CN105403697B - It is a kind of microsphere supported and preparation method thereof - Google Patents
It is a kind of microsphere supported and preparation method thereof Download PDFInfo
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- CN105403697B CN105403697B CN201510325843.3A CN201510325843A CN105403697B CN 105403697 B CN105403697 B CN 105403697B CN 201510325843 A CN201510325843 A CN 201510325843A CN 105403697 B CN105403697 B CN 105403697B
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
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- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/551—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being inorganic
- G01N33/553—Metal or metal coated
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/531—Production of immunochemical test materials
- G01N33/532—Production of labelled immunochemicals
- G01N33/535—Production of labelled immunochemicals with enzyme label or co-enzymes, co-factors, enzyme inhibitors or enzyme substrates
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Abstract
The invention discloses a kind of Magnetic Microspheres-Carrier and preparation method thereof, the Magnetic Microspheres-Carrier uses the magnetic microsphere of micron or submicron-scale as mother bulb, there is the bulbec of abundant functional group on its surface by Chemical Crosslinking Methods assemble nanometer yardstick, surface, obtain the assembling magnetic microsphere with regular topological structure.The Magnetic Microspheres-Carrier of the present invention can realize the efficient capture to target molecule to be checked and detection as the carrier of identification, capture with manipulating target molecule to be checked.Compared with traditional smooth, spherical magnetic microsphere with similar face functional group, this kind of magnetic microsphere with regular topological structure is remarkably improved the detection sensitivity of in-vitro diagnosis application process.Also, its assembling process is simple, the regular appearance of obtained assembling microballoon, and the surface area of microballoon greatly improved in the formation of its topological structure, ensure that the efficient fixation to target organism probe molecule during subsequent in vitro diagnostic application.
Description
The application is the divisional application of following application：The applying date：On 2 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 its preparation and application, more particularly to a kind of in-vitro diagnosis that improves to detect
Magnetic Microspheres-Carrier of sensitivity and preparation method thereof.Belong to technical field of biological materials.
Background technology
Solid phase carrier of the magnetic microsphere as in-vitro diagnosis, play identification, capture, control and transport target molecule to be checked
Carrier function.Because it is suspended in sample to be checked in whole course of reaction, compared with traditional porous plate carrier, its
Whole reaction is substantially at liquid or quasi- liquid condition, and the probability that molecular collision occurs between carrier and thing to be checked greatly increases.
Further, since the larger surface area of magnetic microsphere improves the efficiency of load target molecule to be checked, so that its biological respinse
Efficiency greatly improves compared with conventional porous plate mode；Biological respinse on general porous plate was needed between 1~3 hour, and was based on
The biological respinse of magnetic microsphere only needs more than ten minutes.Further, since the superparamagnetic characteristic that magnetic microsphere is remarkable so that use entirely certainly
Dynamic Magnetic Isolation mode can be realized easily with detection.
At present, the Magnetic Microspheres-Carrier of in-vitro diagnosis field application be mainly particle diameter below 100nm magnetic-particle with
And particle diameter more than 1 μm (be usually several microns) the class of magnetic microsphere two.Particle diameter below 100nm magnetic-particle, due to tool
There is great specific surface area, identification can be significantly improved with capturing the kinetics and capture mesh of target biological molecules to be checked
The ability of biomolecule is marked, and then improves the detection sensitivity and detection efficiency of in-vitro diagnosis.But the magnetic of nanoscale
The responsiveness of grain external magnetic field is low, is not easy to be manipulated by magnetic field；And easily occur to reunite and do not have in actual application
Nanoscale effect, so as to make in the full-automatic vitro diagnostic test systems of current main flow as efficient solid phase carrier
With.On the other hand, the magnetic microsphere of micro-meter scale, because it includes substantial amounts of magnetic-particle, although can conveniently realize complete
Automatic detection, but microsphere supported surface area ratio nano magnetic particle substantially reduces, detection during so as to using it as carrier are clever
Sensitivity is also greatly lowered with reaction efficiency.
Found through the literature search to prior art, Matsunaga etc. exists《Analytica Chimica Acta》597th
" the magnetic microsphere detection for full-automatic immune detection based on beads on beads compounds that 331-339 pages of volume is delivered
PSA specific antigens " (Fully automated immunoassay for detection of prostate-specific
Antigen using nano-magnetic beads and micro-polystyrene bead composites, '
Beads on Beads ' ") in report using beads on beads structures, the magnetic-particle of nanoscale is passed through into biology
Element is attached on the polystyrene microsphere for the micro-meter scale that surface is connected with Avidin, on the one hand improves the surface of magnetic microsphere
Product, the problems such as on the other hand overcoming the weak response of the magnetic-particle external magnetic field of nanoscale again and easily reunite.But
Being this article, topological structure not special to this beads on beads diagnoses in vitro with smooth micro-meter scale microballoon
Detection sensitivity carry out comparative study, i.e., the inspection of in-vitro diagnosis whether can be improved without this special topological structure of explaination
Survey sensitivity.Further, since package technique is the affinity interaction of Streptavidin and biotin used by this report, therefore need
To pass through up to more than ten times repeatedly repeatedly assembling could realize microballoon of the magnetic-particle of nanoscale on micro-meter scale compared with
High Density Packaging, whole preparation process is complicated, and costly.
