CN114314561A

CN114314561A - Carbon dots with hawthorn as carbon source and preparation method thereof

Info

Publication number: CN114314561A
Application number: CN202210038944.2A
Authority: CN
Inventors: 谢燕; 田瑞; 王峥涛; 郭雨凡; 罗发珍
Original assignee: Shanghai University of Traditional Chinese Medicine
Current assignee: Shanghai University of Traditional Chinese Medicine
Priority date: 2022-01-13
Filing date: 2022-01-13
Publication date: 2022-04-12

Abstract

The application relates to the field of nano materials, in particular to a carbon dot taking hawthorn as a carbon source and a preparation method thereof. The average particle size of the carbon dots is 1.68-3.02nm, the carbon dots have good size uniformity, and good pH detection and cell imaging effects are shown through photoluminescence characteristics of the carbon dots when the carbon dots are used. The carbon dots are controlled to have narrow size distribution, namely the size difference of the carbon dots is small, so that the luminous purity can be ensured, and the method has important significance on the properties and the application of the carbon dots; meanwhile, the carbon dots with small and uniform sizes have better and more stable fluorescence performance and higher fluorescence intensity.

Description

Carbon dots with hawthorn as carbon source and preparation method thereof

Technical Field

The application relates to the field of nano materials, in particular to a carbon dot taking hawthorn as a carbon source and a preparation method thereof.

Background

Carbon dots, also called carbon quantum dots, are a new class of zero-dimensional carbon-based nanomaterials, typically spherical particles with diameters less than 10 nm. In recent years, due to the advantages of low toxicity, high chemical stability, good biocompatibility, small size, easy modification, low cost, large-scale production, excellent optical performance, low photobleaching and the like of carbon dots, people attract extensive attention. Compared with other fluorescent carbon nano particles, the carbon dots also have the characteristics of higher Quantum Yield (QY), better water solubility, easy synthesis and the like. In view of the many advantages of carbon dots, they have been developed for use in bioimaging, biosensing, drug and gene delivery, antimicrobial agents, and the like.

The synthetic precursor of the carbon dots can be artificially synthesized precursors such as graphite, candle ash, carbon nano tubes, fullerene and the like; or natural substances such as orange juice, coffee, potatoes, egg shells and the like, and traditional Chinese medicinal materials are also commonly used as precursors for green synthesis of carbon dots such as turmeric, kudzu root, pseudo-ginseng and the like.

Due to the advantages of low toxicity, excellent water solubility, biocompatibility, photoluminescence and the like of the carbon dots, the carbon dots gradually replace other metal nanoparticles and semiconductor quantum dots, and are widely applied to biological imaging, sensors and antibacterial agents. Although many methods for preparing fluorescent carbon dots have been developed, the size, distribution and optical properties of the fluorescent carbon dots cannot be precisely controlled, which is a problem that needs to be solved in further development of carbon dots.

Disclosure of Invention

The application provides carbon dots taking hawthorn as a carbon source and a preparation method thereof, which aim to solve the technical problem of uneven size distribution of the carbon dots during application.

In a first aspect, the present application provides carbon dots using hawthorn as a carbon source, wherein the average particle size of the carbon dots is 1.68-3.02 nm.

Optionally, the fluorescence intensity of the carbon dots is pH sensitive. Optionally, the fluorescence intensity and pH of the carbon dot satisfy the following relationship: 172.21X +596.62, R²When the fluorescence intensity was 0.994, Y was the fluorescence intensity, and X was the pH.

Optionally, the pH value ranges from 0.5 to 4.5.

In a second aspect, the present application provides a method for preparing the carbon dots according to the first aspect, the method comprising the steps of:

carrying out pyrolysis reaction on hawthorn to obtain carbonized powder;

crushing the carbonized powder, then carrying out at least one time of water bath and suction filtration to obtain a concentrated solution, wherein the concentrated solution contains part of the carbonized powder;

and (3) filtering, dialyzing and centrifuging the concentrated solution at a high speed gradient to purify the concentrated solution to obtain the carbon point.

Optionally, the dialysis method comprises dialyzing for 24-48 h by using a 1000-3500 Da dialysis bag.

