CN1231760C - Rare earth nano particle for biological material label and its preparing method and use - Google Patents
Rare earth nano particle for biological material label and its preparing method and use Download PDFInfo
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- CN1231760C CN1231760C CN 01109551 CN01109551A CN1231760C CN 1231760 C CN1231760 C CN 1231760C CN 01109551 CN01109551 CN 01109551 CN 01109551 A CN01109551 A CN 01109551A CN 1231760 C CN1231760 C CN 1231760C
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Abstract
The present invention relates to a rare earth nanometer particle used for marking biologic materials. The particle diameter of the particle is between 3 nm and 400 nm. The particle can be connected with the biologic material, and can emit fluorescence during excitation. The components of the particle are selected from one of or a plurality of the following compound substances: SrAl2O4: Eu<2+>; SrAl2O4: Eu<2+>+Dy<3+>; Y2O3: Eu<3+>; CaS: Eu<3+>; CaS: Eu<3+>+X, and X=Tm<3+>, Y<3+> or Al<3+>; ZnS: Y, and Y=Eu<2+>, Mn<2+>, Cu<2+> and/or Au<+>; Gd2O3: Eu<3+>; La2O3: Eu<3+>; MgB5O10: Ce<3+>+Gd<3+>; SrB4O7: Sm<2+> and/or Gd3 (PO4)2: Eu<3+>. In order to prevent the hydrolyzation of the rare earth nanometer particle, the surface of the particle can be coated with a layer of polymer film or an inorganic matter film. The particle diameter of the particle which is coated is preferably between 5 and 100 nm.
Description
The present invention relates to a kind of rare-earth nanometer particles material, Preparation Method And The Use that can be used for biological material label.Specifically, the present invention relates to a kind of can be used for rare-earth nanometer particles material, Preparation Method And The Use biological material label, that be aggregated thing bag quilt.
Biomaterial such as DNA, polypeptide, protein, cell and tissue etc. itself lack the character that can be used for detecting.In life science or clinical diagnosis, often need carry out mark, so that it has character such as radioactivity, fluorescence or chemiluminescence to these materials.Fluorescent marker method is a kind of method that generally adopts at present.At present, fluorescent material commonly used mainly is an organic dyestuff, as fluorescein isothiocyanate (fitc) (FITC), RB 200 (RB 200) etc.The shortcoming of organic dyestuff fluorescence spectrum is that excitation peak is narrow, and Stokes shift is little, and fluorescent lifetime is short, is easy to photobleaching, and this makes the application of organic fluorescence labelling technique be very limited.
At present, there are many reports to utilize the fluorescent marker of fluorescent microsphere as antibody (antigen) molecule.For example, with perylene microballoon (0.8-1 μ m) as fluorescent marker, in the water-soluble solution that links to each other with antibody afterwards, concentration that can detection by quantitative antigen.And the quantum dot nano microballoon is because its quantum limitation effect, has that excitation of spectra peak width, emission peak are narrow, fluorescent lifetime is difficult for being suitable as a kind of desirable fluorescence labeling material by characteristics such as photobleachings.Chan has reported the mark that respectively different quantum dot nano microballoons is used for biomolecule with Marcel.But, because the condition of preparation quantum dot Nano microsphere is very harsh, and the cost costliness, make its level of application also be subjected to very big restriction.
U.S. Pat 5,990 has proposed to be used as fluorescence probe with a kind of nano crystal semiconductor in 479.The semiconductor that it adopted is MgS, MgSe, MgTe, CaS, CaSe, CaTe, SrS, SrSe, SrTe, BaS, BaSe, BaTe, ZnS, ZnSe, ZnTe, CdS, CdSe, CdTe, HgS, HgSe and HgTe etc.
As everyone knows, terres rares solid material fluorescence intensity height, fluorescence lifetime long (life-span of a lot of materials can reach millisecond to the number of minutes magnitude), Stokes shift is big, and various in style, simple synthetic method, with low cost, be the extremely wide fluorescent material of a class usable range.But rare earth material is normally synthesized by high temperature at present, mostly is bulk material.Wherein some composition is water-soluble, as its borate, aluminate etc., can cause these phosphors to be partially dissolved in water when using in aqueous solution and loses the characteristic of sending fluorescence, and is therefore unsuitable directly as the fluorescent marker of biomaterial.Simultaneously, also there are inorganic rare earth material and biomaterial strength of joint problem, and the activity problems that how to keep biomaterial after connecting.
