CN1475805A - Magnetic fluorescence microsphere and its preparation method and method of proceeding biomolecule detection using said magnetic fluorescence microsphere - Google Patents
Magnetic fluorescence microsphere and its preparation method and method of proceeding biomolecule detection using said magnetic fluorescence microsphere Download PDFInfo
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- CN1475805A CN1475805A CNA021393656A CN02139365A CN1475805A CN 1475805 A CN1475805 A CN 1475805A CN A021393656 A CNA021393656 A CN A021393656A CN 02139365 A CN02139365 A CN 02139365A CN 1475805 A CN1475805 A CN 1475805A
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Abstract
A magnetic fluorescent microsphere is prepared through adding magnetic nanoparticles and fluorescent dyeing in preparing high-molecular microspheres. Various biological moleculae can be bound on the surface of said magnetic fluorescent microphere for reacting on relative target moleculae in specimen in order to qualitatively and quantitatively detect the biologic macro moleculae.
Description
The present invention relates to a kind of magnetic fluorescent microspheres and preparation method thereof and adopt this magnetic fluorescent microspheres to carry out the method for biomolecule detection.
Since Horan in 1977 utilizes flow cytometer that polymer microsphere is applied to immunology detection first, be penetrated into every field such as cell biology, molecular biology, immunologic assay, medical science with polymer microsphere as the Measurement for Biotechnique of carrier, demonstrated wide application prospect.Since flow cytometer have can according to microspherulite diameter and microballoon is carried out the ability of specific recognition with the characteristic fluorescence signal, therefore how to utilize polymer microsphere to set up high flux, the macromolecular detection technique of automatic biological, become the new research focus of present microballoon technology in conjunction with flow cytometer.
By mixing the fluorescence polymer microballoon that one or more fluorescent materials are prepared at polymer microsphere surface or inside, the accurate matter microballoon of fluorescence that is used as calibration flow cytometer and fluorescent microscope at first, and be applied to cell marking, biomolecular labeling and the spike under reactive conditions gradually, but also fixing protein molecules and follow the tracks of its functionalization process.Enter the nineties, offshore company continually develops out the micro-sphere array with characteristic fluorescence mark, the flow cytometer and the corresponding analyzing software system thereof that can carry out specific recognition to microballoon, and produced fluorescent microsphere technology on this basis with multiple analysis ability, its range of application further be extended to Ag-Ab detect,
The fields such as genotyping of enzyme-substrate reactions, nucleic acid hybridization, amplification, order-checking and nucleic acid.Fluorescent microsphere can be realized by three approach: [1]. the covalent bond by fluorescent dye and microsphere surface functional group makes microsphere surface have one or more fluorescent materials, referring to United States Patent (USP) U.S.Pat.No.5194300; U.S.Pat.No.4774189; [2]. in the polymer microsphere polymerization process, mix one or both fluorescent materials, referring to United States Patent (USP) U.S.Pat.No.5073498; U.S.Pat.No.4717655; [3]. after microballoon forms, make the microballoon mark fluorescent, referring to United States Patent (USP) U.S.Pat.NO.5723218 by physisorption.Can obtain the fluorescent microsphere array that fractions does not have different fluorescent characteristicss by preceding method, wherein each microballoon can both be with its characteristic fluorescence by the flow cytometer specific recognition.Difference according to target molecule to be checked, the functional group that utilizes microsphere surface is with antigen, antibody, zymolyte, receptor-ligand, oligonucleotides etc. carry out immobilization, will be through the microballoon and the sample mix to be checked of above-mentioned processing, make corresponding target thing molecular reaction in these biomolecule and the sample, reporter molecules and fluorescent microsphere-target thing compound by a kind of fluorescent material mark are hatched, after flow cytometer detect, can discern the different biological reaction that takes place on each fluorescent microsphere, analyze, and provide testing result, thereby make fluorescent microsphere have the feature of synchronous detection target substances of multiple types in same test tube.
Though existing fluorescent microsphere technology has more advantage, but still there is following defective: owing to the detection that will in same reaction system, finish the sample target substances of multiple types, the reaction system complexity, therefore need purification steps such as the cleaning of multistep, centrifugal or enzyme processing, remove the material of disturbance reponse, as two anti-, primer, enzyme etc., make that to detect step comparatively loaded down with trivial details, sense cycle is longer; In the cleaning of multistep, centrifugal process, easily cause losing of fluorescent microsphere, influence experiment or testing result, even can cause the failure of testing, in actual applications can only be by increasing the consumption of microballoon, the consumption that makes microballoon lose the influence to the result to prevent microballoon, but this must increase the detection cost considerably beyond the actual detected requirement.
Magnetic microsphere is that grow up the early 1980s a kind of is used to separate the new function material with purifying, and it adopts the material complex technique, with organic monomer or macromolecular material and magnetic particle Fe
3O
4Be combined with each other, utilize macromolecule direct embedding magnetic particle or in the presence of magnetic fluid high molecular polymerizations such as the emulsion polymerization by monomer, suspension polymerization, dispersin polymerization form magnetic macromolecular microsphere, i.e. magnetic microsphere.Magnetic microsphere and antibody or antigen are formed immune magnetic microspheres with methods such as physisorption, chemical couplings, and the separating of the detection, isolation and purification, cell marking that can be widely used in various soluble antigens and identification, nucleic acid, PCR detects etc.But the shortcoming of this technology is: it is not high to detect flux.
The objective of the invention is to avoid above-mentioned weak point of the prior art, and provide a kind of and detect that flux is big, highly sensitive, high specificity, the detection step is comparatively easy, sense cycle short, testing result accurately, detect the preparation method of the low magnetic fluorescent microspheres of cost and adopt it to carry out the method for biomolecule detection.
