A kind of hypochlorous acid fluorescent probe and its preparation method and application

Abstract

The present invention relates to a fluorescent probe for detecting hypochlorous acid in a liver injury process, a preparation method and an application thereof, and relates to the technical field of fluorescent probes. Specifically, the probe of the present invention can be used to measure, detect or screen hypochlorous acid, and the probe can achieve at least one of the following technical effects: highly specific identification of hypochlorous acid; rapid and highly sensitive response to hypochlorous acid, and the ability to achieve quantitative analysis of hypochlorous acid; high biocompatibility, and can be used for biological cell imaging, and by constructing alcoholic liver injury and drug-induced liver injury models, the changes in endogenous hypochlorous acid can be dynamically monitored, providing ideas for the detection of human liver injury; the required materials are simple to synthesize, stable in nature, and can be stored and used for a long time.

Description

Technical Field

[0001] The present invention relates to the technical field of fluorescent probes, and in particular to a hypochlorous acid fluorescent probe and a preparation method and application thereof. Background Art

[0002] As a core organ of the human body, the liver carries out basic functions to maintain life, such as metabolism, detoxification, protein production and bile excretion. However, its health status is easily disturbed by internal and external environmental factors, leading to damage, which in turn poses a major threat to the health of the whole body. Liver failure caused by liver damage often leads to circulatory dysfunction, hypotension and multiple organ failure. In addition, it may also induce cerebral edema, increased intracranial pressure, and ultimately lead to death. Accurate and timely detection of liver damage lesions is crucial for timely intervention and treatment. In particular, liver damage leads to increased levels of oxidative stress, which increases the production of endogenous reactive oxygen species (ROS), including oxidizing substances such as hypochlorous acid. Hypochlorous acid can induce apoptosis of hepatocytes, and by activating apoptotic signaling pathways (such as the caspase pathway), it leads to programmed death of hepatocytes, further aggravates the degree of liver damage, and even leads to cirrhosis and fibrosis, highlighting the importance of monitoring its dynamic changes.

[0003] Traditional detection methods, including chemiluminescence, chromatography, mass spectrometry and spectroscopy, generally have problems such as complex operation, long detection cycle and insufficient sensitivity in the determination of hypochlorous acid, which greatly hinders the effective application of this indicator in clinical and scientific research fields, especially the identification and intervention of early liver damage.

[0004] Fluorescent probe technology has become a hot topic in the field of biological monitoring due to its rapid response, real-time monitoring, high sensitivity, and ease of labeling and imaging, providing a new approach for real-time detection of hypochlorous acid. Unfortunately, existing hypochlorous acid-specific fluorescent probe technology still faces challenges: slow response, susceptibility to interference from other oxidizing substances, insufficient sensitivity, and complex synthesis procedures, which limit its potential in precise biological imaging and disease diagnosis.

[0005] In view of the above limitations, developing an innovative fluorescent probe system with the characteristics of single excitation wavelength, high selectivity, and ultra-high sensitivity, which is specifically targeted at real-time monitoring of hypochlorous acid and easy to use in vivo, is particularly critical and urgent for promoting the development of early diagnosis and treatment strategies for liver damage. This will not only fill the gap in existing technologies, but also open up new paths for a deeper understanding of the pathogenesis of liver disease and improving treatment effects. Summary of the invention

[0006] In view of this, the purpose of the present invention is to provide a hypochlorous acid fluorescent probe, as well as its preparation method and use. The fluorescent probe has the advantages of simple synthesis, good selectivity, high sensitivity, and the ability to quickly detect hypochlorous acid, and can be used for fluorescent imaging of hypochlorous acid in cells.

[0007] Specifically, the present invention provides a fluorescent probe for measuring, detecting or screening hypochlorous acid, the chemical structure of which is shown in formula (I): .

[0008] The present invention also provides a method for preparing the fluorescent probe of formula (I), comprising the following steps:

[0009] The fluorescent probe shown is prepared by reacting 4-(4-thiomorpholinyl) and 2-(4-cyano-2,2,5-trimethyl-3-furylidene)-malononitrile. The reaction formula is as follows:

[0010] .

