biosensors
Article
A Urea Potentiometric Biosensor Based on a
Thiophene Copolymer
Cheng-Yuan (Kevin) Lai, Peter J. S. Foot *, John W. Brown and Peter Spearman
Materials Research Centre & School of LSPC, Faculty of Science, Engineering and Computing,
Kingston University London, Penrhyn Road, Kingston upon Thames, Surrey KT1 2EE, UK;
[email protected] (C.-Y.L.); [email protected] (J.W.B.); [email protected] (P.S.)
* Correspondence: [email protected]; Tel.: +44-20-8417-2485
Academic Editor: Jeff D. Newman
Received: 11 January 2017; Accepted: 27 February 2017; Published: 3 March 2017
Abstract: A potentiometric enzyme biosensor is a convenient detector for quantification of urea
concentrations in industrial processes, or for monitoring patients with diabetes, kidney damage or
liver malfunction. In this work, poly(3-hexylthiophene-co-3-thiopheneacetic acid) (P(3HT-co-3TAA))
was chemically synthesized, characterized and spin-coated onto conductive indium tin oxide
(ITO) glass electrodes. Urease (Urs) was covalently attached to the smooth surface of this
copolymer via carbodiimide coupling. The electrochemical behavior and stability of the modified
Urs/P(3HT-co-3TAA)/ITO glass electrode were investigated by cyclic voltammetry, and the bound
enzyme activity was confirmed by spectrophotometry. Potentiometric response studies indicated that
this electrode could determine the concentration of urea in aqueous solutions, with a quasi-Nernstian
response up to about 5 mM. No attempt was made to optimize the response speed; full equilibration
occurred after 10 min, but the half-time for response was typically <1 min.
Keywords: urea; urease; biosensors; potentiometry; polythiophene; conducting polymer
1. Introduction
Enzyme-based electrochemical biosensors have achieved great commercial importance since the
first use of glucose oxidase in an amperometric sensor for glucose [1] in 1962. All such systems require
the incorporation of the biocatalytic element onto (or into) the sensing electrode structure, and this
has been achieved by physical adsorption [2] or entanglement [3], DNA intercalation [4] and a wide
variety of covalent bonding techniques [5–8].
Conducting polymers may be considered as good transducers which can help to convert
biochemical signals into electronic signals in enzyme biosensors [9]. They have the important
advantage of being mixed conductors (allowing both electronic and ionic/molecular transport) [10]
as well as having greater biocompatibility than many inorganic transducers. Immobilization of
an enzyme stably onto conducting polymer electrodes is an important type of technology for the
fabrication of efficient and enduring enzyme biosensors [11,12]. Electrochemical co-deposition has
been utilized widely to entrap enzymes into polypyrrole or its derivatives during the process of
polymerization [9,12–15]. However, the effects of harsh chemical conditions on covalent linkages within
the enzyme proteins can result in denaturation of the active material. Alternatively, immobilizing
the enzyme directly onto the conducting polymers can avoid the enzyme experiencing aggressive
conditions. The carbodiimide coupling reaction provided by 1-ethyl-3-(3-dimethylaminopropyl)
carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) has previously been used
to create a peptide bond between amides and carboxylic acids in aqueous solution at room
temperature [16]. This method has been applied for the immobilization of enzymes on modified
conducting polymers under moderate conditions [17].
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The concentration
of urea in blood serum is used to monitor diabetes and also to indicate the
onset
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2017, 7, 13
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of kidney failure and liver malfunction. A potentiometric conducting polymer biosensor is a convenient
concentration
of urea in blood
is potential
used to monitor
diabetes
and
to indicate
device toThe
quantify
urea concentration,
sinceserum
its rest
varies when
urea
is also
hydrolyzed
by the
urease,
onset
of
kidney
failure
and
liver
malfunction.
A
potentiometric
conducting
polymer
biosensor
is a
−
causing a p[OH ] change in the analyte [9,12]. Many studies have investigated the immobilization
convenient device to quantify urea concentration, since its rest potential varies when urea is
of enzymes on polypyrrole because of− its high biocompatibility and low electropolymerization
hydrolyzed by urease, causing a p[OH ] change in the analyte [9,12]. Many studies have investigated
potential [11,17–21]. However, a drawback is the frequent poor morphology of polypyrrole film,
the immobilization of enzymes on polypyrrole because of its high biocompatibility and low
which
may cause the detection to be irreproducible; the polymer is also susceptible to oxidative
electropolymerization potential [11,17–21]. However, a drawback is the frequent poor morphology
damage.
Other conducting
polymers
such the
as polyaniline
more stable biosensor
properties
of polypyrrole
film, which
may cause
detection toshow
be irreproducible;
the polymer
is also[22],
and susceptible
have been used
in
potentiometric
[23]
and
amperometric
[24]
urea
biosensors,
but
polyaniline
to oxidative damage. Other conducting polymers such as polyaniline show more stable has
potential
toxic
hazards. [22],
A copolymer
of glycidyl
ferrocene [25][24]
hasurea
shown
biosensor properties
and have been
used inmethacrylate
potentiometricand
[23]vinyl
and amperometric
some
promise for
electroanalysis.
biosensors,
buturea
polyaniline
has potential toxic hazards. A copolymer of glycidyl methacrylate and
vinyl
ferrocene [25]
has been
shown
some promiseneglected
for urea electroanalysis.
Polythiophenes
have
comparatively
in the literature on electrochemical conducting
have been
comparatively
neglected
in the literature
on electrochemical
polymerPolythiophenes
biosensors, although
a glucose
sensor based
on polythiophene
was reported
as long ago as
conducting
polymer
biosensors,
although
a
glucose
sensor
based
on
polythiophene
was
reported
as
1996 [26]. This neglect may have been due to the high electropolymerization potential of thiophenes
long
ago
as
1996
[26].
This
neglect
may
have
been
due
to
the
high
electropolymerization
potential
and the low conductivity of polythiophenes under the typical conditions of biosensor use, but of
some
thiophenes and the low conductivity of polythiophenes under the typical conditions of biosensor
polythiophene-based sensors for glucose [27–31], lactate [32,33], choline [34], glutamate [35], ascorbic
use, but some polythiophene-based sensors for glucose [27–31], lactate [32,33], choline [34],
acid [36] and H2 O2 [37,38] have successfully been produced.
glutamate [35], ascorbic acid [36] and H2O2 [37,38] have successfully been produced.
