An aggregation-induced emission fluorescent probe, and a preparation method and use thereof

Abstract

The application discloses a preparation method of an aggregation-induced emission fluorescent probe, and relates to the technical field of fluorescent probes, and comprises the following steps: synthesis of TPE-4NM, placing bis(4-(dimethylamino)phenyl) ketone and zinc powder in a double-neck flask, vacuumizing, filling argon, adding anhydrous tetrahydrofuran, slowly injecting titanium tetrachloride into the solution through a syringe, filtering, collecting the filtrate, concentrating under reduced pressure, separating through column chromatography, and obtaining TPE-4NM; synthesis of TPE-4NMB, dispersing TPE-4NM in anhydrous acetonitrile, then adding p-bromomethylphenylboronic acid, stirring and reacting overnight at room temperature, collecting the filter cake, redissolving after adding water, removing insoluble substances through centrifugation, freeze-drying the filtrate, and obtaining TPE-4NMB. ‑ The fluorescent probe can specifically detect and remove ONOO ‑ in a solution or cells, and after activation, exhibits typical aggregation-induced emission characteristics in an aqueous solution, has the advantages of strong water solubility, small toxicity, strong anti-interference ability and excellent selective removal of ONOO

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of fluorescent probe, and particularly relates to an aggregation-induced emission fluorescent probe and a preparation method and application thereof. BACKGROUND

[0002] ONOO - is an active substance with strong oxidizing property, which is mainly generated by diffusion reaction of nitric oxide and superoxide anion under non-enzyme catalysis in the body. Low concentration ONOO - can participate in the signal transduction process in the organism, maintain normal physiological functions, but when the concentration is too high, it can destroy the structure and function of biological macromolecules such as enzymes, proteins, lipids and DNA. Since mitochondria are the main organelles for its generation, excessive ONOO - often leads to cell metabolic disorders and energy depletion, causes cell apoptosis, leads to autoimmune diseases, inflammatory lesions and the like, for example, it is often closely related to atherosclerosis, ischemia-reperfusion injury, Alzheimer's disease, cancer occurrence and metastasis and the like. Therefore, monitoring the dynamic changes of ONOO - can provide important enlightenment for monitoring the occurrence of related diseases and predicting pathological progression, and timely removal of excessive ONOO - can provide an effective way to prevent related lesions.

[0003] However, ONOO - has the properties of high reactivity, short half-life and difficulty in capture, which makes it extremely challenging to directly detect and remove it. At present, the detection methods for ONOO - include ultraviolet spectrophotometry, electrochemical analysis, fluorescence spectrophotometry and the like, among which the fluorescence probe has the advantages of high sensitivity, fast response speed and simple operation, and is suitable for online monitoring of ONOO - . However, the existing fluorescence probes are mostly traditional fluorescent molecular cores, such as coumarin and the like, and such fluorescent molecules have the property of aggregation-induced quenching, that is, the fluorescence intensity is high in a dilute solution or a single dispersion state, but in a high concentration or aggregation state, the excited state molecules often return to the ground state in a non-fluorescent emission manner, resulting in fluorescence quenching, and further causing the detection sensitivity to be reduced. In addition, the existing probes are easily interfered by active oxygen substances such as ·OH (for example, BBoxiProbe® O72 which is commercialized), resulting in poor selectivity of the probe detection and reduced accuracy. From the perspective of ONOO - removal, the currently reported ONOO - fluorescence detection probes only have the detection function and do not have the ability to selectively remove ONOO - , which makes it necessary to additionally apply therapeutic drugs for removing ONOO -, not only increases the pain of patients, but also increases the cost of treatment; and the existing active oxygen scavenger also faces the bottleneck of being unable to selectively remove ONOO - , which will remove the active oxygen components (such as H2O2) that are beneficial to maintain normal physiological functions at the same time.

[0004] Therefore, it is necessary to develop an aggregation-induced emission fluorescence probe with the ability of specific detection and removal of ONOO - . SUMMARY

[0005] The purpose of the present application is to provide an aggregation-induced emission fluorescence probe and a preparation method and use thereof, to solve the problems in the background art.

