iron oxide-chitosan nanobiocomposite for urea sensor

Sensors and Actuators B 138 (2009) 572–580
Contents lists available at ScienceDirect
Sensors and Actuators B: Chemical
journal homepage: www.elsevier.com/locate/snb
Iron oxide-chitosan nanobiocomposite for urea sensor
Ajeet Kaushik
a,b
, Pratima R. Solanki
a
, Anees A. Ansari
a
, G. Sumana
a
, Sharif Ahmad
b
,
Bansi D. Malhotra
a,∗
a
Department of Science & Technology Centre on Biomolecular Electronics, National Physical Laboratory, Dr. K. S. Krishnan Marg, New Delhi 110012, India
b
Materials Research Laboratory, Department of Chemistry, Jamia Millia Islamia, New Delhi 110025, India
article info
Article history:
Received 29 December 20 08
Received in revised form 4 February 2009
Accepted 6 February 2009
Available online 20 February 2009
Keywords:
Chitosan
Fe

3
O
4
nanoparticles
Nanobiocomposite
Urea Biosensor
abstract
Urease (Ur) and glutamate dehydrogenase (GLDH) have been co-immobilized onto superparamegnatic
iron oxide (Fe
3
O
4
) nanoparticles-chitosan (CH) based nanobiocomposite film deposited onto indium-tin-
oxide (ITO) coated glass plate via physical adsorption for urea detection. The magnitude of magnetization
(60.9 emu/g) of Fe
3
O
4
nanoparticles (∼22 nm) estimated using vibrating sample magnetometer (VSM)
indicates superparamagnetic behaviour. It is shown that presence of Fe
3
O
4
nanoparticles results in
increased active surface area of CH-Fe
3
O
4
nanobiocomposite for immobilization of enzymes (Ur and
GLDH), enhanced electron transfer and increased shelf-life of nanobiocomposite electrode. Differential

pulse voltammetry (DPV) studies show that Ur-GLDH/CH-Fe
3
O
4
/ITO bioelectrode is found to be sensitive
in the 5–100 mg/dL urea concentration range and can detect as low as 0.5 mg/dL. A relatively low value
of Michaelis–Menten constant (K
m
, 0.56 mM) indicates high affinity of enzymes (Ur and GLDH) for urea
detection.
© 2009 Elsevier B.V. All rights reserved.
1. Introduction
The increasing demand for clinical diagnostics relating to kid-
ney and liver diseases has necessitated evolution of new methods
for faster and accurate estimation of urea in desired samples
including urine and blood samples. The increase d urea concen-
tration (normal level in serum is 8–20 mg/dL) causes renal failure
(acute or chronic), urinary tract obstruction, dehydration, shock,
burns and gastrointestinal bleeding. Moreover, decreased urea con-
centration causes hepatic failure, nephritic syndrome, cachexia
(low-protein and high-carbohydrate diets) [1–3]. The conven-
tional methods for urea detection including gas chromatography,
calorimetry and fluorimetric analysis suffer from complicated
sample pre-treatment and are unsuitable for on-line monitor-
ing. Electrochemical biosensors have been considered to provide
interesting alternatives due to their simplicity, low cost and high
sensitivity [3,4].
Biosensors reported for urea detection are generally based on
urease (Ur) that is often present in most biological systems [1–7].
Ur catalyzes decomposition of urea into hydrogen bicarbonate

and ammonium ions (NH
4
+
). NH
4
+
ions are known to be unsta-
ble and easily disperse in the environment. Keeping this in view,
glutamate dehydrogenase (GLDH) along with Ur has been utilized
for urea detection since GLDH immediately catalyzes the reaction
∗
Corresponding author. Tel.: +91 11 45609152; fax: +91 11 45609310.
E-mail address: (B.D. Malhotra).
between NH
4
+
, ␣-ketoglutarate (␣-KG) and nicotinamide adenine
di-nucleotide (NADH) to produce NAD
+
and l-glutamate [5–7].
Immobilization of Ur onto a suitable matrix is crucial for
the development of an electrochemical urea sensor [1–6].In
this context, organic–inorganic hybrid nanobiocomposites have
attracted much interest as a new class of materials that uti-
lize the synergy of organic and inorganic components to obtain
improved biosensing characteristics. In hybrid nanobiocompos-
ites, surface fuctionalization of nanoparticles allows their covalent
attachment and self-assembly on surfaces that can be used for
loading of desired biomolecules in a favourable microenviron-
ment for development of a biosensor [8,9]. In this context, metal

oxide nanoparticles-chitosan (CH) based hybrid composites have
attracted much interest for the development of a desired biosen-
sor [6–8]. Metal oxide nanoparticles such as iron oxide (Fe
3
O
4
)
[10–12], zinc oxide (ZnO) [13,14], cerium oxide (CeO
2
) [15,16] etc.
have been suggested as promising matrices for the immobiliza-
tion of desired biomolecules. These nanomaterials exhibit large
surface-to-volume ratio, high surface reaction activity, high cat-
alytic efficiency and strong adsorption ability that can be helpful to
obtain improved stability and sensitivity of a biosensor. Moreover,
nanoparticles have a unique ability to promote fast electron transfer
between electrode and the active site of an enzyme. Among various
metal oxide nanoparticles, Fe
3
O
4
nanoparticles due to biocom-
patibility, strong superparamagnetic behaviour and low toxicity
have been considered as interesting for immobilization of desired
biomolecules [17–19]. Immobilization of bioactive molecules onto
surface charged superparmagnetic nanoparticles (size ∼25 nm) is
0925-4005/$ – see front matter © 2009 Elsevier B.V. All rights reserved.
doi:10.1016/j.snb.2009.02.005
A. Kaushik et al. / Sensors and Actuators B 138 (2009) 572–580 573
of special interest, since magnetic behaviour of these bioconju-

