Sensors and Actuators B 221 (2015) 172–178

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Sensors and Actuators B: Chemical

journal homepage: www.elsevier.com/locate/snb

Synthesis of Mn3 O4 nanoparticles/nitrogen-doped graphene hybrid

composite for nonenzymatic glucose sensor
Suling Yang a,∗ , Gang Li a , Guifang Wang a , Junhong Zhao a , Xiuhuan Gao a , Lingbo Qu a,b
a
College of Chemistry and Chemical Engineering, Anyang Normal University, Anyang 455002, PR China
b
Chemistry and Chemical Engineering School, Henan University of Technology, Zhengzhou 450001, PR China

a r t i c l e i n f o a b s t r a c t

Article history: Through a simple method, a new kind of Mn3 O4 nanoparticles (Mn3 O4 NPs) decorated nitrogen-doped
Received 5 February 2015 graphene (N-GR) was synthesized firstly, and then a Mn3 O4 NPs/N-GR modified carbon paste electrode
Received in revised form 27 May 2015 (CPE) was fabricated. Several techniques, such as X-ray diffraction (XRD), scanning electron microscopy
Accepted 16 June 2015
(SEM), transmission electron microscopy (TEM), X-ray photoelectron spectroscopy (XPS) and electro-
Available online 25 June 2015
chemistry, were performed to characterize Mn3 O4 NPs/N-GR. A large amount of N on the graphene
surface (about 9.86% of atomic ratio) was detected by XPS. Mn3 O4 nanoparticles (50–80 nm) homo-
Keywords:
geneously coated on N-GR. Mn3 O4 NPs/N-GR/CPE was used for sensitive non-enzymatic glucose sensing,
Mn3 O4 nanoparticles
Nitrogen-doped graphene
which exhibited the response current of glucose ca. 10-fold higher than that of pure CPE. Under the opti-
Glucose mum determination conditions, Mn3 O4 NPs/N-GR/CPE provided glucose with a broader detection range
Electro-analysis of 2.5–529.5 ␮M with a low detection limit (1.0 ␮M, S/N = 3). This novel method was successfully used to
determine glucose in human urine sample.
© 2015 Elsevier B.V. All rights reserved.

1. Introduction and realize direct electro-catalytic oxidation of glucose at elec-

Glucose widely takes part in human body’s metabolism [1] and
its determination has important significance in clinical diagno-
sis, health protection and control of food production processes.
The exploitation of efficient glucose sensors characterized by high
sensitivity, reliability, fast response, and excellent selectivity has
received special attention from analytical scientists. In generally,
high sensitivity and stability, fast response and excellent selectiv-
ity are of importance in the measurement, and these properties
are at the forefront of glucose sensor research. Among different
glucose sensors, amperometric sensors and optical sensors for glu-
cose determination occupy important positions [2–5]. Moreover,
among different amperometric sensors for glucose determination,
enzymatic sensors are promising due to their simple equipment,
convenient operation, good selectivity and high sensitivity for glu-
cose detection. However, owing to the intrinsic nature of enzymes,
enzymatic glucose sensors can easily be affected by tempera-
ture, pH value, humidity, and toxic chemicals. As an alternative
strategy, enormous interest has been paid on the employment of
enzyme-free glucose sensors, which would provide convenience
and advantages to avoid the drawbacks in enzyme electrode,
∗ Corresponding author. Tel.: +86 03722900040; fax: +86 3722900040.
E-mail address: yang suling@163.com (S. Yang).
trode surface [6–10]. For example, several nanomaterials, such as
Au–Ag particles/reduced graphene oxide [7], copper–cobalt nanos-
tructures/reduced graphene oxide [8], gold nanoparticles [9] and
CuO nanowire [10], have been designed and employed as sensing
materials for fabricating non-enzymatic glucose sensors, which
overcome the disadvantages of enzymatic electrochemical glucose
sensors to some extent.
Graphene, a novel carbon nanomaterial with a one-atom-thick
planar sheet carbon structure arranged in a two dimensional
honeycomb lattice, has attracted scientists’ considerable interests
because of its unique high surface area, rapid electron transport
capability and thermal stability. It has been widely used as the elec-
trode material in the electrochemical sensing. In the past decades,
nitrogen-doped graphene, as a kind of novel carbon nanomate-
rial derived from graphene has received considerable interests due
to the excellent properties, such as the widely available nitrogen
sources, biocompatible C N microenvironment, the low produc-
tion cost, high electrical conductivity and many chemically active
sites [11]. Furthermore, the latest studies indicated that the intro-
duction of nitrogen atoms in carbon grid of graphene not only
increases the active site of adsorption for the metal nanoma-
terials to graphene, but also enhances the interaction between
graphene and nanomaterials, and improves their catalytic activity
[12]. According to Jiang’s report [13], Cu N-GR exhibited significant
enhancement in the catalytic activity for glucose electro-oxidation