[0006] To achieve the above-mentioned purpose, the present application provides the following technical solutions:

[0007] An aggregation-induced emission fluorescence probe, the fluorescence probe has the following structural formula:

[0008]

[0009] Among them, anion X - is an anion with one or more charges, X - is F - , Cl - , Br - , I - , AcO - , PF6 - , BF4 - , CF3COO - , SO4 2- , CO3 2- , SO3 2- , ClO4 - or PO4 3- .

[0010] On the basis of the above technical solutions, the present application further provides the following optional technical solutions:

[0011] In an optional solution, the fluorescence probe has the following structural formula:

[0012] .

[0013] A preparation method of an aggregation-induced emission fluorescence probe, comprising the following steps:

[0014] Step S1: Synthesis of TPE-4NM: Bis(4-(dimethylamino)phenyl) ketone and zinc powder were placed in a double-necked flask, evacuated and purged with argon, and then anhydrous tetrahydrofuran was added. The solution was then cooled to -80°C, and titanium tetrachloride was slowly injected into the solution through a syringe. After the reaction solution cooled to room temperature, it was filtered, the filtrate was collected, concentrated under reduced pressure, and separated by column chromatography to obtain TPE-4NM.

[0015] Step S2: Synthesis of TPE-4NMB. TPE-4NM was dispersed in anhydrous acetonitrile, followed by the addition of p-bromomethylphenylboronic acid. The mixture was stirred overnight at room temperature. After the reaction was complete, the mixture was filtered through a nylon microporous membrane, and the filter cake was collected. After redissolving in water, the insoluble matter was removed by centrifugation. The filtrate was then lyophilized to obtain TPE-4NMB. The structural formula of this fluorescent probe is as follows: .

[0016] In one alternative: after titanium tetrachloride is added to the solution in step S1, the solution is transferred to room temperature and stirred for 30-60 min and then refluxed and stirred overnight.

[0017] In one alternative: in step S2, the pore size of the nylon microporous filter membrane is 0.45 μm.

[0018] In one alternative: in step S1, the concentration of TPE-4NM is 5~200 μM.

[0019] In one alternative: in step S2, the concentration of TPE-4NMB is 5~200 μM.

[0020] The aggregation-induced emission fluorescent probes described above are used in specific detection solutions or intracellular ONOO. - Applications.

[0021] The aggregation-induced emission fluorescent probes described above can be used to specifically clear ONOO from solutions or cells. - Applications.

[0022] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0023] The fluorescent probe provided by this invention can specifically detect and remove ONOO in solution or cells. - After activation, it exhibits typical aggregation-induced emission characteristics in aqueous solutions. It possesses high water solubility, low toxicity, strong anti-interference ability, and selective scavenging of ONOO. - Advantages of superior abilities. Attached Figure Description

[0024] Figure 1 The TPE-4NM in Embodiment 1 of this invention 1H-NMR spectrum.

[0025] Figure 2 The image shows the HR-MS plot of TPE-4NM in Embodiment 1 of the present invention.

[0026] Figure 3 The TPE-4NMB in Embodiment 1 of this invention 1 H-NMR spectrum.

[0027] Figure 4 The TPE-4NMB in Embodiment 1 of this invention 13 C-NMR spectrum.

[0028] Figure 5 The UV-Vis scanning spectra of TPE-4NM and TPE-4NMB in the range of 350~800 nm are shown in Example 2 of this invention.

[0029] Figure 6 The fluorescence emission spectra of TPE-4NM in Example 2 of this invention in different proportions of tetrahydrofuran / water mixed solvent are shown.

[0030] Figure 7 The fluorescence emission spectra of TPE-4NMB in different proportions of tetrahydrofuran / water mixed solvent in Example 2 of the present invention are shown.

[0031] Figure 8 The TPE-4NMB and different concentrations of ONOO in Example 1 of this invention - Fluorescence emission spectrum after incubation.

[0032] Figure 9 The TPE-4NMB and ONOO in Embodiment 1 of this invention - Fluorescence emission spectra after co-incubation for different times.

[0033] Figure 10 The relative fluorescence intensity at 532 nm of TPE-4NMB in Example 1 of this invention after incubation with different substances for 30 min is shown.

