CN114578042A

CN114578042A - Antibody-modified magnetic affinity material, preparation and application thereof

Info

Publication number: CN114578042A
Application number: CN202011383812.0A
Authority: CN
Inventors: 许国旺; 常蒙蒙; 石先哲; 刘心昱
Original assignee: Dalian Institute of Chemical Physics of CAS
Current assignee: Dalian Institute of Chemical Physics of CAS
Priority date: 2020-12-01
Filing date: 2020-12-01
Publication date: 2022-06-03

Abstract

The invention belongs to the technical field of inorganic materials and analysis, and particularly relates to preparation of an antibody modified magnetic graphene oxide composite material (MagG @ PD @ Avidin @ TOM20) and application of the antibody modified magnetic graphene oxide composite material in separation of mitochondria in different hepatocytes. Firstly, a magnetic graphene oxide composite material MagG is synthesized in one step by a solvothermal method, and then is dispersed in an alkaline dopamine solution to realize coating of polydopamine. The obtained matrix material MagG @ PD can be prepared into the magG @ PD @ Avidin by connecting Avidin Avidin to the surface of the material through an amine-catechol addition reaction. Subsequently, biotinylated antibody TOM20 was coupled to MagG @ PD @ Avidin surface by high affinity between Avidin-biotin to make antibody-modified magnetic graphene oxide composite material MagG @ PD @ Avidin @ TOM 20. The material integrates controllable external magnetic field of ferroferric oxide and large graphene surfaceAreal, polydopamine vs NH₂The reaction activity of the cell and the high specificity recognition of the antibody to the outer membrane protein of the mitochondria, and the like, and can separate the mitochondria in cells such as LO2, HuH7, HepG2, and the like.

Description

Antibody-modified magnetic affinity material, preparation and application thereof

Technical Field

The invention belongs to the technical field of inorganic materials and analysis, and particularly relates to a method for separating mitochondria in different hepatocytes based on an antibody-modified magnetic affinity material and application thereof.

The magnetic affinity material provided by the invention can separate mitochondria in homogeneous liquid of liver cells LO2, HuH7 and HepG 2. The material has good application prospect in the research field based on mitochondria.

Background

Mitochondria, an important organelle ubiquitous in eukaryotic cells, are Ca²⁺Plays a key role in the regulation of homeostasis, cellular energy metabolism, biosynthesis, and cell death. The abnormal function of mitochondria is closely related to the occurrence and development of various diseases such as cardiomyopathy, neurodegenerative diseases, cancer and the like. The comprehensive analysis of the changes of mitochondria in genome, proteome and metabolome is beneficial to better understand the relationship between the structure and the function of the mitochondria. It is noteworthy that mitochondria play an important role in the synthesis and breakdown processes of the tricarboxylic acid cycle, beta-oxidation, urea cycle, etc. Based on the central role of mitochondria in metabolism, metabolic disorder of mitochondria is closely related to occurrence and development of a plurality of physiological and pathological diseases, and the deep understanding of metabolic change of mitochondria in the development of diseases is beneficial to the diagnosis and treatment of mitochondrial diseases.

It is undeniable that obtaining mitochondria with intact structure and high purity is a prerequisite for mitochondrial research. Subcellular analysis has enjoyed the 20 th century for the 50 s, with the increasing demand for research into certain organelles, various strategies have emerged in analytical chemistry. Centrifugation is a traditional and reliable method of separating mitochondria that primarily involves differential centrifugation and density gradient centrifugation. Differential centrifugation is a simple and rapid analytical strategy that can collect large numbers of intact mitochondria, but the resulting mitochondria often also adulterate other organelles. As a more specific separation method, the density gradient method can obtain mitochondria with higher purity. However, the time-consuming, laborious and low throughput resulting from multiple resuspension and supernatant transfer operations is an inevitable drawback of density gradient methods, which limits subsequent analysis, especially when the sample size is limited.