http://dx.doi.org/10.1016/j.snb.2015.06.110
0925-4005/© 2015 Elsevier B.V. All rights reserved.
 S. Yang et al. / Sensors and Actuators B 221 (2015) 172–178
Scheme 1. Illustration of the synthesis of the Mn3 O4 NPs/N-GR nanocomposite.
reaction compared to pure Cu. Luo et al. [14] developed a sensor
for electrochemical determination of glucose based on Ag–N-GR,
which exhibited better electro-catalytic performance for glucose
than Ag/graphene. All these reports indicated that N-GR has great
potential as enhanced materials for preparing the electrochemical
sensor, and up to now, it is just the beginning of this fantastic field.
Manganese oxide is an important material, which is widely used
in various fields, such as electrochemistry, soft magnetism, and
catalysis [15,16]. Typically, the properties of materials are closely
related to the specific surface area. To achieve a high performance,
catalytic materials have been synthesized as small as possible to
get a high accessible surface area. Nevertheless, the ultra-small size
nanoparticles (NPs) usually are unstable and easily aggregate into
larger particles due to their high surface energy, which will result
in the decrease of the specific surface area. To avoid the aggrega-
tion, the most commonly used method is to stabilize the NPs by
some capping agents, for example surfactants, polymers or ligands
[17–19]. However, the employment of capping agent, which could
effectively restrain NPs from aggregating, may severely limit the
chemical activities of the NPs, hindering their practical use. To the
best of our knowledge, anchoring Mn3 O4 NPs on GR sheets not only
block aggregation to retain their high specific surface area but also
improve their electrochemical activity [20–23].
In this paper, according to the design, for the first time, man-
ganese oxide nanoparticles (Mn3 O4 NPs) decorated nitrogen-doped
grapheme were produced through a facile thermal treatment by
using N-GR as a novel substrate material. The processes of the
synthesis of Mn3 O4 NPs/N-GR are shown in Scheme 1. In the stir-
ring process, Mn2+ was uniformly dispersed on the surface of the
GO with the assistance of the surface oxygen-containing groups,
and Mn2+ was partially oxidized to Mn3+ by GO and oxygen from
the air [20,22]. After the addition of ethylenediamine (EDA), Mn2+
and Mn3+ was converted to Mn(OH)x . Finally, the above solu-
tion was subjected to the hydrothermal reduction to produce the
Mn3 O4 NPs/N-GR. A possible reaction mechanism for the formation
of Mn3 O4 NPs/N-GR composite is described as follows
Mn2+ (partial) + GO → Mn3+ Mn2+ (partial) + O2 → Mn3+
Mn2+ , Mn3+ + EDA → Mn3 O4
GO + ne− → N-dopedgraphene
Due to the integration of N-GR, the as-prepared Mn3 O4 NPs/N-
GR displayed enhanced electro-catalytic activity to glucose
oxidation, which showed the oxidation peak current ca. 10-fold
higher than that of pure CPE. Furthermore, combining the excellent
properties of N-GR and Mn3 O4 NPs, a novel non-enzymatic glucose
sensor was fabricated, which showed good performances for glu-
cose detection, such as high sensitivity, good stability, fast response,
wide detection range and excellent selectivity.
173
2. Experimental
2.1. Apparatus and reagents
CHI 660E electrochemical system (Shanghai Chenhua Instru-
ment Co., Ltd., Shanghai, China) was used for all the electrochemical
measurement. A conventional three-electrode system was used for
