CN109897634B - pH-sensitive long-wavelength fluorescent carbon dot and biological application thereof - Google Patents
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Abstract
The invention discloses a pH-sensitive long-wavelength fluorescent carbon dot and biological application thereof, belonging to the field of manufacturing of functional luminescent carbon materials. The invention directly carries out solid-phase calcination carbonization treatment on a specific carbon source at the temperature of 150-300 ℃ to prepare the pH response red carbon dot material. The carbon dot material prepared by the invention effectively overcomes the defects of weaker penetrating power, larger cell damage, easiness in influence of autofluorescence of a cell matrix and the like of the existing pH response probe, can be simply prepared into a large batch of carbon dot materials with excellent colorimetric/fluorescent dual-mode pH response long wavelength, is applied to cell pH detection, and has high industrial situation.
Description
Technical Field
The invention belongs to the field of functional luminescent carbon material manufacturing, and particularly relates to a pH-sensitive long-wavelength fluorescent carbon dot and biological application thereof.
Background
The pH value in the cell is an important parameter of metabolism, and plays an important role in regulating physiological and pathological processes of the cell, and important life processes such as multidrug resistance, cell proliferation and apoptosis, signal transduction, endocytosis, ion transport, muscle contraction and the like all depend on the maintenance of normal pH value. Small fluctuation of intracellular pH value deviating from a normal range can affect the normal function of a nervous system, hinder signal conduction processes such as synaptic transmission, neuron excitation and intercellular gap transmission, and even the pH deviation of 0.1-0.2 unit can possibly cause diseases such as cancer, tumor, senile dementia and the like; while a greater degree of pH deviation may be fatal. Therefore, the sensitive and accurate monitoring of intracellular pH changes plays an important role in cell analysis or diagnosis.
At present, methods for detecting the pH of cells mainly comprise a weak acid and weak base distribution method, a microelectrode method, a nuclear magnetic resonance method and a fluorescence probe method. Weak acid and weak base distribution methods usually use weak electrolytes such as methylamine, nicotine, DMO, butyric acid and the like, which have membrane permeability when not in a dissociated form, slowly metabolize after entering cells, and generally do not change the pH value of the cells after the concentration inside and outside the cells reaches distribution balance, thereby achieving the purpose of measuring the pH value. The method is simple and convenient to operate, needs few materials, has moderate resolution (reaching 0.1-0.2 pH unit), and can be used for measuring cells with smaller volume. However, the weak electrolyte requires a long time to reach a distribution equilibrium inside and outside the cell, a change in pH cannot be detected quickly and in real time, the time resolution is poor, and the method may damage the cell tissue and cause a deviation in the measurement result. Nuclear magnetic resonance method31P NMR technique using intracellular31The chemical shift of the P spectrum determines the change in pH. The method has high sensitivity (up to 0.06 pH unit), does not need exogenous molecules, has small damage to cells, and can avoid measurement errors caused by cell damage and internal metabolism change; however, this method requires a relatively high concentration of cells to be measured, is time-consuming, and has an intracellular pH that is acidic (<5.5) or more basic>7.5), the measurement results largely vary. The microelectrode method has high detection accuracy, and the resolution can reach 0.02-0.05 pH unit; the time resolution is high, the time resolution can be changed continuously in the experimental process, and the method is suitable for continuously recording the pH value in the cell and the instantaneous change of the pH value. However, the method is complicated and difficult to operate, and is not suitable for measuring cells with small volume and living cells.
Compared with the methods, the fluorescence probe method introduces the probe into the cell, reflects the physiological state of the cell by utilizing the change of the fluorescence property of the probe along with the change of the pH value, has the sensitivity of 0.01 pH unit, has the spatial accuracy of 200nm and the time accuracy of ms level, and can be combined with the confocal imaging technology, so the fluorescence probe method has more advantages in the aspect of visual real-time monitoring. In addition, the method has the advantages of high sensitivity, good selectivity, low detection limit, simple and convenient operation, capability of realizing non-invasive detection and the like. Therefore, the fluorescence probe method is an important means for monitoring intracellular pH change and regional distribution in real time and in situ at a molecular level.
