Achieving Direct Electrical Connection to Glucose Oxidase Using Aligned Single Walled Carbon Nanotube Arrays

Direct electron transfer between an electrode and the redox active centre of glucose oxidase, flavin adenine dinucleotide (FAD), is probed using carbon nanotube modified gold electrodes. Gold electrodes are first modified with a self‐assembled monolayer of cysteamine and then shortened single walled carbon nanotubes (SWNT) are aligned normal to the electrode surface by self‐assembly. The electrochemistry of these aligned nanotube electrode arrays is initially investigated using potassium ferricyanide which showed SWNT act as nanoelectrodes with the ends of the tubes more electrochemically active than the walls. Subsequently the nanotubes are plugged into the enzymes in one of two ways. In the first method, native glucose oxidase is covalently attached to the ends of the aligned tubes which allowed close approach to FAD and direct electron transfer to be observed with a rate constant of 0.3 s^?1. In the second strategy, FAD was attached to the ends of the tubes and the enzyme reconstituted around the surface immobilized FAD. This latter approach allowed more efficient electron transfer to the FAD with a rate constant of 9 s^?1.     

Breaking the barrier to fast electron transfer

A study of the electron transfer for a non-glycosylated redox variant of GOx is reported, immobilised onto an electrode via a polyhistidine tag. The non-glycosylated variant allows the enzyme to be brought closer to the electrode, and within charge transfer distances predicted by Marcus' theory. The enzyme-electrode-hybrid shows direct very fast reversible electrochemical electron transfer, with a rate constant of ~ 350 s^− 1 under anaerobic conditions. This is 2 orders of magnitude faster than the enzyme-free flavin adenine dinucleotide (FAD). These results are discussed in the context of the conformation of FAD in the active site of GOx. Further data, presented in the presence of oxygen, show a reduced electron transfer rate (~ 160 s^− 1) that may be associated with the oxygen interaction with the histidines in the active site. These residues are implicated in the proton transfer mechanism and thus suggest that the presence of oxygen may have a profound effect in attenuating the direct electron transfer rate and thus moderating ‘short-circuit’ incidental electron transfer between proteins.     

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.     

Feasibility Study of Introducing Redox Property by Modification of PMBN Polymer for Biofuel Cell Applications

In this study, the feasibility of introducing redox property to an amphiphilic phospholipid polymer (PMBN) was investigated. The active ester group in the side chain of the polymer was used to react with pyrroloquinoline quinine (PQQ). Redox peaks that corresponded to PQQ redox potentials were observed after the modification. Glucose oxidase was immobilized to the modified polymer. Electrochemical oxidation of glucose was carried out with the polymer electrode. The oxidation current increased with elevating glucose concentration indicating electron transfer established between the electrode and enzyme. It suggests that by modification, PMBN is possible to use for enzyme electrode for bioelectronics.     

A molecular wire modified glassy carbon electrode for achieving direct electron transfer to native glucose oxidase

Here we describe a strategy for achieving direct electron transfer to native glucose oxidase (GOx), an enzyme in which the redox active centre is buried deep within the glycoprotein. To achieve this a glassy carbon electrode is modified with a mixed monolayer of 4-carboxyphenyl and a 20 Å long oligo(phenylethynyl) molecular wire (MW), assembled from the respective aryl diazonium salts. Subsequently GOx is adsorbed to the interface, followed by covalent attachment. The redox chemistry of the active centre of glucose oxidase, flavin adenine dinucleotide, was observed at an E_1/2 of –443 mV (vs. Ag|AgCl). The enzyme was shown to retain its activity. Most importantly, in the absence of oxygen the electrode was still able to biocatalytically turn over glucose at −400 mV, thereby demonstrating that the enzyme was being recycled back to its catalytically active oxidized form from its inactive reduced form. The rate of enzyme turnover was 1.1 s⁻¹.     

