Fluorescent probe for detecting hypochlorite as well as preparation and application of fluorescent probe

Abstract

The invention discloses a fluorescent probe for detecting hypochlorite as well as preparation and application of the fluorescent probe. The fluorescent probe disclosed by the invention has a structure as shown in a formula I. The fluorescent probe disclosed by the invention is simple in preparation method, and has relatively high sensitivity and selectivity to hypochlorite in an environmental water sample. Along with the increase of the concentration of hypochlorite in a solution, the fluorescence intensity of probe molecules at 470 nm is remarkably enhanced, the increase multiple of a fluorescence signal reaches 66 times, meanwhile, the color of the solution is changed from purple to yellow, and according to the phenomenon, the probe can also be used for detecting hypochlorite in the environment through a visual method. Therefore, the probe molecule provided by the invention has potential application value and can be applied to determination of hypochlorite in an actual water sample.

Description

Technical Field

[0001] The invention relates to a fluorescent probe for detecting hypochlorite, preparation and application thereof, and belongs to the field of organic small molecule fluorescent probes. Background Art

[0002] Reactive oxygen species (ROS) play a vital role in regulating intracellular redox homeostasis, ensuring normal cell proliferation, metabolism and signal transduction. As one of the most important reactive oxygen species (ROS) in organisms, hypochlorite (ClO − ) plays an important role in the regulation of apoptosis and the prevention of inflammation, and hypochlorite has high oxidative properties and can bleach paper or industrial textiles and kill invading bacteria in drinking water. However, the large-scale use of hypochlorite in industry for disinfection purposes has contaminated groundwater storage, and drinking this hypochlorite-contaminated water can lead to its excessive storage in the human body. Excessive hypochlorite levels may induce a variety of physical diseases related to oxidative stress, such as cardiovascular disease, tissue damage, cardiovascular disease, Alzheimer's disease, ischemia-reperfusion injury, atherosclerosis, etc. Endogenous hypochlorite is produced by myeloperoxidase (MPO) catalyzing hydrogen peroxide and chloride ions and is highly oxidative and neurotoxic. Therefore, sensitive and selective monitoring of exogenous and endogenous hypochlorite is crucial for human health and the environment.

[0003] To date, a variety of analytical techniques, including electron paramagnetic resonance (EPR), high performance liquid chromatography (HPLC), optical analysis methods, mass spectrometry (MS), electrochemical methods, and iodometric methods, have been applied to detect hypochlorite. However, these traditional techniques have limitations such as high daily maintenance costs and cumbersome procedures. Compared with the above methods, fluorescent probes have attracted attention due to their simple operation, high sensitivity, rapid response, good selectivity, low toxicity, and great potential for real-time bioimaging. In recent years, an increasing number of fluorescent probes for hypochlorite have been developed. Although these probes have shown many outstanding capabilities in hypochlorite imaging, most of them still have disadvantages in detecting hypochlorite in the environment, such as high detection limits, poor anti-interference ability, and inability to be used for visual detection. Therefore, it is of great significance to construct a fluorescent probe with a simple synthetic route, fast response, good selectivity, high sensitivity, and visual detection of ClO in the environment. − New fluorescent probes are crucial. Summary of the invention

[0004] In view of some problems existing in the fluorescent probe for detecting hypochlorite in the prior art, such as low signal-to-noise ratio, poor selectivity and inability to be used for visual detection, the present invention aims to provide a fluorescent probe for detecting hypochlorite under neutral conditions.

[0005] Another object of the present invention is to provide a method for preparing the above-mentioned fluorescent probe for detecting hypochlorite, which has readily available raw materials, simple synthesis steps and high yield.

[0006] Another object of the present invention is to provide a use of the above fluorescent probe for detecting hypochlorite in preparing a reagent for detecting the hypochlorite content in water.

[0007] To achieve the above purpose, the present invention adopts the following technical solution.

