CN106399251A - Antibody-coupled bionic immune magnetic sphere and preparation method thereof - Google Patents
Antibody-coupled bionic immune magnetic sphere and preparation method thereof Download PDFInfo
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Abstract
The invention relates to an antibody-coupled bionic immune magnetic sphere and a preparation method of the antibody-coupled bionic immune magnetic sphere, belonging to the technical field of nanometer materials. According to the bionic immune magnetic sphere, the water-soluble Fe3O4 nanocluster is taken as the core, a cell membrane coats the core, and an antibody with a modification group is bonded outside the cell membrane through covalent coupling. The preparation method of the antibody-coupled bionic immune magnetic sphere comprises the following steps: preparing the Fe3O4 nanocluster, namely, MNC; preparing cell membrane fragments modified by choline azide, namely, M; reacting MCN and M to obtain a bionic magnetic sphere, and reacting the modification group with the antibody to obtain the antibody with the cyclo-ethynylation modification group; and coupling bionic magnetic sphere solution and the antibody with the cyclo-ethynylation modification group, thus obtaining the bionic immune magnetic sphere provided by the invention. The bionic immune magnetic sphere is good in dispersibility, rapid in magnetic response, and high in identifying efficiency when used for capturing tumor cells, no white blood cells exist in the background, and the captured tumor cells are good in activity. For the preparation method, the raw materials are easily available and have universality in the construction of the bionic immune magnetic sphere.
Description
Technical field
The present invention relates to bionical biomolecular of a kind of coupled antibody and preparation method thereof, belong to nano material technology neck
Domain.
Background technology
According to World Health Organization's issue in 2014《World's cancer report》, have within 2012 14000000 people to be diagnosed trouble
Cancer will be it is predicted that this data in 2035 will increase to 24,000,000.Cancer has become the serious class disease threatening human life and health,
Come from metastases more than the death of 90% patient.During metastases, circulating tumor cell (CTC) plays very heavy
The role wanting, the extremely low cancer cell of this kind of abundance comes off from primary tumors and enters Peripheral Circulation.Detection CTC can be swollen
Tumor metastasis, tumor prognosis, curative effect monitoring provide reference.But separate and detect that CTC is very challenging, CTC is in blood
Probability of occurrence is about 1 CTC/109Individual haemocyte (leucocyte：3×106mL-1～10 × 106mL-1, red blood cell：3×109mL-1
～9 × 109mL-1, blood platelet：2.5×108mL-1～4 × 108mL-1).
CTC beneficiation technologies mainly include immunomagnetic beads enrichment, density gradient centrifugation and membrane filtration etc., wherein, immune magnetic
Pearl beneficiation technologies are most widely used.Compared with the micron magnetic bead of early stage, nanometer magnetic bead specific surface area is high, good dispersion, and
Cell activation can be avoided, need not dissociate magnetic bead, can directly carry out subsequent experimental.Wherein, superparamagnetic nanometer magnetic bead is additional
Under magnetic fields, there is magnetic, and there is no after externally-applied magnetic field removes magnetic, this makes it is in single dispersing state, no in the solution
Easily reunite.Superparamagnetic particulates good dispersion, is the big advantage that superparamagnetic nanometer magnetic bead efficiently catches CTC.In order to ensure magnetic
The superparamagnetism of grain, traditional magnetic grain diameter little (about 10nm), magnetic is weak and unhandy, and commercial system is such as(Veridex company) and(German Mei Tian Ni company) needs special instrument additional to improve
Magnetic field intensity, thus realize Magneto separate.Additionally, also there is the non-specific adsorption problem of leucocyte in magnetic enrichment technology.For example：System is uniquely to be ratified for metastatic breast cancer, colorectal cancer or front by food and medicine surveillance authority (FDA)
Row gland cancer detects the technology of CTC, and by the EpCAM on the magnetic fluid targeting CTC surface of binding specificity antibody, (epithelial cell sticks
Molecule) antigen, then in the presence of specific magnetic fields, separate CTC, butSystem has significantly non-to leucocyte
Specific adsorption (1000～3000/7.5mL), is unfavorable for the CTC analysis in downstream.
Content of the invention
The defect existing for prior art, an object of the present invention is to provide a kind of bionical immunity of coupled antibody
Magnetic ball, described bionical biomolecular can efficiently catch extremely low-abundance circulating tumor cell, simultaneously in complicated blood environment
Reduce leucocyte background, provide strong means for the detection of circulating tumor cell, research.
The second object of the present invention is to provide a kind of preparation method of the bionical biomolecular of coupled antibody.
For realizing the purpose of the present invention, provide technical scheme below.
A kind of bionical biomolecular of coupled antibody, described bionical biomolecular is with water-soluble Fe3O4Nanocluster is core,
Its outer cladding cell membrane, has the antibody with modification group in the outer covalent coupling of cell membrane；
Described Fe3O4Nanocluster is by Fe3O4Nano crystal and polyethyleneimine (polyethyleneimine, PEI) group
Become, positively charged, particle diameter is 30nm～196nm；
Described antibody has the antibody of lysine residue for Fc end；
Described modification group is DBCO-PEGn- NHS, n=1～2000, preferably n=4；
Described modification group passes through the amino coupled on cycloalkyne modification and antibody.
Wherein, preferably described Fe3O4The particle diameter of nanocluster is 81nm；
Preferably described cell membrane is the cell membrane of leukon J774A.1.
A kind of preparation method of the bionical biomolecular of coupled antibody of the present invention, described preparation method step is as follows：
Step one, Fe3O4The preparation of nanocluster (referred to as MNC)
(1) under oxygen-free environment, NaOH is completely dissolved in diethylene glycol (Diethylene glycol, DEG), system
Standby obtain NaOH storing liquid；
Wherein, oxygen-free environment can be by being realized using inert gas deoxidation treatment and protection；
Can be heated to 100 DEG C～150 DEG C makes NaOH be completely dissolved in DEG, stops heating when NaOH is completely dissolved；
NaOH storing liquid can be maintained at 50 DEG C～100 DEG C and store for future use；
Being preferably heated to 120 DEG C makes NaOH be completely dissolved in DEG；
Preferably NaOH storing liquid is maintained at 70 DEG C and stores for future use；
Preferably NaOH storing liquid is adopted and is prepared with the following method：
DEG is done in the environment of inert gas deoxidation treatment, after oxygen is removed completely, NaOH is dissolved in DEG,
Heat simultaneously, be warming up to 120 DEG C, stop heating when NaOH is completely dissolved, described preparation process whole process all needs inert gas
Protection, obtain pale yellow solution be NaOH storing liquid, be maintained at store at 70 DEG C standby.
