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J. Singh et al. / Process Biochemistry 48 (2013) 1724–1735
the excess SPEEK and hydrazine monohydrate. The final product
was designated as SPEEK-functionalized graphene (SPG).
as hydroxyl, epoxy, and carboxyl, which interact with the water
molecules through hydrogen bonding, leading to the formation of a
stable dispersion. However, the water dispersion stability of chem-
ically reduced GO is very poor due to the removal of all the oxygen
functionalities from its surfaces and sediments at the bottom of the
vial or floating on the top of the water. However, the noncovalently
functionality (−SO3H groups in SPEEK) on the surface of graphene
helps in the preparation of a stable dispersion of the graphene in
an aqueous medium, and can easily prevent the agglomeration of
graphene by restacking [43,44].
ing high sensitivity to electronic and crystallographic structures.
It has therefore been extensively applied to the structural inves-
tigation of carbon materials, particularly nanotubes. The Raman
spectrum of SPG (Fig. 1(a)) exhibits G (graphite) bands at around
1582 cm−1 corresponding to the first order scattering of the E2g
due to the second-order two-phonon mode. The D (diamond) band
at 1342 cm−1 is due to the out-of-plane breathing mode of the sp2
atoms of A1g symmetry, which is attributed to local defects and
disorder [45,46]. The intensity ratio (ID/IG) of the D band to the G
band is correlated with the average size of the sp2 domains. The
shape, position, and intensity of the G band relative to those of the
D band for this peak depend markedly on the number of layers. The
observed ratio of SPG is ID/IG = 1.17, indicating the physical adsorp-
tion of the SPEEK polymer chains on the surface of graphene. This
kind of physical adsorption through – interactions precludes
the formation of defects due to the surface treatment of graphene.
The presence of defects is suitable for biosensor applications. The
defects, and it is thus not suitable for the amperometric detection
of biomolecules. It has been reported that the appropriate chemical
rials on it are important, as functional groups can create defects on
the surface of graphene [9]. The appearance of a broad peak at 2693
few-layer graphene [43].
Fig. 1(b) shows an SEM image of SPG, which suggests the for-
mation of a layered structure. The SPG dispersed in water was
characterized by TEM. Fig. 1(c) shows the large sheet of SPG, which
can be seen on the top of the grid. HR-TEM images were taken to
examine the number of layers at multiple locations. The edges of
obtained. The lateral dimension of the SPG was found to be in the
range of 1.8–2 m. The folding of one or two layers at the edges of
the graphene films appears as one or two dark lines, respectively
[47]. The appearance of a single dark line in Fig. 1(d) indicates the
formation of few-layer graphene.
Fig. S2 (supplementary file) shows the XRD spectra indicating
the highly crystalline nature of the as-prepared AuNPs. Five peaks
were observed at 38 2◦, 44.5◦, 64.4◦, 77.5◦, and 81.7◦ in the 2ꢁ
range of 20–90◦, which can respectively be indexed to the (1 1 1),
(2 0 0), (2 2 0), (3 1 1), and (2 2 2) reflections of the face-centered
cubic structure of metallic gold (JCPDS, card No. 04-0784) [48].
absorption peak around 532 nm. AuNPs exhibit strong absorption
that is dependent on the size and shape of particles. For spher-
ical nanoparticles, the absorption band maximum generally falls
between about 520 and 532 nm [49]. To further investigate the
formation and morphology of the AuNPs, FE-SEM and TEM obser-
vations are presented in Fig. S4 (supplementary file). The FE-SEM
2.2.4. Preparation of SPG–AuNPs–CH nanocomposite film
One gram of CH powder was added to 100 mL of 0.1 M acetic
acid, and the mixture was stirred to form a clear 1 wt% CH solu-
tion. The optimal amounts of CH (1 wt%) and SPG (1 mg/mL)
(2:1) were mixed, and the mixture was ultra-sonicated for 2 h
to obtain a homogeneous dispersion of SPG in the CH matrix.
The calculated amount of AuNPs (1 mg/mL) was dispersed in
the matrix containing SPG and CH with stirring at room tem-
perature, followed by ultra-sonication for about 2 h to obtain a
highly viscous SPG–CH solution with uniformly dispersed AuNPs.
A nanocomposite thin film was fabricated by uniformly spread-
ing 15 L of the SPG–AuNPs–CH nano-biocomposite solution onto
an ITO-coated glass surface (0.25 cm2), and then dried for 12 h at
room temperature and washed with de-ionized water to remove
any unbound particles. It was found that the optimized ratio of
SPG, CH, and AuNPs was 1:2:1, and that 15 L was the opti-
mum amount of SPG–AuNPs–CH solution, standardized for the
dispersion, to deposit onto the ITO surface when preparing the
SPG–AuNPs–CH/ITO film, in order to achieve the maximum amper-
ometric current.
2.2.5. Immobilization of GOx on SPG–AuNPs–CH/ITO
nanocomposite film
A freshly prepared solution of GOx (2 mg/mL) in phosphate
buffer solution (50 mM, pH 7.0) was uniformly spread (10 L)
onto a desired SPG–AuNPs–CH/ITO nanocomposite electrode. The
GOx/SPG–AuNPs–CH/ITO bioelectrode was kept undisturbed in a
humid chamber for about 24 h at room temperature. This bio-
electrode (GOx/SPG–AuNPs–CH/ITO) was washed thoroughly with
phosphate buffer (50 mM, pH 7.0) containing 0.9% NaCl to remove
any unbound enzyme, and stored at 4 ◦C when not in use.
2.3. Characterization
The Raman spectra of SPG were obtained on a Nanofinder
30 (Tokyo Instruments Co., Osaka, Japan). The Fourier trans-
form infrared (FT-IR) spectra of all of the samples were obtained
with a Nicolet 6700 spectrometer (Thermo Scientific, USA) over
the wavenumber range of 4000–400 cm−1. Transmission electron
microscopy (TEM) was carried with a JEM-2200 FS (JEOL, Japan).
The field emission scanning electron microscopy (FE-SEM) of SPG
and the other samples was carried out on a JSM-6701F (JEOL,
Japan). UV–visible spectroscopy was carried out using a UVS-
2100 SCINCO spectrophotometer. The cyclic voltammetry (CV),
electrochemical impedance spectroscopy (EIS), and amperomet-
ric measurements were recorded on a CH660D electrochemical
workstation (CH Instruments Inc, USA). The electrochemical mea-
surements were conducted on a three-electrode system with a
GOx/SPG–AuNPs–CH/ITO bioelectrode as the working electrode, a
platinum (Pt) wire as the counter electrode, and a saturated Ag/AgCl
electrode as a reference electrode in phosphate buffer (50 mM, pH
7.0, 0.9% NaCl) containing 5 mM [Fe(CN)6]3−/4− as a mediator.
3. Results and discussion
3.1. Structural and morphological studies
The water dispersion stability of SPG is comparable to that of GO
due to the presence of hydrophilic group (–SO3H) on the surface of
graphene. Fig. S1 (supplementary file) shows the water-dispersion
observation of GO, chemically reduced GO, and SPG. In contrast,
GO can be homogeneously dispersed in water, forming a brown-
ish dispersion, due to the presence of oxygen functionalities, such