Supported planar lipid bilayers (s-BLMs) as

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Supported planar lipid bilayers (s-BLMs) aselectrochemical biosensors

H. T. Tiena and A. L. Ottovaa,b*

aMembrane Biophysics Lab (Giltner Hall), Department of Physiology, Michigan State University,

East Lansing, MI 48824, U.S.A.bCenter for Interface Sciences, Department of Microelectronics, Slovak Technical University, Bratislava,

Slovak Republic

(Received 3 October 1997; in revised form 17 February 1998)

AbstractThis paper presents a description of current research on the use of metal and hydrogel supportedbilayer lipid membranes (s-BLMs and sb-BLMs) in the area of biosensor development. Simple andstraight-forward experimental techniques for making these types of probes are given in some details.Emphasis is placed on the potential applications of these planar lipid bilayer-based probes. Among thetopics covered include ion sensors, antigenantibody interactions via electrical detection, probes for mol-ecular species, supported BLMs doped with fullerenes and photoelectric eects in C60-containing BLMs.# 1998 Elsevier Science Ltd. All rights reserved

Key words: lipid bilayer, BLM, supported BLMs biosensors, antigen, fullerenes, photoelectric eects, gel,

membranes.

INTRODUCTION

Planar lipid bilayers (bilayer lipid membranes or

BLMs for short) along with spherical vesicles (lipo-

somes) have been used for decades as models of

biomembranes. They provide a natural environment

for embedding a host of compounds such as ion

carriers, peptides, proteins, pigments, receptors,

membrane/tissue fragments and even whole cells for

studying membrane functions and for elucidatingthe mechanisms of ligandreceptor interactions.

One of the main tasks of biomembrane functions is

molecular recognition which entails selectivity and

specicity. Thus, understanding the principles that

lie behind the structuralfunctional relationship of

the cell membrane should therefore help to provide

the insights of much of the cell's biochemistry.

There have been many approaches in achieving this

understanding. The biophysicist's approach is to

nd simple model systems that mimic just a few of

the chemical physical properties of biomembranes.

This approach was used by Rudin and his associ-

ates in the late 1950s that resulted in the reconstitu-

tion of a bilayer lipid membrane (BLM) in

vitro [1, 2]. The background of the membrane bio-

physics work described has been focusing in under-

standing of the living organisms in physical and

chemical terms. The initial discovery of planar

BLMs and later developed supported BLMs have

made it possible for the rst time to study directly

the electrical properties and transport phenomena

across a lipid bilayer separating two interfaces. A

BLM is viewed as a dynamic system that changes

as a function of time in response to environmental

stimuli. A functional biomembrane system, based

on a self-assembled lipid bilayer and its associated

proteins, carbohydrates and their complexes, is also

in a liquid-crystalline and dynamic state. In molecu-

lar and electronic terms; a functional membrane

system can facilitate both ion and electron transport

and is the site of cellular activities in that it func-

tions as a ``device'' for either energy conversion or

signal transduction. Such a system, as we know

intuitively, must act as some sort of a transducer

capable of gathering information, processing it andthen delivering a response based on this received in-

formation. With the availability of supported

Electrochimica Acta, Vol. 43, No. 23, pp. 35873610, 1998# 1998 Published by Elsevier Science Ltd. All rights reserved

Printed in Great Britain00134686/98 $19.00 + 0.00PII: S0013-4686(98)00107-8

*Author to whom correspondence should be addressed. E-mail: [email protected]

3587


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BLMs, a host of compounds may be embedded in

the ultrathin lipid bilayer for detecting their

counterparts present in the environment. Owing to

its long-term stability, ease of formation and lowcost in its construction, a supported BLM oers an

approach especially advantageous in the research

and development of lipid bilayer-based sensors and

devices.

In terms of physical properties, the BLM, analo-

gous somewhat to a ``black'' soap lm, is an ultra-

thin, uid structure mainly consisting two

amphiphilic lipid molecules separating two phases.

A typical planar lipid bilayer (BLM) system may be

represented as follows:

Aqueous solution jBLMj Aqueous solution 1

where v line denotes an interface. A free standingBLM separating two aqueous solutions is truly amiraculous structure whose thickness is only about

5 nm. Thus, it is not surprising that the BLM is

extremely fragile and susceptible to rupture. To

work with a conventional BLM, requires patience

and tenacity; it could be an extremely frustrating

experience to the investigator. Yet, in spite of its

poor mechanical stability, the BLM has been exten-

sively studied, since its rst report in 1961, as evi-

denced by the vast literature that now exists [39].

The main reason for the sustained interest in exper-

imental planar lipid bilayers (BLMs) is two-fold: (a)

basic scientic studies and (b) potential practical

applications. The former is because these cellular

functions are membrane-bound, and the lipid

bilayer is the central structural component of all

biomembranes [59]. Further, the vast majority of

physiological/biochemical reactions involve a high

degree of molecular recognition. These reactions are

collectively known as ligandreceptor interactions,

of which the BLM system has often been

used [10, 11]. Some of these include antigenanti-

body binding, substrateenzyme reaction, ion-car-

rier selectivity, ion-channel specicity, hormone-

gated channels, light- and redox-species-induced

reactions. Further, the BLM system allows the

exquisite investigation of electrical properties (mem-brane potentials, resistance, currentvoltage curves

and membrane capacitance). Unlike most other

model membranes, BLMs are dynamic, ultrathin

and liquid-crystalline. Indeed, studies of the BLMs

facilitate the initial testing of working hypothesis,

which have generated guidelines for a better choice

of reconstituted membrane experiments and have

led to potential applications [5, 7, 10, 118, 119].

Concerning potential applications, however, there

is one major shortcoming of conventional BLMs in

that they have very limited lifetime, rarely lasting

more than 1/2 of a day. This long-term stability

problem of conventional BLMs to practical appli-cations was nally solved by forming planar lipid

bilayers on either newly cleaved metal surfaces or

hydrogel supports [11]. These novel types of planar

lipid bilayers, dubbed respectively as s-BLMs

(metal-supported) and sb-BLMs (agar salt-bridge-

supported) have been found to be very long-lasting

and yet possessing properties similar to those of

conventional BLMs, except in one respect, in that

one of the contacting interfaces of the planar lipid

bilayer has been now replaced by either a rigid met-

allic substrate or a soft hydrogel. The s-BLM and

sb-BLM systems, respectively, may be represented,

as follows:

Aqueous solution js BLMj metallic substrate 2

and

Aqueous solution jsb BLMj hydrogel support

3

where as before the symbol v denotes an interface.This paper focuses on use of supported planar lipid

bilayers (s-BLMs and sb-BLMs) as electrochemical

biosensors. Sucient details for the preparation and

Table 1.

Self-assembling amphiphilic interface-active systems

Type Interface Refs.

(1) Soap lms air vsoap lmv air [12](2) Monolayers (Langmuir type) air vmonolayerv water* [13](3) Multilayers (LangmuirBlodgett type) air vmultilayerv solid substrate6 [14](4) Planar lipid bilayers (BLMs) water vBLMv water [15](5) Nucleopore supported BLMs water vBLMs7v water [16](6) Gold supported monolayers air vthio monolayer}v Au [1720](7) Metal supported BLMs (s-BLMs) water vBLMv metal} [21](8) Salt-bridge supported BLMs (sb-BLMs) water vBLMv hydrogel + KCl6 [22,23]

*aqueous solution6such as glass7tens of thousands of BLMs in a polycarbonate lter

}such as Au}such as Pt, stainless steel6such as agar in 3 M KCl.

H. T. Tien and A. L. Ottova3588


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investigations of these two types of planar lipid

bilayers are given. Key references related to the sup-

ported BLMs are also provided. Table 1 presents a

list of self-assembling systems that have been inves-tigated to date together with their key

references [16, 120].

It should be mentioned at the outset that self-

assembled and supported BLMs dier from the

much studied LangmuirBlodgett (LB) lms and

other thin lm systems [13, 14, 1720] in several

major respects. First, a BLM is uid and self-seal-

ing and resisting to puncture. Second, the formation

of a self-assembled BLM, either on metal or hydro-

gel, is exceedingly simple. Unlike LB lms for

which elaborate setup such as lm balance, clean

room, etc., is usually required, whereas supported

BLMs may be formed without any special tool (see

Section 2). Third, self-assembled BLMs are recon-

stituted lipid bilayers as existed in nature; thus they

possess all the desired properties and are ideal for

ligandreceptor interactions experiments. Supported

planar BLMs, therefore, are one of the ideal system

for biosensor technology.

METHODS OF INVESTIGATION

The conventional BLM formed by spreading a

lipid solution across an aperture in a hydrophobic

septum that separates two aqueous solutions is an

extremely fragile structure. Therefore, it is of lim-

ited use for protracted studies and for practical ap-plications such as biosensors and molecular

electronic devices. Some BLMs supported on solid

substrates such as stainless steel, silver and Pt wires

(s-BLMs) have the desired dynamic properties as

well as the requisite mechanical stability. However,

the presence of a solid support on one side of the

BLM precludes the ion translocation across the

membrane. To overcome this and other shortcom-

ings, supported BLMs can be formed on salt

bridges made of hydrogels in Teon tubing (sb-

BLMs). These sb-BLMs possess similar electrical

properties to those of conventional BLMs and s-

BLMs. Additionally, sb-BLMs are as stable as s-

BLMs. Thus sb-BLMs overcome the shortcomings

of both conventional BLMs and s-BLMs.

