CN102120168B - Multifunctional core-shell structure fluorescent coding magnetic microspheres and preparation method thereof - Google Patents
Multifunctional core-shell structure fluorescent coding magnetic microspheres and preparation method thereof Download PDFInfo
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Abstract
The invention belongs to the technical field of nano materials and biomedicine, in particular to multifunctional core-shell structure fluorescent coding magnetic microspheres and a preparation method thereof. On the basis of ferroferric oxide nanoparticles synthesized by a hydrothermal process, pre-prepared coupled product of fluoresceins and amino propyl trimethoxysilane and ethyl orthosilicate are subjected to cohydrolysis in ammonia water to form a multifunctional fluorescent magnetic nano composite material with fluorescent and magnetic properties and high biological stability and biological adaptability. By regulating the mixing ratio of fluoresceins, namely fluorescein isothiocyanate (FITC) and rhodamine B isothiocyanate (RBITC), various fluorescent coding magnetic microspheres can be prepared, and the particle size of obtained material can be regulated according to the different ratios of the added ethyl orthosilicate to the ferroferric oxide. In addition, the obtained multifunctional composite nano material is subjected to amino silanization modification, so the biological application range of the novel fluorescent coding magnetic microspheres is further expanded and the novel fluorescent coding magnetic microspheres have bright application prospect in fields of biomedicine technology, medicine development, suspension chip and the like.
Description
Technical field
The invention belongs to nano material and field of biomedicine technology, be specifically related to nano-magnetic microsphere of a kind of double fluorescence labeling and preparation method thereof.
Background technology
At present, magnetic Nano silicon ball composite has obtained greatly development in the biotechnology research field, multiple magnetic microsphere is based on its efficient separative efficiency, simple sample process process, harmless operating condition and be easy to the advantage such as functional modification has been widely used in Protein Separation, cell marking, cell detection, cell imaging etc.In addition; based on the good paramagnetism of this magnetic material of tri-iron tetroxide and be easy to synthesize, the character of functionalization; make it to become the important research and development object of separation, enrichment, NMR imaging, immunoassay; and silicon nanosphere particle is based on its good biocompatibility; stability; water-soluble and be easy to modify again ability has also obtained greatly paying close attention to and development.So synthetic magnetic Nano silicon ball composite has been preserved the multiple advantageous property of the two, such as superparamagnetism, large specific area, good biocompatibility, water-soluble character with being easy to modify etc.The range of application of magnetic microsphere composite nano materials has greatly been widened in the introducing of fluorescence especially, so that the real-time detection under nanoscale becomes possibility.
In addition, along with biomedical development, the magnetic microsphere composite of the multiple codified of exigence more on biomarker, biological diagnosis, fluorescence-encoded micro-beads becomes a kind of main solution.Fluorescence-encoded micro-beads the has referred to a kind of load novel microballoon of two or more fluorescent material, the proportioning by regulating fluorescent material and then realize optical encoding to microballoon.So, can realize the protein in the complex sample (antigen), gene, cell are carried out specific recognition and diagnosis by further functional modifications such as coupling antibody.The main method for preparing fluorescence-encoded micro-beads has at present: 1, by the method for swelling, quantum dot is wrapped in microsphere surface.2, the method by layer by layer self assembly is adsorbed in microsphere surface with quantum dot.3, utilize the micro emulsion method, and teos hydrolysis parcel fluorescent material synthetic composite material (brilliant far away, Tu Chifeng, Yang Yunhua, CN1948383A. Sun Kang, Dou Hongjing, pottery can, CN101037205A. big vast rosy clouds, white jade is white, Li Jun etc., CN1524925A. the leaf tinkling of pieces of jade, Yu Jingxian, Wang Nan etc., CN101671554A.).And the preparation of fluorescence-encoded magnetic microsphere is the multifunctional nano microballoon of a kind of integrated magnetic and photoluminescent property, than traditional fluorescence-encoded polystyrene microsphere (mostly being micron order), possessed more advantage, comprise that the nanometer specific area is large, superparamagnetism, be easy to functional modification etc., thereby greatly widened the application of composite.Here, quantum dot and organic fluorescent dye all can be used as the fluorescence source of coding magnetic microsphere nano composite material, and quantum dot also has plurality of advantages, such as wide excitation spectrum, narrow emission spectrum is difficult for photobleaching etc., yet quantum dot also has some self shortcoming, such as the general sensitiveness to environment, intrinsic (Zhou L, Gao C, the Hu X such as bio-toxicity, et al. ACS Appl. Mater. Interfaces. 2010,2 (4): 1211-1219. King-Heiden T C, Wiecinski P N, Mangham A N, et al. Environ. Sci. Technol. 2009,43 (5): 1605-1611.).Comparatively speaking, the organic molecule dyestuff has good biocompatibility and avirulence, thereby still shows good using value.
