Analytica Chimica Acta 369 (1998) 123128

Amperometric biosensor for uric acid based on

uricase-immobilized silk broin membrane
Yu-Qing Zhanga,*, Wei-De Shena, Ren-Ao Gub, Jiang Zhuc, Ren-Yu Xuec
a
Biotechnology Laboratory for Silkworm and Silk, School of Sericulture, Suzhou University, Suzhou 215151, China
b
Department of Chemistry, Suzhou University, Suzhou 215151, China
c
Department of Biology, Suzhou University, Suzhou 215151, China

Received 12 December 1997; received in revised form 30 March 1998; accepted 3 April 1998

Abstract

An amperometric urate sensor based on a uricase-immobilized silk broin membrane and an oxygen electrode in ow
injection analysis are described in the present paper. The biosensor shows that recoveries of uric acid in human serum and
urine are in the range of 94.2102.6% and 92.597.9%, respectively. The relative standard deviations (RSDs) for repeatedly
monitoring standard urate solution, human serum and urine are 2.37, 3.72 and 2.95%, respectively, based on 100
measurements. The urate sensor based on the uricase-immobilized membrane is capable of detecting 6070 human serum
samples per hour. Moreover, a piece of uricase-immobilized broin membrane used at the sensor could not only be stored for
over 2 years, but also repeatedly monitored more than 1000 times for biosamples such as human serum or urine. # 1998
Elsevier Science B.V. All rights reserved.

Keywords: Enzyme electrode; Flow injection analysis; Tailing inhibitor; Urate sensor

1. Introduction As earlier methods for the detection of uric in

Uricase is an enzyme participating in the nal step
of purine degradation. Uric acid represents the major
catabolite of purine breakdown in humans and for this
reason remains an important marker molecule for
disorders associated with purine metabolism, most
notably gout, hyperuric aemia and the LeschNyhan
syndrome [1]. Methods for rapidly and reliably mon-
itoring urate levels in human uids and sera in clinical
diagnoses are desirable.
*Corresponding author. Tel.: +86 512 5390341; fax: +86 512
5391606.
human uids and sera were time-consuming or needed
some toxic compounds, attempts have been made to
develop a reagentless biosensor based on an enzyme
electrode for determining this substance. There were
several reports on electrochemical biosensors for
determining urate level in human serum or urine.
Uchiyama et al. [2] described a concentration-step
amperometric sensor for uric acid using uricase.
Keedy et al. [3] used enzyme electrode for measuring
urate in undiluted whole blood. Motonaka et al. [4]
also reported a microenzyme-sensor with an osmium
complex and porous carbon for measuring uric acid.
Gilmartin et al. [5] described a convenient method

0003-2670/98/$19.00 # 1998 Elsevier Science B.V. All rights reserved.

PII S0003-2670(98)00236-0
124 Y.Q. Zhang et al. / Analytica Chimica Acta 369 (1998) 123128

