Synthesis of reduced graphene oxide-iron nanoparticles with superior enzyme-mimetic activity for biosensing application

Article abstract of DOI:10.1016/j.jallcom.2015.03.176

Development of enzyme-mimetic catalysts with sustainability and environmental benignancy has gained considerable attention with the growing demands for large-scale applications in recent years. Here, we demonstrate that the reduced graphene oxide (RGO)-iron nanoparticles (INs) can be utilized as the highly active and cost-effective enzyme-mimetic catalysts for the first time, which have been successfully synthesized by a facile iron-self-catalysis process at room temperature. Benefitting from synergetic effects between RGO and INs, the RGO-INs could efficiently catalyze the oxidization of 3,3′,5,5′-tetramethylbenzidine (TMB) in the presence of H₂O₂ to produce a typical color reaction, showing the much better peroxidase-like activity than that of each individual part. The mechanistic insight into the enhanced peroxidase-like activity of the RGO-INs was investigated systematically. On the basis of the enzyme-mimetic activity of the RGO-INs, the simple, sensitive, selective and cost-effective colorimetric assays for the detection of hydrogen peroxide and glucose with naked eyes were successfully established. The RGO-INs showed several prominent advantages, such as facile preparation, low cost, tunability in catalytic activity, and low detection limit, over natural peroxidase or other nanomaterial-based alternatives, holding great potential as enzymatic mimics for biosensing applications.

Full text of DOI:10.1016/j.jallcom.2015.03.176

Journal of Alloys and Compounds 639 (2015) 470–477
Contents lists available at ScienceDirect
Journal of Alloys and Compounds
journal homepage: www.elsevier.com/locate/jalcom
Synthesis of reduced graphene oxide-iron nanoparticles with superior
enzyme-mimetic activity for biosensing application
⇑
⇑
Lili Li, Chunmei Zeng, Lunhong Ai , Jing Jiang
Chemical Synthesis and Pollution Control Key Laboratory of Sichuan Province, College of Chemistry and Chemical Engineering, China West Normal University, Nanchong 637002,
PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
Development of enzyme-mimetic catalysts with sustainability and environmental benignancy has gained
considerable attention with the growing demands for large-scale applications in recent years. Here, we
demonstrate that the reduced graphene oxide (RGO)-iron nanoparticles (INs) can be utilized as the highly
active and cost-effective enzyme-mimetic catalysts for the first time, which have been successfully syn-
thesized by a facile iron-self-catalysis process at room temperature. Benefitting from synergetic effects
between RGO and INs, the RGO-INs could efficiently catalyze the oxidization of 3,30,5,50-tetramethylben-
zidine (TMB) in the presence of H2O2 to produce a typical color reaction, showing the much better peroxi-
dase-like activity than that of each individual part. The mechanistic insight into the enhanced
peroxidase-like activity of the RGO-INs was investigated systematically. On the basis of the enzyme-
mimetic activity of the RGO-INs, the simple, sensitive, selective and cost-effective colorimetric assays
for the detection of hydrogen peroxide and glucose with naked eyes were successfully established. The
RGO-INs showed several prominent advantages, such as facile preparation, low cost, tunability in cat-
alytic activity, and low detection limit, over natural peroxidase or other nanomaterial-based alternatives,
holding great potential as enzymatic mimics for biosensing applications.
Received 28 January 2015
Received in revised form 19 March 2015
Accepted 22 March 2015
Available online 27 March 2015
Keywords:
Iron nanoparticles
Reduced graphene oxide
Peroxidase-like activity
H2O2
Glucose
Ó 2015 Elsevier B.V. All rights reserved.
