Chemical reaction type fluorescent probe HClO-1 and preparation and application thereof in hypochlorous acid detection

Abstract

The present application relates to a kind of preparation method of chemical reaction type fluorescent probe for detecting hypochlorous acid and its application in hypochlorous acid detection.The present application utilizes 4-formylbenzoic acid and acetyl acetate methyl ester and ammonia water reaction, obtains the molecular probe HClO-1 of blue fluorescence emission.The fluorescent probe can selectively occur addition reaction with hypochlorous acid molecule, causes the blue fluorescence of fluorescent probe to be quenched, so as to be used for the rapid high-sensitivity high-selectivity detection of hypochlorous acid.

Description

Technical Field

[0001] This invention relates to the field of chemical detection technology, and specifically to a chemically reactive fluorescent probe for detecting hypochlorous acid, and more particularly to a method for preparing a chemically reactive fluorescent probe and its application in the detection of hypochlorous acid. Background Technology

[0002] Hypochlorous acid is an important reactive oxygen species (ROS) and is widely used in daily life for disinfecting domestic water due to its strong oxidizing power. Hypochlorous acid is widely used for indoor and outdoor disinfection and sterilization. This leads to residual hypochlorous acid entering rivers and lakes through sewers. Excessive hypochlorous acid poses a potential threat to human and animal health, causing kidney disease, lung damage, and cancer. The maximum free chlorine concentration in water distribution systems should not exceed 4 mg / L. Therefore, developing a highly selective and rapid sensing analysis method to detect the concentration of hypochlorous acid in actual water samples is of great significance.

[0003] In recent years, real-time detection methods based on fluorescent probes have been widely developed in the analysis of environmental and biological samples. Fluorescent probes offer advantages such as high sensitivity and non-destructive real-time detection, enabling highly sensitive detection of abnormal changes in analyte samples. Currently reported fluorescent probes for hypochlorous acid detection mainly utilize the oxidizing power of hypochlorous acid, using methoxyaniline, methoxyphenol, oximes, selenides, thioethers, and thiols as reaction sites. Simultaneously, hypochlorous acid also possesses electrophilic addition ability, capable of undergoing electrophilic addition reactions with olefin double bonds. This invention designs an organic fluorescent molecule with double bonds, not previously reported in the literature, and utilizes the electrophilic addition ability of hypochlorous acid to develop a chemical reaction-based method for the preparation and analysis of fluorescent probes for the detection of hypochlorous acid. Summary of the Invention

[0004] This invention provides a fluorescent molecular probe for detecting hypochlorous acid and its preparation method. The invention utilizes the reaction of 4-formylbenzoic acid with methyl acetoacetate and ammonia to obtain a blue fluorescent molecular probe HClO-1. This fluorescent probe can selectively undergo an addition reaction with hypochlorous acid molecules, resulting in the quenching of the blue fluorescence of the probe, thus enabling rapid, highly sensitive, and highly selective detection of hypochlorous acid. Furthermore, the probe preparation method of this invention is simple and easy to manufacture. Specifically, this invention is achieved using the following technical solution:

[0005] Firstly, this invention provides a fluorescent probe molecule for detecting hypochlorous acid, named HClO-1, with the following chemical structural formula:

[0006]

[0007] Secondly, this invention also provides a method for preparing the fluorescent probe HClO-1, which involves refluxing 4-formylbenzoic acid, methyl acetoacetate, and ammonia in ethanol under an inert atmosphere, followed by separation and purification. The preferred molar ratio of 4-formylbenzoic acid to methyl acetoacetate is 1:(6.0-6.6), more preferably 1:(6.0-6.3); the preferred molar ratio of ammonia to 4-formylbenzoic acid is (6.0-6.6):1, more preferably (6.0-6.3):1; the preferred volume / mass ratio of ethanol to 4-formylbenzoic acid is (20-60):1, mL / g; the concentration of ethanol is not particularly critical, but 50-95% is preferred. The preferred reflux reaction time is 4-6 hours. The separation and purification step can be performed by evaporating the solvent after the reaction is complete, separating the residue by column chromatography, and then purifying it by recrystallization with anhydrous ethanol or 95% ethanol to obtain the crystalline product fluorescent probe molecule HClO-1.

