CN107525920B - Poly ion liquid magnetic nanocomposites and its application to trace enriching specificity of circulating tumor cell and detection - Google Patents
Poly ion liquid magnetic nanocomposites and its application to trace enriching specificity of circulating tumor cell and detection Download PDFInfo
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Abstract
A kind of application the invention discloses poly ion liquid magnetic nanocomposites and its to trace enriching specificity of circulating tumor cell and detection, the present invention provides one kind to have high sensitivity, high special and high accuracy magnetic Nano bioprobe, and for in the separation and enrichment of CTCs.Magnetic bead surfaces carboxyl functional group abundant can improve by being chemically bonded with the amino of antibody and significantly improve the bioaccumulation efficiency to CTCs in conjunction with the biggish specific surface area of magnetic bead itself and good dispersibility with the efficiency of EpCAM antibody coupling.Meanwhile the biocompatibility that poly ion liquid itself is excellent, it is significantly reduced the toxicity to cell, to improve the survival rate of cell after enrichment.
Description
Technical field
The invention belongs to medical sciences, and in particular to a kind of poly ion liquid magnetic nanocomposites and its application.
Background technique
One of the main reason for malignant tumour is threat human health and leads to death.Tumour cell has to surrounding tissue
Invasion or even the characteristic for spreading and shifting to whole body.It is reported that about 90% tumor patient dies of invasion and metastasis of tumor.Molecule
Biology and clinical study results show that metastases just have already appeared in tumorigenic early stage.However the hair of current tumour
The monitoring of imageological examination and traditional tumour marker is now still relied on clinical diagnosis, it is difficult in turning for early stage i.e. discovery tumour
It moves or recurs.Therefore, it in order to further increase the curative effect of oncotherapy, needs to find more effective curative effect and prognosis Testing index
And therapy target.The study found that detecting circulating tumor cell in the peripheral blood of patient during metastases
(Circulating Tumor Cells, CTCs) is therefore likely to become the early detection and detection of such tumour cell
It was found that a kind of effective means of tumour early stage transfer, to the early diagnosis of tumour, Index for diagnosis and treatment effectiveness evaluation etc.
With important reference value.
CTCs be usually by primary tumor tumour cell enter blood after change from.Its quantity in peripheral blood is extremely rare,
The concentration of CTCs only has 1~10/mL in the blood of usual tumor patient.Thus the detection of CTCs is generally first had to through excessive
Pre-treatment step from enrichment, to remove normal cell in most of blood.Physics is broadly divided into the separation and enrichment of CTCs
Method and biochemical process.Physical method mainly utilizes the difference of CTCs and blood normal cell in terms of physical property, such as size, close
Degree, suspending power size etc.；Biochemical process often relies on the antigen of cell membrane surface and the antibody being coupled on carrier of separating
It combines, achievees the purpose that separating trap CTCs.
CellSearch system be it is currently the only by U.S. Food and Drug Administration (FDA) approval be applied to clinic
The instrument of separation and concentration CTCs, cardinal principle are exactly to utilize the magnetic particle and tumor cell surface for being coupled EpCAM antibody
Antigen combines, and then separates under the influence of a magnetic field.It is common to have molecular biology method (as quantitatively to the CTCs that enrichment obtains
PCR it) is identified with cell biology method (such as immunofluorescence, immunocytochemical stain).However, the main of the system lacks
Falling into is that CTCs group is heterologous, and some CTCs do not express EpCAM.In addition, within the system, the CTCs of capture can lose original
Vigor.
Ferroso-ferric oxide (Fe3O4) magnetic nano-balls be the separation of current cell and enrichment aspect one of magnetic Nano material.
Compared to nano particle, Fe3O4Response speed of the nanosphere (MNs) during Magnetic Isolation faster, in the orientation of externally-applied magnetic field
Under control, by cleaning and desorption operations, can by object, quick separating is come out from multiple groups time-sharing environment, eliminate centrifugation, mistake
The cumbersome steps such as filter bring great convenience for the separation of complex system.But the Fe of nano-scale3O4MNs is because of its partial size
The features such as small, magnetic strong, easily reunite, makes it difficult to apply in terms of the separation of CTCs and enrichment.
Summary of the invention
The purpose of the present invention is the prior art on the basis of, using Magnet Treatment, with carboxyl poly ion liquid (PIL)
Ferroso-ferric oxide (Fe3O4) magnetic nano-balls (MNs) of functionalization are carrier of separating, with epithelial cell adhesion molecule antibodies
(anti-EpCAM) specific recognition molecules as CTCs in blood, it is immune by the way that it is carried out Bioconjugation preparation with MNs
Magnetic bead (IMNs), constructing one kind can be directly used for efficiently dividing trace CTCs in the tumour cells such as breast cancer, cervical carcinoma, liver cancer
From, enrichment, identification and detection nanometer bio probe, for malignant tumour early stage transfer diagnose and prognosis evaluation provide it is important
Reference frame.
It is rich in trace circulating tumor cell specificity that it is a further object of the present invention to provide a kind of above-mentioned nanometer bio probes
The application of collection and context of detection.
The purpose of the present invention can be achieved by the following measures:
It is a kind of can to the poly ion liquid magnetic nanocomposites of trace enriching specificity of circulating tumor cell or detection,
It is characterized in that it is prepared by following steps:
(A) synthesis of Carboxyl-functional Ionic Liquid monomer: bromoacetic acid and 1- vinyl imidazole are heated instead in a solvent
It answers, obtains Carboxyl-functional Ionic Liquid monomer；
(B)Fe3O4The synthesis of/SH magnetic nano-balls: by Fe3O4Magnetic nano-balls disperse in a solvent, 3- sulfydryl third to be added
Base trimethoxy silane is reacted, and Fe is obtained3O4/ SH magnetic nano-balls；
(C) synthesis of poly ion liquid functionalization magnetic bead: by Fe3O4/ SH magnetic nano-balls and Carboxyl-functional Ionic Liquid
After monomer and crosslinking agent mixing, initiator is added and carries out polymerization reaction, obtains poly ion liquid functionalization magnetic bead MNs；
(D) preparation of poly ion liquid magnetic nanocomposites: by MNs be scattered in the PBS solution containing EDC and NHS into
Row activation is resuspended in PBS solution then by the MNs of magnetic field separation activation, and EpCAM antibody is added and is incubated
It educates, repeatedly washs, obtain poly ion liquid magnetic nanocomposites IMNs.
