A fluorescent probe for detecting polarity changes, preparation method thereof, and detection kit

Abstract

The present invention provides a fluorescent probe for detecting polarity changes, a preparation method thereof and a detection kit, wherein the fluorescent probe is (Z)-N, N-bis(2-chloroethyl)-4-(2-(5,5-difluoro-1,7,9-trimethyl-10-phenyl-5H-4-14,5-14-dipyrrolo[1,2-c:2',1'-f][1,3,2]diazaborin-3-yl)vinyl)aniline. The fluorescent probe and the detection kit of the present invention have high sensitivity, and the color development reaction occurs only under the condition of the presence of a low-polarity solvent, and other common active oxygen, amino acids, and enzyme species do not cause interference.

Description

Technical Field

[0001] The invention relates to the technical field of bioanalysis, and in particular to a fluorescent probe for detecting polarity changes, a preparation method thereof and a detection kit. Background Art

[0002] Polarity is an important microenvironmental factor in biological systems, because a variety of complex physiological and pathological processes such as protein denaturation, enzyme catalysis, peptide polymerization, membrane fusion and signal transduction are heavily dependent on a balanced microenvironment. Changes in the cell's microenvironment, especially subcellular changes, are associated with most pathologies. Polarity also governs cell apoptosis, migration, differentiation and activation of immune responses. Microenvironmental abnormalities can be found in many diseases, such as polycystic kidney disease and type 2 diabetes. Therefore, detecting changes in polarity in the microenvironment has important research value and significance.

[0003] Literature disclosed in the prior art: The invention patent with application number 2019104270252 discloses a fluorescent probe for detecting polarity of the endoplasmic reticulum and its application, which has good fluorescence emission spectrum characteristics (415-700nm). By drawing a standard curve to measure the polarity of the endoplasmic reticulum of cells, the purpose of rapid and accurate detection of the polarity of the endoplasmic reticulum of normal cells and cancer cells can be achieved. The fluorescent probe has high specificity and is not interfered by other components during the detection of polarity of different solvents. It can be used for real-time determination of endoplasmic reticulum polarity in living cells. The invention patent with application number 2020104888084 discloses a class of dehydroabietic acid triarylamine D-π-A compounds as fluorescent probes. This class of compounds can be used as fluorescent probes to detect the water content in 1 to 3 polar solvents. The fluorescence intensity decreases with the increase of water content. When the water content is within 0-1%, the fluorescence intensity is linearly related to the water content. The invention patent with application number 2021110279919 discloses a polar fluorescent probe, a preparation method and application thereof, and provides a fluorescent probe for detecting polarity by wavelength change. The probe structure contains a DAD structure, and its emission wavelength gradually red-shifts as the polarity of the system increases, and the emission wavelength has a good linear relationship with the polarity range of 0.020-0.287. It solves the problem that the fluorescent probe that detects polarity by changes in fluorescence intensity is easily interfered by probe concentration and environmental factors in practical applications. The invention patent with application number 2022108614951 provides a polarity-responsive small molecule probe based on benzothiadiazole, a preparation method and application thereof. A polarity-responsive small molecule fluorescent probe based on benzothiadiazole as the parent structure is prepared by a simple one-step synthesis method. The morpholine group it has can be located in the lysosome. The probe is not affected by the environmental pH, and shows an excellent polar solvent colorization effect. It has significant photostability, large Stokes shift and bright emission light. The invention patent with application number 2022105278319 discloses a fluorescent probe for targeting lipid droplets to detect polarity, and its preparation method and application. The fluorescent probe has a simple synthesis process, can target lipid droplets, is sensitive to polarity, has low cytotoxicity, and can be imaged in cells and tissues. It is a new type of near-infrared probe.

[0004] After analysis and verification, the fluorescent probes in the existing technology have the following technical defects: the color development time for detecting polarity changes is long, it is easily interfered by inorganic salts, active nitrogen, active oxygen, etc., and is not suitable for detecting changes in the polarity of the cell microenvironment under physiological conditions. Summary of the invention

[0005] In response to the above technical problems, the present invention provides a fluorescent probe for detecting polarity changes, a preparation method thereof, and a detection kit, to achieve the following invention objectives: shortening the color development time for detecting polarity changes, being able to detect changes in the polarity of the cell microenvironment under physiological conditions, and not being interfered by inorganic salts, reactive nitrogen, reactive oxygen, etc.

