Cell nucleus dye molecule with aggregation-induced emission activity as well as preparation method and application of cell nucleus dye molecule

Abstract

The invention provides a cell nucleus dye molecule with aggregation-induced emission activity as well as a preparation method and application thereof, and relates to the technical field of fluorescence labeling. The cell nucleus dye molecule with the aggregation-induced emission activity is a fluorescent molecule dye material which is modified by pyridinium and hydroxamic acid based on triphenylamine or triphenylamine aryl as a mother nucleus and has the aggregation-induced emission activity, and can be used for cell nucleus specific fluorescent labeling of biological tissues such as brain and the like. The dye molecule can rapidly, simply and conveniently carry out specific fluorescence labeling on a cell nucleus, fluorescence signal emission is obviously enhanced, low-background fluorescence is provided, and meanwhile, the dye molecule has a washing-free function.

Description

Technical Field

[0001] The invention relates to the technical field of fluorescent labeling, and in particular to a cell nucleus dye molecule with aggregation-induced luminescence activity and a preparation method and application thereof. Background Art

[0002] The cell nucleus is a highly specialized subcellular organelle and the largest and most important cell structure in eukaryotic cells. As the "heart" of the cell, the cell nucleus is the storage place for genetic information and the control center of the cell. The cell nucleus is composed of the nuclear membrane, nuclear lamina, chromatin, nucleolus and nuclear body. The nuclear membrane is composed of a double layer containing protein and phospholipid molecules, and there are a large number of nuclear pores on the surface of the membrane. The nuclear pore is a selective hydrophilic channel. Small molecules (such as ions, metabolites, etc.) pass through the nuclear pores by simple diffusion, while macromolecules (such as proteins, RNA, ribosomal subunits, etc.) need to be transported by active transport under the guidance of nuclear translocation signals and transport carriers. Chromatin is the existing form of genetic material in the cell, mainly composed of DNA and histones. Chromatin also contains some RNA, which is equally important for the normal function of chromatin. The nucleolus is mainly composed of RNA and protein. Its functional changes are manifested as abnormal ribosome biogenesis or nucleolar stress, which can induce a variety of diseases. The various biological molecules contained in the cell nucleus are closely related to normal life activities, the occurrence and development of diseases.

[0003] In recent years, fluorescence imaging has attracted more and more attention due to its advantages such as intuitiveness and in situ. The development of fluorescent probes for high-resolution imaging of the cell nucleus and detection of biological molecules in the cell nucleus is of great value for understanding the normal physiological processes of cells or organisms and for disease diagnosis and treatment. Aggregation-induced emission (AIE) is different from conventional luminophores. Typical AIE luminophores have a propeller-shaped non-planar structure. In dilute solutions, AIE molecules rotate and consume energy through non-radiative pathways, making them non-luminescent. When bound to and wrapped in the cell nucleus, the free rotation of the dye molecules is limited due to mutual constraints from adjacent molecules, thereby inducing high-efficiency fluorescence activity. This unique AIE property provides a responsive fluorescence lighting analysis strategy in chemical sensing and visual biological imaging. AIE dye molecules can be used for labeling to obtain wash-free imaging labels, making the related labeling operation process simple. Many AIE fluorescent dye systems have been proven to be materials with high brightness, low background, low toxicity, good cell permeability and good photostability.

[0004] In order to observe the fluorescent labeling signal of three-dimensional thick tissue samples, traditional technologies usually first slice the tissue and then image the slice, or first image the tissue surface and then cut it, and reconstruct the captured two-dimensional image in three dimensions to finally obtain three-dimensional information of tissues and organs. However, these methods require a lot of time and effort, and have high deformation rates and difficulty in tissue reconstruction. Therefore, tissue clearing technology came into being. It is an emerging technology. Its principle is to use physical or chemical methods to match the refractive index to make the tissue optically transparent. The transparent tissue is conducive to the penetration of light and can be combined with fluorescence microscopy imaging technology to achieve thicker and complete three-dimensional imaging of tissues and organs. Tissue clearing technology combined with fluorescence imaging and three-dimensional reconstruction technology can perform three-dimensional observation and analysis of the contents and structure of tissues at the cellular level.

