CN108530446B - Fluorescent probe for identifying thiophenol - Google Patents
Fluorescent probe for identifying thiophenol Download PDFInfo
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- CN108530446B CN108530446B CN201810609532.3A CN201810609532A CN108530446B CN 108530446 B CN108530446 B CN 108530446B CN 201810609532 A CN201810609532 A CN 201810609532A CN 108530446 B CN108530446 B CN 108530446B
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- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
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Abstract
The invention discloses a fluorescent probe capable of rapidly identifying thiophenol (PhSH) and application thereof, belonging to the technical field of chemical analysis and detection, wherein the molecular structural formula is as follows:
the probe has the advantages of good selectivity, high sensitivity, strong anti-interference capability, red light emission and large Stokes displacement. While long wavelength emission (red or near infrared) has good tissue penetration and less background interference; meanwhile, the large Stokes shift can reduce the interference of self-absorption and autofluorescence so as to improve the detection sensitivity. These excellent properties indicate that the fluorescent probe has important application values in the fields of environment, biology and the like.
Description
Technical Field
The invention belongs to the technical field of chemical analysis and detection, and particularly relates to preparation of a fluorescent probe for detecting thiophenol and application of the probe in detecting thiophenol.
Background
Thiophenol, also known as mercaptobenzene, is an important chemical raw material and widely applied to the synthesis process of pesticides, high polymer materials and medicines. Thiophenol, on the other hand, has a foul odor and is a highly toxic compound. Studies have shown that the lethal median concentration (LC50) for fish is 0.1-0.4mM, and the hemilethal dose (LD50) for mice is 46.2mg/kg (oral) or 2.15mg/kg (subcutaneous injection). When exposed to thiophenol, it has strong irritation to eyes, mucous respiratory tract and skin, and can cause death due to larynx, bronchospasm, edema, chemical pneumonia and pulmonary edema after inhalation. The poisoning may be manifested by burning sensation, cough, asthma, laryngitis, short breath, headache, nausea, and emesis. Therefore, it is of great interest to develop a method for detecting thiophenol.
The fluorescence detection method is an important detection method due to high sensitivity, high detection speed and high resolution. In recent years, there are some reports on thiophenol fluorescent probes, but most thiophenol fluorescent probes have slow response speed (>10min), short emission wavelength (in the blue and yellow regions), or small stokes shift (<100 nm). Long wavelength emission (red or near infrared) has good tissue penetration, less background interference, while large stokes shift can reduce interference from self-absorption and autofluorescence to improve detection sensitivity. At present, thiophenol fluorescent probes having long wavelength emission, large stokes shift and rapid correspondence are rarely reported. Therefore, the development of the thiophenol fluorescent probe with the characteristics has great significance.
Disclosure of Invention
In order to overcome the defects of the prior art, the invention aims to provide the fluorescent probe which has high sensitivity and good selectivity, can emit red light and has large Stokes displacement and can quickly detect thiophenol.
The molecular structure of the fluorescent probe is as follows:
the fluorescent probe is prepared by 4 steps of reaction, and the synthetic route is as follows:
the specific synthesis method comprises the following steps: (a) dissolving a compound 1 in ethylene glycol monomethyl ether at room temperature, continuously heating to 115 ℃ until a reaction solution becomes clear, then dropwise adding hydrazine hydrate (80%) into the mixed solution, continuously heating the mixed solution at 115 ℃ for reflux reaction for 3 hours after dropwise adding is finished, cooling to room temperature after the reaction is finished, precipitating a large amount of orange-yellow precipitates, filtering and washing to obtain a compound 2; (b) dissolving the compound 2 in 3-methyl-2-butanone, slowly dropwise adding concentrated sulfuric acid, refluxing and stirring the reaction mixed solution under the protection of nitrogen, performing reduced pressure rotary evaporation on the residual 3-methyl-2-butanone to obtain a crude product, and performing separation and purification by using a silica gel column layer to obtain a light yellow solid product 3; (c) dissolving the compound 3 and p-hydroxybenzaldehyde in toluene, uniformly stirring, sequentially dropwise adding glacial acetic acid and piperidine into the mixed solution, refluxing and stirring the mixed solution under the protection of nitrogen after dropwise adding is finished, removing toluene by reduced pressure distillation after reaction is finished to obtain a crude product, and separating and purifying by using a silica gel column layer to obtain a red solid product 4; (d) dissolving the compound 4 and 2, 4-dinitrofluorobenzene in acetone, adding anhydrous potassium carbonate, refluxing and stirring, obtaining a crude product after the reaction is finished, and separating and purifying by using a silica gel column layer to obtain an orange solid product 5, namely a probe molecule.
