CN102676157B - Fluorescent microsphere marker and preparation method thereof - Google Patents
Fluorescent microsphere marker and preparation method thereof Download PDFInfo
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- CN102676157B CN102676157B CN201210150231.1A CN201210150231A CN102676157B CN 102676157 B CN102676157 B CN 102676157B CN 201210150231 A CN201210150231 A CN 201210150231A CN 102676157 B CN102676157 B CN 102676157B
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- 239000004005 microsphere Substances 0.000 title claims abstract description 74
- 239000003550 marker Substances 0.000 title claims abstract description 41
- 238000002360 preparation method Methods 0.000 title claims abstract description 20
- 239000000126 substance Substances 0.000 claims abstract description 30
- 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 claims abstract description 18
- PPBRXRYQALVLMV-UHFFFAOYSA-N Styrene Chemical group C=CC1=CC=CC=C1 PPBRXRYQALVLMV-UHFFFAOYSA-N 0.000 claims abstract description 17
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 62
- 238000006243 chemical reaction Methods 0.000 claims description 34
- 239000004793 Polystyrene Substances 0.000 claims description 25
- 239000002105 nanoparticle Substances 0.000 claims description 25
- 229920002223 polystyrene Polymers 0.000 claims description 25
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 claims description 24
- 238000003756 stirring Methods 0.000 claims description 21
- 229960004756 ethanol Drugs 0.000 claims description 19
- FZHAPNGMFPVSLP-UHFFFAOYSA-N silanamine Chemical class [SiH3]N FZHAPNGMFPVSLP-UHFFFAOYSA-N 0.000 claims description 18
- 238000005406 washing Methods 0.000 claims description 14
- 229910052757 nitrogen Inorganic materials 0.000 claims description 12
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 12
- 238000005576 amination reaction Methods 0.000 claims description 11
- 150000001282 organosilanes Chemical class 0.000 claims description 10
- 238000002604 ultrasonography Methods 0.000 claims description 9
- 239000006185 dispersion Substances 0.000 claims description 8
- 239000007788 liquid Substances 0.000 claims description 8
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 claims description 7
- 229960000935 dehydrated alcohol Drugs 0.000 claims description 7
- 239000001301 oxygen Substances 0.000 claims description 7
- 229910052760 oxygen Inorganic materials 0.000 claims description 7
- 239000000725 suspension Substances 0.000 claims description 7
- 229920000036 polyvinylpyrrolidone Polymers 0.000 claims description 6
- 239000001267 polyvinylpyrrolidone Substances 0.000 claims description 6
- 235000013855 polyvinylpyrrolidone Nutrition 0.000 claims description 6
- 235000019394 potassium persulphate Nutrition 0.000 claims description 5
- 239000004159 Potassium persulphate Substances 0.000 claims description 4
- USHAGKDGDHPEEY-UHFFFAOYSA-L potassium persulfate Chemical compound [K+].[K+].[O-]S(=O)(=O)OOS([O-])(=O)=O USHAGKDGDHPEEY-UHFFFAOYSA-L 0.000 claims description 4
- 229910052761 rare earth metal Inorganic materials 0.000 abstract description 26
- 150000002910 rare earth metals Chemical class 0.000 abstract description 22
- 238000001514 detection method Methods 0.000 abstract description 5
- 230000009286 beneficial effect Effects 0.000 abstract description 2
- 239000000243 solution Substances 0.000 description 11
- 238000000034 method Methods 0.000 description 10
- 239000000463 material Substances 0.000 description 9
- -1 rare earth ion Chemical class 0.000 description 9
- JGVWCANSWKRBCS-UHFFFAOYSA-N tetramethylrhodamine thiocyanate Chemical compound [Cl-].C=12C=CC(N(C)C)=CC2=[O+]C2=CC(N(C)C)=CC=C2C=1C1=CC=C(SC#N)C=C1C(O)=O JGVWCANSWKRBCS-UHFFFAOYSA-N 0.000 description 9
- 150000001875 compounds Chemical class 0.000 description 8
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 description 8
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N silicon dioxide Inorganic materials O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 7
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 6
