CN110484234A - A kind of fluorescent microsphere and its preparation and fluorescence-encoded method - Google Patents
A kind of fluorescent microsphere and its preparation and fluorescence-encoded method Download PDFInfo
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- CN110484234A CN110484234A CN201810458170.2A CN201810458170A CN110484234A CN 110484234 A CN110484234 A CN 110484234A CN 201810458170 A CN201810458170 A CN 201810458170A CN 110484234 A CN110484234 A CN 110484234A
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- 239000004005 microsphere Substances 0.000 title claims abstract description 130
- 238000002360 preparation method Methods 0.000 title claims abstract description 46
- 238000000034 method Methods 0.000 title abstract description 17
- 239000007850 fluorescent dye Substances 0.000 claims abstract description 111
- 239000003153 chemical reaction reagent Substances 0.000 claims abstract description 87
- BLRPTPMANUNPDV-UHFFFAOYSA-N Silane Chemical compound [SiH4] BLRPTPMANUNPDV-UHFFFAOYSA-N 0.000 claims abstract description 47
- 229910000077 silane Inorganic materials 0.000 claims abstract description 46
- 230000004913 activation Effects 0.000 claims abstract description 42
- 238000005859 coupling reaction Methods 0.000 claims abstract description 33
- 239000002904 solvent Substances 0.000 claims abstract description 15
- 229920001296 polysiloxane Polymers 0.000 claims abstract description 14
- 239000004593 Epoxy Substances 0.000 claims abstract description 13
- 150000004678 hydrides Chemical class 0.000 claims abstract description 13
- FALRKNHUBBKYCC-UHFFFAOYSA-N 2-(chloromethyl)pyridine-3-carbonitrile Chemical compound ClCC1=NC=CC=C1C#N FALRKNHUBBKYCC-UHFFFAOYSA-N 0.000 claims abstract description 12
- 229940014800 succinic anhydride Drugs 0.000 claims abstract description 12
- 239000005046 Chlorosilane Substances 0.000 claims abstract description 10
- KOPOQZFJUQMUML-UHFFFAOYSA-N chlorosilane Chemical compound Cl[SiH3] KOPOQZFJUQMUML-UHFFFAOYSA-N 0.000 claims abstract description 10
- BUZRAOJSFRKWPD-UHFFFAOYSA-N isocyanatosilane Chemical class [SiH3]N=C=O BUZRAOJSFRKWPD-UHFFFAOYSA-N 0.000 claims abstract description 8
- FHIMHLLFPNMXKI-UHFFFAOYSA-N S(=O)(=O)=[SiH]C1=CC=CC=C1.[Cl] Chemical compound S(=O)(=O)=[SiH]C1=CC=CC=C1.[Cl] FHIMHLLFPNMXKI-UHFFFAOYSA-N 0.000 claims abstract description 7
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims description 66
- 239000000377 silicon dioxide Substances 0.000 claims description 33
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 20
- 239000000975 dye Substances 0.000 claims description 11
- 238000003756 stirring Methods 0.000 claims description 11
- 235000019441 ethanol Nutrition 0.000 claims description 10
- BOTDANWDWHJENH-UHFFFAOYSA-N Tetraethyl orthosilicate Chemical compound CCO[Si](OCC)(OCC)OCC BOTDANWDWHJENH-UHFFFAOYSA-N 0.000 claims description 9
- 229920000036 polyvinylpyrrolidone Polymers 0.000 claims description 9
- 235000013855 polyvinylpyrrolidone Nutrition 0.000 claims description 9
- 239000001267 polyvinylpyrrolidone Substances 0.000 claims description 9
- 239000000460 chlorine Substances 0.000 claims description 8
- 229910052801 chlorine Inorganic materials 0.000 claims description 8
- 239000011806 microball Substances 0.000 claims description 8
- 238000002156 mixing Methods 0.000 claims description 5
- 239000007787 solid Substances 0.000 claims description 5
- 239000006228 supernatant Substances 0.000 claims description 5
- KSCAZPYHLGGNPZ-UHFFFAOYSA-N 3-chloropropyl(triethoxy)silane Chemical group CCO[Si](OCC)(OCC)CCCCl KSCAZPYHLGGNPZ-UHFFFAOYSA-N 0.000 claims description 4
- OWIKHYCFFJSOEH-UHFFFAOYSA-N Isocyanic acid Chemical compound N=C=O OWIKHYCFFJSOEH-UHFFFAOYSA-N 0.000 claims description 4
- XLJMAIOERFSOGZ-UHFFFAOYSA-N anhydrous cyanic acid Natural products OC#N XLJMAIOERFSOGZ-UHFFFAOYSA-N 0.000 claims description 4
- CWAFVXWRGIEBPL-UHFFFAOYSA-N ethoxysilane Chemical group CCO[SiH3] CWAFVXWRGIEBPL-UHFFFAOYSA-N 0.000 claims description 4
- 150000002240 furans Chemical class 0.000 claims description 4
- 125000001436 propyl group Chemical group [H]C([*])([H])C([H])([H])C([H])([H])[H] 0.000 claims description 4
- 150000001335 aliphatic alkanes Chemical class 0.000 claims description 3
- -1 chlorine sulfonyl-phenyl silicon Chemical compound 0.000 claims description 3
- 239000000463 material Substances 0.000 claims description 3
- ARYZCSRUUPFYMY-UHFFFAOYSA-N methoxysilane Chemical compound CO[SiH3] ARYZCSRUUPFYMY-UHFFFAOYSA-N 0.000 claims description 3
- FRGPKMWIYVTFIQ-UHFFFAOYSA-N triethoxy(3-isocyanatopropyl)silane Chemical group CCO[Si](OCC)(OCC)CCCN=C=O FRGPKMWIYVTFIQ-UHFFFAOYSA-N 0.000 claims description 3
- 238000005119 centrifugation Methods 0.000 claims description 2
- 125000000956 methoxy group Chemical group [H]C([H])([H])O* 0.000 claims description 2
- GWEVSGVZZGPLCZ-UHFFFAOYSA-N Titan oxide Chemical compound O=[Ti]=O GWEVSGVZZGPLCZ-UHFFFAOYSA-N 0.000 claims 2
- XUIMIQQOPSSXEZ-UHFFFAOYSA-N Silicon Chemical compound [Si] XUIMIQQOPSSXEZ-UHFFFAOYSA-N 0.000 claims 1
- 125000001495 ethyl group Chemical group [H]C([H])([H])C([H])([H])* 0.000 claims 1
- 229910052710 silicon Inorganic materials 0.000 claims 1
- 239000010703 silicon Substances 0.000 claims 1
- 239000004408 titanium dioxide Substances 0.000 claims 1
- 239000011325 microbead Substances 0.000 abstract description 34
- 239000004698 Polyethylene Substances 0.000 description 47
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 25
- 239000000243 solution Substances 0.000 description 23
- 239000008367 deionised water Substances 0.000 description 18
- 229910021641 deionized water Inorganic materials 0.000 description 18
- 125000003700 epoxy group Chemical group 0.000 description 10
- 230000008878 coupling Effects 0.000 description 9
- 238000010168 coupling process Methods 0.000 description 9
- MHMNJMPURVTYEJ-UHFFFAOYSA-N fluorescein-5-isothiocyanate Chemical compound O1C(=O)C2=CC(N=C=S)=CC=C2C21C1=CC=C(O)C=C1OC1=CC(O)=CC=C21 MHMNJMPURVTYEJ-UHFFFAOYSA-N 0.000 description 9
- XEEYBQQBJWHFJM-UHFFFAOYSA-N iron Substances [Fe] XEEYBQQBJWHFJM-UHFFFAOYSA-N 0.000 description 8
- 230000004048 modification Effects 0.000 description 8
- 238000012986 modification Methods 0.000 description 8
- 239000002994 raw material Substances 0.000 description 7
- 230000008859 change Effects 0.000 description 6
