Potentiometric urea biosensor based on carbon nanotubes and polyion complex film

Article abstract of DOI:10.1166/jnn.2015.9327

Enzymatic electrode for a potentiometric urea sensor was prepared by sequential coating of carbon nanotube (CNT), urease (Urs) and polyion complex (mixture of poly-L-lysine hydrobromide and poly (sodium 4-styrenesulfonate), PIC) on an ITO glass. The prepared electrode (ITO/CNT/Urs/PIC) was characterized by potentiometric measurements at different urea concentrations in Tris-HCl buffer (pH 7.0). The potentiometric response of the electrode was linear in the range of 1 × 10^-5 to 3 × 10^-3 M with a correlation coefficient of 0.999 and a sensitivity of 59.1 mV/decade. It was found that the addition of CNT caused considerable improvement of the sensitivity of the electrode to urea. The response time was approximately 60-90 s. A half of the initial sensitivity was retained for 15-17 d at room temperature.

Full text of DOI:10.1166/jnn.2015.9327

Article
Journal of
Nanoscience and Nanotechnology
Vol. 15, 1150–1153, 2015
Copyright © 2015 American Scientific Publishers
All rights reserved
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Potentiometric Urea Biosensor Based on Carbon
Nanotubes and Polyion Complex Film
∗
Linh Thi My Nguyen and Hyon Hee Yoon
Department of Chemical and Biological Engineering, Gachon University, Seongnam, Gyeonggi-do 406-701, S. Korea
Enzymatic electrode for a potentiometric urea sensor was prepared by sequential coating of carbon
nanotube (CNT), urease (Urs) and polyion complex (mixture of poly-L-lysine hydrobromide and poly
(sodium 4-styrenesulfonate), PIC) on an ITO glass. The prepared electrode (ITO/CNT/Urs/PIC) was
characterized by potentiometric measurements at different urea concentrations in Tris-HCl buffer (pH
−
5
−3
M
7
.0). The potentiometric response of the electrode was linear in the range of 1×10 to 3×10
with a correlation coefficient of 0.999 and a sensitivity of 59.1 mV/decade. It was found that the
addition of CNT caused considerable improvement of the sensitivity of the electrode to urea. The
response time was approximately 60–90 s. A half of the initial sensitivity was retained for 15–17 d
at room temperature.
Keywords: Urea Sensor, Urease, Carbon Nanotube, Polyion Complex.
Delivered by Publishing Technology to: McMaster University
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Copyright: American Scientific Publishers
1
. INTRODUCTION
carbon nanotubes are also used as a component in the bio-
electrode architecture due to their large surface area, high
Urea is the waste product of protein metabolization in
human body and its analysis has been an important opera-
tion in a clinical chemical laboratory because the level of
urea in blood is a measure of renal functioning. Monitoring
of urea levels is also performed in food and environmen-
1
7ꢀ18
electrical conductivity, and chemical stability.
Electro-
chemical property of CNTs for detecting pH and ammonia
1
9
was reported earlier.
For the enzymatic electrode fabrication, electrode archi-
tecture components such as enzyme and CNT should
be well dispersed and integrated on the electrode sur-
face without leakage during use to achieve fast response
and stability. Most immobilization methods are based on
adsorption, covalent binding, entrapment, encapsulation,
1
ꢀ2
tal industries. The concentration of urea can be mea-
3
ꢀ4
sured by chromatographic and spectroscopic methods.
However, these methods require a complicated sample pre-
treatment and are inapplicable to on-site monitoring. As
alternative methods, various types of biosensors based on
urease enzyme have been developed due to their simplicity
and capability for on-site urea analysis.
2
0
and/or cross-linking. Recently, polyion complex (PIC)
has been used as a matrix for the enzyme immobilization
2
1ꢀ22
in the preparation of bioelectrodes.
tive membrane and has molecular sieving ability.
It is a permselec-
Urea is converted enzymatically by urease to ammo-
nia and carbon dioxide. The concentration of urea is
2
3
+
−
In this study, we attempted to combine the CNT
and urease together on a base electrode using PIC
to prepare an enzyme electrode. The simply integrated
CNT/Urease/PIC-layered electrodes were characterized for
the application in urea sensors.
then measured by monitoring the NH4 and HCO3 ions
in the sample. These ions can be detected using various
5
ꢀ6
7ꢀ8
tranducers including amperometric,
potentiometric,
9
ꢀ10
11ꢀ12
thermal,13ꢀ14 and piezo-
conductometric,
electric tranducers.
optical,
1
5ꢀ16
Since the enzymatic reaction of
urea is not electroactive, potentiometric urea biosensors
using pH-sensitive conducting polymers are widely used.
However, the slow response for steady state potential val-
ues is often problem for its practical applications. To
enhance speed and sensitivity of the urea biosensors,
2. EXPERIMENTAL DETAILS
2
.1. Chemicals and Materials
Urease (Type III, 31660 units/g solid), poly-L-lysine
PLL), and poly-sodium-4-styrensulfonate (PSS) were pur-
(
chased from Sigma–Aldrich. Carbon nanotube (CNT)
(multiwalled, > 95 wt% purity, 10–15 nm in diameter) was
∗
Author to whom correspondence should be addressed.
1
150
J. Nanosci. Nanotechnol. 2015, Vol. 15, No. 2
1533-4880/2015/15/1150/004
doi:10.1166/jnn.2015.9327

