CN100593546C - Polymer microsphere containing inorganic nano microparticles, and its preparing method and use - Google Patents
Polymer microsphere containing inorganic nano microparticles, and its preparing method and use Download PDFInfo
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- CN100593546C CN100593546C CN200510130313A CN200510130313A CN100593546C CN 100593546 C CN100593546 C CN 100593546C CN 200510130313 A CN200510130313 A CN 200510130313A CN 200510130313 A CN200510130313 A CN 200510130313A CN 100593546 C CN100593546 C CN 100593546C
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Abstract
The invention relates to a method for connecting nanoparticles and polymer spheres with chemical bonding in the polymer microspheres and its preparation and biomedical applications, which recombines the water-soluble inorganic nano-particles into hydrophobic polymers with reactive surfactant through emulsion polymerization. In this way, the multi-functional polymer microspheres can be obtained through the recombination of different types of nanoparticles on the same microspheres. The positive charge on the microsphere surface can couple the biological molecules onto it by the electrostatic interaction to get a microsphere fluorescent probe used in immunodetection, chromosome analysis of multiple genes, protein array, DNA sequencing and simultaneous detection in different regions of gene chips and cell or biological tissues.
Description
Technical field
The invention belongs to the polymer microballoon field of containing nanoparticle, being particularly related between nanoparticle and the polymer spheres is to link to each other by chemical bond, more specifically says so to obtain microballoon of being compounded to form by inorganic nano-particle and polymkeric substance and preparation method thereof and in bio-medical applications by emulsion polymerisation process.
Background technology
Because its unique dimensional effect is compared with corresponding bulk material, nanoparticle has very unique physicochemical property, is with a wide range of applications in various fields.At present, purposes the most widely nanoparticle mainly contain semi-conductor, precious metal and metal oxide.Nanoparticle is compound to nanoparticle and extraneous chemical environment are isolated, make its peculiar property not be subjected to the influence of environment; And can give microballoon more new function, thereby obtain the novel material of class extensive application in fields such as photonic crystal, transmitter, high throughput testing and life science.Utilize the fluorescence of inorganic nano-particle or polymer microballoon that magnetic obtains to show wide application at aspects such as molecule and biological studies.Wherein, the unique fluorescence size adjustable of II-VI family fluorescence semiconductor nanoparticle (being called quantum dot again) makes it have a extensive future at aspects such as molecule and biological studies as a kind of marker material.A large amount of fluorescence semiconductor nanoparticles is compound to the fluorescent microsphere that can obtain high fluorescent in the same microballoon; In addition, with different types of fluorescent nano particles be compound to by a certain percentage can realize microballoon in the same microballoon fluorescence-encoded, thereby obtain in the high-throughput bioanalysis and the fluorescence labeling material that has significant application value in detecting.The inorganic nano-particle that will have different physical properties simultaneously, being integrated into same microballoon inside as fluorescent particle and magnetic nanometer particles, can also to realize having multi-functional biomarker and parting material (brilliant far away etc., magnetic fluorescence material and preparation method and application, number of patent application: 200510109318.4).
At present, the preparation method who contains the polymer microballoon of nanoparticle mainly is included in the spheroid original position synthesizing nano-particle or by static, hydrogen bond and hydrophobic interaction the nanoparticle for preparing is compound in the microballoon.But the former can not control the pattern of nanoparticle and the surface tissue of degree of crystallinity and nanoparticle (J.Am.Chem.Soc.2004,126,7908); The latter is not owing to have enough strong interaction between nanoparticle and the polymer spheres, and nanoparticle is separated out (Adv.Mater.2005,17 (3), 267) easily from matrix under certain condition.Letex polymerization also is used for preparing the polymer microballoon that contains nanoparticle, but trend (J.Am.Chem.Soc.2002,124,13864.Chem.Mater.2005,17,1346) appears inevitably assembling and separating out in the nanoparticle in the microballoon.
Summary of the invention
One of purpose of the present invention provides the polymer microballoon that contains inorganic nano-particle, and microballoon has further and can modify processing characteristics.
Two of purpose of the present invention provides the polymer fluorescent microspheres that contains the semiconductor nano particulate.
Three of purpose of the present invention provides the polymer fluorescent microspheres that contains the fluorescence semiconductor nanoparticle, and its fluorescence efficiency is not less than 20%.
Four of purpose of the present invention provides a kind of simple method the water-soluble inorganic nanoparticle is compound in the polymkeric substance, prepares the polymer microballoon that contains inorganic nano-particle, links to each other by chemical bond between nanoparticle and the polymeric matrix.
Five of purpose of the present invention provides the preparation method of the polymer fluorescent microspheres that contains the fluorescence semiconductor nanoparticle.
Six of purpose of the present invention provides the purposes of the polymer fluorescent microspheres that contains the semiconductor nano particulate, can be used in fields such as fluorescence-encoded and biological detection.
The present invention adopts two kinds of reactable tensio-active agents, by method of emulsion polymerization the water-soluble inorganic nanoparticle is compound in the polymkeric substance, thereby obtaining multi-functional polymer microballoon, is to link to each other by chemical bond between nanoparticle and the matrix, and nanoparticle can not separated out from polymeric matrix.The polymer microballoon surface has positive charge, easily biomolecules is coupled to microsphere surface by electrostatic interaction, thereby carries out the detection of biological aspect.
The present invention utilizes a kind of reactable tensio-active agent that the water-soluble inorganic nanoparticle is transferred to the oil-soluble monomer from water at first at the clear and definite inorganic nano-particle of aqueous phase preparation property then; With water is external phase, and monomer solution is a disperse phase.Another kind of reactable tensio-active agent is that emulsifying agent forms emulsion, adopts the initiator initiated polymerization at a certain temperature.Separate the polymer microballoon that obtains having inorganic nano-particle.Change the consumption of reactable tensio-active agent, particle diameter that to a certain extent can the controlling polymers microballoon.
The polymer microballoon that contains inorganic nano-particle of the present invention, the water-soluble inorganic nanoparticle is embedded in the inside of polymer microballoon, and the water-soluble inorganic nanoparticle combines with chemical bond with polymer molecular chain by the carbon-carbon double bond on the reactable surfactant molecule chain;
The particle diameter of described polymer microballoon is between 200 nanometers to 5 micron.
The particle diameter of described inorganic nano-particle is between 1～100 nanometer.
Described inorganic nano-particle is selected from one or more the mixture in semiconductor nano particulate, noble metal nano particulate or the metal oxide nanoparticles.
Described semiconductor nano particulate is selected from one or more the mixture in Cadmium Sulfide, zinc sulphide, zunsober, lead sulfide, cadmium selenide, zinc selenide, lead selenide, cadmium telluride, tellurium mercury, lead telluride or the silicon nanometer particle.
Described noble metal nano particulate is selected from one or more the mixture in gold and silver, iron, platinum, nickel, cobalt, palladium, the manganese, or contains the alloy nano particles of above-mentioned metallic element.
Described metal oxide nanoparticles is selected from one or more the mixture in Z 250, ferric oxide, manganese oxide, cobalt oxide, nickel oxide, the zinc oxide.
Described polymkeric substance is selected from one or more the multipolymer in polystyrene, polydivinylbenezene, polyethylene glycol dimethacrylate, polyvinyl acetate, polyacrylate(s), the polymethacrylate.
Described polyacrylate(s) is a kind of in polymethyl acrylate, polyethyl acrylate, polyacrylic acid propyl ester, the butyl polyacrylate.
Described polymethacrylate is a kind of in polymethylmethacrylate, polyethyl methacrylate, polypropylmethacryla,es, the poly-n-butyl methacrylate.
