Three dimensional porous graphene-chitosan composites from ice-induced assembly for direct electron transfer and electrocatalysis of glucose oxidase

Article abstract of DOI:10.1039/c4ra07707e

Three dimensional porous graphene-chitosan (GR-CS) composites via ice-induced assembly for glucose oxidase (GOD) immobilization were reported for the first time. By adjusting the GR amount, the porous GR-CS composites containing different GR content could be produced. It was found that the GR amount played an important role in their morphologies. Higher GR amount resulted in more pores appearing in the GR-CS composites. When the GR amount was 70 wt%, the GR-CS composites (GR70-CS) had good flexibility and interpenetrating porous structures. The GR70-CS composite showed good stability, and still kept a porous structure even after dispersing and coating on the substrates. Current response from the GR70-CS composite modified glassy carbon electrode (GR70-CS/GCE) was about two times that from the bare GCE with Fe(CN) ₆^3-/4- as a probe. The direct electrochemical behavior was observed when GOD was immobilized onto the GR70-CS/GCE. The GOD modified GCE also showed good electrocatalytic activity for glucose. Using ferrocenecarboxylic acid as a mediator, a linear relationship from 0.14 to 7.0 mM (R = 0.995) between currents and the glucose concentration with a detection limit of 17.5 μM was obtained. the Partner Organisations 2014.

Full text of DOI:10.1039/c4ra07707e

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Three dimensional porous graphene–chitosan
composites from ice-induced assembly for direct
electron transfer and electrocatalysis of glucose
oxidase
Cite this: RSC Adv., 2014, 4, 38273
*
Lei Qian and Lu Lu
Three dimensional porous graphene–chitosan (GR–CS) composites via ice-induced assembly for glucose
oxidase (GOD) immobilization were reported for the first time. By adjusting the GR amount, the porous GR–
CS composites containing different GR content could be produced. It was found that the GR amount played
an important role in their morphologies. Higher GR amount resulted in more pores appearing in the GR–CS
composites. When the GR amount was 70 wt%, the GR–CS composites (GR70–CS) had good flexibility and
interpenetrating porous structures. The GR70–CS composite showed good stability, and still kept a porous
structure even after dispersing and coating on the substrates. Current response from the GR70–CS
composite modified glassy carbon electrode (GR70–CS/GCE) was about two times that from the bare
3ꢀ/4ꢀ
GCE with Fe(CN)6
as a probe. The direct electrochemical behavior was observed when GOD was
Received 28th July 2014
Accepted 6th August 2014
immobilized onto the GR70–CS/GCE. The GOD modified GCE also showed good electrocatalytic
activity for glucose. Using ferrocenecarboxylic acid as a mediator, a linear relationship from 0.14 to 7.0
mM (R ¼ 0.995) between currents and the glucose concentration with a detection limit of 17.5 mM was
obtained.
DOI: 10.1039/c4ra07707e
www.rsc.org/advances
reaction, self-assembly and centrifugal evaporation. Among
these methods, hydrothermal reaction is usually used to
1. Introduction
prepare 3D porous GR or composites. A graphene oxide (GO)
solution was put into a Teon-lined stainless steel autoclave,
heated at high temperature, and freeze dried to obtain 3D
porous GR. 3D porous GR or their composites with metallic
nanoparticles have been produced by this method.14 Yan
et al.16 proposed a mild method to produce 3D porous GR and
GR–nanoparticle architectures via self-assembly and reduc-
tion in aqueous suspension. Seo et al.18 reported that GR
sponges and lms were produced by the centrifugal evapora-
tion in a speed vacuum concentrator. Controlling different
temperature resulted in formation of GR sponges and lms.
However, these proposed methods still have some disadvan-
tages. For example, hydrothermal method requires high
temperature and subsequent drying process. Self-assembly
method need consume long time (about seven days) due to
dialysis and lyophilization.
