Rhodamine-coumarin-based fluorescent probes, fluorescent sensitive membranes, their preparation methods and applications

Abstract

This application relates to a rhodamine-coumarin-based fluorescent probe, a fluorescently sensitive membrane, its preparation method, and its applications. The rhodamine-coumarin-based fluorescent probe provided in this application comprises the compound shown in Formula I. The fluorescently sensitive membrane provided in this application comprises a chitosan-based membrane and the rhodamine-coumarin-based fluorescent probe. The fluorescently sensitive membrane of this application can be used for ultrasensitive detection of metal ions, especially mercury ions, in biological tissues and environmental wastewater.

Description

Technical Field

[0001] This application belongs to the field of thin-film sensitive materials and the technology of heavy metal detection and removal, and particularly relates to a rhodamine-coumarin-based fluorescent probe and a fluorescent sensitive membrane for detecting and removing mercury ions in water. This application also relates to a method for preparing the fluorescent probe and the fluorescent sensitive membrane. Background Technology

[0002] Mercury (Hg) and its compounds are global environmental pollutants, among which Hg... 2+ (Divalent mercury ions) are of particular concern due to their high toxicity and bioaccumulation. Hg 2+ Mercury can accumulate through the food chain, leading to various health problems, including kidney damage, nervous system dysfunction, and even death, posing a serious threat to human health. Furthermore, mercury pollution can disrupt the balance of ecosystems, harming aquatic life and wildlife. Therefore, detecting Hg in the aquatic environment is crucial. 2+ Concentration is crucial for environmental protection and public health.

[0003] Hg in water sample 2+ There are many traditional quantitative methods for Hg analysis, mainly including atomic absorption spectrometry, cold vapor atomic fluorescence spectrometry, gas chromatography, high-performance liquid chromatography, and electrochemistry. However, due to the complexity of sample pretreatment and the high cost of instruments, traditional methods are difficult to use for real-time, in-situ detection of Hg. 2+ Recently, fluorescence assays have attracted widespread attention due to their advantages such as convenient detection, short response time, and high sensitivity. However, some small organic molecule fluorescent probes have very low detection limits and cannot detect Hg in pure water. 2+ It will also pollute the environment.

[0004] Fluorescent membranes, containing specific molecular recognition units, can form stable complexes with mercury ions through covalent bonds or coordination interactions. This specific interaction ensures high selectivity for detection. Fluorescent membranes are generally environmentally friendly, introducing no additional harmful substances. More importantly, their preparation and use are usually simple, allowing for convenient application in natural environments and biological systems, facilitating rapid on-site detection and monitoring. Fluorescent membranes can also be integrated into different detection platforms, such as membranes, films, or fibers, providing diverse detection methods. Their effective operation in complex environmental samples offers new strategies for practical environmental monitoring and biological sample analysis. In summary, fluorescent membranes offer a series of advantages over traditional organic probes in detection, including higher sensitivity and selectivity, rapid response, ease of application, cost-effectiveness, and environmental friendliness. These advantages make fluorescent membranes a powerful tool in environmental monitoring and biological detection. Therefore, providing high selectivity and high sensitivity for Hg... 2+ Fluorescent sensitive membranes are of great significance. Summary of the Invention

[0005] To address the problems in the prior art, this application provides a rhodamine-coumarin-based fluorescent probe and a fluorescent sensitive membrane, along with their preparation method and applications. The fluorescent sensitive membrane of this application can be used for ultrasensitive detection of metal ions, especially mercury ions, in biological tissues and environmental wastewater.

[0006] In a first aspect, this application provides a rhodamine-coumarin-based fluorescent probe comprising the compound shown in Formula I:

[0007]

[0008] In Formula I, R1 and R2 may be the same or different, and each is independently selected from C1-C6 alkyl groups; R 13 and R 14 Whether the alkyl groups are the same or different, each is independently selected from C1-C6 alkyl groups;

[0009] R3-R 12 R 15 -R 23 Each is an independent hydrogen, halogen, or C1-C6 alkyl group.

[0010] The rhodamine-coumarin-based fluorescent probe of this application uses the rhodamine-coumarin fluorophore as the fluorescent backbone and introduces thiourea as Hg. 2+ The recognition group, using Hg 2+ It can promote the ring-opening of ronolamide, resulting in a change in fluorescence from blue-green to orange-yellow, enabling specific fluorescence detection and strong anti-interference ability.

[0011] In some embodiments, R1 and R2 may be the same or different, each independently selected from C1-C4 alkyl groups, such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, or tert-butyl.

[0012] In some implementations, R 13 and R 14 They may be the same or different, each independently selected from C1-C4 alkyl groups, such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, or tert-butyl.

[0013] In some implementations, R3-R 12 R 15 -R 23 Each is independently selected from hydrogen, fluorine, chlorine, bromine or C1-C4 alkyl groups, such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl or tert-butyl.

