Vol.2, No.2, 106-111 (2010) Natural Science
http://dx.doi.org/10.4236/ns.2010.22017
Optical nonlinearity in measurement of urea
and uric acid in blood
A. N. Dhinaa, P. K. Palanisamy
Department of Physics, Anna University Chennai, Chennai, India;
Received 23 November 2009; revised 15 December 2009; accepted 30 December 2009.
ABSTRACT
The Z-scan technique is a simple and effective
tool for determining nonlinear optical properties
of materials. This technique is utilized in meas-
urement of urea and uric acid in blood. The
nonlinear refractive index of urea and uric acid
are found to vary linearly with concentration.
Hence by calculating the nonlinear refractive
index it is possible to measure their concentra-
tion in the sample. The results of this method
are found to be in good agreement with the
conventional colorimetric method.
Keywords: Z-Scan Technique;
Nonlinear Refractive Index; Urea; Uric Acid
1. INTRODUCTION
Urea and Uric acid are the metabolic nitrogenous wastes
present in the body that can be measured in blood and
urine. Serum uric acid reflects the interactions of four
major processes: dietary purine intake, endogenous
purine metabolism, urinary urate excretion, and intesti-
nal uricolysis. Uric acid is a metabolite of purines, nu-
cleic acids and nucleoproteins. Consequently, abnormal
levels may be indicative of a disorder in the metabolism
of these substances. Hyperuricaemia may be observed in
renal dysfunction, gout, leukemia, polycythaemia, athe-
rosclerosis, diabetes, hypothyroidism, or in some genetic
diseases. Decreased levels are present in patients with
Wilson’s disease and Fanconis syndrome [1]. The exist-
ing methods could be conveniently divided into two
groups: reductive and enzymatic. The reductive methods
are non-specific and involve the oxidation of uric acid
with phosphotungstate reagent to allantoin with resultant
blue coloring of tungstate solution.
The enzymatic methods are specific. They involve the
catalytic oxidation of uric acid with the enzyme uricase
to allantoin with the formation of hydrogen peroxide [2].
The peroxide concentration which is directly propor-
tional to the concentration of uric acid could then be
determined by a number of methods [3-11]. The amount
of urea nitrogen is a breakdown product of protein me-
tabolism. Urea formed in the liver as the end product of
protein metabolism enters in to the blood and is ulti-
mately eliminated in the urine by the kidneys. Most kid-
ney diseases affect urea excretion so that blood urea ni-
trogen (BUN) levels increase in the blood. It may also
increase the dehydration or bleeding in the stomach
and/or intestines or result in any side effect of some
medications. Raised levels may also be seen in any state
causing hypovolemia, heart failure, starvation and uri-
nary tract obstruction, etc. Urea is one of the first sub-
stances to be determined in biological fluids as one of
the parameters of liver function tests. In routine proce-
dures urea determination in biological fluids is carried
out with chemical reagents and enzymatic methods
[12,13].
The Z-scan technique was extending to study the op-
tical nonlinearity of vitreous humor in human and rabbit
[14], retinal derivatives [15]. In this present investigation
enzymatic reacted urea and uric acid blood sample are
subjected to the Z-scan technique to calculate the non-
linear refractive index (n2). Already work has been done
on measurement of glucose [16], total cholesterol and
triglycerides [17].
The single beam Z-scan analysis, which was devel-
oped by Mansoor Sheik Bahae et al. [18], is a simple
and effective tool for determining nonlinear optical pro-
perties of materials [19-22]. Nonlinear refractive index is
proportional to the real part of the third-order suscepti-
bility Re [x(3)]. Basically, the Z-scan method consists in
translating a nonlinear sample through the focal plane of
a tightly focused Gaussian laser beam and monitoring
the changes in the far field intensity pattern. For a purely
refractive nonlinearity, the light field induces an inten-
sity dependent nonlinear phase and, as consequence of
the transverse Gaussian intensity profile, the sample
presents a lens-like behavior. The induced self-phase
modulation has the tendency of defocusing or re-colli-
mating the incident beam, depending on its Z position
with respect to the focal plane. By monitoring the trans-
mittance change through a small circular aperture placed

