A Hybrid DNA-Templated Gold Nanocluster for Enhanced Enzymatic Reduction of Oxygen

Article abstract of DOI:10.1021/jacs.5b05338

We report the synthesis and characterization of a new DNA-templated gold nanocluster (AuNC) of ～1 nm in diameter and possessing ～7 Au atoms. When integrated with bilirubin oxidase (BOD) and single walled carbon nanotubes (SWNTs), the AuNC acts as an enhancer of electron transfer (ET) and lowers the overpotential of electrocatalytic oxygen reduction reaction (ORR) by ～15 mV as compared to the enzyme alone. In addition, the presence of AuNC causes significant enhancements in the electrocatalytic current densities at the electrode. Control experiments show that such enhancement of ORR by the AuNC is specific to nanoclusters and not to plasmonic gold particles. Rotating ring disk electrode (RRDE) measurements confirm 4e^- reduction of O₂ to H₂O with minimal production of H₂O₂, suggesting that the presence of AuNC does not perturb the mechanism of ORR catalyzed by the enzyme. This unique role of the AuNC as enhancer of ET at the enzyme-electrode interface makes it a potential candidate for the development of cathodes in enzymatic fuel cells, which often suffer from poor electronic communication between the electrode surface and the enzyme active site. Finally, the AuNC displays phosphorescence with large Stokes shift and microsecond lifetime.

Full text of DOI:10.1021/jacs.5b05338

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Article
A Hybrid DNA-Templated Gold Nanocluster
For Enhanced Enzymatic Reduction of Oxygen
Saumen Chakraborty, Sofia Babanova, Reginaldo C. Rocha, Anil Desireddy,
Kateryna Artyushkova, Amy E Boncella, Plamen Atanassov, and Jennifer S Martinez
J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.5b05338 • Publication Date (Web): 19 Aug 2015
Downloaded from http://pubs.acs.org on August 24, 2015
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Journal of the American Chemical Society
A Hybrid DNA-Templated Gold Nanocluster For Enhanced
Enzymatic Reduction of Oxygen
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Saumen Chakraborty,||,$ Sofia Babanova,#,$ Reginaldo C. Rocha, Anil Desireddy, Kateryna
||
||
#
||,±
#,
||,,
Artyushkova, Amy E. Boncella, Plamen Atanassov, Jennifer S. Martinez
||Center for Integrated Nanotechnologies, Institute for Materials Science, Los Alamos National
Laboratory, PO BOX 1663, Los Alamos, NM 87545, USA
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Center for Micro-Engineered Materials (CMEM) and Department of Chemical & Biological
Engineering, The University of New Mexico, Advanced Materials Laboratory, 1001 University Blvd. SE,
Albuquerque, NM 87106, USA
Abstract: We report the synthesis and characterization of a new DNA-templated gold nanocluster (AuNC)
of ~1 nm in diameter and possessing ~7 Au atoms. When integrated with bilirubin oxidase (BOD) and
carbon nanotubes (CNTs), the AuNC acts as an enhancer of electron transfer (ET) and lowers the
overpotential of electrocatalytic oxygen reduction reaction (ORR) by ~15 mV as compared to the enzyme
alone. In addition, the presence of AuNC causes significant enhancements in the electrocatalytic current
densities at the electrode. Control experiments show that such enhancement of ORR by the AuNC is
specific to nanoclusters and not to plasmonic gold particles. Rotating ring disk electrode (RRDE)
−
measurements confirm 4e reduction of O
2
to H
2
O with minimal production of H
2
O , suggesting that the
2
presence of AuNC does not perturb the mechanism of ORR catalyzed by the enzyme. This unique role of
the AuNC as enhancer of ET at the enzyme-electrode interface makes it a potential candidate for the
development of cathodes in enzymatic fuel cells, which often suffer from poor electronic communication
between the electrode surface and the enzyme active site. Finally, the AuNC displays phosphorescence
with
large
Stokes
shift
and
microsecond
lifetime.
Introduction: With sources of fossil fuels dwindling,
there is an urgent need to find cheap, renewable, and
alternate forms of energy using naturally abundant
resources such as sunlight, air, and water.
Nanostructured materials and enzymatic fuel cells are
gold nanoparticles (AuNPs) have been used as
substrates for immobilization of laccase, which
showed enhanced oxygen reduction kinetics by DET.
However, the electrochemical output of this system
still remained poor.
4
1
showing great promise in this respect. In enzymatic
fuel cells, both the anodic and cathodic reactions are
carried out by enzymes acting as bio-electrocatalysts.
The enzymes oxidize fuels at the anode while
Atomically precise metal nanoclusters (NCs)
with a diameter of less than 2 nm and consisting of
~2-200 atoms arranged in well-defined and stable
reduction of O
2
takes place at the cathode, typically
geometric structures are showing
important
2
catalyzed by multi-copper oxidases (MCOs). The
efficiency of these systems depends on how
effectively the enzymes communicate with the
electrode surface via direct electron transfer (DET) at
applications across multidisciplinary fields such as
sensing, bio-imaging, electronics, photovoltaics, and
5
catalysis. Owing to their ultra-small size, NCs
possess discrete molecule-like electronic, optical, and
electrochemical properties as well as specific packing
3
potentials close to the redox potential of the enzyme.
6
of atoms on NC surface and the metallic core. These
Although MCOs have been used as ORR
catalysts on various electrode surfaces they suffer
from low conversion efficiency primarily due to the
lack of effective ET between the electrode surface
and the enzyme active site. In addition, there is a
need for engineering suitable material architectures
that provide a large surface area for good electrical
connectivity, substrate accessibility to the enzyme,
and yet still retain a biocompatible environment for
enzyme immobilization. Overcoming these limitations
can enable widespread utilization of enzymatic fuel
cells as simplified devices for single-compartment
operation under neutral reaction conditions and
integration into various scalable systems. To this end,
unique electronic and structural aspects of NCs play
critical roles in fine-tuning their characteristics and
bestow them with size-dependent properties that are
quite different from those of bulk metals, metal
complexes, and metal nanoparticles.
