Fluorescent probe applied to hypochlorous acid detection of allergic diseases and preparation method of fluorescent probe

Abstract

The invention relates to a fluorescent probe applied to hypochlorous acid detection of allergic diseases as well as a preparation method and application of the fluorescent probe, and belongs to the field of fluorescent probes. The preparation method of the probe comprises the following steps: mixing 1, 3-indandione and 6-(dimethylamino)-naphthaldehyde with an organic solvent, carrying out a condensation reaction under the catalysis of a basic catalyst and under the heating reflux condition of 60-100 DEG C, and treating after the reaction is finished to obtain the fluorescent probe with the structure shown in the formula (I). The fluorescent probe prepared by the invention is high in specificity, strong in anti-interference capability, good in selectivity to hypochlorite ions, good in cell membrane penetrability and biocompatibility, capable of entering living cells in a lossless manner and relatively low in toxicity; the hypochlorous acid concentration (chemical signal) in inflammatory cells can be detected, the viscosity change (physical microenvironment) in the inflammatory cells can also be detected, the dual-function effect is achieved, and a powerful tool is provided for studying the pathological process of allergic diseases.

Description

Technical Field

[0001] This invention belongs to the field of fluorescent probes, specifically relating to a fluorescent probe for detecting hypochlorous acid in allergic diseases and its preparation method. Background Technology

[0002] Allergic diseases are mainly IgE-mediated type I hypersensitivity reactions or allergic reactions, including allergic rhinitis, allergic bronchial asthma (referred to as "asthma"), atopic dermatitis (AD), etc. They have become a major global public health problem, affecting the quality of life and health of hundreds of millions of people. They have been listed as one of the six most common chronic diseases and have become a key area for research and prevention.

[0003] These diseases are essentially an excessive immune response of the body's immune system to harmless antigens (allergens). Despite their undeniable clinical importance, gaps remain in our understanding of the key molecular events driving their chronicity and tissue damage, which limits the development of more effective diagnostic and treatment strategies.

[0004] In recent years, scientific research has gradually revealed that in allergic inflammation, activated immune cells (such as eosinophils and mast cells) produce large amounts of highly reactive hypochlorous acid (HClO) through the myeloperoxidase (MPO) pathway. Hypochlorous acid is not only a weapon against pathogens, but it has also been found to play a key pathogenic role in the allergic environment: it can oxidize and modify its own proteins to form new antigenic epitopes, thereby maintaining and amplifying the immune response; it directly damages the epithelial barrier function, increasing the permeability of allergens; and it leads to tissue dysfunction and chronic inflammation by inducing oxidative stress. Therefore, changes in intracellular hypochlorous acid are considered a key indicator for measuring the severity and activity of allergic diseases.

[0005] Despite the increasing demand for detecting intracellular hypochlorous acid, existing technologies have significant limitations. For example, traditional biochemical methods such as enzyme-linked immunosorbent assay (ELISA) or chromatography typically require cell lysis, which cannot achieve real-time monitoring of live cells in situ, thus damaging cell integrity and losing the temporal and spatial dynamic information of hypochlorous acid production. Existing fluorescent probes exhibit the following characteristics: (1) poor specificity: many commercially available reactive oxygen species (ROS) probes (such as DCFH-DA) respond to multiple ROS, failing to specifically distinguish hypochlorous acid from other ROS, leading to inaccurate detection results and low signal-to-noise ratio; (2) weak anti-interference ability: high concentrations of reducing substances (such as glutathione) within cells can quench probe signals or compete with hypochlorous acid, resulting in decreased sensitivity and false negatives; (3) limited functionality: existing probes are typically designed only for detecting hypochlorous acid. However, in allergic reactions, physical microenvironment parameters such as cell viscosity also change significantly. Currently, there is a lack of tools that can simultaneously provide multiple complementary information to comprehensively assess the stress state of cells. (4) Biocompatibility and localization issues: Some probes may have problems such as high cytotoxicity, difficulty in penetrating cell membranes, or inability to target specific organelles (such as mitochondria, the main site of oxidative stress).

