ANALYTICAL SCIENCES APRIL 2014, VOL.

30 501
2014 The Japan Society for Analytical Chemistry

Covalent Immobilization of Uricase Inside a Plastic Vial for Uric

Acid Determination in Serum and Urine
Nidhi CHAUHAN,* PREETI,** PINKY,** and C. S. PUNDIR*

*Department of Biochemistry, M. D. University, Rohtak, 124001, Haryana, India

**Centre for Biotechnology, M. D. University, Rohtak, 124001, Haryana, India

Uricase from Candida species was immobilized covalently onto the inner wall of a plasticized polyvinyl chloride
(PVC/plastic) vial through glutraldehyde coupling with a 65.23% retention of its initial activity and a conjugation yield
of 0.37 mg/cm2. The vial-bound enzyme showed the optimum activity at pH 7.2, when incubated at 45 C for 5 min.
There was a linear relationship between the immobilized uricase activity and the uric acid concentration in the range of
0.01 to 1.2 mM with an apparent Km for uric acid of 0.17 mM. The vial-bound enzyme was employed for an enzymic
colorimetric determination of uric acid in serum and urine. The minimum detection limit of the method was 0.01 mM.
The analytical recoveries of added uric acid in serum (10 and 20 mM) were 98.0 and 96.5%, respectively. Within and
between assays, the coefficients of variation (CVs) for urate in sera determinations were 5.6 and 4.7%, respectively. A
good correlation (r = 0.997) was obtained between the serum uric acid values by the standard enzymic colorimetric
method using free enzyme and the present method. The vial was reused 200-times over a period of 4 months, when
stored at 4C.

Keywords Covalent immobilization, PVC vial, serum, uric acid, uricase, urine

(Received September 28, 2013; Accepted January 25, 2014; Published April 10, 2014)

membrane,23 cellulose acetate membrane,24 ZnO nano

Introduction
Uric acid is an end product of purine metabolism in human. A
number of diseases and pathological disorders are related to
variations of the uric acid concentration in serum and urine such
as gout, arthritis,1 cardiovascular disease2 and neurological
diseases.3 Methods based on uricase and mass fragmentography
have been proposed as reference methods. However, mass
fragmentography is not suitable for routine clinical practice, and
offers only a certified reference method for calibration purpose.4
In other methods, such as electrochemical,5 HPLC,6,7
chemiluminescence8 and fluorescence methods,9 either
substances coexisting with uric acid can affect the determination,
or the related sample preparation procedure is time consuming,
and certain amounts of uric acid may also become oxidized.10
Nevertheless enzymic colorimetric/spectrophotometric methods
based on uricase are much more popular in clinical practice,
since the enzyme has a unique ability of molecular
recognition.11,12 However, the enzymes are expensive for routine
purposes. The immobilization of the enzymes onto insoluble
supports not only permits their easy separation off the reaction
medium, but may also improve their stability and selectivity1315
and often reduces the cost of the procedure.12
Uricase from different sources has been immobilized onto
different supports, such as nylon tubing,16 polyamide tubing,17
elastin,18 the inner wall of glass beaker,19 hornblende,20 protamine
glass beads,21 dextran and polyethylene glycol,22 silk fibroin
tetrapods,25 polystyrene membrane,26 alkylamine/arylamine
glass beads,12 egg shell membrane,27 epoxy resin biocomposite
membrane,28 polyethylene terephthalate membrane,29 silk fibroin
nanoparticles,30 zinc oxide nanoflakes (ZnO-NF)31 and
Naon/cellulose inner membrane.32 The enzyme has been
immobilized onto these supports mostly either through
absorption or entrapment, which allows its leakage during their
washing for reuse. We have reported on the covalent
immobilization of oxalate oxidase onto a plasticized polyvinyl
chloride (PVC) surface, which is one of the cheapest, widely
available, chemically inert, corrosion free, weather resistant,
tough, light weight and maintenance free support, as it has a
high strength-to-weight ratio.33,34 This report describes for the
first time, the covalent immobilization of uricase on the inner
wall of a plasticized PVC vial and its application for the
determination of uric acid in serum and urine. The proposed
plasticized PVC vial is expected to find its immediate use in the
determination of serum/urine uric acid in clinical
laboratories/hospitals for the diagnosis and medical management
of gout patients and stone formers.
Experimental
Reagents and solutions
Recombinant uricase from Candida species expressed in
E.coli, 4-aminophenazone, glutaraldehyde, uric acid,
DEAE-Sephacel and Sephadex G-100 from Sigma Aldrich Co.

