Visual fluorescence detection of H2O2 and glucose based on “molecular beacon”-hosted Hoechst dyes

Abstract

In this work, a label-free molecular beacon (MB)-like biosensor is designed for the determination of H₂O₂ and glucose based on the fluorescence regulation of Hoechst dyes hosted by the designed AT-rich single-stranded DNA (ssDNA), in which Hg²⁺ and cysteine (Cys) act as activators. The designed AT-rich ssDNA (ATprobe) can be directed to form a hairpin with an Hg²⁺-induced T–Hg²⁺–T complex, which provides a medium for enhancing the fluorescence of Hoechst dyes significantly. On the other hand, Cys can effectively grab Hg²⁺ from the T–Hg²⁺–T complex by thiol–Hg²⁺ interactions, destructing the hairpin and then switching the Hoechst dyes to the fluorescence “off” state. Combined with these properties, we have demonstrated its application for label-free fluorescence “turn on” detection of H₂O₂. The sensing mechanism is based on the specific reaction between H₂O₂ and Cys catalyzed by I⁻, the resulting disulfide reverses the Cys-mediated fluorescence decrease of the MB-hosted Hoechst dyes. The approach achieves a low detection limit of 0.1 μM for H₂O₂. Moreover, this method is further applied to the noninvasive detection of glucose in artificial saliva and urine samples, combining with glucose oxidase (GOx) for the oxidation of glucose and formation of H₂O₂. Compared to traditional methods, the proposed design is cost-effective, simple to prepare and manipulate without fluorescence labeling or chemical modification.