Water-soluble AIE fluorescent probe, preparation method and application thereof

Abstract

The application discloses a water-soluble AIE fluorescent probe, and a structure formula is shown in the following formula (I), wherein R1 and R2 are independently selected from alkyl alcohol with carbon number of 1-6; X is selected from water-soluble anions, and M is selected from water-soluble cations. The fluorescent probe has AIE characteristics, can realize rapid and efficient staining of cytoplasmic membranes, and does not need to be cleaned; the fluorescent probe has good water solubility, small cytotoxicity, and can be used in in vivo cell fluorescence imaging; the fluorescent probe has orange red fluorescence color, large Stokes shift, and strong anti-self-fluorescence interference capability.

Description

Technical Field

[0001] This invention relates to the technical field of fluorescent probes, and more particularly to a water-soluble AIE fluorescent probe, its preparation method, and its application in the field of live cell fluorescence imaging. Background Technology

[0002] The cell membrane participates in various cellular processes and biological functions, such as cell signaling, cell adhesion, endocytosis, exocytosis, and selective permeation of substances. Therefore, the cell membrane is indispensable to cells, and observing the cell membrane can yield much information related to cell state and disease. It is well known that imaging techniques using fluorescent probes to label and trace the cell membrane are important tools for studying cell membrane structure and function, and have significant practical value.

[0003] As a powerful non-invasive imaging technique, fluorescence bioimaging technology has made significant progress. Organic small-molecule fluorescent chromophore compounds, a major class of fluorescent chromophores used in this field, have also seen substantial development, such as commercially available cell membrane staining probe dyes (e.g., DiI, DiO).

[0004] Currently, commercially available cell membrane fluorescent staining probes generally suffer from the following common problems during application: 1) They all require long staining times and cumbersome post-staining washing procedures. Long staining times are not only time-consuming but also frequently lead to non-specific luminescence of cell components. Furthermore, frequent post-washing is not only cumbersome but also easily alters the cell's surrounding environment and causes cell loss during washing, failing to meet the requirements for continuous monitoring of biological processes. In fact, long cell staining times and cumbersome post-washing procedures have long been unsolved technical problems in the field of cell fluorescence imaging. 2) Due to π-π stacking and the existence of other non-radiative decay pathways, traditional NIR chromophores can only emit weak fluorescence or no fluorescence at high concentrations or in aggregated states. The main reason for this fluorescence quenching is related to the formation of aggregates, hence it is often referred to as aggregation-caused quenching (ACQ). This phenomenon is very common in bioimaging and analysis and has become a major obstacle to practical applications: due to the high hydrophobicity of its luminescent center, organic molecules spontaneously aggregate in biological media, resulting in a high quenching constant of the fluorescent dye, which in turn leads to rapid internalization of the dye within the cell, resulting in a very narrow imaging time window.

[0005] In 2001, Tang Benzhong's research group discovered a peculiar phenomenon: some thiophene molecules hardly emit light in solution, but their luminescence is greatly enhanced in the aggregated state or under solid film conditions. Because this enhanced luminescence is caused by aggregation, it is figuratively called aggregation-induced emission (AIE).

[0006] In fact, luminescent systems with aggregation-induced emission (AIE) properties avoid fluorescence quenching of common fluorescent molecules under high concentration conditions. Simultaneously, the aggregated state of fluorescent molecules reduces cytotoxicity and enhances their resistance to enzymatic degradation in vivo. Therefore, fluorescent probes with AIE properties are more suitable for applications in fluorescence bioimaging.

[0007] Water-soluble AIEgens (molecules with AIE properties), especially anti-autofluorescence water-soluble AIEgens applicable to the field of fluorescence bioimaging, have been a long-sought goal. This is because currently commercially available cell fluorescence probes are mostly dissolved in organic solvents such as DMSO, DMF, or ethanol, which pose potential problems such as cytotoxicity and membrane fusion. However, the exploration of anti-autofluorescence water-soluble AIEgen fluorescent probes for application in fluorescence bioimaging remains extremely challenging. In addition, the selective permeability of living cell membranes makes the development of rapidly transmembrane water-soluble AIEgen fluorescent probe molecules even more challenging. Summary of the Invention

[0008] To address the aforementioned problems in the existing technology, this invention discloses a fluorescent probe with AIE properties, enabling rapid and efficient staining of the cell membrane without the need for washing; the fluorescent probe has good water solubility, low cytotoxicity, and can be used in live cell fluorescence imaging; the fluorescent probe has an orange-red fluorescence color and a large Stokes shift, exhibiting strong resistance to autofluorescence interference.