[0014] In some embodiments, the rhodamine-coumarin-based fluorescent probe is selected from the following compounds:

[0015]

[0016] Secondly, this application provides a method for preparing the rhodamine-coumarin-based fluorescent probe described in the first aspect, which includes one or more of the following steps:

[0017] Step 1: React compound a with compound b to produce compound 1.

[0018]

[0019] Step 2: React compound 1 with hydrazine hydrate to generate compound 2.

[0020]

[0021] Step 3: React compound 2 with compound c to generate compound 3.

[0022] Step 4: React compound 3 with compound d to generate compound 4.

[0023]

[0024] Among them, the definitions of each element in equations 1-3 and 'ad' are the same as those in equation I.

[0025] In some embodiments, in formula a, R1 and R2 may be the same or different, each independently selected from C1-C4 alkyl groups, such as methyl, ethyl, n-propyl, isopropyl, or n-butyl.

[0026] In some embodiments, in formula a, R3, R4 and R5 are each independently selected from hydrogen, fluorine, chlorine, bromine or C1-C4 alkyl groups, such as methyl, ethyl, n-propyl, isopropyl or n-butyl.

[0027] In some embodiments, in formula a, R6, R7, R8 and R9 are each independently selected from hydrogen, fluorine, chlorine, bromine or C1-C4 alkyl groups, such as methyl, ethyl, n-propyl, isopropyl or n-butyl.

[0028] In some embodiments, the compound of formula a is 2-(4-(diethylamino)-2-hydroxybenzoylbenzoic acid or 2-(4-(dimethylamino)-2-hydroxybenzoylbenzoic acid.

[0029] In some implementations, in formula b, R 10 R 11 and R 12 Each compound is independently selected from hydrogen, fluorine, chlorine, bromine, or C1-C4 alkyl groups, such as methyl, ethyl, n-propyl, isopropyl, or n-butyl. In some embodiments, compound b is 3-(piperazin-1-alkyl)phenol.

[0030] In some embodiments, the compound of formula 1 is

[0031] In some embodiments, the compound of formula 2 is

[0032] In some implementations, in equation c, R 13 and R 14 They may be the same or different, each independently selected from C1-C4 alkyl groups, such as methyl, ethyl, n-propyl, isopropyl, or n-butyl.

[0033] In some implementations, in equation c, R 15 R 16 R 17 and R 18 Each is independently selected from hydrogen, fluorine, chlorine, bromine or C1-C4 alkyl groups, such as methyl, ethyl, n-propyl, isopropyl or n-butyl.

[0034] In some embodiments, the compound of formula c is 7-(diethylamino)coumarin-3-carboxylic acid or 7-(dimethylamino)coumarin-3-carboxylic acid.

[0035] In some embodiments, the compound of formula 3 is

[0036] In some implementations, R in equation d 19 R 20 R 21 R 22 R 23 Each is independently selected from hydrogen, fluorine, chlorine, bromine or C1-C4 alkyl groups, such as methyl, ethyl, n-propyl, isopropyl or n-butyl.

[0037] In some embodiments, the compound of formula d is phenyl isothiocyanate.

[0038] In some embodiments, the compound of formula 4 is

[0039]

[0040]

[0041] In some embodiments, in step 1, the molar ratio of compound a to compound b is 1:1 to 1:3, for example, 1:1.5, 1:2 or 1:2.5.

[0042] In some embodiments, the reaction (condensation reaction) temperature in step 1 is 80°C-120°C, for example, 85°C, 90°C, 95°C, 100°C, 105°C, 110°C, or 115°C. In some embodiments, the reaction temperature is 90°C-100°C.

[0043] In some embodiments, the reaction time in step 1 is 5h-50h, for example 10h, 12h, 18h, 24h, 30h, 36h, 42h, or 48h. In some embodiments, the reaction time is 10h-24h.

[0044] In some embodiments, in step 1, the reaction is carried out in a solvent preferably trifluoroacetic acid.

[0045] In some embodiments, in step 2, the molar ratio of the compound of formula 1 to hydrazine hydrate is 1:1.5-1:10, for example, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8 or 1:9.

[0046] In some embodiments, the reaction (methoxy substitution reaction) temperature in step 2 is 70°C-100°C, for example, 75°C, 80°C, 85°C, 90°C, or 95°C. In some embodiments, the reaction temperature is 75°C-90°C.

[0047] In some implementations, the reaction time in step 2 is 5h-24h, for example 6h, 10h, 12h or 18h.

[0048] In some embodiments, in step 3, the molar ratio of compound 2 to compound c is 1:1 to 3:1, for example, 1.5:1, 2:1 or 2.5:1.

