Research Article
www.acsami.org
M13 Virus-Incorporated Biotemplates on Electrode Surfaces To
Nucleate Metal Nanostructures by Electrodeposition
Shanmugam Manivannan,† Inhak Kang,† Yeji Seo,† Hyo-Eon Jin,‡,§ Seung-Wuk Lee,‡
and Kyuwon Kim*,†
†
Electrochemistry Laboratory for Sensors & Energy (ELSE) Department of Chemistry, Incheon National University, Incheon
406-772, Republic of Korea
‡
Department of Bioengineering, University of California, Berkeley, California 94720, United States
S Supporting Information
*
ABSTRACT: We report a virus-incorporated biological template (biotemplate) on electrode surfaces and its use in electrochemical nucleation of metal
nanocomposites as an electrocatalytic material for energy applications. The
biotemplate was developed with M13 virus (M13) incorporated in a silicate
sol−gel matrix as a scaffold to nucleate Au−Pt alloy nanostructures by
electrodeposition, together with reduced graphene oxide (rGO). The phage
when engineered with Y3E peptides could nucleate Au−Pt alloy nanostructures, which ensured adequate packing density, simultaneous stabilization of
rGO, and a significantly increased electrochemically active surface area.
Investigation of the electrocatalytic activity of the resulting sol−gel composite
catalyst toward methanol oxidation in an alkaline medium showed that this
catalyst had mass activity greater than that of the biotemplate containing wildtype M13 and that of monometallic Pt and other Au−Pt nanostructures with
different compositions and supports. M13 in the nanocomposite materials
provided a close contact between the Au−Pt alloy nanostructures and rGO. In addition, it facilitated the availability of an OH−rich environment to the catalyst. As a result, efficient electron transfer and a synergistic catalytic effect of the Au and Pt in the
alloy nanostructures toward methanol oxidation were observed. Our nanocomposite synthesis on the novel biotemplate and its
application might be useful for developing novel clean and green energy-generating and energy-storage materials.
KEYWORDS: biotemplate, electrodeposition, M13 virus, Au−Pt alloy, methanol oxidation
1. INTRODUCTION
Developing clean and green renewable energy sources is one of
the most critical needs for mankind. Direct methanol fuel cells
(DMFCs) have attracted wide attention as power sources for
portable electronic devices and fuel cell vehicles owing to their
quick start-up, compactness and lightweight nature, high power
density, and simplicity.1,2 Moreover, DMFCs operating in
alkaline media show advantageous features such as improved
reaction kinetics and a less corrosive environment for catalysts.
DMFCs in alkaline media can also operate at relatively low
potentials.2−5 However, wide application of the DMFC still
requires addressing multiple issues including its high cost, low
durability of its platinum (Pt) catalyst, and the relatively easy
passage of methanol through membranes.5−7 To decrease the
amount of Pt that needs to be loaded into DMFCs and to
increase their efficiency levels, a secondary metal such as gold
(Au),2 ruthenium (Ru),8,9 copper (Cu),10 and palladium (Pd)11
has been introduced. Pt-based bimetallic (alloy and core−shell)
catalysts have been explored to develop DMFCs that operate in
alkaline media and represent an alternative way to minimize the
amount of pure Pt-based catalyst loaded.2,12,13 Furthermore,
considerable efforts have been devoted to employing Au−Pt
© 2017 American Chemical Society
alloy nanostructures to take advantage of the synergistic effect
between Au and Pt that had already been detemined.14,15
Reduced graphene oxide (rGO) is, due to its unique electronic
and chemical properties arising from its 2D structure,16 a
promising support for loaded metallic nanostructures in fuel
cell applications,2,17−22 and it is crucial to prevent its individual
sheets from aggregating. In this regard, several approaches have
been used to prevent aggregation between individual rGO
sheets using biomolecules such as DNA,23 proteins,24 and
genetically modified viruses.25−27 The resulting biomolecule−
rGO composites have found significant applications in various
biosensors28−30 and energy devices.31,32 In addition, substantial
research is being pursued to develop rGO as support materials
for metal nanostructures.12,13,18 Such a combination can
provide a highly active surface area, enhanced catalytic activity,
and durability for long durations. In this regard, there is a great
need to develop a synthesis of composites of Au−Pt alloy
nanostructures with low-density packing and suitable solid
Received: May 10, 2017
Accepted: September 5, 2017
Published: September 5, 2017
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Figure 1. (a) Schematic illustration of M13wild, M13Y3E, and M134E. (b) Schematic representation of SSG/M13Y3E/rGO/(Au−Pt) catalyst
fabrication.
DMFCs. On the other hand, biomineralization through
electrodeposition could secure electrical pathways by connecting the nanostructures on M13. Therefore, M13 viruses with
nanostructures electrodeposited on their surfaces might provide
excellent biotemplates to synthesize DMFC materials with
engineered catalysts. To the best of our knowledge, electrodeposition-based biomineralization on viruses-modified surfaces
has never been reported.
Here we report a novel fabrication of a virus-templated
silicate sol−gel matrix (SSG) with an rGO dispersion to
nucleate Au−Pt alloy nanostructures by electrodeposition. We
demonstrated the use of the nanostructures as efficient
electrocatalysts of the methanol oxidation reaction (MOR)
for DMFC applications. The density of the nanostructures at
the electrode surface was controlled with the incorporated
viruses and its genetically modified peptide surfaces, which
offered a convenient way to synthesize relatively less densely
packed metal nanostructures on the biotemplates. We
demonstrated the density and shape of the alloy nanostructures
that affected the MOR performance to be highly influenced by
the peptide sequences on the major coat protein of the virus.
Interestingly, the incorporated viruses in the biotemplate did
not change the electronic conductivity of the template with
rGO and the alloy nanostructures. In addition, the M13 virus
infusion improved the colloidal stability of the rGO and
contributed to the generation of an OH−-rich environment for
an efficient MOR.
supports to ensure both the performance and durability of
DMFCs.
