CN114736675A - Carbon quantum dot two-photon fluorescent dye for ultrafast cell staining - Google Patents
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Abstract
The invention discloses a carbon quantum dot two-photon fluorescent dye for ultrafast cell dyeing, which is prepared by the following method: 1) preparing a carbon quantum solution by using m-phenylenediamine as a precursor through a one-step solvothermal method; 2) separating a green fluorescent component from the carbon quantum prepared in the step 1) by adopting a silica gel column chromatography, namely the carbon quantum dot two-photon fluorescent dye. The carbon quantum dot two-photon fluorescent dye for ultrafast cell staining provided by the invention has the absolute quantum efficiency as high as 58.65%, shows good biocompatibility and also shows excellent imaging effect even at low concentration; it can enter cells in 10 seconds, which provides a solid foundation for rapid fluorescence imaging; and it can also be used for two-photon imaging, using near-infrared excitation light source, the G-CDs also show strong green fluorescence and show excellent imaging performance in 4T1 cells.
Description
Technical Field
The invention relates to the field of nano materials, in particular to a carbon quantum dot two-photon fluorescent dye for ultrafast cell dyeing.
Background
Biological imaging is an important research means for understanding the tissue structure of an organism and elucidating various physiological functions of the organism. Biological imaging has been widely used in scientific research and biomedical diagnosis fields due to its advantages of high sensitivity, high resolution, intuitive imaging, fast imaging speed, nondestructive testing, etc. The biological imaging also has important practical application value in the aspects of exploring pathogenesis, clinical manifestation and genetic lesion of diseases, understanding corresponding physiological and pathological information, diagnosing diseases, developing new medical methods and the like. Currently, biological imaging includes fluorescence imaging, Magnetic Resonance Imaging (MRI), Computed Tomography (CT), Emission Computed Tomography (ECT), photothermal imaging (PTI), Raman Imaging (RI), ultrasound imaging (USI), and photoacoustic imaging (PAI). Among them, fluorescence imaging has higher signal intensity, longer duration, lower experimental cost, and feasibility of imaging from in vivo to ex vivo, and thus has been widely used.
Among the reported fluorescent labeling molecules, carbon quantum dots (CDs) have attracted great attention due to their inherent advantages of easy preparation, unique optical properties, excellent light stability, and good biocompatibility. Thus, CD is widely used as a novel fluorescent probe in cell imaging. However, the current CDs imaging has the disadvantages of low absolute quantum efficiency, long fluorescence imaging time, incapability of two-photon imaging and the like. Low absolute quantum efficiency means that if a high CDs concentration is required to obtain a clear and bright image, this will directly affect cell viability. Furthermore, most cellular imaging using CDs requires 1-24 hours, and the cellular environment changes rapidly, which makes rapid imaging and detection of intracellular activity difficult. In addition, single photon fluorescence imaging has the characteristics of low spatial distribution, poor tissue permeability, shallow imaging depth and the like. To meet the spatial and temporal resolution required for tissue depth imaging and better imaging performance, two-photon imaging (TP) using CDs seems to be a good approach to solve this problem. Two-photon absorption/excitation refers to a process in which a molecule absorbs two photons simultaneously under strong light excitation, and transits from a ground state to an excited state in which the photon energy is twice. In two-photon fluorescence imaging, absorbed near-infrared light causes ultraviolet light emission. The long-wave infrared light is not easy to scatter by cells, penetrates into the sample and can be used for detecting thicker samples. In addition, long wave light sources cause less optical damage to the organism. The absorbed long wavelength photon has twice the wavelength of the single photon excitation light of the fluorescent molecule. Thus, fluorescent molecules requiring single photon uv excitation can be excited by two photons in the near infrared and even infrared wavelength ranges. Therefore, the two-photon technique can significantly reduce phototoxicity in the detection of living samples. But now lack a reliable two-photon fluorescent dye.
Disclosure of Invention
The invention aims to solve the technical problem of providing a carbon quantum dot two-photon fluorescent dye for ultrafast cell dyeing aiming at the defects in the prior art.
In order to solve the technical problems, the invention adopts the technical scheme that: a carbon quantum dot two-photon fluorescent dye for ultra-fast cell staining is prepared by the following steps:
1) preparing a carbon quantum solution by using m-phenylenediamine as a precursor through a one-step solvothermal method;
2) separating a green fluorescent component from the carbon quantum prepared in the step 1) by adopting silica gel column chromatography, namely the carbon quantum dot two-photon fluorescent dye.
