CN109507418A

CN109507418A - With imitating cyto-architectural magnetic nano-particle, immune magnetic nano particle and the preparation method and application thereof

Info

Publication number: CN109507418A
Application number: CN201811260412.3A
Authority: CN
Inventors: 吴尧; 周小熙; 易强英; 张宇佳; 康珂
Original assignee: Sichuan University
Current assignee: Sichuan University
Priority date: 2018-10-26
Filing date: 2018-10-26
Publication date: 2019-03-22
Anticipated expiration: 2038-10-26
Also published as: CN109507418B

Abstract

The invention discloses have to imitate cyto-architectural magnetic nano-particle, immune magnetic nano particle and the preparation method and application thereof.The present invention, which has, imitates cyto-architectural magnetic nano-particle with superparamagnetism Fe₃O₄Nanoparticle is as kernel, and surface is coated with leucocyte film, therefore the magnetic nano-particle not only shows excellent magnetic responsiveness energy, and its surface coated leucocyte film and leucocyte have homology, can effectively weaken the non-specific adsorption to leucocyte；By antibody through in the mediation modification to the surface coated leucocyte film of magnetic nano-particle of lipid molecular-polyethylene glycol-biotin molecule and Avidin, obtain with imitate cyto-architectural immune magnetic nano particle, it can be achieved that the enrichment of CTCs cell high-efficient rate, high-purity with separate.

Description

With imitating cyto-architectural magnetic nano-particle, immune magnetic nano particle and its system Preparation Method and application

Technical field

The invention belongs to bionical immune magnetic Nano field of material technology, be related to have imitate cyto-architectural magnetic material with The application of preparation method and the magnetic material in terms of circulating tumor cell enrichment.

Background technique

Circulating tumor cell (Circulating tumor cells, CTCs) refers to that falling to blood from entity tumor follows Tumour cell in ring, CTCs diffuse to the other tissues or organ of body by the circulatory system, in metastases and recurrence It plays an important role.By the detection to CTCs, can provide for treatment of cancer, cancer metastasis and cancer prognosis etc. with indicative Information.Therefore, the detection of circulating tumor cell has important clinical value in peripheral blood.

However, concentration is extremely low (serious to hinder only containing one to dozens of CTCs) in 1mL blood in blood by CTCs The counting of CTCs and deeper into research.Currently, CTCs enrichment and separation technology can be divided into two classes according to its separation principle: (1) difference, including density, size, morphotropism of physical property based on CTCs and blood cell etc. are such as based on the separation principle The uncommon Er Shi density gradient centrifugation solution (FicollHypaque density centrifugation) of design, microwell chips (Micropore Chip) etc.；But since CTCs and the density of haemocyte, size and morphotropism etc. have biggish overlapping, institute It is lower with the CTCs purity obtained based on physical property differential enrichment；(2) it is had based on CTCs from blood cell surface different CTCs is enriched with and is divided from blood by modifying the corresponding antibody of the antigen or aptamer etc. on isolation medium by antigen Separate out come, such as based on the separation principle design immune Magneto separate platform (e.g., CellSearch system), CTC chip, MagSweeper equipment etc..Wherein, magnetic separation technique is immunized due to modifying convenient, easy to operate, capture effect with functional component The advantages that rate is higher, and receive significant attention.Although immune magnetic separation technique has prospect very much, by its anti-leucocyte absorbability It is poor, cause the CTC purity being enriched in sample lower, greatly limits the downstream research of CTCs.For example, up to the present, FDA The only approved CTCs separation platform --- in the sample of CellSearch system enrichment, still there are thousands of leucocytes, CTCs's Purity is only 0.1~1.4%.

In order to improve anti-leucocyte absorbent properties, non-ionic hydrophilic polyethylene glycol under the premise of not influencing arresting efficiency It is modified on CTCs separation platform with artificial lipid's bilayer similar with membrane structure.However, result is unlike expected It is satisfactory like that, when blood sample is added in only fewer number of CTCs, still have in sample after enrichment tens of to thousands of white Cell.Then, people gradually recognize, being mainly made of protein and phospholipid bilayer, can protect cells from the external world A kind of natural fine after birth of the influence of environment, it may be possible to material for the homologous cell that material can preferably be disguised oneself as.For white thin Platform after after birth camouflage, it may be possible to repel in blood a large amount of existing, influence leucocyte (every milliliter of blood of CTCs targeting Middle about 10⁷It is a), to create the environment for being advantageously implemented high-purity C TCs separation.For example, Xiong et al. is first by cell Film azide functionalization, then cell membrane is extracted, and cell membrane is coated on Fe₃O₄On nanocluster, finally by click chemistry by two In the antibody connection of benzo octyl modification (Xiong et cl.Advanced Material 2016,28,7929-7935.DOI: 10.1002/adma.201601643).Rao et al. is extracted and is merged leucocyte film and platelet membrane, by way of coextrusion Hybrid films are coated on magnetic bead, antibody (the Rao et cl.Advanced of the upper selectively targeted CTCs of energy of finally modification Functional Materials.2018,1803531.DOI:10.1002/adfm.201803531).Although both CTCs Separation platform obtains good anti-leucocyte performance, but in order to extract purpose membrane component, it is necessary to it is broken in conjunction with hyposmosis, machinery A series of bad and gradient centrifugations and etc., removal does not rupture cell and cellular content.Further, it is necessary to again by ultrasound and/or The means such as multiple mechanical presses could combine n cell membrane component with magnetic-particle.In short, traditional cell membrane Extract and integration process of the cell membrane with magnetic nano platform involved in complex process, period length, and it also requires use it is special The equipment such as extruder.Therefore, needing to develop one kind can easily realize that cell membrane extracts and its whole with magnetic nano-particle The method of conjunction.

Summary of the invention

The purpose of the present invention is intended to provide a kind of cyto-architectural with imitating for above-mentioned problems of the prior art Magnetic nano-particle, by the surface coated leucocyte film of magnetic nano-particle, the anti-leucocyte for enhancing magnetic nano-particle is inhaled Attached characteristic.

Another object of the present invention be intended to provide it is above-mentioned have imitate cyto-architectural magnetic nano-particle preparation method.

Third object of the present invention be intended to provide it is a kind of have imitate cyto-architectural immune magnetic nano particle, realize pair Circulating tumor cell high efficiency, the enrichment and separation of high-purity.

Fourth object of the present invention be intended to provide it is a kind of have imitate cyto-architectural immune magnetic nano particle preparation side Method.

5th purpose of the invention be intended to provide it is above-mentioned have imitate cyto-architectural immune magnetic nano particle and be enriched with Application in circulating tumor cell.

It is provided by the invention have imitate cyto-architectural magnetic nano-particle, be by Fe₃O₄Magnetic nano-particle successively wraps It is overlying on Fe₃O₄High polymer layer, graphene layer and the leucocyte film on magnetic nano-particle surface are constituted.This has imitative cell The magnetic nano-particle of structure is with the Fe of superelevation saturation magnetization₃O₄Nanoparticle is kernel (57.21emu g^-1), so that magnetic Property nanoparticle entirety saturation intensity reaches finally obtained (50.27emu g^-1), saturation magnetization with higher, thus Having good magnetic responsiveness energy to externally-applied magnetic field, (all magnetic nanometers almost can be adsorbed all by magnet within 2min Firmly).Fe₃O₄The high polymer layer of magnetic nano-particle outer cladding is transition zone, the high molecular polymer used in the present invention For polyallylamine hydrochloride or polyethyleneimine.This kind of high molecular polymer is positively charged, can be used for realizing the absorption to graphene. The graphene of high polymer layer outer cladding is carboxylated graphene, and it is nanometer chip architecture, can be by buying outside market It arrives；The graphene is inserted into leucocyte film, scrapes a large amount of phospholipid molecules, so that leucocyte film is adsorbed in Fe₃O₄It is magnetic On nanoparticle.On the one hand the magnetic nano-particle of leucocyte film cladding has homology with leucocyte due to it, and by white thin Born of the same parents repel, and unspecif iotac leukocyte absorption is suppressed significantly；On the other hand, good biocompatibility, can be more stable in blood Ground exists.Therefore it is above-mentioned have imitate cyto-architectural magnetic nano-particle and modifying upper corresponding antibody or aptamer etc., can be with For targeting in complex samples (such as whole blood, serum, artificial whole blood etc.), enrichment and separation target cell, excretion body etc..

Invention further provides the above-mentioned preparation method for having and imitating cyto-architectural magnetic nano-particle, technology structures Think for by superparamagnetism Fe₃O₄It can be inserted into nanoparticle (MNs) surface modification and extract a large amount of phosphatide from cell membrane Graphene nanometer sheet, so that the magnetic nano-particle for being quickly obtained leucocyte film cladding, which has, imitates cyto-architectural magnetic Nano Particle (abbreviation BMNs).Specifically, first in Fe₃O₄Magnetic nano-particle surface passes through layer-by-layer (Layer- By-Layer, LbL) successively coated high molecular polymeric layer and graphene layer；Due to graphene ribbon negative electricity, the present invention is with band Polyallylamine hydrochloride or polyethyleneimine of positive electricity etc. are used as high polymer layer, and graphene is coated by electrostatic interaction In Fe₃O₄On magnetic nano-particle.Then the magnetic nano-particle and leucocyte self assembly obtained is incubated for, and obtains leucocyte packet The magnetic nano-particle covered, it is simple process, easy to operate relative to conventional cell film extracting method.It, should in specific implementation With the preparation method for imitating cyto-architectural magnetic nano-particle, steps are as follows:

(1) by electrostatic interaction, in Fe₃O₄Magnetic nano-particle surface successively coated high molecular polymeric layer and graphene Layer obtains product L1；

(2) by product L1 and the evenly dispersed culture medium for having cell after mixing in 37 DEG C, carbon dioxide volumetric concentration It is incubated for 1.5~2h under the conditions of 5%, Magneto separate is carried out to gained reaction solution later and collects the solid product isolated, then to solid Body product is washed to obtain the magnetic nano-particle of leucocyte film cladding product L1, that is, is had and imitated cyto-architectural magnetic Nano Particle.