The preparation method who contains the polymer microballoon of inorganic nano-particle of the present invention, this method may further comprise the steps:
(1) the water-soluble inorganic nanoparticle prepares according to existing document.The semiconductor nano particulate is with semi-conductor cadmium telluride nanoparticle (the M.Y.Gao et al of II-VI family, J.Phys.Chem., 1998,102,8360) as follows for the example detailed process: that cadmium salt (comprising Cadmium chloride fine powder, cadmium acetate, cadmium perchlorate) is mixed with 0.001mol/L～0.15mol/L, solution between preferred 0.005mol/L～0.05mol/L, (mol ratio of cadmium salt and sulfydryl modification agent is between 1: 1～1: 3 to add the sulfydryl modification agent subsequently, between preferred 1: 2.0～1: 2.5), regulate its pH value and transfer to 10～13, preferred 10.5～11.4.With excessive dilution heat of sulfuric acid and tellurium aluminium (Al
2Te
3) H that generates of reaction
2Te directly feeds in the above-mentioned cadmium solution (mol ratio of cadmium and tellurium between 1: 0.2～1: 0.8, preferred 1: 0.4～1: 0.6), stirs 10～20 minutes post-heating this solution that refluxes.Control return time, obtain the cadmium telluride nanoparticle of fluorescent emission centre wavelength any modulation between 430 nanometer to 650 nanometers.The noble metal nano particulate is representative (Michael J.Natan et al with the gold sol, Anal.Chem.1995,67,735), detailed process is as follows: the hydrochloro-auric acid of 0.15 milliliter of 2wt% is joined in 30 ml waters, add 0.45 milliliter of 2wt% sodium citrate aqueous solution during boiling, refluxing just obtained gold sol in 30 minutes.Metal oxide is representative (M.Y.Gao et al, Adv.Mater.2005,17,1001) with the Z 250.Detailed process is as follows: with the 10mmol ferric acetyl acetonade, the two carboxyl polyvinyl alcohol of 5mmol join in the 100mL alpha-pyrrolidone, and refluxing just obtained the ferriferrous oxide nano particulate in 5～20 hours.
(2) (J.ControlledRelease 1990 for Aoyagi, T.et al according to the document preparation for the reactable tensio-active agent, 13,63), detailed process is as follows: equimolar tertiary amine and 4-vinyl Bian Ji chlorine are dissolved in acetone, refluxed 2～48 hours at 40 ℃, obtain white solid; The recrystallization final vacuum is drying to obtain the reactable tensio-active agent.
(3) the reactable surfactant A with step (2) is dissolved in the oil-soluble monomer, the water-soluble inorganic nanoparticle aqueous solution that in this monomer solution, adds step (1), stirring and segregation obtains inorganic nano-particle/monomer solution, then, initiator is dissolved in inorganic nano-particle/monomer solution; The concentration of reactable surfactant A in monomer is 0.1%～10wt%, and the concentration of the inorganic nano-particle aqueous solution is 2.5 * 10
-7～2.5 * 10
-3Between the mol/L (number with nanoparticle calculates), the inorganic nano-particle aqueous solution and monomeric volume ratio are between 1: 0.2～1: 2, and initiator and monomeric mass ratio are between 1: 20～1: 1000.
(4) the reactable surfactant B is soluble in water, inorganic nano-particle/the monomer solution that is dissolved with initiator that step (3) is obtained joins in the above-mentioned aqueous solution, stirs, and forms emulsion oil-in-water, logical rare gas element deoxygenation, emulsion system was 70～80 ℃ of following polyreactions 5～12 hours; Wherein the ratio of the amount of substance of reactable surfactant B and water is between 1: 250～1: 10000, and the mass ratio of inorganic nano-particle/monomer solution and water is between 1: 4～1: 100.After reaction finished, centrifugation went out to contain the polymer microballoon of inorganic nano-particle, after unnecessary reactable surfactant B is removed in water washing, is dispersed in aqueous phase again again, forms the stable polymer microballoon aqueous dispersion that contains inorganic nano-particle.
When inorganic nano-particle is fluorescent nano particles, described inorganic nano-particle/the monomer solution that is dissolved with initiator of step (4) is the polymer monomer solution that contains the water-soluble nano particulate of different fluorescent emission wavelength, and what obtain is the polymer fluorescent microspheres that contains the nanoparticle of various different fluorescent emission wavelength.
When inorganic nano-particle is fluorescent nano particles and magnetic nanometer particles, described nanoparticle/the monomer solution that is dissolved with initiator of step (4) is to contain the water-soluble semi conductor nanoparticle of different fluorescent emission wavelength and the polymer monomer solution of magnetic nanometer particles, and what obtain is the magnetic polymer fluorescent microsphere that contains the semiconductor nano particulate of various different fluorescent emission wavelength.
For semiconductor nano particulate/monomer solution, be several semiconductor nano particulate/monomer solutions with single emission wavelength to be mixed by arbitrary proportion obtain with different fluorescent emission wavelength.
The described polymer fluorescent microspheres that contains the semiconductor nano particulate of various different fluorescent emission wavelength, the fluorescence intensity of the semiconductor nano particulate of its different fluorescent emission wavelength are to realize by the ratio between semiconductor nano particulate/monomer solution of controlling various different fluorescent emission wavelength.
Described inorganic nano-particle is selected from one or more the mixture in semiconductor nano particulate, noble metal nano particulate or the metal oxide nanoparticles.
Described monomer is selected from vinylbenzene, Vinylstyrene, ethylene glycol dimethacrylate, vinyl-acetic ester, esters of acrylic acid, a kind of or any two kinds of monomeric mixtures in the methyl acrylic ester.
Described semiconductor nano particulate is selected from one or more the mixture in Cadmium Sulfide, zinc sulphide, zunsober, lead sulfide, cadmium selenide, zinc selenide, lead selenide, cadmium telluride, tellurium mercury, lead telluride or the silicon nanometer particle.
The mixture of one or more in described sulfydryl modification agent selected from mercapto acetate, thiohydracrylic acid, the Thioctic Acid.
Described noble metal nano particulate is selected from one or more the mixture in gold and silver, iron, platinum, nickel, cobalt, palladium, the manganese, or contains the alloy nano particles of above-mentioned metallic element.
Described metal oxide nanoparticles is selected from one or more the mixture in Z 250, ferric oxide, manganese oxide, cobalt oxide, nickel oxide, the zinc oxide.
Described tertiary amine is selected from monoalkyl chain dimethyl amine, two alkyl chain methylamine, and one or more in the trialkylamine, the number of carbon atom is between 6～18 in the alkyl chain.
Described initiator is selected from azo-bis-isobutyl cyanide, a kind of in benzoyl peroxide or other oil-soluble initiator.
Described reactable surfactant A is two alkyl chain methyl to vinyl Bian ammonium chloride or trialkyl to vinyl Bian ammonium chloride, and the number of carbon atom is between 8～18 in the alkyl chain.
Described reactable surfactant B be monoalkyl chain dimethyl to vinyl benzyl chlorination ammonium, the carbon atom number of alkyl chain is between 6～18.
Described rare gas element is argon gas, nitrogen, helium etc.
The polymer fluorescent microspheres surface of containing the fluorescence semiconductor nanoparticle of the present invention is because polymerizable cationic tensio-active agent and positively charged, method by electrostatic adhesion directly is adsorbed onto the surface of fluorescent microsphere with biomolecules, obtains the microballoon fluorescent probe that can discern living things system.The microballoon fluorescent probe of method for preparing is used for biological detection: the microballoon fluorescent probe can be used for the fluorescence synchronous detection of different zones in joint-detection, dna sequencing, gene chip and the cell of immunodetection, polygene genome analysis, protein chip or the biological tissue.The microballoon fluorescent probe can also be used for highly sensitive immunodetection, the concrete practice is as detecting carrier with the substrate that is modified with antibody, substrate is successively immersed in solution to be measured and the fluorescent probe solution, when having antigen to occur in the solution to be measured, the microballoon fluorescent probe can be adsorbed onto the solid substrate surface by antibody-antigenic specific recognition effect automatically, makes solid substrate produce fluorescence.Because contain a large amount of fluorescent particles in the microballoon, the use of microballoon fluorescent probe will improve the sensitivity of immunodetection widely.
