阅读说明：本技术 基于bodipy染料专一响应次氯酸的荧光探针、制备及应用 (Fluorescent probe based on specific response of BODIPY dye to hypochlorous acid, preparation and application ) 是由 张健 赵伟利 范冠文 鲁小艳 苏慧慧 王楠楠 陶远芳 秦淑春 于 2021-07-12 设计创作，主要内容包括：本发明提供了一种基于BODIPY染料专一响应次氯酸的荧光探针、制备及应用,探针结构式为：。本探针选择BODIPY染料为荧光团,N,N-二甲氨基硫代甲酸酯基团为识别基团。检测机制是N,N-二甲氨基硫代甲酸酯基团被次氯酸氧化离去,引起电子转移,酚盐进一步发生自消除反应释放出荧光团,从而引起荧光信号变化。上述探针检测ClO～(－)且不受其他活性氮和活性氧类物质的干扰,操作过程简便、快速、灵敏,检测限为1.8nM。更重要的是该探针可以检测细胞和活体中的ClO～(－),在生物监测领域具有良好应用前景。(The invention provides a BODIPY dye-based specific response hypochlorous acid fluorescent probe, preparation and application, wherein the structural formula of the probe is as follows: . The probe selects BODIPY dye as a fluorophore and N, N-dimethylamino-thiocarbamate group as a recognition group. The detection mechanism is that the N, N-dimethylamino thiocarbamate group is oxidized and departed by hypochlorous acid to cause electron transfer, and phenolate further undergoes self-elimination reaction to release a fluorophore, thereby causing the change of a fluorescence signal. ClO detection by the above-mentioned probe － And free from other reactive nitrogen and reactive oxygen speciesInterference, simple, convenient, rapid and sensitive operation process, and the detection limit is 1.8 nM. More importantly, the probe can detect ClO in cells and living bodies － And has good application prospect in the field of biological monitoring.)

1. The BODIPY dye-based fluorescent probe specifically responding to hypochlorous acid is characterized in that the structural formula of the probe is as follows:

。

2. the method for preparing a fluorescent probe according to claim 1, characterized by comprising the steps of:

（1）N₂under protection, dissolving p-hydroxybenzyl alcohol in 1, 2-dichloroethane, adding diisopropylethylamine in ice bath, adding N, N-dimethylamino-thiocarbonyl chloride, stirring, and reacting at 50 deg.CPerforming dichloromethane extraction for 24 hours, and performing column chromatography separation to obtain a compound 1;

（2）N₂under protection, dissolving the compound 1 prepared in the step (1) in dichloromethane, adding phosphorus tribromide, stirring at room temperature, quenching with ice water, extracting with dichloromethane, and performing column chromatography separation to obtain a compound 2;

（3）N₂dissolving acetone-based p-toluenesulfonate oxime ester and 6-methoxy-1-tetralone in a mixed solvent in a protective ice water bath, reacting for 2-3 h at 50 ℃, quenching with ice water, extracting with dichloromethane, and performing column chromatography separation to obtain a compound 3;

（4）N₂under protection, dissolving the compound 3 and 4-pyridine formaldehyde in anhydrous DCM, adding a drop of trifluoroacetic acid, and standing overnight at 30 ℃; adding tetrachlorobenzoquinone into the reaction system, then adding diisopropylethylamine and boron trifluoride diethyl etherate under ice bath, and stirring for 2-3 h at room temperature; washing with saturated sodium bicarbonate solution, extracting with dichloromethane, and performing column chromatography to obtain compound 4;

(5) and dissolving the compound 2 and the compound 4 in toluene, refluxing at 120 ℃, and recrystallizing to obtain the probe BDP-R-ClO.

