AIE fluorescent probe for detecting HSO3 <-> by targeting lysosome

Abstract

The invention provides an aggregation-induced emission (AIE) fluorescent probe (Z)-3-[4-(bis {4-[2-morpholinyl ethyoxyl] phenyl} amino) phenyl]-2-(4-quinolyl) acrylonitrile for targeted lysosome detection of HSO3 <->, namely a fluorescent probe MoTAQ as well as a preparation method and application thereof in the aspect of detection. The weak alkalinity of the morpholine ring group in the probe and the weak acidity in the cell lysosome attract each other to realize lysosome targeting; according to the fluorescent probe, a molecular structure is modified, so that the molecular structure can realize specific detection of HSO3 <->, and meanwhile, the wavelength of a fluorophore in the fluorescent probe is shifted from red light to blue light before and after HSO3 <-> detection, so that effective observation can be performed through color change during fluorescence detection; secondly, MoTAQ has good fluorescence emission spectrum characteristics, and the fluorescent probe has high signal-to-noise ratio in vivo and in vitro; in addition, the fluorescent probe disclosed by the invention also has good biocompatibility.

Description

Technical Field

[0001] This invention relates to the field of organic fluorescent probe molecules, specifically to a targeted lysosomal detection method for HSO3. - Preparation methods and applications of AIE fluorescent probes. Background Technology

[0002] Lysosomes, as highly dynamic acidic digestive centers in eukaryotic cells, are responsible not only for the degradation and recycling of biomolecules and the regulation of metabolic homeostasis, but also for participating in cell fate determination through the regulation of ion balance and signal transduction. The HSO3- within their lumen... - As a key sulfur-containing metabolic intermediate, lysosomal enzyme activity, membrane permeability, and autophagy-apoptosis balance are directly affected. Abnormal fluctuations in its concentration have been proven to be closely related to pathological processes such as lysosomal storage disorders, neurodegenerative diseases (e.g., Alzheimer's disease, Huntington's disease), tumor drug resistance, and immune metabolic disorders. Due to the unique acidic microenvironment (pH 4.5-5.5) and highly active hydrolytic enzyme system of lysosomes, traditional detection probes are prone to protonated fluorescence quenching or enzymatic degradation, making it difficult to detect HSO3. - Precise in-situ quantification is crucial. Therefore, developing novel detection tools that combine lysosomal targeting specificity, acidic environment stability, and dynamic response sensitivity is urgently needed to elucidate the molecular mechanisms of lysosomal-related diseases and advance targeted therapy strategies.

[0003] In the field of lysosomal microenvironment probe design, existing technologies generally face three major challenges: First, conventional probes lack lysosomal membrane penetration and precise subcellular organelle localization, easily resulting in non-specific distribution in the cytoplasm; second, acid-induced molecular protonation significantly alters the probe's electronic structure, leading to absorption / emission spectral shifts and decreased fluorescence quantum efficiency; third, HSO3... - Other biothiols (such as glutathione and cysteine) and reactive sulfur species (such as H2S and SO3) 2⁻ Complex redox crosstalk exists between HSO3 and other organelles, making specific recognition difficult with traditional probes. In recent years, functional materials with AIE properties have offered a new path to overcome these bottlenecks due to their unique advantage of "hydrophobic aggregation enhancing luminescence." Compared to planar conjugated ACQ-type probes, AIE probes maintain high brightness in the acidic, water-rich environment of lysosomes through intramolecular motion restriction, while achieving precise subcellular organelle localization through targeting group modification. More importantly, by rationally designing the molecular rotor structure and response sites of the probe, specific recognition of HSO3 can be achieved. - The specifically activated fluorescence signal is amplified and simultaneously sensed multidimensional microenvironment parameters such as lysosomal viscosity and pH.

