The flexibility of DNA is of central importance in biology, medicine, materials science, and mechanical engineering. In this study, we report an unprecedented electrochemical approach for investigating the flexibility of a short (typically 20-base), surface end-tethered single-stranded synthetic DNA oligonucleotide and of its postformed DNA duplex, taking as an example the homopolymer (dT)20 sequence in the regime of very high ionic strength ( approximately 1 M).

In the first part of the study, PEG(3400)-Fc linear polymeric chains bearing an electrochemically active ferrocene (Fc) head at their loose end are terminally attached at the gold electrode surface. The surface concentration of grafted polymeric chains is measured electrochemically under good solvent (water) conditions, and in the present work, it appears that the electrode is covered with terminally attached PEG chains in the mushroom configuration. The dynamics of the terminally attached chain can be completely characterized according to a model of elastic bounded diffusion which requires the knowledge of the diffusion coefficient D of the loose end and a spring constant, k(spr), that is entropic in nature. Both D and k(spr) are determined. In the second part, the electrode is coated with terminally attached PEG(3400) and PEG(20000) chains which are not electrochemically active. The rate constants of penetration and escape of mobile HO-PEG(3400)-Fc chains in and out of the polymeric coating were determined together with the partition coefficient of HO-PEG(3400)-Fc between the coating and the surrounding aqueous solution.

A new approach for the self-assembly of supported and tethered lipid membranes of large surface area is proposed. The template is a microporous electrode made by anodic etching of aluminum and covered with a monolayer of streptavidin. We show that spontaneous fusion of biotinylated lipid vesicles on the affinity layer is a slow process despite abundant accumulation of lipid material at the template surface. To increase dramatically the efficiency of the self-assembly, fast fusion is provoked with the help of a fusogen solution of poly(ethylene glycol). The extent of fusion is assessed by electrochemical monitoring of the long-range lateral mobility of ubiquinone (coenzyme Q10) in the supported bilayer. Finally, the geometrical characterization of the honeycomb structure at key steps of the self-assembly procedure is performed by electrochemical measurement of the porosity. As expected, the formation of the supported bilayer causes a decrease in the apparent inner diameter of the pores. It is expected that the type of supported lipid membrane built according to the present approach can be adequate for the incorporation of transmembrane proteins in structures that would mimic the membrane stacking found in chloroplasts or mitochondria.

A method for fabricating submicrometer-sized gold electrodes of conical or spherical geometry is described. By generating an electric arc between an etched gold microwire and a tungsten counter electrode, the very end of the gold microwire can be melted and given an overall spherical or conical shape a few hundred nanometers in size. The whole wire is subsequently insulated via the cathodic deposition of electrophoretic paint. By applying a high-voltage pulse to the microwire, the film covering its very end can then be selectively removed, thus exposing a submicrometer-sized electrode surface of predefined geometry. The selective exposure of the preformed end of the microwire is demonstrated by cyclic voltammetry, scanning electron microscopy, and metal electrodeposition experiments. The electrophoretic paint coating provides a low-capacitance, robust insulating film allowing exploration of a very wide potential window in aqueous solution. The submicrometer-sized electrodes can easily be turned into probes suitable for combined scanning electrochemical-atomic force microscopy by bending and flattening the gold microwire so that the tip is borne by a flexible enough arm. The good agreement between theoretical and experimental scanning electrochemical microscopy approach curves thus obtained confirms that only the very end of the tip, of predefined geometry, is exposed to the solution.

