© Copyright 1998
American Chemical Society
AUGUST 18, 1998
VOLUME 14, NUMBER 17
Letters
Ion-Selective Lipid Bilayers Tethered to Microcontact
Printed Self-Assembled Monolayers Containing Cholesterol
Derivatives
A. Toby A. Jenkins, Richard J. Bushby, Neville Boden, Stephen D. Evans,*
Peter F. Knowles, Quanying Liu, Robert E. Miles, and Simon D. Ogier
Centre for Self-Organising Molecular Systems, University of Leeds, Leeds, LS2 9JT, U.K.
Received May 15, 1998
Microcontact printing has been used to prepare patterned self-assembled monolayers (SAMs) of
cholesterylpolyethylenoxy thiol. These patterned SAMs were used as supports for the formation of integral
supported lipid bilayers. Biofunctionality was confirmed by addition of valinomycin ionophores and
gramicidin ion channels. Impedance spectroscopy of the resulting bilayer structures, incorporating
valinomycin and the gramicidin, showed the expected ion selectivity.
The attachment of a lipid bilayer to a solid substrate,
in such a way that it retains its biomimetic properties,
has generated an increasing interest over recent years.1-17
* To whom correspondence should be addressed. E-mail:
s.d.evans@leeds.ac.uk.
(1) Stelzle, M.; Sackmann, E. Biochim. Biophys. Acta 1989, 981, 35142.
(2) Brink, G.; Schmitt, L.; Tampé, R.; Sackmann, E. Biochim. Biophys.
Acta 1994, 1196, 227-230.
(3) Kühner, M.; Tampé, R.; Sackmann, E. Biophys. J. 1994, 67, 217226.
(4) Stelzle, M.; Weismüller, G.; Sackmann, E. J. Phys. Chem. 1993,
97, 2974-2981.
(5) Plant, A.; Gueguetchkeri, M.; Yap, W. Biophys. J. 1994, 67, 11261133.
(6) Plant, A.; Brigham-Burke, M.; Petrella, E.; O’Shannessy, D. Anal.
Biochem. 1995, 226, 342-348.
(7) Terrettaz, S.; Stora, T.; Duschl, C.; Vogel, H. Langmuir 1993, 9,
1361-1369.
(8) Duschl, C.; Liley, M.; Corradin, G.; Vogel, H. Biophys. J. 1994,
67, 1229-1237.
(9) Lang, H.; Duschl, C.; Vogel, H. Langmuir 1994, 10, 197-210.
(10) Lindholm-Sethson, B. Langmuir 1996, 12, 3305-3314.
(11) Kalb, E.; Frey, S.; Tamm, L. K. Biochim. Biophys. Acta 1992,
1103, 307-316.
(12) Nakashima, N.; Yamaguchi, Y.; Eda, H.; Kunitake, M.; Manabe,
O. J. Phys. Chem. 1997, 101, 215-220.
(13) Nelson, A. Langmuir 1996, 12, 2058-2067.
(14) Sackmann, E. Science 1996, 271, 43-48.
(15) Williams, L. M.; Evans, S. D.; Flynn, T. M.; Marsh, A.; Knowles,
P. F.; Bushby, R. J.; Boden, N. Langmuir 1997, 13, 751-757.
(16) Williams, L. M.; Evans, S. D.; Flynn, T. M.; Marsh, A.; Knowles,
P. F.; Bushby, R. J.; Boden, N. Supramol. Sci. 1997, 4, 513-517.
(17) Cheng, Y.; Boden, N.; Bushby, R. J.; Clarkson, S.; Evans, S. D.;
Knowles, P. F.; Marsh A. Langmuir 1998, 14, 839-844.
