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An electrochemical glucose biosensor based on

graphene composites: use of dopamine as reducing
Cite this: RSC Adv., 2014, 4, 43624
monomer and as site for covalent immobilization of
enzyme†
Huiping Liu,ab Cheng-an Tao,a Zhihong Hu,a Sida Zhang,a Jianfang Wang*a
and Yonggong Zhan*b

Received 26th May 2014
Accepted 8th September 2014
DOI: 10.1039/c4ra04975f
www.rsc.org/advances
A new graphene-based electrochemical biosensor was constructed and applied for the simple, rapid and
highly selective determination of glucose. The modified glassy carbon electrode (GCE) was prepared
using dopamine (DA) as a reducing agent for graphene oxide (GO) and as a capping agent to stabilize
and coat the resulting reduced graphene oxide (RGO) surface as it can polymerize to form
polydopamine (PDA). PDA-RGO composites were dropped onto a glassy carbon electrode surface, and
glucose oxidase (GOx) was immobilized on the surface of the composites by Michael addition. Finally, Pt
nanoparticles (PtNPs) were electrodeposited on the modified electrode. Such modified electrodes
(denoted as GCE/PDA-RGO/GOx/PtNPs) were characterized by UV-visible spectroscopy, atomic force
microscopy, X-ray power diffractometry, Fourier-transform infrared spectroscopy, and scanning electron
microscopy. Electrochemical behaviors were investigated by cyclic voltammetry and electrochemical
impedance spectroscopy. The fabricated biosensor exhibited a high sensitivity of 33 mA mM
1 cm
2 over
a wide linear range of 0.2 mM to 1 mM, good stability, excellent repeatability, and a detection limit of
0.10 mM.

transfer of redox proteins.11–16 Graphene and its derivatives can

1 Introduction
Fast and efficient methods for the determination of glucose are
becoming increasingly important in biology, chemistry, and the
food industry; glucose determination is also important in the
medical eld for controlling diabetes. Diabetes is a worldwide
public health problem and one of the leading causes of death
and disability in the world.1–4 Numerous studies have been
performed to develop simple, sensitive, and accurate
approaches for glucose detection, especially those using glucose
oxidase (GOx), which has high selectivity and sensitivity.5–7
However, direct electron transfer (DET) without the assistance
of mediators for GOx is extremely difficult because of the deep
embedding of the redox center of GOx in a protective protein
shell.8–10 Several materials have been used to promote electron
a
College of Science, National University of Defense Technology, Changsha 410073, P.
R. China. E-mail: wangjianfang@nudt.edu.cn; Fax: +86 731 8457 4250; Tel: +86
731 8457 4241
b
College of Chemistry and Chemical Engineering, Hunan University, Changsha,
410081, P. R. China. E-mail: ygzhan2006@hnu.edu.cn; Tel: +86 731 88821449
† Electronic supplementary information (ESI) available: The UV-Vis absorption
spectra of PDA-RGO. The size of PtNPs. CVs of different modied electrodes in
the absence and in the presence of glucose. The characterization of
reproducibility, selectivity, and long-term stability of biosensor. See DOI:
10.1039/c4ra04975f
facilitate DET of metalloproteins.17–20 Moreover, the excellent
conductivity and small band gap of graphene are favorable for
transferring electrons from biomolecules.21–23 Reported proce-
dures for fabricating biosensors, which include restoration of
the sp2 structure of graphene, preparation of polymer–graphene
composites, and immobilization of GOx, are relatively compli-
cated. GOx is generally immobilized by simple physical
adsorption, which results in enzyme loss during determination
and limits the performance and further application of the
enzyme.17,19,24 To develop a more simple and stable fabrication
method is very important in the biosensor eld.
Dopamine (DA), as a biogenic species, has an important
physiological function as a chemical messenger in mammals.