[0006] In summary, existing technologies lack an ideal tool that can specifically and sensitively detect hypochlorous acid associated with allergic diseases in living cells and complex biological systems, and may also have multi-parameter detection capabilities. Summary of the Invention

[0007] In order to overcome the shortcomings and deficiencies of the prior art, the present invention aims to provide a hypochlorous acid fluorescent probe with good specificity, high sensitivity and good biocompatibility.

[0008] To solve the above-mentioned technical problems, the technical solution adopted by the present invention is as follows: A first aspect of the present invention is to provide a fluorescent probe for the detection of hypochlorous acid in allergic diseases, the fluorescent probe having the molecular structure shown in formula (I): .

[0009] A second aspect of the present invention provides a method for preparing a fluorescent probe for the detection of hypochlorous acid in allergic diseases, comprising the following steps:

[0010] 1,3-indanedione and 6-(dimethylamino)-naphthal were mixed with an organic solvent and subjected to a condensation reaction under reflux at 60–100 °C catalyzed by an alkaline catalyst. After the reaction was completed, the fluorescent probe of the structure of formula (I) was obtained by post-treatment.

[0011] Preferably, the fluorescent probe is obtained by column chromatography separation and purification, wherein the mobile phase of column chromatography separation is dichloromethane-methanol with a volume ratio of (10-30):1.

[0012] A third aspect of the present invention is to provide a fluorescent probe for the detection of hypochlorous acid in allergic diseases, and its application in the research, diagnosis and drug screening of allergic diseases.

[0013] Preferably, the fluorescent probe is used in detecting hypochlorous acid content in allergic diseases.

[0014] Preferably, the allergic disease is a type I allergic disease.

[0015] Compared with the prior art, the technical solution of the present invention has the following advantages: The fluorescent probe prepared by this invention has high specificity and strong anti-interference ability. It can specifically recognize hypochlorite ions even in complex cellular environments and has good selectivity for hypochlorite ions. The probe has high sensitivity and low detection limit. The fluorescence is extremely weak before reacting with hypochlorite, but the fluorescence intensity can be enhanced by hundreds of times after the reaction. The probe has good cell membrane penetration and biocompatibility, and can enter living cells without damage. It has low toxicity. In addition to detecting the concentration of hypochlorite (chemical signal) in inflammatory cells, the probe of this invention can also detect the viscosity changes (physical microenvironment) in inflammatory cells. It has a dual function and provides a powerful tool for studying the pathological process of allergic diseases. Attached Figure Description

[0016] Figure 1 The fluorescence emission spectra of the fluorescent probe prepared in this invention in different polar solvents are shown. Figure 2 The fluorescence emission spectra of the fluorescent probe prepared in this invention in hypochlorous acid solutions of different polarities are shown. Figure 3 The graph shows the fluorescence intensity variation of the fluorescent probe prepared in this invention at 530 nm for different concentrations of hypochlorous acid. Figure 4 The results show the specific response of the fluorescent probe prepared in this invention to hypochlorous acid. Figure 5 The graph shows the fluorescence intensity variation of the fluorescent probe prepared in this invention at 650 nm for solutions of different viscosities. Figure 6 The results of the cytotoxicity test of the fluorescent probe prepared in this invention are shown. Figure 7 The fluorescence changes of the fluorescent probe prepared in this invention in inflammatory cells with different concentrations of hypochlorous acid; Figure 8The fluorescence changes of the fluorescent probe prepared in this invention in inflammatory cells of different viscosities. Detailed Implementation

[0017] To enable those skilled in the art to better understand the present application, the technical solutions in the embodiments of the present application will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present application, and not all embodiments. Based on the embodiments in the present application, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present application.

