Dual amplification DNA reactions for ultrasensitive electrochemical biomolecular detection using impedimetric biosensor.

Detecting biomolecules, particularly disease markers, is an important issue for practical diagnosis and clinical therapies. Herein, we proposed an all-DNA hydrogel-based electrochemical impedance sensing platform for ultrasensitive detection of miRNA-21 by synergistic integration of dual amplification circuits: catalytic hairpin assembly (CHA) and clamped hybridization chain reaction (C-HCR). A hairpin DNA complementary to miRNA-21 was designed to initiate CHA, resulting in the self-assembly of dsDNA products upon target addition. This process generates numerous trigger sequences that activate the subsequent C-HCR to form DNA hydrogels on the electrode surface. The insulating DNA hydrogel film significantly enhances impedance responses, allowing us to establish a highly sensitive impedimetric biosensor for miRNA-21 analysis. Our dual signal amplification strategy using DNA hydrogels as the amplification element showed higher electrochemical response, broader dynamic range (10 aM ∼ 10 nM), and lower detection limit (6.5 aM). In addition, this electrochemical impedimetric biosensor with excellent selectivity, non-destructivity, and label-free feature is suitable for practical analytes, highlighting its great potential in clinical diagnostics.