WO2022027174A1 - Cell nucleus fluorescent dye and dyeing method therefor - Google Patents

Cell nucleus fluorescent dye and dyeing method therefor Download PDF

Info

Publication number
WO2022027174A1
WO2022027174A1 PCT/CN2020/106563 CN2020106563W WO2022027174A1 WO 2022027174 A1 WO2022027174 A1 WO 2022027174A1 CN 2020106563 W CN2020106563 W CN 2020106563W WO 2022027174 A1 WO2022027174 A1 WO 2022027174A1
Authority
WO
WIPO (PCT)
Prior art keywords
fluorescent dye
nuclear
staining
group
cells
Prior art date
Application number
PCT/CN2020/106563
Other languages
French (fr)
Chinese (zh)
Inventor
龚萍
周理华
张鹏飞
蔡林涛
Original Assignee
深圳先进技术研究院
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 深圳先进技术研究院 filed Critical 深圳先进技术研究院
Priority to PCT/CN2020/106563 priority Critical patent/WO2022027174A1/en
Publication of WO2022027174A1 publication Critical patent/WO2022027174A1/en

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C211/00—Compounds containing amino groups bound to a carbon skeleton
    • C07C211/62—Quaternary ammonium compounds
    • C07C211/64—Quaternary ammonium compounds having quaternised nitrogen atoms bound to carbon atoms of six-membered aromatic rings
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N1/00—Sampling; Preparing specimens for investigation
    • G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
    • G01N1/30—Staining; Impregnating ; Fixation; Dehydration; Multistep processes for preparing samples of tissue, cell or nucleic acid material and the like for analysis

