Thin Solid Films 354 (1999) 201±207

www.elsevier.com/locate/tsf

Mediated electron transfer across supported bilayer lipid

membrane (s-BLM)
Kinji Asaka a,1, Angelica Ottova b, H. Ti Tien a,*
a
Membrane Biophysics Laboratory (Giltner Hall), Physiology Department, Michigan State University, East Lansing, MI 48824, USA
b
Center for Interface Sciences, Microelectronics Department, Slovak Technical University, Bratislava, Slovakia

Abstract
The mediated electron transfer of the planar bilayer lipid membrane on solid support (s-BLM) was studied by means of cyclic voltammetry
(CV) in order to obtain basic data such as conductance and capacitance useful for biosensor development. Electron mediators tested were
TTF (tetrathiafulvalene), TCNQ (tetracyanoquinodimethane), 2,2 0 -dipyridyne, ferrocene, C60, and redox compounds used were ferricyanide,
ferrocyanide, H2O2, and ascorbic acid. From the membrane capacitance values obtained, it is shown that the self-assembled lipid membrane
has a bilayer structure. The combinations of C60 and other mediators are most effective in enhancing the s-BLM's sensitivity towards redox
species. One conclusion that may be drawn from this ®nding is that the s-BLM probe could be designed for `almost' any species for which a
receptor exists. This opens a new path for the use of metal supported bilayer lipid membranes (s-BLMs) in the area of biosensor development.
q 1999 Elsevier Science S.A. All rights reserved.
Keywords: electron transfer; supported bilayer lipid membrane; cyclic voltammetry

1. Introduction proteins and pigments embedded in lipid bilayer-bound

Much attention has been paid to the research of charge
transfer processes of self-assembled membranes made of
amphiphilic molecules, such as planar bilayer lipid
membranes (BLM) [1±5], LB ®lms [6], liposomes [7], and
monolayers on metallic surface [8]. The purported purpose
is two-fold: the understanding of membrane bioenergetics
and the development of practical biosensors. In this connec-
tion the use of conventional BLMs as models of biomem-
branes, and for analytical applications may be traced to the
early days of BLM research. The interested reader is
referred to a number of recent publications for further
details [9±11]. In this paper we are mainly concerned with
electronic processes in metal-supported BLMs containing
fullerene C60 in conjunction with a number of well-estab-
lished electron mediators.
First, why are we interested in charge transfer processes?
A cogent answer to this question is that ion transfer and
redox reactions play central roles in membrane bioener-
getics [12]. These processes are mediated by specialized
* Corresponding author. Tel.: 11-517-355-6475; fax: 11-517-355-
5125.
E-mail addresses: asaka@onri.go.jp (K. Asaka); ottova@pilot.msu.edu
(A. Ottova); tien@pilot.msu.edu (H.T. Tien)
1
address: Osaka National Research Institute, AIST, 1±8±31, Midori-
gaoka, Ikeda, Osaka 563, Japan.
subcellular organelles called chloroplasts and mitochondria.
One approach to gain some qualitative understanding of
these membrane-bound reactions in the early 1980s was
resorting to membrane reconstitution, using conventional
BLMs containing electron mediators such as TCNQ (tetra-
cyanoquino-dimethane), TTF (tetrathiafulvalene), bipyri-
dine, etc. [1±3]. TCNQ and TTF were often 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 [13]. Two possible mechanisms may be responsi-
ble for the observed transmembrane redox reactions: (1)
electron exchange between redox components bound in
the vicinity of the opposite BLM-solution interface, and
(2) transmembrane processes of the redox components.
These early experiments demonstrated that diffusion in the
aqueous phase is rate limiting and therefore electron `jump-
ing' or `tunneling' is a more probable occurrence in the lipid
bilayer. Thus, the transverse movement of electrons and
charged species across the modi®ed bilayer results in a
BLM which functions as a bipolar redox electrode [13].
To investigate this kind of reactions in BLMs, experimen-
tally, the technique of choice is cyclic voltammetry (CV)
[1±3]. The various parameters, i.e., background current,