[0011] In some specific embodiments of the present invention, the preparation method of the fluorescent probe of formula (I) is as follows: 4-(thiomorpholin-4-yl)benzaldehyde and 2-(4-cyano-2,2,5-trimethyl-3-furanylidene)-malononitrile are dissolved in anhydrous ethanol, piperidine is introduced dropwise, and the reaction solution is reacted under an argon atmosphere; after the reaction is completed, it is cooled to room temperature, and the mixture is concentrated to remove the solvent to obtain a purple-black crude product; and further separated by a chromatographic column, using a mixed system of petroleum ether and ethyl acetate as an eluent to obtain a pure fluorescent probe of formula (I).

[0012] In some specific embodiments of the present invention, the reaction temperature of 4-(thiomorpholin-4-yl)benzaldehyde and 2-(4-cyano-2,2,5-trimethyl-3-furylidene)-malononitrile is 90°C.

[0013] In some specific embodiments of the present invention, the molar ratio of 4-(thiomorpholin-4-yl)benzaldehyde to 2-(4-cyano-2,2,5-trimethyl-3-furylidene)-malononitrile is 1:1.

[0014] In some specific embodiments of the present invention, the reaction time of 4-(thiomorpholin-4-yl)benzaldehyde and 2-(4-cyano-2,2,5-trimethyl-3-furylidene)-malononitrile is 18 hours.

[0015] The present invention also provides a fluorescent probe composition for measuring, detecting or screening hypochlorous acid, which comprises a fluorescent probe of formula (I).

[0016] The present invention also provides a method for detecting the presence of hypochlorous acid in a sample or measuring the hypochlorous acid content in a sample, comprising:

[0017] a) contacting the fluorescent probe of formula (I) with a sample to form a fluorescent compound;

[0018] b) Determine the fluorescence properties of fluorescent compounds.

[0019] In some specific embodiments of the present invention, the sample is a chemical sample or a biological sample or an aqueous environment sample.

[0020] In some specific embodiments of the present invention, the sample is a biological sample including water, blood, microorganisms, or animal cells or tissues.

[0021] The present invention also provides the use of the fluorescent probe of formula (I) in cell fluorescence imaging.

[0022] The present invention also provides the use of the fluorescent probe of formula (I) in liver injury imaging.

[0023] The present invention also provides the use of the fluorescent probe of formula (I) in fluorescent imaging of mouse liver tissue.

[0024] The present invention also provides the use of the fluorescent probe of formula (I) in preparing a liver disease diagnosis or monitoring reagent.

[0025] The present invention also provides a kit or test paper for measuring, detecting or screening hypochlorous acid, which comprises a fluorescent probe of formula (I).

[0026] Compared with the prior art, the present invention has the following significant advantages and effects:

[0027] (1) Quick response

[0028] The fluorescent probe of the present invention can quickly respond to hypochlorous acid, which is beneficial to the rapid and real-time detection of hypochlorous acid.

[0029] (2) Good selectivity

[0030] The fluorescent probe of the present invention can selectively react specifically with hypochlorous acid to generate a product with fluorescence change. Compared with other common metal ions and other substances in the body, including but not limited to bisulfite ions, sulfur ions, bisulfate ions, nitrite ions, sulfate ions, acetate ions, bicarbonate ions, iodine ions, nitrate ions, carbonate ions, bromide ions, chloride ions, superoxide radicals, sodium ions, iron ions, ferrous ions, zinc ions, copper ions, potassium ions, calcium ions, glutathione, cysteine, ascorbic acid, singlet oxygen, hydroxyl radicals, hypochlorous acid, etc., the fluorescent probe of the present invention can eliminate the interference of the above substances and show excellent selectivity.

[0031] (3) High sensitivity

[0032] The fluorescent probe of the present invention reacts very sensitively with hypochlorous acid and can detect hypochlorous acid at a low concentration level (the minimum detection limit is as low as 26 nM).

[0033] (4) Low biological toxicity

[0034] The fluorescent probe of the present invention has the characteristic of low toxicity and can be applied to the detection of hypochlorous acid and fluorescence imaging in cell samples for a long time.