Definitions

  • the invention relates to the technical field of fluorescence imaging, in particular to a nuclear fluorescent dye and a dyeing method thereof.
  • Bioimaging generally refers to methods of visualizing and recording target molecules or processes in cells, tissues, or the body.
  • Biological imaging technology includes fluorescence imaging technology, Raman imaging technology, nuclear magnetic resonance technology, photoacoustic imaging technology, ultrasonic imaging technology, X-ray imaging technology and positron emission tomography technology.
  • fluorescence imaging techniques including confocal microscopy, two-photon microscopy, and super-resolution microscopy, have won extensive research interest due to their low damage to samples, excellent sensitivity, and high spatial-temporal resolution.
  • many luminescent bioprobes have been designed and applied in biomolecular detection, cell imaging, bacterial imaging, cell tracking, vascular imaging, in vivo tumor imaging and therapy, etc.
  • a cell is the smallest unit of life, and it mainly contains organelles such as cell membrane, nucleus, mitochondria, lysosome, endoplasmic reticulum, and Golgi apparatus.
  • organelles such as cell membrane, nucleus, mitochondria, lysosome, endoplasmic reticulum, and Golgi apparatus.
  • the function of these organelles is closely related to the health of the cell, and their loss or abnormality often leads to human diseases. Therefore, visual observation of the structural and functional changes of these organelles is of great significance for analyzing the causes of diseases and guiding drug development.
  • the nucleus is where most of the genetic material in the cell is stored, and it controls the metabolism, growth, and differentiation of the cell.
  • ASCP a probe that produces corresponding fluorescence color changes to changes in the intracellular microenvironment.
  • ASCP has specific selective ability for mitochondria and nucleolus of cells, showing yellow and red fluorescence, respectively.
  • Intracellular mitochondria and nucleoli can be imaged using different fluorescence acquisition channels.
  • MitoTracker green and SYTO RNASelect ASCP exhibits significantly improved photostability and little cytotoxicity. Enhancing the number of positive charges in the molecule helps improve the entry of the molecule into the nuclear region.
  • the nuclear localization signal is a signal peptide, generally consisting of four to eight amino acids, that helps nucleophile proteins enter cells.
  • the probe consists of two targeting peptides, a cell penetrating peptide and a nuclear localization signal peptide and a yellow aggregation-inducing molecule.
  • the probe With the help of the nuclear localization signal peptide, after the probe enters the cell, it finally reaches the nuclear region to light up the nucleus.
  • the probe has low cytotoxicity, can stably exist in the nuclear region and can be used for long-term tracking observation of the nucleus, and the effect is better than the commercial nuclear blue dye Hoechst 33258.
  • some research groups Based on the commonly used copper-catalyzed click method, some research groups have also developed aggregation-induced luminescence fluorescent reagents that can be used to detect DNA synthesis in S phase.
  • the commonly used fluorescent dyes for cell nucleus include the following categories: acridine orange, ethidium bromide and propidium iodide, DAPI, Hoechst dye, EthD III, 7-AAD, RedDot1, RedDot2, etc.
  • the commonly used nuclear fluorescent dyes on the market, the dyes permeable to the membrane are as follows:
  • Acridine orange It has membrane permeability, can penetrate the cell membrane, and stain nuclear DNA and RNA into green and red, respectively, so that the nucleus is green or yellow-green fluorescence.
  • Ethidium bromide a highly sensitive fluorescent dye that excites an orange-red signal at the standard 302nm.
  • DAPI a blue fluorescent dye that can penetrate the cell membrane. It can generate fluorescence more than 20 times stronger than DAPI itself when combined with DNA, but has no fluorescence enhancement when combined with single-stranded DNA.
  • Hoechst dyes a class of fluorescent dyes that label DNA in microscopic observation, the two most common are Hoechst33342 and Hoechst33258. Both dyes are excited at UV 350 nm and emit cyan/blue fluorescence near the emission maximum at 461 nm.
  • Hoechst33342 has an ethyl group added, which has stronger lipophilicity, so it can better penetrate the intact cell membrane and has less cytotoxicity.
  • RedDot 1 Dye Superior nuclear selectivity. RedDot1 dyes can be excited by several common lasers and can fluoresce in the far infrared. The red near-infrared fluorescence of RedDot1 effectively distinguishes it from other commonly used fluorescent probes.
  • Membrane-impermeable dyes such as: PI propidium iodide: cannot pass through living cell membranes, but can pass through dead cell membranes to stain nuclei.
  • PI is the first choice as a red fluorescent counterstain. PI is often used in combination with fluorescent probes such as Calcein-AM or FDA to distinguish dead/live cells. EthD III, 7-AAD, RedDot 2: can not penetrate the living cell membrane, but can distinguish necrotic cells; more suitable for the detection of apoptosis and necrosis experiments.
  • the optimal excitation wavelength of traditional nuclear-targeting dyes such as DAPI series and Hoechst series is only about 360 nm, which cannot be effectively excited by existing commercial lasers, and the light energy of this wavelength is high, which not only has high phototoxicity to cells, but also At the same time, it is easy to cause photobleaching of dyes. This is because after the dye is excited to the excited state by light, it will enter the triplet state by means of intersystem crossing. The molecules in the triplet state are easily attacked by oxygen molecules in the air, resulting in the destruction of the dye structure, thus isolating the dye molecules from oxygen. Photobleaching can be effectively avoided, such as imaging experiments in nitrogen atmosphere, fluorophore wrapping with cucurbituril, etc. However, there are some compounds with good properties that have been used in other fields for a long time, but they have not been found to be applied in nuclear fluorescence staining and have excellent effects.
  • the present invention provides a nuclear fluorescent dye and a dyeing method and application thereof.
  • the nuclear fluorescent dye of the invention has good membrane permeability, high sensitivity, fast nuclear dyeing efficiency, good photostability, good biocompatibility, longer wavelength excitation light and emission light, and can be applied to cell nucleus fluorescent staining.
  • R 1 is C0-C30 linear or branched alkylene
  • R 2 is C0-C30 linear or branched substituted alkylene
  • R 3 is C5-C30 aryl or substituted aryl
  • R 4 is any one of C5-C30 alkylene aryl, substituted alkylene aryl, arylene alkyl, substituted arylene alkyl
  • R 5 is unsaturated alkyl, heteroalkyl , cycloalkyl, heterocycloalkyl, aryl, heteroaryl, carboxyl, amino, sulfonic acid.
  • nuclear fluorescent dye has the following general structural formula:
  • R 1 -R 5 The main part of the general structural formula including R 1 -R 5 is positively charged, and the positive charge is important for its nuclear staining function, and R 6 is the negative ion part of the compound, which is negatively charged, and the whole compound is regarded as a salt.
  • R 1 is C0-C30 linear or branched alkylene
  • R 2 is C0-C30 linear or branched substituted alkylene
  • R 3 is C5-C30 aryl or substituted aryl
  • R 4 is any one of C5-C30 alkylene aryl, substituted alkylene aryl, arylene alkyl, substituted arylene alkyl
  • R 5 is unsaturated alkyl, heteroalkyl , cycloalkyl, heterocycloalkyl, aryl, heteroaryl, carboxyl, amino, sulfonic acid group
  • R 6 is PF 6 - , BF 4 - , SbF 5 - , CH 3 COO - , CF 3 COO - , CO 3 2- , SO 4 2- , SO 3 2- , CF 3 SO 2 - , TsO - , ClO 4 - , F - , Cl - , Br - , I - , (F
  • nuclear fluorescent dye has the following structural formula:
  • Compound (III) is a nuclear fluorescent dye specifically used in the examples of the present invention.
  • the present invention also provides a method for dyeing the cell nucleus fluorescent dye, which can dye the cell nucleus with color, and the cell nucleus fluorescent dye is used.
  • the dyeing method of the nuclear fluorescent dye includes the following steps:
  • the staining solution completely covers the cells or tissues to be stained, and incubate for 1 to 10 minutes.
  • the staining solution is a buffer solution containing the nuclear fluorescent dye.
  • the concentration of the nuclear fluorescent dye in the staining solution is 0.01-10 mg/mL.
  • the step of directly observing under a fluorescence microscope or observing under a fluorescence confocal microscope after mounting is included.
  • the detection wavelength of the fluorescence microscope or fluorescence confocal microscope is 600-900 nm.
  • the cells or tissues to be stained include living cells or cultured tissues, fixed cells or tissues.
  • the fixative should be removed before mixing with the staining solution.
  • step of immunofluorescence staining may be included between the step of removing the fixative and the step of mixing with the staining solution.
  • the present invention achieves the following technical effects:
  • the present invention provides a new application of nuclear fluorescent dyes, and expands the types of nuclear fluorescent dyes.
  • the nuclear fluorescent dye involved in the present invention has good membrane permeability, high sensitivity, fast nuclear dyeing efficiency, good photostability, good biocompatibility, longer wavelength excitation light and emission light .
  • the nuclear fluorescent dye provided by the present invention is a kind of aggregation-induced luminescent dye, which has the advantages of more aggregation and brighter, the dyeing procedure method is simple, no washing is required, and it is easy to operate.
  • Fig. 1 shows the ultraviolet absorption spectrum of the nuclear fluorescent dye of the present invention
  • Fig. 2 shows the fluorescence emission spectrum of the nuclear fluorescent dye of the present invention bound to DNA
  • Figure 3 shows the absorption and emission spectra of Hoechst33342 dye
  • Fig. 4 is a diagram showing the effect of staining the nucleus with the nuclear fluorescent dye of the present invention by confocal microscopy. From left to right are Hoechst 33342, the nuclear fluorescent dye of the present invention, the co-staining stack of Hoechst 33342 and the nuclear fluorescent dye of the present invention, and Hoechst 33342. Co-staining of 33342 and the nuclear fluorescent dye of the present invention and the staining effect of bright field superposition;
  • Fig. 5 is a graph showing the effect of confocal microscope observation of the nuclear fluorescent dye of the present invention staining cell nucleus at a low concentration
  • Fig. 6 shows the fluorescence spectrum of free DNA detected by the micro-nucleus fluorescent dye of the present invention
  • Fig. 7 shows the absorption spectrum diagram of long-time laser irradiation of the nuclear fluorescent dye of the present invention
  • FIG. 8 shows the result of staining of MCF-7 living cells with the nuclear fluorescent dye of the present invention
  • Figure 9 shows the result of staining COS-7 dead cells with the nuclear fluorescent dye of the present invention.
  • Fig. 10 is a graph showing the results of nuclear staining of onion epidermal cells with the nuclear fluorescent dye of the present invention.
  • Fluorescent dyes emit fluorescence, and the so-called fluorescence refers to the fluorescence of visible light with longer wavelengths emitted by substance molecules after absorbing visible light with shorter wavelengths.
  • Membrane permeability The ease with which substances pass through a biomembrane. In the present invention, it refers specifically to the degree of difficulty of the fluorescent dyes permeating the cell membrane.
  • Sensitivity The effect of staining with low concentrations of fluorescent dyes. Low concentrations of fluorescent dyes also have a good nuclear effect, that is, high sensitivity.
  • Nuclear staining efficiency The amount of time a fluorescent dye takes to stain cells. Fast staining means high nuclear efficiency.
  • Light stability Long-term laser irradiation has no effect on the absorption of fluorescent dyes, indicating that its light stability is good.
  • Biocompatibility refers to the ability of cells to react to inactive materials. After the fluorescent dye enters the cell, it will have an impact and effect on the cell, and the cell will also have an impact and effect on the fluorescent dye. Cycling continues until equilibrium is reached.
  • Excitation wavelength is to excite fluorescence with a certain wavelength of light, which can be ultraviolet light or visible light or other light.
  • the excitation wavelength has a significant effect on stray light and signal-to-noise ratio.
  • Emission wavelength The wavelength of fluorescence emitted by a certain light, the wavelength of general visible light can be roughly judged by the naked eye.
  • Excitation light and emission light with longer wavelengths The excitation light and emission light have long wavelengths, so they have peaks with a certain bandwidth. In practical applications, the excitation wavelength range is smaller than the emission wavelength, and the excitation wavelength is longer to obtain a high excitation rate The material form can indirectly improve the sensitivity, and the longer emission wavelength can directly improve the sensitivity, so that a good signal-to-noise ratio can be obtained.
  • a kind of nuclear fluorescent dye provided by the present invention has the following general structural formula:
  • R 1 is C0-C30 linear or branched alkylene
  • R 2 is C0-C30 linear or branched substituted alkylene
  • R 3 is C5-C30 aryl or substituted aryl
  • R 4 is any one of C5-C30 alkylene aryl, substituted alkylene aryl, arylene alkyl, substituted arylene alkyl
  • R 5 is unsaturated alkyl, heteroalkyl , cycloalkyl, heterocycloalkyl, aryl, heteroaryl, carboxyl, amino, sulfonic acid.
  • nuclear fluorescent dye has the following general structural formula:
  • the main part containing R 1 -R 5 in the general structural formula is positively charged, and the positive charge is of great significance to its nuclear staining function.
  • the positively charged compound can be combined with the negatively charged anion part, and after the combination, the Nuclei are better stained.
  • R 1 is C0-C30 linear or branched alkylene
  • R 2 is C0-C30 linear or branched substituted alkylene
  • R 3 is C5-C30 aryl or substituted aryl
  • R 4 is any one of C5-C30 alkylene aryl, substituted alkylene aryl, arylene alkyl, substituted arylene alkyl
  • R 5 is unsaturated alkyl, heteroalkyl , cycloalkyl, heterocycloalkyl, aryl, heteroaryl, carboxyl, amino, sulfonic acid group
  • R 6 is the negative ion part of the compound, negatively charged, the whole compound is taken as a salt
  • R 6 is PF 6 - , BF 4 - , SbF 5 - , CH 3 COO - , CF 3 COO - , CO 3 2- , SO 4 2- , SO 3 2- , CF 3 SO 2 - , TsO - , ClO
  • nuclear fluorescent dye has the following structural formula:
  • the nuclear fluorescent dye of the present invention can not only stain living cells or cultured tissues, but also can stain fixed cells or tissues, and has different staining methods for living cells and dead cells:
  • staining solution For adherent cells or tissue sections, add a small amount of staining solution to cover the sample.
  • concentration of nuclear fluorescent dye in the staining solution is 0.01-10 mg/mL, diluted with buffer solution.
  • For suspension cells add at least 3 times the volume of the sample to be stained with staining solution and mix well. Place at room temperature for 3-5min.
  • staining solution Add an appropriate amount of staining solution, which must fully cover the sample to be stained. Usually, 1 mL of staining solution should be added to one well of a 6-well plate, and 100 ⁇ L of staining solution should be added to one well of a 96-well plate. The concentration of nuclear fluorescent dye in the staining solution is 0.01-10 mg/mL, diluted with buffer solution.
  • the application of the nuclear fluorescent dye provided by the present invention and the reagents or instruments used in the method for staining the nucleus can be purchased from the market.
  • the present invention will be further described below with reference to the examples.
  • the compound structure of the nuclear fluorescent dye used in the examples is the above-mentioned structural formula (III).
  • the dyeing method is:
  • the preparation stock solution is 1 mg/mL of the PBS buffer solution of the nuclear fluorescent dye of the present invention.
  • the staining solution of the nuclear fluorescent dye of the present invention at a concentration of 100 ⁇ g/mL to cover the sample.
  • For suspension cells add at least 3 times the volume of the sample to be stained with staining solution and mix well. Place at room temperature for 3-5min. Without washing, observe directly under a fluorescence microscope or under a fluorescence confocal microscope after mounting.
  • staining solution of the nuclear fluorescent dye of the present invention with a concentration of 100 ⁇ g/mL, which must fully cover the sample to be stained.
  • 1 mL of staining solution should be added to one well of a 6-well plate, and 100 ⁇ L should be added to one well of a 96-well plate. staining solution.
  • Test Example 1 Absorption spectrum and emission spectrum detection of the nuclear fluorescent dye of the present invention
  • an ultraviolet-visible spectrometer is used to detect the absorption spectrum of the nuclear fluorescent dye of the present invention.
  • the absorption light wavelength range of the nuclear fluorescent dye of the present invention is 250-850 nm, and the general nuclear dye absorbs light.
  • the wavelength range is 250-450nm.
  • the fluorescence emission spectrum of the nuclear fluorescent dye of the present invention combined with DNA was detected by a fluorescence spectrometer.
  • the fluorescence emission wavelength range of the nuclear fluorescent dye of the present invention combined with DNA is 600-900 nm. The range is 300-600nm.
  • Figure 3 shows the spectrum of the traditional nuclear dye Hoechst33342, with the absorption spectrum on the left and the fluorescence emission spectrum on the right.
  • the spectrum of traditional dyes is blue-shifted, while the spectrum of the nuclear fluorescent dye of the present invention is red-shifted.
  • the optimal excitation wavelength of Hoechst33342 is blue, and the excitation wavelength is short, which cannot be effectively excited by existing commercial lasers, and the light energy of this wavelength is high, which not only has high phototoxicity to cells, but also easily causes photobleaching of dyes.
  • the nuclear fluorescent dye of the present invention overcomes this shortcoming well, and will not cause serious cell damage during imaging.
  • the PBS buffer of the nuclear fluorescent dye of the present invention with a concentration of 1 mg/mL in the mother solution was added to the MCF-7 cells, so that the final concentration of the nuclear fluorescent dye in the cell culture medium was 100 ⁇ g/mL, and placed at room temperature for 5min , followed by fluorescence confocal imaging, and the nuclei were treated with the commercial nuclear dye Hoechst33342.
  • the dyeing effect is shown in Figure 4.
  • the test results show: from left to right, the first picture is the nuclear staining effect of Hoechst33342, the second picture is the nuclear staining effect of the nuclear fluorescent dye of the present invention, and the third picture is Hoechst and the nuclear fluorescence of the present invention.
  • the effect of dye co-staining nuclear effect is the superposition of the nuclear effect and bright field of the two. It can be seen from the figure that the nuclear staining effect of the nuclear fluorescent dye of the present invention and the nuclear staining effect of the commercial nuclear staining dye Hoechst33342 are superimposed very obviously, and the overlap is highly consistent. It shows that the nuclear fluorescent dye of the present invention has the same or even better nuclear staining effect compared with commercial nuclear staining dyes.
  • the PBS buffer of the nuclear fluorescent dye of the present invention with a concentration of 1 mg/mL in the stock solution was added to the MCF-7 cells, so that the final concentration of the nuclear fluorescent dye in the cell culture medium was 5 ⁇ g/ml, compared with 100 ⁇ g
  • the concentration of the working solution/mL was diluted by 20 times and placed at room temperature for 5 min, followed by fluorescence confocal imaging. The staining effect is shown in Figure 5.
  • test results show that the nuclei of MCF-7 cells exhibit red fluorescence, the cell structure is complete, the fluorescence edge is clear and the brightness is high, indicating that the very low concentration of the nuclear fluorescent dye of the present invention also has a very good nuclear staining effect.
  • the concentration of which is 1, 2, 5, 10, 20, 50 ⁇ M, respectively binds to free DNA, and it can be seen that there is a good fluorescence peak shape and linearity, as shown in Figure 6, It shows that the nuclear fluorescent dye of the present invention has high sensitivity.
  • the results of measuring the absorption spectrum of the nuclear fluorescent dye of the present invention show that the optical The stability test is shown in Figure 7, the abscissa is the wavelength, and the ordinate is the UV absorption value.
  • Test Example 5 The results of staining of living MCF-7 cells with the nuclear fluorescent dye of the present invention
  • MCF-7 Staining of live cells for MCF-7. After the MCF-7 cells were cultured, they were stained with the PBS buffer solution of the nuclear fluorescent dye of the present invention with a concentration of 1 mg/mL, so that the final concentration of the nuclear fluorescent dye in the cell culture medium was 5 ⁇ g/mL, and placed at room temperature for 5 minutes, and then fluorescence was carried out. Confocal imaging.
  • the leftmost column is the fluorescence channel
  • the nucleus shows red fluorescence
  • the middle column is the brightfield channel
  • the rightmost column is the superposition of the fluorescence channel and the brightfield channel
  • the lower row is a partial enlarged view of the upper row.
  • Test Example 6 The result of staining of COS-7 dead cells with the nuclear fluorescent dye of the present invention
  • COS-7 cells were cultured, they were fixed with paraformaldehyde, and then stained with the PBS buffer solution of the nuclear fluorescent dye of the present invention with a concentration of 1 mg/mL, so that the final concentration of the nuclear fluorescent dye in the cell culture medium was 5 ⁇ g/ml, at room temperature. After standing for 5 min, fluorescence confocal imaging was performed immediately.
  • Test Example 7 The results of nuclear staining of onion epidermal cells with the nuclear fluorescent dye of the present invention
  • the steps of taking onion epidermal cells for nuclear staining with the nuclear fluorescent dye of the present invention are as follows:
  • the test results are shown in Figure 10.
  • the nuclei of the onion epidermal cells are clearly dyed red, which is completely consistent with the position of the nuclei observed in the bright field, indicating that the nuclear fluorescent dye of the present invention can be applied not only to animal cells or tissues, but also to plant cells. Or the same applies to organizations.