0040-6090/99/$ - see front matter q 1999 Elsevier Science S.A. All rights reserved.
PII: S 0040-609 0(99)00520-9
202 K. Asaka et al. / Thin Solid Films 354 (1999) 201±207

redox potential peaks, stability of the mediator within the properties. With these desired properties in mind, we
BLM during the electrochemical measurements, and repro- wanted to explore further whether C60, when used in combi-
ducibility of the amperometric response, were considered at nation with other known redox mediators mentioned above,
length [1±3,14]. Thus, the past work provides a good start- could have synergistic effects on the electrical properties of
ing point on the use of TCNQ, TTF, etc. for the present s-BLMs. If true, this information might be useful for biosen-
development on supported BLM-based biosensors, which sor applications. As evidenced by our results described
is usually done by embedding the species of interest (e.g. below, we have found that, indeed, fullerene C60, beside
enzyme) together with electron mediators in the BLM-form- greatly enhancing the stability of supported BLMs, also
ing solution before starting the self-assembling process on dramatically increases the sensitivity of s-BLMs to a
the substrate [15]. The embedding of appropriate active number of tested redox species in solution.
molecules (modi®ers) into the matrix of the lipid bilayer
should be able to impart the functional characteristics to
the planar lipid bilayer. The conventional BLM method is 2. Experimental
an ideal system as a tool of basic research. However, the
2.1. Materials
membrane of the thickness of about 5 nm separating two
aqueous solutions is too fragile to be applied for practical
The BLM-forming solution was phosphatidylcholine
applications. Besides, the conventional BLM rarely lasts
(PC) in squalene (2% PC, Sigma Chemicals, St. Louis,
more than few hours; it is very sensitive to electrical and
MO). This lipid solution was used to contain fullerene C60
mechanical disturbances. In order to overcome these disad-
which was prepared as follows: 50 mg of Buckminsterful-
vantages, a novel, yet very simple method was found in the
lerene C60 ( . 99.5% pure, Strem Chemicals, MA) was ®rst
late 1980s for forming planar lipid bilayers on solid support
dissolved in 4 ml toluene, 2 ml of which was then added to
(s-BLMs) [15], and later on agar gel in the form of salt
the PC solution (20 ml). As a metallic substrate, Te¯on-
bridge support (sb-BLMs) [16,17]. Additionally, when
coated stainless-steel needles (ss, 0.35 mm diameter) was
compared with other self-assembled membrane systems,
used. Other reagents of analytical reagent grade were
the supported BLMs have several advantages: (i) the elec-
obtained commercially and were used without further puri-
tron mediators can be easily incorporated into the lipid
®cation. Double-distilled water was used for making all the
bilayer due to its ¯uidity, (ii) the electric parameters of
aqueous solution in this study.
both sides of the membrane are capable of being controlled
or measured, (iii) the composition of aqueous solutions of 2.2. Electrochemical measurements
both sides are easily de®ned, and (iv) there are no pin-holes
present in the ultrathin structure due to its self-sealing prop- Fig. 1 shows the schematic representation of the setup for
erty [18]. Both s-BLMs and sb-BLMs have a long-life time
and are very stable as well as not affected by normal elec-
trical or mechanical disturbances. Hence, these supported
BLMs have been utilized in various biosensor investigations
[9±11]. For examples TCNQ modi®ed s-BLMs show
promise for the development of glucose sensors [19],
whereas TTF modi®ed s-BLMs can be effectively used as
a hydrogen peroxide probes [20,21]. Here, mention should
also be made that as it has been reported recently both
conventional BLMs and supported BLMs containing
TCNQ, TTF, ferrocene or other modi®ers are excellent
systems for investigating transport and mediated electroca-
talytic redox reactions [11,22±28].
In this study we have made an extensive use of fullerene
C60, a geodesic form of carbon. Our interest in fullerenes as
a BLM modi®er is owing to their most unique properties,
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. Further, C60 is
a good electron mediator and behaves as an n-type semi-
conductor (Bandgap ˆ 1:6 eV) [29]. Recently we have
investigated electron transfer across a BLM containing
C60, which can act both as an electron mediator and a photo-
sensitizer [30]. The presence of C60 greatly strengthens the Fig. 1. (a) Schematic representation of the setup for the electrochemical
stability of s-BLMs and dramatically alters their electrical measurement. (b) Enlarged view of the s-BLM.
 K. Asaka et al. / Thin Solid Films 354 (1999) 201±207 203

