Strategies for "wiring" redox-active proteins to electrodes and applications in biosensors, biofuel cells, and nanotechnology

In this tutorial review the basic approaches to establish electrochemical communication between redox-active proteins and electrodes are elucidated and examples for applications in electrochemical biosensors, biofuel cells and nanotechnology are presented. The early stage of protein electrochemistry is described giving a short overview over electron transfer (ET) between electrodes and proteins, followed by a brief introduction into experimental procedures for studying proteins at electrodes and possible applications arising thereof. The article starts with discussing the electrochemistry of cytochrome c, the first redox-active protein, for which direct reversible ET was obtained, under diffusion controlled conditions and after adsorption to electrodes. Next, examples for the electrochemical study of redox enzymes adsorbed on electrodes and modes of immobilization are discussed. Shortly the experimental approach for investigating redox-active proteins adsorbed on electrodes is outlined. Possible applications of redox enzymes in electrochemical biosensors and biofuel cells working by direct ET (DET) and mediated ET (MET) are presented. Furthermore, the reconstitution of redox active proteins at electrodes using molecular wire-like units in order to "wire" the proteins to the electrode surface and possible applications in nanotechnology are discussed.     

Infrared Spectroscopy During Electrocatalytic Turnover Reveals the Ni‐L Active Site State During H2 Oxidation by a NiFe Hydrogenase

A novel in situ IR spectroscopic approach is demonstrated for the characterization of hydrogenase during catalytic turnover. E. coli hydrogenase 1 (Hyd‐1) is adsorbed on a high surface‐area carbon electrode and subjected to the same electrochemical control and efficient supply of substrate as in protein film electrochemistry during spectral acquisition. The spectra reveal that the active site state known as Ni‐L, observed in other NiFe hydrogenases only under illumination or at cryogenic temperatures, can be generated reversibly in the dark at ambient temperature under both turnover and non‐turnover conditions. The observation that Ni‐L is present at all potentials during turnover under H₂ suggests that the final steps in the catalytic cycle of H₂ oxidation by Hyd‐1 involve sequential proton and electron transfer via Ni‐L. A broadly applicable IR spectroscopic technique is presented for addressing electrode‐adsorbed redox enzymes under fast catalytic turnover.     

ZnO/Cu nanocomposite: a platform for direct electrochemistry of enzymes and biosensing applications

Unique structured nanomaterials can facilitate the direct electron transfer between redox proteins and the electrodes. Here, in situ directed growth on an electrode of a ZnO/Cu nanocomposite was prepared by a simple corrosion approach, which enables robust mechanical adhesion and electrical contact between the nanostructured ZnO and the electrodes. This is great help to realize the direct electron transfer between the electrode surface and the redox protein. SEM images demonstrate that the morphology of the ZnO/Cu nanocomposite has a large specific surface area, which is favorable to immobilize the biomolecules and construct biosensors. Using glucose oxidase (GOx) as a model, this ZnO/Cu nanocomposite is employed for immobilization of GOx and the construction of the glucose biosensor. Direct electron transfer of GOx is achieved at ZnO/Cu nanocomposite with a high heterogeneous electron transfer rate constant of 0.67 ± 0.06 s(-1). Such ZnO/Cu nanocomposite provides a good matrix for direct electrochemistry of enzymes and mediator-free enzymatic biosensors.     

Amperometric Biosensors Based on Direct Electron Transfer Enzymes

The accurate determination of analyte concentrations with selective, fast, and robust methods is the key for process control, product analysis, environmental compliance, and medical applications. Enzyme-based biosensors meet these requirements to a high degree and can be operated with simple, cost efficient, and easy to use devices. This review focuses on enzymes capable of direct electron transfer (DET) to electrodes and also the electrode materials which can enable or enhance the DET type bioelectrocatalysis. It presents amperometric biosensors for the quantification of important medical, technical, and environmental analytes and it carves out the requirements for enzymes and electrode materials in DET-based third generation biosensors. This review critically surveys enzymes and biosensors for which DET has been reported. Single- or multi-cofactor enzymes featuring copper centers, hemes, FAD, FMN, or PQQ as prosthetic groups as well as fusion enzymes are presented. Nanomaterials, nanostructured electrodes, chemical surface modifications, and protein immobilization strategies are reviewed for their ability to support direct electrochemistry of enzymes. The combination of both biosensor elements—enzymes and electrodes—is evaluated by comparison of substrate specificity, current density, sensitivity, and the range of detection.     

