Fluorescent probe JHG-1 for detecting mercury ions as well as preparation method and application of fluorescent probe JHG-1

Abstract

The invention discloses a fluorescent probe JHG-1 for detecting mercury ions and a preparation method and application of the fluorescent probe JHG-1. The probe is designed mainly based on a thioacetal structure by utilizing intramolecular charge transfer. In various ions, the probe has specificity in recognition of Hg < 2 + >, shows fluorescence response different from that of other ions, has excellent anti-interference capability in a solution system in which the probe and other ions coexist, and can recognize Hg < 2 + > in a solution. The reaction ratio of the probe to Hg < 2 + > is 1: 1, and verification is carried out through mass spectrometry. In addition, under physiological conditions, cell imaging results also show that the probe can realize recognition of Hg < 2 + > in living cells.

Description

Technical Field

[0001] The present invention belongs to the field of chemical technology, and further to the field of chemical detection technology, and specifically relates to a fluorescent probe JHG-1 for detecting mercury ions, and a preparation method and application thereof. Background Art

[0002] Mercury is a heavy metal element and a rare metal, commonly known as quicksilver. Its overall content in nature is very small, and its main forms are mercury, mercury salts, and inorganic mercury. Mercury exists in the air, water, and soil, and is found all over the world. Although the stock of mercury is small, it is a highly toxic element with bioaccumulation and can accumulate in the food chain, especially in fish and other types of aquatic products. Mercury can be methylated to become methylmercury, which is also highly toxic. The culprit of Minamata disease, which once caused a sensation in the world, is methylmercury. In mercury poisoning, the enzyme that maintains glutamate stability will be affected, and the glutamate level will also be affected, resulting in neurotoxic effects. Mercury can cause nephrotoxicity, induce apoptosis of renal tubular epithelial cells, inhibit the normal function of transporters, and lead to renal insufficiency. In addition to possible oral damage, it is also immunopathogenic. Mercury is irritating and poses a great safety hazard to the human body. Therefore, the development of a rapid and effective detection method for mercury ions and the realization of qualitative and quantitative research on mercury ions are of great value to multiple disciplines such as environmental science, biomedicine, and chemical sensing. Summary of the invention

[0003] Traditional detection methods for mercury include atomic absorption spectroscopy, inductively coupled plasma atomic emission spectroscopy, inductively coupled plasma mass spectrometry, high performance liquid chromatography, enzyme-linked immunosorbent assay, etc. Most of these methods have high selectivity and excellent sensitivity and have been greatly developed, but they are not conducive to real-time monitoring of mercury due to the high cost and professional technical means required. In recent years, due to the high selectivity, high sensitivity, low detection limit, visualization and relatively low price advantages of small molecule fluorescent probes, they have aroused great research interest in the development of optical imaging and analytical sensors, and have become a hot field in biomarker detection and environmental pollution monitoring.

[0004] As the field of mercury ion recognition and corresponding fluorescent probes becomes more mature, there is a higher requirement for synthesizing probes with low toxicity that can be used in vivo.

[0005] Therefore, based on the above research, the present invention proposes a low-toxic fluorescent probe JHG-1 for detecting mercury ions and its synthesis route, which utilizes the combination of mercury ions and sulfur to produce a recognition effect on mercury ions.

[0006] The technical problem to be solved by the present invention is to synthesize a novel molecule capable of detecting and identifying Hg by molecular design. 2+The mercury ion fluorescent probe JHG-1 has a simple synthesis method, is easy to operate, has a high yield, high selectivity, strong specificity, good anti-interference, low economic cost, high sensitivity, low cytotoxicity, cell membrane permeability, and can detect Hg in living cells. 2+ This has practical significance and application value in the field of environment and biological imaging.

[0007] The first object of the present invention is to provide a fluorescent probe JHG-1 for detecting mercury ions; the second object is to provide a method for preparing the fluorescent probe JHG-1 for detecting mercury ions; and the third object is to provide an application of the fluorescent probe JHG-1 for detecting mercury ions.

[0008] The first object of the present invention is achieved in that the fluorescent probe JHG-1 for detecting mercury ions is 2-(6-methoxynaphthalen-2-yl)-1,3-dithiopentene, and its molecular formula is: 14 H 14 OS2, the structural formula is as follows: .

[0009] The second object of the present invention is achieved by using compounds 6-(dimethylamino)-2-naphthaldehyde and 1,3-propanedithiol as template substrates, BF3 as a catalyst, and dichloromethane as a solvent, and reacting for 6 to 10 hours at room temperature. The synthesis route is as follows: .

