Article

pubs.acs.org/IECR

The Immobilization of Glucose Oxidase at Manganese Dioxide

Particles-Decorated Reduced Graphene Oxide Sheets for the
Fabrication of a Glucose Biosensor
A. T. Ezhil Vilian, Veerappan Mani, Shen-Ming Chen,*, Bose Dinesh, and Sheng-Tung Huang*,

Department of Chemical Engineering and Biotechnology, National Taipei University of Technology, No.1, Section 3, Chung-Hsiao
East Road, Taipei 106, Taiwan, R.O.C

Department of Materials Science, School of Chemistry, Madurai Kamaraj University, Madurai, Tamilnadu 625 021, India
S Supporting Information
*

ABSTRACT: Here, we describe a simple approach for the ecient immobilization of poly-L-lysine (PLL) impregnated glucose
oxidase (GOx) at a manganese dioxide (MnO2)-decorated chemically reduced graphene oxide (CRGO) composite lm modied
electrode. The MnO2/CRGO lm was analyzed by various techniques such as scanning electron microscopy, UVvisible
spectroscopy, Fourier transform infrared spectroscopy, X-ray diraction, and electrochemical impedance spectroscopy. MnO2
particles are uniformly deposited at the CRGO surface through an easily adoptable electropolymerization method. The asprepared (GOx-PLL/MnO2/CRGO) composite lm exhibited a surface coverage concentration () of 2.68 1010 mol/cm2,
indicative of high GOx loading. The heterogeneous rate constant for the redox reaction of GOx has been calculated to be 4.92
s1. A glucose biosensor was fabricated which exhibits a wide linear range from 0.04 to 10 mM with a limit of detection of 0.02
mM (S/N = 3). The proposed inexpensive, simple, and sensitive sensing approach based on the MnO2/CRGO nanocomposite
electrode can be a promising method for the determination of glucose present in the clinical and environmental food analysis.
sensors.1214 The positively charged biomolecules improve the
microenvironment to provide a suitable enzyme immobilization
matrix. This method can be adapted to nd better enzyme
immobilization strategies.15
Graphene oxide (GO), an oxygenated derivative of graphene
is the adoptable precursor for the preparation of graphenebased electrodes owing to its signicant advantages such as
inexpensive production from graphite, easy processing in
aqueous dispersion, and available sites for functionalization.16,17
GO can be prepared by the oxidation of graphite with
subsequent exfoliation through ultrasonication.18 Manganese
dioxide (MnO2) and its derivatives have attracted enormous
attention because of their excellent physicochemical properties,
and they nd widespread applications in diverse research
areas.19,20 Dierent preparation methods could produce
dierent morphologies which are highly useful for various
kinds of electrochemical applications.21 The development of
graphene and MnO2 composites was reported recently for highperformance electrochemical capacitors22 and supercapacitors2325 applications. However, very few reports are available
about the electrodeposition of MnO2 particles at the graphene
surface. Nevertheless, electrodeposition is an attractive and easy
strategy for the preparation of highly stable and uniformly
arranged particles on the electrode surface. Herein, we have
used a simple electrodeposition approach for the preparation of
MnO2 particles at the chemically reduced graphene oxide

1. INTRODUCTION
In the past few years, the incidence of diabetes mellitus has
been rising, and it has now become a serious public health
problem worldwide. To facilitate rapid response to variations in
the bodys glucose levels and detection of this condition, highly
sensitive and selective biosensors are being developed.1 Recent
interest has been focused toward development of glucose
oxidase (GOx) incorporated biosensors for the determination
of glucose concentration, and this continues to be the chief
model for the fabrication of next generation sensors.2 However,
at the present time, the development of amperometric
biosensors remains challenging because of the issues associated
with enzyme immobilization and ensuring ecient electrical
communication between the redox sites of enzymes and
electrodes.3,4 Electron transport between the active sites of
GOx and the electrode is dicult since the avin adenine
dinucleotide (FAD) group is deeply buried within the interior
of the protein backbone5 The direct electrocatalytic oxidation
of glucose that occurs in enzyme free glucose sensors is thus
stimulating keen interest.6
Recently, eorts have been focused on the use of poly-Llysine (PLL) as electrode material for various applications.7
PLL is a polycationic homopolymer and it has been shown to
be a good support for the protection of biomolecules.7,8 The
successful attachment of PLL with the graphene sheets is
possible via a covalent approach. PLL-based nanocomposites
are considered to be excellent alternatives to conventional
materials for the manufacture of electrochemical sensors
attributed to the presence of various functional groups.10
They have also been widely utilized to modify the working
electrode used for electrochemical detection11 and in DNA
2014 American Chemical Society

Received:
Revised:
Accepted:
Published:
15582

June 16, 2014

August 13, 2014
September 12, 2014
September 12, 2014
dx.doi.org/10.1021/ie502430d | Ind. Eng. Chem. Res. 2014, 53, 1558215589