[0018] Example 1: Synthesis of Fluorescent Probes A fluorescent probe for detecting hypochlorous acid in allergic diseases is prepared by the following steps:

[0019] 1,3-Indanedione (292.90 mg, 2 mmol) and 6-(dimethylamino)-naphthaldehyde (200.46 mL, 2.2 mmol) were dissolved in 15 mL of ethanol. The mixture was stirred in a round-bottom flask for 15 min, and two drops of piperidine were added. The mixture was refluxed at 85 °C overnight. After the reaction was complete, the mixture was filtered while hot and washed three times with ethanol. The solution was evaporated to dryness using silica gel. The compound (0.49 g, 75% yield) was purified by column chromatography using a mixture of dichloromethane and methanol (20:1 v / v) as the eluent. ¹H NMR (400 MHz, DMSO-) d 6) δ 8.28 – 8.23 ​​(m, 0H), 8.01 (dd, J = 8.9, 1.8 Hz, 0H),7.38 – 7.21 (m, 1H), 7.09 (d, J = 8.8 Hz, 0H), 6.66 (dd, J = 9.1, 2.5 Hz, 0H), 6.36 (d, J = 2.5 Hz, 0H), 2.50 (s, 1H). 13C NMR (100 MHz, DMSO- d 6) δ190.50, 189.47, 151.45, 147.08, 142.29, 139.80, 138.58, 138.16, 136.04,135.86, 131.77, 130.16, 126.84, 126.38, 126.21, 125.42, 123.22, 123.15,116.80, 105.40. HRMS (ESI): calcd for C 22 H 17 NO2 [M + H] + 328.1332, found328.2457.

[0020] Example 2: Fluorescence emission spectroscopy determination of fluorescent probes in different solutions First, prepare 5 mL of solvents of different polarities, including toluene, 1,4-dioxane, tetrahydrofuran, dichloromethane, dimethyl sulfoxide, ethanol, methanol, and buffer solution. Prepare two tubes for each solvent. Dissolve the fluorescent probe in dimethyl sulfoxide to prepare a 10 mM stock solution. Then, add 50 μL of each stock solution to the above solutions to achieve a final probe concentration of 10 μM. Measure the fluorescence emission spectra of different solutions using a fluorescence spectrometer to study the effect of solvents of different polarities on the fluorescent molecules and select the system that exhibits excellent fluorescence ability of the probe. The results are as follows: Figure 1 As shown, by Figure 1 It can be seen that the fluorescent probe exhibits good fluorescence performance in dioxane, toluene, and dichloromethane, and has low polarity in these solvents, indicating that the probe molecule has a certain polar response.

[0021] Example 3: Fluorescence intensity changes of fluorescent probes in hypochlorous acid solutions of different polarities First, the fluorescent probe was dissolved in dimethyl sulfoxide to prepare a 1 mM detection stock solution. Then, solvent systems with dioxane concentrations of 100% (pure dioxane), 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, and 0% (pure PBS) were prepared. Two tubes were prepared for each different solvent ratio. 50 μL of the above detection stock solution was added to 5 mL of each of the above solvents with different dioxane concentrations. Then, 50 μL of sodium hypochlorite aqueous solution was added to each solvent to bring the final concentrations of the probe and hypochlorous acid to 10 μM and 10 μM respectively (experimental group). 50 μL of ultrapure water was added to each solvent as a control group. After mixing, the fluorescence intensity (FI) was measured using a fluorescence spectrometer. A standard curve was constructed with fluorescence intensity as the ordinate and wavelength as the abscissa. By comparing the fluorescence intensity ratio of the experimental group and the control group under different polarity solvents, the effect of solvent polarity on the sensitivity of the probe in detecting hypochlorous acid was evaluated. The results are shown below. Figure 2 As shown.

[0022] from Figure 2 As can be seen, the fluorescence intensity at 660 nm gradually increases and undergoes a blue shift as the polarity of the solution system decreases, indicating that the fluorescent probe has good response characteristics to solvent polarity.

[0023] Example 4: Response of fluorescent probe to different concentrations of hypochlorous acid To further investigate the effect of fluorescent probes on ClO - To assess the detection capability, a corresponding fluorescence titration experiment was performed. The fluorescent probe was dissolved in dimethyl sulfoxide to prepare a 1 mM detection stock solution; a hypochlorous acid standard analyte stock solution (pH=7.0) in the range of 1 mM-8 mM was prepared. 50 μL each of the detection stock solution and the analyte stock solution were added to a 10 mL test tube and diluted to 5000 μL with toluene. After mixing, the fluorescence intensity FI at 530 nm was measured. A standard curve was constructed with fluorescence intensity as the ordinate and wavelength as the abscissa. The results are shown below. Figure 3 As shown.