(2) vacuumize 5min～30min after source of iron, PEI being mixed with solvent, be subsequently filled argon (Ar) gas shielded,
It is heated to 100 DEG C～180 DEG C, adds the NaOH storing liquid of step one (1) gained, the concentration of NaOH is 0.03mol/ to solution
L～0.4mol/L, becomes after solution colour and is heated to 190 DEG C～230 DEG C after black, continues condensing reflux 30min～2h, reaction
Terminate, obtain the reactant liquor containing positively charged MNC；
After the cooling of the reactant liquor containing positively charged MNC, Magneto separate, remove reactant liquor, will be ultrasonic for Magneto separate product
Washing, then Magneto separate, remove reactant liquor, and obtaining Magneto separate end-product is MNC, MNC is suspended in deionized water and prepares
MNC solution；
Wherein, described solvent is DEG, or the mixed liquor of DEG and ethylene glycol (Ethylene gl ycol, EG), described mixed
The volume ratio closing EG and DEG in liquid is 1:14～1:4, preferably 2:13；
Described source of iron is containing Fe2+, stable under the normal temperature, material that can dissolve in described solvent and not react,
It is preferably FeSO4·7H2O or FeCl2·4H2O；
Described PEI uses as crosslinking agent and stabilizer；
Source of iron is 1 with the mass ratio of PEI:60～128:60；
25min is vacuumized after preferably mixing；
It is preferably heated to 160 DEG C, add the NaOH storing liquid of step one (1) gained；
It is preferably heated to 220 DEG C of continuation condensing reflux 1h, reaction terminates；
Preferably Magneto separate product is first scattered in supersound washing in absolute ethyl alcohol, is redispersed in ultrasonic in deionized water washing
Wash；Supersound washing 3 times repeatedly more preferably in absolute ethyl alcohol, supersound washing 5 times repeatedly in deionized water；
Different-grain diameter size can be prepared by adjusting the time vacuumizing or NaOH storing liquid addition as needed
MNC.
Step 2, be modified with nitrine choline cell membrane fragments (referred to as M) preparation
Cell is cultivated 20h～28h in cell culture complete medium, changes to containing 0.1mM～0.4mM nitrine choline
(N3) cell culture complete medium in cultivate 22h～28h, collect cell, be resuspended in cushioning liquid, use refiner
The cell being resuspended in cushioning liquid is carried out with interval broken, after described interval being crushed using SDGC
Cell fragment carry out separation and Extraction, obtain M；M is dissolved in Hepes C buffer solution (being purchased from Thermo Fischer Scient Inc.)
In, obtain M solution.
Wherein, preferably at 37 DEG C, CO2Concentration be 5% constant incubator in cultivate；
24h is cultivated preferably in cell culture complete medium；
Preferably change to containing N3Cell culture complete medium in cultivate 24h；
Preferably described cell culture complete medium is to include 10% volume fraction hyclone (FBS), 60 μ g/mL moulds
Element and the DMEM complete medium of 100 μ g/m streptomysins；
Preferably comprise N3Cell culture complete medium in the N containing 0.1mM3；
Preferably useIt is broken that 2 grades of Dispersers and Shakers refiner carries out interval to the cell collected.
Step 3, the preparation of the bionical biomolecular of coupled antibody
(1) MNC solution, M solution and PBS (PBS) are mixed, anti-in constant incubator with suspension instrument
Answer 4h～24h, Magneto separate, discard reactant liquor, by Magneto separate product, that is, bionical magnetic ball is resuspended in PBS, obtains bionical magnetic ball molten
Liquid；
Wherein, M is excessive with respect to MNC；
Preferably react 12h in 4 DEG C of constant incubators with suspension instrument；
Bionical magnetic ball solution can be stand-by in 4 DEG C of preservations.
(2) modification group and antibody are mixed to get mixed liquor in PBS, mixed liquor is incubated, remove unreacted repairing
Decorations group and antibody small molecule, prepare the solution of the antibody with cycloalkyne modification group.
Wherein, modification group and antibody molar ratio are 10:1～100:1, preferably 50:1；
Preferably mixed liquor is incubated 2h～12h at 4 DEG C；
Remove unreacted modification group and antibody small molecule can adopt ultrafiltration or dialysis, preferably at 4 DEG C, 7000r/min
Under, ultrafiltration 30min in super filter tube；
The solution of the antibody with cycloalkyne modification group can preserve in 4 DEG C.
(3) mix in PBS by bionical magnetic ball solution with the antibody-solutions of cycloalkyne modification group, and 4 DEG C～
, there is click chemistry reaction in 37 DEG C of suspension 1h～4h, bionical magnetic ball is coupled the antibody with cycloalkyne modification group, obtains this
A kind of bionical biomolecular of the described coupled antibody of invention.
Wherein, the relatively bionical magnetic ball of the antibody of cycloalkyne modification group is excessive；
Preferably in 37 DEG C of suspension 1h.
Beneficial effect
1. the invention provides a kind of bionical biomolecular of coupled antibody, described bionical biomolecular good dispersion and magnetic
Response is rapid, and membrane fluidity gives its higher recognition efficiency.Described bionical biomolecular is added in simulation blood sample, incubates for 37 DEG C
Educate 15min, you can catch about 90% tumour cell and background in almost there is no leucocyte, the cytoactive capturing is good, no
Release magnetic ball is needed to may be used for follow-up cultivation and study further；
2., the invention provides a kind of preparation method of the bionical biomolecular of coupled antibody, described preparation method raw material comes
Source extensively, has universality on bionical biomolecular builds, can prepare the bionical biomolecular for different cells.
Brief description
Fig. 1 is the burnt sign of copolymerization of M in embodiment 2.
The characterization result of the bionical magnetic ball that Fig. 2 prepares for embodiment 5 step (1)：A is the transmission electron microscopy of MNC
Mirror figure；B is the transmission electron microscope figure of bionical magnetic ball.
Fig. 3 is the MALDI-TOF figure of the antibody in embodiment 5 step (2) with cycloalkyne modification group.
Fig. 4 is the bionical magnetic ball in embodiment 5, the burnt checking of the copolymerization of the bionical biomolecular of coupled antibody.
Fig. 5 catches the reaction density (A) of CTC and the excellent of time (B) for the IMSs that embodiment 9 prepares to embodiment 5
Change result.
Fig. 6 is the immunostaining identification knot that in embodiment 9 step (3), IMSs catches circulating tumor cell in simulation blood sample
Really.
Fig. 7 is IMSs and commercialization magnetic bead in embodiment 9 step (4)The back-to-back comparative result of beads.A catches
The rate of catching compare (tumour cell concentration be 105Individual/mL, 1 PBS system)；Relatively (tumour cell concentration is 5-1500 to B remolding sensitivity
Individual/mL, 1 PBS system)；C leucocyte background compares.
Fig. 8 is with calcein acetoxymethyl ester (Calcein AM) and red second ingot dimer -1 in embodiment 9 step (5)
(EthD-1) the copolymerization Jiao's picture to the tumour cell catching.
Specific embodiment
Following instance is only used for further illustrating the present invention, but the enforcement that should not be construed as the present invention is confined to these says
Bright, for those skilled in the art, without departing from the inventive concept of the premise, can also make
Some deduction or replace.
Described below Magneto separate using method is specifically：Using magnetic suck, reactant liquor is removed with liquid-transfering gun, obtain remaining
Material is Magneto separate product.