With the recent advent of virus-based bionanotechnology,
various viruses have been utilized as biological templates
(biotemplates) to synthesize functional nanomaterials. Functional nanostructures resulting from virus-based synthesis have
attracted alot of attention as novel nanoscale platforms due to
their advantageous characteristics including their monodispersity, multiple valences, tunable structural features, biocompatibility, stability, and low-cost production.31−33 The M13 virus
(M13) is particularly interesting. This virus, which infects
bacterial host cells, is 880 nm in length and 6.6 nm in diameter,
consists of a single-stranded DNA enclosed by 2700 identical
copies of major coat protein pVIII and capped with five copies
of four different minor coat proteins (pIX, pVII, pVI, and pIII)
at the ends,34 and is stable in a wide range of pH, temperature,
and organic solvent conditions. Moreover, M13 is considered
not harmful in humans and animals. Hence, it has been applied
to various biomedical applications such as drug and gene
delivery as well as tissue engineering.35,36 In addition, the virus
can be engineered to have specific affinity toward selected metal
precursors by having certain of its genes modified, with inserted
DNA sequences.37−40 Recently, the Belcher group and other
research groups have made significant contributions toward the
synthesis of various nanostructures based on engineered M13
for various applications including semiconductor synthesis,
energy storage, electric generators, and tissue engineering
materials.31,32,41−44 The resulting M13 engineered with specific
peptides allows them to bind and organize the precursors and
nucleate metallic nanostructures as biotemplates. Biomineralization of metal nanostructures using peptides expressed on the
virus surfaces has proven to be efficient and highly costeffective. On the one hand, biomineralization carried out using
M13 by typical chemical deposition could lead to a lack of
electrical pathways in electrochemical applications such as
2. EXPERIMENTAL SECTION
2.1. Materials. Graphite (powder <20 μm), gold(III) chloride
hydrate (HAuCl4·3H2O), chloroplatinic acid hexahydrate (H2PtCl6·
6 H 2 O ) , 5% N a fi on, a s co rb ic ac id (A A ) , an d N 1 - ( 3 trimethoxysilylpropyl)diethylenetriamine (silane monomer used to
prepare the SSG; silicate sol−gel matrix) were received from SigmaAldrich. The commercial Pt/C catalyst (20% Pt/C) was received from
Alfa Aesar. Methanol (MeOH) was obtained from DaeJung chemicals.
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Figure 2. (A to C1) SEM images of (A, A1) ITO/SSG/M13Y3E/rGO/Au, (B, B1) ITO/SSG/M13Y3E/rGO/Pt, and (C, C1) ITO/SSG/M13Y3E/
rGO/(Au−Pt) electrodes at different magnifications. (D) EDX analysis of the ITO/SSG/M13Y3E/rGO/(Au−Pt) electrode.
Indium tin oxide (ITO, dimension 2 × 1 cm) and its modified forms
were used as working electrodes. Pt wire was used as a counter
electrode, and Ag/AgCl (in 3 M NaCl solution) was used as a
reference electrode. All the electrochemical experiments were
conducted in a single-compartment three-electrode cell using an
Ivium Technologies electrochemical workstation. Nitrogen (N2) was
bubbled for 30 min prior to each experiment.
2.2. Wild-Type M13 Virus (M13Wild) Preparation. M13Wild was
grown and purified by following standard biochemical protocols
described elsewhere.38,45,46 Briefly, one colony of E. coli XL-1 blue was
grown in 3 mL of LB media to mid log phase (E. coli XL-1 blue
culture) and infected with 10 μL of M13Wild. The culture was
incubated at 37 °C with shaking for 12 h and then centrifuged to
remove E. coli. The M13Wild was collected by PEG/NaCl (20% PEG
and 2.5 mol/L NaCl) precipitation and reconstituted in Tris-buffered
saline (10 mM). The typical yield was ∼20 mg of M13Wild per liter.
The final concentration was determined spectrophotometrically using
an extinction coefficient of 3.84 cm2/mg at 269 nm.45,47
2.3. Engineered Phage with YEEE (M13Y3E) and 4E (M134E)
Peptide and Their Preparation. Attachment of Au binding peptides
at the major coat protein (Gene VIII) of M13 was reported
previously.48 We prepared the engineered phage with YEEE
(M13Y3E) and 4E (M134E) peptides. Briefly, three primers were
designed to insert M13Y3E and M134E into the gene VIII protein: 5′ATATATCTGCAGNKTAYGAAGAGGAANNKGATCCCGCAAAAGCGGCCTTTAACTCCC-3′ (Y3E), 5′-ATATATCTGCAGAAGAGGAAGAGCCCGCAAAAGCGGCCTTTAACTCCC-3′
(4E), and 5′-GGAAGCTGCAGCGAAAGACAGCATCGGAACGAGG-3′ (linearization primer). To engineer the M13Y3E and
M134E phages, the inverse polymerase chain reaction (PCR) cloning
method was performed using the above-mentioned primers (the
linearization primer with M13Y3E and M134E primers, respectively).
The PCR product was purified and digested with PstI, and then
recircularized by ligation. The ligated DNA vector was transformed
into E. coli XL1-blue. Phage plaques were verified by DNA sequencing.
Furthermore, we have amplified the M13Y3E and M134E phages for our
experiments, and the methods were the same as that used for M13Wild
as mentioned in section 2.2a.45,47 Figure 1(a) demonstrates the
structural features of the three different M13 used in this study.
2.4. Synthesis of SSG/M13/rGO Composite. A homogeneous 1
mM SSG solution was prepared49 by adding 10 μL of 1 M SSG silane
monomer into 10 mL of aqueous solution under vigorous stirring, and
stirring was continued for another 60 min. Graphene oxide (GO) was
prepared from graphite by a modified Hummers method.50 The
detailed synthetic procedure of rGO is found elsewhere.51 Briefly, 20
mL of GO (2 mg/mL) was ultrasonicated for 2 h to yield a yellowish
brown dispersion. Subsequently, 80 mg of AA was added to the
reaction mixture. The mixture was stirred at room temperature for 48
h. The resultant black dispersion was centrifuged and washed with
water five times and then dried in a hot air oven. To prepare SSG/
rGO composite, aliquots of rGO (0.1 mg/mL) were added to the SSG
solution and vigorously stirred for 1 h, and the obtained homogeneous
solution was stored in a refrigerator for further use. To prepare the
SSG/M13/rGO composite, an aliquot of M13Wild, M13Y3E, and M134E
(0.1 mg/mL, respectively) was added to the SSG/rGO solution
accompanied by stirring for 1 h, and the obtained homogeneous
solution was stored in a refrigerator for further use.
