A cysteine / homocysteine responsive aie fluorescent probe and a preparation method and application thereof

Abstract

The application discloses a cysteine / homocysteine response AIE fluorescent probe, the fluorescent probe has good AIE effect, after reacting with cysteine (Cys) / homocysteine (Hcy), the fluorescence signal is significantly enhanced, after reacting with glutathione (GSH), the fluorescence signal has no obvious change. The probe can realize selective detection of cysteine Cys / Hcy in the presence of GSH. The probe has very rapid response, high sensitivity and low detection limit, and can realize the purpose of rapid detection.

Description

Technical Field

[0001] This invention belongs to the field of fluorescent probes, specifically relating to a cysteine / homocysteine-responsive AIE fluorescent probe, its preparation method, and its application. Background Technology

[0002] Cysteine ​​(Cys), homocysteine ​​(Hcy), and glutathione (GSH), these small molecules containing thiol functional groups, are important intracellular thiols in organisms. Widely distributed within cells, they are crucial for maintaining redox homeostasis and protein conformation in biological systems. Cys, as a precursor in the synthesis of proteins, acetyl-CoA, glycine, taurine, and inorganic sulfur, is an essential amino acid. Homocysteine ​​is an important intermediate in the metabolism of methionine and homocysteine ​​and is a contributing factor to diseases such as arteriosclerosis and cerebral thrombosis. The amount of GSH in the human body far exceeds that of Cys / Hcy, but due to their similarity in structure and reactivity, it is often difficult to distinguish between Cys, Hcy, and GSH. Therefore, selectively identifying Cys, Hcy, and GSH is a key focus for researchers.

[0003] Traditional bio-thiol fluorescent probes may aggregate in aqueous solutions, leading to fluorescence quenching, a phenomenon known as aggregation-induced quenching (ACQ). This significantly limits the application of fluorescent probes in aqueous solutions. Since Tang's research group discovered a fluorescent material in 2001 that exhibits weak or no emission in solution but is induced to produce strong emission upon aggregation, the concept of aggregation-induced emission (AIE) has come into focus and has flourished in recent years.

[0004] Currently, fluorescent probes based on AIE properties have advantages such as low signal-to-noise ratio, high brightness, and good optical stability, and have been widely studied. Compared with the traditional ACQ fluorescent probes whose applications are limited by the water content in the body, the development of fluorescent probes with AIE properties for the detection of biothiols has broad prospects. Summary of the Invention

[0005] Purpose of the invention: The present invention aims to design and prepare an AIE fluorescent probe with good AIE properties, exhibiting good selectivity for biothiols, short response time, high detection sensitivity, and cysteine / homocysteine ​​response.

[0006] The present invention also provides a method for preparing and applying a cysteine / homocysteine-responsive AIE fluorescent probe.

[0007] Technical solution: To achieve the above objectives, this invention provides a cysteine / homocysteine-responsive AIE fluorescent probe, denoted as TBP-NBD, whose structural formula is shown in Formula I below:

[0008]

[0009] This invention also provides a method for preparing the cysteine / homocysteine-responsive AIE fluorescent probe, the synthetic route of which is as follows:

[0010]

[0011] Specifically, the following steps are included:

[0012] Step 1: 4,7-Dibromobenzothiadiazole and triphenylamine 4-boronic acid were dissolved in an organic solvent and reacted under palladium catalysis and refluxed. After the reaction was completed, the mixture was filtered. The solid obtained after filtration was purified by column chromatography to prepare compound 1.

[0013] Step 2: Compound 1 is dissolved in an organic solvent and reacted with 4-pyridineboronic acid under palladium catalysis and heated. After the reaction is completed, the mixture is filtered and the resulting solid is purified by column chromatography to obtain compound TBP.

[0014] Step 3: Dissolve p-hydroxybenzyl alcohol and NBD-Cl in an organic solvent and reflux at 50-80℃. After the reaction is completed, concentrate under reduced pressure and purify the crude product by column chromatography to obtain compound 2.

[0015] Step 4: Dissolve compound 2 in an organic solvent, add PBr3 for bromination, quench the reaction after the reaction is complete, extract with organic solvent, remove the solvent under reduced pressure to obtain crude product, and purify to obtain compound 3.

