Electrochimica Acta, Vol. 43, No. 23, pp.

3587±3610, 1998
# 1998 Published by Elsevier Science Ltd. All rights reserved
Printed in Great Britain
PII: S0013-4686(98)00107-8 0013±4686/98 $19.00 + 0.00

Supported planar lipid bilayers (s-BLMs) as

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

a
Membrane Biophysics Lab (Giltner Hall), Department of Physiology, Michigan State University,
East Lansing, MI 48824, U.S.A.
b
Center for Interface Sciences, Department of Microelectronics, Slovak Technical University, Bratislava,
Slovak Republic

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

AbstractÐThis paper presents a description of current research on the use of metal and hydrogel supported
bilayer lipid membranes (s-BLMs and sb-BLMs) in the area of biosensor development. Simple and
straight-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 the
topics covered include ion sensors, antigen±antibody interactions via electrical detection, probes for mol-
ecular species, supported BLMs doped with fullerenes and photoelectric e€ects in C60-containing BLMs.
# 1998 Elsevier Science Ltd. All rights reserved

Key words: lipid bilayer, BLM, supported BLMs biosensors, antigen, fullerenes, photoelectric e€ects, gel,
membranes.

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

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 elucidating
the mechanisms of ligand±receptor interactions.
One of the main tasks of biomembrane functions is
molecular recognition which entails selectivity and
speci®city. Thus, understanding the principles that
lie behind the structural±functional 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-
*Author to whom correspondence should be addressed. E-
mail: ottova@pilot.msu.edu
3587
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 and
then delivering a response based on this received in-
formation. With the availability of supported
3588 H. T. Tien and A. L. Ottova
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 low
cost in its construction, a supported BLM o€ers 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 …
where v line denotes an interface. A free standing
BLM separating two aqueous solutions is truly a
miraculous 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 [3±9].
The main reason for the sustained interest in exper-
imental planar lipid bilayers (BLMs) is two-fold: (a)
basic scienti®c 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 [5±9]. Further, the vast majority of
physiological/biochemical reactions involve a high
degree of molecular recognition. These reactions are
collectively known as ligand±receptor interactions,
of which the BLM system has often been
used [10, 11]. Some of these include antigen±anti-
body binding, substrate±enzyme reaction, ion-car-
Table 1.
Self-assembling amphiphilic interface-active systems
Type
(1) Soap ®lms
(2) Monolayers (Langmuir type)
(3) Multilayers (Langmuir±Blodgett type)
(4) Planar lipid bilayers (BLMs)
(5) Nucleopore supported BLMs
(6) Gold supported monolayers
(7) Metal supported BLMs (s-BLMs)
(8) Salt-bridge supported BLMs (sb-BLMs)
*aqueous solution
$
such as glass
%
tens of thousands of BLMs in a polycarbonate ®lter
}
such as Au
}
such as Pt, stainless steel
6
such as agar in 3 M KCl.
rier selectivity, ion-channel speci®city, hormone-
gated channels, light- and redox-species-induced
reactions. Further, the BLM system allows the
exquisite investigation of electrical properties (mem-
brane potentials, resistance, current±voltage 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
1†
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 ÿ BLMj metallic substrate …2†
and
Aqueous solution jsb ÿ 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. Sucient details for the preparation and
Interface Refs.
air vsoap ®lmv air [12]
air vmonolayerv water* [13]
air vmultilayerv solid substrate$ [14]
water vBLMv water [15]
water vBLMs%v water [16]
air vthio monolayer}v Au [17±20]
water vBLMv metal} [21]
water vBLMv hydrogel + KCl6 [22, 23]
 Supported planar lipid bilayers
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 di€er from the
much studied Langmuir±Blodgett (L±B) ®lms and
other thin ®lm systems [13, 14, 17±20] 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 L±B ®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
ligand±receptor 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 Te¯on 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, 24±30]. For
example, Ladha and colleagues [28±30] 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, which
are amenable to physical constraints, and thus o€er
new opportunities for systematic studies of func-
3589
tion±structural relationships in lipid bilayers. These
techniques developed speci®cally for conventional
BLMs are of obvious interest to the investigator of
supported BLMs, and their applications to such
systems 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 squalene±decane (1:1
v/v), (2) 20 mg PC + 5 mg cholesterol in 1 ml n-
decane, (3) 25±100 mg PC/ml of squalene±butanol
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 unbu€ered 0.1 M KCl (or NaCl) to
0.1 M KCl in 0.1 M Tris±HCl at pH 7 or use 0.1 M
phosphate bu€er. Te¯on-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. Te¯on 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 Te¯on-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.
Speci®cally, the formation of a supported BLM
consists of two-consecutive steps. First, the tip of a
Te¯on-coated Pt (or Ag, stainless steel, Au or other
3590 H. T. Tien and A. L. Ottova

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 [32±34]. The working electrode can be either a s-BLM probe or a sb-BLM probe. In either case, a P±G
(Plateau±Gibbs) border, a torous of lipid solution, surrounds a BLM. The reference electrode can be either an Ag/AgCl
as 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.