In addition, the method for the nanometer magnetic fluorescent microsphere material of the synthetic multifunction of development still is the key of biological nano technical development.The synthetic method of reporting at present mainly comprises take magnetic nano-particle as core, the outsourcing silicon layer, then amination, different thiocyanate and the outer amino of composite by organic dye molecule reacts again, organic dye molecule in the coupling, (Jang J H, Lim H B. Microchem. J. 2010,94 (2): 148-158. Zhang Y, Gong S W Y, Jin L, et al. Chinese Chem. Lett. 2009,20 (8): 969-972.) but this coupling method can cause organic molecule to occupy the surface functional group of composite, and then further reaction of impact, the in-situ preparation method that we propose has well solved this problem.Simultaneously, with respect to the quantum dot fluorescence coding composite of preparation double-nucleocapsid structure (Su Xingguang, crown man. CN 101530766A.), we have realized one step of fluorescence coding, thereby have saved operating procedure, and, with respect to one or both fluoresceins or quantum dot separate marking (Li Zhenkui, Zhao Minghang, Piao Chengfan etc., CN101283276A. Deng Yong brightness, Yang Wuli, high seapeaks etc., CN1523076A.), this double fluorescence labeling can prepare more fluorescence-encoded material.In addition, with respect to the magnetic microsphere that is using at present, adopt be utilize pre-prepared microballoon that the magnetic particle is adsorbed again and prepare (Pang Daiwen thanks to petrel, kingdom's equality, CN1869692A).The magnetic microsphere of our preparation uses original position synthetic, thereby has avoided magnetic particle seepage and unstability.In addition, we only use a kind of exciting light is the 488nm exciting light, has namely realized two fluorescent emission, and to sum up, this fluorescence-encoded magnetic microsphere has more the prospect advantage.
Summary of the invention
The object of the present invention is to provide a kind of nano-magnetic microsphere that can be fluorescence-encoded and preparation method thereof.
Nano-magnetic microsphere that can be fluorescence-encoded provided by the invention is nucleocapsid structure, and its kernel is tri-iron tetroxide (Fe
3O
4) magnetic nanoparticle, this nano particle diameter is less than 250nm, and for example, particle diameter is generally between 50-250 nm; Shell is silicon dioxide layer, contains two kinds of fluorescein(e) dye molecules in this silicon dioxide layer: fluorescein isothiocynate (FITC) and RITC (RBITC); The different quality ratio of fluorescein isothiocynate (FITC) and RITC (RBITC) consists of different fluorescence-encoded magnetic microspheres; The particle diameter of this magnetic microsphere is less than 320nm, and for example, particle diameter is generally between 100-320 nm.
Synthetic this functional fluorescence coding magnetic microsphere, be in microemulsion with two kinds of fluoresceins with different fluorescence: fluorescein isothiocynate (FITC) and RITC (RBITC) mix with the different quality ratio, and with 3-aminopropyl trimethoxysilane (APTMS) coupling reaction, then with ethyl orthosilicate (TEOS) cohydrolysis in ammoniacal liquor, at the Fe of superparamagnetism
3O
4Nanoparticle surface forms the silicon dioxide layer shell that contains two kinds of fluoresceins, namely obtains particle diameter less than 320nm, and fluorescence intensity is high, the fluorescence-encoded magnetic microsphere of good stability.
