WO2011137574A1

WO2011137574A1 - Fluoride-boron dye fluorescent probe for detecting mercury ion

Info

Publication number: WO2011137574A1
Application number: PCT/CN2010/001973
Authority: WO
Inventors: 彭孝军; 樊江莉; 王嵩; 李宏林; 胡明明; 宋克东
Original assignee: 大连理工大学
Priority date: 2010-05-05
Filing date: 2010-12-06
Publication date: 2011-11-10
Also published as: CN101851500A; CN101851500B

Abstract

A fluorescent probe and its combination are disclosed. They have excitation and emission wavelengths within visible range, high sensitivity, and good selectivity for mercury ion when pH is 5-12, and can be used to detect mercury ion in water sample. There is no interference of Na, K, Ca, Mg, Cu, chloride, nitrate and sulfate ions for the detection. In ethanol-HEPES buffer solution, the ion concentration and fluorescence intensity are in good linear relation within mercury ion concentration of 2-12 ppb.

Description

Fluorine-boron dye fluorescent probe for mercury ion detection

Technical field

The invention relates to a fluorescent boron dye fluorescent probe for detecting mercury ions, which belongs to the fluorescent molecular probe suitable for detecting mercury ions in natural water samples and biological cells in the field of fine chemicals.

Background technique

When thermometers and batteries become essential daily necessities for every family, mercury products have penetrated into every corner of people's lives. The use of mercury products due to improper use of mercury products or mercury contamination due to improper handling of mercury leaks has emerged. There are many other ways to cause mercury pollution, such as volcanic eruptions, the consumption of mineral fuels, especially the mining of mercury mines, causing serious mercury pollution, and the problems of harm to the ecological environment and human health have become increasingly serious!

In recent years, fluorescent molecular probe detection technology with high sensitivity, high selectivity, simple and rapid development has developed rapidly, and its application in elemental analysis has become more and more extensive. There are many fluorescent molecular probes capable of detecting mercury ions, such as rhodamine-based fluorescent molecular probes (Chen XQ, Nam SW, Jou MJ, et al., Org. Lett, 2008, 10, 5235-5238.), fluorescence A probe that is a fluorescent precursor and a crown ether is a recognition group (Yoon S, Albers AE, Wong AP, et al., J. Am. Chem. Soc., 2005, 127, 16030-16031.), based on fluorescence resonance Ratio-type fluorescent molecular probe designed by energy transfer mechanism (FRET) (Zhang XL, Xiao Y, Qian X Η, Angew. Chem., Int. Ed" 2008, 47, 8025-8029.) and ion-catalyzed hydrolysis type fluorescence probe Needle (Santra M, Ryu D, Chatterjee A, et al., Chem. Commun., 2009, 21 15-21 17). Due to the sulphur-purifying nature of mercury ions, most of the identification groups of these probes contain sulfur. Although this improves the complexing ability of the ligand with mercury ions, it also reduces the selectivity of the ligand, because the sulfur-containing groups are often complexed with elements such as lead and silver. Secondly, most of these probes are tested. They are all carried out under laboratory conditions, but the actual application in natural environmental conditions is rare because of natural environmental conditions. Miscellaneous, the water solubility, selectivity, and sensitivity of the probe are extremely challenging. In addition, some molecules in the organism contain sulfhydryl groups, which can form stable complexes with mercury ions, but few literatures explore these organisms. The effect of molecules on the identification of mercury ions by probes. Therefore, a new type of structure is simple and easy to prepare, highly selective and sensitive, and can be applied under actual natural environmental conditions and biological fineness. Intracellular fluorescent molecular probes remain a challenge.

Summary of the invention

The invention improves the structure and performance deficiencies of the existing complex-type mercury ion fluorescent probes, designs and synthesizes the low-concentration mercury ion detection and the detection of the living cells in the natural water sample, and has the advantages of simple structure and excellent performance. The probe molecule of the fluoroboron fluorescent dye is for the purpose.

The technical solution adopted by the present invention is: A fluoroboron dye fluorescent probe molecule for mercury ion detection has the following structural formula BH _g:

The probe molecule was formulated into a composition for mercury ion detection using an ethanol-HEPES buffer solution.

