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A simple and sensitive method was developed to determine biothiols in both sulfhydryl (–SH) and disulfide (S–S) forms using a fluorometric sensor composed of citrate–stabilized gold nanoparticles (AuNPs) prepared by electrostatic adsorption of water-soluble fluorophore Rhodamine–6G (Rh–6G) dye onto citrate–stabilized AuNPs resulting in the fluorescence resonance energy transfer in the turn-off mode. Due to the high surface affinities of biothiols to AuNPs, the fluorescence increment at 552 nm (turn-on mode) accompanying the release of Rh–6G indicated the thiol content. This assay also enables the differentiation of oxidized and reduced thiols and the measurement of thiol/disulfide homeostasis as a vital oxidative stress indicator using thiol-alkylating agent N-ethylmaleimide (NEM). After pretreatment with NEM, disulfides were measured in the presence of reduced thiols. The overall biothiol levels and thiol/disulfide ratios were measured using the proposed assay. This method was shown to be selective over various potential interfering compounds (e.g., polyphenols and plasma antioxidants) known to adversely affect redox-based assays. The proposed assay also enabled the naked-eye detection of biothiols by NP agglomeration and may be successfully applied to pharmaceutical samples containing thiols.

analytical letters

Gold–Nanoparticle Based Turn–on Fluorometric Sensor for Quantification of Sulfhydryl and Disulfide Forms of Biothiols: Measurement of Thiol/Disulfide Homeostasis