gates may result in improved delivery and recovery of biomolecules
for desired biosensing applications [9–11]. Besides this, existing
problem of aggregation and rapid biodegradation of Fe
3
O
4
nanopar-
ticles onto a given matrix containing biomolecules can perhaps be
overcome by modifying these nanoparticles using CH by preparing
hybrid nanobiocomposite [9,20–26].
CH (N-deacetylated derivative of chitin) is a linear copoly-
mer of glucosamine and N-acetylglucosamine units and has been
found in the exoskeleton of crustaceans, in fungal cell walls and
in other biological materials. It displays an excellent film-forming
ability, good adhesion, biocompatibility, high mechanical strength
and susceptibility to chemical modification due to the presence
of reactive hydroxyl and amino functional groups. CH has also
been exploited for application in contact lens, as a matrix for
cell and enzyme immobilization, and as an artificial skin (e.g.,
CH-collagen composite) [27–30]. Efforts have recently been made
to improve optical and electrical properties of CH for biosensor
application by dispersing superparamagnetic Fe
3
O
4
nanoparticles
[9,26].
Lin et al. (2005) have fabricated CH-Fe
3
O

4
nanocomposite based
chemical sensor for cathodic estimation of hydrogen peroxide
(H
2
O
2
). Harbac et al. (2007) have reported carbon electrode mod-
ified by nanoscopic Fe
3
O
4
particles to assemble chemical sensor
for the estimation of H
2
O
2
using amperometric technique. Zhang
et al. (2007) have studied direct electrochemistry of hemoglobin
immobilized on carbon coated Fe
3
O
4
nanoparticles foramperomet-
ric estimation of H
2
O
2
. Zhao et al. (2006) have reported multi-layer
thin film of CH-Fe

3
O
4
nanocomposite that has been found to pro-
mote direct electron transfer of hemoglobin. Liang et al. (2007) have
synthesized polysaccharide modified iron oxide nanoparticles as
an effective magnetic adsorbent for bovine albumin serum. CH-
Fe
3
O
4
nanocomposite has recently been utilized for the estimation
of ferritin and glucose sensing [9,21].
In this manuscript, we report results of studies relating to the
immobilization of Ur and GLDH onto CH-Fe
3
O
4
nanobiocomposite
film for urea sensor.
2. Materials and method
2.1. Reagents and preparation of solutions
Urease (Ur), glutamate dehydrogenase (GLDH), nicotinamide
adenine dinucleotide (NADH), ␣-ketoglutarate (␣-KG) and chi-
tosan have been procured from Sigma–Aldrich (USA). Ferrous
chloride, ferric chloride and triethyl amine have been purchased
from Sigma–Aldrich and have been used for preparation of Fe
3
O
4

nanoparticles. Indium-tin-oxide (ITO) coated glass plates have been
obtained from Balzers, UK. All chemicals used are of molecular biol-
ogy grade. The deionized water (Milli Q 10 TS) has b een used for
the preparation of reagents. All the solutions and glass wares are
autoclaved prior to being used.
2.2. Preparation of CH-Fe
3
O
4
hybrid nanobiocomposite
Fe
3
O
4
nanoparticles (∼22 nm) prepared using co-precipitation
method [9] are dispersed into 10 mL of CH (0.5 mg/mL) solution in
acetate buffer of 0.05 M at pH 4.2 under continuous stirring at room
temperature after which it is sonicated for about 4 h. Finally, vis-
cous solution of CH with uniformly dispersed Fe
3
O
4
nanoparticles
is obtained. CH-Fe
3
O
4
hybrid nanobiocomposite films have been
fabricated by uniformly dispersing 10 ␮L solution of CH-Fe
3

O
4
com-
posite onto an ITO surface (surface area is 0.25 cm
2
) and allowing
it to dry at room temperature for 12 h. These solution cast CH-
Fe
3
O
4
hybrid nanobiocomposite films are washed repeatedly with
deionized water to remove any unbound particles.
2.3. Immobilization of Ur and GLDH onto CH-Fe
3
O
4
hybrid
nanobiocomposite film
10 ␮L of bienzyme solution containing Ur (10 mg/ml) and
GLDH (1 mg/ml) in 1:1 ratio [prepared in Tris buffer (5 mM)] is
immobilized onto CH-Fe
3
O
4
nanobiocomposite/ITO electrode. The
Ur-GLDH/CH-Fe
3
O
4