In principle, it should be possible to construct biosensors
that are highly analyte specific, utilizing the specificity
found naturally in cellular biology,18 in effect utilizing
Nature’s evolutionary designed biosensing systems. There
are several problems in achieving this. First, one needs
to create a lipid bilayer, on a solid surface, that is
sufficiently flexible and defect free such that ion channel
activity can actually be measured. Second, the lipid
membrane must be in a physical state in which incorporated biological molecules function as if in a natural
system. Third, one needs to entrap water on the inner
surface of the membrane to facilitate the incorporation of
transmembrane proteins. Experiments on randomly selfassembled mixtures of cholesterylpolyethylenoxy thiol
(CPEO3) and mercaptoethanol on gold have shown that
although supported bilayers were formed, they were not
sufficiently blocking to be characterized by electrochemistry. The move to using microcontact printed SAMs as
described here has two perceived benefits. First, the area
over which an integral bilayer has to span is reduced
significantly. Second, as suggested by Groves et al.,19
(18) Darszon, A.; Vandenberg, C. A.; Scönfeld, M.; Ellisman, M. H.;
Spitzer, N. C.; Montal, M. Proc. Natl. Acad. Sci. U.S.A. 1980, 77, 239243.
(19) Groves, J. T.; Wulfing, C.; Boxer, S. G. Biophys. J. 1996, 71,
2716-2723.
(20) Boden, N.; Bushby, R. J.; Clarkson, S.; Evans, S. D.; Knowles,
P. F.; Marsh A. Tetrahedron 1997, 53, 10939-10952.
S0743-7463(98)00581-2 CCC: $15.00 © 1998 American Chemical Society
Published on Web 07/25/1998
4676 Langmuir, Vol. 14, No. 17, 1998
transmembrane proteins will self-locate in supported
bilayer structures on micropatterned substrates.
In this communication, we demonstrate that it is
possible to form phospholipid bilayers supported on a
patterned, lipophilic SAM.8,15-17 As in our previous work,
the tethered bilayers have been characterized by surface
plasmon resonance and atomic force microscopy. We now
describe how bilayer formation can be monitored using ac
impedance spectroscopy, which was also used to demonstrate the ion transport selectivity of the ionophore
valinomycin and the ion channel forming peptide, gramicidin.
A lipophilic tether molecule consisting of cholesterol
derivatized with a thiol-terminated trisethylenoxy chain
(Figure 1), was synthesized to anchor the lipid to a gold
substrate.20 A patterned CPEO3 SAM, with 15 µm × 15
µm square holes, was produced on the gold21 using
microcontact printing (µCP).22 The “bare” wells thus
created were subsequently filled with a mercaptoethanol
SAM (Figure 1). All SAM solutions were 1 mM in HPLC
grade dichloromethane.
Supported bilayers of egg-phosphatidyl (egg-pc) choline
were formed by the incubation of large unilamellar
vesicles, produced by extrusion, with the SAM patterned
substrate.11 Immediately before addition of the vesicles,
the micropatterned SAM was measured by impedance
spectroscopy. On addition of vesicles, during the incubation, impedance measurements were made every minute
for the first 10 measurements (when the impedance
changes most quickly) and then at longer intervals for a
period of up to 18 h. All impedance measurements were
made at room temperature, 19 °C, well above the egg-pc
phase transition temperature of around 1 °C. Each
(21) The gold substrate was formed by evaporation of 5 nm of
chromium followed by 150 nm of gold onto high quality glass microscope
slides at a pressure of 2 × 10-6 mb. The gold substrate (working electrode)
is connected by a 1 mm wide gold track to the instruments leads.