[0034] Figure 11 The cytotoxicity of TPE-4NM and TPE-4NMB on mouse fibroblast L929 cells in Example 1 of this invention.

[0035] Figure 12 The cytotoxicity of TPE-4NM and TPE-4NMB on mouse breast cancer cells 4T1 in Example 1 of this invention.

[0036] Figure 13 In Example 1 of this invention, TPE-4NMB was co-incubated with 4T1 cells for 4 h, and then different concentrations of ONOO were added.- The fluorescence photo of TPE-4NMB in Example 1 of the present application after co-incubation with mouse macrophage RAW264.7 cells treated with LPS (16 h) and PMA (1 h) for 4 h; the control group is RAW264.7 cells without LPS (16 h) and PMA (1 h) treatment; the RAW264.7 cells after being treated with LPS (16 h) and PMA (1 h) and then adding NAC.

[0037] Figure 14 The fluorescence photo of TPE-4NMB in Example 1 of the present application after co-incubation with 4T1 cells for 4 h, and then adding different stimulants for 2 h.

[0038] Figure 15 The fluorescence photo of TPE-4NMB in Example 1 of the present application after co-incubation with 4T1 cells for 4 h, and then adding different stimulants for 2 h.

[0039] Figure 16 The fluorescence photo of TPE-4NMB in Example 1 of the present application after co-incubation with different concentrations of ONOO - After co-incubation for 30 min, continue to co-incubate with the reduced glutathione kit for 5 min, and then scan the ultraviolet-visible light spectrum in the range of 300-600 nm.

[0040] Figure 17 The fluorescence photo of TPE-4NMB in Example 1 of the present application after co-incubation with different concentrations of ONOO

[0041] Figure 18 The fluorescence photo of TPE-4NMB in Example 1 of the present application after co-incubation with different concentrations of ONOO - The fluorescence photo of TPE-4NMB in Example 1 of the present application after co-incubation with different concentrations of ONOO - The fluorescence photo of TPE-4NMB in Example 1 of the present application after co-incubation with different concentrations of ONOO - The fluorescence photo of TPE-4NMB in Example 1 of the present application after co-incubation with different concentrations of ONOO - The fluorescence photo of TPE-4NMB in Example 1 of the present application after co-incubation with different concentrations of ONOO - The fluorescence photo of TPE-4NMB in Example 1 of the present application after co-incubation with different concentrations of ONOO DETAILED DESCRIPTION

[0042] In order to make the purpose, technical scheme and advantages of the present application more clear, the present application is further described in detail below in combination with the drawings and examples; each example listed in the present application is only used to illustrate the present application, and is not used to limit the scope of the present application. Any obvious modification or change made to the present application does not deviate from the spirit and scope of the present application. Example 1

[0043] In the embodiments of the present application, an aggregation-induced emission fluorescent probe is provided, which has the following structural formula:

[0044]

[0045] wherein the anion X - is an anion with one or more charges, X - is F - , Cl - , Br - , I - , AcO - , PF6 - , BF4 - , CF3COO - , SO4 2- , CO3 2- , SO3 2- , ClO4 - or PO4 3- . Example 2

[0046] In the embodiments of the present application, a preparation method of an aggregation-induced emission fluorescent probe is provided, which comprises the following steps:

[0047] Take 2.0 g of bis (4- (dimethylamino) phenyl) ketone and 1.2 g of zinc powder into a double-necked flask, then vacuumize and fill with argon, add 50 mL of anhydrous tetrahydrofuran, cool the solution to-80℃, then slowly add 680 μL of titanium tetrachloride into the solution using a syringe, stir for 30 min, then transfer the solution to room temperature and continue to stir for 1 h, then reflux overnight, after the reaction is completed, add 20 mL of 10% K2CO3 aqueous solution to quench the reaction, remove the insoluble substances by filtration, collect the filtrate, extract with dichloromethane three times, combine the organic phases, vacuum reduce and concentrate, and separate by column chromatography to obtain TPE-4NM; 1 H NMR (400 MHz, DMSO- d 6) δ6.75 (d, J = 8.3 Hz, 8H), 6.44 (d, J= 8.6 Hz, 8H), 2.82 (s, 24H). HR-MS, m / z calcd. for C 34 H 40 N4 + :505.3287; found, 505.3312.