[0009] The specific technical solution is as follows:

[0010] A water-soluble AIE fluorescent probe has the following structural formula (Ⅰ):

[0011]

[0012] In the formula, R1 and R2 are independently selected from alkyl alcohols having 1 to 6 carbon atoms; X is selected from water-soluble anions; and M is selected from water-soluble cations.

[0013] Preferred:

[0014] R1 = R2;

[0015] X is selected from halide anions.

[0016] M is selected from alkali metal cations.

[0017] Further optimization:

[0018] R1 = R2, selected from one or more of CH2OH, CH2CH2OH, and CH2CH2CH2OH;

[0019] X is selected from F - Cl - ,Br - One or more of the following;

[0020] M is selected from Na + and / or K + .

[0021] The water-soluble AIE fluorescent probe prepared in this invention has an emission wavelength of 590–610 nm.

[0022] This invention also discloses a method for preparing the water-soluble AIE fluorescent probe, comprising:

[0023] (1) Using N,N-dialkylolamine and p-fluorobenzaldehyde as raw materials, intermediate product A was prepared by nucleophilic substitution reaction;

[0024] (2) Mix 4-methylpyridine, 2-haloethyl sulfonate and organic solvent A, and heat to reflux temperature to carry out alkylation reaction to prepare intermediate product B;

[0025] (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 water-soluble AIE fluorescent probe.

[0026] In step (1):

[0027] 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;

[0028] 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.

[0029] The temperature for the nucleophilic substitution reaction is 110–160 °C, preferably 110–130 °C.

[0030] 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.

[0031] Preferably, the amount of catalyst added is 1 to 10 wt% based on the total mass of N,N-dialkylolamine and p-fluorobenzaldehyde.

[0032] In step (2):

[0033] The 2-haloethylsulfonate is selected from one or more of sodium 2-bromoethylsulfonate, sodium 2-bromoethylsulfonate, and sodium 2-chloroethylsulfonate.

[0034] The organic solvent A is selected from one or more of N,N-dimethylformamide, N,N-dimethylacetamide, dimethyl sulfoxide, methanol, and ethanol;

[0035] The molar ratio of 4-methylpyridine to 2-haloethylsulfonate is 1 to 20:1; preferably 5 to 15:1, more preferably 11.2:1.

[0036] The specific reflux temperature should be adjusted according to the type of organic solvent A used.

[0037] In step (3):

[0038] The molar ratio of intermediate product A to intermediate product B is 1.0 to 1.2:1, preferably 1.1:1;

[0039] The organic solvent B is selected from one or more of N,N-dimethylformamide, N,N-dimethylacetamide, dimethyl sulfoxide, methanol, and ethanol;

[0040] The nucleophilic addition reaction is carried out in the presence of a catalyst, which is selected from one or more of piperidine, pyridine, and potassium tert-butoxide.

[0041] The amount of catalyst added is 1-3 wt% based on the mass of intermediate product B.

[0042] The specific reflux temperature should be adjusted accordingly based on the type of organic solvent B used.

[0043] The inert atmosphere used is a common atmosphere in the art, such as nitrogen, argon, helium, etc.

[0044] Preferably, in step (3), the product after nucleophilic addition reaction needs to be precipitated and washed.

[0045] Precipitation refers to dissolving the target crude product, adding it dropwise to a poor solvent to precipitate and purify the product. Conventional experimental methods can be used to obtain the target crude product, such as removing the organic solvent by rotary evaporation after extraction. Preferably, the poor solvent is selected from anhydrous diethyl ether.

[0046] The washing process involves using a poor organic solvent, such as anhydrous diethyl ether, to further remove impurities that adhered to the target product during precipitation.

[0047] This invention investigated the photophysical properties, AIE properties, and cytotoxicity of the prepared water-soluble AIE fluorescent probe. The results showed that:

[0048] The fluorescent probe prepared by this invention has good water solubility, low cytotoxicity, and is a compound that emits (orange) red fluorescence with AIE properties.

[0049] This invention also discloses the application of the water-soluble AIE fluorescent probe in the field of live cell fluorescence imaging, especially in cell membrane-targeted fluorescence imaging.

[0050] Experiments have shown that the water-soluble AIE fluorescent probe disclosed in this invention can achieve rapid and efficient staining of the cytoplasmic membrane. It can also overcome the selective permeability of the living cell membrane, smoothly pass through the cell membrane and enter the cell to stain the cytoplasm and nucleus, observe the overall morphology of the cell, and may have potential applications in nucleolar staining.

