Rhodamine-coumarin-based fluorescent probe, fluorescent sensitive film and preparation method and application thereof

Abstract

The invention relates to a rhodamine-coumarin-based fluorescent probe, a fluorescent sensitive film as well as a preparation method and application of the rhodamine-coumarin-based fluorescent probe. The rhodamine-coumarin-based fluorescent probe provided by the invention comprises a compound as shown in a formula I which is described in the specification. The fluorescent sensitive film provided by the invention comprises a chitosan-based film and the rhodamine-coumarin-based fluorescent probe. The fluorescent sensitive membrane can be used for ultra-sensitive detection of metal ions, especially mercury ions, in biological tissues and environmental sewage. # imgabs0 #

Description

Technical Field

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

[0002] Mercury (Hg) and its compounds are global environmental pollutants. 2+ (divalent mercury ion) is of particular concern due to its high toxicity and bioaccumulation. 2+ It can accumulate through the food chain and cause a variety of health problems, including kidney damage, nervous system damage and even death, posing a serious threat to human health. In addition, mercury pollution can also affect the balance of the ecosystem and cause harm to aquatic organisms and wildlife. Therefore, the detection of Hg in the water environment 2+ Concentrations are critical for environmental protection and public health.

[0003] Hg in water samples 2+ There are many traditional quantitative analysis methods, including atomic absorption spectroscopy, cold vapor atomic fluorescence spectroscopy, gas chromatography, high performance liquid chromatography and electrochemistry. Traditional methods are difficult to detect Hg in real time due to complex sample pretreatment and expensive instruments. 2+ Recently, fluorescence detection has attracted wide attention due to its advantages such as convenient detection, short response time and high sensitivity. However, some organic small molecule fluorescent probes have very low detection limits and cannot detect Hg in pure water. 2+ , and will pollute the environment.

[0004] Since the fluorescent sensitive membrane contains specific molecular recognition units, it can form a stable complex with mercury ions through covalent bonds or coordination. This specific interaction ensures high selectivity of detection. Fluorescent sensitive membranes are usually environmentally friendly and do not introduce additional harmful substances. More importantly, the preparation and use of fluorescent sensitive membranes are usually simple, and can be easily applied to natural environments and biological systems, which is conducive to rapid on-site detection and monitoring. Fluorescent sensitive membranes can also be integrated into different detection platforms, such as sensitive membranes, films or fibers, to provide a variety of detection methods. It works effectively in complex environmental samples, which provides a new strategy for practical environmental monitoring and biological sample analysis. In summary, fluorescent sensitive membranes provide a series of advantages over traditional organic probes in detection, including higher sensitivity and selectivity, rapid response, easy application, cost-effectiveness and environmental friendliness. These advantages make fluorescent sensitive membranes a powerful tool in the field of environmental monitoring and biological detection. Therefore, it provides high selectivity and high sensitivity to select Hg 2+ The fluorescence sensitive membrane is of great significance. Summary of the invention

[0005] In order to solve the problems in the prior art, the present application provides a rhodamine-coumarin-based fluorescent probe and a fluorescent sensitive membrane and a preparation method and application thereof. The fluorescent sensitive membrane of the present application can be used for ultrasensitive detection of metal ions, especially mercury ions, in biological tissues and environmental sewage.

[0006] In a first aspect, the present application provides a rhodamine-coumarin-based fluorescent probe, which includes a compound shown in formula I:

[0007]

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

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

[0010] The rhodamine-coumarin-based fluorescent probe of the present application uses the rhodamine-coumarin fluorophore as the fluorescent skeleton and introduces thiourea as Hg 2+ The recognition group of Hg 2+ It can promote the ring-opening of rolactam and realize the change of fluorescence from blue-green to orange-yellow. It has specific fluorescence detection and strong anti-interference ability.

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

[0012] In some embodiments, R 13 and R 14 The same or different, each independently selected from C1-C4 alkyl, for example methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl or tert-butyl.

[0013] In some embodiments, R3-R 12 , R 15 -R 23 Each is independently selected from hydrogen, fluorine, chlorine, bromine or C1-C4 alkyl, 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] In a second aspect, the present application provides a method for preparing the rhodamine-coumarin-based fluorescent probe according to the first aspect, which comprises one or more of the following steps:

[0017] Step 1: reacting a compound of formula a with a compound of formula b to produce a compound of formula 1,

[0018]

[0019] Step 2: reacting the compound of formula 1 with hydrazine hydrate to generate a compound of formula 2,

[0020]

[0021] Step 3: reacting the compound of formula 2 with the compound of formula c to generate the compound of formula 3,

[0022] Step 4: reacting the compound of formula 3 with the compound of formula d to generate the compound of formula 4,

[0023]

[0024] Wherein, the definitions of the components in Formulas 1-3 and ad are the same as those in Formula I.

