CN106381286A - Folic acid immunomagnetic beads and preparation method thereof - Google Patents
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Abstract
The invention provides folic acid immunomagnetic beads and a preparation method thereof. The and preparation method thereof comprises a magnetic microsphere part and a FA-PEG part, a structural formula of immunomagnetic beads is A-NH-N=CH-B, wherein, A expresses the magnetic microsphere, B expresses FA-PEG, or A expresses FA-PEG, B expresses magnetic microsphere. The immunomagnetic beads can be used for capturing tumor cells, have the advantages of good specificity, good sensitivity, rapid magnetic response, short enrichment time, and high capture efficiency, and have the characteristics of stable property, small particle size, good magnetic responsiveness and good dispersibility. THE preparation method is simple, and has strong practicality.
Description
Technical field
The present invention relates to the preparation field of immunomagnetic beadses, specifically a kind of folic acid immunity magnetic bead and preparation method thereof.
Background technology
Folic Acid (folate, FA) is Bao Kuo needed for a lot of bioprocesss including DNA synthesis, DNA reparation and cell division
A kind of essential vitamin.In addition to the peripheral blood macrophage being activated on a small quantity, normal cell surface expression quantity is relatively
Three few folacin receptors, and in the universal overexpression of tumor cell middle period acid acceptor.Folacin receptor (folate receptor,
FR) it is found universal overexpression early in the tumor cell surface of ovarian cancer and renal carcinoma, Nunez in 2012 et al. finds to exist
In 75.7% nonsmall-cell lung cancer (non-smallcelllungcancer, NSCLC) patient, FR also occurs significantly to express
Raise.Publication date is on July 22nd, 2015, and the Chinese patent of Publication No. CN104790216A is received folacin coupled in functionalization
Rice fiber surface, to capture tumor cell, has the advantages that time-consuming short, specificity, but relies on microfluidic device, and nanometer
The high cost of fiber.
Immunological magnetic bead sorting method (Immunomagnetic separation, IMS) is that one kind of rising in recent years is used for carefully
The method of born of the same parents' sorting, it is to be coated the immunoreactive antibody of tool in magnetic bead surfaces to carry out antigen-antibody reaction, in cell surface
Form " Ag-Ab-magnetic bead " immune complex.The cell that these combine magnetic bead is once placed under powerful magnetic field, will
Displacement, makes immune complex and other not combined cells divide group.After the magnetic bead having superparamagnetism departs from magnetic field, magnetic
Property disappear immediately, thus reach positive or negative select specific cells purpose.For example utilize immunomagnetic beadses Solid phase in jump,
Leukocyte in blood is removed thus enrichment cycles tumor cell (circulating tumor using leucocyte removal magnetic bead
Cells, CTC), then captured with being coupled the oligonucleotide probe having folate ligand, by oligonucleotide probe is entered with performing PCR amplification
Carry out the detection by quantitative that CTCs is amplified, but the detection that actually probe portion carried out of the method is it is impossible to enrichment
CTCs carry out follow-up cellular immunology analysis and foranalysis of nucleic acids.(in jump. the circulating tumor cell of folate receptor-positive can be made
For new pulmonary carcinoma auxiliary diagnosis mark [D]. Beijing Union Medical College Chinese Academy of Medical Sciences, 2014.)
The sorting that immunological magnetic bead sorting method is applied to tumor cell is the technology of this area rising in recent years.Immunomagnetic beadses
Sorting rule can be divided into positive separating method and negative two kinds of separating method, respectively has its advantage to be located.Positive sorting refers to immunomagnetic beadses
By antigen or receptor binding with tumor surface, directly sub-elect tumor cell.Negative sorting then pass through immunomagnetic beadses with white
Other untargeted cells such as leukocyte is removed, to reach richness after the antigen on the other such as cell untargeted cells surface or receptor binding
The purpose of collection tumor cell.But sub-elect few the swelling of quantity from the complex environments such as blood using immunological magnetic bead sorting method
Oncocyte, needs immunomagnetic beadses to have specific biomarker and sensitivity, and stability, good dispersion are it is impossible to roll into a ball
Poly-.Meanwhile, the particle diameter of immunomagnetic beadses can not be excessive, crosses conference compressing tumor cell；The particle diameter of immunomagnetic beadses nor too small, grain
The too small magnetic responsiveness in footpath is also little, is easily unable to reach the effect of tumor cell sorting.
