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Recent developments in urea biosensors

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 Biochemical Engineering Journal 44 (2009) 42–52

Contents lists available at ScienceDirect

Biochemical Engineering Journal

journal homepage: www.elsevier.com/locate/bej

Review

Recent developments in urea biosensors

Gunjan Dhawan, Gajjala Sumana ∗ , B.D. Malhotra
Biomolecular Electronics & Conducting Polymer Research Group, National Physical Laboratory, Dr. K. S. Krishnan Marg, New Delhi 110012, India

a r t i c l e i n f o a b s t r a c t

Article history: This paper reviews recent developments in urea biosensors, as reported in the literature. The advantages
Received 10 March 2008 and roles of various matrices, different strategies for biosensor construction, analytical performance and
Received in revised form 16 May 2008 applications are discussed. The prospects of urea biosensors for medical applications are also discussed.
Accepted 1 July 2008
© 2008 Elsevier B.V. All rights reserved.

Keywords:
Urea
Urease
Matrix
Biosensor

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 42
2. Historical background . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 43
3. Urea biosensors based on polymeric matrices . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 44
3.1. Urea biosensors based on non-conducting polymer matrices . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 44
3.2. Urea biosensors based on conducting polymer matrices . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 45
3.3. Sol–gel-based urea biosensors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 46
4. Urea biosensors based on Langmuir–Blodgett films . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 47
5. Urea biosensors based on nanomaterials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 47
5.1. Nanoparticles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 47
5.2. Quantum dots (QDs) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 48
5.3. Nanofibers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 48
5.4. Nanoporous silicon technology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 48
6. Miscellaneous matrices for urea biosensing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 48
7. ISFET-based urea biosensors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 49
8. Demands, challenges and commercialization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 49
9. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 50
Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 50
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 50

levels. The normal level of urea in serum is from 15 to 40 mg/dl

1. Introduction
(2.5–7.5 mM/l). In patients suffering from renal insufficiency, urea
concentrations in serum vary from 180 to 480 mg/dl and, at ele-
Urea is widely distributed in nature and its analysis is of con-
vated levels above 180 mg/dl, hemodialysis is required. Apart from
siderable interest in clinical and agricultural chemistry [1,2]. It
clinical applications, there is a growing demand for robust, reli-
is known to be an important marker for evaluating uremic toxin
able instrumentation toward the estimation of urea in other fields
(e.g., food science and environmental monitoring). As the principal
component of non-protein nitrogen in cow milk, urea is utilized
∗ Corresponding author. Tel.: +91 011 25734273. as an indicator of protein-feeding efficiency [3]. Improved feeding
E-mail address: sumanagajjala@gmail.com (G. Sumana). practices through appropriate urea testing over a period of several