To whom correspondence should be addressed. (St. Louis, MO) and uric acid from SRL (Mumbai, India) were
E-mail: pundircs@rediffmail.com used. All other chemicals were of analytical reagent grade.
502
Plasticized PVC vial (plastic vial) was from a local market.
Colorimetric assay of free/native uricase
It was based on the measurement of H2O2 generated from the
aerobic oxidation of uric acid by uricase, using a color reaction
consisting of 4-aminophenazone, p-hydroxybenzoic acid and
horse-radish peroxidase as chromogen.12 In a 15-mL test tube
wrapped with black carbon paper, the reaction mixture
containing 1.8 mL, 0.05 M glycine buffer (pH 8.8) and 0.1 mL
enzyme was preincubated at 37 C in a temperature-controlled
water bath shaker for 40 min. The reaction was started by
adding 0.1 mL of uric acid (1 mM). After 10 min of incubation
at 37C, 1.0 mL of a color reagent was added, and kept at 37 C each) from 20 apparently healthy adults and gout patients were
for 15 min to develop the color. A520 was read in Spectronic-20
(Thermo Scientific, USA), and the content of H2O2 generated in
the reaction was determined from the standard curve of H2O2.
The color reagent consisted of 61 mg of 4-aminophenazone,
1.035 g of p-hydroxybenzoic acid and 1 mg (100 U) of horse-
radish peroxidase in 100 mL of 0.4 M sodium phosphate buffer
(pH 7.0). It was stored in an amber-colored bottle at 4 C, and
prepared fresh every week. One unit of uricase activity was
defined as the amount of enzyme required to generate 1.0 nmol
H2O2/mL min1.
Immobilization of uricase in plasticized PVC vial
Uricase in a solution (15 U/mL or 25 mg/mL) was immobilized
onto the inner wall of a plasticized PVC/plastic vial through
covalent coupling, as described.33 The plasticized PVC surface
was first treated with nitrating acid (mixture of conc. nitric acid
and sulfuric acid in a 5:1 ratio) for 6 h to cleave plasticized PVC
polymers oxidatively into small chain polymers having
protruding ends toward the surface. This acid-treated plasticized
PVC vial was washed with distilled water (DW), and then
incubated with a 2.5% (w/v) glutaraldehyde solution in a 0.05 M
sodium phosphate buffer (pH 7.0) for 7 h at 30 5 C. The
glutaraldehyde-treated surface was washed with DW twice to
remove excess of glutaraldehyde. The glutaraldehyde activated
plasticized PVC surface vial was incubated with 1.5 mL of
dissolved uricase in 50 mM sodium phosphate buffer (pH 7.0)
at 4C for 24 h in the dark. Any excess of enzyme was decanted
off and tested for remaining activity and protein concentration.
The plasticized PVC vial was tested for activity of the enzyme.
Assay of immobilized uricase
The assay of immobilized uricase was carried out in the same
plasticized PVC vial in which it was immobilized. The vial was
termed as reaction vial. The colorimetric assay of the
immobilized enzyme was done in this reaction vial in the same
manner as that described for its free/native form, except that the
free enzyme was replaced by 0.1 mL of the reaction buffer, and
was kept under constant stirring during incubation. After
incubation, the reaction mixture was transferred from the
reaction vial to a test tube (capacity, 15 mL) containing 1 mL of
the color reagent, and then incubated it at 37 C for 15 min to
develop the color in the dark. A520 of pink color was read in a
Spectronic-20. The vial containing immobilized enzyme was
washed with DW 3 4 times followed by the reaction buffer
before its use in next assay. The conjugation yield and %
retention of enzyme activity after immobilization were measured
as follow:
mg protein/enzyme immobilized
Conjugation yield = ,
cm 2 of PVC wall
ANALYTICAL SCIENCES APRIL 2014, VOL. 30
Specific activity of immobilized enzyme
Retention (%) = 100.
Specific activity of native/free enzyme
Application of the plasticized PVC vial for serum/urine uric acid
determination
Fresh serum samples from apparently healthy adults (both
male and female) and gout patients were collected from a
hospital of local Pt. BDS P.G. Institute of Medical Science, and
analyzed for uric acid using the same procedure as that described
for the assay of immobilized enzyme, except that uric acid was
replaced by serum. The first morning urine samples (1 mL
collected. Uric acid content was determined in these urine
samples in the reaction vial in the same manner as described
for the assay of immobilized enzyme, under the optimized
working conditions, except that the uric acid solution was
replaced by urine. The serum/urine samples were stored at 4 C
when not in use. The uric acid concentration in these biological
fluids was determined from the standard curve between uric
acid concentrations vs. A520 prepared under optimal conditions
(Fig. S1, Supporting Information).
Evaluation of plasticized PVC vial
The method was evaluated by studying the analytical recovery
and the precision. The serum uric acid values obtained by the
present method (y) were compared with those by the standard
enzymic colorimetric method employing free uricase (x); their
correlation coefficient (r) was calculated using the regression
equation. The interfering effect of various metals and
metabolites found in blood, such as iron, copper, zinc, potassium,
sodium, urea, ascorbic acid, cholesterol and glucose, were
studied at their physiological concentrations.
Reusability and storage stability of plasticized PVC vial bound
uricase
The long-term storage and stability of the plasticized PVC
vial bound enzyme was investigated over a 4-months period,
when the plasticized PVC vial was stored dry in a refrigerator at
4C. Plasticized PVC vial bound uricase activity was measured
once in a week.
Results and Discussion
Immobilization of uricase onto plasticized PVC/plastic vial
Commercial uricase from Candida was immobilized
covalently onto the inner wall of a plasticized PVC vial with a
conjugation yield of 0.37 mg/cm2 and a 65.23% retention of its
initial activity. During immobilization, vinyl polymers of
plasticized PVC material were broken down by treating it with
a HNO3 and H2SO4 mixture, which introduced nicks in the
long-chain polymer, and generated free ends protruding from
the polymer surface.33 When free ends of the treated polymer
were reacted with the aldehyde group of a bifunctional
cross-linking agent, such as glutaraldehyde, one aldehyde group
of a glutraldehyde reacted with the free end of the vinyl chain to
form a C=CH bond between the plasticized PVC sheet of
glutraldehyde, and thus led to activation of the surface for
covalent immobilization of the enzymes. The NH2 group on
the surface of the enzyme was attached covalently onto another
CHO group of glutraldehyde, already attached to the plasticized
PVC sheet, through Schiff-base formation. This led to the
covalent immobilization of uricase with the plasticized PVC
vial surface35 (Scheme 1).
ANALYTICAL SCIENCES APRIL 2014, VOL. 30 503

