Miranda N Hurst received her Bachelor of Science in Cell and Molecular Biology from Missouri State University in 2015. She is currently working as a Research Assistant under Dr. Robert Delong at the Nanotechnology Innovation Center of Kansas State (NICKS). Her current research focuses on the stabilization and delivery of RNA via nanoparticles into human and mouse cell lines. Her research interests include the relationship between structure and function of RNA and protein in the complex cellular environment, specifically for the development of cancer therapies. She has 2 publications in peer reviewed journals and 1 patent submitted.
Two dimensional fluorescence difference spectroscopy (2D FDS) is an innovative technique to detect nanoparticles and validate interaction upon surface functionalization and biomolecule loading. 2D FDS detects emission while scanning multiple excitation wavelengths, generating a contour plot with fluorescent intensity reflected as color per an excitation and emission intersect, also called a spectral signature. Initially metal oxide nanoparticles of various compositions were compared, where 2D FDS revealed a unique spectral signature per material composition. To gauge detection of surface functionalization, zinc oxide nanoparticles (ZnO NP) were loaded with one of three polymers: glycol chitosan, polyacrylic acid (PAA), and methoxy polyethylene glycol (mPEG). 2D FDS revealed a shift in the spectral signature in the presence of the polymer. ZnO NP was loaded with one of three RNAs: Torula Yeast RNA (TYRNA), polyinosinic: polycytidylic acid (pIC), and splice switching oligonucleotide (SSO), to determine the efficacy of 2D FDS in identifying biomolecular interactions. The greatest spectral shift was observed for pIC, followed by SSO and TYRNA. Extending this technique from nanoparticles to proteins, Ras binding domain (RBD) is a derivative of B-Raf protein capable of binding Ras and thus a potential cancer therapeutic. RBD was shown to give a spectral signature, where a shift in the optimal excitation and emission intersect elucidates protein conformation. In the presence of ZnO NP and iron oxide nanoparticle (Fe2O3 NP), a shift was observed for both particles. Altogether, these data support 2D FDS as a novel technique in identifying nanoparticles and their surface interactions.
Ashley Gasiorowski is currently pursuing her Bachelor of Science in Biology at Kansas State University. She is enrolled in the Veterinary Scholars Early Admission Program and Honors Program. She has previously worked as a Laboratory Assistant in the Department of Plant Pathology, as well as at Kansas State’s Olathe campus. She is continuing her passion for research as a student researcher under Dr. Rob DeLong in the Department of Anatomy and Physiology. Her research interests include veterinary science and medicine with hopes of becoming a veterinarian.
Luciferase (Luc) and β-Galactosidase (β-gal) serve as model enzymes where we study the effects of physiologically relevant nanoparticles on the structure and function of proteins. The objective is to examine the extent to which each nanoparticle will enhance or deter the performance of β-gal and Luc. Two dimensional fluorescence difference spectroscopy (2D FDS) determined the optimal intensity per excitation and emission wavelengths to view a change in the intrinsic fluorescence of both enzymes in the presence of nanoparticles, indicative of protein conformation. The intrinsic fluorescence spectra were obtained for both enzymes in the presence of copper (Cu) and boron carbide (B4C) nanoparticles revealing fluorescence quenching as result of interaction. Luc activity was measured by photoluminescence through the conversion D-Luciferin substrate to oxyluciferin, where Cu alters and B4C eliminates this catalytic reaction. Limited proteolytic digestion experiments were conducted on Luc where Cu increased degradation and B4C protected against it. Luc allows for rapid kinetic analysis where Cu showed delayed onset of the reaction and iron oxide was less inhibitory initially. β-gal activity was measured by absorbance as a result of cleaving the substrate ortho-nitrophenyl- β-galactoside (ONPG). The nanoparticles effect on both enzymes was compared to determine if the effect was specific or a general phenomenon.