(22) Wilbur, J. L.; Kumar, A.; Biebuyck, H. A.; Kim, E.; Whitesides,
G. M. Nanotechnology 1996, 7, 452-457.
(23) The impedance of the bare gold, in the absence of a redox couple,
is dominated by the double layer capacitance of the substrate, with an
experimentally determined value of 40 µF cm-2. This result is higher
than that typically quoted for smooth bare gold and is probably due to
the roughness of the surface.24 The adsorption of a single component
CPEO3 from solution (nonstamped) onto a bare gold substrate presents
a blocking SAM. The capacitance of such a monolayer derives from the
dielectric properties of the SAM and has a value of 0.85 µF cm-2 in 0.1
M KCl. Measurements on single-component stamped (nonpatterned)
CPEO3 SAMs gives a capacitance of 2.5 µF cm-2, significantly higher
than the nonstamped CPEO3 SAMs. This higher capacitance can be
readily ascribed to the presence of defects in the stamped films. Assuming
the defect regions exhibit the same capacitance as bare gold would
equate to a 4% defect concentration (i.e., R ) 0.96). The adsorption of
a pure mercaptoethanol SAM on gold gave capacitance values of around
9.2 µF cm-2 in 0.1 M KCl. Theoretically, a perfect blocking SAM of
mercaptoethanol would have a capacitance of 4 µF cm-2 (assuming a
thickness of 5 Å and r to be 2.25). Hence, an approximate calculation
would suggest that 70% of the available surface sites must be blocked
(θ ) 0.7). The micropatterned SAM consists of two components: the
CPEO3 system and mercaptoethanol. The capacitance of these SAMs
derives from three separate contributions: the areas of CPEO3 SAM,
areas of mercaptoethanol SAM, and areas of gold at the bottom of defects.
Typical capacitance values of such patterned SAMs are close to 6.0 µF
cm-2 in 0.1 M KCl. These experimentally determined values are a factor
of 2.4 greater than those predicted theoretically (based on the values
obtained for the stamped single component surfaces). Note, this
calculation assumes that all defects within the CPEO3 layer are
“backfilled” with mercaptoethanol. However if only 90% of these defects
were back filled, then we would expect to obtain a value close to that
observed. Unilamellar egg-pc lipid vesicles made by extrusion were
added to single component unstamped (solution adsorbed) CPEO3
SAMs, and the impedance of the system was measured over a period
of 3 h. The final capacitance of the SAM and lipid monolayer was 0.58
µF cm-2. This is close to the value predicted theoretically for an egg-pc
monolayer-CPEO3 of around 0.44 µF cm-2, suggesting that the SAMlipid system is blocking. Measurements of egg-pc vesicles unrolling on
the single component stamped CPEO3 SAMs give a final capacitance
of 0.95 µF cm-2 suggesting a higher defect concentration in this system.
Letters
impedance measurement was made over the frequency
range 50 kHz to 300 mHz, with a 20 mV peak-peak ac
signal at the open circuit potential of the cell. The lipid
was dispersed in 0.1 M BaCl2, which was used as the
electrolyte to follow the formation of the bilayer. For ion
selectivity measurements, the electrolyte was exchanged
with four times the cell volume of the new electrolyte. The
molar concentration of the electrolyte was maintained at
0.1 M in each case.
To understand the impedance spectra of the lipidpatterned SAM system, the impedance of the “individual”
components making up the system were independently
measured, where possible. These results are presented
in footnote 23. The capacitance values were determined
by fitting the high-frequency (50 kHz to 1 kHz) region of
the impedance spectra. A semicircle (modeling a series
RC circuit) was fitted to the data points obtained by
plotting real admittance, divided by frequency (Y′/ω),
against imaginary admittance, divided by frequency (Y′′/
ω) (Figure 2b), as described by Lingler et al.25 Using the
Solartron Z plot software to fit data gave estimated
capacitance value errors of 4% or less.
The adsorption kinetics of egg-pc lipid on the patterned
SAM, as followed by the change in capacitance, was similar
to those reported by Lingler et al.25 The initial rapid
decrease in capacitance is attributed to the adsorption
and spontaneous rupture of the vesicles on the surface.
The slower decay of the capacitance for times greater than
20 min is attributed to bilayer spreading and selforganization of the lipids on the surface. Such a two-step
adsorption process is in agreement with SPR results on
vesicle interactions with nonpatterned SAMs, carried out
by ourselves and other workers.15,25 The final capacitance
of the lipid-SAM system was between 0.9 and 1.0 µF
cm-2.