[0035] (5) Can be applied at physiological levels

[0036] The fluorescent probe of the present invention can detect, measure and screen hypochlorous acid under physiological conditions, and can perform cell bioimaging, and can realize the fluorescent signal response to the changes of hypochlorous acid in cells and its real-time visual detection, and also provides the possibility for detecting the changes of hypochlorous acid in the process of liver damage.

[0037] (6) Application in the diagnosis of liver damage

[0038] The fluorescent probe of the present invention can be used as a non-invasive tool to monitor the occurrence and development of liver damage. This application prospect makes the present invention have great potential in the field of medical diagnosis and is expected to improve the early diagnosis rate and treatment effect of liver diseases.

[0039] (7) Simple synthesis

[0040] The fluorescent probe of the present invention is simple to synthesize and is conducive to commercial and industrial promotion and application. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] In order to more clearly explain the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings that may be involved. It should be emphasized that the drawings described below are only some embodiments of the present invention, and for ordinary technicians working in this field, other embodiments can be obtained based on these drawings without performing creative work.

[0042] Figure 1 This is the hydrogen nuclear magnetic resonance spectrum of the synthetic fluorescent probe of Example 1;

[0043] Figure 2 This is the carbon nuclear magnetic resonance spectrum of the synthetic fluorescent probe of Example 1;

[0044] Figure 3 is the mass spectrum of the synthetic fluorescent probe of Example 1;

[0045] Figure 4 It is the absorption spectrum of the fluorescent probe before and after adding hypochlorous acid;

[0046] Figure 5 It is the fluorescence spectrum of the fluorescent probe before and after adding hypochlorous acid;

[0047] Figure 6 It is the response time spectrum of the fluorescent probe before and after adding hypochlorous acid;

[0048] Figure 7 It is the fluorescence spectrum of the fluorescent probe after adding different concentrations of hypochlorous acid;

[0049] Figure 8 It is the linear relationship diagram between the fluorescence intensity of the fluorescent probe at 630 nm and the concentration of hypochlorous acid;

[0050] Fig. 9 is the effect of different ion analytes (glutathione and cysteine ​​are 1 mM, hypochlorous acid is 5 μM, and the others are 100 μM) on the fluorescence intensity of the probe. The bar graph represents the fluorescence intensity of the probe at 630 nm in the presence of different analytes;

[0051] Fig.10 This is the toxicity test spectrum of different concentrations of fluorescent probe on L-02 cells, the concentrations are: 0 μM, 5 μM, 10 μM, 20 μM;

[0052] Fig.11 This is a cell imaging image of a fluorescent probe detecting changes in endogenous hypochlorous acid in ferroptotic cells;

[0053] Fig.12 It is the relative fluorescence statistics of the fluorescent probe detecting the change of endogenous hypochlorous acid in ferroptotic cells;

[0054] Fig.13 This is a cell imaging image of the fluorescent probe detecting the changes in endogenous hypochlorous acid concentration caused by different substances in L-02 cells;

[0055] Fig.14 It is a relative fluorescence statistical graph of the fluorescent probe detecting the change of endogenous hypochlorous acid concentration caused by different substances in L-02 cells;

[0056] Fig.15 This is a tissue imaging diagram of the fluorescent probe detecting the changes of endogenous hypochlorous acid in liver tissue in a mouse model of drug-induced liver injury;

[0057] Fig.16 This is a tissue imaging image of a fluorescent probe detecting changes in endogenous hypochlorous acid in liver tissue in a mouse model of alcoholic liver injury. DETAILED DESCRIPTION

[0058] In conjunction with the drawings in the embodiments of the present invention, the technical solutions in the embodiments of the present invention will be clearly and completely described below. It should be clear that the described embodiments only represent a part of the implementation methods of the present invention and do not limit the protection scope of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without performing creative work belong to the protection scope of the present invention.

