Advances in Planar Lipid Bilayers and Liposomes
Visit the link below to download the full version of this book:
https://medipdf.com/product/advances-in-planar-lipid-bilayers-and-liposomes-3/
Click Download Now
PREFACE
“Model Membrane Systems” is a central theme of Volume 20 of Advances in
Planar Lipid Bilayers and Liposomes (APLBL) which includes eight chapters.
An assortment of subjects is covered under this theme such as chemical and
electrostatic interactions of biomolecules (DNA, antimicrobial peptides)
with model biomembranes, structural evolution and phase transitions in
self-assembling systems, and biological significance of self-assembling sys-
tems. Model systems comprise lipid monolayers, Langmuir–Blodgett films,
supported lipid bilayers, vesicles, as well as nonlamellar nanostructures.
Recent advances in these fields are nicely presented by an amalgamation
of theoretical and experimental approaches. The overall content of this vol-
ume is thus potentially useful for wide scientific community working on
model lipid systems and their biotechnological implications.
We would like to thank all authors who contributed their chapters to the
Volume 20—Jacek Lipkowski, Natalia Wilke, Sarah Rachel Dennison,
Daniela Uhrı́ková, Vernita D. Gordon, Tanja Pott, Theyencheri
Narayanan, Marko Marhl, and their coauthors. We would like to thank
all members of the Editorial Board. We also thank our Technical and
Publishing Team of APLBL Volume 20, especially Shellie Bryant, Kate
Newell, and Preeta Kumaraguruparan.
ALEŠ IGLIČ AND CHANDRASHEKHAR V. KULKARNI
xi
CHAPTER ONE
Biomimetic Membrane Supported
at a Metal Electrode Surface:
A Molecular View
Jacek Lipkowski1
University of Guelph, Guelph, Ontario, Canada
1
Corresponding author: e-mail address:
[email protected]Contents
1. Introduction 2
2. sBLM Preparation Methods 3
2.1 Vesicle fusion 3
2.2 Langmuir–Blodgett and Langmuir–Schaefer deposition 8
3. Potential Drop Across the Membrane and an Estimate of the Electric Field Acting on
the Membrane 10
4. Effect of the Potential Applied to the Gold Electrode on the Membrane Stability:
AFM, NR, and Surface-Enhanced Infrared Absorption Spectroscopy Studies 14
5. Imaging Aggregation of Antibiotic Peptides in a Lipid Membrane 27
6. Potential-Controlled Changes in the Orientation and Conformation of Peptides and
Peripheral Proteins: IR Studies of Gramicidin and Cholera Toxin B 34
7. Summary and Conclusions 41
Acknowledgment 43
References 43
Abstract
This chapter reports on recent advances in the application of spectroscopic and surface
imaging techniques to provide molecular level information about the structure of gold-
supported phospholipid bilayers. It describes methods used to deposit biomimetic
membrane at the gold electrode surface. It provides information about the structure
of the membrane deposited at the gold electrode surface and its changes as a function
of the applied potential obtained with the help of techniques such as scanning electron
microscopy or atomic force microscopy, neutron reflectivity, and infrared reflection
absorption spectroscopy. These experimental approaches provided unique molecular
level information about the interactions of the membrane components with the metal,
orientation, and conformation of molecules within the membrane, water content in the
supported bilayer, and the structure of water molecules within the supported bilayer.
The interactions of the bilayer with the metal restrict mobility of the membrane. From
biomimetic point of view, this is an unwelcomed effect. However, the ability to
Advances in Planar Lipid Bilayers and Liposomes, Volume 20 # 2014 Elsevier Inc. 1
ISSN 1554-4516 All rights reserved.
http://dx.doi.org/10.1016/B978-0-12-418698-9.00001-0
2 Jacek Lipkowski
immobilize phospholipid matrix on a conductive support provides unique opportunity
to employ scanning tunneling microscopy to acquire molecular resolution images of
channels formed by antibiotic peptides and in this way to provide direct evidence
and molecular information of their action and their biocidal activity. The metal-
supported model membranes find applications as biosensors. Proteins incorporated
into such membranes constitute the sensing element and act as transducers of chem-
ical to electrical information. This chapter includes a review of IRRAS studies of the
potential induced changes in the orientation and conformation of membrane and
peripheral proteins incorporated into the gold-supported bilayers.