DA-level abnormalities can result in serious diseases, such as
Parkinson's disease.25 Two phenolic hydroxyl groups in DA
provide it with reducing power. DA at micromolar-scale
concentrations has been used to chemically reduce graphene
oxide (GO) into graphene.26,27 Auto-polymerization of DA
through air-driven chemical polymerization may also occur to
form polydopamine (PDA) at weakly alkaline pH. This charac-
teristic has been used to prepare multifunctional and biocom-
patible PDA coatings on various surfaces, including
graphene.28–34 The functional groups present on the surface of
PDA coatings allow covalent immobilization of proteins and

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enzymes through Michael addition and Schiff base reaction.28 If
DA can simultaneously act as both a reductant and monomer to
synthesize highly biocompatible PDA-reduced GO composites
(PDA-RGO) and if enzymes can be covalently immobilized on
the surface of the resultant composites, the preparation of
graphene-based biosensors will be conveniently facilitated.
In the present study, DA was used simultaneously as a
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reducing agent for GO and as a capping agent to stabilize and
coat the RGO surface and form biocompatible PDA-RGO
composites. A graphene-based electrochemical glucose
biosensor was fabricated by modifying the composites on a
glassy carbon electrode (GCE) surface. GOx was immobilized on
the surface of the composites by Michael addition, and Pt
nanoparticles (PtNPs) were electro-deposited on the modied
electrode. The fabricated biosensor showed great analytical
performance for the detection of glucose, including a high
sensitivity of 33 mA mM
1 cm
2 over a wide linear range of 0.2
mM to 1 mM, good stability, excellent repeatability, and a
detection limit of 0.10 mM. The proposed fabrication method
can promote the preparation of other graphene-based
biosensors.
2 Experimental section
2.1 Reagents and apparatus
GOx (50 kU) was purchased from Shanghai Shenggong Reagent
Co. Graphite powder and dopamine were purchased from
Aladdin. All chemicals were of analytical grade and used as
received. All solutions were prepared using ultrapure water.
Electrochemical experiments were performed using a
CHI660a electrochemical analyzer (Chen Hua Instruments,
Shanghai, China). All experiments were carried out at room
temperature with a three-electrode system; here, a GCE
(F ¼ 3 mm), a Ag/AgCl (0.3 M KCl solution) electrode, and a
platinum wire served as the working electrode, reference elec-
trode, and auxiliary electrode, respectively. Electrochemical
impedance spectroscopy (EIS) was performed in 0.1 M KCl solu-
tion containing 5 mM K3[Fe(CN)6]/K4[Fe(CN)6] (1 : 1) at frequen-
cies ranging from 0.01 Hz to 10 kHz at 0.20 V. The amplitude of
the applied sine wave potential in each case was 5 mV.
UV-Vis absorption spectra were obtained using a UV-1201
UV-Vis spectrophotometer (China). Atomic force microscopy
(AFM) images were acquired in tapping mode using a NanoMan
VS instrument (Veeco, USA). X-ray diffractometry (XRD)
measurements were performed using a Ttr III type X-ray
diffractometer (Rigaku, Japan) equipped with a Cu Ka X-ray
radiation (1.54 A) source at q between 6 and 60 . Scanning
electron microscopy (SEM) observations were carried out with a
LEO-1503 eld emission scanning electron microscope
(German). Prior to SEM, the samples were xed on aluminum
stubs and coated with gold powder. X-ray photoelectron spec-
troscopy (XPS) were carried out with K-Alpha 1063 (Thermo
Fisher Scientic, UK); XPSPEAK v4.0.0.0 soware (Chemistry,
CUHK) was used to perform curve tting. Fourier transform
infrared (FT-IR) spectra were obtained using a Bruker Tensor 27
spectrophotometer within the spectral range of 4000 cm
1 to
400 cm
1 using the KBr pellet method.
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2.2 Preparation of PDA-RGO composites
The GO used in this experiment was synthesized from natural
graphene powder by a modied Hummers method35,36 and
characterized using UV-Vis spectroscopy, optical photography,
AFM, XRD and FT-IR spectroscopy. Briey, 10 mg of GO was
suspended in 25 mL of 0.01 M Tris–HCl buffer (pH 8.0).
