阅读说明：本技术 一种快速识别汞和甲基汞的荧光探针及其制备方法、应用 (Fluorescent probe for rapidly identifying mercury and methyl mercury as well as preparation method and application thereof ) 是由 那娜 付强 欧阳津 于 2020-05-07 设计创作，主要内容包括：本发明属于分析化学领域,涉及一种快速识别汞和甲基汞的荧光探针及其制备方法、应用。所述荧光探针的结构式如下：<Image he="298" wi="200" file="100004_DEST_PATH_IMAGE001.GIF" imgContent="drawing" imgFormat="GIF" orientation="portrait" inline="no"></Image>。本发明制备的荧光探针在不加任何其他附加材料的条件下,提高了检测灵敏性,并且避免了填加附加材料,减少附加材料的消耗以及减少了在检测中的误差来源；在检测中成功检测了待测物在活细胞、斑马鱼体内的成像,适用于细胞和斑马鱼等活体内的实时监测区分,这种方法是在之前的方法中没有能够做到的。成像的实现对于区分汞和甲基汞的深入研究起到很大的推动作用。(The invention belongs to the field of analytical chemistry, and relates to a fluorescent probe for rapidly identifying mercury and methyl mercury, and a preparation method and application thereof. The structural formula of the fluorescent probe is as follows: . The fluorescent probe prepared by the invention improves the detection sensitivity without adding any other additional material, avoids adding the additional material, reduces the consumption of the additional material and reduces the error source in the detection; successfully detects the imaging of the object to be detected in living cells and zebra fish bodies in the detection, and is suitable for the in vivo detection of the cells, the zebra fish and the likeThe discrimination is monitored in real time, which was not possible in previous methods. The realization of imaging has a great driving role in the intensive research of distinguishing mercury from methylmercury.)

1. A fluorescent probe for rapidly identifying mercury and methyl mercury is characterized in that the structural formula of the fluorescent probe is as follows:

。

2. the method for preparing the fluorescent probe according to claim 1, which comprises the following steps:

(1) dissolving 2-bromo-4' -hydroxyacetophenone, 4-dimethylaminobenzaldehyde and sodium carbonate in ethanol, then adding water, stirring for 6 hours at 60 ℃, adding hydrochloric acid to adjust the pH value to be neutral after the reactant is cooled, filtering out solids, washing with ethanol, removing insoluble substances in the solution, and then performing rotary evaporation on the solution to remove the solvent to obtain orange-red solid BHDP;

(2) BHDP, 4-formylphenylboronic acid, potassium carbonate, palladium (II) acetate and triphenylphosphine are dissolved in a mixed solvent of 1, 4-dioxy and water, then the mixture is refluxed for 6h at 80 ℃ under the protection of nitrogen, the solvent is removed under reduced pressure, and then the mixture of petroleum ether and ethyl acetate is used as eluent to carry out column chromatography purification on 300-400-mesh silica gel to obtain the red solid DPAHB.

3. The production method according to claim 2, wherein in the step (1), the molar ratio of the 2-bromo-4' -hydroxyacetophenone, the 4-dimethylaminobenzaldehyde and the sodium carbonate is 1: 1: 2; the concentration of the 2-bromo-4' -hydroxyacetophenone in ethanol is 0.25 mol/L; the volume ratio of the ethanol to the water is 4: 1.

4. the production method according to claim 2 or 3, wherein in the step (2), the molar ratio of BHDP, 4-formylphenylboronic acid, potassium carbonate, palladium (II) acetate and triphenylphosphine is 1: 1: 14: 0.05: 0.05; the concentration of the BHDP in the mixed solvent is 0.04 mol/L; the volume ratio of the 1, 4-dioxy to water is 4: 1.

5. the method according to claim 4, wherein the volume ratio of petroleum ether to ethyl acetate in the eluent is 20: 1.

6. the method according to claim 4, wherein the DPAHB efficacy index of the ratiometric probe is as follows:

the detection sensitivity is that the mercury detection limit is 9.8 nM;

the detection limit of methyl mercury is 0.35 nM;

the optical mechanism index is as follows: has the functions of fluorescence and red shift and blue shift absorption.