The catalytic response of an immobilized redox enzyme connected to the electrode by a freely diffusing mediator (cosubstrate) may be kinetically controlled by the substrate and/or the cosubstrate. How the electrochemical responses are related to the rate constants, to the amount of enzyme on the electrode, to the substrate and cosubstrate concentration and to the mass transport parameter is systematically analyzed in the framework of cyclic voltammetry and steady state techniques (SST). Because of its frequent occurrence in practice, emphasis is put on the case of a fast enzymatic process, as compared to the diffusion of the cosubstrate, provision being made for Michaelis–Menten behavior for both substrate and cosubstrate. Within this framework, two situations of particular interest are discussed, namely the case of a negligible consumption of the substrate in the enzyme coating and the opposite case where the consumption of the substrate is so important that its diffusion toward the electrode controls the current. In the first case, plateau-shaped responses, independent of scan rate, are obtained in cyclic voltammetry, and likewise mass transport independent waves, in SST. In the second case, a curve exhibiting a sharp, discontinuous peak is obtained in cyclic voltammetry with a peak current proportional to the substrate concentration and square root of the scan rate. A discontinuity also appears in SST between the rising portion of the wave and the plateau current while the current is likewise proportional to substrate concentration. The combination of these two regimes accounts for a biphasic variation of the electrochemical signal with the substrate concentration. The relationships between the electrochemical responses and the kinetic characteristics of the enzymatic reaction form the bases of procedures for ascertaining the mechanism and measuring the key rate constants. In this connection, strategies for determining Michaelis–Menten characteristics of both the substrate and cosubstrate reactions are discussed.

The target ferrocene-labeled dideoxynucleotide compound 5-[N-(beta-ferrocenyl-propanoyl)3-amino-propyn-1-yl]-2',3'-dideoxyuridine 5'-triphosphate, Fc-ddUTP, was synthesized and tested with terminal deoxynucleotidyl transferase for enzymatic 3'-redox-active end-labeling of 5'-phosphorylated single-stranded oligodeoxynucleotides. Starting from readily available 5-iodouridine and 3-ferrocenylpropanoic acid, the synthetic strategy elaborated here follows a mild multistep route. Each step involves reliable methods, and all ferrocene intermediates can be easily purified. Enzymatic 3'-ferrocene end-labeling of 5'-phosphorylated oligonucleotides is remarkably efficient, and 3'-ferrocene-labeled oligonucleotides can thus be prepared in sufficient amounts for further use in surface modifications.

The functionality of the membrane-bound, ubiquinone-dependent pyruvate oxidase from the respiratory chain of Escherichia coli was reconstituted with a supported lipidic structure. The artificial structure was especially designed to allow the electrochemical control of the quinone pool through the lateral mobility of the ubiquinone (Q(8)) molecules. The kinetic coupling of the enzyme bound to the lipid structure with the quinone pool was ensured by the regeneration of the oxidized form of ubiquinone at the electrochemical interface. Such an experimental approach enabled us to carry out an unprecedented determination of the kinetic parameters controlling the reaction between the enzyme bound and the electron carrier under conditions taking rigorously into account the fact that the freedom of motion is restricted to two dimensions. The kinetic constants we found show that the activated enzyme can be efficiently regulated by the oxidation level of the quinone pool in natural membranes.

Molecular monolayers of immunoglobulins bearing terminally attached ferrocene poly(ethylene glycol) chains (IgG-PEG-Fc) were self-assembled at an electrode surface in a step-by-step manner involving antigen-antibody recognition reactions. The total number N of assembled IgG-PEG-Fc monolayers and the number of spacers n(i) separating two successive IgG-PEG-Fc monolayers were controlled and varied. Electron transport through the protein assembly involves the dynamics of the terminally attached PEG chains and isotopic electron exchange between ferrocene heads belonging to successive IgG-PEG-Fc monolayers. The model of elastic bounded diffusion enabled us to analyze quantitatively the dependence of the rate of electron transport on N, n(i), and the rate constant (k(e)) of isotopic electron exchange. Wiring of a molecular monolayer of redox enzyme is also quantitatively characterized.

