Mimicking horseradish peroxidase and oxidase using ruthenium nanomaterials

Article abstract of DOI:10.1039/c7ra10370k

Although important progress has been achieved for the study of noble metal-based enzyme-like catalysts, there are rare reports on the enzyme mimicking applications of ruthenium nanoparticles (Ru NPs). In this work, we investigated the horseradish peroxidase (HRP) and oxidase mimetic activity of Ru NPs. Mimicking HRP, Ru NPs could catalyze the oxidation of substrates 3,3,5,5-tetramethylbenzidine (TMB), o-phenylenediamine (OPD) and dopamine hydrochloride (DA) in the presence of exogenously added H₂O₂ to generate the products with blue, yellow and orange colors, respectively. We also report the first evidence that Ru NPs possess intrinsic oxidase-like activity, which could catalyze the oxidization of TMB and sodium l-ascorbate (NaA) by dissolved oxygen. The HRP-like and oxidase-like activities of Ru NPs were found to be related to the concentrations of Ru NPs. The catalytic mechanism was analyzed by electron spin resonance spectroscopy (ESR), which suggested that the enzyme mimicking activities of the Ru NPs might originate from their characteristic of accelerating electron transfer between substrates and H₂O₂ or O₂. Our findings offer a better understanding of enzyme-mimicking Ru NPs and should provide important insights for future applications.

Full text of DOI:10.1039/c7ra10370k

RSC Advances
View Article Online
View Journal | View Issue
PAPER
Mimicking horseradish peroxidase and oxidase
using ruthenium nanomaterials†
Cite this: RSC Adv., 2017, 7, 52210
Gao-Juan Cao, Xiumei Jiang, Hui Zhang, Timothy R. Croley and Jun-Jie Yin *b
ab
b
b
b
Although important progress has been achieved for the study of noble metal-based enzyme-like catalysts, there
are rare reports on the enzyme mimicking applications of ruthenium nanoparticles (Ru NPs). In this work, we
investigated the horseradish peroxidase (HRP) and oxidase mimetic activity of Ru NPs. Mimicking HRP, Ru
NPs could catalyze the oxidation of substrates 3,3,5,5-tetramethylbenzidine (TMB), o-phenylenediamine
2 2
(OPD) and dopamine hydrochloride (DA) in the presence of exogenously added H O to generate the
products with blue, yellow and orange colors, respectively. We also report the first evidence that Ru NPs
possess intrinsic oxidase-like activity, which could catalyze the oxidization of TMB and sodium L-ascorbate
(
NaA) by dissolved oxygen. The HRP-like and oxidase-like activities of Ru NPs were found to be related to the
concentrations of Ru NPs. The catalytic mechanism was analyzed by electron spin resonance spectroscopy
ESR), which suggested that the enzyme mimicking activities of the Ru NPs might originate from their
characteristic of accelerating electron transfer between substrates and H or O . Our findings offer a better
Received 18th September 2017
Accepted 6th November 2017
(
DOI: 10.1039/c7ra10370k
2
O
2
2
rsc.li/rsc-advances
understanding of enzyme-mimicking Ru NPs and should provide important insights for future applications.
like activity and show potential to replace natural peroxidase in
bioassays, substantial research is directed toward the develop-
Introduction
14
Natural enzymes, which have efficient catalytic activity and ment of nanomaterial catalysts that mimic functions of peroxi-
substrate specicity under mild conditions, are the true bio- dase, oxidase or other natural enzymes with the advantages of low
catalysts mediating each biological process in living organisms. cost, controlled synthesis, tunable catalytic activities, and high
1
9
They are able to accelerate chemical reactions up to 10 times stability even under severe reaction conditions. Till now, a variety
for specic substrates and reactions and have been commer- of nanoscale materials have been developed as the efficient
15–29
cially used in various areas such as sewage treatment, textile enzyme alternatives
and their diverse applications cover from

nishing, household product preparation, and energy conver- sensing, imaging, and therapeutics, to pollutant removal, water
1–5
30–32
sion.
However, the widespread practical applications of treatment and beyond.
Among these, much work is centered
natural enzymes have been severely hampered by their intrinsic on the noble metal-based nanozymes, particularly gold, platinum,
drawbacks, i.e., low stability, high costs in preparation, diffi- palladium, and iridium, because of their well-developed synthesis
culty in purication, and high sensitivity of catalytic activity to techniques, easy modication of surface, excellent catalytic
3
3–48
environments. Therefore, a lot of efforts have been made to activity, good bio-compatibility and so on.
extend the natural enzymes to enzyme mimetics.
For instance, gold
6–8
nanoparticles with different surface modications or their
As a promising candidate for articial enzymes, nanomaterials combined nanomaterials have been reported to demonstrate
with intrinsic enzyme-like activity known as nanozymes ignite different enzyme-like activities. Platinum nanostructures are
intensive research interest due to their ability to replace specic outstanding catalysts and have been found to possess diverse
9–13
enzymes in enzyme-based applications. The design and devel- functional activities similar to peroxidase, catalase, superoxide
opment of novel nanostructured materials mimicking the catalytic dismutase, polyphenol oxidase, ascorbate oxidase and ferroxidase.
