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.

中文翻译：

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智能双重淬灭策略增强了细胞内弗林蛋白酶的检测灵敏度

用于成像活细胞中酶活性的敏感荧光“开启”策略的开发对疾病具有重要的诊断意义，但仍具有挑战性。本文中，通过点击缩合反应和单淬灭探针Cys（StBu）-Lys（Gly-Lys（DABCYL）-Gly-Gly-Arg-Arg-Val-Arg-Gly-FITC）-CBT的合理设计（1），我们开发了一种“智能”双重淬灭策略，并将其应用于检测细胞内弗林蛋白酶的活性，并具有更高的灵敏度。在生理条件下，对1进行还原控制的缩合反应以形成1-NP，并且其荧光强度进一步下降至其原始强度的1 / 2.8。弗林蛋白酶体外裂解后，双淬灭的1-NPs。荧光“打开”对比度比单个淬灭对照探针FITC-Gly-Arg-Val-Arg-Arg-Gly-Gly-Lys（DABCYL）-Gly-OH（1-P）高11倍。活细胞成像结果表明，1表示“胞内弗林蛋白酶”活性的荧光“打开”对比度是1-P的6.3倍。我们设想，通过用其他酶可裂解的底物替代RVRR底物，我们的通用“智能”双重淬灭策略可以很容易地调整，以提高灵敏度地检测（或成像）其他细胞内酶的活性。