Purpose of the present invention can reach by following measure:
1]. the synthetic magnetic fluorescence polymer microsphere that does not have characteristic fluorescence on the same group;
2]. in biomolecule such as antibody that the microsphere surface stationary phase should be not required on the same group, oligonucleotides;
3]. after microballoon after will fixing and the biological sample to be checked reaction, add fluorescently-labeled reporter group, hatch;
4]. with flow cytometry qualitative or quantitative test is carried out in the reaction that microsphere surface takes place.
A kind of magnetic fluorescent microspheres, its special character is: it is to comprise that the magnetic nano-particle and the magnetic macromolecular microsphere of polymer microsphere formation and the diameter of fluorescence molecule are the magnetic fluorescence polymer microsphere of 0.7～30 μ m, described magnetic nano-particle accounts for 10～50% of magnetic fluorescence polymer microsphere general assembly (TW), and fluorescence molecule accounts for 0.01～20% of magnetic fluorescence polymer microsphere general assembly (TW).
Above-mentioned magnetic nano-particle mixes fluorescence molecule by the fluorescent dye copolymerization in polymer microsphere inside, or promptly constitutes the magnetic fluorescence polymer microsphere by adsorbing or being chemically bound in microsphere surface mark fluorescent molecule.
The particle diameter of above-mentioned magnetic nano-particle is good with 5～12nm.
Above-mentioned magnetic macromolecular microsphere can be that particle diameter is that 0.7～2 μ m, magnetic content are the magnetic high-molecular seed microballoon of magnetic macromolecular microsphere general assembly (TW) 10～20%.
A kind of preparation method of magnetic fluorescent microspheres as mentioned above, its special character is: the preparation of this method comprises
1]. prepare metal oxide magnetic nano particle by chemical coprecipitation with ferric iron and divalent transition metal salt mixed aqueous solution with superparamagnetism;
2]. adopt the high molecular polymerization legal system to be equipped with polymer microsphere;
3]. the mark fluorescent dyestuff;
4]. last, obtain the magnetic fluorescence polymer microsphere.
The divalent transition metal salt of above-mentioned preparation magnetic nano-particle can be Fe (II), Mn (II), Ni (II), Zn (II) or Cu (II) slaine etc.
The preparation of above-mentioned superparamagnetism metal oxide magnetic nano particle can add surfactant, forms the surface and has hydrophobic magnetic nano-particle.
Above-mentioned magnetic macromolecular microsphere can be that particle diameter is the magnetic high-molecular seed microballoon of 0.7～2 μ m, and the preparation method of this magnetic high-molecular seed microballoon can adopt the microsuspension polymerization method, and it comprises:
1]. with spreading agent the magnetic nano-particle for preparing is carried out surface treatment, make it have hydrophobicity, and be distributed in the organic phase that is dissolved with monomer molecule, then initiating agent and crosslinking chemical are dissolved in the system;
2]. the adding of potpourris such as above-mentioned magnetic nano-particle, monomer, initiating agent, crosslinking chemical is contained in the water of suspending agent, through high speed shear emulsification, form steady suspension by beating action;
3]. heat up, initiated polymerization, preparing particle diameter is that 0.7～2 μ m, magnetic content are 10～20% magnetic high-molecular seed microballoon.
Above-mentioned spreading agent can adopt polyglycol; Described initiating agent can adopt benzoyl peroxide (BPO) or dodecanoyl superoxide.
Above-mentioned magnetic macromolecular microsphere can adopt the preparation of suspension polymerization, and it comprises:
1]. with long-chain fatty acid magnetic Nano level particle is carried out the oleophylic processing, make its surface form one deck oleophilic layer; The magnetic Nano level particle of oleophylic processing is mixed with the lipophilicity monomer solution, form oil phase;
2]. dissolve at least a surfactant and in aqueous solution, form water, disperse oil phase to form oil-in-water suspensions in aqueous phase;
3]. the thermal-initiated polymerization reaction forms magnetic macromolecular microsphere.
Above-mentioned polymer microsphere can be formed by organic monomer molecule aggregation or copolymerization, and described organic monomer molecule can adopt that styrene, acrylic acid, propylene are fine, acrylamide, acryl aldehyde, butadiene, acetate lactone, ethylene glycol terephthalate, isoprene, divinylbenzene or methyl methacrylate etc.
Above-mentionedly can comprise: when described magnetic seeds polymer microsphere is carried out the secondary swelling the magnetic macromolecular microsphere labeling dye, in sweller, mix two kinds of different fluorescent dyes and functional comonomer, make fluorescent material mix magnetic microsphere, prepare magnetic fluorescent microspheres by copolymerization.
Above-mentioned method to magnetic macromolecular microsphere mark fluorescent dyestuff can comprise:
1]. at first preparing active swelling emulsion, is that the magnetic seeds microballoon of 0.7～2 μ m carries out the swelling first time to particle diameter, forms the seed latax after the active swelling, adds stabilizing agent then; Stabilizing agent is advisable with the aqueous solution of emulsifying agent to adopt alkalinous metal halogenide to be dissolved in swelling, and concentration can be 0.001～0.1M; Alkalinous metal halogenide can be the chloride, bromide, iodide of potassium or caesium etc., is best with potassium chloride;
2]. contain two kinds of hydrophobic fluorescent dyes of different proportion, reaction monomers and initiating agents and surfactant, etc. under the condition that exists of composition, add above-mentioned seed latax and carry out the secondary swelling, monomer and initiating agent etc. are infiltrated in the seed latex particle, obtain containing the swelling granulate mixture of fluorescent dye; Heat up at last, cause copolymerization and obtain the equally distributed magnetic fluorescent microspheres of particle diameter; Described two kinds of fluorescent dyes be have overlapping excitation wavelength, have different emission wavelengths, not only can have been excited by the laser of same wavelength, but also the fluorescent dye that can be discerned by flow cytometer respectively.
