Water-soluble fluorescent probe for detecting hypochlorite and its preparation method and application

Abstract

The invention discloses a water-soluble fluorescent probe for detecting hypochlorite and its preparation method and application, and relates to the technical field of fluorescent probes. The preparation method of the fluorescent probe comprises the following steps: 4-methyl-7-methoxycoumarin is dissolved in an organic solvent, N-bromosuccinimide and dibenzoyl peroxide are added and refluxed in a water bath, filtered after the reaction is completed, and the filtrate is concentrated to obtain 4-bromomethyl-7-methoxycoumarin; 4-bromomethyl-7-methoxycoumarin and 1-methylimidazole are added to tetrahydrofuran, stirred under reflux, cooled, filtered and washed to obtain a water-soluble fluorescent probe for detecting hypochlorite. The fluorescent probe has good water solubility and a wide pH detection range, and has good specificity, and common metal ions have little effect on its fluorescent signal.

Description

Technical Field

[0001] The present invention relates to the technical field of fluorescent probes, and in particular to a water-soluble fluorescent probe for detecting hypochlorite, and a preparation method and application thereof. Background Art

[0002] Hypochlorite, one of several reactive oxygen species, plays a crucial role in numerous biological processes, including pathogen responses, aging, and anti-inflammatory regulation. It is closely associated with innate immunity and is typically produced by the enzyme myeloperoxidase, which catalyzes the reaction of chloride ions with hydrogen peroxide, which in turn kills pathogens. Hypochlorite reacts with a variety of biomolecules, contributing to diseases such as obesity, cancer, and diabetes. Fluorescent probes, with their high sensitivity and fast response, are excellent molecular sensors. However, most sensors are insoluble in water. Therefore, the development of new water-soluble fluorescent probes for hypochlorite detection is of great significance. Summary of the Invention

[0003] In order to solve the above technical problems, the purpose of the present invention is to provide a water-soluble fluorescent probe for detecting hypochlorite, and its preparation method and application. The fluorescent probe has good water solubility and a wide pH detection range, and has good specificity. Common metal ions have little effect on its fluorescent signal.

[0004] The present invention solves the above-mentioned technical problem with the following technical solution: providing a water-soluble fluorescent probe for detecting hypochlorite, the chemical structure of which is as follows:

[0005]

[0006] The present invention also provides a method for preparing a water-soluble fluorescent probe for detecting hypochlorite, comprising the following steps:

[0007] (1) Dissolve 4-methyl-7-methoxycoumarin in an organic solvent, add N-bromosuccinimide and dibenzoyl peroxide in sequence, then reflux in a water bath. After the reaction is complete, filter and concentrate the filtrate to obtain 4-bromomethyl-7-methoxycoumarin;

[0008] (2) Adding 4-bromomethyl-7-methoxycoumarin and 1-methylimidazole obtained in step (1) to tetrahydrofuran, refluxing with stirring, then cooling to room temperature, filtering and washing, and obtaining a water-soluble fluorescent probe for detecting hypochlorite.

[0009] Furthermore, in step (1), the organic solvent is carbon tetrachloride.

[0010] Furthermore, in step (1), the molar volume ratio of 4-methyl-7-methoxycoumarin, N-bromosuccinimide, dibenzoyl peroxide and organic solvent is 8-10 mmol:10 mmol:1 mmol:10 mL.

[0011] Furthermore, in step (1), the mixture is refluxed in a water bath at 70-90° C. for 3-4 hours.

[0012] Furthermore, in step (2), the mass volume ratio of the 4-bromomethyl-7-methoxycoumarin, 1-methylimidazole and tetrahydrofuran is 0.4-0.6 g:1 mL:10 mL.

[0013] Furthermore, in step (2), the mixture is refluxed and stirred at 70-90° C. for 0.5-1.5 h.

[0014] The present invention also provides an application of a water-soluble fluorescent probe for detecting hypochlorite in in vitro detection of hypochlorite.

