A two-photon fluorescent carbon dot and a preparation method and application thereof

Abstract

The application provides a two-photon fluorescent carbon dot and a preparation method and application thereof, and the preparation method comprises the following steps: subjecting a carbon source to a hydrothermal reaction with sulfuric acid to obtain the two-photon fluorescent carbon dot. The application synthesizes a novel two-photon fluorescent carbon dot through the reaction of the carbon source with sulfuric acid, simplifies the preparation process, saves the production cost, and the use of sulfuric acid makes the two-photon fluorescent carbon dot surface have abundant sulfonic acid groups; the introduction of the sulfonic acid groups enhances the fluorescence intensity and stability of the two-photon fluorescent carbon dot, so that the two-photon fluorescent carbon dot obtains near-infrared luminescence which does not change with the excitation wavelength; meanwhile, the introduction of the sulfonic acid groups can increase the negative charge density on the surface of the two-photon fluorescent carbon dot, thereby improving the biocompatibility and stability in cells of the two-photon fluorescent carbon dot; when the addition amount of the two-photon fluorescent carbon dot reaches 15 mu g / mL, the cell activity can still reach 90%, and the two-photon fluorescent carbon dot can be applied to live cell imaging.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of cell imaging, and particularly relates to a two-photon fluorescent carbon dot and a preparation method and application thereof. BACKGROUND

[0002] Polymer carbon nanodots (CPDs) are a new kind of fluorescent nanoprobes emerging in recent years, which have excellent luminescence, good biocompatibility, low toxicity, easy chemical modification and functional integration, etc. Therefore, polymer carbon nanodots have broad application potential in many fields such as photocatalysis, biosensing, optoelectronic devices, biological imaging and photodynamic therapy. In 2006, Sun's group of Clemson University reported a method for preparing carbon nanodots by physical cracking and formally proposed the concept of carbon dots. Since then, the preparation and functional research of carbon dots have developed rapidly. At present, many research results have been achieved for blue or green fluorescent carbon dots, while red and near-infrared fluorescent carbon dots with long-waveband are still rarely seen. After 2015, CPDs independent of excitation light emission were reported. The emission peak of such CPDs does not move with the change of excitation wavelength, and the luminescence of CPDs under ultraviolet light is more complex and diverse, and even obvious fluorescence can be seen under sunlight. Ding et al. prepared CPDs with multi-color fluorescent emission by hydrothermal reaction using phenylenediamine and urea as raw materials. The obtained CPD crude product was separated and purified by silica gel column chromatography, different polar eluents were used, and eight samples with different fluorescent properties from blue to red emission were obtained without excitation light dependence (ACS Nano, 2015, 10(1): 484-491). In addition, they obtained a series of CPDs with adjustable emission wavelength from 443-745 nm by using o-phenylenediamine and L-glutamic acid as precursors and changing the solvent type (Small, 2018, 14(22): 1800612). Two-photon absorption is a process in which two photons of medium are absorbed, and each single photon does not have enough energy to excite the molecule (in medium) to the excited state, but two photons work together to reach the excited state from the ground state through a virtual state. This process absorbs two photons in total, hence the name two-photon absorption. Since two-photon absorption mainly occurs at the focal point of the ultrastrong laser generated by a pulsed laser, the laser intensity is not enough to produce two-photon absorption at other places on the light path, and since the wavelength used is longer and the energy is lower, the corresponding single-photon process cannot occur. Therefore, the two-photon process has good spatial selectivity and less damage to the sample.

[0003] The current two-photon fluorescent carbon dot synthesis method needs complex synthesis conditions or needs to use toxic and harmful chemical reagents, and the operation condition is relatively complex, which not only increases the production cost, but also limits the large-scale production and application. Therefore, it is necessary to simplify the process preparation process, reduce the use of toxic raw materials to reduce the pollution to the environment, and at the same time improve the biocompatibility and stability in cells, so that it can be applied to live cell imaging. SUMMARY

[0004] In view of the deficiencies of the prior art, the purpose of the present application is to provide a two-photon fluorescent carbon dot and a preparation method and application thereof, which simplifies the process flow, saves production cost, and makes the obtained two-photon fluorescent carbon dot obtain near-infrared luminescence that does not change with excitation wavelength, has good biocompatibility, high stability in cells, and can be applied to live cell imaging.