2.5. Fabrication of Modified Electrodes. A known amount (50
μL) of SSG or SSG/rGO or SSG/M13/rGO was drop-casted on a
cleaned ITO electrode surface and allowed to dry in an incubator at 37
°C for 2 h. The dried electrode was soaked for 10 min in an electrolyte
solution (mixture of 1.5 mM H2PtCl6 and HAuCl4, 3 mM H2PtCl6,
and 3 mM HAuCl4 in 0.5 M H2SO4 for depositing Au−Pt, Pt, and Au
nanostructures, respectively), Au−Pt or Pt or Au nanostructures were
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Figure 3. SEM images of (A) ITO/SSG/M13Y3E/rGO/(Au−Pt), (B) ITO/SSG/M13Wild/rGO/(Au−Pt), and (C) ITO/SSG/M134E/rGO/(Au−
Pt) electrodes.
electrodeposited by applying a potential of −0.2 V (Ag/AgCl) for 500
s. Fabricated electrodes were denoted as ITO/SSG/(Au−Pt) or ITO/
SSG/rGO/(Au−Pt) or ITO/SSG/M13/rGO/(Au−Pt). Current was
monitored during deposition, and corresponding charge was used to
estimate specific mass (M) of Pt as follows; from the EDAX analysis,
percent composition of Au and Pt were derived and the same percent
of charge for Pt from the total charge was used to calculate the mass of
Pt using eq 1:
M = Q × MW/nFA
rGO. As shown in Figure 1(b), at the electrode modified with
the SSG/M13/rGO composite, metal precursors were observed
to be nucleated by one of the M13 components that had been
functionalized with Au-binding peptide sequences. This
observation indicated that these sequences provided specific
nucleation sites and enhanced the interaction between rGO and
the electrodeposited Au−Pt nanostructures.
Scanning electron microscopy (SEM) was used to analyze
the morphology of the differently modified electrodes, and the
SEM images are shown in Figures 2 and S1−S7. Figure 2 shows
the electrodeposited Au (Figure 2(A,A1)), Pt (Figure 2(B,B1))
and Au−Pt (Figure 2(C,C1)) nanostructures at the ITO/SSG/
M13Y3E/rGO electrode. The Au−Pt nanostructures were
observed to be spherical and well separated. In contrast, the
Au nanostructures showed irregular shapes with randomly
grown dendritic structures, and we suggest that this nucleation
and growth were directed by the Au-binding regions of
M13Y3E.54 Pt nanostructures typically showed an interconnected porous base with a spherical top and were observed at
the ITO/SSG/M13Y3E/rGO/Pt electrode (Figure 2B1).
Elemental mappings from energy-dispersive X-ray (EDX)
analyses of the ITO/SSG/M13Y3E/rGO/(Au−Pt) (Figure
2D), ITO/SSG/M13Y3E/rGO/Au (Figure S2), and ITO/
SSG/M13Y3E/rGO/Pt (Figure S2) electrodes showed that Au
and Pt were found in the same nanostructures, which indicated
that the nanostructures were composed of an Au and Pt alloy
state. Furthermore, the monometallic Au and Pt nanostructures
(Figure S2) were identified in their elemental forms, and there
was no salt contamination. In addition, to analyze the role of
each component of the SSG/M13Y3E/rGO composite in the
nucleation and growth, the Au−Pt nanostructures were
electrodeposited on different supports (ITO (Figure S3(A to
A2), ITO/SSG (Figure S3(B to B2), ITO/SSG/M13Y3E
(Figure S3(C to C2), and ITO/SSG/rGO (Figure S3(D to
D2)), and they were compared with the ITO/SSG/M13Y3E/
rGO/(Au−Pt) electrode. Interestingly, highly dense growth of
Au−Pt alloy nanostructures was indicated to have occurred at
the ITO/SSG/rGO electrode (Figure S3(D to D2)). The
formation of the Au−Pt nanostructure may have been due to
the nucleating ability of rGO, specifically due to the functional
groups (−OH and COO−) of rGO, which was further
supported by SEM and EDX analyses, shown in Figure S4, of
the same ITO/SSG/rGO electrode shown in Figure S3D.
These analyses indicated a greater density of Au and Pt for the
Au−Pt alloy nanostructures with rGO than without rGO.
Furthermore, Au−Pt alloy nanostructures at three different
compositions were electrodeposited on the ITO/SSG/M13Y3E/
rGO electrode, and the results of SEM and EDX analyses of
this electrodeposition are summarized in Figure S5. For
comparison, Au−Pt nanostructures were also deposited on
(1)
where M is specific mass after electrodeposition, Q is charge consumed
for electrodeposition, MW is the molecular mass of Pt, n is number of
electrons (4) transferred for electrodeposition, F is Faraday constant,
and A is the geometrical area (0.44 cm2) of the electrode (O ring).
The mass of Pt was used to calculate the electrochemically active
surface area (ECSA) and normalize the current to obtain current
density plots.
2.6. Electrochemical Studies. The ECSA of modified electrodes
were determined using the curve-fitting tool of the MATLAB software
package by integrating Q of hydrogen adsorption curve after
eliminating nonfaradaic current, which was recorded in 0.5 M
H2SO4 solution (saturated with nitrogen) scanned at 50 mV/s from
−0.4 to 1.2 V. The electrocatalytic performance of the modified
electrodes toward MOR was studied by recording cyclic voltammograms (CV) in 0.1 M KOH and 0.1 M CH3OH solution scanned at 50
mV/s from −1 to 0.6 V. The stability of the catalysts was evaluated by
a continuous cycling test and an amperometric i−t curve technique
under an applied potential of −0.3 V for 1500 s. Also electrochemical
durability (in terms of ECSA values) of catalysts was measured by
recording the potential CV cycles.
