Bioelectrocatalytic oxidation of glucose by hexose oxidase directly wired to graphite

Glucose-oxidizing enzymes are widely used in electrochemical biosensors and biofuel cells; in most applications glucose oxidase, an enzyme with non-covalently bound FAD and low capability of direct electronic communications with electrodes, is used. Here, we show that another glucose-oxidizing enzyme with a covalently bound FAD center, hexose oxidase (HOX), adsorbed on graphite, exhibits a pronounced non-catalytic voltammetric response from its FAD, at − 307 mV vs. Ag/AgCl, pH 7, characterized by the heterogeneous electron transfer (ET) rate constant of 29.2 ± 4.5 s^− 1. Direct bioelectrocatalytic oxidation of glucose by HOX proceeded, although, with a 350 mV overpotential relative to FAD signals, which may be connected with a limiting step in biocatalysis under conditions of the replacement of the natural redox partner, O₂, by the electrode; mediated bioelectrocatalysis was consistent with the potentials of a soluble redox mediator used. The results allow development of HOX-based electrochemical biosensors for sugar monitoring and biofuel cells exploiting direct ET of HOX, and, not the least, fundamental studies of ET non-complicated by the loss of FAD from the protein matrix.     

Electrochemical catalysis with redox polymer and polyion-protein films

Supramolecular redox-active assemblies on electrodes are of fundamental interest and can be used to create functioning devices such as sensors, biosensors, and bioreactors. The ability of redox-active films to mediate electron transfer reactions in 3-D dramatically increases the sensitivity with which target molecules can be determined. Metallopolyion hydrogel films immobilized on electrode surfaces exhibit many properties that are reminiscent of those shown by redox-active proteins. This review discusses the electrochemical properties and applications of such films, including mediating electron transfer between electrodes and oxidase enzymes. In addition, polyion-protein films grown layer by layer have certain advantages in device fabrication, including facilitating direct electron transfer for many proteins, mechanical stability, use of tiny amounts of protein, and control of film architecture. This review presents examples of iron heme proteins in films grown layer by layer by alternate electrostatic adsorption for catalytic reduction of hydrogen peroxide and trichloroacetic acid and for oxidation of styrene.     

Bioelectroanalysis with nanoelectrode ensembles and arrays

This review deals with recent advances in bioelectroanalytical applications of nanostructured electrodes, in particular nanoelectrode ensembles (NEEs) and arrays (NEAs). First, nanofabrication techniques, principles of function, and specific advantages and limits of NEEs and NEAs are critically discussed. In the second part, some recent examples of bioelectroanalytical applications are presented. These include use of nanoelectrode arrays and/or ensembles for direct electrochemical analysis of pharmacologically active organic compounds or redox proteins, and the development of functionalized nanoelectrode systems and their use as catalytic or affinity electrochemical biosensors.     

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.     

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。     

蛋白质直接电化学研究及其应用

蛋白质直接电化学研究在生物电化学中具有重要地位,对于蛋白质结构．功能研究、蛋白质电子传递过程的热力学和动力学研究都有着重要意义,而且是研制第三代电化学生物传感器的基础。本文对在裸电极、分子自组装修饰电极和模拟生物膜修饰电极上进行蛋白质直接电化学的研究及相关应用进行简要综述。     

蛋白质直接电化学研究及其应用

蛋白质直接电化学研究在生物电化学中具有重要地位,对于蛋白质结构-功能研究、蛋白质电子传递过程的热力学和动力学研究都有着重要意义,而且是研制第三代电化学生物传感器的基础。本文对在裸电极、分子自组装修饰电极和模拟生物膜修饰电极上进行蛋白质直接电化学的研究及相关应用进行简要综述。     

Membrane-bound dehydrogenases from Gluconobacter sp.: Interfacial electrochemistry and direct bioelectrocatalysis

Although membrane-bound dehydrogenases isolated from Gluconobacter sp. (mainly PQQ-dependent alcohol and fructose dehydrogenase) have been used for preparing diverse forms of bioelectronic interfaces for almost 2 decades, it is not an easy task to interpret an electrochemical behaviour correctly. Recent discoveries regarding redox properties of membrane-bound dehydrogenases along with extensive investigations of direct electron transfer (DET) or direct bioelectrocatalysis with these enzymes are summarized in this review. The main aim of this review is to draw general conclusions about possible electronic coupling paths of these enzymes on various interfaces via direct electron transfer or direct bioelectrocatalysis. A short overview of the metabolism and respiration chain in Gluconobacter relevant to interfacial electrochemistry is given. Biosensor devices based on DET or direct bioelectrocatalysis using membrane-bound dehydrogenases from Gluconobacter sp. are described briefly with the emphasis given on practical applications of preparing enzymatic biofuel cells. Moreover, interfacial electrochemistry of Gluconobacter oxydans related to the construction of microbial biofuel cells is also discussed.     

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.     

Electrochemistry and electroanalytical applications of carbon nanotubes: a review.

This review addresses recent developments in electrochemistry and electroanalytical chemistry of carbon nanotubes (CNTs). CNTs have been proved to possess unique electronic, chemical and structural features that make them very attractive for electrochemical studies and electrochemical applications. For example, the structural and electronic properties of the CNTs endow them with distinct electrocatalytic activities and capabilities for facilitating direct electrochemistry of proteins and enzymes from other kinds of carbon materials. These striking electrochemical properties of the CNTs pave the way to CNT-based bioelectrochemistry and to bioelectronic nanodevices, such as electrochemical sensors and biosensors. The electrochemistry and bioelectrochemistry of the CNTs are summarized and discussed, along with some common methods for CNT electrode preparation and some recent advances in the rational functionalization of the CNTs for electroanalytical applications.     

