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
The method of immobilization of a protein has a great influence on the overall conformation, and hence, functioning of the protein. Thus, a greater understanding of the events undergone by the protein during immobilization is key to manipulating the immobilization method to produce a strategy that influences the advantages of immobilization while minimizing their disadvantages in biosensor design. In this, the second paper of a two-part series, we have assessed the kinetic parameters of thin-film laccase monolayers, covalently attached to SAMs differing in spacer-arm length and lateral density of spacer arms. This was achieved using chronoamperometry and an electroactive product (p-benzoquinone), which was modeled in a non-linear regressional fashion to extract the relevant parameters. Finally, comparisons between the kinetic parameters presented in this paper and the rheological parameters of laccase monolayers immobilized in the same manner (Part I of this two paper series) were performed. Improvements in the maximal enzyme-catalysed current, i max, the apparent Michaelis-Menten constant, K m and the apparent biosensor sensitivity were noted for most of the surfaces with increasing linker length. Decreasing the lateral density of the spacer-arms brought about a general improvement in these parameters, which is attributed to the decrease in multiple points of immobilization undergone by functional proteins. Finally, comparisons between rheological data and kinetics data showed that the degree of viscosity exhibited by protein films has a negative influence on attached protein layers, while enhanced protein hydration levels (assessed piezoelectrically from data obtained in Paper 1) has a positive effect on those surfaces comprising rigidly bound protein layers. © 2011 Elsevier Inc.
Biosensor design; Bound proteins; Electroactive; Film parameters; Functional proteins; Immobilization method; Immobilized enzyme; Laccases; Michaelis-Menten constant; Multiple points; Negative influence; Non-linear; P-benzoquinone; Positive effects; Protein films; Protein hydration; Protein layers; QCM-D; Rheological data; Rheological parameter; Sams; Spacer arms; Two-part series; Biosensors; Chronoamperometry; Kinetic parameters; Monolayers; Proteins; Rheology; Viscosity; Enzyme immobilization; 1,4 benzoquinone; laccase; self assembled monolayer; amperometry; article; biofilm; catalysis; covalent bond; density; electrochemical analysis; electrode; enzymic biosensor; flow kinetics; hydration; Michaelis Menten kinetics; piezoelectricity; protein analysis; protein immobilization; quartz crystal microbalance; Biosensing Techniques; Electrochemical Techniques; Enzymes, Immobilized; Kinetics; Laccase; Nonlinear Dynamics; Protein Conformation; Quartz Crystal Microbalance Techniques; Rheology; Viscosity