Content of the invention
The main object of the present invention is to provide a kind of folic acid immunity magnetic bead and its preparation in view of the shortcomings of the prior art
Method, the folic acid immunity magnetic bead particle diameter of the present invention is little, and magnetic responsiveness is good, is used for carrying out tumor cell capture, not only capture rate
Height, enrichment time is short, and capturing the tumor cell obtaining can be further analyzed, and the preparation process of immunomagnetic beadses is simple
Single, low cost.
In one aspect of the invention, the present invention is achieved through the following technical solutions：A kind of folic acid immunity magnetic bead, including
Magnetic microsphere part and FA-PEG part, the structural formula of described immunomagnetic beadses is：A-NH-N=CH-B, wherein A represent that magnetic is micro-
Ball, B represents FA-PEG, or A represents that FA-PEG, B represent magnetic microsphere, and described FA-PEG is after being coupled FA and PEG
Obtain.Wherein, the coupling of FA and PEG can be coupled using EDC/NHS system, and this coupling process is those skilled in the art's root
Can complete according to common knowledge.
Preferably, described magnetic microsphere is the magnetic microsphere of the inorganic or organic polymer parcel of nucleocapsid structure.Such as two
Magnetic ferroferric oxide of the magnetic ferroferric oxide of silicon oxide-wrapped or glucosan parcel etc..Most preferably, described magnetic
Microsphere part is the magnetic ferroferric oxide of Silica-coated.
Preferably, described PEG is Polyethylene Glycol, and molecular weight is 2000～6000.
Preferably, the particle diameter of described folic acid immunity magnetic bead is 200～300nm.
In a second aspect of the present invention, there is provided a kind of method preparing above-mentioned folic acid immunity magnetic bead, its step includes：
S1. the preparation of magnetic Nano cluster；
S2. the preparation of amido modified magnetic microsphere；
S3. the preparation of the part A that diazanyl is modified；
S4. the preparation of aldehyde group modified part B；
S5. the preparation of immunomagnetic beadses：The part A that modify diazanyl described in step s3 and aldehyde group modified B described in step s4
Part mixes, and carries out coupling reaction 2～24 hours, obtain described immunomagnetic beadses at 4～25 DEG C.
In one embodiment of the invention, amido modified magnetic microsphere is carried out aldehyde group modified, and to FA-
PEG carries out diazanyl modification.Or carry out diazanyl modification to amido modified magnetic microsphere, and carry out aldehyde radical to FA-PEG repairing
Decorations, all can realize the immunomagnetic beadses of said structure.Because FA molecule is less, and the NH on FA molecule2Activity not high, difficult
To carry out diazanyl modification, after therefore FA being connected with PEG space arm, or directly buy commercially available FA-PEG, then to PEG
On amino carry out diazanyl modification.
Further, the part A that the diazanyl in described step s3 is modified is to pass through：By amido modified magnetic microsphere or
FA-PEG molar equivalent is to obtain after 10～30 times of SANH carries out diazanyl modification.Most preferably, the molar equivalent of SANH
For amido modified magnetic microsphere or FA-PEG 20 times.Described SANH is that p- third hydrazone group pyridine carboxylic acid N- hydroxysuccinimidyl acyl is sub-
Amine ester (CAS：362522-50-7), at ambient temperature can be gentle amino is converted to diazanyl.Described SANH typically dissolves
DMF solution is reacted, concentration can be adjusted according to the concentration of magnetic microsphere or FA-PEG, do not affected reaction knot
Really.This course of reaction can be carried out under 15～25 DEG C of room temperature conditions, and the response time is according to the conventional inspection of those skilled in the art
Survey technology judges, the present invention general using the modification reaction time 16～24h to magnetic microsphere, during to the modification reaction of FA-PEG
Between 2～4h.