With US5,990,479 semiconductor nano fluorescence probe differences, the present invention proposes a kind of rare earth or the rare earth doped nano particle fluorescence probe as biomaterial.Its luminescence mechanism is relevant with the deformation of the f layer electronics of rare earth.
Purpose of the present invention just provides a kind of rare-earth nanometer particles in the aqueous solution, be connected the good marking biomaterial with biomaterial that can be applicable to.
Another object of the present invention provides a kind of method for preparing above-mentioned rare-earth nanometer particles.
A further object of the invention is the detection that above-mentioned rare-earth nanometer particles is applied to biomaterial.
In the present invention, the rare-earth nanometer particles that can be used as the class fluorescence probe can be the potpourri that a kind of rare earth or several rare earth are formed, also can be rear-earth-doped in other non-rare earth formed potpourri, as be doped in the materials such as sulfide, phosphate, aluminate, borate.Specifically, the composition of rare-earth nanometer particles of the present invention can be selected from a kind of in following each compound substance or several: SrAl
2O
4: Eu
2+SrAl
2O
4: Eu
2++ Dy
3+Y
2O
3: Eu
3+CaS:Eu
3+CaS:Eu
3++ X, wherein X=Tm
3+, Y
3+Or Al
3+ZnS:Y, wherein Y=Eu
3+, Mn
2+, Cu
2+, Ag
+And/or Au
+Gd
2O
3: Eu
3+La
2O
3: Eu
3+MgB
5O
10: Ce
3++ Gd
3+SrB
4O
7: Sm
2+And/or Gd
3(PO
4)
2: Eu
3+In above-mentioned each expression formula, M
N+The expression Doped Rare Earth.
Rare earth ion of the present invention has very strong fluorescence intensity, and its shape can be spherical, bar-shaped, cube, rectangular parallelepiped or preceding several combination in any shape.Particle size can be in 3 nanometers between 400 nanometers, and suitable particle size is between the 5-100 nanometer.Best scope is that the size of particle is between the 5-100 nanometer after following bread is processed.The fluorescent nano particles size distribution is narrow, size distribution in ± 20%, be preferably in ± 15% within.
Among the present invention, for making the rare-earth nanometer particles fluorescent material, thereby cause fluorescence intensity to descend, should wrap quilt the rare-earth nanometer particles material not by solution corrosion or dissolving in institute's application system.Promptly at the surperficial coated last layer polymer film or the inorganics film of said rare-earth nanometer particles, with the protection rare-earth nanometer particles.
The method of bag quilt has three kinds basically: organic monomer polymerization, organosilane Hydrolyze method and absorption method.
Utilize the surface of the polymer film of organic monomer polymerization gained to need through functionalization, make different functional groups be connected in the surface of particle, thereby the rare-earth nanometer particles after the coating can be connected effectively with biomaterial as amino, carboxyl, hydroxyl, aldehyde radical and/or epoxy radicals etc.
Total organic monomer that the bag tegillum of organic monomer polymerization is made up of one or more components in the following various monomers gets by suspension polymerization:
1. coating monomer: only contain a carbon-to-carbon double bond that can carry out free radical polymerization in the molecule;
2. crosslinking chemical: contain the carbon-to-carbon double bond that can carry out free radical polymerization more than 2 or 2 in the molecule.The purpose that adds crosslinking chemical is to make to contain reticulate texture in the pan coating layer, but the adding of this monomer is chosen wantonly;
3. functionalized reagent: contain the carbon-to-carbon double bond and the functionalization group that can carry out free radical polymerization in the molecule simultaneously.These functionalization groups can be: amino, carboxyl, hydroxyl, aldehyde radical or epoxy radicals etc.By these functional groups, the rare-earth nano-fluorescent probe can be connected with biomaterial easily;
4. coupling agent: an end contains the phosphinylidyne oxygen groups of strong polarity, has strong complexing with rare earth, and the other end contain long-chain and can with two keys of other monomer copolymerization.Because the surface polarity of rare-earth nanometer particles is strong, and the polarity of polymkeric substance a little less than, can be coated on the surface of rare-earth nanometer particles fully in order to make polymkeric substance, can also add above-mentioned coupling agent.Like this, foregoing polymerization single polymerization monomer can be adsorbed in microparticle surfaces earlier as coating monomer, crosslinking chemical, functionalized reagent etc., carries out polymerization again, and the result is the polymer covering layer that can obtain being evenly distributed.Pure polymerization single polymerization monomer then seldom forms new polymer beads.
