Electrical characterizations of biomimetic molecular layers on gold and silicon substrates.

Electrical impedance technology was used to characterize DNA recognition in a monolayer containing single-stranded DNA probes immobilized on a gold substrate using thiol self-assembly chemistry. Recognition of targeted complementary DNA was principally correlated with an eight-fold increase in the conductance of the monolayer and attributed to electron conduction through double helices formed upon the binding of the DNA targets to the probes. The high recognitive sensitivity was possible without the use of the redox labels or large bias voltages required for recognition using cyclic and Osteryoung square wave voltammetry. The impedance technology also provided atomic resolution of a hybrid bimolecular lipid membrane formed by deposition of a phospholipid:cholesterol monolayer onto a hydrophobic alkyl monolayer covalently attached to a silicon substrate via silicon-carbon bonds. Atomic resolution was achieved through preparation of membranes on surfaces approaching atomic flatness and the performance of impedance measurements over precisely defined areas of the surface in contact with solutions. Principally capacitive properties distinguished between the immobilized (octadecyl) and more fluidic (lipid:cholesterol) leaflets of the hybrid membrane. The lipid:cholesterol leaflets were structurally similar to those leaflets in free-standing bimolecular lipid membranes. The hybrid membrane therefore provides a highly stable and physiologically relevant surface for studying biomolecular interactions with membrane surfaces.

Effects of singlet oxygen on membrane sterols in the yeast Saccharomyces cerevisiae.

Photodynamic treatment of the yeast Saccharomyces cerevisiae with the singlet oxygen sensitizer toluidine blue and visible light leads to rapid oxidation of ergosterol and accumulation of oxidized ergosterol derivatives in the plasma membrane. The predominant oxidation product accumulated was identified as 5alpha, 6alpha-epoxy-(22E)-ergosta-8,22-dien-3beta,7a lpha-diol (8-DED). 9(11)-dehydroergosterol (DHE) was identified as a minor oxidation product. In heat inactivated cells ergosterol is photooxidized to ergosterol epidioxide (EEP) and DHE. Disrupted cell preparations of S. cerevisiae convert EEP to 8-DED, and this activity is abolished in a boiled control indicating the presence of a membrane associated enzyme with an EEP isomerase activity. Yeast selectively mobilizes ergosterol from the intracellular sterol ester pool to replenish the level of free ergosterol in the plasma membrane during singlet oxygen oxidation. The following reaction pathway is proposed: singlet oxygen-mediated oxidation of ergosterol leads to mainly the formation of EEP, which is enzymatically rearranged to 8-DED. Ergosterol 7-hydroperoxide, a known minor product of the reaction of singlet oxygen with ergosterol, is formed at a much lower rate and decomposes to give DHE. Changes of physical properties of the plasma membrane are induced by depletion of ergosterol and accumulation of polar derivatives. Subsequent permeation of photosensitizer through the plasma membrane into the cell leads to events including impairment of mitochondrial function and cell inactivation.

Electrical impedance tomography study of biological processes in a single cell.

An in vivo electrical impedance tomography (EIT) study of single plant cells of Chara corallina is reported. When these aquatic cells grow in alkaline conditions, proton-translocating ATP synthases in the plasma membrane operate in reverse, utilizing ATP to translocate protons against an electrochemical gradient to the periplasm and creating localized acidic regions along the cell's cylindrical surface. These acidic regions, which appear as radial bands, approximately 5 mm long, between narrower alkaline bands, facilitate the uptake of bicarbonate, the plant's source of inorganic carbon for photosynthesis in the carbon dioxide-depleted alkaline conditions. Our EIT study of cell ultrastructure in the acidic and alkaline regions provides evidence that the plasma membrane is folded in localized regions (e.g., charasomes) in the acidic bands. The very low frequency capacitance dispersions were very similar to those of double fixed-charge structures. Such charge distributions are known to be present in the membrane-bound F0 portion of the ATP synthase. The theoretical dependence of the fixed-charge concentrations on pH in the proteins is shown to broadly account for the observed correlations between pH, membrane potential, conductance, and capacitance in these regions. In synthetically formed double fixed-charge membranes, electric field-induced dissociation of water into H+ and OH- occurs. This leads to the speculation that H+/OH- fluxes in ATP synthases located in the alkaline regions of Chara cells might also involve the electric field-induced dissociation of water.

Differential effects of cholesterol and oxidised-cholesterol in egg lecithin bilayers.

Low frequency impedance measurements of pure egg lecithin (phosphatidylcholine) bilayers have revealed the presence of four layers which can be attributed to the acyl chain, carbonyl, glycerol bridge and phosphatidylcholine regions of the lecithin molecule. Measurements on bilayers formed in the presence of unoxidised-cholesterol revealed that cholesterol molecules were located in the hydrocarbon region of the bilayer with its hydroxyl groups aligned with the carbonyl region of the lecithin molecules. Measurements of oxidised-cholesterol lecithin bilayers revealed that these molecules protruded less into the hydrocarbon region and their polar hydroxyl group aligned with the glycerol bridge region of the lecithin molecule.