Electrode-supported Lipid Bilayer Membrane Research Articles

Aptasensor for adenosine triphosphate based on electrode–supported lipid bilayer membrane

We have developed an aptasensor for adenosine triphosphate (ATP) based on an electrode-supported lipid bilayer membrane. The assay is based on a conformational change that is induced after binding the target which modulates the electron transfer rate in the conductive path. The method is highly sensitive, stable, and repeatable. The detection limit for ATP is 50 nM, and the dynamic range extends to 3.2 μM, which covers the concentration range of ATP in cell lysates (from 0.1 to 1 μM). The method also holds promise in that it may be transferred to submicro- or nano-scale electrodes so to enable intracellular monitoring of ATP.

Immobilization of cytochrome c oxidase into electrode-supported lipid bilayer membranes for in vitro cytochrome c sensing

Blood and tissue biochemical oxidation-reduction (redox) reactions are ubiquitous and are reflective of many important biological processes in the body, ranging from the state of cellular oxygenation to the overall antioxidant status. It is likely that, similar to acid-base balance, the body optimally operates within a narrow redox potential range made possible by various homeostatic mechanisms, and that deviation from this range will occur in tissue damage. A means to monitor the redox potential of blood or tissue would be valuable in both the diagnosis and treatment of diseases or conditions that adversely affect the body's redox potential. The biosensor described herein involves the immobilization of bovine cytochrome c oxidase (CCO) into electrode-supported lipid bilayer membranes. As a first proof of concept, the biosensor was used to potentiometrically monitor the concentration ratio of a redox pair (oxidized and reduced cytochrome c) in an in vitro system. The response of this modified electrode is reproducible and exhibits Nernstian behavior consistent with the four-electron reduction of the CCO. The oxidase-modified electrode can also operate as an amperometric biosensor for the detection of solution-resident ferrocytochrome c at concentrations as low as 0.1 /spl mu/M. Because this biosensor uses an electron sensor native to the body, it may be of future value to explore the biosensor as a point of care test to measure blood redox potential or perhaps as an implantable sensor to measure tissue redox potential in many settings.

Characterization of cyanide binding to cytochrome c oxidase immobilized in electrode-supported lipid bilayer membranes

Bovine cytochrome c oxidase has been successfully immobilized in electrode-supported lipid bilayer membranes to investigate the effect of cyanide binding on the oxidation of ferrocytochrome c and the electroreduction of dioxygen. Cyanide binding to oxidase was found to be reversible and exhibited 1:1 stoichiometry. Binding constants ( K i) were also determined for binding of cyanide to the reduced (62 μM) and oxidized (195 μM) forms of the oxidase. The cytochrome c oxidase-modified electrodes described here could potentially be used as an amperometric biosensor for the detection of cyanide.

Electroreduction of Oxygen by Cytochrome c Oxidase Immobilized in Electrode‐Supported Lipid Bilayer Membranes

Cytochrome c oxidase is the terminal enzyme in mammalian respiration, and one of its main functions is to catalyze the reduction of oxygen under physiological conditions. Direct reduction of oxygen at electrodes requires application of substantial overpotentials. In this work, bovine cytochrome c oxidase has been immobilized in electrode-supported lipid bilayer membranes to investigate the electroreduction of oxygen under flow conditions. The effect that temperature, solution pH, and solution composition have on the reduction of oxygen by this novel enzyme-modified electrode is reported. Results indicate that the electroreduction of oxygen is most pronounced at low pH (6.4) and elevated temperature (38 degrees). At an applied potential of -350 mV vs. Ag/AgCl (1M KCl), a current density of ca. 7 microA/cm2 was obtained. The current responses obtained at these electrodes are stable over a period of ca. 10-14 days (10-15% decrease in response). The cytochrome c oxidase-modified electrodes described here could potentially be used for the direct electroreduction of oxygen to water in a biofuel cell.

Steady-state oxidation of cholesterol catalyzed by cholesterol oxidase in lipid bilayer membranes on platinum electrodes

Cholesterol oxidase is immobilized in electrode-supported lipid bilayer membranes. Platinum electrodes are initially modified with a self-assembled monolayer of thiolipid. A vesicle fusion method is used to deposit an outer leaflet of phospholipids onto the thiolipid monolayer forming a thiolipid/lipid bilayer membrane on the electrode surface. Cholesterol oxidase spontaneously inserts into the electrode-supported lipid bilayer membrane from solution and is consequently immobilized to the electrode surface. Cholesterol partitions into the membrane from buffer solutions containing cyclodextrin. Cholesterol oxidase catalyzes the oxidation of cholesterol by molecular oxygen, forming hydrogen peroxide as a product. Amperometric detection of hydrogen peroxide for continuous solution flow experiments are presented, where flow was alternated between cholesterol solution and buffer containing no cholesterol. Steady-state anodic currents were observed during exposures of cholesterol solutions ranging in concentration from 10 to 1000μM. These data are consistent with the Michaelis–Menten kinetic model for oxidation of cholesterol as catalyzed by cholesterol oxidase immobilized in the lipid bilayer membrane. The cholesterol detection limit is below 1μM for cholesterol solution prepared in buffered cyclodextrin. The response of the electrodes to low density lipoprotein solutions is increased upon addition of cyclodextrin. Evidence for adsorption of low density lipoprotein to the electrode surface is presented.

