A red fluorescent carbon dot and a preparation method and application thereof

Abstract

The application discloses a kind of red fluorescent carbon dots, it includes the following weight parts of raw materials: 0.5~1.5 parts fluorescein hydrazide, 0.5~1.5 parts citric acid and 250~700 parts ethanol, wherein, the mass ratio of fluorescein hydrazide and citric acid is 1:1, and the mass fraction of ethanol is 500 times of the mass fraction of fluorescein hydrazide.In the application, by selecting fluorescein hydrazide as a raw material, fluorescein hydrazide is a non-fluorescent fluorescein derivative, which uses fluorescein molecular derivatives as precursors, making it easier to synthesize a luminescent domain with high quantum yield, thereby achieving a high quantum yield, thereby solving the problem of low quantum yield of red fluorescent carbon dots synthesized and applicable to the field of biological imaging in the prior art.The red fluorescent carbon dots prepared in the application can be applied to the field of biological imaging, and the quantum yield is more than 26%, which is much higher than the quantum yield of red fluorescent carbon dots synthesized by existing technical means.

Description

TECHNICAL FIELD

[0001] The application relates to the technical field of nanomaterial synthesis, in particular to a red fluorescent carbon dot and a preparation method and application thereof. BACKGROUND

[0002] Since carbon dots were first discovered in 2004, they have been widely studied due to their unique optical, chemical and physical properties. As the most promising substitutes for commercial semiconductor quantum dots and organic dyes, carbon dots are widely used in fluorescence sensing, biological imaging and photodynamic therapy. However, most of the carbon dots prepared at present have fluorescence emission concentrated in the blue to green light region. The biological samples such as cells and tissues have strong autofluorescence in this region, and the radiation will cause damage to the biological samples, which greatly limits the application of carbon dots in the biological field. Although people have begun to study the red and near-infrared light-emitting fluorescent carbon dots, compared with the carbon dots emitting blue-green fluorescence, the quantum yield of the red fluorescent carbon dots synthesized by the existing technical means and applied in the biological imaging field is low, generally below 20%. SUMMARY

[0003] In order to solve the problem of low quantum yield of the red fluorescent carbon dots synthesized by the existing technical means and applied in the biological imaging field in the prior art, the application provides a red fluorescent carbon dot, which has a simple synthesis process, and the quantum yield of the synthesized red fluorescent carbon dot is 26%, which is greatly improved compared with the quantum yield of the red fluorescent carbon dots synthesized in the prior art.

[0004] To achieve the above object, the technical scheme adopted by the application is as follows: a red fluorescent carbon dot, which comprises the following raw materials in parts by weight: 0.5-1.5 parts of fluorescein hydrazide, 0.5-1.5 parts of citric acid and 250-700 parts of ethanol, wherein the mass ratio of the fluorescein hydrazide to the citric acid is 1:1, and the mass fraction of the ethanol is 500 times that of the fluorescein hydrazide.

[0005] As a further improvement of the above scheme, a red fluorescent carbon dot comprises the following raw materials in parts by weight: 1 part of fluorescein hydrazide, 1 part of citric acid and 500 parts of ethanol.

[0006] As a further improvement of the above scheme, the fluorescein hydrazide is prepared from fluorescein and hydrazine hydrate.

[0007] As a further improvement of the above scheme, the mass content of C in the red fluorescent carbon dot is 75%-76%, the mass content of N is 6%-7%, and the mass content of O is 18%-19%.

[0008] As a further improvement of the above-mentioned scheme, the particle size distribution of the red fluorescent carbon dots is 2.5nm-5.0nm.

[0009] A preparation method of the red fluorescent carbon dots as mentioned above, comprising the following steps: S1, adding fluorescein hydrazide and solid citric acid into ethanol, ultrasonic dissolving, then transferring to a reaction kettle with a polytetrafluoroethylene liner and carrying out reaction in the reaction kettle, and taking supernatant in the reaction kettle after the reaction is completed;

[0010] S2, sequentially purifying, rotary evaporating, and drying the supernatant taken out in the step S1 by column chromatography to obtain brown-red solid powder, i.e. the red fluorescent carbon dots are prepared.

[0011] As a further improvement of the above-mentioned scheme, the reaction temperature of the reaction kettle in the step S1 is 200℃, and the reaction time is 8h-12h.