- SZVJSHCCFOBDDC-UHFFFAOYSA-N ferrosoferric oxide Chemical compound O=[Fe]O[Fe]O[Fe]=O SZVJSHCCFOBDDC-UHFFFAOYSA-N 0.000 description 6
- 230000008961 swelling Effects 0.000 description 6
- 238000001514 detection method Methods 0.000 description 5
- 239000002253 acid Substances 0.000 description 4
- 238000010791 quenching Methods 0.000 description 4
- 230000000171 quenching effect Effects 0.000 description 4
- VHUUQVKOLVNVRT-UHFFFAOYSA-N Ammonium hydroxide Chemical compound [NH4+].[OH-] VHUUQVKOLVNVRT-UHFFFAOYSA-N 0.000 description 3
- 108010053210 Phycocyanin Proteins 0.000 description 3
- 108010004729 Phycoerythrin Proteins 0.000 description 3
- 229920002873 Polyethylenimine Polymers 0.000 description 3
- 239000000908 ammonium hydroxide Substances 0.000 description 3
- SBRXLTRZCJVAPH-UHFFFAOYSA-N ethyl(trimethoxy)silane Chemical compound CC[Si](OC)(OC)OC SBRXLTRZCJVAPH-UHFFFAOYSA-N 0.000 description 3
- IQPQWNKOIGAROB-UHFFFAOYSA-N isocyanate group Chemical group [N-]=C=O IQPQWNKOIGAROB-UHFFFAOYSA-N 0.000 description 3
- 229920000642 polymer Polymers 0.000 description 3
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 description 3
- 238000002444 silanisation Methods 0.000 description 3
- 239000000758 substrate Substances 0.000 description 3
- 238000012360 testing method Methods 0.000 description 3
- PKDCQJMRWCHQOH-UHFFFAOYSA-N triethoxysilicon Chemical compound CCO[Si](OCC)OCC PKDCQJMRWCHQOH-UHFFFAOYSA-N 0.000 description 3
- MYMOFIZGZYHOMD-UHFFFAOYSA-N Dioxygen Chemical compound O=O MYMOFIZGZYHOMD-UHFFFAOYSA-N 0.000 description 2
- 241000790917 Dioxys <bee> Species 0.000 description 2
- 229910003978 SiClx Inorganic materials 0.000 description 2
- 230000009471 action Effects 0.000 description 2
- 230000003213 activating effect Effects 0.000 description 2
- 239000007864 aqueous solution Substances 0.000 description 2
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 2
- 239000012472 biological sample Substances 0.000 description 2
- 125000001309 chloro group Chemical group Cl* 0.000 description 2
- 238000005253 cladding Methods 0.000 description 2
- 239000007822 coupling agent Substances 0.000 description 2
- 229910001882 dioxygen Inorganic materials 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 230000007062 hydrolysis Effects 0.000 description 2
- 238000006460 hydrolysis reaction Methods 0.000 description 2
- 238000007885 magnetic separation Methods 0.000 description 2
- 238000012775 microarray technology Methods 0.000 description 2
- 239000005416 organic matter Substances 0.000 description 2
- 230000003647 oxidation Effects 0.000 description 2
- 238000007254 oxidation reaction Methods 0.000 description 2
- 239000001301 oxygen Substances 0.000 description 2
- 229910052760 oxygen Inorganic materials 0.000 description 2
- 150000003254 radicals Chemical class 0.000 description 2
- 239000000523 sample Substances 0.000 description 2
- RINCXYDBBGOEEQ-UHFFFAOYSA-N succinic anhydride Chemical group O=C1CCC(=O)O1 RINCXYDBBGOEEQ-UHFFFAOYSA-N 0.000 description 2
- VAXLYCYJSUHKRM-UHFFFAOYSA-N 3-isocyanatopropoxysilane Chemical compound [SiH3]OCCCN=C=O VAXLYCYJSUHKRM-UHFFFAOYSA-N 0.000 description 1
- VGGSQFUCUMXWEO-UHFFFAOYSA-N Ethene Chemical compound C=C VGGSQFUCUMXWEO-UHFFFAOYSA-N 0.000 description 1
- 239000005977 Ethylene Substances 0.000 description 1
- WHNWPMSKXPGLAX-UHFFFAOYSA-N N-Vinyl-2-pyrrolidone Chemical compound C=CN1CCCC1=O WHNWPMSKXPGLAX-UHFFFAOYSA-N 0.000 description 1
- DQMUQFUTDWISTM-UHFFFAOYSA-N O.[O-2].[Fe+2].[Fe+2].[O-2] Chemical compound O.[O-2].[Fe+2].[Fe+2].[O-2] DQMUQFUTDWISTM-UHFFFAOYSA-N 0.000 description 1
- 239000004793 Polystyrene Substances 0.000 description 1
- GLBSVKUVHOJJCG-UHFFFAOYSA-N S(=O)(=O)=C1CC=CC=C1.[Cl] Chemical compound S(=O)(=O)=C1CC=CC=C1.[Cl] GLBSVKUVHOJJCG-UHFFFAOYSA-N 0.000 description 1
- FQOUHJLOCJLNOH-UHFFFAOYSA-N [SiH4].N#CO Chemical compound [SiH4].N#CO FQOUHJLOCJLNOH-UHFFFAOYSA-N 0.000 description 1
- 239000011805 ball Substances 0.000 description 1
- 238000006555 catalytic reaction Methods 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 238000007334 copolymerization reaction Methods 0.000 description 1
- 238000002242 deionisation method Methods 0.000 description 1
- 238000003745 diagnosis Methods 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 125000001301 ethoxy group Chemical group [H]C([H])([H])C([H])([H])O* 0.000 description 1
- 229940056319 ferrosoferric oxide Drugs 0.000 description 1
- 238000007667 floating Methods 0.000 description 1
- 238000007306 functionalization reaction Methods 0.000 description 1
- 238000007689 inspection Methods 0.000 description 1
- 238000002372 labelling Methods 0.000 description 1
- 239000010410 layer Substances 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 238000011056 performance test Methods 0.000 description 1
- 229920003229 poly(methyl methacrylate) Polymers 0.000 description 1
- 229920002627 poly(phosphazenes) Polymers 0.000 description 1
- 238000006116 polymerization reaction Methods 0.000 description 1
- 239000004926 polymethyl methacrylate Substances 0.000 description 1
- 229920006389 polyphenyl polymer Polymers 0.000 description 1
- 229920002223 polystyrene Polymers 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 239000011241 protective layer Substances 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- PYWVYCXTNDRMGF-UHFFFAOYSA-N rhodamine B Chemical compound [Cl-].C=12C=CC(=[N+](CC)CC)C=C2OC2=CC(N(CC)CC)=CC=C2C=1C1=CC=CC=C1C(O)=O PYWVYCXTNDRMGF-UHFFFAOYSA-N 0.000 description 1
- 235000012239 silicon dioxide Nutrition 0.000 description 1
- 230000006641 stabilisation Effects 0.000 description 1
- 238000011105 stabilization Methods 0.000 description 1
- 239000004094 surface-active agent Substances 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
- C09K—MATERIALS FOR MISCELLANEOUS APPLICATIONS, NOT PROVIDED FOR ELSEWHERE
- C09K11/00—Luminescent, e.g. electroluminescent, chemiluminescent materials
- C09K11/02—Use of particular materials as binders, particle coatings or suspension media therefor
- C09K11/025—Use of particular materials as binders, particle coatings or suspension media therefor non-luminescent particle coatings or suspension media
-
- C—CHEMISTRY; METALLURGY
- C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
- C09K—MATERIALS FOR MISCELLANEOUS APPLICATIONS, NOT PROVIDED FOR ELSEWHERE