1. INTRODUCTION
Model lipid membranes supported at a metal electrode surface allow
transduction of chemical changes taking place in the membrane to electrical
signal such as current or changes of the membrane capacitance and resis-
tance. The transduction of chemical to electrical information allows devel-
opment of biosensors that find applications for fast drug screening and
selective detection of ions and molecules in general [1,2]. These systems
constitute also ideal platforms for a broad range of biomedical research such
as studies of implant biocompatibility, cell adhesion and fusion, drug screen-
ing, and amyloid plaque formation [3,4].
The supported bilayer lipid membrane (sBLM) is a planar bilayer with
one leaflet physically adsorbed to a solid surface and the other leaflet freely
exposed to solution. The planar geometry and long-term mechanical stabil-
ity of this design allow one to investigate the relationship between the struc-
ture and properties of the bilayer using a wide range of surface sensitive
techniques, such as IR spectroscopy [5–20], scanning tunneling microscopy
(STM) [20–22], atomic force microscopy (AFM) [23,24], Raman spectros-
copy [25] and neutron reflectivity (NR) [26–28].
There are several recent reviews that report on properties of sBLM at
metal surfaces [29–34]. Therefore, the scope of this chapter is to describe
recent advances in the application of spectroscopic and surface imaging tech-
niques to provide molecular level information about the structure of gold-
supported bilayers and on how the structure depends on the potential
applied to the electrode surface. We also discuss how the structure and prop-
erties of sBLM depend on the interaction between the lipid molecules and
the substrate and describe distribution of water molecules within the
supported bilayer. The interactions of the bilayer with the metal restrict
Biomimetic Membrane Supported at a Metal Electrode Surface 3
mobility of the membrane. From biomimetics point of view, this is an
unwelcomed effect. However, the ability to immobilize phospholipid
matrix on a conductive support provides unique opportunity to employ
STM to acquire molecular resolution images of channels formed by antibi-
otic peptides and in this way to provide direct explanation of their biocidal
activity. At metal surface linearly polarized IR photons are interacting with
metal surfaces, the incident and reflected beams enter into destructive inter-
ference when the electric field of the photon is oriented parallel to the sur-
face and into constructive interference when the electric field is located in
the plane of incidence that is normal to the surface. Therefore, by taking a
difference between the two signals one is able to determine the absorption
spectrum of molecules in the supported bilayer. We discuss how to use such
polarization modulation to determine orientation and conformation of mol-
ecules in the supported membrane and how these properties are affected by
the potential drop across the membrane.
2. sBLM PREPARATION METHODS
The most common procedures used to form sBLMs at solid surfaces
are the vesicle fusion (VF) and Langmuir–Blodgett (LB) and Langmuir–
Schaefer (LS) transfer methods, which are discussed below.
2.1. Vesicle fusion
Vesicles are closed lipid bilayers that encapsulate an aqueous solution. The
procedure for VF consists of the adhesion and fusion of small unilamellar
vesicles (50 nm in diameter) at a solid substrate from aqueous vesicle dis-
persion. At hydrophilic surfaces such as glass, quartz, or mica, VF involves
adsorption, deformation, and rupture followed by sliding of a single bilayer
or rolling of two juxtaposed bilayers in a tank tread-type motion on a thin
lubricating film of the solvent. A theory depicting the adhesion, fusion, and
rupture of vesicles at solid surfaces was developed by Lipowsky and Sei-
fert [35]. The validity of this theory was confirmed by Reviakine and
Brisson [36] who with the help of AFM showed images of adsorbed and rap-
tured vesicles at a solid surface. Unilamellar vesicles also fuse at an atomically
smooth surface of gold to form a bilayer [20,21]. STM studies of pure
1,2-dimyristoyl-sn-glycero-3-phosphatidylcholine (DMPC) and mixed
DMPC–cholesterol vesicles fusion at a Au(111) surface [20] demonstrated
that the mechanism of the bilayer formation at the gold surface is distinctly
different from that on hydrophilic surfaces of glass or quartz. The molecules
4 Jacek Lipkowski
released by rupture of a vesicle initially self-assemble at the metal surface into
a well-ordered monolayer. The self-assembly is controlled by the interaction
between the acyl chain and the metal surface. When more molecules accu-
mulate at the surface, the monolayer is transformed into a hemimicellar state.