The described magnetic fluorescence complex microsphere that contains magnetic nanometer particles and fluorescent nano particles is used for the high-sensitivity detection of fluorescence synchronous detection, bacterium and the virus of different zones in joint-detection, dna sequencing, gene chip and the cell of immunodetection, polygene genome analysis, protein chip or the biological tissue.
Describedly remove wherein cancer cells when containing the magnetic nanometer particles polymer microballoon and being used for immunodetection, autologous bone marrow transplantation, separate other karyocytes, isolated cell device, purify DNA are conjugated protein, dna sequence analysis, solid phase clone, vitro mutagenesis.
Preparation process of the present invention is simple, and is easy to operate, high quality water dissolubility inorganic nano-particle can be compound in the polymer microballoon, obtains multi-functional polymer microballoon.Link to each other by chemical bond between nanoparticle and the polymeric matrix.Polymer microballoon can further be modified, and being convenient to has very big application prospect with the biomolecules coupling on biological detection.
Description of drawings
The fluorescence spectrum figure of Fig. 1 embodiment of the invention 1 gained water soluble cadmium telluride semiconductor nano particulate.
The light field of Fig. 2 A embodiment of the invention 10 resulting polymers fluorescent microspheres.
The fluorescence photo of Fig. 2 B embodiment of the invention 10 resulting polymers fluorescent microspheres.
The fluorescence spectrum figure of Fig. 3 embodiment of the invention 10 resulting polymers fluorescent microspheres.
The light field of Fig. 4 A embodiment of the invention 11 resulting polymers fluorescent microspheres.
The fluorescence photo of Fig. 4 B embodiment of the invention 11 resulting polymers fluorescent microspheres.
The light field of Fig. 5 A embodiment of the invention 12 resulting polymers fluorescent microspheres.
The fluorescence photo of Fig. 5 B embodiment of the invention 12 resulting polymers fluorescent microspheres.
The light field of Fig. 6 A embodiment of the invention 15 resulting polymers fluorescent microspheres.
The fluorescence photo of Fig. 6 B embodiment of the invention 15 resulting polymers fluorescent microspheres.
The absorption and the fluorescence spectrum of Fig. 7 embodiment of the invention 15 resulting polymers fluorescent microspheres supernatant liquid after toluene swell.
The light field of Fig. 8 A embodiment of the invention 21 resulting polymers fluorescent microspheres.
The fluorescence photo of Fig. 8 B embodiment of the invention 21 resulting polymers fluorescent microspheres.
The light field of Fig. 9 A embodiment of the invention 22 resulting polymers fluorescent microspheres.
The fluorescence photo of Fig. 9 B embodiment of the invention 22 resulting polymers fluorescent microspheres.
The fluorescence spectrum figure of Figure 10 embodiment of the invention 22 resulting polymers fluorescent microspheres.
The light field of Figure 11 A embodiment of the invention 23 resulting polymers fluorescent microspheres.
The fluorescence photo of Figure 11 B embodiment of the invention 23 resulting polymers fluorescent microspheres.
Figure 12 A embodiment of the invention 27 gained contain the stereoscan photograph of the polymer microballoon of gold.
Figure 12 B embodiment of the invention 27 gained contain the ultrathin section(ing) transmission electron microscope photo of the polymer microballoon of gold.
Figure 13 A embodiment of the invention 28 gained contain the stereoscan photograph of the polymer microballoon of Z 250.
Figure 13 B embodiment of the invention 28 gained contain the ultrathin section(ing) transmission electron microscope photo of the polymer microballoon of Z 250.
Embodiment
Embodiment 1:
Take by weighing 1.315g cadmium perchlorate (Cd (ClO
4)
26H
2O) join in the secondary water of 150mL deoxygenation, add the 0.55mL Thiovanic acid subsequently, its pH value is transferred to 11.2, form the solution that contains sulfhydryl compound and cadmium ion with the sodium hydroxide solution of 1M as modifier.On the other hand, getting 30mL 0.5M sulphuric acid soln is injected into and fills 0.39g tellurium aluminium (Al
2Te
3) flask in, with the H that generates
2Te all feeds in the above-mentioned cadmium-ion solution, stirs after 15 minutes, and reflux 1 minute to 10 days obtains that Thiovanic acid is stablized, the cadmium telluride semiconductor nano particulate aqueous solution of fluorescent emission centre wavelength between 430～650 nanometers.Fluorescence spectrum is seen Fig. 1.
Embodiment 2:
Take by weighing 1.031g Cadmium chloride fine powder (CdCl
22.5H
2O) join in the secondary water of 150mL deoxygenation, add the 0.55mL thiohydracrylic acid subsequently, its pH value is transferred to 11.2, form the solution that contains sulfhydryl compound and cadmium ion with the sodium hydroxide solution of 1M as modifier.All the other steps are with embodiment 1, obtain that thiohydracrylic acid is stable, the cadmium telluride semiconductor nano particulate aqueous solution of fluorescent emission centre wavelength between 430～650 nanometers.
Embodiment 3:
Take by weighing 1.031g Cadmium chloride fine powder (CdCl
22.5H
2O) join in the secondary water of 150mL deoxygenation, add the 0.65mL Thioctic Acid subsequently, its pH value is transferred to 11.2, form the solution that contains sulfhydryl compound and cadmium ion with the sodium hydroxide solution of 1M as modifier.All the other steps are with embodiment 1, obtain that Thioctic Acid is stable, the cadmium telluride semiconductor nano particulate aqueous solution of fluorescent emission centre wavelength between 430～650 nanometers.
Embodiment 4:
Take by weighing 1.315g cadmium perchlorate (Cd (ClO
4)
26H
2O) join in the secondary water of 150mL deoxygenation, add the 0.55mL Thiovanic acid subsequently, its pH value is transferred to 11.2, form the solution that contains sulfhydryl compound and cadmium ion with the sodium hydroxide solution of 1M as modifier.On the other hand, getting 30mL 0.5M sulphuric acid soln is injected into and fills 0.26g aluminum selenide (Al
2Se
3) flask in, with the H that generates
2Se all feeds in the above-mentioned cadmium-ion solution, obtains the stable cadmium selenide nano particulate aqueous solution of Thiovanic acid.
Embodiment 5
Get 10mmol octadecyl dimethyl amine and 10mmol 4-vinyl Bian Ji chlorine is dissolved in 5mL acetone, refluxed two hours at 40 ℃; Cooled solid recrystallization final vacuum drying makes the octadecyl dimethyl to vinyl Bian ammonium chloride.
Embodiment 6:
Get 10mmol hexadecyldimethyl benzyl ammonium amine and 10mmol 4-vinyl Bian Ji chlorine is dissolved in 5mL acetone, refluxed two hours at 40 ℃; Cooled solid recrystallization final vacuum drying makes hexadecyldimethyl benzyl ammonium to vinyl Bian ammonium chloride.
Embodiment 7:
Get 10mmol two (octadecyl) methylamine and 10mmol 4-vinyl Bian Ji chlorine is dissolved in 5mL acetone, refluxed 48 hours at 40 ℃.Cooled solid recrystallization final vacuum drying makes two (octadecyl) methyl to vinyl benzyl chlorination ammonium.
Embodiment 8:
Get 10mmol didecyl alkyl methyl amine and 10mmol 4-vinyl Bian Ji chlorine is dissolved in 5mL acetone, refluxed 48 hours at 40 ℃.Cooled solid recrystallization final vacuum drying makes the didecyl alkyl methyl to vinyl benzyl chlorination ammonium.
Embodiment 9:
Get 10mmol trioctylphosphine amine and 10mmol 4-vinyl Bian Ji chlorine is dissolved in 5mL acetone, refluxed 48 hours at 40 ℃.Cooled solid recrystallization final vacuum drying makes trioctylphosphine to vinyl benzyl chlorination ammonium.
