Preparation and application of water-soluble hypochlorous acid fluorescent probe

Abstract

The invention relates to preparation and application of a water-soluble hypochlorous acid fluorescent probe. The structural formula of the probe is shown in the specification. The invention provides a preparation method for synthesizing the fluorescent probe by taking benzoindole and xanthene as raw materials. The fluorescent probe is a hypochlorous acid fluorescent probe with high sensitivity, high selectivity and short response time; firstly, the fluorescent probe shows response to hypochlorous acid in a range of 1.0 mu M to 50 mu M, and has relatively good sensitivity; secondly, the fluorescent probe is not influenced by other inorganic ions, active oxygen, active sulfur and biological mercaptan, and only shows good selectivity to hypochlorous acid; moreover, when the pH value is 7.0-9.0, the fluorescent probe has good response to hypochlorous acid, and hypochlorous acid can be detected under physiological conditions; in addition, the fluorescent probe can be applied to detection of the hypochlorous acid content in an actual water sample.

Description

Technical Field

[0001] This invention belongs to the field of fluorescent probe technology, specifically relating to the preparation and application of a water-soluble hypochlorous acid fluorescent probe. Background Technology

[0002] Hypochlorous acid (HClO) is a strong oxidizing agent widely used for disinfection of domestic water and for both indoor and outdoor disinfection and sterilization. This leads to residual HClO entering rivers and lakes through sewers (Qian Z, Di Z, Zeyu Z, et al. Detection and application of hypochlorous acid in both aqueous environments and living organisms, Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy, 2024, 314, 124-225.). Excessive HClO poses a threat to human health, causing kidney disease, lung damage, and cancer (Chunpo G, Pengcheng Z, Xiaoyu W, et al. Development of a fast-response NIR fluorescent probe for hypochlorous acid detection and its applications in bioimaging and environmental analysis, Microchemical Journal, 2025, 214: 114-095.). Therefore, developing an analytical method that can accurately monitor HClO in actual water samples is crucial.

[0003] Fluorescence methods have advantages such as high sensitivity, good selectivity and fast response speed, and have shown great application potential. To date, several fluorescent probes have been developed for the real-time detection of hypochlorous acid concentration in environmental samples (Hao Y, Zhang Y, Sun Q, et al. Phenohiazine-coumarin-pyridine hybrid as an efficient fluorescent probe for ratiometric sensing hypochlorous acid, Microchemical Journal, 2021, 171:106851. Li S, Wang P, Liu Y, et al. Amitochondrial-targeted near-infrared fluorescent probe for visualizing the fluctuation of hypochlorite acid in idiopathic pulmonary fibrosis mice, Analytica Chimica Acta, 2023, 1239:340731. Huang T, Ji H, Yan S, et al. A hypochlorite-activated strategy for realizing fluorescence turn-on, type I and type II ROS-combined photodynamic tumor ablation, Biomaterials, 2023, 297:122108. He Q, Guo T,Lan M,et al.Dual-ratiometric fluorescent probes formonitoring ClO - and polarity dynamics in ferroptosis, Sensors and actuators. B, Chemical, 2024, 415:136030.). However, these fluorescent probes suffer from poor water solubility. Therefore, the design and synthesis of fluorescent probes with good water solubility is urgently needed.

[0004] Benzoindole-oxanthracene dyes are currently one of the most widely used dyes in the field of fluorescent probes, possessing advantages such as high molar absorptivity and high fluorescence quantum yield. It has been reported that oxanthracene fluorescent probes have been used to detect many target analytes, such as Cys, HClO, H2O2, and Hg.2+(Ojida A, Takashima I, Kohira T, et al. Turn-on fluorescence sensing of nucleoside polyphosphates using axanthene-based Zn(II) complex chemosensor. Journal of the American Chemical Society, 2008, 130: 12095-12102, Ma S, Wang Y, She M, et al. Design strategies and progress on xanthene-based fluorescent probe for metal ions, Reviews in Analytical Chemistry, 2017, 36: 1231-1242. Kumar R, Han J, Lim H J, et al. Mitochondrial induced and self-monitored intrinsic apoptosis by antitumor theranostic prodrug: in vivo imaging and precise cancer treatment, Journal of the American Chemical Society, 2014, 136: 17836-1784). However, the poor water solubility of benzindole-xanthene dyes limits their application in environmental systems.Introducing adamantane into benzoindole-oxanthracene dyes and utilizing its binding with β-cyclodextrin holds promise for improving the dye's water solubility and fluorescence intensity. Therefore, designing and synthesizing a water-soluble hypochlorous acid fluorescent probe as an effective tool for detecting HClO in environmental samples is essential. Summary of the Invention

[0005] Based on the requirements, the inventors conducted in-depth research and, after a great deal of creative work, provided a water-soluble hypochlorous acid fluorescent probe.