Table 1
Combinations of the mediators and redox substances tested

Redox substances TTF TCNQ Dipyridine Ferocene C60 TTF 1 C60 TCNQ 1 C60 Dipyridine 1 C60 Ferrocene 1 C60 Control
a
H2O2 W ± W £ W W ± W W W
[Fe(CN)6] 32 W ± W £ W W ± W W W
Ascorbic acid ± W W W W ± W W W W
[Fe(CN)6] 42 ± W W W W ± W W W W
a
(W) tested; (±) not tested; (£) the s-BLM was not stable.

the electrochemical measurement, together with that of the acetate or a bicarbonate buffer solution was used for stabi-
s-BLM. The potentiostat used was J1-100A (Jinke Electro- lizing the pH of the test solutions.
nics Co. Ltd., PRC), that was controlled by a personal
computer (DHK system). The measurements were
conducted at a scan rate of 50 mV/s in the range of ^700
3. Results and discussion
mV by using two electrode con®guration. The reference
electrode used was homemade Ag±AgCl with 3 M KCl
Table 1 presents a list of various redox species in combi-
agar salt bridge. The membrane capacitance (Cm) is given
nation with electron mediators tested. The other lipid solu-
approximately by Eq. (1) [18]. In our study, both Cm and
tions used for making modi®ed BLMs were those saturated
resistance (Rm) were estimated from the central part of
with each mediator. Forming solutions containing a mixture
cyclic voltammograms. The capacitance was estimated
of C60 and other mediators were the lipid solutions to
from the current change (I) from If during forward scan to
contain C60 (which was made as mentioned before) satu-
Ir during reverse scan, DI ˆ Ir 2 Ib as given by the follow-
rated with each mediator. It has been reported that TTF
ing equation
responded to H2O2, but there was very little response to
Cm ˆ DI=2n ù 1o 1A=tm …1† ascorbic acid. In the case of TCNQ, the opposite has been
found to be true. These results were not entirely unexpected
where n is the scan rate, A is the membrane area, 1 o, 1 and tm since TTF is an electron donor, whereas TCNQ is an accep-
are the permitivity of vacuum, the relative permitivity and tor. Hence, TTF was tested only for its sensitivity towards
thickness of the hydrocarbon portion of the planar lipid H2O2, and K3Fe(CN)6, whereas TCNQ was tested only for
bilayer, respectively. The resistance was estimated from its sensitivity towards ascorbic acid and K4Fe(CN)6.
the slope of the cyclic voltammogram [14]. All measure- Fig. 2 shows the cyclic voltammograms of s-BLMs (lines
ments were carried out at the room temperature (22 ^ 1)8C. a,b) and those modi®ed by C60 (lines c,d) with or without 6
mM K3Fe(CN)6 present in 0.1 M KCl. The s-BLMs could
2.3. Method of s-BLM formation withstand a voltage in excess of ^700 mV, while it has been
reported that the conventional BLM was broken with the
Supported BLMs (planar lipid bilayers) were prepared as
application of a voltage of about 200 mV [18]. This is a very
described elsewhere by Ottova et al. [15]. Brie¯y, the good property for the various applications of the s-BLM.
formation method of the s-BLM was as follows: (i) the tip
The s-BLM was found to be insensitive to electric and
of a Te¯on-coated ss-probe (encased in wax in a plastic
enclosure) was cut with a clipper or wire cutter, (ii) a freshly
fractured metal surface was then immersed in the lipid solu-
tion for several minutes, and (iii) the metal wire on the
surface of which had been adsorbed the lipid layer, was
then soaked in the aqueous solution, and the BLM was
spontaneously formed as monitored electrically. When the
thick layer of lipid membrane is formed on the surface of the
electrode (probe), the capacitance and conductance decrease
as compared to the bare electrode. Then, both values
increase as the membrane thickness decreases, and ®nally
reach a stable value when an s-BLM is formed. Hence, the
monitoring of the formation process of the s-BLM was
easily carried out by means of CV [1±3,14].
After the BLM had formed in 0.1 M KCl solution an
aliquot of the concentrated solution of the redox reagent Fig. 2. Cyclic voltammogram of s-BLMs (lines a,b) and those of modi®ed
was added to the test solution. After the current had reached with C60 (lines c,d); lines a,c: in 0.1 M KCl, lines b,d: with 6 mM K3Fe(CN)6
a stable value, the measurement was carried out. Either an present in 0.1 M KCl.
204 K. Asaka et al. / Thin Solid Films 354 (1999) 201±207

