Preparation and application of fluorescent probe for detecting hypochlorous acid based on rhodamine derivative

Abstract

The invention relates to a novel fluorescent probe RH based on a rhodamine derivative, which is specially used for rapidly and sensitively detecting hypochlorous acid (HOCl) in an aqueous solution. The fluorescent probe RH disclosed by the invention can effectively eliminate interference of common active small molecules and ensure that accurate detection is realized in a complex physiological environment. Experimental results show that the probe RH can achieve remarkable fluorescence enhancement on HOCl under the fluorescence emission wavelength of 552 nm, the detection limit is as low as 18.6 nM, the response time is short, the response time is 2 minutes, and the good response characteristic is kept when the probe RH and other interfering substances coexist. The invention provides a novel and efficient detection technology for real-time monitoring of HOCl, has a wide application prospect, and can provide a more reliable tool for related research and practical application especially in the fields of biological imaging, environmental monitoring, public health and the like.

Description

Technical Field

[0001] The present invention belongs to the technical field of organic small molecule fluorescent probes, and in particular, relates to a fluorescent probe for detecting hypochlorous acid based on rhodamine derivatives, and a preparation method and application thereof. Background Art

[0002] Hypochlorous acid (HOCl) is an important oxidant widely used in the fields of physiological environment, food safety and environmental monitoring. In the physiological environment, HOCl is a key component of the body's immune system. White blood cells produce HOCl through enzymatic reactions, which effectively kill bacteria and viruses and enhance immune defense. However, excessive HOCl may cause oxidative stress and damage cell health. In food safety, HOCl is used as a disinfectant to remove microorganisms in food and extend the shelf life, but its residues may also pose a threat to human health. In environmental monitoring, HOCl is used as a disinfectant in water treatment to effectively remove pathogenic microorganisms in water, but its residues are potentially toxic to aquatic organisms and affect the ecological balance. Therefore, it is of great significance to develop efficient and sensitive detection methods to achieve real-time monitoring of HOCl.

[0003] Traditional HOCl detection methods mainly rely on chemical analysis techniques such as colorimetry, spectroscopy and electrochemistry. These methods usually require complex experimental steps, professional equipment, and have certain limitations in field applications. Therefore, it is particularly important to find a fast, sensitive and easy-to-operate detection technology. Fluorescent probes have become ideal tools for detecting HOCl due to their high sensitivity, selectivity and real-time monitoring capabilities. In recent years, detection technologies based on fluorescent probes have made significant progress in the fields of bioimaging, environmental monitoring and disease diagnosis. As a widely used fluorescent dye, rhodamine has good photostability and strong fluorescence properties, making it a preferred material for designing fluorescent probes.

[0004] Although some rhodamine-based fluorescent probes have been developed for detecting different biomolecules and small molecules, there is still a lack of dedicated probes for HOCl. The probes in the prior art still have room for improvement in terms of response speed, selectivity and sensitivity. Therefore, the development of a new type of fluorescent probe based on rhodamine derivatives that can efficiently and selectively detect HOCl will provide new solutions for related research and practical applications. By optimizing the structure and function of the probe and improving its responsiveness to HOCl, the development of HOCl monitoring technology will be greatly promoted, providing a more reliable tool for environmental monitoring and public health protection. Summary of the invention

[0005] The present invention provides a fluorescent probe for HOCl detection based on rhodamine derivatives, and a preparation method and application thereof. The fluorescent probe can be referred to as RH for short. The probe can realize rapid and sensitive detection of HOCl in aqueous solution at a fluorescence emission wavelength of 552 nm.

[0006] In a first aspect, the present invention provides a fluorescent probe based on a rhodamine derivative, the molecular formula of which is C 37 H 35 NO8S has the following structural formula: .