[0049] In some embodiments, the reaction (amide reaction) temperature in step 3 is 0°C-30°C, for example, room temperature. In some embodiments, the reaction time in step 3 is 0.5h-24h, for example, 6h, 10h, 12h or 18h.

[0050] In some embodiments, in step 3, the carboxyl group of compound c is first activated, and the activated compound c is mixed with triethylamine for a reaction.

[0051] In some embodiments, carboxyl activation includes dissolving the compound of formula c in an organic solvent such as N,N-dimethylformamide and activating it under EDC (1-ethyl-(3-dimethylaminopropyl)carbodiimide) and NHS (N-hydroxysuccinimide).

[0052] In some embodiments, in step 4, the molar ratio of compound 3 to compound d is 1:1.5 to 1:4, for example 1:2, 1:2.5, 1:3 or 1:3.5.

[0053] In some embodiments, the reaction temperature in step 4 is 5°C-35°C, for example, 10°C, 15°C, 20°C, 25°C, or 25°C. In some embodiments, the reaction temperature is 20°C-30°C. In some embodiments, the reaction temperature is room temperature.

[0054] In some embodiments, the reaction time in step 4 is 5-50 hours, for example, 10 hours, 12 hours, 18 hours, 24 hours, 30 hours, 36 hours, 42 hours, or 48 hours. In some embodiments, the reaction time is 24-48 hours.

[0055] In some embodiments, step 4 is carried out in the presence of a catalyst, such as triethylamine. In some embodiments, step 4 is carried out under an inert atmosphere, such as nitrogen. In some embodiments, step 4 is carried out in an organic solvent, such as DMF.

[0056] In some embodiments, the preparation method of the rhodamine-coumarin-based fluorescent probe includes the following specific steps:

[0057] 1) Synthesis of compound 1: 3-(piperazin-1-alkyl)phenol and 2-(4-(diethylamino)-2-hydroxybenzoic acid were amidated in trifluoroacetic acid. The solvent was removed under reduced pressure to give a red residue, which was purified by silica gel column chromatography (elution: 10% EtOH) to give compound 1.

[0058] 2) Synthesis of compound 2: Compound 1 was added to ethanol and heated to reflux, and excess hydrazine hydrate was added. After cooling to room temperature, the solvent was removed under reduced pressure to obtain a red residue, which was purified by silica gel column chromatography (elution buffer: CH2Cl2:CH3OH = 10:1) to obtain compound 2.

[0059] 3) Synthesis of compound 3: 7-(diethylamino)coumarin-3-carboxylic acid was dissolved in N,N-dimethylformamide (DMF) and the carboxyl group was activated under EDC (1-ethyl-(3-dimethylaminopropyl)carbodiimide) and NHS (N-hydroxysuccinimide). Then, compound 2 and triethylamine were added. The mixture was washed with water and then extracted with dichloromethane. The organic layer was collected, dried with anhydrous sodium sulfate, and the solvent was removed by vacuum drying. The yellow solid was purified by column chromatography to obtain compound 3.

[0060] 4) Synthesis of the monomer FRET-R: Compound 3 and phenyl isothiocyanate were dissolved in dry DMF solution, and a small amount of triethylamine was used as a catalyst. The reaction was carried out under a nitrogen atmosphere, and the solvent was removed under reduced pressure. The black-red solid probe FRET-R was purified by column chromatography.

[0061] In the above technical solution, in step 1), the molar ratio of 3-(piperazine-1-alkyl)phenol and 2-(4-(diethylamino)-2-hydroxybenzoic acid) is 1:1 to 1:3, the condensation reaction temperature is 90-110℃, and the reaction time is 24-48h.

[0062] In the above technical solution, in step 2), the molar ratio of compound 1 to hydrazine hydrate is 1:1.5 to 1:10, the methoxy substitution reaction temperature is 75 to 90°C, and the reaction time is 5 to 24 h.

[0063] In the above technical solution, in step 3), the molar ratio of compound 2 and 7-(diethylamino)coumarin-3-carboxylic acid is 1:1 to 3:1, the amide reaction temperature is 0 to 30°C, the initial reaction temperature is 0 to 5°C, and the reaction time is 0.5 to 2 h; the later reaction temperature is 20 to 30°C, and the reaction time is 12 to 24 h.

[0064] In the above technical solution, in step 4), the molar ratio of compound 3 and phenyl isothiocyanate is 1:1.5 to 1:4, the reaction temperature is 20 to 30°C, and the reaction time is 24 to 48 h.

[0065] Thirdly, this application provides a fluorescently sensitive membrane comprising a chitosan-based membrane and the rhodamine-coumarin-based fluorescent probe described in the first aspect or prepared by the preparation method described in the second aspect.

