Richard D. Berlin Center for Cell Analysis and Modeling , University of Connecticut Health Center , Farmington , Connecticut 06030 , United States.
ACS Sens. 2018 Dec 28;3(12):2621-2628. doi: 10.1021/acssensors.8b01032. Epub 2018 Dec 7.
Voltage sensitive dyes (VSDs) are used for in vitro drug screening and for imaging of patterns of electrical activity in tissue. Wide application of this technology depends on the availability of sensors with high sensitivity (percent change of fluorescence per 100 mV), high fluorescence quantum yield, and fast response kinetics. A promising approach uses a two-component system consisting of anionic membrane permeable quenchers with fluorophores labeling one side of the membrane; this produces voltage-dependent fluorescence quenching. However, the quencher must be kept at low concentrations to minimize pharmacological effects, thus limiting sensitivity. By developing tethered bichromophoric fluorophore quencher (TBFQ) dyes, where the fluorophore and quencher are covalently connected by a long hydrophobic chain, the sensitivity is maximized and is independent of VSD concentration. A series of 13 TBFQ dyes based on the aminonaphthylethenylpyridinium (ANEP) fluorophore and the dipicrylamine anion (DPA) quencher have been synthesized and tested in an artificial lipid bilayer apparatus. The best of these, TBFQ1, shows a 2.5-fold change in fluorescence per 100 mV change in membrane potential, and the response kinetics is in the 10-20 ms range. This sensitivity is an order of magnitude better than that of commonly used VSDs. However, the fluorescence quantum yield is only 1.6%, which may make this first generation of TBFQ VSDs impractical for in vivo electrical imaging. Nevertheless, the design principles established here can serve as foundation for improved TBFQ VSDs. We believe this approach promises to greatly enhance our ability to monitor electrical activity in cells and tissues.
电压敏感染料（VSDs）用于体外药物筛选和组织电活动模式成像。这项技术的广泛应用取决于具有高灵敏度（荧光每 100 mV 变化的百分比）、高荧光量子产率和快速响应动力学的传感器的可用性。一种有前途的方法是使用由阴离子膜可渗透猝灭剂和荧光标记膜一侧组成的双组分系统；这会产生电压依赖性荧光猝灭。然而，为了最小化药理学效应，猝灭剂必须保持在低浓度，从而限制了灵敏度。通过开发连接双生色荧光团猝灭剂（TBFQ）染料，其中荧光团和猝灭剂通过长疏水性链共价连接，可最大限度地提高灵敏度，并且与 VSD 浓度无关。已经合成了基于氨基萘乙烯吡啶鎓（ANEP）荧光团和二硝酰基苯胺（DPA）猝灭剂的 13 种 TBFQ 染料，并在人工脂质双层装置中进行了测试。其中最好的是 TBFQ1，其荧光在膜电位每变化 100 mV 时变化 2.5 倍，响应动力学在 10-20 ms 范围内。这种灵敏度比常用的 VSD 高一个数量级。然而，荧光量子产率仅为 1.6%，这可能使第一代 TBFQ VSD 不适合体内电成像。尽管如此，这里建立的设计原则可以作为改进 TBFQ VSD 的基础。我们相信这种方法有望极大地提高我们监测细胞和组织电活动的能力。