Above-mentionedly can prepare magnetic fluorescent microspheres by physisorphtion to magnetic macromolecular microsphere mark fluorescent dyestuff, magnetic macromolecular microsphere is added the chloroformic solution that contains two kinds of fluorescent dyes, stirred 2-5 hour down at 25-55 ℃, till magnetic ball absorption fluorescent dye reaches capacity, vacuumize and removed fluorescent dye solution, product dilutes, stores with phosphate buffer.
Above-mentionedly can comprise: prepare nano level non magnetic polymer microsphere (5-100nm) with microemulsion polymerization method to magnetic macromolecular microsphere mark fluorescent dyestuff, make two groups of nanoscale microsphere surfaces adsorb two kinds of different fluorescent dyes respectively, the Nano microsphere that will have fluorescent dye again is coupled at the magnetic micrometer level microsphere surface that makes by covalent bond, regulate two kinds of ratios of adsorbing the Nano microsphere of different fluorescent materials on micron order magnetic microsphere surface, to regulate the fluorescent characteristics of magnetic microsphere; Described two kinds of fluorescent dyes be have overlapping excitation wavelength, have different emission wavelengths, not only can have been excited by the laser of same wavelength, but also the fluorescent dye that can be discerned by flow cytometer respectively.
Above-mentionedly can comprise: have the macromolecule shell that has functional group that forms by monomer copolymerization on the fluorescent magnetic microspheres surface magnetic macromolecular microsphere mark fluorescent dyestuff.
A kind of method that adopts above-mentioned magnetic fluorescent microspheres to carry out biomolecule detection, its special character is: the detection step of this method comprises:
1]. at magnetic fluorescent microspheres surface coupling bioactivator
Magnetic fluorescent microspheres is coupled at its surface with multiple bioactivator with covalent bond by its surperficial functional group;
2]. by the bioactivator of magnetic fluorescent microspheres surface coupling, corresponding molecule trapping to microsphere surface in the sample to be checked is reacted, make microballoon become the solid phase carrier of biologically;
3]. carry out magnetic resolution by magnetic separator, the reactant that is captured to microsphere surface is carried out enrichment, separation and purifying;
4]. by the microballoon that flow cytometer identification has different fluorescent characteristicss, the biologically that every kind of microsphere surface took place is distinguished; By to molecule to be checked or can with the detection of the third fluorescent material of mark on the molecule that molecular specific to be checked combines, obtain treating the qualitative and quantitative analyzing and testing result of sample product.
Above-mentioned bioactivator in magnetic fluorescent microspheres surface coupling can be antibody, the method of its coupling be with the surface with the magnetic fluorescent microspheres 30mg of carboxyl and carbodiimide (EDC) solution mixed after, add 1.5mg antibody, at room temperature stirred 24 hours, add bovine serum albumin(BSA) 30mg/ml, hatch 4 hours cessation reactions; Magnetic resolution is abandoned supernatant, washes once with the PBS of PH7.4, is suspended among the PBS again and preserves.
Above-mentioned can be oligonucleotide probe at magnetic fluorescent microspheres surface coupling bioactivator, its step is to use carbodiimide as coupling agent, adds the 1.5mg oligonucleotide probe, at room temperature stirs 24 hours, add bovine serum albumin(BSA) 30mg/ml, hatch 4 hours cessation reactions; Magnetic resolution is abandoned supernatant, washes once with the PBS of PH7.4, is suspended among the PBS again and preserves.
Above-mentioned bioactivator can be Ag-Ab, its detection method be with sample to be checked with the bag mixed by the magnetic fluorescent microspheres of specific antigen, lucifuge effect 10 minutes, with corresponding antibody capture to be checked to microsphere surface; Separate with magnetic separator, with microballoon-antigen-antibody complex purifying, enrichment; Add two of FITC mark and resist in above-mentioned compound, lucifuge effect 10 minutes detects with flow cytometer.
The present invention compares with existing fluorescent microsphere technology has following advantage:
The present invention will have the nano particle of superparamagnetism and introduce the fluorescence polymer microballoon, prepare some groups and have the magnetic fluorescence polymer microsphere array that different fluorescent characteristicss possess good magnetic responsiveness simultaneously again, be called for short magnetic fluorescent microspheres, with its solid phase carrier as bioactivator and biologically, the sample product be can treat and multiple, qualitative, quantitative test carried out, and the automaticity height.Because microballoon possesses superparamagnetism, thereby do not need through centrifugal and other purification step, only need just can carry out enrichment, separation and purifying to the reactant that is captured to microsphere surface by simple magnetic resolution of a step, thereby improve the sensitivity and the specificity that detect, shortened detection time simultaneously; Magnetic resolution can also be avoided losing of microballoon in addition, saves the consumption of microballoon, reduces the detection cost of this technology.The present invention can be widely used in the research of immunology detection, gene diagnosis, SNP Genotyping and interaction of biomacromolecules etc.
The invention will be further described below in conjunction with specific embodiment:
The preparation method of magnetic fluorescent microspheres of the present invention is divided into three steps:
Step 1: the magnetic Nano level particle that preparation has superparamagnetism
Magnetic Nano level particle can adopt the chemical coprecipitation preparation.This method Fe
3+And Fe
2+Reaction obtains Fe under alkali condition
3O
4Crystal settling.Adopt other divalent transition metal salt, be used for the preparation of magnetic Nano level particle as Mn (II), Ni (II), Zn (II), Cu also alternative Fe (II) such as (II).If will make magnetic Nano level particle disperse better, can in medium, add surfactant or change pH value of aqueous solution.The particle diameter of magnetic nano-particle is to be advisable less than 30nm, with best between 5～12nm.