7. Use of a fluorescent probe prepared according to the preparation method of claim 1 or any one of claims 2 to 6 for qualitatively and quantitatively detecting mercury and methylmercury in a biological sample, wherein the fluorescent probe is suitable for qualitatively distinguishing mercury from methylmercury in cells and zebrafish bodies; the method is suitable for real-time monitoring and distinguishing of mercury and methyl mercury in a biological sample; the device is suitable for real-time observation and tracking in living organisms.

8. The use of claim 7, wherein the fluorescent probe detection step of distinguishing between mercury and methylmercury comprises:

preparing solution

Preparing a probe stock solution: accurately weighing a fluorescent probe, dissolving the fluorescent probe in anhydrous acetonitrile, and preparing a probe stock solution with the concentration of 60 uM;

the fluorescence reaction system for Hg (II) and MeHg is 1ml, and the system comprises: probe DPAHB 10 μ M, pH =7.4 PBS buffer 20mM, reaction time was completed within 1 min;

the UV reaction system for Hg (II) and MeHg was 2ml, comprising: probe DPAHB 50 μ M, pH =7.4 PBS buffer 20mM, reaction time was complete within 1 min.

Technical Field

The invention belongs to the field of analytical chemistry, and relates to a fluorescent probe for rapidly identifying mercury and methyl mercury, and a preparation method and application thereof.

Background

The study on Hg (II) and MeHg is mostly indirectly reported in an in vitro anoxic environment. Methods for monitoring hg (ii) and MeHg are mostly indirect methods for separating or derivatizing both in vitro, and pretreatment and routine chromatographic separation are time consuming, which makes in vivo and real-time detection of both ions impossible. Fluorescence imaging provides a powerful method support for studying the physiology and pathology of ions in their natural environment with minimal interference to biological systems. Therefore, fluorescence imaging using biocompatible dual-response fluorescent probes is an ideal way to detect and distinguish hg (ii) or MeHg. However, the probes reported so far show that the emission of hg (ii) or MeHg is always at the same wavelength, making it difficult to detect selective signals of both ions simultaneously in vivo. Therefore, it is crucial to design and synthesize a water-soluble and biocompatible fluorescent probe that simultaneously distinguishes between hg (ii) and MeHg emission for the in vivo visual monitoring of these two ions. This is also a prerequisite for monitoring the dynamic conversion of both ions in a biological environment. The research and development of a novel fluorescent probe which has high selectivity and high sensitivity and can mature and selectively detect and identify mercury and methyl mercury becomes a problem to be solved urgently.

Disclosure of Invention

Aiming at the defects of the prior art and the biological field, the invention provides the fluorescent probe for rapidly identifying mercury and methyl mercury, the fluorescent probe can be applied to biological sample detection, and the probe has good and rapid response characteristics, so the fluorescent probe can be applied to living bodies for real-time observation and tracking. The method has the advantages of high precision, obvious and easily observed phenomenon, high accuracy and the like, is convenient and easy to operate, has strong operability, and is particularly suitable for large-data research such as large-batch sample combination screening.

The invention also provides a preparation method of the fluorescent probe for rapidly identifying mercury and methyl mercury.

The invention also provides application of the fluorescent probe in biological samples.

The technical scheme adopted by the invention for realizing the purpose is as follows:

the invention provides a fluorescent probe for rapidly identifying mercury and methyl mercury, which has the following structural formula:

。

the invention also provides a preparation method of the fluorescent probe, which comprises the following steps:

(1) dissolving 2-bromo-4' -hydroxyacetophenone, 4-dimethylaminobenzaldehyde and sodium carbonate in ethanol, then adding water, stirring for 6 hours at 60 ℃, adding hydrochloric acid to adjust the pH value to be neutral after the reactant is cooled, filtering out solids, washing with ethanol, removing insoluble substances in the solution, and then performing rotary evaporation on the solution to remove the solvent to obtain orange-red solid BHDP;

(2) BHDP, 4-formylphenylboronic acid, potassium carbonate, palladium (II) acetate and triphenylphosphine are dissolved in a mixed solvent of 1, 4-dioxy and water, then the mixture is refluxed for 6h at 80 ℃ under the protection of nitrogen, the solvent is removed under reduced pressure, and then the mixture of petroleum ether and ethyl acetate is used as eluent to carry out column chromatography purification on 300-400-mesh silica gel to obtain the red solid DPAHB.

