Preparation method and application of cellulose-based pH / hclo dual-responsive fluorescent probe

Abstract

The application discloses a cellulose-based pH / HClO dual-response fluorescent probe, and steps of a preparation method thereof are as follows: a deep red solid product compound 1 is synthesized; a light yellow-green compound 2 is synthesized; and modified cellulose is obtained by modifying cellulose. After the modified cellulose is completely dissolved in DMSO, the compound 1 and the compound 2 are added, stirring is carried out at 100 DEG C for 6 hours, freeze-drying is carried out, washing with ethanol is carried out until the product is colorless, and after drying, a red-brown fluorescent probe is obtained. The application takes natural polymer cellulose as a substrate, grafts a fluorescent probe, realizes pH and HClO dual-response, and has good application potential in multitasking processing.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of chemical technology, in particular to a preparation method of a cellulose-based pH / HClO dual-responsive fluorescent probe and application thereof. BACKGROUND

[0002] Renewable energy mainly includes solar energy, wind energy, hydro energy, biomass energy, tidal energy, sea wave energy, and ocean temperature difference energy. Cellulose is not only one of the most abundant and widely distributed natural polymers on earth, but also a green renewable biomass resource. Due to the good biodegradability and biocompatibility of cellulose, cellulose-based composite materials have attracted extensive attention. Cellulose is a polymer in which β-D-glucopyranose units are linked together by 1-4-glycosidic bonds. Various chemical modifications such as esterification, etherification, crosslinking and graft copolymerization can be carried out using the -OH groups on the cellulose molecular chain. These modifications not only improve their processing performance, but also introduce some new functional groups to obtain new properties. Modified cellulose is widely used in adsorption separation, sewage purification, biomedical engineering and other fields, far beyond the scope of cellulose utilization. The high mechanical strength, structural flexibility and good biocompatibility of cellulose make it extremely attractive in the manufacture of various fluorescent probes and their functional applications. Therefore, it is desirable to develop cellulose-based fluorescent probes.

[0003] Hypochlorous acid (HClO) is one of the most important reactive oxygen species (ROS) in the human body, 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 due to its antioxidant properties and low price, such as drinking water disinfection, household bleach and antibacterial agents. 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. Cell processes such as cell adhesion, cell growth, endocytosis and ion transport are largely dependent on intracellular pH. Abnormal intracellular pH can lead to serious dysfunction, such as cell necrosis without distinction, apoptosis, and disease-like inflammation. Tumor cells are kept in a more acidic environment than normal cells. Therefore, monitoring the dynamic changes of intracellular pH has important significance for identifying pathological conditions and studying common physiological processes.

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

[0005] Based on the technical problems existing in the background art, the present application proposes a preparation method of a cellulose-based pH / HClO dual-response fluorescence probe and its application. The probe has good biocompatibility and can realize dual-response to pH and HClO.

[0006] The preparation method of the cellulose-based pH / HClO dual-response fluorescence probe proposed by the present application comprises the following steps:

[0007] S1: 1,6-dihydroxynaphthalene and 3-hydroxy-4-nitroso-N,N-diethyl aniline are added to a round-bottom flask containing DMF. Reaction is carried out under nitrogen protection at 140 DEG C until the raw materials are completely consumed. After cooling to room temperature, washing and purification are carried out. After purification, 1-fluoro-4-nitrobenzene and potassium carbonate are added to DMF, and reaction is carried out at 40 DEG C overnight. After cooling to room temperature, washing and purification are carried out to obtain a dark red intermediate product. The purified intermediate product and iron powder are added to a round-bottom flask containing glacial acetic acid. The mixture is stirred at room temperature for 16 h under nitrogen protection. After the reaction is completed, saturated sodium bicarbonate solution is added to neutralize the glacial acetic acid, and dichloromethane is extracted. After washing, drying and purification, a dark red solid product compound 1 is obtained.

[0008] S2: tert-butyl (2-aminoethyl) carbamate is added to an ethanol suspension of 4-bromo-1,8 naphthalene anhydride, and refluxing is carried out at 80 DEG C for 4 h to obtain a white solid compound. Then the compound is reacted with N-(2-aminoethyl) morpholine and anhydrous potassium carbonate in DMF, and reaction is carried out at 50 DEG C overnight. After washing and purification, 2 ml of trifluoroacetic acid is added to 8 ml of dichloromethane-dissolved compound, and the mixture is stirred at room temperature for 4 h. After evaporation of the solvent, a light yellow-green compound 2 is obtained.

