阅读说明：本技术 一种pH近红外荧光探针的制备和应用 (Preparation and application of pH near-infrared fluorescent probe ) 是由 李春艳 佘遵攀 于 2019-10-30 设计创作，主要内容包括：本发明涉及了一种pH近红外荧光探针的制备和应用,该荧光探针的结构式为：<Image he="286" wi="617" file="DDA0002253979420000011.GIF" imgContent="drawing" imgFormat="GIF" orientation="portrait" inline="no"></Image>本发明提供了以1,1,2-三甲基-1H-苯并[e]吲哚、环己酮、碘乙烷等为原料合成该荧光探针的制备方法；该荧光探针是一种pH近红外荧光探针；首先,该荧光探针在酸性环境下表现出很高的灵敏度；其次,该荧光探针对pH表现出较高的选择性,不受其他无机离子的干扰；并且,该荧光探针对pH反应非常迅速；此外,该荧光探针应用于区别正常细胞与癌细胞。(The invention relates to a preparation method and application of a pH near-infrared fluorescent probe, wherein the structural formula of the fluorescent probe is as follows: the invention provides a method for preparing 1,1, 2-trimethyl-1H-benzo [ e]A preparation method for synthesizing the fluorescent probe by taking indole, cyclohexanone, ethyl iodide and the like as raw materials; the fluorescent probe is a pH near-infrared fluorescent probe; firstly, the fluorescent probe shows high sensitivity in an acidic environment; secondly, the fluorescent probe shows higher selectivity for pH and is not interfered by other inorganic ions; also, the fluorescent probe reacts very rapidly to pH; in addition, the fluorescent probe is applied to distinguish normal cells from cancer cells.)

1. A pH near-infrared fluorescent probe has the following structure:

2. the method for preparing the pH near-infrared fluorescent probe according to claim 1, characterized in that the reaction steps are as follows:

dissolving 1 equivalent of cyanine CyCl and 2 to 3 equivalents of sodium acetate in 5 to 10mL of N, N-dimethylformamide in a 100mL round-bottom flask, stirring for 10 to 14 hours under the protection of nitrogen, stopping the reaction, cooling the reaction mixture to room temperature, extracting the reaction mixture with dichloromethane, washing an organic layer with saturated saline, drying the organic layer with anhydrous sodium sulfate, removing the solvent through reduced pressure distillation, and using CH with the volume ratio of 100:1 to 20:1 as a crude product₂Cl₂/CH₃CH₂And performing column chromatography by using an OH eluant to obtain a red solid product, namely the fluorescent probe.

3. The use of the pH NIR fluorescent probe according to claim 1, wherein the fluorescent probe is used to distinguish between normal cells and cancer cells.

Technical Field

The invention belongs to the technical field of fluorescent probes, and particularly relates to preparation and application of a cyanine dye-based pH near-infrared fluorescent probe.

Background

Intracellular pH plays a key role in a number of cellular events, including cell growth and apoptosis, ion transport and homeostasis, calcium regulation, endocytosis and cell adhesion, among others (Tang B, Yu F, Li P, et al. journal of the American Chemical Society,2009,131: 3016-. Under normal physiological conditions, extracellular hydrogen ion concentrations are kept within a very narrow range, and minor changes will cause many diseases (Lagadic-Gossmann D, Rissel M, Galisteo M, et al. British journal of pharmacology,1999,128: 1673-. Previous evidence suggests that swellingThe internal environment of the tumor has been acidified (Schornack P A, Gillies RJ. Neopalasia, 2003,5: 135-145). With increased glucose metabolism, H in cancer⁺Production and excretion usually also increase. The cellular pH of malignant tumors is lower (pH 6.5-6.9) under physiological conditions compared to normal tissue (pH 7.2-7.4) (Stubbs M, McSheehy P M J, Griffiths J R, et al. molecular media today,2000,6: 15-19; Van Sluis R, Bhujwalla Z M, Raghuanand N, et al. magnetic Resonance in Medicine: An Official Journal of the International Society for magnetic Resonance in Medicine,1999,41: 743-. Therefore, we can consider pH as an effective cancer biomarker and consider it as a breakthrough in early cancer detection, which makes it urgently necessary to design an effective method to accurately detect it.

The fluorescence detection method has attracted extensive attention because of its advantages of high sensitivity, simple operation, and applicability to biological imaging. In recent years, there have been many fluorescent probes designed and developed to detect pH, such as: coumarin-based probes (Dong B, Song X, Wang C, et al analytical chemistry,2016,88: 4085-. However, these probes have shorter excitation and emission wavelengths (<550nm), resulting in excessive autofluorescence and shallower penetration depths, thereby reducing the sensitivity of the probes and preventing their use in biological systems. In contrast, Near Infrared (NIR) fluorescent probes have little photodamage and can penetrate deep into tissue, thereby minimizing interference from background fluorescence and benefiting biological imaging. Therefore, it is very meaningful to design and synthesize a near infrared fluorescent probe having long wavelength emission.

