Sensors and Actuators B 140 (2009) 260–266

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Sensors and Actuators B: Chemical

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Potentiometric biosensor for urea determination in milk

U.B. Trivedi a , D. Lakshminarayana a , I.L. Kothari b , N.G. Patel c , H.N. Kapse d ,
K.K. Makhija a , P.B. Patel a , C.J. Panchal e,∗
a
Department of Electronics, Sardar Patel University, Vallabh Vidyanagar 388120, Gujarat, India
b
Department of Bio-Sciences, Sardar Patel University, Vallabh Vidyanagar 388120, Gujarat, India
c
Department of Chemistry and Physics, University of North Florida, Jacksonville, FL, USA
d
Institute of Science and Technology for Advanced Studies and Research (ISTAR), Vallabh Vidyanagar 388120, Gujarat, India
e
Applied Physics Department, Faculty of Technology and Engineering, M.S. University of Baroda, Vadodara 390001, Gujarat, India

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

Article history: A potentiometric urea sensitive biosensor, using a NH4 + ion sensitive electrode in double matrix mem-
Received 3 March 2009 brane (DMM) technology as the transducer, has been described. Thick film screen printing technique
Received in revised form 3 April 2009 was utilized for the fabrication of the basal conducting track of the potentiometric electrode and also for
Accepted 9 April 2009
the development of Ag/AgCl reference electrode. The ion sensitive polymer matrix membrane could be
Available online 24 April 2009
formed in the presence of an electrochemically inert filter paper. The electrochemical response behavior
of the NH4 + ion sensitive electrode has been studied initially. Later, a urea biosensor has been devel-
Keywords:
oped by immobilizing the urease enzyme, through entrapping, onto the ion sensitive membrane using
Potentiometry
Biosensor
a polymer matrix of poly(carbamoylsulphonate) (PCS) and polyethyleneimine (PEI). The urea biosensor
Urea responded rapidly and in a stable manner to the changes in the test urea concentrations. The sensor
Ion sensitive electrode exhibited a detection limit of 2.5 × 10−5 mol/L. The average slope in the linear range has been found to be
Urease 51.7 ± 0.5 mV/decade. The biosensor system has, then, been tested for the detection of urea levels in the
Polymer matrix real samples of milk. A good agreement could be observed between the urea concentrations detected by
Milk the developed biosensor and the spectrophotometric technique.
© 2009 Elsevier B.V. All rights reserved.

1. Introduction Urea, as mentioned above, is a normal constituent of milk and it

Urea, [CO(NH2 )2 ], an end product of nitrogen metabolism, has
great significance in clinical chemistry and dairy industry [1–6].
The food industry has the requirement of real time and accurate
analysis of dairy products during manufacture and quality control.
In this regard, urea biosensors could prove to be a valuable tool for
monitoring the urea content of milk [7,8]. Milk is an emulsion of fat
in watery solution of sugars, mineral salts and proteins in colloidal
solutions. The typical concentration of urea in milk is 18–40 mg/dl
[9,10]. High urea content in milk, which occurs from the unbalanced
feeding of cows, is known to influence the milk production and fer-
tility [11,12]. Excessive nitrogen (N) in the feed causes high systemic
urea N without a corresponding increase in milk protein [13]. Deter-
mination of values of urea and true protein in milk may be useful
to assess the nutritional program of cows and particularly lactat-
ing dairy cows [14]. It is reported [15,16] that when the milk urea N
(MUN) reaches above 20 mg/dl or so, it may indicate the underlying
pathological problems in cows. Milk urea level is also linked to the
thermal stability of milk at higher temperatures [17].
∗ Corresponding author. Tel.: +91 9825094761; fax: +91 265 2423898.
E-mail address: cjpanchal msu@yahoo.com (C.J. Panchal).
forms a major part (55%) of the non-protein nitrogen of milk [18].