[0006] In order to achieve the above invention object, the technical solution adopted by the present invention is as follows:

[0007] A fluorescent probe for detecting polarity changes, the fluorescent probe is (Z)-N, N-bis(2-chloroethyl)-4-(2-(5,5-difluoro-1,7,9-trimethyl-10-phenyl-5H-4l4,5l4-dipyrrolo[1,2-c:2',1'-f][1,3,2]diazaborin-3-yl)vinyl)aniline, and its structural formula is as follows:

[0008] .

[0009] A method for preparing a fluorescent probe for detecting polarity changes comprises the following steps:

[0010] Step 1: Condensation reaction

[0011] At room temperature, 2,4-dimethylpyrrole and benzaldehyde are added to tetrahydrofuran and mixed to carry out condensation reaction. The reaction time is 12 hours and the catalyst is trifluoroacetic acid.

[0012] Step 2: Oxidation reaction

[0013] 2,3-Dichloro-5,6-dicyanobenzoquinone was added to the solution after the condensation reaction for oxidation, and the reaction time was 4 hours.

[0014] Step 3: Coordination reaction

[0015] Triethylamine was added to the solution after the oxidation reaction; then, boron trifluoride ether was added in an ice bath state, and the solution was stirred for 4 hours at room temperature to carry out coordination reaction.

[0016] Step 4: Separation

[0017] After the coordination reaction is completed, the reaction solution is filtered and the filtrate is retained. The filtrate is poured into a mixed solution of water and ethyl acetate for extraction, and the extraction is repeated three times to extract the organic layer; the organic layer is decompressed to remove volatiles through a rotary evaporator, and the temperature of the low-temperature cooling circulation pump is controlled to be -2.5°C, and the temperature of the rotary evaporator is controlled to be 56°C. When no volatiles are evaporated, the evaporation is stopped to obtain a crude product A.

[0018] The obtained crude product A was separated and purified by silica gel column chromatography, and eluted with a mixed solution of petroleum ether and ethyl acetate to obtain orange-yellow crystals of boron dipyrrole fluoride.

[0019] Step 5: Obtain the final product

[0020] Dissolve fluoroboron dipyrrole, 4-[bis(β-chloroethyl)amino]benzaldehyde and catalyst in 15 mL organic solvent, heat to 120°C under inert gas protection for reaction, and the reaction time is 4 hours. Remove the solvent by rotary evaporation under reduced pressure to obtain crude product B. The crude product B is separated and purified by silica gel column chromatography, eluted with a mixed solution of petroleum ether and ethyl acetate to obtain red crystals, which are the final product. The catalyst is at least one of an organic base and an inorganic base; the organic base is at least one of triethylamine and pyridine; the inorganic base is at least one of potassium carbonate, sodium acetate, sodium carbonate, sodium bicarbonate and magnesium perchlorate. The molar ratio of fluoroboron dipyrrole, 4-[bis(β-chloroethyl)amino]benzaldehyde and catalyst is 1:4:0.5-4, preferably 1:4:1-4, more preferably 1:4:4. The organic solvent is at least one of acetic anhydride, ethanol, acetonitrile, toluene and glacial acetic acid.

[0021] The present invention also provides a detection kit for detecting polarity changes, comprising a fluorescent probe for detecting polarity changes; a buffer solution, which is a phosphate buffer solution with a pH value of 6.0 to 8.0 and a phosphate concentration of 0.01 to 0.5 M; and a solvent, which is dimethyl sulfoxide.

[0022] The application of the polarity detection kit provided by the present invention in measuring polarity changes, especially in detecting polarity changes in cells in biological systems and zebrafish, all fall within the protection scope of the present invention.

[0023] The present invention has the following beneficial effects:

[0024] 1) The fluorescent probe of the present invention is blue and has weak fluorescence; as the polarity decreases, it can show light purple and emit strong red fluorescence.

[0025] 2) The fluorescent probe and detection kit of the present invention have a fast reaction speed during the detection process and can develop color within 20 minutes.

[0026] 3) The color development reaction of the fluorescent probe and detection kit of the present invention only occurs in the presence of a low-polarity solvent during the detection process, and other common inorganic salts, active nitrogen, active oxygen, amino acids, vitamins, and biological thiol species do not interfere.

[0027] 4) The present invention has a long fluorescence emission wavelength (678 nm), which can be detected by fluorescence spectroscopy and can be used to detect changes in the polarity of the cell microenvironment under physiological conditions.

