CN101787163B - Magnetic fluorescent microspheres and preparation method thereof - Google Patents
Magnetic fluorescent microspheres and preparation method thereof Download PDFInfo
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- 238000002360 preparation method Methods 0.000 title claims abstract description 28
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- 230000008961 swelling Effects 0.000 claims abstract description 16
- 239000004793 Polystyrene Substances 0.000 claims abstract description 15
- 229920002223 polystyrene Polymers 0.000 claims abstract description 15
- XDTMQSROBMDMFD-UHFFFAOYSA-N Cyclohexane Chemical compound C1CCCCC1 XDTMQSROBMDMFD-UHFFFAOYSA-N 0.000 claims abstract description 13
- 239000002122 magnetic nanoparticle Substances 0.000 claims abstract description 11
- 239000003795 chemical substances by application Substances 0.000 claims abstract description 9
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- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 8
- 238000004042 decolorization Methods 0.000 claims abstract description 7
- 239000006228 supernatant Substances 0.000 claims abstract description 7
- 230000005284 excitation Effects 0.000 claims abstract description 5
- 239000000203 mixture Substances 0.000 claims abstract description 3
- 239000002244 precipitate Substances 0.000 claims abstract 2
- 239000002096 quantum dot Substances 0.000 claims description 19
- 238000001132 ultrasonic dispersion Methods 0.000 claims description 11
- 230000005415 magnetization Effects 0.000 claims description 10
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 claims description 7
- 238000000034 method Methods 0.000 claims description 6
- 239000011259 mixed solution Substances 0.000 claims description 6
- LRHPLDYGYMQRHN-UHFFFAOYSA-N N-Butanol Chemical group CCCCO LRHPLDYGYMQRHN-UHFFFAOYSA-N 0.000 claims description 4
- PPBRXRYQALVLMV-UHFFFAOYSA-N Styrene Chemical compound C=CC1=CC=CC=C1 PPBRXRYQALVLMV-UHFFFAOYSA-N 0.000 claims description 3
- 240000007594 Oryza sativa Species 0.000 claims description 2
- 235000007164 Oryza sativa Nutrition 0.000 claims description 2
- 239000005083 Zinc sulfide Substances 0.000 claims description 2
- UHYPYGJEEGLRJD-UHFFFAOYSA-N cadmium(2+);selenium(2-) Chemical compound [Se-2].[Cd+2] UHYPYGJEEGLRJD-UHFFFAOYSA-N 0.000 claims description 2
- 239000002131 composite material Substances 0.000 claims description 2
- 239000002105 nanoparticle Substances 0.000 claims description 2
- 235000009566 rice Nutrition 0.000 claims description 2
- DRDVZXDWVBGGMH-UHFFFAOYSA-N zinc;sulfide Chemical compound [S-2].[Zn+2] DRDVZXDWVBGGMH-UHFFFAOYSA-N 0.000 claims description 2
- 239000002075 main ingredient Substances 0.000 claims 3
- VTLYFUHAOXGGBS-UHFFFAOYSA-N Fe3+ Chemical compound [Fe+3] VTLYFUHAOXGGBS-UHFFFAOYSA-N 0.000 claims 1
- 235000013334 alcoholic beverage Nutrition 0.000 claims 1
- JEIPFZHSYJVQDO-UHFFFAOYSA-N iron(III) oxide Inorganic materials O=[Fe]O[Fe]=O JEIPFZHSYJVQDO-UHFFFAOYSA-N 0.000 claims 1
- 229920003145 methacrylic acid copolymer Polymers 0.000 claims 1
- 229940117841 methacrylic acid copolymer Drugs 0.000 claims 1
- 229910052984 zinc sulfide Inorganic materials 0.000 claims 1
- 238000006243 chemical reaction Methods 0.000 abstract description 9
- 238000001514 detection method Methods 0.000 abstract description 7
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- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 description 3
- 239000000975 dye Substances 0.000 description 3
- 238000005516 engineering process Methods 0.000 description 3
- 239000007850 fluorescent dye Substances 0.000 description 3
- OKKJLVBELUTLKV-UHFFFAOYSA-N methanol Natural products OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 3
- 229920000642 polymer Polymers 0.000 description 3
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- 125000004123 n-propyl group Chemical group [H]C([H])([H])C([H])([H])C([H])([H])* 0.000 description 2
- 238000006116 polymerization reaction Methods 0.000 description 2
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- GDCRSXZBSIRSFR-UHFFFAOYSA-N ethyl prop-2-enoate;2-methylprop-2-enoic acid Chemical compound CC(=C)C(O)=O.CCOC(=O)C=C GDCRSXZBSIRSFR-UHFFFAOYSA-N 0.000 description 1
- 238000000684 flow cytometry Methods 0.000 description 1
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- 125000001449 isopropyl group Chemical group [H]C([H])([H])C([H])(*)C([H])([H])[H] 0.000 description 1
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- Luminescent Compositions (AREA)