The lipid-coated CPEO3 functionalized regions (region
1, Figure 1b) may be represented as a capacitance, C1
(equation 2a), which can be considered as having contributions from an ideal film plus defects (C1 has a value of
0.95 µF cm-2).23 In the “well” regions (region 2, Figure
1b) the capacitance C2, given by eq 2b, can be considered
to have two main contributions, one from the bilayercovered portions of the surface, Cb, and the second
originating from the mercaptoethanol-coated substrate,
Cs. If φ represents the surface coverage of CPEO3 regions,
then (1 - φ) will represent the area of the “wells” and the
net capacitance is given by eq 1.
Ctotal ) φC1 + (1 - φ)C2
(1)
In order minimize the number of parameters introduced
in our model, we assume that the fractional lipid bilayer
coverage in the mercaptoethanol regions and the fractional
lipid monolayer coverage in the CPEO3 regions are the
same, and we denote this as β. Thus the capacitance, C1,
has three contributions: the first is from the lipid
monolayer (Clip) in series with the CPEO3 SAM (CSAM),
the second the CPEO3 SAM without adsorbed lipid (CSAM),
and the third from defects in the CPEO3 SAM, the gold
double layer capacitance, (1 - R)Cau, where R is the
coverage of CPEO3 SAM. Equation 2b describes the
capacitance of the lipid bilayer adsorbed on the mercaptoethanol SAM, region 2. In this case the capacitance has
contributions from the bilayer (Cb) in series with a defect
containing mercaptoethanol SAM (Cmer), where θ repre(24) Piela, B.; Wrona, P. K. J. Electroanal. Chem. 1995, 388, 69-79.
(25) Lingler, S.; Rubinstein, I.; Knoll, W.; Offenhäusser, A. Langmuir
1997, 13, 7085-7091.
Letters
Langmuir, Vol. 14, No. 17, 1998 4677
Figure 1. (a) Construction of patterned SAM, formation of egg-pc lipid bilayer on a cholesterylpolyethylenoxy thiol stamped SAM
on gold. (b) The graphical and electrical models of the CPEO3-lipid system.
sents the surface coverage of the mercaptoethanol and
the capacitance contribution from the defects is given by
(1 - θ)Cau.
C1 ) β(Cchol-1 + Clip-1)-1 + (1 - β)Cchol
where:
Cchol ) RCsam + (1 - R)CAu
C2 ) β(Cb-1 + Cs-1)-1 + (1 - β)Cs
where:
(2a)
(2b)
Cs ) θCmer + (1 - θ)CAu
The total capacitance Ctotal can be represented by the
equivalent circuit shown in Figure 1b. This decreases
linearly with increasing lipid coverage β, eq 3.
Ctotal ) φC1 + (1 - φ)(β(Cb-1 + Cs-1)-1 + (1 - β)Cs)
(3)
Using “ideal” values we estimate Ctotal to be 0.47 µF cm-2.
Experimentally, for lipids adsorbed on microcontact
printed SAMs, we find values between 0.9 and 1.0 µF
cm-2, which would correspond to a β (lipid coverage)
variation between 0.84 and 0.88.
Using the Boukamp impedance analysis software,26 the
expected impedance response for the theoretical circuit
shown in Figure 1 has been simulated. It was found that
for sufficiently high values of Rlip, Rd, and Rval (>200 kΩ
cm-2) only one time constant on the Nyquist (Z′, -jZ′′)
plot was observed, this is in agreement with our experimental data, Figure 2b.
The resistance of the system was determined by fitting
the impedance spectrum, obtained between 50 kHz and
300 mHz, to a parallel RC circuit. The fitting procedure
extrapolated the data plotted in the complex plane (Z′ vs
-jZ′′) to the real axis (Z′), Figure 2a. This provides an
estimate of the resistance of the system at very low
frequencies (<100 mHz), that is, such that the capacitive
(26) Boukamp, B. A. Faculty of Chemical Technology, University
Twente, PO Box 217, 7500 AE Enschede, Netherlands.
