A ratiometric fluorescent probe for detecting ClO- based on pyranoquinoline

Abstract

The present invention relates to detection analysis, in particular to a method for detecting ClO with pyranoquinoline as the parent ‑ The ratio-type fluorescent probe has the structural formula #imgabs0# The present invention provides a ratio-type fluorescent probe for detecting ClO based on pyranoquinoline. ‑ The ratiometric fluorescent probe can eliminate the errors caused by factors such as the concentration of the probe molecule itself and increase the dynamic range of fluorescence measurement. At the same time, the probe has fast detection speed, high sensitivity, strong selectivity, and more accurate experimental results.

Description

Technical Field

[0001] The invention relates to detection analysis, in particular to a ratiometric fluorescent probe for detecting ClO- with pyranoquinoline as a matrix. Background Art

[0002] Hypochlorous acid (HOCl) is a reactive oxygen species and a strong antibacterial substance in the human immune defense system. It is mainly produced by the peroxidation of hydrogen peroxide and chloride ions in white blood cells under the catalysis of the heme enzyme myeloperoxidase (MPO). It plays an important role in many biological and pathological processes of organisms. However, the normal concentration of hypochlorous acid in the human body is generally within the range of 200μM. When the concentration of hypochlorous acid is too high, it will cause tissue damage and some diseases, such as inflammation, Alzheimer's disease, cancer, etc.; At present, among the various HClO detection methods studied, fluorescent probe retrieval is the most popular method used by researchers. One of the most widely used methods, due to its instrumental simplicity, excellent sensitivity, low background signal and fast instrumental response, fluorescent probes with different fluorescent groups and reaction sites for detecting hypochlorous acid have been continuously reported. For example, Lin (Chem.-Eur. J., 15 (2009) 2305-2309) et al. developed a new fluorescent probe for tracking hypochlorite through deoxygenation reaction, which was successfully used in the test to detect hypochlorite with ratiometric and fluorescent responses and showed high selectivity compared with other interfering species; Wu (ACS) Appl.Mater.Inter.,8(2016)1511-1519) and his colleagues developed a new pyrene derivative fluorophore probe Py-Cy, and realized the ratio detection of hypochlorite through red or near-infrared fluorescence; however, they still have some limitations, the synthesis route is relatively complicated and the synthesis yield is low. In addition, there are problems such as small Stokes shift, aggregation-induced quenching, poor detection limit and oxidation reaction time delay. Therefore, it is imperative to study a ratiometric fluorescent probe for detecting ClO- based on pyranoquinoline. Summary of the invention

[0003] In view of the above situation, in order to overcome the defects of the prior art, the purpose of the present invention is to provide a ratio-type fluorescent probe based on pyranoquinoline for detecting ClO-. The ratio-type fluorescent probe based on pyranoquinoline for detecting ClO- provided by the present invention can eliminate the errors caused by factors such as the concentration of the probe molecule itself, increase the dynamic range of fluorescence measurement, and at the same time, the probe has a fast detection speed, high sensitivity, strong selectivity, and more accurate experimental results.

[0004] The technical solution provided by the present invention is a ratiometric fluorescent probe for detecting ClO- based on pyranoquinoline, and its structural formula is:

[0005] Its synthesis method comprises the following steps:

[0006] 1. Add salicylaldehyde and 3-bromopropyne into a flask, add K2CO3 to provide an alkaline environment, then add the first DMF solvent, rotate at room temperature for 10 hours, and after the reaction is completed, extract with DCM to obtain a light brown solid, namely compound 2;

[0007] 2. Add compound 2 and 4-aminobenzyl alcohol to a flask, add CuI and (CF3SO3)3La as catalysts, add CH3CN dropwise, and add enough to cover the drugs. Put the flask in an oil bath, stir magnetically and reflux under condensation at 80°C for 5-6h, filter to remove the caked matter, remove the acetonitrile in the flask with a rotary evaporator at 45°C, and purify the obtained crude product by 100-200 mesh silica gel column chromatography to obtain a light yellow solid, i.e., compound 3;

[0008] 3. Compound 3 and MnO2 were added to a reaction flask, and then CH2Cl2 was added to the reaction flask. The mixture was reacted at room temperature for 5 h, wherein MnO2 was added in small amounts several times. After the reaction was completed, the mixture was filtered with diatomaceous earth and washed with dichloromethane to obtain a crude product. The solvent was removed under reduced pressure on a rotary evaporator, and the crude product was purified by 100-200 mesh silica gel column chromatography to obtain a light yellow solid, i.e., compound 4.