Above-mentioned to have the preparation method for imitating cyto-architectural magnetic nano-particle, the purpose of step (1) is inhaled by electrostatic It is attached to act on Fe₃O₄Successively coated high molecular polymeric layer and graphene layer, specific implementation walk on magnetic nano-particle surface It is rapid as follows:

(11) there is Fe for evenly dispersed₃O₄After the aqueous solution mixing of the suspension and high molecular polymer of magnetic nano-particle Reaction system is formed, 1~3h is reacted under oscillating condition, Magneto separate is carried out to gained reaction solution later and what collection was isolated consolidates Body product, then solid product is washed to obtain the Fe for being coated with high polymer layer₃O₄Magnetic nano-particle is denoted as production Object L11；Fe in the reaction system₃O₄The mass ratio of magnetic nano-particle and high molecular polymer is 1:(0.08-0.4)；

(12) it is formed after mixing the evenly dispersed suspension for having product L11 and the evenly dispersed suspension for having graphene anti- System is answered, 1~3h is reacted under oscillating condition, Magneto separate is carried out to gained reaction solution later and collects the solid isolated producing Object, then solid product is washed to obtain in Fe₃O₄Magnetic nano-particle surface successively coated high molecular polymeric layer and graphite Alkene layer obtains product L1；The mass ratio of product L11 and graphene is 1:(0.1-0.11 in the reaction system).

Above-mentioned high molecular polymer is polyallylamine hydrochloride (PAH) or polyethyleneimine (PEI), graphene are carboxylated Graphene can be obtained by market outsourcing.It is evenly dispersed described in step (11) and (12) to have Fe₃O₄Magnetic nano-particle hangs Fe in supernatant liquid₃O₄Concentration be 1~1.25mg/mL, in the aqueous solution of high molecular polymer the concentration of high molecular polymer be 1~ 2mg/mL, the concentration of product L11 is 0.25~0.33mg/mL in the evenly dispersed suspension for having product L11, is dispersed with graphene Suspension in graphene concentration be 1~1.1mg/mL.The purpose of washing in step (11) and (12) be removal it is uncoated The high molecular polymer on magnetic nano-particle surface, graphene raw material wash obtained solid product using deionized water ?.

Above-mentioned to have the preparation method for imitating cyto-architectural magnetic nano-particle, the purpose of step (2) is by leucocyte film It is coated on the outer surface of product L1, is by obtaining product L1 with cell incubation.The used evenly dispersed training for having cell Feeding base is will to obtain in the evenly dispersed serum free medium of cell, and the cell is drenched from mouse monokaryon macrophage or people T Bar cell leukemia cell (Jurkat cell) etc..The mass volume ratio of the product L1 and culture medium is 1:(2~3), quality In terms of mg, volume is in terms of mL, and the number of cell is at least 1 × 10 in every 1mL culture medium⁵It is a.The preparation process of the cell are as follows: Cell is cultivated under the conditions of 37 DEG C, carbon dioxide volumetric concentration 5%, reaches 90% or more to cell confluency, digestion obtains The cells such as mouse monokaryon macrophage or human T lymphocyte leukaemia cell's (Jurkat cell) buffer gained cell with PBS After liquid cleans 2~3 times, cell is finally dispersed in high glycoform DMEM (the dulbecco's modified eagle of serum-free Medium) culture medium.The purpose of the washing of step (2) be removal be coated on it is other miscellaneous other than the leucocyte film on the surface product L1 Matter can directly adopt deionized water and wash 8~20 times.

Above-mentioned to have the preparation method for imitating cyto-architectural magnetic nano-particle, the reaction in step (1)~(2) is to shake It is carried out under the conditions of swinging, the revolving speed of oscillator used by oscillating operation is 300~450 revs/min.

Have invention further provides one kind and imitate cyto-architectural immune magnetic nano particle, is by composition institute It states with insertion lipid molecular-polyethylene glycol-biotin molecule on the leucocyte film for imitating cyto-architectural magnetic nano-particle, then Avidin is modified, antibody is finally modified and obtains.Lipid molecular-polyethylene glycol-biotin molecule of the present invention are as follows: monoacyl rouge Matter (C18)-polyethylene glycol-biotin (C18-PEG₂₀₀₀- Biotin), cholesterol-polyethylene glycol-biotin (cholesterol-PEG₂₀₀₀- Biotin) or distearoylphosphatidylethanolamine acylphosphatidyl ethanolamine-polyethylene glycol- Biotin (DSPE-PEG₂₀₀₀-Biotin)；The Avidin is Streptavidin or avidin (Avidin)；Antibody For the anti-epithelial cell adhesion molecule antibody (anti-EpCAM-Biotin) of biotin modification.This imitates cyto-architectural immune magnetic Property nanoparticle due to being modified with anti-EpCAM-Biotin antibody, it can be achieved that the selectively targeted of circulating tumor cell, and Leucocyte film can inhibit to leucocyte non-specific adsorption, to greatly improve the purity of enrichment cycles tumour cell.

Invention further provides it is above-mentioned have imitate the preparation method of cyto-architectural immune magnetic nano particle, in order to Simply, lipid molecular-polyethylene glycol-biotin molecule and antibody are fast and efficiently introduced, the poly- second of lipid molecular-is primarily based on Lipid molecular is similar to the phospholipid bilayer in leucocyte film in glycol-biotin molecule, using hydrophobic effect, by lipid point In son-polyethylene glycol-biotin molecule insertion leucocyte film, thus successfully non-with resisting in modification on magnetic nano-particle The polyethylene glycol of specific adsorption, and the biotin introduced for subsequent antibody.Avidin is combined on biotin again, is convenient for Cyto-architectural immune magnetic nano particle (BIMNs) is imitated in the antibody modification of the subsequent selectively targeted CTCs of energy, acquisition.It is specific real In existing mode, this, which has, imitates the preparation method of cyto-architectural immune magnetic nano particle steps are as follows:

(31) by it is described have imitate cyto-architectural magnetic nano-particle with containing lipid molecular-polyethylene glycol-biotin point Reaction system is formed after the PBS buffer solution mixing of son, 2~5h is reacted under oscillating condition, magnetic is carried out to gained reaction solution later The solid product isolated is separated and collected, then solid product is washed to obtain product L31；Have in the reaction system It imitates cyto-architectural magnetic nano-particle and lipid molecular-polyethylene glycol-biotin molecule mass ratio is 5000:(1~3)；

(32) reaction system is formed after mixing product L31 with the PBS buffer solution containing Avidin, is reacted under oscillating condition 2~5h carries out Magneto separate to gained reaction solution later and collects the solid product isolated, then wash to solid product To product L32；The mass ratio of product L31 and Avidin is 5000:(1~3 in the reaction system)；

(33) reaction system is formed after mixing product L32 with the PBS buffer solution containing antibody, in 4~7 under oscillating condition DEG C 6~12h of reaction, carries out Magneto separate to gained reaction solution later and collects the solid product isolated, then to solid product into Row washing, which obtains having, imitates cyto-architectural immune magnetic nano particle；The quality of product L32 and antibody in the reaction system Than for 1000:(0.5~1.5).

It is above-mentioned that there is the preparation method for imitating cyto-architectural immune magnetic nano particle, the lipid molecular-polyethylene glycol- Biotin molecule are as follows: monoacyl lipid (C18)-polyethylene glycol-biotin (C18-PEG₂₀₀₀- Biotin), the poly- second two of cholesterol- Alcohol-biotin (cholesterol-PEG₂₀₀₀- Biotin) or distearoylphosphatidylethanolamine-polyethylene glycol-biotin (DSPE-PEG₂₀₀₀-Biotin)；It is described containing lipid molecular-in lipid molecular-polyethylene glycol-biotin molecule PBS buffer solution Polyethylene glycol-biotin molecule concentration is 10~30 μ g/mL；The Avidin is Streptavidin or avidin (Avidin) etc., the concentration of Avidin is 10~30 μ g/mL in the PBS buffer solution containing Avidin；Antibody is repaired for biotin The anti-epithelial cell adhesion molecule antibody (anti-EpCAM-Biotin of biotin modification) of decorations, in the PBS buffer solution containing antibody Antibody concentration is 0.25~0.75 μ g/ μ L.The purpose of washing in step (31)-(33) is that removal modification is coated to leucocyte film Biotin, Avidin and the antibody on magnetic nano-particle surface wash obtained solid product using deionized water.

It is above-mentioned that there is the preparation method for imitating cyto-architectural immune magnetic nano particle, in order to reduce to other than target cell Immune magnetic nano particle and bovine serum albumin are incubated for by the non-specific adsorption of cell altogether in PBS buffer solution, to reduce Non-specific adsorption.Specific implementation are as follows: in step (33), have to gained and imitate cyto-architectural immune magnetic nano particle It carries out following post-processing: gained being had and imitates cyto-architectural immune magnetic nano particle (BIMNs) and is scattered in bovine serum albumin Mass concentration is in the PBS buffer solution of 0.5%-1.0%, in 4~7 DEG C of 20~30min of reaction under oscillating condition, later to institute Reaction solution carries out Magneto separate and collects the solid product isolated, then solid product is washed, that is, completed imitative to having The post-processing of cyto-architectural immune magnetic nano particle.Here the purpose washed to solid product is that removing gained produces The extra bovine serum albumin on object surface washs obtained solid product using deionized water.To treated BIMNs It is resuspended in PBS buffer solution, is placed at 4~7 DEG C and saves backup.

It is above-mentioned that there is the preparation method for imitating cyto-architectural immune magnetic nano particle, the reaction in step (31)~(33) It is to be carried out under oscillating condition, the revolving speed of oscillator used by oscillating operation is 300~450 revs/min.

It is above-mentioned have imitate application of the cyto-architectural immune magnetic nano particle in enrichment cycles tumour cell, this is immune Magnetic nano-particle can be used for circulating tumor cell efficiently, high-purity enrichment and separation.Epithelial cell adhesion molecule (EpCAM) Exist on epitheliated type tumour cell with cytokeratin family member (CK8, CK18 and CK19), is but not present on haemocyte； Therefore, epithelial cell adhesion molecule (EpCAM) and cytokeratin family member (CK8, CK18 and CK19) have become detection " goldstandard " of epithelial phenotype patient CTCs；Therefore it is modified with the immune magnetic nano particle of anti-EpCAM antibody, it can be special Ground targets and combines CTCs cell.And the surface coated leucocyte film of magnetic nano-particle can inhibit and inhale to leucocyte non-specificity It is attached, to greatly improve the enrichment purity of circulating tumor cell.