the electrochemical detection, including a Mn3 O4 NPs/N-GR/CPE as
working electrode, an Ag/AgCl (3 M KCl) as reference electrode and
a platinum wire electrode as auxiliary electrode. The scanning elec-
tron microscopy (SEM) was carried out on a Hitachi S-4800 field
emission scanning electron microscope (http://www.hitachi-hta.
com). Transmission electron microscope (TEM) was examined with
a JEM-2000 (JEOL Japan). X-ray diffraction (XRD) analyses were
executed on a Siemens D500 diffractometer with a Cu K␣ source
(1.54056 Å). X-ray photoelectron spectroscopy (XPS) was recorded
on a ThermoFisher-VG Scientific (ESCALAB 250Xi) photoelectron
spectrometer. All the pH values were measured with a PHS-3C pre-
cision pH meter (Leici Devices Factory of Shanghai, China), which
was calibrated with standard buffer solution every day.
Spectral graphite (average particle size 30 ␮m, Shanghai Colloid
Chemical Plant, China) was purchased from Shanghai Carbon Co.,
Ltd. Glucose, dopamine, ascorbic acid and uric acid were purchased
from Alfa Aesar. Ethylenediamine, MnCl2 ·4H2 O, KCl, K4 Fe(CN)6 ,
K3 Fe(CN)6 , NaOH and NaCl were obtained from Beijing Chemical
Company. All the other chemical agents used in this paper were of
analytical grade. All electrochemical experiments were carried out
at room temperature 25 ± 0.1 ◦ C.
2.2. Preparation of Mn3 O4 NPs/N-GR
Firstly, GO was prepared from spectral graphite by a modi-
fied Hummers’ method [24]. Secondly, according to the reported
technique [25] with a slight change, Mn3 O4 NPs/N-GR compos-
ite was obtained in a typical synthetic process. Briefly, 125 mg
of MnCl2 ·4H2 O was added into 5 mL of a 1 mg/mL GO suspen-
(1)
sion followed by intense agitation for 50 min. Then, 0.125 mL of
ethylenediamine (EDA) was added dropwise for 30 min; subse-
(2)
quently, the as-obtained brown suspension was transferred into
(3) a high temperature and high pressure autoclave and subjected
to the hydrothermal reduction at 180 ◦ C for 24 h. Afterward, the
resulting dark precipitates were collected by centrifugation, and
washed with ultrapure water (18.2 M). Finally, the purified pre-
cipitate was dried at 60 ◦ C, and the Mn3 O4 NPs/N-GR was obtained
for further characterization and preparation of sensor. For com-
parison, Mn3 O4 without N-GR was prepared following the same
procedure above but without adding GO at the beginning. N-GR
without Mn3 O4 NPs was synthesized following the same procedure
above but without adding MnCl2 ·4H2 O at the beginning.
174 S. Yang et al. / Sensors and Actuators B 221 (2015) 172–178
Fig. 1. SEM (A) and TEM (B) images of the Mn3 O4 NPs/N-GR nanocomposite.
2.3. Fabrication of modified electrodes
Modified carbon paste electrode (CPE) was prepared by a con-
ventional method. In brief, the above collected Mn3 O4 NPs/N-GR
composite was hand ground with 0.8 g spectral graphite and suit-
able amount of silicone oil in a mortar for 1 h to get the final
homogeneous paste. A portion of the obtained carbon paste was
filled into one end of a glassy tube (d = 3 mm), and then a copper
wire was inserted through the opposite end to fabricate an electri-
cal contact. Finally, the Mn3 O4 NPs/N-GR/CPE was constructed. The
electrode surface was smoothed on a piece of weighing paper just
before use. For comparison, a bare CPE, Mn3 O4 /CPE and N-GR/CPE
were also fabricated by the similar procedure.
3. Results and discussion
