Hypochlorous acid fluorescent probe as well as preparation method and use method thereof

Abstract

The invention discloses a fluorescent probe for detecting hypochlorous acid as well as a preparation method and a use method of the fluorescent probe. The probe takes isophorone as a fluorescent mother group and 4-aminophenol as a recognition group of hypochlorous acid, and can detect the concentration level of hypochlorous acid in a water phase with high selectivity and high sensitivity. The probe shows orange fluorescence, under the condition that hypochlorous acid exists, hypochlorous acid and 4-aminophenol on probe molecules are subjected to a specific oxidation-hydrolysis reaction, an amino isophorone product is generated, the ICT effect of the molecules is enhanced, the emission wavelength of the probe molecules is subjected to red shift, and red fluorescence is emitted. According to the scheme, the probe obtains'ratio 'type fluorescence response on hypochlorous acid, and high-sensitivity and specific detection on hypochlorous acid is realized.

Description

Technical Field

[0001] This invention belongs to the field of organic small molecule fluorescent probes, specifically relating to a fluorescent probe for use as hypochlorous acid. E Preparation and usage of 2-(3-(4-aminostyryl)-5,5-dimethylcyclohex-2-ene-1-yl)malononitrile. Background Technology

[0002] Hypochlorous acid (HOCl / ClO⁻) is an important reactive oxygen species (ROS) in living organisms, mainly produced by the reaction of chloride ions and hydrogen peroxide catalyzed by myeloperoxidase (MPO). It plays a crucial physiological role in maintaining cellular redox homeostasis and participating in immune defense. However, excessive hypochlorous acid can lead to severe oxidative stress, damaging biomolecules (such as proteins, nucleic acids, and lipids) and cellular structures, and is closely related to various inflammatory diseases, neurodegenerative diseases, and tissue damage. Therefore, specific and sensitive detection of hypochlorous acid levels in living organisms is of great significance for a deeper understanding of its related physiological and pathological mechanisms.

[0003] Currently, methods for detecting hypochlorous acid mainly include electrochemical methods, ultraviolet-visible spectrophotometry, chemiluminescence methods, and chromatography. However, these conventional methods generally suffer from drawbacks such as cumbersome operation, the need for large and precise instruments, high detection costs, long processing times, and insufficient sensitivity, making it difficult to meet the needs for in-situ, real-time, and dynamic monitoring of complex biological samples (such as living cells and tissues). In contrast, fluorescent probe technology, with its advantages of non-invasiveness, high sensitivity and selectivity, ease of operation, rapid response, and the ability to achieve spatiotemporal resolution imaging, shows great potential in the field of biomedical detection, especially suitable for real-time visual analysis of hypochlorous acid in vivo. Most of the reported hypochlorous acid fluorescent probes are "turn-on" type (single-channel enhanced type). The fluorescence signal intensity of these probes is easily affected by various factors such as the local concentration distribution of the probe, fluctuations in instrument efficiency, environmental factors (such as light source stability and photobleaching), and background interference from the sample itself, leading to reduced reliability and accuracy of the detection results. Ratio-modified fluorescent probes quantify fluorescence by measuring the ratio of fluorescence intensity at two different wavelengths. They possess internal reference calibration capabilities, effectively overcoming the limitations of single-channel probes and significantly improving the anti-interference ability and accuracy of detection. Although some ratio-modified hypochlorous acid fluorescent probes have been reported, existing probes still exhibit false-positive signals to coexisting interfering substances such as other reactive oxygen species (e.g., H₂O₂, •OH) or biothiols (e.g., GSH, Cys) in vivo, limiting their reliable application under physiological / pathological conditions (see review Nahyun Kwon, Yahui Chen, Xiaoqiang Chen, MyungHwa Kim, Juyoung Yoon,Dyes and Pigments (2022, 200, 110132.). Therefore, developing novel ratiometric hypochlorous acid fluorescent probes with high selectivity and good biocompatibility to meet the need for accurate and reliable detection in complex biological systems remains a pressing technical challenge, and is of great significance for the study of the mechanisms of related diseases and early diagnosis. Summary of the Invention

[0004] To overcome the aforementioned shortcomings of existing technologies, this invention proposes a ratiometric fluorescent probe (IPH) for detecting hypochlorous acid. This invention allows for the quantitative detection of hypochlorite content in samples.

