CN110187099A - A kind of super paramagnetic microsphere and its preparation and application - Google Patents
A kind of super paramagnetic microsphere and its preparation and application Download PDFInfo
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- CN110187099A CN110187099A CN201910429936.9A CN201910429936A CN110187099A CN 110187099 A CN110187099 A CN 110187099A CN 201910429936 A CN201910429936 A CN 201910429936A CN 110187099 A CN110187099 A CN 110187099A
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
- G01N21/76—Chemiluminescence; Bioluminescence
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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
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
- G01N33/54326—Magnetic particles
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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
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
- G01N33/54346—Nanoparticles
Abstract
The invention belongs to magnetic bionanoparticles application and technical field of medical examination, more particularly to a kind of super paramagnetic microsphere and its preparation and application, the super paramagnetic microsphere, including polymer drops and the bulbec being embedded in inside the polymer drops, the bulbec includes the aggregation of the superparamagnetic nanoparticle of partial size 5-30nm and the protective layer for being coated on the aggregation volume surrounding, the bulbec partial size is 200-500nm, the super paramagnetic microsphere partial size is 2-20 μm, the superparamagnetic nano particle diameter is uniform, form aggregation bulbec, it embeds into polymer drops, superparamagnetism can be good, the super paramagnetic microsphere regular shape, good dispersion, the bulbec surface has parents' protective layer, both it is easy to wrap up superparamagnetic nanoparticle, there is compatibility well with polymer drops again, maintain the stabilization of super paramagnetic microsphere Property.
Description
Technical field
The invention belongs to magnetic bionanoparticles application and technical field of medical examination, in particular to a kind of super paramagnetic microsphere and
It is prepared and application.
Background technique
Chemiluminescence immunoassay (CLIA) belongs to one kind of labelled antibody technology, it is with chemiluminescent agent, catalytic luminescence
Enzyme or product participate in the labelled antibodies such as the substance of luminescence-producing reaction, antigen or other molecules with specific recognition function indirectly,
After specific recognition combines, luminous substrate is by luminous agent, catalyzing enzyme or participates in product effect, chemically reacts, in reaction
It discharges visible light or reaction excitation fluorescent material shines, finally detected with luminometer, chemiluminescence immunoassay is surveyed
Determine technology have specificity is high, sensibility is high, separation is easy, quickly, reagent is nontoxic and the advantage of safety and stability, in section now
In skill development process, with the development of fully automatic technique, artificial intelligence and big data technology, chemiluminescence immunoassay can oneself
Dynamicization prospect has attracted many concerns.
Chemiluminescence immunoassay technology is all utilized in flow cytometry and liquid-phase chip detection method, in recent years, there is
Person utilizes fluorescent microsphere, in conjunction with flow cytometry, collect laser technology, photoelectric measurement technology, computer technology, hydrodynamics and
Cellular immunofluorescence chemical technology, monoclonal antibody technique are in one, so that fluorescent microsphere flows in liquid stream, pass through inspection one by one
Area is surveyed, and records it with high sensitivity detector and scatters light and various fluorescence signals, to carry out multi parameter analysis to microballoon.
Wherein the common raw material of microballoon is polymer, in actual use, in order to avoid in sample background component to detection
As a result influence is needed to separate the fluorescent microsphere for having occurred and that specific binding with sample background ingredient, therefore is taken
Measure usually have: 1) handle sample, isolate in sample detection and be worth low ingredient；2) fluorescent microsphere is separated, is carried out complicated
Elution program, the operating process that the consequence of generation is often is more complicated, and testing result sensitivity is lower.In order to solve this
Problem replaces polymer microballoon using magnetic microsphere, under the action of external magnetic field, can be realized magnetic microsphere and complex sample
It separates and is enriched with while carrying out, simplify operating process, increase detection sensitivity.
Existing Chinese patent literature CN108948557A discloses a kind of preparation method of fluorescent magnetic microspheres, passes through selection
Suitable solvent and initiator, the mediator warming temperature for controlling reaction achieve the effect that uniform particle diameter and partial size are wide in range and controllable,
The microballoon of preparation can be used for marking, tracer and detection still in actual application, need to apply stronger magnetic field ability
It realizes separation and magnetic microsphere is avoided to assemble, and magnetic microsphere prepares reaction condition complexity, at high cost, shape size is difficult
With control, it is difficult to realize industrialized production.
Summary of the invention
Therefore, the technical problem to be solved in the present invention is that the prior art is easy hair for chemiluminescence detection fluorescent microsphere
Raw aggregation, the uncontrollable technical problem of form size, and then a kind of super paramagnetic microsphere and its preparation and application are provided, it is described super
Paramagnetic microsphere is not susceptible to assemble, regular shape, uniform particle diameter.
The invention discloses a kind of super paramagnetic microspheres, including polymer drops and the son being embedded in inside the polymer drops
Ball, the bulbec include the aggregation of the superparamagnetic nanoparticle of partial size 5-30nm and the protection that is coated on outside the aggregation
Layer, the bulbec partial size are 200-500nm, and the super paramagnetic microsphere partial size is 2-20 μm.