The Carboxyl-functional Ionic Liquid monomer of the invention is by bromoacetic acid and 1- vinyl imidazole in 50 in toluene
It reacts and is made at~60 DEG C.Further, the carboxylic ions liquid the preparation method comprises the following steps: bromoacetic acid is dissolved in toluene after, delay
It is slow that 1- vinyl imidazole is added, it is stirred to react at 50~60 DEG C, solid is isolated after reaction, be drying to obtain.It is a kind of more specific
Synthetic method are as follows: take bromoacetic acid and toluene to be added in round-bottomed flask, 1.1mmol 1- vinyl imidazole be slowly added dropwise.At 60 DEG C
Reflux, magnetic agitation react 12h.It filters and is dried in vacuo through decompression, obtain white solid powder, i.e. carboxyl-functional ionic liquid
Body monomer (IL-Br).
Crosslinking agent of the invention, which is reacted by 1- vinyl imidazole with 1,4- dibromobutane, to be made.Further, the crosslinking
The synthetic method of agent BVD are as follows: anti-in 50~60 DEG C of stirrings after mixing 1- vinyl imidazole and Isosorbide-5-Nitrae-dibromobutane in methyl alcohol
It answers, it is after reaction that reaction mixture is cooling, it is then added in ether, postpones separation product for resulting translucent solution is cold, wash
Wash drying.
Fe of the invention3O4Fe in the prior art can be used in magnetic nano-balls3O4Magnetic nano-balls, it can also be direct
By FeCl3It is made after being reacted after being dissolved in ethylene glycol and water with sodium acetate and polyethylene glycol.Further, the Fe3O4It is magnetic
The synthetic method of nanosphere are as follows: take FeCl3, ethylene glycol and water mix to dissolution, sodium acetate and polyethylene glycol is then added
High-speed stirred is carried out, clear solution is obtained, is then reacted in autoclave in 160~210 DEG C, by products therefrom after reaction
Cooling, washing, it is dry to get.
In step (B), it is preferred that the solvent is toluene, Fe3O4Magnetic nano-balls and 3- mercaptopropyi trimethoxy
The amount ratio of silane is 1:0.1~5g/Ml, and preferably 1:0.1~3g/mL, reaction temperature is 60~100 DEG C；Product after reaction
After cooling, toluene, methanol, deionized water and ethanol washing are successively used, is then dried, it is spare under conditions of being placed in 1~5 DEG C.
In step (C), it is preferred that Fe3O4/ SH magnetic nano-balls and Carboxyl-functional Ionic Liquid monomer and crosslinking agent
It mixes in ethanol；Reaction temperature is 60~80 DEG C；After cooling reactant, cleaning, drying will be obtained after reaction, it is placed in 1~5 DEG C
Under conditions of it is spare；Fe3O4/ SH magnetic nano-balls are distinguished equal with the amount ratio of Carboxyl-functional Ionic Liquid monomer and crosslinking agent
For 1:0.01~0.04g/mol, the initiator is AIBN.
In step (D), it is preferred that first disperse MNs in the solution of the PBS of the pH 6.7~6.9 containing EDC and NHS
It is activated, the MNs then activated by magnetic field separation, then is washed with the PBS of pH 7.3~7.5, be resuspended in pH
In 7.3~7.5 PBS solution, EpCAM antibody is added and is incubated for, is then washed with the PBS of pH 7.3~7.5, finally by it
It is stored in the PBS of pH 7.3~7.5, is saved under conditions of being placed in 1~5 DEG C.
In step (D), it is preferred that the mass ratio of MNs and EpCAM antibody is 1:0.05~0.2, activation and incubation temperature
It is 1~5 DEG C.
Poly ion liquid magnetic nanocomposites IMNs of the invention has good stability in complex biological system, Neng Gouying
For the enrichment of trace CTCs in whole blood, it is especially applied to the specific enrichment and/or detection to trace circulating tumor cell
Aspect.
Circulating tumor cell of the invention includes but is not limited to MCF-7 Human Breast Cancer Cells, person monocytic cell's type lymthoma
One or more of THP-1 cell, human hepatoma HepG2 cell, typeⅡ pneumocyte, HeLa Cells.
The content of signified trace circulating tumor cell is 5~3000cell/mL in the present invention, may further reach 5
~400cell/mL, further can achieve 5~200cell/mL.
Spy is carried out to trace circulating tumor cell using poly ion liquid magnetic nanocomposites the present invention provides a kind of
Specific enrichment and/or the method for detection comprising following steps: on being added in the buffer of Xiang Hanyou trace circulating tumor cell
The poly ion liquid magnetic nanocomposites IMNs stated, or it is above-mentioned by being added after the whole blood dilution containing circulating tumor cell
Poly ion liquid magnetic nanocomposites IMNs, stationary incubation after mixing, then in buffer or whole blood under magnetic fields
Circulating tumor cell separated and captured, dyeing identification and/or detection is carried out to the cell captured.
In the method for being enriched with or detecting, buffer is preferably PBS buffer solution；The time of stationary incubation is 2~30min,
Preferably 2~25min or 5~25min, most preferably 20min.
In the method for being enriched with or detecting, the levels after cell are added in poly ion liquid magnetic nanocomposites IMNs
For 0.1-0.5mg/mL.The concentration of IMNs has duality to the influence that CTCs is enriched with.On the one hand, the concentration of IMNs is higher, nothing
It by being the quantity of EpCAM antibody or the speed of Magnetic Isolation and efficiency, all can significantly increase, thus be to the enrichment of CTCs
It is advantageous；However, due to the collision between nanoparticle, magnetic bead is easy to happen autohemagglutination, thus can shadow if the excessive concentration of IMNs
The absorption between IMNs and cell is rung, the enrichment of CTCs is unfavorable for.Discovery is tested repeatedly, and the levels after IMNs is added are
Bioaccumulation efficiency can be made to reach best when 0.2-0.4mg/mL, especially 0.3mg/mL.
Spy is carried out to trace circulating tumor cell using poly ion liquid magnetic nanocomposites the present invention provides a kind of
The method of specific enrichment and detection: the imidazole ion liquid monomer (IL) and Fe of carboxyl-functional are prepared first3O4Magnetic Nano
Ball.On this basis, by the way that 3-mercaptopropyi trimethoxy silane is added, carboxylated poly ion liquid (PIL) functionalization is prepared
Fe3O4Magnetic nano-balls, and it is named as magnetic bead (MNs).The carrier for separating and being enriched with as CTCs.By MNs's and CTCs
Immunomagnetic beads are made in specific recognition molecules-signal transduction factor antibody (1.0mg/mL) Bioconjugation at room temperature
(IMNs)。
IMNs produced by the present invention is added in Mouse whole blood, after centrifuge washing 5 times, measures it at 580nm
Absorbance, and in addition before dulling luminosity ratio compared with the Biostatic of IMNs of the invention can be investigated out to calculate the rate of recovery
Property.