Embodiment 10:
The didecyl alkyl methyl that takes by weighing embodiment 8 is to vinyl benzyl chlorination ammonium 0.0150g, be dissolved in the 2mL vinylbenzene, get cadmium telluride nanoparticle (fluorescent emission centre wavelength is in the 630 nanometers) aqueous solution 1mL of the thiohydracrylic acid finishing of embodiment 2, concentration 0.0125mol/L, under agitation join in the above-mentioned monomer, separate obtaining cadmium telluride/styrene solution.Take by weighing the 0.0300g azo-bis-isobutyl cyanide, be dissolved in 1.5g cadmium telluride/styrene solution.The octadecyl dimethyl that takes by weighing embodiment 5 is dissolved in the middle of the 20mL water vinyl Bian ammonium chloride 0.1200g.Cadmium telluride/the styrene solution that is dissolved with azo-bis-isobutyl cyanide is added to aqueous phase, stirs and form emulsion.Led to nitrogen 20 minutes to this emulsion, in 80 ℃ water-bath, reacted 6.5 hours then.After finishing reaction, centrifugal, wash with water and remove unnecessary emulsifying agent, be dispersed in aqueous phase at last.Contrast as can be known by fluorescence photo and light field photo (Fig. 2 A and Fig. 2 B), made the polystyrene fluorescent microsphere that contains cadmium telluride semiconductor nano particulate.Polymerization as can be seen from Figure 3, polymerization process do not have influence on the photoluminescent property of cadmium telluride, and promptly fluorescent emission centre wavelength is constant.The fluorescence quantum yield of cadmium telluride is about 26% in the polymer microballoon.The particle diameter of polymer fluorescent microspheres is 1.5 microns.
Embodiment 11:
The didecyl alkyl methyl that takes by weighing embodiment 8 is dissolved in the 2.0mL vinylbenzene vinyl benzyl chlorination ammonium 0.2000g.Get cadmium telluride semiconductor nano particulate (fluorescent emission centre wavelength is in the 580 nanometers) aqueous solution 10mL of the Thiovanic acid finishing of embodiment 1, concentration 0.00125mol/L under agitation joins in the above-mentioned monomer, separates to obtain cadmium telluride/styrene solution.Take by weighing the azo-bis-isobutyl cyanide of 0.0150g, be dissolved in 1.5g cadmium telluride/styrene solution.The octadecyl dimethyl that takes by weighing embodiment 5 is dissolved in the middle of the 20mL water vinyl Bian ammonium chloride 0.1000g, obtains being dissolved with the aqueous solution of tensio-active agent.Cadmium telluride/styrene solution is joined in the aqueous solution, stir and form emulsion.In 80 ℃ water-bath, reacted 8 hours then about 20 minutes to logical nitrogen in the middle of this emulsion.After finishing reaction, centrifugal, wash with water and remove unnecessary emulsifying agent, be dispersed in aqueous phase at last.Contrast as can be known by fluorescence photo and light field photo (Fig. 4 A and Fig. 4 B), made the polystyrene fluorescent microsphere that contains the cadmium telluride nanoparticle.
Embodiment 12:
The didecyl alkyl methyl that takes by weighing embodiment 8 is to vinyl benzyl chlorination ammonium 0.2000g, be dissolved in the 2.0mL methyl methacrylate, get cadmium telluride semiconductor nano particulate (fluorescent emission centre wavelength is in the 630 nanometers) aqueous solution 1.0mL of the thiohydracrylic acid finishing of embodiment 2, concentration 0.0130mol/L, under agitation join in the above-mentioned monomer, separate obtaining being dissolved with cadmium telluride/methyl methacrylate solution.Take by weighing the 0.0300g azo-bis-isobutyl cyanide, be dissolved in 1.5g cadmium telluride/methyl methacrylate solution.The octadecyl dimethyl that takes by weighing embodiment 5 is dissolved in the middle of the 20mL water vinyl Bian ammonium chloride 0.1000g, obtains being dissolved with the aqueous solution of tensio-active agent.Cadmium telluride/methyl methacrylate the solution that is dissolved with azo-bis-isobutyl cyanide is added in the aqueous solution, stirs and form emulsion., in 80 ℃ water-bath, reacted 6.5 hours then about 20 minutes to the logical nitrogen of this emulsion.After finishing reaction, centrifugal, wash with water and remove unnecessary emulsifying agent, be dispersed in aqueous phase at last.Contrast as can be known by fluorescence photo and light field photo (Fig. 5 A and Fig. 5 B), made the polymethylmethacrylate fluorescent microsphere that contains the cadmium telluride nanoparticle.
Embodiment 13:
Two decyls that take by weighing embodiment 8 are dissolved in the 2.0mL methyl methacrylate vinyl benzyl ammonio methacrylate 0.0150g.Get cadmium telluride semiconductor nano particulate (fluorescent emission centre wavelength is in the 600 nanometers) aqueous solution 1.0mL of the Thiovanic acid finishing of embodiment 1, concentration 0.0150mol/L, join in the methyl methacrylate solution, separate obtaining cadmium telluride/methyl methacrylate solution.Take by weighing the 0.0150g azo-bis-isobutyl cyanide, be dissolved in 1.0g cadmium telluride/methyl methacrylate.The octadecyl dimethyl that takes by weighing embodiment 5 is dissolved in the 20mL water vinyl Bian ammonium chloride 0.3000g.Cadmium telluride/methyl methacrylate the solution that is dissolved with azo-bis-isobutyl cyanide is added to aqueous phase, stirs and form emulsion.Logical nitrogen reacted 6.5 hours in 80 ℃ water-bath about 20 minutes then in this emulsion.After finishing reaction, centrifugal, wash with water and remove unnecessary emulsifying agent, be dispersed in aqueous phase at last, made the polymethylmethacrylate fluorescent microsphere that contains the cadmium telluride nanoparticle.The particle diameter of fluorescent microsphere is 500 nanometers.
Embodiment 14:
Two (octadecyl) methyl that takes by weighing embodiment 7 is to vinyl benzyl chlorination ammonium 0.3000g, be dissolved in the 2mL ethylene glycol dimethacrylate monomer, get cadmium telluride nanoparticle (fluorescent emission centre wavelength is in the 600 nanometers) aqueous solution 5mL of the Thiovanic acid finishing of embodiment 1, concentration 0.0025mol/L, under agitation join in the above-mentioned monomer, separate obtaining cadmium telluride/ethylene glycol dimethacrylate solution.Take by weighing the 0.0300g azo-bis-isobutyl cyanide, be dissolved in 1.5g cadmium telluride/ethylene glycol dimethacrylate solution.The hexadecyldimethyl benzyl ammonium that takes by weighing embodiment 6 is dissolved in the middle of the 20mL water vinyl Bian ammonium chloride 0.1400g.Cadmium telluride/ethylene glycol dimethacrylate the solution that is dissolved with azo-bis-isobutyl cyanide is added to aqueous phase, stirs and form emulsion.Led to nitrogen 20 minutes to this emulsion, in 80 ℃ water-bath, reacted 10 hours then.After finishing reaction, centrifugal, wash with water and remove unnecessary emulsifying agent, be dispersed in aqueous phase at last, obtained containing the polyethylene glycol dimethacrylate fluorescent microsphere of cadmium telluride nanoparticle.
