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This paper describes a batch-type coulometric D-fructose biosensor based on direct electron transfer reaction of D-fructose dehydrogenase (FDH) adsorbed on a porous carbon electrode surface. The adsorbed-FDH electrodes catalyzed the electrochemieal two-electron oxidation of D-fructose to 5-keto-D-fructose without a mediator. Nanostructured carbon particle-modified electrodes were used for the coulometric D-fructose biosensor to enhance the catalytic current density. The electric charge for the D-fructose oxidation gained by the biocoulometric measurement was in good agreement with the theoretical value corresponding to D-fructose amount in the range from 1 to 100 mM with a sample volume of I [micro]L. This method is also applicable to the determination of several oligo/ palysaccharides containing the D-fructose unit, in combination with specific hydrolases to yield D-fructose. An example was demonstrated by sucrose determination in which the electrode modified with FDH and invertase was used as a working electrode. To address the problem of electroactive interferences such as ascorbate, the electric charge at the FDH-free electrode was subtracted from the total charge obtained at the FDH-adsorbed electrode. The D-fructose concentrations in several beverages were successfully determined with this method. 10.1021/ac901771t

The catalytic reaction of glucose oxidase (GOx) mediated by 3-(10-phenothiazyl)propionic acid (PT-PA) and phenothiazine-labeled poly(ethylene oxide) (PT-PEO1000) that are covalently bonded to Au(111) electrodes has been investigated. The PT-PA and PT-PEO1000 are reacted with 2-aminoethanethiol (AET), followed by the formation of a self-assembled monolayer (SAM) onto the Au surface. The PT group immobilized on the SAM of AET acts as an effective mediator for the electron transfer (ET) between the electrode and the FAD center of freely diffusing GOx in solution. The ET rate constant estimated from the catalytic current using a newly derived equation is larger by 1 order of magnitude for the PT-PA-modified system (1.1 x [l0.sup.5] d[m.sup.3] [mo1.sup.-1] [s.sup.-l]) than for the PT-PEO1000 system (1.4 x [10.sup.4] d[m.sup.3] [mol.sup.-1] [s.sup.-l]). The order of the magnitude of the ET rate constant clearly contrasts with the GOx hybrid systems that we previously investigated (Anal. Chem. 2003, 75, 910-917), in which the presence of the PEO spacer enhances the ET reaction rate. The reduction in the apparent PT concentration at the electrode interface due to the high mobility of the PEO chain, leading to low efficiency in the formation of an enzyme-mediator complex, is a possible reason for the lower mediation ability of PT-PEO1000 than that of PT-PA for the ET between the FAD group and P[T.sup.+] immobilized on the electrode. Inhibition of the penetration of GOx molecules into the monolayer and of the accessibility of some part of PT groups to GOx molecules could also be reasons for the lower mediation ability of PT-PE01000 thickly modified on the electrode.

Mediated biosensors consisting of an oxidase and peroxidase (POx) have attracted increasing attention because of their wide applicability. However, since most of oxidases utilize artificial electron acceptors in place of dioxygen, the competition between [O.sub.2] and the electron acceptor in the mediated sensors is anticipated. This has been evidenced with a glucose oxidase (GOx)- and POx-coentrapped and ferrocene-embedded carbon paste electrode, which exhibits peak-shaped current-time curves at increased concentrations of glucose and also gives a peak-shaped calibration curve. Digital simulation has been applied to clarify the cause of such unusual responses, by taking into account the ping-pong enzyme kinetics on two- and three-substrate models for POx and GOx, respectively. The simulation has well reproduced such unusual responses and has clearly revealed that the depletion of [O.sub.2] in the enzyme layer is the most important factor responsible for such unusual responses. To overcome such a drawback of oxidase/POx bienzyme sensors, it is expected to be essential to decrease the rate of the oxidase reaction. In contrast, increase in the POx activity is useful to improve the sensitivity. According to the simulation-based expectation, the GOx and POx concentrations in the bienzyme sensor are adjusted to exhibit normal behavior with high sensitivity.

