Department of Biochemistry, Microbiology and Biotechnology, Rhodes University, P.O. Box 94, Grahamstown, South Africa
Fogel, R., Department of Biochemistry, Microbiology and Biotechnology, Rhodes University, P.O. Box 94, Grahamstown, South Africa; Limson, J.L., Department of Biochemistry, Microbiology and Biotechnology, Rhodes University, P.O. Box 94, Grahamstown, South Africa
Enzyme immobilization is an ever-growing research-area for both analytical and industrial applications. Of critical importance in this area are the effects of immobilization procedures upon the functionality of the immobilized biomolecules. Both beneficial and detrimental effects can be conferred through the selection and tuning of the immobilization procedure. Quartz-crystal microbalance with dissipation (QCM-D) has been previously used to great effect in tracking alterations to thin films of biomolecules immobilized onto quartz transducers. In this study, we investigate the ability of QCM-D to track and monitor film parameters of a monolayer of laccase immobilized on a series of self-assembled monolayers (SAMs), differing in lateral density of binding residues on the SAM and height of the SAM from the quartz surface. Both mass gains and rheological parameters for these varying surfaces were measured and trends later compared to the apparent enzyme kinetics of the immobilized laccase films, assessed electroanalytically (Paper II in this two part study). For covalent attachment of proteins, both shear and viscosity were increased relative to physically adsorbed proteins. An increase in lateral density of protein-binding surface of the SAM components was shown to increase the shear/viscosity of the resultant film while an increase in distance from the electrode (through incorporation of lysine linkers) was shown to decrease the shear/viscosity while simultaneously increasing the wet mass gain of the films. Shear and viscosity may be indicative of both enzyme denaturation and increased lateral protein packing within the film structure hence it is assumed that less distortion occurs with the inclusion of linkers which allow for more optimal protein immobilization. © 2011 Elsevier Inc.
Binding residues; Covalent attachment; Detrimental effects; Enzyme denaturation; Film parameters; Film structure; Laccases; Mass gain; Protein immobilization; Protein-binding; QCM-D; Quartz crystal microbalance with dissipation; Quartz surfaces; Quartz transducer; Rheological measurements; Rheological parameter; Amino acids; Biomolecules; Biosensors; Enzyme kinetics; Industrial applications; Industrial research; Proteins; Quartz; Quartz crystal microbalances; Rheology; Self assembled monolayers; Viscosity; Enzyme immobilization; laccase; mercaptamine; mercaptoethanol; self assembled monolayer; adsorption kinetics; article; biofilm; chemical modification; covalent bond; crystal structure; density; electrode; enzyme denaturation; enzyme immobilization; enzymic biosensor; flow kinetics; nanofabrication; protein binding; protein immobilization; protein structure; quartz crystal microbalance; rigidity; shear stress; structure analysis; surface property; viscosity; Biosensing Techniques; Elastic Modulus; Enzymes, Immobilized; Laccase; Molecular Weight; Protein Binding; Protein Denaturation; Quartz Crystal Microbalance Techniques; Rheology; Surface Properties; Viscosity
