Selective electrochemical biosensors from state-switching of bilayer and monolayer lipid membranes by lectin-polysaccharide complexes

Interaction of the lectin concanavalin A with the polysaccharide glycogen can provide rapid spontaneous transients of the surface potential at bilayer and monolayer lipid membranes. The selective binding process can cause large, rapid potassium ion current fluctuations across bilayer membranes in a manner that is periodic and reproducible. The frequency of these transient ion current signals was shown to be related to sub-nanomolar concentrations of the reactive agents in aqueous solution. The physical mechanism responsible for ion current modulation was investigated by fluorescence methods using lipid vesicles, by the thermal dependence of the potassium ion current across planar bilayers and by pressure-area and dipolar potential measurements of lipid monolayers at an air-water interface. The mechanism is primarily associated with physical perturbations of lipid membranes by lectin-polysaccharide aggregates, resulting in the formation of localised domains of variable electrostatic potential and conductivity.     

Biosensors for the Rapid Repetitive Detection of Adrenaline Using Stabilized Bilayer Lipid Membranes (BLMs) with Incorporated Calix[4]resorcinarene Receptor

This work uses lipid film based biosensors with incorporated calix[4]resorcinarene receptor (lipophilic macrocyclic host molecule) for the rapid electrochemical detection of adrenaline. Freely‐suspended and metal supported BLMs (composed of egg phosphatidylcholine (PC) and 35% (w/w) dipalmitoyl phosphatidic acid) modified with the resorcin[4]arene receptor were used as one shot sensors to rapidly detect this catecholamine. The interactions of this compound with freely‐suspended BLMs were found to be electrochemically transduced in the form of a transient current signal with duration of seconds, which reproducibly appeared about 14 s after exposure of the membranes to adrenaline. The response time for these BLMs without incorporated receptor for adrenaline was about 1.5 min. The magnitude of the transient current signal was related to the concentration of adrenaline in bulk solution in the micromolar range. Differential scanning calorimetric (DSC) experiments were performed to explore the mechanism of interactions of BLMs with incorporated receptor with adrenaline. The interactions of adrenaline with surface‐stabilized bilayer lipid membranes (sBLMs) with incorporated receptor produced electrochemical ion current increases, which reproducibly appeared within a few seconds after exposure of the membranes to the stimulant. The use of the receptor in sBLMs increased the sensitivity of the method 6‐fold. The current signal increases were related to the concentration of adrenaline in bulk solution in the micromolar range. Stabilized lipid membranes formed by polymerization on glass fiber microfilters were used as practical chemical biosensors for the rapid detection of adrenaline. The interactions of polymerized lipid films with adrenaline were also found to provide transient current signals similar to those of freely‐suspended BLMs. The magnitude of the transient current signal was also related to the concentration of the stimulating agent in bulk solution in the micromolar range and these stabilized lipid films can be used again after storage in air. No interferences from ascorbic acid were noticed because of the negatively charged lipids in membranes. The effect of other compounds such as proteins and other compounds closely related to adrenaline was also investigated. Results of recovery experiments using human urine have shown recoveries ranged between 94 to 105%, which shows no interferences from matrix effects due to the presence of urine constituents. The present sensor based on stabilized lipid films can be used for the rapid repetitive detection of this pharmaceutical substance and keep prospects for the selective determination of catecholamines in biofluids.     

Fluorescent lipids: functional parts of fusogenic liposomes and tools for cell membrane labeling and visualization

In this paper a rapid and highly efficient method for controlled incorporation of fluorescent lipids into living mammalian cells is introduced. Here, the fluorescent molecules have two consecutive functions: First, they trigger rapid membrane fusion between cellular plasma membranes and the lipid bilayers of their carrier particles, so called fusogenic liposomes, and second, after insertion into cellular membranes these molecules enable fluorescence imaging of cell membranes and membrane traffic processes. We tested the fluorescent derivatives of the following essential membrane lipids for membrane fusion: Ceramide, sphingomyelin, phosphocholine, phosphatidylinositol-bisphosphate, ganglioside, cholesterol, and cholesteryl ester. Our results show that all probed lipids could more efficiently be incorporated into the plasma membrane of living cells than by using other methods. Moreover, labeling occurred in a gentle manner under classical cell culture conditions reducing cellular stress responses. Staining procedures were monitored by fluorescence microscopy and it was observed that sphingolipids and cholesterol containing free hydroxyl groups exhibit a decreased distribution velocity as well as a longer persistence in the plasma membrane compared to lipids without hydroxyl groups like phospholipids or other artificial lipid analogs. After membrane staining, the fluorescent molecules were sorted into membranes of cell organelles according to their chemical properties and biological functions without any influence of the delivery system.     