In solutions with high vesicle concentrations, the hemimicellar state is trans-
formed further into a bilayer. This point is illustrated in Fig. 1.1 which shows
STM images of the Au(111) surface; image (A) 3 min and image (B) 30 min
after addition of vesicles into the solution of an electrochemical STM (EC-
STM) cell. Figure 1.1A shows rows formed by acyl chains of DMPC mol-
ecules lying flat on the surface. However, the images of flat-lying molecules
could be observed only in dilute vesicle solutions and during a short period
of time after the injection of vesicles to the cell. Figure 1.1B shows that after
about 30 min, the film transforms into totally different structure. The nature
of this structure was identified with the help of complementary AFM exper-
iments. Figure 1.2A is an AFM image of the gold surface acquired after about
70 min of incubation in a solution of DMPC vesicles. The contrast in this
image shows film with a corrugated surface with the periodicity of the cor-
rugation similar to that in the film imaged in Fig. 1.1B by STM. In the case of
A B
150
100
50
0
0 10.0 20.0 0 50 100 150
nM
Figure 1.1 (A) STM image of a Au(111) surface acquired 3 min after injection of a solu-
tion of DMPC vesicles showing individual DMPC molecules flat lying on the surface. (B)
The corrugated structure of a film of DMPC molecules at a Au(111) electrode acquired
30 min after injection of vesicles (electrode potential +200 mV vs. Ag/AgCl electrode
saturated with KCl; supporting electrolyte in 50 mM KClO4 with in 0.04 M total DMPC
concentration). Imaging conditions: It ¼ 1.00 nA, Etip ¼ 150 mV. Adapted from Ref. [20].
Biomimetic Membrane Supported at a Metal Electrode Surface 5
Figure 1.2 Comparison of AFM images of a Au(111) electrode surface covered by
bilayer formed by vesicle fusion: (A) pure DMPC bilayer in 50 mM NaF solution con-
taining 0.1 mg/mL DMPC vesicles and (B) DMPC–Chol (7:3) DMPC–Chol bilayer in
1 mM NaF solution containing 0.07 mg/mL of DMPC. Images acquired 70 min after
injection of vesicles to the AFM cell at a temperature of 20 1 C. Adapted from Ref. [24].
AFM, the film can also be characterized by the force–distance curves
recorded in approach of the AFM tip to the film covered surface.
Representative force–distance curves are shown in Fig. 1.3A. Curve 1
displays a characteristic discontinuity when DMPC molecules are present
in the solution. This discontinuity is absent in curve 2 when it is recorded
in the pure supporting electrolyte. The discontinuity corresponds to the
penetration of the tip across the film of DMPC molecules and could be used
as a measure of the film thickness. The measured values of the film thickness
are plotted in Fig. 1.3B as a function of temperature. The thickness of the
film formed by fusion of unilamellar vesicles is equal to about 4.5 nm at
20 C. This corresponds to the expected thickness of DMPC bilayer in
the gel state [37–39]. At temperatures above 24 C, the thickness decreases
to a value 3.8 nm which is expected for the liquid-crystalline state [37].
These data indicate that at sufficiently long incubation times, the bilayer
of DMPC is formed at the gold electrode surface by fusion of unilamellar
vesicles.
The temperature dependence of the bilayer thickness shown in Fig. 1.3B
indicates that transition between gel and liquid-crystalline states is observed
between 20 and 22 C. In DMPC vesicles the phase transition is observed at
24 C [37]. IR experiments on hybrid bilayers with one leaflet composed of
hydrogen and the second with deuterium-substituted acyl chains indicated
that in the bilayer supported at gold the two leaflets are poorly coupled [10].
This poor coupling explains the observed shift of the phase transition to
lower temperatures.