Optionally, the high-speed gradient centrifugation comprises: a first stage centrifugation, a second stage centrifugation and a third stage centrifugation; the target speed range of the first-stage centrifugation is 10000-20000 g, and the time of the first-stage centrifugation is 10-30 min; the target speed range of the second-stage centrifugation is 80000-100000 g, and the second-stage centrifugation time is 0.5-1 h; the target speed range of the third-stage centrifugation is 150000-200000 g, and the time of the third-stage centrifugation is 1-3 h.

Optionally, the filtering includes: filtering with 0.22 μm, 0.45 μm microporous membrane.

Optionally, the water bath includes a first water bath, and during the first water bath, the ratio of the carbonized powder to water is 1: 10-1: 30.

compared with the prior art, the technical scheme provided by the embodiment of the application has the following advantages:

the carbon dots provided by the embodiment of the application have the advantages that the average particle size is 1.68-3.02nm, the average particle size is mainly 2.35 +/-0.67 nm, the size uniformity is good, and when the carbon dots are used, the pH detection and cell imaging effects are good through the photoluminescence characteristics of the carbon dots. The fluorescence characteristics of carbon dots are generally considered to be related to the mechanisms of size-dependent quantum confinement effect, energy trap on the surface of the carbon dots, and the like, so that the optical properties of the carbon dots are influenced by the factors of size, form, composition, crystallinity and the like. As the overall size of the carbon dot is reduced, the movement of photogenerated electrons and holes is limited, the kinetic energy is increased, the effective band gap of the carbon dot is increased, and the fluorescence property is changed. The carbon dots have narrow size distribution, are important ways for ensuring the luminous purity of the carbon dots, and have important significance on the properties and the application of the carbon dots, so that the carbon dots with small and uniform sizes have better and more stable fluorescence performance and higher fluorescence intensity. In the embodiment of the application, natural substances are used as carbon sources, and the contained elements are various, so that the surface structure of carbon points can be enriched, and the carbon points have better fluorescence characteristics.

Drawings

The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate embodiments consistent with the invention and together with the description, serve to explain the principles of the invention.

In order to more clearly illustrate the embodiments or technical solutions in the prior art of the present invention, the drawings used in the description of the embodiments or prior art will be briefly described below, and it is obvious for those skilled in the art to obtain other drawings without inventive exercise.

FIG. 1 is a representation of carbon dots produced by hawthorn fruits provided in the examples of the present application;

FIG. 2 is a photoelectron spectrum of a carbon dot provided in an example of the present application;

FIG. 3 is a graph showing a particle size distribution of carbon dots before purification according to an example of the present application;

FIG. 4 is a particle size distribution diagram of carbon dots provided in the examples of the present application after purification;

FIG. 5 is a graph of the fluorescence spectrum of a carbon dot provided in the examples of the present application before purification;

FIG. 6 is a fluorescence spectrum of a carbon dot after purification according to an example of the present application;

FIG. 7 shows fluorescence intensities of carbon dots at different pH values according to examples of the present application;

FIG. 8 is a graph of fluorescence intensity versus pH for carbon dots provided in examples of the present application;

FIG. 9 shows the reversibility of fluorescence of carbon points with repeated pH changes between 0.5 and 4.5, as provided in the examples of the present application;

FIG. 10 is a fluorescence image of carbon dots in human breast cancer MCF-7/ADR cells provided in the examples of the present application:

FIG. 11 is a fluorescence image of carbon dots in Caco-2 cells of human colon cancer cells provided in the examples of the present application:

FIG. 12 is a schematic flow chart of a method for preparing carbon dots by using hawthorn fruits as a carbon source according to an embodiment of the present application;

FIG. 13 is a graph showing the effect of fluorescence intensity of carbon dots before and after purification by high-speed gradient centrifugation according to an embodiment of the present application.

Detailed Description

In order to make the objects, technical solutions and advantages of the embodiments of the present application clearer, the technical solutions in the embodiments of the present application will be clearly and completely described below with reference to the drawings in the embodiments of the present application, and it is obvious that the described embodiments are some embodiments of the present application, but not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present application.

In a first aspect, the present application provides carbon dots using hawthorn as a carbon source, wherein the average particle size of the carbon dots is in a range of 1.68-3.02 nm.