Embodiment 15:
The didecyl alkyl methyl that takes by weighing embodiment 8 is dissolved in the mix monomer of 2.0mL vinylbenzene and 0.2mL Vinylstyrene vinyl benzyl chlorination ammonium 0.0150g.Get cadmium telluride semiconductor nano particulate (fluorescent emission centre wavelength is in the 630 nanometers) aqueous solution 1.5mL of the thiohydracrylic acid finishing of embodiment 2, concentration 0.0135mol/L joins in the monomer solution, separates to obtain cadmium telluride/monomer solution.Take by weighing the azo-bis-isobutyl cyanide of 0.0150g, be dissolved in 2.0g cadmium telluride/monomer solution.The octadecyl dimethyl that takes by weighing embodiment 5 is dissolved in the middle of the 20mL water vinyl Bian ammonium chloride 0.1000g, obtains emulsifier aqueous solution.Cadmium telluride/the monomer solution that is dissolved with azo-bis-isobutyl cyanide is joined in the aqueous solution, stir and form emulsion., in 80 ℃ water-bath, reacted 8 hours then about 20 minutes to the logical nitrogen of this emulsion.After finishing reaction, centrifugal, wash with water and remove unnecessary emulsifying agent, be dispersed in aqueous phase at last.Contrast (Fig. 6 A and Fig. 6 B) as can be known by fluorescence photo and light field photo, made the fluorescent microsphere of the vinylbenzene/divinyl benzene copolymer that contains cadmium telluride semiconductor nano particulate.With toluene swell microballoon 24 back centrifugations, the fluorescence spectrum of the upper strata stillness of night and absorption spectrum show that the cadmium telluride nanoparticle is retained in the polymer spheres fully as shown in Figure 7, thereby are to link to each other by chemical bond between explanation nanoparticle and the polymer drops.
Embodiment 16:
Two decyls that take by weighing embodiment 8 are dissolved in the 2.0mL methyl methacrylate vinyl benzyl ammonio methacrylate 0.0150g.Get cadmium telluride semiconductor nano particulate (fluorescent emission centre wavelength is in the 600 nanometers) aqueous solution 1.0mL of the Thiovanic acid finishing of embodiment 1, concentration 0.0150mol/L, join in the methyl methacrylate solution, separate obtaining cadmium telluride/methyl methacrylate solution.Take by weighing the 0.0150g azo-bis-isobutyl cyanide, be dissolved in 1.0g cadmium telluride/methyl methacrylate.The octadecyl dimethyl that takes by weighing embodiment 5 is dissolved in the 20mL water vinyl Bian ammonium chloride 0.1000g.Cadmium telluride/methyl methacrylate the solution that is dissolved with azo-bis-isobutyl cyanide is added to aqueous phase, stirs and form emulsion.Logical nitrogen reacted 6.5 hours in 80 ℃ water-bath about 20 minutes then in this emulsion.After finishing reaction, centrifugal, wash with water and remove unnecessary emulsifying agent, be dispersed in aqueous phase at last, made the polymethylmethacrylate fluorescent microsphere that contains cadmium telluride semiconductor nano particulate.
Embodiment 17:
The didecyl alkyl methyl that takes by weighing embodiment 8 is to vinyl benzyl chlorination ammonium 0.0150g, be dissolved in the 2mL vinyl cyanide, get cadmium telluride semiconductor nano particulate (fluorescent emission centre wavelength is in the 640 nanometers) aqueous solution 1mL of the thiohydracrylic acid finishing of embodiment 2, concentration 0.0125mol/L, under agitation join in the above-mentioned monomer, separate obtaining cadmium telluride/acrylonitrile solution.Take by weighing the 0.0300g azo-bis-isobutyl cyanide, be dissolved in 1.5g cadmium telluride/acrylonitrile solution.The octadecyl dimethyl that takes by weighing embodiment 5 is dissolved in the middle of the 20mL water vinyl Bian ammonium chloride 0.1200g.Cadmium telluride/the acrylonitrile solution that is dissolved with azo-bis-isobutyl cyanide is added to aqueous phase, stirs and form emulsion.Led to nitrogen 20 minutes to this emulsion, in 80 ℃ water-bath, reacted 6.5 hours then.After finishing reaction, centrifugal, wash with water and remove unnecessary emulsifying agent, be dispersed in aqueous phase at last.Made the polyacrylonitrile fluorescent microsphere that contains cadmium telluride semiconductor nano particulate.
Embodiment 18
Two decyls that take by weighing embodiment 8 are dissolved in the 2.0mL methyl acrylate vinyl benzyl ammonio methacrylate 0.0150g.Get cadmium selenide semiconductor nano particulate (fluorescent emission centre wavelength is in the 610 nanometers) aqueous solution 1.0mL of the Thiovanic acid finishing of embodiment 4, concentration 0.0150mol/L joins in the methyl acrylate solution, separates to obtain cadmium selenide/methyl acrylate solution.Take by weighing the 0.0150g azo-bis-isobutyl cyanide, be dissolved in 1.0g cadmium selenide/methyl acrylate.The octadecyl dimethyl that takes by weighing embodiment 5 is dissolved in the 20mL water vinyl Bian ammonium chloride 0.1000g.Cadmium selenide/methyl acrylate the solution that is dissolved with azo-bis-isobutyl cyanide is added to aqueous phase, stirs and form emulsion.Logical nitrogen reacted 6.5 hours in 80 ℃ water-bath about 20 minutes then in this emulsion.After finishing reaction, centrifugal, wash with water and remove unnecessary emulsifying agent, be dispersed in aqueous phase at last, made the polymethyl acrylate fluorescent microsphere that contains cadmium selenide semiconductor nano particulate.
Embodiment 19
Two decyls that take by weighing embodiment 8 are dissolved in the 2.0mL vinylbenzene vinyl benzyl ammonio methacrylate 0.0150g.Get cadmium sulfide nano particulate (fluorescent emission centre wavelength is in the 550 nanometers) aqueous solution 1.0mL of Thiovanic acid finishing, concentration 0.0150mol/L joins in the vinylbenzene, separates to obtain Cadmium Sulfide/styrene solution.Take by weighing the 0.0150g azo-bis-isobutyl cyanide, be dissolved in 1.0g Cadmium Sulfide/vinylbenzene.The octadecyl dimethyl that takes by weighing embodiment 5 is dissolved in the 20mL water vinyl Bian ammonium chloride 0.1000g.Cadmium Sulfide/the styrene solution that is dissolved with azo-bis-isobutyl cyanide is added to aqueous phase, stirs and form emulsion.Logical nitrogen reacted 6.5 hours in 80 ℃ water-bath about 20 minutes then in this emulsion.After finishing reaction, centrifugal, wash with water and remove unnecessary emulsifying agent, be dispersed in aqueous phase at last, made the polystyrene fluorescent microsphere that contains the CdS semiconduct nanoparticle.
Embodiment 20:
The trioctylphosphine that takes by weighing embodiment 9 is to vinyl benzyl chlorination ammonium 0.0163g, be dissolved in the 2mL butyl methacrylate, get cadmium telluride semiconductor nano particulate (fluorescent emission centre wavelength is in the 630 nanometers) aqueous solution 1mL of the thiohydracrylic acid finishing of embodiment 2, concentration 0.0250mol/L, under agitation join in the above-mentioned monomer, separate obtaining cadmium telluride/butyl methacrylate solution.Take by weighing the 0.0221g benzoyl peroxide, be dissolved in 1.5g cadmium telluride/butyl methacrylate solution.The octadecyl dimethyl that takes by weighing embodiment 5 is dissolved in the middle of the 20mL water vinyl Bian ammonium chloride 0.1000g.Cadmium telluride/butyl methacrylate the solution that is dissolved with benzoyl peroxide is added to aqueous phase, stirs and form emulsion.Led to nitrogen 20 minutes to this emulsion, in 75 ℃ water-bath, reacted 8 hours then.After finishing reaction, centrifugal, wash with water and remove unnecessary emulsifying agent, be dispersed in aqueous phase at last, made the poly-n-butyl methacrylate fluorescent microsphere that contains cadmium telluride semiconductor nano particulate.