Although
bulk
gold
is
inert,7
gold
nanoparticles (AuNPs) larger than 2 nm in diameter
have been shown to possess interesting catalytic
properties when dispersed as ultrafine particles on
8
metal oxide supports. Among others, CO and
hydrocarbon oxidation, hydrogenation, and reduction
of nitrogen oxides and oxygen are the most notable
examples where AuNPs have been employed as
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catalysts.8a,9 The high catalytic activity of small Au
Herein we report synthesis and thorough
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particles compared to bulk metal has been attributed
to several factors, including high surface density of
low coordination number Au atoms and less electron
density in small Au particles compared to bulk
characterization of a new DNA-templated AuNC. We
demonstrate the application of this novel material in
enzyme-based biofuel cells as facilitator of ET at the
enzyme-electrode interface. Composites of the AuNC
integrated with carbon nanotubes (CNTs) and bilirubin
oxidase (BOD) were immobilized on electrode
surfaces for ORR assays. Bilirubin oxidase from
Myrothecium verrucaria was chosen as the desired
MCO due to its known structure and ready
commercial availability. BOD has an ET T1 Cu site,
and a catalytic T2/T3 Cu site where the reduction of
10
metal. Although several studies examined the effect
8,9c,11
of NP size on catalytic activity,
it was only
recently discovered that the catalytically active
species in CO oxidation is a bilayer of 10-atom gold
12
nanoclusters (AuNC), ~0.5 nm in diameter.
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Subsequently, several research groups reported
catalytic activity of atomically monodisperse,
ultrasmall AuNCs (<2 nm) in solution towards
25
O takes place. The relatively high redox potential of
2
1
3
13a,14
26
oxidation of organic substrates, hydrogenation,
BOD
makes it advantageous for improving its
towards electrocatalytic oxygen
electrocatalytic reduction of CO
,15 and ORR. While
16
performance
2
the reported AuNCs showed a strong size effect on
reduction. This unique application of the AuNC as
facilitator of ET for ORR demonstrates the beneficial
aspects of NC size effects and opens up many
possibilities for technology developments in the long
term, including biosensors, actuators, and biological
fuel cells.
ORR activity, unfortunately onset potentials (Eonset)
for ORR were low [e.g. −0.1 V for Au11, −0.16 V for
Au25, −0.2 V for Au55, and −0.25 V for Au140 (vs.
Ag/AgCl)] indicating a high overpotential for the
16
reaction.
In addition, these experiments were
exclusively performed in alkaline media. Therefore,
efficient ORR catalysts need to be designed with low
overpotential and which operate under more
environmentally benign aqueous conditions.
Results and Discussion:
Electronic and secondary structural changes
during AuNC formation: DNA-templated AuNC was
synthesized
according
to
Scheme
1
Ligands are critical for the synthesis,
stabilization and control of electronic properties of
17
metal nanoclusters. Over the last decade, DNA has
1
8
been increasingly used as a ligand to prepare silver,
copper,19 and platinum nanoclusters with interesting
20
5
d,21
luminescent, detection, and catalytic properties.
Scheme 1. Synthetic scheme of the AuNC. Black
curves represent DNA backbone, pink lines represent
DNA bases, individual yellow spheres represent Au(III),
while AuNC is shown as the cluster of yellow spheres.
Because DNA is a natural nanoscale material with
strong affinity for metal cations,22 DNA can template
23
and localize metals to form and stabilize NCs. In
addition, exquisite control of NC size and the resulting
electronic and optical properties has made DNA a
natural ligand choice for NC synthesis and their
various applications. Finally, the chemistry of DNA-
templated NCs can be performed in water and neutral
conditions, which is a green and desirable method for
technology development as opposed to organic
solvents or acidic/alkaline reaction conditions.
(see Materials and Methods for details). To monitor
electronic changes occurring during AuNC formation,
UV-vis absorption spectroscopy was employed.
Incubation of DNA with Au(III) causes a red shift in
the λmax of DNA from 261 nm to 266 nm (Figure 1),
indicating complexation of Au(III) ions to the functional
groups of DNA (likely binding to nucleobases by
Lewis acid-base interactions). Upon reduction of
Au(III) and formation of nanoclusters, further spectral
changes occur and the λmax subsequently blue-shifts
from 266 nm to 264 nm (Figure 1), indicating different
electronic transitions in the DNA when the AuNC is
formed, compared to the initial Au(III)-DNA complex.
Similar trend in spectral shifts was observed during
Using these advantages of DNA as a ligand
for NC synthesis having well-defined materials
architectures, and with the wide variety of catalytic
applications of AuNCs, we set out to synthesize
stable AuNCs using DNA as the ligand and
investigate their applications as facilitators of ET in
enzymatic fuel cells. While a few examples of DNA-
18a
the formation of a DNA-templated AgNC.
Upon
2
4
templated gold nanoclusters have been reported,
subtracting the DNA contribution from the spectrum of
AuNC, the presence of a broad shoulder centered
~394 nm (3.15 eV) was observed (Figure 1, inset).
Discrete molecule-like electronic transitions in the
range 330-390 nm have been reported for small gold
their potential applications have been unexplored due
to a lack a thorough characterization. We hypothesize
that due to the small size, electrochemical activity and
unique properties of the AuNCs the DNA-templated
AuNC could facilitate ET to the enzyme active site
2
7
28
clusters
(e.g.
[S(CH
Au10-12SG10-12
,
Au11
[S(CH
C
2
12
,
29
where reduction of O
2
takes place and thus lower the
Au13[PPh
3
]
4
2
)
11CH
3
]
2
30
Cl
2
,
Au13[PPh
PAMAP ), the spectral features of
which depend on various factors such as ligand type,
]
3 4
)
11
-
2
9
overpotential while increasing the electrocatalytic
current density for ORR.
CH
3
]
4
,
and Au
8
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Journal of the American Chemical Society
formation of AuNC (Figure 2), suggesting secondary
geometry, core size, and oxidation states of the
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clusters. Therefore, it is likely that some or all of
structural changes in the DNA during these
processes. Spectral shifts in both the absorption and
CD spectra suggest changes in the electronic
transitions and secondary structure of DNA upon
Au(III) coordination and subsequent cluster formation
process.
these factors contribute to the broadness of this
shoulder feature in the spectra of the DNA-templated
AuNC reported here.
The AuNC is a small cluster with ~7 Au atoms:
Transmission electron microscopy (TEM) analysis
was further performed to determine the size of AuNC.
The TEM micrograph of the AuNC shows the
presence of many small particles with average size of
~0.9 nm in diameter (Figure 3), which is characteristic
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of small gold clusters.
The observed apparent
polydispersity due to the presence of a few larger
particles is originating from electron beam damage of
the nanoclusters that causes sintering of the metal,
which is a widely observed phenomenon while
33
imaging such small particles.