Methods that meet different needs have been developed for the isolation of mitochondria, benefiting from advances in technology. Therein, a centrifuge device consisting of two key components is designed for rapid separation of intact mitochondria. First, the cells are disrupted as they flow through the microfluidic device designed by centrifugal force, and the resulting cell lysate is collected in a stainless steel container for subsequent differential centrifugation. Although the centrifugal apparatus is designed to achieve complete mitochondrial separation within 30 minutes, the purity of mitochondria separated by the principle of differential centrifugation is relatively low. In addition, centrifugal-based differential migration microfluidic chips were developed to effectively distinguish mitochondria from nuclei and cellular debris in small amounts of biological samples. Mitochondria are smaller in size compared to nuclei and cell debris, and thus, the mitochondria tend to migrate to the outer channel of the chip, while the nuclei and cell debris migrate to the inner channel of the chip, thereby achieving effective separation thereof. The limited recovery of mitochondria in small biological samples and potential contamination of other similarly sized organelles limits the usefulness of this approach.

Notably, affinity purification of organelles has received much attention due to the high and specific affinity interaction of antibodies with specific proteins on the surface of the organelles. Affinity purification relies primarily on the recognition by antibodies of organelle surface proteins or epitope tags fused to surface proteins. For example, commercial Miltenyi immunomagnetic beads that modify TOM22 (a subunit of the mitochondrial outer membrane transporter) antibodies successfully achieved complete mitochondrial purification under optimized conditions. But the efficiency of separation of Miltenyi magnetic beads is currently controversial. Furthermore, the availability of highly purified mitochondria by means of fusion of the HA epitope tag on the outer mitochondrial membrane is a recent work representative of epitope tag purification of mitochondria. Accordingly, streptavidin tags (Strep) and green fluorescent protein tags (GFP) were also fused to the outer mitochondrial membrane proteins to achieve affinity purification of mitochondria in a certain cell line. The purification strategy of the epitope tag requires that it be exclusively located on the outer membrane of mitochondria and exhibit proper expression, and thus, this requires a certain degree of expertise. Accordingly, the universality of epitope tag purification strategies is limited. In addition, the availability of commercial magnetic beads coated with certain antibodies also limits the types of stable cell lines constructed subsequently; meanwhile, the particle size of the magnetic beads also affects the efficiency of affinity purification. At present, a general, detailed and efficient strategy for preparing immunomagnetic beads to realize specific separation of mitochondria has not been reported yet.

Disclosure of Invention

The invention aims at the requirements of related researches on mitochondria, and establishes a method for separating mitochondria in hepatocytes by using a magnetic composite material modified by an antibody, which is universal and can specifically separate mitochondria, and application thereof.

The technical route of the invention is as follows:

firstly, synthesizing a magnetic graphene oxide composite matrix material MagG in one step by adopting a solvothermal method: with 0.08-0.32g FeCl₃·6H₂0.015-0.06g of C with O as iron source₆H₅Na₃O₇·2H₂Adding 15-60mL of glycol into O as a stabilizer for dissolution, taking 0.015-0.06g of graphene as a magnetic nucleus growth substrate, carrying out ultrasonic treatment for 1-3h under the condition of ice bath (0-4 ℃), and then adding 0.35-1.4g of CH₃CO₂Na is stirred for 0.5 to 1.0 hour, the mixed solution is transferred into a reaction kettle and reacts for 6 to 10 hours at the temperature of 220 ℃, and the product is sequentially washed by ethanol and water and dried to obtain a magnetic graphene oxide composite matrix material MagG;

secondly, synthesizing a polydopamine-coated MagG @ PD by adopting oxidative polymerization of dopamine: weighing 15-60mg of MagG material prepared in the first step, dispersing in 30-120mL of ethanol, 15-60mL of 10-20mM Tris-HCl buffer solution and 20-90mL of H₂Adding 60-240mg of dopamine into the mixed solution of O, mechanically stirring for 6-12h at 25-60 ℃, carrying out solid-liquid separation on the product by using a magnet, collecting the solid product, sequentially washing with water and ethanol, and drying to obtain polydopamine-coated MagG @ PD;