[0010] After the reaction was completed, the obtained crude product was purified by silica gel column using dichloromethane / petroleum ether as eluent to obtain the pure product with a yield of 43%.

[0011] The third object of the present invention is achieved by: the fluorescent probe JHG-1 for detecting mercury ions specifically identifies and monitors Hg in organic chemistry and analytical chemistry. 2+ Application in.

[0012] The beneficial effects of the present invention are as follows: 1. The prepared 2-(6-methoxynaphthalene-2-yl)-1,3-dithiopentene has a simple synthesis method, is easy to operate, has a high yield, has strong selectivity for mercuric ions, is not easily interfered by other metal cations, has high sensitivity, and can be used to detect Hg in living cells. 2+ This has practical significance and application value in the field of environment and biological imaging.

[0013] 2. Compound JHG-1 was applied to the detection of intracellular mercury ions. The cytotoxicity of compound JHG-1 was explored by CCK-8. The results showed that compound JHG-1 had low cytotoxicity and was permeable to cell membranes. It also achieved Hg ion permeability in HepG2 cells. 2+ Fluorescence microscopy imaging.

[0014] 3. The present invention provides a fluorescent probe JHG-1 for detecting mercury ions and its synthesis path. The preparation method is simple, easy to operate, has a high yield, good biocompatibility, high selectivity, strong specificity, low detection limit, good anti-interference ability, low economic cost, and high sensitivity. It provides a fast, convenient, and effective method for detecting mercury ions in organisms or the environment, and has high practical value. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 The H NMR spectrum of 2-(6-methoxynaphthalen-2-yl)-1,3-dithiolene ( 1 H NMR); Figure 2 The carbon NMR spectrum of 2-(6-methoxynaphthalen-2-yl)-1,3-dithiolene ( 13 C NMR); Figure 3 It is the fluorescence spectrum of the probe JHG-1 of the present invention after adding different metal ions; Figure 4 It is the fluorescence response spectrum of the probe JHG-1 of the present invention under ultraviolet light after adding different metal ions; Figure 5 The probe JHG-1 of the present invention is added with Hg 2+ And add the fluorescence histogram of other interfering ions; Figure 6 The probe JHG-1 of the present invention is added in different pH buffers / Hg 2+ Fluorescence intensity change diagram when Figure 7 The probe HJG-1 of the present invention was added with different concentrations of Hg 2+ Fluorescence spectrum and fluorescence intensity diagram of the reaction; Figure 8 The probe JHG-1 of the present invention was added with different concentrations of Hg 2+ Fluorescence intensity graph of the reaction at 528 nm; Fig. 9 The probe JHG-1 of the present invention was added with different concentrations of Hg 2+ Fluorescence intensity graph of the reaction at 436 nm; Fig.10 It is the Job-Plot working curve diagram of the probe JHG-1 of the present invention; Fig.11 The probe JHG-1 of the present invention and Hg 2+ Response mechanism diagram; Fig.12 The probe JHG-1 of the present invention and Hg 2+ High-resolution mass spectra after the reaction; Fig.13 It is a graph of cell survival rate after the probe JHG-1 of the present invention is treated with different concentrations of JHG-1 (0-50 μM) in HepG2 cells for 24 hours; Fig.14 The probes of the present invention JHG-1 (20 μM) and JHG-1 (20 μM) + Hg 2+ (20 μM) Cell imaging in the blue channel; Fig.15 The probes of the present invention JHG-1 (20 μM) and JHG-1 (20 μM) + Hg 2+ (20 μM) Cell imaging under the green channel. DETAILED DESCRIPTION

[0016] The present invention is further described below in conjunction with the embodiments, but the present invention is not limited in any way. Any changes or substitutions made based on the teachings of the present invention belong to the protection scope of the present invention.

[0017] The fluorescent probe JHG-1 for detecting mercury ions described in the present invention is 2-(6-methoxynaphthalene-2-yl)-1,3-dithiopentene, and its molecular formula is: 14 H 14 OS2, the structural formula is as follows: .

[0018] The preparation method of the fluorescent probe JHG-1 for detecting mercury ions of the present invention is to use the compound 6-(dimethylamino)-2-naphthaldehyde and 1,3-propanedithiol as template substrates, BF3 as a catalyst, and dichloromethane as a solvent, and react for 6 to 10 hours at room temperature. The synthesis route is as follows: .

[0019] The application of the present invention is that the fluorescent probe JHG-1 for detecting mercury ions can specifically identify and monitor Hg in organic chemistry and analytical chemistry. 2+ Application in.