[0016] Step 5: Dissolve compound 3 and TBP in an organic solvent and reflux at 80-90°C to obtain an AIE fluorescent probe that responds to cystine / homocysteine.

[0017] Beneficial effects

[0018] Compared with the prior art, the present invention has the following advantages:

[0019] (1) The fluorescent probe has a low detection limit for cysteine / homocysteine. After responding to a low concentration of cysteine / homocysteine, it releases the fluorophore TBP, and the fluorescence intensity is enhanced by 50-100 times, which is obvious.

[0020] (2) It responds quickly, and the fluorescence signal can be turned on after 5 minutes of incubation, enabling rapid detection of cysteine / homocysteine.

[0021] (3) The fluorescent probe itself does not exhibit fluorescence. After reacting with glutathione, the fluorescence signal is negligible. However, in the presence of glutathione, the fluorescence intensity is significantly enhanced after reacting with cysteine / homocysteine, thereby achieving selective detection of biothiols. Therefore, the fluorescent probe in this invention effectively detects cysteine / homocysteine ​​in biothiols. Attached Figure Description

[0022] Figure 1 This is a high-resolution mass spectrum of the fluorescent probe TBP-NBD prepared in one embodiment of the present invention;

[0023] Figure 2 This is a high-resolution mass spectrum of the fluorescent probe TBP-NBD prepared in one embodiment of the present invention;

[0024] Figure 3 This is a high-resolution mass spectrum of the fluorescent probe TBP-NBD prepared in one embodiment of the present invention;

[0025] Figure 4 (A) shows the fluorescence emission spectrum of the fluorescent probe TBP-NBD of the present invention after incubation with cysteine, homocysteine ​​and glutathione in a DMSO-PBS mixed solution, respectively; (B) shows the fluorescence emission spectrum of the fluorescent probe TBP-NBD after co-incubation with cysteine ​​and glutathione in a DMSO-PBS mixed solution.

[0026] Figure 5 This is an imaging image of the fluorescent probe TBP-NBD in HeLa cells according to one embodiment of the present invention. Detailed Implementation

[0027] The preferred embodiments of the present invention will now be described in detail with reference to specific examples. It should be understood that the following examples are given for illustrative purposes only and are not intended to limit the scope of the invention. Those skilled in the art can make various modifications and substitutions to the present invention without departing from its spirit and essence.

[0028] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0029] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0030] One embodiment of this application provides a cysteine / homocysteine-responsive AIE fluorescent probe, the fluorescent probe having the following structural formula:

[0031]

[0032] This application provides a method for preparing the cysteine / homocysteine-responsive AIE fluorescent probe, characterized in that...

[0033] The synthesis route is as follows:

[0034]

[0035] Specifically, the following steps are included:

[0036] Step 1: 4,7-Dibromobenzothiadiazole and triphenylamine 4-boronic acid were dissolved in an organic solvent and reacted under palladium catalysis and refluxed. After the reaction was completed, the mixture was filtered. The solid obtained after filtration was purified by column chromatography to prepare compound 1.

[0037] Step 2: Compound 1 is dissolved in an organic solvent and reacted with 4-pyridineboronic acid under palladium catalysis and heated. After the reaction is completed, the mixture is filtered and the resulting solid is purified by column chromatography to obtain compound TBP.

[0038] Step 3: Dissolve p-hydroxybenzyl alcohol and NBD-Cl in an organic solvent and reflux at 50-80℃. After the reaction is completed, concentrate under reduced pressure and purify the crude product by column chromatography to obtain compound 2.

[0039] Step 4: Dissolve compound 2 in an organic solvent, add PBr3 for bromination, quench the reaction after the reaction is complete, extract with organic solvent, remove the solvent under reduced pressure to obtain crude product, and purify to obtain compound 3.

[0040] Step 5: Dissolve compound 3 and TBP in an organic solvent and reflux at 80-90°C to obtain an AIE fluorescent probe that responds to cystine / homocysteine.

[0041] In one embodiment, the molar ratio of 4,7-dibromobenzothiadiazole to triphenylamine 4-boronic acid in step 1 is 1:1, the organic solvent is anhydrous acetonitrile / tetrahydrofuran, and the heating and reflux reaction is carried out under the reaction conditions of 80-90°C and N2 protection for 8-10 hours.