The TEM image
further proves that the material under study is truly
Figure 1. Electronic changes occur during the AuNC
formation. UV-vis absorption spectra of solutions
containing 15 μM DNA (black line), 15 μM DNA + 225 μM
HAuCl
4
(blue line), and the as synthesized AuNC (red line)
, pH 7. The top
in 20 mM phosphate buffer, 1 mM Mg(OAc)
2
inset shows the clear shifts in the λmax of DNA upon Au(III)
complexation and subsequent AuNC formation. The bottom
inset shows the spectrum of AuNC after subtraction of the
absorption by DNA alone.
To probe whether secondary structural
changes occur in the DNA molecule during AuNC
formation, we used circular dichroism spectroscopy,
which is sensitive to changes in the chirality of ribose
Figure 3. TEM shows small gold clusters. TEM image of
the AuNC showing the presence of small clusters with
average size of ~0.9 nm in diameter. Scale bar: 10 nm.
nanocluster in nature and not plasmonic AuNPs (>2
nm in diameter). To determine the number of Au
atoms present in the AuNC we performed MALDI-MS
of the DNA and the AuNC in both positive and
negative ionization modes. The observed molecular
weights of the AuNC are 10400 Da and 10524 Da in
positive and negative ionization modes, respectively;
while those of DNA are 9054 Da and 9196 Da (Figure
S1). After subtracting the corresponding DNA, the
total number of Au atoms present in the AuNC was
calculated to be ~7 in both positive and negative
ionization modes, suggesting that the AuNC is a 7-
atom cluster ligated by a single DNA molecule.
Although the widths of the AuNC peak in the MALDI
spectra are greater than that of DNA alone, it is likely
that the extent of ionization of DNA and AuNC are
different, giving rise to differences in the observed
resolution. Additionally, given that even well
characterized and atomically precise thiol-protected
Figure. 2. Secondary structural changes occur during
the AuNC formation. CD spectra of solutions containing
1
00 μM DNA (black line); 150 μM DNA + 2250 μM HAuCl
4
(blue line); and the synthesized AuNC (red line).
sugars. DNA alone shows two positive CD bands at
2
2
79 and 219 nm and two negative bands at 244 and
09 nm (Figure 2), respectively. Similar to the
electronic absorption spectrum, the CD spectrum also
changes upon Au(III) complexation to DNA and
subsequent reduction of Au(III) leading to the
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gold clusters exhibit broad MALDI spectra, the
observation of such spectral broadening in the DNA-
XPS to investigate whether the clusters possessed
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only Au(0) or both Au(0) and Au(I) oxidation states.
The Au 4f XPS spectrum of the AuNC sample (Figure
4, blue line) shows an intense and sharp peak at ~85
eV, a less intense and broader peak at ~88.5 eV, and
a small peak at ~91 eV. The sharp peak at ~85 eV
corresponds to the Au4f7/2 component and the other
two peaks correspond to the Au4f5/2 components.
Deconvolution of the spectral envelope yielded
individual Au species corresponding to different
oxidation states. The Au4f7/2 line consists of
contributions from a Au(0) species at 84.2 eV (Figure
4, black line) and a Au(I) species at 85 eV (Figure 4,
green line) present at a relative population of 0.27:1,
respectively. These data therefore show that the
DNA-templated AuNC has characteristics of
nanoclusters with both Au(0) and Au(I) oxidation
states (i.e., a mixed-valence cluster). In addition to
Au(I) and Au(0), a small fraction of residual Au(III)
may still remain in the sample as observed from the
Au 4f7/2 peak at 87.1 eV (red dotted line).
16,34
protected AuNC is not unusual.
The observed
number of Au atoms in the AuNC is less than the
initial molar ratio of 1:15 (DNA:Au) as some of the
added Au(III) produces plasmonic Au particles upon
reduction (see Materials and Methods). Furthermore,
it is commonly found for DNA-templated nanoclusters
that the metal-ligand stoichiometry of the reaction
mixture
is
not
maintained
in
the
final
product.1
8a,18c,18d,35
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To further probe the composition of the
AuNC, we performed energy dispersive X-ray
spectroscopy (EDX) analysis to calculate the Au atom
count (Figure S2). From intensities of the Au Lα
(9.712 keV) and P Kα (2.013 keV) lines, the atomic
percentages of P and Au were obtained. From this
analysis the number of Au atoms present in one DNA
molecule was found to be ~7 (see Materials and
Methods for details). While small clusters of 3-13 Au
atoms protected by ligands other than DNA have
been reported,36 a rigorous analysis of AuNC size and
atom count has not been previously performed for any
DNA-templated AuNC.
Based upon NMR and EXAFS data of Ag-
coordinated DNA as well as Raman data on DNA-
bound metal ions it is suggested that metals bind to
DNA through the N7 of purines and N3 of
18a,37
To find out the number of gold atoms present
in the AuNC, we inspected the P 2p and Au 4f X-ray
photoelectron spectra (XPS). From these data the
relative atomic % of P and Au are found to be 3.1%
and 0.74%, respectively. Analysis of the data yielded
a ~7 atom Au cluster (see Materials and Methods),
which is also consistent with the MALDI-MS and EDX
data (vide supra) in suggesting the presence of ~7 Au
atoms in the AuNC.
pyrimidines.
Here, we examined the N1s XPS
data to gain insight into whether nitrogen atoms of
DNA bases are ligating to Au in the AuNC. The N1s
XPS data show that the speciation of nitrogen has
changed in the AuNC sample compared to metal-free
DNA (Figure S3). Specifically, the relative ratio of
amine (398.8 eV) and amide (400.3 eV) peaks
changes between the DNA and the AuNC samples. In
addition, protonated nitrogen species (401.3 eV) from
the DNA bases changes significantly when the AuNC
is formed. Measuring the pH of the DNA-only and the
AuNC samples (both at pH ~7.0) ensured that
deprotonation of the nitrogen was not due to a
difference in pH. Although the identity of specific DNA
bases that bind to the Au cannot be determined,
these data suggest that the AuNC is preferentially
formed with ligation from the nitrogens of the DNA
bases, for which the chemical environment changes
upon binding of gold.
