Simple approach for the immobilization of horseradish peroxidase on poly-L-histidine modified reduced graphene oxide for amperometric determination of dopamine and H2O2

Article abstract of DOI:10.1039/c4ra09011j

In this work, immobilization of horseradish peroxidase (HRP) on poly-L-histidine (P-L-His) modified reduced graphene oxide (RGO) was demonstrated. The HRP/P-L-His-RGO bio-nanocomposite film was prepared through layer-by-layer (LBL) assembly. Scanning electron microscopy, Fourier transform infrared spectroscopy, electrochemical impedance spectroscopy, and UV-Vis spectroscopy were adopted to monitor the uniformity of the LBL assembly of the HRP/P-L-His-RGO bio-nanocomposite film. The immobilized HRP exhibited excellent electrocatalytic activity towards the reduction of hydrogen peroxide (H₂O₂). The catalysis currents showed a linear relationship with H₂O₂ concentration, ranging from 0.2 to 5000 μM. The detection limit (S/N = 3) of H₂O₂ was 0.05 μM. The apparent Michaelis-Menten constant (K_m) was calculated to be 1.2 mM. Moreover, the modified electrode displayed a rapid response (5 s) to H₂O₂ with good stability and reproducibility. Based on the HRP/P-L-His-RGO bio-nanocomposite film, a third-generation reagentless biosensor was constructed for the determination of H₂O₂.

Full text of DOI:10.1039/c4ra09011j

RSC Advances
View Article Online
View Journal | View Issue
PAPER
Simple approach for the immobilization of
horseradish peroxidase on poly-L-histidine
modified reduced graphene oxide for
amperometric determination of dopamine and
H2O2†
Cite this: RSC Adv., 2014, 4, 55867
*
A. T. Ezhil Vilian and Shen-Ming Chen
In this work, immobilization of horseradish peroxidase (HRP) on poly-L-histidine (P-L-His) modified reduced
graphene oxide (RGO) was demonstrated. The HRP/P-L-His-RGO bio-nanocomposite film was prepared
through layer-by-layer (LBL) assembly. Scanning electron microscopy, Fourier transform infrared
spectroscopy, electrochemical impedance spectroscopy, and UV-Vis spectroscopy were adopted to
monitor the uniformity of the LBL assembly of the HRP/P-L-His-RGO bio-nanocomposite film. The
immobilized HRP exhibited excellent electrocatalytic activity towards the reduction of hydrogen
peroxide (H2O2). The catalysis currents showed a linear relationship with H2O2 concentration, ranging
from 0.2 to 5000 mM. The detection limit (S/N ¼ 3) of H2O2 was 0.05 mM. The apparent Michaelis–
Menten constant (Km) was calculated to be 1.2 mM. Moreover, the modified electrode displayed a rapid
response (5 s) to H2O2 with good stability and reproducibility. Based on the HRP/P-L-His-RGO bio-
nanocomposite film, a third-generation reagentless biosensor was constructed for the determination of
H2O2.
Received 21st August 2014
Accepted 6th October 2014
DOI: 10.1039/c4ra09011j
www.rsc.org/advances
abovementioned methods have some disadvantages, such as
time consuming, high costs, low sensitivity and complicated
pretreatments.11 On the other hand, electrochemical methods
have been actively developed owing to their high sensitivity,
rapid response time, low cost and simplicity of use.12 Recently,
considerable attention has been focused on the development of
biosensors due to their advantages of easy preparation, fast
detection, rapidity and high sensitivity.13
Hydrogen peroxide (H2O2) is a well-known oxidizing agent,
which plays an important role in the production of textiles,
cleaning products, organic compounds, and food industry.14
Owing to the increased usage of H2O2, it has been released into
the environment in large amounts. Thus, developing a simple,
rapid, reliable and highly sensitive method for the determina-
tion of trace amounts of H2O2 at physiological pH has become a
necessity.15 Furthermore, H2O2 is also a vital intermediate that
is formed in oxidase enzymes and its accurate and rapid
determination is a key factor for the development of efficient
biosensors.16 In recent years, there have been numerous
attempts to develop enzyme-based electrochemical biosensors
for the detection of H2O2. However, efforts have been limited by
the problems of poor stability, high cost of enzymes, compli-
cated immobilization procedures and critical operational
conditions due to sensitivity of enzymes to pH, temperature,
humidity, ionic detergents and toxic chemicals. It has become
necessary to develop a simple enzyme immobilization strategy
1. Introduction
The study of the electrochemical behaviour of dopamine (DA)
has received considerable attention from researchers and has
been extensively studied since the 1950s.1 DA is a crucial cate-
cholamine neurotransmitter existing in the brain and the
peripheral nervous system of mammals.2,3 It has been found
that a loss of DA-containing neurons may occur in some serious
diseases such as Parkinson's, schizophrenia, epilepsy, and HIV
infection to name a few.4,5 However, DA has a close oxidation
potential with some interference, which can cause poor selec-
tivity and sensitivity in the electrochemical determination of DA
in brain uids if it coexists with many interfering compounds.6
It is necessary to develop a simple and rapid analytical method
for the determination of DA that will overcome these problems.7
Over the past few years, several methods have been applied for
the analytical determination of DA in pharmaceutical and
clinical preparations, including, spectrophotometry,8 chem-
iluminescence,9 high performance liquid chromatography,10
and electrochemical methods. However, most of the
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).