3. The method of claim 2, wherein: in the step (1), p-hydroxybenzyl alcohol and N, N-dimethylamino-thiocarbonyl chloride are mixed according to the ratio of equivalent weight of 1: (1-2), stirring, and heating for reaction for 24 hours;

the structural formula of the compound 1 in the step (1) is as follows:。

4. the method of claim 2, wherein: in the step (2), the compound 1 and phosphorus tribromide are mixed according to an equivalent of 1: (1-2) adding the raw materials in proportion, and stirring at room temperature for 1-2 hours;

the structural formula of the compound 2 in the step (2) is as follows:。

5. the method of claim 2, wherein: the mixed solvent in the step (3) is obtained by adding sodium hydride into a tetrahydrofuran solvent and mixing, wherein 6-methoxy-1-tetralone and acetonyl p-toluenesulfonate oxime ester are mixed according to the equivalent weight of 1: (1-2) dissolving the mixture in a mixed solvent according to the proportion, and reacting for 3 hours at 50 ℃;

the structural formula of the compound 3 in the step (3) is as follows:。

6. the method of claim 2, wherein: in the step (4), the compound 3, the 4-pyridinecarboxaldehyde and the chloranil are mixed according to the ratio of equivalent weight of 1: (0.5-1): adding the mixture according to the proportion of 0.5, and stirring the mixture for 3 hours at room temperature;

the structural formula of the compound 4 in the step (4) is as follows:。

7. the method of claim 2, wherein: in the step (5), the compound 2 and the compound 4 are added according to the proportion of 1 (2-3) equivalent weight, and are heated and stirred for 48 hours.

8. The fluorescent probe prepared by the preparation method of any one of claims 2 to 7, wherein the structural formula of the probe is:

。

9. the method for specific detection of ClO using the fluorescent probe of claim 9^－Application in reagents.

10. Use according to claim 9, characterized in that: the detection limit of the fluorescent probe is 1.8 nM.

Technical Field

The invention relates to the field of fluorescent probes, in particular to a fluorescent probe based on BODIPY dye specific response hypochlorous acid, preparation and application.

Background

Hypochlorous acid (HClO), an important weakly acidic biologically active oxygen (ROS), consists of H in vivo₂O₂And Cl^－Produced under catalysis of Myeloperoxidase (MPO). HClO/ClO with a certain concentration in organism^－Plays an important role in maintaining normal physiological functions of cells, cell signal transduction, maintaining homeostasis and immune defense systems. However, excess HClO/ClO^－Can cause apoptosis and tissue damage, and further increase the occurrence risk of Alzheimer disease, neurodegenerative disease, diabetes, cardiovascular disease, rheumatoid arthritis, cancer and other diseases. Thus, a rapid and sensitive detection method for identifying HClO/ClO in the detection environment and in the living body^－Has important significance.

To date, various approaches have been explored for ClO^‒The detection includes electrochemical analysis, immunohistochemistry, photoluminescence, chemiluminescence, and other methods. Among the methods, the fluorescence probe method has the advantages of high response speed, high sensitivity, capability of in-situ imaging, low biological traumatism and the like. Has been widely used in chemical analysis, biological analysis and medical research. In recent years, these chemically reactive fluorescent probes are mainly based on the following mechanisms: oxidation of chalcogen, breaking of carbon-carbon double bonds, oxidation of hydrazide, intramolecular cyclization and other reaction strategies. Despite the reported fluorescent probesNow to ClO^‒But still have problems such as low selectivity (some clos)^‒Probes may also be paired with H₂O₂And ONOO^‒Response), shorter fluorescence emission wavelength (in the uv-vis region) and poor in vivo imaging applications, etc., so that construction for ClO is made^‒Research on novel long-wavelength fluorescent probes having high selectivity and enabling imaging of cells and living bodies is still imminent. BODIPY is an excellent fluorophore, and has good light stability, high molar absorption, high fluorescence quantum yield and strong tolerance to environmental factors, so that BODIPY is introduced into various probes in recent years to obtain a good fluorescence effect. Thus, the BODIPY-based fluorescent dye specifically responds to ClO in biological cells^‒The long-wavelength fluorescent probe and the realization of the application at the living body level have important significance.

Disclosure of Invention

The invention provides a BODIPY dye-based specific response hypochlorous acid fluorescent probe, preparation and application thereof^－The specificity of (3).