Industrial & Engineering Chemistry Research

Article

wt %). Finally, the solid suspension consigned as graphite oxide

was washed with a 2 M HCl solution and then washed 34
times with ethanol and dried overnight. The graphite oxide
dispersion (1 mg/mL in water) was exfoliated to GO through
ultrasonication for 1 h. The unexfoliated graphite oxide was
removed by centrifugation at 3000 rpm for 30 min. Then 400
mg of NaBH4 was slowly added to the GO dispersion (20 mL,
1 mg/mL) with vigorous stirring, and then the reaction mixture
remained stirred for 20 h. Thereafter, the CRGO was separated
through ltration, washed with water and ethanol, and dried at
60 C for 48 h in a vacuum oven. Finally, the as-prepared
CRGO powder was redispersed in DMF (1 mg/mL) through
ultrasonic agitation.
2.4. Fabrication of the Glucose Biosensors. Before
starting each experiment, GCE was carefully polished utilizing
0.05 mm alumina slurry, and then subjected to ultrasonication
in ethanol, and water for 5 min. A 5 L aliquot of CRGO (1
mg/mL) was spread at the GCE surface and dried at room
temperature. The electrochemical deposition of MnO2 particles
onto the CRGO-modied electrode surface was also performed
following a previously reported procedure.27 In a typical
preparation process, MnO2 was deposited at the CRGO/GCE
from 10 mM of KMnO4 (5 mL) present in the 0.04 M H2SO4
aqueous solution. The scan was performed between the
potentials of 0.5 V and 0.3 V at the scan rate of 2 mV s1
for eight cycles. After electrodeposition, a 5 L dispersion of
GOx (10 mg/mL in PBS, pH 7) was vigorously mixed together
with PLL in PBS (pH 7) solution and stored in a refrigerator
for at least 24 h. Then, 5 L from the mixture of GOx and PLL
(1:10) was dropped onto the MnO2/CRGO/GCE and allowed
to dry at ambient conditions. Finally, 5 L (0.5%) of naon
solution was drop casted on the GOx-PLL/MnO2/CRGO lm
modied electrode and allowed to dry. The control electrodes
such as bare GCE, GOx-PLL/MnO2/GCE, GOx-PLL/CRGO/
GCE, CRGO/GCE, and CRGO/MnO2 /GCE are also
prepared accordingly. A schematic illustration of the entire
electrode fabrication procedure has been given as Scheme 1.

(CRGO) surface. The electrodeposition method reported

involves very simple, fast, and easily adoptable protocols for
the preparation of highly ordered and uniform MnO2 particles
and does not require high temperature, any chemical agents,
dicult protocols, or additional equipment.
In this present study, for the rst time, we have prepared
MnO2/CRGO lm modied GCEs, where GOx-PLL was
adsorbed on the nanocomposite lm. We found that the
MnO2/CRGO/GCE nanocomposite lm provided a greatly
favorable microenvironment for GOx to retain its redox activity.
The GOx-PLL/MnO2/CRGO/GCE lm modied electrode
has the advantage of facilitating electrochemical detection for
glucose sensing as demonstrated by the improved linear range,
low detection limit, high sensitivity, rapid response time,
reproducibility, selectivity, and stability, which were all
investigated in detail.

2. EXPERIMENTAL SECTION
2.1. Materials. GOx (type x-s from Aspergillus niger),
Na2HPO4, and NaH2PO4, N, N-dimethylformamide (DMF),
and PLL were purchased from Sigma and used without further
purication. KMnO4 reagent was acquired from Shimakyu,
Osaka, Japan. The GOx stock solution was prepared by using a
0.05 M phosphate buer solution (PBS, pH 7) and stored at 4
C. Phosphate buer solution (PBS, 0.05 M) was prepared
from Na2HPO4 and NaH2PO4 and used as supporting
electrolyte. Double-distilled water was used for the preparation
of all the solutions.
2.2. Equipment. Cyclic voltammetry and dierential pulse
voltammetry experiments were performed using CHI electrochemical workstation (CHI 405A). A three-electrode system
comprised modied GCE (area 0.07 cm2) as working electrode,
Pt as a counter electrode, Ag/AgCl (saturated KCl) as a
reference electrode. Electrochemical impedance spectroscopy
(EIS) analysis was performed at a frequency range of 0.1 Hz to
1 MHz with a ZAHNER instrument (Kroanch, Germany). The
structures of the samples were characterized by SEM
measurements with a Hitachi S-3000 H, and energy-dispersive
X-ray spectroscopy (EDX) was recorded using a HORIBA
EMAX X-ACT model 51-ADD0009. XRD analysis was
performed with an XPERT-PRO diractometer (PANalytical
B.V., The Netherlands) using Cu K radiation (k = 1.54 ).
Fourier transform infrared (FT-IR) measurements were
performed using a Perkin Elmer spectrophotometer RXI.
Hitachi U-3300 spectrophotometer was used to carry out
UVvisible absorption spectroscopy measurements.
2.3. Preparation of GO. The GO was prepared by
following the modied Hummers method26 whereby 1 g of
graphite (graphite powder, < 20 m, Aldrich) was suspended in
2.5 g of K2S2O8 and 46 mL of H2SO4 and then stirred in a
round-bottom ask at 0 C for 15 min. Next, 2.5 g of P2O5 was
added into the mixture over 15 min in order to avoid a
temperature spike, and the mixture was left to be stirred
vigorously for 6 h at 20 C. On completion, the mixture was
poured, diluted by adding it to 1000 mL of water, and ltered.
Afterward, 6 g of KMnO4 and 1 g of NaNO3 in 31.2 mL of
water was added gradually, while the temperature was managed
to be less than 20 C. The reaction occurred as the mixture was
stirred at 35 C for 2 h. Then, 500 mL of distilled water was
added, and the temperature was maintained to be less than 50
C. The solution was kept at this temperature for 2 h. The
color of the resulting solution was changed to brilliant yellow
upon the addition of 250 mL of water and 6 mL of H2O2 (30

3. RESULTS AND DISCUSSION

3.1. Structural Characterization of the MnO2/CRGO
and GOx-PLL/MnO2/CRGO Composites Film. The XRD
Scheme 1. Schematic Representation of the Preparation of
the GOx-PLL/MnO2/CRGO/GCE and Its Application for
the Determination of Glucose

