Preparation method and application of cellulose-based pH / HClO dual-response fluorescent probe

Abstract

The invention discloses a cellulose-based pH / HClO dual-response fluorescent probe. A preparation method of the cellulose-based pH / HClO dual-response fluorescent probe comprises the following steps: synthesizing a deep red solid product compound 1; synthesizing a light yellow green compound 2; and modifying cellulose to obtain modified cellulose. And after the modified cellulose is completely dissolved in DMSO, adding a compound 1 and a compound 2, stirring at 100 DEG C for 6 hours, freeze-drying, washing with ethanol until the product is colorless, and drying to obtain the reddish brown fluorescent probe. Natural polymer cellulose is used as a substrate, a fluorescent probe is grafted, pH and HClO dual response is achieved, and the method has good application potential in the multi-task processing process.

Description

Technical Field

[0001] The invention relates to the technical field of chemical engineering, and in particular to a preparation method and application of a cellulose-based pH / HClO dual-response fluorescent probe. Background Art

[0002] Renewable energy sources mainly include solar energy, wind power, hydropower, biomass energy, tidal energy, wave energy, ocean temperature difference energy, etc. Cellulose is not only one of the most abundant and widely distributed natural polymers on the earth, but also a green and renewable biomass resource. Due to its good biodegradability and biocompatibility, cellulose-based composite materials have received widespread attention. Cellulose is a polymer composed of β-D-pyranose units connected by 1-4-glycosidic bonds. Various chemical modifications such as esterification, etherification, cross-linking and graft copolymerization can be carried out using the -OH groups on the cellulose molecular chain. These modifications can not only improve their processing properties, but also introduce some new functional groups to obtain new properties. Modified cellulose is widely used in adsorption separation, sewage purification, biomedicine and other fields, far beyond the scope of cellulose utilization. The high mechanical strength, structural flexibility and good biocompatibility of cellulose make it very attractive in the manufacture of various fluorescent probes and their functional applications. Therefore, the development of cellulose-based fluorescent probes is very desirable.

[0003] Hypochlorous acid (HClO) is one of the most important reactive oxygen species (ROS) in the human body. It is mainly produced by the reaction of hydrogen peroxide and chloride ions catalyzed by myeloperoxidase (MPO) and plays an important role in maintaining the normal function of the human immune system. Hypochlorous acid is widely used in industrial production and daily life, such as drinking water disinfection, household bleaching agent and antibacterial agent due to its antioxidant properties and low price. Excessive HClO is harmful to organisms and the environment, and is also associated with cell or tissue damage in neurodegeneration, periodontal and cardiovascular diseases. Hypochlorous acid is closely related to human health and disease, so it is crucial to detect hypochlorous acid in the environment and the human body. Cellular processes such as cell adhesion, cell growth, endocytosis and ion transport are largely dependent on intracellular pH. Abnormal intracellular pH can lead to severe functional disorders, such as indiscriminate cell necrosis, apoptosis, and disease-like inflammation. Tumor cells are kept in a more acidic environment than normal cells. Therefore, monitoring the dynamic changes of cellular pH is of great significance for identifying pathological states and studying common physiological processes.

[0004] Traditional detection methods for HClO and pH include electrochemical analysis, high performance liquid chromatography, fluorescence spectroscopy, etc. Among them, fluorescent probes have attracted the attention of many researchers due to their high sensitivity, high selectivity, fast response time, and low toxicity. Fluorescent probes composed of different organic molecules, different kinds of nanoparticles, or various fluorescent proteins have been the driving force for labeling, sensing, and detecting biologically or environmentally important species. Although great progress has been made in the development of HClO or pH probes in the past decade, the main problem with these probes is that most of them are only used to detect a single type of ion. Therefore, multifunctional probes that can detect both ions will be of great significance in some occasions that require multitasking. Summary of the invention

[0005] Based on the technical problems existing in the background technology, the present invention proposes a preparation method and application of a cellulose-based pH / HClO dual-response fluorescent probe, which has good biocompatibility and can achieve dual response to pH and HClO.

