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Probing fundamental film parameters of immobilized enzymes—Towards enhanced biosensor performance. Part II—Electroanalytical estimation of immobilized enzyme performance

Paper ID Volume ID Publish Year Pages File Format Full-Text
17399 42666 2011 7 PDF Available
Title
Probing fundamental film parameters of immobilized enzymes—Towards enhanced biosensor performance. Part II—Electroanalytical estimation of immobilized enzyme performance
Abstract

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, imax, the apparent Michaelis–Menten constant, Km 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.

Keywords
QCM-D, crystal microbalance with dissipation; SAM, self-assembled monolayer; SLB, succinic-lactic acid buffer, pH 4.5; Glut, glutaraldehyde; Lys, lysin; β-ME, β-mercaptoethanol; Cys, cysteamine; CV, coefficient of variation (C.V.)Enzyme; Immobilization; K
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Probing fundamental film parameters of immobilized enzymes—Towards enhanced biosensor performance. Part II—Electroanalytical estimation of immobilized enzyme performance
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Publisher
Database: Elsevier - ScienceDirect
Journal: Enzyme and Microbial Technology - Volume 49, Issue 2, 10 July 2011, Pages 153–159
Authors
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Subjects
Physical Sciences and Engineering Chemical Engineering Bioengineering
Get Full-Text Now
Don't Miss Today's Special Offer
Price was $35.95
You save - $31
Price after discount Only $4.95
100% Money Back Guarantee
Full-text PDF Download
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