Hydroxyphenyl benzothiazole dye-based fluorescence probe specifically responding to hclo and preparation method and application thereof

Abstract

The application discloses a hydroxyphenyl benzothiazole dye-based fluorescent probe for responding to HClO and a preparation method and application thereof, and belongs to the technical field of fluorescent probes.The hydroxyphenyl benzothiazole dye-based fluorescent probe for responding to HClO has a chemical molecular formula of C 17 H 13 BrN2O2S2.The fluorescent probe can detect HClO through an ultraviolet and fluorescence spectrometer and is not interfered by active nitrogen and active oxygen, the detection process is simple, rapid and sensitive, and the detection limit is 1.7nM.More importantly, the probe can detect HClO in cells and living bodies, and has a good application prospect in the field of biological monitoring.

Description

Technical Field

[0001] This invention relates to the field of fluorescent probe technology, and more specifically to fluorescent probes based on hydroxyphenylbenzothiazole dyes that specifically respond to HClO, their preparation methods, and applications. Background Technology

[0002] Endogenous hypochlorous acid (HClO) is produced by the reaction of hydrogen peroxide and chloride ions under the catalysis of myeloperoxidase (MPO). Because HClO can exist stably within cells, it can serve as a representative indicator for studying changes in intracellular reactive oxygen species (ROS). Fluctuations in intracellular HClO levels are closely related to excessive oxidative stress, leading to a range of diseases such as arthritis, cardiovascular disease, neurodegenerative diseases, and cancer. Therefore, developing a method for detecting cellular HClO and exploring its role in disease diagnosis and various pathophysiological characteristics is crucial.

[0003] To date, methods for detecting HClO mainly include iodometric titration, colorimetry, chromatography, fluorescent probe methods, and electrochemical methods. Among these, small-molecule fluorescent probes have attracted significant attention from researchers due to their non-invasiveness, real-time assessment, and ease of operation. Numerous recognition groups for HClO detection have been reported, including thioethers, thiocarbamates, p-aminophenyl ethers, unsaturated C=C bonds, oximes, selenides, acylhydrazides, and other recognition groups. Despite the existence of many reported fluorescent probes for HClO detection, ultra-fast fluorescent probes for detecting fluctuations in HClO levels during oxidative stress remain scarce. Therefore, the development of novel long-wavelength fluorescent probes with high specificity for HClO and their application in cellular and in vivo imaging remains an urgent priority.

[0004] Hydroxyphenylbenzothiazole, as a typical ESIPT fluorescent dye, has advantages such as low raw material cost, simple synthesis, multiple modifiable sites, high fluorescence quantum yield, and good photostability. More importantly, hydroxyphenylbenzothiazole fluorescent dyes have attracted widespread attention due to their relatively small molecular weight, good biocompatibility, easy penetration into living cells, and suitability for bioluminescence imaging. Therefore, the preparation of fluorescent probes based on the specific response of hydroxyphenylbenzothiazole fluorescent dyes to HClO in biological cells and their application in vivo is of great significance.

[0005] Therefore, providing fluorescent probes based on hydroxyphenylbenzothiazole dyes that specifically respond to HClO, along with their preparation methods and applications, is a problem that urgently needs to be solved by those skilled in the art. Summary of the Invention

[0006] In view of this, the present invention provides a fluorescent probe based on hydroxyphenylbenzothiazole dye that specifically responds to HClO, its preparation method and application. The preparation method of the fluorescent probe is convenient to operate, the raw materials are readily available, and it can achieve specific detection of HClO in cells and zebrafish.