[0042] In one embodiment, the molar ratio of compound 1 to 4-pyridineboronic acid in step 2 is 1:2, the organic solvent is anhydrous acetonitrile / tetrahydrofuran, and the heating reaction in step 2 is carried out at 80-90°C under a N2 atmosphere for 8-10 hours.

[0043] In one embodiment, in step 3, p-hydroxybenzyl alcohol and 4-chloro-7-nitro-2,1,3-benzoxoxadiazole (NBD-Cl) are dissolved in anhydrous dichloromethane at a molar ratio of 1:1 and reacted under stirring and reflux at 50-80°C under N2 protection for 6-12 hours.

[0044] In one embodiment, in step 4, compound 2 is dissolved in anhydrous acetonitrile and PBr3 / CBr4 (1.0 eq) is slowly added dropwise at 0°C. The reaction is carried out overnight. After the reaction is completed, the solvent is removed by vacuum distillation to obtain a crude product. The crude product is purified by column chromatography to obtain compound 3.

[0045] In one embodiment, in step 5, compound 2 and compound TBP are dissolved in acetonitrile / toluene at a molar ratio of 1:1, and refluxed at 80-110°C for 8-12 hours under N2 protection. After the reaction is complete, the solvent is removed by vacuum distillation, and the crude product is purified by column chromatography to obtain the fluorescent probe TBP-NBD.

[0046] One embodiment of this application provides the application of the cysteine / homocysteine-responsive AIE fluorescent probe in the responsive detection of cysteine / homocysteine.

[0047] In one embodiment, cysteine, homocysteine, and glutathione solutions were added to the reaction system of an AIE fluorescent probe containing cysteine / homocysteine ​​responses, respectively. The reaction solutions were mixed evenly and incubated at 30-40°C. The ultraviolet absorption spectrum and fluorescence emission spectrum were measured, and the cysteine / homocysteine ​​was qualitatively and / or quantitatively analyzed based on the spectral results.

[0048] In one embodiment, HeLa cells were cultured in a laser confocal dish, and a cysteine / homocysteine-responsive AIE fluorescent probe was added to the culture dish for co-incubation. The cells were then imaged using a laser confocal microscope, and the cysteine / homocysteine ​​ratio was qualitatively and / or quantitatively analyzed based on the imaging results.

[0049] Example 1

[0050] The specific synthesis process of the fluorescent probe of this invention is as follows:

[0051] (1) Synthesis of Compound 1: A mixture of 4,7-dibromo-2,1,3-benzothiadiazole (10 mmol), 4-(diphenylamino)phenylboronic acid (10 mmol), and Pd(PPh3)4 (0.5 mmol) was heated in acetonitrile at 80 °C under N2 protection for 10 h. After the reaction was completed, the mixture was filtered, purified by column chromatography (methanol:dichloromethane = 1:10), and dried under vacuum to give an orange-red solid, which was Compound 1. Yield: 87%

[0052] (2) Synthesis of the fluorophore TBP: A mixture of compound 1 (2.18 mmol), 4-pyridineboronic acid (4.36 mmol), and Pd(PPh3)4 (0.065 mmol) was refluxed overnight in anhydrous acetonitrile at 80 °C under a N2 atmosphere. After the reaction was complete, the mixture was filtered and purified by column chromatography (ethyl acetate: petroleum ether = 3:10). The purified solid, TBP, was obtained by vacuum drying. Yield: 80%

[0053] 1H NMR (600MHz, CDCl3) δ7.21–7.12(m,2H),7.00–6.85(m,2H),6.51–6.37(m,1H) ,6.18(ddd,J=22.2,17.7,11.4Hz,1H),5.94–5.81(m,1H),4.47–4.32(m,2H).