[0015] The present invention has the following beneficial effects:

[0016] 1. Since the structure of this probe contains the fluorescent group coumarin and the water-soluble imidazolium ion, it combines the characteristics of both to become a water-soluble fluorescent compound. After the addition of hypochlorite, the coumarin skeleton is oxidized and destroyed, and the fluorescence of the probe compound is quenched.

[0017] 2. The water-soluble fluorescent probe for detecting hypochlorite of the present invention has good selectivity for hypochlorite ions, and its fluorescence signal decreases as the concentration of hypochlorite ions increases, and has good linearity.

[0018] 3. The probe of the present invention has a wide pH detection range and good specificity, and common metal ions have little effect on its fluorescence signal. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 The water-soluble fluorescent probe for detecting hypochlorite prepared in Example 4 1 HNMR spectrum;

[0020] Figure 2 The water-soluble fluorescent probe for detecting hypochlorite prepared in Example 4 13 CNMR spectrum;

[0021] Figure 3 is a graph showing the change in fluorescence intensity of a water-soluble probe for detecting hypochlorite as a function of hypochlorite concentration;

[0022] Figure 4 This is the standard curve diagram for hypochlorite detection in Test Example 2;

[0023] Figure 5is the fluorescence intensity graph of the fluorescent probe in the pH range of 3-11;

[0024] Figure 6 The fluorescence intensity diagram of the fluorescent probe under different interfering ions. DETAILED DESCRIPTION

[0025] The principles and features of the present invention are described below. The examples are intended only to illustrate the present invention and are not intended to limit the scope of the present invention. In the examples, where specific conditions are not specified, conventional conditions or manufacturer-recommended conditions were used. Reagents or instruments used where the manufacturer is not specified are conventional products that can be purchased commercially.

[0026] Example 1

[0027] A water-soluble fluorescent probe for detecting hypochlorite, the chemical structure of which is as follows:

[0028]

[0029] Example 2

[0030] A method for preparing a water-soluble fluorescent probe for detecting hypochlorite comprises the following steps:

[0031] (1) Dissolve 8 mmol of 4-methyl-7-methoxycoumarin in 10 mL of carbon tetrachloride, add 10 mmol of N-bromosuccinimide and 1 mmol of dibenzoyl peroxide in sequence, and then reflux in a water bath at 70°C for 3 h. After the reaction is complete, filter and concentrate the filtrate to obtain 4-bromomethyl-7-methoxycoumarin.

[0032] (2) 0.4 g of 4-bromomethyl-7-methoxycoumarin and 1 mL of 1-methylimidazole were added to 10 mL of tetrahydrofuran, refluxed and stirred at 70°C for 0.5 h, then cooled to room temperature, filtered and washed to obtain a water-soluble fluorescent probe for detecting hypochlorite.

[0033] Example 3

[0034] A method for preparing a water-soluble fluorescent probe for detecting hypochlorite comprises the following steps:

[0035] (1) Dissolve 9 mmol of 4-methyl-7-methoxycoumarin in 10 mL of carbon tetrachloride, add 10 mmol of N-bromosuccinimide and 1 mmol of dibenzoyl peroxide in sequence, then reflux in a 90°C water bath for 4 h. After the reaction is complete, filter and concentrate the filtrate to obtain 4-bromomethyl-7-methoxycoumarin.

[0036] (2) 0.6 g of 4-bromomethyl-7-methoxycoumarin and 1 mL of 1-methylimidazole were added to 10 mL of tetrahydrofuran, refluxed at 90°C with stirring for 1.5 h, then cooled to room temperature, filtered, and washed to obtain a water-soluble fluorescent probe for detecting hypochlorite.

[0037] Example 4

[0038] A method for preparing a water-soluble fluorescent probe for detecting hypochlorite comprises the following steps:

[0039] (1) Dissolve 10 mmol of 4-methyl-7-methoxycoumarin in 10 mL of carbon tetrachloride, add 10 mmol of N-bromosuccinimide and 1 mmol of dibenzoyl peroxide in sequence, then reflux in a water bath at 80°C for 3.5 h. After the reaction is complete, filter and concentrate the filtrate to obtain 4-bromomethyl-7-methoxycoumarin.