The method for synthesizing a fluoroboron dye fluorescent probe molecule for mercury ion detection comprises the following steps: 1) reacting 2,4-dimethylpyrrole with 3-hydroxy-4nitro-benzaldehyde: 2, 4- Dimethylpyrrole and 3-hydroxy-4-nitro-benzaldehyde were dissolved in dichloromethane, and a drop of trifluoroacetic acid was added dropwise, followed by stirring at room temperature for 5 hours; the solvent was evaporated under reduced pressure, and dichlorodicylidene was added and stirred. After 15 minutes, a solution of triethylamine and boron trifluoride diethyl ether was added, and stirring was continued for 45 minutes. The reaction solution was washed with water and extracted with dichloromethane, and dichloromethane was evaporated under reduced pressure. Obtained intermediate I:

2) Reduction of the intermediate I to obtain the compound BH _g: Dissolving the intermediate I in ethanol, adding a hydrazine hydrate and a palladium carbon catalyst, and refluxing for 2 hours; after cooling, filtering the solution, and evaporating the ethanol under reduced pressure. It is then redissolved with methylene chloride. The organic layer was washed twice with deionized water; the organic layer solution was dried over anhydrous sodium sulfate, dichloromethane was distilled off under reduced pressure, and the objective product was purified by column chromatography to give a red solid:

Fluorescent dyes obtained by the above technical solutions can be recovered by separation and purification techniques well known in the art to achieve the desired purity. The various starting materials used are either commercially available or can be readily prepared from materials well known in the art by methods well known to those skilled in the art or as disclosed in the prior art. The present invention provides not only a composition comprising the above compound BH _g , which is used for the detection of mercury ions. The composition may be present as an ethanol-HEPES buffer solution, or may be present in other suitable forms prepared as a solution with an ethanol-HEPES buffer solution prior to use; and a composition using the above compound BHg or BHg to detect mercury is also provided. the method of ion, which comprises reacting a compound comprising mercury ions or BH test sample composition in _G BH.

The beneficial effects of the present invention are: The above probe molecules have extremely important application value. In particular, the probe molecule has high detection sensitivity, is insensitive to pH changes, and has excellent anti-interference ability to various metal ions and anions. It can be used not only to detect mercury ions in a sulfur-rich environment, but also to be applied in actual nature. The detection of mercury ions in water samples and the detection of the presence of mercury ions in living cells make such probes extremely useful as reagents for determining changes in mercury ion concentration. From the above description and common knowledge well known to those skilled in the art, various advantages of BODIPY dye molecular fluorescent probes can be understood, including but not limited to the following:

(1) Fluorescence probe molecular excitation and emission spectra in the visible region, high fluorescence quantum yield, solvent Polarity is not sensitive and chemical/light stability is good.

(2) The design of the fluorescent probe molecule is based on the mechanism of the complexation of o-aminophenol with mercury ions. The fluorescence emission of the probe molecules before and after complexing with mercury ions is about 20 times higher. Fluorescent probe molecules have good selectivity for mercury ions, and metal ions such as sodium, potassium, calcium, magnesium, and copper do not interfere with detection. In addition, the fluorescent probe molecules are not sensitive to pH changes, and pH changes have little effect on fluorescence emission in the pH range of 5-12.

(3) Fluorescent probe molecules can detect ppb mercury ion concentration and have a good linear relationship.

(4) Fluorescent probe molecules can detect mercury ions in a sulfur-rich environment without interference.

(5) Fluorescent probe molecules can be used to detect mercury ions in actual natural water samples.

(6) The fluorescent probe has good cell permeability, and has little toxic and side effects on the cells, and can detect mercury ions in living cells.

DRAWINGS

Figure 1 is in ethanol-HEPES (N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid) buffer solution (20 mM HEPES, 100 mM NaN0 ₃ , 1:1, v/v, pH 7.2) The fluorescence intensity of the fluorescent molecular probe BHg is plotted as a function of mercury ion concentration. The concentration of the fluorescent probe molecule BHg is 10, and the concentration of mercury ions changes from 0 to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 20/^4. The abscissa is the wavelength (nm) and the ordinate is the fluorescence intensity. The instrument used was a fluorescence spectrophotometer, model: LS55.

Figure 2 is a diagram showing the selective fluorescence emission of mercury ion by fluorescent molecular probe BHg. The concentration of the fluorescent probe molecule BHg is 10, and the concentration of each metal ion is a fluorescence emission spectrum at a 5-fold equivalent. The abscissa is the wavelength (nm) and the ordinate is the fluorescence intensity. The instrument used was a fluorescence spectrophotometer, model: LS55.

Figure 3 is a fluorescence emission diagram of the fluorescence intensity of the fluorescent probe molecule BHg as a function of pH in an ethanol-HEPES buffer solution (20 mM HEPES, 100 mM NaN0 ₃ , 1:1, v/v, pH 7.2). The abscissa is pH and the ordinate is fluorescence intensity. The concentration of the fluorescent probe molecule BHg is 10. The pH was adjusted with NaOH (1 M) and HCl (1 M). The instrument used was a fluorescence spectrophotometer, model: LS55.

