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Graphene and its variants exhibit excellent electrical properties for the construction of enzymatic interfaces. In particular, the direct electron transfer of glucose oxidase on the electrode surface is a very important issue in the development of enzyme-based bioelectrodes. However, the number of studies conducted to assess how pristine graphene forms different interfaces with other carbon materials is insufficient. Enzyme-based electrodes (formed using carbon materials) have been extensively applied because of their low manufacturing costs and easy production techniques. In this study, the characteristics of a single-walled carbon nanotube/graphene-combined enzyme interface are analyzed at the atomic level using molecular dynamics simulations. The morphology of the enzyme was visualized using an elastic network model by performing normal-mode analysis based on electrochemical and microscopic experiments. Single-carbon electrodes exhibited poorer electrical characteristics than those prepared as composites with enzymes. Furthermore, the composite interface exhibited 4.61- and 2.45-fold higher direct electron efficiencies than GOx synthesized with single-carbon nanotubes and graphene, respectively. Based on this study, we propose that pristine graphene has the potential to develop glucose oxidase interfaces and carbon-nanotube–graphene composites for easy fabrication, low cost, and efficient electrode structures for enzyme-based biofuel cells. [ABSTRACT FROM AUTHOR]

A disposable paper-based glucose biosensor with direct electron transfer (DET) of glucose oxidase (GOX) was developed through simple covalent immobilization of GOX on a carbon electrode surface using zero-length cross-linkers. This glucose biosensor exhibited a high electron transfer rate (ks, 3.363 s−1) as well as good affinity (km, 0.03 mM) for GOX while keeping innate enzymatic activities. Furthermore, the DET-based glucose detection was accomplished by employing both square wave voltammetry and chronoamperometric techniques, and it achieved a glucose detection range from 5.4 mg/dL to 900 mg/dL, which is wider than most commercially available glucometers. This low-cost DET glucose biosensor showed remarkable selectivity, and the use of the negative operating potential avoided interference from other common electroactive compounds. It has great potential to monitor different stages of diabetes from hypoglycemic to hyperglycemic states, especially for self-monitoring of blood glucose. [ABSTRACT FROM AUTHOR]

A glucose biosensor was layer-by-layer assembled on a modified glassy carbon electrode (GCE) from a nanocomposite of NAD(P)+-dependent glucose dehydrogenase, aminated polyethylene glycol (mPEG), carboxylic acid-functionalized multi-wall carbon nanotubes (fMWCNTs), and ionic liquid (IL) composite functional polymers. The electrochemical electrode was denoted as NF/IL/GDH/mPEG-fMWCNTs/GCE. The composite polymer membranes were characterized by cyclic voltammetry, ultraviolet-visible spectrophotometry, electrochemical impedance spectroscopy, scanning electron microscopy, and transmission electron microscopy. The cyclic voltammogram of the modified electrode had a pair of well-defined quasi-reversible redox peaks with a formal potential of −61 mV (vs. Ag/AgCl) at a scan rate of 0.05 V s−1. The heterogeneous electron transfer constant (ks) of GDH on the composite functional polymer-modified GCE was 6.5 s−1. The biosensor could sensitively recognize and detect glucose linearly from 0.8 to 100 µM with a detection limit down to 0.46 μM (S/N = 3) and a sensitivity of 29.1 nA μM−1. The apparent Michaelis–Menten constant ( K m app ) of the modified electrode was 0.21 mM. The constructed electrochemical sensor was compared with the high-performance liquid chromatography method for the determination of glucose in commercially available glucose injections. The results demonstrated that the sensor was highly accurate and could be used for the rapid and quantitative determination of glucose concentration. [ABSTRACT FROM AUTHOR]

The electrochemical enzyme sensors based on direct electron transfer (DET)-type oxidoreductase-based enzymes are ideal for continuous and in vivo monitoring. However, the number and types of DET-type oxidoreductases are limited. The aim of this research is the development of a versatile method to create a DET-type oxidoreductase complex based on the SpyCatcher/SpyTag technique by preparing SpyCatcher-fused heme c and SpyTag-fused non-DET-type oxidoreductases, and by the in vitro formation of DET-type oxidoreductase complexes. A heme c containing an electron transfer protein derived from Rhizobium radiobacter (CYTc) was selected to prepare SpyCatcher-fused heme c. Three non-DET-type oxidoreductases were selected as candidates for the SpyTag-fused enzyme: fungi-derived flavin adenine dinucleotide (FAD)-dependent glucose dehydrogenase (GDH), an engineered FAD-dependent d-amino acid oxidase (DAAOx), and an engineered FMN-dependent l-lactate oxidase (LOx). CYTc-SpyCatcher (CYTc-SC) and SpyTag-Enzymes (ST-GDH, ST-DAAOx, ST-LOx) were prepared as soluble molecules while maintaining their redox properties and catalytic activities, respectively. CYTc-SC/ST-Enzyme complexes were formed by mixing CYTc-SpyCatcher and SpyTag-Enzymes, and the complexes retained their original enzymatic activity. Remarkably, the heme domain served as an electron acceptor from complexed enzymes by intramolecular electron transfer; consequently, all constructed CYTc-SC/ST-Enzyme complexes showed DET ability to the electrode, demonstrating the versatility of this method. [ABSTRACT FROM AUTHOR]