Depending on the intended purpose, sb-BLMs are

believed to be a better system for biomembrane

research and may also be useful in biosensor devel-

opment.

Since the earlier days of BLM studies, a com-

bined electrical and optical methods have been used

by a number of investigators [3, 8,2430]. For

example, Ladha and colleagues [2830] reported

recently the use of a combined electrical and optical

chamber for uorescence recovery after photo-

bleaching from stable BLMs and pointed out the

feasibility of such experiments with BLMs, whichare amenable to physical constraints, and thus oer

new opportunities for systematic studies of func-

tionstructural relationships in lipid bilayers. These

techniques developed specically for conventional

BLMs are of obvious interest to the investigator of

supported BLMs, and their applications to suchsystems are anticipated.

Preparation of metal-supported BLMs (s-BLMs)

Materials. For BLM-forming solutions, any one

of the following are suitable: (1) 1% egg phospha-

tidylcholine (=PC) by wt. in squalenedecane (1:1

v/v), (2) 20 mg PC + 5 mg cholesterol in 1 ml n-

decane, (3) 25100 mg PC/ml of squalenebutanol

mixture (1:3 v/v), (4) 0.5% PC and 0.5% cholesterol

in squalene:butanol (v/v), (5) 1% PC and 1% oxi-

dized cholesterol in squalene:n-octane (1:1 v/v), (6)

1% glycerol dioleate (GDO) in squalene:n-decane

(1:1 v/v), (7) 20 mg PC + 4.4 mg per ml in n-decan-

e:chloroform (3:1 v/v). Supported BLMs, as in con-

ventional BLMs, can also be formed from glycerol

mono-oleate and/or l,a-dipalmitoyl-phosphatidyl-

choline in halogenated alkane solvents [31].

Depending on experimental requirements and situ-

ations, various aqueous solutions can be used ran-

ging from unbuered 0.1 M KCl (or NaCl) to

0.1 M KCl in 0.1 M TrisHCl at pH 7 or use 0.1 M

phosphate buer. Teon-coated Pt and stainless

steel (ss) wires are commonly used (from 0.02 to

0.5 mm diameter and larger), although other metal

wires such as Au, Al, Cu and Ag and even safety

fuse have been also used. Teon is the material of

choice for insulating these wires, however polyethy-lene has also found to be suitable. Reference elec-

trodes are typically either Ag/AgCl or saturated

calomel electrodes. A loop of Pt wire is used as an

auxiliary electrode in cyclic voltammetry.

Techniques of formation

A number of methods are now available for

forming supported planar lipid bilayers. The essen-

tial idea is very simple. An insulated metallic wire

(e.g. a Teon-coated Pt wire) is cut while immersed

in a BLM-forming solution (Fig. 1). The hydrophi-

lic polar groups of lipid molecules form a mono-

layer on the freshly cut wire tip, which serves as a

base for the second monolayer to assemble. Note

here that the surface of a lipid monolayer-coated

metallic surface is no longer hydrophilic but hydro-

phobic, as seen by other lipid molecules in the

attached lipid droplet. This is because of the hydro-

carbon parts of the phospholipids are sticking out

from the sorbed rst monolayer. The second mono-

layer of phospholipid molecules is therefore formed

with an orientation mirror image of the rst one

(see Fig. 1). Upon immersing of the lipid-coated

wire tip into an aqueous solution, excess lipid sol-

ution is drained away leading to the formation of a

self-assembled, supported BLM.

Specically, the formation of a supported BLMconsists of two-consecutive steps. First, the tip of a

Teon-coated Pt (or Ag, stainless steel, Au or other

Supported planar lipid bilayers 3589


4/24

metals) wire is cut in the presence of a BLM-form-

ing solution. The hydrophilic part of the phospholi-

pids adheres to the hydrophilic surface of the metal,

thereby allowing the rst monolayer to be formed.

By cutting the metal wire under the lipid solution,

it prevents the oxidation of the metal by air. In the

second step, the lipid-solution coated tip is

immersed into an aqueous solution (e.g. 0.1 M

KCl), excess lipid solution on the tip is allowed todiuse away leaving a BLM to self-assemble on the

metallic tip. The nal BLM is surrounded by a

PlateauGibbs border, as is in the case of a conven-

tional BLM, as is shown in Fig. 1.

Alternatively, an s-BLM can be formed in the

following manner. A Te on-coated Pt wire

immersed in a BLM-forming solution is cut as

before. This produces a fresh Pt tip with a lipid sol-

ution. The wire tip is then moved through a fresh

lipid droplet oating on the surface of the bathing

solution (i.e. a lipid monolayer with a lens at theairwater interface). By doing so, a supported BLM

is assembled at the wire tip [35, 36].

Fig. 1. Upper: Schematic illustration of a cell assembly used for determining the electrical properties of supported planar

lipid bilayers (s-BLMs and sb-BLMs). For cyclic voltammetry investigations, an auxiliary electrode made from a Pt wire

loop is also used [3234]. The working electrode can be either a s-BLM probe or a sb-BLM probe. In either case, a PG

(PlateauGibbs) border, a torous of lipid solution, surrounds a BLM. The reference electrode can be either an Ag/AgClas shown or a saturated calomel electrode. Lower: Enlarged views of supported BLM-based probes. Showing on the left

is an s-BLM on a metal substrate and on the right is a sb-BLM supported on agar gel.



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For the best cutting of metal wires, a miniature

guillotine was later constructed and used (Fig. 2),

where the sharp knife is moved vertically onto the

wire placed on a at surface and immersed in a

lipid solution [35, 36]. In comparison with the orig-

inal technique, the guillotine method of formation

of s-BLMs allows a more precise control of the

ratio of surface supported bilayer to the Plateau

Gibbs border. Therefore it is easier to obtain the

same electrical parameters of the system.

Preparation of supported BLMs on interdigitated

electrodes

As has been demonstrated by a number of

investigators [2123, 3749] the solid supported

BLM system (s-BLM) not only possesses the advan-

tages of a conventional BLM structure but ad-

ditionally gains new important properties such as

(a) an anisotropic, highly ordered, yet very dynamic

liquid-like structure, (b) two asymmetric interfaces

and (c) this type of probe is destined for microelec-

tronics fabrication. On this last mentioned property,

we have extended the experiments carried out with

s-BLMs to the interdigitated structures (IDS). IDS

are nger-like electrodes made by microelectronics

technologies and used in microchip applications [50].

Forming of supported BLMs on planar chips with

thin-lm electrodes have opened broad possibilitiesfor the development of miniaturized biosensors as

well as for basic research of electrical and mechan-

ical properties of biological membranes. More

details may be found in recent publications [5154].

Figure 3 provides the fabrication detail for making

a s-BLM probe.

Preparation of hydrogel-supported BLMs (sb-BLMs)

Although s-BLMs on metallic substrates are

attractive for certain purposes (e.g. certain biosen-

sors), the metallic substrate however precludes ion

translocation across the lipid bilayer. Therefore,

until a few years ago the pursuit of a simple method

for obtaining long-lived, planar BLMs separating

two aqueous media has been an elusive

one [22,23, 55]. A brief description of forming a

planar BLM on the agar or agarose gel is givenbelow.

Materials. Teon tubing (0.5 mm diameter), agar

or agarose, KCl, PC in n-decane:squalene (5:2 v/v).

BLM-forming solution consists of 20 mg/ml sol-

vent.

Techniques of formation

An overview for forming a planar BLM on agar

or agarose gel is as follows. A small diameter

Teon tubing is lled with a hot hydrogel solution.

For electrical connection as well as serving as a

reference electrode, an Ag/AgCl wire is inserted at

the one end. The other end of the agar-lled Teontubing is cut in air and then the cut end immedi-

ately immersed in the lipid solution. The next step

Fig. 2. The miniature guillotine used for cutting metal wires. With the wire in place, a small droplet of lipid solution is

placed on it where it is to be cut [35,36]. For wires of diameter less than 0.1 mm, a nger nail cutter could do a good

job as well.



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is to put the lipid-coated tip into an aqueous sol-

ution for a sb-BLM to self-assemble. Figure 1 illus-

trates a planar BLM on a hydrogel substrate

[22, 23].

