CN109021971A - A kind of nuclear targeting fluorescent carbon point and its application and method in nuclei images - Google Patents

A kind of nuclear targeting fluorescent carbon point and its application and method in nuclei images Download PDF

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CN109021971A
CN109021971A CN201810996309.9A CN201810996309A CN109021971A CN 109021971 A CN109021971 A CN 109021971A CN 201810996309 A CN201810996309 A CN 201810996309A CN 109021971 A CN109021971 A CN 109021971A
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carbon point
fluorescent carbon
nuclear targeting
nucleus
water
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CN201810996309.9A
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CN109021971B (en
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李朝辉
刘海芳
孙远强
杨杰
屈凌波
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Zhengzhou University
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Zhengzhou University
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    • CCHEMISTRY; METALLURGY
    • C09DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
    • C09KMATERIALS FOR MISCELLANEOUS APPLICATIONS, NOT PROVIDED FOR ELSEWHERE
    • C09K11/00Luminescent, e.g. electroluminescent, chemiluminescent materials
    • C09K11/08Luminescent, e.g. electroluminescent, chemiluminescent materials containing inorganic luminescent materials
    • C09K11/65Luminescent, e.g. electroluminescent, chemiluminescent materials containing inorganic luminescent materials containing carbon
    • BPERFORMING OPERATIONS; TRANSPORTING
    • B82NANOTECHNOLOGY
    • B82YSPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y20/00Nanooptics, e.g. quantum optics or photonic crystals
    • BPERFORMING OPERATIONS; TRANSPORTING
    • B82NANOTECHNOLOGY
    • B82YSPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y40/00Manufacture or treatment of nanostructures
    • CCHEMISTRY; METALLURGY
    • C01INORGANIC CHEMISTRY
    • C01BNON-METALLIC ELEMENTS; COMPOUNDS THEREOF; METALLOIDS OR COMPOUNDS THEREOF NOT COVERED BY SUBCLASS C01C
    • C01B32/00Carbon; Compounds thereof
    • C01B32/15Nano-sized carbon materials
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64Fluorescence; Phosphorescence
    • G01N21/6486Measuring fluorescence of biological material, e.g. DNA, RNA, cells

Abstract

A kind of nuclear targeting fluorescent carbon point, the carbon dots, which are adopted, to be prepared by the following steps: 1) folic acid and m-phenylene diamine (MPD) being dissolved in water heating reaction;2) after reacting, silica gel column chromatography is crossed in centrifugation, is collected yellow-green fluorescence part, is transferred in water, is lyophilized after revolving to obtain the final product.Carbon dots prepared by the present invention emit green light under burst of ultraviolel, have biggish Stokes shift, and best excitation position and 405 existing commercial lasers coincide, and can be advantageously applied to laser confocal microscope.

