Direct electron transfer and biosensing of glucose oxidase immobilized at multiwalled carbon nanotube-alumina-coated silica modified electrode

Investigations are reported regarding the direct electrochemical performance of glucose oxidase (GOD) immobilized on a film of multiwalled carbon nanotube-alumina-coated silica (MWCNT-ACS). The surface morphology of the GOD/MWCNT-ACS nanobiocomposite is characterized by scanning electron microscopy. In cyclic voltammetric response, the immobilized GOD displays a pair of well-defined redox peaks, with a formal potential (E°′) of ? 0.466 V versus Ag/AgCl in a 0.1 M phosphate buffer solution (pH 7.5) at a scan rate of 0.05 V s^? 1; also the electrochemical response indicates a surface-controlled electrode process. The dependence of formal potential on solution pH indicates that the direct electron transfer reaction of GOD is a reversible two-electron coupled with a two-proton electrochemical reaction process. The glucose biosensor based on the GOD/MWCNT-ACS nanobiocomposite shows a sensitivity of 0.127 A M^? 1 cm^? 2 and an apparent Michaelis–Menten constant of 0.5 mM. Furthermore, the prepared biosensor exhibits excellent anti-interference ability to the commonly co-existed uric acid and ascorbic acid.     

Determination of the potential of zero charge of Au(111) modified with thiol monolayers

A new method is proposed for the determination of the potential of zero charge of gold electrodes modified with thiol monolayers. It makes use of the immersion technique, in combination with a vapor deposition protocol to build the thiol monolayers. As compared to previous methods, the present approach provides more accurate results, particularly in the case of long-chain alkanethiol monolayers, and it is applicable to any monolayer irrespective of its degree of hydrophilicity. Results are presented for a series of 12 alkanethiol monolayers and for 11-mercaptoundecanol and 11-mercaptoundecanoic acid monolayers. Good agreement is found between the variation of the potential of zero charge along the alkanethiol series with the corresponding change of the surface work function. The potential of zero charge of the 11-mercaptoundecanoic acid monolayer is shown to depend on the extent of dissociation of the acid, thus opening the possibility of applying this type of measurements to the study of surface ionization processes.     

Redox reactions at titanium electrodes covered with doped passive films

The rate of electron transfer reactions at titanium electrodes covered with thin platinum-doped passive films was investigated. The doping enhances the current considerably. It is suggested that the platinum atoms in the film serve as intermediate states in an electronic resonance-tunnelling mechanism. The corresponding model calculations show a satisfactory agreement with the experimental data. From these calculations the energy levels of the platinum states in the film are estimated.     

Amperometric determination of total cholesterol at gold electrodes covalently modified with cholesterol oxidase and cholesterol esterase with use of thionin as an electron mediator

Immobilization of cholesterol oxidase (EC 1.1.3.6) (ChOx) on a gold electrode was attempted by cross-linking using glutaraldehyde between ChOx molecules and a self-assembled monolayer of 2-aminoethanethiolate. The resulting electrode (ChOx/Au) exhibits an amperometric response to free cholesterol in the presence of thionin as an electron mediator, and a steady-state response is obtained approximately 60 s after injection of cholesterol into the electrolyte solution. Coimmobilization of cholesterol esterase (EC 3.1.1.13) (ChE) and ChOx (ChE/ChOx/Au) allows the amperometric determination of both esterified cholesterol and free cholesterol. Cyclic voltammetry of the ChE/ChOx/Au and the dependence of the amperometric response to cholesterol on the concentration of thionin suggest that thionin is encapsulated in the enzyme film on the electrode surface. Apparent Michaelis constants of the ChOx/Au and the ChE/ChOx/Au electrodes suggest that the amperometric response was controlled by penetration of the reaction substrate into the films of the enzyme(s). The concentration of total (free and esterified) cholesterol in human serum samples, determined by using the techniques developed in the present study, is in good agreement with that determined by the well-established technique using colorimetry.     