Preferably, the superparamagnetic nanoparticle is Fe3O4Nanoparticle, partial size 10-20nm.
Preferably, the monomer of the material of the polymer drops is ethoxylated trimethylolpropane triacrylate.
Preferably, the protective layer is parents' molecular layer, and parents' molecule includes stearyl phosphatidyl ethanol amine-poly- second
Glycol 2000- amino and phosphatidyl-ethanolamine-polyethylene glycol 2000-maleimide.
Preferably, the super paramagnetic microsphere is by 3- aminopropyl triethoxysilane and butanedioic anhydride modification.
The invention also discloses a kind of methods for preparing the super paramagnetic microsphere, comprising the following steps:
Prepare superparamagnetic nanoparticle: hydro-thermal method prepares Fe3O4Nanoparticle is simultaneously modified；
Preparation bulbec: protective layer is coated outside superparamagnetic nanometer particle congery, and is freeze-dried crushing；
It prepares super paramagnetic microsphere: super paramagnetic microsphere is made in the bulbec and polymer.
Preferably, super paramagnetic microsphere is prepared by microballoon generating means.
The invention also discloses a kind of fluorescence-encoded micro-beads, including the super paramagnetic microsphere or the super paramagnetic microsphere
The super paramagnetic microsphere of preparation method preparation.
It preferably, further include fluorescence signal molecule and biological identification molecule I.
Preferably, the fluorescence signal molecule is selected from the different fluorescent molecule of center emission wavelength.
Preferably, the biological identification molecule I is selected from antigen molecule, antibody molecule or nucleic acid molecules.
The invention also discloses a kind of fluorescence detection products, including the fluorescence-encoded micro-beads and contain fluorescent reporter group
Solution；The fluorescent reporter group is the biological identification molecule II for being marked with fluorescent reporter molecule, the fluorescent reporter molecule
With center emission wavelength difference >=30nm of fluorescence signal molecule.
Preferably, the biological identification molecule I and biological identification molecule II can specific recognition target molecules.
The invention also discloses superparamagnetics prepared by a kind of super paramagnetic microsphere or the super paramagnetic microsphere preparation method
The application of microballoon, the fluorescence-encoded micro-beads or the fluorescence detection product in fluorescence analysis field.
The invention also discloses superparamagnetics prepared by a kind of super paramagnetic microsphere or the super paramagnetic microsphere preparation method
The application of microballoon, the fluorescence-encoded micro-beads or the fluorescence detection product in flow cytometry field.
Technical solution of the present invention has the advantages that
1. super paramagnetic microsphere of the present invention, including polymer drops and the bulbec being embedded in inside the polymer drops, institute
The superparamagnetic nanometer particle congery and the protective layer being coated on outside the aggregation that bulbec includes partial size 5-30nm are stated, it is described
Bulbec partial size is 200-500nm, and the super paramagnetic microsphere partial size is 2-20 μm；Superparamagnetic nanoparticle of the partial size between 5-30nm
Son does not apply and does not assemble because itself is magnetic under external magnetic field state, while having magnetism after applying external magnetic field, wraps up through PEI,
With good dispersibility, coated after through parents' molecule, can form the aggregation of superparamagnetic nanoparticle, formation it is described
It is coated with parents' protective layer outside aggregation, embeds into polymer drops, paramagnetic performance is good, the polymer microballoon shape rule of formation
Then, good dispersion.
2. the bulbec surface embedded in super paramagnetic microsphere of the present invention has parents' protective layer, both it is easy to wrap up superparamagnetic
Nanoparticle, and there is compatibility well with polymer, maintain the stability of super paramagnetic microsphere.
3. super paramagnetic microsphere preparation method of the present invention is simple, reaction process is easily controllable.
4. superparamagnetic coding microball of the present invention is equipped with signaling molecule of the fluorescence signal molecule as separation analysis, lead to
The method for crossing fluorescence analysis is used for flow cytometer, and in conjunction with magnetic separation technique, detection sensitivity is high, and accuracy is good, operation letter
It is single, there is excellent practicability, it is easy to accomplish automatic detection.
5. detection kit of the present invention, including the superparamagnetic fluorescence-encoded micro-beads and fluorescent reporter group, can
Suitable biological identification molecule is connected according to the target molecule of required detection, practicability is good, and easy to use, detection is quick.
Specific embodiment
There is provided following embodiments is to preferably further understand the present invention, it is not limited to the best embodiment party
Formula is not construed as limiting the contents of the present invention and protection scope, anyone under the inspiration of the present invention or by the present invention and its
The feature of his prior art is combined and any and identical or similar product of the present invention for obtaining, all falls within of the invention
Within protection scope.
Specific experiment step or condition person are not specified in embodiment, according to the literature in the art described routine experiment
The operation of step or condition can carry out.Reagents or instruments used without specified manufacturer, being can be by commercially available acquisition
Conventional products.
