Modification of the optoelectronic properties of membranes via insertion of amphiphilic phenylenevinylene oligoelectrolytes

Article abstract of DOI:10.1021/ja1016156

We report on the modification of membranes by incorporation of phenylenevinylene oligoelectrolytes with the goal of tailoring their optical and electronic properties and their applications. A watersoluble distyrylstilbene oligoelectrolyte (DSSN+), capped

Full text of DOI:10.1021/ja1016156

Published on Web 07/07/2010
Modification of the Optoelectronic Properties of Membranes via
Insertion of Amphiphilic Phenylenevinylene Oligoelectrolytes
Logan E. Garner,† Juhyun Park,‡ Scott M. Dyar,† Arkadiusz Chworos,†
James J. Sumner,§ and Guillermo C. Bazan*,†
Department of Chemistry and Biochemistry, UniVersity of California, Santa Barbara,
California 93106, U.S. Army Research Laboratory, Sensors and Electron DeVices Directorate,
Adelphi, Maryland 20783, and School of Chemical Engineering and Materials Science,
Chung-Ang UniVersity, 221 Heukseok-Dong, Dongjak-Gu, Seoul, Korea
Received February 24, 2010; E-mail: bazan@chem.ucsb.edu
Abstract: We report on the modification of membranes by incorporation of phenylenevinylene oligoelec-
trolytes with the goal of tailoring their optical and electronic properties and their applications. A water-
soluble distyrylstilbene oligoelectrolyte (DSSN+), capped at each end with nitrogen bound, terminally charged
pendant groups, was synthesized. The photophysical and solvatochromatic properties of DSSN+ and the
shorter distyrylbenzene analogue DSBN+ were probed and found to be useful for characterizing insertion
into membranes based on phospholipid vesicle systems. A combination of UV/visible absorbance and
photoluminescence spectroscopies, together with confocal microscopy, were employed to confirm membrane
incorporation. Examination of the emission intensity profile in stationary multilamellar vesicles obtained
with a polarized excitation source provides insight into the orientation of these chromophores within lipid
bilayers and indicates that these molecules are highly ordered, such that the hydrophobic electronically
delocalized region positions within the inner membrane with the long molecular axis perpendicular to the
bilayer plane. Cyclic voltammetry experiments provide evidence that DSSN+ and DSBN+ facilitate
transmembrane electron transport across lipid bilayers supported on glassy carbon electrodes. Additionally,
the interaction with living microorganisms was probed. Fluorescence imaging indicates that DSSN+ and
DSBN+ preferentially accumulate within cell membranes. Furthermore, notable increases in yeast microbial
fuel cell performance were observed when employing DSSN+ as the electron transport mediator.
porated into FETs via different deposition methods.3 The
accumulated effort has yielded insight not only into device
Introduction
Conjugated oligomers can be described by a select number
of repeat units extracted from a polymer containing an electroni-
cally π-delocalized backbone. Homologous progressions of these
molecules, and related systems with extended electronic delo-
calization, have been useful in fundamental studies with a focus
on understanding how molecular connectivity influences optical
and electronic properties and in the development of emerging
technologies.1 One well-appreciated opportunity involves inte-
gration as the semiconducting component in field effect transis-
tors (FETs) relevant for plastic electronics.2 A wide range of
structural variations have been designed, developed, and incor-
optimization but also on how weak intermolecular forces can
be coordinated to yield desirable morphologies at interfaces and
how intermolecular arrangements mediate charge carrier trans-
port.4 More recently, thin films of conjugated oligomers bearing
pendant groups with ionic functionalities, i.e., conjugated
oligoelectrolytes (COEs), were demonstrated to be effective for
reducing charge injection barriers at metal/organic interfaces.5
(3) (a) Bao, Z.; Rogers, J. A.; Katz, H. E. J. Mater. Chem. 1999, 9, 1895–
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† University of California.
‡ Chung-Ang University.
§ U.S. Army Research Laboratory.
(1) (a) Martin, R. E.; Diederich, F. Angew. Chem., Int. Ed. 1999, 38, 1350–
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10042 J. AM. CHEM. SOC. 2010, 132, 10042–10052
10.1021/ja1016156  2010 American Chemical Society

Modification of Optoelectronic Properties of Membranes
A R T I C L E S
Scheme 1. Structure of OPV Oligoelectrolytes
While more than one mechanism may be operating, for example,
ion motion and/or the formation of a spontaneously aligned
dipole layer, the simplicity of incorporating COE injection layers
via solution methods opens the opportunity to reduce the
operating voltages of polymer-based light emitting diodes.
Several structural types of conjugated oligomers have also
been used in molecular transconductance studies that assess
charge transfer across a single or a few molecules. For instance,
comparison of oligophenylenevinylene (OPV) and oligophe-
nyleneethynylene (OPE) structures that span two gold contacts
reveals better conductance across the OPV framework, relative
to OPE.6 Such experimental findings are corroborated by
theoretical calculations and have revealed that structural pa-
rameters that affect HOMO-LUMO energy levels, such as
planarity and bond length alternation, influence charge transport
efficiency.7,8 Electrochemical measurements have also been used
to demonstrate that OPVs of various lengths facilitate tunneling
between a gold surface and a tethered redox species.9 The work
to date regarding single molecule transconductance has offered
much insight into the factors that influence charge transport and
has laid the foundation for the design and development of
molecular wires that may play a role as charge transporting
components in new technologies.
Oligomers broadly described by a D-π-D structure, where
D is an electron-donating group and π refers to a π-delocalized
linker, have been immensely instructional for understanding and
optimizing two photon absorption processes in organic materials.