[0066] The fluorescently sensitive membrane recognition monomer FRET-R of this application uses a rhodamine-coumarin fluorophore as the fluorescent backbone and introduces thiourea as Hg. 2+ The recognition group, using Hg 2+ This method can promote the ring-opening of loramide, resulting in a fluorescence change from blue-green to orange-yellow, exhibiting specific fluorescence detection and strong anti-interference capabilities. Simultaneously, the fluorescence-sensitive membrane of this application recognizes the polymerization of monomers and chitosan into a membrane. The high porosity and adsorption properties of the chitosan membrane can promote a rapid response between the analyte and the probe, improving the probe's sensitivity. This fluorescence-sensitive membrane can selectively detect Hg in a pure water system. 2+ FRET-type fluorescence activation occurs, enabling quantitative detection of Hg. 2+ The detection limit is 0.5 ppb, and the identification is almost unaffected by other ions in the environment.

[0067] In some embodiments, the chitosan-based membrane is physically cross-linked with the rhodamine-coumarin-based fluorescent probe.

[0068] In some embodiments, the fluorescent probe is loaded onto the chitosan-based membrane at a concentration of 5-30 mg / g, for example, 5 mg / g, 5.5 mg / g, 6 mg / g, 6.5 mg / g, 7 mg / g, 7.5 mg / g, 8 mg / g, 8.5 mg / g, 9 mg / g, 9.5 mg / g, 10 mg / g, 11 mg / g, 12 mg / g, 13 mg / g, 14 mg / g, 15 mg / g, 16 mg / g, 17 mg / g, 18 mg / g, 19 mg / g, 20 mg / g, 22 mg / g, 24 mg / g, 26 mg / g, or 28 mg / g. In some embodiments, the fluorescent probe is loaded onto the chitosan-based membrane at a concentration of 7 mg / g-20 mg / g.

[0069] In some embodiments, the thickness of the fluorescent sensing film is 3-30 μm, for example, 4 μm, 5 μm, 6 μm, 7 μm, 8 μm, 9 μm, 10 μm, 11 μm, 12 μm, 13 μm, 14 μm, 15 μm, 16 μm, 17 μm, 18 μm, 19 μm, 20 μm, 21 μm, 22 μm, 23 μm, 24 μm, 25 μm, 26 μm, 27 μm, 28 μm, or 29 μm. In some embodiments, the thickness of the fluorescent sensing film is 10-25 μm.

[0070] In some embodiments, the pore size of the fluorescent sensitive membrane is 100-5000 nm, for example, 200 nm, 500 nm, 1000 nm, 1500 nm, 2000 nm, 2500 nm, 3000 nm, 3500 nm, 4000 nm or 4500 nm.

[0071] Fourthly, this application provides a method for preparing a fluorescent sensitive membrane, which includes reacting the rhodamine-coumarin-based fluorescent probe described in the first aspect or the rhodamine-coumarin-based fluorescent probe prepared by the method described in the second aspect with a solution containing a chitosan-based membrane.

[0072] In some embodiments, the mass ratio of the rhodamine-coumarin-based fluorescent probe to chitosan is 0.005-0.1, for example, 0.006, 0.007, 0.008, 0.009, 0.01, 0.011, 0.012, 0.013, 0.014, 0.015, 0.016, 0.017, 0.018, 0.019, 0.02, 0.023, 0.025, 0.027, 0.03, 0.033, 0.035, 0.037, 0.04, 0.043, 0.045, or 0.047. In some embodiments, the mass ratio of the rhodamine-coumarin-based fluorescent probe to chitosan is 0.005-0.05.

[0073] In some embodiments, the mass ratio of the rhodamine-coumarin-based fluorescent probe to the chitosan-based membrane is 1:350 to 3:350.

[0074] In some embodiments, the reaction temperature is 5°C-35°C, for example, 10°C, 15°C, 20°C, 25°C, or 30°C. In some embodiments, the reaction temperature is 10°C-30°C. In some embodiments, the reaction temperature is room temperature.

[0075] In some embodiments, the reaction time is 5h-30h, for example 7h, 10h, 13h, 15h, 17h, 20h, 23h, 25h, or 27h. In some embodiments, the reaction time is 10h-20h.

[0076] In some embodiments, the solution containing the chitosan-based membrane is obtained by:

[0077] Chitosan is dissolved in an acidic solution to obtain an acidic chitosan solution; the acidic chitosan solution is mixed with a polyol and then subjected to a prepolymerization reaction to obtain the solution containing the chitosan-based membrane.

[0078] In some embodiments, the acid solution is selected from one or more of acetic acid, lactic acid, and propionic acid.

[0079] In some embodiments, the acid solution contains 0.5-5% by mass, for example, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, or 4.5% by mass. In some embodiments, the acid content is 0.5-2% by mass.

[0080] In some embodiments, the polyol is selected from one or more C2-C6 polyols. In some embodiments, the polyol is selected from ethylene glycol or glycerol.