3.1. Characterization of the Mn3 O4 NPs/N-GR nanocomposite
In order to verify the composition of the synthesized
Mn3 O4 NPs/N-GR samples, SEM, TEM, XRD, and XPS were used to
characterize Mn3 O4 NPs/N-GR samples.
The morphologies of the prepared Mn3 O4 NPs/N-GR nanocom-
posite are depicted in Fig. 1A and B. One can see in Fig. 1A
that the Mn3 O4 nanoparticles are uniformly coated on the sur-
face of graphene, although a few aggregates were observed. The
TEM observation gives more detailed structural characteristics of
the Mn3 O4 NPs/N-GR nanocomposite. From Fig. 1B, it can be seen
that the Mn3 O4 nanoparticles are coated homogeneously on the
transparent graphene, and further indicates the size of Mn3 O4
nanoparticles ranging from 50 to 80 nm.
Fig. 2. XRD pattern of GO, N-GR and Mn3 O4 NPs/N-GR composite.
XRD patterns of the GO and N-GR are shown in Fig. 2. The fea-
ture diffraction peak of GO appeared at 9.9◦ (0 0 2) as the AB stacking
order was displayed in graphite oxide with layer-to-layer distance
(d-spacing) of 0.849 nm. After the GO is chemically reduced and
doped with N, the diffraction peak located at the 2 value of ∼24◦
was ascribed to the hexagonal structure ((0 0 2) plane) of N-G,
similar to the reported value [15]. For Mn3 O4 /N-GR, the peak of
GO at 9.9◦ disappears, all the diffraction peaks can be indexed to
hausmannite structure Mn3 O4 (JCPDS No. 18-0803). The diffrac-
tion peaks of N-GR are dimly visible, which may be that graphene
sheets sufficiently separated by the loaded Mn3 O4 nanoparticles
[20–22].
XPS spectrum of Mn3 O4 NPs/N-GR is displayed in Fig. 3. With a
survey region of 0–1200 eV, carbon, manganese, nitrogen and oxy-
gen are detected (Fig. 3a). From the C 1s peak (Fig. 3b), we can
see the presence of non-oxygenated carbon, C O (carbonyl groups)
and O C O (carboxyl groups) at 285.0 eV, 287.3 eV and 290.5 eV,
respectively. It is worth noting that the appearance of the peak
at 285.6 eV corresponding to C N confirms the formation of the
covalent bonds between graphene sheets and nitrogen [26,27]. For
the XPS N 1s spectrum (Fig. 3c), the peaks at 399.2, 400.1, 401.5,
and 404.5 eV are assigned to pyridinic N, pyrrolic N, graphitic N,
and oxidized N, respectively [28]. Also, the manganese oxidation
state is confirmed by detecting the high resolution Mn 2p region. As
shown in Fig. 3d, In the Mn 2p region, the binding energy of 653.25
and 641.36 eV attributed to Mn2p3/2 and Mn2p1/2 , respectively. The
splitting width of 11.89 eV is in well agreement with other ear-
lier reports about Mn3 O4 [29–31], which further reveals that the
Mn3 O4 nanoparticles have formed in Mn3 O4 NPs/N-GR composites.
3.2. Electrochemical characteristics of Mn3 O4 NPs/N-GR/CPE
Electrochemical impedance spectroscopy (EIS) is an effective
technique to prove the interfacial changes of modified electrodes.
Generally speaking, the semicircle diameter equals to the electron
transfer resistance (Ret ). Fig. 4 shows the EIS of Mn3 O4 NPs/N-
GR/CPE (a), N-GR/CPE (b), CPE (c), and Mn3 O4 /CPE (d) in 1.0 mM
Fe(CN)6 3−/4− and 0.1 M KCl solution in the frequency range of
0.1–105 Hz. The AC voltage amplitude was 5 mV. The applied poten-
tial was 0.250 V. In order to fit the experimental data, a Randles
equivalence circuit model was shown in Fig. 4 (inset), which
included the ohmic resistance of the electrolyte (Rs ), the electron
transfer resistance (Ret ), the double layer capacitance (Cdl ), and
