Article

pubs.acs.org/ac

SnO2 Quantum Dots-Reduced Graphene Oxide Composite for

Enzyme-Free Ultrasensitive Electrochemical Detection of Urea
Dipa Dutta,† Sudeshna Chandra,† Akshaya K. Swain,‡ and Dhirendra Bahadur*,†
†
Department of Metallurgical Engineering & Materials Science, Indian Institute of Technology Bombay, Mumbai, Maharashtra
400076, India
‡
IITB Monash Research Academy, Department of Metallurgical Engineering & Materials Science, Indian Institute of Technology
Bombay, Mumbai, Maharashtra 400076, India
*
S Supporting Information

ABSTRACT: Most of the urea sensors are biosensors and

utilize urease, which limit their use in harsh environments.
Recently, because of their exceptional ability to endorse faster
electron transfer, carbonaceous material composites and
quantum dots are being used for fabrication of a sensitive
transducer surface for urea biosensors. We demonstrate an
enzyme free ultrasensitive urea sensor fabricated using a SnO2
quantum dots (QDs)/reduced graphene oxide (RGO)
composite. Due to the synergistic effect of the constituents,
the SnO2 QDs/RGO (SRGO) composite proved to be an
excellent probe for electrochemical sensing. The morphology
and structure of the composite was characterized by various
techniques, and it was observed that SnO2 QDs are decorated
on RGO layers. Electrochemical studies were performed to evaluate the characteristics of the sensor toward detection of urea.
Amperometry studies show that the SRGO/GCE electrode is sensitive to urea in the concentration range of 1.6 × 10−14−3.9 ×
10−12 M, with a detection limit of as low as 11.7 fM. However, this is an indirect measurement for urea wherein the analytical
signal is recorded as a decrease in the amperommetric and/or voltammetric current from the solution redox species ferrocyanide.
The porous structure of the SRGO matrix offers a very low transport barrier and thus promotes rapid diffusion of the ionic
species from the solution to the electrode, leading to a rapid response time (∼5 s) and ultrahigh sensitivity (1.38 μA/fM). Good
analytical performance in the presence of interfering agents, low cost, and easy synthesis methodology suggest that SRGO can be
quite promising as an electroactive material for effective urea sensing.

C omposite materials containing two or more constituents

are of great interest as they can jointly exhibit unique
physical and chemical properties for varied applications.
Quantum dots are zero dimensional materials having size-
dependent properties which make them quite interesting.
Among the wide band gap semiconductors, tin dioxide
quantum dots (SnO2 QDs) have attracted enormous research
interest for their widespread applications in electronics and
optics due to their excellent electrical and electrochemical
properties.1,2 On the other hand, graphene has also attracted
great attention due to its outstanding electrical, thermal, and
optical properties and a theoretical high surface area.3,4 The
number of graphene layers in reduced graphene oxide (RGO)
significantly influences the properties of RGO. However, these
graphene layers tend to aggregate in solution due to lack of
oxygen-containing groups which can be overcome by
incorporating different metal oxide nanoparticles between the
layers.4
Metal oxide/graphene composites are expected to exhibit
excellent material properties of parent components, due to the
synergistic effect of both. Uniform loading of metal oxide
nanocrystals on the graphene surface is quite challenging due to
the uneven distribution of functional groups on its surface.4,5
Presently, tin oxides/graphene composites have aroused the
interest of researchers, since they are efficient composites for
lithium ion batteries,4,6 supercapacitors,7 dye degradation,8 and
biosensing.9 The SnO2/graphene composites can be used to
construct electrochemical sensors due to high active surface
area, high electrocatalytic activity, chemical stability, and
interface dominated properties. In line with this, one may
think of using the composites for sensing small molecules like
urea which is an end product of protein degradation and
nitrogen metabolism. Urea is also a critical indicator of liver and
kidney malfunction in the human body.10 The normal
concentration of urea in blood serum is 3−7 mM (15−40
mg/dL).11 The presence of urea in the environment is also a
matter of great concern for ecosystem conservation and human
health protection.12 Due to its relatively higher water solubility,
Received: February 25, 2014
Accepted: May 15, 2014
Published: May 15, 2014