Some of these molecular systems have been utilized for three-
dimensional fabrication10 and for two-photon microscopy of
biological systems.11-13 Molecules possessing D-π-D struc-
tures typically undergo intramolecular charge transfer excitation
that results in large two-photon absorption cross sections.14 One
specific example is the molecule 1,4-bis(4′-(N,N-bis(6′′-(N,N,N-
trimethylammonium)hexyl)amino)-styryl)benzene tetraiodide (DS-
BN+), the structure of which is shown in Scheme 1. This
molecule incorporates charged groups that increase solubility
in highly polar organic solvents and water. The distyrylbenzene
(DSB) conjugated region is capped at each end with two
nitrogen-bound, six-carbon pendant groups containing terminal
quaternary ammonium salts. By examination of optical proper-
ties in different solvents, and in combination with neutral
derivatives, it is possible to examine how the dielectric constant
of the medium perturbs linear and two-photon spectral re-
sponses.15 These studies have highlighted the challenges in
predicting a priori how the molecular features and the environ-
ment combine to yield different optical properties. It was also
found that molecules such as DSBN+ display much larger
emission quantum efficiencies and two-photon absorption cross
sections upon incorporation into micelles from an aqueous
environment.16 Association with the interior of the micelle is
likely a consequence of the hydrophobic DSB framework.
On the basis of the background delineated above and a
consideration of the molecular dimensions of DSBN+, it
occurred to us that it would be possible to intercalate this
molecule into lipid bilayer membranes in an ordered orientation.
A schematic cartoon of the general concept is provided in Figure
1. The dependence of optical features as a function of the
medium provides a useful characterization handle. Furthermore,
the precedence of using OPVs in single-molecule charge
transport measurements argues in favor of these molecules being
able to mediate transmembrane charge transfer.
This manuscript is organized as follows. We begin with the
synthesis of a longer water-soluble oligoelectrolyte analogue
of DSBN+, namely 4,4′-bis(4′-(N,N-bis(6′′-(N,N,N-trimethy-
lammonium)hexyl)amino)-styryl)stilbene tetraiodide (DSSN+
in Scheme 1). Successful incorporation into lipid bilayer vesicles
and membranes is demonstrated by a variety of techniques
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M. W. Nat. Biotechnol. 2003, 21, 1369–1377.
Figure 1. Cartoon representation of a DSBN+ modified lipid bilayer
depicting the predicted orientation when intercalated within a phospholipid
bilayer. The long molecular axis is normal to the plane of the membrane
with the hydrophobic conjugated region within the nonpolar inner membrane
and the polar pendant group terminals oriented outward toward the polar
aqueous environment on either side of the lipid bilayer.
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A R T I C L E S
Garner et al.
Scheme 2. Synthetic Procedures for Preparation of DSSN and DSSN+a
a Reagents and conditions: (i) Zn, TiCl4, THF, 24 h. (ii) NBS, AIBN, benzene, reflux, 24 h. (iii) C46H65Cl2N2PRu (Grubbs catalyst, second generation),
t
CH2Cl2, reflux, 24 h. (iv) P(OEt)3 neat, reflux, 24 h. (v) BuONa, THF, RT, 24 h. (vi) a. NMe3, THF, RT, 24 h; b. NMe3, MeOH, RT, 24 h.
including confocal microscopy. Polarized excitation sources
demonstrate that DSBN+ and DSSN+ are immobilized within
the bilayers in an orientation such that the hydrophobic long
molecular axis is perpendicular to the plane of the membrane
and the polar pendant group terminals are positioned at the outer
surfaces. Favorable interaction with living systems revealed
spontaneous insertion within cell membranes. Results obtained
from cyclic voltammetry blocking experiments are in agreement
with significant improvements of transmembrane electron
transport. Lastly, notable improvements in yeast microbial fuel
cell performance were observed when employing DSBN+ and
DSSN+ as electron transport mediators. These findings provide
the basis for improving charge extraction from living microor-
ganisms and for developing new membrane-specific optical
labels for a variety of imaging techniques.
reaction. Alternatively, 3 can be prepared in two steps (Pathway
2, Scheme 2) beginning with the metathesis condensation of
4-vinylbenzylchloride to yield 2B, followed by an Arbuzov
reaction. The second pathway removes one step, while ef-
fectively circumventing a low-yielding radical bromination at
the negligible cost of a slightly lower reaction yield (50% and
55% yield of 3 for the two- and three-step pathways, respec-
tively). As stated above, a subsequent Horner-Wadsworth-
Emmons reaction affords the neutral chromophore DSSN in
reasonable yield (76%). Preparation of DSSN+ (73% yield) was
completed by quaternization of DSSN using trimethylamine.
Each synthetic target and intermediate was characterized by
1H and 13C NMR spectroscopy, mass spectrometry, and
elemental analysis. Evidence indicating all-trans conformations
of DSSN and DSSN+ was provided by 1H NMR spectroscopy.
Signals corresponding to protons of the center vinylic linkage
are observed as a singlet, indicating a high degree of molecular
symmetry, while signals corresponding to protons of the outer
vinylic linkages are observed as a doublet with a coupling
constant indicative of a trans conformation (J ) ∼16 Hz).
Results and Discussion
Synthesis and Characterization. Compound DSBN+ was
prepared as previously reported.15 Scheme 2 shows the synthesis
of DSSN+. This route is based upon the assembly of the desired
end-capped π-system by coupling a bis(methylene)phosphonate
(for example, 3 in Scheme 2) with the amino-functionalized
benzaldehyde 415 via a trans-selective Horner-Wadsworth-
Emmons reaction. Compound 3 may be accessed via two
different pathways. The three-step pathway (Pathway 1, Scheme
2) begins with preparation of 1 in good yield via a McMurry
coupling of p-tolualdehyde followed by a low-yielding (29%)
Wohl-Ziegler radical bromination that affords 2A.17 The
desired bisphosphonate 3 can then be generated by an Arbuzov
Optical Characterization. General photophysical and solva-
tochromatic features of the neutral (DSSN, DSBN) and charged
(DSSN+, DSBN+) versions of each chromophore were probed
by using UV-vis absorption and photoluminescence (PL)
spectroscopies. As expected, these structural analogues exhibit
similar sensitivities to the polarity of the surrounding medium.