role of natural enzymes has emerged as a promising eld for both
Compared with other noble metal-based enzyme mimics,
fundamental and applied research. Since the discovery by Yan and there is a large space for us to explore the potential of Ru NPs as
coworkers that Fe
O
3 4
nanoparticles possess intrinsic peroxidase- nanozymes, which is limited by the relatively less research
activities. Recently, Ru nanoframes are found to show
49
peroxidase-like properties for the rst time. Very recently, our
group reported that Ru NPs have catalase-like and superoxide
dismutase-like activities by scavenging hydrogen peroxide and
a
Department of Applied Chemistry, College of Life Sciences, Fujian Agriculture and
Forestry University, Fuzhou, Fujian 350002, China
b
Division of Analytical Chemistry, Office of Regulatory Science, Center for Food Safety
and Applied Nutrition, U.S. Food and Drug Administration, College Park, Maryland superoxide, which may play important roles in maintaining
2
0740, USA. E-mail: junjie.yin@fda.hhs.gov
redox balance in living organisms by scavenging excess reactive
oxygen species. In this work, we discovered that Ru NPs
†
Electronic supplementary information (ESI) available: Scheme, table and
50

gures. See DOI: 10.1039/c7ra10370k
52210 | RSC Adv., 2017, 7, 52210–52217
This journal is © The Royal Society of Chemistry 2017

View Article Online
Paper
RSC Advances
possess intrinsic HRP-like and oxidase-like mimetic activities. or absence of H
2
O
2
to produce a blue-colored product with
ꢀ1
ꢀ1
The Ru NPs could catalyze the oxidation of the substrates maximum absorbance at 652 nm (3 ¼ 39 000 M cm ). The
,3,5,5-tetramethylbenzidine (TMB), o-phenylenediamine HRP-like activity of Ru NPs was studied by catalyzing the
OPD) and dopamine hydrochloride (DA) by H O to produce oxidation of TMB in the presence of H O . For a typical oxida-
3
(
2
2
2 2
the color products in aqueous solution. More interestingly, we tion reaction, the time-dependent absorbance of the solution of
ꢀ1
found that Ru NPs could catalyze the oxidation of the colorless 0.1 mM TMB and 0.1 mM H
substrate TMB to the blue-colored product in the absence of concentrations of Ru NPs (2.5, 5, 10, 15, 20 mg mL ) was
. This study also demonstrated that the Ru NPs mimicking recorded for 15 min. The control experiments were carried out
2 2
O with 1 mg mL HRP or variable
ꢀ1
2 2
H O
ascorbic acid oxidase (AAO) can oxidize NaA under ambient in the absence of Ru NPs. The steady-state kinetic assays were
condition, as measured by using both UV-vis and ESR spec- conducted at room temperature in a reaction solution with
ꢀ1
troscopy. It was speculated that the nature of enzyme 10 mg mL Ru NPs as catalyst in the presence of H O and TMB.
2
2
mimicking activities of Ru NPs were attributed to their ability The kinetic assays of Ru NPs with TMB as the substrate were
of facilitating electron transfer between substrates and H performed with 0.1 mM H and different amounts of TMB
or O (0.05, 0.075, 0.1, 0.15 mM) aqueous solution. The kinetic assays
of Ru NPs with H as the substrate were performed with
.1 mM TMB and different amounts H solution (0.05, 0.1,
2
O
2
2 2
O
2
.
2 2
O
0
0
2 2
O
Experimental
Chemicals and materials
.15, 0.2 mM). Reaction systems containing 0.1 mM TMB and
ꢀ1
Ru NPs at different concentrations (10, 25, 50, 75, 100 mg mL
)
All chemicals were from commercial suppliers without further were used to show the chromogenic reactions implying oxidase-
ꢀ
1
purication unless otherwise mentioned. Ruthenium nano- like activity. The steady-state kinetic assays of 50 mg mL Ru
powder (Ru NPs, 20–30 nm) was purchased from US Research NPs with TMB (0.05, 0.1, 0.2, 0.5 mM) as the substrate were
Nanomaterials Inc. (Houston, TX). Hydrogen peroxide (H
3
3
2
O
2
,
performed at room temperature. The kinetic parameters were
0%), horseradish peroxidase (HRP), ascorbate oxidase (AAO), calculated using Lineweaver–Burk plots of the double reciprocal
,3,5,5-tetramethylbenzidinedihydrochloride (TMB$2HCl), o- of Michaelis–Menten equation: 1/v ¼ (K /Vmax) ꢁ {1/[S] + 1/K },
m
m
phenylenediamine (OPD), dopamine hydrochloride (DA), where v is the initial velocity, Vmax is the maximal reaction
sodium L-ascorbate (NaA), 3-carbamoyl-2,5-dihydro-2,2,5,5-tet- velocity, [S] is the concentration of substrate and K
ramethyl-1H-pyrrol-1-yloxyl (CTPO) were all purchased from Michaelis constant.
Sigma-Aldrich (St. Louis, MO). 5-tert-Butoxycarbonyl-5-methyl-
is the
m
1
-pyrroline-N-oxide (BMPO) was purchased from Bioanalytical
Labs (Sarasota, FL). 5,5-Dimethyl-1-pyrroline-N-oxide (DMPO)
was obtained from Dojindo Molecular Technologies, Inc. The HRP-like activity of Ru NPs was also investigated by the
Rockville, MD). PBS 7.4 buffer, 1-hydroxy-3-carboxy-2,2,5,5- catalytic oxidation of the HRP substrate OPD in the presence of
tetramethylpyrrolidine hydrochloride (CPH) were obtained
. The absorbance of the color reaction at 417 nm for OPD
OPD and DA oxidation studies
(
2 2
H O
from Enzo Life Sciences (Farmingdale, NY). The concentration was recorded to express the HRP-like activity. For a typical
of buffer stock solution (pH 1.12 HCl–KCl, pH 3.2 HAc–NaAc, oxidation reaction, the time-dependent absorbance of the
ꢀ
1
pH 10.96 KOH–KCl) was 0.1 M.
solution of 0.2 mM OPD and 1 mM H
2 2
O with 1 mg mL HRP or
ꢀ
1
variable concentrations of Ru NPs (10, 25, 50, 75, 100 mg mL
)
was recorded during 20 min. Steady-state kinetics assays of Ru
NPs toward OPD oxidation were carried out with varied
Characterization
The hydrodynamic size (dynamic light scattering, DLS) and
surface charge (zeta potential, mV) were detected using a Zeta-
Sizer Nano series Nano-ZS (Malvern, UK). Scanning electron
microscopy (SEM) and transmission electron microscopy (TEM)
images were captured on a Hitachi SU-70 SEM (Hitachi, USA)
and a Jeol JEM 2100 TEM (Jeol, USA), respectively. The dispersity
of Ru NPs is not good but Ru NPs are commercially used (see
Table S1 and images in Fig. S1 and S2†). UV-vis absorption
spectra were obtained using a Varian Cary 300 spectropho-
tometer (Santa Clara, CA) (Fig. 1–5 and S3†). Electron spin
resonance (ESR) measurements were carried out using a Bruker
EMX ESR spectrometer (Billerica, MA) (Fig. 6, 7 and S7†).
