CN105403697A - Microsphere carrier and preparation method thereof - Google Patents
Microsphere carrier and preparation method thereof Download PDFInfo
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- CN105403697A CN105403697A CN201510325843.3A CN201510325843A CN105403697A CN 105403697 A CN105403697 A CN 105403697A CN 201510325843 A CN201510325843 A CN 201510325843A CN 105403697 A CN105403697 A CN 105403697A
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- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
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Abstract
The present invention discloses a magnetic microsphere carrier and a preparation method thereof, wherein the magnetic microsphere carrier adopts micron-scale or submicron-scale magnetic microspheres as mother balls, and a chemical cross-linking method is used to assemble nano-scale son balls having rich functional groups on the surface so as to obtain the assembled magnetic microspheres having regular topology structure. According to the present invention, the magnetic microspheres are adopted as the carrier for identification, capture and regulation of target molecules to be detected so as to achieve the efficient capture and detection of the target molecules to be detected; and compared with the traditional smooth spherical magnetic microspheres, the magnetic microspheres having the regular topology structure have the following characteristics that the detection sensitivity of the in vitro diagnostic application process can be significantly increased, the assembly process is simple, the obtained assembled microspheres have the regular morphology, the surface area of the microspheres is substantially improved with the topology structure forming, and the efficient immobilization of the target biological probe molecules during the subsequent in vitro diagnostic application process is ensured.
Description
The application is the divisional application of following application: the applying date: on February 27th, 2013; Application number: 201310062308.4; Denomination of invention: " a kind of Magnetic Microspheres-Carrier and preparation method thereof ".
Technical field
The present invention relates to a kind of bioabsorbable carrier material and Synthesis and applications thereof, particularly relate to and a kind ofly improve Magnetic Microspheres-Carrier of in-vitro diagnosis detection sensitivity and preparation method thereof.Belong to technical field of biological materials.
Background technology
Magnetic microsphere, as the solid phase carrier of in-vitro diagnosis, plays identification, catches, controls the carrier function of the molecule to be checked with transport target.Because it is all be suspended in sample to be checked in whole course of reaction, compared with traditional porous plate carrier, its whole reaction is in liquid or accurate liquid condition substantially, and the probability that molecular collision occurs between carrier and thing to be checked increases greatly.In addition, because surface area that magnetic microsphere is larger improves the efficiency of load target molecule to be checked, thus make its biological respinse efficiency comparatively conventional porous plate mode greatly improve; Biological respinse on general porous plate needed between 1 ~ 3 hour, and only needed tens minutes based on the biological respinse of magnetic microsphere.In addition, due to the superparamagnetic characteristic of magnetic microsphere brilliance, make to adopt Full-automatic magnetic separate mode and detect and can realize like a cork.
At present, the Magnetic Microspheres-Carrier of in-vitro diagnosis field application is mainly magnetic microsphere two class that particle diameter (is generally several microns) at magnetic-particle and the particle diameter of below 100nm more than 1 μm.Particle diameter is at the magnetic-particle of below 100nm, owing to having great specific surface area, can significantly improve and identify and catch the reaction kinetics of target biological molecules to be checked and the ability of target acquisition biomolecule, and then improve detection sensitivity and the detection efficiency of in-vitro diagnosis.But the responsiveness of the magnetic-particle external magnetic field of nanoscale is low, is not easily manipulated by magnetic field; And very easily occur reunite and in actual application, do not have nanoscale effect, thus cannot use as efficient solid phase carrier in the full-automatic vitro diagnostic test systems of current main flow.On the other hand, the magnetic microsphere of micro-meter scale, because it comprises a large amount of magnetic-particles, although can realize fully-automated synthesis easily, but microsphere supported surface area ratio nano magnetic particle reduces greatly, thus detection sensitivity when being carrier with it and reaction efficiency also greatly reduce.
Through finding the literature search of prior art, Matsunaga etc. are at " AnalyticaChimicaActa " the 597th " magnetic microsphere for full-automatic immune detection based on beadsonbeads compound detects PSA specific antigen " (Fullyautomatedimmunoassayfordetectionofprostate-specific antigenusingnano-magneticbeadsandmicro-polystyrenebeadco mposites of delivering of volume 331-339 page, ' BeadsonBeads ' ") in report and utilize beadsonbeads structure, the magnetic-particle of nanoscale is attached to surface by biotin to be connected with on the polystyrene microsphere of the micro-meter scale of Avidin, improve the surface area of magnetic microsphere on the one hand, overcome again on the other hand weak reponse and the very easily problem such as reunion of the magnetic-particle external magnetic field of nanoscale.But the detection sensitivity that this article does not have the special topological structure of this beadsonbeads and smooth micro-meter scale microballoon are diagnosed in vitro carries out comparative study, does not namely explain the detection sensitivity whether this special topological structure can improve in-vitro diagnosis.In addition, the package technique adopted due to this report is the affinity interaction of Streptavidin and biotin, therefore the microballoon higher density assembling of magnetic-particle on micro-meter scale that could realize nanoscale through repeatedly repeatedly assembling up to more than ten times is needed, whole preparation process is complicated, and cost intensive.
