Aie fluorescent probe for detecting mercury ions in red and near-infrared light and preparation and application thereof

Abstract

The present invention discloses an AIE fluorescent probe for detecting mercury ions using red light and near-infrared light, as well as its preparation and application. The molecular structure of the AIE fluorescent probe is shown in formula (I). The present invention is based on fluorenone dyes and uses thiolation reaction and aggregation-induced fluorescence to identify Hg 2+ A novel AIE fluorescent probe was constructed, which can detect Hg with high selectivity and sensitivity in the red to near-infrared range. 2+ The fluorescence emission intensity of the probe is significantly enhanced at 630 nm, and its fluorescence intensity is similar to that of Hg 2+ The concentration is linear within a certain range and can detect Hg in living cells and plants. 2+ Implement detection.

Description

Technical Field

[0001] The present invention relates to the preparation and application of a novel fluorenone fluorescent dye, in particular to a red to near infrared Hg 2+ The invention relates to the construction of aggregation-induced emission (AIE) fluorescent probe and its bioimaging application, belonging to the field of organic fluorescence sensing technology. Background Art

[0002] Mercury is a highly toxic, non-essential element, originating from both natural processes and human activities, such as coal combustion, mining, chemical production, and cement production. Trace amounts of mercury eventually enter freshwater and marine systems. Due to its bioaccumulative nature, long-term biochemical degradation, and high affinity for proteins containing sulfur, thiols, and selenium, mercury can have harmful effects on ecosystems, humans, and other organisms. In severe cases, methylmercury poisoning can lead to Minamata disease, which can cause cerebral palsy in fetuses. Therefore, the development of an accurate and rapid analytical method for the detection of mercury ions is crucial.

[0003] Currently, various technologies are available for the quantitative detection of mercury ions, such as direct mercury analyzers (DMA), inductively coupled plasma mass spectrometry (ICP-MS), and atomic absorption spectroscopy (AAS). However, due to the complex detection process, high cost, and high requirements for professional skills, these methods have difficulties in the real-time detection of mercury ions. In contrast, fluorescence detection methods have many advantages, including fast and convenient detection, rapid response, high sensitivity, and good selectivity, making fluorescence detection one of the effective tools for mercury ion detection in complex environments. In recent years, much work has been devoted to the development and design of small molecule fluorescent probes for the detection of mercury ions. As typical reactive fluorescent probes, the use of response mechanisms such as spirolactam ring opening reaction, hydroxymercury reaction, desulfurization / desalting reaction, hydrolysis reaction, and mercury ion-induced displacement reaction combined with various high-performance organic fluorescent substances is a necessary condition for achieving accurate identification of mercury ions in complex systems using fluorescent probes.

[0004] More importantly, the fluorescent group, as the entity that provides the fluorescent signal response, directly affects the sensitivity of the probe sensing detection and its imaging potential in biological applications. Since small molecule organic fluorescent probes may aggregate when detecting mercury ions in water and biological systems, the fluorescence signal is quenched. In recent years, the discovery of fluorophores with aggregation-induced emission (AIE) characteristics has solved the quenching problem of fluorescent probe detection in water. To date, several fluorescent probes have been developed based on the typical AIE fluorophore tetraphenylethylene (TPE) as a fluorescent carrier to detect mercury ions, but the autofluorescence background signal generated in the visible light region in complex biological systems may interfere with the detection results. In addition, a fluorescence response with a large Stokes shift (>100nm) can further reduce the interference caused by the excitation light source and self-absorption, and improve the signal-to-noise ratio of imaging detection. Therefore, it is challenging to develop a fluorescent probe with AIE characteristics that can detect mercury ions in the red to near-infrared region (>600nm). Summary of the Invention

[0005] The present invention provides a novel AIE fluorescent probe, a preparation method thereof and its detection of Hg 2+ Fluorescence detection technology was used.

