CN106405075B - A kind of immunomagnetic beads and preparation method thereof - Google Patents
A kind of immunomagnetic beads and preparation method thereof Download PDFInfo
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- CN106405075B CN106405075B CN201610780209.3A CN201610780209A CN106405075B CN 106405075 B CN106405075 B CN 106405075B CN 201610780209 A CN201610780209 A CN 201610780209A CN 106405075 B CN106405075 B CN 106405075B
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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
- G01N33/531—Production of immunochemical test materials
Abstract
The present invention provides a kind of immunomagnetic beads and preparation method thereof, including magnetic microsphere and biological aglucon, the structure of the immunomagnetic beads to be from the inside to the outside：Magnetic microsphere biotinstreptatin biotin biology aglucon.The immunomagnetic beads property of the present invention is stablized, and magnetic good, and grain size is small.The present invention uses the connection type of biotinstreptatin biotin, first passes through amidation process and obtains biotinylation magnetic microsphere, is then combined biotinylation magnetic microsphere with Streptavidin.Due to affinity high and specificity the feature of the combination of biotinstreptatin, so that magnetic microsphere is oriented combination with biological aglucon, be not easy to be crosslinked, be not easy that Streptavidin structure is caused to change.Meanwhile preparation method of the invention is simple, reaction condition is mild, and biological aglucon is allow to retain bioactivity.
Description
Technical field
The present invention relates to the preparation field of immunomagnetic beads, specifically a kind of immunomagnetic beads and preparation method thereof.
Background technology
Immunomagnetic beads (Immonumagnetic beads, abbreviation IMB), be by magnetic microsphere and biological aglucon in conjunction with and
At.The combination of magnetic microsphere and biological aglucon generally has physical bond and the method for chemical coupling.Chemical coupling method, which has, to be combined
Capacity is big, and stability is good, and the immunomagnetic beads being prepared can usually preserve the several months.But antibody inactivates in order to prevent, chemistry
Coupling method can only generally use comparatively gentle condition, therefore the coating efficiency of antibody can be caused bad；And chemical coupling meeting
Using excessive antibody activity group, degree of controllability is low thus antibody can be caused to inactivate.Physical adsorption process is earliest by John
The method of the uses such as Ugelstad has coating simply, the mild feature of reaction condition, but it mainly utilizes the hydrophobic of albumen
Property be coated on magnetic microsphere surface, magnetic microsphere before coating must have hydrophobicity, the immunomagnetic beads holding time is not
Can be too long, magnetic bead surfaces active material is easy to fall off, and protein stability and activity can be affected.
By the combination of biotin-avidin magnetic bead come to prepare immunomagnetic beads be the current high object of most common compatibility
Reason combines coating mode, rapid reaction, product to stablize, and specificity is strong, the shortcomings that overcoming general physical absorption, and with height
Compatibility, can be with the activity and specificity of biometric safeguard matrix.Avidin background itself is high, and common physical bond includes
The method of biotin-Streptavidin connection, specifically first by after one layer of Streptavidin of magnetic microsphere surface modification, then with life
Biotin-Streptavidin that the biological aglucon of object element label is oriented combines, and obtains immunomagnetic beads.Due to each strepto- parent
The package amount of magnetic microsphere surface biological aglucon can be improved with plain molecule in conjunction with the biotin of 4 molecules.Such as Publication No.
The Chinese patent of CN103443626A《Streptavidin combination magnetic particle and its manufacturing method》Disclose a kind of protein knot
Magnetic particle, that is, immunomagnetic beads are closed, it is to connect to obtain with biotinylated protein matter by Streptavidin combination magnetic particle.Its
Middle Streptavidin combination magnetic particle is by making surface have the magnetic particle of amino and Streptavidin and glutaraldehyde anti-
It is obtained after should restoring.Every peptide chain of Streptavidin contains 159 amino acid residues, the wherein alkalinity such as lysine, arginine
The content of amino acid is fewer than general avidin, more containing acidic amino acid, isoelectric point 6.0, therefore on Streptavidin
Amino, which reacts away, excessive to be easy to cause isoelectric point and changes or inactivate；And this method also needs to addition when being restored by force
Reducing agent (such as sodium borohydride or sodium cyanoborohydride) easily causes albumen isoreactivity substance inactivation, and glutaraldehyde can be led
It causes magnetic particle itself and protein therein to crosslink, is unfavorable for the coupling of protein.
