A composite electrode made by association of gold and porous aluminum oxide has been used as template for a supported lipid bilayer. An oxide pore size of 170nm was chosen so that lipid vesicles or proteoliposomes can enter in the pore and cover the pore wall. Triggered vesicle fusion produced a stable and continuous lipid bilayer supported on the large surface area of the honeycomb structure. Continuity of the supported bilayer was attested by electrochemical measurement of the lateral mobility of ubiquinone, a water-insoluble electroactive marker, dissolved at low concentration in the hydrophobic leaflet of the bilayer.

31P and 1H solid-state nuclear magnetic resonance (NMR) experiments have been designed with the aim of studying directly the formation of supported bilayers tethered inside nanoporous aluminum oxide supports as a model of biomimetic membranes. The static and magic angle spinning 31P NMR spectra of the supported bilayers have been compared with the experimental and simulated spectra of a simpler model with cylindrical geometry, namely a phospholipid bilayer adsorbed on an oriented polymer sheet. The broadening observed for the nanoporous model is most likely due to the presence of paramagnetic ions in the aluminum oxide. A phospholipid lateral diffusion coefficient of (2.8 ± 0.4) × 10−8 cm2/s has been measured for the tethered bilayer on a spherical support, indicating a good fluidity as compared with adsorbed membrane models.

A general procedure for the formation ofsolid-supported artificial membranes containing transmembrane proteins is reported. The main objective was to directly use the pool of proteins of the native biomembrane (here the inner membrane from mitochondria of human carcinogenic hepatic cells) and to avoid purification steps with detergent. Proteoliposomes of phospholipid-enriched inner membranes from mitochondria were tethered and fused onto a tailored surface via a streptavidin link. The failure of some preliminary experiments on membrane formation was attributed to strong nonspecific interactions between the solid surface and the protuberant hydrophilic parts of the transmembrane complexes. The correct loading of uniform membranes was performed after optimization of a tailored surface, covered with a grafted short-chain poly(ethylene glycol), so that nonspecific interactions are reduced. Step-by-step assembly of the structure and triggered fusion of the immobilized proteoliposomes were monitored by surface plasmon resonance and fluorescence photobleaching recovery, respectively. The long-range lateral diffusion coefficient (at 22 degrees C) for a fluorescent lipid varies from 2.5 x 10(-8) cm2 s(-1) for a tethered lipid bilayer without protein to 10(-9) cm2 s(-1) for a tethered membrane containing the transmembrane proteins of the respiratory chain at a protein area fraction of about 15%. The decrease in the diffusion coefficient in the tethered membrane with increase in protein area fraction was too pronounced to be fully explained by the theoretical models of obstructed lateral diffusion. Covalent tethering links with the solid are certainly involved in the decrease of the overall lateral mobility of the components in the supported membrane at the highest protein-to-lipid ratios.

A light-activated electron-transfer chain was assembled using solubilized cyanobacterial photosystem I as photoactive enzyme, cytochrome c(6) (also from cyanobacteria) as electron donor, and methyl viologen as electron acceptor. The photocatalytic activity of the ensemble was measured by direct and reversible electrochemistry of cytochrome c(6) at a surface-modified gold electrode. Analysis of the electrochemical response with an appropriate model for the reaction mechanism allowed the relation of the overall catalytic reaction rate to the individual steps of the catalytic cycle. Second-order rate constants were determined for the first time under steady-state conditions. The results validate this approach as an efficient method for the study of electron transfer between photoactive enzymes and their redox partners.

We have used fluorescence microscopy, fluorescence photobleaching recovery (FPR), and atomic force microscopy (AFM) to investigate the formation of tethered lipid bilayers on plane aluminum oxide or glass surfaces. The bilayers were assembled with the help of a two-step methodology recently proposed for microporous templates (Proux-Delrouyre et al. J. Am. Chem. Soc. 2001, 123, 8313). The first step consists of the accumulation of intact biotinylated vesicles (PC + DOPE) on a streptavidin sublayer itself immobilized on the substrate. The second step, clearly time separated, is the deliberate triggering of bilayer formation with the help of poly(ethylene glycol) (PEG), a fusion agent of lipidic vesicles. AFM and FPR measurements confirm that the vesicles do not spontaneously fuse during the first step provided that the streptavidin sublayer is present on the substrate. On the contrary, the treatment with PEG provokes the fast formation of a continuous lipid bilayer, as attested at the hundred nanometer scal...

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.

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.