Preferably, the step 1) includes: adding m-phenylenediamine into absolute ethyl alcohol, then adding hydrochloric acid, carrying out hydrothermal treatment on the obtained mixture in a high-pressure kettle, cooling to room temperature after the reaction is finished, centrifuging the product, and removing the precipitate to obtain a carbon quantum solution.
Preferably, the mass concentration of the hydrochloric acid added in the step 1) is as follows: 36 to 38 percent.
Preferably, the mass concentration of the hydrochloric acid added in the step 1) is as follows: 36.5 percent.
Preferably, the autoclave in step 1) is a stainless steel autoclave lined with polytetrafluoroethylene.
Preferably, in the step 1), the temperature of the hydrothermal treatment is 180 ℃.
Preferably, the step 1) includes: adding 0.2g of m-phenylenediamine into 20mL of absolute ethyl alcohol, then adding 672 mu L of hydrochloric acid, carrying out hydrothermal treatment on the obtained mixture in a stainless steel autoclave with a polytetrafluoroethylene lining at 180 ℃ for 2 hours, cooling to room temperature after the reaction is finished, centrifuging the product at 10000rpm for 20 minutes, and removing the precipitate to obtain a carbon quantum solution.
Preferably, the step 2) includes: purifying the carbon quantum prepared in the step 1) by silica gel column chromatography using a mixture of dichloromethane and methanol as an eluent, selecting a green fluorescent component, then evaporating the solvent and drying under vacuum, and collecting solid powder, namely the carbon quantum dot two-photon fluorescent dye.
Preferably, the volume ratio of dichloromethane to methanol in the eluent in the step 2) is 10: 1.
Preferably, the carbon quantum dot two-photon fluorescent dye is used as a fluorescent dye for fluorescence imaging and two-photon imaging.
The beneficial effects of the invention are: the carbon quantum dot two-photon fluorescent dye for ultra-fast cell staining provided by the invention has the absolute quantum efficiency as high as 58.65%, shows good biocompatibility and also shows excellent imaging effect even at low concentration; most importantly, the carbon quantum dot two-photon fluorescent dye can enter cells within 10 seconds, which provides a solid foundation for rapid fluorescence imaging; and it can also be used for two-photon imaging, using a near infrared (920nm) excitation light source, the G-CDs also exhibit strong green fluorescence and show excellent imaging performance in 4T1 cells; the carbon quantum dot two-photon fluorescent dye synthesized by the invention can provide an excellent biological imaging tool for early diagnosis and clinical treatment.
Drawings
FIG. 1 is a transmission electron micrograph of a carbon quantum dot two-photon fluorescent dye prepared in example 1 of the present invention;
FIG. 2 is an X-ray diffraction pattern of a carbon quantum dot two-photon fluorescent dye prepared in example 1 of the present invention;
FIG. 3 is a graph of the UV absorption spectrum and the fluorescence spectrum of the carbon quantum dot two-photon fluorescent dye prepared in example 1 of the present invention;
FIG. 4 is an infrared spectrum of a carbon quantum dot two-photon fluorescent dye prepared in example 1 of the present invention;
FIG. 5 is an X-ray photoelectron spectrum of a carbon quantum dot two-photon fluorescent dye prepared in example 1 of the present invention;
FIG. 6 shows the spectra of the carbon quantum dot two-photon fluorescent dye prepared in example 1 of the present invention under different excitation lights;
FIG. 7 is a full spectrum scan of a carbon quantum dot two-photon fluorescent dye prepared in example 1 of the present invention;
FIG. 8 is a CIE chromaticity diagram of a carbon quantum dot two-photon fluorescent dye prepared in example 1 of the present invention;
FIG. 9 is a result of a pH stability test of a carbon quantum dot two-photon fluorescent dye prepared in example 1 of the present invention;
FIG. 10 shows the results of an ion interference test of a carbon quantum dot two-photon fluorescent dye prepared in example 1 of the present invention;
FIG. 11 shows cytotoxicity test results of a carbon quantum dot two-photon fluorescent dye prepared in example 1 of the present invention;
FIG. 12 shows fluorescence imaging results of the carbon quantum dot two-photon fluorescent dye prepared in example 1 of the present invention in 4T1 cell (A) and HeLa cell (B) cultured for 10 seconds, 1 minute, 10 minutes and 180 minutes;
FIG. 13 shows the results of carbon quantum dot two-photon fluorescent dye prepared in example 1 of the present invention counterstaining two cells with DAPI;
fig. 14 shows the two-photon imaging effect of the carbon quantum dot two-photon fluorescent dye prepared in example 1 of the present invention in 4T1 cells.