Figure 4 is an interference experiment of various metal ions on fluorescent probe molecule BHg-mercury ion complex in ethanol-HEPES buffer solution (20 mM HEPES, 100 mM NaN0 ₃ , 1:1, v/v, pH 7.2). Add mercury ions to the metal ions and probes other than mercury ions. Fluorescent probe molecule The concentration of BHg is 10. The ion concentration on the abscissa is 5 times the concentration of the probe molecule, and the ordinate is the fluorescence intensity. The instrument used was a fluorescence spectrophotometer, model: LS 55.

Figure 5 is an interference experiment of various anions on the fluorescent probe molecule BHg-mercury ion complex in ethanol-HEPES buffer solution (20 mM HEPES, 100 mM NaN0 ₃ , 1:1, v/v, pH 7.2). Mercury ions are added after adding various anions and probes. The concentration of the fluorescent probe molecule BHg is 10, Μ. The various ion concentrations on the abscissa are 5 times the concentration of the probe molecule, and the ordinate is the fluorescence intensity. The instrument used was a fluorescence spectrophotometer, model: LS 55.

Figure 6 is a graph showing the relationship between the ppb-level concentration of mercury ions and the fluorescence intensity using the fluorescent probe molecule BH _g . The concentration of the fluorescent probe molecule BH _g is 5. The abscissa is the mercury ion concentration and the ordinate is the fluorescence intensity. The instrument used was a fluorescence spectrophotometer, model: LS 55.

Figure 7 is a graph showing the relationship between the fluorescence enhancement factor of BH _{g and} the concentration of mercury ions. The concentration of the fluorescent probe molecule BHg is 10. The abscissa is the mercury ion concentration and the ordinate is the fluorescence enhancement factor. The instrument used was a fluorescence spectrophotometer, model: LS 55.

Figure 8 shows the fluorescence changes of BHg after adding 50ppb mercury ions to each of the three water samples. The abscissa is a different water source and the ordinate is the fluorescence enhancement factor. The instrument used was a fluorescence spectrophotometer, model: LS 55.

Figure 9 shows the study of BHg for identifying mercury ions in a sulfur-rich environment. The concentration of the fluorescent probe molecule BHg was 10^. The abscissa is a different system and the ordinate is the fluorescence intensity. The instrument used was a fluorescence spectrophotometer, model: LS 55.

Figure 10 is an image of the identification of mercury ions in Osteoblasts cells using the fluorescent probe molecule BHg. Figure (a) is a bright field image of BHg added to cultured Osteoblasts cells after incubation for 30 minutes at 37 ° C in medium; Figure (b) shows BHg added to cultured Osteoblasts at 37 ° C An image after incubation for 30 minutes in the medium; (c) An image obtained by adding mercury ions to the probe-containing cell culture solution and incubating at 37 ° C for 30 minutes. The fluorescent probe molecule has a BHg concentration of 10 and a mercury ion concentration of 10 μM. The instrument is Olympus 1X70-131.

detailed description

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C.6100/010ZN3/X3d tLSL£l/U0Z OAV Example 2 The relationship between the fluorescence intensity of the probe BHg and the concentration of mercury ions:

Add BHg to ethanol-HEPES (N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid) buffer solution (20 mM HEPES, 100 mM NaN0 ₃ , 1:1, v/v, pH 7.2). Formulated into a 10 M concentration solution. When mercury ions are not added, the BH fluorescence is weak, and then the concentration of mercury ions is gradually increased by 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 20/^M, BHg fluorescence is also gradually enhanced, and titration to saturation fluorescence is approximately 20 times enhanced (Fig. 1). The instrument used was a fluorescence spectrophotometer, model: LS55.

Example 3 Probe BH _g selectivity to mercury ions and anti-interference ability:

10 compound BHg was added to a 5-fold excess of various metal ions in ethanol-HEPES (N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid) buffer solution (20 mM HEPES, 100 mM NaN0 ₃ , 1:1, v/v, pH 7.2), probe excitation wavelength is _{49Q nm} , probe emission wavelength is _{513 nm} , and the test results are shown in Fig. 2. As can be seen from the figure, the probe BHg has a high selectivity for mercury ions, and only the addition of mercury ions can produce significant fluorescence enhancement. In addition, it can be seen from Fig. 4 and Fig. 5 that metal ions such as sodium, potassium, calcium, magnesium, copper, and anions such as chloride ions, nitrate ions, and sulfate ions have little or no interference with the entire recognition process. The instrument used was a fluorescence spectrophotometer, model: LS55.