[0012] As a further improvement of the above-mentioned scheme, the eluent in the column chromatography in the step S2 is a mixed solution of dichloromethane and methanol, and the volume ratio of dichloromethane to methanol is 100:1.

[0013] As a further improvement of the above-mentioned scheme, the preparation method of the fluorescein hydrazide in the step S1 comprises the following steps:

[0014] S11, weighing a proper amount of fluorescein and placing it in a flask, adding ethanol solution, ultrasonic dissolving, then adding excess hydrazine hydrate, and carrying out reflux reaction in an oil bath pot;

[0015] S12, rotary evaporating the mixture in the step S11, then washing with water, centrifuging, and vacuum drying to obtain white solid, i.e. fluorescein hydrazide.

[0016] As a further improvement of the above-mentioned scheme, the excess hydrazine hydrate in the step S11 refers to that the molar equivalent of the hydrazine hydrate is more than 50 times of the fluorescein.

[0017] Further, the ultrasonic time in the step S11 is 5-15 minutes, the temperature of the oil bath pot is 80℃-100℃, and the reflux reaction time is 15h-25h.

[0018] An application of the red fluorescent carbon dots prepared as mentioned above, the red fluorescent carbon dots are used as imaging reagents of lipid droplets in living cells.

[0019] Further, the living cells are common tumor cells in laboratories, including HeLa, HepG2, A549, etc.

[0020] Compared with the prior art, the application has the beneficial effects that:

[0021] (1) The raw material fluorescein hydrazide used in the preparation process of the red fluorescent carbon dots of the present application is a non-fluorescent fluorescein derivative. The use of fluorescein molecular derivatives as precursors makes it easier to synthesize a luminescent domain with high quantum yield, thereby obtaining a high quantum yield, thereby solving the problem of low quantum yield of the synthesized red fluorescent carbon dots in the prior art.

[0022] (2) The fluorescein hydrazide used in the red fluorescent carbon dots of the present application is a non-fluorescent fluorescein derivative, which can avoid the interference of dye fluorescence on carbon dot fluorescence, and is also more conducive to the subsequent separation and purification of red fluorescent carbon dots.

[0023] (3) The red fluorescent carbon dots in the present application have a simple preparation method, are easy to repeat, and are prepared from inexpensive and environmentally friendly materials.

[0024] (4) The quantum yield of the red fluorescent carbon dots in the present application is generally above 26%, which is much higher than the quantum yield of the red fluorescent carbon dots synthesized in the prior art, which is below 20%. The quantum yield is greatly improved, and the red fluorescent carbon dots have good anti-photobleaching performance and good biocompatibility.

[0025] (5) The red fluorescent carbon dots prepared in the present application have an oil-water partition coefficient (LogP value) of 1.4-1.5, good lipophilicity, and lipid droplet targeting specificity. The red fluorescent carbon dots have strong anti-photobleaching performance, little toxicity to cells, excellent light stability, and are suitable for imaging of live cell lipid droplets. BRIEF DESCRIPTION OF DRAWINGS

[0026] Figure 1 A brief synthesis diagram of the red fluorescent carbon dots in the present application.

[0027] Figure 2 An FT-IR spectrum of the red fluorescent carbon dots prepared in the present application.

[0028] Figure 3 An ultraviolet-visible spectrum and an excitation emission spectrum of the red fluorescent carbon dots in the present application.

[0029] Figure 4 A fluorescence emission spectrum of the red fluorescent carbon dots in the present application under excitation of different excitation lights.

[0030] Figure 5 A fluorescence intensity change graph of the red fluorescent carbon dots in the present application under different ultraviolet irradiation times.

[0031] Figure 6 A TEM graph of the red fluorescent carbon dots in the present application.

[0032] Figure 7 A size distribution graph of the red fluorescent carbon dots in the present application.

[0033] Figure 8 XPS spectrum of red fluorescent carbon dots in the present application.

[0034] Figure 9 Cell toxicity analysis chart of red fluorescent carbon dots in the present application.

