Building Multicolor Color pH Modulation Sensing Platforms for Time Resolved and Multistep Sensing

This DURIP award provides funds for the Principal Investigator to purchase an inverted fluorescence microscope with a digitally triggered filter wheel, and a dynamic light scattering instrument with zeta potential measurement capabilities. Both instruments will facilitate the Principal Investigator measuring the fluorescence images of two or more dye molecules nearly simultaneously in pH modulation sensing assays, a sensing technique that the Principal Investigator designed under ONR funding since 2008 for the sensing of proteins, peptides, small molecules, and ions, but established with only single dye sensing. (The Principal Investigator~s current award number is N000141410792 with a period of performance from 13-JUN-14 to 12-JUN-17.) Two color pH modulation sensing measurements enable time resolved information to be obtained not just on analyte binding to lipid membranes, but also with complementary information on parameters such as translocation across the membrane, membrane leakage, protein-protein dissociations, membrane protein dimerization, bilayer solubilization, and transmembrane signaling events. In this case, a pH sensitive dye will be used to sense the first binding process and a second fluorophore will be used to detect the complementary process. To get near simultaneous responses from the two dyes in the same platform, it is necessary to excite both dyes at their respective frequencies. The easiest and most efficient way to do that is to use a digitally triggered filter wheel that rapidly switches between two different filter sets, and the LED light sources and the triggered filtered wheel on the proposed inverted fluorescence microscope are well suited for that task. The Principal Investigator intends to develop three sensing platforms with the proposed equipment: (1) A double bilayer system to study the binding of cell penetrating, or antimicrobial, peptides to membranes and subsequent cell penetration; (2) Giant unilamelar vesicles to study, for example, the binding of ligands to G-protein coupled receptors and subsequent transmembrane signaling events that such binding events trigger, monitoring gated ion channels or monitoring ligand binding and protein dimerization or oligimerization on the membrane surface; and (3) Liquid-order domains and liquid-disordered domains for studying the binding of ibuprofen and the subsequent leakage and solubilization of the membranes. The proposed dynamic light scattering instrument will replace a similar 15 year old instrument that has exceeded its lifetime, since the software for using the instrument is no longer compatible with modern computers and data has to be recorded by hand. The requested version of this instrument enables the determination of vesicle size distributions even for vesicle populations with complex diameter distributions, which is necessary in the preparation of supported bilayer systems that the Principal Investigator uses in his experiments. The Principal Investigator will also use this instrument to determine the potential at the slip plane for lipid bilayer/aqueous interfaces. Such measurements are important for optimizing the dye chemistry employed in two color pH modulation sensing measurements. Beyond its value to the specific research efforts in which it will be used, the proposed equipment will also play an important role in the science and technology education of the undergraduate students, graduate students, and postdoctoral fellows performing the research activities with the equipment. The Principal Investigator estimates that the proposed equipment will serve 25-40 undergraduates, Ph.D. students, and postdoctoral fellows over the next five years. Additionally, the requested microscope will be used in an upper level undergraduate physical chemistry laboratory course at The Pennsylvania State University.