bilayer lipid membrane-based electrochemical biosensors

Journal of Clinical Laboratory Analysis 2:256-264 (1988)

Bilayer Lipid Membrane-Based Electrochemical Biosensors

H. Ti Tien Membrane Biophysics Laboratory, Michigan State University,

East Lansing, Michigan

This paper presents a brief review on the chemical assay of drugs, as well as light- bilayer lipid membrane (BLM) system and sensitive BLMs for optical sensors. Sug- its potential applications as a chemicallbio- gestions for improvements in the deve- sensor. The applications reviewed include lopment of BLM-based electrochemical substrate-enzyme complexes, antigen- biosensors for the clinical setting are also antibody-complement interactions, electro- presented.

Key words: membranes, sensors, antigen, antibody, monolayers, Langrnuir films, immunology

INTRODUCTION

The bilayer lipid membrane (BLM) postulated as the basic structural matrix of biomembranes is widely accepted. This ultrathin structure, together with cytoskeletal systems, de- fines and determines the shape of the cell (1-3). The BLM similarly surrounds the various organelles and vesicles within the cell that contain compounds essential for life processes. Of equal importance to life is intercellular communication, which takes place across as well as between cell membranes (e.g., through gap junctions). This usually en- tails the sending, receiving, and decoding of signals. These signals may be chemical and/or electrical in nature predi- cated upon the presence of membranes. As to the membranes themselves, they are made of, besides the universal lipid bilayer, proteins and lipid-protein-carbohydrate complexes, which are believed to constitute the signal transducers in organisms.

Because of complex structural and environmental factors associated with biomembranes, it is realized that approaches using experimental model systems are essential to an under- standing of the fundamental life processes in physical and chemical terms. Thus, resorting to model systems has led to the discovery of a method for forming experimental BLM, whose physical and chemical properties resemble closely those of biomembranes (43). The attractiveness of the planar BLM together with its closely related liposome system, unlike any other membrane models thus far devised, lies in the fact that it has made possible, for the first time, the study of electrical events and material transport across an ar- tificially created ultrathin BLM separating two aqueous solutions (5,6). Basic studies of the self-assembly properties of BLMs opened the way to the present BLM technology. In this paper current planar BLM methods together with relevant past work on potential biosensor application will be briefly reviewed. In the last section a number of specific ex-

0 1988 Alan R. Liss, Inc.

amples on BLM-based biosensor devices will be described and some of their possible clinical applications will be given to illustrate the new approaches in electrochemical/bio-sensor development.

Techniques have been developed to incorporate a wide variety of compounds into BLMs to endow them with desired properties. These incorporated membrane-active compounds can be divided into six categories-(7), namely, those I ) altering the ionic electrical properties, 2 ) conferring ion specificity, 3) inducing electrical excitability, 4) changing the mechanical properties, 5) generating photoelectrical effects, and 6) imparting electronic characteristics.

Two crucial parameters are responsible for these interesting phenomena: the ultrathinness of BLMs (less than 10 nm thick) and two associated anisotropic interfaces. On the molecular scale, the interface acts as an external field on con- stituent molecules. The BLM itself may be properly viewed as a molecular junction (or molecular reactor in two dimen- sions) free from the usual rigid bulk phase effects.

There is a growing interest in clinical chemistry in moving chcmical analysis closer to the patient and to the physician’s office. To meet this need, many new approaches are being suggested. Among these, the electrochemical/bio-sensor is the most unique and offers many potential advantages in- cluding simple and inexpensive instrumentation, small probe, minimum sample pretreatment, and fast response time. Thus, there is ample accumulated evidence that the BLM system is ripe for exploitation of the particular order- ing at the interface for molecular manipulation (8-10).

Received June 17, 1988: accepted June 23, 1988.

Address reprint requests to H . Ti Tien, Professor of Biophysics and Physiology, Department of Physiology, Michigan State University, East Lansing, MI 48824.

2 100 1 f" - - - - --

BLM-Based Electrochemical Biosensors 257

THEBLMSYSTEM

BLMs of planar configuration separating two aqueous solutions are formed by using two basic techniques: self-assembling from a thin lipid layer or by apposing two preformed monolayers (5) . The aim of this section is to dc- scribe in sufficient detail how to set up a simple BLM system by using the self-assembling technique. For more details, a number of comprehensive papers may be con- sulted (5,6,8,11,12).

