ENHANCING T7 DNA POLYMERASE IMMOBILIZATION FOR REUSABLE BIOSENSORS

The immobilization of DNA-modifying enzymes onto solid surfaces is a complex yet essential task in biotechnology, especially for reusable biosensors for DNA analysis [1]. One of the unique challenges in immobilizing these enzymes is their structural sensitivity and the risk of denaturation, distinguishing them from more robust industrial enzymes that can function effectively while attached to solid supports. This study focuses on enhancing the reusability of T7 DNA polymerase by optimizing its immobilization process for sustainable biotechnological applications. DNA-modifying enzymes often require precise conditions, such as specific ion concentrations (e.g., Mg²⁺ for polymerases) and stable temperatures [2]. To monitor the real-time immobilization of T7 DNA polymerase and evaluate the analytical signals produced during enzymatic activity, we employed quartz crystal microbalance (QCM) technology. QCM does not require additional labeling, which reduces potential interference and helps maintain enzyme activity. It can detect mass changes at the nanogram level, making it ideal for monitoring thin molecular layers and facilitating targeted DNA detection. We explored covalent attachment strategies using self-assembled monolayers of different thiol compositions on a gold-based QCM sensor (QSense-disk) to enhance enzyme stability and functionality. Additionally, we implemented an active-site protection strategy [2,3] for exonucleases to reduce the risk of inactivation. Our findings demonstrate that effective immobilization allows DNA detection while preserving enzyme functionality, paving the way for cost-effective, reusable biosensors. These results underscore the potential of immobilized DNA-modifying enzymes in real-time, label-free biosensing technologies, offering significant advantages [4,5] for continuous DNA analysis and sustainable biotechnological applications.