AuNC is a mixed-valence cluster: Next, we used
The AuNC displays large Stokes shift with
microsecond lifetime: Having established the size
and composition of DNA-templated AuNC, we
explored whether it was luminescent. At relatively
high concentrations (~1 mM), the AuNC showed
luminescence with an emission peak at 650 nm
(
4
Figure 5, blue line) resulting from a photoexcitation at
70 nm (Figure 5, green line). The large Stokes shift
Figure 4. The AuNC consists of both Au(0) and Au(I)
oxidation states. Au 4f XPS spectra showing the
presence of both Au(0) and Au(I). Blue line: experimental
spectrum; red solid line: fitted spectrum; black line: Au4f7/2
component of Au(0); green line: Au4f7/2 component of Au(I);
red dotted line: Au4f7/2 component of residual Au(III); gray
lines: corresponding Au4f5/2 components.
of 180 nm suggests that the primary origin of this
emission is phosphorescence, which was supported
by lifetime measurements. Analysis of the
luminescence decay curves showed the presence of
two emission components with microsecond lifetimes
[
4.2 μs (89%) and 0.6 μs (11%); Figure S4)]. Such
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large Stokes shifts and microsecond lifetimes have
also been observed in luminescent Au(I)
complexes, as well as in ligand-protected
The
AuNC
is
electrochemically
active:
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Electrochemical properties of the AuNC were
assessed by cyclic voltammetry (CV) and differential
pulse voltammetry (DPV). Although the CV scans
show two poorly defined redox processes (Figure 6a,
marked as *), DPV shows two resolved processes
occurring at 0.155 V and 0.210 V vs. Ag/AgCl,
27a,38
luminescent AuNCs with glutathione (AuNC@GSH:
27a

(
ex = 365 nm, em = 610 nm),
D-penicillamine
5a
AuNC@D-Pen: ex = 400 nm, em = 610 nm), and
dihydrolipoic acid (AuNC@DHLA: ex = 490 nm, em
=
5b,5c
-3
650 nm).
A quantum yield of 2.6x10 determined
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Figure 6. The AuNC is electrochemically active. a) CV
scans at a scan rate of 50 mV/s. b) DPV scans in the
anodic direction (pulse period = 250 ms, pulse width = 25
ms, amplitude = 25 mV, increment = 2 mV). The
concentration of sample solutions were ~0.5 mM AuNC in
5
2
0 mM phosphate buffer, 1 mM Mg(OAc) , pH 7.
respectively (Figure 6b). From electrochemical
studies of monolayer protected AuNCs of various
Figure 5. The luminescent AuNC shows large Stokes
shift. Excitation spectrum (green line; λem = 650 nm) and
emission spectrum (blue line; λex = 470 nm) of ~1 mM
44
45
sizes (e.g. Au25(SC6)18
,
Au38(SC
Au67(SR)35 ), multiple redox processes have been
assigned to sequential one-electron
2
H
4
Ph)24
,
and
46
2
AuNC in 20 mM phosphate buffer, 1 mM Mg(OAc) , pH 7.
oxidation/reduction of the various charge states of the
clusters. Here, the two closely spaced potentials are
likely associated with two successive one-electron
oxidations at the AuNC (i.e. two Au(0)/Au(I)
processes). As with polynuclear charge-transfer
molecular complexes, it is possible that the first
oxidation introduces an electronic perturbation (in
addition to the change in overall charge) that causes
( = 2.8x10-2 in air-saturated
using [Ru(bpy)
3
]Cl
2
9
aqueous solution)3 suggests that the AuNC is weakly
luminescent. However, the luminescence quantum
yield is comparable to or, in some cases, several
orders of magnitude greater than those of ligand-
protected gold clusters with ligands such as
-
3
5f
-3 40
glutathione ( = 3.5x10 ), tiopronin ( = 3x10 ),
47
-6
41
the shift of the second process.
dimarcaptosuccinic acid (
dodecanethiol ( = 4.4x10 , <3x10 ).
=
1x10 ),
and
-5
-7 42
Oxygen reduction activity
of
AuNC/BOD
composites: Motivated by the electrochemical
activity and small size of the AuNC, we investigated
whether these unique properties can be utilized for
enhanced ET between the electrode and the enzyme.
BOD was used as an enzyme of choice as it is a well-
known enzyme for catalyzing ORR. To test our
hypothesis, the AuNC was integrated with BOD by
using single-walled carbon nanotubes (SWNT) as a
support material. SWNT was dispersed via
tetrabutylammonium bromide (TBAB) modified
Nafion. TBAB modification causes exchange of the
proton from Nafion sulfonic acid group and affords the
TBAB salt of Nafion. This modification results in an
increase in the pore size of the Nafion polymer
allowing easy diffusion of substrates and ions to the
enzyme active site, and reduces acidity of Nafion,
thus making it a more biocompatible polymer for
immobilization of the enzyme on the electrode
It has been recently proposed that the origin
of luminescence in AuNCs can be attributed to the
presence of large fraction of Au(I), and that the
AuNCs can be present as a mixed-valence species
lying in between luminescent Au(I) complexes and
non-luminescent AuNPs.43 To test whether the
luminescence in the DNA-templated AuNC is due to
the presence of Au(I), the spectral changes were
monitored upon reducing the Au(I) in the luminescent
AuNC. Addition of NaBH (1.0 equivalent with respect
4
to gold concentration) to a solution of AuNC caused a
significant decrease in emission at 650 nm, with
~90% loss of the initial luminescence (Figure S5).
This observation suggests that the luminescence of
DNA-templated AuNC is associated with the presence
of Au(I). Consequently, no luminescence was
4
8
observed when NaBH
4
was used instead of
dimethylamine borane (DMAB) as the reductant
during the AuNC synthesis. This result corroborates
the hypothesis that the presence of Au(I) is critical to
the appearance of luminescence in the DNA-
templated AuNC.
48
surface. DNA-templated AuNC was then mixed with
the suspension of SWNT to allow for stacking of the
DNA to the SWNT by non-covalent π-π stacking
interactions. Next, 1-pyrenebutanoic acid succinimidyl
5
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Journal of the American Chemical Society
Page 6 of 13
ester (PBSE) was added to the mixture followed by DNA/SWNT control composite (Figure 7, blue line)
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
BOD and incubated overnight. While the pyrene
groups of PBSE tether to the SWNT by π-π stacking
interactions, the succinimidyl ester groups covalently
attach to the surface amine groups of the BOD via
succinimidyl ester-amine cross-linking chemistry to
prepare the final composite material BOD-
showed a cathodically shifted Eonset at ~1.125 V. In
addition, E1/2 decreased to ~1.040 V in conjunction
with the catalytic current density, which was reduced
2
2
to ~197 A/cm from ~257 A/cm observed in the
BOD/SWNT composite (Table 1). These results
suggest that DNA can hinder the interfacial ET from
the electrode to the enzyme and thus be detrimental
to the ORR (supported by lower current densities with
increase in DNA concentration, Figure S7).