A. N. Dhinaa et al. / Natural Science 2 (2010) 106-111
107
at the far field position, it is possible to determine the
nonlinear refractive index. In this present study, we have
measured urea and uric acid levels in blood by calculat-
ing the nonlinear refractive index (n2) value using a sin-
gle beam Z-scan method.
2. EXPERIMENTAL
2.1. Urea Sample Preparation
For sample preparation (Urea by Berthelot method – a
kit supplied by Aspen Laboratories, Baddi, Himachal
Pradesh, India) was used. To 10 l of the serum first 50
l of urease enzyme was added and incubated for 5 min-
utes at 37 o C. Then 1.5 ml of phenol reagent and 1.5 ml
of hypochlorite reagent were added and incubated for 20
minutes at 37 o C. The principles involved for this reac-
tion are follows:
Urea + H2O Ammonia + CO
2
Ammonia + Phenol + Hypochlorite
Indophenol
Urease hydrolyses urea into ammonia and carbon di-
oxide. In alkaline conditions, ammonia reacts with hy-
pochlorite and phenol in the presence of nitropruside to
form Indophenol colored complex. The intensity of the
color is directly proportional to the concentration of urea
in the sample.
2.2. Uric Acid Sample Preparation
1 ml of uricase enzyme was added to 20 l of serum
(Uricase based on POD - a kit supplied by Merck,
Mumbai, India). The solution was mixed well and incu-
bated at 37 o C for 10 minutes. The principle involved in
this reaction is represented as:
Uric acid + H2O + O2 Allantoin + CO2 +H2O2
4- Aminoantipyrine + TBHBA + 2H2O2
Quinoneimine dye + 3H2O
Uric acid is hydrolyzed by Uricase to allontoin and
hydrogen peroxide. Hydrogen peroxide so formed reacts
with 4-aminoantipyrine and 2, 4, 6-tribromo-3- hydroxy
benzoic acid (TBHBA) in the presence of enzyme per-
oxidase (POD) to produce Quinoneimine dye compound.
The intensity of the color is directly proportional to the
concentration of uric acid in the sample.
2.3. Nonlinearity Measurements
Z-scan experiments were performed using a 532 nm Nd:
YAG (SHG) CW laser beam (COHERENT –Compass
215M-50 diode-pumped laser) focused by a lens of 35
mm focal length. The experimental set up is shown in
Solution
Laser Beam
A
p
erture
Detecto
r
-Z +Z
Figure 1. Experimental setup for Z-scan technique.
Figure 1. A typical closed-aperture Z-scan curves for the
standard urea and uric acid solution at incident intensity
Iο = 7.824 kW/cm2, are shown in Figure 3. This normal-
ized transmittance curves are characterized by a
pre-focal peak followed by a post-focal valley. This im-
plies that the nonlinear refractive indices of urea, uric
acid are negative (n2 < 0). The defocusing effect shown
in Z- scan curve can be attributed to a thermal nonlinear-
ity resulting from absorption of radiation at 532 nm.
Localized absorption of a tightly focused beam propa-
gating through an absorbing sample medium produces a
spatial distribution of temperature in the sample solution
and consequently, a spatial variation of the refractive
index, that acts as a thermal lens resulting in phase dis-
tortion of the propagating beam.
The nonlinear refractive index (n2) is calculated using
the standard relations [18].
0.25
0
0.406(1 )
pv
TS
Ф (1)
where pv T

 can be defined as the difference between
the normalized peak and valley transmittances (Tp – Tv),
׀Ф0׀ is the on-axis phase shift at the focus.
The linear transmittance of the aperture is given by
22
1exp(2/)
aa
Srw  (2)
where ra is the radius of the aperture and wa is the beam
radius at the aperture.
0
2
0eff
nkI L Ф (3)
where n2 is the nonlinear refractive index, k is the wave
number (2
k


) and
1
L
eff
e
L





02
0
2
W
P
I

 is defined as the peak intensity within the
sample at the focus. L is the thickness of the sample, α is
the linear absorption coefficient.
The results are checked by performing conventional
colorimetric method following the standard procedure of
Urease
Nitropruside
POD
Uricase

