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，其溶液具有有趣的性质，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。新的合成进展已经允许制备越来越复杂的大分子，其可以通过荧光在分子水平上进行探测。一种可以应用于在分子水平上探测大分子的荧光技术是基于芘激基缔合物的形成。芘是一种染料，形成一种被称为激基缔合物的激发复合物。激基缔合物是两种芘染料相遇的结果。通过将芘连接到大分子上，可以通过荧光来探测两个芘染料之间的激基缔合物形成过程。这些荧光实验提供了关于芘激基缔合物形成发生的时间尺度和芘染料的状态的信息。由于芘部分共价连接到大分子上，芘激基缔合物形成的时间尺度提供了关于大分子的柔性和变形性的信息。芘染料的状态表明芘是孤立的，通过扩散形成激基缔合物，还是聚集的，这描述了染料如何分布在大分子上。这些效果将用于本提案中提出的三个主题。 第一个主题的目的是开发一个程序来估计所经历的力的线性链，其两端已被荧光标记的芘，使它们可以锚定在两个表面活性剂胶束。表面活性剂是聚集成胶束的分子。胶束具有油性内部，其可以容纳芘，而链是水溶性的，保持在水中，并且如果足够长，可以跨越两个胶束。可以调节溶液条件以允许两个胶束更接近，使得它们可以通过较短的链桥接。如果两个末端位于同一胶束内，则两个芘标记物将形成激基缔合物，而如果两个末端位于两个不同的胶束中，则不会形成激基缔合物。荧光将用于确定在同一胶束中具有其两个末端的链的分数。反过来，该信息将被应用于估计由表面活性剂胶束施加的力，因为它们将两个链端拉开。 第二个主题将探讨与DNA聚集体中涉及的表面活性剂的流动性。DNA-表面活性剂聚集体用于基因递送，其中一段DNA被转运穿过细胞膜。研究已经证实，有效的基因递送关键取决于表面活性剂与聚集体内的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聚集体中表面活性剂排列的函数。 最后，将采用荧光测量来探测聚合物在临界温度以上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，因为它在蛋白质折叠过程中遇到。将制备在设定温度以上塌陷的温敏聚合物并用芘标记。通过监测聚合物坍塌时芘聚集的程度和芘激基缔合物形成的速率，应该可以获得关于温度响应性聚合物坍塌的时间尺度的信息。

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.