血红蛋白直接电化学的界面设计与传感研究进展

血红蛋白为人体红细胞中的一种主要蛋白质,是血液中运输氧气的主要物质。由于其类酶的性质、确定的结构以及分布的广泛性,长期以来一直是氧化还原蛋白质直接电化学以及生物传感研究的理想模型。近年来,受益于材料科学与信息技术的快速发展,血红蛋白直接电化学的界面设计以及生物传感已成为当前的研究热点。为此,本文对近年来这方面的研究进展进行综述。通过介绍血红蛋白直接电化学界面设计的基本方法以及生物传感的原理和研究现状,以探索今后的发展趋势。     

Surface reconstitution of glucose oxidase onto a norbornylogous bridge self-assembled monolayer

An electrode construct was fabricated in which a self-assembled monolayer containing a novel norbornylogous bridge was covalently attached to flavin adenine dinucleotide (FAD), the redox active centre of several oxidase enzymes. The electrochemistry of the construct was investigated before and after the reconstitution of glucose oxidase around the surface bound FAD. Rapid rates of electron transfer were observed both before and after the reconstitution of biocatalytically active enzyme. However, no biocatalytic activity was observed under anaerobic conditions suggesting the a lack of enzyme turnover through direct electron transfer. It is proposed that a decrease in the electronic coupling between the redox active FAD and the electrode following reconstitution of the glucose oxidase – a probable consequence of the FAD being immersed in a protein environment – was responsible for the inability of the enzyme to be turned over under anaerobic conditions.     

Direct Electrochemistry of Cytochrome c Covalently Immobilized on ω-Carboxyalkanethiol Monolayer Electrode and its Biosensor Applications

The study of direct electron transfer (ET) between solid electrodes and proteins or enzymes has been attracting considerable research interest for several decades since it represents a basic feature for the application of biocatalysts in chemical sensors and other electrical devices. We have been focusing our research interest on the use of SAMs for the study of diffusionless, direct electrochemistry of cytochromes. In the present paper, we report electrochemistry of cytochrome c covalently immobilized on ω-carboxyalkanethiol monolayer electrodes. A carboxylic acid terminated monolayer was utilized to provide an uniform surface for attaching cytochrome c, and characterization of the redox reaction of the protein was made with using cyclic voltammetric and electrochemical impedance measurements.     

Fast second order electron transfer reactions coupled to redox protein electrochemistry: Experiment and digital simulation

Chemical modification of metal surfaces by chemisorption provides a versatile method for the production of electrode interfaces which can be selective for the direct electrochemistry of one redox protein over another. The electrochemistry of a mixture of horse heart cytochrome c and spinach plastocyanin has been investigated at gold surfaces made selective for first one and then the other protein. The resulting cyclic voltammetry is quite unusual, containing pre-shoulders to both reduction current and reoxidation current peaks. The results have been interpreted in terms of fast second order electron transfer reactions taking place between the two proteins in homogeneous solution. This rationalisation has been corroborated by an explicit digital simulation of the proposed reaction scheme, using second order RKI. There are three independently variable parameters to the simulation: forward kinetic parameter, reverse kinetic parameter, and concentration ratio of non-electrode-active species to electrode-active species. The simulation has been used to explore a number of interesting trends in these parameters. Five such sequences of simulated cyclic voltammograms are reported, together with peak current and potential data in most cases. Attention is drawn to the possibility for further interesting experimental mixed redox protein electrochemistry at selective surfaces.     

In situ and operando electrochemistry of redox enzymes

Understanding the biocatalytic or the interfacial electron transfer processes of redox enzymes is decisive to develop high-performance biofuel cells, mimetic catalysts, bioelectrosynthesis reactors, biosensors, and bioelectronic devices. The state-of-art of redox enzyme electrochemistry lies in using in situ and operando instrumentation, in which protein electrochemistry is resourcefully coupled to or hyphenated with numerous analytical techniques. Nevertheless, there is still a lot to research about the manipulation of redox proteins in the unusual sample holding environments, and bioelectrodes engineering emerges as a key. Here, we discuss these challenges in detail, focusing on contemporary instrumentation setups.     