© 2014 American Chemical Society 5914 dx.doi.org/10.1021/ac5007365 | Anal. Chem. 2014, 86, 5914−5921
Analytical Chemistry
a considerable amount of the applied urea herbicides is washed
out in the aquatic environment. It can pollute the surface and
the groundwater into which it drains.13 It is therefore, essential
to analyze urea in the environment, drinking water, and food.12
Electrochemical analysis has inherent advantages of simplicity,
high sensitivity, and relatively low cost over other sophisticated
methods like high performance liquid chromatography,
chemiluminescence, fluorimetry, and the like.
Most of the urea sensors are biosensors, which utilize urease
(Urs) as a sensing element. However, their complicated
immobilization procedures, activity, stability, high cost of
enzymes, and critical operating condition have some
limitations, and they are not suitable in harsh environments.14
Recently, metal oxide matrices such as NiO nanoparticles,10
zinc oxide nanostructures,15 metal oxide−chitosan composite,16
carbonaceous materials composites,17 and quantum dots18 have
been used for fabrication of a sensitive transducer surface for
urea biosensors because of their exceptional ability to endorse
faster electron transfer between the electrolyte and the
electrode. So far, no attempt has been made to fabricate
enzymeless chemical sensors for lower level urea detection.
SnO2 QDs (SQDs) and RGO composite based chemical
sensors can be used as potential probes for detection of urea in
environmental samples.
Herein, we report a new strategy for fabrication of SQDs/
RGO (SRGO) from a hybrid complex [(N2H4)m(SnCl4)n].
Different morphological characterization techniques were used
to confirm the formation of the SRGO composite. Electro-
chemical studies were performed to investigate its performance
as a proposed enzymeless chemical sensor for urea. It showed a
detection limit which is much lower than the previously
reported sensors that are based on different matrixes such as
metal oxides, carbon materials, and quantum dots.16−18 SRGO
has synergistic effects of the QDs and the two matrixes, namely,
SnO2 and RGO, which results in highly sensitive sensor
material.
■ EXPERIMENTAL SECTION
Materials Preparation. A hybrid SRGO composite was
fabricated in two simple steps. In the first step, graphite oxide
(GO) was prepared from natural graphite power, and in the
second step, it was reduced to form reduced graphene oxide.
Natural graphite powder (trace metals <100.0 ppm, purity
>99.99%, average particle size <45 μm), SnCl4·5H2O (98%),
and hydrazine hydrate (98%) from Sigma-Aldrich and H2SO4
(98%), HCl, KMnO4, H3PO4, and H2O2 from Thomas Baker
were used for this synthesis. All reagents were used without
further purification. Natural graphite powder was oxidized by a
modified Hummers method.19,20
40 mg of GO was added to 100 mL of Milli-Q water and
sonicated for 30 min to form a brown homogeneous
suspension. 1 mL of N2H4·H2O (98%) and 10 mL of
NH4OH (25%) were added to this suspension and maintained
at 100 °C. In an alkaline medium, N2H4·H2O serves as a strong
reductant which reduces GO to RGO. For the synthesis of
SRGO composites, a hybrid complex (N2H4)m(SnCl4)n was
added to the mixture after 90 min and maintained at 100 °C for
another 18 h. The preparation of this hybrid complex is
reported in our previous work.21 The product, thus obtained,
was centrifuged at 10 000 rpm (9838g) for 10 min and washed
with ethanol followed by deionized water; this washing is
repeated six times and then dried overnight at 60 °C in air. The
proposed mechanism of the formation of the SRGO composite
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can be explained with the fact that the surface of the GO is
covered with oxygen-containing groups, such as hydroxyl and
epoxy at basal plane and carbonyl and carboxyl acid at the
edges.22 Along with the reduction of GO, the hybrid complex
((N2H4)m(SnCl4)n) simultaneously decomposes to Sn4+,23
which gets attached to the surface of RGO through the
oxygen-containing groups. The attached Sn4+ then get
converted to SnO2 in an aqueous medium.24
The bare RGO was synthesized by the same method
described above without addition of the hybrid complex
[(N2H4)m(SnCl4)n]. The methodology for the preparation of
bare SQDs has been described elsewhere.21
Materials Characterization. The crystallinity and phase
purity of the products were verified by X-ray diffraction (XRD)
patterns using a Philips powder diffractometer PW3040/60
with Cu Kα (1.5406 Å) radiation. The surface compositions
and chemical states of the samples were determined by X-ray
photoelectron spectroscopy (XPS) (MULTILAB from Thermo
VG Scientific) using monochromatic Al Kα X-rays (1486.6 eV).
The size and morphology of the samples were investigated
using a high resolution transmission electron microscopy
(HRTEM), JEOL JEM 2100F (200 kV), and field emission
scanning electron microscope (FESEM, JEOL, JSM-7600F).
Raman scattering measurement was performed at room
temperature on Horiba Jobin Yvon LabRam series HR(800)
spectrometer using an excitation of 514.5 nm from an Ar laser.
The porosity and surface area measurement were performed
after thoroughly degassing the samples at 150 °C for 4 h, using
an N2 absorption desorption isotherm in a surface area and
porosity analyzer (Micromeritics ASAP 2020).
Electrochemical Studies. Electrochemical behavior of
