Antibody-modified magnetic affinity material and its preparation and application

Abstract

The present invention belongs to the field of inorganic materials and analytical technology, and in particular to the preparation of antibody-modified magnetic graphene oxide composite materials (MagG@PD@Avidin@TOM20) and the application of separating mitochondria in different hepatocytes. First, the magnetic graphene oxide composite material MagG is synthesized in one step by a solvothermal method, and then dispersed in an alkaline dopamine solution to achieve the coating of polydopamine. The obtained matrix material MagG@PD can be connected to the material surface by the addition reaction of amine-catechol to obtain MagG@PD@Avidin. Subsequently, the biotinylated antibody TOM20 is coupled to the surface of MagG@PD@Avidin by the high affinity between avidin-biotin to obtain the antibody-modified magnetic graphene oxide composite material MagG@PD@Avidin@TOM20. This material integrates multiple advantages, such as the controllable external magnetic field of ferroferric oxide, the large surface area of ​​graphene, the reactivity of polydopamine to NH2, and the high specificity of antibodies to recognize mitochondrial outer membrane proteins. It can separate mitochondria in LO2, HuH7, HepG2 and other cells.

Description

Technical Field

[0001] The invention belongs to the field of inorganic materials and analysis technology, and in particular relates to a method for separating mitochondria in different liver cells based on antibody-modified magnetic affinity materials and an application thereof.

[0002] The magnetic affinity material proposed by the present invention can separate mitochondria in the homogenized solution of liver cells LO2, HuH7 and HepG2, and has good application prospects in the research field based on mitochondria. Background Art

[0003] Mitochondria are an important organelle that is ubiquitous in eukaryotic cells. 2+ It plays a key role in the regulation of homeostasis, cellular energy metabolism, biosynthesis, and cell death. Mitochondrial dysfunction is closely related to the occurrence and development of many diseases such as cardiomyopathy, neurodegenerative diseases, and cancer. Comprehensive analysis of mitochondrial changes in the genome, proteome, and metabolome is beneficial to a better understanding of the relationship between mitochondrial structure and function. It is worth noting that mitochondria play an important role in the synthesis and decomposition processes of the tricarboxylic acid cycle, β-oxidation, and urea cycle. Based on the central role of mitochondria in metabolism, mitochondrial metabolic disorders are closely related to the occurrence and development of many physiological and pathological diseases. A deep understanding of the metabolic changes of mitochondria in the development of diseases is conducive to the diagnosis and treatment of mitochondrial diseases.

[0004] It is undeniable that obtaining mitochondria with intact structure and high purity is a prerequisite for mitochondrial research. Subcellular analysis emerged in the 1950s. With the increasing demand for studying certain organelles, various strategies have emerged in analytical chemistry. Centrifugation is a traditional and reliable method for isolating mitochondria, which mainly includes differential centrifugation and density gradient centrifugation. Differential centrifugation is a simple and rapid analytical strategy that can collect a large number of intact mitochondria, but the mitochondria obtained are often mixed with 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-yield operations caused by multiple resuspension and supernatant transfer operations are inevitable disadvantages of the density gradient method, which restricts subsequent analysis, especially when the sample amount is limited.

[0005] Benefiting from the advancement of technology, methods that meet different needs have been developed for the separation of mitochondria. Among them, a centrifugal device consisting of two key components is designed for the rapid separation of intact mitochondria. First, cells are destroyed when they flow through the designed microfluidic device by centrifugal force, and the resulting cell lysate is collected in a stainless steel container for subsequent differential centrifugation. Although the designed centrifugal device can achieve the separation of intact mitochondria within 30 minutes, the purity of mitochondria separated by the principle of differential centrifugation alone is relatively low. In addition, a microfluidic chip based on centrifugal differential migration has been developed to effectively distinguish mitochondria from cell nuclei and cell fragments in a small amount of biological samples. Compared with cell nuclei and cell fragments, mitochondria are smaller in size, so mitochondria tend to migrate to the external channels of the chip, while cell nuclei and cell fragments migrate to the internal channels of the chip, thereby achieving their effective separation. However, the limited recovery of mitochondria in a small amount of biological samples and the potential contamination of other organelles of similar size limit the practicality of this method.