[0040] (2) 0.5 g of 4-bromomethyl-7-methoxycoumarin and 1 mL of 1-methylimidazole were added to 10 mL of tetrahydrofuran, refluxed at 80°C with stirring for 1 h, then cooled to room temperature, filtered and washed to obtain a water-soluble fluorescent probe for detecting hypochlorite.

[0041] Test Example 1

[0042] The water-soluble fluorescent probe for detecting hypochlorite prepared in Example 4 1 HNMR and 13 The CNMR spectra are as follows Figure 1 and Figure 2 As shown, 1 HNMR and 13 The CNMR spectrum analysis is shown in Table 1.

[0043] Table 1 1 HNMR and 13 CNMR spectrum analysis

[0044]

[0045] Test Example 2

[0046] (1) The water-soluble fluorescent probe for detecting hypochlorite prepared in Example 4 was dissolved in distilled water to prepare a 1.0×10 -5 mol / L fluorescent probe solution, respectively detecting 0 and 11.3×10 -6 mol / L, 22.6×10 -6 mol / L、34.0×10 -6 mol / L、45.2×10 -6 mol / L、56.6×10 -6 mol / L and 67.6×10-6 mol / L hypochlorite standard solution, record the change of fluorescence intensity at room temperature (excitation wavelength is 337nm), the fluorescence intensity of the probe changes with the change of hypochlorite concentration as shown in the figure. Figure 3 As shown, the concentration of hypochlorite gradually increases from the top to the bottom of the curve.

[0047] Depend on Figure 3 It can be seen that as the concentration of hypochlorite increases, the fluorescence intensity of the probe decreases.

[0048] According to the change in fluorescence intensity (I0-I) at the maximum emission wavelength of 415nm and the hypochlorite concentration, the standard curve for hypochlorite detection can be obtained as shown below: Figure 4 shown.

[0049] Depend on Figure 4 It can be seen that in 0-67.6×10 -6 In the range of mol / L hypochlorite concentration, (I0-I)415nm is linearly related to the concentration of hypochlorite (y=7902.46-67.65×106[ClO - ], R=0.9915), therefore, the fluorescent probe of the present invention can be used for the detection of hypochlorite.

[0050] (2) Effect of pH on probe fluorescence intensity

[0051] Take 1.0mL of fluorescent probe solution, add 2mL of phosphate buffer solution with different pH (3-11), and test the fluorescence intensity of the probe. The results are as follows: Figure 5 shown.

[0052] Depend on Figure 5 It can be seen that there is no obvious difference in the fluorescence intensity of the probe within a wide pH range (pH 3-11), indicating that the optical behavior of the probe is very stable.

[0053] (3) Effects of different interfering ions on fluorescence intensity

[0054] Prepare 0.1 mol / L solutions of CuCl2, HgCl2, BaCl2, AlCl3, NiSO4, ZnCl2, MgCl2, CoCl2, MnCl2, CrCl3, Pb(NO3)2 and Sr(NO3)2 in distilled water as interfering ion solutions, add 3.0 mL of 1.0×10 -5 mol / L probe solution, and then added 1ml of different interfering ion solutions, and used the same concentration of hypochlorite ion solution as a comparison, and recorded the changes in fluorescence intensity each time at room temperature (excitation wavelength 337nm, emission wavelength 415nm). The results are as follows Figure 6 shown.

[0055] Depend on Figure 6 It can be seen that Cu 2+ 、Hg 2+ 、Ba 2+ 、Al 3+ 、Ni 2+ 、Zn 2+ Mg 2+ 、Co 2+ 、Mn 2+ Cr 3+ , Pb 2+ and Sr 2+ The quenching effect of plasma on fluorescence was poor, indicating that these ions had little interference with the detection of hypochlorite by this fluorescent probe.

[0056] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. Application of a water-soluble fluorescent probe for detecting hypochlorite in in vitro detection of hypochlorite for non-diagnostic purposes; The chemical structural formula of the water-soluble fluorescent probe for detecting hypochlorite is as follows: 。

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