Analytica Chimica Acta 974 (2017) 54e62

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Analytica Chimica Acta

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

Trace level and highly selective determination of urea in various real

samples based upon voltammetric analysis of diacetylmonoxime-urea
reaction product on the carbon nanotube/carbon paste electrode
Taher Alizadeh a, *, Mohammad Reza Ganjali a, b, Faride Rafiei a
a
Department of Analytical Chemistry, Faculty of Chemistry, University College of Science, University of Tehran, P.O. Box 14155-6455, Tehran, Iran
b
Biosensor Research Center, Endocrinology & Metabolism Molecular-Cellular Sciences Institute, Tehran University of Medical Sciences, Tehran, Iran

h i g h l i g h t s g r a p h i c a l a b s t r a c t

Diacetylmonoxime reaction with

urea led to an electroactive product.

Urea was indirectly determined by
square wave voltammetry of the re-
action product.

A graphite/MWCNTs paste electrode
was used as the electrochemical
transducer.

Reaction condition and electrode
composition was very effective on
the method.

Linear range of 107-102 mol L1
and detection limit of 52 nmol L1
were obtained.

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

Article history: In this study an innovative method was introduced for selective and precise determination of urea in
Received 21 October 2016 various real samples including urine, blood serum, soil and water. The method was based on the square
Received in revised form wave voltammetry determination of an electroactive product, generated during diacetylmonoxime re-
5 April 2017
action with urea. A carbon paste electrode, modified with multi-walled carbon nanotubes (MWCNTs) was
Accepted 10 April 2017
Available online 26 April 2017
found to be an appropriate electrochemical transducer for recording of the electrochemical signal. It was
found that the chemical reaction conditions influenced the analytical signal directly. The calibration
graph of the method was linear in the range of 1  107- 1  102 mol L1. The detection limit was
Keywords:
Urea
calculated to be 52 nmol L1. Relative standard error of the method was also calculated to be 3.9% (n ¼ 3).
Carbon nanotube The developed determination procedure was applied for urea determination in various real samples
Voltammetric method including soil, urine, plasma and water samples.
Diacetylmonoxime © 2017 Elsevier B.V. All rights reserved.

1. Introduction
Urea is one of the most important nitrogen containing organic
compounds. It is a side product of protein degradation in living
* Corresponding author.
E-mail addresses: talizadeh@ut.ac.ir, taa_55@yahoo.com (T. Alizadeh).
organs [1,2]. The created urea is constantly released in blood and its
proper balance in blood is necessary for renal health and overall
wellbeing. The normal concentration of urea in blood varies from
1.7 to 8.3 mM. However, sometimes urea level in blood is increased
up to 100 mM under pathophysiological conditions [3,4].
More than 90% of urea, produced in the world, is utilized as a
nitrogen-containing fertilizer. For this reason, the presence of urea