Further, in the step (1), the molar ratio of the 2-bromo-4' -hydroxyacetophenone, the 4-dimethylaminobenzaldehyde and the sodium carbonate is 1: 1: 2; the concentration of the 2-bromo-4' -hydroxyacetophenone in ethanol is 0.25 mol/L; the volume ratio of the ethanol to the water is 4: 1.

further, in the step (2), the molar ratio of BHDP, 4-formylphenylboronic acid, potassium carbonate, palladium (II) acetate and triphenylphosphine was 1: 1: 14: 0.05: 0.05; the concentration of the BHDP in the mixed solvent is 0.04 mol/L; the volume ratio of the 1, 4-dioxy to water is 4: 1.

in the eluent, the volume ratio of the petroleum ether to the ethyl acetate is 20: 1.

the DPAHB effect judgment indexes of the ratiometric probe prepared by the invention are as follows:

the detection sensitivity is that the mercury detection limit is 9.8 nM; the detection limit of methyl mercury is 0.35 nM;

the optical mechanism index is as follows: has the functions of fluorescence and red shift and blue shift absorption.

The invention also provides an application of the fluorescent probe prepared by the preparation method in qualitative and quantitative detection of mercury and methyl mercury in a biological sample, and the fluorescent probe is suitable for qualitative differentiation of mercury and methyl mercury in cells and zebra fish bodies in a biological living body; the method is suitable for real-time monitoring and distinguishing of mercury and methyl mercury in a biological sample; the device is suitable for real-time observation and tracking in living organisms.

Further, the specific steps of the fluorescent probe detection for distinguishing mercury from methyl mercury comprise:

preparing solution

Preparing a probe stock solution: accurately weighing a fluorescent probe, dissolving the fluorescent probe in anhydrous acetonitrile, and preparing a probe stock solution with the concentration of 60 uM;

the fluorescence reaction system for Hg (II) and MeHg is 1ml, and the system comprises: probe DPAHB 10 μ M, pH =7.4 PBS buffer 20mM, reaction time was completed within 1 min;

the UV reaction system for Hg (II) and MeHg was 2ml, comprising: probe DPAHB 50 μ M, pH =7.4 PBS buffer 20mM, reaction time was complete within 1 min.

The synthetic route of the DPAHB provided by the invention is as follows:

the probe prepared by the invention is based on the extension of a chalcone mother body loop. A dual-response fluorescent probe (E) -4 ' - (3- (4- (dimethylaminophenyl) acryloyl) -3 ' -hydroxy- [1, 1' -biphenyl ] -3-carbaldehyde (DPAHB) is synthesized and used for simultaneously distinguishing and detecting Hg (II) and MeHg.

The fluorescent probe prepared by the invention is applied to qualitative and quantitative analysis of mercury and methyl mercury differentiation in a biological sample, and has sensitive, accurate and quick detection; the biological sample mainly comprises living cells, zebra fish and the like, and can be applied to analytical chemistry and life organic analytical chemistry.

The detection mechanism of the fluorescent probe prepared by the invention is as follows:

the design scheme of the fluorescent probe has the following principle: the optical properties of DPAHB and the changes after addition of hg (ii) and MeHg were explained by Density Functional Theory (DFT) and time-dependent density functional theory (TDDFT) calculation methods. Stokes shift calculated absorption of DPAHB of about 242nm (pi-pi electrons transition from HOMO to LUMO, fig. 1A a-b). This is based on the efficient Excited State Proton Transfer (ESPT) process initiated by the phenolic hydroxyl groups in DPAHB. This is essentially consistent with the Stokes shift of 220 nm (emission from 460 nm to 680 nm) in the experiment. Furthermore, by changing the excited state, from 440 nm (maximum absorption of DPAHB) to 480nm, the emission at 680nm decreased and the emission slightly increased at 595nm (fig. 4). According to calculation, this can be done by₂Abovedescribed to eliminate ESPT and produce an absorption at 576 nm (based on 595nm experimental data). After addition of hg (ii) to DPAHB, the most energetically favorable binding site is N1 (fig. 1A-a), which ensures multiple binding sites with parallel stacking of molecules.