6 Jacek Lipkowski
A B 5.5
Corrected thickness (nm)
2.0 5.0
DMPC-LB-LS
1.5
4.5
Force (nN)
1.0
1 4.0
0.5
3.5
0.0
DMPC - VF
2
-0.5 3.0
0 5 10 15 20 25 30 35 40 45 50 12 16 20 24 28 32 36
Tip-substrate distance (nm) Temperature (⬚C)
C 5.5
Corrected thickness (nm)
DMPC + cholesterol
5.0
4.5
4.0 DMPC
3.5
3.0
12 16 20 24 28 32 36
Temperature (⬚C)
Figure 1.3 (A) Force–distance curves recorded at E ¼ 0.2 V, curve 1 and solid circles for
0.1 M NaF + 0.1 mg/mL DMPC vesicle solution; curve 2 and open squares for pure
supporting electrolyte; (B) Dependence of the thickness of the film of DMPC at the gold
electrode surface as a function of temperature; solid squares: DMPC bilayer formed by a
combination of the Langmuir–Blodgett and Langmuir–Schaefer methods; open
squares: DMPC bilayer formed by a spontaneous fusion of small unilamellar vesicles
from 0.1 M NaF + 0.2 mg/mL DMPC vesicle solution; E ¼ 0.2 V versus Ag/AgCl electrode
saturated with KCl; (C) Temperature dependence of the bilayer thickness, squares for the
mixed 70% DMPC + 30% cholesterol and circles for pure DMPC bilayer formed by the
LB-LS method. Adapted from Ref. [23].
The differences between mechanisms of spreading unillamelar vesicles at
hydrophilic surfaces and at a gold are particularly well illustrated by the
example of spreading mixed DMPC–cholesterol vesicles [21]. When vesi-
cles rapture and their material is released onto the surface, strong lipid–metal
interactions are causing segregation of the film into pure cholesterol and
pure DMPC domains seen in the STM image (Fig. 1.4A) recorded
36 min after injection of vesicles. The zigzag-like features in this contrast
correspond to an ordered domain formed by flat-lying cholesterol
Biomimetic Membrane Supported at a Metal Electrode Surface 7
Figure 1.4 Evolution of the film structure with time. Figures (A) and (B) are images of a
Au(111) electrode surface in a solution of mixed DMPC/cholesterol (7:3) vesicles
acquired 36 and 45 min after the potential step from 0.6 to +0.2 V, respectively. Imag-
ing conditions: Etip ¼ 0.15 V, it ¼ 0.25 nA. The estimated total concentration of DMPC/
cholesterol in the solution is 3.1 102 mM. The 0.05 M KClO4 was used as the
supporting electrolyte. The dark holes correspond to defects in the bilayer. Adapted
from Ref. [21].
molecules [21]. These features disappear in the image (Fig. 1.4B) acquired
45 min after injection of vesicles indicating that at longer incubation times a
homogeneous film is formed. This is confirmed by the AFM image of the
film acquired 70 min after vesicles injection and shown in Fig. 1.2B. The
force–distance curves measured for this film allowed to determine its thick-
ness. The thickness of the film formed by fusion of mixed DMPC–Chol ves-
icles is equal to 4 nm and is consistent with the thickness expected for the
bilayer providing additional evidence that the lipid bilayer may be formed
spontaneously on the gold surface by fusion of unilamellar vesicles [24].
The mixed DMPC–Chol bilayer is smooth and free from the corruga-
tion observed in the images of the pure DMPC film. The shape of aggregates
formed by lipids is governed by the packing parameter p defined as [40]:
V
p¼
pr 2o h
where V is the volume of the cylinder circumcised by the motion of the tails
to the motion of the volume of a cylinder with radius ro corresponding to the
radius of the head group and h is the length of the molecule. A planar bilayer
is formed when p ¼ 1. Figure 1.5 shows that the DMPC molecule is conical
and its packing parameter is 0.79 [41]. It has intrinsic curvature and the
bilayer has a spontaneous tendency to curve. When spread on a planar
8 Jacek Lipkowski
ro
V
p=
pro2 h
N
O
O P O
O OH
O
O
O
O
DMPC chol
½<p<1 p>1
Figure 1.5 Shape of DMPC and cholesterol and estimate of their packing parameters for
a bilayer.
surface it is stressed. The stress is released by forming a ripple phase seen as
corrugations in the AFM and STM images [23]. In contrast, cholesterol has
the shape of an inverted cone, and its packing parameter is 1.21. The packing
parameter of the 70% DMPC and 30 mol% cholesterol mixture is equal to
0.9 [41] and is close to the value expected for a planar bilayer.
The improved value of the packing parameter explains why insertion of
cholesterol releases the stress in the bilayer and its surface becomes flat.