[0006] It is worth noting that affinity purification of organelles has attracted widespread attention due to the high and specific affinity of antibodies to specific proteins on the surface of organelles. Affinity purification mainly relies on the recognition of antibodies on organelle surface proteins or epitope tags fused to surface proteins. For example, commercial Miltenyi immunomagnetic beads modified with TOM22 (a subunit of the mitochondrial outer membrane transporter) antibodies can successfully purify intact mitochondria under optimized conditions. However, there is currently controversy over the separation efficiency of Miltenyi magnetic beads. In addition, obtaining high-purity mitochondria by relying on the HA epitope tag fused to the mitochondrial outer membrane is a representative work of recent epitope tag purification of mitochondria. Based on this, streptavidin tags (Strep) and green fluorescent protein tags (GFP) have also been fused to mitochondrial outer membrane proteins to achieve affinity purification of mitochondria in a certain cell line. The purification strategy of epitope tags requires that they are exclusively located on the outer membrane of mitochondria and show appropriate expression, so this requires certain professional skills. Accordingly, the universality of epitope tag purification strategies is limited. In addition, the availability of commercial magnetic beads coated with certain antibodies also restricts the types of stable cell lines that can be constructed subsequently; at the same time, the particle size of the magnetic beads also affects the efficiency of affinity purification. At present, a universal, detailed, and efficient strategy for preparing immunomagnetic beads to achieve specific isolation of mitochondria has not yet been reported. Summary of the invention

[0007] In response to the needs of mitochondria-related research, the present invention establishes a method for separating mitochondria in hepatocytes based on an antibody-modified magnetic composite material that is universal and can specifically separate mitochondria, and its application.

[0008] The technical route of the present invention is as follows:

[0009] The first step is to synthesize the magnetic graphene oxide composite matrix material MagG in one step by a solvothermal method: 0.08-0.32g of FeCl3·6H2O is used as an iron source, 0.015-0.06g of C6H5Na3O7·2H2O is used as a stabilizer, 15-60mL of ethylene glycol is added to dissolve, 0.015-0.06g of graphene is used as a matrix for magnetic nucleus growth, ultrasonic treatment is performed under ice bath (0-4°C) conditions for 1-3h, and then 0.35-1.4g of CH3CO2Na is added, stirred for 0.5-1.0h, and the mixed solution is transferred to a reactor, reacted at 160-220°C for 6-10h, and the product is washed with ethanol and water in turn, and dried to obtain the magnetic graphene oxide composite matrix material MagG;

[0010] The second step is to synthesize polydopamine-coated MagG@PD by oxidative polymerization of dopamine: 15-60 mg of the MagG material obtained in the first step is weighed and dispersed in a mixture of 30-120 mL of ethanol, 15-60 mL of 10-20 mM Tris-HCl buffer and 20-90 mL of H2O, 60-240 mg of dopamine is added, and mechanical stirring is performed at 25-60 ° C for 6-12 hours. The product is separated from the solid by a magnet, and the solid product is collected, washed with water and ethanol in turn, and dried to obtain polydopamine-coated MagG@PD;

[0011] The third step is to synthesize MagG@PD@Avidin coated with avidin by using the addition reaction of amine-catechol: take 0.02-1.5 mg of MagG@PD material prepared in the second step, add 100-400 μL Tris-HCl solution containing 0.1-0.4 mg avidin, incubate with shaking at a reaction temperature of 4-37°C for 2-12 hours, separate the product by solid-liquid separation using a magnet, collect the solid product, wash it with Tris-HCl Buffer and PBS solution in turn, and obtain MagG@PD@Avidin coated with avidin;

[0012] Step 4: Biotinylation modification of antibody TOM20 or IgG: 60-160 μg of TOM20 antibody or IgG was transferred, 5-10 μL of 4-10 mM biotinylation reagent EZ-Link Sulfo-NHS-LC-LC-Biotin (Thermo Scientific, 21338) was added, and the mixture was incubated with shaking at a reaction temperature of 4-37°C for 0.5-2 h. The biotinylated TOM20 antibody or IgG was transferred into a desalting column Zeba Spin Desalting Columns (Thermo Scientific, 89889) pre-washed with PBS. After centrifugation and desalting, the flow-through was collected to obtain the biotin-modified antibody TOM20 or IgG.