[0009] 4. Compound 5 (4-bromo-1,8-naphthalene dicarboxylic anhydride) and n-butylamine were mixed in ethanol, refluxed for 10 h, cooled to room temperature, filtered to obtain an off-white precipitate, and the filter cake was washed with ethanol to obtain the product on the filter paper, i.e., compound 6;

[0010] 5. Dissolve compound 6 in the second DMF, then add H2N-NH2·H2O dropwise, reflux at 110°C for 3 h, add water after the reaction to precipitate the product, filter the product on the filter paper, and dry in a vacuum oven at 45°C to obtain an orange solid, i.e., compound 7;

[0011] 6. Add compound 4 and anhydrous ethanol to a single-necked flask and heat to 80°C. When the solid is completely dissolved, add compound 7 to the single-necked flask and add glacial acetic acid at the same time. The mixture is refluxed at 80°C for 4 hours. After the reaction is completed, take the single-necked flask out of the oil bath, and the product is recrystallized in ethanol. When the solution is cooled to room temperature, solids precipitate. After suction filtration, the filter cake is washed with ethanol to obtain an orange-red solid, i.e., Probe 1.

[0012] The molar volume ratio of salicylaldehyde, 3-bromopropyne, K2CO3, compound 2, 4-aminobenzyl alcohol, CuI, (CF3SO3)3La, compound 3, MnO2, compound 5, n-butylamine, compound 6, compound 4, compound 7, the first DMF, CH2Cl2, ethanol, the second DMF, H2N-NH2·H2O, anhydrous ethanol, and glacial acetic acid is 10:11:10:5:5:5:0.5:0.7:9.8:3.6:3.6:1.5:0.25:0.25:5:10:20:4:3:15:1, and the molar volume ratio refers to the solid being measured in mmol and the liquid being measured in ml.

[0013] The eluent for the chromatographic purification in step 2 is dichloromethane / ethyl acetate (volume ratio: 7 / 1).

[0014] The eluent for the chromatographic purification in step 3 is petroleum ether / dichloromethane (volume ratio: 1 / 1).

[0015] The method for synthesizing the probe molecule of the present invention is simple, the synthesis yield is as high as 84.7%, the selectivity is good, the probe has a large Stokes shift (137nm), a low detection limit (5.1nM), a fast response time (<4min) and a high synthesis yield (84.7%), and the probe has low toxicity and good solubility. The probe has been successfully applied to imaging of cells and zebrafish, and can perform quantitative detection of sodium hypochlorite (NaClO). The pH application range is wide, and detection can be performed from pH=4.0 to pH=10.0. The probe has strong anti-interference ability, low toxicity, and good cell membrane penetration. The probe has fast detection speed, high sensitivity, more accurate experimental results, and significant economic and social benefits.

[0016] Instruction Manual

[0017] Figure 1 This is a diagram of the synthesis process of the fluorescent probe of the present invention;

[0018] Figure 2 Diagram of the detection mechanism of the fluorescent probe of the present invention;

[0019] Figure 3 The figure shows the change of ultraviolet absorption spectrum of the fluorescent probe (10.0 μM) of the present invention after being acted on with different concentrations of ClO- in PBS buffer solution (10 mM, pH=7.4, 1.0 mM CTAB). The horizontal axis is the wavelength and the vertical axis is the absorption.

[0020] Figure 4 The fluorescence spectrum changes of the fluorescent probe (10.0 μM) of the present invention after being acted on with different concentrations of ClO- in a PBS buffer solution (10 mM, pH=7.4, 1.0 mM CTAB), the abscissa is the wavelength, and the ordinate is the fluorescence intensity.