1. Introduction
been discovered to possess intrinsic peroxidase-like activity and
shown promising potential in environmental remediation [17]
Natural enzymes are well-known class of biological catalysts
with high substrate specificities and high activity under mild reac-
tion conditions, which have found significant and widespread
applications in medicine, chemical industry, food processing and
agriculture [1,2]. However, they intrinsically own some drawbacks,
including limited natural sources, inherent instability, and requir-
ing expensive and time-consuming purification, restricting their
practical applications greatly. To overcome these limitations of
natural enzymes while maintain the high reactivity, numerous
efforts have been focused on the design and construction of artifi-
cial enzymes that can mimic the complexities and functions of
natural enzymes. Particularly, the merging of nanotechnology with
biology has ignited research interest for designing and seeking
functional nanomaterials mimicking catalytically active enzymes
[3,4]. For instance, inorganic metal oxides (Fe3O4 [5], MnO2 [6],
Co3O4 [7], CuO [8], TiO2 [9], V2O5 [10]), metal sulfides (CuS [11],
FeS [12], CdS [13], MoS2 [4]) and carbon (carbon nanodots [14],
graphene oxide [15], carbon nanotube [16]) nanomaterials have
and biosensing applications. Meanwhile, noble metal nanoparticles
have been emerging as another type of the most promising mate-
rials with excellent peroxidase-mimetic activity because of their
unique physical and chemical properties. For example, Au [18], Pt
[19], Ag [20] nanoparticles have been reported separately to pos-
sess an intrinsic peroxidase-like activity. Their nanostructured
alloys such as Au@Pt [21], AgM (M = Au, Pd, Pt) [22], Au@PtAg
[23], and AuPd [24] achieve excellent ability toward peroxidase
mimicry for catalytic acceleration of specific reaction as well.
However, their practical large-scale applications are restricted dra-
matically by the inevitable disadvantages of high price and scar-
city. Towards this end, it is of considerable interest and great
significance to develop cheap and earth-abundant alternatives to
noble metal catalyst to achieve high efficiency at low cost.
Iron is an earth abundant metal, and its oxides have been
demonstrated as highly active peroxidase mimetics [25,26].
Apparently, utilization of metallic iron as peroxidase mimetics
for biosensing application is remarkably attractive because of the
anticipated sustainability and environmental benignancy. Very
recently, Huang and his coworkers have been demonstrated a
non-noble metal-based bimetallic Fe–Co nanoparticles indeed
⇑
Corresponding authors. Tel./fax: +86 817 2568081.
E-mail addresses: ah_aihong@163.com (L. Ai), 0826zjjh@163.com (J. Jiang).
http://dx.doi.org/10.1016/j.jallcom.2015.03.176
0925-8388/Ó 2015 Elsevier B.V. All rights reserved.

L. Li et al. / Journal of Alloys and Compounds 639 (2015) 470–477
2.3. Characterization
471
possesses peroxidase-like activity which exhibits high affinity to
H2O2 and found that the combination with metallic cobalt gener-
ates the synergistic effect to enhance the peroxidase-like activity
of monometallic iron [27]. However, to a certain extent, it is still
not fully understood or very limitedly what dominates the catalytic
mechanism and how metallic iron interacts with substrates during
biosensing application. More surprisingly, to the best of our knowl-
edge, there are no reports in the literature on the peroxidase mim-
icking behaviors of other metallic iron-based nanoparticles.
To maximize the reactivity and stability of metal NPs, a robust
support is usually required to protect them against dissolution
and aggregation. Owing to its large specific surface area, great
mechanical strength, and low manufacturing cost, graphene has
been considered a promising candidate as a new two-dimensional
(2D) carrier to support metal nanoparticles [28,29]. Moreover, gra-
phene-family materials represent the interesting properties which
would be favorable for the design of nanomaterial-based enzyme
mimics [30]. The opened surface areas of graphene are readily
accessible to substrates with a small diffusion and transport bar-
rier, while its rich surface chemistry ensures the stability of the
supported systems. Thus, it is reasonably expected that graphene
would be an effective 2D carrier for loading metal nanoparticles
to create highly active enzyme mimetics and to further realize
the cooperatively enhanced performances by the combination of
the respective properties of each component.
The powder X-ray diffraction (XRD) measurements were recorded on a Rigaku
Dmax/Ultima IV diffractometer with monochromatized Cu
Ka radiation
(k = 0.15418 nm). The morphology was observed with a JEOL JSM-6510LV scanning
electron microscope (SEM) and transmission electron microscope (TEM, FEI Tecnai
G20). The elemental composition of the samples were characterized by energy-dis-
persive X-ray spectroscopy (EDS, Oxford instruments X-Max). The Fourier trans-
form infrared (FTIR) spectrum was measured on
a Nicolet 6700 FTIR
spectrometric Analyzer using KBr pellets. Raman measurements were carried out
by a confocal laser micro-Raman spectrometer (Thermo DXR Microscope, USA).
The laser was 633 nm with a 5 mW.
2.4. Peroxidase-like catalytic activity of the RGO-INs
To evaluate the peroxidase-like catalytic activity of the RGO-INs, the catalytic
oxidation of the peroxidase substrate TMB in the presence of H2O2 was tested.