[0004] Current research in this field still faces significant technical limitations: existing probe synthesis strategies often rely on complex, multi-step modifications, making it difficult to simultaneously optimize the lysosomal targeting module, signal response unit, and AIE luminescent core. Furthermore, traditional detection systems often require exogenous stimuli (such as hydrogen peroxide pretreatment or ion carrier intervention) to enhance probe sensitivity. Such operations easily disrupt lysosomal membrane integrity, leading to ion homeostasis imbalance and ultimately distorting the detection signal. Therefore, developing a class of AIE probes that can adapt to the acidic environment of lysosomes, possess in-situ activation properties, and require no external intervention will be crucial for overcoming existing technological barriers and achieving lysosomal HSO3 luminescence detection. - A key breakthrough in the precise analysis of metabolic networks. Summary of the Invention

[0005] Objective of the invention: To address the shortcomings and defects of existing technologies, this invention provides a targeted lysosomal detection method for HSO3. - The preparation method and application of an AIE fluorescent probe are described. The weakly basic nature of the morpholine ring group in this probe attracts the weakly acidic environment of lysosomes, enabling lysosomal targeting. This fluorescent probe, through molecular structure modification, allows for targeting of HSO3-. - This allows for specific detection, thus exhibiting high selectivity; simultaneously, the fluorescent group in the fluorescent probe detects HSO3. - The wavelength shifts from red light to blue light, thus allowing for effective observation through color changes during fluorescence detection.

[0006] Technical solution: A targeted lysosomal detection method for HSO3 according to the present invention - The AIE fluorescent probe is characterized by the following chemical structural formula: .

[0007] The weakly basic morpholine ring group in this probe attracts the weakly acidic environment of lysosomes, enabling lysosomal targeting. This fluorescent probe, through molecular structure modification, allows it to target HSO3. - This allows for specific detection, thus exhibiting high selectivity; simultaneously, the fluorescent group in the fluorescent probe detects HSO3. - The wavelength shifts from red to blue light, allowing for effective observation through color change during fluorescence detection. This invention enables the detection of HSO3 using a fluorescent probe. - The specific procedure for concentration is as follows: Select HSO3 - The Michael addition reaction involved is a probe reaction, which assesses HSO3 in food, live cells, and zebrafish by detecting changes in the fluorescence of the reaction product over a specific time period. - The actual concentration of HSO3. -The fluorescence properties of the reaction probe before and after the reaction show significant differences under the same conditions. Therefore, the fluorescence change of the reaction probe can be used to detect HSO3 in various biological systems. - The concentration and distribution of [the substance] were evaluated.

[0008] The present invention provides a targeted lysosomal detection method for HSO3. - The preparation method of the AIE fluorescent probe is characterized by: placing 4-bis(4-hydroxyphenyl)aminobenzaldehyde (TTBH), 4-(2-chloroethyl)morpholine, potassium iodide, and cesium carbonate in a reaction flask, adding ethanol to the reaction flask, heating the mixture to not less than 85°C under nitrogen protection, cooling the reaction solution to room temperature, extracting and combining the organic phases, rotary evaporating the mixed reaction solution, adding 2-(quinolin-4-yl)acetonitrile and piperidine, adding ethanol to the reaction flask, heating the mixture to not less than 85°C under nitrogen protection, cooling the reaction solution to room temperature, extracting and combining the organic phases, drying and vacuum concentration to obtain a crude product, using dichloromethane / methanol as the eluent, purifying the crude product by column chromatography to obtain the fluorescent probe (Z)-3-[4-(bis{4-[2-morpholinylethoxy]phenyl}amino)phenyl]-2-(4-quinolinyl)acrylonitrile, i.e., the fluorescent probe MoTAQ.

[0009] Among them, the targeted lysosomal detection of HSO3 - The method for preparing the AIE fluorescent probe is characterized in that the molar ratio of 4-bis(4-hydroxyphenyl)aminobenzaldehyde (TTBH) to 4-(2-chloroethyl)morpholine is 1:1 to 1:10.

[0010] The aforementioned targeted lysosomal detection of HSO3 - The method for preparing the AIE fluorescent probe is characterized in that the molar ratio of 4-bis(4-hydroxyphenyl)aminobenzaldehyde (TTBH) to 2-(quinolin-4-yl)acetonitrile is 1:0.5~1:10.

[0011] The method for preparing the lysosome-targeted aggregation-induced luminescence ionic probe is characterized in that the reaction time is at least 1 hour.

[0012] The method for preparing the lysosome-targeted aggregation-induced luminescence ionic probe is characterized in that the ratio of dichloromethane / methanol as eluent is 1:1 to 100:1.

[0013] The present invention provides a targeted lysosomal detection method for HSO3. - Applications of AIE fluorescent probes in detection.