Further, the aldehyde group modified part B in described step s4 is to pass through：By amido modified magnetic microsphere or
FA-PEG molar equivalent be 5～20 times of SFB carry out aldehyde group modified after obtain.Most preferably, the molar equivalent of SFB is ammonia
Magnetic microsphere that base is modified or 10 times of FA-PEG.Described SFB is 4- carbamoyl benzoate N- succinimide ester (CAS：60444-
78-2), at ambient temperature can be gentle amino is converted to aldehyde radical.Described SFB is typically dissolved in DMF solution and carries out instead
Should, concentration can be adjusted according to the concentration of magnetic microsphere or FA-PEG, does not affect reaction result.This course of reaction can be
Carry out under 15～25 DEG C of room temperature conditions, the response time judges, the present invention one according to the conventional detection technique of those skilled in the art
As using modification reaction time 16～24h, the modification reaction time 2～4h to FA-PEG to magnetic microsphere.
Magnetic Nano cluster in described step s1 is to be prepared by hydro-thermal method, solvent-thermal method or coprecipitation, also may be used
To adopt commercial goods.Prepare the technology that the method for magnetic Nano cluster is well known to those skilled in the art, the product obtaining is only
Need to meet and there is good magnetic, and nucleocapsid structure can be formed with inorganic or organic polymer.
As preferred, the magnetic Nano cluster in described step s1 is to be prepared via a method which to obtain：
1) in the aqueous solution of divalent iron salt, in atmosphere, add ammonia, then stirring makes solution become black, obtains black
Color Fe3O4Granule；
2) to step 1) middle addition Oleic acid, it is transferred to after mix homogeneously in airtight reactor, heat at 60～130 DEG C
Reaction 3～5 hours, you can obtain described magnetic Nano cluster.
Further, the amido modified magnetic microsphere in described step s2 is the magnetic of the Silica-coated of nucleocapsid structure
Property microsphere, effect more be better than directly carry out the amido modified magnetic microsphere obtaining in magnetic Nano cluster particle surface, can adopt
With commercial goods or according to conventional method preparation known to those skilled in the art, all do not affect the result of the present invention.Permissible
Make nano-cluster be gathered in silica interior, form nucleocapsid structure, increase particle diameter, and increase magnetic and stability.Excellent
Choosing, the present invention adopts the magnetic microsphere preparing amido modified Silica-coated with the following method：To containing magnetic Nano cluster
Solution in add ammonia, silylating reagent and amino silicane coupling agent, it is micro- that reaction obtained amido modified magnetic after 1～3 day
Ball portion；Described magnetic Nano cluster, ammonia, silylating reagent, the mass ratio of amino silicane coupling agent addition are：1:(12.5
～40):(2～8):(0.5～3).The mass percentage concentration of wherein ammonia is 25～28%.Wherein, silylating reagent can be
Tetraethyl orthosilicate (CAS：562-90-3), amino silicane coupling agent can be (3- aminopropyl) triethoxysilane (CAS：
919-30-2).Those skilled in the art, can be former to reaction when selecting other silylating reagents with amino silicane coupling agent
The ratio of material is adjusted, all without departing from protection scope of the present invention.
Preferably, in described step s5, the mass ratio of magnetic microsphere and FA-PEG is 1：(2～10).
In a third aspect of the present invention, there is provided a kind of test kit for capturing tumor cell, contain in described test kit
There is above-mentioned folic acid immunity magnetic bead.Described tumor cell is capable of the cancer cell of overexpression folacin receptor for surface, including ovum
The cancers such as nest cancer, pulmonary carcinoma, renal carcinoma, carcinoma of endometrium, breast carcinoma, the brain cancer, nasopharyngeal carcinoma, colon cancer and hemopoietic pastern bone myelocyte cancer
Tumor cell.
Beneficial effects of the present invention are：
(1) immunomagnetic beadses of the present invention are used for capturing tumor cell, not only specificity, sensitivity are good, and magnetic response
Rapidly, enrichment time is short, and capture rate is high.Avoid the tumor cell damage of capture, tumor cell can be used for further
Analysis and culture.Can be used for tumor cell capture and the analysis of excretion body, body fluid or biopsy.
(2) immunomagnetic beadses of the present invention, its magnetic microsphere is connected by hydrazone key structure with FA-PEG, the immunomagnetic beadses obtaining
(in blood) stable in properties under weak basic condition, particle diameter is little simultaneously, magnetic responsiveness is good, good dispersion, be difficult reunite.