This review focuses on the development of microbial biofuel cells to demonstrate how similar principles apply to the development of bioelectronic devices. The low specificity of microorganism-based amperometric biosensors can be exploited in designing microbial biofuel cells, enabling them to consume a broader range of chemical fuels. Charge transfer efficiency is among the most challenging and critical issues while developing biofuel cells. Nanomaterials and particular redox mediators are exploited to facilitate charge transfer between biomaterials and biofuel cell electrodes. The application of conductive polymers (CPs) can improve the efficiency of biofuel cells while CPs are well-suitable for the immobilization of enzymes, and in some specific circumstances, CPs can facilitate charge transfer. Moreover, biocompatibility is an important issue during the development of implantable biofuel cells. Therefore, biocompatibility-related aspects of conducting polymers with microorganisms are discussed in this review. Ways to modify cell-wall/membrane and to improve charge transfer efficiency and suitability for biofuel cell design are outlined. [ABSTRACT FROM AUTHOR]

Nitrate (NO3−-N) and nitrites (NO2−-N) are common pollutants in various water bodies causing serious threats not only to aquatic, but also to animals and human beings. In this study, we developed a strategy for efficiently reducing nitrates in microbial fuel cells (MFCs) powered by a granular activated carbon (GAC)-biocathode. GAC was developed by acclimatizing and enriching denitrifying bacteria under a redox potential (0.3 V) generated from MFCs. Thus, using the formed GAC-biocathode we continued to study their effect on denitrification with different cathode materials and circulation speeds in MFCs. The GAC-biocathode with its excellent capacitive property can actively reduce nitrate for over thirty days irrespective of the cathode material used. The stirring speed of GAC in the cathode showed a steady growth in potential generation from 0.25 V to 0.33 V. A rapid lag phase was observed when a new carbon cathode was used with enriched GAC. While a slow lag phase was seen when a stainless-steel cathode was replaced. These observations showed that effective storage and supply of electrons to the GAC plays a crucial role in the reduction process in MFCs. Electrochemical analysis of the GAC properties studied using electrochemical impedance spectroscopy (EIS), cyclic voltammetry (CV), and zeta potential showed distinct properties with different abiotic and biocathode conditions. We found that the enrichment of electrotrophic bacteria on GAC facilitates the direct electron transfer in the cathode chamber for reducing NO3−-N in MFCs as observed by scanning electron microscopy. [ABSTRACT FROM AUTHOR]

Enzymatic biosensors based on glucose oxidase has been proven to be one of the effective strategies for the detection of glucose and contributed to health improvements. Therefore, research and debates to date are ongoing in an attempt to find the most effective way to detect this analyte using this enzyme as the recognition center. The 3rd generation biosensors using direct electron transfer (DET) type enzymes are a great way towards practical devices. In this work, we developed a simple method for the functionalization of glucose oxidase with redoxable ferrocene groups in chloroform. The enzyme retained its activity after storage in this organic solvent and after the functionalization procedures. This enzyme functionalization strategy was employed to develop the biosensing monolayer-based platforms for the detection of glucose utilizing the quasi-DET mechanism. As a result of an electrochemical regeneration of the catalytic center, the formation of harmful H2O2 is minimized during enzymatic electrocatalysis. [ABSTRACT FROM AUTHOR]

