CN103333675A - Fluorescent microsphere suitable for rapid immunology detection and preparation method thereof - Google Patents
Fluorescent microsphere suitable for rapid immunology detection and preparation method thereof Download PDFInfo
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- CN103333675A CN103333675A CN2013100869722A CN201310086972A CN103333675A CN 103333675 A CN103333675 A CN 103333675A CN 2013100869722 A CN2013100869722 A CN 2013100869722A CN 201310086972 A CN201310086972 A CN 201310086972A CN 103333675 A CN103333675 A CN 103333675A
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- Investigating, Analyzing Materials By Fluorescence Or Luminescence (AREA)
Abstract
The invention relates to a fluorescent microsphere in the field of biology engineering technology, and especially relates to a fluorescent microsphere suitable for rapid immunology detection and a preparation method thereof. In the first step, polymer particles comprising fluorescence molecules, and an olefin monomer having functional groups that have modification functions, are used as raw materials for the inner core and the outer shell respectively; and polymer microspheres, external surfaces of which have functional groups having modification functions, are prepared through a microemulsion polymerization method. In the second step, according to a ratio of 1% (w/v) to 5% (w/v), the polymer microspheres prepared in the first step are slowly injected into an acetone or dichloromethane solution comprising fluorescence molecules; the polymer microspheres are swelled and the fluorescence molecules penetrate into the polymer microspheres; and then the fluorescent microspheres are prepared after steps of centrifuging to remove supernate, cleaning, adding water and suspending. The preparation method is simple. The product can be used for preparing precise and quantified immune chromatography test strips, and is suitable for detection technology fields of medical diagnosis, biological detection, environmental detection, food safety monitoring, and detection of different-species.
Description
Technical field
The present invention relates to fluorescent microsphere of a kind of technical field of bioengineering and preparation method thereof, especially a kind of fluorescent microsphere that is applicable to the immunology rapid detection and preparation method thereof.
Background technology
In biological and medical science detect, often use immunoassay, it comprises methods such as radioimmunoassay, enzyme immunoassay and immunochromatography.Radioimmunoassay and enzyme-linked immunosorbent assay need operator and the complicated operations step of expensive equipment, specialty, are difficult to obtain fast detected result.Characteristics such as immunochromatographic method is simple to operate because having, reach fast at the bottom of the cost, and be usually used in fast qualitative or half-quantitative detection.
Immunochromatography technique is the immunoassay mode that comes across a kind of uniqueness at the initial stage eighties, it is solid phase with the fibre strip chromatographic material usually, make sample solution swimming on chromatography strip by wicking action, and make on determinand in the sample and the chromatographic material immune response that high special, high-affinity takes place at determinand acceptor (as antibody or antigen) simultaneously.The common trace particle of immunochromatography technique has Radioactive colloidal gold, latex, electroselenium, gelatin etc., wherein using the most successful marker is Radioactive colloidal gold, has for example occurred detecting the colloidal gold strip of Clenbuterol hydrochloride, hepatitis B surface antigen, aflatoxin and metakentrin etc.
Yet the sensitivity of colloidal gold immunochromatographimethod technology for detection is lower, can only carry out qualitative or sxemiquantitative to detecting thing in actual detected, can't be accurately quantitative.At present, existing relevant patent report be that the mark thing that collapses carries out immunochromatography and detects with the fluorescent nano particles, as publication number be CN1645146A patent disclosure a kind of with fluorescent rare earth nanometer particle the serve as a mark immune chromatography method of thing and the preparation of test strip thereof; Publication number be CN1866012A patent disclosure a kind of quantitatively, immunologic detection method and isolated plant thereof fast, this method is combined fluorescent substance inner complex Eu31-, SrTi3+, Tb3+, Dy3+ with the organic polymer nanoparticle, be prepared into fluorescent particle, undertaken quantitatively by fluoroscopic examination, but these fluorescent microspheres all are to excite under ultraviolet source, ultraviolet source the cost that has increased detecting instrument is set.
Letex polymerization refers to the effect by emulsifying agent, and under mechanical stirring or vibration, monomer forms emulsion and the polymerization carried out in water. emulsion polymerization product is latex, can directly use, also can destroy latex, through postprocessing working procedures such as washing, dryings, get powdery or needle-like polymkeric substance.Letex polymerization can obtain the polymkeric substance of higher molecular weight under higher speed of response, the viscosity of material is low, is easy to conduct heat and mix, and produces control easily, and residual monomer is removed easily.The shortcoming of letex polymerization is that emulsifying agent of adding in the polymerization process etc. influences product properties.For obtaining solid polymer, consume through technological processs such as cohesion, separation, washings.
