Fluorescent probe for detecting metal mercury ions as well as preparation method and application of fluorescent probe

Abstract

The invention discloses a fluorescent probe for detecting metal mercury ions and a preparation method and application thereof, the molecular formula of the probe is C21H20O5N2S, and the structural formula is shown in the specification. The invention designs a fluorescent probe for detecting metal mercury ions (Hg < 2 + >) aiming at environmental Hg < 2 + > monitoring requirements. The probe can rapidly react with Hg < 2 + > (response time is 1t, 10 s), and has nanomole-level detection sensitivity (detection limit is 1.0 nM) and excellent anti-interference capability. The method is applied to detection of mercury ions (Hg < 2 + >) in real water samples, crop soil, plant root systems and in-vitro living cells. And a novel tool is provided for providing intermediate information for pathological research of Hg < 2 + > related diseases and evaluating environmental toxicity.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of fluorescent probes, and particularly relates to a fluorescent probe for detecting metal mercury ions and a preparation method and application thereof. BACKGROUND

[0002] Mercury ion (Hg 2+ ) is one of the most toxic heavy metal pollutants in the environment, mainly from chlor-alkali industrial wastewater, gold mining, coal combustion and the use of mercury-containing pesticides. Hg 2+ The bioaccumulation effect in the ecosystem can cause food chain disorder and pose a serious threat to environmental safety (such as the Minamata disease incident in Japan). The surface water environmental quality standard (GB 3838-2002) of China stipulates that the Class I water quality limit of Hg 2+ is 0.00005 mg / L, therefore, it is of great significance to develop a high-sensitivity and high-selectivity Hg 2+ detection method.

[0003] At present, the conventional detection methods of Hg 2+ in environmental samples include: (1) Instrument analysis method: atomic fluorescence spectrometry (AFS), cold vapor atomic absorption spectrometry (CVAAS) and the like, although the detection limit can reach ng / L level, but it needs large instruments, and the sample pretreatment (such as acid digestion, distillation) is complex, and real-time detection on site cannot be realized. (2) Electrochemical method: anodic stripping voltammetry (ASV) has the advantage of portability, but it is easily interfered by other heavy metal ions (such as Cu 2+ , Pb 2+ ) in the environment, and the detection stability is insufficient in complex matrix (such as water and soil leaching liquid containing humic acid); (3) Test strip method: although the test strip based on colloidal gold or enzyme coloration is simple to operate, the sensitivity is low (the detection limit is usually >10 μg / L), and it cannot meet the monitoring demand of trace Hg 2+ . In addition, the traditional method is difficult to realize the dynamic monitoring of the spatial distribution of Hg 2+ (such as pollution gradient analysis of river section and positioning of soil mercury pollution hot spot), which limits the accuracy of environmental risk assessment.

[0004] The fluorescence detection technology has become a new means of environmental Hg 2+ monitoring due to the following characteristics: high sensitivity and selectivity: the fluorescent probe is specifically combined with Hg 2+ through molecular recognition mechanism, and the detection limit can reach nM level (such as 1nM = 0.201 μ g / L), which meets the requirements of surface water and drinking water quality monitoring. However, the existing environmental Hg 2+ fluorescent probe still has technical bottlenecks: insufficient anti-matrix interference ability: high concentration of Cl - , SO4 2-and humus is easy to compete with the probe to bind Hg 2+ , resulting in the attenuation of the detection signal; stability defect: traditional fluorescent probes are prone to photobleaching or structural degradation under natural light, redox conditions or pH fluctuations (such as acid mine drainage, alkaline soil); poor field applicability: most probes need to be used in laboratory conditions, and there is a lack of anti-interference detection scheme suitable for field sampling (such as in-situ detection of Hg 2+ in sediments).