Enzymatic Biofuel Cell (EBC) represents a promising green source since it is capable of harvesting electricity from renewable and abundantly available biofuels using enzymes as catalysts. Nevertheless, nowadays long-term stability and low power output are currently the main concerns. To this end, several research studies focus on using complex tridimensional and highly expensive nanostructures as electrode support for enzymes. This increases cell performance whilst drastically reducing the economic feasibility needed for industrial viability. Thus, this paper analyzes a novel flow-based EBC consisting of covalent immobilized GOx (bioanode) and Laccase (biocathode) on a commercial flat conductive polymer. A suitable immobilization technique based on covalent ligands is carried out to enhance EBC durability. The experimental characterization demonstrates that the cell generates power over three weeks, reaching 590 mV and 2.41 µW cm−2 as maximum open circuit voltage and power density, respectively. The most significant contributions of this configuration are definitely ease of implementation, low cost, high scalability, and reproducibility. Therefore, such a design can be considered a step forward in the viable EBC industrialization process for a wide range of applications. [ABSTRACT FROM AUTHOR]

The FAD-dependent glucose dehydrogenase from Burkholderia cepacia (FADGDH) is a hetero-oligomeric enzyme that is capable of direct electron transfer (DET) with an electrode. The cytochrome c (cyt c) subunit, which possesses three hemes (heme 1, heme 2, and heme 3, from the N-terminal sequence), is known to enable DET; however, details of the electron transfer pathway remain unknown. A mutagenesis investigation of the heme axial ligands was carried out to elucidate the electron transfer pathway to the electron mediators and/or the electrode. The sixth axial ligand for each of the three heme irons, Met109, Met263, and Met386 were substituted with His. The catalytic activities of the wild-type (WT) and mutant enzymes were compared by investigating their dye-mediated dehydrogenase activities and their DET abilities toward the electrode. The results suggested that (1) heme 1 with Met109 as an axial ligand is mainly responsible for the electron transfer with electron acceptors in the solution, but not for the DET with the electrode; (2) heme 2 with Met263 is responsible for the DET-type reaction with the electrode; and (3) heme 3 with Met386 seemed to be the electron acceptor from the catalytic subunit. From these results, two electron transfer pathways were proposed depending on the electron acceptors. Electrons are transferred from the catalytic subunit to heme 3, then to heme 2, to heme 1 and, finally, to electron acceptors in solution. However, if the enzyme complex is immobilized on the electrode and is used as electron acceptors, electrons are passed to the electrode from heme 2. [ABSTRACT FROM AUTHOR]

An impedimetric-based biosensor constructed using gold nanoparticles (AuNP) entrapped within titanium dioxide (TiO2) particles for hydrogen peroxide (H2O2) detection is the main feature of this research. The matrix of the biosensor employed the surface of TiO2, which was previously modified with an amine terminal group using 3-Aminopropyltriethoxysilane (APTS) at a low temperature to create a ready to immobilise surface for the biosensor application. Hemoglobin (Hb), which exhibits peroxidase-like activity, was used as the bioreceptor in the biosensor to detect H2O2 in solution. The analysis was carried out using an alternative impedance method, in which the biosensor exhibited a wide linear range response between 1 × 10-4 M and 1.5 × 10-2 M and a limit of detection (LOD) of 1 × 10-5 M without a redox mediator. [ABSTRACT FROM AUTHOR]

For the first time a molecularly imprinted polymer (MIP) with direct electron transfer (DET) and bioelectrocatalytic activity of the target protein is presented. Thin films of MIPs for the recognition of a hexameric tyrosine-coordinated heme protein (HTHP) have been prepared by electropolymerization of scopoletin after oriented assembly of HTHP on a self-assembled monolayer (SAM) of mercaptoundecanoic acid (MUA) on gold electrodes. Cavities which should resemble the shape and size of HTHP were formed by template removal. Rebinding of the target protein sums up the recognition by non-covalent interactions between the protein and the MIP with the electrostatic attraction of the protein by the SAM. HTHP bound to the MIP exhibits quasi-reversible DET which is reflected by a pair of well pronounced redox peaks in the cyclic voltammograms (CVs) with a formal potential of -184.4 ± 13.7 mV vs. Ag/AgCl (1 M KCl) at pH 8.0 and it was able to catalyze the cathodic reduction of peroxide. At saturation the MIP films show a 12-fold higher electroactive surface concentration of HTHP than the non-imprinted polymer (NIP). [ABSTRACT FROM AUTHOR]