3. RESULTS AND DISCUSSION
3.1. Surface Characterization of the Modified Electrodes. Employing an appropriate support is essential to prevent
the aggregation of nanostructures and hence to be able to
explore their chemical properties, particularly their active
surface areas and catalytic efficiency levels. Here we had
selected the SSG/M13/rGO composite as a biotemplate onto
which Au−Pt nanostructures were electrodeposited. In this
biotemplate, SSG acted as a solid support and provided a matrix
support for the immobilization of both M13 and rGO.52,53
Surface morphology of the SSG was analyzed (Figure S1) after
incorporating M13, and porous structures due to M13
incorporation were clearly visible. Such porous structures
could promote the efficient nucleation of metal nanostructures
and mass transport at the electrode surface. While the
composite made from rGO has often been observed to suffer
severe aggregation and resulting poor water dispersity, rGO
from the SSG/M13/rGO composite was found to be highly
stable in aqueous solutions and to show a highly stable aqueous
dispersion, apparently as a result of the 3D matrix of the SSG
and noncovalent binding between the engineered M13 and
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due to the interdiffusion of Pt that altered the size of the Au
unit cell in the Au−Pt alloy nanostructures.
Figure S8 shows an X-ray photoelectron spectroscopy (XPS)
analysis of the ITO/SSG/M13Y3E/rGO/(Au−Pt) electrode.
The survey spectrum revealed the presence of Pt, Au, Si, C, N,
and O at this modified electrode. In brief, the C 1s spectrum
(Figure S8B) was fitted to three components, corresponding to
carbon atoms bound in the in-plane sp2 carbon: CC (284.4
eV), C−OH (285.2 and 285.8 eV), C−N (286.4 eV), and C−O
(287.4 eV) of rGO. The N 1s spectrum was fitted with one
component at 400.1 eV, indicating the presence of NH2 groups
of SSG and M13Y3E (from Au-binding peptides). As shown in
Figure S8D, the electrode also yielded an XPS doublet peak for
zero-valence Pt at 70.9 eV (Pt 4f7/2) at 74.2 eV (Pt 4f5/2),
and another for zero-valence Au57 at 84.3 eV (Au 4f7/2) and
87.7 eV (Au 4f5/2), which confirmed that both Pt and Au were
in their zero oxidation states. These results demonstrated the
successful deposition of Pt and Au from its precursor to form
Au−Pt nanostructures on the ITO/SSG/M13Y3E/rGO electrode and that Pt and Au were mixed with each other on the
atomic level with no phase separation.
3.2. Electrocatalytic Studies. The MOR was used as a
model system to explore the electrocatalytic activity of the
SSG/M13Y3E/rGO/(Au−Pt) catalyst and to compare it with
the activities of the catalysts containing M13Wild and M134E,
with corresponding monometallic catalysts, and with catalysts
with different supports and compositions. The results are
summarized in Figures 5−8 and S9−S12. As mentioned above,
DMFCs operating in alkaline conditions have advantages such
as improved reaction kinetics and an environment less corrosive
to the electrodes.53,58,59 Recent studies have shown that Au−Pt
and other bimetallic electrocatalysts to be attractive catalysts for
methanol oxidation in an alkaline medium and to constitute an
important alternative to reduce the usage of precious
monometallic Pt- or Pd-based catalysts for DMFCs.59−61 In
addition, as also mentioned above, much effort has been
devoted to employing Au−Pt-based core−shell/alloy nanostructures to take advantage of the synergetic effect between Au
and Pt that had already been determined. Thus, rational
syntheses of Au−Pt alloy nanostructures with suitable solid
supports and having very high ECSA values are in great
demand because they are very promising for enhancing
performance of catalysts for the MOR. Therefore, we expect
our engineered M13-driven Au−Pt alloy nanostructures when
closely interfacing with rGO in the SSG/M13Y3E/rGO/(Au−
Pt) electrode to be of use as an advanced electrocatalyst. To
analyze the electrocatalytic ability of the aforementioned
catalyst, both ECSA (m2/gPt) and specific mass (mgPt/cm2)
values were derived and are shown in Table 1. The ECSA
measurement was taken by carrying out CV experiments in 0.5
M H2SO4 (Figures 5A, 6A, 7A, and S10A) using eq 2:
the SSG/M13Wild/rGO composite-modified and SSG/M134E/
rGO composite-modified electrodes, and the results of SEM
and EDX analyses of these electrodepositions are shown in
Figures 3, S6, and S7. The Au−Pt nanostructures grown from
M13Wild and M134E were observed to be, respectively, more
densely packed than and as similarly packed as those grown
from M13Y3E. This observation indicated the ability of
engineered M13 (M13Y3E and M134E) to yield a less dense
packing of Au−Pt nanostructures at the SSG/M13/rGO
biotemplate (Figure 3). This trend clearly demonstrated that
the tyrosine moieties at the major coat protein of M13Y3E
actively contributed to the nucleation and growth of the
nanostructures. In general, polymerized catechol compounds
behave like adherents and have affinity toward inorganic
materials.55 The role of tyrosine in the MOR is discussed
below.
X-ray diffraction (XRD) patterns of the modified electrodes
are shown in Figure 4. For all of the electrodes, XRD peaks at
Figure 4. XRD patterns of (a) ITO/SSG/M13Y3E/rGO, (b) ITO/
SSG/M13Y3E/rGO/Au, (c) ITO/SSG/M13Y3E/rGO/Pt, and (d)
ITO/SSG/M13Y3E/rGO/(Au−Pt) electrodes.