[0006] The chemical structure of the fluorescent probe of the present invention is shown in formula (I):

[0007]

[0008] The novel AIE fluorescent probe of the present invention is characterized by Hg 2+ The probe is induced to undergo thiometallate deprotection reaction and molecular aggregation changes, realizing red and near-infrared fluorescence response detection. Since the spatial arrangement of methyl thioglycolate affects the aggregation state between probe molecules, the probe has almost no fluorescence emission. 2+ Induced thioketone reaction, the probe reacts with Hg 2+ The combination generates a fluorenone dye structure, which triggers the AIE fluorescence of the fluorenone dye in a high water content system for rapid detection of Hg 2+ In the test system CH3CN / H2O (v / v, 3 / 7), the probe has strong orange fluorescence at 630nm and has strong detection selectivity and sensitivity, and can be used for naked eye qualitative identification and fluorescence quantitative detection.

[0009] The present invention also provides a method for preparing the novel AIE fluorescent probe, comprising the following steps:

[0010] (1) 3,6-Dibromofluorenone and 4,4'-dimethyldiphenylamine are catalyzed and treated after the reaction to obtain a fluorenone dye.

[0011] (2) Fluorenone dye and methyl thioglycolate are reacted by addition reaction and then treated to obtain a probe.

[0012] The preparation chemical reaction formula is as follows

[0013]

[0014] The present invention also provides a novel AIE fluorescent probe for Hg 2+ spectral response and cell imaging applications.

[0015] The novel AIE fluorescent probe of the present invention is in CH3CN / H2O (v / v, 3 / 7), such as Figure 4 As shown, when Hg 2 + After that, the fluorescence response is turned on and a new peak of fluorescence emission appears at 630nm. 2+ In addition, if Figure 5 As shown, the emission intensity (I 630nm ) and Hg 2+ The concentration range was 1 to 6 μM, and the detection limit was 17 nM according to the 3σ / k method, indicating that the probe DS was sensitive to Hg. 2+ The detection has high sensitivity.

[0016] The novel AIE fluorescent probe of the present invention has high selectivity. In CH3CN / H2O (v / v, 3 / 7), different interfering substances (including Zn 2+ , K + 、Cu 2+ 、Ni 2+ , Ca 2+ Mg 2+ 、Co 2+ 、Bi 2+ 、Cd 2+ 、In 3+ 、Na + 、Fe 3+ 、Mn 2+ 、Li + 、Ag + , Cys) to detect its selectivity, such as Figure 7 As shown. Among them, Hg 2+ The fluorescence of the probe was significantly enhanced, while other interfering substances did not cause significant changes in fluorescence intensity, indicating that DS has a significant effect on Hg 2+ The selectivity is higher than other interferences.

[0017] The novel AIE fluorescent probe of the present invention has high competitiveness. In CH3CN / H2O (v / v, 3 / 7), different interfering substances (including Zn 2+ , K + 、Cu 2+ 、Ni 2+ , Ca 2+ Mg 2+ 、Co 2+ 、Bi 2+ 、Cd 2+ 、In 3+ 、Na + 、Fe 3+ 、Mn 2+ 、Li + 、Ag + , Cys) and then Hg 2+ To test its competitiveness. Figure 8 As shown, the probe can still accurately identify Hg in the presence of interferences 2+ The fluorescence of the probe was significantly enhanced, indicating that DS was sensitive to Hg 2+ There are specific tests.

[0018] The novel AIE fluorescent probe of the present invention detects Hg in living cells 2+ Detection imaging application. After the probe (10μM) was incubated in A549 cells for 30 minutes, a weak fluorescence signal was observed in the red channel (570-670nm). 2+ When the cells were treated with Hg (0-20 μM), and then incubated with the probe (10 μM) for 30 minutes, a strong fluorescence signal was detected. 2+ As the concentration increases, the fluorescence signal increases. Figure 9 shown.

