CN103013492B - A kind of Nano microsphere time-resolved fluorescence probe and its production and use - Google Patents
A kind of Nano microsphere time-resolved fluorescence probe and its production and use Download PDFInfo
- Publication number
- CN103013492B CN103013492B CN201110287991.2A CN201110287991A CN103013492B CN 103013492 B CN103013492 B CN 103013492B CN 201110287991 A CN201110287991 A CN 201110287991A CN 103013492 B CN103013492 B CN 103013492B
- Authority
- CN
- China
- Prior art keywords
- nano microsphere
- rare earth
- earth element
- resolved fluorescence
- fluorescence probe
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
- 239000004005 microsphere Substances 0.000 title claims abstract description 50
- 239000000523 sample Substances 0.000 title claims abstract description 23
- 238000004519 manufacturing process Methods 0.000 title abstract description 4
- 229910052761 rare earth metal Inorganic materials 0.000 claims abstract description 21
- 238000001514 detection method Methods 0.000 claims abstract description 20
- -1 lanthanide ions Chemical class 0.000 claims abstract description 12
- 239000000463 material Substances 0.000 claims abstract description 11
- 150000001875 compounds Chemical class 0.000 claims abstract description 7
- 150000002500 ions Chemical class 0.000 claims abstract description 6
- 229910052747 lanthanoid Inorganic materials 0.000 claims abstract description 6
- 238000002360 preparation method Methods 0.000 claims description 17
- 238000002372 labelling Methods 0.000 claims description 10
- 239000004793 Polystyrene Substances 0.000 claims description 8
- 229920002223 polystyrene Polymers 0.000 claims description 8
- ZMBHCYHQLYEYDV-UHFFFAOYSA-N Trioctylphosphine oxide Chemical group CCCCCCCCP(=O)(CCCCCCCC)CCCCCCCC ZMBHCYHQLYEYDV-UHFFFAOYSA-N 0.000 claims description 5
- DGEZNRSVGBDHLK-UHFFFAOYSA-N [1,10]phenanthroline Chemical compound C1=CN=C2C3=NC=CC=C3C=CC2=C1 DGEZNRSVGBDHLK-UHFFFAOYSA-N 0.000 claims description 5
- 102000004965 antibodies Human genes 0.000 claims description 4
- 108090001123 antibodies Proteins 0.000 claims description 4
- 239000002245 particle Substances 0.000 claims description 4
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 claims description 3
- 239000003814 drug Substances 0.000 claims description 3
- 235000013305 food Nutrition 0.000 claims description 3
- IJVRPNIWWODHHA-UHFFFAOYSA-M 2-cyanoprop-2-enoate Chemical compound [O-]C(=O)C(=C)C#N IJVRPNIWWODHHA-UHFFFAOYSA-M 0.000 claims description 2
- WVVLURYIQCXPIV-UHFFFAOYSA-N 4,4,4-trifluoro-1-naphthalen-2-ylbutane-1,3-dione Chemical compound C1=CC=CC2=CC(C(=O)CC(=O)C(F)(F)F)=CC=C21 WVVLURYIQCXPIV-UHFFFAOYSA-N 0.000 claims description 2
- 239000000427 antigen Substances 0.000 claims description 2
- 102000038129 antigens Human genes 0.000 claims description 2
- 108091007172 antigens Proteins 0.000 claims description 2
- 238000000034 method Methods 0.000 abstract description 10
- 239000000203 mixture Substances 0.000 abstract description 6
- 125000005594 diketone group Chemical group 0.000 abstract 1
- 238000003260 fluorescence intensity Methods 0.000 description 11
- UIIMBOGNXHQVGW-UHFFFAOYSA-M buffer Substances [Na+].OC([O-])=O UIIMBOGNXHQVGW-UHFFFAOYSA-M 0.000 description 9
- JDSHMPZPIAZGSV-UHFFFAOYSA-N Melamine Chemical compound NC1=NC(N)=NC(N)=N1 JDSHMPZPIAZGSV-UHFFFAOYSA-N 0.000 description 8
- 238000006243 chemical reaction Methods 0.000 description 7
- 238000003756 stirring Methods 0.000 description 7
- 239000000020 Nitrocellulose Substances 0.000 description 6
- BTBUEUYNUDRHOZ-UHFFFAOYSA-N borate Chemical compound [O-]B([O-])[O-] BTBUEUYNUDRHOZ-UHFFFAOYSA-N 0.000 description 6
- 238000005516 engineering process Methods 0.000 description 6
- 229920001220 nitrocellulos Polymers 0.000 description 6
- 229940098773 Bovine Serum Albumin Drugs 0.000 description 5
- 108091003117 Bovine Serum Albumin Proteins 0.000 description 5
- 229910052693 Europium Inorganic materials 0.000 description 5
- 239000007850 fluorescent dye Substances 0.000 description 5
- 241001502050 Acis Species 0.000 description 4
- 241000283973 Oryctolagus cuniculus Species 0.000 description 4
- HDTRYLNUVZCQOY-LIZSDCNHSA-N Trehalose Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 HDTRYLNUVZCQOY-LIZSDCNHSA-N 0.000 description 4
- HDTRYLNUVZCQOY-WSWWMNSNSA-N Trehalose Natural products O[C@@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@H](O)[C@@H](O)[C@@H](O)[C@@H](CO)O1 HDTRYLNUVZCQOY-WSWWMNSNSA-N 0.000 description 4
- 230000004913 activation Effects 0.000 description 4
- 150000001718 carbodiimides Chemical class 0.000 description 4