Compared with prior art, the invention has the following advantages:

1, provided by the invention with cyto-architectural magnetic nano-particle is imitated, due to superparamagnetism Fe₃O₄Nanoparticle As kernel, surface is coated with leucocyte film, therefore the magnetic nano-particle not only shows excellent magnetic responsiveness energy, and Its surface coated leucocyte film and leucocyte have homology, can effectively weaken the non-specific adsorption to leucocyte, should It is thin that magnetic nano-particle is applicable to targeting in complex samples (such as whole blood, serum, artificial whole blood etc.), enrichment and separation target Born of the same parents, excretion body etc..

2, it is provided by the invention have imitate in cyto-architectural magnetic nano-particle preparation process, using being coated on Fe₃O₄Magnetic Property nanoparticle surface graphene and phosphatide between dispersion interaction, it can be achieved that dialogue cell membrane rapidly extracting, obtain The magnetic nano-particle of leucocyte film cladding.

3, provided by the invention with cyto-architectural immune magnetic nano particle is imitated, by antibody through the poly- second of lipid molecular- In the mediation modification to the surface coated leucocyte film of magnetic nano-particle of glycol-biotin molecule and Avidin, the immune magnetic Property nanoparticle not only can be to CTCs cell bioaccumulation efficiency with higher, but also since leucocyte film is non-to homologous leucocyte Effective inhibition of specific adsorption substantially increases the purity (purity is up to 96% or more) of the CTCs cell of enrichment, to CTCs Downstream research, as DNA, RNA and protein of in vitro culture, amplification and high-purity extraction in terms of with highly important meaning Justice.

4, provided by the invention with cyto-architectural immune magnetic nano particle is imitated, utilize lipid molecular-polyethylene glycol- The mutual hydrophobic effect of lipid molecular and leucocyte film in biotin molecule, can be quick by lipid molecular-polyethylene glycol-biotin The magnetic nano-particle surface of cladding leucocyte film is modified, polyethylene glycol has the function of anti-non-specific adsorption, and biotin is The Rapid Modification of the antibody of subsequent selectively targeted CTCs lays the foundation.

5, provided by the invention with cyto-architectural immune magnetic nano particle is imitated, it can be within the short time (about 5min) The enrichment and separation to CTCs cell is completed, so that it is guaranteed that the CTCs activity with higher of enrichment, facilitates the downstream of CTCs Research.

6, it is provided by the invention have imitate cyto-architectural magnetic nano-particle and immune magnetic nano particle preparation method, Simple process, reaction condition are mild, have great potential value in clinical test and application aspect, are suitable for leading in biological medicine It is promoted the use of in domain.

Detailed description of the invention

Fig. 1 is the morphology characterization figure that magnetic nano-particle prepared by the embodiment of the present invention 1 is detected by transmission electron microscope；Its In, B, D, F, H are respectively the magnetism of the magnetic nano-particle (MNs@PEI) of PEI cladding prepared by embodiment 1, graphene coated The immune magnetic of nanoparticle (MNs@PEI@G), the magnetic nano-particle (BMNs) of leucocyte film cladding and leucocyte film cladding Property nanoparticle (BIMNs) TEM figure (scale be 100 μm), A, C, E, G are respectively corresponding enlarged drawing (scale is 20 μm).

Fig. 2 is that the preparation of the embodiment of the present invention 1 has the particle surface during imitating cyto-architectural magnetic nano-particle electric Position variation diagram.

Fig. 3 is total focused view and SDS- of the magnetic nano-particle of the preparation of the embodiment of the present invention 1 under 488nm laser excitation PAGE figure (scale is 10 μm)；Wherein A is the J774A.1 cell incubation resulting materials of MNs@PEI@G and fluorescent dye DiO pre-dyed Total focused view under 488nm laser excitation, B are total protein of cell (Cell), epicyte protein (M), have imitative eucaryotic cell structure Magnetic nano-particle (BMNs) contained by leucocyte memebrane protein SDS-PAGE (Coomassie blue stain figure), C be modified with DSPE- PEG₂₀₀₀Gained after the magnetic nano-particle and isosulfocyanic acid fluorescence labelled streptavidin (FITC-SA) of-Biotin is incubated for altogether Total focused view of the magnetic nano-particle under 488nm laser excitation, D are to imitate cyto-architectural immune magnetic nano particle (BIMNs) gained magnetic nano-particle is incubated for the anti-goat antibody of the donkey of marked by fluorescein isothiocyanate in 488nm laser excitation Under total focused view.

Fig. 4 is magnetic nano-particle magnetic property phenogram prepared by the embodiment of the present invention 1；Wherein A is Fe₃O₄Magnetic Nano The magnetic hysteresis imitating cyto-architectural immune magnetic nano particle (BIMNs) and measuring at room temperature prepared by particle and embodiment 1 Loop line, B be UV absorption spectrogram of the various concentration BIMNs aqueous solution 600nm at, C be permanent magnet enrichment BIMNs when it is m- Efficiency chart.

Fig. 5 be in application examples 1 using embodiment 1 prepare to imitate cyto-architectural immune magnetic nano particle (BIMNs) right CTCs cell capture effect picture；Wherein, A is the histogram that BIMNs changes CTCs cell capture efficiency with BIMNs concentration, and B is The histogram that BIMNs changes CTCs cell capture CTCs cell capture efficiency with incubation time, C are BIMNs and unmodified anti- The BIMNs of body, to the capture rate of variety classes cell under optium concentration and best incubation time.

Fig. 6 is to be existed in application examples 2 using cyto-architectural immune magnetic nano particle (BIMNs) of imitating prepared by embodiment 1 Detection limit and average detected efficiency characterize schematic diagram in different systems.

Fig. 7 be application examples 3 in using embodiment 1 prepare imitate cyto-architectural immune magnetic nano particle (BIMNs) with U.S. day Ni commercialization magnetic bead by MCF-7 cell and Jurkat cell (number ratio: 1:1, MCF-7 cell dye red in advance, Jurkat cell dyes green) composition sample in, the anti-leucocyte adsorption capacity comparative effectiveness schematic diagram of the two；Wherein A is to use Copolymerization coke stacking chart (scale be 50 μm) of the products therefrom under laser excitation after U.S. day Ni commercialization magnetic capture, B is to use BIMNs Copolymerization coke stacking chart (scale be 50 μm) of the products therefrom under laser excitation after capture, C is purity before and after MCF-7 cell capture Histogram.

Fig. 8 be application examples 4 in using embodiment 1 prepare imitate cyto-architectural immune magnetic nano particle (BIMNs) from It is dispersed in the whole blood of MCF-7 cell, three colors of the typical MCF-7 cell and the non-specific leucocyte captured that are enriched with Cellular immunity dyes focused view altogether (scale is 10 μm).

Fig. 9 be application examples 5 in using embodiment 1 prepare imitate cyto-architectural immune magnetic nano particle (BIMNs) with U.S. day Ni commercialization magnetic bead resists white in the removal red blood cell blood of the cell containing MCF-7 and the whole blood analog sample of the cell containing MCF-7 Cell adsorption capacity comparative effectiveness schematic diagram；Wherein A, D are to be captured in two kinds of blood samples with U.S. day Ni commercialization magnetic bead Copolymerization coke stacking chart (scale be 50 μm) of the products therefrom under laser excitation, B, E are that BIMNs is captured in two kinds of blood samples Copolymerization coke stacking chart (scale be 50 μm) of the products therefrom under laser excitation, C, F are to be existed with U.S. day Ni commercialization magnetic bead and BIMNs The capture rate and capture purity histogram of MCF-7 cell are captured in two kinds of blood samples.

Figure 10 is that MCF-7 cell dyes focused view (scale is 100 μm) altogether anyway in application examples 6, and wherein A is in experimental group Prepared by embodiment 1 imitates the MCF cell dyeing copolymerization anyway of cyto-architectural immune magnetic nano particle (BIMNs) enrichment capture Jiao Tu, B are that the MCF cell of the not enriched acquisition procedure of control group dyes focused view altogether anyway.

Figure 11 is the MCF-7 cell injuring model figure of experimental group enrichment in Figure 10, and wherein A, B, C are first generation cell culture Stage 4h, for 24 hours, the culture effect figure of 48h, D, E, F are respectively passage for the first time, second pass generation, in the third time passage stage Culture effect figure when fusion rate reaches 90% or so (scale is 100 μm).

Specific embodiment

Clear, complete description is carried out below with reference to technical solution of the attached drawing to various embodiments of the present invention, it is clear that is retouched Stating embodiment is only a part of the embodiments of the present invention, instead of all the embodiments.Based on the embodiments of the present invention, ability Domain those of ordinary skill obtained all other embodiment without making creative work, belongs to the present invention The range protected.

To keep technical solution provided by the invention clearer, is provided below with reference to embodiment and more detailed illustrate to conciliate It releases.

The Fe used in following embodiment₃O₄The specific preparation process of magnetic nano-particle are as follows: be hydrated raw material 1.157g six Iron chloride, 3.303g ammonium acetate and 0.4g sodium citrate are added in the reaction kettle for filling 60mL ethylene glycol, 1~3h of magnetic agitation Make above-mentioned raw materials after mixing, remove stirrer, reaction kettle is put into heating furnace, be warming up to 200 DEG C, reacts 16 hours Afterwards, turn off heating furnace power supply, reaction kettle is kept the temperature 1 hour in heating furnace and again takes out reaction kettle.Room is cooled to reaction kettle Wen Hou carries out Magneto separate 1h or more to reaction solution and collects the solid product isolated；Then ethyl alcohol and deionized water are successively used Solid product is washed repeatedly 5 times or so, until the complete clear of supernatant, collected black magnetic nanoparticle, that is, Fe₃O₄Magnetic Property nanoparticle, it is stand-by to place it in 4~7 DEG C of refrigerators.