In the present invention, the coating monomer is selected from one or more in following group usually: acrylic acid, methacrylic acid, methyl acrylate, methyl methacrylate, 2-hydroxyethyl methacry-late, acrylic acid-2-hydroxyl ethyl ester, glycol monomethyl epoxy acrylate, diethylene glycol monoacrylate, triethylene glycol diacrylate, glycol diacrylate and styrene etc.
Crosslinking chemical of the present invention is selected from one or more in following group usually: divinylbenzene, glycol diacrylate, diethylene glycol diacrylate, triethylene glycol diacrylate, trimethylolpropane triacrylate, pentaerythritol diacrylate, pentaerythritol triacrylate etc.
Functionalized reagent of the present invention is selected from one or more in following group usually: acrylic acid, methacrylic acid, methacrolein, acrylamide, 3-aminopropyltriethoxywerene werene, 3-aldehyde radical propyl-triethoxysilicane, 3-aminocarbonyl propyl triethoxysilane, 3-sulfydryl propyl-triethoxysilicane, 2-hydroxyethyl methacry-late, acrylic acid-2-hydroxyl ethyl ester, methacrylic acid-2-amine ethyl ester, ethylene glycol bis be oxygen mono acrylic ester, 3-epoxypropyl triethyl silicane etc. also.
The selected coupling agent of the present invention can be phosphoric acid two-(trimethylolpropane diacrylate), phosphoric acid is two-(2-hydroxyethyl methacry-late) and/or phosphoric acid is two-(pentaerythritol triacrylate) etc.
Be aggregated the thing layer except above-mentioned employing polymerization wraps on rare-earth nanometer particles, another kind of method of wrapping quilt on rare-earth nanometer particles is the organosilane Hydrolyze method.This method at first forms silicon dioxide layer through hydrolysis with alkyl silicate on rare-earth nanometer particles, and then carry out functionalization with organosilane such as triethoxy-3-aminopropyl silane, triethoxy-3-aldehyde radical propyl silane, triethoxy-3-mercaptopropylsilane and/or tetraethoxysilane etc. so that bag by after rare-earth nanometer particles be connected better with the biomaterial continuation.
In addition, can also directly on the rare-earth nanometer particles surface, adsorb glycosaminoglycan, polymethylacrylic acid, epoxy glucosan and/or phosphatide etc., form the bag tegillum.
When adopting the preparation of organic monomer polymerization to be used for the rare-earth nanometer particles of label biological materials, mainly comprise the steps:
(1) rare-earth nanometer particles of preparation particle diameter between 3-400nm;
(2) rare earth ion with step (1) preparation joins in the organic solvent;
(3) in the potpourri of step (2) gained, add total organic monomer and polymerization initiator, carry out suspension polymerization, make the rare-earth nanometer particles surface coat the one layer of polymeric film.
Rare-earth nanometer particles between the 3-400nm in the step of the present invention (1) can utilize multiple preparation nano particle method to make, for example sol-gel process, the precipitation method, firing method, hydro-thermal method, the high temperature precipitation method, microwave method etc.In order to obtain the strongest fluorescence intensity, the nano particle that makes heats under higher temperature makes the nano particle lattice arrangement neat.In order to obtain the particle of narrower particle size range, can carry out grain size segregation to the particulate of gained.
Polyreaction involved in the present invention is finished by suspension polymerization in organic solvent usually.The selected organic solvent of step (2) can be toluene, dimethylbenzene and/or tetrahydrofuran etc.
For the particle that makes in the step (1) is fully disperseed, also to add surfactant usually in above-mentioned organic solvent.Surfactant can be non-ionic surfactant and/or anionic surfactant, as APES (OP-10, OP-15), lauryl sodium sulfate, neopelex etc.The content of surfactant in organic solvent is 0～5 volume %, and preferred content is 0.1～5 volume %.
In the step (3) of above-mentioned preparation process because the surface polarity of rare-earth nanometer particles is strong, and the polarity of polymkeric substance a little less than, be coated on the rare earth microparticle surfaces fully for making polymkeric substance, can add coupling agent.Added coupling agent can be phosphoric acid two-(trimethylolpropane diacrylate), phosphoric acid is two-(methacrylic acid 2-hydroxyl ethyl ester) and/or phosphoric acid is two-(pentaerythritol triacrylate) etc.
