TY - THES A1 - Dronov, Roman T1 - Multi-component protein films by layer-by-layer : assembly and electron transfer T1 - Multikomponenten-Proteinfilme mittels Layer-by-Layer-Technik : Design und Elektronentransfer N2 - Electron transfer phenomena in proteins represent one of the most common types of biochemical reactions. They play a central role in energy conversion pathways in living cells, and are crucial components in respiration and photosynthesis. These complex biochemical reaction cascades consist of a series of proteins and protein complexes that couple a charge transfer to different forms of chemical energy. The efficiency and sophisticated optimisation of signal transfer in these natural redox chains has inspired engineering of artificial architectures mimicking essential properties of their natural analogues. Implementation of direct electron transfer (DET) in protein assemblies was a breakthrough in bioelectronics, providing a simple and efficient way for coupling biological recognition events to a signal transducer. DET avoids the use of redox mediators, reducing potential interferences and side reactions, as well as being more compatible with in vivo conditions. However, only a few haem proteins, including the redox protein cytochrome c (cyt.c), and blue copper enzymes show efficient DET on different kinds of electrodes. Previous investigations with cyt.c have mainly focused on heterogeneous electron transfer of monolayers of this protein on gold. An important advance was the fabrication of cyt.c multilayers by electrostatic layer-by-layer self-assembly. The ease of fabrication, the stability, and the controllable permeability of polyelectrolyte multilayers have made them particularly attractive for electroanalytical applications. With cyt.c and sulfonated polyaniline it was for the first time possible that fully electro-active multilayers of the redox protein could be prepared. This approach was extended to design an analytical signal chain based on multilayers of cyt.c and xanthine oxidase (XOD). The system does not need an external mediator but relies on an in situ generation of a mediating radical and thus allows a signal transfer from hypoxanthine via the substrate converting enzyme and cyt.c to the electrode. Another kind of a signal chain is based on assembling proteins in complexes on electrodes in such a way that a direct protein-protein electron transfer becomes feasible. This design does not need a redox mediator in analogy to natural protein communication. For this purpose, cyt.c and the enzyme bilirubin oxidase (BOD, EC 1.3.3.5) are co-immobilized in a self-assembled polyelectrolyte multilayer on gold electrodes. Although these two proteins are not natural reaction partners, the protein architecture facilitates an electron transfer from the electrode via multiple protein layers to molecular oxygen resulting in a significant catalytic reduction current. Finally, we describe a novel strategy for multi-protein layer-by-layer self-assembly combining cyt.c with an enzyme sulfite oxidase (SOx) without use of any additional polymer. Electrostatic interactions between these two proteins with rather separated pI values during the assembly process from a low ionic strength buffer were found sufficient for the layer-by-layer deposition of the both biomolecules. It is anticipated that the concepts described in this work will stimulate further progress in multilayer design of even more complex biomimetic signal cascades taking advantage of direct communication between proteins. N2 - Elektronentransferphänomene in Proteinen stellen den häufigsten Typ biochemischer Reaktionen dar. Sie spielen eine zentrale Rolle bei der Energieumwandlung in der Zelle und sind entscheidende Komponenten in der Atmung und Photosynthese. Diese komplexen Kaskaden biochemischer Reaktionen setzen sich aus einer Reihe von Proteinen und Proteinkomplexen zusammen, die den Energietransfer an verschiedene Formen chemischer Energie koppeln. Die große Effektivität und Selektivität des Signaltransfers in diesen natürlichen Redoxketten war Vorbild für die Entwicklung künstlicher Architekturen, die die wesentlichen Eigenschaften ihrer natürlichen Analoga nachahmen. Die Implementierung des direkten Elektronentransfers (DET) von Proteinen mit Elektroden war ein Durchbruch im Bereich der Bioelektronik. Sie lieferte einen einfachen und effizienten Weg für das Koppeln biologischer Erkennungsereignisse an einen Signalumwandler. Durch den DET können Redoxmediatoren vermieden und damit potentielle Grenzflächen und Nebenreaktionen reduziert werden. Ebenso wird damit die Kompatibilität für in vivo Bedingungen