Invention content
The main object of the present invention is to provide a kind of immunomagnetic beads and preparation method thereof in view of the shortcomings of the prior art,
The immunomagnetic beads being prepared with this method, biological aglucon are connected with magnetic microsphere orientation, will not lead to internal crosslinking and albumen
Matter inactivates, and biological aglucon package amount is big, and bioactivity is good.
In one aspect of the invention, the present invention is achieved through the following technical solutions：A kind of immunomagnetic beads, including magnetism
Microballoon and biological aglucon, the structure of the immunomagnetic beads are from the inside to the outside：Magnetic microsphere-biotin-Streptavidin-biology
The biological aglucon of element-.
Preferably, the biological aglucon is one kind in BSA, enzyme, antibody, DNA or RNA.
Preferably, the magnetic microsphere is the magnetic Nano cluster of the inorganic or organic polymer package of nucleocapsid.Such as
The magnetic ferroferric oxide etc. of magnetic ferroferric oxide or the glucan package of Silica-coated, most preferably dioxy
The magnetic ferroferric oxide of SiClx package.
In another aspect of this invention, a kind of method preparing above-mentioned immunomagnetic beads is provided, step includes：
S1. the preparation of magnetic Nano cluster；
S2. the preparation of amido modified magnetic microsphere；
S3. the preparation of biotinylation magnetic microsphere；
S4. biotinylation magnetic microsphere is combined with Streptavidin；
S5. the preparation of immunomagnetic beads：The biotinylation magnetic microsphere and biology of Streptavidin will be combined in step s4
Plain metaplasia object aglucon is in conjunction with obtaining the immunomagnetic beads.
In one embodiment of the invention, the preparation of the biotinylation magnetic microsphere in the step s3 is by amino
The magnetic microsphere of modification is dispersed in dimethyl sulfoxide or n,N-Dimethylformamide, and biotin activity ester is then added, and carries out acyl
It is obtained after aminating reaction, wherein the mass ratio of amido modified magnetic microsphere and biotin activity ester is 1:(0.25~1).By
In active ester in water phase easy in inactivation, the reaction time is short, can be conducive under alkaline condition improve reaction efficiency prepare biology
Elementization magnetic microsphere.The present invention can prevent the inactivation of active ester using dimethyl sulfoxide or n,N-Dimethylformamide as solvent.Together
The Shi Yanchang reaction time improves the bioid on magnetic microsphere surface, preferably reacts 10~24 hours to the reaction of biotin activity ester
Completely.As well known to a person skilled in the art in the step s3, Magneto separate, magnetic should be carried out after the completion of amidation process
Isolated biotinylation magnetic microsphere is washed with the pH PBS solutions for being 7.2 and is dispersed in PBS solution.
Preferably, the step s4 is that biotinylation magnetic microsphere is reacted 60~90 points with Streptavidin at room temperature
It is obtained after clock, wherein the additive amount of Streptavidin is the 0.1~0.6 of biotinylation magnetic microsphere quality.
Preferably, the biotinylation magnetic microsphere and biotin of Streptavidin are combined in the step s5 in step s4
The mass ratio of metaplasia object aglucon is 1：(0.01~1).Since the compatibility of Streptavidin and biotin is very high, do not need
Consider concentration and ratio problems, as long as the biotinylation magnetic microsphere for combining Streptavidin is unsaturated, biotinylation biology
Aglucon can be coupled with magnetic microsphere.It is contemplated that the cost factor of biological aglucon, it is 1 that the present invention, which takes mass ratio,：
(0.01~0.2).