15583

dx.doi.org/10.1021/ie502430d | Ind. Eng. Chem. Res. 2014, 53, 1558215589

Industrial & Engineering Chemistry Research

Article

Figure 1. (A) XRD patterns of (a) MnO2/ITO, (b) MnO2/CRGO/ITO composite; (B) FTIR spectra of (a) GO, (b) MnO2/CRGO composite;
and (C) FTIR spectra of (a) GOx, (b) GOx-PLL/MnO2/CRGO modied electrode; (D) UVvis absorption spectra of (a) GOx, (b) GOx-PLL, (c)
GOx-PLL/MnO2/CRGO composite.

for NH stretching, and two peaks centered at 1650 and 1544

cm1 are assigned to the characteristic amide I and II
absorption bands of GOx. The peak appearing around 1082
cm1 was assigned to the stretching mode of CO of GOx
(Figure 1Ca). The FT-IR spectrum of GOx-PLL/MnO2/
CRGO nanocomposites (see Figure 1 Cb) also presents two
typical adsorption bands of GOx at the wavenumber of 1648
and 1558 cm1 revealing the successful immobilization of
GOx.30
UVvisible and FT-IR spectroscopy were utilized to examine
the structural information about the GOx. Figure 1D shows the
typical UVvisible absorption spectra for GOx (a), GOx-PLL
(b), and GOx-PLL/MnO2/CRGO (c). There are sharp peaks
at 380 nm and 453 nm indicating the presence of GOx.31,32 It
can be seen in Figure 1Dc that the positively charged GOx-PLL
was adsorbed on the MnO2/CRGO surface, suggesting that the
GOx-PLL was immobilized in the MnO2/CRGO lm and
retained its native structure.
3.2. Surface Morphological Characterization of MnO2/
CRGO Composite. The morphology and microstructure were
examined through scanning electron microscope (SEM)
experiments. The SEM image of CRGO shows the characteristic crumpled and wrinkled sheet-like morphology of CRGO
(Figure 2A). Figure 2B shows typical SEM images of the MnO2
particles showing the spherical shape, which was very consistent
with previous results obtained with the electrochemical
approach. The inset shows that the results of the EDX analysis
conrm that the MnO2 particles consisted of Mn and O.
Furthermore, Figure 2C displays a representative SEM image of

patterns of the MnO2 particles are shown in Figure 1A,a. The

XRD patterns of the as-prepared samples exhibit three
characteristic diraction peaks around 39.7, 46.2, and 67.4
corresponding to (100), (101), and (102), respectively, and the
(110) of MnO2 (JCPDS Card No. 44-0141), which is
conrmed by the SEM micrographs (see Figure 1A). This
can be clearly seen in the XRD patterns of the as-synthesized
MnO2/CRGO composites shown in Figure 1A,b. Graphite
showed a very sharp diraction peak at 22.93 which
corresponds to the (002) suggesting that the CRGO was
fully reduced. The peaks at 2 = 24.4, 32.2, 39.7, 46.2, and
67.4 can be assigned to the (100), (101), (102), and (110)
crystalline plane diraction peaks, respectively, suggesting that
the MnO2 had been successfully deposited on the surface of the
CRGO Sheet.28
FT-IR spectroscopy was used to characterize the samples
further and conrmed the presence of MnO2 in the MnO2/
CRGO nanocomposites. The results of the analysis of GO,
MnO2/CRGO, GOx, and GOx-PLL/MnO2/CRGO nanocomposites by FT-IR are shown in Figure 1B,C. In the FTIR spectrum of GO, the absorption peaks appearing at the
wavenumber of 1743 cm1 and 1637 cm1 are assigned the
CO stretching and CC stretching vibrations, respectively
(see curve a).29 In contrast, the peaks at 1731 and 1183 cm1
are missing from the FTIR spectrum of the MnO2/CRGO
nanocomposites, which indicates a reduction of GO and its
transformation into CRGO (see curve b). The FT-IR spectrum
of GOx exhibits the characteristic peaks of native GOx (see
Figure 1C,a). The intense absorption at 3409 cm1 is assigned
15584

dx.doi.org/10.1021/ie502430d | Ind. Eng. Chem. Res. 2014, 53, 1558215589

Industrial & Engineering Chemistry Research

Article

(d) GOx-PLL/MnO2/CRGO electrodes and (e) GOx-PLL

lms modied GCEs. The semicircular diameter of the EIS
curve corresponds to the electron-transfer resistance (Ret).
According to experimental data based on equivalent circuits
(Supporting Information, Table S1), the Ret of the MnO2/
CRGO/GCE is lower than that of the bare GCE and CRGO/
GCE (curve b). This means that MnO2 particles were
uniformly distributed over the surface of the CRGO surface
which could lead to faster charge transfer in the MnO2/CRGO
lm. After the GOx-PLL was coated on the bare electrode,
there was an increase in Ret (curve e) owing to the
immobilization of GOx-PLL on the bare GCE surface. After
GOx-PLL was coated on the MnO2/CRGO modied electrode,
the minimum resistance value changed (curve d), suggesting
that the MnO2/CRGO accelerated the electron transfer
between electrode surface and the redox probe. The low Ret
might also result from the electrostatic repulsion between the
negatively charged redox probe and positively charged GOxPLL/MnO2/CRGO/GCE. This result shows the successful
fabrication of a modied electrode and conrms immobilization
of GOx on PLL/MnO2/CRGO.
3.4. Direct Electrochemistry of GOx at PLL-MnO2/
CRGO Film Modied Electrode. Figure 3B shows the cyclic
voltammograms (CVs) obtained at GOx-PLL/MnO2/CRGO
and control electrodes in nitrogen saturated PBS. The absence
of observable voltammetric peaks at the bare GCE, CRGO, and
MnO2/CRGO indicating that no redox reaction took place.
The background current of GOx-PLL/MnO2/CRGO lm
modied electrode shows good conductive performance
which assists in faster transportation than that of the MnO2/
CRGO/GCE. This could be attributed to the large surface area
of MnO2/CRGO. In fact, the high electroactive area of MnO2/
CRGO induces a high background current, and MnO2 may
enable the accumulation of negatively charged GOx. GOx-PLL
immobilized on the surface of the MnO2/CRGO displays a