A DNA/chitosan-Fe3O4 magnetic nanoparticle bio-complex film was constructed for the immobilization of horseradish peroxidase (HRP) on a glassy carbon electrode. HRP was simply mixed with DNA, chitosan and Fe3O4 nanoparticles, and then applied to the electrode surface to form an enzyme-incorporated polyion complex film. Scanning electron microscopy (SEM) was used to study the surface features of DNA/chitosan/Fe3O4/HRP layer. The results of electrochemical impedance spectroscopy (EIS) show that Fe3O4 and enzyme were successfully immobilized on the electrode surface by the DNA/chitosan bio-polyion complex membrane. Direct electron transfer (DET) and bioelectrocatalysis of HRP in the DNA/chitosan/Fe3O4 film were investigated by cyclic voltammetry (CV) and constant potential amperometry. The HRP-immobilized electrode was found to undergo DET and exhibited a fast electron transfer rate constant of 3.7 s-1. The CV results showed that the modified electrode gave rise to well-defined peaks in phosphate buffer, corresponding to the electrochemical redox reaction between HRP(Fe(III)) and HRP(Fe(II)). The obtained electrode also displayed an electrocatalytic reduction behavior towards H2O2. The resulting DNA/chitosan/Fe3O4/HRP/glassy carbon electrode (GCE) shows a high sensitivity (20.8 A·cm-2·M-1) toward H2O2. A linear response to H3O2 measurement was obtained over the range from 2 μM to 100 μM (R2 = 0.99) and an amperometric detection limit of 1 μM (S/N = 3). The apparent Michaelis-Menten constant of HRP immobilized on the electrode was 0.28 mM. Furthermore, the electrode exhibits both good operational stability and storage stability. [ABSTRACT FROM AUTHOR]

As electrode nanomaterials, thermally reduced graphene oxide (TRGO) and modified gold nanoparticles (AuNPs) were used to design bioelectrocatalytic systems for reliable D-tagatose monitoring in a long-acting bioreactor where the valuable sweetener D-tagatose was enzymatically produced from a dairy by-product D-galactose. For this goal D-fructose dehydrogenase (FDH) from Gluconobacter industrius immobilized on these electrode nanomaterials by forming three amperometric biosensors: AuNPs coated with 4-mercaptobenzoic acid (AuNP/4-MBA/FDH) or AuNPs coated with 4-aminothiophenol (AuNP/PATP/FDH) monolayer, and a layer of TRGO on graphite (TRGO/FDH) were created. The immobilized FDH due to changes in conformation and spatial orientation onto proposed electrode surfaces catalyzes a direct D-tagatose oxidation reaction. The highest sensitivity for D-tagatose of 0.03 ± 0.002 μA mM−1cm−2 was achieved using TRGO/FDH. The TRGO/FDH was applied in a prototype bioreactor for the quantitative evaluation of bioconversion of D-galactose into D-tagatose by L-arabinose isomerase. The correlation coefficient between two independent analyses of the bioconversion mixture: spectrophotometric and by the biosensor was 0.9974. The investigation of selectivity showed that the biosensor was not active towards D-galactose as a substrate. Operational stability of the biosensor indicated that detection of D-tagatose could be performed during six hours without loss of sensitivity. [ABSTRACT FROM AUTHOR]

Here, we report the development of a novel photoactive biomolecular nanoarchitecture based on the genetically engineered extremophilic photosystem I (PSI) biophotocatalyst interfaced with a single layer graphene via pyrene-nitrilotriacetic acid self-assembled monolayer (SAM). For the oriented and stable immobilization of the PSI biophotocatalyst, an His6-tag was genetically engineered at the N-terminus of the stromal PsaD subunit of PSI, allowing for the preferential binding of this photoactive complex with its reducing side towards the graphene monolayer. This approach yielded a novel robust and ordered nanoarchitecture designed to generate an efficient direct electron transfer pathway between graphene, the metal redox center in the organic SAM and the photo-oxidized PSI biocatalyst. The nanosystem yielded an overall current output of 16.5 µA·cm−2 for the nickel- and 17.3 µA·cm−2 for the cobalt-based nanoassemblies, and was stable for at least 1 h of continuous standard illumination. The novel green nanosystem described in this work carries the high potential for future applications due to its robustness, highly ordered and simple architecture characterized by the high biophotocatalyst loading as well as simplicity of manufacturing. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

This review focuses on the overview of microbial amperometric biosensors and microbial biofuel cells (MFC) and shows how very similar principles are applied for the design of both types of these bioelectronics-based devices. Most microorganism-based amperometric biosensors show poor specificity, but this drawback can be exploited in the design of microbial biofuel cells because this enables them to consume wider range of chemical fuels. The efficiency of the charge transfer is among the most challenging and critical issues during the development of any kind of biofuel cell. In most cases, particular redox mediators and nanomaterials are applied for the facilitation of charge transfer from applied biomaterials towards biofuel cell electrodes. Some improvements in charge transfer efficiency can be achieved by the application of conducting polymers (CPs), which can be used for the immobilization of enzymes and in some particular cases even for the facilitation of charge transfer. In this review, charge transfer pathways and mechanisms, which are suitable for the design of biosensors and in biofuel cells, are discussed. Modification methods of the cell-wall/membrane by conducting polymers in order to enhance charge transfer efficiency of microorganisms, which can be potentially applied in the design of microbial biofuel cells, are outlined. The biocompatibility-related aspects of conducting polymers with microorganisms are summarized. [ABSTRACT FROM AUTHOR]