[0007] To achieve the above objectives, the present invention adopts the following technical solution:

[0008] A fluorescent probe based on hydroxyphenylbenzothiazole dye specifically responding to HClO, the chemical formula of the fluorescent probe being: C 17 H 13 BrN2O2S2, named BTHCl, has the following structural formula:

[0009]

[0010] Furthermore, the specific steps for preparing the fluorescent probe based on hydroxyphenylbenzothiazole dye specifically responding to HClO are as follows:

[0011] (1) 5-bromosalicylaldehyde and 2-aminobenzenethiol were added to anhydrous dimethyl sulfoxide and stirred at 175℃~185℃ for 5~6h to obtain a reaction solution; the reaction solution was cooled to room temperature and H2O was slowly added to the reaction solution, and the precipitate was filtered; the precipitate was washed with water and dried to obtain compound 1;

[0012] The 5-bromosalicylaldehyde, 2-aminobenzylthiol, and anhydrous dimethyl sulfoxide are present in a molar ratio of 1:(1-2):400.

[0013] (2) The compound 1 and hexamethylenetetramine obtained in step (1) were dissolved in acetic acid and refluxed and stirred at 120℃~125℃ for 10~12h. The solvent was removed by rotary evaporation to obtain the crude product. The crude product was separated by silica gel column chromatography using petroleum ether / ethyl acetate as the mobile phase to obtain compound 2.

[0014] The molar ratio of compound 1, hexamethylenetetramine, and acetic acid is (10-20):1:500;

[0015] The volume ratio of petroleum ether to ethyl acetate is 15:1;

[0016] (3) The compound 2, dimethylthiocarbamoyl chloride and potassium carbonate obtained in step (2) are dissolved in acetonitrile and stirred at room temperature for 12-14 h. The solvent is removed by rotary evaporation to obtain the crude product. The crude product is separated by silica gel column chromatography using petroleum ether / ethyl acetate as the mobile phase to obtain the fluorescent probe.

[0017] The molar ratio of compound 2, potassium carbonate, dimethylthiocarbamoyl chloride and acetonitrile is 1:(2-4):(1-2):500;

[0018] The volume ratio of petroleum ether to ethyl acetate is 6:1.

[0019] Furthermore, the amount of H2O used in step (1) is 1 / 2 of the volume of the reaction liquid.

[0020] Furthermore, the structural formula of compound 1 in step (1) is:

[0021]

[0022] Furthermore, the structural formula of compound 2 in step (2) is:

[0023]

[0024] Furthermore, the fluorescent probe described herein or the fluorescent probe prepared by the method described herein is used in the preparation of cell- and in vivo specific detection reagents for HClO.

[0025] Furthermore, the cells are 4T1 cells, MCF-7 cells, MDA-MB-231 cells, LX-2 cells, and HepG2 cells; the living organism is a zebrafish.

[0026] Furthermore, a reagent or kit for detecting HClO in cells or living organisms includes the fluorescent probe described above or the fluorescent probe prepared by the preparation method described above.

[0027] The fluorescent probe uses a hydroxyphenylbenzothiazole derivative as the fluorophore and N,N-dimethylthiocarbamate as the recognition group. The detection mechanism is based on chloride ions (Cl-) released from the decomposition of HClO. + This fluorescent probe can react with N,N-dimethylthiocarbamate to release a fluorophore, causing a change in fluorescence signal. The probe detects HClO using UV and fluorescence spectroscopy without interference from reactive nitrogen and oxygen species. The detection process is simple, rapid, and sensitive, with a detection limit of 1.7 nM. More importantly, this probe can detect HClO in cells and living organisms, showing promising applications in biomonitoring.

[0028] This invention relates to a fluorescent probe based on hydroxyphenylbenzothiazole dye for the specific detection of HClO. In solution testing, the reaction time and concentration dependence of the probe with HClO can be determined using UV and fluorescence spectroscopy. Selectivity and anti-interference tests show that the probe specifically detects HClO, does not react with reactive oxides, and exhibits strong anti-interference capabilities. Furthermore, the probe demonstrates strong pH stability and low cytotoxicity. Fluorescence imaging technology can also be used to detect HClO in MCF-7 cells and live zebrafish.