4678 Langmuir, Vol. 14, No. 17, 1998
Letters
Rtotal ) Relec +
where
R2 )
R1 )
Figure 2. Experimentally determined impedance spectra for
lipid adsorbed on patterned SAM: (a) All data points (50 kHz300 mHz) fitted to a resistor capacitor parallel RC circuit; (b)
High-frequency (50 kHz-1000 Hz) impedance points fitted to
series RC circuit. Dashed line shows fit.
(
(
)
(1 - β - γ)
γ
+
Rd
Rval
((
)
(4)
+ Rmer
(5)
(1 - φ)
φ
+
R1
R2
-1
-1
) ( ))
β
1-β
+
Rsam
(Rlip + Rsam)
-1
(6)
The measured resistance of the system, Rtotal, will contain
contributions from each of the regions 1 and 2; R1 from
the CPEO3-lipid region and R2 from the supported lipid
bilayer region. The resistance R1 is in turn made up of
the resistance of the CPEO3 monolayer (Rsam) and the
adsorbed lipid monolayer (Rlip). The resistance R2 includes
contributions due to the presence of valinomycin or
gramicidin (Rval) and the bilayer (Rd) (Figure 1b). The
finite nature of the measured resistance is due to the
presence of defects within each of the regions.
It is evident from eq 4 that in order for ionophore activity
to be monitored (manifested as a change in resistance,
Rval) the resistance of the bilayer must be sufficiently high
that Rtotal is not dominated by the presence of defects.
Figure 2a shows the experimentally obtained impedance
spectra (and fit) used to determine Rtotal of an adsorbed
lipid layer on a micropatterned SAM. Typically we find
Rtotal to be approximately 0.1 MΩ cm-2. The estimated
errors due to fitting for resistance were no greater than
9%.
Valinomycin selectively transports cations according
to the sequence K+ > Cs+ > Li+ > Na+ > Ba2+; thus we
would expect the conductance of a bilayer containing
functional valinomycin to decrease in the same order.27
Figure 3 shows the experimentally determined conductances for bilayers in the presence (circles) and absence
(triangles) of valinomycin under 0.1 M KCl and 0.1 M
BaCl2 solutions. The control experiment shows no selectivity for potassium or barium while in the presence of
valinomycin we find that the conductance varies by a factor
of 2.
Experiments with the ion channel forming peptide
gramicidin also show the expected selectivity, Cs+ > K+
> Na+ . Ba2+ (all with chloride anions at 0.1 M).28,29
Membranes were found to be stable for a minimum of 24
h.
Figures 2 and 3 both show the successful formation of
a lipid bilayer on microcontact printed SAMs and importantly, for future biosensor applications, the selective
functionality of valinomycin within the bilayer. The latter
implies that conductance is dominated by transport
through the ionophore rather than defects induced in the
bilayer. The combination of the CPEO3 molecules and
microcontact printing has allowed us to develop a unique
new method for supporting lipid bilayers (on solid supports), while retaining the biological functionality of
incorporated ionophores.
Acknowledgment. This work was supported by
funding from the BBSRC and the Defence and Evaluation
Research Agency (DERA).
LA980581U
Figure 3. Ion selectivity for Ba2+ and K+ of a lipid bilayer (1)
without valinomycin and (2) with 0.5 mol % valinomycin.
contribution to the impedance can be effectively ignored.
As a result, we can write
(27) Steinem, C.; Janshoff, A.; Ulrich, W. P.; Sieber, M.; Galla, H. J.
Biochim. Biophys. Acta 1996, 1279, 169-180.
(28) Hill, B. Ionic Channels of Excitable Membranes, 2nd ed.; Sinauer
Associates, 1992; pp 305-312.
(29) Woolley, G. A.; Wallace, B. A. J. Membr. Biol. 1992, 129, 109136.