(3) preparation method of the present invention is simple, and reaction condition is gentle, and the process that amino, aldehyde radical and diazanyl are modified is all permissible
Carry out at room temperature, it is not easy to cause rotten, degraded of folate ligand etc., avoiding simultaneously and using reducing agent, make antibody permissible
At low temperature with cell incubation, keep the activity of antibody and cell.
(4) raw material of the present invention is simple and easy to get, low cost, and processing step is simple, has very strong practicality.
Brief description
Fig. 1 is the tumor cell immunofluorescence figure of folic acid immunity magnetic bead sorting of the present invention.
Specific embodiment
By the following specific examples further illustrate the invention：The experiment of unreceipted actual conditions in the following example
Method, conventionally and condition, or selects according to catalogue.
The preparation of embodiment 1 folic acid immunity magnetic bead
(1) preparation of magnetic Nano cluster：
A. in atmosphere, by 7g FeCl2·4H2O is added in 50mL deionized water, and obtaining concentration is 0.14g/mL's
FeCl2Aqueous solution.To 50mL FeCl2Ammonia 30mL is added, after stirring 20min, color gradually becomes light green color in aqueous solution, then
Become bottle green, eventually become black；
B. add 1.1g Oleic acid in step a, after mix homogeneously, mixed liquor is placed in airtight reactor, at 110 DEG C
Lower reacting by heating 4 hours, then alternately washing is each once for deionized water and ethanol, is dispersed in normal hexane, that is, after Magneto separate
Can get the magnetic Nano cluster Fe of black3O41.
(2) preparation of amido modified magnetic microsphere：To 10mg magnetic Nano cluster Fe3O4125mg ammonia is added in 1 solution
Water, 30mg tetraethyl orthosilicate and 30mg (3- aminopropyl) triethoxysilane, reaction carried out Magneto separate after 1 day, and used
Second alcohol and water replace wash each twice, and to obtain concentration after the phosphate buffer dispersion with the pH 7～8 of 0.1M be 5mmol/L's
Amido modified magnetic microsphere 1.
(3) preparation of the FA-PEG that diazanyl is modified：The DMF solution (concentration is 5mmol/L) of 50 μ L SANH is added to
In 1000 μ L FA-PEG solution (concentration is 10 μm of ol/L), after room temperature reaction 2h, the FA-PEG obtaining diazanyl modification (writes a Chinese character in simplified form
For FA-PEG-SANH) 1.
(4) preparation of aldehyde group modified magnetic microsphere：The amido modified magnetic microsphere 1 of 5 μ L is added to the DMF of SFB
In solution (concentration is 5mmol/L) 50 μ L, after room temperature reaction 20h, carry out Magneto separate purification, obtain aldehyde group modified magnetic micro-
Ball 1.
(5) preparation of immunomagnetic beadses：By in the aldehyde group modified magnetic microsphere in 1mg step (4) and 5mg step (3)
FA-PEG-SANH mix, at 25 DEG C pH value be 6.0 PBS in mixing 2 hours after, carry out Magneto separate and obtain Folic Acid
Conjugated magnetic microsphere, be calculated on every milligram of folic acid immunity magnetic bead 1, coated antibody rate be 99% (amount of antibody of consumption with
The ratio of the antibody total amount adding).
The sensitivity Detection of embodiment 2 folic acid immunity magnetic bead
Collection healthy volunteer's blood sample, carries out PBMCs extraction by human lymphocyte separating liquid, then by human mouth epidermis
Sample cancerous cell KB (source Chinese Academy of Sciences cell bank is bought) suspension is proportionally added in PBMCs, makes the ratio of PBMCs and KB
It is respectively 103:1、104:1、105:1、106:1..Then the immunomagnetic beadses of embodiment 1 are added sequentially to above-mentioned each cell mixing
In suspension, it is incubated 30 minutes at 4 DEG C.Carry out magnetic separation and washed 2-3 time with PBS in 1 minute, obtain being caught with immunomagnetic beadses
The KB cell obtaining and reclaiming.And magnetic separation completed in 1 minute, illustrate that the magnetic responsiveness of immunomagnetic beadses is good.
Repeat the experiment under each concentration, result shows, may detect that KB cell under all concentration, folic acid immunity is described
The sensitivity of magnetic bead is good.