Abstract
本发明涉及一种磁性荧光微球及其制备方法。磁性荧光微球粒径大小在5到10微米，荧光激发波长范围在400-700nm之间，荧光强度在24小时内不降低。将单分散羧基化聚苯乙烯微球加入溶胀剂，将磁性纳米微粒加入上述溶胀体系中；在脱色摇床上震荡12～48小时；用环己烷与乙醇的混合液清洗沉淀物，超声分散后，离心分离至上清液在紫外灯下为无色；将最终沉淀产物保存在1ml酒水中。与现有的磁性荧光微球相比，径均匀可控，荧光稳定性好，制备工艺简单，荧光编码种类多等特点，应用于生物大分子检测，既能对反应物进行快速分离和纯化，又能在一个反应管、孔内对待检样品的多个靶分子同时进行检测，可广泛应用于免疫学检测、核酸杂交、基因型分析等领域。
The invention relates to a magnetic fluorescent microsphere and a preparation method thereof. The particle size of the magnetic fluorescent microspheres is 5 to 10 microns, the fluorescence excitation wavelength range is between 400-700nm, and the fluorescence intensity does not decrease within 24 hours. Add monodisperse carboxylated polystyrene microspheres to the swelling agent, add magnetic nanoparticles to the above swelling system; shake on the decolorization shaker for 12 to 48 hours; wash the precipitate with a mixture of cyclohexane and ethanol, and ultrasonically disperse , and centrifuged until the supernatant was colorless under ultraviolet light; the final precipitated product was stored in 1ml wine water. Compared with the existing magnetic fluorescent microspheres, the diameter is uniform and controllable, the fluorescence stability is good, the preparation process is simple, and there are many types of fluorescence coding. It can also detect multiple target molecules of the sample to be tested in one reaction tube and hole at the same time, and can be widely used in immunological detection, nucleic acid hybridization, genotype analysis and other fields.
Description
Technical field
The present invention relates to a kind of magnetic fluorescent microspheres and preparation method thereof.
Background technology
Magnetic fluorescent microspheres is meant introduces magnetic nano-particle and fluorescent substance in the polymer microsphere, make microballoon have magnetic property and characteristic fluorescence simultaneously, and can be by the flow cytometry discriminance analysis.The magnetic fluorescent microspheres surface can be in conjunction with various biomolecules, corresponding target thing molecular reaction in these biomolecules and the sample, with the reporter group as the detection of biological reaction such as fluorescently-labeled mensuration substrate, target thing molecule is carried out qualitative and quantitative analysis by the measured value of fluorescent signal.Magnetic fluorescent microspheres has boundless biomedical applications scope.The applied magnetic fluorescent microsphere carries out biomacromolecule detection, can carry out sharp separation and purifying to reactant, the a plurality of target molecules that can treat the sample product in a reaction tubes, hole detect simultaneously again, can be widely used in fields such as immunology detection, nucleic acid hybridization, gene type assay.
Said magnetic fluorescent microspheres mainly refers to introduce magnetic nano-particle and prepares magnetic microsphere in the synthetic macromolecule microballoon, mix organic fluorescent dye more therein, thus the preparation magnetic fluorescent microspheres.This magnetic fluorescence technology main difficult technical and problem are, and be as follows:
1, preparation process is too complicated.In the preparation process, need earlier magnetic particle to be aggregated in the polymer microsphere, again organic dye is compound in the microballoon through behind the purifying then.Whole process need is many through step, has caused the increase of production cost, has limited the application of magnetic fluorescent microspheres.
2, Zhi Bei microballoon use properties is relatively poor.The index that influences the magnetic fluorescent microspheres use mainly is size, the homogeneity of microspherulite diameter, the magnetic property of microballoon and fluorescence property.Common preparation method is owing to add magnetic nanoparticle in the polymerization process of microballoon, cause that the microsphere surface for preparing is coarse, uniform particle diameter is relatively poor, has reduced the accuracy of signal in the fluidic cell testing process.The organic fluorescent dye poor stability of Shi Yonging simultaneously, shortcomings such as the easy cancellation of fluorescence make the fluorescence property of magnetic fluorescent microspheres reduce greatly.
Summary of the invention
The objective of the invention is to design and prepare a kind of magnetic fluorescent microspheres and preparation thereof of new functionization.
