CN1265197C - Beta-diketone-trivalent europium complex nano fluorescent probe, its preparation and use thereof - Google Patents
Beta-diketone-trivalent europium complex nano fluorescent probe, its preparation and use thereof Download PDFInfo
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Abstract
The present invention relates to a beta-diketone-trivalent europium complex nanometer fluorescent probe, a preparing method thereof and an application thereof. Monomers which can be copolymerized with silicate ester generate covalent bonding reaction with trivalent europium-beta-diketone fluorescent complexes in an organic solvent and then are copolymerized with silicate ester to form functional nanometer rare earth fluorescent particles. The mole ratio of trivalent europium ions to beta-diketone organic ligands to the monomers which can be copolymerized with silicate ester to silicate ester is 1:2 to 3:10 to 100:350 to 450. The obtained particles have strong fluorescent light and stable properties, and the surfaces of particles have active functional groups which can be directly used for biological labeling. If used for time-resolved fluorimetric determination, the fluorescent probe is free from the interference of various kinds of scattered light and fluorescence with a short life. The fluorescent probe of the present invention has important application value in the biochemical detection technical fields of immunological detection of time-resolved fluorescence, microscopic imaging, DNA hybridization measurement, biological chip measurement, etc.
Description
Technical field
The present invention relates to the nano rare earth fluorescence probe, specifically a kind of beta-diketon-trivalent europium complex nanometer (rare earth) fluorescence probe and preparation and application.
Background technology
The develop rapidly of medical science and life science has proposed a large amount of new problems to analytical chemistry, the assay determination of nucleic acid, polypeptide, protein and other, bio-pharmaceutical, microorganism, ultramicron bioactivator etc. is to medical science and the important effect of the positive play more and more of development of life science, so the bioanalysis chemistry has become one of most important forward position, current international analysis science field research field; Required based on above-mentioned situation, many new analytical approachs and technology constantly are developed, as namo fluorescence probe technology, biochip technology, nanochannel technology, molecular imprinting, molecular beacons technology etc., wherein the namo fluorescence probe technology is one and has that widespread use is worth and supersensitive bioanalysis detection technique.
The Molecular Spectral Analysis method is maximum method of using in medical science and the life science, since fluorescence analysis method highly sensitive, selectivity good, required sample size is few, thereby be widely used in based on fluorimetric bioanalytical method in the analyzing and testing of various medical science and life science.Fluorometry is commonly used to measure the content of some composition in the biological sample, because the high complexity of living things system, in order to obtain more valuable information, for employed fluorescence probe very high requirement is arranged, as light stability preferably, be difficult for by photobleaching or photodissociation, probe itself is highly stable, luminous continuing do not glimmered, activity influence to analyzed biotic component is little, possesses good fluorescence quantum efficiency, and spectral signature is outstanding, preferably class line spectrum, and maximum excitation peak wants less etc. to the interference of maximum emission peak.
But namo fluorescence probe is meant the nano luminescent particle that mark uses as fluorescent detection probe on the biomolecule, and the namo fluorescence probe that has developed at present mainly contains following several:
(1) golden nanometer particle (collaurum) probe (document 1:W.P.Faulk, Immunochem., 1971,8,1081-1083): the research of golden nanometer particle probe starts from the sixties in 20th century, 1971, Faulk and Taylor reported first immunogold staining; When the antigen that is adsorbed on the collaurum surface or antibody run in tissue or the cell corresponding antibody or antigen, special association reaction will take place, because it is golden nanometer particle has higher electron density, therefore clear and legible under Electronic Speculum; But obtain higher sensitivity, just need higher concentration and probe concentration, and need larger-diameter gold particle, therefore often cause the label instability, automatic aggegation easily takes place in standing storage.At the problems referred to above, developed silver staining afterwards again, its principle is that the developer solution that will contain silver ion joins in the golden nanometer particle that reaction has taken place, because a large amount of charge attraction of gold particle, silver ion is adsorbed in around the gold particle, is reduced into simple substance silver then, the result presents the pewter of argent, signal is further amplified, and its sensitivity often can reach the pg/ml level.Utilize HAuCl in addition
4And NH
2OHHCl further increases the gold particle of mark, thereby strengthen the method for detection signal, the detection sensitivity that this method is used for human immunoglobulin(HIg) (IgG) is also high than silver staining, even can compare (document 2:Z.Ma with fluorescence immunoassay, radio-immunity and chemiluminescence immunoassay method, S.Sui, Angew.Chem.Int.Ed., 2002,41,2176-2179).