The technical scheme for realizing the invention is as follows:

specific response ClO based on BODIPY dye^－The chemical formula of the fluorescent probe is as follows: c₄₄H₄₂BF₂N₄O₃S⁺Named BDP-R-ClO, the structural formula of the probe is as follows:

。

the preparation method of the fluorescent probe comprises the following steps:

（1）N₂under protection, p-hydroxybenzyl alcohol is dissolved in 1, 2-dichloroethane, diisopropylethylamine and N, N-dimethylaminothiocarbonyl chloride are added in an ice-water bath according to an equivalent weight of 1: (1-2), stirring, reacting at 50 ℃ for 24 hours, extracting with dichloromethane, and performing column chromatography separation to obtain a compound 1;

compound (I)1 structural formula:；

(2) dissolving the compound 1 prepared in the step (1) in dichloromethane, and reacting phosphorus tribromide in a molar ratio of 1: (1-2), stirring at room temperature for 1-2h, adding water for quenching, extracting with dichloromethane, and performing column chromatography separation to obtain a compound 2;

the structural formula of compound 2:；

（3）N₂under the protection of ice water bath, adding sodium hydride into a tetrahydrofuran solvent to be mixed to obtain a mixed solvent, dissolving acetonyl p-toluenesulfonate oxime ester and 6-methoxy-1-tetralone into the mixed solvent according to the proportion of 1 (1-2) equivalent weight, reacting for 3 hours at 50 ℃, quenching with ice water, extracting with dichloromethane, and performing column chromatography separation to obtain a compound 3;

the structural formula of compound 3:；

（4）N₂under protection, compound 3 and 4-pyridinecarboxaldehyde are mixed according to an equivalent weight of 1: (0.5-1) was dissolved in anhydrous DCM, and a drop of trifluoroacetic acid was added thereto, followed by reaction overnight at 30 ℃. Then tetrachlorobenzoquinone with the equivalent weight of 0.5 is added into the reaction system and stirred for 30min, and then diisopropylethylamine with the equivalent weight of 0.01 and boron trifluoride diethyl etherate are added under ice bath and stirred for 3h at room temperature. Washing with saturated sodium bicarbonate solution, extracting with dichloromethane, and performing column chromatography to obtain compound 4;

the structural formula of compound 4:；

(5) compound 2 and compound 4 were added in an equivalent of 1: and (2-3) dissolving in toluene, refluxing at 120 ℃ for 48h, and recrystallizing to obtain the probe BDP-R-ClO.

The fluorescent probe prepared by the invention can specifically detect ClO in the preparation of cells/living bodies^‒In the reagentThe application is as follows.

The synthetic route of the fluorescent probe is as follows:

specific detection of ClO based on BODIPY dye^‒The probe can be used for judging the probe and the ClO by utilizing ultraviolet and fluorescence spectra in solution test^‒Reaction time, concentration dependence relationship; the probes can specifically detect the ClO observed through the tests of selectivity and anti-interference capability^‒The anti-interference performance is strong without reaction with active oxide; and the probe has strong pH stability and small cytotoxicity. ClO in Hela cells and mice can also be obtained by fluorescence imaging technology^‒And (4) detecting.

The invention has the beneficial effects that:

(1) the invention relates to a method for detecting ClO by utilizing specificity of BODIPY dye^‒The fluorescent probe has simple synthesis method and convenient operation;

(2) the detection method of the invention can realize ClO^‒Specific detection is carried out, and interference of other active nitrogen and active oxides is avoided;

(3) the invention has obvious detection signals and is a near-infrared-like fluorescence enhanced fluorescent probe.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, it is obvious that the drawings in the following description are only some embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to the drawings without creative efforts.

FIG. 1 is a nuclear magnetic hydrogen spectrum diagram of a fluorescent probe BDP-R-ClO.

FIG. 2 is a nuclear magnetic carbon spectrum of a fluorescent probe BDP-R-ClO.

FIG. 3 shows fluorescent probes BDP-R-ClO and ClO^‒When actingAlternating ultraviolet.

FIG. 4 shows fluorescent probes BDP-R-ClO and ClO^‒Time of action fluorescence change.

FIG. 5 shows the determination of ClO by fluorescent probe BDP-R-ClO^‒Concentration titration experiment fluorescence change.

FIG. 6 shows the emission wavelength of the strongest fluorescence of 590 nm and ClO^‒Is linearly fitted to the concentration of (a).