Pass through Fe obtained by the above method₃O₄Magnetic nano-particle can be very good to be dispersed in water, and be formed stable super suitable Magnetic nanoparticle suspension.Through analyzing, Fe₃O₄Magnetic nano-particle partial size is in 300nm or so.

Oscillating operation is completed in common oscillators in following embodiment, and the revolving speed of used oscillator is 300 ~450 revs/min.

The antibody used in following embodiment is the anti-epithelial cell adhesion molecule antibody (anti-of biotin modification EpCAM-Biotin), which is market outsourcing, and producer is R&D Systems, and article No. is BAF960。

The graphene used in following embodiment is market outsourcing for carboxylated graphene, and producer is that Nanjing Ji Cang receives Rice Science and Technology Ltd., article No. JCG-1-50n-COOH, which is about 50nm, and thickness is about 0.8~1.2nm, about 8% carbon atom is by carboxylated.

Embodiment 1

The present embodiment have imitate cyto-architectural immune magnetic nano particle preparation the following steps are included:

(1) graphene and PEI cladding Fe are prepared₃O₄Magnetic nano-particle (being denoted as product L1, MNs@PEI@G), including with Under step by step:

(11) by Fe₃O₄Magnetic nano-particle (MNs) is evenly spread in deionized water, obtains Fe₃O₄Magnetic nano-particle Concentration is the suspension of 1mg/mL, and polyethyleneimine (PEI) is dissolved in the PEI for obtaining that concentration is 1.5mg/mL in deionized water Aqueous solution；There is Fe for 5mL is evenly dispersed₃O₄After suspension and 0.5mL PEI the aqueous solution mixing of magnetic nano-particle, vibrating Under the conditions of react 2h, Magneto separate is carried out to gained reaction solution and collects the solid product isolated, then to solid product spend from Sub- water washing 3 times coats Fe to get to PEI₃O₄Magnetic nano-particle (MNs@PEI), be denoted as product L11；

(12) product L11 is evenly spread in deionized water, obtains the suspension that product L11 concentration is 0.33mg/mL, Graphene uniform is distributed in deionized water, the suspension that graphene concentration is 1mg/mL is obtained；Have 15mL is evenly dispersed After the evenly dispersed suspension mixing for having graphene of the suspension and 0.5mL of product L11,2h is reacted under oscillating condition, to institute It obtains reaction solution to carry out Magneto separate and collect the solid product isolated, then solid matter with deionized water is washed 3 times and obtains stone The magnetic nano-particle of black alkene cladding product L11, i.e. product L1 (MNs@PEI@G)；

(2) preparation have imitate cyto-architectural magnetic nano-particle (BMNs), including it is following step by step:

(21) fusion rate is reached to 90% mouse monokaryon macrophage (J774.1, about 2~3 × 10⁶It is a) utilize cell Scraper scrapes, and the cell scraped is cleaned after 3 times with PBS buffer solution evenly spreads to 10mL high glycoform serum free medium DMEM (Gibco | Life Technologies (Grand Island, USA)) in, obtain the evenly dispersed culture medium for having cell；

(22) 5mg product L1 is added in the evenly dispersed culture medium for having cell after mixing in 37 DEG C, titanium dioxide Carbon volumetric concentration is incubated for 2h under the conditions of being 5%, reacts later gained and carries out Magneto separate and collect the solid product isolated, then Solid matter with deionized water is washed 10 times, the magnetic nano-particle of leucocyte film cladding product L1 is obtained, that is, is had imitative thin The magnetic nano-particle (BMNs) of born of the same parents' structure；

(3) preparation, which has, imitates cyto-architectural immune magnetic nano particle (BIMNs), comprising the following steps:

(31) by DSPE-PEG₂₀₀₀- Biotin (large biology of ancient India), which is dissolved in PBS buffer solution, obtains DSPE-PEG₂₀₀₀- Biotin concentration is the PBS buffer solution of 20 μ g/mL；Have to imitate in cyto-architectural magnetic nano-particle BMNs to 5mg and be added 0.1mLDSPE-PEG₂₀₀₀The PBS buffer solution of-Biotin reacts 3h under oscillating condition, reacts gained carry out magnetic point later From and collect the solid product isolated, then solid matter with deionized water is washed 3 times, obtains product L31；

(32) Streptavidin (Aladdin, No. CAS: 9013-20-1, product number S103034) is dissolved in PBS buffer solution In obtain Streptavidin concentration be 20 μ g/mL PBS buffer solution；0.1mL Streptavidin is added into 5mg product L31 PBS buffer solution reacts 3h under oscillating condition, carries out Magneto separate to gained reaction solution later and collect the solid isolated to produce Object, then solid matter with deionized water is washed 3 times, obtain product L32；

(33) by the anti-epithelial cell adhesion molecule antibody (anti-EpCAM- of biotin modification of biotin modification Biotin) it is distributed to the PBS buffer solution for obtaining that anti-EpCAM-Biotin concentration is 0.5 μ g/ μ L in PBS buffer solution；To 5mg The PBS buffer solution of 10 μ Lanti-EpCAM-Biotin is added in product L32, it is right later in 4 DEG C of reaction 10h under oscillating condition Gained reaction solution carries out Magneto separate and collects the solid product isolated, then washs 3 times to solid matter with deionized water, obtains With imitating cyto-architectural immune magnetic nano particle；

(34) gained is had to imitate cyto-architectural immune magnetic nano particle and be scattered in bovine serum albumin volumetric concentration and is In 0.5% PBS buffer solution, in 4 DEG C of reaction 30min under oscillating condition, Magneto separate is carried out to gained reaction solution later and is received Collect the solid product isolated, then solid matter with deionized water is washed 3 times, what is post-processed is cyto-architectural with imitating Immune magnetic nano particle (BIMNs), and be resuspended in PBS buffer solution, it is placed in spare in 4 DEG C of refrigerators.

Pattern, microstructure and magnetism testing analysis are carried out to the product that the present embodiment obtains:

1, morphology analysis

Shape is carried out to magnetic nano particle MNs@PEI, MNs@PEI@G, BMNs and BIMNs using transmission electron microscope Looks analysis, obtained TEM shape appearance figure is as shown in Figure 1A-H.Comparing Figure 1A and C can be seen that magnetic nano-particle surface in Fig. 1 C More lower and flaky substances of contrast, illustrate that graphene nanometer sheet is successfully modified in magnetic nano particle MNs The surface PEI.After magnetic nano particle MNs@PEI@G and J774A.1 cell incubation be can be seen that from Fig. 1 E and F, the magnetic Nano One layer of contrast of particle surface covering is lesser dizzy, illustrates that cell membrane is scraped by graphene, obtains target cell film cladding Magnetic nano-particle BMNs.Find out from Fig. 1 G and H, by the way that further modification can be selectively targeted on magnetic nano-particle BMNs After the anti-epithelial cell adhesion molecule antibody (anti-EpCAM) of the biotin modification of CTCs, gained magnetic nano-particle BIMNs Shallower dizzy of surface contrast thickens, and illustrates that imitating cyto-architectural immune magnetic nanometer BIMNs is successfully constructed.

2, Micro-Structure Analysis

Using Zetasizer Nano ZS90 type Particle Size Analyzer to magnetic nano particle MNs, MNs@PEI, MNs@PEI@ G, BMNs surface potential is tested, and analysis result is as shown in Fig. 2, material surface current potential is with autonomous layer by layer as we can see from the figure The variation of dress process, Fe₃O₄Magnetic nano-particle surface potential is -20mV, successively coats positively charged PEI and negatively charged thereon Graphene after, magnetic nano-particle surface potential deflects, and becomes+37mV and -10.5mV respectively, further illustrates PEI Successfully it is coated on magnetic nano-particle surface.After magnetic nano particle MNs@PEI@G and J774A.1 cell are incubated for altogether, magnetic Property nanoparticle surface charge becomes -27mV, illustrates that electronegative cell membrane is successfully scraped, is coated on magnetic nano particle sublist Face obtains imitating cyto-architectural magnetic nano-particle BMNs.

J774A.1 cell after by washing in step (21) uses a kind of cell membrane green fluorescence probe (DiO) (lipophilic Property dyestuff, in conjunction with the phospholipid bilayer of cell membrane after issue green fluorescence under 488nm laser excitation) pre-dyed, pre-dyed Cell and magnetic nano particle MNs@PEI@G afterwards is incubated for altogether obtains imitating cyto-architectural magnetic nano-particle BMNs, and BMNs exists Green fluorescence is issued under 488nm laser excitation, as shown in Figure 3A, illustrates that the lipid molecular on cell membrane is successfully scraped to magnetism The surface nanoparticle MNs@PEI@G.

By SDS-PAGE Coomassie blue stain method compare J774A.1 total protein of cell, J774A.1 epicyte protein and Albumen contained by cyto-architectural magnetic nano-particle BMNs is imitated, as shown in Figure 3B, as can be seen from the figure imitates cyto-architectural magnetic Property nanoparticle BMNs contained by albumen it is similar to epicyte protein, the magnetic Nano of cell membrane cladding prepared by explanation the method Particle and leucocyte have biggish similitude, thus imitate cyto-architectural magnetic nano-particle BMNs due to its homology and by Leucocyte repels, and nonspecific leucocyte absorption can be suppressed significantly.

When the Streptavidin marked by fluorescein isothiocyanate that will be used in step (32), it is modified with DSPE-PEG₂₀₀₀- Gained magnetic magnetic nano-particle (produces after the magnetic nano-particle (product L31) and the Streptavidin after dyeing of Biotin is incubated for Object L32) green fluorescence is issued under 488nm laser excitation, as shown in Figure 3 C, illustrate DSPE-PEG₂₀₀₀- Biotin molecule and chain Mould Avidin has successfully been modified to be imitated on cyto-architectural magnetic nano-particle BMNs.