Embodiment 21:
The didecyl alkyl methyl that takes by weighing embodiment 8 is dissolved in the mix monomer of 2.0mL vinylbenzene and 1.0mL methyl methacrylate vinyl benzyl chlorination ammonium 0.0150g.Get cadmium telluride semiconductor nano particulate (fluorescent emission centre wavelength is in the 630 nanometers) aqueous solution 1.5mL of the thiohydracrylic acid finishing of embodiment 2, concentration 0.0135mol/L joins in the monomer solution, separates to obtain cadmium telluride/monomer solution.Take by weighing the azo-bis-isobutyl cyanide of 0.0150g, be dissolved in 2.0g cadmium telluride/monomer solution.The octadecyl dimethyl that takes by weighing embodiment 5 is dissolved in the middle of the 20mL water vinyl Bian ammonium chloride 0.1000g, obtains emulsifier aqueous solution.Cadmium telluride/the monomer solution that is dissolved with azo-bis-isobutyl cyanide is joined in the aqueous solution, stir and form emulsion., in 80 ℃ water-bath, reacted 8 hours then about 20 minutes to the logical nitrogen of this emulsion.After finishing reaction, centrifugal, wash with water and remove unnecessary emulsifying agent, be dispersed in aqueous phase at last.Contrast (Fig. 8 A and Fig. 8 B) as can be known by fluorescence photo and light field photo, made the fluorescent microsphere of the styrene/methacrylic acid methyl terpolymer that contains cadmium telluride semiconductor nano particulate.
Embodiment 22:
The didecyl alkyl methyl that takes by weighing embodiment 8 is dissolved in the 1.0mL styrene monomer vinyl benzyl chlorination ammonium 0.0150g.Get cadmium telluride semiconductor nano particulate (fluorescent emission centre wavelength is in the 630 nanometers) aqueous solution 5mL of the Thiovanic acid finishing of embodiment 1, concentration 0.0025mol/L, join in the monomer solution, separate the cadmium telluride/styrene solution that obtains being dissolved with red fluorescence.The didecyl alkyl methyl that takes by weighing embodiment 8 is dissolved in the 2.0mL vinylbenzene vinyl benzyl chlorination ammonium 0.0300g.Get cadmium telluride semiconductor nano particulate (fluorescent emission centre wavelength is in the 580 nanometers) aqueous solution 2.0mL of the thiohydracrylic acid finishing of embodiment 2, concentration 0.0125mol/L, join in the styrene solution, separate the cadmium telluride/styrene solution that obtains being dissolved with green fluorescence.Get the cadmium telluride/styrene solution 225 μ L that are dissolved with red fluorescence, the cadmium telluride/styrene solution that is dissolved with green fluorescence with 1.8mL is mixed.Take by weighing the 0.0250g azo-bis-isobutyl cyanide, be dissolved in the above-mentioned styrene solution of 1.6g.The octadecyl dimethyl that takes by weighing embodiment 5 is dissolved in the 20mL water vinyl Bian ammonium chloride 0.1000g, obtains emulsifier aqueous solution.The styrene solution that is dissolved with azo-bis-isobutyl cyanide is joined in the aqueous solution, stir and form emulsion., in 80 ℃ water-bath, reacted 6.5 hours then about 20 minutes to the logical nitrogen of this emulsion.After finishing reaction, centrifugal, wash with water and remove unnecessary emulsifying agent, be dispersed in aqueous phase at last.Fig. 9 A and Fig. 9 B are the fluorescence photo and the light field photo of polystyrene fluorescent microsphere, and fluorescent microsphere shows yellow fluorescence; The contrast fluorescent microsphere fluorescence spectrum figure (Figure 10) as can be known, the cadmium telluride of two kinds of different fluorescent emission wavelength has been compound in the polystyrene microsphere simultaneously.
Embodiment 23:
The didecyl alkyl methyl that takes by weighing embodiment 8 is dissolved in the 1.0mL styrene monomer vinyl benzyl chlorination ammonium 0.0150g.Get cadmium telluride semiconductor nano particulate (fluorescent emission centre wavelength is in the 630 nanometers) aqueous solution 6mL of the Thiovanic acid finishing of embodiment 1, concentration 0.00125mol/L, join in the monomer solution, separate the cadmium telluride/styrene solution that obtains being dissolved with red fluorescence.The didecyl alkyl methyl that takes by weighing embodiment 7 is dissolved in the 2.0mL vinylbenzene vinyl benzyl chlorination ammonium 0.0300g.Get cadmium telluride semiconductor nano particulate (fluorescent emission centre wavelength is in the 580 nanometers) aqueous solution 2.0mL of the thiohydracrylic acid finishing of embodiment 2, concentration 0.0125mol/L, join in the styrene solution, separate the cadmium telluride/styrene solution that obtains being dissolved with green fluorescence.Get the cadmium telluride/styrene solution 450 μ L that are dissolved with red fluorescence, the cadmium telluride/styrene solution that is dissolved with green fluorescence with 1.8mL is mixed.Take by weighing the 0.0250g azo-bis-isobutyl cyanide, be dissolved in the above-mentioned styrene solution of 1.6g.The octadecyl dimethyl that takes by weighing embodiment 5 is dissolved in the 20mL water vinyl Bian ammonium chloride 0.1000g, obtains emulsifier aqueous solution.The styrene solution that is dissolved with azo-bis-isobutyl cyanide is joined in the aqueous solution, stir and form emulsion., in 80 ℃ water-bath, reacted 6.5 hours then about 20 minutes to the logical nitrogen of this emulsion.After finishing reaction, centrifugal, wash with water and remove unnecessary emulsifying agent, be dispersed in aqueous phase at last.Figure 11 A and Figure 11 B are the fluorescence photo and the light field photo of polystyrene fluorescent microsphere, fluorescent microsphere shows reddish-brown fluorescence, show that emission center wavelength has increased at the cadmium telluride content of 630 nanometers in the fluorescent microsphere, increase corresponding with this kind particulate loading in the initial feed ratio.
Embodiment 24:
Fluorescent microsphere and anti-CEA chimeric antibody composite liberation that embodiment 11 is obtained obtain anti-CEA chimeric antibody-fluorescent microsphere coupling matter solution.Colorectal carcinoma carninomatosis people perform the operation the sample paraffin section through taking off of routine is cured go into water treatment after, dripping corresponding antibodies concentration is antibody-fluorescent microsphere coupling matter of 10mg/L, 37 degree were hatched 30 minutes, wash 3 times after transparent, mounting, the power and the position of CEA male fluorescence represented in microscopically observation.The fluorescent microsphere of different emission and corresponding antibody coupling connection can be used for many antigens and detect simultaneously.
Embodiment 25:
The aqueous solution of chloraurate 0.15mL that gets 2wt% joins in the 30mL water, is heated to boiling; Add the sodium citrate aqueous solution 0.45mL of 2wt% during boiling, refluxed 30 minutes, cooling obtains gold sol.
Embodiment 26:
Get the two carboxy polyethylene glycol of 5 mmole ferric acetyl acetonades and 2.5 mmoles and join in the 50mL alpha-pyrrolidone, refluxed 10 hours, cooling obtains the ferriferrous oxide nano particulate.
Embodiment 27:
The didecyl alkyl methyl that takes by weighing embodiment 8 is dissolved in the 2.0mL vinylbenzene vinyl benzyl chlorination ammonium 0.2000g.Get the gold sol 30mL of embodiment 25, under agitation join in the above-mentioned monomer, separate obtaining gold/styrene solution.Take by weighing the azo-bis-isobutyl cyanide of 0.0150g, be dissolved in 1.5g gold/styrene solution.The octadecyl dimethyl that takes by weighing embodiment 5 is dissolved in the middle of the 20mL water vinyl Bian ammonium chloride 0.1000g, obtains being dissolved with the aqueous solution of tensio-active agent.Gold/styrene solution is joined in the aqueous solution, stir and form emulsion.In 80 ℃ water-bath, reacted 8 hours then about 20 minutes to logical nitrogen in the middle of this emulsion.After finishing reaction, centrifugal, wash with water and remove unnecessary emulsifying agent, be dispersed in aqueous phase at last.Throw electromicroscopic photograph (12B) as can be known by scanning electron microscope (12A) and ultrathin section(ing), made the polystyrene microsphere that contains gold nano particulate.