http://dx.doi.org/10.1016/j.aca.2017.04.039
0003-2670/© 2017 Elsevier B.V. All rights reserved.
 T. Alizadeh et al. / Analytica Chimica Acta 974 (2017) 54e62
in soil and water is growing due to agricultural activities and
municipal wastewaters. Therefore, urea measurement is important
in various fields including foods science, agricultural processes,
environmental monitoring and medical diagnosis [5,6].
Most of the analytical methodologies, applied for urea deter-
mination, are based on the measurement of ammonia, produced by
catalyzed hydrolysis of urea via urease enzyme. Different analytical
methods including potentiometry [7e10], spectrophotometry [11],
amperomety [12e17] and conductometry [18e20] have already
been used for measuring the ammonia produced during the
described enzymatic reaction [21e23]. Although, potentiometric
based transducers are the most commonly used with universal
acceptance, these still have some problems like low sensitivity and
metal ion interference effects [2,24]. Low stability due to desorp-
tion of enzyme from the immobilizing material during measure-
ment, for those approaches that utilize incorporation of urease into
a polymer film by the immobilization, is the other essential limi-
tation of this approach [2]. Also, ammonia could be found in bio-
logical and other samples and act as interference in urea
measurement with all of the above mentioned methods [25]. The
other urease based approaches also suffer from the same
limitations.
Urea can also be measured by direct methods like Infrared
spectrometry [26,27] and high performance liquid chromatography
[28e30]. Urea measuring is usually performed in the complex
samples and thus is affected by the presence of different in-
terferences. These interferences must be removed by some pre-
treatments for precise measurement [1,31]. High dilution of the
samples before the analysis has been also proposed for urea anal-
ysis in the complex samples. However, such an approach needs a
determination method of very low level detection limit [32].
One of the most well-known and simple methods, utilized
usually for urea determination, is a colorimetric method, proposed
by Fearon et al. [33]. In this approach urea reacts with diacetyl
monoxime in the presence of an oxidizing agent in strong acidic
medium. The urea is determined by measurement of the absor-
bance of formed colored product. This method has some serious
limitations such as changing color of reaction product with time
and light, low sensitivity and non-linear calibration curve [13].
Many researchers tried to eliminate this limitation [34]. However,
the modified methods have been only applied for the samples
containing high amount of urea like urine and blood, meaning that,
they are not applicable for samples with low urea concentration
[35].
In this research a new and innovative method was proposed for
urea measurement at low levels in different real samples with high
precision and selectivity. The proposed method is based on the
electrochemical determination of the electroactive product, created
as a result of urea reaction with diacetylmonoxime. Thanks to big
electrochemical signal of the reaction product and utilizing a car-
bon nanotube-modified carbon paste electrode, the developed
method was capable of determination of urea at very low amounts.
The proposed method was led to achievement to lower detection
limit, compared to the colorimetric method, based on the same
chemical reaction. Furthermore, the intrinsic selectivity of the
electrochemical technique led to highly selective urea determina-
tion method.
2. Experimental
2.1. Instruments and reagents
Urea and graphite powder were supplied from Fluka (Buchs,
Switzerland). Diacetylmonoxime and sulfuric acid was purchased
from Sigma (Munich, Germany). All other reagents were of
55
analytical grade and supplied by sigma (Munich, Germany). Elec-
trochemical data were obtained using a potentiostat/galvanostat
model PGSTAT302, Metrohm. The measurements were performed
in a three electrode system: working electrode (The CNT-modified
carbon paste electrode), counter electrode (platinum) and refer-
ence electrode (Ag/AgCl). Standard solutions of urea were freshly
prepared before the measurements.
2.2. Reaction procedure
Diacetylmonoxime (76 mg) was added to a volumetric flask
containing sulfuric acid. Then, 2.5 mL of urea stock solution was
added to the prepared solution. The volume of the reaction solution
was adjusted to 50 mL in which the concentration of sulfuric acid
was 4 mol L1. The volumetric flask was shaken for a few seconds in
order to mix the contents. Then, it was sealed and placed in a water
bath (fixed at 80  C) for 30 min. Then, the flask was removed from
the water bath and cooled to room temperature.
2.3. Preparation of the working electrode
To prepare the working electrode, 8.5 mg of MWCNTs was added
to 100 mg of graphite and homogenized in a mortar for 10 min.
Subsequently, 30 mg of n-eicosane (used as the binder) was melted
at 45e50  C. Then, the graphite/CNT blend was added to the melted
binder and mixed with it by a stainless steel spatula. The final paste
was used to fill a hole (2.00 mm in diameter, 3 mm in depth) at the
end of an electrode body, previously heated at 45  C. After cooling
at room temperature, the excess of solidified material was removed
by the aid of a paper sheet. The electrode can be reused after each
experiment by rubbing out a thin layer of the electrode surface on
an ordinary paper sheet.
2.4. Electrochemical measurements
The electrodes were inserted into an electrochemical cell con-
taining 20 ml of reaction solution. Then, the square wave voltam-
metry (SWV) technique was applied for the determination of urea.
For this aim, the square wave potential having amplitude of 150 mV
and frequency of 250 Hz was scanned in the range of 0.4e1.3 V and
the oxidative current signal obtained (was recorded and used for
the quantitative determination of urea, with respect to the cali-
bration curve, established previously using determined concen-
trations of urea.
2.5. The measurement of urea in real samples
In order to analyze urea content of urine sample, 1 ml of urine
was transferred to a 50-mL volumetric flask containing sulfuric acid
(4 mol L1) and then, 76 mg of diacetylmonoxim was added to the
solution. The flask was then transferred to a water bath (80  C) and
kept there for 30 min.
For blood serum analysis, the proteins contents of the samples
were first precipitated by adding 1.0 ml of sulfuric acid solution to
0.2 ml of serum. After centrifugation, 0.2 ml of supernatant fluid
was taken for urea analysis according to the procedure described
above.
For analysis of soil samples, 1 g of each soil sample was accu-
rately weighed and introduced into a 30 mL-centrifuge tube. Next,
15 mL of water was added and the mixture was shaken for 15 min.
After centrifugation at 6000 rpm and for 10 min, the supernatant
was filtered through a filter paper and used for its urea content
analysis. The soil samples having no urea were also spiked with
determined amount of urea.
Three kind of water samples including sewage, aqueduct and
56 T. Alizadeh et al. / Analytica Chimica Acta 974 (2017) 54e62

