Dual-detection of miRNAs in living cells via hybridization chain reaction on DNA tetrahedron

Monitoring intracellular miRNA levels is essential for early cancer diagnosis. However, it remains challenging to analyze miRNAs at the cellular level with high accuracy, high sensitivity, and fast kinetics due to the small size and low abundance of miRNAs. Herein, we employed 3D hybridization chain reaction (HCR) on a binary DNA tetrahedron (DTN) system for multiplexed detection of miR-21 and miR-203. This system comprised two nanoprobes, DH-13 and DH-24, each containing two pairs of HCR hairpins labeled with fluorophores and corresponding quenchers. In the absence of targets, hairpins remained folded and exhibited weak fluorescence. However, the presence of targets could activate 3D-HCR between DTN nanoprobes, separating fluorophores from quenchers and restoring fluorescence. By recording FAM and Cy3 signals, the quantification of miR-21 and miR-203 could be realized in buffer solutions, FBS solutions, and living cells. The linear responses for both targets ranging from 0.1 to 1.0 nM, and the detection limits were 1.4 pM and 2.0 pM for miR-21 and miR-203, respectively. The proposed strategy combined the advantages of multiplexed detection and 3D-HCR to achieve accurate, sensitive, and rapid detection of intracellular miRNAs, which is anticipated to provide a potent diagnostic tool for cancer.