[0014] Specifically, for in vitro cells, the detection process of the fluorescent probe for lysosomal localization in cells is as follows: the fluorescent probe is dissolved in DMEM culture medium at a concentration of 10 μM; HepG2 live cells are co-incubated with culture medium containing the fluorescent probe and a commercially available lysosomal localization probe (Lyso-Tracker green) for 30 min, and the co-localization effect of the two is observed under confocal microscopy as an evaluation index of the fluorescent probe's localization of lysosomes.

[0015] The fluorescent probe of this invention, (Z)-3-[4-(bis{4-[2-morpholinylethoxy]phenyl}amino)phenyl]-2-(4-quinolinyl)acrylonitrile, has a specific molecular structure that allows it to react only with HSO3. - A Michael addition reaction occurs, thus enabling the reaction of HSO3. - The specific detection of (Z)-3-[4-(bis{4-[2-morpholinylethoxy]phenyl}amino)phenyl]-2-(4-quinolinyl)acrylonitrile's carbon-carbon double bond can react with HSO3. - The product (2S,3S)-3-[4-bis(4-(2-morpholinoethoxy)phenyl)aminophenyl]-3-di(hydroxy)(oxo)-λ is formed via an intramolecular 1,4-conjugated addition reaction. 6 2-Thio-(quinolin-4-yl)propionitrile (MoTAQ-HSO3) exhibits strong blue fluorescence emission upon photoexcitation, while MoTAQ shows red fluorescence in aqueous solution. The concentration of HSO3 in different biological samples is determined by detecting the color change before and after the reaction over a specific time period. - Concentration and distribution.

[0016] The reaction formula is:

[0017] Beneficial Effects: Compared with existing technologies, the present invention has the following significant advantages: 1. The fluorescent probe of the present invention, through modification of its molecular structure, enables the molecular structure to specifically target lysosomes in living cells, thereby exhibiting high selectivity. 2. The fluorescent group in the fluorescent probe detects HSO3... - The wavelength shifts from red to blue light, allowing for effective observation through color changes during fluorescence detection; 3. MoTAQ exhibits excellent fluorescence emission spectral characteristics (590-800nm), thus the fluorescent probe of this invention has a high signal-to-noise ratio both in vivo and in vitro; 4. The fluorescent probe of this invention has good biocompatibility; after incubating cells with a medium containing MoTAQ (80μM) for 24 hours, cell viability remains greater than 85%; 5. The fluorescent probe of this invention can be used in food, in solutions of live cells, and in live zebrafish containing HSO3. - Its determination has a wide range of applications. Attached Figure Description

[0018] Figure 1 This is a synthetic route diagram of the fluorescent probe of the present invention; Figure 2 This is the normalized ultraviolet absorption (black) and fluorescence emission (red) spectrum of the fluorescent probe of the present invention; Figure 3 The fluorescent probe of this invention reacts with HSO3 - High-resolution mass spectrum of the reaction following the binding reaction; Figure 4 The spectrum shows the maximum fluorescence intensity ratio of the fluorescent probe of the present invention in mixed solvents of different proportions of dimethyl sulfoxide / toluene; Figure 5 The UV absorption spectra of the fluorescent probe of the present invention after adding different concentrations of NaHSO3 are shown. Figure 6 The fluorescent probe of this invention, after adding different concentrations of NaHSO3, exhibits λ EX Fluorescence spectrum at 290 nm (A) and at λ EX = Fluorescence spectrum at 390 nm (B) (Inset: Photographs of MoTAQ and MoTAQ+NaHSO3 under 365 nm UV light); Figure 7 The emission wavelength I of the fluorescent probe of this invention after adding 100 μm concentration of NaHSO3 is... 390nm with I 630nm One of the photostability factors of the ratio; Figure 8 The emission wavelength I of the fluorescent probe of this invention after adding 100 μm concentration of NaHSO3 is... 390nm with I 630nm The second aspect of photostability of the ratio; Figure 9 The illustration shows the fluorescence selectivity spectra of the fluorescent probe of the present invention under different interfering analytes. The illustration shows photographs of each interfering analyte + MoTAQ under a UV lamp.