[0048] Synthesis of TPE-4NMB:

[0049] 50 mg TPE-4NM and 106 mg 4-bromomethylphenylboronic acid were precisely weighed into a round bottom flask, then 20 mL acetonitrile was added, stirred at room temperature under argon protection overnight, after the reaction was completed, the solvent was concentrated under reduced pressure, filtered using a 0.45 μm nylon microporous filter, the filter cake was collected, redissolved with water and centrifuged to remove insoluble matter, and the filtrate was freeze-dried to obtain TPE-4NMB;

[0050] 1 H NMR (600 MHz, D2O) δ 7.47 (d, J = 7.9 Hz, 2H), 7.39 (d, J = 8.6 Hz,2H), 7.23 (d, J = 8.8 Hz, 2H), 6.82 (d, J = 7.9 Hz, 2H), 4.86 (s, 2H), 3.56(s, 6H). 13 C NMR (150 MHz, D2O) δ 144.33, 141.96, 139.07, 133.72, 132.88,131.60, 129.47, 126.76, 121.28, 73.34, 53.23.

[0051] UV-Vis spectrum scanning of TPE-4NM and TPE-4NMB

[0052] 20 μM dimethyl sulfoxide solutions of TPE-4NM and TPE-4NMB were prepared respectively, and their spectra were scanned in the wavelength range of 350~800 nm, and the results are shown in Figure 5 .

[0053] Testing of the aggregation-induced emission effect of TPE-4NM and TPE-4NMB

[0054] TPE-4NM is readily soluble in tetrahydrofuran but poorly soluble in water. In this embodiment, it was used to test the aggregation-induced emission effect. TPE-4NM and TPE-4NMB were weighed separately and dissolved in tetrahydrofuran or water to prepare a 1 mM stock solution. Then, 10 μL of each stock solution was added to centrifuge tubes, along with predetermined volumes of tetrahydrofuran and water, to achieve water volume percentages of 99%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, and 0%. The fluorescence emission wavelength of each sample in the range of 400–800 nm was recorded using 365 nm as the excitation wavelength. The fluorescence emission scan results for TPE-4NM are shown below. Figure 6 As shown, with increasing water content in the solution, the fluorescence emission intensity of TPE-4NM at around 532 nm significantly increases. The fluorescence emission intensity at 99% water content is 220 times that at 0% water content, exhibiting a typical aggregation-induced emission effect. The fluorescence emission scanning results of TPE-4NMB are shown below. Figure 7 As shown, its fluorescence remains low within the water content range of 0% to 99%. This property may be due to its strong water and lipid solubility, which helps to reduce false positives caused by autofluorescence and improve the sensitivity of probe detection.

[0055] The TPE-4NMB obtained in Example 1 for ONOO - Detection performance

[0056] A 20 μM TPE-4NMB aqueous solution was mixed with ONOO solutions of concentrations of 0.1, 1, 2, 5, 10, 20, 50, and 100 μM, respectively. - After co-incubation at 37℃ for 30 min, the fluorescence emission spectrum between 400 and 800 nm was scanned using a fluorescence spectrophotometer with an excitation wavelength of 365 nm. The results are as follows: Figure 8 The fluorescence emission intensity of the incubation solution at 532 nm at various concentrations was linearly fitted, y = 3.0260x - 11.1047, R0 2 = 0.9749, indicating that the TPE-4NMB obtained in Example 1 is effective against ONOO. - It exhibits strong detection sensitivity; the fluorescence emission intensity after activation is similar to that of ONOO in the incubation solution. - The concentration of is linearly related.

[0057] The TPE-4NMB obtained in Example 1 for ONOO - Time-dependent activation:

[0058] Mix a 20 μM TPE-4NMB aqueous solution with a 30 μM ONOO solution. -After co-incubation for different times, the fluorescence emission spectrum in the range of 400-800 nm was scanned using a fluorescence spectrophotometer with 365 nm as the excitation wavelength. The results are as follows: Figure 9 As shown, the fluorescence intensity emitted after TPE-4NMB activation was time-dependent within 30 min, and remained essentially unchanged after 30 min, indicating that TPE-4NMB and ONOO... - The reaction is rapid, and the reaction is complete within 30 minutes.

