HRP@COFs-driven electrochemical biosensor for high-performance miRNA detection

An ultrasensitive biosensor has been developed for miR-375 detection. • HRP is embedded into COFs to form stable HRP@COFs co-precipitated nanospheres. • The HRP@COFs preserved enzymatic activity and enhanced catalytic efficiency. • The biosensor has been successfully validated using clinical samples. The dysregulated expression of microRNA (miRNA) in various cancers has attracted considerable attention owing to their crucial roles in tumor initiation and progression. Among them, miR-375 has been recognized as a prospective biomarker for early malignancy detection and therapeutic monitoring. In this study, a highly sensitive electrochemical biosensor has been developed based on horseradish peroxidase (HRP)-embedded covalent organic framework (HRP@COFs) co-precipitated nanospheres for the detection of miR-375. The HRP@COFs nanocomposite effectively preserves the enzymatic activity of HRP while providing the structural stability and high loading capacity of COFs, thereby enhancing catalytic efficiency and signal amplification. In the presence of target miR-375, the azide-modified H1 hairpin probes on the HRP@COFs surface are activated, leading to the formation of a rigid H1-H2 duplex structure through competitive hybridization with H2 hairpins. The resulting H2-H1-HRP@COFs complexes are subsequently anchored onto the electrode surface via click chemistry. Upon the addition of H 2 O 2 , HRP catalyzes aniline polymerization and deposition, resulting in a significant electrochemical response. This biosensor achieves ultrasensitive detection of miR-375 with a detection limit as low as 230 aM. It has been successfully validated using clinical samples, demonstrating great potential for reliable breast cancer diagnosis. This work highlights the synergistic advantages of integrating HRP with COFs, providing a robust and efficient platform for microRNA detection and expanding the applications of COFs-based materials in biosensing and clinical diagnostics. Download: Download high-res image (151KB) Download: Download full-size image