- 239000008367 deionised water Substances 0.000 description 4
- 150000002148 esters Chemical class 0.000 description 4
- 239000007788 liquid Substances 0.000 description 4
- NBIIXXVUZAFLBC-UHFFFAOYSA-N phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 description 4
- 238000001556 precipitation Methods 0.000 description 4
- 230000035945 sensitivity Effects 0.000 description 4
- XUIMIQQOPSSXEZ-UHFFFAOYSA-N silicon Chemical compound [Si] XUIMIQQOPSSXEZ-UHFFFAOYSA-N 0.000 description 4
- 229910052710 silicon Inorganic materials 0.000 description 4
- 239000010703 silicon Substances 0.000 description 4
- PXIPVTKHYLBLMZ-UHFFFAOYSA-N sodium azide Chemical compound [Na+].[N-]=[N+]=[N-] PXIPVTKHYLBLMZ-UHFFFAOYSA-N 0.000 description 4
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 4
- 210000004080 Milk Anatomy 0.000 description 3
- 229910052771 Terbium Inorganic materials 0.000 description 3
- 230000001276 controlling effect Effects 0.000 description 3
- 239000008267 milk Substances 0.000 description 3
- 235000013336 milk Nutrition 0.000 description 3
- 102000005614 monoclonal antibodies Human genes 0.000 description 3
- 108010045030 monoclonal antibodies Proteins 0.000 description 3
- 239000000178 monomer Substances 0.000 description 3
- 150000002910 rare earth metals Chemical class 0.000 description 3
- 229920001213 Polysorbate 20 Polymers 0.000 description 2
- CSCPPACGZOOCGX-UHFFFAOYSA-N acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 description 2
- 229910000147 aluminium phosphate Inorganic materials 0.000 description 2
- 230000015572 biosynthetic process Effects 0.000 description 2
- 210000004027 cells Anatomy 0.000 description 2
- 125000004122 cyclic group Chemical group 0.000 description 2
- OGPBJKLSAFTDLK-UHFFFAOYSA-N europium Chemical compound [Eu] OGPBJKLSAFTDLK-UHFFFAOYSA-N 0.000 description 2
- 238000005755 formation reaction Methods 0.000 description 2
- 238000004108 freeze drying Methods 0.000 description 2
- 238000003317 immunochromatography Methods 0.000 description 2
- 238000007689 inspection Methods 0.000 description 2
- 239000012528 membrane Substances 0.000 description 2
- 239000002105 nanoparticle Substances 0.000 description 2
- IJGRMHOSHXDMSA-UHFFFAOYSA-N nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- 239000003960 organic solvent Substances 0.000 description 2
- 239000008363 phosphate buffer Substances 0.000 description 2
- 238000006116 polymerization reaction Methods 0.000 description 2
- 235000010486 polyoxyethylene sorbitan monolaurate Nutrition 0.000 description 2
- 238000004321 preservation Methods 0.000 description 2
- 239000000376 reactant Substances 0.000 description 2
- 239000000741 silica gel Substances 0.000 description 2
- 229910002027 silica gel Inorganic materials 0.000 description 2
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 2
- PPBRXRYQALVLMV-UHFFFAOYSA-N styrene Chemical compound C=CC1=CC=CC=C1 PPBRXRYQALVLMV-UHFFFAOYSA-N 0.000 description 2
- 238000005406 washing Methods 0.000 description 2
- HTIQEAQVCYTUBX-UHFFFAOYSA-N Amlodipine Chemical compound CCOC(=O)C1=C(COCCN)NC(C)=C(C(=O)OC)C1C1=CC=CC=C1Cl HTIQEAQVCYTUBX-UHFFFAOYSA-N 0.000 description 1
- FBPFZTCFMRRESA-KAZBKCHUSA-N D-Mannitol Natural products OC[C@@H](O)[C@H](O)[C@H](O)[C@H](O)CO FBPFZTCFMRRESA-KAZBKCHUSA-N 0.000 description 1
- CZMRCDWAGMRECN-UGDNZRGBSA-N D-sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 description 1
- NNMXSTWQJRPBJZ-UHFFFAOYSA-K Europium(III) chloride Chemical compound Cl[Eu](Cl)Cl NNMXSTWQJRPBJZ-UHFFFAOYSA-K 0.000 description 1
- 241000287828 Gallus gallus Species 0.000 description 1
- 229940088597 Hormone Drugs 0.000 description 1
- 210000004698 Lymphocytes Anatomy 0.000 description 1
- FBPFZTCFMRRESA-KVTDHHQDSA-N Mannitol Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)[C@H](O)CO FBPFZTCFMRRESA-KVTDHHQDSA-N 0.000 description 1
- 229920000877 Melamine resin Polymers 0.000 description 1
- 108020004711 Nucleic Acid Probes Proteins 0.000 description 1
- RCEAADKTGXTDOA-UHFFFAOYSA-N OS(O)(=O)=O.CCCCCCCCCCCC[Na] Chemical compound OS(O)(=O)=O.CCCCCCCCCCCC[Na] RCEAADKTGXTDOA-UHFFFAOYSA-N 0.000 description 1
- 229940092253 Ovalbumin Drugs 0.000 description 1
- 108010058846 Ovalbumin Proteins 0.000 description 1
- 229940100684 PENTYLAMINE Drugs 0.000 description 1
- DPBLXKKOBLCELK-UHFFFAOYSA-N Pentylamine Chemical compound CCCCCN DPBLXKKOBLCELK-UHFFFAOYSA-N 0.000 description 1
- USHAGKDGDHPEEY-UHFFFAOYSA-L Potassium persulfate Chemical compound [K+].[K+].[O-]S(=O)(=O)OOS([O-])(=O)=O USHAGKDGDHPEEY-UHFFFAOYSA-L 0.000 description 1