thirdly, synthesizing Avidin-coated MagG @ PD @ Avidin by adopting an amine-catechol addition reaction: taking 0.02-1.5mg of the MagG @ PD material prepared in the second step, adding 100-400 mu L of Tris-HCl solution containing 0.1-0.4mg of Avidin Avidin, oscillating and incubating for 2-12h at the reaction temperature of 4-37 ℃, performing solid-liquid separation on the product by using a magnet, collecting a solid product, and sequentially washing the solid product by using Tris-HCl Buffer and PBS solution to obtain Avidin coated MagG @ PD @ Avidin;

fourth step, biotinylation modification of antibody TOM20 or IgG: removing 60-160 μ g of TOM20 antibody or IgG, adding 5-10 μ L of biotinylation reagent EZ-Link Sulfo-NHS-LC-LC-Biotin (Thermo Scientific,21338) with the concentration of 4-10mM, shaking and incubating for 0.5-2h at the reaction temperature of 4-37 ℃, transferring the biotinylated TOM20 antibody or IgG into a Desalting column Zeba Spin desaling Columns (Thermo Scientific,89889) pre-washed by PBS, centrifuging and Desalting, and collecting the flow-through liquid to obtain Biotin-modified antibody TOM20 or IgG;

fifthly, synthesizing an antibody TOM20 or IgG modified magnetic graphene oxide composite material: adding 100-500 mu L of biotinylated antibody TOM20 or IgG with the mass of 20-100 mu g into 1-5mg of MagG @ PD @ Avidin, oscillating and incubating for 0.5-2h at the reaction temperature of 4-25 ℃, washing for 1-5 times by PBS, adding 1-5mg/mL of PBS solution of BSA with the volume of 500-1000 mu L, oscillating and incubating for 1-4h at the reaction temperature of 4-25 ℃, performing solid-liquid separation on the product by using a magnet, and collecting a solid product to obtain the MagG @ PD @ Avidin @ TOM20 or MagG @ PD @ Avidin @ IgG.

Due to the adoption of the technical scheme, compared with the prior art, the invention has the following advantages: the magnetic graphene oxide composite material is synthesized in one step by a solvothermal method, a polydopamine shell layer is coated on the surface of a substrate material by utilizing oxidative polymerization of dopamine, avidin can be modified on the surface of the obtained material through amine-catechol addition reaction, and the avidin-modified substrate material can be efficiently coupled with a biotinylated antibody. The method is a preparation strategy of the immunoaffinity material with simple and convenient operation and strong universality, the obtained affinity material has the characteristics of controllable external magnetic field and high antibody immobilization capacity, and has good practical value and application prospect in the research field based on mitochondria.

The material integrates controllable external magnetic field of ferroferric oxide, large surface area of graphene and poly-dopamine-to-NH₂Has multiple advantages of reactivity, high specificity recognition of antibodies to mitochondrial outer membrane proteins and the like, and can separate LO2, HuH7 and HepG2Etc. in the cell. The material has good practical value and application prospect in the research field based on mitochondria.

Description of the drawings:

FIG. 1 is a flow chart of the preparation of a material.

FIG. 2 is a schematic flow diagram of material isolation of mitochondria in cells.

FIG. 3 is a transmission electron micrograph of the material at various stages of its preparation. (a) GO, (b) MagG, (c) MagG @ PD. It can be seen from the figure that Fe is dispersed on the graphene oxide of the sheet layer₃O₄Granules, and after polydopamine coating, Fe₃O₄The surface appeared as a thin shell, showing a more opaque character to MagG @ PD than GO and MagG.

FIG. 4 is a representation of various characteristics of a material: (a) infrared spectra of MagG and MagG @ PD. Characteristic peak of MagG after polydopamine coating (1718 cm)^-1The stretching vibration absorption peak of carbonyl C ═ O; 1216cm^-1Stretching vibration peak of epoxy group C-O; 1085cm^-1The stretching vibration peak of alkoxy C-O) is obviously weakened, 1219cm^-1Characteristic peaks of (a) indicate coating of dopamine. (b) Immobilization of biotinylated IgG by MagG @ PD @ Avidin. From the figure, it can be seen that MagG @ PD @ Avidin can achieve about 570 μ g/mg of IgG immobilization. (c) The immobilization of IgG was compared to commercial material. It can be seen from the figure that the material MagG @ PD @ Avidin has an order of magnitude higher solid loading for IgG than commercial.

