Development of non-enzymatic strip for simple and selective determination of urea in water and biological samples

Article abstract of DOI:10.1007/s13738-011-0048-1

A simple and selective method for the determination of urea based on the paptode technique is described. The sensor was constructed by immobilizing an ionophore on a TLC strip. The procedure is based on the nucleophilic displacement of urea with tetrachlorop-benzoquinone (chloranil) as an ionophore, and the formed violet-color product was detected using a flatbed scanner. The color of each spot was analyzed to red (R), green (G) and blue (B) values from 0 to 255 using a program written in visual basic (VB) programming language. The calibration graph obtained with the proposed sensor was linear over the range of 0.05-10.00 mg L^-1 with a detection limit of 0.01 mg L^-1 for urea. Parameters such as pH and concentration of chloranil were optimized. The proposed sensor was successfully applied for the determination of urea in bovine serum, urine and tap water samples.

Full text of DOI:10.1007/s13738-011-0048-1

J IRAN CHEM SOC (2012) 9:383–388
DOI 10.1007/s13738-011-0048-1
ORIGINAL PAPER
Development of non-enzymatic strip for simple and selective
determination of urea in water and biological samples
A. Abbaspour
•
F. Norouz-Sarvestani E. Mirahmadi
•
Received: 26 May 2011 / Accepted: 29 September 2011 / Published online: 3 January 2012
Ó Iranian Chemical Society 2012
Abstract A simple and selective method for the deter-
mination of urea based on the paptode technique is
described. The sensor was constructed by immobilizing an
ionophore on a TLC strip. The procedure is based on the
nucleophilic displacement of urea with tetrachloro-
p-benzoquinone (chloranil) as an ionophore, and the
formed violet-color product was detected using a flatbed
scanner. The color of each spot was analyzed to red (R),
green (G) and blue (B) values from 0 to 255 using a program
written in visual basic (VB) programming language. The
calibration graph obtained with the proposed sensor was
in food and water sources [3]. Thus, there is a great demand
for determination of urea in other fields, such as food sci-
ence and environmental monitoring as well as clinical
applications.
Methods for the determination of urea can be grouped
into three categories: enzymatic hydrolysis, color-forming
reactions, and chromatographic separations [4]. These
methods include conductance [5, 6], potentiometry [7, 8],
voltammetry [9], fluorometric [10], amperometric [11],
spectrophotometry [12, 13], and chromatography [14].
However, some of these methods suffer from complicated
sample pretreatment steps and lack of selectivity and short
linear dynamic range. In addition, the methods that are based
on enzymatic systems have a drawback of requiring large
amounts of expensive enzymes and spectrophotometric
methods are time consuming and labor intensive. On the
other hand, the methods such as chromatography require
sophisticated and expensive instruments.
-
1
linear over the range of 0.05–10.00 mg L with a detection
-
1
limit of 0.01 mg L for urea. Parameters such as pH and
concentration of chloranil were optimized. The proposed
sensor was successfully applied for the determination of urea
in bovine serum, urine and tap water samples.
Keywords Sensor ꢀ Paptode ꢀ Scanner ꢀ Urea ꢀ Chloranil ꢀ
RGB value
Test strips (dry reagent pad strips) are becoming more
popular due to rapid quantitative determination of clini-
cally relevant parameters in urine, serum, plasma and
whole blood. Our group has previously introduced paptode
methodology for construction of a full-range pH sensor
[15] and speciation of iron (II), iron (III) [15, 16], deter-
mination of ascorbic acid in Vitamin C Tablets [17],
development of a dopamine [18], and hydrazine sensor
[19].
Introduction
Urea is a molecule that temporarily stores the nitrogen
produced by the metabolism of proteins and it is the major
end product of nitrogen excretion in mammals [1]. Indeed,
an increase of urea concentration in blood and its reduced
level in urine are strong indication of renal dysfunction [2].