The reaction of cytochrome c with bovine and Bacillus stearothermophilus cytochrome c oxidase immobilized in electrode-supported lipid bilayer membranes

Stable lipid bilayer membranes containing cytochrome c oxidase from two sources (i.e. bovine and Bacillus stearothermophilus) have been immobilized on gold quartz crystal microbalance (QCM) electrodes. The immobilized oxidase can be characterized using direct electrochemical methods. Amperometric flow injection analysis data of reduced cytochrome c reacting at oxidase-modified electrodes indicate that the kinetics are non-hyperbolic, which agrees with previous results found in the literature. The kinetics of the cytochrome c oxidase/cytochrome c redox couple are characterized by a high and low affinity regime each having a distinct K m value. The rate of mediated electron transfer from solution-resident reduced cytochrome c to the electrode is also affected by ionic strength. Electrochemical and QCM results suggest that the fastest rates of electron transfer are observed when the ionic strength is low enough to allow facile binding between the protein and the enzyme but high enough to allow the protein to dissociate from the oxidase.

Detection of Cholesterol through Electron Transfer to Cholesterol Oxidase in Electrode-Supported Lipid Bilayer Membranes

Cholesterol oxidase is immobilized in lipid bilayer membranes assembled on tin-doped indium oxide electrode surfaces to sequester cholesterol from solution and to follow cholesterol oxidation via electrochemical reduction of hydrogen peroxide. The inner leaflet of the bilayer is chemically bound to the electrode surface through a thiol functionality at the polar headgroup end of the lipid. The outer lipid leaflet, containing cholesterol oxidase, is formed using a deoxycholate dialysis procedure. Continuous solution flow experiments, where the flow is changed from buffer solution containing no cholesterol to a buffer solution containing cholesterol, show currents for the reduction of hydrogen peroxide generated by the enzyme. The data indicate that cholesterol oxidase is immobilized on the electrode in an active state. The data are also consistent with energetically favored collection of cholesterol from solution by the electrode-supported lipid bilayer membrane.

The reaction of cytochrome c from different species with cytochrome c oxidase immobilized in an electrode supported lipid bilayer membrane

In past work the direct electron transfer reactions of bovine cytochrome c oxidase in an electrode-supported lipid bilayer membrane have been studied. Its reaction with cytochrome c in solution was also studied and found to be consistent with previous solution studies. In this work it is shown that the electron transfer reactions of cytochrome c oxidase in this electrode-supported lipid bilayer membrane depend on the source of cytochrome c. This property has also been widely studied for solution samples. The differences in the electron transfer reaction rates correlate with the differences in the amino acid sequence for the cytochrome c molecules studied. Electrochemical results suggest that the dissociation of the cytochrome c/cytochrome c oxidase reaction complex is the rate-controlling step in this electron transfer mechanism for cytochrome c from some sources. Moreover, the electron transfer reaction mechanism exhibits biphasic reaction kinetics, which is consistent with earlier work on reactions between solubilized cytochrome c oxidase/cytochrome c samples. These results indicate that the cytochrome c oxidase modified electrodes described herein could be used to distinguish amino acid sequence variations in proteins such as cytochrome c, and this has potential relevance as a diagnostic for disease states.

Temperature and pH effects on cytochrome c oxidase immobilized in an electrode-supported lipid bilayer membrane

Bovine cytochrome c oxidase has been successfully immobilized in an electrode-supported lipid bilayer membrane. The surface confined oxidase is capable of directly transferring electrons to the underlying electrode making characterization of the enzyme with direct electrochemical methods facile. Cyclic voltammetry indicates that the immobilized oxidase is capable of transferring electrons to the solid electrode over a wide range of pH and temperature values. Furthermore, the oxidase maintains its electron transfer properties at temperatures in excess of 80°C. Upon cooling the electrode back to room temperature, it was found that the enzyme still retained its electron transfer capabilities revealing the robust nature of the enzyme is this environment. In addition the oxidase continues to be capable of mediating electron transfer from solution-resident, reduce cytochrome c to the underlying metal electrode. The use of oxidase-modified electrodes enables the detection of solutions of reduced cytochrome c at concentrations as low as 0.25 μM.

Scanning Force Microscopy Images of Cytochrome c Oxidase Immobilized in an Electrode-Supported Lipid Bilayer Membrane

Scanning force microscopic images of cytochrome c oxidase immobilized within an electrode-supported lipid bilayer membrane are reported. These images represent the first direct evidence that the microscopic architecture of this important model system effectively mimics the microstructure proposed for the inner mitochondrial membrane. The images reveal (1) that the oxidase is present as monomers and small aggregates within the supported membrane and (2) that the oxidase constitutes ∼20% of the lipid bilayer membrane. The latter finding allows an estimation of the lower limit for the minimum turnover rate (∼0.5 electrons/s) required for the transition of the oxidase from its resting to pulsed kinetic state.

Observation of the Resting and Pulsed States of Cytochrome c Oxidase in Electrode-Supported Lipid Bilayer Membranes

Cytochrome c oxidase immobilized in lipid bilayer membranes on gold electrodes mediates electron transfer between reduced cytochrome c in solution and the electrode. Under flow conditions, fixed potential amperometry of anaerobic solutions of reduced cytochrome c shows changes in the rate of enzyme mediated electron transfer from cytochrome c to oxidase modified electrodes. These data are consistent with literature that describes both activation of the resting oxidase state to the pulsed oxidase state and subsequent decay of the pulsed oxidase state to the resting oxidase state.