A fluorescent probe applied to hypochlorous acid detection, a preparation method and application thereof
By preparing fluorescent probe molecules, the problems of complex and slow response of hypochlorous acid detection methods were solved, and rapid, simple and efficient hypochlorous acid detection was achieved, which is suitable for real-time and on-site applications.
Patent Information
- Application Number
- CN202411563760.3
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2024-11-05
- Publication Date
- 2025-10-24
- Estimated Expiration
- 2044-11-05
AI Technical Summary
The existing detection methods for hypochlorous acid are complex to operate, have slow responses, are difficult to implement in real time and on-site, and pose health risks.
A fluorescent probe molecule was designed and synthesized through the nucleophilic addition reaction of 4-dimethylaminocinnamaldehyde and 1,3-indandione to prepare a fluorescent probe that can be used directly in water, has high selectivity and sensitivity, and can achieve rapid detection.
The method realizes rapid, simple, sensitive and efficient detection of hypochlorous acid, can specifically identify hypochlorite ions in a variety of ionic environments, and is suitable for real-time monitoring and field applications.
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Figure CN119431167B_ABST
Abstract
Description
TECHNICAL FIELD
[0001] The application belongs to the field of fluorescent probes, and particularly relates to a fluorescent probe for hypochlorous acid detection and a preparation method and application thereof. BACKGROUND
[0002] Hypochlorous acid (ClO - ) is widely used as a common disinfectant. Although hypochlorous acid can effectively inactivate bacteria and pathogenic microorganisms at appropriate concentrations, excessive use or residues may have adverse effects on human health. Hypochlorous acid can irritate the eyes and respiratory tract at high concentrations, causing acute reactions such as eye pain and difficulty breathing. Long-term exposure or excessive intake of hypochlorous acid can lead to chronic health problems, such as respiratory diseases, skin allergies, or even toxic effects on internal organs; moreover, hypochlorous acid can also react with organic compounds in water to generate carcinogenic substances such as chloroform and carbon tetrachloride, seriously endangering human health.
[0003] Fluorescent probes can emit fluorescent signals through specific reactions with the sample to be measured, and the intensity of the fluorescent signals is proportional to the concentration of hypochlorous acid. Compared with traditional analysis methods such as chemical analysis or chromatographic analysis, fluorescent probes have the advantages of simple operation and rapid response, and can realize real-time detection and on-site application. Therefore, it is very important to design and synthesize a fluorescent probe that can efficiently and rapidly detect hypochlorous acid in real time for detecting the concentration of hypochlorous acid in a solution. SUMMARY
[0004] In order to overcome the shortcomings and deficiencies of the prior art, the purpose of the present application is to provide a fluorescent probe molecule and a preparation method thereof, which has the advantages of simple and convenient detection process, rapid and efficient detection, and high sensitivity for hypochlorous acid.
[0005] To solve the above technical problems, the technical scheme adopted by the present application is as follows:
[0006] A fluorescent probe for hypochlorous acid detection, the fluorescent probe has the following formula (I) or formula (II) shown in the molecular structure:
[0007]
[0008] The present application provides a preparation method of a fluorescent probe for hypochlorous acid detection, the preparation method of the fluorescent probe with formula (I) shown in the molecular structure comprises the following steps:
[0009]
[0010] The fluorescent probe with formula (I) structure is synthesized by nucleophilic addition reaction of 4-dimethylaminocinnamaldehyde and 1,3-indan dione.
[0011] Preferably, the preparation method of the fluorescent probe of the molecular structure shown in formula (II) comprises the following steps:
[0012] Step 1:
[0013]
[0014] Compound 1 is obtained by reacting 1,3-indanedione and malononitrile under the catalysis of sodium acetate;
[0015] Step 2:
[0016]
[0017] Then, compound 1 and 4-dimethylaminocinnamaldehyde are subjected to a nucleophilic addition reaction to synthesize the fluorescent probe of the structure shown in formula (II).
[0018] Preferably, the molar ratio of 4-dimethylaminocinnamaldehyde and 1,3-indanedione is 1:1.