Landscapes

  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Biomedical Technology (AREA)
  • Physics & Mathematics (AREA)
  • Molecular Biology (AREA)
  • Analytical Chemistry (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • General Physics & Mathematics (AREA)
  • Immunology (AREA)
  • Pathology (AREA)
  • Engineering & Computer Science (AREA)
  • Investigating Or Analysing Biological Materials (AREA)

Abstract

Provided are a cell nucleus fluorescent dye, a staining method thereof and use thereof. A novel fluorescent dye for cell nucleus staining is proposed. The involved cell nucleus fluorescent dye has good membrane permeability, high sensitivity, rapid nuclear dyeing efficiency, good light stability, good biocompatibility, and excitation light and emission light with longer wavelengths, and can be applied to cell nucleus fluorescent dyeing.

Description

一种细胞核荧光染料及其染色方法A kind of nuclear fluorescent dye and dyeing method thereof 技术领域technical field
本发明涉及荧光成像技术领域，特别涉及一种细胞核荧光染料及其染色方法。The invention relates to the technical field of fluorescence imaging, in particular to a nuclear fluorescent dye and a dyeing method thereof.
背景技术Background technique
生物成像通常指对细胞、组织或体内的目标分子或过程进行可视化和记录的方法。生物成像技术包含荧光成像技术、拉曼成像技术、核磁共振技术、光声成像技术、超声成像技术、X射线照相技术和正电子发射断层扫描技术等。其中，包含共聚焦显微镜、双光子显微镜和超分辨显微镜在内的荧光成像技术以其对样品的低损伤、出色的灵敏度和很高的空间-时间分辨率赢得了广泛的研究兴趣。在生物成像中，针对不同的生物分子和生物过程，许多发光生物探针被设计并应用于生物分子检测、细胞成像、细菌成像、细胞追踪、血管成像、体内肿瘤成像与治疗等方面。Bioimaging generally refers to methods of visualizing and recording target molecules or processes in cells, tissues, or the body. Biological imaging technology includes fluorescence imaging technology, Raman imaging technology, nuclear magnetic resonance technology, photoacoustic imaging technology, ultrasonic imaging technology, X-ray imaging technology and positron emission tomography technology. Among them, fluorescence imaging techniques, including confocal microscopy, two-photon microscopy, and super-resolution microscopy, have won extensive research interest due to their low damage to samples, excellent sensitivity, and high spatial-temporal resolution. In bioimaging, for different biomolecules and biological processes, many luminescent bioprobes have been designed and applied in biomolecular detection, cell imaging, bacterial imaging, cell tracking, vascular imaging, in vivo tumor imaging and therapy, etc.
细胞是组成生命的最小单位，细胞内主要含有细胞膜、细胞核、线粒体、溶酶体、内质网、高尔基体等细胞器。这些细胞器的功能和细胞的健康状态息息相关，它们的功能缺失或异常通常会导致人类的疾病。因此，可视化观察这些细胞器结构和功能变化对分析疾病的诱因和指导药物开发具有重要意义。考虑到聚集诱导发光探针的低背景、高亮度、高光稳定性的特点，科学家已经开发了靶向到生物分子、脂滴、线粒体、细胞核、细胞膜、溶酶体、细胞内的酶、细胞内的理化性质(pH值、黏度)等的发光探针。A cell is the smallest unit of life, and it mainly contains organelles such as cell membrane, nucleus, mitochondria, lysosome, endoplasmic reticulum, and Golgi apparatus. The function of these organelles is closely related to the health of the cell, and their loss or abnormality often leads to human diseases. Therefore, visual observation of the structural and functional changes of these organelles is of great significance for analyzing the causes of diseases and guiding drug development. Considering the characteristics of low background, high brightness, and high photostability of aggregation-induced luminescent probes, scientists have developed targets targeting biomolecules, lipid droplets, mitochondria, nuclei, cell membranes, lysosomes, intracellular enzymes, intracellular Luminescent probes for physicochemical properties (pH value, viscosity), etc.
细胞核是细胞中大多数遗传物质贮存的地方，它控制着细胞的代谢、生长、分化。通过简单的偶联反应和强D-π-A电子推拉结构，有研究组制备了对细胞内的微环境变化产生相应的荧光颜色改变的探针(ASCP)。 ASCP对细胞的线粒体和核仁具有特异的选择能力，分别表现出黄色和红色荧光。采用不同的荧光采集通道分别可以对细胞内的线粒体和核仁进行成像。与目前商业化探针MitoTracker green和SYTO RNASelect相比，ASCP的光稳定性显著提高，几乎没有细胞毒性。增强分子中正电荷数目，有助于提高分子进入核区。最近，有报道显示具有三个吡啶盐取代的三苯胺衍生物可以选择性地靶向到细胞的核区，而具有两个或一个吡啶盐的三苯胺衍生物对核区的靶向性较差。核定位信号是一种信号肽，一般由四个至八个氨基酸组成，可以帮助亲核蛋白进入细胞。此外，还有利用核定位信号设计的靶向细胞核区的黄色聚集诱导发光探针。该探针由两个靶向多肽，细胞穿透多肽和核定位信号肽和黄色的聚集诱导分子组成。在核定位信号肽的帮助下，探针进入细胞后，最终到达细胞核区点亮细胞核。实验还表明该探针的细胞毒性低、可以稳定地存在于核区并应用于细胞核的长期追踪观察，效果优于商业细胞核蓝色染料Hoechst 33258。基于常用的铜催化点击方法，有研究组还开发出了可用来检测S期DNA合成的聚集诱导发光荧光试剂。利用具有叠氮基团的聚集诱导分子TPE-Py-N3和Cy-Py-N3与参与DNA合成的5-乙炔基-2'-脱氧尿苷的乙炔的生物正交的点击反应，可实现对S期的DNA合成进行检测，对细胞的增殖能力进行评估。实验结果还表明，AIE染料与已经商业化的Alexa647-azide相比，具有更好的光稳定性和更高的灵敏度。The nucleus is where most of the genetic material in the cell is stored, and it controls the metabolism, growth, and differentiation of the cell. Through a simple coupling reaction and a strong D-π-A electronic push-pull structure, some research groups have prepared a probe (ASCP) that produces corresponding fluorescence color changes to changes in the intracellular microenvironment. ASCP has specific selective ability for mitochondria and nucleolus of cells, showing yellow and red fluorescence, respectively. Intracellular mitochondria and nucleoli can be imaged using different fluorescence acquisition channels. Compared with the current commercial probes MitoTracker green and SYTO RNASelect, ASCP exhibits significantly improved photostability and little cytotoxicity. Enhancing the number of positive charges in the molecule helps improve the entry of the molecule into the nuclear region. Recently, it has been reported that triphenylamine derivatives with three pyridinium salt substitutions can selectively target the nuclear region of cells, while triphenylamine derivatives with two or one pyridinium salt are less targeted to the nuclear region . The nuclear localization signal is a signal peptide, generally consisting of four to eight amino acids, that helps nucleophile proteins enter cells. In addition, there are yellow aggregation-induced luminescent probes designed to target the nuclear region using nuclear localization signals. The probe consists of two targeting peptides, a cell penetrating peptide and a nuclear localization signal peptide and a yellow aggregation-inducing molecule. With the help of the nuclear localization signal peptide, after the probe enters the cell, it finally reaches the nuclear region to light up the nucleus. Experiments also show that the probe has low cytotoxicity, can stably exist in the nuclear region and can be used for long-term tracking observation of the nucleus, and the effect is better than the commercial nuclear blue dye Hoechst 33258. Based on the commonly used copper-catalyzed click method, some research groups have also developed aggregation-induced luminescence fluorescent reagents that can be used to detect DNA synthesis in S phase. Utilizing the bioorthogonal click reaction of the aggregation-inducing molecules TPE-Py-N3 and Cy-Py-N3 with azide groups and acetylene of 5-ethynyl-2'-deoxyuridine involved in DNA synthesis, the DNA synthesis in S phase was detected and the proliferative capacity of the cells was assessed. The experimental results also show that the AIE dye has better photostability and higher sensitivity than the commercialized Alexa647-azide.
目前细胞核常用荧光染料有以下几大类：吖啶橙、溴化乙锭和碘化丙啶，DAPI、Hoechst染料、EthD III、7-AAD、RedDot1、RedDot2等。目前市场上常用的细胞核荧光染料，透膜的染料如下：At present, the commonly used fluorescent dyes for cell nucleus include the following categories: acridine orange, ethidium bromide and propidium iodide, DAPI, Hoechst dye, EthD III, 7-AAD, RedDot1, RedDot2, etc. At present, the commonly used nuclear fluorescent dyes on the market, the dyes permeable to the membrane are as follows:
1，吖啶橙：具有膜通透性，能透过细胞膜，将核DNA和RNA分别染成绿色和红色，因此使细胞核呈绿色或黄绿色荧光。2，溴化乙锭：一种高度灵敏的荧光染色剂，在标准302nm处激发出橙红色信号。3，DAPI：一种可以穿透细胞膜的蓝色荧光染料，其与DNA结合后可以产生比DAPI自 身强20多倍的荧光，而与单链DNA结合无荧光的增强。DAPI对双链DNA的染色灵敏度要高于EB和碘化丙啶(PI)，荧光强度比Hoechst低，但光稳定性高于Hoechst。4，Hoechst染料：一类在显微观察中标记DNA的荧光染料，最常见的两种是Hoechst33342，Hoechst33258。这两种染料都在紫外350nm处被激发，在461nm处最大发射光附近发射青/蓝色荧光。与DAPI相比，Hoechst33342加有乙基，具有更强的亲脂性，因此能更好的透过完整的细胞膜，并且细胞毒性更小。RedDot 1染料：超强的细胞核选择性。RedDot1染料可被几种常见的激光激发并可在远红外区激发荧光。RedDot1的红色近红外荧光有效的与其他常用荧光探针区分开来。不透膜的染料，如下：PI碘化丙啶：不能通过活细胞膜，但却能穿过死细胞膜而对核染色。PI作为红色荧光复染剂首选，PI经常与Calcein-AM或者FDA等荧光探针合用，区分死/活细胞。EthD III、7-AAD、RedDot 2：不能透过活细胞膜，但能将坏死细胞区分开来；更适合凋亡坏死实验的检测。1. Acridine orange: It has membrane permeability, can penetrate the cell membrane, and stain nuclear DNA and RNA into green and red, respectively, so that the nucleus is green or yellow-green fluorescence. 2. Ethidium bromide: a highly sensitive fluorescent dye that excites an orange-red signal at the standard 302nm. 3. DAPI: a blue fluorescent dye that can penetrate the cell membrane. It can generate fluorescence more than 20 times stronger than DAPI itself when combined with DNA, but has no fluorescence enhancement when combined with single-stranded DNA. The sensitivity of DAPI to double-stranded DNA was higher than that of EB and propidium iodide (PI), the fluorescence intensity was lower than that of Hoechst, but the photostability was higher than that of Hoechst. 4. Hoechst dyes: a class of fluorescent dyes that label DNA in microscopic observation, the two most common are Hoechst33342 and Hoechst33258. Both dyes are excited at UV 350 nm and emit cyan/blue fluorescence near the emission maximum at 461 nm. Compared with DAPI, Hoechst33342 has an ethyl group added, which has stronger lipophilicity, so it can better penetrate the intact cell membrane and has less cytotoxicity. RedDot 1 Dye: Superior nuclear selectivity. RedDot1 dyes can be excited by several common lasers and can fluoresce in the far infrared. The red near-infrared fluorescence of RedDot1 effectively distinguishes it from other commonly used fluorescent probes. Membrane-impermeable dyes, such as: PI propidium iodide: cannot pass through living cell membranes, but can pass through dead cell membranes to stain nuclei. PI is the first choice as a red fluorescent counterstain. PI is often used in combination with fluorescent probes such as Calcein-AM or FDA to distinguish dead/live cells. EthD III, 7-AAD, RedDot 2: can not penetrate the living cell membrane, but can distinguish necrotic cells; more suitable for the detection of apoptosis and necrosis experiments.
DAPI系列、Hoechst系列等传统靶向细胞核的染料的最佳激发波长仅为360nm左右，无法被现有的商业化激光器有效激发，且该波长的光能量较高，不仅细胞的光毒性较大，同时也容易造成染料的光漂白现象。这是由于染料被光激发到激发态后，会通过系间窜越的方式进入三线态，处于三线态的分子容易受到空气中氧分子的进攻致使染料结构被破坏，因而将染料分子与氧气隔离可以有效地避免光漂白现象，例如在氮气氛围中进行成像实验、用葫芦脲包裹荧光团等。然而，有一些性能良好的化合物长期应用于其他领域，但是并未曾发现它们可以应用在细胞核荧光染色方面且具有优良的效果。The optimal excitation wavelength of traditional nuclear-targeting dyes such as DAPI series and Hoechst series is only about 360 nm, which cannot be effectively excited by existing commercial lasers, and the light energy of this wavelength is high, which not only has high phototoxicity to cells, but also At the same time, it is easy to cause photobleaching of dyes. This is because after the dye is excited to the excited state by light, it will enter the triplet state by means of intersystem crossing. The molecules in the triplet state are easily attacked by oxygen molecules in the air, resulting in the destruction of the dye structure, thus isolating the dye molecules from oxygen. Photobleaching can be effectively avoided, such as imaging experiments in nitrogen atmosphere, fluorophore wrapping with cucurbituril, etc. However, there are some compounds with good properties that have been used in other fields for a long time, but they have not been found to be applied in nuclear fluorescence staining and have excellent effects.