[0006] The method for preparing the cellulose-based pH / HClO dual-response fluorescent probe proposed in the present invention is as follows: S1: Add 1,6-dihydroxynaphthalene and 3-hydroxy-4-nitroso-N,N-diethylaniline to a round-bottom flask containing DMF. React at 140°C under nitrogen protection until the raw materials are completely consumed, cool to room temperature, and wash and purify. After purification, add it to DMF with 1-fluoro-4-nitrobenzene and potassium carbonate and react overnight at 40°C. Cool to room temperature, wash and purify to obtain a dark red intermediate. Add the purified intermediate and iron powder to a round-bottom flask containing glacial acetic acid. Stir the mixture at room temperature for 16 h under nitrogen protection. After the reaction is completed, add saturated sodium bicarbonate solution to the reaction solution to neutralize the glacial acetic acid, and extract with dichloromethane. Wash, dry and purify to obtain a dark red solid product compound 1.

[0007] S2: Add tert-butyl (2-aminoethyl)carbamate to the ethanol suspension of 4-bromo-1,8-naphthoic anhydride, mix and reflux at 80°C for 4 hours to obtain a white solid compound. Then react the compound with N-(2-aminoethyl)morpholine and anhydrous potassium carbonate in DMF, react at 50°C overnight, and wash and purify. Add 2 ml of trifluoroacetic acid to the above compound dissolved in 8 ml of dichloromethane, stir the mixture at room temperature for 4 hours, evaporate the solvent, and obtain a pale yellow-green compound 2.

[0008] S3: The cellulose was dried under vacuum at 80°C for 24 h. Under N2 protection, the cellulose was slurried in DMAC at 160°C. An appropriate amount of anhydrous LiCl was added at 100°C and stirred thoroughly, and then cooled to room temperature. DMAC and triethylamine were added at 8°C, and DMAC dissolved with p-toluenesulfonyl chloride was added dropwise. The reaction was continued for 24 h to obtain a reddish brown product, which was poured into ice water for precipitation, washed with deionized water and ethanol in turn, and dried under vacuum at 50°C to obtain modified cellulose.

[0009] S4: After the modified cellulose is completely dissolved in DMSO, compound 1 and compound 2 are added, stirred at 100°C for 6 hours, freeze-dried, washed with ethanol until the product is colorless, and dried to obtain a reddish-brown fluorescent probe. The present invention uses natural polymer cellulose as a substrate, grafts fluorescent probes, and achieves dual response of pH and HClO, which has good application in multi-task processing.

[0010] Preferably, the molar ratio of 1,6-dihydroxynaphthalene, 3-hydroxy-4-nitroso-N,N-diethylaniline and 1-fluoro-4-nitrobenzene in S1 is 2:2:1.

[0011] Preferably, the amount of (2-aminoethyl)carbamic acid tert-butyl alcohol, 4-bromo-1,8-naphthoic anhydride and N-(2-aminoethyl)morpholine in S2 is 2:1:2.5.

[0012] Preferably, the molar ratio of cellulose to p-toluenesulfonyl chloride in S3 is 1:1.5.

[0013] Preferably, the molar ratio of the modified cellulose, compound 1 and compound 2 in S4 is 10:1:1.

[0014] The invention provides a cellulose-based pH / HClO dual-response fluorescent probe prepared by the method.

[0015] The invention discloses an application of a cellulose-based fluorescent probe in pH and HClO detection.

[0016] The method for detecting pH and HClO using a cellulose-based fluorescent probe is as follows: S1: Solution preparation: Prepare a 1 mg / L probe solution stock solution by dissolving in DMSO, prepare a 10 mM sodium hypochlorite stock solution in deionized water, and prepare PBS stock solutions of different pH values.

