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DOI: 10.4172/2155-9872.S7-011

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Analytical & Bioanalytical Techniques

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ISSN: 2155-9872

Research Article Open Access

Spectrophotometric Determination of Uric Acid in Urine Based-Enzymatic

Method Uricase with 4-Aminodiphenylamine Diazonium Sulfate (Variamine
Blue RT Salt)
Hairul Hisham Hamzah1,3*, Zainiharyati Mohd Zain2, Nor Lailatul Wahidah Musa3, Yun-Chun Lin1 and Emma Trimbee1
1
School of Chemistry, University of Southampton, Highfield Campus, SO17 1BJ, Southampton, UK
2
Faculty of Applied Sciences, Universiti Teknologi MARA (UiTM), 40450, Shah Alam Selangor, Malaysia
3
Faculty of Applied Sciences, Universiti Teknologi MARA Pahang, 26400, Jengka Pahang, Malaysia

Abstract
A simple spectrophotometric method based-uricase enzyme for the detection of uric acid in normal urine and gout
patient’s urine samples has been developed based on the reaction of hydrogen peroxide (H2O2) with yellow color of
4-Aminodiphenylamine Diazonium Sulfate (variamine blue RT salt) to yield a pale yellow-green coloured solution.
The absorbance of products as the output of enzymatic reaction and variamine blue RT salt were detected using
uv-visible spectrophotometer with maximum absorbance at 269 nm. The response time of this proposed method
was 20 minutes. The optimum pH activity on enzymatic and variamine blue RT salt reactions was foud to be pH 9.
The relative standard deviation (RSD) of the reproducibility of this method was very good at 0.7%. The calibration
plot of different concentrations of uric acid was found to be between 0.5-13 mM with a detection limit of 0.58 mM.
The kinetic parameters the Michaelis-Menten constant (KM) and the maximum abosrbance (Absmax) in the presence
of different concentrations of uric acid were evaluated using the Linewaver-Burk and Hofstee plots respectively. The
enzymatic-spectrophotometric method was compared with established clinical method and satisfactory agreement
was achieved.

Keywords: Biosensor; Uric acid; Spectrophotometric determination;
Uricase enzyme; Urine samples
Introduction
Uric acid is the primary end-product of purine metabolism and
excreted in the urine. It is derived from purines arising from the
catabolism of dietary and endogenous nucleic acid stem from increased
catabolism dysfunction of one of the shunt pathways which leads to
increased urate production [1]. The assay of uric acid in body fluids
(e.g: serum and urine) is a clinically valuable diagnostic indicator.
Normally, uric acid is present in the blood in concentration range 0.15-
0.45 mmol/L and excreted in urine in 1.19-2.98 mmol/day [2]. The
presence of elevated uric acid levels is a sign of gout, hyperuricemia, or
Lysch-Nyhan syndrome. Similarly, elevated uric acid levels are related
to other conditions including increased alcochol consumption, obesity,
diabeties, high cholestrol, kidney disease, and heart diseases. Many
epidemiological studies have suggested that serum uric acid is also a
risk factor for cardiovascular disease [3]. Therefore, it is necessarily to
develop a reliable analytical method for the detection and quantification
of uric acid in urine. Monitoring uric acid in the urine is important
because it can be used as a powerful indicator for an early warning sign
of kidney diseases [4].
There are many standard analytical methods that have been
reported in the literatures for uric acid determination. The levels of uric
acid in different biological matrices such as urine and serum have been
determined by numerous standard analytical methods such as reversed-
phase liquid chromotography [5], potentiometric enzyme electrode [6]
and flow injection analysis system with tubular amperometric detector
[7]. All the analytical methods are well established and exhibit a large
range of advantages. However, these methods also have weaknesses
such as they are still under development, time-consuming, expensive,
cannot be performed easily outside the laboratory and need very highly
skilled technicians. On the other hand, early-warning systems are
needed for scientists to react in case of contamination pollution.
Biosensor technology is a powerful alternative to coventional
analytical techniques, harnessing the specificity and sensitivity of
biological systems in small and low cost devices. Biosensors are defined
as measurement devices that utilize chemical or biological reactions
to detect and quantify a specific analyte. Biosensors consists of an
analyte-specific bioreceptor (enzyme, antibody, nucleic acid, tissue,
cell or microorganism) and a physical transducer element(electrode,
optical, mass, or quartz) which converts the change in the receptor to
a detectable signal [8]. In enzyme-based determination, the enzyme
reacts selectively with its substrate associated with or intergrated
within a physico-chemical transducers may be optical, electrochemical,
thermometric, piezoelectric or magnetic [9].
The spectrophotometric method is the quantitative measurement
of the reflection or transmission properties of a material as a
function of wavelength. It is more specific than the general terms of
electromagnetic spectroscopy in that spectrophotometry deals with
visible light, near-ultraviolet, and near-infrared, but does not cover
time-resolved spectroscopic techniques. The Spectrophotometric
method involves the use of a spectrophotometer. A spectrophotometer
is a photometer (a device for measuring light intensity) that can measure
intensity as a function of the light source wavelength. Important
*Corresponding author: Hairul Hisham Hamzah, PhD, School of Chemistry,
University of Southampton, Highfield Campus, SO17 1BJ, Southampton UK,
E-mail: hhh1v12@soton.ac.uk
Received March 30, 2013; Accepted June 05, 2013; Published June 07, 2013
Citation: Hamzah HH, Zain ZM, Musa NLW, Lin YC, Trimbee E (2013)
Spectrophotometric Determination of Uric Acid in Urine Based-Enzymatic Method
Uricase with 4-Aminodiphenylamine Diazonium Sulfate (Variamine Blue RT Salt). J
Anal Bioanal Tech S7: 011. doi:10.4172/2155-9872.S7-011
Copyright: © 2013 Hamzah HH, et al. This is an open-access article distributed
under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the
original author and source are credited.