E-mail: smchen78@ms15.hinet.net; Fax: +886 2270 25238; Tel: +886 2270 17147
† Electronic supplementary information (ESI) available. See DOI:
10.1039/c4ra09011j
This journal is © The Royal Society of Chemistry 2014
RSC Adv., 2014, 4, 55867–55876 | 55867

View Article Online
RSC Advances
Paper
to achieve a device capable of sensing H2O2 with high sensi-
tivity.17 Many techniques, such as chemiluminescence,18 spec-
trophotometry,19 titrimetry,20 and electroanalytical methods,21,22
have been employed for the detection and determination of
H2O2. Among these methods, enzyme-based electroanalytical
methods are more attractive because of their low cost, opera-
tional simplicity, high sensitivity and real-time detection.23
Based on the efficient catalyzing ability of horseradish peroxi-
dase (HRP) to decompose H2O2, various types of biosensors
have been developed.24
HRP is a common enzyme widely used in H2O2 biosensors
due to its easy availability, and low cost.25 HRP contains a
heme prosthetic group, which catalyzes the oxidation of
different substrates as a consequence of the protein active
site with the resting state of the heme-iron Fe(II).26 However,
some of the methods for enzyme immobilization are prone to
problems such as low reproducibility, poor enzymatic
activity, and poor spatial control during the deposition
process. The immobilization of HRP on the electrode surface
is difficult.27 In recent years, immobilization of HRP through
several methods, including absorption,28 cross linking,29
layer by layer assembly,30 sol–gel entrapment,31 and electro-
polymerization, have been proposed. Among these methods,
the physical entrapment of enzymes is needed for the
production of a more stable sensor with a reduced loss of
enzyme during measurement as well as a simpler and less-
time consuming fabrication process, while still ensuring
high stability and sensitivity, and biocompatibility, which
can affect the activity of the enzyme.32
Specically, graphene is a two-dimensional nanomaterial
that has received intense attention due to its unique physical
and chemical properties, such as low resistivity, outstanding
mechanical properties and large specic surface area.33,34
With the advantages of graphene substrate and active metal
nanoparticle components, a new class of hybrid materials,
such as graphene supported metal nanocomposites, have
been developed.35 They have shown great potential for
extensive applications in many advanced elds such as
memory electronics, optoelectronic transistors,36 catalysis,37
energy storage,38 and electrochemical sensors and
biosensors.39,40
2. Experimental procedure
2.1 Reagents
The graphite powder (<20 mm) used in the experiments was
purchased from Sigma-Aldrich. L-Histidine, H2O2 (30%), dopa-
mine hydrochloride, NaH2PO4 and Na2HPO4 were supplied by
Sigma-Aldrich. Horseradish peroxidase (E.C. 1.11.1.7 type-VI-A-
S/5 mg, Armoracia rusticana source, 1840 U mgꢀ1) was provided
by Sigma. Phosphate buffer solution (PBS) was prepared by
mixing standard stock solutions of Na2HPO4 and NaH2PO4. The
supporting electrolyte was a 0.05 M phosphate buffer solution
(PBS). All experiments were performed using PBS at room
temperature (approximately 25 ꢁC). All other chemicals were of
analytical grade and were used as received. The water used
throughout the experiments was puried by a Milli-Q water
system (resistivity 18.25 MU cm, Millipore).
2.2 Apparatus
All electrochemical experiments were performed on a CHI 405A
electrochemical work station with
a conventional three-
electrode system comprised of a platinum wire as the auxil-
iary electrode, an Ag/AgCl electrode as the reference electrode,
and a modied glassy carbon electrode (GCE) as the working
electrode. Rotating disc electrode (RDE) and amperometric
measurements were performed using a CHI 405A electro-
chemical workstation with an analytical rotator AFMSRX (PINE
Instruments, USA, Japan). Scanning electron microscopy (SEM)
was performed using a Hitachi S-3000 H instrument (Hitachi,
Japan). Energy dispersive X-ray analysis (EDX) was recorded
using a HORIBA EMAX X-ACT (Model 51-ADD0009) instrument.
UV absorption spectra were collected using a Model UV-3300
spectrophotometer (Hitachi, Japan). Fourier transform
infrared (FT-IR) measurements were carried out using a Perkin
Elmer spectrophotometer RXI. X-Ray photoelectron spectros-
copy (XPS) was performed using a PHI 5000 Versa Probe
equipped with an Al Ka X-ray source (1486.6 eV). Electro-
chemical impedance spectroscopy (EIS) measurements were
carried out in the frequency range from 0.1 Hz to 1 MHz (IM6ex
ZAHNER Kroanch, Germany).
To the best of our knowledge, there has been no report on
the fabrication of HRP/P-L-His-RGO nanocomposites on a glassy
2.3 Synthesis of GO
carbon electrode (GCE) surface for the determination of DA and The graphene oxide (GO) used in this work was prepared using
H2O2. In this work, a novel amperometric biosensor based on the modied Hummers method.41 Specically, graphite powder
the immobilization of HRP on the electrochemically polymer- (5.0 g) and 25 g of KMnO4 were sequentially added to concen-
ized L-His modied RGO has been proposed. The amine groups trated H2SO4 (150 mL) with constant stirring under ice-cold
in P-L-His provide positive surface charges on the P-L-His-RGO, conditions. The resulting mixture was then continuously stir-
enabling a favourable microenvironment for the immobiliza- red at 35 ꢁC for 5 h. Then, it was diluted with 600 mL of
tion of HRP. This simple immobilization strategy demonstrates deionized water and the temperature was kept under 50 ꢁC.
the great potential for P-L-His-RGO composite lms to be used Subsequently, 30 mL of 30% H2O2 was added drop wise into this
as a biomatrix for proteins in new generation biosensors. The mixture. The obtained solid product was repeatedly ltered and
proposed HRP/P-L-His-RGO biosensor has excellent sensitivity, washed with a 1 : 10 HCl aqueous solution (1000 mL) to remove
wide linearity, favourable stability, good reproducibility, low the metal ions, and then further washed with 1000 mL of
detection limit and a low Michaelis–Menten constant. In addi- deionized water until the solution pH became neutral (pH 6.5).
tion, the HRP/P-L-His-RGO composite lm was applied for the Finally, the obtained dark-yellow solid was dried in a vacuum
determination of H2O2 and DA in real samples.
oven at 40 ꢁC for 48 h and then ground to ne powder. The as-
55868 | RSC Adv., 2014, 4, 55867–55876
This journal is © The Royal Society of Chemistry 2014

View Article Online
Paper
RSC Advances
obtained powder was dispersed in water and sonicated for 1 reduction of the functional groups in GO, the deposition of
hour to obtain well-exfoliated GO.
highly conducting material onto the GCE electrode and the
electrochemical generation of the RGO + P-L-His lm on the
GCE. Then, a fresh HRP solution was prepared by dissolving 10
mg of HRP in 1 mL of 0.05 M PBS at pH 7.5 mL of HRP solution
was drop casted onto the surface of the RGO + P-L-His modiꢁed
GCE electrode using a microsyringe and allowed to dry at 4 C
for 24 h. HRP was immobilized onto the RGO + P-L-His modied
GCE through the electrostatic interactions between the posi-
tively charged P-L-His and negatively charged HRP molecules.
Aer each modication step the electrode surface was rinsed
with PBS. Finally, 5 mL of 0.1% Naon was coated on the
modied electrode surface to form a protective lm. The as-
prepared enzyme modied electrode was dried at 4 ꢁC until
used further.
2.4 Fabrication of HRP/P-L-His-RGO/GCE
Before modication, the GCE electrode (3 mm) was rst pol-
ished carefully with 0.05 mm of alumina slurry, and washed
ultrasonically in water. Then, it was electrochemically cleaned
in 0.1 M H2SO4 by cyclic potential scanning between ꢀ0.2 and
1.2 V until a standard cyclic voltammogram was obtained.
Finally, the GCE electrode was sonicated in water and absolute
ethanol before being dried in a nitrogen stream before use.