Summary of the invention
The objective of the invention is to, provide that particle diameter is stable, spectrum is applicable to fluorescent microsphere that is applicable to the immunology rapid detection of visible radiation and preparation method thereof.
The preparation method that the present invention solves the fluorescent microsphere that its technical problem adopts is as follows:
The first step: be to contain the polymer particles of fluorescence molecule and the olefinic monomer of the functional group that shell contains rhetorical function is raw material with kernel, prepare the polymer microballoon that has rhetorical function functional group on the outside surface by microemulsion polymerization method;
Second step: the polymer microballoon that the first step is obtained is in 1%(w/v) to 5%(w/v) ratio slowly inject acetone or the dichloromethane solution that contains fluorescence molecule, polymer microballoon generation swelling, fluorescence molecule enters in the polymer microballoon, centrifugal then, remove supernatant liquor, clean, add aqueous suspension, obtain fluorescent microsphere.
During arranging, this because microemulsion polymerization method is known technology, does not therefore do concrete process description.
The further setting of the present invention is: described polymer microballoon is the microballoon of the multipolymer that forms of the microballoon of polymethylmethacrylate or methyl methacrylate and other monomer copolymerization, the structural unit number average of methyl methacrylate and described other monomer is more than or equal to 50, wherein the weight of methyl methacrylate units accounts for 50% of fluorescent microsphere gross weight at least, and the weight of described fluorescence molecule accounts for the 0.05%-5% of fluorescent microsphere gross weight.
The further setting of the present invention is: described other monomer is to be selected from vinyl fluoride, vinylchlorid, bromine ethene, ethene iodate, vinylbenzene and the vinylformic acid one or more.
The further setting of the present invention is: described functional group is for being selected from carboxyl, ethanol amido, hydroxyl, amido, amino, imido grpup, epoxy group(ing), isocyanate group, a kind of in metal alkoxide and the polyethylene glycol groups.
The further setting of the present invention is: described fluorescence molecule is one or more in rhodamine, fluorescein and derivative thereof, bodipy dye and the fluorine boron jade-like stone, and the weight of described fluorescence molecule accounts for 0.2%-5% of fluorescent microsphere gross weight.
Of the present inventionly further provide a kind of fluorescent microsphere of immunology rapid detection that is applicable to be: to comprise nucleocapsid structure, the particle diameter of described fluorescent microsphere is 200~500nm, its kernel has one or more functional groups of modifying on the described micro polymer outer surface of ball for including fluorescence molecule on the shell.
Functional group described in this arranges directly is connected with the covalent linkage of part or biomacromolecule reaction formation microsphere surface, or carries out covalent labeling with part or biomacromolecule after activation.
The further setting of the present invention is: the particle diameter of described fluorescent microsphere is 380nm.
Above-mentioned fluorescent microsphere adopts based on the Polymerization of Methyl thing as encapsulation matrix, its long service life, be easy to make, preparation is stable and have a good monodispersity, can carry out modification with various functional groups and living species to the surface of microballoon at an easy rate, can both be connected a large amount of fluorescence molecules on each microballoon, it is the carrier that extraordinary fluorescent signal amplifies, be applied in the immunochromatography detection, can improve detection sensitivity greatly, and fluorescent microsphere is highly stable, be subjected to ectocine little, fluorescence is stable, and this provides good condition for carrying out detection by quantitative by fluorescence, therefore, fluorescent microsphere of the present invention be in the detection technique of fluorescence high-sensitivity analysis detect and quantitative test in ideal mark.In addition, fluorescent microsphere of the present invention can excite and obtains by simple method with visible light source, reduces and detects analysis cost.
And above-mentioned preparation method is simple, in the fluorescent microsphere that obtains, fluorescence molecule is evenly distributed in the polymer microballoon, microspherulite diameter is even, stable, can be used for preparing the accurate quantification immuno-chromatographic test paper strip, be applicable in the detection technique field of medical diagnosis, biological detection, environment measuring and food safety monitoring and different plant species.
Description of drawings
In order to be illustrated more clearly in the embodiment of the invention or technical scheme of the prior art, to do to introduce simply to the accompanying drawing of required use in embodiment or the description of the Prior Art below, apparently, accompanying drawing in describing below only is some embodiments of the present invention, for those of ordinary skills, under the prerequisite of not paying creative work, can also obtain other accompanying drawing according to these accompanying drawings.
