Graphene quantum dot solution for imaging of hela cells and preparation method thereof

Abstract

The application discloses a preparation method of a graphene quantum dot solution for HeLa cell imaging, and comprises the following steps: taking waste silicon material and waste fluorinated plastic as a basic precursor, taking sodium chloride as a diluent, and preparing graphite nanometer carbon balls by applying a certain intensity of current and voltage to initiate a self-driven chemical reaction; and after the graphite nanometer carbon balls are purified, oxidation peeling and dialysis are performed to obtain the graphene quantum dot solution. The preparation method is simple, low in cost, clear in imaging effect, stable in luminescence property, and capable of controlling the diameter of quantum dots by adjusting the adding amount of the diluent, so that the fluorescence of different colors can be controlled, and can be used for multicolor labeling imaging of cells. The graphene quantum dots prepared by the application can be well attached to the surface of HeLa cells and present significant fluorescence characteristics. Compared with other quantum dot synthesis processes, the graphene quantum dots prepared by the application are wide in raw material sources, economical, efficient, adjustable in quantum dot particle size, and high in yield.

Description

Technical Field

[0001] This invention belongs to the fields of resource recycling and cell imaging technology. More specifically, this invention relates to a graphene quantum dot solution for HeLa cell imaging and its preparation method. Background Technology

[0002] HeLa cells are a widely used human cervical cancer cell line in biological and medical research. They possess unlimited proliferative capacity and are an important model for studying cell biology, cancer mechanisms, and drug screening. In the field of cell imaging, HeLa cells are often used to evaluate the targeting and biocompatibility of novel probes. With the development of nanotechnology, the development of highly efficient, low-toxicity, and high-fluorescence imaging agents has become a key focus in cell labeling and tracking research, which is of great significance for improving the level of disease diagnosis and treatment monitoring.

[0003] Graphene quantum dots (GQDs) have become ideal candidates for novel fluorescent imaging agents due to their excellent optical properties, good water solubility, low toxicity, and outstanding biocompatibility. Compared with traditional organic dyes and metal quantum dots, GQDs exhibit higher photostability and lower cytotoxicity, making them suitable for long-term cell imaging research.

[0004] Graphene quantum dots (GQDs) have become ideal materials for developing fluorescent probes. Common synthesis methods include chemical oxidation, hydrothermal synthesis, electrochemical synthesis, and microwave-assisted synthesis. These methods typically require high temperature, high pressure, or strong acid / base conditions, making them difficult to operate and demanding on equipment. Furthermore, existing processes generally suffer from expensive raw materials and low yields, resulting in high unit costs that hinder industrialization. Poor batch-to-batch product quality reproducibility further complicates widespread application. Therefore, optimizing synthesis routes, improving yields, and reducing costs while maintaining GQD performance has become a crucial research direction in this field. Summary of the Invention

[0005] One object of the present invention is to solve at least the above-mentioned problems and / or defects, and to provide at least the advantages described below.

[0006] To achieve these objectives and other advantages of the present invention, a method for preparing a graphene quantum dot solution for HeLa cell imaging is provided, comprising the following steps:

[0007] Step 1: The waste silicon material and waste fluorinated plastics are ball-milled, acid-washed, alcohol-washed and vacuum-dried respectively, and then screened to produce silicon material and organic fluorides with a particle size of 50~200 mesh.

[0008] Step 2: Add silicon material, organic fluoride and sodium chloride to anhydrous ethanol, and after ultrasonication, mechanical stirring, filtration and drying, prepare reaction precursor. Then place the reaction precursor in a reactor, apply a certain intensity of current and voltage to initiate a self-driven reaction, and obtain graphite carbon nanospheres.

[0009] Step 3: The graphite carbon nanospheres are acid-washed, water-washed, filtered and dried to obtain purified graphite carbon nanospheres;

[0010] Step 4: The purified graphite carbon nanospheres are oxidized and exfoliated, centrifuged, and dialyzed to obtain a graphene quantum dot solution.

[0011] Preferably, in step one, the waste silicon material comes from high-purity silicon materials used in the photovoltaic industry, semiconductor manufacturing industry, chip waste, and other precision industries.

[0012] Preferably, in step one, the waste fluorinated plastics originate from one or more of the following: new energy fields, waste medical equipment fields, and waste materials, waste products and equipment, and waste electronic products from industrial manufacturing processes.

[0013] Preferably, in step one, the waste fluorinated plastics include one or more of polytetrafluoroethylene, polyvinylidene fluoride, and polychlorotrifluoroethylene, with a molecular weight between 50,000 and 1,000,000.