erhöht. Jedoch zeigen nur einige Hämproteine wie das Redoxprotein Cytochrom c (Cyt c) und blaue Kupferproteine einen effizienten DET auf verschiedenen Elektrodentypen. Bisherige Untersuchungen mit Cyt c konzentrierten sich hauptsächlich auf den heterogenen Elektronentransfer von Monoschichten dieses Proteins auf Gold. Ein wichtiger Fortschritt war die Herstellung von Cyt c Multischichten durch die elektrostatische Layer-by-Layer-Technik. Die einfache Herstellung, die Stabilität sowie die kontrollierbaren Permeationseigenschaften von Polyelektrolyt-Multischichten machte sie besonders attraktiv für elektroanalytische Anwendungen. So gelang es auch zum ersten Mal vollständig elektroaktive Multischichten aus Cyt c und Polyanilinsulfonsäure zu präparieren. Dieser Ansatz wurde hier erweitert, um eine analytische Signalkette auf der Basis von Multischichten aus Cyt c und Xanthinoxidase zu entwerfen. Das System bedarf keinen externen Mediator, es hängt jedoch von der in situ Generierung eines vermittelnden Radikals ab und erlaubt daher einen Signaltransfer von Hypoxanthin über ein substratumwandelndes Enzym und Cyt c zur Elektrode. Eine andere Art von Signalketten basiert auf der Assemblierung von Proteinen in Komplexen auf Elektroden in solcher Art und Weise, daß ein direkter Protein-Protein-Elektronentransfer möglich wird. Dieser Ansatz benötigt keinen Redoxmediator in Analogie zu Beispielen aus dem biologischen Signaltransfer. Zu diesem Zweck werden Cyt c und das Enzym Bilirubinoxidase mit einem selbst-assemblierenden Polyelektrolyten auf einer Goldelektrode koimmobilisiert. Obwohl diese zwei Proteine keine natürlichen Reaktionspartner sind, unterstützt die Protein-Architektur einen Elektronentransfer von der Elektrode über mehrere Proteinschichten zu molekularem Sauerstoff und ergibt einen signifikanten katalytischen Reduktionsstrom. Schließlich wird eine neue Strategie beschrieben für eine Selbstassemblierung von Proteinen ohne zusätzlichen Polyelektrolyten - am Beispiel der Kombination von Cyt c mit Sulfitoxidase. Es stellte sich heraus, daß die elektrostatische Wechselwirkung zwischen diesen zwei Proteinen mit ziemlich weit voneinander entfernt liegenden pI-Werten während des Assemblierungsprozesses durch einen Puffer mit geringer Ionenstärke ausreicht um die beiden Biomoleküle nach dem Layer-by-Layer-Prinzip auf einer Elektrode abzuscheiden. Es wird erwartet, daß das entwickelte Konzept von Multiprotein-Assemblaten auf Elektroden weitere Fortschritte bei dem Entwurf von Multischichten und sogar noch komplexeren biomimetischen Signalkaskaden anregen wird und dabei der Vorteil der direkten Kommunikation zwischen Proteinen genutzt wird. KW - Protein Multilayer KW - Electron transfer KW - Signal transfer chain KW - Cytochrome c KW - Polyelectrolyte KW - Bilirubin oxidase KW - Surface characterization KW - Raman Y1 - 2007 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:kobv:517-opus-17281 ER - TY - JOUR A1 - Bosserdt, Maria A1 - Gajovic-Eichelman, Nenad A1 - Scheller, Frieder W. T1 - Modulation of direct electron transfer of cytochrome c by use of a molecularly imprinted thin film JF - Analytical & bioanalytical chemistry N2 - We describe the preparation of a molecularly imprinted polymer film (MIP) on top of a self-assembled monolayer (SAM) of mercaptoundecanoic acid (MUA) on gold, where the template cytochrome c (cyt c) participates in direct electron transfer (DET) with the underlying electrode. To enable DET, a non-conductive polymer film is electrodeposited from an aqueous solution of scopoletin and cyt c on to the surface of a gold electrode previously modified with MUA. The electroactive surface concentration of cyt c was 0.5 pmol cm(-2). In the absence of the MUA layer, no cyt c DET was observed and the pseudo-peroxidatic activity of the scopoletin-entrapped protein, assessed via oxidation of Ampliflu red in the presence of hydrogen peroxide, was only 30 % of that for the MIP on MUA. This result indicates that electrostatic adsorption of cyt c by the MUA-SAM substantially increases the surface concentration of cyt c during the electrodeposition step, and is a prerequisite for the productive orientation required for DET. After template removal by treatment with sulfuric acid, rebinding of cyt c to the MUA-MIP-modified electrode occurred with an affinity constant of 100,000 mol(-1) L, a value three times higher than that determined by use of fluorescence titration for the interaction between scopoletin and cyt c in solution. The DET of cyt c in the presence of myoglobin, lysozyme, and bovine serum albumin (BSA) reveals that the MIP layer suppresses the effect of competing