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 to
di€use away leaving a BLM to self-assemble on the
metallic tip. The ®nal BLM is surrounded by a
Plateau±Gibbs 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 the
air±water interface). By doing so, a supported BLM
is assembled at the wire tip [35, 36].
 Supported planar lipid bilayers
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.
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 [21±23, 37±49] 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 possibilities
for the development of miniaturized biosensors as
well as for basic research of electrical and mechan-
3591
ical properties of biological membranes. More
details may be found in recent publications [51±54].
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 given
below.
Materials. Te¯on 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
Te¯on 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 Te¯on
tubing is cut in air and then the cut end immedi-
ately immersed in the lipid solution. The next step
3592 H. T. Tien and A. L. Ottova
Fig. 3. Construction details and procedure for making an s-BLM probe on a metal wire tip.
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 Te¯on tubing (00.5 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 Te¯on tubing are then glued together with
wax at the point of insertion. In this way an Ag/
AgCl±Te¯on tubing salt bridge (sb) is constructed
(Fig. 1). In the second step, the tip of the other end
of the Te¯on salt bridge is cut in situ with a razor
blade while immersed in a BLM-
forming solution, as done with the s-BLM
technique [21, 37]. In the third and last step, the
Ag/AgCl±Te¯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/AgCl±Te¯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 identi®ed as RE
(reference electrode) and WE (working electrode),
thereby eliminating the inner Te¯on beaker as has
been done in a conventional BLM setup [3].
Hydrogel-supported sb-BLMs formed in this
 Supported planar lipid bilayers
manner exhibit an average Rm=1.4  1011 O,
Cm=0.45 mF cmÿ2 having excellent mechanical
stability and is able to withstand an applied voltage
on the order of 21.5 V.
Electrical measurements
Over the years a number of techniques have been
developed for characterizing the liquid/BLM/liquid
biface, including the following [3±5, 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 electri®ed as a result of the
charge transfer, separation, and/or redox reactions.
The charges present on BLM interfaces create elec-
tric ®elds which greatly in¯uence 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 and
pointed toward possible technological applications
of BLMs. Ions may be transported through the
membrane either by a carrier or by di€usion
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 membrane±solution 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-di€uses 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 [3±5, 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 side
facing the negative electrode and reduction on the
side of the anode. The formation of BLMs now
3593
makes it possible to examine the phenomenon of
electrostenolysis [3] in a structure similar to bio-
membranes.
All biomembranes are composed of a lipid bilayer
intercalated 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
(01 mF cmÿ2) 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 di€usion 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 substrate
and an aqueous solution (i.e. metal vBLMv aqueous
solution or gel vBLMv aqueous solution) can be
easily 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 [32±34], a block containing two adja-
cent 2 cm diameter chambers (one of which holds a
10 ml Te¯on cup), was commonly used. The Te¯on
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 con®guration: one reference electrode
(SCE or Ag/AgCl) is placed in the Te¯on cup and
two other reference electrodes are on the outside.
The voltammograms of the BLM are obtained
using an X±Y 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
3594 H. T. Tien and A. L. Ottova
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]).
voltage on the current being measured. This means
that the current is measured under ``voltage clamp''
with accuracy 21 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 ÿ0.8 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'' pro®le 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, 32±34]. The current (i) for the
transfer of charges from one side of the BLM to
the other side is given by
i ˆ kn3=2 AD1=2 1=2 c …4†
where k is a constant, n is the charge of the species,
A is the interfacial area, D is the di€usion 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 di€erent s-
BLMs and the acceleration e€ect 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 of 2V0 with the scan rate A (mV
sÿ1) is the
input from the circuit. The current in nanoamperes
or picoamperes is measured. There are two com-
ponents in the current through the membrane,
 Supported planar lipid bilayers

Fig. 5. Equivalent circuits of conventional and supported BLMs. Left: Showing a 3D view of a BLM modi®ed with electron mediators such as TCNQ, TTF, ferrocene, iodine, fullerene C60,
etc. (see Fig. 6 for other BLM modi®ers). Right: Top, the usual representation of a conventional BLM consisting a resistor and a capacitor in parallel. Bottom, the improved circuit for the
supported BLM [32±34, 46, 48, 49].
3595
3596 H. T. Tien and A. L. Ottova
namely the charging current ic and resistance cur-
rent ir. The former is determined by the capacitance
as follows:
dV
ic ˆ C m ˆ Cm A …5†
dt
It can be shown that the capacitance current ic
through 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.
V e€ect of Rn on the shape of I/V cycle is carried out.
ir ˆ …6†
Rm
So the net current passing through the membrane
can be expressed as
V biomembrane. From the obtained curves, the simi-
i ˆ ir ‡ ic ˆ ‡ Cm A …7†
Rm
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 I±V response. ic will jump to its negative
values suddenly (ÿic) 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-
di®ed s-BLM has a di€erent shape when compared
with parallelograms obtained for the planar BLMs.
The di€erence 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 I±V 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 Cs
is 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 a€ected by the proposed non-mem-
brane resistance Rn, whose composition and e€ect
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 e€ect of be
achieved 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 and
Rn 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 Cm
constant.
An improved equivalent circuit has been devel-
oped, where both Rn and Cs are involved in the
consideration [46]. A detailed observation of the
In our experiment, Rn varies in a wide range from
100 to 109 O, while the other membrane properties
are simulated holding Rm at 109 O, Cm at 3 nF con-
stant, both of which are still within the range of
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 108 O
(10.1Rm), 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(1Rm), 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. In
this last case, The intercept can no longer re¯ect 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 a€ection [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 metal±lipid interface (inter-
face resistance). Other important factors in Rn
include the electrode, electrolyte and solution impe-
dance, and any other components which greatly
e€ect the measurement accuracy.
Simulation of the e€ect of Rn on the shape of the
I±V 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 most
important feature is that, when Rn reaches the same
order as Rm, the voltammogram does not display a
 Supported planar lipid bilayers 3597