Schematic illustration shown in Scheme 1 provides an overview
of the fabrication process to prepare the HRP biosensor. Prior to
use, the as-synthesized GO (1 mg) was dissolved by sonication in
1 mL of water for 1 h. Then, 5 mL of the GO suspension was
dropped onto the GCE surface, followed by evaporation of the
solvent under an infrared lamp. The GO modied GCE was
transferred to an electrochemical cell containing the 0.05 M PBS
electrolyte at pH 5 and 5 mM of l-His. 30 cycles were recorded
over a potential range of 0.0 to ꢀ1.5 V vs. Ag/AgCl as displayed in
Fig. S1.† During the rst cathodic potential scan, a large
cathodic peak appears at ꢀ1.0 V with an onset potential of
ꢀ0.75 V, which is attributed to the reduction of the oxygen
containing functional groups in GO. The cathodic current
decreased continuously for each cycle, suggesting the effective
3. Results and discussion
3.1 Surface studies and elemental analysis
Fig. 1 illustrates the SEM images of the as-prepared (A)
P-L-His-RGO and (C) HRP/P-L-His-RGO bio-nanocomposites.
The P-L-His-RGO contains at, wrinkled sheets that are
stacked with each other. Small plate and rod-like P-L-histidine
molecules are also formed on the RGO sheets. Upon modi-
cation with HRP, several bead-like, globular structures are
formed on the P-L-His coated RGO sheets, which can be
attributed to the electrostatic interactions between the nega-
tively charged HRP and the positively charged P-L-His coated
RGO. The EDX spectrum of the P-L-His-RGO sheets (c) reveals
the presence of C (50%), O (40%) and N (10%) related to the
amino groups of histidine (see Fig. 1B). SEM results conrm
the formation of HRP/P-L-His-RGO on the ITO surface, sug-
gesting that the interaction between RGO/P-L-His and HRP
indeed occurs, and may even inuence the morphology of the
dry lms. Furthermore, the terminal functional groups of the
positively charged P-L-His-RGO provide more active sites for
the immobilization of the HRP biocomposite, leading to facile
and efficient electron transfer.
Scheme
1 Schematic illustration of the procedure used for the Fig. 1 (A) SEM image of P-L-His-RGO, (B) EDX spectra of the P-L-His-RGO
preparation of HRP/P-L-His-RGO film modified glassy carbon nanocomposite, and (C) SEM image of the HRP/P-L-His-RGO
electrode.
bio-nanocomposite films.
This journal is © The Royal Society of Chemistry 2014
RSC Adv., 2014, 4, 55867–55876 | 55869

View Article Online
RSC Advances
Paper
3.2 Characterization of the HRP/P-L-His-RGO modied lm
Fig. S2(a)† shows the FT-IR spectra of (a) GO, and (b) P-L-His-
RGO. FT-IR spectrum of GO shows the characteristic peaks of
–OH (n ¼ 3440 cmꢀ1 and 1398 cmꢀ1), aromatic –C]C (n ¼ 1623
cmꢀ1), epoxy –C–O (n ¼ 1288 cmꢀ1), carboxyl –C]O and –C–O (n
¼ 1741 cmꢀ1) and alkoxy –C–O (n ¼ 1076 cmꢀ1) groups,
revealing the existence of oxygen-containing functional groups
on the GO nanosheets.43 For P-L-His-RGO (Fig. S2(b)†), a new
band appears at 1656 cmꢀ1, which corresponds to amide I (the
–C¼ O stretch of P-L-His). The band at 1536 cmꢀ1 is assigned to
amide II (the –C–N stretch and –C–N–H deformation).
Compared with RGO, the C]O stretch at 1623 cmꢀ1 for the
carboxyl group disappeared, indicating the binding of P-L-His
with the RGO surface.
To examine the nature of the interactions between the HRP
and P-L-His-RGO, we recorded the FT-IR spectra (Fig. 3A(a)). The
two characteristic peaks centered at 1630 and 1567 cmꢀ1 are
assigned to the typical amide I and II absorption bands of HRP.
The peak around 1082 cmꢀ1 corresponds to the stretching
vibration of C–O in HRP.44 The biocompatibility of the P-L-His-
RGO lm gives it great potential to be used as a matrix for the
immobilization of the HRP enzyme for biosensor fabrication. In
addition, as can be seen from Fig. 3A(b), the position of the
amide I and II bands of HRP and that of the P-L-His-RGO lm
appear at the same position, suggesting that the HRP retains its
native structure in the HRP/P-L-His-RGO lm. In short there has
been no denaturation of HRP because it was stabilized through
the electrostatic interactions.
The Soret absorption bands associated with the heme groups
Fig. 2 TEM images of the (A) RGO and (B) P-L-His-RGO nano-
composite films. (C) XPS survey of P-L-His-RGO. (D) C1s XPS spectra may provide information about the conformational integrity of
of the P-L-His-RGO nanocomposite film. (E) N1s XPS spectra of the
P-L-His-RGO nanocomposite film.
the heme proteins and the possible denaturation of
Fig. 2(A&B) shows TEM images of the as-prepared (A) RGO
and (B) P-L-His-RGO nanocomposite lms. Fig. 2A shows the as-
prepared reduced graphene was thin and transparent, which
consist of a few layers of sheet-like structures and (B) shows the
P-L-His particles have been deposited as a distorted shaped thin
sheet-like structure, which were uniformly dispersed in the RGO
sheets. The presence of poly-L-His is clearly evident in the EDX
analysis shown in Fig. 1B.
The elemental composition of the P-L-His-RGO lm aer
various stages of surface modication was ascertained by XPS
(Fig. 2C). It is well known that the bands centered at 284.8 and
531.0 eV are associated with C1s and O1s, respectively. It can
be clearly seen that there is a signicant decrease in the
intensity of the O1s peaks of RGO compared with that of the
P-L-His/RGO, which can be attributed to the loss of oxygen in
GO as a result of electrochemical reduction (Fig. 2D). A new
Fig. 3 (A) FT-IR spectra of (a) HRP and (b) HRP-P-L-His-RGO film. (B)
peak observed at 404.2 eV corresponds to N1s, which can be
assigned to the nitrogen atoms in the amino groups of P-L-His
(Fig. 2E). In addition, the new peak observed at 286 eV can be
attributed to the C–N of P-L-His.42 The XPS results therefore
validate the electrodeposition of the nitrogen containing
P-L-His on RGO.
UV-Vis spectra of (a) HRP and (b) HRP immobilized onto different
bilayers (n ¼ b ꢀ d) of the P-L-His-RGO films. (C) Nyquist plots for (a) HRP,
(b) HRP/P-L-His, (c) RGO/GCE, (d) bare GCE, and (e) P-L-His-RGO/GCE
electrodes for 5 mM [Fe(CN)6]3ꢀ/4ꢀ in a 0.05 M PBS (pH 7.0) solution
containing 0.05 M KCl. Amplitude: 5 mV, frequency: 0.1 Hz to 1 MHz.