Preferably, the magnetic Nano cluster in the step s1 is prepared by hydro-thermal method, solvent-thermal method or coprecipitation
It arrives, commercial goods can also be used.The technology that the method for preparing magnetic Nano cluster is well known to those skilled in the art, obtains
Product, which only needs to meet, has good magnetism, and can form nucleocapsid with organic or inorganic macromolecule.
Further, the amido modified magnetic microsphere part in the step s2 is the Silica-coated of nucleocapsid
Magnetic Nano cluster, effect more be better than directly carry out amido modified obtained magnetic microsphere in magnetic Nano cluster particle surface,
Commercial goods may be used or prepared according to conventional method known to those skilled in the art, do not influence the knot of the present invention
Fruit.Nano-cluster can be made to be gathered in silica interior, form nucleocapsid, increase grain size, and increase magnetic and stability
.Preferably, the present invention prepares the magnetic Nano cluster of amido modified Silica-coated with the following method：To containing magnetic
Property nano-cluster solution in ammonium hydroxide, silylating reagent and amino silicane coupling agent is added, reaction obtains amido modified after 1~3 day
Magnetic microsphere；The magnetic Nano cluster, ammonium hydroxide, silylating reagent, amino silicane coupling agent addition mass ratio be：1:
(12.5~40):(2~8):(0.5~3).The mass percentage concentration of the ammonium hydroxide is 25~28%.Wherein, silylating reagent can
Think tetraethyl orthosilicate (CAS：562-90-3), amino silicane coupling agent can be (3- aminopropyls) triethoxysilane
(CAS：919-30-2).Those skilled in the art, can be right when the other silylating reagents of selection are with amino silicane coupling agent
The ratio of reaction raw materials is adjusted, without departing from protection scope of the present invention.
In the third aspect of the present invention, a kind of application of above-mentioned immunomagnetic beads in cell sorting is provided.
Beneficial effects of the present invention are：
(1) the present invention provides a kind of immunomagnetic beads, the structure of immunomagnetic beads is from the inside to the outside：Magnetic microsphere-biology
Element-Streptavidin-biotin-biology aglucon, obtained immunomagnetic beads property are stablized, and magnetic good, while grain size is 200
~400nm.
(2) present invention uses the connection type of biotin-Streptavidin-biotin, first passes through amidation process and obtains
Then biotinylation magnetic microsphere is combined biotinylation magnetic microsphere with Streptavidin.Due to biotin-Streptavidin
Combination is affinity high and the feature of specificity, so that magnetic microsphere is oriented combination with biological aglucon, be not easy to be crosslinked, no
Streptavidin structure is easily caused to change.The higher isoelectric point (isoelectric point 10-10.5) of Avidin and the high structure containing sugar cause poly-
When being combined on styrene plate and nitrocellulose filter or with histocyte DNA, it is also easy to produce a degree of non-specific binding, is made
At higher colour developing background, and Streptavidin (being free of glycosyl, isoelectric point 5-6) overcomes this disadvantage in the application.Meanwhile
, can be in conjunction with four biotin molecules since a Streptavidin molecule is there are four binding site, and the effect of steric hindrance
It can make Streptavidin after being combined with biotinylation magnetic microsphere, still have 1~3 remaining site can be with biotin metaplasia
Object aglucon combines, and makes to be coated on the biological aglucon package amount on the outside of magnetic microsphere and increases.
(3) preparation method of the invention is simple, and reaction condition is mild, and reaction can carry out at room temperature, it is not easy to make
It at the rotten of biological aglucon, degradation etc., while avoiding using reducing agent, and is that biology can be maintained to match by compatibility combination
The bioactivity and specificity of base.
(4) raw material of the present invention is simple and easy to get, at low cost, and processing step is simple, has very strong practicability.
Description of the drawings
Fig. 1 is shape appearance figure under the light microscope of CD45 antibody immune magnetic beads 1 of the present invention；
Fig. 2 is shape appearance figure under the fluorescence microscope of CD45 antibody immune magnetic beads 1 of the present invention；
Fig. 3 is the CTC cellular immunofluorescence figures of EpCAM immunological magnetic bead sortings of the present invention.