(2) semiconductor nano (claiming quantum dot again) (document 3:W.C.W.Chan, S.Nie, Science, 1998,281,2016-2018; Document 4:Y.W.Cao, Angew.Chem.Int.Ed., 1999,38,3692-3694): the nano luminescent particulate frontier that thing uses that serves as a mark has been started in the invention of quantum dot; The quantum dot of bibliographical information mainly is the compound of II-VI family and III-V family element at present, as CdSe, and InP and InAs etc.; Quantum dot exciting light wide ranges, emission peak is sharp-pointed, the Stokes displacement is big, the quantum yield of indivedual quantum dots, nanocrystalline even can reach 100% as CdSe/CdS, under the exciting of exciting light, the color of quantum dot light emitting changes with the size of its size, almost can obtain luminous from blueness to red all wavelengths; Quantum dot after the finishing is combined with biomolecule, can be used as the fluorescence probe of laser scanning imaging and immunoassay; The shortcoming of quantum dot fluorescence probe is luminous instability, easily flicker, and do not possess enough good monodispersity.
(3) include the namo fluorescence probe of fluorescence molecule: wherein a class is high molecule plastic namo fluorescence probe (document 5:H.H rm , T.Soukka, T.L vgren, Clin.Chem., 2001,47,561-568; Document 6: Zhao Yiqiang, high seapeak, Yang Wuli, the mansion longevity is wide, fluorescently-labeled high molecular particle and preparation method thereof, Chinese patent, open day January 3 calendar year 2001, publication number CN1278534A), it is the nano plastic fluorescent particle with bag beta-diketon-europium fluorescence complex in the polystyrene, such high molecule plastic particulate is easily aggegation in water, and in organic solvent the easy swelling and cause that fluorescence molecule leaks in the particulate of macromolecule; Also having a kind of is to be the particulate main body with polystyrene, polymethacrylate, the nano fluorescent particulate of surface bond or absorption fluorescein (as: fluorescein isothiocynate), rhodamine (as: rhodamine 6G), cyanine fluorescent materials such as (Cy dyestuffs).Because these microparticle surfaces bondings or absorption is conventional fluorescent material, except the shortcoming that the high molecule plastic particulate exists, the problem of various light at random and background fluorescence interference measurement when also having fluorometric assay.
Another kind of is nanometer silica gel particulate (document 7:S.Santra, P.Zhang, K.Wang, R.Tapec, W.Tan, Anal.Chem., 2001,73, the 4988-4993 of interior bag three (bipyridine) ruthenium fluorescence complex; Document 8:S.Santra, K.Wang, R.Tapec, W.Tan, Journal of BiomedicalOptics, 2001,6,160-166; Document 9: Tan Weihong, Wang Kemin, Xiao Dan, nm-class core-and-shell particles, Chinese patent, open day on April 3rd, 2002, publication number CN1342515A), this method is a nuclear with three (bipyridine) ruthenium fluorescence complex, and silica gel is that shell has prepared nano fluorescent particulate of uniform size; Compare with classic method, resistance to oxidation, the light fastness stability of the nanoparticle that this method makes all are significantly increased, and are applied to discern SmIgG in the human peripheral
+The measurement result of bone-marrow-derived lymphocyte is with close with the detected value of marked by fluorescein isothiocyanate antibody method.Remained conventional fluorescence because luminous kernel-three (bipyridine) the ruthenium fluorescence complex of this type of nano fluorescent particulate sends, can't solve the problem that various light at random and short life fluorescence disturb fluorometric assay.
Also having a class is a kind of functional nano rare-earth fluorescent particulate of applicant's exploitation recently and preparation thereof and application (document 10: Yuan Jingli, Tan Mingqian, Ye Zhiqiang, Wang Guilan, Chinese patent application number: 02144517.6).The great advantage of time-resolved fluorescence Measurement for Biotechnique is exactly to eliminate various light at random and the influence of short life fluorescence to measuring fully, measures sensitivity thereby greatly improve; This type of nano rare earth fluorescence probe has the advantage of rare-earth fluorescent, and 1. fluorescence lifetime is extremely long, and the fluorescence lifetime of organic fluorescent compounds is usually in 10 nanoseconds, and the fluorescence lifetime of nano rare earth fluorescence probe is more than hundreds of microseconds; 2. the Stokes displacement is big, and the maximum excitation optical wavelength of nano rare earth fluorescence probe is usually in the ultraviolet region of 310-370nm, and the maximum fluorescence emission wavelength is more than 500nm, can be as big as 10 times than the Stokes displacement of common fluorescent dye fluorescence; 3. the energy of fluorescence radiation is very concentrated, and the half-peak breadth at fluorescence radiation peak is below 15nm; 4. higher light stability is difficult for by photobleaching or photodissociation.Because this type of nano rare earth fluorescence probe is to adopt the method for silica gel parcel that some rare-earth fluorescent complexs are wrapped in the silica gel nanoparticle to be prepared from, in the process of finishing and mark biomolecule, the rare-earth fluorescent complex that may cause owing to the reasons such as modification of silica gel shell being wrapped up leaks, make its fluorescent weakening, and then reduced mensuration sensitivity.For addressing this is that, it is more stable to develop a kind of chemical property, and fluorescence radiation is stronger, very necessary in conjunction with more firm nano rare earth fluorescence probe.