parallelogram shape any longer, and it is very simi- stant term CsA in equation (9) will be negative at
lar to that observed for the s-BLM system. The the very moment when the sweep potential reaches
intercept with the i axis drops sharply. The slope its maximum and begins to reverse. So, the height
decreases during potential sweeping and it is much 2h corresponds to twice the value of the distribution
lower than the slope in the case of BLM without capacitance Cs. So from the on-line computer
Rn. In this last case, the intercept can no longer acquire and parameters determination, the height
re¯ect the membrane capacitance and it is the same 2h can be read out and Cs is determined by
as the slope of membrane resistance [46]. Cs ˆ h=A
Here, we propose a new method for determining
Rm, Cm, Cs and Rn accurately from the on-line s- The di€erentiation of equation (9) is given by
BLM cyclic voltammogram. To make clear the di 1 2Rm
e€ect of Rn on the I±V response, the quantitative ˆ ‡
dV Rm ‡ Rn Rn …Rm ‡ Rn †
relationship of the membrane current with the
 
sweep wave potential should be derived rigorously …Rm ‡ Rn †…V ‡ V0 †
exp ÿ …10†
from the solution of coupled equations based on ARn Rm Cm
the equivalent circuit. The set of di€erential
equations is as follows: The potential of the triangular sweep wave moves
… from ÿV0 to +V0 (from a to b). At the half-cycle
1 t terminal b, the exponential term can be rationally
i ˆ ir ‡ ic ‡ is ir R m ˆ ic dt
Cm 0 omitted and the slope of the tangent line CD at the
… point b can be expressed as
1 t
V ˆ …ir ‡ ic †Rn ‡ c dt is ˆ Cs A …8† 1 1
Cm 0 ˆ …11†
D Rm ‡ Rn
where i is the total current recorded, is, which is
de®ned as the current through Cs, and all other par- The intercept of CD with the current i-axis is y1
ameters are de®ned above. Here, the ®nal (de®nite) which is
solution i = f(V) is presented, where Rn and Cs AR2m Cm
have been included: y1 ˆ …12†
 …Rm ‡ Rn †2
ARm Cm V Rm 2Rm
iˆ ‡ ÿ The intercept of the voltammogram with the cur-
Rm ‡ Rn ARm Cm Rm ‡ Rn Rm ‡ Rn
  rent i-axis is y2; therefore
…Rm ‡ Rn †…V ‡ V0 †  
exp ÿ ‡ Cs A …9† Rm V0
ARn Rm Cm Dy ˆ y1 ÿ y2 ˆ 2y1 exp ÿ …13†
y1 DRn
The initial condition for equation (9) is
Thus without any time-wasting ®tting procedures,
Vtˆ0 ˆ ÿV0 ‡ At ˆ ÿV0 all the membrane parameters can be calculated
During the half-cycle sweep from a to b, we can see cycle by cycle from the on-line data acquired as fol-
from equation (9) that there are three components lows:
included in membrane current: (1) the time-linearly  2
m y1 1
dependent term which is the resistance current Rm ˆ D Cm ˆ ‡1
m‡1 A m
obeying Ohm's law; (2) the time-exponentially    
dependent term in which Rn, Rm and Cm should be 1 h 2y1 D
Rn ˆ D Cs ˆ m ˆ ln y1 …14†
taken into account and (3) the constant term result- m‡1 A Dy V0
ing from the distributing capacitance Cs for the
entire circuit. In concluding this analysis, the only parameters
The traditional way for parameter determination that need to be acquired directly for a recorded vol-
is to vary each of the parameter values while hold- tammogram of an s-BLM are D, y1, Dy and 2h.
ing all other parameters constant and to select a ®t- From the relationship described in equation (14), it
ting curve with a minimal deviation. However, the is now possible to calculate accurate values of the
complexity of the calculation is the major impedi- membrane electrical parameters for the s-BLM sys-
ment in ®nding a group of e€ective suitable values tem. In this section, an unusual method is proposed
to ®t the calculated curve. Despite the aid of com- which can determine the properties accurately with-
puter, the calculation still requires much more time, out any iteration [46]. So the real-time measurement
which can not be utilized for on-line parameter de- can be carried out for the dynamic analysis.
termination. So it is necessary to derive an accurate
expression for the parameters Rm, Cm, Rn and Cs REVIEW OF PAST WORK
from the original solution of equation (9).
Thus, the method for determining Rm, Cm, Rn Before presenting the research ®ndings on the use
and Cs has been obtained [46]. First of all, the con- of supported BLMs as electrochemical biosensors,
3598 H. T. Tien and A. L. Ottova
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 vital
functions 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 bene®tted 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 speci®city 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
speci®c substrates. These types of enzyme±substrate
reactions (a sort of ligand±receptor interactions)
require a transducer which converts the interaction
into a quanti®able 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 de®ned 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
receptor±ligand, host±guest, enzyme±substrate, etc.
interactions. The molecule that is doing the recog-
nition is called a host (lock, receptor), which has a
con®guration that is complementary to that of the
guest (key, ligand). Ideally, one would like to
develop a liquid vBLMv liquid (solid) biface system
that function as a transducer±actuator. That is a
suitably modi®ed 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 vBLMv
liquid (solid) biface system that will self-assemble to
function as a transducer±actuator. 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
speci®c modi®ers, which assume their active confor-
mations only in the lipid bilayer environment.
Further, the presence of the lipid bilayer greatly
reduces the interference and e€ectively excludes
hydrophilic electroactive compounds from reaching
the detecting surface, which may cause undesired
reactions. Thus, from the speci®city, 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 di€erence 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
 Supported planar lipid bilayers 3599