 S. Yang et al. / Sensors and Actuators B 221 (2015) 172–178
Fig. 3. XPS survey scan (a), C 1s spectrum (b), N 1s spectrum (c), and Mn 2p spectrum (d) of Mn3 O4 NPs/N-GR composite.
Warburg impedance (Zw ). The bare CPE had a big semicircle diam-
eter with a Ret value of 290.9 . While the N-GR/CPE showed low
Ret of 269.2 , due to the presence of conductive N-GR nanosheets
in the electrode that could increase the electron transfer rate of
Fe(CN)6 3−/4− . Due to the presence of semiconductive Mn3 O4 , the
resistance of Mn3 O4 /CPE is 300.0 , which is higher than CPE. This
further confirms the successful modification of Mn3 O4 in the CPE.
However, Mn3 O4 NPs/N-GR hybrid modified CPE showed a very low
Ret of 108.9 , lower than the values of both the N-GR/CPE and CPE.
The results confirmed the prominent capability of Mn3 O4 NPs/N-GR
hybrid to accelerate electron transfer.
3.3. Electrochemical response of Mn3 O4 NPs/N-GR/CPE to glucose
In order to evaluate the catalytic properties of Mn3 O4 NPs/N-
GR/CPE to the oxidation of glucose, cyclic voltammetries (CVs)
Fig. 4. The EIS of Mn3 O4 NPs/N-GR/CPE (a), N-GR/CPE (b), CPE (c), and Mn3 O4 /CPE
(d). EIS condition: frequency range: 105 –0.1 Hz; potential: 0.250 V; perturbation
amplitude: 5 mV; solution: 1.0 mM Fe(CN)6 3−/4− and 0.1 M KCl solution.
175
were recorded in Fig. 5A. Fig. 5A shows the CVs of Mn3 O4 NPs/N-
GR/CPE (a), Mn3 O4 /CPE (b), N-GR/CPE (c), and CPE (d) in 0.1 M
NaOH containing 5.0 mM glucose and Mn3 O4 NPs/N-GR/CPE (e)
without glucose, respectively. As can be seen in Fig. 5, the responses
of Mn3 O4 /CPE, N-GR/CPE, and CPE toward glucose is very weak,
which are due to the very slow electrode kinetic. In contrast,
the Mn3 O4 NPs/N-GR/CPE exhibits obvious electro-oxidation of
glucose with a remarkable enhancement of the oxidation cur-
rent starting from 0.70 to 1.20 V upon the addition of 5.0 mM
glucose. Comparing the CV response of glucose to the above
mentioned electrodes, it is clear that the Mn3 O4 NPs/N-GR/CPE
showed the largest electro-catalytic response for glucose oxi-
dation. As elegantly explained by Kim et al. [8], this is due
to glucose oxidation to gluconolactone catalyzed by conversion
of MnO2 to MnOOH (redox pair III/IV): 2MnO2 + C6 H12 O6 (glu-
cose) → 2MnOOH + C6 H10 O6 (gluconolactone).
To further prove the electro-catalytic activity of Mn3 O4 NPs/N-
GR/CPE, current–time curve was also studied. As shown in Fig. 5B,
compared to Mn3 O4 NPs/N-GR/CPE (a), Mn3 O4 /CPE (b), N-GR/CPE
(c) and CPE (d), the highest faradic current on Mn3 O4 NPs/N-GR/CPE
is obtained, which fully indicates the exciting electro-catalytic
activity of Mn3 O4 NPs/N-GR/CPE toward the oxidation of glucose.
Such enhanced electro-catalytic performance of the Mn3 O4 NPs/N-
GR hybrids may be ascribed to a synergistic effect between N-GR
and the loaded Mn3 O4 NPs, which includes high catalytic active
sites for the glucose oxidation offered by the well-distributed and
high-loading amount of Mn3 O4 NPs and fast electron transfer chan-
nel provided by an efficient electrical network through Mn3 O4 NPs
directly anchoring on the surface of N-GR. Here, the effect of N-
GR in hybrid materials forms an efficient electrical network for
immobilization of highly distributed Mn3 O4 NPs. The large surface
area and high conductivity of N-GR makes the electron transfer rate
176 S. Yang et al. / Sensors and Actuators B 221 (2015) 172–178