- C09K11/00—Luminescent, e.g. electroluminescent, chemiluminescent materials
- C09K11/06—Luminescent, e.g. electroluminescent, chemiluminescent materials containing organic luminescent materials
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
- G01N15/10—Investigating individual particles
- G01N15/14—Optical investigation techniques, e.g. flow cytometry
- G01N15/1434—Optical arrangements
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- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Materials Engineering (AREA)
- Organic Chemistry (AREA)
- Dispersion Chemistry (AREA)
- Physics & Mathematics (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Investigating Or Analysing Materials By The Use Of Chemical Reactions (AREA)
- Investigating, Analyzing Materials By Fluorescence Or Luminescence (AREA)
Abstract
A kind of fluorescent microsphere and its preparation and fluorescence encoding method, comprising the following steps: microballoon is mixed with silane reagent and solvent and is activated, activation microballoon is obtained；Wherein, silane reagent is at least one of epoxy radicals silicone hydride reagent, chlorosilane reagent, isocyanatosilanes reagent, succinic anhydride base silane reagent and chlorine sulfonyl-phenyl silane reagent；Coupling reaction is occurred into for activation microballoon and fluorescent dye in solvent, obtains fluorescent microsphere.The preparation method is coupled one or more fluorescent dyes in activation microsphere surface, so that it may obtain one or more different fluorescence signals.Adjust the mass volume ratio that every kind of fluorescent dye is added again simultaneously, so that it may obtain the microballoon of one or more different fluorescence varying strengths to get fluorescence-encoded micro-beads are arrived.Fluorescent microsphere obtained has many advantages, such as that fluorescent dye space encoder is big, and fluorescent dye intensity is high, and the fluorescence intensity for avoiding fluorescence-encoded micro-beads made from traditional preparation methods is unable to satisfy the shortcomings that requiring.
Description
Technical field
The present invention relates to biomolecular labeling technical fields, compile more particularly to a kind of fluorescent microsphere and its preparation and fluorescence
The method of code.
Background technique
Based on fluorescent microsphere and its fluorescence-encoded suspension microarray technology have in same sample multiple protein,
The progress such as cell factor are screened simultaneously and quantitative ability, have very high research and application value in medical diagnosis on disease field.It is outstanding
The core technology of floating microarray technology is the fluorescence-encoded micro-beads in detection process with unique tag signal, in order to compile fluorescence
Code microballoon can be applied to high-throughput multiple determination system, this requires fluorescence-encoded micro-beads to have high fluorescent, size
Uniform, surface is easy to the features such as functionalization, good biocompatibility.
Tradition prepares fluorescence-encoded micro-beads and mainly uses swelling method, which is that fluorescent dye swelling is entered polyphenyl
It is obtained during ethylene etc. is polymer microsphere supported.Furthermore also there is the preparation side that the fluorescent dye of silanization is coated on to microsphere surface
Method.But the fluorescent dye space encoder of these preparation methods is small, the fluorescent dye intensity of label is low, is unable to satisfy biochemistry detection
Demand.
In addition, fluorescent dye essence is the organic matter containing unsaturated bond, this type organic is easy by air or water
Dioxygen oxidation, so as to cause fluorescent quenching.When the fluorescent microsphere or fluorescence-encoded micro-beads of conventional method preparation are exposed to biology, water
In the samples such as solution, since these samples are usually present a large amount of oxygen molecules, it is easy to cause fluorescence to be quenched, and then influence instrument
Device detects fluorescence signal.
Summary of the invention
Based on this, the present invention provides a kind of fluorescent microsphere that fluorescence intensity is high and its preparation and fluorescence-encoded methods.
A kind of preparation method of fluorescent microsphere, comprising the following steps:
Step 1: microballoon being mixed with silane reagent and solvent and is activated, and obtains activation microballoon, wherein the silane
Reagent is epoxy radicals silicone hydride reagent, chlorosilane reagent, isocyanatosilanes reagent, succinic anhydride base silane reagent and chlorine sulphonyl benzene
At least one of base silane reagent；
Step 2: coupling reaction is occurred into for the activation microballoon and fluorescent dye in solvent, obtains the fluorescent microsphere.
The type of the fluorescent dye in the coupling reaction is two or more in one of the embodiments, is made
There must be the fluorescent microsphere of multicolor fluorescence.
It in one of the embodiments, further include that the fluorescent microsphere is subjected to coated with silica.
Aiming at the problem that fluorescent quenching in traditional fluorescent microsphere preparation method, the present invention also provides a kind of fluorescent microspheres
Coated with silica method, creatively in the surface coated silica of fluorescent microsphere or fluorescence-encoded micro-beads as protection
Layer, avoids fluorescent dye from being exposed in the samples such as biology, aqueous solution and cause fluorescent quenching, it is strong to further improve dye fluorescence
The stability of degree guarantees that it meets the needs of detection for a long time.It is activated in addition, also helping and being modified again in silica surface, into
One step is used for the label of different biological samples.
The step of fluorescent microsphere is carried out coated with silica in one of the embodiments, is as follows:
After the fluorescent microsphere, ethyl alcohol and polyvinylpyrrolidone are mixed, centrifugation, which is stood, removes supernatant, solid
It is mixed again with ethyl alcohol, and ethyl orthosilicate stirring is added to get the fluorescent microsphere for arriving coated with silica.