The details of the formation procedure consists

of three steps. In the rst step, a chlorided Ag wire

(Ag/AgCl) is inserted into Teon tubing (H0.5 mm

diameter) which has been previously lled with a

solution of agar (e.g. 0.3 g agar in 15 ml 3 M KCl

saturated with AgCl) The AgCl electrode and the

lled Teon tubing are then glued together with

wax at the point of insertion. In this way an Ag/

AgClTeon tubing salt bridge (sb) is constructed

(Fig. 1). In the second step, the tip of the other end

of the Teon salt bridge is cut in situ with a razor

blade while immersed in a BLM-

forming solution, as done with the s-BLMtechnique [21, 37]. In the third and last step, the

Ag/AgClTe on salt bridge with the tip freshly

coated with lipid solution is then immersed in, for

example, 0.1 M KCl solution in a chamber where

the experiment is to be carried out. Alternatively,

the second step described above may be carried out

in air and then the freshly cut end of the salt bridge

is immediately immersed in the lipid solution for a

few minutes. In either case, the cell chamber lled

with an appropriate aqueous solution (e.g. 0.1 M

KCl) contains an Ag/AgCl reference electrode and

an Ag/AgClTe on salt-bridge with a self-

assembled BLM at its end. The lead wires of the

two electrodes are connected to the measuring

instrumentation. In this connection, it should be

noted that the two electrodes with salt-bridges (sb)

shown in Fig. 1 (top) may be identied as RE

(reference electrode) and WE (working electrode),

thereby eliminating the inner Teon beaker as hasbeen done in a conventional BLM setup [3].

Hydrogel-supported sb-BLMs formed in this

Fig. 3. Construction details and procedure for making an s-BLM probe on a metal wire tip.



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manner exhibit an average Rm=1.4 1011O,

Cm=0.45 mF cm2 having excellent mechanical

stability and is able to withstand an applied voltage

on the order of21.5 V.

Electrical measurements

Over the years a number of techniques have been

developed for characterizing the liquid/BLM/liquid

biface, including the following [35, 24, 25]:

. Optical method for thickness determination.

. Maximum bubble method for bifacial tension

measurements.

. Electrical methods such as dc, ac impedance

and cyclic voltammetry (CV). The CV method is

based on monitoring the current of the liquid/

BLM/liquid biface as a function of time.

. Photoelectrospectrometry.

Here, after a brief consideration about the inter-

face, our attention will be focused on the dc electri-

cal method. Interfaces between the BLM and its

aqueous environment are heterogeneous and as

such they are usually electried as a result of the

charge transfer, separation, and/or redox reactions.

The charges present on BLM interfaces create elec-

tric elds which greatly inuence the movement of

ions, electrons, holes and redox species [3, 5, 24,25].

Two of the principal functions of the biomembrane

are the selective transport of ions and redox reac-

tions across as well as within the membrane.

Studies of these phenomena have greatly enhanced

our understanding of these processes in nature andpointed toward possible technological applications

of BLMs. Ions may be transported through the

membrane either by a carrier or by diusion

through pores (channels). The carrier-mediated

transport begins with the association of an ion in

the solution and a carrier in the membrane. This

takes place at the membranesolution interface.

The charged complex moves across the membrane,

driven by the electrochemical potential gradient.

Dissociation takes place at the other interface and

the unloaded carrier back-diuses under its own

concentration gradient. Permeation of ions through

pores is regulated by the kinetics of activation and

inactivation of the permeability pathways. The car-

rier-mediated ion transport is regulated by selective

binding, whereas the ow through ionic channels is

regulated by a selective exclusion of molecules. In

electrostenolysis, a direct current is passed through

a high impedance membrane separating two aqu-

eous solutions [35, 9, 24, 25]. The advantages of the

BLM are its exceedingly high electrical resistance

and high capacitance. A high resistive and capaci-

tive BLM means, respectively, a low background

noise and a high current electrode, both of these

properties are useful in the biosensor development.

Transport of electrons across the membrane can

also take place resulting in oxidation on the sidefacing the negative electrode and reduction on the

side of the anode. The formation of BLMs now

makes it possible to examine the phenomenon of

electrostenolysis [3] in a structure similar to bio-

membranes.

All biomembranes are composed of a lipid bilayerintercalated with other constituents such as pro-

teins, carbohydrates and their complexes of lipids.

Insofar as can be determined, biomembranes are

liquid-like and in a dynamic state. Thus, it is not

surprising that today BLMs and liposomes are the

most used model systems for biomembrane

studies [6, 9]. Electrically speaking, both BLMs and

biomembranes possess very high capacitance

(H1 mF cm2) and dielectric breakdown strength

(>200,000 V/cm). By virtue of its molecular organ-

ization, a BLM possesses a very high electrical re-

sistance which is important in minimizing the high

background noise associated with an ion-conducting

aqueous environment. Since an ion or a charged

species is not able to penetrate a metallic surface, a

redox reaction must take place in order to observe

a current. Concerning the thickness of the coating,

the molecular dimension of a BLM (lipid bilayer)

greatly shortens the diusion pathway of electroac-

tive species towards the electrode surface as com-

pared with other thin lms and coatings (e.g.

polymers, carbon pastes).

Measurements of electrical properties

The electrical properties of a planar BLM separ-

ating two aqueous solutions (i.e. aqueous solution

vBLMv aqueous solution) or a metal/gel substrateand an aqueous solution (i.e. metal vBLMv aqueoussolution or gel vBLMv aqueous solution) can beeasily determined. Figure 4 illustrates an experimen-

tal arrangement used in our lab. Generally, the

BLM resistance (Rm) is several orders of magnitude

higher than those of the combined resistance of the

contacting electrodes and aqueous solutions [3]. A

good electrometer with a 109 input impedance

together with a picoammeter should be adequate.

For BLM experiments involving cyclic

voltammetry [3234], a block containing two adja-

cent 2 cm diameter chambers (one of which holds a

10 ml Teon cup), was commonly used. The Teon

cup was referred to as the inside, and the other

chamber as the outside. A three-electrode system

for obtaining voltammograms has been used in the

following conguration: one reference electrode

(SCE or Ag/AgCl) is placed in the Teon cup and

two other reference electrodes are on the outside.

The voltammograms of the BLM are obtained

using an XY recorder fed by a picoammeter and

the voltage generator (e.g. Princeton Applied

Research, Universal Programmer, Model 175). The

voltage from the programmer is applied through

the potentiometer to the reference electrode

immersed in the inside solution. Another reference

electrode immersed in the outside solution is con-nected to the picoammeter. The important feature

of the setup is a very weak dependence of its input



8/24

voltage on the current being measured. This means

that the current is measured under ``voltage clamp''

with accuracy21 mV. In voltammetry the potential

of the cell is varied and the corresponding current

is monitored. The graph with the current plotted on

the vertical axis vs the potential on the horizontal

axis is called the voltammogram, which is character-ized by several parameters. The working electrode

(WE) may be made of Pt, Au or carbon paste,

glassy carbon, semiconductor SnO2 or in our case

one side of a BLM. An Ag/AgCl or a saturated

calomel electrode (SCE) is used as the reference

electrode (RE). The potential scan or sweep is car-

ried out between two potential values of interest

(e.g. from about 1.2 to 0.8 V vs SCE). The scan

rates can be anywhere from 0.1 mV to 100 V or

more per second but values between 10 to 400 mV/s

are frequently used. The current response of the

processes at a metal electrode are indicative of the

nature of the redox reaction at the interface.

Experimental results derived from measurements of

this kind permit the elucidation of mechanism and

the thermodynamic parameters of the process (e.g.

charge transfer reaction). Frequently a ``duck-

shaped'' voltammogram is obtained for redox reac-

tions. The underlying physical mechanism respon-

sible for the ``duck-shaped'' prole is based on the

interplay between the kinetics of the charge transfer

process and the mass transport of the charge car-

riers (oxidants and reductants).

Cyclic voltammetry of supported BLMs

The basics of CV (cyclic voltammetry) and its ele-gance and simplicity, as described above, are well

known to electrochemists. According to our knowl-

edge, this powerful CV technique was applied for

the rst time to membrane studies in 1984 with

interesting results [5, 3234]. The current (i) for the

transfer of charges from one side of the BLM to

the other side is given by

i kn3a2AD1a2#1a2c 4

where k is a constant, n is the charge of the species,

A is the interfacial area, D is the diusion coe-

cient, n is the scan rate (mV/s) and c is the concen-

tration of the redox species in solution (Fig. 5). An

improved equivalent circuit for the s-BLM has been

recently proposed, which is based on the solution of

the coupled equations [46]. This approach greatly

facilitates the determination of the s-BLM electrical

properties by CV. The curve-tting procedure for

testing the reliability of this method has been elabo-

rated using a newly designed BLM simulator as

well as a 50-point goodness-of-t test. The mech-

anics and characteristics of formation of dierent s-BLMs and the acceleration eect of the CV poten-

tial on the s-BLM formation will be discussed in

Section 2.9 using the newly proposed s-BLM

model.

Parameters of planar lipid bilayers (BLMs) as

determined from voltammograms

The typical equivalent circuit of a traditional pla-

nar BLM system is represented by a membrane re-

sistance Rm in parallel with a membrane

capacitance Cm. The triangular sweep wave in the

range of2V0 with the scan rate A (mVs1) is the

input from the circuit. The current in nanoamperesor picoamperes is measured. There are two com-

ponents in the current through the membrane,

Fig. 4. Schematic representation of the experimental setup for investigating cyclic voltammetry of supported BLMs.