Detailed Description
The present invention is further described in detail below with reference to examples so that those skilled in the art can practice the invention with reference to the description.
It will be understood that terms such as "having," "including," and "comprising," as used herein, do not preclude the presence or addition of one or more other elements or groups thereof.
Example 1
The carbon quantum dot two-photon fluorescent dye for ultrafast cell staining of the embodiment is prepared by the following method:
1) preparing a carbon quantum solution by using m-phenylenediamine as a precursor through a one-step solvothermal method:
adding 0.2g of m-phenylenediamine into 20mL of absolute ethyl alcohol, then adding 672 mu L of concentrated hydrochloric acid (mass concentration: 36.5%), carrying out hydrothermal treatment on the obtained mixture in a stainless steel autoclave with a polytetrafluoroethylene lining at 180 ℃ for 2 hours, cooling to room temperature after the reaction is finished, centrifuging the product at 10000rpm for 20 minutes, and removing non-fluorescent precipitate to obtain a carbon quantum solution;
2) separating a green fluorescent component from the carbon quantum prepared in the step 1) by adopting a silica gel column chromatography, namely the carbon quantum dot two-photon fluorescent dye:
a mixture of dichloromethane and methanol was used as eluent (CH)2Cl2/CH3OH, 10:1v/v), purifying the carbon quantum prepared in the step 1) through silica gel column chromatography, selecting a strong green fluorescent component, then evaporating a solvent and drying under vacuum, and collecting solid powder, namely the carbon quantum dot two-photon fluorescent dye.
The invention synthesizes strong green fluorescent carbon quantum dots (G-CDs) by using m-phenylenediamine (m-PD) and concentrated hydrochloric acid through a one-step method: namely, the carbon quantum dot two-photon fluorescent dye forms G-CDs with the absolute quantum efficiency as high as 58.65%, shows good biocompatibility and also shows excellent imaging effect even at low concentration; most importantly, G-CDs can enter cells in 10 seconds, which provides a solid foundation for rapid fluorescence imaging; G-CDs can also be used for two-photon imaging. The G-CDs also exhibited intense green fluorescence using a near infrared (920nm) excitation light source and showed excellent imaging performance in 4T1 cells. The G-CDs of the invention can provide excellent biological imaging tools for early diagnosis and clinical treatment.
Example 2
The carbon quantum dot two-photon fluorescent dye prepared in example 1 was subjected to a correlation performance test, and the following results were obtained.
1. Referring to fig. 1, which is a Transmission Electron Microscope (TEM) photograph of the carbon quantum dot two-photon fluorescent dye, it can be seen that the carbon dots are spherical, the crystal lattice is obvious, and the lattice spacing is 0.21 nm.
2. Referring to fig. 2, which is an X-ray diffraction pattern of the carbon quantum dot two-photon fluorescent dye, it can be seen that the X-ray diffraction 2 θ angle of the carbon dot is 22 °, which coincides with TEM.
3. Referring to fig. 3, it is a graph of the uv absorption spectrum and the fluorescence spectrum of the carbon quantum dot two-photon fluorescent dye, which can be seen from the graph, has a maximum absorption band at 248nm, which represents pi-pi transition of aromatic C ═ C bond; the weak absorption peak at about 300nm is due to N-pi electron transition, indicating the presence of C ═ N and C ═ O on the surface of the two-photon fluorescent dye at the carbon quantum dot; in addition, the fluorescence emission peak is located at 500nm, and the corresponding excitation peak is located at 460 nm.
4. Referring to fig. 4, the infrared spectrum of the carbon quantum dot two-photon fluorescent dye is analyzed for the transmission peak intensity, and the surface of the dye is rich in hydroxyl, amino and other groups; is located at 3324cm-1The characteristic absorption bands around are due to-NH2Caused by stretching vibration of the radicals; located at 2935cm-1The absorption peak of (a) is due to C-H bond stretching vibration; at 1619cm-1The peak of (a) is caused by stretching vibration belonging to C ═ C; is positioned at 1508cm-1Is considered to belong to the group of-NO2Telescopic vibration of the key; at 1331cm-1The absorption peak of (A) is due to the C-N-C group stretching vibration; these groups enhance the hydrophilicity and stability of the carbon quantum dot two-photon fluorescent dye.