SRGO and its constituents (RGO and SQDs) were studied
in detail. Cyclic voltammetry (CV), amperometry, and
electrochemical impedance spectroscopy (EIS) analysis were
conducted on a CH Instruments Model 660D electrochemical
analyzer (CH Instruments Inc., US) using a three-electrode
system with modified glassy carbon electrode (GCE) as the
working electrode (3.14 mm2), a platinum wire as the counter
electrode, and Ag/AgCl as the reference electrode in 0.1 M
potassium ferricyanide/potassium ferrocyanide ([Fe-
(CN)6]3−/4−) couple solution. The EIS was performed in 0.1
M [Fe(CN)6]3−/4− solution at a potential of 0.32 V in a
frequency range of 10−106 Hz at a signal amplitude of 10 mV.
GCEs were polished with slurries of 1, 0.3, and 0.05 μm
alumina powders and rinsed thoroughly with doubly distilled
water. Then, the electrodes were sonicated alternatively in
methanol and distilled water for 15 min, and the whole process
was repeated six to seven times. A thin film of the materials (40
μg) was deposited on the surface of the working electrodes by
the drop-casting method. The electrodes were left overnight to
dry at room temperature. The electrodes were polished and
cleaned before every deposition step.
■ RESULTS AND DISCUSSION
Characterization of the Samples. XRD patterns of the
SRGO composite as well as GO and RGO are shown in Figure
S-1(Supporting Information). The XRD of GO shows an
intense peak at 2θ = 10.9°, which corresponds to (002)
reflection of stacked GO. After reduction, the sharp peak of GO
disappeared and a broad peak appeared at 2θ = 23° indicating
reduction of GO to RGO.8 The XRD analysis of bare SQDs
(Figure S-2, Supporting Information) and SRGO reveals that
all diffraction peaks can be indexed to the tetragonal SnO2
dx.doi.org/10.1021/ac5007365 | Anal. Chem. 2014, 86, 5914−5921
Analytical Chemistry
Figure 1. Core level XPS spectra of C 1s for (a) GO and (b) SRGO. (c) Low magnification HRTEM image of the SRGO. SAED pattern of SRGO
(inset of (c)), showing both the diffraction rings of SQDs and elongated diffraction spots of RGO. (d) HRTEM image of SRGO exhibiting
crystalline SQDs with sizes of 2−3 nm, adhered on the RGO. (e) Elemental mapping of SRGO depicting the even distribution of C, O, and Sn.
structure (ICDD file no. 41-1445). The values of crystallite size
(D) of pristine SQDs and QDs in SRGO were estimated from
Williamson and Hall theorem.21 Pristine SQDs and QDs in
SRGO exhibit a single phase rutile structure with an average
crystallite size of ∼2 and ∼2.2 nm (averaged from (110), (101),
and (211) peaks), respectively.
The surface chemistry of the SRGO was investigated by XPS
analysis. Carbon 1s core-level XPS signals of GO and RGO are
deconvoluted into four components, as shown in Figure 1a,b,
respectively. The main peak of GO and RGO due to graphitic
sp2 carbon atoms is centered at about 284.6 eV. The other
peaks of GO at 285.8 (hydroxyl carbon, C−OH), 287.0
(carbonyl carbon, CO), and 288.7 eV (carboxylate carbon,
OC−OH) are due to carbon atoms connecting with oxygen
containing groups.3,8 The C 1s XPS spectrum of the RGO also
has four components: nonoxygenated ring C (284.6 eV), C−
OH species (285.5 eV), CO species (287.0 eV), and
carboxylate carbon (289.3 eV). The intensity of the peaks of
RGO due to oxygen containing groups decreases as compared
to GO peaks, indicating the reduction of GO to RGO.22 The
XPS results show that most oxygen containing functionalities
were removed during the reactions. Only small amounts of
residual oxygen containing functionalities are left on RGO,
which is also confirmed by the O 1s XPS peaks at 531.7 and
532.2 eV (Figure S-3, Supporting Information). Remnant
oxygen containing groups on RGO are believed to be helpful
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for maintaining an even dispersion of SQDs on the RGO sheets
through electrostatic attraction and hydrogen bonding.22,25
The core-level XPS signals of Sn 3d as shown in Figure S-4
(Supporting Information) exhibit Sn 3d5/2 and 3d3/2 doublet at
487 and 495.4 eV, respectively, which corresponds to Sn4+ in
the tetragonal rutile structure. This suggests the formation of
SnO2 on the surface of RGO sheets. The separation between
the Sn 3d5/2 and Sn 3d3/2 levels (8.4 eV) and the area ratio
(1.5) of these two peaks are exactly the same as reported
earlier.21,25 The formation of SnO2 on the surface of RGO
sheets is further confirmed by the O 1s peak at binding energy
of 530.8 eV in the oxygen 1s spectra of SRGO (Figure S-3,
Supporting Information) which implies the presence of the O
1s core level of SnO2.25 The oxygen 1s spectra of SRGO
exhibited an asymmetric peak which is deconvoluted into three
peaks.
Figure 1c,d presents HRTEM micrographs of the composite
indicating SQDs decorated on the RGO sheet and their average
particle size which is in the range of 2−3.3 nm; this is in
agreement with the average crystallite size estimated from XRD
line broadening. At higher magnification, SQDs (red circle) can
be seen on the wrinkled RGO surface. The SQDs show lattice
spacing of 0.35 nm which corresponds to the (110) plane of
rutile SnO2. The number of wrinkled layers at higher
magnification, marked as red lines in Figure 1d, indicates the
number of graphene layers in RGO. Restacking of carbona-
ceous materials is a hindrance in the fabrication of such
dx.doi.org/10.1021/ac5007365 | Anal. Chem. 2014, 86, 5914−5921
Analytical Chemistry Article