[0006] The technical solution of this invention is a water-soluble hypochlorous acid fluorescent probe, the structural formula of which is as follows:

[0007]

[0008] A method for preparing a water-soluble hypochlorous acid fluorescent probe. The steps are as follows:

[0009] In a 100 mL round-bottom flask, 1 equivalent of compound ABX-OH and 2–5 equivalents of dimethylaminothiocarbamoyl chloride were added to 10–15 mL of dichloromethane, followed by 2–3 equivalents of N,N-diisopropylethylamine. The mixture was then stirred under nitrogen protection at room temperature for 10–12 h. The reaction was stopped, and the solvent was removed by vacuum distillation. The crude product was subjected to column chromatography using CH2Cl2 / CH3OH at a volume ratio of 100:1–100:5 as the eluent to obtain a blue solid product, which is the fluorescent probe ABX-HClO.

[0010] The beneficial effect of this invention is the excellent spectral response performance of a water-soluble hypochlorous acid fluorescent probe. First, the fluorescence spectral properties of the probe were studied. The probe itself did not exhibit significant fluorescence emission at 735 nm; however, after the addition of HClO, significant emission appeared at 735 nm. Furthermore, the fluorescence intensity continuously increased with increasing HClO concentration. When 50 μM HClO was added, the fluorescence intensity increased 22-fold. The detection range of this probe is from 1 μM to 50 μM, with a detection limit of 0.35 μM, indicating that the probe can detect HClO with high sensitivity. Next, the ultraviolet absorption spectrum of the probe was studied. The probe itself had no absorption band near 700 nm, but a new absorption band appeared near 700 nm after the addition of HClO. Then, the selectivity of the probe was studied. The selectivity of the probe with inorganic ions (Na₂O₃) was investigated. + Mg 2+ ,K + Cu 2+ ,Zn 2+ NH4 + ,Cl - ,Br - ,I - HCO3 -NO3 - SO4 2- ), reactive oxygen species (H2O2, ONOO) - ), active sulfur (HS) - HSO3 - The fluorescence response of the fluorescent probe to biothiols (Cys, Hcy, GSH) and the analyte hypochlorous acid (HClO) was investigated. The results showed that only HClO caused a change in the fluorescence spectrum; other analytes had no significant effect on the probe's fluorescence spectrum. Finally, the effect of pH on the determination of HClO by the fluorescent probe was studied. A pH between 7.0 and 9.0 did not affect the determination of HClO by the fluorescent probe. Furthermore, the fluorescent probe exhibited a rapid response, with a response time within 300 s.

[0011] Application of a water-soluble hypochlorous acid fluorescent probe. HClO is commonly found in water, and lake water and tap water were used to evaluate the application of the fluorescent probe in real samples. To ensure experimental accuracy, the obtained lake water and tap water samples were first allowed to stand for 2 hours, and then the pH of both was adjusted to 7.4 before use. Next, samples containing different concentrations of HClO were prepared and fluorescence tests were performed. Recovery rates of 98.81%–106.35% were obtained in lake water, and 95.00%–106.75% were obtained in tap water. These results demonstrate that the probe can detect HClO in real samples, providing a reliable method for monitoring HClO in water. Attached Figure Description

[0012] Figure 1 This is the synthetic route for the fluorescent probe.

[0013] Figure 2 The fluorescence spectra are shown after the fluorescent probe reacts with different concentrations of HClO.

[0014] The x-axis represents wavelength, and the y-axis represents fluorescence intensity. The concentration of the fluorescent probe is 10.0 μM, and the concentrations of HClO are 0, 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, and 50 μM. The emission wavelength range is 725-800 nm, and the corresponding excitation wavelength is 700 nm.

[0015] Figure 3 This is a linear fluorescence response diagram of the fluorescent probe to different HClO concentrations.

[0016] Figure 4 The images show the UV-Vis absorption spectra of the fluorescent probe and its reaction with HClO.

[0017] The x-axis represents wavelength, and the y-axis represents absorbance. The concentration of the fluorescent probe is 10.0 μM, and the concentration of HClO is 50.0 μM.

[0018] Figure 5 This is a selectivity diagram of the fluorescent probe.

[0019] The concentration of the fluorescent probe was 10.0 μM, the concentration of HClO was 50.0 μM, and the concentration of other analytes was 50.0 μM.

