Hybrid Nanoparticle Platform for Nanoscale Scintillation Proximity Assay

β-Particle emitting radionuclides, such as ³H, ¹⁴C, ³²P, ³³P, and ³⁵S, are important molecular labels due to their small size and the prevalence of these atoms in biomolecules but are challenging to selectively detect and quantify within aqueous biological samples and systems. Here, we present a core-shell nanoparticle-based scintillation proximity assay platform (nanoSPA) for the separation-free, selective detection of radiolabeled analytes. nanoSPA is prepared by incorporating scintillant fluorophores into polystyrene core particles and encapsulating the scintillant-doped cores within functionalized silica shells. The functionalized surface enables covalent attachment of specific binding moieties such as small molecules, proteins, or DNA that can be used for analyte-specific detection. nanoSPA was demonstrated for detection of ³H-labeled analytes, the most difficult biologically relevant β-emitter to measure due to the low energy β-particle emission, using three model assays that represent covalent and noncovalent binding systems that necessitate selectivity over competing ³H-labeled species. In each model, nmol quantities of target were detected directly in aqueous solution without separation from unbound ³H-labeled analyte. The nanoSPA platform facilitated measurement of ³H-labeled analytes directly in bulk aqueous samples without surfactants or other agents used to aid particle dispersal. Selectivity for bound ³H-analytes over unbound ³H analytes was enhanced up to 30-fold when the labeled species was covalently bound to nanoSPA, and 4- and 8-fold for two noncovalent binding assays using nanoSPA. The small size and enhanced selectivity of nanoSPA should enable new applications compared to the commonly used microSPA platform, including the potential for separation-free, analyte-specific cellular or intracellular detection.