Subsequently, 5 mg of DA powder was added to the GO
dispersion aer the solution had been deaerated with high-
purity nitrogen gas for 10 min. The suspension was ultra-
sonicated for 10 min in an ice bath and then reacted for 24 h at
60  C to obtain a black suspension of PDA-RGO. UV-Vis spec-
troscopy was used to monitor the reduction of GO. The product
was obtained by at least three cycles of centrifugation. Finally,
the PDA-RGO composite was suspended in 20 mL of pure water.
2.3 Construction of GCE/PDA-RGO/GOx/PtNPs modied
electrode
Prior to modication, the GCE was carefully polished with 1.0,
0.3, and 0.05 mm g-alumina powders in sequence and rinsed
thoroughly with pure water between each polishing step. The
electrode was then ultrasonicated in pure water, ethanol, and
pure water for 5 min and dried with nitrogen gas. Six microliters
of PDA-RGO suspension was cast onto the surface of the GCE
and air-dried. The modied electrode was immersed in GOx in
phosphate buffered saline (PBS) solution (10 mg mL
1, pH 8.0)
and kept at 4  C for 4 h to covalently link the GOx to the elec-
trode surface. Aer washing with pure water, the electrode was
immersed into 0.5 M H2SO4 solution containing 2 mM H2PtCl6.
Electro-deposition of Pt was carried out by potential scanning
from
0.3 V to 0.4 V for 30 cycles.37 The electrode was then
washed with pure water and stored at 4  C prior to use. For
comparison, GCE/PDA, GCE/GOx, GCE/PDA-RGO, GCE/PDA-
RGO/GOx, and GCE/PDA-RGO/PtNP electrodes (“/” represents
which are used to modify electrodes in successive steps, “-”
means which are mixed rst, and then use to modify electrodes)
were prepared using a similar method.
3 Results and discussion
3.1 Characterization of the nanocomposites
3.1.1 Characterization of the PDA-RGO composite. DA
acted simultaneously as a reductant and as a monomer to
enable synthesis of a highly biocompatible PDA-RGO
composite. The reduction process was monitored by UV-Vis
spectroscopy, as shown in Fig. 1A. The absorption peaks of
DA at 220 and 280 nm as well as that of GO at 230 nm (Fig. 1B)
disappeared as the reaction proceeded. A new peak at 262 nm
appeared and gradually became stronger with reaction time,
which indicates that GO was converted to RGO. The color of the
dispersion nally turned black from brown (Fig. 1C and D),
which conrms the presence of RGO. DA polymerization also
took place simultaneously, and the biocompatible PDA was
coated on the surface of the RGO. The resultant PDA-RGO
suspension exhibited good stability and could stand for at
least one month without signicant degradation. XPS was also
used to conrm the reduction of GO. The C1s XPS spectra of GO
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Fig. 1 UV-Vis absorption spectra of (A) PDA-RGO composites
obtained after different reaction time; (B) DA, PDA, GO, and PDA-RGO
composites. Photographs of (C) GO and (D) PDA-RGO dispersions.
The C1s XPS spectra of (E) GO and (F) PDA-RGO.
and PDA-RGO are shown in Fig. 1E and F. Curve tting of the
C1s spectra was performed using a Gaussian–Lorentzian peak
shape. The peaks of C1s spectra of GO are assigned to four
components that correspond to carbon atoms in different
functional groups: the nonoxygenated ring C(C–C), the C in C–O
bonds (C–O), the carbonyl C (C]O), and the carboxylate carbon
(C(O)O). Aer reduced and composite with PDA, although the
C1s XPS spectrum of PDA-RGO composite still exhibits these
same oxygen-containing groups, their peak intensities are much
smaller than those in GO. Moreover, there is an additional
component at 285.7 eV corresponding to C–N bond.38 These
observation indicates considerable de-oxygenation by DA
reduction, which is consistent with the UV-Vis results.