Amorphous carbon film copper mesh is D200, reaches Zico instrument Science and Technology Ltd. purchased from Beijing；
Hepes B buffer solution and Hepes C buffer solution are purchased from Thermo Fischer Scient Inc.；
Protease inhibitors is purchased from Roche Holding Ag；
Described cell culture complete medium be include 10% volume fraction hyclone (FBS), 60 μ g/mL penicillin and
The DMEM complete medium of 100 μ g/mL streptomysins, purchased from Gibco；
The dyestuff DBCO-Fluor-525 of cycloalkyne is purchased from Click Chemistry Tools；
Anti-EpCAM antibody is purchased from Biolegend；
FITC fluorescently-labeled two resists purchased from Abcam company；
Anti-human (anti-human) CK18 (tumour cell mark) antibody of PE mark, the anti-human (anti-of FITC mark
Human) CD45 (leukocyte surface markers thing) antibody is purchased from Santa Cruze；
Hoechst 33342 is purchased from Mai Chen Science and Technology Ltd.；
LIVE/DEAD Viability/Cytotoxicity Kit is purchased from Invitrogen；
Transmission electron microscope (TEM) adopts JEM-2100F or JEM-1400 of Jeol Ltd. (JEOL)；
Energy disperse spectroscopy (EDS) adopts the Aztec of Oxford Instruments；
X-ray diffraction (XRD) instrument adopts XPert PRO MPD, PANalytical, Holland；
Infrared spectrum (FTIR) instrument adopts the FT/IR660 of JASCO company；
Refiner is using German IKA companyDispersers and Shakers refiner,T18；
Centrifuge and centrifuge tube are purchased from Beckman Coulter Inc. of the U.S. (Beckman Coulter, Inc.), from
Scheming adopts Beckman Coulter L-100XP Ultracentrifuge；
MALDI-TOF adopts AXIMA-Pertomance, SHIMADZU, Japan；
Laser confocal microscope adopts UltraVIEW VoX, PerkinElmer, the U.S.；
Flow cytometer adopts Beckman Coulter, cyan；
Beads is purchased from German Mei Tian Ni company.
The preparation of embodiment 1MNC
(1) by DEG in the environment of Ar gas, after oxygen is removed completely, solid NaOH is dissolved in DEG, simultaneously plus
Heat, is warming up to 120 DEG C, stops heating when solid NaOH is completely dissolved, and described preparation process whole process all uses Ar gas shielded, system
For obtaining pale yellow solution, it is NaOH storing liquid, concentration is 1g/mL, is maintained at and stores for future use at 70 DEG C；
(2) by FeSO4·7H2O, PEI are vacuumized after being mixed with DEG, fill Ar gas shielded afterwards；It is heated to
160 DEG C, add the NaOH storing liquid of step one (1) gained, the molar concentration of NaOH is 0.03mol/L, about 3min to solution
Solution colour becomes black afterwards, then heats to 220 DEG C, continues condensing reflux 1h, reaction terminates, and obtains containing positively charged
The reactant liquor of MNC；
After the cooling of the reactant liquor containing positively charged MNC, Magneto separate, remove reactant liquor, Magneto separate product is disperseed
Supersound washing in absolute ethyl alcohol, 3 times repeatedly, Magneto separate, Magneto separate product is scattered in deionized water, supersound washing 5 times,
Obtain Magneto separate end-product, be MNC；MNC is dissolved in 10mL deionized water, obtains MNC solution, concentration is 0.0128g/
mL；Wherein, FeSO4·7H2The mass ratio of O and PEI is 1:60.
Can as needed, in the case that other preparation conditions are constant, by adjusting the different grain of time preparation vacuumizing
The MNC of footpath size, as shown in table 1：
Table 1
The MNC solution preparing is carried out with test sign experiment as follows：
Ith, each 10 μ L of MNC solution of the different-grain diameter that difference Example 1 is obtained, are dispersed in 1mL deionized water respectively,
MNC solution after being diluted, takes the MNC solution after 10 μ L dilutions to drop in amorphous carbon film copper mesh carrier respectively, after drying,
Observed by transmission electron microscope (JEM-2100F), can be observed the pumpdown time be 30min, 25min,
When 20min, 15min and 5min, obtain MNC average grain diameter and be followed successively by 31nm, 81nm, 110nm, 140nm and 196nm；Meanwhile, may be used
Observe the favorable dispersibility of described MNC and particle diameter is more uniform；Knowable to the MNC for 81nm for the average grain diameter is observed, averagely
Particle diameter is the MNC of 81nm is not a complete single crystal grain, but is about the Fe of 11nm by many particle diameters3O4Monocrystalline is constituted
Cluster, simultaneously it can also be observed that outside fuzzy PEI layer, be 0.29nm by the spacing that measures adjacent crystal planes, with the center of area
The Fe of cubic structure3O4The spacing of lattice of crystal (220) crystal face is consistent.
IIth, the MNC energy spectrometer analysis for 81nm by average grain diameter, result its element visible consists of C, O, Fe, has not
The peak marking derives from amorphous carbon film copper mesh carrier.
The each 0.5g of MNC of the different-grain diameter that Example 1 is obtained, is respectively adopted X-ray diffraction (XRD) instrument and carries out X respectively
X ray diffraction is tested, and using the Cu-K alpha ray gathered data for 0.15406nm for the wavelength, the angle of diffraction 2 θ is 25 °～70 °, obtains phase
The diffracting spectrum result answered, result shows the MNC diffraction that average grain diameter particle diameter is 31nm, 81nm, 110nm, 140nm and 196nm
The 2 θ angles characteristic peak in curve respectively are 30.4 °, 35.6 °, 43.4 °, 53.4 °, 57.4 ° and 62.7 °, with JCPDS
Card proves after contrasting that the peak type of this described characteristic peak is Fe3O4The characteristic diffraction peak of crystal, corresponding Fe respectively3O4Crystal face
It is followed successively by (220), (311), (400), (422), (511) and (440), does not have miscellaneous peak, diffraction maximum is more sharp, show described MNC
Degree of crystallinity is higher.
IIIth, by average grain diameter, the MNC for 81nm is vacuum dried, takes a small amount of and KBr crystal grinding, makes after compressing tablet process
Obtain sample, sample is carried out infrared spectrum analysis using infrared spectrum (FTIR) instrument, by knowable to analysis result in wave number be
586.70cm-1There is absworption peak at place, and peak type is sharp, is Fe-O key characteristic absorption peak；1613.76cm-1Locate curved for N-H key in amino
Bent vibration absorption peak, 1053.07cm-1For C-N key stretching vibration absworption peak；Free amine group is in 3500cm-1～3400cm-1Place should
There are two obvious absorption bands, but in 3418.20cm-1Place only one of which width and strong absworption peak it was demonstrated that PEI is wrapped in MNC table
Face causes it to change change and lead to two absworption peaks to be changed into one.
IVth, described MNC has very strong magnetic navigability, takes the MNC of described different-grain diameter to be dispersed in water respectively, respectively
By magnet Magneto separate, only about 10s gets final product complete concentration and separation, and solution becomes clarification by black.
Vth, (300K) under normal temperature, by the MNC of described different-grain diameter, after vacuum drying, carry out vibrating specimen magnetometer
(VSM) obtain magnetization curve after testing, average grain diameter is the ratio corresponding to MNC of 31nm, 81nm, 110nm, 140nm and 196nm
Saturation magnetization is followed successively by 61.99emu/g, 71.39emu/g, 73.05emu/g, 76.37emu/g and 80.78emu/ respectively
G, numerical value increases with the increase of average grain diameter；Simultaneously it can be seen that magnetization curve is all " S " type, remanent magnetism and coercivity are all
0, illustrate, although MNC particle diameter is in more than 24.8nm under normal temperature, but still performance superparamagnetism.
