Sven Christian Feifel

Silica nanoparticles for the layer-by-layer assembly of fully electro-active cytochrome c multilayers

BackgroundFor bioanalytical systems sensitivity and biomolecule activity are critical issues. The immobilization of proteins into multilayer systems by the layer-by-layer deposition has become one of the favorite methods with this respect. Moreover, the combination of nanoparticles with biomolecules on electrodes is a matter of particular interest since several examples with high activities and direct electron transfer have been found. Our study describes the investigation on silica nanoparticles and the redox protein cytochrome c for the construction of electro-active multilayer architectures, and the electron transfer within such systems. The novelty of this work is the construction of such artificial architectures with a non-conducting building block. Furthermore a detailed study of the size influence of silica nanoparticles is performed with regard to formation and electrochemical behavior of these systems.ResultsWe report on interprotein electron transfer (IET) reaction cascades of cytochrome c (cyt c) immobilized by the use of modified silica nanoparticles (SiNPs) to act as an artificial matrix. The layer-by-layer deposition technique has been used for the formation of silica particles/cytochrome c multilayer assemblies on electrodes. The silica particles are characterized by dynamic light scattering (DLS), Fourier transformed infrared spectroscopy (FT-IR), Zeta-potential and transmission electron microscopy (TEM). The modified particles have been studied with respect to act as an artificial network for cytochrome c and to allow efficient interprotein electron transfer reactions. We demonstrate that it is possible to form electro-active assemblies with these non-conducting particles. The electrochemical response is increasing linearly with the number of layers deposited, reaching a cyt c surface concentration of about 80 pmol/cm2 with a 5 layer architecture. The interprotein electron transfer through the layer system and the influence of particle size are discussed.ConclusionsThis study demonstrates the ability to construct fully electro-active cyt c multilayer assemblies by using carboxy-modified silica nanoparticles. Thus it can be shown that functional, artificial systems can be build up following natural examples of protein arrangements. The absence of any conductive properties in the second building block clearly demonstrates that mechanisms for electron transfer through such protein multilayer assemblies is based on interprotein electron exchange, rather than on wiring of the protein to the electrode.The construction strategy of this multilayer system provides a new controllable route to immobilize proteins in multiple layers featuring direct electrochemistry without mediating shuttle molecules and controlling the electro-active amount by the number of deposition steps.

Advanced unidirectional photocurrent generation via cytochrome c as reaction partner for directed assembly of photosystem I

Conversion of light into an electrical current based on biohybrid systems mimicking natural photosynthesis is becoming increasingly popular. Photosystem I (PSI) is particularly useful in such photo-bioelectrochemical devices. Herein, we report on a novel biomimetic approach for an effective assembly of photosystem I with the electron transfer carrier cytochrome c (cyt c), deposited on a thiol-modified gold-surface. Atomic force microscopy and surface plasmon resonance measurements have been used for characterization of the assembly process. Photoelectrochemical experiments demonstrate a cyt c mediated generation of an enhanced unidirectional cathodic photocurrent. Here, cyt c can act as a template for the assembly of an oriented and dense layer of PSI and as wiring agent to direct the electrons from the electrode towards the photosynthetic reaction center of PSI. Furthermore, three-dimensional protein architectures have been formed via the layer-by-layer deposition technique resulting in a successive increase in photocurrent densities. An intermittent cyt c layer is essential for an efficient connection of PSI layers with the electrode and for an improvement of photocurrent densities.

High photocurrent generation by photosystem I on artificial interfaces composed of π-system-modified graphene

Photosystem I (PSI) is a key component of the oxygenic photosynthetic electron transport chain because of its light-induced charge separation and electron transfer (ET) capabilities. We report the fabrication of an efficient graphene-biohybrid light-harvesting electrode consisting of cyanobacterial trimeric PSI complexes immobilized onto π-system-modified graphene electrodes. Based on the strong interaction between conjugated aromatic compounds and the graphene material via π–π-stacking, we have designed a simple but smart platform to fabricate light-driven photoelectrochemical devices. Due to the possibility of surface property adaptation and the excellent conductivity of graphene, the modified biohybrid electrodes exhibit a well-defined photoelectrochemical response. In particular, the PSI–graphene electrode applying pyrene butyric acid NHS ester displays a very high photocurrent output of 23 μA cm−2 already at the open circuit potential which can be further increased by an overpotential and the use of an electron acceptor (methyl viologen) under air saturation up to 135 μA cm−2. Comparing the graphene–PSI biohybrid systems based on different π-system-modifiers reveals that the pyrene derivatives result in higher current outputs compared to the anthracene derivatives and that the covalent fixation during immobilization appears more efficient compared to simple adsorption. Interestingly, the pyrene-based PSI electrodes also display a nearly unidirectional photocurrent generation, establishing the feasibility of conjoining these nanomaterials as potential constructs in next-generation photovoltaic devices.