[0007] The synthetic route of the fluorescent probe described in the present invention is: In a second aspect, the present invention provides a method for preparing the fluorescent probe based on rhodamine derivatives, comprising: Step 1: Compound R1, triethylamine and dichloromethane are mixed, and triphosgene is added dropwise under the protection of nitrogen and ice bath. After the addition is complete, the reaction is stirred at room temperature for 6 hours. After the reaction is complete, the solvent is removed to obtain compound R2; Step 2: Compound R3 and triethylamine were dissolved in dichloromethane, and compound R2 was added after stirring for 10 minutes. After stirring at room temperature for 12 hours, the solvent was removed and the crude product was purified by column chromatography to obtain a white solid compound, which was the fluorescent probe RH.

[0008] In some embodiments, in step 1, the molar ratio of compound R1 to triethylamine is 1:1.

[0009] In some embodiments, in step 1, the molar ratio of compound R1 to triphosgene is 3:1.

[0010] In some embodiments, in step 1, the solvent is removed by rotary evaporation to obtain compound R2 which is directly used in step 2 without further treatment.

[0011] In some embodiments, in step 2, the molar ratio of compound R3 to compound R2 is 1:1.

[0012] In some embodiments, in step 2, the molar ratio of compound R3 to triethylamine is 1:1.

[0013] In some embodiments, in step 2, the solvent is removed by rotary evaporation.

[0014] In some embodiments, in step 2, the elution solvent used in column chromatography is petroleum ether:ethyl acetate (volume ratio 3:1).

[0015] In some embodiments, the method for preparing the fluorescent probe comprises: Step 1: Add compound R1 (1.0 mmol), triethylamine (1.0 mmol) and dichloromethane (10 ml) into a 50 ml round-bottom flask, and under the protection of nitrogen and ice bath, add triphosgene (0.33 mmol) dropwise. After the addition is complete, stir the mixture at room temperature for 6 hours. After the reaction is complete, remove the solvent by rotary evaporation to obtain compound R2; Step 2: Compound R3 (1 mmol) and triethylamine (1.0 mmol) were dissolved in 10 ml of dichloromethane. After stirring for 10 minutes, compound R2 (1 mmol) was added. After the mixed solution was stirred at room temperature for 12 hours, the solvent was removed by rotary evaporation. The crude product was purified by column chromatography to obtain a white solid compound, which was the fluorescent probe RH.

[0016] In a third aspect, the present invention provides the use of the fluorescent probe based on rhodamine derivatives in detecting HOCl in a physiological environment.

[0017] In some embodiments, the suitable pH range for said application is 5-10.

[0018] In some embodiments, the minimum detection limit of the rhodamine derivative-based fluorescent probe for HOCl is 18.6 nM.

[0019] In some embodiments, the response time of the rhodamine derivative-based fluorescent probe to HOCl is less than 2 minutes.

[0020] The fluorescent probe of the present invention is used for identifying HOCl for the purpose of specific non-disease diagnosis.

[0021] In a fourth aspect, the present invention provides the use of the fluorescent probe based on rhodamine derivatives in the fluorescence imaging and detection of HOCl in plant cells.

[0022] The present invention has the following beneficial effects: (1) The probe RH of the present invention has a unique structural design, a simple synthesis process, and is easy to separate and purify, which can effectively eliminate the interference of common active small molecules on the detection results. The probe shows high selectivity and sensitivity to HOCl and can achieve accurate identification in complex physiological environments.

[0023] (2) The mechanism of the fluorescent probe RH of the present invention in recognizing HOCl is as follows Figure 1 shown.

[0024] The recognition mechanism of the probe of the present invention is to utilize the strong oxidizing property of HOCl to thioacetal. After the probe reacts with HOCl, the response site leaves, releasing the fluorophore, causing a significant enhancement of fluorescence at 550 nm. This reaction site is proposed for the first time in the present invention and has not been reported in other HOCl probes. The present invention is of great significance to the development of HOCl probes.