30.5°, 35.3°, 50.9°, and 60.4° (JCPDS card number 39-1058)
were observed; these peaks are derived from the ITO electrode
and were therefore neglected in the analysis. In addition, the
electrodes yielded a peak at 21.2°, which was due to the
presence of rGO.56 SSG/M13Y3E/rGO/Au (Figure 4(b)) also
yielded XRD peaks at 38.1°, 44.3°, 64.6°, and 77.6°, which we
attributed to the presence of Au with an fcc structure with
lattice planes (1 1 1), (2 0 0), (2 2 0), and (3 1 1), respectively
(JCPDS card number 65-2870). For SSG/M13Y3E/rGO/Pt,
peaks were observed at 39.9°, 46.5°, and 67.9° (JCPDS card
number 04-0802), which were attributed to the (1 1 1), (2 0 0),
and (2 2 0) crystalline planes, respectively, of the fcc crystal
structure of Pt (Figure 4(c)). The XRD pattern of the SSG/
M13Y3E/rGO/(Au−Pt) electrode showed peaks at 38.6°, 45.3°,
65.9°, and 78.9°, which were assigned to the (1 1 1), (2 0 0), (2
2 0), and (3 1 1) planes of the Au−Pt nanostructures.
Comparison of the Au (Figure 4(b)) and Pt (Figure 4(c))
patterns with the Au−Pt pattern (Figure 4(d)) clearly show
shifts in the peaks derived from the (1 1 1), (2 0 0), (2 2 0),
and (3 1 1) planes of Au from 38.1 to 38.6°, 44.3° to 45.3°,
64.6° to 65.9°, and 77.6° to 78.9°, respectively, from the
monometallic Au to the Au−Pt alloy nanostructures, which was
ECSA = QH/QH*
(2)
In eq 2, QH is the charge collected from the hydrogen
adsorption region after a double-layer correction and QH* is
the standard value associated with the adsorption of a hydrogen
monolayer at a polycrystalline Pt surface. QH was determined
using the curve-fitting tool of the MATLAB software package,
and a value of 210 μC cm−2 was used for QH*.62 The obtained
ECSA values are summarized in Table 1 To obtain the mass
activities, the percent compositions of Pt throughout the
bimetallic Au−Pt alloy nanostructures with different supports
and precursor concentrations were derived from the SEM-EDX
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Figure 5. CVs of (a) ITO/(Au−Pt), (b) ITO/SSG/(Au−Pt), (c) ITO/SSG/M13Y3E/(Au−Pt), (d) ITO/SSG/rGO/(Au−Pt), and (e) ITO/SSG/
M13Y3E/rGO/(Au−Pt) electrodes in (A) 0.5 M H2SO4 and in (B) 0.1 M CH3OH and 0.1 M KOH at a scan rate of 50 mV/s. (C) Amperometric i−t
curves observed for A(a−e) in 0.1 M CH3OH and 0.1 M KOH at an applied potential of −0.3 V. (D) Comparison of ITO/SSG/M13Y3E/rGO/
(Au−Pt) electrode in the (a) absence and (b) presence of 0.1 M CH3OH in 0.1 M KOH at a scan rate of 50 mV/s.
Table 1. Electrochemical Parameters of MOR Derived from Various Modified Electrodes
modified electrodes
specific mass
(mgPt/cm2)
ECSA
(cm2)
ECSA
(m2/gPt)
onset potential
(V)
anodic peak
potential (V)
mass activity
(A/mgPt)
If/Ib
ITO/(Au62.5−Pt37.5)
ITO/SSG/(Au63.3−Pt36.7)
ITO/SSG/M13Y3E/(Au62.3−Pt37.7)
ITO/SSG/rGO/(Au60.3−Pt39.7)
ITO/SSG/M13Y3E/rGO/(Au64−Pt36)
ITO/SSG/M13Y3E/rGO/(Au58.9−Pt41.1)
ITO/SSG/M13Y3E/rGO/(Au57.7−Pt42.3)
ITO/SSG/M13wild/rGO/(Au57.7−Pt42.3)
ITO/SSG/M134E/rGO/(Au61.1−Pt38.9)
ITO/SSG/M13Y3E/rGO/Au100
ITO/SSG/M13Y3E/rGO/Pt100
commercial Pt/C
0.00532
0.00437
0.00463
0.00322
0.00210
0.00107
0.00090
0.00269
0.00230
0.01333
0.01006
0.0051
1.18
1.36
1.38
1.31
0.36
1.63
0.44
1.22
1.08
0.25
2.14
-
22.19
31.06
29.93
40.81
17.2
153.23
48.89
45.53
47.20
1.88
21.30
-
−0.590
−0.570
−0.574
−0.644
−0.345
−0.642
−0.525
−0.516
−0.479
−0.570
−0.563
−0.010
0.238
0.038
0.296
0.003
−0.082
0.021
0.061
−0.102
0.152
−0.563
0.1278
0.9798
0.2760
1.3093
0.1336
1.5428
1.0470
0.6840
0.7790
0.4060
0.2389
10.10
14.94
11.13
10.66
5.59
10.37
6.04
3.81
8.07
Figure 6. (A, B) CVs of (a) ITO/SSG/M13Y3E/rGO/Au, (b) ITO/SSG/M13Y3E/rGO/Pt and (c) ITO/SSG/M13Y3E/rGO/(Au−Pt) electrodes in
(A) 0.5 M H2SO4 and in (C) 0.1 M CH3OH and 0.1 M KOH at a scan rate of 50 mV/s. (C) Amperometric i−t curves observed for A(b,c)
electrodes in 0.1 M CH3OH and 0.1 M KOH at an applied potential of −0.3 V.
(Au58.9−Pt41.1) catalyst (made using a concentration of 1.5 mM
for each precursor) was determined to be 153.23 m2/gPt, a
analysis and are indicated in the subscript of the name of each
catalyst (Table 1). The ECSA for the SSG/M13Y3E/rGO/
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Figure 7. CVs obtained at (a) ITO/SSG/M13Wild/rGO/(Au−Pt), (b) ITO/SSG/M134E/rGO/(Au−Pt), and (c) ITO/SSG/M13Y3E/rGO/(Au−
Pt) electrodes in (A) 0.5 M H2SO4 and in (B) 0.1 M CH3OH and 0.1 M KOH at the scan rate of 50 mV/s.
Figure 8. (A) ECSAs, (B) ECSAs per Pt gram, and (C) mass activities of mono- and bimetallic catalysts.