nanobiocomposite/ITO bioelectrodes are kept
undisturbed for about 12 h at 4
◦
C. Finally, the dry bioelectrode is
immersed in 50 mM PBS (pH 7.0) in order to wash out any unbound
enzymes from the electrode surface.
2.4. Characterization
The structure and particle size of Fe
3
O
4
nanoparticles have
been investigated using X-ray diffraction (XRD) studies. The super-
paramagnetism of the Fe
3
O
4
nanoparticles has been measured
using vibrating sample magnetometer (VSM, Lakeshore-7304). FTIR
(PerkinElmer, Spectrum BX II) spectrophotometer has been used to
characterize CH-Fe
3
O
4
hybrid nanobiocomposite and itsinteraction
with Ur. The surface morphological studies have been investi-
gated using scanning electron microscopy (LEO-440). The cyclic
voltammetry (CV), electrochemical impedance spectroscopy (EIS)
and differential pulse voltammetry (DPV) measurements have been
recorded on an Autolab Potentiostat/Galvanostat (Eco Chemie,

Netherlands). The electrochemical measurements have been con-
ducted on a three-electrode cell in phosphate buffer (50 mM,
pH 7.0, 0.9% NaCl containing 50 mM [Fe(CN)
6
]
3−/4−
as the elec-
trolyte.
3. Results and discussion
3.1. Characterization of Fe
3
O
4
nanoparticles
X-ray diffraction (XRD, Fig. 1A) pattern of synthesized Fe
3
O
4
nanoparticles reveals reflection planes that are consistent with
Fe
3
O
4
. However, the broad reflection planes are perhaps due to
the nano-size of Fe
3
O
4
particles. The average particle size of Fe
3

O
4
nanoparticles estimated using Scherrer formula has been estimated
as 22 nm. However, broadening of the reflection peak indicates for-
mation of fine nanocrystalline particles [9,31–32].
The superparamagnetism of Fe
3
O
4
nanoparticles and CH-Fe
3
O
4
has been estimated using Langevin equation as
M = M
s
[(Coth(mH/k
b
T) − k
b
T/mH], (1)
where M
s
is the saturated magnetization of the nanoparticles, m
is the average magnetic moment of an individual particle in the
sample, H is the applied magnetic field, T is absolute tempera-
ture and k
b
is the Boltzmann constant [31]. The VSM plot between
magnetizatioin M and applied magnetic field H (Fig. 1B) reveals

superparamagnetic hysteresis of Fe
3
O
4
nanoparticles (curve a). It
can be seen that the coercive force (Hc) is small (65.84 Oe and Hc
is 500–800 Oe for bulk Fe
3
O
4
), and this value does not decrease
to zero due to small particle size indicating super paramagnetic
behaviour. Interestingly, anisotropy energy is less than the heat dis-
turbance energy of ions indicating that magnetized direction is no
longer fixed and the movement of ions is random [31,32].Itmay
be noted that the value of M
s
is considerably lower (60.91 emu/g)
than that of bulk magnetite particle (65–84 emu/g) and low reten-
tivity of 4.88 emu/g. However, the value of Hc, M
s
and retentivity
are found to decrease to 60.93, 48.67 and 3.98 emu/g, respectively
for CH-Fe
3
O
4
nanobiocomposite (curve b). These results reveal that
Fe
3

O
4
nanoparticles embedded in CH matrix in which each Fe
3
O
4
nanoparticle acts as single magnetic domain with least aggregates
with a little change in magnetization and significant decrease in the
retentivity.
574 A. Kaushik et al. / Sensors and Actuators B 138 (2009) 572–580
Fig. 1. (A) X-ray diffraction pattern of Fe
3
O
4
nanoparticles. (B) Magnetization curve
of Fe
3
O
4
nanoparticles and CH-Fe
3
O
4
nanobiocomposite.
3.2. Mechanism of immobilization of Ur-GLGH onto CH-Fe
3
O
4
nanobiocomposite film
The proposed mechanism relating to the preparation of CH-

Fe
3
O
4
nanobiocomposite film and immobilization of Ur-GLDH onto
CH-Fe
3
O
4
nanobiocomposite film is shown in Fig. 2. It can be seen
that surface charged Fe
3
O
4
nanoparticles [33] interact with cationic
biopolymer matrix of CHvia electrostatic interactions and hydrogen
bonding with –NH
2
/OH group to form hybrid nanobiocomposite
[22]. These electrostatic interactions between Fe
3
O
4
nanoparti-
cles and CH have been confirmed by FTIR spectroscopic studies
[9].
Ur is a multi-active sites enzyme composed of 3:3 (␣:␤)sto-
ichiometry with a 2-fold symmetric structure and GLDH is a
branch-point enzyme composed of 18 ␣ helices and ␤ sheet
that are both parallel and anti-parallel. The Ur-GLDH molecules

exist in anionic form at pH 7 because pH of solution is above
isoelectric point (5.5) of Ur-GLDH molecules that facilitate [34]
interactions with positively charged CH of nanobiocomposite via
electrostatic interactions (Fig. 2). In the nanobiocomposite, pres-
ence of Fe
3
O
4
nanoparticles results in increased electroactive
surface area of CH for loading of the enzymes due to affinity of the
Fe
3
O
4
nanoparticles towards oxygen atoms of enzymes. This sug-
gests that Ur-GLDH molecules easily bind with charged CH-Fe
3
O
4
hybrid nanobiocomposite matrix via electrostatic interactions and
this is further confirmed by optical and electrochemical stud-
ies.
3.3. Characterization of CH-Fe
3
O
4
nanobiocomposite and
Ur-GLDH/CH-Fe
3
O