[0013] The fifth step is to synthesize the magnetic graphene oxide composite material modified with antibody TOM20 or IgG: take 100-500 μL of 20-100 μg of biotinylated antibody TOM20 or IgG, add it to 1-5 mg of MagG@PD@Avidin, incubate with shaking at a reaction temperature of 4-25°C for 0.5-2 h, wash with PBS 1-5 times, add 500-1000 μL of 1-5 mg / mL BSA in PBS solution, incubate with shaking at a reaction temperature of 4-25°C for 1-4 h, separate the product into solid and liquid by a magnet, collect the solid product, and obtain MagG@PD@Avidin@TOM20 or MagG@PD@Avidin@IgG.

[0014] Due to the adoption of the above technical solution, compared with the prior art, the present invention has the following advantages: a magnetic graphene oxide composite material is synthesized in one step by a solvothermal method, a polydopamine shell is coated on the surface of the matrix material by oxidative polymerization of dopamine, the obtained material can be modified with avidin on the surface by an amine-catechol addition reaction, and the matrix material modified with avidin can be efficiently coupled with biotinylated antibodies. The method is a preparation strategy for immunoaffinity materials with simple operation and strong universality, and the obtained affinity material has the characteristics of controllable external magnetic field and high antibody immobilization capacity, and has good practical value and application prospects in the field of mitochondria-based research.

[0015] This material integrates the advantages of controllable external magnetic field of ferroferric oxide, large surface area of ​​graphene, reactivity of polydopamine to NH2, high specificity of antibody to mitochondrial outer membrane protein, etc. It can separate mitochondria in cells such as LO2, HuH7, and HepG2. This material has good practical value and application prospects in the field of mitochondria-based research. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 It is a flow chart of material preparation.

[0017] Figure 2 It is a schematic diagram of the process of separating mitochondria from cells.

[0018] Figure 3 The following are transmission electron micrographs of different stages in the material preparation process. (a) GO, (b) MagG, (c) MagG@PD. From the figure, we can see that Fe3O4 particles are dispersed on the graphene oxide sheets, and after polydopamine coating, a thin shell appears on the surface of Fe3O4. Compared with GO and MagG, MagG@PD is more opaque.

[0019] Figure 4Figure 1 shows various characterizations of the materials: (a) Infrared spectra of MagG and MagG@PD. After polydopamine coating, the characteristic peak of MagG (1718 cm -1 , carbonyl C=O stretching vibration absorption peak; 1216cm -1 , stretching vibration peak of epoxy CO; 1085cm -1 , the stretching vibration peak of alkoxy CO) is significantly weakened, 1219cm -1 The characteristic peaks indicate the coating of dopamine. (b) The immobilization capacity of biotinylated IgG by MagG@PD@Avidin. It can be seen from the figure that the immobilization capacity of IgG by MagG@PD@Avidin can reach about 570μg / mg. (c) The immobilization capacity of IgG compared with commercial materials. It can be seen from the figure that the immobilization capacity of IgG by the material MagG@PD@Avidin is one order of magnitude higher than that of the commercial material.

[0020] Figure 5 It is the characteristic protein of different organelles analyzed by Western blotting. It can be seen from the figure that after separation by material MagG@PD@Avidin@TOM20, the characteristic proteins of mitochondria VDAC and PDH-E1α were significantly enriched.

[0021] This method not only has a simple preparation process and strong universality, but also makes the material have the characteristics of controllable external magnetic field and high antibody immobilization capacity. This material has good practical value and application prospects in the field of mitochondria-based research. DETAILED DESCRIPTION

[0022] The method for separating mitochondria in hepatocytes based on antibody-modified magnetic affinity materials and its application are elaborated in detail through specific examples.

[0023] Example 1. Preparation of antibody-modified magnetic affinity materials.

[0024] In the first step, the magnetic graphene oxide composite matrix material MagG was synthesized by solvothermal method: 0.16g FeCl3·6H2O, 0.03g trisodium citrate dihydrate, and 0.03g graphene were weighed into a 100mL beaker, 30mL ethylene glycol was added to dissolve, and ultrasonic treatment was performed for 3h under ice bath conditions; 0.7g sodium acetate was added to the beaker, mechanical stirring was performed at room temperature for 0.5h, and the mixed solution was transferred to a reactor and reacted at 190℃ for 6h. After the temperature was cooled to room temperature, the material was collected by a magnet, and the product was washed with ethanol and water in turn, and vacuum dried.

[0025] In the second step, polydopamine-coated MagG@PD was synthesized by oxidative polymerization of dopamine: 30 mg of the MagG material obtained in the first step was weighed and ultrasonically dispersed in a mixed solution consisting of 60 mL of ethanol, 30 mL of 10 mM Tris Buffer, and 45 mL of water, 120 mg of dopamine was added, and mechanical stirring was performed at room temperature for 8 h. The product was collected with a magnet, washed with water and ethanol, and vacuum dried to obtain polydopamine-coated MagG@PD.