Fig. 1 is that the fluorescent microsphere among the embodiment 2 detects collection of illustrative plates;
Fig. 2 is the microballoon sem test collection of illustrative plates among the embodiment 2;
Embodiment
Embodiment 1 surface functional group is the preparation of the fluorescent microsphere of carboxyl
According to present embodiment, the preparation of fluorescent microsphere comprises the steps:
(l), the preparation surface has the poly (methyl methacrylate) micro-sphere of carboxyl
Get the polymethylmethacrylate particulate (2.7% that Polysciences company produces, 0.324um) 5ml, clean centrifugal back and in the lOml ultrapure water, suspend ultrasonicly, add and contain the 3mg methacrylic acid monomer 1ml water of (being used for forming carboxyl at microsphere surface), mix 2min, add the 0.5ml water that contains the 15mg polyvinyl alcohol, stir 2min, add the 0.5ml water that contains the 8mg sodium lauryl sulphate then, mix 2min, add 5ml ethanol then, stir 5min, obtain poly (methyl methacrylate) micro-sphere.
(2), preparation fluorescent microsphere
In step (l) gained poly (methyl methacrylate) micro-sphere, slowly add the 750 microlitre methylene dichloride that contain the 1mg rhodamine by syringe, the joining day can surpass 5min, continues to stir 5min.Add water 25ml by syringe pump with 0.5ml/min speed, air draught is removed some solvent constant volumes to 30ml then.Microballoon is carried out centrifugal removal supernatant liquor, clean three times with 90% ethanol, the 30ml aqueous suspension of the microballoon after the cleaning namely gets fluorescent microsphere.
The sign of embodiment 2 fluorescent microspheres
Get the lOul fluorescent microsphere of EXAMPLE l preparation, be suspended in the 750ul water and detect, collect the laser spectrum of microballoon at the 6lOnm place, emmission spectrum is gathered at the 580nm place, and spectrogram as shown in Figure 1.
Microballoon is carried out sem test, and microspherulite diameter is between 370-390nm, and particle diameter is even, and spectrogram is shown in Figure 2.
Utilize above-mentioned fluorescent microsphere can also prepare the preparation that is applicable to quantitative immunochromatographydetecting detecting test strip, can adopt the preparation method of conventional immunochromatographydetecting detecting test strip to prepare, specifically comprise the steps:
(l), the preparation of fluorescent microsphere pad: use with the fluorescent microsphere of mark determinand monoclonal antibody according to the amount of 4uL/cm be sprayed into glass fibre membrane (on 30 * 0.8cm), 25 ℃ of vacuum-drying 1~2h, it is standby to be put in dry environment.
(2), the preparation of nitrocellulose filter (NC film): use the 0.01MpH7.4PBS(phosphate buffered saline buffer, wherein comprise 5% sucrose and 0.05% tween 20) regulate determinand antibody two anti-(concentration is 0.4mg/mL), the gained solution spraying is formed detection line at the NC film; The concentration of regulating Quality Control antibody with 0.OIMpH7.2PBS (phosphate buffered saline buffer wherein comprises 3% sucrose and 0.05% polysorbas20) is 0.5mg/mL, and institute's solution spraying is formed the Quality Control district at the NC film.The Quality Control offset after 37 ℃ away from NC film one end 2mm oven dry are spent the night, is preserved standby be under the drying at room temperature environment.
(3), the preparation of fluorescent micro-ball immune chromatography test card: assembling test strip: overlap joint ground is pasted successively on the PVC base plate: 1) filter paper and sample pad, sample pad are a kind of glass fibre membrane of handling through 5%Tween-20; 2) be coated with the fluorescent microsphere pad of the determinand monoclonal antibody of fluorescent microsphere mark; 3) be coated with the nitrocellulose filter in detection zone and counterfeit control district; 4) thieving paper is assembled the width that cuts into 4mm after finishing, and namely becomes immuno-chromatographic test paper strip.An immune chromatography test paper held a memorial ceremony for be fixed on the plastic bottom card, the test paper surface compresses with the face card, and face is stuck in the sample pad of corresponding test strip and the position of NC film is reserved well and viewing window respectively.Pack into after immunochromatographydetection detection card assembles in the aluminium foil bag, seal preservation behind the adding siccative, under the drying at room temperature environment, can preserve at least 1 year.
Utilize product of the present invention to carry out the detection by quantitative of sample:
In sample well, add and detect serum, behind the 15min, adopt that excitation wavelength is 400, the immunochromatography detection by quantitative instrument scanning district to be checked of (visible light) and the fluorescence signal intensity in Quality Control district in the 700nm scope, thereby draw the content of determinand in serum, by fluorescent microsphere of the present invention, can improve detection sensitivity, for example can reach the level of 40PG/'ML for the detection sensitivity of the PRO-BNP of heart failure index, be better than like product (domestic similar detection at present is merely able to reach 1OOpg/ml substantially).
Obviously, above-described embodiment only be for explanation clearly do for example, and be not restriction to embodiment.To those of ordinary skill in the art, can also make other changes in different forms on the basis of the above description.Here need not also can't give all embodiments exhaustive.And the apparent variation of being extended out thus or change still are in protection scope of the present invention.