1369-703X/$ – see front matter © 2008 Elsevier B.V. All rights reserved.
doi:10.1016/j.bej.2008.07.004
 G. Dhawan et al. / Biochemical Engineering Journal 44 (2009) 42–52
months can result in lower feed costs, greater milk-protein yield,
increased reproductive performance, and minimal nitrogen excre-
tion into the environment [4,5]. Urea also plays a strategic role in
the marine nitrogen cycle [6,7], including sources of excretion by
invertebrates and fish, and bacterial decomposition of nitrogenous
material and terrestrial drainage. Urea estimation is important dur-
ing environmental monitoring. Annual world production of urea
exceeds 100 million metric tonnes, the majority of which is used
as fertilizer. Excessive nitrogen fertilizer applications can lead to
pest problems by increasing the birth rate, longevity and overall
fitness of certain pests. Urea can be responsible for reduction in
soil pH. Many methods are available for urea estimation, including
gas chromatography [8], calorimetry [9], and fluorimetry [10]. How-
ever, these methods suffer from complicated sample pretreatment
steps and are unsuitable for on-site monitoring. It is envisaged that
biosensors can be utilized to overcome some of these problems.
Biosensors are analytical devices that incorporate biological
materials such as enzymes, nucleic acids (DNA and RNA), micro-
organisms, whole cells, antibodies, cell receptors or biologically
derived materials, which in most cases are specific to an analyte in
intimate contact with a physico-chemical transducer. Transducers
are components that convert a biochemical signal into a measur-
able signal. The transducing microsystem may be electrochemical,
thermometric, optical, piezoelectric or magnetic. Owing to their
simplicity, high sensitivity and potential ability for real-time and
on-site analysis, biosensors have been widely applied in various
fields, including industrial processes, clinical detection and envi-
ronmental control [11,12]. The immobilization technique and the
support to impart stability, physical and chemical conditions (e.g.,
pH, temperature, contaminants and thickness) play a major role
in the performance of a biosensor. These interesting biodevices
have been predicted to play a critical role towards the implanta-
tion of a chip into a body to monitor all biological parameters of
clinical importance, as well as for point-of-care devices [13]. There-
fore, biosensors are presently the subject of extensive research
for applications like clinical diagnosis, food technology, military,
industrial, biomedical and environmental monitoring using various
biomolecules (e.g., enzymes, antibodies, nucleic acids, receptors,
bio-mimetic synthetic or semisynthetic molecules). The schematic
of a biosensor is shown in Fig. 1.
Fig. 1. Schematic of biosensor.
43
Enzymatic biosensors utilize the biospecificity of an enzymatic
reaction. In the fabrication of a urea biosensor, urease is most
commonly used as the biosensing element. Enzymatic urea biosen-
sors utilize biochemical reactions. In other words, analyte (urea)
and enzyme (urease) result in a product (ammonium ion) that
can be detected and quantified using a transducer (amperomet-
ric/potentiometric/optical thermal/piezoelectric). The enzymatic
reaction of urea with urease is shown in the following equation:
Urease
NH2 CONH2 + 3H2 O −→ 2NH4 + + HCO−3 + OH− (1)
Many matrices (e.g., polymers, sol–gels, conducting poly-
mers, Langmuir–Blodgett films, nanomaterials and self-assembled
monolayers (SAMs)) have been used to (i) provide support; (ii)
impart stability to biomolecules towards variations in temperature,
pH and ionic strength; (iii) increase shelf-life; and (iv) reduce cost
for the fabrication of a urea biosensor. For optimum biostability
and reaction efficiency, the preferred host matrix appears to be the
one that isolates the desired biomolecule, protecting it from self-
aggregation and microbial attack, while providing essentially the
same local aqueous microenvironment as in biological media. In
recent years, nanomaterials such as carbon nanotubes (CNTs), metal
nanoparticles and semiconducting nanoparticles have emerged
as new classes of nanomaterials that are receiving considerable
interest owing to their unique structure, high chemical stability
and high surface-to-volume ratio. These properties make them
extremely attractive for fabricating chemical sensors [14,15] (Lim
et al., 2005; Wang et al., 2003). Recently, composite materials
based on conducting polymers, redox mediators, metal nanoparti-
cles, nanocomposites and nanoclusters have been used to combine
properties of the individual components with a synergistic perfor-
mance in biosensor fabrication. However, efforts are being made to
fabricate a cost-effective urea biosensor with improved sensitivity
and stability. The present article is an update on recent develop-
ments concerning urea biosensors as reported in the literature.
2. Historical background
In 1962, Professor Leland C. Clark Jr., father of the biosensor con-
cept, published a paper illustrating an experiment in which glucose
oxidase was entrapped at a Clark oxygen electrode using a dialy-
sis membrane, resulting in a glucose biosensor. His development
became the basis of numerous variations on this basic design, as
well as many affinity-based biosensors (e.g., immunosensors and
nucleic acid biosensors), ultimately aiming to achieve lab-on-a-
chip microarrays [16]. Fig. 2 shows the schematic of a typical urea
biosensor.
Guilbault and Montalvo were the first to fabricate a potentio-
metric urease enzyme electrode for the estimation of urea through
Fig. 2. Schematic of a typical urea biosensor.
44 G. Dhawan et al. / Biochemical Engineering Journal 44 (2009) 42–52
Fig. 3. Immobilization techniques used for the development of urea biosensors.
its enzyme-catalyzed hydrolysis [17,18]. The analytical signal of this
early biosensor was the potential of a cation-selective glass elec-
trode for detection of ammonium ion activity by urease-catalyzed
hydrolysis. The urea biosensor was found to be stable for 3 weeks at
25 ◦ C. Efforts were made to improve the applicability of a urea elec-
trode prepared in pioneering work by the same group in the late
sixties [19–23]. Many different versions of this biosensor have been
described. The originally used glass electrode-based sensing ele-
ment was soon replaced with a more selective, neutral carrier-type
ion-selective ammonium electrode [24,25], with Severinghaus-
type ammonium [26] or carbon dioxide [22] gas measuring cells.
Miniaturized urea electrodes, several of them using ISFET transduc-
ers [27,28], have been constructed. Improvements on the enzyme
layer have been achieved [29], resulting in higher enzyme stability
and lower limits of urea detection. The following sections contain
details on various urea biosensors reported to date.
3. Urea biosensors based on polymeric matrices
3.1. Urea biosensors based on non-conducting polymer matrices
Polymers are finding wide usage in the field of electronic mea-
suring devices, especially sensors owing to their ability to have
their chemical and physical properties tailored over a wide range
of characteristics [30]. The suitably chosen polymeric matrices
are found to be biocompatible, flexible, and cost-effective. Besides
this, they can be obtained in the form of free-standing films for
the fabrication of biosensors. Extensive efforts have been made
to fabricate urea biosensors using different polymer matrices,
such as phosphine oxide polyether [31], polygalacturonate [32],
polyurethane-acrylate [33], acrylonitrile [34], hydroxyapatite [35],
alginate [36], bovine serum albumin [37], poly(vinyl ferrocenium)
[38,39], polyethylenimine [40], synthetic latex polymer, activated
polyvinyl alcohol (PVA) [41], polyvinyl chloride (PVC) [42–44],
and chitosan [45]. These materials provide mechanical strength
and long-term stability as supports for the immobilization of the
enzymes. There are a variety of methods by which enzymes can be
immobilized, ranging from covalent chemical bonding to physical
entrapment. The different techniques for enzyme immobilization
are depicted in Fig. 3.
A potentiometric enzymatic biosensor, with a detection
