An aie fluorescent dye, and a preparation method and application thereof

Abstract

The application discloses an AIE fluorescent dye, a structural formula of which is shown in the following formula (I), wherein R1 and R2 are independently selected from alkyl alcohol with carbon number of 1-6; and X is selected from water-soluble anions. The fluorescent dye disclosed by the application has AIE characteristics, can realize rapid staining of living cells, and the staining depth can be deep to the nucleolus of the cell nucleus, so that the morphology of the whole cell tissue is clear and visible; is expected to be applied in the field of living cell fluorescence imaging, and further used for preparing a fluorescent kit for rapid diagnosis of cancer cells; the fluorescent probe has good water solubility, small cytotoxicity and good biocompatibility; the fluorescent color is orange red, the Stokes shift is large, and the anti-self-fluorescence interference ability is strong.

Description

Technical Field

[0001] This invention relates to the technical field of fluorescent probes, and more particularly to an AIE fluorescent dye, its preparation method, and its application in the field of live cell fluorescence imaging, as well as its further use in preparing fluorescent reagent kits for rapid diagnosis of cancer cells. Background Technology

[0002] In cancer diagnosis, imaging methods can only provide a rough overview of tumor morphology, density, growth pattern, and other basic information. However, only one method is widely used for rapid clinical pathological diagnosis of tumors: the hematoxylin-eosin (HE) staining method, considered the "gold standard" by the international medical community and used for over a century. HE staining uses a combination of hematoxylin and eosin dyes. Hematoxylin, being alkaline, stains basophilic structures of the tissue (such as ribosomes, cell nuclei, and ribonucleic acid in the cytoplasm) blue-purple; eosin, being acidic, stains eosinous structures of the tissue (such as intracellular and intercellular proteins, including Lewy bodies, alcohol bodies, and most of the cytoplasm) pink, thus making the morphology of the entire cellular tissue clearly visible. Typically, malignant tumors exhibit disordered tissue structure, cellular atypia, and abnormal nuclear structure. Currently, the medical community uses the overall morphology and nuclear structure of cellular tissue obtained through HE staining as the basis for clinical cancer diagnosis.

[0003] However, HE staining relies on ordinary optical microscopy for imaging, which has low transmittance / resolution, and the color contrast between blue-violet and pink is not strong. To achieve sufficient contrast and transmittance, tissue sections with a thickness of 4-6 micrometers are generally used for HE staining to observe cell structure and morphology. However, living tumor tissue is soft and cannot be directly cut into such thin sections. Therefore, current HE staining methods generally involve pre-treatment of tumor tissue by cryo-hardening or paraffin hardening before sectioning. Cryo-hardening of living tumor tissue typically uses liquid nitrogen or dry ice to harden the tissue before ultrathin sections are made. Because cryo-hardening is rapid, HE staining with pre-treatment by cryo-hardening is often used for rapid clinical diagnosis of tumors during surgery. However, freezing inevitably forms ice crystals, which damage the size and morphology of cells in the tissue. This distorts the diagnostic information of "disordered tissue structure and cellular atypia" on which the "gold standard" relies, reducing diagnostic accuracy. As a result, some patients with suspected tumor tissue found during intraoperative biopsy require a second surgery for resection after routine pathological confirmation. Paraffin-cured tumor tissue primarily replaces freezing by sealing the tissue with paraffin. The slow curing process avoids damage to cells and preserves the accuracy and authenticity of the information. While HE staining typically achieves a 99% accuracy rate in routine tumor pathological diagnosis, the paraffin curing process is lengthy, usually requiring 5-7 days (hence the term "routine tumor pathological diagnosis"). This extended diagnostic time is far from meeting the time requirements for rapid intraoperative tumor pathological diagnosis.

[0004] In summary, it is clear that intraoperative rapid clinical pathological diagnosis of tumors relies on cell imaging technology. If new dyes can be developed that can achieve rapid, accurate, and realistic cell imaging of cancer cells, it may be possible to eliminate some or even all routine HE staining pathological diagnosis, saving a lot of time, manpower, and resources, with immeasurable economic benefits. Summary of the Invention

[0005] To address the aforementioned problems in existing technologies, this invention discloses a fluorescent dye with AIE properties, enabling rapid staining of living cells to a depth reaching the nucleolus, thus clearly revealing the morphology of the entire cell tissue. It holds promise for applications in the field of live cell fluorescence imaging and further for the preparation of fluorescent reagent kits for rapid diagnosis of cancer cells. This fluorescent probe exhibits good water solubility, low cytotoxicity, and good biocompatibility; its fluorescence color is orange-red, and it exhibits a large Stokes shift, demonstrating strong resistance to autofluorescence interference.