Figure 2. SEM images of (A) CRGO, (B) MnO2, (C) MnO2/CRGO

and (D) GOx-PLL/MnO2/CRGO composite.

the MnO2/CRGO/ITO lm. The spherical morphology

intrinsic to the group can be observed. The surface of the
CRGO sheets is uniformly decorated with MnO2 particles. This
clearly indicates the suitability of the MnO2/CRGO modied
electrode for electroanalysis. The corresponding EDX patterns
for the MnO2 deposited on the CRGO/ITO surface for each
case are shown in Figure 2C (see inset image). From these
results, we can conclude that the MnO2 particles completely
covered and were uniformly distributed on the surface of the
CRGO after this process, and they also maintained a high
surface area. After the immobilization of GOx-PLL on the
MnO2/CRGO there was an obvious change in the corresponding SEM image. The relatively blurry surface, as shown in
Figure 2D, suggests that the GOx-PLL has been eectively
immobilized on the surface of the MnO2/CRGO lm.
3.3. EIS Measurements. Figure 3A displays the Nyquist
plots of (a) bare/GCE, (b) CRGO/GCE, (c) MnO2/CRGO,

Figure 3. (A) EIS spectra of (a) bare/GCE, (b) CRGO/GCE, (c) MnO2/CRGO/GCE, (d) GOx-PLL/MnO2/CRGO/GCE electrodes, (e) GOxPLL/GCE electrodes in 5 mM Fe2 (CN)63/4 in PBS; frequency range, 0.1 Hz to 1 MHz. Inset: Randles equivalence circuit. (B) CVs of (a) bare/
GCE, (b) GOx-PLL/MnO2/GCE, (c) GOx-PLL/CRGO/GCE, (d) CRGO/GCE, (e) CRGO/MnO2/GCE, and (f) GOx-PLL/CRGO-MnO2
electrodes in a 0.05 M deoxygenated PBS (pH 7) at the scan rate of 50 mV s1. (C) CVs of GOx-PLL/CRGO-MnO2/GCE at dierent scan rates
(from inner to outer: 10100 mV s1). (D) Plots of the anodic and cathodic peak currents vs the scan rates.
15585

dx.doi.org/10.1021/ie502430d | Ind. Eng. Chem. Res. 2014, 53, 1558215589

Industrial & Engineering Chemistry Research

Article

Figure 4. (A) CVs of MnO2/CRGO modied GCE in dierent pH solutions from a to d (3, 5, 7, 9), at a scan rate of 0.5 V s1. (B) Plot of the
formal potential vs value. (C) CVs of MnO2/CRGO-modied GCE electrodes in N2-saturated 0.05 M PBS (pH 7.0) with dierent concentrations of
H2O2 (0, 1, 2, 3, mM). Scan rate 0.5 V s1. (D) Typical amperometric response of MnO2/CRGO nanostructure modied electrode after successive
injections of 0.05 mM and 0.5 mM H2O2 to the stirred 0.05 M deoxygenated PBS (pH 7.0). Applied potential: 0.45 V. Inset: plot of response
current versus [H2O2].

either the other modied electrodes or the theoretical

monolayer value (2.86 1012 mol cm2) indicating the
eective immobilization of GOx on the MnO2/CRGO lm.
The electron transfer rate constant (ks) of GOx at the
modied electrode can be calculated using Lavirons equation.34

couple of well-dened quasi-reversible redox peaks with formal

potential (E0) of 0.430 V which revealing the attainment of
direct electrochemistry of GOx. Nevertheless, the utilization of
GOx-PLL on the surface of the MnO2/CRGO/GCE exhibited
a dierent electrochemical behavior. Eventhough, GOx-PLL/
MnO2 and GOx-PLL/CRGO modied electrode are presented
minimal redox peaks, they possess very short period of stability
compared to the nal GOx-PLL/MnO2/CRGO composite.
The enhanced electrochemical responses of GOx at the GOxPLL/MnO2/CRGO should be ascribed to the ecient
immobilization of GOx at this lm. PLL is a cationic lm,
which can facilitate the accumulation of negatively charged
GOx on the electrode surface. Moreover, MnO2/CRGO
contains a negatively charged surface which also help to
immobilize GOx at the electrode surface through electrostatic
interaction.
The CVs obtained at the GOx-PLL/MnO2/CRGO for
various scan rates from 0.01 V s1 to 0.1 V s1 are shown in
Figure 3C. Both anodic peak current (Ipa) and cathodic peak
current (Ipc) of the redox peak were linearly increased when
increasing the scan rate. In addition, E0 of the redox peaks was
also shifted when scan rates increased. Figure 3C implies that
the redox process of the GOx occurring at the GOx-PLL/
MnO2/CRGO/GCE is a surface-controlled process. The
surface coverage concentration of GOx () can be calculated
using the following equation:33
Ip = n2F 2vA /4RT

log K s = log(l ) + (l ) log log(RT /nFv)