[0025] (3) The fluorescent probe RH of the present invention has high selectivity and sensitivity for HOCl detection and can achieve specific recognition of HOCl in complex physiological environments. Experimental results show that the probe can still maintain obvious fluorescence enhancement when coexisting with other competitive analytes, and has a rapid response time to different concentrations of HOCl, with a detection limit as low as 18.6 nM. In addition, the probe RH exhibits a relatively stable fluorescence signal in the pH range of 5 to 10, which improves its reliability in practical applications. Through imaging experiments in plant cells, the fluorescent probe RH can effectively detect and image HOCl in plants, showing its wide application potential in the fields of biomedicine and plant science. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0028] Figure 1 The mechanism of the fluorescent probe RH of the present invention recognizing HOCl is shown; Figure 2 This is the RH nuclear magnetic resonance hydrogen spectrum of the fluorescent probe of the present invention (the solvent is CDCl3); Figure 3 This is the RH carbon nuclear magnetic resonance spectrum of the fluorescent probe of the present invention (the solvent is CDCl3); Figure 4 This is the fluorescence selectivity diagram of the fluorescent probe RH of the present invention in recognizing HOCl, with an excitation wavelength of 520 nm; Figure 5 This is the ultraviolet absorption selectivity diagram of the fluorescent probe RH of the present invention for recognizing HOCl, with an excitation wavelength of 520 nm; Figure 6 This is a competitive test diagram of the fluorescent probe RH of the present invention, with an excitation wavelength of 530 nm and an emission wavelength of 550 nm; Figure 7 This is a kinetic curve diagram of the fluorescent probe RH of the present invention recognizing HOCl, with an excitation wavelength of 530 nm and an emission wavelength of 550 nm; Figure 8 This is the fluorescence titration diagram of the fluorescent probe RH of the present invention recognizing HOCl, with an excitation wavelength of 530 nm; Fig. 9This is the minimum detection limit diagram of the fluorescent probe RH of the present invention for identifying HOCl, with an excitation wavelength of 530 nm and an emission wavelength of 550 nm; Fig.10 This is a test diagram of the pH applicable range of the fluorescent probe RH of the present invention for identifying HOCl, with an excitation wavelength of 530 nm and an emission wavelength of 550 nm; Fig.11 is a relationship curve between the fluorescence intensity of the fluorescent probe RH of the present invention at the maximum emission wavelength of 550 nm and the pH value (ranging from 1 to 14); Fig.12 This is a fluorescence imaging diagram of the fluorescent probe RH of the present invention identifying HOCl in onion epidermal cells.

[0029] Specific embodiment The technical solution of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0030] Example 1 The preparation method of the fluorescent probe molecule for HOCl detection in this embodiment comprises the following steps: (1) Preparation of compound R2 Compound R1 (240.0 mg, 1.0 mmol), triethylamine (101.2 mg, 1.0 mmol) and dichloromethane (10 ml) were added to a 50 ml round-bottom flask. Under the protection of nitrogen and ice bath, a dichloromethane solution of triphosgene (296.7 mg, 0.33 mmol) was added dropwise. After the addition was completed, the reaction was stirred at room temperature for 6 hours. After the reaction was completed, the excess solvent was removed by rotary evaporation to obtain compound R2, which was directly subjected to the next step of reaction.

[0031] (1) Preparation of compound RH Compound R3 (387.4 mg, 1 mmol) and triethylamine (101.2 mg, 1.0 mmol) were dissolved in 10 ml of dichloromethane. After stirring for 10 minutes, compound R2 (302.8 mg, 1 mmol) was added. After the mixed solution was stirred at room temperature for 12 hours, the excess solvent was evaporated and the crude product was purified by column chromatography to obtain a white solid compound, which is the fluorescent probe for HOCl detection.