- CZMRCDWAGMRECN-GDQSFJPYSA-N Sucrose Natural products O([C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@H](CO)O1)[C@@]1(CO)[C@H](O)[C@@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-GDQSFJPYSA-N 0.000 description 1
- GFISHBQNVWAVFU-UHFFFAOYSA-K Terbium(III) chloride Chemical compound Cl[Tb](Cl)Cl GFISHBQNVWAVFU-UHFFFAOYSA-K 0.000 description 1
- 239000002250 absorbent Substances 0.000 description 1
- 230000002745 absorbent Effects 0.000 description 1
- NIXOWILDQLNWCW-UHFFFAOYSA-N acrylic acid group Chemical group C(C=C)(=O)O NIXOWILDQLNWCW-UHFFFAOYSA-N 0.000 description 1
- 239000012491 analyte Substances 0.000 description 1
- 229960000070 antineoplastic Monoclonal antibodies Drugs 0.000 description 1
- 230000027455 binding Effects 0.000 description 1
- YBJHBAHKTGYVGT-ZKWXMUAHSA-N biotin Chemical compound N1C(=O)N[C@@H]2[C@H](CCCCC(=O)O)SC[C@@H]21 YBJHBAHKTGYVGT-ZKWXMUAHSA-N 0.000 description 1
- 229960002685 biotin Drugs 0.000 description 1
- 235000020958 biotin Nutrition 0.000 description 1
- 239000011616 biotin Substances 0.000 description 1
- 239000007795 chemical reaction product Substances 0.000 description 1
- 239000003085 diluting agent Substances 0.000 description 1
- 238000004821 distillation Methods 0.000 description 1
- 238000001035 drying Methods 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 239000012467 final product Substances 0.000 description 1
- 238000001917 fluorescence detection Methods 0.000 description 1
- PXGOKWXKJXAPGV-UHFFFAOYSA-N fluorine Chemical compound FF PXGOKWXKJXAPGV-UHFFFAOYSA-N 0.000 description 1
- 238000002290 gas chromatography-mass spectrometry Methods 0.000 description 1
- 239000003365 glass fiber Substances 0.000 description 1
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 description 1
- 238000005469 granulation Methods 0.000 description 1
- 230000003179 granulation Effects 0.000 description 1
- 238000004128 high performance liquid chromatography Methods 0.000 description 1
- 239000005556 hormone Substances 0.000 description 1
- 238000009396 hybridization Methods 0.000 description 1
- 238000003018 immunoassay Methods 0.000 description 1
- 150000002602 lanthanoids Chemical class 0.000 description 1
- 239000003446 ligand Substances 0.000 description 1
- 230000004301 light adaptation Effects 0.000 description 1
- 238000004020 luminiscence type Methods 0.000 description 1
- 230000014759 maintenance of location Effects 0.000 description 1
- 239000000594 mannitol Substances 0.000 description 1
- 235000010355 mannitol Nutrition 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 239000004531 microgranule Substances 0.000 description 1
- 229960000060 monoclonal antibodies Drugs 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- 239000002853 nucleic acid probe Substances 0.000 description 1
- 238000000643 oven drying Methods 0.000 description 1
- MYMOFIZGZYHOMD-UHFFFAOYSA-N oxygen Chemical compound O=O MYMOFIZGZYHOMD-UHFFFAOYSA-N 0.000 description 1
- 239000001301 oxygen Substances 0.000 description 1
- 229910052760 oxygen Inorganic materials 0.000 description 1
- 238000004806 packaging method and process Methods 0.000 description 1
- 150000005041 phenanthrolines Chemical class 0.000 description 1
- 239000004033 plastic Substances 0.000 description 1
- 229920003023 plastic Polymers 0.000 description 1
- 229920001184 polypeptide Polymers 0.000 description 1
- 235000019394 potassium persulphate Nutrition 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 238000004445 quantitative analysis Methods 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 230000001105 regulatory Effects 0.000 description 1
- 238000007789 sealing Methods 0.000 description 1
- 150000003384 small molecules Chemical class 0.000 description 1
- 239000000243 solution Substances 0.000 description 1
- 239000012086 standard solution Substances 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 239000005720 sucrose Substances 0.000 description 1
- 238000001291 vacuum drying Methods 0.000 description 1
Abstract
The invention belongs to detection technique field, be specifically related to a kind of Nano microsphere time-resolved fluorescence probe and its production and use.The invention discloses a kind of Nano microsphere time-resolved fluorescence probe, it it is the Nano microsphere of a macromolecular material parcel rare earth element fluorescence complex, it is characterized in that rare earth element fluorescence complex includes the material of following mol ratio: rare earth element ion: beta diketone body quasi-chelate compound: Fluorescence Increasing synergist=1: 4: 5, wherein said rare earth element ion is Eu3+ and other lanthanide ions are with the double-doped mixture of 100: 1 1000: 1 mol ratios.