The nano material has unique chemical and physical properties due to the size effect, and has unique advantages in disease diagnosis and detection. The carbon dots are used as a part of the nano carbon material, have long-term development by virtue of the advantages of good water solubility, strong chemical stability, high photobleaching resistance, excellent biocompatibility, low cytotoxicity and the like, and show great application potential in the fields of disease diagnosis, drug delivery, biological imaging and the like. Researchers have recently reported that carbon-point based pH probes, such as Xiao et al, produce blue light carbon dots with response pH values in the range of 3.0-13.0. However, practical application of carbon dots faces some problems to be solved, 1) most of the carbon dots emit light in the blue or green region, and in practical application, especially in biomedical research, long-wavelength emitting materials are often needed to avoid background interference caused by self-absorption and autofluorescence of cell matrices. 2) The change in pH is detected based on a slight change in the fluorescence intensity or emission wavelength peak position of the carbon spot, is susceptible to factors such as probe concentration, optical path length, temperature, excitation intensity, and the like, and colorimetric observation is difficult to achieve. 3) The preparation steps of the carbon dots are complicated, the period is long, and the mass preparation is difficult. Therefore, there is a strong market need to develop a long-wavelength carbon dot that is easy and can observe a change in pH compared to color.
Disclosure of Invention
The invention provides a long-wavelength fluorescent carbon dot with a pH colorimetric/fluorescent dual-mode response based on the defects of the prior art, and the long-wavelength fluorescent carbon dot is used for accurately detecting the pH in a cell in real time.
The first purpose of the invention is to provide a preparation method of a pH-sensitive long-wavelength fluorescent carbon dot material, which comprises the following steps:
(1) placing the precursor of the carbon source at the temperature of 150 ℃ and 300 ℃ for solid-phase calcination for 4-8h to obtain a carbonized mixture; the carbon source precursor is an aromatic compound containing amino and derivatives thereof;
(2) and (2) dialyzing and drying the carbonized mixture obtained in the step (1) to obtain the carbon dot material.
In one embodiment of the present invention, the carbon source precursor preferably contains not less than 2 amino-substituted aromatic hydrocarbon compounds and derivatives thereof.
In one embodiment of the present invention, the temperature of the calcination is preferably 200-250 ℃.
In one embodiment of the present invention, the temperature of the calcination is further preferably 200 ℃.
In one embodiment of the invention, the calcining comprises placing the carbon source precursor in a muffle furnace, a tube furnace or an oven for direct heat treatment.
In one embodiment of the present invention, the dialysis is performed by dissolving the carbonized mixture in step (1) in water and dialyzing in a dialysis bag to remove unreacted precursor molecules and large particles.
In one embodiment of the present invention, the dialysis is to remove unreacted precursor molecules by using 500-1000Da dialysis bag, and then to retain large particulate matters by using 3500Da dialysis bag, and to take out the solution outside the dialysis bag.
In one embodiment of the invention, the dialysis time is 30-50 h.
In one embodiment of the present invention, the drying includes any one of freeze drying, vacuum drying, reduced pressure drying or atmospheric drying.
The resulting dark brown powder was dissolved in deionized water and dialyzed in a dialysis bag for 48 hours to remove large particles. The solvent was then removed by distillation under reduced pressure and further freeze-dried to give a purified red carbon dot powder.
The second purpose of the invention is to provide a pH-sensitive long-wavelength fluorescent carbon dot material by using the method.
The third purpose of the invention is to provide a pH probe, wherein the probe contains the pH-sensitive long-wavelength fluorescent carbon dot material.
It is a fourth object of the present invention to provide a method for monitoring pH fluctuation of cells in real time by using the above pH-sensitive long wavelength fluorescent carbon dot material or pH probe.
It is a fifth object of the present invention to provide a method for measuring pH in a cell, which uses the above-mentioned pH sensitive long wavelength fluorescent carbon dot material or pH probe.
The sixth purpose of the present invention is to apply the pH-sensitive long-wavelength fluorescent carbon dot material or the pH probe described above to the biomedical field.
The invention has the following beneficial technical effects:
1) the method has the advantages of simple operation steps, no need of complex instruments and separation processes, no need of high-temperature and oxygen-free environment, no need of additional carbon source, capability of large-scale preparation, low cost and suitability for industrial production.
2) The red carbon dot material prepared by the method has higher quantum yield and longer emission wavelength.
