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Universidade de So Paulo
2012
Amperometric urea biosensors based on theentrapment of urease in
polypyrrole films
REACTIVE & FUNCTIONAL POLYMERS, AMSTERDAM, v. 72, n. 2,
supl. 1, Part 1, pp. 148-152,FEB,
2012http://www.producao.usp.br/handle/BDPI/37151 Downloaded from:
Biblioteca Digital da Produo Intelectual - BDPI, Universidade de So
Paulo
Biblioteca Digital da Produo Intelectual - BDPI
Sem comunidade WoS
http://www.producao.usp.br/handle/BDPI/37151http://www.producao.usp.br/handle/BDPI/37151
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respectively. The electropolymerization of pyrrole was carried
outby cyclic voltammetry in a potentiostat/galvanostat PAR
VersastatII from 1.0 V to 1.0 V vs. SCE up to reach 10 complete
cycles in anaqueous medium containing 0.2 mol L 1 LiClO4 and 0.1
mol L 1
pyrrole. This procedure allowed us to entrap Urs in the PPy
lmsby adding 300 l g mL 1 of Urs into the electrolyte solution(0.2
mol L 1 LiClO4) containing the monomer, pyrrole, at 0.1
mol L 1
. The value of Urs concentration, 300 l g mL 1
, was chosenafter all of the preparation procedures of PPy/Urs
lms had beenoptimized. Chronoamperometry was used as the
transductionmethod for detecting urea in different solutions. The
current den-sity was measured for lms potentiostatically polarized
at a xedpotential of 0.28 V vs. SCE for 160 s, and in a urea
concentrationranging from 1.0 to 10 mmol L 1 in buffer phosphate at
pH 7.0.This value of pH allowed us to achieve a condition of
maximumactivity of Urs.
The UVVIS measurements were carried out with a Hitachi U-2900
spectrophotometer for lms electrodeposited on FTO elec-trodes. The
infrared spectra were obtained by a Thermo Nicolet-Nexus 470/FT-IR
spectrophotometer from 400 cm 1 to 2000 cm 1
after 32 scans for lms electrodeposited on Pt electrodes.
Morphol-ogy studies were carried out with a Digital Scanning
Microscope,DSM 960 (Zeiss West Germany) for lms supported on Al
sub-strates, and then, coated with a layer of Au (about 20 nm).
Commercial urea fertilizers containing different percentages of
total nitrogen (45% and 75%) were comparatively used to detecttheir
amperometric signals from the use of the PPy/Urs lms. Thereal
samples were prepared in aqueous media containing0.06 mg mL 1 of
commercial urea fertilizers (45% and 75%) in0.2mol L 1 LiClO4 . For
the chronoamperometric experiments withreal samples, the
three-electrode electrochemical cell was alsoused. The solutions
containing urea were introduced into the cellusing a needle; after
that, the current density was recorded aftera short period of
stabilization.
3. Results and discussion
Fig. 1 a shows the last voltammetric curves obtained during
thepreparation of the PPy and PPy/Urs lms in an electrolyte
solutioncontaining pyrrole without and with Urs, respectively. The
pres-ence of Urs in the polymerization medium enables the current
den-sity to increase more rapidly and linearly with polymerization
time(number of cycles). This nding is clearly illustrated in the
inset of Fig. 1 a for current densities recorded at a xed potential
(0.4 V vs.SCE). The growth rate appears to be slower during the
preparationof the PPy lm (without the enzyme), as indicated in the
inset of Fig. 1 a. After ten cycles, the total charge transferred
during thepreparation of the PPy lm was 28 mC cm 2, while that of
thePPy/Urs lm was 31 mC cm 2, indicating that the lms growth
was slightly favored in the presence of Urs. The changes in the
cyc-lic voltammograms of the electrodes during the preparation of
thelms, PPy and PPy/Urs, evidence the occurrence of
electrostaticinteractions between negatively charged Urs (IP
5.05.2) and itsmatrix, a positively charged PPy. Fig. 1b
illustrates the voltammet-ric responses of these lms (PPy and
PPy/Urs) in a monomer-freesolution (phosphate buffer).
