2'，7'-二氯二氢荧光素的制备及其在氧化还原测定中的实际应用,Analytical Chemistry

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2'，7'-二氯二氢荧光素的制备及其在氧化还原测定中的实际应用
Analytical Chemistry ( IF 6.7 ) Pub Date : 2017-03-15 00:00:00 , DOI: 10.1021/acs.analchem.7b00043
Megan J. Reiniers _{1,

2} , Rowan F. van Golen ₁ , Sylvestre Bonnet ₃ , Mans Broekgaarden ₁ , Thomas M. van Gulik ₁ , Maarten R. Egmond ₂ , Michal Heger _{1,

2}

Affiliation

氧化应激是细胞内或细胞外氧化剂产生超过抗氧化能力的状态，它是许多疾病的基础。DCFH ₂ -DA（2'，7'-二氯二氢荧光素二乙酸酯）是用于检测一般氧化应激的最广泛使用的荧光探针。然而，与许多其他荧光氧化还原探针一样，DCFH ₂ -DA的使用主要限于体外细胞内氧化应激的检测。为了扩大探针的适用性，开发了一种碱性水解和溶剂萃取程序，可使用基本的实验室设备从DCFH ₂ -DA生成高纯度DCFH ₂（2'，7'-二氯二氢荧光素）。接下来，DCFH ₂的实用程序在多种无细胞和体外氧化还原测定系统中得到了证明，包括过渡金属产生的氧化剂，光动力疗法，活化的巨噬细胞和血小板，以及不同抗氧化剂的抗氧化能力。在细胞中，同时使用DCFH ₂ -DA和DCFH ₂可以通过单个读出参数分别对细胞内和细胞外产生的氧化剂进行测量和分区分析。此外，开发了针对肝细胞的脂质体，以将羧基化衍生物5（6）-羧基-DCFH ₂体内递送至肝细胞。脂质体递送的5（6）-羧基-DCFH ₂支持实时可视化和测量肝脏缺血再灌注过程中肝细胞氧化剂的产生。脂质体5（6）-羧基-DCFH ₂可以靶向氧化应激很重要的其他组织，包括癌症。

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Preparation and Practical Applications of 2′,7′-Dichlorodihydrofluorescein in Redox Assays

Oxidative stress, a state in which intra- or extracellular oxidant production outweighs the antioxidative capacity, lies at the basis of many diseases. DCFH₂-DA (2′,7′-dichlorodihydrofluorescein diacetate) is the most widely used fluorogenic probe for the detection of general oxidative stress. However, the use of DCFH₂-DA, as many other fluorogenic redox probes, is mainly confined to the detection of intracellular oxidative stress in vitro. To expand the applicability of the probe, an alkaline hydrolysis and solvent extraction procedure was developed to generate high-purity DCFH₂ (2′,7′-dichlorodihydrofluorescein) from DCFH₂-DA using basic laboratory equipment. Next, the utility of DCFH₂ was exemplified in a variety of cell-free and in vitro redox assay systems, including oxidant production by transition metals, photodynamic therapy, activated macrophages, and platelets, as well as the antioxidative capacity of different antioxidants. In cells, the concomitant use of DCFH₂-DA and DCFH₂ enabled the measurement and compartmentalized analysis of intra- and extracellularly produced oxidants, respectively, using a single read-out parameter. Furthermore, hepatocyte-targeted liposomes were developed to deliver the carboxylated derivative, 5(6)-carboxy-DCFH₂, to hepatocytes in vivo. Liposome-delivered 5(6)-carboxy-DCFH₂ enabled real-time visualization and measurement of hepatocellular oxidant production during liver ischemia-reperfusion. The liposomal 5(6)-carboxy-DCFH₂ can be targeted to other tissues where oxidative stress is important, including cancer.

更新日期：2017-03-15

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