A. N. Dhinaa et.al. / Natural Science 2 (2010) 106-111
108
450 500 550 600 650 70
0
0.1
0.2
0.3
0.4
0.5 (a)
Absorbance (arb.unit)
Wavelength (nm)
450 500 550 600 650 700
0.00
0.05
0.10
0.15
0.20
0.25
0.30 (b)
Absorbance (arb.unit)
Wavelength (nm)
Figure 2. UV-Vis Spectra of standard (a) urea (b) uric acid with reagent.
-10 -50510
0.7
0.8
0.9
1.0
1.1
1.2
1.3
Normalised Transmittance
Z (mm)
Urea 40(mg/dl)
Urea 50(mg/dl)
Urea 60(mg/dl)
-10 -50510
0.8
0.9
1.0
1.1
1.2
1.3
Normalised Transmittance
Z (mm)
UA (4mg/dl)
UA (6mg/dl)
UA (8mg/dl)
Figure 3. Z-scan data of the standard urea and uric acid (UA).
10 20 30 40 50 60
0.1
0.2
0.3
0.4
0.5
0.6 (a)
Y=0.03625x+0.03901
R2=0.98172
Tp-v
Concentration of urea (mg)
246810
0.1
0.2
0.3
0.4
0.5 (b)
Y=0.0467x+0.045
R2=0.99554
Tp-v
Concentration of uric acid (mg)
10 2030 4050 60
2
4
6
8
10
12
14
16
18
20
(c)
R2=0.99435
Y=0.29907x+0.9584
n2x10-8cm2/W
Concentration of urea (mg)
246810
4
6
8
10
12
14
16
18
(d)
n2x10-8cm2/W
Y=1.4801 3x+1.4 3441
R2=0.99949
Concentration of uric acid (mg)
Figure 4. Linear variation of T p-v and nonlinear refractive index (n2) with concentration of
urea (a,c) and uric acid (b,d) by Z-scan method.

A. N. Dhinaa et al. / Natural Science 2 (2010) 106-111
109
10 20 30 40 50 60
0.05
0.10
0.15
0.20
0.25 (a)
Y=0.0036 6x+0.0286 7
R2=0. 99728
Optical Densit
y
Concentration of urea (mg)
246810
0.04
0.08
0.12
0.16
0.20 (b)
Y=0.02185x -0.0117
R2=0.99511
Optical Density
Concentration of uric acid (mg)
Figure 5. Linear variation of optical density of urea (a) and uric acid (b) by colorimetric method.
A. J. Taylor et al. [23] for urea and P. Fossati et al. [24]
for uric acid. This involves measurement of optical den-
sity variation with respect to concentration as shown in
Figure 5. The results thus obtained are compared with
the results calculated with the Z-scan technique.
2.4. Statistical Analysis
The error involved in the measurements was determined
by t test, P<0.01.These statistical analysis was con-
ducted using SPSS commercial statistical package (SPSS,
version 10.0 for windows, SPSS Inc., Chicago, U.S.A).
3. RESULTS AND DISCUSSION
3.1. Spectral Absorbance Measurements
The absorption spectra were measured using UV-Vis
spectrophotometer (SHIMADZU-UV-2401PC), and the
spectra for both urea and uric acid were found to be
broad banded as depicted in Figure 2. Both have exhib-
ited good absorption at 532 nm. Hence for further study
532 nm Nd: YAG laser was used.
3.2. Nonlinear Refractive Index
The results of typical Z-scan normalized transmittance
measurement for urea and uric acid are shown in Figure
3. As the concentration of the standard urea and uric acid
increases, the normalized transmittance peak increases
whereas the valley decreases respectively.
It is found that ΔTp-v as well as refractive index value
increase linearly with concentration of standard urea and
uric acid as seen in Figure 4(a) to (d). Figure 5(a) and
(b) shows the linear variation of optical density with
concentration of urea and uric acid respectively as
measured with conventional colorimetric method.
The experiments were repeated five times and the
mean value of the nonlinear refractive index (n2) was
calculated from the normalized transmittance values. This
calculated value was assumed to be the standard for
measurement of unknown urea and uric acid content pre-
sent in blood sample. This was arrived by plotting a linear
graph of urea and uric acid concentration Vs non- linear
refractive index. The nonlinear refractive index value was
first measured against the reagent blank solution.
The calibration was made with the conventional col-
orimetric method and the results are tabulated in Table 1
for urea and Table 3 for uric acid. The common urea
level in blood serum is 10 – 50 mg/dl. The common uric
acid level in blood serum is 3.4 – 7.0 mg/dl range for
Males and 2.5-6.0 mg/dl range for Females.
To calculate the urea and uric acid levels, one need not
Table 1. Nonlinear refractive index (n2) values for standard urea.
Standard Urea
Concentration (mg/dl)
Nonlinear refractive index
n2 10-8 (cm2/W)
10 03.79 ± 0.31
20 07.75 ± 0.98
30 09.85 ± 0.51
40 14.55 ± 0.91
50 16.36 ± 0.55
60 17.80 ± 0.68
Table 2. Comparative analysis of serum urea measurement
using colorimetric method and Z-scan method.
Concentration of urea (mg/dl)
Sample
Collection Urea levelColorimetric
method Z-scan method
Male Normal 34.74 35.08
Female Normal 33.82 33.12
Male Normal 42.82 42.24
Female Normal 31.46 32.03
Female Normal 26.64 25.85
Male Normal 38.08 38.99
Each value is the mean of 5 individual observations. The P value (t-test
value) is less than 0.01 at 1% significance level.

