Host-guest confinement in post-metallized COFs for enhanced electrochemiluminescence coupled with Y-shaped DNA amplification for microRNA-499 detection

An ultrasensitive electrochemiluminescence (ECL) biosensor was developed for the detection of the acute myocardial infarction (AMI) biomarker miRNA-499 by synergistically integrating an iridium-metallized covalent organic framework (Ir@TpBpy) with enzyme-assisted Y-shaped DNA amplification. The Ir@TpBpy COF was synthesized via post-metallization of a bipyridine-rich TpBpy framework with Ir(III) complexes. This design significantly enhances the ECL performance not only through material loading but also via a synergistic optimization strategy: (1) the conjugated framework constructs continuous charge transport pathways to accelerate electron transfer; and (2) the porous architecture induces a guest confinement effect that locally enriches the coreactant triethylamine (TEA), facilitating radical kinetics. Concurrently, a Y-shaped DNA amplification strategy, driven by Klenow fragment and Nt. Bbv Cl enzymes, facilitated exponential signal enhancement via cascade strand displacement reactions. The generated output strands triggered the displacement of pre-immobilized WO 3 /CuO quenchers from the electrode surface, thereby restoring the luminescence of Ir@TpBpy and achieving a sensitive “signal-on” readout. Benefiting from this dual-amplification approach, the biosensor achieved an exceptionally low limit of detection (LOD) of 0.81 aM across a linear range from 1 aM to 1 nM in human serum and cellular lysates. Clinical validation using Bland-Altman analysis demonstrated 95% agreement with qRT-PCR results, confirming that this synergy of coordination modulation and guest confinement offers a robust and precise platform for early AMI diagnosis.