[0008] More specifically, in order to better obtain the fluorescent probe HClO-1, this invention, through better experimental optimization, discovered that the following preparation method steps can be adopted:

[0009] In a round-bottom flask equipped with a reflux apparatus, 4-formylbenzoic acid, methyl acetoacetate, and ammonia are added. The mixture is dissolved in ethanol, and then refluxed under an inert atmosphere such as argon. After the reaction is complete, the solvent is removed by rotary evaporation, and the residue is recrystallized or separated by column chromatography to obtain the white solid target product HClO-1. In the preparation of the fluorescent probe HClO-1 of the present invention, preferably, the molar ratio of 4-formylbenzoic acid to methyl acetoacetate is 1:6, and the molar ratio of ammonia to 4-formylbenzoic acid is 6:1. The volume (mL) / mass (g) ratio of ethanol to 4-formylbenzoic acid is preferably 30:1. Preferably, the reaction is heated to reflux with ethanol for 5 hours.

[0010] In the preparation of the fluorescent probe HClO-1 described in this invention, the specific method for the separation and purification steps is as follows: After the reaction is complete, the solvent is removed by rotary evaporation, the residue is extracted with ethyl acetate, and washed successively with saturated brine and ultrapure water. The obtained organic phase is dried with anhydrous sodium sulfate, and then filtered and concentrated under reduced pressure. The concentrate is separated by column chromatography using methanol / dichloromethane (1:20 v / v) as the eluent to obtain a white solid target product probe. It is then purified by recrystallization with anhydrous ethanol to obtain a white crystalline target product probe.

[0011] Thirdly, this invention also investigated the application of the fluorescent probe HClO-1 in the detection of hypochlorous acid. The study found that this fluorescent probe can be used to detect substances containing hypochlorous acid, especially solutions; specifically, it can be used to detect hypochlorous acid in tap water, surface and groundwater, or intracellular fluids.

[0012] The fluorescent probe molecule of this invention is simple to prepare and can be obtained in a single reaction. This molecular probe emits strong blue fluorescence in solvents such as water, methanol, ethanol, n-hexane, and dichloromethane. However, in the presence of hypochlorous acid, the blue fluorescence of the probe is quenched, thus allowing for the selective detection of hypochlorous acid in solution, with a detection limit of [not specified]. The fluorescent probe of this invention is simple to prepare, highly sensitive (detection limit 0.03 μM), and has a wide detection range (0.5-9.0 μM), making it suitable for highly sensitive and selective detection of hypochlorous acid in solution. Attached Figure Description

[0013] Figure 1 This is the proton spectrum of the fluorescent probe HClO-1.

[0014] Figure 2 This is a photostogram of the photostability of the fluorescent probe HClO-1 of this invention.

[0015] Figure 3 This is a graph showing the fluorescence spectrum changes of the fluorescent probe HClO-1 of this invention after adding different concentrations of hypochlorous acid. The fluorescence intensity gradually decreases as the concentration of hypochlorous acid increases.

[0016] Figure 4 The degree of fluorescence attenuation of the fluorescent probe HClO-1 in this invention is linearly related to the concentration of hypochlorous acid.

[0017] Figure 5 This is a graph showing the changes in the absorption spectrum of the fluorescent probe HClO-1 of this invention after adding different concentrations of hypochlorous acid.

[0018] Figure 6 This demonstrates the selectivity of the fluorescent probe HClO-1 of this invention for different species. The final concentration for all species was 8.0 μM.

