How different immobilization techniques affect enzyme performance: a biosensor case study.

The immobilization of enzymes onto support materials is a widely adopted strategy in biosensor development, yet its impact on enzyme activity, stability, and overall performance remains system-dependent. Comparative studies evaluating physical adsorption and covalent binding under identical conditions are scarce. In this work, we systematically investigate the immobilization of glucose oxidase (GOx) and horseradish peroxidase (HRP) onto a hydrophilic polyurethane matrix for the fabrication of a conductometric glucose biosensor. Both immobilization techniques were characterized using thermogravimetric analysis, FTIR spectroscopy, XPS, and confocal microscopy to confirm enzyme attachment and assess enzyme orientation. Biosensor performance was evaluated in terms of sensitivity, selectivity, and stability. The results demonstrate that the spacer length used for enzyme immobilisation plays a critical role in determining sensor performance. Specifically, the shortest linker exhibited superior sensing performance, achieving a linear glucose detection range of 0.01 mM-0.5 mM and a limit of detection of 9 µM. The sensor also proved to exhibit high sensitivity in artificial saliva, enabling the detection of glucose concentrations as low as 10 µM. Furthermore, chemically immobilized enzymes exhibited superior stability, retaining their activity for 21 days while enhancing selectivity. These findings underscore the significance of the immobilization strategy and linker design in optimizing biosensor performance and provide valuable insights for the rational design of enzyme-based sensing platforms.