Fluorescent probe applied to hypochlorous acid detection as well as preparation method and application of fluorescent probe

Abstract

The invention relates to a fluorescent probe applied to hypochlorous acid detection and a preparation method and application thereof, and belongs to the field of fluorescent probes. The preparation method of the probe comprises the following steps: reacting 1, 3-indandione with malononitrile under the catalysis of sodium acetate to obtain a compound 1, and then respectively synthesizing the compound 1 and the 1, 3-indandione with 4-dimethylamino cinnamyl aldehyde through nucleophilic addition reaction to synthesize a fluorescent probe 1 and a fluorescent probe 2. The prepared fluorescent probe can still specifically recognize hypochlorite ions even in various ion environments, has good selectivity to the hypochlorite ions, is high in specificity, simple to operate in the testing process and extremely sensitive in reaction, ensures rapid detection, can be directly used in water, greatly improves the detection convenience, and has a wide application prospect. And real-time monitoring and field application can be realized.

Description

Technical Field

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

[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 contact or excessive intake of hypochlorous acid may lead to chronic health problems such as respiratory diseases, skin allergies, or even toxic effects on internal organs; hypochlorous acid also reacts chemically with organic compounds in water to produce carcinogens such as chloroform and carbon tetrachloride, which seriously endanger human health.

[0003] Fluorescent probes can emit fluorescent signals by reacting specifically with the sample to be tested, and the intensity of the signals is proportional to the concentration of hypochlorous acid. Compared with traditional analytical methods, such as chemical analysis or chromatographic analysis, fluorescent probes have the advantages of simple operation and rapid response, and can achieve real-time detection and field application. Therefore, it is very important to design and synthesize a fluorescent probe that is efficient, fast and can detect hypochlorous acid in real time to detect the concentration of hypochlorous acid in the solution. Summary of the invention

[0004] In order to overcome the above-mentioned shortcomings and deficiencies of the prior art, the object of the present invention is to provide a fluorescent probe molecule and a preparation method thereof, which has the advantages of simple and convenient detection process for hypochlorous acid and has the advantages of fast, efficient and high sensitivity.

[0005] In order to solve the above technical problems, the technical solution adopted by the present invention is:

[0006] A fluorescent probe for hypochlorous acid detection, wherein the fluorescent probe has a molecular structure as shown in the following formula (I) or formula (II):

[0007]

[0008] The present invention provides a method for preparing a fluorescent probe for hypochlorous acid detection. The method for preparing the fluorescent probe having a molecular structure shown in formula (I) comprises the following steps:

[0009]

[0010] A fluorescent probe having a structure of formula (I) is synthesized by reacting 4-dimethylaminocinnamaldehyde with 1,3-indandione through a nucleophilic addition reaction.

[0011] Preferably, the method for preparing the molecular structure of the fluorescent probe formula (II) comprises the following steps:

[0012] Step 1:

[0013]

[0014] 1,3-indandione and malononitrile are reacted under the catalysis of sodium acetate to obtain compound 1;

[0015] Step 2:

[0016]

[0017] Then, compound 1 and 4-dimethylaminocinnamaldehyde are reacted by nucleophilic addition reaction to synthesize a fluorescent probe having a structure of formula (II).

[0018] Preferably, the molar ratio of 4-dimethylaminocinnamaldehyde to 1,3-indandione is 1:1.

[0019] Preferably, the molar ratio of the 1,3-indandione to malononitrile is 1:1; and the molar ratio of the compound 1 to 4-dimethylaminocinnamaldehyde is 5:1.

[0020] Preferably, the fluorescent probe is obtained by column chromatography separation and purification, wherein the mobile phase of the column chromatography separation is dichloromethane-methanol in a volume ratio of (6-10):1.

[0021] The present invention also provides an application of a fluorescent probe for hypochlorous acid detection in measuring, screening or detecting a sample containing hypochlorous acid.

