Dual-wavelength hypochlorite detection probe and preparation method thereof

Abstract

The invention relates to a synthesis method of a novel dual-wavelength off-on fluorescent molecular probe for identifying and detecting hypochlorite and application of the fluorescent probe in detection of hypochlorite. The structure of the fluorescent probe is as shown in a formula I. The fluorescent molecular probe disclosed by the invention is prepared by taking a water sample azine as a raw material and reacting with dimethylaminoformyl chloride. The synthesis method of the probe is simple, reaction conditions are mild, and the yield of the target compound is high. The fluorescent molecular probe is stable in optical performance, high in hypochlorite detection sensitivity and low in lower detection limit, the detection limit is 0.37 mu M, and the response range is 50-180 mu M. The selectivity is good, and no response is caused to fluorine ions, iodide ions, perchlorate ions, hydrogen sulfate ions, sulfate ions, acetate ions, thiocyanate ions, sulfur ions and the like. The fluorescent molecular probe provided by the invention has practical application value in the fields of biochemistry and environmental chemistry, especially detection of hypochlorite ions in water and the like.

Description

Technical Field

[0001] The present invention relates to the technical field of chemical analysis and detection, and in particular to a method for preparing a dual-wavelength hypochlorite fluorescent molecular probe and the application of the fluorescent molecular probe in detecting hypochlorite. Background Art

[0002] Reactive oxygen species (ROS) are deeply involved in numerous critical physiological processes within the body, including signal transduction and immune responses. When the body is exposed to external stimuli, ROS levels rise dramatically, triggering oxidative stress. Hypochlorous acid, a key ROS species in the body, is also a potent bactericidal oxidant. Its antimicrobial mechanism primarily involves disrupting microbial cell membranes, ultimately leading to cell death. Within cells, chloride ions (Cl⁻) and peroxides undergo an oxidation reaction catalyzed by myeloperoxidase to produce HClO, which participates in numerous physiological processes in the body, such as immune responses and inflammation, and is essential for maintaining human health. However, excessive hypochlorous acid can induce a variety of immune disorders, including cancer, rheumatoid arthritis, cardiovascular disease, and neurodegenerative diseases. Therefore, developing highly stable, sensitive, rapid-response, and visual detection methods for hypochlorous acid in living systems and the environment is crucial for both scientific research and practical applications.

[0003] At present, the traditional detection methods of hypochlorous acid include starch-potassium iodide / ferrous sulfate titration, N,N 1,4-Diethyl-1,4-phenylenediamine and tetramethylbenzidine colorimetry, chemical spectroscopy, and electrochemical methods are among them. These methods each have their strengths in specific detection scenarios. However, when used for hypochlorous acid detection in living organisms, their inherent limitations gradually become apparent. Under normal physiological conditions, the amount of hypochlorous acid produced in cells is extremely small. Traditional methods are cumbersome to operate, take a long time to detect, and require specialized equipment. They cannot meet the requirements for detecting low concentrations of hypochlorous acid in biological environments, nor can they perform imaging analysis on living organisms. In view of this, the development of fluorescent probe detection methods with high sensitivity, high selectivity, and high signal-to-noise ratio has become an inevitable choice to overcome the limitations of existing detection technologies. Summary of the Invention

[0004] The purpose of the present invention is to provide a novel dual-wavelength, "off-on" type fluorescent probe for identifying and detecting hypochlorite.

[0005] The novel dual-wavelength fluorescent molecular probe of the present invention has the following structure:

[0006] The above-mentioned method for synthesizing the fluorescent molecular probe comprises the following steps:

[0007] Salicylazine 1, dimethylaminoformyl chloride and alkali are dissolved in an anhydrous organic solvent in a molar ratio of compound 1 to dimethylaminoformyl chloride of 1:3-3:1. The mixture is stirred at room temperature for 6-24 hours, extracted, and the crude product is purified by column chromatography to obtain a light yellow solid probe compound I.