Shown on the left is a BLM simulator (adapted from Refs. [5, 46]).



9/24

Fig.5.

Equivalentcircuitsofconventionalandsup

portedBLMs.Left:Showinga3DviewofaBLM

modiedwithelectronmediators

suchasTCNQ,TTF,ferrocene,iodine,fullereneC60,

etc.(seeFig.6forotherBLM

modiers).Right:Top,theusualrepresentationofaconventionalBLM

consistingaresistorandacapa

citorinparallel.Bottom,theimprovedcircuitforthe

supportedBLM

[3234,46,48,49].



10/24

namely the charging current ic and resistance cur-

rent ir. The former is determined by the capacitance

as follows:

ic CmdV

dt CmA 5

It can be shown that the capacitance current icthrough the membrane capacitance is a constant.

From Ohm's law, the latter component ir of the

membrane current is caused by the membrane re-

sistance, i.e.

ir V

Rm6

So the net current passing through the membrane

can be expressed as

i ir ic VRm

CmA 7

equation (7) shows that the current through the

resistor increases with increasing scan voltage. In

the case of the constant scan rate A, with xed

values of Cm and Rm, the current i has a linear re-

lationship with the sweeping potential V. Thus, the

slope re ects the value of Rm, whereas Cm can be

determined by measuring ic according to the graph

of the IV response. ic will jump to its negative

values suddenly (ic) only at such points where the

sweeping wave reaches its maximum and begins to

reverse. The jump distance 2h equals 2ic, thus Cm

will be calculated by merely measuring h.However, the typical voltammogram of an unmo-

died s-BLM has a dierent shape when compared

with parallelograms obtained for the planar BLMs.

The dierence between them indicates that the

equivalent circuit proposed for the conventional

BLM is no longer valid for the s-BLM system.

Measurement errors will have a great impact on the

accuracy of the parameter determination unless the

circuit is improved.

A typical s-BLM system consists of a supported

BLM, an electrolyte and a reference electrode,

which may be represented by a number of suitably

connected Rm and Cm. According to the IV re-

sponse recorded on the real s-BLM, the improved

equivalent circuit for the s-BLM is developed,

where Rn the non-membrane resistance introduced

in series with the original circuit and the parallel Csis the distributing capacitance for the entire circuit.

The membrane is characterized by high resistance

and a high capacitance. Their measurement accu-

racy is far more aected by the proposed non-mem-

brane resistance Rn, whose composition and eect

on the measurement error will be discussed in

Section 3.

From simulation we have obtained the similar I/

V response by CV with the BLM simulator which

contains relevant electrical circuit. The eect of beachieved by a set of the I/V response generated by

BLM-simulator with and without Rn, where the

regular development of the CV waveform are

shown with varying values of Rm, Cm, Rn and Cs.

A set of voltammograms have been obtained in

which Cm is varied while holding Rm constant andRn at 0 O. From cycle 1 to 3 [46], the intercept with

the i axis rises gradually with an increasing Cm. The

slope drops down as Rm increases while keeping Cmconstant.

An improved equivalent circuit has been devel-

oped, where both Rn and Cs are involved in the

consideration [46]. A detailed observation of the

eect of Rn on the shape of I/V cycle is carried out.

In our experiment, Rn varies in a wide range from

100 to 109 O, while the other membrane properties

are simulated holding Rm at 109O, Cm at 3 nF con-

stant, both of which are still within the range of

biomembrane. From the obtained curves, the simi-

larities of the I/V cycles which have the shape of

the parallelogram even if Rn reaches the value of

107 O. However, when Rn increases to 108O

(I0.1Rm), it begins to be characterized by the char-

ging current. The intercept with the current axis is

also somewhat decreased. The most important fea-

ture is that, when Rn reaches the same order as

Rm(IRm), the voltammogram does not display the

shape of parallelogram any longer, and it is very

similar to that observed for the s-BLMs system [46].

The intercept with the i axis drops sharply. The

slope decreases during the potential sweeping and it

is much lower than the slope in the case of BLM-

simulator without Rn or with a low value of Rn. Inthis last case, The intercept can no longer reect the

membrane capacitance, and it is the same as the

slope of membrane resistance.

Therefore, the measurement error would be

greatly increased if one still considers the intercept

to i-axis as membrane capacitance and the slope as

the resistance. What causes the large value of Rn is

still being discussed. The previous report using

LAPS technique has studied the system by consider-

ation of the solution impedance aection [46].

However the value is not so high that it approaches

to the Rm. Because in our previous research the

non-membrane resistance was not found so high in

conventional planar BLM and the technique for

forming self-organized s-BLMs is based concep-

tually on interactions between a nascent metallic

surface and amphipathic lipid molecules; it is

assumed that a very possible principle composition

of Rn is aroused by the metallipid interface (inter-

face resistance). Other important factors in Rninclude the electrode, electrolyte and solution impe-

dance, and any other components which greatly

eect the measurement accuracy.

Simulation of the eect of Rn on the shape of the

IV cycle indicates that, by gradually increasing the

Rn from 0 to 0.1Rm, the cyclic response begins to

be characterized by the charging current. The mostimportant feature is that, when Rn reaches the same

order as Rm, the voltammogram does not display a



11/24

parallelogram shape any longer, and it is very simi-

lar to that observed for the s-BLM system. The

intercept with the i axis drops sharply. The slope

decreases during potential sweeping and it is muchlower than the slope in the case of BLM without

Rn. In this last case, the intercept can no longer

reect the membrane capacitance and it is the same

as the slope of membrane resistance [46].

Here, we propose a new method for determining

Rm, Cm, Cs and Rn accurately from the on-line s-

BLM cyclic voltammogram. To make clear the

eect of Rn on the IV response, the quantitative

relationship of the membrane current with the

sweep wave potential should be derived rigorously

from the solution of coupled equations based on

the equivalent circuit. The set of dierential

equations is as follows:

i ir ic is irRm 1

Cm

t0

ic dt

V ir icRn 1

Cm

t0

c dt is CsA 8

where i is the total current recorded, is, which is

dened as the current through Cs, and all other par-

ameters are dened above. Here, the nal (denite)

solution i= f(V) is presented, where Rn and Cshave been included:

i ARmCm

Rm Rn

V

ARmCm

Rm

Rm Rn

2Rm

Rm Rn

exp

Rm RnV V0

ARnRmCm

! CsA 9

The initial condition for equation (9) is

Vt0 V0 At V0

During the half-cycle sweep from a to b, we can see

from equation (9) that there are three components

included in membrane current: (1) the time-linearly

dependent term which is the resistance current

obeying Ohm's law; (2) the time-exponentially

dependent term in which Rn, Rm and Cm should be

taken into account and (3) the constant term result-

ing from the distributing capacitance Cs for the

entire circuit.

The traditional way for parameter determination

is to vary each of the parameter values while hold-

ing all other parameters constant and to select a t-

ting curve with a minimal deviation. However, the

complexity of the calculation is the major impedi-

ment in nding a group of eective suitable values

to t the calculated curve. Despite the aid of com-

puter, the calculation still requires much more time,

which can not be utilized for on-line parameter de-

termination. So it is necessary to derive an accurate

expression for the parameters Rm, Cm, Rn and Cs

from the original solution of equation (9).Thus, the method for determining Rm, Cm, Rn

and Cs has been obtained [46]. First of all, the con-

stant term CsA in equation (9) will be negative at

the very moment when the sweep potential reaches

its maximum and begins to reverse. So, the height

2h corresponds to twice the value of the distributioncapacitance Cs. So from the on-line computer

acquire and parameters determination, the height

2h can be read out and Cs is determined by

Cs haA

The dierentiation of equation (9) is given by

di

dV

1

Rm Rn

2Rm

RnRm Rn

exp

Rm RnV V0

ARnRmCm

10

The potential of the triangular sweep wave moves

from V0 to +V0 (from a to b). At the half-cycle

terminal b, the exponential term can be rationally

omitted and the slope of the tangent line CD at the

point b can be expressed as

1

D

1

Rm Rn11

The intercept of CD with the current i-axis is y1which is

y1 AR2mCm

Rm Rn2

12

The intercept of the voltammogram with the cur-

rent i-axis is y2; therefore

Dy y1 y2 2y1 exp

RmV0

y1DRn

13

Thus without any time-wasting tting procedures,

all the membrane parameters can be calculated

cycle by cycle from the on-line data acquired as fol-

lows:

Rm m

m 1D Cm

y1

A

1

m 1

2

Rn 1

m 1D Cs

h

A

m ln

2y1

Dy

y1

D

V0

14

In concluding this analysis, the only parameters

that need to be acquired directly for a recorded vol-

tammogram of an s-BLM are D, y1, Dy and 2h.

From the relationship described in equation (14), it

is now possible to calculate accurate values of the

membrane electrical parameters for the s-BLM sys-

tem. In this section, an unusual method is proposed

which can determine the properties accurately with-

out any iteration [46]. So the real-time measurement

can be carried out for the dynamic analysis.