Specific implementation mode
By the following specific examples further illustrate the invention：The experiment of actual conditions is not specified in the following example
Method according to conventional methods and conditions, or is selected according to product manual.
Embodiment 1
(1) preparation of magnetic Nano cluster：Magnetic Nano cluster Fe is prepared by coprecipitation3O41；
(2) preparation of amido modified magnetic microsphere：To containing 10mg magnetic Nano clusters Fe3O4It is added in 1 solution
125mg ammonium hydroxide, 30mg tetraethyl orthosilicates and 30mg (3- aminopropyls) triethoxysilane, reaction carry out Magneto separate after 1 day,
And it is alternately washed respectively twice with second alcohol and water, obtains amido modified magnetic microsphere 1；
(3) preparation of biotinylation magnetic microsphere：The amido modified magnetic microspheres 1 of 20mg are dispersed in dimethyl sulfoxide,
Then 10mg biotin activity esters are added, carries out amidation process, carries out Magneto separate after reaction overnight, the PBS for being 7.2 with pH is molten
Biotinylation magnetic microsphere 1 is obtained after liquid washing；
(4) biotinylation magnetic microsphere is combined with Streptavidin：By 1mg biotinylations magnetic microsphere 1 and 200 μ L chains
Mould Avidin (a concentration of 0.5 μ g/ μ L) is obtained after 60 minutes of reaction the biotinylation magnetic for combining Streptavidin at room temperature
Property microballoon 1；
(5) preparation of immunomagnetic beads：The life of the biotinylation magnetic microsphere 1 and mark fluorescent of Streptavidin will be combined
Object element antibody CD45 is combined, and is carried out Magneto separate and is obtained the CD45 antibody immune magnetic beads 1.
The concentration with the biological aglucon 1 of biotin modification after reaction before being reacted in solution is detected with nanodrop, is calculated
To addition biotinylated antibody can about be combined completely with 1mg in 50 μ g or so Streptavidin biotinylation magnetism it is micro-
Ball is coupled, and continuously adds biotinylated antibody, by nanodrop detect remaining biotinylated antibody not with combine chain
The reaction was continued for the biotinylation magnetic microsphere of mould Avidin, illustrates that the biotinylation magnetic microsphere for combining Streptavidin reaches
Saturation.
To the progress microscope detection of above-mentioned CD45 antibody immune magnetic beads 1, shape appearance figure such as Fig. 1 institutes under obtained light microscope
Show, shape appearance figure is as shown in Figure 2 under fluorescence microscope.It can be seen that the CD45 antibody immune magnetic beads 1 of the present invention by Fig. 1 and Fig. 2
On, the CD45 for having mark fluorescent is coupled on immunomagnetic beads, coupling efficiency is high.
Embodiment 2
(1) preparation of magnetic Nano cluster：Magnetic Nano cluster Fe is prepared by coprecipitation3O42；
(2) preparation of amido modified magnetic microsphere：To containing 10mg magnetic Nano clusters Fe3O4It is added in 2 solution
250mg ammonium hydroxide, 20mg tetraethyl orthosilicates and 10mg (3- aminopropyls) triethoxysilane, reaction carry out Magneto separate after 2 days,
And it is alternately washed respectively twice with second alcohol and water, obtains amido modified magnetic microsphere 2；
(3) preparation of biotinylation magnetic microsphere：The amido modified magnetic microspheres 2 of 20mg are dispersed in N, N- dimethyl methyls
In amide, 5mg biotin activity esters are then added, carries out amidation process, carries out Magneto separate after reaction overnight, are 7.2 with pH
PBS solution washing after obtain biotinylation magnetic microsphere 2；
(4) biotinylation magnetic microsphere is combined with Streptavidin：By 1mg biotinylations magnetic microsphere 2 and 200 μ L chains
Mould Avidin (a concentration of 1 μ g/ μ L) obtains combining Streptavidin at room temperature biotinylation after reacting 90 minutes is magnetic
Microballoon 2；
(5) preparation of immunomagnetic beads：The life of the biotinylation magnetic microsphere 2 and mark fluorescent of Streptavidin will be combined
Object element antibody CD45 is in conjunction with obtaining the CD45 antibody immune magnetic beads 2.