This preparation method has avoided aforementioned organic dye molecule to be coupled to the problem that silicon surface occupies reaction site, the problem that the while has also been avoided the problem of fluorescence molecule seepage in the aforementioned swelling method and polyelectrolyte weakens the fluorescence molecule signal in the layer assembly method.Here, can reach the change in fluorescence of regulating complex microsphere by the proportioning ratio that changes FITC and RBITC, thereby realize complex microsphere is carried out fluorescence-encoded purpose.Just be based on and thisly in preparation process, added two kinds of different fluorometric reagents, so that this complex microsphere is under the prerequisite that has possessed superparamagnetism, can be fluorescence-encoded to realize by the proportioning of regulating fluorescein, therefore can be referred to as fluorescence-encoded magnetic microsphere.
Fluorescence-encoded magnetic microsphere to above-mentioned preparation, by further finishing, antibody, aptamer, polypeptide, cell factor etc. can be connected, and then immune detection, nucleic acid recognizing, nucleic acid hybridization, genetic analysis, cell recognition, cell imaging etc. can be widely used in.For example modify by silylating reagent, as utilize amino silane reagent to carry out surface amination and process, further coupling antibody is realized bio-identification, mark function.
The concrete steps of the preparation of above-mentioned fluorescence-encoded magnetic microsphere are:
1, the ferriferrous oxide nano-particle of preparation superparamagnetism;
2, the preparation fluorescein comprises FITC and RBITC, under the two different quality conditions of mixture ratios with the performed polymer of APTMS coupling;
3, room temperature, under the miniemulsion conditions, the fluorescein FITC-APTMS of different proportionings and RBITC-APTMS performed polymer and TEOS cohydrolysis under the ammoniacal liquor condition is prepared into the fluorescence-encoded magnetic microsphere with nucleocapsid structure in the magnetic ferroferric oxide surface;
4, with amino silane reagent (such as 3-aminopropyl trimethoxysilane (APTMS), 3-aminopropyl triethoxysilane (APTES)) the fluorescence-encoded magnetic microsphere with nucleocapsid structure that said method is prepared carries out amino to be modified again, obtains can be used for the fluorescence-encoded magnetic microsphere of amino functional of multiple bioanalysis.
Above-mentioned steps 1 can adopt hydro-thermal method to prepare ferriferrous oxide nano-particle, and this particle diameter is less than 250nm.Wherein, by adjusting reaction time and reaction temperature, can regulate the particle diameter of ferriferrous oxide nano-particle.In addition, adding the auxiliary lower of magnet, can carry out easily the magnetic washing, then vacuum drying, for subsequent use.
Above-mentioned steps 2, by regulating the different proportionings of FITC and RBITC, wherein the quality ratio range of FITC and RBITC is 1:1 ~ 10:1, can reach the purpose of regulation and control fluorescence intensity, thereby controls the kind of follow-up synthetic nucleocapsid structure fluorescence-encoded micro-beads.In addition, used reaction vessel needs first the washing through 5% hydrofluoric acid solution, the nucleation site that may bring to remove glass container.
Above-mentioned steps 3, used reaction vessel need first the washing through 5% hydrofluoric acid solution, the nucleation site that may bring to remove glass container., can pass through to regulate the proportioning of fluorescein performed polymer and TEOS herein, and the consumption of TEOS, also have the consumption proportion of TEOS and tri-iron tetroxide etc., can reach thickness, the particle diameter of the silicon shell of regulating the final fluorescence-encoded magnetic microsphere of nucleocapsid structure for preparing.The quality proportioning of fluorescein performed polymer and TEOS is 0.00215:1 ~ 0.0355:1, the quality proportioning of TEOS and tri-iron tetroxide is 0.93:1 ~ 9.3:1, wherein in the certain situation of tri-iron tetroxide amount, every increase by the 1 μ L of the amount of TEOS, grain diameter increases 0.04nm approximately.