[0024] Depend on Figure 3 As can be seen, the maximum fluorescence is observed at 530 nm under 380 nm excitation, and the fluorescence intensity of the fluorescent probe increases with the increase of hypochlorite concentration.

[0025] Example 5: Determination of the specific response of a fluorescent probe to hypochlorite ions First, the fluorescent probe was dissolved in dimethyl sulfoxide to prepare a 1 mM detection stock solution. A solution containing no interfering substances was added to deionized water as the blank group, and solutions containing interfering substances were dissolved in deionized water as the experimental groups. The interfering substances included: F... - Cl - ,Br - I - CH3COO - SCN - HPO4 - ClO4 - HSO3 - Ni 2+ Co 2+ Ag + Ca 2+ Pb 2+ Cys, Zn 2+ Mg 2+ Fe 3+ Cu 2+ ,H2O2,TBHP,·OH,GSH,ClO - The interfering PBS solution was prepared with a concentration of 10 mM.

[0026] Add 50 μL each of the fluorescent probe detection stock solution and the interfering PBS solution to a 10 mL test tube, and dilute to 5000 μL with toluene. After mixing, measure the fluorescence intensity at 530 nm. Plot a standard curve with fluorescence intensity as the ordinate and wavelength as the abscissa. The results are as follows: Figure 4 As shown.

[0027] from Figure 4 As can be seen, the fluorescent probe has high selectivity for hypochlorous acid, can selectively react with hypochlorous acid, and exhibits excellent specific response to hypochlorite ions.

[0028] Example 6: Determination of the response of a fluorescent probe to viscosity First, the fluorescent probe is dissolved in dimethyl sulfoxide to prepare a detection stock solution with a concentration of 1 mM.

[0029] Solvent systems with glycerol concentrations of 100% (pure glycerol), 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, and 0% (pure PBS) were prepared (5 mL for each concentration). 50 μL of the above-mentioned stock solution was added to 5 mL of each solvent concentration to achieve a final probe concentration of 10 μM. The fluorescence emission spectra of different solutions were measured using a fluorescence spectrometer to investigate the effect of different viscosities on the fluorescence intensity of the probe molecules. The results are as follows: Figure 5 As shown.

[0030] Depend on Figure 5 As can be seen, in the glycerol-PBS system, the fluorescence intensity at 650 nm continuously increases with the increase of the glycerol ratio, with the fluorescence intensity increasing by 153 times. The main reason for this is that as the viscosity of the solution increases, the rotation and vibration of the fluorescent molecules are restricted, reducing the possibility of energy loss through non-radiative pathways. This indicates that the probe molecules exhibit a highly sensitive response to viscosity.

[0031] Example 7: Verification of the cytotoxicity of fluorescent probes The cytotoxicity of the fluorescent probe was evaluated using a CCK-8 assay on RBL-2H3 cells.

[0032] When cells are in the logarithmic growth phase, collect them in centrifuge tubes and centrifuge at 1200 rpm for 4 min. Discard the supernatant, wash 2-3 times with sterile PBS, resuspend the cells in culture medium, gently pipette to mix, count the cells, and adjust the cell density to 5 × 10⁶ cells / mL. 4Cells / mL, gently and repeatedly pipet to mix, and seed 100 μL into each well of a 96-well plate. For the control group, add 0.5 μL of DMSO per well. In the experimental groups, add 0.5 μL of different fluorescent probe concentrations (2.5, 5, 10, 20 μM) sequentially, with three replicates for each concentration. Add 100 μL of PBS buffer to the edge wells of the 96-well plate to reduce evaporation. After incubating at 37°C and 5% CO2 for 24 h, add 10 μL of CCK-8 solution to each well and incubate at 37°C and 5% CO2 for 1 h. Measure the absorbance of each well at 450 nm using a microplate reader and save the data. The cell proliferation inhibition experiment was set up with the following three groups: Blank group (Ab): with culture medium and CCK-8 solution, without cells and fluorescent probes.

[0033] Control group (Ac): culture medium containing cells and CCK-8 solution, without fluorescent probe.

[0034] Experimental group (As): culture medium containing cells, fluorescent probe and CCK-8 solution.