Summary of the invention
At above problem, the purpose of this invention is to provide a kind of beta-diketon stable, that luminous intensity is high-trivalent europium complex namo fluorescence probe (Nanoparticle LanthanideFluorescence Probe is called for short NP) and preparation and application.
To achieve these goals, the technical solution used in the present invention is:
By can with trivalent europium-beta-diketone class fluorescence complex the covalent bonding reaction taking place in organic solvent with the monomer (mostly being the compound that contains the tri-alkoxy silicon group) of esters of silicon acis copolymerization, then its again with esters of silicon acis generation copolymerization, the functional nano rare-earth fluorescent particulate of formation; Described trivalent europium ion, beta-diketon organic ligand, can with the monomer and the esters of silicon acis of esters of silicon acis copolymerization, the molar ratio between them is 1: 2～3 (mol ratio of trivalent europium ion and four tooth beta-diketons and bidentate beta-diketon organic ligand was respectively 1: 2 and 1: 3): 10～100: 350～450.
Described can with the monomer of esters of silicon acis copolymerization preferable be 3-aminopropyl tri-alkoxy silicon or 3-sulfydryl propyl trialkoxy silicon etc., the molecular formula of described esters of silicon acis is Si (OR)
4, R wherein is-C
nH
2n+1, n=1～5;
Described beta-diketon organic ligand is bidentate beta-diketone compounds or four tooth beta-diketone compounds preferably;
The bidentate beta-diketone compounds, structural formula can be expressed as follows:
R in the formula
1=C
6F
5Or fat hydrocarbon substituting group C
nH
2n+1, C
nF
2n+1, n=1,2,3,4 or 5 wherein; R
2Be the arene substituting group
One of in; R
3Be chlorosulfonyl (SO
2Cl), different sulphur eyeball base (NCS), amino (NH
2) or hydrazine sulfonyl (SO
2NHNH
2);
Four tooth beta-diketone compounds, structural formula can be expressed as follows:
R in the formula
4Be chlorosulfonyl (SO
2Cl), different sulphur eyeball base (NCS), amino (NH
2) or hydrazine sulfonyl (SO
2NHNH
2); R
5=C
6F
5Or fat hydrocarbon substituting group C
nH
2n+1, C
nF
2n+1, n=1,2,3,4 or 5 wherein.
Beta-diketon-concrete preparation process of trivalent europium complex namo fluorescence probe is:
1) fluorescence precursor: at ambient temperature, in organic solvent, the beta-diketon organic ligand and can with the reaction of the monomer generation covalent bond of ethyl orthosilicate copolymerization, their consumption mol ratio is 1: 5～1: 50, the volumetric molar concentration of beta-diketon organic ligand is 0.05～0.9 μ mol/ml in the system, adds the Eu of beta-diketon organic ligand molar weight 1/2nd then
3+, make the fluorescence precursor;
2) preparation microemulsion: with organic solvent, cosurfactant, surfactant and hydromassage you than being 15～25: 2～7: 1: 6～10 evenly make microemulsion after the mixing;
3) add the fluorescence precursor in microemulsion, room temperature condition stirs after 1 hour down, adds esters of silicon acis, uses the ammoniacal liquor (NH of 3-25% at last
3Account for the percentage by weight of water) initiated polymerization, room temperature reaction is 4～48 hours under stirring, and centrifuging gets final product.
In a word, the present invention is a luminescent substance with trivalent europium and the formed hyperfluorescence complex of beta-diketon class ligand, nano rare earth fluorescence probe particulate adopts the method for chemical covalent bonding the rare-earth fluorescent complex to be securely fixed in the network system of silica gel, be prepared from by reversed-phase emulsion copolymerization granulating technology, and nano-particle surface introduce simultaneously can with the active function groups of biomolecule covalent bonds.
Described beta-diketon organic ligand and can be 1: 7～1: 18 with the consumption mol ratio of the monomer of ethyl orthosilicate copolymerization; Described organic solvent is: benzene, normal hexane, cyclohexane or sherwood oil; Cosurfactant is ethanol, propyl alcohol, isopropyl alcohol, n-hexyl alcohol, ethylene glycol, n-octyl alcohol or Decanol; Surfactant is polyoxyethylene groups octyl phenyl ether (Triton X-100 series) or polyoxyethylene groups nonylplenyl ether (NP-5 series); Organic solvent, cosurfactant and surfactant are 20～25: 3～7 for mol ratio in molar ratio: 1.
Beta-diketon-trivalent europium complex namo fluorescence probe can be used in time-resolved fluorometry.Described time-resolved fluorometry is a time-resolved fluorescent immunoassay, time-resolved fluorescence DNA hybridization assays method, and time-resolved fluorescence microscope imaging determination method, time-resolved fluorescence raji cell assay Raji and time are differentiated the fluorescent biochip determination method.