[0025] In some embodiments, in formula a, R1 and R2 are the same or different, and are 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, 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, 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 embodiments, in formula b, R 10 , R 11 and R 12 Each is independently selected from hydrogen, fluorine, chlorine, bromine or C1-C4 alkyl, such as methyl, ethyl, n-propyl, isopropyl or n-butyl. In some embodiments, the compound of formula b is 3-(piperazine-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 embodiments, in formula c, R 13 and R 14 The same or different, each independently selected from C1-C4 alkyl, for example methyl, ethyl, n-propyl, isopropyl or n-butyl.

[0033] In some embodiments, in formula c, R 15 , R 16 , R 17 and R 18 Each is independently selected from hydrogen, fluorine, chlorine, bromine or C1-C4 alkyl, 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 embodiments, in formula d, R 19 , R 20 , R 21 , R 22 , R 23 Each is independently selected from hydrogen, fluorine, chlorine, bromine or C1-C4 alkyl, 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 the compound of formula a to the compound of formula b is 1:1-1:3, for example, 1:1.5, 1:2 or 1:2.5.

[0042] In some embodiments, in step 1, the reaction (condensation reaction) temperature is 80°C-120°C, such as 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, in step 1, the reaction time is 5 h-50 h, such as 10 h, 12 h, 18 h, 24 h, 30 h, 36 h, 42 h or 48 h. In some embodiments, the reaction time is 10 h-24 h.

[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, in step 2, the reaction (methoxy substitution reaction) temperature 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 embodiments, in step 2, the reaction time is 5 h-24 h, for example, 6 h, 10 h, 12 h or 18 h.

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

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

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

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

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

[0053] In some embodiments, in step 4, the reaction temperature 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, in step 4, the reaction time is 5 h-50 h, such as 10 h, 12 h, 18 h, 24 h, 30 h, 36 h, 42 h or 48 h. In some embodiments, the reaction time is 24 h-48 h.

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

[0056] In some embodiments, the method for preparing the rhodamine-coumarin-based fluorescent probe comprises the following specific steps:

[0057] 1) Synthesis of Compound 1: 3-(piperazine-1-alkyl)phenol and 2-(4-(diethylamino)-2-hydroxybenzoic acid were subjected to amidation reaction in trifluoroacetic acid, and the solvent was removed under reduced pressure to obtain a red residue, which was purified by silica gel (eluent: 10% EtOH) column chromatography to obtain Compound 1;

[0058] 2) Synthesis of Compound 2: Compound 1 was added to ethanol and heated under reflux, and an excess of hydrazine hydrate was added, cooled to room temperature, and the solvent was removed under reduced pressure to obtain a red residue, which was purified by silica gel (eluent: CH2Cl2:CH3OH=10:1) column chromatography 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 in the presence of EDC (1-ethyl-(3-dimethylaminopropyl)carbodiimide) and NHS (N-hydroxysuccinimide), followed by the addition of compound 2 and triethylamine, the mixture was washed with water, and then extracted with dichloromethane, the organic layer was collected, dried over anhydrous sodium sulfate, the solvent was removed by vacuum drying, and purified by column chromatography to obtain a yellow solid, namely compound 3.

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

[0061] In the above technical scheme, 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°C, and the reaction time is 24 to 48 hours.

[0062] In the above technical scheme, 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 hours.

[0063] In the above technical scheme, 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 early reaction temperature is 0 to 5°C, and the reaction time is 0.5 to 2h; the late reaction temperature is 20 to 30°C, and the reaction time is 12 to 24h.

[0064] In the above technical scheme, in step 4), the molar ratio of compound 3 to 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 hours.

[0065] In a third aspect, the present application provides a fluorescence sensitive membrane, which includes a chitosan-based membrane and 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.

