In this study, we report a novel electrochemiluminescence (ECL) biosensor for the ultrasensitive detection of matrix metalloproteinase-2 (MMP-2), an important biomarker associated with tumor invasion and metastasis. The biosensor integrates hairpin-mediated exponential amplification with CRISPR/Cas12a-based trans-cleavage for dual-stage signal amplification. In this design, MMP-2 specifically cleaves a peptide sequence (GPLG↓VRGK) on the DNA hairpin probe (HP1), releasing an initiator peptide nucleic acid (PNA) that triggers hairpin-mediated exponential amplification reaction. The amplified DNA products then activate the Cas12a/gRNA complex, which induces collateral cleavage of ferrocene (Fc)-labeled probes immobilized on a DNA tetrahedron-modified PEI-Ti₃C₂Tx/Ru/AuNPs electrode, thereby generating a strong ECL response. The incorporation of the DNA tetrahedron nanostructure provides a well-defined three-dimensional framework that ensures ordered probe orientation, enhanced hybridization efficiency, and reduced steric hindrance on the electrode surface. This structural organization significantly improves electron transfer and signal stability compared with conventional planar immobilization. Under optimized conditions, the biosensor exhibited a broad linear range from 0.01 fM to 10 nM and an ultralow detection limit of 10 aM. It displayed high specificity against interfering proteins (thrombin, IgG, BSA, lysozyme), excellent stability, and satisfactory recoveries (96.9%-105.0%) in LO2 cell culture supernatants. Overall, this enzyme-responsive, DNA-tetrahedron-assisted, CRISPR-amplified ECL biosensor represents a robust and versatile platform for precise and rapid detection of protease activity, showing great promise for biomedical diagnostics and clinical biomarker monitoring.