[0005] The core of this invention lies in the construction of a classic intramolecular charge transfer (ICT) fluorescent probe using isophorone fluorescent parent groups. Due to its ICT effect, the probe itself exhibits yellow fluorescence. However, in the presence of hypochlorous acid, the hypochlorous acid specifically oxidizes the 4-aminophenol on the probe molecule, exposing aminoisoflurone, thereby modulating the ICT effect of the probe molecule and causing it to emit red fluorescence. Through this scheme, a ratiometric fluorescence response is obtained, achieving highly sensitive and specific detection of hypochlorous acid.

[0006] The hypochlorous acid fluorescent probe described in this invention is named IPH, and its structural formula is shown in formula (I): The preparation method of the above fluorescent probe is as follows: a certain amount of aminoisoflurone (1), triphosgene (2) and triethylamine (3) are dissolved in dichloromethane and reacted at a certain temperature for a period of time to obtain compound ( E )-(4-(2-(3-(dicyanomethylene)-5,5-dimethylcyclohexyl-1-en-1-yl)vinyl)phenyl)carbamoyl chloride (4); tert-butyl (4-hydroxyphenyl)carbamate (5) was dissolved in dichloromethane and added to compound (4), and reacted at a certain temperature for a period of time. After the reaction was complete, the solution was evaporated to dryness and subjected to column chromatography to obtain a yellow solid compound 4-((tert-butyloxycarbonyl)amino)phenyl( E )-(4-(2-(3-(dicyanomethylene)-5,5-dimethylcyclohex-1-en-1-yl)vinyl)phenyl)carbamate (6), then (6) was dissolved in dichloromethane, trifluoroacetic acid (7) was added, and the reaction was carried out at a certain temperature for a period of time. After the reaction was complete, it was extracted with dichloromethane and water, and the organic phase was dried by rotary evaporation to obtain an orange solid ( E )-(4-(2-(3-(dicyanomethylene)-5,5-dimethylcyclohex-1-en-1-yl)vinyl)phenyl)carbamate p-aminophenyl ester (8), i.e. IPH.

[0007] Among them ( EThe reaction of 2-(3-(4-aminostyryl)-5,5-dimethylcyclohexyl-2-en-1-yl)malonadionitrile, triphosgene, and triethylamine in dichloromethane is carried out at a temperature of 0-30°C for 1-24 hours. Tert-butyl (4-hydroxyphenyl)carbamate is then added and reacted for a further period at a temperature of 0-30°C for 1-24 hours. Preferably, the reaction temperature is 25°C and the reaction time is 3 hours. Preferably, ( E The molar ratio of 2-(3-(4-aminostyryl)-5,5-dimethylcyclohexyl-2-en-1-yl)malononitrile, triphosgene, triethylamine, and tert-butyl (4-hydroxyphenyl)carbamate is preferably 1:1~10:1~10:2~10. E The molar ratio of 2-(3-(4-aminostyryl)-5,5-dimethylcyclohex-2-ene-1-yl)malonitrile, triphosgene, triethylamine, and tert-butyl (4-hydroxyphenyl)carbamate is 1:1:1:2.5. in( E The molar volume ratio of 4-(2-(3-(dicyanomethylene)-5,5-dimethylcyclohexyl-1-en-1-yl)vinyl)phenyl)carbamate to dichloromethane and trifluoroacetic acid is 1:6~250:2~70; preferably, the molar / volume ratio is 0.13:3:1. The addition of trifluoroacetic acid to remove the protecting group yields (… E The reaction of p-aminophenyl carbamate with the following reaction temperature is 0-30 degrees Celsius and reaction time is 1-24 hours; preferably, the reaction temperature is 25 degrees Celsius and the reaction time is 1 hour.

[0008] The reaction formula for preparing the above probe is as follows: The usage method of the above-mentioned hypochlorous acid fluorescent probe is as follows: Step 1: Add the same concentration of the compound shown in formula (I) to hypochlorous acid solutions of different concentrations to prepare at least 5 standard solutions containing the compound shown in formula (I) with different hypochlorous acid contents. The concentration of the compound represented by formula (I) in the standard solution shown is 10 μM; The hypochlorous acid content in the standard solutions shown is 5 μM ~ 45 μM; Step 2: Measure the fluorescence emission spectra of the standard solutions respectively, with an excitation wavelength of 420 nm. Plot the hypochlorous acid concentration on the x-axis and Ig on the y-axis. 550 and I 640 Establish a standard curve with the vertical axis as the ordinate; I 550This indicates the fluorescence emission peak intensity value of the standard solution at a wavelength of 550 nm; I 640 This indicates the fluorescence emission peak intensity value of the standard solution at a wavelength of 640 nm; Step 3: Add the compound shown in formula (I) to the sample to be tested, and control its concentration to be equal to the concentration of the compound shown in formula (I) in the standard solution; measure its fluorescence emission spectrum under excitation light with an excitation wavelength of 420 nm, and calculate the hypochlorous acid content of the sample to be tested based on the standard curve.