The bottom right inset shows Randles equivalence circuit for the
abovementioned electrodes.
55870 | RSC Adv., 2014, 4, 55867–55876
This journal is © The Royal Society of Chemistry 2014

View Article Online
Paper
RSC Advances
conformational change in the heme group region.45,46 As can be
seen in Fig. 3B, the Soret band of the HRP molecules appears at
408.8 nm (curve a). HRP immobilized onto different bilayers
(n ¼ b ꢀ d) of the P-L-His-RGO lms exhibits the Soret band at
408.8 nm without any shis, indicating that no signicant
denaturation of the protein occurred even aer being immo-
bilized onto the P-L-His-RGO composite with increased
lm thickness. Thus, HRP retained its native structure in the
P-L-His-RGO lm due to its good lm-forming ability and
excellent biocompatibility.
3.3 Electrochemical characteristics and direct
electrochemistry of HRP on the P-L-His-RGO modied lm
electrode
EIS is a sensitive tool for studying the interface properties of
surfaces aer different steps of modication. The Nyquist plot
of the EIS has a semi-circular portion at higher frequencies
corresponding to a limited electron transfer process and a
linear part at lower frequency ranges representing a diffusion
limited process. Typical Nyquist plots of (a) HRP, (b) HRP/P-L-His,
(c) RGO/GCE, (d) bare GCE, and (e) P-L-His-RGO/GCE electrodes
recorded in [Fe(CN)6]3ꢀ/4ꢀ solution over a frequency range of
0.1 Hz to 1 MHz are shown in Fig. 3C. The inset to Fig. 3C shows
the equivalent circuit comprised of the ohmic resistance of an
electrolyte solution (Rs), Warburg impedance (Zw), capacitance
(C), and apparent charge transfer resistance (Ret). As depicted
in Fig. 3C(d), the bare GCE electrode exhibits a Ret value of
1200 U. Aer being modied with RGO, the Ret value of
the RGO/GCE electrode decreased to 1120 U (see Fig. 3C(c)).
Further modication of RGO/GCE with P-L-His decreased the Ret
value to 730 U, possibly due to the improved electron transport
at the electrode interphase as a result of the electrostatic
attraction between the positive charges in the amino groups of
the L-His-RGO modied GCE electrode and the negatively
charged [Fe(CN)6]3ꢀ/4ꢀ redox probe (see Fig. 3C(e)). When the
HRP enzyme was immobilized on the L-His-RGO modied GCE
electrode, the Ret value increased to 1900 U (see Fig. 3C(b)),
owing to the electrostatic repulsions between the negatively
charged HRP and the negatively charged Fe(CN)6]3ꢀ/4ꢀ redox
electrochemical probe. The EIS data indicates that HRP was
successfully assembled on the P-L-His-RGO surface.
Fig. 4A shows the cyclic voltammetric responses of the (a)
bare GCE, (b) P-L-His-RGO and (c) HRP-L-His-RGO modied
GCE electrodes in 5 mM K3[Fe(CN)6] solution containing 0.1 M
KCl. Fig. 4A shows the cyclic voltammetry (CV) curve of the (a)
bare GCE witnessed a pair of well-dened redox peaks with
anodic (Epa) and cathodic (Epc) peak potentials of 0.201 and
0.110 V, respectively, and redox peaks with a potential differ-
ence of 91 mV, which are attributed to the redox behavior of
[Fe(CN)6]3ꢀ/4ꢀ. Fig. 4A(b) shows an obvious increase in the
redox peak current aer the deposition of P-L-His onto the RGO/
GCE surface. The positively charged P-L-His-RGO lm modied
GCE surface attracted the negatively charged [Fe(CN)6]4ꢀ/3ꢀ
probe due to stronger electrostatic interactions. The peak-to-
peak separation (DEp) was found to be 63 mV. When HRP was
immobilized on the P-L-His-RGO modied GCE electrode, the
Fig. 4 (A) CVs of the different modified electrodes recorded in a 0.05
M PBS (pH 7.0) solution containing 0.05 M KCl and 5 mM [Fe(CN)6]3ꢀ/4ꢀ
:
(a) bare GCE electrode, (b) P-L-His-RGO modified GCE electrode,
and (c) HRP-P-L-His-RGO/GCE modified electrode. (B) CVs of the
HRP/P-L-His-RGO modified GCEs in 0.05 M deoxygenated PBS (pH 7)
at various scan rates (from inner to outer: 10 to 100 mV sꢀ1). Inset: (I)
linear dependence of Ipa and Ipc on the scan rate.
peak currents decreased, whereas the peak-to-peak separation
between the cathodic and the anodic waves increased. Fig. 4A(c)
shows the CV curves of HRP-L-His-RGO modied GCE. There is
a signicant decrease in the peak current, which can be
attributed to the increase in the layer thickness and the elec-
trostatic repulsion between the negatively charged HRP enzyme
on the L-His-RGO electrode surface and the negatively charged
[Fe(CN)6]4ꢀ/3ꢀ
.
Fig. 4B shows the CVs of the HRP/L-His-RGO modied GCE
electrode in PBS at different scan rates (10–100 mV sꢀ1). The
anodic and cathodic currents increase linearly as the scan rate
increases from 10 to 100 mV sꢀ1, revealing a surface conned
redox process. The linear regression equation can be expressed
as follows: Ipa ¼ 1.909v ꢀ 0.228, R ¼ 0.9938; Ipc ¼ ꢀ1.312x +
0.524, R ¼ 0.9962. The effect of the scan rate is shown in Fig. 4B.
The reduction (Ipc) and oxidation (Ipa) peak currents exhibit a
linear relationship with the scan rate in the range from 10 to 100
mV sꢀ1. The obtained results indicate that HRP immobilized on
the L-His-RGO biocomposite modied GCE electrode undergoes
a surface conned electrochemical process. The surface average
This journal is © The Royal Society of Chemistry 2014
RSC Adv., 2014, 4, 55867–55876 | 55871

View Article Online
RSC Advances
Paper
concentration of the electroactive HRP immobilized on the negative potential shi. In addition, the catalytic activity of the
L-His-RGO biocomposite modied GCE electrode can be calcu- HRP/P-L-His-RGO bio-nanocomposite (Fig. S3†) is evident from
lated from the charge integration of the cathodic peak in the a decrease in the over potential as well as an increase in the
cyclic voltammogram according to the formula: Q ¼ nFAG,46 response current for DA oxidation when compared with the bare
where Q is the charge consumed in C, A is the electrode area GCE, RGO/GCE, and P-L-His-RGO/GCE modied electrodes.