5 10 20 30 (TIME, MI N I

Fig. 1. A typical capacitance vs. time curvc during the formation of a BLM (solid line). Also shown is a membrane potential curve as a function of time (dashed line). The BLM was formed from a solution consisting of 3% PE (phosphatidylethanolamine) and 1.3% PS (phosphatidylserine) in n-decane saturated with TCNQ (tetracyanoquinodimethane). The bathing solution was 0.005 M KCI in 0.1 M sodium acetate at pH 5.6. Numbers I and 2 indicate, respectively, the first appearance of a "black hole" and the completion of BLM formation as observed visually.

BLM Cells

A planar BLM is formed by injecting a droplet of lipid solution onto a hole in the wall of a Teflon cup which consti- tutes one of the compartments of the BLM cell. A BLM cell consists of two compartments, the Teflon cup and the outer compartment with two chambers, into one of which the Te- flon cup is placed. The two compartments are connected via the hole in the Teflon cup (see Fig. 2).

A I to instrumentation

AQUEOUS SOLUTION (1) AQUEOUS SOLUTION (2)

Fig. 2. A: Experimental arrangement used in BLM formation an c- trical measuremcnts. (KE)I, (KE)o. and (AE) are saturated calomel electrodes (SCEs) for measuring R,, En,, and C,, of BLM as well as for carrying out cyclic voltammetry. For pH measurements, (RE), and (RE)" are connected to a pH meter via SW 4. R: The equivalent electrical circuit for a BLM as contacted by thc bathing solutions and measured by SCEs. K,,, and C, are, respectively, BLM resistance and capacitance connected in parallel. If assessed by A.C. technique, the R,,, is increasingly short- circuited with increasing frcqucncy.

BLM Formation

Before introducing the lipid droplet, both the Teflon cup and the outer compartment are filled with a bathing solution (e.g., 0.1 M KCI) which connects them via the small hole. Upon introduction of the lipid droplet into the hole, a thin lipid film is formed thereby separating the Teflon cup from the outer compartment. The thin lipid film begins to thin im- mediately, leading eventually under favorable conditions to a self-assembled bimolecular (bilayer) lipid membrane (BLM).

The formation of a BLM may be monitored a number of ways, such as by direct visual observation or by measuring membrane capacitance. For visual observation a low-power (20-60 X ) microscope is used to view the bright interference colors of the light reflected from the thin lipid film. The process of a BLM formation may also be followed by monitoring its capacitance by using an oscilloscope or a capacitance meter. With the oscilloscope set at 1 V/cm and the gain at 1,000 pA/V, a triangular wave of 20 mV peak-to-peak and 100 Hz is used. A capacitance of known value (e.g., 100 pF) is connected in place of the BLM to calibrate the amplitude of the square wave of current in picofarads (e.g., 400 pA for 100 pF). When the two compartments are separated by the presence of a lipid film, a small current should be displayed on the oscilloscope. This is mainly due to the capacitance of the cup. As the lipid film thins, the current amplitude in- creases. After the BLM is formed (absence of any observa- ble interference colors), the final capacitance of the membrane should be about 0.4-0.5 ,uF/cm2. If a capacitance meter is available, the process of BLM formation as a function of time can be dramatically demonstrated, as shown in Figure 1.

258 Tien

BLM-Forming Solutions

Investigators, depending on their degree of interest, have formulated many different lipid solutions for BLMs (5 , 6, 8, 11). The two most common ones are lecithin (1 % lecithin or phosphatidylcholine in n-decane) and oxidized cholesterol in n-octane. The latter is by far the most easy to use and is known to produce long-lasting BLM. The specific details in making oxidized cholesterol solution are given below:

Step 1. Add 12 gm of cholesterol (Distillation Products Division of Eastman Kodak Co., Rochester, NY) to 300 ml of n-octane (practical grade, Eastman Kodak Co.) in a two- neck, I-liter flask.

Step 2. Bubble 0 2 at a rate of of 100-125 cm3 per minute through a gas-dispersing tube (fritted glass, medium porosity).

Step 3 . Reflux the mixture at its boiling point (about 126°C) for 5.5-6.0 hr.

Step 4. Cool in refrigerator. The clear solution may be pippeted off for BLM formation. If the solution is cloudy, it may be filtered or centrifuged to remove the precipitate.