Remarkably, the presence of the AuNC caused
significant changes in the ORR profile displayed by
the BOD-AuNC/SWNT composite (Figure 7, red line).
AuNC/SWNT.
A
schematic of the composite
preparation method is shown in Scheme 2. Control
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
First, Eonset was anodically shifted to ~1.165 V from
~1.150 V observed using the BOD/SWNT sample,
corresponding to a positive shift of ~0.015 V. Second,
the electrocatalytic current density was increased to
2
2
~
412 A/cm , an increase of ~155 A/cm compared
to BOD/SWNT composite. Finally, the E1/2 increased
to ~1.070 V from that of ~1.055 V observed using
BOD/SWNT (Table 1). These exciting results,
therefore, suggest that the presence of AuNC
enhances the ORR activity of the enzyme by lowering
the overpotential by a significant ~0.015 V with
concomitant increase in the kinetics of the reaction,
which leads to higher catalytic current densities.
Scheme 2. Schemeatic representaion of composite
preparation for ORR assays. AuNC: gold nanocluster,
PBSE: 1-pyrenebutanoic acid succinimidyl ester, BOD:
bilirubin oxidase, RDE: rotating disk electrode, RE:
reference electrode, CE: counter electrode
composites consisting of i) SWNT and BOD
(
BOD/SWNT), ii) SWNT, DNA alone, and BOD (BOD-
To investigate whether such enhancement of
ORR activity by the AuNC is specific to nanoclusters,
we performed LSV of BOD-plasmonic Au/SWNT
control composite consisting of the SWNT, plasmonic
Au particles (side product in the cluster synthesis),
and BOD. This data (Figure 7, purple line) shows that
DNA/SWNT), iii) SWNT, plasmonic Au particles (side
product in the AuNC synthesis) and BOD (BOD-
plasmonic Au/SWNT) were prepared using similar
methods as above but without the AuNC. For
electrocatalytic ORR measurements the samples
were drop cast on a rotating disk electrode (RDE),
dried, and their oxygen reduction activity was tested
using linear sweep voltammetry (LSV) with a scan
rate of 10 mV/s.
the Eonset shifts to ~1.120 V as compared to ~1.165 V
observed in the presence of AuNC. Further, the E1/2
also significantly shifts to a lower potential of 0.990 V
as compared to 1.070 V obtained with the BOD-
AuNC/SWNT composite with a concomitant reduction
of electrocatalytic current density at the electrode (74
First,
measurements of BOD-AuNC/SWNT composite
under O depleted, and dissolved O conditions.
Under O -depleted conditions (Figure S6, black line),
very low current was observed due to the small
amount of oxygen present in the electrolyte solution
we
performed
electrochemical
2
2

A/cm compared to 412 A/cm observed with
2
2
AuNC). These data, therefore, convincingly suggest
that the enhancement of ORR (by lowering the
overpotential by ~0.015 V) as well as the
enhancement of catalytic current densities in the
presence of AuNC is unique to nanoclusters, which
improved kinetics and thermodynamics of ORR. Such
enhancement of ORR activity by the AuNC is
unprecedented. The likely mechanism by which the
AuNC enhances the ORR performance is by
facilitating the ET between the electrode surface and
the enzyme active site through a more effective
electronic communication. These findings suggest
that employment of the AuNC as an enhancer of ET
between the electrode surface and the enzyme active
site can potentially remove a significant barrier in
enzymatic fuel cells, which often suffer from poor
2
(
~0.66 mg/L). In the presence of dissolved
atmospheric O (~6.91 mg/L) moderate current was
2
observed for the enzymatic ORR (Figure S6, red line).
Below ~1.040 V (vs. RHE), the current density
decreased because the reaction was limited by the
availability of O in the electrolyte solution.
2
We next tested electrocatalytic activity of
BOD-AuNC/SWNT and two control composites
(
buffer. The BOD/SWNT control composite (Figure 7,
gray line) showed catalytic current with Eonset of
BOD/SWNT and BOD-DNA/SWNT) in O –saturated
2
~
1.150 V (vs. RHE), apparent E1/2 of ~1.055 V, and
performance due to
a
lack of electronic
2
current density of ~257 A/cm [Δi measured as the
difference in current between Eonset and the reductive
current at 0.940 V] (Table 1). In contrast, the BOD-
communication between the electrode and the
enzyme active site.
6
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Journal of the American Chemical Society
materials. For example, platinum nanoparticles of
various sizes (3-7 nm) reduced O with Eonset
.870-0.920 V and E1/2 = 0.750 V vs. RHE,52 platinum
nanoclusters and graphene oxide composites
Pt /gDNA-GO) showed Eonset = 1.010 V and E1/2 =
.900 V vs. RHE, Pt and Pt/Pd nanotubes as well
1
2
3
4
5
6
7
8
9
2
=
0
(
0
n
20b
as graphene supported Pt and Pd catalysts and Pt/Pd
nanodendrites showed ORR with E1/2 = 0.850-0.900
53
V vs. RHE. Furthermore, a recently reported Co
3
O
4
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
nanocrystals on graphene showed ORR activity with
Eonset = 0.880 V and E1/2 = 0.790-0.830 V vs. RHE.54
Further evidence of this unique role of AuNC
was obtained from ORR currents measured using a
different electrode design. In this case, the electrode
material (multi-walled Bucky paper (MWBP)) was first
soaked in AuNC solution, followed by PBSE and BOD
for the immobilization of the various components (see
Materials and Methods for details). The modified
MWBP was then placed on a glassy carbon cap
electrode and the electrode performance towards
Figure 7. LSV of BOD-plasmonic Au/SWNT (purple line);
BOD-DNA/SWNT (blue line); BOD/SWNT (gray line); and
BOD-AuNC/SWNT (red line) composite materials in O -
2
saturated 0.1M phosphate buffer (pH 7.5). Traces and
shaded areas represent the average and standard
deviations, respectively, of the data obtained by testing
ORR was monitored in O -saturated buffer by
2
measuring potentiostatic polarization curves. The
three
different
samples,
prepared
and
tested
independently. Scan rate = 10 mV/s; rotation rate = 1600
rpm. [Note: Although potentials were measured vs.