A. N. Dhinaa et.al. / Natural Science 2 (2010) 106-111
110
Table 3. Nonlinear refractive index (n2) values for standard
uric acid.
Standard Uric acid
concentration (mg/dl)
Nonlinear refractive index
n2 10-8 (cm2/W)
2 04.39 ± 0.09
4 07.34 ± 0.10
6 10.97 ± 0.48
8 13.15 ± 0.18
10 16.46 ± 0.38
Table 4. Comparative analysis of serum uric acid measurement
using colorimetric method and Z-scan method.
Concentration of uric acid (mg/dl)
Sample
collection
Uric acid
level Colorimetric
method Z-scan method
Female Normal 3.40 3.37
Male Normal 5.55 5.61
Male Normal 5.47 5.46
Female Normal 4.05 4.12
Male Normal 6.28 6.26
Female Normal 4.35 4.34
Each value is the mean of 5 individual observations. The P value (t-test
value) is less than 0.01 at 1% significance level.
plot full Z-scan curve every time. Once, experimental
setup explained above is established, one needs to note
down peak and valley values of the transmittance curve
translating the sample holder continuously along Z- axis.
The difference in these two values Tp - Tv, ׀ΔФ0׀
when substituted in Eq.( 3) yields the nonlinear refractive
index value [17].
From the Z-scan results we infer that, the nonlinear
refractive index n2 values for the common level of urea
in blood serum (10-50 mg/dl) are 03.79 ± 0.31 and 16.36
± 0.5510-8 cm2/W respectively.
Likewise, for common level of uric acid in blood se-
rum for males 3.4-7.0 mg/dl and their corresponding n2
values are 6.45 and 11.7810-8 cm2/W respectively. For
females 2.5-6.0 mg/dl and their corresponding n2 values
are 5.15 and 10.97 ± 0.48 10-8 cm2/W respectively.
3.3. Evaluation with Conventional Method
Many trials were performed to measure the urea and uric
acid level with our proposed method. The blood samples
were collected from six volunteers. (Three males and
three female). We could see that the results arrived are in
good agreement with those of the conventional colori-
metric method for urea Table 2 and for uric acid Table 4.
This ascertains that the proposed method is on equality
with the conventional colorimetric method.
4. CONCLUSIONS
We have measured the nonlinear refractive index values
for urea and uric acid present in the serum sample by
Z-scan method with 532 nm Nd:YAG CW laser. The
Z-scan measurements indicate that urea and uric acid in
standard sample and blood sample exhibit nonlinear op-
tical properties. Comparative analysis of these values
with the one obtained by conventional colorimetric
method shows that they are in good agreement. Thus
Z-scan technique is found to be suitable for measure-
ment of bioanalytes.
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