Direct electron transfer of cytochrome P450 2B4 at electrodes modified with nonionic detergent and colloidal clay nanoparticles

A method for construction of biosensors with membranous cytochrome P450 isoenzymes was developed based on clay/detergent/protein mixed films. Thin films of sodium montmorillonite colloid with incorporated cytochrome P450 2B4 (CYP2B4) with nonionic detergent were prepared on glassy carbon electrodes. The modified electrodes were electrochemically characterized, and bioelectrocatalytic reactions were followed. CYP2B4 can be reduced fast on clay-modified glassy carbon electrodes in the presence of the nonionic detergent Tween 80. In anaerobic solutions, reversible oxidation and reduction is obtained with a formal potential between -0.292 and -0.305 V vs Ag/AgCl 1 M KCl depending on the preparation of the biosensor. In air-saturated solution, bioelectrocatalytic reduction currents can be obtained with the CYP2B4-modified electrode on addition of typical substrates such as aminopyrine and benzphetamine. This reaction was suppressed when methyrapone, an inhibitor of P450 reactions, was present. Measurement of product formation also indicates the bioelectrocatalysis by CYP2B4.     

Direct DNA hybridization at disposable graphite electrodes modified with carbon nanotubes

The performance of glassy carbon (GCE) and graphite pencil electrodes (PGE) modified with multiwalled carbon nanotubes (CNTs) are compared, based on the direct electrochemical detection of nucleic acids. This is accomplished by monitoring the differential pulse voltammetry changes of the guanine signal. CNT-modified PGE compares favorably to that of the commonly used CNT-modified GCE owing to the intrinsic improved performance of the supporting PGE. The better intrinsic characteristics of the PGE are related to its composite structure and higher level of porosity compared to GCE. The performance characteristics of the direct DNA hybridization on the disposable CNT-modified PGE are studied in terms of optimum analytical conditions such as probe concentration, target concentration, hybridization time, and selectivity. The new DNA biosensor described here has shown some important advantages such being inexpensive, sensitive, selective, and able to generate reproducible results using a simple and direct electrochemical protocol.     

Multilayer membranes via layer-by-layer deposition of glucose oxidase and Au nanoparticles on a Pt electrode for glucose sensing

We prepared multilayer membranes by the layer-by-layer deposition of glucose oxidase (GOx) and Au nanoparticles (5, 10, or 50 nm φ) on sensor substrates, such as a Pt electrode and a quartz glass plate, to prepare glucose sensors. The enzyme activity of GOx remained even in alternate assemblies, and the activity increased with the increasing number of depositions. The apparent K_m values of the deposited GOx were 28–32 mM, while a reported value in a solution is 33 mM. These results suggest that Au nanoparticles can be used as binders for the deposition of GOx without significant change in the affinity between GOx and glucose.     

Following the biocatalytic activities of glucose oxidase by electrochemically cross-linked enzyme-Pt nanoparticles composite electrodes

An integrated platinum nanoparticles (NPs)/glucose oxidase (GOx) composite film associated with a Au electrode is used to follow the biocatalytic activities of the enzyme. The film is assembled on a Au electrode by the electropolymerization of thioaniline-functionalized Pt NPs and thioaniline-modified GOx. The resulting enzyme/Pt NPs-functionalized electrode stimulates the O 2 oxidation of glucose to gluconic acid and H 2O 2. The modified electrode is then implemented to follow the activity of the enzyme by the electrochemical monitoring of the generated H 2O 2. The effect of the composition of the Pt NPs/GOx cross-linked nanostructures and the optimal conditions for the preparation of the electrodes are discussed.     

Photoelectrochemical reactions of electrodes modified with composites between conjugated polymer or ruthenium complex and single-walled carbon nanotubes

Composite materials between conjugated polymer; poly[2-methoxy-5-(2'-ethylhexyloxy)-1.4-phenylene vinylene] (MEHPPV), or ruthenium(II)-tris(2,2'-bipyridine) (Ru(bpy)₃²⁺)-poly(sodium 4-styrenesulfonate) (PSS) complex and single-walled carbon nanotubes (SWNTs) were fabricated using polymer wrapping method. Formation of SWNT/MEHPPV or SWNT/PSS/Ru(bpy)₃²⁺ composite was confirmed by absorption and fluorescence spectra, and AFM images. Electrode modified with SWNT/MEHPPV or SWNT/PSS/Ru(bpy)₃²⁺ composite was prepared by casting from DMF solution of SWNT/MEHPPV or aqueous solution of SWNT/PSS/Ru(bpy)₃²⁺. The electrode modified with SWNT/MEHPPV or SWNT/PSS/Ru(bpy)₃²⁺ composite showed photocurrent response due to photoexcitation of MEHPPV or Ru(bpy)₃²⁺. The photocurrents are ascribed to photoinduced electron-transfer reaction from excited state of MEHPPV or Ru(bpy)₃²⁺ to SWNT.     