2.2. Langmuir–Blodgett and Langmuir–Schaefer deposition
A combination of the LB and LS methods (LB–LS) is an alternative proce-
dure to assemble supported lipid bilayers [42]. The LB technique involves
the vertical movement of a solid substrate through a compressed monolayer
at the air/solution interface. Figure 1.6 shows the most common case of LB
deposition, where the first monolayer is transferred like a carpet onto the
substrate surface as it is raised through the compressed monolayer. Film
transfer is characterized by measurement of the transfer ratio (or deposition
ratio) defined as the decrease in the area occupied by the monolayer (held at
constant pressure) on the subphase surface, divided by the coated area of the
Biomimetic Membrane Supported at a Metal Electrode Surface 9
Figure 1.6 Schematic diagrams of the LB and LS depositions.
solid substrate. The transfer ratio for a perfectly transferred monolayer should
be 1.0. To complete the bilayer, the second leaflet is transferred using the LS,
or horizontal touch deposition also shown in Fig. 1.6. This procedure allows
for the transfer of the second monolayer onto the surface, which is oriented
such that the hydrocarbon chains face the tails of the first monolayer and the
polar head groups extend into the air. A combination of the LB and LS depo-
sition methods therefore results in a bilayer on the Au(111) surface.
The AFM images of the gold surface supporting bilayers of pure DMPC
and DMPC–Chol assembled by the LB–LS methods show very similar
topography to the films formed by spontaneous fusion of vesicles and pres-
ented in Fig. 1.2A and B. The bilayer made of pure DMPC is corrugated and
the bilayer formed by the DMPC–Chol mixture is smooth [24]. However,
the force–distance curves measurements revealed that thicker bilayers are
formed by the LB–LS methods than by fusion of vesicles. Figure 1.3B
compares changes of the thickness of the bilayers of DMPC formed by
the VF and LB-LS methods as a function of temperature. The bilayers
formed by the LB–LS method are thicker by 0.3–0.4 nm and the phase tran-
sition for these bilayers is sharper. These behaviors indicate that bilayers
formed by the LB–LS methods are more ordered than bilayers formed by
fusion of vesicles. Figure 1.3C compares temperature dependence of the
the thickness of pure DMPC and the mixed DMPC+ cholesterol bilayers
formed by the LB-LS methods. The mixed bilayer is thicker and has less
sharp phase transition. An advantage of the LB–LS deposition over VF is that
it allows for the construction of an asymmetric bilayer. In the LB–LS depo-
sitions, different components can be used for the inner and outer leaflet, for
example, construction of a hydrophilic spacer molecule in only the inner leaf-
let of the bilayer while maintaining more natural phospholipid environment
10 Jacek Lipkowski
in the outer leaflet [43]. On the other hand, an advantage of VF over the LB–
LS depositions is that vesicles may easily incorporate membrane proteins, for-
ming proteoliposomes, and their fusion may be used to incorporate proteins
into the supported bilayer. However, it should be stressed that vesicles fuse
only on the atomically smooth metal surfaces. Quartz crystal microbalance
and surface plasmon resonance studies [44–46] indicated that unilamellar ves-
icles adsorb but do not fuse on a film of vapor deposited gold which consists of
gold nanoparticles 40 nm in size. In that case, the size of vesicles is compa-
rable in magnitude to the size of the nanoparticles. Apparently, spreading of
vesicles into bilayer requires atomically smooth gold surface of dimensions
much larger than the dimension of an individual vesicle.
3. POTENTIAL DROP ACROSS THE MEMBRANE AND AN
ESTIMATE OF THE ELECTRIC FIELD ACTING ON THE
MEMBRANE
The surface of the cell membrane contains various charged or dipolar
groups. The charged groups generate Volta or outer potential (c ). The sur-
face charges are compensated by counterions in solutions on the two sides of
the membrane and the potential drop at the membrane–solution interface
has a long range character. In contrast, oriented surface dipoles generate a
surface potential w which has a local character. Consequently, the value
of the potential at any point is a result of contributions from three electrical
potential differences: the transmembrane potential (Dc ), the potential due
to surface charges (cs), and the dipole potential (wM), as shown in
Fig. 1.7A [47].