(cm2), n is the number of electrons transferred and F is the There is a two-fold increase in the oxidation peak current (Ipa) of
Faraday constant. The surface concentration (G) of the electro- DA on the HRP/P-L-His-RGO bio-nanocomposite over that of the
active HRP/L-His-RGO modied GCE electrode is calculated to other electrodes (bare GCE, RGO/GCE, and P-L-His-RGO/GCE).
be 2.14 ꢂ 10ꢀ10 mol cmꢀ2, which is much larger than for the These experiments clearly indicate that the presence of
HRP immobilized onto a PTMSPA@GNR electrode (4.51 ꢂ P-L-His-RGO provided a favourable microenvironment that
10ꢀ11 mol cm2),47 HRP adsorbed on a graphite electrode (4 ꢂ preserved HRP bioactivity and assisted the electron transfer
10ꢀ11 mol cm2),48 and HRP immobilized onto a silica–hydroxy- process between the protein and the GCE electrode surface.
apatite modied GCE electrode (2.13 ꢂ 10ꢀ11 mol cmꢀ2).49 The This enhanced the signal and reduced the over potential for DA
obtained result indicates the high loading of HRP in multi- electrooxidation, both of which are very useful for practical
layers in the L-His-RGO biocomposite lm.
The heterogeneous electron transfer rate constant (Ks) of electrochemical DA sensor, with less risk of interference.
HRP adsorbed on the L-His-RGO modied GCE electrode is The electrochemical catalytic oxidation of DA at the
estimated (using Laviron's equation) to be about 4.82 sꢀ1, where HRP/P-L-His-RGO/GCE modied electrode was investigated
applications in the fabrication of a sensitive and selective
DEp < 200 mV.50
by linear sweep voltammetry. When DA was added to the PBS
(pH 7.0) solution, a new oxidation peak appeared at about
0.2 V, as shown in Fig. 5A. The new peak originates from the
oxidation of DA. The anodic peak current increased for each
addition of DA into the buffer solution. The abovementioned
experimental results demonstrate the good electrocatalytic
RT
log Ks ¼ a logð1 ꢀ aÞ þ ð1 ꢀ aÞlog a ꢀ log
nFv
að1 ꢀ aÞnF DEp
2:3RT
ꢀ
(1)
where R is the gas constant (8.314 J molꢀ1 Kꢀ1), T is the room
temperature (298.15 K) and DEp is the peak separation of Fe3+/2+
in the active sites of HRP. Here, the a value is assumed to be
0.5 and the number of electrons (n) transferred is considered to
be 2. The Ks value of the HRP immobilized on the L-His-RGO
biocomposite modied GCE electrode has been estimated to be
4.82 sꢀ1 which is comparatively larger than that of 1.13 sꢀ1 for
the HRP/DNA lm electrode,51 3.52 ꢃ 0.34 sꢀ1 for the HRP/
chitosan/sol-gel/CNT lm electrode,52 HRP immobilized onto
graphite electrode (1.3 ꢃ 0.2 sꢀ1) lm electrode,53 HRP/
PTMSPA@GNR lm electrode (3.23 ꢃ 0.1 sꢀ1),47 HRP immobi-
lized onto (2 ꢂ 10ꢀ4 sꢀ1) pyrolitic graphite.54 These ndings
suggest that the L-His-RGO biocomposite modied GCE elec-
trode provides a good microenvironment and high conductivity
that facilitates the direct electron transfer between the HRP and
the electrode surface.
3.4 Electrocatalysis of the HRP/P-L-His-RGO modied lm
The electrocatalytic performances of the (a) bare GCE, (b) RGO/
GCE, (c) P-L-His-RGO/GCE, and (d) HRP/P-L-His-RGO bio-
nanocomposite were evaluated by recording the linear sweep
voltammetry in 0.05 M PBS (pH 7.0) containing 3 mM DA at 50
mV sꢀ1. It can be seen that there is an electrocatalytic reduction
towards DA on the bare GCE and RGO/GCE exhibited by the
lower peak current and higher over potential than that observed
for the HRP/P-L-His-RGO bio-nanocomposite. Under the same
conditions, the P-L-His-RGO/GCE modied electrode exhibits
an Epa of DA with a more negative potential and with an
enhanced peak current (Ipa) response greater than that of the
bare GCE or RGO/GCE. This large decrease in oxidation over
potential corresponds to the presence of high density arrays of
P-L-His-RGO. Their electrocatalytic behaviour indicates that P-L-
Fig.
5 (A) Linear sweep voltammetry of the HRP-P-L-His-RGO
modified GCEs electrode in PBS with DA concentrations of (a) blank,
(b) 1, (c) 2, and (d) 3 mM. (B) Amperometric response of the fabricated
biosensor to successive additions of DA into the PBS solution (0.05 M,
pH 7). Applied potential: +0.23 V. Inset: calibration curve and linear
fitting curve between the current and DA concentration.
His-RGO signicantly facilitates electron transfer with
a
55872 | RSC Adv., 2014, 4, 55867–55876
This journal is © The Royal Society of Chemistry 2014

View Article Online
Paper
RSC Advances
activity of the HRP/P-L-His-RGO/GCE modied electrode towards DA.45 In this work, the Km of the HRP/P-L-His-RGO/GCE
towards the oxidation of DA. Thus, the HRP/P-L-His-RGO/GCE modied electrode was determined to be 2.5 mM. Thus, the
modied electrode showed greater sensitivity and faster P-L-His-RGO modied electrode has an improved affinity for the
catalytic activity towards DA. We only observed a 3% decrease immobilization of HRP to DA. Evidently, the small Km value
in the initial response even aer carrying out 50 continuous revealed the higher affinity of HRP/P-L-His-RGO/GCE for DA.
cyclic scans in the potential range of ꢀ0.1 to 0.4 V at a scan Moreover, the selectivity of the DA biosensor was evaluated
rate of 50 mV sꢀ1, showing its high stability. The mechanism using the amperometric method. The results show that the
of the catalytic oxidation of the immobilized HRP on P-L-His- common interfering species including 100 mM ascorbic acid,
RGO/GCE modied electrode to DA is illustrated in the 100 mM glucose, 100 mM uric acid, and 100 mM cysteine did not
following scheme:55
cause any observable interference in the amperometric
response to 100 mM DA at the HRP/P-L-His-RGO/GCE modied
electrode. As shown in Fig. S5,† the prepared HRP/P-L-His-RGO/
GCE modied electrode allowed high sensitivity and selectivity
for DA detection under normal physiological conditions.
3.5 Electrocatalytic properties towards the reduction of H2O2
In order to investigate the electrocatalytic activity of the
different electrodes: (a) bare GCE, (b) RGO modied GCE, (c)
P-L-His modied GCE, (d) P-L-His-RGO modied GCE, and (e)
HRP/P-L-His-RGO modied GCE towards the reduction of
H2O2, CV was carried out in the presence of H2O2 at the
HRP/P-L-His-RGO bio-nanocomposite modied GCE electrodes.