The preparation of embodiment 2M
Leucocyte J774A.1 clone is placed in 37 DEG C, CO2Concentration is in 5% constant incubator, complete with cell culture
Full medium culture 24h, changes to respectively and continues culture 24h in the cell culture complete medium of the nitrine choline containing 0.1mM,
Collect leucocyte, discard culture medium.
By the leucocyte the collected Hepes B buffer solution Eddy diffusion of pH 7.6, add the protease suppression of 10 μ L/mL
Preparation, with 2 grades of interval smudge cellses of refiner, broken 2min, stops 1min, is repeated 10 times, then 1000r/min centrifugation 3min,
Collect supernatant, precipitation is with having added the Hepes B buffer solution Eddy diffusion of 10 μ L/mL protease inhibitors, thin with homogenate crusher machine
Born of the same parents, broken 2min, stops 1min, is repeated 10 times, then 1000r/min centrifugation 3min, collects supernatant, and supernatant is equally suspended in and contains
In the Hepes B buffer solution of protease inhibitors, supernatant is mixed into last clasmatosis liquid twice, obtains molten in buffering
Leucocyte fragment after crushing in liquid, carries out separation and Extraction using SDGC, obtains M, M is dissolved in Hepes
C buffer solution, obtains M solution.
Described SDGC is specially：In centrifuge tube, laying quality fraction is respectively successively from top to bottom
55%th, 40% and 30% sucrose solution, 4 DEG C of standing 4h；Add M solution, using centrifuge 4 DEG C, 28000g be centrifuged 2h, from
In heart pipe occur three gradients band, be followed successively by from top to bottom mass fraction be 55%, 40% and 30% band, receive respectively
Collect described band, respectively with Hepes C buffer solution Eddy diffusion separate mass fraction be 55%, 40% and 30% sucrose molten
In liquid, with centrifuge at 4 DEG C, 28000g centrifugation 30min carries out desugar process, is precipitated as M.
To M using carrying out protein spotses cross experiment, concrete grammar it is：By described three bands, each takes 5 μ L samples respectively
Product are put successively on polyvinylidene fluoride (PVDF) film, closed overnight with the sheep blood serum that mass fraction is 1%, will close after air-drying
Good pvdf membrane is dipped in the anti-CD45 solution being diluted with PBS, and the volume ratio of CD45 and PBS is 1:200, concussion incubation
Take out after 1h, washed 3 times with the concussion of Tris-Buffered Saline Tween-20 (TBST) buffer solution, then pvdf membrane is soaked
To the rabbit anti-mouse two corresponding anti-solution of horseradish peroxidase-labeled being diluted with PBS, the described two anti-volume ratios with PBS are
1:2000, take out after concussion incubation 1h, washed 3 times with the concussion of TBST buffer solution, finally pvdf membrane is immersed in concussion in DAB solution
Incubation 10min～30min, takes out pvdf membrane afterwards, and deionized water is dried after rinsing, and observes on film in gel imaging instrument
Spot.Three spots from top to bottom in pvdf membrane, be corresponding in turn to mass fraction be 55%, 40% and 30% band it is seen that
Mass fraction is that the color of the 40% corresponding spot of band is the deepest, and effect preferably, therefore selects 40% sucrose gradient to separate
The M cladding MNC extracting.
The M solution preparing is carried out with test sign experiment as follows：
Ith, utilize click chemistry (Click) to react, make the dyestuff (DBCO-Fluor-525) of cycloalkyne mark leucocyte film,
By laser confocal microscope, the Azide of described J774A.1 cell is characterized, comprise the following steps that：By leucocyte
J774A.1 clone is placed in 37 DEG C, CO2Concentration is in 5% constant incubator, cultivates 24h with cell culture complete medium,
The cell culture complete medium changing to the nitrine choline containing 0.1mM respectively continues culture 24h.
Discard nutrient solution, the volume fraction of addition 1mL is 4% paraformaldehyde fixation 15min, removes paraformaldehyde, uses
PBS washes twice；Add 10 μM of DBCO-Fluor-525 dyestuff afterwards, after 37 DEG C of incubation 1h, outwell dyestuff, then PBS washing 2
After secondary, add PBS to obtain sample solution, carry out laser co-focusing fluorescence imaging.
Nitrine choline on the cell membrane of cell of cell culture complete medium culture of the nitrine choline containing 0.1mM
Click chemistry can be occurred to react with DBCO-Fluor-525, therefore laser co-focusing fluorescence imaging has red fluorescent, such as Fig. 1
In right figure (N3- J774A.1+DBCO-Fluor 525) shown in.
Control experiment 1：Leucocyte J774A.1 clone is placed in 37 DEG C, CO2Concentration is in 5% constant incubator, uses
Cell culture complete medium cultivates 48h.
Discard nutrient solution, the volume fraction of addition 1mL is 4% paraformaldehyde fixation 15min, removes paraformaldehyde, uses
PBS washes twice, and adds PBS to obtain sample solution, carries out laser co-focusing fluorescence imaging and flow cytometer sign fluorescence is strong
Degree detection, result shows, laser co-focusing fluorescence imaging figure redfree fluorescence signal, as left figure (J774A.1) institute in Fig. 1
Show.
Control experiment 2：Leucocyte J774A.1 clone is placed in 37 DEG C, CO2Concentration is in 5% constant incubator, uses
Cell culture complete medium cultivates 48h.
Discard nutrient solution, the volume fraction of addition 1mL is 4% paraformaldehyde fixation 15min, removes paraformaldehyde, uses
PBS washes twice；Add 10 μM of DBCO-Fluor-525 dyestuff afterwards, after 37 DEG C of incubation 1h, outwell dyestuff, then PBS washing 2
After secondary, add PBS to obtain sample solution, carry out laser co-focusing fluorescence imaging, result shows because of no nitrine choline on cell membrane,
Click chemistry can not be occurred to react with DBCO-Fluor-525, but because a small amount of non-specific adsorption of DBCO-Fluor-525 is in cell
On, therefore laser co-focusing fluorescence imaging figure has weaker fluorescence signal, the middle graph (J774A.1+DBCO- in such as Fig. 1
Fluor525 shown in).
The preparation of embodiment 3M
Leucocyte J774A.1 clone is placed in 37 DEG C, CO2Concentration is in 5% constant incubator, complete with cell culture
Full medium culture 20h, changes to and continues culture 22h in the cell culture complete medium of the nitrine choline containing 0.1mM, collect
Leucocyte, discards culture medium, and remaining preparation method and control experiment are with embodiment 2.
The M solution preparing and M- solution are carried out with test sign experiment and result is similar to Example 2.Embodiment 4M
Preparation
Leucocyte J774A.1 clone is placed in 37 DEG C, CO2Concentration is in 5% constant incubator, complete with cell culture
Full medium culture 28h, changes to and continues culture 28h in the cell culture complete medium of the nitrine choline containing 0.1mM, collect
Leucocyte, discards culture medium, and remaining preparation method and control experiment are with embodiment 2.