Description
Technical field
The invention belongs to detection technique field, be specifically related to a kind of Nano microsphere time-resolved fluorescence probe and its production and use.
Background technology
Time-resolved fluoroimmunoassay (Time resolved fluoroisnmunoassay, TRFIA) it is a kind of the most novel detection means proposed the eighties in last century, TRFIA is as tracer-labelling protein with rare earth ion, polypeptide, hormone, antibody, nucleic probe or biological activity cell, with its chelating agen, strengthen liquid (some need not) in question response system (such as: antigen-antibody immunoreation, biotin-labeled pentylamine reacts, nucleic acid probe hybridization reacts, the killing reaction etc. of target cell and effector lymphocyte) react after, the fluorescence intensity in end product is measured with time-resolved fluorescence instrument, according to fluorescence intensity and relative intensity of fluorescence ratio, speculate the concentration of analyte in reaction system, reach the purpose of quantitative analysis.TRFIA and chemiluminescence, electrochemiluminescence immunoassay technology referred to as three big super sensitivity detection technology, have relatively broad application at aspects such as food inspection, clinical medicine detection, scientific research detection biology and environment measurings.
Owing to the fluorescence intensity of rare earth element complex is the most relatively low, it is therefore desirable to use Fluorescence Increasing technology to improve detection sensitivity.At present, according to the difference of Signal Enhanced Technology, TRFIA can be divided into three types, and (1) dissociates enhancement techniques (DELFIA) again；(2) Cyber Fluor system；(3) TRFIA (Nano-TRFIA) based on Nano microsphere.Wherein, Nano-TRFIA is a kind of brand-new time-resolved fluorescence detection means, it combines long-life and the signal enlarge-effect of Nano microsphere of rare earth element fluorescence, by rare earth element and coordination compound co-doped thereof in nanometer and microsphere, after surface active, by antibody labeling in label surface, form complex, this complex is used for immune detection, sensitivity can be greatly enhanced, and obtaining the broadest range of linearity, its actual performance is not less than DELIFA technology.
CN02144517 discloses the preparation of a kind of fluorescent both rare-earth fluorescent nanoparticle (Lanthanide Fluorescence Nanoparticles is called for short LFNP) and the application process in Measurement for Biotechnique thereof.It is with fluorescent both rare earth compounding as the centre of luminescence, uses silica gel chemistry parcel granulation technique to be prepared from.
CN03133857 discloses a kind of beta-diketone-trivalent europium complex nano fluorescent probe and preparation thereof and application, it is by can be with the monomer of esters of silicon acis combined polymerization, covalent bonding is occurred to react in organic solvent with trivalent europium-beta-diketone class fluorescence complex, then it is copolymerized with esters of silicon acis again, the functional nano rare-earth fluorescent microgranule of formation；Described trivalent europium ion, beta-diketon organic ligand, can be with the monomer of esters of silicon acis combined polymerization and esters of silicon acis, the molar ratio between them is 1: 2～3: 10～100: 350～450.
But existing rare earth element fluorescent probe still suffers from the defect of fluorescence intensity generation photobleaching relatively low, easy.
Summary of the invention
The technical problem to be solved is the defects such as existing rare earth element fluorescent probe fluorescence intensity is inadequate.
For this, the invention discloses a kind of Nano microsphere time-resolved fluorescence probe, it it is the Nano microsphere of a macromolecular material parcel rare earth element fluorescence complex, it is characterized in that rare earth element fluorescence complex includes the material of following mol ratio: rare earth element ion: beta-diketon body quasi-chelate compound: Fluorescence Increasing synergist=1: 4: 5, wherein said rare earth element ion is Eu3+ and other lanthanide ions are with the double-doped mixture of 100: 1～1000: 1 mol ratios.
In certain embodiments, described macromolecular material is preferably polystyrene or polyalkyl alpha-cyanacrylate, most preferably polystyrene.
In certain embodiments, the particle diameter of described Nano microsphere is 10-200nm, surface charge Surface Charge (μ eq/g) is 170-200, and carboxyl density Parking Area is 25-35.7 for (sq.A/grp).
