Cysteine / homocysteine responsive AIE fluorescent probe as well as preparation method and application thereof

Abstract

The invention discloses a cysteine / homocysteine responsive AIE fluorescent probe, the fluorescent probe has a good AIE effect, and after the fluorescent probe reacts with cysteine (Cys) / homocysteine (Hcy), a fluorescence signal is significantly enhanced; after the fluorescent probe reacts with glutathione (GSH), a fluorescence signal does not change obviously. The probe can realize selective detection of cysteine Cys / Hcy in the presence of GSH (glutathione). The probe is very rapid in response, has relatively high sensitivity and relatively low detection limit, and can achieve the purpose of rapid detection.

Description

Technical Field

[0001] The invention belongs to the field of fluorescent probes, and in particular relates to a cysteine / homocysteine-responsive AIE fluorescent probe and a preparation method and application thereof. Background Art

[0002] Small molecules containing sulfhydryl functional groups, such as cysteine ​​(Cys), homocysteine ​​(Hcy), and glutathione (GSH), are important intracellular thiols in organisms. They are widely distributed in cells and are crucial for maintaining the redox homeostasis of biological systems and the biological conformation of proteins. Cys is an essential amino acid for the human body as a synthetic precursor for proteins, acetyl coenzymes, glycine, taurine, and inorganic sulfur. Homocysteine ​​is an important intermediate in the metabolism of methionine and cysteine ​​and is a trigger for diseases such as arteriosclerosis and cerebral thrombosis. The content of GSH in the human body far exceeds Cys / Hcy, but because they are very similar in structure and reactivity, it is often difficult to distinguish between Cys, Hcy, and GSH. Therefore, selective identification of Cys, Hcy, and GSH is a key focus of scientific researchers.

[0003] Traditional biothiol fluorescent probes may aggregate in aqueous solution, causing the fluorescence signal to extinguish, resulting in aggregation-induced quenching (ACQ), which greatly limits the application of fluorescent probes in aqueous solution. Since 2001, when Tang's group discovered a fluorescent material that exhibited weak or even no emission in solution, but was induced to produce strong emission when aggregated, the concept of aggregation-induced emission (AIE) has entered people's field of vision and has flourished in recent years.

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

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

[0006] The invention also provides a preparation method and application of a cysteine / homocysteine-responsive AIE fluorescent probe.

[0007] Technical solution: In order to achieve the above-mentioned object, the present invention provides a cysteine / homocysteine-responsive AIE fluorescent probe, denoted as TBP-NBD, and its structural formula is shown in the following formula I:

[0008]

[0009] The present invention also provides a method for preparing the cysteine / homocysteine-responsive AIE fluorescent probe, and the synthesis route is as follows:

[0010]

[0011] The specific steps include:

[0012] Step 1, dissolving 4,7-dibromobenzothiadiazole and 4-boric acid triphenylamine in an organic solvent for reaction, heating under reflux for reaction under palladium catalysis, filtering after the reaction, and purifying the solid obtained after filtering by column chromatography to prepare compound 1;

[0013] Step 2, dissolving compound 1 and 4-pyridine boronic acid in an organic solvent for reaction, heating the reaction under palladium catalysis, filtering after the reaction, and purifying the obtained solid by column chromatography to obtain compound TBP;

[0014] Step 3, dissolving p-hydroxybenzyl alcohol and NBD-Cl in an organic solvent, and reacting under reflux at 50-80° C., and concentrating under reduced pressure after the reaction is completed. The crude product is purified by column chromatography to obtain compound 2;

[0015] Step 4, dissolving compound 2 in an organic solvent, adding PBr3 for bromination, quenching the reaction after the reaction is completed, extracting with an organic solvent, and distilling off the solvent under reduced pressure to obtain a crude product, which is purified to obtain compound 3;

[0016] Step 5, compound 3 and TBP are dissolved in an organic solvent, and refluxed at 80-90° C. to obtain an AIE fluorescent probe responsive 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 low concentrations of cysteine / homocysteine, the fluorescent group TBP is released, and the fluorescence intensity is enhanced by 50-100 times, which is a significant phenomenon.

