阅读说明：本技术 一种氧化石墨烯磁珠、抗体偶联氧化石墨烯磁珠及其制备方法和在细胞分选中的应用 (Graphene oxide magnetic bead, antibody-coupled graphene oxide magnetic bead, preparation methods of graphene oxide magnetic bead and antibody-coupled graphene oxide magnetic bead and application of gr) 是由 王明连 梁天亚 王群 于 2020-02-07 设计创作，主要内容包括：本发明提供了一种氧化石墨烯磁珠、抗体偶联氧化石墨烯磁珠及其制备方法和在细胞分选中的应用,属于材料学与生物学交叉技术领域,所述氧化石墨烯磁珠,包括氮化铁磁珠核心和包覆在所述氮化铁磁珠核心表面的石墨烯壳层,所述石墨烯壳层经过插层氧化修饰。本发明所述氧化石墨烯磁珠对石墨烯外壳进行氧化修饰,不需要额外包覆其他材料即可实现抗体偶联,因而没有外包材料阻碍磁响应性,最大限度的利用氮化铁磁珠的性质,进行磁性分离；偶联CD3抗体的免疫磁珠能够实现T细胞的分选,且可刺激所分离T细胞的增殖。所述氧化石墨烯磁珠的石墨烯外壳含有大量含氧基团,亲水性使其便于在水中分散,能够与细胞充分接触,提高细胞回收率。(The invention provides a graphene oxide magnetic bead, an antibody-coupled graphene oxide magnetic bead, a preparation method thereof and application thereof in cell sorting, and belongs to the technical field of crossing materials science and biology. The graphene oxide magnetic beads are used for oxidizing and modifying the graphene shell, and antibody coupling can be realized without additionally coating other materials, so that no coating material is used for hindering magnetic responsiveness, and the properties of the iron nitride magnetic beads are utilized to the maximum extent to perform magnetic separation; CD3 antibody-coupled immunomagnetic beads enable T cell sorting and can stimulate proliferation of isolated T cells. The graphene shell of the graphene oxide magnetic bead contains a large number of oxygen-containing groups, and the graphene shell is convenient to disperse in water due to hydrophilicity, can be fully contacted with cells, and improves the recovery rate of the cells.)

1. The utility model provides a graphite alkene magnetic bead, its characterized in that includes iron nitride magnetic bead core and cladding the graphite alkene shell on iron nitride magnetic bead core surface, the graphite alkene shell is through intercalation oxidation modification.

2. The magnetic graphene oxide bead according to claim 1, wherein the magnetic graphene oxide bead has a particle size of 500nm to 2 μm.

3. The method for preparing graphene oxide magnetic beads according to claim 1 or 2, comprising the steps of:

1) mixing iron nitride magnetic beads coated with graphene, sodium nitrate, sulfuric acid and potassium permanganate to obtain a first mixed feed liquid;

2) ultrasonically dispersing the first mixed feed liquid, and mixing with water to obtain a second mixed feed liquid;

3) carrying out intercalation oxidation on the second mixed feed liquid to obtain graphene oxide magnetic beads; the temperature of the intercalation oxidation is 80-90 ℃, and the time of the intercalation oxidation is 3-8 min.

4. The preparation method according to claim 3, wherein the temperature of the ultrasonic dispersion in the step 2) is 30-40 ℃, and the time of the ultrasonic dispersion is 25-35 min.

5. The preparation method according to claim 3, characterized in that step 3) further comprises the steps of centrifuging, washing and drying after the intercalation oxidation.

6. The method according to claim 5, wherein the rotation speed of the centrifugation is 7000-8000 rpm, and the time of the centrifugation is 8-12 min.

7. An antibody-coupled magnetic graphene oxide bead, wherein a CD3 antibody is coupled to a graphene shell of the magnetic graphene oxide bead according to claim 1 or 2.

8. A method of preparing CD3 antibody-conjugated immunomagnetic beads according to claim 7, comprising the steps of:

1) activating carboxyl on graphene oxide on the surface of the magnetic bead, wherein the activation temperature is 15-25 ℃, and the activation time is 12-18 min

2) Coupling the activated magnetic beads with a CD3 antibody, wherein the coupling temperature is 15-25 ℃, and the coupling time is 2-4 h;

the carboxyl activating agent in the step 1) is EDC and NHS.

