Highly sensitive electrochemical biosensor for MMP-2 detection using multi-stage amplification with PNA T7 RNA polymerase and split CRISPR/Cas12a

[Display omitted] •Novel electrochemical biosensor detects MMP-2 with high sensitivity and specificity.•Combines PNA, T7 RNA polymerase, and split CRISPR/Cas12a for multi-stage amplification.•Detection limit of 0.496 fM ensures ultra-sensitive MMP-2 quantification.•Successfully detects MMP-2 in biological samples, including liver cancer supernatants. Matrix metalloproteinases (MMPs) are critical for the degradation of extracellular matrix components, which is essential for processes such as tissue remodeling, wound healing, and angiogenesis. MMP-2, in particular, degrades type IV collagen and is associated with diseases such as cancer and cardiovascular conditions, making it a significant biomarker. This study presents a novel electrochemical biosensor for the detection of MMP-2. The biosensor combines the specificity of a programmable RNA detection system with the amplification capabilities of T7 RNA polymerase and CRISPR/Cas12a. In the absence of MMP-2, Peptide Nucleic Acid (PNA) adsorbs on the DNA template, preventing RNA amplification and resulting in a low electrochemical signal. When MMP-2 is present, it cleaves a specific peptide, releasing the DNA template and enabling RNA synthesis. This activation leads to the cleavage of single-stranded DNA probes, increasing the electrochemical signal. The biosensor demonstrates high sensitivity with a detection limit of 0.496 fM for MMP-2 and excellent specificity, effectively distinguishing MMP-2 from other proteins. It successfully detected MMP-2 in biological samples, such as liver cancer cell supernatants, indicating its potential for clinical diagnostics. This innovative biosensor offers a powerful tool for the early detection and monitoring of diseases associated with MMP-2 activity.