Catalytically Active Silica Nanoparticle-Based Supramolecular Architectures of Two Proteins - Cellobiose Dehydrogenase and Cytochrome c on Electrodes

Artificial nanobiomolecular architectures that follow natural examples in protein assembly become more and more important from basic and applied points of view. Our study describes the investigation on cellobiose dehydrogenase (CDH), cytochrome c (cyt c), and silica nanoparticles (SiNPs) for the construction of fully catalytically active supramolecular architectures on electrodes. We report on intraprotein, interprotein, and direct electron-transfer reaction cascades of cellobiose dehydrogenase and cytochrome c immobilized in multiple supramolecular layers. Carboxy-modified SiNPs are used to provide an artificial matrix, which enables protein arrangement in an electroactive form. Direct and interprotein electron transfer has been established for a two-protein system with CDH and cyt c in a layered architecture for the first time. We also highlight that the glycosylation of CDH and the silica nanoparticle size play key roles in the mode of operation in such a complex system. The response of the specific substrate, here lactose, can be tuned by the number of immobilized nanobiomolecular layers.

Biohybrid architectures for efficient light-to-current conversion based on photosystem I within scalable 3D mesoporous electrodes

The combination of advanced materials and defined surface design with complex proteins from natural photosynthesis is currently one of the major topics in the development of biohybrid systems and biophotovoltaic devices. In this study transparent mesoporous indium tin oxide (μITO) electrodes have been used in combination with the trimeric supercomplex photosystem I (PSI) from Thermosynechococcus elongatus and the small redox protein cytochrome c (cyt c) from horse heart to fabricate advanced and efficient photobiocathodes. The preparation of the μITO via spin coating allows easy scalability and ensures a defined increase in the electrochemically active surface area with accessibility for both proteins. Using these 3D electrodes up to 40 μm thickness, the immobilization of cyt c and PSI with full monolayer coverage and their electrical communication to the electrode can be achieved. Significant improvement can be made when the heterogenous electron transfer rate constant of cyt c with the electrode is increased by an appropriate surface treatment. The photocurrent follows linearly the thickness of the μITO and current densities of up to 150 μA cm−2 can be obtained without indications of a limitation. The internal quantum efficiency is determined to be 39% which demonstrates that the wiring of PSI via cyt c can be advantageously used in a system with high protein loading and efficient electron pathways inside 3D transparent conducting oxides.

Nanobiomolecular multiprotein clusters on electrodes for the formation of a switchable cascadic reaction scheme.

A supramolecular multicomponent protein architecture on electrodes is developed that allows the establishment of bidirectional electron transfer cascades based on interprotein electron exchange. The architecture is formed by embedding two different enzymes (laccase and cellobiose dehydrogenase) and a redox protein (cytochrome c) by means of carboxy-modified silica nanoparticles in a multiple layer format. The construct is designed as a switchable dual analyte detection device allowing the measurement of lactose and oxygen, respectively. As the switching force we apply the electrode potential, which ensures control of the redox state of cytochrome c. The two signal chains are operating in a non-separated matrix and are not disturbed by the other biocatalyst.