[0059] Example 1: Synthesis of the compound of formula (I)

[0060] The synthetic route is as follows:

[0061] ;

[0062] The specific steps are as follows:

[0063] 4-(Thiomorpholin-4-yl)benzaldehyde (207 mg, 1.0 mmol) and 2-(4-cyano-2,2,5-trimethyl-3-furylidene)-malononitrile (199 mg, 1.0 mmol) were dissolved in 10 mL of anhydrous ethanol. Then, 22 μL of piperidine was added dropwise. The reaction solution was kept at 90°C for 18 hours under an argon atmosphere. After the reaction was completed, it was cooled to room temperature and the mixture was concentrated to remove the solvent, thereby producing a purple-black crude product. The crude product was purified using silica gel column chromatography with an eluent mixture of petroleum ether:ethyl acetate ranging from 2:1 to 1:2. 323 mg of pure compound of formula (I) was obtained with a yield of 79.6%.

[0064] The hydrogen spectrum, carbon spectrum and mass spectrum of the compound of formula (I) synthesized by this method are shown in the attached Figure 1 , Attachment Figure 2 , Attachment Figure 3 shown.

[0065] Example 2: Testing the Absorption Spectrum and Fluorescence Spectrum Changes of the Fluorescent Probe before and after the Reaction with Hypochlorous Acid

[0066] Two fluorescent probe test systems with a concentration of 5 μM were prepared, each with a volume of 10 mL. After shaking, the absorption spectra and fluorescence spectra were measured using an absorption spectrometer and a fluorescence spectrometer, respectively. Next, 5 μM hypochlorous acid was added to each test system, and the mixture was shaken and allowed to stand for 3 minutes. The changes in the absorption and fluorescence spectra were then measured again using an absorption spectrometer and a fluorescence spectrometer. All experiments were conducted in a PBS buffer solution (10 mM, pH = 7.4). The fluorescence spectrum was measured at 25 °C, and the probe used was prepared in Example 1. The test results are shown in the attached figure. Figure 4 and attached Figure 5 shown.

[0067] Depend on Figure 4 It can be clearly seen that after adding hypochlorous acid, the absorption peak of the absorption spectrum of the fluorescent probe at 510 nm is significantly enhanced.

[0068] Depend on Figure 5 It can be clearly seen that after adding hypochlorous acid, the emission peak of the fluorescence spectrum of the fluorescent probe at 630 nm is significantly enhanced.

[0069] Example 3: Time-dependent dynamics of fluorescent probes

[0070] First, a 10 mL test system with a probe concentration of 5 μM was prepared. Then, 5 μM hypochlorous acid was added to the test system, and the fluorescence intensity change was immediately tested using a fluorescence spectrometer after shaking. All experiments were performed in PBS buffer solution (10 mM, pH = 7.4), the fluorescence spectrum was measured at 25 °C, and the probe used was prepared in Example 1. The test results are shown in the attached figure. Figure 6 shown.

[0071] from Figure 6 It can be clearly observed that after the addition of hypochlorous acid, the fluorescence intensity of the probe changes with time. In less than 1 minute, the fluorescence intensity reaches the maximum value and then reaches a stable period. This indicates that the probe has the ability to respond quickly to hypochlorous acid and has good stability, providing an effective analytical method for the rapid detection of hypochlorous acid.

[0072] Example 4: Testing the detection of different concentration gradients of hypochlorous acid by fluorescent probe

[0073] Multiple parallel samples with a probe concentration of 5 μM were prepared in 5 mL cuvettes. Then, different concentrations (0-2.5 μM) of hypochlorous acid were added to each sample, shaken and left to stand for 3 minutes, and the change in fluorescence intensity was tested using a fluorescence spectrometer. All experiments were conducted in PBS buffer solution (10 mM, pH=7.4), the fluorescence spectrum was measured at 25 °C, and the probe used was prepared in Example 1. The test results are shown in the attached figure. Figure 7 and attached Figure 8 shown.

[0074] from Figure 7 It is clearly seen that with the increase of hypochlorous acid concentration, the emission peak of the probe at 630 nm gradually increases. Figure 8 It can be seen that after the probe (10 µM) reacted with hypochlorous acid (0-2.5 µM), its fluorescence intensity showed a good linear relationship with the concentration of hypochlorous acid, indicating that the fluorescent probe can be used for quantitative analysis of hypochlorous acid.