In this paper, a conducting copolymer, poly(3-hexylthiophene-co-3-thiopheneacetic acid 1:1)
In this paper, a conducting copolymer, poly(3-hexylthiophene-co-3-thiopheneacetic acid 1:1)
(P(3HT-co-3TAA))
was
synthesized.
covalentlyattached
attachedtoto
carboxylate
groups
(P(3HT-co-3TAA))
was
synthesized.Urease
Urease was
was covalently
thethe
carboxylate
groups
of of
P(3HT-co-3TAA)
through
the
amine
functionalities
in
the
aminoacids,
via
carbodiimide
coupling
P(3HT-co-3TAA) through the amine functionalities in the aminoacids, via carbodiimide coupling
(Scheme
1), and
thethe
urease
activity
spectrophotometricresponse
response
studies.
(Scheme
1), and
urease
activitywas
wasinvestigated
investigated by
by spectrophotometric
studies.
O
HO
H3CO
HEX
O
CH3OH
HEX
:
1
O
1
OCH3
O
HEX
S
2N NaOH
1
OCH3
HEX
S
*
*
1
S
HEX
S
*
S
1
S
1
*
S
*
+
H2SO4
S
FeCl3
1
O
S
*
1
S
*
1N HCl
1
O-
S
*
1
OH
O
P(3HT-co-3TAA)
HEX
HEX
S
*
1
O
OH
S
1
S
*
*
+
H2N
Urs
EDC + NHS
1
S
*
1
Urs
O
N
H
Scheme
Synthesis of
acid 1:1)acid
(P(3HT-co-3TAA))
and its
Scheme
1. 1.Synthesis
of poly(3-hexylthiophene-co-3-thiopheneacetic
poly(3-hexylthiophene-co-3-thiopheneacetic
1:1) (P(3HT-co-3TAA))
functionalization with urease (Urs) by formation of a peptide linkage. EDC:
and its functionalization with urease (Urs) by formation of a peptide linkage.
EDC:
1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride; NHS: N-hydroxysuccinimide; Urs:
1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride; NHS: N-hydroxysuccinimide;
urease
Urs: urease.
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The modified polymer was used to produce urease (Urs) electrode Urs/P(3HT-co-3TAA)/ITO
glass) biosensors, which were found to give a quasi-Nernstian response to urea concentrations up to
about 5 mM by potentiometric assay. This urease electrode could therefore be used to monitor the level
of urea in blood serum, which is typically 1.3–3.5 mM (8–20 mg/dL) [8].
2. Materials and Methods
2.1. Chemicals
The starting materials were all from Sigma-Aldrich, St. Louis, MO, USA (supplied as Aldrich or
Fluka products, as indicated below). The grades/purities were as follows: 3-hexylthiophene (Aldrich
99%), 3-thiopheneacetic acid (Aldrich, 98%), anhydrous methanol (Aldrich, 99%), anhydrous iron (III)
chloride (Aldrich, 98%), urease (Fluka BioChemika, obtained from Jack beans; activity 100 units mg−1 ),
N-(3-dimethylaminopropyl)-N’-ethyl-carbodiimide hydrochloride (ECD, Fluka 98%), urea (Aldrich,
≥99.5%), TRIS buffer (tris(hydroxymethyl)aminomethane hydrochloride) (Aldrich), thionyl chloride
(Aldrich, +99%), 1,6-diaminohexane (Aldrich, 98%), potassium aluminum sulfate dodecahydrate
(Fluka, ACS puriss.) and N-hydroxysuccinimide (NHS, Fluka 97%).
2.2. Synthetic Procedures
2.2.1. Esterification of 3-thiopheneacetic acid
Firstly, 3-thiopheneacetic acid (1.42 g, 0.01 mol) was dissolved in methanol (20 mL), followed
by the addition of a few drops of conc. H2 SO4 . The solution was stirred for 24 h at 100 ◦ C, and the
excess methanol was then removed by rotary evaporation. Distilled water (10 mL) was poured into
the flask and the shaken mixture was extracted with diethyl ether (10 mL). The organic layers were
collected and dried with anhydrous MgSO4 , and the solvent was removed by rotary evaporation.
The methyl ester product was a yellow oily liquid showing a single component with m/z = 156 by gas
chromatography-mass spectrometry (GC-MS); yield 1.5 g (96%; 9.6 mmol).
2.2.2. Synthesis of poly(3-hexylthiophene-co-methyl 2-(thiophene-3-yl)acetate), (P(3HT-co-MTA))
Firstly, 3-hexylthiophene (1.34 g, 8.0 mmol) and methyl 2-(thiophene-3-yl)acetate (MTA) (1.24 g;
8.0 mmol) were dissolved in CHCl3 (20 mL) with FeCl3 (5.19 g, 32 mmol) and stirred for 24 h at
0 ◦ C under nitrogen atmosphere. The solution was poured into methanol (100 mL) and left for
1 h to form a precipitate. After filtration, the solid was collected on filter paper and the FeCl3 was
removed by washing with methanol in a Soxhlet extractor for 8 h. The product was dried at 60 ◦ C
under reduced pressure for 24 h. The yield of dark-brown powder, poly(3-hexylthiophene-co-methyl
2-(thiophene-3-yl)acetate (P(3HT-co-MTA)), was 1.44 g (56%). Proton nuclear magnetic resonance
(1 H-NMR) (400 MHz, CDCl3 ): δ = 6.96, 3.80, 3.73, 3.69, 2.78, 2.53, 2.20, 1.67, 1.41, 1.32, and 0.88 ppm.
Fourier-transform infrared (FT-IR): 2953, 2923, 2855, 1743, 1516, 1457, 1434, 1377, 1329, 1259, 1197, 1168,
1017, 890, 831 and 725 cm−1 .
2.2.3. Hydrolysis of poly(3-hexylthiophene-co-methyl-2-(thiophene-3-yl) acetate)
P(3HT-co-MTA) (0.5 g) was added to a fivefold excess of 2 M NaOH solution and refluxed at 100 ◦ C
for 24 h. The resulting solid was suspended homogeneously in the solution under vigorous stirring, and
1 M HCl was added until the pH became less than 2. The solid was filtered off and dried at 80 ◦ C under
reduced pressure for 24 h. The brown powder product, poly(3-hexylthiophene-co-3-thiopheneacetic
acid) (P3HT-co-3TAA) (0.36 g, 0.72%) was characterized by 1 H-NMR and FT-IR spectroscopy. 1 H-NMR
(400 MHz CDCl3 ): δ = 6.93, 2.71, 2.50, 1.63, 1.55, 1.36, 1.26, 1.20, 0.82, and 0.00 ppm. FT-IR: 3437, 2954,
2923, 2855, 1713, 1516, 1463, 1377, 1260, 1223, 1099, 1051, 829, 724 cm−1 .