Embodiment 1
Prepare superparamagnetic nanoparticle: by 2.53g FeCl2With 1.62g FeCl3It is dissolved in the distilled water of 250mL, 55
DEG C heating, 1000rpm is stirred under nitrogen protection, and concentrated ammonia liquor is added and is adjusted to pH=9, reacts 2h, is centrifuged, spends after reaction
Ionized water and ethyl alcohol distinguish centrifuge washing, and Fe is made in 80 DEG C of freeze-day with constant temperature3O4Nanoparticle powder takes and is scattered in ethyl alcohol on a small quantity
In, with the particle diameter distribution of Dynamic laser scattering instrument measurement microballoon；It is then dissolved in 100mL water, 40kHz ultrasound 15min, is added
PEI 140mg, 100rpm stirring, removes supernatant, successively uses distilled water and methanol centrifuge washing, be dried in vacuo after centrifugation to obtain the final product
The superparamagnetic nanoparticle of PEI package.
Prepare bulbec: the superparamagnetic nanoparticle that above-mentioned PEI is wrapped up, which is added in 5mL tetrahydrofuran, to be dispersed, then
With 10g distearoylphosphatidylethanolamine-polyethylene glycol 2000- amino and 10g phosphatidyl-ethanolamine-polyethylene glycol 2000-horse
Carry out acid imide to mix, 40kHz ultrasound 10min at 25 DEG C, is added dropwise in 100mL deionized water, will obtain under ultrasound condition
Mixed liquor be placed in the bag filter that molecular weight is 8000~14000D, it is primary to change within every 6 hours water, collects after dialysis 24 hours saturating
The micellar aqueous solution formed in analysis bag is freeze-dried 20h to get bulbec powder；It takes and is scattered in ethyl alcohol on a small quantity, use dynamic laser
The particle diameter distribution of light scattering apparatus measurement bulbec.
It prepares super paramagnetic microsphere: the above-mentioned bulbec powder of 2g being taken to be scattered in three acrylic acid of 20g ethoxylated trimethylolpropane
In ester (ETPTA), ultrasonic disperse is uniform, then by the syringe pumps of microballoon generating means, be injected into mass fraction be 4% it is poly-
In vinyl alcohol aqueous solution, the ETPTA solution droplets containing sub-ball are generated, through ultra-violet curing up to the super paramagnetic microsphere；It takes few
Amount is scattered in ethyl alcohol, with the particle diameter distribution of dynamic laser light scattering instrument measurement super paramagnetic microsphere.
It prepares fluorescence-encoded micro-beads: taking the super paramagnetic microsphere 5g, be scattered in 50mL n-hexyl alcohol, 300rpm stirring is lower to be added
Enter and is scattered in 50mL n-hexyl alcohol after dialysis washing after 0.5g 3- aminopropyl triethoxysilane and 0.3g butanedioic anhydride modification；
By 10mg fluorescein isothiocynate (FITC) (center emission wavelength 518nm) and (center 10mg rhodamine isothiocyanate (RBITC)
Launch wavelength 590nm) it is dissolved in 10ml n-hexyl alcohol as fluorescence signal molecule, it is mixed with the n-hexyl alcohol solution of the super paramagnetic microsphere
It closes, in shaking bed reaction 4h, is packed into bag filter, removes unreacted fluorescence signal molecule, obtain fluorescence super paramagnetic microsphere；It will
The anti-human AFP AFP monoclonal antibody of 200mg mouse is coupled as biological identification molecule I and the fluorescence super paramagnetic microsphere,
It is mixed in phosphate buffer (0.1M, pH=6), 37 DEG C are protected from light lower progress coupling reaction 4h, and centrifugation is removed supernatant, is scattered in
To get the fluorescence-encoded micro-beads in phosphate buffer (0.1M, pH=6).
Fluorescence detection reagent kit: including the fluorescence-encoded micro-beads and containing the solution of fluorescent reporter group；The fluorescence
Reporter group is the biological identification molecule II that mark fluorescent reports molecule, in the fluorescent reporter molecule and fluorescence signal molecule
Heart launch wavelength difference >=30nm；By 120 μ L CdSe/ZnS quantum dots (center emission wavelength 556nm, is dissolved in n-hexane, 30
μM) be marked in the anti-human AFP AFP monoclonal antibody of 100mg mouse to obtain the final product.
Embodiment 2
Prepare superparamagnetic nanoparticle: by 2.53g FeCl2With 1.62g FeCl3It is dissolved in the distilled water of 250mL, 75
DEG C heating, 1500rpm is stirred under nitrogen protection, and concentrated ammonia liquor is added and is adjusted to pH=9, reacts 2h, is centrifuged, spends after reaction
Ionized water and ethyl alcohol distinguish centrifuge washing, and Fe is made in 80 DEG C of freeze-day with constant temperature3O4Nanoparticle powder takes and is scattered in ethyl alcohol on a small quantity
In, with the particle diameter distribution of Dynamic laser scattering instrument measurement microballoon；It is then dissolved in 100mL water, 40kHz ultrasound 15min, is added
PEI 80mg, 300rpm stirring, removes supernatant, successively uses distilled water and methanol centrifuge washing, be dried in vacuo after centrifugation to obtain the final product
The superparamagnetic nanoparticle of PEI package.
