Development of Bioanalytical Assays Using Scintillant Polymer-Core Silica-Shell Nanoparticles
AdvisorAspinwall, Craig A.
MetadataShow full item record
PublisherThe University of Arizona.
RightsCopyright © is held by the author. Digital access to this material is made possible by the University Libraries, University of Arizona. Further transmission, reproduction, presentation (such as public display or performance) of protected items is prohibited except with permission of the author.
EmbargoRelease after 10/04/2019
AbstractIdeal nanosensors of biomolecules are sensitive, selective, stable, minimally invasive, amenable to mass production with low-cost, and applicable for reproducible in vitro and in vivo analyses. The nano scintillation proximity assay (nanoSPA) presented here is based on a composite architecture of polystyrene-core and silica-shell nanoparticles, with a high surface area to volume ratio (ca. 2×107 m-1) and density of approximately 1.6 g/cm3. nanoSPA obviates the need for separation of bound from free radiolabeled molecules prior to measurements, with minimized complexity and maximized versatility. Selected β-emitter radioisotopes were utilized for the development of radioassays for analysis of biological processes using nanoSPA. 35S was employed for thiol/disulfide ratio analysis for the first time. Thiolresponsive nanoSPA was used for quantification of 33S-cysteine and 33S-cystine as models of 35S-thiol and 35S-disulfide. Synthetic samples of 33S-cysteine and 33S-cystine and human embryonic kidney (HEK293) cell lysates were analyzed using thiolresponsive nanoSPA for evaluation of thiol/disulfide ratio as a measure of redox status of the sample. Limit of detection for 35S-thiol analysis was <1.1 pM (<1.1 nCi) with a signal to background ratio over 10-fold. 33P-labeled adenosine triphosphate (ATPγ33P) was utilized for the development of kinase activity assays. Three nanoSPA platforms were developed for kinase activity analysis including adsorption, binding, and immuno-nanoSPA that respond based on electrostatic non-specific adsorption, covalent binding, and antibody-antigen binding, respectively. Signal to background ratio up to 24 was observed using separation-free analyses with nanoSPA, compared to approximately 11.5 using liquid scintillation analysis after many washing steps. 3H emits the lowest energy β-particles and it was utilized with nanoSPA for development of saccharide sensors. Dynamic binding of 3H-D-glucose to nanoSPA functionalized with several monoboronic acids (monoBAs) and diboronic acids (diBAs) was evaluated. The signal to background ratio was up to 2.2-fold that must be improved. Further platforms may be developed based on phospholipid-nanoSPA with minimal nonspecific adsorption and more specificity.
Degree ProgramGraduate College