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The combination of the biocatalytic features of enzymes with the unique physical properties of nanoparticles in a biohybrid system provides a promising approach for the development of advanced bioelectrocatalytic devices. This study describes the construction of photoelectrochemical signal chains based on CdSe/ZnS quantum dot (QD) modified gold electrodes as light switchable elements, and low molecular weight redox molecules for the combination with different biocatalysts. Photoelectrochemical and photoluminescence experiments verify that electron transfer can be achieved between the redox molecules hexacyanoferrate and ferrocene, and the QDs under illumination. Since for both redox mediators a concentration dependent photocurrent change has been found, light switchable enzymatic signal chains are built up with fructose dehydrogenase (FDH) and pyrroloquinoline quinone-dependent glucose dehydrogenase ((PQQ) GDH) for the detection of sugars. After immobilization of the enzymes at the QD electrode the biocatalytic oxidation of the substrates can be followed by conversion of the redox mediator in solution and subsequent detection at the QD electrode. Furthermore, (PQQ) GDH has been assembled together with ferrocenecarboxylic acid on top of the QD electrode for the construction of a funtional biohybrid architecture, showing that electron transfer can be realized from the enzyme over the redox mediator to the QDs and subsequently to the electrode in a completely immobilized fashion. The results obtained here do not only provide the basis for light-switchable biosensing and bioelectrocatalytic applications, but may also open the way for self-driven point-of-care systems by combination with solar cell approaches (power generation at the QD electrode by enzymatic substrate consumption).
A fluidic chip system was developed, which combines a stable generation of superoxide radicals and hydrogen peroxide with their sensorial detection. The generation of both reactive oxygen species was achieved by immobilization of xanthine oxidase on controlled pore glass in a reaction chamber. Antioxidants can be introduced into the fluidic chip system by means of mixing chamber. The detection of both species is based on the amperometric principle using a biosensor chip with two working electrodes. As sensing protein for both electrodes cytochrome c was used. The novel system was designed for the quantification of the antioxidant efficiency of different potential scavengers of the respective reactive species in an aqueous medium. Several model antioxidants such as ascorbic acid or catalase have been tested under flow conditions.
The creation of electron transfer (ET) chains based on the defined arrangement of enzymes and redox proteins on electrode surfaces represents an interesting approach within the field of bioelectrocatalysis. In this study, we investigated the ET reaction of the flavin-dependent enzyme fructose dehydrogenase (FDH) with the redox protein cytochrome c (cyt c). Two different pH optima were found for the reaction in acidic and neutral solutions. When cyt c was adsorbed on an electrode surface while the enzyme remained in solution, ET proceeded efficiently in media of neutral pH. Interprotein ET was also observed in acidic media; however, it appeared to be less efficient. These findings suggest that two different ET pathways between the enzyme and cyt c may occur. Moreover, cyt c and FDH were immobilized in multiple layers on an electrode surface by means of another biomacromolecule: DNA (double stranded) using the layer -by -layer technique. The biprotein multilayer architecture showed a catalytic response in dependence on the fructose concentration, indicating that the ET reaction between both proteins is feasible even in the immobilized state. The electrode showed a defined response to fructose and a good storage stability. Our results contribute to the better understanding of the ET reaction between FDH and cyt c and provide the basis for the creation of all-biomolecule based fructose sensors the sensitivity of which can be controlled by the layer preparation.
We report on the pH-dependent bioelectrocatalytic activity of the redox enzyme xanthine dehydrogenase (XDH) in the presence of sulfonated polyaniline PMSA1 (poly(2-methoxyaniline-5-sulfonic acid)-co-aniline). Ultraviolet-visible (UV-vis) spectroscopic measurements with both components in solution reveal electron transfer from the hypoxanthine (HX)-reduced enzyme to the polymer. The enzyme shows bioelectrocatalytic activity on indium tin oxide (ITO) electrodes, when the polymer is present. Depending on solution pH, different processes can be identified. It can be demonstrated that not only product-based communication with the electrode but also efficient polymer-supported bioelectrocatalysis occur. Interestingly, substrate dependent catalytic currents can be obtained in acidic and neutral solutions, although the highest activity of XDH with natural reaction partners is in the alkaline region. Furthermore, operation of the enzyme electrode without addition of the natural cofactor of XDH is feasible. Finally, macroporous ITO electrodes have been used as an immobilization platform for the fabrication of HX-sensitive electrodes. The study shows that the efficient polymer/enzyme interaction can be advantageously combined with the open structure of an electrode material of controlled pore size, resulting in good processability, stability, and defined signal transfer in the presence of a substrate.