[0029] As can be seen from the above technical solutions, compared with the prior art, the present invention discloses a fluorescent probe based on hydroxyphenylbenzothiazole dye that specifically responds to HClO, its preparation method and application. The fluorescent probe synthesis method is simple and easy to operate; the detection method can achieve specific detection of HClO and is not affected by other reactive oxides; the detection signal is obvious, and it is a fluorescence-enhanced fluorescent probe. Attached Figure Description

[0030] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on the provided drawings without creative effort.

[0031] Figure 1 The attached figure shows the synthesis route of the fluorescent probe of this invention;

[0032] Figure 2 The attached figure shows the hydrogen NMR spectrum of the fluorescent probe HBTCl of this invention;

[0033] Figure 3 The attached figure shows the carbon NMR spectrum of the fluorescent probe HBTCl of this invention;

[0034] Figure 4 The attached figure shows the time-varying fluorescence of the fluorescent probe HBTCl of the present invention reacting with HClO;

[0035] Figure 5 The attached figure shows the absorption changes in a titration experiment of HClO concentration determination using the fluorescent probe HBTCl of this invention;

[0036] Figure 6 The attached figure shows the fluorescence changes in a titration experiment of HClO concentration determination using the fluorescent probe HBTCl of this invention;

[0037] Figure 7 The attached figure is a linear fitting graph of the strongest fluorescence emission wavelength of the present invention (550 nm) and the concentration of HClO.

[0038] Figure 8 The attached figure shows the fluorescence selectivity of the common amino acid pair probe HBTCl for detecting HClO in this invention;

[0039] Figure 9 The attached figure shows the fluorescence interference of the common amino acid pair probe HBTCl in detecting HClO.

[0040] Figure 10 The attached figure shows the changes in maximum fluorescence intensity of the fluorescent probe HBTCl and the probe with added HClO in different pH buffer solutions.

[0041] Figure 11 The attached figure shows the detection of 4T1 cytotoxicity of HClO by the HBTCl probe of this invention;

[0042] Figure 12 The attached figure shows the detection of HClO cytotoxicity in MCF-7 cells using the HBTCl probe of this invention.

[0043] Figure 13 The attached figure shows the detection of MDA-MB-231 cytotoxicity of HClO by the HBTCl probe of this invention;

[0044] Figure 14 The attached figure shows the detection of LX-2 cytotoxicity of HClO by the HBTCl probe of this invention;

[0045] Figure 15 The attached figure shows the detection of HClO cytotoxicity in HepG2 cells using the HBTCl probe of this invention.

[0046] Figure 16 The attached figure shows an imaging diagram of HClO 4T1, MCF-7, MDA-MB-231, LX-2, and HepG2 cells detected by the HBTCl probe of the present invention.

[0047] Among them, (a) is an intuitive diagram; (b) is a statistical chart;

[0048] Figure 17 The attached figure shows a live image of a zebrafish detecting HClO using the HBTCl probe of this invention.

[0049] Among them, (a) is an intuitive diagram; and (b) is a statistical chart. Detailed Implementation

[0050] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0051] Example 1

[0052] Preparation steps of fluorescent probes (synthesis route see...) Figure 1 )as follows:

[0053] (1) Preparation of compound 1

[0054]

[0055] 5-bromosalicylaldehyde, 2-aminobenzenethiol and anhydrous dimethyl sulfoxide were stirred at 180 °C for 6 h to obtain a reaction solution. The reaction solution was cooled to room temperature, and 1 / 2 volume of H2O was slowly added to the reaction solution. The precipitate was filtered, washed with water, and dried to obtain compound 1.

[0056] 5-Bromosalicylaldehyde, 2-aminobenzylthiol, and anhydrous dimethyl sulfoxide were used in a molar ratio of 1:1:400.