SSG/M13Y3E/rGO/(Au58.9−Pt41.1) catalyst in the absence (a)
and presence (b) of 0.1 M CH3OH. We ignored the first 20
cycles of each MOR experiment in our analyses so that the
analyses only consider the MOR at equilibrium conditions. As
shown in Figure 5B and in Table 1, the mass activity (current
normalized with Pt mass) of the SSG/M13Y3E/rGO/(Au58.9−
Pt41.1) catalyst was higher (1.5428 A/mgPt) and the anodic peak
potential was negatively shifted by about 0.38 V, indicating the
advantages of the SSG/M13Y3E/rGO support for the MOR,
compared to other supports. Furthermore, comparison of the
mass activity and ECSA (1.5428 A/mgPt and 153.23 m2/gPt,
respectively) of the SSG/M13Y3E/rGO/(Au58.9−Pt41.1) catalyst
with those of the monometallic SSG/M13Y3E/rGO/Au (no
reaction and 1.88 m2/g Au) and SSG/M13Y3E/rGO/Pt (0.406
A/mgPt and 21.30 m2/gPt) catalysts indicated that both mass
activity and ECSA parameters were not in rational agreement,
and the mass activity of the SSG/M13Y3E/rGO/(Au58.9−Pt41.1)
catalyst was about 3.8 times higher than that of the SSG/
M13Y3E/rGO/Pt catalyst, showing the inclusion of both metals
to be very important for enhancing the MOR and implying a
synergistic electrocatalytic effect (Figure 6B). In addition, the
presence of SSG and rGO (Figure 5B(b,d)) considerably
enhanced the mass activities (Table 1) and indicated their
contribution at the SSG/M13Y3E/rGO/(Au58.9−Pt41.1) catalyst.
Furthermore, at the bare ITO, ITO/SSG, ITO/SSG/M13Y3E,
ITO/SSG/rGO, and ITO/SSG/M13Y3E/rGO electrodes (Figure S9), no considerable catalytic activities toward the MOR
were observed. To evaluate the stability of both the mono- and
bimetallic Pt-based catalysts toward the MOR, their current−
time responses were monitored in a mixture of 0.1 M CH3OH
and 0.1 M KOH at −0.3 V for 1500 s and are shown in Figures
5C and 6C. The results were consistent with the CV data. As
value markedly higher than the ECSA values of other catalysts
(Table 1). The ECSA of the SSG/M13Y3E/rGO/Pt catalyst was
determined to be 2.14 cm2, noteworthy by being considerably
greater, in fact 4.86 times greater, than that of its geometrical
area (0.44 cm2); this discrepancy was attributed to its
interconnected porous base with a spherical top structure
(Figure 2B1). Characteristic Au and Pt redox behaviors were
observed in the CVs of our catalysts (Figures 5A and 6A), in
particular in that of the SSG/M13Y3E/rGO/(Au58.9−Pt41.1)
catalyst (Figure 6A(c)), which showed the characteristic
hydrogen adsorption (forward scan) and desorption (reverse
scan) peaks between −0.2 and 0 V. Moreover, this voltammogram exhibited a single oxidation peak for Au−Pt alloy
nanostructures at 1.19 V in the forward scan, and Au and Pt
reduction peaks at 0.63 and 0.22 V, respectively, in the reverse
scan. When compared to the peak in the voltammogram for
monometallic Pt at 0.48 V (Figure 6A(b)), an approximately
0.26 V negative shift was observed for the corresponding peak
for Pt in the SSG/M13Y3E/rGO/(Au58.9−Pt41.1) catalyst, and
this shift was attributed to the atomic-level mixing of Pt with
Au.
The electrocatalytic activities of the modified electrodes
toward the MOR were evaluated by carrying out CV
experiments in a solution containing 0.1 M CH3OH and 0.1
M KOH. Figures 5B and 6B depict a comparison of the Au−Pt
alloy nanostructures using 1.5 mM of each precursor and
electrodeposited on five different supports (bare ITO (a),
ITO/SSG (b), ITO/SSG/M13Y3E (c), ITO/SSG/rGO (d), and
ITO/SSG/M13Y3E/rGO (e)) and a comparison of the
electrocatalytic activities of the mono- and bimetallic
nanostructures, respectively, toward the MOR. Figure 5D
shows a comparison of the electrocatalytic activities of the
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environment. Hence, an effective reaction between CH3COads
and OHads could have occurred and contributed to the high
electrocatalytic activity. (iv) A synergistic effect between Au and
Pt may have also contributed to the enhanced catalytic activity.
All of these factors clearly suggest that the proposed SSG/
M13Y3E/rGO/(Au58.9−Pt41.1) catalyst can possess advantageous
structural aspects that would impart improved catalytic
performance toward the MOR. Comparison of three different
biotemplates, prepared using M13Wild, M134E, and M13Y3E,
toward the MOR (Figure 7) clearly demonstrated an improved
response resulting from the M13Y3E-applied biotemplate (SSG/
M13Y3E/rGO/(Au−Pt)). Hence, Au-binding peptides of
M13Y3E were evidently contributing to nucleation, growth
(Figure 3), and catalytic activity. In addition, tyrosine (Y)
moieties (catechol compounds) engineered at the M13Y3E
surfaces have been shown to undergo self-polymerization in
an alkaline medium and form a thin and surface-adherent
polytyrosine film.55 Such a film has the tendency to form
covalent and noncovalent interactions with the substrate as well
as the analyte. Polytyrosine coatings can, in turn, serve as a
versatile platform for secondary surface-mediated reactions.