[0035] Figure 10 Co-localization imaging analysis chart of red fluorescent carbon dots in the present application with lipid droplets in living cells. DETAILED DESCRIPTION

[0036] Hereinafter, the present application will be further described in conjunction with specific embodiments, and it should be noted that the following described embodiments or technical features between each other or between each other can be combined to form new embodiments without conflict.

[0037] In the description of the present application, it should be noted that for orientation words, such as the terms "center", "transverse", "longitudinal", "length", "width", "thickness", "upper", "lower", "front", "back", "left", "right", "vertical", "horizontal", "top", "bottom", "inner", "outer", "clockwise", "counterclockwise", etc. The orientation and positional relationship shown in the drawings is based on the orientation or positional relationship shown in the drawings, and is only for the convenience of describing the present application and simplifying the description, and does not indicate or imply that the device or element referred to must have a particular orientation, be constructed and operated in a particular orientation, and cannot be understood as limiting the specific protection scope of the present application. The terms "first", "second" and the like in the specification and claims of the present application are used to distinguish similar objects, and do not necessarily describe a specific order or sequence. The terms "include" and "have" and any variations thereof in the specification and claims of the present application are intended to cover non-exclusive inclusion, for example, a process, method, system, product or device that includes a series of steps or units does not necessarily limit to those clearly listed steps or units, but can include other steps or units that are not clearly listed or inherent to these processes, methods, products or devices.

[0038] As Figure 1As shown, one embodiment of the present application provides a red fluorescent carbon dot, which comprises the following raw materials by weight: 0.5-1.5 parts of fluorescein hydrazide, 0.5-1.5 parts of citric acid, and 250-700 parts of ethanol, wherein the mass ratio of fluorescein hydrazide to citric acid is 1:1, and the mass fraction of ethanol is 500 times that of fluorescein hydrazide. In the present application, fluorescein hydrazide is selected as the raw material for preparation. Fluorescein hydrazide is a non-fluorescent derivative of fluorescein. By using a fluorescein molecule derivative as a precursor, it is easier to synthesize a luminescent domain with high quantum yield, thereby obtaining a high quantum yield, thereby solving the problem of low quantum yield of the red fluorescent carbon dots synthesized in the prior art and capable of being applied in the field of biological imaging. Specifically, the red fluorescent carbon dots prepared in the present application can be applied in the field of biological imaging, and the quantum yield of the red fluorescent carbon dots prepared by the raw materials of the present application is measured by a full-function steady-state transient fluorescence spectrometer to be above 26%, which is much larger than the quantum yield of the red fluorescent carbon dots synthesized by the prior art.

[0039] The mass ratio of fluorescein hydrazide to citric acid is 1:1.

[0040] The mass content of C in the red fluorescent carbon dots is 75%-76%, the mass content of N is 6%-7%, and the mass content of O is 18%-19%.

[0041] In one embodiment, a red fluorescent carbon dot comprises the following raw materials by weight: 1 part of fluorescein hydrazide, 1 part of citric acid, and 500 parts of ethanol.

[0042] The particle size distribution of the red fluorescent carbon dots is 2.5-5.0 nm, and the average particle size is 4.0 nm. Therefore, the red fluorescent carbon dots prepared in the present application have a small particle size, and the small particle size can quickly enter the cell.

[0043] A preparation method of the red fluorescent carbon dots as described above, comprising the following steps: (1) taking a proper amount of fluorescein and placing it in a flask, adding an ethanol solution, and ultrasonically dissolving for 5-15 minutes, then adding excess hydrazine hydrate, and then performing a reflux reaction in an oil bath pot at 80-100 DEG C, and the reflux reaction time is 15-25 hours; (2) performing rotary evaporation on the mixed solution in step (1), then washing with water, centrifuging, and then vacuum drying to obtain a white solid, which is fluorescein hydrazide; (3) adding the fluorescein hydrazide prepared in step (2) and citric acid solid into ethanol, ultrasonically dissolving, and then transferring into a polytetrafluoroethylene-lined reaction kettle, and performing a reaction in the reaction kettle, the temperature of the reaction kettle is 200 DEG C, the reaction time is 8-12 hours, and then taking the supernatant in the reaction kettle; (4) sequentially purifying the supernatant taken out in step (3) by column chromatography, rotary evaporation, and drying to obtain a brownish red solid powder, i.e. the red fluorescent carbon dots are prepared.