using uricase for the indirect measurement of urate
based on an amperometric H2O2-sensing graphite-
epoxy resin composite electrode. As the response time
of the sensor was long (about 15 min), they have
improved the device, and developed an amperometric
sensor for uric acid based on a chemically modied
screen-printed carbon electrode coated with cellulose
acetate and uricase [6]. Recently, Elekes et al. [7]
described a bi-enzyme reactor for electrochemical
detection of low concentrations of urate and glucose.
All these sensors used an enzyme reactor, enzyme-
modied electrodes or complex electrodes, that had
one characteristic in common that they were made
newly before these sensors can run normally. An
amperometric urate sensor described here is based
on an enzyme-immobilized silk broin membrane,
that is, a macromolecular natural protein. It is demon-
strated that peroxidase (POD)-, glucose oxidase
(GOD)- and uricase-immobilized broin membranes,
having a high enzyme activity, can be stored for a long
time [810] and a GOD-immobilized broin mem-
brane was applied to an amperometric glucose bio-
sensor [11]. Here we describe an amperometric urate
sensor in ow injection analysis based on this uricase-
immobilized silk broin membrane and an oxygen
electrode for rapidly determining uric acid in human
serum and urine samples.
2. Materials and methods
2.2. Preparation of uricase-immobilized silk fibroin
membrane
An aqueous solution of silk broin that was dis-
solved in a LiBr solution was prepared as described
previously [8,9]. 0.5 ml Uricase solution (1.0 U) was
added to a given volume of aqueous broin solution,
and mixed gently. The mixture was cast on an acrylic
plate and dried in air with an electric fan at room
temperature for 48 h. The resulting membrane was
immersed into 80% methanol solution to avoid leak-
age of uricase from the membrane. It is pointed out
here that the uricase-immobilized broin membrane
was prepared and stored at 48C for 2 years before it
was used in these experiments.
2.3. Sensor fabrication and electrochemical
apparatus
As shown in Fig. 1, this urate sensor consists of a
sample mixer, a peristaltic pump, a home-made micro-
cell, a heat exchanger, an oxygen electrode (Pt cathode
and Ag anode) coated with an oxygen-permeable
polyethylene lm and a uricase-immobilized broin
membrane, an amplier for the oxygen electrode
(Shanghai Plant Physiology Institute, China) and a
Beckman 427 Integrator (USA). It is necessary to
point out that the oxygen electrode body and reactor
(microcell) were controlled by this heat exchanger so

