阅读说明：本技术 一种单波长激发区分检测GSH和H2Sn(n>1)的荧光探针 (Single-wavelength excitation differential detection GSH and H2Sn(n>1) Fluorescent probe of ) 是由 刘兴江 肜一凡 魏柳荷 于 2020-07-13 设计创作，主要内容包括：本发明公布了一种单激发区分检测GSH和H<Sub>2</Sub>S<Sub>n</Sub>(n>1)的荧光探针,属于化学分析检测技术领域,其分子结构式如下：<Image he="275" wi="520" file="DDA0002581134980000011.GIF" imgContent="drawing" imgFormat="GIF" orientation="portrait" inline="no"></Image>该探针与GSH反应后发出绿色荧光,与H<Sub>2</Sub>S<Sub>n</Sub>反应后发出红色荧光。该探针在单波长激发下即可实现对GSH和H<Sub>2</Sub>S<Sub>n</Sub>(n>1)的同时区分检测,而且具有较好的选择性、高灵敏度、较宽的pH工作范围等特点。同时,在检测过程中该探针能发射出红色荧光。这些优良的性能表明该荧光探针在环境及生物等领域具有重要的应用价值。(The invention discloses a single-excitation differential detection method for GSH and H 2 S n (n>1) The fluorescent probe belongs to the technical field of chemical analysis and detection, and the molecular structural formula is as follows: the probe emits green fluorescence after reacting with GSH, and reacts with H 2 S n After the reaction, red fluorescence was emitted. The probe can realize GSH and H under the excitation of single wavelength 2 S n (n>1) Meanwhile, the kit can distinguish detection, and has the characteristics of good selectivity, high sensitivity, wide pH working range and the like. Meanwhile, the probe can emit red fluorescence during the detection process. These excellent properties indicate that the fluorescent probe is in the environment and biologyAnd the like, and has important application value.)

1. Single-wavelength excitation differential detection GSH and H₂S_n(n>1) The structural formula of the fluorescent probe is as follows:the detection system of the fluorescent probe comprises 10mM PBS buffer solution with pH 7.4 and 1.0mM CTAB, and the fluorescent probe is detected at 25 ℃; the fluorescent probe is used for detecting GSH and H₂S_n(n>1) The maximum emission wavelength is 530nm and 670nm, and the excitation wavelength is 480 nm.

Technical Field

The invention belongs to the technical field of chemical analysis and detection, and particularly relates to a method for simultaneously distinguishing and detecting GSH and H by single-wavelength excitation₂S_n(n > 1) fluorescent probe, and use of the probe in detecting GSH and H₂S_n(n > 1).

Background

Active sulfur species are indispensable in the organism and play an important role in physiological and pathological processes. Glutathione (GSH) and hydrogen polysulphides(H₂S_n(n > 1)) have important roles in antioxidation, apoptosis and signal transduction. With respect to GSH and H₂S_nFor example, GSH is the sulfur source for cells that are enzymatically produced by cystathionine-gamma-lyase (CSE) and cystathionine- β -synthase (CBS). H can be detected in high levels in plasma, cells and tissues of mammals₂S_nThe precursor, glutathione hydrogen sulfide (> 100. mu.M). An abnormal concentration of each active sulfur species within the cell may have an effect on the concentration of other active sulfur species, which can cause serious health problems. To study GSH and H₂S_nComplex biological relationship between GSH and H, development of the reagent for simultaneously distinguishing and detecting GSH and H₂S_nThe fluorescent probe of (2) is very valuable.