The coating of step (3) forms one layer of polymeric bag tegillum by polyreaction and carries out, and it relatively is suitable for containing the rare earth nano particulate of water-soluble component.In this step, add suitable coating monomer and crosslinking chemical, functionalized reagent and coupling agent, make each monomer be distributed to the surface of rare earth nanometer particle.Then under agitation, add initiating agent and improve temperature of reaction, make each monomer form the hydrophilic film of one deck at the surface aggregate of rare-earth nanometer particles.The thickness of this film should not be too thick, and Bao Yuehao needs only and can form continuous overlayer in fact more.If the bag tegillum is too thick, will cause wrapping by after particle volume excessive, be unfavorable for and the combining of biomaterial.The thickness that coats can be regulated with the amount that coats monomer by regulating rare-earth nanometer particles.Fully wash particulate after reaction finishes, remove unreacted matters and surfactant, be dispersed in the phosphate buffered solution it stand-by.
Among the present invention, the consumption of all ingredients can change according to request for utilization when nano particle coated.The total organic monomer (comprising the summation that coats monomer, crosslinking chemical, functionalized reagent, coupling agent) that participates in reaction and the mass ratio of rare-earth fluorescent particulate are in 1: 300～1: 1 scope.The content of various components in total organic monomer is: coating monomer is 5～80 volume %; Functionalized reagent is 5～40 volume %; Crosslinking chemical is 0～80 volume %; Coupling agent 0～10 volume %.The initiating agent that also need add in addition, 1～5 volume %.Preferred each components contents is: coat monomer: 5～80 volume %, functionalized reagent: 5～40 volume %, crosslinking chemical: 5～80 volume %, coupling agent: 1～10 volume %, initiating agent: 1～5 volume %.
Preferably those have the monomer of functional group to be suitable for forming in the step (3) monomer of polymer coating layer, for example: acrylic acid, methacrylic acid, methyl methacrylate, methacrolein, 2-hydroxyethyl methacry-late, glycol monomethyl epoxy acrylate, glycol diacrylate, diethylene glycol monoacrylate, triethylene glycol diacrylate, trimethylolpropane triacrylate, pentaerythritol diacrylate, pentaerythritol triacrylate, styrene, divinylbenzene etc.These monomers can be used alone, but also also mix together, and best and crosslinking chemical is used.
In fact, for not diffluent rare-earth nanometer particles in the water, fluorescence can not descend when these rare-earth nanometer particles were preserved in water.Utilize absorption method wrap by the time, in the aqueous solution of glycosaminoglycan (Aminodextron), polymethylacrylic acid or the phosphatide etc. that can directly rare-earth nanometer particles be dispersed in, make the coated last layer polymer film of surface energy of particle by physisorption.This tunic has extraordinary water wettability, can be connected by physisorption or chemical reaction with biomolecule.
By the rare-earth nanometer particles that is aggregated thing bag quilt that the inventive method makes, can be as the rare-earth nano-fluorescent probe of label biological materials.The biomaterial that can be used to mark comprises DNA, polypeptide, protein (enzyme, antibody, antigen etc.), cell etc.Rare-earth nanometer particles with the inventive method preparation is made fluorescence probe, simple synthetic method, be the label of desirable biomaterial, can be widely used in the fluorescence probe in mark, the immunoassay in the clinical diagnosis, DNA detection and the biochip of the multiple biomaterial in the life science.
The method that rare-earth nanometer particles of the present invention is connected with biomaterial is, will add biomaterial to be marked through coating and the rare-earth nanometer particles of functionalization be dispersed in (w/w, 5%) in the phosphate solution, as DNA, and polypeptide, protein, cell and tissue etc.Reactant liquor should be controlled at alkalescence when epoxy radicals is connected with biomolecule; When being connected with biomolecule, carboxyl adds activating reagent 1-ethyl-3-(3-dimethyl aminopropyl)-carbodiimide; When aldehyde radical was connected with biomolecule, the schiff alkali that the reaction back generates was stable inadequately, needed to add boron sodium cyanide reduction afterreaction and generated stable secondary amine; When amino is connected with biomolecule, adds the glutaraldehyde reaction earlier and generate aldehyde radical, be connected with biomolecule again.All sluggish stirred for several hour, centrifugal, with the phosphate buffered solution washing for several times, obtain the biomolecule of fluorescent rare earth nano particle mark.The biomolecule of these marks obtains fluoroscopic image by the fluorescent spectrophotometer assay light intensity or with laser confocal scanning microscope, can be used for research fields such as life science, clinical disease detection, DNA chip, protein-chip.