The preparation method of a kind of magnetic fluorescent microspheres of the present invention, step is as follows:
1) the monodisperse carboxyl polystyrene microsphere that takes by weighing 90～98mg is put into centrifuge tube, to wherein adding swelling agent, and ultra-sonic dispersion, standby;
2) measure the quantum dot of 1～5mg and the magnetic nanometer particles of 1～5mg, add in the above-mentioned swelling system;
3) with quantum dot, magnetic nanoparticle, monodisperse carboxyl polystyrene microsphere compound system is placed on the decolorization swinging table, shakes 12～48 hours;
4) with hexanaphthene and alcoholic acid mixed solution washing and precipitating thing, behind the ultra-sonic dispersion, centrifugation to supernatant liquor is colourless under ultraviolet lamp;
5) final precipitated product is kept in the drinks.
The correlation parameter of described monodisperse carboxyl polystyrene microsphere is as follows:
Main component: vinylbenzene and Sipacril 2739OF,
Particle diameter: 4～10 microns.
The correlation parameter of described quantum dot nano particle is as follows:
Main component: cadmium selenide or zinc sulphide,
Particle diameter: 5～10 nanometers,
The scope of emission wavelength: 400～700nm.
The correlation parameter of described magnetic rice corpuscles is as follows:
Main component: Z 250,
Particle diameter: 5～10 nanometers,
Saturation magnetization: 60～80emu/g.
Described swelling agent is the propyl carbinol and the trichloromethane of volume proportion 1: 1～1: 3.
Described hexanaphthene is 3: 1 with alcoholic acid mixeding liquid volume ratio.
Described drinks are that volume ratio is 1: 1 second alcohol and water.
Whole process of preparation simple and fast, preparation cycle are 12～48 hours, the productive rate height.Prepared magnetic fluorescent microspheres size 5 to 10 microns controlled, particle diameter is even; Prepared magnetic fluorescent microspheres can pass through the action of a magnetic field sharp separation in 30 minutes; Prepared magnetic fluorescent microspheres, fluorescence exciting wavelength is adjustable, excites scope between 400-700nm, the photochemical stable height, long-term exposure is under ultraviolet lamp, and fluorescence intensity did not reduce in 24 hours.
Effect of the present invention is:
1, uses single stage method to prepare the magnetic response fluorescent microsphere, simplify preparation process.Polymer microsphere by polymerization process elder generation preparation uniform particle diameter mixes magnetic particle and fluorescent substance in the microballoon then together, thereby can simplify separation and purifying number of times in the preparation process greatly.
2, use quantum dot to replace organic fluorescent substance, improve fluorescence property.Quantum dot is called inorganic nanocrystal again, because it has the optics of uniqueness and the extensive concern that electrical properties causes scientists, has become a bright spot of nanotechnology.Quantum dot demonstrates increasing application prospect at biological field, especially is used for aspects such as biomarker, biological detection and bio-imaging as fluorescent probe.Compare with the organic fluorescence molecule, quantum dot has the optical characteristics of many uniquenesses: its emission peak wavelength can be regulated by composition material and size, its excitation wavelength scope is very wide, have bigger stoke shift and narrow symmetric fluorescence spectra, thereby the characteristics that possessed the polynary emission of an elementary excitation provide the foundation for many flux detect.And different luminescent dye molecules need different excitation wavelengths, and emmission spectrum is wide, and are easily overlapped, thereby are difficult to use two or more luminescent dye molecules to carry out multi-color marking simultaneously simultaneously.In addition, quantum dot has good photochemical stability, can tolerate stronger exciting light and longer light emissioning cycle.
Characteristics such as in a word, compare with existing magnetic fluorescent microspheres, the magnetic fluorescent microspheres that the present invention relates to has the particle diameter homogeneous and controllable, and fluorescent stability is good, and preparation technology is simple, and fluorescence-encoded kind is many can be applicable to biomacromolecule detection, can
Reactant is carried out sharp separation and purifying, and a plurality of target molecules that can treat the sample product again in a reaction tubes, hole detect simultaneously, can be widely used in fields such as immunology detection, nucleic acid hybridization, gene type assay.
Description of drawings
Fig. 1: according to the stereoscan photograph of the magnetic fluorescent microspheres of embodiment 1 preparation.
Embodiment
Will the invention will be further elaborated in the following examples, but the invention is not restricted to this.
Embodiment 1:
(1) accurately taking by weighing the 90mg particle diameter is that 4 microns carboxylic polystyrene microsphere is put into centrifuge tube, to wherein add the 5ml volume proportion be 1: 1 propyl carbinol and trichloromethane as swelling agent, ultra-sonic dispersion, standby.
(2) accurately measure the 5mg particle diameter for the 7nm emission wavelength is that quantum dot and the 5mg particle diameter of 500nm is the magnetic nanometer particles of 5nm saturation magnetization 73emu/g with liquid-transfering gun, add in the above-mentioned swelling system.
(3) with quantum dot, magnetic nanoparticle, the carboxylic polystyrene microsphere complex systems is placed on the decolorization swinging table, shakes 36 hours.