[0019] The novel AIE fluorescent probe of the present invention is used for Hg 2+ Detection imaging application. After the probe (10μM) was incubated in the roots of Ophiopogon japonicus and rice for 30 minutes, a weak fluorescence signal was observed in the red channel (570-670nm). 2+ (10 μM) was used to treat Ophiopogon japonicus and rice roots for 1 hour, and then treated with the probe (10 μM) for 30 minutes, and the fluorescence signal was significantly enhanced. Figure 10 shown.

[0020] The beneficial effects of the present invention are: the new AIE fluorescent probe can detect Hg with high selectivity and sensitivity in the red and near-infrared fluorescence regions. 2+ With the Hg 2+ With the increase of concentration, DS showed strong fluorescence, high selectivity and low detection limit (17nM). In addition, the probe DS can detect Hg in living cells and plants in the red and near-infrared range.2+ Capable of significant imaging detection. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 This is the synthesis route of the new AIE fluorescent probe of the present invention.

[0022] Figure 2 The novel AIE fluorescent probe of the present invention is for Hg 2+ responsive sensing mechanism.

[0023] Figure 3 The new AIE fluorescent probe of the present invention is sensitive to different concentrations of Hg 2+ Absorption change spectrum after action.

[0024] Figure 4 The new AIE fluorescent probe of the present invention is sensitive to different concentrations of Hg 2+ Fluorescence emission change spectrum after action.

[0025] Figure 5 The new AIE fluorescent probe of the present invention is sensitive to different concentrations of Hg 2+ (1-6 μM) after the effect of fluorescence ratio changes (I 630nm ) spectrum.

[0026] Figure 6 The novel AIE fluorescent probe of the present invention is 2+ (10μM) after the action of fluorescence emission versus time.

[0027] Figure 7 This is the fluorescence change spectrum of the new AIE fluorescent probe of the present invention after acting on different substances.

[0028] Figure 8 The novel AIE fluorescent probe of the present invention is for Hg 2+ When it interacts with other substances, the fluorescence intensity (I 630nm ).

[0029] Figure 9 This is the fluorescence imaging of the new AIE fluorescent probe of the present invention in A549 cells.

[0030] Figure 10 This is the fluorescence imaging of plants by the new AIE fluorescent probe of the present invention. DETAILED DESCRIPTION

[0031] Example 1

[0032] 2,7-Dibromo-9-fluorenone (1, 1.1 g, 5 mmol) and 4,4'-dimethyldiphenylamine (2, 3.9 g, 20 mmol) were dissolved in 80 mL of toluene. Pd(OAc)2 (60 mg, 0.5 mmol), P-tBu (0.72 g, 0.35 mmol), and NaOtBu (2.89 g, 30 mmol) were added to this solution. The reaction solution was heated at 140°C under Ar protection for 48 hours. The crude product was purified by silica gel column chromatography using petroleum ether / dichloromethane as eluent. The desired dye DO was obtained as a red powder in a 40% yield (1.1 g). 1 HNMR (400MHz, CDCl3) δ7.57(d,J=8.4Hz,2H),7.10(d,J=8.0Hz,8H),7.02(d,J=8.0Hz,8H),6.87(s,2H),6.64(d,J=8.4Hz,2H),2.33(s,12H). 13 C NMR(101MHz, CDCl3)δ190.9,153.6,145.2,144.2,134.1,130.2,127.9,125.5,125.0,120.1,112.3,20.9.MS(ESI):calcd for C 41 H 34 N2O[M+H] + 571.2744,found 571.2742.