When will have the anti-mountain of donkey for imitating cyto-architectural immune magnetic nano particle BIMNs and marked by fluorescein isothiocyanate Goat-anti body is incubated for 2h under oscillating condition, is incubated for gained magnetic nano-particle and issues green fluorescence under 488nm laser excitation, says Bright anti-EpCAM-Biotin successfully modified have imitate on cyto-architectural immune magnetic nano particle.

3, magnetic property detects

Fe is had detected using Model BHV-525 type vibrating specimen magnetometer (VSM) respectively₃O₄Magnetic nano-particle (MNs), it is returned in -18000Oe to the magnetic hysteresis within the scope of 18000Oe with cyto-architectural immune magnetic nano particle (BIMNs) Line, as a result as shown in Figure 4 A, it can be seen from the figure that the hysteresis loop of all samples passes through origin, no remanent magnetism and coercivity, Illustrate that MNs, BIMNs without remanent magnetism and coercivity, have superparamagnetism, and saturation magnetization is higher, respectively 57.21emu/ G and 50.27emu/g.

For the magnetic response time of further test b IMNs, the BIMNs aqueous solution of various concentration absorption intensity at 600nm It is detected, BIMNs concentration-UV absorption strength criterion curve of detection is as shown in Figure 4 B.As can be seen that BIMNs is in 600nm The UV absorption intensity at place is linearly increased with the increase of its concentration.The BIMNs aqueous solution of 100 μ g/mL is adsorbed with permanent magnet, is surveyed The light absorption value of APMNs that different adsorption times are adsorbed by permanent magnet at 600nm is measured, it is strong further according to BIMNs concentration-UV absorption Scale directrix curve calculates the BIMNs percentage that different adsorption times are adsorbed by permanent magnet, and gained BIMNs adsorption percentage is with absorption Time changing curve is as shown in Figure 4 C.When can be seen that Magneto separate 30s from Fig. 4 C, there are about 95% BIMNs to be separated.Explanation The magnetic response ability of BIMNs is very fast,, can be rapidly by it after BIMNs captures CTCs during subsequent captured CTCs It separates.

Embodiment 2

(1) graphene and PAH cladding Fe are prepared₃O₄Magnetic nano-particle (being denoted as product L1, MNs@PAH@G), including with Under step by step:

(11) by Fe₃O₄Magnetic nano-particle (MNs) is evenly spread in deionized water, obtains Fe₃O₄Magnetic nano-particle Concentration is the suspension of 1.25mg/mL, and it is 1mg/mL that polyallylamine hydrochloride (PAH), which is dissolved in deionized water, and obtains concentration PEI aqueous solution；There is Fe for 5mL is evenly dispersed₃O₄After suspension and 0.5mL PAH the aqueous solution mixing of magnetic nano-particle, 1h is reacted under oscillating condition, Magneto separate is carried out to gained reaction solution and collects the solid product isolated, then solid product is used Deionized water washs 3 times and coats Fe to get to PAH₃O₄Magnetic nano-particle (MNs@PAH), be denoted as product L11；

(12) product L11 is evenly spread in deionized water, obtains the suspension that product L11 concentration is 0.33mg/mL, Graphene uniform is distributed in deionized water, the suspension that graphene concentration is 1.1mg/mL is obtained；15mL is evenly dispersed After having suspension and the evenly dispersed suspension mixing for having graphene of 0.5mL of product L11,1h is reacted under oscillating condition, it is right Gained reaction solution carries out Magneto separate and collects the solid product isolated, then washs 3 times and obtain to solid matter with deionized water The magnetic nano-particle of graphene coated product L11, i.e. product L1 (MNs@PAH@G)；

(21) fusion rate is reached to 90% human T lymphocyte leukaemia cell (Jurkat, about 2~3 × 10⁶It is a) it utilizes Cell scraper scrapes, and the cell scraped is cleaned after 2 times with PBS buffer solution evenly spreads to 15mL high glycoform serum free medium DMEM (Gibco | Life Technologies (Grand Island, USA)) in, obtain the evenly dispersed culture medium for having cell；

(22) 5mg product L1 is added in the evenly dispersed culture medium for having cell after mixing in 37 DEG C, titanium dioxide Carbon volumetric concentration is incubated for 1.5h under the conditions of being 5%, reacts progress Magneto separate to gained later and collects the solid product isolated, Solid matter with deionized water is washed 8 times again, obtains the magnetic nano-particle of leucocyte film cladding product L1, that is, is had imitative thin The magnetic nano-particle (BMNs) of born of the same parents' structure；

(31) by C18-PEG₂₀₀₀The large biology of-Biotin (Peng) it is dissolved in PBS buffer solution and obtains C18 (C18)-PEG₂₀₀₀- Biotin concentration is the PBS buffer solution of 10 μ g/mL；Have to imitate in cyto-architectural magnetic nano-particle BMNs to 5mg and be added 0.1mLC18-PEG₂₀₀₀The PBS buffer solution of-Biotin reacts 2h under oscillating condition, reacts gained carry out Magneto separate later And the solid product isolated is collected, then wash 3 times to solid matter with deionized water, obtain product L31；

(32) avidin Avidin (Sigma, No. CAS: 1405-69-2, product number 215-783-6) is dissolved in The PBS buffer solution that Avidin Avidin concentration is 10 μ g/mL is obtained in PBS buffer solution；It is added into 5mg product L31 The PBS buffer solution of 0.1mLAvidin Avidin reacts 2h under oscillating condition, carries out Magneto separate simultaneously to gained reaction solution later The solid product isolated is collected, then solid matter with deionized water is washed 3 times, obtains product L32；

(33) anti-EpCAM-Biotin of biotin modification is distributed in PBS buffer solution and obtains anti-EpCAM- Biotin concentration is the PBS buffer solution of 0.25 μ g/ μ L；It is added 10 μ L anti-EpCAM-Biotin's into 5mg product L32 PBS buffer solution carries out Magneto separate to gained reaction solution later and what collection was isolated consolidates in 7 DEG C of reaction 6h under oscillating condition Body product, then solid matter with deionized water is washed 3 times, it obtains having and imitates cyto-architectural immune magnetic nano particle；

(34) gained is had to imitate cyto-architectural immune magnetic nano particle and be scattered in bovine serum albumin volumetric concentration and is In 1% PBS buffer solution, in 7 DEG C of reaction 20min under oscillating condition, Magneto separate is carried out to gained reaction solution later and is collected The solid product isolated, then solid matter with deionized water is washed 3 times, cyto-architectural exempt from is imitated in having for being post-processed Epidemic disease magnetic nano-particle (BIMNs), and be resuspended in PBS buffer solution, it is placed in spare in 7 DEG C of refrigerators.

Embodiment 3

(11) by Fe₃O₄Magnetic nano-particle (MNs) is evenly spread in deionized water, obtains Fe₃O₄Magnetic nano-particle Concentration is the suspension of 1.25mg/mL, and it is 2mg/mL's that polyethyleneimine (PEI), which is dissolved in, and obtains concentration in deionized water PEI aqueous solution；There is Fe for 2mL is evenly dispersed₃O₄After suspension and 0.5mL PEI the aqueous solution mixing of magnetic nano-particle, shaking 3h is reacted under the conditions of swinging, Magneto separate is carried out to gained reaction solution and collects the solid product isolated, then solid product is spent Ion water washing 3 times coats Fe to get to PEI₃O₄Magnetic nano-particle (MNs@PEI), be denoted as product L11；

(12) product L11 is evenly spread in deionized water, obtains the suspension that product L11 concentration is 0.25mg/mL, Graphene uniform is distributed in deionized water, the suspension that graphene concentration is 1mg/mL is obtained；Have 20mL is evenly dispersed After the evenly dispersed suspension mixing for having graphene of the suspension and 0.5mL of product L11,3h is reacted under oscillating condition, to institute It obtains reaction solution to carry out Magneto separate and collect the solid product isolated, then solid matter with deionized water is washed 3 times and obtains stone The magnetic nano-particle of black alkene cladding product L11, i.e. product L1 (MNs@PAH@G)；

(21) fusion rate is reached to 90% mouse monokaryon macrophage (J774.1, about 2~3 × 10⁶It is a) utilize cell Scraper scrapes, and the cell scraped is cleaned after 2 times with PBS buffer solution evenly spreads to 10mL high glycoform serum free medium DMEM (Gibco | Life Technologies (Grand Island, USA)) in, obtain the evenly dispersed culture medium for having cell；

(22) 5mg product L1 is added in the evenly dispersed culture medium for having cell after mixing in 37 DEG C, titanium dioxide Carbon volumetric concentration is incubated for 2h under the conditions of being 5%, reacts later gained and carries out Magneto separate and collect the solid product isolated, then Solid matter with deionized water is washed 20 times, the magnetic nano-particle of leucocyte film cladding product L1 is obtained, that is, is had imitative thin The magnetic nano-particle (BMNs) of born of the same parents' structure；

(31) by cholesterol-PEG₂₀₀₀The large biology of-Biotin (Peng) it is dissolved in PBS buffer solution and obtaining cholesterol-PEG₂₀₀₀- Biotin concentration is the PBS buffer solution of 30 μ g/mL；Have to 5mg and imitates cyto-architectural magnetism and receive 0.1mLcholesterol-PEG is added in rice corpuscles BMNs₂₀₀₀The PBS buffer solution of-Biotin reacts 5h under oscillating condition, Gained is reacted later and carries out Magneto separate and collects the solid product isolated, then solid matter with deionized water is washed 3 times, Obtain product L31；

(32) Streptavidin (Aladdin, No. CAS: 9013-20-1, product number S103034) is dissolved in PBS buffer solution In obtain Streptavidin concentration be 30 μ g/mL PBS buffer solution；0.1mL Streptavidin is added into 5mg product L31 PBS buffer solution reacts 5h under oscillating condition, carries out Magneto separate to gained reaction solution later and collect the solid isolated to produce Object, then solid matter with deionized water is washed 3 times, obtain product L32；

(33) anti-EpCAM-Biotin of biotin modification is distributed in PBS buffer solution and obtains anti-EpCAM- Biotin concentration is the PBS buffer solution of 0.75 μ g/ μ L；It is added 10 μ L anti-EpCAM-Biotin's into 5mg product L32 PBS buffer solution carries out Magneto separate to gained reaction solution later and what collection was isolated consolidates in 4 DEG C of reaction 12h under oscillating condition Body product, then solid matter with deionized water is washed 3 times, it obtains having and imitates cyto-architectural immune magnetic nano particle；

(34) gained is had to imitate cyto-architectural immune magnetic nano particle and be scattered in bovine serum albumin volumetric concentration and is In 1.0% PBS buffer solution, in 4 DEG C of reaction 30min under oscillating condition, Magneto separate is carried out to gained reaction solution later and is received Collect the solid product isolated, then solid matter with deionized water is washed 3 times, what is post-processed is cyto-architectural with imitating Immune magnetic nano particle (BIMNs), and be resuspended in PBS buffer solution, it is placed in spare in 4 DEG C of refrigerators.

