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

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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.