Koo Juny, Czeslik Claus
Technische Universität Dortmund, Fakultät Chemie, D-44221 Dortmund, Germany.
Rev Sci Instrum. 2012 Aug;83(8):085109. doi: 10.1063/1.4746385.
Total internal reflection fluorescence (TIRF) spectroscopy is a surface sensitive technique that is widely used to characterize the structure and dynamics of molecules at planar liquid-solid interfaces. In particular, biomolecular systems, such as protein adsorbates and lipid membranes can easily be studied by TIRF spectroscopy. Applying pressure to molecular systems offers access to all kinds of volume changes occurring during assembly of molecules, phase transitions, and chemical reactions. So far, most of these volume changes have been characterized in bulk solution, only. Here, we describe the design and performance of a high pressure sample cell that allows for TIRF spectroscopy under high pressures up to 2500 bar (2.5 × 10(8) Pa), in order to expand the understanding of volume effects from the bulk phase to liquid-solid interfaces. The new sample cell is based on a cylindrical body made of Nimonic 90 alloy and incorporates a pressure transmitting sample cuvette. This cuvette is composed of a fused silica prism and a flexible rubber gasket. It contains the sample solution and ensures a complete separation of the sample from the liquid pressure medium. The sample solution is in contact with the inner wall of the prism forming the interface under study, where fluorescent molecules are immobilized. In this way, the new high pressure TIRF sample cell is very useful for studying any biomolecular layer that can be deposited at a planar water-silica interface. As examples, high pressure TIRF data of adsorbed lysozyme and two phospholipid membranes are presented.
全内反射荧光（TIRF）光谱是一种表面敏感技术，广泛用于表征平面液-固界面处分子的结构和动力学。特别是，生物分子系统，如蛋白质吸附物和脂质膜，很容易通过TIRF光谱进行研究。对分子系统施加压力能够研究分子组装、相变和化学反应过程中发生的各种体积变化。到目前为止，大多数这些体积变化仅在本体溶液中得到表征。在此，我们描述了一种高压样品池的设计和性能，该样品池能够在高达2500巴（2.5×10⁸帕）的高压下进行TIRF光谱分析，以拓展从本体相到液-固界面的体积效应的理解。新的样品池基于由尼蒙镍合金90制成的圆柱体，并包含一个压力传递样品比色皿。该比色皿由熔融石英棱镜和柔性橡胶垫圈组成。它容纳样品溶液，并确保样品与液体压力介质完全分离。样品溶液与棱镜的内壁接触，形成正在研究的界面，荧光分子固定在该界面处。通过这种方式，新的高压TIRF样品池对于研究任何可以沉积在平面水-二氧化硅界面的生物分子层非常有用。作为示例，给出了吸附溶菌酶和两种磷脂膜的高压TIRF数据。