Biomimetic membrane arrays on cast hydrogel supports

Lipid bilayers are intrinsically fragile and require mechanical support in technical applications based on biomimetic membranes. Tethering the lipid bilayer membranes to solid substrates, either directly through covalent or ionic substrate-lipid links or indirectly on substrate-supported cushions, provides mechanical support but at the cost of small molecule transport through the membrane-support sandwich. To stabilize biomimetic membranes while allowing transport through a membrane-support sandwich, we have investigated the feasibility of using an ethylene tetrafluoroethylene (ETFE)/hydrogel sandwich as the support. The sandwich is realized as a perforated surface-treated ETFE film onto which a hydrogel composite support structure is cast. We report a simple method to prepare arrays of lipid bilayer membranes with low intrinsic electrical conductance on the highly permeable, self-supporting ETFE/hydrogel sandwiches. We demonstrate how the ETFE/hydrogel sandwich support promotes rapid self-thinning of lipid bilayers suitable for hosting membrane-spanning proteins.     

Agar-supported lipid bilayers — basic structures for biosensor design. Electrical and mechanical properties

The lipid bilayer is widely accepted as the basic structure of all biological membranes. Known as BLM (bilayer lipid membrane), it can be prepared artificially. Suitably modified, the BLM serves as a very appropriate model for biological membranes. Recent investigations have verified the high analytical potential of artificial lipid membranes. With a structure and composition almost identical to the lipid moiety of biomembranes, the BLM may serve as an ideal host for receptor molecules of biological origin, thus becoming a transducer which could “see” the environment the way the living cell does. For the construction of lipid bilayer based biosensors; however, stable, easy to prepare and long-lasting lipid membranes are required. With this aim in mind, we have prepared lipid bilayer membranes which use an agar gel as support. This as-BLM (agar-supported BLM) has been shown to possess the same electrical, mechanical and dynamic properties the conventional BLM is famous for, along with the benefits of long-term stability and considerably elevated breakdown voltages. Its preparation on the tip of an agar-filled Teflon tube of 0.5 mm diameter is easy and can be performed even by less-skilled personnel.

In an attempt of further miniaturization the concept of the as-BLM was applied to thin-film micro-systems manufactured by standard micro-electronic techniques. The result is a lipid bilayer system, which, while preserving all the essential properties of the bilayer lipid membrane, can serve as a basic building block for cheap, disposable biosensoric systems.     

Rapid Detection of Vanillin in Alcoholic Beverages Using Stabilized Polymerized Lipid Film Based Biosensors

This work reports a technique for the rapid detection of vanillin in alcoholic beverages using stabilized lipid membrane based biosensors. Microporous filters composed of glass fibers (nominal pore sizes 0.7 and 1.0 μm) were used as supports for the polymerization of the lipid film and stabilization of these devices. The lipid film is formed on the filter by polymerization prior its use. Methacrylic acid was the functional monomer, ethylene glycol dimethacrylate was the crosslinker and 2,2′‐azobis‐(2‐methylpropionitrile) was the initiator. The response towards vanillin of the present stabilized lipid membrane biosensors composed of phosphatidylcholine was investigated. The stabilized lipid membranes provided artificial ion gating events in the form of transient signals and can be used again after storage in air. This has allowed the practical use of the technique for chemical sensing based on lipid film for the rapid detection of vanillin in wines and alcoholic beverages.     

Construction of a simple optical sensor based on air stable lipid film with incorporated urease for the rapid detection of urea in milk

This work describes the construction of a simple optical sensor for the rapid, selective and sensitive detection of urea in milk using air stable lipid films with incorporated urease. The lipid film is stabilized on a glass filter by polymerization using UV (ultra-violet) radiation prior its use. Methacrylic acid was the functional monomer, ethylene glycol dimethacrylate was the crosslinker and 2,2′-azobis-(2-methylpropionitrile) was the initiator. Urease is incorporated within this mixture prior to the polymerization. The presence of the enzyme in these films quenched this fluorescence and the colour became similar to that of the filters without the lipid films. A drop of aqueous solution of urea provided a “switching on” of the fluorescence which allows the rapid detection of this compound at the levels of 10⁻⁸ M concentrations. The investigation of the effect of potent interferences included a wide range of compounds usually found in foods and also of proteins and lipids. These lipid membranes were used for the rapid detection of urea in milk.     