[0009] This invention has the following characteristics: 1) The fluorescent probe provided by this invention is an orange solid powder with good optical stability.

[0010] 2) The fluorescent probe provided by this invention has a solution that is sensitive to the concentration of hypochlorous acid. As the concentration of hypochlorous acid increases, the fluorescence of its aqueous solution changes from orange to red under a fluorescent lamp.

[0011] 3) The fluorescent probe provided by this invention has an emission wavelength of 550 nm. After the addition of hypochlorous acid, a new emission peak appears at 640 nm, which is a "ratio" type response. This can greatly eliminate the influence of differences in the detection environment on the results and improve the sensitivity of the detection.

[0012] 4) The fluorescent probe provided by this invention has a linear relationship with the concentration of hypochlorous acid and can be used for accurate measurement of hypochlorous acid.

[0013] The isophorone dye-based ratio-type hypochlorous acid probe provided by this invention has a good response to hypochlorous acid solution, enabling sensitive quantitative detection of hypochlorous acid in samples. It has the advantages of simple operation, low cost, sensitive response, and easy promotion and application. Attached Figure Description

[0014] Figure 1 : The proton NMR spectrum of the fluorescent probe IPH.

[0015] Figure 2 Color response of the fluorescent probe IPH to hypochlorous acid solution under visible light.

[0016] Figure 3 Color response of the fluorescent probe IPH to hypochlorous acid solution under fluorescent light.

[0017] Figure 4 UV titration curve of the fluorescent probe IPH in solution with hypochlorous acid, where the probe concentration is 10.0 μM.

[0018] Figure 5The fluorescence titration curve of the fluorescent probe IPH in solution for hypochlorous acid, with an excitation wavelength of 420 nm and a probe concentration of 10.0 μM.

[0019] Figure 6 The fluorescence response of the fluorescent probe IPH to common small molecules is shown in the figure, where the excitation wavelength is 420 nm, the probe concentration is 10.0 μM, and the analyte concentration is 50.0 μM. Detailed Implementation

[0020] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0021] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0022] The compound numbers in the examples correspond to the numbers in the compounds described above.

[0023] Example 1: Synthesis of compound IPH.

[0024] compound ( E Synthesis of p-aminophenyl carbamate (8) of 4-(2-(3-(dicyanomethylene)-5,5-dimethylcyclohex-1-en-1-yl)vinyl)phenyl)carbamate.

[0025] 80 mg (0.27 mmol) E )-2-(3-(4-aminostyryl)-5,5-dimethylcyclohex-2-en-1-yl)malonadionitrile (1), 82 mg (0.27 mmol) triphosgene (2) was dissolved in 2 mL dichloromethane, and then 193 μL (0.27 mmol) triethylamine (3) was added. After reacting at 25 °C for 1 hour, compound ( E )-(4-(2-(3-(dicyanomethylene)-5,5-dimethylcyclohexyl-1-en-1-yl)vinyl)phenyl)carbamoyl chloride (4). 70 mg (0.69 mmol) of tert-butyl (4-hydroxyphenyl)carbamate (5) was dissolved in 1 mL of dichloromethane and slowly injected into the reaction solution of compound (4). After reacting at 25 °C for three hours, rotary cyclohexane chromatography yielded 72 mg of compound 4-((tert-butyloxycarbonyl)amino)phenyl( E )-(4-(2-(3-(dicyanomethylene)-5,5-dimethylcyclohex-1-en-1-yl)vinyl)phenyl)carbamate (6), compound (6) was dissolved in 1.5 mL of dichloromethane, and 0.5 mL of trifluoroacetic acid (7) was added. After reacting at 25 °C for one hour, the mixture was extracted with dichloromethane and water, and the organic phase was evaporated to dryness to give 58 mg of orange solid ( E)-(4-(2-(3-(dicyanomethylene)-5,5-dimethylcyclohexyl-1-en-1-yl)vinyl)phenyl)carbamate p-aminophenyl ester (8), i.e., IPH, yield 50%. NMR spectra are as follows: Figure 1 As shown.