Nano rare earth fluorescence probe of the present invention solves the fluorescent bleach problem except can significantly strengthening the light stability of fluorescence probe, beyond the advantages such as " concentration delustring " phenomenon when avoiding label to use in a large number, also has following advantage:
(1) good stability.The present invention adopts the covalent bonding method to be fixed on the rare-earth fluorescent complex in the Nano microsphere firmly, therefore the nano rare earth fluorescence probe that obtains has very stable structure, fluorescence molecule easily takes place and leaks in the nanometer silica gel fluorescent microsphere that has solved the method preparation of adopting the silica gel parcel, and the problem that causes the nano-probe fluorescence intensity to weaken.
(2) luminous intensity height.Solved beta-diketon class ligand fully owing to be insoluble in water, and caused the more weak problem of nano-probe fluorescence intensity with the preparation of silica gel technique for packing.Because the nanometer silica gel fluorescent particle is prepared from water in oil reversed-phase emulsion, this method has the rare earth compounding of hydrophilic radicals such as more carboxyl or crown ether structure for those, because the rare-earth fluorescent complex mainly concentrates on aqueous phase, thereby prepare the nano fluorescent particulate that contains the higher concentration fluorescence complex easily, promptly easily prepare the bigger nano-probe of fluorescence intensity, but the complex of beta-diketon class ligand and europium is owing to be insoluble in water, with the nano-probe fluorescence intensity of water in oil inverse emulsion polymerization method preparation often a little less than, and earlier the complex of beta-diketon class ligand and europium is covalently bound on the monomer such as tri-alkoxy aminopropyl silane, carry out copolymerization with silica gel again, then can make on the resulting single nano-probe and be fixed with a large amount of beta-diketon-europium complexes, greatly improve the luminous intensity of nano rare earth fluorescence probe.
(3) preparation method is simple.In preparation nanometer silica gel fluorescent particle (in combination, can be excessive with respect to the rare-earth fluorescent complex) with the monomer of ethyl orthosilicate copolymerization, imported once amino grade can with the active function groups of biomolecule covalent bonding, can be directly used in the mark of biomolecule, save the finishing process before loaded down with trivial details nanometer silica gel particulate is used for biomarker.Though there is the silicon hydroxyl on silica gel nano-probe surface, often to adopt the cyanogen bromide (CNBr) of severe toxicity to the surface modification technology of silicon hydroxyl, because the requirement condition harshness can produce greatly inconvenience to operating personnel, even can cause health harm; On the silicon hydroxyl, introduce amino methods such as (with reactions such as silica gel microball continuation and tri-alkoxy aminopropyl silane) and adopt, the one, complicated operation, the 2nd, may make between aerosil particles to be cross-linked with each other, influence the dispersiveness of nano-probe, this invention has then solved this problem well, the nanoparticle probe of preparation not only surface has the reactive group that can be used for mark, and size evenly, favorable dispersibility.
(4) applied range.Biomolecule behind the mark of the present invention can be used for various time-resolved fluorescence Measurements for Biotechnique, as time-resolved fluorescent immunoassay, DNA hybridization assays, cell recognition, microscope imaging mensuration, unicellular in-site detecting, biochip etc., be applicable in the fields such as life science, medical science.
Description of drawings
Fig. 1 is covalent bonding europium complex BHHCT-Eu
3+The transmission electron microscope photo of nano fluorescent particulate.
Fig. 2 is covalent bonding europium complex BHHCT-Eu
3+The conventional fluorescence spectrum and the time resolution fluorescence spectral of nano fluorescent particulate; (wherein:---conventional fluorescence spectrum ,-time resolution fluorescence spectral).
Fig. 3 A is covalent bonding europium complex BHHCT-Eu
3+The fast light bleaching experiment figure as a result of nano fluorescent particulate (NP).
Fig. 3 B is free BHHCT-Eu
3+The fast light bleaching experiment of complex is figure as a result.
Fig. 4 is for using covalent bonding europium complex BHHCT-Eu
3+The preparation nanoparticle with wrap up BHHCT-Eu with silica gel
3+And BHHT-Eu
3+The fluorometric assay sensitivity comparison diagram of the nanoparticle of method preparation; (wherein: ◆ be the nanoparticle of covalent bonding europium complex preparation, is the nanoparticle with the preparation of silica gel parcel, ▲ be BHHT-Eu
3+The nanoparticle of method preparation).
Fig. 5 is for using covalent bonding europium complex BHHCT-Eu
3+The reaction principle figure of nano fluorescent particulate labelled protein.
Fig. 6 is covalent bonding europium complex BHHCT-Eu
3+Nano fluorescent particulate mark streptavidin (SA) be used to measure the hepatitis B surface antibody measuring principle figure of (being called for short HBsAg).