Direct electrochemistry of glucose oxidase immobilized on NdPO4 nanoparticles/chitosan composite film on glassy carbon electrodes and its biosensing application

The direct electrochemistry of glucose oxidase (GOx) immobilized on a composite matrix based on chitosan (CHIT) and NdPO(4) nanoparticles (NPs) underlying on glassy carbon electrode (GCE) was achieved. The cyclic voltammetry and electrochemical impedance spectroscopy were used to characterize the modified electrode. In deaerated buffer solutions, the cyclic voltammetry of the composite films of GOx/NdPO(4) NPs/CHIT showed a pair of well-behaved redox peaks that are assigned to the redox reaction of GOx, confirming the effective immobilization of GOx on the composite film. The electron transfer rate constant was estimated to be 5.0 s(-1). The linear dynamic range for the detection of glucose was 0.15-10 mM with a correlation coefficient of 0.999 and the detection limit was estimated at about 0.08 mM (S/N=3). The calculated apparent Michaelis-Menten constant was 2.5 mM, which suggested a high affinity of the enzyme-substrate. The immobilized GOx in the NdPO(4) NPs/CHIT composite film retained its bioactivity. Furthermore, the method presented here can be easily extended to immobilize and obtain the direct electrochemistry of other redox enzymes or proteins.     

氧化还原蛋白质电化学研究*

研究氧化还原蛋白质与电极之间的电子传递过程不仅为理解代谢过程提供有价值的信息,而且为制备生物传感器奠定基础。本文从蛋白质修饰电极、蛋白质在电极表面的定向固定及蛋白质人工改造三方面,评述了近年来氧化还原蛋白质电化学研究的进展,并提出了今后可能的研究方向。     

碳纳米管促进氧化还原蛋白质和酶的直接电子转移

将血红蛋白(Hb)、辣根过氧化物酶(HRP)和葡萄糖氧化酶(GOx)分别固定在经碳纳米管修饰的玻碳电极(CNT/GC)上,制成Hb CNT/GC、HRP CNT/GC和GOx CNT/GC电极.Hb、HRP和GOx在CNT/GC电极表面均能发生有效和稳定的直接电子转移反应,其相应的循环伏安曲线均显示出一对几近对称的氧化还原峰;在60mV/s下,其式量电位E0'分别为-0.343V、-0.319V和-0.456V(vs.SCE,pH6.9),且不随扫速而变;以上三者在CNT/GC电极表面直接电子转移的表观速率常数ks依次为1.25±0.25、2.07±0.56和1.74±0.42s-1;根据式量电位E0'随缓冲溶液pH值的变化关系,确知在CNT/GC电极上,Hb或HRP发生的直接电化学遵从(1e+1H+)电极过程机理,而GOx发生的直接电化学反应则遵从(2e+2H+)机理.此外,固定在CNT/GC电极表面的Hb、HRP和GOx也同时表现出对各自底物的生物电催化活性.由本文制备的碳纳米管修饰电极及其固定生物蛋白质(酶)的方法具有简单、易于操作等优点,并可用于对其它生物氧化还原蛋白质和酶的直接电子转移测试.     

Protein adsorption on nanoporous TiO2 films: a novel approach to studying photoinduced protein/electrode transfer reactions

We have investigated the use of nanoporous TiO2 films as substrates for protein immobilisation. Such films are of interest due to their high surface area, optical transparency, electrochemical activity and ease of fabrication. These films moreover allow detailed spectroscopic study of protein/electrode electron transfer processes. We find that protein immobilisation on such films may be readily achieved from aqueous solutions at 4 degrees C with a high binding stability and no detectable protein denaturation. The nanoporous structure of the film greatly enhances the active surface area available for protein binding (by a factor of up to 850 for an 8 microns thick film). We demonstrate that the redox state of proteins such as immobilised cytochrome-c (Cyt-c) and haemoglobin (Hb) may be modulated by the application of an electrical bias potential to the TiO2 film, without the addition of electron transfer mediators. The binding of Cyt-c on the TiO2 films is investigated as a function of film thickness, protein concentration, protein surface charge and ionic strength. We demonstrate the potential use of immobilised Hb on such TiO2 films for the detection of dissolved CO in aqueous solutions. We further show that protein/electrode electron transfer may be initiated by UV bandgap excitation of the TiO2 electrode. Both photooxidation and photoreduction of the immobilised proteins can be achieved. By employing pulsed UV laser excitation, the interfacial electron transfer kinetics can be monitored by transient optical spectroscopy, providing a novel probe of protein/electrode electron transfer kinetics. We conclude that nanoporous TiO2 films may be useful both for basic studies of protein/electrode interactions and for the development of novel bioanalytical devices such as biosensors.     

