The behaviour of enzymes on surfaces has been the subject of extensive research in recent years using a wide variety of electrode surfaces, in order to enlarge the understanding of electronic transfer processes [1]. Exploration of spatial organization, ordering, morphology and activity of enzyme molecules in artificial molecular systems is an important target for the development of high-performance enzyme sensors [2]. Enzyme immobilisation onto the electrode surface is a critical step in assembling amperometric biosensors, performed thus far through methods based on simple adsorption, or more complex procedures (e.g. chemical binding). Several techniques such as electrochemical, covalent binding, Langmuir and layer by layer techniques have been developed for the immobilization or stacking the enzymes on various matrices. Recently, layer by layer technique of redox enzymes at electrode surfaces finds application in a variety of areas including biosensors, bioelectronics and bioelectro-synthesis [4]. This technique of constructing multilayer assemblies by consecutively alternating adsorption of anionic and cationic polyelectrolytes was recently developed by Decher and co-workers for polyelectrolytes and later extended to doped conjugated polymers by Rubner et al. This technique has been applied to linear polyanions, bipolar amphiphiles, proteins, DNA, polynucleotides, charged nano-particles, viruses, ceramics and dyes [3,4].
Recently, we used the process developed by A.C. Fou et al. for the fabrication of layer by layer nano-architectures films of polypyrrole (PPY) via in-situ self-assembly [4]. PPY is one of the most extensively studied electronic materials amongst conducting polymers and it has received much interest because of various technological applications [5]. Among redox active enzymes, the electrochemical behaviour of glucose oxidase (GOD) was actively investigated, due to its practical applications in manufacturing biological sensors. The immobilisation of GOD on a conductive polymer (PPY, polyaniline, etc.) allows the construction of glucose responsive biosensors, for which the immobilisation of single or clustered GOD molecules represents a crucial and important step. The development of scanning probe microscopies and related methods yielding molecular or even atomic resolution images of molecules or crystal surfaces recently allowed new conceptual approaches to the study of biological surfaces and/or interfaces.
Beginning with this consideration, the main aim of this work was to analyse the deposition and characterization of glucose oxidase on the in-situ self-assembled films of PPY. In-situ self assembled films of PPY as a function of time on PSS and the layer by layer film of PPY and PSS on such various substrates were fabricated. The results of a Scanning Probe Microscopies studies of the films were analysed to understand the orientation of glucose oxidase on polypyrrole surface and the dependence on the enzyme concentration of the depositing solution. Further, the electrochemical studies were performed to provide the information on electron transfer processes on the spatial arrangement of GOD molecules on PPY surfaces.
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