The fluorescent probe has the following mechanism that the fluorescent probe does not have fluorescence and reacts with thiophenol to cause the 2, 4-dinitrobenzene ring to be partially removed, so that the dye 6 is generated and a red fluorescent signal is emitted. The response process of the probe molecule is as follows:
the fluorescent probe has large Stokes displacement, the maximum absorption is 450nm, the maximum emission is 590nm after the fluorescent probe reacts with thiophenol, and the Stokes displacement is 140 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 after adding 15 equivalents of thiophenol, the fluorescence intensity at 590nm of the maximum emission wavelength increases by a factor of 5. And adding the detection substances (Cys, Hcy, Phe, Trp, Leu, NO)3 -、SO3 2-、S2O3 2-、NO2 -、Ca2+、K+、PO4 3-、SO4 2-、SCN-、Na+、Mg2+、H2O2、Fe3+、S2-、ClO-、Mn2+) After that, there was only little increase in fluorescence.
The fluorescent probe of the invention has strong anti-interference capability, and other detection objects (Cys, Hcy, Phe, Trp, Leu and NO)3 -、SO3 2-、S2O3 2-、NO2 -、Ca2+、K+、PO4 3-、SO4 2-、SCN-、Na+、Mg2+、H2O2、Fe3+、S2-、ClO-、Mn2+) The presence of (a) does not affect the effect of detecting thiophenol.
After 15 times of equivalent of thiophenol is added into the fluorescent probe, the fluorescence is immediately enhanced and reaches the maximum value in 5 min.
The fluorescent probe can carry out quantitative detection on the thiophenol.
The fluorescent probe of the present invention can be applied to the test of actual water samples, and the recovery rate of adding thiophenol in tap water and mineral water is used for the product in the present specification.
The fluorescent probe has low cytotoxicity, the survival rate is more than 95% when the fluorescent probe is cultured for 24 hours within 20 mu M. In addition, the probe has good cell membrane penetrability, and can detect exogenous thiophenol in cells.
The probe molecule has near infrared emission wavelength after response to the thiophenol (PhSH), has large Stokes shift, good selectivity and anti-interference capability, good sensitivity and wider application range, and has practical application value in the fields of biology, chemistry and the like.
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FIG. 1 shows the change of fluorescence spectra of the fluorescent probe of the present invention (10.0. mu.M) in PBS buffer (10mM, pH 7.4,1.0mM CTAB) after the probe was reacted with different concentrations of thiophenol (PhSH), with the wavelength on the abscissa and the fluorescence intensity on the ordinate.
FIG. 2 is a linear relationship of the fluorescence intensity at 590nm with time during the action of the fluorescent probe of the present invention (10.0. mu.M) with thiophenol (PhSH) in a PBS buffer (10mM, pH 7.4,1.0mM CTAB), with time on the abscissa and fluorescence intensity on the ordinate.
FIG. 3 shows the selectivity of the fluorescent probe of the present invention in PBS buffered at 10.0. mu.MThe infusion solution (10mM, pH 7.4,1.0mM CTAB) was mixed with PhSH, Cys, Hcy, Phe, Trp, Leu, NO3 -、SO3 2-、S2O3 2-、NO2 -、Ca2+、K+、PO4 3-、SO4 2-、SCN-、Na+、Mg2+、H2O2、Fe3+、S2-、ClO-、Mn2+The abscissa of the fluorescence spectrum after the action is the wavelength, and the ordinate is the fluorescence intensity.
