Application of graphene as artificial membrane protein in intracellular redox regulation

Abstract

The invention discloses an application of graphene as an artificial membrane protein in intracellular redox regulation. When the graphene interacts with the cells, the graphene tends to be vertically inserted into a cell membrane due to the rough edge, the graphene is structurally similar to transmembrane protein of the cells, and meanwhile, the graphene has good conductivity, so that efficient transmission of electrons can be realized by the graphene; therefore, the graphene is also similar to membrane protein responsible for transmembrane electron transport in function. Therefore, the graphene can be used as an electron transport mediator inside and outside cells to mediate the redox reaction of intracellular and extracellular paraelectric pairs, so that the regulation and control of the redox reaction inside and outside the cells are realized.

Description

Technical Field

[0001] The present invention belongs to the technical field of cell fate regulation, and specifically relates to the application of graphene as an artificial membrane protein in intracellular redox regulation. Background Art

[0002] Membrane proteins have many key functions in life activities, such as energy transmission, transmembrane molecular transport, chemical signal transmission, chemical reaction catalysis, etc. Among them, membrane proteins responsible for transmembrane electron transport are essential for maintaining the redox state of cells, and their dysfunction can lead to the occurrence and development of various diseases such as neurodegenerative diseases. Under pathological conditions, the expression and activity of membrane proteins are variable and uncontrollable. To overcome this problem, inorganic materials can be used to prepare bionic membrane proteins, and their stable properties can achieve more precise regulation. However, international research on inorganic materials as bionic membrane proteins is still in the initial development stage. Therefore, the development of bionic membrane proteins is currently a very important and urgent need. Summary of the invention

[0003] The present invention aims to solve one of the technical problems in the related art at least to a certain extent. To this end, one purpose of the present invention is to propose the application of graphene as an artificial membrane protein in intracellular redox regulation. Graphene can be used as an electron transport mediator inside and outside the cell, mediate the redox reaction of the electron pair inside and outside the cell, and realize the regulation of the redox reaction inside and outside the cell.

[0004] In one aspect of the present invention, the present invention proposes the application of graphene as an artificial membrane protein in intracellular redox regulation.

[0005] The inventors found that when graphene interacts with cells, it tends to vertically insert into the cell membrane due to its rough edges, which is similar to the transmembrane protein of the cell in structure. At the same time, graphene has good conductivity, so graphene can achieve efficient electron transmission, so graphene is also functionally similar to the membrane protein responsible for transmembrane electron transmission. Therefore, graphene can serve as an electron transport mediator inside and outside the cell, mediate the redox reaction of the electric pair inside and outside the cell, and realize the regulation of the redox reaction inside and outside the cell.

[0006] In addition, according to the present invention, the application of the above-mentioned graphene as an artificial membrane protein in intracellular redox regulation can also have the following additional technical features:

[0007] In some embodiments of the present invention, the average size of the graphene is 1 μm to 15 μm, and the thickness of the graphene is no more than 20 nm. Thus, the graphene can be embedded in cells to achieve transmembrane electron transport and regulate redox reactions inside and outside cells.

[0008] In some embodiments of the present invention, the conductivity of the graphene is not less than 4S / cm. Thus, transmembrane electron transport can be achieved, and redox reactions inside and outside the cell can be regulated.

[0009] In some embodiments of the present invention, the oxygen-to-carbon ratio of the graphene is not greater than 0.05. Thus, transmembrane electron transport can be achieved, and redox reactions inside and outside the cell can be regulated.

[0010] In some embodiments of the present invention, the method of using the graphene for intracellular redox regulation includes: (1) mixing a graphene solution, a phospholipid solution, a buffer solution and a cell culture medium to obtain a graphene culture medium mixture; (2) co-culturing the graphene culture medium mixture with a cell solution to insert the graphene into the cells in the cell solution; (3) incubating the cell solution in which the graphene is inserted with a modeling drug solution to put the cells in which the graphene is inserted into a stress state; (4) incubating the cell solution in the stress state with an extracellular auxiliary electrode solution to regulate the cells. Thus, graphene can act as an electron transport mediator inside and outside the cell, mediating the redox reaction of the electrode inside and outside the cell, thereby significantly improving the regulation efficiency of the added drug and greatly alleviating the stress state of the cell.