[0005] Using fluorescent probes to label cell nuclei throughout the brain has a wide range of research value in neuroscience research, especially in understanding brain structure and function, neuronal types and connections, neural circuits, etc. For understanding neuronal types and distribution: By labeling cell nuclei, researchers can identify and classify different types of neurons and study their distribution in the brain. This is essential for understanding the functions of different brain regions and the construction of neural circuits. For understanding neural connections and circuits: Labeling cell nuclei can also help researchers track the connections between neurons and reveal the structure and function of different neural circuits, which helps explain the activity patterns of the brain when performing specific tasks and the changes in the nervous system under different physiological and pathological conditions. For understanding synaptic morphology: By labeling cell nuclei, the morphological characteristics of synapses, such as density, size, and distribution, can be better studied, which is essential for understanding signal transmission between neurons and synaptic plasticity. For understanding neural development and regeneration: Researchers can use cell nucleus labeling technology to track the development of neurons and reveal changes in cell nuclei at different developmental stages. In addition, for the study of neural regeneration, labeling cell nuclei can also be used to study the generation and integration of new neurons. For understanding neurological disease research: By comparing the characteristics and distribution of neuronal nuclei in normal and disease models, it can help researchers understand the pathogenesis of neurological diseases and find potential treatments, so new dye molecules are needed to support the research. Summary of the invention

[0006] In view of the shortcomings of the prior art, the present invention provides a cell nuclear dye molecule with aggregation-induced luminescence activity and a preparation method and application thereof. The new cell nuclear dye molecule can quickly and easily specifically fluorescently label the cell nucleus and significantly enhance the fluorescence signal emission, providing low background fluorescence while having a wash-free function.

[0007] To achieve the above objectives, the technical solution of the present invention is implemented through the following technical solutions:

[0008] A cell nuclear dye molecule with aggregation-induced luminescence activity, wherein the chemical structure of the cell nuclear dye molecule is any one of the following I, II, III, IV, and V:

[0009] Ⅰ: Ⅱ:

[0010] III: IV:

[0011] V:

[0012] And wherein at least one of R, R', R" or R"' is selected from any one of the following three groups: The other groups are selected from any one of hydrogen, heteroatoms, alkyl groups, unsaturated alkyl groups, heteroalkyl groups, cycloalkyl groups, heterocycloalkyl groups, aryl groups and heteroaryl groups.

[0013] Preferably, the cell nuclear dye molecule is PTHA and its derivatives, and the molecular formula of PTHA is C 30 H 27 N4O4, and its compound structure is as follows:

[0014]

[0015] The chemical structure of PTHA derivatives is as follows:

[0016]

[0017] Wherein, R, R' and R" groups are selected from any one of hydrogen, heteroatom, alkyl, unsaturated alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, carboxyl, aryl and heteroaryl.

[0018] The method for preparing a cell nuclear dye molecule having aggregation-induced luminescence activity comprises the following steps:

[0019] S1. Under the protection of an inert gas, any one of 4-[N,N-bis(4-bromophenyl)amino]benzaldehyde, N,N-diphenyl-2-naphthylamine, N,N-dinaphthylaniline, tri(2-naphthyl)amine, and 2-(N,N-diphenylamino)thiophene and pyridine-4-boric acid are dissolved in a first solvent at a molar ratio of 1:1-1.5, and 0.01-0.05 mmol of tetrakistriphenylphosphine palladium is added to carry out a Suzuki coupling reaction, and the mixture is separated and purified by silica gel column chromatography to obtain compound 1 for standby use;

[0020] S2, under the protection of inert gas, dissolving compound 1 and malonic acid in a second solvent at a molar ratio of 1:2-2.5, adding 20-50 mmol of piperidine, and performing Knoevenagel condensation reaction, and washing and purifying with ice water and acetone after the reaction to obtain compound 2;

[0021] S3, under the protection of inert gas, adding compound 2 to an excess of thionyl chloride solvent, after the reaction is completed, the solvent is dried, and then excess methanol is added to carry out esterification reaction, and separated and purified by silica gel column chromatography to obtain compound 3;

[0022] S4, adding compound 3 to methanol, dissolving by ultrasonication, then adding hydroxylamine and 5M sodium hydroxide to carry out acylation reaction, and washing and purifying with ice water and concentrated hydrochloric acid after the reaction to obtain compound 4;

[0023] S5. Compound 4 is added to iodomethane, dissolved by ultrasonication, and subjected to alkylation reaction. After the reaction is completed, the product is cooled to 0°C, washed with water and acetone to purify the final product, and the nuclear dye molecule PTHA is obtained.

[0024] Preferably, the inert gas is one of nitrogen, argon, helium or a mixture of several thereof, and the silica gel column chromatography is used as an eluent with a ratio of petroleum ether to dichloromethane of 5-1:1 for separation.

[0025] Preferably, the first solvent in step S1 is an aqueous solution of 1,4-dioxane, and the second solvent in step S2 is an ethanol solution.