Scheme 1Schematic diagram of the covalent immobilization of

uricase onto a PVC vial.

Evidences for immobilization of uricase on plasticized PVC vial

The SEM of the surface of a chemically modified plasticized
PVC vial with the immobilized enzyme (Fig. 1b) showed folds Fig. 1Scanning electron micrographs of a chemically modified
and clusters along some beaded structures which were not PVC surface without (a) and with (b) immobilized uricase.
present on the surface of the bare plasticized PVC (Fig. 1a).
This change in the surface morphology of the plasticized PVC
support after the immobilization process is evidence of
immobilization of the enzyme.36 The formation of folds instead
of globular beaded structures may be due to a high concentration
of conjugated uricase on the surface of the plasticized PVC vial.

FTIR spectra
The FTIR spectra of bare plasticized PVC sheet (Fig. 2, curve
a) showed the three starching regions characteristics of
plasticized PVC sheet: (i) CCl stretching region in the range
from 600 700 cm1 (ii) CC stretching in the range from
900 1200 cm1 and (iii) 1250 2970 cm1 in plasticized PVC
(numerous CH modes).37 The FTIR spectra of a plasticized
PVC membrane with immobilized uricase (Fig. 2, curve b)
exhibited a peak at 1725 cm1 of the C O bond of aldehyde,
but no peak at 1725 cm1, thus revealing that the C O group of
glutaraldehyde became combined with the NH2 groups on the Fig. 2FTIR spectra of (a) PVC and (b) PVC membrane bound
uricase.
surface of the enzyme to form the N=C bond, and a peak of
the N C bond at 1630 cm1, showing no free aldehyde group
of glutaraldehyde, since it became cross-linked with the enzyme.
These studies confirmed that the enzyme was immobilized
through covalent/glutaraldehyde coupling on the surface of the behavior of the enzyme and the substrate, as well as many other
plasticized PVC membrane. factors that are usually difficult to analyze quantitatively. The
shift of the optimal pH of uricase after immobilization might be
Kinetic properties of an immobilized enzyme on a plasticized due to the loss of NH2 groups present on the surface of the
PVC vial enzyme after glutaraldehyde coupling. No change was observed
The optimal pH of immobilized uricase was 8.0, which is in the optimum temperature of the immobilized enzyme, as
higher than that of the free enzyme (pH, 7.0).38 The pH activity compared to the free enzyme (30 C). The rate of the reaction of
relationship of any given enzyme depends on the acid base immobilized uricase was linear up to 15 min, after which it was
504 ANALYTICAL SCIENCES APRIL 2014, VOL. 30