Preparation bulbec: will PEI package the superparamagnetic nanoparticle be added 5mL tetrahydrofuran in disperse, then with 15g
Distearoylphosphatidylethanolamine-polyethylene glycol 2000- amino and 15g phosphatidyl-ethanolamine-polyethylene glycol 2000-maleimide
Amine mixes, 40kHz ultrasound 10min at 25 DEG C, is added dropwise in 100mL deionized water under ultrasound condition, obtains mixed liquor, will mix
It is primary in the bag filter of 8000~14000D, to change within every 6 hours water that conjunction liquid is placed in molecular weight, and dialysis obtained micella water after 48 hours
Solution is freeze-dried 20h to get bulbec powder；It takes and is scattered in ethyl alcohol on a small quantity, with dynamic laser light scattering instrument measurement bulbec
Particle diameter distribution.
It prepares super paramagnetic microsphere: dispersing 20g ethoxylated trimethylolpropane triacrylate for 5g bulbec powder
(ETPTA) in, ultrasonic disperse is uniform, then passes through the syringe pump of microballoon generating means, is injected into the poly- second that mass fraction is 3%
In enol aqueous solution, the ETPTA solution droplets containing sub-ball are generated, through ultra-violet curing up to the super paramagnetic microsphere；It takes a small amount of
It is scattered in ethyl alcohol, with the particle diameter distribution of dynamic laser light scattering instrument measurement super paramagnetic microsphere.
It prepares fluorescence-encoded micro-beads: taking each 5g of the super paramagnetic microsphere, be scattered in 50mL n-hexyl alcohol, under 300rpm stirring
50mL n-hexyl alcohol is scattered in after dialysis washing after addition 0.8g 3- aminopropyl triethoxysilane and 0.5g butanedioic anhydride modification
In；By 20mg fluorescein isothiocynate (FITC) (center emission wavelength 518nm) and 30mg rhodamine isothiocyanate (RBITC)
(center emission wavelength 590nm) is dissolved in 10ml n-hexyl alcohol, is mixed with the n-hexyl alcohol solution of the super paramagnetic microsphere, on shaking table
3h is reacted, bag filter is packed into, removes unreacted fluorescence signal molecule, obtain fluorescence super paramagnetic microsphere；By the anti-human first of 200mg mouse
Fetoprotein AFP monoclonal antibody is coupled as biological identification molecule I and the fluorescence super paramagnetic microsphere, in phosphate buffer
It is mixed in (0.1M, pH=6), 37 DEG C are protected from light lower progress coupling reaction 3h, and centrifugation removes supernatant, is scattered in phosphate buffer
To get the fluorescence-encoded micro-beads in (0.1M, pH=6).
Fluorescence detection reagent kit: including the fluorescence-encoded micro-beads and containing the solution of fluorescent reporter group；The fluorescence
Reporter group is the biological identification molecule II for being marked with fluorescent reporter molecule, the fluorescent reporter molecule and fluorescence signal molecule
Center emission wavelength difference >=30nm；By 200 μ L CdSe/ZnS quantum dots (center emission wavelength 556nm, is dissolved in n-hexane,
30 μM) it is marked in the anti-human AFP AFP monoclonal antibody of 100mg mouse i.e..
Embodiment 3
Prepare superparamagnetic nanoparticle: by 2.53g FeCl2With 1.62g FeCl3It is dissolved in the distilled water of 250mL, 60
DEG C heating, 1000rpm is stirred under nitrogen protection, and concentrated ammonia liquor is added and is adjusted to pH=9, reacts 2h, is centrifuged, spends after reaction
Ionized water and ethyl alcohol distinguish centrifuge washing, and Fe is made in 80 DEG C of freeze-day with constant temperature3O4Nanoparticle powder takes and is scattered in ethyl alcohol on a small quantity
In, with the particle diameter distribution of dynamic laser light scattering instrument measurement microballoon；It is then dissolved in 100mL water, 40kHz ultrasound 15min, is added
PEI 200mg, 200rpm stirring, removes supernatant, successively uses distilled water and methanol centrifuge washing, be dried in vacuo after centrifugation to obtain the final product
The superparamagnetic nanoparticle of PEI package.
Preparation bulbec: will PEI package the superparamagnetic nanoparticle be added 5mL tetrahydrofuran in disperse, then with 20g
Distearoylphosphatidylethanolamine-polyethylene glycol 2000- amino and 20g phosphatidyl-ethanolamine-polyethylene glycol 2000-maleimide
Amine mixes, 40kHz ultrasound 10min at 25 DEG C, is added dropwise in 100mL deionized water under ultrasound condition, obtains mixed liquor, will mix
It is primary in the bag filter of 8000~14000D, to change within every 6 hours water that conjunction liquid is placed in molecular weight, and dialysis obtained micella water after 36 hours
Solution is freeze-dried 20h to get bulbec powder；It takes a small amount of bulbec powder ultrasonic to be scattered in ethyl alcohol, uses dynamic laser light scattering
The particle diameter distribution of instrument measurement bulbec.