REVIEW OF PAST WORK

Before presenting the research ndings on the use

of supported BLMs as electrochemical biosensors,



12/24

we would like to give the rationale behind the

work. Insofar as is known, in order to make life

viable, the living system must interact with or sense

its surroundings. Thus, sensing is one of the vitalfunctions of life, which include seeing, hearing,

smelling, touching and tasting. From human stand-

points, the signal from the external world must be

transduced into a language that the brain under-

stands. To a very large extent, perhaps exclusively,

this language is encoded in electrical signals. In this

connection bioscientists, in particular membrane

biophysicists, have been investigating for quite

sometime the functional relationship between bio-

membranes and signal transduction [56]. Volkov et

al. have most recently reviewed the thermodynamics

and electrostatics of membranes [57]. Our interest

in membrane biophysics has been focused on exper-

imental lipid bilayers (planar BLMs and spherical

liposomes) and has been greatly benetted by a

cross fertilization of ideas among various branches

of sciences including biochemistry, solid-state phy-

sics, molecular biology and materials science. At the

membrane level a signal, which can be light, chemi-

cal, acoustic, mechanical or electrical, is received by

a receptor embedded in the lipid bilayer of the bio-

membrane. The result of this interaction usually

transduces into a response of some sorts of activi-

ties which have been collectively known as ligand

receptor interactions. In connection with research

and development activities involved in biosensors,

the key component is a sensing element of biologi-cal interest, which is usually embedded in an ultra-

thin lipid bilayer of the nanometer thickness. This

biologically active element endows on the sensor a

great specicity to its counterpart, i.e. a target ana-

lyte, in a complex aqueous environment. For this

purpose, there is a vast variety of biocompounds

that can be used to develop a biosensor. For

examples, enzymes which serve as biological cata-

lysts are commonly used to interact with their

specic substrates. These types of enzymesubstrate

reactions (a sort of ligandreceptor interactions)

require a transducer which converts the interaction

into a quantiable signal for meaningful interpret-

ation.

The main topic of this paper is concerned with

the use of supported bilayer lipid membranes

(BLMs or planar lipid bilayers) as electrochemical

biosensors. A biosensor is commonly dened as a

molecular device that combines a biomaterial-based

sensing element, man-made or natural, with a trans-

ducer embodying a lipid bilayer which converts the

sensed event to an useful signal. The general prin-

ciple of supported BLM-based biosensors is simple;

it is based on the lock-and-key concept of Emil

Fischer of 1894 (see Ref. [56]). Today we speak of

receptorligand, hostguest, enzymesubstrate, etc.

interactions. The molecule that is doing the recog-nition is called a host (lock, receptor), which has a

conguration that is complementary to that of the

guest (key, ligand). Ideally, one would like to

develop a liquid vBLMv liquid (solid) biface systemthat function as a transduceractuator. That is a

suitably modied BLM-biface can receive appropri-ate information, act upon it and initiate a response.

In physiological terms, a ligand interacts on a mem-

brane-bound receptor resulting in a signal. This sig-

nal is transduced from the contact interaction into

an electrical output, which in turn actuates a chemi-

cal or a physical process. For instance, a ligand-

gated channel is an example, where a ligand (i.e. a

hormone) binds with a receptor embedded in the

lipid bilayer of the membrane (see Fig. 6). After

this contact interaction, the ``gates'' of ion-channels

are opened to allow ions to ow into the cell.

The nature and origin of the signal may be elec-

trical, mechanical, optical as well as chemical, as

already mentioned above. In the case of electro-

chemical biosensors, the sensing element is

embedded in the BLM and the signal is usually an

electrical parameter such as potential, current, con-

ductance, capacitance, breakdown voltage or a

combination of these as in a voltammogram. The

challenging task is how to design a liquid vBLMvliquid (solid) biface system that will self-assemble to

function as a transduceractuator. In Sections 3.1,

3.2, 3.3, 3.4, 3.5, 3.6 and 3.7 we will discuss the

results that have been accomplished with supported

BLMs. Before doing so, a short summary of the

basic properties of supported planar lipid bilayers is

in order.

Basic properties of supported BLMs

The development of electrochemical biosensors is

growing at a rapid pace since the early 1980s. In

the biomimetic approach, a lipid bilayer is used [87].

The functions of biomembranes are mediated by

specic modiers, which assume their active confor-

mations only in the lipid bilayer environment.

Further, the presence of the lipid bilayer greatly

reduces the interference and eectively excludes

hydrophilic electroactive compounds from reaching

the detecting surface, which may cause undesired

reactions. Thus, from the specicity, selectivity and

design points of view, the BLM is a natural en-

vironment for embedding a host of materials of

interest for the biosensor development. Hence, the

rationale behind the development of BLM-based

biosensors is remarkably simple: to function in a

biological environment the sensing element must be

biocompatible. The lipid bilayer (BLM) meets this

criterion and is, therefore, an ideal choice upon

which to develop a new class of electrochemical bio-

sensors.

Similar to conventional BLMs, the observed

transmembrane voltage results from a separation of

charges across the BLM. It is the dierence between

the two bulk phases separating by the lipid bilayer.The electric eld that gives rise to this potential lies

principally across the hydrocarbon chains of the



13/24

lipid bilayer. Thus, the origin of the potential across

the biface consists of the following: (a) the surface

potential, c, due to the orientation of dipoles at the

interface and (b) the outer potential, f, due to the

presence of excess free charges separated by the

biface [3, 5, 24,25, 57]. The so-called inner potential,

f (=w + c), where Df is known as Galvani poten-

tial dierence, may be thought as a measure ofchemical anity of the species in the two phases.

Incidentally, Df is also known as the phase-bound-

ary potential, since it exists only in the limited inter-

facial plane. It should be pointed out the so-called

Galvani potential which can not be measured exper-

imentally. The dierence across the biface, however,

is experimentally determinable [3, 5,24,25,57].

Dierent methods for the measurement of the mem-

brane surface potential Dfm, or changes therein

(Dfm), are described and compared with respect totheir sensitivity, generality of application and ease

of use. Examples of the measurement of Dfm for

Fig. 6. The liquid vBLMv liquid (solid or hydrogel) biface. A biface is dened as any two coexisting membranesubstrateinterfaces, through which material, charge and/or energy transfer are possible. In the BLM systems, conventional and

supported, one of the interfaces is always an aqueous solution, whereas the other can be solid (as in s-BLMs), hydrogel

(as in sb-BLMs) or another aqueous solution (as in conventional BLMs). In the BLM (lipid bilayer) domain, a variety

of materials have been embedded. On the outer (left) side, numerous compounds have been tested, some of which are

listed. On the inner (right) side, many aqueous soluble substances have been used and some of which are also given.

Light-induced photoelectric eects in the planar lipid bilayer are shown at the bottom of the gure, where the left side

acts as a cathode for reduction and the right side as a anode for oxidation (modied from Refs. [5, 24, 25]).



14/24

the determination of the surface charge density of

planar lipid bilayers are known [5, 7,59,60,57].

Essentially, it is due the dierence in electrostatic

free energy from a number of sources such as mem-brane constituents, surface and dipolar potentials.

Surface potential results from the potential dier-

ence between the membrane surface and the adja-

cent bathing solution, due to adsorbed or

covalently bonded species. Other potentials involved

in the electrochemistry of BLMs are the Nernst

Planck electrochemical potential and distribution

potential. The latter is due to the unequal distri-

bution of a species across the aqueousBLM inter-

face as a result of the hydrophiliclipophilic

balance (HLB). At thermodynamic equilibrium, an

electrical potential dierence (pd) between the two

phases occurs, so that further dissolution of ions in

both phases is stopped. This kind of potential is

also known as the distribution potential. Further,

at the interface, the law of electroneutrality may

be violated, as discussed by Guggenheim (see

[5, 24, 25]).

Concerning the mechanisms of conduction in

BLMs, one is reminded of the following facts:

(1) Specic resistivity of an unmodied BLM is

exceedingly high, >1015 O-cm.

(2) Permeability to water is fair,H2 104 cm/s,

which is consistent with water solubility in long-

chain hydrocarbon solvents. Thus, even at neutral

pH, [H+] = [OH] = 107 M, these ions will be

participating in electrical conduction.

(3) Redox reactions have been demonstrated in

BLMs containing highly conjugated compounds

and electron mediators such as TCNQ, TTF, ferro-

cenes, fullerenes (e.g. C60), which involve electronic

processes.

(4) Photoelectric eects have been observed in

pigmented BLMs, which imply that electronic pro-

cesses are possible in BLMs by electrons and

``holes''.

In s-BLMs (i.e. planar lipid bilayers on metallic

substrates), because of the highly insulating nature

of the lipid bilayer, it eectively excludes hydrophi-

lic species in the bathing solution from reaching the

metallic surface, thus undesired reactions are pre-

vented. Unlike the cleavage plane of pyrolite graph-

ite that is very hydrophobic, a freshly cut metallic

substrate is highly hydrophilic and attracts the

polar head groups of amphipathic lipid molecules.