[0011] In some embodiments of the present invention, in step (1), the concentration of the graphene in the graphene culture medium mixture is 10 μg / mL to 100 μg / mL. Thus, an appropriate amount of graphene can be inserted into the cells, significantly alleviating the stress state of the cells.

[0012] In some embodiments of the present invention, in step (1), the concentration of graphene in the graphene solution is not less than 5 mg / mL.

[0013] In some embodiments of the present invention, in step (1), the solvent of the graphene solution includes at least one of ethanol and isopropanol.

[0014] In some embodiments of the present invention, in step (1), the buffer comprises at least one of a phosphate buffered saline solution and a bicarbonate buffered saline solution.

[0015] In some embodiments of the present invention, in step (1), the phospholipid concentration in the phospholipid solution is 10 mg / mL to 40 mg / mL.

[0016] In some embodiments of the present invention, in step (2), the cell concentration of the cell solution is 10,000 to 25,000 cells per milliliter. Thus, an appropriate amount of graphene can be inserted into the cells, significantly alleviating the stress state of the cells.

[0017] In some embodiments of the present invention, in step (2), the volume ratio of the graphene culture medium mixture to the cell solution is no greater than 1: 10. Thus, an appropriate amount of graphene can be inserted into the cells, significantly alleviating the stress state of the cells.

[0018] In some embodiments of the present invention, in step (2), the co-culture time is 12 hours to 48 hours. Thus, graphene can be inserted into the cells.

[0019] In some embodiments of the present invention, in step (3), the volume ratio of the modeling drug solution to the cell solution is no greater than 1: 10. Thus, the cells can be placed in a stress state.

[0020] In some embodiments of the present invention, in step (3), the solvent of the modeling drug solution includes at least one of a phosphate buffer and a bicarbonate buffer.

[0021] In some embodiments of the present invention, in step (3), the mixing and incubation time is 6 hours to 12 hours, thereby putting the cells in a stress state.

[0022] In some embodiments of the present invention, in step (3), the modeling drug in the modeling drug solution includes 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine or rotenone.

[0023] In some embodiments of the present invention, in step (4), the volume ratio of the amount of the extracellular auxiliary electrode solution added to the cell solution is not greater than 1:10. Thus, the regulation efficiency of the added drug is improved and the stress state of the cell is alleviated.

[0024] In some embodiments of the present invention, in step (4), the concentration of the extracellular auxiliary electrode in the extracellular auxiliary electrode solution is 300 μmol / L to 500 μmol / L. Thus, the regulation efficiency of the added drug is improved and the stress state of the cells is alleviated.

[0025] In some embodiments of the present invention, in step (4), the solvent of the extracellular auxiliary electrode solution includes at least one of a phosphate buffer and a bicarbonate buffer.

[0026] In some embodiments of the present invention, in step (4), the extracellular auxiliary electrode comprises ascorbic acid or potassium hexachloroiridate.

[0027] In some embodiments of the present invention, in step (4), the mixing and incubation time is no longer than 2 hours, thereby improving the regulation efficiency of the added drug and relieving the stress state of the cells.

[0028] Additional aspects and advantages of the present invention will be given in part in the following description and in part will be obvious from the following description, or will be learned through practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] The above and / or additional aspects and advantages of the present invention will become apparent and easily understood from the description of the embodiments in conjunction with the following drawings, in which:

[0030] Figure 1 is a scanning electron microscope image of the graphene inserted into the cell solution obtained in Example 1 of the present invention;

[0031] Figure 2 is a transmission electron microscope image of graphene inserted into a cell solution obtained in Example 1 of the present invention;

[0032] Figure 3 is a scanning electron microscope image of the size of graphene in Example 1 of the present invention;

[0033] Figure 4 1 is a graph showing the relative ROS content test results of cells in Example 1 of the present invention and Comparative Examples 1-2;

[0034] Figure 5 1 is a graph showing the test results of the NADH content of cells in Example 2 of the present invention and Comparative Examples 3-4;

[0035] Figure 6 is a scanning electron microscope image of the size of graphene in Comparative Example 5 of the present invention;

[0036] Figure 7 It is a graph showing the test results of the relative ROS content of the cells of Example 1, Comparative Examples 1-2 and Comparative Example 5 of the present invention. DETAILED DESCRIPTION

[0037] The embodiments of the present invention are described in detail below, which are intended to explain the present invention but should not be construed as limiting the present invention.