[0019] Figure 10 This is a confocal fluorescence imaging image of the fluorescent probe of the present invention and the commercial lysosomal localization probe (Golgi-Tracker Green) after co-incubation in HepG2 cells; in the figure, A is the colocalization map between MoTAQ and Golgi-Tracker Green; B is a magnified combination map of MoTAQ and Golgi-Tracker Green; C is the overlap coefficient and Pearson colocalization coefficient map between MoTAQ and Golgi-Tracker Green. Figure 11 This is a diagram showing the dark cytotoxicity of the fluorescent probe of the present invention to HepG2 cells; Figure 12 This invention relates to the practical application of the fluorescent probe in food (sugar / salt); Figure 13 The fluorescence images (A) and fluorescence intensity comparison (B) of the fluorescent probe of the present invention after incubation of HepG2 cells with 0 / 50 / 100 μM NaHSO3 are taken. Figure 14 Fluorescence imaging of the fluorescent probe of the present invention in zebrafish; Detailed Implementation

[0020] The technical solution of the present invention will be further described below with reference to the accompanying drawings and specific embodiments.

[0021] The fluorescent probe (Z)-3-[4-(bis{4-[2-morpholinylethoxy]phenyl}amino)phenyl]-2-(4-quinolinyl)acrylonitrile, i.e., the fluorescent probe MoTAQ, of this invention is prepared by the following method, the synthesis method of which is attached. Figure 1 As shown.

[0022] This compound uses hydrophobic triphenylamine as a donor, thiophene as a π-bridge, and a morpholine ring as a lysosomal targeting group to construct an aggregation-induced emission fluorescent probe. The synthetic route is simple, with high yield and low cost. The fluorescent group in the probe is used for the detection of HSO3. - The wavelength shifts from red to blue light, allowing for effective observation through color changes during fluorescence detection. The weakly basic morpholine ring group in the probe attracts the weakly acidic environment of lysosomes, enabling lysosomal targeting. Furthermore, this fluorescent probe can be used in food, in solutions containing live cells, and in HSO3 in live zebrafish. - Its determination has a wide range of applications.

[0023] Specific steps: 1 mmol of TTBH (the TTBH of this invention is prepared using the synthesis method disclosed in the following literature: ACSsensors, 5(1), 225-233.), 1 mmol 4-(2-chloroethyl)morpholine, 0.5 mmol of potassium iodide, and 0.5 mmol of cesium carbonate were placed in a 25 mL two-necked flask. 20 mL of anhydrous acetonitrile was added to the flask. The mixture was heated to at least 85 °C under nitrogen protection. The reaction solution was cooled to room temperature, extracted, and the organic phases were combined. The mixture was then rotary evaporated, and 1 mmol of 2-(quinoline-4-yl)acetonitrile and 200 μL of piperidine were added. 20 mL of ethanol was added to the reaction flask. The mixture was heated to at least 85 °C under nitrogen protection. The reaction solution was cooled to room temperature, extracted, and the organic phases were combined. The mixture was dried and concentrated under reduced pressure to obtain a crude product. The crude product was purified by column chromatography using dichloromethane / methanol as the eluent (eluent: dichloromethane:methanol = 10:1, v:v). A red solid was obtained (yield 81.2%). 1 H NMR (600 MHz, DMSO) δ 8.96 (s, 1H), 8.19 (s, 1H), 8.12 (s, 1H), 7.85 (d, J = 8.9Hz, 3H), 7.64 (d, J = 8.7 Hz, 2H), 7.16 (d, J = 23.9 Hz, 6H), 7.00 (s, 4H), 6.76 (s, 1H), 4.08 (s, 4H), 3.57 (s, 8H), 2.68 (s, 4H), 2.46 (s, 8H). 13 C NMR (151 MHz, DMSO) δ 131.66 (s), 130.39 (d, J = 14.9 Hz), 128.89 (s), 128.41(s), 117.04 (s), 116.30 (d, J = 7.9 Hz), 66.65 (s), 66.05 (s), 57.50 (s), 54.09 (s), 40.16 (s). HR-MS: m / z calcd for C 40 H 32 N3O2S2 682.33486; found,682.34055 [M] + .

[0024] A 10 μM solution of the fluorescent probe MoTAQ in dimethyl sulfoxide was prepared and measured using a UV spectrophotometer. A 10 μM solution of the fluorescent probe MoTAQ in toluene was also prepared and measured using a fluorescence-visible spectrophotometer. The results are shown in the attached figure. Figure 2 .like Figure 2 It can be seen that the maximum absorption peak of the fluorescent probe molecule MoTAQ prepared by this invention is around 415 nm, and the maximum emission peak is around 627 nm.

