A Fluorescent Probe for Rapid Detection of Benzenethiol, Its Preparation, Kit and Detection Method

Abstract

The present invention discloses a rapid detection fluorescent probe for thiophenol, its preparation method, kit and detection method. The technical solution adopted by the present invention uses thiazole hemicyanine dye as the fluorophore and 2,4-dinitrobenzenesulfonyl chloride as the recognition group to synthesize the fluorescent probe HD-B. The probe itself does not have fluorescence. Thiophenol can break the sulfonyl ester bond in the probe and release the fluorophore, thereby enhancing fluorescence. During the reaction of the probe HD-B with thiophenol, the solution changes from colorless to orange; based on this characteristic, a colorimetric kit is designed. The kit contains reagent stock solution a and reagent stock solution b; the stock solution a is a phosphate buffer solution, and the reagent stock solution b is an organic solution of the probe HD-B. This kit has the advantages of fast reaction speed, high sensitivity and visual detection. Moreover, the probe only has the characteristic of specific recognition for thiophenol and is not interfered by other common inorganic salts and biological thiols.

Description

Technical Field

[0001] The present invention relates to the preparation of a fluorescent probe for rapid detection of thiophenol, a kit and a detection method, and belongs to the field of organic small molecule fluorescent probes. Background Art

[0002] Thiophenol is an intermediate in a variety of organic syntheses and plays a crucial role in the production of industrial organic syntheses. It is widely used in the preparation and synthesis of intermediates of fine chemicals such as dyes, cosmetics, pharmaceuticals, pesticides, polymerization inhibitors, antioxidants, etc. However, thiophenol belongs to a highly toxic substance and has a strong irritating effect on eyes, mucous membranes, respiratory tract and skin. Therefore, the use of thiophenol has inevitable problems of high toxicity and high pollution. The median lethal dose (LC50) of thiophenol for fish ranges from 0.01 to 0.40 mmol·L -1 , and the LC50 for mice is 2.15 to 46.2 mg·kg -1 , and it has been added to the priority pollutants by the US Environmental Protection Agency (EPA waste code: P014). Therefore, the detection of the content of thiophenol has a crucial impact on organisms in the human environment.

[0003] At present, methods for detecting thiophenol such as high performance liquid chromatography, gas chromatography, ultraviolet spectrophotometry and sensors based on nanomaterials can be used to detect the concentration of thiophenol. However, these methods have complex sample pretreatment, low sensitivity, are easily interfered, require expensive instruments, and need personnel with a certain professional technical level to operate, and are only suitable for research and analysis in laboratories. Fluorescent probes have been widely used in the detection of thiophenol content due to their simplicity, high sensitivity and good specificity. Reagent test kits have been widely used due to their advantages of being portable, easy to operate quickly and capable of real-time detection. However, the combination of fluorescent probes and test kits for the detection of thiophenol content is extremely rare.

[0004] Among the reported probes for detecting the specificity of thiophenol, most fluorescent probes only have a fluorescent response in the visible region, but the fluorescent response is relatively slow and is easily interfered. In addition, most fluorescent probes only show a fluorescent reaction without any obvious color change, which greatly limits their applications. Therefore, it is of great significance to study a thiophenol fluorescent probe reagent test kit with good selectivity, high sensitivity, simple operation, strong anti-interference ability, visualization and wide application for the on-site detection of thiophenol content. Summary of the Invention

[0005] Aiming at the problems of slow reaction rate, poor specificity and poor visualization of existing probes for detecting thiophenol, the object of the present invention is to provide a fluorescent probe for rapid detection of thiophenol, which has the characteristics of high specificity, high sensitivity and good visualization performance for detection.

[0006] Another object of the present invention is to provide a method for preparing a fluorescent probe for rapid detection of benzenethiol, which has simple synthesis steps and low cost.

[0007] Another object of the present invention is to provide a kit for rapid detection of benzenethiol, which can be used to detect benzenethiol in environmental water samples and analyze the content of benzenethiol in the water samples.