[0038] In one aspect of the present invention, the present invention proposes the application of graphene as an artificial membrane protein in intracellular redox regulation.

[0039] The inventors found that when graphene interacts with cells, it tends to vertically insert into the cell membrane due to its rough edges, which is similar to the transmembrane protein of the cell in structure. At the same time, graphene has good conductivity, so graphene can achieve efficient electron transmission, so graphene is also functionally similar to the membrane protein responsible for transmembrane electron transmission. Therefore, graphene can serve as an electron transport mediator inside and outside the cell, mediate the redox reaction of the electric pair inside and outside the cell, and realize the regulation of the redox reaction inside and outside the cell.

[0040] According to an embodiment of the present invention, the average size of graphene is 1μm to 15μm, and the thickness of graphene is not more than 20nm. The inventors found that if the size of graphene is too small, it will be taken up by cells and cannot realize the construction of embedded membrane structure; if the size of graphene is too large, it will be free around cells and cannot realize the construction of embedded membrane structure. At the same time, the thickness of graphene cannot be too thick, otherwise the construction of embedded membrane structure cannot be realized. Therefore, this application adopts graphene with an average size of 1μm to 15μm and a thickness of no more than 20nm, which can be embedded in cells to realize transmembrane electron transfer and regulate redox reactions inside and outside cells. It can be understood by those skilled in the art that graphene itself is a sheet structure, and the above average size of graphene refers to the length and width dimensions of the graphene of the sheet structure.

[0041] According to an embodiment of the present invention, the conductivity of graphene is not less than 4S / cm. The inventors have found that if the conductivity of graphene is too small, electron transmission cannot be achieved. Therefore, the present application uses graphene with a conductivity of not less than 4S / cm to achieve transmembrane electron transmission and regulate redox reactions inside and outside cells.

[0042] According to an embodiment of the present invention, the oxygen-carbon ratio of graphene is not greater than 0.05. The inventors have found that if the oxygen-carbon ratio of graphene is too large, the conductivity of graphene is low, and thus it cannot conduct electrons well. Therefore, this application uses graphene with an oxygen-carbon ratio of not more than 0.05 to achieve transmembrane electron transport and regulate redox reactions inside and outside cells.

[0043] According to an embodiment of the present invention, a method for using graphene for intracellular redox regulation includes:

[0044] S100: Mix graphene solution, phospholipid solution, buffer and cell culture medium

[0045] In this step, a graphene solution with a graphene concentration of not less than 5 mg / mL, a phospholipid solution with a phospholipid concentration of 10 mg / mL to 40 mg / mL, a buffer solution and a cell culture medium are mixed to obtain a graphene culture medium mixture. Furthermore, the concentration of graphene in the graphene culture medium mixture is 10 μg / mL to 100 μg / mL, so that an appropriate amount of graphene can be inserted into the cell, significantly alleviating the stress state of the cell. It should be noted that the solvent and buffer solution of the graphene solution are conventional reagents in the art, and those skilled in the art can select them according to actual conditions. For example, the solvent of the graphene solution includes at least one of ethanol and isopropanol; the buffer solution includes at least one of a phosphate buffered saline solution and a bicarbonate buffer solution.

[0046] S200: Co-culture the graphene culture medium mixture with the cell solution

[0047] In this step, the graphene culture medium mixture obtained in S100 is co-cultured with the cell solution for 12 hours to 48 hours, so that the graphene is inserted into the cells in the cell solution. Furthermore, the cell concentration of the cell solution is 10,000 to 25,000 cells per milliliter, and the volume ratio of the graphene culture medium mixture to the cell solution is not greater than 1:10, so that an appropriate amount of graphene can be inserted into the cells, significantly alleviating the stress state of the cells.