9. The use of the antibody-coupled graphene oxide magnetic beads according to claim 7 in cell sorting.

10. The use of claim 9, wherein the antibody-coupled graphene oxide magnetic beads are mixed with cells to be sorted, then subjected to magnetic separation, collected and washed; the antibody-coupled graphene oxide magnetic beads have a proliferation stimulating effect on the sorted T cells.

Technical Field

The invention belongs to the technical field of crossing of materials science and biology, and particularly relates to a graphene oxide magnetic bead, an antibody-coupled graphene oxide magnetic bead, a preparation method of the graphene oxide magnetic bead and application of the graphene oxide magnetic bead in cell sorting.

Background

Cell sorting is a technique for separating target cells from a multicellular sample, and is widely used in many fields including basic biological studies of cells, diagnosis of diseases, cell therapy, and the like. The development of cell sorting methods has been driven by the need for a single cell in research work, where T lymphocytes closely related to the human immune system have been the key to medical research and disease treatment. Whether studying the mechanism of T lymphocyte action or advancing clinical use for the treatment of tumors with immune cells as a focus, T lymphocytes need to be isolated from whole blood or other mixed samples and activated to expand.

The current cell sorting methods can be roughly divided into two types, one type is a physical screening method, including density centrifugal sorting, pore screening and the like, the physical screening method is relatively simple and quick, but only can separate cells with large density or size difference, and the cells with similar density or size cannot be separated. For example, mononuclear cells with a density of 1.077 can be initially obtained from whole blood by density centrifugation. The other is an immune screening method, which comprises flow cytometry sorting and immunomagnetic bead sorting, and the immune screening method has the advantages of high precision, high recovery rate and the like compared with a physical sorting method. Among them, the flow sorting must rely on a flow cytometer having a sorting function, and such a large instrument is expensive and complicated to operate. After the cells are marked, the cells receive optical excitation through a microtubule in the instrument, and are damaged to a certain extent, so that the application is limited. Immunomagnetic bead sorting is a method of capturing specific cells in a mixed cell by using immunomagnetic beads, i.e., magnetic particles coupled with antibodies. The specific antibody coupled on the magnetic beads can accurately identify and stably combine with corresponding target cells, and then under an external magnetic field, the cells are gathered around the magnetic poles or are retained in test tubes on a magnetic frame due to the connection with the magnetic beads, so that the separation purpose is achieved. The method has the advantages of convenience, rapidness, simple and convenient operation, no need of large instruments, small influence on cells, good biological activity of the sorted cells and the like, and is more and more widely applied in the field of biomedicine.

Currently, commercially available immunomagnetic beads are Fe₂O₃、Fe₃O₄The ferrite compound or the metal particles are used as the inner core, and the shell is made of silicide or high molecular materials, so that functional groups can be conveniently introduced into the shell. The magnetic responsiveness of the iron nitride magnetic material is far greater than that of the iron oxide material, and the application of the iron nitride magnetic material in biomedicine is not seen at present. Chinese patent ZL201611150118.2(CN106683813B) discloses a graphene-coated magnetic composite material with a core-shell structure, wherein a shell is composed of multiple layers of graphene sheets, and a core is a ferromagnetic nitride particle. Chinese patent ZL201710059934.6(CN106710762B) discloses a method for coating silicon dioxide on the surface of graphene magnetic beads, and additionally coating SiO₂The particle size of the magnetic beads tends to be uniform, but the original shell property of the graphene is changed. Meanwhile, the particle size of the magnetic beads is increased by additional coating, and the magnetic responsiveness is influenced to a certain extent.

Disclosure of Invention

In view of the above, the present invention aims to provide a graphene oxide-coated iron nitride magnetic bead, an antibody-coupled graphene oxide magnetic bead, a preparation method thereof, and applications thereof in cell sorting; the graphene oxide magnetic beads are used for oxidizing and modifying the graphene shells, and antibody coupling can be realized without coating other materials after oxidation, so that no external coating material is used for hindering magnetic responsiveness, the property of iron nitride is utilized to the maximum extent, and efficient separation of cells is carried out. The graphene shell of the graphene oxide magnetic bead contains a large number of oxygen-containing groups, and the graphene shell is convenient to disperse in water due to hydrophilicity, can be fully contacted with cells, and improves the recovery rate of the cells.

In order to achieve the above purpose, the invention provides the following technical scheme:

the invention provides a graphene oxide magnetic bead which comprises an iron nitride magnetic bead core and a graphene shell layer coated on the surface of the iron nitride magnetic bead core, wherein the graphene shell layer is modified through intercalation oxidation.