Application examples

In following application examples by taking MCF-7 or GFP-MCF-7 cell as an example, there is eucaryotic cell structure to what is prepared with embodiment 1 Immune magnetic nano particle BIMNs enrichment CTCs be described in detail.

Human breast cancer cell (MCF-7), human liver cancer cell (HepG2), the white blood of T lymphocyte used in following application examples Sick cell (Jurkat), mouse monokaryon macrophage (J774A.1) visit power biology purchased from Shanghai.

Application examples 1

When the purpose of the application example is to investigate with cyto-architectural immune magnetic nano particle BIMNs concentration, incubation Between to BIMNs enrichment CTCs cell bioaccumulation efficiency influence.The present embodiment is with the highly expressed human milk of epithelial cell adhesion molecule Adenocarcinoma cell (MCF-7) and human liver cancer cell (HepG2) are the model cell of circulating tumor cell, and are expressed or without expression epithelium The T lymphocyte leukaemia cell (Jurkat) of cell adhesion molecule and mouse monokaryon macrophage (J774A.1) are control group Cell.

In the PBS buffer solution that six parts of 1mL contain MCF-7 cell, (MCF-7 cell quantity is 2 × 10⁵It is a) in be separately added into BIMNs (final concentration of BIMNs is as shown in Figure 5A) prepared by different quality embodiment 1, is incubated at room temperature 15min, is incubated for After to be incubated for product carry out Magneto separate, be collected simultaneously supernatant, the cell in supernatant counted with cell counter Number, each sample are surveyed three times, take its average value to be denoted as the cell number in supernatant, and then obtain supernatant cell concentration.According to Non- enrichment of cell sum=supernatant cell concentration × supernatant volume, the available MCF-7 cell number not being enriched with, then root According to bioaccumulation efficiency=(the non-enrichment of cell sum/investment total number of cells of 1-) × 100%, BIMNs can be obtained to the richness of MCF-7 cell Collect efficiency, test result is as shown in Figure 5A, as (for 75 μ g/m to 125 μ g/mL), BIMNs is to MCF-7 for the increase of BIMNs concentration The capture rate of cell rises to 98.5% from 95.4%.But the concentration of BIMNs is further increased to 250 μ g/mL, capture rate 98.5% or so is remained within, is illustrated for 2 × 10⁵A MCF-7 cell, the optimum amount of BIMNs are about 125 μ g.

In the PBS buffer solution that six parts of 1mL contain MCF-7 cell, (MCF-7 cell quantity is 2 × 10⁵It is a) in be separately added into The BIMNs of 125 μ g embodiments 1 preparation, is incubated at room temperature different time (incubation time is as shown in Figure 5 B), right after incubation It is incubated for product and carries out Magneto separate, be collected simultaneously supernatant, the cell in supernatant counted with cell counter, each sample Product are surveyed three times, take its average value to be denoted as the cell number in supernatant, and then obtain supernatant cell concentration.According to non-enrichment of cell Sum=supernatant cell concentration × supernatant volume, the available MCF-7 cell number not being enriched with, further according to bioaccumulation efficiency =(the non-enrichment of cell sum/investment total number of cells of 1-) × 100% can be obtained BIMNs to the bioaccumulation efficiency of MCF-7 cell, survey Test result is as shown in Figure 5 B, and BIMNs can reach 96.8% in 90s to the capture rate of MCF-7 cell；When extending incubation again Between, BIMNs is maintained at 96.5% or so to the capture rate of MCF-7 cell, illustrates for 2 × 10⁵A MCF-7 cell, BIMNs Best incubation time be about 90s.

1mL MCF-7 cell PBS buffer solution (MCF-7 cell quantity be 4 × 10⁵It is a), 1mL HepG2 cell (HepG2 cell quantity is 4 × 10 to PBS buffer solution⁵It is a), (Jurkat cell quantity is for the PBS buffer solution of 1mL Jurkat cell 4×10⁵It is a), (J774A.1 cell quantity is 4 × 10 for the PBS buffer solution of 1mL J774A.1 cell⁵It is a) in be separately added into 250 μ g BIMNs prepared by embodiment 1, is incubated at room temperature 90s；Simultaneously in PBS buffer solution (the MCF-7 cell number of 1mL MCF-7 cell Amount is 4 × 10⁵It is a), (HepG2 cell quantity is 2 × 10 for the PBS buffer solution of 1mL HepG2 cell⁵It is a), 1mL Jurkat cell PBS buffer solution (Jurkat cell quantity be 4 × 10⁵It is a), PBS buffer solution (the J774A.1 cell of 1mL J774A.1 cell Quantity is 4 × 10⁵It is a) in be separately added into 250 μ g embodiments 1 preparation without antibody BIMNs (step (32) products therefrom), in room Temperature is lower to be incubated for 90s；To product progress Magneto separate is incubated for after incubation, it is collected simultaneously supernatant, with cell counter to supernatant Cell in liquid is counted, and each sample is surveyed three times, takes its average value to be denoted as the cell number in supernatant, and then obtain supernatant Liquid cell concentration.According to non-enrichment of cell sum=supernatant cell concentration × supernatant volume, it is not rich that each cell can be obtained The cell number of collection can be obtained further according to bioaccumulation efficiency=(the non-enrichment of cell sum/investment total number of cells of 1-) × 100% BIMNs is and as shown in Figure 5 C with no test result to the bioaccumulation efficiency of each cell.As seen from the figure, anti-is not decorated The magnetic nano-particle of EpCAM-Biotin antibody is all very low (about 5%) to the capture rate of four kinds of cells, and modifies upper anti- The BIMNs of EpCAM-Biotin antibody increases to 95% to the capture rate of MCF-7 cell and HepG2 cell, and to Jurkat And the non-specific capture rate of J774A.1 cell remains within 5% or so.Illustrate that there is cyto-architectural immune magnetic to receive Rice corpuscles BIMNs is to the highly expressed cell of epithelial cell adhesion molecule capture rate with higher, and to Epithelial Cell Adhesion The non-specific adsorption of cell of the molecule without/low expression is lower, has stronger anti-non-specific packet cell adsorption capacity, is it The subsequent utilization in complex samples (such as whole blood) lays the foundation.

Application examples 2

The detection of target cell is limited in different environments in order to probe into BIMNs, the application example is in 1mL PBS buffer solution (pH=7.4), 10⁶It placed 1~150 people for having green fluorescent protein in a Jurkat T cell and Healthy People blood respectively Breast cancer cell (GFP-MCF-7) obtains three groups of artificial samples.125 μ g embodiments 1 are separately added into three groups of artificial samples again After the BIMNs of preparation under the conditions of shaken at room temperature after (200~300 revs/min) incubation 90s, products therefrom will be incubated for and carry out magnetic point From, and Magneto separate products therefrom is collected, then disperse 0.5~1.2mL PBS buffer solution (pH=again for Magneto separate products therefrom 7.4) it after, is separately added into 96 orifice plates, the cell for counting fluoresced green one by one under fluorescence microscope later (is denoted as and captures The GFP-MCF-7 of the green fluorescent protein arrived), statistical result is as shown in Figure 6.

It can be seen from the figure that BIMNs is in PBS buffer solution, 10⁶Detection in the whole blood of a Jurkat T cell and complexity Limit is respectively 1,3,3, it can be seen that, BIMNs is in PBS buffer solution, 10⁶In the whole blood of a Jurkat T cell and complexity Detection limit it is extremely low.BIMNs is in PBS buffer solution, 10⁶Average bioaccumulation efficiency point in the whole blood of a Jurkat T cell and complexity 98.7%, 92.7% and 86.7% (slope of matched curve) can not reached.Show that the present invention was prepared has imitative cell The immune magnetic nano particle BIMNs of structure can be limited in low, complicated blood sample in detection and efficiently be captured CTCs, be had The great potential of CTCs is separated from whole blood sample.

Application examples 3

The application example purpose, which is to investigate, prepared by the present invention there is cyto-architectural immune magnetic nano particle BIMNs to exist Anti- non-specific adsorption ability in cell mixing analog sample.

The application example the following steps are included:

(1) by 2 × 10⁵MCF-7 cell is [by cell tracker Deep Red (producer Invitrogen, the trade mark C7025) pre-dyed takes on a red color under 633nm laser excitation] and 2 × 10⁵Jurkat cell [cell tracker CMFDA (producer Invitrogen, trade mark C34565) pre-dyed, in green under 488nm laser excitation) mixing, two groups of cell mixing samples are configured, all It is dispersed in 1mL PBS buffer solution.

(2) 100uL U.S. day Ni commercialization magnetic bead is wherein added into first part of cell mixing, is incubated for 30 minutes at 4 DEG C, it The Incubating Solution obtained by corresponding Magneto separate post separation afterwards collects after enrichment the component containing target MCF-7 cell and to be scattered in PBS slow It in fliud flushing (pH=7.4), is placed on the ware of glass bottom, obtains being copolymerized burnt stacking chart accordingly under 488nm and 633nm laser excitation, As shown in Figure 7 A.

(3) BIMNs of 125 μ g embodiments 1 preparation is wherein added into second part of cell mixing, after being incubated for 90s, will be incubated for Products therefrom carries out Magneto separate, and collects Magneto separate products therefrom, then disperse PBS buffer solution again for Magneto separate products therefrom (pH=7.4) it in, is placed on the ware of glass bottom, obtains being copolymerized burnt stacking chart accordingly under 488nm and 633nm laser excitation, such as scheme Shown in 7B.