materials like RGO. In our case, SQDs being decorated on

RGO play a pivotal role to prevent the restacking of RGO in
the multilayer. The selected area electron diffraction (SAED)
(inset of Figure 1c) of SRGO also confirms the crystalline
nature of SQDs, which shows three diffraction rings of rutile
SnO2 accompanied by elongated diffraction spots arising from
the RGO sheets.26,27 These findings serve as explicit evidence
confirming the formation of the SRGO composite. FEGSEM
images (Figure S-5, Supporting Information) show uniform
distribution of SQDs on or between the RGO sheets, as is also
evident from the HRTEM study. The energy dispersive
spectrum of this region confirms the presence of C, Sn, and
O (Figure S-6, Supporting Information). The elemental
mapping (Figure 1e) of SRGO confirms uniform decoration
of SQDs on RGO. Red, white, and green represents the
elements C, O, and Sn, respectively.
GO and SRGO composite are investigated by Raman
spectroscopy (Figure S-7, Supporting Information). The
Raman spectrum of GO contains both G (1599 cm−1) and D
(1345 cm−1) bands. The Raman spectra of the SRGO
composites exhibit both G and D bands at 1591 and 1347
cm−1, respectively, along with a weaker band at 570 cm−1,
which is due to the surface defects of SQDs.21 The Raman
results reveal the amalgamation of the SQDs onto the RGO
surface, which is consistent with the HRTEM results.
In order to compare the textural properties such as surface
area and pore volume of SQDs, RGO, and the SRGO
composite, Brunauer−Emmett−Teller (BET) surface area and
pore volume measurements based on the nitrogen adsorption−
desorption isotherms were carried out. Figure 2a−c shows the
N2 physisorption isotherms of the samples and their
corresponding BJH pore size distribution curves. The SRGO
sample exhibits a hysteresis loop at the P/P0 range of 0.42−1.0.
The hysteresis loop of the SRGO resembles the H1 type as per
Figure 2. (a−c) Nitrogen adsorption−desorption isotherm curves of
the IUPAC classification. However, a hysteresis loop in bare SQDs, SRGO, and RGO, respectively, with their corresponding pore
SQDs and RGO is H2 type.28 SRGO has the highest BET size distribution curves.
surface area of 272 m2/g followed by SQDs (214 m2/g) and
RGO (27 m2/g). The surface area of SRGO is larger than the Table 1. SBET, Pore Volume, and Pore Size of SQDs, RGO,
recently reported SQDs and graphene nanosheet composite.29 and SRGO
The higher surface area of SRGO is due to decrease in the
restacking of the RGO sheets, resulting from the decoration of BET
surface single point adsorption BJH desorption
SQDs that have a higher surface to volume ratio. The surface area total pore volume of pores average pore diameter
area of RGO is much less than the composite due to restacking samples (m2/g) (cm3/g) (4 V/A) Å
of graphene layers. The pore size distribution calculated from SQDs 214 0.16 32.4
the desorption branch using the BJH model for SQDs, RGO, RGO 27 0.10 25.5
and SRGO clearly indicates the presence of mesopores SRGO 272 0.67 100.1
according to the IUPAC nomenclature. The presence of
pores in SRGO is attributed to the interstitial space between
the QDs, the interlayer space of RGO, and voids between QDs counterparts (RGO and SQDs), electrochemical measurements
and graphene terrain. The total pore volume (single point of all the three materials were performed. Figure 3 shows the
adsorption) of SRGO is 0.67 cm3/g, which is much larger than cyclic voltammograms of bare GCE, SRGO, RGO, and SQDs
that of RGO and SQDs. The details of texture analysis of at a scan rate of 50 mV/s, within the potential window of −0.8
SQDs, RGO, and SRGO are listed in Table 1. The larger to 1 V, in 0.1 M potassium ferricyanide/ferrocyanide
surface area and pore volume of SRGO can play a significant [Fe(CN)6]3−/4− electrolyte. Well-resolved anodic and cathodic
role in electrochemical analysis as compared to its counterparts. peaks are observed for all three samples. The potential
Electrochemical Detection of Urea. In order to under- separation (ΔE) of the two peaks for SRGO/GCE, RGO/
stand the electrochemical properties of the SRGO composite GCE, and SQDs/GCE are 235, 203, and 563 mV, respectively.
and its use as electrode material in electrochemical sensing of A lower value of ΔE indicates a fast electron transfer process.30
analytes, cyclic voltammetry (CV) was performed. The The SRGO/GCE, RGO/GCE, and SQDs/GCE showed
electroactivity of the composite was tested for selective anodic shift in the peak potential by 242, 261, and 83 mV,
determination of urea using SRGO modified GCE by various respectively, with respect to bare GCE. These two observations
techniques, viz., CV, amperometry, and EIS. To elucidate the aptly suggest that SRGO/GCE and RGO/GCE have a higher
comparative electrochemical performance of SRGO and its electrocatalytic activity than SQDs/GCE.
5917 dx.doi.org/10.1021/ac5007365 | Anal. Chem. 2014, 86, 5914−5921
Analytical Chemistry
Figure 3. Cyclic voltammograms of modified electrodes in 0.1 M
[Fe(CN)6]3−/4− electrolyte at a scan rate of 50 mV/s.
The surface concentration of ionic species per unit surface
area on SRGO/GCE, RGO/GCE, and SQDs/GCE was