J Anal Bioanal Tech Biosensing ISSN:2155-9872 JABT, an open access journal

Citation: Hamzah HH, Zain ZM, Musa NLW, Lin YC, Trimbee E (2013) Spectrophotometric Determination of Uric Acid in Urine Based-Enzymatic
Method Uricase with 4-Aminodiphenylamine Diazonium Sulfate (Variamine Blue RT Salt). J Anal Bioanal Tech S7: 011. doi:10.4172/2155-
9872.S7-011
features of spectrophotometers are spectral bandwidth and linear
range of absorption or reflectance measurement [10]. The UV-visible
spectrophotometer equipment will be adopted for the determination
of uric acid in urine because it is attractive procedure and widely used
in routine analysis around the world. A spectrophotometric technique
is always an acceptable chemical analysis method, due to its acceptable
precision and accuracy. The advantages of this method are low
implementation, low maintenance costs and good analytical frequency
[11]. In spectrophotometric trace analysis, knowledge of the sensitivity
of the colour of the reaction employed of paramount importance and
it is necessary to have a simple method available for the expression of
the sensitivity. The common method of using the spectrophotometer
requires the construction of the standard curve (also called the
analytical reference of calibration curve) for the constituents being the
determined. For this purpose, suitable of the constituent are taken and
treated exactly the same way as the sample solution for the development
of the colour measurement of the transmittance at the optimum
wavelength [12].
Recently, a few reports on spectrophotometric determination of
uric acid using diffrent approaches have been reported [1,13-16]. In
this work, the development of the spectrophotometric method for
the determination of uric acid is based on oxidation of uric acid in
the presence of uricase will produce products of allantoin, CO2, and
H2O2 respectively. Then, the concentration of uric acid can be related
to the yielding and reactions of H2O2 with variamine blue RT to form
a pale yellow-green product. Based on previous studies, there are few
works reported on quantitatively reactions of the pale blue colored
variamine blue B dye with H2O2 to form an intense violet color [17-
19]. Nevertheless, the sensing scheme based upon ezymatic reaction
of uricase with uric acid and Variamine blue RT Salt has not yet been
reported. The sensing scheme is based on the changing of yellow
variamine blue RT solution into the pale yellow-green species, due
to the reaction between hydrogen peroxide and variamine blue RT.
Experimental parameters such as response time study, spectral study,
pH optimization, reproducibility of the method, calibration of the
developed method, kinetic study, interference study, and real sample
analysis were performed. The developed spectrophotometric method
was validated against well established method from clinical laboratory
(Pathlab Sdn Bhd).
Experimental Procedure
Ultraviolet-visible spectrophotometer (Varian-Carry Win) was
used to study on determination of uric acid based on an enzymatic
reaction of uricase with the presence of dye Variamine Blue RT salt. The
measurement of wavelength for absorbance spectra on this procedure
was fixed in the range of from 230-500 nm. Quartz cuvettes were used
in this procedure.
Materials and Methods
Reagent
Uricase enzyme from Bacillus fastidiosus (18.8 units mg ), -1
Uric Acid and Variamine Blue RT Salt were purchased from Sigma
Aldrich. 0.05 M borax buffer solution was prepared and used as buffer
throughout the analysis. 5 mg of Uricase enzyme was dissolved in 100