Immobilization of Prussian Blue nanoparticles onto thiol SAM modified Au electrodes for electroanalytical or biosensor applications

Poly(vinylpyrrolidone) (PVP)-protected Prussian Blue (PB) nanoparticles were prepared by simply mixing FeCI3 and K4Fe(CN)6 with absence or presence of HCI or/and KCI in water solution. The obtained PB nanoparticles were immobilized onto thiol self-assembled monolayer (SAM) modified Au electrodes. L-cysteine (Cys) and 1,8-octanedithiol (ODT) were compared as a bridge between the gold surface and the PB nanoparticles. The results show that PB prepared from the initial solution with KCI gives preferred electrochemical response and that Cys/Au shows improved immobilization effect of PB than ODT/Au. The obtained PB/Cys/Au electrodes exhibit electrocatalytic activity toward H2O2 reduction and DL-homocysteine (HCys) oxidation. Glucose oxidase (GOX) was immobilized onto PB modified electrode to explore the potentials for the design of oxidase-based biosensors. It is possible to anchor PB nanoparticles and develop their application on electroanalysis and biosensing.     

STM investigation of substitute effect on oligothiophene adlayer at Au(111) substrate

Molecular adlayers of a series of oligothiophenes with carboxylic groups and alkyl substitutes, DTDA, TTDA, QTDA, and PTDA, are investigated by STM at Au(111) surface. The effect of molecular structure including alkyl chain and thiophene backbone on the adlayer structure is revealed. DTDA and TTDA with two and three thiophene rings, self-assembled into highly ordered long-range two dimensional structures via hydrogen bondings, while QTDA with four thiophene rings formed short-range ordered structure with hexagonal symmetry. PTDA with five thiophene rings showed disordered pattern due to its strong molecule-substrate interaction. The results are compared with those on HOPG surface, showing that the molecule-substrate interaction plays an important role in the adlayer formation of these oligothiophenes.     

Electrochemistry and electrocatalytic activities of superoxide dismutases at gold electrodes modified with a self-assembled monolayer

In this article, the electrochemical properties and electrocatalytic activity of three kinds of superoxide dismutases (SODs), that is, bovine erythrocyte copper-zinc superoxide dismutase (Cu/Zn-SOD), iron superoxide dismutase from Escherichia coli (Fe-SOD), and manganese superoxide dismutase from E. coli (Mn-SOD), in the SOD family were studied. It was revealed that the direct electron transfer of the three kinds of SODs could be efficiently promoted by a self-assembled monolayer (SAM) of 3-mercaptopropionic acid (MPA) confined on a gold electrode. The electrochemical properties of the SODs at the MPA-SAM electrode vary with the sort of SOD with respect to the formal potential, reversibility of electrode reactions, kinetic parameters, and pH dependence, suggesting different mechanisms for the electrode reactions of the individual SODs. A combination of the facilitated direct electron transfer and the bifunctional enzymatic catalytic activities of the SODs via a redox cycle of their active metals substantially offered a flexible electrochemical route to determination of O(2)(*)(-) where O(2)(*)(-) can be sensed with the SOD-based biosensors in both anodic and cathodic polarizations. Such an intrinsic feature of the SOD-based biosensors successfully enabled a sensitive determination scheme for O(2)(*)(-) free from the interference from some coexisting electroactive species, such as ascorbic acid (AA) and uric acid (UA). Further potential applications for in vivo determination of O(2)(*)(-) is also suggested.     