estimated from the plot of Ip versus scan rate (ν) (Figure S-
8, Supporting Information) using the Brown−Anson model.10
The calculated values of the surface concentration of the ionic
species (Γ) on the SRGO/GCE, RGO/GCE, and bare SQDs/
GCE electrodes are approximately 9.56 × 10−9, 9.75 × 10−9,
and 7.04 × 10−9 mol cm−2, respectively. The larger value of
surface concentration indicates that large numbers of redox
species are available in the electrode surface for oxidation,
resulting in higher Faradaic current. The anodic and the
cathodic peak currents for the three materials increased with
the increase in the scan rate (Figure S-9, Supporting
Information). The peak currents vary linearly with the square
root of the scan rate (ν) over the entire range of 5−1000 mV/s
(Figure S-10, Supporting Information), suggesting diffusion
controlled mass transfer reactions.16,31 Diffusion coefficients are
calculated using the Randles-Sevcik equation.16 The calculated
values are 1.176 × 10−4, 1.242 × 10−4, and 0.667 × 10−4 cm2/s
for SRGO/GCE, RGO/GCE, and SQDs/GCE, respectively.
The sensing response of SRGO/GCE, RGO/GCE, and
SQDs/GCE electrodes toward urea was estimated from the
changes in the anodic peak current in the presence of urea.
Addition of 4 mM urea in the electrolyte solution results in an
abrupt change in the anodic peak current of SRGO/GCE
electrode (inset of Figure 4), while for RGO/GCE and SQDs/
GCE electrodes, the anodic peak currents do not register any
Figure 4. Percentage changes of current with the addition of 4 mM
urea solution for SQDs/GCE, RGO/GCE, and SRGO/GCE electro-
des. Inset shows the cyclic voltammogram of SRGO/GCE with urea
(black) and without urea (red).
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significant change. Figure 4 shows the percentage changes of
current with the addition of urea for the three electrodes as a
bar graph. The anodic peak current changes by 0.89%, 3.5%,
and 48.11% for SQDs/GCE, RGO/GCE, and SRGO/GCE,
respectively.
This observation aptly suggests that SRGO/GCE has the
best electroanalytical applicability toward urea, even though the
surface concentration and diffusion coefficient for RGO/GCE
and SRGO/GCE electrodes are nearly the same. As indicated
by negative zeta potential of SQDs, RGO, and SRGO, urea is
electostatically absorbed on the working electrode. Thus,
electrostatically absorbed urea on the electrodes surface forms
insulating layers. These layers act as a barrier for the interfacial
electron transfer, and the current decreases with the addition of
the urea. Though zeta potential may play a vital role for better
sensing, in this case, the values of zeta potential values are very
similar for all the three materials: −36.9, −35.5, and −38.7 mV
for SQDs, RGO, and SRGO, respectively. Further, the surface
area of the sensing electrode material plays a crucial role as can
be evidenced from the BET surface area of SRGO which is 10
times higher than that of RGO with a high pore volume, which
may allow the larger amount of urea absorption. Moreover, the
pore diameter may also be responsible for the electrochemical
detection of urea as SRGO has the largest pore diameter among
all other materials.
To evaluate the detection limit, sensitivity, and response time
of the fabricated SRGO/GCE system toward sensing of urea,
amperometric measurements were carried out. Figure 5a
describes the amperometric current response as a function of
time, and Figure 5b depicts the steady-state current depend-
ence calibration curve with the urea concentration ranging from
1.6 × 10−14 to 2.5 × 10−13 M and from 3.2 × 10−13 to 3.9 ×
10−12 M. The inset of Figure 5b shows an amplified calibration
curve ranging from 1.6 × 10−14 to 2.5 × 10−13 M. A constant
potential −0.344 V (vs Ag/AgCl) was applied to the SRGO/
GCE electrode.The current was recorded as a function of time
once the steady baseline was reached. The current−time profile
of the amperometric response was studied with successive
addition of 100 μL (1 pM) of urea in 6 mL of 0.1 M
[Fe(CN)6]3−/4− electrolyte at an interval of 100 s with constant
stirring (300 rpm) using a magnetic bead.
Instead of a single calibration curve for the whole
experimental concentration range of urea, two curves were
obtained with good linearity for two different concentration
ranges. Similar observations were also reported by Lian et al.14
for the urea sensor based on molecularly imprinted chitosan
film doped with CdS quantum dots.
The regression equations are
Ip( × 10−4A) = 13.8 × 10−3Curea(fM) − 8.93
(R2 = 0.992, 1.6 × 10−14 to 2.5 × 10−13 M) (1)
Ip( × 10−4A) = 6.87 × 10−4Curea(fM) − 5.68
(R2 = 0.999, 3.2 × 10−13 to 3.9 × 10−12 M) (2)
The developed sensor exhibited a wide linear current
response to the urea concentration ranging from 1.6 × 10−14
to 2.5 × 10−13 M (R2 = 0.992) and 3.2 × 10−13 to 3.9 × 10−12
M (R2 = 0.999). The detection limit is 11.7 fM as calculated
from the signal-to-noise ratio of 3,32 which is much lower than
the previously reported carbonaceous materials and its
composite based urea biosensor.17,33
dx.doi.org/10.1021/ac5007365 | Anal. Chem. 2014, 86, 5914−5921
Analytical Chemistry Article