[0020] The present invention is further described below with specific implementation cases: Example 1 See Figures 1 to 15 As shown, the fluorescent probe JHG-1 used to detect mercury ions, the chemical name of JHG-1 is 2-(6-methoxynaphthalen-2-yl)-1,3-dithiopentene, and its molecular formula is: C 14 H 14 OS2; the structural formula is as follows: The synthetic route of the fluorescent probe JHG-1 for rapid detection of mercury ions is carried out in the following steps:

[0021] The reaction used compound 6-(dimethylamino)-2-naphthaldehyde and 1,3-propanedithiol as template substrates, added BF3 ether solution, and dichloromethane as solvent. The crude product was obtained after 8 hours of reaction at room temperature. The obtained crude product was purified by silica gel column using dichloromethane / petroleum ether as eluent to obtain a pure product of yellow-white solid with a yield of 43%.

[0022] The prepared mercury ion fluorescent probe JHG-1 was structurally identified and characterized by nuclear magnetic resonance spectrometer. Its nuclear magnetic resonance hydrogen spectrum is shown in Figure 1 As shown, the carbon spectrum is Figure 2 As shown in Figure 2, the analysis of the nuclear magnetic resonance spectrum shows that the chemical name of the mercury ion fluorescent probe JHG-1 is 2-(6-methoxynaphthalen-2-yl)-1,3-dithiopentene, and its molecular formula is: C 14 H 14 OS2; The structure is exactly as described above.

[0023] Hydrogen spectrum: 1 H NMR (400 MHz, DMSO-d6), its nuclear magnetic resonance hydrogen spectrum is shown in the figure. The obtained crude product is purified by silica gel column, using dichloromethane / petroleum ether as eluent, and ordinary technicians in this field have not made any invention.

[0024] Carbon spectrum: 13 C NMR (101 MHz, DMSO-d6), and its carbon nuclear magnetic resonance spectrum is shown in the figure. The obtained crude product is purified by silica gel column using dichloromethane / petroleum ether as eluent, and ordinary technicians in this field have not made any invention.

[0025] Example 2 The preparation method of the stock solution of the low-toxic mercury ion fluorescent probe JHG-1 is as follows: Preparation of low-toxic mercury ion fluorescent probe JHG-1 stock solution: Dissolve the mercury ion fluorescent probe JHG-1 in DMSO to obtain a mercury ion fluorescent probe JHG-1 stock solution with a concentration of 10mM. Accurately pipette 1.0ml of the mercury ion fluorescent probe JHG-1 stock solution into a 100ml volumetric flask, dilute to the scale with Tris-HCl-10% DMSO solution (Tris-HCl / DMSO=9:1), and shake well to obtain a mercury ion fluorescent probe JHG-1 solution with a concentration of 10μM.

[0026] The preparation method of the Tris-HCl buffer is as follows: 1.21 g of tris(hydroxymethyl)aminomethane and 2.92 g of sodium chloride are weighed and dissolved in water, and the pH is adjusted to 7.05 with hydrochloric acid to obtain a Tris-HCl buffer with a concentration of 20 mM and a pH of 7.4.

[0027] The Tris-HCl:10% DMSO solution is prepared by mixing Tris-HCl buffer and 10% DMSO in a volume ratio of 9:1.

[0028] Example 3 Preparation of low toxic mercury ion fluorescent probe JHG-1 Metal ion selectivity test, the test method is as follows: Prepare 18 centrifuge tubes, add the same amount of the above probe mother solution to each of them, and then add the stock solution of metal ions prepared with pure water to a concentration of 10mM, including Cu 2+ ,Cr 2+ , Fe 2+ , Sn 4+ , K + , Na + , Ca 2+ , Mn 2+ , Al 3+ , Ba 2+ , Zn 2+ , Ni 2+ , Fe 3+ ,Mg 2+ , Cd 2+ , Pb 2+ , Ag + , Hg 2+ Prepare the stock solution of metal ions such as 2-Hg and 1-Hg ion fluorescent probe JHG-1 as blank sample. Measure the fluorescence spectrum of the prepared solution under a fluorescence spectrophotometer. Add Hg to the solution of probe JHG-1. 2+ When Ag is added, the fluorescence spectrum changes significantly, the spectrum red-shifts from 436nm to 528nm and the fluorescence intensity increases. +When Hg is added, the fluorescence spectrum has similar changes. It can be seen that the peak at 436nm has a downward trend, and there is a very small peak at 528nm, but it is not as good as adding Hg 2+ The phenomenon of Hg is obvious, but there is no such change when other metal ions are added, and there is only a fluorescence peak at 436nm. This shows that the probe JHG-1 is sensitive to Hg 2+ Excellent selectivity, can be used specifically for Hg 2+ Identification and monitoring of fluorescence spectra such as Figure 3 The fluorescence changes of JHG-1 to different ions were observed under a 365nm portable ultraviolet lamp. When other ions showed obvious blue fluorescence, Hg 2+ The system has significant differences, the blue fluorescence becomes a strong yellow-green fluorescence emission, the spectrum is as follows Figure 4 shown.