[0054] (3) Synthesis of Compound 2: p-Hydroxybenzyl alcohol (1.0 mmol) and 4-chloro-7-nitro-2,1,3-benzoxoxadiazole (NBD-Cl) (1.0 mmol) were dissolved in anhydrous dichloromethane (30 ml). The mixture was stirred and refluxed at 50 °C under N2 protection for 12 h. The mixture was concentrated under reduced pressure, and the crude product was purified by column chromatography (ethyl acetate: petroleum ether = 1:2). The product was then dried under vacuum to obtain a yellow solid, which was Compound 2. Yield: 90%

[0055] (4) Synthesis of Compound 3: Compound 2 (1.0 mmol) was dissolved in anhydrous acetonitrile (30 ml), and PBr3 (1.0 mmol) was slowly added dropwise at 0 °C. The reaction was carried out overnight at room temperature in the dark. After the reaction was completed, the reaction was quenched, extracted three times, dried, filtered, and the filtrate was distilled under reduced pressure to obtain the crude product. The crude product was purified by column chromatography (ethyl acetate: petroleum ether = 3:100), and dried under vacuum to obtain a yellow solid, which is compound 3. Yield: 52%

[0056] (5) Synthesis of probe TBP-NBD: Compound 2 (1.0 mmol) and the fluorophore TBP (1.0 mmol) were dissolved in acetonitrile (30 ml) and reacted at 80 °C for 12 h under N2 protection. The mixture was concentrated under reduced pressure, and the crude product was purified by column chromatography (dichloromethane:methanol = 50:1). The product was then dried under vacuum to obtain a dark blue solid, which was probe TBP-NBD. Yield: 65%.

[0057] The prepared fluorescent probe TBP-NBD was detected by high-resolution mass spectrometry. The detection results are shown in the spectrum below. Figure 1 This indicates that the target probe was successfully prepared.

[0058] Example 2

[0059] (1) Synthesis of Compound 1: A mixture of 4,7-dibromo-2,1,3-benzothiadiazole (10 mmol), 4-(diphenylamino)phenylboronic acid (9 mmol), and Pd(PPh3)4 (1.0 mmol) was heated in acetonitrile at 90 °C under N2 protection for 8 h. After the reaction was completed, the mixture was filtered and purified by column chromatography (methanol:dichloromethane = 1:10) to give an orange-red solid, namely Compound 1. Yield: 85%

[0060] (2) Synthesis of the fluorophore TBP: A mixture of compound 1 (2.18 mmol), 4-pyridineboronic acid (4.36 mmol), and Pd(PPh3)4 (0.01 mmol) was refluxed in anhydrous acetonitrile at 90 °C under a N2 atmosphere for 8 h. After the reaction was completed, the mixture was filtered and purified by column chromatography (ethyl acetate: petroleum ether = 3:10). The purified solid, TBP, was obtained by vacuum drying. Yield: 78%

[0061] (3) Synthesis of Compound 2: p-Hydroxybenzyl alcohol (1.0 mmol) and 4-chloro-7-nitro-2,1,3-benzoxoxadiazole (NBD-Cl) (1.0 mmol) were dissolved in anhydrous dichloromethane (30 ml). The mixture was stirred and refluxed at 80 °C under N2 protection for 6 h. The mixture was concentrated under reduced pressure, and the crude product was purified by column chromatography (ethyl acetate: petroleum ether = 1:2). The crude product was dried under vacuum to obtain a yellow solid, which was Compound 2. Yield: 75%

[0062] (4) Synthesis of Compound 3: Compound 2 (1.0 mmol) was dissolved in anhydrous acetonitrile (30 ml), and PBr3 (2.0 mmol) was slowly added dropwise at 0 °C. The reaction was carried out overnight at 0 °C in the dark. After the reaction was completed, the reaction solution was directly extracted, dried, filtered, and the filtrate was distilled under reduced pressure to obtain the crude product. The crude product was purified by column chromatography (ethyl acetate: petroleum ether = 3:100), and dried under vacuum to obtain a yellow solid, which is compound 3. Yield: 45%

[0063] (5) Synthesis of probe TBP-NBD: Compound 2 (1.0 mmol) and the fluorophore TBP (1.0 mmol) were dissolved in acetonitrile (30 ml) and reacted at 90 °C for 8 h under N2 protection. The mixture was concentrated under reduced pressure, and the crude product was purified by column chromatography (dichloromethane:methanol = 50:1). The product was then dried under vacuum to obtain a dark blue solid, which was probe TBP-NBD. Yield: 60%

[0064] The prepared fluorescent probe TBP-NBD was detected by high-resolution mass spectrometry. The detection results are shown in the spectrum below. Figure 2 This indicates that the target probe was successfully prepared.