[0081] In some embodiments, the mass ratio of chitosan to the total volume of the acid solution and polyol is (1-4) g:100 mL, for example, 1.3 g:100 mL, 1.5 g:100 mL, 1.7 g:100 mL, 2.0 g:100 mL, 2.3 g:100 mL, 2.5 g:100 mL, 2.7 g:100 mL, 3.0 g:100 mL, 3.3 g:100 mL, 3.5 g:100 mL, or 3.7 g:100 mL. In some embodiments, the mass ratio of chitosan to the total volume of the acid solution and polyol is (2-3) g:100 mL.

[0082] In some embodiments, the volume ratio of the acid solution to the polyol is (95-99):(1-5), for example, 98:2, 97:3 or 96:4.

[0083] In some embodiments, the preparation method further includes evaporating and cooling the reaction product to obtain a crude film, and washing the crude film to obtain the fluorescence-sensitive film.

[0084] In some embodiments, the evaporation temperature is 40°C-65°C, for example 45°C, 50°C, 55°C or 60°C.

[0085] In some embodiments, the washing includes washing the coarse film with water until it becomes neutral.

[0086] In some embodiments, the preparation of the fluorescence-sensitive film includes the following specific steps:

[0087] (1) Add chitosan and 1% acetic acid to a single-necked flask and stir until the chitosan is completely dissolved. Then, add glycerol for prepolymerization at room temperature to obtain a chitosan-based film solution.

[0088] (2) Rhodamine-coumarin-based fluorescent probe (FRET-R) was added to the chitosan-based membrane solution and the reaction continued. The solution was poured into a glass dish and evaporated to obtain a rough membrane. The membrane was washed three times with water to obtain the fluorescent sensitive membrane FRET-R-Mor.

[0089] Fifthly, this application provides the application of the rhodamine-coumarin-based fluorescent probe described in the first aspect, or the rhodamine-coumarin-based fluorescent probe prepared by the preparation method described in the second aspect, or the fluorescent sensitive membrane described in the third aspect, or the fluorescent sensitive membrane prepared by the preparation method described in the fourth aspect, in the detection of heavy metal ions, especially mercury ions.

[0090] Compared with the prior art, the beneficial effects of this application are as follows:

[0091] 1) The FRET-R-Mor fluorescent membrane exhibits good stability in an aqueous environment, enabling the detection of Hg at the ppb level. 2+The detection;

[0092] 2) Detection of Hg in water based on fluorescence-sensitive membranes 2+ Fluorescence-activated detection provides specific fluorescence detection and has strong anti-interference capabilities.

[0093] 3) The materials used to prepare FRET-R-Mor films are inexpensive, do not cause secondary pollution to the environment, and are easy to integrate, making them applicable to the detection of actual groundwater samples. Attached Figure Description

[0094] Figure 1 The synthesis steps of the rhodamine-coumarin-based fluorescent probe FRET-R of this application are shown.

[0095] Figure 2 These are physical images of FRET-R-Mor films according to some embodiments of this application.

[0096] Figure 3 These are scanning electron microscope (SEM) images of FRET-R-Mor thin films according to some embodiments of this application.

[0097] Figure 4 This is a bar chart showing the fluorescence selectivity and anti-interference of the FRET-R-Mor thin film of Example 1 of this application in the presence of other metal cations.

[0098] Figure 5 The FRET-R-Mor film of Example 1 of this application is effective against Hg. 2+ The fluorescence titration spectrum.

[0099] Figure 6 The fluorescence intensity ratio of the FRET-R-Mor film in Example 1 of this application and Hg 2+ The concentration linear fitting plot. Detailed Implementation

[0100] To make the objectives, technical solutions, and advantages of this application clearer, the following detailed description is provided in conjunction with embodiments and accompanying drawings. The specific embodiments described herein are for illustrative purposes only and are not intended to limit the scope of this application. Furthermore, descriptions of well-known structures and technologies are omitted in the following description to avoid unnecessarily obscuring the concepts of this disclosure. Such structures and technologies have also been described in numerous publications.

[0101] The present application will be further described below through specific embodiments.

[0102] Example 1

[0103] refer to Figure 1 The preparation method of the fluorescent probe FRET-R is as follows:

[0104] (1) Synthesis of Compound 1: 3-(piperazin-1-alkyl)phenol (313 mg, 1.0 mmol) and 2-(4-(diethylamino)-2-hydroxybenzoylbenzoic acid (178 mg, 1.0 mmol) were dissolved in 10 mL of trifluoroacetic acid solution and heated to reflux at 90 °C for 12 h. The reaction solution was cooled to room temperature, the solvent was removed under pressure, and purified by column chromatography (CH2Cl2 solution containing 10% EtOH) to obtain a red solid, which is Compound 1;

[0105] (2) Synthesis of Compound 2: Compound 1 (456 mg, 1.0 mmol) was dissolved in 25 mL of ethanol, and 1.5 mL of hydrazine hydrate was added. The mixture was refluxed in an oil bath at 80 °C for 6 h. The solution color changed from red to yellowish-brown, and the strong orange fluorescence disappeared. The solvent was then removed under pressure. The solution was purified by column chromatography (CH2Cl2:CH3OH = 10:1) to obtain a yellow solid, which was compound 2.