FIG. 5 shows the Western blot WB analysis of characteristic proteins of different organelles. It can be seen from the figure that after the material MagG @ PD @ Avidin @ TOM20 was isolated, the mitochondrial characteristic proteins VDAC and PDH-E1 α were significantly enriched.

The method has the characteristics of simple and convenient preparation process and strong universality, and the material has the characteristics of controllable external magnetic field and high antibody immobilization capacity, and has good practical value and application prospect in the research field based on mitochondria.

Detailed Description

The method for separating mitochondria in hepatocyte based on antibody modified magnetic affinity material and the application thereof are elaborated by the concrete examples.

Example 1. preparation of antibody-modified magnetic affinity materials.

Firstly, synthesizing a magnetic graphene oxide composite matrix material MagG in one step by adopting a solvothermal method, namely sequentially weighing 0.16g FeCl₃·6H₂Adding O, 0.03g of trisodium citrate dihydrate and 0.03g of graphene into a 100mL beaker, adding 30mL of glycol for dissolving, and carrying out ultrasonic treatment for 3h under the ice bath condition; then 0.7g of sodium acetate is added into the beaker, the mechanical stirring is carried out for 0.5h at the room temperature, and the mixed solution is transferred into a reaction kettle and reacts for 6h at 190 ℃. And cooling to room temperature, collecting the material by using a magnet, sequentially washing the product by using ethanol and water, and drying in vacuum.

Secondly, synthesizing a polydopamine-coated MagG @ PD by adopting oxidative polymerization of dopamine: and weighing 30mg of the MagG material prepared in the first step, ultrasonically dispersing the MagG material in a mixed solution consisting of 60mL of ethanol, 30mL of 10mM Tris Buffer and 45mL of water, adding 120mg of dopamine, mechanically stirring at room temperature for 8h, collecting a product by using a magnet, washing with water and ethanol, and drying in vacuum to obtain the polydopamine-coated MagG @ PD.

Thirdly, synthesizing Avidin-coated MagG @ PD @ Avidin by adopting an amine-catechol addition reaction: the MagG @ PD material prepared in the second step was washed three times with 10mM Tris-HCl Buffer, 1.2mL of 1mg/mL Avidin solution in Tris-HCl was added, and incubated at 4 ℃ for 12h with shaking. And washing the magnet collecting material with Tris-HCl Buffer and PBS to obtain the Avidin modified MagG @ PD @ Avidin.

Fourth step, biotinylation modification of antibody TOM20 (one subunit of the mitochondrial outer membrane transporter): 60 mu g of TOM20 was removed, 5. mu.L of biotinylation reagent EZ-Link Sulfo-NHS-LC-LC-Biotin with a concentration of 4mM was added, the mixture was incubated at 25 ℃ for 1 hour with shaking, after completion of the reaction, the biotinylated TOM20 antibody was transferred to a PBS-prewashed column Zeba Spin desaling Columns, centrifuged at 1000g for 2min, and the flow-through was collected to obtain Biotin-modified antibody TOM 20.

Step five, synthesizing a magnetic graphene oxide composite material MagG @ PD @ Avidin @ TOM20 modified by an antibody TOM 20: and (3) adding the biotinylated antibody TOM20 prepared in the fourth step into 2mg of MagG @ PD @ Avidin, performing shake incubation for 0.5h at the reaction temperature of 25 ℃, washing with PBS for three times, adding 1mL of 1mg/mL PBS solution of BSA, performing shake incubation for 1h at the reaction temperature of 25 ℃, collecting the product by using a magnet, and washing with PBS to obtain the MagG @ PD @ Avidin @ TOM 20. The synthetic scheme is shown in figure 1.