It in one of the embodiments, further include with the fluorescent microsphere replacement microballoon of coated with silica and according to step
The step of rapid one is activated, preparation activation fluorescent microsphere, and the activation is replaced with the activation fluorescent microsphere being prepared
Microballoon, and the step of carrying out coupling reaction according to step 2.
The silane reagent has methoxy silane structure or Ethoxysilane structure in one of the embodiments,.
The epoxy radicals silicone hydride reagent is 3- glycydoxy triethoxysilicane in one of the embodiments,
Alkane, the chlorosilane reagent are 3- chloropropyl triethoxysilane, and the isocyanatosilanes reagent is three second of isocyanatopropyl
Oxysilane, the succinic anhydride base silane reagent are dihydro -3- [3- (triethoxy silicon substrate) propyl] furans -2,5- diketone,
The chlorine sulfonyl-phenyl silane reagent is 2- (4- chlorine sulfonyl-phenyl) ethyl trimethoxy silane.
The fluorescent dye is itself to have the work that can be coupled at least one silane reagent in one of the embodiments,
Property group fluorescent dye or be modified with can at least one silane reagent be coupled active group fluorescent dye.
The mass volume ratio concentration in gradient variation of every kind of fluorescent dye of addition is controlled in one of the embodiments,
So that the fluorescent microsphere of the coding of one or more fluorescence varying strengths be made.
A kind of preparation method of fluorescent microsphere, comprising the following steps:
Step 1: after the microballoon with fluorescence is carried out coated with silica, then progress is mixed with silane reagent and solvent
Activation, obtains the activation microballoon with fluorescence, wherein the silane reagent is epoxy radicals silicone hydride reagent, chlorosilane reagent, different
At least one of cyanic acid silane reagent, succinic anhydride base silane reagent and chlorine sulfonyl-phenyl silane reagent；
Step 2: coupling reaction is occurred into for the activation microballoon and fluorescent dye in solvent, obtains the fluorescent microsphere.
The preparation method of the fluorescent microsphere is catalyzed using above-mentioned silane reagent without acid or alkaline condition, can be in neutrality
Condition directly with the acting activating microballoon of microballoon, and microsphere surface formed epoxy group, cl radical, isocyanate group, succinic acid anhydride group
Or chlorine sulfonyl-phenyl, furthermore also avoiding these groups, open loop or hydrolysis cause activation effect bad under acid or alkaline conditions
The problem of；Active microsphere obtained is not necessarily to action of coupling agents, coupling reaction directly can occur with fluorescent dye, obtain above-mentioned fluorescence
Microballoon.The present invention passes through the study found that being coated on microsphere surface compared to traditional swelling method or by the fluorescent dye of silanization
Preparation method, fluorescent microsphere made from the preparation method have fluorescent dye space encoder big, and the fluorescent dye intensity of label is high
The advantages that, be highly convenient for carrying out it is fluorescence-encoded, avoid the fluorescence intensities of fluorescence-encoded micro-beads made from traditional preparation methods without
The shortcomings that method is met the requirements, fluorescence-encoded micro-beads fluorescence intensity obtained is high, can satisfy the demand of detection.
The type of the fluorescent dye in the coupling reaction is two or more in one of the embodiments, is made
There must be the fluorescent microsphere of multicolor fluorescence.
The mass volume ratio concentration in gradient variation of every kind of fluorescent dye of addition is controlled in one of the embodiments,
So that the fluorescent microsphere of the coding of one or more fluorescence varying strengths be made.
Fluorescent microsphere made from the preparation method of the fluorescent microsphere of any of the above-described.
Detailed description of the invention
Fig. 1 is the photo of the fluorescent microsphere of coated with silica made from embodiment 1 under the microscope；
Fig. 2 is the fluorescent microsphere of the different fluorescence intensities obtained using APC coding of embodiment 2 in flow cytometer
Testing result；
Fig. 3 is the fluorescent microsphere of the different fluorescence intensities obtained using two kinds of fluorescent dyes of PE and APC coding of embodiment 6
Testing result in flow cytometer.
Specific embodiment
To facilitate the understanding of the present invention, below will to invention is more fully described, and give it is of the invention compared with
Good embodiment.But the invention can be realized in many different forms, however it is not limited to embodiment described herein.Phase
Instead, purpose of providing these embodiments is makes the disclosure of the present invention more thorough and comprehensive.
Unless otherwise defined, all technical and scientific terms used herein and belong to technical field of the invention
The normally understood meaning of technical staff is identical.Term as used herein in the specification of the present invention is intended merely to description tool
The purpose of the embodiment of body, it is not intended that in the limitation present invention.Term as used herein "and/or" includes one or more phases
Any and all combinations of the listed item of pass.
The preparation method of the fluorescent microsphere of one embodiment, includes the following steps S1~S2.
Step S1: microballoon being mixed with silane reagent and solvent and is activated, and obtains activation microballoon；Wherein, silane reagent
For epoxy radicals silicone hydride reagent, chlorosilane reagent, isocyanatosilanes reagent, succinic anhydride base silane reagent and chlorine sulfonyl-phenyl silicon
At least one of alkane reagent.
It, can be directly acting activating with microballoon in neutrallty condition using above-mentioned silane reagent without the catalysis of acid or alkaline condition
Microballoon, and epoxy group, cl radical, isocyanate group, succinic acid anhydride group or chlorine sulfonyl-phenyl are formed in microsphere surface, furthermore also avoid
These groups problems that open loop or hydrolysis cause the activation effect bad under acid or alkaline conditions；Active microsphere obtained without
Action of coupling agents is needed, coupling reaction directly can occur with fluorescent dye, obtain fluorescent microsphere.
The range of choice of the activated group of above-mentioned silane reagent is wide, enriches the selection of activated group, compensates for current city
The less disadvantage of its active coupling group type of the fluorescent dye sold on face, increases the versatility of microsphere surface label.
Further, solvent is water in step S1.
Specifically, the condition of activation is that 0.5~48h is mixed at 18~80 DEG C.
Silane reagent has methoxy silane structure or Ethoxysilane structure in one of the embodiments,.With first
The silane reagent of oxysilane structure or Ethoxysilane structure, which is easy to slough methoxy or ethoxy, to be hydrolyzed, so as to promote
It is carried out into coupling reaction.
Further, epoxy radicals silicone hydride reagent is 3- glycidyl ether oxypropyltriethoxysilane.
Further, chlorosilane reagent is 3- chloropropyl triethoxysilane.
Further, isocyanatosilanes reagent is isocyanatopropyl triethoxysilane.
Further, succinic anhydride base silane reagent is dihydro -3- [3- (triethoxy silicon substrate) propyl] furans -2,5- bis-
Ketone.