[0017] S2: Fluorescence spectrum test: Using F-7100 fluorescence spectrophotometer (Hitachi Group), add 2 ml of solvent and 20 μl of probe solution into a quartz dish, mix them to make the concentration constant at 10 μg / ml, and scan at 450 nm and 550 nm as excitation wavelengths.

[0018] Beneficial effects: The present invention uses cellulose as a carrier, and obtains a dual-response fluorescent probe that has good biocompatibility and responds to pH and HClO simultaneously through modification and grafting of fluorescent groups. The probe has high sensitivity and can be applied to multi-task processing occasions. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 The synthetic route of the cellulose-based pH / HClO dual-response fluorescent probe proposed in the present invention; Figure 2 The infrared spectra of cellulose a, modified cellulose b, and probe c proposed in the present invention; Figure 3 X-ray diffraction patterns (XRD) of cellulose a, modified cellulose b, and probe c proposed in the present invention; Figure 4 The response and linear relationship of different pH and HClO proposed by the present invention; Figure 5 This is the fluorescence spectrum of the probe in the presence of different ions proposed in the present invention. DETAILED DESCRIPTION

[0020] The present invention will be further explained below in conjunction with specific embodiments.

[0021] Example 1 The method for preparing the cellulose-based pH / HClO dual-response fluorescent probe proposed in the present invention is as follows: S1: Add 1,6-dihydroxynaphthalene and 3-hydroxy-4-nitroso-N,N-diethylaniline to a round-bottom flask containing DMF. React at 140°C under nitrogen protection until the raw materials are completely consumed, cool to room temperature, and wash and purify. After purification, add it to DMF with 1-fluoro-4-nitrobenzene and potassium carbonate and react overnight at 40°C. Cool to room temperature, wash and purify to obtain a dark red intermediate. Add the purified intermediate and iron powder to a round-bottom flask containing glacial acetic acid. Stir the mixture at room temperature for 16 h under nitrogen protection. After the reaction is completed, add saturated sodium bicarbonate solution to the reaction solution to neutralize the glacial acetic acid, and extract with dichloromethane. Wash, dry and purify to obtain a dark red solid product compound 1.

[0022] S2: Add tert-butyl (2-aminoethyl)carbamate to the ethanol suspension of 4-bromo-1,8-naphthoic anhydride, mix and reflux at 80°C for 4 hours to obtain a white solid compound. Then react the compound with N-(2-aminoethyl)morpholine and anhydrous potassium carbonate in DMF, react at 50°C overnight, and wash and purify. Add 2 ml of trifluoroacetic acid to the above compound dissolved in 8 ml of dichloromethane, stir the mixture at room temperature for 4 hours, evaporate the solvent, and obtain a pale yellow-green compound 2.

[0023] S3: The cellulose was dried under vacuum at 80°C for 24 h. Under N2 protection, the cellulose was slurried in DMAC at 160°C. An appropriate amount of anhydrous LiCl was added at 100°C and stirred thoroughly, and then cooled to room temperature. DMAC and triethylamine were added at 8°C, and DMAC dissolved with p-toluenesulfonyl chloride was added dropwise. The reaction was continued for 24 h to obtain a reddish brown product, which was poured into ice water for precipitation, washed with deionized water and ethanol in turn, and dried under vacuum at 50°C to obtain modified cellulose.

[0024] S4: After the modified cellulose is completely dissolved in DMSO, compound 1 and compound 2 are added, stirred at 100°C for 6 hours, freeze-dried, washed with ethanol until the product is colorless, and dried to obtain a reddish-brown fluorescent probe. The present invention uses natural polymer cellulose as a substrate, grafts fluorescent probes, and achieves dual response of pH and HClO, which has good application in multi-task processing.

[0025] The amounts of 1,6-dihydroxynaphthalene, 3-hydroxy-4-nitroso-N,N-diethylaniline and 1-fluoro-4-nitrobenzene in S1 are 2 mol: 2 mol: 1 mol, respectively.

[0026] The amounts of (2-aminoethyl)carbamic acid tert-butyl alcohol, 4-bromo-1,8-naphthoic anhydride and N-(2-aminoethyl)morpholine in S2 are 2 mol: 1 mol: 2.5 mol.

