Fluorescent probe for specifically responding to HClO based on hydroxyphenylbenzothiazole dye as well as preparation method and application of fluorescent probe

Abstract

The invention discloses a fluorescent probe for specifically responding to HClO based on hydroxyphenylbenzothiazole dye as well as a preparation method and application of the fluorescent probe, and belongs to the technical field of fluorescent probes. The invention discloses a fluorescent probe for specifically responding to HClO based on a hydroxyphenyl benzothiazole dye. The chemical formula of the fluorescent probe is C17H13BrN2O2S2. The fluorescent probe detects HClO through ultraviolet and fluorescence spectrometers and is not interfered by active nitrogen and active oxygen, the detection process is simple, convenient, rapid and sensitive, and the detection limit is 1.7 nM. More importantly, the probe can be used for detecting HClO in cells and living bodies, and has a good application prospect in the field of biological monitoring.

Description

Technical Field

[0001] The invention relates to the technical field of fluorescent probes, and more particularly to a fluorescent probe based on a hydroxyphenylbenzothiazole dye that specifically responds to HClO, and a preparation method and application thereof. Background Art

[0002] Endogenous hypochlorous acid (HClO) is produced by the reaction of hydrogen peroxide and chloride ions catalyzed by myeloperoxidase (MPO). Since HClO can exist stably in cells, it can be used as one of the representatives to study the changes of intracellular reactive oxygen species (ROS). Fluctuations in intracellular HClO levels are closely related to excessive oxidative stress, which causes a series of diseases, such as arthritis, cardiovascular disease, neurodegenerative diseases, cancer, etc. Therefore, it is crucial to develop a method to detect cellular HClO and explore its role in disease diagnosis and various pathophysiological characteristics.

[0003] So far, the main methods for detecting HClO include iodine titration, colorimetry, chromatography, fluorescent probe method, electrochemical method, etc. Among them, small molecule fluorescent probes have attracted great attention from researchers due to their advantages of non-invasiveness, real-time evaluation and simple operation. So far, researchers have reported many recognition groups for detecting HClO, including thioethers, thiocarbamates, p-aminophenyl ethers, unsaturated C=C bonds, oximes, selenides, hydrazides and other recognition groups. Although many fluorescent probes for detecting HClO have been reported, there are still few fluorescent probes for super-fast detection of HClO level fluctuations during oxidative stress. Therefore, it is still urgent to construct a new long-wavelength fluorescent probe with high specificity for HClO and realize cell and in vivo imaging.

[0004] Hydroxyphenylbenzothiazole is a typical ESIPT fluorescent dye with the advantages of low raw material cost, simple synthesis, multiple modifiable sites, high fluorescence quantum yield and good photostability. More importantly, hydroxyphenylbenzothiazole fluorescent dyes have relatively small molecular weight, good biocompatibility, easy penetration of living cells, and are very suitable for biological fluorescence imaging, which has attracted widespread attention. Based on this, it is of great significance to prepare fluorescent probes based on hydroxyphenylbenzothiazole fluorescent dyes that specifically respond to HClO in biological cells and realize their application at the living level.

[0005] Therefore, providing a fluorescent probe based on a hydroxyphenylbenzothiazole dye that specifically responds to HClO and a preparation method and application thereof are problems that those skilled in the art need to solve urgently. 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, and a preparation method and application thereof. The preparation method of the fluorescent probe is easy to operate, the raw materials are easily available, and the specific detection of HClO in cells and zebrafish can be achieved.

[0007] In order to achieve the above object, the present invention adopts the following technical solution:

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

[0009]

[0010] Furthermore, the preparation method of the fluorescent probe based on hydroxyphenylbenzothiazole dye that specifically responds to HClO comprises the following specific steps:

[0011] (1) Add 5-bromosalicylaldehyde and 2-aminobenzenethiol to anhydrous dimethyl sulfoxide, stir at 175° C. to 185° C. for 5 to 6 hours to obtain a reaction solution; cool the reaction solution to room temperature, slowly add H2O to the reaction solution, filter the precipitate; wash the precipitate with water, and dry to obtain compound 1;

[0012] The molar ratio of 5-bromosalicylaldehyde and 2-aminobenzenethiol to anhydrous dimethyl sulfoxide is 1:(1-2):400;