The M solution preparing and M- solution are carried out with test sign experiment and result is similar to Example 2.Embodiment 5
The preparation of the bionical biomolecular of coupled antibody
(1) by the average grain diameter that embodiment 1 is obtained be 81nm MNC solution (wherein Fe content be 50 μ g), embodiment 2 makes
The M solution 200 μ L obtaining and PBS 2mL mixing, reacts 12h, Magneto separate with suspension instrument in 4 DEG C of constant incubators, discards reaction
Liquid, by Magneto separate product, that is, bionical magnetic ball is resuspended in PBS, obtains bionical magnetic ball solution；
(2) by DBCO-PEG4- NHS and anti-EpCAM antibody are mixed to get mixed liquor, by mixed liquor at 4 DEG C in PBS
Incubation 4h, then at 4 DEG C, under 7000r/min, ultrafiltration 30min in aperture is for 30KDa super filter tube, remove unreacted DBCO-
PEG4- NHS and anti-EpCAM antibody small molecule, prepare the solution of the antibody with cycloalkyne modification group.
Wherein, DBCO-PEG4- NHS is 50 with the mol ratio of anti-EpCAM antibody:1；
(3) the prepared bionical magnetic ball solution of step (1) and step (2) are carried the solution of the antibody of cycloalkyne modification group
(wherein antibody is 6 μ g) mixes in PBS, and in 37 DEG C of suspension 1h, click chemistry reaction occurs, and bionical magnetic ball is coupled and carries
Cycloalkyne modification group (DBCO-PEG4- NHS) antibody, obtain a kind of bionical biomolecular of coupling anti-EpCAM antibody.
The bionical biomolecular of the coupling anti-EpCAM antibody preparing is carried out with test, and to characterize experiment as follows:
Ith, using transmission electron microscope JEM-1400, the bionical biomolecular preparing is characterized, can by Fig. 2A
Find out, MNC surface irregularity, after parcel leucocyte film, its edge thickens, and the much more obvious layer of transparent of surrounding
Shape material, as shown in Figure 2 B.
IIth, dividing of cycloalkyne antibody is characterized with Matrix-assisted laser desorption ionization (MALDI-TOF)
Son amount, as seen from Figure 3, works as NHS-PEG4- DBCO is 50 with the ratio of antibody:When 1, the antibody average mark that cycloalkyneization is modified
Son amount increased 840.6246, and DBCO-PEG4The relative molecular mass of-NHS is 649.68, so averagely connecting on each antibody
Connect 1～2 cycloalkyne molecule, DBCO-Ab represents with cycloalkyne modification group (DBCO-PEG4- NHS) antibody (Ab).
IIIth, by same concentrations (Fe content：50 μ g) bionical magnetic ball, be coupled anti-EpCAM antibody bionical biomolecular
Fluorescently-labeled with FITC two resist in room temperature lucifuge with 40r/min rotating speed suspension 2h respectively, then wash 2 times, after Magneto separate with PBS
Resuspended with 200 μ L PBS, add copolymerization Jiao's capsule, observe under Laser Scanning Confocal Microscope after standing 30min.As shown in Figure 4, only
The bionical biomolecular being coupled anti-EpCAM antibody by the fluorescently-labeled two anti-identifications of FITC and could mark (Fig. 4 is right), and
Bionical magnetic ball two anti-reflective fluorescently-labeled with FITC can not answer (Fig. 4 is left), illustrates that cycloalkyne antibody has been coupled to bionical magnetic really
Ball surface, the bionical biomolecular being coupled anti-EpCAM antibody is successfully prepared.
The preparation of the bionical biomolecular of embodiment 6 coupled antibody
(1) by the average grain diameter that embodiment 1 is obtained be 81nm MNC solution (wherein Fe content be 50 μ g), embodiment 2 makes
The M solution 200 μ L obtaining and PBS 2mL mixing, reacts 12h, Magneto separate with suspension instrument in 4 DEG C of constant incubators, discards reaction
Liquid, by Magneto separate product, that is, bionical magnetic ball is resuspended in PBS, obtains bionical magnetic ball solution；
(2) by DBCO-PEG70- NHS and anti-EpCAM antibody are mixed to get mixed liquor, by mixed liquor at 4 DEG C in PBS
Incubation 4h incubation, then at 4 DEG C, under 7000r/min, ultrafiltration 30min in aperture is for 30KDa super filter tube, remove unnecessary repairing
Decorations group small molecule, prepares the solution of the antibody with cycloalkyne modification group.
Wherein, DBCO-PEG70- NHS is 50 with the mol ratio of anti-EpCAM antibody:1；
(3) solution (wherein antibody 6 μ g) of the antibody by bionical magnetic ball solution with cycloalkyne modification group is in PBS
Mixing, and in 37 DEG C of suspension 1h, there is click chemistry reaction, bionical magnetic ball is coupled and carries cycloalkyne modification group (DBCO-
PEG70- NHS) antibody, obtain a kind of bionical biomolecular of coupling anti-EpCAM antibody.
Test sign experiment and result and embodiment 5 class are carried out to the bionical biomolecular of the coupled antibody preparing
Seemingly.
The preparation of the bionical biomolecular of embodiment 7 coupled antibody
(1) by the average grain diameter that embodiment 1 is obtained be 81nm MNC solution (wherein Fe content be 50 μ g), embodiment 2 makes
The M solution 200 μ L obtaining and PBS 2mL mixing, reacts 12h, Magneto separate with suspension instrument in 4 DEG C of constant incubators, discards reaction
Liquid, by Magneto separate product, that is, bionical magnetic ball is resuspended in PBS, obtains bionical magnetic ball solution；
(2) by modification group (DBCO-PEG70- NHS) it is mixed to get mixed liquor in PBS, by mixed liquor with anti-Her2
In 4 DEG C of incubation 4h incubations, then at 4 DEG C, under 7000r/min, ultrafiltration 30min in aperture is for 30KDa super filter tube, it is unnecessary to remove
Modification group small molecule, prepare the solution of the antibody with cycloalkyne modification group.
Wherein, modification group (DBCO-PEG70- NHS) it is 50 with the mol ratio of antibody (anti-Her2):1；
(3) by bionical magnetic ball solution (Fe content：50 μ g) and cycloalkyne modification group (DBCO-PEG70- NHS) antibody
(anti-Her2) solution (wherein antibody is 6 μ g) mixes in PBS, and in 37 DEG C of suspension 1h, click chemistry reaction occurs,
Bionical magnetic ball is coupled and carries cycloalkyne modification group (DBCO-PEG4- NHS) antibody (anti-Her2), obtain a kind of coupling
The bionical biomolecular of antibody (anti-Her2).
Test sign experiment and result and embodiment 5 class are carried out to the bionical biomolecular of the coupled antibody preparing
Seemingly.