Description

A kind of nuclear targeting fluorescent carbon point and its application in nuclei images and Method
Technical field
The invention belongs to nano materials and bio-imaging technical field, and in particular to a kind of nuclear targeting fluorescent carbon point And its application and method in nucleus fluorescence imaging.
Background technique
Cell is the important carrier in vital movement, us can be helped to understand cell in cell and allogene Quality Research Transport, metabolism and toxicity.Wherein nucleus is particularly important for many processes of cell, such as metabolism, passage and heredity Deng.However, studying it is opposite lack for the most important property of nucleus.Dyeing to nucleus is First step, he can show that the form of nucleus, and the transport and transhipment reagent that nucleus could be studied after this are arrived The case where nucleus.With the rapid development of fluorescence microscopy, due to its visualization and high-resolution characteristic, to the glimmering of nucleus Light dyeing just becomes extremely widespread.The nucleus fluorescent dye being commonly used now have the defects that it is certain, such as DAPI and Hoechst, they there are self-quenching and easily by photobleaching the shortcomings that, wherein there are also certain cytotoxicities by DAPI.Therefore, it develops The novel fluorescence dyestuff for capableing of specific stain nucleus has great importance.
Carbon dots as a kind of novel fluorescent nano material by wide coverage, and have extremely wide application, such as Cell imaging, bio-sensing, the fields such as photoelectric material.The carbon dots invented at present are when being used for cell imaging, due to material The properties such as size, surface charge are difficult to apply to the imaging of nucleus, and the carbon dots of most of inventions can only enter the cell of cell The positions such as matter, mitochondria, and need longer incubation time.Therefore, develop a kind of quick and specific to nucleus progress The fluorescent dye of dyeing has great importance.
Summary of the invention
Goal of the invention of the invention is to provide a kind of nuclear targeting fluorescent carbon point, while providing it in nuclei dyeing Application and method in color are another goals of the invention of the invention.
For achieving the above object, the technical scheme adopted by the invention is as follows:
A kind of nuclear targeting fluorescent carbon point, the carbon dots, which are adopted, to be prepared by the following steps:
1) folic acid and m-phenylene diamine (MPD) are dissolved in water heating reaction;
2) after reacting, centrifuging and taking upper solution crosses silica gel column chromatography, collects yellow-green fluorescence part, then to rotate removal organic It is transferred in water, is lyophilized after solvent to obtain the final product.
In step 1), the mass ratio of the folic acid and m-phenylene diamine (MPD) is 1:(1~20);Heating reaction temperature be 120~ 240 DEG C, the reaction time is 3~12h.
In step 2, the mobile phase that mistake silica gel column chromatography uses is the mixed liquor of methanol and ethyl acetate;Freeze-drying Concrete operations are as follows: prior to -16 DEG C 24~48h of freezing, after in -60~-75 DEG C of 36~48h of freeze-drying.
The water is deionized water or ultrapure water.
Application of the nuclear targeting fluorescent carbon point in nucleus fluorescence imaging.
The method that the nuclear targeting is used for nucleus fluorescence imaging with fluorescent carbon point, comprising the following steps:
A. fluorescent carbon point is dissolved in water, culture medium is added to be incubated for altogether with cell;
B. after being incubated for, former culture medium is washed off with the phosphate buffered saline solution of 10~100 mM, physiological pH range pH7.2-7.4, and It changes directly total in laser after fresh culture medium carries out fluorescence imaging or is incubated under laser scanning co-focusing fluorescence microscope The fluorescence imaging of nucleus is carried out under confocal fluorescence microscope.
In step A, altogether be incubated for when culture medium in fluorescent carbon point concentration be 5~60ug/mL, altogether incubation time be 1~ 60min。
Compared with prior art, the invention has the following advantages that
1, carbon dots prepared by the present invention emit green light under ultraviolet excitation, there is biggish Stokes shift, and best excitation Position and 405 existing commercial lasers coincide, and can be advantageously applied to laser confocal microscope;
2, extra small nano material is belonged to using fluorescent carbon point prepared by the method for the present invention, still there is good film to seep under lower dosage Permeability can be rapidly introduced into nucleus, and carry out fluorescence imaging to the nucleus of living cells;It can be not only used for conventional glimmering Photoinitiator dye image-forming step, it can also be used to disposable nucleus fluorescence imaging;Cell compatibility with higher, mentions for Bioexperiment Excellent basis is supplied;When encountering hereditary material DNA or RNA, fluorescence intensity can be largely increased, the property of can choose Identify DNA and RNA;Compared to the cell imaging carbon dots having been reported that, when being used for nuclei images, it is only necessary to extremely low incubation Concentration and extremely short incubation time;
3, the device and reagent that the present invention uses are cheap, simple, extremely low compared to cost for the nucleus dyestuff of commercialization, fit Together in using in any Research Center in one's power large and small type hospital, there are very extensive popularization and society and economy value.
Detailed description of the invention
Fig. 1 is the transmission electron microscope picture of fluorescent carbon point of the present invention;
Fig. 2 is the absorption spectrum of fluorescent carbon point of the present invention, excitation spectrum, and the emission spectrum under best excitation;
Fig. 3 is that the nuclei images after the present invention is incubated for fluorescent carbon point are copolymerized burnt microphoto;