However, for commercial benefits chemicals like urea, caustic soda,
refined oil and detergents are sometimes used to adulterate the
milk. The adulteration decreases the nutritive value of the milk and
may pose a great threat to the human health. The mixture of chem-
icals gives the solutions the milk like properties and is termed as
‘Synthetic Milk’ [8,18–20]. A cut off limit for urea concentration in
milk is normally accepted at 70 mg/dl. The presence of urea above
this cut off limit in milk can cause severe health problems for human
beings, such as, indigestion, acidity, ulcers, cancer, malfunctioning
of kidneys etc. Hence, urea detection and its estimation have great
significance in dairy industries, clinical analyses and food process-
ing technology. Moreover urea is widely distributed in the nature
and its analysis is necessary in food chemistry and environmental
monitoring [21]. Because urea concentrations in human blood and
urine are both important indicators of renal health, many sensors
including biosensor types, specific to urea, have been developed
in the biomedical industry. Numerous types of urea sensors have,
thus, been proposed based on conduct metric [22–24], potentio-
metric [25–30], thermometric [31], and optical methods [32]. Most
of these sensors are too delicate for use with raw milk without an
extensive pretreatment [13,33–35]. The biosensors specific to urea
detection in milk will have to be simple, reliable and robust enough

0925-4005/$ – see front matter © 2009 Elsevier B.V. All rights reserved.
doi:10.1016/j.snb.2009.04.022
 U.B. Trivedi et al. / Sensors and Actuators B 140 (2009) 260–266
to withstand repeated exposure to the high lipid and protein con-
centrations in milk.
There are only a few reports available in the literature over the
development of sensors for the specific application of urea detec-
tion in milk [8,18]. The objective of the present investigation is to
develop a low cost screen printed and disposable type urea sensi-
tive enzymatic biosensor system, based on potentiometry, for the
detection and estimation of urea levels in milk samples. Potentiom-
etry is selected for the purpose as it allows the determination of a
wide spectrum of ions and also as it applies portable and inexpen-
sive equipment. This technique is especially useful for carrying out
the tests (urea detection) at remote sites (‘in field’ measurements at
rural milk collection centers, house hold consumer locations etc.).
The sensor designed in the present investigation consisted of a
screen printed disposable NH4 + ion sensitive electrode developed
on the basis of double matrix membrane (DMM) technology [37]
as the transducer with a layer of immobilized urease. The urease
enzyme has been immobilized by entrapping in a unique polymer
matrix of poly(carbamoylsulphonate) (PCS) and polyethyleneimine
(PEI). A screen printed Ag/AgCl reference electrode has formed the
stand alone urea biosensor system for the detection of urea in milk
samples.
2. Experimental
2.1. Chemicals and reagents
The various solutions utilized in the present investigation were
all prepared with analytical reagent grade chemicals and double
distilled water. The various enzymes, polymers, pastes and chemi-
cals used in this investigation are listed below with their makes.
2.1.1. Enzyme
• Urease, Jack bean source [E.C. 3.5.1.5] (Sigma, St. Louis, USA).
2.1.2. Polymers
• Polyethyleneimine (50% (w/v) aqueous solution) (PEI) (Sigma,
USA).
• PCS hydrogel (Sens Lab, Germany).
2.1.3. Pastes for screen printed fabrication of the electrodes
• Silver/silver chloride paste R-414 (DPM-68) (Ag/AgCl) (Ercon,
USA).
• Silver paste (Ted Pella Inc., USA).
2.1.4. Polyester sheets, screen and chemicals
• Polyester foil Melinex type 505 (thickness: 175 ␮m) (Putz, Ger-
many).
• Polyester/polyethylene sheet (thickness: 150 ␮m) (Haltem, Ger-
many).
• Polyester sheet, 68.110/H1, 300 ␮m thick opaque sheet size
297 mm × 420 mm, mesh size 1/77.5 cm (77 wires in 1 cm).
• Screen (MonolenPuls-77T) mesh size 68 ␮m (Berlin, Germany).
• Urea (extra pure, ultra) (Sigma, Deisenhofen, Germany).
• Tetrahydrofuran (THF) (99.5+%, spectrophotometric grade)
(Sigma–Aldrich, St. Louis, USA).
• Cyclohexanone (extra pure) (Merck, Darmstadt, Germany).
• Polyvinylchloride (PVC), high molecular weight (Fluka, Switzer-
land).
• Bis(2-ethylsebacate) (Sigma, Deisenhofen, Germany).
• Disodium hydrogen phosphate 67 mM (Merck, Darmstadt, Ger-
many).
• Sodium dihydrogen phosphate 67 mM (Merck, Darmstadt, Ger-
many).
261
• Potassium chloride 0.1 M KCl (Merck, Darmstadt, Germany).
• Sodium chloride (Merck, Darmstadt, Germany).
• Sodium hydroxide (Merck, Darmstadt, Germany).
• Tris(hydroxymethyl)-aminomethane (analytical grade) (Sigma,
St. Louis, USA).