[0026] 1 H NMR (400 MHz, DMSO- d 6) δ 10.51 (s, 1H), 7.67 (d, J = 8.4 Hz, 2H), 7.54(d, J = 8.4 Hz, 2H), 7.34 – 7.21 (m, 6H), 6.83 (s, 1H), 2.59 (s, 2H), 2.52 (s, 2H), 1.00 (s, 6H). Example 2: Synthesis of compound IPH.

[0027] 80 mg (0.27 mmol) E )-2-(3-(4-aminostyryl)-5,5-dimethylcyclohex-2-en-1-yl)malonadionitrile (1), 410 mg (1.35 mmol) triphosgene (2) was dissolved in 2 mL of dichloromethane, and then 965 μL (1.35 mmol) triethylamine (3) was added. After reacting at 25 °C for 1 hour, compound ( E )-(4-(2-(3-(dicyanomethylene)-5,5-dimethylcyclohexyl-1-en-1-yl)vinyl)phenyl)carbamoyl chloride (4). 140 mg (1.38 mmol) of tert-butyl (4-hydroxyphenyl)carbamate (5) was dissolved in 2 mL of dichloromethane and slowly injected into the reaction solution of compound (4). After reacting at 25 °C for three hours, rotary cyclohexane chromatography was used to obtain compound 4-((tert-butyloxycarbonyl)amino)phenyl( E )-(4-(2-(3-(dicyanomethylene)-5,5-dimethylcyclohex-1-en-1-yl)vinyl)phenyl)carbamate (6), compound (6) was dissolved in 0.5 mL of dichloromethane, and 0.17 mL of trifluoroacetic acid (7) was added. After reacting at 25 °C for one hour, the mixture was extracted with dichloromethane and water, and the organic phase was evaporated to dryness to give 50 mg of orange solid ( E )-(4-(2-(3-(dicyanomethylene)-5,5-dimethylcyclohex-1-en-1-yl)vinyl)phenyl)carbamate p-aminophenyl ester (8), i.e.: IPH, yield 45%.

[0028] Example 3: Synthesis of compound IPH.

[0029] 80 mg (0.27 mmol) E )-2-(3-(4-aminostyryl)-5,5-dimethylcyclohex-2-en-1-yl)malonadionitrile (1), 820 mg (2.7 mmol) triphosgene (2) were dissolved in 2 mL of dichloromethane, and then 1930 μL (2.7 mmol) triethylamine (3) were added. After reacting at 25 °C for 1 hour, compound ( E )-(4-(2-(3-(dicyanomethylene)-5,5-dimethylcyclohexyl-1-en-1-yl)vinyl)phenyl)carbamoyl chloride (4). 280 mg (2.76 mmol) of tert-butyl (4-hydroxyphenyl)carbamate (5) was dissolved in 1 mL of dichloromethane and slowly injected into the reaction solution of compound (4). After reacting at 25 °C for three hours, rotary cyclohexane chromatography was used to obtain compound 4-((tert-butyloxycarbonyl)amino)phenyl( E )-(4-(2-(3-(dicyanomethylene)-5,5-dimethylcyclohexyl-1-en-1-yl)vinyl)phenyl)carbamate (6), compound (6) was dissolved in 15 mL of dichloromethane, and 4.5 mL of trifluoroacetic acid (7) was added. After reacting at 25 °C for one hour, the mixture was extracted with dichloromethane and water, and the organic phase was evaporated to dryness to give 53 mg of orange solid ( E )-(4-(2-(3-(dicyanomethylene)-5,5-dimethylcyclohex-1-en-1-yl)vinyl)phenyl)carbamate p-aminophenyl ester (8) i.e.: IPH.

[0030] Example 3: Color response of compound IPH to hypochlorous acid.

[0031] Prepare a 1 mM dimethyl sulfoxide (DMSO) test stock solution of the fluorescent probe IPH for detecting hypochlorous acid described in this invention. Measure 50 μL of this stock solution and add it dropwise to a hypochlorous acid solution of ethanol:PBS (1:1 ratio), and then dilute to 5 mL with the corresponding ethanol:PBS (1:1 ratio) solution, so that the probe concentration in the test solution is 10.0 μM and the hypochlorous acid concentration is 50.0 μM. Then proceed with the color response test. Figure 2 and 3 As shown, after adding hypochlorous acid solution, the color of the solution changed from light yellow to orange, and the fluorescence of the solution also changed from orange fluorescence to red fluorescence, indicating that the probe IPH has a direct colorimetric and fluorescence response to hypochlorous acid.