A precise determination of the complex mechanism of catalysis and inhibition involved in the reaction of HRP with H(2)O(2) as substrate and an outersphere single electron donor ([Os(bpy)(2)pyCl](+)) as cosubstrate is made possible by a systematic analysis of the cyclic voltammetric responses as a function of the scan rate and of the substrate and cosubstrate concentrations, complemented by spectrophotometric steady-state and stopped-flow experiments. The bell-shaped calibration curve relating the electrochemical response to the concentration of H(2)O(2) is qualitatively and quantitatively explained by taking into account the conversion of the catalytically active forms of the enzyme into the inactive oxyperoxidase in addition to the primary catalytic cycle. These characteristics should be kept in mind in biosensor applications of HRP. The ensuing analysis and data allow one to predict biosensor amperometric responses in all practical cases. From a mechanistic standpoint, conditions may, however, be defined which render inhibition insignificant, thus allowing an electrochemical characterization of the primary catalytic cycle. At very low concentrations of H(2)O(2), its diffusion tends to control the electrochemical response, resulting in proportionality with H(2)O(2) concentration instead of the square root dependence characteristic of the classical catalytic currents. Intriguing hysteresis and trace crossings behaviors are also quantitatively explained in the framework of the same mechanism. As a consequence of the precise dissection of the rather complex reaction mechanism into its various elementary steps, a strategy may be devised for gaining a better understanding of the mechanism and reactivity patterns of each elementary step.

Application of antigen-antibody technology allows the attachment to an electrode surface of an enzyme monolayer structure to which both the enzyme and the mediator are bound. As illustrated with the example of glucose oxidase and a ferrocene mediator, the enzyme preserves its full activity in such structures, which may be easily reproduced. In spite of their fixation to the structure, the mobility of the ferrocene heads is sufficient to ensure that its transport to the enzyme prosthetic group is not rate determining. The reaction is rather controlled by the prior formation of a complex between the ferrocenium ion and the flavin required for electron transfer to occur. The efficiency of this step is affected by steric hindrance and the various observations made with free-moving and attached ferrocene-ended poly(ethylene glycol) chains may be rationalized by the interplay of factors controlling their distribution and shape. Analyzing the dynamics of this system, in comparison with previous systems, was thus an occasion to shed further light on the recognition phenomenon. The enzyme monolayer integrated system is a good starting point for the step-by-step construction of spatially ordered multilayered assemblies with strong catalytic efficiencies. Fast responding systems are expected both in terms of electron transport and electron transfer between the mediator and the enzyme. The spatial order resulting from the step-by-step construction should allow a much more precise analysis of electron transport and electron transfer than in conventional assemblies of redox centers. Mastering both the construction and the functioning of such systems should help the design of more complex systems, integrating additional functionalities electrically controlled by means of their electron transport/electron transfer connection to the electrode surface.

Two methods based on the avidin-biotin technology were developed for the multimonolayer immobilization of Desulfovibrio gigas hydrogenase on glassy carbon or gold electrodes. In both methods the molecular structure of the modified interface was the result of a step-by-step process. The first method alternates monolayers of avidin and biotinylated hydrogenase, the mediator (methyl viologen) being free to diffuse in the structure. In the second method, the avidin monolayers were used to immobilize both the biotinylated enzyme and a long-chain biotinylated viologen derivative. The viologen head of this hydrophilic arm shuttles the electrons between the electrode and the enzyme. The modified electrodes were evaluated for the electroenzymatic oxidation of molecular hydrogen, which has interest for the development of enzymatic fuel cells. The parameters that affect the current density of mediated oxidation of H2 at the modified electrodes was studied. The second structure, which has given typical catalytic currents of 25 μA per cm2 for 10 monolayers, was found clearly less efficient than the first structure (500 μA per cm2 for 10 monolayers). In both methods the catalytic currents increased linearly with the number of monolayers of hydrogenase immobilized, which indicates that the multilayer structures are spatially ordered. © 2000 John Wiley & Sons, Inc. Biotechnol Bioeng 68: 1–10, 2000.