range for urea between 8.9 × 10−5 and 1.1 × 10−3 M, consisting
of urease immobilized on PVA activated with 2-flouro-1-
methylpyridiniumtoluene-4-sulfonate (FMP) was developed by
Sehitogullari and Uslan [41]. This sensor has a detection limit
of only 1 mM and does not cover the physiological range of
urea. Lakard et al. used polyethylenimine films coated onto glass-
sealed, metal microelectrodes for the potentiometric detection
of urea. They state the advantages of chemical immobilization
methods over physical adsorption and entrapment as providing
mechanical strength, long-term stability and overcoming diffu-
sional limitations encountered by substrates and enzymes. Among
the various immobilization techniques used for the fabrication of
urease biosensing electrodes, glutaraldehyde immobilized urease
biosensors have been known to provide many advantages. These
include a short response time (15–30 s), sigmoidal response for urea
concentrations in the working range from 1 × 10−2.5 to 1 × 10−1.5 M
and a lifetime of about 4 weeks. Also included are benefits such as
low diffusional resistance, as well as strong binding forces between
enzyme and matrix, thus reducing the loss of enzymes and increas-
ing stability.
The anion exchange method has been utilized for urease
immobilization onto electroprecipitated poly(vinyl ferrocium) per-
chlorate electrodes to detect urea as low as 1 ␮M in the linear range
from 1 to 250 ␮M with a shelf-life of 29 days. However, these matri-
ces suffer from toxicity, owing to polymer degradation products
[38].
Natural polymers (e.g., polysaccharides like chitin and cellulose)
are biocompatible and have become the materials of choice for
recent technological advances in biosensor fabrication. Chitin and
 G. Dhawan et al. / Biochemical Engineering Journal 44 (2009) 42–52
a deacetylated product, chitosan, have been used for the immo-
bilization of enzymes for biosensors and industrial applications
[47], as they are non-toxic and biocompatible [46]. Enzymatic sen-
sors with chitin and chitosan supports have been reported for the
determination of glucose, lactate, ethanol and urea. Most research
papers reported in the literature toward urease immobilization on
chitosan are dominated by the use of beads. The chitosan-based
potentiometric urea biosensor is founded on the use of solid-state
ammonium electrodes, which utilize four different immobiliza-
tion procedures. These include: (A) adsorption; (B) adsorption
followed by reticulation with dilute aqueous glutaraldehyde solu-
tions; (C) activation with glutaraldehyde followed by contact with
the enzyme solution; and (D) activation with glutaraldehyde, con-
tact with the enzyme solution and reduction of the Schiff base
with sodium borohydride to obtain membranes with high enzyme
loadings with adequate permeability towards ammonium ion and
without significant enzyme leaching [45]. It has been reported that
the biosensor fabricated using method (B) exhibits improved per-
formance with response characteristics including: (i) linearity in
the range 10−4 to 10−2 M; (ii) a slope of up to 56 mV per decade;
(iii) response time between 30 s and 2 min; and (iv) a lifetime of 2
months. However, more work must be done in this field to obtain
a commercial urea biosensor based on natural polymers.
PVC-based ammonium ion-selective electrodes have been
extensively used for the fabrication of potentiometric urea biosen-
sors. These conventional potentiometric urea biosensors, pH
electrodes [48–50] and NH4 + -selective electrodes [51–53] detect
changes in hydrogen ions or ammonium ions, respectively, pro-
duced in an enzymatic reaction. However, they suffer with the
associated problem of strong dependence on sample buffer capac-
ity, which is known to be suppressed by the buffer used, leading
to a narrow dynamic range and loss in sensor sensitivity [48].
PVC membrane electrode-based biosensors [54–57], with suitable
ionophores, offer many advantages that include short response
time, good reproducibility, simple design and longer lifetime, up
to 12 months [58,59].
Functionalized PVCs have been investigated for use as electrode
membrane materials to facilitate efficient enzyme immobiliza-
tion [50,60]. Also, suitable ionophores have been utilized with
PVC ion electrodes to enhance response characteristics, such as
response time, reproducibility, simple design and longer lifetime.
The response characteristics [54,60,61] and potential use of ami-
nated PVC membranes for urea biosensor fabrication has also been
discussed. An ionophore-free PVC–NH2 pH membrane electrode
covalently immobilized with enzyme was applied by Walcerz et al.
for urea estimation by measuring hydronium ions after enzymatic
hydrolysis [62]. Later, in 2002, Tinkilic et al. fabricated a minia-
turized urea sensor by immobilizing urease directly onto an all
solid-state, contact PVC–NH2 membrane, ammonium ion-selective
electrode [44] with improved sensitivity, longer lifetime and low
cost. The biosensors showed a linear range between 5 × 10−2
and 5 × 10−4 M urea in unbuffered solution. However, it has been
reported that sensitivity is highly dependent on the H+ , Na+ and K+
concentrations in the sample solution, as severe interference from
Na+ and K+ ions is observed. Efforts were made to reduce the effect
of interferants to achieve improved performance over carboxylated
PVC-based biosensors. This was in terms of sensitivity, dynamic
stability over 2 months and a response time of 1–2 min. Also,
this concerned the estimation of serum urea concentration using
urease-biosensing electrodes that were based on PVC-membrane
ammonium-selective electrodes containing palmitic acid and non-
actin [43].
An optical biosensor [63] based on an ion-selective optode mem-
brane, which contained nonactin as the ion-selective ionophore
and ETH 5294 chromoionophore in a thin (1 mm) plasticized
45
poly(vinylchloride) film, has been developed. This urea biosen-
sor has been used for flow injection urea measurements in
the 0.01–2 mM concentration range. These sensors suffer from
the influence of interferants such as ammonium, potassium and
sodium ions. In this context, several strategies have been used
to overcome the effect of interferences. For instance, differential
measurements including separation by gas diffusion and electronic
tongues have been used [60,61,64]. Ion-selective-electrode arrays
and multivariate calibration models have proved to be effective for
increasing selectivity, enabling the simultaneous determination of
several species in different types of samples, ranging from biologi-
cal fluids [65] to natural waters [66].
Despite these interesting developments, the development of
simple assay methods is of importance in clinical studies. The
miniaturization of biosensors is particularly important to reduce
the amount of enzyme and reagents needed. More efforts must be
made to minimize the influence of interferants.
3.2. Urea biosensors based on conducting polymer matrices
Conducting polymers have become the materials of choice for
recent technological advances in biotechnology and have been
extensively reviewed by various researchers [67–70]. These matri-
ces are used as supports for biomolecules, resulting in biosensors
that have enhanced speed, sensitivity and versatility in diag-
nostics to measure desired analytes. These devices are finding
ever-increasing use in clinical diagnostics. Conducting polymers are
conjugated polymers that can be synthesized by chemical methods
as well as electrochemical methods, providing easy modulation of
various properties (e.g., film thickness, conductivity, functionaliza-
tion, use of various supporting electrolytes and the ability of serving
as an electrochemical transducer itself). Additional merits are that
enzyme molecules can be entrapped during electro-polymerization
in one step and also that the polymer film uniformly covers the
surface of substrate electrodes of any shape or size [71,72].
Various conducting polymers, like polyaniline (PANi), polypyr-
role and polythiophene, have been used for the fabrication of
biosensors. Among them, polypyrrole is one of the most extensively
used conducting polymers in the fabrication of urea biosensors
[73–75]. The versatility of this polymer is determined by (i) its