Further, chlorine sulfonyl-phenyl silane reagent is 2- (4- chlorine sulfonyl-phenyl) ethyl trimethoxy silane.
It further, also include with the above-mentioned silane reagent with identical active end and at other with oxysilane structure
Silane reagent on the basis of bridge joint coupling or modify that other active groups are obtained secondary or multiple silane reagent.
Specifically, the mass volume ratio of microballoon and silane reagent is (0.01mg~500mg): 1mL.Preferably, microballoon with
The mass volume ratio of silane reagent is (20mg~30mg): 1mL.
Further, microballoon is high molecular polymerization microballoon, magnetic microsphere or silicon dioxide microsphere.Specifically, polyphosphazene polymer
Conjunction microballoon is polystyrene microsphere, poly (methyl methacrylate) micro-sphere or polyethylene methacrylic acid methyl esters copolymerization microsphere.Specifically
Ground, magnetic microsphere are ferroso-ferric oxide microballoon or di-iron trioxide microballoon.Specifically, the surface of magnetic microsphere can be coated with dioxy
SiClx or polymer.
Further, step S1 microballoon is added in the form of microspheres solution, and the mass volume ratio of microspheres solution is (0.01mg
~500mg): 1mL.Preferably, the mass volume ratio of microspheres solution is (20mg~30mg): 1mL.
Specifically, the partial size of microballoon is 2~40 microns.Preferably, the partial size of microballoon is 5~10 microns.
Further, the mixing speed of step S1 mixing is 50~500rpm.
Step S2: coupling reaction is occurred into for activation microballoon and fluorescent dye in solvent, obtains fluorescent microsphere.
Specifically, the condition of coupling reaction is in 18~50 DEG C of 0.5~48h of reaction.Preferably, the condition of coupling reaction is
In 30~40 DEG C react 18~for 24 hours.
More specifically, the mixing speed of step S2 coupling reaction is 50~500rpm.Further, solvent is in step S2
Water.
Fluorescent dye can have the fluorescent dye for the active group that can be coupled at least one silane reagent for itself；It can also
For the fluorescent dye for being modified with the active group that can be coupled at least one silane reagent.Fluorescent dye further includes any glimmering
Photoinitiator dye is by bridge joint or modifies that active group that other can be coupled with silane reagent is obtained secondary or multiple fluorescent dye.
Further, fluorescent dye can for FITC (isocyanic acid fluorescent dye), RBITC (rhodamine), PE (phycoerythrin),
At least one of APC (other phycocyanin) or series connection fluorescent dye.Wherein FITC and RBITC have isocyanate group, PE and
APC has amino.Fluorescent dye be phycoerythrin, other phycocyanin or contain at least one of phycoerythrin and other phycocyanin
Tandem dye when coupling reaction can occurs with any of the above-described kind of silane reagent in it.When fluorescent dye is FITC and RBITC, need
Amino is first modified, or polyethyleneimine is added to bridge silane reagent and fluorescent dye in coupling reaction.
Further, selection has the fluorescent dye of amino.Further, the quality of the corresponding microballoon of active microsphere
Ratio with the quality of every kind of fluorescent dye is 100:(0.15~10).
In step S2, coupling reaction is occurred using two or more fluorescent dye and active microsphere, so that it may
Obtain the signal of a variety of different fluorescence.
Since the activated group on activation microballoon is more, fluorescent dye space encoder is big, by the amount for controlling fluorescent dye
Controllable fluorescence intensity, so that fluorescence intensity is adjustable, thus obtain the microballoon of different fluorescence intensities, i.e., single fluorescence-encoded micro-beads.
If the mass volume ratio for controlling a variety of fluorescent dyes changes in gradient, so that the fluorescence of the coding of a variety of fluorescence varying strengths be made
Microballoon is to get more fluorescence-encoded micro-beads.Here it is the fluorescence encoding methods of fluorescent microsphere.
That is to say, the mass volume ratio for controlling every kind of fluorescent dye of addition changes in gradient, to be made a kind of or more
The fluorescent microsphere of the coding of kind fluorescence varying strength.If the fluorescent dye being added at this time is a variety of, so that it may it is glimmering to obtain a variety of differences
The different fluorescence intensity signals of light, in summary the type of fluorescent dye and mass volume ratio variation, obtain a variety of fluorescence-encoded
Fluorescent microsphere.
Preferably, when using two or more fluorescent dye, a combination thereof can for PE and APC, FITC and
The combination of RBITC, APC and the fluorescent dye APC-Cy7 that connects.The preparation method has the advantages that fluorescent dye space encoder is big,
Therefore a variety of fluorescent dyes are allowed while being encoded, and fluorescence-encoded micro-beads fluorescence intensity obtained is high.Specifically, PE and APC
Mass ratio be 100:(0.01~10000).More specifically, the mass ratio of PE and APC is 100:(20~200).
Specifically, the PE of 1mg is dissolved in 1mL deionized water, obtains PE dye solution.APC is dissolved in 1mL deionization
In water, APC dye solution is obtained.PE and APC according to (80,120), (80,100), (100,100) (80,80), (80,110),
(60,80)、(40,80)、(30,80)、(60,60)、(120,60)、(110,50)、(80,50)、(60,40)、(70,40)、
(60,30) it is combined with (50,50)；Wherein PE and APC refers to according to (80,120) is added 80 μ L of PE solution, and APC solution 120 is added
μ L, it is other similar.
The present invention passes through the study found that being coated on microsphere surface compared to traditional swelling method or by the fluorescent dye of silanization
Preparation method, fluorescent microsphere made from the preparation method has fluorescent dye space encoder big, the fluorescent dye intensity of label
The advantages that high, the fluorescence intensity for avoiding fluorescence-encoded micro-beads made from traditional preparation methods are unable to satisfy the shortcomings that requiring, system
The fluorescence-encoded micro-beads fluorescence intensity obtained is high.
Furthermore swelling method is compared, which also has microsphere supported not easy to stick, size uniformity, and what is be evenly distributed is excellent
Point.
It in one of the embodiments, further include the step that fluorescent microsphere or fluorescence-encoded micro-beads are carried out to coated with silica
Suddenly.Fluorescent dye essence is the organic matter containing unsaturated bond, this type organic is easy by the dioxygen oxidation in air or water, from
And lead to fluorescent quenching.Therefore the present invention creatively makees in the surface coated silica of fluorescent microsphere or fluorescence-encoded micro-beads
For protective layer, fluorescent dye is avoided to be exposed in the samples such as biology, aqueous solution, is led so as to avoid a large amount of oxygen present in sample
It causes fluorescence to be quenched, and then influences the problem of instrument detects fluorescence signal, so further improve the stabilization of dye fluorescence
Property, guarantee that it meets the needs of detection for a long time.In addition, the silane reagent activated again in silica surface modification is also helped,
Label for different biological samples.