Cyanine dyes are near-infrared fluorescent dyes. Fluorescent probes designed based on cyanine dyes have been used to detect Hg²⁺、NO、H₂O₂And ozone, etc. (Guo Z, Zhu W, Zhu M, et al. chemistry-A European journal,2010,16:14424-Soft he American Chemical Society,2005,127: 3684-; yu F, Li P, Song P, equivalent.chemical communications,2012,48: 4980-; xu K, Sun S, Li J, et al chemical communications,2012,48: 684-. However, very few probes based on cyanine dyes are used to detect pH. Therefore, it is necessary to design and synthesize a cyanine dye-based near-infrared fluorescent probe for detecting pH and for distinguishing normal cells from cancer cells.

Disclosure of Invention

In light of the requirements, the inventors have conducted intensive studies to provide a cyanine dye-based pH near-infrared fluorescent probe after a great deal of creative work.

The technical scheme of the invention is that the pH near-infrared fluorescent probe has the following structural formula:

a method for preparing a pH near-infrared fluorescent probe. The method comprises the following steps:

dissolving 1 equivalent of cyanine CyCl and 2 to 3 equivalents of sodium acetate in 5 to 10mLN, N-dimethylformamide in a 100mL round-bottom flask, stirring for 10 to 14 hours under the protection of nitrogen, stopping the reaction, cooling the reaction mixture to room temperature, extracting with dichloromethane, and washing the organic layer with saturated brine. Drying the organic layer with anhydrous sodium sulfate, removing the solvent by distillation under reduced pressure, and using CH with the volume ratio of 100: 1-20: 1 as a crude product₂Cl₂/CH₃CH₂And performing column chromatography by using an OH eluent to obtain a red solid product (the yield is 52 percent), namely the fluorescent probe.

The invention has the beneficial effect that the pH near-infrared fluorescent probe has good spectral response performance. Firstly, the fluorescence spectrum property of the probe is researched, and the fluorescent probe has no near infrared (780nm) fluorescence emission peak under a neutral condition; under acidic conditions, a fluorescence emission peak appears in the near infrared region (780nm), and the near infrared fluorescence intensity of the probe molecule is increased with the increase of the acidic conditions. So that the probe can detectpH under acidic conditions. Secondly, the ultraviolet absorption spectrum of the probe is researched, and the probe has an absorption band at 560nm under a neutral condition; as the acidic condition increased, the absorption peak at 560nm gradually decreased, and a new absorption peak appeared around 760 nm. Next, the selectivity of the probe was investigated, and the probe and the inorganic ion (K) were examined separately⁺,Ca²⁺,Na⁺,Mg²⁺,Fe²⁺,Fe³⁺,Cr³⁺,Hg²⁺,HCO₃ ^-,F^-,Br^-,Ac^-,SO₄ ^2-,NO₃ ^-Fluorescence response in pH 5.0 and pH 7.4 environments. As a result, it was found that only pH caused the change of the fluorescence spectrum, and other inorganic ions did not have a significant effect on the fluorescence spectrum of the probe. In addition, the fluorescent probe responds very rapidly to pH.

An application of a pH near-infrared fluorescent probe. When a fluorescent probe was added to normal cells, little fluorescence was observed, indicating that the pH in normal cells was neutral. By adding a fluorescent probe to the cancer cells, a stronger fluorescence generation can be observed, which indicates a lower pH in the cancer cells. These results indicate that the fluorescent probe can distinguish between normal cells and cancer cells, which provides a reliable means for early detection of cancer.

Drawings

FIG. 1 shows a synthetic route of a fluorescent probe.

FIG. 2 is a graph of UV-VIS absorption spectra of fluorescent probes in buffers of different pH.

The abscissa is wavelength and the ordinate is absorbance. The concentration of the fluorescent probe is 5 μ M, the concentration of SDS is 5mM, and the pH values are respectively: 5.0,5.5,5.9,6.2,6.5,6.8,7.4.

FIG. 3 is a graph of fluorescence spectra of fluorescent probes at different pH buffers.

The abscissa is wavelength and the ordinate is fluorescence intensity. The concentration of the fluorescent probe is 5 μ M, the concentration of SDS is 5mM, and the pH values are respectively: 5.0,5.5,5.9,6.2,6.5,6.8,7.4. The fluorescence excitation wavelength was 720 nm.

FIG. 4 is a graph of the fluorescence linear response of the fluorescent probe in different pH buffers.

The concentration of the fluorescent probe was 5. mu.M, and the concentration of SDS was 5 mM. The fluorescence excitation wavelength was 720 nm.

FIG. 5 is a graph showing selectivity of fluorescent probes.

The concentration of the fluorescent probes was 5. mu.M, the concentration of SDS was 5mM, and the concentration of the other analytes was 100. mu.M, which were: blank,2.K⁺,3.Ca²⁺,4.Na⁺,5.Mg²⁺,6.Fe²⁺,7.Fe³⁺,8.Cr³⁺,9.Hg²⁺,10.HCO₃ ^-,11.F^-,12.Br^-,13.Ac^-,14.SO₄ ^2-,15.NO₃ ^-.

FIG. 6 is a graph showing the change of fluorescence intensity of fluorescent probes with time under different pH buffers.

FIG. 7 human colon cancer cytotoxicity assay. The abscissa is the concentration of the fluorescent probe and the ordinate is the survival rate of the cells.

FIG. 8 is a human colonic mucosal cytotoxicity assay. The abscissa is the concentration of the fluorescent probe and the ordinate is the survival rate of the cells.

FIG. 9 is an image of the fluorescent probe in normal cells and cancer cells.

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.