[0033] Dye DO (275 mg, 0.52 mmol) was dissolved in anhydrous dichloromethane (10 mL), and methyl thioacetate (120 μL, 1.25 mmol) and p-toluenesulfonic acid (49 mg, 0.26 mmol) were added to the solution. The mixture was heated at 50°C for 4 hours. After the reaction was completed, ethyl acetate (50 mL) and water (30 mL) were added. The organic layer was collected, dried over anhydrous sodium sulfate, concentrated under reduced pressure, and purified by silica gel column chromatography using petroleum ether / dichloromethane as eluent to obtain probe DS (white powder, 233 mg, 60% yield). 1 H NMR (400MHz, CDCl3): δ7.41(d,J=8.4Hz,2H),7.20(s,2H),7.05(d,J=7.2Hz,8H),6. 98(d,J=8.4Hz,8H),6.91(d,J=8.4Hz,2H),3.58(s,6H),3.18(s,4H),2.31(s,12H); 13C NMR (101MHz, CDCl3): δ169.8,149.5,145.2,140.2,138.3,132.8,130.0,125.3,124.6,122.9,114.7,62.0,52.4,33.9,20.8.MS(ESI):calcd for C 47 H 44 N2O4S2[M+Na] + 787.2635, found 787.2621.

[0034] Example 2

[0035] The probe reacts to different concentrations of Hg 2+ Spectral changes: The probe was added to the CH3CN / H2O (v / v, 3 / 7) test solution to prepare a solution with a concentration of 10μmol / L, and then different concentrations of Cys aqueous solutions were added. After equilibrium, the absorption and fluorescence emission spectra were measured respectively. The results are shown in Figure 3 and Figure 4 .

[0036] from Figure 3 and 4 Learned that joining Hg 2+ After that, the signal peak of fluorenone dye appeared at around 630nm, and the color of the solution changed from colorless to yellow. At the same time, the fluorescence emission of the probe at 630nm was significantly enhanced, indicating that the probe was sensitive to Hg 2+ There was a clear response.

[0037] Example 3

[0038] The probe was added to the CH3CN / H2O (v / v, 3 / 7) test solution to prepare a solution with a concentration of 10 μmol / L, and then 10 μmol / L of Hg 2+ , record the fluorescence spectra at different times, such as Figure 6 shown.

[0039] The results are Figure 6 It can be seen that as time goes by, the fluorescence intensity of the probe I 630nm The probe can be used to detect Hg 2+ fluorescent probes.

[0040] Example 4

[0041] The detection selectivity of the new AIE fluorescent probe was tested: the probe was added to a CH3CN / H2O (v / v, 3 / 7) test solution to prepare a solution with a concentration of 10 μmol / L, and then various interfering substances were added, including Zn 2+ , K + 、Cu2+ 、Ni 2+ , Ca 2+ Mg 2+ 、Co 2+ 、Bi 2+ 、Cd 2+ 、In 3+ 、Na + 、Fe 3+ 、Mn 2+ 、Li + 、Ag + , Cys, and test the changes in their fluorescence intensity.

[0042] Depend on Figure 7 It can be seen that the fluorescence intensity of the probe is only for Hg 2+ There is a big change, and the influence of other substances is very weak, indicating that the probe is sensitive to Hg 2+ The selectivity is higher than other substances.

[0043] Example 5

[0044] Intracellular fluorescence imaging test: A549 cells were transferred to a glass bottle for confocal imaging and incubated for 24 hours. The experimental group was incubated with the probe (10 μM) solution for 30 minutes, and then washed three times with PBS for confocal cell imaging. 2+ The cells were pretreated with the probe (0-20 μM) solution for 30 minutes, and then incubated with the probe (10 μM) solution for 30 minutes. After each operation, the cells were washed three times with PBS for confocal cell imaging. The red channel of the probe was collected in the emission range of 570-670 nm using 488 nm excitation. Figure 9 shown.

[0045] like Figure 9 As shown in the figure, when the probe is incubated in cells, almost no fluorescence signal is generated. 2+ As the concentration increased, the fluorescence signal in the cells was significantly enhanced.