The biotinylated antibody of addition can about combine the biotin of Streptavidin with 1mg completely in 100 μ g or so
Change magnetic microsphere coupling, and continuously add biotinylated antibody, by nanodrop detect remaining biotinylated antibody not with
Combining the biotinylation magnetic microsphere of Streptavidin, the reaction was continued, illustrates that the biotinylation for combining Streptavidin is magnetic
Microballoon reaches saturation.
Embodiment 3
(1) preparation of magnetic Nano cluster：Magnetic Nano cluster Fe is prepared by coprecipitation3O43；
(2) preparation of amido modified magnetic microsphere：To containing 10mg magnetic Nano clusters Fe3O4It is added in 3 solution
125mg ammonium hydroxide, 40mg tetraethyl orthosilicates and 5mg (3- aminopropyls) triethoxysilane, reaction carry out Magneto separate after 3 days,
And it is alternately washed respectively twice with second alcohol and water, obtains amido modified magnetic microsphere 3；
(3) preparation of biotinylation magnetic microsphere：The amido modified magnetic microsphere parts 1 20mg are dispersed in dimethyl sulfoxide
Or in n,N-Dimethylformamide, 20mg biotin activity esters are then added, carries out amidation process, carries out magnetic after reaction overnight
Separation obtains biotinylation magnetic microsphere 3 after being washed with the pH PBS solutions for being 7.2；
(4) biotinylation magnetic microsphere is combined with Streptavidin：By 1mg biotinylations magnetic microsphere 3 and 200 μ L chains
Mould Avidin (a concentration of 3 μ g/ μ L) obtains combining Streptavidin at room temperature biotinylation after reacting 75 minutes is magnetic
Microballoon 3；
(5) preparation of immunomagnetic beads：The biotinylation magnetic microsphere 3 and biotinylated antibody of Streptavidin will be combined
EpCAM is in conjunction with obtaining the EpCAM antibody immune magnetic beads 3.
The biotinylated antibody of addition can about combine the biotinylation of Streptavidin with 1mg completely in 80 μ g or so
Magnetic microsphere is coupled, and continuously adds biotinylated antibody, by nanodrop detect remaining biotinylated antibody not with knot
Having closed the biotinylation magnetic microsphere of Streptavidin, the reaction was continued, illustrates that the biotinylation magnetism for combining Streptavidin is micro-
Ball reaches saturation.
The CTC cell captures of 4 cancer patient of embodiment
Healthy People and the peripheral blood blood of various cancers patient are taken, the CTC in blood is captured.It is required that subject's blood
Conventional wbc value is located at 2 × 106~1.2 × 107Between a/mL, whole blood sample does not occur haemolysis or clot.And by
Examination person's relevant information is complete, and sample collection, store method specification, experimental implementation specification are as follows：Take various cancers sick
EpCAM antibody immune magnetic beads in embodiment 3 are added sequentially in above-mentioned each mixed cell suspension, at 4 DEG C by human blood 3mL
It is lower to be incubated 30 minutes, magnetic separation is then carried out in 1 minute and is washed 2~3 times with PBS, and the CTC for being incubated recycling is subjected to antibody
It dyes and after fixed on glass slide and then progress nucleus DAPI dyeing, mounting, is identified with fluorescence microscope, as a result as schemed
Shown in 3, cell membrane surface shows fluorescence, is green-emitting fluorescent under fluorescence microscope, illustrates that the cell surface captured is in
EpCAM is positive, illustrates for tumour cell.And do not capture tumour cell in the blood of normal person.
If first carrying out erythrocyte splitting to blood, and with after the leucocyte in CD45 antibody immune magnetic beads removal blood
The capture of tumour cell, capture rate higher are carried out with EpCAM antibody immune magnetic beads again.
For those skilled in the art, under the premise of not departing from principle of the embodiment of the present invention, also
Several improvements and modifications can be made, these improvements and modifications are also considered as the protection domain of the embodiment of the present invention.