ml of 0.05 M borax buffer and was stored at 3°C. 0.29 g of variamine
blue RT salt (yellow powder) was dissolved in 100 ml of deionized water.
The stock solution of 9.9×10-3 M variamine blue RT solution was stored
in refrigerator at 3°C. Stock solution of 20 m Muric acid was prepared
by dissolving 1.68 g of uric acid in 500 ml of hot deionized water with
J Anal Bioanal Tech Biosensing
Page 2 of 6
addition of 300 μL of 0.6 M NaOH. A series of different concentrations
of uric acid were prepared by appropriate dilution of the stock solution.
Procedure
A mixture volume of 2 mL Uricase, 1 mL of 10 mM Uric acid, and
1 mL of 0.1 mM Variamine blue RT salt solutions were transferred to a
sample vial. The solution was left for 20 minutes to allow the reaction to
go to completion. The measurements were carried out using a UV-Vis
Spectrophotometer (Varian-Cary Win).
Real sample analysis
Urine samples were obtained from 2 male volunteers. One volunteer
is healthy and another one of volunteer is a gout patient. The urine
samples were collected 24 hours before analysis and kept in refrigerator
at 3°C. The urine samples were centrifuged for 15 min in 4000 rpm. 1
ml of urine samples were 25-fold diluted with Milli-Q water and then
were filtered through 0.45 m pore filter. The uric acid concentration in
the sample was determined using the calibration graph of the developed
method. In addition, for validation study the urine samples were sent
to Pathlab Sdn Bhd in Malaysia for determination of uric acid based on
established clinical method.
Result and Discussion
Spectral study
Figure 1 shows the three absorbance spectras of the different
mixture of uric acid (UA), uricase enzyme and variamine blue RT salt
solutions respectively. Absorbance spectra A was obtained by mixturing
of uric acid and uricase enzyme solutions and no resultant absorption
peak was observed in the absence of variamine blue RT reagent. By
contrast, the absorbance spectras for B (blank) and C were produced
with the presence of variamine blue RT solution. Quantitatively, in the
presence of uricase enyzme, uric acid will be oxidized into allantonin,
carbon dioxide and hyrogen peroxide (H2O2) respectively until the
equlibrium state (Figure 2) [20-22]. The principle measurements for
uric acid concentration is based on the reaction of hydrogen peroxide
with yellow variamine blue RT to a pale yellow-green coloured
products. Furthermore, this response is proportional to the uric acid
concentrations and can be monitored spectrophotometrically.Hence,
the absorbance spectra (C) reveals that the maximum absorption
at wavelength of 269 nm. Thus, this wavelength was chosen as the
optimum wavelength for the following analytical purposes.
0.8
B
0.7
0.6 C
Absorbance A
0.5
0.4
0.3
0.2
0.1 A
0
230 250 270 290 310 330 350
Wvelength (nm)
Figure 1: Absorbance spectra for (A) Uricase enzyme+Uric Acid (10 mM); (B)
Uricase enzyme+Variamine Blue RT Salt (blank); (C) Uricase enzyme+Uric
Acid (10 mM)+Variamine Blue RT Salt.
ISSN:2155-9872 JABT, an open access journal
Citation: Hamzah HH, Zain ZM, Musa NLW, Lin YC, Trimbee E (2013) Spectrophotometric Determination of Uric Acid in Urine Based-Enzymatic
Method Uricase with 4-Aminodiphenylamine Diazonium Sulfate (Variamine Blue RT Salt). J Anal Bioanal Tech S7: 011. doi:10.4172/2155-
9872.S7-011

Page 3 of 6

pH values (6-12) towards detection of uric acid. The pH values were

adjusted to pH 6-12 by adding a few drops of 0.5 M sodium monobasic
Variamine Blue RT-H202
adduct phosphate (NaH2PO4), 0.5 M of sodium dibasic phosphate (Na2HPO4)
0.8
0.7
and 0.5 M of sodium hydroxide (NaOH) into the mixture of solutions