The transmembrane potential Dc is generated by a difference in concen-
tration of ions in the two solutions separated by the membrane and selective
ionic permeability of the membrane to one or several ions (Donnan poten-
tial). The transmembrane potential across a membrane 5–7 nm thick is typ-
ically in the range of 10–250 mV, and imposes an electric field strength of
approximately 2106 to 5107 V m1 on molecules within the cell
membrane [48]. The fixed charges at the membrane surface control the con-
centration of counterions at the membrane–solution interface, resulting in a
diffuse double layer. This diffuse layer gives rise to a potential of approxi-
mately 60 mV, corresponding to an electric field strength of up to
2 107 V m1, within 1 nm from the surface [48]. The third contribution,
the membrane dipole potential (wm), arises from the alignment of dipolar res-
idues of the lipids and/or water dipoles in the region between the aqueous
Biomimetic Membrane Supported at a Metal Electrode Surface 11
A B C
OHP
e = 80 e=2 e = 80 e=2 e = 80 e=2 e = 80
cm,2 cm,2
Metal
Metal
ys,2 Dy2-s Dy2-s
cm,1 Dy cm,1 DyM-s DyM-s
ys,1 DyM-2
Cm Cs
Figure 1.7 The electrical potential profile across: (A) a cell membrane, (B) lipid bilayer
attached to a metal electrode, and (C) lipid bilayer attached to a metal electrode with
omitted surface potentials. The three contributions to the electrical potential are shown;
the transmembrane potential (Dc), the Volta potential (cs), and the dipole potential
(wm). The subscripts 1 and 2 denote the potentials associated with the two leaflets of
the lipid membrane. The dielectric constants (e) for the lipid core (e ¼ 2) and the exterior
sides of the membrane (e ¼ 80) are given. Below (C) an equivalent electric circuit of the
membrane is shown where Cm and Cs represent the capacitance of the membrane and
diffuse layer, respectively. Adapted from Ref. [48].
phases and the hydrocarbon-like interior of the membrane [48]. The dipole
potential cannot be measured and can only be estimated on the basis of extra-
thermodynamic assumptions. For a fully saturated phosphatidylcholine
bilayer, a dipole potential is estimated to be in the range of 220–280 mV,
positive in the interior of the membrane [48]. Since the dipole potential drop
across the head group region of the membrane takes place over a small dis-
tance, the electric field strength produced is very large (108–109 V m1) in
comparison to the other two contributions [48]. A bilayer membrane can be
viewed as a simple parallel-plate capacitor which capacitance (Cd) is defined
by C d ¼ eedo , where e is the dielectric constant, eo is the permittivity of vac-
uum, and d is the thickness of the bilayer. The measured specific capacitance
(capacitance per unit area) of biological membranes is around 1 mF cm2
[47–49].
Figures 1.7B and C show a schematic of the electrical potential profile
across a lipid bilayer supported at the gold surface at the interface with an
electrolyte solution. The metal side of the interface can be charged and
the charge density sM generates a Volta potential drop across the membrane
equal to:
sM
DcM-S ¼ (1.1)
C
12 Jacek Lipkowski
The inner or Galvani potential is defined as f ¼ c + w. Hence, the drop
of Galvani potential across the metal solution interface (DfM-S) is described
by the formula [11]:
sM
DfM-S ¼ + wM (1.2)
C
where wM is the dipole potential of the membrane and C is the total capac-
itance of the interface. The lipid membrane in contact with electrolyte solu-
tion is equivalent to two capacitors connected in series; a capacitor with
capacitance Cs associated with the diffuse layer and a capacitor with capac-
itance Cm representing the membrane (see Fig. 1.7C). Therefore, Eq. (1.2)
can be rewritten as:
1 1
DfM-S ¼ sM + + wM ¼ DcM2 + Dc2S + wM (1.3)
Cm Cs
where
sM
DcM-2 ¼ (1.4)
Cm
is the potential drop across the membrane due to the presence of charge on
the metal and Dc2-S or in brief c2 is the outer Helmholtz plane potential.
The potential c2 can be calculated using the diffuse layer theory and the
expression [50]:
" #
2RT 1 sM
c2 ¼ sinh (1.5)
zF ð8RT ec b Þ1=2
where R is the gas constant, T is the temperature, F is Faraday’s constant, z is
the ionic charge, sM is the charge density at the metal surface covered with
the lipid layer, and cb is the ionic concentration in the bulk of solution.
Independently, the value of DfM-2 can also be calculated using the fol-
lowing expression [11]:
DfM-2 ¼ E E pzc c2 + wM (1.6)
where E and Epzc are the potentials applied to the gold electrode with respect
to a given reference electrode and Epzc is the potential at which charge den-
sity at the gold surface is equal to zero. The first three terms in Eq. (1.6)
are equal to DcM-2 ¼ E Epzc c2. These terms are either experimental
quantities or a quantity that could be calculated from charge density data.