[0022] Preferably, the hypochlorous acid-containing sample is a solution containing hypochlorous acid, and the hypochlorous acid-containing solution is a beverage or tap water.

[0023] Compared with the prior art, the technical solution of the present invention has the following advantages:

[0024] The fluorescent probe prepared by the present invention has high selectivity and low detection limit, can specifically identify hypochlorite ions even in a variety of ion environments, has good selectivity for hypochlorite ions, has strong specificity, is simple to operate in the test process, has extremely sensitive reactions, ensures rapid detection, and can be used directly in water, greatly improving the convenience of detection, and can realize real-time monitoring and on-site application. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 The fluorescence emission spectra of the fluorescent probe 1 prepared in the embodiment of the present invention in solvents of different polarities;

[0026] Figure 2 The fluorescence emission spectra of the fluorescent probe 2 prepared in the embodiment of the present invention in solvents of different polarities;

[0027] Figure 3 This is a graph showing the fluorescence intensity variation of the fluorescent probe 1 prepared in an embodiment of the present invention at 475 nm in response to different concentrations of hypochlorous acid;

[0028] Figure 4 This is a graph showing the fluorescence intensity variation of the fluorescent probe 2 prepared in an embodiment of the present invention at 650 nm in response to different concentrations of hypochlorous acid;

[0029] Figure 5 This is a fluorescence intensity analysis result diagram of the fluorescent probe 1 prepared in an embodiment of the present invention when other active molecules and hypochlorous acid are added at 475 nm;

[0030] Figure 6 This is a fluorescence intensity analysis result diagram of the fluorescent probe 2 prepared in the embodiment of the present invention when other active molecules and hypochlorous acid are added at 650nm;

[0031] Figure 7 This is a color change diagram of the fluorescent probe 1 prepared in an embodiment of the present invention at different hypochlorous acid concentrations;

[0032] Figure 8 This is a linear relationship diagram between the chromaticity of the fluorescent probe 1 prepared in an embodiment of the present invention at 365 nm and the concentration of hypochlorous acid (0-120 μM). DETAILED DESCRIPTION

[0033] In order to enable those skilled in the art to better understand the solution of the present application, the technical solution in the embodiments of the present application will be clearly and completely described below in conjunction 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 of the embodiments. Based on the embodiments in the present application, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of this application.

[0034] Example 1: Synthesis of fluorescent probe 1

[0035] This embodiment provides a method for synthesizing a fluorescent probe for hypochlorous acid detection, and the specific steps are as follows:

[0036]

[0037] 1,3-Indanedione (293.1 mg, 2 mmol) and 4-dimethylaminocinnamaldehyde (350.46 mL, 2 mmol) were dissolved in 20 mL of ethanol, stirred in an eggplant-shaped bottle for 40 min, and two drops of piperidine were added. After stirring, the mixture was refluxed at 85 ° C for 12 h, filtered while hot, and washed with EtOH. A mixture of CH2Cl2 and CH3OH (the volume ratio of CH2Cl2 and CH3OH was 8:1) was used as the eluent for column chromatography separation and purification to obtain 0.55 g of dark red solid, which was the fluorescent probe with a yield of 72%. It was named fluorescent probe 1 and its chemical name was (E)-2-(3-(4-(dimethylamino)phenyl)allyl)-1H-indene-1,3(2H)-dione. It was characterized by hydrogen spectrum, carbon spectrum and mass spectrum. By analyzing the hydrogen spectrum, carbon spectrum and mass spectrum data of fluorescent probe 1, it was confirmed that the structure of the above fluorescent probe 1 was correct.

[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] The compound malononitrile (198.21 mg, 3 mmol) and 1,3-indanedione (439.39 mg, 3 mmol) were dissolved in 20 mL of pure ethanol solution, stirred in a flask for 40 min, then refluxed for 40 min, filtered while hot and washed with anhydrous EtOH 3 times. After drying, it was dissolved in acetic acid and recrystallized, cooled overnight, filtered and dried to obtain 561.09 g of gray-black solid with a yield of 88%. The obtained gray-black solid was 2-(3-oxo-2,3-dihydro-1H-inden-1-ylidene) malononitrile, named compound 1.