[0020] (2) Rapid response: the fluorescence signal can be turned on after incubation for 5 min, thus realizing rapid detection of cysteine / homocysteine.

[0021] (3) The fluorescent probe itself does not have fluorescence. After reacting with glutathione, the fluorescence signal is negligible. In the presence of glutathione, after reacting with cysteine / homocysteine, the fluorescence intensity is significantly enhanced, thereby achieving selective detection of biothiols. Therefore, the fluorescent probe in the present invention effectively detects cysteine / homocysteine ​​in biothiols. BRIEF DESCRIPTION OF THE DRAWINGS

[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) is 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) is the fluorescence emission spectrum of the fluorescent probe TBP-NBD after incubation with cysteine ​​and glutathione in a DMSO-PBS mixed solution;

[0026] Figure 5 This is an imaging diagram of the fluorescent probe TBP-NBD in Hela cells in one embodiment of the present invention. DETAILED DESCRIPTION

[0027] The preferred embodiments of the present invention will be described in detail below in conjunction with examples. It should be understood that the following examples are provided only for the purpose of illustration and are not intended to limit the scope of the present invention. Those skilled in the art may make various modifications and substitutions to the present invention without departing from the purpose and spirit of the present invention.

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

[0029] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0030] One embodiment of the present application provides an AIE fluorescent probe responsive to cysteine / homocysteine, and the structural formula of the fluorescent probe is as follows:

[0031]

[0032] One embodiment of the present application provides a method for preparing the cysteine / homocysteine ​​responsive AIE fluorescent probe, characterized in that:

[0033] The synthetic route is as follows:

[0034]

[0035] The specific steps include:

[0036] Step 1, dissolving 4,7-dibromobenzothiadiazole and 4-boric acid triphenylamine in an organic solvent for reaction, heating under reflux for reaction under palladium catalysis, filtering after the reaction, and purifying the solid obtained after filtering by column chromatography to prepare compound 1;

[0037] Step 2, dissolving compound 1 and 4-pyridine boronic acid in an organic solvent for reaction, heating the reaction under palladium catalysis, filtering after the reaction, and purifying the obtained solid by column chromatography to obtain compound TBP;

[0038] Step 3, dissolving p-hydroxybenzyl alcohol and NBD-Cl in an organic solvent, and reacting under reflux at 50-80° C., and concentrating under reduced pressure after the reaction is completed. The crude product is purified by column chromatography to obtain compound 2;

[0039] Step 4, dissolving compound 2 in an organic solvent, adding PBr3 for bromination, quenching the reaction after the reaction is completed, extracting with an organic solvent, and distilling off the solvent under reduced pressure to obtain a crude product, which is purified to obtain compound 3;

[0040] Step 5, compound 3 and TBP are dissolved in an organic solvent, and refluxed at 80-90° C. to obtain an AIE fluorescent probe responsive to cystine / homocysteine.

[0041] In one embodiment, the molar ratio of 4,7-dibromobenzothiadiazole to 4-boric acid triphenylamine in step 1 is 1:1, the organic solvent is anhydrous acetonitrile / tetrahydrofuran, and the heating reflux reaction is carried out at 80-90° C. under 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 performed at 80-90° C. in a N 2 atmosphere for 8-10 h.

[0043] In one embodiment, in step 3, p-hydroxybenzyl alcohol and 4-chloro-7-nitro-2,1,3-benzoxadiazole (NBD-Cl) are dissolved in anhydrous dichloromethane at a molar ratio of 1:1, and stirred under 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 allowed to proceed overnight. After the reaction is completed, the solvent is evaporated under reduced pressure 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, the solvent is evaporated under reduced pressure, and the crude product is purified by column chromatography to obtain the fluorescent probe TBP-NBD.