[0009] S3: Cellulose is vacuum dried at 80℃ for 24h, and then is slurried in DMAC at 160℃ under N2 protection. After adding appropriate amount of anhydrous LiCl and stirring well at 100℃, the mixture is cooled to room temperature. DMAC and triethylamine are added at 8℃, and then DMAC solution of p-toluenesulfonyl chloride is added dropwise. The reaction is carried out for 24h to obtain a red-brown product, which is precipitated in ice water, washed with deionized water and ethanol in sequence, and dried at 50℃ under vacuum to obtain modified cellulose.

[0010] S4: After the modified cellulose is completely dissolved in DMSO, compound 1 and compound 2 are added, and the mixture is stirred at 100℃ for 6h, and then is freeze-dried and washed with ethanol until the product is colorless. After drying, a red-brown fluorescent probe is obtained. The present application realizes pH and HClO dual response by grafting a fluorescent probe on a natural polymer cellulose, and has good application in multitasking.

[0011] 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.

[0012] Preferably, the molar ratio of (2-aminoethyl)carbamic acid tert-butyl ester, 4-bromo-1,8-naphthalic anhydride and N-(2-aminoethyl)morpholine in S2 is 2:1:2.5.

[0013] Preferably, the molar ratio of cellulose and p-toluenesulfonyl chloride in S3 is 1:1.5.

[0014] Preferably, the molar ratio of modified cellulose, compound 1 and compound 2 in S4 is 10:1:1.

[0015] The cellulose-based pH / HClO dual-response fluorescent probe prepared by the above method of the present application.

[0016] The application of the cellulose-based fluorescent probe in the detection of pH and HClO.

[0017] The method for detecting pH and HClO by using the cellulose-based fluorescent probe, which comprises the following steps:

[0018] S1: Preparation of a solution: a probe solution mother liquor with a concentration of 1 mg / L is prepared by dissolving in DMSO, a sodium hypochlorite stock solution with a concentration of 10 mM is prepared in deionized water, and PBS solution stock solutions with different pH values are prepared.

[0019] S2: Fluorescence spectrum test: a F-7100 fluorescence spectrophotometer (Hitachi Group) is used, 2ml of solvent and 20 μl of probe solution are added into a quartz dish, and the mixture is mixed to have a constant concentration of 10 μg / ml. Scanning is carried out with 450 nm and 550 nm as excitation wavelengths.

[0020] Advantages:

[0021] The cellulose-based pH / HClO dual-response fluorescent probe provided by the application has good biocompatibility, high sensitivity and can be applied to multitasking occasions. BRIEF DESCRIPTION OF DRAWINGS

[0022] Figure 1 A synthesis route diagram of the cellulose-based pH / HClO dual-response fluorescent probe provided by the application;

[0023] Figure 2 Infrared spectrograms of cellulose a, modified cellulose b and probe c provided by the application;

[0024] Figure 3 X-ray diffraction patterns (XRD) of cellulose a, modified cellulose b and probe c provided by the application;

[0025] Figure 4 Response conditions and linear relationships of different pH and HClO provided by the application;

[0026] Figure 5 Fluorescence spectra of the probe in the presence of different ions provided by the application. DETAILED DESCRIPTION

[0027] The application will be further described below in combination with specific embodiments.

[0028] Embodiment 1

[0029] The method steps for preparing the cellulose-based pH / HClO dual-response fluorescent probe provided by the application are as follows:

[0030] S1: 1,6-dihydroxynaphthalene and 3-hydroxy-4-nitroso-N,N-diethyl aniline are added to a round-bottom flask containing DMF. The mixture is reacted at 140°C under nitrogen protection until the raw materials are consumed completely, and then cooled to room temperature. After washing and purification, the purified product is added to DMF together with 1-fluoro-4-nitrobenzene and potassium carbonate, and the mixture is reacted at 40°C overnight. After cooling to room temperature, the mixture is washed and purified to obtain a dark red intermediate product. The purified intermediate product and iron powder are added to a round-bottom flask containing glacial acetic acid. The mixture is stirred at room temperature under nitrogen protection for 16 h. After the reaction is completed, saturated sodium bicarbonate solution is added to neutralize the glacial acetic acid, and dichloromethane is used for extraction. After washing, drying and purification, a dark red solid product compound 1 is obtained.