[0065] Example 3

[0066] (1) Synthesis of Compound 1: A mixture of 4,7-dibromo-2,1,3-benzothiadiazole (10 mmol), 4-(diphenylamino)phenylboronic acid (9 mmol), and Pd(PPh3)4 (0.5 mmol) was heated in tetrahydrofuran at 80 °C under N2 protection for 10 h. After the reaction was complete, the mixture was washed with ethanol, filtered, and directly used in the next step. Yield: 82%

[0067] (2) Synthesis of the fluorophore TBP: A mixture of compound 1 (2.18 mmol), 4-pyridineboronic acid (4.36 mmol), and Pd(PPh3)4 (0.005 mmol) was refluxed in tetrahydrofuran at 90 °C under a N2 atmosphere for 8 h. After the reaction was completed, the mixture was filtered, purified by column chromatography (dichloromethane:methanol = 100:1), and dried under vacuum to obtain a dark red solid, namely TBP. Yield: 70%

[0068] (3) Synthesis of Compound 2: p-Hydroxybenzyl alcohol (1.0 mmol) and 4-chloro-7-nitro-2,1,3-benzoxoxadiazole (NBD-Cl) (2.0 mmol) were dissolved in anhydrous dichloromethane (50 ml). The mixture was stirred and refluxed at 80 °C under N2 protection for 6 h. The mixture was concentrated under reduced pressure, and the crude product was purified by column chromatography (ethyl acetate: petroleum ether = 1:2). The product was then dried under vacuum to obtain a yellow solid, which was Compound 2. Yield: 78%

[0069] (4) Synthesis of Compound 3: Compound 2 (1.0 mmol) was dissolved in anhydrous dichloromethane (30 ml), and CBr4 (1.0 mmol) was slowly added dropwise at 0 °C. The reaction was carried out overnight at room temperature in the dark. After the reaction was completed, the reaction was quenched, extracted three times, dried, filtered, and the filtrate was distilled under reduced pressure to obtain the crude product, which was directly used in the next step. Yield: 50%

[0070] (5) Synthesis of probe TBP-NBD: Compound 2 (1.0 mmol) and the fluorophore TBP (1.0 mmol) were dissolved in toluene (30 mL) and reacted at 110 °C for 8 h under N2 protection. The mixture was concentrated under reduced pressure, and the crude product was purified by column chromatography (dichloromethane:methanol = 100:1). The product was then dried under vacuum to obtain a dark blue solid, which was probe TBP-NBD. Yield: 60%

[0071] The prepared fluorescent probe TBP-NBD was detected by high-resolution mass spectrometry. The detection results are shown in the spectrum below. Figure 3 This indicates that the target probe was successfully prepared.

[0072] Fluorescent probe detection of Cys / Hcy / GSH

[0073] The method for detecting Cys / Hcy / GSH using the fluorescent probe prepared by the method in Specific Example 1 includes the following steps:

[0074] (1) Instrument parameter settings: Set the excitation wavelength of the F-4600 fluorescence spectrophotometer to 420nm, and the emission wavelength to...

[0075] The wavelength range is 500–700 nm, the photomultiplication voltage is 600 V, the slit width is 5 nm, and a four-sided transparent quartz cuvette is used.

[0076] (2) Preparation of probe TBP-NBD stock solution: Weigh 7.26 mg TBP-NBD and dissolve it in 10 ml DMSO to obtain a concentration of 10 mM;

[0077] (3) Preparation of cysteine ​​(Cys) / homocysteine ​​(Hcy) / glutathione (GSH) stock solution: Weigh 1.21mg, 1.35mg, and 3.07mg and dissolve them in 10ml of DMSO at a concentration of 10mM.

[0078] (4) Preparation of reaction solutions: Take the Cys, Hcy, and GSH stock solutions, and add PBS buffer solution and probe stock solution respectively to form a 10 μM reaction solution. Place the centrifuge tubes in a constant temperature water bath and incubate at 37 °C for 0.5 h. Measure the fluorescence emission spectra of the Cys, Hcy, and GSH reaction solutions respectively.