In application aspect, experimental selection human breast carcinoma MCF-7, lung cancer A549, liver cancer HepG2 of the invention, cervical carcinoma
Hela cell simulates CTCs model, using person monocytic cell THP-1 as negative control cell.It, will be certain density at 4 DEG C
Above-mentioned four kinds of cells and IMNs stationary incubation after a certain period of time, are placed in Magnetic Isolation on magnet, supernatant are removed, through PBS (pH
7.4) it after washing 3 times and collecting cleaning solution, is resuspended in PBS (pH 7.4) solution.It to supernatant and is washed using flow cytometer
It washs in liquid and is not counted by the IMNs cell captured, calculate IMNs to the bioaccumulation efficiency of CTCs.DAPI dyestuff pair is used simultaneously
The cell captured by IMNs is dyed, to verify to result.
The present invention further by taking MCF-7 as an example, has investigated the specificity that IMNs is enriched with CTCs, that is, uses identical experiment
THP-1 cell is incubated for altogether with IMNs respectively and MNs and MCF-7 cell is incubated for altogether, calculates separately and compare three kinds by method
Under the conditions of bioaccumulation efficiency, and result is verified using confocal fluorescent micro-imaging.Comparison is with and without functional poly ion
Liquid modifies obtained IMNs to the height of CTCs bioaccumulation efficiency, to illustrate poly ion liquid to the shadow of this method sensitivity
It rings.
The present invention shows the MNs and IMNs that are successfully prepared poly ion liquid functionalization by various test methods.IMNs
It after being centrifuged in Mouse whole blood and washing 5 times, is tested through UV-vis, calculating its rate of recovery is (95.72 ± 0.88) %, the rate of recovery
Well, meet the requirement tested to stability of material.Specificity experiments the result shows that, IMNs is only to human breast carcinoma MCF-7 tumour
Cells show has gone out higher bioaccumulation efficiency, and bioaccumulation efficiency can reach 97% or more, and hardly to person monocytic cell THP-1
((3.8 ± 2.2) %) (n=3) can be identified and be captured, there is significant difference with tumour cell；Meanwhile unmodified EpCAM is anti-
The MNs of body equally also can not almost identify with enriched tumor cell ((3.8 ± 1.4) %, n=3), this illustrates IMNs to CTCs's
Enrichment has high degree of specificity.DAPI immunofluorescence dyeing is carried out respectively to MCF-7 the and THP-1 cell after enrichment, as a result table
Bright MCF-7 cell is that DAPI core dye is positive, and THP-1 shows as feminine gender, and coloration result is consistent with bioaccumulation efficiency quantitative result.Together
When, the IMNs of poly ion liquid modification will be significantly larger than the IMNs of no poly ion liquid modification to the bioaccumulation efficiency of tumour cell
(bioaccumulation efficiency of the two is respectively (97.6 ± 1.1) %, (18.4 ± 2.3) %).IMNs prepared by the present invention to MCF-7,
The bioaccumulation efficiency of tetra- kinds of tumour cells of A549, HepG2 and Hela can respectively reach (97.6 ± 1.1) %, (97.6 ±
1.3) %, (95.2 ± 1.1) % and (91.1 ± 4.1) % (n=3).
IMNs of the present invention is in three kinds of media (PBS (pH 7.4) buffer, MCF-7 and THP-1 cell mixture, whole blood)
In, good linear relationship is presented to the MCF-7 cell in 5~400cell/mL concentration range and higher enrichment is imitated
Rate, equation of linear regression are respectively y=0.96x+0.65 (R2=0.999), y=0.97x+0.17 (R2=0.999), y=
0.97x–1.57(R2=0.999).The result shows that IMNs is in arbitrary medium, it can the certain density CTCs of efficiently concentrating.
Three kinds of colouring methods the tumour cell in the simulation clinical sample being enriched to is identified after the result shows that,
MCF-7 cells show is DAPI nuclei staining positive, and anti-CD45 is negative staining, anti-CK19 stained positive；The leucocyte of surrounding is anti-
CD45 stained positive, anti-CK19 are negative staining.Cell activity experimental result is shown, most thin after being incubated for IMNs
Born of the same parents are still able to maintain greater activity after enrichment, and cell survival rate is about 98.8%.
The present invention provides one kind to have high sensitivity, and high special and high accuracy magnetic Nano bioprobe is used in combination
In the separation and enrichment to CTCs.Magnetic bead surfaces carboxyl functional group abundant, can by being chemically bonded with the amino of antibody,
It improves and is significantly improved with the efficiency of EpCAM antibody coupling in conjunction with the biggish specific surface area of magnetic bead itself and good dispersibility
To the bioaccumulation efficiency of CTCs.Compared with a variety of reported methods, this work is higher to the bioaccumulation efficiency of CTCs, can reach
97% or more.Molecule of this experimental selection EpCAM antibody as specific recognition CTCs.The antibody can efficient identification CTCs,
Under same experimental conditions, only has (3.8 ± 2.2) % to the bioaccumulation efficiency of negative control cell THP-1, well below breast cancer
MCF-7 ((97.6 ± 1.1) %).The experimental results showed that this method has preferable potential applicability in clinical practice, it can be clinical tumor
The detection of trace CTCs and tumour early stage transfer diagnosis provide important reference frame in blood samples of patients.
Detailed description of the invention
Fig. 1 is the preparation of IMNs of the present invention and the schematic diagram to tumor cell enrichment process.
Fig. 2 is the resulting Fe of embodiment3O4The TEM of nanosphere (A), MNs (B) and IMNs (C) schemes；
It is seen that the Fe without functionalization3O4Nanosphere dispersibility is preferable, average grain diameter 210nm.When
Carboxyl-functional poly ion liquid is wrapped up to Fe3O4After nanometer ectosphere, obtained functionalization magnetic bead can be significantly observed
Partial size become larger, be computed, average diameter reaches 290nm；And after EpCAM antibody further modifies magnetic bead surfaces, due to
The covering of large biological molecule film, the partial size of obtained immunomagnetic beads continue to increase to 320nm or so.Above-mentioned three kinds of materials are average
Partial size incrementally increases, and also illustrates poly ion liquid and antibody in Fe3O4The successful modification of nanometer ball surface.It is worth noting
, to Fe3O4Before and after nanosphere is modified, above-mentioned three kinds of materials maintain good dispersion performance in ethanol.Magnetic
Pearl or immunomagnetic beads can keep good dispersion performance in blood sample, contact it sufficiently with cell, this is that CTCs successfully divides
From essential condition.Thus this result haves laid a good foundation for the Magnetic Isolation of CTCs in subsequent experimental.