Above-mentioned steps 4, described preparation functional fluorescence coding magnetic microsphere is the silylating reagent that adds in the fluorescence-encoded magnetic microsphere solution of nucleocapsid structure with amino, carries out functional modification at composite material surface.Specifically under 20 ℃ ~ 40 ℃ temperature, make magnetic microsphere solution (such as the ferroferric oxide nano granules of 10 ~ 50mg preparation in step 3, in the FITC-APTMS and RBITC-APTMS mixed solution of 100 ~ 150 μ L variable concentrations proportionings, and 50 ~ 100 μ L ethyl orthosilicate and 30 ~ 60 μ L mass fractions be the fluorescence-encoded magnetic microsphere solution for preparing under 20 ~ 30% the ammoniacal liquor condition) in add (such as 10 ~ 50 μ L) amino silane reagent, continue to stir 19 ~ 24h, then add again a small amount of acetone breakdown of emulsion, stop to stir, under the help that adds magnet, use ethanol, the washing of deionized water magnetic repeatedly namely obtains the fluorescence-encoded magnetic microsphere of amino functional.
Employed water is two ionized waters that boil off in the above-mentioned steps.
The silylating reagent with amino carries out the amination processing to fluorescence-encoded magnetic microsphere after, can increase the biological applicability of this kind composite, but owing to the silicon hydroxyl of the meeting of the amino in the modification and silica surface is had an effect, so can have influence on the charge density of silica surface, and the dispersive property of this kind composite is exactly based on the repulsion performance of this electric charge.So excess of ammonia base reagent can reduce complex microsphere in the dispersiveness of aqueous phase, and then causes gathering.At this moment, can consider to add three hydroxyls silica-based-3-propyl group methylphosphine acid esters (THPMP), three hydroxyls that add are silica-based-and 3-propyl group methylphosphine acid esters is that 1:1 ~ 2:1 gets final product with mass ratio with the silylating reagent of amino, this is a kind of inert stable agent that contains methyl phosphorodithioate, amino meeting and the combination of methyl acid phosphate ester group on fluorescence-encoded magnetic microsphere surface, thus reach the effect that stops complex microsphere to be reunited.
The invention has the beneficial effects as follows, the fluorescence-encoded magnetic microsphere of preparation has good fluorescence property and magnetic property, and biological stability and water-soluble, and further functional modification, be applicable to biomedical sector, such as medicament research and development, suspension chip, biomarker etc., and by applying externally-applied magnetic field, can realize separation and purifying to analyzed identification molecule, cell.
Description of drawings
The present invention is further described below in conjunction with drawings and Examples.
Fig. 1 is the involved chemical equation of preparation Multifunctional core-shell structure fluorescent coding magnetic microspheres.Wherein:
(a) be depicted as FITC and RBITC respectively at APTMS reaction preparation performed polymer;
(b) be depicted as FITC and TEOS and under the ammoniacal liquor condition, cohydrolysis reaction occur;
(c) be depicted as RBITC and TEOS and under the ammoniacal liquor condition, cohydrolysis reaction occur;
(d) be respectively FITC and TEOS cohydrolysis product and APTES shown in and carry out the amino functional modification, RBITC and TEOS cohydrolysis product and APTES carry out amino functional and modify.
Fig. 2 is the scanning electron microscope diagram of the synthetic tri-iron tetroxide of hydro-thermal method.
Fig. 3 is the transmission electron microscope figure of the fluorescence-encoded magnetic microsphere of nucleocapsid structure.
Fig. 4 is the separately Fourier infrared spectrum figure of ferriferrous oxide nano-particle and the fluorescence-encoded magnetic microsphere of nucleocapsid structure.
Fig. 5 is the X-ray diffraction figure of the fluorescence-encoded magnetic microsphere of nucleocapsid structure.