Bioelectrocatalytic Activity of Immobilized Alcohol Oxidase on 4‐(pyrrole‐1‐yl) Benzoic Acid Modified Carbon Nanotubes

A novel bioelectrocatalytic system was prepared by immobilizing alcohol oxidase (AOx) onto multiwalled carbon nanotubes (MWCNT) modified with 4‐(pyrrole‐1‐yl) benzoic acid (PyBA). Functional carboxylic groups from PyBA create covalent amide linkages with amine groups from the enzyme molecule and provide an anchor for the effective immobilization of AOx improving the stability of the whole system. The immobilized enzyme displayed a pair of reversible redox peaks of flavin adenine dinucleotide (FAD) cofactor with the formal potential E^0’=?0.451 V. The response showed a surface‐controlled electrode process with the heterogeneous electron transfer rate constant k_s=2.7 s^?1. Under aerobic conditions AOx(FADH₂) can be oxidized to AOx(FAD) by oxygen, which then reacts with ethanol decreasing the cathodic response, which could be used for ethanol detection with a high sensitivity 13.1 μA mM^?1 cm^?2. The lack of bioactivity towards ethanol in anaerobic conditions suggests the presence of two types of AOx molecules in the system: active with oxygen maintaining the direct electron transfer feature and not active without a redox mediator, due to the deeply embedded FAD cofactor. The polarization curve showed that the electrooxidation current of ethanol appears at ?410 mV and reaches 2.0 µA cm^?1 at ?300 mV. In this case, the bioactivity of AOx to ethanol can be observed offering promising solution for the development of mediatorless systems for application to biosensors and biofuel cells.     

Glucose Oxidase/Cellulose–Carbon Nanotube Composite Paper as a Biocompatible Bioelectrode for Biofuel Cells

Biofuel cells are devices for generating electrical energy directly from chemical energy of renewable biomass using biocatalysts such as enzymes. Efficient electrical communication between redox enzymes and electrodes is essential for enzymatic biofuel cells. Carbon nanotubes (CNTs) have been recognized as ideal electrode materials because of their high electrical conductivity, large surface area, and inertness. Electrodes consisting entirely of CNTs, which are known as CNT paper, have high surface areas but are typically weak in mechanical strength. In this study, cellulose (CL)–CNT composite paper was fabricated as electrodes for enzymatic biofuel cells. This composite electrode was prepared by vacuum filtration of CNTs followed by reconstitution of cellulose dissolved in ionic liquid, 1-ethyl-3-methylimidazolium acetate. Glucose oxidase (GOx), which is a redox enzyme capable of oxidizing glucose as a renewable fuel using oxygen, was immobilized on the CL–CNT composite paper. Cyclic voltammograms revealed that the GOx/CL–CNT paper electrode showed a pair of well-defined peaks, which agreed well with that of FAD/FADH_2, the redox center of GOx. This result clearly shows that the direct electron transfer (DET) between the GOx and the composite electrode was achieved. However, this DET was dependent on the type of CNTs. It was also found that the GOx immobilized on the composite electrode retained catalytic activity for the oxidation of glucose.     

Amperometric glucose biosensor based on mediated electron transfer between immobilized glucose oxidase and plasma-polymerized thin film of dimethylaminomethylferrocene on sputtered gold electrode.

We propose an electron transfer-mediated amperometric enzyme biosensor based on plasma-polymerized thin film of dimethylaminomethylferrocene (DMAMF) on a sputtered gold electrode. The DMAMF plasma-polymerized film is deposited directly onto the surface of the electrode under dry conditions. The resulting thin film not only has redox sites but also is extremely thin (approximately 20 nm), adheres well onto the substrate (electrode), has a flat surface and a highly-crosslinked network structure, and is hydrophilic in nature. Glucose oxidase is densely immobilized onto the surface of DMAMF plasma-polymerized film on the gold electrode. From the electrochemical measurement, the biosensor can cover the wide range of glucose concentration (1.3 - 81 mM) at +350 mV of applied potential. The current response of the glucose biosensor was decreased by less than 5% in an aerobic solution as compared to that in an anaerobic solution. These show that the DMAMF plasma-polymerized films play a role as the electron transfer mediators between the reaction center of enzyme and the electrode.     

