Laccase-Mediated Catalyzed Fluorescent Reporter Deposition for Live-Cell Imaging.
Laccase-Mediated Catalyzed Fluorescent Reporter Deposition for Live-Cell Imaging.
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DOI：
10.1002/cbic.201900593
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发表时间：
2020-01-15
期刊：
影响因子：
--
通讯作者：
Devaraj NK
中科院分区：
文献类型：
--
作者：
Cisneros BT;Devaraj NK
催化报告沉积 (CARD) 是一种广泛使用的标记使用显微镜分析的生物样品的方法。辣根过氧化物酶通常在 CARD 中用于放大报告信号，需要添加过氧化氢，这可能会干扰活细胞显微镜中使用的样品。在此，我们描述了使用漆酶进行 CARD 的替代方法，该方法不需要外源过氧化氢。漆酶是一种氧化酶，可通过还原分子氧对酚类及相关化合物进行单电子氧化。我们通过使用荧光标记的阿魏酸衍生物作为漆酶报告基因底物对牛血清白蛋白进行非靶向共价标记来证明该技术的概念。我们通过使用抗体缀合的漆酶针对表面结合的靶标进行活细胞 CARD，进一步证明了这种方法的可行性。使用漆酶的 CARD 通过标记细胞产生放大的荧光信号，无需外源过氧化氢。漆酶可作为报告酶进行催化报告沉积以荧光标记细胞。当漆酶与抗体一起靶向细胞时，它可以将荧光标记的阿魏酸衍生物共价沉积在细胞表面。该方法无需过氧化氢即可放大活细胞的荧光标记。
Catalyzed reporter deposition (CARD) is a widely established method for labeling biological samples analyzed using microscopy. Horseradish peroxidase, commonly used in CARD to amplify reporter signals, requires the addition of hydrogen peroxide which may perturb samples used in live-cell microscopy. Herein we describe an alternative method of performing CARD using a laccase enzyme, which does not require exogenous hydrogen peroxide. Laccase is an oxidative enzyme which can carry out single-electron oxidations of phenols and related compounds by reducing molecular oxygen. We demonstrate proof-of-concept for this technique through the non-targeted covalent labeling of bovine serum albumin using a fluorescently-labeled ferulic acid derivative as the laccase re-porter substrate. We further demonstrate the viability of this approach by performing live-cell CARD with an antibody-conjugated laccase against a surface bound target. CARD using laccase produces an amplified fluorescence signal by labeling cells without the need for exogenous hydrogen peroxide. Laccase can be used as a reporter enzyme to carry out catalyzed reporter deposition for fluorescent labeling of cells. When laccase is targeted to cells with an antibody, it can covalently deposit a fluorescently-labeled ferulic acid derivative on the surface of cells. This method enables the amplified fluorescent labeling of live cells without the need for hydrogen peroxide.
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