- VEJHUGGPLQWGPR-UHFFFAOYSA-N 4,7-bis(3-chloro-2-sulfophenyl)-1,10-phenanthroline-2,9-dicarboxylic acid Chemical compound C=12C=CC3=C(C=4C(=C(Cl)C=CC=4)S(O)(=O)=O)C=C(C(O)=O)N=C3C2=NC(C(=O)O)=CC=1C1=CC=CC(Cl)=C1S(O)(=O)=O VEJHUGGPLQWGPR-UHFFFAOYSA-N 0.000 description 4
- 239000000178 monomer Substances 0.000 description 4
- 230000035945 sensitivity Effects 0.000 description 4
- 239000000377 silicon dioxide Substances 0.000 description 4
- 229910052693 Europium Inorganic materials 0.000 description 3
- 239000000872 buffer Substances 0.000 description 3
- 239000000460 chlorine Substances 0.000 description 3
- 229910052801 chlorine Inorganic materials 0.000 description 3
- LOKCTEFSRHRXRJ-UHFFFAOYSA-I dipotassium trisodium dihydrogen phosphate hydrogen phosphate dichloride Chemical compound P(=O)(O)(O)[O-].[K+].P(=O)(O)([O-])[O-].[Na+].[Na+].[Cl-].[K+].[Cl-].[Na+] LOKCTEFSRHRXRJ-UHFFFAOYSA-I 0.000 description 3
- 230000000694 effects Effects 0.000 description 3
- 230000005284 excitation Effects 0.000 description 3
- 229910010272 inorganic material Inorganic materials 0.000 description 3
- 239000011147 inorganic material Substances 0.000 description 3
- 239000002953 phosphate buffered saline Substances 0.000 description 3
- 229920000642 polymer Polymers 0.000 description 3
- 238000006116 polymerization reaction Methods 0.000 description 3
- 229960001866 silicon dioxide Drugs 0.000 description 3
- 235000012239 silicon dioxide Nutrition 0.000 description 3
- 238000013329 compounding Methods 0.000 description 2
- 238000006073 displacement reaction Methods 0.000 description 2
- GNBHRKFJIUUOQI-UHFFFAOYSA-N fluorescein Chemical compound O1C(=O)C2=CC=CC=C2C21C1=CC=C(O)C=C1OC1=CC(O)=CC=C21 GNBHRKFJIUUOQI-UHFFFAOYSA-N 0.000 description 2
- 238000001917 fluorescence detection Methods 0.000 description 2
- ZMZDMBWJUHKJPS-UHFFFAOYSA-N hydrogen thiocyanate Natural products SC#N ZMZDMBWJUHKJPS-UHFFFAOYSA-N 0.000 description 2
- GRHBQAYDJPGGLF-UHFFFAOYSA-N isothiocyanic acid Chemical compound N=C=S GRHBQAYDJPGGLF-UHFFFAOYSA-N 0.000 description 2
- 230000001795 light effect Effects 0.000 description 2
- 108020004707 nucleic acids Proteins 0.000 description 2
- 102000039446 nucleic acids Human genes 0.000 description 2
- 150000007523 nucleic acids Chemical class 0.000 description 2
- 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 2
- NHBSKWFWXDCHNF-UHFFFAOYSA-N 4,7-dichloro-3-phenyl-5-sulfo-1,10-phenanthroline-2,9-dicarboxylic acid Chemical compound ClC1=C(C(=NC2=C3N=C(C=C(C3=CC(=C12)S(=O)(=O)O)Cl)C(=O)O)C(=O)O)C1=CC=CC=C1 NHBSKWFWXDCHNF-UHFFFAOYSA-N 0.000 description 1
- NIAXQHHPSYAYHU-UHFFFAOYSA-N C(CC(=O)CC=O)CF Chemical compound C(CC(=O)CC=O)CF NIAXQHHPSYAYHU-UHFFFAOYSA-N 0.000 description 1
- 229910052692 Dysprosium Inorganic materials 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 108020004711 Nucleic Acid Probes Proteins 0.000 description 1
- 229910052772 Samarium Inorganic materials 0.000 description 1
- BLRPTPMANUNPDV-UHFFFAOYSA-N Silane Chemical compound [SiH4] BLRPTPMANUNPDV-UHFFFAOYSA-N 0.000 description 1
- 229910052771 Terbium Inorganic materials 0.000 description 1
- DGEZNRSVGBDHLK-UHFFFAOYSA-N [1,10]phenanthroline Chemical class C1=CN=C2C3=NC=CC=C3C=CC2=C1 DGEZNRSVGBDHLK-UHFFFAOYSA-N 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- FJFLGYOUAGKKNQ-UHFFFAOYSA-N benzene 1,10-phenanthroline Chemical group N1=CC=CC2=CC=C3C=CC=NC3=C12.C1=CC=CC=C1 FJFLGYOUAGKKNQ-UHFFFAOYSA-N 0.000 description 1
- 230000004071 biological effect Effects 0.000 description 1
- 238000004061 bleaching Methods 0.000 description 1
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 description 1
- 229910052681 coesite Inorganic materials 0.000 description 1
- 238000009833 condensation Methods 0.000 description 1
- 230000005494 condensation Effects 0.000 description 1
- 229910052906 cristobalite Inorganic materials 0.000 description 1
- 238000012674 dispersion polymerization Methods 0.000 description 1
- KBQHZAAAGSGFKK-UHFFFAOYSA-N dysprosium atom Chemical compound [Dy] KBQHZAAAGSGFKK-UHFFFAOYSA-N 0.000 description 1
- 238000005530 etching Methods 0.000 description 1
- OGPBJKLSAFTDLK-UHFFFAOYSA-N europium atom Chemical compound [Eu] OGPBJKLSAFTDLK-UHFFFAOYSA-N 0.000 description 1
- 230000002349 favourable effect Effects 0.000 description 1