Claims (8)
1. a kind of immunomagnetic beads for capturing tumour cell in peripheral blood blood, including magnetic microsphere and biological aglucon, special
Sign is：The structure of the immunomagnetic beads is from the inside to the outside：Magnetic microsphere-biotin-Streptavidin-biotin-biology is matched
Base, the magnetic microsphere are the magnetic Nano cluster that the inorganic or organic polymer of nucleocapsid wraps up, and the biology is with base
One kind in BSA, enzyme, antibody, DNA or RNA.
2. a kind of method prepared described in claim 1 for capturing the immunomagnetic beads of tumour cell in peripheral blood blood,
Step includes：
S1. the preparation of magnetic Nano cluster；
S2. the preparation of amido modified magnetic microsphere；
S3. the preparation of biotinylation magnetic microsphere；
S4. biotinylation magnetic microsphere is combined with Streptavidin；
S5. the preparation of immunomagnetic beads：The biotinylation magnetic microsphere and biotinylation of Streptavidin will be combined in step s4
Biological aglucon is in conjunction with obtaining the immunomagnetic beads.
3. it is used to capture the preparation method of the immunomagnetic beads of tumour cell in peripheral blood blood according to claim 2, it is special
Sign is：The preparation of biotinylation magnetic microsphere in the step s3 is that amido modified magnetic microsphere is dispersed in diformazan Asia
In sulfone or n,N-Dimethylformamide, biotin activity ester is then added, is obtained after progress amidation process, wherein amino is repaiied
The magnetic microsphere of decorations and the mass ratio of biotin activity ester are 1:(0.25~1).
4. it is used to capture the preparation method of the immunomagnetic beads of tumour cell in peripheral blood blood according to claim 2, it is special
Sign is：The step s4 is obtained after reacting biotinylation magnetic microsphere 60~90 minutes at room temperature with Streptavidin
It arrives, wherein the additive amount of Streptavidin is the 0.1~0.6 of biotinylation magnetic microsphere quality.
5. it is used to capture the preparation method of the immunomagnetic beads of tumour cell in peripheral blood blood according to claim 2, it is special
Sign is：The biotinylation magnetic microsphere of Streptavidin is combined in step s4 and biotinylation biology is matched in the step s5
The mass ratio of base is 1：(0.01~1).
6. it is used to capture the preparation method of the immunomagnetic beads of tumour cell in peripheral blood blood according to claim 2, it is special
Sign is：Magnetic Nano cluster in the step s1 is prepared by hydro-thermal method, solvent-thermal method or coprecipitation.
7. it is used to capture the preparation method of the immunomagnetic beads of tumour cell in peripheral blood blood according to claim 2, it is special
Sign is：Amido modified magnetic microsphere in the step s2 is the magnetic Nano cluster of the Silica-coated of nucleocapsid,
It is prepared via a method which to obtain：It is even that ammonium hydroxide, silylating reagent and amino silane are added into the solution containing magnetic Nano cluster
Join agent, reaction obtains amido modified magnetic microsphere after 1~3 day；The magnetic Nano cluster, ammonium hydroxide, silylating reagent, amino silicone
The mass ratio of alkane coupling agent addition is：1:(12.5~40):(2~8):(0.5~3).
8. immunomagnetic beads the answering in cell sorting described in a kind of claim 1 for capturing tumour cell in peripheral blood blood
With.
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| CN112007620B (en) * | 2019-05-30 | 2023-08-01 | 苏州海狸生物医学工程有限公司 | Preparation method of streptavidin magnetic microsphere |
| CN114152741B (en) * | 2021-11-18 | 2022-08-19 | 上海北昂医药科技股份有限公司 | dELISA sample for improving effective microbead proportion and preparation and detection method thereof |
| CN117607463B (en) * | 2024-01-23 | 2024-04-09 | 杭州华得森生物技术有限公司 | EGFR (epidermal growth factor receptor) immune chromogenic detection reagent for circulating tumor cells |
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