Fig. S4† shows the CVs of 3 mM H2O2 in a pH 7 PBS buffer
solution for the unmodied (a) bare GCE, (b) RGO modied
GCE, (c) P-L-His modied GCE, (d) P-L-His-RGO modied GCE,
and (e) HRP/P-L-His-RGO modied GCE electrodes. The results
demonstrate the superior performance of the as-obtained
HRP/P-L-His-RGO bio-nanocomposite. The HRP/P-L-His-RGO
bio-nanocomposite modied GCE shows a greater background
current than that of the other modied electrodes, which can be
attributed to the large surface area of the P-L-His-RGO lm. The
nanostructures in this material provide a large surface area
such that the composite P-L-His-RGO matrix has a higher
capability for enzyme immobilization. Therefore, there was a
two-fold increase in the reduction peak current of H2O2 at
HRP/P-L-His-RGO modied GCE electrodes. These results show
that the P-L-His-RGO modied GCE electrodes can improve the
catalytic activity of HRP with more effective electrocatalytic
activity toward H2O2 than the other modied electrodes.
Fig. 6A shows the CVs of the HRP/P-L-His-RGO/GCE recorded
in the presence of different concentrations of H2O2. The elec-
trocatalytic responses of both the bare GCE and HRP/P-L-His-
RGO/GCE towards the electrocatalytic reduction process of
H2O2 have also been shown in Fig. 6A. However, no reduction
peak was observed using the bare GCE. The intercalated HRP
molecules produce good electrocatalytic activity due to the
interactions between the HRP and the P-L-His-RGO/GCE.
It is evident that the reduction current of H2O2 increased
when increasing the H2O2 concentrations from 1 mM to 3 mM.
These observations clearly indicate that the HRP/P-L-His-RGO/
GCE exhibits excellent catalytic activity towards the reduction
of H2O2. The electrocatalytic process can be expressed as
follows:27,46,57
Fig. 5B illustrates a typical current-time plot obtained at the
HRP/l-His-RGO modied rotating disc electrode for each
successive addition of DA into the 0.05 M PBS (pH 7.0) solution.
The electrode potential was held at +0.23 V vs. Ag/AgCl. When
the background current became stable, a certain amount of DA
was added into the solution and stirred at 1200 rpm. Fig. 5B
shows that the amperometric peak current was linearly
proportional to the concentration of DA. A stable and fast
amperometric response could be observed for each successive
injection of DA into the PBS solution. The time required to
reach the steady state was less than 5 s, which indicates the
rapid response of this sensor for DA. The inset to Fig. 5B shows
the calibration plot for DA obtained over the concentration
range of 0.2–12 000 mM. The corresponding regression equa-
tions for the linear plot are expressed as follows:
I(mA) ¼ 56.624 + 2.486, R ¼ 0.9952
(2)
The sensitivity of the sensor to DA was determined to be 16.2
mA mMꢀ1 cmꢀ2; and the detection limit was found to be 0.42 mM
at a signal-to-noise ratio of 3. Further comparison of the
HRP/P-L-His-RGO/GCE modied electrode developed in this
study with other modied electrodes for the determinations of
DA are listed in Table S1.† Because amperometry is more sensitive
than cycle voltammetry, we used the amperometric method
to determine the affinity of DA to the HRP/P-L-His-RGO/GCE
modied electrode.
1
1
Km
¼
þ
(3)
Iss Imax Imax
C
As shown in Fig. 5B, the apparent Michaelis–Menten
constant (Km) can be obtained from the Lineweaver–Burk
equation to evaluate the affinity of the immobilized enzyme
HRP (Fe3+) + H2O2 / Compound I (Fe4+ ¼ O) + H2O (4)
Compound I (Fe4+ ¼ O) + eꢀ + H+ / Compound II
(5)
This journal is © The Royal Society of Chemistry 2014
RSC Adv., 2014, 4, 55867–55876 | 55873

View Article Online
RSC Advances
Paper
(Km), which is an indicator of the enzyme-substrate kinetics was
calculated from the Lineweaver–Burk equation, where Iss is the
steady-state current attained aer the addition of the substrate,
Imax is the maximum current measured under saturated
substrate conditions and C is the bulk concentration of the
substrate. The Km value was found to be 1.2 mM, which suggests
an apparent affinity between immobilized HRP and L-His-RGO/
GCE. Furthermore, the effect of possible interfering species,
such as uric acid (UA), glucose (Glu), and ascorbic acid (AA), on
H2O2 detection was also investigated. The amperometric
responses of the HRP/l-His-RGO/GCE to the continuous addi-
tion of 100 mM UA, 100 mM AA, 100 mM glucose and 100 mM
H2O2 at working potentials of ꢀ0.2 V in PBS (0.05 M, pH 7.0)
were studied. As shown in Fig. S6† there is clear indication that
the interference from UA or AA is negligible at an applied
potential of ꢀ0.2 V.
3.6 Real sample analysis
We investigated the response of the HRP/P-L-His-RGO/GCE
biosensor for the measurement of H2O2 in real eye drop
samples. The eye drop sample was obtained from a phar-
macy. 1 mL of the sample was dissolved in PBS (pH 7) prior
to measurement. The analytical applicability of the
HRP/P-L-His-RGO/GCE biosensor was evaluated by deter-
mining the recovery towards different concentrations of
hydrogen peroxide using the standard addition method. The
obtained results are given in Table 2, which indicate that this
method is reliable and could be effectively used in future
applications. We further explored the practicality of the
proposed HRP/P-L-His-RGO/GCE biosensor for the determi-
nation of DA in human urine and serum samples. Sampling
was performed using the standard additional method. All the
urine and serum samples (1 mL) were dissolved in PBS (pH 7)
prior to use. The analytical results are shown in Table S2.†
These results indicate the effectiveness of this method for
practical applications.
Fig. 6 (A) Bioelectrocatalysis of the HRP/P-L-His-RGO modified GCEs
towards H2O2 in PBS with a scan rate of 0.5 V sꢀ1 and H2O2
concentrations of (a) are GCE, (b) blank, (c) 1, (d) 2, and (e) 3 mM. (B)
Typical current-time response curves for the HRP/P-L-His-RGO
modified GCEs after successive additions of H2O2 into pH 7 PBS.
Applied potential: ꢀ0.2 V (vs. Ag/AgCl). Inset: calibration curve
showing variation of the biosensor response with H2O2 concentration.