[0005] To achieve this purpose, the present application adopts the following technical solutions:

[0006] In a first aspect, the present application provides a preparation method of a two-photon fluorescent carbon dot, which comprises: hydrothermal reaction of a carbon source with sulfuric acid to obtain the two-photon fluorescent carbon dot.

[0007] The two-photon fluorescent carbon dot provided by the present application, the use of sulfuric acid makes the surface of the synthesized two-photon fluorescent carbon dot exist rich sulfonic acid groups, so as to change the chemical environment of the surface of the two-photon fluorescent carbon dot, and then affect its fluorescent properties, and at the same time, the introduction of sulfonic acid groups can enhance the fluorescence intensity and stability of the carbon dot, and also can regulate the fluorescence wavelength and emission spectrum, so that it obtains near-infrared luminescence that does not change with excitation wavelength; in addition, the sulfonic acid group is a functional group with strong hydrophilicity, which can enhance the water solubility of the two-photon fluorescent carbon dot when introduced onto the surface of the two-photon fluorescent carbon dot, and the introduction of the sulfonic acid group can increase the negative charge density on the surface of the two-photon fluorescent carbon dot, thereby improving its biocompatibility and stability in cells.

[0008] The following is a preferred technical solution of the present application, but not as a limitation to the technical solution provided by the present application, through the following preferred technical solution, the purpose and beneficial effects of the present application can be better achieved and realized.

[0009] As a preferred technical solution, the carbon source comprises any one or a combination of at least two of o-phenylenediamine, m-phenylenediamine, catechin, anthocyanin, aniline, amino acid, glucuronic acid, glucosamine or citric acid.

[0010] Preferably, the mass ratio of the carbon source to sulfuric acid is 1:(12-310), for example, it can be 1:12, 1:20, 1:50, 1:80, 1:100, 1:120, 1:140, 1:160, 1:180, 1:200, 1:220, 1:250, 1:280, 1:300 or 1:310, etc.

[0011] Preferably, the sulfuric acid participates in the reaction in the form of a sulfuric acid solution after being dissolved in water.

[0012] Preferably, the concentration of the sulfuric acid solution is 2-10 mol / L, for example, it can be 2 mol / L, 3 mol / L, 4 mol / L, 5 mol / L, 6 mol / L, 7 mol / L, 8 mol / L, 9 mol / L or 10 mol / L, and specific point values between the above point values, limited to the length and for the sake of simplicity, the present application will not be listed again. The specific point values included in the range.

[0013] Preferably, the time of the hydrothermal reaction is 8-16h, for example, it can be 8h, 9h, 10h, 11h, 12h, 13h, 14h, 15h or 16h, and specific point values between the above point values, limited to the length and for the sake of simplicity, the present application will not be listed again. The specific point values included in the range.

[0014] Preferably, the temperature of the hydrothermal reaction is 150-210℃, for example, it can be 150℃, 160℃, 170℃, 180℃, 190℃, 200℃ or 210℃, and specific point values between the above point values, limited to the length and for the sake of simplicity, the present application will not be listed again. The specific point values included in the range.

[0015] Preferably, the pressure of the hydrothermal reaction is 0.5-2.4MPa, for example, it can be 0.5MPa, 0.8MPa, 1MPa, 1.2MPa, 1.4MPa, 1.6MPa, 1.8MPa, 2MPa, 2.2MPa or 2.4MPa, and specific point values between the above point values, limited to the length and for the sake of simplicity, the present application will not be listed again. The specific point values included in the range.

[0016] Preferably, after the completion of the hydrothermal reaction, the step of washing the reaction product is further included.

[0017] Preferably, the reagent for the washing includes a combination of water and ethanol.