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2.2.4. Immobilization
Immobilization of
of Urease
Urease on
on the
the Surface
Surface of
of P(3HT-co-3TAA)
P(3HT-co-3TAA)Using
UsingaaCarbodiimide
Carbodiimide Coupling
2.2.4.
Reaction
Coupling Reaction
P(3HT-co-3TAA) (0.10
was
spin-coated
onto
ITOITO
glass
(20
P(3HT-co-3TAA)
(0.10 g)
g) dissolved
dissolvedininchloroform
chloroform(5(5mL)
mL)
was
spin-coated
onto
glass
◦
mm
×
15
mm)
and
dried
in
an
oven
at
60
°C
under
reduced
pressure
for
24
h;
the
thickness
of
the
film
(20 mm × 15 mm) and dried in an oven at 60 C under reduced pressure for 24 h; the thickness
was
400 about
± 100 nm,
as100
estimated
from the intensity
the ultraviolet-visible
(UV-vis)
of
thetypically
film wasabout
typically
400 ±
nm, as estimated
from the of
intensity
of the ultraviolet-visible
absorption
peak.
The
ITO
glass
was
dipped
into
a
TRIS-HCl
buffer
solution
(50
mM,
(UV-vis) absorption peak. The ITO glass was dipped into a TRIS-HCl buffer solution (50 mM,pH
pH == 7)
7)
containing
urease
(1
mg/mL).
ECD
(0.0573
g,
0.3
mmol)
and
NHS
(0.0693
g,
0.6
mmol)
were
added
to
containing urease (1 mg/mL). ECD (0.0573 g, 0.3 mmol) and NHS (0.0693 g, 0.6 mmol) were added
thethe
buffer
solution
slowly
washed with
with
to
buffer
solution
slowlyand
andstirred
stirredfor
for33h.
h. Thus-modified
Thus-modifiedITO
ITO electrodes
electrodes were
were washed
◦
TRIS-HCl
buffer
(2
mL),
dried
at
room
temperature
and
stored
in
a
freezer
(−18
°C).
TRIS-HCl buffer (2 mL), dried at room temperature and stored in a freezer (−18 C).
Immobilized on
on the
the Surface
Surface of
of P(3HT-co-3TAA)
P(3HT-co-3TAA)
2.3. Spectrophotometric Assay of the Urease Immobilized
Using Nessler’s reagent to form a complex with ammonia products is a well-known method to
assay the activity of urease. By the enzymatic hydrolysis of urea (Scheme 2), ammonia is ultimately
and is
is reacted
reacted with
withNessler’s
Nessler’s reagent
reagent(K
(K22Hg
HgIIIII44). The
produced, and
The absorption
absorption of
of the amide complex
NH22Hg22II33at
at385
385nm
nmisisobserved
observedby
byUV-visible
UV-visible spectroscopy.
spectroscopy.
Scheme 2.
2. The
The reactions
reactions forming
forming ammonia
ammonia and
and its
its complex
complex with
with Nessler’s
Nessler’s reagent.
reagent.
Scheme
A test for possible leaching of urease from the sensor film (Urs/copolymer/ITO glass) was set up
A test for possible leaching of urease from the sensor film (Urs/copolymer/ITO glass) was set up
by dipping an electrode into TRIS-HCl buffer solution (1.0 mM; pH = 7) (5 mL) and shaking for 20
by dipping an electrode into TRIS-HCl buffer solution (1.0 mM; pH = 7) (5 mL) and shaking for 20 min.
min. The electrode was removed from the solution and then urea solution (10 mM; 1 mL) with
The electrode was removed from the solution and then urea solution (10 mM; 1 mL) with Nessler’s
Nessler’s reagent (200 μL) was added. If there were any urease leaching from the film, an absorption
reagent (200 µL) was added. If there were any urease leaching from the film, an absorption peak of the
peak of the amide complex would be found at 385 nm in the UV-vis spectrum; no such absorption
amide complex would be found at 385 nm in the UV-vis spectrum; no such absorption was detected.
was detected.
The response time of a sensor was investigated by determining the profile of its UV-visible
The response time of a sensor was investigated by determining the profile of its UV-visible
absorbance against time. Firstly, six sample vials, each containing 5 mM urea solution, were marked
absorbance against time. Firstly, six sample vials, each containing 5 mM urea solution, were marked
with specific times (1.5, 2, 2.5, 3, 4, 5 and 6 min). The Urs/copolymer/ITO slice was dipped into each
with specific times (1.5, 2, 2.5, 3, 4, 5 and 6 min). The Urs/copolymer/ITO slice was dipped into each
solution for the corresponding time and then removed. Nessler’s reagent (200 µL) was then added
solution for the corresponding time and then removed. Nessler’s reagent (200 μL) was then added to
to each vial, and the solutions were left for 30 min. The sample absorbances were then measured at
each vial, and the solutions were left for 30 min. The sample absorbances were then measured at 385
385 nm vs. an ITO glass blank.
nm vs. an ITO glass blank.
The lifetime of urease immobilized on the copolymer and stored at −18 ◦ C was examined
The lifetime of urease immobilized on the copolymer and stored at −18 °C was examined using
using the same method. The Urs/copolymer/ITO electrode was placed into 5 mM urea solution for
the same method. The Urs/copolymer/ITO electrode was placed into 5 mM urea solution for 10 min
10 min after storage in the freezer for each month. Nessler’s reagent (200 µL) was added after the
after storage in the freezer for each month. Nessler’s reagent (200 μL) was added after the
Urs/copolymer/ITO electrode was removed, and the solution absorbance at 385 nm was measured.
Urs/copolymer/ITO electrode was removed, and the solution absorbance at 385 nm was measured.
2.4. Potentiometric Assay Using the Electrochemical Biosensor System
2.4. Potentiometric Assay Using the Electrochemical Biosensor System
For the potentiometric experiments, the Urs/copolymer/ITO glass was the working electrode,
potentiometric
the Urs/copolymer/ITO
glass
was sensor
the working
electrode,
with For
a Ptthe
counter
electrode experiments,
and an Ag/AgCl
reference electrode.