[0008] A fluorescent probe for detecting hypochlorite, the structural formula of which is shown in Formula I:

[0009] A method for preparing the above-mentioned near-infrared fluorescent probe for detecting thiophenol comprises the following steps: (1) Dissolve compound 1 and para-acetaminobenzaldehyde in an organic solvent at room temperature and in an Ar2 atmosphere, and heat the mixture under reflux under alkaline conditions. After the reaction is completed, filter, wash, and dry the precipitate to obtain a red solid compound 2;

[0010] (2) Dissolving compound 2 in an organic solvent at 0°C and reacting under acidic conditions for a period of time, and obtaining compound 3 after the reaction is completed;

[0011] (3) Compound 3 was dissolved in HCl at 0°C, and then an aqueous solution of NaNO2 was added and stirred at 0°C for reaction. Finally, an HCl solution of anhydrous SnCl2 was added to the mixture and stirred for reaction. After the reaction was completed, the product was extracted, dried, desolvated, and separated and purified by column chromatography to obtain a red solid compound 4;

[0012] (4) Compound 5 is added to an alcohol organic solvent and heated to dissolve it. Compound 4 and glacial acetic acid are then added to a bottle and the mixture is heated under reflux for reaction. After the reaction is completed, the precipitate is filtered, washed, and dried to obtain a deep purple solid fluorescent probe formula I.

[0013] The fluorescent probe is used to prepare a reagent for detecting hypochlorite in water.

[0014] The mechanism of the fluorescent probe of the present invention responding to hypochlorite is as follows: The probe formula I and ClO were verified by HRMS. − The reaction mechanism ( Figure 7). Excess hypochlorite was added to the PBS buffer containing the probe. After the reaction was completed, the reactants were detected by HRMS. As shown in the figure, Figure 8 554.2527 is the mass spectrum peak of probe formula Ⅰ before reaction. Fig. 9 284.3389 is the mass spectrum of the reaction mixture. From the mass spectra before and after the reaction, it can be inferred that after the probe formula I reacts with hypochlorite, the HC=N bond is destroyed by hypochlorite, and a dicyanoisophorone derivative (HN) and the corresponding aldehyde coumarin (CQ) are produced. Since the hydrazine group on HN has a strong electron-donating ability, HN does not emit fluorescence, while the released CQ has a strong blue fluorescence.

[0015] Compared with the shortcomings and deficiencies of the prior art, the present invention has the following beneficial effects: Probe type I has almost no fluorescence in neutral PBS buffer. − After that, the fluorescence intensity at 470 nm increased rapidly and reached stability in 25 min. Compared with most other reported fluorescent probes, it has three main advantages: first, the probe type I is similar to ClO − After the reaction, the fluorescence signal increased by 66 times. − After the reaction, the solution color changes from purple to yellow, thus achieving the − Third, the synthesis steps of the fluorescent probe are simple, the yield is high, and the stability is good. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 This is the structural diagram of fluorescent probe type Ⅰ.

[0017] Figure 2 This is the high-resolution mass spectrum of probe type I.

[0018] Figure 3 This is the NMR spectrum of probe formula Ⅰ.

[0019] Figure 4 Probe I reacts with ClO at room temperature − Absorption spectra (a) and fluorescence spectra (b) before and after the reaction.

[0020] Figure 5 (a) Fluorescence of probe type I changes with ClO − ; (b) ClO − The linear relationship between the concentration and the fluorescence intensity of probe type I; (c) Under fluorescent light, the probe type I and different concentrations of ClO − Color change diagram after reaction; (d) Under 365 nm ultraviolet light, probe formula I reacts with different concentrations of ClO − Fluorescence changes after the reaction.

[0021] Figure 6 (a) Fluorescence spectra of probe type I (10.0 μmol·L-1) and different analytes, (b) fluorescence intensity at 470 nm in (a) (Na + ,Mg 2+ , Mn 2+ , K + , HSO3 − , NO3 − , HS − ,CO3 2− , Cl − , SO4 2− ,Br − , S2O3 2− , AA, Cys, GSH, ONOO − , 1 O2, •OH, TBHP, TBO• and ClO − , where ClO − The concentration is 0.12 mmol·L -1 ,ONOO − The concentration is 0.6mmol·L -1 , the concentration of other substances was 1.2 mmol·L -1 ).