The voltammetric response of the PPy lm shows the redoxwaves
typically attributed to the PPy structure, and which have al-ready
been documented in the literature [16] . However, when Ursis
entrapped in the PPy matrix, the broad processes at 0.45 V, 0.0
V,and 0.58V vs. SCE (peaks A, B, C), typical of PPy, disappear
while anew, well dened redox couple is established at 0.28 V,
and
0.64V vs. SCE (CC0 peak). Although the possible
electroactive
nature of Urs has been briey considered in studies on the
electro-chemical response of Hg electrodes with adsorbed Urs [17] ,
it can
be assumed here that this peculiar voltammetric behavior of
thePPy/Urs lm cannot be attributed to the redox response of an
elec-troactive enzyme. In fact, it can be attributed to the nature
of thepolymeric matrix itself, PPy, which is a well known ion
exchangeand size exclusion membrane [18,19] . This nding is also
consis-tent with previous results [11] , which demonstrated that
PPy/Urslms prepared galvanostatically on Au electrodes exhibit a
redoxresponse similar to the one exhibited by our PPy/Urs lms. In
ourcase, the voltammogram of the PPy/Urs lm shows an even
betterdenition for the redox couple CC 0. This type of
electroactive re-sponse has also been detected for PPy lms prepared
in media con-taining large dopants with limited mobility, such as
sodiumdodecylbenzenesulfonate [20] . Therefore, it can be inferred
thatthe presence of entrapped Urs may give rise to an ion
exchangeprocess during the redox scanning of the PPy/Urs lm.
Instead of anions entering the PPy structure and neutralizing the
charge, cat-ions are incorporated into the PPy/Urs lm, particularly
Li + ions,
thus dening the ion-exchangeable nature of the PPy matrix.
Theconcept of this mechanism was rst described by Adeloju et
al.,who prepared PPy lms galvanostatically in pyrrole/NaNO 3
solu-tions with incorporated Urs [11] . The change in the
electroactivenature of the PPy lm after enzyme entrapment can be
related di-rectly to the existence of electrostatic interactions
between abulky, negatively charged enzyme entrapped in a
positivelycharged polymer matrix, where the insertion of cations
into thelm becomes well established to ensure the electroneutrality
of the PPy matrix. A detailed description of the differences found
inthe voltammetric responses of pure PPy lms in solutions
contain-ing different anions and cations has been made previously
[18] .
The addition of an analyte, in this case, urea, to the buffer
solu-tion at different concentrations causes the voltammetric
response
of the PPy/Urs lm to change signicantly ( Fig. 2 a). Urea was
usedin a wide range of concentrations (from 1.0 10 2 mol L 1 to
-1.0 -0.5 0.0 0.5 1.0
-2
0
2
4
(b)
E/V vs SCE
PPy
PPy/Urs
-4
0
4
8
12
16
A
C
without Urs
(a)
j / m
A c m
- 2
B
with Urs
-3
-2
-1
0
1
0 2 4 6 8 10
0
2
4
6
PPy/Urs
j / m
A c m
- 2
Number of cycles
PPy
Fig. 1. Cyclic voltammograms: (a) obtained during the
preparation of the PPy andPPy/Urs lms in an aqueous 0.1 molL 1
pyrrole and 0.2 mol L 1 LiClO4 solutionwithout and with 3.0 mg of
Urs, respectively and (b) obtained for the PPy and PPy/Urs lms in
phosphate buffer at pH 7. The inset indicates the current density
vs.number of cycles obtained at a xed potential (0.4 V vs. SCE)
from the voltammo-grams of the preparation of the PPy and PPy/Urs
lms.
J.C. Soares et al. / Reactive & Functional Polymers 72
(2012) 148152 149
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1.0 10 6 mol L 1), enabling us to obtain the current densities
atthe region of the peak oxidation potential (inset of Fig. 2a).
Thepeak oxidation potential shifted continuously to more
negativevalues after increasing the urea concentration, from 0.28 V
to
0.33 V vs. SCE, therefore dening an optimal operating
potentialrange for the activation of Urs within the PPy matrix. At
a relativelylow applied potential (chosen in this study as 0.28 V),
one can ex-pect minimum effects of the interference of species that
are onlyoxidizable at higher potentials. Fig. 2b shows that the
current den-sity increases linearly in response to increasing urea
concentra-tions at the peak oxidation potential. As expected, the
pH varies
only locally in response to variations in the concentration of
urea,since H + ions are consumed and ammonia is formed during
thehydrolysis of urea catalyzed by Urs. Since it is well known
thatPPy is a pH-sensitive polymer [2123] , the polymer matrix
re-sponds with a negative shift of its anodic potential when the
ureaconcentration is increased. As related to the role of the
immobi-lized Urs, the enzyme responds with an increase of the
currentdensity when in the presence of urea added into the
solution, aneffect not seen if only PPy lms are used.
Another technique used here to characterize the PPy and PPy/Urs
lms was UVVIS spectroscopy ( Fig. 3 ). The spectrum of thePPy lm
shows a well-dened band at about 400 nm and a free car-rier tail
after about 680 nm, which are typically seen in the spectraof PPy
lms with conductive properties [24] . For the PPy/Urs lm,
the band at about 400 nm is visible as a shoulder in the
spectrum,and a new band appears at about 283 nm due to the presence
of
peptide links (tryptophan and tyrosine residues) of Urs [25] ,
alsoconrming the entrapment of Urs in the PPy matrix.