Therefore, determination of urea in these matrices is an
essential task in clinical laboratory. Urea is also abundant
In paptode, instead of immobilizing of an ionophore on
a hydrophobic or hydrophilic polymer as in optodes, sim-
ply a paper or other ordinary porous material such as cotton
or clay and even TLC can be used as a substrate support for
reagents. CCD camera or flatbed scanner is used for
obtaining data, which makes it possible to use non-trans-
parent supporting substrates. Unlike visible spectroscopy in
which the absorption or emission of radiation is measured,
A. Abbaspour (&) ꢀ F. Norouz-Sarvestani ꢀ E. Mirahmadi
Chemistry Department, College of Sciences,
Shiraz University, 71456-85464 Shiraz, Iran
e-mail: abbaspour@chem.susc.ac.ir
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J IRAN CHEM SOC (2012) 9:383–388
in paptode reflection properties of color spots are measured
using a flatbed scanner or CCD camera. So, turbid samples
which are generally not suitable for optical spectroscopy
can be analyzed by means of paptode in which the analytes
are liable to undergo precipitation reaction with ligand can
be measured easily. The output data streams composed of
red, green, and blue (RGB) light intensities.
sodium hydroxide. All standard solutions were prepared by
diluting an appropriate volume of stock solution with
buffer of desired pH in a 10-ml volumetric flask.
Chloranil stock solution was prepared by dissolving
appropriate amount of reagent in tetrahydrofuran (THF)
and diluting to the mark with buffer solution of desired pH.
In this method, not only experimental data, but also
images of supporting substrate or sample solutions (i.e. the
colorful complex) can be saved in a computer to be ana-
lysed with upcoming more powerful programs in future. In
addition, the porosity of supporting substrates in paptode
leads to shorter response times in these sensors compared
with that of optode. Other advantages such as simplicity,
usability and ease of reagent immobilization are consid-
erable in this method.
Procedure
Urea forms a highly violet color product with chloranil.
-
1
First, urea solutions (from 0.01 to 20.0 mg L ) were
placed into the water bath to adjust its temperature to
65 °C. Then, different aliquots of known concentrations of
urea (4 ll) and chloranil solution (4 ll) were injected on
TLC strip (5 9 10 cm). This strip was placed into an oven
whose temperature was adjusted to 90 °C. After 2 min, the
violet spots were formed on top of the strip due to reaction
of analyte with reagent. The strip was scanned and images
were digitized by the written software to R, G and B values.
RGB values vary from 0 to 255 for each of three colors
from spot to spot depending on the urea concentration.
Experimental
Apparatus and software
A (Canoscan-4200F) scanner was used for scanning the
TLC strip. Resolution of the scanner was regulated at
Results and discussion
1
50 dpi. A program written in VB programming language
was developed for analyzing color spots to digital RGB
values [18]. An Eppendorf micropipette was used for
injecting sample solutions on TLC strips. The pH values of
buffer solutions were measured using a 780 Metrohm pH
meter.
Urea reacts with chloranil to give violet color products, the
structures of which depend on the reaction medium. In
general, these reactions involve nucleophilic displacement
of one or two of the chlorine atoms by amino groups.
Chloranil reacts with neutral aqueous solutions of urea
on heating to give 2,5-dichloro-3,6-diamino-6-p-benzo-
quinone (Scheme 1) [20]. This reaction is sensitive to urea
Chemicals and reagents
-
1
in which as little as 0.03 mg L of urea could be detected.
The participation of urea in these reactions can be
explained on the basis of its decomposition to ammonia. In
aqueous solutions, ammonia entities are available as
nucleophile.
All solutions were prepared using reagents of analytical
purity and doubly distilled water. Urea was purchased from
Fluka and tetrachloro-p-benzoquinone (chloranil) was
obtained from Merck. All other reagents were from Merck
and of analytical grade except when specified differently.
Effective intensity for each component of RGB color
was calculated as follow:
-
1
A stock solution of 2.5 g L urea was prepared by
dissolving the required amount of the sample in double
distilled water. Fresh working solutions were prepared
daily. Universal buffer solutions were prepared using acetic
acid (PanreakQuimicaSa), phosphoric acid, boric acid and
Ar ¼ ꢁLogðRs=RbÞ
Ag ¼ ꢁLogðGs=GbÞ
A ¼ ꢁLogðB =B Þ
b
s
b
Scheme 1 Reaction between
chloranil and urea
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J IRAN CHEM SOC (2012) 9:383–388
385
where, A , A and Ab variables refer to the effective
r
g
intensities of R, G and B respectively. R , G and B rep-
s
s
s
resent R, G and B values of sample and R , G and B
b
b
b
represent those of blank, To obtain calibration curves,
effective intensities of R, G and B values were plotted
versus analyte concentration.
Effect of the injection volume
Effect of sample injection volume on the effective intensity
measurements was investigated. The optimum sample
injection volume was obtained to be 4 lL. At high injec-
tion volumes, spot spreading due to diffusion occurred
during the course of the experiment, and consequently the
intensity of colors was decreased.