Fig. 7 is for using covalent bonding europium complex BHHCT-Eu
3+Nano fluorescent particulate mark streptavidin measure the working curve of hepatitis B surface antibody (HBsAg) in the human serum.
Embodiment
The invention will be further described below by embodiment.
Covalent bonding 4,4 '-two (1 ", 1 ", 1 " and, 2 ", 2 " and, 3 ", 3 " and-seven fluoro-4 ", 6 "-acetyl butyryl-6 "-yl) preparation and the sign of the nano fluorescent particulate of chlorosulfonyl-adjacent diphenyl benzene (be called for short BHHCT)-europium complex:
(1) covalent bonding BHHCT-Eu
3+The preparation of the nano fluorescent particulate of complex:
(synthetic method is referring to document 11:J.Yuan to add BHHCT in the cyclohexane, K.Matsumoto, H.Kimura, Anal.Chem., 1998,70,596-601) with aminopropyl-triethoxy silicon (being called for short APS), the mol ratio of BHHCT and APS 1: 5～1: 50, preferred 1: 7～1: 18, the volumetric molar concentration of BHHCT is 0.05～0.9 μ mol/ml in the system, and stirring reaction is 2 hours under the room temperature, adds the europium chloride (EuCl of BHHCT molar weight 1/2nd then
3), supersonic oscillations 20 minutes, the APS-BHHCT-Eu for preparing
3+Combination is preserved standby.(mol ratio is 15～25: 2～7: 1: 6～10 make microemulsion after evenly mixing by a certain percentage with cyclohexane, n-octyl alcohol and Triton X-100 and water; The APS-BHHCT-Eu that adds 30 μ l again
3+The ethyl orthosilicate of combination solution and 200 μ l (TEOS) is with 6% ammoniacal liquor (NH
3Account for the water percentage by weight) initiated polymerization, stirring reaction is 24 hours under the room temperature, promptly obtains containing covalent bonding BHHCT-Eu after the centrifuging
3+The nano rare earth fluorescent particle of complex.
(2) covalent bonding BHHCT-Eu
3+The property representation of the nano fluorescent particulate of complex:
(a) form of nano fluorescent particulate and size dimension
Fig. 1 is covalent bonding BHHCT-Eu
3+The transmission electron microscope photo of the nano fluorescent particulate of complex, as seen with the nanoparticle of the method for covalent bonding preparation and the nanoparticle for preparing with the silica gel packaging method clearly different are arranged from photo, in the nanoparticle that makes with packaging method because europium ion can be focused on the particulate center, therefore can clearly see the formed stain of nanoparticle center europium ion on the transmission electron microscope photo, and with having can't see stain in the center of the nanoparticle of the method for covalent bonding preparation, and the nanoparticle color is even, there is not visible light and shade difference, this explanation rare-earth europium ion has not been to resemble the main center that accumulates in particulate the nanoparticle that makes with the method for wrapping up, but is evenly distributed in the middle of the whole nanoparticle.The nanoparticle size that makes with this method is even, and grain size is 37 ± 3nm.The size of particle diameter can wait by adjusting water and the ratio of surfactant, the kind of cosurfactant, the concentration of ammoniacal liquor, amount and the reaction time of TEOS.
(b) the fluorescence spectrum characteristic of nano rare earth fluorescent particle
Fig. 2 is covalent bonding BHHCT-Eu
3+The fluorescence spectrum and the time resolution fluorescence spectral of the nano fluorescent particulate aqueous solution of complex.There is serious at random smooth Interference Peaks in the glow peak of the fluorescence spectrum of visible NP among the figure, and the glow peak of its time resolution fluorescence spectral does not then thoroughly have the existence of Interference Peaks.Free complex B HHCT-Eu
3+Fluorescence spectrum on its maximum emission wavelength be 612nm, the maximum emission wavelength of its fluorescence spectrum also is 612nm behind the nano fluorescent particulate and make, wavelength does not change.The fluorescence lifetime of the nano fluorescent particulate that this method makes is 0.376 second after measured, and the fluorescence that the nano fluorescent particulate is described has still kept the feature of the long fluorescence lifetime of europium complex.
(c) light stability of nano rare earth fluorescent particle experiment
Covalent bonding BHHCT-Eu
3+The light stability experimental result of the nano fluorescent particulate of complex as shown in Figure 3, used light source is the 25W deuterium lamp, sample is apart from deuterium lamp 12cm, measures an emission spectrum in per 5 minutes.Through strong UV-irradiation after 60 minutes, free BHHCT-Eu
3+The fluorescence intensity of complex itself decays to for 40.1% when initial, and the fluorescence intensity of nano fluorescent particulate only decays to for 84.2% when initial, illustrates that the anti-photobleaching ability of nanoparticle is better than free BHHCT-Eu far away
3+Fluorescence complex.