In some embodiments, the fluorescence intensity of the carbon dots is pH sensitive. In some embodiments, the fluorescence intensity and pH of the carbon dot satisfy the following relationship: 172.21X +596.62, R²When the fluorescence intensity was 0.994, Y was the fluorescence intensity, and X was the pH.

In some embodiments, the carbon dot, when used, has a pH in the range of 0.5 to 4.5.

carrying out pyrolysis reaction on hawthorn to obtain carbonized powder;

specifically, the hawthorn decoction pieces can be placed in a crucible and put in a muffle furnace for pyrolysis at the high temperature of 300-450 ℃ for 30-90 min.

specifically, it can be pulverized by pulverizer, and extracted with water bath for 2 times. Suction filtration was carried out to obtain a concentrated solution, which was subjected to suction filtration in order to remove the residue.

High-speed gradient centrifugation can remove particulate matters with different sizes in the solution step by step, thereby obtaining particles with a certain size range. And (4) taking the supernatant to carry out high-speed gradient centrifugation, and taking the supernatant obtained by the final high-speed gradient centrifugation as a carbon dot solution so as to obtain the carbon dots.

In the embodiment of the application, the preparation method of the carbon dots is simple to operate, mild and safe in reaction, and green and environment-friendly. The obtained carbon dots are safe, non-toxic and reproducible, have uniform size, good fluorescence characteristics, pH sensitivity and biocompatibility, and have good application in pH detection and cell imaging.

In some embodiments, the dialysis method comprises dialyzing for 24-48 h by using a 1000-3500 Da dialysis bag.

In some embodiments, the high-speed gradient centrifugation comprises: a first stage centrifugation, a second stage centrifugation and a third stage centrifugation; the target speed range of the first-stage centrifugation is 10000-20000 g, and the time of the first-stage centrifugation is 10-30 min; the target speed range of the second-stage centrifugation is 80000-100000 g, and the second-stage centrifugation time is 0.5-1 h; the target speed range of the third-stage centrifugation is 150000-200000 g, and the time of the third-stage centrifugation is 1-3 h.

Preferably, the ultra high speed gradient centrifugation comprises: centrifuging at 20000g for 0.5h, collecting supernatant, centrifuging at 100000g for 1.5h, collecting supernatant, centrifuging at 200000g for 3h, and collecting supernatant as carbon dot solution.

In order to realize the purpose of removing large granular substances and purifying solution, the separation of particles with different sizes is realized by controlling different centrifugal speeds, and 20000g is centrifuged for 0.5h to remove large granular impurities; the 100000g centrifugation mainly removes particles with the size of more than 100nm, the particles removed by the centrifugation gradually increase along with the prolonging of the centrifugation time, and the particles with the size of more than 100nm can be completely removed after 1.5 h; 150000 ~ 200000g mainly get rid of the size and be less than 100 nm's granule, compare in direct centrifugation, get rid of the large granule through gradient centrifugation, carbon point purification effect is better.

In some embodiments, the filtering comprises: filtering with 0.22 μm, 0.45 μm microporous membrane.

In order to remove the large-particle carbonized powder, a 0.22 mu m microporous filter membrane is selected for filtration.

In some embodiments, the water bath comprises two water baths, wherein the ratio of the carbonized powder to water in the water bath is 1: 10-1: 30.

in order to sufficiently extract carbon dots contained in the carbonized powder, the ratio of the carbonized powder to water is 1: 20.

The process of the present invention will be described in detail below with reference to examples, comparative examples and experimental data.

Example 1

1. And (4) preparing carbon dots.

Putting hawthorn decoction pieces into a crucible, putting into a muffle furnace, and pyrolyzing at 400 ℃ for 1 h. And crushing the sample subjected to high-temperature pyrolysis by using a crusher. Weighing 60g of sample powder subjected to high-temperature pyrolysis, adding 20 times of deionized water, heating in 100 ℃ water bath for 1h, roughly filtering with filter paper, continuously adding 20 times of deionized water into the medicine residue, heating in 100 ℃ water bath for one time, roughly filtering with filter paper, and combining the filtrates. The filtrate was concentrated to 120mL using a rotary evaporator, filtered through a 0.22 μm microporous membrane, and placed in a 1000Da dialysis bag and dialyzed in a beaker containing deionized water for 24 h. Separating and purifying the obtained dialysate by ultra-high speed gradient centrifugation, specifically centrifuging the dialysate at 20000g for 0.5h, centrifuging the supernatant at 100000g for 1.5h, centrifuging the supernatant at 200000g for 3h, and collecting the supernatant as carbon dot solution with uniform particle size.