The preparation of the bionical biomolecular of embodiment 8 coupled antibody
(1) by the average grain diameter that embodiment 1 is obtained be 81nm MNC solution (wherein Fe content be 50 μ g), embodiment 2 makes
The M solution 200 μ L obtaining and PBS 2mL mixing, reacts 12h, Magneto separate with suspension instrument in 4 DEG C of constant incubators, discards reaction
Liquid, by Magneto separate product, that is, bionical magnetic ball is resuspended in PBS, obtains bionical magnetic ball solution；
(2) by modification group (DBCO-PEG4- NHS) it is mixed to get mixed liquor in PBS with antibody (anti-Her2), will
Mixed liquor is incubated in 4 DEG C of incubation 4h, then at 4 DEG C, under 7000r/min, ultrafiltration 30min in aperture is for 30KDa super filter tube, removes
Go unnecessary modification group small molecule, prepare the solution of the antibody with cycloalkyne modification group.
Wherein, modification group (DBCO-PEG4- NHS) it is 100 with the mol ratio of antibody (anti-Her2):1；
(3) by bionical magnetic ball solution (Fe content：50 μ g) and cycloalkyne modification group (DBCO-PEG4- NHS) antibody
The solution (wherein antibody 6 μ g) of ((anti-Her2)) mixes in PBS, and in 25 DEG C of suspension 4h, click chemistry reaction occurs,
Bionical magnetic ball is coupled and carries cycloalkyne modification group (DBCO-PEG4- NHS) antibody (anti-Her2), obtain a kind of coupling
The bionical biomolecular of antibody (anti-Her2).
Test sign experiment and result and embodiment 5 class are carried out to the bionical biomolecular of the coupled antibody preparing
Seemingly.
The bionical biomolecular of embodiment 9 coupled antibody catches CTC
(1) the bionical biomolecular of the coupled antibody of preparation in embodiment 5 is carried out the optimization of reaction density
It is respectively the enforcement of 10 μ g/mL, 25 μ g/mL, 50 μ g/mL, 100 μ g/mL, 150 μ g/mL and 200 μ g/mL to concentration
About 1 × 10 is put in the bionical biomolecular of coupled antibody that example 5 is obtained5Individual MCF-7 cell (Hoechst 33342 contaminates core),
37 DEG C, 40r/min suspension 1h；After Magneto separate, collect supernatant, use the resuspended substrate of 1mL PBS, counted with cell counter respectively
Cell in cleer and peaceful substrate.
(2) the bionical biomolecular of the coupled antibody of preparation in embodiment 5 is carried out the optimization in reaction time
About 1 × 10 is put in the bionical biomolecular of the coupled antibody being obtained to 50 μ g/mL embodiments 55Individual MCF-7 (people
Breast cancer) cell, at 37 DEG C, 40r/min is suspended different time respectively：5min, 15min, 30min and 60min.After Magneto separate,
Collect supernatant, use the resuspended substrate of 1mL PBS, count the cell in supernatant substrate with cell counter respectively.
(3) the bionical biomolecular using the coupled antibody of preparation in embodiment 5 catches and immunostaining in simulation blood sample
Identification circulation and tumour cell
1000 MCF-7 cells are separately added in 1mL people's whole blood, add preparation in embodiment 5 in above system
Bionical biomolecular (the Fe content of coupled antibody：50 μ g), be suspended at 37 DEG C incubation 15min.Magnetic separation simultaneously washs capture with PBS
CTCs, the fixing cell 10min of 37 DEG C of the paraformaldehyde solution with 4%.Add volume fraction 0.1% in substrate after magnetic separation
Triton-X 100 solution is in 37 DEG C of permeabilized cells 10min；Mass fraction is added to be the 37 DEG C of closings of 1%BSA solution after magnetic separation
Cell 30min.The anti-human CK18 antibody of Hoechst33342, PE mark, the anti-human CD45 of FITC mark is added to resist after magnetic separation
Body, at 37 DEG C, lucifuge incubation 30min, carries out immunocytochemical stain；Magnetic separation simultaneously be washed once with PBS, finally will be thin
Born of the same parents are resuspended in 300 μ L PBS and are placed in copolymerization Jiao's capsule, under confocal fluorescent microscope, cell are observed.
(4) by embodiment 5 preparation the bionical biomolecular of coupled antibody and commercialization magnetic bead (beads)
Carry out back-to-back comparison, the bionical biomolecular comprehensively investigating coupled antibody is enriched with the effect 1. CTCs catch rate contrast of CTC
Beads is used for catching 105Individual MCF-7 cell, concrete operation step is by being given in product description
Method for separating and MACS magnetic bead suggestion consumption.
2. CTCs test limit contrast
The MCF-7 cell of the Dio mark of 5～1500 is added the coupling using preparation in embodiment 5 in PBS respectively to resist
The bionical biomolecular of body,Beads catches；Magneto separate, the cell capturing is added 96 orifice plates, by rotating disk
Copolymerization Jiao count to MCF-7.
3. leucocyte background value contrast
To about 105Individual MCF-7 cell adds in the Healthy People cracking blood of 1mL, with the coupled antibody of preparation in embodiment 5
Bionical biomolecular,Beads catches MCF-7 cell respectively；Magnetic separation simultaneously washs the CTCs capturing with PBS, uses
4% paraformaldehyde solution fixes cell 20min in room temperature.Then use 0.1%Triton-X 100 solution penetrating thin at 37 DEG C
Born of the same parents 10min, is subsequently incubated 1h with the anti-human CD45 antibody and cell of FITC mark at 4 DEG C, with marking with PE after PBS washed cell again
The anti-human CK18 antibody of note is incubated 1h at 4 DEG C, finally cell is resuspended in 500 μ L PBS and carries out streaming and common focus analysis.
(5) LIVE/DEAD Viability/Cytotoxicity Kit is used to detect the coupled antibody of preparation in embodiment 5
The activity of MCF-7 cell that captures of bionical biomolecular.First prepare Calcein-AM/EthD-1 dye liquor：To 1mL DPBS
The EthD-1solution 2 μ L of Calcein AM solution 0.5 μ L and 2mM of middle addition 4mM.Then by dye liquor and thin
Born of the same parents act on 20min at 37 DEG C.Calcein AM solution can make living cells send green fluorescence (494/517nm),
EthD-1solution can make dead cell send red fluorescence (528/617nm).
Bionical biomolecular (abbreviation IMSs) to the coupled antibody that embodiment 5 prepares catches CTC effect experimental knot
Fruit is as follows:
Ith, from Fig. 5 A, when IMSs concentration is 50 μ g/mL, catch rate has reached more than 90%, but when IMSs exceedes
During finite concentration, catch rate has almost no change.
IIth, be can be seen that after IMSs and MCF-7 reaction 15min by Fig. 5 B, catch rate has just reached 95.85%, continues to prolong
Long incubation time, catch rate is not significantly improved, and illustrates that the combination speed of IMSs and MCF-7 is very fast.
IIIth, CK18 be tumour cell mark, CD45 be leukocyte surface markers thing, only CK18+/CD45-/
The cell of Hoechst 33342+ just can be considered CTCs, and the cell of CK18-/CD45+/Hoechst33342+ is leucocyte
(Leukocytes).About 1000 MCF-7 cells are added in 1mL people's whole blood, obtains simulating clinical sample.Catch it with IMSs
In CTCs and carry out ICC (three-color immunocytochemistry) identification.As shown in fig. 6, in Fig. 6, by left-hand
The right side is followed successively by Hoechst, CK18, CD45 and Merge；Wherein, Hoechst in figure has cell with the presence of blue explanation；CK18 schemes
In have CTCs with the presence of green explanation, there is no any display in CD45, illustrate not have leucocyte presence, Merge is Hoechst,
The result Overlapping display of CK18 and CD45, illustrates that IMSs can capture CTCs in people's whole blood, and almost thinless in vain in the visual field
Born of the same parents, illustrate that IMSs can efficiently catch CTCs in whole blood and significantly reduce the non-specific adsorption to leucocyte.