Table 2 support material is different, it is hypothesized that the same

Capacitance and resistance of s-BLMs doped with various electron media- situation occurred in our system.
tors
It can be clearly seen from Fig. 2 that s-BLM without
Mediator Cm (10 25 F cm 22) Rm (10 4 V cm 2) mediators did not respond to K3Fe(CN)6 in aqueous solu-
tion, while C60 doped ones responded remarkably. The
Control 5 7
response time was not accurately measured, but it has esti-
TTF 16 2
TCNQ 16 5 mated to be within 1 min. We attribute the increased current
Ferrocene 8 5 observed to be a redox current through the s-BLM, which is
Dipyridyl 34 3 mediated by C60. Similar experiments were carried out with
C60 13 3 s-BLMs doped with different mediators in aqueous solutions
TTF 1 C60 19 4
containing different redox materials at various concentra-
TCNQ 1 C60 6 6
Ferrocene 1 C60 10 3 tions. Each s-BLM tested had a different membrane area,
Dipyridyl 1 C60 45 2 since it was dif®cult to cut the probe resulting in the same
surface roughness and area (see Eq. (1)). Hence the results
of the different s-BLMs were compared by their normalized
value. The sensitivity is de®ned by the ratio of the current at
mechanical noises. It easily lasted for more than 4 days in each concentration of redox substances with respect to that
our routine studies. at 0 mM concentration (control). The current values adopted
From the cyclic voltammogram, we can estimate the are shown in Fig. 3. Fig. 4a±d shows the dependence of
values of the capacitance and that of the resistance of the various modi®ed s-BLM's sensitivity as a function of
s-BLMs [14]. The values of s-BLMs in 0.1 M KCl without redox compounds at different concentrations. The character-
redox substances are summarized in Table 2. It can be seen istics are that the slope decreases at the point of 1 or 2 mM.
from the table that the mediators in the planar lipid bilayer The orders of the sensitivities toward various dopants are as
have opposite effects on the membrane resistance (Rm) and follows:H2O2:(C60 1 TTF) . Dipyridyl . (Ferrocene 1
capacitance (Cm), respectively. It causes Rm to decrease and C60) ù (Dipyridyl 1 C60) . C60 . TTF . Control
Cm to increase. It is apparent that the decrease in resistance K3Fe(CN)6: (Dipyridyl 1 C60) . C60 . (TTF 1 C60) .
is due to the increase in the concentration of the electron Dipyridyl . (Ferrocene 1 C60) . TTF . Control Ascorbic
mediators, i.e. due to the increase in the conductivity of the acid: (TCNQ 1 C60) . Dipyridyl 1 C60) . Dipyridyl .
s-BLM. The membrane capacitance depends on various Ferrocene ù (Ferrocene 1 C60) ù Control . C60 . TCNQ
factors, such as the surface charge density of the BLM, K4Fe(CN)6: (TCNQ 1 C60) . C60 . Ferrocene . TCNQ .
the nature of the hydrocarbon chains and the embedded Control . (Dipyridyl 1 C60) . (Ferrocene 1 C60) . Dipyr-
modi®ers. It is inferred that the increase in the capacitance idyl. The s-BLMs doped by some mediators in high concen-
is due to the increase in the dielectric constant (see Eq. (1)) tration of redox materials were not stable (see Table 2).
[18]. The values of the membrane capacitance are repre- These data therefore are not shown in Fig. 4. For most
sented by the value per the geometric area (9:6 £ 1024 cases, the s-BLM became stable in the presence of C60.
cm 2). They are much larger than that of the conventional The combination of C60 together with other mediators was
BLM because of the roughness of the electrode surface. It is found to be the most effective in stabilizing the s-BLM in all
known that the capacitance value of the conventional BLM cases.
made from squalene solution was the same as that of the
solvent free one, which has been reported to be 0.72 mF
cm 22 [18]. The electric double layer capacitance of the
stainless steel in HClO4 solution was reported [31] to be
70 mF cm 22, which is assumed to be the value as in KCl
solution. From the cyclic voltammograms, the capacitance
values of the bare ss-probe and that of the s-BLM were 3.8
mF cm 22 and 50 mFcm 22, respectively. In consideration of
the surface roughness, the capacitance value of the s-BLM is
calculated to be 0.92 mF cm 22. Hence, albeit there is some
uncertainty of the structure of the lipid layer assembled on
the substrate, it is inferred that the membrane has a bilayer
structure, since it has a thickness on the order of bilayer, and
thermodynamically, the bilayer structure is considered to be
the most stable one [18]. Recently, Chiang et al. [32]
measured the thickness of the BLM on the Ag support by
Fig. 3. Cathodic current Ic and anodic current Ia for calculating the sensi-
means of ellipsometry. They con®rmed the spontaneous tivity. Ic is the value at the potential of 600 mV and Ia is the anodic peak
thinning of lipid ®lms to a bilayer thickness. Although the current.
 K. Asaka et al. / Thin Solid Films 354 (1999) 201±207 205