Fig. 6 is the fluorescence spectrum figure of the fluorescence-encoded magnetic microsphere of nucleocapsid structure.
Fig. 1 by the detailed explanation of chemical equation how to prepare fluorescein-APTMS performed polymer, TEOS and FITC-APTMS and RBITC-APTMS cohydrolysis how under the ammoniacal liquor condition, and how to be implemented in fluorescence-encoded magnetic microsphere surface and to carry out amination and modify this chemical process.Fig. 2 has illustrated that the present invention is monodispersed in order to the ferriferrous oxide nano-particle as magnetic core, (250nm) of uniform grading.Fig. 3 has presented the fluorescence-encoded magnetic microsphere of this nucleocapsid structure, particle diameter ~ 300nm, and peripheral silicon layer has well wrapped up magnetic kernel.Fig. 4 compares by the Fourier infrared spectrum of ferriferrous oxide nano-particle and the infrared spectrum of fluorescence-encoded magnetic microsphere, is not difficult to find out, at 582cm
-1Absworption peak is the Fe-O vibration peak in the tri-iron tetroxide, and the two all has this absworption peak, and the 1090cm of fluorescence-encoded micro-beads
-1Infrared absorption peak then is the stretching vibration peak of Si-O, has further proved thus really to contain silicon dioxide layer in the fluorescence-encoded micro-beads.Fig. 5 is the X-ray diffraction spectrogram of fluorescence-encoded magnetic microsphere, (220) from figure, (311), (400), (422), (400) diffraction maximum, the comparison document can determine that this is the characteristic peak of paramagnetic tri-iron tetroxide crystal formation, (100) diffraction maximum has illustrated that then silicon layer exists, and this silica silicon layer is the indefinite form state.Fig. 6 is the fluorescence spectrum figure of fluorescence-encoded magnetic microsphere, and under the 488nm excitation, at 515nm, 580nm has respectively the emission spectrum of FITC, RBITC.
The specific embodiment
To be further described in more detail the present invention hereinafter.
Fluorescence-encoded magnetic composite nano material of the present invention contains the paramagnetism ferriferrous oxide nano-particle that is positioned at material internal, and parcel silica shell thereon, contain the organic fluorescent dye (FITC and RBITC) of two kinds of different proportionings in this shell, and can be silylated reagent and carry out finishing.Therefore, fluorescence-encoded magnetic nanometer composite material of the present invention namely possesses optical property and also possesses magnetic performance.
Fluorescence-encoded magnetic microsphere of the present invention can prepare by following steps:
1, preparation SPIO nano particle.
The synthetic employing hydro-thermal method of ferriferrous oxide nano-particle.At first, take by weighing Iron(III) chloride hexahydrate (FeCl
36H
2O) 1.35g is dissolved in the 40mL ethylene glycol, and magnetic agitation 30min until ferric trichloride dissolves fully, obtains the solution of yellow transparent.Then add successively the 3.6g anhydrous sodium acetate, 1.0g polyethylene glycol, magnetic agitation 30min then is transferred to gained solution in the 50mLTeflon-Lined stainless steel cauldron, react 8 ~ 16h under 200 ℃ of conditions, the taking-up reactor naturally cooled to room temperature after reaction was finished.The gained nano material, utilize the suction-operated of strong magnet, use ethanol and deionized water repeatedly to clean repeatedly, to remove the water-solubility impurities such as sodium acetate and ethylene glycol, then vacuum drying is for subsequent use under 60 ℃ of conditions, and gained ferriferrous oxide nano-particle particle diameter is ~ 250nm.
2, preparation FITC-APTMS, the RBITC-APTMS performed polymer.
The reaction bulb that reacts used uses 5% hydrofluoric acid clean 5min in advance, then removes washing lotion, and uses deionized water rinsing, places vacuum drying chamber dry.