[0013] (2) dissolving the compound 1 and hexamethylenetetramine obtained in step (1) in acetic acid, stirring under reflux, stirring at 120° C. to 125° C. for 10 to 12 hours, and removing the solvent by rotary evaporation to obtain a crude product; separating the crude product by silica gel column chromatography using petroleum ether / ethyl acetate as the mobile phase to obtain compound 2;

[0014] The molar ratio of the 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) dissolving the compound 2 obtained in step (2), dimethylthiocarbamoyl chloride and potassium carbonate in acetonitrile, stirring at room temperature for 12 to 14 hours, and removing the solvent by rotary evaporation to obtain a crude product; separating the crude product by silica gel column chromatography using petroleum ether / ethyl acetate as the mobile phase to obtain a fluorescent probe;

[0017] The molar ratio of the 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 solution.

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

[0021]

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

[0023]

[0024] Furthermore, the fluorescent probe or the fluorescent probe prepared by the preparation method is used in the preparation of a reagent for specifically detecting HClO in cells or living bodies.

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

[0026] Furthermore, a reagent or kit for detecting HClO in cells or living bodies comprises the fluorescent probe or the fluorescent probe prepared by the preparation method.

[0027] The fluorescent probe uses hydroxyphenylbenzothiazole derivatives as the fluorophore and N,N-dimethylthiocarbamate as the recognition group. The detection mechanism is that the chloride ions (Cl + ) can undergo an oxidation reaction with N,N-dimethylthiocarbamate, releasing a fluorophore and causing a change in the fluorescence signal. The fluorescent probe detects HClO through ultraviolet and fluorescence spectrometers and is not interfered by its reactive nitrogen and reactive oxygen species. The detection process is simple, rapid, and sensitive, with a detection limit of 1.7nM. More importantly, the probe can detect HClO in cells and living bodies, and has good application prospects in the field of biological monitoring.

[0028] The present invention is a fluorescent probe for specifically detecting HClO based on hydroxyphenylbenzothiazole dye. In solution testing, the probe can determine the reaction time and concentration dependence of the probe and HClO by using ultraviolet and fluorescence spectra. Through selectivity and anti-interference ability tests, it is observed that the probe can specifically detect HClO, does not react with active oxygen and has strong anti-interference ability. In addition, the probe has strong pH stability and low cytotoxicity. The purpose of detecting HClO in MCF-7 cells and living zebrafish can also be achieved through fluorescence imaging technology.

[0029] It can be seen from the above technical scheme that, compared with the prior art, the present invention discloses a fluorescent probe based on hydroxyphenylbenzothiazole dye that specifically responds to HClO, and a preparation method and application thereof. The synthesis method of the fluorescent probe is simple and easy to operate; the detection method can realize the specific detection of HClO and is not interfered by other active oxides; the detection signal is obvious, and it is a fluorescence-enhanced fluorescent probe. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the provided drawings without paying creative work.

[0031] Figure 1 The accompanying drawing is a synthetic route of the fluorescent probe of the present invention;

[0032] Figure 2 The accompanying drawing is a hydrogen NMR spectrum of the fluorescent probe HBTC1 of the present invention;

[0033] Figure 3 The accompanying drawing is a nuclear magnetic carbon spectrum of the fluorescent probe HBTC1 of the present invention;

[0034] Figure 4 The accompanying drawing shows the fluorescence change of the fluorescent probe HBTC1 of the present invention when it interacts with HClO;

[0035] Figure 5 The accompanying drawing shows the absorption change of the fluorescent probe HBTC1 of the present invention in the titration experiment of measuring HClO concentration;

[0036] Figure 6 The accompanying drawing shows the fluorescence change of the fluorescent probe HBTC1 in the titration experiment of HClO concentration;

[0037] Figure 7 The accompanying drawing is a linear fitting diagram of the strongest fluorescence emission wavelength of 550nm and the concentration of HClO of the present invention;

[0038] Figure 8 The accompanying drawing shows the fluorescence selectivity of the common amino acid pair probe HBTC1 in detecting HClO of the present invention;

[0039] Fig. 9 The accompanying drawing shows the fluorescence interference of common amino acids in the present invention on the detection of HClO by probe HBTC1;

[0040] Fig.10 The accompanying drawings are graphs showing the maximum fluorescence intensity changes of the fluorescent probe HBTC1 and the probe plus HClO in buffer solutions of different pH values;

[0041] Fig.11 The accompanying drawing shows the detection of 4T1 cell toxicity of HClO by the probe HBTC1 of the present invention;