city water were analyzed with this method. Aqueduct water sam-
ples were collected from near agricultural lands. The sewage
sample was diluted 10 times with water and the other samples
were used without dilution. Since, the city water had not urea it
was spiked with determined amount of urea. After, the rest of the
procedure was applied as described above.

3. Results and discussion

3.1. Electrochemical behavior of the reaction product

The reaction of urea with diacetylmonoxime produces a yellow-

colored compound with an absorbance band at about 489 nm
which can be used for the colorimetric determination of urea
(Fig. 1). According to the literature [33e35] this compound (colored
reaction product) is 3-hydroxy-5,6-dimethyl-1,2,4triazine (HDMT).
However, the method sensitivity is low and thus trace level deter-
mination of urea can not be possible by the method. This is because
of low absorption coefficient of the formed colored product of
HDMT. However, we found that the colored compound, created as a
result of urea reaction with diacetyl monoxime (3-hydroxy-5,6-
dimethyl-1,2,4 triazine), is an electroactive compound which can
be detected by means of an appropriate electrochemical transducer.
Fig. 2 shows the cyclic voltammetry response of 3-hydroxy-5,6-
dimethyl-1,2,4triazine, recorded in urea-diacetylmonoxime reac- Fig. 2. Cyclic voltammetry response of 3-hydroxy-5,6-dimethyl-1,2,4triazine recorded
tion media at various scanning rates. Moreover, based on the cyclic in urea-diacetylmonoxime reaction media at various scan rates of 10 (a), 50(b), 100(c),
150 (d), 350 (e), 800(f) and 1000(g) mVs1; magnified zone of the cathodic peaks of
voltammetry results, some electron transfer reactions, coupled with
the depicted voltammograms at the cited scan rates (inset); urea
different chemical reactions, depicted in Fig. 3, are proposed as a concentration ¼ 104 M, obtained with carbon paste electrode (potential scan
mechanism for the cyclic voltammetry behavior of the urea- range: þ0.4  þ1.3 V and vice versa).
diacetylmonoxime reaction product. As can be seen in Fig. 2, the
cyclic voltammetry response of HDMT (see as example curve “b”)
exhibits two overlapping peaks, at about 0.75 and 0.86 V, respec-
tively. The first peak is likely related to the oxidation of 3-hydroxy- HMDT via two electron/two proton process. Since, the reduction
peaks are absent in low scan rates; it seems that both (3a) and (2b)
5,6-dimethyl-1,2,4 triazine (1a) to 3-hydroxy-5,6-dimethyl-
1,2,4triazine 2-oxide (3a) which occurs at þ 0.75 V vs. Ag/AgCl, are instable products, hydrolyzed in aqueous media, producing
another chemical compound being electro-inactive. It must be
supposed to be a two-electron/two-proton process (two successive
one-electron involved electrochemical oxidation). Likely, the radical mentioned herein that the mechanism represented in Fig. 3 and
described above is not imminent. It is only a guess and shall by
compound (2a), produced during the first step of the electro-
chemical oxidation, undergoes a dimerization reaction, leading to investigated precisely via performing another separate study.
another electroactive compound (1b). Therefore, the peak at þ 0.86 V
may be assigned to the two-electron oxidation of the HDMT dimers. 3.2. Evaluation of the electrochemical transducer type and
However, we think that as described radical compound can also voltammetric method kind
further oxidized via one-electron process as competitive route to
dimerization reaction, leading to a product (2b) which may be Electrochemical oxidation of urea-diacetylmonoxime reaction
instable and is converted to an electro-inactive compound (3b) with product was investigated on various electrochemical transducers
a definitive rate constant. As can be seen in Fig. 2, increasing of scan including, carbon paste electrode (CP), carbon paste electrode
rate of CV leads to the appearance of two new reduction peaks at modified with MWCNTs (CP-CNT) and carbon paste electrode
about þ0.60 and 0.35 V. Both of these peaks increase with increasing modified with graphene (CP-graphene). Fig. 4 shows the square
of the potential scanning rate. This is an indication for the presence of wave voltammetry responses of HDMT on different electrodes at
a chemical reactions coupled with the electron transfer reactions. the same conditions. According to the voltammograms depicted in
The first reduction peak is assigned to reduction of oxidized dimer Fig. 4, a significant response is generated for HDMT on CP electrode.
product (2b) and the second reduction peak is likely assigned to the Furthermore, the HDMT signal increases by the modification of CP
reduction of 3-hydroxy-5,6-dimethyl-1,2,4triazine 2-oxide (3a) to electrode either with graphene or CNT. However, it is clear that the