Effect of chloranil concentration
Fig. 2 Effect of pH on color intensities of strips in fixed urea and
chloranil concentrations
To study the effect of chloranil concentration initially,
solutions of different concentrations of chloranil were
prepared and immobilized on TLC strip. Then 4 ll of urea
-1
.10 mg L urea solutions at different pH values were
0
-
1
solution (4.0 mg L ), pre-adjusted to 65 °C, was injected
on the TLC strip surface, and the strip was placed into an
oven at 90 °C for 2 min to complete the complexation
reaction. After the TLC strip has cooled down to room
temperature, images of violet spots on TLC strips were
recorded using a flatbed scanner for analysis
prepared and their effective intensities were recorded after
complexation reaction. As shown in Fig. 2, the maximum
color intensity was obtained at pH 12, but at this pH, the
sensor could not discriminate between samples with dif-
ferent urea contents, since the concentration of hydroxyl
ions is sufficiently high. In the highly basic media,
hydroxyl reacts with chloranil and produce 2,5-dichloro-3,
Figure 1 shows the plot of effective color intensities
versus chloranil concentrations. Since the effective inten-
6
dihydroxy-p-benzoquinone which develops a violet color
-
1
sity was maximum in 30 mg mL of chloranil, this con-
centration was chosen as the optimum concentration for
subsequent experiments.
[
21]. In addition, color spots were not uniform in pH 12
Fig. 3). The most uniform color distribution was observed
(
at pH 6.3 as illustrated in Fig. 3 and the color intensities at
this pH were higher than the other media (Fig. 2), so this
pH was chosen for the rest of the experiment.
Effect of pH
Finally, pH was optimized to find the range at which urea
would gain the maximum possible sensitivity. A series of
Calibration curves
Changes in color intensity of each color spot was analyzed
using a programm written in VB and the data stream
composed of R, G and B values was obtained. Each pixel
(elements of CCD) represents three values in the range of
0–255 corresponding to R, G, and B light intensities. The
assigned R, G, B values for black color are 0, 0, 0 and for
white color are 255, 255, 255 and for other colors, they are
between these extreme values. The average RGB values of
pixels of each color spot’s picture were taken as the ana-
lytical signals. Figure 4 shows the calibration curves of
urea at optimum conditions. This figure shows a linear
relationship between effective intensities of RGB colors
with urea concentration, and the obtained linear range was
-
1
0
.05–10.00 mg L . In addition, as illustrated in Fig. 4, the
Fig. 1 Effect of chloranil concentration on color intensities of strips
at fixed urea concentrations
variation in color intensity of R and B components with
123

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J IRAN CHEM SOC (2012) 9:383–388
-1
-1
Fig. 3 Color spots of 0.1 mg L urea and 30 g L chloranil at different pH values: 6.3, 8.0, 9.0, 10.0, 11.0 and 12.0
-1
Fig. 4 Calibration graph of urea at optimum condition (3 g L of
chloranil and pH 6.3)
-
1
Fig. 5 Reproducibility of the system at optimum condition (3 L of
chloranil and pH 6.3)
respect to urea concentration is insignificant, while the
color intensity of G component changes rapidly on
changing the urea concentration. Therefore G value was
chosen as an analytical signal and for obtaining a calibra-
tion curve.
intensity. The response time of 2.0 min was achieved in
this study (Fig. 6).
To study the stability of a color spot, an aliquot of urea
-
5.00 mg L ) was injected on top of the TLC strip under
1
(
the optimum experimental condition. Scanning of the strip
was done in the time period of 5, 60, 120, 180, 240, 300,
Reproducibility, repeatability, response time, stability
and detection limit of the system
3
60, 420, 480, 540, 600 and 660 min. No significant
changes in color intensity were observed after exposure for
40 min. Therefore, the sensor is stable at least for 9 h
The reproducibility of the system was studied by means of
three independent TLC strips on which chloranil was
immobilized. The results of reproducibility assessment of
the method are shown in Fig. 5, in which the results are
promising.
5
after injection of the sample.
The detection limit (DL) was calculated according to the
following equation:
ꢀ
ꢁ
Repeatability of the system was studied by means of five
independent spots on TLC strip on which chloranil has
been immobilized. Relative standard deviation (RSD) of
the system was obtained as 1.7%.
KSbk
DL ¼
=
m
where K is the confidence factor which is generally taken to
be 3, Sbk is standard deviation of the blank measurement
(n = 9) and m is the calibration slope. The DL of this
The response time of the system was evaluated under the
optimum experimental conditions at a fixed concentration
-
1
method was 0.01 mg L for G factor. Since R and B
factors have no contribution in calibration curve, we
neglected to determine DL for these factors.