[0020] Figure 6 This is a graph showing the effect of pH on fluorescent probes.

[0021] Figure 7 The graph shows the change in fluorescence intensity over time after the fluorescent probe reacts with HClO at concentrations of 10.0, 30.0, and 50.0 μM. Detailed Implementation

[0022] The present invention will now be described in detail with reference to the accompanying drawings and specific embodiments, but is not limited thereto.

[0023] Example 1:

[0024] Synthesis of fluorescent probes

[0025] Synthetic routes such as Figure 1 In a 100 mL round-bottom flask, 1 equivalent of compound ABX-OH and 2 equivalents of dimethylaminothiocarbamoyl chloride were added to 10 mL of dichloromethane, followed by 2 equivalents of N,N-diisopropylethylamine. The mixture was then stirred under nitrogen protection at room temperature for 10 h. The reaction was stopped, and the solvent was removed by vacuum distillation. The crude product was subjected to column chromatography using CH2Cl2 / CH3OH at a volume ratio of 100:3 as the eluent to obtain a blue solid product (yield of 65%), which is the fluorescent probe ABX-HClO. 1H NMR(400MHz,Chloroform-d)δ8.10(dt,J=7.5,1.5Hz,1H),8.03–7.95(m,2H),7.94–7.89(m,1H),7.66(d,J=15.0Hz,1H), 7.53(td,J=7.4,1.6Hz,1H),7.39(td,J=7.5,1.5Hz,1H),7.33(d,J=7.5Hz,1H),7.30–7.22(m,2H),7.14(dd,J=7.5,1.5H z,1H),6.90–6.84(m,2H),4.62(t,J=7.1Hz,2H),3.49(q,J=7.0Hz,2H),3.27(s,4H),2.79(td,J=7.1,1.1Hz,2H),2.76–2 .71(m,1H),2.71–2.64(m,1H),2.07–1.96(m,3H),1.93–1.88(m,6H),1.73(t,J=7.0Hz,6H),1.55(s,6H).MS(TOF):712.3.

[0026] Example 2:

[0027] Preparation of fluorescent probe and HClO solution

[0028] A certain amount of fluorescent probe ABX-HClO solid was weighed and dissolved in DMSO to prepare 1.0 × 10⁻⁶ ppm. -3 mol·L -1 ABX spare solution. Preparation of HClO solution: Dissolve a certain amount of HClO in double-distilled water to obtain a concentration of 1.0 × 10⁻⁶. - 2 mol·L -1 HClO. Add 50 μL LABX-HClO stock solution, 1.2 mL DMSO, and different volumes of HClO stock solution to a 5 mL volumetric flask, and dilute to volume with PBS buffer to obtain a concentration of 1.0 × 10⁻⁶. -5 mol·L -1 Fluorescent probe and 1.0 × 10 -6 ~5.0×10 -5 mol·L -1 The HClO was mixed with the solution to be tested.

[0029] Example 3:

[0030] Determination of fluorescence spectrum of the interaction between fluorescent probe and HClO

[0031] Figure 2The fluorescence spectrum is shown for the reaction of the fluorescent probe with HClO. The concentration of the fluorescent probe was 10 μM, and the concentrations of HClO were 0, 5.0, 10.0, 15.0, 20.0, 25.0, 30.0, 35.0, 40.0, 45.0, and 50.0 μM, respectively. The excitation wavelength used in the experiment was 700 nm, and the emission wavelength range was 725–800 nm. The slit width was 10.0 nm / 10.0 nm, and the fluorescence measurement instrument used was a Hitachi F4600 fluorescence spectrophotometer. Figure 2 As can be seen, before the addition of HClO, the probe itself had almost no emission peak due to the quenching effect of thioisocyanate; with the addition of HClO, the emission peak at 735 nm was enhanced. Furthermore, the fluorescence intensity of the probe continuously increased with the increase of HClO concentration. Figure 3 This is a linear response graph of the probe to different HClO concentrations. The fluorescence intensity shows a linear relationship with the HClO concentration. The detection range of this probe is from 1.0 μM to 50.0 μM, and the detection limit is 0.35 μM. This indicates that the probe can detect HClO with high sensitivity.

[0032] Example 4:

[0033] Determination of UV-Vis absorption spectra of the reaction between fluorescent probes and HClO

[0034] Figure 4 The image shows the UV-Vis absorption spectrum of the fluorescent probe after reaction with HClO. The concentration of the fluorescent probe was 10.0 μM, and the amount of HClO added was 20.0 μM. The UV-Vis absorption spectroscopy was performed using an Agilent Cary 60 UV-Vis spectrophotometer. Figure 4 As can be seen, the probe itself has no absorption band at 700 nm; after adding HClO, a new absorption band appears at 700 nm.