4
nanobiocomposite/ITO bioelectrodes
3.3.1. Optical studies
The FTIR spectra (Fig. 3A) of pure CH (curve a,) display bands at
3200–3400 cm
−1
due to the stretching vibration mode of OH and
NH
2
groups. The peaks seen at 2950–3000 and 2870 cm
−1
are due
to sp
3
and sp
2
hybridization, respectively (corresponding to C–H
group). The peak due to C–O stretching along with N–H deformation
mode has been observed at 1615 cm
−1
. The bands at 1550, 1430 and
1340 cm
−1
are attributed to N–H deformation, symmetrical defor-
mation of CH
3
and CH
2
group, COO
−

group in carboxylic acid salt.
The IR peak s seen at 1150 and 925 cm
−1
are assigned to ␤ (1–4)
glucosidic band in the polysaccharide unit, 1080 cm
−1
is attributed
to stretching vibration mode of the hydroxyl group and 1020 cm
−1
stretching vibration is due to C–O–C in the glucose unit of CH [9,35].
The IR bands corresponding to –NH/OH stretching modes are
found to be shifted in the IR spectra of CH-Fe
3
O
4
hybrid nanobio-
composite film (curve b) as compared to that of pure CH. This
indicates that amine group of CH binds with Fe
3
O
4
nanoparticles
via electrostatic interactions. The presence of IR band at 580 cm
−1
(curve b) pertaining to the stretching vibration mode and the tor-
sional vibration mode of Fe–O bonds in the tetrahedral sites and
octahedral site reveal the formation of complex between surface
charged Fe
3
O

4
nanoparticles and cationic CH matrix, indicating
the formation of CH-Fe
3
O
4
hybrid nanobiocomposite [9]. However,
shape of absorption peak in the CH-Fe
3
O
4
hybrid nanobiocompos-
ite becomes broader due to overlapping of the functional groups of
Ur-GLDH and CH-Fe
3
O
4
nanobiocomposite film (curve c) indicating
immobilization of Ur-GLDH onto hybrid nanobiocomposite matrix.
The surface morphologies of CH-Fe
3
O
4
nanobiocomposite/ITO
electrode and Ur-GLDH/CH-Fe
3
O
4
nanobiocomposite/ITO bioelec-
trode have been investigated using scanning electron microscopy

(SEM, Fig. 3B). Globular porous morphology of CH-Fe
3
O
4
nanobio-
composite reveals incorporation of the Fe
3
O
4
nanoparticles in
CH, indicating the formation of CH-Fe
3
O
4
hybrid nanobiocompos-
ite. This may be attributed to electrostatic interactions between
cationic CH and the surface charged Fe
3
O
4
nanoparticles. However,
after the immobilization of Ur-GLDH onto CH-Fe
3
O
4
nanobiocom-
posite/ITO (image d) electrode, the globular morphology changes
to regular form. This suggests that Fe
3
O

4
nanoparticles provide
a favourable environment for high loading of Ur-GLDH moieties.
These results are further supported by electrochemical and FTIR
studies.
3.3.2. Electrochemical studies
Fig. 4A shows cyclic voltammograms of CH/ITO electrode, CH-
Fe
3
O
4
nanobiocomposite/ITO electrode and Ur-GLDH/CH-Fe
3
O
4
nanobiocomposite/ITO bioelectrode recorded in phosphate buffer
saline (PBS, 50 mM, pH, 7.0, 0.9% NaCl) containing 5 mM [Fe
(CN)
6
]
3−/4−
and in the potential range, −0.3 to 0.6 V at 20 mV/s rate.
CH/ITO shows a well-defined redox behaviour (curve a) at 0.257 V
(Epc) and the anodic peak potential (Epa) at 0.052 V. This may be
due to cationic CH that accepts electrons from ferricyanide species
(that are negatively charged in PBS) resulting in enhanced redox
current. The magnitude of current response for CH-Fe
3
O
4

nanobio-
composite/ITO electrode (curve b) increases in comparison to that
of CH/ITO electrode. This may be due to the induced magnetiza-
tion of magnetic domains (Fe
3
O
4
nanoaparticles) on application of
electrical field. It appears that the electric field induces alignment
of magnetic nanoparticles in a particular direction and facilitates
electron flow resulting in increased value of current.
These results suggest that the presence of Fe
3
O
4
superparamag-
netic nanoparticles results in increased electroactive surface area
of CH and enhanced electron transfer. In the CH-Fe
3
O
4
nanobio-
composite, the electrocatalytic activity ofFe
3
O
4
superparamagnetic
A. Kaushik et al. / Sensors and Actuators B 138 (2009) 572–580 575
Fig. 2. Proposed mechanism for preparation of CH-Fe
3