In recent years, freeze drying has been used to prepare
porous materials. Pores in materials are formed by removing
ice in frozen samples during freeze-drying. We previously
reported preparation of porous polymeric and inorganic
monolith, nanobers and porous microspheres by freeze-
drying or combining freeze-drying with emulsion-templating
method.20–23 Moreover, the pore structures can be adjusted by
controlling the experimental conditions such as solvent type,
Graphene (GR) is a two-dimensional monolayer of carbon
atoms bonding with sp2 hybrid orbitals. It exhibits many
interesting properties including high electron mobility, high
conductivity, good mechanical strength and thermal stability,
and biocompatibility.1–3 GR has received much attention and
has been a research focus in various elds such as physics,
material, chemistry and biology. Due to strong planar stacking
of sheets from the p–p* interaction, GR tends to self-assemble
and form two dimensional (2D) GR agglomerates. So fabrication
of 2D GR including GR lms and layered paper-like GR is
relative simple and easy to realize.4–8 However, the strong planar
stacking between sheets results in the decrease of surface area
and catalytic sites. These drawbacks limit the applications of 2D
GR in some elds.
Compared with 2D GR, 3D porous GR materials have some
advantages such as good porous structures, high mass transfer
speed, effectively active surface, high conductivity and loading
capacity.9–19 Thus development of 3D porous GR is very
signicant and it has potential applications. It has been
reported that 3D porous GR can be prepared by hydrothermal
Key Laboratory for Liquid-Solid Structural Evolution and Processing of Materials
(Ministry of Education), Shandong University, 17923 Jingshi Road, Jinan 250061,
China. E-mail: qleric@sdu.edu.cn; Tel: +86-531-88393396
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solution concentration, freezing temperature, etc. Compared
with other methods, freeze drying is simple, low-cost and easy
for large-scale production. Freeze drying has been reported to
prepare porous GR, GO and their composites.24–29 Gao
et al.26,28 produced various materials of GO and GR foams
based on freeze drying. These porous materials were mostly
used for adsorption of oil, gas and metallic ions due to their
good porous structure and high surface area. Chitosan (CS)
shows good biocompatibility, biodegradability, lm-forming
ability and high stability in neutral and alkaline aqueous
solutions. It can be used for immobilization of biomolecules
to prepare biosensors. However, there are few papers repor-
ted on freeze-dried porous GR–CS composites as electrode
materials. Only recently, Li et al.30 fabricated hierarchically
porous chitosan-vacuum stripped GR scaffolds for a bioanode
of microbial fuel cell. It was found that the porous scaffolds
could produce a maximum power of 1530 mW mꢀ2. The good
performance was attributed to high specic surface area,
affinitive contact between biolm and anode, etc. Consid-
ering porous GR–CS composites combine high surface area,
effective mass transfer, good conductivity and biocompati-
bility from GR and CS, it will be a good matrix for enzyme
immobilization. Here, we rst reported that ice-induced
assembly of 3D porous GR–CS composites from freeze-drying
as a matrix for enzyme immobilization (Fig. 1). Field emis-
sion scanning electron microscopy (FESEM), X-ray diffraction
(XRD) and ultraviolet-visible (UV-vis) spectroscopy were used
to characterize GR and porous GR–CS composites. The effect
of GR amounts on morphologies of the GR–CS composites
was carefully investigated. The 3D porous GR–CS composites
were coated on an electrode to study their electrochemical
properties. At last, glucose oxidase (GOD) was immobilized
onto the porous GR–CS composite, and its direct electro-
chemical properties and electrocatalysis for glucose were
examined.
2. Experimental
2.1 Chemicals and reagents
Graphene oxide powder was purchased from Nanjing JCNano.
Tech. Co., Ltd. Chitosan (CS, medium molecular weight),
glucose oxidase (Type X–S, lyophilized powder, from Aspergillus
niger), ferrocenecarboxylic acid (FMCA, purum $ 97.0%) and
Naon (5% in a mixture of lower aliphatic alcohols and water)
were purchased from Sigma-Aldrich. Hydrazine (N2H4$H2O,
80%) was from Tianjing Guangfu development Co. Ltd. D-
Glucose was from Shanghai Sangon Biotechnology. Na2HPO4
and NaH2PO4 were obtained from Tianjin Damao Chemical
Reagent Factory. All chemicals and reagents were analytical
grade, and all solutions were prepared with distilled water. The
glucose solution was kept to mutorotate at room temperature
overnight. The phosphate buffer solution (PBS, pH 7.0) was
used as the supporting electrolyte by mixing solution Na2HPO4
and NaH2PO4.
2.2 Apparatus
Morphologies of composites were observed by SU-70 FESEM
and before observation the samples were coated with platinum
using a sputter-coater. UV-vis absorption spectra were obtained
on a Shimadzu UV1700 spectrometer. XRD measurements were
operated on Rigaku Dmax-2500 X-ray diffractometer with Cu Ka
radiation operated at 50 KV and 80 mA. Cyclic voltammetric and
amperometric experiments were acquired on an electro-
chemical workstation (CHI 660D, Chen Hua Instruments Co.