[0029] Example 4 The low-toxicity mercury ion fluorescent probe JHG-1 was prepared for metal ion interference test. The test method is as follows: Prepare 18 centrifuge tubes, add the same amount of the above probe stock solution and add Hg 2+ Then, other metal ions Cu at a concentration of 10 mM were added to each vial. 2+ , Ca 2+ 、Cd 2+ , Pb 2+ , Fe 2+ , Fe 3+ , Mn 2+ 、Zn 2+ Cr 3+ 、Ag + Mg 2+ Sn 4+ , K + 、Ni 2+ 、Al 3+ , Ba 2+ 、Na + The aqueous solution of compound JHG-1-Hg 2+ The solution was used as a blank sample. The fluorescence spectrum of the solution prepared above was measured under a fluorescence spectrophotometer. Figure 5 As shown, when other ions exist alone, I 528 / I 436 is at a very low level, and surprisingly, among other ions and Hg 2+ In the coexistence system of Hg, I528 / I436 increased significantly. This shows that in the complex system, the probe JHG-1 can still identify Hg 2+ , for Hg 2+Has higher selectivity.

[0030] Example 5 The preparation and stability of the low-toxic mercury ion fluorescent probe JHG-1 under different pH conditions were investigated as follows: The pH value of the mercury ion fluorescent probe JHG-1 solution was adjusted by hydrochloric acid and sodium hydroxide solution, and solutions with pH values ​​of 2-11 were obtained. The mercury ion fluorescent probe JHG-1 solution within this pH range was measured under a fluorescence spectrophotometer for changes in fluorescence spectrum. By testing the fluorescence spectrum of the mercury ion fluorescent probe JHG-1 solution under different pH conditions, such as Figure 6 As shown, in the pH range of 2-11, I 528 / I 436 The value is very small, indicating that the probe is relatively stable to changes in pH. 2+ After joining, I 528 / I 436 The value increased significantly, and the fluorescence was stable and strong in the pH range of 4-7. Therefore, the pH of our working buffer was also neutral.

[0031] Example 6 Preparation of low-toxic mercury ion fluorescent probe JHG-1 for fluorescence titration test. The test method is as follows: Prepare 18 centrifuge tubes, add the same amount of the above probe stock solution to each of them, and then add different concentrations of Hg to the compound JHG-1 solution. 2+ Aqueous solution (0-20 μM) was prepared, and a 10 μM mercury ion fluorescent probe JHG-1 solution was prepared as a blank sample. The fluorescence spectrum of the prepared solution was measured under a fluorescence spectrophotometer. Figure 7 As shown, in the absence of Hg 2+ In the case of Hg 2+ With the addition of Hg 2+ The increase of 528nm gradually decreases to a gentle level, and the 528nm peak gradually increases and tends to be stable. It can be clearly seen that there is a crossover point in the graph, which means that new substances are generated. Figure 8 ) It can be concluded that when the concentration of JHG-1 is 10 μM, as Hg 2+ When Hg 2+ The concentration is also 10 μM, that is, when the amount of substance reaches 1:1, the fluorescence intensity reaches the peak, and there is no obvious increase afterwards. Observe the fluorescence intensity at 436 nm ( Fig. 9 ), also reached similar conclusions.

[0032] Example 7 Low toxicity mercury ion fluorescent probe JHG-1 and Hg 2+ The research method of fluorescence sensing mechanism is as follows: JHG-1 and Hg 2+ The total concentration of Hg was set to 20 μM and kept constant. 2+ The ratio of 528nm fluorescence intensity to Hg 2+ The proportion of changes is as follows ( Fig.10 )Job's curve, in each proportional relationship, we can see that when C （Hg） When it accounts for half of the total concentration, the fluorescence intensity value reaches the highest, which means that the ratio of Hg to JHG-1 is 1:1.