O
4
nanobiocomposite and immobilization of Ur-GLDH onto CH-Fe
3
O
4
nanobiocomposite film.
Fig. 3. (A) FTIR spectra of CH/ITO electrode (a), CH-Fe
3
O
4
nanobiocomposite/ITO electrode (b) and Ur-GLDH/CH-Fe
3
O
4
nanobiocomposite/ITO bioelectrode. (B) Scanning
electron microscopy images of CH-Fe
3
O
4
nanobiocomposite/ITO electrode (a) Ur-GLDH/CH-Fe
3
O
4
nanobiocomposite/ITO electrode.
576 A. Kaushik et al. / Sensors and Actuators B 138 (2009) 572–580
Fig. 4. (A) Cyclic voltammograms of CH/ITO electrode (a), CH-Fe
3
O
4

nanobiocomposite/ITO electrode (b) and Ur-GLDH/CH-Fe
3
O
4
nanobiocomposite/ITO bioelectrode (c).
(B) Cyclic voltammograms of Ur-GLDH/CH-Fe
3
O
4
nanobiocomposite/ITO bioelectrode at different scan rate. (C) Electrochemical impedance spectroscopic studies of CH/ITO
electrode (a), CH-Fe
3
O
4
nanobiocomposite/ITO electrode (b) and Ur-GLDH/CH-Fe
3
O
4
nanobiocomposite/ITO bioelectrode (c). (D) Differential pulse voltammograms of CH/ITO
electrode (a), CH-Fe
3
O
4
nanobiocomposite/ITO electrode (b) and Ur-GLDH/CH-Fe
3
O
4
nanobiocomposite/ITO bioelectrode (c).
nanoparticles and the accumulation ability of CH due to amino
groups of the negatively charged ferricyanide ions have been found

to be responsible for enhanced electrochemical properties due
to increased surface concentration. The surface concentrations of
redox species onto CH/ITO, CH-Fe
3
O
4
nanobiocomposite/ITO elec-
trodes have been estimated using Eq. (2) [36].
i
p
= 0.227 nFA C
∗
0
k
0
exp

−˛n
a
F
RT
(E
p
− E

0
)

(2)
where, i

p
is the anodic peak current, n is the number of electrons
transferred (1), F is the Faraday constant (96,485.34 C mol
−1
), A is
surface area (0.25cm
2
), R is the gas constant (8.314 Jmol
−1
K
−1
),
C
∗
0
is surface concentration of the ionic species of film surface
(mol cm
−2
), E
p
is the peak potential and E

0
is the formal potential.
–␣n
a
F/RT and k
0
(rate constant) corresponding to the slope and
intercept of ln (i

p
) verses E
p
− E

0
curve at different scan rates. It
may be noted that surface concentration of redox species onto CH-
Fe
3
O
4
nanobiocomposite/ITO bioelectrode (2.6 × 10
−6
mol cm
−2
)is
higher than that of CH/ITO (1.25 × 10
−6
mol cm
−2
) revealing the
interaction between CH and Fe
3
O
4
nanoparticles that increase
the electroactive surface area of CH-Fe
3
O

4
nanocomposite. The
increased surface concentration of redox species onto CH-Fe
3
O
4
electrode reveals that larger number of redox moieties are avail-
able for oxidation leading to higher faradic current [37] wherein,
the presence of Fe
3
O
4
nanoparticles results in increased electron
transport between redox species and electrode. However, after the
immobilization of Ur-GLDH onto CH-Fe
3
O
4
nanobiocomposite/ITO
electrode, the magnitude of the response current decreases (curve
c), indicating strong binding of Ur-GLDH with CH-Fe
3
O
4
nanobio-
composite/ITO electrode that blocks transport of charge carriers.
This may be attributed to the insulating characteristics of enzymes
(Ur-GLDH) that may perturb the transfer of electrons between
medium and electrode.
Fig. 4B shows cyclic voltammograms of Ur-GLDH/CH-Fe

3
O
4
nanobiocomposite/ITO bioelectrode in PBS (50mM, pH 7.0, 0.9%
NaCl) containing 5 mM [Fe(CN)
6
]
3−/4−
as afunction of scan rate from
10 to 10 0 mV s
−1
. It can be seen that both cathodic (I
p
) and anodic
(I
c
) peak currents of the electrode increase linearly and are pro-
portional to the scan rate (inset, Fig. 4B) according to Eqs. (3) and
(4).
I
p
(A) = 0.77 ␮A(s/mV) × scan rate [mV/s] − 0.11 ␮(A)
with value of regression coefficient as 0.999 (3)
I
c
(A) = 0.6 ␮A(s/mV) × scan rate (mV/s) − 0.514 ␮(A)
with value of regression coefficient as 0.996 (4)
It has been shown that Ur-GLDH adsorbed onto CH-Fe
3
O

4
nanobio-
composite/ITO electrode undergoes reversible electron transfer
with nanobiocomposite film. It is found that both catiodic (E
p
) and
anodic (E
c
) peak potentials increase linearly and are proportional
A. Kaushik et al. / Sensors and Actuators B 138 (2009) 572–580 577
to the logarithm of scan rates and obey Eqs. (5) and (6):
E
p
(V) = 0.489 (s) × scan rate (mV/s) + 0.518 (V)
with value of regression coefficient as 0.996 (5)
E
c
(V) =−0.043 (s) × scan rate (mV/s) + 0.143 (V)
with value of regression coefficient as 0.993 (6)
The values of heterogeneous electron transfer rate constant (k
s
)
of Ur-GLDH immobilized CH-Fe
3
O
4
nanobiocomposite/ITO elec-
trode have been calculated using the Laviron model [38].
k
s