Ltd.). A three-electrode system was used with an Ag/AgCl
(saturated KCl) electrode as reference electrode, a platinum wire
as counter electrode and a glassy carbon electrode (GCE) as
working electrode. The electrochemical impedance spectros-
copy (EIS) was recorded within the frequency range of 0.1–105
Hz with the direct current potential limited at 0.25 V.
2.3 Procedures
2.3.1 Preparation of GR by reducing GO with hydrazine. GR
was prepared according to the reported method with hydrazine
as a reductant. 100 mg GO was dispersed into 400 ml distilled
water with ultrasonication. 91 ml hydrazine and 1.4 ml ammonia
were added into the resulted brown dispersion. The mixed
dispersion was heated at 95 ꢁC in a water bath to reduce GO to
GR. Aer 1 h, the dispersion became black indicating the
formation of GR. GR was separated and obtained by ltering the
black dispersion and thoroughly washing twice with distilled
water. Finally, the obtained GR was dried in a vacuum oven at
ꢁ
80 C for 12 h.
2.3.2 Preparation of 3D porous GR–CS composites by
freeze drying. 200 mg CS powder was dissolved into 20 ml 0.6%
HAc solution to prepare a 1 wt% CS solution. Different amount
of GR was dispersed into the 4 ml 1 wt% CS solution by vigorous
ultrasonication for at least 30 min. Then the black dispersion
was immersed into liquid nitrogen to freeze it. Aer that, the
frozen sample was rapidly put in a freeze drier (FD-18 Beijing
Detianyou Company) for 36 h to completely dry the sample.
Finally, the porous GR–CS composites were obtained.
Fig. 1 Schematic representation for the fabrication of porous GR–CS
composites and immobilization of GOD onto the GR–CS modified
GCE.
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2.3.3 Fabrication of GOD modied GCE. The porous GR–
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effectively reduced to GR.31 UV-vis spectroscopy was also used to
monitor the formation of GR, and the corresponding result is
shown in the inset of Fig. 2. A strong peak of 220 nm appeared
for the GO dispersion, attributed to the p–p* transition in the
GO structure. For the GR dispersion, the absorbance peak red-
shied to 260 nm because the p conjugation system of the GR
sheet was partly restored during the reducing process.32,33
As a biopolymer, CS has backbone structure of glucose with
primary amines. It usually has positive charges in aqueous
solution and been used to stabilize carbon materials, including
carbon nanotubes and graphene.34,35 In this experiment, the
stable GR–CS dispersion was rst obtained. Then the black
porous GR–CS composites were obtained aer freeze-drying the
dispersion. Fig. 3 gives the FESEM images of the produced CS
and GR–CS composites. Layered structure and few pores were
observed for the freeze-dried CS (Fig. 3A). The structure was
oen obtained by freeze-drying polymer solutions and it was
similar with that reported in the literature.20 When the GR–CS
dispersion was freeze dried, the resulted composites gave
different morphologies. Some pores formed in the composites,
as shown in Fig. 3B. From the pore wall, it was observed that GR
was immobilized into the CS matrix. The pore amount
increased with the GR content from 12.5 to 70 wt% (Fig. 3B–D).
In addition, more and more GR were immobilized into the CS
matrix until large amounts of GR and few CS were observed for
70 wt% GR. The resulted GR70–CS composite showed inter-
penetrating porous architectures, which was different from
other freeze-dried samples. Formation of the porous GR–CS
composites was easily explained according to freeze drying
process. For the GR–CS dispersion, many CS molecules adsor-
bed onto the GR surfaces and stabilized them in aqueous
solution. When the GR–CS dispersion was immersed into liquid
nitrogen, ice crystal grew while the GR and CS molecules were
excluded from the ice crystals. These excluded GR and CS
CS composite containing 70 wt% GR (GR70–CS) was used to
immobilize GOD. Before fabrication of GR70–CS modied
electrodes, the GR70–CS was rstly washed with ethanol and
dried in an oven at 60 ꢁC overnight to remove the le HAc and
improve the stability of composites in aqueous solution. Then
the GR70–CS was dispersed into water to prepare the GR70–CS
dispersion. The GCE was polished with 1.0, 0.3 and 0.05 mm
alumina powder respectively, and then it was washed with
ethanol and distilled water to clean the electrode and dried in
nitrogen. The 5 ml GR70–CS dispersion was dropped onto the
GCE electrode and dried at room temperature. Then the
modied GCE was immersed into 10 mg mlꢀ1 GOD solution
(PBS, pH 7.0) overnight to immobilize GOD onto the GR70–CS/
GCE (GOD/GR70–CS/GCE). The other method was also used to
prepare the GOD modied GCE. 15 ml 10 mg mlꢀ1 GOD solution
was rstly mixed with 5 ml 10 mg mlꢀ1 GR–CS dispersion. Then
5 ml of the mixture was dropped onto the GCE and dried at 4 ꢁC.
