Anal Bioanal Chem (2004) 379 : 347–350 DOI 10.1007/s00216-004-2611-4

TRENDS

Marek Trojanowicz · Ashok Mulchandani

Analytical applications of planar bilayer lipid membranes

Published online: 14 April 2004 © Springer-Verlag 2004

Introduction In recent decades significant research effort in chemistry has been oriented toward development of chemical and biochemical sensing devices. Many of these sensors are destined for applications in miniaturized systems in which conventional analytical instruments and detectors cannot be used easily. They can be more easily employed for insitu and in-vivo measurements, and are important research and diagnostic instruments for biology, physiology, clinical, and environmental analysis. The most advanced investigation of biosensors is conducted in the field of biomimetics, in which structures, functions, and phenomena found in biological systems are transferred into synthetic systems. The artificial formation of bilayer lipid membranes (BLM) is one of the most fascinating examples of self-assembly phenomena on a molecular level. Employed widely in Nature, BLM can be mimicked for design of chemical sensors and biosensors. They are planar nanostructures (in contrast with spherical liposomes, which are not discussed here) 5 to 10 nm thick, depending on the biomolecules, for example proteins or transmembrane entities, incorporated into the lipid layer. BLM are held together by non-covalent hydrophobic interactions of amphiphilic lipids with hydrophilic and lipophilic parts, forming a kind of two-dimensional fluid structure for other membrane constituents.

M. Trojanowicz (✉) Department of Chemistry, Warsaw University, Pasteura 1, 02-093 Warsaw, Poland e-mail: [email protected] M. Trojanowicz Institute of Nuclear Chemistry and Technology, Dorodna 16, 03–195 Warsaw, Poland A. Mulchandani Department of Chemical and Environmental Engineering, University of California at Riverside, 92521 Riverside, CA, USA

Since the beginning of modern science BLM have been the subject of extensive biophysical and chemical research [1]. The analytical applications of BLM, developed since the mid-sixties, are based on direct interaction of analytes with BLM structure, resulting in changes of the electrical and structural properties of membrane, or on interactions of analytes with other components, e.g. biomolecules, incorporated for this purpose into the BLM structure, leading to similar changes. A variety of such systems has been already reported in numerous original research papers and several reviews [2, 3, 4, 5]. The analytical applications of planar BLM developed by various research groups deal both with various methods for their artificial formation, leading to enhanced stability, and with finding various chemical and physicochemical interactions to obtain sensitivity for a given analyte.

Methods of preparation of planar BLM Besides having satisfactory selectivity and sensitivity chemical and biochemical sensors should have sufficient longterm stability or should be inexpensive in mass production, so they can be used as single-shot disposable devices or be regenerated quickly, simply, and at low cost. These conditions, of course, apply also to sensors based on the fragile structures of bilayer lipid membranes, and numerous methods have been developed for forming freely suspended and solid- or gel-supported BLM. A free-suspended planar BLM between two aqueous phases can be formed across a small window (diameter 1 mm or less) made in a hydrophobic septum, by spreading a lipid solution with a thin brush; this can be achieved with commercially available devices [6]. Such membranes can be also assembled in automatic preparation devices by use of an inkjet mechanism [7]. The “tip–dip” technique enables formation of BLM at the tip of a capillary by folding from monolayers; this technique has also been further improved by appropriate use of the Langmuir–Blodgett technique. Very often the method employed is based on use of a vessel containing a hydrophobic membrane with

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Fig. 1 Schematic diagrams of (A) a cell used in measurements with a hybrid bilayer membrane supported on a polycarbonate membrane covered with Au and octadecanethiol, and (B) the membrane formed [11]

a small, 0.25–1 mm, aperture and formation of BLM is achieved by lowering the level of a solution with a lipid layer on the surface [8]. Lipids most commonly used for formation of artificial BLM include components extracted from a variety of tissues of living organisms and synthetic lipids and other amphiphilic compounds. Freely suspended BLM are extremely fragile. To improve their mechanical stability compounds such as cholesterol, stearylamine, or saccharides are added; for these different mechanisms of stabilization have been postulated. A similar effect can be also achieved by blending BLM with hydrophobic polymers, polymerizable lipids, or phospholipids with highly branched hydrophobic chains. The high resistivity of freely suspended BLM can be reduced by incorporation of electron mediators or conducting polymers into the BLM. For design of analytical sensors BLM formation on different supports such as membranes, solid materials, or gels is desired to extend sensor life-time and to obtain a larger surface area. BLM can be formed on polycarbonate or glass micro-fiber membranes. For the same purpose smooth layers of crystalline cell-surface protein lattices (S-layers) can be used [9], deposited on a Nylon membrane [10]. BLM assembly in the holes of polycarbonate membranes previously covered by gold and self-assembled monolayer of octadecanethiol has also been reported [11] (Fig. 1). Formation of BLM on inorganic solid surfaces (metals, oxides, optoelectronic devices) provides a natural environment for immobilization of biomolecules (Fig. 2). The formation of stable lipid layers on the surface of metals is

attributed to interaction of lipid molecules with a highly hydrophobic nascent metallic surface. Conducting polymers [12] and gold electrodes of a quartz crystal microbalance [13] have also been used as solid supports. A hybrid nanoscale electrode array with supported BLM was recently produced by electrodeposition of platinum nanoparticles through BLM [14]. BLM on gels are formed by “tip–dip” procedure with capillaries filled with gel. Because solid-supported BLM are not suitable for monitoring trans-membrane current for analytical purposes, the bilayer can be fixed by a molecular spacer in a tethered BLM configuration [15]. Such systems function as fluid