[0032] Example 4: Ultraviolet titration detection of compound IPH with different concentrations of hypochlorous acid.

[0033] Prepare a 1 mM dimethyl sulfoxide (DMSO) test stock solution of the fluorescent probe IPH for detecting hypochlorous acid described in this invention. Measure 50 μL of this stock solution and add it dropwise to a hypochlorous acid solution of ethanol:PBS (1:1 ratio), and then dilute to 5 mL with the corresponding ethanol:PBS (1:1 ratio) solution to achieve a probe concentration of 10.0 μM and a hypochlorous acid concentration of 0-45.0 μM. Perform absorption spectroscopy measurements. Obtain the UV absorption curves for each system and establish a standard curve for absorbance versus hypochlorous acid concentration. Figure 4 As shown, with the increase of hypochlorous acid concentration, the absorbance at 419 nm gradually decreases and the absorbance at 500 nm gradually increases. When the hypochlorous acid concentration is 30.0 μM, the absorbance in the reaction system reaches equilibrium.

[0034] Example 5: Fluorescent titration detection of compound IPH with different concentrations of hypochlorous acid.

[0035] Prepare a 1 mM dimethyl sulfoxide (DMSO) test stock solution of the fluorescent probe IPH for detecting hypochlorous acid described in this invention. Measure 50 μL of this stock solution and add it dropwise to a hypochlorous acid solution of ethanol:PBS (1:1 ratio), and then dilute to 5 mL with the corresponding ethanol:PBS (1:1 ratio) solution to achieve a probe concentration of 10.0 μM and a hypochlorous acid concentration of 0-45.0 μM for fluorescence detection. (λex = 420 nm, λem1 = 550 nm, λem2 = 640 nm). Measure the fluorescence intensity in each system and establish a standard curve of fluorescence intensity versus hypochlorous acid concentration. Figure 5 As shown, with increasing hypochlorous acid concentration, the fluorescence intensity at 550 nm gradually decreases, while the fluorescence intensity at 640 nm gradually increases. When the hypochlorous acid concentration reaches 30.0 μM, the fluorescence intensity in the reaction system reaches equilibrium.

[0036] Example 6: Selectivity of compound IPH for different common reactive oxygen species.

[0037] Prepare a 1 mM test stock solution of dimethyl sulfoxide (DMSO) for the detection of hypochlorous acid using the fluorescent probe IPH described in this invention. Prepare 10 mM solutions of various small reactive oxygen species (ROS) for testing. Measure 50 μL of this stock solution and add it dropwise to ethanol:PBS (1:1) solutions of different small molecule analytes, and then dilute to 5 mL with the corresponding ethanol:PBS (1:1) solutions to achieve a probe concentration of 10.0 μM and an analyte concentration of 50.0 μM in the test solution for fluorescence detection (λex = 420 nm, λem1 = 550 nm, λem2 = 640 nm). Measure the fluorescence intensity in each system and establish a fluorescence intensity (Ig) measurement system. 640 / I 550 A bar chart showing the relationship between the analytes and the various test objects. (e.g.) Figure 6 As shown, other common small molecules to be tested have almost no effect on the fluorescence of the probe IPH.

Claims

1. A fluorescent probe for detecting hypochlorous acid, characterized in that: Its molecular formula C 26 H 24 N4O2, abbreviated as IPH, has the structural formula (I). 。 2. The method for preparing a fluorescent probe for detecting hypochlorous acid according to claim 1, characterized in that, The synthesis steps are as follows: Will( E 2-(3-(4-aminostyryl)-5,5-dimethylcyclohex-2-en-1-yl)malonitrile, triphosgene, and triethylamine were dissolved in dichloromethane. After reacting for a certain time, tert-butyl (4-hydroxyphenyl)carbamate was added and reacted for another certain time. Column chromatography yielded 4-(tert-butyloxycarbonyl)amino)phenyl ( E )-(4-(2-(3-(dicyanomethylene)-5,5-dimethylcyclohex-1-en-1-yl)vinyl)phenyl)carbamate, finally dissolved in dichloromethane, and deprotected by adding trifluoroacetic acid to obtain ( E p-Aminophenyl carbamate, denoted as IPH, is a carbamate derived from 4-(2-(3-(dicyanomethylene)-5,5-dimethylcyclohexyl-1-en-1-yl)vinyl)phenyl)carbamate.

