CN109320490A - 一种近红外特异性检测半胱氨酸的荧光探针 - Google Patents

一种近红外特异性检测半胱氨酸的荧光探针 Download PDF

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CN109320490A
CN109320490A CN201811254762.9A CN201811254762A CN109320490A CN 109320490 A CN109320490 A CN 109320490A CN 201811254762 A CN201811254762 A CN 201811254762A CN 109320490 A CN109320490 A CN 109320490A
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cysteine
probe
fluorescence
fluorescence probe
detection
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吕正亮
路亚男
范春华
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University of Jinan
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D311/00—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only hetero atom, condensed with other rings
    • C07D311/02—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only hetero atom, condensed with other rings ortho- or peri-condensed with carbocyclic rings or ring systems
    • C07D311/78—Ring systems having three or more relevant rings
    • C07D311/80—Dibenzopyrans; Hydrogenated dibenzopyrans
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    • C09K11/06—Luminescent, e.g. electroluminescent, chemiluminescent materials containing organic luminescent materials
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • 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
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • 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
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
    • G01N21/643—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" non-biological material
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • 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
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/6486—Measuring fluorescence of biological material, e.g. DNA, RNA, cells
    • C—CHEMISTRY; METALLURGY
    • C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
    • C09K—MATERIALS FOR MISCELLANEOUS APPLICATIONS, NOT PROVIDED FOR ELSEWHERE
    • C09K2211/00—Chemical nature of organic luminescent or tenebrescent compounds
    • C09K2211/10—Non-macromolecular compounds
    • C09K2211/1018—Heterocyclic compounds
    • C09K2211/1025—Heterocyclic compounds characterised by ligands
    • C09K2211/1088—Heterocyclic compounds characterised by ligands containing oxygen as the only heteroatom

Abstract

本发明公开了一种检测半胱氨酸的荧光探针，属于分析化学技术领域。该探针由2‑((6‑羟基‑2,3‑二氢‑1H‑呫吨‑4‑基)亚甲基)丙二腈和丙烯酰氯在二氯甲烷中室温搅拌得到。本发明的荧光探针使用方便，对半胱氨酸具有较好的选择性、灵敏性、检测限低等优点，可应用于细胞中的半胱氨酸的检测。

