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

利用微型红外波导传感器分析电压引起的细胞膜组件结构变化

• 批准号: 9406681
• 负责人: Mark Braiman
• 金额: $ 31万
• 依托单位: University of Virginia Main Campus
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 1994
• 资助国家: 美国
• 起止时间: 1994-07-01 至 1998-06-30
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=9406681&HistoricalAwards=false
• 关键词: Analysis; Voltage; Induced; Structural; Changes

9406681 Braiman Miniature waveguide sensors will be used to detect IR absorbance changes associated with voltage-induced conformational transitions in membrane proteins. The motivation for miniaturization of the sensors is to permit in situ observation of IR spectra from membranes of intact individual cells. This, in turn, will allow IR spectroscopy to be performed as a function of cell membrane potential, which can be controlled by using a whole-cell voltage clamp. Sensitive voltage-dependent IR difference spectra should thus reveal how the living cell's membrane components responds to transient shifts in transmembrane voltage. A particular goal of this approach is to allow investigation of a variety of ion channel proteins expressed at high levels in single frog oocytes. These IR spectroscopic studies of biomembranes will depend heavily on the development of evanescent-wave sensors based on thin-film dielectric waveguides. A living cell's plasma membrane will be held in contact with a thin film of Ge deposited on ZnS or CaF2 substrate. A portion of IR light passing through the waveguide is carried in an evanescent wave that can be absorbed by molecules in the membrane. The wavelength dependence of this absorption will provide information about the vibrational modes, and thus the structures, of the lipid and protein molecules in the membrane. %%% Miniaturized biomembrane sensors based on waveguides that conduct infrared light will be developed. These waveguide sensors, consisting of thin films of infrared-transparent materials will conduct and concentrate infrared light into a small tapered region where the waveguide can be held in close contact with the membrane of a single cell, such as a 1-mm-dia. frog egg cell. Although most of the infrared light passing through the waveguide will be confined to the interior of the germanium film, a significant fraction of the light energy will be conducted in a micron-thick region outside the waveguide. This s o-called evanescent wave must therefore pass through the membrane of the cell. 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 (responsible, e.g., for neuronal signaling) change in structure in response to the varying transmembrane voltages found in living cells. 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 and industrial process control. ***

Braiman微型波导传感器将用于检测膜蛋白中与电压诱导的构象转变相关的红外吸收变化。传感器小型化的动机是允许原位观察完整单个细胞膜的红外光谱。反过来，这将允许红外光谱作为细胞膜电位的函数来执行，这可以通过使用全细胞电压钳来控制。因此，敏感的电压依赖性红外差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。这种方法的一个特殊目标是允许研究在单个青蛙卵母细胞中高水平表达的各种离子通道蛋白。这些生物膜的红外光谱研究将在很大程度上依赖于基于薄膜介质波导的倏逝波传感器的发展。活细胞的质膜将与沉积在ZnS或CaF2衬底上的Ge薄膜接触。通过波导的一部分红外光以一种可被膜中的分子吸收的倏逝波的形式携带。这种吸收的波长依赖性将提供有关膜中脂质和蛋白质分子的振动模式和结构的信息。基于传导红外光的波导的小型化生物膜传感器将被开发。这些由红外透明材料薄膜组成的波导传感器将传导并将红外光集中到一个小的锥形区域，在这个区域中，波导可以与单个细胞的膜紧密接触，例如直径为1毫米的细胞。青蛙卵细胞。虽然大多数通过波导的红外光将被限制在锗膜的内部，但很大一部分光能将在波导外的微米厚区域内传导。因此，这种所谓的倏逝波必须穿过细胞膜。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。一个特别的目标是基本了解各种离子通道蛋白（负责，例如神经元信号传导）如何响应活细胞中不同的跨膜电压而改变结构。然而，这项研究也应该提高我们国家在各种微小样品的敏感化学分析方面的技术能力，这对于环境监测和工业过程控制等各种任务可能是重要的。＊＊＊