Enhanced-electrochemiluminescence biosensor for detecting miRNA-21 based on a CuO-mediated click reaction and catalytic hairpin self-assembly

MicroRNAs (miRNAs) are closely associated with cancer and have been considered cancer biomarkers. Herein, we propose an electrochemiluminescence (ECL) biosensor for detecting miRNA-21 based on target-induced catalytic hairpin self-assembly (CHA) and CuO-mediated azide-alkyne cycloaddition. Two hairpin DNAs were employed: one was immobilized on magnetic beads (HP2) and another was labeled with CuO (HP1-CuO). HP1 and HP2 formed a duplex through CHA induced by miRNA-21, resulting in the immobilization of CuO on magnetic beads and in the recycling of miRNA-21. After magnetic separation, CuO was treated with hydrochloric acid to release Cu²⁺, which concentration is quantitatively proportional to the target concentration. Subsequently, Cu²⁺ was reduced to Cu⁺, which catalyzed the click reaction between Fc-C CH and SH-DNA-N₃⁺ immobilized on a Au/g-C₃N₄ modified electrode. Thus, the ECL of Au/g-C₃N₄ was quenched by Fc, and miRNA-21 was indirectly detected through a change in ECL intensity. Benefiting from the amplification effect of CuO nanoparticle loading, CHA-based target recycling, and the catalytic effect of click reaction, the proposed ECL biosensor showed high sensitivity. Experimental results indicate that the ECL biosensor proposed for detecting miRNA-21 exhibits a wide linear range from 1 fM to 1 nM and a low detection limit of 0.26 fM (3σ/S). Furthermore, the ECL sensor was capable of measuring miRNA-21 in real serum with high selectivity, indicating its notable applicable potential in biomedicine and clinical diagnosis.