[0035] The absorbance of each well was obtained using a microplate reader. Cell viability was calculated by dividing the difference in absorbance between the experimental and blank wells by the difference between the control and blank wells, as shown in the following formula:

[0036] Where As is the absorbance of the experimental group, Ab is the absorbance of the blank group, and Ac is the absorbance of the control group.

[0037] The degree of drug toxicity to cells was evaluated based on the obtained cell viability (CV%), and the results were as follows: Figure 6 As shown.

[0038] from Figure 6 The results showed that when RBL-2H3 cells were incubated with different doses (2.5-20 μM) of the probe molecules, the probe did not exhibit any cytotoxicity within the 10 μM range. This indicates that the probe has very low cytotoxicity to cells and is highly safe within this concentration range.

[0039] Example 8: Ability of fluorescent probes to detect endogenous hypochlorous acid in an inflammatory cell model RBL-2H3 mast cells were digested, centrifuged, resuspended, and counted before being seeded into 96-well blackboard cells (1×10⁻⁶ cells per well). 4 After the cells have adhered to the well, discard the culture medium and group them as follows (three replicates per group).

[0040] Blank control group: Unsensitized and unstimulated cells + probe.

[0041] Low-dose model group: IgE sensitization + 1 ng / ml antigen stimulation + probe.

[0042] Medium-dose model group: IgE sensitization + 10 ng / ml antigen stimulation + probe.

[0043] High-dose model group: IgE sensitization + 100 ng / ml antigen stimulation + probe.

[0044] Inhibitor group: IgE sensitization + MPO inhibitor (ABAH) + 100 ng / ml antigen stimulation + probe.

[0045] Positive low-dose control group: 0.5 μM sodium hypochlorite + probe.

[0046] Positive medium-dose control group: 2μM sodium hypochlorite + probe.

[0047] Positive high-dose control group: 5μM sodium hypochlorite + probe.

[0048] The specific procedure was as follows: 0.5 μL of IgE / mL was added to both the model group and the inhibitor group, bringing the final IgE concentration to 500 ng / mL. The cells were then incubated together in an incubator for at least 6 hours to ensure complete sensitization. After incubation, the culture medium was removed, and the cells were washed once with PBS. Then, different concentrations of DNP-HSA solution were added to the model group, bringing the final concentrations to 1 ng / mL, 10 ng / mL, and 100 ng / mL, respectively. The inhibitor group was pretreated with 10 μM ABAH for 1 hour, then the culture medium was removed, and the cells were washed once with PBS. DNP-HSA solution was then added to bring the final concentration to 100 ng / mL. The cells were then incubated in an incubator for 4 hours. Simultaneously, the positive control group was incubated with 1 μM, 2 μM, and 5 μM sodium hypochlorite for 15 minutes. Each group was repeated three times.

[0049] After incubation, the culture medium for all groups was discarded, aspirated, and washed away with 4°C PBS to remove any remaining medium. 100 μL of fluorescent probe was added to each well to a final concentration of 10 μM, and incubated at room temperature in the dark for 0.5 hours. After incubation, the culture medium was aspirated, and the plates were washed three times with 4°C PBS for 10 min each time. Finally, 100 µL of PBS was added to each well. The 96-well plate was placed in a microplate reader. The fluorescence intensity of each well was acquired at 530 nm. The acquired data were processed and analyzed, and the results are as follows: Figure 7 As shown.

[0050] from Figure 7As can be seen, in the positive control group, the fluorescence intensity of the probe increased with increasing hypochlorous acid concentration, indicating that the probe still has good cell membrane penetration and intracellular stability in the cellular environment. In the model group, after stimulation with the IgE / antigen complex, the fluorescence intensity of RBL-2H3 mast cells was significantly higher than that of the blank control group, and the fluorescence intensity increased with increasing antigen concentration, indicating that the probe can detect different concentrations of hypochlorous acid in cells of allergic diseases. In the inhibitor group, the fluorescence signal was significantly suppressed after pretreatment with the MPO inhibitor ABAH, indicating that the signal originated from hypochlorous acid produced by MPO catalysis.