Fig. 3 is Fe obtained by embodiment3O4The XRD spectrum of nanosphere and MNs；
Wherein upper curve be MNs, it can be seen that 2 θ be 30.1 °, 35.5 °, 43.1 °, 57 °, 62.6 ° of 5 characteristic peaks
Corresponding is standard ferroso-ferric oxide { 220 }, { 311 }, { 400 }, { 511 } and { 440 } 5 planes.Meanwhile MNs
Characteristic peak and Fe3O4The characteristic peak of nanosphere is completely the same.This shows to be successfully prepared with cubic spinel structure
Fe3O4, and the functional modification of poly ion liquid can't destroy original Fe3O4Crystalline structure.
Fig. 4 is Fe3O4Nanosphere (a), MNs (b), the full spectrum analysis of surface composition of IMNs (c) and above-mentioned three kinds of nanometer materials
The high-resolution XPS spectrum figure (d) of C1s in material；
Since the precursor structure of poly ion liquid is vinyl imidazole, containing N element, and in preparation MNs and IMNs process
In used-SH, therefore scheme compared to a, occur the characteristic absorption peak of apparent N1s and S2p in b and c figure, respectively position
It can be at 401.8 and 167.6eV in combination.Fe3O4The characteristic peak of middle Fe2p at 710.7eV, with nanometer ectosphere gathered from
Sub- liquid and antibody gradually cover, and the intensity at the peak also gradually weakens, and completely disappear in IMNs.The high-resolution energy of the C1s of d figure
Spectrum the result shows that, Fe3O4The C1s characteristic peak of C element in nanosphere and MNs is located at 284.5eV (black and green curve),
And the characteristic peak of C1s is located at 286.0eV at (red curve) in IMNs, confirms after Literature Consult, this absorption peak at two
C=C the and C-N key that can be respectively belonging in aromatic rings38, this is also absolutely proved ,-the NH of antibody surface2With in MNs-
COOH has passed through ammonia carboxylic reaction bonded together.The result of XPS further demonstrates that poly ion liquid and antibody are successfully modified
To Fe3O4Nanometer ball surface, has successfully been made the immunomagnetic beads that can be used in CTCs separation and enrichment.
Fig. 5 is the hydraulics grain size curve that IMNs is added to (A) and rear (B) before incubation in Mouse whole blood；
Before incubation, the average grain diameter of IMNs is 321.3nm or so.After incubation, partial size does not almost change
(339.6nm).It can be seen that IMNs can resist nonspecific absorption, keep stable shape in complicated biotic environment
Specific recognition occurs for state and CTCs.
Fig. 6 is bright after IMNs (A, B) and MNs (C, D) mark goat anti-mouse lgG to be incubated for 30min altogether with FITC respectively
Field and fluorogram.
Fig. 7 is (A) IMNs to MCF-7, the bioaccumulation efficiency of THP-1, comparison of the MNs to the bioaccumulation efficiency of MCF-7；(B)
IMNs is to HepG2, A549, the bioaccumulation efficiency of HeLa cell；
Wherein, cell concentration: 200cells/mL.Fig. 9 A shows that designed IMNs is thin to the MCF-7 of 200cell/mL
The bioaccumulation efficiency of born of the same parents can achieve 97% or more ((97.6 ± 1.1) %, n=3).Meanwhile it being enriched with to verify IMNs to CTCs
Specificity, choose person monocytic cell THP-1 as negative control cell, and under same experimental conditions, carried out MNs respectively
To MCF-7 cell and IMNs to the enrichment experiment (Fig. 9 A) of THP-1 cell.With IMNs to the bioaccumulation efficiency phase of MCF-7 cell
Than MNs is to MCF-7 cell almost without accumulation ability ((3.8 ± 1.4) %, n=3), and this is mainly due to the spies for lacking CTCs
Opposite sex identification antibody.Meanwhile IMNs also only has (3.8 ± 2.2) % (n=3) to the bioaccumulation efficiency of THP-1 cell, well below
MCF-7 cell, it was demonstrated that IMNs is high degree of specificity to the enrichment of MCF-7 cell.On this basis, it also further investigates
Bioaccumulation efficiency of the IMNs to human lung cancer A549, liver cancer HepG2 and cervical cancer Hela cells.It is similar to MCF-7 cell results, IMNs
It still is able to above-mentioned three kinds of tumour cell efficiently concentratings, bioaccumulation efficiency is respectively (97.6 ± 1.3) %, (95.2 ± 1.1) %
(91.1 ± 4.1) % (n=3) (Fig. 9 B).
Fig. 8 is the DAPI fluorescent staining figure of the MCF-7 (A, B, C) and THP-1 cell after IMNs enrichment；
Wherein, A and D: light field figure；B and E: fluorescent staining figure；C and F: the stacking chart of light field and fluorescent staining.In light field figure
In, it can be observed, the IMNs particle after capturing cell becomes coherent condition (figure A) centered on cell, from dispersed.Exist simultaneously
In fluorescent staining figure, the peculiar sapphirine nucleus of DAPI dyeing is showed, and cellular morphology preferably preserves (figure B),
Illustrate that IMNs successfully captures MCF-7 cell；It is most of in light field however in the IMNs after THP-1 cell enrichment
IMNs particle is individually present (figure D), and the sapphirine for not occurring DAPI dyeing in the full visual field under fluorescence microscope is thin
Karyon (figure E), shows that IMNs can not identify and be enriched with the cell.Result above absolutely proves, identification of the IMNs to tumour cell
There is specificity with enrichment.
Fig. 9 is influence of (A) enrichment time to IMNs and MCF-7 cell enrichment efficiency；(B) IMNs concentration to IMNs with
The influence of MCF-7 cell enrichment efficiency；
Wherein, MCF-7 cell concentration: 200cells/mL.Obtained IMNs is to MCF-7 cell under 5 enrichment times
Bioaccumulation efficiency be respectively (77.9 ± 3.5) %, (90.9 ± 4.2) %, (92.5 ± 3.0) %, (97.0 ± 2.3) %, (97.6
± 2.1) % (n=3).It is right by the IMNs (0.1,0.2,0.3,0.4,0.5mg/mL) of 5 concentration as shown in (B) of Fig. 9
The bioaccumulation efficiency of CTCs, it can be found that bioaccumulation efficiency also increases with the increase of IMNs concentration, but when IMNs concentration is super
When crossing 0.3mg/mL, bioaccumulation efficiency declines instead.