- 238000001879 gelation Methods 0.000 description 1
- 230000003301 hydrolyzing effect Effects 0.000 description 1
- 238000002372 labelling Methods 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 238000012703 microemulsion polymerization Methods 0.000 description 1
- 239000005543 nano-size silicon particle Substances 0.000 description 1
- 239000002086 nanomaterial Substances 0.000 description 1
- 239000002853 nucleic acid probe Substances 0.000 description 1
- 230000003287 optical effect Effects 0.000 description 1
- 239000002245 particle Substances 0.000 description 1
- 238000005498 polishing Methods 0.000 description 1
- 239000002861 polymer material Substances 0.000 description 1
- 230000001737 promoting effect Effects 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 230000000171 quenching effect Effects 0.000 description 1
- YGSDEFSMJLZEOE-UHFFFAOYSA-N salicylic acid Chemical class OC(=O)C1=CC=CC=C1O YGSDEFSMJLZEOE-UHFFFAOYSA-N 0.000 description 1
- KZUNJOHGWZRPMI-UHFFFAOYSA-N samarium atom Chemical compound [Sm] KZUNJOHGWZRPMI-UHFFFAOYSA-N 0.000 description 1
- 229910000077 silane Inorganic materials 0.000 description 1
- 238000003980 solgel method Methods 0.000 description 1
- 238000001228 spectrum Methods 0.000 description 1
- 239000003381 stabilizer Substances 0.000 description 1
- 229910052682 stishovite Inorganic materials 0.000 description 1
- 238000010189 synthetic method Methods 0.000 description 1
- 230000002123 temporal effect Effects 0.000 description 1
- GZCRRIHWUXGPOV-UHFFFAOYSA-N terbium atom Chemical compound [Tb] GZCRRIHWUXGPOV-UHFFFAOYSA-N 0.000 description 1
- 238000012719 thermal polymerization Methods 0.000 description 1
- 229910052905 tridymite Inorganic materials 0.000 description 1
- 125000000391 vinyl group Chemical group [H]C([*])=C([H])[H] 0.000 description 1
- 229920002554 vinyl polymer Polymers 0.000 description 1
- 230000004304 visual acuity Effects 0.000 description 1
Landscapes
- Investigating, Analyzing Materials By Fluorescence Or Luminescence (AREA)
- Investigating Or Analysing Materials By The Use Of Chemical Reactions (AREA)
Abstract
The invention relates to a fluorescent microsphere marker and a preparation method thereof. The invention aims to provide a fluorescent microsphere marker. Compared with the traditional fluorescent marker, the fluorescent microsphere marker disclosed by the invention has the advantages of higher fluorescence intensity and higher stability, is easier to mark and is simpler to prepare. The technical scheme of the fluorescent microsphere marker is to provide the fluorescent microsphere marker. The fluorescent microsphere marker comprises a carrier, wherein the carrier is a microsphere polymerized by a styrene monomer, and the microsphere is connected with a fluorescence complex. The fluorescent microsphere marker disclosed by the invention has the beneficial effects that the preparation method is simpler and easier because the microsphere polymerized by the styrene monomer is used as the carrier. The microsphere disclosed by the invention not only can be used for marking the rare earth fluorescence complex but also can be used for marking common fluorescence substance, such as FITC (fluorescein isothiocyanate), the microsphere with various fluorescence properties can be manufactured, and the requirement of multiple detection can be satisfied.
Description
Technical field
The present invention relates to a kind of fluorescent microsphere marker and preparation method thereof.
Background technology
Conventional fluorescent marker is as fluorescein isothiocyanate (FITC), rhodamine-6-G (R-6-G) etc., and fluorescence lifetime is shorter, is difficult to overcome the interference of background fluorescence, and therefore detection sensitivity is lower.The rare-earth complex that utilization the has long fluorescence lifetime thing that serves as a mark, adopts temporal resolution detection technique, can effectively overcome the interference of background fluorescence, can make sensitivity have great raising.This technology is called as time resolved fluorescence detection technique, has become one of common technique in current biomedical research and clinical biochemical detection.