Further retrieval is found, Tan etc. exists《ANALYTICAL AND BIOANALYTICAL CHEMISTRY》Volume 383
738-746 pages of " spherical biological function core shell nanoparticles microstructured layers improve reaction surface area in protein arrays " delivered
(Microstructured layers of spherical biofunctional core-shell nanoparticles
Provide enlarged reactive surfaces for protein microarrays) in report in flat plate substrate
The upper microballoon by assemble nanometer yardstick, then in this, as solid phase carrier, carry out the detection to model proteins, as a result find detection
Sensitivity is highly improved.But the solid phase carrier due to using is flat plate substrate, it is complete with intermolecular reaction to be checked
Realized by the diffusion of molecule so that kinetics is greatly lowered compared with microballoon.
The content of the invention
In view of the drawbacks described above of prior art, the technical problems to be solved by the invention are to provide a kind of preparation method letter
Magnetic Microspheres-Carrier single, that in-vitro diagnosis detection sensitivity can be improved.
To achieve the above object, the invention provides it is a kind of improve in-vitro diagnosis detection sensitivity Magnetic Microspheres-Carrier and
Its preparation method.
The present invention uses the magnetic microsphere of micron or submicron-scale to pass through Chemical Crosslinking Methods group on its surface for mother bulb
Filling surface has the nanoscale bulbec of abundant functional group, so as to obtain the assembling magnetic microsphere with regular topological structure.
Carrier using the obtained assembling magnetic microsphere with regular topological structure as identification, capture and manipulation target molecule to be checked,
So as to realize efficient capture and the detection to target molecule to be checked.With traditional smooth, spherical magnetic with similar face functional group
Property microballoon is compared, and the magnetic microsphere with regular topological structure of the invention is remarkably improved the detection of in-vitro diagnosis application process
Sensitivity.The preparation method of the present invention realizes the bulbec of nanoscale in micro-meter scale mother bulb by a chemical crosslink reaction
On High Density Packaging, its assembling process is simple, obtain assembling microballoon regular appearance；And the formation of topological structure is very big
Ground improves the surface area of microballoon, ensure that during subsequent in vitro diagnostic application to target organism probe molecule (nucleic acid or egg
Efficient fixed and detection in vain).
The present invention is that solve above-mentioned technical problem by the following technical programs：
On the one hand, the invention provides a kind of Magnetic Microspheres-Carrier for being remarkably improved in-vitro diagnosis detection sensitivity.Tool
Body, the component and content/physicochemical characteristic of Magnetic Microspheres-Carrier of the invention are：With with micron or submicron-scale
Mother bulb is core, and particle diameter is that the bulbec of nanoscale is shell, is closely connected by chemical covalent bonds between bulbec and mother bulb, and mother bulb
Activity functional groups are carried with the surface of bulbec.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 nanoparticle, and the particle diameter of bulbec is 30nm~200 nanometer.
Preferably, the activity functional groups are amino, carboxyl, sulfydryl or hydroxyl isoreactivity functional group.
In the better embodiment of the present invention, mother bulb or bulbec of the surface with activity functional groups refer to have
Single dispersing particle diameter, and surface grafting has the mother bulb or bulbec of amino, carboxyl, sulfydryl or hydroxyl isoreactivity functional group.
In the embodiment of the present invention, magnetic mother microballoon is by ferroferric oxide nano granules and polymer or oxygen
SiClx microballoon forms, and non-magnetic sexupara microballoon is polymer or silicon oxide microsphere.The sub- microballoon of magnetic Nano is received by ferroso-ferric oxide
Rice grain forms with polymer or silicon oxide microsphere, and the non magnetic sub- microballoon of nanometer is polymer or silicon oxide microsphere.
In another better embodiment of the present invention, described magnetic mother's microballoon refers to：With single dispersing particle diameter, surface
It is grafted with the functional groups such as amino, carboxyl, sulfydryl, hydroxyl and the internal polyphenyl second containing ferriferrous oxide nano magnetic-particle
Alkene, polystyrene and poly- (methyl) acrylic copolymer or silicon oxide magnetic microspheres.
Described non magnetic mother bulb refers to：There are the work(such as amino, carboxyl, sulfydryl, hydroxyl with single dispersing particle diameter, surface grafting
Can the polystyrene of group, polystyrene and poly- (methyl) acrylic copolymer or silicon oxide microsphere.
Described non magnetic nanometer bulbec refers to：There are amino, carboxyl, sulfydryl, hydroxyl with single dispersing particle diameter, surface grafting
Polystyrene, polystyrene and poly- (methyl) acrylic copolymer or silicon oxide microsphere Deng functional group.