Fig. 6. The liquid vBLMv liquid (solid or hydrogel) biface. A biface is de®ned as any two coexisting membrane±substrate
interfaces, 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 e€ects 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 (modi®ed from Refs. [5, 24, 25]).

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 di€erence, may be thought as a measure of
chemical anity 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 di€erence across the biface, however,
is experimentally determinable [3, 5, 24, 25, 57].
Di€erent methods for the measurement of the mem-
brane surface potential Dfm, or changes therein
(Dfm), are described and compared with respect to
their sensitivity, generality of application and ease
of use. Examples of the measurement of Dfm for
3600 H. T. Tien and A. L. Ottova
the determination of the surface charge density of
planar lipid bilayers are known [5, 7, 59, 60, 57].
Essentially, it is due the di€erence in electrostatic
free energy from a number of sources such as mem-
brane constituents, surface and dipolar potentials.
Surface potential results from the potential di€er-
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 aqueous±BLM inter-
face as a result of the hydrophilic±lipophilic
balance (HLB). At thermodynamic equilibrium, an
electrical potential di€erence (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) Speci®c resistivity of an unmodi®ed BLM is
exceedingly high, >1015 O-cm.
(2) Permeability to water is fair, 02  10ÿ4 cm/s,
which is consistent with water solubility in long-
chain hydrocarbon solvents. Thus, even at neutral
pH, [H+] = [OHÿ] = 10ÿ7 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 e€ects 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 e€ectively 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, 59±63] have used the electrostric-
tion method to study the elasticity modulus perpen-
dicular to the lipid bilayer plane (E_), dynamic
viscosity coecient (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 the
membrane. 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. The
parameters 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. Signi®cant di€erences between s-BLMs
and BLMs are very probably due to di€erences 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 e€ects. In another paper, Hianik et
al. [62] have reported the binding of streptavidin to
biotin-modi®ed 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. Modi®cation of s-BLM by strepta-
vidin leads to reduced changes of E_ with the rate
of 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-
modi®ed 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 reached 0290 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 in¯uence
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 system
in biosensor development was explored. Parameters
to be measured were the resistance as well as the
 Supported planar lipid bilayers
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 e€ect at
a molar ratio of 4:1 to 1:3 (PC:cholesterol). The
e€ect 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 e€ect
of cholesterol was con®rmed for the s-BLM system.
Both the pH in the range 5.0±8.0 (silver and stain-
less steel supports) and the ionic strength (for all
types of supports) in the range from 0.025 to
0.1 mol
lÿ1 at pH 7.0 were found not to be
crucial [64].
Supported planar lipid bilayers modi®ed 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 diculties. 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) [40±44]. This
pH-sensitive s-BLM o€ers prospects for ligand-
selective probe development using microelectronics
technologies [50]. The main obstacle to use such
modi®ed 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
3601
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 basic
constituents 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 Te¯on 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 di€erence
between an experimental system to demonstrate a
sensory behavior of a suitably modi®ed 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) as a pH
sensor. The electrical parameters such as conduc-
tance and breakdown voltage of modi®ed s-BLMs
have been studied. The electrical conductance of the
s-BLMs was in the range from 2  10ÿ2 to
2  10ÿ1 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. [66±68] consisting of a
thin-®lm Ag/AgCl reference electrode and a s-BLM.
They have studied the in¯uence 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 di€erent
kinds of pH-sensitive or ion selective electrodes has
been constructed.
Supported planar lipid bilayers modi®ed with ion
carriers
A hydrated ion cannot be transported across the
lipid bilayer owing to its very energy barrier (from
3602 H. T. Tien and A. L. Ottova
aqueous solution to the lipid bilayer, dielectric con-
stant, e, changes from 80 to about 2±5. An iono-
phore, such as valinomycin when complexed with
an ion, provides it a hydrophobic coat; it thus
becomes ``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
di€erent 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 the
concentration range of 10ÿ4 to 101 M having a
Nernstian slope. The speci®city for three alkali
metal cations and NH+
4 of ®ve di€erent kinds of
bis-crown ethers in BLMs were investigated. The
order of speci®city for most of these bis-crown
ethers was found to follow hydrated radii of cat-
ions, i.e. NH+ + + + Syringotoxin, a cyclic lipodepsipeptide produced
4 >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, SCNÿ,
ClOÿ4 , etc. did not interfere with the sensor response
characteristics. Factors in¯uencing s-BLM sensors'
reproducibility and stability were also investigated
[40±42]. In another report the e€ect of valinomycin
on a di€erent kind of s-BLM has been investigated
[40±42]. An increase in concentration of valinomy-
cin from 6  10ÿ6 to 6  10ÿ4 mol
lÿ1 increases the
conductivity of the system after addition of potass-
ium ions. A remarkable e€ect of the bu€er medium
on the electrode response has been observed, a sol-
ution of 0.1 M Tris±HCl 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  10ÿ3 to 1.3  10ÿ1 mol
lÿ1.
The e€ect of pH, temperature and possibly interfer-
ing substances are reported [40±42].