Deglycosylation of glucose oxidase to improve biosensors and biofuel cells

We demonstrate that a more efficient redox hydrogel structure can be achieved by engineering the size and the surface charge of the bioelectrocatalyst. Deglycosylated glucose oxidase (GOx) modified electrode exhibits higher current density than native GOx, for the same molar composition of the hydrogel. This improvement is very likely due to a more efficient hydrogel structure rather than a better intrinsic electron transfer between the FAD/FADH₂ redox center and the redox mediator.     

1,10-Phenanthroline-5,6-dione and 9,10-phenanthrenequinone as redox mediators for amperometric glucose biosensors

In this study, two ortho-quinoidal compounds, 1,10-phenanthroline-5,6-dione (PD) and 9,10-phenanthrenequinone (PQ), were examined as electron transfer mediators suitable for amperometric glucose biosensors. The dependences of the electrochemical responses of PD- and PQ-based amperometric glucose biosensors on varied concentrations of glucose were investigated under aerobic and anaerobic conditions. The PD-modified graphite rod (GR) electrode revealed a current response seven times higher than that of the PQ-modified GR electrode. The reactivity indices of ortho-quinoidals assessed by means of B3LYP functional method applying 6-311G(D) basis set showed that the electron-accepting potency for PD was markedly higher as compared with that of PQ. Compared to PQ, considerably higher reactivity of PD has been defined in the reactions with NADP⁺-ferredoxin reductase (FNR, EC 1.18.1.2) as a model single-electron transfer FAD-dependent enzyme, which provided an additional evidence for PD as a more efficient mediator compared to PQ. This study illustrates that PD can be applied as a redox mediator for glucose oxidase and it could be more suitable for a reagent-less biosensor design than PQ.     

Direct Electrochemistry of Glucose Oxidase at a Gold Electrode Modified with Graphene Nanosheets

In this work, we report the direct electrochemistry of glucose oxidase (GOD) observed at a gold electrode modified with graphene nanosheets. Initially, graphene nanosheets were synthesized and conjugated to the enzyme GOD and immobilized on to a gold electrode surface. Cyclic voltammetry was then performed using Gold-Graphene-GOD modified electrodes in a pH 7.2 phosphate buffered saline (PBS). A pair of well-defined redox peaks was obtained for GOD with the reduction peak centered at +180 mV and a peak separation of 70 mV in PBS under physiological conditions. Moreover, the electron transfer rate of GOD redox reaction was greatly enhanced and the peak potential was found to be pH dependent at the graphene-GOD surface. Further, the performance of the Gold-Graphene-GOD was found to be stable and excellent under physiological conditions indicating the possibility of employing this platform for real time analysis. The observed results indicated that the 2D-graphene holds great promise for conjugation ability with a variety of enzymes. Further, our results also confirmed that graphene is capable of holding the enzyme GOD in a favorable position and retains its original structure and functionality that are essential for biosensing.     

Separation-free amperometric enzyme immunoassay

A new technique for conducting a separation-free amperometric enzyme immunoassay is described using DNP-aminocaproic acid as the analyte. The technique is based on the combined use of a recently described separation-free enzyme immunoassay (19) and an electrode system that senses H₂O₂. Oxidation of glucose to gluconate and H₂O₂ by the enzyme reconstituted from DNP-conjugate apoglucose oxidase (DPN-CAGO) and FAD was continuously measured amperometrically. The reconstitution was inhibited by preincubation with anti-DNP antibody before adding FAD. This antibody-induced inhibition of the reconstituting of the holoenzyme was reversed by adding DNP-amino caproic acid to DNP-CAGO before adding the antibody to DNP-CAGO. Based on (a) the antibody-induced inhibition of holoenzyme reconstitution, (b) a specific ligand-induced reversal of the inhibition, and (c) an electrochemical system that measures H₂O₂, we developed a separation-free (homogeneous) amperometric enzyme immunoassay.     

