Development of Bioanalytical Assays Using Scintillant Polymer-Core Silica-Shell Nanoparticles

Abstract

Ideal 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.