Further retrieval finds, Tan etc. report the microballoon by assemble nanometer yardstick on flat plate substrate in volume 738-746 page " spherical biological function core shell nanoparticles microstructured layers improves reaction table area in protein arrays " (the Microstructuredlayersofsphericalbiofunctionalcore-shelln anoparticlesprovideenlargedreactivesurfacesforproteinmic roarrays) that deliver at " ANALYTICALANDBIOANALYTICALCHEMISTRY " the 383rd, again in this, as solid phase carrier, carry out the detection to model proteins, found that detection sensitivity is highly improved.But because the solid phase carrier adopted is flat plate substrate, itself and intermolecular reacting completely to be checked are realized by the diffusion of molecule, make reaction kinetics comparatively microballoon greatly reduce.
Summary of the invention
Because the above-mentioned defect of prior art, technical matters to be solved by this invention is to provide the Magnetic Microspheres-Carrier that a kind of preparation method is simple, can improve in-vitro diagnosis detection sensitivity.
For achieving the above object, the invention provides and a kind ofly improve Magnetic Microspheres-Carrier of in-vitro diagnosis detection sensitivity and preparation method thereof.
The present invention adopts the magnetic microsphere of micron or submicron-scale to be mother bulb, has the nanoscale bulbec enriching functional group, thus obtain the assembling magnetic microsphere with regular topological structure on its surface by Chemical Crosslinking Methods assembling surface.Using the assembling magnetic microsphere with regular topological structure obtained as identifying, catching and the carrier manipulating target molecule to be checked, thus the efficient capture realized target molecule to be checked and detection.Compared with traditional smooth, spherical magnetic microsphere with similar face functional group, the magnetic microsphere with regular topological structure of the present invention can significantly improve the detection sensitivity of in-vitro diagnosis application process.Preparation method of the present invention achieves the High Density Packaging of bulbec on micro-meter scale mother bulb of nanoscale by chemical crosslink reaction, and its assembling process is simple, the regular appearance of the assembling microballoon obtained; And the formation of topological structure drastically increases the surface area of microballoon, ensure that in subsequent in vitro diagnostic application process the efficient fixing of target organism probe molecule (nucleic acid or albumen) and detection.
The present invention solves above-mentioned technical matters by the following technical programs:
On the one hand, the invention provides a kind of Magnetic Microspheres-Carrier significantly improving in-vitro diagnosis detection sensitivity.Particularly, component and the content/physicochemical characteristic of Magnetic Microspheres-Carrier of the present invention are: to have the mother bulb of micron or submicron-scale for core, particle diameter is the bulbec of nanoscale is shell, by chemical covalent bonds compact siro spinning technology between bulbec and mother bulb, and the surface of mother bulb and bulbec is all with activity functional groups.Wherein, mother bulb is magnetic microsphere or non-magnetic microspheres, and the particle diameter of mother bulb is 500nm ~ 20 μm.Bulbec is magnetic Nano microsphere or non magnetic Nano microsphere, and the particle diameter of bulbec is 30nm ~ 200 nanometer.
Preferably, described activity functional groups is amino, carboxyl, sulfydryl or hydroxyl isoreactivity functional group.
In better embodiment of the present invention, described surface refers to have single dispersing particle diameter with the mother bulb of activity functional groups or bulbec, and surface grafting has mother bulb or the bulbec of amino, carboxyl, sulfydryl or hydroxyl isoreactivity functional group.
In the specific embodiment of the present invention, the female microballoon of magnetic is made up of ferroferric oxide nano granules and polymkeric substance or silicon oxide microsphere, and non-magnetic sexupara microballoon is polymkeric substance or silicon oxide microsphere.The sub-microballoon of magnetic Nano is made up of ferroferric oxide nano granules and polymkeric substance or silicon oxide microsphere, and the sub-microballoon of non magnetic nanometer is polymkeric substance or silicon oxide microsphere.