3) The red carbon dot material prepared by the method has high pH sensitivity, and the change of the fluorescence emission wavelength and the ultraviolet and visible light absorption wavelength along with the change of pH is large, so that the red carbon dot material can generate color change which can be judged by naked eyes along with the change of pH, and has excellent pH colorimetric/fluorescent dual-mode response.
4) The carbon dot material prepared by the invention has good dispersibility, the particle size range is 1.2-3.6nm, and the average particle size is 2.3 nm; the probe has the advantages of good stability, high biocompatibility, no toxicity and dual-mode pH response, overcomes the defects of weaker penetrating power, larger cell damage, easy influence of autofluorescence of cell matrixes and the like of the conventional pH response probe, and has application value and wide prospect in cell pH detection.
Drawings
FIG. 1 is a Transmission Electron Microscope (TEM) photograph of a red carbon dot of example 1, with the inset being a high resolution TEM photograph;
FIG. 2 is a graph showing UV absorption, fluorescence excitation, and emission spectra of red carbon dots of example 1;
FIG. 3 is a UV-VIS absorption spectrum of the red carbon dot of example 1 with pH from 4.0 to 8.0;
FIG. 4 is a fluorescence emission spectrum of red carbon dots of example 1 with pH from 4.0 to 8.0;
FIG. 5 is a photograph of the red carbon dot solution of example 1 at a pH of from 4.0 to 8.0 under fluorescent light and under 365nm UV light;
FIG. 6 is a graph of the fluorescence intensity of red carbon dots of example 2 at different pH's in the presence of different concentrations of sodium chloride;
FIG. 7 is a graph of the activity of Hela cells incubated with different concentrations of red carbon spots in example 2;
fig. 8 is a graph showing the application of red carbon dots in the bio-imaging of example 2: typical laser scanning confocal microscope images of Hela cells incubated with red carbon dots in three different pH environments;
FIG. 9 is a UV-VIS absorption spectrum of the red carbon dot of example 6 with pH from 4.0 to 8.0;
FIG. 10 is a fluorescence emission spectrum of the red carbon spot of example 6 with pH from 4.0 to 8.0;
FIG. 11 is a photograph of the red carbon dot solution of example 6 at pH 4.0 to 8.0 under fluorescent light and under 365nm UV light;
FIG. 12 is a UV-VIS absorption spectrum of the red carbon dot of example 7 with pH from 4.0 to 8.0;
FIG. 13 is a fluorescence emission spectrum of red carbon dots with pH from 4.0 to 8.0 of example 7;
FIG. 14 is a photograph of the red carbon dot solution of example 7 at pH 4.0 to 8.0 under fluorescent light and under 365nm UV light.
Detailed Description
The invention is further described with reference to specific examples.
Transmission electron microscopy: tecnai GI F20U-TWIN Transmission Electron microscope (200KV accelerating voltage).
A fluorescence spectrometer: horiba JobinYvon Fluoromax 4C-L (France) spectrophotometer.
The quantum yield determination method comprises the following steps: the quantum yield of red carbon dots was determined by relative measurement. Rhodamine B (quantum yield in water 89%, λ ex ═ 495nm) was selected as a reference. The quantum yield calculation method is as follows:
wherein
Is the quantum yield of the test sample, I is the integrated emission intensity of the test sample, n is the refractive index (1.33 for water and 1.36 for ethanol), and a is the optical density. The symbol (') refers to a reference. To obtain more accurate results, a series of carbon dots and reference solutions were prepared, the concentrations of which were adjusted so that the light absorption values were between 0 and 0.1 at 495nm excitation. Its photoluminescence spectrum was measured and its intensity was integrated. The quantum yield was determined by comparing the integrated photoluminescence intensity with the absorbance curve (refractive index, n, must also be taken into account).
Example 1 preparation of colorimetric/fluorescent Dual-mode response pH-sensitive Red carbon dots
Weighing 1g of 1,2, 4-triaminobenzene, and placing in a temperature program tubular furnace at 5 deg.C for min-1Heating to 200 ℃ and keeping the temperature for 6 h. After cooling to room temperature, the dark brown powder obtained by carbonization was dissolved in deionized water, unreacted precursor molecules were removed using a 1000Da dialysis bag, and then dialyzed for 48 hours in a 3500Da dialysis bag to remove large particles. The solvent was then removed by distillation under reduced pressure and further freeze-dried to give a purified red carbon dot powder. The quantum yield of the obtained carbon dots was 9.93%.
