CN109021971B - Fluorescent carbon dot for cell nucleus staining and application and method thereof in cell nucleus imaging - Google Patents
Fluorescent carbon dot for cell nucleus staining and application and method thereof in cell nucleus imaging Download PDFInfo
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Abstract
A fluorescent carbon dot for cell nucleus staining, which is prepared by the following steps: 1) dissolving folic acid and m-phenylenediamine in water, and heating for reaction; 2) after reaction, centrifuging, passing through silica gel column chromatography, collecting yellow-green fluorescence part, transferring to water after rotary evaporation, and freeze-drying to obtain the final product. The carbon dots prepared by the invention emit green light under ultraviolet excitation, have larger Stokes displacement, are matched with the current commercial 405 laser at the optimal excitation position, and can be well applied to a laser confocal microscope.
Description
Technical Field
The invention belongs to the technical field of nano materials and biological imaging, and particularly relates to a fluorescent carbon dot for cell nucleus staining and application and a method thereof in cell nucleus fluorescence imaging.
Background
Cells are important carriers in life activities, and the study of cells and foreign substances can help us to understand the transportation, metabolism and toxicity of cells. The cell nucleus is particularly important for many processes of cells, such as metabolism, passage, heredity and the like. However, their study is relatively lacking with respect to the vital nature of the nucleus. Staining of the nucleus is the first step, which reveals the morphology of the nucleus, after which transport and transport of reagents to the nucleus can be studied. With the rapid development of fluorescence microscopy, fluorescent staining of cell nuclei has become extremely common due to its visualization and high resolution properties. The nuclear fluorescent dyes currently in common use have certain disadvantages, such as DAPI and Hoechst, which have the disadvantages of self-quenching and susceptibility to photobleaching, with DAPI also having certain cytotoxicity. Therefore, the development of novel fluorescent dyes capable of specifically staining cell nuclei is of great significance.
Carbon dots, a novel fluorescent nano material, have been widely reported and have extremely wide applications in the fields of cell imaging, biosensing, photoelectric materials and the like. When the carbon dots are used for cell imaging, the carbon dots are difficult to be applied to the imaging of cell nucleus due to the properties of the size, the surface charge and the like of materials, most of the carbon dots can only enter the cytoplasm, the mitochondria and other parts of cells, and long incubation time is needed. Therefore, it is of great interest to develop a fluorescent dye that stains cell nuclei rapidly and specifically.
Disclosure of Invention
The invention aims to provide a fluorescent carbon dot for nuclear staining, and also provides an application and a method of the fluorescent carbon dot in nuclear staining.
In order to realize the purpose, the invention adopts the technical scheme that:
a fluorescent carbon dot for cell nucleus staining, which is prepared by the following steps:
1) dissolving folic acid and m-phenylenediamine in water, and heating for reaction;
2) after reaction, centrifuging, taking the upper layer solution, performing silica gel column chromatography, collecting a yellow-green fluorescent part, performing rotary evaporation to remove the organic solvent, transferring the organic solvent to water, and performing freeze-drying to obtain the fluorescent material.
In the step 1), the mass ratio of folic acid to m-phenylenediamine is 1: (1-20); the heating reaction temperature is 120-240 ℃, and the reaction time is 3-12 h.
In the step 2), the mobile phase adopted by the silica gel column chromatography is the mixed solution of methanol and ethyl acetate; the freeze-drying operation comprises the following specific steps: freezing for 24-48 h at-16 ℃ and then freeze-drying for 36-48 h at-60 to-75 ℃.
The water is deionized water or ultrapure water.
The fluorescent carbon dots for cell nucleus staining are applied to cell nucleus fluorescence imaging.
The method for using the fluorescent carbon dots for nuclear staining in nuclear fluorescence imaging comprises the following steps:
A. dissolving the fluorescent carbon dots in water, and adding a culture medium to incubate with the cells;
B. after incubation, washing off the original culture medium by using a phosphate buffer saline solution with the concentration of 10-100 mM and the physiological pH value of 7.2-7.4, and replacing the original culture medium with a fresh culture medium to perform fluorescence imaging under a laser scanning confocal fluorescence microscope or directly performing fluorescence imaging of cell nuclei under the laser confocal fluorescence microscope after incubation.
In the step A, the concentration of the fluorescent carbon dots in the culture medium is 5-60 ug/mL during co-incubation, and the co-incubation time is 1-60 min.
Compared with the prior art, the invention has the following advantages:
1. the carbon dots prepared by the invention emit green light under the excitation of ultraviolet light, have larger Stokes displacement, and the optimal excitation position is matched with the existing commercial 405 laser, so that the carbon dots can be well applied to a laser confocal microscope;
2. the fluorescent carbon dots prepared by the method belong to ultra-small nano materials, still have good membrane permeability under a lower dosage, can quickly enter cell nucleuses, and carry out fluorescence imaging on the cell nucleuses of living cells; the method can be used for the conventional fluorescent dye imaging step and can also be used for the washing-free cell nucleus fluorescence imaging; the cell compatibility is high, and a good foundation is provided for biological experiments; when genetic material DNA or RNA is encountered, the fluorescence intensity can be greatly improved, and the DNA and the RNA can be selectively identified; when used for nuclear imaging, only extremely low incubation concentrations and extremely short incubation times are required, compared to the reported cell imaging carbon spots;
3. the device and the reagent adopted by the invention are cheap and simple, the cost is extremely low compared with the commercialized nuclear dye, and the invention is suitable for any research institute and large and small hospitals, and has wide popularity, social value and economic value.