[0021] Figure 5 When the fluorescent probe (10.0 μM) of the present invention responds to ClO- (35 μM-60 μM) in a PBS buffer solution (10 mM, pH=7.4, 1.0 mM CTAB), the fluorescence intensity ratio I 419nm / I 556nm The linear relationship with the concentration change is that the horizontal axis is the concentration of ClO- and the vertical axis is I 419nm / I 556nm The fluorescence intensity ratio.

[0022] Figure 6 The fluorescence intensity at 556 nm changes with time during the interaction of the fluorescent probe (10.0 μM) of the present invention with ClO- in a PBS buffer solution (10 mM, pH=7.4, 1.0 mM CTAB). The horizontal axis is time and the vertical axis is fluorescence intensity.

[0023] Figure 7 The fluorescence intensity at 419 nm changes with time during the interaction of the fluorescent probe (10.0 μM) of the present invention with ClO- in a PBS buffer solution (10 mM, pH=7.4, 1.0 mM CTAB). The horizontal axis is time and the vertical axis is fluorescence intensity.

[0024] Figure 8 It is the fluorescence intensity of the fluorescent probe (10.0 μM) of the present invention before and after the action with ClO- in buffer solutions with different pH values, the horizontal axis is pH, and the vertical axis is fluorescence intensity.

[0025] Fig. 9 To demonstrate the selectivity of the fluorescent probe of the present invention, the fluorescent probe (10.0 μM) was reacted with ClO- and other analytes (ONOO-, 1 O2, OH, Na + ,K + ,Ca 2+ ,PO4 3- ,H2PO4-,HPO4 2- Mg 2+ ,Zn 2+ ,SO4 2- ,SO3 2- ,HSO3 - ) after the action of .

[0026] Fig.10 The anti-interference property of the fluorescent probe of the present invention is that ClO- and analyte (ONOO-, 1 O2, OH, Na+ ,K + ,Ca 2 + ,PO4 3- ,H2PO4-,HPO4 2- Mg 2+ ,Zn 2+ ,SO4 2- ,SO3 2- ,HSO3 - ) coexisted, the fluorescence intensity ratio of the fluorescent probe (10.0 μM) after the action of ClO- in PBS buffer solution (10 mM, pH = 7.4, 1.0 mM CTAB) (I 419nm / I 556nm ) bar chart.

[0027] Fig.11 The cytotoxicity test of the fluorescent probe of the present invention was carried out using the MTT method, where the abscissa represents the probe concentration (0.0, 1.0, 2.0, 5.0, 10.0, 20.0 μM) and the ordinate represents the cell survival rate.

[0028] Fig.12 The confocal cell imaging of the fluorescent probe of the present invention to detect ClO- in Hela cells. A1-A3 are the imaging effects of the probe (10.0 μM) incubated in cells at 37°C for 1 hour, and B1-B3 are the imaging effects of the cells first incubated with ClO- at 37°C. - C1-C3 are the imaging effects of cells being first incubated with PBS buffer solution (containing 1% CTAB) containing lipopolysaccharide (LPS, 1.0 μg / ml) and amphetamine acetate (PMA, 1.0 μg / ml) for 1 hour at 37°C, and then incubated with the probe (10.0 μM) for 1 hour.

[0029] Fig.13 The present invention is used to detect confocal cell imaging of ClO- in zebrafish using the fluorescent probe of the present invention. A1-A3 are blank groups, and confocal microscopy imaging is performed on 3-day-old zebrafish without any treatment. B1-B3 are probe groups, and imaging effects are shown after only adding 10.0 μM probe solution and incubating with 3-day-old zebrafish for 1 hour. C1-C3 are exogenous groups, and imaging effects are shown after 3-day-old zebrafish are pretreated with 60.0 μM ClO- solution for 1 hour and then adding 10.0 μM probe solution and incubating for 1 hour. D1-D3 are endogenous groups, and imaging effects are shown after 3-day-old zebrafish are pretreated with LPS and PMA for 1 hour and then adding probe 1 (10.0 μM) and incubating for 1 hour. DETAILED DESCRIPTION

[0030] The specific implementation modes of the present invention are described in detail below in combination with embodiments and specific situations.