[0106] (3) Synthesis of Compound 3: 7-(diethylamino)coumarin-3-carboxylic acid (261 mg, 1.0 mmol) was dissolved in 25 mL of dry DMF, and 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) (286.5 mg, 1 mmol) and N-hydroxysuccinimide (NHS) (172.5 mg, 1.0 mmol) were added. The mixture was activated in an ice bath at 5 °C for 1 h. Subsequently, Compound 2 (562.8 mg, 1.2 mmol) and a catalytic amount of triethylamine were added and stirred overnight at room temperature. The reaction solution was extracted and washed with dichloromethane and water. The organic layer was collected, dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure. The solution was purified by column chromatography (CH2Cl2:CH3OH = 10:1) to give a yellow solid, namely Compound 3.

[0107] (4) Synthesis of FRET-R: Compound 3 (712 mg, 1.0 mmol) and phenyl isothiocyanate (202.5 mg, 1.5 mmol) were dissolved in dry DMF solution, with a small amount of triethylamine as a catalyst, and reacted at room temperature for 24 h under a nitrogen atmosphere. The solvent was then removed from the reaction solution under reduced pressure, and the solution was purified by column chromatography to obtain a blackish-red solid, which was the rhodamine-coumarin-based fluorescent probe FRET-R.

[0108]

[0109] 1H NMR(400MHz,Chloroform-d)δ8.03(d,J=7.8Hz,1H),7.90(s,1H),7.67(t,J=7.5Hz,1H),7.61(d,J=7.4Hz,1H ),7.50(s,1H),7.32(d,J=7.9Hz,1H),7.19(t,J=7.4Hz,2H),7.12(s,1H),7.06(d,J=8.7Hz,2H),6.90(s,1H), 6.69(s,1H),6.61(dd,J=9.0,2.4Hz,1H),6.53(s,2H),6.52–6.47(m,2H),6.44(s,1H),6.30(dd,J=8.9,2.6Hz ,1H),5.29(s,1H),3.90(s,2H),3.60–3.39(m,7H),3.39–3.23(m,7H),1.24–1.20(m,6H),1.19–1.13(m,6H));

[0110] 13C NMR(101MHz,Chloroform-d)δ182.31,166.91,164.93,158.97,157.15,153.91,153.75,152 .04,151.58,149.55,149.23,145.33,137.32,134.24,129.76,129.07,128.82,128.13,127 .48,127.42,125.96,124.77,124.52,123.77,115.60,112.10,109.22,108.44,107.55,103.64,103.01,98.05,96.71,66.69,48.22,47.76,46.71,44.76,44.20,41.80,12.31,12.20.

[0111] Preparation of fluorescent sensitive membrane:

[0112] Chitosan (1.75 g) and 1% acetic acid (97 mL) were added to a single-necked flask and stirred until the chitosan was completely dissolved. Then, glycerol (3 mL) was added and prepolymerized at room temperature for 4 h to obtain a chitosan-based membrane solution. Finally, the fluorescent probe FRET-R (20 mg) was added, and the reaction was continued for 12 h. The reaction solution was poured into a glass dish, evaporated, and cooled to obtain a rough membrane. The rough membrane was washed three times with water to obtain a fluorescently sensitive membrane (FRET-R-Mor film).

[0113] Application Example 1

[0114] The FRET-R-Mor films prepared in Example 1 were cut into 6 mm diameter circular films and fixed onto glass slides. The moisture on the films was absorbed with filter paper, and then 10 μL of deionized water was added to ensure consistent water content. The glass slides containing the films were then immersed in 20 mL of each test solution. The initial fluorescence intensity of each 6 mm diameter circular film was measured first, followed by the fluorescence intensity in the presence of different metal ions, and finally, the Hg... 2+ Fluorescence intensity when coexisting with other metal ions.

[0115] Each test solution contained 1 ppm of NaCl, KCl, CaCl2, CdCl2, ZnCl2, NiCl2, CuCl2, CoCl2, PbCl2, FeCl3, and 100 ppb of HgCl2.