[0019] Preferably, the molar ratio of 1,3-indanedione and malononitrile is 1:1; and the molar ratio of compound 1 and 4-dimethylaminocinnamaldehyde is 5:1.
[0020] Preferably, the fluorescent probe is purified by column chromatography separation, wherein the mobile phase of the column chromatography separation is dichloromethane-methanol, and the volume ratio is (6-10):1.
[0021] The application also provides an application of the fluorescent probe for hypochlorous acid detection in measuring, screening or detecting a sample containing hypochlorous acid.
[0022] Preferably, the sample containing hypochlorous acid is a solution containing hypochlorous acid, and the solution containing hypochlorous acid is a beverage or tap water.
[0023] Compared with the prior art, the technical scheme of the application has the following advantages:
[0024] The fluorescent probe prepared by the application has high selectivity and low detection limit, can specifically recognize hypochlorite ions even in a variety of ionic environments, has good selectivity for hypochlorite ions, has strong specificity, has a simple testing process, has a very sensitive reaction, ensures rapid detection, and can be directly used in water, greatly improves the convenience of detection, and can realize real-time monitoring and on-site application. BRIEF DESCRIPTION OF DRAWINGS
[0025] Figure 1 The fluorescence emission spectrum of the fluorescent probe 1 prepared in the embodiment of the application in different polar solvents is shown in the following figure;
[0026] Figure 2 The fluorescence emission spectrum of the fluorescent probe 2 prepared in the embodiment of the application in different polar solvents is shown in the following figure;
[0027] Figure 3 The fluorescence intensity change diagram of the fluorescent probe 1 prepared in the embodiment of the present application at 475nm for different concentrations of hypochlorous acid;
[0028] Figure 4 The fluorescence intensity change diagram of the fluorescent probe 2 prepared in the embodiment of the present application at 650nm for different concentrations of hypochlorous acid;
[0029] Figure 5 The fluorescence intensity result analysis diagram of the fluorescent probe 1 prepared in the embodiment of the present application at 475nm for adding other active molecules and hypochlorous acid;
[0030] Figure 6 The fluorescence intensity result analysis diagram of the fluorescent probe 2 prepared in the embodiment of the present application at 650nm for adding other active molecules and hypochlorous acid;
[0031] Figure 7 The color change diagram of the fluorescent probe 1 prepared in the embodiment of the present application under different concentrations of hypochlorous acid;
[0032] Figure 8 The linear relationship diagram of the colorimetric value of the fluorescent probe 1 prepared in the embodiment of the present application at 365nm and the concentration (0-120μM) of hypochlorous acid. DETAILED DESCRIPTION
[0033] In order to enable persons skilled in the art to better understand the scheme of the present application, the technical scheme in the embodiments of the present application will be described clearly and completely in combination with the drawings in the embodiments of the present application. Obviously, the described embodiments are only part of the embodiments of the present application, not all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by persons skilled in the art without creative labor are within the scope of protection of the present application.
[0034] Embodiment 1: Synthesis of fluorescent probe 1
[0035] The embodiment provides a synthesis method of a fluorescent probe applied to hypochlorous acid detection, and the specific steps are as follows:
[0036]
[0037] 1,3-Indandione (293.1 mg, 2 mmol) and 4-dimethylaminocinnamaldehyde (350.46 mL, 2 mmol) were dissolved in 20 mL of ethanol and stirred in an eggplant-shaped flask for 40 minutes. Two drops of piperidine were then added. After stirring, the mixture was refluxed at 85°C for 12 hours, filtered while hot, and washed with EtOH. Column chromatography using a mixture of CH2Cl2 and CH3OH (CH2Cl2:CH3OH, volume ratio 8:1) as the eluent yielded 0.55 g of a dark red solid, the fluorescent probe. The yield was 72%, designated fluorescent probe 1, and its chemical name was (E)-2-(3-(4-(dimethylamino)phenyl)allyl)-1H-indene-1,3(2H)-dione. The product was characterized by proton, carbon, and mass spectrometry. Analysis of the proton, carbon, and mass spectrometry data confirmed the structure of fluorescent probe 1.