[0018] Preferably, the mass ratio of the water to ethanol is (3-8):1, for example, it can be 3:1, 3.5:1, 4:1, 4.5:1, 5:1, 5.5:1, 6:1, 6.5:1, 7:1, 7.5:1 or 8:1, etc.

[0019] Preferably, the mass ratio of the carbon source to the reagent is 1:(38.5-52.5), for example, it can be 1:38.5, 1:40, 1:41, 1:42, 1:43, 1:44, 1:45, 1:46, 1:47, 1:48, 1:49, 1:50, 1:51 or 1:52.5, etc.

[0020] Preferably, the washing is followed by a step of drying the reaction product.

[0021] Preferably, the drying temperature is 30-50℃, for example, it can be 30℃, 32℃, 34℃, 36℃, 38℃, 40℃, 42℃, 44℃, 46℃, 48℃ or 50℃, and specific point values between the above point values, limited to the length and for the sake of simplicity, the present application will not be listed again. The specific point values included in the range.

[0022] Preferably, the drying time is 8-12h, for example, it can be 8h, 8.5h, 9h, 9.5h, 10h, 10.5h, 11h, 11.5h or 12h, and specific point values between the above point values, limited to the length and for the sake of simplicity, the present application will not be listed again. The specific point values included in the range.

[0023] Preferably, the preparation method specifically comprises: the carbon source is subjected to a hydrothermal reaction with a sulfuric acid solution, the reaction product is washed with a mixture of water and ethanol after the completion of the hydrothermal reaction, and then the reaction product is dried to obtain the two-photon fluorescent carbon dots.

[0024] The mass ratio of the carbon source to the sulfuric acid is 1:(12-310).

[0025] The concentration of the sulfuric acid solution is 2-10mol / L.

[0026] The hydrothermal reaction time is 8-16h.

[0027] The hydrothermal reaction temperature is 150-210℃.

[0028] The hydrothermal reaction pressure is 0.5-2.4MPa.

[0029] The mass ratio of the water to the ethanol is (3-8):1.

[0030] The mass ratio of the carbon source to the reagent is 1:(38.5-52.5).

[0031] The drying temperature is 30-50℃.

[0032] The drying time is 8-12h.

[0033] In a second aspect, the present invention provides a two-photon fluorescent carbon dot, which is prepared by the preparation method described in the first aspect.

[0034] Preferably, the fluorescence emission wavelengths of the two-photon fluorescent carbon dots are 600 nm and 650 nm.

[0035] Preferably, the two-photon fluorescent carbon dots are red fluorescent carbon dots.

[0036] Thirdly, the present invention provides an application of two-photon fluorescent carbon dots as described in the second aspect, wherein the two-photon fluorescent carbon dots are used in cell imaging agents or fluorescent probes.

[0037] Compared with the prior art, the present invention has the following beneficial effects:

[0038] The present invention provides a method for preparing two-photon fluorescent carbon dots, which uses a hydrothermal method to synthesize a novel two-photon fluorescent carbon dot. This method simplifies the preparation process and saves production costs. The use of sulfuric acid results in abundant sulfonic acid groups on the surface of the two-photon fluorescent carbon dots. The introduction of sulfonic acid groups enhances the fluorescence intensity and stability of the two-photon fluorescent carbon dots, enabling them to exhibit near-infrared emission that does not change with the excitation wavelength. At the same time, the introduction of sulfonic acid groups can increase the negative charge density on the surface of the two-photon fluorescent carbon dots, thereby improving their biocompatibility and stability in cells. When the amount of two-photon fluorescent carbon dots added reaches 15 μg / mL, the cell viability can still reach 90%, making it applicable to live-cell imaging. Attached Figure Description

[0039] Figure 1 This is a TEM scan image of the two-photon fluorescent carbon dots provided in Example 1;

[0040] Figure 2 The Fourier transform infrared absorption spectrum of the two-photon fluorescent carbon dot provided in Example 1;

[0041] Figure 3 The X-ray photoelectron spectrum of the two-photon fluorescent carbon dots provided in Example 1;

[0042] Figure 4 The steady-state UV-Vis absorption spectrum and PL spectra of two-photon fluorescent carbon dots with different excitation wavelengths are provided in Example 1.