The measurements were carried out by monitoring the absorbance change of
TMB at 652 nm. In
(1 mg mLꢁ1) was mixed in 1600
adding 400 L of TMB solution (1 mM, ethanol solution). Then, 20
a
typical experiment, 80
L of NaAc buffer solution (pH 3.0), followed by
L of H2O2 with
lL of the RGO-INs dispersion
l
l
l
various concentrations was added into the mixture. The mixed solution was incu-
bated at 40 °C for 30 min. For comparison, the control experiments were also con-
ducted under the same conditions by using bare RGO, INs or their physical mixture
as catalysts. In addition, the influences of reaction buffer pH and incubation tem-
perature on the peroxidase-like catalytic activity of the RGO-INs were also
investigated.
2.5. Bioassay
Kinetic measurements were carried out in time course mode by monitoring the
absorbance change at 652 nm. To investigate the mechanism, assays were carried
out by varying concentrations of TMB at a fixed concentration of H2O2 or vice versa.
Bearing these issues in mind, we herein report a facile and
rational synthesis of reduced graphene oxide-iron nanoparticles
(RGO-INs) by an iron-self-catalysis process and demonstrate the
resulting RGO-INs can be utilized as the highly active and cost-
effective enzyme-mimetic catalysts for the first time. The synthetic
strategy is simple, inexpensive and scalable, and the whole pro-
cessing is completely at room temperature. Benefitting from syner-
getic effects between INs and RGO, the RGO-INs could catalyze the
oxidation of different peroxidase substrates in the presence of
H2O2 to produce typical color reactions, which showed the better
peroxidase-like activity than that of each individual part. Based
on the peroxidase-like behavior of the RGO-INs, the simple, sensi-
tive, selective and cost-effective colorimetric assays for the detec-
tion of H2O2 and glucose with naked eyes were successfully
established.
Experiments were performed using 38 l lL of reaction buf-
g mLꢁ1 RGO-INs in 1600
fer (0.2 M NaAc, pH 3.0) with 0.09 mM TMB as substrate, or 0.47 mM H2O2, unless
otherwise stated. The apparent kinetic parameters were calculated using
Lineweaver–Burk plots of the double reciprocal of the Michaelis–Menten equation:
1/v = Km/Vmax(1/[S] + 1/Km), where v is the initial velocity, Vmax is the maximal reac-
tion velocity, [S] is the concentration of substrate, Km is the Michaelis constant
[14,31].
2.6. Detection of glucose using the RGO-INs as peroxidase-like mimetics
Glucose detection was carried out as follows: firstly, 100
solution (1.0 mg mLꢁ1) and 100
L of -glucose with various concentration were
mixed in 500 L of NaH2PO4 buffer (0.5 mM, pH 7.0) and incubated at 37 °C for
1 h; then 200
lL of GOx aqueous
l
D
l
l
L of TMB (5 mM, ethanol solution), 100 lL of the RGO-INs stock solu-
tion (1 mg mLꢁ1) and 4.00 mL of NaAc buffer (0.2 M, pH 3.0) were successively
added to the glucose reaction solution; finally, the mixed solution was incubated
at 40 °C for 30 min for standard curve measurement.
2. Experimental
3. Results and discussion
2.1. Materials
Graphite powder, KMnO4, NaNO3, H2SO4 (98%), FeCl3ꢀ6H2O, NaBH4, acetic acid
(HAc), sodium acetate (NaAc) and H2O2 (30 wt%) were purchased from Kelong
Chemical Reagents Company (Chengdu, China). Na2HPO4, KH2PO4, polyvinylpyrroli-
done (PVP) and 3,30,5,50-tetramethylbenzidine dihydrochloride (TMB) were pur-
chased from Aladin Ltd (Shanghai, China). Glucose, fructose, lactose, maltose, and
Our synthesis strategy was based on the in situ simultaneous
reduction of Fe3+ and GO at room temperature. Graphite oxide with
abundant oxygenous groups was highly negatively charged, which
was firstly liquid exfoliated by ultrasonication to form a stable
aqueous solution. When Fe3+ ions were introduced, the electro-
static interactions between positively charged Fe3+ and GO pro-
vided a necessary driving force for the effective enrichment of
Fe3+ onto GO. During the chemical reduction process, iron nanopar-
ticles (INs) were then in situ formed on the surface of the reduced
GO. Importantly, reduction of GO in our case can be achieved in a
mild condition (e.g. room temperature and short reaction time). As
previously reported, the reactive hydrogen atoms released from
the NaBH4 hydrolysis can reduce the oxygenous groups on the
GO, which is a critical step toward the chemical reduction ability
of NaBH4 [32]. It is believed that in situ formed INs can act as the
catalyst to accelerate the NaBH4 hydrolysis to release active
hydrogen. To support this hypothesis, we further monitored the
amounts of hydrogen generation during the synthesis process
(Fig. 2). It is interestingly found that GO was incapable for hydro-
gen generation, while both the INs and RGO-INs could induce
in situ fast hydrogen generation, confirming that the formation of
glucose oxidase (GOx, 340 U mgꢁ1
) were purchased from Sangon Biochemical
Engineering Technology Co., Ltd (Shanghai, China). All chemicals used in this study
were analytical reagent grade. Freshly deionized water was used to prepare all solu-
tions and conduct all of the tests. Graphite oxide was synthesized from oxidation of
nature graphite powder by a modified Hummers method.