[0031] Embodiment 1:

[0032] A ratiometric fluorescent probe for detecting ClO- based on pyranoquinoline, the structural formula of which is:

[0033]

[0034] Its synthesis method comprises the following steps:

[0035] 1. Add salicylaldehyde (1.220 g, 10 mmol) and 3-bromopropyne (1.298 g, 11 mmol) into a flask, then add K2CO3 (1.382 g, 10 mmol) to provide an alkaline environment, then add 5 ml of the first DMF solvent, rotate at room temperature for 10 h, and after the reaction is completed, extract with DCM to obtain a light brown solid, namely compound 2;

[0036] 2. Add compound 2 (0.900 g, 5 mmol) and 4-aminobenzyl alcohol (0.615 g, 5 mmol) into a flask, add CuI (0.952 g, 5 mmol) and (CF3SO3)3La (0.293 g, 0.5 mmol) as catalysts, add CH3CN dropwise, and add enough to cover the drugs. Put the flask into an oil bath, stir magnetically and reflux under condensation at 80°C for 5-6 h, filter to remove the caking material, remove the acetonitrile in the flask with a rotary evaporator at 45°C, and purify the crude product by 100-200 mesh silica gel column chromatography to obtain a light yellow solid, i.e., compound 3;

[0037] 3. Compound 3 (0.2 g, 0.7 mmol) and MnO2 (0.925 g, 9.8 mmol) were added to a reaction flask, and then 10 ml of CH2Cl2 was added to the reaction flask. The reaction was carried out at room temperature for 5 h, wherein MnO2 was added in small amounts several times. After the reaction was completed, the mixture was filtered with diatomaceous earth and washed with dichloromethane to obtain a crude product. The solvent was removed under reduced pressure on a rotary evaporator, and the crude product was purified by 100-200 mesh silica gel column chromatography to obtain a light yellow solid, i.e., compound 4.

[0038] 4. Compound 5 (4-bromo-1,8-naphthalene dicarboxylic anhydride) (1.000 g, 3.600 mmol) and n-butylamine (0.26399 g, 3.600 mmol) were dissolved in 20 ml of ethanol, refluxed for 10 h, cooled to room temperature, filtered to obtain an off-white precipitate, and the filter cake was washed with ethanol to obtain the product on the filter paper, i.e., compound 6;

[0039] 5. Dissolve compound 6 (0.516 g, 1.5 mmol) in 4 ml of the second DMF, then add 3 ml of H2N-NH2·H2O dropwise, reflux at 110°C for 3 h, add water after the reaction to precipitate the product, filter the product on the filter paper, and dry in a vacuum oven at 45°C to obtain an orange solid, i.e., compound 7;

[0040] 6. Add compound 4 (0.06925 g, 0.25 mmol) and 15 ml of anhydrous ethanol to a single-necked flask and heat to 80°C. When the solid is completely dissolved, add compound 7 (0.07075 g, 0.25 mmol) to the single-necked flask and add 1 ml of glacial acetic acid. The mixture is refluxed at 80°C for 4 h. After the reaction is completed, the single-necked flask is taken out of the oil bath, and the product is recrystallized in ethanol. When the solution is cooled to room temperature, solids precipitate. After filtration, the filter cake is washed with ethanol to obtain an orange-red solid, i.e., Probe 1.

[0041] Example 2: The optimization of this example compared with Example 1 is reflected in that the eluent in step 2 is dichloromethane / ethyl acetate (volume ratio: 7 / 1).

[0042] Example 3: The optimization of this example compared with Example 1 is reflected in that the eluent in step 3 is petroleum ether / dichloromethane (volume ratio: 1 / 1).