[0043] Figure 5 The steady-state UV-Vis absorption spectrum and PL spectra of two-photon fluorescent carbon dots with different excitation wavelengths are provided in Example 6.

[0044] Figure 6 This is a graph showing the effect of different concentrations of two-photon fluorescent carbon dots on cytotoxicity, as provided in Example 1.

[0045] Figure 7 is a graph showing the influence of different concentrations of two-photon fluorescent carbon dots provided by Comparative Example 1 on cell toxicity;

[0046] Figure 8 is a graph showing the influence of different concentrations of two-photon fluorescent carbon dots provided by Comparative Example 2 on cell toxicity;

[0047] Figure 9 is a live cell imaging graph of one-photon fluorescent carbon dots;

[0048] Figure 10 is a live cell imaging graph of two-photon fluorescent carbon dots provided by Example 1. DETAILED DESCRIPTION

[0049] The technical solutions of the present application will be further described below in conjunction with the accompanying drawings and through specific embodiments. Those skilled in the art should understand that the embodiments are only to help understand the present application and should not be regarded as specific limitations of the present application.

[0050] Example 1

[0051] A two-photon fluorescent carbon dot and a preparation method thereof, the preparation method comprising: adding 0.16 g of o-phenylenediamine to 25 mL of dilute sulfuric acid with a concentration of 4 mol / L, transferring to a polytetrafluoroethylene-lined stainless steel autoclave after ultrasonic dissolution and dispersion, heating to 180℃, reacting under a pressure of 1.1 MPa for 12 h, then cooling to room temperature in an annealing furnace, removing the supernatant to obtain a reaction product, washing the reaction product several times with a mixture of deionized water and ethanol (mass ratio of deionized water to ethanol is 6:1) 7 g until the pH is 7, and finally vacuum drying at 40℃ for 12 h to obtain the two-photon fluorescent carbon dot.

[0052] Example 2

[0053] A two-photon fluorescent carbon dot and a preparation method thereof, which are only different from Example 1 in that o-phenylenediamine is replaced by glucuronic acid in equal amount, and other raw materials, process parameters and steps are the same as those of Example 1.

[0054] Example 3

[0055] A two-photon fluorescent carbon dot and a preparation method thereof, which are only different from Example 1 in that the concentration of dilute sulfuric acid in this embodiment is 6 mol / L, and other raw materials, process parameters and steps are the same as those of Example 1.

[0056] Example 4

[0057] The application discloses a kind of two-photon fluorescent carbon dots and preparation method thereof, the preparation method includes: 0.408g catechin is added to 25mL concentration 2mol / L dilute sulfuric acid, after ultrasonic dissolution and dispersion, it is transferred to polytetrafluoroethylene-lined stainless steel autoclave, heated to 150 DEG C, under the condition that pressure is 0.5MPa, reaction 8h, then it is cooled to room temperature in annealing furnace, remove supernatant to obtain reaction product, the mixture of deionized water and ethanol (the mass ratio of deionized water and ethanol is 6:1) 15g is used to wash the reaction product several times until pH is 7, finally vacuum drying 10h at 30 DEG C, and the two-photon fluorescent carbon dots are obtained.

[0058] Example 5

[0059] The application discloses a kind of two-photon fluorescent carbon dots and preparation method thereof, the preparation method includes: 0.0791g anthocyanin is added to 25mL concentration 10mol / L dilute sulfuric acid, after ultrasonic dissolution and dispersion, it is transferred to polytetrafluoroethylene-lined stainless steel autoclave, heated to 210 DEG C, under the condition that pressure is 2.4MPa, reaction 16h, then it is cooled to room temperature in annealing furnace, remove supernatant to obtain reaction product, the mixture of deionized water and ethanol (the mass ratio of deionized water and ethanol is 6:1) 4g is used to wash the reaction product several times until pH is 7, finally vacuum drying 8h at 50 DEG C, and the two-photon fluorescent carbon dots are obtained.