[0005] Therefore, how to develop a mercury ion fluorescent probe with short reaction time, easy operation and good interference ability is a technical problem to be solved at present. SUMMARY

[0006] The purpose of the present application is to provide a fluorescent probe for detecting metal mercury ions (Hg 2+ ), hereinafter referred to as P-Hg, which has fast response, high sensitivity and high selectivity for detecting mercury ions (Hg 2+ ), and can be applied to the detection of mercury ions (Hg 2+ ) in real water samples, crop soils, plant root systems and living cells.

[0007] To achieve the above technical purpose, the technical scheme adopted by the present application is: A fluorescent probe for detecting metal mercury ions, the probe molecule formula is C 21 H 20 O5N2S, the structural formula is: .

[0008] A preparation method of a fluorescent probe for detecting metal mercury ions, comprising the following steps: (1) synthesis of intermediate compound 2: in a 100ml round-bottom flask, 1.0g 3-hydroxy phthalic anhydride, 0.78ml N-(2-aminoethyl) morpholine and 30ml glacial acetic acid are added, heated to reflux at 120℃ and stirred overnight, then the reaction mixture is cooled, and then the glacial acetic acid is evaporated, and the obtained crude solid is separated by silica gel column chromatography to obtain intermediate compound 2, and the structural formula of compound 2 is: ; (2) synthesis of probe: the round-bottom flask is placed in an ice bath, and 0.380g compound 2 (3-hydroxy-2-(2-morpholinoethyl) isoindoline-1,3-dione), 0.202ml benzylthiochloroformate, 0.243ml triethylamine and 15ml dichloromethane are added in turn, stirred at room temperature for 3h, and then column chromatography is used for separation and purification to obtain white solid, which is the target product probe.

[0009] The reaction route is as follows:

[0010] The present application probe is used for detecting mercury ion (Hg 2+ ) in solution, the fluorescent probe itself has no fluorescence, reacts with Hg 2+ to generate an intermediate, produces strong green fluorescence, the emission wavelength is at 510 nm, the linear range is 0-1.0 μM, the detection limit is 1.0 nM, the probe shows very high sensitivity; the fluorescent probe reacts with mercury ion (Hg 2+ ) rapidly, the response time is within 10 seconds; the fluorescent probe shows very good selectivity to mercury ion (Hg 2+ ) and is not affected by cations and anions.

[0011] The application of a fluorescent probe for detecting mercury ion is used for detecting the content of mercury ion in real water sample, crop soil, plant root system and in-vitro living cells.

[0012] The specific simulation verification application method is as follows: (1) Detection of mercury ion (Hg 2+ ) in real water sample: different concentrations (0, 5, 10, 15, 20, 50 μM) of mercury ion (Hg 2+ ) are added into tap water respectively, then the probe is incubated, and strong green fluorescence is observed.

[0013] (2) Detection of mercury ion (Hg 2+ ) in crop soil: different concentrations of mercury ion (0, 5, 10, 15, 20, 50 μM) are sprayed on the surface of soil, then the probe (10 μM) solution is sprayed, and strong green fluorescence is observed under ultraviolet lamp irradiation.

[0014] (3) Detection of mercury ion (Hg 2+ ) in plant root system: the root systems of peppers, tomatoes and eggplants are soaked in mercury ion (50 μM) solution for 30 min, washed for 2 times, then the root systems of peppers, tomatoes and eggplants are soaked in the probe (10 μM) solution, washed for 2 times, and strong green fluorescence is observed under ultraviolet lamp irradiation.

[0015] (4) Detection of mercury ion (Hg 2+ ) in living cells: different concentrations (0, 5, 10 μM) of mercury ion (Hg 2+ ) are added into cells, then the probe is incubated, and strong green fluorescence is observed.

[0016] These phenomena show that the fluorescent probe can not only detect mercury ion (Hg 2+ ) in water solution, crop soil and plant root system, but also can be applied to detect the content of mercury ion (Hg 2+ ) in in-vitro cells, which is helpful for in-depth study of mercury ion (Hg 2+It is of great significance to study the physiological and pathological processes of organisms by obtaining the intermediate information in the environment and organisms.