Embodiment 28:
The didecyl alkyl methyl that takes by weighing embodiment 8 is dissolved in the 2.0mL vinylbenzene vinyl benzyl chlorination ammonium 0.2000g.Get the Z 250 aqueous solution 3mL of embodiment 26, under agitation join in the above-mentioned monomer, separate obtaining Z 250/styrene solution.Take by weighing the azo-bis-isobutyl cyanide of 0.0150g, be dissolved in 1.5g Z 250/styrene solution.The octadecyl dimethyl that takes by weighing embodiment 5 is dissolved in the middle of the 20mL water vinyl Bian ammonium chloride 0.1000g, obtains being dissolved with the aqueous solution of tensio-active agent.Z 250/styrene solution is joined in the aqueous solution, stir and form emulsion.In 80 ℃ water-bath, reacted 8 hours then about 20 minutes to logical nitrogen in the middle of this emulsion.After finishing reaction, centrifugal, wash with water and remove unnecessary emulsifying agent, be dispersed in aqueous phase at last.Throw electromicroscopic photograph (14B) as can be known by scanning electron microscope (13A) and ultrathin section(ing), made the polystyrene microsphere that contains the ferriferrous oxide nano particulate.
Embodiment 29:
The didecyl alkyl methyl that takes by weighing embodiment 8 is dissolved in the 2.0mL vinylbenzene vinyl benzyl chlorination ammonium 0.2000g.Get the gold sol 10mL of embodiment 25, under agitation join in the above-mentioned monomer, separate obtaining gold/styrene solution.The didecyl alkyl methyl that takes by weighing embodiment 8 is dissolved in the 2.0mL vinylbenzene vinyl benzyl chlorination ammonium 0.2000g.Get citric acid and receive stable silver nano-particle aqueous solution 10mL, under agitation join in the above-mentioned monomer, separate obtaining silver/styrene solution.Two kinds of solution mixing are contained gold and silver nano-particle/styrene solution simultaneously.Take by weighing the azo-bis-isobutyl cyanide of 0.0150g, be dissolved in 1.5g and contain in gold and the silver nano-particle/styrene solution.The octadecyl dimethyl that takes by weighing embodiment 5 is dissolved in the middle of the 20mL water vinyl Bian ammonium chloride 0.1000g, obtains being dissolved with the aqueous solution of tensio-active agent.To contain gold and silver nano-particle/vinylbenzene and join in the aqueous solution, and stir and form emulsion.In 80 ℃ water-bath, reacted 8 hours then about 20 minutes to logical nitrogen in the middle of this emulsion.After finishing reaction, centrifugal, wash with water and remove unnecessary emulsifying agent, be dispersed in aqueous phase at last.Obtained containing the polystyrene microsphere of gold and silver nano-particle.
Embodiment 30:
The didecyl alkyl methyl that takes by weighing embodiment 8 is dissolved in the 2.0mL vinylbenzene vinyl benzyl chlorination ammonium 0.2000g.Get platinum iron nanoparticle aqueous solution 3mL, under agitation join in the above-mentioned monomer, separate obtaining platinum iron/styrene solution.Take by weighing the azo-bis-isobutyl cyanide of 0.0150g, be dissolved in 1.5g platinum iron/styrene solution.The octadecyl dimethyl that takes by weighing embodiment 5 is dissolved in the middle of the 20mL water vinyl Bian ammonium chloride 0.1000g, obtains being dissolved with the aqueous solution of tensio-active agent.Platinum iron/styrene solution is joined in the aqueous solution, stir and form emulsion.In 80 ℃ water-bath, reacted 8 hours then about 20 minutes to logical nitrogen in the middle of this emulsion.After finishing reaction, centrifugal, wash with water and remove unnecessary emulsifying agent, be dispersed in aqueous phase at last.Made the polystyrene microsphere that contains platinum iron nanoparticle.
Embodiment 31
The didecyl alkyl methyl that takes by weighing embodiment 8 is dissolved in the 2.0mL vinylbenzene vinyl benzyl chlorination ammonium 0.2000g.Get the Z 250 aqueous solution 3mL of embodiment 26, under agitation join in the above-mentioned monomer, separate obtaining Z 250/styrene solution.The didecyl alkyl methyl that takes by weighing embodiment 8 is dissolved in the 2.0mL vinylbenzene vinyl benzyl chlorination ammonium 0.2000g.Get the cobalt oxide nanometer 3mL that the two carboxy polyethylene glycol in surface are modified, under agitation join in the above-mentioned monomer, separate obtaining cobalt oxide/styrene solution.Two kinds of solution are mixed.Take by weighing the azo-bis-isobutyl cyanide of 0.0150g, be dissolved in the 1.5g mixing solutions.The octadecyl dimethyl that takes by weighing embodiment 5 is dissolved in the middle of the 20mL water vinyl Bian ammonium chloride 0.1000g, obtains being dissolved with the aqueous solution of tensio-active agent.To contain Z 250 and cobalt oxide nanoparticle/styrene solution joins in the aqueous solution, and stir and form emulsion.In 80 ℃ water-bath, reacted 8 hours then about 20 minutes to logical nitrogen in the middle of this emulsion.After finishing reaction, centrifugal, wash with water and remove unnecessary emulsifying agent, be dispersed in aqueous phase at last.Obtained containing the polystyrene microsphere of Z 250 and cobalt oxide nanoparticle.
Embodiment 32:
The didecyl alkyl methyl that takes by weighing embodiment 8 is dissolved in the 2.0mL vinylbenzene vinyl benzyl chlorination ammonium 0.2000g.Get the Z 250 aqueous solution 3mL of embodiment 26, under agitation join in the above-mentioned monomer, separate obtaining Z 250/styrene solution.Get cadmium telluride semiconductor nano particulate (fluorescent emission centre wavelength is in the 630 nanometers) aqueous solution 5mL of the Thiovanic acid finishing of embodiment 1, concentration 0.0025mol/L, join in the monomer solution, separate the cadmium telluride/styrene solution that obtains being dissolved with red fluorescence.Two kinds of nanoparticles/styrene solution are mixed, take by weighing the azo-bis-isobutyl cyanide of 0.0150g, be dissolved in the mixing solutions that 1.5g obtains.The octadecyl dimethyl that takes by weighing embodiment 5 is dissolved in the middle of the 20mL water vinyl Bian ammonium chloride 0.1000g, obtains being dissolved with the aqueous solution of tensio-active agent.The mixed monomer solution that is dissolved with initiator is joined in the aqueous solution, stir and form emulsion.In 80 ℃ water-bath, reacted 8 hours then about 20 minutes to logical nitrogen in the middle of this emulsion.After finishing reaction, centrifugal, wash with water and remove unnecessary emulsifying agent, be dispersed in aqueous phase at last.The multi-functional microballoon that is had fluorescence and magnetic simultaneously.
Embodiment 33:
At first four kinds of different antibody (anti-HER2, anti-CEA chimeric antibody, monoclonal antibody 9E10, monoclonal antibody 1E2) are respectively fixed to KRYSTAL
TMOn the hole wall in four holes in (Britain Porvair company) black 24 orifice plates, then, the adding cell culture fluid is stand-by.To mix overnight incubation according to magnetic fluorescent microspheres (emission wavelength 530 nanometers) and the anti-HER2 that embodiment 32 prepares; The magnetic fluorescent microspheres (emission wavelength 650 nanometers) and the anti-CEA chimeric antibody of embodiment 32 preparations are mixed, and overnight incubation is carried out magnetic respectively and is separated, and can obtain antibody-magnetic fluorescent microspheres conjugate.Anti-HER2-magnetic fluorescent microspheres coupling matter and anti-CEA-magnetic fluorescent microspheres coupling matter mixed solution are splashed into gastric carcinoma cells N87 (surface of cell membrane high expression level HER2 antigen), in human colon cancer cell LS180 (surface of cell membrane high expression level CEA antigen) and other normal gastric mucosas and the small intestinal mucosa cell mixture, through 37 ℃ of overnight incubation, again through magnetic separation and concentration antibody-magnetic fluorescent microspheres-cancerous tumor cell (surface of cell membrane high expression level corresponding antigens) mixture, next, the rich long-pending pregnant solution of material through disperseing again to obtain splash into respectively in the above micropore that is fixed with antibody, after hatching 30 minutes, with the PBS solution that is dissolved with 0.25%BSA purge 3 times repeatedly, at last, porous plate places under the fluorescent microscope and observes: the position of fixing anti-HER2, green fluorescence occurs, show to occur gastric carcinoma cells N87 in the cell mixing liquid; Red fluorescence appears in the position of fixing anti-CEA, shows to occur human colon cancer cell LS180 in the cell mixing liquid.