AFM, XRD and FT-IR were used to further conrm the
formation of PDA-RGO composites. Fig. 2 shows a typical AFM
Fig. 2 Typical AFM images of (A) GO and (B) PDA-RGO. Cross-
sectional analysis was performed along the lines shown in the AFM
images.
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image obtained in tapping mode displaying the morphology
and thickness of GO and PDA-RGO. Compared with GO, the
large RGO sheet of the composite was coated with numerous
small and uniformly distributed island-like nanostructures,
which are PDA coatings. The corresponding line-scan indicated
that the thickness of PDA-RGO is 1.76 nm, much larger than
that of GO (1 nm). These results could be attributed to the
presence of a PDA coating on both sides of the RGO sheets.
The XRD patterns of PDA-RGO composite are shown in
Fig. 3A. For comparison, the XRD patterns of DA, PDA and GO
are also displayed. DA powder showed many peaks. While once
it has polymerized to be PDA, only a wide diffraction peak exist
around 24 due to the stacking of the benzene rings of DA
molecules. GO has the characteristic peak at 10.0 , while the
PDA-RGO composite only showed a wide peak at 24 , which is
very similar to that of pure PDA and demonstrates the DA has
successfully self-polymerized. The disappearance of the peak at
10.0 also suggests that GO was thoroughly converted to RGO.
The FT-IR spectra of DA, PDA and PDA-RGO are displayed in
Fig. 3B. DA shows broad and strong bands in the 3000–3400
cm
1 region (aromatic O–H stretching vibration). Peaks at 1519
cm
1 (NH2 scissoring vibration), 1627 cm
1 (C]C stretching
vibration), 1340 cm
1 (CH2 bending vibration), 1175 cm
1 (C–C
stretching vibration), 1260 cm
1 (C–O–H symmetry vibration),
and 1190 cm
1 (C–O symmetry vibration) were also observed.37
Aer polymerization, one broad band was observed at the 3000–
3400 cm
1 region and the peaks at 1519, 1340, 1175, 1260, and
1190 cm
1 disappeared. A new peak at 1707 cm
1 (C]O)
appeared. These results demonstrate that DA underwent intra-
molecular cyclization to form indole derivatives (Fig. S1†). The
IR spectra of PDA-RGO are similar to those of DA; the absence of
peaks at 1731 cm
1 (C]O), 1226 cm
1 (C–O–C), and 1054 cm
1
(C–O) was observed, which further implies that reduction and
polymerization simultaneously occurred to successfully yield
the PDA-RGO composites.
The surface morphology of the PDA-RGO composite was
examined by SEM, as illustrated in Fig. 4. PDA-RGO (Fig. 4B) has
a typical crumpled and wrinkled sheet structure, very similar to
that of GO (Fig. 4C). The SEM image of PDA (Fig. 4A) shows a
very rough surface featuring several irregular cracks.39 This
result indicates that the PDA layer coating RGO is very thin and
therefore cannot affect the morphology of RGO signicantly.
Fig. 3 (A) XRD patterns of (1) DA, (2) PDA, (3) PDA-RGO, and (4) GO. (B)
FT-IR spectra of (1) DA, (2) PDA, (3) PDA-RGO, (4) GO, (5) PDA-RGO/
GOx, and (6) GOx.
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Fig. 4 SEM images of (A) PDA, (B) PDA-RGO, (C) GO, (D) PDA-RGO/
GOx on a Si substrate, and (E) PDA-RGO/GOx/PtNPs on the GCE
electrode. (F) EDX spectra of PDA-RGO/GOx/PtNPs (1 bar ¼ 10 mm).