2. Characterization of carbon points.

A series of characterizations were performed on carbon dots prepared using hawthorn as a carbon source, and fig. 1 includes: (A) transmission electron microscopy, (B) high resolution transmission electron microscopy, (C) carbon dot size distribution, (D) ultraviolet-visible light absorption spectra, photographs of carbon dots under white light (left) and ultraviolet light (right), (E) infrared spectroscopy, (F) fluorescence excitation and emission spectra.

The observation of carbon dots prepared by using hawthorn as a carbon source by using a transmission electron microscope (figure 1A) shows that the carbon dots have uniform particle size and good dispersibility, the particle size distribution of the carbon dots is shown in figure 1C, the average particle size is 2.35 +/-0.67 nm, and the high-purity carbon dots with small size and narrow distribution range are obtained after the carbon dots are subjected to ultrahigh-speed gradient centrifugal purification; the observation of hawthorn prepared carbon dots by a high-resolution electron microscope shows that (figure 1B), the carbon dots have an obvious graphite crystal form, and the lattice spacing is 0.21 nm; fourier infrared spectroscopy was used to analyze the surface structure of carbon dots prepared from hawthorn (FIG. 1E), 3387cm^-1The peak at (A) is due to O-H/N-H stretching vibration, 1571cm^-1，1387cm^-1The peak at (A) is due to COO-, 2927cm^-1The peak at (a) is attributed to C-C stretching vibration, and indicates that a functional group characteristic to a carboxyl group, a hydroxyl group, or the like is present on the surface of the carbon dot.

The carbon dots have an optimal excitation wavelength of 352nm and an optimal emission wavelength of 445nm (FIG. 1F), and are yellowish in white light (left inset in FIG. 1D) and fluoresce blue-green in 365nm UV light (right inset in FIG. 1D).

The X-ray photoelectron spectroscopy (fig. 2) shows that the carbon dots prepared by using hawthorn as a carbon source mainly contain elements such as C, O, N, wherein C, O, N corresponds to 285.29eV, 532.29eV and 399.29eV in the XPS spectrogram respectively. In fig. 2B, 284.67eV in the C1s spectrum corresponds to sp2/sp3 carbon (C-C, C ═ C); 286.26eV corresponds to C-N/C-OH; 288.0eV for C ═ O/C-O-C; in FIG. 2C, 531.78eV of the spectrum of O1s corresponds to C-O-C/C-OH; 532.9eV for C ═ O; in FIG. 2D, 399.45eV and 400.15eV of the spectrum of N1s correspond to C-N-C and N-C, respectively₃(ii) a The result shows that hydroxyl and carboxyl are mainly present on the surface of the carbon dot prepared by taking hawthorn as a carbon source.

Example 2

The purification method for preparing carbon dots by taking hawthorn as a carbon source comprises the following steps: the carbon dots prepared by using hawthorn fruits as carbon sources before ultrahigh-speed gradient centrifugal purification are obtained according to the preparation method of the carbon dots described in the example 1. The conditions of ultra high speed gradient centrifugation were selected according to the conditions in Table 1, and the particle size distribution was examined by DLS.

Table 1 particle size distribution under different centrifugation conditions.

Table 1 shows that a carbon dot solution with a uniform particle size can be obtained by centrifuging at 20000g for 0.5h, collecting the supernatant and centrifuging at 100000g for 1.5h, collecting the supernatant and centrifuging at 200000g for 3 h. When the gradient centrifugation method is not used, the carbon spot cannot be effectively purified even if the rotation speed or time is increased.

The results of Dynamic Light Scattering (DLS) measurement of the particle size of carbon dots prepared using hawthorn as a carbon source before and after ultrahigh-speed gradient centrifugation (fig. 3 and 4) show that the particle size distribution of the carbon dots prepared using hawthorn as a carbon source before ultrahigh-speed gradient centrifugation is not uniform, large-particle impurities having a particle size of more than 100nm are present, and the overall particle size is too large. After ultrahigh-speed gradient centrifugation, the particle size distribution of carbon dots prepared by taking hawthorn as a carbon source is uniform, and impurities larger than 100nm do not exist, so that the ultrahigh-speed gradient centrifugation can effectively improve the size distribution of the carbon dots prepared by taking the hawthorn as the carbon source, and the effects of impurity removal and uniform size are achieved.