[0075] Example 5: Sensitivity of the method for detecting hypochlorous acid provided by the present invention

[0076] The detection limit calculation formula for hypochlorous acid in the method for detecting hypochlorous acid provided by the present invention is:

[0077] Detection limit = 3 σ / k

[0078] σ is the standard deviation of the blank experiment, k The fluorescence intensity F is measured by fluorescence concentration titration experiment 630 The slope of the straight line obtained by linear fitting of hypochlorous acid concentration (see Appendix Figure 8 ).

[0079] It is calculated that the detection limit of the method for detecting hypochlorous acid provided by the present invention is 26 nM.

[0080] Example 6: Testing the selectivity of fluorescent probes

[0081] Prepare multiple parallel samples with a probe concentration of 5 μM in 10 mL colorimetric tubes, and then add different analytes (the analytes are bisulfite ion, sulfur ion, bisulfate ion, nitrite ion, sulfate ion, acetate ion, bicarbonate ion, iodide ion, nitrate ion, carbonate ion, bromide ion, chloride ion, superoxide radical, sodium ion, iron ion, ferrous ion, zinc ion, copper ion, potassium ion, calcium ion, glutathione, cysteine, ascorbic acid, singlet oxygen, hydroxyl radical, hypochlorous acid, corresponding to the attached Fig. 9 az; wherein the concentrations of cysteine ​​and glutathione are 1mM, the concentration of hypochlorous acid is 5 μM, and the concentrations of other analytes are all 100 μM) are added to the test system, shaken evenly, left to stand for 3 minutes, and then the fluorescence intensity change is tested by a fluorescence spectrometer. The above determination is carried out in a PBS buffer solution (10 mM, pH=7.4) system, the measurement temperature of the fluorescence spectrum is 25 ℃, and the probe used is the one prepared in Example 1. The test results are shown in the attached Fig. 9 shown.

[0082] from Fig. 9 The results show that only when hypochlorous acid is added, the fluorescence intensity of the probe changes significantly, while the influence of other analytes is almost negligible. This proves that the probe of the present invention has high selectivity for hypochlorous acid and can be used as a specific identification and analysis tool for hypochlorous acid in the environment and in organisms.

[0083] Example 7: Toxicity test of fluorescent probe on L-02 cells

[0084] The MTT method was used to detect the cytotoxicity of different concentrations of probes to L-02 cells. 4The cells were seeded into the culture medium of a 96-well plate at a density of 100 μM, and then the probes were added at concentrations of 0, 5, 10, and 20 μM. The cells were cultured in an incubator at 37 °C, 5% CO2, and 95% air for 24 hours. Finally, 100 μL of MTT (0.5 mg mL -1 ) and then cultured for 4 hours. Finally, the culture medium was replaced with DMSO and quantified spectrophotometrically at a wavelength of 490 nm. The test results are shown in the attached Fig.10 shown.

[0085] from Fig.10 It can be clearly seen that the probe has the characteristics of low toxicity and can be used for real-time detection of hypochlorous acid in cell samples for a long time. The spectrophotometric measurement temperature is 25°C, and the probe used is the one prepared in Example 1.

[0086] Example 8: Fluorescent probe testing of changes in endogenous hypochlorous acid in ferroptotic cells

[0087] L-02 cells were divided into 3 groups. Group a was the blank group, which was used as the control group and was directly incubated with the probe (1 μM) for 30 minutes. Group b was the Erastin group, which was first incubated with Erastin (a small molecule compound, one of the compounds that induce ferroptosis, 10 μM) for 2 hours, and then incubated with the probe (1 μM) for 30 minutes. Group c was the Erastin+Fer-1 group, which was first incubated with Erastin (1 μM) and Fer-1 (an effective ferroptosis inhibitor, 20 μM) for 3 hours, and then incubated with the probe (1 μM) for 30 minutes. Finally, confocal microscopy imaging was performed on the three groups of cells. The test results are shown in the attached figure. Fig.11 and 12 shown.

