CN109913207B

CN109913207B - Fluorescent probe for detecting long-wave emission of thiophenol

Info

Publication number: CN109913207B
Application number: CN201910290756.7A
Authority: CN
Inventors: 吕正亮; 王云; 蒋绪川; 路亚男; 范春华
Original assignee: University of Jinan
Current assignee: University of Jinan
Priority date: 2019-04-11
Filing date: 2019-04-11
Publication date: 2020-05-12
Anticipated expiration: 2039-04-11
Also published as: CN109913207A

Abstract

The invention discloses a fluorescent probe for detecting thiophenol, belonging to the technical field of analytical chemistry. The probe is prepared from 2- ((6-hydroxy-2, 3-dihydro-1H-xanthen-4-yl) methylene) malononitrile and 2, 4-dinitrofluorobenzene by stirring overnight in dichloromethane at room temperature. The fluorescent probe is convenient to use, has the advantages of good selectivity, sensitivity, low detection limit and the like for the thiophenol, and can be applied to qualitative and quantitative detection of the thiophenol in the environment and cells.

Description

Fluorescent probe for detecting long-wave emission of thiophenol

Technical Field

The invention provides a fluorescent probe for detecting thiophenol, and belongs to the technical field of fluorescent probes.

Technical Field

Thiophenol is a highly toxic environmental pollutant and is widely used in the preparation of chemical products and pesticides, such as the synthesis of local anesthetics, organophosphorus pesticides, high molecular resin vulcanizing agents and the like. The low-concentration thiophenol has strong stimulation effect on skin, eyes and mucous membrane, and can cause throat and bronchospasm, myasthenia and chemical pneumonia; at high concentrations, it can paralyze the central nervous system or cause pulmonary edema, and even death. Therefore, the development of a high-efficiency thiophenol detection method is of great significance. The detection of thiophenol includes high performance liquid chromatography, gas chromatography-mass spectrometry, ultraviolet spectrophotometry, fluorescent probe analysis, etc., wherein the fluorescent probe analysis attracts attention because of its advantages of high sensitivity, high selectivity, simple operation, rapidity, less destruction, etc.

The long-wave emitting fluorescent probe shows excellent characteristics such as effectively avoiding the interference of the background fluorescence of the fluorescent substance with short wavelength in the environment or biological sample, improving the tissue penetrating power, reducing the light damage to the sample and the like. Small molecule mercapto substances in environmental or biological samples, such as hydrogen sulfide, hemisarcosine, glutathion and the like, have similar properties with thiophenol and are possible interfering species, so that the design of the thiophenol fluorescent probe has better selectivity and anti-interference capability; furthermore, thiophenol is a volatile substance, trace volatilization or leakage causes damage to the environment, and the change of the concentration of thiophenol is greatly influenced by environmental factors or test conditions, so that a probe is required to be capable of rapidly and sensitively detecting trace thiophenol, and the detection is ensured to have high timeliness.

The disadvantage of the fluorescent probes currently in use is that most probes can only identify and detect thiophenol in a short excitation or emission region, for example, the fluorescent probe provided in patent CN 106543202A has an emission wavelength of 550 nm. Therefore, the design and development of the fluorescent probe with excellent performances such as long-wave emission, high selectivity, quick response and the like have extremely important theoretical significance and application value.

Disclosure of Invention

The invention relates to a fluorescent probe for detecting thiophenol, which is characterized in that the chemical structure of the fluorescent probe is as shown in the formula

Shown in the figure:

the fluorescent probe for detecting thiophenol is prepared by the following method:

synthesis of Compound I

Under the conditions of nitrogen protection and ice bath, 0.28 g of compound II is dissolved in anhydrous dichloromethane, then 0.18 g of 2, 4-dinitrofluorobenzene is added, and 0.2 ml of triethylamine is added into the reaction system in a dropwise manner. Stir at room temperature overnight. The crude product was obtained and purified by column chromatography to give compound I in 85% yield.

According to the invention, the compound II, 2, 4-dinitrofluorobenzene and triethylamine in the step are preferably in a molar ratio of 1:4: 4;

according to the invention, preferably, compound I is carried out under nitrogen protection;

the fluorescent probe can be used for detecting thiophenol in an aqueous solution.

Further preferably, the fluorescent probe is used for detecting a fluorescent signal at a pH of 7.4 and a volume ratio of 2: 8 quick detection of thiophenol in DMF and water mixture. The detection limit of this probe was 90 nM.

The fluorescent probe is used for detecting the fluorescence intensity of a fluorescent probe at a pH value of 7.4 and a volume ratio of 2: the para-thiophenol in the mixed solution of the DMF of 8 and the water has specific response performance. The invention is verified by experiments that the fluorescent probe has the characteristics that the pH value is 7.4, the volume ratio is 2: in the mixed solution of DMF and water of 8, a light source having a wavelength of 560nm was used as excitation light, and the solution hardly emitted light. With the addition of thiophenol, the solution fluoresces strongly red at 645nm when excited by 560nm light. The addition of various other interfering ions has no effect on the fluorescence of the probe. Therefore, the fluorescent probe compound of the invention can specifically detect thiophenol.