Calculation of Hg (II) and H₂The absorption of O coordinated simultaneously on N1 (simplified model) shifts blue. This is due to the fact that the stabilization of HOMO (via Hg (II) linkage to DPAHB at N1) results in an increase in the energy gap of HOMO-LUMO (FIGS. 1A-a). Furthermore, hydrolysis of hg (ii) may eliminate the ESPT process by deprotonation of DPAHB. This resulted in a Stokes shift (91 nm) less than DPAHB, which is consistent with the experimental results (430-350 nm). And the most favorable binding site for MeHg is O2. This may reduce the pKa of the adjacent phenolic hydroxyl groups, inhibiting ESPT by deprotonation. Subsequently, we calculated the Stokes shift of MeHg- (O2) DPAHB complex, which was 108nm, consistent with the experimental data (595-490 nm). Furthermore, it has been observed in this experiment that the small HOMO-LUMO energy gap of the meag- (O2) DPAHB complex leads to red-shifted absorption. Thus, selective optical changes in hg (ii) and MeHg are caused by different ESPT inhibition and changes in HOMO-LUMO energy gaps at different binding sites. As shown in fig. 1B, hg (ii) combined with DPAHB at N1, blue emission at 430 nm. And MeHg is bonded to DPAHB at O2In combination, a yellow signal is produced at 595 nm.

The technical scheme of the invention has the beneficial effects that:

1) the detection sensitivity is improved:

under the condition of not adding any other additional material, the detection sensitivity is improved, the additional material is prevented from being added, the consumption of the additional material is reduced, and the error source in the detection is reduced.

2) Imaging of biological samples is diverse:

the invention successfully detects the imaging of the object to be detected in living cells and zebra fish in the detection, is suitable for real-time monitoring and distinguishing of the cells, the zebra fish and other living bodies, and cannot be realized in the prior method. The realization of imaging has a great driving role in the intensive research of distinguishing mercury from methylmercury.

Drawings

FIG. 1 shows Hg (II) -H₂Molecular orbits (A) and sensing strategies (B) of O- (N1) DPAHB (a), DPAHB (B) and MeHg- (O2) DPAHB (c);

FIG. 2 is a graph showing the H spectrum (A) and C spectrum (B) of BHDP, a compound prepared in example 1;

FIG. 3 is a graph of the H spectra (A) and C spectra (B) of DPAHB compound prepared in example 1;

FIG. 4 is a graph of fluorescence spectra of probe DPAHB in response to Hg (II) and MeHg. A) Hg (ii) a) fluorescence spectra at different hg (ii) concentrations. Inset is fluorescence signal intensity I₄₃₀/I₆₈₀Linear relationship to concentration. b) Selective detection of hg (ii) (Ex =410 nm); B) MeHg. a) fluorescence spectra at different MeHg concentrations. Inset is fluorescence signal intensity I₅₉₅Linear relationship to concentration. b) Selective detection of MeHg (Ex =480 nm);

FIG. 5 is a fluorescence confocal image of HeLa cells with added Hg (II) and MeHg; wherein, the channel 1: the fluorescence collection wavelength is 640-740nm (excitation wavelength: 408 nm); and (3) a channel 2: a fluorescence collection wavelength of 410-470nm (excitation wavelength of 408 nm); and (3) passage: the fluorescence collection wavelength is 550-630nm (excitation wavelength: 488 nm); and (4) passage: bright field; passage 5: and (4) overlapping.

FIG. 6 is a fluorescence confocal image of zebrafish with added Hg (II) and MeHg. Wherein, the channel 1: the fluorescence collection wavelength is 640-740nm (excitation wavelength: 408 nm); and (3) a channel 2: a fluorescence collection wavelength of 410-470nm (excitation wavelength of 408 nm); and (3) passage: the fluorescence collection wavelength is 550-630nm (excitation wavelength: 488 nm); and (4) passage: bright field; passage 5: and (4) overlapping.

