谷胱甘肽和半胱氨酸/同型半胱氨酸基于荧光的检测和区分的广泛应用策略：体内和体外的演示,Analytical Chemistry

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谷胱甘肽和半胱氨酸/同型半胱氨酸基于荧光的检测和区分的广泛应用策略：体内和体外的演示
Analytical Chemistry ( IF 6.7 ) Pub Date : 2016-03-14 00:00:00 , DOI: 10.1021/acs.analchem.5b04333
Wenqiang Chen _{1,

2} , Hongchen Luo ₁ , Xingjiang Liu ₁ , James W. Foley ₃ , Xiangzhi Song ₁

Affiliation

谷胱甘肽（GSH），半胱氨酸（Cys）和高半胱氨酸（Hcy）是存在于健康哺乳动物的所有细胞和细胞外液中的小生物分子硫醇。众所周知，每种物质在人类生理学中都起着至关重要的作用，并且每种物质的异常水平都可以预测多种不同的疾病状态。尽管已开发出许多基于荧光的方法来检测含有巯基部分的生物分子，但很少能够区分GSH和Cys / Hcy。在本报告中，我们演示了可用于实现此目标的荧光探针设计的广泛适用方法。我们采用的策略是共轭荧光猝灭7-硝基-2,1,3-苯并恶二唑（NBD）通过巯基不稳定的醚键与选定的荧光团（Dye）结合，得到非荧光的NBD-O-Dye。在存在GSH或Cys / Hcy的情况下，醚键断裂，同时产生非荧光NBD-SR衍生物和具有特征性强发射带（B1）的荧光染料。在Cys / Hcy的特殊情况下，NBD-S-Cys / Hcy裂解产物可进行进一步的分子内Smiles快速重排，形成新的高荧光NBD-N-Cys / Hcy化合物（B2带）。由于几何约束，GSH衍生了NBD-S-GSH派生对象不能进行Smiles重排。因此，单个B1或双B1 + B2签名的存在可分别用于检测和区分GSH与Cys / Hcy。通过在我们的研究中加入黄酮，Bodipy和香豆素染料家族的成员，我们证明了该方法的广泛适用性。特别是，在水溶液和活细胞中，在检测Cys / Hcy上的GSH时，可以将单个激发波长应用于探针NBD-OF。

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Broadly Applicable Strategy for the Fluorescence Based Detection and Differentiation of Glutathione and Cysteine/Homocysteine: Demonstration in Vitro and in Vivo

Glutathione (GSH), cysteine (Cys), and homocysteine (Hcy) are small biomolecular thiols that are present in all cells and extracellular fluids of healthy mammals. It is well-known that each plays a separate, critically important role in human physiology and that abnormal levels of each are predictive of a variety of different disease states. Although a number of fluorescence-based methods have been developed that can detect biomolecules that contain sulfhydryl moieties, few are able to differentiate between GSH and Cys/Hcy. In this report, we demonstrate a broadly applicable approach for the design of fluorescent probes that can achieve this goal. The strategy we employ is to conjugate a fluorescence-quenching 7-nitro-2,1,3-benzoxadiazole (NBD) moiety to a selected fluorophore (Dye) through a sulfhydryl-labile ether linkage to afford nonfluorescent NBD-O-Dye. In the presence of GSH or Cys/Hcy, the ether bond is cleaved with the concomitant generation of both a nonfluorescent NBD-S-R derivative and a fluorescent dye having a characteristic intense emission band (B1). In the special case of Cys/Hcy, the NBD-S-Cys/Hcy cleavage product can undergo a further, rapid, intramolecular Smiles rearrangement to form a new, highly fluorescent NBD-N-Cys/Hcy compound (band B2); because of geometrical constraints, the GSH derived NBD-S-GSH derivative cannot undergo a Smiles rearrangement. Thus, the presence of a single B1 or double B1 + B2 signature can be used to detect and differentiate GSH from Cys/Hcy, respectively. We demonstrate the broad applicability of our approach by including in our studies members of the Flavone, Bodipy, and Coumarin dye families. Particularly, single excitation wavelength could be applied for the probe NBD-OF in the detection of GSH over Cys/Hcy in both aqueous solution and living cells.

更新日期：2016-03-14

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