[0019] Figure 7 This describes the fluorescence quenching mechanism of the fluorescent probe HClO-1 in this invention. Detailed Implementation

[0020] The following embodiments are further illustrations of the present invention and serve as explanations of the technical content of the present invention. However, the essence of the present invention is not limited to the embodiments described below. Those skilled in the art can and should know that any simple changes or substitutions based on the spirit of the present invention should fall within the protection scope claimed by the present invention.

[0021] Example 1

[0022] Synthesis of fluorescent probe HClO-1

[0023] The chemical reaction formula for the synthesis of the fluorescent probe of this invention is as follows:

[0024]

[0025] In a round-bottom flask equipped with a reflux apparatus, 4-formylbenzoic acid (0.90 g, 6.0 mmol), methyl acetoacetate (4.20 g, 36.2 mmol), and ammonia (2.70 mL, 36.2 mmol) were added. The mixture was dissolved in 25 mL of ethanol, and then refluxed under an argon atmosphere. After the reaction was complete, the solvent was removed by rotary evaporation. The residue was separated by column chromatography using methanol / dichloromethane (1:20 v / v) as the eluent to give the target product HClO-1 (1.16 g, 3.36 mmol, yield 56.0%) as a white solid. This solid could be recrystallized from a small amount of anhydrous ethanol to give a crystalline solid. 1 H NMR (400MHz, MeOD) δ7.88(d,J=6.5Hz,2H),7.33(d,J=6.5Hz,2H),5.04(s,1H),3.64(s,6H),2.33(s,6H).( Figure 1 )ESIMScalculated 345.1212,found 346.1213[M+H] + .

[0026] Example 2

[0027] UV-Vis and fluorescence spectra were performed on the fluorescent molecular compound HClO-1 prepared in Example 1:

[0028] Preparation of stock solution of fluorescent molecular compound HClO-1 (probe) (2 mmol / L): Weigh 3.45 mg of fluorescent molecular compound HClO-1, place the compound in a 10 mL round bottom flask, measure 5 mL of anhydrous ethanol with a pipette, stir to dissolve and obtain the stock solution, and store in a refrigerator for later use.

[0029] Preparation of the test solution for the fluorescent molecule compound HClO-1: Add 4 μL of the HClO-1 stock solution to 2000 μL of PBS 7.4 buffer solution, mix thoroughly, and then measure its UV-Vis absorption and fluorescence spectra. The parameters of the fluorescence spectrophotometer are set as follows: excitation wavelength 365 nm; emission spectral scanning range 390-600 nm; slit width for both excitation and emission light is 5 nm.

[0030] Photostability test of the fluorescent molecule HClO-1: 4 μL of the HClO-1 stock solution was added to 2000 μL of PBS 7.4 buffer solution, mixed thoroughly, and the fluorescence intensity change of the molecule under UV irradiation was measured. The parameters of the fluorescence spectrophotometer were set as follows: excitation wavelength 365 nm; emission wavelength 450 nm; continuous irradiation time 1800 s (30 min). Figure 2 As shown, the fluorescence intensity of the fluorescent probe at 450 nm did not change significantly, proving that it has good photostability.

[0031] Example 3

[0032] Fluorescence spectral change test of the fluorescent probe HClO-1 prepared in Example 1 for hypochlorous acid detection: In this example, the response of the probe HClO-1 prepared in Example 1 to hypochlorous acid was detected by fluorescence spectroscopy. Specifically, 4 μL of the probe stock solution prepared in Example 2 was added to 2000 μL of PBS 7.4 (50 mM, pH 7.4) buffer solution and mixed thoroughly. Then, 2 μL of hypochlorous acid solutions with final concentrations of 0, 0.5, 1.0, 2.0, 3.0, 4.0, 5.0, 6.0, 7.0, and 8.0 μM were added to the mixed solution, and the fluorescence spectra were recorded. The solution was measured under constant temperature conditions of 25°C, using an excitation wavelength of 365 nm and a scan rate of 500 nm / min, within the fluorescence spectrum range of 390-600 nm. Figure 3 As shown in the figure, after adding hypochlorous acid to the system, the fluorescence intensity of the probe molecule solution gradually decreased. Upon adding 8 μM hypochlorous acid, the fluorescence of the probe molecule was quenched. The degree of fluorescence quenching was linearly related to the concentration of hypochlorous acid added, as shown in the figure. Figure 4 As shown.