biocompatibility, (ii) capability to transduce energy arising from
the interaction of analytes and analyte recognizing sites into
electrical signals that are easily monitored, (iii) capability to pro-
tect electrodes from interfering material, and (iv) easy ways for
electro-chemical deposition on the surface of any type of electrode.
Among other conducting polymers, polyaniline is often used as the
immobilizing substrate for biomolecules and as an efficient electro-
catalyst. However, the necessity to detect bioanalytes in neutral pH
ranges leads to electro-inactivity of the deposited film, discouraging
the use of polyaniline and polythiophene as biosensing materials
for the detection of urea.
Adeloju et al. have fabricated amperometric, flow injection, urea
biosensors using electrochemically entrapped urease in polypyr-
role [76–78]. The influence of flow rate and various other analytical
parameters (e.g., applied potential and polymerization time) have
been studied. Under optimized conditions, the amperometric
response was linear between 3 and 15 mg/l of urea. The sensi-
tivity and detection range were further improved by increasing
the enzyme loading and utilizing pulsed amperometric detec-
tion [79]. Using pulsed amperometric flow injection sensors based
on polypyrrole, detection limits of 60 mg/l and a linearity in the
100–450 mg/l range has been observed. However, a shelf-life of only
2 weeks could be achieved using this technique. This sensor suf-
fers from a limitation of quantitative urea estimation in biological
samples, as it needs pretreatment with anion exchange separators
46 G. Dhawan et al. / Biochemical Engineering Journal 44 (2009) 42–52
for the removal of interferants and needs further improvement to
minimize the interferant effect [79].
Efforts have been made to improve the sensitivity of the
polypyrrole-based urea sensor by using composite films with
polyion complexes. Komaba et al. have reported a potentiometric
urea sensor based on nucleophilic electrolytes and urease, indi-
cating improved urea response with a slope of 31.8 mV per decade
[74]. However, it has been found that only a small number of urease
molecules could be immobilized using the electro-polymerization
process. To overcome this problem, the polyanion complexes,
polyacrylic acids, have been used to enhance the degree of immobi-
lization and the response was found to increase to 53 mV per decade
[80]. It has been demonstrated that the sensitivity can be further
increased by using polyion complexes (i.e., polycation polystyrene
sulphonate and polyanion polyacrylic acid) and, using a polyion
complex, the sensitivity was found to be enhanced to 110 mV per
decade. This high sensitivity has been attributed to effective immo-
bilization of the large amount of urease by electro-polymerization
of the electrode with a precoated polyion complex [81]. However,
the experiments on shelf-life and sensor reproducibility have not
been reported.
To minimize enzyme desorption from the desired immo-
bilization material and to obtain increased enzyme electrode
stability, various approaches and immobilization methods have
been used. A urea biosensor achieving the electrochemical entrap-
ment of urease in a polypyrrole matrix, including a stability of
around 2 months, has been reported [78]. This biosensor, how-
ever, cannot be reused. A functionalized copolymer of pyrrole and
N-3-aminopropylpyrrole [75] has been used for the fabrication
of thin film urea biosensors. In this system, urease was cova-
lently immobilized onto the copolymer matrix. The sensor acted
via covalent binding with free –NH2 groups of the polymer. This
sensor exhibited improved characteristics, such as 2-month sta-
bility at 4–6 ◦ C and urea detection in the range of 6.3 × 10−6 to
4.07 × 10−4 M, with electrode sensitivity at 27.5 mV/dl. Sensor sta-
bility of this system has been attributed to a porous morphology,
owing to the large, inserted PTS dopant. This results in high enzyme
loading and covalent binding with copolymers having free –NH2
groups.
A polypyrrole-based urea biosensor has been reported that pro-
vides advantages via eliminating the need for an optical indicator,
dye and pH sensitive reagent. Polypyrrole itself acts as a pH sensitive
indicator in the near-IR range, with the change in urea concentra-
tion at the linear range of 0.06–1 M urea [73].
Polyaniline, among other conducting polymers, is known to
have a broad range of tunable properties, desirable electrochem-
ical activity, chemical stability, structural flexibility and, above all,
two redox couples in a convenient potential range. A pH urea sen-
sor has been reported in both aqueous and non-aqueous media via
electro-polymerization of polyaniline in dry 0.5 M acetonitrile. This
sensor retains its reproducibility for more than 9 months. However,
the sensor has not yet been used for clinical samples [82].
A copolymer of polyaniline-poly(n-butyl-methacrylate) (Pn-
PBMA) homogeneous composite films has been obtained using
poly(vinyl methyl ether) (PVME) and poly(vinyl ethyl ether)
(PVEE) as dispersants. This copolymer has been character-
ized for its morphological, electrical and mechanical properties
[83]. These composites have been used for the detection of
H2 O2 , NH3 and toward the fabrication of urea and uric acid
biosensors. Polyaniline–Nafion composites have been used to
develop an amperometric urea biosensor that is based on
polyaniline–perfluorosulfonated ionomer composite electrodes.
This system uses a flow injection analysis system, has been fab-
ricated and is reported to have a detection limit of 0.5 ␮M as well
as a response time of 40 s [84].
Polyaniline–Nafion-based composite films have been immobi-
lized with urease via electrochemical and casting methods for
the fabrication of amperometric urea biosensors [75]. Urease
immobilization on these composite films has been achieved by
electrochemical immobilization and the casting method. It was
found that the sensitivity of composite electrodes immobilized
with the casting method is greater than that of the electrochemical
immobilization method. The sensitivity and detecting limit of this
urea sensor were found to be 0.7 ␮A (mg dl−1 )−1 cm2 and 6 mg dl−1 ,
respectively, when urease was immobilized by a glutaraldehyde
(GA) cross-linker; and 5.27 (mg dl−1 )−1 cm2 and 0.3 mg/dl, when
immobilized by a Nafion network [85]. However, no studies have
been carried out for the stability of urease and the estimation of
urea in clinical samples.
For practical applications of urea biosensors, problems like
reproducibility, long-term stability and clinical sample measure-
ments (e.g., in urine and serum) remain the major thrust. Although,
conducting polymer-based urea sensors have a lot of advantages
(e.g., stability, tunable properties, flexibility and biocompatibility)
they cannot work properly in high ionic strength samples. Fur-
thermore, the response of these sensors is highly dependent on
the buffer capacity of the sample, resulting in a narrow dynamic
range and low sensitivity. It may be noted that the use of additional
perm-selective membranes on top of the enzymatic membrane
could substantially reduce the dependence of sensor response on
buffer concentration and significantly extend its dynamic range.
For example, the use of Nafion and polyvinylene phenylene mem-
brane on top of enzymatic layers has been reported to substantially
improve the dynamic range of urea biosensors and reduce the
dependence of buffer concentrations [86].
Current developments in conducting polymer-based biosensors
show that electrochemical affinity sensors, based on molecularly
imprinted conducting polymers, have a great potential for direct
electrochemical sensing. This is because they enable deliberate con-
trol of the molecular structure at the electrode surface, resulting
in the additional advantage of higher affinity to the target ana-
lyte. Therefore, nanostructured conducting polymers and tailored,
molecularly imprinted conducting polymers are likely to be the
best choice to obtain enhanced stability and reproducibility in urea
biosensors.
3.3. Sol–gel-based urea biosensors
Sol–gel matrices have been known for their rigidness, chemical