Embodiment 3 simulates the capture of tumor cell in blood
Collection healthy volunteer's blood sample, KB cell is mixed with the peripheral blood of Healthy People, is made into mixed cell suspension, adjusts
The concentration of KB cell is 1,10,20,50,500,1000/mL, then immunomagnetic beadses is added sequentially to above-mentioned each cell mixing
In suspension, it is incubated 30 minutes at 4 DEG C.Carry out magnetic separation and washed 2-3 time with PBS in 1 minute, obtain being caught with immunomagnetic beadses
The KB cell obtaining and reclaiming.The KB cell that statistics reclaims, still can capture KB when the concentration of KB cell is 1/mL thin
Born of the same parents.
From the point of view of the capture result of embodiment 2-3, the capture rate of (embodiment 2) can in simple environment for immunomagnetic beadses
It is accurate to 1：106, immunomagnetic beadses (embodiment 3) in complex environment may also detect that the KB cell of 1/mL, and sensitivity can
To meet the requirement of current liquid biopsy.
Resuspended with the PBS of no calcium and magnesium to the magnetic bead after above-mentioned incubation, the magnetic bead affording-cell is used
PicoPure RNA Isolation Kit test kit (Thermo Cat No：KIT0214) extraction purification total serum IgE, employs
PrimeScriptTMRT reagent Kit with gDNA Eraser(TAKARA,Cat No：RR047A), by specification will
Seek removal genomic DNA and carry out cDNA chain synthesis.Next it is related to corresponding Taqman primer, carry out qPCR detection, with
GAPDH compares qPCR testing result for internal standard, under the same conditions, consistent with the qPCR testing result of KB cell.Illustrate with this
Invention immunomagnetic ca pture tumor cell will not damaging cells, can be used for subsequent cell analysis.And illustrate, use the present invention
Immunomagnetic ca pture to cell substantially there is no PBMCs cell, PBMCs cell is not then combined with folic acid immunity magnetic bead substantially.
The breast cancer tumor cells capture of embodiment 4 cancer patient
Take Healthy People and the peripheral blood blood of different breast carcinoma cancer patient, the tumor cell in blood is captured.
Experimenter's routine blood test wbc value is required to be located at 2 × 106～1.2 × 107Between individual/mL, haemolysis or clot in whole blood sample
Condense.And experimenter's relevant information is complete, sample collection, store method specification, experimental implementation specification comprises the following steps that：
Take various cancers patient blood 3ml, add folic acid immunity magnetic bead to be added sequentially in above-mentioned each mixed cell suspension, at 4 DEG C
Incubation 30 minutes, then carried out magnetic separation in 1 minute and is washed 2-3 time with PBS, and the tumor cell that incubation is reclaimed is resisted
Body dyeing and on microscope slide fixing then carry out nucleus DAPI dyeing, after mounting, with fluorescence microscope identification, result is such as
Shown in Fig. 1, surface of cell membrane shows fluorescence, is green-emitting fluorescent, illustrates that the cell surface capturing is under fluorescence microscope
Folate receptor-positive, illustrates as tumor cell.And in the blood of normal person, do not capture tumor cell.
If first blood is carried out with erythrocyte splitting, and removed after the leukocyte in blood with CD45 antibody immune magnetic beads
Carry out the capture of tumor cell again with folic acid immunity magnetic bead, capture rate is higher.
Embodiment described above is one kind preferably scheme of the present invention, not the present invention is made any pro forma
Limit, for those skilled in the art, without departing from embodiment of the present invention principle and claim
Under the premise of, some improvements and modifications can also be made, these improvements and modifications are also considered as the protection domain of the embodiment of the present invention.
Claims (10)
1. a kind of folic acid immunity magnetic bead, including magnetic microsphere part and FA-PEG part it is characterised in that：Described immunomagnetic beadses
Structural formula is：A-NH-N=CH-B, wherein A represent magnetic microsphere, and B represents FA-PEG, or A represents that FA-PEG, B represent magnetic
Microsphere, described FA-PEG is obtained after being coupled FA and PEG.
2. folic acid immunity magnetic bead according to claim 1 it is characterised in that：Described magnetic microsphere part is nucleocapsid structure
The magnetic microsphere that inorganic or organic polymer wraps up.