[0025] A 5 mM dimethyl sulfoxide (DMSO) stock solution of the fluorescent probe MoTAQ was prepared by adding a certain amount of 5 mM MoTAQ DMSO stock solution to a 10 μM NaHSO3 aqueous solution to achieve a MoTAQ concentration of 10 μM. After reacting for 10 minutes, the mixed solution was evaporated to dryness, and a certain amount of methanol was added. High-resolution mass spectra were then obtained, and the results are shown in the attached figure. Figure 3 .like Figure 3 It can be seen that the mass-to-charge ratio of the fluorescent probe molecule MoTAQ prepared in this invention is 764.31711 after the reaction, which is the same as the theoretical calculation result, verifying the progress of this reaction.

[0026] 10 μM fluorescent probe MoTAQ was prepared in mixed solutions of dimethyl sulfoxide / toluene at different ratios, and the results were measured using a fluorescence-visible spectrophotometer. The results are shown in the attached figure. Figure 4 .from Figure 4 It can be seen that the fluorescence emission intensity in the MoTAQ mixed solution significantly increases with the increase of the proportion of poor solvent. Meanwhile, the maximum emission peak in the toluene solvent is located at around 625 nm, exhibiting significant infrared fluorescence emission performance.

[0027] The maximum fluorescence intensity ratio (I / I0) of the 10 μM fluorescent probe MoTAQ in mixed solutions of dimethyl sulfoxide / toluene at different ratios is shown in the attached figure. Figure 5 .from Figure 5 It can be seen that the fluorescent probe MoTAQ has obvious AIE performance, that is, the fluorescence intensity gradually increases with the increase of molecular aggregation degree.

[0028] A 5 mM dimethyl sulfoxide stock solution of the fluorescent probe MoTAQ was prepared. NaHSO3 aqueous solutions of different concentrations (0, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 μM) were prepared. The 5 mM MoTAQ stock solution was added to the NaHSO3 aqueous solutions of different concentrations to make the MoTAQ concentration 100 μM. The results were measured using a UV spectrophotometer and are shown in the attached figure. Figure 6 .like Figure 6It can be seen that the absorption peak of the fluorescent probe molecule MoTAQ prepared in this invention at the position of 295 nm continuously increases with the increase of NaHSO3 concentration, while the absorption peak at the position of 395 nm continuously decreases with the increase of NaHSO3 concentration.

[0029] A 5 mM dimethyl sulfoxide stock solution of the fluorescent probe MoTAQ was prepared. NaHSO3 aqueous solutions of different concentrations (0, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 μM) were prepared. The 5 mM MoTAQ stock solution was added to the NaHSO3 aqueous solutions of different concentrations to make the MoTAQ concentration 100 μM. The results were measured using a fluorescence-visible spectrophotometer, and are shown in the attached figure. Figure 7 and Figure 8 .like Figure 7 and Figure 8 It can be seen that the fluorescence emission peak of the fluorescent probe molecule MoTAQ prepared in this invention at 390 nm continuously increases with the increase of NaHSO3 concentration. Figure 7 The fluorescence emission peak at 630 nm gradually weakens with increasing NaHSO3 concentration. Figure 7 It can be seen that the color of the aqueous solution can change from red to blue as the concentration of NaHSO3 changes. Therefore, it is possible to study the color of NaHSO3. - The content of [the substance] was effectively detected.

[0030] A 5 mM dimethyl sulfoxide stock solution of the fluorescent probe MoTAQ was prepared by adding a certain amount of the 5 mM MoTAQ dimethyl sulfoxide stock solution to a 10 μM NaHSO3 aqueous solution to achieve a MoTAQ concentration of 10 μM. Measurements were taken at different time intervals using a fluorescence-visible spectrophotometer, and the results are shown in the attached figure. Figure 9 .pass Figure 9 It can be seen that, as time increases, the emission peak is I. 390nm With the launch peak being I 630nm The ratio continuously increased and stabilized after 4 minutes, indicating that it can effectively treat HSO3. - The detection of its content can be completed in just 4 minutes, which greatly shortens the detection time compared to traditional methods.

