CN108690011A - 一种检测半胱氨酸的荧光探针 - Google Patents

一种检测半胱氨酸的荧光探针 Download PDF

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CN108690011A
CN108690011A CN201810767818.4A CN201810767818A CN108690011A CN 108690011 A CN108690011 A CN 108690011A CN 201810767818 A CN201810767818 A CN 201810767818A CN 108690011 A CN108690011 A CN 108690011A
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吕正亮
路亚男
范春华
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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‑甲酰基喹啉基丙烯酸酯和邻氨基苯硫醇在乙醇中室温搅拌30min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一质谱联用法、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 。其中双光子有机小分子荧光探针具有1、长波激发；2、即可以实现生物组织内深层成像、又不会损伤生物组织；3、即能有效避免生物体自身荧光物质的背景光的干扰，实现暗场成像，而且还能降低光漂白、光毒性的优点。因此，找到一种在生物样品内检测Cys含量的双光子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 106946801 A中所提供的荧光探针检测半胱氨酸的响应时间为45分钟，而我们所提供的一种新型的双光子的荧光探针可以在10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实时检测。
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：
化合物I的合成
将化合物II和邻氨基苯硫醇溶解在乙醇中，接着将双氧水滴加入反应体系中，然后在滴加浓盐酸，室温搅拌2小时，抽滤得到化合物I。
根据本发明，优选的，在化合物I的合成中，所述化合物II、邻氨基苯硫醇、浓盐酸和双氧水的摩尔比为1:1:3:10；
根据本发明，优选的，化合物I的合成全程在氮气保护下进行；
本发明所述的荧光探针可用于水溶液中的半胱氨酸的检测。
进一步优选的，所述荧光探针用于在pH7.4的DMSO与PBS缓冲液混合液中的半胱氨酸的快速检测。该探针的检测限为3.73×10-7 mol/L。
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，将半胱氨酸加入到所述荧光探针在pH7.4的DMSO与PBS缓冲液混合液中并以420nm波长的光源作为激发光，溶液在542nm处发出强烈的黄色荧光。其它种类的氨基酸的加入对探针的荧光没有影响。因此，本发明的荧光探针化合物可以特异性的检测半胱氨酸。
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：将化合物I 加入到Hela细胞中培养15分钟，更换培养基，再加半胱氨酸缓冲溶液，细胞发出强烈的荧光。实验表明，化合物I对细胞内的半胱氨酸有良好的成像作用。
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：该探针能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；抗干扰能力强，灵敏度高。
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
将0.22克化合物II和0.12克邻氨基苯硫醇溶解在乙醇中，在把0.09mL双氧水滴加入反应体系中，然后在滴加0.18mL浓盐酸，室温搅拌2小时，抽滤得到化合物I，产率80%。图1为该探针的核磁图（400MHz,DMSO-d6）。
实施例2 探针化合物I选择性分析
在含有半胱氨酸的体积比为3:7的DMSO / PBS缓冲液中加入5 μM的化合物I，检测结果如图2，当激发光波长为420nm时，探针化合物I对半胱氨酸在542nm出有强烈的荧光响应，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
在含有不同浓度的半胱氨酸（0 - 200 μM）的缓冲液中加入5 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。
实施例4 化合物I在细胞检测中的应用
在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，然后再分别加入半胱氨酸，洗涤后成像结果如图4。显然，当细胞与化合物I培养后再加入半胱氨酸后细胞发出明亮的黄色荧光。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，其特征在于，所述荧光探针的化学结构式如I所示
。
2.根据权力要求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细胞中的半胱氨酸检测。
CN201810767818.4A 2018-07-13 2018-07-13 一种检测半胱氨酸的荧光探针 Pending CN108690011A (zh)

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Cited By (4)

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CN109320490A (zh) * 2018-10-26 2019-02-12 济南大学 一种近红外特异性检测半胱氨酸的荧光探针
CN110156695A (zh) * 2019-05-16 2019-08-23 济南大学 一种检测半胱氨酸的荧光探针及其应用
CN113354676A (zh) * 2021-06-28 2021-09-07 香港理工大学深圳研究院 一种半胱氨酸荧光探针及其制备方法与应用
CN114349717A (zh) * 2022-02-25 2022-04-15 明士新材料有限公司 一种基于苯并噻唑衍生物半胱氨酸的荧光探针及其制备方法

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Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109320490A (zh) * 2018-10-26 2019-02-12 济南大学 一种近红外特异性检测半胱氨酸的荧光探针
CN110156695A (zh) * 2019-05-16 2019-08-23 济南大学 一种检测半胱氨酸的荧光探针及其应用
CN113354676A (zh) * 2021-06-28 2021-09-07 香港理工大学深圳研究院 一种半胱氨酸荧光探针及其制备方法与应用
CN113354676B (zh) * 2021-06-28 2023-09-05 香港理工大学深圳研究院 一种半胱氨酸荧光探针及其制备方法与应用
CN114349717A (zh) * 2022-02-25 2022-04-15 明士新材料有限公司 一种基于苯并噻唑衍生物半胱氨酸的荧光探针及其制备方法

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