Supported planar lipid bilayers modi®ed with ion-
channels
For transporting ions in biomembranes, ion-
channels embedded in the lipid bilayer are the key
to our understanding [3±5, 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.
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 of
this 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 di€erent conducting states. From
plots of frequency vs single-channel conductance
histogram, and the current±voltage curve, a linear
relationship was obtained. In the latter case, the
slope conductance was found to be 125 pS for the
S-toxin-modi®ed 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 0.43 mF cmÿ2.
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 of
between 21500 mV. A similar percentage of the
membrane stayed intact under vigorous stirring. In
 Supported planar lipid bilayers
a preliminary report [40±42] 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 [75±77], 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 ÿ60 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 modi®ed 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 L±B ®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 NH3
generating enzymes are able to convert many sub-
strates into electroactive species. As a speci®c
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 a
reference electrode constituted the basic unit of a
glucose biosensor. Operationally, the GOD-coated
3603
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
GOD
ÿ gluconic acid ‡ H2 O2
glucose ÿ
ÿ4 …15†
H2 O2 ÿ4 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 avidin±glucose
oxidase (A±GOD) 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 sÿ1, which is close to that for GOD reaction in
homogeneous solution. The Michaelis constant for
an enzymatic electrode KME=(0.66 2 0.18) mM was
close to that for the enzymatic reaction of ferrocene
modi®ed 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-modi®ed 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 [17±20, 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
di€erent 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 of
lipids and as a function of temperature by means of
cyclic voltammetry and potential relaxation after
3604 H. T. Tien and A. L. Ottova
charge injection. They could show that these s-
BLMs exhibit an extraordinarily high speci®c resis-
tivity which, depending on the lipid, may be as high
as 109 O cmÿ2. They could also show that due to
this low conductivity, an electrical polarization
across the lipid bilayer relaxes with characteristic
time constants of up to 20 min. Speci®c conduc-
tance and speci®c 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,K±ATPase, H,K±ATPase and
Ca±ATPase 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 [84±86]. 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
current±voltage curves show that the direct electro-
chemistry of cytochrome c at the metal electrode
occurs quite eciently. An adsorption equilibrium
constant for cytochrome at the BLM surface, as
well as an electron transfer rate constant between
the protein and the modi®ed electrode have been
estimated from these results. The values of both
parameters are much higher than those reported
with other types of electrode modi®cations, 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 ÿ0.05 2 0.01, ÿ0.07 2 0.01 and
ÿ0.06 2 0.01 V, whereas the second one has values
of ÿ0.48 2 0.01, ÿ0.39 2 0.01 and ÿ0.45 2 0.01 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 di€usion of free species to
and from the electrode, and that strongly adsorbed
species do not signi®cantly contribute at the scan
rates 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 modi®ed s-BLMs provide a biocompati-
ble and direct means of eciently 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-e€ectiveness is preferred above other detection
techniques for urease reaction (measuring of NH3,
NH+ 4 , 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 [32±34, 88±91]. 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 attenuated
reversibly 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 receptor±ligand contact
 Supported planar lipid bilayers
interactions. This paper reports the electrochemical
transduction of an antigen±antibody (Ag±Ab) reac-
tion by an s-BLM. The antigen (HBs-Ag) is incor-
porated into an s-BLM, which then can interact
with its corresponding antibody (HBs-Ab) in the
bathing solution. This Ag±Ab 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 Ag±Ab
interaction via s-BLM as a transducing device for
the detection of ligand±receptor contact reaction is
proposed.
Supported planar lipid bilayers modi®ed 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, 32±34]. The embedding
of appropriate active molecules (modi®ers) into the
matrix of the lipid bilayer should be able to impart
the functional characteristics to s-BLMs. TCNQ
and TTF have been chosen as modi®ers because of
their properties as typical electron acceptor and
donor molecules, respectively. With TCNQ or TTF
modi®ed 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 membrane±solution interface and (2)
transmembrane di€usion of the redox components.
These earlier experiments demonstrated that di€u-
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 modi®ed 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, 32±34]. The past work provides a
good starting point on the use of TCNQ, TTF, etc.
for the present development on supported BLM-
3605
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 on
the 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 modi®ed s-BLMs can
be e€ectively used as a hydrogen peroxide sensor
[96, 97], whereas TCNQ modi®ed s-BLMs show
promise for the development of glucose probes [100],
as described in some details below.
Supported bilayer lipid membranes (s-BLMs)
were formed by the self-assembly method on freshly
fractured metallic wires [94, 95]. The in¯uence of a
host of compounds in membrane forming solutions
on s-BLMs were examined and the e€ect of pH on
the function of the s-BLM studied. Additionally,
the redox reactions of a s-BLM containing vinyl-
ferrocene as an electron mediator have been investi-
gated by cyclic voltammetry. The results have