[0066] The fluorescence sensitive membrane recognition monomer FRET-R of the present invention is based on rhodamine-coumarin fluorophore as the fluorescent skeleton, and thiourea is introduced as Hg 2+ The recognition group of Hg 2+ It can promote the ring opening of rolactam and realize the change of fluorescence from blue-green to orange-yellow. It has specific fluorescence detection and strong anti-interference ability. At the same time, the fluorescent sensitive membrane of the present application will polymerize the recognition monomer and chitosan into a membrane. The high porosity and adsorption of the chitosan membrane can promote the rapid response of the analyte and the probe and improve the sensitivity of the probe. The fluorescent sensitive membrane can detect Hg in a pure water system with high selectivity. 2+ FRET-type fluorescence is turned on to achieve quantitative detection of Hg 2+ The detection limit is 0.5 ppb, and the identification is almost not interfered by other ions in the environment.

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

[0068] In some embodiments, the loading amount of the fluorescent probe on the chitosan base film is 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 loading amount of the fluorescent probe on the chitosan base film is 7 mg / g-20 mg / g.

[0069] In some embodiments, the thickness of the fluorescence sensitive 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 fluorescence sensitive film is 10-25 μm.

[0070] In some embodiments, the pore size of the fluorescence 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] In a fourth aspect, the present application provides a method for preparing a fluorescent sensitive membrane, comprising reacting 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 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 1:350-3:350.

[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 comprising chitosan-based membrane is obtained by:

[0077] The chitosan is dissolved in an acid solution to obtain an acid solution of chitosan; the acid solution of chitosan 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 has an acid content of 0.5-5%, such as 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4% or 4.5%. In some embodiments, the acid content is 0.5-2%.

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

[0081] In some embodiments, the ratio of the mass of the chitosan to the total volume of the acid solution and the 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 ratio of the mass of the chitosan to the total volume of the acid solution and the 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 comprises evaporating and cooling the reaction product to obtain a crude film, and washing the crude film to obtain the fluorescent 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 comprises washing the crude membrane with water until it is neutral.

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

[0087] (1) Chitosan and 1% acetic acid were added to a single-necked flask and stirred until the chitosan was completely dissolved. Then, glycerol was added for prepolymerization at room temperature to obtain a chitosan-based membrane solution.

[0088] (2) Adding rhodamine-coumarin-based fluorescent probe (FRET-R) to the chitosan-based membrane solution, continuing the reaction, pouring the solution into a glass dish and evaporating it to obtain a crude membrane, which was then rinsed with water three times to obtain a fluorescent sensitive membrane FRET-R-Mor.

[0089] In the fifth aspect, the present application provides the use 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:

[0091] 1) The fluorescence sensitive membrane FRET-R-Mor shows good stability in water environment and can detect Hg at ppb level. 2+Detection;

[0092] 2) Detection of Hg in water based on fluorescence sensitive membrane 2+ Fluorescence detection, with specific fluorescence detection and strong anti-interference ability;

[0093] 3) The materials used to prepare the FRET-R-Mor film are low-cost, will not cause secondary pollution to the environment, and are easy to integrate, and can be applied to the detection of actual groundwater samples. BRIEF DESCRIPTION OF THE DRAWINGS

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

[0095] Figure 2 is a physical picture of the FRET-R-Mor film according to some embodiments of the present application.

[0096] Figure 3 is a scanning electron microscope (SEM) image of the FRET-R-Mor film according to some embodiments of the present application.

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

[0098] Figure 5 The FRET-R-Mor film of Example 1 of the present application is 2+ Fluorescence titration spectrum of .

[0099] Figure 6 is the fluorescence intensity ratio of the FRET-R-Mor film of Example 1 of the present application and Hg 2+ Linear fitting diagram of the concentration. DETAILED DESCRIPTION

[0100] In order to make the purpose, technical scheme and advantages of the present application clearer, the present application is further described in detail below in conjunction with the embodiments and drawings. The specific embodiments described herein are only used to explain the present application and are not intended to constitute any limitation to the present application. In addition, in the following description, the description of known structures and technologies is omitted to avoid unnecessary confusion of the concepts of the present disclosure. Such structures and technologies are also described in many publications.

[0101] The present application is further described below through specific examples.

