New methods for the production and analysis of nanostructured self-assembled lipid mesophases with bicontinuous cubic topology as supported thin films

这项研究旨在扩展我们对一类新出现的称为QII或反双连续立方相的材料的理解，通过在平坦基底上以薄膜的形式制备它们。当脂质分子与水混合时，它们自发地组装成各种有序结构，包括三种不同形状的QII相。每一个细胞都含有分支的水通道网络，大小为十亿分之一米，由一个单一的脂质双层隔开，就像生物细胞中的脂质双层一样。我们可以通过改变样品的含水量或温度来精确控制水通道的大小。通过进一步改变条件，我们还可以诱导从一种形状到另一种形状的相变。水通道的可控纳米尺度尺寸，脂质双层的大面积包装成小体积，以及双层包含类似于生物细胞膜的环境的事实，使QII相具有广泛的应用。在某些情况下，双层用另一种材料模板化，以制造分子筛，电极或传感器。在其他情况下，它们可以直接用作药物递送的载体，用于基于膜结合蛋白的生物传感器中，或作为结晶膜蛋白以解析其结构的方法。此外，QII相存在于自然界中，发挥着各种生物学作用;了解它们的形成可以帮助我们更普遍地了解细胞膜分裂或融合时发生的情况。目前有很多研究致力于了解QII相的产生和相互转换的过程，并开发新材料来利用它们的特性。然而，这一研究受到以下事实的阻碍：对QII材料的实验是在多畴样品上进行的，其中常规的3D有序仅在单个微米尺寸的畴内延伸。一个QII样本将包含数十亿个这样的域，所有这些域都以随机方向取向。这减少了从实验中获得的信息，引入了额外的影响，由于域之间的边界，并限制了材料的技术潜力。在这里，我们的目标是开发方法来生产一种新形式的QII样品，作为20和200纳米之间的薄膜厚度。为了实现这一点，我们将开始，使一堆双层支持在一个非常平坦的表面上，使用成熟的方法。在已知的多结构域脂质样品相图的指导下，我们将改变样品环境到不同的温度和/或湿度，在那里它将经历到QII相的转变。这将是一个单一的深度领域，我们将能够应用，第一次，一系列的技术，可以调查一个区域只有一个域跨。其中包括原子力显微镜，它探测样品的表面，分辨率高到足以可视化QII相中的单个水通道，以及X射线散射，它告诉我们所采用的规则结构的几何形状，方向和重复间隔。这些新的样品制备和分析方法将产生大量关于QII相的信息。首先，我们将能够测试未经证实的模型和预测，以确定一个相转变为另一个相的几何路径。其次，我们将了解如何控制畴的大小，并了解畴边界在相变中的作用。第三，我们将能够产生和分析不对称的QII相，这种结构迄今为止从未在实验室中制造过，其中构成双层的两个单层在脂质组成上不同。这些材料将具有新的特性和应用，并将提供更好的细胞膜类似物。最后，这项工作将成为进一步项目的基础，使用QII薄膜作为电化学，膜蛋白研究和一系列基于QII阶段的其他纳米技术应用的更好的控制系统。

This research aims to extend our understanding of a newly-emerging class of materials known as QII or inverse bicontinuous cubic phases, by producing them in the form of thin films on flat substrates.When lipid molecules are mixed with water, they spontaneously assemble into a variety of ordered structures, including three different shapes of QII phase. These each contain branching networks of water channels, billionths of a metre in size, separated by a single lipid bilayer which is just like the one in a biological cell. We can precisely control the size of the water channels by varying the water content or temperature of the sample. By changing conditions further, we can also induce a phase transition from one shape into another.The controllable nanometer-scale size of the water channels, the large area of lipid bilayer packed into a small volume, and the fact that the bilayer contains an environment similar to a biological cell membrane, give QII phases a wide range of applications. In some, the bilayer is templated with another material, to make molecular sieves, electrodes or sensors. In other cases they may be used directly, as a vehicle for drug delivery, in biosensors based on membrane-bound proteins, or as a method of crystallizing membrane proteins in order to solve their structure. Furthermore, QII phases exist in nature, performing various biological roles; understanding their formation can help us to understand more generally what happens when cell membranes divide or fuse.There is much current research towards understanding the processes that produce and inter-convert QII phases, and towards developing new materials to exploit their properties. However, this research is hampered by the fact that experiments on QII materials are carried out on polydomain samples, where the regular 3D ordering only extends within a single micron-sized domain . A QII sample will contain billions of these domains, all oriented in random directions. This reduces the information obtained from experiments, introduces additional effects due to the boundaries between domains, and limits the technological potential of the material.Here, we aim to develop ways to produce a new form of QII sample, as thin films between 20 and 200nm thick. To achieve this we will begin by making a stack of bilayers supported on an extremely flat surface, using proven methods. Guided by phase diagrams that are already known for polydomain lipid samples, we will then change the sample environment to a different temperature and/or humidity, where it will undergo a transition into a QII phase. This will be a single domain in depth, and we will be able to apply, for the first time, a range of techniques that can investigate an area only one domain across. These include atomic force microscopy, which probes the surface of the sample with a resolution high enough to visualize single water channels in the QII phase, and x-ray scattering, which tells us the geometry, orientation and repeat spacing of the regular structures adopted. These new methods of sample preparation and analysis will produce a wealth of information on QII phases.First, we will be able to test unconfirmed models and predictions for the geometric pathways by which one phase turns into another. Secondly, we will find out how to control the sizes of the domains, and see the role that domain boundaries play in phase transitions. Thirdly, we will be able to produce and analyse asymmetric QII phases, structures that so far have never been made in a laboratory, where the two monolayers making up the bilayer differ in lipid composition. Such materials would have new properties and applications, and would offer better analogs of cell membranes. Finally, the work will form the basis for further projects, using supported thin films of QII as a better controlled system for electrochemistry, membrane protein research,and a range of other nanotechnological applications based on QII phases.