Table 1Serum uric acid level in apparently healthy adults and

gout patients, as measured by PVC/plastic vial bound uricase

Serum uric acid (mM)

Mean SEa

Sex Age in yr Healthy person Age in yr Sex Gout patient

M 21 0.28 0.03 38 F 0.49 0.02

M 25 0.34 0.02 40 F 0.51 0.05
F 32 0.22 0.02 41 F 0.45 0.02
F 27 0.27 0.04 37 M 0.60 0.03
F 35 0.34 0.05 39 F 0.49 0.05
M 33 0.29 0.03 45 M 0.54 0.01
F 22 0.24 0.05 48 M 0.63 0.03
M 34 0.31 0.02 55 M 0.49 0.02
Fig. 3Correlation between urine uric acid values determined by the M 23 0.33 0.03 49 F 0.62 0.04
standard colorimetric method (x-axis) and the PVC vial bound uricase F 37 0.23 0.01 57 F 0.56 0.02
enzyme (y-axis).
a. SE = standard error.

Table 2Urine uric acid level of diseased persons, as measured

constant. A linear relationship was obtained between the uric by PVC/plastic vial bound uricase
acid concentrations, ranging from 0.01 to 1.2 mM and A520 Urine uric acid (mM/24 h)
(Fig. S1, Supporting Information), which is better than those Mean SEa
employing an immobilized enzyme on an epoxy resin
biocomposite membrane,28 polyethylene terephthalate Sex Age in yr Healthy person Age in yr Sex Gout patient
membrane,29 egg shell membrane27 and fibroin membrane.23
M 21 6.28 0.03 38 F 7.39 0.02
Immobilized uricase had an apparent Km value of 0.17 mM for M 25 6.35 0.04 40 F 8.41 0.05
uric acid, which is lower than that for the native enzyme F 32 5.22 0.02 41 F 7.45 0.03
(0.80 mM) Vmax of immobilized enzyme was 0.66 mM/H2O2 F 27 5.27 0.02 37 M 8.50 0.03
min, which is higher than that for the native enzyme F 35 6.31 0.05 39 F 9.49 0.05
(0.084 mM).39 The decrease in Km of uricase after immobilization M 33 6.29 0.05 45 M 8.55 0.01
indicates the increased affinity of the enzyme after F 22 5.24 0.05 48 M 8.63 0.01
immobilization. The changes in Km and Vmax of an enzyme after M 34 5.32 0.01 55 M 7.49 0.02
covalent immobilization on a solid support are controlled by the M 23 6.34 0.03 49 F 8.62 0.04
F 37 5.23 0.01 57 F 8.57 0.02
changes in the enzyme confirmation, steric effects, its
microenvironment and bulk and diffusibilities of substrate and a. SE = standard error.
products.40 The immobilized uricase maintained its original
activity in the presence of interferents, such as iron chloride,
urea, ascorbic acid, potassium nitrate, sodium chloride,
cholesterol, copper sulphate, zinc sulfate and glucose at their the method was tested by studying its correlation with the
physiological concentration, revealing a stable conformation of standard enzymic colorimetric method employing a free enzyme.
the immobilized enzyme. PVC material is a highly inert The uric acid level in 19 sera samples from apparently healthy
material, and the enzyme is covalently attached to it, which adults and gout patients, as determined by the present method
provides stability to the immobilized enzyme. (y) were compared with those by the standard enzymatic
colorimetric kit method (x). The values obtained by both
Evaluation of plasticized PVC vial for uric acid determination methods agreed with each other, showing a good correlation
The minimum detection limit of the method was 0.01 mM (in (r = 0.997) (regression equation being y = 1.0148x + 0.0072)
reaction mixture), which is lower than that by an epoxy resin (Fig. 3). These results are comparable to those using epoxy
biocomposite membrane28 and a polyethylene terephthalate resin biocomposite membrane (r = 0.98)28 and polyethylene
membrane.29 To check the reliability of this method, the terephthalate membrane (r = 0.98).29
analytical recovery of added uric acid in six serum samples was
determined. The mean analytical recoveries of added uric acid Determination of serum/urine uric acid by a plasticized PVC vial
(10 and 20 mM) in serum were 98.0 and 96.5%, respectively The plasticized PVC vial was employed to measure uric acid
(Table S1, Supporting Information), which are comparable to in serum and the urine of apparently healthy adults and gout
those by an epoxy resin biocomposite membrane28 and this patients. The uric acid in serum, as measured by the present
polyethylene terephthalate membrane.29 To study the method in apparently 10 healthy adults, was 0.22 to 0.34 mM
reproducibility and reliability of the method, the uric acid with a mean of 0.28 mM, which is within the normal established
content in six serum samples in one run (within-batch) and after range of 0.21 0.36 mM and 0.49 to 0.63 mM with a mean of
storage at 25 C for one week (between-batch) were determined. 0.53 mM in gout patients (Table 1). The urine uric-acid level in
The results showed that the uric acid value of these determination healthy adults and gout patients was 5.22 to 6.35 mM/24 h with
agreed with each other; the within-batch and between-batch a mean of 5.78 mM/24 h (within the normal established range
coefficients of the variation (CVs) were 5.6 and 4.7% (Table S2, <6.0 mM/24 h) and 7.39 to 8.63 mM/24 h with a mean of
Supporting Information), which is quite close to the results of 8.31 mM/24 h, respectively (Table 2). The levels of serum/urine
earlier methods using an epoxy resin biocomposite membrane28 uric acid in gout patients were significantly higher than those of
and a polyethylene terephthalate membrane.29 The accuracy of apparently healthy adults (p <0.01).
ANALYTICAL SCIENCES APRIL 2014, VOL. 30 505