Described magnetic Nano bulbec refers to：There are amino, carboxyl, sulfydryl, hydroxyl etc. with single dispersing particle diameter, surface grafting
The functional group and internal polystyrene containing ferriferrous oxide nano magnetic-particle, polystyrene and poly- (methyl) propylene
Acid copolymer or silica nano-magnetic microsphere.
On the other hand, the above-mentioned magnetic for being remarkably improved in-vitro diagnosis detection sensitivity is prepared present invention also offers a kind of
Microsphere supported method, comprises the following steps：
The first step, first, lives to the functional group of magnetic or nonmagnetic sub-micron or micro-meter scale mother bulb surface
Change is handled；Then add that excessive nanoscale is non magnetic or magnetic Nano bulbec, stirring reaction more than 2 hours, by what is obtained
Microballoon removes unreacted bulbec by magnetic field or centrifugation, and finally giving surface-assembled has nanoscale, and with rule
The magnetic microsphere of whole topological structure.
Second step, first, what will be obtained has the functional group activation on the assembling magnetic microsphere surface of regular topological structure,
Then probe biomolecule is added, is reacted more than 2 hours, washing removes unreacted probe biomolecule, obtains surface connection
There is the magnetic assembling of probe biomolecule microsphere supported.Magnetic assembling is microsphere supported to have bioactivity, can specific recognition
Some targets biomolecule to be checked.
3rd step, the above-mentioned magnetic assembling with bioactivity microsphere supported is added to the mesh containing various concentrations gradient
Mark in biomolecule sample system, carry out biological compatible reaction or hybridization reaction；Calculate, detection sensitivity.
In the better embodiment of the present invention, in step 1, when mother bulb surface is carboxyl, using EDC/NHS reagents
Activation；When mother bulb surface is amino, using NHS-PEGnThe difunctional activator activation of-NHS；When mother bulb surface is hydroxyl,
Activated using CDI reagents；When mother bulb surface is sulfydryl, activated using maleimide reagent.Preferably, the activator
NHS-PEGnPEG repeat units n is 2~20 in-NHS.But the work in the preparation method of the present invention to mother bulb surface functional group
Change method is not limited to upper type, and EDC/NHS, NHS-PEGnThe activators such as-NHS, CDI or maleimide it is specific
Activation method is method well-known in the art, and this is not particularly limited the present invention.Also, to assembling magnetic in step 2
The method that the functional group of microsphere surface is activated is identical with the activation method described in step 1.
In the present invention, described probe biomolecule is following several reagents commonly used in the art, but is not limited to this：
Antibody, Streptavidin, oligonucleotide probe or albumen aglucon etc..
On the other hand, present invention also offers divided according to the Magnetic Microspheres-Carrier that method made above obtains in detection biology
Purposes in son.Application especially in some target biological molecules are detected.
Preferably, described target biological molecules refer to antigen to be detected, biotin labeling biological molecule to be checked, treat
Target nucleic acid or acceptor molecule to be checked etc. are examined, but is not limited to this.
In the embodiment of the present invention, calculated in step 3, the method for detection sensitivity reference international standard EP-
17A；And the sensitivity being calculated is adopted with smooth, spherical magnetic microsphere that surface has same composition and functional group structure
With identical biological respinse and detection sensitivity comparative analysis is carried out, it was found that, Magnetic Microspheres-Carrier of the invention compares smooth ball
Shape magnetic microsphere has higher bioactivity and detection sensitivity.
The present invention uses the assembling magnetic microsphere with regular topological structure as carrier, on the one hand, due to using nanometer chi
The minimicrosphere of degree is assembled into the big microsphere surface of micro-meter scale, significantly improves the surface area of micro-meter scale microballoon so that it is solid
Surely can the probe biomolecule ability of specific recognition target molecule to be checked greatly improve, so as to significantly improve the inspection of in-vitro diagnosis
Survey sensitivity.On the other hand, because assembling microballoon uses simple Chemical Crosslinking Methods, its preparation condition is gently, simply；And group
The compound with regular structure, controllable of microballoon is filled, ensure that the detection stability of in-vitro diagnosis.In addition, with conventional porous plate solid phase carrier phase
Than, it is of the invention it is microsphere supported there is higher kinetics, be a kind of new high-performance in-vitro diagnosis with microsphere supported.
Design, concrete structure and the caused technique effect of the present invention are described further below with reference to accompanying drawing, with
It is fully understood from the purpose of the present invention, feature and effect.
Brief description of the drawings
Fig. 1 is the SEM photograph of the resulting magnetic assembling microballoon of embodiments of the invention 1；
Fig. 2 is that the resulting assembling magnetic microsphere of embodiments of the invention 1 is examined for carrier applied to chemiluminescence immunoassay
The chemiluminescence intensity of survey is to HbsAg concentration curves.
Embodiment
Embodiments of the invention are elaborated below, the present embodiment is carried out lower premised on technical solution of the present invention
Implement, give detailed embodiment and specific operating process, but protection scope of the present invention is not limited to following implementation
Example.