[0042] Fig.12 The accompanying drawing shows the MCF-7 cell cytotoxicity of HClO detected by the probe HBTC1 of the present invention;

[0043] Fig.13 The accompanying drawing shows the detection of HClO cytotoxicity in MDA-MB-231 cells by the probe HBTC1 of the present invention;

[0044] Fig.14 The accompanying drawing shows the detection of HClO cytotoxicity in LX-2 cells by the probe HBTC1 of the present invention;

[0045] Fig.15 The accompanying drawing shows the detection of HClO cytotoxicity in HepG2 cells by the probe HBTC1 of the present invention;

[0046] Fig.16 The accompanying drawings are imaging images of the probe HBTC1 of the present invention detecting HClO 4T1, MCF-7, MDA-MB-231, LX-2, and HepG2 cells;

[0047] Among them, (a) intuitive graph; (b) statistical graph;

[0048] Fig.17 The accompanying drawing shows the in vivo imaging of zebrafish using the probe HBTC1 of the present invention to detect HClO;

[0049] Among them, (a) intuitive graph; (b) statistical graph. DETAILED DESCRIPTION

[0050] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0051] Example 1

[0052] Preparation steps of fluorescent probe (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, and the precipitate was washed with water and dried to obtain compound 1;

[0056] 5-bromosalicylaldehyde, 2-aminobenzenethiol and anhydrous dimethyl sulfoxide in a molar ratio of 1:1:400;

[0057] (2) Preparation of Compound 2

[0058]

[0059] The compound 1 obtained in step (1) and hexamethylenetetramine (HMTA) were dissolved in acetic acid (ACOH) and refluxed with stirring at 120° C. for 10 h, and the solvent was removed by rotary evaporation to obtain a crude product; the crude product was separated by silica gel column chromatography using petroleum ether / ethyl acetate (15:1, v / v) as a 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] The compound 2 prepared in step (2), dimethylthiocarbamoyl chloride and potassium carbonate were dissolved in acetonitrile (CH3CN), stirred at room temperature for 12 hours, and the solvent was removed by rotary evaporation to obtain a crude product; the crude product was separated by silica gel column chromatography using petroleum ether / ethyl acetate (6:1, v / v) as a mobile phase to obtain a fluorescent probe BTHCl;

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

[0065] The H NMR spectrum of the fluorescent probe BTHCl is shown in Figure 2 ; The NMR carbon spectrum of the fluorescent probe BTHCl is shown in 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-bromosalicylaldehyde, 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, and the precipitate was washed with water and dried to obtain compound 1;

[0072] 5-bromosalicylaldehyde, 2-aminobenzenethiol and anhydrous dimethyl sulfoxide in a molar ratio of 1:1.5:400;

[0073] (2) Preparation of Compound 2

[0074] The compound 1 obtained in step (1) and hexamethylenetetramine were dissolved in acetic acid, and the mixture was refluxed and stirred at 125° C. for 11 h, and the solvent was removed by rotary evaporation to obtain a crude product; the crude product was separated by silica gel column chromatography using petroleum ether / ethyl acetate (15:1, v / v) as a 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] The compound 2 prepared in step (2), dimethylthiocarbamoyl chloride and potassium carbonate were dissolved in acetonitrile, stirred at room temperature for 13 hours, and the solvent was removed by rotary evaporation to obtain a crude product; the crude product was separated by silica gel column chromatography using petroleum ether / ethyl acetate (6:1, v / v) as a mobile phase to obtain a 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 hours 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, and the precipitate was washed with water and dried to obtain compound 1;

[0083] 5-bromosalicylaldehyde, 2-aminobenzenethiol and anhydrous dimethyl sulfoxide in a molar ratio of 1:2:400;

[0084] (2) Preparation of Compound 2

[0085] The compound 1 and hexamethylenetetramine prepared in step (1) were dissolved in acetic acid and refluxed with stirring at 122° C. for 12 h, and the solvent was removed by rotary evaporation to obtain a crude product; the crude product was separated by silica gel column chromatography using petroleum ether / ethyl acetate (15:1, v / v) as a 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] The compound 2 prepared in step (2), dimethylthiocarbamoyl chloride and potassium carbonate were dissolved in acetonitrile, stirred at room temperature for 14 hours, and the solvent was removed by rotary evaporation to obtain a crude product; the crude product was separated by silica gel column chromatography using petroleum ether / ethyl acetate (6:1, v / v) as a mobile phase to obtain a fluorescent probe BTHCl;

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

[0090] Test example

[0091] Performance test of the fluorescent probe prepared in Example 1

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

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

[0094] The fluorescent probe BTHCl (10 μM) was reacted with HClO (80 μM) in a solution system of acetonitrile / PBS buffer (1:99, v / v, 10 mm, pH 7.4). The results are shown in Figure 4 After adding HClO, the response time of the fluorescent probes BTHCl and HClO was ultra-fast (about 15 s).