IVth, by 105Individual MCF-7 cell is dispersed in PBS solution, uses respectivelyBeads and even IMSs carries out richness
Collection, the rate of recovery of cell is respectively 79.08%, 95.85% it is clear that IMSs has higher catch rate, as shown in Figure 7 A.
The MCF-7 cell of the Dio mark of 5～1500 is added in PBS.Respectively use IMSs,beads
Catch.Magneto separate, the cell capturing is added 96 orifice plates, and by rotating disk copolymerization, Jiao counts to MCF-7.Cancer in clinic
In patient's blood sample, the number of CTCs is usually 0～50/mL.In order to detect that IMSs is used for the feasibility of clinical sample, will be by Dio
Mark MCF-7 cell add PBS, preparation concentration be 5～1500/mL cell series of samples, more respectively use IMSs andBeads catches MCF-7 therein, and as shown in Figure 7 B, regression equation in PBS for the IMSs is y=to result
0.9331x(R2=1.000), average catch rate is 93.31% (scope：84.62%～100%)；Beads exists
Regression equation in PBS is y=0.6069x (R2=0.9887), average catch rate is 60.69% (scope：25%～90%),
It can be seen that IMSs shows higher sensitivity.
The purity of beads enrichment of cell can reach 90%.Thin in vain in order to more intuitively assess IMSs reduction
The effect of born of the same parents' background, puts into 10 in 1mL Healthy People cracking blood5Individual MCF-7 cell, uses respectivelyBeads and IMSs
Enrichment, statistics catches the number of white blood cells in corresponding background during same number MCF-7 cell.From Fig. 7 C, CK18+/CD45-
For MCF-7 cell (green), CK18-/CD45+ is the leucocyte (yellow) in cracking blood.Knowable to streaming quantitative result, useWhen beads captures 10329 MCF-7 cells, the leucocyte number of detection is 340 (i.e. about 3400 leucocytes
Background value/1mL cracking blood sample).In sharp contrast, when catching equal number of MCF-7 cell with IMSs, it is not detected by
Leucocyte background.Focusing results have been also demonstrated that IMSs significantly reduces the background of leucocyte altogether.
VIth, the acetoxymethyl ester lipophilicity of Calcein-AM very high so as to permeable cell membrane, by the ester in living cells
Enzyme effect, and then slough AM base, produces the Calcein (calcein) sending out green fluorescence strong, therefore Calcein-AM is only to work
Cell dyeing.And red second ingot dimer -1 (EthD-1) can not pass through living cells film, can only enter dead cell, and intracellular
Genetic fragment with reference to after fluorescence occurs, can be used for detect dead cell.As shown in Figure 8, the overwhelming majority being captured by IMSs is thin
Born of the same parents are in green fluorescence, cytoactive are described very well, IMSs is little to the mechanical damage of CTCs.
The bionical biomolecular of embodiment 10 coupled antibody catches CTC
(1) the bionical biomolecular of preparation in embodiment 6 is carried out the optimization of reaction density
The bionical biomolecular of coupled antibody is that embodiment 6 is obtained, and remaining is with embodiment 9 step (1)
(2) the bionical biomolecular of the coupled antibody of preparation in embodiment 6 is carried out the optimization in reaction time
The bionical biomolecular of coupled antibody is that embodiment 6 is obtained, and remaining is with embodiment 9 step (2)
(3) the bionical biomolecular using the coupled antibody of preparation in embodiment 6 catches and immunostaining in simulation blood sample
Identification circulation and tumour cell
The bionical biomolecular of coupled antibody is that embodiment 6 is obtained, and remaining is with embodiment 9 step (3)
(4) by embodiment 6 preparation coupled antibody bionical biomolecular and commercialization magnetic bead (
Beads) carry out back-to-back comparison, the bionical biomolecular comprehensively investigating coupled antibody is enriched with the effect of CTC
The bionical biomolecular of coupled antibody is that embodiment 6 is obtained, and remaining is with embodiment 9 step (4)
(5) the bionical biomolecular of coupled antibody is that embodiment 6 is obtained, and remaining is with embodiment 9 step (4)
The bionical biomolecular of the coupled antibody that embodiment 6 prepares is carried out catch CTC effect experimental and result with
The effect experimental of embodiment 9 and result are similar to.
Claims (9)
1. a kind of coupled antibody bionical biomolecular it is characterised in that：Described bionical biomolecular is with water-soluble Fe3O4Nanometer
Cluster is core, its outer cladding cell membrane, has the antibody with modification group in the outer covalent coupling of cell membrane；
Described Fe3O4Nanocluster is by Fe3O4Nano crystal and polyethyleneimine composition, positively charged, particle diameter be 30nm～
196nm；
Described antibody has the antibody of lysine residue for Fc end；
Described modification group is DBCO-PEGn- NHS, n=1～2000；
Described modification group passes through the amino coupled on cycloalkyne modification and antibody.
2. a kind of coupled antibody according to claim 1 bionical biomolecular it is characterised in that：Described modification group is
DBCO-PEGn- NHS, n=4；Described Fe3O4The particle diameter of nanocluster is 81nm；Described cell membrane is leukon J774A.1's
Cell membrane.
3. a kind of preparation method of the bionical biomolecular of coupled antibody as claimed in claim 1 or 2 it is characterised in that：System
Preparation Method step is as follows：
Step one, Fe3O4Nanocluster, i.e. the preparation of MNC
(1) under oxygen-free environment, NaOH is completely dissolved in DEG, prepares NaOH storing liquid；
(2) vacuumize 5min～30min after source of iron, PEI being mixed with solvent, be subsequently filled argon gas protection, be heated to 100
DEG C～180 DEG C, add NaOH storing liquid, the concentration of NaOH is 0.03mol/L～0.4mol/L to solution, solution colour becomes
It is heated to 190 DEG C～230 DEG C after black, continue condensing reflux 30min～2h, obtain containing positively charged MNC reactant liquor；
After the cooling of the reactant liquor containing positively charged MNC, Magneto separate, remove reactant liquor, by Magneto separate product supersound washing,
Magneto separate again, removes reactant liquor, and obtaining Magneto separate end-product is MNC, MNC is suspended in deionized water to prepare MNC molten
Liquid；
Wherein, described solvent is DEG, or the mixed liquor of DEG and EG, and in described mixed liquor, the volume ratio of EG and DEG is 1:14～
1:4；
Described source of iron is containing Fe2+, stable under the normal temperature, material that can dissolve in described solvent and not react；
Source of iron is 1 with the mass ratio of PEI:60～128:60；
Step 2, it is modified with the cell membrane fragments of nitrine choline, i.e. the preparation of M
Cell is cultivated 20h～28h in cell culture complete medium, changes to thin containing 0.1mM～0.4mM nitrine choline
Cultivate 22h～28h in born of the same parents' culture complete medium, collect cell, be resuspended in cushioning liquid, with refiner to again hanging
Float on the cell in cushioning liquid to carry out intermittently crushing, the cell after described interval being crushed using SDGC is broken
Piece carries out separation and Extraction, obtains M；M is dissolved in Hepes C buffer solution, obtains M solution；
Step 3, the preparation of the bionical biomolecular of coupled antibody
(1) MNC solution, M solution and PBS are mixed, react 4h～24h with suspension instrument in constant incubator,
Magneto separate, discards reactant liquor, and by Magneto separate product, that is, bionical magnetic ball is resuspended in PBS, obtains bionical magnetic ball
Solution；
(2) modification group and antibody are mixed to get mixed liquor in PBS, mixed liquor is incubated, remove not anti-
The modification group answered and antibody small molecule, prepare the solution of the antibody with cycloalkyne modification group；
Modification group and antibody molar ratio are 10:1～100:1；
(3) antibody-solutions by bionical magnetic ball solution with cycloalkyne modification group mix in PBS, and
In 4 DEG C～37 DEG C suspension 1h～4h, obtain a kind of bionical biomolecular of described coupled antibody.