Drawings
FIG. 1 is a transmission electron micrograph of a fluorescent carbon dot of the present invention;
FIG. 2 is an absorption spectrum, an excitation spectrum, and an emission spectrum under an optimal excitation of a fluorescent carbon dot according to the present invention;
FIG. 3 is a confocal micrograph of nuclei after incubation with fluorescent carbon spots according to the present invention;
FIG. 4 is a confocal micrograph of nuclei after incubation with fluorescent carbon spots according to the present invention;
FIG. 5 is a confocal micrograph of a nucleus incubated with fluorescent carbon spots taken with pbs wash and no-wash operation in accordance with the present invention;
FIG. 6 is a confocal micrograph of HeLa cells after incubation with fluorescent carbon spots and treatment with DNA and RNA digesting enzyme, respectively, in accordance with the present invention.
Detailed Description
The present invention will be further described with reference to the following detailed description.
Example 1
A fluorescent carbon dot for cell nucleus staining, which is prepared by the following steps:
1) dissolving 5mg of folic acid and 13mg of m-phenylenediamine in 5000mg of deionized water, and placing the solution in a hydrothermal reaction kettle to heat the solution to 200 ℃ for reaction for 12 hours; in other examples, the mass ratio of folic acid to m-phenylenediamine is in the range of 1: (2-20) the dosage of the deionized water can meet the requirement of dissolving the raw materials;
2) after reaction, centrifuging (removing large-particle aggregates), taking the upper-layer solution, performing silica gel column chromatography, collecting a yellow-green fluorescent part, performing rotary evaporation (removing an organic solvent), transferring the yellow-green fluorescent part into distilled water, and performing freeze-drying to obtain a fluorescent carbon dot; the mobile phase adopted when passing through the silica gel column chromatography is methanol and ethyl acetate according to the volume ratio of 1: 1; the freeze-drying operation comprises the following specific steps: freezing at-16 deg.C for 30 hr, and freeze-drying at-60 deg.C for 48 hr; in other embodiments, the freezing is performed for 24-48 h at-16 ℃ and then the freeze-drying is performed for 36-48 h at-60-75 ℃.
The method for fluorescence imaging in cell nucleus by using the fluorescent carbon dots for cell nucleus staining comprises the following steps:
A. dissolving the fluorescent carbon dots with distilled water to obtain a 1mg/mL carbon dot solution, adding an RPMI1640 culture medium to co-incubate with HeLa cells, wherein the concentration of the fluorescent carbon dots in the culture medium is 15ug/mL during incubation, and the incubation time is 10 min; in other embodiments, the concentration is 5-60 ug/mL, and the incubation time is 1-60 min;
B. after incubation, the fluorescence imaging of the cell nucleus is directly carried out under a laser confocal fluorescence microscope, and a laser with the wavelength of 405 nm is adopted during imaging. In other embodiments, after incubation, the original medium can be washed out with 10-100 mM phosphate buffered saline at physiological pH ranging from pH7.2 to 7.4 and replaced with fresh medium for fluorescence imaging under a laser scanning confocal fluorescence microscope.
It should be noted that the culture medium and cells described in the above embodiments are merely illustrative of the present invention as specific embodiments, and the scope of the present invention is not limited thereby, and the key point of the present invention lies in the application of fluorescence imaging of fluorescent carbon dots in cell nucleus, that is, in other embodiments, other cells and culture medium can be replaced, and the inventive effect can be achieved, but it should be noted that the culture medium used must be that required by the cell.
Performance testing
The fluorescent carbon dots obtained in example 1 were subjected to the relevant performance tests, and the results are shown in fig. 1 to 6.
Wherein, FIG. 1 shows a transmission electron microscope image of the fluorescent carbon dots of the present invention, and it can be seen from the image that the carbon dots obtained by the present invention are relatively uniform and ultra-small in size, the particle size is 1.6-4nm, and the average particle size is 2.4 nm.
Fig. 2 shows an absorption spectrum, an excitation spectrum and an emission spectrum under the optimal excitation of the fluorescent carbon dot of the present invention, and it can be seen from the figure that the ultraviolet absorption position is at 395 nm, the emission peak under the optimal excitation is at 535 nm, and the excitation position is matched with the existing commercial laser, so that the fluorescent carbon dot can be well used in a confocal laser microscope.
FIG. 3 shows a confocal photomicrograph of nuclear imaging after incubation with fluorescent carbon spots, in which the carbon spots can well enter the nucleus and the nucleus is subjected to a fluorescence image to well show the microstructure of the nucleus.