[0026] Preferably, the temperature of the coupling reaction in step S1 is 60-80°C, and the reaction time is 12-20h; the temperature of the condensation reaction in step S2 is 60-80°C, and the reaction time is 15-24h; the temperature of the esterification reaction in step S3 is 0-10°C, and the reaction time is 2-6h; the temperature of the acylation reaction in step S4 is 0-10°C, and the reaction time is 4-6h; the temperature of the alkylation reaction in step S5 is 40-60°C, and the reaction time is 48-72h.

[0027] Preferably, the cell nuclear dye molecules are used to mark cell nuclei of whole brain tissue and to perform three-dimensional fluorescence labeling and analysis of whole brain tissue.

[0028] The method for applying the cell nucleus dye molecule to mark the cell nuclei of whole brain tissue comprises the following steps:

[0029] (1) dissolving the nuclear dye molecules in a dimethyl sulfoxide solution, mixing with a PBS solution to form a stock solution, and taking out a portion of the stock solution and diluting it to prepare a working solution;

[0030] (2) taking the animal brain, obtaining the whole brain or brain slices of the animal as samples;

[0031] (3) Using working solution to incubate and label sample cell nuclei;

[0032] (4) The labeled samples were dehydrated, degreased, permeabilized, and refractive index matched using the UDISCO clearing method;

[0033] (5) Using laser to irradiate the sample and perform fluorescence imaging.

[0034] Preferably, when the sample is a brain slice, fluorescence imaging is performed using a super-resolution laser confocal microscope.

[0035] Preferably, when the sample is a whole brain, three-dimensional fluorescence imaging is performed using a light sheet fluorescence microscope.

[0036] The present invention provides a cell nucleus dye molecule with aggregation-induced luminescence activity and a preparation method and application thereof, which have the following advantages over the prior art:

[0037] The present invention provides a fluorescent molecular dye material with aggregation-induced luminescence activity, which is based on triphenylamine or triphenylamine aromatics as a parent core and is modified by pyridinium salt and isohydroxyoxime acid. The electrostatic interaction between the positively charged pyridinium salt in the dye molecule and the negatively charged DNA and RNA in the cell nucleus and the hydrogen bonding between the isohydroxyoxime acid and the bases in the DNA and RNA restricts the movement of the dye molecules, thereby generating a phenomenon of significantly enhanced aggregation-induced luminescence. The main feature of the invention is that the dye molecules do not emit light after being first dissolved in dimethyl sulfoxide and then in PBS solution, but can be efficiently excited to emit fluorescence when combined with the cell nucleus, thereby serving as a simple and efficient method for fluorescent lighting marking of cell nuclei. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 A schematic diagram of the process for applying the method of fluorescently labeling cell nuclei;

[0039] Figure 2 This is the synthetic route for preparing the PTHA compound in Example 1;

[0040] Figure 3 The ultraviolet absorption and fluorescence emission spectra of the PTHA compound prepared in Example 1;

[0041] Figure 4 is the hydrogen nuclear magnetic resonance spectrum of the PTHA compound prepared in Example 1;

[0042] Figure 5 is the nuclear magnetic resonance mass spectrum of the PTHA compound prepared in Example 1;

[0043] Figure 6 Middle: a is a 1 mm mouse brain slice that was not stained and transparentized; b is a 1x10 -5mol / L PTHA / DAPI molecules co-stained non-transparent 1mm mouse brain sections; c is the concentration used is 1x10 -5 mol / L PTHA / DAPI molecules co-stained cleared 1mm mouse brain sections;

[0044] Figure 7 For the 405 nm channel using a 10x confocal microscope, the concentration was 1x10 -5 mol / L DAPI molecules fluorescently labeled the nuclei of 1 mm rat brain sections, scale bar is 1000 μm;

[0045] Figure 8 For the 488 nm channel using a 10x confocal microscope, the concentration was 1x10 -5 mol / LPTHA molecules fluorescently labeled nuclei in 1 mm rat brain sections, scale bar is 1000 μm;

[0046] Fig. 9 To use the confocal 10x merge channel, the concentration is 1x10 -5 mol / LPTHA / DAPI molecules fluorescently labeled the nuclei of 1 mm rat brain sections, scale bar is 1000 μm;

[0047] Fig.10 The middle a is the 488nm channel using a confocal 63x microscope, with a concentration of 1x10 -5 mol / LPTHA molecules fluorescently labeled the nuclei of 1 mm rat brain slices; b was using a confocal 63x microscope at 405 nm channel, with a concentration of 1x10 -5 mol / L DAPI molecules fluorescently labeled the nuclei of 1mm mouse brain slices; c is the merge channel using a confocal 63x microscope, with a concentration of 1x10 -5 mol / LPTHA / DAPI molecules fluorescently label the nuclei of 1 mm rat brain slices; scale bars are 20 μm;

[0048] Fig.11 For the 488 nm channel using a 10x confocal microscope, the concentration was 1x10 -5 mol / LPTHA molecules fluorescently labeled nuclei in 1 mm rat brain sections, scale bar is 1000 μm;

[0049] Fig.12 The 639 nm channel of the confocal microscope at 10x was used to fluorescently label the neuronal nuclei of 1 mm rat brain sections with the primary antibody NeuN and the secondary antibody Alexa fluor 647. The scale bar is 1000 μm.