(l )nF Ep/2.3RT

(2)

where ( 0.5), R and T are representing the charge transfer

coecient, universal gas constant, and temperature. By
substituting all the parameters into eq 2, the value of ks was
calculated to be 4.92 s1. The ks value of 4.92 s1 for GOx-PLL/
MnO2/CRGO is comparatively larger than that obtained for
GOx immobilized on common graphene (2.83 s1),35 GOx/
RGO (4.8 s1),36 CNT (2.76 s1),37 multiwalled carbon
nanotubes/electrochemically reduced graphene oxide (ERGO)
(3.02 s1),33 mesoporous carbon (4.09 s1),38 mesoporous
silica (3.89 s1),39 graphene/naon (3.42 s1),40 CdS nanoparticles (1.56 s1),41 ERGO/poly L-lysine (3.27 s1),42 or
graphene/naon/Au (1.96 s1).43 From these results, it can be
concluded that the GOx-PLL/MnO2/CRGO lm provides an
ecient platform for the immobilization of large quantities of
GOx.
3.5. Eect of pH. The pH of the electrolyte is one of the
crucial parameters aecting the activity and stability of the
resulting modied electrode. Therefore, we have investigated
the eect of pH on the redox reaction of GOx. The CVs of
GOx-PLL/MnO2/CRGO show well-dened redox peaks, and
both Ipa and Ipc were shifted negatively with an increase in pH
(see Figure 4A). A plot of E0 versus pH shows a linear
relationship as can be seen from Figure 4B. The linear
regression equation can be expressed as E0 = 0.054 pH
0.057, R2 = 0.994. The obtained slope value is very close to the
expected slope value of 0.058 V given by the Nernst equation

(1)

where, n, A, and represent the number of electrons, area of

the electrode (cm2), and scan rate (V s1). The constants R, T,
and F have their usual meanings. The values for GOx at the
GOx-PLL/MnO2/CRGO/GCE and GOx-PLL/CRGO/GCE
electrodes were estimated to be 2.68 1010 mol/cm2 and
1.42 1010 mol/cm2, respectively. The value of GOx
observed at the GOx-PLL/MnO2/CRGO/GCE is higher than
15586

dx.doi.org/10.1021/ie502430d | Ind. Eng. Chem. Res. 2014, 53, 1558215589

Industrial & Engineering Chemistry Research

Article

Figure 5. (A) CVs of GOx-PLL/MnO2/CRGO/GCE in oxygen saturated 0.05 M pH 7.0 PBS containing (a) 0 mM, (b) 1 mM, (c) 2 mM, (d) 3
mM, (e) 4 mM, (f) 5 mM, and (g) 6 mM glucose at a scan rate of 50 mV s1; (B) DPVs obtained at GOx-PLL/MnO2/CRGO/GCE in oxygen
saturated PBS (pH 7) in the absence (a) and presence of various concentrations of glucose (from a to j); (C) [glucose] vs electrocatalytic current.

3.7. Determination of Glucose. The biosensor activity of

the fabricated biosensor has been studied through oxygen
reductive detection of glucose. Figure 5A presents the CVs
obtained at the GOx-PLL/MnO2/CRGO/GCE in oxygen
saturated PBS (pH 7) in the absence (a) and presence of
glucose (b to g; each 1 mM). When glucose was added to the
oxygen saturated PBS, the Ipc was decreased because the
reduction of oxygen occurred via oxidation of glucose catalyzed
by the GOx as expressed in eq 57. Further additions of
glucose lead to the linear decrease of Ipc which is suitable for the
determination of glucose,

for the quasireversible reaction involving two electrons and two

protons.33 We choose pH 7 to carry out electrochemical studies
since neutral pH is suitable for the practicality analysis in the
biological samples.
3.6. Electrocatalytic Reduction of Hydrogen Peroxide
at MnO2/CRGO/GCE. The electrocatalytic behavior of the
MnO2/CRGO/GCE toward reduction of hydrogen peroxide
(H2O2) has been investigated. The CVs obtained at the MnO2/
CRGO/GCE in the deoxygenated PBS (pH 7) with and
without the addition of H2O2 are shown in Figure 4C. With the
gradual addition of H2O2, there was a steady increase in the Ipc
and decrease in the Ipa of the MnO2/CRGO modied
electrode. A large Ipc was observed for the MnO2/CRGO/
GCE in comparison with the bare electrode (Figure 4C). The
MnO2/CRGO exhibited signicantly enhanced electrocatalytic
ability for the reduction of H2O2 which might be attributed to
the excellent synergy between MnO2 and CRGO. The
electrochemical pathway responsible for catalyzing the H2O2
at the MnO2/CRGO electrode can be described as follows,44
MnO2 + H 2O2 MnO + H 2O + O2

(3)

MnO MnO2+2e

(4)

glucose + GOx(FAD)
gluconolactone + GOx(FADH 2)

(5)

GOx(FADH 2) + O2 GOx(FAD) + H 2O2

(6)

O2 + 4H+ + 4e 2H 2O

(7)

To investigate the role of MnO2 in the determination of

glucose, we have fabricated an electrode without incorporating
MnO2 (GOx-PLL/CRGO/GCE) and tested its biosensing
ability toward determination of glucose (Supporting Information, Figure S1). As evident from the Figure S1, minimal
decrease in the Ipc was observed at GOx-PLL/CRGO/GCE for
the each addition of glucose, however greatly improved
response was observed at GOx-PLL/CRGO/GCE (Figure 5).
These discussions are clearly revealing that MnO2 play critical
role in both direct electrochemistry of GOx and also in the
biosensor performance. Figure 5B illustrates typical dierential
pulse voltammetry (DPV) curves obtained at the GOx-PLL/
CRGO/GCE upon each sequential addition of glucose to the
oxygen saturated PBS (pH 7) in the absence and presence of
glucose. The Ipc responsible for the reduction of oxygen was
increase for each sequential addition of glucose. A calibration
plot was made between concentration of glucose and response
current which exhibited linear behavior (Figure 5A), while the