(4) stopped reaction, centrifugation, with 2ml hexanaphthene alcohol mixed solution (hexanaphthene and alcoholic acid volume ratio are 3: 1), washing and precipitating thing, behind the ultra-sonic dispersion, centrifugation to supernatant liquor is colourless under ultraviolet lamp.
(5) final precipitated product is kept in the drinks (volume ratio of ethanol and water is 1: 1).The magnetic fluorescent microspheres particle diameter of preparation is 5 microns, and particle diameter is even, and emmission spectrum is 501nm, half-peak width, and the saturation magnetization that records is 61emu/g.(Figure 1 shows that the magnetic fluorescent microspheres of preparation)
Embodiment 2:
(1) accurately taking by weighing the 98mg particle diameter is that 8 microns carboxylic polystyrene microsphere is put into centrifuge tube, to wherein add the 5ml volume proportion be 1: 2 propyl carbinol and trichloromethane as swelling agent, ultra-sonic dispersion, standby.
(2) accurately measure the 1mg particle diameter for the 4nm emission wavelength is that quantum dot and the 1mg particle diameter of 610nm is the magnetic nanometer particles of 7nm saturation magnetization 60emu/g with liquid-transfering gun, add in the above-mentioned swelling system.
(3) with quantum dot, magnetic nanoparticle, the carboxylic polystyrene microsphere complex systems is placed on the decolorization swinging table, shakes 24 hours.
(4) stopped reaction, centrifugation, with 2ml hexanaphthene alcohol mixed solution (hexanaphthene and alcoholic acid volume ratio are 1: 1) washing and precipitating thing, behind the ultra-sonic dispersion, centrifugation to supernatant liquor is colourless under ultraviolet lamp.
(5) final precipitated product is kept in the drinks (volume ratio of ethanol and water is 1: 1).The magnetic fluorescent microspheres particle diameter of preparation is 8.2 microns, and particle diameter is even, and emmission spectrum is 610nm, half-peak width, and the saturation magnetization that records is 54.1emu/g.
Embodiment 3:
(1) accurately taking by weighing the 96mg particle diameter is that 10 microns carboxylic polystyrene microsphere is put into centrifuge tube, to wherein add the 5ml volume proportion be 1: 3 propyl carbinol and trichloromethane as swelling agent, ultra-sonic dispersion, standby.
(2) accurately measure the 2mg particle diameter for the 10nm emission wavelength is that quantum dot and the 2mg particle diameter of 400nm is the magnetic nanometer particles of 8nm saturation magnetization 80emu/g with liquid-transfering gun, add in the above-mentioned swelling system.
(3) with quantum dot, magnetic nanoparticle, the carboxylic polystyrene microsphere complex systems is placed on the decolorization swinging table, shakes 48 hours.
(4) stopped reaction, centrifugation, with 2ml hexanaphthene alcohol mixed solution (hexanaphthene and alcoholic acid volume ratio are 2: 1) washing and precipitating thing, behind the ultra-sonic dispersion, the supernatant liquor after centrifugation extremely separates is colourless under ultraviolet lamp.
(5) final precipitated product is kept in the drinks (volume ratio of ethanol and water is 1: 1).The magnetic fluorescent microspheres particle diameter of preparation is 10 microns, and particle diameter is even, and emmission spectrum is 403nm, half-peak width, and the saturation magnetization that records is 76.8emu/g.
Embodiment 4:
(1) accurately taking by weighing the 93mg particle diameter is that 6 microns carboxylic polystyrene microsphere is put into centrifuge tube, to wherein add the 5ml volume proportion be 1: 1 propyl carbinol and trichloromethane as swelling agent, ultra-sonic dispersion, standby.
(2) accurately measure the 4mg particle diameter for the 8.6nm emission wavelength is that quantum dot and the 3mg particle diameter of 700nm is the magnetic nanometer particles of 10nm saturation magnetization 65.4emu/g with liquid-transfering gun, add in the above-mentioned swelling system.
(3) with quantum dot, magnetic nanoparticle, the carboxylic polystyrene microsphere complex systems is placed on the decolorization swinging table, shakes 12 hours.
(4) stopped reaction, centrifugation, with 2ml hexanaphthene alcohol mixed solution (hexanaphthene and alcoholic acid volume ratio are 3: 1) washing and precipitating thing, behind the ultra-sonic dispersion, the supernatant liquor after centrifugation extremely separates is colourless under ultraviolet lamp.
(5) final precipitated product is kept in the drinks (volume ratio of ethanol and water is 1: 1).The magnetic fluorescent microspheres particle diameter of preparation is 6.4 microns, and particle diameter is even, and emmission spectrum is 703nm, half-peak width, and the saturation magnetization that records is 60.8emu/g.
Claims (7)
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| CN1693416B (en) * | 2005-04-29 | 2010-09-22 | 同济大学 | A kind of fluorescent microsphere and its spray-drying preparation method and application |
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