性能优良细胞核荧光染料应具有好的膜通透性，高灵敏度，快速的染核效率，良好的光稳定性，良好的生物相容性细胞毒性更小，波长更长的激发光和发射光等。仔细分析目前国内外市场上常用的细胞核荧光染料，满足以上要素的化合物体系为数不多，且有些染色程序方法复杂，需要洗 涤，不便于操作。探究新的优良的细胞核荧光染料是目前研究人员的共识，便于进一步深层研究细胞核内的生物机制。Excellent performance Nuclear fluorescent dyes should have good membrane permeability, high sensitivity, fast nuclear staining efficiency, good photostability, good biocompatibility, less cytotoxicity, longer wavelength excitation light and emission light, etc. . After careful analysis of the nuclear fluorescent dyes commonly used in the domestic and foreign markets, there are not many compound systems that meet the above requirements, and some of the dyeing procedures are complicated, require washing, and are inconvenient to operate. It is the consensus of researchers to explore new and excellent nuclear fluorescent dyes, which is convenient for further in-depth study of biological mechanisms in the nucleus.
发明内容SUMMARY OF THE INVENTION
针对现有技术中的缺陷，本发明提出了一种细胞核荧光染料及其染色方法和应用。本发明的细胞核荧光染料具有良好的膜通透性，高灵敏度，快速的染核效率，良好的光稳定性，良好的生物相容性，波长更长的激发光和发射光，可以应用于细胞核的荧光染色。Aiming at the defects in the prior art, the present invention provides a nuclear fluorescent dye and a dyeing method and application thereof. The nuclear fluorescent dye of the invention has good membrane permeability, high sensitivity, fast nuclear dyeing efficiency, good photostability, good biocompatibility, longer wavelength excitation light and emission light, and can be applied to cell nucleus fluorescent staining.
为了实现上述发明目的，本发明提供以下技术方案：In order to achieve the above-mentioned purpose of the invention, the present invention provides the following technical solutions:
一种细胞核荧光染料，具有如下结构通式：A nuclear fluorescent dye with the following general structural formula:
Figure PCTCN2020106563-appb-000001
Figure PCTCN2020106563-appb-000001
其中，R 1为C0-C30的直链或支链的亚烷基，R 2为C0-C30的直链或支链的取代亚烷基，R 3为C5-C30的芳香基或取代芳香基，R 4为C5-C30的亚烷基芳香基、取代亚烷基芳香基、亚芳香基烷基、取代亚芳香基烷基中的任一种；R 5为不饱和烷基、杂烷基、环烷基、杂环烷基、芳基、杂芳基、羧基、氨基、磺酸基。 Wherein, R 1 is C0-C30 linear or branched alkylene, R 2 is C0-C30 linear or branched substituted alkylene, R 3 is C5-C30 aryl or substituted aryl , R 4 is any one of C5-C30 alkylene aryl, substituted alkylene aryl, arylene alkyl, substituted arylene alkyl; R 5 is unsaturated alkyl, heteroalkyl , cycloalkyl, heterocycloalkyl, aryl, heteroaryl, carboxyl, amino, sulfonic acid.
更进一步的，所述的细胞核荧光染料，具有如下结构通式：Further, the nuclear fluorescent dye has the following general structural formula:
Figure PCTCN2020106563-appb-000002
Figure PCTCN2020106563-appb-000002
所述的结构通式中包含R 1-R 5的主体部分带正电，带正电对于其细胞核染色功能有重要意义，R 6为化合物的负离子部分，带负电，把整个化合物作为一个盐。其中，R 1为C0-C30的直链或支链的亚烷基，R 2为C0-C30的直链或支链的取代亚烷基，R 3为C5-C30的芳香基或取代芳香基，R 4为C5-C30的亚烷基芳香基、取代亚烷基芳香基、亚芳香基烷基、取代亚芳香基烷基中的任一种；R 5为不饱和烷基、杂烷基、环烷基、杂环烷基、芳基、杂芳基、羧基、氨基、磺酸基；R 6为PF 6 -、BF 4 -、SbF 5 -、CH 3COO -、CF 3COO -、CO 3 2-、SO 4 2-、SO 3 2-、CF 3SO 2 -、TsO -、ClO 4 -、F -、Cl -、Br -、I -、(F 3CSO 2)N -、PO 4 3-中的任一种。 The main part of the general structural formula including R 1 -R 5 is positively charged, and the positive charge is important for its nuclear staining function, and R 6 is the negative ion part of the compound, which is negatively charged, and the whole compound is regarded as a salt. Wherein, R 1 is C0-C30 linear or branched alkylene, R 2 is C0-C30 linear or branched substituted alkylene, R 3 is C5-C30 aryl or substituted aryl , R 4 is any one of C5-C30 alkylene aryl, substituted alkylene aryl, arylene alkyl, substituted arylene alkyl; R 5 is unsaturated alkyl, heteroalkyl , cycloalkyl, heterocycloalkyl, aryl, heteroaryl, carboxyl, amino, sulfonic acid group; R 6 is PF 6 - , BF 4 - , SbF 5 - , CH 3 COO - , CF 3 COO - , CO 3 2- , SO 4 2- , SO 3 2- , CF 3 SO 2 - , TsO - , ClO 4 - , F - , Cl - , Br - , I - , (F 3 CSO 2 )N - , PO Any of 4 3- .
更进一步的，所述的细胞核荧光染料，具有如下结构式：Further, the nuclear fluorescent dye has the following structural formula:
Figure PCTCN2020106563-appb-000003
Figure PCTCN2020106563-appb-000003
化合物(Ⅲ)为本发明实施例中具体采用的细胞核荧光染料。Compound (III) is a nuclear fluorescent dye specifically used in the examples of the present invention.
本发明还提供一种细胞核荧光染料的染色方法，能够使细胞核染上颜色，使用所述的细胞核荧光染料。The present invention also provides a method for dyeing the cell nucleus fluorescent dye, which can dye the cell nucleus with color, and the cell nucleus fluorescent dye is used.
所述的细胞核荧光染料的染色方法，包括如下步骤：The dyeing method of the nuclear fluorescent dye includes the following steps:
将待染色细胞或组织与染色液混合，染色液完全覆盖住待染色细胞或组织，培养1～10分钟。Mix the cells or tissues to be stained with the staining solution, the staining solution completely covers the cells or tissues to be stained, and incubate for 1 to 10 minutes.
更进一步的，所述染色液为含有所述细胞核荧光染料的缓冲溶液。Further, the staining solution is a buffer solution containing the nuclear fluorescent dye.
更进一步的，所述染色液中所述细胞核荧光染料的浓度为0.01～10mg/mL。Further, the concentration of the nuclear fluorescent dye in the staining solution is 0.01-10 mg/mL.
更进一步的，所述培养1～10分钟后还包括直接在荧光显微镜下观察或封片后在荧光共聚焦显微镜下观察的步骤。所述荧光显微镜或荧光共聚焦显微镜的检测波长为600～900nm。Furthermore, after culturing for 1-10 minutes, the step of directly observing under a fluorescence microscope or observing under a fluorescence confocal microscope after mounting is included. The detection wavelength of the fluorescence microscope or fluorescence confocal microscope is 600-900 nm.
更进一步的，所述待染色细胞或组织包括活细胞或培养的组织、固定的细胞或组织。Further, the cells or tissues to be stained include living cells or cultured tissues, fixed cells or tissues.
更进一步的，所述待染色细胞或组织为固定的细胞或组织时，与染色液混合之前要先去除固定剂。Further, when the cells or tissues to be stained are fixed cells or tissues, the fixative should be removed before mixing with the staining solution.
更进一步的，所述去除固定剂的步骤和所述与染色液混合的步骤之间可以包括免疫荧光染色的步骤。Further, the step of immunofluorescence staining may be included between the step of removing the fixative and the step of mixing with the staining solution.
综上，与现有技术相比，本发明达到了以下技术效果：To sum up, compared with the prior art, the present invention achieves the following technical effects:
1、本发明提供了一种细胞核荧光染料的新应用，拓展了细胞核荧光染料的种类。1. The present invention provides a new application of nuclear fluorescent dyes, and expands the types of nuclear fluorescent dyes.
2、本发明所涉及的细胞核荧光染料有具有好的膜通透性，高灵敏度，快速的染核效率，良好的光稳定性，良好的生物相容性，波长更长的激发光和发射光。2. The nuclear fluorescent dye involved in the present invention has good membrane permeability, high sensitivity, fast nuclear dyeing efficiency, good photostability, good biocompatibility, longer wavelength excitation light and emission light .
3、通过进一步的实验研究了本发明的细胞核荧光染料的细胞核染色过 程，为其应用奠定了良好的理论基础。3. The nuclear staining process of the nuclear fluorescent dye of the present invention is studied through further experiments, which lays a good theoretical foundation for its application.
4、本发明提供的细胞核荧光染料是一种聚集诱导发光染料，具有越聚越亮的优势，染色程序方法简单，无需洗涤，易于操作。4. The nuclear fluorescent dye provided by the present invention is a kind of aggregation-induced luminescent dye, which has the advantages of more aggregation and brighter, the dyeing procedure method is simple, no washing is required, and it is easy to operate.
附图说明Description of drawings
为了更清楚地说明本发明实施例的技术方案，下面将对实施例中所需要使用的附图作简单地介绍，应当理解，以下附图仅示出了本发明的某些实施例，因此不应被看作是对范围的限定，对于本领域普通技术人员来讲，在不付出创造性劳动的前提下，还可以根据这些附图获得其他相关的附图。In order to illustrate the technical solutions of the embodiments of the present invention more clearly, the following briefly introduces the accompanying drawings used in the embodiments. It should be understood that the following drawings only show some embodiments of the present invention, and therefore do not It should be regarded as a limitation of the scope, and for those of ordinary skill in the art, other related drawings can also be obtained according to these drawings without any creative effort.
图1示本发明的细胞核荧光染料的紫外吸收光谱；Fig. 1 shows the ultraviolet absorption spectrum of the nuclear fluorescent dye of the present invention;
图2示本发明的细胞核荧光染料结合DNA的荧光发射光谱；Fig. 2 shows the fluorescence emission spectrum of the nuclear fluorescent dye of the present invention bound to DNA;
图3示Hoechst33342染料的吸收和发射光谱图；Figure 3 shows the absorption and emission spectra of Hoechst33342 dye;
图4示共聚焦显微镜观察本发明的细胞核荧光染料染细胞核的效果图，从左到右依次为Hoechst 33342、本发明的细胞核荧光染料、Hoechst 33342和本发明的细胞核荧光染料的共染叠加、Hoechst 33342和本发明的细胞核荧光染料的共染以及明场叠加的染色效果；Fig. 4 is a diagram showing the effect of staining the nucleus with the nuclear fluorescent dye of the present invention by confocal microscopy. From left to right are Hoechst 33342, the nuclear fluorescent dye of the present invention, the co-staining stack of Hoechst 33342 and the nuclear fluorescent dye of the present invention, and Hoechst 33342. Co-staining of 33342 and the nuclear fluorescent dye of the present invention and the staining effect of bright field superposition;
图5示共聚焦显微镜观察本发明的细胞核荧光染料在低浓度下的染细胞核效果图；Fig. 5 is a graph showing the effect of confocal microscope observation of the nuclear fluorescent dye of the present invention staining cell nucleus at a low concentration;
图6示本发明的微量细胞核荧光染料检测游离DNA荧光光谱图；Fig. 6 shows the fluorescence spectrum of free DNA detected by the micro-nucleus fluorescent dye of the present invention;
图7示长时间激光照射本发明的细胞核荧光染料的吸收光谱图；Fig. 7 shows the absorption spectrum diagram of long-time laser irradiation of the nuclear fluorescent dye of the present invention;
图8示MCF-7活细胞用本发明的细胞核荧光染料染色的结果图；FIG. 8 shows the result of staining of MCF-7 living cells with the nuclear fluorescent dye of the present invention;
图9示COS-7死细胞用本发明的细胞核荧光染料染色的结果图；Figure 9 shows the result of staining COS-7 dead cells with the nuclear fluorescent dye of the present invention;
图10示洋葱表皮细胞用本发明的细胞核荧光染料进行细胞核染色的结果图。Fig. 10 is a graph showing the results of nuclear staining of onion epidermal cells with the nuclear fluorescent dye of the present invention.
具体实施方式detailed description
为了使本技术领域的人员更好地理解本发明方案，下面将结合本发明实施例中的附图，对本发明实施例中的技术方案进行清楚、完整地描述，显然，所描述的实施例仅仅是本发明一部分的实施例，而不是全部的实施例。基于本发明中的实施例，本领域普通技术人员在没有做出创造性劳动的前提下所获得的所有其他实施例，都应当属于本发明保护的范围。In order to make those skilled in the art better understand the solutions of the present invention, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only Embodiments are part of the present invention, but not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by persons of ordinary skill in the art without creative work shall fall within the protection scope of the present invention.