Embodiment 34
The magnetic microsphere that contains Z 250 that obtains is combined with Anti-HER 2, inject then by gastric carcinoma cells N87 (surface of cell membrane high expression level HER2 antigen) and other normal gastric mucosas and small intestinal mucosa cell mixture, through 37 ℃ of overnight incubation, separate through magnetic again and just gastric carcinoma cells N87 is separated from other normal cell.
From above-mentioned each embodiment and remaining experiment, we can draw to draw a conclusion: (1) utilizes two kinds of response type surfactant actives, the inorganic nano-particle of the different surfaces stablizer being modified by radical polymerization is compound in the polymkeric substance, obtains the functional polymer microballoon.(2) the semiconductor nano particulate of different fluorescent emission wavelength can be compound in the same polymer microballoon, be launched the microballoon of different colours fluorescence simultaneously; Adjust feed ratio, different colours intensity of fluorescence in the may command fluorescent microsphere is expected to realize fluorescence-encoded.(3) multi-functional microballoon is directly mixed with antibody, obtain the fluorescent microsphere that coupling is associated with antibody, can be used for highly sensitive biological detection by electrostatic adsorption.
Claims (12)
1. polymer microballoon that contains inorganic nano-particle, it is characterized in that: the water-soluble inorganic nanoparticle is embedded in the inside of polymer microballoon, and the water-soluble inorganic nanoparticle combines with chemical bond with polymer molecular chain by the carbon-carbon double bond on the reactable surfactant molecule chain;
Described polymkeric substance is selected from polystyrene, polydivinylbenezene, polyethylene glycol dimethacrylate, polyvinyl acetate, polyacrylic ester, the multipolymer of one or more in the polymethacrylate;
The two alkyl chain methyl of the number that described reactable tensio-active agent is a carbon atom in the alkyl chain between 8～18 to vinyl benzyl chlorination ammonium or trialkyl to vinyl benzyl chlorination ammonium; Perhaps the monoalkyl chain dimethyl of the carbon atom number of alkyl chain between 6～18 is to vinyl benzyl chlorination ammonium;
Described inorganic nano-particle is selected from one or more the mixture in semiconductor nano particulate, noble metal nano particulate or the metal oxide nanoparticles.
2. microballoon according to claim 1 is characterized in that: the water-soluble semi conductor nanoparticle that includes different fluorescent emission wavelength in the described polymer microballoon that contains the semiconductor nano particulate.
3. microballoon according to claim 1 and 2 is characterized in that: described semiconductor nano particulate is selected from one or more the mixture in Cadmium Sulfide, zinc sulphide, zunsober, lead sulfide, cadmium selenide, zinc selenide, lead selenide, cadmium telluride, tellurium mercury, lead telluride or the silicon nanometer particle.
4. microballoon according to claim 1 is characterized in that: described noble metal nano particulate is selected from one or more the mixture in gold and silver, iron, platinum, nickel, cobalt, palladium, the manganese, or contains the alloy nano particles of above-mentioned metallic element.
5. microballoon according to claim 1 is characterized in that: described metal oxide nanoparticles is selected from one or more the mixture in Z 250, ferric oxide, manganese oxide, cobalt oxide, nickel oxide, the zinc oxide.
6. microballoon according to claim 1 is characterized in that: the described particle diameter that contains the polymer microballoon of inorganic nano-particle is between 200 nanometers to 5 micron.
7. according to claim 2,4,5 or 6 described microballoons, it is characterized in that: the particle diameter of described particulate is between 1～100 nanometer.
8. microballoon according to claim 3 is characterized in that: the particle diameter of described particulate is between 1～100 nanometer.
9. preparation method according to each described microballoon of claim 1～8, this method comprises water-soluble inorganic nanoparticle, reactable tensio-active agent, is dissolved with the preparation of the inorganic nano-particle/monomer solution of initiator, it is characterized in that:
(1) the reactable surfactant A is dissolved in the oil-soluble monomer, in this monomer solution, add the water-soluble inorganic nanoparticle aqueous solution, stirring and segregation obtains inorganic nano-particle/monomer solution, then, initiator is dissolved in inorganic nano-particle/monomer solution; The concentration of reactable surfactant A in monomer is 0.1%～10wt%; Number with inorganic nano-particle calculates, and the concentration of the inorganic nano-particle aqueous solution is 2.5 * 10
-7～2.5 * 10
-3Between the mol/L; The inorganic nano-particle aqueous solution and monomeric volume ratio are between 1: 0.2～1: 2; Initiator and monomeric mass ratio are between 1: 20～1: 1000;
(2) the reactable surfactant B is soluble in water, inorganic nano-particle/the monomer solution that is dissolved with initiator that step (1) is obtained joins in the above-mentioned aqueous solution, stirs, and forms emulsion oil-in-water, logical rare gas element deoxygenation, emulsion system carries out polyreaction; Wherein the ratio of the amount of substance of reactable surfactant B and water is between 1: 250～1: 10000, and the mass ratio of inorganic nano-particle/monomer solution and water is between 1: 4～1: 100; After reaction finished, centrifugation went out to contain the polymer microballoon of inorganic nano-particle;
Described monomer is selected from vinylbenzene, Vinylstyrene, ethylene glycol dimethacrylate, vinyl-acetic ester, the monomeric mixture of one or more in the acrylate;
Described reactable surfactant A is two alkyl chain methyl to vinyl benzyl chlorination ammonium or trialkyl to vinyl benzyl chlorination ammonium, and the number of carbon atom is between 8～18 in the alkyl chain;
Described reactable surfactant B be monoalkyl chain dimethyl to vinyl benzyl chlorination ammonium, the carbon atom number of alkyl chain is between 6～18;
Described inorganic nano-particle is selected from one or more the mixture in semiconductor nano particulate, noble metal nano particulate or the metal oxide nanoparticles.
10. method according to claim 9, it is characterized in that: when inorganic nano-particle is fluorescent nano particles, nanoparticle/the monomer solution that is dissolved with initiator of described step (2) is the nanoparticle/monomer solution that contains different fluorescent emission wavelength, obtains containing the polymer fluorescent microspheres of the semiconductor nano particulate of various different fluorescent emission wavelength.
11. method according to claim 10, it is characterized in that: the described polymer fluorescent microspheres that contains the semiconductor nano particulate of various different fluorescent emission wavelength, the fluorescence intensity of the semiconductor nano particulate of its different fluorescent emission wavelength are to realize by the ratio between semiconductor nano particulate/monomer solution of controlling various different fluorescent emission wavelength.
12. one kind according to each described purposes that contains the polymer microballoon of inorganic nano-particle of claim 1～8, it is characterized in that: the polymer fluorescent microspheres that contains the semiconductor nano particulate be used for preparing the joint-detection that is used for immunodetection, protein chip, and cell or biological tissue in the microballoon fluorescent probe of fluorescence synchronous detection of different zones; Or
The magnetic fluorescence complex microsphere that contains magnetic nanometer particles and fluorescent nano particles is used for preparing the reagent of the high-sensitivity detection of the joint-detection that is used for immunodetection, protein chip, the fluorescence synchronous detection that reaches different zones in cell or the biological tissue, bacterium and virus; Or
Contain that the magnetic nanometer particles polymer microballoon is used for preparing when being used for immunodetection, autologous bone marrow transplantation the cancer cells removed wherein, separates other karyocytes, and the reagent of isolated cell device.
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