Specifically, the step of fluorescent microsphere or fluorescence-encoded micro-beads being subjected to coated with silica include the following steps: by
Fluorescent microsphere or fluorescence-encoded micro-beads, ethyl alcohol and polyvinylpyrrolidone (PVP) are stood after 20~70 DEG C of 0.5~48h of stirring
Remove supernatant, solid mixes with ethyl alcohol again, and be added ethyl orthosilicate (TEOS) in 20~70 DEG C of 0.5~48h of stirring to get
To the fluorescent microsphere of coated with silica or the fluorescence-encoded micro-beads of coated with silica.PVP is as surfactant, with dioxy
SiClx and fluorescent dye all have good affinity, are conducive to coated with silica.
Preferably, fluorescent microsphere or fluorescence-encoded micro-beads, ethyl alcohol and polyvinylpyrrolidone stir to 2 in 30~50 DEG C~
After 4h, stand and remove supernatant, solid mix with ethyl alcohol again, and be added ethyl orthosilicate and ammonium hydroxide in 30~50 DEG C of stirrings 2~
4h to get coated with silica fluorescent microsphere or fluorescence-encoded micro-beads.
Specifically, above-mentioned fluorescent microsphere or fluorescence-encoded micro-beads correspond to the quality and polyvinylpyrrolidone of raw material microballoon
The ratio of quality is 0.5~4:1.Preferably, above-mentioned fluorescent microsphere or fluorescence-encoded micro-beads correspond to the quality of raw material microballoon and gather
The ratio of the quality of vinylpyrrolidone is 2:1.
Specifically, above-mentioned fluorescent microsphere or fluorescence-encoded micro-beads correspond to the quality of raw material microballoon and the volume of ethyl orthosilicate
Ratio be (10mg~30mg): 1mL.Preferably, above-mentioned fluorescent microsphere or fluorescence-encoded micro-beads correspond to the quality of raw material microballoon
Ratio with the volume of ethyl orthosilicate is 20mg:1mL.
Specifically, above-mentioned fluorescent microsphere or fluorescence-encoded micro-beads correspond to the ratio of the quality of raw material microballoon and the volume of ammonium hydroxide
For (10mg~30mg): 1mL.Preferably, above-mentioned fluorescence-encoded micro-beads correspond to the ratio of the quality of raw material microballoon and the volume of ammonium hydroxide
Value is 20mg:1mL.
Specifically, when microballoon is magnetic microsphere, removing supernatant can be used magnetic separation, and solid and solvent are separated.
It further, further include the microballoon replaced with the microballoon of coated with silica in above-mentioned steps S1, and according to step
S1 is activated, the microballoon of the coated with silica activated, and carries out coupling reaction according to step S2.Specifically, the work
The step of change and coupling reaction, can be identical as step S1~S2, can also be not limited to above-mentioned raw materials.
The present invention also provides the preparation methods of the fluorescent microsphere of an embodiment.The preparation method is glimmering using having had
The microballoon of light, after first carrying out coated with silica, then the microballoon substituted in above-mentioned preparation method carries out step S1 and step S2.
It is understood that wherein having had the microballoon of fluorescence can be made for either step in the preparation method of above-mentioned fluorescent microsphere
The microballoon with fluorescence, can also be the existing or other modes microballoon obtained with fluorescence.
It is understood that step S1, step S2 and other steps can be similar with above-mentioned preparation method in present embodiment.Such as:
The type of fluorescent dye in coupling reaction is two or more, and the fluorescent microsphere with multicolor fluorescence is made.Such as: control
The mass volume ratio concentration in gradient variation for making every kind of fluorescent dye being added, so that one or more fluorescence varying strengths be made
Coding fluorescent microsphere.
Further, the present invention also provides made from the preparation method of the fluorescence-encoded micro-beads of any of the above-described embodiment
Fluorescence-encoded micro-beads.
Fluorescence-encoded micro-beads made from the preparation method have fluorescent dye space encoder big, and the fluorescent dye of label is strong
Spend the advantages that high.
The following are specific embodiments.
Embodiment 1
Microsphere surface activation: the 20mg/mL Fe of 30mL deionized water, 1mL is added in 250mL three-necked flask3O4Microballoon
Solution and 1mL epoxy radicals silicone hydride reagent (3- glycidyl ether oxypropyltriethoxysilane), in the revolving speed of 200rpm and 50
It is stirred at DEG C for 24 hours to get to epoxy group modified activation microballoon.Fe3O4The partial size of microballoon is 5 microns.
Microballoon is coupled fluorescent dye: 30mL deionized water, above-mentioned epoxy group modified work being added in 250mL three-necked flask
Change microballoon and the 80 μ L of PE fluorescent dye of 1mg/mL is added, is stirred at the revolving speed of 200rpm and 30 DEG C for 24 hours to get to PE
The microballoon of fluorescence.
Embodiment 2
It is substantially the same manner as Example 1, the difference is that fluorescent dye is APC, that is, the APC dye solution of 1mg/mL is added
10 μ L, 30 μ L, 60 μ L, 120 μ L, obtaining tool, there are four types of the microballoons of different APC fluorescence intensities.
Embodiment 3,4
Embodiment 3,4 and embodiment 1,2 are essentially identical, the difference is that Fe3O4The concentration of microspheres solution is respectively
10mg/mL and 30mg/mL, equally in different Fe3O4It is available with PE or APC fluorescence under conditions of microspheres solution concentration
Microballoon.
Embodiment 5
It is substantially the same manner as Example 1, the difference is that carrying out silica to the obtained PE fluorescent microsphere of embodiment 1
Cladding, its step are as follows:
Above-mentioned fluorescent microsphere is added in 250mL three-necked flask, 30mL ethyl alcohol and 10mg polyvinylpyrrolidone are added
(PVP), 2h is stirred at 30 DEG C, is redissolved after magnetic separation into 30mL ethyl alcohol, and 1mL ethyl orthosilicate TEOS is added, in
Stirring 2h is at the revolving speed of 200rpm and 30 DEG C to get the fluorescent microsphere for arriving corresponding coated with silica.
Embodiment 6
Microsphere surface activation: the 20mg/mL Fe of 30mL deionized water, 1mL is added in 250mL three-necked flask3O4Microballoon
Solution and 1mL epoxy radicals silicone hydride reagent (3- glycidyl ether oxypropyltriethoxysilane), in the revolving speed of 200rpm and 50
It is stirred at DEG C for 24 hours to get to epoxy group modified activation microballoon.Fe3O4The partial size of microballoon is 5 microns.