Figure 10 is various concentration MCF-7 cell in PBS (pH 7.4), THP-1 cell (106/ mL) mixed liquor and whole blood
In bioaccumulation efficiency.
Figure 11 is three kinds of immunocytochemical techniques to MCF-7 in the simulation clinical blood sample after IMNs enrichment and white
The identification of cell；
In figure, DAPI: core dye, excitation wavelength 405nm, launch wavelength 447nm；CK19(Alexa594):
Excitation wavelength is 605nm, launch wavelength 685nm；CD45(Alexa488): excitation wavelength 488nm, launch wavelength
For 525nm.Combination chart is the stacking chart of three kinds of fluorescent stainings.
Figure 12 is the superposition fluorogram of the DAPI (A) of the MCF-7 cell after IMNs enrichment, PI (B) and two kinds of dyeing
(C)。
Specific embodiment
Below in conjunction with drawings and examples, the present invention will be further described, but protection scope of the present invention is not limited to
Following embodiment.
The preparation of 0.01M PBS buffer solution (pH 6.8): precision weighs 0.6119g NaH respectively2PO4, 2.621g
Na2HPO4·12H2O is dissolved in the distilled water of certain volume, with 1000.00mL is settled to after 1M HCl tune pH to 6.8, at room temperature
It saves.
The preparation of 0.01M PBS buffer solution (pH 7.4): PBS dry powder is dissolved in certain volume distilled water, takes 5mL bis-
Residual powder in water dissolution bag is steamed by solvent and washing lotion in 2000.00mL volumetric flask constant volume, to save at room temperature in triplicate.
The preparation of 0.1%Triton-X 100: taking 1mL Triton-X 100 to be dissolved in 900mL PBS (pH 7.4), fixed
Hold to 1000.00mL, 4 DEG C of preservations.
The preparation of 1%BSA: taking 1g Triton-X 100 to be dissolved in 100mL PBS (pH 7.4), 4 DEG C of preservations.
Embodiment 1: the synthesis of carboxylic ions liquid (IL)
Take bromoacetic acid 10mmol and 10mL toluene to be added in dry round-bottomed flask, it is to be dissolved completely after, be slowly added dropwise
1.1mmol 1- vinyl imidazole.It flows back at 60 DEG C, magnetic agitation reacts 12h.After reaction, decompression, which filters, obtains white admittedly
Body, 50 DEG C of vacuum drying for 24 hours, obtain white solid powder.It is spare to be placed in 4 DEG C of refrigerators.The structural formula of gained ionic liquid is as follows:
M/z 153.0 is the cation peak of the electrospray ionization mass spectrum obtained in the positive-ion mode, with ionic liquid IL-Br's
Molecular formula matches.Using DMSO as solvent, ionic liquid monomer IL-Br is carried out1HNMR analysis, spectral data analysis such as table
Shown in 1.
1. ionic liquid monomer IL-Br's of table1HNMR analyzes result
Embodiment 2: the synthesis of crosslinking agent B VD
Take 1- vinyl imidazole (2.82g, 30mmol) and Isosorbide-5-Nitrae-dibromobutane (13g, 60mmol) in 5mL methanol, 60
DEG C stirring 20h.Reaction mixture is cooled to room temperature, is then added in 250mL ether.Resulting translucent solution is placed in ice
5h in case.Solid product supernatant is separated by decantation, is washed for several times with ether, 50 DEG C of vacuum drying for 24 hours, are placed in 4 DEG C of refrigerators
It is spare.
Embodiment 3:Fe3O4The synthesis of magnetic nano-balls
Take FeCl3(0.405g, 25mmol) and ethylene glycol (20.0mL), water (0.270g, 270 μ L) mixing, stirring until
FeCl3It is completely dissolved.Then, anhydrous addition sodium acetate (1.8g), polyethylene glycol (0.5g) high-speed stirred 30min, obtains transparent
Solution.Acquired solution is transferred to 200 DEG C of reaction 16h in autoclave.Products therefrom is cooling, and with hot water and ethanol washing number
It is secondary.50 DEG C of vacuum drying for 24 hours, it is spare to be placed in 4 DEG C of refrigerators.Gained Fe3O4Infrared, the TEM of nanosphere, XPS, the methods of XRD are carried out
Characterization.
Embodiment 4:Fe3O4The synthesis of/SH magnetic nano-balls
Take Fe3O4Magnetic nano-balls (3.0g) are dispersed in dry toluene (20.0mL), and excessive 3- mercaptopropyi three is added
Methoxy silane (3.0mL).By suspension mechanical stirring 12h under reflux.Products therefrom is cooling, successively use toluene, methanol,
Deionized water and ethanol washing are for several times.50 DEG C of vacuum drying for 24 hours, it is spare to be placed in 4 DEG C of refrigerators.
Take Fe3O4Magnetic nano-balls (0.382g) are dispersed in dry toluene (20.0mL), and excessive 3- mercaptopropyi is added
Trimethoxy silane (2.0mL).By suspension mechanical stirring 12h under reflux.Products therefrom is cooling, successively use toluene, first
Alcohol, deionized water and ethanol washing are for several times.50 DEG C of vacuum drying for 24 hours, it is spare to be placed in 4 DEG C of refrigerators.Embodiment 5: magnetic bead (MNs)
Preparation
By 0.1g Fe3O4The IL and BVD of/SH and 0.002mol is mixed in 8.0mL ethyl alcohol.After 1.0%AIBN is added, mixing
Object dynamic respons 10 hours at 70 DEG C.It is cooling that reactant will be obtained, successively cleaned three times with deionized water and ethyl alcohol, 50 DEG C true
Sky is dry for 24 hours, and it is spare to be placed in 4 DEG C of refrigerators.
Compare Fe3O4The Fourier infrared absorption spectrogram of nanosphere and MNs, is compared to Fe3O4Nanosphere, MNs exist
3380cm-1Place has increased an absorption peak newly.Meanwhile 1625cm-1The absorption peak strength at place is remarkably reinforced.Above-mentioned two absorption peak
- the OH and C=O in COOH group are corresponded respectively to, is thus proved, in Fe3O4Nanometer ball surface has successfully modified carboxyl-functional
Poly ion liquid.
The synthesis of embodiment 6:IMNs
It disperses the MNs of 5mg in the solution of 0.01M pH 6.8PBS of 1mL EDC containing 50mM and 50mM NHS, room temperature
30min is gently shaken to be activated.By the MNs of magnet separation activation, and washed three times with 0.01M PBS (pH 7.4).It will
In its PBS for being resuspended in 1mL (pH 7.4), 50 μ g EpCAM antibody are added, room temperature, which is shaken, is incubated for 4h.Gained IMNs PBS (pH
7.4) washing is stored in PBS (pH 7.4) for several times to remove superfluous antibody, it is spare to be placed in 4 DEG C of refrigerators.