[0014] Preferably, in step one, the ball milling speed is 600~900 r / min; the acid washing uses 1~3 mol / L hydrochloric acid solution; the alcohol washing uses anhydrous ethanol to wash 2~4 times; and the drying temperature is 50~80℃.

[0015] Preferably, in step two, the mass ratio of silicon material to organofluorine compound is 3:1 to 1:3.

[0016] Preferably, in step two, the amount of sodium chloride added is 1% to 10% of the total mass of silicon material and organofluorine compound; the mass-to-volume ratio of organofluorine compound and anhydrous ethanol is 50 to 150 g: 1 L.

[0017] Preferably, in step two, when polytetrafluoroethylene (PTFE) and polyvinylidene fluoride (PVDF) are used, the mass ratio of PTFE to PVDF is 10:1 to 1:10.

[0018] Preferably, in step two, the ultrasonic power is 300~800W, and the mechanical stirring lasts for 4~8 hours.

[0019] Preferably, in step two, the applied voltage is 220V and the current is 0.5~9A.

[0020] Preferably, in step three, the pickling uses a hydrofluoric acid solution with a concentration of 0.5~2M.

[0021] Preferably, in step four, the specific method for oxidative stripping is as follows: the purified graphite carbon nanospheres are added to a potassium permanganate-sulfuric acid solution cooled in an ice bath, and then the temperature is raised to 35-40°C and magnetically stirred for 3-5 hours to oxidize and strip the graphite carbon nanospheres; wherein the concentration of potassium permanganate in the potassium permanganate-sulfuric acid solution is 0.5-2M, and the concentration of the concentrated sulfuric acid solution is 60-75wt%; the mass-to-volume ratio of the purified graphite carbon nanospheres to the potassium permanganate-sulfuric acid solution is 1g:10-50mL.

[0022] Preferably, in step four, the specific method for oxidative exfoliation is as follows: the purified graphite carbon nanospheres are added to a potassium permanganate-sulfuric acid solution cooled in an ice bath, then the temperature is raised to 35-40°C, and the mixture is magnetically stirred for 1-2 hours. Then, under continuous stirring, the mixture is irradiated with a high-pressure mercury lamp for 10-20 minutes, followed by treatment with an alternating electric field for 10-30 minutes, thereby oxidizing and exfoliating the graphite carbon nanospheres. The concentration of potassium permanganate in the potassium permanganate-sulfuric acid solution is 0.5-2M, and the concentration of the concentrated sulfuric acid solution is 60-75wt%. The mass-to-volume ratio of the purified graphite carbon nanospheres to the potassium permanganate-sulfuric acid solution is 1g:10-50mL.

[0023] Preferably, the specific method for applying the alternating electric field is as follows: the reaction liquid is placed between two parallel copper plates with a plate spacing of 10~30cm, an electric field strength of 100~1000V / m, and a frequency of 100~500Hz; the power of the high-pressure mercury lamp is 300~1000W, and the wavelength is 200~400nm.

[0024] Preferably, in step four, the molecular weight cutoff of the dialysis bag is ≤3500 Da, the dialysis solution is ultrapure water, and the concentration of the obtained graphene quantum dot solution is controlled to be ≤0.1 mg / mL.

[0025] Application of a graphene quantum dot solution prepared by the method described above in HeLa cell imaging.

[0026] The present invention has at least the following beneficial effects: The present invention prepares a water-soluble graphene quantum dot with controllable particle size, economy and good fluorescence effect, which can be applied to HeLa cell imaging; The present invention uses waste silicon material and waste fluorinated plastic as basic precursors, and pre-treats the waste silicon material and waste fluorinated plastic through ball milling, acid washing, alcohol washing and drying to ensure the uniformity of raw material particle size. Sodium chloride is used as a diluent to produce graphene carbon nanospheres through a self-driven chemical reaction. After acid washing to remove impurities, a graphene quantum dot solution of a certain concentration is obtained by dialysis, which is the prepared graphene quantum dot;

[0027] The preparation method of this invention is simple and low in cost, with clear imaging effect and stable luminescence properties. The diameter of graphene quantum dots can be controlled by adjusting the amount of diluent added, so as to achieve the regulation of fluorescence of different colors, which is very useful for multicolor labeling imaging of cells.