Get the reaction bulb that 1mL ethanol places dry cleansing, the FITC, the RBITC that then get different quality are dissolved in the ethanolic solution, add 10 μ LAPTMS under the magnetic agitation condition, dry, airtight, lucifuge, stirring reaction 24h.Obtain FITC-APTMS and RBITC-APTMS performed polymer.
3, the fluorescence-encoded magnetic microsphere of preparation nucleocapsid structure.
Before the reaction, used reaction bulb uses 5% hydrofluoric acid clean 5min equally in advance, then removes washing lotion, and uses deionized water rinsing, places vacuum drying chamber dry.
In the 50mL round-bottomed flask, add the 7.5mL cyclohexane, 1.77mLTriton X-100,1.8mL n-hexyl alcohol, machinery strong stirring 30min makes it mixing, then drip the 250 μ L aqueous solution, continue to stir and make it mixing, the ferroferric oxide nano granules that then adds the aforementioned preparation of 10 ~ 50mg, auxiliary is ultrasonic, so that tri-iron tetroxide well is dispersed in the solution, continues to stir 12 ~ 15min, then the FITC-APTMS and the RBITC-APTMS mixed solution that add 100 ~ 150 μ L variable concentrations proportionings, continue to stir 5 ~ 8min, adding 50 ~ 100 μ L ethyl orthosilicates and 30 ~ 60 μ L mass fractions are 20 ~ 30% ammoniacal liquor, continue to stir 20 ~ 24h under the lucifuge condition, add afterwards 10 μ L acetone breakdowns of emulsion, stop to stir.Add outside under the suction-operated of strong magnet, use respectively repeatedly cleaning reaction product of ethanol and deionized water, namely obtain the fluorescence-encoded magnetic microsphere of nucleocapsid structure, particle diameter is at ~ 300nm, according to adding TEOS and Fe
3O
4Measure the difference of ratio, can regulate the particle diameter of resulting materials.
4, the fluorescence-encoded magnetic microsphere of preparation amino functional.
Abovementioned steps is with above-mentioned 3, at the same fluorescein of ethyl orthosilicate-APTMS performed polymer cohydrolysis after the 24h of tri-iron tetroxide surface, add amino silane reagent, such as APTES, 10 ~ 50 μ LAPTES join above-mentioned mixed solution, continue to stir 19 ~ 24h, then add again a small amount of acetone (10 μ L) breakdown of emulsion, stop to stir.Auxiliary lower in the suction-operated that adds magnet, repeatedly clean products therefrom with ethanol and deionized water respectively, namely obtain the fluorescence-encoded magnetic microsphere with nucleocapsid structure of amino functional.Be the fluorescence-encoded magnetic microsphere of the amination that obtains fine dispersion, can when adding APTES, add simultaneously 30 ~ 100 μ LTHPMP.
Embodiment 1:
The fluorescence-encoded magnetic microsphere of nucleocapsid structure of preparation fluorescence proportioning (mass ratio) FITC:RBITC=1:1.
A, preparation SPIO nano particle.
Take by weighing Iron(III) chloride hexahydrate (FeCl
36H
2O) 1.35g is dissolved in the 40mL ethylene glycol, magnetic agitation 30min, and ferric trichloride dissolves fully.Then add successively the 3.6g anhydrous sodium acetate, 1.0g polyethylene glycol, behind the magnetic agitation 30min gained solution is moved in the 50mLTeflon-Lined stainless steel cauldron, under 200 ℃ of conditions, react 8 ~ 16h, the taking-up reactor naturally cooled to room temperature after reaction was finished, use ethanol and deionized water repeatedly to clean repeatedly, then vacuum drying is for subsequent use under 60 ℃ of conditions.
B, preparation FITC-APTMS, the RBITC-APTMS performed polymer.
Get 1mL ethanol and place dry cleansing, in the pretreated reaction bulb of 5% hydrofluoric acid, then getting 1mgFITC, 1mgRBITC is dissolved in the ethanolic solution, add 10 μ LAPTMS under the magnetic agitation condition, dry, airtight, lucifuge, stirring reaction 24h obtain the performed polymer that FITC-APTMS and RBITC-APTMS mix.