Figure 7 is a screen of antidotal agents by confocal imaging of probe DPAHB after dosing. (A) Blank; (B)0.5 h 5. mu.M Hg (II); (C) dimercaptopropanol; (D) acetyl racemic penicillamine; (E) sodium dimercaptopropanesulfonate; (F) dimercaptosuccinic acid sodium salt. Channel 1: 640-740nm (excitation wavelength: 408 nautical miles); and (3) a channel 2: bright field; and (3) passage: a pseudo-color image; and (4) passage: flow cells, regions Q1, Q2, Q3, Q4 are necrotic, late apoptotic, viable, early apoptotic cells, respectively.

FIG. 8 is a comparison of 6 groups of cells in detoxification experiments. (A) Blank; (B)0.5 h 5. mu.M Hg (II); (C) dimercaptopropanol; (D) acetyl racemic penicillamine; (E) sodium dimercaptopropanesulfonate; (F) dimercaptosuccinic acid sodium salt.

FIG. 9 is a graph of optimization of conditions for Hg (II) and MeHg detection operating temperature (A) and pH (B).

FIG. 10 is a real-time fluorescence detection of DPAHB for Hg (II) and MeHg.

FIG. 11 shows the experiment of detecting the cytotoxicity of DPAHB probe to Hela cells in vivo. 1-5 of DPAHB concentration: 0.5, 10, 20 and 30 μ M.

Detailed Description

By describing the small molecule dual response probe of the present invention in conjunction with more specific embodiments, various alternatives or modifications according to the general technical knowledge and the technical means commonly used in the research, while surrounding the technical idea described in the present invention, are included in the scope of the present invention.

The fluorescence detection in the embodiment of the invention is carried out by using an FLS980 fluorescence spectrometer, the excitation wavelength is 410nm/480nm, the emission wavelength is 430 nm and 595nm, the excitation and emission slit widths are both 10.0 nm, and the scanning speed is 2400 nm/min. The UV-Vis spectra were performed by a UV2600 UV-Vis spectrometer with a scanning range of 350-700 nm. Fluorescence imaging observations were performed by nikon A1R fluorescence confocal microscope. The separation and purification of the compound are realized by adopting a thin-layer chromatography silica gel column.

Cytotoxicity experiments: to assess the toxicity of the probe DPAHB in cells, we performed a methylthiazoline tetrazole (MTT) assay. Healthy hela cells were incubated in 10% fetal bovine serum in a 5% carbon dioxide incubator at 37 ℃. Then, different concentrations of probe DPAHB (0. mu.M 5. mu.M 10. mu.M 20. mu.M 30. mu.M) were added to the cells and incubation continued for 4 h. Next, 25 μ LMTT (5mg/mL) was added to each dish and incubation continued for 4 h. Assays were performed using the MTT cytotoxicity assay. All experiments were performed in 8 replicates. Cell viability is expressed as mean ± Standard Deviation (SD).

Cell experiments: hella cells were obtained from the China academy of sciences type culture Collection Committee (Shanghai, China), in DMEM medium supplemented with 10% fetal bovine serum (FBS, Invitrogen), at 37 deg.C, in 5% CO₂The cells were placed in a petri dish, left for 24 hours before imaging, then washed with DMEM medium, cultured with different concentrations of hg (ii) and MeHg or with DMEM medium supplemented with VB12, fluorescence imaging was performed with objective (× 40) confocal laser scanning microscope.

Zebra fish experiment: zebrafish are supplied by Nanjing Ezerlan Bio Inc. All animal experiments were performed in full compliance with international ethical guidelines. Zebrafish imaging employs 7-day old juvenile fish. Zebra fish were incubated in E3 medium (15mM NaCl, 0.5 mM KCl, 1mM MgSO)₄， 1mM CaCl₂， 0.15 mM KH₂PO₄， 0.05 mMNa₂HPO₄And 0.7 mM NaHCO₃pH 7.5), incubation temperature 28 ℃, then, fluorescence imaging of young fish using confocal laser scanning microscope with objective lens (× 10) the medium was refreshed every day.