Aer that, 5 ml 1% Naon was coated onto the GCE and dried in
a freezer and nally the modied electrode was obtained (GOD–
Naon/GR70–CS/GCE). For the electrochemical and chro-
noamperometry experiments, the phosphate buffer solutions
were rstly purged with nitrogen for at least 50 min.
3. Results and discussion
3.1 Preparation of porous GR–CS composites from freeze
drying
Hydrazine is oen used to reduce oxygen-containing groups
and prepare GR from GO. Here the produced GR was charac-
terized with XRD and UV-vis adsorption spectroscopy. Fig. 2
shows the XRD patterns and UV-vis spectra of GO and GR. From
the XRD patterns, it was found that GO gave a strong peak at 2q
of 12ꢁ (curve a) ascribed to the (002) interplanar spacing
between the GO sheets. However, aer reduction it was
observed that the peak disappeared and only a broad peak at 2q
of 23ꢁ (curve b) was observed. This suggested that GO was
Fig. 3 FESEM images of the porous CS and GR–CS composites by
freeze drying. (A) from CS solution; (B)–(D) from 12.5, 50 and 70 wt%
GR in CS dispersion. The scale bar was 10 mm.
Fig. 2 XRD patterns of GO (curve a) and GR (curve b). Inset: UV-vis
spectroscopy of the GO (black line) and GR dispersion (red line).
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molecules were assembled to the GR–CS composites between dispersion, GR and CS were randomly dispersed in the solution.
the ice crystals. Aer removing ice crystals via freeze drying, the The strong interaction of GR sheets and good lm-forming
porous GR–CS composites were produced. So in fact these ability of CS resulted in the formation of GR–CS composite lms
porous GR–CS composites resulted from the ice-induced on the substrate. However, for the GR–CS composite from
assembly of GR and CS. These results indicated that the pres- freeze-drying, the stable composite was formed and GR was
ence of GR could inuence the composite structures aer freeze immobilized in the CS matrix, which reduced the p–p* inter-
drying. Addition of GR was in favor of the pore formation in the action between GR, so porous structure still was remained and
GR–CS composites, and the porous GR–CS composites with observed on the surface. These results showed that stable
different GR content could be produced by adjusting the GR porous GR–CS composites could be successfully produced by
concentration in the dispersion.
freeze drying. They still exhibited porous structure on the
It was also noticed that the GR amount could obviously affect surface, and could be used to prepare the porous electrodes. It
mechanical property of the GR–CS composites. When the GR was expected that the porous GR70–CS could increase active
content was above 70 wt%, the mechanical strength was weak surface area and loading amount of biomolecules.
and the composite exhibited exibility. Aer removing the le
HAc, the porous GR–CS composite was soaked into PBS (pH 7.0) studied with Fe(CN)6
The electrochemical properties of the GR70–CS/GCE were
3ꢀ/4ꢀ
as the probe. Fig. 5A gives cyclic
in order to examine its stability in aqueous solution. No obvi- voltammograms of the bare and the GR70–CS modied GCE in
ously change was observed even aer 1 month and this was 5 mM Fe(CN)63ꢀ/4ꢀ solution containing 0.1 M KCl at a scan rate
attributed to the fact that CS was stable and not dissolved in of 100 mV sꢀ1. A couple of redox peak at 0.235 V was observed
solutions (pH > 6.0). This also suggested that the resulted from the bare GCE. For the GR70–CS/GCE, the redox peaks were
porous GR–CS composites were very stable and could be used in
neutral and alkaline solutions.
