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.BbvCl enzymes, facilitated exponential signal enhancement via cascade strand displacement reactions. The generated output strands triggered the displacement of pre-immobilized WO3/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.