Three
electrodes
were
with
a
Pt
counter
electrode
and
an
Ag/AgCl
reference
electrode.
Three
sensor
electrodes
immersed in 1.0 M KCl (40 mL) and stirred slowly; the rest potential was measured until itwere
had
immersed
in 1.0
KCl Then,
(40 mL)
and
stirred
slowly;
the was
rest added
potential
was
measured
until ait urea
had
become
stable
for M
5 min.
1.0 M
urea
solution
(40 µL)
to the
electrolyte
(giving
become stable in
forthe
5 min.
Then,
1.0 M
ureaThe
solution
(40 μL) was
was measured
added to the
electrolyte
(giving
a
concentration
solution
of 0.99
mM).
rest potential
each
minute for
10 min.
urea
concentration
in
the
solution
of
0.99
mM).
The
rest
potential
was
measured
each
minute
for
10
Urea was also prepared in the following concentrations: 2.99, 3.98, 4.97, 5.96 and 6.95 mM, and the rest
min. Urea were
was also
prepared
in thesolution.
following concentrations: 2.99, 3.98, 4.97, 5.96 and 6.95 mM, and
potentials
measured
for each
the rest potentials were measured for each solution.
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2.5. Electrochemical Analysis by Cyclic Voltammetry
2.5. Electrochemical
Analysis
by Cyclic
Voltammetry glass (20 mm × 15 mm) working electrodes were
Urs/copolymer/ITO
glass
and copolymer/ITO
prepared.
A platinum counter electrode and a sealed aqueous Ag/AgCl/3.4 M KCl reference
Urs/copolymer/ITO glass and copolymer/ITO glass (20 mm × 15 mm) working electrodes
electrode (eDAQ type ET072-1) were used. The cyclic voltammetry experiments were run in a 0.1 M
were prepared. A platinum counter electrode and a sealed aqueous Ag/AgCl/3.4 M KCl reference
LiClO4/propylene carbonate electrolyte. The scan region was from −1000 mV to 2000 mV, starting
electrode (eDAQ type ET072-1) were used. The cyclic voltammetry experiments were run in a 0.1 M
from the rest potential, and a non-aqueous solvent was required in order to permit such a wide
LiClO4 /propylene carbonate electrolyte. The scan region was from −1000 mV to 2000 mV, starting
potential range; acetonitrile was found to give very erratic results, so propylene carbonate was
from the rest potential, and a non-aqueous solvent was required in order to permit such a wide
chosen as the solvent. The cycles began in the positive direction, and the scan rate was 10 mV·s−1.
potential range; acetonitrile was found to give very erratic results, so propylene carbonate was chosen
as the solvent. The cycles began in the positive direction, and the scan rate was 10 mV·s−1 .
2.6. Polymer Film Morphology
2.6. Polymer
Film of
Morphology
Spun films
the copolymers P(3HT-co-MTA), P(3HT-co-TAA) and Urs-P(3HT-co-TAA) were
examined
by
scanning
microscopy
using a Zeiss
EVO 50 instrument
with 20 kV accelerating
Spun films of the electron
copolymers
P(3HT-co-MTA),
P(3HT-co-TAA)
and Urs-P(3HT-co-TAA)
were
potential
after
sputter-coating
with
Au-Pd.
The
films
were
remarkably
smooth,
showed no
examined by scanning electron microscopy using a Zeiss EVO 50 instrument with 20and
kV accelerating
discernible
morphological
features.
potential
after
sputter-coating
with Au-Pd. The films were remarkably smooth, and showed no
discernible morphological features.
3. Results and Discussion
3. Results and Discussion
3.1. 1H-NMR Spectra for P(3HT-co-MTA) and P(3HT-co-3TAA)
3.1. 1 H-NMR
Spectra for P(3HT-co-MTA) and P(3HT-co-3TAA)
The 1H-NMR spectra confirmed the successful formation and hydrolysis of P(3HT-co-MTA). In
the spectrum
of that
polymer
(Figure
the proton
on theand
thiophene
rings
was observed atIn6.97
The 1 H-NMR
spectra
confirmed
the1a),
successful
formation
hydrolysis
of P(3HT-co-MTA).
the
ppm, andofthe
peaks
for –CH
2 closest
toproton
the thiophene
ring at 2.73
ppm. at
The
spectrum
that
polymer
(Figure
1a), the
on the thiophene
ringsand
was2.50
observed
6.97-OCH
ppm,3
resonance
wasfor
at–CH
3.702 ppm,
thethiophene
other peaks
to 0.82
ppm
towas
the
and
the peaks
closestand
to the
ringatat1.63
2.73ppm
and 2.50
ppm.
Thewere
-OCHattributed
3 resonance
protons
on the
alkyl
After
the proton
resonance
on the
thiophene
became
at
3.70 ppm,
and
the groups.
other peaks
atacidification,
1.63 ppm to 0.82
ppm were
attributed
to the
protons ring
on the
alkyl
sharp and
the acidification,
-OCH3 peak vanished
to its being
changed
to -OH
1b).
A new
due to3
groups.
After
the protondue
resonance
on the
thiophene
ring(Figure
became
sharp
andpeak
the -OCH
-COOH
was observed
0.00 changed
ppm. Hence
it was
concluded
that peak
the synthesis
of P(3HT-co-3TAA)
peak
vanished
due to itsatbeing
to -OH
(Figure
1b). A new
due to -COOH
was observed
had
been
achieved
copolymerizing
and MTA and then
hydrolyzing
at
0.00
ppm.
Hence itbywas
concluded that3-hexylthiophene
the synthesis of P(3HT-co-3TAA)
had been
achievedthe
by
product.
copolymerizing 3-hexylthiophene and MTA and then hydrolyzing the product.
1 H-NMR spectra of (a) poly(3-hexylthiophene-co-methyl 2-(thiophene-3-yl)acetate
Figure
Figure 1.1. 1H-NMR
spectra of (a) poly(3-hexylthiophene-co-methyl 2-(thiophene-3-yl)acetate
(P(3HT-co-MTA))
(P(3HT-co-MTA)) and
and (b)
(b)P(3HT-co-3TAA).
P(3HT-co-3TAA).