shown that ferrocene can be very easily immobilized
in s-BLMs. This suggests that s-BLM systems o€er
a novel approach to the electrode modi®cation by a
simple way of immobilization of compounds within
BLM. For instance, a TTF modi®ed s-BLM has
been found that it can not only improve the stab-
ility, but increased the range of s-BLM's sensitivity
to hydrogen peroxide [96, 97]. In contrast, TCNQ-
containing s-BLMs showed little response to H2O2.
This was not entirely unexpected since TCNQ
should behave as an electron acceptor. In another
report, when TCNQ was embedded in the s-BLM
forming solution, the resulting s-BLM was able to
detect the presence of ascorbic acid, which is con-
sistent with the ®ndings obtained with conventional
BLMs [22, 23, 32±34]. In terms of the mechanical
stability and conductivity, another promising system
for biosensor development seems to be the modi®-
cation of the s-BLM with the electron mediator
nathraquinone-2-sulfonic acid [48, 49]. Additionally,
the lead ion sensitivity of a solid-supported BLM
system containing TCNQ as an electron mediator
in 4,4'-n-octyl-cyanobiphenyl (8CB, a liquid crystal)
dissolved in squalene, with a Te¯on coated plati-
num wire as a solid support, has been investigated
with cyclic voltammetry [98]. These results have
shown that TCNQ in a 8CB-containing s-BLM pro-
duces a unique system that increases by three orders
of magnitude the lead ion sensitivity of the plati-
num electrode. Cyclic voltammograms have also
shown that a charge-transfer complex between
liquid crystal and TCNQ molecules is created and
participates in redox reactions. The electrochemical
behavior of TCNQ modi®ed s-BLM has been inves-
tigated through capacitor measurement and cyclic
voltammetry (CV) which shows the surface wave
behavior of the TCNQ redox form. The voltamme-
3606 H. T. Tien and A. L. Ottova
try CV has shown di€erent peaks at di€erent scan
rates and a possible explanation is provided [99].
Generally speaking, TTF is a better mediator for
the development of peroxide biosensor compared to
TCNQ and substituted ferocenes. On the other
hand, TCNQ is a better mediator for the develop-
ment of a glucose sensor. In this connection, a glu-
cose minisensor based on a biotinylated, s-BLM has
been reported [100]. This was carried out as follows.
First, glucose oxidase (GOX) was immobilized by
coupling an avidin±GOX complex to a s-BLM
formed from biotinylated crude ox brain extract.
Second, the BLM was supported on the metal tip
of a Te¯on-coated stainless steel wire (diameter
0.33 mm). Third, the determination of glucose was
based on detection of enzymatically generated
hydrogen peroxide at a potential of +670 mV.
There was an almost linear increase of membrane
current with increasing glucose concentration up to
10 mM and a saturation e€ect above 30 mM glu-
cose. A lower voltage for detection of glucose was
made possible by modi®cation of the membrane by
TCNQ. The capacitance relaxation method was
used to determine the changes of dielectric relax-
ation times (reorientation of dipole moments of
polar groups of individual lipid molecules or lipid
clusters) following addition of avidin±GOX. The
above-mentioned results clearly demonstrate the in-
teraction of the embedded enzyme with the
bilayer [100].
Concerning ferrocenes, Tai and co-workers [70]
reported that the biferrocene-containing Schi€ base
complexes were synthesized and embedded in Pt-
wire s-BLMs. The data obtained from cyclic vol-
tammetry provide an oxidation mechanism of elec-
trocatalysis of ascorbic acid.
Recent advances in supported planar lipid bilayers
modi®ed with fullerenes
Within the last decade fullerenes (C60) have been
of great interest in materials science, since it has
been shown that C60 is a good electron mediator
and behaves as an n-type semiconductor
(bandgap = 1.6 eV). Our interest in fullerenes as a
BLM modi®er is owing to their most unique prop-
erties, namely, unmodi®ed C60 and C70, are water-
insoluble and highly hydrophobic. Hence, the lipid
bilayer is an ideal environment for these compounds
to reside. Recently we have investigated electron
transfer across a BLM containing C60, which can
act both as an electron mediator [101] and a
photosensitizer [102]. The presence of C60 greatly
strengthens the stability of s-BLMs and dramati-
cally alters their electrical properties. The cyclic vol-
tammograms contain distinct redox peaks, which
are not symmetrical. Further, the C60 in the lipid
bilayer has the opposite e€ects on membrane resist-
ance (Rm) and capacitance (Cm), respectively; it
causes the Rm to decrease and Cm to increase. The
presence of iodine in conjunction with C60 in the s-
BLMs further accentuated the e€ects. It is apparent
that both C60 and iodine, when embedded in
BLMs, facilitate electrical conduction, thereby low-
ering the Rm. Since the dielectric constant (e) of a
typical, unmodi®ed BLM is about 2, the presence
of C60 and iodine should exert a great in¯uence on
e. Since Cm depends on a number of factors such as
the surface charge of the BLM, the nature of hy-
drocarbon chains and embedded modi®ers, an
increase in e and consequently in Cm, is therefore
expected. The modi®ed probe increased the detec-
tion limit for Iÿ by 100 times with a linear response
in the range from 10ÿ8 to 10ÿ2 M. Thus, C60 facili-
tates the discharge of Iÿ
3 at the metal surface, which
demonstrates clearly that the embedded C60 is
indeed an excellent electron mediator [102].
Concerning fullerenes, such as C60, as photonab-
sorbers, mentioned should be made once more
about electron transfer processes in the membranes
of photosynthesis and mitochondrial respiration,
both of which have been reported in a number of
BLM studies [16, 24, 25, 89±91, 101±103]. In the mid
1980s, electron transfer was demonstrated in
BLMs [24, 25]. These phenomena were explained in
terms of the light-induced charge generation and
separation, ®eld-driven charge transport and sub-
sequent redox reactions on opposite sides of the
BLM. In the absence of light, the theory of electron
tunneling was invoked (see Section 3.6). When a s-
BLM doped with Zn±phthalocyanine was excited
by light, a voltage and a current were recorded,
with the action spectra paralleled closely to that of