荧光染料会发出荧光，所谓荧光是指物质分子吸收波长较短的可见光后发出的波长较长的可见光荧光。Fluorescent dyes emit fluorescence, and the so-called fluorescence refers to the fluorescence of visible light with longer wavelengths emitted by substance molecules after absorbing visible light with shorter wavelengths.
膜通透性：物质通过生物膜的难易程度。在本发明中特指荧光染料透过细胞膜的难易程度。Membrane permeability: The ease with which substances pass through a biomembrane. In the present invention, it refers specifically to the degree of difficulty of the fluorescent dyes permeating the cell membrane.
灵敏度：低浓度的荧光染料染色的效果。低浓度的荧光染料也有很好的染核效果即为灵敏度高。Sensitivity: The effect of staining with low concentrations of fluorescent dyes. Low concentrations of fluorescent dyes also have a good nuclear effect, that is, high sensitivity.
染核效率：荧光染料使细胞染上颜色的时间。能快速染色即为染核效率高。Nuclear staining efficiency: The amount of time a fluorescent dye takes to stain cells. Fast staining means high nuclear efficiency.
光稳定性：长时间的激光照射对荧光染料的吸收没有影响，说明其光稳定性好。Light stability: Long-term laser irradiation has no effect on the absorption of fluorescent dyes, indicating that its light stability is good.
生物相容性：生物相容性是指细胞对非活性材料产生反应的一种性能，荧光染料进入细胞后，对细胞产生影响和作用，细胞对荧光染料也会产生影响和作用，两者的循环作用一直持续，直到达到平衡。Biocompatibility: Biocompatibility refers to the ability of cells to react to inactive materials. After the fluorescent dye enters the cell, it will have an impact and effect on the cell, and the cell will also have an impact and effect on the fluorescent dye. Cycling continues until equilibrium is reached.
激发波长：激发波长是用某种波长的光激发出荧光，这种波长的光可以是紫外光或者可见光也可以是其他光。激发波长对于杂散光和信噪比的影响非常显著。Excitation wavelength: Excitation wavelength is to excite fluorescence with a certain wavelength of light, which can be ultraviolet light or visible light or other light. The excitation wavelength has a significant effect on stray light and signal-to-noise ratio.
发射波长：某种光发射出来的荧光的波长，一般的可见光的波长用肉眼就能大致判断出来。Emission wavelength: The wavelength of fluorescence emitted by a certain light, the wavelength of general visible light can be roughly judged by the naked eye.
波长更长的激发光和发射光：激发光和发射光的波长长，就具有一定 带宽的峰，在实际应用的时候，激发波长的范围要小于发射波长，激发波长更长可以获得高激发率的物质形态，间接提高灵敏度，发射波长更长可以直接提高灵敏度，从而可以得到良好的信噪比的结果值。Excitation light and emission light with longer wavelengths: The excitation light and emission light have long wavelengths, so they have peaks with a certain bandwidth. In practical applications, the excitation wavelength range is smaller than the emission wavelength, and the excitation wavelength is longer to obtain a high excitation rate The material form can indirectly improve the sensitivity, and the longer emission wavelength can directly improve the sensitivity, so that a good signal-to-noise ratio can be obtained.
本发明提供的一种细胞核荧光染料，具有如下结构通式：A kind of nuclear fluorescent dye provided by the present invention has the following general structural formula:
Figure PCTCN2020106563-appb-000004
Figure PCTCN2020106563-appb-000004
其中，R 1为C0-C30的直链或支链的亚烷基，R 2为C0-C30的直链或支链的取代亚烷基，R 3为C5-C30的芳香基或取代芳香基，R 4为C5-C30的亚烷基芳香基、取代亚烷基芳香基、亚芳香基烷基、取代亚芳香基烷基中的任一种；R 5为不饱和烷基、杂烷基、环烷基、杂环烷基、芳基、杂芳基、羧基、氨基、磺酸基。 Wherein, R 1 is C0-C30 linear or branched alkylene, R 2 is C0-C30 linear or branched substituted alkylene, R 3 is C5-C30 aryl or substituted aryl , R 4 is any one of C5-C30 alkylene aryl, substituted alkylene aryl, arylene alkyl, substituted arylene alkyl; R 5 is unsaturated alkyl, heteroalkyl , cycloalkyl, heterocycloalkyl, aryl, heteroaryl, carboxyl, amino, sulfonic acid.
更进一步的，所述的细胞核荧光染料，具有如下结构通式：Further, the nuclear fluorescent dye has the following general structural formula:
Figure PCTCN2020106563-appb-000005
Figure PCTCN2020106563-appb-000005
所述的结构通式中包含R 1-R 5的主体部分带正电，带正电对于其细胞核染色功能有重要意义，带正电的化合物可以与带负电的负离子部分结合，结合之后从而使细胞核更好的染上颜色。其中，R 1为C0-C30的直链或支链的亚烷基，R 2为C0-C30的直链或支链的取代亚烷基，R 3为C5-C30的芳香基或取代芳香基，R 4为C5-C30的亚烷基芳香基、取代亚烷基芳香基、亚芳香基烷基、取代亚芳香基烷基中的任一种；R 5为不饱和烷基、杂烷基、环烷基、杂环烷基、芳基、杂芳基、羧基、氨基、磺酸基；R 6为化合物的负离子部分，带负电，把整个化合物作为一个盐，R 6为PF 6 -、BF 4 -、SbF 5 -、CH 3COO -、CF 3COO -、CO 3 2-、SO 4 2-、SO 3 2-、CF 3SO 2 -、TsO -、ClO 4 -、F -、Cl -、Br -、I -、(F 3CSO 2)N -、PO 4 3-中的任一种。 The main part containing R 1 -R 5 in the general structural formula is positively charged, and the positive charge is of great significance to its nuclear staining function. The positively charged compound can be combined with the negatively charged anion part, and after the combination, the Nuclei are better stained. Wherein, R 1 is C0-C30 linear or branched alkylene, R 2 is C0-C30 linear or branched substituted alkylene, R 3 is C5-C30 aryl or substituted aryl , R 4 is any one of C5-C30 alkylene aryl, substituted alkylene aryl, arylene alkyl, substituted arylene alkyl; R 5 is unsaturated alkyl, heteroalkyl , cycloalkyl, heterocycloalkyl, aryl, heteroaryl, carboxyl, amino, sulfonic acid group; R 6 is the negative ion part of the compound, negatively charged, the whole compound is taken as a salt, R 6 is PF 6 - , BF 4 - , SbF 5 - , CH 3 COO - , CF 3 COO - , CO 3 2- , SO 4 2- , SO 3 2- , CF 3 SO 2 - , TsO - , ClO 4 - , F - , Cl Any of - , Br - , I - , (F 3 CSO 2 )N - , PO 4 3- .
更进一步的，所述的细胞核荧光染料，具有如下结构式：Further, the nuclear fluorescent dye has the following structural formula:
Figure PCTCN2020106563-appb-000006
Figure PCTCN2020106563-appb-000006
本发明的细胞核荧光染料既能对活细胞或培养的组织进行染色，也可以对固定的细胞或组织进行染色，对于活细胞和死细胞具有不同的染色方法：The nuclear fluorescent dye of the present invention can not only stain living cells or cultured tissues, but also can stain fixed cells or tissues, and has different staining methods for living cells and dead cells:
1.对于固定的细胞或组织：1. For fixed cells or tissues:
a.细胞或组织样品，固定后，适当洗涤去除固定剂。随后如果需要进行免疫荧光染色，则先进行免疫荧光染色，染色完毕后再按后续步骤进行细胞核染色。如果不需要进行其它染色，则直接进行后续的细胞核染色。a. Cell or tissue samples, after fixation, wash appropriately to remove fixative. Then, if immunofluorescence staining is required, immunofluorescence staining is performed first, and after the staining is completed, the nucleus staining is performed according to the subsequent steps. If no other staining is required, proceed directly to subsequent nuclear staining.
b.对于贴壁细胞或组织切片，加入少量染色液，覆盖住样品即可。染色液中细胞核荧光染料的浓度为0.01～10mg/mL，用缓冲溶液稀释。对于悬浮细胞，至少加入待染色样品体积3倍的染色液，混匀。室温放置3-5min。b. For adherent cells or tissue sections, add a small amount of staining solution to cover the sample. The concentration of nuclear fluorescent dye in the staining solution is 0.01-10 mg/mL, diluted with buffer solution. For suspension cells, add at least 3 times the volume of the sample to be stained with staining solution and mix well. Place at room temperature for 3-5min.
c.不需洗涤，直接在荧光显微镜下观察或封片后荧光共聚焦显微镜下观察，检测波长为600～900nm。c. No need to wash, observe directly under a fluorescence microscope or under a fluorescence confocal microscope after mounting, and the detection wavelength is 600-900 nm.
2.对于活细胞或培养的组织：2. For live cells or cultured tissue:
a.加入适量染色液，必须充分覆盖住待染色的样品，通常对于6孔板一个孔需加入1mL染色液，对于96孔板一个孔需加入100μL染色液。染色液中细胞核荧光染料的浓度为0.01～10mg/mL，用缓冲溶液稀释。a. Add an appropriate amount of staining solution, which must fully cover the sample to be stained. Usually, 1 mL of staining solution should be added to one well of a 6-well plate, and 100 μL of staining solution should be added to one well of a 96-well plate. The concentration of nuclear fluorescent dye in the staining solution is 0.01-10 mg/mL, diluted with buffer solution.
b.在适宜于细胞培养的温度培养1-10min，不需洗涤，直接在荧光显微镜下观察或封片后荧光共聚焦显微镜下观察，检测波长为600～900nm。b. Incubate for 1-10 minutes at a temperature suitable for cell culture, without washing, observe directly under a fluorescence microscope or observe under a fluorescence confocal microscope after mounting, and the detection wavelength is 600-900 nm.
本发明提供的细胞核荧光染料的应用及细胞核的染色方法中所用试剂或仪器均可由市场购得。The application of the nuclear fluorescent dye provided by the present invention and the reagents or instruments used in the method for staining the nucleus can be purchased from the market.
下面结合实施例，进一步阐述本发明，实施例中采用的细胞核荧光染料的化合物结构为上述结构式(Ⅲ)。The present invention will be further described below with reference to the examples. The compound structure of the nuclear fluorescent dye used in the examples is the above-mentioned structural formula (III).
实施例1 对于固定的细胞或组织的染色Example 1 Staining of fixed cells or tissues
染色方法为：The dyeing method is:
配制母液为1mg/mL的本发明的细胞核荧光染料的PBS缓冲溶液。The preparation stock solution is 1 mg/mL of the PBS buffer solution of the nuclear fluorescent dye of the present invention.
a.细胞或组织样品，固定后，适当洗涤去除固定剂。加入浓度为100μg/mL的本发明的细胞核荧光染料的染色液，覆盖住样品即可。室温放置3-5min。不需洗涤，直接在荧光显微镜下观察或封片后荧光共聚焦显微镜下观察。a. Cell or tissue samples, after fixation, wash appropriately to remove fixative. Add the staining solution of the nuclear fluorescent dye of the present invention at a concentration of 100 μg/mL to cover the sample. Place at room temperature for 3-5min. Without washing, observe directly under a fluorescence microscope or under a fluorescence confocal microscope after mounting.
b.对于贴壁细胞或组织切片，加入浓度为100μg/mL的本发明的细胞核荧光染料的染色液，覆盖住样品即可。对于悬浮细胞，至少加入待染色样品体积3倍的染色液，混匀。室温放置3-5min。不需洗涤，直接在荧光显微镜下观察或封片后荧光共聚焦显微镜下观察。b. For adherent cells or tissue sections, add the staining solution of the nuclear fluorescent dye of the present invention at a concentration of 100 μg/mL to cover the sample. For suspension cells, add at least 3 times the volume of the sample to be stained with staining solution and mix well. Place at room temperature for 3-5min. Without washing, observe directly under a fluorescence microscope or under a fluorescence confocal microscope after mounting.
实施例2 对于活细胞或培养的组织的染色Example 2 Staining of live cells or cultured tissue
a.加入浓度为100μg/mL的本发明的细胞核荧光染料的染色液，必须充分覆盖住待染色的样品，通常对于6孔板一个孔需加入1mL染色液，对于96孔板一个孔需加入100μL染色液。a. Add the staining solution of the nuclear fluorescent dye of the present invention with a concentration of 100 μg/mL, which must fully cover the sample to be stained. Usually, 1 mL of staining solution should be added to one well of a 6-well plate, and 100 μL should be added to one well of a 96-well plate. staining solution.
b.在适宜于细胞培养的温度培养5min，即可进行荧光检测。b. Incubate for 5 minutes at a temperature suitable for cell culture, and then perform fluorescence detection.
试验例1 本发明的细胞核荧光染料的吸收光谱和发射光谱检测Test Example 1 Absorption spectrum and emission spectrum detection of the nuclear fluorescent dye of the present invention