The most important ®nding in this work is that C60 or the means that the electron transfer reaction rate through the
combination of C60 and other mediators were effective to membrane depends on the reaction rate at the interface
every redox species tested. One conclusion that may be between the BLM and the solutions. The reaction rate at
drawn from this ®nding is that the s-BLM probe could be the interface is expected to depend on the species of the
designed for `almost' any species for which a receptor exists mediators.
[33]. The receptor could be an antibody, a DNA fragment, The combination of C60 and other mediators is expected
an enzyme, a binding protein, etc. Although s-BLM- to give the different redox species as the following equations
containing C60 appears to be an excellent foundation for a show:
new class of novel biosensors, there are still many open
questions to be answered regarding its light sensitivity and M 1 C60 ! M1 ±C2
60 …2†
chemical reactivity. For instance, the characteristics of C60
as an electron mediator in s-BLMs need to be considered. TCNQ 1 C60 ! TCNQ2 ±C1 …3†
60
There are three possibilities of the electron transfer mechan-
isms through the s-BLM, namely. hopping, tunneling, and where M ˆ TTF, ferrocene or dipyridyl, M 1±C2 60 and
mediator diffusion [1±5,9,30]. In any case, the observed TCNQ 2±C60 1
are sorts of complexes, and C2 1
60 and C60 are,
redox current depends on the concentration of the mediator, respectively, a somewhat reduced and an oxidized form of
the transfer rate of the electron or the carrier in the lipid C60. Hence, the oxidation of ascorbic acid and [Fe(CN)6] 42
bilayer, and the reaction rates of the redox species at both are enhanced by incorporating C60 into the TCNQ doped s-
interfaces. From the experimental results, the order of the BLM, and the reduction of H2O2 and [Fe(CN)6] 32 are
response was found to depend on the redox species, which enhanced by incorporating C60 into doped s-BLM. It was