Embodiment 1
The first step, to use particle diameter first for 700nm magnetic polymer microsphere be mother bulb, there is Dispersed precipitate inside the mother bulb
50wt% nano ferriferrous oxide granule, magnetic microsphere matrices are polystyrene, and surface is that polystyrene is total to polyacrylic acid
Polymers.The magnetic microsphere 80mg that carboxyl is contained on this surface is taken, is distributed to equipped with MES (2- (the N- morpholines that 20mL concentration is 10mM
Generation) ethyl sulfonic acid) in solution in the three-necked flask of (polysorbas20 containing 0.05wt% in solution), under stirring, add EDC/NHS and live
Agent, make the final concentration of 50mg/mL of both.Stirring reaction 15min, magnetic field help under wash unreacted EDC with
NHS, the magnetic mother bulb after being activated.Then the magnetic mother bulb after activation is distributed to (pH in 20mL phosphate buffer solutions
=7.6, the polysorbas20 containing 0.05wt%).Secondly, the magnetic mother bulb dispersion liquid after activation is added dropwise to 80mL and made in advance
With amino, silicon oxide microsphere aaerosol solution that particle diameter is 80nm, (silicon oxide particle is distributed to described slow on the surface that is dispersed with got ready
Rush the suspension formed in solution) in, stirring reaction is respectively washed after 3 hours under being helped in magnetic field with deionized water, absolute ethyl alcohol
3 times, remove the unassembled silica bulbec to magnetic mother bulb surface.Obtained surface is had to the magnetic group of regular topological structure
Dress microballoon is stored in standby in absolute ethyl alcohol.The SEM photograph of resulting magnetic assembling microballoon as shown in figure 1, as shown in Figure 1,
The regular High Density Packaging of amino oxygen SiClx microballoon of nanoscale is to the magnetic microsphere surface of submicron-scale.
Second step, the above-mentioned magnetic of 2mg is taken to assemble microballoon in absolute ethyl alcohol system with 3 microgram NHS-PEG4- NHS is lived
Change 2 hours, then the assembling magnetic microsphere after activation is distributed in 400 microlitres of phosphate buffer solutions and (pH=7.6, contained
0.05wt% polysorbas20), washing is three times.Add 200 microgram Streptavidins in the assembling magnetic microsphere system again, 37 DEG C
Mix revolving reaction 2 hours, after washing 3 times with closed reagent (containing inert protein) under being helped in magnetic field, closed at 4 DEG C
Night, obtain to surface be connected with Streptavidin, can be with the Streptavidin magnetic of biotinylation biomolecule specific reaction
Assemble microballoon.This Streptavidin magnetic assembling microballoon is stored in 200 microlitres of phosphate buffer solutions and (pH=7.6, contained
0.05wt% polysorbas20,0.1wt%BSA) it is standby.
3rd step, it is used as using the chemiluminescence detection of hepatitis B surface antigen and assesses magnetic and assemble microsphere supported detection sensitivity
Method.By the hepatitis B surface antigen and horseradish peroxidating of biotinylation anti-hepatitis B surface antigen antibody and various concentrations gradient
Another anti-hepatitis B surface antigen antibody of thing enzyme mark forms immune complex in 37 DEG C of incubation reactions 20 minutes.Then exist
The assembling magnetic microsphere that the surface that 10 micrograms are obtained by step 2 is connected with Streptavidin is added in each concentration gradient sample,
The incubation reaction 10min at 37 DEG C again, the immune complex and mistake of not assembled magnetic microsphere capture are washed under being helped in magnetic field
The enzyme labelled antibody of amount, adds chemical luminous substrate in system, the chemiluminescence intensity signal value tested under each concentration gradient.
Then it is mark of the chemiluminescence intensity to HbsAg concentration that carrier is applied to chemiluminescence immunoassay detection to draw assembling magnetic microsphere
Directrix curve figure, as shown in Fig. 2 wherein RMMBs is the standard curve of obtained assembling magnetic microsphere, CMMBs is that contrast is smooth micro-
The standard curve of ball.According to international standard EP-17A documentation requirements, the obtained assembling magnetic microsphere of detection is calculated to second by Fig. 2
The detection of liver surface antigen is limited to 0.90ng/mL.And identical mother bulb is used, the amino oxygen SiClx shell of pan coating same thickness
Smooth microballoon be carrier, using the reagent same with the 3rd step and pass through identical detection process, it is to hepatitis B surface antigen
Detection be limited to 2.6ng/mL.
Embodiment 2
For the first step with second step except the carboxyl magnetic microsphere that mother bulb particle diameter is 5 μm, bulbec is 200 nanometers in the present embodiment
Outside aminopolystyrene microballoon, remaining operating method is same as Example 1.
In 3rd step, in addition to each concentration gradient adds 15 micrograms assembling magnetic microsphere, remaining step is identical.Count after testing
The obtained detection of the detection to hepatitis B surface antigen is limited to 1.2ng/mL, the identical coated surface amino groups silica shell of mother bulb
Smooth magnetic microsphere its detection be limited to 3.1ng/mL.