[0043] The fluorescent probe prepared by the present invention is designed to detect ClO- by hybridizing pyranoquinoline and naphthalimide fluorophores. The amino group on the naphthalimide reacts with the aldehyde group on the pyranoquinoline to form a C=N bond as a recognition site. Due to the FRET mechanism, Probe 1 displays a green fluorescent naphthalimide part, and the reaction site C=N is oxidatively cleaved by ClO-, interrupting the FRET process, thereby releasing quinoline (CQ) that emits blue fluorescence and a non-fluorescent naphthalimide derivative (HN). HN does not generate fluorescence because the hydrazine group has a strong electron donor ability, which inhibits the photoinduced electron transfer effect.

[0044] The method for synthesizing the probe molecule of the present invention is simple, and the synthesis yield is as high as 84.7%.

[0045] The fluorescent probe of the present invention is a single-wavelength excitation type fluorescent probe for distinguishing and detecting ClO-, and the excitation wavelength is set to 350nm during the test process.

[0046] The fluorescent probe of the present invention is a ratiometric fluorescent probe, and its maximum emission peak is at 556 nm before ClO- is added, and after responding to ClO-, it blue-shifts to 419 nm.

[0047] After the fluorescent probe of the present invention responds to ClO-, it has a large Stokes shift (137nm).

[0048] After the fluorescent probe of the present invention acts on a 60.0 μM ClO- solution, the fluorescence intensity at 556 nm decreases immediately and reaches an equilibrium state in 15 seconds.

[0049] After the fluorescent probe of the present invention acts on a 60.0 μM ClO- solution, the fluorescence intensity at 419 nm is immediately enhanced and reaches a maximum value at 225 s.

[0050] The fluorescent probe of the present invention has good selectivity. The test system of the probe molecule is a 10mM PBS buffer solution with a pH of 7.4 containing 1.0mM CTAB, and the test is performed at 25°C. The probe molecule itself is in green fluorescence. After adding 60.0μM ClO- solution, the fluorescence intensity at 556nm decreases, the fluorescence intensity at 419nm increases, and the fluorescence intensity at 1 410nm / I 547nm The fluorescence emission intensity ratio reached the maximum ratio, increasing from 0.004 to 6.714, an increase of 1678 times.

[0051] The fluorescent probe of the present invention can quantitatively detect sodium hypochlorite (NaClO). Probe 1 has a good linear relationship with ClO- (35-60 μM), and the linear correlation coefficient can be calculated as: R 2 =0.97434, slope k is 0.24167. After scanning the blank group 5 times, σ=0.00041 (σ is the standard deviation of the blank sample) was calculated, and the detection limit was 5.1 nM according to LOD=3σ / k.

[0052] The probe of the present invention has a wide application range of pH, and can detect from pH=4.0 to pH=10.0.

[0053] The fluorescent probe of the present invention has good selectivity. After adding 60.0 μM ClO- solution, it can respond quickly, and after adding other detection substances (ONOO-, 1 O2, OH, Na + ,K + ,Ca 2+ ,PO4 3- ,H2PO4-,HPO4 2- Mg 2+ ,Zn 2+ ,SO4 2- ,SO3 2- ,HSO3 - ) after RT, the fluorescence hardly changed.

[0054] The fluorescent probe of the present invention has strong anti-interference ability and can detect other objects (ONOO-, 1 O2, OH, Na+ ,K + ,Ca 2+ ,PO4 3- ,H2PO4-,HPO4 2- Mg 2+ ,Zn 2+ ,SO4 2- ,SO3 2- ,HSO3 - ) and ClO- have almost no effect on the response of Probe 1 to ClO-.

[0055] The fluorescent probe of the present invention has low toxicity. The investigation of probe solutions and cells with different concentrations (0.0, 1.0, 2.0, 5.0, 10.0, 20.0 μM) shows that when the concentration of the probe does not exceed 20 μM, the survival rate of the cells is as high as 90%.

[0056] The fluorescent probe of the present invention has good cell membrane penetration and can be used for endogenous and exogenous ClO- detection in Hela cells and zebrafish.

