Selectivity and specificity of small molecule fluorescent dyes/probes used for the detection of Zn2+ and Ca2+ in cells.
Selectivity and specificity of small molecule fluorescent dyes/probes used for the detection of Zn2+ and Ca2+ in cells.
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DOI：
10.1039/c3mt00283g
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发表时间：
2014-02
期刊：
影响因子：
--
通讯作者：
Caruso J
中科院分区：
文献类型：
--
作者：
Figueroa JA;Vignesh KS;Deepe GS Jr;Caruso J
荧光染料被广泛用于检测活细胞中不稳定（游离或交换）的Zn2+和Ca2+。然而，它们对其他阳离子的特异性和检测细胞中不稳定金属与蛋白结合金属的选择性仍不清楚。我们在细胞环境中表征了常用的Zn2+和Ca2+染料的这些重要性质。通过紫外可见光谱和尺寸排除色谱-电感耦合等离子质谱（SEC-ICP-MS）串联追踪荧光发射信号，我们证明了在用于Zn2+的染料中，Zinpyr-1在含有不稳定Zn2+的低分子质量（LMM）区域发出荧光，但在检测锌离子的不同分子质量区域也发出荧光。然而，FluoZin™-3 AM、Newport Green™DCF和Zinquin乙酯显示弱荧光，缺乏金属特异性，在高分子质量（HMM）区域反应强烈。在不受干扰的细胞环境中研究了四种Ca2+染料，其中两种染料在细胞内Ca2+释放刺激下的结合行为进行了测试。通过SEC-ICP-MS检测，大多数Ca2+处于不稳定状态，但细胞中来自Calcium Green-1™AM、Oregon Green®488 BAPTA-1、Fura red™AM和Fluo-4 NW染料的荧光不对应于游离Ca2+检测。相反，染料在含有Zn、Fe和Cu的中、高分子质量区域显示非特异性荧光。对其中一个常见荧光区域的蛋白质组学分析表明，一些染料可能识别与金属硫蛋白-2结合的锌和铜。这些研究表明，Zn2+和Ca2+结合染料表现出的荧光响应不是唯一的识别不稳定金属和结合其他金属，导致不理想的特异性和选择性。
Fluorescent dyes are widely used in the detection of labile (free or exchangeable) Zn2+ and Ca2+ in living cells. However, their specificity over other cations and selectivity for detection of labile vs. protein-bound metal in cells remains unclear. We characterized these important properties for commonly used Zn2+ and Ca2+ dyes in a cellular environment. By tracing the fluorescence emission signal along with UV-Vis and size exclusion chromatography-inductively coupled plasma mass spectrometry (SEC-ICP-MS) in tandem, we demonstrated that among the dyes used for Zn2+, Zinpyr-1 fluoresces in the low molecular mass (LMM) region containing labile Zn2+, but also fluoresces in different molecular mass regions where zinc ion is detected. However, FluoZin™-3 AM, Newport Green™ DCF and Zinquin ethyl ester display weak fluorescence, lack of metal specificity and respond strongly in the high molecular mass (HMM) region. Four Ca2+ dyes were studied in an unperturbed cellular environment, and two of these were tested for binding behavior under an intracellular Ca2+ release stimulus. A majority of Ca2+ was in the labile form as tested by SEC-ICP-MS, but the fluorescence from Calcium Green-1™ AM, Oregon Green® 488 BAPTA-1, Fura red™ AM and Fluo-4 NW dyes in cells did not correspond to free Ca2+ detection. Instead, the dyes showed non-specific fluorescence in the mid- and high-molecular mass regions containing Zn, Fe and Cu. Proteomic analysis of one of the commonly seen fluorescing regions showed the possibility for some dyes to recognize Zn and Cu bound to metallothionein-2. These studies indicate that Zn2+ and Ca2+ binding dyes manifest fluorescence responses that are not unique to recognition of labile metals and bind other metals, leading to suboptimal specificity and selectivity.
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影响因子：
4
作者：
Devinney, MJ;Reynolds, IJ;Dineley, KE
通讯作者：
Dineley, KE
影响因子：
64.8
作者：
Finch, EA;Augustine, GJ
通讯作者：
Augustine, GJ
DOI：
10.1007/978-1-61779-382-0_14
发表时间：
2012-01-01
期刊：
MITOCHONDRIAL BIOENERGETICS: METHODS AND PROTOCOLS
影响因子：
--
作者：
Davidson, Sean M.;Duchen, Michael R.
通讯作者：
Duchen, Michael R.
影响因子：
5.4
作者：
Kaltenberg, Jennifer;Plum, Laura M.;Haase, Hajo
通讯作者：
Haase, Hajo
DOI：
10.1038/nrmicro2836
发表时间：
2012-07-16
期刊：
Nature reviews. Microbiology
影响因子：
--
作者：
通讯作者：
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