Research Mentor(s): Yuji Mishina, Professor
Research Mentor School/College/Department: Biological and Material Sciences, School of Dentistry
Presentation Date: Thursday, April 22, 2021
Session: Session 2 (11am – 11:50am)
Breakout Room: Room 11
Fluorescent probes are excitable by specific frequencies of light and emit bright visible electromagnetic radiation upon their relaxation to a ground state energy. These probes can be chemically modified to tune their properties and impart specific reactivity. In the biomedical sciences, fluorescent probes are widely used in applications including determining the presence and quantity of a specific protein, determining enzyme activity in real-time assays, determining gene expression, and elucidating physicochemical conditions both intracellularly and extracellularly. However, current probes present a significantly high cost, modest fluorescent intensity, and lack chemical functionality for important uses such as site-specific protein labelling, orthogonal click chemistry, and for use in biomaterial constructs. In this study, we aimed at designing high fluorescent yield, tunable pH-sensitive probes which could be synthesized using inexpensive common reagents to illuminate microenvironmental changes in pH, a critical factor in biologic processes including endosomal uptake, lysosome processing, and extracellular matrix resorption. Utilizing commercially available Rhodamine 6G (R6G), Rhodamine 110G (R110G), and Rhodamine B (RhB) bases, we designed and synthesized a library of fluorescent probes from small-molecule ligands. In this project, a computational approach was employed to explain the properties of these probes at a molecular level and predict the properties of additional hypothetical probes. We hypothesized that the energy difference between spirolactam (unprotonated) and quinone (protonated) molecular structures would drive pH sensitivity, and ligand properties would drive pKa (“turn-on pH”). Theoretical minimum enthalpy energies were computed on geometry-optimized structures of modified rhodamine bases (n=24) in a protonated and deprotonated state using Avogadro computational software, in an iterative approach. Our data supports experimental evidence that unmodified R6G, RhB, and R110G bases are not pH-sensitive, but certain modified structures are pH-sensitive based on the energy difference between protonated and deprotonated forms. All molecular derivatives of R6G and RhB in this study are pH fluorescent, where the difference in energy correlates similarly to differences in pKa. Conversely, R110G derivatives are not pH-sensitive, consistent with experimental evidence. These data suggest that pH-sensitivity results when the protonated molecules are more thermodynamically favorable and would respond to changes in pH. In agreement, we demonstrate that these molecules fluoresce below an adjustable threshold pH (threshold < 7.35 pH) since only the protonated structure is aromatic and fluorescent. Hence, these fluorescent probes "turn off" above this threshold pH in their deprotonated structures. Normal background physiological cytosol pH (~7.34 pH) does not activate fluorescence in these probes, therefore enhancing visual pH juxtaposition for acidic microenvironments within the cell that related to proton-producing biochemical phenomena that do prompt probe fluorescence. Finally, we demonstrate that these small-molecule probes can be further functionalized for incorporation into a wide variety of molecular biology tools, including biodegradable polymers, while maintaining their pH-sensitive properties. This technology has great potential for use as a biomedical research tool to determine differences in pH within biologic microenvironments as these probes could bind to nanoparticles, proteins, or antibodies to act as pH-reporters. With promising theoretical and experimental data, further research will focus on specific applications to validate its use in biologic systems.
Authors: Seth Woodbury, Miranda Eberle, Ben Swanson, Yuji Mishina
Research Method: Laboratory Research