Fig. 1. Chemical reaction of urea with diacetyl monoxime to produce 3-hydroxy-5,6-dimethyl-1,2,4triazine as an electroactive compound.
 T. Alizadeh et al. / Analytica Chimica Acta 974 (2017) 54e62 57

Fig. 3. Probable mechanism for the electron transfer reactions coupled with different chemical reactions, assumed to be responsible for the cyclic voltammetry behavior of the urea-
diacetylmonoxime reaction product.

transducer for the recording of indirect signal of urea. The bigger

signal on the CP-CNT can be attributed to the increase of the surface
area of the CP electrode in the presence of the CNT electrode.
Fig. 5 displays scanning electron microscopy (SEM) image of the
carbon paste electrode modified with MWCNTs. In this image, one
can see the graphite sheets on which the MWCNTs are extensively

Fig. 4. Comparison of square wave voltammetry responses of 3-hydroxy-5,6-dimethyl-

1,2,4triazine, created in the urea-diacetylmonoxime reaction media, on CP, CP-CNT and
CP-CNT electrodes; diacetylmonoxime/urea ¼ 30/1, H2SO4 concentration ¼ 2 M,
amplitude ¼ 0.15 V, frequency ¼ 150 Hz, reaction time ¼ 60 min, reaction
temperature ¼ 80  C; electrodes composition: binder ¼ 21.5%, graphite ¼ 74.5%, CNT
and graphen ¼ 4%, blank: SWV signal of the CP-CNT electrode in the absence of urea.