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

[0047] In one embodiment, cysteine, homocysteine ​​and glutathione solutions are respectively added to the reaction system of the AIE fluorescent probe responsive to cysteine / homocysteine, the reaction solutions are mixed evenly, incubated at 30-40°C, and the ultraviolet absorption spectrum and fluorescence emission spectrum are measured, and the cysteine / homocysteine ​​is qualitatively and / or quantitatively analyzed according to the spectral results.

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

[0049] Example 1

[0050] The specific synthesis process of the fluorescent probe of the present 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, the mixture was filtered and purified by column chromatography (methanol: dichloromethane = 1:10), and dried in vacuo to obtain an orange-red solid, namely Compound 1. Yield: 87%

[0052] (2) Synthesis of fluorophore TBP: A mixture of compound 1 (2.18 mmol), 4-pyridine boronic acid (4.36 mmol), and Pd(PPh3)4 (0.065 mmol) was refluxed in anhydrous acetonitrile at 80°C under N2 atmosphere overnight. After the reaction, the mixture was filtered and purified by column chromatography (ethyl acetate: petroleum ether = 3:10), and vacuum dried to obtain a dark red solid, namely TBP. 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-benzoxadiazole (NBD-Cl) (1.0 mmol) were dissolved in anhydrous dichloromethane (30 ml), stirred and refluxed at 50° C. under N2 protection for 12 h, and concentrated under reduced pressure. The crude product was separated and purified by column chromatography (ethyl acetate: petroleum ether = 1:2), and dried in vacuo to obtain a yellow solid, namely 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 allowed to react overnight in the dark at room temperature. After the reaction was completed, the reaction was quenched, extracted three times, dried, filtered, and the filtrate was distilled under reduced pressure to obtain a crude product. The crude product was separated and purified by column chromatography (ethyl acetate: petroleum ether = 3:100), and vacuum dried to obtain a yellow solid, namely Compound 3. Yield: 52%

[0056] (5) Synthesis of probe TBP-NBD: Compound 2 (1.0 mmol) and 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 separated and purified by column chromatography (dichloromethane:methanol=50:1), and dried in vacuo to obtain a dark blue solid, namely the probe TBP-NBD. Yield: 65%.

[0057] The prepared fluorescent probe TBP-NBD was subjected to high-resolution mass spectrometry detection. The detection result spectrum is shown in Figure 1 , indicating that the target probe is 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, the mixture was filtered and purified by column chromatography (methanol: dichloromethane = 1:10) to obtain an orange-red solid, namely Compound 1. Yield: 85%

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

[0061] (3) Synthesis of Compound 2: p-Hydroxybenzyl alcohol (1.0 mmol) and 4-chloro-7-nitro-2,1,3-benzoxadiazole (NBD-Cl) (1.0 mmol) were dissolved in anhydrous dichloromethane (30 ml), stirred and refluxed at 80° C. under N2 protection for 6 h, and concentrated under reduced pressure. The crude product was separated and purified by column chromatography (ethyl acetate: petroleum ether = 1:2), and dried in vacuo to obtain a yellow solid, namely 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 allowed to react overnight in the dark at 0°C. After the reaction was completed, the reaction solution was directly extracted, dried, filtered, and the filtrate was distilled under reduced pressure to obtain a crude product. The crude product was separated and purified by column chromatography (ethyl acetate: petroleum ether = 3:100), and vacuum dried to obtain a yellow solid, namely Compound 3. Yield: 45%

[0063] (5) Synthesis of probe TBP-NBD: Compound 2 (1.0 mmol) and 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 separated and purified by column chromatography (dichloromethane:methanol=50:1), and dried in vacuo to obtain a dark blue solid, namely the probe TBP-NBD. Yield: 60%