FIG. 7 shows the detection of ClO by BDP-R-ClO probe with common amino acid pair^‒Fluorescence selectivity of (2).

FIG. 8 shows the detection of ClO by BDP-R-ClO probe with common amino acid pair^‒Of (3) fluorescence interference.

FIG. 9 shows fluorescent probe BDP-R-ClO and probe plus ClO^‒Graph of maximum fluorescence intensity change in different pH buffer solutions.

FIG. 10 is a probe BDP-R-ClO for detecting ClO^‒The cytotoxicity of (a).

FIG. 11 shows the detection of ClO by the fluorescent probe BDP-R-ClO^‒RAW264.7 cytogram.

FIG. 12 shows the detection of ClO by the fluorescent probe BDP-R-ClO^‒Mice were imaged in vivo.

Detailed Description

The technical solutions of the present invention will be described clearly and completely with reference to the following embodiments of the present invention, and it should be understood that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be obtained by a person skilled in the art without inventive effort based on the embodiments of the present invention, are within the scope of the present invention.

Example 1

The preparation steps of the probe are as follows:

(1) preparation of Compound 1

N₂Under protection, dissolving p-hydroxybenzyl alcohol in 1, 2-dichloroethane, adding diisopropylethylamine and N, N-dimethylaminoThiocarbonyl chloride was added in an equivalent of 1:1, heating to react for 24 hours at 50 ℃, extracting by dichloromethane, and carrying out column chromatography separation to obtain a compound 1;

(2) preparation of Compound 2

Dissolving the compound 1 prepared in the step (1) in dichloromethane, and reacting phosphorus tribromide in a molar ratio of 1:1.2, stirring at room temperature for 1-2h, adding water for quenching, extracting with dichloromethane, and performing column chromatography separation to obtain a compound 2;

(3) preparation of Compound 3

N₂Under the protection of ice water bath, adding sodium hydride into a tetrahydrofuran solvent for mixing to obtain a mixed solvent, dissolving acetonyl p-toluenesulfonate oxime ester and 6-methoxy-1-tetralone in the mixed solvent according to the equivalent weight of 1:1.2, reacting for 3 hours at 50 ℃, quenching with ice water, extracting with dichloromethane, and performing column chromatography separation to obtain a compound 3;

(4) preparation of Compound 4

N₂Under protection, compound 3 and 4-pyridinecarboxaldehyde are mixed according to an equivalent weight of 1: 0.5 was dissolved in anhydrous DCM, and one drop of trifluoroacetic acid was added to react overnight at 30 ℃. Then adding tetrachlorobenzoquinone with the equivalent of 0.5 into the reaction system, stirring for 30min, then adding diisopropylethylamine with the equivalent of 0.01 and boron trifluoride diethyl etherate with the equivalent of 0.01 respectively under ice bath, and stirring for 3h at room temperature. Washing with saturated sodium bicarbonate solution, extracting with dichloromethane, and performing column chromatography to obtain compound 4;

1H NMR (400 MHz,CDCl₃) δ 8.69 (t, J = 7.4 Hz, 4H), 7.29 (d, J = 5.5 Hz, 2H), 6.88 (dd, J = 8.9, 2.5 Hz, 2H), 6.71 (d, J = 2.2 Hz, 2H), 3.78 (s, 6H), 2.76 (t, J = 6.9 Hz, 4H), 2.43 (t, J = 6.9 Hz, 4H), 1.26 (s, 6H)。¹³C NMR (101 MHz, CDCl₃) δ 160.67, 150.65, 150.48, 145.06, 143.09, 134.65, 133.84, 132.40, 131.53, 130.64, 130.52, 130.40, 124.46, 121.33, 114.47, 114.18, 112.34, 55.30, 30.82, 20.44, 12.71, 12.28.

(5) preparation of BDP-R-ClO

Compound 2 and compound 4 were added in an equivalent of 1:2, refluxing for 48h at 120 ℃, and recrystallizing to obtain the probe BDP-R-ClO.