Fig. 4 is that the nucleus three-dimensional imaging after the present invention is incubated for fluorescent carbon point is copolymerized burnt microphoto;
Fig. 5 is that the copolymerization coke that the nuclei images pbs after the present invention is incubated for fluorescent carbon point is washed and disposable operation obtains is micro- Photo;
Fig. 6 is after the present invention is incubated for fluorescent carbon point, and thin with DNA and RNA zymolase treated HeLa cell respectively Karyon is copolymerized burnt microphoto.
Specific embodiment
Using specific embodiment, the present invention will be further described below.
Embodiment 1
A kind of nuclear targeting fluorescent carbon point, the carbon dots, which are adopted, to be prepared by the following steps:
1) 5mg folic acid and 13mg m-phenylene diamine (MPD) are dissolved in the deionized water of 5000mg, are placed in hydrothermal reaction kettle and are heated to 200 DEG C of reaction 12h;In other embodiments, the mass ratio of folic acid and m-phenylene diamine (MPD) is in 1:(2-20), the use of deionized water Amount meets dissolution of raw material;
2) after reacting, centrifugation (removal bulky grain agglutinator) takes upper solution to cross silica gel column chromatography, collects yellow-green fluorescence Part is transferred in distilled water after rotating (removal organic solvent), is lyophilized up to fluorescent carbon point;It is adopted when crossing silica gel column chromatography Mobile phase is the mixed liquor of methanol and ethyl acetate 1:1 by volume;The concrete operations of freeze-drying are as follows: freezed prior to -16 DEG C 30h, after in -60 DEG C of freeze-drying 48h;In other embodiments, prior to -16 DEG C 24 ~ 48h of freezing, after in -60 ~ 75 DEG C freeze Dry 36 ~ 48h.
Utilize the method for nuclear targeting fluorescent carbon point fluorescence imaging in nucleus, comprising the following steps:
A. first add distilled water to dissolve to obtain the carbon dots solution of 1mg/mL fluorescent carbon point, add RPMI1640 culture medium and HeLa is thin Born of the same parents are incubated for altogether, and the concentration of fluorescent carbon point is 15ug/mL in culture medium when incubation, and incubation time is 10 min;In other embodiments In, concentration is 5~60ug/mL, and incubation time is 1~60min;
B. after being incubated for, the fluorescence imaging of nucleus is carried out directly under laser confocal fluorescence microscope, and wavelength is used when imaging For the laser of 405 nm.In other embodiments, after incubation, the phosphorus of 10-100 mM, physiological pH range pH7.2-7.4 can be used Acid buffering salting liquid washes off former culture medium, and change fresh culture medium carried out under laser scanning co-focusing fluorescence microscope it is glimmering Light imaging.
It should be pointed out that culture medium described in above-described embodiment and cell are only as specific embodiment to this hair The explanation of bright progress, can not limit the scope of protection of the present invention according to this, and key point of the invention is fluorescent carbon point thin The application of fluorescence imaging in karyon, that is, in other embodiments, also can be replaced other cells and culture medium, it equally can be real Existing invention effect, but need to be intended that, the culture medium of use must be required for the cell.
Performance test
Correlated performance test is carried out to the fluorescent carbon point that embodiment 1 obtains, shown in the result is shown in Figure 1-Fig. 6.
Wherein, Fig. 1 show the transmission electron microscope picture of fluorescent carbon point of the present invention, it can be seen from the figure that the present invention obtained Carbon dots are more uniform super-small, and partial size is in 1.6-4nm, average grain diameter 2.4nm.
Fig. 2 show the absorption spectrum, excitation spectrum and the emission spectrum under best excitation of fluorescent carbon point of the present invention, It can be seen from the figure that UV absorption position is located at 395 nm, the lower emission peak of best excitation is located at 535 nm, excite position with Existing commercialization laser extremely matches, and can be good at for laser confocal microscope.
Fig. 3 show the nuclei images after the present invention is incubated for fluorescent carbon point and is copolymerized burnt microphoto, and cytological map is shown Carbon dots can enter nucleus well out, and carry out fluoroscopic image to nucleus, show the microstructure of nucleus well.
Fig. 4 show the nucleus three-dimensional imaging after the present invention is incubated for fluorescent carbon point and is copolymerized burnt microphoto, three-dimensional thin Born of the same parents go out carbon dots as the result is shown and are almost only distributed in nuclear fractions in entire cell, and have carried out fluorescence shadow to nucleus Picture.
Fig. 5 show the nuclei images pbs washing after the present invention is incubated for fluorescent carbon point and is total to what disposable operation obtained Focus microphoto, it was demonstrated that normal operating and disposable operation can obtain good nuclei images result.
Fig. 6 is shown after the present invention is incubated for fluorescent carbon point, and respectively with DNA and RNA zymolase treated HeLa The nucleus of cell is copolymerized burnt microphoto, and the control for digesting nucleic acid further demonstrates carbon dots and carries out fluoroscopic image to nucleus Principle: it is DNA and RNA in nucleus that carbon dots, which enter after cell combine, and shows the result of fluorescence enhancement.
Comparative test
In order to illustrate invention effect, other conditions are identical, and it is different with dividing to change the m-phenylene diamine (MPD) (mpd) in embodiment 1 into its structure Structure body: o-phenylenediamine (opd) and p-phenylenediamine (ppd) prepare fluorescent carbon point, and carry out fluorescence imaging analysis to obtained carbon dots.
The results show that cannot achieve using the carbon dots that o-phenylenediamine (opd) and p-phenylenediamine (ppd) obtain to nucleus Fluorescence imaging, but shown using opd to the imaging of other different parts (such as cytoplasm and mitochondria) of cell.