Preferably, the particle size of the graphene oxide magnetic beads is 500nm to 2 μm.

The invention provides a preparation method of graphene oxide magnetic beads, which comprises the following steps:

1) mixing iron nitride magnetic beads coated with graphene, sodium nitrate, sulfuric acid and potassium permanganate to obtain a first mixed feed liquid;

2) ultrasonically dispersing the first mixed feed liquid, and mixing with water to obtain a second mixed feed liquid;

3) carrying out intercalation oxidation on the second mixed feed liquid to obtain graphene oxide magnetic beads; the temperature of the intercalation oxidation is 80-90 ℃, and the time of the intercalation oxidation is 3-8 min.

Preferably, the temperature of the ultrasonic dispersion in the step 2) is 30-40 ℃, and the time of the ultrasonic dispersion is 25-35 min.

Preferably, the step 3) further comprises the steps of centrifuging, washing and drying after the intercalation oxidation.

Preferably, the rotating speed of the centrifugation is 7000-8000 rpm, and the time of the centrifugation is 8-12 min.

Preferably, the graphene magnetic beads prepared by the method are homogenized, so that the agglomeration of the magnetic beads is reduced. The invention provides an antibody-coupled graphene oxide magnetic bead, wherein a graphene shell of the graphene oxide magnetic bead is coupled with a CD3 antibody, and the antibody-coupled graphene oxide magnetic bead comprises the following steps:

1) activating carboxyl on graphene oxide on the surface of the magnetic bead, wherein the activation temperature is 15-25 ℃, and the activation time is 12-18 min; 2) coupling the activated magnetic beads with a CD3 antibody, wherein the coupling temperature is 15-25 ℃, and the coupling time is 2-4 h; the carboxyl activating agent in the step 1) is EDC and NHS, and the mass ratio of the activating agent added is EDC: NHS ═ 3: 4.

The invention provides application of the antibody-coupled graphene oxide magnetic beads in cell sorting.

Preferably, the antibody-coupled graphene oxide magnetic beads and the cells to be sorted are mixed and then subjected to magnetic separation, the magnetic beads are collected and washed, and the washing solution contains the T cells.

Preferably, the concentration of the mixed cells to be sorted is 10⁷～10⁸cell/mL, and the co-incubation time of the mixed cells and the magnetic beads is 8-12 min; placing the magnetic separation on a magnetic pole for 8-12 min; washing for 2-4 times, wherein the washing time is 3-7 min each time.

The invention has the beneficial effects that: the invention provides a graphene oxide magnetic bead, which reduces the obstruction of an outer coating material to the magnetic responsiveness of a core by oxidizing a graphene shell layer, utilizes the property of an iron nitride core to the maximum extent and can efficiently separate T cells; the sorted T cells are in a normal form of conglomerate growth, and the cell activity is not influenced.

Drawings

Fig. 1 shows the effect of the homogenized graphene oxide magnetic beads and the measured average particle size, wherein the left image is a photograph of the graphene oxide magnetic beads, and the right image is average particle size data;

FIG. 2 is a comparison of an infrared spectrum of a graphene oxide magnetic bead provided by the present invention and a commercially available graphene oxide;

fig. 3 is a TEM image of a graphene oxide magnetic bead provided by the present invention, which is a magnetic bead with two visual fields, and both the magnetic bead shell-core structure can be seen;

FIG. 4 is a value of absorbance detected by an enzyme-linked immunosorbent assay for TMB color development, and it can be seen that the value of the magnetic bead group is significantly higher than that of the blank group and is similar to that of the antibody control group;

FIG. 5 is a photograph of a green fluorescent antibody-coupled graphene oxide magnetic bead (not homogenized) under a confocal microscope at a magnification of 10X on the left and 60X on the right, wherein green fluorescence is visible on the surface;

FIG. 6 is a micrograph of antibody-coupled graphene oxide magnetic beads sorted MT-4 cells (CD3 expression: 34%) and cell proliferation, from left to right, the cell morphology on the day of sorting and 1 day of culture, and cell aggregation proliferation is visible;

FIG. 7 is a standard curve obtained by quantification using BCA protein;

FIG. 8 is an absorbance value detected by an enzyme-labeling instrument when detecting the activity of an antibody coupled with a CD3 antibody, and it can be seen that the absorbance value of a magnetic bead group is higher than that of a blank group after 100 times of dilution;

FIG. 9 shows the proliferation and morphological change of MT-4 cells sorted by magnetic beads from left to right, during which the magnetic beads are gradually removed by magnetic separation, and from left to right, the cell morphology on the day of sorting, 3 days of culture, and 5 days of culture, respectively, shows that the cells proliferate normally.