The resulting powder was dispersed in ultrapure water and subjected to transmission electron microscopy, as shown in fig. 1, with the red carbon dots well dispersed in water with no observable aggregation or large particles, their size distribution was in the range of 1.2 to 3.6nm, and the average size was about 2.3 nm. In high resolution TEM images, a well resolved lattice spacing of 0.21nm corresponds to the (100) lattice plane of the graphene carbon, indicating successful preparation of carbon dots.
The optical properties were measured by uv-vis spectrophotometer and fluorescence spectrometer, and the results are shown in fig. 2, and the prepared material has two distinct absorption peaks at 348nm and 505nm, which are attributed to pi-pi of C ═ C bond*N-pi of transition and C ═ N/C-N bond*The optimum excitation and emission wavelengths of transition, red carbon point are respectively located507nm and 640 nm.
The pH response study of the red carbon dot was demonstrated by the following experiments:
as can be seen in FIG. 3, the broad 444nm absorption peak of the UV-visible spectrum of the red carbon spot gradually shifted to 510nm as the pH increased from 4.0 to 8.0, while the absorption peak at 348nm was insensitive to pH changes.
The pH-dependent fluorescence response of the red carbon dot solution is shown in fig. 4, with a red shift of the fluorescence emission from 585nm to 650nm as the pH is lowered from 8.0 to 4.0.
As shown in fig. 5, when the pH is 4.0-5.0, the color of the red carbon dot solution is red under the fluorescent lamp, and the color of the fluorescent light is purple under the ultraviolet lamp; when the pH value is 5.5-7.0, the color of the red carbon dot solution is changed into orange under a fluorescent lamp, and the fluorescent color is changed into orange under an ultraviolet lamp; when the pH value is 7.5-8.0, the color of the red carbon dot solution is changed into yellow under a fluorescent lamp, and the color of the fluorescent light is correspondingly changed into yellow under an ultraviolet lamp.
The above results indicate that pH probes based on red carbon dots have excellent colorimetric/fluorescent dual mode response, which facilitates pH measurement and determination in living cells.
Example 2 biological applications
The red carbon dots with pH bimodal response prepared according to example 1 were further used to monitor pH fluctuations of living cells in an organism.
The effect of salt ionic strength was first tested by varying the concentration of NaCl in red carbon dot solutions of different pH values. As shown in fig. 6, the intensity change of the red carbon dot was less than 20% at pH 4.0 and pH 8.0 under NaCl (1M), indicating that the red carbon dot has excellent stability in biomedical applications.
To evaluate the cytotoxicity of the red carbon dot, MTT assay was performed on Hela cells containing different concentrations of the red carbon dot. As shown in FIG. 7, the viability of Hela cells remained nearly 83% even after incubation for 24h at a red carbon spot concentration of 500. mu.g/mL. These indicate that the red carbon dot of example 1 shows excellent biocompatibility and has no adverse effect on Hela cells.
At the same time, we explored the ability of fluorescent red carbon spots to monitor intracellular pH fluctuations. Three samples containing 20 μ g/mL red carbon spots were tested at different environmental pH (4.0, 6.0 and 8.0) and as shown in FIG. 8, the fluorescence signal was detected not only in the cytoplasm but also in the nucleus, indicating that it could be used for cell imaging. When the pH increased from 4.0 to 8.0, the corresponding color changed from red to orange to yellow. Thus, a pH responsive red carbon dot may be used for real-time pH monitoring at the sub-cellular level.
Example 3: influence of carbon Source precursor
Referring to example 1, the carbon source precursor was replaced with p-phenylenediamine, 1,3, 5-triaminobenzene from 1,2, 4-triaminobenzene, and the quantum yield and optical properties of the resulting carbon dots were as shown in table 1.
TABLE 1 Quantum yield and optical Properties of the carbon dots obtained from different carbon Source precursors
Example 4: influence of solid phase calcination temperature
Referring to example 1, the quantum yields and optical properties of the carbon dots obtained by replacing the calcination temperature with 200 ℃ to 150 ℃, 250 ℃, 300 ℃ and 350 ℃ are shown in table 2.
TABLE 2 Quantum yield and optical Properties of the carbon dots obtained at different calcination temperatures
Example 5: influence of solid phase calcination time
Referring to example 1, the calcination time was replaced with 4h, 8h, and 10h from 6h, and the quantum yield and optical properties of the obtained carbon dots are shown in table 3.