signal enhancement effect of CNT is better than that of graphene. Fig. 5. Scanning electron microscopy image of the carbon paste electrode modified
Therefore, CP-CNT was chosen as an appropriate electrochemical with MWCNTs.
58 T. Alizadeh et al. / Analytica Chimica Acta 974 (2017) 54e62
lied, leading to a relatively homogenous surface with increased
effective area. Such an increased surface area may be responsible
for higher electrochemical responses of the CP-CNT, compared to
the CP electrode.
In order to investigate the effect of voltammetric method kind
on the aimed analytical signal, two highly sensitive voltammetric
techniques including pulse voltammetry (DPV) and square wave
voltammetry (SWV), were tested in this study. The experiments
showed that the magnitude of current signal of the reaction
product, obtained via the SWV, was greater than that recorded
using DPV, at the same conditions. Therefore, the first method was
selected for the determination of the reaction product. The effect of
the excitation wave pulse amplitude of the SWV on the reaction
product signal intensity was also evaluated. The effect of this
operating variable was studied over the range of 50e200 mV and it
was concluded that in order to assure maximum peak current,
150 mV pulse amplitude was the ideal choice for this operational
parameter. It was also found that the maximum peak height was
observed at the frequency of 250 Hz.
3.3. Optimization of the electrode composition
For the optimization of the CNT content of the electrode, the CP-
CNT electrode was prepared with the fixed amount of carbon and
binder, while the amount of CNT was altered. The resulting elec-
trodes were then applied for indirect determination of urea. The
obtained results are presented in Fig. 6. It can be seen that, the
current response of the sensor to urea increases with increasing of
the CNT amount of the electrode. In order to maintain the current
intensity characteristic of the CNT-based electrode, the electrode
having 0.005 g (6% w/w) of CNT was chosen as the best electrode
for the electrode preparation. It was also found that the presence of
n-eicosane, higher than 0.03 g, led to a considerable decrease in the
electrode response to urea. Therefore, the optimum amount of CNT,
carbon and n-eicosane, were chosen to be 0.0048 g, 0.1016 g and
0.03 g, respectively.
Fig. 6. The effect of CNT content of the CP-CNT electrode on the SWV signal magnitude
eof hydroxy-5,6-dimethyl-1,2,4triazine created as a result of urea reaction with diac-
etylmonoxime; condition: diacetylmonoxime/urea ¼ 30/1, H2SO4 concentration ¼ 2 M,
amplitude ¼ 0.15 V, frequency ¼ 150 Hz, reaction time ¼ 60 min, reaction
temperature ¼ 80  C.
3.4. The effect of urea-diacetylmonoxime reaction conditions on the
electrochemical signal
To achieve maximum efficiency of the reaction of urea with
diacetylmnoxim and thus optimal electrochemical signal, the fac-
tors influencing the chemical reaction including diacetylmonoxim
and sulfuric acid amount, reaction time and reaction temperature
were investigated according to one at a time strategy.
Diacetylmonoxim is the main reactant in this procedure which
takes part in a reaction with urea. In order to investigate the effect
of diactetylmonoxim amount on the finally obtained analytical
signal, various ratios of diactetylmonoxim/urea were checked in the
reaction. As shown in Fig. 7(I), the analytical signal of the method
increases as the diacetylmonoxime/urea molar ratio is increased
from 1 to 30 and then it was relatively kept constant. The aim of this
research was to measure urea in the samples containing unknown
amount of urea. Therefore it is necessary to add diacetylmonoxime
more enough to be sure that all urea molecules will react with
diacetylmonoxime. It is well-known fact that two factors including
stoichiometric molar ratio of the reactants and reaction time in-
fluence the reaction product creation efficiency which affect thus
the analytical signal. Based on our studies, the kinetic of this re-
action is slow. On the other hand, because of the analytical
importance, the reaction is conducted in very low concentration of
urea (down to about 107 M). It means that the probability of the
molecular collision between urea and diacetylmonoxim is low that
makes the reaction time longer. By increasing the diac-
etylmionoxim/urea mole ratio to 30 the probability of molecular
collision is getting high enough to complete the reaction that
produce the electroactive product. Based on the result obtained and
the above mentioned interpretations, it was decided to fix the
diacetylmonoxime/urea mole ratio equal to 30 or even higher
values in the chemical reaction, conducted to create the analytical
signal of urea.
It was also observed that the increase in the sulfuric acid con-
centration (H2SO4), led to current signal enhancement. This
continued to about 4 mol L1 of H2SO4. However, at concentrations
higher than this amount, the signal decreased considerably
(Fig. 7(II)). This means that an optimal concentration of H2SO4 have
to be used in the reaction. The optimal point observed in the curve
related to the optimization of H2SO4 concentration (Fig. 7 (II)), roots
from two distinct role of sulfuric acid in the method developed. As
the first role, sulfuric acid has positive effect on the chemical re-
action efficiency. This means that the presence of high concentra-
tion of H2SO4 is essential requirement in the initiation and
conduction of the chemical reaction, involved in this analytical
method. Such a harsh media is required really to get the described
electroactive product from the reaction of urea with diac-
etylmonoxime; otherwise, no reaction happens. This is not only our
finding in our study but also has been reported in different papers,
dedicated to colorimetric analysis of urea-diacetylmonoxime re-
action product [13,36,37]. Also, visual inspection of the reaction
product at high concentration of urea revealed that the colored
compound of HDMT is created efficiently only at enough concen-
tration of sulfuric acid; meaning that, for example, at 2 M of acid the
product was really pale; this was also the case with acid concen-
tration of 5 M.
As the second role, it functions as supporting electrolyte in the
voltammetric analysis step; since, the electrochemical determina-
tion is carried out directly in the reaction media, just after
completion of the chemical reaction. In other words, the electro-
analysis condition is exactly dictated by chemical reaction condi-
tion which is harsh in nature, as mentioned above. The reaction that
gives HMDT (as the electroactive representation of urea) involves a
dehydration step which is conducted via sulfuric acid, added to the
 T. Alizadeh et al. / Analytica Chimica Acta 974 (2017) 54e62
Fig. 7. The effect of different reaction factors including diacetylmonoxime/urea molar ratio (I), sulfuric acid concentration (II), reaction time (III) and reaction temperature (IV) on
the indirect analytical signal of urea; SWV condition: amplitude ¼ 0.15 V, frequency ¼ 150 Hz.
reaction media. When the concentration of sulfuric acid is low, the
dehydration step cannot be done completely, leading to small
analytical signal. However, by increasing of the acid concentration,
the dehydration is performed completely and thus the signal in-
creases. On the other hand, in the electrochemical reaction, sulfuric
acid functions as the source of conducting ions, required for the
performing of the voltammetric determination procedure.
Increasing of sulfuric acid concentration decreases the ionization of
sulfuric acid as result of decreasing of relative permittivity of the
media (H2SO4/H2O mixture). This, results in declining in the vol-
tammetric signal. Therefore, the optimum amount of 4 mol L1 was
adopted for acid sulfuric concentration, according to the results.
As we have described within the text, the indirect electroanal-
ysis of urea, which was performed by SWV analysis of the product
of urea with diacetylmonoxime, is executed exactly in chemical
reaction media which is highly concentrated H2SO4. In other
words, the electroanalysis condition is exactly dictated by chemical
reaction condition which is harsh in nature as you mentioned. Such
59
a harsh media is required really to get the described electroactive
product from the reaction of urea with diacetylmonoxime; other-
wise, no reaction happens. This is not only our finding in our study
but also has been reported in different papers, dedicated to color-
imetric analysis of urea-diacetylmonoxime reaction product. Also,
visual inspection of the reaction product at high concentration of
urea revealed that the colored compound of HDMT is created effi-
ciently only at enough concentration of sulfuric acid; meaning that,
for example, at 2 M of acid the product was really pale; this was also
the case with acid concentration of 5 M.
The effect of the reaction time on the analytical signal is shown
in Fig. 7(III). According to this figure, the increase in the reaction
time leads to an intensive growth in the current intensity till about
30 min and afterwards, the response of the electrode does not grow
by increasing of the reaction time. The increased current intensity
as result of reaction time enhancement can be explained by kinetic
of the reaction which seems to be significantly slow. In order to
achieve maximum efficiency, 30 min was selected for the reaction
60 T. Alizadeh et al. / Analytica Chimica Acta 974 (2017) 54e62