Thus, the SSG/M13Y3E/rGO biotemplate served as an ideal
platform for accommodating less densely packed Au−Pt alloy
nanostructures and thus facilitating the efficient electron
transfer for the oxidation of MeOH to CO2, as illustrated in
Figure 9.
expected, the initial current was much higher for the SSG/
M13Y3E/rGO/(Au58.9−Pt41.1) catalyst than for the other
catalysts (Figures 5C(e) and 6C(b)), followed by a slow
decay in current when compared to other catalysts up to 600 s,
after which a fast decay occurred. In addition, to reveal the role
of the mass percent of Pt at the SSG/M13Y3E/rGO/(Au−Pt)
catalyst, three different compositions were prepared by keeping
the Au precursor at a constant concentration (of 1.5 mM) and
testing various Pt precursor concentrations, specifically 1.0, 1.5,
and 2.0 mM; the percent compositions were then derived from
EDX analyses, the fabricated electrodes are referred to as SSG/
M13Y3E/rGO/(Au64−Pt36), /(Au58.9−Pt41.1), and /(Au57.3−
Pt42.3), respectively, and their activities toward the MOR are
shown in Figure S10. The SSG/M13Y3E/rGO/(Au58.9−Pt41.1)
catalyst (Figure S10B(b)) showed high catalytic activity in
terms of onset potential and peak current while the other two
catalysts showed lower values. Thus, the mass of Pt in the Au−
Pt alloy nanostructures played an important role in determining
the catalytic activities. The ECSAs, ECSAs per gram of Pt, and
mass activities of the mono- and bimetallic catalysts are listed in
Figure S11.
The ratio between forward peak current (If) and backward
peak current (Ib) is a measure of the poisoning effect resulting
from adsorption of reaction intermediates on the catalyst
surface.5,62 Thus, the If/Ib ratio is inversely proportional to the
rate of such adsorption. An eventually high value of this ratio in
our case would indicate a better MOR. The values of this ratio
for the different modified electrodes are listed in Table 1. To
investigate the kinetics of the MOR when using the SSG/
M13Y3E/rGO/Pt and SSG/M13Y3E/rGO/(Au58.9−Pt41.1) catalysts, the anodic peak current (jp) and peak potential (Ep)
values at various scan rate (ν) values obtained from forward CV
scans were determined and are shown in Figure S12. The SSG/
M13Y3E/rGO/Pt (a) and SSG/M13Y3E/rGO/(Au58.9−Pt41.1)
(b) catalysts (Figure S12C) yielded a linear relationship
between log jp and log ν, with slopes of approximately 0.203
and 0.162, respectively. That the slopes were between 0 and 0.5
indicated that the MOR using both catalysts was a diffusioncontrolled process.62,63 In addition, the linear relationship
(Figure S12D) between Ep and log (ν) indicated that the MOR
was involved in an irreversible charge transfer process.62,63
The better electrochemical response of the SSG/M13Y3E/
rGO/(Au58.9−Pt41.1) catalyst than that of the others can be
explained in various ways. (i) From a structural perspective,
compared to the other supports, the alloy nanostructure
particles were well enough separated from one another (Figure
S3) to realize an improved ECSA. (ii) Regarding the poisoning
effect, CH3COads intermediate species have been shown to
form on Au−Pt alloy nanostructures during the MOR in an
alkaline medium, with their reaction with OHads being a ratedetermining step. The observed catalytic enhancement may
have been in part due to electronic effects of the two types of
the metals on each other, which can alter the work functions of
both metals and increase the catalytic activity via strong binding
of biotemplate with MeOH and hence enhance the oxidation of
the MeOH. In addition, Au atoms may have periodically
renewed the nearby poisoned (CH3COads) Pt surface during
the MOR by catalytically oxidizing poisoning species into CO2.
(iii) At the biotemplate, M13Y3E ensured a close contact
between Au−Pt alloy nanostructures and rGO. Furthermore,
functional groups of rGO, the protein coat (gene VIII) of
engineered M13Y3E, and the porosity of SSG could have
effectively attracted OHads, thereby facilitating an OHads-rich
Figure 9. Schematic representation of MOR at ITO/SSG/M13Y3E/
rGO/(Au−Pt) electrode.
CVs of the commercial Pt/C and SSG/M13Y3E/rGO/
(Au58.9−Pt41.1) catalysts of the MOR obtained in a solution
of 0.1 M CH3OH and 0.1 M KOH at a scan rate of 50 mV/s
are shown in Figure 10A. A description of how the working
electrode for Pt/C was fabricated is given in Supporting
Information. The peak current densities of the Pt/C and SSG/
M13Y3E/rGO/(Au58.9−Pt41.1) catalysts during the forward scan
reached 0.23 A/mgPt and 1.54 A/mgPt, respectively. The MOR
mass current density of the SSG/M13Y3E/rGO/(Au58.9−Pt41.1)
catalyst was 6.5-fold higher than that of the commercial Pt/C
catalyst. In addition, the If/Ib ratio for the SSG/M13Y3E/rGO/
(Au58.9−Pt41.1) catalyst was 10.37, higher than the 8.07 value for
the commercial Pt/C catalyst, indicating an improved poison
tolerance performance of the SSG/M13Y3E/rGO/(Au58.9−
Pt41.1) catalyst. The amperometric i−t curves (Figure 10B)
recorded at −0.3 V indicated that the current density of the
SSG/M13Y3E/rGO/(Au58.9−Pt41.1) catalyst was higher than that
of the commercial Pt/C catalyst up to 800 s, after which decay
occurred. The long-term poisoning rate (δ) was calculated by
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Figure 10. (A) CVs of (a) ITO/Pt/C and (b) ITO/SSG/M13Y3E/rGO/(Au−Pt) electrodes in (A) 0.1 M CH3OH and 0.1 M KOH at a scan rate of
50 mV/s. (B) Amperometric i−t curves observed for (a) ITO/Pt/C and (b) ITO/SSG/M13Y3E/rGO/(Au−Pt) electrodes in 0.1 M CH3OH and 0.1
M KOH at an applied potential of −0.3 V. (C) CVs obtained at ITO/SSG/M13Y3E/rGO/(Au−Pt) electrode in 0.1 M CH3OH and 0.1 M KOH at a
scan rate of 50 mV/s with 1 to 100 cycles. (D) Corresponding calibration plot.