Electrical Measurements

For a basic BLM system, a high-impedance (> 10130) electrometer and a low-impedance picoammeter are essential. A variable voltage source made of a 1.5-V dry cell and a precision 10-turn potentiometer (Helipot) is useful to provide a selection of voltages (+800 mV) to the BLM. A dia- grammatic representation of a simple BLM setup is shown in Figure 2.

The equivalent circuit of a BLM is represented by a parallel combination of membrane resistance (R,) and capacitance (C,) as shown in Figure 2B. The relationship between R, and input resistance (Ri) is given by

where E, is the voltage drop across the BLM divided by the current density. A is the membrane area in cm2. R, is thus expressed in !J/cm2. When a known voltage (Vi) is applied to the BLM system (BLM, electrodes, and bathing solution), the voltage developed across the BLM is measured by the electrometer (E) and is given by

Vi R, + Ri

Em = I,R,; I, =

where I, is membrane current. From Eq. (2) one can solve for R,,,, which is given by

(3)

To measure C,, a voltage is applied to charge up the membrane capacitance. Upon opening the switch to stop the charging process, the potential decays as a function of time and is given by

(4)

Where V , is the voltage at time t = 0 and R is the parallel resistance (R,Ri/(R,, + Ri) through which the capacitance discharges. By measuring the time constant t = r when Em/ V, = l/e (0.37), C, is given by

C, can be obtained by solving Eq. (4) and the result is expressed as ,uF/cm2. Alternatively, the BLM capacitance is given by

where eo and E , denote, respectively, the vacuum permittiv- ity (8.85 x 10-I2Fm-') and the BLM dielectric constant ( - 2.5-5), A, = the area of BLM, and t, is the lipid bilayer thickness.

Many new electrochemical methods have been developed and applied to membrane research in recent years. Among them, cyclic voltammetry (CV) turned out to be a very pow- erful method (13). The basics of CV consist of cycling the potential of a working electrode in an unstirred solution and measuring the resulting current. The potential of the working electrode is controlled relative to a reference electrode which is provided by a triangular potential waveform generator. The instrumentation used with BLMs can be much simpler than that used in conventional CV. This is owing to the fact that the high resistance of BLMs can be studied with a two-electrode setup. Thus, a picoammeter together with a voltage waveform generator is all that is required.

If an x-y-displaying device is available (e.g., x-y re- corder), the currentholtage (IN) curves may be obtained, which are known as voltammograms. From such voltammograms, information about thermodynamic and kinetic parameters of the BLM system may be obtained, thereby providing insights into the mechanism of the membrane process under investigation (12,13).

BLM Modifiers

Unmodified BLMs (i.e., a BLM formed from common phospholipids or oxidized cholesterol dissolved in n-octane)in 0.1 M KC1 will typically have the following electrical properties: membrane resistance (Rd greater than lo8 O cm2, membrane capacitance (C,) of about 0.4 ,uF/cm2, membrane potential (Em) about 0, breakdown voltage (V,)


tact with the BLM was added to the system; 2) the presence of urea at high concentration (>0.2 mglml) blocked the effects; and 3) the responses of chymotrypsin-treated BLM wcre inhibited by Cu2+ and Hg2+ (at lop5 M). It was concluded that they were dealing with the effects of an interaction between the enzyme-modified BLM and the added substrate molecules, which could be used for the detection of ES interactions.

Other types of contact interactions involving membranes are generally referred to as ligand-receptor interactions such as between antigen and antibody in immunology and between hormone and receptor in neurophysiology (Table 1). We shall review membrane immunology in the next section. Here we will consider only hormone and neuromediator effects (3,16).

The BLM has been used in studies on the mechanism of hormone-membrane receptor binding and on its impact on membrane properties. By measuring the membrane conductance, Chatelain et al. (17) showed that thc luteinizing hormone may interact specifically with GT gangliosides which were incorporated in BLM formed from GMO. The same BLM system was used for demonstration of a specific reaction between GM ganglioside with the hormone, follitropin (18). The changes of the membrane electrical propertics ac- companying this reaction may be important for the hor- monal regulation mechanism involving the membrane-bound adenylate cyclase system.