[0079] (5) Results are as follows Figure 4 As shown in (A), the fluorescence signal of the fluorescent probe TBP-NBD was significantly enhanced after incubation with Cys / Hcy, while the fluorescence signal was negligible after incubation with GSH.

[0080] Detection of Cys / Hcy with fluorescent probes under GSH interference

[0081] The method for detecting Cys / Hcy using the fluorescent probe prepared by the method in Specific Example 1 under GSH interference includes the following steps:

[0082] (1) Preparation of reaction solution: Take the Hcy and GSH stock solutions, add PBS buffer solution and incubate with the probe at 37℃ for 0.5h. Measure the fluorescence emission spectrum of the reaction solution.

[0083] (2) Figure 4 As shown in Figure (B), the fluorescence signal of the fluorescent probe TBP-NBD was significantly enhanced after co-incubation with Hcy and GSH. This indicates that the fluorescent probe prepared in this invention can achieve selective detection of Cys / Hcy under GSH interference and has a fast response speed.

[0084] Detection of Cys / Hcy / GSH with fluorescent probes in live cells

[0085] The method for detecting Cys / Hcy / GSH in live cells using the fluorescent probe prepared by the method in Specific Example 1 includes the following steps:

[0086] (1) Cell Culture: HeLa cells were placed in culture flasks and cultured in an incubator (37℃, 5% CO2). After 24 hours, the cells were removed and 2 mL of trypsin digestion solution was added to the culture flasks to digest the cells. After digestion, 2 mL of culture medium (MEM medium containing 10% FBS) was added to stop the digestion. The cell suspension was then centrifuged, the supernatant was discarded, and 1 mL of culture medium was added. The cells were resuspended by pipetting. The cell suspension was transferred to a laser confocal dish and incubated overnight in a 5% CO2, 37℃ incubator. The culture medium was discarded, and the cells were washed with PBS buffer and then 2 mL of culture medium was added.

[0087] (2) Detection of endogenous and exogenous Cys: The probe TBP-NBD (20 μM) prepared in Example 1 was used for confocal fluorescence imaging of HeLa live cells. The cells were divided into three groups. The first group was incubated with 40 μL of the TBP-NBD stock solution prepared in Example 2 for 2 h. The second group was first treated with Cys (1 mM / L) for 2 h, then 40 μL of the TBP-NBD stock solution prepared in Example 2 was added, and the cells were incubated for another 0.5 h. The third group was first treated with NEM for 0.5 h, then 40 μL of the TBP-NBD stock solution prepared in Example 2 was added to the cells and the cells were incubated for another 2 h. After washing the treated cells three times with PBS buffer, the cells were fixed with paraformaldehyde, washed three times with PBS buffer, stained with DAPI, and then washed with PBS buffer for confocal imaging.

[0088] (3) Figure 5 As shown, the first group exhibits weak green fluorescence, indicating that TBP-NBD can detect endogenous biothiols in cells. The second group exhibits strong green fluorescence, indicating that TBP-NBD can detect exogenous Cys, and the concentration of exogenous Cys is significantly higher than that of endogenous Cys. The third group did not show obvious fluorescence, indicating that NEM inhibits the expression of biothiols in cells, further confirming that TBP-NBD reacts with Cys / Hcy and can detect endogenous Cys / Hcy. Therefore, TBP-NBD can detect both endogenous and exogenous Cys / Hcy in living cells.

[0089] The results fully demonstrate that the fluorescent probe in this invention can effectively detect cysteine / homocysteine ​​in biothiols.