5. Referring to fig. 5, it can be seen that the X-ray photoelectron spectrum of the carbon quantum dot two-photon fluorescent dye comprises three elements, namely, carbon, nitrogen and oxygen, which account for 71.92%, 14.10% and 13.97% respectively; further proves that the surface of the carbon quantum dot two-photon fluorescent dye has rich amino, hydroxyl and other groups.
6. Referring to fig. 6, it can be seen that the carbon dots exhibit excitation-independent characteristics for the spectra of the carbon quantum dot two-photon fluorescent dye under different excitation lights;
7. referring to fig. 7, a full spectrum scan of the two-photon fluorescent dye for the carbon quantum dots can determine the light emitting area of the carbon dots;
8. referring to fig. 8, the CIE chromaticity diagram of the carbon quantum dot two-photon fluorescent dye shows that the coordinates of the CIE chromaticity diagram are (0.169, 0.502), and in summary, the carbon dot is an excitation independent carbon dot and emits green fluorescence.
9. Referring to fig. 9, it can be seen from the pH stability test results of the carbon quantum dot two-photon fluorescent dye that the light intensity is hardly affected in the range of pH4.0 to 9.0.
10. Referring to fig. 10, it can be seen that the carbon quantum dot two-photon fluorescent dye shows good stability to common metal ions and some amino acids, and the light intensity is hardly affected, for the ion interference test result of the carbon quantum dot two-photon fluorescent dye.
11. Referring to FIG. 11, the cytotoxicity test results of the carbon quantum dot two-photon fluorescent dye in this example were evaluated by WST-1 cell proliferation and cytotoxicity test kit, and 40. mu.g mL of the carbon dot cytotoxicity test kit was added-1The viability of the cells was hardly affected at all with the carbon quantum dot two-photon fluorescent dye of (1), which demonstrates that there is hardly any toxicity at this concentration; after adding 100. mu.g mL of-1After the carbon quantum dot two-photon fluorescent dye is used, the cell viability is slightly reduced but still more than 80%. The carbon quantum dot two-photon fluorescent dye is proved to have low toxicity and good biocompatibility.
12. Referring to FIG. 12, the carbon quantum dot two-photon fluorescent dye was applied to 4T1 cells (A)) Fluorescence imaging results at 10 seconds, 1 minute, 10 minutes and 180 minutes of incubation with HeLa cells (B) were 40. mu.g mL of carbon quantum dot two-photon fluorescent dye-1The scale bar is 20 μm. The carbon quantum dot two-photon fluorescent dye can enter cells within 10 seconds, so that clear and rapid imaging can be realized; considering that most of conventional carbon dots need to be incubated for 1-24 hours for cell imaging, the imaging capability of the carbon quantum dot two-photon fluorescent dye in 10s remarkably accelerates the process, and is helpful for timely researching the physiological state of cells. In addition, the cell imaging of the carbon quantum dot two-photon fluorescent dye of the invention has little dependence on the culture time. From time gradient cell imaging experiments of two types of cells, the embodiment determines that the fluorescence intensity under a 1min cell fluorescence imaging field is not obviously different from the fluorescence intensities of 10min and 180min, and all imaging results are clear and bright. The main reasons are that: the carbon quantum dot two-photon fluorescent dye has small particle size, extremely high absolute quantum efficiency and low cytotoxicity, can penetrate cells in a short time, and shows strong green fluorescence, so the carbon quantum dot two-photon fluorescent dye has huge application potential in the aspects of short-term and long-term imaging.
13. Referring to FIG. 13, the results of the carbon quantum dot two-photon fluorescent dye and DAPI counterstaining of two cells were shown, and the concentration of the carbon quantum dot two-photon fluorescent dye was 40. mu.g mL-1The scale bar is 20 μm. In this example, the carbon quantum dot two-photon fluorescent dye (40. mu.g mL)-1) After incubating with 4T1 cells and HeLa cells for 60min, counterstaining with DAPI, distinguishing carbon dots from DAPI with green and blue, respectively, and finally obtaining the confocal picture shown in FIG. 13. As can be seen from fig. 13, there is a large overlap of the green and blue regions. Analyzing the fluorescence co-localization result by using two software packages, namely Color2 and JACoP, and obtaining Pearson coefficients of 13(A) and 13(B) which are respectively 0.80 and 0.84 by using Color2 software; using JACoP software, Pearson coefficients of 0.803 and 0.838, respectively, and overlap coefficients of 0.962 and 0.994, respectively, were obtained for 13(a) and 13 (B). In conclusion, the carbon quantum dot two-photon fluorescent dye has stronger target nucleic acid characteristics, and the application of the carbon quantum dot two-photon fluorescent dye in the biomedical field is widened.