Finally, EIS was used to study the interface properties of
electrodes modied with the PDA-RGO composites (Fig. 5).40
Compared with GCE, when GO is coated on the electrode, a rapid
increase in the diameter of the semicircle is obtained because of
the ability of GO to block electron transfer. When the PDA-RGO
composite was deposited on the GCE, the Rct dramatically
decreased to 3000 U, which suggests that PDA-RGO can greatly
improve the conductivity of the resultant product and promote
electron transfer. These observations are attributed to the unique
properties of RGO.
Fig. 5 Nyquist plots of the EIS spectra of (a) GCE/GOx, (b) GCE/PDA-RGO,
(c) GCE/PDA-RGO/PtNPs, (d) GCE/PDA-RGO/GOx, and (e) GCE/PDA-
RGO/GOx/PtNPs electrodes in 0.1 M KCl aqueous solution containing
5.0 mM Fe(CN)63
/4
(1 : 1). The frequency range is from 100 mHz to
100 kHz. Insets show the EIS of (f) bare GCE and (g) GCE/GO.
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3.1.2 Characterization of PDA-RGO/GOx. FT-IR spectros-
copy, SEM, and EIS were used to investigate the process of
immobilization of GOx on the PDA-RGO-modied electrode. FT-
IR spectrum of PDA-RGO/GOx, scraped from the electrode, is
shown in Fig. 3B. For comparison, the spectrum of GOx is also
shown. The characteristic absorption band of the NH2 group of
GOx in the 3500–3000 cm
1 region and the peak at 1707 cm
1
(C]O) of PDA-RGO disappeared, which implies successful
immobilization of GOx through Michael addition (Fig. S2†). The
SEM image of PDA-RGO/GOx (Fig. 4D) shows no obvious
difference from that of PDA-RGO (Fig. 4B), likely because the
GOx particles are too small in size to be observed under the
present conditions. To conrm the immobilization of GOx, we
studied the EIS spectra of GCE/GOx and GCE/PDA-RGO/GOx
electrodes. As shown in Fig. 5, the Rct of GOx (1000 U) is
larger than that of the bare GCE, which demonstrates inhibition
of electron transfer by GOx. When GOx is covalently linked to
the surface of GCE/PDA-RGO, the Rct increases from 3000 U to
5000 U. It illustrates that GOx is steadily immobilized on the
modied electrode.
3.1.3 Characterization of GCE/PDA-RGO/GOx/PtNPs. PtNPs
were electrodeposited on the GCE/PDA-RGO/GOx electrodes.
The SEM image in Fig. 4E shows numerous spherical nano-
particles on the surface of the electrodes; these nanoparticles
are composed of Pt (Fig. 4F). The average diameter of the PtNPs
is about 260 nm (Fig. S3†). All of the results conrm the
successful deposition of PtNPs. EIS was also applied to
demonstrate the presence of PtNPs further (Fig. 5). Aer
deposition of PtNPs on the GCE/PDA-RGO and GCE/PDA-RGO/
GOx electrodes, Rct values decreased to 400 U and 2000 U,
respectively, because of the superior conductivity of the PtNPs
and the synergistic effects of PtNPs and RGO.
3.2 Direct electrochemistry of GOx immobilized on the GCE/
PDA-RGO/GOx/PtNP electrode
DET of GOx-modied electrodes was studied by cyclic voltam-
metry (CV) in PBS solution without O2 (Fig. 6). No current peak
was observed in the voltammograms of GCE/PDA, GCE/PDA-
RGO and GCE/PDA-RGO/PtNPs. The absence of a peak indi-
cates that PDA, PDA-RGO and PDA-RGO/PtNPs composites are
inactive electrochemically. The background current of the RGO-
modied electrodes obviously increased, which can be ascribed
Fig. 6 CV curves of the GCEs modified with (a) GOx, (b) PDA, (c) PDA-RGO,
(d) PDA-RGO/PtNPs, (e) PDA-RGO/GOx, and (f) PDA-RGO/GOx/PtNP
films in PBS saturated with N2 at a scan rate of 0.1 V s
1.