Statistics capture before, captured using BIMNs after, using in the cell mixing system after U.S. day Ni commercialization magnetic capture MCF-7 cell quantity obtains the MCF-7 cell purity of capture front and back, as seen in figure 7 c.It can be seen that capturing institute using BIMNs MCF-7 cell purity is up to 99.4%, without Jurkat cell in whole visual field.And it uses obtained by U.S. day Ni commercialization magnetic capture MCF-7 cell purity is lower to can only achieve 77.5%, and there are also more Jurkat cells in institute's enriched product.It can be seen that It is provided by the invention that there is cyto-architectural immune magnetic nano particle BIMNs can effectively inhibit to cell mixing analog sample The absorption of middle leucocyte considerably increases the purity of MCF-7 cell in enrichment sample, lays for the research of the subsequent downstream CTCs good Basis.

Application examples 4

The application example is identified using three cytochrome immunizations to epithelial phenotype cancer patient and healthy Healthy Volunteers whole blood The number of middle enrichment CTCs cell.

Into cancer patient (patient information is shown in Table 1) whole blood (1.5mL) plus having for 37.5 μ g embodiments 1 preparation is imitated Cyto-architectural immune magnetic nanometer BIMNs is incubated for 5 minutes, will be incubated for products therefrom and is carried out Magneto separate, and collects Magneto separate Products therefrom.Add the preparation of 25 μ g embodiments 1 into healthy volunteer's (healthy volunteer's information is shown in Table 2) whole blood (1mL) With cyto-architectural immune magnetic nanometer BIMNs is imitated, it is incubated for 5 minutes for (200~300 revs/min) under the conditions of shaken at room temperature, Products therefrom will be incubated for and carry out Magneto separate, and collect Magneto separate products therefrom.By the cell being collected into 4% (mass volume ratio, Quality is in terms of mg, and volume is in terms of mL) PBS buffer solution of paraformaldehyde (Suo Laibao Co., Ltd, producer, trade mark P1110) fixes 10 Minute, it is cleaned three times after fixed with PBS buffer solution；Triton X-100 (the factory for being again 0.1% with volumetric concentration Suo Laibao Co., Ltd, family, CAS:9002-93-1, cat:T8200) PBS buffer solution be incubated for 10 minutes, used after incubation PBS buffer solution is cleaned three times；Then with 1% bovine serum albumin (Wuhan (mass volume ratio, quality is in terms of mg, and volume is in terms of mL) Biology Co., Ltd, Google, cat:G5001, Lot:160862) PBS buffer solution be incubated for 30 minutes, it is slow with PBS after incubation Fliud flushing is cleaned three times；30 μ g mL are added later^-1Alexa Fluor 647-CK18,30 μ g mL^-1Alexa Fluor 488- CD45,20mM Hochest 33324 is dyed 12 hours in 4 DEG C, is cleaned after dyeing with PBS buffer solution and obtain three three times Solid product after cytochrome immunostaining.After dyeing course, disperse staining cell in PBS buffer solution again, It is placed on the ware of glass bottom, in 405nm, 488nm, under 633nm laser excitation, obtains corresponding focused view altogether and be copolymerized burnt stacking chart (as shown in Figure 8).The above experiment in triplicate, counts the CTCs number detected every time in whole blood sample, is shown in Table 1 and 2 institute of table Show.

From figure 8, it is seen that the cell with blue-fluorescence signal and red fluorescent is considered as CTCs, and band Having the cell of blue-fluorescence signal and green florescent signal is considered as leucocyte, illustrates to may be implemented using trichrome staining This method can be used to distinguish circulating tumor cell and leucocyte for differentiation to CTCs cell and leucocyte, subsequent experimental.

The number statistical table of CTCs in 1. cancer patient's Basic Information Table of table and cancer patient's whole blood

^aAverage RSD:8.7 ± 5.6%

2. healthy volunteer's essential information of table and circulating tumor cell number statistical table

The CTCs cell number statistics found in cancer patient's blood sample and healthy volunteer's blood sample is shown in Table shown in 1 and table 2, and 8 There are 2 to 48 circulating tumor cells to be detected in the example every 1.5 milliliters of blood of cancer patient, and is not sent out in 5 healthy samples Existing circulating tumor cell, this explanation is prepared by the present invention to be had and imitates cyto-architectural magnetic nano-particle BIMNs and can successfully answer For in true clinical blood sample.In addition, the count results three times of circulating tumor cell are not much different in same sample, show Show good reproducibility (average relative standard's deviation 8.7 ± 5.6%), show it is prepared by the present invention have imitate cyto-architectural magnetic Property nanoparticle BIMNs has good reproducibility and reliability, there is the prospect of very big clinical application.

Application examples 5

The application example purpose, which is to investigate, prepared by the present invention there is cyto-architectural immune magnetic nano particle BIMNs to exist Anti- non-specific adsorption ability in whole blood analog sample.

The application example the following steps are included:

(1) 1 × 10 is first added in red whole blood to 100uL whole blood/go⁴MCF-7 cell obtains two parts of whole blood analog samples, The BIMNs of 125 μ g embodiments 1 preparation is added into two parts of whole blood analog samples respectively, (100~300 under the conditions of shaken at room temperature Rev/min) be incubated for 90s after, products therefrom will be incubated for and carry out Magneto separate, and collect Magneto separate products therefrom.

(2) 1 × 10 is first added in red whole blood to 100uL whole blood/go⁴After MCF-7 cell, two parts of whole blood simulation samples are obtained This, is added 100uL U.S. day Ni magnetic bead into two parts of whole blood analog samples respectively, is incubated for 30 minutes at 4 DEG C, later with corresponding magnetic The separating obtained Incubating Solution of splitter, and collect the solid product isolated.

(3) by step (1) and step (2) from going in red whole blood analog sample Magneto separate obtained solid product to carry out three colors After cellular immunity dyeing, the solid product after gained dyeing is dispersed in PBS buffer solution (pH=7.4), is placed on the ware of glass bottom, Under 405nm, 488nm and 633nm laser excitation, obtain being copolymerized burnt stacking chart accordingly, as shown in figs. 9 a-b.

(4) by Magneto separate obtained solid product carries out three cytochromes from whole blood analog sample in step (1) and step (2) After immunostaining, the solid product after gained dyeing is dispersed in PBS buffer solution (pH=7.4), is placed on the ware of glass bottom, Under 405nm, 488nm and 633nm laser excitation, obtain being copolymerized burnt stacking chart accordingly, such as Fig. 9 D-E.

Above-mentioned three cytochromes immunostaining operation is as follows: three color ICC methods of standard are generally included in energy selective binding Chrotoplast (the anti-cell Keratin 18 monoclonal antibody that Alexa Fluor 647 is marked, Alexa Fluor 647-CK18, 633nm laser excitation, takes on a red color), leucocyte (Alexa Fluor 488 mark anti-leukocyte common antigen, Alexa Fluor 488-CD45 (producer abcam, trade mark ab197730), 405nm laser excitation, in green) probe and nucleus Dyestuff (Hochest 33324 (producer abcam, trade mark ab206269), 405nm laser excitation are blue).Three cytochromes are immune Staining procedure is as follows: (mass volume ratio, quality is in terms of mg, body with 4% for the cell that step (1) is collected into from whole blood sample Product is in terms of mL) PBS buffer solution of paraformaldehyde (Suo Laibao Co., Ltd, producer, trade mark P1110) fixes 10 minutes, and fixation terminates It is cleaned three times with PBS buffer solution afterwards；Triton X-100 (the limited public affairs of producer Suo Laibao of volumetric concentration 0.1% are used again Department, CAS:9002-93-1, cat:T8200) PBS buffer solution be incubated for 10 minutes, clean three with PBS buffer solution after incubation It is secondary；Then with 1% (mass volume ratio, quality is in terms of mg, and volume is in terms of mL) bovine serum albumin (limited public affairs of Wuhan Google biology Department, cat:G5001, Lot:160862) PBS buffer solution be incubated for 30 minutes, cleaned three times after incubation with PBS buffer solution； 30 μ g mL are added later^-1Alexa Fluor 647-CK18,30 μ g mL^-1Alexa Fluor 488-CD45,20mM Hochest 33324, is dyed 12 hours in 4 DEG C, clean after dyeing with PBS buffer solution and is obtained three cytochromes three times and be immunized Solid product after dyeing.By three cytochrome immunostainings, statistics calculates step (1), (2) using commercial magnetic bead, BIMNs From the capture rate of capture MCF-7 cell and capture purity in red whole blood analog sample and whole blood analog sample is gone, such as Fig. 8 C, figure Shown in 8F.Capture rate is by the MCF-7 cell number that captures compared with the MCF-7 cell number being put into whole blood sample It arrives.Capture purity is obtained compared with the whole cell numbers captured by the MCF- cell number captured.

Fig. 9 A is to go that 1 × 10 is added in red whole blood to 100uL⁴After MCF-7 cell, with commercial magnetic capture cell, through three After cytochrome immunostaining, gained laser co-focusing stacking chart.Fig. 9 B is to go that 1 × 10 is added in red whole blood to 100uL⁴MCF-7 After cell, cell, after three cytochrome immunostainings, gained laser co-focusing stacking chart are captured with BIMNs.Fig. 9 C is commercial magnetic Pearl, BIMNs are in the capture rate gone in red blood sample containing MCF-7 cell and capture MCF-7 cell purity statistical chart.Fig. 9 D For 1 × 10 is added into 100uL whole blood⁴After MCF-7 cell, with commercial magnetic capture cell, after three cytochrome immunostainings, Laser co-focusing stacking chart.Fig. 9 E is that 1 × 10 is added into 100uL whole blood⁴After MCF-7 cell, captured with BIMNs thin Born of the same parents, after three cytochrome immunostainings, laser co-focusing stacking chart.Fig. 9 F is containing MCF-7 for commercial magnetic bead, BIMNs Capture rate and capture MCF-7 cell purity statistical chart in the whole blood sample of cell.