Ag/AgCl, here they are converted and referred to against
RHE due its appropriateness to ORR; for the conversion,
0
0
E
RHE = EAg/AgCl + 0.059*pH + E Ag/AgCl (E Ag/AgCl = 0.197 V)].
2
Table 1. Electrochemical results obtained from LSVs in O -
saturated buffer. Potentials vs. RHE in parenthesis.
a
Sample
E
onset
E
1/2
Δi
V vs. Ag/AgCl
V vs. Ag/AgCl
(V vs. RHE)
(A/
2
(V vs. RHE)
cm )
BOD-plasmonic
Au/SWNT
0.480
(1.120)
0.355
(0.995)
74
BOD-DNA/SWNT
0.485
(1.125)
0.510
0.400
(1.040)
0.415
(1.055)
0.430
197
257
412
BOD/SWNT
Figure 8. The AuNC enhances ORR by BOD.
Potentiostatic polarization curves for BOD/MWBP (black
line); and BOD-AuNC/MWBP (red line) carried out in 0.1
M phosphate buffer (pH 7.5). Standard deviations were
calculated from data obtained by testing three different
samples, prepared and tested independently.
(
1.150)
0.525
1.165)
BOD-AuNC/SWNT
(
(1.070)
aΔi is the differential current between the onset potential
(
Eonset) and the reductive current at 0.940 V.
sample containing both AuNC and BOD caused an
While the Eonset of the BOD-AuNC/SWNT
2
increase in the ORR current density to 735 μA/cm
(
composite is comparable (Table 1) to that of a BOD
on air breathing gas diffusion electrode (GDE) (1.160
V vs. RHE), the observed apparent E1/2 in the current
system (Table 1) is higher than that of the GDE
2
Figure 8, red line) from that of 493 μA/cm obtained
using BOD alone (Figure 8, black line), amounting to
an increase in current density of ~50%. Therefore,
these results also demonstrate that the AuNC is
enhancing the performance of BOD by acting as a
facilitator of the ET between the electrode surface and
the enzyme active site.
(0.920
V
vs. RHE).49 In addition, the BOD-
AuNC/SWNT composite showed higher Eonset and
E1/2 compared to many reported in literature including
BOD on spectrographic graphite (Eonset=1.136 V,
−
−
1/2=1.036 V vs. RHE),50 and BOD on CNTs (Eonset =
.149 V, E1/2 = 0.950 V vs. RHE).51 The present
system also displays better thermodynamic
parameters (Table 1) compared to many Pt-based
Mechanistic insight on 4e vs. 2e reduction: To
understand whether the presence of the AuNC
perturbs the mechanism of ORR by BODs with
E
1
−
−
regards to 2e vs. 4e processes, we performed mass
7
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Page 8 of 13
(Fisher
and charge balance analysis of rotating ring disk
electrode (RRDE) data obtained using the BOD-
AuNC/SWNT composite in -saturated buffer
Figure S8). From this analysis the number of
electrons (n) transferred during O reduction can be
phosphate buffer (pH 7), 1 mM Mg(OAc)
Scientific) and equilibrated for 24 h with inverted
mixing at room temperature (RT, 23±2 C). After
2
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
o
O
2
(
equilibration, the solution became yellow. Reduction
of Au(III) was initiated by addition of 2.25 mM
dimethylamine borane (DMAB, Sigma-Aldrich)
followed by equilibration at RT for 16 h. At this point, a
purple solution was formed indicating the presence of
plasmonic Au particles. This solution was then
purified by spin filtration using 30 KDa MWCO
membranes (Millipore). A yellow solution of the AuNC
was collected in the filtrate while the plasmonic Au
particles were retained in the membrane. The AuNC
solution was stored at 4°C before further use.
Whenever necessary, the as-synthesized AuNC was
concentrated using 10 KDa MWCO membranes. The
highest yield of AuNC was obtained at a maximum
reaction volume of ~5 mL. At higher reaction volumes
the yield of the AuNC significantly decreased and
plasmonic Au particles were formed at a greater
extent. For energy dispersive X-ray spectroscopy
(EDX) measurements (see below) and to determine
the P 2p atomic % from XPS, the AuNC was
2
calculated using the following equation:
4
n =
ꢀ1ꢁ
iR
1
+ (
)
η ∗ iD
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
where i
R
is the ring current, i
D
is the disk current, and
55
η is the collection efficiency at the electrode. For
56
RRDE, the collection efficiency is known to be 37%.
The calculated number of electrons transferred during
reduction by BOD-AuNC/SWNT composite was
found to be 3.9±0.1. This result indicates that less
O
2
−
than 3% of O
(
2
was partially reduced by 2e to H
+2e +2H  H ), while almost all of O was
reduced to 4e reduction process
O). These observations lead to
the conclusion that the presence of the AuNC did not
2
O
2
−
+
O
2
2
O
by a
2
2
−
H
2
O
−
+
(
O
2
+4e +4H  2H
2
57
perturb the mechanism of O
that the BOD-AuNC/SWNT composite material
cleanly reduced O to H O with minimal production of
2
reduction by BOD, and
2
2
synthesized in a solution of 50 mM NH
(pH 5.5), 1 mM Mg(OAc) to avoid error in measuring
OAc buffer
4
reactive oxygen species (ROS).
2
the relative ratio of Au:P arising from the presence of
P in phosphate buffer.
Conclusions:
In conclusion, a new DNA-templated AuNC has been
synthesized and thoroughly characterized. While the
TEM, MALDI, EDX, and XPS analyses show that the
AuNC is ~1 nm in diameter and consists of ~7 Au
atoms, XPS also suggests the presence of both Au(0)
and Au(I) oxidation states. The AuNC shows weak
photoluminescence with microsecond lifetime and
large Stokes shift. The observed phosphorescence
can be attributed to the presence of high fraction of
Au(I) in the cluster. The AuNC is electrochemically
active and enhances the performance of BOD
catalyzed enzymatic ORR by lowering the
overpotential by ~15 mV, and improving the electronic
communication between the electrode and the
enzyme active site. RRDE analysis showed that the
presence of the AuNC did not perturb the mechanism
UV-vis and fluorescence spectroscopy. UV-vis
spectra were collected at RT using a Cary 5000
(
Agilent)
UV-vis
NIR
spectrophotometer.