concentrations of the substrate OPD or H
2 2
O at room temper-
ature. The control experiments were carried out in the absence
ꢀ1
of Ru NPs. The kinetic assays of 75 mg mL RuNPs with OPD as
the substrate were performed with 1 mM H and different
amounts of OPD aqueous solution (0.05, 0.1, 0.15, 0.2, 0.25,
2 2
O
ꢀ
1
0
2 2
.3 mM). The kinetic assays of 25 mg mL Ru NPs with H O as
substrate were performed with 0.2 mM OPD and different
amounts H O solution (1, 2, 3, 4, 6 mM).
2
2
The Ru NPs were then applied as catalyst for the oxidation of
DA to aminochrome (AC) by H . The time-scan mode
measurements were performed by monitoring the absorbance
2 2
O
ꢀ
1
change of DA at 480 nm. In a typical experiment, 1 mg mL HRP
ꢀ
1
or various concentrations of Ru NPs (10, 25, 50, 75, 100 mg mL
)
TMB oxidation studies
were respectively reacted with 0.1 mM DA and 1 mM H . The
2 2
O
In order to investigate the enzyme-like activities of Ru NPs, the control experiments were carried out in the absence of Ru NPs.
absorbance variation of the reaction solution was monitored in The kinetics behavior of Ru NPs was also evaluated by applying
ꢀ1
a time-scan mode at 652 nm. Similar to HRP and oxidase, Ru the same procedure described. The kinetic assays of 25 mg mL
NPs can catalyze the oxidation of substrate TMB in the presence Ru NPs with DA as the substrate were performed with 1 mM
This journal is © The Royal Society of Chemistry 2017
RSC Adv., 2017, 7, 52210–52217 | 52211

View Article Online
RSC Advances
Paper
2 2
H O and different amounts of DA (0.25, 0.5, 0.75, 1, 2 mM) in
ꢀ1
10 mM PBS 7.4 buffer solution. The kinetic assays of 25 mg mL
Ru NPs with H O as the substrate were performed with 0.1 mM
2
2
DA and different amounts H O solution (0.5, 1, 2, 3 mM).
2
2
NaA oxidation studies
The oxidation of NaA catalyzed by AAO (1 U mL ) or Ru NPs
ꢀ1
ꢀ
1
(
25, 50, 75, 100, 150 mg mL ) in 10 mM PBS 7.4 were performed
at room temperature. The absorbance variation of the reaction
solution was monitored in a time-scan mode at l (265 nm,
¼ 54 200 M
max
ꢀ1
ꢀ1
3
cm ). The apparent steady-state kinetic
parameters were determined by varying the concentration of NaA
25, 50, 100, 150, 200 mM) in presence of 50 mg mL Ru NPs.
ꢀ
1
(
ESR spectroscopic measurements
All ESR measurements were carried out using a Bruker EMX ESR
ꢂ
spectrometer (Billerica, MA) at ambient temperature (27 C).
5
0 mL of aliquots of control or sample solutions were taken in
Scheme 1 Ru NPs catalyzed oxidation of TMB (a), OPD (b), DA (c) and
ascorbate (d).
glass capillary tubes with internal diameters of 1 mm and
sealed. The capillary tubes were inserted in the ESR cavity, and
the spectra were recorded at selected times.
The oxidation of NaA can also be monitored by ESR spec-
troscopy. The ascorbyl radical is an intermediate formed during
the oxidation of NaA by molecular oxygen. ESR can directly
detect this radical which has a 10 min half-life. Ru NPs at
different concentrations were added to 5 mM NaA to start the
reaction. The peak-to-peak value of the rst line of ascorbyl
radical ESR spectrum was recorded to indicate the amount of
ascorbyl formed. Spectra were recorded under the following
conditions: 20 mW microwave power, 1 G eld modulation, and
Oxidases that can catalyze the oxidation of substrates by simply
employing molecular oxygen as the electron acceptor are more
facile, economic and environment friendly. In this study, the
catalytic activity of Ru NPs and their ability of mimicking HRP
were evaluated using the chromogenic substrates TMB, OPD
and DA (Scheme 1a–c). We also report the rst evidence that Ru
NPs possess intrinsic oxidase-like activity, which could catalyze
the oxidization of TMB and NaA by dissolved oxygen (Scheme 1a
and d).
25 G scan width.
The catalytic mechanism of Ru NPs was rst evaluated in the
H
2
O
2
/DMPO or H
2 2
O /BMPO system in the presence and
Enzyme-like activity of Ru NPs during oxidation of TMB
absence of Ru NPs. Samples containing 50 mM DMPO, 10 mM
2 2
H O , and different concentrations of the Ru NPs (25, 50, 75, The Ru NPs were subjected to reaction conditions typical for
ꢀ
1
1
00 mg mL ) were prepared in pH 1.12, 3.2, 7.4 and 10.96 HRP and oxidase. TMB is a chromophoric substrate commonly
buffer, then transferred to a quartz capillary tube and placed in used in HRP mimetic studies and was rstly selected as
the ESR cavity for spectra recording. The H O /BMPO system a substrate to be oxidized in this study. The Ru NPs with varied
2
2
was applied the same procedure but varying the BMPO concentrations can quickly catalyze the oxidation of TMB to
concentration (25 mM). The instrument settings (20 mW produce a typical blue color either in the presence or absence of
microwave power, 1 G modulation amplitude, and 100 G scan H O . Control reactions in the absence of Ru NPs were per-
2
2
range) were used for the present ESR experiment.