In certain embodiments, described beta-diketon body quasi-chelate compound is β-naphthoyltrifluoroacetone.
In certain embodiments, described Fluorescence Increasing synergist is trioctyl phosphine oxide and phenanthroline, and wherein mol ratio is trioctyl phosphine oxide: phenanthroline=2: 2.
In certain embodiments, other lanthanide ions described are Sm3+、Tb3+、Nd3+Or Dy3+。
On the other hand, the invention discloses the preparation method of above-mentioned Nano microsphere time-resolved fluorescence probe, comprise the following steps:
The preparation of high molecular nano-microsphere；
The preparation of nanometer fluorescent microspheres；
The labelling of nanometer fluorescent microspheres antibody (or antigen).
On the other hand, the invention discloses above-mentioned Nano microsphere time-resolved fluorescence probe as tracer-labelling purposes in terms of food inspection, clinical medicine detection, scientific research detection biology and environment measuring.
Nano microsphere time-resolved fluorescence probe of the present invention has the advantages such as fluorescence intensity height, good stability, strong stress resistance, detection sample be compatible big, and can combine with immunochromatography technique, it is achieved quickly with quantitative perfect adaptation.
Detailed description of the invention
Below in conjunction with specific embodiment, the present invention is expanded on further.These embodiments are merely to illustrate the present invention rather than limit the scope of the present invention.The experimental technique of unreceipted actual conditions in the following example, generally according to normal condition or according to the condition proposed by manufacturer.Unless otherwise defined, the same meaning that all specialties used in literary composition are familiar with one skilled in the art with scientific words.Additionally, any method similar or impartial to described content and material all can be applicable in the inventive method.Preferable implementation described in literary composition only presents a demonstration with material and is used.
The preparation of embodiment 1 carboxylic polystyrene Nano microsphere
Take 10mm styrene monomer and 0.95mm acrylic monomers is dissolved in the 10ml deionized water containing 0.45mm dodecyl sodium sulfate, add in round-bottomed flask, stir by magnetic stir bar, then with high pure nitrogen, air in round-bottomed flask is eliminated, heated sealed is to 70 DEG C, add the potassium peroxydisulfate of 0.5ml 0.15mm, after sealing oxygen barrier stirring reaction 8h, it is down to room temperature, then reactant liquor whatman 2v filter paper (aperture 8 μm) is filtered, , finally with bag filter (molecular cut off 30, 000Da) deionized water is dialysed 5 days, collect liquid in bag filter, add the sodium azide 4 DEG C preservation of 0.05%.
By measuring, a diameter of 190 ± 10nm of prepared carboxylic polystyrene Nano microsphere, surface charge Surface Charge (μ eq/g) are 170-200, and carboxyl density Parking Area is 25-35.7 for (sq.A/grp).
The preparation of embodiment 2 nanometer fluorescent microspheres
Take the polystyrene microsphere of the 190nm of preparation in a small amount of embodiment 1, add in 10ml deionized water and acetone mixture (v/v=1: 1), make the density of polystyrene microsphere in reactant liquor be about 1 × 1014Individual, stir, add the europium chloride of 100ul 0.1M, 1 μ l 0.1M terbium trichloride, 400 μ l 0.1M beta-diketon (β-NTA), 300 μ l trioctyl phosphine oxide (TOPO), 100 μ l phenanthrolines, first it is heated to 60 DEG C of constant temperature stirring lucifuge reaction 10h, is then down to room temperature reaction 2h, finally by decompression distillation mode, the organic solvent in solution is removed, deionized water is dialysed 5 days, remove remaining residue small-molecule substance, collect liquid in bag filter, add the sodium azide 4 DEG C preservation of 0.05%.
By test and calculating, in average each nanometer fluorescent microspheres, the quantity of the europium ion chelate of parcel is about 18-20 ten thousand.
The comparison of the different packaging method nanometer fluorescent microspheres fluorescence intensity of embodiment 3
Prepare undoped p terbium ion respectively with reference to the formula of embodiment 2 and step, doping terbium ion does not uses phenanthroline, undoped p terbium ion to use phenanthroline nanometer fluorescent microspheres, carries out the comparison of fluorescence intensity, the results are shown in Table 1.
Table 1
Note: the definition of (1) fluorescence intensity after exciting by a Nano microsphere produced fluorescence intensity be equivalent to single free europium ion chelate the multiple of generation fluorescence intensity；
(2) the fluorescent microsphere particle diameter of commercialization is 0.2 μm, purchased from Thermo Fisher Scientific, trade name Fluoro-Max Carboxylate-Modified and Streptavidin-Coated Europium Chelate Particles.