Enzymatic electrocatalysis, triggered and monitored by means of cyclic voltammetry, enabled us to achieve quantitative analysis of the kinetics of the hydrogenase catalyzed process, in the 7.8-10.0 pH range, in the presence of an electrochemically generated redox mediator. The quantitative analysis can be carried out by use of a quite simple SRC model. The simplicity of the SRC model is compatible with the existence of multiple redox microstates, which can be combined in a potential adjustable triangular mechanism consisting of three catalytic cycles, which are formally identical from the kinetic point of view. The steps involved in the kinetic control of the reversible process are H2 uptake or production at the Ni-Fe catalytic site and the intermolecular electron transfer between the mediator and the distal [4Fe-4S] cluster. The related rate constants have been determined. For the two accompanying intramolecular electron transfers which proceed at equilibrium, the equilibrium constants were found to be in very good agreement with previously published data.

ADVERTISEMENT RETURN TO ISSUEPREVNoteNEXTIs There a Deuterium Kinetic Isotope Effect in the One-Electron Transfer from 1-Benzyl-1,4-dihydronicotinamide to 9-Fluorenylidenemalononitrile?Agnès Anne, Jacques Moiroux, and Jean-Michel SavéantView Author Information Laboratoire d'Electrochimie Moléculaire, Unité Mixte de Recherche Université-CNRS No 7591, Université de Paris 7 - Denis Diderot, 2 place Jussieu, 75251 Paris Cedex 05, France [email protected]Cite this: J. Org. Chem. 2000, 65, 21, 7213–7214Publication Date (Web):September 15, 2000Publication History Received27 April 2000Published online15 September 2000Published inissue 1 October 2000https://pubs.acs.org/doi/10.1021/jo000651dhttps://doi.org/10.1021/jo000651dbrief-reportACS PublicationsCopyright © 2000 American Chemical SocietyRequest reuse permissionsArticle Views180Altmetric-Citations3LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InRedditEmail Other access optionsGet e-Alertsclose SUBJECTS:Anions,Hydride transfer,Molecules,Redox reactions,Transfer reactions Get e-Alerts

An electrochemical method is proposed for investigating the dynamics of recognition between a biomolecule and an immobilized receptor. It involves redox labeling of the solute molecule and monitoring the binding by the electrochemical response of the electrode onto which the receptor is immobilized. With large biomolecules, as, for example, antigens and antibodies, leading to small surface concentrations, simple redox labeling may prove insufficient to obtain detectable responses. Redox enzymes are then advantageously used as labels thanks to the signal amplification offered by their catalytic properties. The applicability of the method is illustrated by the reaction of an immobilized monolayer of goat IgG antigen (or of one Fab fragment) with an antigoat antibody labeled with glucose oxidase. Particular care is taken to free the kinetic data from the effect of diffusion. The latter factor may interfere whatever the detection technique. A full account of the combination between recognition kinetics and diffusion is therefore given in terms of a kinetic zone diagram leading to diagnostic criteria and data processing procedures that allow a proper extraction of the recognition thermodynamic and kinetic constants. The theory applies as well to the dynamics of adsorption of nonbiological molecules on surfaces.

Avidin-biotin technology is used to achieve the step-by-step construction of electrode coatings in which two monomolecular layers of biotinylated hexokinase are immobilized on top of five monomolecular layers of biotinylated glucose oxidase. The two enzymes compete for the consumption of glucose. Because the reaction of hexokinase with glucose depends on the presence and concentration of ATP in the solution, the electrochemical response is sensitive to the ATP concentration. Such a system illustrates the possibility of translating the catalytic activity of a nonredox enzyme into an electrical signal within a spatially ordered structure. Full kinetic analysis of the electrochemical responses allowed a description of the diffusion controlled communication between the two enzymes over distances that are comparable to those involved in enzyme coupling in cell cytoplasmic systems.