[0033] Example 4

[0034] Test of absorption spectrum changes of the fluorescent probe HClO-1 prepared in Example 1 for hypochlorous acid detection: In this example, the response of the probe HClO-1 prepared in Example 1 to hypochlorous acid was detected by UV-Vis spectroscopy. Specifically, 4 μL of the probe stock solution prepared in Example 2 was added to 2000 μL of PBS 7.4 (50 mM, pH 7.4) buffer solution and mixed thoroughly. Then, 2 μL of hypochlorous acid solution with final concentrations of 0, 1.0, 2.0, 3.0, 4.0, 5.0, 6.0, 7.0, and 8.0 μM was added to this solution, and the absorption spectra were recorded. Figure 5 As shown.

[0035] Example 5

[0036] Selectivity test of fluorescent probe: 4 μL of the probe stock solution prepared in Example 2 was added to 2000 μL of PBS 7.4 (50 mM, pH 7.4) buffer solution, mixed thoroughly, and then some common analytes were added (control - blank control, O2). - ·- Superoxide anion, OH·- Hydroxyl radical, NO- Nitric oxide, TBO·- Peroxy-tert-butyl radical, TBHP- Peroxy-tert-butanol, ONOO — Peroxynitrosyl radical (H₂O₂ - hydrogen peroxide) was used to bring the final concentration of the analyte to 8 μM. Then, under isothermal conditions of 25 °C, fluorescence spectra in the 390-600 nm range were recorded using an excitation wavelength of 365 nm and a scan rate of 500 nm / min to measure the solution. Figure 6 As shown, only hypochlorous acid has a high fluorescence quenching effect on this fluorescent probe, meaning that this fluorescent probe only has a high selective response to hypochlorous acid.

[0037] It should be noted that the above-described technical content of this invention is merely an explanation and clarification to enable those skilled in the art to understand the technical essence of this invention, and therefore is not intended to limit the scope of protection of this invention. The scope of protection of this invention should be determined by the claims. Those skilled in the art should understand that any modifications, equivalent substitutions, and improvements made based on the essential spirit of this invention should be within the scope of protection of this invention.

Claims

1. The application of a chemically reactive fluorescent probe in the detection of hypochlorous acid, wherein the hypochlorous acid detection is for the detection of non-disease diagnostic or therapeutic methods, and the fluorescent probe is named HClO-1, and its chemical structural formula is as follows: ； The fluorescent probe is prepared by reacting 4-formylbenzoic acid, methyl acetoacetate and ammonia in ethanol under an inert atmosphere, followed by separation and purification.

2. The application as described in claim 1, characterized in that, The molar ratio of 4-formylbenzoic acid to methyl acetoacetate is 1:(6.0-6.6), and the molar ratio of ammonia to 4-formylbenzoic acid is (6.0-6.6):

1.

3. The application as described in claim 1, characterized in that, The volume / mass ratio of the solvent ethanol to 4-formylbenzoic acid used is (20-60):1, mL / g.

4. The application as described in claim 1, characterized in that, The reflux reaction time is 4-6 h.

5. The application as described in claim 1, characterized in that, The separation and purification steps are as follows: after the reaction is complete, the solvent is evaporated, and the residue is separated by column chromatography to obtain a white solid powder product, which is then recrystallized with anhydrous ethanol or 95% ethanol to obtain a crystalline product.

6. The application as described in claim 1, characterized in that, The hypochlorous acid detection refers to the detection of hypochlorous acid in tap water or surface / groundwater.

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