[0017] Beneficial effects: The present application is directed to environmental Hg 2+ Monitoring needs, a fluorescent probe for detecting metal mercury ions (Hg 2+ ) is designed. The probe can quickly (response time <10 s) after reaction with Hg 2+ It has nanomolar detection sensitivity (detection limit of 1.0 nM) and excellent anti-interference ability. It is applied to the detection of mercury ions (Hg 2+ ) in real water samples, crop soils, plant root systems and isolated living cells. It provides a new tool for the pathological study of Hg 2+ related diseases and environmental toxicity evaluation. BRIEF DESCRIPTION OF DRAWINGS

[0018] Figure 1 The single crystal graph of the probe of the present application is shown in the figure; Figure 2 The hydrogen spectrum of the probe is shown in the figure; Figure 3 The carbon spectrum of the probe is shown in the figure; Figure 4 The selective fluorescence spectrum of the probe of the present application for different dry ions is shown in the figure; Figure 5 The selective fluorescence column chart of the probe of the present application for different ions is shown in the figure; Figure 6 The fluorescence response graph of the probe of the present application under different pH conditions is shown in the figure; Figure 7 The anti-interference test graph of the probe of the present application is shown in the figure; Figure 8 The fluorescence spectrum of the probe of the present application after interaction with different concentrations of mercury ions (Hg 2+ ) is shown in the figure; wherein the abscissa is wavelength and the ordinate is fluorescence intensity. The concentration of the fluorescent probe is 2 μM, and the concentration of mercury ions (Hg 2+ ) is 0, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0 μM, respectively. The fluorescence excitation wavelength is 330 nm; Figure 9 The linear fitting graph of the fluorescence response value of the probe for mercury ions (Hg 2+ ) and the concentration of mercury ions (Hg 2+ ) is shown in the figure; Figure 10 The time response fluorescence spectrum graph of the probe of the present application before and after adding mercury ions (Hg 2+ ) is shown in the figure; Figure 11 The fluorescence spectrum of the probe of the present application and mercury ions (Hg2+ ) Mechanism of action (A) and the probe and mercury ions (Hg 2+ ) Mass spectrometry changes before and after the action (B) ; Figure 12 The probe of the present application and different concentrations of mercury ions (Hg 2+ ) Effect after the action under visible light and ultraviolet light; Figure 13 The probe of the present application and different concentrations of mercury ions (Hg 2+ ) Fluorescence under ultraviolet light after the action; Figure 14 The probe of the present application and different concentrations of mercury ions (Hg 2+ ) Fluorescence under ultraviolet light after the action; Figure 15 The probe of the present application and different concentrations of mercury ions (Hg 2+ ) Fluorescence imaging of the probe in liver cancer Huh7 cells and exogenous ions. DETAILED DESCRIPTION

[0019] The technical solutions of the present application will be further described below in conjunction with specific examples, but are not limited thereto.

[0020] Example 1 Probe synthesis: A preparation method of a fluorescent probe for detecting mercury ions, comprising the following steps: (1) Synthesis of intermediate compound 2: 3-hydroxyphthalic anhydride (1.0 g, 0.006 mol), N-(2-aminoethyl) morpholine (0.78 mL, 0.006 mol) and 30 mL of glacial acetic acid were added to a 100 mL round-bottom flask, heated to reflux at 120°C and stirred overnight, then the reaction mixture was cooled, and the glacial acetic acid was evaporated, and the obtained crude solid was separated by silica gel column chromatography to obtain intermediate compound 2, and the structural formula of compound 2 is: , yield 83%; (2) Synthesis of probe: the round-bottom flask was placed in an ice bath, and compound 2 (0.380 g, 0.0013 mol) (3-hydroxy-2-(2-morpholinoethyl) isoindoline-1,3-dione), phenylthiochloroformate (0.202 mL, 0.0015 mol), triethylamine (0.243 mL, 0.0016 mol) and 15 mL of dichloromethane were added in turn, stirred at room temperature for 3 h, and purified by column chromatography to obtain white solid, which is the target product probe, with a yield of 61%. The single crystal graph of the target probe P-Hg is shown in Figure 1 , and the carbon spectrum and hydrogen spectrum are shown in Figures 2-3 .