Figure 5. (a) Current−time profile of the amperometric response of the developed sensor. (b) Calibration plots with the concentration of urea
ranging from 1.6 × 10−14 to 2.5 × 10−13 M and from 3.2 × 10−13 to 3.9 × 10−12 M. The inset shows an amplified calibration curve with the
concentration of urea ranging from 1.6 × 10−14 to 2.5 × 10−13 M.

The detection limit is around 100 times lower than the

recently reported urea electrochemical sensor based on other
QDs composite.14 A rapid response time (∼5 s) is observed for
detection. The porous structure of the SRGO matrix offers a
very low transport barrier and, thus, provides a rapid diffusion
of ionic species from the solution to the electrode, which leads
to a rapid response time. The sensitivity of the SRGO/GCE
electrode calculated from the slope of the calibration curve has
been found to be 1.38 μA/fM, which is much larger than the
urea sensors’ sensitivity reported previously.10,16 Successive
addition of urea probably blocks the pores of SRGO, which
prevents further absorption of urea and hinders electron
transfer between the electrode and the electrolyte.This also
decreases the sensitivity (68.7 μA/pM), as can be seen from the
calculations of the slope value from the second calibration
curve. This result confirms that the textural properties of SRGO
play a vital role in the sensing of urea. We also obtained a new
set of calibration data with decreasing urea concentrations
ranging from 40 to 10 fM (Figure S-11, Supporting
Information). The nature of the calibration curve remains the
same as that obtained earlier with increasing concentrations of
urea; it rules out the possibility of irreversible electrode fouling
due to urea adsorption.
To have further insight into the detection of urea by the
fabricated electrode system, electrochemical impedance spec-
troscopy was performed. With regard to sensors, EIS is
particularly well-suited for the analysis of the binding events on
the transducer surface and detection of molecules. Besides this,
EIS can also be used to characterize surface modifications which
occur during the recognition process on the transducers.34 In
this work, EIS was used to monitor the impedance changes of
the SRGO-modified electrode surface and its interfacial
properties in the presence of different concentrations of urea.
EIS was performed between 106 and 10 Hz at AC amplitude of
10 mV and at DC potential (Eocp = 0.32 V) in 0.1 M
[Fe(CN)6]3−/4− electrolyte. The corresponding semicircles
represent the best-fit curves on the equivalent circuit modeling
of R(CR(QR))(CR) as shown in Figure 6. The good fit implies
that the equivalent circuit model reasonably reveals the
electrochemical processes occurring on the modified electrodes.
The electrochemical parameters of the electrode/electrolyte
system were evaluated with the ZSimpWin program. The
process at the electrode/electrolyte interface is represented by
an equivalent circuit composed of resistance R, capacitance C,
5919
Figure 6. Nyquist plots of SRGO modified electrodes with varying
concentration of urea; the bottom shows the equivalent circuit diagram
used to model impedance data.
and constant phase elements (Q), combined in parallel or in
series. The solution resistance Rs in the range of 4.535−6.749
Ω, is very low, and is unaffected by the applied potentials. The
diameter of the semicircular region in the Nyquist plot (Figure
6) increases with increasing urea concentration. In presence of
urea, the modified electrode shows higher values of resistance
R3. The components C1 and C2 are the double layer capacitance
of the system. However, the impedance response of the double
layer at a modified electrode does not show the ideal behavior
as pure capacitance. It rather shows a constant phase element
(CPE) with a phase angle less than 90°. This infers that the
impedance of such a nonideal double layer is represented by a
constant phase element Q which is usually employed in a model
in place of a capacitor to compensate for the nonhomogeneity
of the system. For an ideal electrode system, Q is equal to
double layer capacitance and n = 1. However, in this case, the
value of n was found to be 0.88. As this value lies between 0.5
and 1, the effect may be attributed to surface roughness or due
to surface heterogeneity caused by the deposition of urea.35
Such surface occurs as a result of surface coupling between
nonhomogeneous double layer capacitance and the electrolyte
resistance. R1 and R2 are in the parallel connection in the
circuit, and R2 is the charge transfer resistance of the system. R3
dx.doi.org/10.1021/ac5007365 | Anal. Chem. 2014, 86, 5914−5921
Analytical Chemistry Article

Table 2. Sensing Characteristics of Urea Sensors Reported in Recent Literature Compared to Present Results
response
electrode material methods sensitivity linear range detection limit time, s ref
Urs-GLDH/CH-ZnO/ cyclic voltammetry 0.13 μA/mM cm2 5−100 mg/dL 0.5 mM 10 16
ITO
Ur/NiO-NP/ITO/glass cyclic voltammetry 21.3 μA/mM cm2 0.83−16.65 mM 0.88 mM 5 10
Urs/MWCNTs/SiO2/ potentiometry 23 mV/decade/cm2 2.18 × 10−5−1.07 × 10−3 M 10−25 33
ITO
Urs-GLDH/Nano-ZnO/ cyclic voltammetry 1.44 μA/mg/dL cm2 10−80 mg/dL 2.25 mM 15
ITO
Urs-GLDH/MLG/ITO cyclic voltammetry 5.43 μA/mgdL/cm−2 10−100 mg/dL 0.65 mM 10 17
Urs/H40−Au/ITO amperometry 7.48 nA/mM 0.01−35 mM 1 × 10−5 M 3 37
Ur-GLDH/CH-Fe3O4/ differential pulse 12.5 μA/(mMcm−2) 5−100 mg/dL 8.3 × 10−5 M 10 38
ITO voltammetry
CdS QDs−MIP differential pulse 5.0 × 10−12−7.0 × 10−8 M 1.0 pM 300 14
voltammetry