In another better embodiment of the present invention, the female microballoon of described magnetic refers to: there is single dispersing particle diameter, the functional group such as surface grafting has amino, carboxyl, sulfydryl, hydroxyl and inner polystyrene containing ferriferrous oxide nano magnetic-particle, polystyrene with gather (methyl) acrylic copolymer or silicon oxide magnetic microspheres.
Described non magnetic mother bulb refers to: have single dispersing particle diameter, the polystyrene of the functional group such as surface grafting has amino, carboxyl, sulfydryl, hydroxyl, polystyrene and poly-(methyl) acrylic copolymer or a silicon oxide microsphere.
Described non magnetic nanometer bulbec refers to: have single dispersing particle diameter, the polystyrene of the functional group such as surface grafting has amino, carboxyl, sulfydryl, hydroxyl, polystyrene and poly-(methyl) acrylic copolymer or a silicon oxide microsphere.
Described magnetic Nano bulbec refers to: there is single dispersing particle diameter, the functional group such as surface grafting has amino, carboxyl, sulfydryl, hydroxyl and inner containing ferriferrous oxide nano magnetic-particle polystyrene, polystyrene with gather (methyl) acrylic copolymer or monox nanometer magnetic microsphere.
On the other hand, present invention also offers and a kind ofly prepare the above-mentioned method significantly improving the Magnetic Microspheres-Carrier of in-vitro diagnosis detection sensitivity, comprise the following steps:
The first step, first, activation process is carried out to the functional group on magnetic or nonmagnetic sub-micron or micro-meter scale mother bulb surface; Then the non magnetic or magnetic Nano bulbec of excessive nanoscale is added, stirring reaction more than 2 hours, the microballoon obtained is removed unreacted bulbec by magnetic field or centrifugation, and finally obtaining surface-assembled has nanoscale, and has the magnetic microsphere of regular topological structure.
Second step, first, by the functional group activation with the assembling magnetic microsphere surface of regular topological structure obtained, then probe biomolecule is added, react more than 2 hours, washing remove unreacted probe biomolecule, obtain surface be connected with probe biomolecule magnetic assembling microsphere supported.The assembling of this magnetic is microsphere supported has biologically active, can some targets of specific recognition biomolecule to be checked.
3rd step, by above-mentioned have bioactive magnetic assembling the microsphere supported target biological molecules sample system joined containing variable concentrations gradient in, carry out biocompatible reaction or hybridization reaction; Calculating, detection sensitivity.
In better embodiment of the present invention, in step one, when mother bulb surface is carboxyl, adopt the activation of EDC/NHS reagent; When mother bulb surface is amino, adopt NHS-PEG
nthe difunctional activator activation of-NHS; When mother bulb surface is hydroxyl, adopt the activation of CDI reagent; When mother bulb surface is sulfydryl, adopt maleimide reagent activation.Preferably, described activator NHS-PEG
nin-NHS, PEG repetitive n is 2 ~ 20.But in preparation method of the present invention, the activation method of mother bulb surface functional group is not limited to upper type, and EDC/NHS, NHS-PEG
nthe concrete activation method of the activators such as-NHS, CDI or maleimide is method well-known in the art, and the present invention is not particularly limited this.Further, identical to the activation method described in the Methods and steps one that activates of functional group on assembling magnetic microsphere surface in step 2.
In the present invention, described probe biomolecule is following several reagent that this area is commonly used, but is not limited to this: antibody, Streptavidin, oligonucleotide probe or albumen aglucon etc.
On the other hand, present invention also offers the Magnetic Microspheres-Carrier obtained according to above preparation method and detect the purposes in biomolecule.Especially the application in some target biological molecules is being detected.
Preferably, described target biological molecules refers to antigen to be detected, biotin labeled biology molecule to be checked, target nucleic acid to be checked or acceptor molecule to be checked etc., but is not limited to this.
In the specific embodiment of the present invention, in step 3, the method for calculating, detection sensitivity is with reference to international standard EP-17A; And the smooth, spherical magnetic microsphere that the sensitivity calculated and surface have same composition and functional group structure is adopted identical biological respinse and carries out detection sensitivity comparative analysis, relatively find, Magnetic Microspheres-Carrier of the present invention has higher biologically active and detection sensitivity than smooth, spherical magnetic microsphere.