(d) covalent bonding BHHCT-Eu
3+The nano fluorescent particulate of complex and the BHHCT-Eu for preparing with direct silica gel packaging method
3+And BHHT-Eu
3+Complex [4,4 '-two (1 ", 1 ", 1 ", 2 ", 2 ", 3 ", 3 "-seven fluoro-4 ", 6 "-acetyl butyryl-6 "-yl)-adjacent diphenyl benzene-europium complex] the nano fluorescent particulate the fluorometric assay remolding sensitivity
In order relatively to adopt covalent bonding BHHCT-Eu
3+Complex method and the difference of nanoparticle aspect fluorescence intensity that adopts the direct packaging method preparation of silica gel, we adopt same reaction system and identical initial complex concentration (0.164 μ mol/ml), prepared the nano rare earth fluorescent particle with covalent bonding method and silica gel packaging method respectively, and tested the fluorometric assay sensitivity of the nanoparticle that two kinds of methods make, concrete measurement result sees the following form:
| The rare earth compounding kind | The particle diameter of nanoparticle | The lowest detection lower limit | |
| Covalent bonding | BHHCT-Eu 3+ | 37 | 0.41 |
| The silica gel pack | BHHCT-Eu 3+ | 33 | 87.5 |
| BHHT-Eu 3+ | 31 | 382.6 |
Europium complex concentration when the preparation nanoparticle in the reaction system is identical, but the result as seen from table, the fluorometric assay lowest detection lower limit of the nano fluorescent particulate of covalent bonding method and the preparation of silica gel packaging method but differs greatly, lowest detection lower limit with the nanoparticle of covalent bonding method preparation can reach 0.41ng/ml (weight/liquor capacity of nano particle), and can only reach 87.5 and 382.6ng/ml with the lowest detection lower limit of the nano fluorescent particulate of silica gel packaging method preparation, and the particle diameter of nanoparticle is close, can learn that thus fluorescence intensity with the nanoparticle of covalent bonding method preparation will be far longer than the fluorescence intensity with the nanoparticle of silica gel packaging method preparation, three kinds of nanoparticle dilute solutions are fluorimetric to the results are shown in Figure 4.
Embodiment 2
Use covalent bonding BHHCT-Eu
3+The serve as a mark time-resolved fluorescent immunoassay of thing of the nano fluorescent particulate of complex is measured hepatitis B surface antibody (being called for short HBsAg) in the human serum.
(1) utilizes nano rare earth fluorescent particle mark streptavidin (being called for short SA)
Get in the 0.1mol/L phosphate buffer solution (pH=7.1) that the 1mg nanoparticle is distributed to 1.0ml, with supersonic oscillations after 20 minutes, add 5mg bovine serum albumin(BSA) (being called for short BSA), stir 1% glutaraldehyde (phosphate buffer solution) that adds 300 μ l down, room temperature reaction 24 hours, centrifugal collecting precipitation.Precipitation is washed in 1% glutaraldehyde (phosphate buffer solution) that joins 200 μ l after twice with 200 μ l phosphate buffers, adds 1mg SA then, and room temperature reaction added 0.5mg NaBH after 24 hours
4Reduction, room temperature reaction 2 hours contains 0.2%BSA-0.1%NaN with being suspended in after the phosphate buffer solution washing for several times
3Standby in the 0.05mol/L Tris-HCl buffer solution (buffer solution 1) of the pH value 7.8 of-0.9%NaCl, the reaction principle of this labelling method is seen Fig. 5.
(2) preparation of the anti-people HBsAg of biotin labeling cavy antibody
1.4ml the anti-people HBsAg of cavy (0.5mg/ml) antibody (Shanghai Xiamen Kehua) at 4 ℃, contained twice dialysis of aqueous solution of 0.9%NaCl in 24 hours to 3L after, add the NaHCO of 12mg
3With the NHS-LC-biotin (Pierce Chemical Co.) of 3mg, stirring at room was placed 24 hours down for 4 ℃ after 1 hour.Reactant liquor is at 4 ℃, 3L contained 0.25g NaN in 24 hours
30.1mol/L NaHCO
3Add the BSA of 10mg and the NaN of 20mg after twice dialysis of aqueous solution
3, place-20 ℃ standby.When being used for immunoassays, after diluting 1000 times, usefulness (buffer solution 1) uses.
(3) mensuration of HBsAg in the human serum
(a) the bag quilt of 96 microwell plates: will contain the 0.1mol/L of anti-people HBsAg monoclonal antibody (Shanghai Xiamen Kehua, 10 μ g/ml), the NaHCO of pH value 9.6
3Buffer solution 50 μ l dispensings are in each hole of 96 microwell plates, 4 ℃, behind 24 hours bag quilts, with the 0.05mol/L that contains 0.05%Tween 20, the Tris-HCl buffer solution of pH value 7.8 (buffer solution 2) is washed twice, use 0.05mol/L again, the Tris-HCl buffer solution of pH value 7.8 (buffer solution 3) is washed 1 time, and the microwell plate behind the bag quilt can be preserved more than 1 month under-20 ℃.