[0090] The above are merely preferred embodiments of the present invention. It should be noted that, for those skilled in the art, numerous improvements and modifications can be made without departing from the principles of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A cysteine / homocysteine-responsive AIE fluorescent probe, characterized in that, The fluorescent probe has the following structural formula: 。 2. A method for preparing the cysteine / homocysteine-responsive AIE fluorescent probe according to claim 1, characterized in that, The synthesis route is as follows: Specifically, the following steps are included: Step 1: 4,7-Dibromobenzothiadiazole and triphenylamine 4-boronic acid were dissolved in an organic solvent and reacted under palladium catalysis and refluxed. After the reaction was completed, the mixture was filtered. The solid obtained after filtration was purified by column chromatography to prepare compound 1. Step 2: Compound 1 is dissolved in an organic solvent and reacted with 4-pyridineboronic acid under palladium catalysis and heated. After the reaction is completed, the mixture is filtered and the resulting solid is purified by column chromatography to obtain compound TBP. Step 3: Dissolve p-hydroxybenzyl alcohol and NBD-Cl in an organic solvent and reflux at 50-80℃. After the reaction is completed, concentrate under reduced pressure and purify the crude product by column chromatography to obtain compound 2. Step 4: Compound 2 is dissolved in an organic solvent, PBr3 is added for bromination, the reaction is quenched after completion, extracted with an organic solvent, and the solvent is removed by vacuum distillation to obtain the crude product, which is then purified to obtain compound 3; Step 5: Dissolve compound 3 and TBP in an organic solvent and reflux at 80-90°C to obtain an AIE fluorescent probe that responds to cystine / homocysteine.

3. The method for preparing a cysteine / homocysteine-responsive AIE fluorescent probe according to claim 2, characterized in that, In step 1, the molar ratio of 4,7-dibromobenzothiadiazole to triphenylamine 4-boronic acid is 1:1, the organic solvent is anhydrous acetonitrile / tetrahydrofuran, and the heating and reflux reaction is carried out at 80-90℃ under N2 protection for 8-10 hours.

4. The method for preparing a cysteine / homocysteine-responsive AIE fluorescent probe according to claim 2, characterized in that... In step 2, the molar ratio of compound 1 to 4-pyridineboronic acid is 1:2, the organic solvent is anhydrous acetonitrile / tetrahydrofuran, and the heating reaction in step 2 is carried out at 80-90°C under a N2 atmosphere for 8-10 h.

5. The method for preparing a cysteine / homocysteine-responsive AIE fluorescent probe according to claim 2, characterized in that, In step 3, p-hydroxybenzyl alcohol and 4-chloro-7-nitro-2,1,3-benzoxoxadiazole (NBD-Cl) are dissolved in anhydrous dichloromethane at a molar ratio of 1:1 and reacted under stirring and reflux at 50-80°C under N2 protection for 6-12 h.

6. The method for preparing a cysteine / homocysteine-responsive AIE fluorescent probe according to claim 2, characterized in that, In step 4, compound 2 is dissolved in anhydrous acetonitrile and PBr3 / CBr is slowly added dropwise at 0°C. 4， PBr3 and CBr4 were reacted in a 1:1 equivalence ratio overnight. After the reaction was completed, the solvent was removed by vacuum distillation to obtain the crude product, which was then purified by column chromatography to obtain compound 3.

7. The method for preparing a cysteine / homocysteine-responsive AIE fluorescent probe according to claim 2, characterized in that, In step 5, compound 3 and compound TBP were dissolved in acetonitrile / toluene at a molar ratio of 1:1 and refluxed at 80-110℃ for 8-12 h under N2 protection. After the reaction was completed, the solvent was removed by vacuum distillation, and the crude product was purified by column chromatography to obtain the fluorescent probe TBP-NBD.

8. An application of the cysteine / homocysteine-responsive AIE fluorescent probe of claim 1 in the responsive detection of cysteine / homocysteine, wherein the application is not for the purpose of disease diagnosis and treatment.

9. The application of the cysteine / homocysteine-responsive AIE fluorescent probe according to claim 8 in the detection of cysteine / homocysteine, characterized in that, Add cysteine, homocysteine, and glutathione solutions to the reaction system of the AIE fluorescent probe containing cysteine / homocysteine ​​response, respectively. Mix the reaction solutions thoroughly and incubate at 30-40℃. Measure the ultraviolet absorption spectrum and fluorescence emission spectrum. Perform qualitative and / or quantitative analysis of cysteine / homocysteine ​​based on the spectral results.

10. The application of the cysteine / homocysteine-responsive AIE fluorescent probe according to claim 8 in the detection of cysteine / homocysteine, characterized in that, HeLa cells were cultured in laser confocal dishes, and AIE fluorescent probes responsive to cysteine / homocysteine ​​were added to the culture dishes for co-incubation. The cells were then imaged using a laser confocal microscope, and the cysteine / homocysteine ​​ratio was qualitatively and / or quantitatively analyzed based on the imaging results.

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