[0006] The specific technical solution is as follows:

[0007] An AIE fluorescent dye has the following structural formula (Ⅰ):

[0008]

[0009] In the formula, R1 and R2 are independently selected from alkyl alcohols with 1 to 6 carbon atoms; X is selected from water-soluble anions, such as halogen anions, nitrate ions, etc.

[0010] Preferred:

[0011] R1 = R2;

[0012] X is selected from halide anions.

[0013] Further optimization:

[0014] R1 = R2, selected from one or more of CH2OH, CH2CH2OH, and CH2CH2CH2OH;

[0015] X is selected from F - Cl - ,Br - One or more of them.

[0016] The fluorescence emission wavelength of the AIE fluorescent dye prepared in this invention is 590–620 nm.

[0017] This invention also discloses a method for preparing the aforementioned AIE fluorescent dye, comprising:

[0018] (1) Using N,N-dialkylolamine and p-fluorobenzaldehyde as raw materials, intermediate product A was prepared by nucleophilic substitution reaction;

[0019] (2) Mix 4-methylpyridine, 3-halopropyltrimethylammonium salt and organic solvent A, and heat to reflux temperature to carry out alkylation reaction to prepare intermediate product B;

[0020] (3) Under an inert atmosphere, intermediate product A and intermediate product B are mixed with organic solvent B and heated to reflux temperature to carry out a nucleophilic addition reaction to obtain the AIE fluorescent dye.

[0021] In step (1):

[0022] The N,N-dialkylolamine is selected from one or more of diethanolamine, diethanolamine, N,N-di(3-hydroxypropyl)amine, N,N-di(4-hydroxybutyl)amine, N,N-di(5-hydroxypentyl)amine, and N,N-di(6-hydroxyhexyl)amine;

[0023] The nucleophilic substitution reaction is carried out in the presence of a catalyst, which is selected from one or more of aluminum trichloride, ferric trichloride, and titanium tetrachloride.

[0024] The temperature for the nucleophilic substitution reaction is 110–160 °C, preferably 110–130 °C.

[0025] Preferably, the molar ratio of N,N-dialkylolamine to p-fluorobenzaldehyde is 1 to 8:1, more preferably 3 to 6:1, and even more preferably 4.5:1.

[0026] Preferably, the amount of catalyst added is 1 to 10 wt% based on the total mass of N,N-dialkylolamine and p-fluorobenzaldehyde.

[0027] In step (2):

[0028] The organic solvent A is selected from one or more of N,N-dimethylformamide, N,N-dimethylacetamide, dimethyl sulfoxide, methanol, and ethanol;

[0029] The molar ratio of 4-methylpyridine to 3-halopropyltrimethylammonium salt is 1.0 to 1.2:1; preferably 1.0 to 1.1:1.

[0030] The specific reflux temperature should be adjusted according to the type of organic solvent A used.

[0031] In step (3):

[0032] The molar ratio of intermediate product A to intermediate product B is 1.0 to 1.1:1; preferably, they are added in equal molar amounts.

[0033] The organic solvent B is selected from one or more of N,N-dimethylformamide, N,N-dimethylacetamide, dimethyl sulfoxide, methanol, and ethanol;

[0034] The nucleophilic addition reaction is carried out in the presence of a catalyst, which is selected from one or more of piperidine, potassium tert-butoxide, and pyridine.

[0035] The amount of catalyst added is 1-3 wt% based on the mass of intermediate product B.

[0036] The specific reflux temperature should be adjusted accordingly based on the type of organic solvent B used.

[0037] The inert atmosphere used is a common atmosphere in the art, such as nitrogen, argon, helium, etc.

[0038] Preferably, in step (3), the product after nucleophilic addition reaction needs to be post-processed, including rotary evaporation and purification operations. The purification operation refers to silica gel column separation operation, and the eluent is ethanol.