In the present invention, rare-earth nanometer particles with polymer overmold and functional modification after, can be scattered in preferably in the aqueous solution, be difficult for precipitation and flocculation mutually, coat and modification can not cause the remarkable decline of fluorescence intensity.Rare-earth nanometer particles after handling like this can be connected preferably with biomaterial, and the biologically active of biomaterial can significantly not descend yet simultaneously, thereby can be used as the fluorescence probe of biomaterial such as protein, polypeptide, DNA, cell and tissue etc.
Below in conjunction with accompanying drawing, further specify the present invention with embodiment.But should be appreciated that these embodiment do not limit the scope of the present invention, they just are used for describing.
Fig. 1 is rare-earth nanometer particles CaS:Eu
3+Electromicroscopic photograph;
Fig. 2 is rare-earth nanometer particles CaS:Eu
3+Fluorescence spectrum figure;
Fig. 3 is that bag is by front and back rare-earth nanometer particles SrAlO
4: Eu
2+Fluorescence spectrum figure:
(A) before the bag quilt; (B) behind the bag quilt;
Fig. 4 is the fluoroscopic image of Scanner Array 4000 scannings;
Fig. 5 is the fluorescence intensity of rare-earth nanometer particles and the concentration relationship of determined antigen.
Embodiment 1 nano particleCaS
:Eu
3+ Preparation
Prepared sizes are the CaS:Eu about 15 nanometers
3+Particle, the preparation method is as follows: with 1.3875gCaCl
2(0.0125mol) be dissolved in the 250ml absolute ethyl alcohol, add Eu (NO) again
3(0.111g) with the 0.027g histidine, ultrasonic 30min obtains storing solution a.Take by weighing 0.24g Na
2S is dissolved in the absolute ethyl alcohol of 250ml, and ultrasonic 30min obtains storing solution b.Storing solution a 50ml is put into there-necked flask, and logical nitrogen with the quick agitating solution of magnetic stirring apparatus, injects there-necked flask with syringe with storing solution b with the airtight back of flask mouth fast, stops reaction after 3 hours.After centrifugal, remove supernatant liquid, use deionized water wash solid 3 times, use absolute ethanol washing again 3 times.After carrying out drying under the solid vacuum, be placed under 675 ℃ sample calcination 3h, and logical nitrogen protection.After treating the sample cool to room temperature, just obtain the rare earth ion of particle diameter about 15 nanometers, the sem photograph of nano particle is seen accompanying drawing 1, and fluorescence spectrum figure sees Fig. 2.
Embodiment 2 nano particlesSrAlO
4 :Eu
2+ Preparation
Prepare nano level rare earth particulate SrAlO
4: Eu
2+, the proportioning of participating in compound of reaction is SrCO
3: Al
2O
3: Eu
2O
3=1: 1: 0.005, add an amount of 1mol/l nitric acid pressed powder is dissolved just, then with after the urea liquid of 1mol/l mixes, directly move in the muffle furnace that is heated to 500 ℃ in advance.After treating sample combustion, can obtain white foam shape long-persistence nano material, particle size is in the 10-15 nanometer.
Embodiment 3 nano particlesCaS
:Eu
3+ +Sr
2+ Preparation
Prepared sizes are the CaS:Eu about 10 nanometers
3++ Sr
2+Particle, the preparation method is as follows: with 0.712g CaCl
2With 0.625g SrCl
2Be dissolved in the 250ml absolute ethyl alcohol, add Eu (NO) again
3(0.111g) with the 2.5ml mercaptoacetic acid, ultrasonic 30min obtains storing solution a.Take by weighing 0.25g Na
2S is dissolved in the absolute ethyl alcohol of 250ml, and ultrasonic 30min obtains storing solution b.Storing solution a 50ml is put into there-necked flask, and logical nitrogen with the quick agitating solution of magnetic stirring apparatus, injects there-necked flask with syringe with storing solution b with the airtight back of flask mouth fast, stops reaction after 3 hours.After centrifugal, remove supernatant liquid, use deionized water wash solid 3 times, use absolute ethanol washing again 3 times.After carrying out drying under the solid vacuum, be placed under 675 ℃ sample calcination 3h, and the mixed gas of logical nitrogen and hydrogen (95/5, V/V) protection.After treating the sample cool to room temperature, just obtain the rare earth ion of particle diameter about 10nm.