[0043] The compound 1 was characterized by hydrogen spectrum, and the results were as follows: 1 H NMR (400MHz, Chloroform-d) δ8.69 (d, J = 7.8Hz, 1H), 8.07–8.00 (m, 1H), 7.98–7.83 (m, 2H), 3.82–3.71 (m, 2H).

[0044] (Bi) 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 an eggplant-shaped bottle for 40 min, and two drops of DIPEA were added. After stirring, the mixture was refluxed at 85°C for 12 h, filtered while hot, and washed with EtOH. Column chromatography was performed using a mixture of CH2Cl2 and CH3OH (the volume ratio of CH2Cl2 and CH3OH was 8:1) as the eluent to separate and purify the mixture, and 0.52 g of a blue-black solid was obtained, which was the fluorescent probe with a yield of 69%. It was named fluorescent probe 2 and its chemical name was (E)-2-(3-(4-(dimethylamino)phenyl)allyl)-1H-indene-1,3(2H)-dione. The hydrogen spectrum, carbon spectrum and mass spectrum of the compound were characterized, and the structure of the fluorescent probe 2 was confirmed to be correct by analyzing the hydrogen spectrum, carbon spectrum and mass spectrum data of the compound.

[0046] The characterization results of fluorescent probe 2 are as follows:

[0047] 1 H NMR(400MHz, CDCl3)δ8.65(m,2H),8.50(d,J=11.8Hz,1H),7.86(m,1H),7.71(m,2H), 7.65(t,J=9.9Hz,2H),7.39(d,J=14.7Hz,1H),6.75(d,J=8.8Hz,2H),3.13(s,6H).13C NMR (101MHz, CDCl3) δ189.75,160.22,155.89,152.91,148.88,139.94,137.31,134.62,134.00,132. 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 spectra of fluorescent probes in different solutions

[0049] First, prepare 5 mL of solvents of different polarities such as toluene, 1,4-dioxane, tetrahydrofuran, dichloromethane, dimethyl sulfoxide, ethanol, methanol, and buffer. Prepare two tubes of each solvent, dissolve fluorescent probe 1 and fluorescent probe 2 in dimethyl sulfoxide and prepare a mother solution with a concentration of 1 mM, then take 50 μL of each and add them to the above solutions to make the final concentration of the probe 10 μM. Use a fluorescence spectrometer to test the fluorescence emission spectra of different solutions, study the effects of solvents of different polarities on fluorescent molecules, and select a system that can show the excellent fluorescence ability of the probe. The results are as follows: Figure 1 and Figure 2 As shown, Figure 1 is the fluorescence emission spectra of fluorescent probe 1 in solvents of different polarities; Figure 2 The fluorescence emission spectra of fluorescent probe 2 in solvents of different polarities.

[0050] Depend on 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] Depend on Figure 2 It can be seen that since the fluorescent probe 2 has better fluorescence performance in ethanol, ethanol is used as the test solution for the fluorescent probe 2.

[0052] Example 4: Detection of fluorescence intensity changes in the reaction of fluorescent probes with hypochlorite solutions of different concentrations

[0053] (1) Detection of hypochlorite solutions with different concentrations by fluorescent probe 1

[0054] First, the fluorescent probe 1 was dissolved in dimethyl sulfoxide to prepare a detection mother solution with a concentration of 1 mM; hypochlorous acid test mother solutions in the range of 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) were prepared, 50 μL of each of the above detection mother solution and the test mother solution were added to a 10 mL test tube and the volume was made up to 5000 μL with dimethyl sulfoxide, and the fluorescence intensity FI at 475 nm was measured after mixing, and a standard curve was prepared with the fluorescence intensity as the ordinate and the wavelength as the abscissa. The results are shown in FIG. Figure 3 shown.