(b) time-resolved fluorescent immunoassay of HBsAg: the reaction principle of this mensuration as shown in Figure 6.With containing 0.01%BSA-0.9%NaCl-0.1%NaN
3The 0.05mol/L Tris-HCl buffer solution of pH value 7.8 inject above-mentioned bag by after each hole of microwell plate, 37 ℃, the reaction back washed twice with buffer solution 2 in 1 hour, buffer solution 3 is washed 1 time.With containing 5%BSA-0.9%NaCl-0.1%NaN
30.05mol/L Tris-HCl buffer solution (buffer solution 4) the dilution people HBsAg of pH value 7.8 (Dakopatts Denmark) makes the HBsAg standard solution.With this standard solution and blood serum sample respectively 50 μ l inject above-mentioned bag by after each hole of microwell plate, 37 ℃, after the reaction in 1 hour, with buffer solution 2, buffer solution 3 is cleaned, add the biotin labeled anti-HBsAg antibody-solutions of 50 μ l then, 37 ℃, after the reaction in 1 hour, with buffer solution 2, buffer solution 3 is cleaned, and adds the SA solution of 50 μ l NP marks again, 37 ℃, after the reaction in 1 hour, with the 0.05mol/L that contains 0.05%Tween 20, the Tris-HCl damping fluid of pH value 9.1 is washed 4 times, carries out the solid phase time-resolved fluorometry then.Measuring with instrument is WALLAC VICTOR 1420 multiple labeling calculating instruments (PerkinElmerLife Science company products), and condition determination is: excitation wavelength, 340nm; Detect wavelength, 615nm; Delay time, 0.2ms; The window time, 0.4ms; Cycling time, 1.0ms.
The working curve of measuring HBsAg with this method as shown in Figure 7,3 times of the standard deviation (SD) of the fluorescence signal during with zero-dose (background) are calculated the minimum lower limits that detect that HBsAg measures, minimum detecting down that must this law is limited to 30pg/ml.The working curve upper limit can reach 10ng/ml.
This method is used for human serum sample HBsAg and adds the measurement result of the recovery and measure precision as shown in the table, average measurement result of adding the recovery is 95.9%, average relative standard deviation is 6.20%, illustrates that the mensuration that method of the present invention is used for human serum HBsAg has extraordinary accuracy and precision.
| HBsAg addition (ng/ml) | HBsAg measured value (ng/ml) | The recovery (%) | Relative standard deviation (%, n=6) |
| | 0.456 | ---- | 2.76 |
| 2.500 | 2.656 | 88.0 | 5.39 |
| 5.000 | 4.823 | 87.3 | 8.24 |
| Sample 2 | 0.158 | ---- | 1.41 |
| 0.250 | 0.416 | 103.2 | 8.43 |
| 2.500 | 2.528 | 94.8 | 6.93 |
| Sample 3 | 0.411 | ---- | 6.35 |
| 0.500 | 0.817 | 81.2 | 9.39 |
| 2.500 | 3.184 | 110.9 | 9.44 |
| 5.000 | 5.342 | 98.6 | 11.71 |
| Sample 4 | 0.169 | ---- | 2.42 |
| 0.500 | 0.612 | 88.6 | 5.16 |
| 2.500 | 2.583 | 96.6 | 6.87 |
| 5.000 | 5.651 | 109.6 | 2.36 |
Claims (7)
1. beta-diketon-trivalent europium complex namo fluorescence probe, it is characterized in that: its be by can with the monomer of esters of silicon acis copolymerization, the covalent bonding reaction takes place in organic solvent with trivalent europium-beta-diketone class fluorescence complex, then its again with esters of silicon acis generation copolymerization, the functional nano rare-earth fluorescent particulate of formation; Described trivalent europium ion, beta-diketon organic ligand, can with the monomer and the esters of silicon acis of esters of silicon acis copolymerization, the molar ratio between them is 1: 2～3: 10～54: 350～450.
2. according to the described beta-diketon of claim 1-trivalent europium complex namo fluorescence probe, it is characterized in that: described can be 3-aminopropyl tri-alkoxy silicon or 3-sulfydryl propyl trialkoxy silicon with the monomer of esters of silicon acis copolymerization.
3. according to the described beta-diketon of claim 1-trivalent europium complex namo fluorescence probe, it is characterized in that: the molecular formula of described esters of silicon acis is Si (OR)
4, R wherein is-C
nH
2n+1, n=1～5.