It disperses the MNs of 5mg in the solution of 0.01M pH 6.8PBS of 1mL EDC containing 50mM and 50mM NHS, room temperature
30min is gently shaken to be activated.By the MNs of magnet separation activation, and washed three times with 0.01M PBS (pH 7.4).It will
In its PBS for being resuspended in 1mL (pH 7.4), 100 μ g EpCAM antibody are added, room temperature, which is shaken, is incubated for 4h.Gained IMNs PBS
(pH 7.4) washing is stored in PBS (pH 7.4) for several times to remove superfluous antibody, it is spare to be placed in 4 DEG C of refrigerators.
50 μ g IMNs and MNs are respectively taken, are scattered in the FITC- goat anti-mouse IgG solution of 100 μ L, 5 μ g/mL respectively, 4
DEG C be incubated for half an hour.It is adsorbed with magnet, PBS is cleaned three times, and under the microscope in fluorescence microscopy, result figure 6 has shown EpCAM antibody
Modify the surface MNs.
The investigation of the stability of embodiment 7:IMNs
Partial size test: 0.3mg IMNs made from Example 6 is scattered in 1mL deionized water, in particle size analyzer
Detection.
The rate of recovery investigates (UV-VIS spectrophotometry): taking 0.3mg IMNs to be scattered in 1mL rat blood, 4 DEG C incubate
20min, magnetic-adsorption 5min are educated, gained IMNs is washed for several times with PBS (pH 7.4), and is scattered in 1mLPBS (pH 7.4),
Its absorbance at 580nm is tested, the dulling luminosity ratio before being added in blood with IMN is compared with being calculated as follows the recycling of IMNs
Rate:
Recovery=Ac/A0× 100%
Wherein AcIt is that IMNs is added in blood surveyed absorbance, A after soldier's incubation0It is to be surveyed before IMN is added in blood
Absorbance.
This experiment the result shows that IMNs has good stability in complex biological system, can be applied to trace in whole blood
The enrichment of CTCs.
The application of embodiment 8:IMNs
Cell culture and processing:
Cell line used in testing includes human breast carcinoma cell lines MCF-7, lung cancer cell line A549, liver cancer HepG2, son
Cervical cancer tumer line HeLa, person monocytic cell's type lymphoma cell, THP-1；Wherein MCF-7 is adherent growth cell line.THP-1
For suspension cell.
Cell culture operations:
MCF-7, A549, THP-1 cell culture are in being added to 10% fetal calf serum (FBS) and dual anti-(100U/mL mould
Element, 100U/mL streptomysin) 1640 culture mediums in.
HeLa, HepG-2 cell culture in be added to 10% fetal calf serum (FBS) and it is dual anti-(100U/mL penicillin,
100U/mL streptomysin) DMEM culture medium in.
MCF-7 cell culture is in being added to 10% fetal calf serum (FBS) and dual anti-(100U/mL penicillin, 100U/mL chain
Mycin) 1640 culture mediums in.
Cell is placed in 37 DEG C, 5%CO in culture bottle2It is cultivated in incubator.
Cell cryopreservation and recovery:
Cell recovery: by the cryopreservation tube of required cell, tank is taken out in liquid nitrogen, is immediately placed in 37 DEG C of water-baths quickly
Agitation dissolves cells frozen storing liquid, and frozen stock solution is transferred to 15mL centrifuge tube, supplements 3~4mL culture medium, 1500R/min centrifugation
5min discards supernatant liquid, and 5mL culture medium is taken gently to blow and beat cell precipitation, mixes, is inoculated in 25mm2In culture bottle, it is placed in 37
DEG C, 5%CO2It is cultivated in incubator.
Cell cryopreservation: collect logarithmic growth phase cell suspension, 1500r/min be centrifuged 5min, abandon supernatant, take culture medium and
DMSO 9:1 prepares cells frozen storing liquid, cell precipitation is resuspended with cells frozen storing liquid, concentration is about 1 × 107/ mL, gently
Piping and druming mixes, and suspension is transferred in steril cell cryopreservation tube (1mL/ pipe), and closing is placed on 4 DEG C (30min), and -20 DEG C
(30min), is finally placed in liquid nitrogen container and saves by -80 DEG C (overnight).
Attached cell changes liquid and passage:
Replacement in culture medium every two days is primary, cleans cell with appropriate PBS when changing liquid；When adherent cell growth is extremely under microscope
70% or more needs to be passed on.Culture medium is discarded first, and pancreatin 37 DEG C digestion of the 1mL containing EDTA are then added in cell bottle
Appropriate time；Culture medium is added immediately when cell periphery is mellow and full luminous and terminates digestion, is blown and beaten cell uniformly with pasteur pipet,
Gained cell suspension 1:3 is transferred into new culture bottle, 4mL culture medium is added, is placed in cell incubator.
Suspension growth cell changes liquid and passage:
Cell suspension 1000R/min is centrifuged 3min, precipitating 1:3 is resuspended in fresh medium.
Cell count:
By blood counting chamber cotton ball soaked in alcohol wiped clean, 75% alcohol rinse, natural air drying are used；Cover plate is placed in meter
On number plate, count block is covered；It is (existing using capillary from tally count block sample-adding respectively that 10 μ L cell suspensions are drawn using pipettor
Count block is infiltrated through as filling suspension voluntarily)；It is placed under inverted microscope and is counted；To each of upper and lower two region when counting
4 middle lattice (inside having 4 × 4 small lattice) are counted, and the calculating of crimping individual instances, which is taken, removes, and take the left original for not taking the right side
Then.
Cell is fixed and is dyed:
Cell is fixed: discarding cell/magnetic bead culture medium or PBS (pH 7.4), 4% paraformaldehyde is added, stands at room temperature
10min；Cell is washed twice with PBS；(the room cell 5min after being fixed with the PBS solution permeabilization of the X-100 containing 0.1%Triton
Temperature), PBS (pH 7.4) is washed cell 3 times.
DAPI dyeing: discarding cell/magnetic bead culture medium or PBS (pH 7.4), with 10mM PBS, pH7.4, cleaning one
It is secondary, then it is added dropwise to the DAPI dye liquor of 100 μ L, room temperature is protected from light 5~15min of dyeing, discards dyeing liquor, PBS (pH 7.4) rinsing one
It is secondary.Glycerol or Buffer A is added dropwise, fluorescence microscope is taken pictures with 340/380nm burst of ultraviolel, observation.