TABLE 3 Quantum yield and optical Properties of the carbon dots obtained from the different carbon source precursors
Example 6 preparation of colorimetric/fluorescent Dual-mode response pH-sensitive Red carbon dots
0.5g of 1,2, 4-triaminobenzene is weighed and directly kept in an oven for 8 hours at the temperature of 200 ℃. Cooling to room temperature, dissolving the carbonized black brown powder in deionized water, removing unreacted precursor molecules by using a 1000Da dialysis bag, and dialyzing for 48 hours by using a 3500Da dialysis bag to remove large particles; and vacuum drying to obtain purified red carbon dot powder. The quantum yield of the obtained carbon dots was 9.32%.
The resulting powder was dispersed in ultrapure water and tested for pH response by uv-vis spectrophotometer and fluorescence spectrometer, as shown in fig. 9, with the pH increasing from 4.0 to 8.0, the broad absorption peak at 505nm of the uv-vis spectrum of the red carbon dot gradually shifted to 439nm and the narrow absorption peak at 257nm shifted to 269 nm. The pH-dependent fluorescence response of the red carbon dot solution is shown in fig. 10, with a red shift of the fluorescence emission from 593nm to 646nm as the pH is lowered from 8.0 to 4.0. As shown in fig. 11, when the pH is 4.0 to 6.5, the color of the red carbon dot solution is red under the fluorescent lamp, and the color of the fluorescent light is red under the ultraviolet lamp; when the pH value is 7.0, the color of the red carbon dot solution is changed into orange under a fluorescent lamp, and the fluorescent color is changed into orange under an ultraviolet lamp; when the pH value is 7.5-8.0, the color of the red carbon dot solution is changed into yellow under a fluorescent lamp, and the color of the fluorescent light is correspondingly changed into yellow under an ultraviolet lamp.
Example 7 preparation of colorimetric/fluorescent Dual-mode response pH-sensitive Red carbon dots
Weighing 1g of 1,2, 4-triaminobenzene, preserving heat for 6h at 200 ℃ in a muffle furnace, cooling to room temperature, dissolving carbonized blackish brown powder in deionized water, removing unreacted precursor molecules by using a 1000Da dialysis bag, and dialyzing for 48h by using a 3500Da dialysis bag to remove large particles; and freeze-drying to obtain purified red carbon dot powder. The quantum yield of the resulting carbon dots was 9.28%.
The resulting powder was dispersed in ultrapure water and tested for pH response by uv-vis spectrophotometer and fluorescence spectrometer, as shown in fig. 12, the 497nm broad absorption peak of the uv-vis spectrum of the red carbon dot was gradually shifted to 436nm as the pH was increased from 4.0 to 8.0. The pH-dependent fluorescence response of the red carbon dot solution is shown in fig. 13, with a red shift of the fluorescence emission from 600nm to 653nm as the pH is lowered from 8.0 to 4.0. As shown in fig. 14, when the pH is 4.0 to 6.0, the color of the red carbon dot solution is red under the fluorescent lamp, and the color of the fluorescent light is red under the ultraviolet lamp; when the pH value is 6.5-7.0, the color of the red carbon dot solution is changed into orange under a fluorescent lamp, and the fluorescent color is changed into orange under an ultraviolet lamp; when the pH value is 7.5-8.0, the color of the red carbon dot solution is changed into yellow under a fluorescent lamp, and the color of the fluorescent light is correspondingly changed into yellow under an ultraviolet lamp.
Comparative example 1 preparation of carbon dots by Water-soluble method
Weighing 1g of 1,2, 4-triaminobenzene, dissolving the 1g of 1,2, 4-triaminobenzene in 10mL of ultrapure water, uniformly stirring, putting the solution into a reaction kettle, and moving the reaction kettle into an oven to keep the temperature at 200 ℃ for 8 hours. Unreacted precursor molecules were removed using a 1000Da dialysis bag and then dialyzed against a 3500Da dialysis bag for 48 hours to remove large particles. The solvent was then removed by distillation under reduced pressure and further freeze-dried to give a purified red carbon dot powder. The quantum yield of the obtained carbon dots was 8.63%.