-
1
of urea (5.00 mg L ). This was calculated by measuring
the time required to achieve a 90% value of steady color
1
23

J IRAN CHEM SOC (2012) 9:383–388
387
Interference studies
To study the selectivity of the proposed method, the effect
of various nitrogen-containing species on the determination
-
1
of 1.0 mg L urea was tested under the optimum condi-
tions. The tolerance limit was defined as the concentration
of added species that causes less than ± 2% relative error.
-
1
The results indicated that 100 mg L of amino benzoic
acid, thiourea, citrate, glucose, 2,4-dinitro phenyl hydra-
-
1
0
zine, toloiden and 50 mg L of 4,4 -diamino-diphenyl
methane, 3,4 diamino toluene and acetate did not interfere
in the determination of urea (Table 1).
Analysis of real samples
The standard addition method was used for calculating the
concentration of urea in a tap water and a bovine serum
samples which were spiked with the analyte. Furthermore,
urea concentration of urine was measured by the proposed
method and compared with a commercial method
Fig. 6 Response time of the system at optimum condition
(
-
5.00 mg L urea)
1
(Autoanalyzer, Hitachi, Japan). The results of these
determinations are given in Table 2, which confirm that
urea can be determined quantitatively in the region
Table 1 Interference study
Interference species
Concentration
relative to
urea (folds)
Relative
error %
assessed. It should be noted that the normal values of urea
-1
in serum and urine are 150–400 mg L
-1
and about
15.0 g L , respectively (it depends on the diet). So these
Amino benzoic acid, thiourea,
citrate, glucose, 2,4-dinitro
phenyl hydrazine, toloiden
0
100
50
\2
\2
real samples were diluted to appropriate concentration
with buffer solution before measurement. This dilution
step simplifies the sample matrix and reduces any possible
interference. Therefore, it leads to more reliable and
precise results.
4
,4 -Diamino-diphenyl methane,
3
,4 diamino toluene and acetate
Table 2 Determination of urea
in real samples
-1
)
-1
)
b
Actual (g L
-1
)
a
Recovery (%)
Real sample
Spiked (mg L
Found (mg L
Tap water
2.00
5.00
2.3 ± 0.13
5.5 ± 0.32
–
–
115
110
Bovine serum
a
Recovery (%) = (Ccalculated
Actual) 9 100
/
-1
)
-1
)
b
Actual (g L
-1
)
C
Real sample
Urine
Spiked (mg L
–
Found (g L
Recovery (%)
117
b
Calculated by urine
autoanalyzer (Hitachi, Japan)
17.6 ± 0.2
15.0
Table 3 Comparison of the proposed urea sensor with some reported methods
Reagent
Technique
Linear range
(
Responsetime
(min)
Detection limit
-1
References
-
1
mg L
)
(mg L
)
–
Potentiometric
Potentiometric
Potentiometric
Amprometric
1.90–600
5.34–66.60
0.06–6.00
1.20–12.00
0.5–15.00
60.00–300.00
60–300
Few second
2 min
20 min
3 min
–
1.50
[5]
–
[7]
Acid urease column
–
[8]
–
[10]
[19]
[20]
[20]
This work
1
2
2
-Phenyl-1,2-propanodione-2-oxime
Spectrophotometric
Conductometric
Spectrophotometric
Paptode
0.10
15.60
16.80
0.01
-Oxoglutarate
-Oxoglutarate
Chloranil
0.05–10
2 min
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J IRAN CHEM SOC (2012) 9:383–388
Comparative study
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method as a test strip for determination of urea in tap
water, urine and serum samples. The proposed method is
based on complexation reaction of chloranil with urea at
elevated temperature (65 °C).Violet spots formed after
reaction of analyte with reagent on top of the strips were
scanned and images were analyzed using a program
written in VB programming language to convert color
intensities to relative R, G and B values. The sensor
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responds linearly in the range of 0.05–10.00 mg L urea
and can be successfully applied for the determination of
urea in complex samples. The described method is a
simple, robust and rapid technique which does not
require expensive apparatus and is superior to optrode
method since it is not just limited to transparent sub-
strates for analysis. No need for determination of kmax (as
in visible spectrophotometry) is another considerable
advantage.
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Acknowledgments We gratefully acknowledge the support of
Shiraz University Research Council for this study.
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