Surface Plasmon-coupled Fluorescence Microscope to Study Ion Channel Dynamics

描述(申请人提供)：该项目的长期目标是开发一种基于表面等离子体共振的光学检测系统，以研究膜蛋白的动力学，重点是电压门控离子通道，如负责神经冲动产生和传播的Na和K通道。使用半胱氨酸化学方法在特定位置用荧光探针标记通道蛋白，并通过所提出的光学装置来检测荧光。由猝灭或能量转移产生的荧光变化是局部环境变化的指示器，因此当通道经历从关闭状态到开放状态的转变时，它们跟随蛋白质内的构象变化。该光学设备使用半球形透镜，将入射激光耦合到玻璃芯片上，该玻璃芯片具有薄(50 Nm)的镀银层，其中生物制剂由薄(10 Nm)的氧化硅层分隔。正确的激发角度诱导金属中的等离子体共振，并增强标记通道的荧光团的荧光。检测从生物制剂侧或从激发侧进行。在第二种情况下，信噪比预计要大得多，因为耦合发射来自受限于20 nm的区域，而且由于它是方向性的，所以特殊设计的光学元件收集了光电探测器上的大部分光。光学系统的测试是在哺乳动物细胞或支持的双层中表达的标记离子通道上进行的。在第二种情况下，所支持的双层由含有纯化的标记通道的脂质体制成。利用等离子激元芯片的银层改变双分子层上的电压。还提出了光学系统的改进，以成像生物制剂，以跟踪响应于改变通道构象的电压脉冲的单个分子的荧光的时间进程。了解离子通道蛋白的构象动力学是设计改善或治愈离子通道功能异常所致的多种神经系统疾病所需的药物或治疗方法的关键步骤。在这一应用中开发的光学系统旨在开发一种新的显微镜，该显微镜专门设计用于检测离子通道的构象变化，具有比现有设备更高的分辨率、更高的灵敏度和对虚假荧光的更好的抑制。

DESCRIPTION (provided by applicant): The long term objective of this project is the development of an optical detection system based on surface plasmon resonance to study the dynamics of membrane proteins with special emphasis in voltage gated ion channels such as the Na and K channels that are responsible for the generation and propagation of the nerve impulse. Channel proteins are labeled in specific sites with fluorescent probes using cysteine chemistry and the fluorescence is detected by the proposed optical setup. Fluorescence changes, produced by quenching or energy transfer are indicators of local environmental changes and thus they follow conformational changes within the protein as the channel undergoes transitions from the closed to the open state. The optical apparatus uses a hemispherical lens that couples an incoming laser beam on a glass chip that has a thin (50 nm) siver layer where the biological preparation lies separated by a thin (10 nm) layer of silicon oxide. The correct angle of excitation induces plasmon resonance in the metal and enhances the fluorescence of fluorophores labeling the channel. The detection is done from the biological preparation side or from the excitation side. In the second case the signal to noise ratio is expected to be much larger because the coupled emission comes from a region limited to 20 nm and, as it is directional, a specially designed optics collects most of the light on a photodetector. The testing of the optical sytem is done on labeled ion channels expressed in mammalian cells or in supported bilayers. In the second case, the supported bilayer is made with liposomes containing purified labeled channels. The voltage across the bilayer is changed taking advantage of the silver layer of the plasmon chip. A modification of the optical system is also proposed to image the biological preparation to follow the time course of the fluorescence of individual molecules in response to voltage pulses that change the conformation of the channel. The understanding of conformational dynamics of channel proteins is a crucial step in the design of drugs or therapies needed to ameliorate or cure several neurological deseases produced by abnormal function of ion channels. The optical system developed in this application is aimed at developing a new microscope that is especially designed to detect conformational changes of ion channels with improved resolution, higher sensitivity and improved rejection of spurious fluorescence than presently available devices.