1H NMR (500 MHz, DMSO-d6) δ 9.48 (s, 2H), 8.63 – 8.49 (m, 4H), 7.67 – 7.56 (m, 2H), 7.29 – 7.12 (m, 2H), 7.07 – 6.92 (m, 4H), 6.02 (s, 2H), 3.85 (s, 6H), 3.32 (s, 4H), 2.84 (s, 4H), 1.30 (s, 6H)。¹³C NMR (126 MHz, DMSO) δ 186.50, 161.27, 154.92, 153.22, 150.72, 146.41, 144.06, 134.79, 132.42, 132.26, 131.43, 131.16, 130.38, 130.29, 130.12, 129.99, 124.23, 120.59, 114.71, 113.13, 63.58, 55.91, 43.34, 40.51, 40.34, 39.68, 39.51, 39.04, 30.33, 20.20, 13.04.

Example 2

The preparation steps of the probe are as follows:

(1) preparation of Compound 1

N₂Under protection, p-hydroxybenzyl alcohol is dissolved in 1, 2-dichloroethane, diisopropylethylamine and N, N-dimethylaminothiocarbonyl chloride are added in an ice-water bath according to an equivalent weight of 1:2, heating at 50 ℃ for reaction for 24 hours, extracting with dichloromethane, and performing column chromatography separation to obtain a compound 1;

(2) preparation of Compound 2

Dissolving the compound 1 prepared in the step (1) in dichloromethane, and reacting phosphorus tribromide in a molar ratio of 1:1, stirring at room temperature for 1-2h, adding water for quenching, extracting with dichloromethane, and performing column chromatography separation to obtain a compound 2;

(3) preparation of Compound 3

N₂Under the protection of ice water bath, adding sodium hydride into a tetrahydrofuran solvent for mixing to obtain a mixed solvent, dissolving acetonyl p-toluenesulfonate oxime ester and 6-methoxy-1-tetralone in the mixed solvent according to the proportion of 1:1 of equivalent weight, reacting for 3 hours at 50 ℃, quenching with ice water, extracting with dichloromethane, and performing column chromatography separation to obtain a compound 3;

(4) preparation of Compound 4

N₂Under protection, compound 3 and 4-pyridylaldehyde are dissolved in anhydrous DCM at an equivalent ratio of 1:0.8, and a drop of trifluoroacetic acid is added to react at 30 ℃ overnight. Then adding tetrachlorobenzoquinone with the equivalent weight of 0.5 into the reaction system, stirring for 30min, then adding diisopropylethylamine and boron trifluoride with the equivalent weight of 0.01 under ice bath, and stirring for 3h at room temperature. Extracting with dichloromethane, and performing column chromatography to obtain compound 4;

(5) preparation of BDP-R-ClO

Compound 2 and compound 4 were added in an equivalent of 1: 3, refluxing for 48h at 120 ℃, and recrystallizing to obtain the probe BDP-R-ClO.

Example 3

The preparation steps of the probe are as follows:

(1) preparation of Compound 1

N₂P-hydroxybenzyl alcohol is dissolved in 1, 2-dichloroethane under protection, and N, N-dimethylaminothioformyl chloride is added in an amount of 1 equivalent: 1.5, adding diisopropylethylamine into an ice water bath, heating to react for 24 hours at 50 ℃, extracting by dichloromethane, and carrying out column chromatography separation to obtain a compound 1;

(2) preparation of Compound 2

Dissolving the compound 1 prepared in the step (1) in dichloromethane, and reacting phosphorus tribromide in a molar ratio of 1:2, adding alkali, stirring at room temperature for 1-2h, adding water for quenching, extracting by dichloromethane, and performing column chromatography separation to obtain a compound 2;

(3) preparation of Compound 3

N₂Under the protection of ice water bath, adding sodium hydride into tetrahydrofuran solvent to obtain mixed solvent, dissolving acetone-based p-toluenesulfonate oxime ester and 6-methoxy-1-tetralone in the mixed solvent according to the equivalent weight of 1:2, reacting for 3h at 50 ℃, and quenching with ice waterExtracting with dichloromethane, and separating by column chromatography to obtain compound 3;