Nguyen and Yoon
Potentiometric Urea Biosensor Based on Carbon Nanotubes and Polyion Complex Film
3
3
50
00
purchased from Iljin Nanotech Co., Ltd. and used without
purification. All other chemicals were of reagent grade.
Indium tin oxide (ITO) glass (< 20 X/square) was used
as a base electrode. ITO glass was cut to a rectangular
shape of 5 mm in width and 30 mm in length. The working
area of the ITO glass for the electrochemical tests was
Immobilization
Free
250
2
1
1
00
50
00
2
controlled to 0.25 cm using a masking tape.
2
.2. Preparation of Enzyme Electrodes
CNT/Urs/PIC-layered electrodes were prepared on the ITO
glass. First, the ITO was coated with 15 ꢁL of CNT
suspension (0.335 mg/mL in N,N-dimethylformaide) and
dried at room temperature. Onto the CNT layer, ure-
ase solution of 10 mg/mL in a Tris-HCl buffer solution
5
0
0
0
20
40
60
80
100 120 140 160 180
Time [min]
(
Tris, pH 7.0) was dripped and dried at room tempera-
Figure 1. The comparison of enzymatic activities of free urease and
immobilized urease on electrode (ITO/CNT/Urs/PIC) as measured by UV
absorbance (ꢄ = 380 nm).
ture. Finally, 5 ꢁL of PLL aqueous solution (60 mmol/L
monomer unit) and 5 ꢁL of PSS aqueous solution (60
mmol/L monomer unit) were successively dropped on the
surface and dried in air to form the PIC film. It was store
−
4
3
×10 M, the correlation coefficient for linear curve was
0.992.
The effect of urease enzyme and CNT loading and PIC
ꢀ
at 4 C.
loading (film thickness) on the electrochemical response
was investigated to optimize the composition of the elec-
trode architecture. As shown in Figure 3, the potential
2
.3. Analysis
Ammonia concentrations were analyzed by UV-Vis spec-
trophotometer (UV/Vis, Varian) using Nessler’s reagent.
Potentiometric measurements were carried out using a
potentiostat (VSP, Bio-Logic) with a conventional three-
2
response of the electrode with 0.75 mM/cm of PIC load-
ing was highest at the early stage of the measurement,
however, it decreased later on, indicating the PIC film is
electrode system: a working electrode, an Ag/AgCl (3M
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too thin and thus unstable for the entrapment of urea.
On the other hand, the electrode with 3.0 mM/cm of
Copyright: American Scientific Publishers