• Ethylenediaminetetraacetic acid (EDTA) (disodium salt) (analyti-
cal grade) (Sigma, St. Louis, USA).
• Phenol (Fluka, Switzerland).
• Sodium hypochloride dihydrate (Fluka, Switzerland).
2.1.5. Apparatus
• Micropipette (Eppendorf, Germany).
• Micro fiber matrix (MFM) material filter paper (110 mm diameter)
(Whatman, USA).
• Multi-Channel ISE/pH/mV/ORP/Temperature bench top meter
(Thermo Orion, USA).
• Vacuum Coating Unit (Hind Hi Vacuum, India).
2.2. NH4 + ion sensitive electrode fabrication
A potentiometric biosensor system was developed in the present
investigation for the detection of urea. The potentiometric biosen-
sors make use of ion selective electrodes in order to transduce the
biological reaction into an electrical signal. Initially, a NH4 + ion
sensitive electrode was fabricated and it was, later, used for the
detection of urea by immobilizing urease enzyme on the NH4 + ion
sensitive membrane of the electrode.
The method of screen printing that was utilized for the fabrica-
tion of amperometric electrodes and reported earlier by the authors
[37] was again adopted for the development of NH4 + ion sensitive
electrodes and also for the Ag/AgCl reference electrode. Silver (Ag)
was selected as the base conducting track of the potentiometric
electrode. The silver paste was screen printed using screens hav-
ing a screen mesh size of 68 ␮m. The base transducer used was,
accordingly, a screen printed silver electrode on a Melinex sheet of
150 ␮m thickness. The basic steps for the preparation of screen or
mask normally involved the processes of layout, artwork genera-
tion, mask frame preparation, image transfer and mounting [38].
The electrodes were produced in a batch process and after each
screen printing the sheets were kept at 100 ◦ C for 1 h annealing.
Then, in a separate experimental process, high purity thin silver
film was thermally deposited on one side of a filter paper in a
high vacuum (10−4 Pa). From the silver coated filter paper, circu-
lar shaped 3 mm diameter discs were separated. The filter paper
disc with the silver deposited side was then attached to one end
of the previously screen printed silver basal conducting track. The
contact between the silver conducting track and the silver coated
side of the filter paper disc was made with a high purity silver
paste. Later, the silver conducting track with the polyester sheet
base was laminated using a laminating machine (SANON, CR-309,
India), leaving the 3 mm diameter unsilvered filter paper without
any lamination. Later, a NH4 + ion sensitive membrane was pre-
pared on the unlaminated and unsilvered filter paper, following
the procedure described below. The other end of the conducting
track was utilized for the electrical connection purpose. The entire
lamination process was carried out at 413 K. A large number of sil-
ver based filter paper tipped electrodes were fabricated in a similar
way.
The NH4 + sensitive DMM electrode was then prepared following
the procedure described by Eggenstien et al. [36,39]. A mixture of
1% nonactine, 66.8% bis(2-ethylsebacate) and 32.2% PVC was pre-
pared first. From this mixture, 400 mg was dissolved in 1.5 ml THF
(99.5%, spectrophotometric grade) and 0.5 ml cyclohexanone. The
components were allowed to dissolve overnight and shaken vigor-
ously until a homogenous solution was obtained. A micropipette
262 U.B. Trivedi et al. / Sensors and Actuators B 140 (2009) 260–266
(Eppendorf, Germany) was used to dispense 10 ␮l solution over the
filter paper tip. The NH4 + ion sensitive electrodes thus prepared,
in a batch of 60, were all stored overnight at 4 ◦ C. These working
electrodes were now ready on one hand for the NH4 + measure-
ments and on the other hand, for the immobilization of the urease
enzyme for urea detection.