Construction of a Novel Glucose Biosensor Based on Covalent Immobilization of Glucose Oxidase on Poly(glycidyl methacrylate‐co‐vinylferrocene)

A novel glucose biosensor was constructed via direct covalent attachment of glucose oxidase onto epoxy group containing polymeric electron transfer mediator, Poly(glycidyl methacrylate‐co‐vinylferrocene). A copolymer of glycidyl methacrylate (GMA) and vinylferrocene (VFc) with different molar ratios has been prepared by free radical copolymerization. These copolymers have been utilized as polymeric mediators for amperometric glucose sensing. The catalytic electrochemistry of the enzyme electrode with the copolymer was investigated. Copolymer acts as an electron transfer mediator between the redox center of Glucose oxidase (GOx) and the electrode. The stability, reusability, pH and temperature response of the biosensor as well as its kinetic parameter have also been studied.     

Biocatalytic anode for glucose oxidation utilizing carbon nanotubes for direct electron transfer with glucose oxidase

Covalently linked layers of glucose oxidase, single-wall carbon nanotubes and poly-l-lysine on pyrolytic graphite resulted in a stable biofuel cell anode featuring direct electron transfer from the enzyme. Catalytic response observed upon addition of glucose was due to electrochemical oxidation of FADH₂ under aerobic conditions. The electrode potential depended on glucose concentration. This system has essential attributes of an anode in a mediator-free biocatalytic fuel cell.     

Electrochemical,microscopic, and EQCM studies of cathodic electrodeposition of ZnO/FAD and anodic polymerization of FAD films modified electrodes and their electrocatalytic properties

Two kinds of chemically modified electrodes were prepared. In the first type of electrodes, zinc oxide (ZnO) and flavin adenine dinucleotide (FAD) molecules were deposited onto the glassy carbon-, gold-, and SnO₂-coated glass electrodes by using cyclic voltammetry from the bath solution containing aqueous 0.1 M zinc nitrate, 0.1 M sodium nitrate, and 1 × 10⁻⁴ M FAD. It was called as ZnO/FAD modified electrodes. The second type of modified electrode was prepared by the electropolymerization method. Electrochemical polymerization of FAD was carried out from the acidic solution containing 1 × 10⁻⁴ M FAD monomers onto electrode surfaces. This poly(FAD)-modified electrode yields a new redox couple in addition to the monomers redox couple. The influence of the concentrations, pH, and electrocatalytic properties of the ZnO/FAD- and poly(FAD)-modified electrodes are investigated by means of the in situ technique electrochemical quartz–crystal microgravimetry (EQCM) combined with cyclic voltammetry and the ex situ technique scanning electron microscopy. From these studies, it appears that the cathodic deposition of ZnO/FAD-modified electrodes gives only one redox couple, and the anodically polymerized FAD film-modified electrodes gives two reversible redox couples. The pH dependence of the redox responses were investigated and the kinetics of electron transfer was evaluated. In addition, the EQCM technique was employed to follow the deposition process of both kinds of modified electrodes in real time as well as the characteristics of the charge transfer associated with the surface-confined redox-active couples. The electrocatalytic activity of the poly(FAD)-modified electrode towards the reduction of hydrogen peroxide and the oxidation of dopamine and ascorbic acid was explored. The important electrocatalytic properties of poly(FAD)-modified electrode were observed for simultaneous separation of dopamine and ascorbic acid in neutral solution. This poly(FAD)-modified electrode has several advantages than the previously reported FAD-modified electrodes.     