It prepares super paramagnetic microsphere: dispersing 20g ethoxylated trimethylolpropane triacrylate for 3g bulbec powder
(ETPTA) in, ultrasonic disperse is uniform, then passes through the syringe pump of microballoon generating means, is injected into the poly- second that mass fraction is 4%
In enol aqueous solution, the ETPTA solution droplets containing sub-ball are generated, through ultra-violet curing up to the super paramagnetic microsphere；It takes a small amount of
It is scattered in ethyl alcohol, with the particle diameter distribution of dynamic laser light scattering instrument measurement super paramagnetic microsphere.
It prepares fluorescence-encoded micro-beads: taking each 5g of the super paramagnetic microsphere, be scattered in 50mL n-hexyl alcohol, under 300rpm stirring
Dialysis is washed after 0.3g 3- aminopropyl triethoxysilane and 0.3g butanedioic anhydride modification is added, and is scattered in 50mL n-hexyl alcohol；
By through 120 modified μ LCdSe/ZnS quantum dots of TEOS and 3- aminopropyl triethoxysilane (center emission wavelength 556nm, it is molten
In n-hexane, 30 μM) and 120 μ LCdSe/ZnS quantum dots (center emission wavelength 680nm, is dissolved in n-hexane, 30 μM)
It is dissolved in 10ml n-hexyl alcohol, is mixed with the n-hexyl alcohol solution of the super paramagnetic microsphere, shake bed reaction 4h, be packed into bag filter, remove
Unreacted fluorescence signal molecule is removed, fluorescence super paramagnetic microsphere is obtained；By the anti-human AFP AFP monoclonal antibody of 200mg mouse
It is mixed in phosphate buffer (0.1M, pH=6) as biological identification molecule I and the fluorescence super paramagnetic microsphere, 37 DEG C are kept away
Coupling reaction 4h is carried out under light, centrifugation is removed supernatant, is scattered in phosphate buffer (0.1M, pH=6) to get described glimmering
Pumped FIR laser microballoon.
Fluorescence detection reagent kit: including the fluorescence-encoded micro-beads and containing the solution of fluorescent reporter group；The fluorescence
Reporter group is the biological identification molecule II for being marked with fluorescent reporter molecule, the fluorescent reporter molecule and fluorescence signal molecule
Center emission wavelength difference >=30nm；By the 120 μ L CdSe/ZnS quantum modified through TEOS and 3- aminopropyl triethoxysilane
Point (center emission wavelength 630nm, is dissolved in n-hexane, 30 μM) is marked on the anti-human AFP AFP monoclonal antibody of 100mg mouse
On to obtain the final product.
Embodiment 4
Prepare superparamagnetic nanoparticle: by 2.53g FeCl2With 1.62g FeCl3It is dissolved in the distilled water of 250mL, 75
DEG C heating, 1200rpm is stirred under nitrogen protection, and concentrated ammonia liquor is added and is adjusted to pH=9, reacts 2h, is centrifuged, spends after reaction
Ionized water and ethyl alcohol distinguish centrifuge washing, and Fe is made in 80 DEG C of freeze-day with constant temperature3O4Nanoparticle powder takes and is scattered in ethyl alcohol on a small quantity
In, with the particle diameter distribution of dynamic laser light scattering instrument measurement microballoon；It is then dissolved in 100mL water, 40kHz ultrasound 15min, is added
PEI 140mg, 100rpm stirring, removes supernatant, first use distilled water and methanol centrifuge washing, vacuum drying are after centrifugation
?.
Preparation bulbec: the superparamagnetic nanoparticle is added in 5mL tetrahydrofuran and is dispersed, then with 25g distearyl
Phosphatidyl-ethanolamine-polyethylene glycol 2000-amino and the mixing of 25g phosphatidyl-ethanolamine-polyethylene glycol 2000-maleimide,
40kHz ultrasound 10min at 25 DEG C is added dropwise in 100mL deionized water under ultrasound condition, obtains mixed liquor, mixed liquor is set
Primary in the bag filter of 8000~14000D, to change within every 6 hours water in molecular weight, dialysis obtained micellar aqueous solution after 24 hours,
20h is freeze-dried to get bulbec powder；It takes and is scattered in ethyl alcohol on a small quantity, with the partial size point of Dynamic laser scattering instrument measurement bulbec
Cloth.
It prepares super paramagnetic microsphere: dispersing 20g ethoxylated trimethylolpropane triacrylate for 2g bulbec powder
(ETPTA) in, ultrasonic disperse is uniform, then passes through the syringe pump of microballoon generating means, is injected into the poly- second that mass fraction is 3%
In enol aqueous solution, the ETPTA solution droplets containing sub-ball are generated, through ultra-violet curing up to the super paramagnetic microsphere；It takes a small amount of
It is scattered in ethyl alcohol, with the particle diameter distribution of dynamic laser light scattering instrument measurement super paramagnetic microsphere.