[0028] Based on the intramolecular charge transfer mechanism of the fluorescent probe itself, the present invention uses 4-[bis(β-chloroethyl)amino]benzene as the electron donor part (D), the double bond is a π structure, and boron dipyrrole fluoride is an electron acceptor (A), and establishes a "D-π-A" structure with ICT effect. It is a near-infrared fluorescent probe with ICT effect. The fluorescence intensity of the probe itself is very weak. After the polarity of the environment is reduced, the fluorescence of the solution is significantly enhanced and the color changes from blue to lavender, indicating that the method can be used to detect polarity changes. In order to verify the practicality of the method and apply it to the quantitative determination of polarity changes in organisms, a polarity detection kit is prepared by applying this principle. The present invention has the advantages of simple operation, low cost, rapidity, high efficiency and sensitivity, and is easy to promote and apply. The kit is a polarity defense detection device with excellent performance and convenient use, which will become a powerful research tool for related fields such as modern biology, physiology and medicine. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] 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 some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative labor.

[0030] Figure 1 is the chemical structural formula of a fluorescent probe for detecting polarity changes;

[0031] Figure 2 It is the chemical structural formula of 2,4 dimethylpyrrole;

[0032] Figure 3 is the chemical structural formula of boron dipyrrole fluoride;

[0033] Figure 4 A chemical reaction equation for preparing a fluorescent probe for detecting polarity changes according to the present invention;

[0034] Figure 5 In the figure, Figure A is the fluorescence spectrum of the fluorescent probe of the present invention reacting in a water and 1,4-dioxane system; Figure B is a photograph of the change of the fluorescent probe in a mixed solution of 1,4-dioxane and water in different proportions (0%-100%) under visible light and ultraviolet light;

[0035] Figure 6 Figure A shows the fluorescence spectra of the fluorescent probe in solvents of different polarities; Figure B shows the maximum emission wavelength of the fluorescent probe and E T Correlation curve of (30);

[0036] Figure 7 The fluorescence emission spectra of the fluorescent probes that selectively react with different biomarkers;

[0037] Figure 8 Figure A is a fluorescence image of H9c2 cells treated with fluorescent probes and different concentrations of lipopolysaccharide by confocal imaging; Figure B is a relative fluorescence intensity analysis of different channels of the fluorescence image (Figure A). Figure C is a flow cytometry analysis of H9c2 cells treated with fluorescent probes and different concentrations of lipopolysaccharide;

[0038] Fig. 9 The middle is the result of the polarity detection kit for detecting lipid droplets;

[0039] Fig.10 Results of polarity detection kit to detect polarity changes in zebrafish;

[0040] Fig.11 The polarity detection kit was used to detect the polarity changes in mice with septic cardiomyopathy;

[0041] Fig.12 The hydrogen spectrum (400 MHz, CDCl3, 298 K) of the final product prepared in Example 1;

[0042] Fig.13 Carbon spectrum (100 MHz, CDCl3, 298 K) of the final product prepared in Example 1;

[0043] Fig.14 This is a high-resolution mass spectrum of the final product prepared in Example 1. DETAILED DESCRIPTION

[0044] The present invention is described below by means of specific embodiments, but the present invention is not limited thereto.

[0045] The experimental methods described in the following examples are conventional methods unless otherwise specified; the reagents and biological materials described are commercially available unless otherwise specified.

[0046] Example 1 A fluorescent probe for detecting polarity changes

[0047] A fluorescent probe for detecting polarity changes is (Z)-N,N-bis(2-chloroethyl)-4-(2-(5,5-difluoro-1,7,9-trimethyl-10-phenyl-5H-4l4,5l4-dipyrrolo[1,2-c:2',1'-f][1,3,2]diazaborin-3-yl)vinyl)aniline, the structural formula of which is shown in the attached figure. Figure 1 shown.

[0048] A method for preparing a fluorescent probe for detecting polarity changes is as follows:

[0049] According to the attached Figure 4 The preparation is carried out according to the chemical reaction flow chart shown in the figure, and the operation steps are as follows:

[0050] Step 1: Condensation reaction

[0051] At room temperature, 2,4-dimethylpyrrole and benzaldehyde were first added to 15 mL of tetrahydrofuran and mixed; then 120 μL of trifluoroacetic acid was added, and the condensation reaction was carried out under inert gas protection conditions with stirring for 12 hours. The amount of 2,4-dimethylpyrrole added was 498 μL, i.e. 4.8 mmol; the amount of benzaldehyde added was 254 μL, i.e. 2.4 mmol.

[0052] The chemical structure of 2,4-dimethylpyrrole is as shown in the attached Figure 2 shown.

[0053] Step 2: Oxidation reaction

[0054] 540 mg (2.4 mmol) of 2,3-dichloro-5,6-dicyanobenzoquinone was added to the solution after the condensation reaction, and the mixture was stirred for 4 hours at room temperature for oxidation reaction.