2.3. Urea detection electrode fabrication
For urea detection, the urease enzyme was immobilized by
entrapping it in a PCS + PEI polymer matrix on the surface of the
NH4 + ion sensitive membrane electrode. For the urease immobi-
lization, a PCS solution was prepared first, by dissolving 150 mg
PCS pre-polymer in 400 ␮l deionized water. Next, a PEI solution
was prepared by dissolving 2.5 mg PEI pre-polymer in 60 ␮l deion-
ized water. A mixture of PCS and PEI solution was prepared later
by slowly adding PEI solution using a micropipette (Eppendorf,
Germany) into the acidic PCS solution. The PEI (2.5% in aque-
ous solution) was added till the pH value of the mixture solution
reached 6.8. A urease enzyme stock solution was prepared by
using 60 mg/ml of the enzyme with the buffer solution and then
stored at −10 ◦ C. Then, 30 ␮l of urease stock solution was added
to the mixed solution of PCS and PEI in a proportion of 1:1
(urease:PCS + PEI) and gently stirred at the room temperature of
303 K.
Using the micropipette, 2 ␮l mixture of urease and PCS + PEI
was dropped on the NH4 + ion sensitive membrane of the working
electrode at the room temperature of 303 K. A batch of 30 work-
ing electrodes was prepared accordingly and were all kept at 4 ◦ C
overnight. Fig. 1 shows a schematic of a typical working electrode
for urea biosensor for use in potentiometric measurements. In the
figure, the layer formation at the electrochemically active end is
shown on the right hand side with arrows (i.e. filter paper/NH4 +
membrane/urease:PCS + PEI).
2.4. Ag/AgCl reference electrode fabrication
Eggenstien et al. [36] reported the preparation of the Ag/AgCl
reference electrodes following the way described by Diekmann et
al. [40], leading to a combination of the reference electrode with
the urea sensitive working electrode. In the present investigation,
the authors, however, adopted the simple thick film screen print-
ing technique for the fabrication of a separate Ag/AgCl reference
electrode for use with the working electrode in the potentiometric
setup. The Ag/AgCl paste was screen printed on a Melinex sheet
of 150 ␮m thickness using a screen with a screen mesh size of
68 ␮m. The screen printing procedure was similar to the above
described way of the fabrication of the silver conducting basal
track.
Fig. 1. Schematic of a typical fabricated working electrode for potentiometric urea
detection.
2.5. Measurements
The potentiometric measurements were made at room temper-
ature (303 K) using a Multi-Channel ISE/pH/mV/ORP/Temperature
ion sensitive meter (Thermo Orion-920A, USA). The standard stock
solutions of NH4 Cl and urea were duly prepared to check the ion
selective nature of the stand alone NH4 + electrode and the urea
detection capability of the urease immobilized enzyme electrode
(having the NH4 + sensitive membrane) respectively.
2.6. Principle of sensor operation
The urea sensor operation is based on the enzymatic decompo-
sition of urea by urease:
Urease
CO(NH2 )2 + H2 O −→ CO2 + 2NH3
In the pH region where the enzyme is active (around pH 7), the
products of the above enzymatic reaction dissociate as:
CO2 + H2 O  HCO3 − + H+
NH3 + H2 O  NH4 + + OH−
Thus, it is possible to determine the urea concentration potentio-
metrically using a variety of transducers such as the pCO2 electrode,
the pNH3 electrode, the pH electrode, and the pNH4 + electrode. Ure-
ase can be physically entrapped in a polymer gel and placed on the
tip of an electrode, which is sensitive to ammonium ions. This basic
transducer has the drawback of being highly sensitive to H+ , K+ ,
and Na+ ions, which considerably restricts the use of the sensor
in environments containing monovalent cations. A buffer solution
is therefore, needed such that its constituents do not interfere
with the response of the enzyme electrode [41]. The potentiomet-
ric urea biosensor, therefore, needs an ion sensitive electrode in
combination with an immobilized urease enzyme. Hence, a NH4 +
ion sensitive electrode was first prepared and characterized. Later,
this NH4 + sensitive electrode was used for the detection of urea
by immobilizing the urease enzyme onto the NH4 + ion sensitive
membrane of the electrode.
3. Results and discussion
3.1. Characterization of NH4 + ion sensitive electrode
At first, the NH4 + sensitive electrodes were characterized to eval-
uate whether they could serve as transducers in sensing urea. Fig. 2
shows the response behavior of the NH4 + sensitive electrode against
the Ag/AgCl reference electrode. The electrode responded rapidly
and in a stable manner to the additions of the ammonium chloride
stock solution. The response time was found to be the in the range
of a few seconds. The detection limit determined from the cali-
bration graph was found to be 3.0 × 10−5 mol/L. The inset of Fig. 2
shows the calibration curve of the NH4 + electrode. The average slope
in the linear range was found to be 56.7 ± 0.4 mV/decade, indicat-
ing a high reproducibility. It could, therefore, be concluded that
the developed NH4 + ion sensitive electrode was a suitable trans-
ducer for the potentiometric urea sensor fabrication. The next step
was then, the immobilization of the urease enzyme onto the NH4 +
sensitive electrode.