Table 1 shows a summary and comparison of the spectral
characteristics for the neutral and charged versions of each
chromophore in solvents of varying polarities. Figure 2 shows
the corresponding PL spectra. General trends expressed by both
structural analogue pairs are as follows: absorbance maxima
(λabs) occur in the 406-436 nm range and exhibit relatively
small shifts in different solvents. Large hypsochromic shifts in
PL maxima (λem) and increased quantum efficiencies (η) are
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A R T I C L E S
Table 1. Summary of UV-vis and PL Spectra
toluene
THF
DMSO
water
a
)
λabs (εmax
λem (η)b
λabs (εmax
)
λem (η)
λabs (εmax
)
λem (η)
λabs (εmax
)
λem (η)
DSSN
DSSN+
DSBN
425 (10.7)
410 (8.4)
476 (0.95)
426 (10.6)
410 (8.5)
526 (0.89)
436 (9.6)
435 (9.8)
419 (7.8)c
419 (8.2)
602 (0.73)
596 (0.71)
516 (0.81)
511 (0.86)
412 (6.6)
406 (6.0)
594 (0.06)
566 (0.33)
454 (0.98)
466 (0.92)
DSBN+
a Molar extinction coefficients (εmax) were measured at λmax and are reported in units of L mol-1 cm-1 × 10-4
.
b Quantum efficiency (η) values were
c
measured relative to a fluorescein standard at pH 12. εmax value abstracted from ref 15.
in toluene for DSSN and DSBN, respectively). This shift is
accompanied by the appearance of vibronic structure.19 The η
values of each analogue are similar for a given solvent and are
considerably greater in toluene (0.95 and 0.98 for DSSN and
DSBN, respectively) than in water (0.06 and 0.33 for DSSN+
and DSBN+, respectively). Figure 2 shows the PL spectra of
DSSN/DSSN+ (Figure 2A) and DSBN/DSBN+ (Figure 2B);
peak areas are proportional to the magnitude of η in Table 1.
The difference in η values of DSSN+ and DSBN+ in water
may be due to a greater degree of DSSN+ aggregation caused
by its larger hydrophobic component, which may lead to
increased self-quenching.20,21
Incorporation of COEs into Lipid Bilayers. A model vesicle
system was chosen to examine the incorporation of DSBN+
and DSSN+ into phospholipid bilayers, develop a characteriza-
tion methodology, and ascertain molecular orientation. Vesicles
are excellent model systems for membranes due to their ease
of formation via self-assembly, structural integrity, large surface
area, and high degree of order with respect to spherical shape
and orientation of the molecular constituents.22 The specific
lipids employed were 1,2-dimyristoyl-sn-glycero-3-phospho-
choline (DMPC) and 1,2-dipalmitoyl-sn-glycero-3-phosphocho-
line (DPPC), the tails of which contain 14 and 16 carbons,
respectively. These lipids were chosen due to ease of vesicle
formation under the conditions employed in this study23 and
the tendency of phosphatidylcholine lipid bilayers to be tolerant
of added components with respect to maintenance of their
microheterogeneous structures.24 Lipid bilayers composed of
DMPC have thicknesses on the order of ∼31-34 Å, while
DPPC lipid bilayers are slightly thicker (∼34-37 Å).25 The
thickness of DMPC and DPPC bilayer membranes is close to
the molecular lengths of DSBN+ and DSSN+ (∼35-40 Å).
Figure 2. PL spectra of 5-7 µM DSSN (A, solid lines), DSSN+ (A,
dashed lines), DSBN (B, solid lines), and DSBN+ (B, dashed lines) in
various solvents. Peak areas shown are proportional to η values. General
trends exhibited by both chromophores: λem decreases and η increases with
decreasing solvent polarity. Excitation wavelengths were chosen to match
the optical density of the fluorescein reference sample.
observed as the solvent polarity decreases. Compared to the
distyrylbenzene chromophore (DSBN and DSBN+), the distyr-
ylstilbene counterpart (DSSN and DSSN+) has red-shifted λabs
and λem and a larger molar extinction coefficient (εmax),
consistent with its more extended conjugation length and greater
size.18 It is important to note that when comparing the
solvatochromatic properties of a given neutral and charged
chromophore pair the difference in pendant group terminals
(alkyliodide in DSSN and DSBN vs trimethyl ammonium iodide
in DSSN+ and DSBN+) has a negligible effect on the electronic
structure of the chromophore.15 This conclusion is supported
by the nearly identical UV-vis and PL features of each
structural analogue pair when dissolved in DMSO (Table 1,
Figure 2).
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As seen in Figure 2, the λem values of each chromophore
exhibit a ∼110-120 nm hypsochromic shift as the solvent is
shifted from polar (λem ) 594 and 566 nm in water for DSSN+
and DSBN+, respectively) to nonpolar (λem ) 476 and 454 nm
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Garner et al.
Figure 3. (A) Cryo-TEM image of unilamellar DMPC vesicles modified with 5 mol % of DSBN+ relative to lipid (16 mg/mL). (B) Confocal microscopy
image of 5 mol % of DSBN+/DMPC multilamellar vesicles (2 mg/mL) obtained following 488 nm excitation. Each image demonstrates successful vesicle
formation as well as a maintained liposomal microstructure upon addition of DSBN+. The confocal microscopy image is generated by the fluorescence
response of DSBN+ and demonstrates oligoelectrolyte association with lipid bilayers.
Such similarities were anticipated to favor the type of membrane
intercalation shown in Figure 1.