SRGO/GCE amperometry 1.38 μA/fM 1.6 × 10−14−3.9 × 10−12 M 11.7 fM 5 present

work

may be considered as the ionic/electronic charge transfer
resistance of the system or surface charge transfer resistance
caused due to Faradaic reaction at the electrode/electrolyte
interface.
This type of resistance (R3) appears due to absorbed
chemical species from the electrolyte,36 and it has been found
that R3 increases linearly with urea concentration in the range
of 7.9 × 10−15 to 0.3 × 10−12 M. This indicates migration and
absorption of urea from the electrolyte to the SRGO electrode/
electrolyte interface. The presence of urea leads to an additional
interfacial impedance which can be treated as a platform to
determine the sensitivity of the system toward detection of the
analyte. The limit of detection (LOD) was calculated to be 23.7
fM (defined as S/N = 3). The regression equation is R3 (ohm)
= 0.63 × Curea (fM) + 227.97, where C is the concentration of
urea in fM. The correlation coefficient is 0.974.
Stability and Selectivity of the SRGO/GCE Electrode.
Shelf life of the electrode was studied for more than 2 months
by measuring the current with respect to time at an interval of 1
week. No considerable change in response characteristics (data
not shown) was observed even after 2 months which confirms
storage capability for a long period, stability, and reproduci-
bility.
Table 2 shows a comparative analysis of previously reported
urea sensors with the proposed enzymeless sensor taking into
account various electrochemical methods and their perform-
ance.
■ CONCLUSION
In conclusion, a two-step simple method has been developed to
fabricate the SRGO composite, in which RGO sheets are
loaded with SnO2 QDs. These QDs prevent the restacking of
graphene layers which always remains as a big challenge to a
fabricator. Decrease in the restacking of graphene sheets results
in a higher surface area for SRGO, which is a favorable criterion
for any good sensor. The SRGO based enzyme-free sensor has
been successfully exploited for the understanding of a proficient
sensor for urea. It showed improved characteristics, such as a
linear current response (R2 = 0.992), to the urea concentration
ranging from 1.6 × 10−14 to 3.9 × 10−12 M and a detection limit
as low as 11.7 fM (S/N = 3) by amperometry. The sensor
exhibited an ultrahigh sensitivity of 1.38 μA/fM with a rapid
respose time of 5 s. Besides this, we provide a thorough
understanding of the mechanism of detection of urea using the
SRGO electrode by impedance spectroscopy. The low
detection limit promises its practical use in the detection of
urea in environmental samples with great ease.

■
The selectivity of the SRGO/GCE electrode with respect to
other interfering agents, viz., ascorbic acid, uric acid, and
ASSOCIATED CONTENT
glucose, has been studied by comparing the shape and
magnitude of the anodic current response (Figure S-12, *
S Supporting Information

Supporting Information). The voltammograms of urea (4
mM) were obtained in the presence of various interfering
agents of 40 mM each (10 times higher concentration than
urea). The interference concentration used for this study is
XRD of GO, RGO, SRGO and SnO2 QDs, XPS data of the
oxygen 1s spectra and Sn 3d of SRGO, SEM image of the
SRGO, EDS of SRGO, Raman spectra of GO and SRGO, and
electrochemical data of the composite. This material is available
free of charge via the Internet at http://pubs.acs.org.

■
much more as compared to their normal physiological values in
human serum. No substantial changes in peak position and
AUTHOR INFORMATION
shape of the voltammograms were observed in the presence of
ascorbic acid and uric acid; however, a negligible decrease in Corresponding Author
current intensity is observed. In the case of glucose, the current *E-mail: dhirenb@iitb.ac.in. Fax: (022)25723480. Tel: (022)
intensity of the voltammogram of urea remains the same but a 25767632.
slight change in the peak position is observed. This may be due Author Contributions
to the competitive effect of the electrostatic interaction of the The manuscript was written through contributions of all
SRGO with the −OH groups of glucose. When one takes into authors. All authors have given approval to the final version of
account the low concentration of the interfering agents in the manuscript.
human serum, the proposed sensor can be used for detection of Notes
urea in real samples. The authors declare no competing financial interest.
5920 dx.doi.org/10.1021/ac5007365 | Anal. Chem. 2014, 86, 5914−5921
Analytical Chemistry

■
Article

ACKNOWLEDGMENTS
We gratefully acknowledge the financial support by nano-
mission of DST, Govt. of India. D.D. acknowledges CSIR,
India, for the award of Senior Research Fellowship (SRF).
A.K.S. acknowledges IITB-Monash Research Academy for
financial support. We acknowledge the central characterization
facilities at IIT Bombay.
(29) Chen, C.; Wang, L.; Liu, Y.; Chen, Z.; Pan, D.; Li, Z.; Jiao, Z.;
Hu, P.; Shek, C.-H.; Wu, C. M. L.; Lai, J. K. L.; Wu, M. Langmuir
2013, 29, 4111−4118.
(30) Yan, J.; Sun, W.; Wei, T.; Zhang, Q.; Fan, Z.; Wei, F. J. Mater.
Chem. 2012, 22, 11494−11502.
(31) Prakash, A.; Chandra, S.; Bahadur, D. Carbon 2012, 50, 4209−
4219.
(32) Chandra, S.; Arora, K.; Bahadur, D. Mater. Sci. Eng., B 2012,

