Reaction-Based Probes for Imaging Mobile Zinc in Live Cells and Tissues.
Reaction-Based Probes for Imaging Mobile Zinc in Live Cells and Tissues.
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DOI：
10.1021/acssensors.5b00022
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发表时间：
2016-01-22
期刊：
影响因子：
8.9
通讯作者：
Lippard SJ
中科院分区：
文献类型：
--
作者：
Zastrow ML;Radford RJ;Chyan W;Anderson CT;Zhang DY;Loas A;Tzounopoulos T;Lippard SJ
可螯合的或可移动的锌在许多生物过程中发挥着关键的信号作用。阐明可移动的锌(II)在复杂生物环境中的作用需要灵敏的工具来可视化、跟踪和操纵锌(II)离子。基于光诱导电子转移(PET)的合成锌(II)荧光传感器已经有了很大的工具箱，但由于锌的低灵敏度和由于质子干扰而导致的低动态范围，许多这些探针的适用性受到限制。我们在这里提出了一种通用的方法，用于乙酰化包含各种荧光团和锌结合单元的基于PET的探针。新传感器大幅提高了锌的灵敏度，并允许活细胞和组织切片与NM探针浓度孵育，与可测量荧光信号通常所需的μM浓度相比，这是一个重大改进。乙酰化有效地减少或完全猝灭了无金属传感器中的背景荧光。锌(II)的结合选择性和快速地介导了乙酰基的水解性裂解，提供了一个大的荧光响应。使用乙酰化的蓝香豆素传感器对金属结合和金属促进的乙酰水解性进行了详细的分析。在低浓度(∼50-100 nm)下，具有不同锌结合部位的乙酰化苯并二氢呋喃红色发光探针可以有效地检测活细胞内的锌离子。我们用绿色的二乙酰化锌(DA-ZP1)对小鼠耳蜗核背侧核(DCN)分子层内的内源性移动锌(II)进行了成像，证实了乙酰化是制备生物系统中对移动锌高度特异和敏感的传感器的合适方法。
Chelatable, or mobile, forms of zinc play critical signaling roles in numerous biological processes. Elucidating the action of mobile Zn(II) in complex biological environments requires sensitive tools for visualizing, tracking, and manipulating Zn(II) ions. A large toolbox of synthetic photoinduced electron transfer (PET)-based fluorescent Zn(II) sensors are available, but the applicability of many of these probes is limited by poor zinc sensitivity and low dynamic ranges owing to proton interference. We present here a general approach for acetylating PET-based probes containing a variety of fluorophores and zinc-binding units. The new sensors provide substantially improved zinc sensitivity and allow for incubation of live cells and tissue slices with nM probe concentrations, a significant improvement compared to the μM concentrations that are typically required for a measurable fluorescence signal. Acetylation effectively reduces or completely quenches background fluorescence in the metal-free sensor. Binding of Zn(II) selectively and quickly mediates hydrolytic cleavage of the acetyl groups, providing a large fluorescence response. An acetylated blue coumarin-based sensor was used to carry out detailed analyses of metal binding and metal-promoted acetyl hydrolysis. Acetylated benzoresorufin-based red-emitting probes with different zinc-binding sites are effective for sensing Zn(II) ions in live cells when applied at low concentrations (∼50–100 nM). We used green diacetylated Zinpyr1 (DA-ZP1) to image endogenous mobile Zn(II) in the molecular layer of mouse dorsal cochlear nucleus (DCN), confirming that acetylation is a suitable approach for preparing sensors that are highly specific and sensitive to mobile zinc in biological systems.