Absorbanoc (A)
Uric Acid 0.6
0.5 respectively. Figure 4 illustrates the effect of pH for the detection of
Variamine Blue RT
uric acid. The pH value of 9 was selected for the following analytical
0.4
Salt 0.3
O2 0.2
0.1
0 procedure. This value is in good agreement with previously reported
Detector
results [21,24]. At pH 9, the enzyme catalyzed the reaction at the
Light Source 230 280 330 380
Uricase
Wavelength (nm)

Allantoin
+ (b)
maximum rate and greatest amount of oxygen was produced. The
CO2
+ amount of hydroxide ion pH 9 did not affect the charge of the tertiary
H2O2
(a)
structure and the charges of amino acids within the active site.
Reproducibility study
Figure 2: Reaction scheme for oxidation of Uric Acid by Uricase enzyme in
the presence of Variamine Blue RT (a). Absorption spectra of the enzymatic Reproducibility refers to the successive runs made using the same
reaction uricase-uric acidin the absence and presence of Variamine Blue RT
salt (b).
developed method to evaluate discrepancies between its responses.
Figure 5 depicts the reproducibility of the enzymatic reactions of
uricase, uric acid and variamine blue RT is based on ten measurements
(n=10). The RSD for reproducibility of the developed method was
0.55 calculated to be 0.7%. A small RSD value was observed for this method
indicating a good precision of the method developed. The precision
0.54
of an analytical method was dependent on the degree of agreement
0.53 among individual test results when the method was applied repeatedly
0.52 to the multiple samplings of the homogenous samples. This is usually
Absorbance

expressed as the standard deviation or the relative standard deviation

0.51
(coefficient of variation) [25]. The reproducibility data was obtained
0.5

0.49 0.600
0.48 0.550
0.47 0.500
0 5 10 15 20 25 30 35
Absorbance

0.450
Time (Minutes)

Figure 3: Response time of the enzymatic reaction of uricase with uric acid and
0.400
variamine blue RT salt.
0.350

0.300
The response time of this method relied on enzymatic reaction of
uricase with uric acid and hydrogen peroxide with Variamine Blue RT 0.250
respectively at different times (2-20 minutes) (Figure 3). The response 5 6 7 8 9 10 11 12 13
time was determined by steady signal that produced in 20 minutes pH
when absorbance was constant. From this study, it can be concluded
that the entire enzymatic reaction required 20 minutes to attain an Figure 4: Effect of different pH on the ezymatic spectrophotometric
determination of uric acid.
equilibrium state. This response time was chosen as the acquisition
time for all analytical purposes.
pH study
pH is a measure of concentration of hydrogen ions in a solution.
The higher the hydrogen ion concentration, the lower the pH. Most
enzymes function is only efficient over a narrow pH range. A change
in pH above or below this range reduces the rate of enzyme reaction
considerably. Changes in pH leads to the breaking of the ionic character
of the side chain of the amino acid that hold the tertiary structure of
the enzyme in place. The enzyme begins to lose its functional moiety,
particularly the active site. When the substrate no longer is recognized
by the active site, the enzyme is said to be denatured [23]. Also changes
in pH, affect the charges on the amino acids within the active site such
that the enzyme will not be able to form a specific enzyme-substrate
complex. A suitable pH of enzyme plays an important role to provide an
appropriate environment for the enzymatic reaction and pH at which
Figure 5: Reproducibility of the developed ezymatic spectrophotometric
enzyme catalysis a reaction at maximum rate is called the optimum pH. method.
The Optimum pH was determined by studying the effect of different

J Anal Bioanal Tech Biosensing ISSN:2155-9872 JABT, an open access journal

Citation: Hamzah HH, Zain ZM, Musa NLW, Lin YC, Trimbee E (2013) Spectrophotometric Determination of Uric Acid in Urine Based-Enzymatic
Method Uricase with 4-Aminodiphenylamine Diazonium Sulfate (Variamine Blue RT Salt). J Anal Bioanal Tech S7: 011. doi:10.4172/2155-
9872.S7-011

Page 4 of 6

by repeatedly analyzing, in one laboratory within one day. The RSD

600
value was important to show the degree of variation expected when the
analytical procedure was repeated several times in a standard situation. 500