2.1. Materials

Uricase (EC 1.7.3.3, Type I; 2.0 U/ml) from porcine

liver was obtained from Sigma. Uric acid, LiCO3,
LiBr, urea, ascorbic, glutamic acid, glucose, sucrose
and lactose were of analytical reagent grade.
A standard urate solution was prepared daily and
immediately wrapped in aluminum foil to prevent
photothermal degradation of the biomolecules. Uric
acid was dissolved in 100 ml 0.45% (g/v) LiCO3 at
708C. Then it was stored at 48C until use.
The electrolyte concentration used in the oxygen
electrode was 0.5 mol/l KCl solution. The 0.2 mol/l
phosphate buffer was adjusted to the required pH with
0.5 M HCl or NaOH solution. This buffer was then
diluted with deionized distilled water to 0.02 mol/l
phosphate solution prior to use. Fig. 1. Diagram of urate sensor in flow injection analysis.
 Y.Q. Zhang et al. / Analytica Chimica Acta 369 (1998) 123128
the body's temperature was in accordance with that for
enzymatic reaction of the base of the electrode to abate
the measuring error induced by the uctuation of the
temperature of the surroundings. Moreover, a tailing
inhibitor in proportion of 0.5% (v/v) was added into
the phosphate buffer system to eliminate the tailing of
the peak of biosamples at the sensor based on enzyme
membrane and electrode in ow injection analysis as
described previously [11]. All the experiments were
performed under the following conditions unless sta-
ted otherwise: buffer, 0.02 mol/l phosphate solution,
pH 8.50; ow rate, 1.50 ml/min; temperature,
32.00.18C; polarizing voltage, 0.640 V; sample
volume per injection, 50 ml.
3. Results and discussion
3.1. Polarizing voltage
In order to determine the optimum polarizing vol-
tage of the uricase electrode-based sensor, its differ-
ential current response (
i) was investigated with
1.0 mmol/l urate solution at a different level of work-
ing potential (Fig. 2). The response current rose lin-
early, and reached a maximum value at about 0.600 V,
then went slightly down with increase of the working
Fig. 2. Effect of electrode voltage on the current response of the
device observed with 1.0 mmol/l uric acid solution. Sample
volume, 50 ml; flow rate, 1.50 ml/min 0.02 mol/l phosphate buffer
pH 8.50; reaction temperature, 328C (0.18C). The values in the
figure are the relative standard deviations for repeated measure-
ments (n3).
125
Fig. 3. Effect of flow rate of buffer on the current response of the
device. Polarizing voltage, 0.640 V; sample volume, 50 ml
1.0 mmol/l, 0.02 mol/l phosphate buffer pH 8.50; reaction
temperature, 328C (0.18C).
voltage. Thus, in all tests below, the selected polariz-
ing voltage for the enzyme electrode is 0.640 V.
3.2. Flow rate of buffer
The ow rate of the buffer in the ow injection
analysis system inuences directly the response cur-
rent of the sensor. The differential current response of
this urate sensor decreased gradually, and response
time shortened considerably with acceleration of the
buffer ow (Fig. 3). When the ow rate was 1.50 ml/
min, the response time of 3.0 mmol/l urate solution at
the sensor was about 50 s. This implies that the sensor
can detect over 60 urate samples per hour. In all
experiments below, the ow rate of the buffer is
1.50 ml/min.
3.3. pH Effect
For the urate sensor in the ow injection analysis
system, the effect of pH on the response current of the
urate solution at the sensor was not as evident as it was
for the glucose sensor described in a previous paper
[11]. Even though the optimum pH for a standard uric
acid solution is about 9.00, the uctuation of response
current is less than 6.0 nA in the range of pH 8.00
10.00 and less than 1.0 nA in the range of pH 8.00
9.50 (Fig. 4). Therefore, in the determination of urate
126 Y.Q. Zhang et al. / Analytica Chimica Acta 369 (1998) 123128
Fig. 4. Effect of pH on the current response of the biosensor.
Sample volume, 50 ml 1.0 mmol/l. Other conditions for determina-
tion were the same as in Fig. 3.
sample, the urate sensor could run well in alkaline
buffer.
3.4. Effect of temperature on urate sensor
The urate sensor based on the enzyme electrode is
very sensitive to temperature changes. Fig. 5 shows
the effect of temperature on the response current of the
urate sensor. We observed that the response current
linearly increases with the temperature of the enzy-
0.99802, respectively
Fig. 5. Effect of temperature of enzymatic reaction on the response
of 1.0 mmol/l urate standard solution at the urate biosensor.
Polarizing voltage, 0.640 V; sample volume, 50 ml, 1.0 mmol/l;
flow rate: 1.5 ml/min; buffer, 0.02 mol/l phosphate pH 8.50.
matic reaction. In the present experiment, we selected
a temperature of 32.00.18C for heat exchanger,
reaction solution and electrode body.
3.5. Effect of O2 concentration in sample solution on
response current of urate sensor
Since the partial pressure of oxygen differs in
various biosample solutions such as human blood,
serum or urine, it is necessary to determine whether
the current response of urate sample on the sensor
suffers from the O2 concentration. The results
obtained in the experiment indicated that the increased
response current of deaerated 1.0 mmol/l urate solu-
tion (deaerated for 20 min) is about 5% higher than
control urate solution (O2-satured for 20 min). In fact,
a large amount of O2 in the continuously owing
phosphate buffer acts as a buffer against changes in
the concentration of O2 in 50 ml volume of urate
solution. Therefore, the difference of O2 concentration
in various biosamples is not sufcient to induce a
signicant inuence in the urate measurement on the
sensor.
3.6. Calibration curve
When a series of urate standard solutions were
injected into the sample mixer at sensor, their current
response peaks were recorded by a Beckman 427
Integrator. The calibration curve of the current
response of uric acid standard solution on urate sensor
showed that its linearity is not good in the range of
0.25.0 mmol/l, while in the range of 0.21.0 mmol/l,
its linearity is good, their linear equation and regres-
sion coefcient for the region (0.21.00 mmol/l) being
y5.479715.621x, and
(Fig. 6).
3.7. Recovery tests
The accuracy of the urate sensor was evaluated by
performing recovery studies by measuring the urate
level in human serum or urine and standard additions
of uric acid. The percentage recovery was obtained by
calculating the ratio of urate detected and urate added
in human serum or urine. Table 1 indicates that
recoveries of urate in human serum and urine are in
 Y.Q. Zhang et al. / Analytica Chimica Acta 369 (1998) 123128 127

monitoring varieties of biosamples 100 times. The

relative standard deviations (RSDs) for monitoring
urate solution, human serum and urine were 2.37,
3.72 and 2.95%, respectively (Table 2). The sensor
is able to detect 100 serum samples within 94 min. The
response time per serum sample is about 50 s. There-
fore, this urate sensor is capable of detecting over 60
serum samples per hour.