It prepares fluorescence-encoded micro-beads: taking each 5g of the super paramagnetic microsphere, be scattered in 50mL n-hexyl alcohol, under 300rpm stirring
Dialysis is washed after 0.5g 3- aminopropyl triethoxysilane and 0.3g butanedioic anhydride modification is added, and is scattered in 50mL n-hexyl alcohol；
By through 150 modified μ L CdSe/ZnS quantum dots of TEOS and 3- aminopropyl triethoxysilane (center emission wavelength 580nm, it is molten
In n-hexane, 30 μM) and 200 μ L CdSe/ZnS quantum dots (center emission wavelength 630nm, is dissolved in n-hexane, 30 μM)
It is dissolved in 10ml n-hexyl alcohol, is mixed with the n-hexyl alcohol solution of the super paramagnetic microsphere, in shaking bed reaction 4h, be packed into bag filter,
Unreacted fluorescence signal molecule is removed, fluorescence super paramagnetic microsphere is obtained；The anti-human AFP AFP monoclonal of 200mg mouse is resisted
Body mixes in phosphate buffer (0.1M, pH=6) as biological identification molecule I, with the fluorescence super paramagnetic microsphere, and 37 DEG C
It is protected from light lower progress coupling reaction 4h, is centrifuged, is removed supernatant, be scattered in phosphate buffer (0.1M, pH=6) to get described
Fluorescence-encoded micro-beads.
Fluorescence detection reagent kit: including the fluorescence-encoded micro-beads and containing the solution of fluorescent reporter group；The fluorescence
Reporter group is the biological identification molecule II for being marked with fluorescent reporter molecule, the fluorescent reporter molecule and fluorescence signal molecule
Center emission wavelength difference >=30nm；By the 300 μ L CdSe/ZnS quantum modified through TEOS and 3- aminopropyl triethoxysilane
The anti-human AFP AFP monoclonal of 100mg mouse that point (center emission wavelength 680nm, is dissolved in n-hexane, 30 μM) is marked on is anti-
On body to obtain the final product.
Embodiment 5
Prepare superparamagnetic nanoparticle: by 2.53g FeCl2With 1.62g FeCl3It is dissolved in the distilled water of 250mL, 65
DEG C heating, 1400rpm is stirred under nitrogen protection, and concentrated ammonia liquor is added and is adjusted to pH=9, reacts 2h, is centrifuged, spends after reaction
Ionized water and ethyl alcohol distinguish centrifuge washing, and Fe is made in 80 DEG C of freeze-day with constant temperature3O4Nanoparticle powder takes and is scattered in ethyl alcohol on a small quantity
In, with the particle diameter distribution of dynamic laser light scattering instrument measurement microballoon；It is then dissolved in 100mL water, 40kHz ultrasound 15min, is added
PEI 250mg, 100rpm stirring, removes supernatant, successively uses distilled water and methanol centrifuge washing, be dried in vacuo after centrifugation to obtain the final product
The superparamagnetic nanoparticle of PEI package.
Preparation bulbec: will PEI package the superparamagnetic nanoparticle be added 5mL tetrahydrofuran in disperse, then with 30g
Distearoylphosphatidylethanolamine-polyethylene glycol 2000- amino and 30g phosphatidyl-ethanolamine-polyethylene glycol 2000-maleimide
Amine mixes, 40kHz ultrasound 10min at 25 DEG C, is added dropwise in 100mL deionized water under ultrasound condition, obtains mixed liquor, will mix
It is primary in the bag filter of 8000~14000D, to change within every 6 hours water that conjunction liquid is placed in molecular weight, and dialysis obtained micella water after 24 hours
Solution is freeze-dried 20h to get bulbec powder；It takes and is scattered in ethyl alcohol on a small quantity, with dynamic laser light scattering instrument measurement bulbec
Particle diameter distribution.
It prepares super paramagnetic microsphere: dispersing 15g ethoxylated trimethylolpropane triacrylate for 4g bulbec powder
(ETPTA) in, ultrasonic disperse is uniform, then passes through the syringe pump of microballoon generating means, is injected into the poly- second that mass fraction is 5%
In enol aqueous solution, the ETPTA solution droplets containing sub-ball are generated, through ultra-violet curing up to the super paramagnetic microsphere；It takes a small amount of
It is scattered in ethyl alcohol, with the particle diameter distribution of dynamic laser light scattering instrument measurement super paramagnetic microsphere.
It prepares fluorescence-encoded micro-beads: taking each 3g of the super paramagnetic microsphere, be scattered in 50mL n-hexyl alcohol, under 300rpm stirring
50mL n-hexyl alcohol is scattered in after dialysis washing after addition 0.1g 3- aminopropyl triethoxysilane and 0.1g butanedioic anhydride modification
In；By the 120 μ L CdSe/ZnS quantum dot (center emission wavelengths modified through TEOS and 3- aminopropyl triethoxysilane
580nm is dissolved in n-hexane, 30 μM) and 120 μ L CdSe/ZnS quantum dots (center emission wavelength 630nm, is dissolved in n-hexane
In, 30 μM) it is dissolved in 10ml n-hexyl alcohol, it mixes with the n-hexyl alcohol solution of the super paramagnetic microsphere, in shaking bed reaction 4h, is packed into
Bag filter removes unreacted fluorescence signal molecule, obtains fluorescence super paramagnetic microsphere；By the anti-human AFP AFP list of 200mg mouse
Clonal antibody mixes in phosphate buffer (0.1M, pH=6) as biological identification molecule I and the fluorescence super paramagnetic microsphere
Even, 37 DEG C are protected from light lower progress coupling reaction 4h, and centrifugation is removed supernatant, is scattered in phosphate buffer (0.1M, pH=6), i.e.,
Obtain the fluorescence-encoded micro-beads.