[0051] Compared with the prior art, the present invention has the following advantages:

[0052] 1) The fluorescent probe prepared by this invention has AIE properties and can be used as a cell membrane fluorescent staining probe in cell fluorescence imaging, which can avoid the aggregation fluorescence quenching (ACQ) phenomenon of conventional organic light-emitting dyes.

[0053] 2) The fluorescent probe prepared by this invention is an orange-red fluorescent molecule, while biological autofluorescence is mostly blue-green. When the water-soluble fluorescent probe of this invention is applied to the field of cell fluorescence imaging, it has strong resistance to autofluorescence interference.

[0054] 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.

[0055] 4) Most importantly, the fluorescent probe prepared by this invention can rapidly and uniformly stain the cell membrane as a cell membrane fluorescent staining probe. The staining of the cell membrane can be completed within 3 minutes, and no post-washing operation is required, thus realizing targeted fluorescence imaging of the cell membrane. Attached Figure Description

[0056] Figure 1 The 1H NMR spectrum of the product prepared in Example 1;

[0057] Figure 2 The carbon NMR spectrum of the product prepared in Example 1;

[0058] Figure 3 The UV-Vis absorption spectrum was measured after the product prepared in Example 1 was dissolved in water;

[0059] Figure 4 The photoluminescence spectrum was measured after dissolving the product prepared in Example 1 in water;

[0060] Figure 5 The fluorescence spectrum (PL) intensity of mixed solutions with different THF contents varies with emission wavelength.

[0061] 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.

[0062] Figure 7 The fluorescence spectrum (PL) intensity of mixed solutions with different glycerol contents varies with emission wavelength.

[0063] 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.

[0064] Figure 9 The results of the cytotoxicity test of the product prepared in Example 1;

[0065] Figure 10 The images show fluorescence (PL), bright field (DIC), and merge images of HeLa cells after staining with 200 μM water-soluble AIE fluorescent probe solution.

[0066] Figure 11 Images of PL, DIC, and Merge in HeLa cells after staining with 800 μM water-soluble AIE fluorescent probe solution. Detailed Implementation

[0067] 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.

[0068] Example 1

[0069] (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).

[0070] The 1H NMR spectrum data of compound 3 are as follows: 1 H NMR (400MHz, CDCl3): δ / ppm:

[0071] 9.67(1H),7.70(2H),6.73(2H),3.93(4H),3.70(6H).

[0072]

[0073] (2) First, 4-methylpyridine (16 mL, 161.8 mmol), sodium 2-bromoethanesulfonate (3.07 g, 14.5 mmol), and a magnetic flux were added to a 250 mL flask. Then, 50 mL of ethanol was added as a solvent, and the mixture was reacted at reflux temperature for 5 h. After the white solid completely disappeared, heating was stopped, and the solvent was removed by vacuum distillation to obtain the crude product. After washing three times with diethyl ether, a white solid, namely compound 6 (2.0 g), was obtained, with a yield of 49.3%. The reaction formula is shown in formula (2) above.

[0074] (3) Compound 6 (0.437 g, 2.17 mmol) and compound 3 (0.5 g, 2.39 mmol) were added to a 250 mL flask, followed by 50 mL of methanol as a solvent and 0.2 mL of piperidine. The mixture was heated under reflux for 8 h. Product 9 was obtained as red needle-like crystals, namely 4-(4-(N,N-dihydroxyethylamine)styryl)-N-(2-sulfonate ethyl)pyridine ammonium bromide (0.348 g), with a yield of 34.0%. The reaction formula is shown in formula (3) above.

[0075] The 1H and 1C NMR spectra of product 9 are as follows: Figure 1 and 2 As shown, the specific data is as follows:

[0076] 1H NMR (500MHz, DMSO): δ / ppm: 8.73(2H), 7.98(2H), 7.90(1H), 7.56(2H), 7.14(1H), 6.80(2H), 4.81(2H), 4.66(2H), 3.58, (4H), 3.52(4H), 3.33(2H).

[0077] 13 C NMR (126MHz, DMSO): δ / ppm: 153.61, 150.22, 144.13, 141.80, 130.27, 122.06, 121.59, 116.79, 111.58, 58.08, 56.39, 53.08, 50.58, 48.58, 39.48.

[0078] Example 2

[0079] The preparation process is basically the same as in Example 1, except that:

[0080] In step (1), the reaction temperature for nucleophilic substitution is replaced with 130℃ and the reaction time is replaced with 70h;

[0081] In step (3), the time for the nucleophilic addition reaction is replaced with 12 hours.