The fluorescent probe technology has become an effective analysis and detection method due to the advantages of convenient operation, nondestructive detection, high space-time resolution and the like. In 2017, Chen et al (Analytical Chemistry, 2017,89(23), 12984-₂S_n(n > 1) fluorescent probes, but the fluorescent probes emit shorter wavelengths in the blue and green regions; in addition, dual wavelength excitation is required during the application. The dual-wavelength excitation type probe needs to set related instrument parameters such as different excitation wavelengths according to different analysis targets, so that the operation is complicated, unnecessary background interference can be caused by multi-wavelength excitation, and the accuracy of a detection result is influenced. On the contrary, the single-wavelength excitation type probe works under the excitation of single wavelength, so that the fluorescence detection and the biological imaging are more convenient and efficient, the background interference caused by the excitation of multiple wavelengths can be reduced, the fluorescence interference generated by the cell can be effectively reduced during the cell imaging, and the accuracy is higher. In addition, the red light or the near infrared light has good tissue penetrability and small background interference so as to improve the detection accuracy. Currently, single wavelength excitation with long wavelength emission is developed for the differential detection of GSH and H₂S_nFluorescent probes of (n > 1) remain challenging.

Disclosure of Invention

In order to overcome the defects of the prior art, the invention aims to provide a single-wavelength excitation method for distinguishing and detecting GSH and H₂S_n(n > 1) and a fluorescent probe having red light emission.

The molecular structure of the fluorescent probe is as follows:

the fluorescent probe is prepared by the following reaction, and the synthesis process is as follows:

the specific synthesis method comprises the following steps: compound 4 and compound 8 are dissolved in anhydrous dichloromethane and 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride and 4-dimethylaminopyridine are added. Reacting at room temperature under the protection of nitrogen to obtain a crude product, and separating and purifying by using a silica gel column layer to obtain an orange-yellow solid product, namely the probe.

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

when the fluorescent probe reacts with GSH, the phenylselenol part (Site 1) is replaced by sulfhydryl of GSH due to nucleophilicity of sulfhydryl, and green fluorescence is emitted. And H₂S_nDuring reaction, nucleophilic substitution reaction is firstly carried out on the probe site 1, and then intramolecular cyclization reaction is carried out on the probe site 2, so that red fluorescence is emitted.

Selection of fluorescent Probe of the present invention for Na₂S₂As H₂S_nDue to the source of H₂S₂With other hydrogen polysulphides (H)₂S_nAnd n > 2) are in dynamic equilibrium.

The fluorescent probe of the invention is a single-wavelength excitation type differential detection GSH and H₂S_n(n > 1) fluorescent probes, set stimuli during the testThe emission wavelength was 480 nm.

The maximum emission peak of the fluorescent probe of the invention after reaction with GSH is 530nm, and Na₂S₂The maximum emission peak after the reaction was 670 nm.

The fluorescent probe has good selectivity. The probe molecules were tested in 10mM PBS buffer containing 1.0mM CTAB at pH 7.4 at 25 ℃. The probe molecule itself is in a fluorescence quenching state, and the fluorescence intensity increases 2.7 times at the maximum emission wavelength of 530nm after 4.0 equivalent Glutathione (GSH) is added. And adding the detection substances (NaCl, KCl, CaCl)₂、ZnCl₂、MgCl₂、NaClO、 Na₂S₂O₃、Na₂SO₃、NaHSO₃、Na₂SO₄、Na₂S、H₂O₂L-Pro, L-Ser, L-Glu, DL-Tyr, Cys and Hcy), there was almost no increase in fluorescence.

The fluorescent probe has good selectivity. The probe molecules were tested in 10mM PBS buffer containing 1.0mM CTAB at pH 7.4 at 25 ℃. After 18 times equivalent of sodium persulfate (Na) is added₂S₂) After that, the fluorescence intensity at the maximum emission wavelength of 670nm was increased by 8 times. And adding the detection substances (NaCl, KCl, CaCl)₂、ZnCl₂、MgCl₂、NaClO、Na₂S₂O₃、Na₂SO₃、NaHSO₃、Na₂SO₄、Na₂S、H₂O₂Almost no increase in fluorescence was observed after addition of L-Pro, L-Ser, L-Glu, DL-Tyr, Cys and Hcy.