[0046] Example 6

[0047] Plant fluorescence imaging test: Ophiopogon rhizomes in the presence of 10 μM Hg 2+ Soak in aqueous solution for 1 hour. The middle part of the rhizome was cut into thin slices and soaked in the probe solution (10μM) for 30 minutes. The slices were washed three times with PBS and imaged on an Olympus FV3000 confocal fluorescence microscope. Fluorescence channel signals were collected from 570nm to 670nm under 488nm excitation. The rice seeds were first washed three times with deionized water. After soaking in water for 6 hours, the seeds were placed on wet filter paper on a culture dish and germinated in a dark incubator at 28°C for 48 hours. The germinated seeds were transferred to the mud and grown until ready for imaging application. Rice roots were exposed to 0 and 10μM Hg2+ The seeds were incubated in the solution for 1 hour. After washing with PBS, they were incubated in the probe solution for 1 hour and then confocal imaging was performed. Figure 10 shown.

[0048] like Figure 10 As shown, the plants treated with the probe showed almost no fluorescence signal. 2+ The treated plants showed strong fluorescence signals, proving that the probe can be used for Hg 2+ imaging.

Claims

1. An AIE fluorescent probe based on fluorenone dye, characterized in that: The structural formula of the AIE fluorescent probe is as follows: 。 2. A method for preparing the AIE fluorescent probe according to claim 1, characterized in that: The following steps are involved: (1) 3,6-dibromofluorenone and 4,4'-dimethyldiphenylamine are reacted by substitution and then treated to obtain a fluorenone dye; (2) Fluorenone dye and methyl thioglycolate undergo addition reaction and then undergo treatment after the reaction to obtain the AIE fluorescent probe.

3. Use of the AIE fluorescent probe according to claim 1 in the preparation of a mercury ion detection reagent.

4. The use of the AIE fluorescent probe according to claim 3 in preparing a mercury ion detection reagent, characterized in that The mercury ion detection reagent is 2+ spectral response.

5. Use of the AIE fluorescent probe according to claim 3 or 4 in preparing a mercury ion detection reagent, characterized in that, The mercury ion detection reagent is used for Hg 2+ The specific method for qualitative detection is as follows: The AIE fluorescent probe is configured into a test solution, and then the sample to be tested is added. Then, the change in the signal peak intensity around 630 nm is detected, and combined with the change in the solution color, it is determined whether Hg is present in the test solution. 2+ .

6. Use of the AIE fluorescent probe according to claim 3 or 4 in preparing a mercury ion detection reagent, characterized in that The mercury ion detection reagent is effective for Hg 2+ The specific method for detecting the spectral response is as follows: (1) Prepare the fluorescent probe into a test solution and add different concentrations of Hg 2+ , observe the changes in its fluorescence spectrum; (2) Calculate Hg 2+ The detection limit of the fluorescent probe for Hg was calculated based on the ratio of the concentration to the fluorescence intensity at 630 nm and the 3σ / k method. 2+ sensitivity; (3) The fluorescent probe is prepared into a test solution, and then the sample to be tested is added, and the Hg content in the sample to be tested is obtained according to the change of its fluorescence spectrum. 2+ content.

7. Use of the AIE fluorescent probe according to claim 6 in preparing a mercury ion detection reagent, characterized in that The test solution was prepared as follows: A mixed solvent of CH3CN / H2O was added to the fluorescent probe to prepare a test solution with a concentration of 10 μmol / L; The volume ratio of CH3CN and H2O in the mixed solvent is 3:

7.

8. Use of the AIE fluorescent probe according to claim 3 in preparing a mercury ion detection reagent, characterized in that The mercury ion detection reagent is used for intracellular fluorescence imaging testing, and the specific method is as follows: the cells to be tested are incubated with fresh FBS-free culture medium containing the probe, then washed three times with PBS, and subjected to confocal cell imaging detection; For confocal cell imaging, the excitation wavelength was 488 nm, and the red channel collection wavelength was 570–670 nm; Under 488 nm excitation, if a strong fluorescent signal is displayed in the cell, it indicates that Hg is present in the cell. 2+ .

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