[0060] Example 6

[0061] The application discloses a kind of two-photon fluorescent carbon dots and preparation method thereof, which are only different from the embodiment 1 in that the concentration of dilute sulfuric acid is 0.5mol / L, and the rest of raw materials, process parameters and steps are the same as those of the embodiment 1.

[0062] Comparative example 1

[0063] The application discloses a kind of two-photon fluorescent carbon dots and preparation method thereof, which are only different from the embodiment 1 in that 25mL concentration 4mol / L dilute sulfuric acid is replaced by 25mL concentration 4mol / L dilute hydrochloric acid, and the rest of raw materials, process parameters and steps are the same as those of the embodiment 1.

[0064] Comparative example 2

[0065] The application discloses a kind of two-photon fluorescent carbon dots and preparation method thereof, 200mg o-phenylenediamine is dissolved in 15mL ultrapure water by ultrasonic for 30min to obtain a clear and transparent solution, which is transferred to a Teflon reactor for heating at 220 DEG C for 6h, and then the obtained solution is taken out and centrifuged at 8000r·min -1The precipitate obtained after centrifugation and the precipitate adsorbed in the reaction kettle were dissolved with dilute sulfuric acid, and filtered using a water-based filter membrane with a pore size of 0.22 μm. The solution obtained after filtration was neutralized with sodium bicarbonate, placed in a dialysis bag with a molecular weight cut-off of 500D, dialyzed in deionized water for 6 h, and freeze-dried to obtain the two-photon fluorescent carbon dots.

[0066] Material characterization and performance testing:

[0067] (1) The two-photon fluorescent carbon dots provided in Example 1 were tested using a transmission electron microscope (model: Talos F200X G2, manufacturer: Thermo scientific), and the test results are shown in FIG. 1. Figure 1 The TEM scanning test diagram of the two-photon fluorescent carbon dots provided in Example 1 is shown in the figure, and it can be seen from the figure that the carbon quantum dots have a spherical morphology and have a monodisperse size.

[0068] (2) Infrared spectroscopy characterization: Fourier transform infrared absorption spectroscopy test was performed on the two-photon fluorescent carbon dots provided in Example 1 using a Fourier transform infrared spectrometer (model: Nicolet iS50, manufacturer: Thermo Fisher), and the test results are shown in FIG. 2. Figure 2 The Fourier transform infrared absorption spectrum of the two-photon fluorescent carbon dots provided in Example 1 is shown in the figure, and it can be seen from the figure that there are C-H vibration modes at about 3000 cm -1 , C=C at 1477 cm -1 , C=O / C=N at 1583 cm -1 , C-N at 1307 cm -1 , and C-N-C at 1060 cm -1 , and the FTIR peaks of C-SO3 and C-S are located at 1140 cm -1 and 731 cm -1 , respectively, proving that there are sulfonic acid groups and thiol groups on the surface of the two-photon fluorescent carbon dots provided in Example 1.

[0069] (3) X-ray photoelectron spectroscopy test: The two-photon fluorescent carbon dots provided in Example 1 were tested using an X-ray photoelectron spectrometer (model: XPS Escalab Xi+, manufacturer: Thermo Fisher Scientific (China) Co. Ltd), and the test results are shown in FIG. 3. Figure 3 The X-ray photoelectron spectrum of the two-photon fluorescent carbon dots provided in Example 1 is shown in the figure, and it can be seen from the figure that there are four different peaks corresponding to S-C and C-SO3 bonds in the 2p 1 / 2 and 2p 3 / 2 orbitals, proving that there are abundant sulfonic acid groups on the surface of the two-photon fluorescent carbon dots provided in Example 1.