[0039] This invention investigated the photophysical properties, AIE properties, cytotoxicity, and concentration response of the prepared AIE fluorescent dye. The results showed that:

[0040] The fluorescent probe prepared by this invention has good water solubility, low cytotoxicity, and good biocompatibility; it is also a compound that emits (orange) red fluorescence with AIE properties and has strong resistance to biological auto-background fluorescence; and it responds to both DNA and RNA, with the fluorescence intensity increasing with the increase of DNA and RNA content.

[0041] The present invention also discloses the application of the aforementioned AIE fluorescent dye in the field of live cell fluorescence imaging.

[0042] Experiments have shown that the AIE fluorescent dye prepared in this invention can achieve rapid and efficient staining of living cells, completing the staining process within 3 minutes. The staining depth can reach the nucleolus of the cell nucleus, allowing for precise observation of the morphology of the entire cell tissue.

[0043] Preferably, the AIE fluorescent dye is used in combination with a blue fluorescent nuclear dye.

[0044] Experiments also revealed that when the AIE fluorescent dye prepared in this invention stains the cell nucleus, the fluorescence intensity of the nucleolus is significantly stronger than that of the surrounding nucleoplasm, making it impossible to accurately observe the nucleoplasm. However, when used in conjunction with a blue fluorescent nuclear dye, all details within the cell nucleus can be accurately observed. Furthermore, due to the high contrast between red and blue colors, the professional requirements for diagnostic personnel are greatly reduced.

[0045] The blue fluorescent nuclear dye can be selected from commercially available dyes commonly used in the field, such as DAPI (4',6-diamidinyl-2-phenylindole) and Hoechst 33342.

[0046] Based on the above experiments, it can be seen that the AIE fluorescent dye prepared by this invention can achieve rapid staining of living cells, and the staining depth can reach the nucleolus of the cell nucleus, making the morphology of the entire cell tissue clearly visible. It can quickly, accurately and clearly observe tissue structure disorder, cell aberration, cell nuclear structure abnormality, etc., and is expected to be used in the preparation of fluorescent reagent kits for rapid diagnosis of cancer cells.

[0047] Preferably, the AIE fluorescent dye, when used in combination with a blue fluorescent nuclear dye, can be better applied to the preparation of fluorescent reagent kits for rapid diagnosis of cancer cells, thereby improving the accuracy of diagnosis.

[0048] Compared with the prior art, the present invention has the following advantages:

[0049] 1) The fluorescent probe prepared by this invention has AIE properties and can be used in cell fluorescence imaging, which can avoid the aggregation fluorescence quenching (ACQ) phenomenon of conventional organic light-emitting dyes;

[0050] 2) The fluorescent probe prepared by this invention is an orange-red fluorescent molecule, while biological autofluorescence is mostly blue-green. When the fluorescent probe of this invention is applied to the field of cell fluorescence imaging, it has strong resistance to autofluorescence interference.

[0051] 3) The fluorescent probe prepared by this invention is an ionic fluorescent dye with good water solubility; and the cytotoxicity experiment shows that it has low cytotoxicity and good biocompatibility, so it can be applied to the field of live cell fluorescence imaging.

[0052] 4) Most importantly, the fluorescent probe prepared by this invention can rapidly stain living cells, and the staining depth can reach the nucleolus of the cell nucleus, making the morphology of the entire cell tissue clearly visible. It can quickly, accurately and clearly observe tissue structure disorder, cell aberration, cell nuclear structure abnormality, etc., and can be used to prepare fluorescent reagent kits for rapid diagnosis of cancer cells. Attached Figure Description

[0053] Figure 1 The 1H NMR spectrum of the product prepared in Example 1;

[0054] Figure 2 The carbon NMR spectrum of the product prepared in Example 1;

[0055] Figure 3 The UV-Vis absorption spectrum was measured after the product prepared in Example 1 was dissolved in water;

[0056] Figure 4 The photoluminescence spectrum was measured after dissolving the product prepared in Example 1 in water;

[0057] Figure 5 The fluorescence spectrum (PL) intensity of mixed solutions with different THF contents varies with emission wavelength.

[0058] Figure 6 The curves show the relative PL intensity (I / I0) of mixed solutions with different THF contents as a function of THF volume fraction.

[0059] Figure 7 The fluorescence spectrum (PL) intensity of mixed solutions with different glycerol contents varies with emission wavelength.

[0060] Figure 8 The curves show the relative PL intensity (I / I0) of mixed solutions with different glycerol contents as a function of glycerol volume fraction.