[0027] The molar ratio of cellulose to p-toluenesulfonyl chloride in S3 is 1:1.5.

[0028] The molar ratio of modified cellulose, compound 1 and compound 2 in S4 is 10:1:1.

[0029] The structure characterization and performance study of the cellulose-based pH / HClO dual-response fluorescent probe prepared in this embodiment are as follows: Characterization of fluorescent probes (1) Infrared spectroscopy characterization The Fourier transform infrared spectrometer (KBr pellet method at 400-4000 cm) was used. -1 The infrared spectra of cellulose, modified cellulose and probe were tested by scanning within the range of Figure 2 The infrared spectra of cellulose a, modified cellulose b, and probe c are shown. In the FT-IR spectrum ( Figure 2 b, 2c), originally located at about 3300 cm -1 The hydroxyl OH stretching vibration peak in the region disappears, indicating that the hydroxyl groups in cellulose are effectively replaced. Figure 2 b) and probe ( Figure 2c) averaged at 1360cm -1 The CH in-plane bending vibration of the methyl group (-CH3) appears at 1180 cm -1 The vibration peaks of sulfur-oxygen double bond and sulfur-oxygen bond appeared at 1560 cm -1 The characteristic peak of NH bending vibration appears at 830cm -1 and 680cm -1 The peak appearing at indicates the CH out-of-plane bending vibration of the benzene ring, which is an important marker for identifying aromatic compounds, indicating the successful preparation of the cellulose-based fluorescent probe.

[0030] (2) Infrared spectroscopy characterization The X-ray diffractometer was used to test cellulose, modified cellulose and probe in the range of 5-80. Figure 3 The XRD patterns of cellulose a, modified cellulose b, and probe c are shown. The XRD pattern shows the unique diffraction region of cellulose I: the three characteristic peaks of the cellulose I crystal structure at 2θ=14.9°, 16.4°, and 22.5° are attributed to the planes with Miller indices of (1-10), (110), and (200), respectively ( Figure 3 a). From the XRD graph, it can be seen that after cellulose modification and grafting of fluorescent small molecules, the cellulose hydroxyl groups are replaced, the hydrogen bond network between cellulose molecules is destroyed, the cellulose diffraction peak disappears, and the cellulose changes from crystalline to amorphous ( Figure 3 b, 3c).

[0031] Fluorescence sensing of pH / HClO by probe (1) Fluorescence spectrum test S1: Solution preparation: Prepare a 1 mg / L probe solution stock solution by dissolving in DMSO, prepare a 10 mM sodium hypochlorite stock solution in deionized water, and prepare PBS stock solutions of different pH values.

[0032] S2: Fluorescence spectrum test: Using F-7100 fluorescence spectrophotometer (Hitachi Group), add 2 ml of solvent and 20 μl of probe solution into a quartz dish, mix them to make the concentration constant at 10 μg / ml, and scan at 450 nm and 550 nm as excitation wavelengths.

[0033] (2) Fluorescence sensing of pH / HClO by probe Figure 4 As shown in Figure 2, as the pH increases from 3 to 9, the fluorescence intensity of the probe gradually decreases ( Figure 4 a), with the increase of HClO concentration, the fluorescence intensity of the probe gradually increased ( Figure 4c). There is a good linear relationship between the fluorescence intensity of the probe and pH and HClO, with linear correlation coefficients of 0.99738 and 0.99864, respectively ( Figure 4 b,4d).

[0034] The probe reacts with other ions and amino acids (Fe 3+ , Zn 2+ , Ag + , Al 3+ , Cu 2+ Mg 2+ , HSO3 - , NO3 - , S 2- ,S2O3 2− , cysteine ​​(Cys), leucine (Leu), threonine (Thr), tryptophan (Trp)) have weak or almost no response ( Figure 5 ).