Embodiment 4 nano particlesZnS
:Eu
3+ Preparation
Rare-earth nanometer particles ZnS:Eu
3+0.25M the preparation method as follows: with 1mol/l (in the solution of the 0.01mol/l) ZnSO of 4.3ml
4Eu (NO with the 0.05mol/l of 1.76ml
3)
3Be injected into fast in Tris (1mol/l) solution of 0.25mol/l histidine of 35.6ml, logical nitrogen in solution simultaneously, and fast behind the agitating solution 20min, add the Na of the 1mol/l of 4.4ml
2S solution, solution at room temperature react 60min.0 ℃ absolute ethyl alcohol dropwise is added drop-wise in the reactant liquor, the ZnS:Eu of white in solution, occurs
3+The time stop to drip ethanol; On hydro-extractor that reaction solution is centrifugal, remove supernatant, white depositions is dissolved in the 1mol/l Tris solution again, repeat above operation twice, then with the solid that obtains ambient temperature overnight drying in a vacuum.The diameter of nano particles that obtains is about 10nm.
Embodiment 5 rare-earth nanometer particlesSrAl
2O
4 :Eu
2+ Method for coating one
With silester reagent with nano particle SrAl
2O
4: Eu
2+The surface coats one deck SiO
2Film is used multiple silylating reagent then and is made it connect upward hydrophilic functional group.Concrete grammar is as follows: with 70mg SrAl
2O
4: Eu
2+Be added in the 50ml isopropyl alcohol, add the NH of 1.6ml 50% again
3H
2O and 1.5ml water, ultrasonic 20min is distributed in the solution nano particle equably.Then reactant liquor is put into 40 ℃ water-bath, and added 160 μ l Si (OC
2H
5)
4, behind the reaction 1h, add 100 μ l 3-aminopropyl triethyl silicanes again, continue reaction 40min.Then that nano particle is centrifugal, behind isopyknic absolute ethanol washing three times, wash twice with water, use absolute ethanol washing twice again, at 80 ℃ of vacuum drying 6h, coating finishes then.
Embodiment 6 rare-earth nanometer particlesSrAl
2O
4 :Eu
2+ Method for coating two
Take by weighing 100mg rare-earth nanometer particles SrAl
2O
4: Eu
2+Join in the 20ml toluene and add the 0.1g neopelex, after grinding with agate mortar with particle SrAl
2O
4: Eu
2+Fully be distributed in the toluene, add 400 μ l methacrylic acids and 150 μ l trihydroxy methyl three propane triacrylate and 5mg benzoyl peroxides, 80 ℃ are reacted after 10-12 hour down, toluene is removed in centrifugal back, after using 20ml absolute ethyl alcohol and deionized water wash respectively, be distributed in the 2ml phosphate buffer solution rare earth ion stand-by.Like this, the rare earth ion surface is surrounded by the one layer of polymeric film, can prevent that rare earth ion is dissolved in water and loses fluorescence.Simultaneously, carboxyl is contained on the surface, can be connected with biomaterial.Bag is seen accompanying drawing 4 by the fluorescence spectrum figure of front and back, wraps little by the change in fluorescence of front and back.
The rare earth ion coupling of embodiment 7 antibody immunoglobulin IgG and surperficial coated polymer
Get Immunoglobulin IgG and 10mg 1-ethyl-3-(3-dimethyl aminopropyl)-carbodiimide that solution among the 500 μ l embodiment 6 adds 500 μ l 2.4mg/ml, slow stirring reaction 24 hours under the room temperature.Then, under the 4000r/min centrifugal 5 minutes, remove supernatant liquor, use phosphate buffer solution (PBS) washing secondary again after, it is stand-by to add 1000ml PBS.
Embodiment 8 preparation can sessile antibody matrix and the application aspect immunoassay
Before antibody is fixed in glass plate, need be with glass surface treatment.At first clean glass sheet, with glass sheet (25mm * 75mm) in washing lotion, soaked 2 hours, with N behind a large amount of deionized water rinsings
2Dry up.Then they are put into normal hexane, acetone and absolute ethyl alcohol after each ultrasonic 15 minutes more successively, be placed in 80 ℃ the baking oven baking and use immediately after 5 minutes.Glass surface carries out the fixing amino group of going up of Silanization reaction then.The 3-aminopropyl trimethoxysilane is dissolved in the diisopropyl ethyl amine that the diformazan benzo adds catalytic amount.Mechanical arm becomes the array shape with silylating reagent at the glass sheet point, and each is put about skin and rises magnitude, and size is 300 μ m, and distance between points is 800 μ m.At last 80 ℃ of placements of glass sheet are spent the night, with after the ethyl acetate rinse and dry immediately and to use.At last that glass sheet is fixing NH
2Become aldehyde radical by schiff base reaction.Amidized glass is dipped among 5% the glutaraldehyde PBS 5 hours, dries up with flowing down at nitrogen behind the deionized water rinsing.Glass sheet surface has aldehyde radical like this, can sessile antibody.