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3.2. FT-IR Spectra of P(3HT-co-MTA), P(3HT-co-3TAA) and Urs/P(3HT-co-3TAA)
Biosensors 2017, 7, 13
6 of 13
Spectra of P(3HT-co-MTA), P(3HT-co-3TAA) and Urs/P(3HT-co-3TAA)
In3.2.
theFT-IR
FT-IR
spectrum of the copolymer P(3HT-co-MTA) (Figure 2a), the C-H stretching vibrations
1 P(3HT-co-3TAA)
In 2953,
the
FT-IR
spectrum
of −
the
copolymer
P(3HT-co-MTA)
2a), was
the seen
C-H at
stretching
3.2. FT-IR
Spectra
of P(3HT-co-MTA),
and
absorbed
at
2928
and
2854 cm
.A
strong peak
dueUrs/P(3HT-co-3TAA)
to C=O(Figure
stretching
1740 cm−1 ,
−1
1 The
vibrations absorbed
2953,
2928 andring
2854atcm
. A 1454
strongand
peak
duecm
to −C=O
stretching
was seen at
and vibrational
modes
ofatthe
thiophene
1575,
1377
C-O
absorption
In the
FT-IR spectrum
of the copolymer
P(3HT-co-MTA)
(Figure .2a),
the
C-H
stretchingof the
−1, and vibrational
−1. The C-O
−
1
1740
cm
modes
of
the
thiophene
ring
at
1575,
1454
and
1377
cm
acetate
ester
was
at
1327
cm
.
It
would
be
expected
that
the
–OH
stretching
vibration
atseen
3400atcm−1
−1
vibrations absorbed at 2953, 2928 and 2854 cm . −1
A strong peak due to C=O stretching was
absorption
of
the
acetate
ester
was
at
1327
cm
.
It
would
be
expected
that
the
–OH
stretching
would
be undetectable
in the spectrum
P(3HT-co-MTA),
moisture
wasC-O
present
1740
cm−1, and vibrational
modes ofofthe
thiophene ringbut
at some
1575, inadvertent
1454 and 1377
cm−1. The
vibration at 3400 cm−1 would be undetectable−1 in the spectrum of P(3HT-co-MTA), but some
of the acetate
ester wasan
at –OH
1327 stretching
cm . It would
expected
thatAfter
the –OH
stretching of
in theabsorption
KBr disk samples,
and therefore
peakbe
was
observed.
the acidification
inadvertent moisture −1was present in the KBr disk samples, and therefore
an –OH stretching peak
−1ofand
vibration at the
3400C=O
cm stretching
would be vibration
undetectable
in the
spectrum
P(3HT-co-MTA),
butofsome
P(3HT-co-MTA),
shifted
to
1710
cm
the
C-O
stretch
the ester
was observed. After the acidification of P(3HT-co-MTA), the C=O stretching vibration shifted to 1710
inadvertent
moisture
was
present
in
the
KBr
disk
samples,
and
therefore
an
–OH
stretching
peak
vanished
from
the
spectrum
of
P(3HT-co-3TAA)
(Figure
2b).
It
was
thus
concluded
that
the
cm−1 and the C-O stretch of the ester vanished from the spectrum of P(3HT-co-3TAA) (Figure 2b).acetate
It
was observed. After the acidification of P(3HT-co-MTA), the C=O stretching vibration shifted to 1710
ester in
thethus
copolymer
converted
-COOH.
was
concludedhad
thatbeen
the acetate
ester to
in the
copolymer had been converted to -COOH.
cm−1 and the C-O stretch of the ester vanished from the spectrum of P(3HT-co-3TAA) (Figure 2b). It
was thus concluded 0.20
that the acetate ester in the copolymer had been converted to -COOH.
P(3HT-co-3TAA)
P(3HT-co-MTA)
P(3HT-co-3TAA)
P(3HT-co-MTA)
0.18
0.20
0.16
0.18
C=O 1710 cm
0.14
0.16
Abs Abs
0.12
0.14
0.10
0.12
C=O 1710 cm
(b)
-1
(b)
0.08
0.10
C=O 1740 cm
0.06
0.08
0.04
0.06
0.02
0.04
0.00
0.02
0.00 4000
-1
C=O 1740 cm
-1
-1
(a)
(a)
3500
3000
2500
2000
1500
1000
500
-1
4000
3500
3000
Wavenumber
(cm ) 1500
2500
2000
1000
500
-1
Figure 2. FT-IR spectra of copolymers
(a) P(3HT-co-MTA);
(b) P(3HT-co-3TAA).
Wavenumber
(cm )
Figure 2. FT-IR spectra of copolymers (a) P(3HT-co-MTA); (b) P(3HT-co-3TAA).
Figure 2. FT-IR spectra of copolymers (a) P(3HT-co-MTA); (b) P(3HT-co-3TAA).
In the FT-IR spectrum of urease immobilized on the P(3HT-co-3TAA) by peptide bonds (Figure
very strong
peaks immobilized
for N-C=O stretching
vibrations, the symmetric
asymmetric
In3b),
thethere
FT-IRare
spectrum
of urease
on the P(3HT-co-3TAA)
by peptideand
bonds
(Figure 3b),
In the FT-IR spectrum of urease immobilized
on the P(3HT-co-3TAA) by peptide bonds (Figure
−1. Furthermore, a very sharp peak appeared at 3200
deformations
being
seen
at
1600
and
1552
cm
there3b),
arethere
veryare
strong
peakspeaks
for N-C=O
stretching
thesymmetric
symmetric
asymmetric
very strong
for N-C=O
stretchingvibrations,
vibrations, the
andand
asymmetric
−1 . Furthermore,
cm−1, characterizing
the
N-Hand
stretching
vibration.
Therefore, ait very
can be
concluded
that a peptide
bond −1
deformations
being
seen
at
1600
1552
cm
sharp
peak
appeared
at
−1
deformations being seen at 1600 and 1552 cm . Furthermore, a very sharp peak appeared at3200
3200cm ,
between
urease
and
(3HT-co-3TAA)
was
created
via
the
carbodiimide
coupling
reaction.
characterizing
the N-H stretching
vibration.
Therefore,
it can be
concluded
that athat
peptide
bondbond
between
cm−1, characterizing
the N-H stretching
vibration.
Therefore,
it can
be concluded
a peptide
urease
and (3HT-co-3TAA)
was createdwas
viacreated
the carbodiimide
couplingcoupling
reaction.
between
urease and (3HT-co-3TAA)
via the carbodiimide
reaction.
Figure 3. FT-IR spectra of (a) P(3HT-co-3TAA); (b) Urs/P(3HT-co-3TAA).