■
177, 1531−1537.
(33) Ahuja, T.; Kumar, D.; Singh, N.; Biradar, A. M.; Rajesh. Mater.
REFERENCES Sci. Eng., C 2011, 31, 90−94.
(1) Kilic, C.; Zunger, A. Phys. Rev. Lett. 2002, 88, 095501-1. (34) Lisdat, F.; Schäfer, D. Anal. Bioanal. Chem. 2008, 391, 1555−
(2) Cui, H.; Liu, Y.; Ren, W.; Wang, M.; Zhao, Y. Nanotechnology 1567.
2013, 24, 345602. (35) Ates, M.; Uludag, N. J. Appl. Polym. Sci. 2012, 124, 4655−4662.
(3) Zhu, C.; Fang, Y.; Wen, D.; Dong, S. J. Mater. Chem. 2011, 21, (36) Sujaya, C.; Sashikala, H. D.; Umesh, G.; Hegde, A. C. J.
Electrochem. Sci. Eng. 2012, 2, 19−31.
16911−16917.
(37) Tiwari, A.; Aryal, S.; Pilla, S.; Gong, S. Talanta 2009, 78, 1401−
(4) Zhang, L.-S.; Jiang, L.-Y.; Yan, H.-J.; Wang, W. D.; Wang, W.;
1407.
Song, W.-G.; Guo, Y.-G.; Wan, L.-J. J. Mater. Chem. 2010, 20, 5462−
(38) Kaushik, A.; Solanki, P. R.; Ansari, A. A.; Suman, G.; Ahmad, S.;
5467.
Malhotra, B. D. Sens. Actuators, B 2009, 138, 572−580.
(5) Wang, X. R.; Tabakman, S. M.; Dai, H. J. J. Am. Chem. Soc. 2008,
130, 8152−8153.
(6) Su, Y.; Li, S.; Wu, D.; Zhang, F.; Liang, H.; Gao, P.; Cheng, C.;
Feng, X. ACS Nano 2012, 6, 8349−8356.
(7) Li, F.; Song, J.; Yang, H.; Gan, S.; Zhang, Q.; Han, D.; Ivaska, A.;
Niu, L. Nanotechnology 2009, 20, 455602−455607.
(8) Seema, H.; Kemp, K. C.; Chandra, V.; Kim, K. S. Nanotechnology
2012, 23, 355705−355712.
(9) Zhu, C.; Fang, Y.; Wen, D.; Dong, S. J. Mater. Chem. 2011, 21,
16911−16917.
(10) Tyagi, M.; Tomar, M.; Gupta, V. Biosens. Bioelectron. 2013, 41,
110−115.
(11) Ansari, S. G.; Wahab, R.; Ansari, Z. A.; Kim, Y.-S.; Khang, G.;
Al-Hajry, A.; Shin, H.-S. Sens. Actuators, B 2009, 137, 566−573.
(12) Velichkova, Y.; Ivanov, Y.; Marinov, I.; Ramesh, R.; Ramudu
Kamini, N.; Dimcheva, N.; Horozova, E.; Godjevargova, T. J. Mol.
Catal. B: Enzym. 2011, 69, 168−175.
(13) Luong, J. H. T.; Bouvrette, P.; Male, K. B. Trends Biotechnol.
1997, 15, 369−377.
(14) Lian, H.-T.; Liu, B.; Chen, Y.-P.; Sun, X.-Y. Anal. Biochem. 2012,
426, 40−46.
(15) Ali, A.; Ansari, A. A.; Kaushik, A.; Solanki, P. R.; Barik, A.;
Pandey, M. K.; Malhotra, B. D. Mater. Lett. 2009, 63, 2473−2475.
(16) Solanki, P. R.; Kaushik, A.; Ansari, A. A.; Sumana, G.; Malhotra,
B. D. Appl. Phys. Lett. 2008, 93, 163903−163903.
(17) Srivastava, R. K.; Srivastava, S.; Narayanan, T. N.; Mahlotra, B.
D.; Vajtai, R.; Ajayan, P. M.; Srivastava, A. ACS Nano 2012, 6, 168−
175.
(18) Ruedas-Rama, M. J.; Hall, E. A. H. Anal. Chem. 2010, 82, 9043−
9049.
(19) Marcano, D. C.; Kosynkin, D. V.; Berlin, J. M.; Sinitskii, A.; Sun,
Z.; Slesarev, A.; Alemany, L. B.; Lu, W.; Tour, J. M. ACS Nano 2010, 4,
4806−4814.
(20) Swain, A. K.; Li, D.; Bahadur, D. Carbon 2013, 57, 346−356.
(21) Dutta, D.; Bahadur, D. J. Mater. Chem. 2012, 22, 24545−24551.
(22) Zhang, J.; Xiong, Z.; Zhao, X. S. J. Mater. Chem. 2011, 21,
3634−3640.
(23) Zhu, H.; Yang, D.; Yu, G.; Zhang, H.; Yao, K. Nanotechnology
2006, 17, 2386−2389.
(24) Liang, J.; Wei, W.; Zhong, D.; Yang, Q.; Li, L.; Guo, L. ACS
Appl. Mater. Interfaces 2012, 4, 454−459.
(25) Song, H.; Zhang, L.; He, C.; Qu, Y.; Tian, Y.; Lv, Y. J. Mater.
Chem. 2011, 21, 5972−5977.
(26) Zhong, C.; Wang, J.; Chen, Z.; Liu, H. J. Phys. Chem. C 2011,
115, 25115−25120.
(27) Wang, X.; Zhou, X.; Yao, K.; Zhang, J.; Liu, Z. Carbon 2011, 49,
133−139.
(28) Salmas, C. E.; Androutsopoulos, G. P. Langmuir 2005, 21,
11146−11160.

5921 dx.doi.org/10.1021/ac5007365 | Anal. Chem. 2014, 86, 5914−5921