Embodiment 3
For the first step with second step except the carboxyl magnetic microsphere that mother bulb particle diameter is 10 μm, bulbec is 200nm's in the present embodiment
Outside aminopolystyrene microballoon, remaining operating method is same as Example 1.
In 3rd step, in addition to each concentration gradient adds 30 micrograms assembling magnetic microsphere, remaining step is identical.Count after testing
The obtained detection of the detection to hepatitis B surface antigen is limited to 1.5ng/mL, the identical coated surface amino groups silica shell of mother bulb
Smooth magnetic microsphere its detection be limited to 3.8ng/mL.
Embodiment 4
Except the oxidation silicono microballoon that the mother bulb particle diameter of selection is 20 μm in the present embodiment, sub- microballoon is from 100nm's
Magnetic amino microballoon, while wash removal the bulbec in micron carboxyl mother bulb over-assemble is not removed using 1000 turns of centrifugal methods
Outside supernatant, remaining is same as Example 1.
In 3rd step, in addition to each concentration gradient adds 30 micrograms assembling magnetic microsphere, remaining step is identical.Count after testing
The obtained detection of the detection to hepatitis B surface antigen is limited to 2.1ng/mL, the identical coated surface amino groups silica shell of mother bulb
Smooth magnetic microsphere its detection be limited to 4.5ng/mL.
Embodiment 5
The first step, to use particle diameter first for 1000nm magnetic silicon oxide microballoon be mother bulb, there is Dispersed precipitate inside mother bulb
70wt% nano ferriferrous oxide granule, magnetic microsphere matrices are silica, and surface is to be modified with amino-functional group.Take this
The magnetic microsphere 80mg of amino group is contained on surface, is distributed to equipped with 20mL ethanol solutions.Under stirring, by above-mentioned mother bulb
Suspension be added dropwise it is well prepared in advance be dispersed with surface amino groups, particle diameter suspends for the absolute ethyl alcohol of 30 nano silicon oxide microballoons
In solution (150mL).Added after being added dropwise to complete dissolved with 150 microgram NHS-PEG20- NHS ethanol solution, stirring are anti-
It should stay overnight, respectively be washed 3 times with deionized water, absolute ethyl alcohol under being helped in magnetic field, remove the unassembled oxygen to magnetic mother bulb surface
SiClx bulbec.It is standby in absolute ethyl alcohol that there is the assembling magnetic microsphere of regular topological structure to be stored on the surface most obtained at last.
Second step, take group shape microsphere surface amino that 2mg obtains in absolute ethyl alcohol system with 3 microgram NHS-PEG4-NHS
Activation 2 hours is carried out, then the assembling microballoon after activation is distributed in 400 microlitres of phosphate buffer solutions and (pH=7.6, contained
Have 0.05wt% polysorbas20), washing is three times.Add 200 microgram Streptavidins in above-mentioned assembling microballoon system again, 37 DEG C
Mix revolving reaction 2 hours, after washing 3 times with closed reagent (containing inert protein) under being helped in magnetic field, closed at 4 DEG C
Night, obtain to surface be connected with Streptavidin, can be with the Streptavidin magnetic of biotinylation biomolecule specific reaction
Assemble microballoon.This Streptavidin magnetic assembling microballoon is stored in 200 microlitres of phosphate buffer solutions and (pH=7.6, contained
0.05wt% polysorbas20,0.1%BSA) it is standby.
3rd step, using the chemiluminescence detection of hepatitis B surface antigen be used as assess Magnetic Microspheres-Carrier detection sensitivity side
Method.By biotinylation anti-hepatitis B surface antigen antibody and the hepatitis B surface antigen and horseradish peroxidase of various concentrations gradient
Another anti-hepatitis B surface antigen antibody of mark forms immune complex in 37 DEG C of incubation reactions 20 minutes.Then each
The assembling magnetic microsphere that the surface that 10 micrograms are obtained by upper step is connected with Streptavidin is added in concentration gradient sample, then 37
Incubation reaction 10min at DEG C, the immune complex of not assembled magnetic microsphere capture and excessive enzyme are washed under being helped in magnetic field
Labeling antibody, chemical luminous substrate is added in system, the chemiluminescence signal value tested under each concentration gradient.Then mark is drawn
Directrix curve, according to international standard EP-17A documentation requirements, calculate detection and be limited to 0.92ng/mL.And identical mother bulb is used, surface bag
Smooth magnetic microsphere by the amino oxygen SiClx shell of same thickness is carrier, using the reagent same with the 3rd step and passes through phase
Same detection process, its detection to hepatitis B surface antigen are limited to 2.7ng/mL.