3.2. Urea biosensor
The fabricated enzyme based urea biosensors were used as the
working electrodes with the screen printed Ag/AgCl as the refer-
ence electrodes. The electrodes were suitably dipped into 30 ml of
potassium phosphate buffer solutions of varied pH values and then
 U.B. Trivedi et al. / Sensors and Actuators B 140 (2009) 260–266
Fig. 2. Response curve of the NH4 + ion sensitive electrode. The inset shows the
calibration graph.
connected to the Multi-Channel ISE/pH/mV/ORP/Temperature ion
sensitive meter (Thermo Orion-920A, USA). 1 M urea stock solution
of 10 ml was added to the buffer solutions of different pH values.
The solutions were then continuously stirred for about 15–18 min
to get the stable base line. Then, solutions of known urea concentra-
tions were carefully added, as the test samples, to the above buffers
to study the urea testing ability of the sensor and its pH depen-
dence. The measurements were all made at the room temperature
of about 303 K.
3.2.1. pH dependence
Fig. 3 shows the pH dependence of buffer solutions on the poten-
tiometric response of the fabricated urea biosensor. In the present
investigation, the highest response could be observed at pH 7.5
which was subsequently utilized in further experimental investi-
gations.
It has been reported [42] that there is no consistent pattern
so far as the effect of buffer ions on the urease kinetics are con-
Fig. 3. Influence of buffer solution pH on the response of urea biosensor.
263
Fig. 4. Response curve of the urea biosensor with screen printed reference electrode.
The inset shows the calibration graph.
cerned. The inhibition of urease by phosphate buffer is of partially
mixed type while that by citrate is uncompetitive [43]. The inhibi-
tion by phosphate increases rapidly with decreasing pH [44]. Below
pH 7.0, urease activity was not influenced by the presence of elec-
trolytes in a wide range of both concentration and type of ions. At
pH value higher than 7.0, urease activity was reported to decrease
more significantly with increasing electrolyte concentration than
at low pH values [45]. In order to obtain urea sensors which would
show Nernstian potentiometric response over a wide pH range, dif-
ferent organic functional groups as the hydrogen ion carriers were
studied by Yuan et al. [46]. These functional groups were found to
be capable of creating pH response in acidic and alkaline regions. In
the present investigation, as mentioned above, a pH 7.5 was found
to offer the optimum response.
3.2.2. Response behavior of urea biosensor
The response time of biosensor is normally the time taken for it
to reach the steady state when there is no further variation in the
signal. The response time depends upon the analyte, co-substrate,
product transport through different membranes, membrane per-
meability etc. The response time also depends upon the activity of
the molecular recognition system. Fig. 4 shows the electrochem-
ical response behavior of the fabricated urea biosensor system.
The system responded rapidly and in a stable manner to the addi-
tions of known concentration of urea from the stock solution.
The behavior shows that the screen printed Ag/AgCl reference
electrode provides a suitable reference electrode leading to the
development of a complete low cost and disposable type of
system.
The present simple urea biosensor system exhibited a detection
limit of 2.5 × 10−5 mol/L, on par with the detection limit reported
by Eggenstien et al. [36] with their more sophisticated version. The
urea biosensor system was also found to function in the analytically
useful range with a response time of 30–40 s. The inset in Fig. 4
shows the calibration curve obtained with the disposable type of
electrode system. The average slope in the linear range was found
to be 51.7 ± 0.5 mV/decade with a high degree of reproducibility.
The urea biosensor exhibited a good linear least squares fit (linear
regression analysis) with a good correlation coefficient (R2 = 0.989,
n = 8). The noise range amounted to about 1.0 mV. The slope showed
264 U.B. Trivedi et al. / Sensors and Actuators B 140 (2009) 260–266

Fig. 5. Effect of temperature on the response of urea biosensor. Fig. 6. Response curves for biosensors in batch process.

no significant decline over a period of more than 24 h of operation.
This shows that the fabrication technology utilized here is suitable
for the development of a disposable urea biosensor. Also, the inves-
tigation clearly indicates the suitability of PCS + PEI polymer matrix
for the immobilization of urease enzyme.
3.2.3. Temperature dependence
Urea biosensors use enzymatic reactions and so an increase in
temperature will also increase the catalytic activity and hence, the
rate of reaction. The effect of temperature of the buffer solution
on the response of urea biosensor was studied in the range of
280–330 K.