Example 4 Probe BHg is insensitive to pH:

Adjust with compound BHg (10 _μM ethanol-HEPES buffer solution (20 mM HEPES, 100 mM NaN0 ₃ , 1:1, v/v, pH 7.2) with NaOH solution (1 M) or HCl solution (1 M). The pH of the solution was measured and the fluorescence intensity was measured. The corresponding change in fluorescence intensity was recorded. The test results are shown in Figure 3. It can be seen from the figure that the probe BH _{g is} in the range of pH 5-12, and the pH change has little effect on the fluorescence emission. Therefore, the probe can be used for the detection of mercury ions in this pH range. The instrument used is a fluorescence spectrophotometer, model: LS55.

Example 5 Sensitivity of Probe BHg to Mercury Ion Detection - Compound BHg

Add mercury ions at a concentration of 2-12 ppb to ethanol-HEPES buffer solution (20 mM HEPES, 100 mM NaN0 ₃ , 1:1, v/v, pH 7.2) and record the corresponding changes in fluorescence intensity. Figure 6. It can be seen from the figure that the probe BH _g has a significant increase in fluorescence intensity in the range of 2-12 ppb and a good linear relationship between fluorescence intensity and mercury ion concentration. Therefore, the probe can be used for the detection of low concentrations of mercury ions. The instrument used was a fluorescence spectrophotometer, model: LS55.

Example 6 Detection of Mercury Ions by Probe BH _g in Natural Water Samples We selected three different sources of water: Yellow Sea Water (Dalian), Pool Water and Tap Water. BH _g was added to the three samples to form a 10 _M concentration solution, which in turn increased the amount of mercury ions added. It can be seen from Fig. 7 that as the concentration of mercury ions increases, the fluorescence intensity of the three water samples increases significantly and has a good linear relationship. Especially after adding 50ppb mercury ions, the fluorescence intensity of the three water samples increases respectively. 3.9 times, 4.5 times, 2.9 times (Fig. 8), the effect is obvious, indicating that BH _g can be applied to the detection of mercury ions in actual natural water samples.

Example 7 Detection of mercury ions in BHg in a sulfur-rich environment

In ethanol-HEPES buffer solution (20 mM HEPES, 100 mM NaNO ₃ , 1 : 1, v/v, pH 7.2), the probes BHg ( 10 ), cysteine (5(VM), mercury ions were added in sequence. (50 _M ) , the change of fluorescence intensity was recorded and compared with the change of fluorescence intensity of direct addition of mercury ion (50^) BHg. According to Figure 9, cysteine did not affect the process of probe identification of mercury ions, indicating The probe BHg can be used to detect mercury ions in a sulfur-rich environment.

Example 8 Probes Detection of mercury ions in living cells by the BHg series:

BHg was added to the cultured Osteoblasts cells and cultured in the medium at 37 ° C for 30 minutes, at which time the fluorescence of BHg in the living Osteoblasts cells was weak (Fig. 10(b)). Mercury ions were added to the above probe-containing cell culture medium and incubated for 30 minutes under yVC conditions, at this time in the living

Fluorescence in Osteoblasts cells became very strong (Fig. 10(c)). Bright field imaging demonstrated that Osteoblasts containing BHg and mercury ions were observed throughout the process (Fig. 10(a)). The instrument used was Olympus, 1X70-131.

Claims

A fluoroboron dye fluorescent probe for mercury ion detection, characterized in that the probe molecule has the following structural formula BHg:

The fluoroboron dye fluorescent probe for mercury ion detection according to claim 1, wherein the probe molecule is formulated into a composition for mercury ion detection using an ethanol-HEPES buffer solution.

3. The method for synthesizing a fluoroboron dye fluorescent probe for mercury ion detection according to claim 1, wherein the method for synthesizing the probe molecule comprises the following steps:

1) Reaction of 2,4-dimethylpyrrole with 3-hydroxy-4nitro-benzaldehyde: 2,4-dimethylpyrrole and ₃ -hydroxy-4-nitro-benzaldehyde are dissolved in dichloromethane A drop of trifluoroacetic acid was added dropwise, and then stirred at room temperature for 5 hours; the solvent was evaporated under reduced pressure, and then dichlorodicyanobenzene was added. After stirring for 15 minutes, triethylamine and boron trifluoride diethyl ether solution were added and stirring was continued for 45 minutes. The reaction solution was washed with water and extracted with methylene chloride; dichloromethane was distilled off under reduced pressure, and column chromatography was used to obtain intermediate I:

2) Reduction of the intermediate I to obtain the compound BH _g: Dissolving the intermediate I in ethanol, adding a hydrazine hydrate and a palladium carbon catalyst, and refluxing for 2 hours; after cooling, filtering the solution, and evaporating the ethanol under reduced pressure. It was then redissolved in dichloromethane. Washing the organic layer twice with deionized water; organic layer solution Drying with anhydrous sodium sulfate, dichloromethane was distilled off under reduced pressure, and the title product was purified by column chromatography to give a red solid: