Optical nonlinearity in measurement of urea and uric acid in blood

doi:10.4236/ns.2010.22017

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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; [email protected]

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

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

erture

Detecto

-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

0.406(1 )

Ф (1)

where pv



 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

1exp(2/)

Srw  (2)

where ra is the radius of the aperture and wa is the beam

radius at the aperture.

0eff

nkI L

Ф (3)

where n2 is the nonlinear refractive index, k is the wave

number (2





) and

eff









 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

0.1

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

(c)

R2=0.99435

Y=0.29907x+0.9584

n2x10-8cm2/W

Concentration of urea (mg)

246810

(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

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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