[0050] Fig.13 The merge channel was acquired using a 10x confocal microscope, and the scale bar is 1000 μm;

[0051] Fig.14 The middle a is the 488nm channel using a confocal 63x microscope, with a concentration of 1x10 -5 mol / LPTHA molecules were used to fluorescently label the nuclei of 1mm rat brain slices; b was the use of the 647nm channel of the confocal 63x microscope, the primary antibody NeuN, and the secondary antibody Alexa fluor 647 molecules to fluorescently label the neuronal nuclei of 1mm rat brain slices; c was the use of the merge channel of the 63x microscope; the scale bars are all 10μm;

[0052] Fig.15 For the 488 nm channel using a 10x confocal microscope, the concentration was 1x10 -5 mol / LPTHA molecules fluorescently labeled (AD) 1 mm rat brain section cell nuclei, scale bar 1000 μm;

[0053] Fig.16 The 647 nm channel of the confocal microscope at 10x was used, the primary antibody NeuN and the secondary antibody Alexa fluor 647 were used to fluorescently label the neuronal nuclei of 1 mm rat brain sections (AD). The scale bar is 1000 μm.

[0054] Fig.17 The merge channel was acquired using a 10x confocal microscope, and the scale bar is 1000 μm;

[0055] Fig.18 The middle a is the 488nm channel using a confocal 63x microscope, with a concentration of 1x10 -5 mol / LPTHA molecular fluorescence labeling (AD) of 1mm rat brain slice cell nuclei; b is the use of 647nm channel of confocal 63x microscope, primary antibody NeuN, secondary antibody Alexa fluor647 molecular fluorescence labeling (AD) of 1mm rat brain slice neuron cell nuclei; c is the use of confocal 63x microscope merge channel; scale bar is 20μm;

[0056] Fig.19 For the 488 nm channel using a 10x confocal microscope, the concentration was 1x10 -5 mol / LPTHA molecules fluorescently labeled Thy1-GFP 1mm mouse brain section cell nuclei, scale bar is 1000μm;

[0057] Fig. 20 For the 488 nm channel using a 63x confocal microscope, the concentration was 1x10 -5 mol / LPTHA molecules fluorescently labeled Thy1-GFP 1mm mouse brain section cell nuclei, scale bar is 20μm;

[0058] Fig.21 a is the whole brain of the mouse that has not been stained and transparentized; b is the whole brain of the mouse that has been treated with 1x10-5 mol / L PTHA molecules were used to stain the whole brain of non-transparent rats; c is the concentration used is 1x10 -5 mol / L PTHA molecule staining of cleared mouse brain;

[0059] Fig. 22 For light sheet microscopy, the concentration was 1x10 -5 mol / LPTHA molecules fluorescently labeled the whole mouse brain, scale bar is 1500μm. DETAILED DESCRIPTION

[0060] In order to make the purpose, technical solution and advantages of the embodiments of the present invention clearer, the technical solution in the embodiments of the present invention is clearly and completely described below in combination with the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0061] Embodiment 1:

[0062] The steps for synthesizing PTHA dye molecules are:

[0063] 1. Synthesis of compound 1:

[0064] Place the corresponding 4-[N,N-bis(4-bromophenyl)amino]benzaldehyde (3.0 g, 7.89 mmol), pyridine-4-boric acid (1.45 g, 11.83 mmol), Pd(PPh3)4 (0.091 g, 0.079 mmol) and K2CO3 (6.54 g, 47.34 mmol) in a 500 mL two-necked round-bottom flask.

[0065] The flask was evacuated under vacuum and flushed with dry nitrogen three times. A mixture of 200 mL of 1,4-dioxane, 50 ml of deionized water was added to the reaction mixture. The reaction mixture was refluxed at 95 °C for 16 h. The reaction mixture was cooled to room temperature. The mixture was poured into water. The reaction mixture was extracted with dichloromethane three times. The organic layer was dried over anhydrous sodium sulfate. The organic layer was filtered and the solvent was evaporated.