2.2. Preparation of reduced graphene oxide-iron nanoparticles (RGO-INs)
The preparation procedure for the RGO-INs was schematically illustrated in
Fig. 1. In a typical synthesis, the required amount of graphite oxide was dispersed
in deionized water by ultrasonication for 1 h. 5 mL of aqueous solution containing
FeCl3ꢀ6H2O (0.6757 g) and PVP (0.111 g) was poured into GO solution and sonicated
for another one hour. Afterward, 20 mL of NaBH4 (1.25 g) solution was added drop-
wise into the above mixture solution under constant stirring at room temperature.
After the reaction of 3 h, the black solid was collected by filtration, washed with
ethanol several times, and dried in flowing nitrogen. The catalysts with different
ratios of RGO to INs for the designed RGO-INs were listed in Table S1 (see
Supporting Information). Among all the samples, the RGO-INs-5% showed the best
peroxidase-like activity; therefore, if no further notification is provided, the RGO-
INs notation in this study refers to RGO-INs-5%.

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L. Li et al. / Journal of Alloys and Compounds 639 (2015) 470–477
Fig. 1. Schematic diagrams of (a) the synthesis process of the RGO-INs and (b) colorimetric detection of glucose using the RGO-INs as peroxidase-like mimetics.
of nanonecklace-like iron and crumpled silk-like morphology of
graphene. The nanonecklaces had diameters of 20–50 nm and
lengths of several micrometers and were well distributed onto
the surface of the flexible graphene nanolayers. Moreover, no free
nanoparticles were found outside of the graphene nanolayers dur-
ing the TEM observation, indicating a perfect combination between
the RGO and the INs. The HRTEM image (Fig. 3c) of the RGO-INs
further revealed that the core–shell structure of INs, which can
be ascribed to the surface iron combined with oxygen of atmo-
sphere to form oxide layers during the catalyst preparation and
storage process, consistent with previous reports [33–35].
Fig. S1 shows the XRD patterns of the GO, bare INs and RGO-INs.
In the XRD pattern of GO, a sharp and intense diffraction peak was
observed at 2h = 10.3°, which is associated with the interplanar
spacing of GO sheets, corresponding to the characteristic (001)
reflection of GO. The RGO-INs exhibit face-centered cubic (fcc)
Fe0 phase (JCPDS no. 88-2324) similar to the pure INPs, where a
Fig. 2. Hydrogen generation during the sample synthesis process at room
broad characteristic diffraction peak centered at 2h = 44.9° was
observed. In addition, the disappearance of the peak at 2h = 10.3°
confirms that the graphite oxide has been exfoliated to GO and
some oxygen-containing groups in GO have been removed during
the chemical reduction process. The composition of the RGO-INs
was determined by X-ray energy-dispersive spectroscopy (EDS),
as shown in Fig. S2, which unambiguously demonstrates the coex-
istence of Fe, O and C elements in the product. The oxygen element
could come from the residual oxygen-containing functional groups
on RGO and the oxide layer of INPs. Fig. 4a shows the FTIR spectra
of GO and RGO-INs. The characteristic absorption peaks of GO were
observed at 3439 cmꢁ1 (the OAH stretching mode), 1730 cmꢁ1 (the
C@O stretching mode), 1630 cmꢁ1 (the C@C stretching mode),
1389 cmꢁ1 (the CAOH stretching mode), and 1068 cmꢁ1 (the
CAOAC stretching mode) [36,37]. It can be seen clearly that the
disappearance of C@O peak and the remarkable decrease in
intensity of CAOH and CAOAC peak for GO in the spectrum of
temperature: (a) GO, (b) INs and (c) RGO-INs.
the RGO-INs at room temperature had undergone an iron-self-
catalysis reduction process.