[0070] (4) UV-Vis absorption spectrum and photoluminescence spectrum test: UV-Vis absorption spectrum and photoluminescence spectrum of the two-photon fluorescent carbon dots provided by Example 1 and Example 6 were tested by using UV-Vis spectrophotometer (model: Lambda 1050+, manufacturer: PerkinElmer) and steady-state transient fluorescence spectrometer (model: FS5, manufacturer: Edinburgh Instruments), and the test results are shown in Figure 4 the steady-state UV-Vis absorption spectrum and PL spectrum of different excitation wavelengths of the two-photon fluorescent carbon dots provided by Example 1 and Figure 5 the steady-state UV-Vis absorption spectrum and PL spectrum of different excitation wavelengths of the two-photon fluorescent carbon dots provided by Example 6, as shown in the figures, it can be seen from the figures that the photoluminescence wavelength band of the two-photon fluorescent carbon dots provided by Example 1 does not change with the change of excitation wavelength, which is a non-excitation wavelength dependent two-photon fluorescent carbon dot, Figure 5 which shows that the two-photon fluorescent carbon dots provided by Example 6 are blue two-photon fluorescent carbon dots.

[0071] (5) Cell toxicity evaluation test: CCK-8 detection kit (Dojindo Laboratories Tokyo, Japan) was used to evaluate cell toxicity. 2.0 x 10 4 cells / well were seeded in a 96-well plate. After the cells adhered to the bottom of the well, the culture medium was discarded and replaced with fresh culture medium mixed with different concentrations of two-photon fluorescent carbon dots (different amounts of two-photon fluorescent carbon dots were dissolved in ethanol to prepare, and the concentrations of two-photon fluorescent carbon dots were 0 μg / mL, 2 μg / mL, 4 μg / mL, 6 μg / mL, 8 μg / mL, 10 μg / mL, 12.5 μg / mL, 15 μg / mL, 18 μg / mL and 20 μg / mL) for 24 h. Then 100 μL of fresh culture medium was mixed with 10 μL of CCK-8 solution to change the culture medium, and after incubation at 37°C for 1 hour, the plate was read at 450 nm by using an enzyme-labeled instrument (pectraMax M5, Molecular Device, USA). The test results are shown in Figure 6 、 Figure 7 and Figure 8 the figure showing the effect of different concentrations of two-photon fluorescent carbon dots provided by Example 1, Comparative Example 1 and Comparative Example 2 on cell toxicity, as shown in the figure, when the amount of carbon dots added reaches 15 μg / mL, the cell activity treated with the two-photon fluorescent carbon dots provided by Example 1 can still reach 90%, indicating that the two-photon fluorescent carbon dots provided by the present application do not significantly weaken the cell activity when entering the cell interior, while the carbon dots provided by Comparative Example 1 and Comparative Example 2 have a greater impact on cell activity, with cell activities of 74% and 76% respectively, which are not suitable for cell imaging.

[0072] (6) Cell imaging:

[0073] a. Single-photon fluorescent carbon dot imaging: bEnd.3 cells grown on a confocal dish were cultured in a medium containing 15 μg / mL of carbon quantum dots for 12 h, washed three times with phosphate buffered saline (PBS), incubated in a culture solution containing a Hoechst 33342 cell nucleus staining kit (10 μL / mL, kit) at 37°C for 10 min, washed three times with PBS after nucleus staining, and the cells remained normal. Fluorescent images were taken using a Nikon A1 confocal microscope;

[0074] b. Two-photon fluorescent carbon dot imaging: bEnd.3 cells grown on a confocal dish were cultured in a medium containing 15 μg / mL of carbon quantum dots for 12 h, then soaked in 4% paraformaldehyde (PFA) at 4°C for 10 min. After washing three times with PBS, the cells were stained with diphenylamino-4,6-diamino-2-indolyl (DAPI) (Solarbio, China) for 5 min. Fluorescent images were taken using a Nikon A1 confocal microscope.