=
mnF
RT
(7)
where m is peak-to-peak separation, F is Faraday constant,  is
scan rate (mV/s), n is the number of transferred electrons and R
is gas constant. The value of k
s
obtained as 4.2 s
−1
(T =298K, n =1,
m =0.103 V and  = 100 mV) is higher than that of other nanopar-
ticles based bioelectrodes [39–42] indicating fast electron transfer
between immobilized Ur-GLDH and electrode due to the presence
of Fe
3
O
4
nanoparticles in the CH-Fe
3
O
4
nanobiocomposite.
Electrochemical impedance spectroscopy (EIS) technique mea-
sures impedance of the electrode surface as a function of frequency
due to variation in interfacial properties of the interface of the
electrode and solution (R
s
), including electrode impedance (W),
capacity of theelectric double layer (C

dl
), and surface electron trans-
fer resistance (R
CT
). The modification of electrode surface results
in change in the value of R
CT
. In the EIS, semicircle part corre-
sponds to electron-transfer limited process as its diameter is equal
to the electron transfer resistance (R
CT
) that controls electron trans-
fer kinetics of the redox probe at the electrode interface [15,26].
Fig. 4C shows the Nyquist diagrams of CH/ITO electrode, CH-Fe
3
O
4
nanobiocomposite/ITO electrode and Ur-GLDH/CH-Fe
3
O
4
nanobio-
composite/ITO bioelectrode in PBS solution [50 mM, pH, 7.0, 0.9%
NaCl) containing 5 mM [Fe(CN)
6
]
3−/4−
in the frequency range from
0.01to10
5

Hz.
It can be seen (Fig. 4C) that R
CT
value (1.31 K, curve a) obtained
from the semicircle of CH/ITO electrode, characteristic of a diffu-
sion limiting step of the electrochemical process, decreases for the
CH-Fe
3
O
4
nanobiocomposite/ITO electrode (R
CT
=1.11K curve c).
This result suggests that electron transfer in the CH-Fe
3
O
4
nanobio-
composite film is easier between solution and the electrode i.e.
Fe
3
O
4
nanoparticles not only provide the hydrophilic surface, but
also act as a nanoscale electrode and promote electron transfer
due to permeable structure of CH/ITO. However, after the immo-
bilization of Ur-GLDH onto and CH-Fe
3
O
4

nanobiocomposite/ITO,
R
CT
is found to increase to 1.47 . This suggests that immobilized
Ur-GLDH molecules strongly bind with hybrid nanobiocomposite
and block charge carriers in the nanobiocomposite matrix. These
results clearly indicate the immobilization of Ur-GLDH onto CH-
Fe
3
O
4
nanobiocomposite/ITO electrode.
Fig. 4D shows differential pulsevoltammograms (DPV) of CH/ITO
electrode (curve a), CH-Fe
3
O
4
nanobiocomposite/ITO electrode
(curve b) and Ur-GLDH/CH-Fe
3
O
4
nanobiocomposite/ITO bioelec-
trode (curve c) in PBS solution {50 mM PBS (pH 7, 0.9% NaCl)
containing 5 mM [Fe(CN)
6
]
3−/4−
}. CH/ITO electrode (curve a) shows
well-defined DPV characteristics suggesting that CH promotes elec-

tron transfer due to cationic characteristics and accepts electrons
from the medium and transfer these to the electrode. Moreover,
magnitude of the current peak further increases for the CH-Fe
3
O
4
nanobiocomposite/ITO electrode (curve b) suggesting that Fe
3
O
4
nanoparticles promote electron transfer due to uniform disper-
sion throughout the CH network on the electrode [26]. Moreover,
Fe
3
O
4
nanoparticles provide favourable environment for the immo-
bilization of Ur-GLDH onto electrode resulting in enhanced electron
transfer phenomena. The magnitude of current response is found
to decrease for Ur-GLDH/CH-Fe
3
O
4
nanobiocomposite/ITO bioelec-
trode (curve c) due to insulating characteristics of Ur-GLDH that
hinders transfer of electrons at bioelectrode.
The effect of pH on Ur-GLDH/CH-Fe
3
O
4

nanobiocomposite/ITO
bioelectrode has been carried out using DPV. It is observed that
magnitude of the current is maximum at pH 7 (inset Fig. 4D).
This value of pH is optimum for catalytic activity of Ur for the
decomposition of urea [35]. This suggests that Ur-GLDH/CH-Fe
3
O
4
nanobiocomposite/ITO bioelectrode shows maximum activity at pH
7 at which Ur retains its natural structure and is responsible for low
detection limit and high sensitivity for urea detection. The results of
electrochemical experiments repeated at least 20 times have been
found to be reproducible.
3.4. Electrochemical response studies of Ur-GLDH/CH-Fe
3
O
4
nanobiocomposite/ITO bioelectrode
Electrochemical response studies of Ur-GLDH/CH-Fe
3
O
4
nanobiocomposite/ITO bioelectrode have been carried out as a
function of urea concentration in the presence of 30 ␮L of nicoti-
namide adenine dinucleotide (NADH, 3.7 mg/dL) and 70 ␮Lof
␣-Keto glutamate (␣-KG, 47.5 mg/dL) using DPV in PBS solution
{50 mM PBS (pH 7, 0.9% NaCl) containing 5 mM [Fe(CN)
6
]
3−/4−