Hianik et al. [37, 5963] have used the electrostric-

tion method to study the elasticity modulus perpen-

dicular to the lipid bilayer plane (E_), dynamic

viscosity coecient (Z), electrical capacitance (C)

and membrane potential (DFm) of s-BLMs formed

from soybean phosphatidylcholine as a function of

length of hydrocarbon chain of the solvent, choles-

terol concentration and dc voltage applied to themembrane. They found that E_ of s-BLMs is one

order of magnitude less than that for conventional

BLMs formed in the aqueous phase. Unlike that

for BLMs, E_ of s-BLMs did not depend on the

length of hydrocarbon chain of the solvent or the

cholesterol concentration in the lipid solution. Theparameters E_, Z and C of s-BLMs showed a com-

plicated behavior as a function of the amplitude,

polarity and rate of change of applied dc voltage.

In addition, s-BLMs are considerably more stable

than BLMs: their electrical breakdown voltage can

reach 1.5 V. Signicant dierences between s-BLMs

and BLMs are very probably due to dierences in

bilayer structure. Further, they have found [61] that

Cm decreases almost monotonically but irregularly

under these conditions. An increase in the scan rate

leads to a shift toward higher voltages of the value

at which the marked changes of E_ and Cm take

place. Repeated cyclic scans of the s-BLM lead to

the stepwise stabilization of E_ and Cm, i.e. the

changes of these parameters become less pro-

nounced. Analysis of experimental results suggests

that solvent redistribution between support-induced

inhomogeneities in s-BLM may be the main cause

of the observed eects. In another paper, Hianik et

al. [62] have reported the binding of streptavidin to

biotin-modied s-BLM and resulted in a slight

decrease of membrane capacitance, increase of E_and increase of Z, while DFm did not change. The

value of E_ of s-BLMs was found to change con-

siderably with increasing dc voltage and the rate of

voltage change. Modication of s-BLM by strepta-

vidin leads to reduced changes of E_ with the rateof dc voltage change and it made s-BLM extremely

stable even at an external dc voltage of 2 V. The

results reported in Refs. [59, 60, 62] indicate that

streptavidin considerably stabilized s-BLM by

means of the formation of a complex with biotin-

modied phospholipids. In connection with E_, the

properties of both conventional BLMs and s-BLMs

formed from archaeal lipids and/or from egg phos-

phatidylcholine (egg PC) as a function of dc applied

voltage were investigated [63]. The values of E_ for

BLM and s-BLM of archaeal lipids were approxi-

mately 1.4 times higher in comparison with BLMs

from egg PC. The values of breakdown voltage of

BLM from archaeal lipids were 2.3 times higher

than for egg PC membranes and reachedH290 mV.

s-BLM from both lipid composition were consider-

ably more stable than BLM and their breakdown

voltage well over 1 V. Also, the measured value of

E_ was found to change considerably with increas-

ing dc voltage, the rate of change of voltage and on

successive cycles of changing the voltage after mem-

brane formation [59, 60]. In an earlier paper on s-

BLM studies, Otto et al. [47] studied the inuence

of lipid concentration, lipid composition, pH and

temperature on the stability and function of metal-

supported phospholipid bilayer membranes (s-

BLM) and the potential applicability of this systemin biosensor development was explored. Parameters

to be measured were the resistance as well as the



15/24

amperometric response of the system to oxygen and

hydrogen peroxide. Stability and function of the s-

BLM system are inversely related to the lipid con-

centration. Cholesterol exerts a stabilizing eect ata molar ratio of 4:1 to 1:3 (PC:cholesterol). The

eect of stearylamine on membrane stability

depends on the sign of the polarizing voltage.

Ascorbic acid, but not citric acid nor glutathion nor

uric acid interferes with measurements of hydrogen

peroxide. Further, it was found that the s-BLM is

remarkably stable in urine. Its use as a simple but

sensitive oxygen sensor for measuring the respir-

ation rate of rat liver mitochondria was also

demonstrated. Consistent with the ndings of con-

ventional BLMs, the well-known stabilizing eect

of cholesterol was conrmed for the s-BLM system.

Both the pH in the range 5.08.0 (silver and stain-

less steel supports) and the ionic strength (for all

types of supports) in the range from 0.025 to

0.1 moll1 at pH 7.0 were found not to be

crucial [64].

Supported planar lipid bilayers modied with pH-

sensitive compounds

It was demonstrated in the earlier years of BLM

research that embedding the H+ carrier 2,4-dinitro-

phenol (DNP) into conventional BLMs results in

increased sensitivity to H+ ions [3]. In numerous

experiments supported BLMs have proved to pro-

vide suitable base for incorporation of various

kinds of substance isolated from biological mem-branes as well as of non-biological origin. This way

membrane systems could be prepared with distinc-

tive selective properties. Currently, for hydronium

ion detection, the pH glass electrode is routinely

used. However, the large size and fragility of pH

glass electrodes preclude their use in many situ-

ations such as in vivo cell studies and in monitoring

membrane boundary potentials. For example, the

hydrolysis of membrane lipids by phospholipid

enzymes (lipases A and C) could alter the boundary

potential of BLM because of a local pH change.

These ndings suggest that s-BLMs can be used as

a pH probe in membrane biophysical research and

in biomedical elds where the conventional glass

electrode presents many diculties. To test this con-

cept, a number of quinonoid compounds (chlora-

nils) have been embedded in s-BLMs and found

that, indeed, s-BLMs containing either TCoBQ (tet-

rachloro-o-benzoquinone) or TCpBQ (tetrachloro-p-

benzoquinone) responded to pH changes with a

nearly theoretical slope (55 + 3 mV) [4044]. This

pH-sensitive s-BLM oers prospects for ligand-

selective probe development using microelectronics

technologies [50]. The main obstacle to use such

modied bilayers as sensors for the eld application

was their poor mechanical stability. With the avail-

ability of the supported lipid bilayer system (s-BLM), the preparation of lipid bilayer membranes

with dramatically improved lifetime has become

possible. Preserving all the unique features of the

conventional lipid bilayer like the membrane thick-

ness in the nanometer scale, the bimolecular

arrangement of phospholipid molecules (the basicconstituents of all biological membranes), the s-

BLM opens the way to biosensor development.

Using a rather simple lipid preparation, Ziegler et

al. [43,44] have shown that a s-BLM prepared on

the cross section of a freshly cut Teon coated

stainless steel wire displays pH sensing properties

with a fair reproducibility and a sensitivity compar-

able to commercially manufactured macroelec-

trodes. Even when working on the same physico

chemical base, there is of course a dierence

between an experimental system to demonstrate a

sensory behavior of a suitably modied s-BLM and

the use of its sensory properties for practical pur-

poses. To pave the way from laboratory results to

sensor chips produced in larger amounts, they have

checked conditions to restore sensory properties

observed with the experimental set-up in the s-BLM

prepared on support structures manufactured by

microelectronics technologies and used in micro-

chip applications [50]. This paper reports the use of

a supported bilayer lipid membrane (s-BLM) con-

taining chloranils (TCoBQ and TCpBQ) a s a p H

sensor. The electrical parameters such as conduc-

tance and breakdown voltage of modied s-BLMs

have been studied. The electrical conductance of the

s-BLMs was in the range from 2 102 to

2 101

pS as estimated from their respective cur-rent/voltage curves. s-BLMs prepared from lipids

containing TCoBQ were used to monitor light-

induced pH changes in the vicinity of Nitella sp.

algae. The extent of light-induced pH changes was

strongly dependent on the distance between the sur-

face of algae cells and the tip of the s-BLM. After

adding DCCD, an inhibitor, to the algae cell, light-

induced pH changes were no longer observed [40

44, 65].

Most recently, a measuring ``microcell'' was

developed by Rehacek et al. [6668] consisting of a

thin-lm Ag/AgCl reference electrode and a s-BLM.

They have studied the inuence of the thin-lm sup-

ports (platinum, or gold) and the pH-sensitivity

mediating molecules (TCNQ and/or TCmBQ,

TCoBQ, TCpBQ) to the BLM-forming solution on

the electrical properties (capacitance, conductance)

and pH-response of the probe. Additionally, the

dependence of the essential Ag/AgCl electrode par-

ameters on the condition of its preparation are

shown. A simple portable pH-meter/voltameter

with an universal input adaptable for dierent

kinds of pH-sensitive or ion selective electrodes has

been constructed.