[0026] The third step is to synthesize MagG@PD@Avidin coated with avidin by the addition reaction of amine-catechol: 90 μg of MagG@PD material prepared in the second step is taken, washed three times with 10 mM Tris-HCl Buffer, and 1.2 mL of 1 mg / mL Avidin Tris-HCl solution is added, and incubated at 4°C for 12 h with shaking. The material is collected by magnet, washed with Tris-HCl Buffer and PBS, and MagG@PD@Avidin modified with avidin is obtained.

[0027] Step 4. Biotinylation modification of antibody TOM20 (a subunit of mitochondrial outer membrane transporter): 60 μg of TOM20 was transferred, 5 μL of 4 mM biotinylation reagent EZ-Link Sulfo-NHS-LC-LC-Biotin was added, and the reaction temperature was 25°C for 1 hour. After the reaction was completed, the biotinylated TOM20 antibody was transferred to the desalting column Zeba SpinDesalting Columns pre-washed with PBS, centrifuged at 1000 g for 2 minutes, and the flow-through was collected to obtain the biotin-modified antibody TOM20.

[0028] Step 5: Synthesize the magnetic graphene oxide composite material MagG@PD@Avidin@TOM20 modified with antibody TOM20: Take the biotinylated antibody TOM20 prepared in step 4, add it to 2 mg of MagG@PD@Avidin, incubate it at 25°C for 0.5 h, wash it with PBS three times, add 1 mL of 1 mg / mL BSA in PBS, incubate it at 25°C for 1 h, collect the product with a magnet, wash it with PBS, and obtain MagG@PD@Avidin@TOM20. Synthesis diagram is shown in Figure 1 .

[0029] Example 2. The immobilization capacity of the matrix material MagG@PD@Avidin was evaluated using biotinylated IgG as a model antibody.

[0030] (1) Biotinylation of IgG: 600 μL of IgG was transferred to 22.3 μL of 10 mM biotinylation reagent EZ-Link Sulfo-NHS-LC-LC-Biotin, and incubated with shaking at 25°C for 1 h. After the reaction was completed, the biotinylated IgG was transferred to a PBS-prewashed desalting column Zeba Spin Desalting Columns, centrifuged at 1000 g for 2 min, and the flow-through was collected to obtain biotin-modified IgG.

[0031] (2) Modification of equal amounts of matrix material MagG@PD@Avidin with different concentrations of biotinylated IgG: The biotinylated IgG was diluted 4 times, 8 times, 20 times, 40 times, 80 times, 200 times, and 400 times, respectively. Each concentration point was repeated three times. 140 μL of each sample was incubated with 15 μg of MagG@PD@Avidin at 25°C for 0.5 h with shaking. The IgG solution was collected after incubation. The material was collected with a magnet, washed with PBS, dispersed in PBS, and stored at 4°C.

[0032] (3) BCA protein quantification method was used to evaluate the biotinylated IgG immobilization capacity of MagG@PD@Avidin: Take the incubated IgG solution collected in (2), and take 20 μL of the biotinylated IgG incubation solution diluted 8 times, 20 times, 40 times, 80 times, 200 times, and 400 times, and mix it with 200 μL of BCA working solution; take 5 μL of the biotinylated IgG incubation solution diluted 4 times, add 15 μL PBS, and mix it with 200 μL of BCA working solution; take 2 μL of the biotinylated IgG stock solution, add 18 μL PBS, and mix it with 200 μL of BCA working solution; place it at 37°C for 30 minutes, and measure the absorbance at a wavelength of 562 nm using an enzyme reader. The biotinylated IgG concentration in each sample was calculated based on the standard curve of BSA from the same batch and the sample volume used. The biotinylated IgG immobilization capacity of the material MagG@PD@Avidin was calculated based on the corresponding concentration. For specific results, see Figure 4 (b) and (c).

[0033] Example 3. The material MagG@PD@Avidin@TOM20 prepared in Example 1 was used to separate mitochondria from hepatocytes LO2, HuH7, and HepG2.