Efforts to modify the vesicle membranes with DSBN+ and
DSSN+ followed modified literature procedures.26 First, a lipid
stock solution containing the desired fraction of either DSSN+
or DSBN+, typically 1-5 mol % relative to lipid, was prepared
in methanol. This lipid/COE stock solution was then heated and
held above the transition temperature of the lipid (23 and 41
°C for DMPC and DPPC, respectively) as the solvent was
removed via Argon flushing, followed by vacuum drying to a
constant weight. The resulting lipid/COE solid was then
suspended in pH ) 7.3 HEPES buffer to the desired concentra-
tion (1-10 mg/mL). Multilamellar vesicles were then formed
by self-assembly upon sonication of the resulting solution.
Unilamellar vesicles were prepared by filtration (using 0.45 or
0.2 µm pore size syringe filters) or by extrusion.27
Confirmation of vesicle formation and that the microhetero-
geneous liposomal structure is maintained upon modification
with DSBN+ or DSSN+ was provided by cryogenic transmis-
sion electron microscopy (cryo-TEM) and confocal microscopy.
The cryo-TEM image of unilamellar DMPC vesicles containing
membrane embedded DSBN+ shown in Figure 3A demonstrates
Figure 4. Normalized UV-vis absorbance and PL spectra of (A) DSSN+
successful vesicle formation, as well as unperturbed liposomal
and (B) DSBN+ in pH 7.3 HEPES buffer (absorbance ) blue, emission )
microstructure. Figure 3B is a confocal microscopy image of
red) and while embedded within DMPC vesicle membranes (absorbance
multilamellar DMPC vesicles prepared in the presence of
DSBN+. The image was generated by DSBN+ fluorescence
emission following excitation at 488 nm. The layers that
compose the vesicles can be seen, demonstrating that these
oligoelectrolytes readily associate with the membranes. Subse-
quent characterization described in more detail below will
demonstrate that, indeed, the chromophores span the width of
the membranes.
Optical Characterization of PV Oligoelectrolytes within
Lipid Bilayers. Several indicative changes in UV-vis and PL
properties of DSSN+ and DSBN+ are observed upon associa-
tion with phospholipid bilayer membranes. A comparison of
the UV-vis and PL spectra of DSSN+ and DSBN+ in water
) green, emission ) orange). Both molecules exhibit an indicative red shift
in λabs (21 and 16 nm for DSSN+ and DSBN+, respectively) and a blue
shift in λem (62 and 92 nm for DSSN+ and DSBN+, respectively) due to
the difference in environmental polarity between an aqueous solvent and
the inner region of a lipid bilayer.
and lipid bilayers is shown in Figure 4; the corresponding
summary of the data is provided in Table 2. Compared to the
spectroscopic features of the chromophores in water, one
observes a bathochromic shift in λabs (21 and 16 nm for DSSN+
and DSBN+, respectively) and a large hypsochromic shift in
λem (62 and 92 nm for DSSN+ and DSBN+, respectively) when
associated with the lipid bilayers. Each shift is accompanied
by the appearance of vibronic structure. When compared to the
(25) (a) Kucˇerka, N.; Uhr´ıkova´, D.; Teixeira, J.; Balgavy´, P. Phys. B:
Condens. Matter 2004, 350, e639-e642. (b) Lewis, B. A.; Engelman,
D. M. J. Mol. Biol. 1983, 166, 211–217.
(27) (a) Olson, F.; Hunt, C. A.; Szoka, F. C.; Vail, W. J.; Papahadjopoulos,
D. Biochim. Biophys. Acta 1979, 557, 9–23. (b) Socaciu, C.; Bojarski,
P.; Aberle, L.; Diehl, H. A. Biophys. Chem. 2002, 99, 1–15. (c) Szoka,
F.; Olson, F.; Heath, T.; Vail, W.; Mayhew, E.; Papahadjopoulos, D.
Biochim. Biophys. Acta 1980, 601, 559–571.
(26) Lin, A. J.; Slack, N. L.; Ahmad, A.; George, C. X.; Samuel, C. E.;
Safinya, C. R. Biophys. J. 2003, 84, 3307–3316.
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Modification of Optoelectronic Properties of Membranes
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Table 2. Comparison of UV-vis Absorbance and PL Spectroscopy
of DSSN+ and DSBN+ in Water and Lipid Bilayers
Figure 5 displays four confocal microscopy images of
modified multilamellar vesicles, each corresponding to one of
the four lipid/oligoelectrolyte combinations (DMPC/DSBN+,
DPPC/DSBN+, DMPC/DSSN+, and DPPC/DSSN+; each 5
mol % of COE). These images, obtained following excitation
of the chromophores, illustrate preferential accumulation inside
the vesicle membranes. These images also suggest excellent
structural diversity; i.e., the short/long COEs can both be
incorporated into bilayers composed of lipids of varying tail
lengths.
water
lipid bilayer
a
)
λabs(εmax
λ
em(η)b
λ
abs(εmax
)
λem(η)
DSSN+
DSBN+
412 (6.6)
406 (6.0)
594 (0.06)
566 (0.33)
433 (8.4)
422 (4.8)
532 (0.85)
474 (0.59)
a εmax values are reported in units of L mol-1cm-1 × 10-4
.
b η values
were obtained relative to a fluorescein standard at pH 12.