[0008] Furthermore, in the synthesis step, the organic solvent is preferably dimethyl sulfoxide.

[0009] Furthermore, in the synthesis step, the base is preferably potassium carbonate.

[0010] In order to solve the technical problem of the present invention, the following technical solution is adopted: Preparation of a dimethyl sulfoxide solution dispersed with fluorescent probe molecule I; Determine the detection limit of fluorescent probe molecules; Plot and calculate the concentration of hypochlorite in the solution; Determine and calculate the concentration of hypochlorite in the solution.

[0011] The specific process of the test is as follows: Prepare a 90% dimethyl sulfoxide-water mixed solution with a probe molecule concentration of 0.1 mM; Take 1-3 mL of a series of dimethyl sulfoxide dispersion solutions of fluorescent probe molecules and add different volumes of mother solution containing hypochlorite to make the concentration of hypochlorite in the test solution 0, 20, 50, 70, 90, 120, 140, 160, 180, 200, 250, 300, 350, 400, and 450 μM, respectively. After standing for 5 minutes, measure and calculate the fluorescence intensity at 494 and 599 nm, respectively. Prepare a working curve based on the relationship between fluorescence intensity and hypochlorite concentration.

[0012] The beneficial effects of the present invention are: (1) The fluorescent probe molecules provided by the present invention are specific for hypochlorite, with fluorescence gradually changing from none to present, from weak to strong, and the solution gradually turning from colorless to yellow. The detection process is not affected by probe molecule photobleaching, instrumentation, or environmental factors.

[0013] (2) The fluorescent probe molecules provided by the present invention exhibit dual-wavelength emission under the stimulation of hypochlorite, with a high signal-to-noise ratio, effectively reducing background interference.

[0014] (3) The fluorescent probe molecule provided by the present invention has a low detection limit for hypochlorite, which can reach the micromolar level. BRIEF DESCRIPTION OF THE DRAWINGS Figure 1The following graph shows the response of the probe to hypochlorite concentration. Fluorescence intensity is plotted on the y-axis, and wavelength is plotted on the x-axis. The curves from bottom to top represent hypochlorite concentrations of 0, 30, 50, 70, 90, 120, 140, 160, 180, 200, 250, 300, 350, 400, and 450 μM, respectively. Figure 2 The standard curve of fluorescence intensity at 494 nm and hypochlorite concentration is plotted on the ordinate, and on the abscissa, against fluorescence intensity. Figure 3 This is a graph showing the response of fluorescent probes to different molecules or ions. The fluorescence intensity is the vertical axis and the wavelength is the horizontal axis. DETAILED DESCRIPTION

[0015] The present invention is further described in detail below through specific examples, but it should not be understood that the scope of the above subject matter of the present invention is limited to the following examples. All technologies realized based on the above content of the present invention belong to the scope of the present invention.

[0016] Instruments and reagents used in the present invention: Nuclear magnetic resonance instrument: Bruker AV-II 500 MHz NMR, TMS as internal standard, CDCl3 as solvent; melting point apparatus: XT-4 melting point tester.

[0017] All reagents used were commercially available chemically pure or analytically pure. Example

[0018] Synthesis of the fluorescent probe of the present invention The preparation method of the fluorescent probe of the present invention is as follows:

[0019] Salicylazine 1, dimethylaminoformyl chloride and potassium carbonate were dissolved in anhydrous dimethyl sulfoxide in a molar ratio of 1:2. The mixture was stirred at room temperature for 24 hours and extracted. The crude product was purified by column chromatography to obtain a light yellow solid probe compound I.