[0034] (1) Cultivation of hepatocytes LO2, HuH7, and HepG2: LO2, HuH7, and HepG2 were cultured in DMEM (containing a final volume concentration of 10% FBS + 1% pen / stre) at 37°C in an atmosphere of 5% CO2 to a confluence of 90-100%.

[0035] (2) MagG@PD@Avidin@TOM20 was used to separate mitochondria from hepatocytes LO2, HuH7, and HepG2: LO2 cells were first washed twice with 10 mL PBS, digested with 5 mL trypsin (0.25% Trypsin-EDTA) at 37°C for 5 min, and digested with 5 mL DMEM (containing a final volume concentration of 10% FBS + 1% pen / stre) medium. After centrifugation at 800 rpm for 3 min, LO2 cells were collected and washed with PBS precooled at 0-4°C and mitochondrial isolation buffer (210 mM mannitol, 70 mM sucrose, 1 mM EDTA, 5 mM Tris, pH 7.5) in sequence. Subsequently, LO2 cells were resuspended in 3 mL of mitochondrial isolation buffer precooled at 0-4°C containing 1 mM PMSF and transferred to a Potter-Elvejhem homogenizer. The homogenizer was placed on ice, and 50% of the cells were broken by pushing the pestle in the Potter-Elvejhem homogenizer vertically downward 40 times. 100 μL of the homogenate was taken out as the whole cell fraction for subsequent analysis, and the remaining homogenate was centrifuged twice at 1000g, 4°C for 5 min to remove cell nuclei, cell debris and unbroken cells. Subsequently, the collected supernatant was centrifuged at 10000g, 4°C for 10 min, and the mitochondria-rich pellet was resuspended in 0-4°C pre-cooled mitochondrial isolation buffer and incubated with 1 mg of MagG@PD@Avidin@TOM20 at 4°C for 0.5 h. After incubation, the solid material with captured mitochondria on the surface was collected by magnet, and the material was washed three times with mitochondrial isolation buffer.

[0036] The isolation procedures of mitochondria in HuH7 and HepG2 cells are the same as the above-mentioned isolation procedures of LO2. Figure 2 .

[0037] (3) Identification of characteristic proteins of mitochondria isolated from the material: Take 100 μL of RIPA lysis buffer containing 1 mM PMSF and add it to 1 mg of mitochondrial material obtained in (2) or 100 μL of whole cell fraction taken out, mix by pipetting, and incubate on ice for 15 min. Collect the supernatant under magnet assistance or 12000g centrifugation conditions, take out a small amount of supernatant to measure protein concentration by BCA method, add SDS-PAGE protein loading buffer (5×) (Beyotime, P0015) to the rest of the supernatant, mix, and denature at 97℃ for 10 min. After cooling to room temperature, load the mitochondrial sample or whole cell fraction of the material with equal protein amount, and separate the proteins by SDS-PAGE. Subsequently, transfer the proteins to the PVDF membrane. After the transfer, the membrane was blocked with 5% skim milk (5g skim milk: 100mL TBST) at room temperature for 1h, and the membrane was washed three times with 10mL TBST (20mM Tris-HCl, 500mM NaCl, 0.05% Tween-20, pH7.5), and incubated with 5mL of rabbit anti-VDAC, mouse anti-PDH-E1α, mouse anti-Histone H3 and mouse anti-Actin at a volume ratio of 1:1000 (antibody: antibody diluent (5g BSA+100mL PBS+20mgNa3N)) at 4°C overnight. The membrane was washed three times with TBST, and then incubated with 5mL of 5% skim milk at a volume ratio of 1:5000 peroxide horseradish enzyme cross-linked secondary antibody at room temperature for 1h, and exposed to chemiluminescence. For specific results, see Figure 5 .