Reconstitution of supramolecular organization involved in energy metabolism at electrochemical interfaces for biosensing and bioenergy production

How the redox proteins and enzymes involved in bioenergetic pathways are organized is a relevant fundamental question, but our understanding of this is still incomplete. This review provides a critical examination of the electrochemical tools developed in recent years to obtain knowledge of the intramolecular and intermolecular electron transfer processes involved in metabolic pathways. Furthermore, better understanding of the electron transfer processes associated with energy metabolism will provide the basis for the rational design of biotechnological devices such as electrochemical biosensors, enzymatic and microbial fuel cells, and hydrogen production factories. Starting from the redox complexes involved in two relevant bacterial chains, i.e., from the hyperthermophile Aquifex aeolicus and the acidophile Acidithiobacillus ferrooxidans, examination of protein–protein interactions using electrochemistry is first reviewed, with a focus on the orientation of a protein on an electrochemical interface mimic of a physiological interaction between two partners. Special attention is paid to current research in the electrochemistry of essential membrane proteins, which is one mandatory step toward the understanding of energy metabolic pathways. The complex and challenging architectures built to reconstitute a membrane-like environment at an electrode are especially considered. The role played by electrochemistry in the attempt to consider full bacterial metabolism is finally emphasized through the study of whole cells immobilized at electrodes as suspensions or biofilms. Before the performances of biotechnological devices can be further improved to make them really attractive, questions remain to be addressed in this particular field of research. We discuss the bottlenecks that need to be overcome in the future.     

Direct electrochemistry of heme multicofactor-containing enzymes on alkanethiol-modified gold electrodes

Direct electrochemistry of heme multicofactor-containing enzymes, e.g., microbial theophylline oxidase (ThOx) and D-fructose dehydrogenase (FDH) from Gluconobacter industrius was studied on alkanethiol-modified gold electrodes and was compared with that of some previously studied complex heme enzymes, specifically, cellobiose dehydrogenase (CDH) and sulphite oxidase (SOx). The formal redox potentials for enzymes in direct electronic communication varied for ThOx from -112 to -101 mV (vs. Ag|AgCl), at pH 7.0, and for FDH from -158 to -89 mV, at pH 5.0 and pH 4.0, respectively, on differently charged alkanethiol layers. Direct and mediated by cytochrome c electrochemistry of FDH correlated with the existence of two active centres in the protein structure, i.e., the heme and the pyrroloquinoline quinone (PQQ) prosthetic groups. The effect of the alkanethiols of different polarity and charge on the surface properties of the gold electrodes necessary for adsorption and orientation of ThOx, FDH, CDH and SOx, favourable for the efficient electrode-enzyme electron transfer reaction, is discussed.     

酶直接电化学与第三代生物传感器

本文详细地评述并展望了酶直接电化学与第三代生物传感器这个领域已取得的成果，主要内容涉及生物电催化的三个发展阶段，实现酶与电极之间的直接电子转移方法和相应机理、以及第三代酶传感器的研制。     

Sensing nitrite through a pseudoazurin-nitrite reductase electron transfer relay.

Nitrite is converted to nitric oxide by haem or copper-containing enzymes in denitrifying bacteria during the process of denitrification. In designing an efficient biosensor, this enzymic turnover must be quantitatively assessed. The enzyme nitrite reductase from Alcaligenes faecalis contains a redox-active blue copper centre and a nonblue enzyme-active copper centre. It can be covalently tethered to modified gold-electrode surfaces in configurations in which direct electron transfer is possible. A surface cysteine mutant of the enzyme can be similarly immobilised on bare electroactive gold substrates. Under such circumstances, however, electron transfer cannot be effectively coupled with substrate catalytic turnover. In using either the natural redox partner, pseudoazurin, or ruthenium hexammine as an "electron-shuttle" or "conduit" between enzyme and a peptide-modified electrode surface, the coupling of electron transfer to catalysis can be utilised in the development of an amperometric nitrite sensor.     