Another kind of regulator of cell function is the prostaglandins, most of which are also acting through mechanisms involving binding to glycoprotein membrane receptors and subsequent influence on adenylate cyclase. The prostaglandins PGEl, PGA, , and PG12 induce a conductance increase of BLMs from different phospholipids and phospholipid- ganglioside mixtures (19).

The effects of neuromediators such as acetylcholine and dopamine have been investigated in BLM systems. The dopamine permeability of BLM is enhanced significantly by the Ca2+-ionophore lasalocid X-537A, which may be related to the ionophore effect in the native system, where it induces a releasc of the catecholamine neurotransmitters from sym- pathetic nerve terminals (20). This carboxylate antibiotic forms charged or neutral complexes with different biogcnic amines (amphetamine, epinephrine, dopamine, tyramine, ctc.)-both kinds of complexes probably playing a role for the transport process (21).

200 & 50 mV, and I/V curves obeying Ohm's law. However, electrical properties of the BLM can be drastically altered by incorporating a host of materials such as dyes, polypeptides, membrane proteins, etc. (5,8,11). Some of these will be dis- cussed in this paper. It will suffice to say that substances in- teracting with the BLM change its properties, which results in a change of the I/V characteristics of the membrane. In order to understand how these processes occur, it is necessary to understand what kind of mechanisms are involved in the interaction. If the modifier is water soluble, it may be added to the bathing solution so that its molecules can then approach the membrane interface and 1) randomly adsorb on the surface, 2) adsorb on surface, creating an ordered layer, 3) penetrate the membrane to a certain depth, and 4) penetrate across the membrane.

Modifiers which remain in the membrane may alter its in- trinsic structure by 1) creating channels (pores) for ions, 2 ) closing some existing channels, 3) closing or opening the channels depending on the electric field direction, 4) altering the permeability of channels, and 5) enabling a redox reaction to take place across the membrane.

Review of Relevant Work

The interaction of biophysics with cell physiology and molecular biology has given rise to an exciting area of research termed membrane biophysics, which integrates up-to-date findings on molecules and processes involved in inter- and intracellular recognition and communication. Knowledge of the ideas and findings resulting from such an interdisciplinary research are now being used for practical applications in analytical chemistry, immunology, photo- biology, chemical/bio-sensors and transducers, and molecular electronics. In this section a summary of the work on BLMs relevant to clinical laboratory analysis is presented.

Enzyme-Substrate Interactions

One of the bcst-described contact interactions is between two reactants-an enzyme (E) and a substrate (S); the formation of an enzyme-substrate (ES) complex is the first essential step. Of the two reactants, the substrate, being the much smaller one, can bind specifically to a tiny patch (active site) on the surface of the very large enzyme. The active site can accept only substrate molecules having an exact fit, which is often described by the so-called key-and-lock model (14). This unique and sensitive ES contact interaction can be monitored by a marked change in electrical parameters of BLM such as conductance andlor capacitance. Using an A.C. method, Del Castillo et al. (15) found the time course of the transient changes in impedance which followed the addition of substrate (ovalbumin) to the compartment containing the enzyme (chymotrypsin). From this and other experiments, Del Castillo and associates summarized their results as follows: 1) such impedance changes could only be observed if' an appropriate substrate for the enzyme in con-

Drugs and Other Biologically Active Compounds

The possibility of using thc BLM system as a tool for drug- membrane interaction studies was mentioned in the late 1960s (5 ,22) . Since that time, many investigatons have been carried out (see references 3, 6 , and 11). Only recent experiments relevant to the topic will be dealt with here.

The effects of cardioactive drugs and local anesthetics on biomembranes were tested on BLMs from PC, cholesterol,

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and other lipids (16,23). These compounds induced significant potentials, the side to which the effector was added being negative. The influences of pH, CaC12, and NaCl were investigated. Changes of the dipole moments of molecules around the supposed channels are suggested to explain the way of action of the local anesthetics as well as of the &ad- renoreceptor blocking agents. In this connection Shen et al. (23) have initiated an investigation of the potentials induced by calcium blockers (verapamil, diltiazem, micardipine) and P-adrenergic blockers (propranolol, sotaolol, labetalol, lo- pressor, transicor) on BLMs in order to elucidate the mechanisms of interaction between simple lipid membranes and these drugs. After the addition of the drug, a potential was usually developed across the BLM. The sign of potentials on the side to which the drug was added was negative. The magnitude of potentials was found to be dependent on the composition and concentration of the membrane-forming solution. Dose-response curves show the difference in the intensity of interaction between the membrane and the drugs. The pH curve for verapamil shows clearly the potential change as a function of Hf ion concentrations. For most cases, the potentials induced by the addition of drugs to solutions containing CaC12 were much less than those containing NaC1. Furthermore, in solutions containing both CaClz and NaCl, the drug-induced potentials showed no significant change when the salt concentration was varied from 0 to 0.1 M, but it decreased dramatically from 0.1 to 1 M. The results obtained may provide insight into the membrane-drug interaction. The BLM used in this study lacks specific receptors. It should be of interest to incorporate receptor proteins and to repeat the experiments so that the membrane- drug interaction may be clearly delineated (16).

