Surface Display of a Redox Enzyme and its Site-Specific Wiring to Gold Electrodes (original) (raw)
Related papers
Langmuir, 2012
Achieving efficient electrochemical communication between redox enzymes and various electrode materials is one of the main challenges in bioelectrochemistry and is of great importance for developing electronic applications. Cellobiose dehydrogenase (CDH) is an extracellular flavocytochrome composed of a catalytic FAD containing dehydrogenase domain (DH CDH), a heme b containing cytochrome domain (CYT CDH), and a flexible linker region connecting the two domains. Efficient direct electron transfer (DET) of CDH from the basidiomycete Phanerochaete chrysosporium (PcCDH) covalently attached to mixed self-assembled monolayer (SAM) modified gold nanoparticle (AuNP) electrode is presented. The thiols used were as follows: 4-aminothiophenol (4-ATP), 4-mercaptobenzoic acid (4-MBA), 4-mercaptophenol (4-MP), 11mercapto-1-undecanamine (MUNH 2), 11-mercapto-1-undecanoic acid (MUCOOH), and 11-mercapto-1-undecanol (MUOH). A covalent linkage between PcCDH and 4-ATP or MUNH 2 in the mixed SAMs was formed using glutaraldehyde as cross-linker. The covalent immobilization and the surface coverage of PcCDH were confirmed with surface plasmon resonance (SPR). To improve current density, AuNPs were cast on the top of polycrystalline gold electrodes. For all the immobilized PcCDH modified AuNPs electrodes, cyclic voltammetry exhibited clear electrochemical responses of the CYT CDH with fast electron transfer (ET) rates in the absence of substrate (lactose), and the formal potential was evaluated to be +162 mV vs NHE at pH 4.50. The standard ET rate constant (k s) was estimated for the first time for CDH and was found to be 52.1, 59.8, 112, and 154 s −1 for 4-ATP/4-MBA, 4-ATP/4-MP, MUNH 2 /MUCOOH, and MUNH 2 /MUOH modified electrodes, respectively. At all the mixed SAM modified AuNP electrodes, PcCDH showed DET only via the CYT CDH. No DET communication between the DH CDH domain and the electrode was found. The current density for lactose oxidation was remarkably increased by introduction of the AuNPs. The 4-ATP/4-MBA modified AuNPs exhibited a current density up to 30 μA cm −2 , which is ∼70 times higher than that obtained for a 4-ATP/4-MBA modified polycrystalline gold electrode. The results provide insight into fundamental electrochemical properties of CDH covalently immobilized on gold electrodes and promote further applications of CDHs for biosensors, biofuel cells, and bioelectrocatalysis.
Langmuir, 2005
A novel electrochemical approach is described for redox-active membrane proteins. A total membrane extract (in the form of vesicles) of Bacillus subtilis is tethered onto gold surfaces modified with cholesterol based thiols. The membrane vesicles remain intact on the surface and do not rupture or fuse to form a planar bilayer. Oxidation/reduction signals are obtained of the natural co-enzyme, menaquinone-7, located in the membrane. The membrane protein, succinate menaquinone oxidoreductase (SQR), remains in the vesicles and is able to reduce fumarate using menaquinone as mediator. The catalysis of the reverse reaction (oxidation of succinate), which is the natural catalytic function of SQR, is almost absent with menaquinone. However, adding the co-enzyme ubiquinone, which has a reduction potential that is about 0.2 V higher, restores the succinate oxidation activity.
Assembling nanostructured connections in bio-electrochemical systems
2016
Microbial fuel cells (MFCs) have been used in a variety of applications to date, however the power output of an MFC is its biggest limitation. The power output of an MFC can be improved by improving the electron transfer from the bacteria to the electrode. This project was interested in improving the electron transfer using gold nanoparticles. Gold nanoparticles were incorporated in MAgarose which showed good conductive properties and when used in a bio-electrochemical cell with Shewanella oneidensis MR-1 as the bacteria, a current enhancement of ~ 29 times was recorded when compared to plain veil. Gold macrostructures were also electrodeposited in thiolated agarose gels and used as electrodes in the bio-electrochemical cell, but the current enhancement was not very significant compared to the control and reached a saturation point over time. Thiol group functionalised PAMAM dendrimer protected gold nanoparticles, were attached to So surface and used as the biocatalysts in a bio-ele...
