ratiometric detection of nucleic acid using hybridization chain reaction and color-switching DNA-templated silver nanoclusters

Enzyme-free nucleic acid amplification techniques, such as hybridization chain reaction (HCR), are often coupled with various nanomaterials to fabricate biosensors for nucleic acid amplification and detection, thereby reducing reliance on sophisticated instruments and complicated protocols. A typical HCR system consists of a pair of DNA hairpins (H1 and H2), which unfold and self-assemble into DNA concatemers in the presence of a target nucleic acid. While the versatility of HCR is demonstrated by a variety of available designs, the design process can be tedious, requiring multiple rounds of iteration and optimization to achieve the desired performance. Developing ‘universal’ hairpin designs can significantly streamline this process, reducing the need to redesign HCR hairpin probes for each target. In this work, we developed an enzyme-free and label-free HCR-silver nanoclusters (AgNCs) biosensor using a capture probe (CP-S15) and ‘universal’ hairpins (H1-S13 and H2-S13) for DNA target detection. CP-S15 was designed as a hairpin with a toehold region for DNA target recognition and a loop region to initiate HCR between H1 and H2, which host color-switching fluorescent AgNCs. Notably, H1 and H2 function as ‘universal’ hairpins, as they do not share sequences with or complement the target, making them adaptable for other targets when the CP-S15 loop region remains unchanged. After isothermal HCR incubation with the target and a simple one-pot synthesis of AgNCs at room temperature, the biosensor generated a fluorescence signal, perceived as a color change from red to yellowish orange. The detection range of the biosensor was 50–400 nM, with a detection limit of 41 nM.