Fig. 6. Amperometric responses of Mn3 O4 NPs/N-GR/CPE upon the successive addi-

tions of 100 ␮M glucose at different applied potentials.

in Fig. 7B). Based on the signal to noise ratio of 3, the detection

limit of 1.0 ␮M was obtained. For comparison, nonenzyme glucose
sensing performances based on different nanomaterials from pre-
vious reports and our present study are listed in Table 1. As shown
in Table 1, the performance of the present Mn3 O4 NPs/N-GR-based
sensor is comparable to other similar Mn-based non-enzyme sen-
Fig. 5. (A) CVs of Mn3 O4 NPs/N-GR/CPE (a), Mn3 O4 /CPE (b), N-GR/CPE (c), and CPE sors (such as Refs. [33,34]) in view of sensitivities and linear range.
(d) in the presence of 5.0 mM glucose and Mn3 O4 NPs/N-GR/CPE (e) without glucose
in 0.1 M MaOH; insert: different electrodes in 0.1 M MaOH. Scan rate: 100 mV/s. (B)
i-t curves of Mn3 O4 NPs/N-GR/CPE (a), Mn3 O4 /CPE (b), N-GR/CPE (c) and CPE (d) for
two successive addition 50 ␮M glucose in 0.1 M MaOH at an applied potential of
0.90 V.

of Mn3 O4 NPs/N-GR/CPE obviously higher than that of Mn3 O4 /CPE,

which facilitates the electro-oxidation of glucose correspondingly.

3.4. Effect of applied potentials

Prior to nonenzymatic glucose detection, experimental param-

eters possibly influence the analytical performance. To further
verify the effects of applied potentials on the response of glucose,
the effect of applied potentials was studied on the amperometric
response of Mn3 O4 NPs/N-GR/CPE. Constant potential chronoam-
perometry was realized at the potential range from +0.70 to +0.95 V
on Mn3 O4 NPs/N-GR/CPE for successive additions of 100 ␮M glu-
cose (Fig. 6). Obviously, the current response of 100 ␮M glucose
increases notably from 0.70 to 0.95 V, and then keeps nearly the
equilibrium at 0.90 V. In order to obtain a larger and stable current,
0.90 V was thus used as the optimum applied potential.

3.5. Amperometric sensing of glucose

Under the optimized conditions, amperometric response of

Mn3 O4 NPs/N-GR/CPE (holding at 0.90 V) upon successive addi-
tions of glucose into constantly stirred 0.1 M NaOH solution was
recorded. As can be seen in Fig. 7, well-defined amperometric
currents are increasing stepwise with the level of glucose. The
Mn3 O4 NPs/N-GR/CPE exhibits sensitive and rapid current response
to the addition of glucose, achieving a steady-state current in less
than 6 s. These experimental results reveal efficient catalytic abil-
ity of the Mn3 O4 NPs/N-GR hybrids for glucose electro-oxidation.
Fig. 7. (A) Amperometric response of the Mn3 O4 NPs/N-GR/CPE (holding at 0.90 V)
The inset of Fig. 7A displays the relationship between the amper-
upon addition of glucose to increasing concentrations in 0.1 M NaOH, the inset is the
ometric response of glucose and its concentration. The oxidation relationship between the amperometric response of glucose and its concentration.
peak current of glucose increased with its concentration in the (B) The linear relationship between the current and concentration of glucose in the
range from 2.5 to 529.5 ␮M with two linear ranges (as shown range of 2.5–119.5 ␮M; the inset is the i vs. C glucose in the range of 119.5–529.5 ␮M.
 S. Yang et al. / Sensors and Actuators B 221 (2015) 172–178 177

Table 1
Comparison of glucose sensing performances based on various nanomaterials.