Figure
3. FT-IR
spectraofof(a)
(a)P(3HT-co-3TAA);
P(3HT-co-3TAA); (b)
Figure
3. FT-IR
spectra
(b)Urs/P(3HT-co-3TAA).
Urs/P(3HT-co-3TAA).
Biosensors 2017, 7, 13
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2017, 7, 13
3.3. Cyclic
Voltammetry
(CV) of P(3HT-co-3TAA) and Urs/P(3HT-co-3TAA) in 0.1 M
LiClO4 /Propylene Carbonate
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3.3. Cyclic Voltammetry (CV) of P(3HT-co-3TAA) and Urs/P(3HT-co-3TAA) in 0.1 M LiClO4/Propylene
In
the cyclic voltammogram of P(3HT-co-3TAA) (Figure 4a), the main oxidation and reduction
Carbonate
peaks appeared
at +1390 and +300 mV respectively in the first cycle, and at +1290 and +390 mV
In the cyclic voltammogram of P(3HT-co-3TAA) (Figure 4a), the main oxidation and reduction
respectively
in
the
cycles. This
electrochemical
to be a
peaks appeared second
at +1390and
andsubsequent
+300 mV respectively
in the
first cycle, and process
at +1290 appeared
and +390 mV
partially-reversible
doping-dedoping
reaction.
However,
the observed process
reversibility
increased
respectively in the
second and subsequent
cycles.
This electrochemical
appeared
to be a after
immobilizing
urease ondoping-dedoping
the surface of P(3HT-co-3TAA),
and
peak potentials
decreased
partially-reversible
reaction. However,
thethe
observed
reversibility
increasedsomewhat,
after
immobilizing
urease
on
the
surface
of
P(3HT-co-3TAA),
and
the
peak
potentials
decreased
becoming about +1260 and +295 mV for oxidation and reduction respectively (Figure 4b). A possible
somewhat,
becoming
about
+1260
and held
+295 mV
oxidation
and
reduction respectively
(Figure
reason
is that the
thiophene
rings
were
in afor
more
planar
configuration
as a result
of the4b).
urease
A
possible
reason
is
that
the
thiophene
rings
were
held
in
a
more
planar
configuration
as
a
result
of are
functionalization. In addition, the enzyme-modified polymer lost the oxidation shoulders
which
the urease functionalization. In addition, the enzyme-modified polymer lost the oxidation shoulders
a notable
feature in the CV of the unmodified copolymer.
which are a notable feature in the CV of the unmodified copolymer.
It will be seen in Section 3.6 that the potential range associated with the biosensing action
It will be seen in Section 3.6 that the potential range associated with the biosensing action of the
of theenzyme-modified
enzyme-modified
electrodes
(+100~300
is the
below
theassociated
region associated
with redox
the above
electrodes
(+100~300
mV) ismV)
below
region
with the above
redoxprocesses;
processes;
thisimplies
implies
that
modified
copolymer
electrodes
would
a largely-undoped
this
that
thethe
modified
copolymer
electrodes
would
be inbeainlargely-undoped
semiconducting
state
when
functioning
as
a
potentiometric
biosensor.
semiconducting state when functioning as a potentiometric biosensor.
Figure 4. Cyclic voltammetry (CV) plots of (a) P(3HT-co-3TAA); (b) Urs/P(3HT-co-3TAA) in 0.1 M
Figure 4. Cyclic voltammetry (CV) plots of (a) P(3HT-co-3TAA); (b) Urs/P(3HT-co-3TAA) in 0.1 M
LiClO4/PC (Potentials are vs. Ag/AgCl/3.4 M KCl reference.).
LiClO4 /PC (Potentials are vs. Ag/AgCl/3.4 M KCl reference.).
3.4. UV-Visible Absorption Spectra of P(3HT-co-3TAA) and Urs/P(3HT-co-3TAA)
3.4. UV-Visible Absorption
Spectra of P(3HT-co-3TAA) and Urs/P(3HT-co-3TAA)
*
The π-π transition peak of a spin-coated film of P(3HT-co-3TAA) on ITO glass was observed at
* transition
about
437
nm (2.84 eV)
(Figure
After immobilizing
on the film, the
The π-π
peak
of a5).spin-coated
film of urease
P(3HT-co-3TAA)
on π-π
ITO* transition
glass wasshifted
observed
at about 437 nm (2.84 eV) (Figure 5). After immobilizing urease on the film, the π-π* transition
Biosensors
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2017, 7,
7, 13
13
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88 of
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13
8 of 13
slightly to 453 nm (2.74 eV). Such a shift suggests that the P(3HT-co-3TAA) was slightly better
conjugated as a result of coupling with the urease, presumably due to the thiophene units being
shifted slightly to 453 nm (2.74 eV). Such a shift suggests that the P(3HT-co-3TAA) was slightly better
forced into a more coplanar configuration as a result of successful attachment of urease to the
conjugated as a result of coupling with the urease, presumably due to the thiophene units being forced
P(3HT-co-3TAA).
into a more coplanar configuration as a result of successful attachment of urease to the P(3HT-co-3TAA).
0.26
0.24
0.22
0.20
Abs
0.18
0.16
0.14
0.12
0.10
P(3HT-co-3TAA)
Urs/P(3HT-co-3TAA)
0.08
0.06
1.5
2.0
2.5
3.0
3.5
4.0
Energy (eV)
Figure
absorption spectra
spectra of
of P(3HT-co-3TAA)
P(3HT-co-3TAA) and
andUrs/P(3HT-co-3TAA).
Urs/P(3HT-co-3TAA).
Figure 5.
5. UV-visible
UV-visible absorption
3.5. Response
of Urs/P(3HT-co-3TAA)/ITO
Urs/P(3HT-co-3TAA)/ITOGlass
Glassby
bySpectrophotometric
SpectrophotometricStudies
Studies
3.5.
Response Time
Time of
To observe
observe the
P(3HT-co-3TAA),
thethe
variation
of
To
the kinetic
kinetic behavior
behaviorofofurease
ureaseimmobilized
immobilizedononthe
the
P(3HT-co-3TAA),
variation
absorbance
was
measured
as
a
function
of
time
during
the
enzyme-catalyzed
hydrolysis
of
4.97
mM
of absorbance was measured as a function of time during the enzyme-catalyzed hydrolysis of
ureamM
solution.
In Figure
6, the 6,
first
was was
obtained
at at
1.51.5
min,
increased
4.97
urea solution.