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

[0096] The spectral properties of the probe BTHCl (10 μM) and 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) were 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 increased, the absorption of the probe at 225 nm and 306 nm gradually increased, and a gradually increasing red-shifted absorption peak appeared at 440 nm ( Figure 5 ); and the fluorescence peak at 550nm gradually increased, and its fluorescence intensity reached the maximum when the HClO concentration reached 80μM and increased by about 171 times (the fluorescence value of the probe BTHCl (10μM) at 550nm was 0.528359, and the fluorescence value of the probe BTHCl (10μM) + HClO (80μM) at 550nm was 90.94859) ( Figure 6 ).

[0097] With HClO concentration as the horizontal axis and the fluorescence intensity of the probe BTHCl at 550nm as the vertical axis, a graph was drawn and linear fitting was performed to obtain the linear regression equation of the probe: Y = 1.742X + 2.012, and the linear correlation coefficient R 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 fluorescence cuvettes, 4 mL of acetonitrile / PBS buffer (1:99, v / v, 10 mm, pH 7.4) and fluorescent probe stock solution were added respectively. The results are shown in Figure 8When the probe BTHCl (40 μM) was added with active nitrogen and active oxides (HClO (40 μM), ONOO - (50μM), H2O2(50μM), 1 O2(50μM), O t Bu (50 μM), ·OH (50 μM), O2 - (50μM), TBHP (50μM), HS - (50μM), SO3 2- After reacting with analytes such as HCl (50μM), Hcy (50μM), Cys (50μM), and GSH (50μM), the probe BTHCl can specifically identify HClO and show bright yellow fluorescence at 550nm, while no fluorescence appears after reacting with other types of analytes. Therefore, the probe can achieve specific response to HClO. Blank does not add reactive nitrogen and reactive oxides.

[0100] When BTHCl (10 μM) was added to the above analytes and then 80 μM HClO was added for 12 seconds, it can be seen that when HClO coexists with various analytes, BTHCl can still specifically detect HClO in a complex solution system with a strong fluorescence. The experiment proves that BTHCl can respond to HClO without interference from other substances (see Fig. 9 ).

[0101] (4) pH responsiveness 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 the reaction of BTHCl (10 μM) and BTHCl (10 μM) and HClO (80 μM) were studied.

[0103] The results are as follows Fig.10 As shown in the figure, the fluorescence intensity of the fluorescent probe BTHCl remains basically unchanged in solutions with a pH value of 2.0-12.0; after adding HClO, as the pH value continues to increase, its fluorescence at 550nm gradually increases, and the fluorescence intensity is strongest at a pH value in the physiological range of 7.0-8.0. The experiment proves that the fluorescent probe BTHCl can adapt to the pH environment in the body.

[0104] (5) MTT cytotoxicity assay

[0105] Under the conditions of 37°C, DMEM medium, and 5% CO2, MTT experiments were performed in 96-well plates to evaluate the cytotoxicity of the probe BTHCl (0-20 μM). Different concentrations of the probe BTHCl were used to treat different cells (4T1 cells, MCF-7 cells, MDA-MB-231 cells, LX-2 cells, and HepG2 cells) for three groups of parallel experiments and continued to culture for 24 hours, then washed 3 times with PBS, added 0.5 mg / L of MTT and continued to incubate for 4 hours, aspirated the medium, and added 100 μL of DMSO (about 15 minutes). Each experiment was performed in triplicate. The ultraviolet absorption at 490 nm was measured using an ELISA instrument, and the cell viability was calculated based on the following equation: Cytotoxicity (%) = (average absorbance of the experimental group / absorbance of the control group) × 100%.

[0106] Results Fig.11 , Fig.12 , Fig.13 , Fig.14 and Fig.15 , the cell survival rate can be as high as over 90% at low concentrations of the probe, and the probe has almost no cytotoxicity.