[0061] Figure 9 The results of the cytotoxicity test of the product prepared in Example 1;

[0062] Figure 10 The curve shows the change in fluorescence intensity of the test solution as a function of emission wavelength.

[0063] Figure 11 The curve shows the relative PL intensity (I / I0) of the test solution as a function of the DNA solution volume.

[0064] Figure 12 The curve shows the change in fluorescence intensity of the test solution as a function of emission wavelength.

[0065] Figure 13 The curve shows the change in relative PL intensity (I / I0) of the test solution with the volume of RNA solution;

[0066] Figure 14 Fluorescence imaging and overlay images of HeLa cells stained with mixed dyes at different excitation wavelengths;

[0067] Figure 15 The images show fluorescence (PL), bright field (DIC), and merge images of HeLa cells after staining with 500 μM AIE fluorescent dye solution for different time periods. Detailed Implementation

[0068] The specific implementation methods of the present invention will be further described below with reference to examples. It should be noted that the specific implementation methods described herein are only for illustration and explanation of the present invention and are not intended to limit the scope of protection of the present invention.

[0069] Example 1

[0070] (1) Diethanolamine (40.7 g, 387.1 mmol), p-fluorobenzaldehyde (10.7 g, 86.2 mmol), and a magnetic particle were placed in a 250 mL flask, and then anhydrous aluminum chloride (0.61 g, 4.57 mmol) was added. After reacting at 120 °C for 42 h, 50 mL of water was added for dilution. The mixture was then neutralized with 10% hydrochloric acid and extracted four times with ethyl acetate at 60 °C. The organic phases were combined and dried under vacuum. The crude product was purified by column chromatography by elution with ethyl acetate / n-hexane (2 / 1, v / v). After vacuum drying, a grayish-white solid, compound 3 (6.25 g), was obtained with a yield of 30.4%, and the reaction formula is shown in equation (1).

[0071] The 1H NMR spectrum data of compound 3 are as follows: 1 H NMR (400MHz, CDCl3): δ / ppm:

[0072] 9.67(1H),7.70(2H),6.73(2H),3.93(4H),3.70(6H).

[0073]

[0074]

[0075] (2) 4-Methylpyridine (0.5 mL, 5.1 mmol, compound 5) and (3-bromopropyl)trimethylammonium bromide (1.3 g, 5 mmol, compound 4) and a magnetic precipitator were added to a 250 mL flask, followed by the addition of 10 mL of ethanol as a solvent. The mixture was reacted at reflux for 4 h, and heating was stopped after the white solid had completely disappeared. After the reaction was complete, the crude product was dissolved in methanol and precipitated by adding it dropwise to diethyl ether. After washing three times with diethyl ether, a grayish-white solid (1.52 g), namely compound 6, was obtained, with a yield of 83%. The reaction formula is shown in formula (2) above.

[0076] (3) Compound 3 (0.706 g, 3.38 mmol) and Compound 6 (0.5 g, 3.38 mmol) were added to a 250 mL flask, followed by 10 mL of ethanol as a solvent, and then 0.2 mL of piperidine. The mixture was heated at reflux for 8 h. The crude products were combined and dried under vacuum, and then purified by column chromatography with ethanol elution. After vacuum distillation and drying, a red solid with a metallic luster (1.1 g) was obtained, namely Compound 7, 4-(4-(N,N-dihydroxyethylamino)styryl)-N-(3-trimethylaminopropyl)pyridine diammonium salt, with a yield of 59.9%. The reaction formula is shown in formula (3) above.

[0077] The proton and carbon NMR spectra of the product are as follows: Figure 1 and 2 As shown, the specific data is as follows:

[0078] 1 H NMR (400MHz, DMSO): δ / ppm: 8.86 (2H), 8.13 (2H), 8.0 (1H), 7.60 (2H), 7.22 (1H), 6. 83(2H),4.85(2H),4.55(2H),3.59(4H),3.54(4H),3.46(2H),3.12(9H),2.45(2H).

[0079] 13 C NMR(101MHz,DMSO):δ / ppm:154.16,150.43,143.53,142.50,130.45,122.26 ,122.00,116.61,111.66,61.76,58.05,55.90,53.04,52.43,48.55,24.07.