Conventional rare earth ion mainly contains: europium, samarium, dysprosium and terbium, they can form the complex compound with intense fluorescence with some specific title complex.Rare earth fluorescence complex has the following advantages: (1) long fluorescence lifetime.The life-span of general fluorescent complex and background fluorescence was about for 1～10 nanosecond, and the fluorescence lifetime of rare earth fluorescence complex is generally 10～1000 microseconds, and both differ 5～6 orders of magnitude.Therefore adopt time resolved fluorescence detection technique, can effectively overcome the interference of nonspecific background fluorescence, greatly put forward high measurement sensitivity.(2) wider exciting light bands of a spectrum.This is conducive to increase excitation energy, improves the specific activity of marker.(3) narrower emission band.Peak width at half height is less than 15 nanometers, is conducive to improve resolving power.(4) larger Stokes displacement.The maximum excitation optical wavelength of rare-earth complex is conventionally in the ultraviolet region of 300～380 nanometers, and maximum emission wavelength is more than 500 nanometers, and Stokes displacement reaches 250～350 nanometers, can effectively get rid of the interference of diversity of settings fluorescence, strengthens the specificity and the sensitivity that detect.(5) excitation wavelength has the characteristic of part, changes with the variation of part, and emission wavelength has the character of rare earth ion, does not change with the variation of part.
Rare earth ion or rare earth compounding and the silica-doped existing many reports of luminescent material, Cannasa (Cannasa.C, etal, Mat.Res.Soc.Symp.Proc.676 (Y3) 18.1-18.6,2001) describe always with directly doping Eu of sol-gel method preparation
3+y2O3-SiO2 nano luminescent material.Moran etc. (C.E.Moranetal, Langmuir2001,17,8376-8379) have synthesized and have mixed Pr always
3+and Er
3+nano silicon particles.(the Cheng Yanling etc. such as Cheng Yanling, applied chemistry 2008,25 (10), 1217-1220) with polyvinylpyrrolidone used as stabilizers, monomer styrene is made to the Properties of Polystyrene Nano Particles of diameter in 80nm left and right by ultrasound wave irradiation dispersion polymerization.
CN01133257.3 patent discloses one mixes silicon-dioxide, high polymer monomer and part rare earth fluorescence complex, first produces gel, then carries out high molecular polymerization, can obtain block luminescent material, has good mechanical property, can grind polishing.But also exist preparation method loaded down with trivial details, the shortcoming that suitability for industrialized production difficulty is larger.
CN01133258 patent discloses the synthetic method of a kind of solvent-free human-like superpolymer/rare earth chemical combination without nanometer hybridized luminous material, adopt Sol-Gel reaction, presoma tetraethoxy, vinyl monomer and the rare earth compounding of inorganic component are all joined in sol system, hydrolytic condensation under the existence of acetic acid, acetic acid is to regulate pH value to play again the object of promoting two-phase intermiscibility at this.Stirring synthesizes homogeneous sol, then gelation.Utilize afterwards the polymerization of thermal polymerization method trigger monomer, thereby obtain organic/inorganic nanometer hybridized luminous material.There is equally the shortcoming of complicated process of preparation in the method, and the wayward nano material preparing size and all once.
CN1631951 patent discloses a kind of preparation method of polymer hollow particles, this microballoon is the surface chemistry keyed jointing one layer of polymeric at inorganic materials Nano microsphere by microemulsion polymerization method, form polymer/inorganic material nano compound nucleus shell structure microballoon, then send out and remove inorganic materials acquisition by strong acid etching, it is of a size of micron to nano level.The microballoon size that the method prepares is larger, is not suitable for being applied to biomedical sector.
Existing rare earth fluorescence complex marker, rare earth ion is mainly Eu
3+, Sm
3+, Dy
3+and Tb
3+form complex compound with some organic coordination compounds as institutes such as beta-diketone compound, o-phenanthroline compounds, salicylic acid compounds, bipyridyliums compounds, although obtained application in various degree at present, be all difficult to meet following requirement: (1) has strong fluorescence simultaneously.(2) good stability: comprise Thermodynamically stable and fast light bleaching etc.(3) be easy to be cross-linked with the biomacromolecule such as antibody, nucleic acid, after being cross-linked, can keep the high fluorescent and the biological activity that is labeled thing of marker simultaneously.(4) easily synthetic.
Summary of the invention
The object of this invention is to provide a kind of fluorescent microsphere marker, compared with conventional fluorescent marker, have higher fluorescence intensity, higher stability, is easier to mark, and preparation is simple.
Technical scheme of the present invention, for a kind of fluorescent microsphere marker is provided, comprises carrier, and described carrier is the microballoon being polymerized by styrene monomer, on described microballoon, is connected with fluorescent complex.
Preferably, fluorescent complex described in such scheme is rare earth fluorescence complex.
Preferably, the chemical bonding of microballoon described in such scheme has the organosilane with active group.
Preferably, active group described in such scheme is amino, carboxyl or sulfydryl.
Preferably, the fluorescent rare earth complex compound described in such scheme is the rare earth part with active group, and described rare earth fluorescence complex is connected with the organosilane microballoon with active group by chemical bonding.
Preferably, fluorescent complex described in such scheme is 4,7-dichloro sulphophenyl-1,10-phenanthroline-2,9-dicarboxylic acid-europium, 4,4,-bis-(1,1,1,2,2,3,3,-seven is fluoro-4,6 ,-hexanedione base) one or several of chlorine sulfo group-o-terphenyl-europium, fluorescein isothiocyanate, tetraethylrhodamine, TRITC.