[0032] H NMR spectroscopy: 1H NMR (400 MHz, CDCl3) 8.04 (d, J = 7.4 Hz, 1H), 7.73 – 7.59 (m, 2H), 7.46 – 7.34 (m, 2H), 7.22 (d, J = 7.5 Hz, 1H), 7.12 (d, J = 2.2 Hz, 1H), 6.92 – 6.86 (m, 2H), 6.86 – 6.76 (m, 2H), 6.61 (s, 1H), 6.48 (d, J = 2.5 Hz, 1H), 6.39 (dd, J = 8.9, 2.6 Hz, 1H), 4.48 – 4.23 (m, 3H),4.04 – 3.90(m, 1H), 3.82 (s, 4H), 3.49 – 3.31 (m, 4H), 3.28 – 3.13 (m, 1H), 3.14 – 3.02 (m, 1H), 2.74 – 2.46(m, 2H), 1.20 (t, J = 7.0 Hz, 6H). Carbon NMR spectroscopy determination: 13 C NMR (101 MHz, CDCl3) δ 169.5, 159.0, 153.0,152.7, 149.7, 136.9, 134.9, 129.6, 129.1, 128.9, 127.1, 126.6, 124.9,124.1,117.3, 116.3, 113.7, 109.7, 108.6, 104.8, 97.6, 96.4, 83.6, 70.7, 65.8, 55.3,44.5, 42.5, 34.3.

[0033] H NMR spectrum Figure 2 As shown, the carbon NMR spectrum is as follows Figure 3 shown.

[0034] Example 2 Accurately weigh the probe RH prepared in Example 1 and dissolve it in dimethyl sulfoxide (DMSO) solution to prepare a 1 mM solution for later use. Preparation of HOCl (10 mM) solution: Measure NaOCl (13.7) and dissolve it in 10 ml of deionized water, transfer it to a 10 mL volumetric flask to make up the volume, and store it at -20°C (prepare it for immediate use). Use a fluorescence spectrometer and a UV spectrophotometer to test the fluorescence and UV absorption of the PBS solution containing the probe RH (10 μM).

[0035] like Figure 4 and Figure 5As shown in the figure, in PBS buffer solution, the probe alone has no fluorescence emission at 550 nm and no UV absorption peak at 520 nm. When other analytes are added, the emission peak and absorption peak of the probe will not change. Only after the addition of HOCl (100 μM), there is a significant fluorescence enhancement at 550 nm, and the absorption peak at 520 nm increases significantly. This shows that the probe RH has a high selectivity in response to HOCl.

[0036] Example 3 The probe RH concentration in Example 1 was kept at 10 μM, and the effects of other competitive analytes on the probe's response to HOCl were investigated.

[0037] like Figure 6 As shown in the figure, after adding HOCl and other competitive analytes to the PBS solution containing the probe RH (10 μM), the fluorescence of the probe at 550 nm increased significantly, and the addition of the analyte did not induce a decrease in fluorescence. This shows that the probe can achieve specific recognition of HOCl under complex physiological conditions.

[0038] Example 4 The concentration of the probe RH in Example 1 was kept at 10 μM, and the response time of the probe to HOCl was examined.

[0039] HOCl (100 μM) was added to the PBS solution containing the probe RH (10 μM), and the fluorescence change of the probe at 550 nm was recorded. Figure 7 As shown in the figure, when HOCl was not added, the fluorescence of the probe RH at 550 nm hardly changed from 0 to 30 minutes. However, after the addition of HOCl, its fluorescence signal increased significantly within 2 minutes. This experimental result shows that the probe has a fast response time for the detection of HOCl.

[0040] Example 5 The concentration of the probe RH in Example 1 was kept at 10 μM, and the changes in the ultraviolet absorption and fluorescence emission of the probe to different concentrations of HOCl were investigated.

[0041] like Figure 8 and Fig. 9 As shown in Figure 2, the probe alone has almost no absorption peak and fluorescence emission at 520 nm and 550 nm. However, after adding different concentrations of HOCl (0-100 μM) solution, the absorption peak at 520 nm and the fluorescence emission at 550 nm are significantly enhanced. Fig.10As shown in the figure, by linearly fitting the fluorescence intensity of the probe RH at 550 nm and the added HOCl concentration, the linear equation Y=68.76X+24.58 was obtained, and a high linear correlation was shown. According to the minimum detection limit equation LOD=3σ / k, the detection limit was calculated to be 18.6 nM. This shows that the probe RH has a high sensitivity for the detection of HOCl.