Heme proteins take part in a number of fundamental biological processes, including oxygen transport and storage, electron transfer, catalysis and signal transduction. The redox chemistry of the heme iron and the biochemical diversity of heme proteins have led to the development of a plethora of biotechnological applications. This work focuses on biosensing devices based on heme proteins, in which they are electronically coupled to an electrode and their activity is determined through the measurement of catalytic currents in the presence of substrate, i.e., the target analyte of the biosensor. After an overview of the main concepts of amperometric biosensors, we address transduction schemes, protein immobilization strategies, and the performance of devices that explore reactions of heme biocatalysts, including peroxidase, cytochrome P450, catalase, nitrite reductase, cytochrome c oxidase, cytochrome c and derived microperoxidases, hemoglobin, and myoglobin. We further discuss how structural information about immobilized heme proteins can lead to rational design of biosensing devices, ensuring insights into their efficiency and long-term stability. [ABSTRACT FROM AUTHOR]

Charge transfer (CT) is a very important issue in the design of biosensors and biofuel cells. Some nanomaterials can be applied to facilitate the CT in these bioelectronics-based devices. In this review, we overview some CT mechanisms and/or pathways that are the most frequently established between redox enzymes and electrodes. Facilitation of indirect CT by the application of some nanomaterials is frequently applied in electrochemical enzymatic biosensors and biofuel cells. More sophisticated and still rather rarely observed is direct charge transfer (DCT), which is often addressed as direct electron transfer (DET), therefore, DCT/DET is also targeted and discussed in this review. The application of conducting polymers (CPs) for the immobilization of enzymes and facilitation of charge transfer during the design of biosensors and biofuel cells are overviewed. Significant attention is paid to various ways of synthesis and application of conducting polymers such as polyaniline, polypyrrole, polythiophene poly(3,4-ethylenedioxythiophene). Some DCT/DET mechanisms in CP-based sensors and biosensors are discussed, taking into account that not only charge transfer via electrons, but also charge transfer via holes can play a crucial role in the design of bioelectronics-based devices. Biocompatibility aspects of CPs, which provides important advantages essential for implantable bioelectronics, are discussed. [ABSTRACT FROM AUTHOR]

Interfacial electron transfer between redox enzymes and electrodes is a key step for enzymatic bioelectrocatalysis in various bioelectrochemical devices. Although the use of carbon nanomaterials enables an increasing number of redox enzymes to carry out bioelectrocatalysis involving direct electron transfer (DET), the role of carbon nanomaterials in interfacial electron transfer remains unclear. Based on the recent progress reported in the literature, in this mini review, the significance of carbon nanomaterials on DET-type bioelectrocatalysis is discussed. Strategies for the oriented immobilization of redox enzymes in rationally modified carbon nanomaterials are also summarized and discussed. Furthermore, techniques to probe redox enzymes in carbon nanomaterials are introduced. [ABSTRACT FROM AUTHOR]

Self-assembled molecular monolayers (SAMs) have long been recognized as crucial "bridges" between redox enzymes and solid electrode surfaces, on which the enzymes undergo direct electron transfer (DET)—for example, in enzymatic biofuel cells (EBFCs) and biosensors. SAMs possess a wide range of terminal groups that enable productive enzyme adsorption and fine-tuning in favorable orientations on the electrode. The tunneling distance and SAM chain length, and the contacting terminal SAM groups, are the most significant controlling factors in DET-type bioelectrocatalysis. In particular, SAM-modified nanostructured electrode materials have recently been extensively explored to improve the catalytic activity and stability of redox proteins immobilized on electrochemical surfaces. In this report, we present an overview of recent investigations of electrochemical enzyme DET processes on SAMs with a focus on single-crystal and nanoporous gold electrodes. Specifically, we consider the preparation and characterization methods of SAMs, as well as SAM applications in promoting interfacial electrochemical electron transfer of redox proteins and enzymes. The strategic selection of SAMs to accord with the properties of the core redox protein/enzymes is also highlighted. [ABSTRACT FROM AUTHOR]