formed and showed negligible color changes over the same time
ESR spectra were recorded from the sample mixture, con- period. The TMB cation free radical, a one-electron oxidation
taining spin probe 200 mM CPH and Ru NPs at different product, would be formed during the reaction procedure, which
ꢀ1
concentration (25, 50, 75, 100 mg mL ). The ESR measurements is responsible for the blue color (maximum absorbance at
were carried out using the following settings: 20 mW microwave 652 nm), similar to the phenomena observed for the commonly
power, 100 G scan range, and 1 G eld modulation.
used HRP enzyme. Time- and dose-dependent absorbance
changes at 652 nm were measured as shown in Fig. 1a and b, 2a
and b. The observed color changes also support UV-vis spectral
analysis. The results showed that Ru NPs could catalyze the
Results and discussion
Noble metal nanoparticles were reported to act as catalysts oxidation of TMB by H
mimicking the native enzyme. Currently, research is highly colored product, suggesting HRP-like and oxidase-like activity.
focused on mimicking peroxidases and oxidase. HRP, a widely In order to regulate enzyme activity and understand the
recognized peroxidase, has been largely explored as a sensor for relationship between nanomaterials and enzyme kinetic
detection, a tracer in enzyme-linked immunosorbent parameters, the Michaelis–Menten behaviors of Ru NPs were
2 2 2
O or dissolved O to produce a blue
2 2
H O
assay (ELISA), and for detection of some peroxidase inhibitors. studied with H
2
O
2
and TMB as substrates (Fig. 1c, d, 2c, d, S4
52212 | RSC Adv., 2017, 7, 52210–52217
This journal is © The Royal Society of Chemistry 2017

View Article Online
Paper
RSC Advances
presence and absence of H
2
O
2
were calculated. Typical
Michaelis–Menten curves were received in a certain concentra-
tion range of TMB and H O (Fig. S4 and S5†). Lineweaver–Burk
2
2
plots of 1/v vs. Vmax were constructed and tted to the Michae-
lis–Menten equation to calculate the Michaelis constant K and
the maximal reaction velocity Vmax. From the Lineweaver–Burk
plots, the kinetic parameters Vmax and K were obtained and
shown in Table 1, Fig. 1d and 2d. In natural enzyme, K is an
m
m
m
indicator of affinity between the enzyme and substrate where
a lower Km value indicates a higher affinity and catalytic activity.
In the case of the nanostructure enzyme mimetics, the Km value
is oen used to compare the enzyme-like performance of the
nanoparticles. The K
m
value of Ru NPs with TMB as the
49
42
substrate is higher than that of Ru frames, Pd nanoplates
14
and Fe
3
O
4
NPs. The K
m
value of Ru NPs with H
2
O
2
as the
substrate is much lower than that of Ru frames and Fe
and comparable with Pd nanoplates. These results indicated
3 4
O NPs,
Fig. 1 HRP-like activity of Ru NPs during oxidation of TMB. (a) Time-
dependent absorbance changes upon oxidation of TMB to oxTMB by
ꢀ1
that Ru NPs had a low binding affinity towards TMB, and high
2
H O
2
using 1 mg mL HRP or variable concentrations of Ru NPs in
presence of 0.1 mM TMB and 0.1 mM H . Insets are color evolution
of TMB oxidation by 0.1 mM H
and different concentrations of Ru oxidase-like activity of Ru NPs was much lower than its HRP-like
binding affinity towards H O . It is also obvious that the
2
O
2
2 2
2
O
2
ꢀ1
NPs or 1 mg mL HRP. (b) Time-dependent absorbance spectra of activity.
oxTMB generated upon the oxidation of 0.1 mM TMB in presence of
ꢀ
1
2
0 mg mL Ru NPs and 0.1 mM H
activity in the presence of 10 mg mL Ru NPs, 0.1 mM H
different concentrations of TMB. (d) Lineweaver–Burk plot of the
2 2
O . (c) Lineweaver–Burk plot of the
ꢀ1
2
2
O and Enzyme-like activity of Ru NPs during oxidation of OPD and
DA
ꢀ1
activity in the presence of 10 mg mL Ru NPs, 0.1 mM TMB and
different concentrations of H
2
O .
2
Typically, HRP as the catalyst via H O for the oxidation of OPD
2
2
to 2,3-diaminophenazine (DAP) has been successfully applied
51,52
for more than a century.
In the presence of HRP and H
2 2
O ,
OPD is oxidized to DAP by H
2 2
O
, resulting in a yellow solution.
and S5†). A series of experiments were performed by changing
the concentration of one substrate and keeping the other
constant. The reaction rates of TMB oxidation both in the
The oxidation of chromogenic OPD has been employed for
evaluating the HRP-like activity of Ru NPs, which is also inves-
tigated by UV-vis spectroscopy (Fig. 3). Similar to the enzymatic
peroxidase activity observed from the HRP, OPD can be oxidized
to a yellow reaction product in the presence of H O2 upon
2
addition of Ru NPs catalysts. Moreover, the color deepened with
increasing reaction time and the concentration of Ru NPs. This
process was also conrmed by absorption spectra. The catalytic
reaction can be monitored by following the changing of absor-
bance at 417 nm, which originates from the oxidation products
DAP. Fig. 3a and b presented the time course curves of the
absorbance at 417 nm within 20 min. The reaction was also
Table 1 Comparison of the kinetic parameters of different catalysts
ꢀ
1
Catalyst
Ru NPs
Substrate
TMB
K
m
(mM)
V
max (mM min
)
Reference
0.234
2.206
0.0603
318
4.95
34.96
8.04
This work
This work
49
H
2
O
2
Ru frames
TMB
H O
Fig. 2 Oxidase-like activity of Ru NPs during oxidation of TMB. (a)
4.45
2
2
Time-dependent absorbance changes upon oxidation of TMB using Pd nanoplates
variable concentrations of Ru NPs in presence of 0.1 mM TMB. Insets
TMB
0.1098
4.398
0.098
154
54.92
0.6
3.492
3.906
2.064
5.868
0.57
16.2
2.331
12
42
14
2
H O
2
are color evolution of TMB oxidation by different concentrations of Ru Fe O NPs
TMB
3
4
NPs. (b) Time-dependent absorbance spectra of oxTMB generated
2
H O
2
ꢀ1
upon the oxidation of 0.1 mM TMB in presence of 100 mg mL Ru NPs. Ru NPs
TMB
TMB
NaA
This work
38
This work
40
(
5
c) Time-dependent absorbance changes corresponding to the Pt NPs
ꢀ1
0 mg mL
Ru NPs-catalyzed oxidation of TMB using variable Ru NPs
0.155
0.022
concentrations of TMB. (d) Lineweaver–Burk plot of the activity in the Pt NPs (30 nm) NaA
ꢀ1
presence of 50 mg mL Ru NPs and different concentrations of TMB.