Compound II + eꢀ + H+ / HRP (Fe3+) + H2O
The overall reaction would be:
(6)
3.7 Stability and reproducibility of the sensor
H2O2 + 2eꢀ + 2H+ / 2H2O
(7)
The as-prepared HRP/P-L-His-RGO/GCE modied electrode
showed an acceptable reproducibility with a relative standard
deviation of 3.6% for the current determined at a DA
concentration of 50 mM. The reproducibility of the different
modied electrodes was also examined. Compared with the
HRP/P-L-His-RGO/GCE, only RGO and P-L-His-RGO lms have
an RSD of 3.22% and 4.22%, respectively. In addition, the
stability of the HRP/P-L-His-RGO/GCE modied electrode was
We then investigated the amperometric response of the
HRP/P-L-His-RGO/GCE modied rotating disc electrode towards
each successive addition of H2O2 into 0.05 M PBS (pH 7.0) at an
applied potential of ꢀ0.2 V as shown in Fig. 6B. Upon addition
of aliquots of H2O2 into the 0.05 M PBS (pH 7.0) solution, the
prepared HRP/P-L-His-RGO/GCE sensor responded rapidly and
achieved the maximum steady-state current within 5 s. As
shown in Fig. 6B a linear dependence of the peak current with
the concentration of H2O2 was observed in the range of 0.2 to
5000 mM with a sensitivity of 266.4 mA mMꢀ1 cmꢀ2. The
detection limit was estimated to be 0.05 mM at a signal-to-noise
ratio of 3. The electrocatalytic efficiency of the proposed
HRP/P-L-His-RGO/GCE electrode for the determination of H2O2
was compared with other types of HRP modied electrodes as
shown in Table 1. The apparent Michaelis–Menten constant
ꢁ
evaluated aer being stored in 0.05 M PBS (pH 7.0) at 4 C.
The storage stability was evaluated by periodically checking
its relative response current in PBS containing 50 mM of DA.
Aer a storage period of 20 days, the HRP/P-L-His-RGO/GCE
modied electrode showed an 8% loss of activity. Thus, the
good repeatability, reproducibility and selectivity of the
developed biosensor make it attractive for practical
applications.
55874 | RSC Adv., 2014, 4, 55867–55876
This journal is © The Royal Society of Chemistry 2014

View Article Online
Paper
RSC Advances
Table 1 Comparison of the performance of the reported HRP modified electrodes for the detection of H2O2
Modied electrodes
Linear range mM
Sensitivity mA mMꢀ1 cmꢀ2
LOD mM
Ref.
Au/graphene sheet/HRP/chitosan
Sonogel-carbon electrode/HRP
MWNT–silica–polyaniline–NW–HRP
GCE/silica–hydroxyapatite/HRP
HRP–Naon-screen printed electrode
ZnO/GNPs/Naon/HRP
Clay/HRP/Au/CS
Au/CS/HRP
CMCS/Au/HRP
HRP–ZrO2/Au
HRP/P-L-His-RGO/GCE
5–5130
—
1.7
1.6
27
45
47
56
58
59
60
61
4.0–100
12.8
58.1
0.051
0.143
—
—
0.013
—
1 ꢂ 10ꢀ6–1000
1 ꢂ 10ꢀ6
0.35
0.48
9
1–100
5.98–35.36
15–1100
39–3100
12.2–2430
5–1400
9
6.3
0.401
2
0.05
62
63
20–9450
0.2–5000
—
266.4
This work
Table 2 Determination of H2O2 in real eye drop samples obtained
using the HRP/P-L-His-RGO modified glassy carbon electrode
4 U. Chandra, B. E. Kumara Swamy, O. Gilbert and
B. S. Sherigara, Electrochim. Acta, 2010, 55, 7166.
5 J. H. Jin, E. Cho and S. Jung, Biotechnol. Lett., 2010, 32, 413.
6 R. Suresh, K. Giribabu, R. Manigandan, S. Praveen Kumar,
S. Munusamy, S. Muthamizh, A. Stephen and
V. Narayanan, Sens. Actuators, B, 2014, 202, 440–447.
7 Y. Wu, L. Cui, Y. Liu, G. Lv, T. Pu, D. Liu and X. He, Analyst,
2013, 138, 1204–1211.
Samples
Added [mM]
Found [mM]
Recovery rate (%)
1
2
3
5
10
20
4.8
9.6
18.2
101%
99.5%
98%
8 Q. M. Li, J. Li and Z. J. Yang, Anal. Chim. Acta, 2007, 583, 147–
152.
4. Conclusions
9 Z. H. Guo and S. J. Dong, Electroanalysis, 2005, 17, 607.
10 S. Sarre, Y. Michotle, P. Herregodts, D. Deleu, N. De Klippel
and G. Ebinger, J. Chromatogr., 1992, 585, 207.
11 S. Thiagarajan, R. Yang and S. Chen, Bioelectrochemistry,
2009, 75, 163–169.
12 Y. Wang, Y. Li, L. Tang, J. Lu and J. Li, Electrochem. Commun.,
2009, 11, 889–892.
13 M. Rajkumar, B. Devadas and S. Chen, Electrochim. Acta,
2013, 105, 439–446.
14 S. Liu, B. Yu and T. Zhang, RSC Adv., 2014, 4, 544–548.
15 B. Yu, J. Feng, S. Liu and T. Zhang, RSC Adv., 2013, 3, 14303–
14307.
16 Q. Lu, X. Dong, L. Li and X. Hu, Talanta, 2010, 82, 1344–1348.
17 E. Ferapontova, K. Schmengler, T. Borchers, T. Ruzgas and
L. Gorton, Biosens. Bioelectron., 2002, 17, 953–963.
18 K. Nakashima, K. Maki, S. Kawaguchi, S. Akiyama,
Y. Tsukamoto and K. Imai, Anal. Sci., 1991, 7, 709–713.
19 C. Matsubara, N. Kawamoto and K. Takamala, Analyst, 1992,
117, 1781–1784.
In summary, a biocompatible nanocomposite electrode based
on HRP/P-L-His-RGO lm was proposed for the determination
of H2O2 and DA. We have demonstrated the feasibility of this
novel design for an enzymatic electrochemical H2O2 and DA
sensor based on a HRP/P-L-His-RGO/GCE modied electrode.
The as-prepared HRP/P-L-His-RGO/GCE modied electrode
exhibited very strong and sensitive amperometric responses to
H2O2 and DA. The ease of fabrication, fast response and high
sensitivity and long stability show the potential of the enzymatic
H2O2 and DA sensor, proposed in this study, for the detection of
H2O2 and DA in real clinical and wastewater samples. The HRP/
P-L-His-RGO/GCE modied electrode provides a promising
platform for the design of reagent-less biosensors owing to the
advantages of simplicity, rapidity and ease of operation.
Acknowledgements
20 E. C. Hurdis and J. H. Romeyn, Anal. Chem., 1954, 26, 320–
325.
21 S. Liu, J. Tian, L. Wang and X. Sun, Carbon, 2011, 49, 3158–
3164.
The authors gratefully acknowledge the nancial support
received from the Ministry of Science and Technology, Taiwan
(ROC).