Electrochemical methods offer the simple characterization of the synthesis of molecularly imprinted polymers (MIPs) and the readouts of target binding. The binding of electroinactive analytes can be detected indirectly by their modulating effect on the diffusional permeability of a redox marker through thin MIP films. However, this process generates an overall signal, which may include nonspecific interactions with the nonimprinted surface and adsorption at the electrode surface in addition to (specific) binding to the cavities. Redox-active low-molecular-weight targets and metalloproteins enable a more specific direct quantification of their binding to MIPs by measuring the faradaic current. The in situ characterization of enzymes, MIP-based mimics of redox enzymes or enzyme-labeled targets, is based on the indication of an electroactive product. This approach allows the determination of both the activity of the bio(mimetic) catalyst and of the substrate concentration. [ABSTRACT FROM AUTHOR]

Glucose oxidase (GOx) has been widely utilized for monitoring glycemic levels due to its availability, high activity, and specificity toward glucose. Among the three generations of electrochemical glucose sensor principles, direct electron transfer (DET)-based third-generation sensors are considered the ideal principle since the measurements can be carried out in the absence of a free redox mediator in the solution without the impact of oxygen and at a low enough potential for amperometric measurement to avoid the effect of electrochemically active interferences. However, natural GOx is not capable of DET. Therefore, a simple and rapid strategy to create DET-capable GOx is desired. In this study, we designed engineered GOx, which was made readily available for single-step modification with a redox mediator (phenazine ethosulfate, PES) on its surface via a lysine residue rationally introduced into the enzyme. Thus, PES-modified engineered GOx showed a quasi-DET response upon the addition of glucose. This strategy and the obtained results will contribute to the further development of quasi-DET GOx-based glucose monitoring dedicated to precise and accurate glycemic control for diabetic patient care. [ABSTRACT FROM AUTHOR]

Direct electron transfer (DET)-capable oxidoreductases are enzymes that have the ability to transfer/receive electrons directly to/from solid surfaces or nanomaterials, bypassing the need for an additional electron mediator. More than 100 enzymes are known to be capable of working in DET conditions; however, to this day, DET-capable enzymes have been mainly used in designing biofuel cells and biosensors. The rapid advance in (semi) conductive nanomaterial development provided new possibilities to create enzyme-nanoparticle catalysts utilizing properties of DET-capable enzymes and demonstrating catalytic processes never observed before. Briefly, such nanocatalysts combine several cathodic and anodic catalysis performing oxidoreductases into a single nanoparticle surface. Hereby, to the best of our knowledge, we present the first review concerning such nanocatalytic systems involving DET-capable oxidoreductases. We outlook the contemporary applications of DET-capable enzymes, present a principle of operation of nanocatalysts based on DET-capable oxidoreductases, provide a review of state-of-the-art (nano) catalytic systems that have been demonstrated using DET-capable oxidoreductases, and highlight common strategies and challenges that are usually associated with those type catalytic systems. Finally, we end this paper with the concluding discussion, where we present future perspectives and possible research directions. [ABSTRACT FROM AUTHOR]

One of the processes most studied in bioenergetic systems in recent years is the oxygen reduction reaction (ORR). An important challenge in bioelectrochemistry is to achieve this reaction under physiological conditions. In this study, we used bilirubin oxidase (BOD) from Myrothecium verrucaria, a subclass of multicopper oxidases (MCOs), to catalyse the ORR to water via four electrons in physiological conditions. The active site of BOD, the T2/T3 cluster, contains three Cu atoms classified as T2, T3α, and T3β depending on their spectroscopic characteristics. A fourth Cu atom; the T1 cluster acts as a relay of electrons to the T2/T3 cluster. Graphite electrodes were modified with BOD and the direct electron transfer (DET) to the enzyme, and the mediated electron transfer (MET) using an osmium polymer (OsP) as a redox mediator, were compared. As a result, an alternative resting (AR) form was observed in the catalytic cycle of BOD. In the absence and presence of the redox mediator, the AR direct reduction occurs through the trinuclear site (TNC) via T1, specifically activated at low potentials in which T2 and T3α of the TNC are reduced and T3β is oxidized. A comparative study between the DET and MET was conducted at various pH and temperatures, considering the influence of inhibitors like H2O2, F−, and Cl−. In the presence of H2O2 and F−, these bind to the TNC in a non-competitive reversible inhibition of O2. Instead; Cl− acts as a competitive inhibitor for the electron donor substrate and binds to the T1 site. [ABSTRACT FROM AUTHOR]