[0080] Performance testing:

[0081] 1. Spectral testing:

[0082] The product prepared in Example 1 was dissolved in water to prepare a solution with a concentration of 10.-5 The test solution was prepared at a concentration of mol / L. The solution was then transferred to the sample holder of the UV-Vis spectrometer (Shimadzu UV-1800 UV-Vis absorption spectrometer) for spectral analysis. The measured UV-Vis absorption spectrum is shown below. Figure 3 As shown.

[0083] observe Figure 3 It can be observed that the wavelength of the highest energy absorption peak of the AIE fluorescent dye prepared in this embodiment in water is 472 nm.

[0084] The prepared sample was transferred to the sample holder of the fluorescence spectrometer (Shimadzu RF-5301PC photoluminescence spectrometer), and the spectral measurement was performed at an excitation wavelength of 472 nm. The normalized photoluminescence spectrum is shown below. Figure 4 As shown in the figure, the photoluminescence spectrum of the water-soluble AIE fluorescent dye prepared in this embodiment after normalization in the solid state is also given.

[0085] observe Figure 4 It can be observed that the maximum fluorescence emission wavelength of the AIE fluorescent dye prepared in this embodiment is 598 nm in water and 651 nm in solid state, with an orange-red fluorescence color. Since biological autofluorescence is mostly blue-green, the AIE fluorescent dye prepared in this embodiment has strong resistance to autofluorescence interference. Compared with the maximum absorption wavelength in ultraviolet light (472 nm), the Stokes shift is relatively large, which is beneficial for biological detection and fluorescence imaging.

[0086] 2. Luminescence property test

[0087] 2.1 The product prepared in Example 1 was mixed with mixed solutions of tetrahydrofuran (THF) and water in different volume fractions to prepare a solution with a concentration of 10. -5 A mol / L THF / H₂O mixed solution was prepared, with THF volume contents of 0, 10, 20, 30, 40, 50, 60, 70, 80, and 90%. The fluorescence spectrum (PL) intensity of the above mixed solutions was tested as a function of emission wavelength. (Specific details are as follows...) Figure 5 As shown ( Figure 5 The THF content corresponding to different curves increases sequentially from bottom to top; then, at the maximum emission wavelength (598 nm), the relative PL intensity (I / I0) of the mixed solution is tested as a function of the THF volume fraction, as detailed below. Figure 6 As shown.

[0088] observe Figure 5 It can be observed that the maximum emission wavelength of the AIE fluorescent dye prepared in Example 1 is 598 nm in THF / H2O mixed solutions prepared with different THF volume fractions.

[0089] Combination Figure 5 and Figure 6 As can be seen from the changes, the fluorescence intensity of the AIE fluorescent dye prepared in Example 1 continuously increases with the increase of THF content. When the THF content increases to 80% or above, the fluorescence emission intensity increases sharply. When the THF volume content reaches 90%, the fluorescence intensity is about 6 times that of the initial value. The reason for this may be that the compound aggregates due to the decrease in solubility and precipitates out of the solvent system. This proves that the product prepared in Example 1 has the characteristics of typical AIE molecules and has aggregation-induced emission properties.

[0090] 2.2 The product prepared in Example 1 was mixed with mixed solutions of glycerol and water of different volume fractions to prepare a solution with a concentration of 10. -5 A glycerol / water mixed solution with a volume content of 0%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, and 99% was prepared. The PL intensity of the above mixed solutions was tested as a function of emission wavelength. (Specific details are as follows...) Figure 7 As shown ( Figure 7 The THF content corresponding to different curves increases sequentially from bottom to top; then, at the maximum emission wavelength (598 nm), the relative PL intensity (I / I0) of the mixed solution is tested as a function of the glycerol volume fraction, as detailed below. Figure 8 As shown.

[0091] observe Figure 7 It can be observed that the water-soluble AIE fluorescent dyes prepared in Example 1 all have a maximum emission wavelength of 598 nm in a volume fraction of glycerol / water mixed solution.

[0092] Combination Figure 7 and Figure 8 As can be seen from the changes, the fluorescence intensity of the AIE fluorescent dye prepared in Example 1 continuously increases with the increase of glycerol content. When the glycerol content increases to more than 60%, the fluorescence emission intensity increases sharply. When the glycerol volume content is 99%, the fluorescence intensity of the AIE fluorescent dye is about 30 times that in pure water. The reason for this may be that the viscosity of the system increases sharply, and the movement of the AIE fluorescent probe in the glycerol solvent system is restricted, thus exhibiting typical aggregation-induced emission (AIE) characteristics and having aggregation-induced emission properties.