Embodiment 6
The first step, to use particle diameter first for 1000nm magnetic silicon oxide microballoon be mother bulb, there is Dispersed precipitate inside mother bulb
70wt% nano ferriferrous oxide granule, magnetic microsphere matrices are silica, and surface is to be modified with amino-functional group.Take this
The magnetic microsphere 80mg of amino group is contained on surface, is distributed to equipped with 20mL MES (pH=5.0, the tween containing 0.05wt%
20) in cushioning liquid.Under stirring, by above-mentioned mother bulb suspension be added dropwise shift to an earlier date ready 150mL be dispersed with surface carboxylic
Base, particle diameter is in the MES aaerosol solutions of 100 nanometer polymer microballoons.The MES that 30mL EDC/NHS are added after being added dropwise to complete delays
Solution is rushed, making EDC/NHS, each concentration is 50mg/mL, stirring reaction 3 hours, deionized water, anhydrous second is used under being helped in magnetic field
Alcohol respectively washing 3 times, remove the unassembled nanometer polymer bulbec to magnetic mother bulb surface.The surface most obtained at last has regular
The assembling magnetic microsphere of topological structure, it is stored in standby in phosphate buffer solution.
Second step, the assembling microballoon that the surface for taking 2mg to obtain is carboxyl are distributed to phosphate and (pH=7.6, contained
0.05wt% polysorbas20) in cushioning liquid, activation 15min is carried out with 50mg/mL EDC/NHS in system, then will activation
Assembling microballoon afterwards is distributed in 400 microlitres of phosphate buffer solutions (pH=7.6, the polysorbas20 containing 0.05wt%), washing
Three times.200 microgram Streptavidins are added in above-mentioned assembling microballoon system again, 37 DEG C mix revolving reaction 2 hours, in magnetic field
After washing 3 times with closed reagent (containing inert protein) under helping, closed overnight at 4 DEG C, obtain to surface and be connected with strepto- parent
With element, microballoon can be assembled with the Streptavidin magnetic of biotinylation biomolecule specific reaction.By this Streptavidin
Magnetic assembling microballoon be stored in 200 microlitres of phosphate buffer solutions (pH=7.6, the polysorbas20 containing 0.05wt%,
It is 0.1wt%BSA) standby.
3rd step, using the chemiluminescence detection of hepatitis B surface antigen be used as assess Magnetic Microspheres-Carrier detection sensitivity side
Method.By biotinylation anti-hepatitis B surface antigen antibody and the hepatitis B surface antigen and horseradish peroxidase of various concentrations gradient
Another anti-hepatitis B surface antigen antibody of mark forms immune complex in 37 DEG C of incubation reactions 20 minutes.Then each
The assembling magnetic microsphere that the surface that 10 micrograms are obtained by upper step is connected with Streptavidin is added in concentration gradient sample, then 37
Incubation reaction 10min at DEG C, the immune complex of not assembled magnetic microsphere capture and excessive enzyme are washed under being helped in magnetic field
Labeling antibody, chemical luminous substrate is added in system, the chemiluminescence signal value tested under each concentration gradient.Then mark is drawn
Directrix curve, according to international standard EP-17A documentation requirements, calculate detection and be limited to 0.95ng/mL.And identical mother bulb is used, surface bag
Smooth magnetic microsphere by the carboxyl oxygen SiClx shell of same thickness is carrier, using the reagent same with the 3rd step and passes through phase
Same detection process, its detection to hepatitis B surface antigen are limited to 2.85ng/mL.
Embodiment 7
The first step, to use particle diameter first for 1000nm magnetic silicon oxide microballoon be mother bulb, there is Dispersed precipitate inside mother bulb
70wt% nano ferriferrous oxide granule, magnetic microsphere matrices are silica, and surface is to be modified with sulfydryl functional group.Take this
The magnetic microsphere 80mg of mercapto groups is contained on surface, is distributed to equipped with 20mL phosphate buffer solutions.Under stirring, by above-mentioned mother
The phosphorus for shifting to an earlier date that ready 150mL is dispersed with surface amino groups, particle diameter is 50 nano silicon oxide microballoons is added dropwise in ball suspension
In hydrochlorate cushioning liquid.Phosphate buffer solution (the pH=of the sulfo-SMCC dissolved with 50mg/mL is added after being added dropwise to complete
7.6), stirring reaction is overnight, is washed 3 times with deionized water, absolute ethyl alcohol under being helped in magnetic field, and removal is unassembled to arrive magnetic mother bulb
The silica bulbec on surface.The assembling magnetic microsphere that the surface most obtained at last has regular topological structure is stored in absolute ethyl alcohol
In it is standby.
Second step, take assembling magnetic microsphere surface amino groups that 2mg obtains in absolute ethyl alcohol system with 3 microgram NHS-
PEG4-NHS carries out activation 2 hours, and the assembling microballoon after activation then is distributed into (pH in 400 microlitres of phosphate buffer solutions
=7.6, the polysorbas20 containing 0.05wt%), washing is three times.200 microgram strepto-s parent is added in above-mentioned assembling microballoon system again
And element, 37 DEG C mix revolving reaction 2 hours, after washing 3 times with closed reagent (containing inert protein) under being helped in magnetic field, 4
At DEG C close overnight, obtain to surface be connected with Streptavidin, can be with the strepto- of biotinylation biomolecule specific reaction
Avidin magnetic assembles microballoon.This Streptavidin magnetic assembling microballoon is stored in (pH in 200 microlitres of phosphate buffer solutions
=7.6, the polysorbas20 containing 0.05wt%, 0.1wt%BSA) it is standby.