}.Itis
observed that magnitude of current obtained for the Ur-GLDH/CH-
Fe
3
O
4
nanobiocomposite/ITO bioelectrode increases on addition
of urea (Fig. 5A). The response time of the Ur-GLDH/CH-Fe
3
O
4
nanobiocomposite/ITO bioelectrode found to be about 10 s is
attributed to faster electron communication feature of CH-Fe
2
O
3
nanobiocomposite. It is revealed that Ur-GLDH/CH-Fe
3
O
4
nanobio-
composite/ITO bioelectrode (inset Fig. 5A) can be used to estimate
urea from 5 to 100 mg/dL. The sensitivity of the Ur-GLDH/CH-Fe
3
O
4
nanobiocomposite/ITO bioelectrode calculated from the slope of
curve has been found to be 12.5 ␮A/(mM cm
−2
).

It has been shown that good linearity is found in the low
concentration range (5–40 mg dL
−1
) and the current varies as I
(A)=186␮A +293 ␮A (mg/dL) × urea concentration (mg/dL) with
the value of correlation coefficient as 0.996 (inset b, Fig. 5A)
and standard deviation of 0.46 ␮A/mg/dL. When urea concentra-
tion is larger than 40 mg/dL, the response current starts to level
off. The observed sluggish increase in the current for solutions
at higher concentration is likely to be due to restriction of the
enzymatic reaction. At higher urea concentration, the original first-
order enzymatic reaction appears to have changed to 0th order
reaction at which the reaction rate becomes independent of sub-
strate concentration [5]. Interestingly, in the concentration range
(60–100 mgdL
−1
), the variation of current follows I (A) = 2.82 ␮A
+0.9 ␮A (dL/mg) × urea concentration (mg/dL) with correlation
coefficient of 0.999 and standard deviation of 0.46 ␮A/mg/dL. The
detection limit of Ur-GLDH/CH-Fe
3
O
4
nanobiocomposite/ITO bio-
electrode is estimated to be about 5 mg/dL. The reproducibility of
response of the bioelectrode has been investigated at 10 mg/dL urea
concentration. No significant decrease in current is observed after
using at least 6 times (data not shown). This bioelectrode achieves
95% of steady state current in less than 10 s indicating fast electron
exchange between Urs-GLDH and CH-Fe

3
O
4
nanobiocomposite/ITO
electrode.
The proposed biochemical reaction during the urea detection is
shown in Fig. 5B. Ur catalyzes hydrolysis of urea to carbamine acid
that gets hydrolyzed to ammonia (NH
3
) and carbon dioxide (CO
2
).
GLDH catalyzes the reversible reaction between ␣-KG and NH
3
to
NAD
+
and linked oxidative deamination of l-glutamate in two steps.
The first step involves a Schiff base intermediate being formed
between NH
3
and ␣-KG. The second step involves the Schiff base
intermediate being protonated due to the transfer ofthe hydride ion
from NADH resulting in l-glutamate. NAD
+
is utilized in the forward
578 A. Kaushik et al. / Sensors and Actuators B 138 (2009) 572–580
Fig. 5. (A) Electrochemical response of Ur-GLDH/CH-Fe
3
O

4
nanobiocomposite/ITO bioelectrode as a function of urea concentration (5–100 mg/dL). (B) Biochemical reaction
during electrctrochemical detection of urea using Ur-GLDH/CH-Fe
3
O
4
nanobiocomposite/ITO bioelectrode. (C) The effect of interferents on electrctrochemical response of
Ur-GLDH/CH-Fe
3
O
4
nanobiocomposite/ITO bioelectrode. (D) Shelf-life curve for Ur-GLDH/CH-Fe
3
O
4
nanobiocomposite/ITO bioelectrode as a function of time.
reaction of ␣-KG and free NH
3
that are converted to l-glutamate
via hydride transfer from NADH to glutamate. NAD
+
is utilized in
the reverse reaction, involving l-glutamate being converted to ␣-
KG and free (NH
3
) via oxidative deamination reaction. The electrons
generated from the biochemical reactions are transferred to the CH-
Fe
3
O

4
/ITO electrode through the Fe(III)/Fe(IV) couples that help in
amplifying the electrochemical signal resulting in increased sensi-
tivity of the sensor.
The value of the apparent Michaelis–Menten constant (K
m
) has
been calculated to show suitability of the enzyme in the hybrid
nanobiocomposite matrix to urea. Using Lineweaver–Burke plot
(1/I versus 1/[C]), K
m
value has been found to be 0.56 mM for the
immobilized Ur-GLDH indicating maximal catalytic activity of the
enzyme at low substrate concentration.
The selectivity of Ur-GLDH/CH-Fe
3
O
4
nanobiocomposite/ITO
bioelectrode has been determined by measuring its electrochemical
response by adding normal concentration of different interferents
(Fig. 5C) such as cholesterol (5 mM), ascorbic acid (0.05 mM), uric
acid (0.1 mM) and glucose (100 mg/dL) along with urea (1 mM) in
phosphate buffer (50 mM, pH 7, 0.9% NaCl). It can be seen that
value of the electrochemical response current remains nearly same
except for lactic acid wherein there is a decrease of about 6%.
The shelf-life of Ur-GLDH/CH-Fe
3
O
4