[0093] 3. Cytotoxicity test

[0094] HeLa cells in the culture medium were transferred into 96-well plates and cultured for one day. After the cells reached the appropriate confluence, the culture medium was washed with PBS buffer. The AIE fluorescent dye prepared in Example 1 was mixed with DMEM medium containing 1 vol% antibiotics and 10 vol% FBS to prepare solutions with concentration gradients of 200 μM, 400 μM, 600 μM, 800 μM, and 1000 μM.

[0095] Culture medium containing different concentrations of AIE fluorescent dye was added to well plates, with a blank control group included. After culturing for 24 hours, the culture medium was washed off with PBS buffer, and then CCK-8 was added. Cells were cultured again for 2 hours, and cell viability was detected using a microplate reader. The cytotoxicity test results are as follows: Figure 9 As shown. By Figure 9 The cytotoxicity test results show that as the concentration of AIE fluorescent dye gradually increases, the activity of co-cultured HeLa cells gradually decreases. When the concentration reaches 600 μM, the cell activity decreases significantly, but as the concentration increases to 1000 μM, the cell activity increases again. The overall cell activity remains above 75%, indicating that the AIE fluorescent dye prepared in this invention has low molecular cytotoxicity and has the potential to be applied to live cell staining.

[0096] 4. DNA and RNA response experiments

[0097] The AIE fluorescent dye prepared in Example 1 was dissolved in water to prepare a solution with a concentration of 10. -3 The mother liquor of M was then used. A certain amount of DNA and RNA were dissolved in appropriate amounts of water to prepare DNA solutions with concentrations of 637.2 ng / μL and RNA solutions with concentrations of 136.2 ng / μL, respectively.

[0098] 30 μL of the stock solution was mixed with different volumes of DNA solution, and then water was added to bring the total volume to 3 mL to obtain the test solution. The fluorescence intensity of the test solution as a function of emission wavelength was measured on a fluorescence spectrometer. The test results are as follows: Figure 10 As shown ( Figure 10 The DNA solution volumes corresponding to different curves increase sequentially from bottom to top: 30, 60, 90, 120, 150, 180, 210, 240, 270, 300, and 330 mL. Then, at the maximum emission wavelength (598 nm), the relative PL intensity (I / I0) of the test solution is measured as a function of DNA solution volume. (Specific details are as follows...) Figure 11 As shown.

[0099] 30 μL of the stock solution was mixed with different volumes of RNA solution, and then water was added to bring the total volume to 3 mL to obtain the test solution. The fluorescence intensity of the test solution as a function of emission wavelength was measured on a fluorescence spectrometer. The test results are as follows: Figure 12As shown ( Figure 12 The RNA solution volumes corresponding to different curves are increased sequentially from bottom to top: 0, 30, 60, 90, 120, 150, 180, 210, and 240 mL. Then, at the maximum emission wavelength (598 nm), the relative PL intensity (I / I0) of the test solution is measured as a function of the RNA solution volume. (Specific details are as follows...) Figure 13 As shown.

[0100] observe Figure 10 and 11 It can be seen that as the DNA content in the test solution increases, the fluorescence intensity also increases. Initially, the relationship is linear, but the upward trend becomes gradual. The reason for this may be that the AIE fluorescent dye and DNA interact by charge, causing the dye to aggregate on the DNA and produce enhanced fluorescence. In the end, due to the high DNA content, the AIE fluorescent dye molecules are saturated with DNA, so the upward trend becomes gradual.

[0101] observe Figure 12 and 13 As the RNA content increases, the fluorescence of the test solution gradually increases, but because the initial concentration of RNA is low, the fluorescence intensity increases slowly when it is first added.

[0102] The above tests verified that the AIE fluorescent dye prepared in this invention can interact with DNA and RNA in cells to lead to enhanced fluorescence, and that the genetic material in the cell is enriched in the nucleolus in the cell nucleus, proving that this dye has the potential to be used in the diagnosis of cancer cells by staining the nucleolus.