Table 3Comparison of the analytical performance of uricase immobilized onto PVC and other membranes

Ref. 27 25 28 29 Present report

Support for Egg shell ZnO nanorods Epoxy resin Polyethylene terephthalate PVC plasticized
immobilization membrane (PET) membrane sheet

Method of immobilization Encapsulated Crosslinking Encapsulated Covalent Covalent

Optimum pH 8.0 NR 8.5 7.0 7.2

Linearity/mM 0.04 0.64 0.005 1.0 0.025 to 0.1 0.075 to 0.13 0.01 to 1.2

Detection limit/mM 0.02 2 25 75 0.01

Response time 100 s NR 10 12 s NR 5 min

Stability (days) 90 20 60 60 120

NR = not reported.

Storage stability and reusability of the plasticized PVC vial
The plasticized PVC vial bound enzyme lost 50% of its initial
activity, after its 200-times regular use over a period of 4 months,
when stored in sodium phosphate buffer (pH 7.0) at 4
(Fig. S2, Supporting Information). This reusability of a
plasticized PVC vial of immobilized enzyme was higher than
that for an epoxy resin biocomposite membrane,28 polyethylene
terephthalate membrane,29 egg shell membrane27 and fibroin
membrane23 (Table 3).
Conclusions
The covalent immobilization of uricase on a plasticized PVC
vial not only allowed for repeated use in a colorimetric
determination of uric acid in urine and serum, but also resulted
in its improved analytical performance and storage stability.
The immobilized enzyme showed no interference by a number
of serum substances during uric acid determination. The
plasticized PVC vial could also be used as a support for the
covalent immobilization of other enzymes employed in various
diagnostic kits.
Acknowledgements
We are thankful to Dr. Vikas Hooda, Centre for Biotechnology,
M. D. University, Rohtak for providing uricase, and allowing
two of the authors (Preety and Pinky) to work in the laboratory
of the corresponding author.
Supporting Information
This material is available free of charge on the Web at http://
www.jsac.or.jp/analsci/.
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