3rd step, using the chemiluminescence detection of hepatitis B surface antigen be used as assess Magnetic Microspheres-Carrier detection sensitivity side
Method.By biotinylation anti-hepatitis B surface antigen antibody and the hepatitis B surface antigen and horseradish peroxidase of various concentrations gradient
Another anti-hepatitis B surface antigen antibody of mark forms immune complex in 37 DEG C of incubation reactions 20 minutes.Then each
The assembling magnetic microsphere that the surface that 10 micrograms are obtained by upper step is connected with Streptavidin is added in concentration gradient sample, then 37
Incubation reaction 10min at DEG C, the immune complex of not assembled magnetic microsphere capture and excessive enzyme are washed under being helped in magnetic field
Labeling antibody, chemical luminous substrate is added in system, the chemiluminescence signal value tested under each concentration gradient.Then mark is drawn
Directrix curve, according to international standard EP-17A documentation requirements, calculate detection and be limited to 0.85ng/mL.And identical mother bulb is used, surface bag
Smooth magnetic microsphere by the amino oxygen SiClx shell of same thickness is carrier, using the reagent same with the 3rd step and passes through phase
Same detection process, its detection to hepatitis B surface antigen are limited to 2.5ng/mL.
Embodiment 8
The first step, to use particle diameter first for 1000nm magnetic silicon oxide microballoon be mother bulb, there is Dispersed precipitate inside mother bulb
70wt% nano ferriferrous oxide granule, magnetic microsphere matrices are silica, and surface is silicone hydroxyl functional group.Take this magnetic
Microballoon 80mg, it is distributed to equipped with 20mL tetrahydrofuran solutions.Under stirring, standard in advance has been added dropwise in above-mentioned mother bulb suspension
The tetrahydrofuran that the 150mL got ready is dispersed with surface amino groups, particle diameter is 150 nano silicon oxide microballoons is in suspension.After being added dropwise to complete
The tetrahydrofuran solution of the CDI dissolved with 50mg/mL is added, stirring reaction is overnight, with tetrahydrofuran, nothing under being helped in magnetic field
Water-ethanol washs 3 times respectively, removes the unassembled silica bulbec to magnetic mother bulb surface.The surface most obtained at last has rule
The assembling magnetic microsphere of whole topological structure is stored in standby in ethanol solution.
Second step, take group shape microsphere surface amino that 2mg obtains in absolute ethyl alcohol system with 3 microgram NHS-PEG4-NHS
Activation 2 hours is carried out, then the assembling microballoon after activation is distributed in 400 microlitres of phosphate buffer solutions and (pH=7.6, contained
Have 0.05wt% polysorbas20), washing is three times.Add 200 microgram Streptavidins in above-mentioned assembling microballoon system again, 37 DEG C
Mix revolving reaction 2 hours, after washing 3 times with closed reagent under being helped in magnetic field, closed overnight at 4 DEG C, obtain surface company
Be connected to Streptavidin, microballoon can be assembled with the Streptavidin magnetic of biotinylation biomolecule specific reaction.By this
Streptavidin magnetic assembling microballoon is stored in 200 microlitres of phosphate buffer solutions (pH=7.6, telling containing 0.05wt%
Temperature 20,0.1wt%BSA) it is standby.
3rd step, using the chemiluminescence detection of hepatitis B surface antigen be used as assess Magnetic Microspheres-Carrier detection sensitivity side
Method.By biotinylation anti-hepatitis B surface antigen antibody and the hepatitis B surface antigen and horseradish peroxidase of various concentrations gradient
Another anti-hepatitis B surface antigen antibody of mark forms immune complex in 37 DEG C of incubation reactions 20 minutes.Then each
The assembling magnetic microsphere that the surface that 10 micrograms are obtained by upper step is connected with Streptavidin is added in concentration gradient sample, then 37
Incubation reaction 10min at DEG C, the immune complex of not assembled magnetic microsphere capture and excessive enzyme are washed under being helped in magnetic field
Labeling antibody, chemical luminous substrate is added in system, the chemiluminescence signal value tested under each concentration gradient.Then mark is drawn
Directrix curve, according to international standard EP-17A documentation requirements, calculate detection and be limited to 0.93ng/mL.And identical mother bulb is used, surface bag
Smooth magnetic microsphere by the amino oxygen SiClx shell of same thickness is carrier, using the reagent same with the 3rd step and passes through phase
Same detection process, its detection to hepatitis B surface antigen are limited to 2.85ng/mL.
Preferred embodiment of the invention described in detail above.It should be appreciated that the ordinary skill of this area is without wound
The property made work can makes many modifications and variations according to the design of the present invention.Therefore, all technician in the art
Pass through the available technology of logical analysis, reasoning, or a limited experiment on the basis of existing technology under this invention's idea
Scheme, all should be in the protection domain being defined in the patent claims.