The FTIR spectra of the PPy and PPy/Urs lms are quite
similar,and exhibit the main bands of a predominant PPy matrix, in
accor-dance with the literature [26,27] . For purposes of
comparison, tworegions in the spectra of the PPy and PPy/Urs lms
are presented inFig. 4 . From 1250 cm 1 to 1750 cm 1, the two bands
at 1460 cm 1
and 1543 cm 1 seen in the spectrum of the PPy lm can be
-1.0 -0.5 0.0 0.5 1.0
-2.0
-1.0
0.0
1.0
10 -6 10 -5
10 -4 10 -3
10 -2
-0,3 0,0 0,3 0,6 0,90,0
0,2
0,4
0,6
0,8
1,0
10 -3
10 -4
10-2
10 -5
j /
A c m
- 2
E/V vs . SCE
buffer
10 -6
without urea
j /
A c m
- 2
E/V vs . ECS
(a)
10 -6 10 -5 10 -4 10 -3 10 -20.3
0.4
0.5
0.6
0.7
0.8
[Urea] mol L -1
(b)
j /
A c m
- 2
Fig. 2. (a) Cyclic voltammograms of the PPy/Urs lm in phosphate
buffer at pH 7without and with urea at different concentrations and
(b) values of currentdensities vs. urea concentration obtained at
the peak oxidation potential.
400 600 800 1000
0.0
0.2
0.4
0.6
PPy
400
283
A b s o r
b a n c e / a . u .
Wavelength (nm)
PPy
PPy/Urs
Fig. 3. UVVIS spectra for the PPy and PPy/Urs lms (dotted line:
deconvolutioncurve).
500 600 700 800 900 1000 1100 1200
PPy/Urs
PPyPPy
1 2 1 1
1 1 5 4
1 0 8 4
9 6 6
7 7 7
6 6 5
1 2 1 3
1 1 5 2
1 0 8 2
1 0 2 7
9 6 8
9 1 3
7 7 3
6 6 7
% R
e f l e c t a n c e
5 3 8
6 2 5
PPy/Urs
1300 1400 1500 1600 1700
PPy
PPy/Urs
PPy/Urs
1 6 8 8
1 7 0 0
1 6 2 1
1 5 4 2
1 4 4 7
1 4 6 6
1 4 5 8
1 4 0 3
1 3 8 5
1 2 9 3
1 2 9 4
% R
e f l e c t a n c e
Wavenumber (cm -1)
PPy
Wavenumber (cm -1)
Fig. 4. FTIR spectra of the PPy/Urs and PPy lms at two
wavelength ranges.
150 J.C. Soares et al. / Reactive & Functional Polymers 72
(2012) 148152
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Urs lm is selective only to the presence of urea solutions,
partic-ularly in commercial proportions.
4. Conclusions
Amperometric biosensors for the determination of urea
weresuccessfully built by immobilizing urease (Urs) in
electrochemi-cally prepared polypyrrole (PPy) lms. The response of
these bio-sensors to different concentrations of urea in standard
solutionswas linear between 0.0 and 3.0 mmol L 1 . Based on the
chrono-amperometric curves, urea was detected at a relatively high
ef-ciency up to a concentration of 3.0 mmol L 1, i.e., a
sensitivity of 2.4 l A cm 2 mmol 1 L and a response time of about 3
min. Whencommercial fertilizers were tested, the PPy/Urs lms were
able todetect differences in the current density at concentrations
of 45%and 75% of urea. Therefore, the PPy/Urs lms provide a
suitablemeans of obtaining efcient and low-cost biosensors, since
they re-quire only the presence of pyrrole and Urs in the
preparation med-ium and involve only a few fabrication steps.
Acknowledgements
The authors acknowledge nancial support of FAPESP, CAPESand
CNPq. They are also grateful to Beatrice Allain for
manuscriptcorrections and suggestions.
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500 1000 1500 2000-50
-45
-40
-35
-30-25
-20
-15
-10
-5
0
Urea 75%
UreaUrea 45%
Buffer
(a) (b) (c)(d)
j / A
c m - 2
Time (s)
Fig. 7. Chronoamperometric curves for the PPy/Urs lm after
adding successivealiquots of fertilizer I (45% urea) (10 l L of0.1
molL 1 fertilizer I) (a), urea (100 l L of 0.1 mol L 1 urea) (b),
fertilizer II (75% urea) (10 l L of 0.1 mol L 1 fertilizer II)
(c),and phosphate buffer (100 l L of 0.1 mol L 1 phosphate buffer)
(d).
152 J.C. Soares et al. / Reactive & Functional Polymers 72
(2012) 148152