Fig. 4. Dependence of the sensitivity on the concentration of redox substances. (a) H2O2, (b) K3Fe(CN)6, (c) ascorbic acid, (d) K4Fe(CN)6.
206 K. Asaka et al. / Thin Solid Films 354 (1999) 201±207
reported that ferrocinium ion was not hydrohopbic [34].
Hence, in Eq. (2), ferrocinium ion moves to the aqueous
phase, and there are C60 molecules in the BLM, which are
the electron donors. It may be the reason of the remarkable
decrease in the response of ferrocene doped s-BLM in addi-
tion to C60 (see Fig. 4d).
Another reason for the enhanced electron transfer
mediated by C60 may be explained in terms of electron
transfer mechanisms through the lipid bilayer. As already
mentioned, there are three possibilities about the electron
transfer mechanism; hopping, tunneling, mediator diffusion.
In tunneling, electrons must tunnel through the potential
barrier separating the mediator molecules embedded in the
BLM, the probability of which is a function of both the
barrier height and width. The height of the barrier is in
turn determined by the electronic state of the mediator
molecule. Concerning the hopping mechanism, the aggre-
gates of the mediator molecules have a chain structure in the
BLM. That is C60 molecules are lined up spanning the BLM
and electrons may hop from one C60 molecule to the next
[30]. The mediator diffusion mechanism where the electrons
are transported with diffusion of charging mediator, is also a
plausible one. Presently, we do not have any speci®c infor-
mation regarding above proposed mechanisms. Additional
experiments such as temperature dependence of conduc-
tance studies are needed for the elucidation of the details
of the electron transfer mechanism [1±3,30].
The cyclic voltammogram of the mediator modi®ed BLM
in redox species has shown redox peaks which is similar to
metal electrodes. Cheng et al. [5] studied the transmem-
brane electron transfer reaction of the ubiquinone doped
BLM by CV. The mediator transfer instead of the electron
tunneling explained the results. The cyclic voltammograms
contain distinct redox peaks, which are not symmetrical.
Further, the C60 in the lipid bilayer has the opposite effects
on membrane resistance (Rm) and capacitance (Cm), respec-
tively; 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 effects. It is apparent that both C60
and iodine, when embedded in BLMs, facilitate electrical
conduction, thereby lowering the Rm. Since the dielectric
constant (1 ) of a typical, unmodi®ed BLM is about 2.1,
the presence of C60 and iodine should exert a great in¯uence
on 1 . Since Cm depends on a number of factors such as the
surface charge of the BLM, the nature of hydrocarbon
chains, and embedded modi®ers, an increase in 1 , and
consequently in Cm, is therefore expected. The modi®ed
probe increased the detection limit for I 2 by 100 times
with a linear response in the range from 10 28 to 10 22 M.
Thus, C60 facilitates the discharge of I32 at the metal surface,
which demonstrates clearly that the embedded C60 is indeed
an excellent electron mediator [30].
In this paper, we have studied the electron transfer reac-
tion of the s-BLM by means of CV in order to obtain basic
data useful for applying the s-BLM to bioenergetics and to
sensor research. Five different kinds of mediators and four
redox materials were tested. The electron mediators tested
were TTF, TCNQ, 2,2-dipyridyne, ferrocene and C60. The
redox materials tested were ferricyanide, ferrocyanide,
H2O2 and ascorbic acid. The mediators tested were all
well-known molecular electronics devices [13] or mediators
for biosensors. We especially focused on C60, which has
unique properties [16,17], namely, hydrophobicity, semi-
conductivity (Eg ˆ 1:6 eV) [29], and being very easily
reduced. Hence, it is one of the ideal electron mediators.
Electronic processes of the C60-doped BLM such as the
photo-induced current [35] and recognition of I2 have
been reported [30]. The electrochemical characterization
of the s-BLM is carried out by means of the CV. The evalua-
tion of the electron transfer through the s-BLM doped by
various mediators are conducted from the results of the CV,
compared with each other, and the transfer mechanism has
been discussed.
4. Conclusion
Much attention has been paid to the study of self-assem-
bly ultrathin layers of amphiphilic molecules for the purpose
of bioenergetics studies, and sensor applications research.
This paper describes the electron transfer study of the
bilayer lipid membrane on the solid support (s-BLM) by
means of cyclic voltammetry (CV). Five mediators and
four redox substances were tested. Especially, we have
focused on C60. From the membrane capacitance values, it
is inferred that the lipid membrane has a bilayer structure.
We have found that a combination of C60 and other media-
tors (TTF,TCNQ) is the most effective way in enhancing the
sensitivity of s-BLMs to the tested redox species. One
conclusion that may be drawn from this ®nding is that the
s-BLM probe could be designed for `almost' any species for
which a receptor exists. The receptor could be an antibody, a
DNA fragment, an enzyme, a binding protein, etc. Thus,
with the availability of supported BLMs, a host of
compounds may now be embedded in the ultrathin lipid
bilayer for detecting their counterparts present in the envir-
onment. Owing to their ease of formation, long-term stabi-
lity, and their naturalness for modi®cation, supported BLMs
offer an approach especially advantageous in the research
and development of lipid bilayer-based sensors and devices
[33].
Acknowledgements
K.A. acknowledges ®nancial support from New Energy
and Industrial Technology Development Organization
(NEDO) of Japan.
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