From fig. 9, it can be seen that BIMNs and is being gone in red blood sample whole blood sample, capture rate is 81.2% respectively, 84.2%, capture purity is 96.7%, 96.9% respectively, illustrates to have and imitates cyto-architectural immune magnetic nano particle extremely High efficiency, the CTCs capture of high-purity are still able to achieve in complicated blood.However, commercial magnetic bead is in whole blood sample and removes red blood In sample, capture rate is respectively 10.7%, 69.5%, and capture purity is 44.3%, 72.5%.Obviously, a large amount of in whole blood Red blood cell largely reduced the capture rate and capture purity of business magnetic bead.Result above is confirmed with cyto-architectural immune The antileukocytic ability of the superior function of magnetic nano-particle BIMNs, especially whole blood.Therefore, for more commercial magnetic bead, complete More accurate result directly can be provided using BIMNs in blood.

Application examples 6

The application example purpose, which is to investigate, has cyto-architectural immune magnetic nano particle using prepared by the present invention The CTCs cell activity of BIMNs enrichment.

The application example the following steps are included:

(A) to 1 × 10⁶Having for 625 μ g embodiments 1 preparation is added in MCF-7 cell to imitate cyto-architectural immune magnetic and receive Rice corpuscles BIMNs under the conditions of shaken at room temperature after (200~300 revs/min) incubation 90s, will be incubated for products therefrom and carry out magnetic point From, and collect Magneto separate products therefrom.After taking 1/5 Magneto separate solid product to be dispersed in 1mL PBS buffer solution (pH=7.4), It is added AO/PI cell life or death dye liquor (Suo Laibao Co., Ltd, producer, AO article No.: CA1143, PI article No.: P8080), is incubated for 5 points Zhong Hou washed once using PBS buffer solution, solid product be collected by centrifugation with 1200 revs/min, the solid product of collection divides again It is dispersed in 1mL PBS buffer solution, is placed on the ware of glass bottom, at 488nm, 543nm laser excitation, obtain being copolymerized burnt superposition accordingly Figure, such as Figure 10 A.

(B) by 2 × 10⁵After MCF-7 cell is dispersed in 1mL PBS buffer solution, AO/PI cell life or death dye liquor is added, incubates It educates after five minutes, washed once using PBS buffer solution, solid product, the solid product of collection is collected by centrifugation with 1200 revs/min Again it is dispersed in 1mL PBS buffer solution, is placed on the ware of glass bottom, at 488nm, 543nm laser excitation, is copolymerized accordingly Burnt stacking chart, as shown in Figure 10 B.

(C) to 1 × 10⁶Having for 625 μ g embodiments 1 preparation is added in MCF-7 cell and imitates cyto-architectural BIMNs, incubates After educating 90s, products therefrom will be incubated for and carry out Magneto separate, and collect Magneto separate products therefrom.Magneto separate solid product is dispersed in In 2mL complete medium (DMEM, high sugar), it is placed in 6 orifice plates, is cultivated in 37 DEG C, the incubator of 5% carbon dioxide, to When cell confluency is up to 85% or more, passed on.Entire culture and succeeding generations keep sterile.Not same order is passed in cell The light field cell state figure that section obtains, as shown in figure 11.

Acridine orange (AO) is a kind of fluorescent dye with membrane permeability, which can be in conjunction with DNA/RNA, in 488nm Under laser excitation, yellow-green fluorescence is issued.And propidium iodide (PI) can also issue in conjunction with DNA, under 543nm laser excitation Red fluorescence.But the dyestuff is without membrane permeability, therefore only the DNA of dead cell can be in combination.By Figure 10 A-B it is found that by Entire enrichment and separation process, the activity of MCF-7 cell quite (~98%), illustrate incubation and separation process pair with control group MCF-7 cell activity influences smaller, this explanation, which has, imitates cyto-architectural immune magnetic nanometer BIMNs and can be used for high activity The acquisition of CTCs.

By enrichment of cell in vitro culture Figure 11 it is found that after incubation and separation process, the adherent ability of MCF-7 cell and Proliferative capacity is not affected, and changes without obvious pattern.Illustrate to be incubated for and separation process to MCF-7 cell Proliferation function and Pattern influence is smaller, this explanation has the circulating tumor cell for imitating cyto-architectural immune magnetic nanometer BIMNs enrichment can be directly Tap into row in vitro culture.

Claims

1. having and imitating cyto-architectural magnetic nano-particle, it is characterised in that be by Fe₃O₄Magnetic nano-particle is successively coated on Fe₃O₄High polymer layer, graphene layer and the leucocyte film layer on magnetic nano-particle surface are constituted.

2. having imitate cyto-architectural magnetic nano-particle according to claim 1, it is characterised in that the high molecular polymerization Object is polyallylamine hydrochloride or polyethyleneimine；The graphene is carboxylated graphene.

3. having described in claim 1 and imitating cyto-architectural magnetic nano-particle preparation method, it is characterised in that steps are as follows:

(1) by electrostatic interaction, in Fe₃O₄Successively coated high molecular polymeric layer and graphene layer obtain on magnetic nano-particle surface To product L1；

(2) by product L1 and the evenly dispersed culture medium for having cell after mixing in 37 DEG C, carbon dioxide volumetric concentration be 5% Under the conditions of be incubated for 1.5~2h, Magneto separate is carried out to gained reaction solution later and collects the solid product isolated, then solid is produced Object is washed to obtain the magnetic nano-particle of leucocyte film cladding product L1, that is, is had and imitated cyto-architectural magnetic nano particle Son.

4. having imitate cyto-architectural magnetic nano-particle preparation method according to claim 3, it is characterised in that the step Suddenly (1) it is as follows step by step:

(11) there is Fe for evenly dispersed₃O₄It is formed after the aqueous solution mixing of the suspension and high molecular polymer of magnetic nano-particle Reaction system reacts 1~3h under oscillating condition, carries out Magneto separate to gained reaction solution later and collect the solid isolated to produce Object, then solid product is washed to obtain the Fe for being coated with high polymer layer₃O₄Magnetic nano-particle is denoted as product L11；Fe in the reaction system₃O₄The mass ratio of magnetic nano-particle and high molecular polymer is 1:(0.08-0.4)；

(12) reactant is formed after mixing the evenly dispersed suspension for having product L11 and the evenly dispersed suspension for having graphene 1~3h reacts under oscillating condition in system, carries out Magneto separate to gained reaction solution later and collects the solid product isolated, then Solid product is washed to obtain in Fe₃O₄Magnetic nano-particle surface successively coated high molecular polymeric layer and graphene layer Obtain product L1；The mass ratio of product L11 and graphene is 1:(0.1-0.11 in the reaction system).

5. having according to claim 3 or 4 and imitating cyto-architectural magnetic nano-particle preparation method, it is characterised in that described High molecular polymer is polyallylamine hydrochloride or polyethyleneimine；The graphene is carboxylated graphene；It is described uniformly to divide Dissipating has the cell in the culture medium of cell from mouse monokaryon macrophage or human T lymphocyte leukaemia cell.

6. one kind, which has, imitates cyto-architectural immune magnetic nano particle, it is characterised in that by tool as claimed in claim 1 or 2 Have on the leucocyte film for imitate cyto-architectural magnetic nano-particle successively modify upper lipid molecular-polyethylene glycol-biotin molecule, Avidin and antibody obtain.

7. having imitate cyto-architectural immune magnetic nano particle according to claim 6, it is characterised in that the lipid point Son-polyethylene glycol-biotin molecule is C18-PEG₂₀₀₀-Biotin、cholesterol-PEG₂₀₀₀- Biotin or DSPE- PEG₂₀₀₀-Biotin；The Avidin is Streptavidin or avidin；The antibody is resisting for biotin modification Epithelial Cell Adhesion Molecule antibody.

8. having described in requiring 6 and imitating cyto-architectural immune magnetic nano particle preparation method, it is characterised in that steps are as follows:

(31) by it is as claimed in claim 1 or 2 have imitate cyto-architectural magnetic nano-particle with containing lipid molecular-polyethylene glycol- Reaction system is formed after the PBS buffer solution mixing of biotin molecule, 2~5h is reacted under oscillating condition, gained is reacted later Liquid carries out Magneto separate and collects the solid product isolated, then is washed to obtain product L31 to solid product；The reactant Have that imitate cyto-architectural magnetic nano-particle and lipid molecular-polyethylene glycol-biotin molecule mass ratio be 5000 in system: (1~3)；

(32) form reaction system after mixing product L31 with the PBS buffer solution containing Avidin, under oscillating condition react 2~ 5h carries out Magneto separate to gained reaction solution later and collects the solid product isolated, then washed to obtain to solid product Product L32；The mass ratio of product L31 and Avidin is 5000:(1~3 in the reaction system)；

(33) reaction system is formed after mixing product L32 with the PBS buffer solution containing antibody, it is anti-in 4~7 DEG C under oscillating condition 6~12h is answered, Magneto separate is carried out to gained reaction solution later and collects the solid product isolated, then solid product is washed It washs to obtain to have and imitates cyto-architectural immune magnetic nano particle；The mass ratio of product L32 and antibody is in the reaction system 1000:(0.5~1.5).

9. having imitate cyto-architectural immune magnetic nano particle preparation method according to claim 8, it is characterised in that institute Stating lipid molecular-polyethylene glycol-biotin molecule is C18-PEG₂₀₀₀-Biotin、cholesterol-PEG₂₀₀₀- Biotin or DSPE-PEG₂₀₀₀-Biotin；The Avidin is Streptavidin or avidin；The antibody is biotin modification Anti- epithelial cell adhesion molecule antibody；

It imitates cyto-architectural immune magnetic nano particle to having obtained by step (33) and can carry out following post-process: will have imitative thin The immune magnetic nano particle of born of the same parents' structure is scattered in the PBS buffer solution that bovine serum albumin quality concentration is 0.5%~1.0%, In 4~7 DEG C of 20~30min of reaction under oscillating condition, Magneto separate is carried out to gained reaction solution later and what collection was isolated consolidates Body product, then solid product is washed.

10. having described in claim 6 or 7 and imitating cyto-architectural immune magnetic nano particle in enrichment cycles tumour cell Using.