Fluorescence turn-on assay for glutathione reductase activity based on a conjugated polyelectrolyte with multiple carboxylategroups

Abstract

In this work, an anionic conjugated polyelectrolyte (CPE) that features four carboxylate groups on each repeat unit, PPE-(COOK)₄, has been designed and synthesized by integrating the two synthetic strategies for conjugated poly(phenylene ethynylene) and dendritic polyamidoamine. Through the installation of multiple functional groups, the proposed CPE has been greatly improved in many aspects, such as the good water solubility, slight aggregation, and relatively high quantum yield. Thanks to the close resemblance in chemical structures between the polymer's pendant groups and EDTA, the resulting PPE-(COOK)₄ has also been found to show stronger complexation with metal ions, and has further been demonstrated to be an excellent fluorescent probe for Cu²⁺ with both high sensitivity and remarkable specificity. Moreover, by taking advantage of the distinct fluorescence recovery rates of PPE-(COOK)₄/Cu²⁺ caused by GSH and by GSSG, a novel fluorescence turn-on assay for glutathione reductase (GR) has successfully been developed. With a minimum detectable enzyme concentration of 0.2 mU/mL, this newly proposed GR activity assay is highly sensitive and robust as compared to most spectrophotometric and fluorescent methods.