Claims (7)
1. method for preparing fluorescent microsphere, its step is as follows:
The first step: be to contain the polymer particles of fluorescence molecule and the olefinic monomer of the functional group that shell contains rhetorical function is raw material with kernel, prepare the polymer microballoon that has rhetorical function functional group on the outside surface by microemulsion polymerization method;
Second step: the polymer microballoon that the first step is obtained is in 1%(w/v) to 5%(w/v) ratio slowly inject acetone or the dichloromethane solution that contains fluorescence molecule, polymer microballoon generation swelling, fluorescence molecule enters in the polymer microballoon, centrifugal then, remove supernatant liquor, clean, add aqueous suspension, obtain fluorescent microsphere.
2. according to the described fluorescent microsphere that is applicable to the immunology rapid detection of claim 1, it is characterized in that: described polymer microballoon is the microballoon of the multipolymer of poly (methyl methacrylate) micro-sphere or methyl methacrylate and the formation of other monomer copolymerization, the structural unit number average of methyl methacrylate and described other monomer is more than or equal to 50, wherein the weight of methyl methacrylate units accounts for 50% of fluorescent microsphere gross weight at least, and the weight of described fluorescence molecule accounts for the 0.05%-5% of fluorescent microsphere gross weight.
3. according to the described fluorescent microsphere that is applicable to the immunology rapid detection of claim 2, it is characterized in that: described other monomer is to be selected from vinyl fluoride, vinylchlorid, bromine ethene, ethene iodate, vinylbenzene and the vinylformic acid one or more.
4. according to the described fluorescent microsphere that is applicable to the immunology rapid detection of claim 3, it is characterized in that: described functional group is for being selected from carboxyl, ethanol amido, hydroxyl, amido, amino, imido grpup, epoxy group(ing), isocyanate group, a kind of in metal alkoxide and the polyethylene glycol groups.
5. according to the described fluorescent microsphere that is applicable to the immunology rapid detection of claim 5, it is characterized in that: described fluorescence molecule is one or more in rhodamine, fluorescein and derivative thereof, bodipy dye and the fluorine boron jade-like stone, and the weight of described fluorescence molecule accounts for 0.2%-5% of fluorescent microsphere gross weight.
6. fluorescent microsphere that is applicable to the immunology rapid detection that is prepared by claim 1~5 either party method, comprise nucleocapsid structure, it is characterized in that: the particle diameter of described fluorescent microsphere is 200~500nm, kernel includes fluorescence molecule, has one or more functional groups of modifying on the described micro polymer outer surface of ball on the shell.
7. according to the described fluorescent microsphere that is applicable to the immunology rapid detection of claim 6, it is characterized in that: the particle diameter of described fluorescent microsphere is 380nm.
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CN104212087A (en) * | 2014-09-19 | 2014-12-17 | 哈尔滨工业大学 | Preparation method of monodisperse fluorescent microspheres |
CN105181662A (en) * | 2015-08-24 | 2015-12-23 | 西北大学 | Qualitative detection method for distribution of functional nanometer particles carried in polysaccharide microspheres |
CN105693906A (en) * | 2015-01-20 | 2016-06-22 | 于乐 | Zwitterionic polymer microspheres and preparing method thereof |
CN106566528A (en) * | 2016-11-07 | 2017-04-19 | 合肥亨纳生物科技有限公司 | Up-conversion fluorescence microsphere capable of being coded and preparation method thereof |
CN113667048A (en) * | 2021-08-16 | 2021-11-19 | 安徽为臻生物工程技术有限公司 | Monodisperse polymer color microsphere and preparation method and application thereof |
CN115746825A (en) * | 2022-10-26 | 2023-03-07 | 苏州星烁纳米科技有限公司 | Water-soluble fluorescent material-resin compound, preparation method thereof and tracer prepared from compound |
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CN105181662A (en) * | 2015-08-24 | 2015-12-23 | 西北大学 | Qualitative detection method for distribution of functional nanometer particles carried in polysaccharide microspheres |
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CN106566528A (en) * | 2016-11-07 | 2017-04-19 | 合肥亨纳生物科技有限公司 | Up-conversion fluorescence microsphere capable of being coded and preparation method thereof |
CN113667048A (en) * | 2021-08-16 | 2021-11-19 | 安徽为臻生物工程技术有限公司 | Monodisperse polymer color microsphere and preparation method and application thereof |
CN115746825A (en) * | 2022-10-26 | 2023-03-07 | 苏州星烁纳米科技有限公司 | Water-soluble fluorescent material-resin compound, preparation method thereof and tracer prepared from compound |
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