[0048] S300: Mix and incubate the graphene-inserted cell solution and the modeling drug solution

[0049] In this step, the cell solution with graphene inserted obtained in S200 and the modeling drug solution are mixed and incubated for 6h to 12h, so that the cells with graphene inserted are in a stressed state. Furthermore, the volume ratio of the modeling drug solution to the cell solution is not greater than 1:10, so that the cells can be in a stressed state. It should be noted that the modeling drug is a conventional preparation in the field, and those skilled in the art can select it according to actual conditions. For example, the modeling drug in the modeling drug solution includes 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine or rotenone; the solvent of the modeling drug solution includes at least one of a phosphate buffer and a bicarbonate buffer.

[0050] S400: Mix the stressed cell solution and the extracellular auxiliary electrode solution for incubation

[0051] In this step, the cell solution obtained in S300 in which the cells are in a stressed state and the extracellular auxiliary electrode solution are mixed and incubated for no more than 2 hours to relieve the stress state of the cells. Furthermore, the concentration of the extracellular auxiliary electrode in the extracellular auxiliary electrode solution is 300 μmol / L to 500 μmol / L, and the volume ratio of the added amount of the extracellular auxiliary electrode solution to the cell solution is not greater than 1:10, thereby improving the regulation efficiency of the added drug and relieving the stress state of the cells. It should be noted that the extracellular auxiliary electrode is a conventional reagent in the art, and those skilled in the art can select it according to actual conditions, for example, the extracellular auxiliary electrode includes ascorbic acid or potassium hexachloroiridate; the solvent of the extracellular auxiliary electrode solution includes at least one of a phosphate buffer and a bicarbonate buffer.

[0052] Therefore, graphene can act as an electron transport mediator inside and outside cells, mediating the redox reactions of electrons inside and outside cells, thereby significantly improving the regulatory efficiency of externally added drugs and greatly alleviating the stress state of cells.

[0053] The present invention is described below with reference to specific embodiments. It should be noted that these embodiments are merely illustrative and do not limit the present invention in any way.

[0054] Example 1

[0055] (1) A 5 mg / mL graphene solution and 1 mL of a 40 mg / mL phospholipid solution were prepared with ethanol, 100 μL of the graphene solution was added to 62 μL of the phospholipid solution, vortexed for 5 seconds, and finally 838 μL of phosphate buffered saline was added to obtain a graphene microplatelet dispersion with a graphene concentration of 500 μg / mL, wherein the average size of the graphene was about 1 to 5 μm, the thickness was about 15 nm to 20 nm, the conductivity of the graphene was 5 to 10 S / cm, and the carbon-oxygen ratio was 0.02.

[0056] (2) Dilute the graphene microsheet dispersion with cell culture medium to a solution with a graphene concentration of 10 μg / mL. Plant the cells on the well plate and attach them to the wall overnight under appropriate culture conditions of 37°C and 5% CO2. The cell concentration of the cell solution is 15,000 cells per milliliter. Add the diluted graphene dispersion and incubate for 12 hours. The volume ratio of the diluted graphene dispersion to the cell solution is 1:10, so that the graphene is inserted into the cells in the cell solution.

[0057] (3) The neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) is a commonly used neurotoxin for inducing PD models, and its induction model has obvious similarities with human Parkinson's disease. MPTP interferes with the activity of the mitochondrial electron transport chain complex, causing mitochondrial dysfunction, leading to increased intracellular ROS levels, and ultimately inducing cell apoptosis. A 1mmol / L MPTP solution is prepared with phosphate buffer and diluted with cell culture medium to a 300μmol / L MPTP solution. The original medium containing graphene microsheets is aspirated, and the un-membrane-inserted graphene in the cell solution obtained in step (2) is removed, and then 300μmol / L MPTP is added to the cell solution obtained in step (2), and incubated at 37 degrees Celsius and 5% CO2 for 6 hours to increase intracellular ROS and put the cells into an oxidative stress state, wherein the volume ratio of 300μmol / L MPTP solution to the cell solution obtained in step (2) is 1:10.

[0058] (4) Finally, 400 μmol / L ascorbic acid prepared in phosphate buffer was added to the cell solution of cells in a stressed state obtained in step (3) as an extracellular regulatory factor, and incubated for 1 hour, wherein the volume ratio of 400 μmol / L ascorbic acid to the cell solution of cells in a stressed state obtained in step (3) was 1:10.