This journal is © The Royal Society of Chemistry 2017
RSC Adv., 2017, 7, 52210–52217 | 52213

View Article Online
RSC Advances
Paper
Fig. 3 HRP-like activity of Ru NPs during oxidation of OPD. (a) Time-
dependent absorbance changes upon oxidation of OPD by H
1
2
O
2
using
mg mL HRP or variable concentrations of Ru NPs in presence of
.2 mM OPD and 1 mM H . Insets are color evolution of 0.2 mM
and different concentrations of Ru NPs
or 1 mg mL HRP. (b) Time-dependent absorbance spectra generated
ꢀ1
Fig. 4 HRP-like activity of Ru NPs during oxidation of DA. (a) Time-
2 2
dependent absorbance changes upon oxidation of DA by H O using 1
0
2 2
O
ꢀ1
mg mL HRP or variable concentrations of Ru NPs in presence of 1 mM
2 2
OPD oxidation by 1 mM H O
ꢀ1
DA and 1 mM H O . Insets are color evolution of DA oxidation by 1 mM
2
2
ꢀ1
ꢀ
1
2 2
H O and different concentrations of Ru NPs or 1 mg mL HRP. (b)
upon the oxidation of 0.2 mM OPD in presence of 75 mg mL Ru NPs
and 1 mM H . (c) Time-dependent absorbance changes corre-
sponding to the 75 mg mL Ru NPs-catalyzed oxidation of OPD using
variable concentrations of OPD in presence of 1 mM H . (d) Time-
Time-dependent absorbance spectra generated upon the oxidation of
2 2
O
ꢀ
1
ꢀ1
1 mM DA in presence of 100 mg mL Ru NPs and 1 mM H O . (c) Time-
2 2
ꢀ1
dependent absorbance changes corresponding to the 50 mg mL Ru
2 2
O
ꢀ1
NPs-catalyzed oxidation of DA using variable concentrations of OPD in
dependent absorbance changes corresponding to the 25 mg mL Ru
NPs-catalyzed oxidation of 0.2 mM OPD using variable concentrations
of H O .
2 2
presence of 1 mM H
corresponding to the 50 mg mL Ru NPs-catalyzed oxidation of
.2 mM DA using variable concentrations of H
2 2
O . (d) Time-dependent absorbance changes
ꢀ1
0
2 2
O .
monitored while changing the substrate concentration of OPD
or H O and xing the other two concentrations (Fig. 3c and d).
Dopamine (DA) is a neurotransmitter in the catecholamine
and phenethylamine families that plays a number of important
2
2
damage by many oxidants. Numerous studies have shown the
54–56
benecial effects of NaA on human health.
However, NaA
can be oxidized very slowly by dioxygen to produce ascorbyl
roles in the brain and body of animals. HRP/H
2 2
O couple is
commonly selected as a biomimetic oxidizing agent to examine
53
the early stages of dopamine oxidation. The Ru NPs can
catalyze the H -driven oxidation of DA to aminochrome (AC),
2 2
O
in analogy to HRP. The time-dependent absorbance change of
AC, generated upon oxidation of dopamine, was shown in Fig. 4.
Fig. 4a and b depicted the absorption spectra corresponding to
AC generated within a xed time interval using HRP or Ru NPs
2 2
in the presence of DA and H O . There was an obvious increase
of absorbance intensity at 480 nm with the increasing of the Ru
NPs concentration, accompanying the progression of the
orange color. With the increase of the concentration of Ru NPs,
the oxidation reaction proceeded faster, resulted in the
enhanced oxidation of dopamine. Fig. 4c illustrated the time-
dependent absorbance changes of AC upon oxidation of dopa-
mine using variable concentrations of dopamine and a xed
concentration of Ru NPs and H
2 2
O . Evidently, the Ru NPs
catalyzed oxidation of dopamine was controlled by the Fig. 5 AAO-like activity of Ru NPs during oxidation of NaA. (a) Time-
ꢀ
1
2 2
concentration of H O
, and as its concentration increased, the dependent absorbance changes upon oxidation of NaA using 1 U mL
AAO or variable concentrations of Ru NPs in presence of 100 mM NaA.
b) Time-dependent absorbance spectra generated upon the oxida-
tion of 100 mM NaA in presence of 150 mg mL Ru NPs. (c) Time-
dependent amount of NaA remaining in different NaA dispersions
containing 50 mg mL Ru NPs. (d) Lineweaver–Burk plot of the activity
in the presence of 50 mg mL Ru NPs and different concentrations of
rate of the oxidation of dopamine was enhanced (Fig. 4d).
(
ꢀ
1
Enzyme-like activity of Ru NPs during oxidation of NaA
ꢀ
1
ꢀ
1
NaA is an essential nutrient and a well-known antioxidant that
protects other important biological structures against oxidative NaA.