[0028] The graphene quantum dots prepared by this invention can adhere well to the surface of HeLa cells and exhibit significant fluorescence characteristics. Compared with other quantum dot synthesis processes, this invention has a wide range of raw material sources, is economical and efficient, has adjustable quantum dot particle size, and high yield. This is of great significance for imaging other cell lines such as Vero cells and A549 cells, and also shows potential application prospects for dynamic detection in fields such as drug sustained release and gene therapy.

[0029] Other advantages, objectives and features of the present invention will become apparent in part from the following description, and in part from those skilled in the art through study and practice of the invention. Attached Figure Description

[0030] Figure 1 This is a scanning electron microscope image of the graphite carbon nanospheres prepared in Example 1 of the present invention;

[0031] Figure 2 a is an image of HeLa cells without attached graphene quantum dots; Figure 2 b is a HeLa cell imaging image of the graphene quantum dot solution prepared in Example 1;

[0032] Figure 3 The particle size diagrams are of the graphene quantum dots prepared in Examples 1-5 of this invention. Detailed Implementation

[0033] The present invention will now be described in further detail with reference to the accompanying drawings, so that those skilled in the art can implement it based on the description.

[0034] It should be understood that terms such as “having,” “comprising,” and “including” as used herein do not exclude the presence or addition of one or more other elements or combinations thereof.

[0035] Example 1

[0036] A method for preparing a graphene quantum dot solution for HeLa cell imaging includes the following steps:

[0037] Step 1: At room temperature, waste silicon material from the semiconductor manufacturing industry, waste polyvinylidene fluoride (molecular weight of 1 million) from the new energy field, and waste polytetrafluoroethylene (molecular weight of 1 million) from the industrial manufacturing process are respectively subjected to ball milling at 800 r / min, acid washing with 1 mol / L hydrochloric acid solution, washing three times with anhydrous ethanol, and vacuum drying at 70℃. After that, they are sieved to produce silicon material and organic fluorides (including polyvinylidene fluoride and polytetrafluoroethylene) with a particle size of about 100 mesh.

[0038] Step 2: Add 500g of silicon material, 500g of organic fluoride (where the mass ratio of polytetrafluoroethylene and polyvinylidene fluoride is 1:1), and 10g of analytical grade sodium chloride to 5L of anhydrous ethanol. Sonicate for 1 hour at a power of 500W, then mechanically stir for 6 hours. After filtration and drying, the reaction precursor is prepared. The precursor is then placed in a sealed self-driven reactor and ignited by applying a 220V voltage and 6A current to induce a self-driven reaction, yielding graphite carbon nanospheres. The scanning electron microscope image is shown below. Figure 1 As shown;

[0039] Step 3: Add the graphite carbon nanospheres to a 1M hydrofluoric acid solution for acid washing, then wash with deionized water to remove unreacted silicon and other impurities, then filter and dry to obtain purified graphite carbon nanospheres.

[0040] Step 4: Add the purified graphene carbon nanospheres to a 1M potassium permanganate-sulfuric acid solution (68wt%) cooled in an ice bath. The mass-to-volume ratio of the purified graphene carbon nanospheres to the potassium permanganate-sulfuric acid solution is 1g:25mL. Then, heat to 38℃ and magnetically stir for 4 hours to oxidize and exfoliate the graphene carbon nanospheres. After centrifugation, transfer the solution to a dialysis bag with a molecular weight cutoff of 3000 Da. Use ultrapure water as the dialysate and perform cyclic dialysis to obtain a graphene quantum dot solution with a concentration of 0.08 mg / mL.

[0041] HeLa cells were cultured in DuPont modified Eagle medium at 37°C and 5% CO2 concentration using 10% fetal bovine serum and 1% penicillin / streptomycin as additives. The cultured HeLa cells were then seeded into 96-well cell culture plates at a cell density of 1×10⁻⁶ cells / well. 3 pcs / cm 2 HeLa cells were then subjected to a 24-hour cell adhesion experiment with 120 μL of a 0.08 mg / mL graphene quantum dot solution prepared in Example 1. The cells were then washed multiple times with phosphate buffer solution to obtain graphene quantum dot-coated HeLa cells. Fluorescence imaging was performed, and the results are as follows: Figure 2As shown, the results indicate that the graphene quantum dot solution of this embodiment can adhere well to the surface of HeLa cells, emit green fluorescence under 570nm excitation light, and produce clear images, making it applicable to cell imaging.

[0042] Example 2

[0043] This embodiment is basically the same as Embodiment 1, except that: this embodiment uses 500g of silicon material, 500g of organic fluoride and 20g of analytical grade sodium chloride.