Direct electron transfer of heme- and molybdopterin cofactor-containing chicken liver sulfite oxidase on alkanethiol-modified gold electrodes

Direct heterogeneous electron transfer (ET) of sulfite oxidase (SOx), a heme- and molybdopterin cofactor-containing intermembrane enzyme, was studied on alkanethiol-modified Au electrodes both with SOx entrapped between the modified Au electrode and a permselective membrane and with SOx adsorbed at the electrode surface, in the absence of any membrane. SOx in direct electronic communication with the electrode surface gave a quasi-reversible electrochemical signal with a midpoint potential of--120 mV vs Ag/AgCl corresponding to the redox transformations of the heme domain of SOx and with a heterogeneous ET constant in the order of 15 s(-1). The efficiency of the bioelectrocatalytic 2e- oxidation of sulfite catalyzed by SOx in direct ET exchange with the electrode was shown to depend essentially on the nature of the alkanethiol layer. Adsorption and orientation of SOx on an 11-mercapto-1-undecanol (MuD-OH) self-assembled monolayer, i.e., terminally functionalized with OH groups, provided efficient catalytic oxidation of sulfite, contrary to nonfunctionalized alkanethiols, e.g., 1-decanethiol, or alkanethiol layers terminally functionalized with NH2 groups. Comparative studies with short-chain alkanethiols, e.g., cysteamine and 2-mercaptoethanol, revealed an evidently different mode of adsorption of SOx on these layers, onto which SOx was not catalytically active. Coadsorption of MuD-OH and 11-mercapto-1-undecanamine improved the surface properties of the SAM, resulting in a higher surface coverage with bioelectrocatalytically active SOx but not in an increased apparent catalytic rate constant, kcat, ranging in the order of 18-24 s(-1) at pH 7.4. The achieved efficiency of SOx bioelectrocatalysis in direct ET reaction between the modified electrode and the enzyme approached the rates characteristic for the catalysis mediated by cytochrome c, the natural redox partner of SOx, thus implying the retention of the biological function of SOx under the heterogeneous electrode reaction conditions. Results obtained enable the development of a third-generation biosensor for sulfite monitoring.     

Electrooxidation mechanism of biogenic amines at amine oxidase modified graphite electrode

Amine oxidase (AO, EC. 1.4.3.6) was previously shown to be a very efficient biological recognition element of amperometric biosensors for monitoring biogenic amines. The enzyme was effectively working in both mono- and bienzyme electrode designs, based on either a direct or a mediated electron-transfer pathway. This work focuses on the elucidation of the electron-transfer mechanism of the monoenzymatic unmediated AO-modified biosensor. The observed unmediated catalytic currents were assumed to be caused by (i) a direct electron-transfer process, (ii) the electrooxidation of the formed product, or (iii) their combination. Experiments supporting these assumptions are discussed in detail.     

Catalytic oxidation and flow detection of carbohydrates at ruthenium dioxide modified electrodes

Ruthenium dioxide (RuO2) containing carbon paste electrodes exhibiting electrocatalytic response toward carbohydrates are described. The electrocatalytic behavior is exploited for developing a highly stable and sensitive flow detection scheme for carbohydrates at a low and fixed potential (+0.4 V vs Ag/AgCl). The effects of pH, flow rate, operating potential, surface "loading", concentration, and other variables are explored. The electrode response was stable for more than 48 h, with a signal loss of less than 10% over this period. The detection limits at the picomole level and a relative standard deviation of 1.2% (n = 72) are reported. Electrocatalytic oxidation is described also for related polyhydroxyl compounds (aldonic and aldaric acids and alditols).     

The nature of the interaction layer formed at the Fe-(111) Au interface

Iron was vacuum deposited onto (111) gold platelets at substrate temperatures of approximately 300 °C. Diffraction spots which were not predicted by epitaxial relationships for iron-gold layers revealed the existence of a new phase in the Fe-(111) Au bicrystals. The structure of this new phase was positively identified as face-centred cubic by tilting diffraction experiments. The lattice parameter of the structure was determined and was found to be a function of the overgrowth thickness with a limiting value of $\text{3.875±0.001}\text{A}\text{?}$. The origin of the new phase is discussed and is indicated to be in the form of a thin interaction layer at the interface between the iron and the gold.