Hybrid Nanoparticles for Enhanced Sensitivity in Biological Labeling and Biomolecular Sensing

Abstract

Nanoparticles (nPs) demonstrate significant advantages over other sensor and marker technologies. The most useful optical nanosensor and label platform for biological samples would be non-toxic, hydrophilic, resistant to non-specific protein interactions and degradation over time or under harsh conditions, highly retentive of entrapped components, and easily functionalized for target specificity. The work described here is part of an investigation into the fabrication and application of polyacrylamide, polyacrylamide/silica hybrid, and polystyrene-core silica-shell nPs. Polyacrylamide (PA) nP nitric oxide (NO) sensors were made by co-entrapping 4, 5-diaminofluorescein (DAF-2) and Texas Red dextran in 60 nm PAnPs. Sensors were used to measure NO produced by a diazeniumdiolate NO donor in solution, and have a response time of 30 seconds or less. Entrapped DAF-2 was protected from non-specific interactions with bovine serum albumin (BSA). Sensor response to NO in FBS solutions was reduced compared to buffer, although improvement over free dyes was observed. The sensors were applied to J477A.1 macrophages as well as a HT1080 cell line (HTRiNOS) in preliminary studies for measuring intracellular NO production. Polyacrylamide/silica hybrid nPs were fabricated and nP architecture was evaluated by transmission electron microscopy. Isopycnic centrifugation of nP samples indicates that the hybrid nPs have a density between 1.70 and 1.76 g/cm³. Silica in the hybrid nPs was covalently labeled with Texas Red, suggesting that the hybrid nPs may be used as ratiometric or possibly multiplexed sensors. Hybrid nPs coated with 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) exhibit reduced adsorption of TRITC-BSA compared to uncoated hybrid nPs. Hybrid nP pH sensors were prepared and responded reproducibly and reversibly to changes in pH, nominally from pH 6.0 to 8.0. Core-shell nPs for scintillation proximity assay (SPA) were fabricated by entrapping the scintillants p-terphenyl and 4-bis(4-methyl-5-phenyl-2oxyzolyl)benzene in polystyrene, onto which silica shells were subsequently added. Core-shell nPs were found to have a scintillation response similar to that of shell-less polystyrene cores, indicating that the presence of the silica shells does not reduce scintillation efficiency. Preliminary studies using core-shell nPS for biotin-streptavidin binding SPA do not indicate an enhancement in scintillation efficiency, although this may be due to high nP:radiolabeled analyte ratios.