GelRed/[G3T]5/Tb3+ hybrid: A novel label-free ratiometric fluorescent probe for H2O2 and oxidase-based visual biosensing

A novel label-free ratiometric fluorescent probe is developed for the detection of H2O2 based on GelRed/[G3T]5/Tb3+ hybrid, in which GelRed (a nucleic acid dye) intercalated into the designed single-stranded DNA [G3T]5 (i.e. GelRed/[G3T]5) acts as a stable build-in reference with red emission and Tb3+ sensitized by [G3T]5 (i.e. [G3T]5/Tb3+) as a sensitive response signal with green fluorescence. With the successive addition of Hg2+ and cysteine (Cys) to GelRed/[G3T]5/Tb3+, the fluorescence of [G3T]5/Tb3+ can be effectively quenched and recovered respectively, while the fluorescence of GelRed/[G3T]5 remains unchangeable. Combined with these properties, we have demonstrated its application for label-free ratiometric fluorescence detection of H2O2 with self-calibration. The sensing mechanism is based on the specific reaction between H2O2 and Cys, the resulting disulfide reverses the Cys-mediated fluorescence changes of [G3T]5/Tb3+. This method is further applied to the monitoring of oxidase-related reactions. As the glucose oxidase (GOx)-biocatalyzed oxidation of glucose and the acetylcholine esterase/choline oxidase (AChE/ChOx) cascade yield H2O2, such two biocatalytic processes are successfully examined utilized our proposed method, indicating its potential broad applications in biomedical analysis. In addition, we have also demonstrated its feasibility for oxidase-based visual biosensing, in which only a handheld UV lamp is used. Importantly, coupled with our proposed visual biosensing, a realtime quantitative scanometric assays has been also demonstrated by using a smartphone with easy-to-access color-scanning APP as the detection platform. Compared to traditional methods, the proposed design is cost-effective, simple to prepare and easy-to-use without fluorescence labeling or chemical modification.