emission features of each chromophore in toluene (Figure 2),
it can be seen that the emission of DSSN+ and DSBN+ within
lipid bilayer membranes is consistent with a nonpolar environ-
ment. The εmax and η values of DSSN+ and DSBN+ embedded
in lipid bilayers (8.4 and 4.8 L mol-1cm-1 × 10-4 for εmax, and
0.85 and 0.59 for η of DSSN+ and DSBN+, respectively) are
greater than those of these chromophores in water (6.6 and 6.0
L mol-1cm-1 × 10-4 for εmax, and 0.06 and 0.33 for η of
DSSN+ and DSBN+, respectively), with the exception of the
εmax of DSBN+. These values are also approaching the
Oligoelectrolyte Molecular Orientation within Lipid Bilay-
ers. Molecular orientations of DSBN+ and DSSN+ were probed
via confocal microscopy by examination of the emission
intensity profile of the COEs within stationary vesicles following
excitation using a polarized source. Oligo(phenylenevinylenes)
possess a primary transition dipole (µ) that is oriented along
the long molecular axis.29 The probability of generating an
emissive excited state using a polarized excitation source
depends on the orientation of µ with respect to the plane of
polarization of the excitation light.30 An immobilized membrane
with DSBN+ or DSSN+ in the expected molecular orientation
should exhibit regions with greater and less emission upon
excitation with a polarized excitation source. This concept has
been previously employed to investigate local order in smectic
liquid crystals.31
characteristic values in toluene (10.7 and 8.4 L mol-1cm-1
×
10-4 for εmax, and 0.95 and 0.98 for η of DSSN and DSBN,
respectively). The observed UV-vis and PL features of DSSN+
and DSBN+ indicate that these chromophores are embedded
within the lipid bilayers and experience the more hydrophobic
interior environment of the membrane.28
Figure 5. Confocal microscopy fluorescence images of multilamellar vesicles containing 5 mol % of oligoelectrolyte (10 mg/mL). (A) DMPC/DSBN+. (B)
DPPC/DSBN+. (C) DMPC/DSSN+. (D) DPPC/DSSN+. Images were collected by excitation at 488 nm.
9
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Garner et al.
Figure 6. (A) Image of a stationary multilamellar DMPC vesicle (diameter ) ∼15 µm) containing 3 mol % of DSBN+ that exhibits an equatorial extinction
line (black line) perpendicular to the plane of the 488 nm Ar laser excitation light. (B) A closer view within the extinction line region in which DSBN+
molecules are oriented with their transition dipoles (denoted by red arrows) perpendicular to the plane of the excitation light (blue arrow) resulting in
attenuated emission (small green arrow). (C) A closer view within the regions above and below the extinction line in which DSBN+ transition dipoles are
oriented parallel to the plane polarized excitation light (blue arrow) resulting in a greater number of excited states and stronger emission (larger green arrow).
Figure 6A provides the confocal fluorescence image of a
stationary vesicle obtained upon excitation of membrane-
embedded DSBN+ with a polarized excitation source. The
vesicle image contains an equatorial region (highlighted by a
black line) with attenuated intensity perpendicular to the plane
of the polarized light. These intensity distributions can be used
to determine the preferred orientations of µ. Specifically, less
intense emission is anticipated in regions where µ is perpen-
dicular to the plane of polarized excitation light, as illustrated
in Figure 6B. Conversely, as shown in Figure 6C, the alignment
of chromophores such that µ is parallel to the plane of incident
polarized light favors excitation, leading to greater emission
intensity. In Figures 6B and 6C, the emission output is
schematically represented by the size of the green arrows. The
results indicate that DSBN+ and DSSN+ exist within lipid
bilayers in an ordered orientation with the long axis perpen-
dicular to the plane of the membrane. This molecular orientation
is fully consistent with the integration of π-segments and polar
groups as shown in Figure 1.
a metallic electrode to a tethered redox species.9 We have chosen
a variation of this approach in which supported bilayer
membranes (sBLM) containing DSSN+, DSBN+, and other
control molecules were prepared on the surface of a glassy
carbon electrode surface. The sBLM-modified electrodes were
then employed in a series of “blocking” experiments in which
cyclic voltammetry was used to monitor the reversible oxidation
of aqueous ferricyanide. The unmodified sBLM acts as an
insulating layer between the working electrode and the solution
containing ferricyanide thereby greatly suppressing the redox
current.32 DSBN+ and DSSN+ within an sBLM are oriented
with their long axes perpendicular to the surface of the electrode,
thus potentially forming a transmembrane molecular wire
through which electrons tunnel to and from redox species at
the water-membrane interface.
In our experiments, a bare glassy carbon electrode and glassy
carbon electrodes supporting DMPC and DPPC BLMs contain-
ing no additional components, 2 mol % of tridodecylamine
(TDA), 2 mol % of DSSN+, and 2 mol % of DSBN+ (relative
to lipid) were used to collect voltammograms in the presence
of an aqueous 0.5 M KCl solution containing 2 mM ferricyanide.
The surface of the electrode was pretreated, according to
literature protocols, to promote bilayer formation.33 Specifically,
Transmembrane Electron Transport. OPVs within a satu-
rated alkane thiol monolayer can facilitate charge tunneling from
(28) Bose, D.; Ghosh, D.; Das, P.; Girigoswami, A.; Sarkar, D.; Chatto-
padhyay, N. Chem. Phys. Lipids 2010, 163, 94–101.
(29) (a) Gierschner, J.; Ehni, M.; Egalhaaf, H.-J.; Medina, B. M.; Beljonne,
D.; Benmansour, H.; Bazan, G. C. J. Chem. Phys. 2005, 123, 144914.
(b) Spano, F. C. Chem. Phys. Lett. 2000, 331, 7–13.
(32) (a) Wiegand, G.; Arribas-Layton, N.; Hillebrandt, H.; Sackmann, E.;
˙
Wagner, P. J. Phys. Chem. B. 2002, 106, 4245–4254. (b) Zebrowska,
(30) (a) Bur, A. J.; Roth, S. C.; Thomas, C. L. ReV. Sci. Instrum. 2000, 71,
1516–1523. (b) Bur, A. J.; Lowry, R. E.; Roth, S. C.; Thomas, C. L.;
Wang, F. W. Macromolecules. 1992, 25, 3503–3510.
A.; Krysin´ski, P.; Łotowski, Z. Bioelectrochemistry 2002, 56, 179–
184.