[0064] The prepared fluorescent probe TBP-NBD was subjected to high-resolution mass spectrometry detection. The detection result spectrum is shown in Figure 2 , indicating that the target probe is 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, the mixture was washed and filtered with ethanol and directly used in the next step. Yield: 82%

[0067] (2) Synthesis of fluorophore TBP: A mixture of compound 1 (2.18 mmol), 4-pyridine boronic acid (4.36 mmol), and Pd(PPh3)4 (0.005 mmol) was refluxed in tetrahydrofuran at 90°C under N2 atmosphere for 8 h. After the reaction, the mixture was filtered and purified by column chromatography (dichloromethane:methanol=100:1), and vacuum dried 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-benzoxadiazole (NBD-Cl) (2.0 mmol) were dissolved in anhydrous dichloromethane (50 ml), stirred and refluxed at 80° C. under N2 protection for 6 h, and concentrated under reduced pressure. The crude product was separated and purified by column chromatography (ethyl acetate: petroleum ether = 1:2), and dried in vacuo to obtain a yellow solid, namely 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 allowed to react 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 a 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 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 separated and purified by column chromatography (dichloromethane:methanol=100:1), and dried in vacuo to obtain a dark blue solid, namely the probe TBP-NBD. Yield: 60%

[0071] The prepared fluorescent probe TBP-NBD was subjected to high-resolution mass spectrometry detection. The detection result spectrum is shown in Figure 3 , indicating that the target probe is 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 of specific embodiment 1 comprises the following steps:

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

[0075] The range is 500-700nm, the photomultiplier voltage is 600V, the slit width is 5nm, and a quartz cuvette with four sides of light 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 a concentration of 10 mM;

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

[0078] (4) Preparation of reaction solution: Take the Cys, Hcy and GSH stock solutions, add PBS buffer solution and probe stock solution respectively, to form a reaction solution with a concentration of 10 μM. Place the centrifuge tube 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) The results are as follows Figure 4 As shown in (A), after the fluorescent probe TBP-NBD was incubated with Cys / Hcy, the fluorescence signal was significantly enhanced, while after incubation with GSH, the fluorescence signal was weak and negligible.

[0080] Fluorescent probe for detecting Cys / Hcy under the interference of GSH

[0081] The method for detecting Cys / Hcy under the interference of GSH using the fluorescent probe prepared by the method of specific embodiment 1 comprises the following steps:

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

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

[0084] Fluorescent probes for detecting Cys / Hcy / GSH in living cells

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

[0086] (1) Cell culture: Place Hela cells in a culture flask and culture them in an incubator (37°C, 5% CO2). Take them out after 24 hours, add 2 mL of trypsin digestion solution to the culture flask to digest the cells, and add 2 mL of culture medium (MEM culture medium containing 10% FBS) to terminate the digestion after digestion is completed. Then centrifuge the cell suspension, discard the supernatant after centrifugation, add 1 mL of culture medium, and use a pipette to blow to resuspend the cells. Transfer the cell suspension to a laser confocal dish, place it in a 5% CO2, 37°C incubator overnight to adhere to the wall, discard the culture medium, wash with PBS buffer, and add 2 mL of culture medium.

[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 living cells. The cells were divided into three groups. The first group was added with 40 μL of the TBP-NBD stock solution prepared in Example 2 and incubated for 2 hours. The second group was first treated with Cys (1 mM / L) for 2 hours, and then 40 μL of the TBP-NBD stock solution prepared in Example 2 was added, and then incubated for 0.5 hours. The third group was first treated with NEM for 0.5 hours, and then 40 μL of the TBP-NBD stock solution prepared in Example 2 was added to the cells and incubated for 2 hours. After washing the treated cells three times with PBS buffer, paraformaldehyde was added to fix the cells, washed three times with PBS buffer solution, stained with DAPI, and then washed with PBS buffer solution for confocal imaging.