Kinetic determination of protein redox potentials with a mediated continuous-flow column electrolytic spectro-electrochemical technique (CFCESET) is described. In this method, the redox state of the mediator is completely regulated by the continuous-flow column electrolysis, and the homogeneous redox reaction between the mediator and a protein sample in the column is monitored spectroscopically at the downstream of the column. The protein/mediator reaction is in the pseudo-first-order kinetics, and then the rate equation is analytically solved. The kinetic analysis provides the protein redox potential as well as the homogeneous rate constant. In the kinetic measurements, equilibration of the system within the column is not required, which allows the use of increased kinds of mediators. This method was successfully applied to quinoprotein methylamine dehydrogenase containing tryptophan tryptophylquinone (TFQ) as a prosthetic group. The kinetic aspect is also valuable for the thermodynamic analysis with the mediated CFCESET. The half-life time of the kinetics can be utilized to optimize the system for the attainment of the equilibrated state within the column and can provide the assurance that the system is in equilibrium.

An electrode modified with immobilized whole cells of Desulfovibrio vulgaris (Hildenborough) produces an S-shaped voltammogram with both cathodic- and anodic-catalytic-limiting currents in a methyl viologen-containing buffer saturated with [H.sub.2]. Methyl viologen penetrates into the bacterial cells to serve as an electron carrier in the reversible reaction of hydrogenase in the cells and functions as an electron-transfer mediator between the bacterial cells and the electrode, thus producing the catalytic currents for the evolution and consumption of [H.sub.2]. An equation for the catalytic current that takes into account the reversible hydrogenase reaction explains well the shape of the voltammogram. The potential at null current on the voltammogram agrees with the potential determined by potentiometry with the same electrode, which is equal to the redox potential of the [H.sup.-]/[H.sub.2] couple in the solution-the standard potential of a hydrogen electrode at the pH of the solution. When D. vulgaris cells are suspended in an argon-saturated buffer containing methyl viologen, the suspension produces a catalytic current at a bare glassy carbon electrode for the evolution of [H.sub.2]. Analysis of the current by a theory for a catalytic current for a unidirectional nonlinear enzyme catalysis allows us to determine the kinetic parameters of the reaction between methyl viologen and hydrogenase in intact D. vulgaris cells. Thus we obtain the apparent Michaelis constant for methyl viologen cation radical, [K[prime].sub.[MV.sup.+]] = 0.16 mM, and the apparent catalytic constant (that is, the turnover number per D. vulgaris cell), [zk.sub.cat,[H.sup.+]] = 1.2 x [10.sup.7] [s.sup.-1], for the [H.sub.2] evolution reaction at pH 5.5 and at 25 [degrees] C, z being the number of hydrogenases contained in a D. vulgaris cell. The bimolecular reaction rate constant, [k.sub.cat,[H.sup.+]]/[K[prime].sub.[MV.sup.+]], of the reaction between methyl viologen cation radical and oxidized hydrogenase in intact D. vulgaris cells is estimated as 4.2 x [10.sup.7] [M.sup.-1] [s.sup.-1]. Similarly, the bimolecular reaction rate constant, [k.sub.cat,[H.sub.2]]/[K[prime].sub.[MV.sup.2]], of the reaction between methyl viologen and reduced hydrogenase is estimated to be 1.2 x [10.sup.7] [M.sup.-1] [s.sup.-1] at pH 9.5 and 25 [degrees] C. Both rate constants are large enough for the reactions to be diffusion-limited processes.