[0057] (2) Preparation of compound 2

[0058]

[0059] Compound 1 and hexamethylenetetramine (HMTA) obtained in step (1) were dissolved in acetic acid (ACOH) and refluxed with stirring. The mixture was stirred at 120°C for 10 h. The solvent was removed by rotary evaporation to obtain the crude product. The crude product was separated by silica gel column chromatography using petroleum ether / ethyl acetate (15:1, v / v) as the mobile phase to obtain compound 2.

[0060] The molar ratio of compound 1, hexamethylenetetramine, and acetic acid is 10:1:500;

[0061] (3) Preparation of probe BTHCl

[0062]

[0063] Compound 2, dimethylthiocarbamoyl chloride and potassium carbonate obtained in step (2) were dissolved in acetonitrile (CH3CN), stirred at room temperature for 12 h, and the solvent was removed by rotary evaporation to obtain crude product; the crude product was separated by silica gel column chromatography using petroleum ether / ethyl acetate (6:1, v / v) as mobile phase to obtain fluorescent probe BTHCl;

[0064] The molar ratio of compound 2, potassium carbonate, dimethylthiocarbamoyl chloride, and acetonitrile is 1:2:1:500.

[0065] The 1H NMR spectrum of the fluorescent probe BTHCl is shown below. Figure 2 The carbon NMR spectrum of the fluorescent probe BTHCl is shown below. Figure 3 .

[0066] 1H NMR (500MHz, DMSO) δ9.99,8.74,8.73,8.22,8.21,8.20,7.99,7.97,7.63,7.63,7.62,7.62 ,7.61,7.60,7.60,7.56,7.56,7.54,7.54,7.54,7.53,7.53,7.28,7.26,7.26,3.60,3.43.

[0067] 13 C NMR (126MHz, DMSO) δ188.21,184.91,159.66,152.16,151.30,136.70,135.78,1 34.97,132.57,130.32,127.47,126.70,123.77,122.86,120.39,119.83,43.94.

[0068] Example 2

[0069] The preparation steps of the fluorescent probe are as follows:

[0070] (1) Preparation of compound 1

[0071] 5-bromosalicylic acid aldehyde, 2-aminobenzenethiol and anhydrous dimethyl sulfoxide were stirred at 175 °C for 5.5 h to obtain a reaction solution. The reaction solution was cooled to room temperature, and 1 / 2 volume of H2O was slowly added to the reaction solution. The precipitate was filtered, washed with water, and dried to obtain compound 1.

[0072] 5-Bromosalicylaldehyde, 2-aminobenzylthiol, and anhydrous dimethyl sulfoxide were mixed in a molar ratio of 1:1.5:400.

[0073] (2) Preparation of compound 2

[0074] Compound 1 and hexamethylenetetramine obtained in step (1) were dissolved in acetic acid and refluxed with stirring at 125°C for 11 h. The solvent was removed by rotary evaporation to obtain the crude product. The crude product was separated by silica gel column chromatography using petroleum ether / ethyl acetate (15:1, v / v) as the mobile phase to obtain compound 2.

[0075] The molar ratio of compound 1, hexamethylenetetramine, and acetic acid is 15:1:500;

[0076] (3) Preparation of compound 3

[0077] Compound 2, dimethylthiocarbamoyl chloride and potassium carbonate obtained in step (2) were dissolved in acetonitrile and stirred at room temperature for 13 h. The solvent was removed by rotary evaporation to obtain the crude product. The crude product was separated by silica gel column chromatography using petroleum ether / ethyl acetate (6:1, v / v) as the mobile phase to obtain the fluorescent probe BTHCl.

[0078] The molar ratio of compound 2, potassium carbonate, dimethylthiocarbamoyl chloride and acetonitrile is 1:3:1.5:500.