[0107] (6) Detection performance test of fluorescent probe for 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 with 0.2% (w / v) trypsin-EDTA solution and dispersed into cell culture medium, and then the cell suspension was transferred to the confocal dish to allow it to grow on the bottom of the dish. In order to study the ability of the probe BTHCl to detect exogenous HClO in cells, the probe BTHCl (10μM, 0.5h) was first incubated, and then different concentrations of HClO (0μM, 5μM, 10μM, 30μM, 50μM, 0.5h) were incubated, and then washed 3 times with PBS before confocal imaging. Control means no addition of probe BTHCl and HClO; BTHCl means only adding the probe BTHCl without adding HClO.

[0109] The results are as follows Fig.16 As shown, after the cells were first incubated with the probe, almost no fluorescence was observed in the cells, and as the concentration of HClO added increased, the fluorescence in the cells gradually increased. This result also shows that the fluorescent probe of the present invention has a good application prospect for detecting HClO in different cells.

[0110] (7) Testing the detection performance of fluorescent probes for HClO in zebrafish

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

[0112] like Fig.17 As shown in the figure, zebrafish incubated with the probe alone showed almost no fluorescence. However, after incubating zebrafish with HClO or PMA+LPS and then incubating zebrafish with the probe, bright fluorescence signals appeared in the zebrafish. This shows that the probe can quickly monitor the production of endogenous HClO in living zebrafish in situ.

[0113] The above description of the disclosed embodiments enables one skilled in the art to implement or use the present invention. Various modifications to these embodiments will be apparent to one 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 present invention. Therefore, the present invention will not be limited to the embodiments shown herein, but rather to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A fluorescent probe based on hydroxyphenylbenzothiazole dye that specifically responds 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 that specifically responds to HClO according to claim 1, characterized in that: The specific steps are as follows: (1) Add 5-bromosalicylaldehyde and 2-aminobenzenethiol to anhydrous dimethyl sulfoxide, stir at 175° C. to 185° C. for 5 to 6 hours to obtain a reaction solution; cool the reaction solution to room temperature, slowly add H2O to the reaction solution, filter the precipitate; wash the precipitate with water, and dry to obtain compound 1; The molar ratio of 5-bromosalicylaldehyde and 2-aminobenzenethiol to anhydrous dimethyl sulfoxide is 1:(1-2):400; (2) dissolving the compound 1 and hexamethylenetetramine obtained in step (1) in acetic acid, stirring under reflux, stirring at 120° C. to 125° C. for 10 to 12 hours, and removing the solvent by rotary evaporation to obtain a crude product; separating the crude product by silica gel column chromatography using petroleum ether / ethyl acetate as the mobile phase to obtain compound 2; The molar ratio of the compound 1, hexamethylenetetramine and acetic acid is (10-20):1:500; The volume ratio of petroleum ether to ethyl acetate is 15:1; (3) dissolving the compound 2 obtained in step (2), dimethylthiocarbamoyl chloride and potassium carbonate in acetonitrile, stirring at room temperature for 12 to 14 hours, and removing the solvent by rotary evaporation to obtain a crude product; separating the crude product by silica gel column chromatography using petroleum ether / ethyl acetate as the mobile phase to obtain a fluorescent probe; The molar ratio of the 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 that specifically responds to HClO according to claim 2, characterized in that: The amount of H2O used in step (1) is 1 / 2 of the volume of the reaction solution.

4. The method for preparing a fluorescent probe based on hydroxyphenylbenzothiazole dye that specifically responds to HClO according to claim 2, characterized in that: The structural formula of compound 1 in step (1) is:

5. The method for preparing a fluorescent probe based on hydroxyphenylbenzothiazole dye that specifically responds to HClO according to claim 2, characterized in that: The structural formula of compound 2 in step (2) is:

6. Use of the fluorescent probe according to claim 1 or the fluorescent probe prepared by the preparation method according to any one of claims 2 to 5 in preparing a reagent for specifically detecting HClO in cells or living bodies.

7. The use according to claim 6, characterized in that: The cells are 4T1 cells, MCF-7 cells, MDA-MB-231 cells, LX-2 cells, and HepG2 cells; and the living organism is zebrafish.

8. A reagent or kit for detecting HClO in cells or living bodies, characterized in that: The invention comprises the fluorescent probe according to claim 1 or the fluorescent probe prepared by the preparation method according to any one of claims 2 to 5.

Images