FIG. 4 shows a confocal micrograph of nuclei after incubation with fluorescent carbon spots according to the present invention, the three-dimensional cell result shows that the carbon spots are distributed almost only in the nuclear part of the whole cells and the nuclei are subjected to fluorescence imaging.
FIG. 5 shows confocal micrographs obtained by pbs washing and no-wash operation of nuclear imaging after incubation with fluorescent carbon spots according to the invention, demonstrating that good nuclear imaging results can be obtained with both normal and no-wash operation.
FIG. 6 shows the confocal photomicrographs of the nuclei of HeLa cells after incubation with fluorescent carbon spots and treatment with DNA and RNA digesting enzymes, respectively, the control of the digested nucleic acids further confirms the principle of the fluorescent image of the carbon spots on the nuclei: the carbon dots, upon entering the cell, bind to DNA and RNA in the nucleus and exhibit fluorescence enhancement.
Comparative test
To illustrate the effect of the invention, the m-phenylenediamine (mpd) in example 1 is replaced by its structural isomer under otherwise identical conditions: o-phenylenediamine (opd) and p-phenylenediamine (ppd) were used to prepare fluorescent carbon dots, and the resulting carbon dots were analyzed by fluorescence imaging.
The results show that fluorescence imaging of the nucleus cannot be achieved with either o-phenylenediamine (opd) or p-phenylenediamine (ppd) carbon dots, but that other different parts of the cell (e.g. cytoplasm and mitochondria) are imaged with opd.
Claims (4)
1. The application of the fluorescent carbon dots for cell nucleus staining in cell nucleus fluorescence imaging is characterized in that the fluorescent carbon dots are prepared by the following steps:
1) dissolving folic acid and m-phenylenediamine in water, and heating to react, wherein the mass ratio of the folic acid to the m-phenylenediamine is 1: 2-20; the heating reaction temperature is 120-240 ℃, and the reaction time is 3-12 h;
2) after the reaction, centrifuging to take the upper layer solution, passing through a silica gel column chromatography chromatogram, collecting a yellow-green fluorescence part, removing the organic solvent by rotary evaporation, transferring into water, and freeze-drying to obtain the fluorescent carbon dot, wherein the mobile phase adopted by passing through the silica gel column chromatography chromatogram is a mixed solution of methanol and ethyl acetate, and the specific operation of freeze-drying is as follows: freezing for 24-48 h at-16 ℃ and then freeze-drying for 36-48 h at-60 to-75 ℃.
2. The use of a fluorescent carbon dot for nuclear staining in nuclear fluorescence imaging as claimed in claim 1 wherein the water is deionized or ultrapure water.
3. The use of fluorescent carbon dots for nuclear staining in nuclear fluorescence imaging as claimed in any of claims 1-2, wherein the method for nuclear fluorescence imaging using fluorescent carbon dots for nuclear staining comprises the steps of:
A. dissolving the fluorescent carbon dots in water, and adding a culture medium to incubate with the cells;
B. after incubation, washing off the original culture medium by using a phosphate buffer saline solution with the concentration of 10-100 mM and the physiological pH value of 7.2-7.4, and replacing the original culture medium with a fresh culture medium to perform fluorescence imaging under a laser scanning confocal fluorescence microscope or directly performing fluorescence imaging of cell nuclei under the laser confocal fluorescence microscope after incubation.
4. The use of the fluorescent carbon dots for nuclear staining in nuclear fluorescence imaging as claimed in claim 3, wherein in step A, the concentration of the fluorescent carbon dots in the culture medium during co-incubation is 5-60 ug/mL, and the co-incubation time is 1-60 min.
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| CN112461807B (en) * | 2020-11-26 | 2021-11-19 | 山西大学 | Application of carbon quantum dots in targeted nucleolus wash-free imaging |
| CN113583657B (en) * | 2021-09-01 | 2022-07-19 | 郑州大学 | Cell nucleus targeting carbon dot, preparation and application |
| CN114605989B (en) * | 2022-02-28 | 2023-12-01 | 东南大学 | Green fluorescent carbon dot and preparation method and application thereof |
| CN115386370A (en) * | 2022-08-30 | 2022-11-25 | 东南大学 | Application of green fluorescent carbon dot Mis-mPD-CDs in detecting pH values of cell nuclei, microorganisms, animals and solutions |
| CN116285969A (en) * | 2022-12-19 | 2023-06-23 | 重庆国科医创科技发展有限公司 | Super-bright green carbon quantum dot fluorescent probe, preparation method thereof and application thereof in DNA imaging |
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| CN106566539A (en) * | 2016-10-21 | 2017-04-19 | 上海纳米技术及应用国家工程研究中心有限公司 | Preparation method for carbon quantum dots obtained through phenylenediamine and folic acid |
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| CN106610376A (en) * | 2015-10-27 | 2017-05-03 | 中国科学院宁波材料技术与工程研究所 | Application of fluorescent carbon dots in living cell nucleolus imaging or RNA labeling or display |
| CN106566539A (en) * | 2016-10-21 | 2017-04-19 | 上海纳米技术及应用国家工程研究中心有限公司 | Preparation method for carbon quantum dots obtained through phenylenediamine and folic acid |
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