The fluorescent probe has strong anti-interference capability and can be used for detecting other detection objects (NaCl, KCl and CaCl)₂、 ZnCl₂、MgCl₂、NaClO、Na₂S₂O₃、Na₂SO₃、NaHSO₃、Na₂SO₄、Na₂S、H₂O₂L-Pro, L-Ser, L-Glu, DL-Tyr, Cys and Hcy) hardly affect the detection of Glutathione (GSH) and sodium persulfate (Na)₂S₂) The effect of (1).

After the fluorescent probe of the invention acts with Glutathione (GSH) with 4.0 times of equivalent respectively, the fluorescence is immediately enhanced and reaches the maximum value in 4.5 minutes. 18 times equivalent of sodium persulfate (Na) was added₂S₂) After this time, fluorescence immediately increased to a maximum at 45 minutes.

The fluorescent probe can be used for treating Glutathione (GSH) and sodium persulfate (Na)₂S₂) And respectively carrying out quantitative detection. The fluorescent probe has good linearity, and the linear correlation coefficients are respectively as follows: glutathione (GSH) R ═ 0.9959, sodium persulfate (Na)₂S₂)R＝0.9976。

The fluorescent probe has good cell membrane penetrability, and can be used for Glutathione (GSH) and sodium persulfate (Na) in cells₂S₂) Detection of (3).

The probe has wide pH application range, and can detect pH 6.0 to pH 9.0.

The probe molecule of the invention is sodium persulfate (Na)₂S₂) And has red emission after response.

The probe molecule can realize the treatment of Glutathione (GSH) and sodium persulfate (Na) under the excitation of single wavelength₂S₂) While distinguishing between detections.

Drawings

Fig. 1 shows the change of fluorescence spectrum of the fluorescent probe of the present invention (10.0 μ M) after reacting with Glutathione (GSH) at different concentrations in a PBS buffer solution (10mM, pH 7.4,1.0mM CTAB), with the wavelength on the abscissa and the fluorescence intensity on the ordinate.

FIG. 2 is a linear relationship of fluorescence intensity at 530nm with concentration during the action of the fluorescent probe of the present invention (10.0. mu.M) with Glutathione (GSH) in PBS buffer (10mM, pH 7.4,1.0mM CTAB), with concentration on the abscissa and fluorescence intensity on the ordinate.

FIG. 3 shows fluorescent probes of the invention (10.0. mu.M) in PBS buffer (10mM, pH 7.4,1.0mM CTAB) with different concentrations of sodium peroxosulfide (Na)₂S₂) The fluorescence spectrum after the action changes, the abscissa is the wavelength, and the ordinate is the fluorescence intensity.

FIG. 4 shows the reaction of a fluorescent probe of the present invention (10.0. mu.M) with hemithionic sodium (Na) sulfide in PBS buffer (10mM, pH 7.4,1.0mM CTAB)₂S₂) The linear relation of the fluorescence intensity at 670nm along with time in the action process, the abscissa is concentration, and the ordinate is fluorescence intensity.

FIG. 5 shows the selectivity of the fluorescent probes of the present invention in PBS buffer (10mM, pH 7.4,1.0mM CTAB) with GSH and other analytes (NaCl, KCl, CaCl)₂、ZnCl₂、MgCl₂、NaClO、Na₂S₂O₃、Na₂SO₃、NaHSO₃、Na₂SO₄、Na₂S、 H₂O₂The ratio of fluorescence intensities (I) after the actions of L-Pro, L-Ser, L-Glu, DL-Tyr, Cys and Hcy)_Probe+others/I_Probe) A histogram.

FIG. 6 shows the selectivity of the fluorescent probe of the present invention in PBS buffer (10mM, pH 7.4,1.0mM CTAB) and Na (10.0. mu.M)₂S₂And other analytes (NaCl, KCl, CaCl)₂、ZnCl₂、MgCl₂、NaClO、Na₂S₂O₃、Na₂SO₃、NaHSO₃、Na₂SO₄、Na₂S、 H₂O₂The ratio of fluorescence intensities (I) after the actions of L-Pro, L-Ser, L-Glu, DL-Tyr, Cys and Hcy)_Probe+others/I_Probe) A histogram.