[0038] The fluorescent probe 1 was characterized, and the results were as follows:
[0039] 1 H NMR(400MHz, CDCl3)δ8.25(d,J=12.6Hz,1H),7.91(s,2H),7.66(m,5H),7.32(s,1H),6.70(s,2H),3.08(s,6H).13C NMR (101MHz, CDCl3) δ191.13,153.69,152.48,146.56,142.07,140.76,134.48,134.3 3,131.37,124.43,123.72,122.64,122.40,119.36,111.90,40.13.HRMS(ESI):calcd for C 20 H 17 NO2[M+H]+304.1293,found 304.1332.
[0040] Example 2: Synthesis of fluorescent probe 2
[0041] (I) Synthesis of compound 1
[0042] Malononitrile (198.21 mg, 3 mmol) and 1,3-indanedione (439.39 mg, 3 mmol) were dissolved in 20 mL of pure ethanol and stirred in a flask for 40 minutes, then refluxed for 40 minutes. The mixture was filtered while hot and washed three times with anhydrous EtOH. After drying, the mixture was dissolved in acetic acid and recrystallized. The mixture was cooled overnight, filtered, and dried to obtain 561.09 g of a gray-black solid with an 88% yield. The resulting gray-black solid was 2-(3-oxo-2,3-dihydro-1H-inden-1-ylidene)malononitrile, designated Compound 1.
[0043] The compound 1 was characterized by hydrogen spectrum, and the results were as follows: 1 H NMR (400 MHz, Chloroform-d) δ 8.69 (d, J = 7.8 Hz, 1H), 8.07 - 8.00 (m, 1H), 7.98 - 7.83 (m, 2H), 3.82 - 3.71 (m, 2H).
[0044] (ii) Synthesis of fluorescent probe 2
[0045] 2-(3-oxo-2,3-dihydro-1H-inden-1-ylidene)malononitrile (388.39 mg, 10 mmol) and 4-dimethylaminocinnamaldehyde (349.80 mg, 2 mmol) were dissolved in 20 mL of ethanol, stirred in a flask for 40 min, and two drops of DIPEA were added. After stirring, it was refluxed at 85°C for 12 h, filtered while hot and washed with EtOH. Column chromatography was performed for purification with a mixture of CH2Cl2and CH3OH (volume ratio of CH2Cl2and CH3OH was 8:1) as eluent, and 0.52 g of blue-black solid was obtained, which was fluorescent probe 2, with a yield of 69%, named fluorescent probe 2, and chemically named (E)-2-(3-(4-(dimethylamino)phenyl)allyl)-1H-indene-1,3(2H)-dione. It was characterized by hydrogen spectrum, carbon spectrum and mass spectrum. Through analysis of the hydrogen spectrum, carbon spectrum and mass spectrum data of the compound, it was confirmed that the structure of the above fluorescent probe 2 was correct.
[0046] The characterization results of fluorescent probe 2 were as follows:
[0047] 1 H NMR (400 MHz, CDCl3) δ 8.65 (m, 2H), 8.50 (d, J = 11.8 Hz, 1H), 7.86 (m, 1H), 7.71 (m, 2H), 7.65 (t, J = 9.9 Hz, 2H), 7.39 (d, J = 14.7 Hz, 1H), 6.75 (d, J = 8.8 Hz, 2H), 3.13 (s, 6H).13C NMR (101 MHz, CDCl3) δ 189.75, 160.22, 155.89, 152.91, 148.88, 139.94, 137.31, 134.62, 134.00, 132.46, 125.10, 124.44, 123.35, 122.68, 120.40, 115.17, 115.07, 112.50, 67.68, 40.45. HRMS (ESI): calcd for C 23 H 17 N3O[M+H]+352.1405, found 352.1442.