time. The results obtained are summarized in Table 1. It is worth noticing

The effect of reaction temperature on the electrode signal was
also investigated. The obtained results are shown in Fig. 7 (IV). As
can be seen, the increase in the reaction temperature leads to an

intensive growth of reaction efficiency up to temperature of 80 C
and afterwards, the response of the electrode does not grow further
by increasing of the reaction temperature. The first increase can be
attributed to the slow kinetic of the reaction that is enhanced by
increasing of temperature, leading to higher amount of the elec-
troactive product and thus greater signal. However, urea is sensitive
to temperature and starts to decompose to ammonia and CO2 at
higher degrees of temperature, leading to a distinct decrease in the
analytical signal.
that the tolerance limit was adopted as the maximum concentra-
tion of the examined species that caused a relative error of 5% in the
analytical signal of urea. According to the depicted results, over
1000-fold concentration excess of glucose, thiourea, uric acid,
ammoniac do not interfere with the urea response. Furthermore,
the obtained results indicate that tryptophan, cysteine, arginine
have no interference effect on the urea determination until 500,
400 and 200-fold molar excess, respectively. The interference effect
of some usual cationic and anionic species were also checked and
according to the results obtained (shown in Table 1) most of them
are not interfering agent in this method up to 500-fold molar
excess.