[0059] The TPE-4NMB obtained in Example 1 affects ONOO in solution. - Selective activation

[0060] A 20 μM TPE-4NMB aqueous solution was reacted with 1 mM cysteine ​​(Cys), sodium bisulfite (NaHSO3), sodium ascorbate (VC), anhydrous copper chloride (CuCl2), and superoxide anion (…). 1 O2 - After co-incubating with tert-butanol peroxide (tBuOOH), sodium nitroprusside (SNP), sodium hypochlorite, hydroxyl radicals, hydrogen peroxide, sodium thiosulfate, reduced glutathione, and N-acetylcysteine ​​for 30 min, the fluorescence emission intensity of the incubation solution at 532 nm was recorded using a fluorescence spectrophotometer at an excitation wavelength of 365 nm. The relative fluorescence intensity of each group and the PBS group at this wavelength was calculated. The results are as follows: Figure 10 As shown, when TPE-4NMB and ONOO - During co-incubation, the fluorescence intensity increased by approximately 120-fold, while the fluorescence intensity of other groups remained within a 10-fold increase, indicating that the TPE-4NMB probe is effective against ONOO. - It has good selectivity.

[0061] Cytotoxicity of TPE-4NM and TPE-4NMB obtained in Example 1

[0062] TPE-4NM and TPE-4NMB obtained in Example 1 were prepared at concentrations of 5, 10, 20, 50, 100, and 200 μM using serum-free DMEM medium, and were set aside for later use; mouse breast cancer cells 4T1 or mouse fibroblasts L929 were cultured at a concentration of 5 × 10⁻⁶ μM. 3The cells were seeded at a density of approximately 80% in 96-well plates. The culture medium was then discarded and replaced with the previously prepared medium containing different concentrations of TPE-4NM and TPE-4NMB. The plates were incubated at 37°C for 24 h. Subsequently, 10 μL of MTT was added to each well under dark conditions, and the plates were incubated for another 4 h in a cell culture incubator. The culture medium was then discarded, and 100 μL of dimethyl sulfoxide was added to each well to dissolve the formazan within the cells. The absorbance of each well at 490 nm was measured using a microplate reader. The relative viability (%) of each test well relative to the control group was calculated using the following formula:

[0063] Cell viability (%) = (Absorbance value of test well at 490 nm / Absorbance value of control group at 490 nm) × 100%

[0064] Test results are as follows Figure 11 and Figure 12 As shown, TPE-4NM exhibited low cytotoxicity against mouse 4T1 cells and mouse fibroblast L929 cells in the range of 5–100 μM, with moderate cytotoxicity against 4T1 cells only at 200 μM. The TPE-4NMB obtained in Example 1 showed selective cytotoxicity against 4T1 cells, but did not show significant cytotoxicity against normal L929 cells in the range of 5–200 μM. This may be related to the difference in the uptake capacity of TPE-4NMB between tumor cells and normal cells. Therefore, TPE-4NMB and TPE-4NM have good biocompatibility with normal cells.

[0065] The TPE-4NMB obtained in Example 1 showed exogenous ONOO at the cellular level. - Detection:

[0066] Mouse breast cancer cells 4T1 were administered at a rate of 1×10⁻⁶. 5 The cells were seeded at a density of / well in 6-well plates. When the cell density reached approximately 60%, the medium was discarded and replaced with DMEM medium containing 50 μM TPE-4NMB. After incubation in a cell culture incubator for 4 h, the medium was discarded and replaced with DMEM medium containing 0, 1, 5, and 10 μM NMB, respectively. - The mixture was incubated in DMEM medium for another 2 hours, followed by observation and photography using a fluorescence microscope. The results are as follows: Figure 13 As shown: With ONOO - As the concentration increases, the intracellular fluorescence intensity increases in a concentration-dependent manner, indicating that TPE-4NMB can be converted into exogenous N-type ... - Concentration-dependent activation.

