Novel micromachined silicon sensor for continuous glucose monitoring

The construction and the application properties of a micro-machined silicon sensor for continuous glucose monitoring are presented. The sensor uses the conventional enzymatic conversion of glucose with amperometric detection of H(2)O(2). The innovation is the precise diffusion control of the analyte through a porous silicon membrane into a silicon etched cavity containing the immobilised enzyme. A variation of the number and size of the membrane pores allows to adjust the linear range of the sensor to the respective requirement. The sensor was tested in vitro as well as in clinical studies, being supplied with interstitial fluid. The cavity sensor was designed for a linear range between 0.5 and 20 mM. A signal response time of below 30 s and a signal stability exceeding 1 week is shown. By using a double cavity sensor falsification of the glucose signal by interfering substances can be compensated. In clinical trials the sensor measured continuously in interstitial fluid for up to 18 h without any signal drift and with good correlation to blood glucose reference values.     

Photoelectrospectrometry of bilayer lipid membranes

Three different bilayer lipid membrane systems were studied under visible and ultraviolet illumination. The first system consisted of a bilayer lipid membrane formed with a mixture of phospholipids and cholesterol, to one side of which purple membrane fragments from Halobacterium halobium were added. The second system consisted of a membrane formed from spinach chloroplast extract. When either of these membrane systems was illuminated with ultraviolet and visible radiation, photopotentials were observed and photoelectric action spectra were recorded (the technique is termed photoelectrospectrometry). Each spectrum had a definite structure which was characteristic of each of the modified membranes. The third system studied consisted of an otherwise photoinactive membrane formed with a mixture of phospholipids and cholesterol, to one side of which chymotrypsin was added. When the membrane was illuminated with visible light no photoresponse was observed. On the other hand, a photopotential which increased with incubation time was observed when the membrane was illuminated with ultraviolet light. Since, in our systems, the photoresponses have been observed to be due to certain species incorporated into the membrane, it appears that photoelectrospectrometry is a useful tool for studying lipid-protein interactions, constituent organization and energy transfer in membranes.     

Membrane on a chip: a functional tethered lipid bilayer membrane on silicon oxide surfaces

Tethered membranes have been proven during recent years to be a powerful and flexible biomimetic platform. We reported in a previous article on the design of a new architecture based on the self-assembly of a thiolipid on ultrasmooth gold substrates, which shows extremely good electrical sealing properties as well as functionality of a bilayer membrane. Here, we describe the synthesis of lipids for a more modular design and the adaptation of the linker part to silane chemistry. We were able to form a functional tethered bilayer lipid membrane with good electrical sealing properties covering a silicon oxide surface. We demonstrate the functional incorporation of the ion carrier valinomycin and of the ion channel gramicidin.     

Phosphoinositides alter lipid bilayer properties

Phosphatidylinositol-4,5-bisphosphate (PIP₂), which constitutes ∼1% of the plasma membrane phospholipid, plays a key role in membrane-delimited signaling. PIP₂ regulates structurally and functionally diverse membrane proteins, including voltage- and ligand-gated ion channels, inwardly rectifying ion channels, transporters, and receptors. In some cases, the regulation is known to involve specific lipid–protein interactions, but the mechanisms by which PIP₂ regulates many of its various targets remain to be fully elucidated. Because many PIP₂ targets are membrane-spanning proteins, we explored whether the phosphoinositides might alter bilayer physical properties such as curvature and elasticity, which would alter the equilibrium between membrane protein conformational states—and thereby protein function. Taking advantage of the gramicidin A (gA) channels’ sensitivity to changes in lipid bilayer properties, we used gA-based fluorescence quenching and single-channel assays to examine the effects of long-chain PIP₂s (brain PIP₂, which is predominantly 1-stearyl-2-arachidonyl-PIP₂, and dioleoyl-PIP₂) on bilayer properties. When premixed with dioleoyl-phosphocholine at 2 mol %, both long-chain PIP₂s produced similar changes in gA channel function (bilayer properties); when applied through the aqueous solution, however, brain PIP₂ was a more potent modifier than dioleoyl-PIP₂. Given the widespread use of short-chain dioctanoyl-phosphoinositides, we also examined the effects of diC₈-phosphoinositol (PI), PI(4,5)P₂, PI(3,5)P₂, PI(3,4)P₂, and PI(3,4,5)P₃. The diC₈ phosphoinositides, except for PI(3,5)P₂, altered bilayer properties with potencies that decreased with increasing head group charge. Nonphosphoinositide diC₈ phospholipids generally were more potent bilayer modifiers than the polyphosphoinositides. These results show that physiological increases or decreases in plasma membrane PIP₂ levels, as a result of activation of PI kinases or phosphatases, are likely to alter lipid bilayer properties, in addition to any other effects they may have. The results further show that exogenous PIP₂, as well as structural analogues that differ in acyl chain length or phosphorylation state, alters lipid bilayer properties at the concentrations used in many cell physiological experiments.     