[0059] The cell morphology of the graphene obtained in step (2) of Example 1 inserted into the cell solution is shown in the figure below: Figure 1 (SEM) and Figure 2 (TEM) shows that Figure 1 and Figure 2It can be seen that the graphene microsheets are embedded in the cell membrane to obtain a biomimetic membrane protein with a transmembrane structure. Figure 3 As shown, the average size of the graphene in Example 1 is about 1 μm to 5 μm.

[0060] Comparative Example 1

[0061] (1) Cells were seeded on a well plate and allowed to adhere overnight under appropriate culture conditions of 37°C and 5% CO2. The cell concentration of the cell solution was 15,000 cells per ml.

[0062] (2) Prepare 1mmol / L MPTP solution with phosphate buffer and dilute it to 300μmol / L MPTP solution with cell culture medium. Aspirate the original cell culture medium, then add 300μmol / L MPTP to the cell solution obtained in step (1), incubate at 37 degrees Celsius and 5% CO2 for 6 hours to increase intracellular ROS and put the cells into an oxidative stress state, wherein the volume ratio of 300μmol / L MPTP solution to the cell solution obtained in step (1) is 1:10.

[0063] Comparative Example 2

[0064] (1) Cells were seeded on a well plate and allowed to adhere overnight under appropriate culture conditions of 37°C and 5% CO2. The cell concentration of the cell solution was 15,000 cells per ml.

[0065] (2) Prepare 1mmol / L MPTP solution with phosphate buffer and dilute it to 300μmol / L MPTP solution with cell culture medium. Aspirate the original cell culture medium, then add 300μmol / L MPTP to the cell solution obtained in step (1), incubate at 37 degrees Celsius and 5% CO2 for 6 hours to increase intracellular ROS and put the cells into an oxidative stress state, wherein the volume ratio of 300μmol / L MPTP solution to the cell solution obtained in step (2) is 1:10.

[0066] (3) Finally, 400 μmol / L ascorbic acid prepared in phosphate buffer was added to the cell solution of cells in a stressed state obtained in step (2) as an extracellular regulatory factor, and incubated for 1 hour, wherein the volume ratio of 400 μmol / L ascorbic acid to the cell solution of cells in a stressed state obtained in step (1) was 1:10.

[0067] Taking the normally cultured cells as the control group, the cells obtained in the control group and Example 1 and Comparative Examples 1-2 were treated with ROS fluorescent dye DCFH-DA, and the relative content of intracellular reactive oxygen species was quantitatively detected using flow cytometry.

[0068] The test results of ROS levels of cells in the control group, Example 1 and Comparative Examples 1-2 are shown in Figure 4 .from Figure 4 It can be seen that the ROS content of Comparative Example 1 is greatly increased, indicating that the cell oxidative stress modeling is successfully achieved; the ROS content of Example 1 is greatly reduced compared with that of Comparative Example 2, and there is a significant difference, indicating that graphene, as an artificial membrane protein, plays a role in transmembrane electron transport, and utilizes the oxidation reaction of the extracellular regulatory factor ascorbic acid to induce the reduction reaction of intracellular ROS, thereby reducing the ROS content and achieving the regulation of cell oxidative stress.

[0069] Example 2

[0070] The preparation method of Example 2 is the same as that of step (1) and step (2) of Example 1, with the main difference being that the average size of the graphene used in Example 2 is about 7 to 12 μm, the thickness is about 15 nm to 20 nm, the electrical conductivity of the graphene is 7 to 11 S / cm, and the carbon-oxygen ratio is 0.02.

[0071] (3) Rotenone disrupts the normal functioning of the mitochondrial respiratory chain by inhibiting the electron transfer of NADH-Q oxidoreductase (complex I) in mitochondria. The addition of rotenone will cause a decrease in mitochondrial oxygen consumption and a decrease in NADH / NAD + The ratio increases, thereby causing NADH reduction stress, leading to metabolic disorders in cells, and cell growth will also be inhibited. A 1mmol / L rotenone solution is prepared with a phosphate buffer, and diluted with a cell culture medium to a 250nmol / L rotenone solution. The original culture medium containing graphene microsheets is sucked clean, and the graphene without membrane insertion in the cell solution obtained in step (2) is removed, and then a 250nmol / L rotenone solution is added to the cell solution obtained in step (2), and incubated for 12 hours to increase intracellular NADH and the cells are in a reduction stress state, wherein the volume ratio of the 250nmol / L rotenone solution to the cell solution obtained in step (2) is 1:10.