Claims

1. A cellulose-based pH / HClO dual-response fluorescent probe, characterized in that: Its molecular structure is as follows: 。 2. A method for preparing the cellulose-based pH / HClO dual-response fluorescent probe according to claim 1, characterized in that: The following steps are involved: S1: 1,6-dihydroxynaphthalene and 3-hydroxy-4-nitroso-N,N-diethylaniline were added to a round-bottom flask containing DMF, reacted at 140°C under nitrogen protection until the raw materials were completely consumed, cooled to room temperature, washed and purified; after purification, it was added to DMF with 1-fluoro-4-nitrobenzene and potassium carbonate, reacted at 40°C overnight; cooled to room temperature, washed and purified to obtain a dark red intermediate; the purified intermediate and iron powder were added to a round-bottom flask containing glacial acetic acid, and the mixture was stirred at room temperature for 16 h under nitrogen protection. After the reaction, saturated sodium bicarbonate solution was added to the reaction solution to neutralize the glacial acetic acid, extracted with dichloromethane, washed, dried and purified to obtain a dark red solid product compound 1. S2: Add tert-butyl (2-aminoethyl)carbamate to an ethanol suspension of 4-bromo-1,8-naphthoic anhydride, mix and reflux at 80°C for 4 hours to obtain a white solid compound; then react the compound with N-(2-aminoethyl)morpholine and anhydrous potassium carbonate in DMF, react at 50°C overnight, and wash and purify; add 2 ml of trifluoroacetic acid to the above compound dissolved in 8 ml of dichloromethane, stir the mixture at room temperature for 4 hours, evaporate the solvent, and obtain a pale yellow-green compound 2; S3: The cellulose was vacuum dried at 80°C for 24 h. Under N2 protection, the cellulose was slurried in DMAC at 160°C. An appropriate amount of anhydrous LiCl was added at 100°C and stirred thoroughly, and then cooled to room temperature. DMAC and triethylamine were added at 8°C, and DMAC dissolved with p-toluenesulfonyl chloride was added dropwise. The reaction was continued for 24 h to obtain a reddish brown product, which was poured into ice water for precipitation, washed with deionized water and ethanol in turn, and vacuum dried at 50°C to obtain modified cellulose. S4: After the modified cellulose was completely dissolved in DMSO, compound 1 and compound 2 were added, stirred at 100°C for 6 h, freeze-dried, washed with ethanol until the product was colorless, and dried to obtain a reddish-brown fluorescent probe.

3. The preparation method according to claim 2, characterized in that: The molar ratio of 1,6-dihydroxynaphthalene, 3-hydroxy-4-nitroso-N,N-diethylaniline and 1-fluoro-4-nitrobenzene in S1 is 2:2:

1.

4. The preparation method according to claim 2, characterized in that: The amounts of (2-aminoethyl)carbamic acid tert-butyl alcohol, 4-bromo-1,8-naphthoic anhydride and N-(2-aminoethyl)morpholine in S2 are 2:1:2.

5.

5. The preparation method according to claim 2, characterized in that: The molar ratio of cellulose to p-toluenesulfonyl chloride in S3 is 1:1.

5.

6. The preparation method according to claim 2, characterized in that: The molar ratio of modified cellulose, compound 1 and compound 2 in S4 is 10:1:

1.

7. Use of a cellulose-based pH / HClO dual-response fluorescent probe prepared by the preparation method according to claims 2-6 in pH and HClO detection.

8. The use according to claim 7, characterized in that: The detection method steps are as follows: S1: Solution preparation: Prepare 1 mM probe solution stock solution by dissolving in DMSO, prepare 10 mM sodium hypochlorite stock solution in deionized water, and prepare PBS stock solutions of different pH values; S2: Fluorescence spectrum test: Using F-7100 fluorescence spectrophotometer, add 2 ml of solvent and 20 μl of probe solution into a quartz dish, mix them to make the concentration constant at 10 μg / ml, and scan at 450 nm and 550 nm as excitation wavelengths.

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