[0116] Figure 4 This is a bar graph showing the fluorescence selectivity and anti-interference performance of the FRET-R-Mor film in the presence of other metal cations, as described in the example. From... Figure 4 It can be seen from this that Hg 2+ The addition of Hg will cause fluorescence activation, while the addition of other metal ions has almost no effect on the fluorescence of FRET-R-Mor. 2+ The addition of [a specific ingredient] can reduce the fluorescence intensity at 474 nm and turn on the fluorescence at 594 nm in the FRET-R-Mor film.

[0117] The FRET-R-Mor film is effective against Hg. 2+ The ions exhibit high selectivity and good anti-interference ability, making them suitable for handling trace amounts of Hg in complex environments. 2+ The detection.

[0118] Application Example 2

[0119] The FRET-R-Mor film prepared in Example 1 was cut into circular film pieces with a diameter of 6 mm and fixed onto a glass slide. The moisture on the film was then absorbed with filter paper. 10 μL of deionized water was then added to ensure a consistent water content in the film. The glass slide containing the film was then immersed in 20 mL of water containing 5–60 ppb Hg. 2+ In the test solutions (5 ppb, 10 ppb, 20 ppb, 30 ppb, 40 ppb, 50 ppb, 60 ppb), after 2 minutes, the change in fluorescence intensity is detected on a fluorescence spectrophotometer to obtain the result. Figure 5 The corresponding concentration-fluorescence intensity standard working curves are shown.

[0120] from Figure 5 The standard curve shows that as the Hg in the test solution increases... 2+With increasing concentration, the fluorescence of the sensitive membrane undergoes a FRET change, meaning that coumarin fluorescence gradually turns off while rhodamine fluorescence gradually turns on. When Hg... 2+ When the ion concentration is 5 ppb, it contains Hg. 2+ The fluorescence intensity of the ion-sensitive membrane is significantly different from that of the blank membrane, meaning that when using fluorescence emission spectroscopy to detect Hg... 2+ At that time, the actual detection limit of the sensitive membrane prepared by the present invention can be as low as 5 ppb.

[0121] Figure 6 The fluorescence intensity ratio and Hg of the FRET-R-Mor film are shown. 2+ The concentration linear fitting curve. From Figure 6 Hg can be calculated from 2+ The detection limit for the ion is 0.5 ppb.

[0122] The preferred embodiments of this application have been described in detail above; however, this application is not limited thereto. Within the scope of the technical concept of this application, various simple modifications can be made to the technical solution of this application, including combining various technical features in any other suitable manner. These simple modifications and combinations should also be considered as the content disclosed in this application and are all within the protection scope of this application.

Claims

1. A fluorescence-sensitive membrane comprising a chitosan-based membrane and a rhodamine-coumarin-based fluorescent probe, wherein the rhodamine-coumarin-based fluorescent probe comprises a compound represented by Formula I: In Formula I, R1 and R2 may be the same or different, and each is independently selected from C1-C6 alkyl groups; R 13 and R 14 Whether the alkyl groups are the same or different, each is independently selected from C1-C6 alkyl groups; R3-R 12 R 15 -R 23 Each is an independent hydrogen, halogen, or C1-C6 alkyl group.

2. The fluorescent sensitive film according to claim 1, characterized in that, R1 and R2 may be the same or different, and each is independently selected from C1-C4 alkyl groups; R 13 and R 14 Whether the alkyl groups are the same or different, each is independently selected from C1-C4 alkyl groups; R3-R 12 R 15 -R 23 Each is independently selected from hydrogen, fluorine, chlorine, bromine, or C1-C4 alkyl groups.

3. The fluorescent sensitive film according to claim 1, characterized in that, The rhodamine-coumarin-based fluorescent probe is selected from the following compounds:

4. The fluorescent sensitive film according to any one of claims 1-3, characterized in that, The method for preparing the rhodamine-coumarin-based fluorescent probe includes one or more of the following steps: Step 1: React compound a with compound b to produce compound 1. Step 2: React compound 1 with hydrazine hydrate to generate compound 2. Step 3: React compound 2 with compound c to generate compound 3. Step 4: React compound 3 with compound d to generate compound 4. Among them, the definitions of each element in equations 1-3 and 'ad' are the same as those in equation I.

5. The fluorescent sensitive film according to claim 4, characterized in that, In step 1, the molar ratio of compound a to compound b is 1:1 to 1:3, the reaction temperature is 80℃ to 120℃, and the reaction time is 5h to 50h; and / or In step 2, the molar ratio of compound 1 to hydrazine hydrate is 1:1.5-1:10, the reaction temperature is 70℃-100℃, and the reaction time is 5h-24h; and / or In step 3, the molar ratio of compound 2 to compound c is 1:1-3:1, the reaction temperature is 0℃-30℃, and the reaction time is 0.5h-24h; and / or In step 4, the molar ratio of compound 3 to compound d is 1:1.5-1:4, the reaction temperature is 5℃-35℃, and the reaction time is 5h-50h.