[0048] Example 3: Fluorescence emission spectrum determination of fluorescent probes in different solutions
[0049] First, toluene, 1,4-dioxane, tetrahydrofuran, dichloromethane, dimethyl sulfoxide, ethanol, methanol, buffer and other different polarity solvents 5 mL, each solvent is configured two tubes, the fluorescent probe 1 and the fluorescent probe 2 are respectively dissolved in dimethyl sulfoxide and configured into a 1 mM mother liquor, and then 50 μL of each is added to the above solution, so that the final concentration of the probe is 10 μM. The fluorescence emission spectrum of different solutions is tested by fluorescence spectrometer to study the effect of different polarity solvents on fluorescent molecules, and the system that can exhibit excellent fluorescence ability of the probe is selected. The results are shown in Figure 1 and Figure 2 , wherein, Figure 1 is the fluorescence emission spectrum of fluorescent probe 1 in different polarity solvents; Figure 2 is the fluorescence emission spectrum of fluorescent probe 2 in different polarity solvents.
[0050] From Figure 1 , it can be seen that since the fluorescent probe 1 has good fluorescence performance in dimethyl sulfoxide, dimethyl sulfoxide is used as the test system solution of the fluorescent probe 1.
[0051] From Figure 2 , it can be seen that since the fluorescent probe 2 has good fluorescence performance in ethanol, ethanol is used as the test solution of the fluorescent probe 2.
[0052] Example 4: Fluorescence intensity change detection of fluorescent probe and different concentrations of hypochlorite solution
[0053] (1) Detection of fluorescent probe 1 on different concentrations of hypochlorite solution
[0054] First, the fluorescent probe 1 is dissolved in dimethyl sulfoxide to configure a 1 mM detection mother liquor; prepare a 1 mM-20 mM (1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 mM) interval of hypochlorite test mother liquor, and then 50 μL of the above detection mother liquor and test mother liquor is added to a 10 mL test tube and diluted with dimethyl sulfoxide to 5000 μL. After mixing, the fluorescence intensity FI at 475 nm is measured, and the standard curve is prepared with the fluorescence intensity as the vertical coordinate and the wavelength as the horizontal coordinate, as shown in Figure 3 .
[0055] From Figure 3 , it can be seen that as the concentration of hypochlorite increases, the fluorescence intensity at 475 nm gradually increases, indicating that the fluorescence intensity of the fluorescent probe 1 increases with the increase of the concentration of hypochlorite.
[0056] (2) Fluorescent probe 2 detection of different concentrations of hypochlorite solution
[0057] Fluorescent probe 2 was dissolved in dimethyl sulfoxide to prepare a detection mother liquor with a concentration of 1 mM. A 1 mM-8 mM interval of hypochlorite standard test mother liquor was prepared. 50 μL of the above detection mother liquor and test mother liquor were added to a 10 mL test tube and diluted with ethanol to 5000 μL. After mixing, the fluorescence intensity FI at 650 nm was measured. The fluorescence intensity was used as the vertical coordinate and the wavelength as the horizontal coordinate to prepare a standard curve, as shown in Figure 4 .
[0058] As can be seen from Figure 4 , with the increase of the concentration of hypochlorite, the fluorescence intensity at 650 nm gradually increases, indicating that the fluorescence intensity of fluorescent probe 2 increases with the increase of the concentration of hypochlorite.
[0059] Example 5: Specific response determination of fluorescent probe to hypochlorite
[0060] First, fluorescent probe 1 and fluorescent probe 2 were respectively dissolved in dimethyl sulfoxide to prepare a detection mother liquor with a concentration of 1 mM. In deionized water without any solution containing interfering substances as a blank group, and in deionized water respectively dissolved in a solution containing interfering substances as an experimental group, the interfering substances include: - , Cl - , Br - , I - , CH3COO - , SCN - , HPO4 - , ClO4 - , HSO3 - , Ni 2+ , Co 2+ , Ag 2+ , Ca 2+ , Pb 2+ , Hg 2+ , Zn 2+ , Mg 2+ , Fe 3+ , Cu 2+ , H2O2, TBHP, ·OH, ROO·, S 2+ , ClO - , and a 10 mM interfering substance PBS solution was prepared.