Electrode materials Method Applied potential (V) Sensitivity (␮A/␮M) Linear range (␮M) LOD (␮M) Reference

g-C3 N4 -GOD Amperometry −0.81 – 1000–12,000 11 [32]

MnCu/MWCNT/GO DPV 0.05 0.0593 1000–32,000 1 [33]
MnO2 /MWCNT Amperometry 0.3 0.0332 10–28,000 – [34]
NiCPNP/rGO Amperometry 0.45 – 10–8750 0.14 [6]
Nafion–Cu–N-G Amperometry 0.5 0.04813 4–4500 1.3 [13]
Ag-NG Amperometry 0.65 0.00482 100–15,000 20 [14]
GOD/AgNPs/PANINFs Amperometry −0.58 – 1000–12,000 250 [35]
GOD/AgNP-TPTNB Amperometry −0.59 – 3000–20,000 190 [36]
Chitosan/GOD/AgNPs–G Amperometry −0.5 – 2000–10,000 100 [37]
GOD/AgNPA/F-SiO2 /GO Amperometry −0.62 – 2000–12,000 310 [38]
GP-GOD Amperometry −0.79 – 2000–22,000 20 [39]
Mn3 O4 NPs/N-GR Amperometry 0.90 0.1011 2.5–529.5 1.0 This work

For example, the present sensitivity is higher than that based on

MnCu/MWCNT/GO and MnO2 /MWCNT [33,34].
These results indicated that the Mn3 O4 NPs/N-GR exhibited
excellent electro-catalytic activity for the oxidation of glucose
as expected. Such remarkable electro-catalytic activity may be
attributed to synergistic effect of the introduction of Mn3 O4 NPs and
N-GR. The electrical network formed through Mn3 O4 NPs directly
distributing on the surface of N-GR, which not only can keep
their intrinsic wonderful electrical conductivity but also facilitate
Mn3 O4 NPs easily accessing to glucose. Therefore, the electrochem-
ical response toward glucose oxidation can be greatly enhanced.
The stability and reproducibility of the biosensor are the
important properties. The Mn3 O4 NPs/N-GR/CPE was used for five
measurements continuously, and the relative standard deviation Fig. 8. Amperometric responses of Mn3 O4 NPs/N-GR/CPE to successive additions of
(RSD) of the current responses upon 0.1 mM glucose in 0.1 M NaOH 1.0 mM glucose, 0.05 mM UA, 0.05 mM AA, 0.05 mM DA and 0.5 M NaCl in 0.1 M
solution was 6.9%, which suggested that this biosensor was highly NaOH at 0.90 V.
repeatable. When not in use, the Mn3 O4 NPs/N-GR/CPE was stored
in air at room temperature. Experimental results displayed that the Table 2
steady-state response current decreased by 7.6% after 7 days (as Determination of glucose in a human urine sample (n = 5).
shown in Fig. S1), suggesting that the biosensor was considerably Sample Spiked (␮M) Detected (␮M) Recovery (%) RSD (%)
stable.
1 20.0 18.6 ± 0.12 93.0 6.2
2 40.0 39.2 ± 0.31 98.0 3.1
3.6. Interference studies and detection of glucose in urine sample 3 60.0 60.1 ± 0.23 100.2 5.2
4 100.0 98.1 ± 0.51 98.1 4.6