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to the large specic surface area and extraordinary electron
transfer conductivity of the material. No redox peak current
attributable to GOx was observed on the GCE/GOx electrode,
which suggests that achieving DET in the electrodes is extremely
difficult because of the deep embedding of the redox center of
GOx in a protective protein shell.8–10 The CV curves of GOx on the
GCE/PDA-RGO/GOx electrode displays a pair of stable redox
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peaks, which is attributable to the redox reaction of the elec-
troactive center of GOx. This nding indicates that the large
surface-to-volume ratio and high conductivity of RGO works
together with the good biocompatibility of PDA to promote DET
between GOx and the surface of the electrodes.41 Two well-
dened redox peaks were obtained for the GCE/PDA-RGO/GOx/
PtNPs electrode with Epa ¼
0.546 V and Epc ¼
0.617 V.
The peak-to-peak separation (DEp) was approximately 71 mV,
much smaller than that observed for the GCE/PDA-RGO/GOx
electrode (104 mV). This result reveals that a larger number of
reversible electron transfers in the redox active center of the GOx
were achieved, which may be attributed to the excellent
conductivity of PtNPs and the synergistic effects of PtNPs and
RGO. In the presence of O2, glucose standard was added to the
electrodes, among which the GCE/PDA-RGO/GOD/PtNP elec-
trode showed the best responses (Fig. S4†).
The inuence of scan rate on the peak current was also
investigated, as shown in Fig. 7. As the scan rate increased from
20 mV s
1 to 300 mV s
1, the redox peak currents linearly
increased, which implies that the electrochemical kinetics is a
surface-controlled process. According to Faraday's law, the
average surface concentration of electroactive GOx (G*) immo-
bilized on the GCE/PDA-RGO/GOx/PtNPs electrode may be
estimated to be 3.1  10
9 mol cm
2, which is a value charac-
teristic of a small molecule (FAD) in contrast to the enzyme
which should be around 10
11 mol cm
2.10,42 It demonstrates
that free FAD is extruded from the enzyme and remains
adsorbed on the electrode surface and GOx is immobilized on
the surface of PDA-RGO through covalent linkage. These results
are in agreement with the FT-IR analysis.
3.3 Glucose detection using the GCE/PDA-RGO/GOx/PtNP
electrode
Fig. 8A shows CV curves of the GCE/PDA-RGO/GOx/PtNP elec-
trode in 0.01 M PBS solution containing different concentrations
Fig. 7 (A) CV curves of GCE/PDA-RGO/GOx/PtNPs electrode in 0.01
M PBS (pH 7.4) at scan rates of 0.02, 0.05, 0.08, 0.1, 0.15, 0.2, 0.25, 0.3
V s
1. (B) Plot of redox peak current against scan rate. The correlation
coefficient (R2) is 99.84% and 99.19% for oxidation and reduction peak
current, respectively. The relative standard deviation (RSD) of each
point is less than 3.8%.
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Fig. 8 (A) Cyclic voltammograms of GCE/PDA-RGO/GOx/PtNPs
electrode obtained with successive addition of 1 mM glucose in 10 mM
PBS (pH 7.4) at a rate of 0.1 V s
1. Inset: enlarged image of the vol-
tammogram from
0.61 V to
0.7 V. (B) Calibration curve of the linear
dependence of the cathodic peak current on the glucose
concentration.
of glucose and saturated with air. Successive addition of glucose
resulted in a gradual decrease in reduction current. This trend
could be explained by the fact that addition of glucose triggers
the enzyme-catalyzed reaction of GOx and glucose. This reaction
causes a decrease in the amount of oxidized GOx on the GCE/
PDA-RGO/GOx/PtNPs electrode and reduces the electrode
reduction current (see ESI†). The response current was directly
proportional to the concentration of glucose in the range of 0.2–
1 mM, with a correlation coefficient (R2) of 0.9957 (Fig. 8B). The
relative standard deviation of each point is less than 0.65%. The
biosensor also displayed a high sensitivity of approximately 33
mA mM
1 cm
2.