[0055] from Figure 3 It can be seen that with the increase of hypochlorite concentration, the fluorescence intensity at 475nm gradually increases, indicating that the fluorescence intensity of fluorescent probe 1 increases with the increase of hypochlorite concentration.

[0056] (2) Detection of hypochlorite solutions with different concentrations by fluorescent probe 2

[0057] Dissolve the fluorescent probe 2 in dimethyl sulfoxide to prepare a detection mother solution with a concentration of 1mM; prepare a hypochlorous acid standard test mother solution in the range of 1mM-8mM, take 50μL of the above detection mother solution and the test mother solution, add them to a 10mL test tube and dilute to 5000μL with ethanol, mix well and measure the fluorescence intensity FI at 650nm, and prepare a standard curve with fluorescence intensity as the ordinate and wavelength as the abscissa. The results are shown in Figure 4 shown.

[0058] from Figure 4 It can be seen that with the increase of hypochlorite concentration, the fluorescence intensity at 650nm gradually increases, indicating that the fluorescence intensity of fluorescent probe 2 increases with the increase of hypochlorite concentration.

[0059] Example 5: Determination of the specific response of fluorescent probe to hypochlorite

[0060] First, fluorescent probe 1 and fluorescent probe 2 were dissolved in dimethyl sulfoxide to prepare a detection mother solution with a concentration of 1mM. No solution containing any interfering substances was added to deionized water as a blank group, and solutions containing interfering substances were dissolved in deionized water as experimental groups. The interfering substances included: F - , 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 - , prepared into a PBS solution with a concentration of 10 mM interferor.

[0061] (1) Specific response determination of fluorescent probe 1

[0062] 50 μL of fluorescent probe 1 detection mother solution and interference PBS solution were added to 10 mL test tubes and the volume was adjusted to 5000 μL with dimethyl sulfoxide. After mixing, the fluorescence intensity at 475 nm was measured. A standard curve was prepared with the fluorescence intensity as the ordinate and the wavelength as the abscissa. The results are shown in the figure. Figure 5 shown.

[0063] from Figure 5 It can be seen that fluorescent probe 1 has a high selectivity for hypochlorous acid, can react selectively with hypochlorous acid, and exhibits an excellent specific response to hypochlorite ions.

[0064] (2) Determination of specific response of fluorescent probe 2

[0065] 50 μL of the fluorescent probe 2 detection mother solution and the interference PBS solution were added to a 10 mL test tube and diluted to 5000 μL with ethanol. After mixing, the fluorescence intensity at 650 nm was measured. A standard curve was prepared with the fluorescence intensity as the ordinate and the wavelength as the abscissa. The results are shown in the figure. Figure 6 shown.

[0066] from Figure 6 It can be seen that fluorescent probe 2 has a high selectivity for hypochlorous acid, can react selectively with hypochlorous acid, and exhibits an excellent specific response to hypochlorite ions.

[0067] Example 6: Relationship between the chromaticity of fluorescent probe 1 and different hypochlorite concentrations

[0068] The specific process of determination is: Preparation of ClO - The concentrations of the aqueous solution were 0, 20, 40, 60, 80, 100 and 120 μM. Then the fluorescent probe 1 (10 μM) was added and the color changed after standing for 30 minutes. Figure 7 shown.

[0069] Then, the chromaticity of the solution is read and analyzed under ultraviolet light by a detection device (you can choose to use the mobile phone APP ColorAssist for RGB readings), and the ratio of the solution chromaticity R to B R / B is used to calculate the ClO - The linear relationship of concentration was plotted with R / B value as the ordinate and concentration value as the abscissa. Figure 8 shown.

[0070] Figure 7 It is the color of the hypochlorite solution at different concentrations. From Figure 7It can be seen that as the concentration of hypochlorite increases (from left to right), the color of the solution changes from red at the beginning (the color of the fluorescent probe 1 itself is red fluorescence, and after reacting with hypochlorous acid, its own red fluorescence can be changed to green fluorescence) to green gradually. The color change of the test system can be clearly observed, indicating that the presence of hypochlorous acid can be detected by the naked eye, which is conducive to its commercial application.