The incorporation of influenza virus proteins into BLM and the effects of antiviral drugs such as ramantddine and amantadine on BLM conductance were studied by Karad- aghi and colleagues (24). From the results, they suggest that thcsc antiviral drugs may modify the cell membrane, thus affecting virus penetration into the cell. For a different class of compound, Arndt et al. (25) have reported the effect of miconazole, a potent antimycotic agent with a wide spec-

TABLE 1. An Assortment of Ligand-Receptor Contact Interactions'

Ligand Receptor

Substrate Hapten or antigen Hormones Drugs Ions Ions Electron donor Electron acceptor

Enzyme Antibody (immunoglobulins) Receptor synapse sites Membrane Carriers Channels Redox species Redox spCcies

'Ligand usually represents a much smaller species in the solution, whereas receptor is membranc-bound most often with active sites.

trum of antimicrobial activity, on oxidized cholesterol BLM. According to Arndt and co-workers, the antimycotic drug induces a significant increase in BLM conductivity, which appears to be reversible after removal of miconazole by washing the BLM.

The BLM system has been proposed as a sensitive method for assay of antibiotics (9). The rapid and reversible changes of BLM resistance correlated with the concentration of the antibiotics (e.g., amphotericin B and nystatin). The low limit of detection of the technique was demonstrated to be about

M of the drug. The effects of fungicidal agents have been also tested on BLM. It was shown that the antimycotic compound miconazole nitrate, affecting the growth of der- matophytes, yeasts, and gram-positive bacteria, provokes significant increase of the conductance of BLMs made from yeast lipids or oxidized cholesterol (26).

The organochlorine insecticide DDT and its analog DDE, which interfere with the active transport processes in biomembranes, were found to influence the conductance and to slightly influence the capacitance of BLMs (27). An increase of membrane fluidity was proposed to explain the observed effects. The toxic herbicide 2,6-dichloro- phenoxyacetic acid (2,4-D), which acts as plant growth regulator and as uncoupler of oxidative phosphorylation, can induce an increase of cation transport across BLM (27). It was suggested that this effect is due to adsorption of 2,4-D moleculcs on the membrane surface with their dipole moments directed toward the polar phase (16).

The high electrical resistance can also be reduced by a number of other interesting compounds. Rudnev et al. (28) reported thc effect of hemolysin on BLM-containg sphin- gomeylin. A single channel has an asymmetric current-voltage curve with maximal conductivity at positive potential on the side containing the toxin. Blumenthal and Habig (29) reported the ion selectivity of tetanolysin (a cytolytic toxin) in BLM and concluded that the permeability pathway induced by tetanolysin is caused by lipid perturbation rather than by formation of structural channels.

TOWARD BIOSENSOR DEVICES DEVELOPMENT

It should be stated at the outset that the BLM system described in this paper has not yet been developed into practical devices for use in clinical laboratories. However, the potential applications of ultrathin lipid films, in the form of BLM, were recognized as early as 1966, 4 years after the publication of the seminal paper in 1962 (4). As already described in the preceding section, Del Castillo and co-workers were the first to demonstrate the usefulness of the BLM system for monitoring ES and antigen-antibody interactions (15). Over the years, the advances made in the fields of sensory physiology and membrane biology, especially in the measurement of electrical properties of membranes, have provided the impetus for practical applications. The following is our rationale for future use of the BLM system for biosensor development applicable to the clinical laboratory.