[0102] Example 1

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

[0104] (1) Synthesis of Compound 1: 3-(Piperazine-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, 1.5 mL of hydrazine hydrate was added, and the mixture was refluxed in an oil bath at 80 °C for 6 h. The color of the solution changed from red to earthy yellow, and the strong orange fluorescence disappeared. The solvent was then removed under pressure. Column chromatography was used for purification (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, 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) (286.5 mg, 1 mmol) and N-hydroxysuccinimide (NHS) (172.5 mg, 1.0 mmol) were added and activated in an ice bath at 5°C for 1 h. Compound 2 (562.8 mg, 1.2 mmol) and a catalytic amount of triethylamine were then added and stirred at room temperature overnight. 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 mixture was purified by column chromatography (CH2Cl2:CH3OH=10:1) to obtain 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, a small amount of triethylamine was used as a catalyst, and the reaction was carried out at room temperature under nitrogen atmosphere for 24 h. The solvent in the reaction solution was then removed under reduced pressure and purified by column chromatography to obtain a black-red solid, which is 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 fluorescence 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, propylene glycol (3 mL) was added and prepolymerized at room temperature for 4 h to obtain a chitosan-based film solution. Finally, the above-mentioned 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 and evaporated and cooled to obtain a crude film. The crude film was rinsed with water three times to obtain a fluorescent sensitive film (FRET-R-Mor film).

[0113] Application Example 1

[0114] The FRET-R-Mor film prepared in Example 1 was cut into a circular film with a diameter of 6 mm and fixed on a glass slide. The water on the film was absorbed with filter paper, and then 10 μL of secondary water was dripped into it to ensure that the water content of the film was consistent. Then the glass slide containing the film was immersed in 20 mL of each test solution, and the initial fluorescence intensity of each circular film with a diameter of 6 mm was tested first, and then the fluorescence intensity in the presence of different metal ions was tested, and finally the Hg 2+ Fluorescence intensity in the presence of other metal ions.

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

[0116] Figure 4 : is a histogram of the fluorescence selectivity and anti-interference of the FRET-R-Mor film in the embodiment when other metal cations are present. Figure 4 It can be seen that Hg 2+ The addition of Hg will cause the fluorescence to turn on, and the addition of other metal ions will hardly affect the fluorescence of FRET-R-Mor. 2+ The addition of can achieve a decrease in the fluorescence intensity at 474nm of the FRET-R-Mor film and an increase in the fluorescence at 594nm.

[0117] The FRET-R-Mor film is effective for Hg 2+ Ions have high selectivity and good anti-interference ability and can be used for trace Hg in complex environments 2+ Detection.

[0118] Application Example 2

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

[0120] from Figure 5 As can be seen from the standard curve, as the Hg 2+As the concentration increases, the fluorescence of the sensitive membrane undergoes FRET changes, that is, the coumarin fluorescence gradually turns off and the rhodamine fluorescence gradually turns on. 2+ When the ion concentration is 5ppb, it contains Hg 2+ The fluorescence intensity of the ion-sensitive membrane is obviously different from that of the blank membrane, that is, when using fluorescence emission spectroscopy to detect Hg 2+ When the detection limit of the sensitive membrane prepared by the present invention is lower than 5 ppb, the actual detection limit can be as low as 5 ppb.

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

[0122] The preferred embodiments of the present application are described in detail above, but the present application is not limited thereto. Within the technical concept of the present application, the technical solution of the present application can be subjected to a variety of simple modifications, including combining various technical features in any other suitable manner, and these simple modifications and combinations should also be regarded as the contents disclosed in the present application and belong to the protection scope of the present application.

Claims

1. A rhodamine-coumarin-based fluorescent probe comprising a compound shown in formula I: In Formula I, R1 and R2 are the same or different and are independently selected from C1-C6 alkyl groups; 13 and R 14 The same or different, each independently selected from C1-C6 alkyl; R3-R 12 , R 15 -R 23 Each is independently hydrogen, halogen or C1-C6 alkyl.

2. The fluorescent probe according to claim 1, characterized in that R1 and R2 are the same or different and are independently selected from C1-C4 alkyl groups; R 13 and R 14 The same or different, each independently selected from C1-C4 alkyl; R3-R 12 , R 15 -R 23 Each is independently selected from hydrogen, fluorine, chlorine, bromine or C1-C4 alkyl; preferably, The rhodamine-coumarin-based fluorescent probe is selected from the following compounds:

3. A method for preparing the rhodamine-coumarin-based fluorescent probe according to claim 1 or 2, comprising one or more of the following steps: Step 1: reacting a compound of formula a with a compound of formula b to produce a compound of formula 1, Step 2: reacting the compound of formula 1 with hydrazine hydrate to generate a compound of formula 2, Step 3: reacting the compound of formula 2 with the compound of formula c to generate the compound of formula 3, Step 4: reacting the compound of formula 3 with the compound of formula d to generate the compound of formula 4, in, The definitions of the components in formulas 1-3 and ad are the same as those in formula I.

