A pyridine salt-based hypochlorite detection probe and a preparation method thereof

Abstract

The present application relates to a kind of hypochlorite detection probe based on pyridine salt and preparation method thereof.The fluorescence probe structure is as shown in formula I.The fluorescent molecular probe of the present application is prepared from o-alkynyl aldehyde as raw material, by condensation reaction with pyridine salt.The probe synthesis method is simple, and the reaction condition is mild, and the yield of target compound is high.The fluorescent molecular probe described in the present application has stable optical performance, high sensitivity for hypochlorite detection, low detection lower limit, detection limit is 0.38 μM, response range is 5-200 μM.It has good selectivity, and has no response to fluoride ion, iodine ion, perchlorate ion, bisulfate ion, sulfate ion, acetate ion, thiocyanate ion, sulfur ion, etc.The fluorescent molecular probe described in the present application has practical application value in the field of biochemistry and environmental chemistry, especially the detection of hypochlorite ion in water, etc.

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 pyridinium salt-based fluorescent molecular probe for detecting hypochlorite and the application of the fluorescent molecular probe in detecting hypochlorite. Background Art

[0002] Hypochlorous acid (HOC) / hypochlorite, one of the most important reactive oxygen species in biological systems, is generated from chloride ions and hydrogen peroxide by myeloperoxidase under certain physiological conditions. HOC is a double-edged sword in life. On the one hand, HOC protects human health by killing pathogens and bacteria that invade the immune system. On the other hand, excessive HOC can lead to the oxidation of biomolecules (such as proteins, nucleic acids, lipids, and enzymes), resulting in tissue damage, inflammation, and a range of diseases, including cardiovascular disease, rheumatoid arthritis, liver ischemia-reperfusion injury, and even cancer. Given the extraordinary importance of HO in biology and medicine, the search for highly selective, sensitive, and readily accessible methods for the detection of endogenous HOC has become a hot topic in the biomedical field. Numerous methods exist for the detection of HO, including the starch-potassium iodide method, the N,N-diethyl-p-phenylenediamine colorimetric method, chemiluminescence, spectrophotometry, and fluorescence analysis. Among these detection methods, fluorescent probe technology has outstanding advantages such as good selectivity, high sensitivity, low detection limit, simple operation and low cytotoxicity, making it widely used in chemical and biological analysis.

[0003] Fluorescent probes for detecting hypochlorous acid have been reported one after another, with extensive research based on various fluorophores, recognition groups, and recognition mechanisms. However, these fluorescent probes for hypochlorite suffer from limitations such as lengthy synthesis steps, low detection limits, and long response times. To develop a better method for detecting hypochlorite in aqueous solutions, the present invention designed and synthesized a pyridinium salt-based fluorescent chemical probe that uses changes in fluorescence intensity as a quantitative signal. The probe has been successfully applied to the detection of hypochlorite in aqueous phases. This probe exhibits high sensitivity and selectivity. Summary of the Invention

[0004] The purpose of the present invention is to provide a novel fluorescent probe for identifying and detecting hypochlorite.

[0005] Another object of the present invention is to provide a novel fluorescent probe for detecting hypochlorite for use in detecting hypochlorous acid / hypochlorite in an aqueous phase.

[0006] The novel fluorescent molecular probe of the present invention has the following structure:

[0007]

[0008] The above-mentioned method for synthesizing the fluorescence ratio probe comprises the following steps:

[0009]

[0010] Take o-alkynyl aldehyde 1, pyridinium salt 2 and an organic catalyst and dissolve them in anhydrous ethanol. The molar ratio of compound 1 to pyridinium salt 2 is 1:2-2:1. Heat and stir for 12 hours. The solid is completely precipitated. Filter and recrystallize the crude product with anhydrous ethanol to obtain a red solid probe compound I.

[0011] Furthermore, in the synthesis step, the organic catalyst is preferably piperidine.

[0012] Furthermore, in the synthesis step, the reaction temperature is preferably 80 degrees Celsius.

[0013] To solve the second technical problem, the present invention adopts the following technical solution:

[0014] 1) preparing an aqueous solution in which fluorescent probe molecules I are dispersed;

[0015] 2) Determine the detection limit of the fluorescent probe molecule; Implementation Method

[0016] 3) Plot and calculate the concentration of hypochlorite in the solution;

[0017] 4) Determine and calculate the concentration of hypochlorite in the solution.

[0018] The specific process of the test is as follows: Example

[0019] 1) Prepare a 10% N,N-dimethylformamide-water mixed solution with a probe molecule concentration of 0.1 mM;

[0020] 2) Take 1-3 mL of a series of aqueous dispersions of ratiometric fluorescent probe molecules, add 10-30 μL of different concentrations of hypochlorite (0, 1, 2, 5, 10, 20, 50, 100, 200 μM), let it stand for 1 minute, measure and calculate the fluorescence intensity at 498 nm, and generate a working curve based on the relationship between fluorescence intensity and hypochlorite concentration.

[0021] The beneficial effects of the present invention are: Example

[0022] (1) The fluorescent probe molecules provided by the present invention are specific for hypochlorite, and the fluorescence gradually fades from strong to weak as the solution fades from red. The detection process is not affected by probe molecule photobleaching, instrumentation, or environmental factors.

[0023] (2) The fluorescent probe molecules provided by the present invention are suitable for aqueous solutions containing hypochlorite.

[0024] (3) The fluorescent probe molecule provided by the present invention has a low detection limit for hypochlorite, which can reach the micromole level.

[0025] 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.