ECSAs were generally observed to decrease with more CV
cycles, predominantly during the first 500−750 cycles (Figure
S13D). Specifically, after 250, 500, 750, 1000, and 1250 ADT
cycles, the ECSAs of SSG/M13Y3E/rGO/Pt, SSG/M13Wild/
rGO/(Au57.7−Pt42.3) and SSG/M13Y3E/rGO/(Au58.9−Pt41.1)
catalysts decreased by 5.6, 10.8, and 12.7%, 58.1, 53.4, and
85.2%, 62.6, 55.9, and 81.3%, 65.3, 60.0, and 78.5%, and 67.5,
61.0, and 76.4%, respectively. Note that the ECSAs did not
decrease significantly after 750 cycles. The overall ECSA loss
for the three catalysts was on average about 68% after 1250
ADT cycles. Interestingly, the ECSA of the SSG/M13Y3E/rGO/
(Au58.9−Pt41.1) catalyst (Figure S13C) increased a little bit after
500 cycles, suggesting that new Pt surface features were
generated at that point during the cycling test. Pt
nanostructures in all three catalysts would have been expected
to grow and aggregate during the durability tests owing to
Ostwald ripening,2,7,65 causing the aggregation, coalescence,
and ECSA loss.
Despite our method being very simple, easy to follow, and
very convenient for fabricating nanostructures, the performances we obtained were comparable or superior to the previous
results obtained from more complicated approaches. Moreover,
the use of peptides expressed on the virus surfaces as a
surfactant to fabricate metal nanostructures is highly costeffective not only because M13 is a kind of bacteriophage that
enables low-cost mass production but also because the
corresponding synthetic peptides that give similar effects to
those we found for the fabrication are extremely expensive.
measuring the linear decay of the current for a period of more
than 500 s from Figure 10B by using the following equation:64
δ = 100/I0 × (dI /dt )t > 500s (%s−1)
(3)
where (dI/dt)t>500s is the slope of the linear portion of current
decay and I0 is the current at the start of polarization backextrapolated from the linear current decay. The poisoning rates
were calculated to be 0.0212 and 0.0075% s−1 for the
commercial Pt/C and SSG/M13Y3E/rGO/(Au58.9−Pt41.1) catalysts, respectively. The result further revealed the better
poisoning tolerance of the SSG/M13Y3E/rGO/(Au58.9−Pt41.1)
catalyst.
To determine the stability of the SSG/M13Y3E/rGO/
(Au58.9−Pt41.1) catalyst, 100 continuous cycles of the MOR
were carried out using this catalyst (Figure 10C,D). After the
100 cycles, the peak current decreased by only 10.2%, which
indicated that the proposed SSG/M13Y3E/rGO/(Au58.9−Pt41.1)
catalyst showed good sensitivity and stability toward the MOR.
The durability of the catalyst has been recognized as one of the
most important issues to be addressed before commercializing
DMFCs. Thus, the durability of the SSG/M13Y3E/rGO/
(Au58.9−Pt41.1) catalyst was further evaluated in an accelerated
durability test (ADT) by applying linear potential sweeps
between 0.6 and 1.1 V at 100 mV/s in 0.5 M H2SO4 electrolyte
at room temperature. For comparison, SSG/M13Y3E/rGO/Pt
and SSG/M13Wild/rGO/(Au57.7−Pt42.3) catalysts were also
studied under the same conditions. Figure S13A(a−f), B(a−
f), and C(a−f) show the CVs of the SSG/M13Y3E/rGO/Pt,
SSG/M13Wild/rGO/(Au57.7−Pt42.3), and SSG/M13Y3E/rGO/
(Au58.9−Pt41.1) catalysts, respectively, at initial conditions and
then every 250 ADT cycles up to the 1250th ADT cycle. After
250 cycles, the current densities in the hydrogen adsorption/
desorption potential regions (−0.2 to 0 V) dropped
dramatically with additional CV cycles for all three catalysts.
4. CONCLUSIONS
In summary, we have demonstrated the preparation of an
engineered M13 virus-incorporated biotemplate on electrode
surfaces and its use in the electrodeposition of highly efficient
Au−Pt alloy nanostructures for the MOR. The alloy
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nanostructures on the biotemplate incorporating specifically
M13Y3E exhibited significantly higher electrocatalytic activity
than did the monometallic counterparts or those incorporating
M13Wild or M134E. The SSG/M13Y3E/rGO/(Au58.9−Pt41.1)
catalyst showed a larger ECSA and better MOR performance
than did the commercial Pt/C catalyst, which we attributed to
the improved dispersion of Au−Pt alloy nanostructures on the
biotemplate that ensured fast mass transport during the
reactions. But in terms of durability, the present catalyst
suffered after 800 s when compared to commercial Pt/C
catalyst. The combination of the biotemplate and electrodeposition could offer a convenient way to control the density
of the nanostructures at the electrode surface by allowing for
the peptide functionalities and the incorporated virus
concentration to be selected, which are essential for optimizing
the electrocatalytic performances. The use of peptides expressed on the virus surfaces as a surfactant to fabricate metal
nanostructures is highly cost-effective because corresponding
synthetic peptides able to give similar effects to the fabrication
are extremely expensive. This simple approach can be extended
to other metals and matrixes, and the fabricated templates
should provide highly promising scaffolds for sensors and
energy applications including batteries, supercapacitors, and
fuel cells.
■
ASSOCIATED CONTENT
* Supporting Information
S
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acsami.7b06545.
SEM, SEM-EDX, and XPS analysis of different modified
electrodes; controlled experiments for MOR; comparison
of ECSA and mass activities; comparison of different
composition effect of Au−Pt nanostructures toward
MOR; MOR at different scan rate study; electrochemical
durability studies (PDF)
■
AUTHOR INFORMATION
Corresponding Author
*E-mail: kyuwon_kim@inu.ac.kr.
ORCID
Shanmugam Manivannan: 0000-0001-7751-717X
Seung-Wuk Lee: 0000-0002-0501-8432
Kyuwon Kim: 0000-0002-9252-0737
Present Address
§
College of Pharmacy, Ajou University, Suwon 16499, Republic
of Korea.
Notes
The authors declare no competing financial interest.
■
■
ACKNOWLEDGMENTS
This work was partially supported by the Incheon National
University (International Cooperative) Research Grant in 2012.
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