[0088] (3) Figure 5 As shown, the first group showed weak green fluorescence, indicating that TBP-NBD can detect endogenous biothiols in cells. The second group showed 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 inhibited 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 endogenous and exogenous Cys / Hcy in living cells.

[0089] The results fully demonstrate that the fluorescent probe of the present invention is effective in detecting cysteine / homocysteine ​​in biothiols.

[0090] The above are only preferred embodiments of the present invention. It should be pointed out that, for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the protection scope of the present invention.

Claims

1. A cysteine / homocysteine-responsive AIE fluorescent probe, characterized in that: The fluorescent probe structural formula is as follows:

2. A method for preparing the cysteine / homocysteine-responsive AIE fluorescent probe according to claim 1, characterized in that: The synthetic route is as follows: The specific steps include: Step 1, dissolving 4,7-dibromobenzothiadiazole and 4-boric acid triphenylamine in an organic solvent for reaction, heating under reflux for reaction under palladium catalysis, filtering after the reaction, and purifying the solid obtained after filtering by column chromatography to prepare compound 1; Step 2, dissolving compound 1 and 4-pyridine boronic acid in an organic solvent for reaction, heating the reaction under palladium catalysis, filtering after the reaction, and purifying the obtained solid by column chromatography to obtain compound TBP; Step 3, dissolving p-hydroxybenzyl alcohol and NBD-Cl in an organic solvent, and reacting under reflux at 50-80° C., and concentrating under reduced pressure after the reaction is completed. The crude product is purified by column chromatography to obtain compound 2; Step 4, dissolving compound 2 in an organic solvent, adding PBr3 for bromination, quenching the reaction after the reaction is completed, extracting with an organic solvent, and distilling off the solvent under reduced pressure to obtain a crude product, which is purified to obtain compound 3; Step 5, compound 3 and TBP are dissolved in an organic solvent, and refluxed at 80-90° C. to obtain an AIE fluorescent probe responsive 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 4-boric acid triphenylamine is 1:1, the organic solvent is anhydrous acetonitrile / tetrahydrofuran, and the heating reflux reaction is carried out at 80-90° C. 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 N 2 atmosphere for 8-10 hours.

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-benzoxadiazole (NBD-Cl) are dissolved in anhydrous dichloromethane at a molar ratio of 1:1, and stirred and refluxed at 50-80° C. under N2 protection for 6-12 hours.

6. The method for preparing the AIE fluorescent probe responsive to cysteine / homocysteine ​​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 an equivalent ratio of 1:1 and the reaction was carried out overnight. After the reaction was completed, the solvent was evaporated under reduced pressure to obtain a crude product, and the crude product was 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 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 h under N2 protection. After the reaction, the solvent is evaporated under reduced pressure, and the crude product is purified by column chromatography to obtain the fluorescent probe TBP-NBD.

8. Use of the cysteine / homocysteine-responsive AIE fluorescent probe according to claim 1 in responsive detection of cysteine / homocysteine.

9. Use of the cysteine / homocysteine-responsive AIE fluorescent probe according to claim 8 in cysteine / homocysteine ​​detection, characterized in that: Cysteine, homocysteine ​​and glutathione solutions are respectively added to the reaction system of the AIE fluorescent probe responsive to cysteine / homocysteine, the reaction solution is mixed evenly, incubated at 30-40° C., the ultraviolet absorption spectrum and fluorescence emission spectrum thereof are measured, and cysteine / homocysteine ​​is qualitatively and / or quantitatively analyzed according to the spectral results.

10. Use of the cysteine / homocysteine-responsive AIE fluorescent probe according to claim 8 in cysteine / homocysteine ​​detection, characterized in that: Hela cells are cultured in a laser confocal dish, an AIE fluorescent probe responsive to cysteine / homocysteine ​​is added to the dish for co-incubation, and imaging is performed using a laser confocal microscope. According to the imaging results, qualitative and / or quantitative analysis of cysteine / homocysteine ​​is performed.

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