[0079] Example 3

[0080] The preparation steps of the fluorescent probe are as follows:

[0081] (1) Preparation of compound 1

[0082] 5-bromosalicylaldehyde, 2-aminobenzenethiol and anhydrous dimethyl sulfoxide were stirred at 185 °C for 5 h to obtain a reaction solution. The reaction solution was cooled to room temperature, and 1 / 2 volume of H2O was slowly added to the reaction solution. The precipitate was filtered, washed with water, and dried to obtain compound 1.

[0083] 5-Bromosalicylaldehyde, 2-aminobenzylthiol, and anhydrous dimethyl sulfoxide were used in a molar ratio of 1:2:400.

[0084] (2) Preparation of compound 2

[0085] Compound 1 and hexamethylenetetramine obtained in step (1) were dissolved in acetic acid and refluxed with stirring. The mixture was stirred at 122°C for 12 h. The solvent was removed by rotary evaporation to obtain the crude product. The crude product was separated by silica gel column chromatography using petroleum ether / ethyl acetate (15:1, v / v) as the mobile phase to obtain compound 2.

[0086] The molar ratio of compound 1, hexamethylenetetramine, and acetic acid is 20:1:500;

[0087] (3) Preparation of compound 3

[0088] Compound 2, dimethylthiocarbamoyl chloride and potassium carbonate obtained in step (2) were dissolved in acetonitrile and stirred at room temperature for 14 h. The solvent was removed by rotary evaporation to obtain the crude product. The crude product was separated by silica gel column chromatography using petroleum ether / ethyl acetate (6:1, v / v) as the mobile phase to obtain the fluorescent probe BTHCl.

[0089] The molar ratio of compound 2, potassium carbonate, dimethylthiocarbamoyl chloride, and acetonitrile is 1:4:2:500.

[0090] Test case

[0091] Performance testing was performed using the fluorescent probe prepared in Example 1.

[0092] (1) Reaction time test of BTHCl, a fluorescent probe for detecting HClO in solution.

[0093] Prepare a fluorescent probe stock solution of 1 mM BTHCl using acetonitrile;

[0094] The fluorescent probe BTHCl (10 μM) reacted with HClO (80 μM) in an acetonitrile / PBS buffer solution (1:99, v / v, 10 mL, pH 7.4). The results are shown in the figure. Figure 4 After the addition of HClO, the response time of the fluorescent probes BTHCl and HClO is extremely fast (approximately 15 seconds).

[0095] (2) Concentration titration test and linear relationship of BTHCl, a fluorescent probe for detecting HClO in solution.

[0096] The spectral performance of the probe BTHCl (10 μM) with different concentrations of HClO (0, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80 μM) was tested in acetonitrile / PBS (1:99, v / v, 10 mm, pH 7.4) at 37 °C. It was found that as the concentration of HClO gradually increased, the probe showed gradually enhanced absorption at 225 nm and 306 nm, and a gradually increasing red-shifted absorption peak appeared at 440 nm. Figure 5 Furthermore, the fluorescence peak at 550 nm gradually increases, with the fluorescence intensity reaching its maximum and increasing by approximately 171 times when the HClO concentration reaches 80 μM (the fluorescence value of probe BTHCl (10 μM) at 550 nm is 0.528359, and the fluorescence value of probe BTHCl (10 μM) + HClO (80 μM) at 550 nm is 90.94859). Figure 6 ).

[0097] Plotting HClO concentration on the x-axis and the fluorescence intensity of the probe BTHCl at 550 nm on the y-axis, a graph was constructed and linearly fitted. The linear regression equation for this probe was obtained as: Y = 1.742X + 2.012, with a linear correlation coefficient R0. 2 =0.994 and the detection limit was calculated to be 1.7 nM (see Figure 7 ).