Supported planar lipid bilayers modied with ion

carriers

A hydrated ion cannot be transported across the

lipid bilayer owing to its very energy barrier (from



16/24

aqueous solution to the lipid bilayer, dielectric con-

stant, e, changes from 80 to about 25. An iono-

phore, such as valinomycin when complexed with

an ion, provides it a hydrophobic coat; it thusbecomes ``oil-soluble'' enabling it to cross the lipid

bilayer and be discharged on the other side of the

membrane. The overall driving force is provided by

the concentration gradient of the ions. With the

availability of crown ethers, in particular, natural

occurring valinomycin, an antibiotic which is able

to bind K+ and disrupts the normal functioning of

certain bacterial membranes. s-BLMs containing six

dierent kinds of crown ethers were synthesized

and investigated using CV [69, 70]. In particular, s-

BLMs formed from a liquid crystalline aza-18-

crown-6 ether and cholesterol-saturated n-heptane

solution was found to be sensitive to K

+

in theconcentration range of 104 to 101 M having a

Nernstian slope. The specicity for three alkali

metal cations and NH4+ of ve dierent kinds of

bis-crown ethers in BLMs were investigated. The

order of specicity for most of these bis-crown

ethers was found to follow hydrated radii of cat-

ions, i.e. NH4+>K+>Na+>Li+. The results

obtained with these s-BLMs compare favorably

with conventional BLMs containing related com-

pounds such as valinomycin [3]. It is interesting to

note that Minami et al. [71] earlier have made a

comparative study on the potentiometric responses

between a valinomycin-based conventional BLM

and a polymeric membrane. In this connection

some electrical properties were studied on potential

responses of self-assembled s-BLMs. Gramicidin

doped s-BLM sensors responded to hydrogen ion

concentrations linearly with the slope of 144 mV/

decade in the range of 0.4 to 4 M; monensin doped

s-BLM sensors responded to sodium ion, which has

a slope of about 40 mV/decade in the concentration

range of 0.3 to 30 mM; valinomycin doped s-BLM

sensors have nearly Nernstian response in the con-

centration range of 1.0 to 30 mM/[K+]; ferrocene

doped s-BLM sensors responded linearly to the fer-

ricyanide ion, whose slope is 146 mV/decade in the

concentration range of 0.03 to 30 mM/[K3Fe(CN)6].Many ions such as ferrocyanide, citrate, SCN,

ClO4, etc. did not interfere with the sensor response

characteristics. Factors inuencing s-BLM sensors'

reproducibility and stability were also investigated

[4042]. In another report the eect of valinomycin

on a dierent kind of s-BLM has been investigated

[4042]. An increase in concentration of valinomy-

cin from 6 106 to 6 104 moll1 increases the

conductivity of the system after addition of potass-

ium ions. A remarkable eect of the buer medium

on the electrode response has been observed, a sol-

ution of 0.1 M TrisHCl being most suitable. The

assembled s-BLM system is stable for about 120 h.A linear response toward potassium has been found

in the range from 2.5 103 to 1.3 101 moll1.

The eect of pH, temperature and possibly interfer-

ing substances are reported [4042].

Supported planar lipid bilayers modied with ion-channels

For transporting ions in biomembranes, ion-

channels embedded in the lipid bilayer are the key

to our understanding [35, 24, 25, 56]. There are two

main classes of ion-channels, voltage-gated and

ligand-gated, both of which operate by opening and

closing to regulate the ow of ions. As ions move

across the ion-channels embedded in the lipid

bilayer, they alter its electrical potential. This trans-

membrane potential results from a separation of

charge across the liquid/BLM/liquid biface. The

electric eld that gives rise to this transmembrane

potential is due primarily to the ultrathin hydro-

phobic portion of the lipid bilayer. One of well-stu-

died ion-channels embedded in conventional BLMs

is gramicidin [3, 4]. Steinem et al. [72] have carried

out an impedance analysis of ion transport through

gramicidin channels incorporated in solid supported

lipid bilayers.

Syringotoxin, a cyclic lipodepsipeptide produced

by certain strains of the phytopathogenic bacterium

Pseudomonas syringae pv. syringse, is known to

form anion selective, voltage-sensitive channels in

conventional BLMs [73]. With the availability of

highly stable sb-BLMs, it was decided to embed cer-

tain phytotoxic substances such as syringotoxin (S-

toxin) into the lipid bilayer, since the dimensions ofthis S-toxin are almost in the same range as the

lipid bilayer thickness [58, 73]. Indeed, the single ion

channel activities of this S-toxin-containing sb-BLM

were observed, which appear as a square-shaped

current uctuation during the transitions of the

channels between dierent conducting states. From

plots of frequency vs single-channel conductance

histogram, and the currentvoltage curve, a linear

relationship was obtained. In the latter case, the

slope conductance was found to be 125 pS for the

S-toxin-modied sb-BLM, which is in accord with

known values. Thus, we have established the useful-

ness of sb-BLMs in membrane channel reconstitu-

tion studies. Also using agar-supported planar lipid

bilayers, Uto and colleagues [55] have investigated

BLMs made of lecithin and cholesterol. The

membranes exhibited an average electric resistance

of 135 pS and a capacitance of 0.43mF cm2.

Gramicidin, known to form a channel in uni-lamel-

lar lipid bilayers, reduced the electric resistance

to a pS level, thus showing the membranes to

be of an uni-lamellar bilayer type. The BLM

stability was investigated against perturbation

with electric potentials and against mechanical

agitation in the contacted aqueous solution. About

80% of the membrane preparations remained

intact after applying electric potentials ofbetween 21500 mV. A similar percentage of the

membrane stayed intact under vigorous stirring. In



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a preliminary report [4042] some electrical proper-

ties of gramicidin doped s-BLM sensors were stu-

died, which responded to hydrogen ion

concentrations linearly with the slope of 144 mV/decade in the range of 0.4 to 4 M. In this connec-

tion Cornell et al. [74] most recently reported a sup-

ported planar lipid bilayer sensor using the ion-

channel gramicidin. The element of recognition is

provided by antibodies linked to the gramicidin

molecules. It is stated that the sensor has been

demonstrated in the detection of drugs, bacteria

and viruses, and in blood typing. Mention should

be made here that in earlier reports [7577], ion-

channels incorporated in s-BLMs have been stu-

died. For instance, Stenger et al. [77] discussed Pt-

supported phospholipid bilayers containing voltage-

dependent anion channels (VDAC) that were used

to physically model a chemically-sensitive ampero-

metric biosensor. An ac electrical technique was

used to measure admittance changes caused by

channel gating. The magnitude of the responses dif-

fered from those observed in conventional BLMs.

However, the supported BLMs were mechanically

stable and permitted reproducible gating of the

VDAC conductance in response to dc bias voltages

in the range of 0 to 60 mV. The authors concluded

that the technique may be generally suitable for

fabrication of durable biosensors using chemically-

sensitive protein channels.

Supported planar lipid bilayers modied with peptides

and enzymes

In the last few years a variety of enzyme-based

biosensor have been developed [11,78]. For

example, Schulmann et al. [79, 80] have reported im-

mobilization of enzymes on LB lms via a mem-

brane-bound receptor and suggested their possible

applications for amperometric biosensors. For such

biosensors, electrochemical techniques are often

used because of their sensitivity, rapid responses

and ease of interfacing with personal computers

(PC's). Presently, glucose sensors using electrical

techniques as a detection method are among the

most developed. In general, the principle of electro-

chemical transduction methods is simple. There

must be an intimate contact between the sensing el-

ement and transducer. Enzyme-based biosensors

using mainly oxidases, dehydrogenases and NH3generating enzymes are able to convert many sub-

strates into electroactive species. As a specic

example, the much studied glucose sensor using

amperometric detection be described as follows.

The enzyme glucose oxidase (GOD) is usually in-

corporated into a layer of thickness ranging from

tens of mm to a few nm (from carbon paste, var-

ious thin lms, to BLMs). The thin layer doped

with GOD in close contact with a substrate (e.g.

the surface of a metallic electrode) together with areference electrode constituted the basic unit of a

glucose biosensor. Operationally, the GOD-coated

metal working probe is held at a constant potential

with respect to the reference electrode (e.g. for

example, at 600 mV vs Ag/AgCl), glucose oxidase

converts glucose to gluconic acid and hydrogen per-oxide as is given by

glucose 4GOD

gluconic acid H2O2 15

H2O2 4 O2 2H 2e 16

The amount of H2O2 is proportional to the glucose

concentration. In equation (16), the direct transfer

of electrons from GOD to the transducing electrode

is not an easy task. For this purpose, s-BLM-based

probes containing TCNQ and TTF have been stu-

died, which will be covered in Section 3.6.

In connection with glucose sensors, Hianik and

co-workers [62, 63] have developed a mini-glucose

sensor based on immobilization of avidinglucose

oxidase (AGOD) complex onto the biotinylated

bilayer lipid membranes formed on a s-BLM. Using

the simple amperometric method the authors have

studied the glucose oxidation by GOD and deter-

mined several important constants of enzymatic

reaction. The enzyme turnover was approximately

1.1 s1, which is close to that for GOD reaction in

homogeneous solution. The Michaelis constant for

an enzymatic electrode KME=(0.6620.18) mM was

close to that for the enzymatic reaction of ferrocene

modied GOD immobilized onto glassy-carbon

electrode. The results are consistent with their ear-lier work [47]. Regarding this, Rehak and

colleagues [81] have earlier reported an ampero-

metric xanthine minibiosensor based on a supported

biotinylated phospholipid membrane. To this mem-

brane streptavidin-modied xanthine oxidase was

coupled. The fabricated biosensor corresponds in

shape and size to a needle of 0.3 mm in diameter.

The assay is based on the electrochemical detection

of enzymatically generated hydrogen peroxide. The

response to xanthine was linear up to 1 mmol/l with

a detection limit of 0.02 mmol/l. The response time

was less than 1 min and the biosensor was stable

for at least 5 days.