Step 2: preparation magnetic macromolecular microsphere
Method 1: adopt the microsuspension polymerization legal system to be equipped with magnetic high-molecular seed microballoon
(1). the magnetic nano-particle that step 1 is obtained with spreading agent carries out making it have hydrophobicity after hydrophobic surface is handled, and is distributed in the organic phase that is dissolved with monomer, then initiating agent and crosslinking chemical is dissolved in the system;
(2). the adding of potpourris such as above-mentioned magnetic nano-particle-monomer is contained in the water of suspending agent, through high speed shear emulsification, form steady suspension by beating action;
(3). heat up, initiated polymerization, preparing particle diameter is 0.7～2 μ m, magnetic content is 10～20% magnetic macromolecular microsphere.Because this kind magnetic macromolecular microsphere particle diameter is less, so claim magnetic high-molecular seed microballoon again.
Spreading agent of the present invention can adopt polyglycol (PEG); Initiating agent can adopt benzoyl peroxide (BPO), dodecanoyl superoxide (LPO) etc.
Method 2: adopt the suspension polymerization magnetic macromolecular microsphere
(1). with long-chain fatty acid magnetic Nano level particle is carried out the oleophylic processing, make its surface form one deck oleophilic layer; The magnetic Nano level particle of oleophylic processing is mixed with the lipophilicity monomer solution, to form oil phase;
(2). dissolve at least a surfactant and in aqueous solution, form water, disperse oil phase to form oil-in-water suspensions in aqueous phase;
(3). the thermal-initiated polymerization reaction forms magnetic macromolecular microsphere.Specifically can be referring to Chinese patent CN1253147A; Number of patent application 98124516.1.
Be applicable to organic monomer of the present invention:, acrylamide fine, acryl aldehyde, butadiene, acetate lactone, ethylene glycol terephthalate, isoprene, divinylbenzene, methyl methacrylate etc. as styrene, acrylic acid, propylene.For obtain the surface contain functional group as-COOH ,-NH
2,-OH ,-CHO ,-SO
3The polymer microsphere of H etc. can adopt two or more organic monomer copolymerization that has functional group, obtains the surface as acrylic acid and styrene or methyl methacrylate and methacrylic acid copolymerization and has carboxylic microballoon; With hydroxyethyl methylacrylate HEMA and acryl aldehyde respectively with the styrene copolymerized microballoon that obtains hydroxyl-OH and aldehyde radical-CHO.
Step 3: to magnetic macromolecular microsphere mark fluorescent dyestuff
Method 1: the magnetic seeds polymer microsphere that obtains in the step 2-method 1 is carried out the secondary swelling, in sweller, mix simultaneously two kinds of different fluorescent dyes and functional comonomer, make fluorescent material mix magnetic microsphere inside, prepare magnetic fluorescent microspheres by copolymerization.Concrete steps are as follows
(1). at first using the 1-chlorododecane as a sweller, is that the magnetic seeds microballoon of 2 μ m carries out the swelling first time to particle diameter, forms the seed latax after the active swelling, adds stabilizing agent then so that the seed microballoon keeps stable in swelling and polymerization process.Other selectable sweller comprises: 1-chlorononane breast, 1-bromo-dodecane, cetyl alcohol etc.
(2). under the condition that contains two kinds of compositions such as the hydrophobic fluorescent dye of different proportion, organic monomer and initiating agent and surfactant existence, add above-mentioned seed microballoon and carry out the secondary swelling, monomer and initiating agent etc. are infiltrated in the seed microballoon, obtain containing the swelling granulate mixture of fluorescent dye; Heat up at last, cause copolymerization.Obtain the equally distributed magnetic fluorescent microspheres of particle diameter.
By changing the ratio of two kinds of fluorescent dyes, the microballoon that can obtain having different fluorescent characteristicss.Microballoon with identical fluorescent characteristics constitutes one group, and the microballoon with different fluorescent characteristicss can constitute many groups, and every group of microballoon can wrap respectively by different biomolecule.Many group microballoons can satisfy the demand of the different target molecules of synchronous detection.
Method 2: prepare magnetic fluorescent microspheres: the magnetic macromolecular microsphere adding is contained in the organic solvent of two kinds of different proportion fluorescent dyes by physisorphtion, stirred 2-5 hour down at 25～55 ℃, till magnetic ball absorption fluorescent dye reaches capacity, with the magnetic-type separation vessel post reaction mixture is separated, remove fluorescent dye unnecessary in the dereaction and other organic solvent, then magnetic fluorescent microspheres is stored in 4 ℃ of refrigerators with phosphate buffer cleaning and dilution.
Method 3: prepare nanoscale polymer microsphere (5～100nm) with microemulsion polymerization method, make two groups of nanoscale microsphere surfaces adsorb two kinds of different fluorescent dyes respectively, the Nano microsphere that will have fluorescent dye again is coupled at the magnetic macromolecular microsphere surface by covalent bond, and the ratio of the Nano microsphere of two kinds of different fluorescent materials of absorption by regulating micron order magnetic microsphere surface can reach the purpose of the fluorescent characteristics of accurate adjusting magnetic microsphere.For preventing that nanometer fluorescent microspheres from coming off from the magnetic macromolecular microsphere surface, can form the macromolecule shell that has functional group on the fluorescent magnetic microspheres surface by monomer copolymerization.