The dyeing of Anti-CK19 antibody: will fix cell and be scattered in the Anti-CK19 antibody of 100 μ L, 5 μ g/mL, and 4 DEG C
It is incubated overnight.Glycerol is added dropwise, fluorescence microscope is excited with 590/617nm, is observed, is taken pictures.
The dyeing of Anti-CD45 antibody: will fix cell and be scattered in the Anti-CD45 antibody of 100 μ L, 20 μ g/mL, and 4
It DEG C is incubated overnight.Glycerol is added dropwise or Buffer A, fluorescence microscope are excited with 488/519nm, observes, takes pictures.
Identification of the IMNs to MCF-7 and THP-1 cell
The resulting IMNs of embodiment 6 is to the enrichment process of CTCs as shown in schematic diagram 1.By MCF-7 cell, THP-1 cell,
After collecting and counting being resuspended, it is diluted to 2000/mL using PBS (pH 7.4) buffer, is placed in 1.5mL centrifuge tube, and point
Not Jia Ru same concentrations IMNs, then 4 DEG C of stationary incubation 20min after mixing divide the cell in suspension using magnet
From with capture (5min).After capture, Aspirate supernatant is moved in another clean centrifuge tube, and the cell that will be captured PBS (pH
7.4) buffer be resuspended and is washed twice, and is merged with the supernatant drawn for the first time, molten to this using drain cell analyzer
It is not counted by the IMNs cell captured in liquid.The cell that IMNs is captured simultaneously is fixed, and DAPI dyeing is carried out, and fluorescence is aobvious
Micro- microscopic observation.As a result see Fig. 7.
Identification of the MNs and IMNs to MCF-7 cell
After collecting by MCF-7 cell and counting is resuspended, it is diluted to about 2000/mL with PBS (pH 7.4) buffer, is placed in
In 1.5mL centrifuge tube, and it is separately added into the IMNs and MNs of same concentrations, then 4 DEG C of stationary incubation 20min after mixing use magnetic
Iron is separated and is captured to the cell in suspension.After capture, Aspirate supernatant is moved in another clean centrifuge tube, and will capture
To cell be resuspended with PBS (pH 7.4) buffer and wash twice, and merge with the supernatant drawn for the first time, using streaming
Cytoanalyze counts cell at large in the solution.Calculating bioaccumulation efficiency, i.e. cell capture rate=(capture
Number of cells/stock Cell number) × 100%.The cell that will be captured simultaneously is fixed, and DAPI dyeing, fluorescence microscope are carried out
Lower observation.As a result see Fig. 8.
Capture of the IMNs to other type tumour cells
After collecting by HepG2, A549, HeLa cell and counting is resuspended, 2000 are diluted to using PBS (pH 7.4) buffer
A/mL is placed in 1.5mL centrifuge tube, and is separately added into the IMNs of same concentrations, 4 DEG C of stationary incubation 20min after mixing, then
The cell in suspension is captured using magnet.After capture, Aspirate supernatant is moved in another clean centrifuge tube, and will capture
To cell be resuspended with PBS (pH 7.4) buffer and wash twice, and merge with the supernatant drawn for the first time, using streaming
Cytoanalyze counts cell at large in the solution.Cell capture rate=(number of cells/stoste of capture is thin
Born of the same parents' number) × 100%.As a result see Fig. 8.
Concentration used when IMNs is with MCF-7 cell incubation time and cell incubation
After collecting by MCF-7 cell and counting is resuspended, it is diluted to 2000/mL using PBS (pH 7.4) buffer, is placed in
In 1.5mL centrifuge tube, and it is separately added into the IMNs of same concentrations, 4 DEG C of stationary incubations 2min, 5min, 10min after mixing,
20min, 30min are measured in parallel 3 times.Then the cell in suspension is captured using magnet.After capture, Aspirate supernatant
It moves in another clean centrifuge tube, and the cell that will be captured be resuspended with PBS (pH 7.4) buffer and be washed twice, and with
The supernatant drawn for the first time merges, and is counted using stream type cell analyzer to cell at large in the solution.Cell
Capture rate=(number of cells of capture/stock Cell number) × 100%.As a result see Fig. 9.
After collecting by MCF-7 cell and counting is resuspended, it is diluted to 2000/mL using PBS (pH 7.4) buffer, is placed in
In 1.5mL centrifuge tube, and it is separately added into the IMNs of 0.1,0.2,0.3,0.4,0.5mg/mL, 4 DEG C of stationary incubations after mixing
Then 20min captures the cell in suspension using magnet.After capture, Aspirate supernatant moves to another clean centrifuge tube
In, and the cell that will be captured be resuspended with PBS (pH 7.4) buffer and be washed twice, and is closed with the supernatant drawn for the first time
And cell at large in the solution is counted using stream type cell analyzer.The cell capture rate=(cell of capture
Number/stock Cell number) × 100%.As a result see Fig. 9.
The range of linearity that IMNs captures MCF-7 cell-specific
Respectively at PBS (pH 7.4), THP-1 cell suspending liquid after MCF-7 cell suspension in normal growth is counted
(106A/mL, PBS (pH 7.4)) and whole blood in, and be diluted to the concentration of 5,50,100,200,300,500/mL respectively.Benefit
With stream type cell analyzer to the cell stoste accurate counting of above-mentioned several concentration after, sequentially add certain density IMNs, it is quiet
It sets and is incubated for 20min.Then the cell in suspension is captured using magnet.After capture, Aspirate supernatant moves to another clean
In centrifuge tube, and the cell that will be captured be resuspended with PBS (pH 7.4) buffer and be washed twice, and upper with drawing for the first time
Clear liquid merges, and is counted using stream type cell analyzer to cell at large in the solution.Cell capture rate=(capture
Number of cells/stock Cell number) × 100%.
In figure 12 it can be seen that in PBS (pH 7.4) solution, with the increase of cell original liquid concentration, by IMNs richness
The number of cells collected is also linearly increasing, and equation of linear regression is y=0.96x+0.65 (R2=0.999)；When 5 MCF-7 are thin
Born of the same parents are added in immunomagnetic beads, can be enriched to 4, and the cell of this 4 captures all keeps activity, bioaccumulation efficiency is
83%；In addition to the concentration, IMNs is to the bioaccumulation efficiency of concentration MCF-7 cell larger in the concentration range 92% or more；
Meanwhile IMNs to the accumulation ability of the MCF-7 of same concentrations in MCF-7 and THP-1 cell mixture and whole blood medium also compared with
Height, 90% or more, equation of linear regression is respectively y=0.97x+0.17 (R2=0.999) (n=3), y=0.97x-
1.57(R2=0.999) (Figure 12).And statistical result shows that bioaccumulation efficiency of the MCF-7 in three kinds of media does not have conspicuousness
Difference (P > 0.05).