[0008] A fluorescent probe for rapid detection of benzenethiol, whose structural formula is shown as formula HD-B:

[0009]

[0010] A method for preparing a fluorescent probe for rapid detection of benzenethiol includes the following steps:

[0011] Step 1: Mix 3-ethyl-2-methylbenzothiazol-3-ium iodide, 4-hydroxybenzaldehyde and absolute ethanol, heat the mixture for reaction, and after the reaction is completed, filter to obtain a solid product HD.

[0012] Step 2: Dissolve 2,4-dinitrobenzenesulfonyl chloride in anhydrous dichloromethane to obtain solution A; drop solution A into the solid product HD, then add triethylamine to the mixture, stir the reaction at low temperature for a period of time, then heat and stir, and react overnight to obtain a crude product C; after column chromatography of the crude product C, a pure product probe HD-B is obtained.

[0013] The synthesis steps included in this route are as follows:

[0014]

[0015] The results of the structural characterization data of this fluorescent probe are as follows:

[0016] 1H NMR (400 MHz, DMSO): δ (ppm) 9.14 (d, 1H, J = 2.3 Hz), 8.64 (t, 1H, J = 8.8, 2.3 Hz), 8.47 (d, 1H, J = 8.1 Hz), 8.39 (t, 1H, J = 8.6, 2.3 Hz), 8.33 (t, 2H, J = 8.1 Hz), 8.16 (d, 2H, J = 8.4 Hz), 8.10–8.06 (m, 1H), 7.91 (t, 1H, J = 7.8 Hz), 7.82 (t, 1H, J = 7.7 Hz), 7.41 (d, 2H, J = 8.4 Hz), 4.98 (d, 2H, J = 7.3 Hz,), 1.47 (t, 3H, J = 7.1 Hz).

[0017] A fluorescent probe for rapid detection of benzenethiol can be used for quantitative analysis of the content of benzenethiol in water samples in the laboratory. The specific detection method is as follows:

[0018] Using 490 - 530 nm as the excitation wavelength, measure the fluorescence intensities of a series of different benzenethiol standard sample solutions at the emission wavelength of 550 - 590 nm, denoted as F. Plot a standard curve with the benzenethiol concentration as the abscissa and the fluorescence intensity value F as the ordinate. The different concentrations of benzenethiol standard samples are prepared in the laboratory.

[0019] The principle of detecting benzenethiol by a rapid detection benzenethiol fluorescence probe of the present invention is as follows:

[0020] The probe HD - B of the present invention uses a thiazole hemicyanine structure as the fluorophore and 2,4 - dinitrobenzenesulfonyl chloride as the specific response group. After the fluorophore reacts with 2,4 - dinitrobenzenesulfonyl chloride, the fluorescence is quenched, which is due to the occurrence of a photoinduced electron transfer (PET) process between the two. When the probe HD - B reacts with benzenethiol, the recognition group detaches, and the probe releases the fluorophore with enhanced fluorescence, and the solution color changes from colorless to orange. To explore the recognition mechanism of the probe HD - B with benzenethiol, the product obtained by reacting the probe HD - B with benzenethiol was characterized by mass spectrometry. The results show that: a new peak at m / z = 282 appears in the mass spectrometry data (corresponding to HD[M + H] + ), and the peak at m / z = 512 disappears (corresponding to HD - B[M + H] + ). The mass spectrometry results indicate that when the probe HD - B reacts with benzenethiol, the sulfonyl ester bond in the probe breaks, releasing the fluorophore HD, thereby enhancing the fluorescence of the probe solution, and the solution color changes from colorless to orange, so as to achieve specific recognition and visual detection of benzenethiol.

[0021] The beneficial effects of the present invention are:

[0022] A rapid detection benzenethiol fluorescence probe provided by the present invention and its preparation, kit and detection method have the advantages that the preparation method of the fluorescence probe is simple, the kit is easy to operate and inexpensive; it has high sensitivity, strong anti - interference ability and fast reaction speed with benzenethiol; the concentration of benzenethiol can be quickly determined by colorimetry; the content of benzenethiol can be quantitatively detected in the laboratory; it has broad application prospects in environmental water samples such as rivers, lakes and tap water. Description of the Drawings

[0023] Figure 1 It is a schematic diagram of the reaction mechanism of the probe HD - B.