Microballoon is coupled fluorescent dye: 30mL deionized water, above-mentioned epoxy group modified work being added in 250mL three-necked flask
Change microballoon, PE and APC, is stirred at the revolving speed of 200rpm and 30 DEG C for 24 hours to get the microballoon for arriving fluorescent dye PE and APC.PE and
APC fluorescent dye is combined according to different quality, carries out coupling fluorescent dye step respectively, it is different to obtain PE with APC fluorescence intensity
Coding microball.The proportion of the PE and APC fluorescent dye of different quality volume ratio combination is as follows:
The PE fluorescent dye of 1mg is dissolved in 1mL deionized water, PE dye solution is obtained.By APC be dissolved in 1mL go from
In sub- water, APC dye solution is obtained.PE and APC according to (80,120), (80,100), (100,100), (80,80), (80,110),
(60,80)、(40,80)、(30,80)、(60,60)、(120,60)、(110,50)、(80,50)、(60,40)、(70,40)、
(60,30) and (50,50) combination, wherein PE and APC refers to according to (80,120) is added 80 μ L of PE fluorescent dye solution, is added
APC fluorescent dye solution 120 μ L, it is other similar.
Embodiment 7
Microsphere surface activation: the 20mg/mL Fe of 30mL deionized water, 1mL is added in 250mL three-necked flask3O4Microballoon
Solution and 1mL chlorosilane reagent (3- chloropropyl triethoxysilane), are stirred for 24 hours, i.e., at the revolving speed of 200rpm and 50 DEG C
Obtain the activation microballoon of chloro modification.
Microballoon is coupled fluorescent dye: the activation of 30mL deionized water, the modification of above-mentioned chloro being added in 250mL three-necked flask
Microballoon, PE and APC fluorescent dye are stirred for 24 hours at the revolving speed of 200rpm and 30 DEG C to get to PE and APC fluorescent dye microballoon.
PE and APC fluorescent dye carries out coupling fluorescent dye step respectively, it is different to obtain PE with APC fluorescence intensity according to various combination
Coding microball.The proportion of the PE and APC fluorescent dye of different quality volume ratio combination is same as Example 5.
Embodiment 8
Microsphere surface activation: the 20mg/mL Fe of 30mL deionized water, 1mL is added in 250mL three-necked flask3O4Microballoon
Solution and 1mL isocyanatosilanes reagent (isocyanatopropyl triethoxysilane), are stirred at the revolving speed of 200rpm and 50 DEG C
For 24 hours to get the activation microballoon modified to isocyanic acid.
Microballoon is coupled fluorescent dye: the work of 30mL deionized water, the modification of above-mentioned isocyanic acid being added in 250mL three-necked flask
Change microballoon, PE and APC fluorescent dye, is stirred at the revolving speed of 200rpm and 30 DEG C for 24 hours to get the microballoon for arriving PE and APC fluorescence.
PE and APC fluorescent dye is combined according to different quality, is carried out coupling fluorescent dye step respectively, is obtained PE and APC fluorescence intensity
Different coding microballs.The proportion that different quality combines PE and APC fluorescent dye is same as Example 6.
Embodiment 9
Microsphere surface activation: the 20mg/mL Fe of 30mL deionized water, 1mL is added in 250mL three-necked flask3O4Microballoon
Solution and 1mL succinic anhydride base silane reagent (dihydro -3- [3- (triethoxy silicon substrate) propyl] furans -2,5- diketone), in
Stirring 20h is at the revolving speed of 200rpm and 60 DEG C to get to the activation microballoon of succinic anhydride modification.
Microballoon is coupled fluorescent dye: 30mL deionized water being added in 250mL three-necked flask, above-mentioned succinic anhydride is modified
Microballoon, PE and APC fluorescent dye are activated, stirs 18h at the revolving speed of 200rpm and 40 DEG C to get PE and APC fluorescent microsphere is arrived.
PE and APC are combined according to different quality, are carried out coupling fluorescent dye step respectively, are obtained the different volume of PE with APC fluorescence intensity
Code microballoon.The proportion that different quality combines PE and APC fluorescent dye is same as Example 6.
Embodiment 10
Microsphere surface activation: the 20mg/mL Fe of 30mL deionized water, 1mL is added in 250mL three-necked flask3O4Microballoon
Solution and 1mL chlorine sulfonyl-phenyl silane reagent (2- (4- chlorine sulfonyl-phenyl) ethyl trimethoxy silane), in the revolving speed of 200rpm
Stirring 18h is at 70 DEG C to get to the activation microballoon of chlorine sulfonyl-phenyl modification.
Microballoon is coupled fluorescent dye: 30mL deionized water, the modification of above-mentioned chlorine sulfonyl-phenyl being added in 250mL three-necked flask
Activation microballoon, PE and APC fluorescent dye, at the revolving speed of 200rpm and 35 DEG C stir 20h to get to PE and APC fluorescence it is micro-
Ball.PE and APC fluorescent dye is combined according to different quality, carries out coupling fluorescent dye step respectively, it is strong to obtain PE and APC fluorescence
Spend different coding microballs.The proportion that different quality combines PE and APC fluorescent dye is same as Example 6.
Embodiment 11
Microsphere surface activation: the 20mg/mL Fe of 30mL deionized water, 1mL is added in 250mL three-necked flask3O4Microballoon
Solution and 1mL epoxy radicals silicone hydride reagent (3- glycidyl ether oxypropyltriethoxysilane), in the revolving speed of 200rpm and 50
It is stirred at DEG C for 24 hours to get to epoxy group modified activation microballoon.
Microballoon is coupled fluorescent dye: 30mL deionized water, above-mentioned epoxy group modified work being added in 250mL three-necked flask
Change microballoon, FITC and RBITC fluorescent dye, 50mg polyethyleneimine (PEI), stirred for 24 hours at the revolving speed of 200rpm and 30 DEG C,
Up to FITC and RBITC fluorescent microsphere.FITC and RBITC fluorescent dye is combined according to different quality, carries out coupling fluorescence respectively
Dyestuff step obtains the different coding microball of FITC with RBITC fluorescence intensity.Different quality combines FITC and RBITC fluorescence dye
The proportion of material is same as Example 6.
Embodiment 12
Microsphere surface activation: the 20mg/mL Fe of 30mL deionized water, 1mL is added in 250mL three-necked flask3O4Microballoon
Solution and 1mL epoxy radicals silicone hydride reagent (3- glycidyl ether oxypropyltriethoxysilane), in the revolving speed of 200rpm and 50
It is stirred at DEG C for 24 hours to get to epoxy group modified activation microballoon.
Microballoon is coupled fluorescent dye: 30mL deionized water, above-mentioned epoxy group modified work being added in 250mL three-necked flask
Change microballoon, APC and APC-Cy7 connect fluorescent dye, stirred at the revolving speed of 200rpm and 30 DEG C for 24 hours to get to APC and APC-
Cy7 fluorescent microsphere.APC and APC-Cy7 fluorescent dye is combined according to different quality, is carried out coupling fluorescent dye step respectively, is obtained
The coding microball different to APC with APC-Cy7 fluorescence intensity.The proportion of the APC and APC-Cy7 fluorescent dye of different quality combination
It is same as Example 6.
Embodiment 13
In Examples 1 to 4 and embodiment 6~12, method same as Example 5 can be used and carry out silica packet
It covers to get the fluorescence-encoded micro-beads of corresponding coated with silica are arrived.