[0103] Application examples

[0104] I. After treating HeLa cells to improve cell membrane permeability, the cells were co-stained with the AIE fluorescent dye prepared in Example 1 and the commercial nuclear dye 4',6-diamidinyl-2-phenylindole (DAPI). The fluorescence images of the cells were then observed using a laser scanning confocal microscope to examine the staining effect of the dye on the cell nucleus after the change in cell membrane permeability. The specific operating steps are as follows:

[0105] 1) HeLa cells were cultured at 37°C in DMEM medium containing 1 vol% antibiotics and 10 vol% FBS, with a humidity of 5%. The medium was changed every other day.

[0106] 2) After passaged into confocal culture dishes and cultured for 24 h, the culture medium was washed off with PBS, 4% paraformaldehyde (PFA) was added to fix the cells for 10 min, then washed with PBS buffer, and 0.2 wt% Triton X-100 was added to permeate the cell membrane for 5 min, and then washed with PBS buffer to remove excess Triton X-100 and PFA.

[0107] 3) Stain with a mixed dye prepared by mixing AIE fluorescent dye solution (500 μM, 200 μL, PBS buffer) and DAPI aqueous solution (2 μg / mL, 200 μL). After staining for 5 minutes, observe under a laser confocal microscope.

[0108] Fluorescence images of cells were observed using a laser scanning confocal microscope (excitation wavelength of AIE fluorescent dye: 561 nm, emission band: 578–700 nm; excitation wavelength of DAPI: 350 nm, emission band: 380–480 nm; scale bar: 7.5 μm).

[0109] Figure 14 The images show fluorescence imaging of HeLa cells stained with mixed dyes at an excitation wavelength of 561 nm and an emission band of 578–700 nm (denoted as PL), fluorescence imaging of HeLa cells at an excitation wavelength of 350 nm and an emission band of 380–480 nm (denoted as DAPI), and a superimposed image of the two images (denoted as Merge).

[0110] DAPI dye is a commercially available dye for staining cell nuclear regions. It is a traditional fluorescent material, which confirms... Figure 14 The DAPI-stained area represents the HeLa cell nucleus, but it's also noticeable that DAPI stains the entire nucleus, failing to highlight nucleus details. Combining the nucleus's location in the DAPI image with a comparative analysis... Figure 14 The PL image shows that AIE fluorescent dyes can record bright red emission light in the cell membrane, cytoplasm, and nucleus within the spectral window of 578–700 nm. Of particular note is the staining pattern in the nucleus: several bright spots in the nucleus exhibit significantly stronger fluorescence intensity than the surrounding nucleoplasm. Combined with the AIE fluorescent dye's response to DNA and RNA, this confirms that the red bright spots in the nucleus are located in the nucleolar region. Further observation... Figure 14 The Merge diagram shows that after co-staining with AIE fluorescent dye, the details in the cell nuclei, which were not prominent after DAPI staining, were fully revealed. Furthermore, due to the strong contrast between the two fluorescent colors, the morphology of the entire cell tissue became more clearly visible.

[0111] 2. HeLa cells were co-cultured with the AIE fluorescent dye prepared in Example 1 to stain the HeLa cells, and then the fluorescence images of the cells were observed using a laser scanning confocal microscope for further study. The specific operation steps are as follows:

[0112] 1) HeLa cells were cultured at 37°C in DMEM medium containing 1 vol% antibiotics and 10 vol% FBS, with a humidity of 5%. The medium was changed every other day.

[0113] 2) After passaged into confocal culture dishes and cultured for 24 h, the culture medium was washed off with PBS buffer, and then 500 μM AIE fluorescent dye solution (PBS buffer) was added for staining.

[0114] 3) After staining for 3 min, 5 min and 10 min, observe under a laser confocal microscope.

[0115] Fluorescence images of cells were observed using a laser scanning confocal microscope (excitation wavelength: 561 nm, emission band: 578–700 nm, scale bar: 20 μm).

[0116] Figure 15 The images show fluorescence (PL), bright field (DIC), and merge images of HeLa cells after staining with 500 μM AIE fluorescent dye solution for different time periods. Figure 15 The AIE fluorescent dye prepared according to this invention can complete live cell staining within 3 minutes. Within the spectral window of 578–700 nm, bright red emission light can be recorded around the cell membrane and nucleolus, indicating that the AIE fluorescent dye has been successfully internalized into living HeLa cells. Careful examination of the fluorescence image details shows that the red emission originates from the nucleolar region of the cell, where the fluorescence intensity is stronger than the surrounding nucleoplasm. This allows the location, number, and morphology of the nucleoli within each cell to be clearly displayed. Normally, a normal cell nucleus contains only one nucleolus, while… Figure 15 The cluster of cells shown clearly has more than one nucleolus, because typical cancer cells—HeLa cells—were used. Even after the staining time was extended to 10 minutes, the staining remained uniform and stable, indicating that the AIE fluorescent dye prepared in this invention is stable.