The detection limit of the biosensor was 0.10 mM based on a
signal-to-noise ratio of 3 (inset in Fig. 8A). Results indicate that
the modied electrode has satisfactory performance and may be
practically applied in the determination of human blood sugar
concentrations.
3.4 Reproducibility, selectivity, and long-term stability of the
biosensor
The prepared biosensor displayed good reproducibility. The
RSD of the current response to 0.2 mM glucose was 2.7% in ve
successive measurements (Fig. S5†). Aer 50 cycles of potential
scanning between
0.8 and 0 V, the cathodic current remained
almost unchanged, with an RSD of 0.8% (Fig. S6a and b†). The
RSDs of the peak current response of four independent elec-
trodes to 0.2 mM glucose was 3.3% (Fig. S6†). The effect of
interferences on the glucose response was further studied to
evaluate the selectivity of the sensor (Fig. S7†). Ascorbic acid
(AA) and DA are common reducing substances in blood and
they were chosen as the interference molecules. It's found that
both of AA and DA has little effect on the glucose response.
When there are only 0.2 mM glucose in the PBS solution, the
current response reached to 40.39 mA. When ten-fold concen-
tration of AA (2 mM) co-exists in the solution, the current
response only changed a little (retained 98%). DA got the similar
results. These results indicate that the proposed biosensor has
high selectivity.
The stability of the biosensor was nally investigated. The
response current maintained 91% of its initial electrochemical
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response aer storage at 4  C for one week (Fig. S6c†). This
stability is attributed to the unique properties of RGO, efficient
covalent linkages between GOx and PDA, and the synergistic
effects of PtNPs and RGO. These results further indicate that the
PDA-RGO composite could efficiently immobilize GOx, retain its
bioactivity, and prevent biomolecules from leaking out. These
actions are attributable to chemical interactions between GOx
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and the electrode, rather than pure physical adsorption.
4 Conclusions
An electrochemical glucose biosensor based on polymer–
graphene/enzyme/Pt composites was fabricated. Here, DA
served as both a reducing monomer and site for covalent
immobilization of enzyme. DA simultaneously reduced GO and
capped the resultant RGO to form PDA-RGO composites, which
could easily and covalently immobilize enzymes through
Michael addition. The biosensor exhibited good sensitivity,
excellent stability, and superior repeatability toward glucose.
These characteristics are attributed to the large surface area of
the electrode, the fast electron transfer of RGO, the synergistic
effects of PtNPs and RGO, and the stable immobilization of
GOx. The proposed procedure for fabricating graphene-based
biosensors is convenient and the performance of the resultant
sensor is excellent. These ndings indicate that the proposed
biosensor has potential applications in glucose sensing.
Acknowledgements
This work was supported by the National Natural Science
Foundation of China (61171020, 21203247, 61376125).
Notes and references
1 A. L. Ghindilis, P. Atanasov and E. Wilkins, Electroanalysis,
1997, 9, 661.
2 P. Pandey, S. P. Singh and S. K. Arya, Langmuir, 2007, 23,
3333.
3 R. W. Keay and C. J. McNeil, Biosens. Bioelectron., 1998, 13, 963.
4 J. J. Xu and H. Y. Chen, Anal. Chim. Acta, 2000, 423, 101.
5 N. S. Oliver, C. Toumazou, A. E. Cass and D. G. Johnston,
Diabetic Med., 2009, 26, 197.
6 J. Wang, Chem. Rev., 2008, 108, 814.
7 C. Chen, Q. Xie and D. Yang, RSC Adv., 2013, 3, 4473.
8 A. Guiseppi-Elie, C. Lei and R. H. Baughman,
Nanotechnology, 2002, 13, 559.
9 O. Nadzhafova, M. Etienne and A. Walcarius, Electrochem.
Commun., 2007, 9, 1189.
10 J. M. Goran, S. M. Mantilla and K. J. Stevenson, Anal. Chem.,
2013, 85, 1571.
