Cholesterol-modified DNA nanostructures enable a cascaded CHA-HCR system for enhanced miRNA detection in liquid biopsies.

Accurate detection of tumor-associated microRNAs (miRNAs) is crucial for early cancer diagnosis and prognosis. However, detecting low-abundance miRNAs with high sensitivity and specificity in complex biological fluids remains a significant challenge. Here, we report a novel detection system that integrates catalytic hairpin assembly (CHA) and hybridization chain reaction (HCR) for the sensitive detection of miR-21, a well-established oncomiR. Our strategy employs cholesterol-modified DNA (Chol-DNA) to self-assemble into micellar nanostructures, which stabilize the DNA probes and create a spatially confined environment to boost reaction efficiency. In the presence of miR-21, a CHA reaction is initiated, triggering a downstream HCR cascade that generates long DNA polymers. This dual-amplification mechanism produces a robust fluorescence signal via Förster resonance energy transfer (FRET) between Cy3 and Cy5 fluorophores. The system demonstrates exceptional sensitivity with a limit of detection (LOD) of 10 pM and exhibits high specificity, effectively discriminating miR-21 from single-base mismatched sequences and other interfering miRNAs. Operating isothermally and without enzymes, the assay maintains its performance in complex biological media, including human serum, making it ideal for liquid biopsy applications. The Chol-DNA modification not only prevents probe degradation but also accelerates signal amplification through the confinement effect. This CHA-HCR system represents a simple, portable, and reliable tool for miRNA analysis, showing immense potential for non-invasive cancer diagnostics in clinical environments.