在本发明中，用紫外-可见光谱仪检测了本发明的细胞核荧光染料的吸收光谱，如图1所示，本发明的细胞核荧光染料的吸收光波长范围在250-850nm，一般的核染料吸收光波长范围在250-450nm。同时用荧光光谱仪检测了本发明的细胞核荧光染料结合DNA后的荧光发射光谱，如图2所示，本发明的细胞核荧光染料结合DNA后的荧光发射光波长范围在600-900nm，一般的核染料范围在300-600nm。图3所示为传统细胞核染料Hoechst33342的光谱图，左边是吸收光谱，右边是荧光发射光谱。传统染料的光谱是偏蓝光，而本发明的本发明的细胞核荧光染料的光谱偏红光。Hoechst33342最佳激发波长偏蓝光，激发波长短，无法被现有的商业化激光器有效激发，且该波长的光能量较高，不仅细胞的光毒性较大，同时也容易造成染料的光漂白现象，而本发明的细胞核荧光染料很好的克服了这一缺点，在成像中不会造成严重的细胞损伤。In the present invention, an ultraviolet-visible spectrometer is used to detect the absorption spectrum of the nuclear fluorescent dye of the present invention. As shown in FIG. 1 , the absorption light wavelength range of the nuclear fluorescent dye of the present invention is 250-850 nm, and the general nuclear dye absorbs light. The wavelength range is 250-450nm. At the same time, the fluorescence emission spectrum of the nuclear fluorescent dye of the present invention combined with DNA was detected by a fluorescence spectrometer. As shown in FIG. 2 , the fluorescence emission wavelength range of the nuclear fluorescent dye of the present invention combined with DNA is 600-900 nm. The range is 300-600nm. Figure 3 shows the spectrum of the traditional nuclear dye Hoechst33342, with the absorption spectrum on the left and the fluorescence emission spectrum on the right. The spectrum of traditional dyes is blue-shifted, while the spectrum of the nuclear fluorescent dye of the present invention is red-shifted. The optimal excitation wavelength of Hoechst33342 is blue, and the excitation wavelength is short, which cannot be effectively excited by existing commercial lasers, and the light energy of this wavelength is high, which not only has high phototoxicity to cells, but also easily causes photobleaching of dyes. However, the nuclear fluorescent dye of the present invention overcomes this shortcoming well, and will not cause serious cell damage during imaging.
试验例2 染色效果检测Test example 2 Dyeing effect detection
参照实施例1的方法对MCF-7细胞加入母液浓度为1mg/mL的本发明的细胞核荧光染料的PBS缓冲液，使其细胞培养液的细胞核荧光染料的终浓度为100μg/mL，室温放置5min，随即进行荧光共聚焦成像，并同时使用商业化的染核染料Hoechst33342处理染核。染色效果如图4所示。With reference to the method of Example 1, the PBS buffer of the nuclear fluorescent dye of the present invention with a concentration of 1 mg/mL in the mother solution was added to the MCF-7 cells, so that the final concentration of the nuclear fluorescent dye in the cell culture medium was 100 μg/mL, and placed at room temperature for 5min , followed by fluorescence confocal imaging, and the nuclei were treated with the commercial nuclear dye Hoechst33342. The dyeing effect is shown in Figure 4.
试验结果显示：从左至右，第1张图为Hoechst33342的染核效果图，第2张图为本发明的细胞核荧光染料的染核效果图，第3张图为Hoechst和本发明的细胞核荧光染料共染染核效果图，第4张图是两者的染核效果和明场的叠 加。从图中可以看出，本发明的细胞核荧光染料的染核效果和商业化的染核染料Hoechst33342的染核效果荧光叠加非常明显，重合高度一致。说明本发明的细胞核荧光染料和商业化的染核染料相比有同等甚至更好的染核效果。The test results show: from left to right, the first picture is the nuclear staining effect of Hoechst33342, the second picture is the nuclear staining effect of the nuclear fluorescent dye of the present invention, and the third picture is Hoechst and the nuclear fluorescence of the present invention. The effect of dye co-staining nuclear effect, the fourth picture is the superposition of the nuclear effect and bright field of the two. It can be seen from the figure that the nuclear staining effect of the nuclear fluorescent dye of the present invention and the nuclear staining effect of the commercial nuclear staining dye Hoechst33342 are superimposed very obviously, and the overlap is highly consistent. It shows that the nuclear fluorescent dye of the present invention has the same or even better nuclear staining effect compared with commercial nuclear staining dyes.
试验例3 灵敏度检测Test example 3 Sensitivity detection
参照实施例1的方法对MCF-7细胞加入母液浓度为1mg/mL的本发明的细胞核荧光染料的PBS缓冲液，使其细胞培养液的细胞核荧光染料的终浓度为5μg/ml，相比100μg/mL的工作液浓度又稀释了20倍，室温放置5min，随即进行荧光共聚焦成像，染色效果如图5所示。Referring to the method of Example 1, the PBS buffer of the nuclear fluorescent dye of the present invention with a concentration of 1 mg/mL in the stock solution was added to the MCF-7 cells, so that the final concentration of the nuclear fluorescent dye in the cell culture medium was 5 μg/ml, compared with 100 μg The concentration of the working solution/mL was diluted by 20 times and placed at room temperature for 5 min, followed by fluorescence confocal imaging. The staining effect is shown in Figure 5.
试验结果显示，MCF-7细胞的细胞核呈现红色荧光，细胞结构完整，荧光边缘清晰且亮度很高，说明极低浓度的本发明细胞核荧光染料也有非常好的染核效果。The test results show that the nuclei of MCF-7 cells exhibit red fluorescence, the cell structure is complete, the fluorescence edge is clear and the brightness is high, indicating that the very low concentration of the nuclear fluorescent dye of the present invention also has a very good nuclear staining effect.
此外，用微量的本发明的细胞核荧光染料，浓度分别为1，2，5，10，20，50μM结合游离的DNA可以看到有很好的荧光峰型和线性规律，如图6所示，说明本发明的细胞核荧光染料有很高的灵敏度。In addition, with a trace amount of the nuclear fluorescent dye of the present invention, the concentration of which is 1, 2, 5, 10, 20, 50 μM, respectively, binds to free DNA, and it can be seen that there is a good fluorescence peak shape and linearity, as shown in Figure 6, It shows that the nuclear fluorescent dye of the present invention has high sensitivity.
试验例4 光稳定性检测Test Example 4 Light Stability Detection
通过对本发明的细胞核荧光染料长时间660nm激光照射(0、10、20、30、60、120s)，测出本发明的细胞核荧光染料的吸收光谱的结果所示，本发明的细胞核荧光染料的光学稳定性检测如图7，橫坐标为波长，纵坐标为紫外吸收值。By irradiating the nuclear fluorescent dye of the present invention with a 660 nm laser for a long time (0, 10, 20, 30, 60, 120 s), the results of measuring the absorption spectrum of the nuclear fluorescent dye of the present invention show that the optical The stability test is shown in Figure 7, the abscissa is the wavelength, and the ordinate is the UV absorption value.
结果显示，长时间的激光照射对本发明的细胞核荧光染料的吸收没有影响，即使照射时间长达120s，本发明的细胞核荧光染料的紫外吸收峰也没有明显的降低，说明本发明的细胞核荧光染料的光学稳定性好。The results show that long-time laser irradiation has no effect on the absorption of the nuclear fluorescent dye of the present invention. Even if the irradiation time is as long as 120 s, the ultraviolet absorption peak of the nuclear fluorescent dye of the present invention is not significantly reduced, which shows that the nuclear fluorescent dye of the present invention has no obvious effect. Good optical stability.
试验例5 MCF-7活细胞用本发明的细胞核荧光染料染色的结果Test Example 5 The results of staining of living MCF-7 cells with the nuclear fluorescent dye of the present invention
MCF-7活细胞的染色。MCF-7细胞培养后，用浓度为1mg/mL的本发明的细胞核荧光染料的PBS缓冲液染色，使其细胞培养液的细胞核荧光染料的 终浓度为5μg/mL，室温放置5min，随即进行荧光共聚焦成像。Staining of live cells for MCF-7. After the MCF-7 cells were cultured, they were stained with the PBS buffer solution of the nuclear fluorescent dye of the present invention with a concentration of 1 mg/mL, so that the final concentration of the nuclear fluorescent dye in the cell culture medium was 5 μg/mL, and placed at room temperature for 5 minutes, and then fluorescence was carried out. Confocal imaging.
结果如图8所示，最左边一列是荧光通道，细胞核呈现红色荧光，中间一列为明场通道，最右边的一列为荧光通道和明场通道的叠加，下面的一行是上面一行的局部放大图。通过本发明的细胞核荧光染料染色，可以看到活细胞的核，还能看到核的有丝***过程。染色体聚集，然后细胞核***，染色体一分为二的整个过程都可以通过染料的染色清晰看到，以上结果说明本发明的细胞核荧光染料对于活细胞的细胞核染色具有良好效果。The results are shown in Figure 8, the leftmost column is the fluorescence channel, the nucleus shows red fluorescence, the middle column is the brightfield channel, the rightmost column is the superposition of the fluorescence channel and the brightfield channel, and the lower row is a partial enlarged view of the upper row. . By staining with the nuclear fluorescent dye of the present invention, the nucleus of living cells can be seen, and the mitotic process of the nucleus can also be seen. The whole process of chromosome aggregation, then nucleus division, and chromosome division into two can be clearly seen by dye staining. The above results show that the nuclear fluorescent dye of the present invention has a good effect on the nuclear staining of living cells.
试验例6 COS-7死细胞用本发明的细胞核荧光染料染色的结果Test Example 6 The result of staining of COS-7 dead cells with the nuclear fluorescent dye of the present invention
COS-7细胞培养后用多聚甲醛固定，然后使用浓度为1mg/mL的本发明的细胞核荧光染料的PBS缓冲液染色，使其细胞培养液的细胞核荧光染料的终浓度为5μg/ml，室温放置5min，随即进行荧光共聚焦成像。After COS-7 cells were cultured, they were fixed with paraformaldehyde, and then stained with the PBS buffer solution of the nuclear fluorescent dye of the present invention with a concentration of 1 mg/mL, so that the final concentration of the nuclear fluorescent dye in the cell culture medium was 5 μg/ml, at room temperature. After standing for 5 min, fluorescence confocal imaging was performed immediately.
结果如图9所示，可以观察到COS-7的细胞核呈现出边缘清晰的红色，图中以灰度表示，以上结果说明本发明的细胞核荧光染料对于死细胞的细胞核染色同样具有良好效果。The results are shown in Figure 9. It can be observed that the nucleus of COS-7 presents a clear red edge, which is represented by grayscale in the figure. The above results show that the nuclear fluorescent dye of the present invention also has a good effect on the nucleus staining of dead cells.
试验例7 洋葱表皮细胞用本发明的细胞核荧光染料进行细胞核染色的结果Test Example 7 The results of nuclear staining of onion epidermal cells with the nuclear fluorescent dye of the present invention
取洋葱表皮细胞用本发明的细胞核荧光染料进行细胞核染色的步骤如下：The steps of taking onion epidermal cells for nuclear staining with the nuclear fluorescent dye of the present invention are as follows:
(1)用洁净的纱布把载玻片和盖玻片擦拭干净。(1) Wipe the slide and cover glass with a clean gauze.
(2)把载玻片放在实验台上，用滴管在载玻片的中央滴一滴PBS缓冲液。(2) Put the slide glass on the laboratory table, and drop a drop of PBS buffer in the center of the slide glass with a dropper.
(3)用镊子从洋葱鳞片内侧撕取一小块透明薄膜——内表皮。(3) Use tweezers to tear a small piece of transparent film - the inner epidermis from the inside of the onion scale.
(4)把撕下的内表皮浸入载玻片上的水滴中，用镊子把它展平。(4) Immerse the torn inner epidermis in a drop of water on the glass slide and flatten it with tweezers.
(5)用镊子夹起盖玻片，使它的一边先接触载玻片上的水滴，然后缓缓地放下，盖在要观察的材料上，避免盖玻片下面出现气泡影响观察。(5) Pick up the cover glass with tweezers, make one side touch the water droplets on the glass slide first, then put it down slowly, and cover it on the material to be observed, so as to avoid bubbles under the cover glass from affecting the observation.
(6)把一滴本发明的细胞核荧光染料滴在盖玻片的一侧。(6) Drop a drop of the nuclear fluorescent dye of the present invention on one side of the cover glass.
(7)用吸水纸从盖玻片的另一侧吸引，使染液浸润标本的全部。(7) Suction from the other side of the cover glass with absorbent paper to infiltrate the entire specimen with the dye solution.
(8)制作好的样本用荧光显微镜观察并拍照。(8) The prepared samples were observed with a fluorescence microscope and photographed.
试验结果如图10所示，洋葱表皮细胞的细胞核被清晰的染上红色，与明场所观察到的细胞核位置完全吻合，说明本发明的细胞核荧光染料不仅可以应用于动物细胞或组织，对植物细胞或组织同样适用。The test results are shown in Figure 10. The nuclei of the onion epidermal cells are clearly dyed red, which is completely consistent with the position of the nuclei observed in the bright field, indicating that the nuclear fluorescent dye of the present invention can be applied not only to animal cells or tissues, but also to plant cells. Or the same applies to organizations.
以上所述仅为本发明的较佳实施例，并不用以限制本发明，凡在本发明的精神和原则之内，所作的任何修改、等同替换、改进等，均应包含在本发明的保护范围之内。The above descriptions are only preferred embodiments of the present invention, and are not intended to limit the present invention. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection of the present invention. within the range.