[0044] Example 3

[0045] This embodiment is basically the same as Embodiment 1, except that: this embodiment uses 500g of silicon material, 500g of organic fluoride and 50g of analytical grade sodium chloride.

[0046] Example 4

[0047] This embodiment is basically the same as Embodiment 1, except that: this embodiment uses 500g of silicon material, 500g of organic fluoride and 80g of analytical grade sodium chloride.

[0048] Example 5

[0049] This embodiment is basically the same as Embodiment 1, except that: this embodiment uses 500g of silicon material, 500g of organic fluoride and 100g of analytical grade sodium chloride.

[0050] Graphene quantum dots are obtained by freeze-drying a graphene quantum dot solution. Figure 3 The figures show the particle size distribution of the graphene quantum dots prepared in Examples 1-5. In Examples 1-5, the amount of NaCl added accounted for 1%, 2%, 5%, 8%, and 10% of the total mass of the silicon material and organofluorine compound, respectively. This invention uses sodium chloride as a diluent, and by adjusting the amount of diluent added, the diameter of the graphene quantum dots is controlled to achieve modulation of different colors of fluorescence, which can be used for multicolor labeling imaging of cells.

[0051] Example 6

[0052] This embodiment is basically the same as Embodiment 1, except that: this embodiment uses 900g of silicon material, 300g of organic fluoride and 15g of analytical grade sodium chloride.

[0053] Example 7

[0054] This embodiment is basically the same as Embodiment 2, except that: this embodiment uses waste silicon material from the semiconductor manufacturing industry, waste polyvinylidene fluoride (molecular weight of 500,000) from the new energy field, and waste polytetrafluoroethylene (molecular weight of 500,000) from the industrial manufacturing process.

[0055] Example 8

[0056] This embodiment is basically the same as embodiment 3, except that: this embodiment uses waste silicon material from the semiconductor manufacturing industry, waste polyvinylidene fluoride (molecular weight of 100,000) from the new energy field, and waste polytetrafluoroethylene (molecular weight of 200,000) from the industrial manufacturing process.

[0057] Example 9

[0058] This embodiment is basically the same as Embodiment 1, except that in step four, the purified graphite carbon nanospheres are added to a 1M potassium permanganate-sulfuric acid solution (68wt%) cooled in an ice bath, then heated to 38°C and magnetically stirred for 1.5 hours. Then, under continuous stirring, the solution is irradiated with a high-pressure mercury lamp for 15 minutes, followed by treatment with an alternating electric field for 20 minutes to oxidize and exfoliate the graphite carbon nanospheres. After centrifugation, the solution is transferred to a dialysis bag with a molecular weight cutoff of 3000 Da and ultrapure water as the dialysate. Circulation dialysis is performed to obtain a graphene quantum dot solution with a concentration of 0.08 mg / mL. The specific method for applying the alternating electric field is as follows: the reaction solution is placed between two parallel copper plates with a plate spacing of 20 cm, an electric field strength of 500 V / m, and a frequency of 300 Hz; the high-pressure mercury lamp has a power of 500 W and a wavelength of 365 nm.

[0059] In this embodiment, during the oxidative exfoliation of purified graphene nanospheres, high-pressure mercury lamp irradiation and alternating electric field treatment are used to promote the oxidation reaction and exfoliation, thereby improving reaction efficiency and shortening the reaction cycle. The alternating electric field and high-pressure mercury lamp irradiation have a synergistic effect, enhancing the reactivity of the reaction solution while preventing the graphene nanospheres from agglomerating, ensuring sufficient contact with the solution. Furthermore, the exfoliation of the graphene nanospheres is promoted through vibration and other mechanisms, further improving reaction efficiency and reducing reaction time. The graphene quantum dots obtained in this embodiment have a uniform size distribution of approximately 20 nm, smaller than the 60 nm obtained in Example 1. This invention can further control the diameter of the graphene quantum dots by adjusting the process parameters of the alternating electric field.

[0060] Example 10

[0061] The alternating electric field strength in this embodiment is 100V / m, and the frequency is 500Hz; the remaining steps are the same as in embodiment 9.

[0062] The graphene quantum dots obtained in this embodiment have a uniform size distribution of approximately 15 nm.

[0063] Example 11

[0064] The alternating electric field strength in this embodiment is 1000V / m, and the frequency is 100Hz; the remaining steps are the same as in embodiment 9.

[0065] The graphene quantum dots obtained in this embodiment have a uniform size distribution of approximately 22 nm.