Fig. 3a shows a typical SEM image of the RGO-INs. The iron
nanoparticles present the nanonecklace-like morphology formed
through connection of nanospheres one by one, looking like ‘‘pearl
necklaces’’. The diameters of these nanoparticles were in the range
of 20–50 nm. Meanwhile, they were effectively decorated on the
surface of graphene nanolayers. It should be noted that the
graphene nanolayers hybridized randomly with nanoparticles
endow them with good connection and intimate contact, which
is beneficial to the subsequent catalysis applications. The detailed
morphology of the RGO-INs was further investigated by transition
electron microscopy (TEM). Fig. 3b shows a typical TEM image of
the RGO-INs, which further confirms the structure characteristic
Fig. 3. SEM (a), TEM (b) and HRTEM (c) images of the RGO-INs.

L. Li et al. / Journal of Alloys and Compounds 639 (2015) 470–477
473
O 1s could be fitted by four peaks (Fig. 5c), which are attributed
to the oxygen components on the RGO (OH, CAO: 531.9 eV, C@O:
533.0 eV, and OAC@O: 534.0 eV) [46] and the FeAO bonds
(530.8 eV) of Fe2O3 [45], respectively. Fig. 5d shows the high-
resolution XPS spectrum of the C 1s, which can be deconvoluted
into three surface components, corresponding to the CAC
(284.6 eV), CAO (286.0 eV) and OAC@O (289.5 eV) carbon compo-
nents on the RGO [47]. This result reveals that the graphene in the
RGO-INs has a very high degree of reduction.
The peroxidase-like activities of the as-prepared RGO-INs were
evaluated by the catalytic oxidation of a typical peroxidase sub-
strate 3,30,5,50-tetramethylbenzidine (TMB) that could generate a
blue color reaction in the presence of H2O2. As shown in Fig. 6A,
the RGO-INs present a remarkable catalytic activity toward the oxi-
dation of TMB substrate in the presence of H2O2, which can
develop a typical blue-green color with an intense characteristic
absorbance at 652 nm (curve e). In contrast, the RGO-INs (curve
c) or H2O2 (curve d) alone was incapable to produce a significant
change of the solution color, as compared to that of the blank
solution containing only TMB (curve a). To further confirm the
characteristic peroxidase-like activity of the RGO-INs, other typical
peroxidase substrates such as o-phenylenediamine (OPD) and
1,2,3-trihydroxybenzene (THB) were employed as replacements
for the TMB to proceed the identical catalytic reaction. As shown
in Fig. S3 (see Supporting Information), the RGO-INs can also cat-
alyze the OPD and THB to produce the typical color reactions in
the presence of H2O2. All these observations suggest that the
RGO-INs possess excellent peroxidase-like catalytic activity.
Noticeably, the previously reported results have shown that the
leaching solution from the catalysts could affect the catalytic activ-
ity [31,48]. In order to rule out the possibility that the observed
catalytic activity results from the RGO-INs rather than from the
free leaching ions, we further performed a leaching experiment.
The RGO-INs was incubated in the reaction buffer (pH 3.0) for
2 h and then centrifuged to collect the supernatant. The leaching
solution was then tested towards the TMB oxidation reaction
(Fig. 6B). Evidently, the leaching solution had no catalytic activity,
confirming that the observed peroxidase-like activity was due to
intact RGO-INs. In addition, we further compared the catalytic
activity of the RGO-INs under aerobic and anaerobic conditions
(Fig. 6C). It is clear that both conditions show the comparable
absorbance at 652 nm, indicating that the activity of the RGO-INs
could not be affected by the molecular oxygen in air.
Fig. 4. FTIR (a) and Raman spectra (b) of the GO and RGO-INs.
the RGO-INs, indicating most of oxygen-containing functional
groups in the GO have been removed. Fig. 4b shows the Raman
spectra of GO and RGO-INs. For the GO, two prominent bands at
around 1350 and 1590 cmꢁ1 can be observed, corresponding to
the local defects or disorder atomic arrangement of sp3-carbon
(D-band) and the plane vibration of the sp2-carbon in the two-
dimensional lattice (G-band), respectively [38]. In comparison with
GO (ID/IG = 0.93), it is evident that RGO-INs displays the increased
intensity ratio of the D- to G-band (ID/IG = 1.27), confirming the
efficient reduction of GO during in situ preparation process [39].