[0031] A 5 mM dimethyl sulfoxide stock solution of the fluorescent probe MoTAQ was prepared, and the MoTAQ was diluted with purified water to a final concentration of 10 μM. The probe was then used to detect HSO3 in vitro. - At that time, the fluorescent probe monitors HSO3 by detecting changes in the fluorescence spectrum. -Interfering substances (1 blank; 2 CuBr2; 3 CaCl2; 4 CsCO3; 5 GSH; 6 Cys; 7 H2O2; 8 K2CO3; 9 KI; 10 KSCN; 11 MgCl2; 12 Na2S; 13 Na2SO3; 14 NaAC; 15 NaCl; 16 NaCIO; 17 NaF; 18 NaNO2; 19 NAS2O3; 20 TBHP; 21 ZNSO4; 22 NaHSO3) were added to MoTAQ solution, and the results are shown in the attached figure. Figure 10 .pass Figure 10 It can be seen that in MoTAQ aqueous solutions with different added substances, the emission peak I in NaHSO3+MoTAQ aqueous solution is... 390nm With the launch peak being I 630nm The ratio was as high as 58, far exceeding that of other groups, indicating that MoTAQ can effectively target HSO3. - The detection has specific identification capabilities.

[0032] Confocal fluorescence imaging of 10 μM MoTAQ fluorescent probe co-incubated with a commercial lysosomal probe (Lyso-tracker Green) in HepG2 cells. Figure 11 It can be seen that the Pearson coefficient for co-localization of the fluorescent probe MoTAQ with commercial fluorescent probes is 0.943, indicating that the fluorescent probe MoTAQ has a very high lysosomal localization ability.

[0033] Cellular toxicity assay using the fluorescent probe MoTAQ. Cell viability was determined by the MTT assay (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide). HepG2 cells (human liver cancer cells) were first seeded in 96-well plates, and 100 μL of medium containing 10% fetal bovine serum (FBS) was added. The cells were incubated at 37°C for 24 hours in 5% CO2. The medium was then replaced with 100 μL of medium containing different concentrations of MoTAQ (0, 1, 5, 10, 20, 40, 80 μM) and incubated for another 24 hours. The medium was then removed, and the cells were washed with 100 μL of PBS. 100 μL of medium containing 0.05% MTT was added to each well, and the cells were incubated for another 4 hours. After incubation, the medium was removed, and 150 μL of dimethyl sulfoxide was added to each well. The plates were then shaken slowly for 10 minutes to dissolve the formazan formed in the wells. Finally, the absorbance of each well at 490 nm was measured using a microplate reader and compared with the absorbance of the control group (100% survival rate) to obtain the relative cell viability at different concentrations of the fluorescent probe. Figure 12 It is evident that the fluorescent probe of this invention has good biocompatibility.

[0034] Example 1: Application of the fluorescent probe MoTAQ in food A 5 mM dimethyl sulfoxide stock solution of the fluorescent probe MoTAQ was prepared, and the MoTAQ was diluted with purified water to a final concentration of 10 μM. The probe was then used to detect HSO3 in vitro. - At that time, the fluorescent probe monitors HSO3 by detecting changes in the fluorescence spectrum. - Different concentrations (10, 20, 30, 50, 55, 60) of interfering substances (sugar / salt) were added to MoTAQ solutions, and the results are shown in the attached figure. Figure 10 .pass Figure 10 It can be seen that in MoTAQ aqueous solutions with different added substances, the emission peak I in NaHSO3+MoTAQ aqueous solution is... 390nm With the launch peak being I 630nm The ratio deviates from the theoretical value by no more than 3%, and the accuracy meets the requirements.

[0035] Example 2: Fluorescence Imaging Experiment of Fluorescent Probe MoTAQ in Cells HepG2 cells were incubated in DMEM medium containing 10% fetal bovine serum and cultured in a constant temperature incubator (37℃ and 5% CO2). For the exogenous SO2 derivative detection experiment, the experiment was divided into three groups: Group 1 was incubated with MoTAQ (10 μM) for 0.5 hours; Groups 2 and 3 were pretreated with 50 / 100 μM HSO3⁻, respectively, and then incubated with MoTAQ for 0.5 hours. For the endogenous SO2 detection experiment, the experiment was divided into three groups: Group 1 was incubated with MoTAQ (10 μM) for 0.5 hours; Groups 2 and 3 were pretreated with 50 / 100 μM Cys, respectively, and then incubated with MoTAQ for 0.5 hours. Both experiments were performed using confocal fluorescence microscopy to observe fluorescence imaging in the red / blue channels and to verify the effectiveness of MoTAQ in detecting in vitro / in vivo SO2 derivatives. Figure 12 It is evident that the MoTAQ fluorescent probe of this invention can effectively detect HSO3 within cells. - The content of [something] changed, and the color changed significantly.