The amperometric performance of the MnO2/CRGO lm

modied rotating disc electrode (with a rotation speed of 1300
rpm) is measured after each successive addition of H2O2 to PBS
(pH 7) at an applied potential (Eapp) of 0.45 V (Figure 4D).
The amperometric responses are linearly increased with H2O2
concentration. The linear range was found to be from 0.05 M
to 7 mM and the sensitivity was calculated to be 46.36 mAcm2
mM1. The limit of detection (LOD) was calculated to be as
low as 0.01 M (signal-to-noise ratio, S/N = 3). The proposed
MnO2/CRGO sensor exhibited higher sensitivity, lower LOD,
wide working linear range, and less overpotential. The excellent
performance of the MnO2/CRGO/GCE shows promise for the
development of electrochemical sensors for H2O2 detection.
15587

dx.doi.org/10.1021/ie502430d | Ind. Eng. Chem. Res. 2014, 53, 1558215589

Industrial & Engineering Chemistry Research

Article

linear regression equation can be expressed as, I (A) = 12.288

+ 0.986Cg (mM), R = 0.9936, where I is the response current
and Cg is the concentration of glucose. The linear concentration
range was found between 0.04 mM and 10 mM of glucose. The
LOD was calculated to be 0.02 M (S/N = 3), and the
sensitivity was calculated to be 28.8 A/mM cm2. The
important analytical parameters were compared with those
obtained for other reported sensors available in the literature
(Supporting Information, Table S2). As can be seen from Table
S2, the sensor performance of the GOx-PLL/CRGO/GCE was
quite comparable with previous reports and revealed the good
capability of the sensor toward the determination of glucose.
This can be attributed to the excellent electrocatalytic ability of
the GOx-PLL/CRGO/GCE modied electrode leading to the
ecient reduction of oxygen, which indirectly assists in
monitoring the concentration of glucose.
Selectivity of the GOx-PLL/CRGO-MnO2/GCE has been
investigated in the presence of interferences including
dopamine, uric acid, ascorbic acid, and acetaminophen. DPV
experiments have been carried out in oxygen-saturated PBS
containing glucose (1 mM) and the above-mentioned
interferences (1 mM). The amount of interference percentage
has been calculated from the ratio between response current
obtained in the presence of glucose and that obtained in the
presence of respective interfering agents. As can be seen from
Supporting Information, Table S3, all the interference agents
have shown less than 5% interference with the glucose
determination at the GOx-PLL/CRGO-MnO2/GCE revealing
the appreciable selectivity of the modied electrode.
3.8. Real Sample Analysis. The practicality of the
biosensor was tested by determining the amount of glucose
present in human blood serum sample. Fresh human serum
samples were supplied from the local hospital, Taipei, Taiwan.
The real samples were diluted with a known amount of PBS
(pH 7), and the DPV experiments were carried out using
similar experimental conditions of the lab sample. The glucose
content present in the serum sample was calculated from the
DPVs, and the results are summarized in Supporting
Information, Table S4. The found and recovery results are
satisfactory and agree very closely with the hospital data. This
indicates that the GOx-PLL/MnO2/CRGO/GCE lm is a
suitable candidate for the determination of glucose present in
clinical samples.
3.9. Reproducibility, Repeatability, and Stability
Determination. This biosensor shows acceptable reproducibility with a relative standard deviation (R.S.D.) of 5.2% for the
determination of glucose (1 mM) at four dierent modied
electrodes. In addition, the biosensor shows appreciable
repeatability with an R.S.D. of 3.1% for the ve repetitive
measurements. The storage stability of the sensor was studied
over 20 days by monitoring the response currents toward the
detection of glucose. Only 4.02% of the initial response current
was decreased even after continuous scanning for 100 cycles,
indicating good stability of the sensor.

mM to 10 mM and low LOD of 0.02 mM. The biosensor

possesses appreciable reproducibility, repeatability, and longterm stability. Additionally, a nonenzymatic amperometric
sensor was demonstrated at MnO2/CRGO/GCE for the
sensitive determination of H2O2. The preparation protocols
for the biosensor fabrication are simple and convenient, not
requiring toxic reagents and not involving any complex
multistep process. The results demonstrate that the GOxPLL/MnO2/CRGO-modied electrode is a promising architecture for further development of microelectronic devices.

ASSOCIATED CONTENT

S Supporting Information
*

The cyclic voltammetry curves for the determination of glucose

at GOx-PLL/CRGO/GCE (Figure S1), electrochemical
impedance spectroscopy parameters (Table S1), comparison
table with references (Table S2), data for the selectivity studies
(Table S3), and real sample analysis (Table S4). This material
is available free of charge via the Internet at http://pubs.acs.org.

AUTHOR INFORMATION

Corresponding Authors

*S.M.C. E-mail: smchen78@ms15.hinet.net. Tel: +886 2270

17147. Fax: +886 2270 25238.
*S.-T.H. E-mail: ws75624@ntut.edu.tw. Tel.: +886 2271-2171
2525. Fax: +886-02-2731-7117.
Notes

The authors declare no competing nancial interest.

ACKNOWLEDGMENTS
This work was nancially supported by the Ministry of Science
and Technology, Taiwan (Republic of China).