Detailed Description

The invention provides a graphene oxide magnetic bead which comprises an iron nitride magnetic bead core and a graphene shell layer coated on the surface of the iron nitride magnetic bead core, wherein the graphene shell layer is modified through intercalation oxidation.

In the present invention, the particle size of the graphene oxide magnetic beads is preferably 500nm to 2 μm, and more preferably 1 μm.

The invention also provides a preparation method of the graphene oxide magnetic bead, which comprises the following steps: 1) mixing iron nitride magnetic beads coated with graphene, sodium nitrate, sulfuric acid and potassium permanganate to obtain a first mixed feed liquid; 2) ultrasonically dispersing the first mixed feed liquid, and mixing with water to obtain a second mixed feed liquid; 3) carrying out intercalation oxidation on the second mixed feed liquid to obtain graphene oxide magnetic beads; the temperature of the intercalation oxidation is 80-90 ℃, and the time of the intercalation oxidation is 3-8 min.

In the invention, graphene-coated iron nitride magnetic beads, sodium nitrate, sulfuric acid and potassium permanganate are mixed to obtain a first mixed feed liquid. In the present invention, it is preferable to mix the graphene-coated iron nitride magnetic beads with sodium nitrate, and then sequentially mix with sulfuric acid and potassium permanganate. In the present invention, the mass ratio of the graphene-coated iron nitride magnetic beads to sodium nitrate is preferably 1: 2. In the present invention, sulfuric acid is preferably added to the mixed graphene-coated iron nitride magnetic beads and sodium nitrate in an ice bath environment. In the invention, the mass ratio of the volume of the sulfuric acid to the graphene-coated iron nitride magnetic beads is 85-95 mL:1g, more preferably 92mL:1 g; in the present invention, the sulfuric acid is preferably concentrated sulfuric acid, and the concentration and the source of the concentrated sulfuric acid are not particularly limited in the present invention, and commercially available concentrated sulfuric acid that is conventional in the art may be used. In the invention, the mass ratio of the potassium permanganate to the graphene-coated iron nitride magnetic beads is (3-5): 1, and more preferably 4: 1. In the invention, the graphene-coated iron nitride magnetic beads, sodium nitrate, sulfuric acid and potassium permanganate are preferably stirred for 8-12 min, and more preferably 10 min; the stirring speed is not specially limited, and the uniform mixing can be realized.

In the invention, the first mixed feed liquid is subjected to ultrasonic dispersion and then is mixed with water to obtain a second mixed feed liquid. In the invention, the temperature of ultrasonic dispersion is preferably 30-40 ℃, and more preferably 35 ℃; the time for ultrasonic dispersion is preferably 25-35 min, and more preferably 30 min. The power of the ultrasonic dispersion is not particularly limited in the invention, and the power used by the ultrasonic cleaning instrument in the prior art can be adopted. In the invention, after the ultrasonic dispersion, mechanical stirring is preferably carried out, and the rotating speed of the mechanical stirring is preferably 60 rpm; the mechanical stirring time is preferably 25-35 min, and more preferably 30 min. In the present invention, it is preferable to mix with water after the mechanical stirring is completed. In the present invention, the volume of the water is preferably 2 times the volume of the sulfuric acid.

In the invention, the second mixed feed liquid is subjected to intercalation oxidation to obtain graphene oxide magnetic beads. In the invention, the temperature of intercalation oxidation is 80-90 ℃, preferably 85 ℃; the time of intercalation oxidation is 3-8 min, preferably 5 min. In the invention, nitrate radicals in sulfuric acid and sodium nitrate can provide an environment of composite strong acid, and under the environment, a strong oxidant potassium permanganate destroys the structure of graphene to generate oxygen-containing groups. After the intercalation oxidation is finished, preferably adding hydrogen peroxide into an intercalation oxidation system to neutralize excessive potassium permanganate to finish the reaction; the hydrogen peroxide is preferably a hydrogen peroxide solution having a mass concentration of 30%, and bubbles are generated when the hydrogen peroxide is added, and the hydrogen peroxide is preferably added until bubbles are no longer generated in the system.