(4) preparation of Compound 4

N₂Under protection, 4-pyridylaldehyde is mixed with a compound 3 according to an equivalent weight of 1:1 in anhydrous DCM, a drop of trifluoroacetic acid was added and the reaction was carried out overnight at 30 ℃. Then adding tetrachlorobenzoquinone with the equivalent of 0.5 into the reaction system, stirring for 30min, then adding diisopropylethylamine with the equivalent of 0.01 under ice bath, adding boron trifluoride diethyl etherate with the equivalent of 0.01 after 10min, and stirring for 3h at room temperature. Washing with saturated sodium bicarbonate solution, extracting with dichloromethane, and performing column chromatography to obtain compound 4;

(5) preparation of BDP-R-ClO

Compound 2 and compound 4 were added in an equivalent of 1: 2.5, refluxing at 120 ℃ for 48h, and recrystallizing to obtain the probe BDP-R-ClO.

Performance testing Using the Probe of example 1

(1) Detection of ClO in solution^‒Reaction time test of fluorescent Probe BDP-R-ClO

Preparing a fluorescent probe stock solution of 1 mM BDP-R-ClO by using dimethyl sulfoxide (DMSO); probe BDP-R-ClO (10 mu M) and ClO^‒(100. mu.M) the reaction was carried out in a solution system of acetonitrile/PBS buffer (1: 1, v/v, 10mM, pH 7.4), as shown in FIG. 3, and the absorption peak of BDP-R-ClO at 561nm was observed from an ultraviolet absorption spectrum after addition of ClO^－Then, the blue shift is reduced and weak blue shift is generated, and then the blue shift is kept unchanged; the absorption peaks before and after the reaction were blue-shifted by 1 nm. Meanwhile, it was observed on the fluorescence spectrum that a fluorescence emission peak was observed at 661nm and increased by about 20-fold under the excitation at 560nm (shown in FIG. 4).

(2) Detection of ClO in solution^‒The concentration titration test and the concentration linear relation of the fluorescent probe BDP-R-ClO

In concentration titration experiments, it was found that ClO was associated with^‒The concentration of (A) is gradually increased, the fluorescence peak at 661nm is also gradually increased, and the fluorescence intensity is in ClO^‒The concentration reaches the maximum when the concentration reaches 100 mu M. (see FIG. 5).

With ClO^‒Concentration is abscissa, probe BDP-R-ClO is 661nAnd (3) taking the fluorescence intensity at the position m as a vertical coordinate, drawing a graph and performing linear fitting to obtain a linear regression equation of the probe, wherein the linear regression equation is as follows: y = 14.295X + 32.247, linear correlation coefficient R²= 0.993 and a detection limit of 1.8nM was calculated. (see FIG. 6).

(3) Interference and anti-interference ion experiments

4mL acetonitrile/PBS buffer (1: 1, v/v, pH 7.4) and 40. mu.L stock solutions of fluorescent probes were added to different fluorescence cuvettes, as shown in FIG. 7, after the probe BDP-R-ClO was added to the selected active nitrogen and active oxide analyte species (100. mu.M) (0: Blank, 1: ClO), respectively^－, 2:ONOO^－, 3:H₂O₂ , 4:·OH^－, 5:·O^tBu, 6:TBHP , 7:O₂·^－ , 8:¹O₂ , 9:NO , 10:NO₂ ^-, 11:Hcy ,12:GSH, 13:Cys, 14:HS^-, 15:SO₄ ^2-, 16:SO₃ ^2-, 17:Ca²⁺, 18:Mg²⁺, 19:Cu²⁺The probe BDP-R-ClO can be used for ClO^－Specific recognition is carried out, obvious red fluorescence appears at 661nm, no fluorescence appears after reaction with other kinds of analytes, therefore, the probe can realize specific response to ClO^‒. When BDP-R-ClO (10. mu.M) was added to the above-mentioned analyte (0: Blank, 1: ONOO)^－, 2:H₂O₂, 3:·OH^－, 4:·O^tBu, 5:TBHP , 6:O₂·^－ , 7:¹O₂ , 8:NO , 9:NO₂ ^-， 10:Hcy ,11:GSH, 12:Cys, 13:HS^-, 14:SO₄ ^2-, 15:SO₃ ^2-, 16:Ca²⁺, 17:Mg²⁺, 18:Cu²⁺) Then adding 100 mu M ClO^‒After 10 minutes of reaction, ClO was observed^‒BDP-R-ClO can still specifically detect ClO in a complex solution system under the condition of coexistence with various analytes^‒. Experiments prove that BDP-R-ClO can respond to ClO^‒Without interference from other substances (see fig. 8).