Unidirectional Photocurrent of Photosystem I on π-System-Modified Graphene Electrodes: Nanobionic Approaches for the Construction of Photobiohybrid Systems

One major vital element of the oxygenic photosynthesis is photosystem I (PSI). We report on the construction of graphene-based nanohybrid light-harvesting architectures consisting of PSI supercomplexes adsorbed onto π-system-modified graphene interfaces. The light-driven nanophotobioelectrochemical architectures have been designed on a modified carbon surface, on the basis of π-π-stacking interactions between polycyclic aromatic compounds and graphene. As a result of the remarkable features of graphene and the feasibility of purposeful surface property adjustment, well-defined photoelectrochemical responses have been displayed by the nanophotohybrid electrodes. In particular, the PSI-graphene electrodes utilizing naphthalene derivatives provided a suitable surface for the adsorption of PSI and display already at the open circuit potential (OCP) a high cathodic photocurrent output of 4.5 ± 0.1 μA/cm(2). By applying an overpotential and addition of a soluble electron acceptor (methyl viologen), the photocurrent density can be further magnified to 20 ± 0.5 μA/cm(2). On the contrary, the investigated anthracene-based PSI-graphene electrodes exhibit considerably smaller and not very directed photoelectrochemical responses. This study grants insights into the influences of different polycyclic aromatic compounds acting as an interface between the very large protein supercomplex PSI and graphene while supporting the electrochemical communication of the biomolecule with the electrode. It needs to be emphasized that solely the naphthalene-based photoelectrodes reveal unidirectional cathodic photocurrents, establishing the feasibility of utilizing this advanced approach for the construction of next-generation photovoltaic devices.

Electroactive nanobiomolecular architectures of laccase and cytochrome c on electrodes: applying silica nanoparticles as artificial matrix.

Fully electroactive multilayer architectures combining the redox protein cytochrome c and the enzyme laccase by the use of silica nanoparticles as artificial matrix have been constructed on gold electrodes capable of direct dioxygen reduction. Laccase form Trametes versicolor and cytochrome c from horse heart were electrostatically coimmobilized by alternate deposition with interlayers of silica nanoparticles in a multilayer fashion. The layer formation has been monitored by quartz crystal microbalance. The electrochemical properties and performance of the nanobiomolecular entities were investigated by cyclic voltammetry, indicating, that a multistep electron transfer cascade, from the electrode via cytochrome c in the layered system toward the enzyme laccase, and here to molecular dioxygen was achieved. The response of the novel architecture is based on direct electron exchange between immobilized proteins and can be tuned by the assembly process.

Insights into Interprotein Electron Transfer of Human Cytochrome c Variants Arranged in Multilayer Architectures by Means of an Artificial Silica Nanoparticle Matrix

The redox behavior of proteins plays a crucial part in the design of bioelectronic systems. We have demonstrated several functional systems exploiting the electron exchange properties of the redox protein cytochrome c (cyt c) in combination with enzymes and photoactive proteins. The operation is based on an effective reaction at modified electrodes but also to a large extent on the capability of self-exchange between cyt c molecules in a surface-fixed state. In this context, different variants of human cyt c have been examined here with respect to an altered heterogeneous electron transfer (ET) rate in a monolayer on electrodes as well as an enhanced self-exchange rate while being incorporated in multilayer architectures. For this purpose, mutants of the wild-type (WT) protein have been prepared to change the chemical nature of the surface contact area near the heme edge. The structural integrity of the variants has been verified by NMR and UV–vis measurements. It is shown that the single-point mutations can significantly influence the heterogeneous ET rate at thiol-modified gold electrodes and that electroactive protein/silica nanoparticle multilayers can be constructed with all forms of human cyt c prepared. The kinetic behavior of electron exchange for the mutant proteins in comparison with that of the WT has been found altered in some multilayer arrangements. Higher self-exchange rates have been found for K79A. The results demonstrate that the position of the introduced change in the charge situation of cyt c has a profound influence on the exchange behavior. In addition, the behavior of the cyt c variants in assembled multilayers is found to be rather similar to the situation of cyt c self-exchange in solution verified by NMR.

Protein Multilayer Architectures on Electrodes for Analyte Detection

This chapter provides an overview of different assembly methodologies used for the construction of multilayer architectures with biomolecules for application in sensors. Besides the use of bioaffinity interactions and covalent strategies, special attention will be paid to the electrostatic layer-by-layer technique. Different building blocks can be used for the formation of multilayers with a clear preference for polymers and nanoparticles. Among the biomolecules, enzymes and redox proteins are in focus. Because of the high importance of multilayers formed on electrodes, the chapter will concentrate on sensor systems with electrochemical transduction. Particularly advantageous are schemes that can avoid diffusible shuttling molecules between the biomolecule and the electrode and that represent artificial signal chains by exploiting direct protein-protein communication in the immobilized state.