In the invention, the intercalation oxidation also comprises the steps of centrifugation, water washing and drying. In the invention, the rotation speed of the centrifugation is preferably 7000-8000 rpm, and more preferably 7500 rpm; the time for centrifugation is preferably 8-12 min, and more preferably 10 min. After the centrifugation, the precipitate is collected to obtain the graphene oxide magnetic beads. In the invention, the number of washing times is preferably 2-3, and the washing times is not particularly limited, and a conventional washing method in the field can be adopted. In the present invention, the drying is preferably drying, the temperature of the drying is preferably 55 ℃, and the time of the drying is preferably 4 h.

In the present invention, it is preferable that the step of dispersing the graphene oxide magnetic beads is further included after the drying, in the present invention, the dispersing is preferably performed by using a high-pressure homogenizer, and in the implementation process of the present invention, the graphene oxide magnetic beads, ethanol, and water are preferably mixed and homogenized. In the invention, the mass ratio of the graphene oxide magnetic beads to the ethanol to the water is preferably 1:160: 2000; the pressure for homogenization is preferably 350-450 bar, more preferably 400bar, and the time for homogenization is preferably 3-8 min, more preferably 5 min. The method comprises the steps of collecting a homogenized graphene oxide magnetic bead solution, wherein magnetic beads in the graphene oxide magnetic bead solution are uniformly dispersed; the dispersing step in the invention aims to solve the problem of magnetic bead agglomeration caused by the drying step.

The invention also provides an antibody coupled graphene oxide magnetic bead, wherein a graphene shell of the graphene oxide magnetic bead is coupled with a CD3 antibody. In the present invention, the CD3 antibody is an antibody corresponding to the T cell surface antigen CD 3. In the present invention, the CD3 antibody is preferably a custom made antibody with low BSA content. In the present invention, the carboxyl groups on the surface of the graphene oxide magnetic beads are preferably activated by an EDC/NHS method, and the CD3 antibody is preferably coupled to the carboxyl groups activated on the surface of the graphene oxide magnetic beads.

In the present invention, the preparation method of the antibody-coupled graphene oxide magnetic bead preferably includes the following steps: s1) placing the graphene oxide magnetic beads in MES buffer solution for ultrasonic dispersion to obtain graphene oxide magnetic bead solution; s2) mixing and reacting the graphene oxide magnetic bead solution, EDC & HCl and NHS to obtain activated magnetic beads; s3) mixing and incubating the activated magnetic beads with antibodies to obtain antibody-coupled graphene oxide magnetic beads. In the present invention, the graphene oxide magnetic beads in the graphene oxide magnetic bead solution are preferably 1 mg/mL. In the present invention, the mass ratio of the graphene oxide magnetic beads, EDC · HCl and NHS is preferably 20:7.65: 11.5; the mixing reaction time is preferably 10-20 min, and more preferably 15 min. The present invention preferably will perform a wash after obtaining the activated magnetic beads, the wash preferably being performed using a PCS buffer. In the invention, the mass ratio of the antibody to the graphene oxide magnetic beads is preferably 1 (150-250), and more preferably 1: 200. In the invention, the time for mixing and incubating the activated magnetic beads and the antibodies is preferably 2-4 h, and more preferably 3 h; the temperature of the mixed incubation is preferably 20 ℃; the mixing incubation process is preferably accompanied by oscillation, and the frequency of the oscillation is preferably 180 rpm.

The invention also provides application of the antibody-coupled graphene oxide magnetic beads in cell sorting. In the present invention, preferably, the antibody-coupled graphene oxide magnetic beads are mixed with cells to be sorted, and then subjected to magnetic separation, the cells adsorbed by the magnetic poles are washed and placed in a cell culture medium or a buffer solution, and when the magnetic poles are removed, the target cells are dispersed in the culture medium or the buffer solution. In the present invention, the cells to be sorted are preferably dispersed in 3% FBS PBS buffer, and the concentration of the cells to be sorted is preferably 10⁷～10⁸cell/mL. In the invention, the magnetic separation is preferably realized through a magnetic test tube rack, and the time of the magnetic separation is preferably 8-12 min, and more preferably 10 min. After the T cells are magnetically separated, the T cells can be cultured, and the CD3 immunomagnetic beads bound on the T cells have the effect of promoting the proliferation of the T cells.

The technical solutions provided by the present invention are described in detail below with reference to examples, but they should not be construed as limiting the scope of the present invention.