Fluorescence spectra were collected using wither a
Cary Eclipse spectrophotometer or a Horiba Jobin
Yvon Fluoromax
4 spectrofluorometer, with an
excitation/emission band pass of 5 nm. In lifetime
measurements, the spectrofluorometer was coupled
with
a
time-correlated single photon counting
(
TCSPC) system from Horiba Jobin Yvon. The
apparatus was equipped with a pulsed laser diode
source (NanoLED) operating at 1 MHz and with
excitation centered at 452 nm. Analysis of
fluorescence decay profiles was performed with the
Horiba DAS6 software. All measurements were
performed at RT. Quantum yield was determined by
of O
2
reduction, as the BOD-AuNC/SWNT composite
58
the gradient method, using [Ru(bpy)
3
]Cl in air-
2
−
material cleanly reduced O
2
to H O in a 4e pathway.
2
saturated aqueous solution as the standard (λem = 625
Furthermore, we show that the enhancement of ORR
activity is unique to nanoclusters and not to plasmonic
gold nanoparticles. This unique role as ET enhancers
at the enzyme-electrode interface makes the new
AuNC as a potential candidate for the development of
cathodes for enzymatic fuel cells, thus lifting a critical
methodological barrier in biofuel cell design.
-2 39
nm;  = 2.8x10 ). The excitation wavelength (λex
)
was 470 nm, and the absorbance of both standard
and AuNC sample solutions was maintained in the
0.03-0.1 range.
CD spectroscopy. CD spectra were collected on a
JASCO instrument using a 1 mm path length cuvette.
Three scans were collected for each sample.
Materials and Methods:
TEM imaging. Bright-field transmission electron
microscopy (TEM) analysis of the AuNC was
performed using a FEI Tecnai F30 instrument
operating at 200 kV acceleration voltage. A thin
carbon-coated (carbon film thickness <10 nm) copper
TEM grid (Pacific Grid-Tech, 300 mesh, 3.05 mm
Synthesis and purification of the AuNC. In a typical
synthesis, 15 M single-stranded DNA (IDT, standard
desalting)
ACCCGAACCTGGGCTACCACCCTTAATCCCC was
mixed with 225 M HAuCl .3H O (Sigma-Aldrich,
99.9% trace metals basis) in a solution of 20 mM
of
sequence
4
2
≥
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Journal of the American Chemical Society
and thus 31 P atoms), we obtain 0.1 as the
O.D., hole size: ~63 m) was soaked in as-
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
synthesized AuNC solution for 2 h and air-dried
before imaging.
normalization factor (3.1/31=0.1). After normalizing
the atomic % of Au with this normalization factor, the
number of Au atoms present in the AuNC is thus
calculated to be 7.4 (0.74/0.1=7.4).
MALDI-MS. MALDI data were collected on ABsciex
4800 Plus TOF/TOF MALDI mass spectrometer using
both DNA and AuNC samples in both positive and
negative ion modes with sinapinic acid (Sigma-
Aldrich) as matrix. The AuNC was synthesized using
the same DNA stock solution, which was used for
MALDI-MS analysis of the DNA-only sample.
Electrochemistry. Cyclic voltammetry (CV) and
differential pulse voltammetry (DPV) experiments
were performed using a CH Instruments CHI760E
potentiostat. A three-electrode setup consisted of a
glassy carbon working electrode (3.0 mm disk), a Pt
wire auxiliary electrode, and a standard Ag/AgCl
reference electrode. Cyclic voltammograms were
recorded at scan rates of 10-100 mV s− and were let
run for at least ten full cycles. The 20 mM phosphate
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
EDX measurements. Energy dispersive X-ray
spectroscopy (EDX) data were collected at 30 kV
acceleration voltage using a FEI Quanta 400 FEG-E-
SEM instrument equipped with an EDX system
1
buffer solution (pH 7) containing 1 mM Mg(OAc)
used in the AuNC synthesis) was the only electrolyte
2
(
EDAX Inc.). Data processing was performed using
Genesis software. A concentrated sample (~1-2 mM)
of the AuNC synthesized in NH OAc buffer was drop
(
source. Differential pulse voltammograms were
obtained at a pulse period of 250 ms, pulse width of
4
cast and dried on carbon tape. The ratio of gold to
phosphorus in the DNA backbone was calculated
based on the total atomic % of these two elements
determined from the intensities of the Au Lα (9.712
keV) and P Kα (2.013 keV) lines in the EDX spectra
of the sample. Contribution from spectral overlap of
the Au M line (2.120 keV) to the P Kα line was
subtracted. The atomic percentages of these two
elements were calculated to be 14 and 59.2,
respectively. The total atomic percentage of
phosphorus present in the DNA was then used to
calculate the number of DNA molecules, determined
to be 1.9 (59.2/31) by taking the contributions from 31
P atoms (in this study, the DNA is 31 nucleotides
long). As nanoclusters are formed by a single DNA
molecule, the number of Au atoms present in the
AuNC was found to be ~7.4±1.0 (14/1.9).
25 ms, amplitude of 25 mV, and increment of 2 mV.
All sample solutions were first deoxygenated and then
blanketed with an argon atmosphere throughout the
CV and DPV experiments.
Electrochemical measurements for ORR. 1)
Preparation of BOD-AuNC/SWNT composite
materials: First, a suspension of 1% single-walled
carbon nanotubes (SWNT, cheaptubes.com) in 4:1
water:methanol
solution
and
0.1%
tetrabutylammonium bromide (TBAB)-modified Nafion
(provided by Prof. Shelley Minteer, University of Utah)
in absolute ethanol was made and bath sonicated for
30 min at RT to disperse the SWNT. 5 L of the
AuNC solution was added to 40 L of the SWNT-
TBAB-Nafion suspension and left for 1 h to allow for
the stacking of the DNA to the SWNT. Identical
luminescence emission spectra of the AuNC before
and after mixing with SWNT-TBAB-Nafion confirmed
that the integrity of the AuNC remained intact after
stacking with SWNT (Figure S9). Next, 2 L (4
mg/mL) 1-pyrenebutanoic acid succinimidyl ester
(PBSE, Sigma-Aldrich) dissolved in ethanol were
introduced to the SWNT-TBAB-Nafion-AuNC mixture
and incubated for additional 1 h. After the PBSE
adsorption on the SWNT, 2 L of a 200 mg/mL BOD
(Amano Enzyme Inc.) solution in 100 mM phosphate
buffer at pH 7.5 was added and the sample was
XPS measurements and processing. Samples were
drop cast on mica surface and air dried before
measurements. XPS measurements were performed
with a Kratos Axis Ultra DLD X-ray photoelectron
spectrometer using a monochromatic Al Kα source
operating at 225 W. The data were acquired from 3
different areas in the sample. Survey and high
resolution C1s, O1s, N1s, and Au 4f were acquired at
80 and 20 eV pass energy, respectively. Standard
operating conditions for charge compensation were:
bias voltage of 3.1 V, filament voltage of −1.0 V, and
filament current of 2.1 A. Data analysis and
quantification were performed using the CasaXPS
software. A linear background was used for C 1s,
N 1s, O 1s, and Shirley background for Au 4f spectra.