3.2 Electrochemical properties of the GR70–CS/GCE
Considering that GR has good conductivity, the GR70–CS
composites were chosen to examine its electrochemical prop-
erties due to the high GR content. Aer removing the le HAc
with ethanol, the GR70–CS composite was rst dispersed in PBS
(pH 7.0) to modify the GCE. To conrm if the composite still
kept porous structure aer modication, its morphology was
observed by FESEM. Fig. 4A corresponds to the FESEM image of
the GR70–CS composites aer dispersing and coating on the
substrate. It was found the composite still remained porous
structure on the surface which was similar with that without
washing with ethanol and dispersing (Fig. 3D). This also indi-
cated that the porous structure was maintained even washing
with ethanol and drying. The assembly of GR and CS was clearly
observed from the magnication image (the inset in Fig. 4A).
For the control experiment, the GR70–CS dispersion before
freeze drying was also dropped on the surface. As shown
in Fig. 4B, only thin folded lm was observed. Due to the strong
p–p* interaction between GR, thin GR lms were easily formed
aer dropping the GR solution on surfaces. In the GR–CS
Fig. 5 (A) Cyclic voltammograms of the bare GCE (curve a) and GR70–
CS/GCE (curve b) in PBS (pH 7.0, 0.1 M KCl) with 5 mM Fe(CN)63ꢀ/4ꢀ at
Fig. 4 FESEM images of the GR–CS composites on the substrate. (A)
from the freeze-dried GR70–CS after washing with ethanol and
a scan rate of 100 mV sꢀ1. (B) Electrochemical impedance spectra of
drying, scale bar: 10 mm, inset: 2 mm; (B) from the GR70–CS dispersion
before freeze drying, scale bar: 5 mm.
the GR70–CS/GCE (curve a) and bare GCE (curve b) in 5 mM
3ꢀ/4ꢀ
Fe(CN)6
with 0.1 M KCl.
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still observed, which were similar with the bare GCE. However,
the redox peak current was about 2 times that from the bare
GCE. Moreover, the GR70–CS/GCE gave higher capacitance
current. These results were ascribed to porous structure of the
GR70–CS composites, which were consisted with those from the
FESEM images. EIS was used to further investigate the electro-
chemical properties. Fig. 5B shows the Nyquist impedance
spectra of the bare and the GR70–CS/GCE. The diameter of the
semicircle part in the high frequency showed the electron-
transfer resistance at the electrode surface. As shown in Fig.5B,
the semicircle part from the GR70–CS/GCE was 126 U and
smaller than that from the bare GCE (416 U), indicating that the
GR70–CS composite had lower electron transfer resistance at
the GCE surface. This showed that GR70–CS composite
exhibited good conductivity and enhanced the electron transfer
aer modication on the GCE, and the composite could be used
as an effective electrode material.
3.3 The electrochemical properties of GOD/GR70–CS/GCE
CS has good biocompatibility and usually been used to immo-
bilize enzyme molecules. The produced GR70–CS composites
showed porous structure and good conductivity. Thus the
composite was used as the matrix for immobilization of
enzymes to fabricate biosensors. Here, GOD as a model was
immobilized onto the GR70–CS/GCE by soaking in the GOD
solution overnight. For the GOD/GR70–CS/GCE, the direct
electrochemical behavior of GOD was observed, as shown in
Fig. 6A. The GOD/GR70–CS/GCE gave a couple of redox peak at
ꢀ0.382 and ꢀ0.444 V with the DEp of 62 mV at a scan rate of 100
mV sꢀ1 (curve a). However, no redox peak was observed for the
GR70–CS/GCE (curve b). This indicated that the couple of peak
was attributed to the direct electron transfer between the active
center of GOD (FAD/FADH2) and the electrode surface. In this
experiment, the direct electron transfer of GOD at electrode
surface was observed due to the good conductivity and porous
structure. Sun et al.31 fabricated the glucose biosensor via self-
assembled GR–GOD nanostructure. However, the direct elec-
trochemical behavior of GOD was not observed. The DEp was
smaller than or similar with that from the nanographene
platelet and hydrophilic ordered mesoporous carbon modied
electrodes.36,37 According to the Laviron's equation, the surface
average of GOD on the electrode surface was estimated to be 3.5
ꢂ 10ꢀ10 mol cmꢀ2, which was higher than that from the elec-
trodes based on ordered mesoporous carbon, gold nano-
Fig. 6 (A) Cyclic voltammograms of the GOD/GR70–CS/GCE (curve
a) and GR70–CS/GCE (curve b) in nitrogen-saturated 0.1 M PBS (pH
7.0) at a scan rate of 100 mV sꢀ1. (B) Cyclic voltammograms of the
GOD/GR70–CS/GCE in nitrogen-saturated 0.1 M PBS (pH 7.0) with
different scan rate of 20, 40, 60, 80, 100, 200, 300 mV sꢀ1. Inset of (B):
the relationship between cathodic and anodic peak current with the
scan rates.