[0057] The specific application of the probe molecule of the present invention is described below:

[0058] Application of probe molecules to detect hypochlorous acid (ClO-) in Hela cells:

[0059] Endogenous ClO- detection: After pre-treating Hela cells with lipopolysaccharide (LPS) and amphetamine acetate (PMA) for 1 hour, Hela cells were incubated with PBS buffer (containing 1% CTAB) containing the probe (10 μM) for 1 hour. After washing the cells three times with PBS buffer (containing 1% CTAB), cell fluorescence imaging experiments were performed using a laser confocal fluorescence microscope, and blue fluorescence signals could be seen.

[0060] Exogenous ClO- detection: Hela cells were first incubated with PBS buffer (containing 1% CTAB) containing ClO- (60 μM) for 1 hour, and then incubated with PBS buffer (containing 1% CTAB) containing the probe (10 μM) for 1 hour. The cells were rinsed three times with PBS buffer (containing 1% CTAB) and cell fluorescence imaging experiments were performed, and blue fluorescence signals could be seen.

[0061] Fig.12 This is the confocal cell imaging of the fluorescent probe of the present invention to detect ClO- in Hela cells. A1-A3 are the imaging effects of the probe (10.0 μM) incubated in cells at 37°C for 1 hour, with the green channel turned on. B1-B3 are the imaging effects of the cells first incubated with ClO- at 37°C. -(60.0μM) for 1 hour, followed by the probe (10.0μM) for 1 hour. It can be found that the green fluorescence disappears and the cells show blue fluorescence. At this time, the green channel is closed and the blue channel is open. C1-C3 are the imaging effects of cells first incubated with PBS buffer solution (containing 1% CTAB) containing lipopolysaccharide (LPS, 1.0μg / ml) and amphetamine acetate (PMA, 1.0μg / ml) at 37°C for 1 hour, and then incubated with the probe (10.0μM) for 1 hour. At this time, the blue channel is open.

[0062] Application of probe molecules to detect hypochlorous acid (ClO-) in zebrafish:

[0063] Blank group experiment: The zebrafish were not treated in any way and then imaged using a confocal microscope, so no fluorescence was produced by the zebrafish.

[0064] Probe group experiment: After adding only 10.0 μM probe solution and incubating with 3-day-old zebrafish for 1 hour, the cells were washed three times with PBS buffer (containing 1% CTAB), and cell fluorescence imaging experiments were performed using a laser confocal fluorescence microscope, and strong green fluorescence was observed.

[0065] Exogenous ClO- detection: 3-day-old zebrafish were pretreated with 60.0 μM ClO- solution for 1 h, then 10.0 μM probe solution was added and cultured for 1 h. After the cells were washed three times with PBS buffer (containing 1% CTAB), cell fluorescence imaging experiments were performed using a laser confocal fluorescence microscope, and blue fluorescence signals could be seen.

[0066] Endogenous ClO- detection: After pretreatment with lipopolysaccharide (LPS) and amphetamine acetate (PMA) for 1 hour, the probe (10.0 μM) was added and incubated for 1 hour. After washing the cells three times with PBS buffer (containing 1% CTAB), the cell fluorescence imaging experiment was performed using a laser confocal fluorescence microscope, and the blue channel was opened.

[0067] Fig.13The confocal cell imaging of ClO- in zebrafish detected by the fluorescent probe of the present invention, A1-A3 are blank groups, and the 3-day-old zebrafish are not treated with any confocal microscope imaging, so the zebrafish do not produce any fluorescence. B1-B3 are probe groups, and the imaging effect after only adding 10.0 μM probe solution and incubating with 3-day-old zebrafish for 1 hour, strong green fluorescence is observed, and the green channel is open at this time. C1-C3 are exogenous groups, and the imaging effect after 3-day-old zebrafish are pretreated with 60.0 μM ClO- solution for 1 hour, and then adding 10.0 μM probe solution to incubate for 1 hour, the fluorescence color changes from green to blue, and the blue channel is open at this time. D1-D3 are endogenous groups, and the 3-day-old zebrafish are pretreated with LPS and PMA for 1 hour, and the imaging effect after adding probe 1 (10.0 μM) and incubating for 1 hour is the same as the imaging effect of the exogenous group, and the blue channel is open.