[0067] The TPE-4NMB obtained in Example 1 has an effect on endogenous ONOO at the cellular level. -Detection of ONOO

[0068] After incubating mouse macrophage RAW 264.7 with DMEM medium containing 1 μg / mL lipopolysaccharide (LPS) and 100 ng / mL IFN-γ for 16 h, the medium was removed and DMEM medium containing 600 nM phorbol-12-myristate-13-acetate (PMA) was added for further incubation for 1 h, then the medium was removed and DMEM medium containing 50 μM TPE-4NMB was added for further incubation for 4 h, and then the cells were observed and photographed under a fluorescence microscope, with RAW 264.7 cells not treated with the above stimulants as a control; in the inhibition experiment, after the LPS induction was completed, 1 mM NAC was added for incubation for 4 h, and then 50 μM TPE-4NMB was added for incubation for 4 h, and then the cells were observed and photographed, and the results are shown in Figure 14 : RAW 264.7 cells stimulated by LPS and PMA can induce the production of ONOO - , and after incubation with TPE-4NMB, obvious fluorescence can be observed in the cells, while almost no fluorescence can be observed in the original RAW 264.7 cells. Acetylcysteine (NAC) is a natural active nitrogen oxide scavenger, which can scavenge ONOO - produced by LPS / PMA induction, and as can be seen from the figure, after NAC treatment, the fluorescence intensity in the cells decreased obviously, further confirming that TPE-4NMB is caused by endogenous ONOO - .

[0069] Specific detection of ONOO - by TPE-4NMB obtained in Example 1 at the cell level:

[0070] 4T1 cells were inoculated into a 24-well plate at a density of 2×10 4 cells / well, and when the cell density grew to about 60%, the 4T1 cells were incubated with 50 μM TPE-4NMB for 4 h, then washed twice with PBS, and then incubated with culture medium containing different stimulants for 2 h, and then observed and photographed under a fluorescence microscope; the concentration of the stimulant contained in the culture medium was: ONOO - (10 μM), NAC (1 mM), NaClO (100 μM), SNP (100 μM), H2O2 (100 μM); and the results are shown in Figure 15 : The fluorescence intensity in the ONOO - group cells was obviously stronger than that in the other groups, which is consistent with the conclusion obtained in Example 6, and after the addition of NAC, the fluorescence intensity in the cells decreased significantly, further confirming that ONOO - is the main active ingredient for activating TPE-4NMB.

[0071] The selectivity of TPE-4NMB in eliminating ONOO - from solution

[0072] Reduced glutathione is the main reducing component in cells to maintain the redox balance. After co-incubation of TPE-4NMB with ONOO - or hydrogen peroxide, and then co-incubation with reduced glutathione, the ability of TPE-4NMB in eliminating ONOO - or hydrogen peroxide can be indirectly reflected by monitoring the content of residual reduced glutathione. After co-incubation of different concentrations of TPE-4NMB with equal concentrations of ONOO - or hydrogen peroxide for 30 min, the same amount of reduced glutathione was added for co-incubation for 5 min, and then color development was performed according to the instructions of the reduced glutathione detection kit. The ultraviolet-visible light scanning spectrum of each test group was scanned by an ultraviolet spectrophotometer in the range of 300-600 nm. NC is the negative control, representing the test well without reduced glutathione; PC represents the positive control, which is the test well containing 1 mM reduced glutathione; the rest of each group is the test well containing different concentrations of TPE-4NMB; the results are as follows Figure 16 and Figure 17 : The darker the yellow color in the test well, the more residual reduced glutathione. It can be seen that the yellow intensity of the test well in the co-incubation group of TPE-4NMB and ONOO - is significantly stronger than that in the NC group, and it shows a concentration-dependent manner, indicating that TPE-4NMB can selectively eliminate ONOO - , maintaining a high level of reduced glutathione; since TPE-4NMB cannot eliminate hydrogen peroxide, co-incubation of TPE-4NMB with hydrogen peroxide cannot alleviate the consumption of reduced glutathione by hydrogen peroxide; the ultraviolet absorption spectrum also reaches the same conclusion.

