h1

TY  - THES
AU  - Schröper, Florian
TI  - Optimierte Immobilisierung von Redoxproteinen an funktionale Elektrodenoberflächen für die molekulare Bioelektronik
VL  - 4300
CY  - Jülich
PB  - Forschungszentrum Jülich, Zentralbibliothek
M1  - RWTH-CONV-113859
T2  - Berichte des Forschungszentrums Jülich
SP  - III, 224 S. : Ill., graph. Darst.
PY  - 2009
N1  - Zsfassung in engl. und dt. Sprache
N1  - Zugl.: Aachen, Techn. Hochsch., Diss., 2009
AB  - Within the last decade, protein electrochemistry at miniaturized electrodes has gained in importance, not only for the study of the charge transfer properties of redox proteins, but also for developing novel biosensor and bioelectronic devices with enhanced sensitivity and selectivity. The major challenges within this field of research are the directed coupling between electronic and biological compounds without losing biofunctionality and a successful miniaturization of electronic compounds leading from 2D macroelectrodes over 2D micro- and 1D nanoelectrodes up to 0D nano contacts for single molecule approaches. Proteins from biological electron transfer systems like respiratory chain or photosynthesis, are preferably used in the field of protein bioelectronics. These proteins typically have redox active centers accessible to the outer surface and are thus able to efficiently communicate with electrodes. This thesis deals with electrochemical investigations of the charge transfer behavior of redox proteins with regards to electrode dimension and immobilization strategy. The main focus lies on the redox protein cytochrome c (cyt c). The charge transfer behavior of this protein was studied while immobilized on macroscopic, microscopic and nanostructured electrodes. Besides the use of previously reported immobilization strategies, e.g. covalent immobilization or attachment via electrostatic interaction, novel strategies for a directed protein immobilization were developed. The aim was to achieve an optimized orientation of the protein on the electrode allowing a fast and reversible electron transfer between electrode and protein as well as the conservation of functionality, such that the active site remains accessible for enzyme binding and interaction. For this purpose, new strategies to direct immobilization of horse heart cyt c on gold electrode surfaces using bioaffinity tags at specified positions within the protein were developed. By using an expression system for cyt c in E. coli and genetic modifications, His and/or Cys tags were incorporated into the amino acid sequence at strategically defined positions. After successful expression and purification, comparative immobilization studies of different recombinant proteins and native cyt c were performed by means of Surface Plasmon Resonance Spectroscopy (SPR) and Atomic Force Microscopy (AFM). Functionality was proven by a photochemical enzyme test using UV/Vis spectroscopy and electrochemical studies. Thereby it was possible to demonstrate that both His and, moreso, Cys tagged cyt c can be immobilized on electrode surfaces either by electrostatic interaction as well as via its affinity tag, while still providing sufficient and fast electron transfer. Cyclovoltammetric studies demonstrated that tag-based immobilization enabled the proteins to exchange electrons with the supporting electrode and at the same time interact with the enzymes cytochrome c reductase as well as cytochrome c oxidase which were dissolved in the electrolyte. Moreover the combination of two different immobilization strategies, simultaneously using affinity tag and electrostatic immobilization at two different sites, enabled completely new possibilities for bifunctional immobilization of proteins. Bifunctional immobilization of different cyt c mutants was characterized by SPR and AFM. The proteins were first immobilized on a gold surface via an affinity tag, and in a second immobilization step, gold nanoparticles were immobilized on top of this protein layer by electrostatic interaction. In a first approach the recombinant Cys tagged protein was immobilized between two microscopic crossbar electrodes by electrostatic immobilization and via the Cys tag at the same time. Measuring the current-voltage characteristics revealed tunneling currents attributed to the charge transfer through the cyt c molecules. Thus it succeeded to develop different types of functional recombinant cyt c that can be used for bifunctional immobilization on or between electrode surfaces. Such bifunctional immobilized proteins are of special interest in the field of molecular bioelectronics, in particular for the development of novel biosensing devices where proteins can be immobilized between two crossing electrodes or bridging a nano gap within single molecule approaches. Further research at the Institute of Bio- and Nanosystems 2 in Jülich is addressed to the development of such systems.
KW  - Cytochrom c (SWD)
KW  - Immobilisierung (SWD)
KW  - Bioelektronik (SWD)
KW  - Elektrochemie (SWD)
KW  - Elektronentransfer (SWD)
LB  - PUB:(DE-HGF)11 ; PUB:(DE-HGF)3
UR  - https://publications.rwth-aachen.de/record/51579
ER  -