14. Referring to fig. 14, for the two-photon imaging effect of the carbon quantum dot two-photon fluorescent dye in 4T1 cells, in this embodiment, light with a wavelength of 920nm is used to excite the carbon quantum dot two-photon fluorescent dye, so as to obtain a bright and clear imaging result in 4T1 cells, and break through the limitations of low spatial distribution rate and poor tissue permeability of single-photon fluorescence imaging. The carbon quantum dot two-photon fluorescent dye prepared by the invention can show clear green fluorescence under the excitation conditions of single photon and two photons, and provides a powerful tool for the fields of fluorescence imaging, tissue depth imaging, space high-resolution imaging and the like.
While embodiments of the invention have been disclosed above, it is not limited to the applications listed in the description and the embodiments, which are fully applicable in all kinds of fields of application of the invention, and further modifications may readily be effected by those skilled in the art, so that the invention is not limited to the specific details without departing from the general concept defined by the claims and the scope of equivalents.
Claims (10)
1. A carbon quantum dot two-photon fluorescent dye for ultra-fast cell staining is prepared by the following steps:
1) preparing a carbon quantum solution by using m-phenylenediamine as a precursor through a one-step solvothermal method;
2) separating a green fluorescent component from the carbon quantum prepared in the step 1) by adopting a silica gel column chromatography, namely the carbon quantum dot two-photon fluorescent dye.
2. The carbon quantum dot two-photon fluorescent dye for ultrafast cell staining of claim 2, wherein the step 1) comprises: adding m-phenylenediamine into absolute ethyl alcohol, then adding hydrochloric acid, carrying out hydrothermal treatment on the obtained mixture in a high-pressure kettle, cooling to room temperature after the reaction is finished, centrifuging the product, and removing the precipitate to obtain a carbon quantum solution.
3. The carbon quantum dot two-photon fluorescent dye for ultrafast cell staining of claim 3, wherein the mass concentration of the hydrochloric acid added in the step 1) is: 36 to 38 percent.
4. The carbon quantum dot two-photon fluorescent dye for ultrafast cell staining according to claim 4, wherein the hydrochloric acid added in the step 1) has a mass concentration of: 36.5 percent.
5. The carbon quantum dot two-photon fluorescent dye for ultrafast cell staining of claim 2, wherein the autoclave in the step 1) is a stainless steel autoclave lined with polytetrafluoroethylene.
6. The carbon quantum dot two-photon fluorescent dye for ultrafast cell staining of claim 2, wherein the temperature of the hydrothermal treatment in the step 1) is 180 ℃.
7. The carbon quantum dot two-photon fluorescent dye for ultrafast cell staining of claim 2, wherein the step 1) comprises: adding 0.2g of m-phenylenediamine into 20mL of absolute ethyl alcohol, then adding 672 mu L of hydrochloric acid, carrying out hydrothermal treatment on the obtained mixture in a stainless steel autoclave with a polytetrafluoroethylene lining at 180 ℃ for 2 hours, cooling to room temperature after the reaction is finished, centrifuging the product at 10000rpm for 20 minutes, and removing the precipitate to obtain a carbon quantum solution.
8. The carbon quantum dot two-photon fluorescent dye for ultrafast cell staining of claim 1, wherein the step 2) comprises: purifying the carbon quantum prepared in the step 1) by silica gel column chromatography using a mixture of dichloromethane and methanol as an eluent, selecting a green fluorescent component, then evaporating the solvent and drying under vacuum, and collecting solid powder, namely the carbon quantum dot two-photon fluorescent dye.
9. The carbon quantum dot two-photon fluorescent dye for ultrafast cell staining of claim 8, wherein the volume ratio of dichloromethane and methanol in the eluent in the step 2) is 10: 1.
10. The carbon quantum dot two-photon fluorescent dye for ultrafast cell staining according to any one of claims 1 to 9, which is applied as a fluorescent dye for fluorescence imaging and two-photon imaging.
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