A mediator-assisted continuous-flow column electrolytic spectroelectrochemical method was designed to study redox properties of proteins. This method is based on a flow-through column electrolytic control of a redox buffer containing relatively high concentrations of mediators and an accelerated reaction of a protein with the mediators to reach equilibrium within the electrolysis time. A small amount of a protein sample is introduced into the electrochemically regulated redox buffer in the mode of flow injection analysis (FIA). The equilibrated redox state of the protein is evaluated by a highly sensitive flow-through photodiode array detector. Reproducible and stable background spectra and the employment of the FIA mode allow precise background subtraction in spectral analysis. This method was successfully applied to determining the redox potentials of horseradish peroxidase (HRP) using potassium hexachloroiridate as a mediator. The absorbance versus the electrode potential curves for the redox reactions among the ferric form, compound II, and compound I of HRP were well interpreted by a Nernstian equation based on the two-step one-electron-transfer model. The advantage and the kinetic aspects of this method are discussed in detailed.

Separation of hydrophobic cations in capillary electrophoresis under the mixed micellar system composed of SDS and Tween 20 was investigated from the viewpoints of thermodynamics and practical application. Hydrophobic cations interact strongly with the anionic SDS micelle, and this often lead to predominant dissolution of the analytes into the micellar phase, resulting in poor resolution. The ionic interaction was evaluated to be close in strength to the hydrophobic interaction between benzenes and the SDS micelle. Tween 20 as a component to the mixed micelle was found to weaken the attractive ionic interactions between the cationic solutes and SDS. In addition, the polyether chain of Tween 20 serves also as a hydrogen acceptor to cause the attractive hydrogen-bonding interactions with hydrogen-donating analytes. The two different functions of Tween 20 improve the separation of hydrophobic cations remarkably. Separation patterns are well controlled by varying the mixing ratio of the two surfactants. Adrenaline and its six precursors were successfully separated with this mixed micellar system. The improved separation efficiency was not affected by the presence of bovine serum matrix, realizing the direct MEKC analysis of serum sample without deproteination, in which proteins were comigrated with the micelle.

The oxidation of D-glucose and nicotinic acid by intact cells of Gluconobacter industrius and Pseudomonus fluorescens, respectively, is successfully measured by an amperometric method using such compounds as Fe(CN)[6.sup.3-], p-benzoquinone, and dichlorophenolindophenol as electron acceptors. Analysis of the experimental results reveals that the intact cells behave like oxido-reductases whose kinetics follows a Michaelis-Menten-type equation. The catalytic behavior is explained by a model which treats the bacterial cells as bags of enzymes and assumes distribution equilibrium in the concentrations of both the substrate and the electron acceptor between the test solution and the medium within the cells. The catalytic activity can be characterized by three quantities: the maximum reaction rate ([v.sub.B]) and the ratios of the Michaelis constant to the distribution constant for the substrate ([K.sub.s,cell]/[K.sub.s,p]) and to that for the electron acceptor ([K.sub.M,cell]/[K.sub.M,p]). Advanced modification of the model to involve the membrane permeability reveals that the three quantities are effective for explaining the catalytic behavior even when the permeability effect is significant. Thus, the three quantities should be regarded as the parameters which can reflect the permeability effect.

Reversible and quasi-reversible cyclic voltammograms of 9,10-phenanthrenequinone, 1,4-benzoquinone, chloranil, 1,4-naphthoqulnone, 9,10-anthraqulnone, and 5,12-naphthacenequlnone adsorbed on basal-plane pyrolytic graphite electrodes have been theoretically analyzed in view of a one-step two-electron mechanism and a two-step one-electron mechanism. In each mechanism, the Frumkin-type interaction between the adsorbed molecules is taken into account and curvefitting analysis of current-potential curves is presetned. Detalled analysis has revealed that the electrochemistry of the adsorbed quinones can be well interpreted on the basis of the two-step mechanism rather than the one-step mechanism., The theoretical characterization of reversible and quasi-reversible cyclic voltammograms of basal-plane pyrolytic graphite electodes-adsorbed quinone compounds by a one-step two-electron mechanism and a two-step one-electron mechanism is discussed. Comparison of the two mechanisms demonstrate that the two-step mechanism is better suited for adsorbed quinone electrochemistry rather than its one-step counterpart.