Claims (11)

  1. 一种细胞核荧光染料，其特征在于，具有如下结构通式：A nuclear fluorescent dye, characterized in that it has the following general structural formula:
    Figure PCTCN2020106563-appb-100001
    Figure PCTCN2020106563-appb-100001
    其中，R 1为C0-C30的直链或支链的亚烷基，R 2为C0-C30的直链或支链的取代亚烷基，R 3为C5-C30的芳香基或取代芳香基，R 4为C5-C30的亚烷基芳香基、取代亚烷基芳香基、亚芳香基烷基、取代亚芳香基烷基中的任一种；R 5为不饱和烷基、杂烷基、环烷基、杂环烷基、芳基、杂芳基、羧基、氨基、磺酸基。 Wherein, R 1 is C0-C30 linear or branched alkylene, R 2 is C0-C30 linear or branched substituted alkylene, R 3 is C5-C30 aryl or substituted aryl , R 4 is any one of C5-C30 alkylene aryl, substituted alkylene aryl, arylene alkyl, substituted arylene alkyl; R 5 is unsaturated alkyl, heteroalkyl , cycloalkyl, heterocycloalkyl, aryl, heteroaryl, carboxyl, amino, sulfonic acid.
  2. 根据权利要求1所述的细胞核荧光染料，其特征在于，具有如下结构通式：The nuclear fluorescent dye according to claim 1, characterized in that, has the following general structural formula:
    Figure PCTCN2020106563-appb-100002
    Figure PCTCN2020106563-appb-100002
    所述的结构通式中包含R 1-R 5的主体部分带正电，其中，R 1为C0-C30的直链或支链的亚烷基，R 2为C0-C30的直链或支链的取代亚烷基，R 3为 C5-C30的芳香基或取代芳香基，R 4为C5-C30的亚烷基芳香基、取代亚烷基芳香基、亚芳香基烷基、取代亚芳香基烷基中的任一种；R 5为不饱和烷基、杂烷基、环烷基、杂环烷基、芳基、杂芳基、羧基、氨基、磺酸基；R 6为化合物的负离子部分，R 6为PF 6 -、BF 4 -、SbF 5 -、CH 3COO -、CF 3COO -、CO 3 2-、SO 4 2-、SO 3 2-、CF 3SO 2 -、TsO -、ClO 4 -、F -、Cl -、Br -、I -、(F 3CSO 2)N -、PO 4 3-中的任一种。 The main part comprising R 1 -R 5 in the general structural formula is positively charged, wherein R 1 is a C0-C30 straight-chain or branched alkylene group, and R 2 is a C0-C30 straight-chain or branched chain Chain substituted alkylene group, R 3 is C5-C30 aryl group or substituted aryl group, R 4 is C5-C30 alkylene aryl group, substituted alkylene aryl group, arylene alkyl group, substituted arylene group any one of the base alkyl groups; R 5 is an unsaturated alkyl group, a heteroalkyl group, a cycloalkyl group, a heterocycloalkyl group, an aryl group, a heteroaryl group, a carboxyl group, an amino group, a sulfonic acid group; R 6 is a compound Negative ion part, R 6 is PF 6 - , BF 4 - , SbF 5 - , CH 3 COO - , CF 3 COO - , CO 3 2- , SO 4 2- , SO 3 2- , CF 3 SO 2 - , TsO Any of - , ClO 4 - , F - , Cl - , Br - , I - , (F 3 CSO 2 )N - , PO 4 3- .
  3. 根据权利要求2所述的细胞核荧光染料，其特征在于，具有如下结构式：The nuclear fluorescent dye according to claim 2, characterized in that, has the following structural formula:
    Figure PCTCN2020106563-appb-100003
    Figure PCTCN2020106563-appb-100003
  4. 一种细胞核荧光染料的染色方法，能够使细胞核染上颜色，其特征在于，使用权利要求1-3任一项所述的细胞核荧光染料。A method for dyeing cell nucleus fluorescent dye, which can dye cell nucleus with color, characterized in that the cell nucleus fluorescent dye according to any one of claims 1-3 is used.
  5. 根据权利要求4所述的染色方法，其特征在于，包括如下步骤：dyeing method according to claim 4, is characterized in that, comprises the steps:
    将待染色细胞或组织与染色液混合，培养1～10分钟。Mix the cells or tissues to be stained with the staining solution and incubate for 1-10 minutes.
  6. 根据权利要求5所述的染色方法，其特征在于，所述染色液为含有所述细胞核荧光染料的缓冲溶液。The staining method according to claim 5, wherein the staining solution is a buffer solution containing the nuclear fluorescent dye.
  7. 根据权利要求5或6任一项所述的染色方法，其特征在于，所述染色液中所述细胞核荧光染料的浓度为0.01～10mg/mL。The staining method according to any one of claims 5 or 6, wherein the concentration of the nuclear fluorescent dye in the staining solution is 0.01-10 mg/mL.
  8. 根据权利要求5所述的染色方法，其特征在于，所述培养1～10分钟后还包括直接在荧光显微镜下观察或封片后在荧光共聚焦显微镜下观察 的步骤，所述荧光显微镜或荧光共聚焦显微镜的检测波长为600～900nm。The staining method according to claim 5, characterized in that, after culturing for 1-10 minutes, further comprising the step of directly observing under a fluorescence microscope or observing under a fluorescence confocal microscope after mounting the slide, the fluorescence microscope or fluorescence microscope The detection wavelength of confocal microscope is 600-900 nm.
  9. 根据权利要求5所述的染色方法，其特征在于，所述待染色细胞或组织包括活细胞或培养的组织、固定的细胞或组织。The staining method according to claim 5, wherein the cells or tissues to be stained comprise living cells or cultured tissues, fixed cells or tissues.
  10. 根据权利要求5所述的染色方法，其特征在于，所述待染色细胞或组织为固定的细胞或组织时，与染色液混合之前要先去除固定剂。The staining method according to claim 5, wherein when the cells or tissues to be stained are fixed cells or tissues, the fixative must be removed before mixing with the staining solution.
  11. 根据权利要求10所述的染色方法，其特征在于，所述去除固定剂的步骤和所述与染色液混合的步骤之间可以包括免疫荧光染色的步骤。The staining method according to claim 10, wherein a step of immunofluorescence staining can be included between the step of removing the fixative and the step of mixing with the staining solution.
PCT/CN2020/106563 2020-08-03 2020-08-03 Cell nucleus fluorescent dye and dyeing method therefor WO2022027174A1 (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
PCT/CN2020/106563 WO2022027174A1 (en) 2020-08-03 2020-08-03 Cell nucleus fluorescent dye and dyeing method therefor