[0044] In step (4), the eluent in the column chromatography is a mixture of dichloromethane and methanol, and the volume ratio of dichloromethane to methanol is 100:1.

[0045] In step (1), the excess hydrazine hydrate refers to that the molar equivalent of hydrazine hydrate is more than 50 times that of fluorescein.

[0046] In the present application, the synthesis method of the prepared red fluorescent carbon dots is simple and easy to repeat, and the materials used are cheap and environmentally friendly.

[0047] The application of the red fluorescent carbon dots prepared as described above, wherein the red fluorescent carbon dots are used as imaging reagents for lipid droplets in living cells.

[0048] When the red fluorescent carbon dots are used as imaging reagents for lipid droplets in living cells, the culture time of the red fluorescent carbon dots with the living cells is about 10 minutes, and the living cells do not need to be washed with PBS solution and treated by replacing the culture solution before being used for imaging.

[0049] In the present application, fluorescein hydrazide is selected as a raw material for preparation, which is a non-fluorescent fluorescein derivative, thereby avoiding the interference of dye fluorescence on the fluorescence of carbon dots, and facilitating the subsequent separation and purification of carbon dots.

[0050] Further, the living cells are common tumor cells in laboratories, including HeLa, HepG2, A549, etc.

[0051] Among them, the red fluorescent carbon dots prepared in this application are a fluorescent probe for imaging lipid droplets in living cells, which have red fluorescence emission characteristics that are not dependent on excitation light and the ability to locate lipid droplets in living cells.

[0052] Example 1

[0053] (1) Weigh 5 g of fluorescein and place it in a 500 mL round-bottom flask, add ethanol solution, ultrasonicate for 10 minutes until it is completely dissolved, then add excess hydrazine hydrate, and then reflux in an 80°C oil bath for 20 hours; (2) The mixed solution in step (1) is subjected to rotary evaporation, washed with water, centrifuged, and then vacuum-dried to obtain a white solid, which is fluorescein hydrazide; (3) Weigh 0.1 g of the fluorescein hydrazide prepared in step (2) and 0.1 g of citric acid solid, add them to 50 mL of ethanol, and ultrasonicate for 15 minutes. After the fluorescein hydrazide and citric acid are completely dissolved, the mixture is transferred to a polytetrafluoroethylene-lined reactor with a volume of 100 mL and reacted at 200°C for 10 hours, and then naturally cooled to room temperature. After the reaction is completed, the supernatant in the reactor is taken out; (4) the supernatant taken out in step (3) is purified by column chromatography in sequence, and the eluate is a mixture of dichloromethane and methanol, and the volume ratio of dichloromethane to methanol is 100:1. The solution is evaporated and concentrated to obtain a brown solution, which is then placed in an oven and dried at 70°C to obtain a brown-red solid powder, that is, the red fluorescent carbon dots are prepared.

[0054] The surface functional groups of the FH-CDs (ie, red fluorescent carbon dots) synthesized in Example 1 were determined by FI-IR spectroscopy.

[0055] like Figure 2 As shown, the FH-CDs prepared in Example 1 were at 3423 cm -1 The absorption bands near 1608 cm are attributed to -OH and -NH on the surface. -1 and 1450cm -1 The peaks at 2994 cm correspond to the absorption bands of C=O and CO, respectively. -1 ~2910cm -1 The absorption band is attributed to the C-H bond, which verifies the structure of the prepared FH-CDs.

[0056] like Figure 3 and Figure 4 As shown in FIG, the optical properties of the FH-CDs (ie, red fluorescent carbon dots) synthesized in Example 1 were detected by UV-Vis absorption spectroscopy and fluorescence spectroscopy.

[0057] pass Figure 3 It can be seen that the absorption peak at 280nm can be attributed to the π→π generated by the C=C bond of the carbon core, as shown by the Abs line. *transition, and the absorption band from 370 nm to 600 nm can be attributed to sp 2 n→π generated by the C=O / C=N bond at the edge of the hybrid system * The red fluorescent carbon dots prepared in Example 1 have the ability to absorb light. As shown by the Em (540 nm) line, when the optimal excitation wavelength is 540 nm, the corresponding maximum emission peak is 600 nm. Since the emission peak is at 600 nm, it can be verified that the prepared red fluorescent carbon dots can emit red light.