Another technical scheme of the present invention, for a kind of preparation method of fluorescent microsphere marker is provided, comprises the following steps:
A, prepare Properties of Polystyrene Nano Particles, and by it with the organosilane chemistry bonding of active group, form the microballoon of surface with active group;
B, fluorescent complex is doped in above-mentioned microballoon with chemical bonding, obtains the microballoon with fluorescent complex, introduce active group with the organosilicon with active group at microsphere surface more afterwards.
Preferably, such scheme comprises the following steps:
A, prepare Properties of Polystyrene Nano Particles, and by it with the organosilane chemistry bonding of active group, form the microballoon of surface with active group, be specially:
In every 100mL ethanolic soln, add 25g polyvinylpyrrolidone, 0.06g Potassium Persulphate, 10g vinylbenzene, in described ethanolic soln, the volume of ethanol and water is 3:1, the every 100mL dissolve with ethanol solution configuring is placed in ultrasound reactor, oxygen in logical nitrogen eliminating system, open ultrasonic generator and carry out polyreaction, obtain Properties of Polystyrene Nano Particles dispersion liquid, centrifugal collection Properties of Polystyrene Nano Particles, suspend with 5mL dehydrated alcohol, add 5 μ L aminosilanes, stirring reaction 1 hour, to collect after equal-volume absolute ethanol washing;
B, fluorescent complex is doped in above-mentioned microballoon with chemical bonding, obtains the microballoon with fluorescent complex, introduce active group with the organosilicon with active group at microsphere surface more afterwards, be specially:
The BCPDA of 1mg/mL is joined in amination Nano microsphere suspension by equal-volume, stir 15min, centrifugal collection, is suspended in the pH=7.8 phosphate buffered saline buffer of 50mmol/L, adds the EuCl of equal-volume 0.1mol/L
3solution, lucifuge stirring reaction 10min; After centrifugal collection, be suspended in equal-volume ethanol solution, add 5 μ L aminosilanes, stirring reaction 1 hour, to collect after equal-volume absolute ethanol washing, obtains rare-earth fluorescent microballoon marker.
Preferably, such scheme comprises the following steps:
A, prepare Properties of Polystyrene Nano Particles, and by it with the organosilane chemistry bonding of active group, form the microballoon of surface with active group, be specially:
In every 100mL ethanolic soln, add 25g polyvinylpyrrolidone, 0.06g Potassium Persulphate, 10g vinylbenzene, in described ethanolic soln, the volume of ethanol and water is 3:1, the every 100mL dissolve with ethanol solution configuring is placed in ultrasound reactor, oxygen in logical nitrogen eliminating system, open ultrasonic generator and carry out polyreaction, obtain Properties of Polystyrene Nano Particles dispersion liquid, centrifugal collection Properties of Polystyrene Nano Particles, suspend with 5mL dehydrated alcohol, add 5 μ L aminosilanes, stirring reaction 1 hour, to collect after equal-volume absolute ethanol washing;
B, fluorescent complex is doped in above-mentioned microballoon with chemical bonding, obtains the microballoon with fluorescent complex, introduce active group with the organosilicon with active group at microsphere surface more afterwards, be specially:
FITC is mixed with to the ethanolic soln of 1mg/mL, join in amination Nano microsphere suspension by equal-volume, stir 15min, centrifugal collection, after centrifugal collection, be suspended in equal-volume ethanol solution, add 5 μ L aminosilanes, stirring at room temperature reaction 1 hour, to collect after equal-volume absolute ethanol washing, obtain stable FITC fluorescent microsphere marker.
Beneficial effect of the present invention is that fluorescent microsphere marker of the present invention has adopted the microballoon being polymerized by styrene monomer as carrier, and preparation method is simpler, simpler and easy.Microballoon of the present invention both can mark rare earth fluorescence complex, again can the common fluorescent substance of mark (FITC etc.), therefore can make the microballoon of multiple photoluminescent property, and can meet the needs of Multiple detection.The present invention is marked on fluorescent substance on the surface of microballoon, makes this microballoon include multiple fluorescent substances, and fluorescence intensity is greatly improved, and has significantly reduced the quenching effect of external conditions to fluorescence, and the fluorescence intensity of fluorescent substance is further enhanced.The fluorescence intensity of fluorescent microsphere, higher than single fluorescent substance, is had to adopt the trouble of multiple labelling when this has just been avoided the single fluorescent substance of mark.Its stability is higher than conventional fluorescent substance.Introduced active group on its surface, can be cross-linked easily with biomacromolecule, synthetic difficulty is less, and cost is lower.There is the long fluorescence lifetime that conventional fluorescent marker does not have, the conventional fluorescent substance of tool does not have again high fluorescent, stability and easily markup.
