A Mn2+-gated conformational switch-activated fluorescent biosensor for Mucin-1 detection based on a 3D porous Fe3O4@ZIF-90 interface and DNAzyme walker-mediated CRISPR-Cas13a cascade

3D porous Fe 3 O 4 @ZIF-90 interface provides high-density DNA loading sites. • Mn 2+ -gated DNAzyme walker ensures a low-background controllable assay. • DNAzyme walker and CRISPR-Cas13a cascade enable dual signal amplification. • The biosensor demonstrated high accuracy and specificity in human serum samples. The precise quantification of Mucin-1 (MUC1) is vital for the early diagnosis and therapeutic monitoring of epithelial malignancies. In this study, a fluorescent biosensing platform based on a 3D porous metal-organic framework (MOF) and a cascaded signal amplification strategy was developed for MUC1 detection, driven by a target-induced conformational switch and Mn 2+ -dependent catalytic DNAzyme walking. Porous Fe 3 O 4 @ZIF-90 nanocomposites were used to construct the sensing interface, which co-immobilized dormant DNAzyme/aptamer duplexes and a high-density layer of substrate DNA (sub-DNA), serving as a robust platform for the subsequent walking process. Upon specific MUC1 recognition, the aptamer preferentially captures the target MUC1. The thermodynamic driving force induces the dissociation of the MUC1/aptamer complex, liberating the DNAzyme engine. Following magnetic separation to eliminate matrix interferences, the addition of Mn 2+ triggers the processive inter-molecular migration of the DNAzyme walker across the sub-DNA immobilized 3D porous Fe 3 O 4 @ZIF-90 interface via successive hybridization and site-specific cleavage of internal riboadenosine (rA) junctions. This inter-molecular walking process generates a high flux of trigger strands, which subsequently activates the CRISPR-Cas13a system for a robust trans -cleavage of dual-labeled fluorophore-quencher (FQ) reporters. The resulting degradation of FQ reporter enables sensitive MUC1 quantification through exponential fluorescence recovery. By synergizing the structure advantages of the 3D MOF with this cascaded signal amplification, the biosensor achieved a wide linear range from 0.37 nM to 500 nM with a low detection limit of 17.2 pM. Furthermore, the biosensor exhibited reliable specificity and recovery (102.28%–106.94%) in complex human serum assays. The proposed strategy effectively minimizes non-specific background leakage, demonstrating promising potential for the ultra-sensitive detection of MUC1 in complex biological matrices. Schematic presentation of the fluorescent biosensor for the determination of Mucin-1 based on a 3D porous Fe 3 O 4 @ZIF-90 interface and DNAzyme walker-mediated CRISPR-Cas13a cascade. Download: Download high-res image (117KB) Download: Download full-size image