[0088] from Fig.11 It can be clearly seen that when the stimulation leads to ferroptosis, the signal of the red channel is significantly enhanced; when ferroptosis is inhibited, that is, hypochlorous acid is removed, the fluorescence signal of the red channel is significantly reduced. Fig.12 The cell relative fluorescence statistics also showed the same result. The above experimental results show that the probe can detect the dynamic changes of endogenous hypochlorous acid before and after inducing cell ferroptosis. The cell imaging environment temperature is 25 ℃, and the probe used is prepared in Example 1.

[0089] Example 9: Detection of changes in endogenous hypochlorous acid concentration in L-02 cells using fluorescent probes

[0090] L-02 cells were divided into seven groups. Group a was the blank group, which was used as the control group and was directly incubated with the probe (1 μM) for 30 minutes. Group bd was the stimulation group: incubated with 10 mM APAP for 12 hours, 1 μM LPS (lipopolysaccharide) for 8 hours, and 10 μM PMA (phorbol ester) for 8 hours, and then incubated with the probe (1 μM) for 30 minutes. Group eg was the clearance group: incubated with 1 mM NAC (N-acetylcysteine) for 1 hour, 200 μg / mL silymarin for 1 hour, and 200 μM 4ABH (4-aminobutyric acid hydroxamic acid) for 2 hours. Finally, confocal microscopy imaging was performed on the seven groups of cells. The test results are shown in the attached figure. Fig.13 and 14 shown.

[0091] from Fig.14 (ag) It can be clearly seen that when hypochlorous acid is produced by stimulation, the fluorescence signal in the red channel is significantly enhanced, and when hypochlorous acid is removed, the fluorescence signal in the green channel is significantly reduced; Fig.14 The cell relative fluorescence statistics also show the same result. The above experimental results show that the probe can detect the dynamic changes of hypochlorous acid in cells. The cell imaging environment temperature is 25°C, and the probe used is the one prepared in Example 1.

[0092] Example 10: Detection of endogenous hypochlorous acid in liver tissue sections of drug-induced liver injury mouse model using fluorescent probes

[0093] In this example, all experimental mice were adaptively raised for one week at a temperature of 23±2°C, a relative humidity of 55±5%, and a 12 / 12 h light / dark cycle. The experimental mice were randomly divided into three groups, namely a normal group, a drug-induced liver injury group (treated with APAP-acetaminophen, a common antipyretic drug that can cause liver damage), and a clearance + injury group, with 6 mice in each group. Before intraperitoneal injection of the drug, the three groups of mice were fasted for 12 hours. The mice in the injury group were intraperitoneally injected with APAP (the dosage was calculated based on the weight of the mice, 300 mg kg -1 The cleanup + injury group was intragastrically administered with NAC 2 hours before APAP injection (the dosage was calculated based on the mouse body weight, 100 mg kg -1 ) were treated with 4% paraformaldehyde and then injected with APAP. The mice in the normal group were intraperitoneally injected with normal saline.

[0094] 12 hours after drug injection, mice were anesthetized and livers were collected to prepare liver tissue slices with a thickness of about 10 μm for fluorescence microscopy imaging. Subsequently, the tissue slices were incubated with the fluorescent probe of the present invention (20 μM) at 37°C for 1 hour, rinsed three times with PBS (10 mM, pH=7.4), and then images were collected under a fluorescence microscope. The test results are shown in the attached Fig.15 shown.

[0095] from Fig.15 It can be clearly seen that compared with the normal group of mice, the relative fluorescence intensity of the liver of the mice in the drug-induced liver injury group increased significantly; compared with the mice in the drug-induced liver injury group, the relative fluorescence intensity of the liver of the mice in the clearance + injury group decreased significantly. The above experimental results show that the probe can detect the dynamic changes of hypochlorous acid in the liver tissue of drug-induced liver injury, and can perform fluorescence imaging of the damaged liver. The ambient temperature of tissue fluorescence microscopy imaging is 25 ° C, and the probe used is prepared in Example 1.