Embodiment
By describing technology contents of the present invention, structural attitude in detail, being realized object and effect, be explained in detail below in conjunction with embodiment.
Technical scheme of the present invention is for providing a kind of fluorescent microsphere marker.Compared with conventional fluorescent substance, there is higher fluorescence intensity.Higher stability, is easier to mark, and the simple microballoon marker doped with fluorescent substance of preparation.
Fluorescent microsphere marker of the present invention, is a kind of microballoon that is marked with fluorescent substance, and fluorescent substance is wherein the surface that is doped in microballoon with the form of chemical bonding.Chemical bonding carries out after microballoon kernel forms.Microballoon is to be formed by styrene polymerization, and organic silane hydrolyzate product that adulterates.
Fluorescent microsphere marker of the present invention, is characterized in sending the characteristic fluorescence of common fluorescent substance, can send again the characteristic fluorescence of rare earth ion.The fluorescent substance of mark can be 4,7-dichloro sulphophenyl-1,10 phenanthroline-2,9 dicarboxylic acid-europium (BCPDA-Eu3
+), 4,4 ,-bis-(1,1,1,2,2,3,3 ,-seven fluoro-4,6 ,-hexanedione base) chlorine sulfo group-o-terphenyl-europium (BHHCT-Eu3
+), fluorescein isothiocyanate, tetraethylrhodamine, TRITC one or several.
Fluorescent microsphere marker of the present invention, its surface is with active group, and as amino, carboxyl, sulfydryl, these groups are to be introduced by the organosilane with corresponding group.This structure makes the fluorescent microsphere marker of the present invention can be with the effect of chemical bonding by biomacromolecule, as protein, nucleic acid, enzyme etc. are marked on the surface of fluorescent microsphere.Can be used as marker for immunodetection, labeled nucleic acid probe etc.
The principle of specific implementation method of the present invention: polyvinylpyrrolidone (PVP), Potassium Persulphate (KPS), vinylbenzene (St) are joined in ethanol/water medium and dissolves and be placed in ultrasound reactor, polyreaction under the protection of nitrogen.In reaction process, maintaining temperature of reaction is 60 DEG C.After reaction for some time, obtain Properties of Polystyrene Nano Particles dispersion liquid.Utilize and introduce active group with the organosilane of active group at silicon dioxide microsphere, again fluorescent substance is fixed on to the surface of silicon dioxide microsphere by the form of chemical bonding, then again introduces active group at microsphere surface with the organosilane with active group.
Implement example 1 and prepare FITC fluorescent microsphere marker
Fluorescein isothiocyanate (FITC) is a kind of conventional fluorescein, under the exciting light effect that is 490nm, sends the fluorescence that wavelength is 520nm at wavelength, presents bright yellow-green colour.Water-soluble and the alcohol dissolubility of FITC is good, and himself can react with amino with isothiocyanic acid base, forms covalent linkage.Utilize the materials such as vinylbenzene preparation to prepare Properties of Polystyrene Nano Particles kernel, introduce amino with aminosilane at microsphere surface, FITC can be marked on to microsphere surface, make fluorescent nanometer microsphere.
(1) prepare amination Nano microsphere
25gPVP, 0.06gKPS, 10gSt are joined in 100mL ethanol/water (volume is 3:1) medium and dissolve and be placed in ultrasound reactor, and the oxygen in logical nitrogen eliminating system, opens ultrasonic generator and carries out polyreaction.The temperature of reaction that in reaction process, logical recirculated water maintains is 60 DEG C, and keeps constant power 300W and constant nitrogen flow rate.After reaction for some time, obtain Properties of Polystyrene Nano Particles dispersion liquid.
Centrifugal collection Properties of Polystyrene Nano Particles, suspends with 5mL dehydrated alcohol, adds 5 μ L aminosilanes, and stirring at room temperature reaction 1 hour, to collect after equal-volume absolute ethanol washing.
(2) prepare rare-earth fluorescent Nano microsphere
FITC is mixed with to the ethanolic soln of 1mg/mL, join in amination Nano microsphere suspension by equal-volume, room temperature is kept away and is stirred 15min, centrifugal collection, after centrifugal collection, be suspended in equal-volume ethanol solution, add 5 μ L aminosilanes, stirring at room temperature reaction 1 hour, to collect after equal-volume absolute ethanol washing, obtain stable FITC fluorescent microsphere marker.
Implement example 2 and prepare TRITC fluorescent microsphere marker
TRITC (TRITC) is a kind of conventional fluorescein, under the exciting light effect that is 550nm, sends the fluorescence that wavelength is 620nm at wavelength, presents red fluorescence.Water-soluble and the alcohol dissolubility of TRITC is good, and himself can react with amino with isothiocyanic acid base, forms covalent linkage.Utilize the materials such as vinylbenzene preparation to prepare Properties of Polystyrene Nano Particles kernel, introduce amino with aminosilane at microsphere surface, TRITC can be marked on to microsphere surface, make fluorescent nanometer microsphere.
