Electrochemical detection of MMP-2 with enhanced sensitivity: Utilizing T7 RNA polymerase and CRISPR-Cas12a for neuronal studies

Abstract

This study introduces a novel electrochemical biosensor for detecting Matrix Metalloproteinase-2 (MMP-2), a key biomarker in cancer diagnostics and tissue remodeling. The biosensor is based on a dual-amplification strategy utilizing T7 RNA polymerase isothermal amplification and CRISPR-Cas12a technology. The principle involves the release of a DNA template in the presence of MMP-2, leading to RNA synthesis by T7 RNA polymerase. This RNA activates CRISPR-Cas12a, which cleaves a DNA probe on the electrode surface, resulting in a measurable electrochemical signal.The biosensor demonstrated exceptional sensitivity, with a detection limit of 2.62 fM for MMP-2. This high sensitivity was achieved through the combination of transcriptional amplification and the collateral cleavage activity of CRISPR-Cas12a, which amplifies the signal. The sensor was able to detect MMP-2 across a wide dynamic range from 2 fM to 1 nM, showing a strong linear correlation between MMP-2 concentration and the electrochemical signal. In practical applications, the biosensor accurately detected elevated levels of MMP-2 in cell culture supernatants from HepG2 liver cancer cells, distinguishing them from normal LO2 liver cells. The use of an MMP-2 inhibitor confirmed the specificity of the detection. These results underscore the biosensor's potential for clinical diagnostics, particularly in early cancer detection and monitoring of tissue remodeling activities. The biosensor's design allows for rapid, point-of-care testing without the need for complex laboratory equipment, making it a promising tool for personalized healthcare and diagnostic applications.

Keywords: CRISPR-Cas12a; Electrochemical biosensor; MMP-2; T7 RNA.