[0024] Figure 2 It is the 1H - NMR spectrum of the probe HD - B.

[0025] Figure 3 It is the mass spectrum of the probe HD - B.

[0026] Figure 4They are the UV and fluorescence spectra of probe HD-B and probe + benzenethiol.

[0027] Figure 5 It is the fluorescence diagram of the stability of probe HD-B.

[0028] Figure 6 It is the linear diagram and equation for the detection of benzenethiol by probe HD-B.

[0029] Figure 7 It is the anti-interference ability test diagram of probe HD-B. The concentration of probe HD-B is 10 μM, and the concentrations of metal ions (Na + , K + , Mg 2+ , Ni 2+ , Hg 2+ ), anions (HSO3 - , SO3 2- , SO4 2- , ClO - , S2O3 2- , Br - , Cl - , CO3 2- , NO3 - , NO2 - , CH3COO - ) are all 1 mM, the concentrations of transition metal ions (Ca 2+ , Cr 3+ , Mn 2+ , Zn 2+ , Fe 2+ ) are 100 μM, and the concentrations of biological thiols (GSH, Hcy, Cys) and the common oxide H2O2 are all 160 μM.

[0030] Figure 8 It is the mass spectrometry mechanism verification diagram for the detection of benzenethiol by probe HD-B.

[0031] Figure 9 It is a simple diagram for the use of the kit based on probe HD-B.

[0032] Figure 10 It is the colorimetric card made of probe HD-B in the kit. Specific implementation method

[0033] The technical solutions in the embodiments of the present invention will be clearly and completely described below. The described embodiments are only a part of the embodiments of the present invention, not all of the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0034] Example 1: Synthesis of HD

[0035] 3-Ethyl-2-methylbenzothiazol-3-ium iodide (366.2 mg, 1.2 mM) and 4-hydroxybenzaldehyde (146.7 mg, 1.2 mM) were placed in a 25 mL round-bottom flask and added to 4 mL of absolute ethanol. The mixture was reacted at 65 °C for 4 h under an inert gas atmosphere, cooled to room temperature, quenched with diethyl ether, and the crude product was filtered. It was recrystallized from methanol to obtain a red solid HD.

[0036]

[0037] Example 2: Spectral Test of HD-B

[0038] The probe HD-B was prepared as a 1 mM stock solution with dimethyl sulfoxide, and benzenethiol was prepared as a 1 mM stock solution with absolute ethanol.

[0039] To investigate the response of the probe HD-B to benzenethiol, 10 μM of the probe HD-B, (0 - 10 μM) benzenethiol, and 20 mM phosphate buffer (pH = 7.0) were diluted to 5 mL with high-purity water, and the ultraviolet-visible absorption spectrum and fluorescence emission spectrum were measured.

[0040] Example 3: Selectivity Test of HD-B

[0041] 10 μM of the probe HD-B was respectively mixed with 1 mM metal ions (Na + , K + , Mg 2+ , Ni 2+ , Hg 2+ ), anions (HSO3 - , SO3 2- , SO4 2- , ClO - , S2O3 2- , Br - , Cl - , CO3 2- , NO3 - , NO2 - , CH3COO - ) with a concentration of 1 mM each, transition metal ions (Ca 2 + , Cr 3+ , Mn 2+ , Zn 2+ , Fe 2+ ) with a concentration of 100 μM, and biothiols (GSH, Hcy, Cys) and the common oxide H2O2 with a concentration of 160 μM each. Then the fluorescence emission spectrum was measured.

