Novel Experiments Based on Time-Resolved Fluorescence to Probe Complex Physical Phenomena in Macromolecules and their Supramolecular Assemblies

大分子是非常大的分子，其溶液具有有趣的特性，用于各种应用，包括化妆品，药物输送，洗涤剂，油漆或提高石油采收率。反过来，大分子的理想性质是由于它们的分子结构和化学组成。新的合成进展使制备越来越复杂的大分子成为可能，这些大分子可以通过荧光在分子水平上进行探测。一种可以应用于分子水平上探测大分子的荧光技术是基于芘准分子的形成。芘是一种染料，能形成一种被称为准分子的受激复合体。准分子是两种芘染料相遇的结果。通过将芘连接到大分子上，可以用荧光探测两种芘染料之间的准分子形成过程。这些荧光实验提供了有关芘准分子形成发生的时间尺度和芘染料状态的信息。由于芘部分是共价连接到大分子上的，因此芘准分子形成的时间尺度提供了有关大分子的柔韧性和可变形性的信息。芘染料的状态表明芘是分离的，还是通过扩散形成准分子，或者是聚集的，这描述了染料如何在大分子上分布。这些影响将在本提案所提出的三个主题中加以利用。第一个主题旨在开发一种程序来估计线性链所经历的力，其末端被荧光标记为芘，以便它们可以锚定在两个表面活性剂胶束中。表面活性剂是聚集成胶束的分子。胶束有一个油性的内部，可以容纳芘，而链是水溶性的，留在水中，如果它足够长，可以跨越两个胶束。溶液条件可以调整，使两个胶束靠得更近，这样它们就可以用更短的链桥接。如果两端位于同一胶束内，则两个芘标签形成一个准分子，而如果两端位于两个不同的胶束内则不会形成准分子。荧光将用于确定两端在同一胶团中的链的比例。反过来，这些信息将被应用于估计表面活性剂胶束在将两个链端拉开时所施加的力。第二个主题将探讨与DNA聚集在一起的表面活性剂的移动性。DNA表面活性剂聚集体用于基因传递，其中DNA片段通过细胞膜运输。研究证实，有效的基因传递关键取决于表面活性剂与聚集体内DNA的排列。通过使用芘标记的表面活性剂，表面活性剂在表面活性剂- dna聚集体中的迁移率将被确定为表面活性剂在聚集体中排列的函数。最后，荧光测量将用于探测聚合物在超过临界温度的溶液中坍塌时会发生什么。这种现象在聚合物科学中非常重要，因为它是在蛋白质折叠过程中遇到的。将制备一种热响应性聚合物，该聚合物在设定温度以上会坍塌，并用芘进行标记。通过监测芘聚集的程度和聚合物崩溃时芘准分子形成的速率，应该有可能获得有关热响应性聚合物崩溃的时间尺度的信息。

Macromolecules are very large molecules whose solutions have interesting properties that are used in a variety of applications that include cosmetics, drug delivery, detergents, paints, or enhanced oil recovery. In turn, the desirable properties of macromolecules are due to their molecular architecture and chemical composition. New synthetic advances have allowed the preparation of increasingly complex macromolecules which can be probed at the molecular level by fluorescence. One fluorescence technique that can be applied to probe macromolecules at the molecular level is based on pyrene excimer formation. Pyrene is a dye that forms an excited complex called an excimer. The excimer is the result of an encounter between two pyrene dyes. By attaching pyrene onto a macromolecule, the process of excimer formation between two pyrene dyes can be probed by fluorescence. These fluorescence experiments provide information about the time scale over which pyrene excimer formation takes place and the state of the pyrene dyes. Since the pyrene moieties are covalently attached onto the macromolecule, the time scale of pyrene excimer formation provides information about the flexibility, and thus deformability, of the macromolecule. The state of the pyrene dyes indicates whether the pyrenes are isolated, form excimer by diffusion, or are aggregated which describes how the dyes are distributed on the macromolecule. These effects will be put to use in the three themes developed in this proposal. The first theme aims to develop a procedure to estimate the force experienced by a linear chain whose ends have been fluorescently labeled with pyrene so that they can be anchored in two surfactant micelles. Surfactants are molecules that aggregate into micelles. The micelles have an oily interior which can host pyrene while the chain is water-soluble, remains in water, and can span the two micelles if it is long enough. The solution condition can be adjusted to allow the two micelles to come closer so that they can be bridged by a shorter chain. If the two ends are located inside a same micelle, the two pyrene labels will form an excimer whereas no excimer will be formed if the two ends are in two different micelles. Fluorescence will be used to determine the fraction of chains having their two ends in a same micelle. In turn, this information will be applied to estimate the force exerted by the surfactant micelles as they pull the two chain ends apart. The second theme will probe the mobility of surfactants involved in an aggregate with DNA. DNA-surfactant aggregates are used for gene delivery where a piece of DNA is transported across the membrane of a cell. Studies have confirmed that efficient gene delivery depends critically on the arrangement on the surfactants with the DNA inside the aggregate. By using a pyrene-labeled surfactant, the mobility of the surfactant in the surfactant-DNA aggregate will be determined as a function of the surfactant arrangement in the aggregate. Finally, fluorescence measurements will be employed to probe what happens to a polymer as it collapses in solution above a critical temperature. This phenomenon is very important in Polymer Science since it is encountered during protein folding. A thermoresponsive polymer that collapses above a set temperature will be prepared and labeled with pyrene. By monitoring both the extent of pyrene aggregation and the rate of pyrene excimer formation as the polymer collapses, it should be possible to gain information about the time scale over which the thermoresponsive polymer collapses.