[0075] The test results are shown in Figure 9 The live cell imaging images of single-photon fluorescent carbon dots and Figure 10 The live cell imaging images of two-photon fluorescent carbon dots provided in Example 1 are shown in the figures. As can be seen from the figures, compared with the fluorescent imaging effect of single-photon fluorescent carbon dots, the two-photon fluorescent carbon dot imaging is clearer and can be better applied to fluorescent labeling of biological cells and tissues.

[0076] The applicant declares that the detailed process flow of the present application is illustrated by the above examples, but the present application is not limited to the above detailed process flow, i.e. it does not mean that the present application must rely on the above detailed process flow to be implemented. It should be understood by those skilled in the art that any improvement of the present application, equivalent replacement of each raw material of the product of the present application, addition of auxiliary ingredients, selection of specific modes, etc. fall within the protection scope and disclosure scope of the present application.

Claims

1. Use of a two-photon fluorescent carbon dot in a cell imaging agent, characterized in that, The two-photon fluorescent carbon dots are prepared by the following method: a carbon source and sulfuric acid are subjected to a hydrothermal reaction to obtain the two-photon fluorescent carbon dots; the mass ratio of the carbon source to sulfuric acid is 1: (50-310); the two-photon fluorescent carbon dots have a fluorescence emission wavelength of 600 nm and 650 nm. The carbon source is selected from any one or a combination of at least two of o-phenylenediamine, m-phenylenediamine, catechin, anthocyanin, aniline, amino acid, glucuronic acid, glucosamine or citric acid; The sulfuric acid is dissolved in water to form a sulfuric acid solution which participates in the reaction; The concentration of the sulfuric acid solution is 2-10 mol / L; The hydrothermal reaction is performed for 8-16 h; The hydrothermal reaction is performed at a temperature of 150-210 ℃; The hydrothermal reaction is performed at a pressure of 0.5-2.4 MPa. 2.The use of the two-photon fluorescent carbon dots according to claim 1 in a cell imaging agent, characterized in that, The method further comprises a step of washing the reaction product after the hydrothermal reaction is completed. 3.The use of the two-photon fluorescent carbon dots according to claim 2 in a cell imaging agent, characterized in that, The washing reagent comprises a combination of water and ethanol. 4.The use of the two-photon fluorescent carbon dots according to claim 3 in a cell imaging agent, characterized in that, The mass ratio of the water to ethanol is (3-8):

1. 5.The use of the two-photon fluorescent carbon dots according to claim 3 in a cell imaging agent, characterized in that, The mass ratio of the carbon source to the reagent is 1: (38.5-52.5). 6.The use of the two-photon fluorescent carbon dots according to claim 2 in a cell imaging agent. The method further comprises a step of drying the reaction product after the washing is completed. 7.The use of the two-photon fluorescent carbon dots according to claim 6 in a cell imaging agent. The drying is performed at a temperature of 30-50 ℃. 8.The use of the two-photon fluorescent carbon dots according to claim 6 in a cell imaging agent. The drying is performed for 8-12 h. 9.The use of the two-photon fluorescent carbon dots according to claim 1 in a cell imaging agent. The method specifically comprises: the carbon source and the sulfuric acid solution are subjected to a hydrothermal reaction, the reaction product is washed with a mixture of water and ethanol after the hydrothermal reaction is completed, and then the reaction product is dried to obtain the two-photon fluorescent carbon dots; The mass ratio of the carbon source to sulfuric acid is 1: (50-310); The concentration of the sulfuric acid solution is 2-10 mol / L; The hydrothermal reaction is performed for 8-16 h; The hydrothermal reaction is performed at a temperature of 150-210 ℃; The hydrothermal reaction is performed at a pressure of 0.5-2.4 MPa; The mass ratio of the water to ethanol is (3-8):

1. The mass ratio of the carbon source to the mixture of water and ethanol is 1: (38.5-52.5); The drying is performed at a temperature of 30-50 ℃. The drying is performed for 8-12 h. 10.The use of the two-photon fluorescent carbon dots according to claim 1 in a cell imaging agent. The two-photon fluorescent carbon dots are red fluorescent carbon dots.

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