Calibration plot of uric acid 400

[Uric Acid] / Absorbance min-1

300
Figure 6 illustrates the linear response when uric acid concentration Y=40.109x- 12.083
is within the range of 0.5-13 mM. The straight line obtained from this 200 R2=0.998
plot can be fitted by the linear regression (y=0.0031x+0.5549) and the 100
calculated correlation coefficient (R2) was found to be 0.9307. The
0
limit of detection (LOD) is defined as the concentration equivalent to -1 4 9 14
a signal of blank plus three times the standard deviation of the blank -100
was calculated to be 0.58 mM and it is higher than obtained by the -200
electrochemical/optical biosensors-based immobilization uricase Concentration of uric acid (mM)

enzyme which are 1×10-3 mM [21], 8.5×10-4 mM [26], and (0.55 mM Figure 8: Hofstee plot for uric acid towards uricase enzyme activity.
and 0.22 mM) [27] respectively. However, the LOD obtained is still
under permitted levels (1.98-2.98 mM) [2]. Calculation Method Lineweaver-Burk Hofstee
Kinetic study Absmax 0.0254 0.0249
Michaelis Menten 0.313 mM 0.301 mM
The study of the rate at which an enzyme works is called enzyme constant (KM)
kinetics. The kinetics of uric acid on uricase enzyme was expressed and Formula 1/v=-12.385 (1/[S])+39.45 [S]/v=40.109 [S]-12.083
compared by Lineweaver-Burk and Hofstee equations respectively. The Absmax calculation Absmax=1/c Absmax=1/m
relationship of enzyme activity in the presence of different concentration Absmax=1/39.45 Absmax=1/40.19
Absmax=0.0254 Absmax=0.0249
uric acid was shown upon the double-reciprocal Lineweaver-Burk and
KM calculation KM=m x Absmax KM=c x Absmax
Hofstee plots. The plots are expressed as linear plots of the same data KM=12.385 x 0.0254 KM=12.03×0.0249
derived from the enzyme kinetic reactions. The result is shown in KM=0.313 mM KM=0.301 mM
Figures 7 and 8 respectively. Table 1: Comparison of calculation methods for KM and Absmax based on
Lineweaver-Burk and Hofstee equations.

0.57 Sample Clinical Developed Relative Erors

0.56 Method (mM) Method (mM) (%)
0.55 Normal Urine 2.83 ± 2 2.54 ± 5 -11.4
0.54 Gout Patient’s Urine 5.24 ± 0.9 5.02 ± 3 -3.6
Absorbance

0.53 Table 2: Determination of uric acid in urine samples by the developed

spectrophotometric method and comparison with established clinical method.
0.52 y = -0.003x + 0.554
0.51 R² = 0.930 The KM and Absmax values for each plot can then be determined
0.5 by the slope and the intercepts of the vertical and horizontal axes of
0.49 these plots respectively. KM is defined as the concentration of the
0.48 specific substrate at which a given enzyme yields one half its maximum
0 2 4 6 8 10 12 14 velocities. A lower KM indicates that the enzymes requires only a small
amount of substrate to become saturated and also shows greater affinity
Concentration of uric acid (mM)
of the enzymes towards the substrate. Hence, the maximum velocity is
Figure 6: Calibration plot for determination of uric acid. reached at a relatively low substrate [28]. The KM constant and Absmax
were calculated by the use of Lineweaver-Burk and Hofstee equations
(Table 1). In addition, the KM values that were found on literature
42 reviews for uric acid detection-based uricase are 1.57 mM and 0.90
mM [21,29]. Since the enzymatic reactions take place through a carrier
41 medium, the affinity of the enzyme towards the substrate is not the
-0.2 -0.1 0 0.1 0.2 0.3 same in every carrier media [21]. Therefore, it is considered normal
1 / Absorbance min-1

40 different values of KM will be found on several literature reviews.

y = -12.38x + 39.45
39 R² = 0.858 Real sample analysis

38
The proposed method was applied for detection of uric acid in
real samples. The 2-male volunteers’ urine were assayed in order
37 to demonstrate the practical usage of the developed method. The
concentration of uric acid in the samples was calculated using the
36 constructed calibration curve (Figure 6) and the result is listed in Table
Concentrations of uric acid (mM) 2. The results based on the developed method were compared with the
results obtained using a established clinical method. The accuracy of
Figure 7: Lineweaver-Burk plot for the uric acid towards uricase enzyme the developed method was found to be high as verified by the t-test with
activity.
two samples (paired t-test), at a confidence level of 95% (7.36<12.76),