Example 2. evaluation of the immobilization capacity of the matrix material MagG @ PD @ Avidin using a Biotized IgG as model antibody.

(1) Biotinylation of IgG: transferring 600 mu L of IgG, adding 22.3 mu L of 10mM biotinylation reagent EZ-Link Sulfo-NHS-LC-LC-Biotin, incubating for 1h under oscillation at the reaction temperature of 25 ℃, transferring the biotinylated IgG into a Desalting column Zeba Spin desaling Columns prewashed by PBS after the reaction is finished, centrifuging for 2min at 1000g, and collecting the flow-through liquid to obtain the Biotin-modified IgG.

(2) Different concentrations of biotinylated IgG modified equivalent amounts of matrix material MagG @ PD @ Avidin: respectively diluting biotinylated IgG by 4 times, 8 times, 20 times, 40 times, 80 times, 200 times and 400 times, paralleling each concentration point for three times, taking 140 mu L of the biotinylated IgG and 15 mu g of MagG @ PD @ Avidin, performing oscillation incubation for 0.5h at the reaction temperature of 25 ℃, and collecting the incubated IgG solution; the material was collected with a magnet, washed with PBS, dispersed in PBS and stored at 4 ℃.

(3) BCA protein quantification assay to assess the immobilization of MagG @ PD @ Avidin on biotinylated IgG: taking the incubated IgG solution collected in the step (2), and mixing 20 mu L of the incubated solution of biotinylated IgG diluted by 8 times, 20 times, 40 times, 80 times, 200 times and 400 times with 200 mu L of BCA working solution; for the solution after 4-fold dilution of biotinylated IgG incubation, 5. mu.L of the solution was taken, supplemented with 15. mu.L of LPBS, and mixed with 200. mu.L of BCA working solution; for biotinylated IgG stock, 2 μ L was taken, supplemented with 18 μ LPBS, and mixed with 200 μ L BCA working solution; standing at 37 deg.C for 30min, and measuring absorbance at 562nm wavelength with enzyme labeling instrument. The biotinylated IgG concentration in each sample was calculated from the standard curve for the same batch of BSA and the sample volume used. The immobilization of the material MagG @ PD @ Avidin on biotinylated IgG was calculated from the corresponding concentrations. The results are shown in FIGS. 4(b) and (c).

Example 3. the material MagG @ PD @ Avidin @ TOM20 prepared in example 1 isolates mitochondria in hepatocytes LO2, HuH7, HepG 2.

(1) Culture of hepatocytes LO2, HuH7, HepG 2: LO2, HuH7, HepG2 were all in DMEM (10% FBS + 1% pen/stre in final volume concentration) at 37 ℃ with 5% CO in volume concentration₂Under air conditions of (2), culturing to 90-100% coverage.

(2) MagG @ PD @ Avidin @ TOM20 isolates mitochondria in hepatocytes LO2, HuH7, HepG 2: LO2 cells were first washed twice with 10mL PBS, 5mL pancreatin (0.25% Trypsin-EDTA) was digested for 5min at 37 deg.C, and digestion was stopped in 5mL DMMEM (10% FBS + 1% pen/stre at final volume concentration). The cells were centrifuged at 800rpm for 3min and LO2 cells were collected and washed sequentially with 0-4 ℃ pre-chilled PBS, mitochondrial separation buffer (210mM mannitol, 70mM sucrose, 1mM EDTA,5mM Tris, pH 7.5). LO2 cells were then resuspended in 3mL of 0-4 deg.C pre-cooled mitochondrial separation buffer containing 1mM PMSF and transferred to a Potter-Elvejhem homogenizer. The homogenizer was placed on ice and 50% of the cells were disrupted by pushing the pestle vertically down 40 times in a Potter-Elvejhem homogenizer. 100 μ L of the homogenate was taken as a whole cell fraction for subsequent analysis, and the remaining homogenate was centrifuged twice at 1000g at 4 ℃ for 5min to remove nuclei, cell debris and unbroken cells. Subsequently, the collected supernatant was incubated at 10000g, 4 ℃ for 10min, and the resulting pellet enriched in mitochondria was resuspended in 0-4 ℃ pre-cooled mitochondrial isolation buffer and incubated with 1mg of MagG @ PD @ Avidin @ TOM20 at 4 ℃ for 0.5h with shaking. After incubation, the solid material with surface-captured mitochondria was collected with a magnet and washed three times with mitochondrial isolation buffer.