Fluorescence spectra of carbon dots prepared by using hawthorn as a carbon source before and after ultrahigh-speed gradient centrifugal purification (fig. 5 and 6) show that the average size of the carbon dots before purification is about 40nm, a large number of large particles exist, and the size distribution of the carbon dots after purification is 2.35 +/-0.67 nm, as shown in fig. 1C; in fig. 5 and 6, EX represents an excitation spectrum, and EM represents an emission spectrum. The maximum excitation wavelength and the maximum emission wavelength of the carbon dots prepared by taking the hawthorn fruits as the carbon source before and after the ultrahigh-speed gradient centrifugal purification are basically consistent and are respectively 352nm and 445nm, but the fluorescence intensity of the carbon dots prepared by taking the hawthorn fruits as the carbon source after the ultrahigh-speed gradient centrifugal purification is greatly different from that of the carbon dots prepared by taking the hawthorn fruits as the carbon source under 352nm, so that the fluorescence intensity of the carbon dots prepared by taking the hawthorn fruits as the carbon source after the ultrahigh-speed gradient centrifugal purification is improved by 47 percent compared with that of the original liquid, and the ultrahigh-speed gradient centrifugal purification process can improve the particle size uniformity in the carbon dot solution and can also obviously improve the fluorescence intensity of the carbon dots.

In addition, the effect of fluorescence intensity of carbon dots before and after purification by ultracentrifugation with ultra high speed gradient is shown in FIG. 13, the left graph is the fluorescence intensity shown by carbon dots before purification, and the right graph is the fluorescence intensity shown by carbon dots after purification; the fluorescence intensity of the right graph is stronger than that of the left graph, the average size of the carbon dots before purification is about 40nm, and more large particles exist, and the size distribution of the carbon dots after purification is 2.35 +/-0.67 nm, which shows that the carbon dots after purification, which are uniform in size and small in particle size, can improve the fluorescence intensity.

Example 3: application of carbon dots prepared by using method for preparing carbon dots by using hawthorn as carbon source in aspect of pH detection

A carbon dot solution prepared from hawthorn is diluted 1-fold with deionized water, adjusted to

pH

2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 with 1mol/L HCl and NaOH, and measured for fluorescence intensity at an excitation wavelength of 352 nm. FIG. 7 shows that the carbon dots have the highest fluorescence intensity at pH 7, and the fluorescence intensity changes significantly with the pH of the solution, and changes significantly between pH 2 and pH 7, indicating that the carbon dots prepared from hawthorn have certain pH sensitivity.

FIG. 8 shows that the fluorescence intensity of carbon dots prepared from Crataegus pinnatifida ranges from 0.5 to 4.5 in the pH range of the solution, and shows a good linear relationship, wherein the linear formula is that Y is 172.21X +596.62, and R is²When the fluorescence intensity was 0.994, Y was the fluorescence intensity, and X was the pH.

Diluting a carbon dot solution prepared from hawthorn with deionized water by 1 time, adjusting the pH value to 0.5 by using 1mol/L HCl and NaOH, measuring the fluorescence intensity of the carbon dot solution, adjusting the pH value to 4.5 by using 1mol/L HCl and NaOH, measuring the fluorescence intensity of the carbon dot solution, and circulating the operation for 3 times, wherein FIG. 9 shows that the fluorescence intensity of the carbon dot of the hawthorn is changed along with the change of the pH value of the solution in the continuous conversion of the pH value of the solution of 0.5-4.5, and a good switching effect is shown. The carbon dots prepared from the hawthorn have good application potential in pH detection.

Example 4: application of carbon dots prepared by using hawthorn as carbon source in cell imaging

1. Carbon dots prepared from hawthorn were imaged on human breast cancer cells MCF-7/ADR cells.

The carbon spots prepared from hawthorn were lyophilized and dissolved in cell culture medium to a concentration of 1 mg/mL.

