Molecular Beacon-Based Fluorescent Assay for Selective Detection of Glutathione and Cysteine

We report on the development of a fluorescence turn-on molecular beacon probe for the detection of glutathione (GSH) and cysteine (Cys). The method is based on a competitive ligation of Hg2+ ions by GSH/Cys and thymine-thymine (T-T) mismatches in a DNA strand of the self-hybridizing beacon strand. The assay relies on the distance-dependent optical properties of the fluorophore/quencher pair attached to the ends of the molecular beacon DNA strand. In a very selective coordination of Hg2+ to GSH/Cys, the fluor phore/quencher distance increases concomitantly with the dehybridization and dissociation of the beacon stem T-Hg2+-T due to the extraction of Hg2+ ions. This process results in switching the molecular beacon to the on state. The concentration range of the probe is 4-200 nM with the limit of detection (LOD) of 4.1 nM for GSH and 4.2 nM Cys. The probe tested satisfactorily against interference for a range of amino acids including sulfur-containing methionine.