The identification of tumour cell after capture
The MCF-7 cell that above-mentioned experiment captures is resuspended in PBS (pH 7.4), uses DAPI, anti-respectively
CK19, anti-CK45 dye it, and observe under fluorescence microscope, have round cell and DAPI to ellipse
With the CK19 positive, the cell of CD45 feminine gender is accredited as tumour cell.
The result is shown in Figure 11.It can be seen from the figure that (1) MCF-7 tumour cell volume is greater than leucocyte volume (general feelings
Condition, but be not necessary)；(2) MCF-7 tumour cell is shown as DAPI nuclei staining positive, and anti-CD45 is negative staining, anti-CK19 dye
Color is positive；Around leucocyte be DAPI nuclei staining positive, anti-CD45 stained positive, anti-CK19 is negative staining.Meet the above spy
The cell of sign is accredited as CTCs.
Activity of tumor cells after capture is investigated
By collected by trypsinisation of the cell without EDTA, cell is diluted to 2000/mL with PBS (pH 7.4), is taken
500 μ L carry out specific capture.The cell after capture is resuspended with the Binding Buffer of 500 μ L, 5 μ L Annexin are added
V-FITC, 5 μ L Propidium Iodide, mix, and room temperature is protected from light 5~15min, sees under 1h intrinsic fluorescence microscope
It surveys.
The result is shown in Figure 12, it can be seen from the figure that most of MCF-7 cell can be kept higher after enrichment
Activity, the only Apoptosis of only a few, thus minimal amount of red fluorescence (B) can only be observed in PI colored graph.Through counting
It calculates, about 98.8% cell can survive after enrichment.Compared with many similar approach reported at present, institute in the present invention
The enrichment material bio-compatibility used is excellent, there is no toxicity to cell, so cell is able to maintain higher survive
Rate.
Claims (8)
1. one kind can to the poly ion liquid magnetic nanocomposites of trace enriching specificity of circulating tumor cell and detection,
It is characterized in that it is prepared by following steps:
(A) synthesis of Carboxyl-functional Ionic Liquid monomer: by bromoacetic acid and 1- vinyl imidazole in 50~60 DEG C in toluene
Lower reaction, obtains Carboxyl-functional Ionic Liquid monomer；
(B) Fe3O4The synthesis of/SH magnetic nano-balls: by Fe3O4Magnetic nano-balls disperse in a solvent, 3- mercaptopropyi three to be added
Methoxy silane is reacted, and Fe is obtained3O4/ SH magnetic nano-balls；
(C) synthesis of poly ion liquid functionalization magnetic bead: by Fe3O4/ SH magnetic nano-balls and Carboxyl-functional Ionic Liquid monomer
After being mixed in ethanol with crosslinking agent, initiator is added in 60~80 DEG C of progress polymerization reactions, obtains poly ion liquid functionalization
Magnetic bead MNs；
The synthetic method of the crosslinking agent are as follows: after 1- vinyl imidazole and Isosorbide-5-Nitrae-dibromobutane are mixed in methyl alcohol, 50~
60 DEG C are stirred to react, after reaction that reaction mixture is cooling, are then added in ether, postpone resulting translucent solution is cold
Separation product washs drying；
(D) it the preparation of poly ion liquid magnetic nanocomposites: disperses MNs in the PBS solution containing EDC and NHS and lives
Change, then by the MNs of magnetic field separation activation, be resuspended in PBS solution, EpCAM antibody is added and is incubated for, it is more
Secondary washing obtains poly ion liquid magnetic nanocomposites IMNs.
2. poly ion liquid magnetic nanocomposites according to claim 1, it is characterised in that the Fe3O4Magnetic Nano
Ball is by FeCl3It is made after being reacted after being dissolved in ethylene glycol and water with sodium acetate and polyethylene glycol.
3. poly ion liquid magnetic nanocomposites according to claim 1, it is characterised in that described in step (B)
Solvent is toluene, Fe3O4Magnetic nano-balls and the amount ratio of 3-mercaptopropyi trimethoxy silane are 1:0.1~3g/mL, reaction
Temperature is 60~100 DEG C；After product cooling after reaction, toluene, methanol, deionized water and ethanol washing are successively used, is then done
It is dry, it is spare under conditions of being placed in 1~5 DEG C.
4. poly ion liquid magnetic nanocomposites according to claim 1, it is characterised in that in step (c), reaction
It is spare under conditions of being placed in 1~5 DEG C after cooling reactant, cleaning, drying will be obtained afterwards；Wherein Fe3O4/ SH magnetic nano-balls
Amount ratio with Carboxyl-functional Ionic Liquid monomer and crosslinking agent is respectively 1:0.01~0.04g/mol, and the initiator is
AIBN。
5. poly ion liquid magnetic nanocomposites according to claim 1, it is characterised in that in step (D), first will
MNs, which is scattered in the solution of the PBS of the pH 6.7~6.9 containing EDC and NHS, to be activated, and magnetic field separation activation is then passed through
MNs, then washed with the PBS of pH 7.3~7.5, it is resuspended in the PBS solution of pH 7.3~7.5, it is anti-that EpCAM is added
Body is incubated for, and is then washed with the PBS of pH 7.3~7.5, is finally stored in the PBS of pH 7.3~7.5, is placed in 1
It is saved under conditions of~5 DEG C.
6. poly ion liquid magnetic nanocomposites according to claim 1, it is characterised in that in step (D), MNs with
The mass ratio of EpCAM antibody is 1:0.05~0.2, and activation and incubation temperature are 1~5 DEG C.
7. poly ion liquid magnetic nanocomposites according to claim 1, it is characterised in that the carboxyl-functional from
Sub- liquid monomer the preparation method comprises the following steps: bromoacetic acid is dissolved in toluene after, 1- vinyl imidazole is slowly added to, at 50~60 DEG C
It is stirred to react, solid is isolated after reaction, be drying to obtain.
8. poly ion liquid magnetic nanocomposites according to claim 1, it is characterised in that the Fe3O4Magnetic Nano
The synthetic method of ball are as follows: take FeCl3, ethylene glycol and water mix to dissolution, sodium acetate is then added and polyethylene glycol carries out
High-speed stirred obtains clear solution, is then reacted in autoclave in 160~210 DEG C, after reaction that products therefrom is cold
But, wash, it is dry to get.
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