Claims

1. A ratiometric fluorescent probe for detecting ClO- based on pyranoquinoline, characterized in that: Its structural formula is:

2. The method for preparing a ratiometric fluorescent probe for detecting ClO- based on pyranoquinoline according to claim 1, characterized in that: The synthesis method comprises the following steps: (1) Add salicylaldehyde and 3-bromopropyne into a flask, then add K2CO3 to provide an alkaline environment, then add the first DMF solvent, rotate at room temperature for 10 hours, and after the reaction is completed, extract with DCM to obtain a light brown solid, namely compound 2; (2) Compound 2 and 4-aminobenzyl alcohol were added to a flask, CuI and (CF3SO3)3La were added as catalysts, CH3CN was added dropwise in an amount sufficient to cover the drugs, the flask was placed in an oil bath, magnetically stirred and refluxed at 80°C for 5-6h, the caked matter was removed by suction, the acetonitrile in the flask was removed by rotary evaporation at 45°C, and the crude product was purified by 100-200 mesh silica gel column chromatography to obtain a light yellow solid, i.e., Compound 3; (3) Compound 3 and MnO2 were added to a reaction flask, and then CH2Cl2 was added to the reaction flask, and the reaction was carried out at room temperature for 5 hours, wherein MnO2 was added in small amounts several times. After the reaction was completed, the mixture was filtered with diatomaceous earth and washed with dichloromethane to obtain a crude product. The solvent was removed under reduced pressure on a rotary evaporator, and the obtained crude product was purified by 100-200 mesh silica gel column chromatography to obtain a light yellow solid, namely compound 4; (4) Compound 5 (4-bromo-1,8-naphthalene dicarboxylic anhydride) and n-butylamine were mixed in ethanol, refluxed for 10 h, cooled to room temperature, filtered to obtain an off-white precipitate, and the filter cake was washed with ethanol to obtain the product on the filter paper, i.e., compound 6; (5) Compound 6 was dissolved in the second DMF, and then H2N-NH2·H2O was added dropwise, and the temperature was refluxed at 110°C for 3 h. After the reaction was completed, water was added to precipitate the product, and the product on the filter paper was filtered off and dried in a vacuum oven at 45°C to obtain an orange solid, namely compound 7; (6) Compound 4 and anhydrous ethanol were added to a single-necked flask and heated to 80°C. When the solid was completely dissolved, compound 7 was added to the single-necked flask and glacial acetic acid was added at the same time. The mixture was refluxed at 80°C for 4 hours. After the reaction was completed, the single-necked flask was taken out from the oil bath, and the product was recrystallized in ethanol. When the solution was cooled to room temperature, solids precipitated. After suction filtration, the filter cake was washed with ethanol to obtain an orange-red solid, which was a ratiometric fluorescent probe for detecting ClO- based on pyranoquinoline; The molar volume ratio of salicylaldehyde, 3-bromopropyne, K2CO3, compound 2, 4-aminobenzyl alcohol, CuI, (CF3SO3)3La, compound 3, MnO2, compound 5, n-butylamine, compound 6, compound 4, compound 7, the first DMF, CH2Cl2, ethanol, the second DMF, H2N-NH2·H2O, anhydrous ethanol, and glacial acetic acid is: 10:11:10:5:5:5:0.5:0.7:9.8:3.6:3.6:1.5:0.25:0.25:5:10:20:4:3:15:1; The compound 2 is 6-(prop-2-yn-1-oxy)cyclohexa-1,3-diene-1-carbaldehyde, the compound 3 is (6H-chromeno[4,3-b]quinolin-9-yl)methanol, the compound 4 is 6H-chromeno[4,3-b]quinoline-9-carbaldehyde, the compound 6 is 6-bromo-2-butyl-1H-benzo[de]isoquinoline-1,3(2H)-dione, and the compound 7 is 4-hydrazino-N-butyl-1,8-naphthalimide.