The preparation of embodiment 4 nanometer fluorescent microspheres labelling tripolycyanamide monoclonal antibody
Take the nanometer fluorescent microspheres of preparation in a small amount of embodiment 2, be dissolved in the borate buffer of 10ml 0.01M pH8.0, make fluorescent microsphere density be about 1.0 × 1012Individual/mL, 400w supersound process 30 seconds, it is then slowly added into the carbodiimide (EDC) of 200ul 15mg/mL, after room temperature at the uniform velocity stirs incubation 15min, 150000g is centrifuged 10 minutes, collect precipitation, be centrifuged 2 times with the borate buffer cyclic washing of 0.01M pH8.0 i.e. obtain activation fluorescent microsphere.The fluorescent microsphere of activation is redissolved in the borate buffer of 5ml 0.01MpH8.0, add 250 μ g tripolycyanamide monoclonal antibodies, after 4 DEG C of stirring reaction 12h, 12000g is centrifuged 10 minutes, collect precipitation, redissolve in containing 1.5% (m/v) trehalose, the 0.01M pH7.4 of 2% (m/v) bovine serum albumin phosphate buffer in, obtain the tripolycyanamide monoclonal antibody of fluorescent microsphere labelling, be placed in 4 DEG C and save backup.
The preparation of embodiment 5 nanometer fluorescent microspheres labelling rabbit igg
Take the nanometer fluorescent microspheres of preparation in a small amount of embodiment 2, be dissolved in the borate buffer of 10ml 0.01M pH8.0, make fluorescent microsphere density be about 1.0 × 1012Individual/mL, 400w supersound process 30 seconds, it is then slowly added into the carbodiimide (EDC) of 200ul 15mg/mL, after room temperature at the uniform velocity stirs incubation 15min, 150000g is centrifuged 10 minutes, collect precipitation, be centrifuged 2 times with the borate buffer cyclic washing of 0.01M pH8.0 i.e. obtain activation fluorescent microsphere.The fluorescent microsphere of activation is redissolved in the borate buffer of 5ml 0.01MpH8.0, add 600 μ g rabbit iggs, after 4 DEG C of stirring reaction 12h, 12000g is centrifuged 10 minutes, collect precipitation, redissolve in containing 1.5% (m/v) trehalose, the 0.01M pH7.4 of 2% (m/v) bovine serum albumin phosphate buffer in, obtain the rabbit igg of fluorescent microsphere labelling, be placed in 4 DEG C and save backup.
The lyophilizing of embodiment 6 namo fluorescence probe
Fluorescent probe prepared in embodiment 3 and embodiment 4 is diluted 20 times and 30 times with lyophilizing diluent (containing 6% sucrose, 4% bovine serum albumin, the PBPS buffer of 0.05M ph7.4 of 1% mannitol) respectively, then by 1: 1 (v/v) mix homogeneously, during the 100 every holes of μ L are sub-packed in 96 hole detachable micropore plates again, use lyophilisation mode to be dried (lyophilizing technique is shown in Table 2), then seal with silica gel plug.
Table 2 freeze-drying curve
| Temperature | Regulating time | Retention time | Pressure |
| -55℃ | 30min | 240min | Normal pressure |
| -35℃ | 30min | 180min | 0.15mbar |
| -15℃ | 30min | 480min | 0.15mbar |
| -5℃ | 30min | 120min | 0.11mbar |
| 5℃ | 30min | 120min | 0.11mbar |
| 25℃ | 30min | 240min | 0.15mbar |
| 25℃ | 5min | 60min | 0mbar |
Embodiment 7
The preparation of chromatograph test strip
1) preparation of nitrocellulose filter C/T line
Tripolycyanamide and chicken ovalbumin conjugate (MEL-OVA) are dissolved to final concentration 0.1mg/mL with containing 1.5% (m/v) trehalose, 2% (m/v) bovine serum albumin, the phosphoric acid buffer of 0.01M pH7.4 of 0.05% (v/v) tween 20, are sprayed on away from nitrocellulose filter left end 2mm to be formed with Membrane jetter and detect line T line；It is dissolved to final concentration 1.0mg/ml with containing 1.5% (m/v) trehalose, 2% (m/v) bovine serum albumin, the phosphoric acid buffer of 0.01M pH7.4 of 0.05% (v/v) tween 20 by anti-for goat-anti rabbit two, being sprayed on formation nature controlling line C line at nitrocellulose filter right-hand member 4mm with Membrane jetter, nature controlling line is separated by 5mm with detection line.The nitrocellulose filter sprayed is placed in 25 DEG C of vacuum drying oven drying, drying at room temperature environment saves backup.
2) assembling of test strips
Hardboard paper overlap successively and pastes: nitrocellulose filter, glass fibre element pad, drawing and have detection line and the nitrocellulose filter of nature controlling line, filter paper and sample pad and absorbent paper, the wide test strips of 4mm is cut into after assembling, putting into seal in dry plastics keg and preserve, the shelf-life was up to more than 1 year.
Embodiment 8
The detection of tripolycyanamide in milk
Take 100u l milk sample to be measured, adding lyophilizing to have in 96 hole detachable micropores of fluorescent probe, be inserted into chromatograph test strip, sample pad one end is immersed in liquid, inserting after 5min in Portable fluorescence readout instrument is readable, is got final product the testing result of quantitative by built-in standard curve.