[0098] (3) Interference and anti-interference ion experiments

[0099] In different fluorescent cuvettes, 4 mL of acetonitrile / PBS buffer (1:99, v / v, 10 mL, pH 7.4) and fluorescent probe stock solution were added, respectively. The results are shown in the figure. Figure 8When the probe BTHCl (40 μM) was added with active nitrogen and active oxide (HClO (40 μM), ONOO) respectively - (50μM), H2O2 (50μM), 1 O2 (50μM), ·O t Bu (50μM), ·OH (50μM), O2 - (50μM), TBHP (50μM), HS - (50μM), SO3 2- After reacting with analytes such as Hcy (50 μM), Cys (50 μM), and GSH (50 μM), the probe BTHCl specifically recognizes HClO and exhibits bright yellow fluorescence at 550 nm. No fluorescence is observed after reacting with other analytes, thus demonstrating the probe's specific response to HClO. Blank does not contain reactive nitrogen or reactive oxides.

[0100] When 10 μM BTHCl was added to the above analytes and then 80 μM HClO was added and reacted for 12 s, it was observed that BTHCl could still specifically detect HClO in the complex solution system, even with the coexistence of various analytes, and the fluorescence intensity was strong. Experiments demonstrate that BTHCl is unaffected by other substances in its response to HClO (see...). Figure 9 ).

[0101] (4) pH response experiment

[0102] PBS buffer solutions with pH values ​​of 2.0, 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, 10.0, 11.0, and 12.0 were prepared, and the changes in fluorescence intensity after reaction with BTHCl (10 μM) and BTHCl (10 μM) and HClO (80 μM) were investigated.

[0103] The results are as follows Figure 10 As shown, the fluorescence intensity of the fluorescent probe BTHCl remained essentially constant in solutions with pH values ​​ranging from 2.0 to 12.0. Upon addition of HClO, the fluorescence at 550 nm gradually increased with increasing pH, reaching its strongest intensity within the physiological pH range of 7.0-8.0. This experiment demonstrates that the fluorescent probe BTHCl can adapt to the pH environment within organisms.

[0104] (5) MTT cytotoxicity assay

[0105] The cytotoxicity of the probe BTHCl (0-20 μM) was evaluated in 96-well plates under DMEM medium and 5% CO2 conditions using an MTT assay. Different cell types (4T1, MCF-7, MDA-MB-231, LX-2, and HepG2) were treated with different concentrations of BTHCl in triplicate for 24 h. Cells were then washed three times with PBS, incubated with 0.5 mg / L MTT for another 4 h, aspirated, and 100 μL of DMSO was added (approximately 15 min). Each experiment was performed in triplicate. UV absorbance at 490 nm was measured using a microplate reader, and cell viability was calculated based on the following equation: Cytotoxicity (%) = (Average absorbance of experimental group / Absorbance of control group) × 100%.

[0106] See results Figure 11 , Figure 12 , Figure 13 , Figure 14 and Figure 15 At low concentrations of probe, cell survival rates can reach over 90%, and the probe has almost no cytotoxicity.

[0107] (6) Performance test of fluorescent probes for detecting HClO in 4T1 cells, MCF-7 cells, MDA-MB-231 cells, LX-2 cells, and HepG2 cells.

[0108] Before cell imaging, adherent cells were separated using 0.2% (w / v) trypsin-EDTA solution and dispersed in cell culture medium. The cell suspension was then transferred to confocal dishes for growth. To investigate the ability of the probe BTHCl to detect exogenous HClO, cells were first incubated with BTHCl (10 μM, 0.5 h), followed by incubation with different concentrations of HClO (0 μM, 5 μM, 10 μM, 30 μM, 50 μM, 0.5 h). After washing three times with PBS, confocal imaging was performed. Control mode: no BTHCl or HClO added; BTHCl mode: only BTHCl added, no HClO added.

[0109] The results are as follows Figure 16 As shown, after incubating the cells with the probe, almost no fluorescence was observed. However, as the concentration of HClO added increased, the fluorescence within the cells gradually increased. This result also indicates that the fluorescent probe of the present invention has good application prospects for detecting HClO in different cells.