Phase diagrams and lipid domains in multicomponent lipid bilayer mixtures

Understanding the phase behavior of biological membranes is helped by the study of more simple systems. Model membranes that have as few as 3 components exhibit complex phase behavior that can be well described, providing insight for biological membranes. A number of different studies are in agreement on general findings for some compositional phase diagrams, in particular, those that model the outer leaflet of animal cell plasma membranes. These model mixtures include cholesterol, together with one high-melting lipid and one low-melting lipid. An interesting finding is of two categories of such 3-component mixtures, leading to what we term Type I and Type II compositional phase diagrams. The latter have phase regions of macroscopic coexisting domains of {Lα + Lβ + Lo} and of {Lα + Lo}, with domains resolved under the light microscope. Type I mixtures have the same phase coexistence regions, but the domains seem to be nanoscopic. Type I mixtures are likely to be better models for biological membranes.     

Fission of the bilayer lipid tube

The fusion of two black lipid membranes results in the formation of peculiar bilayer lipid tubes (‘cylindrical’) membranes (Neher, E. (1974) Biochim. Biophys. Acta 373, 328–336 and Melikyan, G.B., Abidor, L.G., Chernomordik, L.V. and Chailakhyan, L.M. (1983) Biochim. Biophys. Acta 730, 395–398). The mechanical stability of such tubes has been investigated experimentally and theoretically. With increasing hydrostatic pressure on the outside of the tube the radius of its middle part decreases. After this radius has reached a critical value, which constitutes 0.55 of the radius of the tube base, there occurs a collapse of the tube and its disintegration into two planar bilayers (fission). Expressions are obtained which relate the transmembrane difference of the hydrostatic pressure, causing the collapse, to the geometrical characteristics of the tube (its length and the radius of its base) and to the tension of the lipid bilayer. A method for measuring the membrane tension is proposed on the basis of the phenomenon considered.     

Electrostatics of lipid bilayer bending.

The electrostatic contribution to spontaneous membrane curvature is calculated within Poisson-Boltzmann theory under a variety of assumptions and emphasizing parameters in the physiological range. Asymmetrical surface charges can be fixed with respect to bilayer midplane area or with respect to the lipid-water area, but induce curvatures of opposite signs. Unequal screening layers on the two sides of a vesicle (e.g., multivalent cationic proteins on one side and monovalent salt on the other) also induce bending. For reasonable parameters, tubules formed by electrostatically induced bending can have radii in the 50-100-nm range, often seen in many intracellular organelles. Thus membrane associated proteins may induce curvature and subsequent budding, without themselves being intrinsically curved. Furthermore, we derive the previously unexplored effects of respecting the strict conservation of charge within the interior of a vesicle. The electrostatic component of the bending modulus is small under most of our conditions and is left as an experimental parameter. The large parameter space of conditions is surveyed in an array of graphs.     

Electrostimulated fusion and fission of bilayer lipid membranes

The interaction and fusion of black lipid membranes have been studied. Two planar bilayers were simultaneously inflated towards each other; when they made contact, spontaneous formation of a ‘trilaminar structure’ (one bilayer bounded by two bilayers along the perimeter) was observed. Application of a discrete voltage pulse gave rise to the formation of a cylindrical membrane, that is, to the fusion of two bilayers. It is shown that fusion results from electrical breakdown in the contact region of the ‘trilaminar structure’.     

Water and nonelectrolyte permeability of lipid bilayer membranes

Both the permeability coefficients (Pd's) through lipid bilayer membranes of varying composition (lecithin [L], lecithin:cholesterol [LC], and spingomyelin:cholesterol [SC]) and the n-hexadecane:water partition coefficients (Knc's) of H2O and seven nonelectrolytes (1,6 hexanediol, 1,4 butanediol, n-butyramide, isobutyramide, acetamide, formamide, and urea) were measured. For a given membrane compositiin, Pd/DKnc (where D is the diffusion constant in water) is the same for most of the molecules tested. There is no extraordinary dependence of Pd on molecular weight; thus, given Pd(acetamide), Pd(1,6 hexanediol) is correctly predicted from the Knc and D values for the two molecules. The major exceptions are H2O, whose value of Pd/DKnc is about 10-fold larger, and urea, whose value is about 5-fold smaller than the general average. In a "tight" membrane such as SC, Pd(n- butyramide)/Pd(isobutyramide)=2.5; thus this bilayer manifests the same sort of discrimination between branched and straight chain molecules as occurs in many plasma membranes. Although the absolute values of the Pd's change by more than a factor of 100 in going from the tightest membrane (SC) to the loosest (L), the relative values remain approximately constant. The general conclusion of this study is that H2O and nonelectrolytes cross lipid bilayer membranes by a solubility- diffusion mechanism, and that the bilayer interior is much more like an oil (a la Overton) than a rubber-like polymer (a la Lieb and Stein).     