The present invention adopts the assembling magnetic microsphere with regular topological structure to be carrier, on the one hand, due to the large microsphere surface adopting the minimicrosphere of nanoscale to be assembled into micro-meter scale, significantly improve the surface area of micro-meter scale microballoon, it fixing the probe biomolecule ability of specific recognition target molecule to be checked greatly can be improved, thus significantly improve the detection sensitivity of in-vitro diagnosis.On the other hand, because assembling microballoon adopts simple Chemical Crosslinking Methods, its preparation condition is gentle, simple; And the compound with regular structure, controlled of assembling microballoon, ensure that the detection stability of in-vitro diagnosis.In addition, compared with conventional porous plate solid phase carrier, of the present invention microsphere supportedly have higher reaction kinetics, is that a kind of novel high-performance in-vitro diagnosis is with microsphere supported.
Be described further below with reference to the technique effect of accompanying drawing to design of the present invention, concrete structure and generation, to understand object of the present invention, characteristic sum effect fully.
Accompanying drawing explanation
Fig. 1 is the SEM photo of the magnetic the obtained assembling microballoon of embodiments of the invention 1;
Fig. 2 is the assembling magnetic microsphere obtained of embodiments of the invention 1 is that carrier is applied to the chemiluminescence intensity of chemiluminescence immunoassay detection to HbsAg concentration curve.
Embodiment
Elaborate to embodiments of the invention below, the present embodiment is implemented under premised on technical solution of the present invention, give detailed embodiment and concrete operating process, but protection scope of the present invention is not limited to following embodiment.
Embodiment 1
The first step, first employing particle diameter are the magnetic polymer microsphere of 700nm is mother bulb, and the inner Dispersed precipitate of this mother bulb has the nano ferriferrous oxide granule of 50wt%, and magnetic microsphere matrices is polystyrene, and surface is polystyrene and acrylic copolymer.Get the magnetic microsphere 80mg of this surface containing carboxyl, be distributed in the there-necked flask that (polysorbas20 containing 0.05wt% in solution) in MES (2-(N-morpholino) ethyl sulfonic acid) solution that 20mL concentration is 10mM is housed, under stirring, add EDC/NHS activator, make the final concentration both it be 50mg/mL.Stirring reaction 15min, washs unreacted EDC and NHS under magnetic field helps, and obtains the magnetic mother bulb after activating.Then the magnetic mother bulb after activation is distributed to (pH=7.6, the polysorbas20 containing 0.05wt%) in 20mL phosphate buffered solution.Secondly, magnetic mother bulb dispersion liquid after activation is dropwise joined in the silicon oxide microsphere aaerosol solution (silicon oxide particle is distributed to the suspending liquid formed in described buffer solution) that 80mL previously preparedly good is dispersed with surface band amino, particle diameter is 80nm, stirring reaction is after 3 hours, under magnetic field helps, respectively wash 3 times with deionized water, absolute ethyl alcohol, remove the unassembled monox bulbec to magnetic mother bulb surface.The magnetic assembling microballoon surface obtained with regular topological structure is kept in absolute ethyl alcohol for subsequent use.As shown in Figure 1, as shown in Figure 1, the regular High Density Packaging of amino oxygen SiClx microballoon of nanoscale is surperficial to the magnetic microsphere of submicron-scale for the SEM photo of the magnetic assembling microballoon obtained.
Second step, get 2mg above-mentioned magnetic assembling microballoon in absolute ethyl alcohol system with 3 microgram NHS-PEG
4-NHS carries out activation 2 hours, then the assembling magnetic microsphere after activation is distributed to (pH=7.6, the polysorbas20 containing 0.05wt%) in 400 gel phosphate buffer solution, washs three times.200 microgram Streptavidins are added again in this assembling magnetic microsphere system, 37 DEG C mix revolving reaction 2 hours, wash 3 times with closed reagent (containing inert protein) under magnetic field helps after, at 4 DEG C close spend the night, obtain surface be connected with Streptavidin, microballoon can be assembled with the Streptavidin magnetic of biotinylation biomolecule specific reaction.This Streptavidin magnetic assembling microballoon is kept at (pH=7.6, the polysorbas20 containing 0.05wt%, 0.1wt%BSA) in 200 gel phosphate buffer solution for subsequent use.