The selection of fluorescent dye of the present invention: select oil-soluble fluorescent dye, prepare multiple magnetic fluorescent microspheres array, can select for use two kinds of fluorescent dyes to mix in different ratios with different fluorescent characteristicss.These two kinds of fluorescent dyes are to have overlapping excitation wavelength, and have different emission wavelengths is good, different emission wavelengths differs at least greater than 10nm, with greater than 50nm the best, thereby two kinds of fluorescent materials both can be excited by the LASER Light Source of same wavelength, and the fluorescence intensity of measuring respectively separately at different emission wavelength places is also confirmed fluorescent microsphere by the ratio of two intensity.(Ex=546 is Em=647nm) with orange colour fluorescent material PO-PRO as red fluorescent material 7-Aminoactinomycin D
TM-3iodide (Ex=539, Em=567nm).
Adopt magnetic fluorescent microspheres to carry out the method for biomolecule detection
Step 1: magnetic fluorescent microspheres surface coupling bioactivator (bioactive molecule)
According to detecting needs, magnetic fluorescent microspheres can be coupled at its surface with multiple bioactivator with covalent bond by its surperficial functional group, and these bioactivators comprise: antigen, antibody, hormone receptor, enzyme, nucleic acid, oligonucleotides, haptens etc.
Step 2: carry out Biological Detection with magnetic fluorescent microspheres
The bioactivator of magnetic fluorescent microspheres surface coupling can react with corresponding molecule in the sample to be checked, makes microballoon become the solid phase carrier of biologically; Because of microballoon possesses superparamagnetism, need not centrifugal and other purification step, and only carry out simple magnetic resolution of a step by magnetic separator, just can carry out enrichment, separation and purifying to the reactant that is captured to microsphere surface, not only improve the sensitivity and the specificity that detect, shortened detection time simultaneously; Can obtain some groups of micro-sphere arrays owing to mix two kinds of fluorescent materials in different ratios in the magnetic microsphere with different fluorescent characteristicss, every kind of microballoon all can be discerned by flow cytometer by the characteristic fluorescence of self marker, therefore flow cytometer just can be distinguished the biologically that every kind of microsphere surface took place, and this just makes that using many group magnetic fluorescent microspheres that same sample is carried out multiple analysis becomes possibility in a test tube or reacting hole; Common flow cytometer p.s. can be to 5, data are analyzed and provided to fluorescence signal on 000 microballoon, therefore by to molecule to be checked or other can with the third fluorescent material of mark on the molecule that molecular specific to be checked combines, as the detection of signal intensities such as FITC, PE, can obtain treating the qualitative and quantitative analyzing and testing result of sample product.
Magnetic fluorescent microspheres preparation method's of the present invention instantiation: preparation method embodiment one:
Step 1: chemical coprecipitation prepares magnetic nano-particle
Fe with 0.5mol/l
2+The Fe of solution and 0.5mol/l
3+Liquid joins in the there-necked flask by the sampling of 1: 2 volume ratio, after stirring, adds an amount of spreading agent polyglycol, is warmed up to 70 ℃, slowly drips strong aqua, stirs, and reacts 30 minutes, prepares particle diameter and be the Fe about 20nm
3O
4Particle.Reaction product, is preserved with the suspension state to neutral with the distilled water cyclic washing after magnetic resolution.(prior art)
Step 2: suspension polymerization synthetic polystyrene magnetic seeds microballoon
With the polyglycol spreading agent with Fe
3O
4Carry out being distributed in the styrene after the surface treatment, add initiating agent dodecanoyl superoxide (LPO) and crosslinking chemical divinylbenzene (DVB), then above-mentioned mixed liquor is dispersed in the water, through high speed shear emulsification, form steady suspension, pour in the 250ml four-hole boiling flask that stirrer, thermometer, reflux condensing tube and blanket of nitrogen conduit are housed, flask is moved into carry out the micro emulsion polymerization in the Water Tank with Temp.-controlled, polymerization temperature is 80 ℃, and polymerization time is 2.0h.After polyreaction finishes.Add SDS as spreading agent, can obtain the polystyrene seed magnetic ball of the about 2 μ m of particle diameter after then filtrate being separated through magnetic.
Step 3: poly-(styrene-propene acid methyl esters) magnetic fluorescent microspheres of preparation band carboxyl
2ml 1-chlorododecane (CDD) and 0.25% SDS 5ml aqueous solution mixing are as a sweller, behind mixing in 10ml 10% magnetic polystyrene seed microballoon and the 0.25%50mlSDS solution, add a sweller that has prepared, 30% acetone soln that adds 20ml simultaneously makes the easier infiltration microballoon of CDD, stirs 12 hours; Get the above-mentioned suspending liquid of 2ml and add 20ml distilled water and 40ml0.25%SDS, magnetic resolution obtains the seed microballoon of a swelling.400mg4% benzoyl peroxide initiating agent dissolves in 10ml and contains in styrene (90%) and methyl acrylate (10%) solution, add equal-volume 0.25%SDS again, mixing is pressed different proportion again and is added fluorescent dye 7-aminoactinomycin D (7-Aminoactinomycin D) and PO-PRO
TM-3iodide makes the homogeneous mixed solution of monomer-initiating agent-fluorescent dye, gets the above-mentioned mixed solution of 20ml then and adds above-mentioned seed microballoon after a swelling, feeds nitrogen protection, heats 70 ℃, the rapid polymerization of 2 hours initiation swelling seeds.Obtain the magnetic fluorescent microspheres of particle diameter 5-6 μ m.Preparation method embodiment two:
Step 1: the preparation magnetic nano-particle, with the step 1 of embodiment one.