介孔分子筛上的蛋白质直接电化学

将介孔分子筛用于不同含血红素蛋白质的直接电子传递研究,分别研究了辣根过氧化物酶、血红蛋白和肌红蛋白在六方介孔硅(HMS)上的直接电化学,探讨了介孔分子筛与这些蛋白质间的相互作用,构建了过氧化氢和亚硝酸根的新型的生物传感器. 这些工作扩展了HMS在蛋白质固定、直接电子传递研究和无试剂生物传感器制备方面的应用.     

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.     

Engineering of glucose oxidase for direct electron transfer via site-specific gold nanoparticle conjugation

Optimizing the electrical communication between enzymes and electrodes is critical in the development of biosensors, enzymatic biofuel cells, and other bioelectrocatalytic applications. One approach to address this limitation is the attachment of redox mediators or relays to the enzymes. Here we report a simple genetic modification of a glucose oxidase enzyme to display a free thiol group near its active site. This facilitates the site-specific attachment of a maleimide-modified gold nanoparticle to the enzyme, which enables direct electrical communication between the conjugated enzyme and an electrode. Glucose oxidase is of particular interest in biofuel cell and biosensor applications, and the approach of "prewiring" enzyme conjugates in a site-specific manner will be valuable in the continued development of these systems.     

Spectroscopic analysis of immobilised redox enzymes under direct electrochemical control

This article reviews recent developments in spectroscopic analysis of electrode-immobilised enzymes under direct, unmediated electrochemical control. These methods unite the suite of spectroscopic methods available for characterisation of structural, electronic and coordination changes in proteins with the exquisite control over complex redox enzymes that can be achieved in protein film electrochemistry in which immobilised protein molecules exchange electrons directly with an electrode. This combination is particularly powerful in studies of highly active enzymes where redox states can be controlled even under fast electrocatalytic turnover. We examine examples in which UV-visible, IR, Raman and MCD spectroscopy have been combined with direct electrochemistry to probe redox-dependent chemistry, and consider future opportunities for 'direct' spectroelectrochemistry of immobilised enzymes.     

Electrochemistry of Nucleic Acids at Solid Electrodes and Its Applications

The knowledge of the redox chemistry of nucleic acids (NA) is of paramount importance in cancer and aging research. Charge migration through DNA is also involved in biologically relevant functions such as DNA damage and repair. In the first part of this article the main aspects of the electrochemistry of nucleic acids at solid electrodes are revised, including redox processes, photoelectroactivity and electrical conductivity. In the second part, an overview of its applications is presented. Methods for electrochemical detection of NA, NA‐based biosensors for detection of nonnucleic acid molecules, studies on the nature and dynamics of interactions and structural conformations of NA, are some applications that take advantage of NA electrochemistry at solid electrodes.     

Nucleobase modification as redox DNA labelling for electrochemical detection

Basic aspects of DNA electrochemistry with a strong focus on the use of modified nucleobases as redox probes for electrochemical bioanalysis are reviewed. Intrinsic electrochemical properties of nucleobases in combination with artificial redox-active nucleobase modifications are frequently applied in this field. Synthetic approaches (both chemical and enzymatic) to base-modified nucleic acids are briefly summarized and their applications in redox labelling are discussed. Finally, analytical applications including DNA hybridization, primer extension, PCR, SNP typing, DNA damage and DNA-protein interaction analysis are presented (critical review, 91 references).     

Charge propagation in "ion channel sensors" based on protein-modified electrodes and redox marker ions

The mechanism of charge propagation in "ion channel sensors" (ICSs) consisting of gold electrodes modified with a layer of charged proteins and highly charged redox-active marker ions in solution was investigated by electrochemical techniques, QCM and AFM. The study is based on seven proteins (concanavalin A, cytochrome c, glucose oxidase, lysozyme, thyroglobulin, catalase, aldolase, and EF1-ATPase) in combination with seven electroactive marker ions ([Fe(CN)6]3-, [Fe(CN)6]4-, [Ru(NH3)6]3+, mono-, di-, and trimeric viologens), as well as a series of suppressor and enhancer ions leading to the following general statements: (i) electrostatic binding of charged marker ions to the domains of the protein is a prerequisite for an electrochemical current and (ii) charge propagation through the layer consists of electron hopping along surface-confined marker ions into the pores between adsorbed proteins. It is further shown that (iii) marker ions and suppressor ions with identical charge compete for oppositely charged sites on the protein domain, (iv) electrostatically bound multilayers of marker or enhancer ions with alternating charge form on a charged protein domain, and (v) self-exchange and exergonic ET catalysis between adsorbed marker ions and marker ions in solution take place. In addition to fundamental insight into the mechanism of charge propagation, valuable information for the design, optimization, and tailoring of new biosensors based on the ICS concept is demonstrated by the current findings.     

氧化还原蛋白质在模拟生物膜修饰电极上的直接电化学

评述了氧化还原蛋白在模拟生物膜这种新型的化学修饰电极上的直接电化学研究的进展。对蛋白质在表面活性剂薄膜电极和多层复合薄膜电极上的电化学行为、模拟生物膜的超分子结构以及蛋白质在该类薄膜修饰电极上对不同底物的电催化性质进行了较详细的介绍。     

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.