Embodiment 9
Namo fluorescence probe immuno-chromatographic test paper strip accuracy is tested
1) tripolycyanamide standard solution is added in the fresh milk that high performance liquid chromatography GC-MS detection content of melamine is 0, make its concentration respectively reach 0ng/mL, 10ng/mL, 20ng/mL, 40ng/mL, 80ng/mL, 160ng/mL, 320ng/mL, 640ng/mL, detected by the detection method of embodiment 7
2) above experiment 10 times are repeated.
Experimental result is as shown in table 3 below.
Table 3
Test result indicate that: the fluorescent quantitation test strip of the present invention is quantitatively limited to 10ng/mL to the tripolycyanamide in sample, quantitative linearity scope is in 10-640ng/mL, sample TIANZHU XINGNAO Capsul, all within 80%～120%, fully meets the needs of detection by quantitative.Its remolding sensitivity colloidal gold immuno-chromatography test paper strip prepared by same antigen-antibody raw material wants high more than 10 times.
The scope of the present invention is not limited by the specific embodiments described, and described embodiment is only used as illustrating the single example of various aspects of the present invention, also includes method and the component of functional equivalent in the scope of the invention.It practice, in addition to content as herein described, those skilled in the art can easily grasp the multiple improvement to the present invention with reference to described above.Within described improvement also falls into the scope of the appended claims.Every list of references mentioned above is listed in herein all in full as reference.
Claims (5)
1. a Nano microsphere time-resolved fluorescence probe, is that a macromolecular material parcel rare earth element is glimmering
The Nano microsphere of light coordination compound, it is characterised in that described rare earth element fluorescence complex includes following mol ratio
Material: rare earth element ion: beta-diketon body quasi-chelate compound: Fluorescence Increasing synergist=1:4:5, Qi Zhongsuo
Stating rare earth element ion is Eu3+With other lanthanide ions with the double-doped mixing of 100:1-1000:1 mol ratio
Thing；The particle diameter of described Nano microsphere is 10-400nm, surface charge is 170-200 μ eq/g, and carboxyl is close
Degree is 25-35.7sq.A/grp；Described beta-diketon body quasi-chelate compound is β-naphthoyltrifluoroacetone；Described
Fluorescence Increasing synergist is trioctyl phosphine oxide and/or phenanthroline；Other lanthanide ions described are Sm3+、
Tb3+、Nd3+Or Dy3+。
Nano microsphere time-resolved fluorescence probe the most according to claim 1, it is characterised in that institute
Stating macromolecular material is polystyrene or polyalkyl alpha-cyanacrylate.
Nano microsphere time-resolved fluorescence probe the most according to claim 1, it is characterised in that institute
Stating macromolecular material is carboxylic polystyrene.
4. a preparation method for Nano microsphere time-resolved fluorescence probe described in claim 1, including
Following step:
A) preparation of high molecular nano-microsphere；
B) preparation of nanometer fluorescent microspheres；
C) nanometer fluorescent microspheres antibody or the labelling of antigen.
5. Nano microsphere time-resolved fluorescence probe described in claim 1 as tracer-labelling at food
Purposes in terms of detection, clinical medicine detection, scientific research detection biology or environment measuring.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201110287991.2A CN103013492B (en) | 2011-09-26 | A kind of Nano microsphere time-resolved fluorescence probe and its production and use |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201110287991.2A CN103013492B (en) | 2011-09-26 | A kind of Nano microsphere time-resolved fluorescence probe and its production and use |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN103013492A CN103013492A (en) | 2013-04-03 |
| CN103013492B true CN103013492B (en) | 2016-12-14 |
Family
ID=
Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1493647A (en) * | 2002-11-01 | 2004-05-05 | 中国科学院大连化学物理研究所 | Functional nano-rare earth fluorescent micro particle and its preparation and application |
| CN1778786A (en) * | 2004-11-24 | 2006-05-31 | 上海师范大学 | Rare-earth doped beta-diketone complex fluorescent powder its production and use |
| CN101045200A (en) * | 2007-04-06 | 2007-10-03 | 山东大学 | Method for preparing micro-capsule containing high light-effect rare earth compounding matter |
| CN101705084A (en) * | 2009-11-09 | 2010-05-12 | 东南大学 | Multicolour rare earth nano particle and preparation method thereof |
| CN102002359A (en) * | 2010-11-17 | 2011-04-06 | 同济大学 | Method for preparing beta-diketone functional rare-earth organic luminous gel based on silicon-boron composite network |
| CN102115508A (en) * | 2010-12-03 | 2011-07-06 | 黑龙江大学 | Synthetic method for micro-size polymer fluorescent microspheres |
| CN102153576A (en) * | 2011-01-28 | 2011-08-17 | 阜阳师范学院 | Rare earth complex coated with silicon dioxide and preparation method of rare earth complex |