3rd step, assemble the method for microsphere supported detection sensitivity using the chemiluminescence detection of hepatitis B surface antigen as assessment magnetic.By biotinylation anti-hepatitis B surface antigen antibody and the hepatitis B surface antigen of variable concentrations gradient and another anti-hepatitis B surface antigen antibody of horseradish peroxidase-labeled 37 DEG C of incubation reaction 20 minutes, form immune complex.Then in each concentration gradient sample, add the assembling magnetic microsphere that surface that 10 micrograms obtain by step 2 is connected with Streptavidin, incubation reaction 10min at 37 DEG C again, the immune complex that not assembled magnetic microsphere catches and excessive enzyme labelled antibody is washed under magnetic field helps, in system, add chemical luminous substrate, test the chemiluminescence intensity signal value under each concentration gradient.Then drawing assembling magnetic microsphere is that carrier is applied to the chemiluminescence intensity of chemiluminescence immunoassay detection to the canonical plotting of HbsAg concentration, as shown in Figure 2, wherein RMMBs is the typical curve of obtained assembling magnetic microsphere, and CMMBs is the typical curve contrasting smooth microballoon.According to international standard EP-17A documentation requirements, detect obtained assembling magnetic microsphere by Fig. 2 calculating and 0.90ng/mL is limited to detecting of hepatitis B surface antigen.And adopting identical mother bulb, the smooth microballoon of the amino oxygen SiClx shell of pan coating same thickness is carrier, adopts the reagent same with the 3rd step through identical testing process, it is limited to 2.6ng/mL to detecting of hepatitis B surface antigen.
Embodiment 2
In the present embodiment, the first step and second step remove the carboxyl magnetic microsphere that mother bulb particle diameter is 5 μm, and bulbec is that outside the aminopolystyrene microballoon of 200 nanometers, all the other methods of operating are identical with embodiment 1.
In 3rd step, except each concentration gradient adds 15 microgram assembling magnetic microspheres, all the other steps are identical.Detecting the detection of hepatitis B surface antigen of calculating after testing is limited to 1.2ng/mL, the smooth magnetic microsphere of the surface amino groups monox shell of identical mother bulb bag quilt its detect and be limited to 3.1ng/mL.
Embodiment 3
In the present embodiment, the first step and second step remove the carboxyl magnetic microsphere that mother bulb particle diameter is 10 μm, and bulbec is that outside the aminopolystyrene microballoon of 200nm, all the other methods of operating are identical with embodiment 1.
In 3rd step, except each concentration gradient adds 30 microgram assembling magnetic microspheres, all the other steps are identical.Detecting the detection of hepatitis B surface antigen of calculating after testing is limited to 1.5ng/mL, the smooth magnetic microsphere of the surface amino groups monox shell of identical mother bulb bag quilt its detect and be limited to 3.8ng/mL.
Embodiment 4
Mother bulb particle diameter except selecting in the present embodiment is the monox carboxyl microballoon of 20 μm, the amino microballoon of the magnetic of 100nm selected by sub-microballoon, washing is simultaneously removed the bulbec of not assembling on micron carboxyl mother bulb and is adopted 1000 turns of centrifugal methods to remove outside supernatant, and all the other are all identical with embodiment 1.
In 3rd step, except each concentration gradient adds 30 microgram assembling magnetic microspheres, all the other steps are identical.Detecting the detection of hepatitis B surface antigen of calculating after testing is limited to 2.1ng/mL, the smooth magnetic microsphere of the surface amino groups monox shell of identical mother bulb bag quilt its detect and be limited to 4.5ng/mL.
Embodiment 5
The first step, first employing particle diameter are the magnetic silicon oxide microballoon of 1000nm is mother bulb, and the inner Dispersed precipitate of mother bulb has the nano ferriferrous oxide granule of 70wt%, and magnetic microsphere matrices is monox, and surface is for being modified with amino-functional group.Get the magnetic microsphere 80mg of this surface containing amino group, be distributed to and be equipped with in 20mL ethanol solution.Under stirring, above-mentioned mother bulb suspending liquid is dropwise added and previously preparedly good is dispersed with surface amino groups, in absolute ethyl alcohol aaerosol solution (150mL) that particle diameter is 30 nano silicon oxide microballoons.Add again after being added dropwise to complete and be dissolved with 150 microgram NHS-PEG
20the ethanol solution of-NHS, stirring reaction spends the night, under magnetic field helps, respectively wash 3 times with deionized water, absolute ethyl alcohol, removes the unassembled monox bulbec to magnetic mother bulb surface.The assembling magnetic microsphere that the surface obtained the most at last has regular topological structure is kept in absolute ethyl alcohol for subsequent use.
