Ultrasensitive multiplexed detection of small molecules and enzymes using stimuli-responsive nucleic acids

Small biomolecules and enzymes play essential roles in regulating various physiological functions and are highly correlated with many diseases, making them promising biomarkers for disease diagnosis. However, it remains challenging to achieve multiplexed detection of trace enzymes and small molecules due to the limited signal transformation and amplification capabilities in existing technologies. Here, we developed a signal transformation and amplification strategy based on stimuli-responsive nucleic acids (SR-NAs) by incorporating stimuli-responsive linker in single-stranded nucleic acid via click chemistry. The targets (e.g., enzymes, small molecules) could recognize the responsive linker and trigger the degradation of the SR-NAs, thus transforming the targets into DNA signals by generating new toehold nucleic acid strands, which were further augmented by nucleic acid amplification. We demonstrated that this strategy significantly improved the detection precision for small biomolecules (H₂O₂) and enzymes (esterase, β-galactosidase). Furthermore, the signal transformation function of SR-NA enables multiplexed detection via logical operation with multiple small molecules and enzymes as inputs, strand displacement reaction as Boolean logic-based algorithm, and fluorescence signal as output signal, as demonstrated by multiplexed detection of esterase and H₂O₂. More importantly, this method was capable of realizing ultrasensitive intracellular H₂O₂ detection. We envision that this SR-NA-based strategy could be expanded to detect other kinds of targets by designing self-immolative linkers, and thus provides a powerful toolbox for bioanalytical and biomedical applications.