(33) (a) Huang, W.; Zhang, Z.; Han, X.; Tang, J.; Wang, J.; Dong, S.;
Wang, E. Biophys. J. 2002, 83, 3245–3255. (b) Wu, Z.; Tang, J.;
Cheng, Z.; Yang, X.; Wang, E. Anal. Chem. 2000, 72, 6030–6033.
(31) Smalyukh, I. I.; Shiyanovskii, S. V.; Lavrentovich, O. D. Chem. Phys.
Lett. 2001, 336, 88–96.
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Modification of Optoelectronic Properties of Membranes
A R T I C L E S
Figure 7. (A-C) Results of the cyclic voltammetry blocking experiment that indicate that DSSN+ (A) and DSBN+ (B) facilitate transmembrane electron
transport across insulating sBLMs from the glassy carbon electrode surface to aq ferricyanide. The insulating effect can be seen by comparison of the
prevalent reversible redox couple observed when a bare electrode is employed (A-C, black curves) that is absent in the traces obtained using electrodes
supporting unmodified BLMs (A-C, blue and green curves for DMPC and DPPC sBLMs, respectively). The facilitation of electron transport by DSSN+
and DSBN+ can be seen by the greater current observed when an electrode bearing an sBLM containing 2 mol % of DSSN+ or DSBN+ is employed (red
and purple curves for DMPC and DPPC modified sBLMs, respectively). Little to no transmembrane electron transfer occurs across an sBLM containing 2
mol % of TDA (C, orange and dark red traces for modified DMPC and DPPC sBLMs). (D) Current/potential curves corresponding to DSSN+ and DSBN+
show an oxidation (∼0.55 V vs Ag/AgCl) above that of ferricyanide (∼0.3 V vs Ag/AgCl). These data suggest a charge tunneling process. All traces shown
were obtained using a 200 mV/s scan rate.
membranes were prepared by depositing 3 µL of a 2 mg/mL
lipid stock solution containing either 0 or 2 mol % of DSSN+,
DSBN+, or TDA onto a surface modified electrode, followed
by submerging into pH 7.4 phosphate buffer to allow sBLM
formation. The sBLM containing TDA provides a control
sample to examine whether additional membrane components
and possible structural modifications increase electron transfer
to redox species in solution.
(Figure 7B). This higher current may be attributed to the ability
of DSSN+ to more completely span the membrane structure
due to its more extended conjugation length.
The results obtained from the CV blocking experiments
indicate that transmembrane charge transfer through insulating
sBLMs is facilitated upon incorporation of DSSN+ and
DSBN+. Furthermore, these COEs may act as “molecular wire”
tunneling pathways.
Figure 7 shows the results of the sBLM blocking experiments.
The reversible ferricyanide oxidation (Ep(ox) ) 0.252 V vs SCE)
is observed in the voltammogram obtained by using a bare
glassy carbon electrode (A-C, black curves). The insulating
property of an unmodified sBLM is confirmed by nearly
featureless CV traces (Figure 7A-C, blue and green curves).
Similarly, the CV traces obtained employing an electrode
supporting a BLM containing 2 mol % of TDA is also
featureless (Figure 7C), evidence that the presence of this
component does not perturb the bilayer structure.
CV traces corresponding to sBLMs modified with DSSN+
and DSBN+ (Figure 7A and 7B, purple and red for modified
DMPC and DPPC sBLMs, respectively) display higher currents
with respect to the control experiments, indicating that trans-
membrane electron transfer is facilitated. That observed oxida-
tion occurs at Ep(ox) ) ∼0.3 V vs Ag/AgCl and Ep(red) ) ∼0.1
V vs Ag/AgCl (Figure 7A-C), below the oxidation potential
of DSSN+ and DSBN+ (Figure 7D, Eox ) ∼0.55 V), suggests
a transmembrane tunneling process. The ability of DSSN+ to
better facilitate transmembrane electron transport can be seen
by the greater current response corresponding to runs employing
sBLMs containing DSSN+ (Figure 7A) instead of DSBN+
Interaction with Living Systems. Up to this point, only the
interaction of these COEs with artificial membranes has been
discussed. We now examine interactions with living microor-
ganisms. Our studies focused on yeast due to its application as
an exoelectrogen in microbial fuel cells (see next section). Figure
8 consists of two sets of confocal microscopy fluorescence
images of Baker’s yeast, each stained and imaged in a 100 µM
solution of DSSN+ (Figure 8A) or DSBN+ (Figure 8B) after
1 h of shaking without exchanging the staining media. These
images illustrate the apparent preferential accumulation of the
COEs within cell membranes, in agreement with other reports
in which amphiphilic compounds are introduced to living
systems.34 The lack of background fluorescence is attributed to
the low η of these chromophores when in contact with water.
COE Electron Transport Mediators in Yeast Microbial
Fuel Cells. Microbial fuel cells (MFCs) function on the principle
that electrons from the metabolic cycle of many anaerobic and
facultative organisms known as exoelectrogens can be used to
(34) Reeve, J. E.; Collins, H. A.; Mey, K. D.; Kohl, M. M.; Thorley, K. J.;
Paulsen, O.; Clays, K.; Anderson, H. L. J. Am. Chem. Soc. 2009, 131,
2758–2759.
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Garner et al.
Figure 9. Schematic of the U-tube MFC design40 employed in this study.
The MFC is composed of an anaerobic anode/yeast compartment that is
separated by a proton permeable Nafion membrane from an aerobic cathode
compartment.
that maintains charge balance, much like a salt bridge in a
galvanic cell. The cathode compartment is typically aerobic and
is the site of oxygen reduction, which completes the oxidation-
reduction reaction of the whole cell. Electrons gained from
biofuel oxidation at the anode must flow through a desired
pathway (the lead between the electrodes) to participate in
oxygen reduction at the cathode, thus generating a usable
current.37 In summary, biological fuel is oxidized by the
microorganisms, releasing electrons to the anode and protons
to the solution. Protons diffuse into the cathode compartment,
where they participate in oxygen reduction at the cathode to
form water.38 A schematic of a U-tube type MFC that outlines
the key compartments and components is shown in Figure 9.