J Anal Bioanal Tech Biosensing ISSN:2155-9872 JABT, an open access journal

Citation: Hamzah HH, Zain ZM, Musa NLW, Lin YC, Trimbee E (2013) Spectrophotometric Determination of Uric Acid in Urine Based-Enzymatic
Method Uricase with 4-Aminodiphenylamine Diazonium Sulfate (Variamine Blue RT Salt). J Anal Bioanal Tech S7: 011. doi:10.4172/2155-
9872.S7-011
Substance Interference %
Ascorbic Acid 3.1
Urea 1.5
Glucose 0.3
Note: Interference (%)=[(x-y)/y]×100, where x is the average of absorbance
value for mixed solution of uric acid and foreign substances, y is the average of
absorbance value for uric acid solution only.
Table 3: Effect of possible interfering substances.
demonstrating that the results obtained by both developed method
and clinical method are in excellent agreement. This indicates that it is
possible to apply the developed method for determination of uric acid
in urine samples.
Inteference study
Effects of the presence of interfering substances (ascorbic acid, urea,
and glucose) on detection of uric acid were evaluated using 10 mM uric
acid, and different concentrations of interfering substances. The degree
of the interference of ascorbic acid, urea, and glucose towards detection
of uric acid are listed in Table 3. From the listed results, all the foreign
substances gave very lowest interference with the different signals.
However, previous works by Syed et al. and Fatma, they reported that
there were not any signal interferences detected for ascorbic acid, urea
and glucose respectively [20,21]. In conrast the previous works by Yan
and co-workers reported that slight interferences were seen in their
methods for ascorbic acid, glucose and urea respectively [22].
Conclusion
Some biosensors-based uricase enzyme immobilized and
spectrophotometric methods for uric acid determination have been
reported [13,16,20,21,27,30]. In this work, a simple spectrophotometric
method for detection of uric acid in urine is based on enzymatic
reaction of uricase, hyrogen peroxide and variamine blue RT Salt
has been developed. A favourable use of dye Variamine blue RT salt
(yellow) for determination of uric acid has been described based upon
the formation of a pale yellow-green coloured adduct of Variamine
blue RT-hydrogen peroxide. The optimized pH condition for operation
was at pH 9. The response of the method was linear within substrate
concentrations of 0.5-13 mM at the response time of 20 minutes. A
good reproducibility (RSD=0.7%) was obtained using the developed
method indicating a reliable detection system. The LOD value was 0.58
mM. The kinetic interpretations of the absorbance response of uric acid
upon uricase were plotted using the Lineweaver-Burk and Hofstee plots
respectively. The proposed method is simple, easy to operate, and able
to detect uric acid in urine at the permitted level with satisfactory results.
Acknowledgement
The authors are indebted to the Research Management Institute (RMI),
Universiti Teknologi MARA (UiTM), Shah Alam, Malaysia for funding under research
project No.021000110032 as well as Ministry of Higher Education, Malaysia for
support under Exploratory Research Grant Scheme (ERGS) project No. 600-RM1/
ERGS5/3(13/2012). Also, for Majlis Amanah Rakyat (MARA) for sponsoring Hairul
Hisham Hamzah on his PhD studies at the University of Southampton, Highfield
Campus, Southampton UK.
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Biosensing ISSN:2155-9872 JABT, an open access journal
Citation: Hamzah HH, Zain ZM, Musa NLW, Lin YC, Trimbee E (2013) Spectrophotometric Determination of Uric Acid in Urine Based-Enzymatic
Method Uricase with 4-Aminodiphenylamine Diazonium Sulfate (Variamine Blue RT Salt). J Anal Bioanal Tech S7: 011. doi:10.4172/2155-
9872.S7-011

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Citation: Hamzah HH, Zain ZM, Musa NLW, Lin YC, Trimbee E (2013) Spectrophotometric
Determination of Uric Acid in Urine Based-Enzymatic Method Uricase with
4-Aminodiphenylamine Diazonium Sulfate (Variamine Blue RT Salt). J Anal Bioanal
Tech S7: 011. doi:10.4172/2155-9872.S7-011

J Anal Bioanal Tech Biosensing ISSN:2155-9872 JABT, an open access journal