oxidize glucose in the presence of oxygen according to the
following electrocatalytic reaction:39
Glucose + GODox / gluconolactone + GODred
GODred + O2 / GODox + H2O2
particles–GR–carbon
nanotubes,
GR–GOD
composites,
electrochemical reduction of porous GR.37–40 Effect of scan rates
on the peak currents was also studied (Fig. 6B). A linear rela-
tionship between them was obtained indicating a surface-
conned electrode reaction, and the small shi of peak poten-
tials showed the fast electron transfer rate.
Fig. 7 gives the corresponding cyclic voltammograms of the
GOD–Naon/GR70–CS/GCE in the air-saturated PBS solution
containing different glucose concentration. It was found that a
strong cathodic current peak at ꢀ0.68 V appeared in the
absence of glucose, and it was attributed to the electrochemical
reduction of O2 to H2O. The peak potential was slightly positive
than that from the GR–GOD nanostructure modied electrode
(ꢀ0.79 V).31 When glucose was added, the cathodic current
decreased with the increase of glucose concentration. According
to the electrocatalytic reaction mechanism, more oxygen in the
solution was consumed in the presence of more glucose, and
3.4 Electrocatalytic properties of GOD–Naon/GR70–CS/
GCE for glucose
In order to study the electrocatalytic properties of GOD, GOD
and the GR70–CS composite were mixed to prepare the GOD
modied electrodes. It has been known that GOD can also
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Fig. 7 Cyclic voltammograms of the GOD–Nafion/GR70–CS/GCE in
air-saturated 0.1 M PBS (pH 7.0) at a scan rate of 10 mV sꢀ1 with 0, 0.1,
0.4, 1.6 and 5.2 mM glucose.
this resulted in the low electrocatalytic response of O2 to H2O.
The current at ꢀ0.68 V showed a good linear relationship from
0.1 to 5.2 mM (R ¼ 0.994) with the sensitivity of 5.7 mA mMꢀ1
cmꢀ2, which was higher than the modied electrodes based on
the GR–GOD composite (1.85 mA mMꢀ1 cmꢀ2) and GOD–GR–
gold nanoparticles composites (3.84 mA mMꢀ1 cmꢀ2).38,41
FMCA is also used as a mediator to examine the electro-
catalytic activity of GOD for glucose in nitrogen-saturated
solution. Fig. 8A shows the cyclic voltammograms of the GOD–
Naon/GR70–CS/GCE in PBS (pH 7.0) solution containing 5 mM
FMCA in the absence and presence of 6.2 mM glucose. A couple
peak around 0.35 V was observed from the curve a, due to the
redox process of FMCA. When 6.2 mM glucose was added, the
anodic current obviously increased (a peak at 0.38 V appeared),
and the cathodic peak current disappeared. The anodic
peak current was 2.6 times that in the absence of glucose. This
suggested that the GOD–Naon/GR70–CS/GCE showed good
electrocatalytic activity for the glucose oxidation. The electro-
catalytic process of GOD for glucose could be explained as
follows:35
Fig. 8 (A) Cyclic voltammograms of the GOD–Nafion/GR70–CS/GCE
in nitrogen-saturated 0.1 M PBS (pH 7.0) containing 5 mM FMCA at a
scan rate of 10 mV sꢀ1 without (curve a) and with (curve b) 6.2 mM
glucose. Inset: Cyclic voltammograms of the GOD–Nafion/GR70–Cs/
GCE in air-saturated 0.1 M PBS (pH 7.0) containing 5 mM FMCA at a
scan rate of 10 mV sꢀ1 without (curve c) and with (curve d) 6.2 mM
glucose; (B) The relationship between the current with the glucose
concentration. Inset: Amperometric response of the GOD–Nafion/
GR70–CS/GCE at 0.40 V in a stirring nitrogen-saturated 0.1 M PBS
(pH 7.0) containing 5 mM FMCA.