(1) prepare amination Nano microsphere
25gPVP, 0.06gKPS, 10gSt are joined in 100mL ethanol/water (volume is 3:1) medium and dissolve and be placed in ultrasound reactor, and the oxygen in logical nitrogen eliminating system, opens ultrasonic generator and carries out polyreaction.The temperature of reaction that in reaction process, logical recirculated water maintains is 60 DEG C, and keeps constant power 300W and constant nitrogen flow rate.After reaction for some time, obtain Properties of Polystyrene Nano Particles dispersion liquid.
Centrifugal collection Properties of Polystyrene Nano Particles, suspends with 5mL dehydrated alcohol, adds 5 μ L aminosilanes, and stirring at room temperature reaction 1 hour, to collect after equal-volume absolute ethanol washing.
(2) prepare rare-earth fluorescent Nano microsphere
TRITC is mixed with to the ethanolic soln of 1mg/mL, join in amination Nano microsphere suspension by equal-volume, room temperature is kept away and is stirred 15min, centrifugal collection, after centrifugal collection, be suspended in equal-volume ethanol solution, add 5 μ L aminosilanes, stirring at room temperature reaction 1 hour, to collect after equal-volume absolute ethanol washing, obtain stable TRITC fluorescent microsphere marker.
Implement example 3 and prepare BHHCT-Eu
3+fluorescent nanometer microsphere
4,4 ,-bis-(1,1,1,2,2,3,3-seven fluoro-4,6-hexanedione base) chlorine sulfo group-o-terphenyl (BHHCT) is a kind of trilobal cross beta-diketone compound, can with Eu
3+form stable complex compound, under the exciting of UV-light, can launch strong fluorescence.BHHCT's is water-soluble very poor, favorable solubility in ethanol, and himself is with-SOCl
2group, can react with amino, forms covalent linkage.Utilize the materials such as vinylbenzene preparation to prepare Properties of Polystyrene Nano Particles kernel, introduce amino with aminosilane at microsphere surface, can be by BHHCT-Eu
3+be marked on microsphere surface, make fluorescent nanometer microsphere.
(1) prepare amination Nano microsphere
25gPVP, 0.06gKPS, 10gSt are joined in 100mL ethanol/water (volume is 3:1) medium and dissolve and be placed in ultrasound reactor, and the oxygen in logical nitrogen eliminating system, opens ultrasonic generator and carries out polyreaction.The temperature of reaction that in reaction process, logical recirculated water maintains is 60 DEG C, and keeps constant power 300W and constant nitrogen flow rate.After reaction for some time, obtain Properties of Polystyrene Nano Particles dispersion liquid.
Centrifugal collection Properties of Polystyrene Nano Particles, suspends with 5mL dehydrated alcohol, adds 5 μ L aminosilanes, and stirring at room temperature reaction 1 hour, to collect after equal-volume absolute ethanol washing.
(2) prepare rare-earth fluorescent Nano microsphere
BHHCT is mixed with to 1mg/mL, joins in amination Nano microsphere suspension by equal-volume, room temperature is kept away and is stirred 15min, and centrifugal collection, is suspended in 50mmol/L pH=7.8 phosphate buffered saline buffer, adds equal-volume 0.1mol/LEuCl
3solution, room temperature lucifuge stirring reaction 10min.
After centrifugal collection, be suspended in equal-volume ethanol solution, add 5 μ L aminosilanes, stirring at room temperature reaction 1 hour, to collect after equal-volume absolute ethanol washing, obtains stable rare-earth fluorescent microballoon marker.
Implement example 4 and prepare BCPDA-Eu
3+fluorescent nanometer microsphere
4,7-dichloro sulphophenyl-1,10 phenanthroline-2,9 dicarboxylic acid (BCPDA) are a kind of adjacent benzene phenanthroline compounds with carboxyl, can with Eu
3+form stable complex compound, under the exciting of UV-light, can launch strong fluorescence.Water-soluble and the alcohol dissolubility of BCPDA is all good, and himself is with-SOCl
2group, can react with amino, forms covalent linkage.Utilize the materials such as vinylbenzene preparation to prepare Properties of Polystyrene Nano Particles kernel, introduce amino with aminosilane at microsphere surface, can be by BCPDA-Eu
3+be marked on microsphere surface, make fluorescent nanometer microsphere.
(1) prepare amination Nano microsphere
25gPVP, 0.06gKPS, 10gSt are joined in 100mL ethanol/water (volume is 3:1) medium and dissolve and be placed in ultrasound reactor, and the oxygen in logical nitrogen eliminating system, opens ultrasonic generator and carries out polyreaction.The temperature of reaction that in reaction process, logical recirculated water maintains is 60 DEG C, and keeps constant power 300W and constant nitrogen flow rate.After reaction for some time, obtain Properties of Polystyrene Nano Particles dispersion liquid.