Step 2: the magnetic polystyrene microsphere of preparation band carboxyl.
With above-mentioned Fe
3O
4Nano particle 35g adds in the organic phase of styrene 50g, methacrylic acid (MAA) 5g, divinylbenzene 10g, benzoyl peroxide (BPO) 5g composition, slightly through stirring the oil phase magnetic colloid solution that can form stable dispersion; In 1 liter of cylindrical stirring reactor, add 500ml distilled water and 10g polyvinyl alcohol (PVA), 50 ℃ of constant temperature stir introduces above-mentioned oil phase magnetic colloid after 0.5 hour, regulate stirring rate to 800rpm, be warmed up to 80 ℃ of reactions 6 hours, be warmed up to 95 ℃ of slakings 4 hours again; After the cooling,, can obtain the magnetic polystyrene microsphere of particle diameter at 5-15 μ m band carboxyl through operations such as magnetic resolution, washings.
Step 3: prepare magnetic fluorescent microspheres by physisorphtion
Magnetic macromolecular microsphere is added dimethyl sulfoxide (DMSO) (MDSO) solution that contains two kinds of fluorescent materials, the concentration of general fluorescence molecule is 0.1～1wt% in this adsorption liquid, with 5～10ml concentration is that 0.5～2wt% magnetic microsphere dilution adds among the DMSO of 0.5ml fluorescence molecule, stirred 2-5 hour down at 25-55 ℃, till magnetic ball absorption fluorescent dye reaches capacity, magnetic resolution is gone out unnecessary fluorescent dye solution, and product cleans the back dilution, stores with phosphate buffer.Preparation method embodiment three:
Step 1: the preparation magnetic nano-particle is with embodiment one---step 1;
Step 2: the magnetic polystyrene microsphere of preparation surface band carboxyl is with embodiment two---step 2;
Step 3: the nanoscale fluorescent microsphere of preparation surface band amido
About 1/3 monomers such as styrene, emulsifying agent SDS, assistant for emulsifying agent 1-amylalcohol, deionized water are made into the homogeneous phase microemulsion on a small quantity earlier; This microemulsion is stirred and be warming up to 25～80 ℃, after 5～10 minutes, add initiator potassium persulfate (KPS) and form reaction system to the logical nitrogen of above-mentioned reaction system; With function monomer methacrylic acid amino ethyl ester (AEM), the crosslinking chemical divinylbenzene (DVB) of residual monomer, amino-contained, dropwise add reaction system simultaneously, keep dripping off in 1～6 hour, accompany by 400～650 rev/mins of speed and stir; Dropwise and continue reaction 0.5～3.5 hour, can obtain the Nano microsphere of surface band amido.In the reaction system, emulsifier concentration is 2～40wt%, and monomer concentration is 1～50wt%, and function monomer concentration is 0.2～20wt%, and initiator concentration is 0.5～10mM.The method of Nano microsphere absorption fluorescence molecule is with example 2. (3).
Step 4: preparation magnetic fluorescent microspheres
After the magnetic fluorescent microspheres 30mg of surperficial band carboxyl and carbodiimide (EDC) solution were mixed, the Nano microsphere that the surface band amido of two kinds of different fluorescent dyes is adsorbed on two groups of surfaces that adding mixes by a certain percentage respectively was total to 3.0mg, at room temperature stirs 24 hours.Magnetic resolution is abandoned supernatant.PBS with PH7.4 washes once, and magnetic resolution is abandoned supernatant.Promptly obtain the magnetic microsphere of coupled to Nano fluorescent microsphere.
Effect is after 1 hour in the organic phase that adding styrene 50g, methacrylic acid (MAA) 5g, divinylbenzene (DVB) 10g form in above-mentioned product, be introduced into and fill in the cylindrical stirring reactor of 500ml distilled water, add potassium persulfate 5g, regulate stirring rate and be warming up to 95 ℃ to 800rpm, be warmed up to 80 ℃ of reactions 2 hours, be warmed up to 95 ℃ of slakings 2 hours again, can form the macromolecule shell that has functional group on the magnetic microsphere surface of coupled to Nano fluorescent microsphere.After the cooling, through magnetic resolution, washing, etc. operation, can obtain magnetic fluorescence polystyrene microsphere with carboxyl.Biomolecule detection embodiment one: magnetic fluorescent microspheres surface coupling antibody
After the magnetic fluorescent microspheres 30mg of surface band carboxyl and carbodiimide (EDC) solution were mixed, the pH of regulator solution was about 4.5, adds 1.5mg antibody, at room temperature reacts 1～2 hour, adds bovine serum albumin(BSA) (30mg/ml), hatches 4 hours cessation reactions.Magnetic resolution is abandoned supernatant, washes once with the PBS of pH7.4, is suspended among the PBS again and preserves.Biomolecule detection embodiment two: carboxylic magnetic fluorescent microspheres surface coupling oligonucleotide probe
Still adopt carbodiimide as coupling agent, the antibody in the example 4 is changed to oligonucleotide probe, length can be preferably 20-30mer at 5-500mer.Biomolecule detection embodiment three: applied magnetic fluorescent microsphere technology is carried out the immunology detection of Ag-Ab
Sample to be checked with the bag mixed by the magnetic fluorescent microspheres of specific antigen, lucifuge effect 10 minutes, with corresponding antibody capture to be checked to microsphere surface.Magnetic separator separates, with microballoon-antigen-antibody complex purifying, enrichment.In above-mentioned compound, add two of FITC mark and resist, lucifuge effect 10 minutes, flow cytometer detects.Biomolecule detection embodiment four: applied magnetic fluorescent microsphere technology is carried out the HLA-DNA somatotype and is detected
With HLA allele sequence specific oligonucleotide probes (SSOP) respectively with different microballoon couplings, preparation has the magnetic fluorescent microspheres of different typing probes.
