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

Abstract

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.