inertness, thermal and photochemical stability, negligible swelling
in aqueous solution, tunable porosity and optical transparency.
Besides this, they have been widely used in the fabrication of chem-
ical opto-electronic sensors, as most of the biological materials tend
to retain their activity owing to the attractive low temperature pro-
cess of immobilization for various biomolecules (e.g., enzymes and
antibodies).
A conductometric urea biosensor, based on tetramethyl
orthosilicate sol–gel immobilized urease, on a screen-printed inter-
digitated array (IDA) electrode, has been used to detect urea
in linear dynamic range 0.03–2.5 mM with good reproducibly
using the conductometric screen printed technique [87]. This urea
biosensor exhibits good sensor-to-sensor reproducibility, but has a
poor storage stability of around 25 days. The stability of the sol–gel-
encapsulated urease biosensor decreased over time, which was
attributed to denaturation during immobilization or restriction of
the enzyme in an inactive conformation after encapsulation.
Cu(II) and Cd(II) array-based optical urease biosensors, which
were found on tetra ethoxy orthosilicate, have been fabricated
with a FITC–dextran encapsulated fluorescent dye. Attempts have
been made for its application to biological and environmen-
 G. Dhawan et al. / Biochemical Engineering Journal 44 (2009) 42–52
tal samples [88]. Similarly, urease, acetyl choline esterase and
FITC–dextran co-entrapped TEOS sol–gel matrix have been used to
detect heavy metals and urea (2.5–50 M), with an analytical range
of 2–3 orders-of-magnitude. The applicability of this developed-
array biosensor toward simultaneous multi-analyte determination
has been demonstrated. Although results provide a method for
simultaneously analyzing multiple samples and multi-analytes in
environmental and biological samples, the urease-based system
exhibited relatively poor stability and its initial sensitivity dropped
to 45% after 4 weeks [89].
The performance characteristics of urea biosensors based on
urease immobilization using physical adsorption, a physically
entrapped sandwich and microencapsulation in a tetramethy-
lorthosilicate (TMOS)-derived sol–gel matrix on the surface of
a glass–pH electrode, have been studied [90]. Various parame-
ters for biosensor performance (e.g., detection limit, sensitivity,
linear range, response time and regression) have been studied
using potentiometric techniques. The results indicate that the
microencapsulation technique is a better method for enzyme
immobilization in sol–gel films derived from TMOS. The advan-
tage of microencapsulated biosensors over physically entrapped
sandwich configuration biosensors includes: higher sensitivity
(2.4 (dpH/dp(C))), lower detection limit (4.5 ␮M) and a larger lin-
ear range of urea determination (0.01–30 mM). Microencapsulated
enzyme electrode systems yield more satisfactory results with
blood serum samples. However, microencapsulated enzyme elec-
trode systems have shown interference for various analytes (e.g.,
glucose, FeSO4 , thiourea and HgCl) and suffer from a poor storage
stability of 2 weeks and a longer response time of 5 min. Sol–gel
matrices based on molybdenum trioxide, for the development of
urea biosensors, have been studied. It has been found that ure-
ase retains its activity inside molybdenum trioxide sol–gels that
are sensitive to ammonia. These hybrid nanoporous composites are
reported to be useful for biosensors and fuel cells [91].
Apart from the entrapment of sensing agents without enzyme
leaching, longer biomolecule stability, the advantages of sol–gel
glasses include short response time and easy fabrication of multi-
analyte detection electrodes. Drawbacks, including diffusional
limitations, reproducibility of results, sensitivity, non-conductive
nature, unknown catalyst–matrix interactions and kinetics, chal-
lenge researchers. Attempts and approaches should be directed
toward solving these problems.
4. Urea biosensors based on Langmuir–Blodgett films
Langmuir–Blodgett technology has been considered a conve-
nient tool for designing artificial systems with biological functions
(e.g., biosensors), where ultra-thin films containing biomolecules
are expected to preserve high enzymatic activity. Until now, many
kinds of biosensors (e.g., cholesterol, glucose and urea) have been
designed with LB films as the biosensitive part. However, very little
work has been done in the area of urea biosensors based on LB films
[92,93].
Urea biosensors based on octadecylamine and enzyme urease
on ISFET surfaces have been used to detect urea at levels as low as
0.2 mM, in the dynamic range of 0.2–20 mM, within 15 s. Monolayer
stabilization has been achieved using glutaraldehyde [94].
Highly stable urease monolayers have been immobilized onto
hydrophobic surfaces of octadecyl silane-modified SiO2 /Si sub-
strates using the Langmuir–Blodgett deposition technique. The sen-
sitivity of urea detection in this enzyme/insulator/semiconductor
(EnIS) system was found to be 22 mV/pUrea [95].
Urease-immobilized, mixed monolayers of poly(N-vinyl car-
bazole) (PNVK) and stearic acid (SA) LB monolayers have been
studied. Potentiometric measurements on these urease electrodes
47
were carried out using an ammonium ion analyzer [92]. The detec-
tion limit and sensitivity of these electrodes was found to be 5 mM
and 10 mV/mM, respectively. The shelf-life of these electrodes was
found to be 5 weeks at 4 ◦ C. However, the detection limit does not
cover the physiological range of urea.
It may be noted that most studies discussed above were not
carried out using clinical samples and the influence of interferants
(e.g., potassium and sodium ions) has also not been reported. As
these materials show promise for applications toward the design
and development of biosensors, more emphasis should be given to
explore these advantageous materials for improved characteristics
of urea biosensors.
5. Urea biosensors based on nanomaterials
Nanostructured materials have found a great variety of appli-
cations owing to characteristic properties, such as (i) in catalysis
reactions, due to their high surface area and controlled crystal
surfaces, (ii) in sun screen, hyperthermic cancer treatment and flu-
orescent tags based on optical properties, and (iii) in smoke/fog
screens because of their light scattering effect. Nanomaterials
have found applications in drug delivery (inhalation asthma and
timed drug release), pesticide delivery (fogging and fumigation),
magnetic recording (orient magnetic domain axis, hard drives,
video and audio tapes), pigments, inks and paints. A number of
biosensors based on nanomaterials, such as nanoparticles [96],
nanofibers, conducting polymers, nanofilms/nanocomposites [97],
or self-assembled monolayers [98,99], have recently been reported.
Biomolecules (e.g., enzymes, antigens/antibodies or DNA)
exhibit nanoscale dimensions comparable to the dimensions of
metal or semiconductor nanoparticles. These size similarities pave
the way to combine the unique electronic, photonic, and catalytic
properties of nanoparticles with the nature-evolved, selective bind-
ing and catalytic functions of biomolecules. This can be achieved
by integrating native and synthetic components into hybrid sys-
tems. Such hybrids may reveal new material functions, wherein
the nanoparticles would introduce new electronic, photonic or
opto-electronic properties into biomolecules, or alternatively, the
biomolecules would affect the structure or properties of nanopar-
ticles. Extensive research efforts have been directed in the past
few years towards the development of biomolecule–nanoparticle
hybrid assemblies and their application to construct biosensors,
nanoscale circuitry or nanodevices.
5.1. Nanoparticles
Gold nanoparticles have been studied for biosensor fabrication,
as biological molecules can retain their activity when adsorbed onto
them [100]. Gold nanoparticles can immobilize enzymes based
on chemical adsorption onto self-assembled monolayers [101,102].
Yang et al. have proposed a renewable inhibitive mercury sensor
based on self-assembled gold nanoparticles on an ethylenediamine
poly(vinyl chloride) (PVC–NH2 ) membrane-based pH electrode