Blood sample collection to be checked; The DNA extracting; The specific hypotype dna fragmentation amplification of HLA, the ddNTP of adding FITC mark makes pcr amplification product flag F ITC in amplification system; The magnetic fluorescent microspheres that is combined with the SSOP typing probes is mixed lucifuge hatched 1 hour with pcr amplification product; Magnetic separator separates; Flow cytometer detects, the software analysis result.
The microballoon that the present invention does not make particular determination all refers to macromolecule micron order microballoon.
Claims (20)
1. magnetic fluorescent microspheres, it is characterized in that: it is to comprise that the magnetic nano-particle and the magnetic macromolecular microsphere of polymer microsphere formation and the diameter of fluorescence molecule are the magnetic fluorescence polymer microsphere of 0.7～30 μ m, described magnetic nano-particle accounts for 10～50% of magnetic fluorescence polymer microsphere general assembly (TW), and fluorescence molecule accounts for 0.01～20% of magnetic fluorescence polymer microsphere general assembly (TW).
2. magnetic fluorescent microspheres as claimed in claim 1, it is characterized in that: described magnetic nano-particle mixes fluorescence molecule by the fluorescent dye copolymerization in polymer microsphere inside, or constitutes the magnetic fluorescence polymer microsphere by adsorbing or being chemically bound in microsphere surface mark fluorescent molecule.
3. magnetic fluorescent microspheres as claimed in claim 1 or 2 is characterized in that: the particle diameter of described magnetic nano-particle is 5～12nm.
4. magnetic fluorescent microspheres as claimed in claim 3 is characterized in that: described magnetic macromolecular microsphere is that particle diameter is 0.7～2 μ m, and magnetic content is the magnetic high-molecular seed microballoon of magnetic macromolecular microsphere general assembly (TW) 10～20%.
5. preparation method of magnetic fluorescent microspheres according to claim 1, it is characterized in that: the preparation of this method comprises
1]. prepare metal oxide magnetic nano particle by chemical coprecipitation with ferric iron and divalent transition metal salt mixed aqueous solution with superparamagnetism;
2]. adopt the high molecular polymerization legal system to be equipped with polymer microsphere;
3]. the mark fluorescent dyestuff;
4]. last, obtain the magnetic fluorescence polymer microsphere.
6. the preparation method of magnetic fluorescent microspheres as claimed in claim 5, it is characterized in that: the divalent transition metal salt of described preparation magnetic nano-particle is Fe (II), Mn (II), Ni (II), Zn (II) or Cu (II) slaine.
7. as the preparation method of claim 5 or 6 described magnetic fluorescent microspheres, it is characterized in that: comprise the adding surfactant in the preparation of described superparamagnetism metal oxide magnetic nano particle, form the surface and have hydrophobic magnetic nano-particle.
8. the preparation method of magnetic fluorescent microspheres as claimed in claim 5, it is characterized in that: described magnetic macromolecular microsphere is that particle diameter is when being the magnetic high-molecular seed microballoon of 0.7～2 μ m, the preparation method of this magnetic high-molecular seed microballoon is the microsuspension polymerization method, comprises
1]. with spreading agent the magnetic nano-particle for preparing is carried out surface treatment, make it have hydrophobicity, and be distributed in the organic phase that is dissolved with monomer molecule, then initiating agent and crosslinking chemical are dissolved in the system;
2]. the adding of potpourris such as above-mentioned magnetic nano-particle, monomer, initiating agent, crosslinking chemical is contained in the water of suspending agent, through high speed shear emulsification, form steady suspension by beating action;
3]. heat up, initiated polymerization, preparing particle diameter is that 0.7～2 μ m, magnetic content are 10～20% magnetic high-molecular seed microballoon.
9. the preparation method of magnetic fluorescent microspheres as claimed in claim 8, it is characterized in that: described spreading agent is a polyglycol; Described initiating agent is benzoyl peroxide (BPO) or dodecanoyl superoxide.
10. the preparation method of magnetic fluorescent microspheres as claimed in claim 5 is characterized in that: described magnetic macromolecular microsphere adopts the preparation of suspension polymerization, and it comprises
1]. with long-chain fatty acid magnetic Nano level particle is carried out the oleophylic processing, make its surface form one deck oleophilic layer; The magnetic Nano level particle of oleophylic processing is mixed with the lipophilicity monomer solution, form oil phase;
2]. dissolve at least a surfactant and in aqueous solution, form water, disperse oil phase to form oil-in-water suspensions in aqueous phase;
3]. the thermal-initiated polymerization reaction forms magnetic macromolecular microsphere.
11. preparation method as claim 5 or 10 described magnetic fluorescent microspheres, it is characterized in that: described polymer microsphere is formed by organic monomer molecule aggregation or copolymerization, and described organic monomer molecule is that styrene, acrylic acid, propylene are fine, acrylamide, acryl aldehyde, butadiene, acetate lactone, ethylene glycol terephthalate, isoprene, divinylbenzene or methyl methacrylate.
12. the preparation method as claim 5 or 8 described magnetic fluorescent microspheres is characterized in that: described the magnetic macromolecular microsphere labeling dye is comprised
When described magnetic seeds polymer microsphere is carried out the secondary swelling, in sweller, mix two kinds of different fluorescent dyes and functional comonomer, make fluorescent material mix magnetic microsphere, prepare magnetic fluorescent microspheres by copolymerization.