[0051] Example 9: The ability of fluorescent probes to detect cell viscosity in an inflammatory cell model RBL-2H3 mast cells were digested, centrifuged, resuspended, and counted before being seeded into 96-well blackboard cells (1×10⁻⁶ cells per well). 4 After the cells have adhered to the well, discard the culture medium and group them as follows (three replicates per group).

[0052] Blank control group: probe.

[0053] Model group: IgE / antigen + probe.

[0054] Model + High Viscosity Group: IgE / antigen + Jasplakinolide (actin polymerization inducer) + probe.

[0055] Model + Low viscosity group: IgE / antigen + Nocodazole (microtubule polymerization inhibitor) + probe.

[0056] High viscosity control group: Jasplakinolide + probe.

[0057] Low viscosity control group: Nocodazole + probe.

[0058] The specific procedure was as follows: 0.5 μL of IgE / mL was added to the model group, model + high viscosity group, and model + low viscosity group, respectively, to achieve a final IgE concentration of 500 ng / mL. The cells were then incubated together in an incubator for at least 6 hours to ensure complete cell sensitization. After incubation, the culture medium was removed, and the cells were washed once with PBS. Then, DNP-HSA solution was added to the model group, model + high viscosity group, and model + low viscosity group, respectively, to achieve a final concentration of 100 ng / mL. These groups were then incubated for 6 hours. During the last 2 hours before the end of incubation, 2 µM Jasplakinolide was added to the model + high viscosity group, and 20 µM Nocodazole was added to the model + low viscosity group. Simultaneously, 2 µM Jasplakinolide was added to the cells in the high viscosity control group, and 20 µM Nocodazole was added to the cells in the low viscosity control group. These groups were then incubated for 2 hours. Each group was repeated three times.

[0059] After incubation, the culture medium for all groups was discarded, aspirated, and washed away with 4°C PBS to remove any remaining medium. 100 μL of fluorescent probe was added to each well to a final concentration of 10 μM, and incubated at room temperature in the dark for 0.5 hours. After incubation, the culture medium was aspirated, and the plates were washed three times with 4°C PBS for 10 min each time. Finally, 100 µL of PBS was added to each well. The 96-well plate was placed in a microplate reader. The fluorescence intensity of each well was acquired at 650 nm. The acquired data were processed and analyzed, and the results are as follows: Figure 8 As shown.

[0060] from Figure 8 As can be seen, the probe can detect different viscosities in both high and low viscosity groups. In inflammatory cells, the intensity of the fluorescent probe also changes when the viscosity changes, indicating that the probe can detect viscosity changes in the complex environment of inflammatory cells.

[0061] Although the present invention has been described using the above preferred embodiments, it is not intended to limit the scope of protection of the present invention. Any changes and modifications made by those skilled in the art to the above embodiments without departing from the spirit and scope of the present invention shall still fall within the scope of protection of the present invention.

Claims

1. A fluorescent probe for detecting hypochlorous acid in allergic diseases, characterized in that, The fluorescent probe has the molecular structure shown in formula (Ⅰ): 。 2. The method for preparing a fluorescent probe for detecting hypochlorous acid in allergic diseases according to claim 1, characterized in that, Includes the following steps: ， 1,3-indanedione and 6-(dimethylamino)-naphthal were mixed with an organic solvent and subjected to a condensation reaction under reflux at 60–100 °C catalyzed by an alkaline catalyst. After the reaction was completed, the fluorescent probe of the structure of formula (I) was obtained by post-treatment.

3. The method for preparing a fluorescent probe for detecting hypochlorous acid in allergic diseases according to claim 2, characterized in that, The fluorescent probe was obtained by column chromatography separation and purification, wherein the mobile phase for column chromatography separation was dichloromethane-methanol with a volume ratio of (10-30):

1.

4. The application of the fluorescent probe for hypochlorous acid detection in allergic diseases as described in claim 1 in the research, diagnosis and drug screening of allergic diseases.

5. The application according to claim 4, characterized in that, Application of the fluorescent probe in detecting hypochlorous acid content in allergic diseases.

6. The application according to claim 4 or 5, characterized in that, The allergic disease mentioned is a type I allergic disease.

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