NaCl) electrode as a reference a nI Pd : a6 7p .l 1a t0i n3 u. 1m 9 6w. 2i r5e Oa sn :a Sat, 26 Dec 2015 04:00:06
2
counter electrode. All measurements were performed at
about 25 C in Tris–HCl buffer solution (pH 7.0).
ꢀ
PIC loading showed unstable potential response, probably
due to the a high diffusion barrier of the thick film. There-
2
fore, 1.5 mM/cm was chosen as optimum PIC loading
3
. RESULTS AND DISCUSSION
and used throughout the present study.
Enzymatic activity of the immobilized urease was evalu-
ated by measuring reaction rate of urea hydrolysis. Ammo-
nia is produced by the hydrolysis of urea. The ammonia
then reacts with Nessler’s reagent to form a colored prod-
uct as shown in the following reactions:
CNT loading also affected the potential response of
the ITO/CNT/Urs/PIC electrode as shown in Figure 4.
Comparing CNT loading of 0.32, 0.16 and 0.24 ꢁg/cm ,
2
0
0
0
.8
.6
.4
0
.8
Urease
y = 0.0023x –0.0027
NH CONH ꢂureaꢃ+H O −− −→ 2NH +CO
(1)
(2)
0.6
0.4
2
2
2
2
3
2
R
= 0.992
II 2−
+
4
−
2
NH +2Hg I −→ NH Hg I +NH +5I
3
4
2
2 3
0
0
.2
.0
By measuring the spectrophotometric absorbance for
f
the product NH Hg I (ꢄmax = 380 nm), urea concen-
0
50 100 150 200 250 300
2
2 3
e
Urea concentration [µΜ]
tration are determined. Figure 1 shows reaction rates of
urea hydrolysis catalyzed by free and immobilized ure-
ases. The activity of the immobilized urease, as calcu-
lated from the slope, exhibited about 40% of that of free
enzyme.
0.2
d
c
b
0.0
a
–0.2
In order to analyze the analytical performance of the
ITO/CNT/Urs/PIC electrode, the electrode was immersed
in urea solutions of different concentrations and then the
UV absorbances of the resulting solutions were observed.
Figure 2 depicts the absorbance response to varying con-
centrations of urea. Inset of Figure 2 represents a typi-
3
50
400
450
500
550
600
650
Wavelength [nm]
Figure 2. Spectrophotometric response to varying concentrations of
−5
−5
−5
−4
urea (a) 1 × 10 M, (b) 2 × 10 M, (c) 5 × 10 M, (d) 1 × 10 M,
−4
−4
(
e) 2×10 M, and (f) 3×10 M in the presence of ITO/CNT/Urs/PIC
electrode. Inset: linear regression curve for UV absorbance (ꢄ = 380 nm)
−
5
cal calibration curve for urea. In the range of 1×10 to
versus urea concentration.
J. Nanosci. Nanotechnol. 15, 1150–1153, 2015
1151