C, the fluorescence-encoded magnetic microsphere of preparation nucleocapsid structure.
In the 50mL round-bottomed flask, add the 7.5mL cyclohexane, 1.77mLTriton X-100,1.8mL n-hexyl alcohol, machinery strong stirring 30min makes it mixing, then drip the 250 μ L aqueous solution, continue stirring and make it to add the ferroferric oxide nano granules that 10mg step a prepares behind the mixing, auxiliary is ultrasonic, tri-iron tetroxide is dispersed in the solution, continue to stir 15min, add FITC-APTMS and the RBITC-APTMS mixed solution of 150 μ L step b preparation, continue to stir 5min, then add 100 μ L ethyl orthosilicates and 60 μ L mass fractions and be 28% ammoniacal liquor, under the lucifuge condition, continue to stir 24h, add afterwards 10 μ L acetone breakdowns of emulsion, stop to stir.Add outside under the suction-operated of strong magnet, use respectively repeatedly cleaning reaction product of ethanol and deionized water, namely obtain the fluorescence-encoded magnetic microsphere of nucleocapsid structure.
Embodiment 2:
The fluorescence-encoded magnetic microsphere of nucleocapsid structure of the amino functional of preparation fluorescence proportioning (mass ratio) FITC:RBITC=4:1.
A, preparation SPIO nano particle are with implementing sample 1 step a.
B, preparation FITC-APTMS, the RBITC-APTMS performed polymer.
Get 1mL ethanol and place dry cleansing, in the pretreated reaction bulb of 5% hydrofluoric acid, then getting 4mgFITC, 1mgRBITC is dissolved in the ethanolic solution, add 10 μ LAPTMS under the magnetic agitation condition, dry, airtight, lucifuge, stirring reaction 24h obtain the performed polymer that FITC-APTMS and RBITC-APTMS mix.
C, the fluorescence-encoded magnetic microsphere of preparation nucleocapsid structure.
In the 50mL round-bottomed flask, add the 7.5mL cyclohexane, 1.77mLTriton X-100,1.8mL n-hexyl alcohol, machinery strong stirring 30min makes it mixing, then drip the 250 μ L aqueous solution, continue to stir and make it to add the ferroferric oxide nano granules that 10mg step a prepares behind the mixing, auxiliary is ultrasonic, so that tri-iron tetroxide well is dispersed in the solution, continues to stir 15min, then the FITC-APTMS and the RBITC-APTMS mixed solution that add 150 μ L step b preparation, continue to stir 5min, add 100 μ L ethyl orthosilicates and 60 μ L mass fractions and be 28% ammoniacal liquor, under the lucifuge condition, continue to stir 24h.
The fluorescence-encoded magnetic microsphere of d, preparation amino functional.
Amino silane reagent, such as APTES, 10 μ LAPTES join in the mixed solution behind the step c reaction 24h, continue to stir 19 ~ 24h, are then adding a small amount of acetone (10 μ L) breakdown of emulsion, stop to stir.Auxiliary lower in the suction-operated that adds magnet, repeatedly clean products therefrom with ethanol and deionized water respectively, namely obtain the fluorescence-encoded magnetic microsphere with nucleocapsid structure of amino functional.Wish to get the fluorescence-encoded magnetic microsphere of amination of fine dispersion, only need when adding APTES, to add simultaneously 30 μ LTHPMP.
Claims (7)
1. Multifunctional core-shell structure fluorescent coding magnetic microspheres, it is characterized in that: its kernel is ferroferric oxide magnetic nanoparticle, and this nano particle diameter is less than 250nm; Shell is silicon dioxide layer, contains two kinds of fluorescein(e) dye molecules in this silicon dioxide layer: fluorescein isothiocynate (FITC) and RITC (RBITC); The different quality ratio of fluorescein isothiocynate (FITC) and RITC (RBITC) consists of different fluorescence-encoded magnetic microspheres; The particle diameter of magnetic microsphere is less than 320nm.