FIG. 4 shows the interference resistance of the fluorescent probe of the present invention, thiophenol (PhSH) and analytes (Cys, Hcy, Phe, Trp, Leu, NO)3 -、SO3 2-、S2O3 2-、NO2 -、Ca2+、K+、PO4 3-、SO4 2-、SCN-、Na+、Mg2+、H2O2、Fe3+、S2-、ClO-、Mn2+) In the coexistence, the fluorescence intensity ratio (I/I) of the fluorescent probe after the interaction with thiophenol (PhSH) in PBS buffer (10.0. mu.M, pH 7.4,1.0mM CTAB) with a fluorescent probe (10.0. mu.M)0) A histogram.
FIG. 5 shows the change of fluorescence intensity at 590nm with time in PBS buffer (10mM, pH 7.4,1.0mM CTAB) with time on the abscissa and fluorescence intensity on the ordinate during the reaction of the probe with thiophenol (PhSH) in 10.0. mu.M fluorescent probe of the present invention.
FIG. 6 is confocal cell imaging of fluorescent probes of the invention for detection of thiophenol (PhSH) in Hela cells. A1-A3 is the imaging effect of cells incubated with thiophenol (PhSH) (150.0. mu.M) for 15min at 37 ℃ followed by a probe (10.0. mu.M) for 15 min. B1-B3 is the imaging effect of a probe (10.0. mu.M) incubated at 37 ℃ for 15min in cells.
FIG. 7 is a toxicity test of the fluorescent probe of the present invention on cells. The concentration of the probe molecules is 2.0X 10-5At mol/L, the cell survival rate is more than 95%.
Detailed description of the preferred embodiment
Example 1: synthesis of Compound 2
Dissolving a compound 1(3.32g, 10.0mmol) in 50mL of ethylene glycol monomethyl ether, heating to 115 ℃ until the reaction solution becomes clear, dropwise adding 3.0mL of hydrazine hydrate (80%) into the mixed solution, continuously heating and refluxing the mixed solution for reaction for 3 hours after the dropwise adding is finished, cooling to room temperature after the reaction is finished, separating out a large amount of orange-yellow precipitate, filtering and washing to obtain a compound 2. Yield: 2.32 g; yield: 81.9 percent.
Example 2: synthesis of Compound 3
Dissolving the intermediate 2(1.98g, 7mmol) in 3-methyl-2-butanone (100mL), slowly dropwise adding 3mL concentrated sulfuric acid, refluxing and stirring the reaction mixture under nitrogen for 4 hours, after the reaction is finished, performing reduced pressure rotary evaporation to remove 3-methyl-2-butanone to obtain a crude product, and performing separation and purification by using a silica gel column (petroleum ether/dichloromethane is 1/10, v/v) to obtain a light yellow solid product 3. Yield: 1.13 g; yield: 48.5 percent.
Example 3: synthesis of Compound 4
Dissolving the compound 3(1.0g and 3mmol) and parahydroxybenzaldehyde (0.37g and 3mmol) in 20mL of dry toluene, uniformly stirring, sequentially dropwise adding glacial acetic acid (0.02mL) and piperidine (0.05mL) into the mixed solution, after dropwise adding, refluxing and stirring the mixed solution for 6 hours under the protection of nitrogen, rotationally evaporating the toluene under reduced pressure to obtain a crude product, and separating and purifying by column chromatography (ethanol/dichloromethane is 1/50, v/v) to obtain a red solid product 4. Yield: 0.68 g; yield: 51.7 percent.