Second step, get group shape microsphere surface that 2mg obtains amino in absolute ethyl alcohol system with 3 microgram NHS-PEG
4-NHS carries out activation 2 hours, then the assembling microballoon after activation is distributed to (pH=7.6, the polysorbas20 containing 0.05wt%) in 400 gel phosphate buffer solution, washs three times.200 microgram Streptavidins are added again in above-mentioned assembling microsphere system, 37 DEG C mix revolving reaction 2 hours, wash 3 times with closed reagent (containing inert protein) under magnetic field helps after, at 4 DEG C close spend the night, obtain surface be connected with Streptavidin, microballoon can be assembled with the Streptavidin magnetic of biotinylation biomolecule specific reaction.This Streptavidin magnetic assembling microballoon is kept at (pH=7.6, the polysorbas20 containing 0.05wt%, 0.1%BSA) in 200 gel phosphate buffer solution for subsequent use.
3rd step, method using the chemiluminescence detection of hepatitis B surface antigen as assessment Magnetic Microspheres-Carrier detection sensitivity.By biotinylation anti-hepatitis B surface antigen antibody and the hepatitis B surface antigen of variable concentrations gradient and another anti-hepatitis B surface antigen antibody of horseradish peroxidase-labeled 37 DEG C of incubation reaction 20 minutes, form immune complex.Then in each concentration gradient sample, add 10 micrograms walk the assembling magnetic microsphere that the surface obtained is connected with Streptavidin from above, incubation reaction 10min at 37 DEG C again, the immune complex that not assembled magnetic microsphere catches and excessive enzyme labelled antibody is washed under magnetic field helps, in system, add chemical luminous substrate, test the chemiluminescence signal value under each concentration gradient.Then drawing standard curve, according to international standard EP-17A documentation requirements, calculating detects and is limited to 0.92ng/mL.And adopting identical mother bulb, the smooth magnetic microsphere of the amino oxygen SiClx shell of pan coating same thickness is carrier, adopts the reagent same with the 3rd step through identical testing process, it is limited to 2.7ng/mL to detecting of hepatitis B surface antigen.
Embodiment 6
The first step, first employing particle diameter are the magnetic silicon oxide microballoon of 1000nm is mother bulb, and the inner Dispersed precipitate of mother bulb has the nano ferriferrous oxide granule of 70wt%, and magnetic microsphere matrices is monox, and surface is for being modified with amino-functional group.Get the magnetic microsphere 80mg of this surface containing amino group, be distributed to and be equipped with in 20mLMES (pH=5.0, the polysorbas20 containing 0.05wt%) buffer solution.Under stirring, above-mentioned mother bulb suspending liquid is dropwise added ready 150mL is in advance dispersed with surface carboxyl groups, particle diameter is in the MES aaerosol solution of 100 nanometer polymer microballoons.The MES buffer solution of 30mLEDC/NHS is added again after being added dropwise to complete, make EDC/NHS separately concentration be 50mg/mL, stirring reaction 3 hours, respectively washs 3 times with deionized water, absolute ethyl alcohol under magnetic field helps, and removes the unassembled nanometer polymer bulbec to magnetic mother bulb surface.The surface obtained the most at last has the assembling magnetic microsphere of regular topological structure, is kept in phosphate buffered solution for subsequent use.
Second step, to get surface that 2mg obtains be that the assembling microballoon of carboxyl is distributed to phosphate (pH=7.6, polysorbas20 containing 0.05wt%) in buffer solution, activation 15min is carried out with the EDC/NHS of 50mg/mL in system, then the assembling microballoon after activation is distributed to (pH=7.6 in 400 gel phosphate buffer solution, polysorbas20 containing 0.05wt%), wash three times.200 microgram Streptavidins are added again in above-mentioned assembling microsphere system, 37 DEG C mix revolving reaction 2 hours, wash 3 times with closed reagent (containing inert protein) under magnetic field helps after, at 4 DEG C close spend the night, obtain surface be connected with Streptavidin, microballoon can be assembled with the Streptavidin magnetic of biotinylation biomolecule specific reaction.This Streptavidin magnetic assembling microballoon is kept at (pH=7.6, the polysorbas20 containing 0.05wt%, 0.1wt%BSA) in 200 gel phosphate buffer solution for subsequent use.