Preparation of gold surfaces with biospecific affinity for NAD(H)-dependent lactate dehydrogenase
Sensors and Actuators B: Chemical, 1997
Gold surfaces with biospecific affinity for NAD(H)-dependent lactate dehydrogenase have been prepared by covalent attachment of several triazine dyes to chemisorbed functionalized alkanethiol self-assembled monolayers. The dyes which work as coenzyme analoga build a site-specific affinity complex with the enzyme by binding through its NAD +-binding pocket. This immobilization method implies a biospecific recognition of the enzyme which is expected to yield lactate dehydrogenase self-assembled monolayers in which the enzyme is oriented facing its NAD +-binding pocket (and therefore its active site) towards the metal surface. Self-assembled monolayers with Cibacron Blue F3G-A as anchored ligand showed always the highest affinity for lactate dehydrogenase. However, the biospecific affinity of the ligand-anchored monolayer was strongly dependent on the nature of the alkanethiol underlayer used to bind the triazine dye. Higher enzymatic surface coverages were obtained with mixed self-assembled monolayers providing a low amount of ligand (less than a 10% of a densely packed alkanethiol monolayer) bound to the metal surface through a long and flexible spacer. Lactate dehydrogenase-modified gold electrode surfaces catalyzed the electro-oxidation of lactate only when the biological cofactor (NAD +) was present in the reaction mixture, thus suggesting that the NAD +-binding pocket used to anchor the enzyme to the monolayer is not involved in the enzymatic reaction.
Micromachines
Microbial Fuel Cells (MFCs) are biological fuel cells based on the oxidation of fuels by electrogenic bacteria to generate an electric current in electrochemical cells. There are several methods that can be employed to improve their performance. In this study, the effects of gold surface modification with different thiol molecules were investigated for their implementation as anode electrodes in micro-scale MFCs (µMFCs). Several double-chamber µMFCs with 10.4 µL anode and cathode chambers were fabricated using silicon-microelectromechanical systems (MEMS) fabrication technology. µMFC systems assembled with modified gold anodes were operated under anaerobic conditions with the continuous feeding of anolyte and catholyte to compare the effect of different thiol molecules on the biofilm formation of Shewanella oneidensis MR-1. Performances were evaluated using polarization curves, Electrochemical Impedance Spectroscopy (EIS), and Scanning Electron Microcopy (SEM). The results showed th...
Direct Bioelectrocatalysis at the Interfaces by Genetically Engineered Dehydrogenase
Bioinspired, Biomimetic and Nanobiomaterials, 2014
There is an emerging interest in developing bio-functionalisation routes serving as platforms for assembling diverse enzymes onto material surfaces. Specifically, the fabrication of next-generation, laboratory-on-a-chip-based sensing and energy-harvesting systems requires controlled orientation and organisation of the proteins at the inorganic interfaces. Herein, the authors take the initial steps towards designing multifunctional, enzyme-based platforms by genetically integrating the engineered materialselective peptide tags for tethering redox enzymes onto electrode surfaces. The authors engineered a fusion protein that genetically conjugates gold-binding peptide to formate dehydrogenase derived from Candida methylica. The expressed proteins were tested for both enzyme activity and self-directed gold-surface functionalisation ability. Their findings demonstrate the successful self-immobilisation of the engineered enzyme onto different gold electrodes while retaining the catalytic activity.