The immunoassay step:
Exempt from anti-reaction and adopt traditional antibody-sandwich reaction.The immunoassay experimental procedure is as follows, and the first five step operation steps is similar substantially to the enzyme-linked immuno assay step.
● with the anti-human immunoglobulin(HIg) IgG of the 1.2mg/ml rabbit sodium carbonate buffer (PH9.6A of 100 μ l
280/ 1.44=1mg IgG/ml), is evenly coated on the glass sheet, reacted 2 hours down, note in course of reaction, not making the liquid on the glass sheet to become dry at 37 ℃.
● with phosphate cleansing solution (PBS-T contains 0.05%Tween20 among the PBS) cleaning glass sheet, washed 5 minutes at every turn, wash three times.
● glass sheet is dipped in the BAS of 1% bovine serum albumin(BSA), 37 ℃ of down reactions 1 hour, this step is necessary to eliminating the non-specific adsorption of antigen on glass sheet, and is at last that glass sheet is stand-by with drying up under nitrogen after the PBS-T washing three times.
● human immunoglobulin(HIg) IgG, sheep Immunoglobulin IgG are determined antigen, the determined antigen solution of 100 μ l are coated on the glass sheet 37 ℃ of reactions 1 hour down.
● with PBS-T washing three times, each 5 minutes, use N
2Drying up glass sheet dries up stand-by.
● exempt from anti-human immunoglobulin(HIg) IgG 100 μ l and be coated on the glass sheet fluorescently-labeled, 37 ℃ are reacted after 2 hours down, wash secondary with PBS-T, again with deionized water wash once, dry up glass sheet with nitrogen, use Scanner Array 4000 fluorescence imagings then.With laser instrument is excitation source, and the fluoroscopic image of the rare earth particulate that Scanner Array 4000 scanning glass sheets obtain is seen accompanying drawing 4, and its fluorescence intensity is crossed through Array 4000 software processes, is a relative intensity.
Along with the increase of antigen concentration, fluorescence intensity is seen accompanying drawing 5 also along with increase, rare earth is described on the antibody labeling after, the ability of its specific recognition antigen does not disappear.
Claims (12)
1, a kind of rare-earth nanometer particles that is used for label biological materials, the particle diameter of this particle is between 3nm-400nm, it can be connected with said biomaterial, and when exciting, can send fluorescence, it is characterized in that the composition of said particle is to be selected from a kind of in following each compound substance or several: SrAl
2O
4: Eu
2+SrAl
2O
4: Eu
2++ Dy
3+Y
2O
3: Eu
3+: CaS:Eu
3+CaS:Eu
3++ X, wherein X=Tm
3+, Y
3+Or Al
3+ZnS:Y, wherein Y=Eu
2+, Mn
2+, Cu
2+, Ag
+And/or Au
+Gd
2O
3: Eu
3+La
2O
3: Eu
3+Or Gd
3(PO
4)
2: Eu
3+
2, as the said rare-earth nanometer particles of claim 1, it is characterized in that the surperficial coated last layer polymer film or the inorganics film of said rare earth ion.
As the said rare-earth nanometer particles of claim 2, it is characterized in that 3, the particle diameter of the rare-earth nanometer particles behind the said bag quilt is between 5-100nm.
4, as the said rare-earth nanometer particles of claim 2, it is characterized in that, the surperficial coated last layer polymer film of said rare earth ion, and the surface of this polymer film has following functional group: amino, carboxyl, hydroxyl, aldehyde radical and/or epoxy radicals.
5, as the said rare-earth nanometer particles of claim 4, it is characterized in that, contain the polymkeric substance that one or more coat monomer and one or more functionalized reagents in the polymer film of bag quilt or the inorganics film on the said rare-earth nanometer particles surface, wherein said coating monomer comprises acrylic acid, methacrylic acid, methyl acrylate, methyl methacrylate, 2-hydroxyethyl methacry-late, acrylic acid-2-hydroxyl ethyl ester, glycol monomethyl epoxy acrylate, diethylene glycol monoacrylate, triethylene glycol diacrylate, glycol diacrylate and styrene; Said functionalized reagent comprises acrylic acid, methacrylic acid, methacrolein, acrylamide, 3-aminopropyltriethoxywerene werene, 3-aldehyde radical propyl-triethoxysilicane, 3-sulfydryl propyl-triethoxysilicane, 2-hydroxyethyl methacry-late, acrylic acid-2-hydroxyl ethyl ester, methacrylic acid-2-amine ethyl ester, ethylene glycol bis also oxygen mono acrylic ester and 3-epoxypropyl triethyl silicane.