[0071] Figure 8 is a linear relationship diagram between the chromaticity of fluorescent probe 1 and hypochlorite solutions of different concentrations. Figure 8 It can be seen that with the increase of hypochlorite concentration, the chromaticity value of fluorescent probe 1 (10 μM) at 365 nm showed an obvious change, indicating that there is a good linear relationship between the chromaticity of fluorescent probe 1 and hypochlorite concentration.

[0072] Example 7: Application of Fluorescent Probe 1

[0073] The fluorescent probe 1 was used to detect tap water, Pulse beverage and Nongfu Spring mineral water, respectively. The specific steps were as follows: 50 μL of the fluorescent probe 1 detection mother solution and 2500 μL of dimethyl sulfoxide were added to a 10 mL test tube, and the volume was adjusted to 5000 μL with tap water, Pulse beverage and Nongfu Spring mineral water respectively. After mixing, the detection tool of Example 6 was used for detection, and the "linear relationship diagram of the chromaticity of the fluorescent probe 1 and hypochlorite solutions of different concentrations" obtained in Example 6 was calculated. The results are shown in Table 1 below.

[0074] Table 1 Determination of hypochlorous acid content in some beverages and tap water

[0075]

[0076] It can be seen from Table 1 that the fluorescent probe 1 of the present invention can measure the hypochlorite content in pulse water, tap water and mineral water.

[0077] Although the present invention has been described using the above preferred embodiments, it is not intended to limit the scope of protection of the present invention. Any person skilled in the art who makes various changes and modifications to the above embodiments without departing from the spirit and scope of the present invention still fall within the scope of protection of the present invention.

Claims

1. A fluorescent probe for hypochlorous acid detection, characterized in that: The fluorescent probe has a molecular structure as shown in the following formula (I) or formula (II):

2. The method for preparing a fluorescent probe for hypochlorous acid detection according to claim 1, characterized in that: The method for preparing the molecular structure of the fluorescent probe formula (I) comprises the following steps: A fluorescent probe having a structure of formula (I) is synthesized by reacting 4-dimethylaminocinnamaldehyde with 1,3-indandione through a nucleophilic addition reaction.

3. The method for preparing a fluorescent probe for hypochlorous acid detection according to claim 1, characterized in that: The method for preparing the molecular structure of the fluorescent probe formula (II) comprises the following steps: Step 1: 1,3-indandione and malononitrile are reacted under the catalysis of sodium acetate to obtain compound 1; Step 2: Then, compound 1 and 4-dimethylaminocinnamaldehyde are reacted by nucleophilic addition reaction to synthesize a fluorescent probe having a structure of formula (II).

4. The method for preparing a fluorescent probe for hypochlorous acid detection according to claim 2, characterized in that: The molar ratio of the 4-dimethylaminocinnamaldehyde to 1,3-indandione is 1:

1.

5. The method for preparing a fluorescent probe for hypochlorous acid detection according to claim 3, characterized in that: The molar ratio of the 1,3-indandione to malononitrile is 1:1; the molar ratio of the compound 1 to 4-dimethylaminocinnamaldehyde is 5:

1.

6. The method for preparing a fluorescent probe for hypochlorous acid detection according to claim 2 or 3, characterized in that: The fluorescent probe is obtained by column chromatography separation and purification, wherein the mobile phase of the column chromatography separation is dichloromethane-methanol, and the volume ratio is (6-10):

1.

7. Use of the fluorescent probe for hypochlorous acid detection according to claim 1 in measuring, screening or detecting samples containing hypochlorous acid.

8. The use according to claim 7, characterized in that: The hypochlorous acid-containing sample is a solution containing hypochlorous acid, and the hypochlorous acid-containing solution is a beverage or tap water.

Images