[0082] The 1H NMR spectrum data of the product prepared in this embodiment are as follows:

[0083] 1 H NMR (500MHz, DMSO): δ / ppm: 8.73(2H), 7.98(2H), 7.90(1H), 7.56(2H), 7.14(1H), 6.80(2H), 4.81(2H), 4.66(2H), 3.58, (4H), 3.52(4H), 3.33(2H).

[0084] Performance testing:

[0085] 1. Spectral testing:

[0086] 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.

[0087] observe Figure 3 It can be observed that the wavelength of the highest energy absorption peak of the water-soluble AIE fluorescent probe prepared in this embodiment is 468 nm.

[0088] The prepared sample was transferred to the sample holder of the fluorescence spectrometer (Shimadzu RF-5301PC photoluminescence spectrometer). Spectroscopic measurements were performed at an excitation wavelength of 468 nm. The normalized photoluminescence spectrum is shown below. Figure 4 As shown in the figure, the photoluminescence spectrum of the water-soluble AIE fluorescent probe prepared in this embodiment after normalization in the solid state is also given.

[0089] observe Figure 4 It can be observed that the water-soluble AIE fluorescent probe prepared in this embodiment has a maximum emission wavelength of 594 nm in water and a fluorescence color of orange-red, while biological autofluorescence is mostly blue-green. Therefore, the water-soluble AIE fluorescent probe prepared in this embodiment has strong resistance to autofluorescence interference. Compared with the maximum absorption wavelength of ultraviolet light (468 nm), the Stokes shift is relatively large, which is beneficial for biological detection and fluorescence imaging.

[0090] 2. Luminescence property test

[0091] 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 THF / H₂O mixed solutions with THF volume contents of 0, 10, 20, 30, 40, 50, 60, 70, 80, 90, and 99% were prepared. The fluorescence (PL) intensity of these mixed solutions was measured 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 (594nm), 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.

[0092] observe Figure 5 It can be observed that the maximum emission wavelength of the water-soluble AIE fluorescent probe prepared in Example 1 is 594 nm in THF / H2O mixed solutions prepared with different THF volume fractions.

[0093] Combination Figure 5 and Figure 6 As can be seen from the changes, the fluorescence intensity of the water-soluble AIE fluorescent probe 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 99%, the fluorescence intensity is about 6 times that of the initial value. The reason for this may be that the compound aggregates and precipitates out of the solvent system. This proves that the product prepared in Example 1 has the characteristics of a typical AIE molecule and has aggregation-induced emission properties.

[0094] 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 glycerol volume content of 0%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, and 80% 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 glycerol content corresponding to different curves increases sequentially from bottom to top; then, at the maximum emission wavelength (594nm), 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.

[0095] observe Figure 7 It can be observed that the water-soluble AIE fluorescent probes prepared in Example 1 all have a maximum emission wavelength of 594 nm in a volume fraction of glycerol / water mixed solution.

[0096] Combination Figure 7 and Figure 8 As can be seen from the changes, the fluorescence intensity of the water-soluble AIE fluorescent probe 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 80%, the fluorescence intensity of the water-soluble AIE fluorescent probe 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 water-soluble AIE fluorescent probe in the glycerol solvent system is restricted, thus exhibiting typical aggregation-induced emission (AIE) characteristics and having aggregation-induced emission properties.

[0097] 3. Cytotoxicity test

[0098] 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 water-soluble AIE fluorescent probe prepared in Example 1 was mixed with DMEM medium containing 1 vol% penicillin antibody and 10 vol% FBS to prepare solutions with concentration gradients of 200 μM, 400 μM, 600 μM, 800 μM, and 1000 μM.

[0099] Culture medium containing different concentrations of water-soluble AIE fluorescent probes 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.

[0100] Depend on Figure 9The cytotoxicity test results show that the activity of co-cultured HeLa cells decreased as the concentration of the water-soluble AIE fluorescent probe gradually increased; when the concentration reached 1000 μM, the overall cell activity remained above 80%, indicating that the water-soluble AIE fluorescent probe prepared in this invention has low molecular cytotoxicity and has the potential to be applied to live cell staining.

[0101] Application examples

[0102] HeLa cells were co-cultured with the water-soluble AIE fluorescent probe 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:

[0103] 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.

[0104] 2) After passaged into confocal culture dishes and cultured for 24 h, the culture medium was washed off with PBS buffer, and water-soluble AIE fluorescent probe solutions of different concentrations (with PBS buffer as the solvent) were added for staining.