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
PCT/CN2020/106563 WO2022027174A1 (en) 2020-08-03 2020-08-03 Cell nucleus fluorescent dye and dyeing method therefor

Publications (1)

Publication Number Publication Date
WO2022027174A1 true WO2022027174A1 (en) 2022-02-10

Family

ID=80119129

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2020/106563 WO2022027174A1 (en) 2020-08-03 2020-08-03 Cell nucleus fluorescent dye and dyeing method therefor

Country Status (1)

Country Link
WO (1) WO2022027174A1 (en)

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101004377A (en) * 2007-01-24 2007-07-25 北京望升伟业科技发展有限公司 Kit for rapid diagnosis of sperm concentration, preparation, and method of application
CN105143847A (en) * 2013-02-05 2015-12-09 三路影像公司 Cytological staining compositions and uses thereof

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101004377A (en) * 2007-01-24 2007-07-25 北京望升伟业科技发展有限公司 Kit for rapid diagnosis of sperm concentration, preparation, and method of application
CN105143847A (en) * 2013-02-05 2015-12-09 三路影像公司 Cytological staining compositions and uses thereof

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
DATABASE REGISTRY 16 November 1984 (1984-11-16), ANONYMOUS : "Methanaminium, N-[4-[[4-(dimethylamino)phenyl]phenylmethylene]-2,5- cyclohexadien-1-ylidene]-N-methyl-, chloride (1:1) (CA INDEX NAME) ", XP055894613, retrieved from STN Database accession no. 569-64-2 (+ RN 10309-95-2, RN 2113610-82-3, RN 85188-05-2, RN 61941-41-1, RN 60885-33-8, RN 41272-40-6, RN 4562-43-0, RN 3946-77-8, RN 3737-92-6) *
J. R. LAWTON: "Ultrastructural localization of nucleic acids in plant tissues following the use of malachite green or neutral red in the fixative solution", JOURNAL OF MICROSCOPY, vol. 158, no. 3, 30 June 1990 (1990-06-30), pages 343 - 354, XP009533795, ISSN: 1365-2818, DOI: 10.1111/j.1365-2818.1990.tb03006.x *
LIU JUNYAN, JI WEI-HUA: "Preliminary Exploration Of Making the Stained Specimens of Musca Domestica Ovaries", ZHONGGUO JISHENGCHONGBING FANGZHI ZAZHI - CHINESE JOURNAL OFPARASITIC DISEASE CONTROL, SHANDONG SHENG JISHENGCHONGBING FANGZHI YANJIUSUO, JINING, CN, vol. 18, no. 4, 31 August 2005 (2005-08-31), CN , XP055894610, ISSN: 1001-6627 *

Similar Documents

Publication Publication Date Title
Dmitriev et al. Optical probes and techniques for O 2 measurement in live cells and tissue
JP6112169B2 (en) Fluorescent label for detection of biological materials
EP2613138B1 (en) Biological substance detection method
JP6586070B2 (en) Methods for analyzing cells or other biomaterials containing nucleic acids
WO2016165487A1 (en) Real-time monitoring mitophagy process by fluorescent photostable mitochondrial specific bioprobe with aie characteristics
CN108138043A (en) Red fluorescence AIEgen
Martinić et al. Near-infrared luminescent metallacrowns for combined in vitro cell fixation and counter staining
CN108815537A (en) A kind of tumour cell targeting specific fluorescence probe and the preparation method and application thereof
CN107936945B (en) Fluorescent viscosity probe with aggregation-induced emission characteristic
CN110981842B (en) Fluorescent probe for distinguishing normal cells and cancer cells and specifically detecting lipid droplets and application
WO2019243617A9 (en) Fluorescent particles with molecularly imprinted fluorescent polymer shells for cell staining applications in cytometry and microscopy
CN108982447A (en) It is a kind of for detecting the preparation method and application of the ratio formula fluorescence probe of hydrazine
CN112500386B (en) Near-infrared HClO fluorescent probe based on piroctone olamine, preparation and application thereof
Zhu et al. Fluorescent AIE dots encapsulated organically modified silica (ORMOSIL) nanoparticles for two-photon cellular imaging
CN108329302A (en) A kind of half flower cyanines class near infrared fluorescent probe compound of sulfide specificly-response and its preparation method and application
Zhu et al. Ultra-bright carbon quantum dots for rapid cell staining
Kurutos et al. Organelle-selective near-infrared fluorescent probes for intracellular microenvironment labeling
CN114057604A (en) Cell nucleus fluorescent dye and dyeing method thereof
WO2022027174A1 (en) Cell nucleus fluorescent dye and dyeing method therefor
US8198098B2 (en) Optical measurement method for determining the pH of a medium using ageladine A as a fluorescent pH indicator
Papkovsky et al. O 2-sensitive probes based on phosphorescent metalloporphyrins
CN107576787B (en) Kit for fluorescence labeling cell nucleus and labeling method thereof
NL2032008B1 (en) Fluorescent labeling kit for a tumor cell nucleus and labeling method thereof
CN115028572B (en) Carbazole benzo [ e ] indole hybrid compound, and preparation method and application thereof
Ye et al. Quantitative image analysis and cell imaging of peroxynitrite based on an upconversion luminescence total internal reflection single particle imaging platform

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 20947864

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

32PN Ep: public notification in the ep bulletin as address of the adressee cannot be established

Free format text: NOTING OF LOSS OF RIGHTS PURSUANT TO RULE 112(1) EPC (EPO FORM 1205A DATED 13/04/2023)

122 Ep: pct application non-entry in european phase

Ref document number: 20947864

Country of ref document: EP

Kind code of ref document: A1