Performance test is as follows:
The fluorescent microsphere of coated with silica made from embodiment 1 is observed under the microscope, it is as shown in Figure 1 to obtain photo.
As can be known from Fig. 1, the fluorescence-encoded micro-beads of the coated with silica are sticked without viscous, and size uniformity is evenly distributed.Embodiment 2~12
The fluorescent microsphere or fluorescence-encoded micro-beads and embodiment 5 and the resulting silica of embodiment 13 of uncoated silica obtained
The photo of the fluorescence-encoded micro-beads of cladding under the microscope is similar to Fig. 1.
Inspection of the fluorescent microsphere of the different fluorescence intensities of APC coding in flow cytometer will be used made from embodiment 2
It surveys as a result, as shown in Fig. 2.Abscissa FSC refers to forward scattering (Forward of the microballoon in flow cytometer
Scattering), directly related to the relative size of microspherulite diameter, ordinate is that fluorescent microsphere is measured by flow cytometer
Corresponding APC fluorescence intensity.It can be seen that obtained fluorescent microsphere is compared with known swelling method through the invention, roll into a ball
Cluster is assembled very much, and fluorescence intensity is uniform, and it is larger to can be used to coding fluorescence space.
The fluorescent microsphere that the different fluorescence intensities of two kinds of fluorescent dyes of PE and APC coding are used made from embodiment 6 is existed
Testing result in flow cytometer, as shown in Figure 3.Abscissa and ordinate are that fluorescent microsphere is surveyed by flow cytometer respectively
The fluorescence intensity of the PE and APC that obtain.It can also be seen that the microballoon cluster of two-dimensional fluoroscopic coding is still assembled very much, fluorescence is strong
Degree uniformly, has very big fluorescence-encoded space.
Each technical characteristic of embodiment described above can be combined arbitrarily, for simplicity of description, not to above-mentioned reality
It applies all possible combination of each technical characteristic in example to be all described, as long as however, the combination of these technical characteristics is not deposited
In contradiction, all should be considered as described in this specification.
The embodiments described above only express several embodiments of the present invention, and the description thereof is more specific and detailed, but simultaneously
It cannot therefore be construed as limiting the scope of the patent.It should be pointed out that coming for those of ordinary skill in the art
It says, without departing from the inventive concept of the premise, various modifications and improvements can be made, these belong to protection of the invention
Range.Therefore, the scope of protection of the patent of the invention shall be subject to the appended claims.
Claims (13)
1. a kind of preparation method of fluorescent microsphere, which comprises the following steps:
Step 1: microballoon being mixed with silane reagent and solvent and is activated, and obtains activation microballoon, wherein the silane reagent
For epoxy radicals silicone hydride reagent, chlorosilane reagent, isocyanatosilanes reagent, succinic anhydride base silane reagent and chlorine sulfonyl-phenyl silicon
At least one of alkane reagent；
Step 2: coupling reaction is occurred into for the activation microballoon and fluorescent dye in solvent, obtains the fluorescent microsphere.
2. the preparation method of fluorescent microsphere as described in claim 1, which is characterized in that the fluorescent dye in the coupling reaction
Type be two or more, be made with multicolor fluorescence fluorescent microsphere.
3. the preparation method of fluorescent microsphere as claimed in claim 1 or 2, which is characterized in that further include by the fluorescent microsphere
Carry out coated with silica.
4. the preparation method of fluorescent microsphere as claimed in claim 3, which is characterized in that the fluorescent microsphere is carried out titanium dioxide
The step of silicon coats is as follows:
By the fluorescent microsphere, ethyl alcohol and polyvinylpyrrolidone be mixed after, centrifugation stand remove supernatant, solid again with
Ethyl alcohol mixing, and ethyl orthosilicate stirring is added to get the fluorescent microsphere for arriving coated with silica.
5. the preparation method of fluorescent microsphere as claimed in claim 3, which is characterized in that further include glimmering with coated with silica
The step of light microballoon is replaced the microballoon and is simultaneously activated according to step 1, preparation activation fluorescent microsphere, and with being prepared
Activation fluorescent microsphere replace the activation microballoon, and the step of carrying out coupling reaction according to step 2.
6. the preparation method of fluorescent microsphere as claimed in claim 1 or 2, which is characterized in that the silane reagent has methoxy
Base silane structure or Ethoxysilane structure.
7. the preparation method of fluorescent microsphere as claimed in claim 1 or 2, which is characterized in that the epoxy radicals silicone hydride reagent is
3- glycidyl ether oxypropyltriethoxysilane, the chlorosilane reagent is 3- chloropropyl triethoxysilane, described
Isocyanatosilanes reagent is isocyanatopropyl triethoxysilane, and the succinic anhydride base silane reagent is dihydro -3- [3- (three
Ethyl-silicone) propyl] furans -2,5- diketone, the chlorine sulfonyl-phenyl silane reagent is 2- (4- chlorine sulfonyl-phenyl) ethyl three
Methoxy silane.
8. the preparation method of fluorescent microsphere as claimed in claim 1 or 2, which is characterized in that the fluorescent dye is tool itself
There is the fluorescent dye for the active group that can be coupled at least one silane reagent or be modified with can be even at least one silane reagent
The fluorescent dye of the active group of connection.
9. the preparation method of fluorescent microsphere as claimed in claim 1 or 2, which is characterized in that control every kind of fluorescence dye of addition
The mass volume ratio concentration in gradient of material changes, so that the fluorescent microsphere of the coding of one or more fluorescence varying strengths be made.
10. a kind of preparation method of fluorescent microsphere, which comprises the following steps:
Step 1: after the microballoon with fluorescence is carried out coated with silica, then mixing with silane reagent and solvent and activated,
Obtain the activation microballoon with fluorescence, wherein the silane reagent is epoxy radicals silicone hydride reagent, chlorosilane reagent, isocyanic acid
At least one of silane reagent, succinic anhydride base silane reagent and chlorine sulfonyl-phenyl silane reagent；
Step 2: coupling reaction is occurred into for the activation microballoon and fluorescent dye in solvent, obtains the fluorescent microsphere.
11. the preparation method of fluorescent microsphere as claimed in claim 10, which is characterized in that the fluorescence dye in the coupling reaction
The type of material is two or more, and the fluorescent microsphere with multicolor fluorescence is made.
12. the preparation method of fluorescent microsphere as described in claim 10 or 11, which is characterized in that control every kind of fluorescence of addition
The mass volume ratio concentration in gradient of dyestuff changes, so that the fluorescence that the coding of one or more fluorescence varying strengths is made is micro-
Ball.
13. the preparation method of fluorescent microsphere as described in any one of claims 1 to 9 or such as any one of claim 10~12 institute
Fluorescent microsphere made from the preparation method for the fluorescent microsphere stated.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113281317A (en) * | 2021-05-14 | 2021-08-20 | 北京指真生物科技有限公司 | Coded microsphere containing cyanine compounds, and preparation method and application thereof |
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