readily measured. These electrical properties include potential (Em), resistance (Rnl or G, = l/R,), current (I,), capacitance (Cnl), and I/V curves. The sensors may be developed on the basis of either potentiometric or ampero- metric measurements. In the former, the potential (V,) across the BLM is measured under conditions when no charges flow. In the latter, a current (I,) is measured when a voltage is impressed across the BLM (5,6). Therefore, one can speak of potentiometric BLM-based devices or amper- ometric BLM-based devices. Since the capacitance of a BLM is also readily monitored, the third type is a capacitative BLM-based device. A variation of the above is conducto- metric BLM-based devices in which the conductivity (G, or resistance R,) of the BLM is determined. Further, simultaneous measurements of the current and voltage of a BLM resulting in a voltammogram may contain information useful for sensor application. In the following paragraphs some specific examples of BLM-based biosensors which are being developed will be described.

Ag/AgCl

Electrode

Fig. 3. A BLM-based probe using a polycarbonate membrane (Nucle- pore filter) for support (35). A gel-filled tube is used to enhance the BLM stability. Electrical contact is made through an Ag/AgCI electrode (see text for details).

The sensory transduction in living cells is thought to in- volve a change of electrical parameters across the receptor membrane following a specific binding event at the membrane surface. Because of complex and environmental factors associated with the biomembrane, experimental BLMs (planar BLMs and vesicular liposomes) have been employed in the elucidation of a host of processes at the membrane level. Recent advances in microelectronics and bioteehnol- ogy have aroused interest in the development of BLM-based electrochemical/bio-sensors which incorporate immuno- logic recognition systems. The unique aspects of the BLM system are that the membrane’s electrical properties can be

BLM-Based Biosensors for Immunology

The ability of BLMs to substitute for biomembranes in immune cytolytic studies has been demonstrated by several groups, and some information has already been derived from these studies regarding lytic mechanism (30). These types of experiments were first initiated by Del Castillo (15) and were extensively carried out by Mayer’s group (31,32) and other investigators (33,34). Traditionally, complement (C) is de- tined as a system of nine serum protein components that react in a specific order (Cl , C4, C2, C3, C5, C6, C7, C8, C9) to produce lysis of target cell. The distinguishing feature of this classical pathway is the activation of the complement se- quence by appropriate antigen-antibody complexes on the cell plasma membrane surface. The effect of antibody-complement on BLMs containing Forssman or Cardiolipin antigen has been reported (33). The specificity of Ag-Ab reactions coupled with the sensitivity of electrical measurements developed for the BLM provides not only a useful tool for immunology but also a method for testing various proposed mechanisms for immune mediated processes includ- ing immune cytolysis and cell agglutination.

Mountz and Tien (35) have developed a BLM system which is constructed by filling the smooth circular pores of known diameter and density in a commercially available polycarbonate membrane (Nuclepore). Under an ideal situa- tion this BLM system can be visualized as tens of thousands of micro-BLMs simultaneously generated in the polycarbonate support. The membranes formed in this manner exhibit far superior stability and manipulability in addition to having longer lifetimes and larger areas than can be achieved with conventional BLMs. Since the BLM system has the de- sirable quality of long-term stability, it has been used to repeat some of the difficult and uncertain experiments (35). Thus, a probe for a practical biosensor for clinical labora-

262 Tien

tory that makes use of high specificity in an immunochemi- cal rcaction is proposed in Figure 3. The design of this probe is as follows. A piece of Nuclepore filter is glued over a small hole (I-mm diameter). The tube is filled with a gel (e.g., agar in 0.1 M KCI). Electrical contact to external instrumentation is made via an Ag/AgCl electrode immersed in the gel. With the filled tube placed in a trough containing aqueous solution over the hole, a monolayer of a suitable lipid solution is spread on the surface in the usual manner. After evaporation of the solvent, the monolayer is first slowly lowered over the hole and then raised above it by using an infusion-withdrawal pump. A BLM is thus formed over the hole covered by the Nuclepore filter. Essentially, this method of BLM formation is a combination of the Lang- muir-Blodgett technique and the dipping method first intro- duced in 1965 by Takagi et al. (see ref. 5 , p. 477, Fig. 11- 3). The use of a closed tube has certain advantages. For in- stance, the stability of the BLM should be much enhanced. Further, the above procedure may be repeated as many times as necessary until a BLM coverage is obtained (8). The ex- istence of a completely BLM-covered Nuclepore filter is monitored by observing a change in either electrical capacitance or resistance. It is envisioned that inexpensive plastic materials will be utilized in the proposed construction. The BLM-based probes thus fabricated are ready for use and dis- posable, which in many situations is critical to a successful sensor application.