In situ STM imaging and direct electrochemistry of Pyrococcus furiosus ferredoxin assembled on thiolate-modified Au111 surfaces

We have addressed here electron transfer (ET) of Pyrococcus furiosus ferredoxin (PfFd, 7.5 kDa) in both homogeneous solution using edge plane graphite (EPG) electrodes and in the adsorbed state by electrochemistry on surface-modified single-crystal Au111 electrodes, This has been supported by surface microscopic structures of PfFd monolayers, as revealed by scanning tunneling microscopy under potential control (in situ STM). Direct ET between PfFd in phosphate buffer solution, pH 7.9, and EPG electrodes is observed in the presence of promoters. Neomycin gives rise to a pair of redox peaks with a formal potential of ca -430 mV (vs SCE), corresponding to [3Fe-4S]1+/0. The presence of an additional promoter, which can be propionic acid, alanine, or cysteine, induces a second pair of redox peaks at approximately -900 mV (vs SCE) arising from [3Fe-4S]0/1-. A robust neomycin-PfFd complex was detected by mass spectrometry. The results clearly favor an ET mechanism in which the promoting effect of small organic molecules is through formation of promoter-protein complexes. The interaction of PfFd with small organic molecules in homogeneous solution offers clues to confine the protein on the electrode surface modified by the same functional group monolayer and to address diffusionless direct electrochemistry, as well as surface microstructures of the protein monolayer. PfFd molecules were found to assemble on either mercaptopropionic acid (MPA) or cysteine-modified Au111 surfaces in stable monolayers or submonolayers. Highly ordered (2 radical 3 x 5)R30 degrees cluster structures with six MPA molecules in each cluster were found by in situ STM. Individual PfFd molecules on the MPA layer are well resolved by in situ STM. Under Ar protection reversible cyclic voltammograms were obtained on PfFd-MPA/Au111 and PfFd-cysteine/Au111 electrodes with redox potentials of -220 and -201 mV (vs SCE), respectively, corresponding to the [Fe3S4]1+/0 couple. These values are shifted positively by 200 mV relative to homogeneous solution due to interactions between the promoting layers and the protein molecules. Possible mechanisms for such interactions and their ET patterns are discussed.     

An interface comprising molecular wires and poly(ethylene glycol) spacer units self-assembled on carbon electrodes for studies of protein electrochemistry

The characterization and application of a modified electrode interface for protein electrochemistry is reported. This generic interface is composed of a mixed monolayer of oligo(phenylethynylene) molecular wires (MWs) and poly(ethylene glycol) (PEG) deposited on glassy carbon electrodes by reductive adsorption of the respective aryl diazonium salts. Electrochemistry and scanning electron microscopy demonstrate that the PEG component exhibits a distinct decrease in nonspecific adsorption of blood serum and the proteins bovine serum albumin (BSA) and horseradish peroxidase (HRP) relative to a bare glassy carbon electrode. The ability of the MWs to facilitate efficient electron transfer through the PEG layer to the underlying electrode was demonstrated by covalently attaching ferrocenemethylamine to the end of the MWs. The calculated rate constant for this system was 229 +/- 30 s(-1). Covalent attachment of HRP to the MWs allowed direct electron transfer to the redox protein with almost ideal electrochemistry, indicating a specific interaction between the MW and HRP, with a rate constant of 13.4 +/- 2.3 s(-1). This rate constant is more rapid than previously reported for HRP shown to still be catalytically active. Retained catalytic activity of HRP was demonstrated by the enzyme responding to the addition of hydrogen peroxide. Similarly, by attaching myoglobin to the end of the MWs, a rate constant for this protein of 2 s(-1) was measured. The rigidity of the MWs, as well as it being longer than the PEG diluent, means this generic interface can be employed to investigate the electrochemistry of a wide range of redox proteins.     

Topological and Electron‐Transfer Properties of Yeast Cytochrome c Adsorbed on Bare Gold Electrodes

The redox metalloprotein yeast cytochrome c was directly self‐chemisorbed on “bare” gold electrodes through the free sulfur‐containing group Cys102. Topological, spectroscopic, and electron transfer properties of the immobilised molecules were investigated by in situ scanning probe microscopy and cyclic voltammetry. Atomic force and scanning tunnelling microscopy revealed individual protein molecules adsorbed on the gold substrate, with no evidence of aggregates. The adsorbed proteins appear to be firmly bound to gold and display dimensions in good agreement with crystallographic data. Cyclic voltammetric analysis showed that up to 84 % of the electrode surface is functionalised with electroactive proteins whose measured redox midpoint potential is in good agreement with the formal potential. Our results clearly indicate that this variant of cytochrome c is adsorbed on bare gold electrodes with preservation of morphological properties and redox functionality.