[0072] (4) Finally, a 400 μmol / L potassium hexachloroiridate (IV) solution prepared with phosphate buffer was added to the cell solution in which the cells were in a stressed state obtained in step (3) as an extracellular regulating electrode, and the mixture was incubated for 1 hour, wherein the volume ratio of the 400 μmol / L potassium hexachloroiridate (IV) solution to the cell solution in which the cells were in a stressed state obtained in step (3) was 1:10.

[0073] Comparative Example 3

[0074] (1) Cells were seeded on a well plate and allowed to adhere overnight under appropriate culture conditions of 37°C and 5% CO2. The cell concentration of the cell solution was 15,000 cells per ml.

[0075] (2) A 1 mmol / L rotenone solution is prepared with a phosphate buffer solution, and the rotenone solution is diluted to 250 nmol / L with a cell culture medium. The original culture medium containing graphene microsheets is aspirated, and the graphene without membrane insertion in the cell solution obtained in step (2) is removed, and then 250 nmol / L rotenone solution is added to the cell solution obtained in step (2), and incubated for 12 hours to increase intracellular NADH and put the cells in a reducing stress state, wherein the volume ratio of 250 nmol / L rotenone solution to the cell solution obtained in step (2) is 1:10.

[0076] Comparative Example 4

[0077] (1) Cells were seeded on a well plate and allowed to adhere overnight under appropriate culture conditions of 37°C and 5% CO2. The cell concentration of the cell solution was 15,000 cells per ml.

[0078] (2) A 1 mmol / L rotenone solution is prepared with a phosphate buffer solution, and the rotenone solution is diluted to 250 nmol / L with a cell culture medium. The original culture medium containing graphene microsheets is aspirated, and the graphene without membrane insertion in the cell solution obtained in step (2) is removed, and then 250 nmol / L rotenone solution is added to the cell solution obtained in step (2), and incubated for 12 hours to increase intracellular NADH and put the cells in a reducing stress state, wherein the volume ratio of 250 nmol / L rotenone solution to the cell solution obtained in step (2) is 1:10.

[0079] (3) Finally, a 400 μmol / L potassium hexachloroiridate (IV) solution prepared with phosphate buffer was added to the cell solution in which the cells were in a stressed state obtained in step (2) as an extracellular regulating electrode, and the mixture was incubated for 1 hour, wherein the volume ratio of the 400 μmol / L potassium hexachloroiridate (IV) solution to the cell solution in which the cells were in a stressed state obtained in step (2) was 1:10.

[0080] With normal cultured cells as the control group, the relative changes of NADH content were detected by NADH detection kit, colorimetry and microplate reader.

[0081] The test results of NADH content of cells in the control group, Example 2 and Comparative Examples 3-4 are shown in Figure 5 .from Figure 5 It can be seen that the NADH content of Comparative Example 3 is greatly increased, indicating that the cell reduction stress modeling is successfully achieved; the NADH content of Example 2 is greatly reduced compared with that of Comparative Example 4, and there is a significant difference, indicating that graphene, as an artificial membrane protein, plays a role in transmembrane electron transport, and utilizes the reduction reaction of the extracellular regulatory factor potassium hexachloroiridate (IV) to induce the oxidation reaction of intracellular NADH, thereby reducing the NADH content and achieving the regulation of cell reduction stress.

[0082] Comparative Example 5

[0083] The main difference between Comparative Example 5 and Example 1 is that the average size of graphene is different. Comparative Example 5 uses nano-scale graphene, and the specific average size of graphene is 200nm to 500nm. The electron microscope image is as follows Figure 6 shown.

[0084] Taking the normally cultured cells as the control group, the cells obtained in the control group and Example 1, Comparative Examples 1-2 and Comparative Example 5 were treated with ROS fluorescent dye DCFH-DA, and the relative content of intracellular reactive oxygen species was quantitatively detected using flow cytometer.