52214 | RSC Adv., 2017, 7, 52210–52217
This journal is © The Royal Society of Chemistry 2017

View Article Online
Paper
RSC Advances
radical (AAc). The presence of AAO greatly accelerated this concentrations, the ESR signal slowly increased (Fig. 6c). CTPO
process. Here, we investigated whether Ru NPs possess AAO-like is a stable, water soluble nitroxide and has been widely used for
57
enzyme-mimetic properties by oxidizing NaA (Fig. 5 and S6†). ESR oximetry. The resolution of the super hyperne structure
The oxidation of NaA to AAc is accompanied by a decrease in the of the low-eld line of the ESR spectrum of CTPO depends on
characteristic absorption of NaA at 265 nm. Therefore, we used the oxygen concentration of the sample solution. Thus, CTPO is
the absorbance change of NaA at 265 nm to monitor its oxida- commonly used for detecting changes in the concentration of
tion. A typical set of experiment investigating Ru NPs with NaA oxygen. As Fig. 6d showed, at the same incubation time, the ESR
was shown in Fig. 5a. No evident change at 265 nm was spectra of CTPO in the samples containing Ru NPs showed
observed in Ru NPs-free reference solution during a 20 min more super hyperne splitting than the control sample, indi-
monitoring. While mixing of NaA with Ru NPs (25, 50, 75, 100, cating more consumption of oxygen. A higher increase in super
ꢀ
1
1
50 mg mL ) resulted in decreased absorbance at 265 nm hyperne splitting was observed for the sample containing high
during time monitoring measurements (Fig. 5a and b). It is concentration of Ru NPs. The above results revealed that Ru NPs
obvious that Ru NPs exhibited a signicant ability to oxidize exhibit intrinsic AAO-like activity.
NaA in a time and dose- dependent manner. We determined the
apparent steady-state kinetic parameters by varying the
Catalytic mechanism of Ru NPs mimicking HRP and oxidase
concentration of NaA in the presence of Ru NPs (Fig. 5c). The K
m
value of Ru NPs with NaA as a substrate was higher than that of The enzyme-like catalytic mechanisms of noble metal nano-
40
41,42,48
AAO and Pt NPs (30 nm), suggesting that Ru NPs have a lower particles have been reported in related literature.
Genera-
affinity than NaA (Fig. 5d).
tion of hydroxyl radical and accelerating electron transfer
We further demonstrated that Ru NPs can mimic the activity process are two most possible mechanisms responsible for the
of AAO by using ESR spectroscopy, which is the most reliable enzyme mimic activities of Ru NPs (Scheme S1†). To test the
and direct method for identication and quantication of hypothesis, we applied two models to measure the hydroxyl
short-lived free radicals. The intermediate AAc is an indicator radical generation and electron transfer process. First, Ru/
for the oxidation of the active reducing agent NaA. AAc can be
2 2
H O /DMPO and Ru/DMPO systems were applied to examine
easily detected by ESR spectroscopy at room temperature whether the enzyme mimicking property of Ru NPs was related
without using any spin trap or spin label. The ESR spectra of AAc to the generation of the hydroxyl radical, which could oxidize
were shown in Fig. 6. There was no ESR signal in the pure NaA the substrate molecules. The catalytic mechanism of Ru NPs
solution which indicates that NaA itself has not been oxidized was evaluated in pH conditions of 1.12, 3.2, 7.4 and 10.96,
within 3 min. However, signicant ESR signals were detected respectively. The sample solution contains 5 mM DMPO, and
ꢀ
1
when mixing NaA with different concentrations of Ru NPs or different concentrations of Ru NPs (25, 50, 75, 100, 150 mg mL
1
)
ꢀ1
2 2
U mL AAO. The oxidation of NaA to AAc was dependent on in the absence or presence of 1 mM or 0.1 mM H O . All of these
the concentration of Ru NPs (Fig. 6a and b). At low NaA samples showed no ESR signal of DMPO–OH adduct (Fig. S7a†).
concentrations, the ESR signal rapidly increased. At high NaA The above results are further supported by ESR measurements
ꢀ1
Fig. 6 (a) ESR spectra upon oxidation of NaA using 1 U mL AAO or
variable concentrations of Ru NPs in presence of 5 mM NaA after 3 min
incubation. (b) Effect of Ru NP concentration on NaA oxidation after
incubation for 3 min. (c) Effect of NaA concentration on the oxidation Fig. 7 (a) ESR spectra of CPc obtained from samples containing 20 mM
ꢀ1
in presence of 50 mg mL Ru NPs after incubation for 3 min. (d) ESR CPH and variable concentrations of Ru NPs after mixing for 10 min. (b)
spectra of samples containing 0.1 mM CTPO, 5 mM NaA, 10 mM PBS CPc ESR signal intensity vs. time in the absence and presence of Ru NPs
7.4 and different concentrations of Ru NPs after mixing for 5 min.
with different concentrations.
This journal is © The Royal Society of Chemistry 2017
RSC Adv., 2017, 7, 52210–52217 | 52215

View Article Online
RSC Advances
Paper
using BMPO as a spin trap (Fig. S7b†). Thus, the catalytic
behavior of Ru NPs can not be assumed to the cOH generation.
It is very likely that Ru NPs act as an efficient electron transfer
intermediate and facilitate the electron transfer between
4 T. Matsuo, A. Hayashi, M. Abe, T. Matsuda, Y. Hisaeda and
T. Hayashi, J. Am. Chem. Soc., 2009, 131, 15124–15125.
5 Y. Xianyu, Y. Chen and X. Jiang, Anal. Chem., 2015, 87,
10688–10692.
substrates and H
surface of Ru NPs and donated electrons to Ru NPs, resulting in
increased of electron density on Ru NPs. Then the electrons
transferred from Ru NPs to H O or O and facilitated the
2 2 2
oxidation of substrates. This assumption is supported by ESR
2
O
2
or O
2
. Substrates were absorbed onto the
6 Y. Lin, J. Ren and X. Qu, Acc. Chem. Res., 2014, 47, 1097–1105.
7 Y. Lin, J. Ren and X. Qu, Adv. Mater., 2014, 26, 4200–4217.
8 W. He, W. Wamer, Q. Xia, J.-J. Yin and P. P. Fu, J. Environ. Sci.
Health, Part A: Toxic/Hazard. Subst. Environ. Eng., 2014, 32,
186–211.
measurements using CPH as a spin probe, which is oen used
9 J. Huang, L. Lin, D. Sun, H. Chen, D. Yang and Q. Li, Chem.
Soc. Rev., 2015, 44, 6330–6374.
58,59
in ESR studies of electron transfer in biological system.