Patent Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1493647A (en) * | 2002-11-01 | 2004-05-05 | 中国科学院大连化学物理研究所 | Functional nano-rare earth fluorescent micro particle and its preparation and application |
| CN1778786A (en) * | 2004-11-24 | 2006-05-31 | 上海师范大学 | Rare-earth doped beta-diketone complex fluorescent powder its production and use |
| CN101045200A (en) * | 2007-04-06 | 2007-10-03 | 山东大学 | Method for preparing micro-capsule containing high light-effect rare earth compounding matter |
| CN101705084A (en) * | 2009-11-09 | 2010-05-12 | 东南大学 | Multicolour rare earth nano particle and preparation method thereof |
| CN102002359A (en) * | 2010-11-17 | 2011-04-06 | 同济大学 | Method for preparing beta-diketone functional rare-earth organic luminous gel based on silicon-boron composite network |
| CN102115508A (en) * | 2010-12-03 | 2011-07-06 | 黑龙江大学 | Synthetic method for micro-size polymer fluorescent microspheres |
| CN102153576A (en) * | 2011-01-28 | 2011-08-17 | 阜阳师范学院 | Rare earth complex coated with silicon dioxide and preparation method of rare earth complex |
Non-Patent Citations (3)
| Title |
|---|
| Highly sensitive immunoassay of free prostate-specific antigen in serum using europium(III) nanoparticle label technology;Tero Soukka et al.;《Clinica Chimica Acta》;20030114;45-58 * |
| Zeptomole detection sensitivity of prostate-specific antigen in a rapid microtitre plate assay using time-resolved fluorescence;Harri Harma et al.;《Luminescence》;20000830;351-355 * |
| 光致发光稀土有机配合物材料的研究进展及应用;张泉平等;《化工新型材料》;20060630;1-4 * |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN104280542B (en) | Double; two enhanced chemiluminescence immunoassays that and nanometer particle to mark luminous based on Reinforced by Metal amplifies | |
| Liu et al. | Magnetic graphene nanosheets based electrochemiluminescence immunoassay of cancer biomarker using CdTe quantum dots coated silica nanospheres as labels | |
| CN107817232B (en) | Automated immunoassay system for performing diagnostic assays for allergies and autoimmune diseases | |
| CN104169710A (en) | Method and apparatus for time-resolved fluorescence immunoassay testing | |
| CN102998467B (en) | β human chorionic gonadotrophin magnetic microparticle chemiluminescence immune quantitative detection reagent box and preparation method thereof | |
| CN103868913B (en) | The enzyme-catalyzed chemical luminescence substrate liquid of alkaline phosphatase | |
| Zhang et al. | Highly sensitive thrombin detection by matrix assisted laser desorption ionization-time of flight mass spectrometry with aptamer functionalized core–shell Fe3O4@ C@ Au magnetic microspheres | |
| CN107655864A (en) | A kind of method using nanoparticle time-resolved fluorescence probe in detecting tetracycline | |
| CN103674935B (en) | A kind of method of measuring gibberellin based on hybridization chain reaction signal amplification technique | |
| CN103076455B (en) | Quantitative detection serum amyloid A protein kit and Synthesis and applications thereof | |
| Zhu et al. | Ultrasensitive aptamer-based bio bar code immunomagnetic separation and electrochemiluminescence method for the detection of protein | |
| CN107656079A (en) | A kind of method using nanoparticle time-resolved fluorescence probe in detecting sulfa antibiotics | |
| CN109116038A (en) | Unbonded estriol chemiluminescence immune detection reagent kit and preparation method thereof | |
| CN101539576A (en) | Hepatitis B virus pre S1 antigen chemiluminscence immunoassay kit and preparation method thereof | |
| CN107328938A (en) | Propepsin and helicobacter pylori antibody detection method and its kit | |
| Valanne et al. | Rapid and sensitive HBsAg immunoassay based on fluorescent nanoparticle labels and time-resolved detection | |
| CN107656061A (en) | A kind of method using nanoparticle time-resolved fluorescence probe in detecting gentamicin | |
| Zhong et al. | Multiplex immunoassay of chicken cytokines via highly-sensitive chemiluminescent imaging array | |
| CN113419059A (en) | Blocking agent for chemiluminescence immune analysis method | |
| CN102565020A (en) | Method for quantitatively detecting protein through quantum dot resonant scattering | |
| CN109061198A (en) | inhibin A detection kit and preparation method thereof | |
| CN116773807A (en) | Method for detecting African swine fever virus by using time-resolved fluorescence immunoassay probe | |
| JP2006521549A (en) | Solid phase immunochromatography method | |
| CN103013492B (en) | A kind of Nano microsphere time-resolved fluorescence probe and its production and use | |
| CN107656062A (en) | A kind of method using nanoparticle time-resolved fluorescence probe in detecting chloramphenicol |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| TA01 | Transfer of patent application right |
Effective date of registration: 20151207 Address after: 200120 Shanghai Zhangjiang High Tech Park of Pudong New Area Cailun Road No. 308 Building 2 room 1101 Applicant after: SHANGHAI SUYUAN BIOLOGICAL TECHNOLOGY CO., LTD. Address before: 201616 Shanghai city Songjiang District Guangfulin road 4855 Lane 56 Applicant before: Shanghai Uni Biotech Co.,Ltd. |
|
| GR01 | Patent grant |