REFERENCES

(1) Wang, J. J.; Xu, H.; Chen, Y. A novel glucose biosensor based on
the nanoscaled cobalt phthalocyanine-glucose oxidase biocomposite.
Biosens. Bioelectron. 2005, 20, 13881396.
(2) Ammam, M.; Easton, E. B. High-performance glucose sensor
based on glucose oxidase encapsulated in new synthesized platinum
nanoparticles supported on carbon Vulcan/Nafion composite
deposited on glassy carbon. Sens. Actuators, B 2011, 155, 340346.
(3) Joshi, P. P.; Merchant, S. A.; Wang, Y.; Schmidtke, D. W.
Amperometric biosensors based on redox polymer-carbon nanotubeenzyme composites. Anal. Chem. 2005, 77, 31833188.
(4) Xiao, X.; Zhou, B.; Zhu, L.; Xu, L.; Tan, L.; Tang, H.; Zhang, Y.;
Xie, Q.; Yao, S. An reagentless glucose biosensor based on direct
electrochemistry of glucose oxidase immobilized on poly(methylene
blue) doped silica nanocomposites. Sens. Actuators, B 2012, 165, 126
132.
(5) Barton, S. C.; Gallaway, J.; Atanassov, P. Enzymatic biofuel cells
for implantable and microscale devices. Chem. Rev. 2004, 104, 4867
4886.
(6) Heller, A. Electrical wiring of redox enzymes. Acc. Chem. Res.
1999, 23, 128134.
(7) Edwards, C. R.; Dang, W.; Berger, S. L. Histone H4 Lysine 20 of
Saccharomyces cerevisiae is monomethylated and functions in
subtelomeric silencing. Biochemistry 2011, 50, 1047310483.
(8) Sundlass, N. K.; Raines, R. T. Arginine residues are more effective
than Lysine residues in eliciting the cellular uptake of Onconase.
Biochemistry 2011, 50, 1029310299.
(9) Luz, C. S.; Damosa, F. S.; Tanaka, A. A.; Kubota, L. T. Dissolved
oxygen sensor based on cobalt tetrasulphonated phthalocyanine
immobilized in poly-L-lysine film onto glassy carbon electrode. Sens.
Actuators, B 2006, 114, 10191027.

4. CONCLUSION
A PLL/MnO2/CRGO lm was used as a suitable matrix for the
immobilization of GOx which was found to eciently
accelerate the direct electrochemistry of GOx. The GOx
immobilized at the MnO2/CRGO composite lm exhibited
signicantly improved electrocatalytic ability toward oxidation
of glucose via reduction of oxygen. The proposed GOx-PLL/
MnO2/CRGO sensor exhibited a wide linear range from 0.04
15588

dx.doi.org/10.1021/ie502430d | Ind. Eng. Chem. Res. 2014, 53, 1558215589

Industrial & Engineering Chemistry Research

Article

(10) Monterroso, S. C. C.; Carapuc, H. M.; Duarte, A. C. Mixed

polyelectrolyte coatings on glassy carbon electrodes: Ion-exchange,
permselectivity properties and analytical application of poly-L-lysinepoly(sodium 4-styrenesulfonate)-coated mercury film electrodes for
the detection of trace metals. Talanta 2006, 68, 16551662.
(11) Daz-Gonzalez, M.; de la Escosura-Mu niz, A.; Gonzalez-Garca,
M. B.; Costa-Garca, A. DNA hybridization biosensors using polylysine
modified SPCEs. Biosens. Bioelectron. 2008, 23, 13401346.
(12) Deinhammer, R. S.; Ho, M.; Anderegg, J. W.; Porter, M. D.
Electrochemical oxidation of amine-containing compounds: A route to
the surface modification of glassy carbon electrodes. Langmuir 1994,
10, 13061313.
(13) Shan, C.; Yang, H.; Han, D.; Zhang, Q.; Ivaska, A.; Niu, L.
Water-soluble graphene covalently functionalized by biocompatible
poly-L-lysine. Langmuir 2009, 25, 1203012033.
(14) Some, S.; Ho, S. M.; Dua, P.; Hwang, E.; Shin, Y. H.; Yoo, H.;
Kang, J. S.; Lee, D. K.; Lee, H. Dual functions of highly potent
graphene derivative poly-L-lysine composites to inhibit bacteria and
support human cells. ACS Nano 2012, 6, 71517161.
(15) Czupryniak, J.; Niedzialkowski, P.; Karbarz, M.; Ossowski, T.;
Stojek, Z. Lysine arginine oligopeptides tagged with anthraquinone:
Electrochemical properties. Electroanal 2012, 24, 975982.
(16) Zhou, W.; Zhu, J.; Cheng, C.; Liu, J.; Yang, H.; Cong, C.; Guan,
C.; Jia, X.; Fan, H.; Yan, Q.; Lid, C.; Yu, T. A general strategy toward
graphene@metal oxide coreshell nanostructures for high-performance lithium storage. Energy Environ. Sci. 2011, 4, 49544961.
(17) Mani, V.; Periasamy, A.; Chen, S.-M. Highly selective
amperometric nitrite sensor based on chemically reduced graphene
oxide modified electrode. Electrochem. Commun. 2012, 17, 7578.
(18) Basavaraja, C.; Kim, W. J.; Kim, Y. D.; Huh, D. S. Synthesis of
polyaniline-gold/graphene oxide composite and microwave absorption
characteristics of the composite films. Mater. Lett. 2011, 65, 3120
3123.
(19) Luo, Y. L. Preparation of MnO2 nanoparticles by directly mixing
potassium permanganate and polyelectrolyte aqueous solutions. Mater.
Lett. 2007, 61, 18931895.
(20) Ljukic, B. S.; Compton, R. G. Manganese dioxide graphite
composite electrodes formed via a low temperature method: Detection
of hydrogen peroxide, ascorbic acid and nitrite. Electroanal. 2007, 19,
12751280.
(21) Sun, W.; Wang, X.; Zhu, H.; Sun, X.; Shi, F.; Li, G.; Sun, Z.
Graphene-MnO2 nanocomposite modified carbon ionic liquid
electrode for the sensitive electrochemical detection of rutin. Sens.
Actuators, B 2013, 178, 443449.
(22) Yu, G. H.; Hu, L. B.; Vosgueritchian, M.; Wang, H. L.; Xie, X.;
McDonough, R. J.; Cui, X.; Cui, Y.; Bao, Z. N. Solution-processed
graphene/MnO2 nanostructured textiles for high-performance electrochemical capacitors. Nano Lett. 2011, 11, 29052911.
(23) Qian, Y.; Lu, S. B.; Gao, F. L. Preparation of MnO2/graphene
composite as electrode material for supercapacitors. J. Mater. Chem.
2011, 46, 35173522.
(24) Zhu, C. Z.; Guo, S. J.; Fang, Y. X.; Han, L.; Wang, E. K.; Dong,
S. J. One-step electrochemical approach to the synthesis of graphene/
MnO2 nanowall hybrids. Nano Res. 2011, 4, 648657.
(25) Yu, G. H.; Hu, L. B.; Liu, N.; Wang, H. L.; Vosgueritchian, M.;
Yang, Y.; Cui, Y.; Bao, Z. N. Enhancing the supercapacitor
performance of graphene/MnO2 nanostructured electrodes by
conductive wrapping. Nano Lett. 2011, 11, 44384442.
(26) Marcano, D.; Kosynkin, D.; Berlin, J.; Sinitskii, A.; Sun, Z.;
Jsarev, A.; Alemany, L.; Lu, W.; Tour, J. Improved synthesis of
graphene oxide. ACS Nano 2010, 4, 48064814.
(27) Unnikrishnan, B.; Ru, P.; Chen, S.-M. Electrochemically
synthesized PtMnO2 composite particles for simultaneous determination of catechol and hydroquinone. Sens. Actuators, B 2012, 169,
235242.
(28) Chen, H.; Dong, X.; Shi, J.; Zhao, J.; Hua, Z.; Gao, J.; Ruan, M.;
Yan, D. Templated synthesis of hierarchically porous manganese oxide
with a crystalline nanorod framework and its high electrochemical
performance. J. Mater. Chem. 2007, 17, 855860.