Fig. 5 shows the response of urea biosensor based on DMM
technology against the buffer solution temperature. It is seen that
the potentiometric response initially increases with the tempera-
ture and then decreases later. The response reached a maximum at
about 313 K and the decrease of response after 313 K may be due
to the thermal inactivation of the urease enzyme or the enhanced
disproportionate kinetics of electrochemical reactions. Fig. 5, thus,
indicates a maximum working temperature of 313 K.
3.2.5. Storage stability and shelf life
Most enzymes lose their activity when not stored in refrigerator,
and therefore, storage at low temperatures is one of the most impor-
tant parameters to retain the stability of enzyme based biosensors.
Moreover, in order to attain high water content in the immobilized
layer, the sensors were kept in a closed bottle together with wet
cotton to achieve moisture condition. Fig. 6 compares the storage
stability of the two enzyme sensors stored at two different tem-
peratures. The operational stability of a biosensor response may
vary considerably depending upon the sensor geometry, method of
preparation, biological recognition reactions etc.
From Fig. 7 it can be seen that the performance of the urea sensor
stored at low temperature is higher. A slight decrease in the elec-
trode activity and selectivity was observed over a week of operation.
Thereafter, the decrease in the activity was more significant. The life
time of the enzyme based urea biosensor in the present investiga-

3.2.4. Reproducibility
Reproducibility is a measure of the scatter or the drift in a series
of observations or results performed over a period of time. It is
generally determined for the analyte concentration within a usable
range.
During the present investigation, a large number of urea biosen-
sors were fabricated in a batch process. From a batch of 30 screen
printed DMM based urea sensitive electrodes, the authors ran-
domly selected five electrodes and subjected them for obtaining the
potentiometric response. Fig. 6, thus, shows the response curves
for biosensors fabricated in four batches, with each batch hav-
ing a total of 30 biosensors. It is to be noted that each point
of the curve, corresponding to each batch, is an average value
of five measurements. It is seen from the figure that the results
are reproducible for each batch of process. Hence, the present
low cost screen printing technology can be effectively imple-
mented for the mass/batch production of disposable type of urea
biosensors comprising of enzyme and screen printed Ag/AgCl Fig. 7. Storage stability of urea biosensor: curve (a) corresponds to urea biosensor
electrode. stored at 303 K and curve (b) corresponds to the biosensor stored at 270 K.
 U.B. Trivedi et al. / Sensors and Actuators B 140 (2009) 260–266
tion was found to be in the range of 7–8 days when operated at
room temperature (303 K) and below. This life time is quite suit-
able for disposable type [36] of urea biosensors for use in food and
clinical analysis. An untested urea biosensor was found to remain
intact for about 3 months also. A prudent use (maximum 12 h/day
operation and storage at very low temperatures) however, enabled
the sensor system to function properly up to about 25–30 days only.
Whenever the sensor was not in operation, the electrodes after dis-
connecting from the Multi-Channel ISE/pH/mV/ORP/Temperature
ion sensitive meter (Thermo Orion-920A, USA) were kept at about
4 ◦ C in a refrigerator, along with the buffer solution. That is, the elec-
trodes were always placed in a buffer solution and connected to the
ion sensitive meter whenever the measurements were carried out.
For any subsequent measurements over the time, the electrodes
were cleaned with the deionized water first and later, were placed
into a freshly prepared phosphate buffer solution.
3.2.6. Effect of interferents on the biosensor response
The use of enzyme based potentiometric biosensors for deter-
mining substrates in complex media like biological fluids poses
interference problems. For the determination of urea, especially in
human blood samples for the medical diagnosis purpose, it is neces-
sary to take into account the interference by Na+ and K+ ions [36,47].
These ions are abundantly present in human and animal body flu-
ids. The general problem of NH4 + ion selective electrode based urea
biosensors is that the potentiometric signal can be fouled by the
interferents like Na+ and K+ ions. Hence, in order to gauge the influ-
ence of the interfering Na+ and K+ ions on the response behavior of
the developed urea biosensors, the electrodes were dipped in 20 ml
of the sample buffer solution containing various combinations of
NaCl and KCl solution concentrations. The calibration curves were
then obtained by the addition of standard urea stock solutions.
Fig. 8 shows the influence of increasing Na+ and K+ concentra-
tions and the response of the fabricated urea biosensors. It can be
seen that as the concentration of these ions increased, the lower
detection limit of urea moved towards higher concentrations. Con-
centrations up to a level of 140 mmol/L for Na+ and 4.5 mmol/L for
K+ ions were tested because these are the normal levels of the ions
in human blood and serum. The observed response in the presence
of the interference ions indicates that the fabricated biosensors can
be applied for the urea detection in real samples of milk, blood,
Fig. 8. Influence of Na+ and K+ concentrations on the response of urea biosensor.