Fluorescence detection reagent kit: including the fluorescence-encoded micro-beads and containing the solution of fluorescent reporter group；The fluorescence
Reporter group is the biological identification molecule II that mark fluorescent reports molecule, in the fluorescent reporter molecule and fluorescence signal molecule
Heart launch wavelength difference >=30nm；By through TEOS and 3- aminopropyl triethoxysilane 120 μ L CdSe/ZnS quantum dots (in
Heart launch wavelength 680nm, is dissolved in n-hexane, and 30 μM) it is marked in the anti-human AFP AFP monoclonal antibody of 100mg mouse i.e.
?.
Experimental example 1
A little super paramagnetic microsphere is taken, uses ethyl alcohol as dispersing agent, with the partial size point of dynamic laser light scattering instrument measurement microballoon
Cloth surveys the saturation magnetization of magnetic microsphere with JDM-14D type vibrating specimen magnetometer, specific as shown in table 1.
The particle diameter distribution test result of 1 embodiment 1-5 superparamagnetic nanoparticle of table, bulbec and super paramagnetic microsphere
| Embodiment | 1 | 2 | 3 | 4 | 5 |
| Superparamagnetic nanoparticle (nm) | 5-20 | 10-30 | 10-25 | 5-15 | 5-28 |
| Bulbec (nm) | 200-250 | 300-500 | 200-300 | 300-400 | 250-450 |
| Super paramagnetic microsphere (μm) | 2-10 | 5-15 | 5-15 | 10-20 | 10-20 |
Experimental example 2
780ng/ml human a-fetoprotein (AFP) standard items are taken, using the continuous doubling dilution of serum dilution to 390ng/ml,
48.75ng/ml sample ten is diluted to 4.875ng/ml, 0.4875ng/ by 195ng/ml, 97.5ng/ml, 48.75ng/ml again
Ml, each concentration prepare 5 parts in parallel, are detected using fluorescence detection reagent kit described in embodiment 1-5.
The sample of selected concentration is mixed with fluorescence-encoded micro-beads with the solution containing fluorescent reporter group, is reacted at room temperature
Then 4h is cleaned with PBST solution, and the BSA that 300 μ L mass fractions are 3% is added and carries out closing not connected site, after reaction
Fluorescence-encoded micro-beads injected in capillary by syringe pump with constant speed；Coding microball flows through magnetic material content one by one
Meter, while the fluorescence intensity of coding microball is detected, to obtain the concentration of AFP, as shown in table 2 below.
2 embodiment 1-5 fluorescence-encoded micro-beads examination criteria product result of table.
| Embodiment | 1 | 2 | 3 | 4 | 5 |
| Sample 1 | 385.88 | 380.25 | 390.45 | 389.36 | 398.21 |
| Sample 2 | 189.75 | 192.22 | 199.34 | 192.82 | 200.64 |
| Sample 3 | 93.25 | 92.31 | 96.11 | 99.04 | 94.55 |
| Sample 4 | 48.26 | 45.21 | 51.78 | 55.36 | 43.45 |
| Sample 5 | 4.63 | 4.02 | 3.96 | 3.99 | 4.53 |
| Sample 6 | 0.48 | - | - | - | 0.39 |
Above-described embodiment is only intended to clearly illustrate technical solution of the present invention example, and not to embodiment
Restriction.For those of ordinary skill in the art, other not similar shapes can also be made on the basis of the above description
The variation or variation of formula.There is no necessity and possibility to exhaust all the enbodiments.And it is extended from this aobvious and easy
The variation or variation seen are still within the protection scope of the invention.
Claims (14)
1. a kind of super paramagnetic microsphere, which is characterized in that including polymer drops and the bulbec being embedded in inside the polymer drops, institute
State the aggregation for the superparamagnetic nanoparticle that bulbec includes partial size 5-30nm and the protective layer being coated on outside the aggregation, institute
Stating bulbec partial size is 200-500nm, and the super paramagnetic microsphere partial size is 2-20 μm.
2. super paramagnetic microsphere according to claim 1, which is characterized in that the superparamagnetic nanoparticle is Fe3O4Nanoparticle
Son, partial size 10-20nm.
3. super paramagnetic microsphere according to claim 1 or 2, which is characterized in that the monomer of the material of the polymer drops is
Ethoxylated trimethylolpropane triacrylate.
4. super paramagnetic microsphere according to claim 1-3, which is characterized in that the protective layer is parents' molecule
Layer, parents' molecule includes stearyl phosphatidyl ethanol amine-polyethylene glycol 2000-amino and the poly- second two of phosphatidyl-ethanolamine-
Alcohol 2000- maleimide.
5. super paramagnetic microsphere according to claim 1-4, which is characterized in that the super paramagnetic microsphere is by 3- ammonia third
Ethyl triethoxy silicane alkane and butanedioic anhydride modification.