3.5. Analytical characteristics

Table 1
Interference levels for some tested molecules in the deter-
The calibration graph of the electrode, depicted in Fig. 8 (I),
mination of urea by the developed method
shows a linear relationship over very wide concentration range of (urea ¼ 1  106 mol L-1).
urea (1  107- 1  102 mol L1). However, one can distinguish a
Interference level Species
separate dynamic linear range at lower concentration domain of
the depicted plot (in the concentration range of 1  107- >1000 Ammoniac
1  105 mol L1, shown in Fig. 8 (II) and (III) as the calibration plot >400 Cysteine
>1000 Uric acid
and voltammetric curves, respectively). The detection limit of >200 Arginine
52 nmol L-1 was calculated for the developed based on S/N ¼ 3. >500 Tryptophan
Each point of the calibration graph is the average of three replica- >1000 Thiourea
tions. These results indicate that the electrode exhibits very wide >1000 Glucose
>500 Naþ
determination range as well as excellent detection limit for urea
>500 Kþ
determination. The relative standard deviation of 5 separate de- >500 Cl
terminations was found to be 3.9%. >500 NO3
Following the optimization of the parameters and establish- >500 Ca2þ
ment of the determined method, various species were examined >500 SO2-
4
>200 PO3-
4
with respect to their interference with the determination of urea.

Fig. 8. Calibration curve obtained for the developed method at the optimized conditions (I); linear range of the general calibration curve at lower concentration domain (II) and the
related square wave voltammograms at different concentration of urea: a) 107 M, b) 5  107 M, c) 106 M, d) 5  106 M, e) 105 M (III).
 T. Alizadeh et al. / Analytica Chimica Acta 974 (2017) 54e62 61

Table 2
Determination of urea in different samples by this method (n ¼ 3) and reference Elisa technique (n ¼ 3).

RSD Recovery (%) Found (mmol L1) Added (mmol L1) Elisa Sample

3.25 97.3 230.7 e 237.0 Urine

3.44 101.5 291.0 50 237.0 Urine
4.56 98.3 5.8 e 5.9 Blood serum
3.12 98.7 7.73 2 5.9 Blood serum
3.57 98.3 8.3  103a e 8.4  103a Soil1
1.29 100.4 2.8  106a e 2.8  106a Soil2
3.75 104.2 3.1a 3.0a e Soil3
4.25 84.6 0.09 e 0.11 Water1
3.07 102.2 21.7 e 21.2 Water2
3.67 87.7 8.7  104 1.00  103 e Water3
a
Concentration unit: mgg1.

The fabricated electrode with optimized composition was used
for more than 7 months and no variation was found in the electrode
response (regarding confidence level of 95%). Furthermore, the
reproducibility of the electrode was checked for 10 separately
fabricated electrodes via measurement of a fixed amount of urea
with the constructed electrode and executing the developed mea-
surement procedure. The reproducibility of the electrode was
found to be about 4.1% which is slightly more than the measure-
ment repeatability found for the method.
3.6. Application of the urea analysis method in real samples
The utility of the optimized urea determination method was
checked by the application of the method for the determination of
urea in various real samples. Some of the samples were spiked with
determined amount of urea; however, at such cases the samples
were tested regarding their urea contents, before addition of urea.
The results of urea analysis by the developed procedure in real
samples are summarized in Table 2. The urea contents of the
samples were also measured using a standard method (Elisa) to
investigate the significant of the determination results obtained via
the developed method. The results depicted in the table show the
applicability of this method for determination of urea in various
real samples.
4. Conclusion
In conclusion, an innovative electrochemical method was
developed for trace level determination of urea in different real
samples including urine, blood serum, soil and tap water samples.
The method was based upon SWV determination of an electro-
active product, created as a result of urea reaction with diac-
etylmonoxime. The developed method provided very wide
dynamic linear range and appropriate detection limit. It was found
that all chemical reaction factors including diacetylmonoxime/urea
mole ratio, sulfuric acid concentration, reaction temperature and
reaction time were crucially important in the analytical signal, used
for urea determination. Furthermore the presence of CNT in the CP
electrode improved the indirect urea signal and also the CNT con-
tent of the electrode was demonstrated to be effective on the
analytical signal created.
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