3. The method for preparing a ratiometric fluorescent probe for detecting ClO- based on pyranoquinoline according to claim 2, characterized in that: The synthesis method comprises the following steps: (1) Salicylaldehyde (1.220 g, 10 mmol) and 3-bromopropyne (1.298 g, 11 mmol) were added to a flask, and K2CO3 (1.382 g, 10 mmol) was added to provide an alkaline environment. Then, 5 ml of the first DMF solvent was added and the mixture was rotated at room temperature for 10 h. After the reaction was completed, DCM was used for extraction to obtain a light brown solid, namely compound 2; (2) Compound 2 (0.900 g, 5 mmol) and 4-aminobenzyl alcohol (0.615 g, 5 mmol) were added to a flask, CuI (0.952 g, 5 mmol) and (CF3SO3)3La (0.293 g, 0.5 mmol) were added as catalysts, CH3CN was added dropwise in an amount sufficient to cover the drugs, the flask was placed in an oil bath, magnetically stirred and refluxed at 80°C for 5-6 h, the caked matter was removed by suction, the acetonitrile in the flask was removed by rotary evaporation at 45°C, and the crude product was purified by 100-200 mesh silica gel column chromatography to obtain a light yellow solid, i.e., Compound 3; (3) Compound 3 (0.2 g, 0.7 mmol) and MnO2 (0.925 g, 9.8 mmol) were added to a reaction flask, and then 10 ml of CH2Cl2 was added to the reaction flask. The mixture was reacted at room temperature for 5 h, wherein MnO2 was added in small amounts several times. After the reaction was completed, the mixture was filtered with diatomaceous earth and washed with dichloromethane to obtain a crude product. The solvent was removed under reduced pressure on a rotary evaporator, and the obtained crude product was purified by 100-200 mesh silica gel column chromatography to obtain a light yellow solid, i.e., compound 4. (4) Compound 5 (4-bromo-1,8-naphthalene dicarboxylic anhydride) (1.000 g, 3.600 mmol) and n-butylamine (0.26399 g, 3.600 mmol) were dissolved in 20 ml of ethanol and refluxed for 10 h. The mixture was cooled to room temperature and filtered to obtain an off-white precipitate. The filter cake was washed with ethanol to obtain the product on the filter paper, i.e., Compound 6. (5) Compound 6 (0.516 g, 1.5 mmol) was dissolved in 4 ml of the second DMF, and then 3 ml of H2N-NH2·H2O was added dropwise. The reaction mixture was refluxed at 110°C for 3 h. After the reaction was completed, water was added to precipitate the product. The product on the filter paper was filtered off and dried in a vacuum oven at 45°C to obtain an orange solid, i.e., Compound 7. (6) Compound 4 (0.06925 g, 0.25 mmol) and 15 ml of anhydrous ethanol were added to a single-necked flask and heated to 80°C. When the solid was completely dissolved, compound 7 (0.07075 g, 0.25 mmol) was added to the single-necked flask and 1 ml of glacial acetic acid was added. The mixture was refluxed at 80°C for 4 h. After the reaction was completed, the single-necked flask was taken out of the oil bath and the product was recrystallized in ethanol. When the solution was cooled to room temperature, solid precipitated. After filtration, the filter cake was washed with ethanol to obtain an orange-red solid, which is a ratiometric fluorescent probe for detecting ClO- based on pyranoquinoline.

4. The method for preparing a ratiometric fluorescent probe for detecting ClO- based on pyranoquinoline according to claim 2, characterized in that: The eluent for the chromatographic purification in step 2 is dichloromethane / ethyl acetate in a volume ratio of 7 / 1.

5. The method for preparing a ratiometric fluorescent probe for detecting ClO- based on pyranoquinoline according to claim 2, characterized in that: The eluent for the chromatography purification in step 3 is petroleum ether / dichloromethane in a volume ratio of 1 / 1.

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