Electrophoretic separation method for membrane pore‐forming proteins in multilayer lipid membranes

In this paper, we report on a novel electrophoretic separation and analysis method for membrane pore‐forming proteins in multilayer lipid membranes (MLMs) in order to overcome the problems related to current separation and analysis methods of membrane proteins, and to obtain a high‐performance separation method on the basis of specific properties of the lipid membranes. We constructed MLMs, and subsequently characterized membrane pore‐forming protein behavior in MLMs. Through the use of these MLMs, we were able to successfully separate and analyze membrane pore‐forming proteins in MLMs. To the best of our knowledge, this research is the first example of membrane pore‐forming protein separation in lipid membranes. Our method can be expected to be applied for the separation and analysis of other membrane proteins including intrinsic membrane proteins and to result in high‐performance by utilizing the specific properties of lipid membranes.     

Phospholipid-linked coumarin: a fluorescent probe for sensing hydroxyl radicals in lipid membranes.

A fluorescent probe, DPPEC (1,2-dipalmitoylglycerophosphorylethanolamine labeled with coumarin) was developed for detecting hydroxyl radical (*OH) in lipid membranes. The coumarin moiety contributes to the fluorescent detection of *OH and the phospholipids moiety gives a driving force to localize the probe in lipid membranes. DPPEC in liposomal membranes rapidly reacted with *OH and increased the fluorescence intensity, depending on the concentration of *OH. The increase in the fluorescence intensity induced by *OH was effectively suppressed by the addition of DMSO. The probe exhibited a higher fluorescence response to *OH over other reactive oxygen species, such as hydrogen peroxide, nitric oxide, peroxynitrite, alkylperoxyl radical, and hypochlorite. DPPEC would be useful as a new type of fluorescent probe that can localize in lipid membranes and detect *OH efficiently.     

Bivalent cholesterol-based coupling of oligonucletides to lipid membrane assemblies

By mimicking Nature's way of utilizing multivalent interactions, we introduce in the present work a novel method to improve the strength of cholesterol-based DNA coupling to lipid membranes. The bivalent coupling of DNA was accomplished by hybridization between a 15-mer DNA and a 30-mer DNA, being modified with cholesterol in the 3' and 5' end, respectively. Compared with DNA modified with one cholesterol moiety only, the binding strength to lipid membranes appears to be significantly stronger and even irreversible over the time scale investigated ( approximately 1 hr). First, this means that the bivalent coupling can be used to precisely control the number of DNA per lipid-membrane area. Second, the strong coupling is demonstrated to facilitate DNA-hybridization kinetics studies. Third, exchange of DNA between differently DNA-modified vesicles was demonstrated to be significantly reduced. The latter condition was verified via site-selective and sequence-specific sorting of differently DNA-modified lipid vesicles on a low-density cDNA array. This means of spatially control the location of different types of lipid vesicles is likely to find important applications in relation to the rapid progress currently made in the protein chip technology and the emerging need for efficient ways to develop membrane protein arrays.     

Continuous lipid bilayers derived from cell membranes for spatial molecular manipulation

Progress with respect to enrichment and separation of native membrane components in complex lipid environments, such as native cell membranes, has so far been very limited. The reason for the slow progress can be related to the lack of efficient means to generate continuous and laterally fluid supported lipid bilayers (SLBs) made from real cell membranes. We show in this work how the edge of a hydrodynamically driven SLB can be used to induce rupture of adsorbed lipid vesicles of compositions that typically prevent spontaneous SLB formation, such as vesicles made of complex lipid compositions, containing high cholesterol content or being derived from real cell membranes. In particular, upon fusion between the moving edge of a preformed SLB and adsorbed vesicles made directly from 3T3 fibroblast cell membranes, the membrane content of the vesicles was shown to be efficiently transferred to the SLB. The molecular transfer was verified using cholera toxin B subunit (CTB) binding to monosialoganglioside receptors (G(M1) and G(M3)), and the preserved lateral mobility was confirmed by spatial manipulation of the G(M1/M3)-CTB complex using a hydrodynamic flow. Two populations of CTB with markedly different drift velocity could be identified, which from dissociation kinetics data were attributed to CTB bound with different numbers of ganglioside anchors.     