3. The method for preparing a fluorescent probe for detecting hypochlorous acid according to claim 1, characterized in that: In i), ( E The molar ratio of 2-(3-(4-aminostyryl)-5,5-dimethylcyclohexyl-2-en-1-yl)malononitrile, triphosgene, triethylamine, and tert-butyl (4-hydroxyphenyl)carbamate is 1:1~10:1~10:2~10, wherein ( E The reaction of 2-(3-(4-aminostyryl)-5,5-dimethylcyclohex-2-ene-1-yl)malonitrile, triphosgene, and triethylamine in dichloromethane is carried out at a temperature of 0-30 degrees Celsius for 1-24 hours.

4. The method for preparing a fluorescent probe for detecting hypochlorous acid according to claim 1, characterized in that: Add tert-butyl (4-hydroxyphenyl)carbamate and react for a certain period of time, with the reaction temperature ranging from 0 to 30 degrees Celsius and the reaction time from 1 to 24 hours.

5. The method for preparing a fluorescent probe for detecting hypochlorous acid according to claim 1, characterized in that: The (mentioned) E The molar volume ratio of 4-(2-(3-(dicyanomethylene)-5,5-dimethylcyclohexyl-1-en-1-yl)vinyl)phenyl)carbamate to dichloromethane and trifluoroacetic acid is 1:6~250:2~70; the addition of trifluoroacetic acid to remove the protecting group yields ( E The reaction of p-aminophenyl carbamate with the following reaction temperature is 0~30 degrees and the reaction time is 1~24 hours.

6. The method for preparing a fluorescent probe for detecting hypochlorous acid according to claim 1, characterized in that: i) ( E The molar ratio of 2-(3-(4-aminostyryl)-5,5-dimethylcyclohex-2-ene-1-yl)malonitrile, triphosgene, triethylamine, and tert-butyl (4-hydroxyphenyl)carbamate is 1:1:1:2.

5.

7. The method for preparing a fluorescent probe for detecting hypochlorous acid according to claim 1, characterized in that: The one mentioned is ( E The reaction of 2-(3-(4-aminostyryl)-5,5-dimethylcyclohex-2-en-1-yl)malonitrile, triphosgene, and triethylamine in dichloromethane was carried out at a temperature of 25 degrees Celsius for 3 hours; the reaction of adding tert-butyl (4-hydroxyphenyl)carbamate was carried out at a temperature of 25 degrees Celsius for 3 hours.

8. The method for preparing a fluorescent probe for detecting hypochlorous acid according to claim 1, characterized in that: ( E The molar / volume ratio of 4-(2-(3-(dicyanomethylene)-5,5-dimethylcyclohex-1-en-1-yl)vinyl)phenyl)carbamate to dichloromethane and trifluoroacetic acid was 0.13:3:1; the reaction temperature was 25 degrees Celsius and the reaction time was 1 hour.

9. A method for using a fluorescent probe for detecting hypochlorous acid; characterized in that: 1) Add the same concentration of the compound shown in formula (I) to aqueous solutions of hypochlorous acid of different concentrations to prepare at least 5 standard solutions containing the compound shown in formula (I) with different hypochlorous acid contents. The concentration of the compound represented by formula (I) in the standard solution shown is 1 nM to 10 μM; The hypochlorous acid content in the standard solution shown is 0.1 nM to 1 mM; 2) Measure the fluorescence emission spectra of the standard solutions respectively, with an excitation wavelength of 420 nm. Plot the hypochlorous acid concentration on the x-axis and Ig on the y-axis. 550 and I 640 Establish a standard curve with the vertical axis as the ordinate; I 550 This indicates the fluorescence emission peak intensity value of the standard solution at a wavelength of 550 nm; I 640 This indicates the fluorescence emission peak intensity value of the standard solution at a wavelength of 640 nm; 3) Add the compound of formula (I) to the sample to be tested, and control its concentration to be equal to the concentration of the compound of formula (I) in the standard solution; The fluorescence emission spectrum of the sample was measured under excitation light with an excitation wavelength of 420 nm, and the hypochlorous acid content of the sample was calculated based on the standard curve.

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