Although the MFC concept is relatively simple, much remains
poorly understood about the factors that influence their perfor-
mance at the molecular, cellular, and device level.39
Some exoelectrogens are capable of direct electron transfer
to an electrode, while others require an electron transport
mediator.41 Common mediators employed in bacterial and yeast
fuel cells are diffusion-based redox carriers that are membrane
permeable, such as methylene blue and neutral red.42 These
types of mediators may have undesirable features, such as
cellular uptake that does not favor electron transfer, redox
properties that are not compatible with target species and
substrates,43 and possible diffusion limited kinetics.44 It would
thus be beneficial to modify microorganisms with structural
features that enable transmembrane electron transport and that
do not impede their metabolic function. The accumulated
information on DSSN+ and DSBN+ argues that these molec-
Figure 8. Confocal microscopy fluorescence images of Baker’s yeast
stained with 100 µM DSSN+ (A) and DSBN+ (B) upon excitation at 488
nm. It can be seen that these dyes readily interact with the membranes of
living cells. Note that some cells (that are imaged in the focal plane) possess
the equatorial extinction line that indicates an ordered molecular orientation
within cell membranes.
(37) (a) Ref 35. (b) Walker, A. L.; Walker, C. W., Jr. J. Power Sources
2006, 160, 123–129.
(38) Logan, B. E.; Hamelers, B.; Rozendal, R.; Schro¨der, U.; Keller, J.;
Freguia, S.; Aelterman, P.; Verstraete, W.; Rabaey, K. EnViron. Sci.
Technol. 2006, 40, 5181–5192.
generate a useful electrical current.35 Recent work has shown
that a surprisingly large number of organisms exhibit exoelec-
trogenic character.36 Extracting energy from these organisms
is typically achieved by using a strategy similar to that employed
in a chemical fuel cell. Typically, a community of microorgan-
isms populating a high surface area electrode in an anaerobic
environment oxidize biological fuels such as sugars in an anode
compartment. This anode compartment is commonly separated
from a cathode compartment by a proton exchange membrane
(39) (a) Logan, B. E.; Regan, J. M. EnViron. Sci. Technol. 2006, 40, 5172–
5180. (b) Yazdi-Rismani, H.; Carver, S. M.; Christy, A. D.; Tuovinen,
O. H. J. Power Sources 2008, 180, 683–694. (c) Zhao, F.; Slade,
R. C. T.; Varcoe, J. L. Chem. Soc. ReV. 2009, 38, 1926–1939.
(40) (a) Milliken, C. E.; May, H. D. Appl. Microbiol. Biotechnol. 2007,
73, 1180–1189. (b) Sund, C. J.; Wong, M. S.; Sumner, J. J. Biosens.
Bioelectron. 2009, 24, 3144–3147. (c) Sund, C. J.; McMasters, S.;
Crittenden, S. R.; Harrell, L. E.; Sumner, J. J. Appl. Microbiol.
Biotechnol. 2007, 76, 561–568.
(41) Watanabe, K.; Manefield, M.; Lee, M.; Kouzuma, A. Curr. Opin.
Biotechnol. 2009, 20, 633–641.
(42) Gunawarddena, A.; Fernando, S.; To, F. Int. J. Mol. Sci. 2008, 9, 1893–
1907.
(35) (a) Logan, B. E. Nat. ReV. Microbiol. 2009, 7, 375–381. (b) Schaetzle,
O.; Barriere, F.; Baronian, K. Energy EnViron. Sci. 2008, 1, 607–
620.
(43) Wilkinson, S.; Klar, J.; Applegarth, S. Electroanalysis 2006, 18, 2001–
2007.
(44) (a) Bullen, R. A.; Arnot, T. C.; Lakeman, J. B.; Walsh, F. C. Biosens.
Bioelectron. 2006, 21, 2015–2045. (b) Torres, C. I.; Marcus, A. K.;
Lee, H.-S.; Parameswaran, P.; Krajmalnik-Brown, R.; Rittmann, B. E.
FEMS Microbiol. ReV. 2010, 34, 3–17.
(36) (a) Ref 35. (b) Logan, B. E.; Regan, J. M. Trends Microbiol. 2006,
14, 512–518. (c) Lovley, D. R. Nat. ReV. Microbiol. 2006, 4, 497–
508.
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Figure 10. Plots of yeast MFC voltage production as a function of time
for MFCs containing no mediator (green), 13 µM methylene blue (blue),
190 nM DSBN+ (orange), and 190 nM DSSN+ (red). Voltage values are
obtained by measuring the potential across a 10 kΩ resistor. Note the large
increase in performance afforded by DSSN+ despite a concentration 2 orders
of magnitude lower than that of the common diffusion-based electron
transport mediator methylene blue.
ular species display all the desirable features to accomplish this
modification.
A U-tube type MFC40 (Figure 9) was employed to test and
compare the performance of a series of yeast MFCs employing
nanomolar concentrations of DSSN+ and DSBN+ to the
performance of a series of negative controls with no external
mediator and a series of positive controls employing the
common electron transport mediator methylene blue in micro-
molar concentrations. The details of MFC construction, prepara-
tion, and evaluation are discussed in detail in the Experimental
Section (Supporting Information), but it is important to note
here that MFC performance is evaluated in terms of voltage
instead of current. Voltage readings were obtained by measuring
the potential across a 10 kΩ resistor.38,40b
Figure 10 shows the relative performance of the tested MFCs.