The pH response properties were tested by uv-vis spectrophotometer and fluorescence spectrometer and it was found that the 496nm broad absorption peak of the uv-vis spectrum of the red carbon dot gradually shifted to 528nm as the pH increased from 4.0 to 8.0. When the pH was lowered from 8.0 to 4.0, the fluorescence emission red-shifted from 595nm to 623 nm. With the increase of pH, the color change of the red carbon dot solution under the fluorescent lamp is not obvious, and the change of the pH cannot be judged through color comparison.
Comparative example 2 microwave method for preparing carbon dots
1g of 1,2, 4-triaminobenzene is weighed and then dissolved in 10mL of ultrapure water, and the mixture is stirred uniformly. Placing the solution in a household microwave oven, heating at 750W power for 8min to obtain brown solution, removing unreacted precursor molecules with 1000Da dialysis bag, and dialyzing with 3500Da dialysis bag for 48 hr to remove large particles. The solvent was then removed by distillation under reduced pressure and further freeze-dried to give a purified red carbon dot powder. The quantum yield of the resulting carbon dots was 7.26%.
The pH response properties were tested by an ultraviolet-visible spectrophotometer and a fluorescence spectrometer, and it was found that as the pH was increased from 4.0 to 8.0, the 486nm wide absorption peak of the ultraviolet-visible spectrum of the red carbon dot was gradually shifted to 517 nm. When the pH was lowered from 8.0 to 4.0, the fluorescence emission red-shifted from 584nm to 617 nm. With the increase of pH, the color change of the red carbon dot solution under the fluorescent lamp is not obvious, and the change of the pH cannot be judged through color comparison.
Although the present invention has been described with reference to the preferred embodiments, it should be understood that various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.
Claims (12)
1. A preparation method of a pH-sensitive long-wavelength fluorescent carbon dot material is characterized by comprising the following steps:
(1) placing the precursor of the carbon source at the temperature of 150 ℃ and 250 ℃ for solid-phase calcination for 4-8h to obtain a carbonized mixture; the precursor of the carbon source is p-phenylenediamine, 1,3, 5-triaminobenzene or 1,2, 4-triaminobenzene;
(2) and (2) dialyzing and drying the carbonized mixture obtained in the step (1) to obtain the carbon dot material.
2. The method as claimed in claim 1, wherein the temperature of the calcination is 200-250 ℃.
3. The process according to claim 1 or 2, characterized in that the temperature of the calcination is 200 ℃.
4. The method of claim 1 or 2, wherein the solid phase calcination comprises direct heat treatment of the carbon source precursor in a muffle furnace, tube furnace or oven.
5. The method of claim 3, wherein the solid phase calcination comprises placing the carbon source precursor in a muffle furnace, tube furnace, or oven for direct heat treatment.
6. The method according to any one of claims 1,2 and 5, wherein the dialysis is carried out by dissolving the carbonized mixture in water and dialyzing in a dialysis bag to remove unreacted precursor molecules and large particles.
7. The method of claim 3, wherein the dialysis is performed by dissolving the carbonized mixture in water and dialyzing the solution in a dialysis bag to remove unreacted precursor molecules and large particles.
8. The method of claim 4, wherein the dialysis is performed by dissolving the carbonized mixture in water and dialyzing the solution in a dialysis bag to remove unreacted precursor molecules and large particles.
9. A pH probe comprising the pH-sensitive long-wavelength fluorescent carbon dot material prepared by the method for preparing a pH-sensitive long-wavelength fluorescent carbon dot material according to any one of claims 1 to 8.
10. A method for monitoring pH fluctuation of a cell in real time, wherein the method comprises introducing a pH-sensitive long-wavelength fluorescent carbon dot material prepared by the method for preparing a pH-sensitive long-wavelength fluorescent carbon dot material according to any one of claims 1 to 8 or a pH probe according to claim 9 into a cell.
11. A method for measuring pH in a cell, comprising introducing a pH-sensitive long-wavelength fluorescent carbon dot material prepared by the method for preparing a pH-sensitive long-wavelength fluorescent carbon dot material according to any one of claims 1 to 8 or a pH probe according to claim 9 into a cell.
12. Use of the pH-sensitive long-wavelength fluorescent carbon dot material prepared by the method for preparing the pH-sensitive long-wavelength fluorescent carbon dot material according to any one of claims 1 to 8 or the pH probe according to claim 9 in the field of HeLa cell imaging.
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