6. a kind of method for preparing any one of claim 1-5 super paramagnetic microsphere, which comprises the following steps:
Prepare superparamagnetic nanoparticle: hydro-thermal method prepares Fe3O4Nanoparticle is simultaneously modified；
Preparation bulbec: protective layer is coated outside superparamagnetic nanometer particle congery, and is freeze-dried crushing；
It prepares super paramagnetic microsphere: super paramagnetic microsphere is made in the bulbec and polymer.
7. the preparation method of super paramagnetic microsphere according to claim 6, which is characterized in that prepared by microballoon generating means
Super paramagnetic microsphere.
8. a kind of fluorescence-encoded micro-beads, which is characterized in that including any one of the claim 1-5 super paramagnetic microsphere or right
It is required that the super paramagnetic microsphere of the preparation of super paramagnetic microsphere preparation method described in 6 or 7.
9. fluorescence-encoded micro-beads according to claim 8, which is characterized in that further include fluorescence signal molecule and bio-identification
Molecule I.
10. fluorescence-encoded micro-beads according to claim 8 or claim 9, which is characterized in that the fluorescence signal molecule is selected from center
The different fluorescent molecule of launch wavelength.
11. fluorescence-encoded micro-beads according to claim 8 or claim 9, which is characterized in that the biological identification molecule I is selected from anti-
Original molecule, antibody molecule or nucleic acid molecules.
12. a kind of fluorescence detection product, which is characterized in that including the described in any item fluorescence-encoded micro-beads of claim 8-11 and
Solution containing fluorescent reporter group；The fluorescent reporter group is the biological identification molecule II for being marked with fluorescent reporter molecule, institute
State center emission wavelength difference >=30nm of fluorescent reporter molecule Yu fluorescence signal molecule.
13. a kind of any one of claim 1-5 super paramagnetic microsphere or the super paramagnetic microsphere preparation side of claim 6 or 7
Fluorescence inspection described in the super paramagnetic microsphere of method preparation, any one of the claim 9-11 fluorescence-encoded micro-beads or claim 12
Product is surveyed in the application in fluorescence analysis field.
14. a kind of any one of claim 1-5 super paramagnetic microsphere or the super paramagnetic microsphere preparation side of claim 6 or 7
Fluorescence inspection described in the super paramagnetic microsphere of method preparation, any one of the claim 9-11 fluorescence-encoded micro-beads or claim 12
Product is surveyed in the application in flow cytometry field.
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Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5411730A (en) * | 1993-07-20 | 1995-05-02 | Research Corporation Technologies, Inc. | Magnetic microparticles |
| CN1475805A (en) * | 2002-08-15 | 2004-02-18 | 陕西西大北美基因股份有限公司 | Magnetic fluorescence microsphere and its preparation method and method of proceeding biomolecule detection using said magnetic fluorescence microsphere |
| US20070258907A1 (en) * | 2006-04-24 | 2007-11-08 | Davis Mark E | Polymer-coated paramagnetic particles |
| CN102718931A (en) * | 2012-05-18 | 2012-10-10 | 北京化工大学 | Preparation method of superparamagnetic composite microballoons used in biomedicine |
| CN103143042A (en) * | 2013-02-04 | 2013-06-12 | 厦门大学 | Superpara magnetism polymeric microsphere magnetic resonance imaging (MRI) contrast agent and preparation method thereof |
| CN105693906A (en) * | 2015-01-20 | 2016-06-22 | 于乐 | Zwitterionic polymer microspheres and preparing method thereof |
| CN109481700A (en) * | 2018-12-29 | 2019-03-19 | 广州贝奥吉因生物科技有限公司 | A kind of molecular probe and preparation method thereof for hepatocarcinoma early diagnosis |
-
2019
- 2019-05-22 CN CN201910429936.9A patent/CN110187099A/en active Pending
Patent Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5411730A (en) * | 1993-07-20 | 1995-05-02 | Research Corporation Technologies, Inc. | Magnetic microparticles |
| CN1475805A (en) * | 2002-08-15 | 2004-02-18 | 陕西西大北美基因股份有限公司 | Magnetic fluorescence microsphere and its preparation method and method of proceeding biomolecule detection using said magnetic fluorescence microsphere |
| US20070258907A1 (en) * | 2006-04-24 | 2007-11-08 | Davis Mark E | Polymer-coated paramagnetic particles |
| CN102718931A (en) * | 2012-05-18 | 2012-10-10 | 北京化工大学 | Preparation method of superparamagnetic composite microballoons used in biomedicine |
| CN103143042A (en) * | 2013-02-04 | 2013-06-12 | 厦门大学 | Superpara magnetism polymeric microsphere magnetic resonance imaging (MRI) contrast agent and preparation method thereof |
| CN105693906A (en) * | 2015-01-20 | 2016-06-22 | 于乐 | Zwitterionic polymer microspheres and preparing method thereof |
| CN109481700A (en) * | 2018-12-29 | 2019-03-19 | 广州贝奥吉因生物科技有限公司 | A kind of molecular probe and preparation method thereof for hepatocarcinoma early diagnosis |
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