11 C. Lei and J. Deng, Anal. Chem., 1996, 68, 3344.
12 S. Kroning, F. W. Scheller and U. Wollenberger,
Electroanalysis, 2004, 16, 253.
This journal is © The Royal Society of Chemistry 2014
View Article Online
RSC Advances
13 S. Gaspar, H. Zimmermann and I. Gazaryan, Electroanalysis,
2001, 12, 284.
14 Z. H. Dai, S. Q. Liu and H. X. Ju, Biosens. Bioelectron., 2004,
19, 861.
15 J. S. Long, D. S. Silvester and G. G. Wildgoose,
Bioelectrochemistry, 2008, 74, 183.
16 X. B. Lu, Q. Zhang and L. Zhang, Electrochem. Commun.,
2006, 8, 874.
17 X. Kang, J. Wang and H. Wu, Biosens. Bioelectron., 2009, 25,
901.
18 P. Wu, Q. Shao, Y. Hu and J. Jin, Electrochim. Acta, 2010, 55,
8606.
19 C. Ruan, T. Li and Q. Niu, Electrochim. Acta, 2012, 64, 183.
20 H. Xu, H. Dai and G. Chen, Talanta, 2010, 81, 334.
21 Y. Liu, X. Dong and P. Chen, Chem. Soc. Rev., 2012, 41,
2283.
22 D. Chen, L. Tang and J. Li, Chem. Soc. Rev., 2010, 39, 3157.
23 T. Gan and S. Hu, Microchim. Acta, 2011, 175, 1.
24 K. Mao, D. Wu and Y. Li, Anal. Biochem., 2012, 422, 22.
25 A. Pezzella, M. d'Ischia and A. Napolitano, J. Med. Chem.,
1997, 40, 2211.
26 H. Liu, P. Xi and G. Xie, J. Phys. Chem. C, 2012, 116, 3334.
27 I. Kaminska, M. R. Das and Y. Coffinier, ACS Appl. Mater.
Interfaces, 2012, 4, 1016.
28 L. Q. Xu, W. J. Yang and K.-G. Neoh, Macromolecules, 2010,
43, 8336.
29 H. Lee, S. M. Dellatore and W. M. Miller, Science, 2007, 318,
426.
30 S. Zurcher, D. Wackerlin and Y. Bethuel, J. Am. Chem. Soc.,
2006, 128, 1064.
31 M. A. Watson, J. Lyskawa and C. Zobrist, Langmuir, 2010, 26,
15920.
32 S. Alwarappan, A. Erdem and C. Liu, J. Phys. Chem. C, 2009,
113, 8853.
33 N. G. Shang, P. Papakonstantinou and M. McMullan, Adv.
Funct. Mater., 2008, 18, 3506.
34 Y. Wang, Y. Li, L. Tang and J. Lu, Electrochem. Commun.,
2009, 11, 889.
35 D. C. Marcano, D. V. Kosynkin and J. M. Berlin, ACS Nano,
2010, 4, 4806.
36 C. Tao, J. Wang and Y. Lv, J. Mater. Chem., 2012, 22, 24856–
24861.
37 X. Q. Lin and Y. X. Li, Biosens. Bioelectron., 2006, 22, 253.
38 S. Stankovich, D. A. Dikin, R. D. Piner, K. A. Kohlhaas,
A. Kleinhamme, Y. Jia, Y. Wu, S. T. Nguyen and
R. S. Ruoff, Carbon, 2007, 45, 1558.
39 F. Yu, S. Chen and Y. Chen, J. Mol. Struct., 2010, 982, 152.
40 R. Ehret, W. Baumann and M. Brischwein, Biosens.
Bioelectron., 1997, 12, 29.
41 B. Karlberg, M. Grasserbauer and J. E. T. Andersen, Trends
Anal. Chem., 2009, 28, 515.
42 P. Wu, Q. Shao and Y. J. Hu, Electrochim. Acta, 2010, 55,
8606.
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