Protein multilayers, consisting of cytochrome c (cyt c) and poly(aniline sulfonic acid) (PASA), are investigated by electrochemical quartz crystal microbalance with dissipation monitoring (E-QCM-D). This technique reveals that a four-bilayer assembly has rather rigid properties. A thickness of 16.3 +/- 0.8 nm is calculated with the Sauerbrey equation and is found to be in good agreement with a viscoelastic model. The electroactive amount of cyt c is estimated by the deposited mass under the assumption of 50% coupled water. Temperature-induced stabilization of the multilayer assembly has been investigated in the temperature range between 30 and 45 degrees C. The treatment results in a loss of material and a contraction of the film. The electroactive amount of cyt c also decreases during heating and remains constant after the cooling period. The contraction of the film is accompanied by the enhanced stability of the assembly. In addition, it is found that cyt c and PASA can be assembled at higher temperatures, resulting in the formation of multilayer systems with less dissipation.
We use substituted polyanilines for the construction of new polymer electrodes for interaction studies with the redox protein cytochrome c (cyt c) and the enzyme sulfite oxidase (SO). For these purposes four different polyaniline copolymers are chemically synthesized. Three of them are copolymers, containing 2-methoxyaniline-5-sulfonic acid with variable ratios of aniline; the fourth copolymer consists of 3-amino-benzoic acid and aniline. The results show that all polymers are suitable for being immobilized as thin stable films on gold wire and indium tin oxide (ITO) electrode surfaces from DMSO solution. This can be demonstrated by cyclic voltammetry and UV-Vis spectroscopy measurements. Moreover, cyt c can be electrochemically detected not only in solution, but also immobilized on top of the polymer films. Furthermore, the appearance of a significant catalytic current has been demonstrated for the sulfonated polyanilines, when the polymer-coated protein electrode is being measured upon addition of sulfite oxidase, confirming the establishment of a bioanalytical signal chain. Best results have been obtained for the polymer with highest sulfonation grade. The redox switching of the polymer by the enzymatic reaction can also be analyzed by following the spectral properties of the polymer electrode.
The effect of introducing positive charges (lysines) in human cytochrome c (cyt c) on the redox properties and reaction rates of cyt c with superoxide radicals was studied. The mutated forms of this electron-transfer protein are used as sensorial recognition elements for the amperometric detection of the reactive oxygen radical. The proteins were prepared by site-directed mutagenesis focusing on amino acids near the heme edge. The 11 mutants of human cyt c expressed in the course of this research have been characterized by UV-vis spectroscopy, circular dichroism, and NMR spectroscopy to verify overall structure integrity as well as axial coordination of the heme iron. The mutants are investigated voltammetrically using promoter-modified gold electrodes with respect to redox activity and formal redox potential. The rate constants for the reaction with superoxide have been determined spectrophotometrically. Four mutants show a higher reaction rate with the radical as compared to the wild type. These mutants are used for the construction of superoxide sensors based on thiol-modified gold electrodes and covalently fixed proteins. We found that the E66K mutant-based electrode has a clearly higher sensitivity in comparison with the wild-type-based sensor while retaining the high selectivity and showing a good storage stability.
An efficient electrocatalytic biosensor for sulfite detection was developed by co-immobilizing sulfite oxidase and cytochrome c with polyaniline sulfonic acid in a layer-by-layer assembly. QCM, UV-Vis spectroscopy and cyclic voltammetry revealed increasing loading of electrochemically active protein with the formation of multilayers. The sensor operates reagentless at low working potential. A catalytic oxidation current was detected in the presence of sulfite at the modified gold electrode, polarized at +0.1 V ( vs. Ag/AgCl 1 M KCl). The stability of the biosensor performance was characterized and optimized. A 17-bilayer electrode has a linear range between 1 and 60 mu M sulfite with a sensitivity of 2.19 mA M-1 sulfite and a response time of 2 min. The electrode retained a stable response for 3 days with a serial reproducibility of 3.8% and lost 20% of sensitivity after 5 days of operation. It is possible to store the sensor in a dry state for more than 2 months. The multilayer electrode was used for determination of sulfite in unspiked and spiked samples of red and white wine. The recovery and the specificity of the signals were evaluated for each sample.
The layer-by-layer adsorption technique based on the consecutive deposition of oppositely charged species is for the preparation of protein multilayers with fully electro-active protein molecules. The methodology was established with cytochrome c and the polyelectrolyte sulfonated polyaniline (PASA). The technique is also useful for the construction of bi-protein architectures confining protein-protein communication to an electrode. Following natural examples of protein complexes with defined signal transfer, cytochrome c was arranged with enzymes such as xanthine oxidase, bilirubin oxidase, laccase, and sulfite oxidase in self-assembled multilayer architectures. Thus, biomimetic signal chains from the enzyme substrate via the enzyme and cytochrome c towards the electrode can be established. Communication between proteins immobilised in multiple layers on the electrode can be achieved by in situ generation of small shuttle molecules or more advantageously by direct interprotein electron transfer. This allows the construction of new sensing electrodes, the properties of which can be tuned by the number of deposited protein layers. The mechanism of electron transfer within such protein assemblies on gold electrodes will be discussed.