[0066] The crude product was purified by silica gel column chromatography using a mixture of petroleum ether and dichloromethane in a ratio of 5:1 as the eluent to obtain the product (2.8 g) with a yield of 94%.

[0067] 1H NMR (400MHz, DMSO-d6) δ9.90 (s, 2H), 8.64 (d, J = 5.8Hz, 2H), 7.89 (t, J = 8.3Hz, 6H), 7.7 3(d,J=5.9Hz,2H),7.31(d,J=8.4Hz,2H),7.24(d,J=8.5Hz,4H).HRMS(APCI,m / z)calcd for C25H18N2O2[M] + ,379.14;found:379.139.

[0068] 2. Synthesis of compound 2:

[0069] In a 250mL double-necked round-bottom flask, compound 1 (2.54 g, 6.4 mmol), malonic acid (1.72 g, 20.16 mmol), piperidine (1.72 g, 20.16 mmol) and ethanol as solvent were added in sequence. The reaction mixture was refluxed for 24 hours. After the reaction, the mixture was poured into ice water, the precipitated product was filtered and washed with acetone to purify the product (2.1 g), with a yield of 68%.

[0070] 1 H NMR (400MHz, DMSO-d6) δ12.33(s,2H),8.60(d,J=5.2Hz,2H),7.80(d,J=8.3Hz,2H),7.66(dd,J=16.2,6.7Hz,6H),7 .55(d,J=15.9Hz,2H),7.18(d,J=8.2Hz,2H),7.06(d,J=8.2Hz,4H),6.42(d,J=16.0Hz,2H).HRMS(APCI,m / z)calcd for C 29 H 22 N2O4[M] + ,463.16;found:463.304.

[0071] 3. Synthesis of compound 3:

[0072] Compound 2 (1 g, 2.16 mmol) was added to a 250 mL single-mouth round-bottom flask and dissolved in 80 mL of thionyl chloride. The mixture was stirred at room temperature for 3 h, the solvent was dried by spin drying, 80 mL of methanol was added, the solvent was dried by spin drying, and the product (0.8 g) was purified by column chromatography on a silica gel column with a mixed petroleum ether and dichloromethane as the eluent in a ratio of 1:1. The yield was 75%.

[0073] 1H NMR (600MHz, DMSO-d6) δ8.62(s,2H),7.82(d,J=8.9Hz,2H),7.69(d,J=8.4Hz,6H),7.63(d,J=15.9Hz,2H) ,7.20(d,J=8.9Hz,2H),7.07(d,J=8.9Hz,4H),6.53(d,J=16.1Hz,2H),3.71(s,6H).HRMS(APCI,m / z)calcd for C 31 H 26 N2O4[M] + ,491.19;found:491.136.

[0074] 4. Synthesis of compound 4:

[0075] Compound 3 (0.6 g, 1.22 mmol) was added to a 100 mL single-necked round-bottom flask and dissolved in 30 mL of methanol. The mixture was dissolved by ultrasonication. Then, 1 mL of hydroxylamine and 1 mL of 5 M sodium hydroxide were added. The mixture was stirred at room temperature for 1 hour. Then, 1 mL of hydroxylamine was added and the temperature was raised to 40 ° C. After the reaction was completed, the mixture was cooled to 0 ° C, ice water was added, 2 mL of concentrated hydrochloric acid was added, the solid was precipitated, filtered, and the purified product (0.4 g) was washed with water. The yield was 67%.

[0076] 1 H NMR (400MHz, DMSO-d6) δ10.70(s,1H),9.02(s,1H),8.60(d,J=5.1Hz,2H),7.80(d,J=8.4Hz,2H),7.68(d,J=5.2Hz,2H),7.54(dd,J=1 3.4,8.4Hz,4H),7.42(d,J=15.9Hz,2H),7.17(d,J=8.0Hz,2H),7.08(d,J=8.2Hz,4H),6.37(d,J=15.5Hz,1H).HRMS(APCI,m / z)calcd for C 29 H 24 N4O4[M] + ,493.18;found:493.233.

[0077] 5. Synthesis of compound PTHA:

[0078] Compound 4 (0.2 g, 0.4 mmol) was added to a 50 mL single-necked round-bottom flask, followed by 10 mL of iodomethane, and the mixture was dissolved by ultrasonication. The mixture was heated at 40 °C for 36 hours. After the reaction was completed, the mixture was cooled to 0 °C, 10 ml of ice water was added, and the precipitated solid was filtered, washed with water, and then washed with acetone to purify the product (0.1 g). The yield was 40%.