13. the preparation method of magnetic fluorescent microspheres as claimed in claim 12 is characterized in that: described method to magnetic macromolecular microsphere mark fluorescent dyestuff comprises
1]. at first preparing active swelling emulsion, is that the magnetic seeds microballoon of 0.7～2 μ m carries out the swelling first time to particle diameter, forms the seed latax after the active swelling, adds stabilizing agent then; Described stabilizing agent is dissolved in the aqueous solution of swelling with emulsifying agent for alkalinous metal halogenide, and concentration is 0.001～0.1M; Chloride, bromide or iodide that described alkalinous metal halogenide is potassium or caesium;
2]. under the condition that contains the existence of two kinds of hydrophobic fluorescent dyes of different proportion, reaction monomers and initiating agents and surfactant component, add above-mentioned seed latax and carry out the secondary swelling, monomer and initiating agent etc. are infiltrated in the seed latex particle, obtain containing the swelling granulate mixture of fluorescent dye; Heat up at last, cause copolymerization and obtain the equally distributed magnetic fluorescent microspheres of particle diameter; Described two kinds of fluorescent dyes be have overlapping excitation wavelength, have different emission, not only can have been excited by the laser of same wavelength, but also the fluorescent dye that can be discerned by flow cytometer respectively.
14. the preparation method of magnetic fluorescent microspheres as claimed in claim 11, it is characterized in that: described is to prepare magnetic fluorescent microspheres by physisorphtion to magnetic macromolecular microsphere mark fluorescent dyestuff, it is that magnetic macromolecular microsphere is added the chloroformic solution that contains two kinds of fluorescent dyes, stirred 2-5 hour down at 25-55 ℃, till reaching capacity to magnetic ball absorption fluorescent dye, vacuumize and removed fluorescent dye solution, product dilutes, stores with phosphate buffer.
15. the preparation method of magnetic fluorescent microspheres as claimed in claim 11 is characterized in that: described magnetic macromolecular microsphere mark fluorescent dyestuff is comprised
Prepare the non magnetic polymer microsphere of nanoscale with microemulsion polymerization method, make two groups of nanoscale microsphere surfaces adsorb two kinds of different fluorescent dyes respectively, the Nano microsphere that will have fluorescent dye again is coupled at the magnetic micrometer level microsphere surface that makes by covalent bond, regulate two kinds of ratios of adsorbing the Nano microsphere of different fluorescent materials on micron order magnetic microsphere surface, to regulate the fluorescent characteristics of magnetic microsphere; Described two kinds of fluorescent dyes be have overlapping excitation wavelength, have different emission wavelengths, not only can have been excited by the laser of same wavelength, but also the fluorescent dye that can be discerned by flow cytometer respectively.
16. the preparation method of magnetic fluorescent microspheres as claimed in claim 15 is characterized in that: described magnetic macromolecular microsphere mark fluorescent dyestuff is comprised
Has the macromolecule shell that has functional group that forms by monomer copolymerization on the fluorescent magnetic microspheres surface.
17. a method that adopts magnetic fluorescent microspheres according to claim 1 to carry out biomolecule detection, it is characterized in that: the detection step of this method comprises
1]. at magnetic fluorescent microspheres surface coupling bioactivator
Magnetic fluorescent microspheres is coupled at its surface with multiple bioactivator with covalent bond by its surperficial functional group;
2]. by the bioactivator of magnetic fluorescent microspheres surface coupling, corresponding molecule trapping to microsphere surface in the sample to be checked is reacted, make microballoon become the solid phase carrier of biologically;
3]. carry out magnetic resolution by magnetic separator, the reactant that is captured to microsphere surface is carried out enrichment, separation and purifying;
4]. by the microballoon that flow cytometer identification has different fluorescent characteristicss, the biologically that every kind of microsphere surface took place is distinguished; By to molecule to be checked or can with the detection of the third fluorescent material of mark on the molecule that molecular specific to be checked combines, obtain treating the qualitative and quantitative analyzing and testing result of sample product.
18. employing magnetic fluorescent microspheres as claimed in claim 17 carries out the method for biomolecule detection, it is characterized in that: described bioactivator in magnetic fluorescent microspheres surface coupling is an antibody, the method of its coupling be with the surface with the magnetic fluorescent microspheres 30mg of carboxyl and carbodiimide (EDC) solution mixed after, add 1.5mg antibody, at room temperature stirred 24 hours, add bovine serum albumin(BSA) 30mg/ml, hatch 4 hours cessation reactions; Magnetic resolution is abandoned supernatant, washes once with the PBS of PH7.4, is suspended among the PBS again and preserves.
19. employing magnetic fluorescent microspheres as claimed in claim 17 carries out the method for biomolecule detection, it is characterized in that: described is oligonucleotide probe at magnetic fluorescent microspheres surface coupling bioactivator, its step is as coupling agent with carbodiimide, add the 1.5mg oligonucleotide probe, at room temperature stirred 24 hours, add bovine serum albumin(BSA) 30mg/ml, hatch 4 hours cessation reactions; Magnetic resolution is abandoned supernatant, washes once with the PBS of PH7.4, is suspended among the PBS again and preserves.
20. employing magnetic fluorescent microspheres as claimed in claim 17 carries out the method for biomolecule detection, it is characterized in that: described bioactivator is an Ag-Ab, its detection method is that sample to be checked is mixed by the magnetic fluorescent microspheres of specific antigen with bag, lucifuge effect 10 minutes, with corresponding antibody capture to be checked to microsphere surface; Separate with magnetic separator, with microballoon-antigen-antibody complex purifying, enrichment; Add two of FITC mark and resist in above-mentioned compound, lucifuge effect 10 minutes detects with flow cytometer.
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