[0085] The test results of ROS levels of cells in the control group and Example 1, Comparative Examples 1-2 and Comparative Example 5 are shown in Figure 7 .from Figure 7 It can be seen that the ROS content of Comparative Example 1 is greatly increased, indicating that the cell oxidative stress modeling is successfully achieved; the ROS content of Example 1 is greatly reduced compared with that of Comparative Example 2, indicating that graphene as an artificial membrane protein realizes the regulation of cell oxidative stress; there is no significant difference in the ROS content of Comparative Example 5 compared with that of Comparative Example 2, indicating that small-sized graphene has almost no ability to regulate oxidative stress, that is, size is very important for the construction of artificial membrane proteins.

[0086] In the description of this specification, the description with reference to the terms "one embodiment", "some embodiments", "example", "specific example", or "some examples" etc. means that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described may be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art may combine and combine the different embodiments or examples described in this specification and the features of the different embodiments or examples, without contradiction.

[0087] Although the embodiments of the present invention have been shown and described above, it is to be understood that the above embodiments are exemplary and are not to be construed as limitations of the present invention. A person skilled in the art may change, modify, replace and vary the above embodiments within the scope of the present invention.

Claims

1. Application of graphene as an artificial membrane protein in intracellular redox regulation.

2. The use according to claim 1, characterized in that: The average size of the graphene is 1 μm to 15 μm, and the thickness of the graphene is no more than 20 nm.

3. The use according to claim 1, characterized in that: The electrical conductivity of the graphene is not less than 4S / cm.

4. The use according to claim 1, characterized in that: The oxygen-carbon ratio of the graphene is not greater than 0.

05.

5. The use according to claim 1, characterized in that: The method of using the graphene for intracellular redox regulation comprises: (1) mixing a graphene solution, a phospholipid solution, a buffer solution and a cell culture medium to obtain a graphene culture medium mixture; (2) co-culturing the graphene culture medium mixture with a cell solution so that the graphene is inserted into cells in the cell solution; (3) mixing and incubating the cell solution in which the graphene is inserted and a modeling drug solution, so as to put the cells in which the graphene is inserted into a stress state; (4) Mixing and incubating the cell solution in the stress state with an extracellular auxiliary electric couple solution to regulate the cells.

6. The use according to claim 5, characterized in that: In step (1), the concentration of the graphene in the graphene culture medium mixture is 10 μg / mL to 100 μg / mL; Optionally, the concentration of graphene in the graphene solution is not less than 5 mg / mL; Optionally, the solvent of the graphene solution includes at least one of ethanol and isopropanol; Optionally, the buffer comprises at least one of a phosphate buffered saline solution and a bicarbonate buffer; Optionally, the phospholipid concentration in the phospholipid solution is 10 mg / mL to 40 mg / mL.

7. The use according to claim 5, characterized in that: In step (2), the cell concentration of the cell solution is between 10,000 and 25,000 cells per milliliter.

8. The use according to claim 5, characterized in that: In step (2), the volume ratio of the graphene culture medium mixture to the cell solution is no more than 1:10; Optionally, the co-culture time is 12 h to 48 h.

9. The use according to claim 5, characterized in that: In step (3), the volume ratio of the modeling drug solution to the cell solution is no greater than 1:10; Optionally, the solvent of the modeling drug solution includes at least one of a phosphate buffer and a bicarbonate buffer; Optionally, the mixed incubation time is 6h to 12h; Optionally, the modeling drug in the modeling drug solution includes 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine or rotenone.

10. The use according to claim 5, characterized in that: In step (4), the volume ratio of the added amount of the extracellular auxiliary electrode solution to the cell solution is not greater than 1:10; Optionally, the concentration of the extracellular auxiliary electrode pair in the extracellular auxiliary electrode pair solution is 300 μmol / L to 500 μmol / L; Optionally, the solvent of the extracellular auxiliary electrode solution includes at least one of a phosphate buffer and a bicarbonate buffer; Optionally, the extracellular auxiliary electrode pair includes ascorbic acid or potassium hexachloroiridate; Optionally, the mixing incubation time does not exceed 2 hours.

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