[0055] Step 3: Coordination reaction

[0056] To the solution after the oxidation reaction, 6 ml of triethylamine was added; then, in an ice bath, 6 ml of boron trifluoride ether was added, and the solution was stirred for 4 hours at room temperature to carry out coordination reaction.

[0057] Step 4: Separation

[0058] After the coordination reaction is completed, the solution after the reaction is filtered to filter out the sediment in the reaction process, and the filtrate is retained. The filtrate is poured into a mixed solution of water and ethyl acetate for extraction, and the extraction is repeated three times to extract the organic layer; the organic layer is decompressed to remove volatiles through a rotary evaporator, and the temperature of the low-temperature cooling circulation pump is controlled to be -2.5°C, and the temperature of the rotary evaporator is controlled to be 56°C. When no volatiles are evaporated, the evaporation is stopped to obtain a crude product A.

[0059] The mixed solution of water and ethyl acetate: the volume ratio of ethyl acetate to water is 1:1.

[0060] The obtained crude product A was separated and purified by silica gel column chromatography, and eluted with a mixture of petroleum ether and ethyl acetate to obtain orange-yellow crystals of boron dipyrrole fluoride. The chemical formula is shown in the attached Figure 3 The volume ratio of petroleum ether to ethyl acetate is 70:1.

[0061] Step 5: Obtain the final product

[0062] Dissolve boron dipyrrole fluoride, 4-[bis(β-chloroethyl)amino]benzaldehyde, glacial acetic acid, piperidine and magnesium perchlorate in 15 mL of ultra-dry toluene, wherein the amount of boron dipyrrole fluoride is 32.4 mg, i.e., 0.1 mmol; the amount of 4-[bis(β-chloroethyl)amino]benzaldehyde is 98.5 mg, i.e., 0.4 mmol; the amounts of glacial acetic acid and piperidine are both 0.6 mL; the amount of magnesium perchlorate is 5 mg; under the protection of inert gas, heat to 120°C for reaction, connect the reactor to a water separator, reflux and stir continuously during the reaction until the reaction is complete, and the reaction time is 4 hours.

[0063] The reactant was extracted with a mixed solution of water and ethyl acetate to extract an organic layer. The mixed solution of water and ethyl acetate had a volume ratio of ethyl acetate to water of 1:1.

[0064] The solvent was removed by rotary evaporation under reduced pressure, the circulating pump temperature was low-temperature cooled to -2.5°C, the rotary evaporator temperature was 56°C, and evaporation was stopped when no volatiles were evaporated to obtain a crude product B. The crude product B was separated and purified by silica gel column chromatography and eluted with a mixture of petroleum ether and ethyl acetate to obtain red crystals, which were the final product. The volume ratio of petroleum ether to ethyl acetate was 13:1.

[0065] The structural characterization data of the final product are as follows:

[0066] 1 H NMR (400 MHz, CDCl3, 298 K), δ (ppm): 7.51 (m, 6H), 7.30 (d, J = 6.7Hz, 2H), 7.18 (d, J = 16.2 Hz, 1H), 6.68 (d, J = 8.1 Hz, 2H), 6.59 (s, 1H), 5.98(s, 1H), 3.79 (t, J = 6.7 Hz, 4H), 3.66 (t, J = 6.7 Hz, 4H), 2.59 (s, 3H), 1.42 (s, 6H).

[0067] 13 C NMR (100 MHz, CDCl3, 298 K), δ(ppm): 129.52, 129.05, 128.86,128.32, 117.57, 111.92, 53.42, 40.34, 31.94, 29.72, 29.68, 29.63, 29.38,29.34, 26.92, 22.71, 14.65, 14.30, 14.14.

[0068] ESI HRMS (m / z): calculated for C 30 H 30 BCl2F2N3. [M+H] + 552.1951, found552.1961.

[0069] The hydrogen spectrum (400 MHz, CDCl3, 298 K) of the final product is shown in the attached figure. Fig.12 As shown;

[0070] The carbon spectrum of the final product (100 MHz, CDCl3, 298 K) is shown in the attached figure. Fig.13 As shown;

[0071] The high-resolution mass spectrum of the final product is shown in the attached figure. Fig.14 shown.

[0072] Combined with the structural characterization data of the final product and the Fig.12 , attached Fig.13 , attached Fig.14 It can be seen that the final product is: (Z)-N,N-bis(2-chloroethyl)-4-(2-(5,5-difluoro-1,7,9-trimethyl-10-phenyl-5H-4l4,5l4-dipyrrolo[1,2-c:2',1'-f][1,3,2]diazaborin-3-yl)vinyl)aniline.