The fluorescent probe compound of the present invention has a pH of 7.4 and a volume ratio of 2: 8 in the mixed solution of DMF and water, the concentration of thiophenol and the fluorescence intensity present a linear relationship, so that the content of thiophenol in the solution can be quantitatively determined.

The fluorescent probe disclosed by the invention is applied to detecting thiophenol in cells.

The fluorescent probe can be applied to the detection of thiophenol in cells. The specific detection method comprises the following steps: when SH-SY5Y cells were incubated at 37 ℃ with 10. mu.M of Compound I, the cells showed no fluorescence emission. SH-SY5Y cells were then incubated with 10. mu.M of Compound I at 37 ℃ and washed three times with PBS, the medium was changed and the cells strongly fluoresced after incubation with thiophenol buffer (25. mu.M). Experiments show that the compound I has good imaging effect on thiophenol in cells, can be well used for detecting thiophenol in cells, and has important potential application value in the aspects of biomedicine and the like.

The invention has the beneficial effects that:

the fluorescent probe for detecting thiophenol has the following advantages: 1. the probe can react with thiophenol specifically, so that the fluorescence intensity of the compound is greatly enhanced; 2. the probe is not interfered by other ions in the process of detecting thiophenol, and has good selectivity to the thiophenol; 3. when the probe is used for detecting thiophenol in living cells, the near-infrared fluorescent dye has the advantages of small damage to biological tissues, strong tissue penetrating capability and capability of effectively eliminating tissue fluorescence background interference.

Drawings

FIG. 1 is the nuclear magnetic spectrum of Compound I.

FIG. 2 is a graph of the selectivity spectrum of Compound I.

FIG. 3 is a fluorescence spectrum of compound I as a function of thiophenol concentration.

Detailed Description

The present invention will be described in detail below with reference to the accompanying drawings and specific embodiments, but is not limited thereto. The various materials in the examples were purchased from the market.

EXAMPLE 1 Synthesis of Compound I

Under the conditions of nitrogen protection and ice bath, dissolving the compound II in anhydrous dichloromethane, then adding 2, 4-dinitrofluorobenzene, and dropwise adding triethylamine into the reaction system. Stir at room temperature overnight. Obtaining a crude product, and purifying by column chromatography to obtain a red compound I. FIG. 1 is a nuclear magnetic diagram of the probe.

EXAMPLE 2 Probe Compound I Selectivity assay

In the volume ratio of the contained thiophenol being 2: when 10 μ M of compound I (PBS 20mM, pH = 7.4) is added to DMF and PBS buffer of 8, the detection result is shown in fig. 2, when the excitation light wavelength is 560nm, the probe compound I has a strong fluorescence response to thiophenol at 645nm, and the probe compound I does not have a significant response to other interfering ions, which indicates that the probe compound I has excellent selectivity to thiophenol.

EXAMPLE 3 Probe Compound I analysis of Change in Phenothiol concentration response

The probe compound I with 10 mu M is added into the buffer solution containing thiophenol (0-200 mu M) with different concentrations, the fluorescence response intensity is linearly increased along with the increase of the added thiophenol, the detection result is shown in figure 3, and the result shows that the probe compound I has wider detection range and higher sensitivity on the thiophenol concentration. And (4) making a working curve by taking the maximum value of the fluorescence intensity and the corresponding concentration of thiophenol as coordinates.

EXAMPLE 4 use of Compound I in cellular assays

After 10. mu.M of Compound I was added to the culture broth of SH-SY5Y cells and cultured at 37 ℃ and then thiophenol was added thereto, respectively, and the cells were imaged after washing and emitted bright red fluorescence. Experiments show that the probe compound I has good imaging effect on thiophenol in cells, can be used for detecting thiophenol in cells, and has important potential application value in the aspects of biomedicine and the like.

Example 5 quantitative determination of Phenethiol in Water by Compound I

Compound I was added to a solution containing different concentrations of thiophenol in a volume ratio of 2: 8 in DMF and PBS buffer (PBS 20mM, pH = 7.4), the final probe concentration is 10 μ M, the concentration of thiophenol is 1, 5, 10, 20 and 40 μ M respectively, the fluorescence values of thiophenol with different concentrations are tested, the concentration of thiophenol is calculated according to a working curve, the recovery rate is above 96%, and the probe can quantitatively detect the content of thiophenol in the water body.

Although the present invention has been described with reference to the specific embodiments shown in the drawings, it is not intended to limit the scope of the present invention, and it should be understood by those skilled in the art that various modifications and variations can be made without inventive faculty based on the technical solutions of the present invention.

Claims

1. The fluorescent probe for detecting thiophenol is characterized in that the chemical structure of the fluorescent probe is shown as formula I

。