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.