4. a kind of bionical biomolecular of coupled antibody according to claim 3 preparation method it is characterised in that：Step
In one：
(1) oxygen-free environment passes through to realize using inert gas deoxidation treatment and protection；
Being heated to 100 DEG C～150 DEG C makes NaOH be completely dissolved in DEG, stops heating when NaOH is completely dissolved；
(2), in the mixed liquor of DEG and EG, the volume ratio of EG and DEG is 2:13；
Described source of iron is FeSO4·7H2O or FeCl2·4H2O；
25min is vacuumized after mixing；
It is heated to 160 DEG C, add the NaOH storing liquid of step one (1) gained；
It is heated to 220 DEG C of continuation condensing reflux 1h, reaction terminates；
Magneto separate product is first scattered in supersound washing in absolute ethyl alcohol, is redispersed in supersound washing in deionized water.
5. a kind of bionical biomolecular of coupled antibody according to claim 3 preparation method it is characterised in that：Step
In two：
At 37 DEG C, CO2Concentration be 5% constant incubator in cultivate；
24h is cultivated in cell culture complete medium；
Change to containing N3Cell culture complete medium in cultivate 24h；
Described cell culture complete medium is to include 10% volume fraction hyclone, 60 μ g/mL penicillin and 100 μ g/m chains
The DMEM complete medium of mycin；
WithIt is broken that 2 grades of Dispersers and Shakers refiner carries out interval to the cell collected.
6. a kind of bionical biomolecular of coupled antibody according to claim 3 preparation method it is characterised in that：Step
In three：
(1) react 12h in 4 DEG C of constant incubators with suspension instrument；
(2) modification group and antibody molar ratio are 50:1；
Mixed liquor is incubated 2h～12h at 4 DEG C；
Remove unreacted modification group and antibody small molecule adopts ultrafiltration or dialysis；
(3) in 37 DEG C of suspension 1h.
7. a kind of bionical biomolecular of coupled antibody according to claim 3 preparation method it is characterised in that：Step
In one：
(1) oxygen-free environment passes through to realize using inert gas deoxidation treatment and protection；
Being heated to 100 DEG C～150 DEG C makes NaOH be completely dissolved in DEG, stops heating when NaOH is completely dissolved；
(2), in the mixed liquor of DEG and EG, the volume ratio of EG and DEG is 2:13；
Described source of iron is FeSO4·7H2O or FeCl2·4H2O；
25min is vacuumized after mixing；
It is heated to 160 DEG C, add the NaOH storing liquid of step one (1) gained；
It is heated to 220 DEG C of continuation condensing reflux 1h, reaction terminates；
Magneto separate product is first scattered in supersound washing in absolute ethyl alcohol, is redispersed in supersound washing in deionized water；
In step 2：
At 37 DEG C, CO2Concentration be 5% constant incubator in cultivate；
24h is cultivated in cell culture complete medium；
Change to containing N3Cell culture complete medium in cultivate 24h；
Described cell culture complete medium is to include 10% volume fraction hyclone, 60 μ g/mL penicillin and 100 μ g/m chains
The DMEM complete medium of mycin；
WithIt is broken that 2 grades of Dispersers and Shakers refiner carries out interval to the cell collected；
In step 3：
(1) react 12h in 4 DEG C of constant incubators with suspension instrument；
(2) modification group and antibody molar ratio are 50:1；
Mixed liquor is incubated 2h～12h at 4 DEG C；
Remove unreacted modification group and antibody small molecule adopts ultrafiltration or dialysis；
(3) in 37 DEG C of suspension 1h.
8. a kind of bionical biomolecular of coupled antibody according to claim 3 preparation method it is characterised in that：
In step：
(1) DEG is done in the environment of inert gas deoxidation treatment, after oxygen is removed completely, NaOH is dissolved in DEG,
Heat simultaneously, be warming up to 120 DEG C, stop heating when NaOH is completely dissolved, described preparation process whole process all needs inert gas
Protection, obtains NaOH storing liquid；
(2) supersound washing 3 times repeatedly in absolute ethyl alcohol, supersound washing 5 times repeatedly in deionized water；
In step 2：
Containing N3Cell culture complete medium in the N containing 0.1mM3；
In step 3：
(2), at 4 DEG C, under 7000r/min, in super filter tube, ultrafiltration 30min removes unreacted modification group and antibody small molecule.
9. a kind of bionical biomolecular of coupled antibody according to claim 7 preparation method it is characterised in that：
In step：
(1) DEG is done in the environment of inert gas deoxidation treatment, after oxygen is removed completely, NaOH is dissolved in DEG,
Heat simultaneously, be warming up to 120 DEG C, stop heating when NaOH is completely dissolved, described preparation process whole process all needs inert gas
Protection, obtains NaOH storing liquid；
(2) supersound washing 3 times repeatedly in absolute ethyl alcohol, supersound washing 5 times repeatedly in deionized water；
In step 2：
Containing N3Cell culture complete medium in the N containing 0.1mM3；
In step 3：
(2), at 4 DEG C, under 7000r/min, in super filter tube, ultrafiltration 30min removes unreacted modification group and antibody small molecule.
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| CN109100504A (en) * | 2018-06-25 | 2018-12-28 | 武汉大学 | A kind of platelet-leucocyte hybrid films coating immunomagnetic beads and the preparation method and application thereof |
| CN111097045A (en) * | 2020-01-15 | 2020-05-05 | 华南理工大学 | Bifunctional nanoparticle and preparation method and application thereof |
| CN112011513A (en) * | 2020-09-04 | 2020-12-01 | 四川大学 | Method for capturing high-purity circulating tumor cells based on bioorthogonal chemical method |
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| CN115125205A (en) * | 2022-06-14 | 2022-09-30 | 中国医学科学院生物医学工程研究所 | Leukocyte magnetic ball for modifying EpCAM (EpCAM) antibody and/or CSV (soluble protein kinase) antibody and preparation method thereof |
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