CPH
is an ESR silent probe, and can be oxidized to form CP-nitroxide 10 L. Gao, M. Liu, G. Ma, Y. Wang, L. Zhao, Q. Yuan, F. Gao,
radicals (CPc) with a typical ESR spectrum of three lines with
intensity ratios of 1 : 1 : 1. As shown in Fig. 7a, CPH itself is ESR
R. Liu, J. Zhai, Z. Chai, Y. Zhao and X. Gao, ACS Nano,
2015, 9, 10979–10990.
silent. Upon addition of Ru NPs, a triplet ESR signal appeared 11 Y. Liu, D. L. Purich, C. Wu, Y. Wu, T. Chen, C. Cui, L. Zhang,
with hyperne splitting constant a
oxidation of CPH. The signal intensity increased in a time and
dose-dependent manner (Fig. 7b).
N
¼ 16.2 G, implying of the
S. Cansiz, W. Hou, Y. Wang, S. Yang and W. Tan, J. Am.
Chem. Soc., 2015, 137, 14952–14958.
12 C.-P. Liu, T.-H. Wu, Y.-L. Lin, C.-Y. Liu, S. Wang and S.-Y. Lin,
Small, 2016, 12, 4127–4135.
1
3 Y. Hu, H. Cheng, X. Zhao, J. Wu, F. Muhammad, S. Lin, J. He,
L. Zhou, C. Zhang, Y. Deng, P. Wang, Z. Zhou, S. Nie and
H. Wei, ACS Nano, 2017, 11, 5558–5566.
Conclusions
In summary, this study has introduced Ru NPs mimicking HRP
and oxidase functionalities. Ru NPs could catalyze the oxidation 14 L. Gao, J. Zhuang, L. Nie, J. Zhang, Y. Zhang, N. Gu, T. Wang,
of substrate TMB, OPD and DA in the presence of H
produce the color products. We also report for the rst time that
2
O
2
to
J. Feng, D. Yang, S. Perrett and X. Yan, Nat. Nanotechnol.,
2007, 2, 577–583.
Ru NPs possess intrinsic oxidase-like activity, which could 15 X. Yan, Y. Song, X. Wu, C. Zhu, X. Su, D. Du and Y. Lin,
catalyze the oxidization of TMB and NaA by dissolved molecular Nanoscale, 2017, 9, 2317–2323.
oxygen. The HRP-like and oxidase-like activities of Ru NPs were 16 H. Jia, D. Yang, X. Han, J. Cai, H. Liu and W. He, Nanoscale,
found to be relevant to the concentrations of Ru NPs. The 2016, 8, 5938–5945.
enzyme mimicking activities of the Ru NPs might originate 17 W. Zhang, S. Hu, J.-J. Yin, W. He, W. Lu, M. Ma, N. Gu and
from their characteristic of accelerating electron transfer Y. Zhang, J. Am. Chem. Soc., 2016, 138, 5860–5865.
between substrates and H or O . Our ndings offer a better 18 T. Naganuma, Nano Res., 2017, 10, 199–217.
understanding of enzyme-mimicking activities of Ru NPs and 19 A. A. Vernekar, D. Sinha, S. Srivastava, P. U. Paramasivam,
2
O
2
2
should provide important insights for future applications.
P. D'Silva and G. Mugesh, Nat. Commun., 2014, 5, 5301.
0 S. Wang, R. Cazelles, W.-C. Liao, M. V ´a zquez-Gonz ´a lez,
A. Zoabi, R. Abu-Reziq and I. Willner, Nano Lett., 2017, 17,
2
Conflicts of interest
2043–2048.
There are no conicts of interest to declare.
21 M. V ´a zquez-Gonz ´a lez, W.-C. Liao, R. Cazelles, S. Wang, X. Yu,
V. Gutkin and I. Willner, ACS Nano, 2017, 11, 3247–3253.
2
2 A. M. Fracaroli, P. Siman, D. A. Nagib, M. Suzuki,
H. Furukawa, F. D. Toste and O. M. Yaghi, J. Am. Chem.
Soc., 2016, 138, 8352–8355.
Acknowledgements
G.-J. Cao appreciates the National Natural Science Foundation
of China (Grant No. 21601035) and Natural Science Foundation 23 S. Singh, K. Mitra, A. Shukla, R. Singh, R. K. Gundampati,
of Fujian Province (Grant No. 2016J01043) for partial support.
N. Misra, P. Maiti and B. Ray, Anal. Chem., 2017, 89, 783–791.
This work was also supported by a regulatory science grant 24 Y. Wang, Y. Zhu, A. Binyam, M. Liu, Y. Wu and F. Li, Biosens.
under the FDA Nanotechnology CORES Program. This paper is Bioelectron., 2016, 86, 432–438.
not an official U.S. FDA guidance or policy statement. No official 25 H.-H. Zeng, W.-B. Qiu, L. Zhang, R.-P. Liang and J.-D. Qiu,
support or endorsement by the U.S. FDA is intended or should
be inferred.
Anal. Chem., 2016, 88, 6342–6348.
26 H. Yang, J. Xiao, L. Su, T. Feng, Q. Lv and X. Zhang, Chem.
Commun., 2017, 53, 3882–3885.
2
7 S. Zhang, D. Zhang, X. Zhang, D. Shang, Z. Xue, D. Shan and
X. Lu, Anal. Chem., 2017, 89, 3538–3544.
Notes and references
1
2
3
S. J. Benkovic and S. Hammes-Schiffer, Science, 2003, 301, 28 L. Jiang, S. Fernandez-Garcia, M. Tinoco, Z. Yan, Q. Xue,
1
196–1202.
S. J. Benkovic and S. Hammes-Schiffer, Science, 2006, 312,
08–209.
J. Barber, Chem. Soc. Rev., 2009, 38, 185–196.
G. Blanco, J. J. Calvino, A. B. Hungria and X. Chen, ACS
Appl. Mater. Interfaces, 2017, 9, 18595–18608.
2
29 K. Wang, N. Li, J. Zhang, Z. Zhang and F. Dang, Biosens.
Bioelectron., 2017, 87, 339–344.