Transmembrane Signaling with Lipid‐Bilayer Assemblies as a Platform for Channel‐Based Biosensing

Artificial and natural lipid membranes that elicit transmembrane signaling is are useful as a platform for channel‐based biosensing. In this account we summarize our research on the design of transmembrane signaling associated with lipid bilayer membranes containing nanopore‐forming compounds. Channel‐forming compounds, such as receptor ion‐channels, channel‐forming peptides and synthetic channels, are embedded in planar and spherical bilayer lipid membranes to develop highly sensitive and selective biosensing methods for a variety of analytes. The membrane‐bound receptor approach is useful for introducing receptor sites on both planar and spherical bilayer lipid membranes. Natural receptors in biomembranes are also used for designing of biosensing methods.     

Stabilized Lipid Membrane Based Biosensors with Incorporated Enzyme for Repetitive Uses

This work reports a technique for the stabilization of lipid membrane based biosensors with incorporated enzyme that retains its activity for repetitive uses. Microporous filters composed of glass fibers were used as supports for the stabilization of these sensors. The lipid film is formed on the filter by polymerization using UV (ultraviolet) radiation prior its use. Methacrylic acid was the functional monomer, ethylene glycol dimethacrylate was the crosslinker and 2,2′‐azobis‐(2‐methylpropionitrile) was the initiator. The enzyme (acetylcholinesterase) is incorporated within this mixture prior to polymerization. The polymerization process takes place by using UV irradiation instead of heating at 60 °C the lipid mixture because this temperature might denature the enzyme. This method for preparation of stabilized lipid membranes was investigated using Raman spectroscopy. The results have indicated that the kinetics of polymerization are completed within 4 hours. The retain in activity of the enzyme was studied using electrochemical experiments which have shown that this mild technique of polymerization can now be used to incorporate a protein in these lipid membranes without loss of their activity. This will allow the practical use of the techniques for chemical sensing based on lipid membranes based biosensors and commercialization of these devices.     

Construction of asymmetric cell-sized lipid vesicles from lipid-coated water-in-oil microdroplets

We present a simple, rapid, and robust method for preparing asymmetric cell-sized lipid bilayer vesicles using water-in-oil (W/O) microdroplets transferred through an oil-water interface. The efficiency for producing cell-sized model membranes is elucidated in relation to the vesicular size and the weight of contained water-soluble molecules. We demonstrate the biological asymmetric nature and the formation of lipid raft microdomain structures using fluorescence microscopy.     

Bilayer lipid membrane formation on a chemically modified S-layer lattice

The present paper describes the generation of a biomimetic model lipid membrane on bacterial surface (S-)layer which covered the entire surface of various sensors. The S-layer lattice allows one to be independent from the underlying solid material and provides a biological surface and anchoring structure for lipid membranes. S-layer proteins were chemically modified via binding of two amine-terminated phospholipids. Subsequently, a bimolecular lipid membrane anchored to the previously generated viscoelastic lipid monolayer was generated by the rapid solvent exchange technique. Characterization of the intermediate (monolayer) and final membrane structures (bilayer) was performed by imaging, surface-sensitive, and electrochemical techniques. This bilayer lipid membrane generated on an S-layer lattice revealed a thickness of ～6 nm and constitutes a stable supported model membrane system with highly isolating properties showing a membrane resistance of 8.5 MΩ × cm(2).     

Ion-current signal optimization for lipid membrane-based biosensors

The conductivities of bilayer lipid membranes are greatly affected by their lipid composition. Thus it is possible to prepare membranes having substantially different background or residual ion currents as observed when a direct voltage is applied across the membrane. Physical perturbation of the membrane by phloretin, valinomycin or receptor molecules provides current increases, which can be maximized by proper choice of residual current and membrane lipid chemistry. A compromise between provision of an efficient ion current pathway and minimization of residual current is necessary to optimize signal-to-noise ratio.     

PH effects on drug interactions with lipid bilayers by liposome electrokinetic chromatography

Liposome electrokinetic chromatography (LEKC) provides convenient and rapid methods for studying drug interactions with lipid bilayers using liposomes as a pseudostationary phase. LEKC was used to determine the effects of pH on the partitioning of basic drugs into liposomes composed of zwitterionic phosphatidylcholine (PC), anionic phosphatidylglycerol (PG), and cholesterol, which mimic the composition of natural cell membranes. An increase in pH results in a smaller degree of ionization of the basic drugs and consequently leads to a lower degree of interaction with the negatively charged membranes. From the LEKC retention data, the fractions of drugs distributed in the bulk aqueous and the liposome phase were determined at various pH values. Finally, lipid mediated shifts in the ionization constants of drugs were examined.     