Amperometric enzyme-modified electrodes based on tetrathiafulvalene derivatives for the determination of glucose

A number of tetrathiafulvalene (TTF) derivatives have been synthesized and tested as electron transfer mediators in glucose oxidase-based amperometric biosensors. Using cyclic voltammetry and stationary potential experiments, it is shown that several of these derivatives can effectively mediate electron transfer from the reduced flavin adenine dinucleotide redox centers of glucose oxidase to a conventional carbon paste electrode. An insoluble polymeric electron relay system, based on the covalent attachment of TTF moieties to a highly flexible siloxane polymer, is also shown to facilitate a flow of electrons from the enzyme to the electrode. The resulting glucose biosensors function efficiently over a clinically relevant range of glucose concentrations.     

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.     

Biosensors based on direct electron transfer in redox proteins

In biosensors based on direct electron transfer in redox proteins, efficient electron-transfer pathways between the immobilized redox protein and the electrode surface have to be established so to allow a fast electron transfer and concomitantly avoiding free-diffusing redox species. In this review, prerequisites for the direct electron transfer of redox proteins and immobilization of redox proteins on the electrode surfaces are addressed. Based on the specific nature of different proteins and non-manual immobilization procedures, possible biosensor designs are discussed, namely biosensors based on (1) ferritin; (2) cytochrome c; (3) myoglobin; (4) hemoglobin; (5) horseradish peroxidase; (6) catalase; (7) glucose oxidase; and (8) xanthine oxidase.     

Preparation of the glucose sensor based on three-dimensional ordered macroporous gold film and room temperature ionic liquid

A novel type of glucose sensor was fabricated based on a glucose oxidase (GOD)-N,N-dimethtylformamide (DMF)-[BMIm][BF₄] composites modified three-dimensional ordered macroporous (3DOM) gold film electrode. The immobilized GOD exhibits a pair of well-defined reversible peaks in 50 mM pH 7.0 phosphate buffer solutions (PBS), which could be attributed to the redox of flavin adenine dinucleotide (FAD) in GOD. The research results show that ionic liquid ([BMIm][BF₄]), DMF and 3DOM gold film are crucial for GOD to exhibit a pair of stable and reversible peaks. It is believed that the large active area of 3DOM gold film can increase the amount of immobilized GOD. Simultaneously, the application of IL enhances the stability of GOD and facilitates the electron transfer between GOD and the electrode. The synergetic effect of DMF can help the GOD to maintain its bioactivity better. GOD immobilized on the electrode exhibits the favorable electrocatalytic property to glucose, and the prepared sensor has a linear range from 10 to 125 nM with a detection limit of 3.3 nM at a signal-to-noise ratio of 3σ. The apparent K _m (Michaelis- Menten constant) for the enzymatic reaction is 0.018 mM.     

Effect of Gold Nanoparticles on the Structure and Electron‐Transfer Characteristics of Glucose Oxidase Redox Polyelectrolyte‐Surfactant Complexes

Efficient electrical communication between redox proteins and electrodes is a critical issue in the operation and development of amperometric biosensors. The present study explores the advantages of a nanostructured redox‐active polyelectrolyte–surfactant complex containing [Os(bpy)₂Clpy]²⁺ (bpy=2,2′‐bipyridine, py= pyridine) as the redox centers and gold nanoparticles (AuNPs) as nanodomains for boosting the electron‐transfer propagation throughout the assembled film in the presence of glucose oxidase (GOx). Film structure was characterized by grazing‐incidence small‐angle X‐ray scattering (GISAXS) and atomic force microscopy (AFM), GOx incorporation was followed by surface plasmon resonance (SPR) and quartz‐crystal microbalance with dissipation (QCM‐D), whereas Raman spectroelectrochemistry and electrochemical studies confirmed the ability of the entrapped gold nanoparticles to enhance the electron‐transfer processes between the enzyme and the electrode surface. Our results show that nanocomposite films exhibit five‐fold increase in current response to glucose compared with analogous supramolecular AuNP‐free films. The introduction of colloidal gold promotes drastic mesostructural changes in the film, which in turn leads to a rigid, amorphous interfacial architecture where nanoparticles, redox centers, and GOx remain in close proximity, thus improving the electron‐transfer process.