[0073] Elimination of intracellular ONOO - by TPE-4NMB obtained in Example 1

[0074] The elimination of intracellular ONOO - by TPE-4NMB was monitored by a commercial ONOO - probe.

[0075] Logarithmic growth phase 4T1 cells were taken, and 1×10 5 / well of the cells were inoculated in a 6-well plate, and then the plate was placed in a cell incubator for overnight incubation. When the cell density reached 60%, the original culture medium was removed, and 50 μM TPE-4NMB or blank medium was added for co-incubation for 4 h, and then the medium was removed. Depending on the grouping, ONOO -Continue to incubate for 1 h, and finally add commercialized ONOO - After 1 h of probe incubation, observe and record the intracellular probe activation using a fluorescence microscope, with the group without adding commercialized probe or the group without adding TPE-4NMB as a control. The results are shown in FIG. 2. Figure 18 As shown in FIG. 2: compared with the group of adding commercialized ONOO - probe + ONOO - , the intracellular fluorescence intensity of the group of TPE-4NMB + ONOO - is significantly reduced, indicating that TPE-4NMB can effectively scavenge intracellular ONOO - .

[0076] The above is merely a specific implementation of the present disclosure, but the protection scope of the present disclosure is not limited thereto. Any person skilled in the art can easily think of changes or replacements within the technical range disclosed by the present disclosure, which shall be encompassed within the protection scope of the present disclosure. Therefore, the protection scope of the present disclosure shall be subject to the protection scope of the claims.

Claims

1. An aggregation-induced emission fluorescent probe, characterized in that, The aggregation-induced emission fluorescent probe has the following structural formula: wherein the anion X - is F - , Cl - , Br - , I - , AcO - , PF6 - , BF4 - , CF3COO - or ClO4 - . 2.The aggregation-induced emission fluorescent probe of claim 1, characterized in that, The aggregation-induced emission fluorescent probe has the following structural formula: 。 3. A method of preparing an aggregation-induced emission fluorescent probe according to claim 2, wherein, The method comprises the following steps: Step S1: synthesis of TPE-4NM, double (4-(dimethylamino) phenyl) ketone, zinc powder are placed in a double-neck flask, vacuum is drawn and argon is filled, then anhydrous tetrahydrofuran is added, then the solution is cooled to-80℃, titanium tetrachloride is slowly injected into the solution through a syringe, after the reaction solution is cooled to room temperature, filtration is performed, the filtrate is collected, vacuum concentration is performed, and column chromatography is performed to separate TPE-4NM; Step S2: synthesis of TPE-4NMB, dispersing TPE-4NM in anhydrous acetonitrile, then adding p-bromomethylphenylboronic acid, stirring at room temperature overnight, after the reaction is completed, filtering with a nylon microporous filter, collecting the filter cake, redissolving with water, removing the insoluble material by centrifugation, and freeze-drying the filtrate to obtain TPE-4NMB; wherein, in the preparation method of the aggregation-induced emission fluorescent probe, the reaction formula is as follows: .

4. The method for preparing the aggregation-induced emission fluorescent probe according to claim 3, characterized in that, In step S1, after the addition of titanium tetrachloride into the solution is completed, the solution is transferred to room temperature for continuous stirring for 30-60 min and reflux stirring overnight.

5. The method for preparing the aggregation-induced emission fluorescent probe according to claim 3, characterized in that, In the step S2, the pore size of the nylon microporous filter membrane is 0.45 μm. 6.The method of preparing an aggregation-induced emission fluorescent probe according to claim 3, characterized in that, In step S1, the concentration of the TPE-4NM is 5-200 μM.

7. The method for preparing the aggregation-induced emission fluorescent probe according to claim 3, characterized in that, In step S2, the concentration of the TPE-4NMB is 5-200 μM.

8. Use of the aggregation-induced emission fluorescent probe according to claim 1 or 2 for the specific detection of ONOO in a solution. - of ONOO in a solution.

9. Use of the aggregation-induced emission fluorescent probe according to claim 1 or 2 for the specific scavenging of ONOO - -1.

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