In addition, the 2D band at around 2700 cmꢁ1 is the overtone of
the D band, originated from two phonon double resonance, which
is sensitive to the number of graphene layers [40–44]. A weak and
broad 2D band in RGO-INs (inset), which is an indication of disor-
der and coupling of the stacked multilayers of graphene sheets.
The detailed chemical and electronic states of the RGO-INs were
determined by X-ray photoelectron spectroscopy (XPS). Fig. 5
shows the XPS survey spectrum and high-resolution XPS spectra
of the RGO-INs. The XPS survey spectrum (Fig. 5a) demonstrates
that C, O, and Fe elements existed in the RGO-INs. Fig. 5b shows
the high-resolution XPS spectra of Fe2p. The binding energies of
711.6 and 725.3 eV with a satellite signal at 718.8 eV are character-
istic of Fe3+ in Fe2O3; while a weak peak at a low binding energy of
706.0 eV is typical for pure iron [35,45]. Considering the XPS could
only detect the photoelectrons from the outer surface of 10 nm, the
thickness of the Fe2O3 shell in the nanoparticles should be less than
10 nm because the metallic iron signal was detected, consistent
with TEM observation. The high-resolution XPS spectrum of the
Similar to other NPs-based peroxidase mimetics and HRP, the
catalytic activity of the RGO-INs depends on pH, temperature,
and H2O2 concentration. The peroxidase-like activity of RGO-INs
was measured by varying the pH from 2 to 12, the temperatures
from 20 °C to 70 °C, the H2O2 concentration from 0.095 lM to
545 mM. Under our experimental conditions, the optimal pH and
temperature were 3.0, 40 °C and 10 mM H2O2, respectively
(Fig. S4 in the Supporting Information). Based on the above results,
RGO-INs-based H2O2 detection can be carried out in a relatively
wide range of H2O2 concentration.
The peroxidase-like catalytic activity of the RGO-INs is further
compared with pure RGO and free INs. Fig. S5 shows the UV–vis
spectra of the TMB-H2O2 system catalyzed by different catalysts.
As can be seen, pure RGO did not produce an apparent change of
the solution color, indicating its limited peroxidase-like catalytic
activity; while free INs could generate considerable colorimetric
responses, revealing the intrinsic peroxidase-like catalytic activity
of metallic iron. Interestingly, after in situ combination with RGO,
the RGO-INs became more active, which lead to the absorbance
at 652 nm significantly higher than that of free INs. To insight into
the positive effect in the RGO-INs system, we further quantitatively
compare the absorbance values at 652 nm in the presence of differ-
ent catalysts, as shown in Fig. 6D. Obviously, the RGO-INs were

474
L. Li et al. / Journal of Alloys and Compounds 639 (2015) 470–477
Fig. 5. XPS spectra of RGO-INs: (a) survey, (b) Fe 2p, (c) O 1s and (d) C 1s.
more catalytically active than the pure RGO and free INs, which
were about 6.3 times as high as that of pure RGO, 3.3 times that
of free INs, and even 2.2 times higher than the sum of the
absorbance value of the RGO and INs. Furthermore, we carried
out a control experiment on the physical mixture of RGO with
INs (RGO/INs) and found that their physical mixture showed much
weaker catalytic activity than the RGO-INs. These results imply the
favorable synergistic effect existed in RGO-INs system. In addition,
the peroxidase-like activity of the RGO-INs depends closely on the
content of RGO. Among all the RGO-INs samples, the optimal
catalyst was found to be RGO-INs-5% (Fig. S6 in the Supporting
Information), which may be benefited from the favorable dispersi-
bility of INs on RGO and the synergistically effective interaction
between RGO and INs.
To gain insight into how the RGO interacts with INs to achieve
the synergistic catalysis in the RGO-INs, electrochemical evaluation
of the RGO-INs was investigated by the electrochemical impedance
spectroscopy (EIS). Fig. S7 shows EIS Nyquist plots of free INs and
RGO-INs in 0.1 M KCl containing 5 mM [Fe(CN)6]4ꢁ/3ꢁ (see
Supporting Information). It is known that EIS measurement can
be used to determine the charge-transfer resistance as indicated
by the diameter of the preceding semicircle in Nyquist plot.
Normally, the smaller radius means the better ability to transfer
charge. In comparison with free INs, the decreased radius of the
Nyquist arc of RGO-INs was observed, reflecting that the introduced
RGO is benefit to the improvement of charge transfer efficiency in
INs due to its excellent electrical conductivity.