In this paper, we present a non-fluidic microsystem for the simultaneous visualization and electrochemical evaluation of confined, growing bacteria on solid media. Using a completely automated platform, real-time monitoring of bacterial and image-based computer characterization of growth were performed. Electrochemical tests, using Escherichia coli K-12 as the model microorganism, revealed the development of a faradaic process at the bacteria–microelectrode interface inside the microsystem, as implied by cyclic voltammetry and electrochemical impedance spectrometry measurements. The electrochemical information was used to determine the moment in which bacteria colonized the electrode-enabled area of the microsystem. This microsystem shows potential advantages for long-term electrochemical monitoring of the extracellular environment of cell culture and has been designed using readily available technologies that can be easily integrated in routine protocols. Complementarily, these methods can help elucidate fundamental questions of the electron transfer of bacterial cultures and are potentially feasible to be integrated into current characterization techniques. [ABSTRACT FROM AUTHOR]

Graphene-based composites have been widely explored for electrode and electrocatalyst materials for electrochemical energy systems. In this paper, a novel composite material of the reduced graphene oxide nanosheets (rGON) with gold nanoparticles (NPs) (rGON-AuNP) is synthesized, and its morphology, structure, and composition are characterized by scanning electron microscopy (SEM), high-resolution transmission electron microscopy (HRTEM), X-ray diffraction (XRD), energy dispersive X-ray (EDX), Fourier transform infrared spectroscopic (FTIR), Raman, and UV-Vis techniques. To confirm this material's electrochemical activity, a glucose oxidase (GOD) is chosen as the target reagent to modify the rGON-AuNP layer to form GOD/rGON-AuNP/glassy carbon (GC) electrode. Two pairs of distinguishable redox peaks, corresponding to the redox processes of two different conformational GOD on AuNP, are observed on the cyclic voltammograms of GOD/rGON-AuNP/GC electrode. Both cyclic voltammetry and electrochemical impedance spectroscopy are employed to study the mechanism of direct electron transfer from GOD to GC electrode on the rGON-AuNP layer. In addition, this GOD/rGON-AuNP/GC electrode shows catalytic activity toward glucose oxidation reaction. [ABSTRACT FROM AUTHOR]

Dehydrogenase based bioelectrocatalysis has been increasingly exploited in recent years in order to develop new bioelectrochemical devices, such as biosensors and biofuel cells, with improved performances. In some cases, dehydrogeases are able to directly exchange electrons with an appropriately designed electrode surface, without the need for an added redox mediator, allowing bioelectrocatalysis based on a direct electron transfer process. In this review we briefly describe the electron transfer mechanism of dehydrogenase enzymes and some of the characteristics required for bioelectrocatalysis reactions via a direct electron transfer mechanism. Special attention is given to cellobiose dehydrogenase and fructose dehydrogenase, which showed efficient direct electron transfer reactions. An overview of the most recent biosensors and biofuel cells based on the two dehydrogenases will be presented. The various strategies to prepare modified electrodes in order to improve the electron transfer properties of the device will be carefully investigated and all analytical parameters will be presented, discussed and compared. [ABSTRACT FROM AUTHOR]

The FAD-dependent glucose dehydrogenase from Burkholderia cepacia (FADGDH) is a hetero-oligomeric enzyme that is capable of direct electron transfer (DET) with an electrode. The cytochrome c (cyt c ) subunit, which possesses three hemes (heme 1, heme 2, and heme 3, from the N-terminal sequence), is known to enable DET; however, details of the electron transfer pathway remain unknown. A mutagenesis investigation of the heme axial ligands was carried out to elucidate the electron transfer pathway to the electron mediators and/or the electrode. The sixth axial ligand for each of the three heme irons, Met109, Met263, and Met386 were substituted with His. The catalytic activities of the wild-type (WT) and mutant enzymes were compared by investigating their dye-mediated dehydrogenase activities and their DET abilities toward the electrode. The results suggested that (1) heme 1 with Met109 as an axial ligand is mainly responsible for the electron transfer with electron acceptors in the solution, but not for the DET with the electrode; (2) heme 2 with Met263 is responsible for the DET-type reaction with the electrode; and (3) heme 3 with Met386 seemed to be the electron acceptor from the catalytic subunit. From these results, two electron transfer pathways were proposed depending on the electron acceptors. Electrons are transferred from the catalytic subunit to heme 3, then to heme 2, to heme 1 and, finally, to electron acceptors in solution. However, if the enzyme complex is immobilized on the electrode and is used as electron acceptors, electrons are passed to the electrode from heme 2., Competing Interests: The authors declare no conflict of interest.