The separation of mitochondria in cells HuH7 and HepG2 was the same as that of LO2 described above. The mitochondrial isolation scheme is shown in FIG. 2.

(3) And (3) carrying out characteristic protein identification on mitochondria separated from the material: mu.L of RIPA lysate containing 1mM PMSF was added to 1mg of the mitochondrial material obtained in (2) or 100. mu.L of the whole cell fraction taken out, pipetted and mixed well, and incubated on ice for 15 min. Collecting supernatant under magnet-assisted or 12000g centrifugation conditions, taking out a small amount of supernatant, determining protein concentration by BCA method, and adding SDS-PAGE protein loading buffer to the rest supernatant(5X) (Beyotime, P0015) and denatured at 97 ℃ for 10 min. After cooling to room temperature, the material is separated into mitochondrial sample or whole cell component and other protein amount sample, and SDS-PAGE separates the protein. Subsequently, the proteins were transmembrane coated onto PVDF membranes. After completion of the membrane transfer, 5% skim milk (5g skim milk: 100mL TBST) was blocked for 1h at room temperature, and 10mL TBST (20mM Tris-HCl,500mM NaCl, 0.05% Tween-20, pH 7.5) was washed three times with 5mL of 1: 1000 (antibody: antibody dilution (5g BSA +100mL PBS +20 mgNa)₃N)), murine anti-VDAC, murine anti-PDH-E1 α, murine anti-Histone H3, and murine anti-Actin were incubated at 4 ℃ overnight. Washing the membrane for three times by TBST, and preparing the membrane with 5mL of 5% skimmed milk respectively in a volume ratio of 1: 5000 peroxidase-cross-linked secondary antibodies were incubated for 1h at room temperature and exposed to a chemiluminescence apparatus. The specific results are shown in FIG. 5.

Claims

1. The magnetic graphene oxide composite material modified by the antibody is characterized in that,

the composite material takes a matrix material MagG with ferroferric oxide particles dispersed on graphene as a core, a polydopamine shell layer is coated on the outer surface of the core, and avidin and a biotinylated antibody are sequentially coupled to the surface of the shell layer.

2. The antibody-modified magnetic graphene oxide composite material according to claim 1, wherein the antibody is one or more of an antibody TOM20 or IgG.

3. The antibody-modified magnetic graphene oxide composite material according to claim 1, wherein the ferroferric oxide particle size of the matrix material MagG is 100-200nm, and the graphene sheet size is 500nm-5 μm; the thickness of the polydopamine shell layer coated on the MagG is 5-10 nm.

4. A method for preparing the antibody-modified magnetic graphene oxide composite material according to any one of claims 1 to 3, which is prepared by the following method:

firstly, ferroferric oxide particles are dispersed on graphene through a solvothermal method to synthesize a magnetic graphene oxide composite matrix material MagG in one step, then a polydopamine PD coating is deposited on the surface of the MagG through oxidative polymerization of polydopamine to form MagG @ PD, then Avidin is connected to the surface of the MagG @ PD through amine-catechol addition reaction to synthesize MagG @ PD @ Avidin, and finally biotinylated antibody TOM20 or biotinylated IgG is coupled to the surface of the material through high affinity between Avidin and biotin to form an antibody modified magnetic graphene oxide composite material MagG @ PD @ Avidin @ TOM20 or MagG @ PD @ Avidin @ IgG.