[0117] The above data have demonstrated that the AIE fluorescent dye prepared in this invention possesses aggregation-induced emission properties and responds to both DNA and RNA. Since the nucleolus is a region of highly concentrated genetic material, fluorescent dyes that respond to genetic material will inevitably accumulate in the nucleolus. In the nucleolus, most DNA is tightly bound to histones. Therefore, we infer that the AIE fluorescent dye is more likely to bind to RNA in the nucleolus to produce aggregation-induced emission, demonstrating the potential of this dye for application in staining the nucleolus for cancer cell diagnosis.

[0118] The above description is merely a specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. The specific examples used above to illustrate the present invention are only for the purpose of helping to understand the present invention and are not intended to limit the present invention. Those skilled in the art to which this invention pertains can make several simple deductions, modifications, substitutions, or combinations based on the concept of the present invention. These deductions, modifications, substitutions, or combinations also fall within the scope of the claims of the present invention.

Claims

1. An AIE fluorescent dye, characterized in that, The structural formula is shown in equation (Ⅰ) below: （Ⅰ）； In the formula, R1=R2, which are selected from alkyl alcohols with 1 to 6 carbon atoms; Selected from water-soluble anions.

2. The AIE fluorescent dye according to claim 1, characterized in that: Selected from halogen anions.

3. The AIE fluorescent dye according to claim 1, characterized in that: R1=R2, selected from CH2OH, CH2CH2OH or CH2CH2CH2OH; Selected from F - Cl - ,Br - One of them.

4. A method for preparing an AIE fluorescent dye according to any one of claims 1 to 2, characterized in that, include: (1) Using N,N-dialkylolamine and p-fluorobenzaldehyde as raw materials, intermediate product A was prepared by nucleophilic substitution reaction; (2) Mix 4-methylpyridine, 3-halopropyltrimethylammonium salt with organic solvent A, and heat to reflux temperature to carry out alkylation reaction to prepare intermediate product B; (3) Under an inert atmosphere, intermediate product A and intermediate product B are mixed with organic solvent B and heated to reflux temperature to carry out a nucleophilic addition reaction to obtain the AIE fluorescent dye.

5. The method for preparing the AIE fluorescent dye according to claim 4, characterized in that, In step (1): The N,N-dialkylolamine is selected from one of diethanolamine, N,N-di(3-hydroxypropyl)amine, N,N-di(4-hydroxybutyl)amine, N,N-di(5-hydroxypentyl)amine, and N,N-di(6-hydroxyhexyl)amine; The nucleophilic substitution reaction is carried out in the presence of a catalyst, which is selected from one or more of aluminum trichloride, ferric trichloride, and titanium tetrachloride. The molar ratio of N,N-dialkylolamine to p-fluorobenzaldehyde is 1~8:1, and the temperature for the nucleophilic substitution reaction is 110~160 °C; In step (2): The organic solvent A is selected from one or more of N,N-dimethylformamide, N,N-dimethylacetamide, dimethyl sulfoxide, methanol, and ethanol; The molar ratio of 4-methylpyridine to 3-halopropyltrimethylammonium salt is 1.0~1.2:1; In step (3): The molar ratio of intermediate product A to intermediate product B is 1.0~1.1:1; The organic solvent B is selected from one or more of N,N-dimethylformamide, N,N-dimethylacetamide, dimethyl sulfoxide, methanol, and ethanol; The nucleophilic addition reaction is carried out in the presence of a catalyst, which is selected from one or more of piperidine, potassium tert-butoxide, and pyridine.

6. The application of an AIE fluorescent dye according to any one of claims 1 to 3 in the preparation of live cell fluorescence imaging dyes, characterized in that, The AIE fluorescent dye is used in conjunction with a blue fluorescent nuclear dye.

7. The application of the AIE fluorescent dye according to claim 6 in the preparation of live cell fluorescence imaging dyes, characterized in that, The live cell fluorescence imaging dye is used to prepare a fluorescent reagent kit for rapid diagnosis of cancer cells.

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