2. Multifunctional core-shell structure fluorescent coding magnetic microspheres according to claim 1, it is characterized in that: by further finishing, its surface also is connected with antibody, aptamer, polypeptide or cell factor.
3. the preparation method of a Multifunctional core-shell structure fluorescent coding magnetic microspheres is characterized in that concrete steps are as follows:
1), adopt hydro-thermal method to prepare the ferriferrous oxide nano-particle of superparamagnetism; This nano particle diameter is less than 250nm;
2), under preparation fluorescein FITC and the two different quality conditions of mixture ratios of RBITC with the performed polymer of APTMS coupling;
3), room temperature, under the miniemulsion conditions, with step 2) the FITC-APTMS performed polymer that obtains and the mixed solution of RBITC-APTMS performed polymer, be prepared into the fluorescence-encoded magnetic microsphere with nucleocapsid structure with TEOS cohydrolysis under the ammoniacal liquor condition in the magnetic ferroferric oxide surface; This magnetic microsphere particle diameter is less than 320nm;
4), using amino silane reagent that the fluorescence-encoded magnetic microsphere with nucleocapsid structure of said method preparation is carried out amino modifies, obtains can be used for the fluorescence-encoded magnetic microsphere of amino functional of multiple bioanalysis;
Wherein, FITC is fluorescein isothiocynate, and RBITC is the RITC fluorescein, and APTMS is the 3-aminopropyl trimethoxysilane, and TEOS is ethyl orthosilicate.
4. the preparation method of Multifunctional core-shell structure fluorescent coding magnetic microspheres according to claim 3 is characterized in that described amino silane reagent is 3-aminopropyl trimethoxysilane (APTMS) or 3-aminopropyl triethoxysilane (APTES).
5. the preparation method of Multifunctional core-shell structure fluorescent coding magnetic microspheres according to claim 4, the different quality ratio range that it is characterized in that regulating FITC and RBITC is 1:1 ~ 10:1, can reach the purpose of regulation and control fluorescence intensity, thereby control the kind of follow-up synthetic nucleocapsid structure fluorescence-encoded micro-beads.
6. according to claim 4 or the preparation method of 5 described Multifunctional core-shell structure fluorescent coding magnetic microspheres, the quality proportioning that it is characterized in that fluorescein performed polymer and TEOS is 0.00215:1 ~ 0.0355:1, and the quality proportioning of TEOS and tri-iron tetroxide is 0.93:1 ~ 9.3:1.
7. according to claim 4 or the preparation method of 5 described Multifunctional core-shell structure fluorescent coding magnetic microspheres, it is characterized in that step 4) is under 20 ℃ ~ 40 ℃ temperature, in the solution of the magnetic microsphere that step 3) makes, add amino silane reagent, continue to stir 19 ~ 24h, then add again a small amount of acetone breakdown of emulsion, stop to stir, under the help that adds magnet, use ethanol, deionized water magnetic to wash repeatedly, namely obtain the fluorescence-encoded magnetic microsphere of amino functional.
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| CN1312479C (en) * | 2003-08-08 | 2007-04-25 | 清华大学 | Nano fluorescent magnetic particle and its preparing method |
| KR100821192B1 (en) * | 2005-09-08 | 2008-04-11 | 주식회사바이테리얼즈 | Magnetic nanoparticle having fluorescent and preparation method thereof |
| CN101671554B (en) * | 2008-09-10 | 2012-12-19 | 首都医科大学 | Silica-coated fluorescent magnetic nanoparticle, preparation method and application |
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| US10319502B2 (en) | 2014-10-23 | 2019-06-11 | Corning Incorporated | Polymer-encapsulated magnetic nanoparticles |
| CN106009347A (en) * | 2016-05-24 | 2016-10-12 | 东南大学 | Magnetic content coded polymer carrier microsphere and preparation method thereof |
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