[0031] S2: (2-aminoethyl) tert-butyl carbamate was added to a suspension of 4-bromo-1,8 naphthalene anhydride in ethanol, mixed and refluxed at 80°C for 4h to obtain a white solid compound. Then the compound was reacted with N-(2-aminoethyl) morpholine and anhydrous potassium carbonate in DMF, and the reaction was carried out at 50°C overnight, washed and purified. 2 ml of trifluoroacetic acid was added to 8 ml of dichloromethane dissolved in the above compound, the mixture was stirred at room temperature for 4 h, and the solvent was evaporated to obtain a light yellow-green compound 2.

[0032] S3: The cellulose was vacuum dried at 80°C for 24h, and then the cellulose was slurried in DMAC at 160°C under N2 protection. After stirring at 100°C, an appropriate amount of anhydrous LiCl was added, and the mixture was 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 carried out at 24 h to obtain a red-brown product, which was precipitated in ice water, washed with deionized water and ethanol in turn, and dried at 50°C under vacuum to obtain the modified cellulose.

[0033] S4: After the modified cellulose was completely dissolved in DMSO, compound 1 and compound 2 were added, and the mixture was stirred at 100°C for 6h, and then freeze-dried. The product was washed with ethanol until it was colorless, and then dried to obtain a red-brown fluorescent probe. The present application uses natural polymer cellulose as a substrate, grafts a fluorescent probe, realizes pH and HClO dual response, and has good application in multi-task processing.

[0034] In S1, the molar amounts of 1,6-dihydroxynaphthalene, 3-hydroxy-4-nitroso-N,N-diethyl aniline and 1-fluoro-4-nitrobenzene were 2 mol: 2 mol: 1 mol.

[0035] In S2, the molar amounts of (2-aminoethyl) tert-butyl carbamate, 4-bromo-1,8 naphthalene anhydride and N-(2-aminoethyl) morpholine were 2 mol: 1 mol: 2.5 mol.

[0036] In S3, the molar ratio of cellulose to p-toluenesulfonyl chloride was 1:1.5.

[0037] In S4, the molar ratio of modified cellulose, compound 1 and compound 2 was 10:1:1.

[0038] The cellulose-based pH / HClO dual-response fluorescent probe prepared in the present embodiment was structurally characterized and its performance was studied, and the results were as follows:

[0039] Characterization of the fluorescent probe

[0040] (1) Infrared spectroscopic characterization

[0041] A Frontier FT-IR Spectromete Fourier transform infrared spectrometer (KBr pellet method, 400-4000 cm⁻¹) was used. -1 Infrared spectroscopy was performed on cellulose, modified cellulose, and the probe within the scanning range to obtain the results. Figure 2 The images shown are the infrared spectra of cellulose a, modified cellulose b, and probe c. In the FT-IR spectrum ( Figure 2 b, 2c), originally located at approximately 3300cm -1 The disappearance of the hydroxyl (OH) stretching vibration peak in the region indicates that the hydroxyl groups in cellulose have been effectively substituted. Modified cellulose ( Figure 2 b) and probe ( Figure 2 c) All are at 1360cm -1 In-plane bending vibrations of the methyl group (-CH3) were observed at 1180 cm⁻¹. -1 Vibrational peaks of sulfur-oxygen double bonds and sulfur-oxygen bonds appeared at 1560 cm⁻¹, and the peak values ​​in the probe were significantly weaker than those in the modified cellulose. The peak spectrum of the probe showed a peak at 1560 cm⁻¹. -1 The characteristic peak of NH bending vibration appears at 830 cm⁻¹. -1 and 680cm -1 The peak appearing at this point indicates the out-of-plane bending vibration of the CH group of the benzene ring, which is an important marker for identifying aromatic compounds, indicating the successful preparation of the cellulose-based fluorescent probe.

[0042] (2) Infrared spectroscopy characterization

[0043] X-ray diffraction 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 patterns reveal 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° belong to planes with Miller indices of (1-10), (110), and (200), respectively. Figure 3 a). As can be seen from the XRD pattern, after cellulose modification and grafting with fluorescent small molecules, the cellulose hydroxyl groups were replaced, the hydrogen bond network between cellulose molecules was destroyed, the cellulose diffraction peaks disappeared, and cellulose changed from a crystalline state to an amorphous state. Figure 3 b, 3c).

[0044] Fluorescence sensing of pH / HClO by the probe

[0045] (1) Fluorescence spectroscopy test

[0046] S1: Preparation of solution: 1 mg / L of probe solution mother liquor was prepared in DMSO, 10 mM of sodium hypochlorite stock solution was prepared in deionized water, and PBS solution stock solution with different pH was prepared.