nanobiocomposite/ITO bio-
electrode has been monitored by measuring electrochemical
current response with respect to time, with regular interval of 1
week. It is observed that this bioelectrode retains about 85% of
enzyme (Ur-GLDH) activity even after about 8 weeks when stored
in refrigerated conditions (4
◦
C) after which the current response
decreases to 75% in about 10 weeks (Fig. 5D).
4. Conclusions
Nanobiocomposite of CH and superparamagnetic Fe
3
O
4
, (mag-
netisation: 60.8 emu/g) nanoparticles have been prepared and
used for urea detection via immobilization of Urs and GLDH.
Ur-GLDH/CH-Fe
3
O
4
nanobiocomposite/ITO based urea biosensor
shows linearity of 5–100 mg/dL, lower detection limit of 2 mg/dL,
response time of 10 s and sensitivity of 12.5 ␮A/mM cm
−2
. A rel-
atively low value of the Michaelis–Menten constant obtained as
0.56 mM indicates enhanced enzyme affinity of Ur to urea. The
wide range of detection and high sensitivity may be assigned to
amplification of the magnitude of current due to the alignment

of Fe
3
O
4
nanoparticles in CH-Fe
3
O
4
nanobiocomposite matrix.
This biosensor shows negligible influence of interferents and can
be useful to detect urea in samples containing other analytes.
This interesting CH-superparamagnetic Fe
3
O
4
nanobiocompos-
ite platform should be utilized for estimation of urea in serum
samples and to develop other biosensors. Besides this, efforts
should be made to utilize these materials for in vivo sens-
ing.
A. Kaushik et al. / Sensors and Actuators B 138 (2009) 572–580 579
Acknowledgements
We thank Dr. Vikram Kumar, Director, National Physical Labo-
ratory, New Delhi, India for providing facilities. We are thankful to
Dr. R. K. Kotnala for VSM measurement. PRS and A K are thank-
ful to Council of Scientific Industrial Research (CSIR), India for
the award of Senior Research Associate ship and Senior Research
Fellowship. Financial support received under the Department of
Science and Technology(DST) projects [DST/TSG/ME/2008/18 and
GAP- 070932], in-house project (OLP-070632D) and the DBTproject

(GAP-070832) is gratefully acknowledged.
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Biographies
Mr. Ajeet Kaushik received his M.Sc. degree in chemistry (Organic) from the
Meerut University, Utter Pradesh 2002. He is working as a Senior Research Fel-
low in the Bimolecular Electronics and Conducting Polymer Research Group at the
National Physical Laboratory, New Delhi, India in the area of development of biosen-
sors based on nanomaterials, bionanocomposites and conducting polymers for
healthcare.
Dr. Pratima R. Solanki obtained her M.Sc. and Ph.D degrees from Maharishi
Dayanand University, Rohtak (Haryana) during 1995 and 2000 in Bioscience. She
is working as a Senior Research Associate (CSIR) with the Biomolecular Electronics
and Conducting Polymer Research Group at the National Physical Laboratory, New
Delhi, India. She is actively engaged in the technical development of biosensors for
healthcare.
Dr. Anees A. Ahmed received his M.Sc. in 1998 from Rohel Khand University,
Breily and Ph.D degree from the Jamia Milia University, Delhi (2004) in chemistry
(inorganic). He is working as Senior Research Associate (CSIR) in the Bimolecular
Electronics and Conducting Polymer Research Group at the National Physical Lab-
oratory, New Delhi, India in the area of development of biosensors based on metal
oxide nanoparticles as well as nanocomposites for healthcare.
Dr. G. Sumana received her Ph.D. (1998) from Jiwji University in chemistry. She
is currently working as scientist in biomolecular electronics and conducting poly-
mer research group, National Physical Laboratory, New Delhi. She has a research
experience of 10 years in controlled drug delivery, liquid crystal polymers, polymer
dispersed liquid crystals and biosensors.
Prof. Sharif Ahmad received his M.Sc. degree in chemistry (Organic) from the
Aligarh Muslim, Utter Pradesh 1981. He is working as a Senior Professor in the
Department of Chemistry at Jamia Millia Islamia, New Delhi, India. He has research

experience of about 30 years in the area of corrosion materials based on nanomate-
rials, nanocomposites and conducting polymers has guided 15 Ph.D. students.
580 A. Kaushik et al. / Sensors and Actuators B 138 (2009) 572–580
Dr. B. D. Malhotra received his Ph.D degree in physics from University of Delhi, Delhi,
India in 1980. He has published 159 papers, has filed 9 patents, has edited/co-edited
books on biosensors and polymer electronics, and is currently the Scientist G and
Head of the Biomolecular Electronics & Conducting Polymer Research Group at the
National Physical Laboratory, India. He has research experience of about 25 years in
the field of biomolecular electronics and has guide d 14 Ph.D students till date. His
current activities including biosensors, conducting polymers, Langmuir–Blodgett
films, self-assembled monolayers and nanomaterials, etc.