22 S. Liu, J. Tian, L. Wang, H. Li, Y. Zhang and X. Sun,
Macromolecules, 2010, 43, 10078–10083.
23 J. L. Huang and Y. C. Tsai, Sens. Actuators, B, 2009, 140, 267–
272.
24 H. L. Zhang, G. S. Lai, D. Y. Han and A. M. Yu, Anal. Bioanal.
Chem., 2008, 390, 971–977.
25 B. Liu, Z. Liu, D. Chen, J. Kong and J. Deng, J. Anal. Chem.,
2000, 367, 539–544.
References
1 M. Zhang, C. Liao, Y. Yao, Z. Liu, F. Gong and F. Yan, Adv.
Funct. Mater., 2014, 24, 978–985.
2 M. Rajkumar, B. Devadas, S. Chen and P. Yeh, Colloids Surf.,
A, 2014, 452, 39–45.
3 S. Palanisamy, S. Ku and S. Chen, Electrochim. Acta, 2013,
180, 1037–1042.
This journal is © The Royal Society of Chemistry 2014
RSC Adv., 2014, 4, 55867–55876 | 55875

View Article Online
RSC Advances
Paper
26 D. F. Cao, P. L. He and N. F. Hu, Analyst, 2003, 128, 1268– 45 M. ElKaoutit, I. Naranjo-Rodriguez, M. Dominguez,
1274.
M. P. Hernandez-Artiga and D. Bellido-Milla, Electrochim.
Acta, 2008, 53, 7131–7137.
46 A. Periasamy, S. Yang and S. Chen, Talanta, 2011, 87, 15–23.
27 K. F. Zhou, Y. H. Zhu, X. L. Yang, J. Luo, C. Z. Li and
S. R. Luan, Electrochim. Acta, 2010, 55, 3055–3060.
28 Y. Xiao, H. X. Ju and H. Y. Chen, Anal. Biochem., 2000, 278, 47 S. Komathi, A. Gopalan, S. Kim, G. Anand and K. Lee,
22–28. Electrochim. Acta, 2013, 92, 71–78.
29 B. Utpal, S. Pragya, K. Krishnamoorthy and N. Pradip, 48 L. Wan, Y. Song, H. Zhu, Y. Wang and L. Wang, Int. J.
Journal of Biotechnology, 2006, 126, 220–229. Electrochem. Sci., 2011, 6, 4700–4713.
30 S. Qu, F. Huang, G. Chen, S. N. Yu and J. L. Kong, 49 B. Wang, J. J. Zhang, Z. Y. Pan, X. Q. Tao and H. S. Wang,
Electrochem. Commun., 2007, 9, 2812–2816. Biosens. Bioelectron., 2009, 24, 1141–1145.
31 J. W. Wang, M. Gu, J. W. Di, Y. S. Gao, Y. Wu and Y. F. Tu, 50 E. Laviron, J. Electroanal. Chem., 1979, 101, 19–28.
Bioprocess Biosyst. Eng., 2007, 30, 289–296.
32 A. Morrin, F. Wilbeer, O. Ngamma, S. E. Moulton,
51 X. Chen, C. Ruan, J. Kong and J. Deng, Anal. Chim. Acta, 2000,
412, 89–98.
A. J. Killard, G. G. Wallace and M. R. Smyth, Electrochem. 52 X. Kang, J. Wang, Z. Tang, H. Wu and Y. Lin, Talanta, 2009,
Commun., 2005, 7, 317–322.
78, 120–125.
33 K. S. Novoselov, Science, 2004, 306, 666–669.
34 P. Avouris, Z. H. Chen and V. Perebeinos, Nat. Nanotechnol.,
2007, 2, 605–615.
53 A. Lindgren, T. Ruzgas, L. Gorton, E. Csoregi, G. Bautista
Ardila, I. Y. Sakharov and I. G. Gazaryan, Biosens.
Bioelectron., 2000, 15, 491–497.
35 A. K. Geim and K. S. Novoselov, Nat. Mater., 2007, 6, 183–191. 54 T. Tatsuma, K. Ariyama and N. Oyama, J. Electroanal. Chem.,
36 A. N. Cao, Z. Liu, S. S. Chu, M. H. Wu, Z. M. Ye, Z. W. Cai, 1998, 446, 205–209.
Y. L. Chang, S. F. Wang, Q. H. Gong and Y. F. Liu, Adv. 55 P. Raghu, T. Madhusudana Reddy, P. Gopal, K. Reddaiah
Mater., 2010, 22, 103–106. and N. Y. Sreedhar, Enzyme Microb. Technol., 2014, 57, 8–15.
37 E. Yoo, T. Okata, T. Akita, M. Kohyama, J. Nakamura and 56 B. Wang, J. J. Zhang, Z. Y. Pan, X. Q. Tao and H. S. Wang,
I. Honma, Nano Lett., 2009, 9, 2255–2259. Biosens. Bioelectron., 2009, 24, 1141–1145.
38 H. Bai, C. Li and G. Q. Shi, Adv. Mater., 2011, 23, 1089–1115. 57 L. Qian and X. Yang, Talanta, 2006, 68, 721–727.
39 M. Zhou and S. Dong, Acc. Chem. Res., 2011, 44, 1232–1243. 58 Y. J. Teng, S. H. Zuo and M. B. Lan, Biosens. Bioelectron.,
40 M. Zhou, Y. Zhai and S. Dong, Anal. Chem., 2009, 81, 5603–
5613.
41 W. S. Hummers and R. E. Offeman, J. Am. Chem. Soc., 1958,
80, 1339–1339.
42 Z. Liu, X. Duan, G. Qian, X. Zhou and W. Yuan,
Nanotechnology, 2013, 24, 1–6.
2009, 24, 1353–1357.
59 C. Xiang, Y. Zou, L. Sun and F. Xu, Sens. Actuators, B, 2009,
136, 158–162.
60 X. Zhao, Z. Mai, X. Kang and X. Zou, Biosens. Bioelectron.,
2008, 23, 1032–1038.
61 C. Lei, S. Hu, G. Shen and R. Yu, Talanta, 2003, 59, 981–988.
43 L. Wang, Y. Ye, X. Lu, Z. Wen, Z. Li, H. Hou and Y. Song, Sci. 62 Q. Xu, C. Mao, N. Liu, J. Zhua and J. Sheng, Biosens.
Rep., 2013, 3568, 1–9. Bioelectron., 2006, 22, 768–773.
44 C. Guo, X. Zheng, S. Ng, Y. Lai, Y. Lei and C. Li, Chem. 63 Z. Tong, R. Yuan, Y. Chai, Y. Xie and S. Chen, J. Biotechnol.,
Commun., 2011, 47, 2652–2654. 2007, 128, 567–575.
55876 | RSC Adv., 2014, 4, 55867–55876
This journal is © The Royal Society of Chemistry 2014