3rd step, method using the chemiluminescence detection of hepatitis B surface antigen as assessment Magnetic Microspheres-Carrier detection sensitivity.By biotinylation anti-hepatitis B surface antigen antibody and the hepatitis B surface antigen of variable concentrations gradient and another anti-hepatitis B surface antigen antibody of horseradish peroxidase-labeled 37 DEG C of incubation reaction 20 minutes, form immune complex.Then in each concentration gradient sample, add 10 micrograms walk the assembling magnetic microsphere that the surface obtained is connected with Streptavidin from above, incubation reaction 10min at 37 DEG C again, the immune complex that not assembled magnetic microsphere catches and excessive enzyme labelled antibody is washed under magnetic field helps, in system, add chemical luminous substrate, test the chemiluminescence signal value under each concentration gradient.Then drawing standard curve, according to international standard EP-17A documentation requirements, calculating detects and is limited to 0.95ng/mL.And adopting identical mother bulb, the smooth magnetic microsphere of the carboxyl oxygen SiClx shell of pan coating same thickness is carrier, adopts the reagent same with the 3rd step through identical testing process, it is limited to 2.85ng/mL to detecting of hepatitis B surface antigen.
Embodiment 7
The first step, first employing particle diameter are the magnetic silicon oxide microballoon of 1000nm is mother bulb, and the inner Dispersed precipitate of mother bulb has the nano ferriferrous oxide granule of 70wt%, and magnetic microsphere matrices is monox, and surface is for being modified with sulfydryl functional group.Get the magnetic microsphere 80mg of this surface containing mercapto groups, be distributed to and be equipped with in 20mL phosphate buffered solution.Under stirring, above-mentioned mother bulb suspending liquid is dropwise added ready 150mL is in advance dispersed with surface amino groups, particle diameter is in the phosphate buffered solution of 50 nano silicon oxide microballoons.The phosphate buffered solution (pH=7.6) of the sulfo-SMCC being dissolved with 50mg/mL is added again after being added dropwise to complete, stirring reaction spends the night, under magnetic field helps, use deionized water, absolute ethanol washing 3 times, remove the unassembled monox bulbec to magnetic mother bulb surface.The assembling magnetic microsphere that the surface obtained the most at last has regular topological structure is kept in absolute ethyl alcohol for subsequent use.
Second step, get the assembling magnetic microsphere surface amino groups that 2mg obtains carry out activation 2 hours with 3 microgram NHS-PEG4-NHS in absolute ethyl alcohol system, then the assembling microballoon after activation is distributed to (pH=7.6 in 400 gel phosphate buffer solution, polysorbas20 containing 0.05wt%), wash three times.200 microgram Streptavidins are added again in above-mentioned assembling microsphere system, 37 DEG C mix revolving reaction 2 hours, wash 3 times with closed reagent (containing inert protein) under magnetic field helps after, at 4 DEG C close spend the night, obtain surface be connected with Streptavidin, microballoon can be assembled with the Streptavidin magnetic of biotinylation biomolecule specific reaction.This Streptavidin magnetic assembling microballoon is kept at (pH=7.6, the polysorbas20 containing 0.05wt%, 0.1wt%BSA) in 200 gel phosphate buffer solution for subsequent use.
3rd step, method using the chemiluminescence detection of hepatitis B surface antigen as assessment Magnetic Microspheres-Carrier detection sensitivity.By biotinylation anti-hepatitis B surface antigen antibody and the hepatitis B surface antigen of variable concentrations gradient and another anti-hepatitis B surface antigen antibody of horseradish peroxidase-labeled 37 DEG C of incubation reaction 20 minutes, form immune complex.Then in each concentration gradient sample, add 10 micrograms walk the assembling magnetic microsphere that the surface obtained is connected with Streptavidin from above, incubation reaction 10min at 37 DEG C again, the immune complex that not assembled magnetic microsphere catches and excessive enzyme labelled antibody is washed under magnetic field helps, in system, add chemical luminous substrate, test the chemiluminescence signal value under each concentration gradient.Then drawing standard curve, according to international standard EP-17A documentation requirements, calculating detects and is limited to 0.85ng/mL.And adopting identical mother bulb, the smooth magnetic microsphere of the amino oxygen SiClx shell of pan coating same thickness is carrier, adopts the reagent same with the 3rd step through identical testing process, it is limited to 2.5ng/mL to detecting of hepatitis B surface antigen.
Embodiment 8
The first step, first employing particle diameter are the magnetic silicon oxide microballoon of 1000nm is mother bulb, and the inner Dispersed precipitate of mother bulb has the nano ferriferrous oxide granule of 70wt%, and magnetic microsphere matrices is monox, and surface is silicone hydroxyl functional group.Get this magnetic microsphere 80mg, be distributed to and be equipped with in 20mL tetrahydrofuran solution.Under stirring, above-mentioned mother bulb suspending liquid is dropwise added ready 150mL is in advance dispersed with surface amino groups, particle diameter be the tetrahydrofuran of 150 nano silicon oxide microballoons in suspension.Add the tetrahydrofuran solution of the CDI being dissolved with 50mg/mL after being added dropwise to complete again, stirring reaction spends the night, under magnetic field helps, wash 3 times respectively with tetrahydrofuran, absolute ethyl alcohol, removes the unassembled monox bulbec to magnetic mother bulb surface.The assembling magnetic microsphere that the surface obtained the most at last has regular topological structure is kept in ethanol solution for subsequent use.