[0105] 3) After staining for 3 minutes, observe under a laser confocal microscope.

[0106] Fluorescence images of cells were observed using a laser scanning confocal microscope (excitation wavelength: 561 nm, emission band: 578–700 nm, scale bar: 10 μm).

[0107] Figure 10 The images show the fluorescence (PL), bright-field (DIC), and merge images of HeLa cells after staining with a 200 μM water-soluble AIE fluorescent probe solution.

[0108] Figure 10 The experiment showed that bright red emission light could be recorded around the cell membrane within the spectral window of 578–700 nm. Furthermore, during the experiment, the water-soluble AIE fluorescent probe rapidly accumulated and emitted light near the cell membrane, achieving aggregation on the cell membrane and emitting strong red fluorescence within 3 minutes of staining, thus illuminating the area around the cell membrane. Based on the above cell staining results, it can be concluded that the AIE fluorescent probe (4-(4-(N,N-dihydroxyethylamine)styryl)-N-(2-sulfonate ethyl)pyridine ammonium bromide) prepared in Example 1 is a good fluorescent agent for in vivo cell fluorescence imaging, possessing the ability to rapidly and efficiently stain the cell membrane.

[0109] Figure 11The images show the fluorescence (PL), bright-field (DIC), and merge images of HeLa cells after staining with 800 μM water-soluble AIE fluorescent probe solution.

[0110] Figure 11 Within the spectral window of 578–700 nm, when the concentration was increased, the water-soluble AIE fluorescent probe solution not only illuminated the cell membrane but also the entire cell within 3 minutes, with strong red fluorescence also appearing at the nucleolus. This indicates that the water-soluble AIE fluorescent probe prepared in this invention can not only rapidly stain the cell membrane but also rapidly pass through the cell membrane.

[0111] The results of the cell staining experiments above show that the water-soluble AIE fluorescent probe prepared in this invention is a good fluorescent agent for live cell fluorescence imaging. It has the ability to stain the cell membrane quickly and efficiently, and at the same time, it can prevent the selective permeability of the live cell membrane from affecting it, thereby realizing a rapid transmembrane process. It has important value in exploring and developing water-soluble AIEgen fluorescent probes that resist biological autofluorescence in the field of fluorescence bioimaging.

[0112] 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. A water-soluble AIE fluorescent probe, characterized in that, The structural formula of the water-soluble AIE fluorescent probe is shown in formula (Ⅰ) below: （Ⅰ）； In the formula, R1=R2, which is selected from one of CH2OH, CH2CH2OH, and CH2CH2CH2OH; Selected from water-soluble anions, Selected from water-soluble cations.

2. The water-soluble AIE fluorescent probe according to claim 1, characterized in that: Selected from halide anions; Selected from alkali metal cations.

3. The water-soluble AIE fluorescent probe according to claim 1, characterized in that: Selected from F - Cl - ,Br - One of them; Selected from Na + or K + .

4. A method for preparing a water-soluble AIE fluorescent probe according to any one of claims 1 to 3, characterized in that, include: (1) Intermediate product A was prepared by nucleophilic substitution reaction using N,N-dialkylolamine and p-fluorobenzaldehyde as raw materials; (2) Mix 4-methylpyridine, 2-haloethyl sulfonate and 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 water-soluble AIE fluorescent probe.

5. The method for preparing the water-soluble AIE fluorescent probe according to claim 4, characterized in that, In step (1): The N,N-dialkylolamine is selected from diethanolamine or N,N-di(3-hydroxypropyl)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.

6. The method for preparing the water-soluble AIE fluorescent probe according to claim 4, characterized in that, In step (2): The 2-haloethyl sulfonate is selected from sodium 2-bromoethyl sulfonate or sodium 2-chloroethyl sulfonate. 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 2-haloethylsulfonate is 1~20:

1.

7. The method for preparing the water-soluble AIE fluorescent probe according to claim 4, characterized in that, In step (3): The molar ratio of intermediate product A to intermediate product B is 1.0~1.2:1; 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.

8. The application of a water-soluble AIE fluorescent probe according to any one of claims 1 to 3 in the preparation of a live cell fluorescence imaging probe.

9. The application of the water-soluble AIE fluorescent probe according to claim 8 in the preparation of live cell fluorescence imaging probes, characterized in that, Application of the water-soluble AIE fluorescent probe in the preparation of cell membrane-targeted fluorescent imaging probes.

Images