Journal of the American Chemical Society, 2005
An electrically contacted glucose dehydrogenase (GDH) enzyme electrode is fabricated by the reconstitution of the apo-GDH on pyrroloquinoline quinone (PQQ)-functionalized Au nanoparticles (Au-NPs), 1.4 nm, associated with a Au electrode. The Au-NPs functionalized with a single amine group were attached to the Au surface by 1,4-benzenedithiol bridges, and PQQ was covalently linked to the Au-NPs. The apo-GDH was then reconstituted on the PQQ cofactor sites. The surface coverage of GDH corresponded to 1.4 × 10-12 mol cm-2. The reconstituted enzyme revealed direct electrical contact with the electrode surface, and the bioelectrocatalytic oxidation of glucose occurred with a turnover number of 11 800 s-1. In contrast, a system that included the covalent attachment of GDH to the PQQ-Au-NPs monolayer in a random, nonaligned, configuration revealed lack of electrical communication between the enzyme and the electrode, albeit the enzyme existed in a bioactive structure. The bioelectrocatalytic function of the later system was, however, activated by the diffusional electron mediator 2,6-dichlorophenolindophenol. The results imply that the alignment of GDH on a Au-NP through the reconstitution process leads to an electrically contacted enzyme-electrode, where the Au-NP acts as a charge-transfer mediator.
Chemistry of Materials, 2017
Redox-active materials are an attractive platform for engineering specific interactions with charged species by electrochemical control. We present nanostructured redox-electrodes, functionalized with poly(vinyl)ferrocene embedded in a carbon nanotube matrix, for modulating the adsorption and release of proteins through electrochemical potential swings. The affinity of the interface towards proteins increased dramatically following oxidation of the ferrocenes, and, due to the Faradaic nature of the organometallic centers, the electrodes were maintained at sufficiently low overpotentials to ensure the preservation of both protein structure and catalytic activity. Our system was selective for various proteins based on size and charge distribution, and exhibited fast kinetics (<120 s for a charge-discharge cycle) and high uptake capacities (>200 mg/g) under moderate overpotentials (+0.4 V vs Ag/AgCl), as well as remarkable stability for binding under ferrocene oxidation conditions. The preservation of bioactivity and protein structure at the interface indicates the potential for these redox-mediated surfaces to be used as heterogeneous supports for enzyme catalysis. This work draws on the molecular selectivity of ferrocene-functionalized materials towards organic anion groups, and demonstrates that these smart redox-active materials can be used for modulation of the macroscopic affinity of surfaces for charged bio-macromolecules to enhance processes such as bio-separations, electrochemically-controlled protein purification, biocatalysis, and electrochemically-mediated drug release.
Analytical Chemistry, 1997
Microscopic, enzymatically active spots on self-assembled monolayers (SAMs) of alkanethiolates on gold were obtained by a combination of localized desorption induced using the scanning electrochemical microscope (SECM) followed by chemical derivatization. Starting from a SAM of dodecanethiolate on gold, localized desorption of alkanethiolates creates microscopic areas of an uncovered gold surface surrounded by a dense Au alkanethiolate layer. The renewed gold surface chemisorbs an aminoderivatized disulfide (cystaminium dihydrochloride) in a second step. Periodate-oxidized glucose oxidase was attached covalently to the terminal amino functions to create a stable, catalytically active pattern of the enzyme on the alkanethiolate SAM. The enzymatic activity was mapped using the imaging capabilities of SECM. The generator-collector mode (amperometric H 2 O 2 detection) was advantageously used, as the feedback mode leads to interferences due to concurrence between mediator regeneration by the enzymatic reaction and by the heterogeneous electron transfer at the gold regions from which the blocking dodecanethiolate layer had been desorbed. Rising backgrounds due to H 2 O 2 accumulation in the bulk solution can be prevented by adding minute amounts of the enzyme catalase to the working solution. By catalyzing the H 2 O 2 decomposition, the lifetime of H 2 O 2 is adjusted to prevent its accumulation in the bulk phase yet to allow its diffusion across the gap between the enzyme-modified region and the collecting electrode. Perspectives for creating miniaturized multienzyme structures, which will become accessible by repeating the desorption and covalent enzyme immobilization steps using different enzymes in each cycle, are highlighted.