Fig. 2 Schematic illustration of attachment of bilayer lipid membranes and lipid monolayer to solid support [28]

349 Fig. 3 Recordings of transient signals obtained in detection of dopamine (a) and ephedrine (b) with freely suspended BLM made of egg phosphatidylcholine and 35% (w/w) dipalmitoyl phosphatidic acid in which the receptor resorcin[4]arene was incorporated [8]. A mixture of analytes was injected into the batch measurement cell at the beginning of each recording. Concentration of injected solutes: A. 1.00 µmol L–1 dopamine, 147 µmol L–1 ephedrine; B. 2.67 µmol L–1 dopamine, 267 µmol L–1 ephedrine; C. 4.44 µmol L–1 dopamine, 400 µmol L–1 ephedrine; D. 6.67 µmol L–1 dopamine, 147 µmol L–1 ephedrine

lipid membranes with an aqueous layer separating BLM and support.

Analytical applications of planar BLM Artificially prepared BLM can be employed in the design of a variety of chemical and biochemical sensors. This is based on their sensitivity to various interactions, the possibility of instrumental control of different electrical and optical conditions, and the possibility of using BLM as matrixes for ionophores, enzymes, ion-channel switches, and antibodies. The properties of chemically unmodified BLM can be affected by interaction with a variety of chemical compounds, creating changes of electrical signal which can be proportional to changes of concentration. This has been shown for different classes of compound such as antibodies, pesticides, organometallic compounds, and surfactants. For triazine herbicides, and recently also for biogenic amines such as dopamine and ephedrine, fast transient signals at different times have been demonstrated with BLM containing a resorcin[4]arene receptor [8] (Fig. 3). This response was attributed to rapid adsorption of a given species followed by a slow aggregation process at the surface of the BLM with consequent rapid reorganization of membrane electrostatics. The matrix of three BLM sensors formed automatically in the measurement cell from three different lipids was used for determination of chlorinated organic compounds in underground waters [7]. The incorporation of redox mediators in the BLM structure can modify electron transfer at the working electrode in electrochemical measurements, as has been demon-

strated for various species with stainless-steel-supported BLM [16]. It was also shown that combination of mediators with fullerene C60 in BLM effectively enhances the sensitivity of BLM sensors to different redox species. Lipophilic ionophores are commonly used in the design of potentiometric membrane electrodes and it has also been shown that simultaneous incorporation of an ionophore and an appropriate anionic site generates Nernstian responses of BLM to potassium, sodium, and calcium ions [17]. Outstanding sensitivity for potassium was shown in capacitance measurements with valinomycin-incorporated BLM supported on a gold electrode covered with a self-assembling layer of methyl sulfide [18]. Another way of activating diffusion of hydrophilic species through a BLM, even against a concentration gradient, is by implementing ion-channel activity in the membrane. Incorporation of gramicidin in BLM deposited on to a silicon–silicon dioxide electrode [19] or on polycarbonate membranes covered with Au and an octadecane thiol [11] provides selectivity for monovalent cations. The incorporation of the nicotine acetylcholine receptor in freely suspended BLM sandwiched between two slabs of agarose gel provides an ion-channel biosensor for acetylcholine [20]. An electrochemiluminescence study showed ion-gate behavior of Pt-supported BLM made of synthetic lipid for permeation of Ru(bpy)32+ in the presence of perchlorate ions, which enabled development of a reversible luminescence sensor for perchlorate [21]. Numerous biosensors have been developed by incorporation of enzymes into the BLM structure. Attachment of enzymes to BLM by straptavidin–biotin binding and incorporation of an electron mediator into the BLM results in production of a glucose biosensor working at low polarizing potential [22]. The formation of BLM with in-

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corporated enzymes on a Pt surface modified with different polymers was shown to be effective way of suppressing electroactive interferences [12]. A peptide-tethered BLM incorporating cytochrome c oxidase has been reported for biosensing cytochrome c with direct electron transfer between enzyme and supporting Au electrode [23]. Supported BLM doped with Pt particles has been used for construction of a glucose biosensor [14]. Similarly to enzymes, antibodies also can be immobilized in metal-supported BLM by attachment of its conjugate with avidin to BLM containing biotinylated lipid; this leads to BLM immunosensors [24]. BLM sensors with incorporated singlestranded DNA have been reported for detection of aflatoxin M1 [25]. The optical analog of the gated ion-channel biosensor has been reported; it has a fluorescence system for sensitive detection of cholera toxin with a fluorophore-labeled recognition receptor incorporated into a lipid bilayer formed on glass beads [26]. This self-quenching system can be employed for fabrication of robust sensors using fiber optics technology. Solid-supported lipid bilayers have been also employed in an immunoassay microfluidic system for coating the surface of an array of microchannels [27]. For fluorimetric assay the bilayers contained dinitrophenyl-conjugated lipids for binding with antibodies.

Conclusions As shown by examples from selected papers published recently artificial planar bilayer lipid membranes can be widely used in the design of chemical and biochemical sensors. The process of self-assembly of BLM is relatively fast and in the appropriate configuration BLM can be stable for several weeks. Because of their nature, BLM sensors are miniature devices in which different chemical and physicochemical interactions can be employed to achieve sensitivity to different analytes. Acknowledgments This work was financially supported by a grant from the Polish–US Fulbright Commission to MT.

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