Preparation of a Nile Red-Pd-based fluorescent CO probe and its imaging applications in vitro and in vivo
Preparation of a Nile Red-Pd-based fluorescent CO probe and its imaging applications in vitro and in vivo
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尼罗红-Pd基荧光CO探针的制备及其体内外成像应用
DOI：
10.1038/nprot.2018.013
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发表时间：
2018
期刊：
影响因子：
14.8
通讯作者：
Lin Weiying
中科院分区：
文献类型：
--
作者：
Liu Keyin;Kong Xiuqi;Ma Yanyan;Lin Weiying
一氧化碳（CO）是活细胞和生物体中的关键气体信号分子。该协议说明了一个高灵敏度的尼罗红（NR）-钯为基础的荧光探针，NR-PdA的合成，及其应用检测内源性CO在组织培养细胞，离体器官，和斑马鱼胚胎。在NR-PdA合成过程中，3-二乙胺苯酚与亚硝酸钠在酸性条件下反应，得到5-（二乙氨基）-2-亚硝基苯酚盐酸盐（化合物1），其在高温下进一步用1-萘酚处理，通过环化反应得到NR染料。最后，NR与乙酸钯反应以获得所需的Pd基荧光探针NR-PdA。NR-PdA具有优异的双光子激发和近红外发射特性、高稳定性、低背景荧光和低检测限。除了化学合成程序，我们提供了一步一步的程序，成像内源性CO在RAW 264.7细胞，小鼠器官体内，活斑马鱼胚胎。探针的合成过程需要14 d，生物成像实验需要14 d。
Carbon monoxide (CO) is a key gaseous signaling molecule in living cells and organisms. This protocol illustrates the synthesis of a highly sensitive Nile Red (NR)–Pd-based fluorescent probe, NR-PdA, and its applications for detecting endogenous CO in tissue culture cells,ex vivoorgans, and zebrafish embryos. In the NR-PdA synthesis process, 3-diethylamine phenol reacts with sodium nitrite in the acidic condition to afford 5-(diethylamino)-2-nitrosophenol hydrochloride (compound 1), which is further treated with 1-naphthalenol at a high temperature to provide the NR dye via a cyclization reaction. Finally, NR is reacted with palladium acetate to obtain the desired Pd-based fluorescent probe NR-PdA. NR-PdA possesses excellent two-photon excitation and near-IR emission properties, high stability, low background fluorescence, and a low detection limit. In addition to the chemical synthesis procedures, we provide step-by-step procedures for imaging endogenous CO in RAW 264.7 cells, mouse organsex vivo, and live zebrafish embryos. The synthesis process for the probe requires ∼4 d, and the biological imaging experiments take ∼14 d.