Centrifugal collection Properties of Polystyrene Nano Particles, suspends with 5mL dehydrated alcohol, adds 5 μ L aminosilanes, and stirring at room temperature reaction 1 hour, to collect after equal-volume absolute ethanol washing.
(2) prepare rare-earth fluorescent Nano microsphere
BCPDA is mixed with to 1mg/mL, joins in amination Nano microsphere suspension by equal-volume, room temperature is kept away and is stirred 15min, and centrifugal collection, is suspended in 50mmol/L pH=7.8 phosphate buffered saline buffer, adds the EuCl of equal-volume 0.1mol/L
3solution, room temperature lucifuge stirring reaction 10min.
After centrifugal collection, be suspended in equal-volume ethanol solution, add 5 μ L aminosilanes, stirring at room temperature reaction 1 hour, to collect after equal-volume absolute ethanol washing, obtains stable rare-earth fluorescent microballoon marker.
Fluorescent nanometer microsphere of the present invention (fluorescent microsphere marker).Compared with conventional fluorescent substance, there is higher fluorescence intensity.Higher stability, is easier to mark, and the simple microballoon marker doped with fluorescent substance of preparation.
The foregoing is only embodiments of the invention; not thereby limit the scope of the claims of the present invention; every equivalent structure or conversion of equivalent flow process that utilizes description of the present invention to do; or be directly or indirectly used in other relevant technical fields, be all in like manner included in scope of patent protection of the present invention.
Claims (2)
1. a preparation method for fluorescent microsphere marker, is characterized in that, comprises the following steps:
A, prepare Properties of Polystyrene Nano Particles, and by it with the organosilane chemistry bonding of active group, form the microballoon of surface with active group;
B, fluorescent complex is doped in above-mentioned microballoon with chemical bonding, obtains the microballoon with fluorescent complex, introduce active group with the organosilicon with active group at microsphere surface more afterwards.
2. the preparation method of fluorescent microsphere marker according to claim 1, is characterized in that, comprises the following steps:
A, prepare Properties of Polystyrene Nano Particles, and by it with the organosilane chemistry bonding of active group, form the microballoon of surface with active group, be specially:
In every 100mL ethanolic soln, add 25g polyvinylpyrrolidone, 0.06g Potassium Persulphate, 10g vinylbenzene, in described ethanolic soln, the volume of ethanol and water is 3:1, the every 100mL dissolve with ethanol solution configuring is placed in ultrasound reactor, oxygen in logical nitrogen eliminating system, open ultrasonic generator and carry out polyreaction, obtain Properties of Polystyrene Nano Particles dispersion liquid, centrifugal collection Properties of Polystyrene Nano Particles, suspend with 5mL dehydrated alcohol, add 5 μ L aminosilanes, stirring reaction 1 hour, to collect after equal-volume absolute ethanol washing;
B, fluorescent complex is doped in above-mentioned microballoon with chemical bonding, obtains the microballoon with fluorescent complex, introduce active group with the organosilicon with active group at microsphere surface more afterwards, be specially:
FITC is mixed with to the ethanolic soln of 1mg/mL, join in amination Nano microsphere suspension by equal-volume, stir 15min, centrifugal collection, after centrifugal collection, be suspended in equal-volume ethanol solution, add 5 μ L aminosilanes, stirring at room temperature reaction 1 hour, to collect after equal-volume absolute ethanol washing, obtain stable FITC fluorescent microsphere marker.
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| CN103979595B (en) * | 2014-04-11 | 2015-07-22 | 中国科学院长春应用化学研究所 | Preparation method of rare-earth-ion-doped cerium fluoride hollow nano microsphere |
| CN104569431B (en) * | 2015-01-04 | 2017-01-11 | 深圳市艾瑞生物科技有限公司 | Homogenous phase fluorescence immunoassay reagent group for fast and quantitatively detecting troponin I and preparation method thereof |
| CN106243835A (en) * | 2015-07-20 | 2016-12-21 | 广东壮丽彩印股份有限公司 | A kind of aqueous rare earth compounding complex microsphere fluorescent ink and preparation technology thereof |
| CN105623651A (en) * | 2016-03-07 | 2016-06-01 | 江苏医诺万细胞诊疗有限公司 | Composite microsphere marker for conducting fluorescent marking on rear earth and preparation method of composite microsphere marker |
| CN106018818A (en) * | 2016-07-01 | 2016-10-12 | 安邦（厦门）生物科技有限公司 | Time-resolved immunofluorescent quantitative detection method and kit used by time-resolved immunofluorescent quantitative detection method |
| CN117645871B (en) * | 2023-11-28 | 2024-09-24 | 广东省计量科学研究院（华南国家计量测试中心） | Time-resolved fluorescence organic sensitization ball and preparation method thereof |
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