[0110] (7) Performance test of fluorescent probe for detecting HClO in zebrafish

[0111] First, zebrafish were divided into parallel groups. The first group served as a control (without the addition of probes BTHCl and HClO). The second group was incubated with probe BTHCl (10 μM, 0.5 h). The third group was incubated with HClO (100 μM, 0.5 h) followed by BTHCl (10 μM, 0.5 h). The fourth group was incubated with LPS (1 μg / mL, 16 h) and PMA (1 μg / mL) followed by BTHCl (10 μM, 0.5 h). The fifth group was incubated with LPS (1 μg / mL, 16 h) and PMA (1 μg / mL), followed by NAC (1 mM, 2 h), and finally BTHCl (10 μM, 0.5 h). LPS and PMA were hypochlorous acid inducers; NAC was a hypochlorous acid inhibitor.

[0112] like Figure 17 As shown, zebrafish incubated with the probe alone showed almost no fluorescence. However, when zebrafish were incubated with HClO or PMA+LPS, and then incubated with the probe, bright fluorescent signals appeared. This indicates that the probe can rapidly monitor the production of endogenous HClO in living zebrafish in situ.

[0113] The above description of the disclosed embodiments enables those skilled in the art to make or use the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A fluorescent probe based on hydroxyphenylbenzothiazole dye specifically responding to HClO, characterized in that, The structural formula of the fluorescent probe is: 。 2. The method for preparing a fluorescent probe based on hydroxyphenylbenzothiazole dye specifically responding to HClO as described in claim 1, characterized in that, The specific steps are as follows: (1) 5-Bromosalicylaldehyde and 2-aminobenzenethiol were added to anhydrous dimethyl sulfoxide and stirred at 175℃~185℃ for 5~6 h to obtain a reaction solution; the reaction solution was cooled to room temperature, and H2O was slowly added to the reaction solution, and the precipitate was filtered; the precipitate was washed with water and dried to obtain compound 1; the structural formula of compound 1 is: ； The 5-bromosalicylaldehyde, 2-aminobenzenethiol, and anhydrous dimethyl sulfoxide are present in a molar ratio of 1:(1~2):

400. (2) The compound 1 and hexamethylenetetramine obtained in step (1) were dissolved in acetic acid and refluxed with stirring at 120℃~125℃ for 10~12 h. The solvent was removed by rotary evaporation to obtain the crude product. The crude product was separated by silica gel column chromatography using petroleum ether / ethyl acetate as the mobile phase to obtain compound 2. The structural formula of compound 2 is: ; The molar ratio of compound 1, hexamethylenetetramine, and acetic acid is (10~20):1:500; The volume ratio of petroleum ether to ethyl acetate is 15:1; (3) The compound 2, dimethylthiocarbamoyl chloride and potassium carbonate obtained in step (2) are dissolved in acetonitrile and stirred at room temperature for 12-14 h. The solvent is removed by rotary evaporation to obtain the crude product. The crude product is separated by silica gel column chromatography using petroleum ether / ethyl acetate as the mobile phase to obtain the fluorescent probe. The molar ratio of compound 2, potassium carbonate, dimethylthiocarbamoyl chloride and acetonitrile is 1:(2~4):(1~2):500; The volume ratio of petroleum ether to ethyl acetate is 6:

1.

3. The method for preparing a fluorescent probe based on hydroxyphenylbenzothiazole dye specifically responding to HClO according to claim 2, characterized in that, In step (1), the amount of H2O used is 1 / 2 of the volume of the reaction liquid.

4. The application of the fluorescent probe according to claim 1 in the preparation of a cell- and in vivo specific detection reagent for HClO, characterized in that, The cells were 4T1 cells, MCF-7 cells, MDA-MB-231 cells, LX-2 cells, and HepG2 cells; the living organism was a zebrafish.

5. A reagent or kit for detecting HClO in cells or living organisms, characterized in that, Includes the fluorescent probe as described in claim 1.

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