All of the spectra were charge referenced to the C 1s
at 284.7 eV. Quantification utilized sensitivity factors
that were provided by the manufacturer. A 70%
Gaussian/30% Lorentzian (GL (30)) line shape was
used for the curve fittings.
o
incubated for 16-18 h at 4 C. The BOD-AuNC/SWNT
composite material thus prepared was further used for
the ORR experiments. Controls consisting of
BOD/SWNT, BOD-DNA/SWNT, and BOD-plasmonic
Au/SWNT were prepared using same procedure. The
concentration of DNA in the composites was
estimated according to suitable dilutions from the
DNA concentration in the respective precursor
materials, as determined using an extinction
5
-1
-1
coefficient of 2.78x10 M cm provided by IDT.
Atomic % of P and Au obtained from P 2p
and Au 4f XPS data were found to be 3.1% and
A glassy carbon rotating disk electrode (RDE)
2
0.74%, respectively. Upon normalizing the atomic %
(disk area 0.2475 cm , Pine Instruments) was used.
of P to 1 DNA molecule (which has 31 nucleotides
The RDE was cleaned with alumina of increasingly
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Page 10 of 13
fine grits of 1, 0.3, and 0.05 mm, and rinsed with AuNC/SWNT composite was drop cast on electrode
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deionized water. After cleaning the electrode, 10 L of
surface, dried, and the ORR activity was measured in
100 mM phosphate buffer at pH 7. Using a
bipotentiostat (Pine Instruments), the disk current was
swept from 0.800 to 0 V at a scan rate of 10 mV/s
while the ring was polarized at 0.800 V. Data analysis
was performed according to Eq. 1.
SWNT-TBAB-Nafion suspension were dropped on the
electrode surface and dried under a flow of N gas.
2
Next, 10 L of the composite material (BOD/SWNT,
BOD-DNA/SWNT, BOD-plasmonic Au/SWNT or
BOD-AuNC/SWNT) was drop cast on the RDE and
allowed to air-dry before the electrochemical
measurements.
Associated content:
Supporting Information: MALDI-MS, EDX, N1s XPS,
2
) Preparation of MWBP electrode: Circular pieces
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lifetime measurements, effect of NaBH
4
on
(
0.3 mm diameter) of MWBP were cut, immersed in a
luminescence, effect of DNA concentration on
electrocatalytic current density, RRDE data, and the
effect of SNWT on the luminescence of AuNC. This
material is available free of charge via the Internet at
http://pubs.acs.org.
solution of the AuNC, and left for 1h for attachment of
the AuNC with MWBP. The paper discs were then
washed with DI water and transferred to a 10 mM
solution of PBSE in ethanol. After 1 h, the modified
nanotube paper was washed with DI water and
placed in solution of BOD (10 mg/mL in 100 mM
phosphate buffer, pH 7.5) and incubated at 4°C for 18
h. After enzyme immobilization, the electrodes were
washed again with buffer to remove any unattached
enzyme. The modified MWBP discs were then placed
on a glassy carbon cap electrode and tested in 100
mM phosphate buffer, pH 7.5. A control electrode was
prepared the same way except for the use of AuNC.
Author Information:
$
These authors contributed equally.
Present address: Department of Chemistry, Colorado
±
State University, Fort Collins, CO 80523, USA.
*
Corresponding authors
jenm@lanl.gov
plamen@unm.edu
3)
Electrocatalytic
measurements:
RDE
measurements were performed with a WEB30 Pine
bi-potentiostat and a rotator from Pine Instruments. A
three-electrode setup (glassy carbon working
electrode, Pt wire auxiliary electrode, Ag/AgCl
reference electrode) was used. The electrolyte was a
100 mM phosphate buffer solution at pH 7.5. Enough
time (20 min) was allowed for the system at open
circuit conditions to reach equilibrium. Using linear
sweep voltammetry (LSV) the disk potential was
swept from 0.8 to 0 V at a scan rate of 10 mV/s. At
least three sets of independent ORR data were
collected from three different preparations of
composite samples as well as controls. With each
preparation, the ORR currents were measured in
Notes
The authors declare no competing financial interest.
Acknowledgement:
The authors would like to acknowledge financial
support by the Laboratory Directed Research and
Development (LDRD) program for EDX and TEM
(
A.D.), synthesis, photophysics, and electrochemistry
by the Basic Energy Sciences, Biomolecular Materials
Program, Division of Materials Science & Engineering
(
S.C., R.C.R., J.S.M.). P.A. thanks the Air Force
Office of Scientific Research (Grant FA9550-12-1-
112) and ARO-Multi-University Research Initiative
0
grant W911NF-14-1-0263 to University of Utah for
funding this collaborative project. This work was
performed, in part, at the Center for Integrated
Nanotechnologies, an Office of Science User Facility
operated for the U.S. Department of Energy (DOE)
Office of Science. Los Alamos National Laboratory,
an affirmative action equal opportunity employer, is
operated by Los Alamos National Security, LLC, for
the National Nuclear Security Administration of the
U.S. Department of Energy under contract DE-AC52-
06NA25396. The authors thank Dr. Darrick Williams
for helping with EDX data collection, and Timothy
Sanchez for helping with MALDI data collection.
electrolyte solutions containing dissolved
saturated O (purged for 20 min), and depleted O
purged for 20 min). Potentiostatic polarization curves
of the BOD-AuNC/MWBP and BOD/MWBP
electrodes were carried out by applying a constant
potential for 300 s, starting from open-circuit potential
O
(N
2
,
2
2
2
to 0 V vs. Ag/AgCl, with a step increase of 0.05 V.
0
Potentials measured against Ag/AgCl (E Ag/AgCl
=
59
0.197 V) were converted to RHE using:
E
RHE = EAg/AgCl + 0.059*pH + E0Ag/AgCl
4)
Oxygen reduction reaction current: The
electrochemical current (Δi) was calculated by
determining the difference in the reductive current at
References:
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