[0066] Example 12

[0067] This embodiment does not use high-pressure mercury lamp irradiation, but only applies an alternating electric field for 35 minutes; the remaining steps are the same as in embodiment 9.

[0068] The graphene quantum dots obtained in this embodiment have a uniform size distribution of approximately 43 nm.

[0069] Example 13

[0070] In this embodiment, no alternating electric field is applied; instead, a high-pressure mercury lamp is used for irradiation for 35 minutes. The remaining steps are the same as in Embodiment 9.

[0071] The graphene quantum dots obtained in this embodiment have a uniform size distribution of approximately 55 nm.

[0072] Although embodiments of the present invention have been disclosed above, they are not limited to the applications listed in the specification and embodiments. They can be applied to various fields suitable for the present invention. For those skilled in the art, other modifications can be easily made. Therefore, without departing from the general concept defined by the claims and their equivalents, the present invention is not limited to the specific details and illustrations shown and described herein.

Claims

1. A method for preparing a graphene quantum dot solution for HeLa cell imaging, characterized in that, Includes the following steps: Step 1: The waste silicon material and waste fluorinated plastics are ball-milled, acid-washed, alcohol-washed and vacuum-dried respectively, and then screened to produce silicon material and organic fluorides with a particle size of 50~200 mesh. Step 2: Add silicon material, organofluorine compound, and sodium chloride to anhydrous ethanol, and then perform ultrasonication, mechanical stirring, filtration, and drying to obtain a reaction precursor. Place the reaction precursor in a reactor and apply a certain intensity of current and voltage to initiate a self-driven reaction to obtain graphite carbon nanospheres. The mass ratio of silicon material to organofluorine compound is 3:1 to 1:3; the amount of sodium chloride added is 1% to 10% of the total mass of silicon material and organofluorine compound. Step 3: The graphite carbon nanospheres are acid-washed, water-washed, filtered and dried to obtain purified graphite carbon nanospheres; Step 4: Oxidize and exfoliate the purified graphite carbon nanospheres, centrifuge, and dialyze to obtain a graphene quantum dot solution. The specific method for oxidation and exfoliation is as follows: Add the purified graphite carbon nanospheres to a potassium permanganate-sulfuric acid solution cooled in an ice bath, then heat to 35-40℃ and magnetically stir for 1-2 hours. Then, under continuous stirring, irradiate with a high-pressure mercury lamp for 10-20 minutes, followed by treatment with an alternating electric field for 10-30 minutes to oxidize and exfoliate the graphite carbon nanospheres. The concentration of potassium permanganate in the potassium permanganate-sulfuric acid solution is 0.5-2M, and the concentration of the concentrated sulfuric acid solution is 60-75wt%. The mass-to-volume ratio of the purified graphite carbon nanospheres to the potassium permanganate-sulfuric acid solution is 1g:10-50mL.

2. The method for preparing a graphene quantum dot solution for HeLa cell imaging as described in claim 1, characterized in that, In step one, the waste fluorinated plastics include one or more of polytetrafluoroethylene, polyvinylidene fluoride, and polychlorotrifluoroethylene, with a molecular weight between 50,000 and 1,000,000; the ball milling speed is 600-900 r / min; and the pickling uses a 1-3 mol / L hydrochloric acid solution. Wash with anhydrous ethanol 2-4 times; dry at 50-80℃.

3. The method for preparing a graphene quantum dot solution for HeLa cell imaging as described in claim 1, characterized in that, In step two, the mass-to-volume ratio of the organic fluoride to anhydrous ethanol is 50-150 g: 1 L.

4. The method for preparing a graphene quantum dot solution for HeLa cell imaging as described in claim 1, characterized in that, In step two, the ultrasonic power is 300~800W, and the mechanical stirring lasts for 4~8 hours.

5. The method for preparing a graphene quantum dot solution for HeLa cell imaging as described in claim 1, characterized in that, In step two, the applied voltage is 220V and the current is 0.5~9A.

6. The method for preparing a graphene quantum dot solution for HeLa cell imaging as described in claim 1, characterized in that, In step three, a hydrofluoric acid solution with a concentration of 0.5~2M is used for pickling.

7. The method for preparing a graphene quantum dot solution for HeLa cell imaging as described in claim 1, characterized in that, In step four, the molecular weight cutoff of the dialysis bag is ≤3500 Da, and the dialysis solution is ultrapure water; the concentration of the obtained graphene quantum dot solution is controlled to be ≤0.1 mg / mL.

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