Pyridinium-based hypochlorite detection probe and preparation method thereof

Abstract

The invention relates to a pyridinium-based hypochlorite detection probe and a preparation method thereof. The structure of the fluorescent probe is as shown in a formula I. The fluorescent molecular probe is prepared by taking o-alkynyl aldehyde as a raw material and carrying out condensation reaction on the o-alkynyl aldehyde and pyridinium salt. The probe synthesis method is simple, the reaction condition is 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 0.38 mu M, and the response range is 5-200 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. # imgabs0 #

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 application of the fluorescent molecular probe in detecting hypochlorite. Background Art

[0002] As one of the most important reactive oxygen species in biological systems, hypochlorous acid / hypochlorite is generated from chloride ions and hydrogen peroxide by myeloperoxidase under certain physiological conditions. Hypochlorous acid is a double-edged sword in the life process. On the one hand, hypochlorous acid / hypochlorite can protect human health by killing pathogens and bacteria that invade the biological immune system; on the other hand, excessive hypochlorous acid / hypochlorite can cause oxidation of biological molecules (such as proteins, nucleic acids, lipids and enzymes), leading to tissue damage, inflammation and a series of diseases, such as cardiovascular disease, rheumatoid arthritis, liver ischemia-reperfusion injury, and even cancer. Given the special importance of hypochlorous acid in biology and medicine, exploring a highly selective, highly sensitive and easy-to-detect method for endogenous hypochlorous acid has become a hot topic in the current biomedical field. There are many methods for detecting hypochlorite, such as starch-potassium iodide method, N,N-diethyl-p-phenylenediamine colorimetry, chemiluminescence, spectrophotometry, fluorescence analysis, etc. 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] At present, fluorescent probes for detecting hypochlorous acid have been reported one after another, and there have been extensive studies based on different fluorophores, different recognition groups, and different recognition mechanisms. However, the fluorescent probes for hypochlorite reported now have the disadvantages of long synthesis steps, low detection limit, and long response time. In order to find a better method for detecting hypochlorite in aqueous solution, the present invention designs and synthesizes a fluorescent chemical probe based on pyridinium salt with the change value of fluorescence intensity as a quantitative signal, and successfully applies it to the detection of hypochlorite in aqueous phase. The probe has the characteristics of high sensitivity and high 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 hypochlorite detection fluorescent probe 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 synthesis method of the above-mentioned 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. 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

[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 detection 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 water-dispersed solutions of ratiometric fluorescent probe molecules, add 10-30 μL of hypochlorite of different concentrations (0, 1, 2, 5, 10, 20, 50, 100, 200 μM), respectively, and after standing for 1 minute, measure and calculate the fluorescence intensity at 498 nm, and make 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 have specificity in identifying hypochlorite, and the fluorescence changes from strong to weak, and the solution gradually fades from red. The detection process is not affected by the photobleaching of the probe molecules, instruments and 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. 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. Instruments and reagents used in the present invention: Nuclear magnetic resonance instrument: BrukerAV-II 500MHz NMR, TMS as internal standard, CDCl 3 as solvent; infrared spectrometer: TFS-40, KBr pellet; melting point instrument: XT-4 melting point tester. All reagents used were commercially available chemically pure or analytically pure. Example 1 Synthesis of the fluorescent probe of the present invention The preparation method of the fluorescent probe of the present invention is as follows: 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. Solid is completely precipitated. Filter and recrystallize the crude product with anhydrous ethanol to obtain a red solid probe compound I. 3-(4-(2-(4-diethylaminophenyl)ethynyl)phenylvinyl)pyridinylpropanesulfonate, dark red solid, melting point 237-240℃, 1 HNMR (CDCl 3 ,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). 1 H NMR (CDCl 3 ,100MHz)δ152.9,147.9,144.6,138.5,134.7,133.1,132.5,129.9,127.9,126.8,125.0,12 4.1,123.8,111.4,107.8,98.2,85.3,59.3,47.5,44.3,27.6,12.6.HRMS:m / z:475.2053[M] + (calculatedC28 H 31 N 2 O 3 S:475.2055). Example 2 Fluorescent probe molecular working curve Take 2mL of the prepared water dispersion solution of the ratio 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 1min, the fluorescence spectrometer records the changes in fluorescence intensity at 498nm. Plot with fluorescence intensity as the ordinate and wavelength as the abscissa to obtain the response curve of the probe molecule to hypochlorite concentration. Figure 1 The upper right corner of the figure is a working curve of the fluorescent probe molecule obtained by plotting the fluorescence intensity as the ordinate and the hypochlorite concentration as the abscissa. The relationship between the fluorescence intensity and the hypochlorite concentration under the excitation of 357nm excitation light is shown in the figure below: Figure 1 shown. Example 3 Fluorescent probe molecular selectivity Take 2mL of the prepared aqueous dispersion solution of the ratio fluorescent probe molecule, add 40μL of different metal ions or neutral molecule solutions of a certain concentration, and after standing for 1 minute, the fluorescence spectrometer records the changes in fluorescence intensity at 498nm. Plot with fluorescence intensity as the ordinate and metal ions or molecules as the abscissa to obtain a bar graph of the selectivity of the fluorescent probe molecule. In this embodiment, the selectivity of the fluorescent probe for different ions or molecules is determined. The bar graph of the fluorescence intensity changing with different ions or molecules under 357nm excitation light is shown in the figure below. Figure 2 shown. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] The specific implementation modes of the present invention are further described in detail below with reference to the accompanying drawings.

[0026] Figure 1 The fluorescence spectra of the aqueous dispersion of the ratiometric fluorescent probe molecule prepared in Example 2 of the present invention after adding hypochlorite of different concentrations are shown; Figure 2 The fluorescence intensity 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 respectively under 357 nm excitation light in Example 3 of the present invention is shown.

Claims

1. A novel 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, characterized in that: The steps include: 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. Solid is completely precipitated. Filter and recrystallize the crude product with anhydrous ethanol to obtain a red solid probe compound I.

3. The method for synthesizing a fluorescent probe according to claim 2, characterized in that: 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, characterized in that: 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 Used for identification and content determination of hypochlorite, the specific method is as follows: A 10% N,N-dimethylformamide-water mixed solution of the fluorescent probe of the invention was prepared with a concentration of 10 μM to prepare a standard solvent, and then the prepared standard solvent was used to prepare standard solutions with hypochlorite concentrations of 0, 1, 5, 10, 25, 50, 75, 100, 150, and 200 μM, respectively. After standing for 1 minute, the fluorescence intensity at different hypochlorite concentrations was measured at an excitation wavelength of 357 nm and an emission wavelength of 498 nm, and a standard curve was made to fit and determine the functional relationship between the fluorescence intensity and the hypochlorite concentration.

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