3. folic acid immunity magnetic bead according to claim 2 it is characterised in that：The particle diameter of described folic acid immunity magnetic bead is 200
～300nm.
4. a kind of method preparing folic acid immunity magnetic bead any one of claim 1-3, its step includes：
S1. the preparation of magnetic Nano cluster；
S2. the preparation of amido modified magnetic microsphere；
S3. the preparation of the part A that diazanyl is modified；
S4. the preparation of aldehyde group modified part B；
S5. the preparation of immunomagnetic beadses：The part A that modify diazanyl described in step s3 and aldehyde group modified part B described in step s4
Mixing, carries out coupling reaction at 4～25 DEG C 2～24 hours, obtains described immunomagnetic beadses.
5. according to claim 4 folic acid immunity magnetic bead preparation method it is characterised in that：Diazanyl in described step s3 is repaiied
The part A of decorations is to pass through：Amido modified magnetic microsphere or SANH that FA-PEG molar equivalent is 10～30 times are carried out hydrazine
Base obtains after modifying.
6. according to claim 4 folic acid immunity magnetic bead preparation method it is characterised in that：Aldehyde radical in described step s4 is repaiied
The part B of decorations is to pass through：Amido modified magnetic microsphere or SFB that FA-PEG molar equivalent is 5～20 times are carried out aldehyde radical
Obtain after modification.
7. according to claim 4 folic acid immunity magnetic bead preparation method it is characterised in that：Magnetic in described step s1 is received
Rice cluster is to be prepared via a method which to obtain：
1) in the aqueous solution of divalent iron salt, in atmosphere, add ammonia, then stirring makes solution become black, obtains black
Fe3O4Granule；
2) to step 1) middle addition Oleic acid, it is transferred to after mix homogeneously in airtight reactor, reacting by heating at 60～130 DEG C
3～5 hours, you can obtain described magnetic Nano cluster.
8. according to claim 4 folic acid immunity magnetic bead preparation method it is characterised in that：Amino in described step s2 is repaiied
The magnetic microsphere part of decorations is the magnetic microsphere of the Silica-coated of nucleocapsid structure, is prepared via a method which to obtain：Xiang Han
It is magnetic in the solution of nano-cluster and adds ammonia, silylating reagent and amino silicane coupling agent, reaction obtained amino after 1～3 day
The magnetic microsphere part modified；The mass ratio that described magnetic Nano cluster, ammonia, silylating reagent, amino silicane coupling agent add
For：1:(12.5～40):(2～8):(0.5～3).
9. according to claim 4 folic acid immunity magnetic bead preparation method it is characterised in that：In described step s5, magnetic is micro-
The mass ratio of ball portion and FA-PEG part is 1：(0.01～1).
10. a kind of test kit for capturing tumor cell it is characterised in that：Contain described in claim 1 in described test kit
Folic acid immunity magnetic bead.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107653575A (en) * | 2017-10-24 | 2018-02-02 | 东华大学 | A kind of preparation method for the micro-fluidic chip for embedding hyaluronic acid functionalized nano-fiber film |
| CN113789302A (en) * | 2021-09-15 | 2021-12-14 | 南昌大学第二附属医院 | Enrichment and separation kit for lung cancer circulating tumor cells |
| CN118022690A (en) * | 2024-04-11 | 2024-05-14 | 杭州普望生物技术有限公司 | Surface-modified magnetic bead and preparation method and application thereof |
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| CN107653575A (en) * | 2017-10-24 | 2018-02-02 | 东华大学 | A kind of preparation method for the micro-fluidic chip for embedding hyaluronic acid functionalized nano-fiber film |
| CN107653575B (en) * | 2017-10-24 | 2019-07-23 | 东华大学 | A kind of preparation method for the micro-fluidic chip embedding hyaluronic acid functionalized nano-fiber film |
| CN113789302A (en) * | 2021-09-15 | 2021-12-14 | 南昌大学第二附属医院 | Enrichment and separation kit for lung cancer circulating tumor cells |
| CN118022690A (en) * | 2024-04-11 | 2024-05-14 | 杭州普望生物技术有限公司 | Surface-modified magnetic bead and preparation method and application thereof |
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