the mediator. Inset of Fig. 8B shows the amperometric response of
the GOD–Naon/GR70–CS/GCE to the successive addition of
glucose with the applied potential of 0.40 V. It was found that the
current increased and became stable in less than 5 s aer the
glucose solution was injected. This was attributed to the good
electrocatalytic activity for glucose. The current exhibited a linear
relationship with the glucose concentration from 0.14 to 7.0 mM (R
¼ 0.995), as shown in Fig. 8B. The sensitivity of the modied GCE
was 11.2 mA mMꢀ1 cmꢀ2, and the detection limit was 17.5 mM (S/N
¼ 3), which was lower than that from the electrodes modied with
Ag nanoparticles–GR composite, self-assembled GR–GOD nano-
structures and GR–gold nanoparticles–CS composite.31,43,44 The
good electrochemical response towards glucose resulted from the
3D porous graphene structure and chitosan providing a microen-
vironment for GOD. Table 1 gave the performance of some glucose
biosensors based on GR. The GOD–Naon/GR70–CS/GCE also
showed good stability, and 95% current response toward glucose
was stilled remained aer being stored at 4 ꢁC for 10 days.
Glucose + GODox / gluconolactone + GODred
GODred + 2FMCA+ / GODox + FMCA + 2H+
2FMCA 5 2FMCA+ + 2e
Inset of Fig. 8A gave the cyclic voltammograms of the GOD–
Naon/GR70–CS/GCE in air-saturated PBS (pH 7.0) solution con-
taining 5 mM FMCA without and with 6.2 mM glucose. It was
found that the modied electrode still exhibited electrocatalytic
activity for glucose. However, the anodic peak current was about
1.88 times that without glucose. This also indicated that oxygen
could affect the electrocatalytic properties, so the GOD–Naon/
GR70–CS/GCE was used for glucose detection in nitrogen-satu-
rated aqueous solution. Amperometric experiments were carried
out to determine the glucose concentration with 5 mM FMCA as
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Table 1 Performance of some glucose biosensors based on GR
Linear range
(mM)
Detection limit
(mM)
Sensitivity
Glucose biosensors
(mA mMꢀ1 cmꢀ2
)
Reference
GOD–GR nanostructures
GOD/SPG–Au NPs–CS
GOD/GR–Ag NPs–CS
GOD/AuNPs/GR/CS
GOD/TiO2–GR
2–22
0.5–22.2
2–10
2–14
0–8
20
510
100
180
—
—
6.51
—
0.55
6.2
31
42
43
44
45
GOD/3D porous GR–CS
0.14–7.0
17.5
11.2
This work
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We developed a facile method to fabricate 3D porous GR–CS
composites based on ice-induced assembly by freeze-drying.
UV-vis, XRD, and FESEM were used to characterize GR and GR–
CS composites. The GR mass could inuence morphologies of
the resulted GR–CS composites, and more pores were produced
with the increase of GR amount. Especially for 70 wt% GR, the
GR70–CS composite exhibited good exibility and 3D porous
structures. The GR70–CS composite also showed good stability,
and remained porous structures aer coating on the surfaces.
Electrochemical experiments indicated that the GR70–CS could
be used as a good matrix to modify electrodes and immobilize
GOD. The GR70–CS/GCE gave higher current response and
lower electron transfer resistance compared with the bare GCE.
The direct electrochemical properties were observed from the
GOD/GR70–CS/GCE. Moreover, the modied GCE exhibited
good electrocatalytic activity for glucose in air or nitrogen-
saturated solution. The modied GCE also were used to detect
glucose, and a good linear range from 0.14 to 7.0 mM and a
detection limit of 17.5 mM were obtained with FMCA as the
mediator. These results indicate that the 3D porous GR–CS
composites are good materials for electrode modication and
biomolecule immobilization, which will be used in the eld of
biosensing and biofuel cells. In addition, freeze-drying method
is simple, facile, and also used to prepare other 3D porous GR
materials with different function and composition.
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9049.
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Acknowledgements
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This work was supported by the National Nature Science
Foundation of China (no. 51202130), the Shandong Provincial
Natural Science Foundation, China (no. ZR2012BQ001), the
Scientic Research Foundation for the Returned Overseas
Chinese Scholars, State Education Ministry and State Key
Laboratory of Electroanalytical Chemistry Open Foundation
(no. SKLEAC201304). We would like to acknowledge Runhua
Fan for his help in preparing the revised manuscript.
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