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.

摘要

开发了一种简单而灵敏的方法来测定巯基 (-SH) 和二硫化物 (S-S) 形式的生物硫醇，该方法使用由水溶性荧光团罗丹明的静电吸附制备的柠檬酸盐稳定的金纳米粒子 (AuNPs) 组成的荧光传感器– 6G (Rh–6G) 染料在柠檬酸盐稳定的 AuNPs 上导致荧光共振能量在关闭模式下转移。由于生物硫醇对 AuNPs 的高表面亲和力，伴随 Rh-6G 释放的 552 nm（开启模式）荧光增量表明硫醇含量。该测定法还能够区分氧化和还原硫醇，并使用硫醇烷基化剂 N-乙基马来酰亚胺 (NEM) 测量硫醇/二硫化物稳态作为重要的氧化应激指标。经 NEM 预处理后，在存在还原硫醇的情况下测量二硫化物。使用所提出的测定法测量了总体生物硫醇水平和硫醇/二硫化物比率。该方法被证明对已知会对基于氧化还原的测定产生不利影响的各种潜在干扰化合物（例如，多酚和血浆抗氧化剂）具有选择性。所提出的测定方法还能够通过 NP 凝聚对生物硫醇进行肉眼检测，并可成功应用于含有硫醇的药物样品。