[0033] In view of the above recognition results, we have obtained a recognition mechanism, such as Fig.11 As shown in the figure, the naphthalene ring in the needle is an electron-rich structure that acts as an electron donor and is also a conjugated fluorescent group, while the thioacetal structure on the substituent is also an electron-donating structure, making it difficult for the charge transfer process within the molecule to occur. 2+ It can recognize and remove the sulfur on the thioacetal, turning it into an aldehyde structure, which opens the ICT process, red-shifts the spectrum, and restores fluorescence. Subsequently, we used high-resolution mass spectrometry to verify the reaction mechanism. Fig.12 , a strong peak appeared at 187.07550 m / z, corresponding to the molecular mass of the reactant 6-methoxy-2-naphthaldehyde (calcd: [M+H]+=187.0754).

[0034] Example 8 Low toxicity mercury ion fluorescent probe JHG-1 and Hg 2+ Toxicity studies and cell imaging, the research methods are as follows: Compound JHG-1 was applied to cells, and the toxicity of the compound was explored by CCK-8. The probe concentration was 50 μM, and the cell survival rate was still above 90% after 24 hours ( Fig.13 ), which indicates that the probe JHG-1 has very low cytotoxicity and good biocompatibility.

[0035] In order to show that the probe reacts with Hg 2+ The probe has a strong recognition ability for Hg 2+ Fluorescence imaging effect. Fig.14 As shown in the figure, in the blue channel, there is almost no fluorescence in the control group. After the cells are incubated with the probe, weak blue fluorescence can be seen in the cells. Fig.15As shown in the green channel, the probe itself only exhibits weak green fluorescence. 2+ After adding it to the cell environment and co-culturing for 90 minutes, we can see obvious green fluorescence, suggesting that JHG-1 can be used to degrade Hg in HepG2 cells. 2+ Therefore, the probe JHG-1 has extremely low cytotoxicity and can be successfully applied in Hg 2+ Imaging detection field.

[0036] The low-toxicity mercury ion fluorescent probe JHG-1 is mainly based on the thioacetal structure and is designed using intramolecular charge transfer. The synthesis method is simple, easy to operate, the reaction conditions are mild, and the yield is high. Among various ions, the probe is the most sensitive to Hg 2+ The recognition of Hg is specific, showing a fluorescence response different from other ions. In a solution system coexisting with other ions, it has excellent anti-interference ability and can identify Hg in the solution. 2+ . Probe and Hg 2+ The reaction ratio was 1:1 and was verified by mass spectrometry. In addition, under physiological conditions, cell imaging results also showed that the probe can detect Hg in living cells. 2+ Identification. The identification has high sensitivity and can be used for highly selective detection of mercury ions in organisms or the environment under normal conditions; the present invention provides a fluorescent probe JHG-1 for detecting mercury ions and its synthesis path, which has a simple preparation method, is green and environmentally friendly, has good biocompatibility, high selectivity, strong specificity, low detection limit, good anti-interference ability, and low economic cost, and provides a fast, convenient and effective method for detecting mercury ions in organisms or the environment, and has high practical value.

[0037] In the description of this specification, the description with reference to the terms "one embodiment", "example", "specific example", etc. means that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representation of the above terms does not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described can be combined in any one or more embodiments or examples in a suitable manner.

[0038] The preferred embodiments of the present invention disclosed above are only used to help illustrate the present invention. The preferred embodiments do not describe all the details in detail, nor do they limit the invention to the specific implementation methods described. Obviously, many modifications and changes can be made according to the content of this specification. This specification selects and specifically describes these embodiments in order to better explain the principles and practical applications of the present invention, so that those skilled in the art can understand and use the present invention well. The present invention is limited only by the claims and their full scope and equivalents.

Claims

1. A fluorescent probe JHG-1 for detecting mercury ions, characterized in that: The fluorescent probe JHG-1 used to detect mercury ions is 2-(6-methoxynaphthalen-2-yl)-1,3-dithiopentene, and its molecular formula is: C 14 H 14 OS2, the structural formula is as follows: 。 2. A method for preparing the fluorescent probe JHG-1 for detecting mercury ions according to claim 1, characterized in that: The compound 6-(dimethylamino)-2-naphthaldehyde and 1,3-propanedithiol are used as template substrates, BF3 is used as a catalyst, and dichloromethane is used as a solvent. The reaction is carried out at room temperature for 6 to 10 hours. The synthetic route is as follows: 。 3. An application of the fluorescent probe JHG-1 for detecting mercury ions according to claim 1, characterized in that: The fluorescent probe JHG-1 for detecting mercury ions specifically identifies and monitors Hg in organic chemistry and analytical chemistry. 2+ Application in.

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