Example 4: synthesis of Probe 5
Compound 4(0.483g, 1mmol), 2, 4-dinitrofluorobenzene (0.279g, 1.5mmol) were dissolved in 20mL of dry acetone, anhydrous potassium carbonate (0.276g, 2mmol) was added thereto, the mixture was stirred under reflux for 4 hours, the reaction mixture was suction filtered to obtain a filtrate, the solvent was evaporated under reduced pressure by rotary evaporation to obtain a crude product, which was separated and purified by a column layer (petroleum ether/dichloromethane ═ 1:10, v/v) to obtain an orange solid product 5, i.e., a probe molecule (0.212g, 35.2%).
Example 5: application of probe molecule in detection of thiophenol in actual water sample
Tap water and mineral water were filtered through a filter membrane (0.22 μm), and then prepared into a PBS buffer solution (10mM, pH 7.4,1.0mM CTAB). Probe molecules were dissolved in the above buffer solution to prepare a 10.0. mu.M probe molecule solution, to which thiophenol (0.0, 4.0, 8.0, 12.0, 16.0. mu.M) was added, and good recoveries (96% -105%) were obtained in both tap water and mineral water samples as shown in Table 1, indicating that the probe can be used for the detection of thiophenol in actual water samples.
TABLE 1 recovery of thiophenol in tap water and mineral water with fluorescent probes
Example 6: application of probe molecule in detecting thiophenol in cell
Hela cells were first incubated for 15min with PBS buffer (10.0mM, pH 7.4) containing thiophenol (150.0 μ M), and rinsed 3 times with PBS buffer; then incubated with PBS buffer solution containing probe (10.0 μ M) for 15min, rinsed with PBS buffer solution for 3 times, and subjected to cell fluorescence imaging by laser confocal fluorescence microscope, so that strong fluorescence signals can be seen. In the control experiment, HeLa cells were incubated in PBS buffer (10.0mM, pH 7.4) containing a probe (10.0 μ M) only for 15min, rinsed with PBS buffer, and subjected to fluorescence imaging by a confocal laser fluorescence microscope, and an extremely weak fluorescence signal was observed. These results indicate that the probe can detect thiophenol in living cells.
Claims (1)
1. A fluorescent probe for identifying thiophenol is characterized in that the structural formula is as follows:
the detection system of the fluorescent probe contained 1.0mM MCTAB in 10mM PBS buffer solution with pH 7.4, and the fluorescent probe detected thiophenol at 25 ℃.
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| CN109180561B (en) * | 2018-11-14 | 2021-07-27 | 中国科学院海洋研究所 | Application of chalcone fluorescent probe in detection of thiophenol compounds in aqueous solution |
| CN109810107A (en) * | 2019-03-02 | 2019-05-28 | 郑州大学 | A kind of fluorescence probe and preparation method thereof identifying mercaptoamino acid |
| CN109913207B (en) * | 2019-04-11 | 2020-05-12 | 济南大学 | Fluorescent probe for detecting long-wave emission of thiophenol |
| CN109879851B (en) * | 2019-04-11 | 2021-08-31 | 济南大学 | Near-infrared fluorescent probe for specifically detecting thiophenol |
| CN111138431B (en) * | 2020-01-13 | 2022-02-15 | 商丘师范学院 | Reactive fluorescent probe for detecting thiophenol and synthetic method and application thereof |
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| CN105566942A (en) * | 2015-10-13 | 2016-05-11 | 华东理工大学 | Preparation method of large emission wavelength fluorescent dye |
| CN107325095A (en) * | 2017-07-04 | 2017-11-07 | 济南大学 | A kind of lysosome hypochlorous acid fluorescence probe and its preparation method and application |
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| CN105566942A (en) * | 2015-10-13 | 2016-05-11 | 华东理工大学 | Preparation method of large emission wavelength fluorescent dye |
| CN107325095A (en) * | 2017-07-04 | 2017-11-07 | 济南大学 | A kind of lysosome hypochlorous acid fluorescence probe and its preparation method and application |
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| Development of a unique family of two-photon full-color-tunable fluorescent materials for imaging in live subcellular organelles, cells, and tissues;Chen, Hua; 等;《Journal of Materials Chemistry B: Materials for Biology and Medicine》;20170224;第5卷(第13期);2436-2444 * |
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