BLM-Based Biosensor for Glucose Detection

As far back as 1962 Clark and Lyons suggested that glucose could be determined amperometrically by using membrane-bound glucose oxidase according to the following reaction:

glucose +oxygen -+ gluconic acid + H202 (7)

The reaction described in Eq. (8) can be followed by monitoring one of the concentration variables: I ) H202, 2) pH, or 3) oxygen. Electrically, the measurement of a change in concentration of any one of these parameters may be related to the concentration of glucose which can be easily accomplished. What one’ needs is a sensitive BLM-based electrochemical sensor capable of converting the chemical signal into an electrical signal. Indeed, such an electrochemical sensor has been proposed in conjunction with a biohel cell (36). The key element is an ultrathin electron-conducting BLM doped with glucose oxidase, which serves as a bipolar electrode. According to Eq. (8) the two electrons per H202 should give rise to a membrane current which will be proportional to the concentration of glucose. In this connection, mention should be made of the work reported by Lubrano and Guilbrault (37).

Photoelectric BLMs and Biosensors

A wealth of literature exists on the photoelectric effects of pigmented BLMs containing a variety of photoactive species such as meso-tetraphenylporphyrins, organic pigments, and dyes (38,39). More recently, photoactive BLMs formed from thermotropic liquid crystals have been studied and light-induced capacitance changes have been found (40). These interesting findings could be used as a basis with which to develop a new type of biosensor sensitive to light in conjunction with fiber optic technology (41). The outstanding and unique properties of fiber optics for sensing purposes have been exploited for many years (42). Among these properties are their potential for remote monitoring, free from electrical interference, and their small size. As prac- ticed today, fiber-optic-based biosensors employ spectro- scopic methods. For example, sensors for biomedical use are available to detect pH through the use of dye indicators. It is conceivable that dye-doped BLMs can be similarly exploited as the sensing element in the development of fiber-optic- based biosensors.

Other Types of BLM-Based Biosensors

The principal idea behind the new class of biosensors is quite simple. We envision that, for detection in biological environments (blood and other body fluids), the sensing element should be biocompatible and biomembranelike. Thus, the BLM has great potential and is an ideal choice upon which to develop a new class of electrochemical sensors. It should be stressed, however, that BLM formed by the conventional method has a number of problems, the most seri- ous of which is its extreme fragility. Thus, the use of Nuclepore membrane as a support is highly recommended. For example, if one is interested in a sensor for odor detection, one would isolate receptor proteins from olfactory or- gans for incorporation into Nuclepore-supported BLM as the sensing element (7).

Finally, it should be mentioned that there are reports in the literature (43-45) in which field effect transistors (FETs) in conjunction with lipid layers have been employed in sensor device development. Generally, an FET carries current (“holes” or positive charges) through a silicon substrate from a “source” to a “sink. ” The flow of charges can be altered by a voltage applied to a third terminal (the so-called “gate”) situated between the source and sink but insulated from the substrate since the field alters the distribution of charge carriers. In the conventional FET, the gate is metal- lic, although the same effect can be obtained by using an electret instead. For example, if the gate is replaced by an electroactive BLM (or an equivalent), voltage changes caused by charge accumulation as carriers in the membrane will also change the current, which may be proportional to species concentration, thus allowing its quantitation. This type of sensor is essentially a capacitometric sensor depend-


ing on an electrical double layer situated at the BLM-solution interface. It remains to be seen whether such a device can be developed.

CONCLUDING REMARKS

For more than 10 years there has been an expectation that biosensors will play a major role in clinical laboratory analysis. However, many technical problems must still be over- come before this becomes a reality. Fortunately, basic research has in most respects already been accomplished. The question now is how to transfer biosensor technology from the laboratory prototype to a finished product of com- merce. The product so designed must have a reasonable shelf-life and be easily fabricated, simple to manufacture in large numbers and easy to use. l b meet these challenges, coordinated efforts are essential among basic scientists, cli- nicians, and developmental engineers, as is the input of ven- ture investors. It is hoped that this brief review will spur further development of biosensor technology.

ACKNOWLEDGMENT

Thanks are due to Ms. Debbie Benedict for excellent sec- retarial assistance.

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