In Figure
the absorbance
first absorbance
obtained
min,and
and then
then increased
monotonically
with
time
until
a
plateau
was
reached.
monotonically with time until a plateau was reached.
0.4
Abs
0.3
Abs
Sigmoidal (Boltzmann fit)
0.2
0.1
0.0
0
2
4
6
8
10
Time (mins)
Figure
glass assayed
6. Absorbance
Absorbance (385
(385 nm)
nm) vs.
vs. time
time for
forUrs/P(3HT-co-3TAA)/ITO
Urs/P(3HT-co-3TAA)/ITO glass
Nessler’s
Figure 6.
assayed by
by Nessler’s
reagent
urea solution.
solution.
reagent in
in 55 mM
mM urea
Biosensors 2017, 7, 13
Biosensors 2017, 7, 13
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9 of 13
The
delayfrom
from00toto1.51.5
min
suggests
the urea
was initially
overcoming
the hydrophobic
The delay
min
suggests
thatthat
the urea
was initially
overcoming
the hydrophobic
barrier
barrier
of
P(3HT-co-3TAA).
After
this
point,
the
urease
was
functional
until
the
ureahydrolyzed
had been
of P(3HT-co-3TAA). After this point, the urease was functional until the urea had been
hydrolyzed
completely
6 min.it Therefore
it wasthat
concluded
that in this
biosensor
system,
completely after
about 6after
min.about
Therefore
was concluded
in this biosensor
system,
the reaction
the
reaction
required
about
six
minutes
to
reach
a
steady
state.
required about six minutes to reach a steady state.
3.6.
Glass
3.6. The
The Potentiometric
Potentiometric Assay
Assay of
of Urease/P(3HT-co-3TAA)/ITO
Urease/P(3HT-co-3TAA)/ITO Glass
To
provethat
thaturease/P(3HT-co-3TAA)
urease/P(3HT-co-3TAA)
on ITO
was functional
as a biosensor,
the
To prove
1:11:1
on ITO
glassglass
was functional
as a biosensor,
the potential
potential
was recorded
ureasewith
reacted
with concentrations
different concentrations
of urea.
The
variation variation
was recorded
while thewhile
ureasethe
reacted
different
of urea. The
resulting
resulting
local
change
in
the
p[OH-]
provided
adequate
verification
that
ammonia
was
produced,
local change in the p[OH-] provided adequate verification that ammonia was produced, and therefore
and
therefore
thatbeen
the successfully
urease had immobilized
been successfully
immobilized on The
the average
P(3HT-co-3TAA).
The
that the
urease had
on the P(3HT-co-3TAA).
initial potential
average
initial
potential
in (40
a blank
aqueous
solution
(40decreased
mL) wassuddenly
292 mV,toand
decreased
in a blank
aqueous
solution
mL) was
292 mV,
and this
288 this
mV on
adding
suddenly
to
288
mV
on
adding
1
M
urea
solution
(40
μL)
(Figure
7).
The
potential
reached
an
1 M urea solution (40 µL) (Figure 7). The potential reached an equilibrium value of 173 mV after 10 min,
equilibrium
value
of
173
mV
after
10
min,
and
diminished
further
if
the
concentration
of
urea
was
and diminished further if the concentration of urea was increased. However, this trend ceased when
increased.
However,
this trend
ceased
when
the concentration of urea reached about 5 mM.
the concentration
of urea
reached
about
5 mM.
0
0 .9 9
1 .9 9
2 .9 9
3 .9 8
4 .9 7
5 .9 6
6 .9 4
320
300
280
260
E /mV
240
mM
mM
mM
mM
mM
mM
mM
mM
220
200
180
160
140
120
100
0
1
2
3
4
5
6
7
8
9
10
Tim e (m in s)
Figure 7.7.Evolution
of electrode
potential
(vs. Ag/AgCl)
as a function
time for various
Figure
Evolution
of electrode
potential
(vs. Ag/AgCl)
as a offunction
of timeconcentrations
for various
of
urea
in
non-buffered
solutions.
concentrations of urea in non-buffered solutions.
According
According to
to Nernst’s
Nernst’s formalism,
formalism, the
the half-cell
half-cell equation
equation for
for aa reduction
reduction potential
potential can
can be
expressed as:
Ered = E◦ red + (RT/nF)·ln[ared /aox ]
Ered = E°red + (RT/nF)·ln[ared/aox]
where Ered is the half-cell reduction potential, E◦ is the standard half-cell potential, R is the universal
is the half-cell reduction potential, E° is the standard half-cell potential, R is the universal
where Ered
gas constant (8.314 J·K− 1·mol−1 ) and F is the Faraday constant (96,485 C·mol−1 ). T is the absolute
gas constant (8.314 J·K−1·mol−1) and F is the Faraday constant (96,485 C·mol−1). T is the absolute
temperature and
and n
n is
is the
the number
number of
of electrons
electrons transferred
transferred in
in the
the half-reaction.
half-reaction. Since
Since the
the change
change of
of the
the
temperature
− ] in the solution, the Nernst equation can be
reduction
potential
is
related
to
the
variation
of
p[OH
−
reduction potential is related to the variation of p[OH ] in the solution, the Nernst equation can be
modified thus:
thus:
modified
E = E◦ + (RT/βnF)·ln(aOH − )
E = E° + (RT/βnF)·ln(aOH−)
where E is the potential difference between the working electrode and the reference electrode
where
E is the
difference
thefactor
working
andactivity
the reference
electrode
(Ag/AgCl),
β ispotential
the electron
transferbetween
symmetry
and electrode
aOH − is the
of the hydroxide
−
(Ag/AgCl),
is the
electron transfer
aOH coefficient
is the activity
of the hydroxide
ion
ion (equal toβthe
concentration
of OH−symmetry
multipliedfactor
by theand
activity
α). Therefore,
the potential
− multiplied by the activity coefficient α). Therefore, the potential
(equal
to
the
concentration
of
OH
can be expressed as:
can be expressed as:
E = E◦ + (RT/βnF)·ln{[OH− ]α}
E = E° + (RT/βnF)·ln{[OH−]α}
Biosensors 2017, 7, 13
10 of 13
In addition, the variation of [OH− ] results from the ammonia produced by the enzyme-hydrolysis
of urea
(Scheme
Biosensors
2017,2).