Potentiometric Urea Biosensor Based on Carbon Nanotubes and Polyion Complex Film
Nguyen and Yoon
0
0
0
0
0
0
.45
.40
.35
.30
.25
.20
a
b
a
a
0.75 mM/cm2
0.25
0.20
a
b
b
c
b
2
1
3
.5 mM/cm
mM/cm2
d
c
c
c
e
d
d
d
0
0
.15
.10
f
0.05
0
2
4
6
8
10
12
14
0
5
10
15
20
Time [min]
Time [min]
Figure 5. Chronopotentiometric response of ITO/CNT/Urs/PIC elec-
Figure 3. Effect of PIC loading (film thickness); Chronopotentiometric
−5
−5
trode to the subsequent addition of urea; (a) 1×10 M, (b) 3×10 M,
responses of ITO/CNT/Urs/PIC electrode with different PIC loadings to
(c) 7.5×10− M, (d) 3×10 M, (e) 1×10 M, and (f) 3×10 M.
5
−4
−3
−3
−5
−5
the subsequent addition of urea; (a) 1 × 10 M, (b) 3 × 10 M, (c)
.5×10 M, (d) 3×10 M.
−
5
−4
7
200
considerable improvement of the sensitivity of the electrode
2
to urea was obtained at CNT loading of 0.32 ꢁg/cm . The
150
electrode showed no significant enhancement of the sensi-
2
tivity by further addition of CNT more than 0.32 ꢁg/cm .
1
00
The potentiometric response of the enzyme electrode
was measured for urea analysis. Figure 5 shows a typical
chronopotentiometric response of ITO/CNT/Urs/PIC elec-
y = 59.1x + 302.1
R2 = 0.999
2
5
0
−
5
trode to increasing urea concentration from 1 × 10 to
Delivered by Publishing Technology to: McMaster University
−
3
3
× 10 M. The response time of the enzyme electrode
IP: 67.103.196.25 On: Sat, 26 Dec 2015 04:00:06
0
was in the range of 60–90 s. This re Cs p oo pn ys er i gt ihmt :eAi ms ce or imc a- n Scientific Publishers
8
parable with those of previously reported biosensors. The
–5.0
–4.5
–4.0
–3.5
Log[Urea]/M
–3.0
–2.5
–2.0
calibration curve for the urea concentration is shown in
−
5
−4
Figure 6. It was linear in the range of 1×10 to 3×10
−
3ꢅ5
Figure 6. Calibration curve for response of ITO/CNT/Urs/PIC elec-
(
i.e., 1×10 ꢃ M with a correlation coefficient of 0.999.
trode to urea in the range 1×10− to 1×10
5
−3ꢅ5
M.
This linearity range is well comparable with that obtained
in the spectrophotometric response studies. The slope of
the linearity, i.e., the sensitivity of the enzyme electrode
towards urea concentrations was 59.1 mV per decade.
The electrode was examined for the enzyme stability. As
shown in Figure 7, the potential response to 5 mM of urea
was measured every for 20 d. The electrode exhibited good
operation stability for 10 d. The potential response started
a
0
0
0
0
0
0
0
0
0
.42
.40
.38
.36
.34
.32
.30
.28
.26
b
b
c
b
b
100
b
c
d
8
6
0
0
c
d
c c
d
0.00 µg/cm2
d
.16 µg/cm2
d
40
20
0
0
0
0
0
2
[Urea] = 0.005 M
.24 µg/cm
.32 µg/cm2
.40 µg/cm2
0
2
4
6
8
Time [min]
0
5
10
15
20
Time [day]
Figure 4. Effect of CNT loading; Chronopotentiometric responses of
TO/CNT/Urs/PIC electrodes with different CNT loadings to the subse-
Figure 7. Variation of potential response of ITO/CNT/Urs/PIC elec-
trode to 5 mM of urea with repeated use after storage at room
temperature.
−5
−5
−5
quent addition of urea; (a) 1×10 M, (b) 3×10 M, (c) 7.5×10 M,
and (d) 3×10− M.
4
1
152
J. Nanosci. Nanotechnol. 15, 1150–1153, 2015

Nguyen and Yoon
Potentiometric Urea Biosensor Based on Carbon Nanotubes and Polyion Complex Film
to decrease slowly after 10 d due to the enzyme denatura-
tion and loss of enzyme from the electrode surface. A half
of the initial sensitivity was retained for 15–17 d at room
temperature.
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1
9
(
−5
−4
M
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improvement of the sensitivity of the electrode to urea. The
response time was approximately 60–90 s. A half of the
initial sensitivity was retained for about 15–17 d at room
temperature. The result indicates that an enzymatic elec-
trode for a potentiometric urea sensor can be prepared by
simply entrapping urease with polyion complex.
1
1
1
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Acknowledgment: This work was supported by
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Received: 31 May 2013. Accepted: 14 October 2013.
J. Nanosci. Nanotechnol. 15, 1150–1153, 2015
1153