Biomimetic Membrane Supported at a Metal Electrode Surface 13
To calculate DfM-2 one needs to know the dipole potential wM. The dipole
potential can only be theoretically calculated or estimated indirectly from
experiment [51]. Therefore, in the estimation of the potential drop across
the membrane the contribution from wM has been frequently omitted and
DcM-2 is used as an approximate measure of DfM-2. A schematic of the sim-
plified electrical profile is shown in Fig. 1.7C. According to Eqs. (1.4) and
(1.6), sM/Cm ¼ E Epzc c2. For high electrolyte concentration, Cm C,
and therefore the potential drop across the membrane can be estimated using
either sM/C or (E Epzc c2).
This point is illustrated in Fig. 1.8 which shows the sM/C ratio using C
calculated from the charge density data for a DMPC bilayer at the Au(111)
electrode surface [11]. In addition, the open squares plot the values of
(E Epzc c2) where Epzc is the potential of zero charge for the bilayer-
covered electrode and c2 is the outer Helmholtz plane potential calculated
according to Eq. (1.5). Figure 1.8 shows that the agreement between these
two sets of data is good. The DMPC bilayer is adsorbed at the Au(111) sur-
face in the potential range from 0.4 to 0.45 V (vs. Ag/AgCl), and the
corresponding potential drop across the membrane is 0.4 to
0.2 V. Considering the thickness of the bilayer is 5 nm, the electric field
Figure 1.8 Left-hand side vertical axis: (solid squares) sM/C calculated from the charge
density data for a DMPC bilayer at the Au(111) electrode surface; (open squares) plot of
(E Epzc f2). Right-hand side vertical axis: the electric field across the bilayer
corresponding to sM/Cm or (E Epzc f2)/d. Potentials were measured versus the
Ag/AgCl reference electrode. The bilayer deposited by the LB–LS techniques, temper-
ature 22 2 C. Adapted from Ref. [11].
14 Jacek Lipkowski
across the bilayer changes between 1.0 108 and 2 107 V m1 in the
potential region where the bilayer is adsorbed at the metal surface.
4. EFFECT OF THE POTENTIAL APPLIED TO THE GOLD
ELECTRODE ON THE MEMBRANE STABILITY: AFM, NR,
AND SURFACE-ENHANCED INFRARED ABSORPTION
SPECTROSCOPY STUDIES
The effect of potential on the properties of a bilayer formed at the
Au(111) electrode surface was initially investigated using DMPC [5–11].
The DMPC bilayer can be deposited at Au(111) surface by the VF
[5,7,23] and LB–LS methods [10,11]. However, the LB–LS method allows
one to form bilayers which structure are better controlled than bilayers
formed by the VF method. Therefore, the DMPC bilayer prepared by
the LB–LS method will be used as a model to illustrate what structural infor-
mation could be obtained from combined electrochemical, polarization
modulation infrared reflection absorption spectroscopy (PM IRRAS) and
AFM studies of a biomimetic membrane supported at an electrode surface.
The stability of the biomimetic membrane as a function of potential
applied to the electrode can be conveniently described with the help of
charge density curves. Figure 1.9A plots the charge density curves for the
bare Au(111) electrode in 0.1 M NaF solution and for the electrode with
DMPC bilayer deposited at Au(111) surface by the LB–LS method [11].
The bottom horizontal axis plots the potential applied to the gold electrode
versus the reference electrode. The top horizontal axis plots (E Epzc c2),
which is a measure of potential drop across the membrane, equivalent to the
transmembrane potential. The data illustrate that the DMPC bilayer is
adsorbed on the metal surface when the absolute value of the charge on
the metal is less than 10 mC cm2. The plateau section on the curve
corresponding to the bilayer-covered electrode has a small step at
E 0.35 V versus Ag/AgCl electrode. This behavior suggests that the
adsorbed bilayer exists in different states in the range of potentials between
0.4 < E < 0.1 and 0.1 < E < 0.45 V versus Ag/AgCl (corresponding to
transmembrane potentials between 0.5 and 0.2 V and 0.2 and 0.2 V,
respectively). When the charge density is more negative than 10 mC cm2
(transmembrane potential is more negative than 0.5 V), the DMPC bilayer
begins to desorb from the Au(111) surface. The charge density curve of the
bilayer-covered electrode merges with that of the bare electrode when
E 0.6 V versus Ag/AgCl, indicating that DMPC is completely detached