Claims (18)
1. a kind of carrier for in-vitro diagnosis detection, it is characterised in that the carrier is the assembling with regular topological structure
Magnetic microsphere, including the mother bulb of micron or submicron-scale and the nanometer that the mother bulb surface is connected to by chemical covalent bonds
The bulbec of yardstick.
2. carrier as claimed in claim 1, it is characterised in that the mother bulb is the spherical structure that particle diameter is 500nm~20 μm.
3. carrier as claimed in claim 1, it is characterised in that the bulbec is the spherical structure that particle diameter is 30nm~200nm.
4. carrier as claimed in claim 1, it is characterised in that the material of the mother bulb and the bulbec is polymer or oxidation
Silicon.
5. carrier as claimed in claim 4, it is characterised in that the polymer is polystyrene, polystyrene and poly- (first
Base) acrylic copolymer.
6. carrier as claimed in claim 1, it is characterised in that the mother bulb and the functional base of the surface grafting of the bulbec
Group, the chemical covalent reaction realization between connecting through the functional group between the mother bulb and the bulbec.
7. carrier as claimed in claim 6, it is characterised in that the functional group is in amino, carboxyl, sulfydryl, hydroxyl
It is a kind of.
8. the carrier as described in claim 1-7 is any, it is characterised in that be additionally included in the mother bulb and/or the bulbec
Magnetic nanoparticle.
9. carrier as claimed in claim 8, it is characterised in that the magnetic nanoparticle is ferroferric oxide nano granules.
10. the preparation method of the carrier for being used for in-vitro diagnosis detection as any one of claim 1-8, its feature exist
In：Pass through bulbec described in chemical covalent key connection on the mother bulb surface.
11. preparation method as claimed in claim 10, it is characterised in that comprise the following steps：
Step 1, respectively in the mother bulb and the bulbec surface grafting functional group；
Step 2, the mother bulb for being grafted with the functional group is activated after with being grafted with the bulbec of the functional group
Mixing, the mother bulb and the bulbec realize connection by chemical covalent reaction between the functional group.
12. preparation method as claimed in claim 11, it is characterised in that the functional group is amino, carboxyl, sulfydryl, hydroxyl
One kind in base.
13. preparation method as claimed in claim 10, it is characterised in that the mother bulb is the ball that particle diameter is 500nm~20 μm
Shape structure.
14. preparation method as claimed in claim 10, it is characterised in that the bulbec is the ball that particle diameter is 30nm~200nm
Shape structure.
15. preparation method as claimed in claim 10, it is characterised in that the material of the mother bulb and the bulbec is polymer
Or silica.
16. preparation method as claimed in claim 15, it is characterised in that the polymer be polystyrene, polystyrene with
Poly- (methyl) acrylic copolymer.
17. the preparation method as described in claim 10-16 is any, it is characterised in that wrapped in the mother bulb and/or the bulbec
Containing magnetic nanoparticle.
18. preparation method as claimed in claim 17, it is characterised in that the magnetic nanoparticle is ferriferrous oxide nano
Particle.
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| CN104634915B (en) * | 2013-11-08 | 2017-03-29 | 中国科学院大连化学物理研究所 | A kind of granule of oligonucleotide library modification and its preparation and application |
| EP3115384B1 (en) * | 2014-03-05 | 2020-07-29 | JSR Corporation | Solid support, ligand-bound solid support, detection or separation method for target substance, solid support production method, and ligand-bound solid support production method |
| 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 |
| CN104069809B (en) * | 2014-06-25 | 2016-02-24 | 广西师范大学 | Fe 3o 4the preparation method of/GO magnetic composite |
| CN104610912B (en) * | 2015-02-10 | 2016-06-08 | 武汉大学 | Degradable magnetic Nano material and nanometer bio probe and preparation method thereof |
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| CN107703307B (en) * | 2017-09-30 | 2020-04-14 | 安徽伊普诺康生物技术股份有限公司 | IV-type collagen detection kit and use method thereof |
| CN107741402A (en) * | 2017-09-30 | 2018-02-27 | 安徽伊普诺康生物技术股份有限公司 | A kind of CER detection kit and its application method |
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| CN116559153A (en) * | 2018-08-13 | 2023-08-08 | 科美博阳诊断技术(上海)有限公司 | Homogeneous chemiluminescence detection kit and application thereof |
| CN116626021A (en) * | 2018-08-13 | 2023-08-22 | 上海索昕生物科技有限公司 | Microsphere composition for chemiluminescence analysis and application thereof |
| CN110201613B (en) * | 2019-06-10 | 2021-09-14 | 东莞东阳光科研发有限公司 | Polystyrene magnetic microsphere and preparation method thereof |
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| CN113062126A (en) * | 2021-04-01 | 2021-07-02 | 昆山阿基里斯新材料科技有限公司 | Artificial leather with low impedance on surface and preparation method thereof |
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