[0042] Example 4: Quantitative Detection of Benzenethiol by Probe HD-B

[0043] First, prepare a 10 μM probe solution and add thiophenol at different concentrations (0 - 12 μM). Use 490 - 530 nm as the excitation wavelength, measure the fluorescence intensity at 550 - 590 nm and record it as F. Plot a curve with the thiophenol concentration as the abscissa and the fluorescence intensity value F as the ordinate. The curve equation is y = 79.03 + 114.43x, R 2 = 0.9938; Subsequently, prepare a 10 μM probe solution and add the water sample to be tested. Use 490 - 530 nm as the excitation wavelength, measure the fluorescence intensity at 550 - 590 nm and record it as F'. Substitute it into the above curve equation to obtain the thiophenol concentration in the water sample.

[0044] Example 5: Practical water sample application of the probe HD - B made into a kit

[0045] In the kit made of HD - B, for the determination of thiophenol in the Hanjiang River and Baohe River water samples, pour the reagent stock solution a, reagent stock solution b and the water sample to be tested into a glass sample bottle, shake well and compare with the standard colorimetric card. The water sample with unknown concentration of thiophenol can be detected by the above method.

Claims

1. A fluorescent probe for detecting thiophenol, characterized in that, The structural formula of the fluorescent probe is shown as HD-B:

2. The preparation method of the fluorescent probe according to claim 1, wherein, The synthesis steps are as follows: (1) Mix 3-ethyl-2-methylbenzothiazol-3-ium iodide, 4-hydroxybenzaldehyde with absolute ethanol, heat the mixture for reaction. After the reaction is completed, filter to obtain a solid product HD; (2) Dissolve 2,4-dinitrobenzenesulfonyl chloride in absolute dichloromethane to obtain solution A; drop solution A into the solid product HD, then add triethylamine to the mixture, stir the reaction at low temperature for a period of time, and then heat and stir, react overnight to obtain a crude product; the crude product is subjected to column chromatography to obtain the pure product probe HD-B; 3. The preparation method according to claim 2, characterized in that, In step (1), the molar ratio of 3-ethyl-2-methylbenzothiazol-3-ium iodide to 4-hydroxybenzaldehyde is (2.0 - 2.6):(1.2 - 2.2); The heating reaction temperature is 60 - 80 °C, and the reaction time is 3 - 6 h.

4. The preparation method according to claim 2 or 3, characterized in that, In step (2), the molar ratio of product HD to 2,4-dinitrobenzenesulfonyl chloride is (1.0 - 1.9):(0.8 - 1.6); The low temperature is -10 - 10 °C, the low temperature reaction time is 20 - 60 min, and the heating temperature is 30 - 50 °C; The eluent is dichloromethane:methanol, and the volume ratio is (2:1) - (10:1).

5. A method for rapid detection of thiophenol in the laboratory, characterized in that, It includes the following steps: (1) Prepare a standard curve Using 490 - 530 as the excitation wavelength, measure the fluorescence intensity values of a series of standard sample solutions of benzenethiol with different concentrations at the emission of 550 - 590 nm, denoted as F, and plot a standard curve with the vertical axis; (2) Detect the concentration of benzenethiol in the sample Replace the benzenethiol standard solution in step (1) with the sample to be measured, add the solution of the fluorescent probe HD-B described in claim 1 to it, measure the fluorescence intensity at the same emission wavelength as in step (1) of the sample, denoted as F', and substitute the F' into the standard curve obtained in step (1) to further obtain the concentration of benzenethiol in the sample to be measured.

6. A fluorescence probe kit for detecting thiophenol, characterized in that, It includes a box body, a glass sample bottle, and reagent stock solution a and reagent stock solution b are provided in the box body. Reagent stock solution a is an aqueous phosphate buffer solution with a pH value of 7.0, and reagent stock solution b is an organic solution of the fluorescent probe HD-B described in claim 1.

7. A method for using a fluorescence probe kit for detecting benzenethiol according to claim 6, characterized in that, It includes the following steps: (1) Add reagent stock solution a, reagent stock solution b and the water sample to be measured into the glass sample bottle in sequence and mix well; (2) Compare the solution in the glass sample with a standard colorimetric card to know whether the benzenethiol in the water sample exceeds the standard.

Images