the absorption spectrum of the pigment [21]. This
provides direct evidence that the light-induced cur-
rents are indeed due to the photonabsorber
embedded in the BLM. Thus, a pigmented s-BLM
can function as a light transducer or photon-acti-
vated switch or detector. In this connection, the ex-
periments reported by Rolandi et al. [103],
Yonezawa et al. [104], Lamrabte et al. [105],
Yamaguchi and Nakanishi [106], Bi et al. [107],
Ikematsu and colleagues [108], and more recently
by Gruszecki and colleagues [109] are of great inter-
est. Mention should also be made of the work
reported by Ford and Rodgers [110]. They used
negatively charged SnO2 particles (ca. 4-nm diam-
eter) which serve as supports for BLMs composed
of didodecyldimethylammonium ion in aqueous
sols. An amphiphilic Ru(II) polypyridine complex,
when incorporated into the bilayer, photosensitizes
electron injection into the conduction band of the
SnO2, but only the population of complex dissolved
in inner monolayer, in contact with the support, is
active. The high quantum eciency of charge trans-
fer together with the relatively slow rate of recombi-
nation between the conduction band electron and
the Ru(III) polypyridine complex makes this med-
ium promising for studies of electron-transfer pro-
cesses in surfactant lipid bilayer assemblies.
Concerning the photoelectric e€ects in supported
 Supported planar lipid bilayers
BLMs, the ®ndings are reported in the following
papers [21, 102, 111]. The light-induced voltage and
current changes under continuous illumination have
been investigated in a s-BLM containing Zn±phtha-
locyanine (ZnPc) as a photosensitizer and glycerol-
dioleate (GDO) as a BLM-forming solution [111]
and found that the probe's potential is shifted to
more positive value on light irradiation as well as
to increase the cathodic current across the BLM.
The results indicate a direct electron transfer from
the platinum substrate to hydrogen ion in the elec-
trolyte solution. Furthermore, it has also been
demonstrated a dramatic increase of the photocur-
rent over the time course of BLM formation visua-
lizing a role of the bulk quenching processes which
are signi®cantly diminished in thin lipid membrane
than in pigmented s-BLMs. In another report, the
light-induced voltage and current generated by dop-
ing a s-BLM with buckminsterfullerene C60 were
investigated [102]. Using this s-BLM system, it is
possible to construct sensor probes and to investi-
gate redox reactions and light-induced electron
transfer across the lipid bilayer. The C60-containing
s-BLM, considered basically as a ``molecular
device'', is a light-sensitive diode capable of photo-
induced charge separation which undergoes redox
reactions across the substrate±hydrophobic lipid
bilayer±aqueous solution junctions. Using the cyclic
voltammetry technique, the results show that C60
embedded in the BLM acts as an excellent electron
carrier/mediator and should be useful for electro-
chemical biosensor and molecular electronics device
development. It is concluded that supported BLMs
with excellent stability, possessing the structural
and dynamic properties of conventional planar lipid
bilayers, are excellent models for investigating basic
membrane-mediated ligand±receptor contact inter-
actions. This is because BLMs and s-BLMs rep-
resent the simplest self-assembled structures that
separate two phases (liquid/liquid or liquid/solid).
Our experiments have shown that certain electron
mediators can indeed modify the electrical par-
ameters of the s-BLM. Thus, the insights gained in
our ®ndings may facilitate the future search in the
use of s-BLMs for practical applications. For
instance, in our recent experiments with a sb-BLM
containing fullerenes (C60), we have found that C60,
function both as a mediator and a
photosensitizer [102].
Also using fullerenes (C60) but for a di€erent pur-
pose, Sivak et al. [112] recently reported that a s-
BLM can be used for constructing biosensors. The
stability and reproducibility of electrical and mech-
anical parameters of that s-BLMs depend on the
type and quality of a support as well as on a lipid
composition and chemical modi®cation of the
bilayer. They also studied the in¯uence of conduct-
ing carbon C60 on the stability and mechanical
properties of the s-BLM. Interestingly, the presence
of C60 also stabilized the membrane structure and
3607
resulted in higher values of E_. They also applied
the method of measurement of conductivity ¯uctu-
ation to analyze properties of s-BLMs, and
observed three types of ¯uctuations: (1) large cur-
rent jumps observed as current steps at dc channel;
(2) small current ¯uctuations near zero line with a
spectrum of 1/f type (¯icker noise) and (3) short
current jumps, whose amplitudes are many folds
higher than a mean noise level. The signi®cance of
noise analysis for determination of mechanisms of
membrane conductance is also discussed in this
paper.
Most recently, s-BLM containing a series of elec-
tron carriers such as quinones, ¯avins, iron±sulfur
complexes are being investigated. These electron
carriers are involved in mitochondrial respiration
[56]. In our initial experiments some of these elec-
tron carriers were examined using s-BLMs contain-
ing either C60 or TCNQ or both [113]. One of our
objectives was to ®nd out whether some of these
compounds could be quantitatively assessed using a
s-BLM probe. The long-term goal is to develop an
s-BLM-based probe having a universal receptor
embedded in the lipid bilayer. We have found that
the ¯avin mononucleotide (FMN, ribo¯avin 5'-
phosphate) is of particular interest, insofar as the
shape of voltammograms are concerned. Some typi-
cal results are shown in Fig. 7. Although certain
e€ects were seen on the current of the C60-doped s-
BLM probe as a function of the FMN concen-
tration, a dramatic increase of the current was
observed when both C60 and TCNQ were present in
the lipid bilayer, whereas the presence of only one
alone did not produce the enhanced e€ect.
Nonetheless, both C60- and C60+TCNQ-doped s-
BLM probes, when tested as a function of the
Fig. 7. Cyclic voltammograms of an s-BLM modi®ed by
C60 and by C 60-TCNQ with FMN (8 mM) present in 1 M
NaCl. Scan rate = 40 mV/s [113].
3608 H. T. Tien and A. L. Ottova