Cyclic voltammetry was used for systematic analysis of the diffusion of a bulky mono ferrocene poly(ethylene glycol) ((PEG-Fc, MW ca, 3400 g/mol) redox probe through a film made of N successively self-assembled monolayers of immunoglobulins immobilized at the electrode surface. A membrane-like model of the film is considered. A theoretical approach is developed showing that, in the general case, five parameters are involved in the control of the process. The influence of each parameter and the possible limiting situations are discussed. Quantitative information is derived from the changes appearing in the experimental cyclic voltammograms when N increases and/or when the potential scan rate is varied over 3 orders of magnitude. The PEG-Fc probe is bully enough to sense the presence of the IgG self-assembly. Its diffusion coefficient through the membrane-like film is roughly 2 orders of magnitude smaller than in solution. The diameter of the channels which exist between the self-assembled IgG layers is not much larger than the 4.2 nm hydrodynamic diameter of the PEG coil.

Many enzymes may be inhibited by the products of the reaction they catalyze by means of a Michaelis-Menten kinetic retro-action. Protons, which are involved as products or reactants in a number of cases, may also influence the enzymatic kinetics. The course of the reaction may therefore be altered by the attending production or depletion of protons. These effects are expected to become particularly important in multilayered films developing high catalytic efficiencies. In the case of redox enzymes, connecting the prosthetic group with an electrode by means of a redox cosubstrate allows a quantitative analysis of the kinetics of product inhibition by means of electrochemical techniques such as cyclic voltammetry. This approach is illustrated with the example glucose oxidase electrode coatings obtained by successive antigen-antibody attachment of a series of monomolecular layers. The kinetics of the inhibition by gluconolactone and the depiction of the pH gradients across the enzyme film could thus be derived, after the appropriate theory was established, from the hysteresis exhibited by the forward and reverse current traces. The theory, derived for the case of successive monomolecular layers, can be easily extended to more disordered enzyme assemblies. A general Strategy is thus made available that allows a full description of the space-dependent dynamics of the system nor: only for the primary species of the enzymatic catalysis but also for secondary species chat may be involved in feedback processes.

A model of elastic bounded diffusion is presented for the quantitative analysis of the dynamics of redox probes borne by the loose ends of linear and flexible polymeric chains terminally grafted to building blocks of a self-assembled construction. Experimentally, poly(ethylene glycol) chains bearing a ferrocene probe were terminally attached at the last one of a series of immunoglobulins monolayers successively self-assembled on top of a glassy carbon electrode. In cyclic voltammetry, the creation of a concentration gradient provoked a diffusion-like displacement of the ferrocene probe counterbalanced by the springlike elasticity of the terminally attached polymeric chain. The morphology and the intensity of the signal depend markedly on the distance separating the terminal attachment from the electrode surface and on the potential-scan rate. The observed changes are qualitatively and quantitatively justified by the model. Both the diffusion and the elasticity can be quantitatively characterized.

Electrodes bearing terminally attached PEG(600)-Fc and PEG(3400)-Fc chains exhibit remarkably similar time responses in cyclic voltammetry. Two asymmetrical NHS-PEG(600)-Fc and NHS-PEG(3400)-Fc poly(ethylene glycol) (PEG) chains of markedly different lengths were synthesized, and their activated N-hydroxysuccinimide ester (NHS) end was used for terminal attachment to the surface of a glassy carbon electrode. A similar surface coverage was obtained with both derivatives. At this coverage, the attached NHS-PEG(600)-Fc, which are relatively small, were not constrained to overlap whereas the attached NHS-PEG(3400)-Fc were necessarily stretched. The presence of the ferrocene (Fc) redox probe gave rise to signals in cyclic voltammetry which were analyzed quantitatively. At sufficiently high potential scan rate the peak currents reflect the flexibility of the terminally attached PEG chains. Both types of derivatized electrodes gave remarkably similar peak currents in cyclic voltammetry.