Maximum voltages (Vmax) obtained for the mediator-less MFCs
and those employing 13 µM methylene blue are ∼25 mV (green)
and ∼40 mV (blue), respectively. MFCs employing the shorter
oligoelectrolyte DSBN+ (190 nM) produce slightly greater
voltages (Vmax ) ∼55 mV, orange plots). A notable increase in
performance is afforded by the longer DSSN+ (190 nM), which
Figure 11. (A) Epi-fluorescence image of yeast in growth media containing
25 µM DSSN+ prior to fuel cell inoculation. The yeast is stained such that
the primarily cell membranes can be seen. Inset: example brightfield image
of the sample prior to inoculation. (B) Epi-fluorescence image of yeast
removed from the MFC on day 4 of operation. Although emission is not
very intense, these cells contain DSSN+ despite the large dilution upon
inoculation (to a nanomolar regime) and exponential increase in the number
of total cells over the course of 4 days. Inset: Brightfield image that matches
the day 4 epi-fluorescence image. Note: λex ) ∼480 nm provided by a Hg
lamp and the proper filter. This excitation range is out of the range of
absorbance of the fluorescent amino acids tryptophan, tyrosine, and
phenylalanine (λabs(Trp, Tyr, Phe) ) ∼220-320 nm), thus any observed
emission is attributed to DSSN+.
resulted in a ∼5-fold increase in voltage production (Vmax
)
∼200 mV) compared to methylene blue, despite being 2 orders
of magnitude lower in concentration. Furthermore, the difference
in performance between MFCs employing DSSN+ and DSBN+
is consistent with the difference in transmembrane electron
transport properties observed in the cyclic voltammetry
experiments.
The ability of DSBN+ and DSSN+ to be passed on to or
exist within cell membranes of subsequent yeast generations
born over the course of MFC operation was also investigated.
Aliquots of an MFC using the mediator DSSN+ were taken
before MFC inoculation and after 4 days of MFC operation.
Cells from each of these aliquots were imaged using a confocal
microscope in epi-fluorescence mode and brightfield mode.
Figure 11A corresponds to the yeast sample containing 25 µM
DSSN+ that was used to inoculate the MFC series correspond-
ing to this mediator. This particular staining/cell membrane
modification procedure consisted of light shaking for 3 h in the
presence of micromolar oligomer concentrations. The external
cell membranes are observable due to emission of DSSN+.
Figure 11B is an epi-fluorescence and brightfield image (inset,
lower left) of a small group of yeast cells removed from the
same MFC on the fourth day of operation. These images were
collected using the same setup as the images in Figure 11A
and demonstrate the presence of this mediator in later cell
generations. The emission is much less intense, an effect that
is not surprising considering the exponentially greater number
of cells present after 4 days of cell reproduction. This result
indicates that DSSN+ can be passed on to daughter cells either
during budding reproduction (implying that some cellular
uptake, into cytoplasm, occurs) and/or by an equilibrium
between membrane embedded and aqueous DSSN+.
Conclusion
To summarize, we have demonstrated the incorporation of
DSBN+ and DSSN+ within membranes. Insertion of the two
oligoelectrolytes changes the optical and electronic properties
of the resulting modified membranes. Additionally, the solva-
tochromic features of these chromophores can be used to probe
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A R T I C L E S
Garner et al.
their location and the polarity of the medium. The size and
relative positions of the charges and hydrophobic regions of
DSBN+ and DSSN+ led us to propose the intuitive, ordered
molecular organization shown in Figure 1. This supramolecular
arrangement, in which the rigid π-conjugated region lies within
the inner membrane oriented such that the long molecular axis
is normal to the bilayer plane, was confirmed by confocal
microscopy using a polarized excitation source. We find two
important consequences of integrating molecules such as
DSBN+ and DSSN+. First, the optical properties and affinity
of these chromophores for both synthetic and biological
membranes allow for effective fluorescence imaging of living
organisms. It is also worth noting that large two-photon cross
sections imply that these compounds can be applied to three-
dimensional imaging of biological samples. Second, all the
evidence accumulated thus far is consistent with facilitation of
cross-membrane charge transport by these conjugated oligo-
electrolytes. It is tempting to hypothesize here that the mech-
anism is charge tunneling by virtue of the rigid π-structure. In
other words, DSBN+ and DSSN+ do not serve as simple
shuttles. Observations that (a) there is affinity for the membranes
of living organisms and (b) that transmembrane electron transfer
is facilitated led us to consider that the current generation in
microbial fuel cells could be improved by the addition of
DSBN+ and DSSN+. We envisioned their function as media-
tors that transfer electrons from within the organism to the
electrode. This idea was confirmed by the nearly 5-fold increase
in performance of yeast microbial fuel cells afforded by using
DSSN+ compared to that of the common diffusion-based
electron transport mediator methylene blue, despite 2 orders of
magnitude difference in concentration. These findings not only
are of potential impact within the scope of contacting micro-
organisms with electrode surfaces but also open the possibilities
of intercellular electronic communication.
Acknowledgment. The authors thank the Institute for Col-
laborative Biotechnologies (W911F-09-D-0001), Air Force Office
of Scientific Research (FA9550-08-1-0248), and the National
Science Foundation (DMR0606414, and subcontract from Caltech:
CMMI-0730689) for financial support; Mr. Htet Khant for cryo-
TEM imaging; Dr. Alexander Mikhailovsky for assistance with
photophysical measurements; Dr. Joshua M. Kogot for cell cultures
and helpful consultations; and Dr. Myung Chul (MC) Choi for
stimulating discussions that contributed to this work.
Supporting Information Available: Experimental details for
synthesis, vesicle and supported lipid bilayer preparation, cyclic
voltammetry measurements, cell staining, confocal fluorescence
and cryo-TEM imaging, microbial fuel cell preparation, instru-
mentation, and complete reference 14. This material is available
free of charge via the Internet at http://pubs.acs.org.
JA1016156
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