[103]. Gold nanoparticles have been assembled on the surface
of a PVC–NH2 membrane. Urease was immobilized on the gold
nanoparticles through chemical adsorption. An advantage of using
assembled gold nanoparticles lies in the fact that the inactive
enzyme layers denatured by Hg2+ can be rinsed out via a saline
solution using acid and alkali washes successively.
Gouma et al. [104] have discussed the production of biological
sensors using nanostructured metal oxides. This work was focused
on the synthesis of novel structures of nanocomposite metal
oxide systems by combining sol–gel processing and electrospinning
with an application towards biosensing probes. Encapsulation and
incorporation of these biomolecules into highly surface-porous,
48 G. Dhawan et al. / Biochemical Engineering Journal 44 (2009) 42–52
biocompatible polymer mats enables their higher catalytic activity,
faster response and possibly longer operation lifetime. Urease was
chosen as the model enzyme system in these studies, as it acts as a
catalyst in the hydrolysis of urea to ammonia and carbon dioxide. A
novel approach to biosensor development using hybrid nanomate-
rials systems for the selective detection of biospecies (pathogens) of
interest has been demonstrated, such as electrospun polymer/oxide
nanofiber structures in which proteins or cells are incorporated
(Fig. 2). The result of biochemical reactions between the react-
ing biospecies and bioreceptors is a chemical signal that can be
sensed by the hybrid nanofiber platform through a conductimetric
mode. These new findings open the pathway for numerous appli-
cations of bio-nanocomposite materials, including non-invasive
medical diagnostics tools, bio-fuel cell devices, protective cloth-
ing (electronic and interactive textiles), bioengineering scaffolds
and advanced sensor arrays. Further work must be performed to
exploit these findings toward designing biosensors.
5.2. Quantum dots (QDs)
Quantum dots or semi-conducting nanoparticles are highly
luminescent, photostable fluorophores that have recently been
used for sensing applications. QDs have much higher photolumi-
nescence quantum efficiency than their bulk counterparts. Hybrid
systems containing QDs coupled with various biomolecules have
stimulated research in biotechnology and nanotechnology. A sub-
tle change of the surface property of QDs can result in a dramatic
change in their optical properties. In principle, this novel feature of
QDs can be extended for detecting specific analytes if appropriate
conditions are established. An assay system with urease as catalyst
and CdSe/ZnS quantum dots as indicators has been developed for
the quantitative analysis of urea by Huang et al. [105]. Detection
in this system is based on the enhancement of QD photolumines-
cence intensity, which is correlated to the enzymatic degradation of
urea. By controlling buffer concentration and pH, PL enhancement
due to urea degradation was found to be linear in the 0.01–100 mM
concentration range.
Important advances using semiconductor QDs for biosensing
processes have been reported in recent years. Nevertheless, this
field requires further development, and challenging goals still
require scientific answers. The development of chemical proce-
dures for the preparation of highly fluorescent QDs in aqueous
media, while retaining the photonic communication between the
QDs and the associated dye units, is essential.
5.3. Nanofibers
With increasing demands for nanotechnology, electro-spinning
has become a novel technique for generating composite nanofibers
[106]. Sawickaa et al. [97] have utilized the electro-spinning
technique to prepare nanocomposite fibers of urease and
polyvinylpyrrolidone (PVP). The non-woven mat was found to have
a potential use as a urea biosensor, showing characteristic features
of fast response time, sensitivity to lower concentrations of urea,
and a more versatile design. The immobilized enzyme was found
to remain active inside the polymer solution and reactivity was
maintained inside electrospun non-woven mats. The electrospun
membrane acts as a catalyst for hydrolysis in 0.5–2.5 mM urea solu-
tions.
5.4. Nanoporous silicon technology
Nanoporous silicon technology has been used for the fabrica-
tion of urea biosensors. Jin et al. [106] fabricated three thin film
electrodes patterned on p-type silicon wafers using platinum RF
sputtering and Ag evaporation. The working electrode was com-
prised of urease immobilized onto an organic polymeric conductor
(PPy). The electrochemical properties were characterized using
both amperometry and potentiometry for detection of urea. Yun
et al. [107] used a similar technique for the development of an
amperometric urea biosensor. In their work, the attachment of
urease to a carboxylic-terminated SAM, which was chemisorbed
onto a gold electrode, was achieved using carbodiimide coupling.
The formation of a self-assembled, chemisorbed layer of ure-
ase on gold was studied by X-ray photoelectron spectroscopy
(XPS). The measured sensitivity of the urease/SAM/Au/planar sil-
icon electrode was found to be 11.21 ␮A/(mM cm2 ) and that of the
urease/SAM/Au/PlanarSi electrode was ca. 4.39 ␮A/(mM cm2 ). An
approximately threefold sensitivity increase was observed in the
porous-silicon-based electrode.
Nanopatterned porous alumina has been prepared by electri-
cal anodization in acid solution and has been used as an enzyme
electrode and pH sensor for urea detection. The biocompatibil-
ity of nano-size porous alumina as an immobilization matrix for
biocolloidal systems has stimulated interest for improved sen-
sor sensitivity. The sensor was tailored to enlarge the active
surface area, which in turn increased the sensitivity of the
sensor.
6. Miscellaneous matrices for urea biosensing
Some other supports used for urea biosensor fabrication
include: bilayer lipid membranes [108], carbon paste electrodes
[109], composite hydrogel membranes [110], gelatin membranes
[111], inorganic matrices (e.g., clays, laponite and Zn–Al layered
double hydroxides matrices) [112,113], SiO2 films, glass beads,
diatomite, silica, porous glass, hornblende and molecular sieves
[114–118].
Srivastava et al. [111] have described a urea biosensor utilizing
urease immobilized on gelatin beads. The system was success-
ful in achieving a long storage stability, with a half-life of 240
days. The characteristics of this sensor include a detection limit
from 0.8 to 23 mM and a response time of 6 min. Another sensing
system has been proposed for urease immobilization on porous
glass beads [119]. The sensor set-up proposed by Seki’s group used
urease immobilized on SiO2 films as the pH sensitive layer in light-
addressable potentiometric sensors (LAPS) [120], with a detection
range from 5 to 15 mM. However, more studies must be done in this
field to fabricate a cost-effective, commercial sensor using these
novel materials.
The use of enzyme mixtures can provide improved performance
in urea sensors. Seo et al. [122] determined NH4 + via amperometric
methods, using the immobilization of l-glutamate dehydrogenase
on the immobilon-AV affinity membrane. Incorporation of the
enzyme into a carbon paste was performed by Yang et al. [109]
for the construction of an amperometric enzyme electrode. The
enzyme-modified carbon paste electrode resulted in improved
specificity and sensitivity. This system made use of bi-enzyme
systems involving glutamate dehydrogenase and urease for urea
estimation. Response characteristics of the system were based on
the redox activity of NADH. The oxidation current of NADH was
monitored at +1.10 volt vs. Ag/AgCl. The optimum pH range for cat-
alyzed hydrolysis reactions of urea was found to be between 7.0 and
7.4. The linear response range and detection limit were found to be
2.0 × 10−5 to 2.0 × 10−4 M and 5.0 × 10−6 M, respectively. Liu et al.
[123] have shown that the immobilization of enzymes on ␥-Al2 O3
is well-suited for the preparation of biosensors. Interestingly, the
enzymes can be entrapped in internal surfaces by physical or chem-
ical reaction (e.g., change in temperature, pH, or pretreatment of the
substrate) without using cross-linking chemicals that can partially
 G. Dhawan et al. / Biochemical Engineering Journal 44 (2009) 42–52 49