5. The preparation method of the antibody-modified magnetic graphene oxide composite material according to claim 4, comprising the following specific steps:

the first step is as follows: synthesizing a magnetic graphene oxide composite matrix material MagG in one step by adopting a solvothermal method: with 0.08-0.32g FeCl₃·6H₂0.015-0.06g of C with O as iron source₆H₅Na₃O₇·2H₂Adding 15-60mL of glycol into O as a stabilizer for dissolution, taking 0.015-0.06g of graphene as a magnetic nucleus growth substrate, carrying out ultrasonic treatment for 1-3h under the condition of ice bath (0-4 ℃), and then adding 0.35-1.4g of CH₃CO₂Na is stirred for 0.5 to 1.0 hour, the mixed solution is transferred into a reaction kettle and reacts for 6 to 10 hours at the temperature of 220 ℃, and the product is sequentially washed by ethanol and water and dried to obtain a magnetic graphene oxide composite matrix material MagG;

the second step is that: synthesizing polydopamine-coated MagG @ PD by oxidative polymerization of dopamine: weighing 15-60mg of MagG material prepared in the first step, dispersing in 30-120mL of ethanol, 15-60mL of 10-20mM Tris-HCl buffer solution and 20-90mL of H₂Adding 60-240mg of dopamine into the O mixed solution, mechanically stirring for 6-12h at 25-60 ℃, carrying out solid-liquid separation on the product by using a magnet, collecting the solid product, sequentially washing with water and ethanol, and drying to obtain polydopamine-coated MagG @ PD;

the third step: synthesizing Avidin-coated MagG @ PD @ Avidin by amine-catechol addition reaction: taking 0.02-1.5mg of the MagG @ PD material prepared in the second step, adding 100-400 mu L of Tris-HCl solution containing 0.1-0.4mg of Avidin Avidin, carrying out oscillation incubation for 2-12h at the reaction temperature of 4-37 ℃, carrying out solid-liquid separation on the product by using a magnet, collecting a solid product, and sequentially washing with Tris-HCl Buffer and PBS solution to obtain Avidin coated MagG @ PD @ Avidin;

fourth step, biotinylation modification of antibody TOM20 or IgG: transferring 60-160 mu g of TOM20 antibody or IgG, adding 5-10 mu L of biotinylation reagent EZ-Link Sulfo-NHS-LC-LC-Biotin with the concentration of 4-10mM, oscillating and incubating for 0.5-2h at the reaction temperature of 4-37 ℃, transferring the biotinylated TOM20 antibody or IgG into a Desalting column Zeba Spin desaling Columns prewashed by PBS, centrifuging and Desalting, and collecting flow-through liquid to obtain Biotin modified antibody TOM20 or IgG;

the fifth step: synthesizing an antibody TOM20 or IgG modified magnetic graphene oxide composite material: adding 100-500 mu L of biotinylated antibody TOM20 or IgG with the mass of 20-100 mu g into 1-5mg of MagG @ PD @ Avidin, oscillating and incubating for 0.5-2h at the reaction temperature of 4-25 ℃, washing for 1-5 times by PBS, adding 1-5mg/mL of PBS solution of BSA with the volume of 500-1000 mu L, oscillating and incubating for 1-4h at the reaction temperature of 4-25 ℃, performing solid-liquid separation on the product by using a magnet, and collecting a solid product to obtain the MagG @ PD @ Avidin @ TOM20 or MagG @ PD @ Avidin @ IgG.

6. The method for preparing the antibody modified magnetic graphene oxide composite material according to the claim or the claim, wherein the immobilization amount of MagG @ PD @ Avidin on biotinylated IgG is 70-570 μ g/mg.

7. Use of the antibody-modified magnetic graphene oxide composite material according to any one of claims 1 to 3 as an immunoaffinity material.

8. The application of the antibody-modified magnetic graphene oxide composite material as an immunoaffinity material according to claim 7, wherein the antibody-modified magnetic graphene oxide composite material is characterized in that: for the isolation of mitochondria in cell homogenates.

9. The application of the antibody-modified magnetic graphene oxide composite material as an immunoaffinity material according to claim 7, wherein the antibody-modified magnetic graphene oxide composite material is characterized in that: the magnetic affinity material is directly added into one or more than two homogenate of liver cells LO2, HuH7 and HepG2 to separate mitochondria.