3.9. Interference from other molecules

The bar chart shown in Fig. 7 depicts the interfer-

Fig. 6. Calibration curve of difference current response of urate
standard solutions on the urate biosensor. Polarizing voltage:
0.640 V; buffer: 0.02 mol/l Na2HPO4 (pH 9.00); temperature: 32.0
0.18C; flow rate: 1.5 ml/min; sample: 50 ml urate standard
solutions.
the range of 94.2102.6% and 92.597.9%, respec-
tively.
3.8. Repeatability
In order to understand the analytical error of the
urate sensor, repeatability was tested by repeatedly
ences from other compounds. Results are compared
with a normalized 1.0 mmol/l urate response (taken as
being 100%). We could see that sugars and other
molecules have almost no response except ascorbate
and lactose. There was a clear current response of
1.0 mmol/l ascorbate solution at the sensor. There
were a lot of reports on eliminating the interference
from biosamples to enzyme electrodes, in which an
acetate cellulose lm [6], bacterial cellulose mem-
branes [12], or a new cellulose-based membrane [13]
were used for abating or eliminating the negatively
charged interference. Therefore, further investigations
including the replacement of the polyethylene lm
covering at the base oxygen electrode in the present
experiment with acetate cellulose membrane or appli-
cation of an enzyme-immobilized broin composite
membrane with an acetate cellulose membrane to
avoid or reduce the interference of ascorbate from
biosamples are in progress.

Table 1
Recovery tests for human serum and urine

Sample sort Mixturea (V:V) Expected (mmol/l) Detected (mmol/l) Recovery (%)

Serum 1:1 13.70 12.94 98.25

2:1 9.94 10.20 102.62
3:1 8.05 8.10 100.62
4:1 6.92 6.52 94.22
5:1 6.17 5.97 96.67

Urine 1:1 10.13 9.92 97.63

2:1 9.34 8.64 92.51
3:1 8.94 8.62 96.42
4:1 8.70 8.23 94.60
5:1 8.54 8.16 95.55
a
Human serum (2.4047 mmol/l) or urine (15.5056 mmol/l) was mixed with 25.0 mmol/l uric acid solution in different proportions. The
mixture solutions were diluted 10-folds prior to measurement. All values in the table were averages of three replicate measurements.
128 Y.Q. Zhang et al. / Analytica Chimica Acta 369 (1998) 123128
Table 2
Repeatability analysis for standard uric acid solution, human serum and urine at the urate sensor
Samplea Repeatedly times Arithmetical mean (nA)
Uric acid 100 20.9942
Serum 100 18.5256
Urine 100 31.0315
a
50 ml 1.0 mmol/l uric acid standard solution, 25 ml human serum and 5 ml urine were measured repeatedly for 100 times, respectively.
Fig. 7. Interference from other molecules. The concentrations of
varieties of compound were 1.0 mmol/l. The current response
values of 1.0 mmol/l uric acid solution was set as 100%. Other
experimental conditions are the same as in Fig. 6.
3.10. Sensor stability
The stability of the urate sensor based on the uri-
case-immobilized broin membrane and oxygen elec-
trode focuses mainly on stability of the enzyme
membrane. The uricase-immobilized broin mem-
brane is just as stable as POD- and GOD-immobilized
broin membranes described in previous reports [8,9].
This uricase membrane could be stored for a long time
(over 2 years) and is also stable in phosphate buffer for
34 months. This uricase membrane could be used
repeatedly for monitoring varieties of samples such as
human serum and urine for more than 1000 times. In
the present experiment, the uricase membrane's activ-
ity lost only less than 10% of its original activity when
Sd-1 RSD (%)
0.4766 2.27
0.6892 3.72
0.9154 2.95
this piece of uricase-immobilized broin membrane
(less than 10 mm in diameter) at the urate sensor was
used for monitoring of 558 urate standard samples and
620 human serum or urine samples within 1 month.
Acknowledgements
The authors are grateful to Mr. Shi Xing-Quan at
Suzhou 7th Hospital, China, for providing human
serum samples. This work was supported by Grant-
in-Aid No. JW970054 from the Education committee
of Jiangsu Province, China.
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