[0021] The crystal data of the probe P-Hg are shown in Tables 1-3.

[0022] Table 1 Crystal data of probe P-Hg

[0023] Table 2 shows the crystal bond length data for the probe P-Hg.

[0024] Table 3 shows the crystal bond angle data for the probe P-Hg.

[0025] Example 2 Fluorescent probe and mercury ions (Hg) 2+ Preparation of the stock solution: Accurately weigh the solid probe using an analytical balance and dissolve it in DMSO solution to obtain a 10 mM stock solution. During analytical experiments, dilute the stock solution to a specific concentration as needed.

[0026] Example 3 Fluorescence spectroscopy determination of the interaction between the fluorescent probe and different anions / cations: Fluorescence spectra of the interaction between the fluorescent probe and different anions / cations were measured using 10 mM PBS buffer solution (pH 7.4) as the solvent. The concentration of the fluorescent probe was 10 μM, the concentration of different anions / cations was 30 equivalents, the excitation wavelength was 330 nm, and the fluorescence emission peaks in the range of 400 nm to 650 nm were collected. From the results (… Figure 2 It can be seen that the addition of different anions / cations only affects mercury ions (Hg). 2+ After the addition of [the substance], the emission peak at 510 nm was significantly enhanced. The probe [is effective against] mercury ions (Hg). 2+ It exhibits good selectivity. Figure 4 The fluorescence values ​​of different ions at 510 nm were plotted as the ordinate and the different ions as the abscissa to obtain a fluorescence histogram of the interaction between the fluorescent probe and different anions / cations. Figure 5 ).

[0027] Example 4 Fluorescent probes and mercury ions (Hg) under different pH conditions 2+ Fluorescence spectroscopy of the response: Using 10 mM PBS buffer solution at pH 7.4 as the solvent, the fluorescence probe and mercury ions (Hg) were measured under the same pH conditions. 2+ The fluorescence spectrum of the response is shown in the figure. The concentration of the fluorescent probe is 10 μM, and the mercury ion concentration is 10 μM. 2+ The concentration of ) is 30 equivalents. The pH ranges from 4 to 12. Figure 6 As can be seen, a strong fluorescence response is observed at pH 4-9.

[0028] Example 5 Determination of the anti-interference ability of the fluorescent probe: Other ions (100 μM) were added separately to the fluorescent probe (10 μM) solution, shaken well, and then mercury ions (Hg) were added. 2+ (300 μM), the changes in fluorescence spectrum were measured. The results showed that the probe exhibited strong green fluorescence in the presence of other ions, was unaffected by other ions, and had good anti-interference ability. Figure 7 ) Example 6 Fluorescent titration experiment: Different equivalents of mercury ions (0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0 equivalents) were added to the fluorescent probe (10 μM) solution, and the reaction between the fluorescent probe and mercury ions (Hg) was determined under different equivalents of mercury ions. 2+ The fluorescence spectrum of the response () Figure 8 The fluorescence value at 510 nm was then fitted to the mercury ion concentration. Figure 9 According to the minimum detection limit (LOD) = 3σ / K Calculated, among which, K σ is the slope after fitting, and σ is the standard deviation.