(29) Zhang, Y.; Liu, H.; Zhu, Z.; Wong, K.; Mi, R.; Mei, J.; Lau, W. A
green hydrothermal approach for the preparation of graphene/MnO2 3D network as anode for lithium ion battery. Electrochim. Acta
2013, 108, 465471.
(30) Villalba, P.; Ram, M. K.; Gomez, H.; Kumar, A.; Bhethanabotla,
V.; Kumar, A. GOX-functionalized nanodiamond films for electrochemical biosensor. Mater. Sci. Eng., C 2011, 31, 11151120.
(31) Guascito, M.; Chirizzi, D.; Malitesta, C.; Mazzotta, E. Mediatorfree amperometric glucose biosensor based on glucose oxidase
entrapped in poly(vinyl alcohol) matrix. Analyst 2011, 136, 164173.
(32) Galhardo, K.; Torresi, R.; Susana, I.; Torresi, C. Improving the
performance of a glucose biosensor using an ionic liquid for enzyme
immobilization. On the chemical interaction between the biomolecule,
the ionic liquid, and the cross-linking agent. Electrochim. Acta 2012, 73,
123128.
(33) Mani, V.; Devadas, B.; Chen, S. M. Direct electrochemistry of
glucose oxidase at electrochemically reduced graphene oxide-multiwalled carbon nanotubes hybrid material modified electrode for
glucose biosensor. Biosens. Bioelectron. 2013, 41, 309315.
(34) Laviron, E. General expression of the linear potential sweep
voltammogram in the case of diffusionless electrochemical systems. J.
Electroanal. Chem. 1979, 101, 1928.
(35) Kang, X. H.; Wang, J.; Wu, H.; Aksay, I. A.; Liua, J.; Lin, Y. H.
Glucose oxidasegraphenechitosan modified electrode for direct
electrochemistry and glucose sensing. Biosens. Bioelectron. 2009, 25,
901905.
(36) Unnikrishnan, B.; Palanisamy, S.; Chen, S. M. A simple
electrochemical approach to fabricate a glucose biosensor based on
grapheneglucose oxidase biocomposite. Biosens. Bioelectron. 2013, 39,
7075.
(37) Wen, D.; Liu, Y.; Yang, G. C.; Dong, S. J. Electrochemistry of
glucose oxidase immobilized on the carbon nanotube wrapped by
polyelectrolyte. Electrochim. Acta 2007, 52, 53125317.
(38) Wang, K. Q.; Yang, H.; Zhu, L.; Ma, Z. S.; Xing, S. Y.; Lv, Q.;
Liao, J. H.; Liu, C. P.; Xing, W. Direct electron transfer and
electrocatalysis of glucose oxidase immobilized on glassy carbon
electrode modified with Nafion and mesoporous carbon FDU-15.
Electrochim. Acta 2009, 54, 46264630.
(39) Wang, K. Q.; Yang, H.; Zhu, L.; Liao, J. H.; Lu, T. H.; Xing, W.;
Xing, S. Y.; Lv, Q. Direct electrochemistry and electrocatalysis of
glucose oxidase immobilized on glassy carbon electrode modified by
Nafion and ordered mesoporous silica-SBA-15. J. Mol.Catal. B: Enzym.
2009, 58, 194198.
(40) Zhang, Y.; Fan, Y.; Wang, S.; Tan, Y.; Shen, X.; Shi, Z. Facile
fabrication of a graphene-based electrochemical biosensor for glucose
detection. Chin. J. Chem. 2012, 30, 11631167.
(41) Yin, H. S.; Zhou, Y. L.; Meng, X. M.; Shang, K.; Ai, S. Y. Onestep green preparation of graphene nanosheets and carbon
nanospheres mixture by electrolyzing graphite rob and its application
for glucose biosensing. Biosens. Bioelectron. 2011, 30, 112117.
(42) Hua, L.; Wu, X.; Wang, R. Glucose sensor based on an
electrochemical reduced graphene oxide-poly(L-lysine) composite film
modified GC electrode. Analyst 2012, 137, 57165719.

15589

dx.doi.org/10.1021/ie502430d | Ind. Eng. Chem. Res. 2014, 53, 1558215589