[0096] Example 11: Detection of endogenous hypochlorous acid in liver tissue sections using fluorescent probes in a mouse model of alcoholic liver injury

[0097] In this embodiment, all experimental mice were adaptively raised for one week under the conditions of 23±2℃, 55±5% relative humidity and 12 / 12 h light / dark cycle. The experimental mice were randomly divided into three groups, namely, normal group, alcoholic liver injury group (gavage with 56° Red Star Erguotou), and removal + injury group, with 6 mice in each group. Before gavage, the three groups of mice were fasted for 12 hours. The mice in the normal group were gavaged with distilled water (0.01 ml / g); the mice in the alcoholic liver injury group were gavaged with the same volume of distilled water for 30 minutes, and then given 56° Red Star Erguotou at a dose of 0.12 mL / g; the removal + injury group was intervened by drugs (silybin, 0.06g / kg), each for 30 minutes, and then given 56° Red Star Erguotou at a dose of 0.12 mL / g. The mice were anesthetized 2 hours after the last gavage, and then blood and liver tissue samples were collected to make liver tissue sections with a thickness of about 10 μm for fluorescence microscopy imaging. Subsequently, the tissue sections were incubated with the fluorescent probe of the present invention (20 μM) at 37°C for 1 hour, rinsed three times with PBS (10 mM, pH = 7.4), and then images were collected under a fluorescence microscope. The test results are shown in the attached Fig.16 shown.

[0098] from Fig.16It can be clearly seen that compared with the normal group of mice, the relative fluorescence intensity of the liver of the mice in the alcoholic liver injury group increased significantly; compared with the mice in the alcoholic liver injury group, the relative fluorescence intensity of the liver of the mice in the clearance + injury group decreased significantly. The above experimental results show that the probe can detect the dynamic changes of hypochlorous acid in the liver tissue of alcoholic liver injury and can perform fluorescence imaging of the damaged liver. The ambient temperature of tissue fluorescence microscopy imaging is 25 °C, and the probe used is prepared in Example 1.

[0099] Although the present invention is described using the above embodiments, it should be understood that the present invention may be further modified and varied without departing from the spirit of the present invention, and these modifications and variations are all within the scope of protection of the present invention.

Claims

1. A fluorescent probe for measuring, detecting or screening hypochlorous acid, characterized in that: The chemical structure is shown in formula (I): 。 2. The method for preparing a fluorescent probe according to claim 1, characterized in that: The method comprises the following steps: reacting 4-(thiomorpholin-4-yl) and 2-(4-cyano-2,2,5-trimethyl-3-furylidene)-malononitrile to prepare a fluorescent probe as shown in formula (I), wherein the reaction formula is as follows: 。 3. The preparation method according to claim 2, characterized in that: The steps include: 4-(4-thiomorpholinyl)benzaldehyde and 2-(4-cyano-2,2,5-trimethyl-3-furylidene)-malononitrile are dissolved in anhydrous ethanol, piperidine is introduced dropwise, and the reaction solution is reacted under an argon atmosphere; after the reaction is completed, it is cooled to room temperature and the mixture is concentrated to remove the solvent to obtain a purple-black crude product; it is further separated by a chromatographic column, using a mixed system of petroleum ether and ethyl acetate as an eluent to obtain a pure compound of formula (I).

4. A fluorescent probe composition for measuring, detecting or screening hypochlorous acid, characterized in that: The method comprises the fluorescent probe as claimed in claim 1.

5. A method for detecting the presence of hypochlorous acid in a sample or determining the hypochlorous acid content in a sample, characterized in that: The method is used for non-disease diagnosis and treatment purposes, and comprises: a) contacting the fluorescent probe according to claim 1 with a sample to form a fluorescent compound; b) determining the fluorescence property of the fluorescent compound.

6. The method according to claim 5, characterized in that The sample is a chemical sample, a biological sample or an aqueous environment sample.

7. Use of the fluorescent probe as claimed in claim 1 in detecting the presence of hypochlorous acid in a sample or determining the hypochlorous acid content in a sample, wherein the use is for non-disease diagnosis and treatment purposes.

8. Use of the fluorescent probe as claimed in claim 1 in preparing a reagent for diagnosing or monitoring liver diseases.

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