[0047] S2: Test of fluorescence spectrum: F-7100 fluorescence spectrophotometer (Hitachi Group) was used, 2 ml of solvent and 20 μl of probe solution were added in a quartz dish, mixed to make the concentration constant at 10 μg / ml, and scanning was performed with 450 nm and 550 nm as excitation wavelength.

[0048] (2) Fluorescence sensing of probe to pH / HClO

[0049] Figure 4 As shown in the figure, with the increase of pH in the range of 3 to 9, the fluorescence intensity of the probe gradually decreased (a) with the increase of HClO concentration, the fluorescence intensity of the probe gradually increased (b) and the fluorescence intensity of the probe had good linear relationship with pH and HClO, and the linear correlation coefficients were 0.99738, 0.99864 (c) respectively. Figure 4 Figure 4 Figure 4 b,4d).

[0050] The probe had weak or almost no response to 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)) (d). Figure 5 )​​

Claims

1. A method for preparing a cellulose-based pH / HClO dual-responsive fluorescent probe, characterized in that, Comprising the following steps: S1: 1,6-dihydroxynaphthalene and 3-hydroxy-4-nitroso-N, N-diethyl aniline are added to a round-bottom flask containing DMF, and reacted at 140°C under nitrogen protection until the raw materials are completely consumed, cooled to room temperature, and washed and purified; after purification, 1-fluoro-4-nitrobenzene and potassium carbonate are added to DMF, and reacted at 40°C overnight; cooled to room temperature, washed and purified to obtain a dark red intermediate product; the purified intermediate product and iron powder are added to a round-bottom flask containing glacial acetic acid, and the mixture is stirred at room temperature under nitrogen protection for 16 h; after the reaction is completed, saturated sodium bicarbonate solution is added to neutralize the glacial acetic acid, dichloromethane is extracted, washed, dried and purified to obtain a dark red solid product compound 1; S2: tert-butyl (2-aminoethyl) carbamate is added to a suspension of 4-bromo-1, 8 naphthalene anhydride in ethanol, and refluxed at 80°C for 4 h to obtain a white solid compound; then the compound is reacted with N- (2-aminoethyl) morpholine and anhydrous potassium carbonate in DMF, and reacted at 50°C overnight, washed and purified; 2 ml of trifluoroacetic acid is added to 8 ml of the above compound dissolved in dichloromethane, and the mixture is stirred at room temperature for 4 h, and the solvent is evaporated to obtain a light yellow-green compound 2; S3: cellulose is vacuum dried at 80°C for 24 h, and then slurried in DMAC at 160°C under N2 protection, and after stirring at 100°C, an appropriate amount of anhydrous LiCl is added and stirred well, and then cooled to room temperature; DMAC and triethylamine are added at 8°C, and then DMAC dissolved with p-toluenesulfonyl chloride is added dropwise, and reacted for 24 h to obtain a red-brown product, which is precipitated in ice water, washed with deionized water and ethanol in sequence, and vacuum dried at 50°C to obtain modified cellulose; S4: after the modified cellulose is completely dissolved in DMSO, compound 1 and compound 2 are added, and stirred at 100°C for 6 h, and then freeze-dried, washed with ethanol until the product is colorless, and dried to obtain a red-brown fluorescent probe.

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

1.

3. The preparation method according to claim 1, characterized in that, The molar ratio of tert-butyl (2-aminoethyl) carbamate, 4-bromo-1, 8 naphthalene anhydride and N- (2-aminoethyl) morpholine in S2 is 2:1:2.

5.

4. The method of claim 1, wherein, The molar ratio of cellulose and p-toluenesulfonyl chloride in S3 is 1:1.

5.

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

1.

6. The application of a cellulose-based pH / HClO dual-responsive fluorescent probe prepared by the preparation method of any one of claims 1-5 in pH and HClO detection.

7. Use according to claim 6, characterized in that, The method steps for detection are as follows: S1: solution preparation: prepare a 1 mM probe solution stock solution in DMSO, prepare a 10 mM sodium hypochlorite stock solution in deionized water, and prepare a PBS solution stock solution with different pH values; S2: Test of fluorescence spectrum: F-7100 fluorescence spectrophotometer was used, 2 ml solvent and 20 μl probe solution were added into a quartz dish, mixed to make the concentration constant at 10 μg / ml, and scanning was performed with 450 nm and 550 nm as excitation wavelength.

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