4. The preparation method according to claim 3, characterized in that: In step 1, the molar ratio of the compound of formula a to the compound of formula b is 1:1-1:3, the reaction temperature is 80°C-120°C, preferably 90°C-100°C, and the reaction time is 5h-50h, preferably 10h-24h. Preferably, 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 and / or In step 2, the molar ratio of the compound of formula 1 to hydrazine hydrate is 1:1.5-1:10, the reaction temperature is 70°C-100°C, preferably 75°C-90°C, and the reaction time is 5h-24h. Preferably, the compound of formula 2 is and / or In step 3, the molar ratio of the compound of formula 2 to the compound of formula c is 1:1-3:1, the reaction temperature is 0°C-30°C, and the reaction time is 0.5h-24h. Preferably, the compound of formula C is 7-(diethylamino)coumarin-3-carboxylic acid or 7-(dimethylamino)coumarin-3-carboxylic acid, and the compound of formula 3 is and / or In step 4, the molar ratio of the compound of formula 3 to the compound of formula d is 1:1.5-1:4, the reaction temperature is 5°C-35°C, preferably 20°C-30°C, and the reaction time is 5h-50h, preferably 24h-48h. Preferably, the compound of formula d is phenyl isothiocyanate, and the compound of formula 4 is 5. A fluorescence sensitive membrane comprising a chitosan-based membrane and the rhodamine-coumarin-based fluorescent probe according to claim 1 or 2 or the rhodamine-coumarin-based fluorescent probe prepared by the preparation method according to claim 3 or 4, Preferably, the chitosan-based membrane is physically cross-linked to the rhodamine-coumarin-based fluorescent probe.

6. The fluorescent sensitive film according to claim 5, characterized in that: The loading amount of the fluorescent probe on the chitosan-based membrane is 5 mg / g-30 mg / g, preferably 7 mg / g-20 mg / g; and / or The thickness of the fluorescence sensitive film is 3-30 μm, preferably 10-25 μm; and / or The pore size of the fluorescence sensitive membrane is 100-5000nm.

7. A method for preparing a fluorescence sensitive film, comprising reacting the rhodamine-coumarin-based fluorescent probe according to claim 1 or 2 or the rhodamine-coumarin-based fluorescent probe prepared by the preparation method according to claim 3 or 4 with a solution containing a chitosan-based film.

8. The preparation method according to claim 7, characterized in that: The mass ratio of the rhodamine-coumarin-based fluorescent probe to chitosan is 0.005-0.1, preferably 0.005-0.05; and / or The reaction temperature is 5°C-35°C, preferably 10°C-30°C; and / or The reaction time is 5h-30h, preferably 10h-20h; and / or The solution comprising the chitosan-based membrane is obtained by: Dissolving chitosan in an acid solution to obtain an acid solution of chitosan; mixing the acid solution of chitosan with a polyol, and performing a prepolymerization reaction to obtain the solution containing the chitosan base film; Preferably, the acid solution is selected from one or more of acetic acid, lactic acid and propionic acid. Preferably, the acid content in the acid solution is 0.5-5%, preferably 0.5-2% by mass; Preferably, the ratio of the mass of the chitosan to the total volume of the acid solution and the polyol is (1-4) g:100 mL, preferably (2-3) g:100 mL, and the volume ratio of the acid solution to the polyol is (95-99):(1-5); Preferably, the polyol is selected from one or more of C2-C6 polyols, preferably ethylene glycol or glycerol.

9. The preparation method according to claim 7 or 8, characterized in that: The preparation method further comprises evaporating and cooling the reaction product to obtain a crude film, and washing the crude film to obtain the fluorescent sensitive film; Preferably, the evaporation temperature is 40°C-65°C; Preferably, the washing comprises washing the crude membrane with water until it is neutral.

10. Use of the rhodamine-coumarin-based fluorescent probe according to claim 1 or 2, or the rhodamine-coumarin-based fluorescent probe prepared by the preparation method according to claim 3 or 4, or the fluorescent sensitive membrane according to claim 5 or 6, or the fluorescent sensitive membrane prepared by the preparation method according to any one of claims 7 to 9 in detecting heavy metal ions, especially mercury ions, in water.

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