Molecular mechanism for lipid flip-flops

Transmembrane lipid translocation (flip-flop) processes are involved in a variety of properties and functions of cell membranes, such as membrane asymmetry and programmed cell death. Yet, flip-flops are one of the least understood dynamical processes in membranes. In this work, we elucidate the molecular mechanism of pore-mediated transmembrane lipid translocation (flip-flop) acquired from extensive atomistic molecular dynamics simulations. On the basis of 50 successful flip-flop events resolved in atomic detail, we demonstrate that lipid flip-flops may spontaneously occur in protein-free phospholipid membranes under physiological conditions through transient water pores on a time scale of tens of nanoseconds. While the formation of a water pore is induced here by a transmembrane ion density gradient, the particular way by which the pore is formed is irrelevant for the reported flip-flop mechanism: the appearance of a transient pore (defect) in the membrane inevitably leads to diffusive translocation of lipids through the pore, which is driven by thermal fluctuations. Our findings strongly support the idea that the formation of membrane defects in terms of water pores is the rate-limiting step in the process of transmembrane lipid flip-flop, which, on average, requires several hours. The findings are consistent with available experimental and computational data and provide a view to interpret experimental observations. For example, the simulation results provide a molecular-level explanation in terms of pores for the experimentally observed fact that the exposure of lipid membranes to electric field pulses considerably reduces the time required for lipid flip-flops.     

Rapid Electrochemical Detection of Propranolol and Metoprolol in Pharmaceutical Preparations Using Stabilized Lipid Films

This work reports a technique for the rapid electrochemical detection of propranolol and metoprolol in pharmaceutical preparations using stabilized lipid films. Microporous filters composed of glass fibers (nominal pore sizes 0.7 and 1.0 μm) were used as supports for the formation and stabilization of these devices. The lipid film is formed on the filter by polymerization prior to its use. This stabilized after storage in air. Lipid films composed of phosphatidylcholine were used for the detection of propranolol and metoprolol in pharmaceutical preparations. The stabilized lipid membranes provided artificial ion gating events in the form of transient signals within about 60 and 34 s after exposure of the membranes to propranolol and metoprolol, respectively. The magnitude of the transient current signal was related to the concentration of propranolol and metoprolol in bulk solution in the micromolar range. The mechanism of signal generation was investigated by differential scanning calorimetric studies. These studies revealed that the adsorption of the drug is through the hydrophobic aryl terminal of the compound, whereas the hydrophilic groups were directed towards the electrolyte solution. This adsorption caused a rapid alteration of the electrochemical double layer of the lipid film (i.e., capacitance changes) that resulted in the transient ion current signal. The present technique was used for the rapid detection of propranolol and metoprolol in pharmaceutical preparations and can function for repetitive uses after storage in air. Future research is targeted to the determination of these chemicals in human biofluids such as urine of athletes.     

Flow Injection Analysis of Mixtures of Dopamine,Adrenaline and Ephedrine in Human Biofluids Using Stabilized after Storage in Air Lipid Membranes with a Novel Incorporated Resorcin[4]arene Receptor

This work describes a technique for the rapid, selective and sensitive electrochemical flow injection analysis of mixtures of the stimulating compounds adrenaline, dopamine, and ephedrine using stabilized after storage in air bilayer lipid membranes (BLMs) with incorporated resorcin[4]arene receptor. Injections of the stimulating compounds were made into flowing streams of a carrier electrolyte solution and a transient current signal, with duration of seconds, reproducibly appeared in less than two min after exposure of the lipid membranes to the compounds. The magnitude of this signal was linearly related to the concentration of the compound, which could be determined at micromolar levels. Repetitive cycles of injection of stimulating compounds have shown no signal degradation during each cycle (30 sequential injections). The time of appearance of the transient response was different for each stimulating compound and increased in the order of adrenaline, dopamine and ephedrine. The difference in time of response has allowed selective detection and analysis of these compounds in mixtures. The investigation of the effect of potent interferences included a wide range of compounds usually found in human biofluids, as well as proteins and lipids. The results showed that only proteins (most common in lipid film based biosensors) pose a problem that can be eliminated by modulation of the carrier solution to flow rates that do not allow adsorption of these compounds in the lipid films. The technique was applied in human urine samples.