Some interference, such as dopamine (DA), ascorbic acid (AA)

and uric acid (UA), usually co-exist with glucose in a biological
sample. Although at low concentrations relative to glucose, these
interfering species could be easily oxidized and interfere with
the detection of glucose. In the physiological liquid, glucose level
(4–7 mM) is generally much higher than that of interfering species.
Thus, the influence of 0.05 mM interfering species on the current
response of 1.0 mM glucose was evaluated. Because Cl− can poi-
son most of the enzymeless glucose sensors based on noble metal
and alloys, the influence of high level (0.5 M) of NaCl on the ampero-
metric response of glucose was also investigated. As shown in Fig. 8
and Fig. S2, a remarkable glucose signal was observed comparing to
the other four interfering species. Compared to 1.0 mM glucose, the
interfering species produced current response ranging from 13.3%
(0.05 mM UA), 10% (0.05 mM AA), 16.7% (0.05 mM DA) to 2.4% (0.5 M
NaCl).
In order to investigate the practicability of the biosensor in clin-
ical analysis, it has been employed to determine the concentration
of glucose spiked in human urine sample. The urine sample was
collected from a healthy woman. Glucose was not found in the
blank urine samples. Generally, 100 ␮L of urine sample was added
into 10 mL 0.1 M NaOH solution; subsequently, 20 ␮L standard glu-
cose solution was successively added into the sample with stirring,
and the final concentration was in the linear range of the proposed
method. The results were listed in Table 2. As can be seen from
Table 2, the recovery of the spiked glucose in the urine sample was
in the range of 93.0–100.2%, revealing the accuracy of the proposed
method.
4. Conclusions
In this study, the Mn3 O4 NPs/N-GR was successfully produced by
a facile method via thermal treatment, and a novel non-enzymatic
biosensor was fabricated based on the resulting Mn3 O4 NPs/N-GR,
which provided excellent electro-catalytic activity toward glucose
oxidation than pure Mn3 O4 . The biosensor exhibited notable per-
formances for glucose detection, such as high stability, wide linear
range, low detection limit, perfect selectivity to glucose in the pres-
ence of common interfering species, and avoiding poison by Cl− .
The relatively facile and cheap fabrication of such biosensor makes
it possible as a promising candidate for routine glucose sensing.
Acknowledgements
The authors gratefully acknowledge the financial support
from Nature Science Foundation of Anyang Normal University
and National Natural Science Foundation of China (NSFC, No.
21102005).
178 S. Yang et al. / Sensors and Actuators B 221 (2015) 172–178
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.snb.2015.06.110
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Biographies
Suling Yang lecturer of chemistry at Anyang Normal University, received her
PhD from Department of Chemistry at Zhengzhou University in 2012. Her current
research interests are mainly focused on the design of novel electrochemical sensors
or biosensor and its application in analysis.
Gang Li received his PhD (2008) from Chinese Academy of Sciences of Chemical
Technology. At present he is an associate professor at Anyang Normal University.
His current research interests are the organic–inorganic hybrid nanomaterials, and
metal–organic compounds.
Guifang Wang graduated from Department of Chemistry at Henan Normal Uni-
versity in 1988. He received his Master’s degree from Department of Chemistry
at Shanxi Normal University in 1997. At present he is a professor of chemistry at
Anyang Normal University. His current research focuses on the preparing material
in the application of UV–vis spectrophotometry and sensor.
Junhong Zhao lecturer of chemistry at Anyang Normal University, received her Ph.D.
in polymer chemistry from Shanghai Jiao Tong University. Her research interests are
the synthesis, structure and properties of multifunctional microporous materials.
Xiuhuan Gao is working toward her B.S. in Chemistry at Anyang Normal Univer-
sity. Her main focus is the synthesis and application of novel hexacyanoferrate
nanoparticles.
Lingbo Qu graduated from Department of Technical Physics, Peking University in
1984. He received the PhD from School of Pharmacy, China Pharmaceutical Univer-
sity in June 1997. At present he is a professor of Chemistry at Henan University of
Technology. His current research interests include the development of electrochem-
ical sensors and biosensors for the detection of drug molecular in environmental,
biology and food science.