[0079] 1 H NMR (400MHz, DMSO-d6) δ10.76(s,1H),9.04(s,1H),8.90(s,2H),8.40(s,2H),8.05(d,J=8.5Hz,2H),7.71-7.67(m,4H),7.5 8(d,J=8.6Hz,1H),7.15(d,J=8.9Hz,6H),7.09(d,J=8.0Hz,2H),6.42(d,J=15.7Hz,1H),4.29(s,3H).HRMS(APCI,m / z)calcd forC 30 H 27 N4O4[MI] + ,506.20;found:506.285.

[0080] Embodiment 2:

[0081] Fluorescence labeling experiments:

[0082] 1. Obtaining rat brain and making 1 mm rat brain slices

[0083] C57BL / 6J male mice aged 6-8 weeks were selected, and anesthetics were prepared by dissolving 10 mL PBS in a ratio of chloral:xylazine hydrochloride (mass ratio 8:1), and injected into the abdomen at a dose of 0.5% of the mouse body weight. After 10 minutes of deep anesthesia, 15 mL PBS solution and 15 mL 4% paraformaldehyde solution were perfused through the heart. After the liver faded and the whole body became rigid, the perfusion was stopped, and the mouse brain was fixed with paraformaldehyde solution overnight, and cut into 1 mm mouse brain slices ( Figure 6 a);

[0084] 2. Fluorescence labeling of 1mm mouse brain slices

[0085] Take out the 1 mm brain slices fixed with paraformaldehyde, wash them with PBS solution 3 times, 1 hour each time, and prepare PTHA working solution with a concentration of 1x10 -5 mol / L, incubate 1 mm brain slices for 10 min, and gently shake them in a 37°C constant temperature shaker ( Figure 6 b), there is no need to wash (because AIE molecules have good background removal ability and no-wash function), and the next step is to permeabilize the fluorescently labeled mouse brain, remove water, remove fat and match the refractive index.

[0086] 3. UDISCO clearing method to permeabilize 1 mm rat brain slices

[0087] After fluorescent labeling, 1 mm brain slices were immersed in 2 mL of 30%, 50%, 70%, 80%, 90%, or 100% tert-butyl alcohol aqueous solution, respectively, and shaken at 37°C for 1 hour, and then immersed in a mixed solution of 1.4 mL of benzyl alcohol, 2.8 mL of benzyl benzoate, and 1 mL of diphenyl ether and shaken at 37°C for 4 hours. Figure 6 c).

[0088] 4. Co-staining of PTHA molecules with commercial nuclear dye DAPI

[0089] After the transparent 1mm brain slice sample was made, PTHA molecules and DAPI were co-stained. First, the 1mm brain slice was placed in a concentration of 1x10 -5 mol / L PTHA molecule PBS solution, incubate for 20 minutes without washing, and then place in a 1x10 -5 mol / L DAPI solution, incubated for 1 hour, and then permeabilized using the UDISCO clearing method mentioned above, and then observed the DAPI-labeled cell nuclei using a confocal microscope at 405 nm ( Figure 7 ), PTHA labels the cell nucleus at 488 nm ( Figure 8 ).

[0090] From the whole 1mm mouse brain slice collected under a 10x microscope, the PTHA molecule labeled cell nuclei with wide range, high uniformity, and strong fluorescence brightness ( Fig. 9 ). The fluorescence signal of PTHA molecules for labeling cell nuclei in specific areas of 1mm brain slices, such as the hippocampus, striatum, cortex, thalamus, etc., is strong and uniform enough. Then, a 63x microscope is used to magnify and collect the data. The observable fluorescence signal indicates that the cell nuclei in the mouse brain are effectively labeled ( Fig.10 )

[0091] 5. Co-staining of PTHA molecules with NeuN antibodies (targeting neuronal nuclei)