ADVERTISEMENT RETURN TO ISSUEPREVCommunicationNEXTFormation of Streptavidin-Supported Lipid Bilayers on Porous Anodic Alumina: Electrochemical Monitoring of Triggered Vesicle FusionVanessa Proux-Delrouyre, Jean-Marc Laval, and Christian BourdillonView Author Information Laboratoire de Technologie Enzymatique Unité associée au CNRS #6022 Université de Technologie de Compiègne, B.P. 20529 60205 Compiègne Cedex, France Cite this: J. Am. Chem. Soc. 2001, 123, 37, 9176–9177Publication Date (Web):August 22, 2001Publication History Received9 February 2001Published online22 August 2001Published inissue 1 September 2001https://pubs.acs.org/doi/10.1021/ja010361uhttps://doi.org/10.1021/ja010361urapid-communicationACS PublicationsCopyright © 2001 American Chemical SocietyRequest reuse permissionsArticle Views569Altmetric-Citations34LEARN 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-AlertscloseSupporting Info (1)»Supporting Information Supporting Information SUBJECTS:Electrodes,Interfaces,Oxides,Vesicle fusion,Vesicles Get e-Alerts

Small-angle neutron scattering (SANS) is used to characterize a phospholipid/alkoxysilane hybrid bilayer membrane (HBM), a model of biological membrane, supported in anisotropic porous alumina (Al(2)O(3)). The bilayer is obtained by fusion of phospholipid vesicles with a hydrophobic alkoxysilane monolayer chemically bound to the microporous alumina support. We first characterized the bare alumina material, then the alkoxysilane (OTS) layer bound to alumina, and finally the hybrid bilayer. By orienting the anisotropic support, we show that the intensity can be considerably increased, enabling the scattering to be measured in a wide q range (6 x 10(-4) - 0.5 Angstrom (-1)) corresponding to 9-10 decades in intensity and down to 10(-4) cm(-1). This enables us to cover the structure factor of the oxide at large scale, the wide Porod regime, and the membrane form factor. Analysis of the scattering curves indicates that both the OTS layer and the HBM produce very smooth, uniform, and continuous layers at the alumina/solvent interface. This new approach in the characterization by SANS of a supported membrane in a porous material provides information on the homogeneity, the specific area, the roughness, and the thickness of the bilayer.

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.

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.

The dynamic and steady-state behaviors of three coupled substrate cycles sharing interconversion enzymes are investigated in a homogeneous flow-through reactor (CSTR). Lactate dehydrogenase (LDH) converts pyruvate (Prv) and NADH into lactate (Lac) and NAD, respectively. In turn, NAD [and glucose 6-phosphate (Glc6P)] is recycled into NADH (and gluconolactone 6-phosphate) by glucose 6-phosphate dehydrogenase, and in the presence of ferricyanide (Ferri), Lac is reoxidized into Pry [and ferrocyanide (Ferro)]. Finally, Ferro is reoxidized in turn by a reticulated vitreous carbon (RVC) electrode poised at a controlled potential in a three-electrode configuration. Under thermodynamically open conditions with a constant supply of Pry, Ferri, NADH, and Glc6P, this multienzyme system exhibits irreversible transitions between alternative stable steady states (bistability without hysteresis) when the electrochemical rate of ferrocyanide recycling is varied. This nonlinear behavior results from the strong inhibition of LDH exerted by its substrate Pry. In the absence of an electrochemically driven recycling of ferrocyanide, only reversible bistability (dynamic hysteresis) may be observed [Simonet et al. J. Phys. Chem. 1996, 100, 19148]. The numerical predictions of a simple mathematical model taking into account the coupling between the actual enzyme rate equations, mass transfers, and electrochemical recycling agree both qualitatively and quantitatively with the observed experiments.

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.

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.

The avidin-biotin technology may be used successfully to build stable and spatially ordered assemblies of monomolecular enzyme layers on surfaces as illustrated with the example of glucose oxidase and glassy carbon electrodes. With ferrocene methanol as the cosubstrate, cyclic voltammetry allows a detailed analysis of the catalytic responses, thus providing a demonstration of the spatial order of the multilayer structure and an estimate of the average distance between monomolecular layers. The respective advantages of the avidin-biotin and antigen-antibody technologies are discussed.

The long-range diffusion coefficients of isoprenoid quinones in a model of lipid bilayer were determined by a method avoiding fluorescent probe labeling of the molecules. The quinone electron carriers were incorporated in supported dimyristoylphosphatidylcholine layers at physiological molar fractions (<3 mol%), The elaborate bilayer template contained a built-in gold electrode at which the redox molecules solubilized in the bilayer were reduced or oxidized. The lateral diffusion coefficient of a natural quinone like UQ(10) or PQ(9) was 2.0 +/- 0.4 x 10(-8) cm(2) s(-1) at 30 degrees C, two to three times smaller than the diffusion coefficient of a lipid analog in the same artificial bilayer. The lateral mobilities of the oxidized or reduced forms could be determined separately and were found to be identical in the 4-13 pH range. For a series of isoprenoid quinones, UQ(2) or PQ(2) to UQ(10) the diffusion coefficient exhibited a marked dependence on the length of the isoprenoid chain. The data fit very well the quantitative behavior predicted by a continuum fluid model in which the isoprenoid chains are taken as rigid particles moving in the less viscous part of the bilayer and rubbing against the more viscous layers of lipid heads. The present study supports the concept of a homogeneous pool of quinone located in the less viscous region of the bilayer.