[0058] from Figure 4 It can be seen that as the excitation wavelength increases from 370 nm to 550 nm, the emission wavelength of FH-CDs hardly changes, which proves that the emission wavelength of FH-CDs prepared in Example 1 does not change with the change of the excitation light.

[0059] from Figure 4 In the photo depicted in the inset in the upper right corner, the deep red solution appears bright red under ultraviolet light (365 nm).

[0060] The fluorescence intensity of the red fluorescent carbon dots under different UV irradiation times was tested: the cuvette containing the red fluorescent carbon dots prepared in Example 1 was placed on a GL-3120 desktop UV analyzer for continuous irradiation. The UV analyzer power was 48W and the wavelength was 365nm. The fluorescence intensity of the red fluorescent carbon dots was observed after irradiation for 10min, 20min, 30min, 40min, 50min, 60min, 70min, 80min, 90min, and 100min. Figure 5 Data. Figure 5 It can be seen that with the increase of ultraviolet irradiation time, the fluorescence intensity of the red fluorescent carbon dots does not decrease significantly, which shows that the red fluorescent carbon dots prepared by the present application have excellent photostability.

[0061] The shape of the red fluorescent carbon dots prepared in Example 1 was measured by transmission electron microscopy, and the results were as follows: Figure 6 data, through Figure 6 It can be seen that the shape of the red fluorescent carbon dots prepared in Example 1 is approximately quasi-spherical and is uniformly dispersed in the solvent. Figure 6 The model of the transmission electron microscope is F200 field emission transmission electron microscope.

[0062] The particle size of the red fluorescent carbon dots prepared in Example 1 was measured by a high-sensitivity particle size analyzer, and the results were as follows: Figure 7 data, through Figure 7 It can be seen that the diameters of the red fluorescent carbon dots are distributed in the range of 2.5 nm to 5.0 nm.

[0063] The chemical composition of the red fluorescent carbon dots prepared in Example 1 was analyzed by X-ray photoelectron spectroscopy (XPS), and the results were as follows: Figure 8 data from Figure 8 It can be seen that the red fluorescent carbon dots mainly contain three elements: C, N and O. The content of C is 75.51%, N is 6.77% and O is 17.72%. The high-resolution XPS spectrum of C1s can be fitted with three characteristic peaks at 286.5eV (CC), 288.2eV (CN) and 291.6eV (C=O). The high-resolution XPS spectrum of N1s shows that N mainly exists in the form of CN, CNC and -NH. Figure 8 The data proved that the main atoms of the prepared red fluorescent carbon dots are C, N, O and the forms of these atoms.

[0064] The red fluorescent carbon dots prepared in Example 1 were used for cytotoxicity analysis. The specific operation was as follows: human cervical cancer cells (HeLa) were plated at 1×10 4 Cells were seeded into a 96-well plate and placed in a cell culture incubator under the following conditions: 37°C, 5% CO2, saturated humidity, and cultured for 24 hours to allow them to completely adhere to the wall. Fresh culture medium was then replaced, and 10 μL of fluorescent probe dispersion at different concentrations was added. After 24 hours of culture, MTT solution (5 mg / mL) was added to each well and incubated for another 4 hours. Then, 100 μL of DMSO was added to each well, and the 96-well plate was placed on a horizontal shaking table and shaken for 10 minutes. The wavelength was set to 492 nm on the microplate reader, and the absorbance (OD value) of the solution in each well of the 96-well plate was measured. The cell survival rate was calculated according to the following formula: Cell survival rate = (OD 待测组 -OD 空白组 ) / (OD 细胞组 -OD 空白组 )×100%. Figure 9 data from Figure 9 It can be seen that the fluorescent probe has almost no cytotoxicity in the range of 0-16 μg / mL.