Taking MCF-7/ADR cell suspension (5X 10)⁴one/mL) 2mL of the culture solution was inoculated to a glass plate, and after 2 days of culture, the old solution was aspirated, 2mL of a cell culture medium containing 2mg of carbon dots prepared from hawthorn was added, and the mixture was incubated at 37 ℃ with 5% CO₂After incubation for 2h, the drug solution was aspirated, the cells in the dish were washed 3 times with PBS, the extracellular carbon spots were removed, 1mL of PBS was added to infiltrate the cells, and the cells were observed and photographed under a confocal laser microscope with an excitation wavelength of 405 nm. FIG. 10 shows that fluorescent carbon dots can be entered into MCF-7/ADR cells for cellular imaging, where (A) is a bright field photograph of the cells; (B) fluorescence imaging (excitation wavelength is 405nm) for carbon dot labeled cells; (C) the images are superimposed by bright field and fluorescence images.

2. The carbon point prepared by taking hawthorn as a carbon source images cells in human colon cancer cells Caco-2 cells.

Collecting Caco-2 cell suspension (5X 10)⁴one/mL) 2mL of the culture solution was inoculated to a glass plate, and after 2 days of culture, the old solution was aspirated, 2mL of a cell culture medium containing 2mg of carbon dots prepared from hawthorn was added, and the mixture was incubated at 37 ℃ with 5% CO₂After incubation for 2h, the drug solution was aspirated, the cells in the dish were washed 3 times with PBS, the extracellular carbon spots were removed, 1mL of PBS was added to infiltrate the cells, and the cells were observed and photographed under a confocal laser microscope with an excitation wavelength of 405 nm. FIG. 11 shows that fluorescent carbon dots can be entered into Caco-2 cells for cell imaging, where (A) is a bright field photograph of the cells; (B) fluorescence imaging (excitation wavelength is 405nm) for carbon dot labeled cells; (C) the photographs were superimposed for bright field and fluorescence imaging.

It is noted that, in this document, relational terms such as "first" and "second," and the like, may be used solely to distinguish one entity or action from another entity or action without necessarily requiring or implying any actual such relationship or order between such entities or actions. Also, the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, article, or apparatus. Without further limitation, an element defined by the phrase "comprising an … …" does not exclude the presence of other identical elements in a process, method, article, or apparatus that comprises the element.

The foregoing are merely exemplary embodiments of the present invention, which enable those skilled in the art to understand or practice the present invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments without departing from the spirit or scope of the invention. Thus, the present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. The carbon dots with hawthorn as a carbon source are characterized in that the average particle size of the carbon dots is 1.68-3.02 nm.

2. The carbon dot according to claim 1, wherein the fluorescence intensity of the carbon dot has a pH sensitivity.

3. The carbon dot according to claim 2, wherein the fluorescence intensity and the pH of the carbon dot satisfy the following relationship: 172.21X +596.62, R²When the fluorescence intensity was 0.994, Y was the fluorescence intensity, and X was the pH.

4. The carbon dot of claim 2, wherein the pH is in the range of 0.5 to 4.5.

5. A method for producing the carbon dot as claimed in any one of claims 1 to 4, characterized by comprising the steps of:

carrying out pyrolysis reaction on hawthorn to obtain carbonized powder;

crushing the carbonized powder, then carrying out at least one time of water bath, suction filtration and concentration to obtain a concentrated solution, wherein the concentrated solution contains part of the carbonized powder;

6. The method of claim 5, wherein the dialysis comprises dialysis for 24-48 hours using a 1000-3500 Da dialysis bag.

7. The method of claim 5, wherein the high speed gradient centrifugation comprises: a first stage centrifugation, a second stage centrifugation and a third stage centrifugation; the target speed range of the first-stage centrifugation is 10000-20000 g, and the time of the first-stage centrifugation is 10-30 min; the target speed range of the second-stage centrifugation is 80000-100000 g, and the second-stage centrifugation time is 0.5-1 h; the target speed range of the third-stage centrifugation is 150000-200000 g, and the time of the third-stage centrifugation is 1-3 h.

8. The method of claim 5, wherein the filtering comprises: filtering with 0.22 μm, 0.45 μm microporous membrane.

9. The method according to claim 5, wherein the water bath comprises a first water bath, and the ratio of the carbonized powder to water in the first water bath is 1: 10-1: 30.