Analysis of Voltage-Induced Structural Changes in Cell Membrane Components with Miniature Infrared Waveguide Sensors

本研究的目的是开发微型红外波导，以检测和分析电压诱导的膜结合蛋白的构象变化。传感器小型化的动机，使用5-50米的厚度和0.5-2毫米的宽度，是为了允许原位观察完整的单个细胞膜的红外光谱。这将允许红外光谱作为细胞膜电位的函数来执行，这将使用全细胞电压钳来控制。敏感的电压依赖性红外光谱应该揭示活细胞的膜组分如何响应跨膜电压的瞬态变化。一个特别的目标是通过实验证明一组可行性计算，预测这种方法将允许在青蛙卵母细胞中表达的离子通道上测量电压诱导的差异光谱，或者在重组成巨大脂质体的小通道肽上测量电压诱导的差异光谱。细胞或脂质体将与微型支撑平面锗波导接触。沿着波导传播的部分红外光能量以倏逝波的形式携带，可被其表面附近的分子吸收，包括细胞或脂质体膜中的分子。探测可重复的电压依赖性的倏逝波吸收光谱是该项目的主要目标，因为这种电压依赖性的光谱将提供有关通道打开所涉及的结构变化的信息。一种基于能传导红外光的薄锗波导的生物传感器，将被用于检测单个细胞膜的结构变化。当一个细胞或一个类似细胞的单层生物膜囊泡与锗表面接触时，通过波导传输的光可以在选择的波长上被吸收，并测量光谱。波导将红外光集中在一条路径上，使光与细胞膜上的mol分子的相互作用最大化，相对于光与强吸收红外的大量水的相互作用，后者也必须存在，才能使细胞正常运作。细胞膜组分对不同红外波长的选择性吸收将成为其化学结构光谱分析的基础。一个特别的目标是对各种离子通道蛋白的功能有一个基本的了解。然而，这项研究也应该提高我们国家对各种微小样品进行敏感化学分析的技术能力，这对于环境监测等各种任务可能是重要的。＊＊＊

9722887 Braiman The goal of this research is to develop miniature infrared wave-guide in order to detect an analyze voltage-induced conformational changes in membrane-bound proteins. The motivation for miniaturization of the sensors, using a thickness of 5-50 (m and a width of 0.5-2 mm, is to permit in-situ observation of IR spectra from membranes of intact individual cells. This will allow IR spectroscopy to be performed as a function of cell membrane potential, which will be controlled using a whole-cell voltage clamp. Sensitive voltage-dependent IR differences spectra should thus reveal how the living cell's membrane components respond to transient shifts in transmembrane voltage. A particular goal is to prove experimentally a set of feasibility calculations predicting that this approach will allow voltage-induced difference spectra to be measured on an ion channel expressed in a frog oocyte, or on a small channel peptide reconstituted into a giant liposome. The cell or liposome will be held in contact with a miniature supported planar Ge waveguide. A portion of the IR light energy propagating along the waveguide is carried in a evanescent wave that can be absorbed by molecules near its surface, including those in the cell or liposome membrane. Detection of a reproducible voltage-dependence of the evanescent-wave absorption spectrum is the principal goal of the project, since this voltage-dependent spectrum will provide information about the structural changes involved in channel opening. A kind of biosensor, based on a thin genmanium waveguide that conducts infrared light, will be used to detect structural changes in the membranes of single cells. When a cell, or a single-layer biomembrane vesicle resembling a cell, is placed in contact with the germanium surface, the light transmitted through the waveguide can be absorbed at selective wavelengths, and the spectrum measured. The waveguide concentrates infrared light along a path that maximizes the light's interaction with mol ecules in the cell membrane, relative to the light's interaction with the strongly-IR-absorbing bulk water that must also be present to allow the cell to function normally. Selective absorption of different infrared wavelengths by the cell membrane's components will be the basis for spectroscopic analysis of their chemical structures. A particular goal is a basic understanding of how various ion-channel proteins function. However, this research should also advance our national technical capabilities in the sensitive chemical analysis of a wide variety of tiny samples, which may be of importance for such varied tasks as environmental monitoring. ***