[0029] Example 6 Fluorescent probe and mercury ions (Hg) 2+ Determination of the response time of the fluorescent probe: In order to study the response time of the fluorescent probe to mercury ions (Hg) 2+ The response time of the fluorescent probe (10 μM) to mercury ions (Hg) was investigated. 2+ The changes in fluorescence spectra at (300 μM) were observed, and the results showed that ( Figure 10 This probe is effective against mercury ions (Hg). 2+ The response time is less than 10 s, which meets the response time requirements for real-time monitoring in actual samples. The fluorescence intensity at 510 nm remains essentially unchanged as time increases.

[0030] Example 7 Mercury ion testing in real water samples: Different concentrations of mercury ions (0, 5, 10, 15, 20, 50 μM) were added to tap water, followed by the addition of a probe solution (10 μM). The fluorescence of the different water samples under ultraviolet light was then detected. The results showed ( Figure 12 As the concentration of mercury ions increases, the fluorescence intensity gradually increases.

[0031] Example 8 Mercury ion testing in soil: Different concentrations of mercury ions (0, 5, 10, 15, 20, 50 μM) were sprayed onto the soil surface, followed by the spraying of a probe solution (10 μM). The fluorescence of the soil surface under ultraviolet light was then detected. The results showed (Figure 13 ) The soil fluorescence intensity gradually increased with the increase of mercury ion concentration.

[0032] Example 9 Mercury ion test in vegetable root system: Pepper, tomato and eggplant root system were soaked in mercury ion (50 μM) solution for 30 min, washed twice, and then soaked in probe (10 μM) solution, washed twice. Finally, the fluorescence imaging of different root systems under ultraviolet light was detected, and the results showed that Figure 14 ) Pepper, tomato and eggplant root system showed strong green fluorescence.

[0033] Example 10 Fluorescence imaging study of probe with exogenous mercury ion (Hg 2+ ) in liver cancer cell Huh 7 cell: Before the experiment, dead cells were washed away with PBS buffer solution, and then the probe diluted in PBS buffer solution (10 μM) was incubated with Huh 7 cells for 20 min, and then the cells were washed with PBS buffer solution for 3 times, and then 0, 5, 10 μM of mercury ion (Hg 2 + ) was added respectively, and incubated for 20 min, and finally cell imaging was performed by inverted fluorescence microscope. The results showed that only the probe group was observed very weak green fluorescence. With the increase of exogenous mercury ion (Hg 2+ ) concentration, the green channel fluorescence gradually increased. The experimental results showed that the probe could detect exogenous mercury ion (Hg 2+ ) in Huh 7 cells by fluorescence microscope. Figure 15 ) It should be noted that the above examples are only part of the preferred mode of implementing the present application, not all. Obviously, based on the above examples of the present application, all other examples obtained by those of ordinary skill in the art without creative labor should belong to the scope of protection of the present application.

Claims

1. A fluorescent probe for detecting metallic mercury ions, characterized in that, The probe molecule is of formula C 21 H 20 O5N2S, and has the structure: .

2. A method for preparing the fluorescent probe for detecting metallic mercury ions according to claim 1, characterized by, Comprising the following steps: (1) Synthesis of intermediate compound 2: In a 100 ml round bottom flask was added 1.0 g of 3-hydroxyphthalic anhydride, 0.78 mL of N-(2-aminoethyl)morpholine and 30 mL of glacial acetic acid. The reaction mixture was heated to reflux and stirred overnight at 120 °C. The reaction mixture was cooled and the glacial acetic acid was evaporated. The resulting crude solid was separated by silica gel column chromatography to obtain intermediate compound 2, which has the following structural formula: ; (2) Synthesis of probe: Put the round-bottom flask in ice bath, add 0.380 g of compound 2, 0.202 mL of benzylthiochloroformate, 0.243 mL of triethylamine and 15 mL of dichloromethane in turn, stir at room temperature for 3 h, separate and purify by column chromatography to obtain white solid, which is the target product probe.

3. Use of the fluorescent probe for detecting metallic mercury ions according to claim 1, characterized in that, For detecting the content of mercury ions in real water samples, crop soil, plant root system and in vitro living cells.

Images