Claims

1. A method for preparing an antibody-modified magnetic graphene oxide composite material, characterized in that: The antibody is antibody TOM20 or IgG; The composite material is prepared as follows: First, ferroferric oxide particles are dispersed on graphene by a solvothermal method to synthesize a magnetic graphene oxide composite matrix material MagG, and then a polydopamine PD coating is deposited on the surface of MagG by oxidative polymerization of dopamine to form MagG@PD. Then, avidin Avidin is connected to the surface of MagG@PD by an amine-catechol addition reaction to synthesize MagG@PD@Avidin. Finally, the high affinity between avidin and biotin is used to couple the biotinylated antibody TOM20 or biotinylated IgG to the material surface to form an antibody-modified magnetic graphene oxide composite material MagG@PD@Avidin@TOM20 or MagG@PD@Avidin@IgG. The specific steps include: Step 1: Solvothermal method was used to synthesize the magnetic graphene oxide composite matrix material MagG in one step: 0.08 - 0.32 g of FeCl3·6H2O was used as the iron source, 0.015 - 0.06 g of C6H5Na3O7·2H2O was used as the stabilizer, and 15 - 60 mL of ethylene glycol was added to dissolve. 0.015 - 0.06 g of graphene is used as a matrix for magnetic core growth. Ultrasonic treatment is performed at 0-4 °C ice bath for 1 - 3 h. 0.35 - 1.4 g of CH3CO2Na is then added and stirred for 0.5 - 1.0 h. The mixed solution is transferred to a reactor and reacted at 160 - 220 °C for 6-10 h. The product is washed with ethanol and water in turn and dried to obtain a magnetic graphene oxide composite matrix material MagG. Step 2: Synthesize polydopamine-coated MagG@PD by oxidative polymerization of dopamine: weigh 15-60 mg of the MagG material obtained in the first step and disperse it in a mixture of 30-120 mL of ethanol, 15-60 mL of 10-20 mM Tris-HCl buffer and 20-90 mL of H2O, add 60-240 mg of dopamine, and mechanically stir at 25-60 °C for 6-12 h. Use a magnet to separate the product from the solid and liquid, collect the solid product, wash it with water and ethanol in turn, and dry it to obtain polydopamine-coated MagG@PD; Step 3: Synthesize MagG@PD@Avidin coated with avidin by the addition reaction of amine-catechol: Take 0.02-1.5 mg of MagG@PD material prepared in the second step, add 100-400 µL Tris-HCl solution containing 0.1-0.4 mg avidin, incubate with shaking at 4-37℃ for 2-12 h, separate the product by solid-liquid separation with a magnet, collect the solid product, wash it with Tris-HCl Buffer and PBS solution in turn, and obtain MagG@PD@Avidin coated with avidin; Step 4: Biotinylation of TOM20 or IgG antibody: 60-160 µg of TOM20 antibody or IgG was transferred, and 5-10 µL of 4-10 mM biotinylation reagent EZ-Link Sulfo-NHS-LC-LC-Biotin was added. The mixture was incubated with shaking at 4-37°C for 0.5-2 h. The biotinylated TOM20 antibody or IgG was transferred to the ZebaSpin Desalting Columns pre-washed with PBS. After centrifugation and desalting, the flow-through was collected to obtain the biotinylated TOM20 or IgG antibody. Step 5: Synthesis of magnetic graphene oxide composites modified with antibody TOM20 or IgG: Take 100-500µL of 20-100 µg of biotinylated antibody TOM20 or IgG, add it to 1-5 mg of MagG@PD@Avidin, incubate with shaking at 4-25°C for 0.5-2 h, wash with PBS 1-5 times, add 500-1000µL of 1-5 mg / mL BSA in PBS, incubate with shaking at 4-25°C for 1-4 h, separate the product by solid-liquid separation with a magnet, collect the solid product, and obtain MagG@PD@Avidin@TOM20 or MagG@PD@Avidin@IgG.

2. The method for preparing the antibody-modified magnetic graphene oxide composite material according to claim 1, characterized in that: The particle size of the ferroferric oxide particles of the matrix material MagG is 100-200 nm, and the diameter of the graphene sheet is 500nm-5 µm; the thickness of the polydopamine shell layer coated on MagG is 5-10 nm.

3. The method for preparing the antibody-modified magnetic graphene oxide composite material according to claim 1, characterized in that: The immobilization capacity of biotinylated IgG on MagG@PD@Avidin was 70 - 570 µg / mg.

4. An antibody-modified magnetic graphene oxide composite material prepared by the method for preparing the antibody-modified magnetic graphene oxide composite material according to any one of claims 1 to 3.

5. Use of the antibody-modified magnetic graphene oxide composite material according to claim 4 as an immunoaffinity material.

6. The use of the antibody-modified magnetic graphene oxide composite material as an immunoaffinity material according to claim 5, characterized in that: For the isolation of mitochondria in cell homogenate, the antibody is antibody TOM20.

7. The use of the antibody-modified magnetic graphene oxide composite material as an immunoaffinity material according to claim 6, characterized in that: The magnetic affinity material is directly added to the homogenate of one or more of the liver cells LO2, HuH7, and HepG2 to separate the mitochondria.

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