4. according to the described beta-diketon of claim 1-trivalent europium complex namo fluorescence probe, it is characterized in that: described beta-diketon organic ligand is bidentate beta-diketone compounds or four tooth beta-diketone compounds;
The bidentate beta-diketone compounds, structural formula can be expressed as follows:
R in the formula
1=C
6F
5Or fat hydrocarbon substituting group C
nH
2n+1, C
nF
2n+1, n=1,2,3,4 or 5 wherein; R
2Be the arene substituting group
One of in;
R
3Be chlorosulfonyl (SO
2Cl), different sulphur eyeball base (NCS), amino (NH
2) or hydrazine sulfonyl (SO
2NHNH
2);
Four tooth beta-diketone compounds, structural formula can be expressed as follows:
R in the formula
4Be chlorosulfonyl (SO
2Cl), different sulphur eyeball base (NCS), amino (NH
2) or hydrazine sulfonyl (SO
2NHNH
2); R
5=C
6F
5Or fat hydrocarbon substituting group C
nH
2n+1, C
nF
2n+1, n=1,2,3,4 or 5 wherein.
5. the preparation method of the described beta-diketon of claim 1-trivalent europium complex namo fluorescence probe is characterized in that:
Specific operation process is,
1) fluorescence precursor: at ambient temperature, in organic solvent, the beta-diketon organic ligand and can with the reaction of the monomer generation covalent bond of ethyl orthosilicate copolymerization, their consumption mol ratio is 1: 5～1: 50, the volumetric molar concentration of beta-diketon organic ligand is 0.05～0.9 μ mol/ml in the system, adds the Eu of beta-diketon organic ligand molar weight 1/2nd then
3+, make the fluorescence precursor;
2) preparation microemulsion: with organic solvent, cosurfactant, surfactant and hydromassage you than being 15～25: 2～7: 1: 6～10 evenly make microemulsion after the mixing;
3) adding fluorescence precursor in microemulsion, room temperature condition stirred down after 1 hour, added esters of silicon acis, used the ammoniacal liquor initiated polymerization of weight content 3-25% at last, and room temperature reaction is 4～48 hours under stirring, and centrifuging gets final product.
6. according to the preparation method of the described beta-diketon of claim 5-trivalent europium complex namo fluorescence probe, it is characterized in that: described organic solvent is: benzene, normal hexane, cyclohexane or sherwood oil; Cosurfactant is ethanol, propyl alcohol, isopropyl alcohol, n-hexyl alcohol, ethylene glycol, n-octyl alcohol or Decanol; Surfactant is polyoxyethylene groups octyl phenyl ether or polyoxyethylene groups nonylplenyl ether.
7. the described beta-diketon of the claim 1-application of trivalent europium complex namo fluorescence probe in time-resolved fluorescent immunoassay.
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| CN101093222B (en) * | 2007-07-24 | 2011-11-23 | 山东师范大学 | Fluorescent probe in use for detecting glutathion inside cell, synthesis method and application |
| CN101712866B (en) * | 2008-10-07 | 2013-03-06 | 中国科学院大连化学物理研究所 | Nanometer europium fluorescent particle with performance of visible light excitation, preparation method and application thereof |
| CN101493460B (en) * | 2009-02-25 | 2012-06-27 | 江西中德生物工程有限公司 | Method for producing fluorescent microballoons immune chromatography test paper stripe and quantitative determination method |
| KR101140029B1 (en) * | 2010-02-23 | 2012-06-21 | 한국식품연구원 | Preparation method of antigen-immobilized immuno- fluorescence slide and the immuno-fluoroscence slide made by the method |
| CN102391162A (en) * | 2011-09-28 | 2012-03-28 | 大连理工大学 | Three fluorescence complexes formed on basis of Eu (III) and chlorosulfonation four-tooth Beta-diketone ligands and application thereof |
| CN102654505A (en) * | 2012-05-16 | 2012-09-05 | 江苏省原子医学研究所 | Time-resolved fluorescence immunoassay reagent kit used for detecting interleukin (IL)-2-human serum albumin (HSA) and detection method thereof |
| CN103439489B (en) * | 2013-08-03 | 2015-07-01 | 河南省农业科学院 | Fluorescent silica labeled immunochromatographic test paper for quantitative gentamicin detection and preparation method |
| CN104694117A (en) * | 2013-12-06 | 2015-06-10 | 中国科学院大连化学物理研究所 | Ratio-type fluorescent probe based on carbon dot as well as preparation method and application thereof |
| CN104926698A (en) * | 2014-03-18 | 2015-09-23 | 中国科学院大连化学物理研究所 | Tetradentate beta-diketone ligand-europium fluorescent complex and application thereof |
| CN105044074B (en) * | 2015-09-10 | 2017-10-20 | 深圳市水务(集团)有限公司 | A kind of method that utilization silicon substrate chemical sensing material detects water pollutant concentration |
| CN105891477A (en) * | 2016-04-06 | 2016-08-24 | 上海奥普生物医药有限公司 | Cup type time-resolved fluorescence MPO analysis method and reagent kit based on microspheres |
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