Optical Voltage Sensing Nano-Devices using DNA Self-Assembly

任何活细胞都需要膜电位来实现广泛的功能，包括能量产生、信息处理和传递。膜电压的精确知识在神经科学，特别是脑研究中至关重要。现有的技术严重依赖于膜电位的光学测量，目前受到低灵敏度，低速度和侵入性的限制。遗传编码传感器依赖于不稳定的荧光蛋白，需要对宿主生物体进行遗传修饰。在这个项目中，我们提出了一种新的方法，光学电压传感纳米器件（VSND）的基础上两个基本的设计都使用DNA支架。DNA支架能够对VSND的所有重要功能进行空间和化学控制，包括膜定位，电压传感和生物相容性。附着于DNA支架的亲脂性锚将使VSND靶向膜或甚至靶向膜中。附着在支架上并用荧光染料标记的带电柔性元件将通过在电场中移动而对膜电位的变化做出反应。柔性元件的移动将通过从位于DNA支架上的染料到柔性元件上的染料的单分子双折射-共振-能量转移来检测。我们的VSND的两个互补设计将是这个项目的起点。附接到膜的电压感测筏具有突出到膜中的柔性元件，其提供了侵入性最小的优点。并行地，电压感测孔集成到膜中，并且包含在中心孔中受到保护的传感器，而不受局部环境的干扰。VSND将在定制设置上使用玻璃纳米移液管在模型膜上同时进行电学和光学测量进行测试和校准。这将使跨膜电压的量化，是由定性测量为主的领域。下一步，我们将应用VSNDs以前所未有的空间和时间分辨率量化活细菌和真核细胞的膜电位。成功实施后，我们将证明在活体斑马鱼的膜电压成像的VSNDs的适用性。这些实验将证明，我们的方法具有所需的灵敏度和快速响应，以回答基本问题，细菌膜电位和真核细胞膜和轴突电位的作用。模块化的基于DNA的设计的VSNDs允许直接优化和适应的电压传感问题的通用解决方案，并可能会发现应用范围从视觉测量的神经元功能的离子通道相关的药物识别和筛选。

Any living cell requires membrane potentials for a wide range of functions including energy production and information processing and transmittance. Exact knowledge of membrane voltages is of paramount importance in neuroscience and especially brain research. The available techniques heavily depend on optical measurements of membrane potentials which are currently limited by low sensitivity, low speed and invasiveness. Genetically encoded sensors rely on unstable fluorescent proteins and require genetic modification of the host organism. In this project, we suggest a new approach for optical voltage sensing nano-devices (VSND) based on two fundamental designs both using a DNA scaffold. The DNA scaffold enables spatial and chemical control over all important functions of the VSNDs including membrane positioning, voltage sensing and biocompatibility. Lipophilic anchors attached to the DNA scaffold will target the VSND to or even into the membrane. A charged, flexible element attached to the scaffold and labeled with a fluorescent dye will react to changes of the membrane potential by moving in the electric field. The movement of the flexible element will be detected by single-molecule Fluorescence-Resonance-Energy-Transfer from a dye located on the DNA scaffold to the dye on the flexible element. Our two complementary designs of VSNDs will be the starting point for this project. A voltage sensing raft that attaches to the membrane with the flexible elements protruding into the membrane offers the advantage of being least invasive. In parallel, a voltage sensing pore integrates into the membrane and contains the sensor protected in a central pore without perturbations from the local environment. The VSNDs will be tested and calibrated using simultaneous electrical and optical measurements on model membranes using glass nanopipettes on a custom-built setup. This will enable quantification of transmembrane voltages in a field that is dominated by qualitative measurements. In the next step, we will apply the VSNDs to quantify membrane potentials in living bacterial and eukaryotic cells with unprecedented spatial and temporal resolution. After successful implementation we will demonstrate the in vivo applicability of the VSNDs by imaging membrane voltages in living zebra fish. These experiments will prove that our approach has the required sensitivity and fast responsiveness to answer fundamental questions with respect to the role of bacterial membrane potentials and eukaryotic membrane and axon potentials. The modular DNA-based design of the VSNDs allows straightforward optimization and adaptation for a generic solution of voltage sensing problems and might find applications ranging from visual measurements of neuronal functions to ion-channel related drug identification and screening.