7, 13In view of the stoichiometry of that reaction, the Nernst equation can be10modified
of 13
to include the initial concentration of urea, thus:
In addition, the variation of [OH−] results from the ammonia produced by the
enzyme-hydrolysis of urea (Scheme
view of ·the
stoichiometry of that reaction, the Nernst
(RT/βnF)
ln{2[urea]α}
E = E◦2).+In
equation can be modified to include the initial concentration of urea, thus:
If α is assumed to be for ideal behavior
(α = 1), and the equilibrated potential responses from
E = E° + (RT/βnF)·ln{2[urea]α}
0.99 mM to 4.95 mM at 10 min (Figure 7) are used, the equation can be reduced to:
If α is assumed to be for ideal behavior (α = 1), and the equilibrated potential responses from
0.99 mM to 4.95 mM at 10 min (Figure 7) are used, the equation
can be reduced to:
2
y = −0.0426x − 0.0927 (R = 0.9987)
y = −0.0426x − 0.0927 (R2 = 0.9987)
For convenience,
thethe
average
electrode
are shown
shownininFigure
Figure
8 as
graphs
of electrode
For convenience,
average
electroderesponses
responses are
8 as
graphs
of electrode
potential
(mV)
vs.
the
decimal
logarithm
of
[urea]/1
mM,
so
that
the
slope
of
the
equilibrated
graph in
potential (mV) vs. the decimal logarithm of [urea]/1 mM, so that the slope of the equilibrated graph
Figurein8b
is −42.6
2.303
= −98.1
mV.
Figure
8b is ×
−42.6
× 2.303
= −98.1
mV.
240
10mins
7mins
1min
220
E (mV)
200
180
(a)
160
140
120
100
-3.0
-2.8
-2.6
-2.4
-2.2
-2.0
180
170
10mins
Linear Fit
160
E (mV)
150
140
(b)
130
120
110
100
-3.0
-2.8
-2.6
-2.4
-2.2
-2.0
log[urea]
Figure 8. (a) Graph of electrode potential (w.r.t. Ag/AgCl) against log[urea] for several different
Figure 8. (a) Graph of electrode potential (w.r.t. Ag/AgCl) against log[urea] for several different delays
delays from the moment of addition of urea; (b) Equilibrated data for the 10-min delay, showing
from the moment of addition of urea; (b) Equilibrated data for the 10-min delay, showing linear fit in
linear fit in the region 1–5 mM.
the region 1–5 mM.
This implies that the value of RT/βnF was equal to 0.0426. A single electron transfer equilibrium
−] in the analyte.
was
assumed
because
the variation
of potential
resulted
from the
changeelectron
of p[OHtransfer
This implies that
the value
of RT/βnF
was equal
to 0.0426.
A single
equilibrium
Hence,
the
value
of
β
in
this
half-reaction
was
calculated
to
be
0.598.
The
expected
of
was assumed because the variation of potential resulted from the change of p[OH− ] invalue
the analyte.
ln{2[urea]α} can be calculated from the ratio of ΔE and RT/βnF. The value of the activity coefficient
Hence,
the value of β in this half-reaction was calculated to be 0.598. The expected value of ln{2[urea]α}
(α) can be estimated from the ratio of ln{2[urea]α} values (Table 1) between the predicted and the
can be calculated from the ratio of ∆E and RT/βnF. The value of the activity coefficient (α) can be
Biosensors 2017, 7, 13
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estimated from the ratio of ln{2[urea]α} values (Table 1) between the predicted and the measured
values, although it should be noted that the numbers in parentheses are from data outside the linear
region, and hence not really meaningful.
The experimental values were found to be quite close to the ideal ones in the region of urea
concentration from 0.99 to 4.97 mM. As a result, it can be concluded that Urs/P(3HT-co-3TAA)
successfully transduced a reliable potential variation whilst the urease enzyme hydrolyzed the urea.
However, when the concentration of urea became greater than about 5 mM, this system could no
longer be used as a urea biosensor because the potential response departed from a linear relationship
with ln[urea].
Table 1. Predicted and experimental values of ln{[urea]*α*2} and the activity coefficient (α). (T = 25.1 ◦ C).
[urea] (mM)
E (V)
RT/βnF (n = 1)
ln{[urea]*α*2} (Predicted)
ln{[urea]*2}
α
0.99
1.99
2.99
3.98
4.97
5.96
6.95
0.182
0.152
0.125
0.115
0.105
0.105
0.105
0.0426
0.0426
0.0426
0.0426
0.0426
0.0426
0.0426
−6.3915
−5.6703
−5.0653
−4.8326
−4.5999
−4.5999
−4.5999
−6.2247
−5.5256
−5.1193
−4.8333
−4.6112
−4.4295
−4.2758
0.8462
0.8660
1.0555
1.0007
1.0113
(0.8433)
(0.6338)
4. Conclusions
The semiconducting thiophene copolymer P(3HT-co-3TAA) (1:1) was synthesized and used
successfully in a urea biosensor, since it acts as a matrix that can immobilize urease on its surface.
The covalent immobilization was carried out via the formation of peptide bonds, as confirmed
by FT-IR spectroscopy. The redox peaks in the cyclic voltammograms and the shift of the π-π*
optical transition offered indirect evidence of the successful bonding between P(3HT-co-3TAA) and
urease. The Urs/P(3HT-co-3TAA)/ITO glass electrode reached equilibrium in a 4.97-mM urea solution
after 6 min, as observed by spectrophotometry. The low conductivity of the thiophene polymer
in its largely-undoped form would preclude the use of the electrode for reliable amperometric
bioanalysis, but potentiometric response studies confirmed that this electrode could repeatedly detect
the concentration of urea in aqueous solutions up to a maximum of about 5 mM. The normal level
of urea in blood serum is in the region of 1.3 to 3.5 mM, and so the Urs/P(3HT-co-3TAA)/ITO glass
electrode would be suitable for application in urea biosensors for blood serum analysis, or for process
monitoring in situations where the analyte has a pH fairly close to neutral. The high intrinsic stability of
polythiophenes, in comparison with many other common conducting polymers, would be a significant
advantage in such applications.
Acknowledgments: The authors thank Kingston University for partial support of the project of K.L., and J.-M.P.
for technical advice and assistance. This research did not receive any specific grant from funding agencies in the
public, commercial, or not-for-profit sectors.
Author Contributions: P.J.S.F., J.W.B. and P.S. supervised the project; the co-authors jointly conceived and
designed the experiments, which were performed by K.L.; P.S. contributed analysis tools; K.L. and P.J.S.F. wrote
the paper.
Conflicts of Interest: The authors declare no conflict of interest.
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