52216 | RSC Adv., 2017, 7, 52210–52217
This journal is © The Royal Society of Chemistry 2017

View Article Online
Paper
RSC Advances
3
3
3
0 H.-B. Noh and Y.-B. Shim, J. Mater. Chem. A, 2016, 4, 2720– 46 X. Xia, J. Zhang, N. Lu, M. J. Kim, K. Ghale, Y. Xu,
2
728.
E. McKenzie, J. Liu and H. Ye, ACS Nano, 2015, 9, 9994–
10004.
47 H. Su, D.-D. Liu, M. Zhao, W.-L. Hu, S.-S. Xue, Q. Cao,
X.-Y. Le, L.-N. Ji and Z.-W. Mao, ACS Appl. Mater. Interfaces,
2015, 7, 8233–8242.
1 J. Xie, X. Zhang, H. Wang, H. Zheng, Y. Huang and J. Xie,
TrAC, Trends Anal. Chem., 2012, 39, 114–129.
2 X. Wang, Y. Hu and H. Wei, Inorg. Chem. Front., 2016, 3, 41–
60.
3
3
3 Y. Jv, B. Li and R. Cao, Chem. Commun., 2010, 46, 8017–8019. 48 M. Cui, J. Zhou, Y. Zhao and Q. Song, Sens. Actuators, B, 2017,
4 C. Wang, Y. Shi, Y.-Y. Dan, X.-G. Nie, J. Li and X.-H. Xia,
Chem.– Eur. J., 2017, 23, 6717–6723.
243, 203–210.
49 H. Ye, J. Mohar, Q. Wang, M. Catalano, M. J. Kim and X. Xia,
Science Bulletin, 2016, 61, 1739–1745.
3
3
3
3
3
4
5 M. C. Ortega-Liebana, J. L. Hueso, R. Arenal and
J. Santamaria, Nanoscale, 2017, 9, 1787–1792.
50 G.-J. Cao, X. Jiang, H. Zhang, J. Zheng, T. R. Croley and
J.-J. Yin, J. Environ. Sci. Health, Part C: Environ. Carcinog.
Ecotoxicol. Rev., 2017, DOI: 10.1080/10590501.2017.1391516.
6 W. He, Y. Liu, J. Yuan, J.-J. Yin, X. Wu, X. Hu, K. Zhang, J. Liu,
C. Chen, Y. Ji and Y. Guo, Biomaterials, 2011, 32, 1139–1147.
7 Y.-T. Zhou, W. He, W. G. Wamer, X. Hu, X. Wu, Y. M. Lo and 51 P. J. Tarcha, V. P. Chu and D. Whittern, Anal. Biochem., 1987,
J.-J. Yin, Nanoscale, 2013, 5, 1583–1591. 165, 230–233.
8 W. He, X. Han, H. Jia, J. Cai, Y. Zhou and Z. Zheng, Sci. Rep., 52 C. Zhao, Z. Jiang, R. Mu and Y. Li, Talanta, 2016, 159, 365–
017, 7, 40103. 370.
9 U. Carmona, L. Zhang, L. Li, W. M u¨ nchgesang, E. Pippel and 53 M. V ´a zquez-Gonz ´a lez, R. M. Torrente-Rodr ´ı guez, A. Kozell,
2
M. Knez, Chem. Commun., 2014, 50, 701–703.
W.-C. Liao, A. Cecconello, S. Campuzano, J. M. Pingarr ´o n
0 C. Chen, S. Fan, C. Li, Y. Chong, X. Tian, J. Zheng, P. P. Fu,
and I. Willner, Nano Lett., 2017, 17, 4958–4963.
X. Jiang, W. G. Wamer and J.-J. Yin, J. Mater. Chem. B, 2016, 54 E. Gitto, D.-X. Tan, R. J. Reiter, M. Karbownik,
4
, 7895–7901.
L. C. Manchester, S. Cuzzocrea, F. Fulia and I. Barberi, J.
Pharm. Pharmacol., 2001, 53, 1393–1401.
55 S. J. Padayatty, A. Katz, Y. Wang, P. Eck, O. Kwon, J.-H. Lee,
S. Chen, C. Corpe, A. Dutta, S. K. Dutta and M. Levine, J. Am.
Coll. Nutr., 2003, 22, 18–35.
4
4
4
4
1 L. Jin, Z. Meng, Y. Zhang, S. Cai, Z. Zhang, C. Li, L. Shang and
Y. Shen, ACS Appl. Mater. Interfaces, 2017, 9, 10027–10033.
2 J. Wei, X. Chen, S. Shi, S. Mo and N. Zheng, Nanoscale, 2015,
7
, 19018–19026.
3 T. Wen, W. He, Y. Chong, Y. Liu, J.-J. Yin and X. Wu, Phys. 56 M. Karajibani, M. Hashemi, F. Montazerifar and M. Dikshit,
Chem. Chem. Phys., 2015, 17, 24937–24943. J. Nutr. Sci. Vitaminol., 2010, 56, 436–440.
4 C. Ge, G. Fang, X. Shen, Y. Chong, W. G. Wamer, X. Gao, 57 Y.-T. Zhou, J.-J. Yin and Y. M. Lo, Magn. Reson. Chem., 2011,
Z. Chai, C. Chen and J.-J. Yin, ACS Nano, 2016, 10, 10436–
0445.
5 Q. Wang, L. Zhang, C. Shang, Z. Zhang and S. Dong, Chem.
49, S105–S112.
1
58 S. I. Dikalov, I. A. Kirilyuk, M. Voinov and I. A. Grigor'ev, Free
Radical Res., 2011, 45, 417–430.
4
Commun., 2016, 52, 5410–5413.
59 W. He, H.-K. Kim, W. G. Wamer, D. Melka, J. H. Callahan
and J.-J. Yin, J. Am. Chem. Soc., 2014, 136, 750–757.
This journal is © The Royal Society of Chemistry 2017
RSC Adv., 2017, 7, 52210–52217 | 52217