WO2022027174A1 - Colorant fluorescent de noyau cellulaire et procédé de coloration associé - Google Patents

Colorant fluorescent de noyau cellulaire et procédé de coloration associé 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
English (en)
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/fr
Publication of WO2022027174A1 publication Critical patent/WO2022027174A1/fr

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

La présente invention concerne un colorant fluorescent de noyau cellulaire, un procédé de coloration associé et l'utilisation de celui-ci Un nouveau colorant fluorescent pour la coloration de noyau cellulaire est proposé. Le colorant fluorescent de noyau cellulaire selon l'invention présente une bonne perméabilité membranaire, une sensibilité élevée, une efficacité de coloration nucléaire rapide, une bonne stabilité à la lumière, une bonne biocompatibilité, et une lumière d'excitation et une lumière d'émission avec des longueurs d'onde plus longues, et peut être appliqué à la coloration fluorescente de noyau cellulaire.
PCT/CN2020/106563 2020-08-03 2020-08-03 Colorant fluorescent de noyau cellulaire et procédé de coloration associé WO2022027174A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
PCT/CN2020/106563 WO2022027174A1 (fr) 2020-08-03 2020-08-03 Colorant fluorescent de noyau cellulaire et procédé de coloration associé

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
PCT/CN2020/106563 WO2022027174A1 (fr) 2020-08-03 2020-08-03 Colorant fluorescent de noyau cellulaire et procédé de coloration associé

Publications (1)

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

Family

ID=80119129

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2020/106563 WO2022027174A1 (fr) 2020-08-03 2020-08-03 Colorant fluorescent de noyau cellulaire et procédé de coloration associé

Country Status (1)

Country Link
WO (1) WO2022027174A1 (fr)

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101004377A (zh) * 2007-01-24 2007-07-25 北京望升伟业科技发展有限公司 精子浓度快速诊断试剂盒及其制备和使用方法
CN105143847A (zh) * 2013-02-05 2015-12-09 三路影像公司 细胞染色组合物及其用途

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101004377A (zh) * 2007-01-24 2007-07-25 北京望升伟业科技发展有限公司 精子浓度快速诊断试剂盒及其制备和使用方法
CN105143847A (zh) * 2013-02-05 2015-12-09 三路影像公司 细胞染色组合物及其用途

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 (ja) 生体物質検出用の蛍光標識体
EP2613138B1 (fr) Procédé de détection de substance biologique
JP6586070B2 (ja) 核酸を含有する細胞または別の生体材料を分析する方法
WO2016165487A1 (fr) Procédé de surveillance en temps réel de la mithophagie au moyen d'une sonde biologique spécifique à la mitochondrie à fluorescence photostable dotée de caractéristique d'aie
CN108138043A (zh) 红色荧光AIEgen
Martinić et al. Near-infrared luminescent metallacrowns for combined in vitro cell fixation and counter staining
CN107936945B (zh) 一种具有聚集诱导发光特性的荧光粘度探针
CN110981842B (zh) 一种区分正常细胞和癌细胞的特异性检测脂滴的荧光探针及应用
WO2019243617A9 (fr) Particules fluorescentes à enveloppes polymères fluorescentes à empreintes moléculaires pour applications de coloration cellulaire en cytométrie et microscopie
CN112500386B (zh) 基于吡罗红肟的近红外HClO荧光探针、制备及其应用
Zhu et al. Fluorescent AIE dots encapsulated organically modified silica (ORMOSIL) nanoparticles for two-photon cellular imaging
CN108329302A (zh) 一种硫化物特异性响应的半花菁类近红外荧光探针化合物及其制备方法和应用
Jung et al. Photophysics of New Water‐Soluble Terrylenediimide Derivatives and Applications in Biology
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 (zh) 一种细胞核荧光染料及其染色方法
WO2022027174A1 (fr) Colorant fluorescent de noyau cellulaire et procédé de coloration associé
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 (zh) 荧光标记细胞核用试剂盒及其标记方法
NL2032008B1 (en) Fluorescent labeling kit for a tumor cell nucleus and labeling method thereof
CN115028572B (zh) 一类咔唑苯并[e]吲哚杂合物及其制备方法与应用
Ye et al. Quantitative image analysis and cell imaging of peroxynitrite based on an upconversion luminescence total internal reflection single particle imaging platform
Papkovsky et al. Evolution of cell-penetrating phosphorescent O2 probes

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