Formation and properties of cell-size lipid bilayer vesicles

Hydration of single or mixed phospholipids or lipid protein mixtures at low ionic strength results in the formation of a population of large, solvent free, single bilayer vesicles with included volumes of up to 300 microliters/mumol lipid. Their size ranges from 0.1 to 300 microns and they can be sorted out according to size by centrifugation. When formed in distilled water their internal solution has a conductivity of 20-50 microseconds/cm-1, an osmolarity of 0.5-5 mOsM, and a density of 1.0005-1.001. The osmotic pressure produced by the internal solutes cause a surface stress of 25 dyn/cm for a 20-microns vesicle. Their elastic constant ranges from 75-150 dyn/cm. During formation they can internalize particles such as latex beads or cell nuclei. They can be impaled with microelectrodes, or patch clamped. They can also be sealed to a small Vaseline-treated hole in a thin partition between two aqueous compartments. Sealing occurs in two stages. In the first stage sealing resistance is similar to that seen with patch-clamp pipettes. In the second stage, a much tighter seal is obtained. After sealing, the smaller portion of the sealed vesicle can be selectively broken by an electric shock leaving a single membrane across the hole. The capacitance and resistance of such membranes, in the presence of 10 mM NaCl, are approximately 0.7 microF/cm2 and 10(8) omega cm2 for pure lipid vesicles. Gramicidin increases the membrane conductance and monazomycin induces voltage-dependent gating thus providing further evidence that the vesicles are bounded by a single bilayer.     

The effect of the lipid bilayer state on fluorescence intensity of fluorescein-PE in a saturated lipid bilayer

Fluorescein-PE is a fluorescence probe that is used as a membrane label or a sensor of surface associated processes. Fluorescein-PE fluorescence intensity depends not only on bulk pH, but also on the local electrostatic potential, which affects the local membrane interface proton concentration. The pH sensitivity and hydrophilic character of the fluorescein moiety was used to detect conformational changes at the lipid bilayer surface. When located in the dipalmitoylphosphatidylcholine (DPPC) bilayer, probe fluorescence depends on conformational changes that occur during phase transitions. Relative fluorescence intensity changes more at pretransition than at the main phase transition temperature, indicating that interface conformation affects the condition in the vicinity of the membrane. Local electrostatic potential depends on surface charge density, the local dielectric constant, salt concentration and water organisation. Initial increase in fluorescence intensity at temperatures preceding that of pretransition can be explained by the decreased value of the dielectric constant in the lipid polar headgroups region related in turn to decreased water organisation within the membrane interface. The abrupt decrease in fluorescence intensity at temperatures between 25 degrees C and 35 degrees C (DPPC pretransition) is likely to be caused by an increased value of the electrostatic potential, induced by an elevated value of the dielectric constant within the phosphate group region. Further increase in the fluorescence intensity at temperatures above that of the gel-liquid phase transition correlates with the calculated decreased surface electrostatic potential. Above the main phase transition temperature, fluorescence intensity increase at a salt concentration of 140 mM is larger than with 14 mM. This results from a sharp decline of the electrostatic potential induced by the phosphocholine dipole as a function of distance from the membrane surface.     

Fluorescence coupling across lipid bilayer membranes

Summary An energy transfer between donor and acceptor fluorophores across single lipid bilayer membranes is demonstrated. Anilino-naphthalene sulfonate is used as the donor chromophore: its fluorescence is enhanced by the presence of lipid and thus indicates association with the purely lipid membranes of our preparation of vesicles in suspension. Light emit ted by the donor molecules excites fluorescence of acriflavine, a suitable acceptor enclosed inside the vesicles. Absorption and fluorescence spectra of this system, in its components and as a whole, are presented in evidence for an energy transfer.Supported by a grant from the Medical Research Council of Canada. The results of this work were presented, in part, at the 17th Annual Meeting of the Biophysical Society, February 27–March 2, 1973, Columbus, Ohio.Scholar of the Medical Research Council of Canada.     

Calmodulin interaction with mesocaine-modified lipid bilayer

Calmodulin (CaM) interactions with bilayer lipid membranes (BLM) were studied by measuring modulus of elasticity in direction perpendicular to the membrane plane (E perpendicular) and intramembrane potential delta psi. Upon interaction of CaM with egg phosphatidylcholine and asolectin BLM the parameter E perpendicular grew slightly (not more than by 10% as compared to the respective vale for nonmodified BLM), suggesting a weak effect on the ordering of the hydrophobic moiety of the lipid bilayer. In the presence of mesocaine (Mes), a calmodulin inhibitor, CaM affected the incorporation of Mes into the membrane. It can be concluded that CaM affects the ordering of the polar (superficial) membrane region.     