265
Table 1
Urea detection in milk samples.
Milk sample no. Urea concentration Urea concentration
measured by measured by
biosensor (mg/dl) spectrophotometric
technique (mg/dl)
1. Sample-1 23.4 24.0
2. Sample-2 54.9 55.5
3. Sample-3 30.7 31.4
4. Sample-4 (67 mg/dl natural 84.6 86.3
urea presence detected by
the biosensor + 20 mg/dl
added urea)
5. Sample-5 (48 mg/dl natural 75.3 77.2
urea presence detected by
the biosensor + 30 mg/dl
added urea)
serum, and urine for clinical and food analysis. Probably the size
exclusion and/or ion exclusion capabilities of the unique PCS + PEI
matrix might have resulted in the suppression of the interferents.
The DMM is also known to prevent the interference of Na+ and K+
ions towards urea estimation [8].
3.3. Urea biosensor’s applicability to milk samples
In the present investigation, the raw milk samples of cows were
collected from the cattle farms situated around the countryside.
The samples were all skimmed in the laboratory by centrifugation
and then were subjected to urea detection by the above developed
urea biosensor. A few samples of milk were deliberately mixed with
known amounts of urea to test the efficacy of the sensor system. The
urea concentrations detected by the present biosensor system were
compared with the concentrations obtained by the spectrophoto-
metric technique. The results obtained are summarized in Table 1.
The table shows clearly that the fabricated enzyme based
biosensors are capable of detecting urea presence in milk samples
in general and more importantly, the biosensors can detect if there
is any adulteration in milk with urea. Since urea is one of the ingre-
dients in the manufacturing of ‘Synthetic Milk’ [8,18–20,48], the
present ammonium ion sensitive based and urease immobilized
screen printed biosensor system can detect the urea levels and the
adulteration of milk. Milk samples with and without added urea
were preserved with 0.2–0.4% of the commonly used preservative
of hydrogen peroxide. The samples were stored at 4 ◦ C in the refrig-
erator overnight and then investigated to observe for the changes
in urea concentrations utilizing the DMM based urea biosensor. The
initial results indicated that there was no significant change in the
urea contents of the samples, similar to the observations made by
Bector et al. [49] by their chemical method. However, further work
is still in progress in this aspect and also with other preservatives.
Sharma et al. [18] recently reported a method for the estimation
of urea in milk using a standard ammonia electrode. The present
biosensor system adopted low cost and disposable screen printed
ammonium ion sensitive electrodes and the concentrations of the
urea detected were found to be in good agreement with those
obtained by the sophisticated and expensive spectrophotometric
technique. The average urea levels in skimmed milk were generally
found to be higher than those in the whole milk samples. Also, the
preliminary results of the present investigation indicated that the
urea concentrations present in the buffalo milk samples were lower
than that of cow milk samples.
4. Conclusions
A potentiometric NH4 + ion sensitive electrode and Ag/AgCl
reference electrode system has been developed by a simple screen
printing technology and characterized. The ion sensitive polymer
266 U.B. Trivedi et al. / Sensors and Actuators B 140 (2009) 260–266
matrix membrane was formed in the presence of an additional
electrochemically inert filter paper, with silver coating on one side,
leading to a DMM technology based transducer. A detection limit
of 3.0 × 10−5 mol/L could be observed with a response time in the
range of a few seconds against the reference electrode for the test
additions from ammonium chloride stock solution. To obtain the
urea biosensor, the urease enzyme has been immobilized using a
unique PCS + PEI polymer matrix. Subsequently, a potentiometric
urea sensitive biosensor using the NH4 + ion sensitive disposable
electrode in DMM technology as the transducer could also be
developed.
The developed urea biosensor responded rapidly and in a sta-
ble manner to the changes in the test concentrations of urea with a
detection limit of 2.5 × 10−5 mol/L. The biosensor exhibited a good
linear least squares fit (linear regression analysis) with a good cor-
relation coefficient (R2 = 0.989, n = 8). The noise range amounted to
about 1.0 mV. The maximum buffer solution working temperature
has been found to be 313 K. The present double matrix membrane
sensor system has been found to offer a reasonably effective bar-
rier to the interferents like Na+ and K+ ions. The studies showed a
reasonably good storage stability and shelf life. The designed urea
biosensor system has, then, been tested for the detection of urea
levels in the real samples of milk. The results indicated a close agree-
ment between the urea levels detected in the milk samples by the
present biosensor system with those obtained by the spectropho-
tometric technique. The reported biosensor system, the authors
believe, should also be able to detect any possible contamination of
milk by synthetic milk of a kind.