deactivate enzyme activity. A potentiometric biosensor for urea
was found to be sensitive in the concentration range of 3.0 × 10−5
to 1.4 × 10−2 M, with a detection limit of 10 ␮M. This novel urea
sensor has been applied toward the measurement of urea in
urine.
Kirstein et al. [121] described a urea biosensor in which amper-
ometric pH sensing was realized by pH dependence of hydrazine
electrochemical oxidation in the Tafel region. The sensor showed
a linear range from 0.8 to 35 mM. Disadvantages lie in its low
sensitivity and use of the hazardous compound hydrazine. Piz-
zariello et al. [124] have made use of a pH-sensitive redox-active
mediator, Hematin, for the development of an amperometric urea
biosensor. Urea biosensors are constructed with urease immobi-
lized on the surface of platinum and graphite composite electrodes
and amperometric measurements are done using 0.5 mM hematin.
The amperometric sensor reported by Yoneyama was combined
with a pH-stat method and flow injection analysis. This system
recorded a linear relation between current and urea concentration
within a narrow range of 0–600 ␮M urea solutions [119]. Stedansky
[125] tried to exploit the redox activity of certain mediators (e.g.,
hematein, lauryl gallate, and adsorbed methylene blue) for the con-
struction of cheap and reliable urea biosensors. Under optimum
conditions, the sensors showed a detection limit of 2 ␮M, linear-
ity from 0.002 to 0.75 mM, dynamic range of 4 mM and sensitivity
of 15.2 Ma/Mcm. However, attention should be paid to the selec-
tion of working conditions, such as initial pH, operating potential,
buffer type and capacity. The authors have concluded that further
improvements could be expected with a more systematic search
for pH-sensitive, redox probe molecules.
It is suggested that a large number of pH sensitive compounds
(e.g., quinines, polyphenols, antioxidants, and organic dyes) are
redox active and could serve as probe molecules for monitoring
urea. Conversely, from a practical point of view, a suitable pH-
sensitive redox compound should have physical stability and work
at a potential near 0 mV, to avoid electrochemical interference from
real clinical samples.
Therefore, the use of mixed enzyme systems with cost-
effective matrices may be suitable for the development of urea
biosensors.
7. ISFET-based urea biosensors
In 1980, Caras and Janata described the first pH-ion-selective,
field effect transistor (pH-ISFET)-based, enzyme-modified biosen-
sor sensitive to penicillin [126]. ISFET-based biosensors have many
advantages, such as miniaturization, high sensitivity, high level of
activity, low cost and multi-detection potential, which is especially
important for the fabrication of multi-biosensors. As the enzy-
matic hydrolysis of urea causes an increase in pH of the system,
ISFETs have been widely used for this purpose. Various methods
for urease immobilization on the surface of ISFET transducers have
been reported, such as inclusion in a gel [127,87], confinement
[128], reticulation with a bifunctional agent [129], covalent bond-
ing on an active support [130,131], and Langmuir–Blodgett films
[132]. Bovine serum albumin (BSA) has been widely used for the
immobilization of urease on a transducer surface via chemical
cross-linking [133,134]. Apart from all these methods, silaniza-
tion is most commonly used [135–143]. This procedure enables
the introduction of chemically reactive groups. In the case of
silanization performed with aminosilanes, reactive groups are of
the amine type (NH2 ). These groups are used to form covalent
bonds between enzyme amino groups and glutaraldehyde. Sev-
eral authors have used this technique for the fabrication of urea
biosensors [144–149].
The various detection techniques and matrices for the fabrica-
tion of urea biosensors are listed in Table 1.
8. Demands, challenges and commercialization
According to the National Kidney Foundation, USA, more than
20 million Americans (one in nine adults) have chronic kidney dis-
ease. More than 20 million more are at increased risk for developing
kidney disease and most do not even know it [150]. The major cause
for kidney failure has been attributed to diabetes. In 2003, diabetes
accounted for about 44% of new cases and about 36% of all kid-
ney failure cases in the US [151]. Uncontrolled or poorly controlled
high blood pressure is the second leading cause of kidney failure in
the US, followed by glomerulonephritis, an inflammatory disease
of the kidneys, and polycystic kidney disease. Kidney-related dis-