6. The fluorescent sensitive film according to claim 5, characterized in that, In step 1, the reaction temperature is 90℃-100℃, and the reaction time is 10h-24h; and / or The compound of formula b is 3-(piperazine-1-alkyl)phenol, the compound of formula a is 2-(4-(diethylamino)-2-hydroxybenzoylbenzoic acid or 2-(4-(dimethylamino)-2-hydroxybenzoylbenzoic acid), and the compound of formula 1 is... In step 2, the reaction temperature is 75℃-90℃; and / or Compound of Formula 2 is In step 3, compound c is 7-(diethylamino)coumarin-3-carboxylic acid or 7-(dimethylamino)coumarin-3-carboxylic acid, and compound 3 is... In step 4, the reaction temperature is 20℃-30℃, and the reaction time is 24h-48h; and / or Compound d is phenyl isothiocyanate, and compound 4 is...

7. The fluorescent sensitive film according to any one of claims 1-3, characterized in that, The chitosan-based membrane is physically cross-linked with the rhodamine-coumarin-based fluorescent probe.

8. The fluorescent sensitive film according to any one of claims 1-3, characterized in that, The fluorescent probe is loaded onto the chitosan-based membrane at a rate of 5 mg / g-30 mg / g; and / or The thickness of the fluorescence-sensitive film is 3-30 μm; and / or The pore size of the fluorescent sensitive membrane is 100-5000 nm.

9. The fluorescent sensitive film according to claim 8, characterized in that, The fluorescent probe is loaded at a concentration of 7 mg / g-20 mg / g on the chitosan-based membrane; and / or The thickness of the fluorescent sensitive film is 10-25 μm.

10. A method for preparing a fluorescent sensitive membrane according to any one of claims 1-9, comprising reacting the rhodamine-coumarin-based fluorescent probe with a solution containing a chitosan-based membrane.

11. The preparation method according to claim 10, characterized in that, The mass ratio of the rhodamine-coumarin-based fluorescent probe to chitosan is 0.005-0.1; and / or The reaction temperature is 5℃-35℃; and / or The reaction time is 5 h-30 h; and / or The solution containing the chitosan-based membrane is obtained by the following method: Chitosan is dissolved in an acidic solution to obtain an acidic chitosan solution; the acidic chitosan solution is mixed with a polyol and then subjected to a prepolymerization reaction to obtain the solution containing the chitosan-based membrane.

12. The preparation method according to claim 11, characterized in that, The mass ratio of the rhodamine-coumarin-based fluorescent probe to chitosan is 0.005-0.05; and / or The reaction temperature is 10℃-30℃; and / or The reaction time is 10-20 hours; and / or The acid solution is selected from one or more of acetic acid, lactic acid, and propionic acid; and / or The acid solution contains 0.5-5% acid by mass; and / or The mass ratio of chitosan to the total volume of the acid solution and polyol is (1-4) g:100 mL, and the volume ratio of the acid solution to the polyol is (95-99):(1-5); and / or The polyol is selected from one or more C2-C6 polyols.

13. The preparation method according to claim 12, characterized in that, The acid solution contains 0.5-2% acid by mass; and / or The mass ratio of the chitosan to the total volume of the acid solution and polyol is (2-3) g: 100 mL; and / or The polyol is selected from ethylene glycol or glycerol.

14. The preparation method according to any one of claims 10-13, characterized in that, The preparation method further includes evaporating and cooling the reaction product to obtain a crude film, and washing the crude film to obtain the fluorescent sensitive film.

15. The preparation method according to claim 14, characterized in that, The evaporation temperature is 40℃-65℃; and / or The washing process includes washing the coarse film with water until it becomes neutral.

16. The application of a rhodamine-coumarin-based fluorescent probe, or the fluorescent sensitive membrane according to any one of claims 1-9, or the fluorescent sensitive membrane prepared by any one of claims 10-15, in the detection of mercury ions in water; wherein, The rhodamine-coumarin-based fluorescent probe comprises the compound shown in Formula I: In Formula I, R1 and R2 may be the same or different, and each is independently selected from C1-C6 alkyl groups; R 13 and R 14 Whether the alkyl groups are the same or different, each is independently selected from C1-C6 alkyl groups; R3-R 12 R 15 -R 23 Each is an independent hydrogen, halogen, or C1-C6 alkyl group.

17. The application according to claim 16, characterized in that, R1 and R2 may be the same or different, and each is independently selected from C1-C4 alkyl groups; R 13 and R 14 Whether the alkyl groups are the same or different, each is independently selected from C1-C4 alkyl groups; R3-R 12 R 15 -R 23 Each is independently selected from hydrogen, fluorine, chlorine, bromine, or C1-C4 alkyl groups.

18. The application according to claim 16, characterized in that, The rhodamine-coumarin-based fluorescent probe is selected from the following compounds:

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