[0092] 1mm mouse brain slices were co-stained with PTHA molecules and rabbit-derived NeuN antibodies (targeting neuronal nuclei), and immunofluorescence staining of neuronal nuclei was performed using NeuN antibodies. First, the brain slices were pretreated and initially infiltrated with 50%, 80% methanol / PBS (2mL) solutions, and constant temperature shaking was performed for 1 hour each time. 1mL hydrogen peroxide solution, 1mL dimethyl sulfoxide, and 4mL methanol were added for bleaching, and shaken overnight. 2mL0.2% Triton PBS solution, 30μL dimethyl sulfoxide, and 45mg glycine were added for infiltration, and constant temperature shaking was performed for 9 hours. 2mL0.2% Triton PBS solution, 40μL dimethyl sulfoxide, 30μL goat serum, etc. were added for blocking, and constant temperature shaking was performed for 10 hours. 2mL PBS solution, 20μL Tween 20, and 100mg heparin sodium were added for washing, and constant temperature shaking was performed for 9 hours. For the primary antibody, 2 mL of 0.2% Triton PBS solution, 30 μL of dimethyl sulfoxide, and 30 μL of goat serum were added, and 2.5 μL of NeuN from rabbit primary antibody was incubated for 12 hours, and then 2 mL of PBS solution, 20 μL of Tween 20, and 10 mg of sodium heparin were added for washing 3 times, and each time the temperature was kept constant for 6 hours. For the secondary antibody, 2 mL of 0.2% Triton PBS solution, 30 μL of dimethyl sulfoxide, and 30 μL of goat serum were added, and 5 μL of goat anti-rabbit secondary antibody (Alexa fluor 647) was incubated for 12 hours, and then 2 mL of PBS solution, 20 μL of Tween 20, and 100 mg of sodium heparin were added for washing 3 times, and each time the temperature was kept constant for 6 hours. Then 1x10 -5 mol / LPHTA molecules were incubated for 10 min and finally made transparent using the UDISCO method. The fluorescence signal was collected by a laser confocal microscope with a magnification of 10 times, and the 488nm channel collected the cell nucleus signal ( Fig.11 ), the 639nm channel collects the neuronal nucleus signal ( Fig.12 ), merge channel signal ( Fig.13 ); Signal collected by 63x microscope ( Fig.14 )

[0093] 6. Labeling of cell nuclei in 1mm mouse brain slices in Alzheimer's disease (AD) mouse model

[0094] Fluorescence labeling experiment was performed on the cell nuclei of Alzheimer's disease (AD) mouse model. 1mm brain slices of 16-week-old male APP / PSEN1 transgenic AD mice were selected for PTHA molecular staining. The staining experiment and transparentization method were the same as the previous experiment. The fluorescence signal was collected by laser confocal microscopy with a 10x lens, and the 488nm channel collected the cell nuclear signal ( Fig.15 ), the 639nm channel collects the neuronal nucleus signal ( Fig.16 ), merge channel signal ( Fig.17); Signal collected by 63x microscope ( Fig.18 ).

[0095] 7. Labeling of nuclei in 1 mm brain sections of Thy1-GFP transgenic mouse model

[0096] The nuclei of 1 mm brain slices from 6-8 week old male Thy1-GFP transgenic mice were fluorescently labeled, and the fluorescence signals ( Fig.19 ), signal collected by 488nm channel of 63x microscope ( Fig. 20 ).

[0097] 8. UDISCO clearing method for permeabilization of whole brain tissue of rats

[0098] The whole mouse brain after fluorescence labeling was immersed in 10 mL of 30%, 50%, 70%, 80%, 90%, or 100% tert-butyl alcohol aqueous solution, and shaken at a constant temperature of 37°C for 12 hours, and then immersed in a mixed solution of 5 mL of benzyl alcohol, 10 mL of benzyl benzoate, and 1 mL of diphenyl ether and shaken at a constant temperature for 10 hours.

[0099] 9. Fluorescently labeled nuclei of whole brain of mouse

[0100] After removing the mouse brain fixed overnight ( Fig.21 a) Wash with 10 mL PBS solution at 37°C for 3 times, 2 hours each time, using a concentration of 1x10 -5 mol / L PTHA molecules were incubated for 48 hours, and the PTHA molecule solution was replaced every 6 hours. Fig.21 b) Immerse in 10 mL of 30%, 50%, 70%, 80%, 90%, or 100% tert-butyl alcohol aqueous solution, shake at 37°C for 12 hours, and then immerse in a mixed solution of 5 mL of benzyl alcohol, 10 mL of benzyl benzoate, and 1 mL of diphenyl ether, shake at 37°C for 10 hours. Fig.21 c).

[0101] 10. Imaging with light sheet microscopy

[0102] After the characterization of the experimental part of the 1 mm mouse brain slice was completed, the whole mouse brain was sampled, fixed, stained, transparentized and imaged in three dimensions using a 4x light sheet microscope ( Fig. 22 ).

[0103] The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit the same. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that the technical solutions described in the aforementioned embodiments may still be modified, or some of the technical features may be replaced by equivalents. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A nuclear dye molecule with aggregation-induced emission activity, characterized in that: The chemical structure of the cell nuclear dye molecule is any one of the following Ⅰ, Ⅱ, Ⅲ, Ⅳ, Ⅴ: Ⅰ： Ⅱ： Ⅲ： Ⅳ： Ⅴ： And wherein at least one of R, R', R" or R"' is selected from any one of the following three groups: The other groups are selected from any one of hydrogen, heteroatoms, alkyl groups, unsaturated alkyl groups, heteroalkyl groups, cycloalkyl groups, heterocycloalkyl groups, aryl groups and heteroaryl groups.

