Utilization of parameters developed in layer-by-layer fabrication of protein-containing films for enzyme immobilization

The layer-by-layer (LbL) self-assembly method has found a broad range of applications in biologically important materials. Developed upon experience based on polyelectrolyte systems, various types of biomolecules have been successfully incorporated into ultrathin films with thickness in the nanometer range. We employed orthogonal experimental design to analyze the factors influencing the buildup of protein-containing LbL ultrathin films, first selecting bovine serum albumin as the exemplary protein. Among the factors, we found that the protein concentration was the most influential factor, followed by protein solution pH. In comparison, the counter polyelectrolyte concentration and solution pH play smaller roles in affecting the film structure. In a preliminary attempt, we employed horseradish peroxidase (HRP) to fabricate ultrathin films and tested the enzymatic activity of these films. We found that the total enzymatic activity increased with more HRP incorporated until reaching four bilayers. After that, the total enzymatic activity became retarded, probably due to amplified diffusion resistance by the added assembly components. The immobilized HRP demonstrated diminished enzymatic activity while recycling. The suspected cause was assigned to the enzyme deactivation by relatively high H2O2 concentrations, employed for lower substrate detection limits. When a low H2O2 concentration was applied during the enzymatic activity measurements, the HRP LbL film maintained the activity level even after nine runs.