[0026] Instruments and reagents used in the present invention:

[0027] Nuclear magnetic resonance instrument: Bruker AV-II 500 MHz NMR, TMS as internal standard, CDCl3 as solvent; infrared spectrometer: TFS-40, KBr pellet; melting point apparatus: XT-4 melting point tester.

[0028] All reagents used were commercially available chemically pure or analytically pure.

[0029] Example 1

[0030] Synthesis of the fluorescent probe of the present invention

[0031] The preparation method of the fluorescent probe of the present invention is as follows:

[0032]

[0033] Take o-alkynyl aldehyde 1, pyridinium salt 2 and an organic catalyst and dissolve them in anhydrous ethanol. The molar ratio of compound 1 to pyridinium salt 2 is 1:2-2:1. Heat and stir for 12 hours. The solid is completely precipitated. Filter and recrystallize the crude product with anhydrous ethanol to obtain a red solid probe compound I.

[0034] 3-(4-(2-(4-diethylaminophenyl)ethynyl)phenyl)pyridinylpropanesulfonate, dark red solid, melting point 237-240℃, 1 HNMR(CDCl3,400MHz)δ8.83(d,J=4.0Hz,2H),7.82-7.76(m,3H),7.57(d,J=8.0Hz,2H),7.26-7.24(m,3H),7.17-7.10 (m,3H),6.56(d,J=8.0Hz,2H),4.79(s,2H),4.11(q,J=8.0Hz,4H),2.95(br,2H),2.45(br,2H),1.09(t,J=8.0Hz,6H). 1H NMR(CDCl3,100MHz)δ152.9,147.9,144.6,138.5,134.7,133.1,132.5,129.9,127.9,126.8,125 .0,124.1,123.8,111.4,107.8,98.2,85.3,59.3,47.5,44.3,27.6,12.6.HRMS:m / z:475.2053[M] + (calculatedC 28 H 31 N2O3S:475.2055).

[0035] Example 2

[0036] Fluorescent probe molecular working curve

[0037] Take 2mL of the prepared aqueous dispersion of the ratiometric fluorescent probe molecule, add 20μL of hypochlorite aqueous solution of different concentrations (0, 1, 5, 10, 25, 50, 75, 100, 150, 200μM), and after standing for 1 minute, the fluorescence spectrometer records the changes in fluorescence intensity at 498nm. 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 upper right corner of the figure shows the working curve of the fluorescent probe molecule obtained by plotting the fluorescence intensity as the ordinate and the hypochlorite concentration as the abscissa. In this embodiment, the fluorescence probe is used to measure the hypochlorite concentration. The relationship between the fluorescence intensity and the hypochlorite concentration under 357nm excitation light is shown in the figure below. Figure 1 shown.

[0038] Example 3

[0039] Fluorescent probe molecular selectivity

[0040] Take 2mL of the prepared aqueous dispersion solution of the ratio fluorescent probe molecule, add 40μL of a certain concentration of different metal ions or neutral molecule solutions, and after standing for 1 minute, the fluorescence spectrometer records the changes in fluorescence intensity at 498nm. Plot the fluorescence intensity as the vertical axis and the metal ions or molecules as the horizontal axis to obtain a histogram of the selectivity of the fluorescent probe molecule. In this embodiment, the fluorescence probe is selectively measured for different ions or molecules. The histogram of the fluorescence intensity changing with different ions or molecules under 357nm excitation light is as shown in the figure. Figure 2 shown. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] The specific embodiments of the present invention will be further described in detail below with reference to the accompanying drawings.

[0042] Figure 1The fluorescence spectra of the aqueous dispersion of the ratiometric fluorescent probe molecule prepared in Example 2 of the present invention after adding hypochlorite at different concentrations are shown;

[0043] Figure 2 The fluorescence intensity of Example 3 of the present invention is shown after 1 minute of adding 20 μL of aqueous solutions of fluoride ions, iodide ions, perchlorate ions, hydrogen sulfate ions, sulfate ions, acetate ions, thiocyanate ions, and sulfide ions under 357 nm excitation light excitation.

Claims

1. A fluorescent molecular probe based on pyridinium salt, as shown in Formula I, 2. The method for synthesizing the fluorescent molecular probe according to claim 1, wherein The steps include: Take o-alkynyl aldehyde 1, pyridinium salt 2 and organic catalyst piperidine and dissolve them in anhydrous ethanol. The molar ratio of compound 1 to pyridinium salt 2 is 1:2-2:

1. Heat and stir for 12 hours. The solid is completely precipitated. Filter and the crude product is recrystallized from anhydrous ethanol to obtain a red solid probe compound I.

3. The method for synthesizing a fluorescent probe according to claim 2, wherein: The organic catalyst used in the synthesis step is piperidine, and the molar ratio of compound 1 to pyridinium salt 2 is 1:

1.

4. The method for synthesizing a fluorescent probe according to claim 2, wherein: In the synthesis step, the reaction heating temperature is 60-100 degrees Celsius.

5. The use of the fluorescent molecular probe for detecting hypochlorite according to claim 1, characterized in that Prepare the reagents for identification and determination of hypochlorite as follows: A 10% N,N-dimethylformamide-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, 1, 5, 10, 25, 50, 75, 100, 150, and 200 μM. After standing for 1 minute, the fluorescence intensities of the solutions containing different hypochlorite concentrations were measured at an excitation wavelength of 357 nm and an emission wavelength of 498 nm. A standard curve was drawn and fitted to determine the functional relationship between the fluorescence intensity and the hypochlorite concentration.

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