Table 1

Matrix Transducer Response time Sensitivity Detection limit Stability Ref.

Poly(vinylferrocium) Amperometry 60 s 1 ␮M 29 days [29]

Polyethylenimine Amperometry 15–30 s 4 weeks [30]
Polyvinyl alcohol Potentiometry 2–4 min 10−4 M 1 month [32]
Polyvinyl chloride–palmitic acid Potentiometry 1–2 min 54.3 ± 0.4 mV/Purea 60 days [34]
PVC–NH2 Potentiometry < 10s 48 ± 5 mV/dl 3 × 10−5 M 1 month [35]
Chitosan Potentiometry 30 s–2 min 56 mV/dl 2 month [42]
PVC–nonactin Optical 16–20 s 0.05 absorption unit in 0.08 mM [63]
0.01–2 mM urea
Polypyrrole Potentiometry 31.8 mV/dl [74]
Polypyrrole Amperometry 60 ug/l 2 weeks [75]
Poly pyrrole Amperometry 3 ppm [77]
Poly(N-3-aminopropylpyrrole-co-pyrrole) film Potentiometry 25–50 s 27.5 mV/dl 2 months [79]
Tetraphenyl doped polyaniline Potentiometry 86 mV/pH 20 ␮M 2 months [80]
Tetramethyl ortho silicate Conductometric 204 ␮S/mM 30 ␮M 25 days [86]
TMOS derived sol–gel silicate matrix Glass–pH electrode 5 min dpH/dp(C) = 2.4 52 ␮g/ml; 25 days at 4 ◦ C [89]
Poly(N-vinylcarbazole) and stearic acid Ammonium 10 mV/mM 5 mM 5 weeks at 4 ◦ C [91]
ion-selective electrode
(potentiometry)
Urease onto Si/SiO2 Struct. CV measurements 22 mV/Purea 1 mM Few days [93]
Urease/PVP nanocomposite nanofibers Quantum dots [95]
Optical 0.01 mM [102]
Carbon paste electrode Amperometric 5.0 × 10−6 M 15 days [106]
Zn3 Al–urease layered double hydroxides Impedometric 21 mV/Purea [110]
␥-Al2 O3 Potentiometric 10 ␮M [120]
50 G. Dhawan et al. / Biochemical Engineering Journal 44 (2009) 42–52

orders rank third amongst all life threatening diseases in India as nanostructure synthesis, has great promise in future nanotechnolo-
well, after cancer and cardiac ailments [152]. gies.
There is increasing demand for a more reliable, accurate urea
monitoring system. Urea biosensors face many problems in differ- Acknowledgements
ent terms. For very low concentrations, there is always a chance of
losing ammonia, as it may not be obtained in the dissolved form and We are grateful to Dr. Vikram Kumar, Director, National Phys-
may be liberated into the atmosphere yielding results with errors ical Laboratory, New Delhi, India for his interest in this work.
in terms of reduced urea levels. Another important problem that Gunjan Dhawan is thankful to the Council of Scientific and
needs to be rectified while fabricating urea biosensors, especially Industrial Research (CSIR) and University Grants Commission
potentiometric biosensors, is the effect of interferants. Sodium and (UGC), India, for a Junior Research Fellowship. We thank Dr.
potassium ions present in blood, serum or urine samples interfere Kavita Aora for interesting discussions during the preparation
in urea analyses and hamper accurate monitoring. Hence, there is of this manuscript. We sincerely acknowledge financial sup-
an urgent need for more and better analyzers that can serve the port from DST-sponsored projects: DST/CLP041332 CSIR funded
desired purpose of monitoring urea in clinical samples, be cost- CMM-11, DBT/PR7667/MEP/14/1057/2006, DST/INT/JAP/P-21/17,
effective and also be free of any disadvantages usually occurring in DST/TST/TE/2007/06 and DST/SEN/SI/03.
the present day biosensors.
A commercially available urea biosensor is the i-STAT Portable
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