Michaelis–Menten constant (Km) were obtained from the
Lineweaver–Burk plots and listed in Table S2 (see Supporting
Information). Km is generally identified as an indicator of binding
affinity of the enzyme to the substrates and can be applied simi-
larly here to determine enzyme mimic-substrate interaction. The
lower value of Km represents stronger affinity between the enzyme
(catalysts) and substrate (TMB or H2O2). The apparent Km of the
RGO-INs with H2O2 as the substrate was much higher than that
of HRP, which is in agreement with the previous observations that
a higher H2O2 concentration was required to obtain maximal activ-
ity for the RGO-INs [49,30]. The Km value of the RGO-INs with TMB
as substrate is remarkably lower than that of HRP (Table S2 in the
Supporting Information), suggesting that the RGO-INs have a larger
affinity for TMB than HRP. This may be attributed to synergistic
effects in the RGO-INs system, resulting from the excellent ability
of RGO toward the adsorption and enrichment of TMB, which is
critical to the subsequent interfacial charge transfer process.
Additionally, we measured their activity toward TMB and H2O2
with various concentrations, respectively. Figs. S8c and S8d show
the double reciprocal plots of initial velocity against one substrate
concentration obtained over a range of the second substrate
concentrations, which reveal the characteristic parallel lines of a
ping-pong mechanism were characterized with parallel lines,
similar to HPR [25,50]. The experimental facts indicate that the
RGO-INs bind and react with the first substrate and then release
the first product before reacting with the second substrate.
In light of the above results, we would like to discuss the
enhanced peroxidase-like catalytic activity of the RGO-INs. Since
neither H2O2 nor RGO-INs can effectively oxidize TMB, the interac-
tions between RGO-INs and H2O2 and TMB are critical for the
catalytic reaction. The negligible change observed in aerobic and
anaerobic reactions (Fig. 6C) reveal the molecular oxygen in air
plays minor role in the peroxidase-like catalytic process. Similar
to previous observations [33–35], iron oxide was easily generated
To further investigate the mechanism of the peroxidase-like
catalytic activity of the RGO-INs, we further carried out the
steady-state kinetic assays by changing one substrate concentra-
tion while keeping the other substrate concentration constant. As
shown in Figs. S8a and S8b, in a certain range of substrate concen-
trations, typical Michaelis–Menten curves were obtained for H2O2
or TMB, respectively. The maximum initial velocity (Vmax) and

L. Li et al. / Journal of Alloys and Compounds 639 (2015) 470–477
475
Fig. 6. (A) UV–vis spectra and corresponding photographs (inset) of different reaction systems: TMB solution (a), H2O2/RGO-INs (b), TMB/RGO-INs (c), TMB/H2O2 (d), and
TMB/H2O2/RGO-INs (e) in acetate buffer (pH 3.0). ([TMB]: 0.19 mM; [H2O2]: 10 mM; [RGO-INs]: 38
g mLꢁ1). (B) UV–vis spectra of the TMB oxidation system in the presence
l
of the leaching solution and the RGO-INs. (C) UV–vis absorbance at 652 nm of the TMB oxidation system in the presence of the RGO-INs in air and nitrogen. (D) Quantitative
comparison of the catalytic activity of different catalysts.
and grown on the surface of iron nanoparticles when freshly pre-
pared iron nanoparticles were exposed to water, resulting in the
formation of core–shell structure. Furthermore, no observation
on the peroxidase-like catalytic activity of the leaching solution
solidly confirms the tightly bound iron ions in the RGO-INs.
Based on above consideration, we think the in situ formed oxide
shell could guide the effective iron cycling and simultaneously
produce the highly reactive hydroxyl radicals (ÅOH) to initiate
peroxidase-like catalytic reaction, which is indispensable for the
excellent activity of the RGO-INs. As schematically illustrated in
Fig. 7, the in situ formation of surface bound ferrous ions is
thermodynamically favored by electron transfer from INs core to
electrons via surface FeIII–FeII switch for the sustained production
of OH. On the other hand, the alternative electron transfer from
Å
INs to the RGO nanosheets via the conduction band of the iron
oxide shell to reduce H2O2 could be spontaneously driven by the
Ohmic contact between INs and iron oxide shell, because the work
function of Fe0 (4.5 eV) is lower than that of iron oxide (5.6 eV)
[45]. To validate these assumptions, we used XPS analysis to
characterize Fe species in RGO-INs before and after peroxidase-like
catalytic reaction (Fig. S9 in the Supporting Information). It is clear
that the amount of FeIII is decreased while the proportion of FeII is
significantly increased after catalytic reaction, which infers that
FeIII and FeII can mutually transform one into another during the
catalytic reaction, indicating the possible surface FeIII–FeII switch.