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Biographies
U.B. Trivedi received his M.Sc. degree in electronics in 1999 from Sardar Patel Uni-
versity, Vallabh Vidyanagar, Gujarat. He later secured a Ph.D. in electronics in 2007
from the same university, for his research work on the development of biosensors
for food and clinical analyses. He is presently working as a senior technician in the
Department of Electronics, Sardar Patel University. His current areas of research
interest are biosensors, bioelectronics and instrumental methods of analysis.
D. Lakshminarayana received his M.Sc. and Ph.D. degrees in Physics from Sardar
Patel University, Vallabh Vidyanagar, Gujarat in 1976 and 1983 respectively. He is
presently working as a professor in the Department of Electronic Science, Sardar
Patel University and has guided doctoral theses in the fields of semiconductor thin
films and biosensors. He has several research publications in the fields of semi-
conductor single crystal growth, thin film science, gas sensors and biosensors. His
current research interests are mainly connected with electronic materials science
and devices, biosensors and nanoscience.
I.L. Kothari received his M.Sc. degree in botany in 1968 from M.S. University of
Baroda, Vadodara, Gujarat and later, a Ph.D. degree in Botany from Sardar Patel Uni-
versity, Vallabh Vidyanagar, Gujarat, in 1974. Since 1997 he has been a professor
at the Department of Biosciences, Sardar Patel University and has guided doctoral
theses in the fields of botany, microbiology and biotechnology. His current areas of
research include plant morphogenesis, environmental & agricultural microbiology
and biosensors.
N.G. Patel received his M.Sc. degree in physics in 1978 from Sardar Patel Univer-
sity, Vallabh Vidyanagar, Gujarat, India and later, a Ph.D. degree in physics from the
same university in 1984. He functioned as the professor and head of the Depart-
ment of Electronics, Sardar Patel University, Vallabh Vidyanagar during the period
1990–2002. He later moved to University of North Florida, USA and is presently asso-
ciated with Department of Chemistry and Physics as a faculty. His areas of research
interest are in the fields of semiconductor thin film devices, instrumentation, gas
sensors and biosensors.
H.N. Kapse received his M.Sc. degree in electronics in 1999 from Sardar Patel Uni-
versity, Vallabh Vidyanagar, Gujarat. He later secured a Ph.D. in electronics in 2004
from the same university. He is presently working as a lecturer in instrumentation at
the Institute of Science and Technology for Advanced Studies and Research (ISTAR),
Vallabh Vidyanagar, Gujarat. His current research interests include gas sensors and
biosensors.
K.K. Makhija received his M.Sc. degree in physics in 1968 from the Vikram Uni-
versity, Ujjain (M.P.) and later, a Ph.D. degree in electronics from the Sardar Patel
University, Vallabh Vidyanagar, Gujarat in 1996. Since 1996 he worked as a reader
at Department of Electronics, Sardar Patel University, Vallabh Vidyanagar, before his
retirement from university service in October 2008. He is currently associated with
the Department of Electronics, Sardar Patel University as a visiting faculty. His cur-
rent areas of research include semiconductor thin film active and passive devices,
gas sensors and biosensors.
P.B. Patel received his M.Sc. degree in electronics in 1993 from Sardar Patel Univer-
sity, Vallabh Vidyanagar, Gujarat. He is presently working as a senior lecturer in the
Department of Electronic Science, Sardar Patel University. His current research inter-
ests include electronic materials science and devices, sensors and microprocessor
interfacing circuits.
C.J. Panchal received his M.Sc. degree in applied physics in 1990 from M.S. Univer-
sity of Baroda, Vadodara, Gujarat and Ph.D. degree in electronics in 1995 from Sardar
Patel University, Vallabh Vidyanagar, Gujarat. He is presently working as a reader in
the Applied Physics Department of M.S. University of Baroda, Vadodara. He worked
extensively in the areas of semiconductor thin film active and passive devices. His
current areas of research include semiconducting thin film hetero-structures, sen-
sors and development of high power diode lasers.