ported BLMs. Examples of a number of practical

applications of s-BLMs and sb-BLMs for construct-
ing probes are given in this review.

ACKNOWLEDGEMENTS
We thank our colleagues for much of the work
described in this paper, whose names are given in
joint publications. The research described here was
supported in part by grants from the oce of
USAID and USARO.

Fig. 8. A log±log plot of the peak current (i) response of
the C60 (iC60 /is-BLM) and C60-TCNQ (iC60‡TCNQ /is-BLM) modi-
®ed s-BLM probe vs FMN concentration in 1.0 M
NaCl [113].
FMN concentration, did give straight line on log±
log plots, as shown in Fig. 8. One conclusion that
may be drawn from these preliminary ®ndings is
that a s-BLM-based probe could be designed for
``almost'' any species for which a receptor exists.
The receptor could be an antibody, a DNA frag-
ment, an enzyme, a binding protein,
etc. [7, 10, 87, 114±117]. Clearly, if the approach is
successful, it could open a new route to a class of
novel biosensors that could combine ¯exibility
coupled with ease of fabrication.
CONCLUDING REMARKS
This paper has reviewed the research done on
supported BLMs (planar lipid bilayers) since its
inception. In view of the fact that the lipid bilayer
is the principal structural component of all biomem-
branes, supported BLMs are logical points of
departure in biosensor design and molecular device
development. Supported BLM-based sensors and
devices have a number of practical advantages over
other thin ®lm composite layer-based systems.
These are simplicity of construction, ease of modi®-
cation, electrical methods of detection, fast response
time and the existence of vast literature on conven-
tional BLM systems. Hence, supported planar lipid
bilayers are eminently suited in molecular recog-
nition and signal transduction investigations. The
results thus far obtained on s-BLMs provide possi-
bilities to choose optimal properties of the support
for constructing lipid bilayer-based biosensors.
Analysis of membrane electrical parameters allow
the examination of the dynamic properties of sup-
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