[0036] Example 3: Fluorescence Imaging Experiment of Fluorescent Probe MoTAQ in Zebrafish A 10 μM fluorescent probe, MoTAQ, was prepared. Purchased zebrafish embryos were placed in clean culture dishes and cultured in E3 medium at room temperature for 4 days. Before fluorescence imaging, the zebrafish were cultured in E3 medium containing MoTAQ (10 μM) and NaHSO3 aqueous solution (100 μM) for 60 minutes, and then washed three times with E3 medium. Finally, the fluorescence distribution within the zebrafish was observed under a confocal microscope. The excitation wavelengths of the commercial lysosomal probe and the fluorescent probe MoTAQ were 515 nm and 415 nm, respectively, with fluorescence acquisition ranges of 500-600 nm and 600-700 nm, respectively. Figure 14 It is evident that, compared to commercial lysosomal probes, the fluorescent probe MotAQ of this invention can effectively detect HSO3 in zebrafish. - The distribution pattern of this data has more practical application value.

Claims

1. A targeted lysosomal detection method for bisulfite (HSO3) - The aggregation-induced emission (AIE) fluorescent probe is characterized by: The chemical structural formula of the fluorescent probe is: 。 2. The targeted lysosomal detection method for HSO3 according to claim 1 - The AIE fluorescent probe is characterized by: The fluorescent probe targets HSO3 in in vitro cells. - The detection process is as follows: For in vitro cells, the fluorescent probe is dissolved in a solution containing HSO3. - The fluorescent probe was incubated in a PBS buffer solution with a pH of 7.4–7.5 and a temperature of 37°C. The concentration of the fluorescent probe in the PBS buffer solution was 10 μM. HepG2 live cells were incubated in the medium containing the fluorescent probe, and the reaction rate of the fluorescent probe with HSO3 in the cells was measured after 10 minutes. - The color change produced by the reaction is due to HSO3 - Concentration evaluation indicators.

3. The targeted lysosomal detection method for HSO3 according to claim 1 - The method for preparing AIE fluorescent probes is characterized by: 4-bis(4-hydroxyphenyl)aminobenzaldehyde (TTBH), 4-(2-chloroethyl)morpholine, potassium iodide, and cesium carbonate were placed in a reaction flask. Acetonitrile was added to the flask, and the mixture was heated to at least 85°C under nitrogen protection. The reaction solution was cooled to room temperature, and the organic phases were extracted and combined. The reaction solution was then rotary evaporated, and 2-(quinolin-4-yl)acetonitrile and piperidine were added. Ethanol was added to the reaction flask, and the mixture was heated to at least 85°C under nitrogen protection. The reaction solution was cooled to room temperature, and the organic phases were extracted and combined. The mixture was dried and concentrated under reduced pressure to obtain a crude product. The crude product was purified by column chromatography using dichloromethane / methanol as the eluent to obtain the fluorescent probe (Z)-3-[4-(bis{4-[2-morpholinylethoxy]phenyl}amino)phenyl]-2-(4-quinolinyl)acrylonitrile, i.e., the fluorescent probe MoTAQ.

4. A targeted lysosomal detection method for HSO3 according to claim 6 - The method for preparing AIE fluorescent probes is characterized by: The molar ratio of 4-bis(4-hydroxyphenyl)aminobenzaldehyde (TTBH) to 4-(2-chloroethyl)morpholine is 1:1 to 1:

10.

5. A targeted lysosomal detection method for HSO3 according to claim 6 - The method for preparing AIE fluorescent probes is characterized by: The molar ratio of 4-bis(4-hydroxyphenyl)aminobenzaldehyde (TTBH) to 2-(quinolin-4-yl)acetonitrile is 1:0.5 to 1:

10.

6. The method for preparing the lysosomal targeted aggregation-induced luminescence ionic probe according to claim 6, characterized in that: The reaction time is at least 1 hour.

7. The method for preparing the lysosomal targeted aggregation-induced luminescence ionic probe according to claim 6, characterized in that: The dichloromethane / methanol used as an eluent has a feed ratio of 1:1 to 100:1.

Images