We also employ the well-established fluorescence method to
determine ÅOH radicals in the catalytic system (Fig. S10a). It is clear
that the significant change in fluorescent intensity at 425 nm was
the surface of oxide layer (2FeIII + Fe0 ? 2FeII,
DE = 1.21 eV)
Å
[51,52], which would react with H2O2 to produce OH with strong
oxidative ability under acidic pH conditions (FeII + H2O2 ? FeIII
ÅOH + OHꢁ), thus offering effective transmission pathway of
+
Fig. 7. A schematic diagram to illustrate the enhanced peroxidase-like catalytic activity of the RGO-INs.

476
L. Li et al. / Journal of Alloys and Compounds 639 (2015) 470–477
Fig. 8. (a) A dose–response curve for H2O2 detection using the RGO-INs under the optimum conditions (inset: linear calibration plot for H2O2 detection). (b) UV–vis
absorption spectra of oxidazed TMB using the RGO-INs under the optimum conditions for glucose detection. (c) Linear calibration plot for glucose detection. (d) Selectivity
analysis for glucose detection by monitoring the relative absorbance. The analyte concentrations were as follows: 1 mM lactose, 1 mM fructose, 1 mM maltose and 100 lM
glucose. Inset: the color change of different solutions. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this
article.)
observed for RGO-INs/H2O2 system, indicating the effective
generation of OH radicals. Noticeably, no observable PL intensity
oxygen to produce H2O2, the proposed colorimetric method could
be extended for the sensitive detection of glucose by coupling with
GOx. Fig. 8b and c shows typical absorption profiles and concentra-
tion–response curve for glucose detection. Under the optimized
condition, a linear relationship was achieved between the absor-
Å
was detected in the absence of the RGO-INs. This solidly confirms
Å
that the RGO-INs can catalytically activate H2O2 to produce OH
radicals that could react with TMB to produce the color reaction.
To further support this mechanism, the electrocatalytic activity
of the RGO-INs modified glassy carbon electrodes (RGO-INs/GCE)
towards the electrochemical reduction of H2O2 was studied by
using their amperometric response. The reduction current
increased sharply to a steady-state value for the RGO-INs/GCE
upon the addition of an aliquot of H2O2 (Fig. S10b in the
Supporting Information), which shows a notably better response
to H2O2 than the individual INs and RGO. Based on the above
results, it is believed that the peroxidase-like activity of the RGO-
INs could originate from its catalytic activation of H2O2 through
electron transfer to produce ÅOH radicals by an iron-recycling
process.
bance and the glucose concentration in the range of 2.0–30
lM
(R2 = 0.995), with a very low detection limit of 0.8
lM, showing
the higher sensitivity than those of previously reported nanoma-
terials-based peroxidase mimics (Table S3 in the Supporting
Information). To test the selectivity of the RGO-INs-based colori-
metric assay for glucose, control experiments were carried out
using lactose, fructose, and maltose. The selectivity of the colori-
metric method was shown in Fig. 8d. The absorbance at 652 nm
of oxidized TMB with glucose is obviously higher than the glucose
analogs, although the concentrations of these analogs are about
10-fold higher than that of glucose. The signals of glucose analogs
are as low as that of the glucose, and the color difference can be
observed by the naked eye (inset in Fig. 8d). Thus, the colorimetric
method developed here showed high sensitivity and selectivity
towards glucose.
Taking into account to the aforementioned peroxidase-like
activity of the RGO-INs, we therefore developed a simple colori-
metric method for detection of H2O2 based on RGO-INs-catalyzed
color reaction. Fig. 8a shows
response curve for the RGO-INs, where the H2O2 can be detected
in a linear range from 0.76 to 47
M (R2 = 0.995). The detection
limit is estimated to be 0.2 M, which is much lower than that of
previously reported nanomaterials-based peroxidase mimics
(Table S3 in the Supporting Information). Considering that GOx
can specifically catalyze the glucose oxidation in the presence of
a typical H2O2 concentration–
4. Conclusions
l
l
In summary, we have reported the iron-self-catalysis method
for the synthesis of iron nanoparticles anchored in situ on reduced
graphene oxide (RGO-INs) under mild condition. The newly syn-
thesized RGO-INs can catalyze the oxidization of typical peroxidase

L. Li et al. / Journal of Alloys and Compounds 639 (2015) 470–477
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