Description

一种近红外特异性检测半胱氨酸的荧光探针
技术领域
本发明提供了一种检测半胱氨酸的荧光探针，属于荧光探针技术领域。
技术背景
半胱氨酸(Cys)、同型半胱氨酸(Hcy)、谷胱甘肽(GSH)是细胞内非常重要的三种小分子生物硫醇，维持着生命体的各项生理平衡。研究表明，过高的Cys浓度是心血管疾病以及阿尔茨海默氏症的一大诱因，并且血浆中的Cys浓度与出生缺陷和老年痴呆症有很大关系。因此，实现细胞内半肤氨酸的荧光成像,并给出细胞内半肤氨酸表达与分布的信息，有着重要的生命科学与医学价值。
目前，半胱氨酸含量测定的方法有气相色谱-质谱联用法、高效液相色谱法、全自动的荧光偏振免疫测定、高效毛细管电泳法、有机小分子荧光探针检测法等。
荧光检测方法以其高灵敏度，操作简单，并且可应用于细胞成像等优点，受到了广泛的关注。然而，以紫外/可见光等短波长的光作为发射光会受到生物体自发荧光的干扰，相反，近红外荧光染料具有对生物组织损害小、组织穿透能力强、能有效消除组织荧光背景干扰的优点，因而在生物荧光成像方面具有独特的优势。因此，找到一种在生物样品内检测Cys含量的近红外有机小分子荧光探针有着重要的意义。在现有技术中，专利CN 104447421A中所提供的荧光探针无法区分半胱氨酸和同型半胱氨酸。此外，该探针检测半胱氨酸和同型半胱氨酸的响应时间为30分钟，而我们所提供的一种新型的近红外的荧光探针可以特异性检测半胱氨酸；同时，我们所提供的探针可以在1分钟内完成对半胱氨酸检测，因此，我们可以在更短的时间内实现对半胱氨酸的定量检测。
发明内容
本发明所述的一种检测半胱氨酸的荧光探针，其特征在于，所述荧光探针的化学结构式所示：
。
上述检测半胱氨酸的荧光探针以如下方法制备：在氮气保护、冰浴条件下，将0.28克化合物II溶于无水二氯甲烷中，然后加入0.18克丙烯酰氯，再将0.2毫升的三乙胺滴入反应体系中。室温下搅拌过夜。得到粗产物，经柱层层析提纯得到化合物I，产率65，。
根据本发明，优选的，步骤（5）所述化合物II、丙烯酰氯和三乙胺的摩尔比为1:4:4；
根据本发明，优选的，步骤（5）全程在氮气保护下进行；
本发明所述的荧光探针可用于环境中水溶液的半胱氨酸的检测。
进一步优选的，所述荧光探针用于在pH 7.4的体积比为1:4的DMSO与PBS缓冲液的混合液中的半胱氨酸的快速检测。该探针的检测限为10纳摩尔/升。
本发明所述荧光探针用于在pH7.4的DMSO与PBS缓冲液混合液中对半胱氨酸具有特异性响应性能。本发明通过实验验证，所述荧光探针在pH7.4的DMSO与PBS缓冲液混合液中以580nm波长的光源作为激发光，其溶液几乎不发光。随着半胱氨酸的加入，溶液在580nm的光激发时，溶液在645nm处发出强烈的红色荧光。其它种类的氨基酸的加入对探针的荧光没有影响。因此，本发明的荧光探针可以特异性检测半胱氨酸。
本发明的荧光探针化合物在pH7.4的DMSO与PBS缓冲液混合液中，半胱氨酸的浓度的与荧光强度的工作曲线呈现线性关系，因此，可以定量测定溶液中半胱氨酸的含量。
本发明所述的荧光探针在细胞中检测半胱氨酸的应用。
本发明所述荧光探针可以应用于细胞内半胱氨酸的检测。具体检测方法为：在Hela细胞中加入10 μM的荧光探针化合物I，在37℃下培养，细胞发出强烈的荧光。实验表明，化合物I对内源性半胱氨酸有良好的成像作用，可以用于检测细胞内源性的半胱氨酸，在生物医学等方面具有重要的潜在的应用价值。
本发明的有益效果：
本发明所涉及的一种检测半胱氨酸的荧光探针，具有以下优势：1、该探针能与半胱氨酸特异性反应，导致化合物的荧光强度得到很大的增强；2、该探针在检测半胱氨酸过程中不会受到其他氨基酸的干扰，对半胱氨酸具有良好的选择性；3、该探针在对活细胞中的半胱氨酸进行检测的时候，近红外荧光染料具有对生物组织损害小、组织穿透能力强、能有效消除组织荧光背景干扰的优点；4、可以检测环境中或者细胞内的半胱氨酸进行定量检测。
附图说明
图1是化合物I的1H NMR谱图。
图2是化合物I的选择性光谱图。
图3是化合物I随半胱氨酸浓度变化荧光光谱图。
图4是化合物I与半胱氨酸的生物成像图。
具体实施方式
下面结合附图和具体实施例对本发明进行详细说明，但不限于此。实施例中的各种原料均来自于市场购买。
实施例1
化合物I的合成
（5）在氮气保护、冰浴条件下，将化合物II溶于无水二氯甲烷中，然后加入丙烯酰氯，再将三乙胺滴加至反应体系中。室温下搅拌过夜。得到粗产物，经柱层层析提纯得到红色化合物I。图1为该探针的核磁图。
实施例2 探针化合物I选择性分析
在含有半胱氨酸的DMSO / PBS缓冲液中加入10 μM的化合物I（半胱氨酸的浓度为200μM，pH = 7.4，体积比为1:4的DMSO:PBS缓冲液），检测结果如图2，当激发光波长为580nm时，探针化合物I对半胱氨酸在645 nm处有强烈的荧光响应，而且探针化合物I对其他生物硫醇没有明显响应，说明探针化合物I对半胱氨酸具有优异的选择性。
实施例3 探针化合物I对半胱氨酸浓度变化响应分析以及定量检测个工作曲线
在含有不同浓度的半胱氨酸（0 - 200 μM）的缓冲液中加入10 μM探针化合物I，荧光响应强度随加入半胱氨酸量的增加呈规律性增加，检测结果如图3，结果说明探针化合物I对半胱氨酸浓度检测范围较广且灵敏度较高。以半胱氨酸的浓度对相应的荧光值作图，得较好的直线型的工作曲线，其可用于半胱氨酸的定量检测。
实施例4 化合物I对半胱氨酸响应时间分析
在含有半胱氨酸的DMSO/ PBS缓冲液中加入10 μM的探针化合物I，对探针化合物I与加入半胱氨酸的响应时间进行了检测。结果显示，在加入半胱氨酸1分钟内，探针化合物I对半胱氨酸的荧光响应强度随时间的增加呈线性增强，在1分钟内就能达到良好的荧光强度。结果表明，探针化合物I对半胱氨酸的响应较快速，可应用于半胱氨酸的快速检测。
实施例5 化合物I在细胞检测中的应用
将10 μM的化合物I加入到Hela细胞的培养液中，然后在37℃下培养10分钟，成像结果如图4。细胞发出较强的红色荧光。实验表明，探针化合物I对细胞中的半胱氨酸有良好的成像作用，能用于检测细胞内的半胱氨酸，在生物医学等方面具有重要的潜在应用价值。
上述虽然结合附图对本发明的具体实施方式进行了描述，但并非对发明范围的限制，所述领域技术人员应该明白，在本发明的技术方案的基础上，本领域技术人员不需要付出创造性劳动即可做出的各种修改或变形仍在本发明的保护范围之内。

Claims (2)

1.一种检测半胱氨酸的荧光探针，所述荧光探针的化学结构式如I所示；其可以与半胱氨酸快速发生迈克尔加成，快速释放出可以以580纳米波长激发并发出645纳米的红色荧光；其对半胱氨酸的检测限为10纳摩尔/升；以645纳米的荧光强度与相应的半胱氨酸溶度值做工作曲线，可以定量检测位置浓度的半管氨酸溶液的浓度值
。
2.根据权利要求1中所述的一种检测半胱氨酸的荧光探针，其特征在于，所述荧光探针可以应用于环境中的或者细胞中的半胱氨酸定量定性检测。
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Publication number Priority date Publication date Assignee Title
CN109879851A (zh) * 2019-04-11 2019-06-14 济南大学 一种特异性检测苯硫酚的近红外荧光探针
CN109913207A (zh) * 2019-04-11 2019-06-21 济南大学 一种检测苯硫酚的长波发射的荧光探针
CN109879851B (zh) * 2019-04-11 2021-08-31 济南大学 一种特异性检测苯硫酚的近红外荧光探针
CN111518071A (zh) * 2020-05-20 2020-08-11 湘潭大学 一种半胱氨酸近红外荧光探针的制备和应用
CN112876392A (zh) * 2021-02-05 2021-06-01 新乡医学院 一种基于异硫氰酸酯结构特异性检测半胱氨酸的近红外荧光探针及其制备方法和应用

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Application publication date: 20190212