[0022] Figure 7 It is a fluorescent probe formula Ⅰ and ClO − Response mechanism diagram of the reaction.

[0023] Figure 8 It is probe type I and ClO − HRMS spectrum before reaction.

[0024] Fig. 9 It is probe type I and ClO − HRMS graph after reaction. Example

[0025] The present invention will be further described below in conjunction with embodiments and drawings, but the present invention is not limited by the following embodiments.

[0026] (1) At room temperature and Ar2 atmosphere, compound 1 (1.0 g, 5.4 mmol) and p-acetaminobenzaldehyde (878.0 mg, 5.4 mmol) were added to a 100 mL round-bottom flask, dissolved in 20 mL of anhydrous acetonitrile, and piperidine (0.1 mL) was added to the solution. The system was then heated to 80 °C and refluxed for 2.0 h. After the reaction was completed, acetonitrile was used for recrystallization, and then washed with ice acetonitrile to obtain a red solid compound 2;

[0027] (2) At 0 °C, compound 2 (50 mg, 0.17 mmol) was added to a 100 mL round-bottom flask and dissolved in 30 mL of isopropanol. HCl (6 mmol·L -1 , 10 mL), then heated to 100 °C, and refluxed for 1.5 h. After the reaction was completed, ethyl acetate was used for extraction, dried, and purified by column chromatography to obtain a red solid compound 3;

[0028] (3) At 0 °C, compound 3 (50 mg, 0.17 mmol) was reacted with HCl (6 mmol·L -1 , 3 mL) was added to a 25 mL round-bottom flask, and then an aqueous solution of NaNO2 (32.2 mg, 0.46 mmol) was added dropwise to the mixture and stirred at 0 °C for 1.5 h. Finally, anhydrous SnCl2 (131.4 mg, 0.68 mmol) in HCl (6 mmol·L -1 , 2 mL) solution was added dropwise to the mixture and stirred for 1.5 h. After the reaction was completed, the mixture was neutralized with NaOH solution, washed with water 3 times, extracted, and the organic phase was concentrated and purified by column chromatography to obtain a red solid compound 4;

[0029] (4) Compound 5 (61.3 mg, 0.25 mmol) and 15 mL of anhydrous ethanol were added to a 50 mL round-bottom flask and heated to 80 °C. When compound 5 was completely dissolved, a small amount of compound 4 (76.3 mg, 0.25 mmol) and 1 mL of glacial acetic acid were added to the flask, and the mixture was refluxed at 80 °C for 4 h. After the reaction was completed, the flask was taken out and recrystallized in ethanol to precipitate a solid, which was filtered and dried to obtain a dark purple solid fluorescent probe formula I;

[0030] Example 2 Fluorescent probe formula I for ClO − Feasibility study of response By comparing the addition of ClO − The changes in the UV-visible absorption spectrum and fluorescence spectrum of probe type I before and after were used to verify the detection of ClO by probe type I − feasibility. Figure 4 As shown in a, the absorption spectrum of probe formula I shows two absorption peaks at 400 nm and 540 nm, respectively. −After that, the absorption peak of the probe type I solution at 410 nm increased rapidly, and the absorption peak at 540 nm decreased rapidly. The maximum fluorescence emission peak of the probe type I solution was at 470 nm ( Figure 4 b) Add ClO − After that, the fluorescence intensity at 470 nm increased rapidly. The experimental results show that the probe formula I can detect ClO − Generate a response.