[0061] (1) Specific response determination of fluorescent probe 1
[0062] The fluorescence probe 1 detection mother liquor and the interference PBS solution were respectively added to 10 mL test tubes, and dimethyl sulfoxide was used to make up to 5000 μL, and the fluorescence intensity at 475 nm was measured after mixing. The standard curve was prepared with the fluorescence intensity as the vertical coordinate and the wavelength as the horizontal coordinate, and the results are shown in Figure 5 .
[0063] As can be seen from Figure 5 , the fluorescence probe 1 has high selectivity for hypochlorous acid and can selectively react with hypochlorous acid, and exhibits excellent specific response to hypochlorite ions.
[0064] (2) Specific response determination of fluorescence probe 2
[0065] The fluorescence probe 2 detection mother liquor and the interference PBS solution were respectively added to 10 mL test tubes, and ethanol was used to make up to 5000 μL, and the fluorescence intensity at 650 nm was measured after mixing. The standard curve was prepared with the fluorescence intensity as the vertical coordinate and the wavelength as the horizontal coordinate, and the results are shown in Figure 6 .
[0066] As can be seen from Figure 6 , the fluorescence probe 2 has high selectivity for hypochlorous acid and can selectively react with hypochlorous acid, and exhibits excellent specific response to hypochlorite ions.
[0067] Example 6: Relationship between color and different concentrations of hypochlorite of fluorescence probe 1
[0068] The specific process of determination is as follows: ClO - concentrations of 0, 20, 40, 60, 80, 100 and 120 μM aqueous solution were prepared. Then, the fluorescence probe 1 (10 μM) was added, and the color change after standing for 30 min was as shown in Figure 7 .
[0069] Then, the color of the solution was read and analyzed under ultraviolet light by a detection device (a mobile phone APP ColorAssist can be selected for RGB reading), and the ratio R / B of the color of the solution was calculated. The linear relationship between the R / B value and the concentration of ClO - was calculated. The standard curve was prepared with the R / B value as the vertical coordinate and the concentration value as the horizontal coordinate. The results are shown in Figure 8 .
[0070] Figure 7 The color of the solution was the color of the different concentrations of hypochlorite. As can be seen from Figure 7As can be seen from the above Table 1, the fluorescence probe 1 can be used to determine the content of hypochlorous acid in the pulse, tap water and mineral water.
[0071] Figure 8 The linear relationship diagram of the color of the fluorescence probe 1 and the hypochlorite solution with different concentrations. Figure 8 As can be seen from the above Table 1, the fluorescence probe 1 can be used to determine the content of hypochlorous acid in the pulse, tap water and mineral water.
[0072] Example 7: Application of the fluorescence probe 1
[0073] The fluorescence probe 1 is used to detect tap water, pulse beverage and farmer spring mineral water, and the specific steps are as follows: 50 μL of the fluorescence probe 1 detection mother liquor and 2500 μL of dimethyl sulfoxide are added into a 10 mL test tube, and the tap water, pulse beverage and farmer spring mineral water are respectively used to make up to 5000 μL, and then mixed, and then the detection tool of Example 6 is used for detection, and the linear relationship diagram of the color of the fluorescence probe 1 and the hypochlorite solution with different concentrations obtained in Example 6 is used for calculation, and the results are shown in Table 1.
[0074] Table 1 Determination of the content of hypochlorous acid in some beverages and tap water
[0075]
[0076] As can be seen from the above Table 1, the fluorescence probe 1 can be used to determine the content of hypochlorous acid in the pulse, tap water and mineral water.
[0077] Although the present application has been described by the above preferred embodiments, it is not intended to limit the protection scope of the present application, and any person skilled in the art can make various changes and modifications to the above embodiments without departing from the spirit and scope of the present application, which still belongs to the protection scope of the present application.
Claims
1. Use of a fluorescent probe in measuring, screening or detecting a hypochlorous acid-containing sample, wherein the fluorescent probe has a molecular structure as shown in the following formula (I): ###0001### Formula (I) and wherein the hypochlorous acid-containing sample is a hypochlorous acid-containing solution, and the hypochlorous acid-containing solution is a beverage or tap water. 。 2. Use according to claim 1, characterized in that,
Citation Information
Patent Citations
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