It should be mentioned that many interesting

results and ndings have been achieved with other

assembled systems [1720, 82, 39, 83] (see Table 1).

For examples, Puu and her colleagues [82] reported

the activity of acetylcholinesterase in lipid

monolayers [82]. Mandler and Turyan [83] have

reviewed the electroanalytical chemistry of the

dierent approaches of self-assembled systems.

Florin and Gaub [75] along with Seifert et al. [76]

have reported their measurements on a novel type

of supported BLMs. The formation process was

investigated with surface plasmon resonance mi-

croscopy. The optical and electrical properties of

the s-BLMs were determined for various types oflipids and as a function of temperature by means of

cyclic voltammetry and potential relaxation after



18/24

charge injection. They could show that these s-

BLMs exhibit an extraordinarily high specic resis-

tivity which, depending on the lipid, may be as high

as 109 O cm

2. They could also show that due tothis low conductivity, an electrical polarization

across the lipid bilayer relaxes with characteristic

time constants of up to 20 min. Specic conduc-

tance and specic capacitance of the solid sup-

ported membrane are comparable to those of a

conventional BLM. However, the solid supported

membrane has the advantage of a much higher

mechanical stability. The electrical activity of bac-

teriorhodopsin, Na,KATPase, H,KATPase and

CaATPase on the solid supported lipid bilayer are

measured and compared to signals obtained on a

conventionally prepared BLM. It is shown that

both methods yield similar results. The solid sup-

ported BLM therefore represents an alternative

method for the investigation of electrical properties

of ion translocating transmembrane proteins.

Meanwhile, Steinem and colleagues [72] have

reported an impedance analysis of ion transport

through ion channels incorporated in solid sup-

ported lipid bilayers.

The use of s-BLMs with other polypeptides and

enzymes have also been reported [8486]. Salamon

and Tollin have investigated the reduction of horse

heart cytochrome c at a Pt s-BLM which immobi-

lized vinyl ferrocene as an electron mediator. The

currentvoltage curves show that the direct electro-

chemistry of cytochrome c at the metal electrodeoccurs quite eciently. An adsorption equilibrium

constant for cytochrome at the BLM surface, as

well as an electron transfer rate constant between

the protein and the modied electrode have been

estimated from these results. The values of both

parameters are much higher than those reported

with other types of electrode modications, indicat-

ing that the s-BLM system provides substantial

improvements in electrochemical activity of cyto-

chrome c at metal electrodes. In another paper, the

same authors have reported the electron transfer

reactions between a s-BLM on Au substrate and

oxidized spinach plastocyanin using cyclic voltam-

metry. Plastocyanin was found to interact strongly

enough with the s-BLM. In this connection, the re-

duction of Escherichia coli thioredoxin (EcT), T4

thioredoxin (T4T) and glutathione (GSSG) occurs

at a s-BLM. The rst electron transfer has half-

wave potentials of 0.0520.01, 0.0720.01 and

0.0620.01 V, whereas the second one has values

of 0.4820.01, 0.3920.01 and 0.4520.01 V,

for EcT, T4T and GSSG, respectively. The scan-

rate dependence of the cyclic voltammetry indicates,

for both waves, that the process of electron transfer

is dominated by a bulk diusion of free species to

and from the electrode, and that strongly adsorbed

species do not signicantly contribute at the scanrates used. The voltage separation of the peak cur-

rents indicates a quasi-reversible electron transfer

process with an electrochemical rate constant which

is larger for the second (lower potential) electron

than for the rst one. These are in excellent agree-

ment with literature values obtained from equili-brium measurements of enzyme-catalyzed reactions

involving these species. It is quite clear from these

results that modied s-BLMs provide a biocompati-

ble and direct means of eciently carrying out elec-

trochemical reactions with sulfur-based redox

systems.

Several groups of researchers, including Racek

and associates [87] have proposed a planar conduc-

tometric urea sensor based on a microfabricated

interdigitated electrode arrays. Urease is immobi-

lized on the surface of this device and splits urea to

ammonia and carbon dioxide. These compounds

are ionized in an aqueous milieu increasing thus its

conductivity. This way of measuring seems to be

the most convenient and due to its simplicity and

cost-eectiveness is preferred above other detection

techniques for urease reaction (measuring of NH3,

NH4+, CO2 or pH with special electrodes).

In self-assembling systems related to s-BLMs (see

Table 1), many enzymes have also been investi-

gating by the CV technique [3234, 8891]. Kinnear

and Monbouquette [88] reported a strong cathodic

current response to the presence of fumarate that

provides evidence of direct electron transfer to

membrane-bound E. coli fumarate reductase in

membrane-mimetic alkanethiol monolayers on gold

electrodes. This enzymatic response is attenuatedreversibly by the competitive enzyme inhibitor, oxa-

loacetate. The hydrophobic enzyme is coadsorbed

with alkanethiols on gold in a novel detergent dialy-

sis process. Contino et al. [89] used planar-

supported phospholipid bilayers containing the

transmembrane protein tissue factor (TF). TF com-

plexed with the serine protease, factor VIIa, is the

primary initiator of blood coagulation and found

that the extracellular domain of TF is located on

the inner lea et. Also using supported BLMs,

Naumann and colleagues [90] have most recently

reported that a H+ATPase can be incorporated

into solid-supported lipid bilayers separated from

the gold support by a peptide spacer. The transloca-

tion of protons across the BLM to the inner side is

coupled to the discharge of protons at the gold sur-

face. The overall process is investigated by square

wave voltammetry (SWV) and double potential-

pulse chronoamperometry (CA). As a result, the

formation of a proton gradient is monitored by

SWV whereas currents measured by CA monitor

the stationary state when the enzyme activity is

directly coupled to the charge transfer at the elec-

trode. Further, the authors report that these cur-

rents markedly depend on the number of ATPases

present in the planar bilayer. The application of

cyclic voltammetry to biosensor devices based on s-BLMs was investigated by Wang et al. [91]. s-

BLMs have been used for receptorligand contact



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interactions. This paper reports the electrochemical

transduction of an antigenantibody (AgAb) reac-

tion by an s-BLM. The antigen (HBs-Ag) is incor-

porated into an s-BLM, which then can interactwith its corresponding antibody (HBs-Ab) in the

bathing solution. This AgAb interaction results in

some remarkable changes in the electrical par-

ameters (conductance, potential and capacitance) of

s-BLMs. The magnitude of these changes are

directly related to the concentration of the antibody

in the bathing solution. The use of such an AgAb

interaction via s-BLM as a transducing device for

the detection of ligandreceptor contact reaction is

proposed.

Supported planar lipid bilayers modied with TCNQ,

TTF, and ferrocenes

In bioenergetics, electron transfer and redox reac-

tions play central roles. These processes are

mediated by specialized proteins embedded in lipid

bilayer-bound subcellular organelles called chloro-

plasts and mitochondria. One approach to gain

some quantitative understanding of these mem-

brane-bound reactions in the early 1980s was resort-

ing to membrane reconstitution, using conventional

BLMs containing electron mediators such as TCNQ

(tetracyanoquinodimethane), TTF (tetrathiafulva-

lene), bipyridine, etc. [5, 9, 3234]. The embedding

of appropriate active molecules (modiers) into the

matrix of the lipid bilayer should be able to impart

the functional characteristics to s-BLMs. TCNQand TTF have been chosen as modiers because of

their properties as typical electron acceptor and

donor molecules, respectively. With TCNQ or TTF

modied BLMs, for example, oxidation takes place

on the side of the lipid bilayer facing the cathode,

whereas, reduction occurs on the side facing the

positive electrode. Two possible mechanisms may

be responsible for the observed transmembrane

redox reactions: (1) electron exchange between

redox components bound in the vicinity of the

opposite membranesolution interface and (2)

transmembrane diusion of the redox components.

These earlier experiments demonstrated that diu-

sion in the aqueous phase is rate limiting and there-

fore electron ``jumping'' or ``tunneling'' is a more

probable occurrence in the lipid bilayer. Thus, the

transverse movement of electrons and protons

across the modied bilayer results in a BLM which

functions as a bipolar redox electrode [3, 24, 25]. To

investigate this kind of reactions in BLMs, exper-

imentally, the technique of choice is cyclic voltam-

metry. The various parameters, i.e. background

current, redox potential peaks, stability of the me-

diator within the BLM during the electrochemical

measurements and reproducibility of the ampero-

metric response, were considered at

length [24, 25, 3234]. The past work provides agood starting point on the use of TCNQ, TTF, etc.

for the present development on supported BLM-

based biosensors, which is usually done by embed-

ding the species of interest (e.g. enzyme) together

with electron mediator in the BLM-forming sol-

ution before starting the self-assembling process onthe substrate.

It has been reported in a number of papers [92

97, 48, 49, 98, 99] that s-BLMs and conventional

BLMs containing TCNQ, TTF, or ferrocene are

excellent systems for mediated electrocatalytic redox

reactions. For examples, TTF modied s-BLMs can

be eectively used as a hydroge

Supported planar lipid bilayers (s-BLMs) as

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