Interlayer-interaction for a lipid bilayer model

In order to elucidate the molecular basis of stability of lipid bilayer membranes, a system of two parallel monolayers consisting of oriented hydrocarbons with polar groups, which are oriented outwardly, is investigated. Relying upon the second-order perturbation theory of intermolecular forces between asymmetric molecules, a general expression for interlayer interaction energies is obtained for induced dipole-induced dipole, dipoleinduced dipole and dipole-dipole interactions. Using the polarizability tensor for a hydrocarbon unit C₂H₄, calculated after Smith & Mortensen (1960), and assuming 3 Debye unit for a dipole moment of a polar group, numerical estimations are performed.     

Alamethicin-induced changes in lipid bilayer morphology

We have found that alamethicin, in the absence of an electric field, modifies both the hydrophilic surface and hydrophobic core of lipid bilayers. As shown by freeze-fracture and X-ray diffraction experiments with multiwalled vesicles, alamethicin increases the fluid space between bilayers by as much as 50 nm, and at the same time perturbs the hydrocarbon regions of the bilayers. For suspensions of gel-state lipid treated with alamethicin, uniformly spaced rows of particles cover the fracture faces and corresponding linear arrays of stain-collecting depressions cover the hydrophilic surfaces. In the liquid-crystalline state, alamethicin induces an irregular granular texture on the fracture faces.     

Nonelectrolyte diffusion across lipid bilayer systems

The permeability coefficients of a homologous series of amides from formamide through valeramide have been measured in spherical bilayers prepared by the method described by Jung. They do not depend directly on the water:ether partition coefficient which increases regularly with chain length. Instead there is a minimum at acetamide. This has been ascribed to the effect of steric hindrance on diffusion within the bilayer which increases with solute molar volume. This factor is of the same magnitude, though opposite in sign to the effect of lipid solubility, thus accounting for the minimum. The resistance to passage across the interface has been compared to the resistance to diffusion within the membrane. As the solute chain length increases the interface becomes more important, until for valeramide it comprises about 90% of the total resistance. Interface resistance is also important in urea permeation, causing urea to permeate much more slowly than an amide of comparable size, after allowance is made for the difference in the water:ether partition coefficient. Amide permeation coefficients have been compared with relative liposome permeation data measured by the rate of liposome swelling. The ratios of the two measures of permeation vary between 3 and 16 for the homologous amides. The apparent enthalpy of liposome permeation has been measured and found to be in the neighborhood of 12 kcal mol-1 essentially independent of chain length. Comparison of the bilayer permeability coefficients with those of red cells shows that red cell permeation by the lipophilic solutes resembles that of the bilayers, whereas permeation by the hydrophilic solutes differs significantly.     

Comparison of double layer potentials in lipid monolayers and lipid bilayer membranes

Summary It is shown that the Gouy-Chapman double layer analysis adequately describes the variation of the surface potential of monolayers of acidic natural lipids over a wide range of surface charge density and salt concentration. It is also shown that the potential which initially appears when an electrolyte gradient is rapidly imposed across a bilayer membrane is due to a difference in the double layer potentials on the two sides of the membrane. This conclusion follows from the fact that the observed bilayer potentials arise much more rapidly than can be accounted for by charge migration across the membrane and from the observation that the bilayer membrane concentration potentials, when measured immediately after establishment of a gradient, are equal to the surface potential change observed when the subphase concentration of a monolayer of the same lipid is changed by an amount equal to the gradient across the bilayer. The bilayer potential and monolayer potential changes, so measured, agree in a number of different electrolyte solutions over a wide range of electrolyte concentrations and surface charge densities. Because of this agreement and the applicability of the Gouy theory to monolayers, initial bilayer potentials may be calculated if the composition of the mixture used to form the membrane is known, provided that the pK's and areas of such components are available. In the absence of this information, membrane potentials may be calculated from electrophoretic data on the membrane lipid mixture; the conditions under which the latter approach is possible have been determined. The experimental results indicate that the composition of monolyers and bilayers spread from the same lipid mixture in decane are very similar, that the composition of the two types of film closely resembles the composition of the solution used to generate them, and that bilayer membranes are close-packed. The evidence further indicates that if any hydrocarbon solvent remains in these bilayers, it must be so situated that it contributes little, if anything, to the surface area. The steady state potential in the bilayer membrane system is frequently not identical with the initial potential which supports the hypothesis that in many cases only a fraction of the electrical conductance of unmodified membranes is caused by the ions which constitute the bulk electrolyte. An expression for the relationship between diffusion and double layer potentials has been derived which shows that, in the absence of any intrinsic selectivity of the hydrocarbon region of the membrane for hydrogen, hydroxyl, or impurity, the two potentials should be identical.