2. A nuclear dye molecule with aggregation-induced emission activity according to claim 1, characterized in that: The cell nucleus dye molecule is PTHA and its derivatives, and the molecular formula of PTHA is C 30 H 27 N4O4, and its compound structure is as follows: The chemical structure of PTHA derivatives is as follows: Wherein, R, R' and R" groups are selected from any one of hydrogen, heteroatom, alkyl, unsaturated alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, carboxyl, aryl and heteroaryl.

3. A method for preparing a nuclear dye molecule as claimed in claim 1 or 2, characterized in that: The preparation method comprises the following steps: S1. Under the protection of an inert gas, any one of 4-[N,N-bis(4-bromophenyl)amino]benzaldehyde, N,N-diphenyl-2-naphthylamine, N,N-dinaphthylaniline, tri(2-naphthyl)amine, and 2-(N,N-diphenylamino)thiophene and pyridine-4-boric acid are dissolved in a first solvent at a molar ratio of 1:1-1.5, and 0.01-0.05 mmol of tetrakistriphenylphosphine palladium is added to carry out a Suzuki coupling reaction, and the mixture is separated and purified by silica gel column chromatography to obtain compound 1 for standby use; S2, under the protection of inert gas, dissolving compound 1 and malonic acid in a second solvent at a molar ratio of 1:2-2.5, adding 20-50 mmol of piperidine, and performing Knoevenagel condensation reaction, and washing and purifying with ice water and acetone after the reaction to obtain compound 2; S3, under the protection of inert gas, adding compound 2 to an excess of thionyl chloride solvent, after the reaction is completed, the solvent is dried, and then excess methanol is added to carry out esterification reaction, and separated and purified by silica gel column chromatography to obtain compound 3; S4, adding compound 3 to methanol, dissolving by ultrasonication, then adding hydroxylamine and 5M sodium hydroxide to carry out acylation reaction, and washing and purifying with ice water and concentrated hydrochloric acid after the reaction to obtain compound 4; S5. Compound 4 is added to iodomethane, dissolved by ultrasonication, and subjected to alkylation reaction. After the reaction is completed, the product is cooled to 0°C, washed with water and acetone to purify the final product, and the nuclear dye molecule PTHA is obtained.

4. The preparation method according to claim 3, characterized in that: The inert gas is one of nitrogen, argon, and helium, or a mixture of several thereof, and the silica gel column chromatography method is used for separation with an eluent having a ratio of petroleum ether to dichloromethane of 5-1:

1.

5. The preparation method according to claim 3, characterized in that: In the step S1, the first solvent is a 1,4-dioxane aqueous solution, and in the step S2, the second solvent is an ethanol solution.

6. The preparation method according to claim 3, characterized in that: The temperature of the coupling reaction in step S1 is 60-80°C, and the reaction time is 12-20h; the temperature of the condensation reaction in step S2 is 60-80°C, and the reaction time is 15-24h; the temperature of the esterification reaction in step S3 is 0-10°C, and the reaction time is 2-6h; the temperature of the acylation reaction in step S4 is 0-10°C, and the reaction time is 4-6h; the temperature of the alkylation reaction in step S5 is 40-60°C, and the reaction time is 48-72h.

7. A use of a nuclear dye molecule as claimed in any one of claims 1 to 2, characterized in that: The cell nuclear dye molecules are used for marking cell nuclei of whole brain tissues and for whole brain three-dimensional fluorescence marking and analysis of brain tissues.

8. The use according to claim 7, characterized in that: The method for applying the cell nucleus dye molecule to mark the cell nuclei of whole brain tissue comprises the following steps: (1) dissolving the nuclear dye molecules in a dimethyl sulfoxide solution, mixing with a PBS solution to form a stock solution, and taking out a portion of the stock solution and diluting it to prepare a working solution; (2) taking the animal brain, obtaining the whole brain or brain slices of the animal as samples; (3) Using working solution to incubate and label sample cell nuclei; (4) The labeled samples were dehydrated, degreased, permeabilized, and refractive index matched using the UDISCO clearing method; (5) Using laser to irradiate the sample and perform fluorescence imaging.

9. The use according to claim 8, characterized in that: When the sample is a brain slice, fluorescence imaging is performed using a super-resolution laser confocal microscope.

10. The use according to claim 8, characterized in that: When the sample is a whole brain, three-dimensional fluorescence imaging is performed using light-sheet fluorescence microscopy.

Images