type magnetic particulate kernel
With 0.3mol/L FeCl
2With 0.6mol/L CoCl
3Mixed with concentrated ammonia liquor, react after 10 minutes, adds 100 times of amounts, 8 ℃ water, stirring is 4.5 hours under 20000rpm, the crystallization reaction completion of particle; Under 0.1T magnetic field, crystalline particle is separated, promptly obtain the FeCo that particle diameter is 2~12nm after cleaning
2O
4Nano particle.Get 4.6 gram FeCo
2O
4It is in the mixed solvent of 25%~28% concentrated ammonia liquor that nano particle is dispersed in by 500ml propane diols, 10ml water, 15ml mass percent concentration; At room temperature; Butyl silicate with 500rpm mixing speed dropping 0.1mol/L reacted 12 hours, isolated black solid with the permanent magnet of 0.1T; Solid matter is with distilled water washing 3~5 times, and accomplishing for the first time, silica coats.The magnetic composite that the first time, silica coated is dispersed in the water, at room temperature adds 9 gram hydrazine hydrates and carry out reduction reaction, separate with the permanent magnet of 0.05T, the gained solid fully washs with distilled water, promptly gets magnetic Co nano particle and FeCo
2O
4Nano particle is a kernel.
Embodiment 4: the core-shell type magnetic microparticle shell coats
Claim composite material 1 gram of the silica coating of embodiment 1, embodiment 2 or embodiment 3, be dispersed in 5.0 gram Na
2SiO
3(SiO
2Content is 27%) in the 450ml aqueous solution of dissolving, using the pH value of acetic acid regulator solution is 8.5, at room temperature; Reacted 12 hours in the flask of 1000ml with the 300rpm mixing speed; After reaction finishes, with the permanent magnet separation of 0.08T, with distilled water washing 3~5 times; Obtaining particle diameter is the silica core/shell type magnetic particle of average grain diameter 0.5 μ m; 0.5g silica core/shell type magnetic particle joined in 0.3% the aminopropyl triethoxysilane coupling agent ethanolic solution, under 75 ℃ of temperature, stir, obtain amidized core/shell type magnetic particle.
Embodiment 5: the core-shell type magnetic microparticle shell coats
Claim composite material 2.5 grams of the silica coating of embodiment 1, embodiment 2 or embodiment 3, be dispersed in 7.5 gram Na
2SiO
3(SiO
2Content is 27%) in the 850ml aqueous solution of dissolving, using the pH value of hydrochloric acid conditioning solution is 6.5, at room temperature; Reacted 18 hours in the flask of 1000ml with the 500rpm mixing speed; After reaction finishes, with the permanent magnet separation of 0.08T, with distilled water washing 3~5 times; Obtaining particle diameter is the silica core-shell type magnetic particulate of average grain diameter 1.5 μ m; 1.5g silica core/shell type magnetic particle joined in 1.0% the mercaptopropyltriethoxysilane coupling agent aqueous solution, under 85 ℃ of temperature, stir, obtain the core/shell type magnetic particle of sulfhydrylation.
Embodiment 6: the core-shell type magnetic microparticle shell coats
Claim composite material 5.0 grams of the silica coating of embodiment 1, embodiment 2 or embodiment 3, be dispersed in 10.0 gram Na
2SiO
3(SiO
2Content is 27%) in the 1100ml aqueous solution of dissolving, using the pH value of sulfuric acid regulation solution is 7.0, at room temperature; Reacted 20 hours in the flask of 2000ml with the 800rpm mixing speed; After reaction finishes, with the permanent magnet separation of 0.08T, with distilled water washing 3~5 times; Obtaining particle diameter is the silica core-shell type magnetic particulate of 3.0 μ m; 1g silica core/shell type magnetic particle joined in γ-epoxypropoxy three alcoxyl silane coupler glycerine solution of 0.8%, under 90 ℃ of temperature, stir, obtain the core/shell type magnetic particle of epoxy radicalsization.
Embodiment 7: core-shell type magnetic separation of particles purifying DNA plasmid
Pour 1.5ml Escherichia coli nutrient solution into microcentrifugal tube, the centrifugal supernatant of abandoning is collected thalline; Add the TE buffer solution that 100ul contains 20ug/ml RNA enzyme to thalline; 200ul contains the alkaline lysis liquid of 0.2M NaOH and 1% (m/v) SDS, and the pH value that adds 150ul again is 5 5M potassium acetate solution, mixes; Centrifugal 5 minutes of 12000rpm transfers to supernatant in the new centrifuge tube; In centrifuge tube, add core-shell type magnetic particulate 1.5mg of the present invention, the concussion mixing, incubation 2-5min places centrifuge tube on the magnetic separator frame, inhales and abandons supernatant; Add 500ul 70% ethanol and clean DNA-magnetic particle compound twice, add 50ul TE buffer solution suspension magnetic particle, magnetic separated and collected eluent obtains the highly purified DNA of about 20ug.
Embodiment 8: core-shell type magnetic separation of particles purification bacillus coli gene group DNA
With the Escherichia coli 100 μ L of incubated overnight, centrifugal; Abandon supernatant, deposition is resuspended in TSEl buffer solution (50mM Tris-HCl, pH8.0,20% sucrose, 50mM EDTA, 100 μ g lysozymes), puts 37 ℃ of water-bath 20min; In reaction mixes, add 10 μ L 10%SDS, 5 μ L 20mg/ml Proteinase Ks are put 37 ℃ of water-bath 20min; Add 1mL cracking adsorption liquid (3M GuSCN, 2%TritonX-100) and 1mg core-shell type magnetic particulate, incubated at room 5min; Place on the magnetic separator frame, magnetic liquid separates the back and removes supernatant; Twice of 700 μ L, 70% washing with alcohol; 100 μ L aqua sterilisa wash-outs obtain 1ug bacillus coli gene group DNA.