For the first time a molecularly imprinted polymer (MIP) with direct electron transfer (DET) and bioelectrocatalytic activity of the target protein is presented. Thin films of MIPs for the recognition of a hexameric tyrosine-coordinated heme protein (HTHP) have been prepared by electropolymerization of scopoletin after oriented assembly of HTHP on a self-assembled monolayer (SAM) of mercaptoundecanoic acid (MUA) on gold electrodes. Cavities which should resemble the shape and size of HTHP were formed by template removal. Rebinding of the target protein sums up the recognition by non-covalent interactions between the protein and the MIP with the electrostatic attraction of the protein by the SAM. HTHP bound to the MIP exhibits quasi-reversible DET which is reflected by a pair of well pronounced redox peaks in the cyclic voltammograms (CVs) with a formal potential of -184.4 ± 13.7 mV vs. Ag/AgCl (1 M KCl) at pH 8.0 and it was able to catalyze the cathodic reduction of peroxide. At saturation the MIP films show a 12-fold higher electroactive surface concentration of HTHP than the non-imprinted polymer (NIP).

A DNA/chitosan-Fe₃O₄ magnetic nanoparticle bio-complex film was constructed for the immobilization of horseradish peroxidase (HRP) on a glassy carbon electrode. HRP was simply mixed with DNA, chitosan and Fe₃O₄ nanoparticles, and then applied to the electrode surface to form an enzyme-incorporated polyion complex film. Scanning electron microscopy (SEM) was used to study the surface features of DNA/chitosan/Fe₃O₄/HRP layer. The results of electrochemical impedance spectroscopy (EIS) show that Fe₃O₄ and enzyme were successfully immobilized on the electrode surface by the DNA/chitosan bio-polyion complex membrane. Direct electron transfer (DET) and bioelectrocatalysis of HRP in the DNA/chitosan/Fe₃O₄ film were investigated by cyclic voltammetry (CV) and constant potential amperometry. The HRP-immobilized electrode was found to undergo DET and exhibited a fast electron transfer rate constant of 3.7 s -1 . The CV results showed that the modified electrode gave rise to well-defined peaks in phosphate buffer, corresponding to the electrochemical redox reaction between HRP(Fe (III ) ) and HRP(Fe (II) ). The obtained electrode also displayed an electrocatalytic reduction behavior towards H₂O₂. The resulting DNA/chitosan/Fe₃O₄/HRP/glassy carbon electrode (GCE) shows a high sensitivity (20.8 A·cm -2 ·M -1 ) toward H₂O₂. A linear response to H₂O₂ measurement was obtained over the range from 2 µM to 100 µM ( R ² = 0.99) and an amperometric detection limit of 1 µM (S/N = 3). The apparent Michaelis-Menten constant of HRP immobilized on the electrode was 0.28 mM. Furthermore, the electrode exhibits both good operational stability and storage stability.

The novel highly ordered mesoporous carbon (known as FDU-15), prepared by the organic-organic self-assembly method was been used for first time for the immobilization of hemoglobin (Hb) and its bioelectrochemical properties were studied. The resulting Hb/FDU-15 film provided a favorable microenvironment for Hb to perform direct electron transfers at the electrode. The immobilized Hb also displayed its good electrocatalytic activity for the reduction of hydrogen peroxide. The results demonstrate that mesoporous carbon FDU-15 can improve the Hb loading with retention of its bioactivity and greatly promote the direct electron transfer, which can be attributed to its high specific surface area, uniform ordered porous structure, suitable pore size and biocompatibility. Our present study may provide an alternative way for the construction of nanostructure biofunctional surfaces and pave the way for its application to biosensors.

We have studied the direct electrochemistry of horseradish peroxidase (HRP) coupled to gold nanoparticles (AuNP) using electrochemical techniques, which provide some insight in the application of biosensors as tools for diagnostics because HRP is widely used in clinical diagnostics kits. AuNP capped with (i) glutathione and (ii) lipoic acid was covalently linked to HRP. The immobilized HRP/AuNP conjugate showed characteristic redox peaks at a gold electrode. It displayed good electrocatalytic response to the reduction of H(2)O(2), with good sensitivity and without any electron mediator. The covalent linking of HRP and AuNP did not affect the activity of the enzyme significantly. The response of the electrode towards the different concentrations of H(2)O(2) showed the characteristics of Michaelis Menten enzyme kinetics with an optimum pH between 7.0 to 8.0. The preparation of the sensor involves single layer of enzyme, which can be carried out efficiently and is also highly reproducible when compared to other systems involving the layer-by-layer assembly, adsorption or encapsulation of the enzyme. The immobilized AuNP-HRP can be used for immunosensor applications.