(4) Test for pH response

Dissolving the probe BDP-R-ClO in dimethyl sulfoxide to obtain 10mM probe mother liquor, preparing solutions with pH values of 5.0, 5.5, 6.0, 6.5, 7.0, 7.5 and 8.0, and aligning the probe, the probe and the ClO^‒The change in fluorescence intensity after the reaction was investigated.

As a result, as shown in FIG. 9, the fluorescence intensity of the probe remained substantially unchanged in the solution having a pH of 5.0 to 8.0; adding ClO^‒Thereafter, the fluorescence intensity was almost constant in the solution of pH 5.0 to 6.0, the fluorescence was gradually increased at 661nm in the solution of pH 6.0 to 8.0, and the fluorescence intensity was still stronger in the physiological range of pH 7.2 to 8.0. Experiments prove that the probe BDP-R-ClO can adapt to the pH environment in organisms.

(5) MTT cytotoxicity assay

MTT cytotoxicity test on HeLa cells using the probe BDP-R-ClO, the results are shown in FIG. 10. After incubation of HeLa cells with medium containing different concentrations of probe (0, 1.25, 2.5, 5, 10, 20. mu.M), the percent survival of the cells was calculated. As shown in FIG. 10, the cell viability reached as high as 90% at low concentrations, and the probe showed almost no cytotoxicity.

(6) Detecting ClO^‒Fluorescent probe pair of (1) to ClO in RAW264.7 cells^‒Detection performance test of

Detecting ClO^‒The fluorescent probe BDP-R-ClO is used for ClO in mouse mononuclear macrophage (RAW 264.7) cells^‒Fluorescence confocal imaging. The results are shown in FIG. 11, (a) blank set; (b) DM-BDP-OCl (10. mu.M) was incubated for 30 min; (c) incubating with lipopolysaccharide (LPS, 1.0 μ g/mL) for 16 h, followed by phorbol-12-myristate-13-acetate (PMA, 1.0 μ g/mL) for 60 min, and finally BDP-R-ClO (10 μ M) for 30 min; (d) pre-incubation with hydrazine 4-aminobenzoate (ABAH, 200. mu.M) for 60 min; (e, f, h, g) are superimposed images of the bright field image and (a, b, c, d), respectively, (lambda)_ex=560 nm，λ_em= 600 and 700 nm). ClO detection by confocal Red channel visualization^‒The fluorescent probe of (a) produces bright red fluorescence in RAW264.7 cells; in the control group experiment, cells are incubated with BDP-R-ClO together, and the red channel fluorescence can be observed according to the imaging experiment resultThe response was weak. Thus indicating that the probe can detect the endogenous ClO of the cells^‒. The result also shows that the fluorescent probe of the invention can detect ClO in RAW264.7 cells^‒Has good application prospect.

(7) Detecting ClO^‒Fluorescent probe of (2) against mouse ClO in vivo^‒Detection performance test of

NaClO (5. eq.) was injected into the right flank of the mouse by intraperitoneal injection into the abdomen of the mouse, and BDP-R-ClO (25. mu.L DMSO, 1 mM) was injected in situ 10min later; physiological saline and BDP-R-ClO were injected into the left abdomen of nude mice as a control group. Performing fluorescence monitoring in imaging system at 0min, 10min, 20min, 30min, and 40min after injection, observing through confocal red channel, and detecting ClO^‒The fluorescent probe generates bright red fluorescence in the abdominal cavity of the mouse added with NaClO; in the control experiment, the weak fluorescence response of the red channel can be observed in the result of the right abdominal imaging experiment only incubated by the physiological saline and the probe. Therefore, the probe can rapidly monitor the endogenous ClO of the mouse in situ^‒Is generated.

The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.