[0073] Example 2: Spectral properties of the fluorescent probe prepared in Example 1 reacting with reagents of different polarity

[0074] (1) Weigh 7 mg of the fluorescent probe prepared in Example 1 and add 9.1 mL of dimethyl sulfoxide to prepare a probe stock solution with a final concentration of 1 mM.

[0075] 10 mL of probe mother solution was added dropwise to different volumes of 1,4-dioxane (1,4-Dioxane) low-polarity solvent, and then diluted to 1 mL with water as the reaction system; the volume proportion of 1,4-dioxane was 0%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% and 100% of the total volume of the reaction system, respectively. After reacting at 37 °C for 30 min, its UV-visible absorption spectrum and fluorescence emission spectrum were measured. When measuring the fluorescence emission spectrum, the excitation was 590 nm; the slit width of excitation and emission was 5 nm; the voltage was 700 V. The fluorescence spectra of the fluorescent probe reacting in different proportions of water and 1,4-dioxane systems are shown in the attached figure. Figure 5 As shown in Figure A. The changes of the fluorescent probe under visible light and ultraviolet light in mixed solutions of 1,4-dioxane and water at different ratios (0%-100%) are shown in the attached figure. Figure 5 As shown in Figure B, the upper row of photos in the figure are photos of fluorescent probes in mixed solutions with different 1,4-dioxane contents under visible light. From left to right, the content of 1,4-dioxane is 0%-100%, and the polarity of the solution decreases (Polarity decrease). The lower row of photos, from left to right, are photos of fluorescent probes in mixed solutions with 1,4-dioxane contents from low to high under ultraviolet light. From left to right, the content of 1,4-dioxane is 0%-100%, and the polarity of the solution decreases (Polarity decrease).

[0076] (2) Take 10 μL of fluorescent probe and add it to the following solvents: tetrahydrofuran (THF), toluene, dichloromethane, 1,4-dioxane, acetone, acetonitrile, and dimethyl sulfoxide (DMSO), and prepare the solution into a 1 mL reaction system. After reacting at 37 °C for 30 min, measure its fluorescence emission spectrum. When measuring the fluorescence emission spectrum, de-excitation is performed at 590 nm; the slit width of excitation and emission is 5 nm; the voltage is 700 V. The normalized fluorescence spectra of the fluorescent probe in solvents of different polarities are shown in the attached figure. Figure 6 As shown in Figure A; the maximum emission wavelength of the fluorescent probe and the solvent polarity parameter E T The linear regression curve of (30) is shown in the attached figure. Figure 6 As shown in Figure B.

[0077] According to the attached Figure 5 , 6 It can be seen that the fluorescent probe of the present invention has the following characteristics:

[0078] 1) The fluorescent probe of the present invention is blue in color and has no fluorescence in pure water, but with the addition of 1,4-dioxane solvent, the fluorescence intensity gradually increases, and with the increase of the volume of 1,4-dioxane, its emission wavelength obviously blue-shifts.

[0079] 2) The maximum emission peak of the fluorescent probe of the present invention shows obvious differences in solvents of different polarities, and the wavelength corresponding to the maximum emission peak shows an obvious red-shift trend as the polarity increases.

[0080] 3) The wavelength and polarity parameters corresponding to the maximum emission peak of the fluorescent probe of the present invention E T (30) show a linear relationship between them.

[0081] Example 3 Reaction experiment of fluorescent probe and other substances

[0082] Add the following substances (biomarkers) to PBS buffer to prepare the detection solution:

[0083] (1) blank control group; (2) Ag + (Ag in the test fluid + content is 100 μM); (3) Fe 2+ (Fe in the test solution 2+ content is 100 μM); (4) Fe 3+ (Fe in the test solution 3+ content is 100 μM); (5) Cr 3+ (Cr in the test fluid 3+ content is 100 μM); (6) Hg + (Hg in the test fluid + content is 100 μM); (7) Ca 2+ (Ca in the test solution 2+ The content is 10 μM); (8) K + (K in the test fluid + Content is 10 μM); (9) Cu 2+ (Cu in the test solution 2+ content is 10 μM);(10) Pb 2+ (Pb in the test fluid 2+ content is 10 μM); (11) Mg 2+ (Mg in the test solution 2+ The content is 10 μM); (12) Bi 3 + (Bi in the test solution 3+ The content is 10 μM); (13) Ni 3+ (Ni in the test fluid3+ The content is 10 μM); (14) S 2- (S in the test fluid 2- The content is 10 μM); (15) HSO3 - (HSO3 in the test solution - (100 μM); (16) AACT (AACT content in the test solution is 100 μg / mL); (17) HSA (HSA content in the test solution is 0.1 g / L); (18) APP (APP content in the test solution is 0.9 μg / mL); (19) NTR (NTR content in the test solution is 500 μg / mL); (20) β-Gal (β-Gal content in the test solution is 4 U / mL); (21) BSA (BSA content in the test solution is 0.1 g / L); (22) CES (CES content in the test solution is 0.5 U / mL); (23) ALDH (ALDH content in the test solution is 10 μM); (24) H2O2 (H2O2 content in the test solution is 15 μM); (25) 1 O2(in the test fluid 1 O2 content is 10 μM); (26) HClO (HClO content in the test solution is 10 μM); (27) ·OH (·OH content in the test solution is 10 μM); (28) O2 - (O2 in the test fluid - content is 10 μM); (29) NO2 - (NO2 in the test fluid - The content of Cys in the test solution is 10 μM); (30) Cys (the content of Cys in the test solution is 100 μM); (31) Asp (the content of Asp in the test solution is 100 μM); (32) 1,4-Dioxane (the volume percentage of 1,4-Dioxane in the test solution is 99%).

[0084] Take 1 mL of detection solution (1)-(32) respectively, and add 10 mL of probe mother solution with a concentration of 1 mM (the preparation method of the probe mother solution is the same as that of Example 2). After reacting at 37°C for 30 min, measure its fluorescence emission spectrum. When measuring the fluorescence emission spectrum, the excitation is performed at 590 nm; the slit width of the excitation and emission is 5 nm; the voltage is 700 V. The fluorescence emission spectra obtained when the fluorescent probe (10 mM) is mixed with different substances are shown in the attached figure. Figure 7 shown.

[0085] The experimental results show that only low-polarity solvents can cause the fluorescent probe of the present invention to produce an obvious light signal response, proving that the fluorescent probe of the present invention has a high degree of selectivity for polarity, but has no obvious selectivity for other substances.

[0086] Example 4 A detection kit for detecting polarity changes

[0087] A detection kit for detecting polarity changes (referred to as: polarity detection kit) is composed as follows:

[0088] 1. A fluorescent probe with a concentration of 1 mM (the preparation method is shown in Example 1).

[0089] 2. Solvent: dimethyl sulfoxide.

[0090] 3. Buffer: PBS buffer with a pH value of 7.4 and a concentration of 12 mmol / L. It is prepared by mixing Na2HPO4 and NaH2PO4 solutions of the same molar concentration.

[0091] Example 5 Detection kit for detecting polarity changes quantitatively determines changes in microenvironment polarity in a septic cardiomyopathy cell model.

[0092] 1) Cell culture:

[0093] H9c2 cells were grown in glass-bottom culture dishes (Corning Inc.) in DMEM (Dulbecco's modified eagle media) containing 10% (volume percentage) fetal bovine serum, 100 U / mL penicillin, and 100 mg / mL streptomycin. The ambient temperature was 37°C and the carbon dioxide concentration was controlled at 5% (volume percentage).

[0094] 2) H9c2 cells were inoculated into 4 groups of confocal culture dishes. After the cells were completely attached to the wall, the culture medium was discarded, and the cells were washed 3 times with serum-free culture medium and incubated for 30 min. The 4 groups of cells were then divided into a blank group and experimental groups 1-3.

[0095] The blank group continued to be cultured with culture medium. Experimental group 1: incubated with 1 μg / mL lipopolysaccharide. Experimental group 2: incubated with 6 μg / mL lipopolysaccharide. Experimental group 3: incubated with 10 μg / mL lipopolysaccharide.

[0096] After incubation for 12 h, the cells in the blank group and experimental groups 1-3 were washed three times with PBS buffer and then incubated with the polarity detection kit (fluorescent probe concentration 5 μmol / L) for 30 min. The culture medium was discarded, the cells were washed with PBS buffer, and finally 1 mL of PBS buffer was added for confocal imaging. The confocal excitation wavelength was 561 nm and the emission wavelength was 600-700 nm. The obtained fluorescence images are shown in the attached figure. Figure 8 The relative fluorescence intensity of different channels of the non-fluorescent image is shown in Figure 2A. Figure 8 B shows the flow cytometry analysis of H9c2 cells treated with fluorescent probes and different concentrations of lipopolysaccharide. Figure 8 As shown in C.