FIG. 7 is a graph showing interference resistance of the fluorescent probe of the present invention, Glutathione (GSH) and analytes (NaCl, KCl, CaCl)₂、ZnCl₂、MgCl₂、NaClO、Na₂S₂O₃、Na₂SO₃、NaHSO₃、Na₂SO₄、 Na₂S、H₂O₂L-Pro, L-Ser, L-Glu, DL-Tyr, Cys and Hcy) in the presence of a fluorescent probe (10.0. mu.M) reacted with Glutathione (GSH) in PBS buffer (10mM, pH 7.4,1.0 mMCTAB)_Probe+others/I_Probe+GSH) A histogram.

FIG. 8 shows the interference resistance of the fluorescent probe of the present invention, sodium peroxosulfide (Na)₂S₂) With analytes (NaCl, KCl, CaCl)₂、ZnCl₂、MgCl₂、NaClO、Na₂S₂O₃、Na₂SO₃、NaHSO₃、Na₂SO₄、 Na₂S、H₂O₂L-Pro, L-Ser, L-Glu, DL-Tyr, Cys and Hcy) in the presence of the fluorescent probe, the ratio of the fluorescence intensity after the interaction with homocysteine (Hcy) in PBS buffer (10.0. mu.M, pH 7.4,1.0 mMCTAB) (I._Probe+others/I_Probe+Na2S2) A histogram.

FIG. 9 shows the change of fluorescence intensity at 530nm with time during the reaction of the fluorescent probe (10.0. mu.M) of the present invention with Glutathione (GSH) in PBS buffer (10mM, pH 7.4,1.0mM CTAB), with time on the abscissa and fluorescence intensity on the ordinate.

FIG. 10 shows the fluorescent probe of the present invention (10.0. mu.M) in PBS buffer (10mM, pH 7.4,1.0mM CTAB) with sodium persulfate (Na)₂S₂) The change of the fluorescence intensity at 670nm along with time in the action process, the abscissa is time, and the ordinate is the fluorescence intensity.

FIG. 11 shows fluorescence intensities of a fluorescent probe (10.0. mu.M) of the present invention before and after the interaction with Glutathione (GSH) in buffer solutions of different pH values, with pH on the abscissa and fluorescence intensity on the ordinate.

FIG. 12 shows fluorescent probes of the invention (10.0. mu.M) in buffer solutions of different pH values with sodium persulfate (Na)₂S₂) The abscissa of fluorescence intensity before and after the action is pH, and the ordinate is fluorescence intensity.

FIG. 13 shows the fluorescent probe of the present invention for detecting Glutathione (GSH) and sodium persulfate (Na) in RAW264.7 cells₂S₂) Confocal cell imaging. A1-A3 is the imaging effect of a probe (10.0. mu.M) incubated at 37 ℃ for 30 minutes in cells. B1-B3 is sodium sulfide (Na) used by cells at 37 deg.C₂S₂) Imaging effect of 15 min incubation (220.0. mu.M) followed by 30 min incubation with probe (10.0. mu.M). C1-C3 is prepared by first using N-ethyl maleic acid at 37 deg.CImaging was performed by pre-treating with imide (1mM) for 15 min and then incubating with probe (10.0. mu.M) for 30 min.

FIG. 14 shows the fluorescent probe of the present invention for detecting endogenous sodium persulfate (Na) in RAW264.7 cells₂S₂) Confocal cell imaging. A1-A3 is cells incubated with Lipopolysaccharide (LPS) (1. mu.g/mL) for 8 hours at 37 ℃. Treatment was again carried out with NEM (1mM) for 15 minutes. Then, incubation with cystine (200. mu.M) was performed for 30 minutes. Finally, the imaging effect was incubated with the probe (10.0. mu.M) for 30 minutes. B1-B3 cells were treated with NEM (1mM) for 15 min at 37 ℃. Then, incubation with cystine (200. mu.M) was performed for 30 minutes. Finally, the imaging effect was incubated with the probe (10.0. mu.M) for 30 minutes.

Detailed description of the preferred embodiment