Small thiols such as cysteine play key roles in biological function. Abnormal concentrations of cysteine are observed in patients with diseases such as Alzheimer’s, Parkinson’s, cystinuria, and cystinosis. Sensors with high cell-permeability and low interference are needed for real-time measurement of intracellular thiols and disulfides since the ratio provides insight into the redox network inside cells. Radioisotopes are used in bioanalytical measurements as they provide the lowest perturbation on analyte properties, such as binding kinetics. 35S isotope has higher decay energy than 3H and the resulting beta particles travel a longer distance (ca. 300 μm in water), making 35S a better tracer for sensitive detection of sulfur-containing analytes. In this research, polystyrene-silica core-shell nanoparticles doped with reporter fluorophores were fabricated for scintillation proximity assay (SPA). SPA works based on the conversion of energy released from bound radiolabeled analyte to detectable visible light. Thiol-functionalized nanoparticles were successfully utilized to quantify cysteine at sub-nM concentration. The signal enhancement by specific binding of 35S-cysteine to these nanoparticles was one order of magnitude in a separation free assay. The extent of binding depends on the ratio of cysteine to cystine which is a function of pH, oxidation state of the compounds, and the presence of thiol-reactive agents. We applied nanoSPA sensors to measure thiols and disulfides, by protecting the thiols and reducing disulfides to more thiols. We also indirectly measured thiol-reactive agents such as HNO, which shows promising pharmaceutical properties.

Development and characterization of thiol-responsive scintillation proximity assay core-shell nanoparticles as turn-on biosensors

253rd National Meeting (2017)

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