Balancing sensitivity, speed, and accuracy in miRNA detection with a localized branched catalytic hairpin assembly strategy

Abstract

Developing accurate, sensitive, and rapid methods for microRNA analysis in living cells is crucial for cancer diagnosis. Recent advances in enzyme-free amplification and spatial-confinement techniques have enhanced both the sensitivity and speed of miRNA detection. However, they also introduce a challenging contradiction: while localized amplification increases the sensitivity of miRNA detection, the consequent elevated risk of probe crosstalk can lead to false-positive signals and compromised accuracy. To strike a balance between the sensitivity, speed, and accuracy, we herein propose a localized branched catalytic hairpin assembly (LBCHA) strategy. This method employs three independent nanoprobes, each equipped with a specific branched catalytic hairpin assembly (bCHA) hairpin. The presence of the target initiates localized bCHA among the LRN probes, generating amplified fluorescent signals. Importantly, by anchoring each bCHA hairpin on separate nanospheres, we enhance detection sensitivity and speed while preserving accuracy by minimizing non-specific interactions. The LBCHA system demonstrated exceptional miRNA sensing performance in vitro, reaching a detection limit of 3.66 pM. Further applications in live cells and clinically derived tissues confirmed its potential for accurate cancer discrimination. This innovative LBCHA strategy holds great potential for clinical applications and real-time cancer diagnostics.