[0031] Example 3 Quantitative detection of ClO in water − Under the optimal experimental conditions, the effect of probe type I on different concentrations of ClO − The spectral characteristics of the response. Figure 5 As shown in a, the fluorescence intensity of probe type I increases with the increase of ClO − The fluorescence intensity of probe I at 470 nm was significantly higher than that of ClO − There is a good linear relationship between the concentrations of 2 = 0.9991), the linear range was 20 - 120 μmol·L -1 The detection limit was 0.7 μmol·L -1 (S / N=3), the linear regression equation is Y = 48.3X + 615.5, Y is the relative fluorescence intensity of probe type I, X is ClO − The concentration ( Figure 5 b). The results show that probe type I can detect ClO in water. − At the same time, Figure 5 As shown in c, under fluorescent light, as ClO − With the increase of concentration, the solution color of probe type I changes from purple to yellow, and the blue fluorescence gradually increases ( Figure 5 d).

[0032] In order to study the specificity of probe type I and consider its applicability in practical applications, different analytes (Na + ,Mg 2+ , Mn 2+ , K + , HSO3 − , NO3 − , HS − ,CO3 2− , Cl − , SO4 2− ,Br − , S2O3 2− ,AA,H2O2,Cys,GSH,ONOO − , 1O2, •OH, TBHP, TBO•) to ClO − Detection interference. Figure 6 As shown, only ClO − The fluorescence intensity of probe formula I at λ = 470 nm increased significantly, while other analytes had almost no effect on it. This result shows that the fluorescent probe formula I has high selectivity.

Claims

1. A fluorescent probe for detecting hypochlorite, the structural formula of which is shown in Formula I, 。 2. A method for preparing a fluorescent probe for detecting hypochlorite as claimed in claim 1, characterized in that: The following steps are involved: (1) Compound 1 and para-acetaminobenzaldehyde are dissolved in an organic solvent, and heated and stirred under alkaline conditions to react. After the reaction is completed, the precipitate is filtered, washed, and dried to obtain Compound 2; ； (2) Dissolving compound 2 in an organic solvent at 0°C and reacting under acidic conditions for a period of time, and obtaining compound 3 after the reaction is completed; ； (3) Compound 3 was dissolved in HCl at 0°C, and then an aqueous solution of NaNO2 was added and stirred at 0°C for reaction. Finally, an HCl solution of anhydrous SnCl2 was added to the mixture and stirred for reaction. After the reaction was completed, the product was extracted, dried, desolvated, and separated and purified by column chromatography to obtain a red solid compound 4; ； (4) Compound 5 is added to an alcohol organic solvent and heated to dissolve it. Compound 4 and glacial acetic acid are then added to a bottle and the mixture is heated under reflux for reaction. After the reaction is completed, the precipitate is filtered, washed, and dried to obtain a deep purple solid fluorescent probe formula I. 。 3. The preparation method according to claim 2, characterized in that: The preparation method has the following characteristics: The reaction in step (1) is carried out in the presence of an organic solvent, wherein the solvent is anhydrous acetonitrile; The reaction of step (1) is carried out under alkaline conditions, and the base is piperidine; The reaction heating temperature of step (1) is 80°C; The reaction time of step (1) is 2.0 hours; The reaction in step (2) is carried out in the presence of an organic solvent, wherein the solvent is isopropanol; The reaction in step (2) is carried out under acidic conditions, wherein the acid is hydrochloric acid; The reaction heating temperature of step (2) is 100°C; The reaction time of step (2) is 1.5 hours; Step (3) at 0°C, compound 3 is dissolved in HCl, then an aqueous solution of NaNO2 is slowly added dropwise, and the mixture is stirred and reacted at 0°C for 1.5 hours, and finally an HCl solution of anhydrous SnCl2 is slowly added dropwise to the mixture and stirred and reacted for 1.5 hours; The reaction in step (4) is carried out in an alcohol organic solvent, wherein the solvent is ethanol; The reaction heating temperature of step (4) is 80°C; The reaction time of step (4) is 4.0 hours.

4. Use of the fluorescent probe for detecting hypochlorite according to claim 1 in preparing a reagent for detecting the content of hypochlorite in an actual water sample.

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