[0097] 3) Kit localizes and detects cell lipid droplets

[0098] The cells were treated as follows: the adherent A549 cells were washed three times with DMEM medium containing fetal bovine serum, and then 1 μM BODIPY 493 / 503 lipophilic fluorescent dye and 5 μM fluorescent probe were added and incubated at 37 °C for 30 minutes. After the incubation, the cells were washed three times with PBS buffer (pH 7.4) and then subjected to confocal fluorescence imaging experiments. The fluorescent images of A549 cells labeled with fluorescent probe and BODIPY 493 / 503 confocal fluorescence imaging are shown in the attached figure. Fig. 9 As shown, Figure a is the red channel image of the fluorescent probe (5μM), and Figure b is the green channel image of BODIPY 493 / 503 (2μM). Figure c is the merged image of Figure a and Figure b. Figure d is the bright channel image. Figure e is the merged image of Figure c and Figure d. Figure f is the intensity curve of ROI.

[0099] Depend on Figure 8 It can be seen that with the increase of lipopolysaccharide concentration, the fluorescence intensity in the cells increased significantly, indicating that there was a phenomenon of decreased polarity in the septic cardiomyopathy cell model.

[0100] Depend on Fig. 9 It can be seen that the polarity detection kit of the present invention can locate and detect the changes in polarity in cell lipid droplets.

[0101] Example 6: Experiment on quantitative detection of changes in microenvironment polarity under zebrafish inflammation and hypoxia models using a polarity detection kit

[0102] 1) Zebrafish model establishment and imaging:

[0103] After adaptive feeding of zebrafish for 1 day, 7-day-old zebrafish were transferred to 6-well plates, three per well, and divided into three groups: group a (control group), group b (experimental group 1), and group c (experimental group 2).

[0104] Group a: Zebrafish were raised normally without any treatment.

[0105] Group b: 4 mL of 1 μg / mL lipopolysaccharide solution was added to each well as a hypoxia zebrafish model.

[0106] Group c: 4 mL of 700 μmol / L cobalt chloride solution was added to each well as an inflammation zebrafish model.

[0107] Then, the three groups of zebrafish were cultured at the same time for 12 hours; then, fluorescent probes were added to the three groups of zebrafish respectively, and the concentration of the probes was controlled at 5μmol / L. After incubation for 120 minutes, the zebrafish were transferred to the confocal culture dish and washed three times with zebrafish culture medium to remove background fluorescence. The liquid around the zebrafish was then aspirated for confocal imaging. The red channel was selected as the channel, and the excitation wavelength of the red channel was selected as 561nm and the emission wavelength was 600-700nm. The confocal imaging results of the three groups of zebrafish at 7d a, b, and c are shown in the attached figure. Fig.10 As shown in Figure A. Fig.10 The normalized fluorescence intensity image obtained in Figure A is shown in the attached figure. Fig.10 As shown in Figure B.

[0108] Depend on Fig.10 It can be seen that compared with the normal zebrafish group, the fluorescence intensity in the zebrafish model of inflammation and hypoxia is significantly enhanced, indicating that the polarity of zebrafish is reduced under inflammation and hypoxia conditions.

[0109] Example 6: Using a polarity detection kit to quantitatively detect changes in the polarity of the microenvironment of a septic cardiomyopathy mouse model.

[0110] 1) Establishment of mouse model of septic cardiomyopathy:

[0111] Twelve 7-week-old C57BL / 6J mice (18 ± 2 g) were divided into four groups, three mice in each group. One group was the control group, one was the sham operation group, and two were the experimental groups (experimental group 1 and experimental group 2). The mouse model of septic cardiomyopathy was established by cecal ligation and puncture and intraperitoneal injection of lipopolysaccharide, respectively.

[0112] The control group did not receive any treatment and the mice were fed normally.

[0113] The sham-operated group was injected intraperitoneally with normal saline. After the injection, the mice were closely monitored for any signs of pain, infection, or complications, and their diet and water intake were maintained normally without intervention.

[0114] Experimental group 1: A mouse model of septic cardiomyopathy was established by cecal ligation and puncture. Mice in the cecal ligation and puncture group were anesthetized with sodium pentobarbital and underwent midline dissection. Under sterile conditions, the cecum was gently exposed, and the area below the ileocecal valve was ligated to maintain blood supply and valve function. The ligated cecum was then punctured twice with a 20-gauge needle to ensure minimal tissue damage. A small amount of fecal material was carefully squeezed out from the puncture site to determine that bacteria had infiltrated the peritoneal cavity. The cecum was then repositioned to the peritoneal cavity. The abdominal incision was sutured in two layers with sterile sutures to ensure wound healing. After surgery, mice received subcutaneous fluid resuscitation to maintain hydration and promote recovery. Warm saline 0.9% was injected subcutaneously on both sides with an injection volume of 1 mL.