Claims (7)

1. a kind of nuclear targeting fluorescent carbon point, which is characterized in that the carbon dots, which are adopted, to be prepared by the following steps:
1) folic acid and m-phenylene diamine (MPD) are dissolved in water heating reaction;
2) after reacting, centrifuging and taking upper solution crosses silica gel column chromatography, collects yellow-green fluorescence part, then to rotate removal organic It is transferred in water, is lyophilized after solvent to obtain the final product.
2. nuclear targeting fluorescent carbon point as described in claim 1, which is characterized in that in step 1), the folic acid and The mass ratio of phenylenediamine is 1:(1~20);The temperature of heating reaction is 120~240 DEG C, and the reaction time is 3~12h.
3. nuclear targeting fluorescent carbon point as described in claim 1, which is characterized in that in step 2, cross silica gel column layer The mobile phase that analysis chromatography uses is methanol and ethyl acetate mixtures;The concrete operations of freeze-drying are as follows: prior to -16 DEG C freezings 24~ 48h, after in -60~-75 DEG C of 36~48h of freeze-drying.
4. nuclear targeting fluorescent carbon point as described in claim 1, which is characterized in that the water is deionized water or ultrapure Water.
5. application of any nuclear targeting fluorescent carbon point of claim 1-4 in nucleus fluorescence imaging.
6. the method for utilizing any nuclear targeting of claim 1-4 to be used for nucleus fluorescence imaging with fluorescent carbon point, Characterized by comprising the following steps:
A. fluorescent carbon point is dissolved in water, culture medium is added to be incubated for altogether with cell;
B. after being incubated for, former culture medium is washed off with the phosphate buffered saline solution of 10~100mM, physiological pH range pH7.2-7.4, and change Upper fresh culture medium is directly copolymerized in laser after carrying out fluorescence imaging under laser scanning co-focusing fluorescence microscope or being incubated for The fluorescence imaging of nucleus is carried out under burnt fluorescence microscope.
7. special such as the method that claim 6 utilizes the nuclear targeting to be used for nucleus fluorescence imaging with fluorescent carbon point Sign is, in step A, altogether be incubated for when culture medium in fluorescent carbon point concentration be 5~60ug/mL, altogether incubation time be 1~ 60min。
CN201810996309.9A 2018-08-29 2018-08-29 Fluorescent carbon dot for cell nucleus staining and application and method thereof in cell nucleus imaging Active CN109021971B (en)

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CN113583657A (en) * 2021-09-01 2021-11-02 郑州大学 Cell nucleus targeting carbon dot, preparation and application
CN114605989A (en) * 2022-02-28 2022-06-10 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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Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN112461807A (en) * 2020-11-26 2021-03-09 山西大学 Application of carbon quantum dots in targeted nucleolus wash-free imaging
CN112461807B (en) * 2020-11-26 2021-11-19 山西大学 Application of carbon quantum dots in targeted nucleolus wash-free imaging
CN113583657A (en) * 2021-09-01 2021-11-02 郑州大学 Cell nucleus targeting carbon dot, preparation and application
CN113583657B (en) * 2021-09-01 2022-07-19 郑州大学 Cell nucleus targeting carbon dot, preparation and application
CN114605989A (en) * 2022-02-28 2022-06-10 东南大学 Green fluorescent carbon dot and preparation method and application thereof
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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