Smart Dual Quenching Strategy Enhances the Detection Sensitivity of Intracellular Furin
Smart Dual Quenching Strategy Enhances the Detection Sensitivity of Intracellular Furin
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智能双淬灭策略提高细胞内弗林蛋白酶的检测灵敏度
DOI：
10.1021/acs.analchem.7b05251
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发表时间：
2018-02-06
影响因子：
7.4
通讯作者：
Liang, Gaolin
中科院分区：
文献类型：
--
作者：
Hai, Zijuan;Wu, Jingling;Liang, Gaolin
开发灵敏的荧光“开启”策略来成像活细胞中的酶活性具有疾病诊断的重要性，但仍然具有挑战性。本文采用点击冷凝反应和合理设计单猝灭探针Cys(StBu)-Lys(Gly-Lys(DABCYL)- gly - gly - arg - arg - var - arg - gly - fitc)-CBT(1)，我们开发了一种“智能”双猝灭策略，并将其应用于提高灵敏度的细胞内furin活性检测。在生理条件下，1发生还原控制缩合反应生成1- nps，其荧光强度进一步下降到原来的1/2.8。在体外裂解furin后，双猝灭的1-NPs比单猝灭的对照探针fitc - gly - arg - valg - arg - arg - gly - gly - lys (DABCYL)-Gly-OH （1-P）的荧光“开启”对比度高11倍。活细胞成像结果显示，1的荧光“开启”对比度是1- p的6.3倍，用于检测细胞内furin活性。我们设想，通过用其他酶可切割的底物取代RVRR底物，我们的多功能“智能”双猝灭策略可以很容易地调整，以提高灵敏度检测（或成像）其他细胞内酶的活性。
Development of sensitive fluorescence "Turn-On" strategies for imaging enzyme activity in living cells is of disease-diagnostic importance but remains challenging. Herein, by employing a click condensation reaction and rational design of a single quenched probe Cys(StBu)-Lys(Gly-Lys(DABCYL)-Gly-Gly-Arg-Arg-Val-Arg-Gly-FITC)-CBT (1), we developed a "smart" dual quenching strategy and applied it to detect intracellular furin activity with enhanced sensitivity. At physiological conditions, 1 was subjected to reduction-controlled condensation reaction to form 1-NPs and its fluorescence intensity further dropped to 1/2.8 of its original. Upon furin cleavage in vitro, the dual quenched 1-NPs had fluorescence "Turn-On" contrast 11-fold more than that of single quenched control probe FITC-Gly-Arg-Val-Arg-Arg-Gly-Gly-Lys(DABCYL)-Gly-OH (1-P). Live cell imaging results indicated that 1 showed fluorescence "Turn-On" contrast 6.3-fold of that of 1-P for sensing intracellular furin activity. We envision that, by replacing the RVRR substrate with other enzyme-cleavable ones, our versatile "smart" dual quenching strategy could be easily adjusted for the detection (or imaging) of other intracellular enzymes' activity with enhanced sensitivity.