6, as the said rare-earth nanometer particles of claim 5, it is characterized in that, on the surface of said rare earth ion, also contain in bag polymer film of quilt or the inorganics film phosphoric acid two-(dihydroxy methylpropane diacrylate), phosphoric acid is two-(methacrylic acid 2-hydroxyl ethyl ester) and/or phosphoric acid is two-(pentaerythritol triacrylate).
7, a kind of preparation is used for the method for the rare-earth nanometer particles of label biological materials, and it comprises the steps:
(1) rare-earth nanometer particles of preparation particle diameter between 3-400nm;
(2) rare-earth nanometer particles with step (1) preparation joins in organic solvent toluene, dimethylbenzene or the tetrahydrofuran;
(3) in the potpourri of step (2) gained, add to coat monomer, functionalized reagent, polymerization initiator and coupling agent, carry out suspension polymerization, make rare earth pan coating one layer of polymeric film.
8, as the said method of claim 7, it is characterized in that, in step (2), also add surfactant, make rare-earth nanometer particles be well dispersed in the organic solvent.
9, as claim 7 or 8 said methods, it is characterized in that, in the polyreaction of step (3), add phosphoric acid two-(trimethylolpropane diacrylate), phosphoric acid is two-(methacrylic acid 2-hydroxyl ethyl ester) or phosphoric acid is two-(pentaerythritol triacrylate) make coupling agent.
10, a kind of preparation is used for the method for the rare-earth nanometer particles of label biological materials, and it comprises the steps:
(1) rare-earth nanometer particles of preparation particle diameter between 3-400nm;
(2) rare-earth nanometer particles that step (1) is made joins in the alkyl silicate, and makes alkyl silicate generation hydrolysis, forms silicon dioxide film on the surface of rare-earth nanometer particles;
(3) in the material of step (2) gained, add organosilane, make said silicon dioxide film functionalization.
11, a kind of preparation is used for the method for the rare-earth nanometer particles of label biological materials, and it comprises the steps:
(1) rare-earth nanometer particles of preparation particle diameter between 3-400nm;
(2) rare-earth nanometer particles that step (1) is made joins in glycosaminoglycan, polymethylacrylic acid or the phospholipid solution, makes it be adsorbed on the rare-earth nanometer particles surface and form to wrap tegillum.
12, as the said rare-earth nanometer particles of one of claim 1～6 in the purposes aspect the label biological materials.
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| CN100354388C (en) * | 2002-11-29 | 2007-12-12 | 郑岩 | Nanometer material with convertible color and preparing method thereof |
| WO2004102196A1 (en) * | 2003-04-30 | 2004-11-25 | Chengdu Kuachang Medical Industrial Limited | Apparatus including nanostructures used for separation or analysis, and the preparation and application thereof |
| EP1620728A4 (en) * | 2003-05-02 | 2006-07-05 | Access Bio Inc | Chromatographic assay system |
| CN1312479C (en) * | 2003-08-08 | 2007-04-25 | 清华大学 | Nano fluorescent magnetic particle and its preparing method |
| CN101663050A (en) | 2007-02-07 | 2010-03-03 | 斯帕果图像有限公司 | Visualization of biological material by the use of coated contrast agents |
| FR2933100B1 (en) | 2008-06-25 | 2010-08-13 | Commissariat Energie Atomique | RUMINESCENT RARE EARTH OXIDE PARTICLE DISPERSIONS, VARNISH COMPRISING THESE PARTICLES, PROCESSES FOR PREPARING THE SAME, AND METHOD FOR MARKING SUBSTRATES. |
| CN101402862B (en) * | 2008-11-10 | 2011-07-27 | 潍坊大耀新材料有限公司 | Producing process for zinc sulfide long-afterglow luminescent powder |
| CN105929155A (en) * | 2016-07-08 | 2016-09-07 | 同济大学 | Immuno-chromatographic test paper and detection method thereof |
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| CN107449898B (en) * | 2017-07-11 | 2022-11-25 | 中检国研(北京)科技有限公司 | Kanamycin residue fluorescence immunochromatographic test paper and preparation method thereof |
| CN111829993B (en) * | 2019-04-18 | 2023-09-29 | 中国科学院福建物质结构研究所 | Method for detecting hydrogen peroxide and related targets by CaS nano fluorescent probe |
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