We have investigated biological functionality of immobilized enzyme structures according to the immobilizing routes and the surface properties. Horse radish peroxidase (HRP) was immobilized on various solid surfaces such as gold, SiO(2), sapphire and anodized aluminum oxide (AAO) membrane via non-specific adsorption, avidin-mediated and biotin/avidin-mediated layer-by-layer (LBL) assembly. The catalytic activity as a measure of biological functionality, of the biotin-HRP immobilized by avidin-mediated LBL assembly was found to be better than that of the directly adsorbed HRP on the surfaces of gold, SiO(2), sapphire and AAO due to the easy accessibility of reactants to active sites as well as the retention of three dimensional native structure of enzyme for bioactive functionality. In addition, the catalytic activity of the biotin-HRP in LBL-assembled avidin/biotin-HRP on AAO membrane was found to be highly better than that on other substrates due to the increasing amount of immobilized HRP which can be attributed to the high surface area of the substrate. SEM images show that the functional avidin/biotin-HRP enzyme structures were Successfully realized by a sequential process of non-specific adsorption and LBL assembly via biotin-avidin interaction. (C) 2009 Elsevier B.V. All rights reserved.