[0065] The red fluorescent carbon dots prepared in Example 1 were applied to intracellular co-localization imaging. In the co-localization experiment, human liver cancer cells (HepG2) were first incubated with the fluorescent probe (2 μg / mL) for 10 min, and then incubated with 1 μM lipid droplet staining reagent for 20 min. The images were then taken using a laser confocal microscope, and the following images were obtained: Figure 10 The results are as follows. Figure 10As shown, from left to right, the first picture is a bright field, which can show the morphology and state of the cells, the second picture is a fluorescent field, which is the fluorescent imaging of the red fluorescent carbon dots prepared in Example 1 in the cells, the third picture is also a fluorescent field, but it is an imaging diagram of a commercial dye, the fourth picture is the picture obtained by overlapping the second picture and the third picture, and the fifth picture shows the degree of overlapping of the second picture and the third picture. Through the analysis of the above five pictures, it can be seen that the overlapping coefficient of the red fluorescent carbon dot channel and the lipid droplet commercial dye channel is as high as about 90%, which proves that the red fluorescent carbon dots prepared in the present application can target lipid droplets, and the effect of the red fluorescent carbon dots prepared in the present application in targeting lipid droplets is very good.

[0066] In summary, the red fluorescent carbon dots prepared in the present application can be used in the field of biological imaging, and have a very high quantum yield, generally more than 26%. At the same time, they can be selectively positioned in the lipid droplets of cells and used for live cell lipid droplet imaging, greatly improving their application in the field of biology, and will not cause radiation damage to biological samples. In addition, the red fluorescent carbon dots prepared in the present application have high light stability and good biocompatibility, overcoming the problems of light bleaching and cytotoxicity of lipid droplet commercial dyes.

[0067] The above describes the basic principles, main features and advantages of the present application. Those skilled in the art should understand that the present application is not limited to the above examples, and the above examples and descriptions in the specification are only the principles of the present application. Without departing from the spirit and scope of the present application, various changes and improvements can be made to the present application, and these changes and improvements all fall within the scope of the claimed present application. The scope of protection claimed by the present application is defined by the appended claims and their equivalents.

Claims

1. A red fluorescent carbon dot, characterized in that: The method comprises the following raw materials in parts by weight: 0.5 to 1.5 parts of fluorescein hydrazide, 0.5 to 1.5 parts of citric acid, and 250 to 700 parts of ethanol, wherein the mass ratio of the fluorescein hydrazide to the citric acid is 1:1, and the mass fraction of the ethanol is 500 times the mass fraction of the fluorescein hydrazide; The preparation method of the red fluorescent carbon dots is as follows: S1, adding fluorescein hydrazide and citric acid solid into ethanol, dissolving them by ultrasonication, transferring them into a polytetrafluoroethylene-lined reactor and reacting them in the reactor, and taking out the supernatant in the reactor after the reaction is completed; Wherein, the preparation method of fluorescein hydrazide in step S1 comprises the following steps: S11, weigh an appropriate amount of fluorescein and place it in a flask, add ethanol solution, sonicate until completely dissolved, then add excess hydrazine hydrate, and then reflux in an oil bath; S12, rotary evaporating the mixed solution in step S11, washing with water, centrifuging, and then vacuum drying to obtain a white solid, which is fluorescein hydrazide; S2, purifying the supernatant obtained in step S1 by column chromatography, rotary evaporation, and drying in sequence to obtain a brown-red solid powder, namely, preparing the red fluorescent carbon dots; The red fluorescent carbon dots are used as imaging agents for lipid droplets in living cells.

2. The red fluorescent carbon dot according to claim 1, wherein The method comprises the following raw materials in parts by weight: 1 part of fluorescein hydrazide, 1 part of citric acid and 500 parts of ethanol.

3. The red fluorescent carbon dots according to claim 1, wherein The fluorescein hydrazide is prepared from fluorescein and hydrazine hydrate as raw materials.

4. The red fluorescent carbon dot according to claim 1, wherein The mass content of C in the red fluorescent carbon dots is 75%-76%, the mass content of N is 6%-7%, and the mass content of O is 18%-19%.

5. The red fluorescent carbon dots according to claim 1, wherein The particle size distribution of the red fluorescent carbon dots is 2.5 nm to 5.0 nm.

6. The red fluorescent carbon dots according to claim 1, wherein The eluate in the column layer purification process in step S2 is a mixture of dichloromethane and methanol, and the volume ratio of dichloromethane to methanol is 100:

1.

7. The red fluorescent carbon dots according to claim 1, wherein The excess hydrazine hydrate in step S11 means that the molar equivalent of the hydrazine hydrate is 50 times or more of that of fluorescein.

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