[0035] Example 5:

[0036] Selectivity of fluorescent probe for HClO determination

[0037] Figure 5 This is a graph showing the selectivity of the fluorescent probe for HClO. The determination was investigated by adding HClO (50.0 μM) and inorganic ions (Na+) to a 10.0 μM fluorescent probe. + Mg 2+ ,K + Cu 2+ ,Zn 2+ NH4 + ,Cl - ,Br - ,I - HCO3 - NO3 - SO42- ), reactive oxygen species (H2O2, ONOO) - ), active sulfur (HS) - HSO3 - The fluorescence response of biothiols (Cys, Hcy, GSH). Figure 5 It can be seen that only HClO causes a significant enhancement in the fluorescence spectrum, while other analytes have no significant effect on the fluorescence spectrum of the probe. These results indicate that the fluorescent probe has good selectivity for HClO.

[0038] Example 6:

[0039] Effect of solution pH on the fluorescence properties of HClO determined by a fluorescent probe

[0040] The effect of pH on the fluorescence spectrum of HClO determined by a fluorescent probe was investigated, and the results are as follows: Figure 6 Our study used a pH range of 4.0–10.0, with a fluorescent probe concentration of 10.0 μM and HClO concentration of 50.0 μM. As shown in the figure, the fluorescence intensity of the fluorescent probe remained essentially unchanged with pH variations, indicating that pH has no effect on the probe itself. However, after adding HClO, the fluorescence intensity ratio significantly increased within the pH range of 7.0–9.0. In conclusion, a pH range of 7.0–9.0 does not affect the determination of HClO by the fluorescent probe and is considered a suitable pH range, which is highly beneficial for the use of this probe in the determination of HClO in real samples.

[0041] Example 7:

[0042] Determination of the response time of the fluorescent probe to HClO

[0043] We investigated the response time of the fluorescent probe to HClO, and the results are as follows: Figure 7 As can be seen from the figure, the probe's response time to HClO is 300 s, which meets the requirements for monitoring in actual samples. Figure 7 It can also be seen that after the fluorescence intensity reaches its maximum value, the fluorescence intensity no longer changes over time, which indicates that this fluorescent probe has good photostability.

[0044] Example 8:

[0045] Fluorescent probes are used for the detection of lake water samples.

[0046] Lake water samples were collected from Huameitan Lake at Xiangtan University. The samples were allowed to stand for 2 hours, and the pH of the settled lake water was adjusted to 7.4. Since the lake water samples did not contain HClO, HClO was added externally before fluorescence detection. The results are shown in Table 1. The table shows that the probe's recovery rate for HClO in the lake water ranged from 98.87% to 103.20%.

[0047] Table 1. Determination of HClO spiked recovery rate in lake water

[0048]

[0049] Example 9:

[0050] Fluorescent probes are used for the detection of tap water samples.

[0051] The lake water samples were taken from the tap water faucet in the Chemistry and Chemical Engineering Building of Xiangtan University, and the pH of the tap water was adjusted to 7.4. Since the tap water sample does not contain HClO, HClO needed to be added externally before fluorescence detection. The results are shown in Table 2. The table shows that the probe's recovery rate of HClO in the tap water was 98.80%–102.30%.

[0052] Table 2. Determination of HClO recovery rate in tap water

[0053]

Claims

1. A water-soluble hypochlorous acid fluorescent probe, namely ABX-HClO, characterized in that, The structure is as follows:

2. The method for preparing a water-soluble hypochlorous acid fluorescent probe according to claim 1, characterized in that, The reaction steps are as follows: In a 100 mL round-bottom flask, 1 equivalent of compound ABX-OH and 2–5 equivalents of dimethylaminothiocarbamoyl chloride were added to 10–15 mL of dichloromethane, followed by 2–3 equivalents of N,N-diisopropylethylamine. The mixture was then stirred under nitrogen protection at room temperature for 10–12 h. The reaction was stopped, and the solvent was removed by vacuum distillation. The crude product was subjected to column chromatography using CH2Cl2 / CH3OH at a volume ratio of 100:1–100:5 as the eluent to obtain a blue solid product (yield of 65%), which is the fluorescent probe ABX-HClO.

3. The application of the water-soluble hypochlorous acid fluorescent probe according to claim 1, characterized in that, The fluorescent probe can be used to detect the hypochlorous acid content in actual water samples.

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