[0115] Experimental group 2: The mouse model of septic cardiomyopathy was established by intraperitoneal injection of lipopolysaccharide. Mice in this group received intraperitoneal injection of lipopolysaccharide to induce sepsis. The specified dose of endotoxin lipopolysaccharide (5 mg / kg) was dissolved in sterile saline and aspirated into a sterile syringe to exclude bubbles, and the lower abdomen of the mouse was wiped with alcohol before injection to prevent contamination. The needle was then smoothly inserted into the lower abdomen, taking care not to pierce too deep or damage internal organs. The lipopolysaccharide solution was then gradually injected into the peritoneal cavity. After the injection was completed, the needle was gently withdrawn to prevent internal injuries and bleeding, and the injection site was cleaned again with alcohol. The mice were observed for signs of leakage at the injection site and immediate adverse reactions or discomfort.

[0116] 2) Imaging of the mouse model of septic cardiomyopathy:

[0117] 24 hours after the mouse model was established, the septic cardiomyopathy mice and healthy mice were killed and their hearts were removed. The polarity detection kit of the present invention was used to detect changes in the polarity of the hearts of septic cardiomyopathy mice. After the removed mouse hearts were rinsed with water, the hearts of the four groups of mice were immersed in a PBS solution containing 20 μM fluorescent probe for 1 hour, and then an organ fluorescence imaging experiment was performed using an in vivo PerkinElmer IVIS Spectrum to obtain fluorescence imaging of the hearts of the four groups of model mice. The results of detecting changes in the polarity of the hearts of septic cardiomyopathy mice using the polarity detection kit of the present invention are shown in the attached figure. Fig.11 As shown in A, the fluorescence images of the control group (Control), saline injection group (Sham), cecal ligation and puncture group (CLP), and lipopolysaccharide intraperitoneal injection group (LPS) show that the fluorescence intensity of the heart of the septic cardiomyopathy model mice was significantly enhanced. Fig.11 A obtained normalized fluorescence intensity image, as shown in the attached figure Fig.11 As shown in B. The fluorescence intensity of the heart of the septic cardiomyopathy model mouse was significantly enhanced, indicating that the polarity of the heart was reduced during the septic cardiomyopathy disease process.

[0118] Finally, it should be noted that the above embodiments only cite the use of the fluorescent probe of the present invention to detect polarity changes in septic cardiomyopathy cells and mouse models, as well as polarity changes in a living zebrafish model. Polarity detection of other biological models and biological environments is not listed one by one, but it is not intended to limit the present invention. Any person skilled in the art should be able to make various modifications and changes without departing from the spirit and scope of the present invention.

Claims

1. A fluorescent probe for detecting polarity changes, characterized in that: The structural formula of the fluorescent probe is as follows: 。 2. The method for preparing a fluorescent probe for detecting polarity changes according to claim 1, characterized in that: The following steps are involved: Condensation reaction, oxidation reaction, coordination reaction, separation, and obtaining the final product; the condensation reaction: 2,4-dimethylpyrrole and benzaldehyde are added to tetrahydrofuran and mixed to carry out condensation reaction, and the reaction is carried out under the protection of inert gas, and the reaction time is 12 to 14 hours; The oxidation reaction: adding 2,3-dichloro-5,6-dicyanobenzoquinone to the solution after the condensation reaction for oxidation; The coordination reaction: adding boron trifluoride ether under alkaline conditions in an ice bath, the reaction time is 4 to 6 hours; the separation: filtering the solution after the reaction, extracting, evaporating, separating and purifying the filtrate to obtain boron fluoride dipyrrole; The final product is obtained by mixing boron dipyrrole fluoride and 4-[bis(β-chloroethyl)amino]benzaldehyde in the presence of a catalyst under the protection of an inert gas for 4 to 6 hours to obtain the final product; The catalyst is at least one of an organic base and an inorganic base.

3. The method for preparing a fluorescent probe for detecting polarity changes according to claim 2, characterized in that: The organic base is at least one of triethylamine and pyridine; the inorganic base is at least one of potassium carbonate, sodium acetate, sodium carbonate, sodium bicarbonate and magnesium perchlorate.

4. The method for preparing a fluorescent probe for detecting polarity changes according to claim 2, characterized in that: The molar ratio of boron fluoride dipyrrole, 4-[bis(β-chloroethyl)amino]benzaldehyde and catalyst is 1:4:0.5-4.

5. A detection kit for detecting polarity changes, characterized in that: It includes a fluorescent probe for detecting polarity changes as described in claim 1.

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