proteins. KW - Cytochrome c KW - Molecularly imprinted polymer film KW - Mercaptoundecanoic acid KW - Direct electron transfer KW - Scopoletin (7-hydroxy-6-methoxycoumarin) Y1 - 2013 U6 - https://doi.org/10.1007/s00216-013-7009-8 SN - 1618-2642 VL - 405 IS - 20 SP - 6437 EP - 6444 PB - Springer CY - Heidelberg ER - TY - JOUR A1 - Frasca, Stefano A1 - Richter, Claudia A1 - von Graberg, Till A1 - Smarsly, Bernd M. A1 - Wollenberger, Ursula T1 - Electrochemical switchable protein-based optical device JF - Engineering in life sciences : Industry, Environment, Plant, Food N2 - The present work contributes to the development of reusable sensing systems with a visual evaluation of the detection process related to an analyte. An electrochemical switchable protein-based optical device was designed with the core part composed of cytochrome c immobilized in a mesoporous indium tin oxide film. A color-developing redox-sensitive dye was used as switchable component of the system. The cytochrome c-catalyzed oxidation of the dye by hydrogen peroxide is spectroscopically investigated. When the dye is co-immobilized with the protein, its redox state is easily controlled by application of an electrical potential at the supporting material. This enables to electrochemically reset the system to the initial state and repetitive signal generation. The implemented reset function of the color forming reaction will make calibration of small test devices possible. The principle can be extended to other color forming redox reactions and to coupled enzyme systems, such as rapid food testing and indication of critical concentrations of metabolites for health care. KW - Cytochrome c KW - Electrochemical switch KW - Indium tin oxide KW - mesoporous materials KW - Optical device Y1 - 2011 U6 - https://doi.org/10.1002/elsc.201100079 SN - 1618-0240 VL - 11 IS - 6 SP - 554 EP - 558 PB - Wiley-Blackwell CY - Malden ER - TY - JOUR A1 - Yarman, Aysu A1 - Dechtrirat, Decha A1 - Bosserdt, Maria A1 - Jetzschmann, Katharina J. A1 - Gajovic-Eichelmann, Nenad A1 - Scheller, Frieder W. T1 - Cytochrome c-derived hybrid systems based on moleculary imprinted polymers JF - Electroanalysis : an international journal devoted to fundamental and practical aspects of electroanalysis N2 - Hybrid architectures which combine a MIP with an immobilized affinity ligand or a biocatalyst sum up the advantages of both components. In this paper, hybrid architectures combining a layer of a molecularly imprinted electropolymer with a mini-enzyme or a self-assembled monolayer will be presented. (i) Microperoxidase-11 (MP-11) catalyzed oxidation of the drug aminopyrine on a product-imprinted sublayer: The peroxide dependent conversion of the analyte aminopyrine takes place in the MP-11 containing layer on top of a product-imprinted electropolymer on the indicator electrode. The hierarchical architecture resulted in the elimination of interfering signals for ascorbic acid and uric acid. An advantage of the new hierarchical structure is the separation of MIP formation by electropolymerization and immobilization of the catalyst. In this way it was for the first time possible to integrate an enzyme with a MIP layer in a sensor configuration. This combination has the potential to be transferred to other enzymes, e.g. P450, opening the way to clinically important analytes. (ii) Epitope-imprinted poly-scopoletin layer for binding of the C-terminal peptide and cytochrome c (Cyt c): The MIP binds both the target peptide and the parent protein almost eight times stronger than the non-imprinted polymer with affinities in the lower micromolar range. Exchange of only one amino acid in the peptide decreases the binding by a factor of five. (iii) MUA-poly-scopoletin MIP for cytochrome c: Cyt c bound to the MIP covered gold electrode exhibits direct electron transfer with a redox potential and rate constant typical for the native protein. The MIP cover layer suppresses the displacement of the target protein by BSA or myoglobin. The combination of protein imprinted polymers with an efficient electron transfer is a new concept for characterizing electroactive proteins such as Cyt c. The competition with other proteins shows that the MIP binds its target Cyt c preferentially and that molecular shape and the charge of protein determine the binding of interfering proteins. KW - Molecularly imprinted polymers KW - Microperoxidase-11 KW - Cytochrome c KW - Catalytically active MIPs KW - Epitope imprinting KW - Monoclonal MIPs Y1 - 2015 U6 - https://doi.org/10.1002/elan.201400592 SN - 1040-0397 SN - 1521-4109 VL - 27 IS - 3 SP - 573 EP - 586 PB - Wiley-VCH CY - Weinheim ER -