Second step, get group shape microsphere surface that 2mg obtains amino in absolute ethyl alcohol system with 3 microgram NHS-PEG
4-NHS carries out activation 2 hours, then the assembling microballoon after activation is distributed to (pH=7.6, the polysorbas20 containing 0.05wt%) in 400 gel phosphate buffer solution, washs three times.200 microgram Streptavidins are added again in above-mentioned assembling microsphere system, 37 DEG C mix revolving reaction 2 hours, wash 3 times with closed reagent under magnetic field helps after, at 4 DEG C close spend the night, obtain surface be connected with Streptavidin, microballoon can be assembled with the Streptavidin magnetic of biotinylation biomolecule specific reaction.This Streptavidin magnetic assembling microballoon is kept at (pH=7.6, the polysorbas20 containing 0.05wt%, 0.1wt%BSA) in 200 gel phosphate buffer solution for subsequent use.
3rd step, method using the chemiluminescence detection of hepatitis B surface antigen as assessment Magnetic Microspheres-Carrier detection sensitivity.By biotinylation anti-hepatitis B surface antigen antibody and the hepatitis B surface antigen of variable concentrations gradient and another anti-hepatitis B surface antigen antibody of horseradish peroxidase-labeled 37 DEG C of incubation reaction 20 minutes, form immune complex.Then in each concentration gradient sample, add 10 micrograms walk the assembling magnetic microsphere that the surface obtained is connected with Streptavidin from above, incubation reaction 10min at 37 DEG C again, the immune complex that not assembled magnetic microsphere catches and excessive enzyme labelled antibody is washed under magnetic field helps, in system, add chemical luminous substrate, test the chemiluminescence signal value under each concentration gradient.Then drawing standard curve, according to international standard EP-17A documentation requirements, calculating detects and is limited to 0.93ng/mL.And adopting identical mother bulb, the smooth magnetic microsphere of the amino oxygen SiClx shell of pan coating same thickness is carrier, adopts the reagent same with the 3rd step through identical testing process, it is limited to 2.85ng/mL to detecting of hepatitis B surface antigen.
More than describe preferred embodiment of the present invention in detail.Should be appreciated that the ordinary skill of this area just design according to the present invention can make many modifications and variations without the need to creative work.Therefore, all technician in the art, all should by the determined protection domain of claims under this invention's idea on the basis of existing technology by the available technical scheme of logical analysis, reasoning, or a limited experiment.
Claims (10)
1., for the carrier that in-vitro diagnosis detects, it is characterized in that, comprise mother bulb and be connected to the bulbec on described mother bulb surface by chemical covalent bonds.
2. carrier as claimed in claim 1, is characterized in that, the spherical structure of described mother bulb to be particle diameter be 500nm ~ 20 μm.
3. carrier as claimed in claim 1, is characterized in that, the spherical structure of described bulbec to be particle diameter be 30nm ~ 200nm.
4. carrier as claimed in claim 1, it is characterized in that, the material of described mother bulb and described bulbec is polymkeric substance or monox.
5. carrier as claimed in claim 4, is characterized in that, described polymkeric substance is polystyrene, polystyrene and poly-(methyl) acrylic copolymer.
6. carrier as claimed in claim 1, it is characterized in that, the surface grafting of described mother bulb and described bulbec has functional group, and the connection between described mother bulb and described bulbec is reacted by chemical covalent between described functional group and realized.
7. carrier as claimed in claim 6, it is characterized in that, described functional group is the one in amino, carboxyl, sulfydryl, hydroxyl.
8. the carrier as described in as arbitrary in claim 1-7, is characterized in that, be also included in the magnetic nanoparticle in described mother bulb and/or described bulbec.
9. carrier as claimed in claim 8, it is characterized in that, described magnetic nanoparticle is ferroferric oxide nano granules.
10. for a preparation method for the carrier of in-vitro diagnosis detection, it is characterized in that: connect described bulbec on described mother bulb surface by chemical covalent bonds.
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| CN105219373B (en) | 2014-06-05 | 2019-06-25 | 中翰盛泰生物技术股份有限公司 | A kind of carrier granular and preparation method thereof |
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