Core‐Shell Nanoparticle Probe Scintillation Proximity Assays for Biological Samples

Abstract

To achieve the overall goal of sensitive and selective radioisotope detection in aqueous samples, we have developed a novel polystyrene‐core silica‐shell nanoparticle scintillator platform (nanoSPA). nanoSPA enabled separation‐free quantification of radiolabeled analytes in complex samples as demonstrated by scintillation proximity assays via protein‐tag, biotin, and antibody binding as well as by other methods such as chemical crosslinking and adsorption.β‐particle emitting radionuclides such as 3H, 33P, and 35S are useful molecular labels due to their small size but are challenging to detect and quantify with spatial and temporal resolution in biological samples due to their low energies (Emax ≤ 300 keV) and short penetration depths (≤ 0.6 mm). Activity measurements for these β‐emitters are usually made in milliliter volumes of liquid scintillation cocktail (LSC), a mixture of energy‐absorbing organic solvents, surfactants, and scintillant fluorophores, which is incompatible with living cells and dynamic biological measurements. Alternatively, solid polymer or inorganic crystals can be used, but the orientation of polymer sheets, the large size of polymer particles (> 2μm) and the high density of inorganic scintillators can result in inaccurate counting. nanoSPA avoids the toxicity of LSC as well as many of the limitations of polymer and inorganic crystal scintillators. The polystyrene acts as an absorber for energy from emitted β‐particles and is loaded with scintillant fluorophores to which the energy is transferred, leading to photon emission at visible wavelengths. The silica shell serves as a hydrophilic shield for the polystyrene core and facilitates functionalization with specific binding molecules for scintillation proximity assays. In this way, the short penetration depths of low‐energy β‐particles are used advantageously, as radiolabeled analytes that are not close enough to the nanoparticle surface (primarily bound to the surface during an assay) are less likely to be detected, resulting in low background signal. Selectivity was determined to be up to 30 times greater for bound 3H‐analytes over unbound 3H analytes when the labeled species was covalently bound to nanoSPA, 18 times greater for antibody functionalized nanoSPA, 8 times greater for biotin functionalized nanoSPA, and 4 times greater for ssDNA functionalized nanoSPA. We have also measured and monitored peptide phosphorylation with kinase and 33P ɣ ‐ ATP. nanoSPA not only facilitates measurement of radiolabeled analytes in bulk aqueous solutions, but due to the small diameter and the protection of the hydrophobic polymer core by the silica shell, nanoSPA particles could potentially be used as cellular or intracellular imaging probes.Support or Funding InformationThis work was supported in part by the National Science Foundation under grant number 1807343, the National Institutes of Health via the National Institute of Biomedical Imaging and Bioengineering under grant number R21EB019133 and the National Institute of General Medical Sciences under grant number 1R01GM116946.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.