[0020] ((1E,1'E)-hydrazine-1,2-diylidenebis(methaneylylidene))bis(benzene-4,1,3-triyl) tetrakis(dimethylcarbamate). Melting point 212-213 degrees, 1 H NMR (400 MHz, Chloroform- d ) δ 8.61 (s, 2H),7.81 (d, J= 2.8 Hz, 2H), 7.19 – 7.09 (m, 4H), 3.10 (s, 6H), 3.03 (s, 6H), 2.97 (d, J = 8.0 Hz, 12H). 13 C NMR (101 MHz, CDCl3) δ 156.2, 154.6, 154.3, 148.7, 148.3, 127.3, 125.5, 124.0, 120.2, 36.9,36.8, 36.7, 36.5. Example

[0021] Fluorescent probe molecular working curve Take 2 mL of the prepared dimethyl sulfoxide dispersion solution of the ratiometric fluorescent probe molecule and add different volumes of hypochlorite-containing mother solution to adjust the hypochlorite concentrations in the test solution to 0, 20, 50, 70, 90, 120, 140, 160, 180, 200, 250, 300, 350, 400, and 450 μM, respectively. After 5 minutes, the fluorescence spectrometer records the changes in fluorescence intensity at 494 and 599 nm, respectively. Plot the fluorescence intensity as the ordinate and the wavelength as the abscissa to obtain the response curve of the probe molecule to the hypochlorite concentration. Figure 1 The fluorescence intensity is plotted as the ordinate and the wavelength as the abscissa to obtain the relationship between the fluorescence intensity and the hypochlorite concentration. The bottom curve represents the fluorescence intensity of the probe system when the hypochlorite concentration is 0 μM, and the top curve represents the fluorescence intensity of the probe system when the hypochlorite concentration is 450 μM. The excitation wavelength is 396 nm. Figure 2 The standard curve of fluorescence intensity and chlorate concentration was obtained by plotting the fluorescence intensity as the ordinate and the hypochlorite concentration as the abscissa. Example

[0022] Fluorescent probe molecular selectivity Take 2 mL of the prepared aqueous dispersion solution of the ratiometric fluorescent probe molecule, add 40 μL of different metal ions or neutral molecule solutions of a certain concentration, and after standing for 5 minutes, record the changes in fluorescence intensity using a fluorescence spectrometer. Figure 3 The fluorescence intensity is plotted as the ordinate and the wavelength is plotted as the abscissa to obtain a relationship diagram of the fluorescence intensity of the system in the presence of different anions or neutral molecules, where the excitation wavelength is 396 nm.

Claims

1. A novel dual-wavelength, "off-on" type hypochlorite fluorescent molecular probe, as shown in Formula I, 。 2. The method for synthesizing the fluorescent molecular probe according to claim 1, wherein The steps include: Salicylazine 1, dimethylaminoformyl chloride and alkali are dissolved in an anhydrous organic solvent in a molar ratio of compound 1 to dimethylaminoformyl chloride of 1:3-3:

1. The mixture is stirred at room temperature for 6-24 hours, extracted, and the crude product is purified by column chromatography to obtain a light yellow solid probe compound I.

3. The method for synthesizing a fluorescent probe according to claim 2, wherein: The organic solvent used in the synthesis step is dimethyl sulfoxide, and the molar ratio of compound 1 to dimethylcarbamoyl chloride is 1:

2.

4. The method for synthesizing a fluorescent probe according to claim 2, wherein: In the synthesis step, the base is a carbonate, specifically potassium carbonate.

5. The use of the fluorescent molecular probe for detecting hypochlorite according to claim 1, characterized in that Used for identification and content determination of hypochlorite, the specific method is as follows: A 90% dimethyl sulfoxide-water mixed solution of the fluorescent probe of the present invention was prepared with a concentration of 100 μM to prepare a standard solution. The prepared standard solution was then used to prepare test solutions containing hypochlorite concentrations of 0, 20, 50, 70, 90, 120, 140, 160, 180, 200, 250, 300, 350, 400, and 450 μM. After standing for 5 minutes, the fluorescence intensities of the solutions containing different hypochlorite concentrations were measured at an excitation wavelength of 396 nm and emission wavelengths of 494 and 599 nm. A standard curve was prepared and fitted to determine the functional relationship between fluorescence intensity and hypochlorite concentration.

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