[0042] Example 6 The concentration of the fluorescent probe RH in Example 1 was kept at 10 μM to investigate the pH applicable range of the probe for HOCl. Fig.11 As shown in the figure, the relationship curve between the fluorescence intensity of the probe RH at the maximum emission wavelength of 550 nm and the pH value (ranging from 1 to 14) is shown. Between the pH values ​​of 5 and 10, the fluorescence intensity of the probe RH is relatively stable; and after the addition of HOCl, the fluorescence intensity is significantly enhanced. Therefore, the pH value between 5 and 10 is determined as the working range of the probe RH to detect HOCl.

[0043] Example 7 The fluorescent probe RH in Example 1 was applied to the detection and imaging of HOCl in onion epidermal cells. Fig.12 As shown, 10 μM probe RH solution was added to the culture medium of onion epidermal cells and imaged after culturing at room temperature for 30 minutes. The results showed Fig.12 (A2) has weak fluorescence, indicating that the probe alone has almost no fluorescence emission. Subsequently, under the same conditions, 10 μM probe RH solution was added to the culture medium of onion epidermal cells, and HOCl (100 μM) was added after 30 minutes of culture. After another 60 minutes of culture, imaging was performed to obtain Fig.12 (B2), where the red fluorescence is significantly enhanced. The experimental results of onion epidermal cells show that the new probe RH can effectively realize the fluorescence imaging and detection of HOCl in plant cells.

[0044] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A fluorescent probe based on a rhodamine derivative, the molecular formula of which is C 37 H 35 NO8S has the following structural formula: 。 2. The method for preparing a fluorescent probe based on a rhodamine derivative according to claim 1, comprising: Step 1: Compound R1, triethylamine and dichloromethane are mixed, and triphosgene is added dropwise under the protection of nitrogen and ice bath. After the addition is complete, the reaction is stirred at room temperature for 6 hours. After the reaction is complete, the solvent is removed to obtain compound R2; Step 2: Compound R3 and triethylamine were dissolved in dichloromethane, and compound R2 was added after stirring for 10 minutes. After stirring at room temperature for 12 hours, the solvent was removed and the crude product was purified by column chromatography to obtain a white solid compound, which was the fluorescent probe RH.

3. The preparation method according to claim 2, wherein In step 1, the molar ratio of compound R1 to triethylamine is 1:

1.

4. The preparation method according to claim 2, wherein In step 1, the solvent is removed by rotary evaporation to give compound R2 which is used directly in step 2 without further treatment.

5. The preparation method according to claim 2, wherein: In step 2, the molar ratio of compound R3 to compound R2 is 1:

1.

6. The preparation method according to claim 2, wherein: include: Step 1: Add 1.0 mmol of compound R1, 1.0 mmol of triethylamine and 10 ml of dichloromethane into a 50 ml round-bottom flask, and under the protection of nitrogen and ice bath, dropwise add 0.33 mmol of triphosgene. After the dropwise addition is completed, stir at room temperature for 6 hours. After the reaction is completed, remove the solvent by rotary evaporation to obtain compound R2; Step 2: Dissolve 1 mmol of compound R3 and 1.0 mmol of triethylamine in 10 ml of dichloromethane, stir for 10 minutes, then add 1 mmol of compound R2. After the mixed solution is stirred at room temperature for 12 hours, remove the solvent by rotary evaporation, and purify the crude product by column chromatography to obtain a white solid compound, which is the fluorescent probe RH.

7. Use of the fluorescent probe based on rhodamine derivatives according to claim 1 in detecting HOCl in a physiological environment.

8. The use according to claim 7, wherein: A suitable pH range for such application is 5-10.

9. The use according to claim 7, wherein: The minimum detection limit of the rhodamine derivative-based fluorescent probe for HOCl is 18.6 nM.

10. Use of the fluorescent probe based on rhodamine derivatives according to claim 1 in fluorescence imaging and detection of HOCl in plant cells.

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